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. 2016 Aug 19;43(Suppl 1):1–88. doi: 10.1159/000448382

Joint Congress DGTI & DGI 2016, 7.-10. September 2016, Nürnberg, Abstracts

Reinhold Eckstein 1,2,3, Robert Zimmermann 1,2,3, Bernd Spriewald 1,2,3
PMCID: PMC5396501

Band 43,

Supplement 1, September 2016

Transfusion Medicine and Hemotherapy

Founded in 1973 as «Die Infusionstherapie» by H. Reissigl, H. Grobecker, U. Henneberg, K.H. Bäßler Continued by: H.-D. Viering (1973–1977), A. Grünert (1982–1992), G. Kleinberger (1982–1988), K. Widhalm (1988–1992), G. Wolfram (1990–1992), J. Eckart (1992–1997), H. Forst (1992–1997), K. Meßmer (1992–1997), K. Peter (1992–1997), K.H. Usadel (1992–1997), B. Zwißler (1995–1997), K. Gutensohn (1998–2001), V. Kretschmer (1990–2002), W. Stangel (1992–2005), W Dzik (1998–2005), H.G. Klein (1998–2005), W. Sibrowski (2003–2014)

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Deutsche Gesellschaft für Transfusionsmedizin und Immunhämatologie

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Band 43,

Supplement 1, September 2016

Joint Congress DGTI & DGI

49. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie (DGTI)

24. Jahrestagung der Deutschen Gesellschaft für Immungenetik e. V. (DGI)

7.–10. September 2016, Nürnberg

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Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):IV.

Contents

Transfus Med Hemother 2016;43(suppl 1):IV

JS-01 Mesenchymale Stammzellen 2
JS-02 Regenerative Medizin I 2
JS-03 Stammzellen – regenerative Medizin II 4
JS-05 ATMPs I 5
JS-06 Therapeutische Ahperese I 6
JS-07 ATMPs II 6
JS-08 Therapeutische Apherese II 7
JS-09 Stammzellen – hämatopoietische Stammzellen und ATMPs 8
JS-10 Antikörperdiagnostik 12
JS-11 Antikörper, Eplets, Epitope 13
SI-02 Hämostaseologie – perioperatives Gerinnungsmanagement 14
SI-03 Klinische Transfusionsmedizin – Patient Blood Management 15
SI-04 Hämostaseologie – neue Aspekte der Hämostaseologie I 15
SI-05 Klinische Transfusionsmedizin – EK-Transfusion 17
SI-06 Hämostaseologie – neue Aspekte der Hämostaseologie II 17
SI-07 Klinische Transfusionsmedizin – Thrombozytentransfusion 19
SI-09 Abstractpräsentation – Immungenetik 20
SI-10 Klinische Transfusionsmedizin – neue Ansätze in der klinischen Transfusionsmedizin 24
SI-11 Immunhämatologie I 24
SI-12 Infektionen I 26
SI-13 Immunhämatologie II 28
SI-14 Infektionen II 29
SI-15 Präparative Apherese 31
SI-16 Blutspenderbetreuung 32
Poster
Gruppe I: Erythrozyten-, Granulozyten- und Thrombozytenphysiologie 33
Gruppe II: Immunhämatologie 36
Gruppe III: Herstellung von Blutkomponenten 46
Gruppe IV: Infektionsrisiken und Pathogeninaktivierung 50
Gruppe V: Klinische Hämotherapie und Patient Blood Management 52
Gruppe VI: Hämostaseologie, Koagulopathien, Plasmaderivate 54
Gruppe VII: Spenderrekrutierung, Spendermotivation und Spendersicherheit 57
Gruppe VIII: Organtransplantation 59
Gruppe IX: Stammzelltransplantation und Stammzellbiologie 60
Gruppe X: Zelltherapie und Tissue Engineering 66
Gruppe XI: Sonstige 76
Author Index 84
Imprint II
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Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):2.

JS-01-1 (INV. SPEAKER) Mesenchymal stromal cells: From bench to bedside and back

K Bieback 1

Human mesenchymal stromal cells (MSC) have been an attractive target for translational research in a wide range of therapeutic applications due to their paracrine effects, multi-lineage differentiation potential and their immunomodulatory properties. A number of clinical trials have been initiated. Although clinical trials document safety, outcomes from these clinical trials are not consistent with respect to efficacy. Further, the magnitude of clinical response appears to diminish moving from early stage science to more rigorous double blind studies. There are some thoughts to explain this: a) differences in the quality of the MSC preparation between donors’ b) differences in the protocols for cell manufacturing for clinical trials and c) differences in defining the end product use and quality. Still critical quality measurements are missing which help to predict and optimize the clinical efficacy. Clinical trials are currently the only mechanism to improve our understanding of clinical efficacy and the mode of action. Since the mode of action is highly complex, frequent product iterations appear to be necessary to tailor the product specifications, to enhance reproducibility and efficacy of clinical trials. Continual feedback between clinical (the bedside) and basic researchers (the bench) performing clinical, preclinical and nonclinical testing is needed to provide further insight into in vivo actions and to select and improve in vitro potency tests as predictors of clinical efficacy.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):2.

JS-01-2 (INV. SPEAKER) Harmonized protocol for MSC production for clinical trials

M Rojewski 1,2, R Lotfi 1,2, S Fleury-Cappellesso 3, Daganzo RM Gonzalo 4, H Rouard 5, T Montemurro 6, CG Gjerde 7, M Brennan 8, Eln Mustafa K Babikeir 7, C Ehrnthaller 9, A Ignatius 10, F Gebhard 9, C Avendaño 11, R Giordano 6, S Hellem 7, L Sensebé 12, Barrena E Gomez 13, P Layrolle 8, H Schrezenmeier 1,2

Introduction

The REBORNE (Regenerating Bone Defects using New biomedical Engineering approaches) consortium (call HEALTH-2009–1-4–2 of the European commission) used MSC in 3 clinical trials on bone regeneration produced at 5 manufacturing centers and applied at 6 clinical sites within Europe.

Methods

A harmonized 2-step protocol for GMP compliant production of 100 – 200 millions of MSC of passage 1 has previously been described and established and was validated at all manufacturing centers. The cells were used in the following trials: (i) EudraCT 2011–005441–13: Evaluation of efficacy and safety of autologous MSCs combined to biomaterials to enhance bone healing in patients with delayed consolidation after long bone fracture requiring graft apposition or alternative orthobiologics, (ii) EudraCT 2012–002010–39: Evaluation of safety and feasibility of bone marrow derived autologous MSCs to enhance bone healing in patients with avascular necrosis of femoral head, and (iii) EudraCT 2012–003139–50: Jaw bone reconstruction using a combination of autologous mesenchymal stem cells and biomaterial prior to dental implant placement.

Results

BM-MSC were isolated from 45 ± 18ml of BM aspirate. MSC of passage 0 (passage 1) were harvested after 14 ± 1 (7 ± 1) days at a density of 23.4 ± 18.6 x104 (43.9 ± 17.2 x104) cells/cm2 with 14 ± 2 (3 ± 1) population doublings. Quality controls were done according to table 1.

Tab. 1.

Matrix Ph. Eur BM aspirate MSC passage 0 MSC passage 1 additional in process controls in indicated country
Cell count and viability 2.7.29 + + +
Flow cytometry 2.7.24 CD73, CD90, CD105, CD45 CD73, CD90, CD105, CD45 CD3, CD34, MHC cI, MHC cII
Endotoxin testing 2.6.14 + +
Mycoplasma testing 2.6.7 and 2.6.21 + day 17/18 (D)
Microbial testing 2.6.27 + + + day 7 (D)
Differentiation capacity (osteogenic, chondrogenic, adipogenic) – +
Karyotyping – +
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Conclusion

BM-MSC are a powerful tool to induce healing of critical size bone defects. No therapy related adverse events were observed. 64 patients were treated in the 3 clinical trials.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):2–3.

JS-02-1 (INV. SPEAKER) From embryonic stem cells towards pluripotent stem cell-based replacement therapies

H Klump 1

Generation of transplantable cell, tissue and organ grafts, in vitro, is a highly desirable goal for future replacement therapies in humans. Pluripotent stem cells (PSCs), which include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great promise for achieving this objective and, thus, are expected to become important tools in clinical medicine. PSCs are defined by their capability to indefinitely self-renew in vitro while maintaining the potency to differentiate towards any cell of the adult organism. Autologous iPSCs obtained after reprogramming of a patient's own cells are of particular interest as the use of differentiated, mature progeny cells for transplantation or transfusion is supposed to preclude the adverse effects commonly associated with allogeneic cell therapies.

To date, large steps have been made towards clinical translation of PSC-derived cell therapeutics, exemplified by a number of clinical trials currently performed for a range of diseases such as neurodegeneration (e.g. Huntington disease or ALS), age-related macula degeneration (AMD) or type I diabetes mellitus. However, for the reliable generation of safe and efficient cell therapeutics, protocols for precisely controlled, stepwise and quantitative differentiation of PSCs towards the desired tissues still need to be significantly improved. In this presentation, I will discuss the current state, challenges and chances ahead for the use of pluripotent stem cell-derived therapeutics.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):3.

JS-02-3 Immunosuppressive capacity of mesenchymal stromal cells depends on allogeneic donor T-cells and correlates with metabolic activity

P Nold 1, M Killer 1, K Henkenius 1, L Fritz 1, A Neubauer 1, C Brendel 1, H Hackstein 2

Introduction

Mesenchymal stromal cells (MSC) have entered the clinic as Advanced Therapy Medicinal Product (ATMP) and are currently evaluated in a wide range of studies for tissue regeneration or in autoimmune disorders. Various efforts have been made to standardize and optimize expansion and manufacturing processes but until now reliable potency assays reflecting therapeutic effectiveness of MSC are still lacking. As recent findings suggest superior therapeutic efficacy of freshly administered MSC in comparison to frozen cells we sought to determine the immunosuppression capacity of MSC in relation to their metabolic activity.

Methods

Human MSC were obtained from bone fragments of patients and were employed in co-culture with peripheral blood mononuclear cells (PBMC) in an allogeneic T-cell proliferation assay to measure immunosuppressive function. Metabolic activity of MSC was measured real-time as aerobic glycolysis quantified by the extracellular acidification rate (ECAR) and mitochondrial respiration quantified by the oxygen consumption rate (OCR).

Results

We show that MSC induced suppression of T-cell proliferation varied enormously between different healthy donors of PBMC. Moreover, direct contact of PBMC and MSC increased the metabolic activity of MSC in a PBMC donor dependent manner. Enhanced lactate production as a surrogate parameter for glycolysis and increased oxygen consumption was paralleled by higher T-cell suppression capacities of MSC. The cryopreservative dimethylsulfoxide decreased metabolic and immunosuppressive performance of MSC while valproic acid enhanced metabolism and T-cell suppression.

Conclusion

Metabolic and immunosuppressive capacity of MSC are linked closely but are highly dependent on the predisposition of interacting T-cells which enhance MSC metabolism upon direct contact in co-culture. Hence functional fitness and quality of MSC can be determined by measuring metabolic activity and can be enhanced by exposure to valproic acid.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):3.

JS-02-4 Contact-dependent abrogation of bone-marrow-derived plasmacytoid dendritic cell differentiation by mesenchymal stem cells

H Hackstein 1, I Tschapikow 1, G Bein 1, P Nold 2, C Brendel 2, N Baal 1

Background

Plasmacytoid dendritic cells (pDC) are rare and central regulators of antiviral immunity and unsurpassed producers of interferon-α (IFN-α) in the immune system. Despite their crucial role bridging innate and adaptive immunity, little is known with respect to the modulation of pDC differentiation by other bone marrow cells.

Methods

We have investigated the modulation of pDC differentiation by prospectively purified mesenchymal stem cells (MSC) in Flt-3 ligand (Flt3L) supplemented bone marrow cultures. Highly pure MSC were FACS-isolated from murine bone marrow (BM) on the basis of PDFGRa+ SCA1+ CD45- TER119- (PαS) expression and used after in vitro expansion.

Results

Initial analysis revealed an almost complete inhibition of bone marrow-derived pDC expansion in the presence of >2% MSC. MSC-dependent inhibition of pDC generation was cell contact-dependent and soluble factor independent as indicated by trans-well experiments. The abrogation of functional pDC development by MSC was confirmed after TLR9 stimulation and revealed a complete, contact-dependent suppression of IFN-a producing capacity in Flt3L MSC BM co-cultures. MSC selectively inhibited pDC development in contrast to myeloid DC development as indicated by significantly increased numbers of myeloid DC in Flt3L supplemented BM cultures. The absence of significant MSC-mediated inhibitory effects on myeloid DC differentiation was confirmed by additional experiments in GM-CSF/IL-4 supplemented BM cultures.

Conclusion

We describe a novel contact-dependent immunomodulatory mechanism of MSC targeting bone-marrow derived expansion of functional pDC.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):3–4.

JS-02-5 ATP renders MSCs to immunosuppressive cells inhibiting lymphocyte proliferation and expressing IDO

R Lotfi 1,2, L Steppe 1, R Hang 1, M Rojewski 1, T Zelenski 2, B Jahrsdörfer 1,2, H Schrezenmeier 1,2

Background

Mesenchymal stromal cells (MSCs) act as immuosuppressive cells, partially due to the expression of the enzyme indoleamine dioxygenase (IDO) which converts tryptophan to kynurenine. Decreased concentration of tryptophan and increased kynurenine, both inhibit lymphocyte proliferation. Accumulation of MSCs within tumor tissue is associated with tumor progression. Necrotic cell death with release of damage associated molecular patterns (DAMPs) is a characteristic feature of advanced solid tumors. ATP is a DAMP family member. Hence, unphysiologically increased concentrations of ATP is found around stressed and necrotic (tumor) tissue.

Methods

MSCs were generated from bone marrow of healthy donors. Fibroblastoid-shaped adherent cells with capacity for chrondrogenic, ostegenic, and lipogenic differentiation and positive for CD73, CD90, CD105 and HLA-A, B, C and negative for CD34, CD3, CD45 were referred to as MSCs. In the presence of ATP at concentrations between 62 to 2000 µM, MSCs were cultured for 4 days in DMEM containing 10% human serum and 100µg tryptophan/ml. Supernatants were tested for kynurenine in a colorometric assay, as well as for their capacity to inhibit lymphocyte proliferation. Metabolism was assessed by measuring WST-1 cleavage. MSC proliferation was measured by using CyQuant detecting DNA-content. Intracellular expression of IDO in MSCs was assessed by FACS.

Results

ATP increased dose dependently the expression of IDO in MSCs with subsequent increased kynurenine concentration within the supernatant at about 60%. This effect could be abolished completely in the presence of ATP degrading enzyme (apyrase) or when MSCs were pretreated with a P2X7-receptor antagonist (AZ 11645373). Consistently, supernatants from MSCs stimulated with ATP inhibited lymphocyte proliferation from 65% to 16%. Of note, ATP intensely enhanced MSC metabolism without having any influence on their proliferation.

Conclusion

We characterized ATP as a DAMP family member responsible for necrosis-induced immunomodulation. Given the increased concentration of DAMPs within tumor tissue and the fact that necrotic material / DAMPs can act as chemotattractants to MSCs, our results have implications for therapeutic strategies targeting the tumor microenvironment.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):4.

JS-03-3 Effect of MSC treatment in a blunt chest trauma model

EM Amann 1, M Rojewski 1,2, S Rodi 1,2, D Fürst 1,2, J Fiedler 3, A Palmer 4, S Braumüller 4, R Brenner 3, M Huber-Lang 4, H Schrezenmeier 1,2

Introduction

Severe blunt chest trauma often leads to Acute Respiratory Distress Syndrome (ARDS) with a mortality rate of more than 30% and causal therapeutic approaches to improve regeneration are still missing. Therefore, we have investigated the immunomodulatory and regenerative effects of a single dose of mesenchymal stromal cells (MSC) immediately after the induction of blunt chest trauma in Wistar rats.

Methods

Blunt chest trauma (TxT) was induced by a single blast wave on the Thorax of male Wistar rats. Our study included six groups: sham, TxT rats and rats receiving MSC after TxT (PKH26-stained human (hMSC) or rat MSC (rMSC) with or without IL-1β-priming of MSC). After 24 h and 5 days, the histological score of lung injury and levels of several cytokines in plasma and BAL were determined (e.g. IL-1β, IL-10), and BAL cellular differential count was performed. Distribution of administered MSC in blood, BAL and several organs was investigated by flow cytometry and ALU sequence analysis.

Results

The histological lung injury score was significantly higher in TxT rats than in sham rats after 24 h. A significant reduction of the lung injury score of at least 50% in rats treated with hMSC and primed hMSC was found. The recovery of the PKH26 labelled cells was higher in whole blood than in BAL fluids. PKH positive cells in blood of hMSC treated rats did not express MSC markers (e.g. hCD90) whereas blood of rats treated with rMSC contained a fraction of cells positive for rMSC markers (e.g. rCD29). TxT resulted in an enhanced recruitment of neutrophils into the BAL after 24 h, which was significantly reduced by application of IL-1ß-primed rMSC. Relative proportion of alveolar macrophages in the BAL decreased after TxT, whereas macrophage content did not differ from sham rats in animals treated with IL-1ß-primed rMSC. There was rather a local inflammatory reaction than a systemic one, as evidenced by cytokine analysis of the BAL. After 5 days, cytokine levels and the distribution of inflammatory cells were similar to sham rats.

In conclusion, blunt chest trauma seems a suitable model to study effects of MSC as an acute treatment strategy after trauma. However, our results indicate that the source of MSC has to be carefully considered in the design of the model.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):4.

JS-03-4 Acupuncture-induced potential regeneration in patients with spinal cord injuries: a summary of four case reports

M Wu 1, S Moldenhauer 2, M Dütsch 3, T Scheffel 4, R Hellweg 4, A Holloschi 5, FR Abel 6, W Han 1, A Moldenhauer 7,8

Spinal cord injuries (SCI) result in mostly traumatic destruction of neurological structures perishing sensory and motor function. Acupuncture can be used for inducing functional recovery, however the underlying mechanisms remain unclear. Previously, we demonstrated the mobilization of regenerative factors like potential neuroprogenitors in healthy volunteers. As a logical consequence, we hypothesized that acupuncture could induce the mobilization of CD133+ CD34– cells in patients with SCI. Therefore, four patients were acupunctured 15 times over a period of eight weeks stimulating points for the treatment of SCI. The frequency of CD133 and CD34 cells in peripheral blood and the serum concentrations of matrix metalloproteinase (MMP)-9 and brain-derived neurotrophic factor (BDNF) were determined before and after each treatment. Moreover, the morphology and immune phenotype of cultured cells were observed in vitro. American Spinal Injury Association (ASIA) impairment scale and short-form (SF) health surveys were taken to monitor neurological changes and the quality of life before the first and after the last acupuncture treatment. The number of CD133+ CD34- cells increased with a concomitant increase in serum MMP-9 and decrease of BDNF within the treatment period. Cultured cells could differentiate into neuron-like cells positive for nerve growth factor receptor (NGFR) and, to a lower extent, for Nestin. Motor function determined by ASIA criteria marginally changed to the better, and sensory function continuously improved after acupuncture treatments in all patients. Our results indicate that acupuncture can promote the mobilization of CD133+ CD34- cells in SCI patients accompanied by increasing concentrations of MMP-9 and decreasing serum levels of BDNF including a slight clinical effect.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):4.

JS-03-5 Feasibility of a GMP compliant production process of a cartilage graft derived from human MSC

C Schmidt 1, O Petters 1, S Küchler 1, T Kugel 1, H Holland 2, G Hütter 3, R Schulz 1

Introduction

Current therapies for cartilage defects, like microfracture and (matrix-associated) autologous chondrocyte transplantation (M/ACT) have several disadvantages including formation of inferior fibrocartilage and chondrocyte de-differentiation.

Alternatively, application of mesenchymal stromal cells (MSCs) instead of chondrocytes in MACT-like grafts have shown a beneficial outcome compared to chondrocyte-based MACT in animal models. However, a major challenge is the translation of this basic research approach to a clinical MSC-MACT graft in compliance to the regulatory framework.

Herein, we evaluated the feasibility of a GMP compliant production process of a MSC-based MACT transplant according to the ISCT recommendations and the regulatory requirements.

Methods

Human bone marrow (hBM) samples of the iliac crest and GMP-compliant raw materials were used to evaluate various process variables, like logistics conditions, density gradients and expansion media as well as differentiation protocols within a 3D collagen matrix. The process

parameters were examined concerning the following quality aspects: identity, purity, viability, frequency, proliferation, differentiation capacity and genetic stability of the cells.

Results

The immediate processing of hBM, stored at room temperature, was beneficial concerning the yield and quality of MSC compared to longer transport conditions and lower storage temperature. Whereas, the yield and quality of MSCs displayed no significant variances using different density gradients, the yield and quality of expanded MSCs differs remarkably between the different GMP expansion media. The oxygen concentration and the administered dose of TGF-ß3 affect the chondrogenesis of MSCs in 3D matrix massively.

Conclusion

With this translational in vitro study it becomes evident that every single process variable, especially the logistics, the expansion media composition and the differentiation protocol substantially influences the quality of the product. Nonetheless, the herein optimized production process for MSC-MACT fulfills the ISCT recommendations regarding the quality, identity and purity of cells. In summary, a GMP-compliant production of a MSC-based MACT is feasible and reliable.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):5.

JS-05-1 (INV. SPEAKER) ATMPs from hematopoietic stem cells

H Boenig 1,2

Relatively abundant, well defined and easily accessible, hematopoietic stem cells (HSCs) are a favored tool for many cell therapies. HSC-based medicines immediately become advanced therapy medicinal products (ATMPs) if the cells have undergone more than minimal manipulation or if its therapeutic purpose is anything other than hematopoietic reconstitution, irrespective of the donor-host relationship. Outside the field of transplantation, HSCs are currently mostly used in regenerative medicine and as targets for gene replacement.

HSCs are being explored in cardiovascular disease for regeneration of acute or chronic post-ischemic myocardial damage and for attenuation of peripheral arterial disease, for liver regeneration in toxic liver cirrhosis, for retinal disease as well as for bone regeneration, to name just a few indications. Although many studies suggest arguably meaningful beneficial results, the vast range of diseases/disease stages, cell types, manufacturing processes, timing and modes of application and variable end points considered to this day have hampered definitive assessment of their therapeutic benefit. Sufficiently large studies are for the most part lacking, as the private sector remains indifferent and the public sector unable or unwilling to provide the necessary funding.

Genetic modification of HSCs, conceptually providing a cure for monogenetic hematopoietic or immunological illnesses, has been attempted for more than two decades. While studies in hereditary immunodeficiency syndromes like X-linked SCID, WAS or ADA deficiency demonstrate that, in principle, the approach is reasonable, the same studies have also demonstrated that currently available tools for genetic modification remain too imprecise, as many of the patients have developed malignancies from unfavorable vector integration.

In discussing some examples of either type of therapy, pitfalls and potential future avenues of HSC-based ATMPs will be explored.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):5.

JS-05-2 (INV. SPEAKER) ATMPs: Virus-specific T cells from third party T cell donors

B Eiz-Vesper 1

Introduction

Patients after transplantation are rendered susceptible to infections and reactivations caused by CMV, EBV, HHV6, ADV, and BKV. The shortcomings of conventional therapies have increased the interest in antiviral T-cell transfer. The efficacy and the clinical outcome in high risk patients will be improved by a rapid recruitment of a respective T-cell donor and an established method for fast manufacturing of antiviral T cells.

Material

An allogeneic registry (alloCELL) was established currently recording >1550 HLA-typed donors and complying screening results on antiviral T-cell repertoire of >550 donors. The alloCELL lab further established comprehensive protocols to consider clinical requirements of patients at high risk for viral infections or with failed conventional therapy. The manufacturing license was obtained for generating clinical-grade mono- and multivirus-specific T-cell products according to the German Medicines Act. T-cell donors were defined as eligible if ≥0.03% specific IFN-γ+ T cells are detectable. A related or at least 5/10 HLA-A/B/DR-matched alloCELL donor is chosen, if the stem cell donor is not eligible.

Results

Antiviral T-cell frequencies in patients after transplantation were determined routinely by ELISPOT and multimer staining. 26 out of 88 monitored patients failed to respond to antiviral treatment and were assigned to receive antiviral T-cell products. For those 58 donors were tested: 25 family, 8 stem cell, 25 alloCELL donors and clinical-grade antiviral T cells were generated. T cells applied were monitored to determine frequency and chimerism. Patients received T cells without significant side effects and in 78% antiviral T cells were detectable.

Conclusion

These data underline the need of (i) accurate monitoring of viral load and antiviral T-cell frequencies in patients, (ii) early selection of suitable T-cell donors and (iii) support data about safe and effective application of third-party antiviral T-cell products.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):5.

JS-05-3 (INV. SPEAKER) ITherapeutic potentials of Mesenchymal Stem Cell-derived extracellular vesicles

B Giebel 1

MSCs have been used to treat a variety of different diseases such as myocardial infarction, stroke and graft-versus-host disease (GvHD). Initially, MSCs were thought to replace lost cells in damaged tissues. Despite contrary reports regarding the outcome of MSC treatments, MSCs seem to exert their beneficial effects by the secretion of immunosuppressive factors and/or small extracellular vesicles (EVs, 70–150 nm), such as exosomes and microvesicles, rather than by interacting into affected tissues. After setting up techniques for the characterization and large scale preparation of EVs, we have treated a steroid-refractory GvHD patient with EVs/exosomes enriched from MSC supernatants (MSC-EVs). In addition we investigate the MSC-EVs’ therapeutic potential to exert neuroprotective functions in animal models for ischemic stroke and preterm brain injury. So fare, we observed beneficial effects in all settings. In the murine stroke model we performed a side by side comparison of the therapeutic effect of MSCs and their EVs and did not recognize any differences, both improved the symptoms significantly. At the functional level MSC-EVs were shown to exert immunosuppressive functions in vivo and in vitro.

Resources:

http://www.ncbi.nlm.nih.gov/pubmed/24445866

http://www.ncbi.nlm.nih.gov/pubmed/26339036

http://www.ncbi.nlm.nih.gov/pubmed/26725829

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):6.

JS-06-1 (INV. SPEAKER) Novel concepts in extracorporeal photopheresis

H Hackstein 1

Extracorporeal photopheresis (ECP) is an important cell-based therapy for heterogenous immune-mediated diseases such as cutaneous T cell lymphoma, graft versus host disease (GVHD) and organ allograft rejection. Despite its documented clinical efficacy major mechanistic aspects are unknown, such as the possible induction of immunostimulatory mediators by ECP as well as the blood volume required per treatment to achieve a clinical response.

Recently we have reported that ECP promotes marked release of the prototypic immunostimulatory cytokine IL-1β. ECP primes IL-1β production and activates IL-1β maturation and release in the context of caspase-1 activation in monocytes and myeloid dendritic cells. Interestingly, IL-1β maturation by ECP was fully intact in murine cells deficient for caspase-1, suggesting the predominance of an inflammasome-independent pathway for ECP-dependent IL-1β maturation. Clinically, patient analysis revealed significantly increased IL-1β production in stimulated leukapheresis concentrates and peripheral blood samples after ECP.

With respect to the minimal blood volume to achieve a clinical response we have developed a mini ECP technique (mini-ECP) using only 100–200 ml of whole blood for patients with contraindications for apheresis or low body weight. Mini-ECP is well tolerated and acute and chronic GVHD resolved in a significant amount of patients.

The identification of IL-1β after ECP provides new insight into the immunostimulatory capacity of photopheresis. The clinical results of mini-ECP indicate that processing of low volumes of blood may be sufficient for successful GVHD-therapy in pediatric patients with contraindications for apheresis.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):6.

JS-06-2 (INV. SPEAKER) Clinical use of therapeutic plasmapheresis

J Kößler 1

Plasmapheresis is a procedure used to remove plasma or plasma components from patients for therapeutic purposes. The most common indications for plasmapheresis refer to acute autoimmune-mediated or malignant diseases to reduce the levels of autoantibodies, proteins or other disease-associated agents in the plasma.

In most cases, plasmapheresis is part of a complex therapy regimen requiring the collaboration of treating specialists with the department of transfusion medicine performing the procedure. According to current guidelines of the American society for Apheresis (ASFA), indications for plasmapheresis are classified into different categories. In category I, plasmapheresis is considered to be part of the first-line therapy. Typical disorders of category I are e.g. Guillain-Barré syndrome, myasthenia gravis or thrombotic-thrombocytopenic purpura. In disorders of category II, plasmapheresis may be used as second-line therapy (e.g. in acute demyelinated encephalomyelitis). In category III, the role of plasmapheresis is not established requiring individual therapy decisions. In category IV, plasmapheresis is not recommended.

Before starting plasmapheresis therapy, initial concerns should address venous access, surveillance of patients, anticoagulation, blood count, interfering medication and additional diseases of the patient.

Technical approaches for plasmapheresis comprise plasma exchange or immunoadsorption. In plasma exchange, removed plasma is substituted by replacement fluids, either frozen plasma or solutions including albumin. In immunoadsorption, plasma is directed through columns retaining antibodies or other agents with different selectivity. For anticoagulation, heparin or citrate solutions are used. Although certain levels of standardization are achieved by subject-specific guidelines, decisions of therapy schedules are often rather specific for institutions and dependent of available technical devices and individual experience.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):6.

JS-06-3 Extracorporeal photopheresis in the treatment of GVHD

N Worel 1, H Greinix 2

Graft-versus-host disease (GVHD) is a serious complication of allogeneic hematopoietic cell transplantation causing significant morbidity and mortality. Steroids are the established first-line treatment of GVHD but patients not responding tosteroids have a dismal prognosis. Extracorporeal photopheresis (ECP) has also proven highly effective in the treatment of GVHD and has become a frequent part of the treatment program for selected patients in most centers.

A meta-analysis in 2002 reviewed 31 studies using ECP in 76 patients with acute and 204 patients with chronic GVHD. In acute GVHD patients skin manifestations regressed in 83% (complete response CR in 67%). CR of liver disease was reported in 38%, and of gut disease in 54% of patients. In chronic GVHD response rates were: cutaneous 75% (36% CR), lung 48%, liver 39%, and oral lesions 63%. There was a tendency to better results with earlier treatment. The overall 1-year survival was 79% vs 56–71% with conventional treatment. A subsequent prospective study of acute GVHD in steroid-refractory (SR) patients confirmed the excellent results of ECP. CR of acute GVHD was seen in 82% of patients with cutaneous, in 61% with liver, and in 61% with gut disease. A prospective study of 95 adults with chronic cutaneous GVHD compared conventional therapy to conventional therapy plus ECP and showed CR in 40% of ECP treated patients vs 10% with conventional therapy alone (p < 0.05). A recent retrospective trial compared ECP (n = 57) to anticytokine therapy (n = 41) in patients with SR acute GVHD. ECP was associated with superior survival (hazard ratio [HR], 4.6; p < 0.05) in patients with SR grade II aGVHD.

ECP treatment allows to reduce the steroid dosage and other immunosuppressive drugs resulting in reduced long-term morbidity and mortality in responding patients. Data collected during the last 30 years of the use of ECP demonstrated that the procedure is tolerated well, with no clinically significant side effects.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):6–7.

JS-07-2 (INV. SPEAKER) NK cells in cancer therapy: different approaches in optimization interaction between effector and target cells

A Humpe 1, C Kellner 1, M Gramatzki 1, M Peipp 1

Different areas of interaction between tumor cells, NK-cells and antibodies are of clinical importance. First, antibodies directed against specific antigens on tumor cells might be used for flow cytometry. Second, antibodies detecting potential tumor cells might serve as tools for tumor cell depletion. Such antibodies like the CD96 antibody directed against an antigen predominantly being expressed by tumor stem cells in patients with an AML can be conjugated with paramagnetic beads enabling depletion of AML stem cells in autologous hematopoietic stem cell grafts. Third, the interaction between NK-cells as effector cells and tumor cells as target cells relies in part on the principle of antibody-dependent cell-mediated cytotoxicity (ADCC). Different strategies are available to optimize this interaction on the basis of manipulation of existing antibodies or on the basis of manufacturing new molecules that trigger improved interaction of NK-cells and target cells. First, the antigen binding property of a Fab part can be improved by affinity maturation steps. Second, the interaction with the NK-cell can be optimized by modifications in the Fc part by protein-engineering or glyco-engineering. With respect to CD96 an affinity maturated single chain fragment of the variable regions (scFv), in combination with an engineered Fc part led to the highest rates of lysis compared to all other constructs (PlosOne, 2012). A different approach are so-called tribodies, the combination of two scFvs against antigens on a tumor cell, like the CD20 on lymphoma cells, with a Fab fragment binding to CD16a (FcgRIIIa) on NK-cells. Such a tribody lead to superior lysis of CLL cells compared to Rituximab (Leukemia, 2013). Finally, immunoligands combining a scFv against CD20 with ligands like ULBP2 or B7-H6 against the activation receptors NKG2D or NKp30 on NK-cells effectively result in lysis of tumor cells alone or potentiated in combination strategies (Leukemia, 2012; OncoImmunology 2015).

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):7.

JS-07-3 (INV. SPEAKER) Development of ATMPs and regulatory aspects

K Cichutek 1, R Sanzenbacher 1

This presentation aims to provide an overview on current developments of Advanced Therapy Medicinal Products (ATMP) as well as on the regulatory framework in EU and Germany, also addressing current challenges and upcoming modifications. On the legal basis of Regulation (EC) No. 1394/2007 seven ATMPs marketing authorization applications submitted to the European Agency (EMA) have been successful so far. Several promising investigational products are in the pipeline. Recently, the European Commission has filed a report on the stakeholders experience on the implementation of the ATMP regulation. Subsequently, the discussion on how the regulatory setting can be further improved has been re-opened. Key issues of discussion are the adaptation of GMP for ATMP and the application of the so-called hospital exemption (HE). The HE can be applied on the national level in situations of medical need and when no authorized product might be available to enable patients to receive “non-routine” ATMPs. Additionally, the concept of offering patients ATMP therapies directly at PoC will be discussed.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):7.

JS-08-2 (INV. SPEAKER) ABO-incompatible kidney transplantation

S Zschiedrich 1, B Jänigen 2, P Pisarski 2, A Kramer-Zucker 1

Background

ABO-incompatible kidney transplantation (ABOi KTx) expands the living donor transplantation options. However, long-term outcome data especially in comparison to ABO-compatible kidney transplantation (ABOc KTx) remain limited. Since the first ABOi KTx in Germany on April 1st 2004 at our center, we have followed 100 ABOi KTx over up to 10 years.

Methods and Results

100 ABOi KTx and 248 ABOc KTx from April 1st, 2004 until October 28th, 2014 were analyzed in this observational, single-center study. 3 ABOi KTx and 141 ABOc KTx were excluded because of cyclosporine A based immunosuppression, 1 ABOc KTx was lost to follow-up. Median estimated 10-year patient and graft survival in ABOi KTx was 99% and 94%, respectively and surpassed ABOc KTx patient and graft survival of 80% and 88%, respectively. Incidence of antibody-mediated rejections was 10% and 8%, and incidence of T-cell mediated rejections was 17% and 20% in ABOi KTx and ABOc KTx, respectively. Infectious and malignant complications in ABOi KTx were not more common than in ABOc KTx. However, postoperative lymphoceles occurred more frequently in ABOi KTx. Subgroup analysis of ABOi KTx patients revealed that patients with high-titer isohaemagglutinins before transplantation had equal long-term results compared to low-titer isohaemagglutinin patients.

Conclusions

Taken together, long-term outcome of ABOi KTx is not inferior to ABOc KTx. Incidences of rejection episodes, infectious complications and malignancies are not increased despite the more vigorous immunosuppression in ABOi KTx. Our data provide further evidence that ABOi KTx with living donation is a safe, successful and reasonable option to reduce the organ shortage.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):7.

JS-08-3 ABO incompatibility has an impact on T cell flow cytometry crossmatch results

M Lindemann 1, V Lenz 1, B Nyadu 1, FM Heinemann 1, A Heinold 1, H Guberina 2, U Eisenberger 2, N Lachmann 3, C Schönemann 3, A Kribben 2, A Paul 4, O Witzke 2, PA Horn 1

Aim

This study focuses on the impact of ABO incompatibility on results of the flow cytometry crossmatch (FCXM), a highly sensitive method to assess histocompatibility prior to solid organ transplantation.

Methods

We analyzed 29 ABO incompatible donor-recipient pairs (73 data sets, i.e., on average 2.5 tests per pair) prior to living donor kidney transplantation. As a control group, 89 ABO compatible pairs (175 data sets) were included. All donor-recipient pairs were negative for lymphocytotoxic crossmatches of T and B cells.

Results

Recipients with blood group O (A to O and B to O) displayed significantly (P < 0.05) higher T-FCXM results than those with blood group A and B (A to B, B to A and AB to A), respectively. Furthermore, donor-specific T-FCXM responses (delta MFI values) were significantly higher (P < 0.05) in all ABOi groups than in the ABO compatible group (ABOi recipient with blood group O: 32 ± 6; with blood group A: 19 ± 7; with blood group B: 7 ± 4; with ABO compatibility: 3 ± 2, resp., data represent mean ± SEM). Consistent with the T-FCXM results donor-specific isohemagglutinins (IgG titers) were significantly higher in recipients with blood group O vs. A, both prior to rituximab treatment and plasmapheresis/immune adsorption (P = 0.004) and immediately prior to kidney transplantation, i.e., after rituximab and antibody-depleting therapies (P = 0.04). As expected, IgG titers prior to rituximab treatment and prior to transplantation were significantly correlated (r = 0.40, P = 0.04). IgM titers of donor-specific isohemagglutinins yielded similar results. Recipients with blood group O vs. A displayed significantly higher titers prior to rituximab treatment and prior to transplantation (P = 0.02 each).

Conclusion

Reference values for the T-FCXM have to be re-assessed prior to ABO incompatible transplantation. The mechanism leading to increased T-FCXM results needs to be further elucidated.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):8.

JS-08-4 Correlation of biomarkers with GvHD and response to extracorporeal photopheresis

H Budde 1, S Papert 1, G Wulf 2, J Hasenkamp 2, J Riggert 1, J Legler T 1

Background

Biomarkers are getting more and more important in order to allow personalized therapy. Up to now no biomarker for GvHD occurrence after hematopoietic stem cell transplantation (HSCT) is established in clinics. Furthermore biomarkers which correlate or even predict the response to extracorporeal photopheresis (ECP) require further research.

Methods

As potential cellular biomarkers we analyzed CD3+ T cells, CD3+ CD4+ T-helper cells, CD3+ CD8+ cytotoxic T cells, CD4+ CD25+ FoxP3+ Tregs, CD19+ CD21- precursor B cells and Lin1- CD11c+ HLA-DR+ myeloid dendritic cells. In addition we evaluated the serum levels of soluble IL-2 receptor, soluble TNF receptor and hepatocyte growth factor. Reference ranges were determined in healthy blood donors. Patients with hematopoietic malignancies were investigated before hematopoietic stem cell transplantation and after engraftment. During follow-up for 6 months occurrence and severity of GvHD was monitored. Biomarkers to determine ECP response probability were analyzed from GvHD patients before first ECP, three months and six months after starting with ECP.

Results

The percentage of CD3+ and CD4+ cells increased in GvHD patients after HSCT in contrast to patients without GvHD in which the percentage decreased. CD8+ cells showed a tendency for increased levels which correlated with GvHD severity. The percentage of CD19+ CD21-cells increased after HSCT in GvHD patients. Higher levels of sIL-2R before and after HSCT correlated with reduced survival. The sTNFR levels were reduced in GvHD patients before and after HSCT compared with healthy controls. The number of CD19+ CD21- cells is decreasing during the first six months of ECP while the number of CD8+ cells is increasing.

Conclusions

Our data provide information about several potential cellular and cytokine biomarkers for GvHD in general and ECP therapy in particular. When combining our findings with results from literature, CD3+, CD4+, CD8+ and CD19+ CD21- cells seem to be interesting candidates as future biomarkers in GvHD therapy.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):8.

JS-08-5 Erythrocyte depletion from bone marrow with the Spectra Optia BMP module: A propos of the first 28 preparations

C Poppe 1, M Bunos 1, E Wingenfeld 1, S Beyer 1, E Seifried 1,2, H Boenig 1,2,3

Background

Erythrocyte (RBC) depletion is a standard technique for preparation of ABO-incompatible BM transplants. We previously reported systematic comparative analyses of five competing technologies, i.e. COBE2991, Spectra Optia BMP, Sepax II NeatCell, Miltenyi CliniMACS Prodigy Density Gradient Separation system and manual Ficoll. Based on the outcomes, we established Spectra Optia BMP as the standard technology. We here present outcomes from 28 routine BM RBC-depletion procedures with Spectra Optia BMP.

Methods

BM was harvested from healthy volunteer donors. BM was RBC depleted with Spectra Optia with BMP software, fitted with IDL tubing and BMP accessory kit. Indications were ABO incompatibility or intention to cryopreserve. BM was assessed for total volume (weight corrected for Hct), RBC volume (calculated from Hct (Sysmex 1800 XT) and volume) and CD34+ cell content (SCE single platform) before and after processing. Recovery of CD34+ cells was calculated there from.

Results

28 BM harvests were analyzed. Apheresis proceeded automatically w/out any issues and w/out operator interference except for adjustment of collection preference based on collection flow color. Starting from a volume of 1294 ± 62 ml (mean±SEM), RBC volume of 396 ± 29 ml and viable (7AAD-) CD34+ cell content of 265 ± 25 × 10e6, products were generated with a volume of 178 ± 12 ml, containing 7.7 ± 0.9 ml RBC and 242 ± 24 × 10e6 viable CD34+ cells. Thus 98 ± 0.2% of RBC were removed and 91 ± 2.3% of CD34+ cells recovered. CD34+ cell viability was 94 ± 1% (pre) and 95 ± 1% (post). No de-novo bacterial contamination occurred. For some transplants, engraftment data were available; there was no primary graft failure and engraftment kinetics for neutrophils and platelets were as expected for BM.

Discussion

Spectra Optia BMP is robust and easy-to-use. Outcomes are highly predictable with respect to RBC depletion and CD34+ cell recovery. The selection process is easily integrated into the daily routine of a cell therapy laboratory. Expecting a surge in BM collections due to the increasing adoption of the Baltimore protocol and derivative transplant schemes, the new module is timely.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):8.

JS-09-1 (INV. SPEAKER) Revision of the human hematopoietic tree – a continuously changing model?

A Görgens 1, S Radtke 1, S Vitoriano 1, F Murke 1, M Möllmann 2, J Dürig 2, PA Horn 1, B Giebel 1

Three important goals of hematopoietic stem cell research are to understand how hematopoietic stem cells (HSCs) self-renew, how lineage commitment takes place and how HSCs can be expanded ex vivo. An important prerequisite for uncovering such mechanisms is the existence of a reliable model detailing the hierarchical interrelationship of different hematopoietic progenitor cells and their mature offspring. The prevailing model of the last 30 years was the so-called classical model of hematopoiesis proposing a clear separation of lymphoid and erythromyeloid lineages. Especially during the last decade a number of murine and human progenitor cells have been identified, whose developmental potentials do not correspond to the classical model and have called it into question. By analyzing human HSPC subpopulations by means of their CD133 surface expression, we recently gained evidence for new hematopoietic lineage relationships and postulated a revised model of human hematopoiesis (Görgens et al, Cell Reports 2013). We revealed that CD133+ multipotent progenitors (MPPs) create CD133+ lymphomyeloid (LMPP) and CD133low erythromyeloid (EMP) daughter cells. The LMPP lineage was shown to contain lymphoid and neutrophil potentials, while the EMP lineage mainly creates eosinophils and basophils as well as erythrocytes and megakaryocytes. Regarding lineage specification, we showed for the first time that under conventional culture conditions almost all MPPs divide asymmetrically to create a set of LMPP and EMP daughter cells, resulting in a loss of MPPs after the first cell division (Görgens et al, Stem Cell Reports 2014). Thus, our data suggest that at least under conventional culture conditions asymmetric cell divisions are rather lineage instructive than self-renewing. Within this talk I will summarize and discuss recent findings contributing to a continuous revision of the human hematopoietic tree.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):10.

JS-09-2 (INV. SPEAKER) Conserved lineage development in human and nonhuman primate hematopoiesis

S Radtke 1, JE Adair 1, H-P Kiem 1

For more than three decades, hematopoietic stem cell (HSC) research has been performed based on the classical model of human hematopoiesis. However, this model has been challenged by several groups proposing a great variety of alternative lineage relationships and read-outs for multipotent HSCs. While hematopoietic lineage relationships are still under investigation, consequences of these findings on the development of treatment strategies for hematological diseases and malignancies are rarely discussed. A critical factor for the development of allogeneic and autologous stem cell therapies is the availability of a read-out for multipotent HSCs supporting the development of all blood cell lineages and at the same time the evaluation of long-term engraftment potential.

Due to their close evolutionary relationship with humans, nonhuman primates NHPs are frequently used as a pre-clinical model system to develop HSC gene therapy approaches. Surprisingly however, validation of this large animal model for pre-clinical HSC research comparing hematopoietic subpopulations as well as the hierarchical organization of defined lineages has not been performed between NHPs and humans.

Here we show for the first time a phenotypic mapping strategy in NHP hematopoietic cells predicting a revised model of hematopoiesis with HSCs giving rise to MPPs followed by a simultaneous segregation of lympho-myeloid, erythro-myeloid and megakaryocytic potentials. Furthermore, we identified corresponding hematopoietic subpopulations in human and NHP, which share phenotypical, functional and transcriptional properties in both species, validating the NHP as a clinically relevant model for stem cell biology and transplantation.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):10.

JS-09-3 Legal framework of cell-based therapies for cartilage repair in point-of-care settings – therapy concepts between innovative personalized medicine and missing legal (ATMP) control?

T Faltus 1, R Schulz 2

Question

The project questions the legal handling of cell-based therapies in point-of-care (PoC) settings in the area of musculoskeletal regeneration. Beside numerous case series conducted as non-randomized controlled trials (non-RCT) with stem cell preparations of different origin and specifications actually two RCTs processed allogeneic bone marrow derived mesenchymal stem cells for intraarticular cell injection for the treatment of osteoarthritis. Cell-based PoC therapies are considered to have benefits, e.g. being rapid and/or economic. However, the regulatory frameworks for Advanced Therapy Medicinal Products (ATMP) and medical devices for PoC cell-based therapies leave loopholes which seem to open doors for unsafe, non-efficient or unproven therapies.

Material and Methods

The project evaluates the state of the art in cell-based PoC therapy models for cartilage repair including/based on “stem cells” and analyses the legal handling of such therapy concepts in an interdisciplinary approach. The project extrapolates the current technical development to examine technical advancements of such therapies. Finally, the project examines within a technological impact assessment how such prospective therapies would and should be regulated.

Results

The legal handling of cell-based therapies in PoC settings is incomplete. Depending on the processing of biopsied cells only in some cases a manufacturing licence is mandatory. The lack of regulation causes that some PoC therapies do not have to be manufactured under Good Manufacturing Practice (GMP). Therefore, in many settings such therapies are either unregulated or only partly regulated although these cells have the character of cell-based pharmaceuticals comparable to other regulated cell-based ATMP therapies. Additionally, for cell therapies in PoC settings there is no legal need to proof such therapies by quality studies, non-clinical, and clinical trials.

Discussion

The assurance of safety, efficient, and proven cell therapies in PoC settings needs a legal ATMP and/or medical device framework. The major obstacle is the separation of medical device law and pharmaceutical (ATMP) law. The project proposes changes in legislation where the current pharmaceutical legislation seems to be insufficient to ensure safe and efficient treatments.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):10.

JS-09-4 Analysis of T cell loss in cryopreserved leukapheresis products collected for hematopoietic transplantation

C Berens 1, J Müller 1, J Oldenburg 1, D Wolf 2, B Pötzsch 1, H Rühl 1

Objectives

While CD34+ stem cells tolerate freezing and subsequent thawing well, a significant loss of CD3+ T cells has been commonly observed in cryopreserved leukapheresis products. Aim of this study was to assess how well T cells tolerate freezing and thawing in comparison to other lymphocyte subpopulations and hematopoietic stem cells. Moreover, potential influencing factors of a T cell loss were evaluated, including G-CSF mobilization, transport, and liquid storage time.

Methods

We determined the post-thaw recoveries of CD34+ cells, CD3+CD4+ cells, CD3+CD8+ cells, CD19+ cells, and CD16+CD56+ cells in 90 cryopreserved apheresis products, among which 65 were from G-CSF mobilized donors, and 34 from unrelated donors that underwent transport before cryopreservation in our laboratory. In addition, 10 leukapheresis products from healthy probands were cryopreserved, and the staining pattern of CD3+CD4+ and CD3+CD8+ cells analyzed by flow cytometry in mixing experiments using native as well as cryopreserved cells, and different sets of antibody clones.

Results

Statistically significant differences were observed for CD34+ cell recovery (93.0 ± 20.7%) in comparison to CD3+CD4+ cell (83.1 ± 15.4%, p = 0.014), and CD3+CD8+ cell recovery (83.3 ± 13.9%, p = 0.001). Similarly, CD19+ cell recovery (98.6 ± 15.1%) was higher than CD3+CD4+ cell (p = 2.5 × 10–7) and CD3+CD8+ cell recovery (p = 1.2 × 10–8). Post-thaw recovery rates of all cell populations were not impaired in G-CSF-mobilized products in comparison to non-mobilized products, nor in unrelated compared to related donor products. Similar results were obtained in the mixing experiments with fresh cells and cryopreserved cells, ruling out a potential change of the CD3 epitope induced by freezing and thawing.

Conclusion

Our data suggest a higher sensitivity of CD3+ cells towards cryopreservation and demonstrate that the tolerance towards freezing and thawing is cell-specific. A clinical consequence may be the monitoring of CD3+ cell doses of cryopreserved products, such as donor lymphocyte infusions, after thawing.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):11.

JS-09-5 Photodynamically allo-depleted donor T-cells (ATIR101) improve survival after T-cell depleted mismatched family donor HSCT: Results from a phase 2 trial in high-risk leukemia patients

DC Roy 1, S Lachance 1, J Roy 1, I Walker 2, J Maertens 3, S Cohen 1, R Foley 2, P Lewalle 4, E Olavarria 5, D Selleslag 6, M Ruediger 7, J Velthuis 7, K Reitsma 7, L Gerez 7, J Rovers 7, H Boenig 8, S Mielke 9

Introduction

Use of haploidentical donors can improve accessibility to HSCT for poor-risk leukemia patients, provided the issues of intractable GvHD and excessive mortality due to delayed immune reconstitution can be managed. We developed a technology to deplete allo-reactive T-cells from DLI, potentially allowing GvHD-free adoptive transfer of the entire memory T-cell repertoire, to provide protection against relapse and infections.

Patients and Methods

This open-label, multicenter phase 2 study (CR-AIR-007; NCT01794299) tested the outcome after CD34-selected haplo-identical MMRD-HSCT without prophylactic immunosuppression, supported with single-dose ATIR101 (2 × 10e6 cryopreserved CD3+ cells/kg) on d. 28. 23 patients, aged 41 (21–64) years, with high-risk AML or ALL, received myeloablative conditioning with TBI or melphalan + thiotepa/fludarabine/ATG. A CD34 selected product containing 11 × 10e6/kg stem cells and 2900/kg T-cells (mean) was given on d. 1, followed by ATIR d. 28.

Results

All patients engrafted; kinetics were favorable (d12 for PMN, Plt.). No patients developed °III/IV aGVHD after infusion of ATIR101. Three cases of °II were reported. One had pre-existed at the time of ATIR infusion, the other two occurred relatively late, albeit phenotypically resembling aGVHD. Thus far, only one patient developed severe cGVHD. No patient died w/in 100 d. post HSCT, there were 3 deaths due to TRM (all infections) and 1 to relapse w/in 6 mo post HSCT, for a 100-d., 6-mo and 12-mo survival of 100%, 83% and 64%. Compared to a historic control group (N = 35) treated with a CD34-selected HSCT only (“naked haplo”), TRM was significantly lower and overall survival was higher in ATIR101 patients at 6 (primary outcome) and 12 mo.

Conclusion

The high dose of 2 × 10e6 haplo-identical CD3+ cells/kg, normally expected to cause lethal GvHD, was tolerated well without inducing grade III/IV acute GvHD or need for prophylactic immunosuppressants, indicating efficient allo-depletion. More importantly, the markedly improved outcome, resulting in overall survival in excess of what is expected from matched-donor transplantation, suggests transfer of relevant anti-infectious / anti-leukemic T-cell specificities with ATIR101. A randomized study against haplo-BM+Cy is forthcoming.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):11.

JS-09-6 Cytotoxic capacity of antiviral CD8+ T cells is directly impaired by G-CSF administration

S Tischer 1,2, C Bunse 1, J Lahrberg 1,2, JC Kwoczek 1, M Oelke 3, C Figueiredo 1, S Thomas 4, R Blasczyk 1,2, B Eiz-Vesper 1,2

Introduction

Patients after hematopoietic stem cell transplantation (HSCT) showing a delayed and impaired immune reconstitution, which is associated with increased risk for viral infections and reactivations. Adoptive transfer of antiviral T cells offers an effective immunotherapeutic strategy to reduce or prevent the clinical manifestation of viral infections/reactivations. If available, seropositive stem cell donors may serve as donor for antiviral T cells but Granulocyte-colony stimulating factor (G-CSF)-mobilization has been shown to negatively affect T-cell function in an antigen-dependent and independent manner. The mechanisms of G-CSF mediated impairment of CD8+ T cells are still unknown.

Methods

We analysed the effects of G-CSF on antiviral T cells after in vitro and in vivo treatment. Isolated G-CSF-treated CD8+ T cells were stimulated (1.) antigen-dependently with viral peptide-loaded artificial antigen-presenting cells (aAPCs) and (2.) antigen-independently with anti-CD3/CD28 stimulator beads. CD8+ T-cell functionality was analyzed by expression of activation markers, miRNA expression profiles, secretion of effector molecules and phosphorylation analysis.

Results

In vivo and in vitro G-CSF treatment resulted in direct effects on antiviral CD8+ T cells, leading to a reduction in their cytotoxicity, their secretion of effector molecules and degranulation capacity. Diminished CD8+ T-cell activation after G-CSF-treatment was indicated by decreased phosphorylation of ERK1/2 (−17%), Lck (−33%) and CD3ζ (−20%) as well as a reduced expression of miRNA-155 (−1.5-fold) and T-cell activation markers such as CD25, CD69 and CD137 (up to −3.9-fold). Microarray analysis confirmed the down-regulatory effect of G-CSF on elements of CD8+ T-cell activation like CD25 (−5.1-fold), CD69 (−2.0-fold), CD57 (−5.2-fold) and CD137 (−6.3-fold). These findings were associated with an impaired expression of the effector molecules IFN-γ and granzyme B at mRNA and protein level.

Conclusion

We showed for the first time that G-CSF directly affects essential elements of the CD8+ T-cell activation pathway leading to a reduced cytotoxic functionality. Patients in need of antiviral T-cell therapy after HSCT may benefit when these findings are taken into account in the process of T-cell donor selection.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):11–12.

JS-09-7 Time-related metabolomic analysis for short-term storage of mononuclear cells

PA Steininger 1, M Rauh 2, B Ziehe 1, R Eckstein 1, EF Strasser 1

Introduction

Short-term storage of mononuclear cell (MNC) products yielded by leukapheresis is often inevitable before further bioprocessing or therapeutic application. The analysis of changes in the metabolic network allows the identification of limiting nutrients or accumulating detrimental metabolites for the optimization of storage conditions.

Methods

In this pilot study, the MNC products from seven leukapheresis procedures with the COM.TEC cell separator (autoMNC program) were stored in fluorinated ethylene propylene culture bags for 48 hours at room temperature. For analysis of storage-induced metabolic changes, aliquots (500 µl) of the supernatant were taken after 3 (baseline), 6, 24 and 48 hours and subjected to targeted mass spectrometry. The concentrations of more than 100 metabolites of key metabolic classes (amino acids, biogenic amines, acylcarnitines (ACs), phospho- and sphingolipids) were (semi-) quantified.

Results

The highest depletion of glucoses was observed between 3 and 24 hours (mean, −0.48 ± 0.16 mM per hour), while the concomitant mean increase in the lactate level was 0.12 ± 0.05 mM per hour. The ratio of small-chain ACs to free carnitine as an indicator of β-oxidation activity continuously increased by approximately 40% during storage, which was mainly due to the elevation of the acetylcarnitine level by Δ 1.30 ± 0.80 µM. In contrast, the uptake of fatty acids, as calculated by the ratio of long-chain ACs to free carnitine, increased by approximately 140% within the first 24 hours, but then remained constant. While the level of the measured amino acids increased by a mean factor of 2.0 after 48 hours, the increase in the amount of aspartate and methionine was considerably higher (both more than fourfold). Of the three polyamines measured, the highest elevation after 48 hours of storage was observed for spermine (Δ 1.85 ± 0.95 μΜ), followed by spermidine (Δ 1.56 ± 0.78 μΜ) and putrescine (Δ 0.70 ± 0.32 μM).

Conclusion

Targeted metabolomics is a feasible approach to monitor the activity of key catabolic pathways during the storage of a heterogeneous MNC product. This allows characterizing the effects of medium supplementation in further intervention studies aiming to optimize storage conditions. Thereby, the first 24 hours of storage should be considered as the decisive interval for intervention.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):12.

JS-09-8 Incomplete peripheral blood chimerism does not sensitively reflect incomplete bone marrow chimerism after allogeneic hematopoietic stem cell transplantation

C Bach 1, M Steffen 2, J Winkler 1, W Rösler 1, BM Spriewald 1

Introduction

Quantification of bone marrow (BM) chimerism is currently the gold standard for post-hematopoietic stem cell transplantation (HSCT) chimerism diagnosis. However, BM aspiration is challenging and up to 5% of marrow aspirations result in dry taps, potentially precluding evaluation of BM samples. Therefore, we evaluated the suitability of peripheral blood (PB) chimerism as a surrogate marker for BM chimerism.

Methods

We collected 215 matched pairs of samples from peripheral blood and bone marrow from 47 patients after 56 allogeneic HSCTs performed at the University Hospital Erlangen following reduced intensity (43) or myeloablative conditioning (13). We determined donor chimerism (DC) in those samples utilizing a sensitive quantitative realtime PCR-based detection of recipient-specific short insertion/deletion mutations modified from a method described by Alizadeh et al. 2002. Incomplete DC was defined as DC < 99.8%.

Results

Overall, comparison of matched samples revealed a strong linear correlation between donor chimerism detected in PB and BM (r=0.89, p < 0.0001, figure 1). However, 91 sample pairs (42.3%) displayed differences in DC between PB and BM, with an average absolute difference of 1.13%. Moreover, 42 sample pairs exhibited complete DC in the PB and incomplete DC in the BM sample (false negative). In contrast, only 5 sample pairs showed the reverse situation. Therefore, incomplete PB DC was highly specific (96.1%), but only moderately sensitive (51.2%) in predicting incomplete BM DC, with an overall accuracy of 78.1%.

Conclusion

Incomplete PB DC does not sensitively predict incomplete BM DC. Consequently, PB DC may fail to indicate decreasing BM DC during the critical early relapse period, potentially precluding timely therapeutic intervention. This severely limits the diagnostic value of PB DC for post-HSCT monitoring.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):12.

JS-10-1 (INV. SPEAKER) Immunogenetic diagnostics in transfusion medicine

J Zingsem 1

Blood banks supply the patients in the hospital with blood product like RBC concentrates, FFP, platelets and sometimes WBC concentrates as well as with the related diagnostics. These diagnostics are mainly based on RBC serology. However, in some cases this technology cannot provide satisfactory results. In case an antibody against rare RBC antigens is suspected, sera for the required determination of this blood group system's alleles are not always available. Furthermore, pretransfusions and/or the presence of autoantibodies often impair the determinatin of the patients’ antigens. In most of these cases, the use of DNA based techniques helps to obtain correct results.

Antibodies directed against blood group systems other than RBC blood groups might cause transfusion reactions, e.g. febrile non haemolytic reactions induced by HLA antibodies. This requires the detection and specification of HLA antibodies in the patients’ blood to the same extend as in the case of refractoriness to platelet transfusions. In analogy to the proceeding in case of RBC antibodies, the patients’ HLA type must be determined in case of a positive result, if their HLA type is not yet known. Post transfusion purpura and/or fetal or neonatal alloimmune thrombocytopenia (FNAIT) mainly are caused by alloantibodies against platelet antigens e.g. HPA. These antibodies are detected and specified using cell based as well as solid phase antibody assays. In case of positive results the patients’ HPA type must be determined what usually is done using DNA based techniques. The same applies for TRALI, TRAIN, and neonatal alloimmun neutrocytopenia. The antibody detection usually is performed using different techniques like leukocyte agglutination, immunofluorescence tests solid phase antibody assays and the subsequent HNA typing requires DNA techniques.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):12.

JS-10-2 (INV. SPEAKER) Anti-platelet antibodies: the known and the unknown

UJ Sachs 1,2

Anti-platelet antibodies are involved in different disorders with low platelet counts as the common clinical sign. Detection of these antibodies is the primary task of platelet immunology laboratories which are faced with two central challenges: first, platelet antibody detection often requires in-house methods and is generally poorly standardized; and second, discrimination between clinically relevant and irrelevant antibodies is difficult because many aspect of antibody-mediated thrombocytopenia are still not or not well understood.

The aim of this overview is to critically review: test systems for platelet antibody detection; the relevance of positive test results for the patient and his attending physician; and the relevance of negative results in the same respect. The overview will focus on platelet alloantibodies and the clinical diagnosis of fetal/neonatal alloimmune thrombocytopenia (FNAIT). Different test systems are commercially available, but their sensitivity can be astonishingly low. If antibodies are detected, their specificity and titer are sometimes used to predict the clinical relevance, but numerous data indicate that this may be an inappropriate approach such as, for HPA-1a and the occurrence of intracranial hemorrhage; or HLA and thrombocytopenia. Critical control points for cases in which an antibody is not detected will also be reviewed including, diagnostic algorithms, appropriateness of the methods used, a common pitfalls such as immunization against HPA-3, HPA-9, and low-frequency HPAs. Since platelet autoantibody detection shares some characteristics with alloantibody testing, aspects of test methods and the clinical relevance of positive test results in the diagnostic work-up of patients with immune thrombocytopenia will be mentioned where appropriate.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):12–13.

JS-10-3 Serum anti-HLA IgA contributes to graft failure in renal re-transplantation patients

C Bach 1, M-L Arnold 2, FM Heinemann 3, A Dick 4, BM Spriewald 1

Introduction

Complement fixing anti-human leukocyte antigen (HLA) IgG and IgM antibodies in the serum of solid organ transplant recipients negatively impact on graft survival. Little is known however about the clinical impact of other, non-complement fixing antibody isotypes. Previously, we reported a high prevalence of anti-HLA IgA in the serum of solid organ transplantation candidates leading us to investigate the impact of the presence of these antibodies on graft survival.

Methods

694 kidney re-transplantation candidates from a retrospective multicenter cohort were selected based on serum and data availability. Importantly, the time to first dialysis in months (TTD) after kidney transplantation was used as a surrogate endpoint for loss of graft function. Anti-HLA IgG and IgA were measured in pre-transplantation serum samples using a Luminex microbead-based screening and specification assays.

Results

Of the 694 serum samples 214 were tested positive for anti-HLA IgA and 518 for anti-HLA IgG. Interestingly, the presence of anti-HLA IgA was highly linked to the presence of anti-HLA IgG (odds ratio 2.8, p < 0.0001). As expected, stratification by IgA and IgG status revealed the highest median graft survival in IgA/IgG double negative patients (TTD 118.5 months). Patients tested positive for either IgA or IgG displayed intermediate graft survival (TTD 93.7 and 101.5 respectively), whereas double positive patients had a significantly reduced median graft survival compared to any other subgroup (TTD 77.1, p < 0.05). Importantly, the presence of donor specific anti-HLA IgA in the serum (n = 96) correlated with a significantly worse outcome (TTD 78.0) compared to the total cohort (TTD 102.3, p < 0.0001), as well as compared to anti-HLA IgA positive patients without donor specific anti-HLA IgA (TTD 87.1, p = 0.006).

Conclusion

Presence of serum anti HLA-IgA in conjunction with anti-HLA IgG marks a high risk subgroup of kidney re-transplantation candidates with significantly reduced graft survival. Notably, the detrimental impact of donor specific anti-HLA IgA among total anti-HLA IgA may indicate a novel functional contribution of anti-HLA IgA to graft failure.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):13.

JS-10-5 Strong humoral anti-HLA immune response upon allogeneic arterial vessel transplantation

H Konrad 1, A Wahle 1, W Altermann 1, G Schlaf 1

Introduction

44 patients were grafted using fresh or cryo-preserved allogenic arterial vessels in order to treat infections of synthetic vascular implants. These infections are regarded as highly harmful as they frequently lead to sepsis, amputations and death. Thus, they require urgent medical care.

Methods

Since adequate autologous vessel grafts were not available for the patients of this cohort allogenic vessel grafts were generally used. One patient was allografted using vessels of two different donors. All of the recipients were HLA-typed, and typing results of the vessels’ donors were inquired or geno-typed from residuary vessels’ segments. Since the allografts were chosen randomly i.e. without considering HLA-matching between donors and recipients anti-HLA immune response was determined prior to and after allografting using Dyna Chip- or Luminex-based analyses in order to define PRA-values before and after grafting and to virtually define transplant-/donor-specific antibodies (DSA). Furthermore, clinical follow up diagnoses were performed as well as histological analyses from tissue excisions (n = 15) of the allografts due to various complications.

Results

The rate of patients cured from the underlying disease “bacterial inflammation of the synthetic vascular implant” involved 84% of them with a re-infection rate of only 9%. However, due to the arbitrary selection of allografted vessels without considering HLA-compatibility 96% of the patients developed a humoral anti-HLA immune response which was virtually (i.e. comparing the recipients’ antibody specificities with the HLA-antigens of the donors) characterized as donor-specific in 89% of the patients. A vast average increase in the panel reactivity (PRA %) of 71% per graft was determinable in the recipients due to poor degrees of HLA-matching. Regarding the clinical outcome 18% of the patients exhibited thromboses of the allograft as the most frequent complication. Furthermore, all explanted and histologically evaluated allografts showed chronic progressive degeneration processes by signs of fibrosis and collagenization as a consequence of the alloreactive immune response.

Conclusion

Although not leading to an acute graft dysfunction comparable with differentiated solid organs the allografting of arbitrarily selected arterial vessels leads to a high degree of allo-immunization with consequent chronic degeneration processes highly limiting the overall practicability of this procedure.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):13.

JS-10-6 Distribution of IgM isotype antibodies aginst anti-HLA class II antigens

M-L Arnold 1, C Bach 2, BM Spriewald 2

Introduction

Antibodies (Ab) of the IgM isotype are in general regarded as auto-Abs and are not considered as transplantation (Tx) relevant. These Abs are routinely identified, using a complement dependent cytotoxicity test (CDC) under the measurement with and w/o dithiothreitol (DTT). However, with this CDC test it is unlikely that IgM-Abs, directed against HLA class II Ag are being detected. The aim of this study was to investigate the prevalence of IgM anti-HLA Abs, directed against HLA class II Ag using bead based assays (Luminex).

Methods

A cohort of 879 sera from patients from the kidney wait list of Erlangen/Nürnberg (Germany) were tested in CDC with and w/o DTT as well as in bead based (Luminex) techniques on the generic level (LSmixed) using IgG- and IgM- secondary Ab. In order to identify Ab specificities, LSm positive sera were investigated by Luminex Single Antigen (LSA) using IgG and IgM conjugate.

Results

Of the 879 sera - analysed by Luminex assay - 23% were tested Ab-negative, 13% IgG-Ab-positive, 33% IgM-Ab-positive and 31% as both, IgG- and IgM-Ab-positive. With respect to anti-HLA class II Abs, 119 of the 879 sera (14%) displayed the IgM isotype. This percentage increased on analysis of re-transplant candidates (n = 160). Here, 57 sera of 160 (34%) showed IgM-Ab against HLA class II. In 52 of these IgM-Abs were donor specific (IgM-DSA). 4 sera remain undetermined due to the lack of a donor HLA-DP typing. In one serum IgM-Abs were not identified as IgM-DSA.

Conclusion

In conclusion, in our cohort the prevalence of IgM anti-HLA class II Abs was 14%. In re-Tx candidates the prevalence increased to 34%, with nearly all IgM-Abs being donor specific. These findings raise the question whether the general assumption, that IgM-Abs represent merely irrelevant autoantibodies must be reconsidered.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):13–14.

JS-11-3 Evaluation of the evanescent biosensor technology for platelet allo- and autoantibody detection

W Song 1, M Baade 1, G Rink 1, H Klüter 1, P Bugert 1, TJ Schulze 1

Background

Alloantibodies against platelet antigens can cause both fetal-neonatal alloimmune thrombocytopenia (FNAIT) and refractoriness after platelet transfusion. Platelet autoantibodies can cause immune thrombocytopenia and thus severe bleeding disorders. The Simultaneous Analysis of Specific Platelet antibodies (SASPA) assay and Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) assay are well established for the detection of platelet allo- and autoantibodies. Both tests make use of the trimolecular complex of monoclonal glycoprotein specific antibody, the platelet fragment and the respective allo- or autoantibody. Here, we evaluated the commercially available evanescent biosensor technology (EVA) as a novel method for allo- and autoantibody detection.

Methods

88 sera containing anti-HPA-1a-, anti-HPA-5b-, HLA class I antibodies or autoantibodies - or a combination - that had been analyzed various times before by SASPA were simultaneously analyzed by SASPA and by EVA using the “MAIPA test chip” (Davos Diagnostics, Switzerland, www.davosdiagnostics.com). Altogether 163 tests were performed for anti-HPA-1a (68 tests, 23 patients), for anti-HPA-5b (22 tests, 22 patients), for HLA-class I antibodies (45 tests, 22 patients) and for platelet autoantibodies (21 tests, 21 patients). The sera were incubated with HPA-1a and HPA-5b positive platelets or pools of platelets for the detection of HLA-class I antibodies and platelet autoantibodies, respectively, then a monoclonal antibody to immobilize the antigen was added. The cells were lysed and subjected to simultaneous antibody detection by SASPA in a flow cytometer and by EVA. The results from SASPA and EVA were compared.

Results

EVA revealed false negative results for anti-HPA-1a (4.4%), for anti-HPA-5b (18.6%), for HLA-class I (20%) but not for autoantibodies. The correlation (Pearson) for all test results was r=0,68 (p < 0,0001). The correlation for Anti-HPA-1a was r=0,97 (p < 0,0001), for anti-HPA-5b 0,6 (p = 0,0018), for autoantibodies 0,7 (0,0002) and for HLA class I 0,85 (< 0,0001). All negative controls were found negative in both tests.

Conclusion

In comparison to SASPA EVA lacks sensitivity: false negative results occur for anti-HPA-1a, anti-HPA-5b and HLA-class I antibodies. All test showed good correlation between SASPA and EVA, best correlation was obtained HPA-1a-antibodies and autoantibodies. However, discrepancies concerning false negativity require further investigation.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):14.

JS-11-4 HLA and HPA Typing of platelet donors – experiences with an automated TaqMan based approach (FluoGene)

T Vollmer 1, J Dreier 1, J Diekmann 1, C Knabbe 1

Introduction

Transfusion-induced alloimmunization, platelet refractoriness or neonatal alloimmune thrombocytopenia (NAIT) are special events in the routine transfusion setting. Hence, the adequate supply of these patients often present a significant challenge to the transfusion service and the blood center. The search for HLA-compatible platelets is complicated and time consuming, not at least due to a small number of characterized donors. In consequence, we decided to progressively type our platelet donor collective regarding their HLA- and HPA-antigen profile.

Methods

Total DNA was extracted from negative whole blood using the MaxWell 16 SEV blood kit on the MaxWell 16 automated DNA extraction system (Promega GmbH, Mannheim, Germany). Low resolution HLA-typing and HPA-typing of blood donors was performed using the FluoGene HLA-ABC and FluoGene HPA kits on the FluoVista system (Innotrain, Kronberg, Germany).

Results

Our pool of active platelet donors included approximately 600 - 700 donors. To date, the antigen profile of 583 (HLA) respectively 348 (HPA) donors have been determined with the FluoGene platform. The number of repeated or failed analysis is minimal (< 1%), likewise the occurrence of ambiguities is low (1.3%).

Conclusion

The ability of blood centers to provide HLA/HPA antigen-negative PLT units is tied to having a good-sized pool of active platelet donors who have been typed. The semi-automated FluoGene HLA-and HPA-antigen typing system allows consecutive typing of platelet donors with low to medium throughput with a simple and clear result interpretation.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):14.

JS-11-5 HLA-DRB3*01:01 is a predictor for immunization against human platelet antigen 1a but not for severity of fetal and neonatal alloimmune thrombocytopenia

E-M Papenkort 1, IR König 2, YC Duong 1, K Müller 1, A Giptner 1, H Hackstein 1, S Santoso 1, U Sachs 1, G Bein 1, S Wienzek-Lischka 1

Background

Most cases of fetal and neonatal alloimmune thrombocytopenia (FNAIT) are caused by maternal alloantibodies against paternally inherited human platelet antigen 1a (HPA-1a) expressed on fetal platelets. Alloimmunization occurs mainly in HPA-1a negative mothers who are carriers of the HLA-DRB3*01:01 allele. However, the question whether the presence of HLA-DRB3*01:01 allele may also determine the severity of FNAIT remains unacknowledged. Recently, it has been reported that the combined presence of DRB3*01:01 and DRB4*01:01 (compound heterozygosity) was associated with severe cases of FNAIT. We tested this hypothesis by analysis of a large cohort of cases and controls.

Material and Methods

In total, 101 mothers with a history of FNAIT caused by anti-HPA-1a antibodies were investigated. High resolution HLA-DRB1, DRB3, DRB4, and DRB5 genotypes were determined by Luminex technology and sequence-specific priming. Haplotype frequencies were compared between cases and 100 healthy controls. The platelet count of neonates (nadir) and the presence/absence of intracranial hemorrhage (ICH) was compared between subgroups that were defined by genotype.

Results

99% (100/101) of the HPA-1a immunized mothers carried at least one copy of HLA-DRB3*01:01. Compared to controls, carriage of HLA-DRB3*01:01 was significantly associated with immune response to HPA-1a (odds ratio (allele positivity) 92,3; 95% CI 26,9–317,1; two-sided p = 6,71 · 10^-13).

No associations were found in our cohort between: HLA-DRB4*01:01 and immune response to HPA-1a, HLA-DRB3*01:01 and HLA-DRB4*01:01 alone or in combination with the platelet count (nadir) of the affected newborns or the incidence of ICH, the incidence of ICH and platelet count on the one hand and the newborn's sex on the other, HLA-DRB alleles and FNAIT risk or severity.

Discussion

In contrast to HLA-DRB4*01:01, the inheritance of HLA-DRB3*01:01 is strongly associated with the propensity for mounting a humoral immune response against fetal HPA-1a antigen. However, the gene dose, neither homozygous nor compound heterozygous, does not predict severity of the disease. Testing for the presence of HLA-DRB3*01:01 is very useful in counseling for siblings of women who gave birth to neonates with FNAIT.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):14–15.

SI-02-1 (INV. SPEAKER) Rotembasierte Gerinnungstherapie

J Fechner 1

Thrombelastographie was the first point-of-care test develloped in 1948 by Hertert. Rotational thrombelastometry (ROTEM, TEM International, Munich, Germany) also is a viscoelastic point-of-care test that is based on the principles of thrombelastography, but can be used in the OR or at the bedsite of the patient. The ROTEM test assesses clot formation and dilution kinetics in whole blood. Providing a graphical interface the ROTEM test result graphs can be interpreted by analysing typcal characteristics of the coagulation curves. Parameters as the clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), maximum clot amplitude at 10 (A10) and 20 (A20) minutes and lysis index 30 (LI30) can be uesd to diagnose hypofibrinogenaemia and hyperfibrinolysis at the bedsite within 20 to 30 minutes. Therefore especially in cardiac surgery the ROTEM test has been extensively studied and numerous studies have been published. Special ROTEM-based transfusion or treatment algorithms have been develloped. Bleeding following pediatric or adult cardiac surgery is common and a difficult clinical problem. It has been shown that ROTEM-based treatment protocols improve outcome, can significantly reduce transfusion of allogenic blood products and even reduce treatments costs in these patients (Pearse B.L. et al., Vox Sang 2015).

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):15.

SI-02-2 Intraoperative Hämostase-gesteuerte Hämotherapie in der Herzchirurgie: Das Düsseldorfer SUBITO-Modell

T Hoffmann 1

Die Hämotherapie-Steuerung auf Grundlage vollblutbasierter echtzeitdiagnostischer Messmethoden hält, auch aufgrund neuer Leitlinien-Empfehlungen, zunehmend Einzug in die Akutblutungsbehandlung. Verbreitet werden viskoelastische Methoden als point-of-care Diagnostik im OP-Saal durchgeführt. Dies stellt hohe Anforderungen an die apparativen und personellen Ressourcen, die Durchführungs- und Interpretationsexpertise sowie an die Qualitätssicherung. Am Universitätsklinikum Düsseldorf wurde mit dem interdisziplinären SUBITO-Projekt ein alternatives Modell entwickelt, bei dem unter optdimierten logistischen und Durchführungsbedingungen die Echtzeit-Diagnostik (ROTEM® und Multiplate®, ergänzt durch konventionelle Hämostase-Parameter) zentral im hämostaseologisch-transfusionsmedizinischen Labor erfolgt. Differentialdiagnostisch werden alle wesentlichen Hämostase-Komponenten erfasst, für die spezifische Therapeutika bzw. Substitutionsoptionen verfügbar sind. Anhand standardisiert-individualisierter Algorithmen werden jeder Therapie-Entscheidung neben den labordiagnostischen auch klinische Parameter zugrundegelegt. Grundprinzipien sind (a) die Detektion ausreichender patienteneigener Hämostase-Reserven ohne Substitutionserfordernis und (b) ein iterativer Therapie-Ansatz, welcher unmittelbar nach Protamingabe eine restriktiv indizierte und dosierte blutungsprophylaktische Substitution vorsieht, während alle weiteren blutstillenden Hämotherapie-Interventionen der klinischen Indikationsstellung unterliegen und - gegebenenfalls - nach Art und Umfang labordiagnostisch gesteuert werden.

Die Auswertungen erlauben Charakterisierungen der hämostaseologischen Minimalerfordernisse für intakte Blutstillung, der Interaktionen verschiedener Hämostase-Komponenten und von hämotherapeutischen Dosis-Wirkunsgbeziehungen im setting komplexer chirurgischer Blutungssituationen.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):15.

SI-03-3 (INV. SPEAKER) Clinical studies on storage time of red blood cell transfusions: Where do we stand?

L Van de Watering 1

A large number of observational studies investigating possible effects of storage time of RBC have been published. The conflicting results from these studies have led to controversy and lots of discussions. In an effort to solve these discussions, the design and analyses of the published studies were scrutinized. In this way a lot of bias and confounding was identified within the reported literature. With more appropriate study designs and analyses adjusting for possible confounding, new observational studies seemed to identify a far less important role for the storage time of RBC. Also more awareness grew on possible effect-modification by storage solutions or differences in the RBC production process, which could explain the earlier reported “continental divide”.

Next to the work on observational data, several RCT's were started over the years in different parts of the world. These studies used different definitions of “old” RBC and “fresh” RBC, in different patient groups, looking at different endpoints, using different RBC products. Until now, none of the published RCT's have shown a significant detrimental effect of prolonged RBC storage. For most people, the universal lack of detrimental effects related to prolonged RBC storage reported in these studies came as a surprise. Where some suggested a lack of power for analyzing “extreme” storage (35–42 days), others even hinted at a detrimental effect of the most fresh RBC transfused.

Combining the results of the separate studies, and adding the results of studies not yet finished, may give us a great opportunity to look more specifically at some subgroups of patients or extremes in RBC storage time.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):15.

SI-04-2 mRNAs and protein synthesis in platelets

H Schwertz 1

Platelets get released from megakaryocytes as cells consisting of cytoplasm that lack genomic DNA. Therefore, they are incapable of transcribing nuclear material. This generated the central dogma that human platelets are synthetically quiescent during their 9–11 day circulation period. This limited view, however, is still viewed at very controversial, and has been questioned as emerging evidence demonstrates that megakaryocytes invest platelets with requisite translational machinery that includes ribosomes, initiation and termination factors, microRNAs (miRNAs), and template messenger RNAs (mRNAs). Several studies have characterized mechanisms by which platelets synthesize proteins, have shown that protein synthesis alters the phenotype and functions of platelets, and that synthetic events can be dependent on respective clinical conditions.

Nevertheless, many of these pathways still wait to be characterized in more detail and others continue to emerge. Studying these synthetic events in detail was and still is limited due to technical hurdles. However, using traditional techniques as well as recent advances in RNA deep sequencing, and deep sequencing of ribosome-protected mRNA fragments of platelet samples indicates that platelets express thousands of megakaryocyte-derived mRNAs and, that subsets of these mRNAs are translated into protein.

Therefore, a primary goal of our laboratory is to identify and characterize new functions of platelets that are regulated by protein synthetic events. In addition, we focus on inflammatory and infectious conditions, under which megakaryocytes might invest platelets with a different array of mRNAs, and therefore, change their synthetic capacity and ultimately their function during the course of disease. In addition, constitutive protein synthetic activity and the capacity of newly released platelets to synthesize a differential array of proteins are of greatest interest and will spark more studies for the future.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):16.

SI-04-3 Antibody mediated glycan modification: a potential role in platelet destruction in autoimmune thrombocytopenia

R Jouni 1,2, J Alex 2, L Janzen 2, K Althaus 2, I Marini 1, A Greinacher 2, T Bakchoul 1,2

Introduction

Immune thrombocytopenia (ITP) is a bleeding disorder caused by autoantibodies (AAbs) directed against platelet (PLT) glycoproteins (GP). Fc-independent PLT clearance via Ashwell-Morell receptors (AMRs) has been recently suggested as a novel mechanism of antibody-mediated PLT destruction in mice. AMRs recognize galactose residues of PLTs.

Aim

To analyze the impact of AAbs from ITP patients on the glycan pattern of human PLTs and the subsequent effect on their survival in vivo.

Methods

Sera from ITP patients and healthy donors were analyzed using monoclonal platelet antigen capture assay (MAIPA) and lectin binding assay (LBA). In LBA, sera were incubated with PLTs from healthy donors, and the change in glycan pattern was analyzed by flow cytometry using lectins; Ricinus communis agglutinin (RCA), Erythrina cristagalli lectin (ECL) and Peanut agglutinin (PNA) that bind to galactose, N-acetyllactosamine and N-acetylgalactosamine residues, respectively. The impact of different glycan patterns on the survival of human PLTs was investigated using the NOD/SCID mouse model.

Results

In total 32 sera from ITP patients and 20 sera from healthy donors were investigated in this study. No significant change was induced by sera from healthy donors. Different patterns of lectin binding to PLT surface were observed after incubation with AAbs. A significant increase in PNA binding was observed in 13/32 sera (median fold increase (FI) compared to heathy donors: 1.11, range: 1.08 - 1.20, p < 0.001) and 9/32 sera caused higher ECL binding (median FI: 1.02, range: 1.00 - 1.15, p > 0.5). In contrast, 6/32 sera showed strong decrease in RCA binding (median FI: 0.55, range: 0.50 - 0.59, p = 0.07). Interestingly, not only GP-Ib/IX AAbs but also GPIIb/IIIa AAbs were able to modify glycan pattern. The injection of AAbs resulted in accelerated clearance of human PLTs from the circulation of the NOD/SCID mice. The destruction of human PLTs by ITP-AAbs was reduced but not completely inhibited by a specific neuraminidase inhibitor that prevents glycan changes on PLT surface (survival of human PLTs after 5h: 29%, range 22–40% vs. 48%, range 41–53%, p = 0.014, respectively).

Conclusion

Our data demonstrate that a considerable a subset of AAbs from ITP patients is able to induce cleavage of glycan moieties on the PLT surface in distinct ways. Antibody-mediated modification of glycan patterns seems to contribute to PLT destruction in vivo presumably via neuraminidase activation.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):16.

SI-04-4 The implementation of a panel-based next generation sequencing approach in the diagnosis of inherited platelet disorders

T Bakchoul 1,2, P Bugert 3, W Streif 4, R Knöfler 5, E-M König 6, E Klopocki 6, O Andres 7, H Schulze 8

Background

Correct classification of platelet disorder is essential for optimal treatment and counseling of bleeding patient. Conventional platelet function analyses lack standardization and require technical expertise. Therefore, many patients remain without substantiated diagnosis despite pathological clinical or laboratory findings. Therefore, identifying the underlying genetic defect is indispensable in the diagnostic algorithm of inherited platelet disorders.

Aims

We report on our experience with the implementation of a panel-based next generation sequencing (NGS) approach to identify genetic variants in patients suspected to have inherited platelet disorders (IPD). An NGS panel designed using 6,800 probes that cover 59 selected genes that are known to be responsible for IPD was employed.

Methods

DNA samples from 48 bleeding patients with suspected IPD were processed using by MiSeq Sequencing System. Common SNPs were excluded from further data analysis by a default filter. As some target regions can have low coverage or single gaps, we monitored read coverage for each patient. DNA from five patients with previously well-characterized genetic defects was used for quality control and process validation. The study was conducted in accordance with local Institutional Review Board guidelines.

Results

All five of the previously identified mutations in the quality control samples were identified and confirmed by the NGS approach. 15 samples revealed one or two unclassified genetic variants in the tested 59 genes indicating the diagnosis of 7 cases with defects in cytoskeletal proteins, 2 with membrane receptor mutations, 2 with transcription factors, 3 with genes involved in Hermansky-Pudlak Syndrome and one with a calcium signaling defect, respectively. All NGS-detected mutations were confirmed by Sanger sequencing and subjected to familial segregation analysis. In one case of a novel likely pathogenic variant, segregation analysis ruled out that the variant was associated with the phenotype. Four samples failed the quality control.

Conclusion

Our data suggest that NGS-panel is sensitive enough to identify genetic variants in platelet-specific genes of classified and unclassified patients. This approach will improve genetic testing of patients without substantiated diagnosis using conventional methods.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):16–17.

SI-04-5 Diagnosis of hereditary platelet disorders from standard blood smears

K Althaus 1, C Blumentritt 1, U Strobel 1, T Bakchoul 2, A Greinacher 1

Introduction

Hereditary platelet disorders are more frequent than previously anticipated. Progress in hematology has identified many hereditary causes for thrombocytopenia and/or platelet function defects, but diagnosis is still challenging. It requires fresh blood and laboratory techniques available only in few specialized centers. Further, required blood volumes are usually prohibitive for investigating young children. Next generation sequencing techniques might allow characterization of the underlying genetic defect. However, at present it is not feasible for routine clinical practice, and it remains challenging to correlate the numerous variants within the genome to the phenotype, especially if the underlying platelet defects are poorly characterized. However, many hereditary platelet defects are associated with characteristic changes in the distribution of specific protein.

Methods

We developed a method to narrow down or confirm the diagnosis of many hereditary platelet disorders. Standard blood smears are prepared by the treating physician and are then shipped by regular mail. The use of specific fixation and permeabilization methods, followed by staining with specific antibodies, allows us to “phenotype” platelets by immunofluorescence microscopy.

Results

Assessing blood smears of 1017 patients referred to our laboratory with unclear thrombocytopenia or platelet function disorders, we achieved the diagnosis in 237 (23%) patients: MYH9-disorders, 142 patients; Bernard Soulier syndrome (BSS), 25 patients; gray platelet syndrome, 2 patients; GFI-1b mutation, 3 patients; ß1-tubulin defects, 5 patients; Wiscott-Aldrich syndrome (WAS), 1 patient; Glanzmann thrombasthenia, 9 patients; alpha storage pool defects, 20 patients; delta storage pool defects, 30 patients. Diagnostic sensitivity and specificity of the method was high for MYH9-disorders/related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombasthenia and gray platelet syndrome.

Conclusion

Combining basic and widely-available pre-analytical methods with cutting edge morphological techniques, supported by targeted genetic testing, greatly facilitates the access of patients with suspected hereditary platelet disorders to diagnosis.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):17.

SI-05-3 (INV. SPEAKER) Diagnosis-specific statistics of usage of blood components and human plasma-derived blood products

J Strobel 1, T Ganslandt 2, R Zimmermann 1

Blood components and human plasma derived blood products are of importance for complex treatments of many diseases. Such treatments and their costs are of both medical and economical interest. In the past, we presented a study on diagnosis-specific statistics of blood coagulation factor usage in our tertiary care hospital, the University Hospital of Erlangen. Meanwhile, we established a systematic review of blood product usage combined with case-specific data of recipients. In Erlangen, we collect and analyze all relevant data for interpreting patterns and changes in the usage of blood products since 2010. We integrate the clinical data warehouse components of recipients’ personal data, International Classification of Diseases (ICD), German Classification of Procedures (OPS), and German Diagnosis Related Group (G-DRG) system codes of inpatients, component data from the blood bank IT system, and plasma product data from the pharmacy IT system. The obtained diagnoses, procedures, and DRGs are associated with the data of the component consumption on an individual basis. The results can be grouped by Major Diagnostic Categories (MDCs), by basic DRGs, or by diagnoses and procedures. Fibrinogen and prothrombin complex usage has markedly increased, while fresh frozen plasma and red blood cell concentrate consumption decreased. Platelet concentrate usage has constantly been rising. Our analyses and reports are suited to localize and to explain such major changes in the use of blood products. Comparing our data with data released by the Paul-Ehrlich-Institute showed that at our hospital the decline of red blood cell transfusions started two years before it was noted for the whole of Germany. We found that most changes in the usage of blood products concern to intensive care patients and to a lesser extent to stem cell transplant recipients whereas the patterns of blood product application in other fields of medicine remained unchanged during the past five years.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):17.

SI-06-1 (INV. SPEAKER) Localization of tissue factor in blood vessels

D Weiss 1

Tissue factor (TF) is the main initiator of haemostasis process in vivo and clotting starts when TF is exposed to blood. In cross-sections through a big blood vessel, only the sub-endothelial part of the intima and the vasa vasorum in the media and adventitia can be stained for TF. Many opinions about the origin of TF in the larger blood vessels have been published. Detailed histological examination of the intima shows that microvascular pericytes (Pc) are the main source of TF. Pc of capillaries and venules are known constitutively to express TF. Pc have long been regarded just as companions of microvessels without further dissemination. However, histological studies with enzyme-histochemical and fluorescence-immunological staining showed that Pc accompany the endothelium throughout all vessels. Even in muscular arterioles, the Pc remain as a thin prothrombogenic layer between the endothelium and the smooth muscle cells. Furthermore, this cell type is also permanently resident in the intima. Pc have a multiply ramified pattern and are embedded in the basement membrane of the endothelial cells. In this dense extracellular matrix (ECM) the Pc weave TF-bearing microparticles. Thus, the antithrombogenic surface of the endothelium is a wafer-thin layer on an ECM with TF-expressing cells and microparticles. In de-endothelialized atherosclerotic plaques, the ECM with the widely ramified Pc is exposed to blood. After pathophysiological reconstruction, the Pc can adopt an endotheloid cobblestone pattern. The thin stratification of endothelium and TF-expriming Pc determines a marked vulnerability of the antithrombotic inner lining of the healthy blood vessel. For example, an iatrogenic de-endotheliazation of bypass vessels caused by improper preservation solutions during preparation can damage the endothelium of the grafts with extensively uncovered TF-positive cells and ECM.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):17–18.

SI-06-2 (INV. SPEAKER) FXIII deficiency and treatment options

V Ivaskevicius 1

The plasma circulating zymogenic coagulation factor XIII (FXIII) is a protransglutaminase, which upon activation by thrombin and calcium cross-links preformed fibrin clots and fibrinolytic inhibitors making them mechanically stable and less susceptible to fibrinolysis. The zymogenic plasma FXIII molecule is a heterotetramer composed of two catalytic FXIII-A and two protective FXIII-B subunits.

Inherited severe FXIII deficiency is a rare, autosomal recessive bleeding disorder affecting approximately one in 1–3 million people. Severe FXIII deficiency is characterized by a lifelong bleeding tendency, impaired wound healing and spontaneous

abortions in females. This disorder was first described in 1960 in Switzerland by Duckert et al. On the other hand the heterozygous (only one affected allele) FXIII deficiency is expected to be more common affecting approximately one out of 1000 inhabitants with a reduction of FXIII activity by 50%. Most of the patients with heterozygous FXIII deficiency do not develop bleeding complications in daily life. The majority of the individuals with inherited, severe FXIII deficiency have mutations in the F13A1 gene encoding FXIII-A subunit and only very few in F13B gene encoding FXIII-B subunit. Although, FXIII-B subunit deficiency in the heterozygous status seems to be more present at least in the German population.

Acquired FXIII deficiency may occur in several disorders, like haematologic malignancies, DIC, major surgery, liver diseases, Henoch-Schönlein purpura, chronic inflammatory bowel disease. FXIII activity can be reduced in these disorders by 50% or more. Decreased FXIII levels in these disease states are not due to anti-FXIII inhibitors.

All types of FXIII deficiencies can be treated by plasma derived factor XIII concentrates (pdFXIII). Besides pdFXIII a recombinant FXIII (rFXIII) concentrate has become available and can be used for prophylactic and bleeding treatment of patients with severe FXIII-A deficiency.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):18.

SI-06-3 Macrophage function is De-regulated in hemophilia

LM Knowles 1, D Lessig 1, H Eichler 1, J Pilch 1

Introduction

Hemophilia is associated with spontaneous bleeding events in large joints that can ultimately lead to joint destruction as extravasating red blood cells (RBCs) cause iron-induced synovial inflammation and cartilage damage. Macrophages are specifically equipped to sequester iron by phagocytosing RBCs, which is critical for resolving inflammation and initiating wound healing. We hypothesize that macrophages from hemophilia patients lack functions that are important for wound healing and that these deficiencies contribute to the perpetual inflammation seen in hemophilic arthropathy.

Methods

Monocytes from hemophilia patients (n = 15) and healthy blood donors (n = 16) were treated with M-CSF to induce differentiation into M2 macrophages that promote wound healing and GM-CSF to generate pro-inflammatory M1 macrophages, respectively. Subsequently, macrophages were probed for cell adhesion as well as phagocytosis on plastic and cell invasion in clotted plasma. Macrophage differentiation was assessed by immunocytochemistry/flow cytometry using antibodies against TNFα and the receptor tyrosine kinase Tie2 (CD202b).

Results

Treatment with M-CSF induced a significant shape change of donor macrophages, which exhibited a spread and polarized phenotype on plastic that translated into extensive phagocytosis and clot invasion. GM-CSF was able to induce filopodia formation in donor macrophages but had only very limited effects on the aforementioned functions. Macrophages from hemophilia patients were largely resistant to the mitogenic effects of M-CSF or GM-CSF as they were significantly impaired in generating filopodia and expressing TNFα. Notably, the deficits of hemophilia macrophages were most pronounced with regard to cell functions that are induced by M-CSF such as cell polarization and clot invasion. Accordingly, we saw a reduction of Tie2-expressing M2 monocytes in the blood of patients with hemophilic arthropathy.

Conclusion

Our results demonstrate that hemophilia macrophages are distinct from macrophages isolated from healthy individuals. The differences are reflected in the fact that hemophilia monocytes express reduced levels of Tie2 and that they are largely resistant to the M2-polarizing effects of M-CSF, which mediates functions critical for wound healing such as clot invasion and phagocytosis.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):18.

SI-06-4 Strong complement activation by antibody can override the inhibitory effect of eculizumab

M Anliker 1, CQ Schmidt 2, I von Zabern 1, M Harder 2, B Höchsmann 1, H Schrezenmeier 1, C Weinstock 1

Background

The classical and the alternative pathways of complement activation merge at the stage of C5 to a common terminal sequence. Eculizumab is a monoclonal antibody directed against component C5 that interferes with the assembly of the terminal membrane attack complex and is approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and aHUS. The usefulness of this complement inhibitor for counteracting antibody mediated hemolysis is still poorly defined; therefore, we investigated the effect of eculizumab and other inhibitors on the lytic activity of complement by in vitro experiments.

Study design and Methods

For very sensitive detection of classical pathway activation, RBC of patients with a PNH III clone > 20% were coated with human alloantibodies. Hemolysis was developed with normal human serum with or without the addition of complement inhibitors. Not hemolyzed CD59 deficient RBC were counted by FACS using PE-labelled anti-CD59. Mini FH and CR1-CCP1–3 were prepared as described (Schmidt CQ et al. J Immunol2013;190:5712).

Results

Strong hemolysis of PNH-RBC coated with the alloantibodies anti-PP1Pk, -Lan or -Jk(b) was observed with both normal serum and factor B deficient serum, pointing to the action of the classical pathway of complement. Complete inhibition of hemolysis could be achieved with eculizumab when PNH-RBC were coated with low amounts of complement activating alloantibodies. However, when the antibody load was increased, hemolytic activity remained despite the addition of eculizumab at concentrations far beyond those reached physiologically and in more than 10 fold excess over C5 in human serum. Other complement inhibitors such as mini FH or CR1 CCP1–3 were able to reduce this residual hemolysis when added together with eculizumab.

Conclusion

The C5 inhibitor eculizumab is effective as an inhibitor of cell lysis during mild complement activation but fast hemolysis is not completely prevented. Hence, in pathologic situations such as severe transfusion reactions, eculizumab is likely of benefit but may not be considered always sufficient as a sole therapeutic agent. Our findings may contribute to the understanding of clinical observations: eculizumab is effective in the therapy of PNH, but hemolysis still occurs; cases with antibody mediated hyperhemolysis treated with eculizumab showed a varying response. Novel complement inhibitors are under investigation which can supplement the action of eculizumab.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):18–19.

SI-06-5 Intoxication with rivaroxaban, phenprocoumon and diclofenac

H Pfeiffer 1, L Herbst 2, B Sallaj 3, B Schwarze 4, R Eckstein 1, V Weisbach 1

Introduction

Oral anticoagulants are widely used and can be easily misused. Although phenprocoumon has been in use for a long time and is widely known, it has a very narrow therapeutic range. Rivaroxaban is a newer and direct anticoagulant. Few cases of mixed overdose are known.

Methods

We report the first case of a patient who ingested 1,960 mg rivaroxaban, 31.5 mg phenprocoumon, 1,425 mg diclofenac and 21,000 mg metamizole with the intent of commiting suicide. Upon admission, the PT, given as Quick-value, was lower than 10%, the INR was too high to measure, the aPTT was 128s. The exact plasma concentrations of rivaroxaban and phenprocoumon were evaluated by liquid chromatography tandem mass spectrometry after stabilization of the patient. The phenprocoumon-level was 28 μg/ml (routine anticoagulation: between 1 and 3 μg/ml) twelve hours after ingestion. The rivaroxaban-level was extremely high at the same time (4.4 μg/ml, no routine references available).

Results

The 23-year-old patient was transferred to an intensive care unit and closely monitored. He received prothrombin complex concentrate (PCC) and vitamin K for the phenprocoumon-overdose as well as cholestyramine and pantoprazole as supportive treatment. A specific antidote for rivaroxaban is not available nor is it dialyzable. Despite the massive inhibition of the secondary as well as the primary anticoagulation system, the patient never showed signs of serious bleeding.

Conclusions

Due to the ceiling effect of rivaroxaban absorption, the rivaroxaban plasma levels were comparably low considering the massive ingestion. Elimination occurred according to the known half life of rivaroxaban. The phenprocoumon overdose was well treated by the fast and ample administration of vitamin K and PCC.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):19.

SI-07-1 (INV. SPEAKER) Current controversies in platelet transfusion

R Zimmermann 1, J Zingsem 1, R Eckstein 1

Platelet (PLT) transfusion therapy is the standard of care for patients suffering from hypoproliferative thrombocytopenia. The majority of all platelet concentrates is applied for preventing or treating bleeding in these patients. Recent randomized controlled trials (RCTs) have examined if a therapeutic-only approach is justifiable for certain subgroups of these recipients. The results of these RCTs are interpreted very differently. Other RCTs have investigated if a low-dose PLT transfusion policy is non-inferior to standard-dose or high-dose PLT transfusion strategies. Whereas one trial had to be discontinued, another could be completed and came to the conclusions that a low-dose PLT transfusion strategy does not cause more bleeding events in thrombocytopenic recipients but increases the number of transfusion episodes. Even while these RCTs were performed, the diversity of available PLT concentrates has increased remarkably. Nowadays, PLT concentrates in plasma or in different additive solutions are available. In addition, different methods of pathogen reduction in PLT concentrates are used more and more frequently despite clear indications of at least a partial loss of function of PLTs such treated. Which results of the mentioned RCTs apply in all available PLT components and which do not, has not yet been investigated. In Germany there is fierce dispute as to whether apheresis PLTs and pooled whole-blood derived PLTs are equivalent or not. To all abundance, a recent judgment of the German federal social court has questioned the justification of different rates for different PLT products. Probably, this judgment will cause a shift from apheresis to whole-blood derived PLTs. In summary, despite recent RCTs have extended our knowledge there remains a remarkable lack of evidence with respect to almost all questions regarding optimal timing, optimal dosing, and optimal stocking of PLT concentrates.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):19.

SI-07-3 Quantification of transfused platelets In recipients with droplet digital™ pcr compared to quantitative real-time pcr

A Doescher 1, EK Petershofen 2, T Müller 3

Introduction

Determination of recovery and survival of transfused platelets is possible by using genetic markers located in the mitochondrial genome. Quantitative real-time PCR (qPCR) served as reliable tool for the quantification. Droplet digital PCR (ddPCR) was evaluated as an alternative to qPCR for absolute quantification of transfused platelets.

Methods

Platelet rich plasma was prepared from 5 mL blood sample for automated extraction (MagnaPure Compact®) of mitochondrial DNA (mtDNA). Dilution series were performed to assess the sensitivity of DNA extraction. ddPCR for mitochondrial markers (n = 8 SNP alleles) was tested for linearity, sensitivity and reproducibility. Blood samples from 30 patients with haematological diseases were collected for six days after platelet transfusion. Endogenous and exogenous platelet counts measured by ddPCR were compared to qPCR results from identical samples.

Results

MtDNA levels increased linearly with platelet counts in the range of 1 - 50 plt/nL (r=0.997). Spiking experiments demonstrated a sensitivity of 0.1 plt/nL in a background of 20 plt/nL, using 20 droplets / assay as cut-off value. The coefficients of variation for ddPCR assays were comparable to qPCR: 0.7–3.2% (intra-assay) and 1.7–9.9% (inter-assay) depending on the SNP-allele. Calculated survival times in samples from 30 patients agreed well. The calculated coefficient of correlation for the comparison of platelet counts measured with ddPCR to qPCR was R2 = 0,96 (all samples) and R2 = 0,86 (samples with platelet counts up to 10/nL), respectively.

Conclusion

Our results demonstrate the reliability of ddPCR for quantitative tracking of transfused platelets against a background of endogenous platelets. The limit of detection for transfused platelets is about tenfold lower than for qPCR. The ddPCR introduces an efficient and cost-effective alternative to qPCR for monitoring platelet transfusions.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):19.

SI-07-4 Blood product supply in Germany The impact of apheresis and pooled platelet concentrates

K Berger 1, D Schopohl 1, W Schramm 2, G Wittmann 3, C Rieger 1, H Ostermann 1

Background/Aims

In Germany, about 60% of all produced platelet concentrates (PCs) are apheresis PCs (APCs). Ongoing discussions on APC reimbursement and costs might lead to a potential shift in pooled PC (PPC)/APC production. Objective of this analysis was to build a comprehensive model from the societal perspective to evaluate consequences associated with shifts in platelet supply and demand.

Methods

Literature search, desktop researches on platelet supply and demand. Model calculations, time horizon one year: model input from the Paul-Ehrlich-Institute, data 2013. Base case: 19.2% of annual whole blood donations (WBDs) were used for production of 38.5% PPCs, decay of 46 218 PCs (8.0%). Scenarios calculated: variation in PPC proportion of 10–100%.

Results

Base case: during PPC production 41 957–83 913 red blood cell concentrates (RBCs) are estimated to be lost, which corresponds to 1–2% of annual RBCs in Germany. Scenarios were calculated for a production of 60%-100% PPCs: loss is estimated to be 1.5%-5.0% of annual RBCs (65 430–218 099), decay 54 189–69 022 PCs (9.4%-12.0%).

Conclusion

Production of different blood components is interlinked and sensitive to unidimensional decisions. Increasing PPC proportion has negative impact on the RBC production and on the antigen-matched APC donor pool. Completion of the model calculations to predict the optimal PPC/APC proportion would require evidence on the number of refractory patients, donor pool sizes, incidences of diseases requiring platelet transfusions.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):20.

SI-07-5 In vitro assessment of untreated, UVC-treated and gamma-irradiated plasma reduced platelet concentrates prepared from pools of 5 buffy coats under routine conditions

V Brixner 1, S Dombos 1, I Weber 1, J Leibacher 1, M Schmidt 1, F Tolksdorf 2, P Pohler 3, U Gravemann 3, A Seltsam 3, T Tonn 4

Introduction

Pathogen inactivation technology may enhance microbial safety of platelet transfusion by reducing bacterial and viral contamination. We established the manufacturing of UVC-treated platelet concentrates (PCs) under routine conditions. The objective of this study was to evaluate potential in vitro effects of the THERFLEX UV-Platelets treatment on platelets produced from pools of 5 buffy coats (BCs) in comparison to untreated and gamma-irradiated platelets.

Methods

For this study, leukoreduced and plasma-reduced PCs were prepared from five BCs using 280 mL of SSP+ additive solution (Macopharma). We established the preparation of three different platelet products: PCs treated with the THERAFLEX UV-Platelets system (Macopharma) within 6 h after PC preparation, untreated PCs and gamma-irradiated (30 Gy) PCs. To evaluate the quality of these products, we analyzed 100 PCs for volume, residual erythrocytes and leukocytes, platelet content, protein concentration and sterility. For a smaller number of PCs, CD62P (P-selectin) surface expression, a marker of platelet activation was analyzed by flow cytometry with and without activation by thrombin-receptor activating peptide (TRAP). In vitro parameters were compared by an unpaired Welch's t-test due to different sample size. A p value of < 0.05 was considered statistically significant.

Results

UVC-treated PCs showed no significant differences compared to untreated or gamma-irradiated PCs for residual leukocytes, residual erythrocytes, protein concentration and pH. The volume of the UVC-treated PCs was significantly lower than the volume of the untreated or gamma-irradiated PCs due to volume loss (5–12ml) by the additional transfer into the illumination bag. Differences in CD62P expression between the PC groups varied during storage, depending on the day of storage and the use of TRAP for activation. Tests for bacterial contamination were negative for all tested PCs.

Conclusions

This data indicates that the plasma-reduced, UVC-treated PCs meet the quality standards for PCs products of the German Guidelines. Studies in patients are necessary for the evaluation of safety, tolerance and efficacy of PCs manufactured by the THERAFLEX-UV Platelets system.

Tab. 1.

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Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):20.

SI-09-1 Heme oxygenase-1 inhibits HLA class I antibody-dependent endothelial cell activation

E Zilian 1, H Saragih 1, V Vijayan 1, O Hiller 1, C Figueiredo 1, A Aljabri 1, R Blasczyk 1, G Theilmeier 2, JU Becker 3, J Larmann 2, S Immenschuh 1

Antibody-mediated rejection is a key limiting factor for long-term graft survival in solid organ transplantation. HLA class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (VCAM-1, ICAM-1, interleukin-8 and MCP-1). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of antibody-mediated rejection in solid organ transplantation.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):20–21.

SI-09-2 Persistence of de novo donorspecific HLA-antibodies seems to increase the risk of lung allograft dysfunction

M Schmitzer 1, N Kneidinger 2, C Neurohr 3, R Hatz 1, R Schramm 4, V von Dossow 5, H Winter 1, T Kauke 1,6

Introduction

The impact of donor-specific (DSA) and non-donor-specific (nDSA) anti-HLA-antibodies diagnosed by solid-phase assays on outcome in patients after lung transplantation is still a matter of debate. We hypothesize that differentiating de novo DSA by persistent and transient appearance may offer a better risk assessment.

Methods

We investigated the clinical relevance of HLA-antibodies on lung allograft outcome prospectively in 72 recipients who were transplanted between 2013 and 2015. The presence of HLA-antibodies was analyzed regular prior and after (3 weeks, 3 months, 6 months, 9 months, 12 months and 18 months) transplantation and in case of graft dysfunction. Lung function, patient survival and risk factors for the development of DSA were assessed within a median follow-up of 21 months.

Results

The majority of recipients (83%) were non-immunized at time of transplantation. Two (3%) patients were transplanted with preformed weak DSA. Twenty-three (32%) patients developed de novo DSA and 14 (19%) developed de novo nDSA. In 13 out of 23 (56%) patients DSA disappeared after a median of 114 days. Fourty-four % (10/23) of patients had persistent DSA post-transplant. Time to first DSA appearance was earlier in case of transient DSA compared to persistent DSA (51.9 ± 62.1 vs. 177.3 ± 156.2 days, p = 0,035). Risk factors for DSA development seem to be the concurrent existence of nDSA (p = 0.001) and change in the immunosuppressive regime from Tacrolimus to Cyclosporine A in the first 3 months after transplantation (p = 0.03). DSA impaired patient survival in comparison to controls (1-year patient survival 83% vs. 97%; p = 0.078). Remarkably 1-year survival of patients with persistent antibodies was only 60%. Patients with persistent DSA had significantly reduced survival compared with those without DSA and with transient DSA (p = 0,001).

Conclusion

De novo DSA are associated with an increased risk for impaired graft function. Persistence of DSA in the first year after transplantation seems to be more harmful for lung allograft dysfunction than temporary detection of DSA at an early stage. Further studies are needed for therapeutical strategies early after transplantation to prevent humoral immune response.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):21.

SI-09-3 HLA-antibody testing with LabScreen/Luminex single antigen beads – how do the results match to CDC results

R Kelsch 1, TMC Binder 2, K Schütte-Nütgen 3, B Suwelack 3

In the Eurotransplant program HLA-antibody screening is performed to define inacceptable donor HLA antigens for kidney organ offers. Different laboratory techniques are used including complement dependent cytotoxicity (CDC), ELISA or the Luminex single antigen bead assay (SAB). CDC is supposed to detect HLA-antibodies, which are most relevant. The SAB is more sensitive than the CDC and usually more HLA-antibody specificities are detected. The results and signals are disturbed by prozone effects, where the binding of the detector antibody is blocked by complement C1q present in the serum or by HLA-antibody overload in highly positive sera. This can be diminished by adding EDTA to the serum, or by heat inactivation, DTT-treatment or dilution of the serum. We investigated, how the results of CDC and SAB are comparable and compared sera positive in CDC with T- or B-lymphocytes with the SAB assay with and without the addition of EDTA.

Material and Methods

Sera from 434 patients (kidney waiting list) were tested for HLA-class I and II antibodies by ELISA (AbScreen HLA Class I/II, Bio-Rad). HLA Class I antibody positive sera were further tested in T-CDC (SeraScreen FCT 60, BAG). Sera solely positive for Class II antibodies were tested in B-CDC (LymphoscreenDR30, Bio-Rad). All ELISA positive sera were tested in SAB-1- (LabScreenSingleAntigen HLA-Class 1, OL) and SAB-2 assays in two different modes: 1. Untreated serum (SAB-1/2-IgG), 2. Serum supplemented with 8 mM EDTA (SAB-1/2-IgG/EDTA).

Results

22 of the 62 ELISA Class I (ELISA1) positive sera were reactive in T-CDC. 24 of 68 ELISA2 positive sera were negative in ELISA1. 19 of these 24 sera were reactive in B-CDC. The SAB-IgG testing of the untreated sera showed that the antibody specificities found in CDC only rarely led to the highest signal intensities in the SAB-IgG. However, the addition of EDTA shifted the T- or B-CDC reactive antibody specificities to the highest ranks of MFI-values in the SAB assays and thus reflected much better the CDC results.

Conclusion

CDC positive HLA-antibodies are supposed to be most relevant in transplantation. The comparison of SAB-IgG assays with untreated serum and EDTA supplemented serum revealed that the CDC positive antibody specificities moved to the highest signal intensities in the SAB-IgG with EDTA-treated serum. Thus with EDTA the signal intensities reflect the presence of HLA-antibody specificities in a quantitative manner and more related to the CDC results.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):21–22.

SI-09-4 De novo donor-specific HLA antibodies after kidney transplantation are correlated with the number of predicted indirectly recognizable epitopes

N Lachmann 1, O Staeck 2, M Niemann 3, K Budde 2, P Reinke 4, E Spierings 5, C Schönemann 1

Introduction

De novo donor-specific HLA antibodies (DSA) have been recognized over the past several years as being the leading cause of late renal allograft failure. De novo DSA posttransplant result from HLA mismatches under the impact of inadequate immunosuppression. Determinants of DSA specificity are generated via the indirect allorecognition pathway. We investigated the relevance of predicted indirectly recognizable HLA epitopes (PIRCHE) to predict the development of de novo DSA following kidney transplantation.

Methods

A total of 2,787 consecutive kidney transplants performed between 1995 and 2015 have been enrolled into the study. All patients revealed having no DSA prior to transplant as detected by solid-phase immunoassays. Posttransplant de novo DSA were detected by the Luminex® single antigen assay. HLA epitope mismatches were determined by both the Matchmaker and PIRCHE approach. Therefore, high resolution HLA typing was estimated from National Marrow Donor Program haplotype frequencies. The count of epitope mismatches and the classical antigen mismatches were correlated in uni- and multivariate analyses with renal allograft survival and the incidence of de novo DSA.

Results

The PIRCHE score moderately predicted death-censored 10-year renal allograft survival. However, the predictive value of PIRCHE scores >9 for development of DSA was statistically significant (p < 0.001) (PIRCHE score strata < 9, 9–34.99, 35–89.99 and >90 with 4%, 13%, 22% and 31%). When analyzing the predicted impact of a high or low PIRCHE score on DSA development stratified according to the degree of antigen mismatch at each HLA locus a clear differentiation could be revealed for HLA-DR and DQ and to a lesser extent also for HLA-A and B. Almost no effect could be revealed for HLA-C. In a multivariate cox regression analysis adjusted for antigen mismatch and Matchmaker epitopes the PIRCHE score could be identified as independent risk factor for de novo DSA.

Conclusion

Here, we could confirm previous findings on the PIRCHE score as a strong predictive measure for allorecognition following transplantation. PIRCHE score independently from antigen mismatch and Matchmaker epitopes could be revealed as having a strong predictive value for de novo DSA. Therefore, the PIRCHE score could help to identify acceptable mismatches with relatively reduced risk for development of de novo DSA and thus improve long-term renal allograft survival.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):22.

SI-09-5 The importance of high resolution HLA typing for association studies in glandular autoimmunity: A codon based view

BK Flesch 1, S Göbel 2, N Matheis 2, T Alt-Mayer 3, GJ Kahaly 2

Introduction

Association studies between HLA class II alleles and the presence of monoglandular (MGA) and polyglandular (PGA) autoimmunity require high resolution (2-fields) typing in order to detect all coding differences. A DRβ peptide binding pocket including Tyr26, Leu67, Lys71 and Arg74 has been described as predisposing for PGA and autoimmune thyroid disease (AITD)1 whereas Asp57 within the DQβ chain has been addressed as protective for T1D2. We performed HLA class II high resolution typing in patients with MGA and PGA and healthy controls to test for this hypothesis.

Methods

A total of 449 patients with MGA, suffering either from type 1 diabetes (T1D) or autoimmune thyroid disease (AITD), or with PGA (suffering from two or more autoimmune glandular diseases) were enrolled in this study as well as 121 healthy controls. HLA-DRB1, -DQA1 and -DQB1 typing was performed by commercial PCR-SSO applying the Luminex technology (Immucor, Rödermark, Germany) or high resolution PCR-SSP (Olerup, Vienna, Austria) and data were entered as 4-digit results.

Results

HLA-DRB1*03:01 and *04:01 were significantly (pc< 0.01) over-expressed in patients with T1D and PGA compared to AITD and healthy controls. Both alleles discriminate in a number of nucleotide positions, e.g. codon 74 (Arg vs. Ala), but they share codons 48 - 72, including codon 71 that encodes Lys. In contrast, the *04:04 allele which discriminates from *04:01 only in codons 71 and 86 (Arg71 and Val86) was clearly associated only with a subgroup of PGA patients suffering from Addison disease and AITD. DRB1*15:01, coding for Ala71, was identified as protective in APS and T1D but not in AITD (pc< 0.01). DQB1*03:01 and *06:02, sharing Asp57, were determined as protective in PGA and T1D but not in AITD compared to the controls. Whereas DQA1*05:01 was significantly more frequent in T1D and PGA, the *05:05 allele, completely sharing the binding groove encoding exon 2 with *05:01 was determined as protective.

Conclusions

High resolution typing enables discrimination and assignment of HLA class II alleles to glandular autoimmunity but the contribution and cooperation of single amino acids within the peptide binding groove is complex and requires further studies.

References

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):22.

SI-09-6 Restricted TCR Vβ usage in HLA-A*31:01-CBZ-induced CD8+ T-cells

H Kunze-Schumacher 1, L Beth 1, AA Celik 1, T Huyton 1, R Blasczyk 1, C Bade-Döding 1

Introduction

Certain adverse drug reactions (ADRs) that resemble acute GvHD are linked to distinct HLA alleles. In these instances the drug molecule is able to occupy part of the HLA molecules peptide binding groove and modify the selected peptide repertoire thereby causing a strong T-cell-mediated immune response that is resolved upon withdrawal of medication. HLA-A*31:01-restricted ADRs represent a major problem for patients treated with carbamazepine (CBZ), inducing life-threatening T-cell mediated immunological disorders such as Stevens Johnson Syndrome or Toxic Epidermal Necrolyse. Only few information about A*31:01-associated CBZ-induced ADRs exist, emphasizing the necessity for a comprehensive analysis of the underlying immune response.

Methods

The peptide repertoire, recruited by HLA-A*31:01 in presence of CBZ, was analyzed utilizing soluble HLA technology and compared to that in absence of CBZ.

HLA-A*31:01-CBZ-restricted cytotoxic CD8+ T-cells were generated by CFSE dilution using CBZ treated rAPCs, characterized and clonally expanded. Appreciation of TCR bias was accomplished by sequencing Vβ gene products in HLA-A*31:01-CBZ-naïve and A*31:01-CBZ-restricted CD8+ T-cells.

Results

Exposure of CBZ to LCL 721.221/sHLA-A*31:01 cells was detected to have great impact on the peptidome. 69% of peptides were found to be presented exclusively in presence of CBZ. Only few peptides showed high binding affinities, suggesting interference of strong binding by CBZ. The preference of a C-terminal Arg as anchor residue was discontinued by CBZ. These data imply that CBZ places in the F pocket within the peptide-binding region of A*31:01 leading to the presentation of an altered peptide repertoire and resulting in T cell activation.

HLA-A*31:01-CBZ-exposed CD8+ T-cells were analyzed for IFN-γ secretion and cytotoxicity; their sensitivity and specificity could be verified to be strikingly A*31:01-CBZ-specific. These A*31:01-CBZ-restricted CD8+ T-cells were shown to possess a significantly screwed TCR Vβ repertoire. The TCR clones Vβ3-V5–1 and Vβ8-BV8S1 were over-expressed due to CBZ-exposure in sensitive patients.

Conclusions

These findings represent a first step towards a reliable biomarker for HLA restricted CBZ-hypersensitivity reactions for HLA-A*31:01-positive patients and will guide towards understanding the emergence of T-cell mediated ADRs.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):22–23.

SI-09-7 Residue 156 impacts peptide selection and dominance of self-and viral-peptides during HCMV infection

WC Abels 1, H Kunze-Schumacher 1, T Manadhar 1, R Blasczyk 1, C Bade-Doeding 1

Introduction

Assembly of HLA class I complex is assisted by the peptide loading complex (PLC). The crucial role of PLC components in selecting and loading peptides makes the PLC an ideal target for viral immune escape mechanisms. The immune evasion strategy of HCMV prevents the presentation of viral antigens; consequently only PLC-independent HLA variants are able to constitutively present viral derived peptides. A problem in cellular therapy strategies is the emergence of antiviral T-cell anergy over time. The crucial question is at what stage of HCMV infection viral-peptides are preferentially presented over self-peptides. Two of those PLC-independent HLA molecules are the common variants B*35:01 and 35:08 distinguished by a single mismatch at residue 156, both of them have been described to present viral-peptides. The impact of the 156 mismatch on the selection and presentation of available viral peptides is not investigated, yet.

Methods

To analyze the competitive HLA-B*35:01 and B*35:08 restricted viral- and self-peptide repertoire in the presence of HCMV, we utilized soluble HLA technology. Soluble HLA complexes from HCMV infected BJ/sHLA cells were affinity purified. Kinetic of immune evasions was monitored by mRNA analysis. sHLA-B*35:01 or sHLA-B*35:08 restricted peptides were recovered and analyzed using nano-LC-ESI MS/MS technology.

Results

We found a distinct self-peptide pattern in the presence of HCMV for B*35 variants, the features and anchor motifs remained mainly unaltered in comparison to peptides recruited in the absence of HCMV. However, a different subset of viral peptides from the early to late phase of infection was recruited by B*35:01 and B*35:08. A high frequency and dominance of certain viral peptides over self-peptides was striking.

Conclusion

These findings explain the discrepancy between predicted and naturally presented immunogenic epitopes for establishing anti-viral effector cells. Furthermore, peptide prediction based on available peptide anchors as given in the databases might fail due to structural alteration of HLA variants and support the need of comprehensive peptide recruitment data for personalized and effective cellular therapies.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):23.

SI-09-8 Impact of HLA-DPB1 matching for pediatric hematopoietic stem cell transplantation with malignant and non-malignant indications: experience from a single-center study

K Todorova 1, G Strauß 2, C Schönemann 1, J-S Kühl 3, M Gorelashvili 4, W Ebell 3, H Schulze 1,4

Allogeneic hematopoietic stem cell transplantation (HSCT) from matched unrelated (MURD) or siblings donors (MSD) has become a standard procedure by malignancies like leukemia or lymphoma, or by certain non-malignant disorders (i.e. immune defects or hemoglobinopathies, metabolic disorders). The standard 10/10 HLA match comprises 5 loci (HLA-A,- B,-C,-DRB1,- DQB1) with two alleles each. An increasing body of evidence suggests that HLA-DPB1 plays an important role for the overall survival (OS) after HSCT. We aimed to determine the role of HLA-DP in a cohort of 264 pediatric patients receiving a 10/10 matched HSCT at the Charité University Hospital, Berlin during 2003 and 2013. In a retrospective study we analyzed the overall outcome on patients receiving a 10/10 transplant by a MSD (n = 65) or MURD (n = 97). All donor/recipient pairs were additionally typed for HLA-DP and the match grade was analyzed by a direct (antigen/allele matching) as well as by a direct T-cell epitope (TCE) model for HLA-DP providing a match analysis of three antigenic classes for “permissive” matches that are tolerated or “non-permissive mismatches” that are recognized by T-cells.

Our results show that patients transplantated with a MSD (10/10 or 12/12) show a better 5-year OS (85%) compared to those with a MURD (69.5%, p < 0.05). For children with malignancies, a non-permissive HLA-DP mismatch was slightly better (p = 0.742, survival DP match=63.49%, survival DP mismatch=71.5%), most likely due to a graft-versus-leukemia effect. In contrast, for children with non-malignant indications, a matched HLA-DP donor was better (p = 0.077, survival DP match=86.7%, survival DP mismatch=55.8%) and should thus be considered as a donor, especially considering regimens that will result in a mixed chimerism. TCE-based matching confirmed our findings (non-malignant: survival TCE permissive=74.4%, survival TCE non-permissive=50.0%, p = 0.109; malignant: survival TCE permissive=66.8%, survival TCE non-permissive=83.3%, p = 0.482). The TCE-based grouping of HLA-DP increases the number of potential donors compared to classical matching on antigen/allele level. An online tool to calculate the TCE algorithm allows to specifically select DP-matched or -mismatched donors depending on the underlying indication for HSCT. We conclude that HLA-DP matching should be considered in children when several suitable 10/10 donors are available.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):23.

SI-09-9 Deconstructing HLA-C mismatch in hematopoietic stem cell transplantation

C Tsamadou 1,2, D Fürst 1,2, D Niederwieser 3, D Bunjes 4, C Neuchel 1,2, M Gramatzki 5, R Arnold 6, E Wagner 7, H Einsele 8, H Schrezenmeier 1,2, J Mytilineos 1,2,9

Contrary to other HLA loci, allele vs antigen mismatches in HLA-C, appear to differentially impact HSCT outcome. Aim of this study was to investigate the independent role of other factors characterizing a patient's HLA-C non-shared allele, in HSCT outcome as well as their distribution in allele vs antigen mismatched cases.

288 9/10 HLA-C mismatched unrelated transplant pairs, were additionally genotyped by sequence based typing for rs9264942(C/T) and rs67384697(ins/del), which are considered surrogate markers for HLA-C expression levels. A proxy MFI model as previously described (Petersdorf et al., Blood 2014), was also implemented for the analysis. All patient non-shared alleles were characterized in regard to mismatch level, expression-relevant polymorphisms’ genotype and proxy MFI levels as well as differences in residues 77, 80 (C1/C2 KIR epitopes) and 116 (immunogenic spot). In order to assess the independent impact of each factor on HSCT outcome, overall survival, disease free survival, non-relapse mortality and relapse incidence were set as endpoints. The balance in distribution of the aforementioned parameters in allele vs antigen mismatched cases was estimated by Chi-square and Wilcoxon tests. Statistical significance was set to a p < 0.05.

Data analysis revealed no statistically significant impact of any of the parameters tested individually on HSCT outcome. Their distribution with respect to mismatch level, however, was markedly skewed. Antigen mismatches were associated with more highly-expressed HLA-C alleles according to the MFI proxy model as well as the rs9264942C and rs67384697del genotypes (MFI: p < 0.0001; rs9264942C: 5% vs 37%, p < 0.0001; rs67384697del: 12% vs 41%, p < 0.0001). Likewise, differences in residues 77, 80 and 116 of the patient non-shared allele were much more frequent in the antigen-mismatched cases (C1/C2: 3% vs 61%, p < 0.0001; Res116: 5% vs 69%, p < 0.0001).

Our findings suggest that the differential impact of HLA-C allele vs antigen mismatch on post-HSCT prognosis must be the result of accumulating parameters acting synergistically. Further studies with larger cohorts are warranted in order to clarify if these parameters should be considered in an allele-level HLA-C mismatch context for even better donor selection.

This work was supported by the Deutsche José Carreras Leukämie-Stiftung e.V. (Grant No. DJCLS 11/10).

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):24.

SI-10-1 (INV. SPEAKER) Blood Pharming

T Tonn 1

The replacement of blood products by artificial oxygen carriers has been a long-time sought after goal in transfusion medicine, as it may make blood substitution available for patients with multiple antibodies against common blood groups and, even more visionary, may even allow standardized and donor-independent pharmaceutical production. Here, advances in stem cell technology have opened the door for new approaches that aim to differentiate and expand mature red blood cells and platelets from different sources of stem cells with the aim to provide a source for replacement therapies. Recently, the term “blood pharming” has evolved to describe this innovative field. Since red blood cells and platelets are devoid of the cell nucleus, both cells appear to be ideal cell populations for clinical applications of pluripotent stem cell based therapies. In addition, the two types of cells theoretically do not carry the potential risk for genomic instabilities and tumor formation that are inherent to embryonal and induced pluripotent stem cells. The technical advance in stem cell based approaches comes along with breakthrough technologies in genetic engineering, such as CRISPR/Cas9 and gene knockdown technologies. These advances will ultimately allow the design of universal blood substitutes to lack common immunological antigens, such as blood group antigens on red blood cells and MHC class I antigens on platelets. This is significant for making blood products readily available as a universal source, irrespective of the patient's blood group and HLA phenotype. This presentation will to summarize the current status of development and will also to glimpse into the future with regard to this exciting field of blood farming.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):24.

SI-10-2 (INV. SPEAKER) Genetically engineered blood pharming: Generation of HLA-universal platelets

C Figueiredo 1

The combination of blood pharming with advanced gene therapeutic strategies enables the in vitro production of safe, more effective and readily available blood cells. Refractoriness to platelet (PLT) transfusion caused by alloimmunization against Human Leukocytes Antigens (HLA) class I antigens remains a relevant clinical problem. To reduce the immunogenicity of cell-based therapeutics, we have permanently reduced HLA class I expression using an RNA interference strategy. We have silenced the expression of HLA class I to generate a stable HLA-universal induced pluripotent stem cell (iPSC) line that can be used as a renewable cell source. The expression of HLA class I was silenced by up to 82% and remained stable during iPSC cultivation. In our studies, we have focused on the generation of megakaryocytes (MK) and PLTs from a HLA-universal iPSC source under feeder-free and xeno-free conditions. Differentiation rates of MKs and PLTs with means of 58% and 76% were observed, respectively. HLA-universal iPSC-derived MKs showed DNA contents up to 64n and formed proPLTs. Importantly, differentiated MKs remained silenced for HLA class I expression and produced functional PLTs in vitro. Notably, iPSC-derived HLA-universal MKs were capable to escape antibody-mediated complement- and cellular-dependent cytotoxicity. HLA-expressing or silenced iPSC-derived MKs were used to transfuse NOD/SCID/IL-2Rγc(-/-) mice under refractoriness conditions. In presence of anti-HLA antibodies, HLA-expressing MKs were rapidly cleared from the circulation of mice, while HLA-silenced MKs escaped HLA antibody-mediated cytotoxicity. Importantly, biodistribution assays have shown no MK engraftment in organs such as lung, heart, kidney, spleen or bone marrow. Thus, in vitro produced HLA-universal MKs and PLTs may become an alternative to PLT donation in PLT-based therapies and an important component in the management of severe alloimmunised patients.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):24.

SI-10-3 (INV. SPEAKER) Challenges for circulating tumor cell (CTC)-based liquid biopsies: low CTC frequency and diagnostic leukapheresis as a potential solution

J Fischer 1, D Niederacher 2, NH Stoecklein 3; Disseminated Cancer Cell Network (DCC Net) Düsseldorf

Circulating tumor cells (CTCs) are very attractive surrogate markers for systemic cancer. Currently, major efforts are being made to use these rare cells in the sense of a liquid biopsy to gain molecular information for rational therapeutic decision-making. The advancements in molecular analyses of CTCs down to the single-cell level have been significant in recent years and some applications are ready to be used in clinical studies. A major challenge for translating such molecular CTC-based assays into the clinic is the extremely low frequency of CTCs and the associated problems of their reliable detection and isolation. A potential solution to overcome the low CTC frequency is the recently introduced diagnostic leukapheresis (DLA) that permits screening of liters of blood. DLA is a clinically safe method that enables a reliable detection of CTCs at high frequency even in nonmetastatic cancer patients, and might facilitate the routine clinical use of CTCs as in the sense of a liquid biopsy. Combined with technologies for single-cell molecular genetics or cell biology, it may significantly improve prediction of therapy response and monitoring of early systemic cancer.

Discussed here are the challenges as well as the current efforts implementing this method into clinical workflows to realize these more reliable liquid biopsies.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):24–25.

SI-11-1 (INV. SPEAKER) Red blood cell antibody diagnostics using synthetic proteins

A Seltsam 1

The main drawback of red blood cell (RBC)-based antibody detection assays is that a positive reaction between serum and test cells does not identify the specificity of a given red blood cell antibody. When antibody mixtures or rare RBC antibodies are present, this indirect method reaches its limits. Sufficient numbers of validated and CE-marked recombinant blood group protein (rBGP) specificities are now available to significantly support antibody identification. The advantage of rBGPs over RBCs is that proteins carrying a single antigen can be used in the antibody identification assay, so a positive test directly indicates the presence and specificity of the target antibody. The use of soluble rBGPs in addition to RBCs has been shown to increase the sensitivity and specificity of antibody detection and identification in cases with difficult-to-identify antibodies, resulting in increased efficiency in providing blood to immunized patients. Because rBGPs are particularly helpful in resolving cases with complex antibody reaction patterns, they are a good supplement to laboratories that provide high-level patient care but do not have the capacity for advanced traditional RBC serology. Presently, the serologic work-up of complex reaction patterns relies more on intuition and experience than on defined rational procedures. However, in times of high cost pressure, the immunohaematology specialist required for tricky antibody testing is an endangered species threatened by extinction. Therefore, rBGPs may become a valuable tool for laboratories, which could help them to ensure a viable future for their immunohaematological expertise. The clinical use of rBGPs in pretransfusion antibody screening introduces a paradigm shift that could help to reduce the risk of haemolytic transfusion reaction, one of the leading causes of transfusion-related death.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):25.

SI-11-2 (INV. SPEAKER) Molecular diagnostics in pregnancy – determination of the fetal D status from maternal blood

TJ Legler 1

Knowledge of the fetal RhD status allows personalized monitoring of pregnant women with alloimmunization. Since invasive tests have a high risk for booster reactions, non-invasive testing for the fetal RHD-status (NIPT-RHD) from maternal plasma is recommended. Furthermore, according to the draft of revised German Guidelines (Richtlinien Hämotherapie) antenatal Rh-prophylaxis is not required if the fetus has been tested D-negative.

Larger studies revealed that the sensitivity of NIPT-RHD is at least equivalent to the sensitivity of serological tests for RhD applied in newborns after birth in order to guide postnatal prophylaxis. However, strict adherence to quality assurance measurements is crucial in order to guarantee a high accuracy for each sample in routine laboratories. At least two RHD exons have to be investigated and replicate testing is mandatory to increase the sensitivity for those samples which have an unusual low concentration of fetal DNA. If the fetus is D-negative, confirmation of the presence of fetal DNA by testing for a sex independent fetal marker is recommended. Each run of tests requires positive and negative controls for RHD and the fetal marker. Extraction controls and PCR controls are available as in-process controls for each sample and further increase the quality of tests. However, validation data are required to demonstrate that control reactions do not inhibit the D-specific reaction if multiplex tests are applied. Extensive validation experiments have to be performed to determine the diagnostic sensitivity, specificity, accuracy, reproducibility, limit of detection (LOD), and inter-assay variation of a NIPT-RHD assay before it can be applied for diagnostic purposes. An external quality scheme is established on an international level and a WHO Reference Reagent is commercially available. Adequate reimbursement by the German health care system is needed to allow NIPT-RHD on a larger scale.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):25.

SI-11-3 Red blood cell alloimmunization in neonates and children up to three years of age

T Türkmen 1, D Qiu 2, N Cooper 1, U Sachs 1, W Wößmann 3, D Schranz 4, K-P Zimmer 5, H Ehrhardt 6, H Hackstein 1, G Bein 1

Background

Alloimmune response after red blood cell (RBC) transfusion in neonates is a rare event. The length of the time frame of unresponsiveness against nonself blood group antigens in newborns and toddlers is unknown. This study compared the immunization risk after RBC transfusion in children up to three years of age with the immunization risk in adults older than 45 years of age.

Study design and Methods

A retrospective cohort study was conducted among consecutive transfused patients at a single university medical center. All non-alloimmunized patients who received their first RBC transfusion between 1995 and 2014, and who had at least one antibody screen follow-up between 7 and 365 days after transfusion were included.

Results

A total of 1,641 neonates and children up to three years of age received 5 (median; interquartile range (IQR) 3–15) RBC units. 5 (0.3%) children developed post-transfusion antibodies. The age at first positive antibody screen follow-up was 889 (median, range 181–1039) days. The antibody specificities included four anti-M antibodies and one anti-E antibody. The cumulative incidence of alloimmunization in the control group of 17,084 adult patients older than 45 years of age, who had received 5 (median; IQR 2–12) RBC units, was 3.6%.

Conclusion

Alloimmunization against RBC antigens was not detected within the first 6 months of life. In this cohort, post-transfusion RBC alloantibodies in children older than 6 months and up to three years of age belonged exclusively to antibody specificities that are known as naturally occurring, and that may also be triggered by non-blood born antigens. Repeated antibody screening and cross matching during the first months of life can be safely omitted.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):25–26.

SI-11-4 Inhibition of SP1 by miRNA-331-3p causes glycosyltransferase A repression and may explain certain weak A (Ax01v)

R Kronstein-Wiedemann 1, N Kronstein 1, T Tonn 1,2

Background

More than 100 AB0 subgroup-related variations were detected in the coding region of glycosyltransferases, which may be causative for a weak blood group antigen A or B expression. Most variation in expression is explained genetically by mutations that reduce transferase activity. Only a small minority have not yet been explained, in the absence of analysis of regulatory promotor or enhancer regions. We found that miRNAs play a critical role in the regulation of AB0 blood group antigen. Here we show that the effects of miR-331–3p could be mediated by the inhibition of transcription factor SP1, which in turn is not able to bind to the promotor of the AB0 gene, thus downregulating blood group A antigen expression.

Methods

By distinct complementary approaches, including gene array analysis and overexpression of glycosyltransferase specific miRNAs in primary hematopoietic stem cells (HSCs), we identified that miR-331–3p and -1908–5p directly target glycosyltransferase A and B. Using microR-NA target prediction tools we also identified Sp1 as a potential target gene for miR-331–3p. Furthermore two of the three binding sites for SP1 in the 5'UTR of glycosyltransferases are in addition also binding sites for miR-1908–5p. Therefore we treated HSCs with an inhibitor or a stimulator of SP1 and analyzed blood group A expression by flow cytometry and ID-Card gel method.

Result

Overexpression of miR-331 and -1908 in HSCs leads to a 30–50% reduction of blood group A antigens per cell in differentiated RBCs. Furthermore miR-331–3p and -1908–5p were enhanced in red blood cells of Aweak variants. Sequencing of the 3'UTR of 6 Aweak variants revealed the presence of more miRNA binding sites for miR-1908–5p compared to normal controls. Inhibition of SP1 also leads to 40–70% reduction of blood group A antigen per RBC, in case of blood group A2 even up to 90%. Stimulation of SP1 results in an increase of blood group A antigen per RBC by about 40–70%.

Conclusion

Glycosyltransferase A and B expression is regulated by miR's 331–3p and 1908–5p, by inhibition of the transcription factor SP1. This new concept may provide an explanation of the molecular basis for Ax01v weak blood group phenotypes. We assume that increased expression of miR-1908–5p and −331–3p in Ax variants may lead to downregulation of Sp1 and to a competition of miR-1908–5p with Sp1 for binding sites in the promotor region of the AB0 gene which in turn results in further gene repression.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):26.

SI-11-5 Establishing a standard operating procedure to overcome the interference of anti-CD38 with the pretransfusional serological testing for the ID-card system

K Selleng 1, K Denker 1, A Greinacher 1

Introduction

A new treatment of patients with multiple myeloma has been approved using an anti-CD38 antibody. The CD38 antigen is also expressed on red blood cells (RBCs). Therefore all serological tests before transfusion (antibody screening and cross matching), performed by the indirect antiglobulin test (ICT) become positive. The treatment of screening cells with dithiothreitol (DTT) can solve the anti-CD38 interference. We established a protocol for DTT treatment of screening cells and their storage conditions for routine laboratory work up.

Methods

Different volumes of commercially available sreening cells (ID-DiaCell I-II-III, BioRad) were treated by different volumes of DTT 0.2M (37°C, 30 min), washed with PBS pH 7.3 (4x) and resuspended either in ID-Diluent 2 (BioRad), PBS pH 7.3 or ID-Cellstab (BioRad) for a 0.8–1% cell suspension. The efficacy of the DTT treatment was tested by ICT of K+ cells with anti-K (IgG) before and after DTT treatment. The integrity of DTT insensitive antigens was tested using anti-D (patient serum) in several dilutions. The antiglobulin test was performed using the ID-Card LISS/Coombs system (BioRad). The DTT treated cells were stored at 4°C for 7 days.

Results

The preparation of DTT treated cells was performed within 1h. The use of small volumes (4 drops 0.8% RBC suspension) or of high volumes (2 ml) made no differences for the results. Freshly prepared DTT treated cells could be used in all tested storage solutions. Storage in PBS resulted in rapid onset hemolysis and complete hemolysis after more than 3 days. DTT treatment caused irreversible destruction of the K antigen, while the screening cells tested positive with anti-D for the entire storage period.

Conclusion

DTT treatment of commercially available screening cells is suitable to overcome the ICT interference by anti-CD38 and allows the detection of RBC alloantibodies directed against DTT insensitive antigens using the ID-Card method. Storage of DTT treated test RBCs in commercial storage solutions is possible but the stability of antigens beside the tested antigens requires further investigations.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):26.

SI-12-1 (INV. SPEAKER) German Haemovigilance report 2015

M Heiden 1, A Lohmann 1, R Spranger 1, S Müller 1, S Schönefeld 1, C Witzenhausen 1, M Funk 1

Blood components are lifesaving medicines which enable medical progress in many fields. Despite the current high level of quality and safety of blood components adverse reactions in donor and recipient as well as adverse events can occur.

Haemovigilance comprises collection and assessment of all unexpected or adverse events occurring before during and after administration of blood components with the ultimate goal of prevention of future adverse events. Systematic centralized collection and assessment of data on serious adverse events (SAE) and serious adverse reactions (SAR) in donor and recipient as done at the PEI provides a tool to recognize relevant risks within the transfusion chain and to give advice for optimizing haemotherapy. German haemovigilance data 2015 are presented as periodic update from 1979 to 2015. Comparison of the results with those of other haemovigilance systems (UK and Switzerland) shows that similar standards exists in these countries.

As in the years before, Grade III/IV acute transfusion reactions (ATR) and transfusion transmitted bacterial infections (TTBI) per million units - graded as possible to certain - were mainly accompanied with platelet transfusions. While ATR still showed an increasing tendency there is a plateau in reported TBBI since 2009. TACO again was mainly caused by RBC and FFP. The increase of TRALI cases classified as possible to likely is linked to the new evaluation algorithm. No transmission of HIV, HBV or HCV could be confirmed. 4 out of 10 reported cases of HEV transmission were confirmed as likely to certain and will be presented more in detail. Only a slight decrease is observed for donor initiated HBV look back procedures following introduction of the new assessment of Anti-HBc initially reactive donations in 2014. A steady increase was observed for reports on severe ATR, on donor SAR as well as for reports on SAE, especially of incorrect blood components transfused.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):26.

SI-12-2 (INV. SPEAKER) Human Cytomegalovirus – current knowledge and relevance for transfusion medicine

M Ziemann 1

German guidelines disadvice prophylaxis against transfusion-transmitted Cytomegalovirus-infections (TT-CMV) besides leukodepletion. Nevertheless, “CMV-safe” units are requested by many clinicians, and CMV-specific tests performed in many transfusion services. This lecture gives an overview about the current knowledge about TT-CMV, and deduces strategies to overcome this discrepancy.

Latently infected WBCs are regarded as the main source of TT-CMV. Also cell-bound and free CMV from primarily seropositive donors might cause TT-CMV. Both established strategies (seronegative and NAT-negative), as well as selecting blood products from donors being seropositive for at least one year prevent transfusion of products from primarily seropositive donors.

The few clinical studies using blood products leukoreduced by current methods report rates of TT-CMV between 0 and 6.5%. Mostly, TT-CMV is not definitely proven. The only exception is a study about very low-birth weight infants transfused with both leukoreduced and CMV-seronegative units, detecting no TT-CMV but 29 other CMV-infections in 539 infants. Future studies should prove the source of CMV-infections by

  • – Differentiating the serostatus of all donors into steadily seronegative, seronegative with subsequent seroconversion, newly seropositive, and long-term seropositive.

  • – Determining CMV DNA not only in stored plasma, but also in urinary samples, because urine remains CMV DNA-positive for years after primary infection.

  • – Ideally a comparison of CMV genomes from patient and donor.

  • – Examining urinary samples from suspected contact persons to prove community-acquired CMV-infections.

As the general incidence of CMV-infections is low, large study populations are needed. Alternatively, collecting data from only about 20 thoroughly examined at-risk patients would allow estimation of the relevance of TT-CMV relative to other sources of infection.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):27.

SI-12-3 Rapid bacterial detection in buffy coat derived pool-platelets and apheresis platelets by Bactiflow and NAT, five year experience

K Gubbe 1, K Hourfar 2, K Frank 1, A Karl 1, B Rüster 2, T Tonn 1, U Mayr-Wohlfart 3, H Schrezenmeier 3, E Seifried 2, M Schmidt 2

Background

The residual risk of bacterial contamination of blood components is approximately 1to 2 log periods higher than that for virus infections. In order to improve blood safety the shelf life of platelets was limited to four days in 2008 in Germany.

Aim

The aim of this study was to show the routine experience of the last 5 years by using an inhouse 16s DNA NAT system and to evaluate the Bactiflow method in individual donation testing, in mini-pools of 5 or 10. Validation of Bactiflow method was done using seven transfusion relevant bacterial strains in buffy coat derived pool-platelets (BC-platelets) as well as in apheresis platelets (A-platelets).

Methods

For routine testing (2012–2016) samples from BC-platelets or A-platelets were collected 49h after blood donation and pooled into mini-pools with a maximum pool size of 10 samples per pool. For the Bactiflow validation study BC-platelets as well as A-platelets were screened for bacterial contamination at day 0 by BacT/ALERT and by Bactiflow. Negative platelet bags were spiked in independent experiments with a low bacterial concentration of 0.03 CFU/ml with Bacillus cereus, Klebsiella pneumoniae, Escherichia coli, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes.

Results

Bacterial detection was 100% on individual donation, mini-pools of 5 and mini-pools 10 for BC-platelets and A-platelets without any exception. Data were comparable at all three test sites. Robustness tests demonstrate that test samples can be shipped for 24h at 4°C as well as at 21°C without impacting detection of bacterial contamination. Over the last 5 years in total 60,000 platelets were tested by NAT (Table 1). 5 platelet concentrates were confirmed positive for bacterial contamination, which represents a yield rate of 1 in 12,000.

Conclusions

The rapid bacterial detection method Bactiflow was able to detect transfusion medicine relevant bacterial strain in mini-pools up to a maximum pool size of 10 samples per pool either in BC-platelets as well as in A-platelets and is comparable to the 16s DNA NAT system. The introduction of rapid bacterial detection systems enables blood donor services to detect bacteria in platelets before products are release.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):27.

SI-12-4 A two-step screening approach for the identification of blood donors with highly and broadly neutralizing capacities against human cytomegalovirus

J Falk 1, M Winkelmann 2, H Schrezenmeier 2,3, D Stöhr 1, C Sinzger 1, R Lotfi 2,3

Background

Hyperimmunoglobulins are frequently applied for prophylaxis and treatment of human cytomegalovirus (HCMV) infections but have been shown to be only marginally effective in meta-analyses of clinical studies. This might be partially due to selection of donors according to total anti-HCMV titers rather than to their neutralizing capacity. We aimed to develop a high-throughput screening method for identification of blood donors with antibodies showing highest neutralization capacities against HCMV.

Methods

Using a Gaussia luciferase-expressing reporter virus, 1000 HC-MV-IgG positive plasma samples with known anti-HCMV-Ig titers were analyzed for their neutralization capacity against fibroblast and endothelial cell infection. Highly neutralizing plasma samples identified in that screen were tested by an ELISA-based neutralization assay for their inhibitory capacity against 7 different HCMV strains: Towne, AD169, VHL-E, 40E, Merlin, VR1814, Toledo.

Results

Anti-HCMV titer did not correlate with the neutralization capacity inhibiting HCMV from fibroblasts and endothelial cell infection. Without exception, all plasma samples neutralizing fibroblast infection were also highly effective against infection of endothelial cells at 100 fold lower concentrations, providing the possibility to simplify our screening method by using only fibroblasts as target cells and only one dilution step of plasma samples. Furthermore, the majority of plasma samples with high neutralization capacity against fibroblast infection were highly effective against different HCMV strains and also superior to commercially available hyperimmunoglobulins (Cytotect®) containing high HCMV-Ig titers.

Conclusion

Donors with highly and broadly HCMV neutralizing capacities could be identified by a two step high throughput screening approach paving the way for development of an antibody-based treatment or prophylaxis of HCMV infections.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):28.

SI-12-5 Pathogen inactivation of double platelet concentrates made of eight buffy coats: results from a validation study

K Rosskopf 1, W Helmberg 1, T Fuchs 1, M Sommer 1, C Url 1, B Vasic 2, S Andresen 3, M Bauhaus 3, N Moerman 3, P Schlenke 1

Pathogen inactivation (PI) of platelet concentrates (PC) increase the safety of platelet transfusion. Prior to the implementation of PI technologies a cost-benefit assessment should be integrated. To increase the production efficacy we tested a system for PI of PC made of buffy coat.

We produced PC made of eight buffy coats and using a dual-set for PI with amotosalen/UVA with one bag for treatment and - after splitting - two bags for storage of two units PC. Buffy coats (BC) from whole blood units were carefully tailored so that PC out of 8 BC with additive solution fit the criteria for PC before PI with the dual set. The second centrifugation program („soft spin”) was adapted to volume and hematocrit of the BC pool. PC volume has to be below 420ml, plasma ratio has to be between 32 and 47%, platelet yield should be between 5 to 8 x10E11 before PI. pH and swirling were tested on pathogeninactivated PC until day +7.

Results are presented as mean±SD and min-max. We produced six pools of eight buffy coats with 42 ± 1ml (40–46) each and 280ml additive solution (PAS III). The volume of the double PC after centrifugation and automatic separation before PI was 400 ± 9ml (394–419), plasma ratio was 40 ± 2% (36–42), platelet yield was 6,5 ± 0,5 x10E11 (5,6–7,1). After PI and bag-splitting the platelet yield per bag was 2,9 ± 0,2 x10E11 (2,6–3,2). pH on day +7 reached 7,21 ± 0,11 (7.03–7,31), and swirling phenomenon was maintained for all products at level 3.

To increase the production efficacy and decrease the costs of pathogen inactivation it is safe and feasible to use a dual-set with one bag for treatment and two bags for storage of double BC-PC. Preconditions are optimizing the separation program of the whole blood units to get small buffy coats (e.g. 42ml) and optimizing the centrifugation program for the pool of eight buffy coats.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):28.

SI-13-1 (INV. SPEAKER) MALDI-TOF MS based blood group genotyping

S Meyer 1, N Trost 1, BM Frey 1, C Gassner 1

Introduction

Most blood group antigens are genetically encoded by single nucleotide polymorphisms (SNPs). MALDI-TOF mass spectrometry (MS) has proven its potential in SNP genotyping and was therefore chosen as platform for genotyping of blood groups with high-throughput capability.

Methods

An initial Swiss R&D project started in 2011 and aimed for the molecular detection of the blood groups Rh, KKD, MNSs, a comprehensive collection of low and high frequency antigens (HFA), as well as HPA and HNA antigens. Subsequently, a routine-oriented typing tool for 46 selected blood group antigens and 4 HPAs was implemented for blood donor genotyping.

Results

Approx. 6000 blood donor samples with complete serological prevalues for Rh, KKD and MNSs were tested for 101 blood group antigens, encoded by 170 alleles, respectively. Additionally, 31000 more donors from all over Switzerland were tested for 45 HFA and delivered >400 donors with HFA negative genotypes, now registered with the Swiss Rare Donor File. The routine-oriented typing tool was accredited with the Swiss Accreditation Service for blood donor genotyping in 2014 and used to analyze another 10000 blood donors since. These projects raised available donor antigens for matched transfusions from 0.7 to 3.0 mio at our centre. Genotyping qualitatively outperformed serology for all KKD and MNSs antigens concurrent with lower typing costs. Systemically relevant new alleles, especially within the MNSs system, were discovered. Measuring gene counts for RHD vs. CE and GYPB vs. other GYP genes, offers supplemental potential of the MALDI-TOF genotyping platform.

Conclusion

Facing the growing ethnic complexity of populations and overcoming the drawbacks of serological typing of blood group antigens would lead to a significant optimization of transfusion strategies, and be most relevant in the clinical management of individual cases. Expanded blood group genotyping using MALDI-TOF MS is an appropriate step into this direction.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):28.

SI-13-2 (INV. SPEAKER) Molecular platelet diagnostics

P Bugert 1

Molecular genetic diagnostics is a growing field in laboratory medicine mainly driven by the development of novel analytical technologies and by the identification of novel genetic markers. This is also true for the diagnostic work-up in the field of platelet immunology and platelet function analysis. In case of anti-platelet allo-antibodies genotyping of the human platelet alloantigens (HPA) that are based on a diallelic SNPs in the corresponding receptor genes is essential for identification of the antibody specificity and for optimal treatment. Nowadays, several commercial HPA test systems are available for the routine lab. In case of a suspected platelet function disorder the subsequent molecular diagnosis is usually more complex. When the platelet phenotype is classified (e.g. Glanzmann thrombastenia, Bernard-Soulier or Hermansky-Pudlak syndrome) the corresponding genes are screened for the underlying mutations by Sanger sequencing in specialized laboratories. Next-generation-sequencing (NGS) technologies are indicated when the platelet phenotype is unclassified or the number of candidate genes is high. The ThromboGenomics working group (Cambridge, UK) offers a NGS-based diagnosis platform covering 76 genes known to be involved in coagulation and platelet function disorders. In Germany the ThromKid study group introduced a panel-based NGS approach focused on platelet biogenesis and function including 59 genes. Both platforms enabled the identification of novel unclassified genetic variants. To validate a novel variant as causal mutation familial segregation analysis is performed. Screening for the variant in the healthy population as well as review of the current literature and databases could help to identify novel pathogenic variants. Using a panel-based approach the success rate for classified cases is high. For unclassified cases a whole genome sequencing (WGS) or whole exome sequencing (WES) strategy could be a reasonable diagnostic approach in future.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):28.

SI-13-3 Using lateral flow technique a positive direct antiglobulin test did not interfere with typing of Fya Fyb Jka Jkb S, or s blood group antigens

C Weinstock 1, M Lotsch 1, H Schrezenmeier 1, M Anliker 1

Introduction

The most common technique for red cell phaenotyping is the haemagglutination test. Antiserum is incubated with the red cells to be typed and an agglutination reaction indicates the presence of the blood group antigen. Antisera containing IgM are capable of directly agglutinating red cells, whereas IgG antisera finally require the use of antiglobulin for the agglutination of antigen-positive cells.

Red cells carrying warm-reactive autoantibodies react with antiglobulin (direct antiglobulin test, DAT). Phaenotyping of such cells with IgG antisera is therefore in most cases not possible.

Recently, a lateral flow card for typing red cells for Fya Fyb Jka Jkb S, or s antigens was developed: Red cells are applied onto the center of a membrane and rinsing solution flushes them to its periphery. In six equidistant, linear detection areas the cells meet the blood group specific antibodies, which cause a visible sedimentation of antigen-positive cells. Antigen-negative cells, in contrast, do not sediment and are flushed through the detection area. Because these cards contains both, IgM and IgG antisera, we investigated, whether a reactive DAT interferes with this technique.

Methods

Individuals with a 2+ to 4+ reactive DAT were studied (No transfusion within the last 3 months was reported). 12 EDTA-anticoagulated samples were tested with the MDmulticard Basic Extended Phenotype from Grifols: 100 μl of diluted blood were pipetted onto the card and rinsed with 300 μl of rinsing solution. After 5 minutes the cards were flushed a second time with 300 μl. After 10 minutes the reactions were interpreted. For comparison, samples were phaenotyped by the gel card and the tube method using commercially available antisera. For control all samples were genotyped by SSP.

Results

With the lateral flow card all samples with a reactive DAT were typed correctly, as confirmed by SSP-PCR. In contrast, samples with a reactive DAT gave false positive results with IgG antisera when tested in the tube or gel card method.

Conclusion

The results of this small preliminary study indicate that the lateral flow technique may not be prone to false positive results when phaenotyping red cells with a reactive DAT. However, further studies and the use in routine testing are needed to confirm this finding.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):29.

SI-13-4 Non-invasive fetal platelet and red cell blood group genotyping with the use of targeted massively parallel sequencing of maternal plasma cell-free DNA

S Wienzek-Lischka 1, J Dehl 1, V Fröhner 1, A Krautwurst 1, S Gattenlöhner 2, A Bräuninger 2, C Deisting 3, R Axt-Fliedner 3, J Degenhardt 3, H Hackstein 1, S Santoso 1, U Sachs 1, G Bein 1

In pregnant women with a history feto-maternal incompatibility (fetal and neonatal alloimmune thrombocytopenia (FNAIT) or hemolytic disease of the newborn (HDN)), fetal human platelet antigen (HPA) or blood group genotyping is required to determine whether the fetus is at risk and whether prenatal interventions are required.

Published methods for non-invasive genotyping of fetal blood groups using maternal plasma cell-free DNA do not provide internal controls for exclusion of false-negative results.

Cell-free DNA was isolated from plasma of 3 RhD-negative pregnant women and 6 pregnant women with a history of FNAIT due to anti-HPA-1a (4 cases), anti-HPA-5b or anti-HPA-15a. The gestational age at the time of blood sampling was 24 weeks (median; range 15–32). A primer panel was designed to target sequences flanking single-nucleotide polymorphisms (SNPs)/exonic regions of ITGB3 (HPA-1), ITGA2B (HPA-3), ITGA2 (HPA-5), CD109 (HPA-15), RHD, RHCE, KEL, DARC, SLC14A1, GYPA, GYPB, and SRY. These regions and 14 anonymous SNPs were massively parallel sequenced by semiconductor technology. The implicated non-maternal fetal blood groups (RHD, HPA-1a, HPA-5b, HPA-15a) were correctly identified in all cases. The counting of non-maternal sequences of Y chromosomal regions and autosomal SNPs allowed quantification of fetal load and served as internal control for the presence of fetal DNA. Fetal DNA at 4 (median, range 1–5) additional loci was detected. The fractional fetal DNA concentration was 9.43% (mean; range 4.4%-27.14%).

We propose this method for reliable non-invasive detection of fetal blood group polymorphisms that are frequently involved in FNAIT and HDN.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):29.

SI-13-5 Role of HLA-class I antibodies detected using a bead-based assay in neonatal alloimmune thrombocytopenia: a single center study

B Schmid-Horch 1, R Jouni 1, M Jurisic 1, K Herbert 1, KO Kagan 2, T Bakchoul 1

Introduction

Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelets. Although, immunization against human platelet antigens (HPA) is generally considered to be the most frequent cause of NAIT, several reports described cases with detectable maternal antibodies against HLA-class I antigens in absence of anti-HPA antibodies.

Methods

25 cases with suspected NAIT were retrospectively analyzed. Maternal sera were tested using the monoclonal antibody immobilization of platelet antigens (MAIPA) assay, ELISA (PakPlus, Lifecodes®) and the bead-based assay (BBA) Luminex Single (One lambda® Canoga Park CA). Maternal DNA samples were analyzed using PCR for HPA-typing (PCR-SSP, innotrain®). As control group, samples of mothers who gave birth to healthy neonates (n = 51) were randomly chosen to measure fetal platelet count and test for maternal antibodies.

Results

In our study, antibodies against HPAs were detectable in 3 cases. In addition, antibodies against HLA-class I antigens were found in 10 out 22 cases. No significant difference in platelet count was observed between newborn of HLA-immunized (group I) and non-immunized mothers (group II), median platelet count 88 × 109/L, range 52–228 × 109/L vs. 69 × 109/L, range 5–164 × 109/L, respectively, p = 0.76. Of note, HLA-class I antibodies were also detected in 9 cases of the control cohort (group III) with no decrease in platelet count (median 259 × 109/L, range 220–322 × 109/L). The mean fluorescence intensity of the BBA in group I was higher than in group III (16.440, range 7.500–23.100 vs. 9.522, range 4.100–20.500), with a higher number of detected allospecificities (median 21, range 1–46 vs. 5, range 1–15).

Conclusion

These data suggest that HLA-class I antibodies detected using the highly sensitive BBA are not necessarily causative for clinically relevant NAIT. Further investigations are needed to verify the impact of antibody titer and specificity on fetal/neonatal platelets.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):29.

SI-14-1 (INV. SPEAKER) Emerging viruses

D Juhl 1, H Hennig 1

During the last 2 decades, several viruses, till then less considered, spread from Africa or the Middle East to other parts of the world. These Arthropode-borne viruses (Arboviruses) are naturally transmitted by mosquitos, but transmission by transfusion has either been proven or is quite probable. First, West Nile Virus (WNV) reached North America in 1999, and spread over the entire mainland within 4 years. Already in 2002, cases of WNV transmission by transfusion, sometimes with fatal outcome, occurred in the USA, resulting in the implementation of WNV-RNA screening there. WNV is often observed in South Europe, and already has been detected in Austria by routine blood donor screening. German authorities thus enacted temporary rejection or WNV RNA screening of blood donors returning from endemic countries. However, rejection of such donors seems to be an alternative to RNA screening due to the low number of lost donations.

Other Arboviruses like Chikungunya Virus and, recently, Zika Virus also got more attention due to their furious spread in the Americas since 2013. Unlike Dengue Virus, they seem to have usually a minor pathogenicity, with the limitation of strong evidence, that Zika Virus causes birth deformities, if pregnant women are infected. Both viruses probably have the potential to be transmissible by blood components, and the temporary rejection of donors, returning from endemic countries, has been enacted by the German authorities or is intended in the revised guidelines. Also Hepatitis E Virus (HEV) attracted more interest in the last years. In Europe, it causes probably zoonotic infections by consumption of contaminated food but there are several well-documented cases of transmission by transfusion of single donor blood components. However, relevance for the recipients is still unclear, as HEV infection resolves spontaneously in many cases. Seroprevalence data from our institute showed no evidence for a transmission of HEV by plasma derivatives.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):29–30.

SI-14-2 (INV. SPEAKER) Relevance of infectious disease epidemiology for transfusion safety

R Offergeld 1, C Houareau 1, K Preußel 1

Infectious diseases still pose a threat to transfusion safety. Especially challenging are emerging infections with unknown epidemiological dynamics like Zika virus. But also well-known infections, like HIV and hepatitis E, demand attention.

In Germany and all EU member states, infectious diseases are reportable to national authorities. In Germany, HIV, HCV, HBV and syphilis infections in the donor population are analyzed since 1999. Infections rates for HCV and HBV have declined between 1999 and 2009 in first time (FTD) and repeat donors (RD) and remained stable ever since. While HIV infection rates in FTD did not change significantly over time, a rise in HIV incidence was observed between 2007 (1.5/100,000 RD) and 2013 (2.9/100,000 RD). Syphilis prevalence and incidence reached a peak in 2014 and need further attention.

In comparison to other EU states, for 2012 Belgium and Portugal report a similar HIV incidence (3.3/100,000 RD), whereas Italy and Spain report a higher (5 and 8.6/100,000 RD) and UK, France and the Netherlands a lower HIV incidence (0.6, 1,4, 0.6/100,000 RD). Reasons for changes over time and differences in states warrant further investigation and additional measures: Motivation for donating should be analyzed and specific risks should be addressed directly. Donor selection should be optimized, e.g. by using a standardized questionnaire.

For emerging infections, routine surveillance data from the donor population is missing. Therefore, data from the general population collected by mandatory reporting or sentinel studies can be used for risk assessments. Routine surveillance as well as sentinel studies and second generation surveillance should therefore be an established part of the haemovigilance system. Detailed information on the spread of infections in different donor populations collected by the national Public health Institute helps to assess transfusion risks and is also needed for modelling of transfusion risks.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):30.

SI-14-3 Which infectious blood donors could be caught by the DHQ? – Comparison of infected blood donors with notified cases from general population

K Preußel 1, R Offergeld 1

Potential risks for transfusion transmissible infections are identified by donor history questionnaires (DHQ) and donors with higher risks are deferred from donation. Still, a small proportion of donors is diagnosed with HIV or HCV infections. We assessed to which extend currently used DHQs support the identification of infections by comparing unreported deferrable risks among infected blood donors and probable transmission routes of notified cases from the general population.

We performed an analysis including notified HIV and HCV cases and positive blood donors in Germany, 2006–13. We used logistic regression analysis of possible transmission routes (adjusted for age group, sex and type of residence) to identify relevant risk factors. We estimate the possible effect of improved capture of infection risks for donor selection by calculation of population attributable fractions (PAF).

Data about possible transmission routes of infections were available for 79% of HIV diagnoses in the general population (17399/22044) and 39% of HIV infected blood donors (309/789). 82% of infected blood donors reported sexual transmission risks. Among HIV infected blood donors 43% were MSM compared to 72% of newly diagnosed HIV infections. In contrast 58% of blood donors reported heterosexual risk compared to 26% of newly diagnosed HIV infections. Identification of all heterosexual risk contacts might prevent acceptance of 53% HIV infected donors (PAF), according to our multivariable model.

Possible transmission routes were reported for 55% of HCV diagnoses (22707/41237) and 19% of HCV infected blood donors (674/3469). Infected blood donors were more likely to report heterosexual exposure, imprisonment, and piercing/tattoo in a multivariable analysis than notified HCV cases from the general population. Improved recording of piercing/tattoo could prevent acceptance of 16% HCV infected donors.

The donor selection process should be improved with special attention to the identification of sexual risk factors, invasive procedures like piercing/tattoo and imprisonment. This could be done by well-designed DHQs, effective donor education and confidential environment in all steps of the donor selection process.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):30.

SI-14-4 Elimination of microparticles from human plasma by the THERAFLEX MB-Plasma System

W Handke 1, B Lambrecht 1, C Sumian 2, S Reichenberg 3, A Seltsam 1

Introduction

THERAFLEX MB-Plasma treatment is used to inactivate pathogens in human plasma. It is known that methylene (MB)/light treatment influences plasma proteins and pathogens. Natural degradation products from blood cells can be found in plasma in form of microparticles. In this study, flow cytometric analysis was used to quantify such subcellular particles.

The aim of the study was to investigate if the filtration steps of THERAFLEX MB-Plasma treatment (PLAS-4 and BlueFlex filtation) reduce RBC derived microparticles and whether THERAFLEX MB-Plasma treatment contributes to the formation of microparticles.

Methods

Pathogen inactivation was done with six plasma units using the THERAFLEX MB-Plasma system and the MacoTronic B2 illumination device. Samples were taken before and after filtrations. Flow cytometric analysis was used to detect and quantify subcellular particles in the range of 0.5 to 4 μm (according to particle size reference beads). Origin of the particles were discriminated by antibody based linage markers of the Plas-macount Kit (BD).

Results

It could be shown that the PLAS-4 filtration procedures reduced RBC derived microparticles for >90% (sd= 5%). The formation of additional microparticles by MB/light treatment was not observed. An additional MP reduction of the BlueFlex filter could not be assessed because of the low starting counts of MPs after PLAS-4 filtration.

Conclusion

We conclude that the PLAS-4 filtration step of THERAFLEX MB-Plasma treatment ensures the efficient reduction of RBC derived MPs in THERAFLEX MB-treated plasma that are already present in human plasma. Additional contribution of the BlueFlex filter should be investigated.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):30.

SI-14-5 Screening pooled blood donation samples for West Nile Virus (WNV) by PCR

L Pichl 1, K Paul-Konietzko 1, B Müller 2, T Zeiler 1

Introduction

West Nile fever is caused by a Flavivirus which naturally circulates between birds and mosquitoes. Humans are mainly infected through mosquito bites but West Nile Virus (WNV) is also transmitted via organ transplants and transfused blood products. The average incubation period varies from 3 to 14 days followed by flu-like symptoms. Although most infections are asymptomatic, severe cases are observed among elderly persons and immunocompromised patients. In accordance with current regulations GRC BTS West decided in transmission season 2015 to implement screening blood donations for WNV instead of deferring blood products from donors who returned from areas where West Nile fever was endemic.

Methods

From blood donors who reported a stay in WNV risk areas within 4 weeks before donation EDTA-Plasma samples were collected and pooled (pools of 6). Pools were screened with CE marked Roche cobas WNV PCR kit on cobas 8800 platform with an analytical LOD of 12.9 copies/ml for WNV lineage 1 and 6.2 copies/ml for lineage 2 respectively.

Results

From 06/01/2015 until 12/30/2015 about 500 pools comprising 2,754 donations were screened for WNV. All donations tested were non reactive for WNV RNA. Percentage of initially invalid results was < 0.5%. False positive results as well as critical hardware hints were not observed.

Conclusion

Feasibility of blood product PCR screening for WNV was demonstrated. Loss of blood products due to temporary donor defer was avoided. An update on current screening data will be given for transmission season 2016.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):31.

SI-15-3 (INV. SPEAKER) Modern Aspects of leukocyte and stem cell apheresis

E Strasser 1

The last 50 years show an amazing development of leukocyte collection by apheresis due to technical improvement of apheresis technique and software development. Modern leukapheresis produced from allogenic donors and patients (autologous donation) requires special apheresis programs and instrument settings to optimize the collection efficiencies of the target leukocyte populations. Unstimulated leukapheresis from the allogenic stem cell donors is used for donor lymphocyte infusion (DLI). New T cell therapies are developed based on leukocyte products of high quality. The latter is prerequisite for subsequent preparation steps prior to isolation of CD34+ cells, culture and differentiation of CD14+ monocytes, or expansion of CD3+ T-cells. Monocyte collection has to be optimized for DC culture. High cell yields are required and depend on cell recruitment during apheresis. Platelet contamination may be removed by elutriation. New apheresis technology enables the production of pure leukocyte concentrates with low residual platelets and red blood cells. In stimulated leukapheresis for stem cell collection the content of residual granulocytes should be low. On the other hand, granulocyte collection for selected immunosuppressed, infected patients requires high granulocyte yields within a short collection time. Recently, usage of HES solution has been restricted in ICU patients with renal failure. However, high molecular HES solution is required for efficient granulocyte collection to produce high granulocyte yields. Side effects of leukocyte donors during leukapheresis are rare and easy to treat (e. g. citrate reaction). In stimulated leukapheresis, additional side effects of leukocyte and stem cell stimulating drugs, such as cortisone, granulocyte-colony stimulating factor (G-CSF) or recently developed new drugs may occur and have to be carefully recorded. Before apheresis, donors need an informed consent addressing special risks of leukocyte stimulating drugs or HES. Further, blood checks before and after drug exposure are required.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):31.

SI-15-4 Paired comparison of continuous and discontinuous MNC collection and mDC generation

H Pfeiffer 1, M Aigner 2, S Achenbach 1, R Zimmermann 1, R Eckstein 1, EF Strasser 1

Introduction

Monocytes can be cultured into dendritic cells with addition of autologous plasma and can be primed to react with certain antigens. This experimental therapy is used in many different, widely recognized therapeutic approaches such as autologous cellular immunotherapy. The influence of different collection programs on the MNC composition and the suitability for use as starting material for DC and T-cell based therapies were evaluated.

Methods

13 donors underwent paired MNC collections with the Spectra Optia (continuous and discontinuous collection: IDL- and MNC-set). In a prospectively-paired analysis, donor pre-donation counts of monocytes were analyzed for their predictive value of monocytes in the collected product. Blood cell contamination of the MNC product was investigated. mDCs were generated from seven single-donor pairs. The quality of the primary apheresis product was assessed as well as after different steps of processing (Ficoll, cryoconservation, recovery, purification). In addition, the influence on the generation of mDCs or on T-cell function was analyzed.

Results

Both programs collected viable mononuclear cells, although the yield was slightly higher when using the discontinuous collection (1.307 × 10e9 monocytes, continuous collection 1.179 × 10e9 monocytes). No significant difference concerning the product composition was observed, although the discontinuous collection showed slightly purer apheresis products (erythrocytes 0.333 × 103/µl discontinuous and 0.538 × 103/µl continuous; platelets 2,138 × 103/µl discontinuous and 4,079 × 103/µl continuous). After processing both collection methods allowed for the differentiation of mDCs. No difference concerning the T-cell function was observed.

Conclusions

Both collection methods yielded MNCs suitable for the use in mDC or T-cell based therapies. The discontinuous collection showed slightly purer apheresis products. In case of a low MNC yield the continuous collection, immediately followed by mDC generation, seems preferable. In case of cryoconservation prior to mDC generation, the discontinuous collection showed better results as well as a higher homogeneity of mDC.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):31.

SI-15-5 Cold storage of platelet concentrates in additive solution without a significant impairment of platelet function and survival

K Wehrmann 1, L Janzen 1, J Wesche 1, A Greinacher 1, T Bakchoul 2, T Thiele 1

Introduction

Bacterial growth in platelet concentrates (PCs) remains a significant risk because of the routine PC-storage temperature at 22°C. Storage at lower temperatures may inhibit bacterial growth but could diminish platelet function. The use of additive solution may facilitate PC-storage at lower temperatures. In this study, we compared the in vitro function of platelets stored at 4°C and 22°C in additive solution.

Methods

Buffy coat derived PCs collected in platelet additive solution (SSP+, Macopharma Tourcoing, France) were split into two equal units and stored either at 4°C or at 22°C under agitation for 20 days. Platelet function was assayed by flow cytometry (PAC-1 binding, CD62P expression before and after stimulation with thrombin receptor agonist peptide [TRAP]), by aggregometry (agonists: TRAP, collagen, ADP and ristocetin) and hypotonic shock response. Survival of platelets stored for 7 days was investigated using the NOD/SCID mouse model.

Results

Flow cytometry revealed similar CD62P-expression before and after stimulation with TRAP until day 10 for both storage conditions (n = 7, p > 0.05). Cold stored platelets showed similar reactivity towards ADP, collagen-, TRAP, and ristocetin (n = 4) and similar hypotonic shock response as platelets stored at 22°C (n = 3). Survival of platelets in NOD/SCID mice did not significantly differ between 4°C and RT-stored platelets (n = 3).

Conclusion

Platelets stored at 4°C in additive solution show similar quality parameters compared to the same platelets stored in additive solution at 22°C. Cold storage in additive solution is a promising approach to reduce the risk of bacterial growth and to prolong PC-shelf life.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):32.

SI-15-6 Performance of the continous MNC collection mode on the new automatic cell separator for autologous blood stem cell apheresis in low weight children in comparison with the previous manual device

B Wagner 1, R Buhmann 1, C Wichmann 1, M Karagianni 1, A Scheffler 1, G Wittmann 1, MH Albert 2

Introduction

High dose chemotherapy with autologous stem cell rescue has been established as standard therapy for various malignancies in adults and children. There are only few data on blood stem cell apheresis in low weight children particularly on procedures with the new automatic cell separator Spectra Optia (SO) applying the recently introduced continous MNC collection (cMNC) mode. In our hands in adults the cMNC mode was more efficient than the MNC mode on SO. cMNC performance was compared to that of the previous manual device in small children.

Methods

Data from the records of autologous PBSC collection procedures in 5 patients (mean age 4.5 y, BW 14.7 kg) were compared with those from 20 PBSC harvests in 14 patients (mean age 3.4 y, BW 14.0 kg) using the manual MNC mode of the former model COBE Spectra. Blood counts, total blood volume (TBV), blood volume processed (PBV), procedure time, CD34+ HPC yield and collection efficacy (CE) were analysed. All procedures were run after priming the devices with irradiated RBCs adjusted to the patient's HCT. The double-lumen Hickman catheter in situ was used with a dual anticoagulation protocol (ACD-A+Heparin). Adverse events and the need for an additional venous access were also considered.

Results

At in the mean 96 CD34+ cells/μl in peripheral blood (pB) 15.3*10e6 CD34+ HPCs/kg were harvested by processing the 3.1fold TBV in 237 min. This appeared to be inferior as compared to the previous manual device yielding 13.9*10e6 CD34+ HPCs/kg in 205 min with a PBV of 2.7fold TBV. This could be due to higher CD34+ counts (194/μl) in pB in these patients. PLT attrition was similar with both methods (0.34 vs. 0.31) as well as CE (0.37 vs. 0.43). The major disadvantage of the new device was the slow automatic adjustment of the interface at the start resulting in longer run-times. The adjustment process being highly susceptible to variations in inlet flow, 60% (3/5) of patients required an additional peripheral venous access as compared to 0/14 harvested with the manual device.

Conclusion

The cMNC procedure with SO was safe. The new device was not superior in small children concerning platelet loss and CE which is surprising from our data in adults. The major problems were the slow automatic adjustment of the interface and its high susceptibility to fluctuating inlet flow resulting not only in longer run-times, but in the need of an additional venous line in 3/5 of the children. In our opinion this should be refined.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):32.

SI-16-2 (INV. SPEAKER) Demographic changes and patient blood management – how much blood do we need?

A Greinacher 1

The population structure in most countries in Europe is currently shifting from younger to older age groups (demographic change), due to an increase in life expectancy, ageing of high birth rate cohorts, and lower birth rates. This implies an increased demand for blood transfusions, while at the same time the total population of potential blood donors will substantially decrease. The challenge for the near future is to avoid a supply gap for blood transfusions. Especially the state of Mecklenburg-West Pomerania (MV), faces a more pronounced demographic change and can be seen as a model-region in which demographic changes manifest approximately 10 years earlier than in other regions. The ratio between the population 18–65/>65 years will be 2.3 in MV in 2020 and ten years later in 2030, it will be 2.2 in total Germany, were the most pronounced change in the population structure will occur 2020–2030. However, parallel to the demographic changes, transfusion practise undergoes major revisions summarized by the efforts called “patient blood management” to reduce the numbers of blood transfusions; and the medical progress is further contributing by less invasive treatments. The net effect of these different factors, which will finally determine the blood supply-demand ratio, requires regular monitoring to allow adjustment of blood donor recruitment activities. We started in 2005 an analysis of the patient and the donor population of MV including all in-hospital blood recipients and all blood donors within this jurisdiction. The 2010 and 2015 longitudinal follow ups of this study will be presented, which show a reduced number of red blood cell transfusions per 1,000 inhabitants in the age groups >55 years, nevertheless the total transfusion numbers increased due to the increase in these population age groups. The study shows that the system of blood supply and demand is becoming volatile.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):32.

SI-16-3 Hemovigilance – Data from 1.2 Million donations

E Ulrich 1, S Kießig 2, A Rabe 1

Aim

In compliance to regulatory requirements of the german drug law the development of a simple and effective computer-assisted UE-documentation system which addresses in sufficient detail the numerous facets of UE (unexpected events) was performed.

Methods

Defined local and systemic UE of donors were registered and classified as mild, moderate or severe; technical UE were defined without grading by severity. An UE-module was developed for the plasma management software (PMS) based on a SQL-database and installed on January 1, 2008, in Haema-blood centers using PMS. In total a number of 57.622 donors donating 166.650 whole blood units and 1.,107.846 plasma units served daily by high frequency preparatory plasmapheresis (PPP) programs and whole blood donation (BD). The donor room physicians enter all UE observed daily into PMS. Medical directors review entries quarterly for completeness and plausibility and correct them where necessary.

Results

6.605 UEs were observed during BDs [corrected incidence 4.30% (0.66% local, 1.59% systemic, 2.04% technical UEs)]. 2.96% of BDs were accompanied by one UE and 0.45% by > 1 UE. 6.3% of donors of 1st-time, 3.5% of 2nd-time and 1.9% of ≥3rd-time experienced UEs. Most common UEs were: broken-off collections due to venous access problems, repeated venipuncture and small hematomas. Severe circulatory UEs occurred at a rate of 16 per 100.000 BDs. 66.822 UE were observed during PPP [corrected incidence of 6.55% (1.4% local, 0.55% systemic, 4.6% technical UE)]. 3.36% of PPP were accompanied by one UE and 1.18% by >1 UE. 13.7% of 1st-time, 9.7% of 2nd-time and 4.0% of ≥3rd-time PPP were UE-associated. Most common: repeated venipuncture, broken off collection due to venous access problems and small hematomas. Severe systemic UE occurred at a rate of 36 per 100,000 PPP.

Conclusions

Technical UEs were common with BD and PPP. The incidence in PPP is higher than in BD but the severity is lower. UEs accompanied first and second donations significantly more often. The rate of severe systemic reactions was found much lower in PPP than in BD.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):33.

SI-16-4 Questions to be asked: The uniform German blood donor questionnaire

C Houareau 1, R Deitenbeck 2, A Sümnig 3, M Heiden 4, F Stötzer 5, H Northoff 6, R Offergeld 1

To increase the quality of donor history questionnaires (DHQ) with respect to donor acceptance and effectiveness, a uniform DHQ (UDHQ) was developed in Germany. The UDHQ was superior to locally established DHQs in a multi-centre trial with 6,500 first-time donors. However, the deferral rate increased by 4.6% mainly due to questions related to the detection of acute illness and new sexual partners. To further assess the impact on the donor population tests were performed on a larger scale including repeat donors.

We present retrospectively analysed data on blood donations collected in one large blood establishment with the DHQ or UDHQ. Differences were analysed using Chi2-test according to Pearson. For the comparison of the questionnaires an adjusted multinomial logistic regression was performed. The analyses included 112,176 (DHQ) and 108,330 (UDHQ) blood donations. First-time donations (FTD) and repeat donations (RD) showed higher deferral rates with the UDHQ than with the DHQ (FTD: 38.0% vs. 30.6%, RD: 11.1% vs. 9.3%). With the UDHQ, overall deferral due to a new partner was 1.5% in first-time and 0.2% in repeat donors respectively. The majority of these deferrals occurred in the two youngest age groups. Overall, the most frequent deferral criterion was acute illness (6.2%).

Using the UDHQ repeat donations had a higher deferral risk in the category “disease” (RRUDHQ= 1.6; p < 0.01) and “tattoos” (RRUDHQ= 1.5; p < 0.01). Within the category “disease” men aged 18–24 yrs. had a higher risk of deferral (reference group 35–44 yrs.; RRmale*18–24 yrs. = 1.3; p < 0.05). Within the category “tattoos”, donations of the two youngest age groups had a higher risk of deferral (reference group 35–44 yrs.; RR18–24 yrs. = 3.2; p < 0.01; RR25–34 yrs. = 2.8; p < 0.01).

The DHQ and UDHQ mainly lead to differences in temporary deferrals. The multinomial logistic regression revealed stronger predictors for deferral than the questionnaire version. Especially younger age carries a higher and independent risk for deferral than the UDHQ. The additional 3.1% of deferrals of young first-time donors due to a new sexual partner has to be considered; however, this is outweighed by the advantages of the UDHQ with respect to comprehensibility and effectiveness and may identify those donors with potential sexual risk behaviour who would otherwise not be identified.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):33.

SI-16-5 Implementation of the first barrier-free blood donation service for blind donors in Germany

A Deitmer 1, D Smida 1, G Bein 2, zu Bexten E Meyer 1, H Hackstein 2

Background

Vision-impaired or blind blood donors often encounter difficulties during the blood donation process because several questionnaires have to be completed during the donation process. A major hurdle frequently leading to the rejection of blind voluntary blood donors represented the completion of the mandatory confidential self-deferral questionnaire at the end of the blood donation process.

Methods

We have developed a set of barrier-free PDF forms that can be completed at a standard PC by the blind blood donor. With respect to the confidential self-deferral questionnaire we have developed a solid overlay frame that fits exactly to the self-deferral form. This overlay frame contains all the required information in braille and allows confidential completion of the questionnaire without any external help. Furthermore, after completion of the deferral form and detachment of the questionnaire from the overlay it is no longer visible that this blood donation was made by a vision-impaired or blind blood donor.

Results

We have successfully developed and implemented a set of simple technical devices allowing barrier free blood donation for blind and vision-impaired blood donors. With respect to the german drug law one major advantage of this solution is the fact that the content and wording of the existing questionnaires can remain untouched and the forms are just covered by a template frame.

Conclusion

Barrier-free blood donation will reduce discrimination of minors and increase the acceptance of blood donation in the community.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):33.

P01-1 Proteome changes in platelets during recovery of platelet counts after apheresis – an approach to compare young and old platelets

T Thiele 1, J Braune 2, V Dhople 2, E Hammer 2, C Scharf 3, A Greinacher 1, U Völker 2, L Steil 2

Introduction

Circulating platelets differ in their age. We used platelet apheresis to remove platelets from circulation to detect changes in the platelet proteome related to platelet recovery. We aimed to map candidate proteins which may serve as markers of young platelets.

Methods

A healthy donor underwent three platelet apheresis procedures on three consecutive days. Blood was drawn at day 1 (baseline) and at days 3, 5, 7, 9 and 11 for the analysis of the platelet proteome using LC-ESI-MS/MS and 2D-DIGE.

Results

1017 proteins were identified by LC-ESI-MS/MS, among those 54 changed in quantity throughout the course of the study. Potential markers of young platelets were 40S ribosomal protein SA, COP9 signalosome complex subunit 5 proteins involved in clathrin-mediated endocytosis signaling. 1036 protein spots were observed by 2D-DIGE, among those 45 spots changed in fluorescence intensity. Identified spots contained IQ motif containing GTPase activating protein 2, talin, moesin, myosin regulatory light chain 2 and coronin-1C.

Conclusions

A combination of platelet apheresis and proteomic approaches enables identification of changes in the platelet proteome that are related to platelet de novo synthesis.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):33–34.

P01-2 Evaluation of the new lot 006 of LABScreen Multi for granulocyte antibody detection

U Schulz 1, A Reil 2, R Moog 3

Background

In order to reduce the risk of TRALI, a high number of plasma donors were tested for HLA- and HNA Abs. For HNA-3a Abs detection the gold standard is the Granulocyte Aggregation Test (GAT), however the test is not suitable for a high sample throughput. With the elucidation of the molecular structure of HNA-3a and −3b molecule it was possible to extend the LABScreen Multi for detection of HLA-class I and II and HNA-1a, −1b, −1c and −2 Abs to HNA-3a, −3b Ab and −4, −5a and −5b Abs. In our first study we found that 100% of the HNA-1a,-1b, and −2 antisera but only 90% of the HNA-3a antisera could be detected as true positive using the new LABScreen Multi lot 005. Here, we report the results of our evaluating study of a new lot 006 including special new coated beads.

Methods

For the evaluation of the lot 006 of LABScreen Multi, 34 sera containing well-defined HNA Abs and previously tested with the lot 005 were analyzed. The antibodies were identified in the involved labs by GIFT, GAT, and MAIGA. The test procedure for LABScreen Multi was carried out according to the manufacturer's instruction. The cutoffs were set for the beads HNA-1a, HNA-1b, HNA-1c, HNA-3a, HNA-3b and HNA-4a to a ratio > 5 and for HNA-2 to a ratio > 20. For background study by lot 006 we confirmed 6 false positive sera out of 91 routine samples tested by lot 005, GIFT and GAT. Additionally, we analyzed 241 routine samples negative tested by GIFT and GAT.

Results

The new lot 006 of LABScreen Multi reacts highly specific with higher ratio values for the specificities HNA-1a, −1b, and −2. A big improvement was seen in finding of HNA-1b and HNA-1d Abs. HNA-1b sera showed higher ratio values by the bead named “HNA-1b”. The NBGs by the bead “HNA 1c” only showed one half to third and not more the threefold of the ratio value of the bead “HNA-1b”. But the same HNA-3a and −3b Abs containing sera witch Abs were not found by using lot 005 were also not detected by using lot 006. Background reactions could be reduced from 5.5% to 4.4%.

Conclusion

The new lot 006 of LABScreen Multi showed higher specific ratios values for HNA-1, and HNA-2 Abs. However, we are still not able to detect all HNA-3a Abs, which can cause TRALI. That means the GAT remains the gold standard for a 100% detection of HNA-3a, and −3b Abs. However, especially for samples with low ratios over the cut we still recommend retesting with classical methods GIFT and GAT.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):34.

P01-3 The role of purinergic receptors in reduced adenosine diphosphate (ADP) responsiveness of platelets from apheresis derived platelet concentrates

J Kößler 1, K Weber 1, A Kößler 1, M Böck 1, A Kobsar 1

Introduction

Platelet storage lesion (PSL) is associated with a loss of platelet integrity and function. One typical manifestation of PSL is the affection of ADP induced aggregation in stored platelets. The underlying processes for that phenomenon, however, have not entirely been elucidated yet. Therefore, the aim of this study was to analyse the surface expression and function of purinergic receptors P2Y1, P2Y12 and P2X1 in stored platelets from apheresis derived platelet concentrates (APCs).

Methods

Platelets were obtained from APCs stored for 0, 2 and 5 days and, for comparison, from freshly donated whole blood. ADP induced aggregation was determined by light transmission aggregometry, purinergic receptor expression by flow cytometry and Western Blot analysis. The functional activity was analysed by calcium induced fluorescence (P2Y1 and P2X1) or by flow cytometric measurement of the platelet reactivity index (P2Y12).

Results

ADP induced aggregation was completely diminished after two days of storage. The basal surface expression of all investigated purinergic receptors and the total content of receptor proteins remained unchanged. After an initial reduction after apheresis, P2X1 mediated calcium flux was maintained throughout storage, whereas P2Y1 mediated calcium flux continuously decreased. In contrast, P2Y12 activity was unaffected with comparable values for the platelet reactivity index in fresh and stored platelets.

Conclusion

The functional impairment of the P2X1 and especially of the P2Y1 receptor function indicated by reduced receptor-mediated calcium flux represents an important mechanism contributing to affected ADP responsiveness in stored platelets.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):34.

P01-4 HNA/HPA NGS paneltyping

D Fürst 1, U Schulz 2, E Urban 3, R Moog 2, C Neuchel 1, C Tsamadou 1, H Schrezenmeier 1, T Tonn 3, J Mytilineos 1

Introduction

Human neutrophil antigens (HNA) have been associated with TRALI a potentially life-threatening complication after transfusion of blood products. Alloantibodies against human platelet antigens (HPA) may lead to refractoriness in patients depending on platelet transfusions. Some blood centers maintain registries of typed cell donors for provision of compatible blood products. Broad and cost effective typing of such donors may improve clinical services.

Methods

We developed an NGS targeted sequencing panel for typing of HNA and HPA antigens based on the Illumina Miseq platform. The process is highly automated with minimal hands on time. Variant calling is performed automatically using custom scripting with R and Bioconductor packages. The following antigens are included: HNA-1, HNA-3, HNA-4, HNA-5, HPA-1–11, HPA-15, HPA-21, HPA-23, HPA-27.

Results

Validation was performed using a set of reference samples. For HNA 15 samples and for HPA 23 reference samples were available. All typings were concordant with reference typings.

Preliminary results indicated that multiplexing can be ramped up to 752 samples in one sequencing run. Human hands-on time is calculated with 2 minutes per sample.

Conclusion

NGS high throughput typing for immunologically relevant polymorphisms can be done in a highly efficient manner. However, the demand for laboratory automation is high making it preferentially applicable in centres providing adequate infrastructure.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):34–35.

P01-5 Expression and function of purinergic receptors in washed platelets

J Kößler 1, S Hermann 1, K Weber 1, A Kößler 1, M Böck 1, A Kobsar 1

Background

The procedure of washing platelets is used to provide platelets for experimental research or for plasma-reduced platelet concentrates. It has been observed that functionality of platelets, e.g. aggregation and responsiveness to adenosine diphosphate (ADP), is affected after washing. Molecular mechanisms, however, contributing to that phenomenon are not well understood. Therefore, the aim of the study was to evaluate purinergic receptors in washed platelets.

Methods

Platelets were prepared from platelet-rich-plasma (PRP), washed by CGS buffer (sodium chloride, trisodium citrate, D-glucose, pH 6.5), resuspended in HEPES buffer and stored in Eppendorf tubes without shaking. The surface expression of purinergic receptors P2Y1, P2X1 and P2Y12 were measured by flow cytometry in washed platelets and, for comparison, in PRP for up to 2 hours. P2Y1 and P2X1 receptor function was determined by induced calcium-flux using a fluorescence assay, P2Y12 function expressed as platelet reactivity index by flow cytometry.

Results

In contrast to PRP, the surface expression of all purinergic receptors showed a biphasic variation in washed platelets with an increase during the first 60 minutes and a consecutive reduction. The activity of P2X1 was preserved in PRP and washed platelets. P2Y12 function was partially diminished after washing, but then maintained throughout storage, whereas the activity of P2Y1 continuously decreased in washed platelets, but not in PRP.

Conclusions

The initial increase of surface receptor expression points to preactivation of platelets after the washing procedure, which is followed

by degenerative processes resulting in decreasing expression during storage. Tampered responsiveness to ADP in washed platelets is particularly caused by impaired activity of the P2Y1 receptor associated with disturbed calcium regulation.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):35.

P01-6 Cancer surgery is followed by a postoperative increase of intracellular concentrations of platelet vascular endothelial growth factor (VEGF)

R Zimmermann 1, T Schmieger 1, T Stützer 2, J Fischer 1, M Bosser 1, R Eckstein 1, V Schellerer 3

Introduction

Previous work on elevated concentrations of circulating growth factors in patients with cancer is significantly biased from severe pre-analytical errors. The questions, in what concentrations the growth factors VEGF and PDGF-AB, which circulate freely or stored in platelets, can be found in the blood of patients with cancer and to what extent the measured values are influenced by surgical removal of tumors, have therefore not yet been clarified.

Material and Methods

We examined three consecutive series of patients suffering from colorectal carcinomas (CRC) (n = 40), and head and neck squamous cell carcinomas (HNC) (n = 18), respectively. Blood was taken on admission before surgical removal of the tumor and before discharge of the patients after surgery. Extracellularly circulating levels of vascular endothelial growth factor (VEGF) and platelet-derived growth factor AB (PDGF-AB) were determined from CTAD plasma. Total intra- and extracellularly circulating amounts of these growth factors were measured from lysates of whole blood in 0.5 percent Triton X-100.

Results

The removal of a colorectal or a head and neck cancer resulted in a significant decrease of the red cell count, the hematocrit, and the hemoglobin as a sign of the operation-induced blood loss. At the same time the platelet counts increased in two of the three series. The extracellularly circulating cytokine levels remained unchanged. Surprisingly, however, there was a significant increase of the VEGF concentration in the lysates in all three patient groups.

Conclusions

The significant and in three case series reproducible change of the levels of the cytokine VEGF in platelets was surprising. In the absence of similar findings in the literature, the questions cannot be answered, whether this finding is a specific consequence of a tumor resection, or whether we discovered a general effect of major surgery. These issues must be clarified by further studies.

Tab. 1.

Pre- and postoperative findings in three series of cancer patients

Series 1 Series 2 Series 3
CRC (n = 20) CRC (n = 20) HNC (n = 20)
Pre-OP Post-OP Pre-OP Post-OP Pre-OP Post-OP
WBCs [10e3/µL] 7.7 ± 3.5 7.5 ± 1.5 7.0 ± 2.6 7.8 ± 2.6 8.4 ± 2.5 10.8 ± 3.9
PLTs [10e3/µL] 249 ± 34 376 ± 135 240 ± 76 231 ± 58 258 ± 71 312 ± 154
VEGF in lysates [pg /ml] 437 ± 233 1395 ± 519 556 ± 390 953 ± 450 900 ± 746 1462 ± 935
VEGF in lysates [pg/10e5 PLTs] 0.18 ± 0.12 0.41 ± 0.17 0.24 ± 0.15 0.44 ± 0.21 0.34 ± 0.21 0.49 ± 0.24
PDGF-AB in lysates [µg /ml] 12.9 ± 4.6 5.4 ± 1.4 14.4 ± 5.7 14.3 ± 4.9 12.5 ± 5.4 13.5 ± 7.8
PDGF-AB in lysates [pg/10e5 PLTs] 5.3 ± 1.9 1.6 ± 0.6 6.07 ± 1.94 6.21 ± 1.40 4.38 ± 2.03 3.70 ± 0.83
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Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):35.

P01-7 A cross-sectional study to affirm mild thrombocytopenia in Swiss elderly: SENIORLAB

W Hermann 1, C Grebhardt 2, P Medina Escobar 3, L Risch 3, U Nydegger 2, M Risch 4; SENIORLAB

Background

Deferall age limit for thrombocyte donation, from whole blood of repeat donors has been moved up to as much as 75 years in some countries with thrombocyte count (TC) >150 × 103/µl prior donation. With prolongation of life expectancy, a progressive decline in TC with aging is scrutinized worldwide. In addition, hydrodynamic focusing, fluorescence and impendance resistence technology make platelet analysis beyond mere counting possible.

Methods

The SENIORLAB study (ISRCTN 53778569) recruites healthy senior citizens from the Swiss plateau aiming to establish reference intervals (RI) for several analytes in the elderly. TC, mean platelet volume (MPV), platelet distribution width, PDW, and thrombocrit were measured using Sysmex XE-5000 equipment (Sysmex, Horgen, Switzerland). Participants with ferritin < 30 U/L ng/ml, CRP > 5mg/L and death at folllow up since study entry 7 to 6 years ago were exluded from evaluation.

Results

A total of 1154 individuals aged 60–99 yrs (median 71), interquartile range IQR (66,76) were included into the study. Males had significantly lower TC and thrombocrit (p < 0.001) as well as significantly higher PDW (p = 0.04) than women, a gender difference which was not apparent with MPV. TC and thrombocrit both significantly decreased with aging but MPV and PDW remained random. The RI (in brackets : 90% confidence intervals, CI) for TC in men were as follows : 148 (143–154) to 300 (291–310) x 103/µl at ages 60–69, 135 (129–142) to 291 (282–300) × 103/μl at age 70–79, and 129 (121–138) to 304 (284 325) x 103/ at age >80. The respective RI for TC in women were : 168 (163–174) - 358 (345–370) x 103/µl at age 60–69, 166 (161–172) - 344 (334–355) x 103/µl at age 70–79, and 163 (156–171) - 338 (319–355) x 103/µl at age >80. For PDW, the RI were 10.2 (10.0–10.3) - 16.6 (16.2–16.9) % for men and 10.0 (9.9–10.1) - 16.7 (16.4–17.1)% for women. For MPV the RI values were 9.3 (9.3–9.4) - 12.5 (12.4–12.6) fl.

Conclusions

Influence of progressing age on TC, more and more reported to reduce the TC, are based on cross-sectional studies such as the one reported here. Nevertheless, a mild thrombocytopenia of apparently healthy elderly individuals might become relieved by other participating systems to maintain appropriate haemostasis.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):35–36.

P01-8 Quality control of platelet concentrates with a microparticle and platelet analysis device (ThromboLUX)

F Dormann 1, V Hähnel 1, N Ahrens 1

Objective

Microparticle (MP) content is a parameter indicating increased activation of platelets in platelet concentrates (PC). We tested the applicability of a device for platelet function and MP content operating in single sample mode with a minimum of user hands-on time delivering results within 30 min (ThromboLUX, LightIntegra).

Methods

PC were obtained from healthy donors by apheresis. Samples were taken in PE-tubes without additional anticoagulation, loaded into a capillary tube (100 μL) and measured at a sequence of 37°C, 20°C, and 37°C. The ThromboLUX (TL) determines type, amount, size of particles and the reaction to changes in temperature by dynamic light scattering. The TL score is calculated with values up to 40 (greatest homogeneity). These results were compared with flow cytometric quality control data (CD61-positive MP and CD62P-acitivated platelets).

Results

Data from TL and flow cytometry (n = 14) showed a median TL score of 21.7, CD62P expression of 9.9%, MP content of 3% and CD61-positive MP of 1.7%. The repeatability precision (n = 3) determined as coefficient of variation (CV) was 2%-17% for the TL scores and 18%-29% for MP content. The inter-user precision with three different staff members showed CVs of 6%-15% and 6%-28% for the TL score and MP content, respectively. Storage of samples for up to 4 hours at room temperature and without agitation had little effect on TL scores or MP. During storage of PC for up to 4–5 days the median TL score and CD62P-expression remained largely constant with values of 20.4 to 20.6 and 13.2% and 13.1%. The median MP content did not change (about 8%) whereas the median proportion of CD61-positive MP increased from 1.9% to 2.5%.

Conclusion

CD62P-expression is an accepted parameter of platelet activation in PC and is in agreement with TL-measured MP content and TL score. TL detected consistently more MP than flow cytometry, likely because it identified smaller MP and is not dependent on staining MP with specific antibodies. The data indicate that samples can be stored for up to 4 hours before analysis. The results of shelf life testing showed little deterioration of platelet quality during storage.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):36.

P01-9 Male gender seems to enhance the risk of NIN development

J-C Wolff 1, UJ Sachs 1, G Bein 1

Question/Background

Neonatal alloimmune neutropenia (NIN) is a rare, self-limiting condition of newborns caused by maternal IgG antibodies against paternal human neutrophil antigens (HNA) present on the newborn's neutrophils. While most newborns with NIN are either unaffected or develop mild infections only, more severe clinical courses including sepsis are reported. The aim of this study was to evaluate clinical and serological data from newborns with NIN in order to establish a potential relationship between anti-HNA antibody types, neutrophil counts, and the clinical presentation.

Methods

A total of 95 cases were eligible for analysis, including 68 cases with defined anti-HNA antibodies (patient group) and 27 with defined anti-HLA, but no anti-HNA antibodies (control group). A standardized questionnaire was used to retrieve clinical data from the treating physicians. We received at least partial data of 88 cases. Groups and subgroups were then analyzed statistically.

Results

Male individuals were over-represented in anti-HNA (41M vs. 21F, p = 0.015), but not anti-HLA cases (14M vs. 9F, p = 0,405). Maternal parameters including week of gestation, mode of delivery, and number of pregnancies were not different between the two cohorts, while the reported onset of neutropenia was significantly earlier in patients compared to controls (p = 0.047). Clinical data were insufficient for an adequate statistical approach. Where G-CSF application was necessary (n = 6), therapy was successful within few days.

Conclusion/Outlook

Male gender seems to be a risk factor for the development of NIN. Data were insufficient to retrieve conclusions regarding an association between the anti-HNA antibody type and clinical severity of NIN.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):36.

P01-10 FCGR3B null phenotype in a patient with Coombs-negative hemolytic anemia, thrombocytopenia and suspected collagenosis

C Grabowski 1, K Winterstein 1, K Fenchel 2, P Hoyer 1, S Jorks 1, H Kroll 1

Background

FcγRIIIb (CD16b, HNA-1) is a glycosylphosphatidylinositol (GPI)-anchored low-affinity Fc receptor for IgG which contributes to the clearance of immune complexes from the blood circulation. Copy number variations (CNV) of FCGR3B have been described in autoimmune disorders. Absent or reduced surface expression of GPI-anchored proteins was also found in paroxysmal nocturnal hemoglobinurea (PNH) due to mutation in the PIG-A gene.

Case report

The 79-year-old male patient presented with xerostomia. A biopsy of the lower lip showed lymphocyte infiltrates in small salivary glands. Abdomen sonography showed a slightly enlarged spleen. Laboratory investigations indicated mild anemia (12.8g/dl) with signs of hemolysis, mild thrombocytopenia (100G/l) and elevated rheumatoid factor (350IU/l). The direct Coombs test was negative. Flow cytometric analysis of GPI-anchored proteins was performed to exclude PNH.

Methods

Multiparametric flow cytometry for the detection of GPI-anchored proteins on granulocytes, monocytes, reticulocytes and red blood cells was performed using fluorescence-labeled mabs against CD16b/66b/24/14/48/58/59. HNA-1 genotyping was performed by PCR-SSP. For the detection of abs against FcγRIIIb, the patient's serum was analyzed by granulocyte immunofluorescence and agglutination tests and glycoprotein-specific immunoassay.

Results

Flow cytometric analysis of GPI-anchored proteins on peripheral blood cells showed lack of FcγRIIIb expression on neutrophils. The GPI-anchored proteins CD66b/24 on neutrophils, CD14/48 on monocytes and CD58/59 on reticulocytes were normally expressed. Interestingly,30% of red blood cells showed reduced CD58 and normal CD59 expression. Thus, PNH as the possible cause of anemia was ruled out. Genotyping of the alleles 227A(FCGR3B*01), 266G(FCGR3B*02) and 266A(FCGR3B*03) by PCR-SSP showed negative results, indicating the absence of the FCGR3B gene. No FcγRIIIb-specific isoantibodies were detected by the serologic assays.

Conclusion

We describe a HNA-1null phenotype in a patient with suspected autoimmune disease. Though CNV have been observed, the functional role of FcγRIIIb deficiency in autoimmune disorders is still unknown. Impaired clearance of immune complexes might have contributed to systemic autoimmunity in our patient. However, further studies are required to elucidate the relevance of FCGR3B deficiency in autoimmunity. Genotyping and flow cytometric analysis can be used to identify FCGR3Bnull individuals.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):36–37.

P02-1 Droplet digital™ pcr as a new tool for the quantification of rhd and rhce in samples with weak Rhce reactions in serological testing and Wildtype sequencing results

A Doescher 1, T Müller 2, FF Wagner 2

Introduction

Weak reactions with certain antisera were frequently observed problems in serological Rh-testing. A large number of genetic variants are known as molecular background for these problems. Nevertheless, some samples, especially with weak RhC reaction patterns, remained unresolved with classical molecular typing methods. We developed a droplet digital™ PCR (ddPCR) assay for co-amplification of RHD- and RHCE specific sequences in exon 3 to 6 for further examination of these samples and compared the results to routinely used quantitative real-time PCR.

Methods

Nine samples with unresolved genotypes were tested in dd PCR (QX 200 Bio Rad) and quantitative real-time PCR (7900HT Applied Biosystems).

a) Absolute quantification assay with ddPCR was performed in a mix of ddPCR buffer, 900 nM primer, 300nM probes and samples diluted 100fold. Amplification of RHD- and RHCE-specific sequences was performed in the same tube and compared to known DD- and Dd controls.

b) Quantitative real-time PCR: Four different DNA concentrations per sample were used for co-amplification of RHD and RHCE in the same tube. Threshold cycles (Ct) were manually set and the difference between RHD- and RHCE-specific Ct values was calculated as ΔCt.

Results

Four samples with problems in RhC typing were used for proof of reliability of the method and for each sample a masked RHCE hybrid gene was detected. Another three samples showed weak reactions in RhC / RhD typing and quantification revealed a decreased amount of the CDe haplotype. Examination of the RhC-negative sample from a newborn, given birth by a mother typed as RhC homozygous, revealed a -D- haplotype. No differences were observed between dd- and quantitative real-time PCR.

Conclusion

The quantification of RHD compared to RHCE exons 3 to 6 can give additional information of the molecular genetic background in samples with problems in serological testing and presumably normal genotypes. In cases where a sample for RNA extraction and haplotype-specific sequencing of cDNA are not available, the quantification of RHD and RHCE offer a useful alternative for identification of masked hybrids and reduced amounts of one haplotype. Droplet digital PCR as a new approach in this context offers a reliable and cost-effective alternative to quantitative real-time PCR.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):37.

P02-1 Primary autoimmune neutropenia of infancy in monocygotic twins

A Reil 1, K Bienemann 2, H-J Laws 2, J Bux 3

Introduction

Autoimmune neutropenia is caused by peripheral destruction of neutrophils as a result of granulocyte-specific autoantibodies. Primary autoimmune neutropenia of infancy is not associated with other diseases and typically occurs in infants from 5–15 months. It is the most common cause of chronic neutropenia in infancy. Severe infections are rare and neutropenia usually resolves within a few years. The etiology of the disease is unknown. Neither a hereditary mechanism nor a clear role of an infectious trigger could be demonstrated so far. We report previously healthy monocygotic twins who suffered from recurrent infections of the upper respiratory tract from the age of twelve months and simultaneously developed abscesses of the labia majora at the age of 14 months. Differential blood count at that time showed a complete absence of neutrophils.

Methods

Sera of both infants were tested by granulocyte immunofluorescence test (GIFT), granulocyte agglutination test (GAT) and glycoprotein-specific ELISA (MAIGA). FCGR3B genotyping was performed by PCR-SSP.

Results

GIFT, GAT and MAIGA revealed autoantibodies preferentially binding to HNA-1a positive granulocytes. Reaction strength was almost identical in both twins. Both girls typed homozygous for the HNA-1a encoding allele FCGR3B*01. Under prophylactic antibiotic treatment no further relevant infections occurred despite sustained agranulocytosis. One year after diagnosis, when neutrophils were starting to recover, laboratory tests were repeated. Granulocyte autoantibodies were no longer detectable in one girl and borderline detectable in the other. Normal neutrophil counts were documented 22 months after diagnosis when the girls were 3 years old.

Conclusion

Laboratory findings and clinical course in both girls were characteristic for the diagnosis of primary autoimmune neutropenia. The simultaneous occurrence of granulocyte specific autoantibodies as well as the identical clinical course in this monocygotic twins strongly suggest a genetic predisposition for the disease.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):37.

P02-3 Rhesus variant – D – caused by transcriptional error in RHCE-gene?

K Strathmann 1, M Wessiepe 1, A Doescher 2, G Hutschenreuter 1

Introduction

Antigens of the Rh blood group system are products of RHD and RHCE two tightly linked and highly homologous genes residing on chromosome 1p36.1. RHCE exists in four allelic forms and each allele determines the expression of two antigens in Ce, ce, cE or CE combination. RHD and RHCE genes, each, contain 10 exons and span ~75 kb DNA sequence. Several mutations in the RHCE gene are known which result in decreased or missing expression of the CE antigens. Patient: The patient, a 80-year-old man, was referred to the department of cardiology because of a NSTEMI with cardiogenic shock based on a three vessel coronary disease. In the emergency setting RBC-concentrates had to be transfused. In the routine blood typing the missing antigen expression of CE-antigens was detectable.

Methods

EDTA-blood specimens from the patient were tested. ABO and rhesus antigens were determined by gel card technique (BioRad GmbH, Munich, Germany). RHCE genotyping was done with allele specific PCR (Rh-Type, BAG, Lich, Germany). Sequencing of RHCE gene, including exons 1 to 10 and intron/exon borders, was done by direct taq cycle sequencing using BigDye terminators v1.1 in an ABIprism310 (Applied Biosystems, Darmstadt, Germany).

Results

In the serologic diagnostic the initial blood typing results were A rhesus positive (D pos.). The rhesus antigens for Cc and Ee were not detectable in serologic diagnostic. In the genomic sequencing of RHCE (exon 1 - exon 10) all exons of the RHCE-gene were present, without detection of polymorphisms. The RHCE sequencing of cDNA showed no RHCE specific products. To exclude the presence of residues of RHCE-specific sequences additional real-time PCR of exon 3 to exon 5 was performed. No RHCE-specific product was detectable.

Conclusions

In this case of a -D- the RHCE-gens showed no polymorphisms as an explanation for the missing antigens. No transcription of RNA to cDNA was detectable. This results in the presumption of a transcriptional error of unknown cause. A possible cause could be a mutation of a transcription factor. The transfusion support of patients with -D-phenotype is challenging. In emergency setting only incompatible red cell concentrates are available like ccddee with the risk of alloimmunization. If there is enough time before the blood transfusion the availability of cryo-preserved or fresh -D- red cell concentrates needs to be checked.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):37.

P02-4 A novel variant of the KEL gene associated with a weak expression of the Kell blood group antigen

C Wieckhusen 1, G Rink 1, A Diel 1, S Rothenberger 2, E Richter 2, EA Scharberg 2, P Bugert 1

Background

The Kell blood group system (KEL, ISBT 006) includes several antithetical, high and low prevalence antigens such as K (KEL1), k (KEL2), Kpa (KEL3) and Kpb (KEL4). The KEL gene encoding the KEL glycoprotein encompasses 19 exons distributed over 21.5 kbp of genomic DNA. The reference allele KEL*02 encodes the KEL2 and the high prevalence antigens. Point mutations represent the molecular basis of most KEL antigens as well as the weak expression (Kmod) or lack (Knull) of the antigens. Here, we describe a novel mutation of the KEL gene in a blood donor with weak expression of the KEL1 antigen.

Methods

The Kell (KEL1) and Cellano (KEL2) antigens were analyzed by standard serological methods. Genomic DNA was isolated from a blood sample of the donor. All exons and flanking intron regions were analyzed using next generation sequencing (GS Junior; Roche, Mannheim, Germany). A PCR-SSP method was established for the novel mutation and used for screening of the mutation in blood donors.

Results

The blood donor showed normal expression of the KEL2 antigen, whereas, the KEL1 antigen expression was significantly diminished. As expected from serology, the donor's KEL gene revealed a heterozygosity at position c.578C>T in exon 6 as the molecular basis of the KEL1 and KEL2 antigens. A second heterozygosity was identified at position c.2102T>C in exon 19. The mutation is not listed in the large databases (dbSNP or ExAc). Screening of 9,668 blood donors for the 2102C variant by PCR-SSP was negative.

Conclusion

We assume that the novel 2102T>C mutation of the KEL gene is located on a KEL*01 allele encoding the KEL1 antigen. The caused amino acid change (V701A) may affect the protein conformation, stability or cell surface expression leading to the significantly weakened KEL1 antigen expression.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):38.

P02-5 Evaluation of the evanescent biosensor technology for rapid phenotyping of the human platelet alloantigen 1a

TJ Schulze 1, W Song 1, G Rink 1, H Klüter 1, P Bugert 1

Background

The human platelet alloantigen (HPA) 1a is located on glycoprotein IIIa (CD61) on the platelet surface. In a HPA1a-negative mother the HPA1a-positive fetus can induce an alloimmune reaction which is the most common cause of neonatal alloimmune thrombocytopenia (NAIT) in Caucasian populations. Additionally, alloimmunization against HPA1a is frequently involved in refractoriness after platelet transfusion and HPA1a-negative blood donors are required for compatible platelet supply. The HPA1a and b antigens are defined by a single nucleotide polymorphism (SNP) in the GPIIIa gene and different genotyping methods are in use. However, genotyping is time consuming and costly depending on the method and sample throughput. Here, we evaluated the commercially available evanescent biosensor technology (EVA) as a novel method for rapid phenotyping of the HPA1a antigen.

Material and Methods

Stored (-30°C) EDTA blood samples from 380 blood donors were used for DNA isolation and subsequent HPA1 genotyping by TaqMan-PCR. The same blood samples were subjected to EVA phenotyping of the HPA1a antigen by using the Davos Diagnostics (Davos, Switzerland, www.davosdiagnostics.com) biosensor system and the HPA1a typing test. For each sample a single EVA value was obtained. The results from genotyping and phenotyping were compared.

Results

The HPA1 genotype could be determined for all 380 blood donors with 269 HPA1aa, 106 HPA1ab and 5 HPA1bb. The EVA HPA1a phenotyping revealed a positive result for 375 samples all with a HPA1aa or HPA1ab genotype. All 5 samples with HPA1bb genotype were negative by EVA HPA1a phenotyping. The EVA values of HPA1aa genotypes were significantly higher than HPA1ab genotypes (168.4 ± 71.8 versus 108.4 ± 55.1; p < 0,0001) but, as expected, HPA1a phenotyping could not discriminate between the two genotypes.

Discussion

EVA is a reliable method for rapid phenotyping of the clinically relevant HPA1a platelet antigen. The test can be performed from only 10 μl of fresh or frozen blood samples. The phenotyping of 8 samples with single values on one EVA chip was completed within 10 minutes (3 minutes hands on time).

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):38.

P02-6 YKL-39, TGF-beta inducible factor in human primary macrophages, stimulates monocyte migration and reversely correlates with hematogenous and lymphatic metastasis in human breast cancer

T Liu 1, I Mitrofanova 2, B Song 1, M Buldakov 2,3, M Zavjalova 2,3, N Litviakov 2,3, N Cherdyntseva 2,3, H Klüter 1,4, J Kzhyshkowska 1,2,4

Introduction

Human chitinase-like proteins are considered as a novel biomarker of cancer, inflammation and tissue remodeling. The role of YKL-39 and its associations with tumor progression has not been studied until now. The aim of study was to examine the intracellular trafficking pathways, and secretion mode of YKL-39 in M2 macrophages, to explore the effect of YKL-39 on monocytes migration; and correlation of YKL39 levels with breast cancer progression.

Methods

CD14+ monocytes were isolated from buffy coats, cultured with IL-4, TGF-beta, dexamethasone and M-CSF to obtain M2 macrophages. YKL-39 expression level was measured by RT-PCR. The intracellular distribution of YKL-39 was checked by IF staining and confocal microscopy. YKL-39 secretion was measured by ELISA assay. Monocyte migration was performed with a trans-well system. The correlations of YKL-39 with metastasis in human breast cancer were verified by RT-PCR, IHC and IF staining.

Results

YKL-39 gene level was strongly up-regulated with IL4/TGF-beta in human macrophages: on day 6 (13.4 fold, p < 0.05) and day 12 (62.2 fold, p < 0.05) (n = 6). YKL-39 was detected in the TGN, p62lck positive late endosomes, Lamp-1 positive lysosomes and CD63 positive secretory lysosomes. The extracellular secretion of YKL-39 (1.51 ng/ml) was detected on day 12. Recombinant YKL-39 significantly enhanced the migration of monocytes by 1.9 fold (p < 0.01) in 1 hour, and reached to 4.9 fold (p < 0.01) in 3 hours. In patients with breast cancer, low levels of YKL39 correlated with a higher frequency of lymphatic metastasis (p = 0.036, n = 74) and hematogenous metastasis (p = 0.0337, n = 74). The pre-dominant TAM phenotypes were YKL-39 +/ stabilin-1+ (73%, n = 10) and CD68+/YKL39+ (25%, n = 10).

Conclusions

YKL-39 is secreted by alternatively activated macrophages via lysosomal pathway, stimulates migration of monocytes and demonstrates a reverse correlation with hematogenous and lymphatic metastasis in human breast cancer.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):38–39.

P02-7 Variability and prediction of the frequency of rare donors in Northern Germany

F Wagner 1, R Bittner 1

Introduction

Rare donors frequency may vary by region. Heterozygote frequency may be used to predict rare donor frequency. We used our database of ~100,000 donors typed by pooled capillary electrophoresis (PCE) to discern regional variability and to test the validity of estimates based on heterozygotes.

Methods

Antigen data from our donor database were correlated with ZIPcodes. ZIP codes were grouped as follows: “northern lower saxonia”: 20000 to 25999 and 27000 to 29999; “western lower saxonia”: 26000 to 26999 and 48000 to 49999; “southern lower saxonia”: 30000 to 31999 and 37000 to 38999; “Saxony-Anhalt”: 06000 to 06999 and 39000 to 39999; “Thuringia” : 07000 to 07999 and 98000 to 99999; and “other”: all other ZIP codes. Phenotype and allele frequency calculation was based on donors with both antithecial alleles known. Allele frequencies were determined by allele counting, predicted phenotype frequencies calculated assuming a Hardy-Weinberg equilibrium.

Results

There were 257066 donors with antigen data (excluding k 170305 donors). For 185376 donors there were 251483 serologic antigen typings (1.36 per donor; excluding k typing: 155779 antigens in 95659 donors; 1.63 per donor), for 101435 donors 1489871 antigens were predicted by molecular methods (14.69 per donor). Zip code data were analyzed for 98358 donors. Frequencies per 10000 varied as follows: Co(a-) 19.35 to 29.41; Yt(a-) 16.63 to 57.92; Lu(b-) 7.47 to 22.09; Vel-: 0 to 10.83; Kp(b-) 0 to 2.40. Observed frequencies differed from that based on heterozygotes: Overall frequencies were Co(a-) predicted 20.16, observed 24.46; Yt(a-) predicted 17.93 observed 26.60; Lu(b-) predicted 10.50 observed 9.49; Vel- predicted 2.87 observed 4.30; Kp(b-) predicted 0.75 observed 1.07. In contrast, predictions for Jk(a-), Fy(b-), NN ans SS were close to the observed frequencies (observed / predicted ratio 1.00 to 1.02).

Conclusion

Estimates of rare phenotype frequency based on heterozygotes may be too low if data obtained with pooled capillary electrophoresis are used. Two complementary causes are likely: (i) Regional variation and inbreeding leads to a surplus of homozygous donors with the rare phenotype. (ii) “Low peak size” and “no call”, technical causes for “no result” by PCE may be more frequent in heterozygotes. Regional variation and inbreeding are confounding factors Independent of the method. Donor screening should not be dismissed too early based on low heterozygote frequency.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):39.

P02-8 Antibody to a new low frequency Kell blood group antigen causing a severe hemolytic disease of the newborn

EA Scharberg 1, C Wieckhusen 2, B Luz 3, L Delle-Chiaie 4, P Neuberger 4, A Stürtzel 1, S Rothenberger 1, G Rink 2, E Richter 1, S Seyboth 1, P Bugert 2

Introduction

A pregnant Caucasian woman (gravida 2, para 1) was hospitalized because of the Mirror Syndrome with edema, ascites, massive fetal hydrops and polyhydramnios. Her child was delivered by a Cesarean section in the 25 week of gestation with severe anemia (3.1 g/dl) and a positive direct antiglobulin test (IgG 3+). It weighed 1070 g and was in a critical condition with APGAR score of 1/3/5. Though it was immediately transfused it died 9 days after birth. The plasma of the mother was reactive with red cells of her husband but it showed negative reactions in antibody identification and with multiple red cells positive for low frequency antigens.

Methods

The clinical data and the serological tests suggested an antibody to a clinically relevant low frequency antigen. To determine the specificity of the antibody the family members were tested by serological and molecular methods.

The antibody identification was performed in the indirect antiglobulin test (IAT) in the gel technique using untreated, papain and DTT treated red cells. The red cells of the child's father, the father's mother and of the child's brother were cross-matched with the serum of the mother. As the reaction was enhanced by papain treatment and negative after DTT treatment of the cells an antibody to a Kell blood group antigen was supposed. The KEL gene was analyzed by exon re-sequencing and a PCR-SSP method was established for genotyping of the identified mutation.

Results

Antibodies to following low frequency antigens were excluded: Wra, Dia, Wu, Cob, Ytb, Lu14, Jsa, K17, K25, Cx, DAK, FPTT, Goa, V, VS, JAL, JAHK, PARG, LWb, Lsa, Ula, Tca, Sc2, Vw, Mg, Mia, Hut, Mur, Hil, Miny, He, Mta, Sta, Mit, Vr, Knb, Vil. The serum of the mother was positive with red cells of the child's father, the father's mother and of the child's brother. Sequencing of the father's KEL gene showed a heterozygous 877C>T (Arg293Trp) mutation in exon 8. The father's mother and the child's brother were positive for the mutation defining a new low frequency Kell blood group antigen, we named KEAL. Screening of 11705 blood donors for the 877C>T mutation by PCR-SSP was negative.

Conclusion

We describe a new low prevalence Kell blood group antigen named KEAL. It is characterized by the 877C>T missense mutation in the KEL gene causing a single amino acid change (Arg293Trp) in the KEL protein. Antibody to KEAL caused a severe hemolytic disease of a newborn. KEAL is antithetic to the high prevalence KHUL antigen (KEL37)

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):39.

P02-9 Loss of rhesus heterozygosity in a healthy blood donor

BK Flesch 1, B Just 2, V Schottstedt 3

Introduction

Mixed field haemagglutination reactions can occur after blood transfusion but usually are not observed in healthy blood donors. Besides blood transfusion, in rare cases mixed field reactivity has been reported to result from a loss of heterozygosity (LOH) in some patients1. We describe the case of a healthy female blood donor who donated blood for over 60 times and was regularly typed as ccD.Ee. At her 60th donation mitigated reactivity and mixed field haemagglutination was observed with anti-D and anti-E for the first time.

Methods

At the time of the divergent findings, the female donor was 52 years old, her last delivery (para 5) was 12 years ago. She as well as her physician reported that she was healthy without any medical complications. Standard blood typing was performed by haemagglutination with automated reading (PK 7300, Beckman) and gel card technique (Biorad). Genotyping was performed by commercial PCR-SSP (Innotrain) and DNA sequencing including the 10 exons and flanking intron sequences of both, the RHD and RHCE genes. Rhesus antigen expression on the donors RBC was tested by flow cytometry using a set of different monoclonal Anti-D, Anti-C, Anti-c, Anti-E and Anti-e as well as polyclonal antibodies against k, Fya, Fyb, Jka and Jkb from different suppliers.

Results

DNA sequencing confirmed the ccD.Ee blood group without any variation. Flow cytometry detected smaller RhD, RhE positive (~23% of events each) and larger RhD, RhE negative RBC populations, respectively. Rhc, Rhe, k (cellano), Fya and Jka was expressed on each RBC of the donor whereas her cells were completely lacking Fyb and Jkb. Follow-up of the donor's RhD and RhE expression during the next 12 months showed a constant or slightly reduced percentage of antigen positive cells (~20% positive cells).

Conclusion

The most probable explanation for the donor's differential RhD and RhE expression on her RBC can be seen in a LOH on part of her stem cells with a deletion of the cDE haplotype on one chromosome during replication. The RHD and RHCE gene region on chromosome 1 has been demonstrated as a hot spot for LOH. LOH has been observed in patients with haematopoietic malignancies increasing with their age. To our knowlede this is the first case of an apparently healthy donor with LOH.

Reference

  • 1.Körmözci GF, et al. Blood. 2007;110:2148–2157. doi: 10.1182/blood-2007-01-068106. [DOI] [PubMed] [Google Scholar]
Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):40.

P02-10 Is a RHCE Intron 3 −5T>G exchange responsible for a diminished Ce expression?

A Karnot 1, BK Flesch 2

Introduction

The Rhesus system is characterized by a high number of antigens that base on a complex genetic background. Some mutations are unremarkable in standard phenotyping assays while others exhibit differential reactivity with Rhesus antisera and require molecular methods to explain the underlying mechanism. We present a new mutation of the RHCE gene that was detected in a case of a diminished Rhesus Ce expression.

Methods

The RBC of a young female patient without transfusion history showed only weak reactivity with different monoclonal anti-C and anti-e (Immucor, Optima, Ortho) in the standard indirect antiglobulin test whereas reactivity with standard monoclonal anti-c, anti-E and anti-D was cleary positive. Commercial PCR-SSP (RBC-Ready Gene CDE, Inno-Train, Kronberg) indicated a CcD.Ee Rhesus type which could not explain the diminished Ce expression. The molecular background was determined by Sanger sequencing of the 10 RHCE exons including flanking intron sequences (BigDye Terminator v3.1, ABI Prism 310, Applied Biosystems).

Results

DNA sequencing revealed a RHCE*CcEe genotype with an additional heterozygous −5G>T mutation within intron 3. The second chromosome carried the wild type RHCE intron 3 −5G nucleotide. To our knowledge this was the first description of this mutation which we found as the only reason for the weak Ce expression. This would imply the haplotypes RHCE*Ce intron 3 −5G>T and a wild type RHCE*cE. The mutation was submitted to GenBank (KT732417).

Conclusion

The RHCE intron 3 −5G>T mutation possibly induces an alternative splicing that could explain the diminished Ce expression. Because no data are available concerning a potential immunization risk against the mutated Ce we recommended substitution of the patient with RBC of the ccD.EE Rhesus type.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):40.

P02-11 Limited use of CA 19-9 measurement in pancreatic cancer patients with Lewis negative phenotype

K Wiesinger 1, M Egger-Salmhofer 1, M Haltmayer 1, M Weitzendorfer 2, K Emmanuel 2, B Dieplinger 1

Background

Carbohydrate antigen (CA 19–9) is the most extensively validated biomarker for pancreatic cancer. Even though a major limitation of CA 19–9 testing, with false negative results in patients with Lewis negative phenotype, has been known for several years by experts in the field, this knowlegde has not been transfered into clinical routine in many institutions. The aim of this study was to evaluate the Lewis phenotype in patients, were routinely ordered CA 19–9 plasma concentrations were below the limit of detection, and compare them to patients with advanced pancreatic cancer and increased CA 19–9.

Methods

Plasma concentrations of CA 19–9 were measured with the Abbott Achitect i2000 analyzer. Lewis phenotype was determined with the Biorad gel-system DiaClon Anti-Lea/Leb in 84 consecutive patients with routinely ordered CA 19–9 plasma concentrations < 2 U/mL, and in 5 patients with advanced pancreatic cancer and increased CA 19–9.

Results

Of the 84 patients with routinely ordered CA 19–9 < 2 U/mL, 61 (73%) showed a Lewis (a-/b-), 23 (27%) a Lewis (a-/b+), and non a Lewis (a+/b-) phenotype. Within the group of patients with Lewis (a-/b-) phenotype and CA 19–9 < 2 U/mL there were 8 patients with advanced pancreatic cancer. Serial measurements of CA 19–9 in these advanced pancreatic cancer patients with Lewis negative phenotype during treatment and/or disease progression always revealed plasma concentrations below the reference value (i.e. 37 U/mL). In contrast all 5 patients with advanced pancreatic cancer and an increased CA 19–9 (median 21817, range 1055–65144 U/mL) showed an Lewis positve phenotype.

Conclusions

False negative results of CA-19–9 testing in patients with Lewis negative phenotype is a common problem in clinical routine. In our institution we have now implemented the routine determination of the Lewis phenotype in primary work up of pancreatic cancer patients.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):40.

P02-12 A new GYPB*04(164T>G, Phe55Cys) allele with a phenotypic partial character of small s

C Engström 1, A Caesar 2, S Meyer 1, C Portmann 1, N Trost 1, P Schwind 2, BM Frey 1, C Gassner 1

Background

The S and s antigens of the MNSs blood group system are caused by a single SNP at coding nucleotide (nt) 143C>T (Thr48Met) of GYPB. Antigens SD+ and Mit+ result from SNPs at nts 161G>A (Arg54His) and 173C>G (Pro58Arg) respectively and underline the immunohematological relevance of this area proximal to the trans-membrane region of the protein on the outer RBC surface. In general, new blood group antigens may be discovered by diagnostic antibodies, discrepancies in pheno-/genotyping, or due to responders’ antibodies.

Methods

Human and monoclonal anti-s typing reagents were applied (Medion Grifols Diagnostics, Duedingen, CH; BioRad, Cressier, CH; Merck-Millipore, Darmstadt, DE) using gel-card, in tube and lateral flow-card assays. Genotyping (MNSs, Inno-Train, Kronberg, DE) and sequencing were performed (Meyer S. et al Br J Haematol. 2016). Trans-membrane helix location was predicted using HMMTOP, PHOBIUS, SOSUI, TMPRED, disulphide-bonds using DiANNA, and N-glycosylation using NetOGlyc.

Results

Initial phenotyping discrepancies were observed using human anti-s (gel-card) and monoclonal anti-s (in tube) in two unrelated blood donors (table 1). Samples were heterozygous for GYPB*03/04 (S/s) and both GYPB*04 (s) exons 4 showed the same new mutant allele GYPB*04(164T>G, Phe55Cys). moAB clones P3Y326Bn5 used in tube and P3BER in lateral flow assays, delivered reliable positive reactions for this new s, whereas moAB P3YAN3 failed to detect it. Software did not predict a change in the trans-membrane helix, nor disulphide-bonds. Still, changes in N-glycosylation of the mutated protein were assessed.

Tab. 1.

anti-s antibodies (AB) tested

graphic file with name tmh-0043-0001-gu04.jpg
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Conclusion

We describe a partial s. Anti-s was not detected in either donor. Potentially, family analysis could deliver this pre-requisite for considering GYPB*04(164T>G, Phe55Cys) as a true new MNSs antigen. Still, partial character of the new s and the location of the mutation in a highly antigenetically relevant part of the peptide are of significance.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):41.

P02-13 Novel B variant allele of the ABO blood group gene B101(133S)

R Conradi 1, A Döscher 2, D Marandiuc 1, W Hitzler 1

Introduction

ABO blood group typing for first time donors is routinely done by forward and reverse typing. One sample with discrepant serological results was further tested molecular biologically and a new, to our knowledge, B-allele was found.

Methods

Donor ABO blood group typing was performed serological with an automated method on Immucor Gamma® Neo™ (microplate agglutination technique). Column agglutination tests were done with BIO-RAD ID cards (ABO/Rh, human and DiaClon ABO/Rh for patients, monoclonal). Agglutination tests in tubes and antibody absorption and elution was performed with antibodies from BAG Healthcare (Anti-B, clone F85 3D6; F97 2D6). Standard ABO DNA typing was performed using a PCR-SSP test from BAG Healthcare (BAGene ABO-TYPE variant). ABO sequencing was performed with Taq-polymerase cycle sequencing using fluorescent-labeled dye terminator reactions. The sequencing data was analyzed and compared to known reference sequences with MacVector® Software.

Results

The sample reacted negative to A-, B- and AB-antibodies in the serological forward ABO typing with the automated method, the monoclonal column agglutination test and tube testing. Reverse ABO typing had strong positive reactions with A1 and A2 cells and negative reactions with B and 0 cells. Column agglutination test with human antibodies showed only with anti-AB antibodies a strong positive (3+) reaction. By tube testing with extended incubation time, weak positive reactions were obtained with anti-B (1+) and anti-AB (2+). Anti-B antibodies from BAG were absorbed on the patient cells and afterwards eluted. The eluate reacted positive (4+) with normal B-cells, which showed the existence of antigen B on the donor cells. DNA-SSP typing indicated an ABO*O02/B101 (rare B-variants cannot be found with the method). Sequencing exons 3–7 showed the known nucleotides for O02 and B101 with an addition mutation at position 398 (T>C). Consequently, at the position 133 of ABO transferase, the phenylalanine amino acid is replaced by a serine (F133S). This allele was named ABO*B101(133S).

Conclusion

A new ABO-B variant was found. This variant reacted negative for antigen B with standard agglutination methods, but after extended serologic testing (human antibodies, extended incubation time, elution) the antigen B was detected. The underlying gene sequence (T398C) that encodes an amino acid substitution (F133S) was identified and the new B-allele was described as ABO*B101(133S).

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):41.

P02-14 Identification of epigenetically controlled regulators at CCR2 promoter induced by hyperglycemia in human macrophages

K Moganti 1, F Li 1, B Yard 2, H Klüter 1,3, M Harmsen 4, J Kzhyshkowska 1,3,5

Introduction

Hyperglycaemia is an essential factor leading to micro- and macrovascular diabetic complications. Macrophages are key innate immune regulators of inflammation that undergo 2 major direction of functional polarisation: classically (M1) and alternatively (M2) activated macrophages. M1 are involved in the establishment and progression of insulin resistance in diabetes, whereas M2 contribute to the decrease of hyperglycaemia. However, the effect of hyperglycaemia on differentiation, functional and epigenetic programming of macrophages is poorly understood.

Methods

Monocytes were isolated from buffy coats by magnetic cell sorting using CD14 beads and cultivated in the presence of 5mM and 25mM glucose for 6 days under stimulation with IFNg (M1), IL4 (M2) or without cytokine (M0). Differentially expressed genes induced by high glucose we identified using Affymetrix chips assay and verified by RT-PCR. In vitro transmigration assays was performed using transwell system. Histone marks in vicinity of CCR2 promoter have been identified using chromatin immunoprecipitation (ChIP).

Results

We found that hyperglycaemia induces the elevated expression of CCR2 in M0 and M1. ChIP assay revealed that hyperglycaemic conditions significantly increase the presence of activating in histone marks of H3K4me1 and H3K4me3 at CCR2 promoter. To address the functional relevance of CCR2 expression in high glucose, we performed the migration assays in normal and high glucose condition. Hyperglycemia stimulated migration of M0 (7.6 fold increase) and M1 (11.2 fold increase) towards CCL2 gradient.

Conclusions

We found that hyperglycemia induces expression of CCR2 on primary human macrophages linked to the epigenetic modifications of CCR2 promoter by histone code. Elevated levels of CCR2 resulted in the attraction of pro-inflammatory macrophages to the sites of inflammation in diabetic conditions that can affect progression of vascular complications at very early stages.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):41.

P02-15 RHD*07.02 allele causes discrepant genotyping results for RHCE*c

S König 1, V Van Sandt 2

Introduction

In the human Rh blood group system the c, C, E, e and D antigens are expressed by the two highly homologous genes RHCE and RHD. After D, c is the most immunogenic Rh antigen. The difference between c (307C) and C (307T) is caused by the SNP on position 307 on the RHCE gene. The RHD*07.02 allele (also known as RHD cat VII type 2) carries the SNP 307T>C on the RHD gene and additionally the SNP 329T>C. This RHD*07.02 allele has been described to partially express RHc on the D polypeptide (Faas, Transfusion, 2001). Genotyping was performed to clarify the cause of the weak c expression. Serology of a patient sample (Male, °1938) indicated a partial c phenotype with a CDe.

Methods

RhD and RhCE phenotyping was done by accredited routine protocols (monoclonal AB ID card: Diaclon Rh subgroups, seraclone anti-c). Genotyping was performed with a TaqMan Probe assay (RBC-Flu-oGene vERYfy, inno-train Diagnostik GmbH), SSO (RBC-Lifecodes, Gen-Probe Inc.), in-house SSP-PCR (HILA, Rode Kruis-Vlaanderen) and commercial SSP-PCR (RBC-Ready Gene CDE, inno-train Diagnostik GmbH). Sanger sequencing of the RHD gene was performed using an in-house method (inno-train Diagnostik GmbH).

Results

Discrepant genotyping results were generated by different test systems: the TaqMan Probe based assay showed in repetition a CCee genotype, while the SSO system RBC-Lifecodes predicted in repetition a Ccee phenotype. In SSP-PCR the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. The parallel analysis of the RHD gene with RBC-Ready Gene CDE test system revealed a variant D cat VII RHD allele. Sequencing of the DNA sample identified two SNPs on one of the RHD alleles (307T>C, 329T>C) confirming a RHD*07.02 and one RHD*01 allele.

Conclusions

Usually genotyping provides clear answers for conspicuous serology. However, in a few cases PCR does not offer conformable results e.g. where mutations on the primer binding sites prevent the amplification. As described here there must be differences in the primer and/or probe design (for RHCE*c) of different test systems causing either correct or erroneous genotyping results. In this example the high homology between the RHD and RHCE genes in combination with the presence of the D cat VII SNP 307T>C lead to false positive RHCE*c (SNP 307C) SSO and SSP genotyping result. We therefore suggest to genotype of both RHCE and RHD to resolve the true nature of weak RHCE serology.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):42.

P02-16 Genotyping of human platelet antigens (HPAs)-1, −2, −3, −5 and −15 in Swiss blood donors using a multiplex SSP-PCR approach

P Gowland 1, J Graber 1, C Niederhauser 1, C Henny 1

Introduction

Human platelet antigens (HPAs) are caused by polymorphisms in platelet membrane glycoproteins. Antibodies against HPAs are clinically relevant in immune platelet disorders, such as foetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP) and multitransfusion platelet refractoriness (MRP). In order to supply these patients with adequate blood products we developed a multiplex-SSP-PCR assay to build up a database of HPA genotyped donors. Here we present the screening data of 1383 donors.

Methods

Both apheresis (platelet and plasma), as well as, whole blood donors were selected for genotyping. Donor screening was performed using published and in-house primers in two multiplex-SSP-PCR reactions targeting HPA-1a, −2b, −3a, −5b and −15a or HPA-1b, −2a, −3b, −5a and −15b respectively. PCR products were analysed using capillary electrophoresis (QIAxcel system, Qiagen, Germany)

Results

381 apheresis and 1002 whole blood donors were genotyped for HPA antigens. The targets for the two multiplex reactions were chosen according to their expected frequency. Since every reaction yielded a PCR product in at least one allele the incorporation of an internal control proved unnecessary. Rare donors were identified in 27 donors homozygote for HPA-1b (2.0%), 9 donors homozygote for HPA-2b (0.7%) and 15 donors homozygote for HPA-5b (1.1%). The detected frequencies for the different HPA alleles were as follows: 84.4% for HPA-1a, 15.6% for HPA-1b, 90.6% for HPA-2a, 9.4% for HPA-2b, 65.1% for HPA-3a, 34.9% for HPA-3b, 91.9% for HPA-5a, 8.1% for HPA-5b, 48.3% for HPA-15a and 51.7% for HPA-15b.

Conclusion

Genotyping of blood donors for the different HPA alleles revealed allele frequencies comparable to data reported for Swiss and other Caucasian populations (http://www.ebi.ac.uk/ipd/hpa/freqs_1.html). Multiplex PCR reactions allow fast and cost-efficient typing of a large number of donors. New blood donors with rare HPA allele conformations were found and added to the database. The characterisation of such donors is essential for the adequate supply of patients who are alloimmunized against common HPA-antigens and who often require platelet transfusions.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):42.

P02-17 A new mechanism of autoimmunity induced by anti-platelet factor 4/polyanion antibodies

T-H Nguyen 1,2, N Medvedev 2, M Delcea 2, A Greinacher 1

Introduction

Antibodies (ABS) against the chemokine platelet factor 4 (PF4/CXCL4) in complex with polyanions (P) mediate human bacterial host defense, but when misdirected, they cause the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT), or even life-threatening autoimmune HIT. Under physiological conditions, anti-PF4/P-ABS opsonize Gram-positive and -negative bacteria by binding to PF4/bacterial-surface polyanion-complexes. During treatment with the polyanion heparin, HIT occurs when anti-PF4/P-ABS activate PF4/heparin-bound platelets. Autoimmune HIT occurs even in the absence of polyanion treatment. By unravelling mechanisms underlying these different clinical manifestations of the anti-PF4/P-immune response, we aim to better understand principles of antibody-mediated autoimmunity.

Methods

We isolated anti-PF4 auto-ABS and anti-PF4/P-ABS from patients with autoimmune HIT and HIT, respectively, and measured binding of single antibodies to single PF4/heparin complexes using single-molecule force spectroscopy and Isothermal Titration Calorimetry.

Results

Anti-PF4/P-ABS with binding forces ~60 pN induced platelet activation only in the presence of polyanions, while anti-PF4 auto-antibodies with binding forces ~100 pN induced polyanion-independent platelet activation. Most importantly, by complexing PF4, anti-PF4 autoantibodies exposed neoepitopes on PF4 like polyanions, allowing binding of otherwise heparin-dependent anti-PF4/P-ABS, hereby recruiting them into an autoimmune process.

Conclusion

We conclude that the mechanism of autoimmune HIT is that a small subset of autoantibodies change the conformation of PF4, thereby catalyzing binding of otherwise physiologic anti-PF4/P antibodies, recruiting them into the autoimmune process. This may have major implications for other autoimmune disorders including development of more specific diagnostic tools and treatment strategies.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):42.

P02-18 Haemolytic disease of the newborn due to primary immunization against Rh(D)-antigen during the first pregnancy

B Mayer 1, S Yürek 1, T Bartolmäs 1, A Salama 1

Background and objectives

It is generally accepted that haemolytic disease of the newborn (HDN) may occur following secondary immunisation, but not following primary immunisation related to pregnancy.

Case report

We describe a 31-year old woman who developed anti-D during the first pregnancy. Her medical history was 100% negative for any blood transfusion, previous pregnancies or abortions. Her foetus was found to be Rh-positive by molecular analysis of DNA from maternal plasma and the titre of anti-D increased to a maximum of 2048 during pregnancy.

Post delivery, the neonate's DAT was strongly positive and phototherapy was necessary for hyperbilirubinemia. Furthermore, the baby had to be transfused for marked anaemia at the age of 17 and 31 days.

Conclusions

Pregnancy-induced primary immunisation may cause clinically relevant HDN in isolated cases.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):42–43.

P02-19 Weak RhD phenotype caused by compound heterozygosity for DAU-2 and a new RHD*c393-394 dupGG-mutation

A Karnot 1, SJ Woestmann 1, BK Flesch 2

Introduction

Weak reactions with different Anti-D sera often are the cause of inconclusive identification of RhD. A considerable number of genetic variants are the moleculare background for these findings. We would like to introduce the case of a new RHD variant.

Methods

The serological RhD determination of a 24 year old female patient showed negative to weak reactions with monoclonal standard anti-sera (Immucor, Optima, Ortho) as well as weak positive reactions tested by indirect antiglobulin test (ImmuClone Anti-D duo IgM + IgG). The patient's RBCs reacted positive with all IgG-sera in the D-Screen panel, negative to weakly positive with IgM-sera (Identification kit for category D, DIAGAST). A conclusive assignment of the D category was not possible. PCR-SSP typing for RHD, RHCE and RHD weak D was performed by commercial assays (RBC-ready gene CDE, RBC-ready gene D weak, Inno-train Diagnostik GmbH, Germany) which led to the assumption of a mutation of the DAU cluster. The molecular background should be evaluated by DNA sequencing of the 10 RHD exons including flanking intron sequences.

Results

DNA sequencing demonstrated a RHD*DAU2 allel (RHD 209G>A, 998G>A, 1136G>T) which causes a very weak antigen expression on the RBCs (1). Additionally, a further, as yet unpublished mutation, RHD*c.393–394 dupGG, was found, in which the two G at positions 393 and 394 were duplicated. The frame shift leads to a premature stop (142X). Because each exon was sequenced independently including short flanking intron sequences it cannot be determined whether the mutation is located in trans or cis towards the DAU2. However, due to the very weak RhD expression the mutation is most likely in trans which indicates 2 independent mutations of both RHD genes. This correlates with a DAU2 phenotype. The mutation was submitted to GenBank (KU859401).

Conclusion

The described mutation induces a frame shift and leads to a premature stop which most probably induces a non functional Rhesus molecule. As the patient expresses DAU2 on her second chromosome we cannot exclude the risk of immunization against the D-antigen.

Reference

  • 1.Wagner FF, et al. The DAU allele cluster of the RHD gene. Blood. 2002;100:306–311. doi: 10.1182/blood-2002-01-0320. [DOI] [PubMed] [Google Scholar]
Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):43.

P02-20 Frequency of anti-D and/or anti-K immunization after transfusion of D- and/or K-positive red blood cell concentrates to D- and/or K-negative patients

M Posset 1, M Solleder 1, N Ahrens 1

Background

Antibody development to red blood cell (RBC) antigens is the most frequent side effect of RBC concentrate transfusion. The frequency is known for D, and it is known for K and other antigens. The immunogenicity of K in relation to D, however, is unknown.

Methods

This is a retrospective single center study of all D- or K-negative patients that were given D- or K-positive RBC concentrates between January 2010 and December 2015. All patients were included that had an antibody screening 4 or more weeks after antigenic mismatched transfusion. Patients with allogeneic blood stem cell transplantation were excluded.

Results

A total of 16% of the 45029 patients were D-negative. Of these, 834 patients received D-positive RBC concentrates, and antibody screening revealed a new anti-D in 72 patients (8.6%). Of these, 41 patients developed additional antibodies apart from anti-D (57%).

A total of 92% of the 45029 patients were K-negative. Of these, 2741 patients received K-positive RBC concentrates, and antibody screening revealed a new anti-K in 43 of these (1.6%). Of these, 17 patients developed additional antibodies apart from anti-K (36%).

Conclusion

Limitations of this study include the retrospective data exploration. Antigenic mismatched RBC concentrates might intuitively have been omitted for patients that are more likely to be immunized. In addition, there was no follow-up for all patients. The frequency of antibody development might therefore be higher than 8.6% for D and 1.6% for K. However, these data indicate independently of these limitations that D is about 5.25 times as immunogenic as K.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):43.

P02-21 Detection of IgM autoantibodies by using a highly sensitive flow cytometry-based dual antiglobulin test

T Bartolmäs 1, A Balola 1, B Mayer 1, A Pruß 1, A Salama 1

Background

Incomplete IgM autoantibodies (aab) in patients with autoimmune haemolytic anaemia (AIHA) are usually not detectable by standard serological techniques. Advanced techniques like IgM-flow cytometry (IgM-FC) or the recently published dual antiglobulin test (DDAT) somewhat improved its detection. Here, we present a new test, called DDAT-FC that resulted in a considerable increase in sensitivity.

Patients and Methods

Patients with cold and warm AIHA were included in this study. Patients’ RBCs were analysed for bound aab by direct antiglobulin test (DAT), dual antiglobulin test (DDAT) and IgM flow cytometry (IgM-FC). A sensitive test (DDAT-FC) combining DDAT and flow cytometry has been developed for the detection of IgM aab. For optimising and comparing the test serial diluted commercially available IgM Anti-Jk(a) has been bound to Jk(a+) RBC. The specifity was tested using IgA- or IgG- coated RBC.

Results

All patients had only positive C3d-DAT and negative IgM-DAT and were suspected of immune haemolysis. Additionally, DDAT and IgM-FC were negative. In contrast, IgM aab were detected in all cases by the new DDAT-FC. In addition, IgM aab could be detected on RBCs of patients with significant cold autoimmune haemolytic anaemia (cAIHA) at 37 °C. The DDAT-FC test for IgM was also highly specific as tested with IgG and IgA-coated RBC.

Conclusions

The new dual antiglobulin flow cytometry test is highly specific and allows the detection of otherwise undetectable IgM aab.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):43.

P02-22 Successful high dose immunoglobulin treatment in a pregnancy complicated by anti-K antibodies

B Mayer 1, K Halle 1, A Salama 1

Background

Kell-immunization in pregnancies is challenging as this antibody may not only cause fetal haemolysis but also induce suppression of erythropoiesis. Management of affected pregnancies usually consists of monitoring of fetal anemia via doppler ultrasound assessment of the fetal middle cerebral artery (MCA) and if indicated, intrauterine. Here, we report on a case of Kell-immunization which was successfully treated with high dose intravenous immunoglobulin (IVIgG) during pregnancy.

Case report

A 34-year old women presented in her second pregnancy for monitoring of a newly diagnosed alloimmunization due to Anti-K. The first pregnancy of the woman was normal. The husband was confirmed as KK.

IVIgG treatment at a dose of 1g / kg body weight was commenced at gestational week 15 and continued weekly until delivery at 40th week. The woman tolerated all IVIgG administrations without any side effects. Repeated assessment of MCA showed no signs of fetal anaemia.

Methods

Serological tests including antibody titers were performed using standard techniques. Genotyping for KEL was performed after DNA-extraction using PCR-SSP.

Results

Antibody-titer for anti-K at thirteen weeks’ gestation was 1024 and remained almost unchanged throughout the pregnancy.

After delivery, the direct antiglobulin test (DAT) was strongly positive and maternal Anti-K antibody could be eluted from the neonate's RBCs. The child's blood group was determined as 0 Rh positive, and Kk was confirmed by genotyping. The newborn had a hemoglobin of 15,7 g/dl and a total bilirubin of 12,68 mg/dl and no therapy for hyperbilirubineamia was necessary. However, shortly after delivery the newborn's haemoglobin gradually declined, with a nadir of 6,9 g/dl at the age of seven weeks. Reticulocytes were within normal limits at that time. No therapy was given and two weeks later, there was a spontaneous increase of the haemoglobin to 10,2 g/dl.

Conclusions

Administration of antenatal IVIgG should be considered in pregnancies at risk for severe courses of red blood cell immunization. This is supported by our case and some other cases reported in the literature

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):44.

P02-23 Auto-anti-Jk(a) induces autoimmune hemolytic anemia in a patient with multiple myeloma

N Greger 1, M Leithäuser 2, V Kiefel 1

A 79-year old male patient with previously diagnosed multiple myeloma developed warm-type autoimmune hemolytic anemia (AIHA). The diagnosis of multiple myeloma was made in January 2015. As CRAB criteria were positive, a therapy with Bortezomib was instituted. Due to serious side effects this treatment was stopped two months later. The patient received outpatient care for the next months. Four months later he developed anemia requiring rbc transfusion. An immunohematological examination revealed anti-Jk(a) in the patient's plasma. The direct antiglobulin test was positive and in the eluate prepared from the patient's autologous rbc again anti-Jk(a) was found. Using specific typing sera, the patients’ rbc typed as Jk(a+b+), this was confirmed by PCR-SSP, using a commercial kit. This complement-activating IgG autoantibody specific for Jk(a) induced an extravasal hemolysis. The patient was transfused with two units of Jk(a-b+) packed rbc. Under therapy with prednisolone, signs of hemolysis disappeared and the concentration of the antibody decreased. Approximately three months later, the antibody was detectable only in the eluate. The general condition of the patient worsened and due to decreasing Hb-levels he again received two units packed rbc. At that time no signs of hemolysis were observed, rbc antibodies were not analyzed at that time. The patient died in November 2015 from complications of multiple myeloma and additional morbidities.

Fig. 1.

Fig. 1

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Diagramm_Hb_Bili.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):44.

P02-24 Role of Eryptosis in Autoimmune Haemolytic Anaemia

B Mayer 1, A Salama 1

Background and objectives

Dyserythropoiesis is a rare phenomenon that may escape recognition in patients with autoimmune haemolytic anaemia (AIHA).

Case report

We describe a patient who developed severe and therapy refractory AIHA of warm type following autologous stem cell transplantation. The direct and indirect antiglobulin tests were strongly positive due to autoantibodies of the IgG-class. Eluates from patient RBCs were also strongly reactive with all tested RBCs. The patient had persisting reticulocytopenia and remained therapy refractory to all drugs used. Furthermore, he needed transfusion of 2 units of packed RBCs per week. A re-examination of his bone marrow morphology revealed the presence of a markedly increased dyserythropoiesis (closely apposed erythroblasts / erythroblastic synartesis). Fortunately, a re-transplantation was possible by using cryoconserved autologous stem cells.

Conclusions

Dyserythropoiesis may occur more frequently than has been suggested, and this phenomenon should be excluded in all patients with AIHA and reticulocytopenia.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):44.

P02-25 Intrauterine transfusion to treat fetal anemia: clinical outcomes and immunhematological conseqeunces- a single center experience

S Nowak-Harnau 1, S Enkel 1, B Zimmermann 1, KO Kagan 2, T Bakchoul 1

Introduction

Maternal alloimmunization against fetal red cell antigens is the most frequent cause of fetal anemia, mainly due to Rhesus antibodies. Although a national wide screening and prophylaxis program for immunization against Rh-D is established, clinically relevant cases of morbus haemolyticus neonatorum (MHN) are not a rare finding. The management of fetal anemia includes intrauterine fetal transfusion (IUT) and early delivery when moderate prematurity is at less risk of impact on the status of the newborn.

Methods

To assess the efficacy and safety of IUT in the management of fetal bohemian caused by MHN and non-immunohematological disorders we analyzed retrospectively patients managed for severe fetal anemia at a university hospital.

Results

Twenty-three pregnancies were managed by IUT. Indication and timing for the IUT were based on Doppler measurement of peak systolic velocity in fetal middle cerebral artery to predict severe fetal anemia. A total of 88 IUTs have been performed to treat MHN (group I, n = 8 cases), infection-associated anemia (Group II, n = 15 cases).

In group I, antibody differentiation revealed IgG antibodies against D and C (n = 5) and Kell (n = 3). First IUT was performed on a median gestation age of 21 weeks (range 18- 32 weeks). During pregnancy, fetus required a median of 7 IUTs (range 3–11 IUTs). No significant increase in antibody titer was observed (anti-D: 8 fold increase (FI), anti-C: 2 FI and anti-Kell: 2 FI). Two mothers developed new antibodies against C-antigen. Elective delivery procedure was induced before 39 weeks of gestation (range 36–38 weeks). After birth, hemoglobin values of the newborns ranged from 13.4–16.5 mg/L. Postnatal transfusion was required in two cases. In group II, 18 IUTs were performed (range 1–2 IUTs/case). Gestation age was shorter in this group than in group I (median: 19, range 18–32 weeks of gestation). Procedure-associated adverse events after IUT have been reported in one case with vasooclussion of umbilical artery leading to emergency caesarean section. None of the newborns developed graft versus host disease or transfusion-associated CMV infection after birth.

Conclusion

Intrauterine transfusion seems to be an effective and save procedure to treat fetal anemia. The results of our study provide better insights in the short-term outcome of IUT, and may improve the quality of antenatal consulting.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):44–45.

P02-26 Erythropoietin in the treatment of decompensated autoimmune haemolytic anaemia

B Mayer 1, A Salama 1

Background and objectives

Haemolysis may frequently decompensate in some patients with autoimmune haemolytic anaemia (AIHA) of warm or cold type.

Patients and Results

During the last few years, we have treated several patients with decompensated AIHA of warm or cold type with erythropoietin. In almost all of the treated patients, the haemolysis decreased or could be somewhat prevented, i.e. in patients with cold type AIHA during winter.

Conclusions

Erythropoietin should be considered in patients with decompensated and / or therapy refractory AIHA

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):45.

P02-27 Introduction of the P1/P2 genotyping into blood donor screening and routine diagnosis

J Portegys 1, G Rink 1, L Colic 1, V Klümpers 1, S Rothenberger 2, E Richter 2, EA Scharberg 2, P Bugert 1

Background

Fast and efficient blood group genotyping is an indispensable diagnostic tool for transfusion medicine, as it can prevent severe transfusion reactions. When serologic results are unclear or a high throughput assay is needed, genotyping may be useful to determine blood groups. The aim of the project was the validation of analytical methods for P1/P2 blood group genotyping. The molecular mechanism for the formation of the P1/P2 blood group phenotypes was correlated with the genotypes of two single-nucleotide polymorphisms (SNPs), rs8138197 and rs2143918, in the A4GALT gene. The two SNPs are completely linked, thus, genotyping of one of the two should be sufficient for the correct prediction of the P1/P2 phenotypes.

Methods

For the development and validation of P1/P2 genotyping assays 20 serologically defined blood donor samples were subjected to DNA extraction and subsequent Sanger sequencing of the A4GALT intron 1 region. PCR-SSP and TaqMan-PCR methods were developed for both SNPs. All samples were analyzed by using the PCR assays and the results were confirmed with the serological phenotype.

Results

All genotyping results from sequencing, PCR-SSP and TaqMan-PCR were in full concordance with each other and with the serologically defined P1 and P2 phenotype. I.e., in 7 of the 10 P1 samples both SNPs were heterozygous for the two SNPs and 3 of the 10 samples were homozygous for the major allele. All P2 samples were homozygous for the minor SNP allele.

Conclusion

As expected from previous data, genotyping of either the rs8138197 or the rs2143819 SNP in the A4GALT gene is sufficient to predict the P1/P2 phenotype. We validated PCR-SSP assays that will be implemented into routine diagnosis of patient samples and TaqMan-PCR assays that will be used for screening of blood donors in a high throughput setup.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):45.

P02-28 Limited lifespan of granulocytes: influence on granulocyte-reactive antibody testing

A Tolios 1, M Schönbacher 1, G Körmöczi 1

Background

Granulocyte-reactive antibodies may cause immunohematologic disorders like immune neutropenia and transfusion-related acute lung injury (TRALI). Assays like the microscopic granulocyte immunofluorescence test (GIFT), the granulocyte aggregation test (GAT) and the flow cytometric white blood cell immunofluorescence test (WIFT) are valuable tools for the detection of HNA and HLA antibodies. Since granulocytes have a very limited lifespan, this study evaluated the delay tolerable for test cell isolation.

Materials and Methods

Serum from eight patients with known antibodies against HNA-1b, −2, −3a as well as HLA class I was used. Three antibody-negative samples served as controls. For cell isolation, blood from three healthy probands expressing the corresponding antigens was drawn. Leukocytes were isolated 0, 4, 8 and 24 h after venipuncture using dextran sedimentation. Granulocytes were isolated using Ficoll density gradient centrifugation followed by classical GAT and GIFT testing. WIFT was done as described (Heinzl M. et al. 2015) including 7-amino-actinomycin-D (7-AAD) cell viability testing.

Results

Compared to instantly isolated cells, no significant changes in the isolation efficiency were observed within the first hours (83–85% granulocytes for GAT/ GIFT after 0, 4 or 8 hours), compared to a marked decrease after 24 h (51%). In GAT, aggregation was visibly reduced after 4 and 8 h, although sample evaluation was not impaired, whereas after 24 h, only 7/9 GAT assays falsly showed no aggregation. In GIFT, samples could be correctly identified up to 8 h of isolation delay. After 24 h, 9/12 samples could be identified correctly. Using WIFT, even after 24 h no false-negative samples were observed. An increase in 7-AAD-positive granulocytes was observed in a time-dependent manner (3.9%, 6.5%, 7.2%, 11.0% dead cells after 0, 4, 8 and 24 h, respectively).

Conclusion

The time between blood draw and granulocyte isolation is most critical for GAT, with weaker aggregation already after 4 h. However, correct GAT and GIFT results were obtained when isolation was performed up to 8 h post venipuncture, by WIFT even after 24 h. Hence, blood samples drawn for cell isolation may be collected and shipped to a specialized laboratory within an interval of 8 h.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):45.

P02-29 A case of Anti-Er3

SJ Woestmann 1, I Marais 2, NM Thornton 2, BK Flesch 3

Introduction

Identifying antibodies to antigens of very high prevalence depends on the availability of rare rbc and antisera. The major challenge arises in the recurrent supply of blood units to a patient with chronically progressive anaemia regarding the risk of further immunization. Samples from a 67 year old female patient were investigated when an additional antibody to high prevalence antigen was suspected.

Methods

Antibody tests were carried out by IAT with all test cells in the gel-card technique (Diamed-ID-System and Capture R-Ready-ED) and papainized test cells (Diamed-ID-System). The crossmatches were carried out as tube tests IAT. The patient's blood sample was submitted to the IBGRL in Bristol where LISS tube IAT was used for antibody identification and rbc typing.

Results

The patient is AB, RhD+. Anti-P1 and anti-S were present in the patient's plasma. IBGRL determined the presence of an additional antibody, identified as anti-Er3. All cross matches by tube IAT were weak positive. The patient's plasma was reactive with all screening and identification panel cells, the autologous control and DAT were negative. Only Er(a-b-) [P1-, S-] cells were found to be compatible and Er(a-b+) cells were incompatible with the patient's plasma. The patient's cells were found to be Er(b-). Extremely weak reactions (detected microscopically) were observed with one example each of anti-Era and anti-Er3. Due to the rarity of antibodies and the patient's AB blood group no further anti-sera (anti-Era nor anti-Er3) were available for typing the patient's cells. The molecular basis of the Er antigens is currently unknown, therefore no DNA based test could be utilized to predict phenotype. Autoadsorption/elution studies showed no evidence to indicate an autoantibody.

Conclusion

Anti-Er3 is extremely rare and information regarding clinical significance is limited. The antibody present in the patient's plasma is best described as anti-Er3 since only Er(a-b-) cells were compatible. However, the patient's Er(a+wkb-) Er3+wk phenotype could indicate that the patient's cells lack an undiscovered Er-related high prevalence antigen that alters the Era and Er3 epitopes. The supply of blood products for patient's with anti-Er3 remains extremely difficult as there are no known Er(a-b-) donors on the International Rare Donor Panel.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):46.

P02-30 Role of eryptosis in autoimmune haemolytic anaemia

B Mayer 1, T Bartolmäs 1, A Balola 1, A Salama 1

Background and objectives

Until now, eryptosis has not been described in autoimmune haemolytic anaemia (AIHA).

Patients and Methods

Red Blood Cells (RBCs) from healthy volunteers (control) and from patients with AIHA of warm and cold type have been examined for the presence of phosphatidylserine (PS) by annexin binding flow cytometry assay.

Results

Significant annexin binding on RBCs was demonstrated in all patients with AIHA of cold type and only in isolated cases with AIHA of warm type.

Conclusions

Eryptosis appears to play a key role in AIHA of cold type, and only infrequently in AIHA of warm type. Gruppe III: Herstellung von Blutkomponenten

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):46.

P03-1 Quality of fresh-frozen plasma after rapid thawing and following storage at 4°C

M Störmer 1, V-M Petrescu-Jipa 1, L Oustianskaia 1, S Radojska 1, B Gathof 1

Background

The thawing procedure of fresh frozen plasma (FFP) may cause delay in delivery of fluid plasma ready for transfusion when acute, massive transfusions are demanded. To circumvent the delay in the thawing procedure a technology using radio waves (RWT) and the cold storage of plasma in fluid phase were validated. Two modes of thawing procedures were applied in the present study to compare thawing time and effect on coagulation factors during prolonged storage at 4°C.

Methods

Plasma was collected from 80 blood donors. Twenty identical plasma units were generated by pooling of four units and following splitting into equal aliquots. The plasma units were shock frozen and stored below −30°C for at least one week before thawing by using the plasmatherm (Barkey GmbH & Co. KG, Leopoldshoehe, Germany) and the transfusio-therm 2000 (EIC Umwelt- und Medizintechnik Ltd., Heilbad Heiligenstadt, Germany) followed by 8 day storage at +4°C. Samples were taken before freezing, immediately after thawing (d0), and after storage (d7) at +4°C for analysis of coagulation factors FV, FVII, FVIII, and FXII on the BCS (Siemens Healthcare Diagnostics, Erlangen, Germany). FVIII was additionally analyzed before freezing and on days 0,1,4,5,6,7, and 8 using the COAMATIC Faktor VIII Kit (Chromogenix, Bedford, USA).

Results

The thawing time for two FFPs per run were 8.21 ± 0.46 min using the transfusio-therm and 33.00 ± 3.28 min using the plasmatherm. All thawed FFPs were clear and free from visible aggregates. Moreover, no effect on all analyzed coagulation factors could be observed immediately after thawing. The factor levels were comparable to the values before freezing in both groups. After storage of 7 days FVIII showed a loss of ~40%, FV and FVII of ~20%, and FXI showed no loss of activity. Most of the FVIII loss could be observed already after one day of storage at 4°C followed by a steady decline during storage.

Conclusion

The radio frequency heating technique represents an attractive process in comparison to thermal transfer using heating pads due to rapid and uniform heating with small temperature gradients. The delay in delivery of fluid plasma can be tremendously reduced without having influence on the plasma quality.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):46.

P03-2 Cutting-edge of cord blood banking in Germany: Closed Sepax® system in routine

S Raab 1, A Schmidt 1, A Platz 1

Introduction

The DKMS Cord Blood Bank (total Cord Blood Unit (CBU) inventory March 2016: 8825) is the first Cord Blood Bank in Germany processing Cord Blood in a closed system using the cell processing device Sepax®/Biosafe. Furthermore the procedure is applied without additives like HES. A cleanroom is not required anymore as it was performing in an open system.

Methods

Analysis of 844 cryopreserved CBUs from two years (02/2014–02/2016) using the single-use kit CS-540.4 focusing on recoveries dependent on input volume. Comparison of recoveries to the open system using Sepax®/Biosafe with the single-use kit CS-430.1.

Results

A total nucleated cell (TNC) recovery of 77.8 ± 10.2% and mononucleated cell (MNC) recovery of 89.9 ± 6.8% could be achieved with the closed Sepax® system. Performance differs slightly compared to the open Sepax® system (TNC recovery: 72.9 ± 11.4%, MNC recovery: 86.8 ± 7.8%, n = 458).

The analysed CBU processed in the closed system have a mean input volume of 124.8 ± 24.4ml. The higher the input volume the less the TNC recovery (see table):

Conclusion

We could demonstrate reliable und reproducible results using Sepax® with a closed system without a sedimentation accelerator. The method is stable and shows acceptable values. Comparison of the TNC recoveries between the open and closed system show a slight improvement of the new method. Always keep in mind that the smaller the input volume, the better TNC recovery can be achieved.

Tab. 1.

Dependency input volume - recovery

volume [ml] amount of CBU TNC recovery [%] MNC recovery [%]
< 100 115 85.0 90.1
≥100, <140 523 78.9 90.4
≥140 206 71.1 88.5
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Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):46–47.

P03-3 In vitro function of triple dose apheresis platelet components suspended in 40% plasma/60% PAS after photochemical treatment using a triple storage (TS) set

F Kahlenberg 1, A Wahler 1, B Brys 1, JM Payrat 2, S Andresen 2, J Thierbach 1, B Baumann-Baretti 1

Introduction

Although the risk of viral transfusion-transmitted infections (TTI) from recognized pathogens is low today, bacterial contamination and emerging pathogens remain a threat to transfusion safety. The INTERCEPT™ Blood System was developed to prevent TTI by inactivating pathogens utilizing amotosalen in combination with UVA light. The triple storage (TS) set has been designed to treat double or triple dose platelet donations and is currently in development. This study evaluated whether the residual amotosalen levels and in vitro function of pathogen inactivated platelets processed using the TS set were functional when compared to untreated controls over 7 days storage.

Methods

12 triple dose platelet donations from normal volunteer donors were collected using Trima 6.0 automated blood collection system (Terumo BCT). Platelets were suspended in 40% plasma/60% SSP+, pooled and split to represent a triple dose (9 to 11 × 1011 platelets) for INTERCEPT treatment and a paired untreated platelet single (~ 3 × 1011 platelets) as control. Platelet function was evaluated on Day 1, 5 and 7.

Results

Platelets treated with amotosalen had in vitro function comparable to control. The pH (22°C) was 6.79 ± 0.14 (d1) to 7.03 ± 0.07 (d7) in test but slightly lower compared to control. The level of pO2 was stable, while the level of pCO2 dropped consistently during storage (pCO2 treated platelets 44.83 ± 1.55 mmHg [d1] to 17.33 ± 2.58 mmHg [d7] and control 25.33 ± 2.66 mmHg [d1] to 17.67 ± 1.97 mmHg [d7]). The concentration of HCO3- decreased during storage but more profoundly in the treated group (treated platelets 6.33 ± 0.52 mmol/l [d1] to 4.33 ± 1.03 mmol/l [d7] and control 7.17 ± 0.41 mmol/l [d1] to 7.17 ± 0.75 mmol/l [d7]).

The aerobic metabolism pathway (ATP) remained intact (treated platelets 10.30 ± 1.4 µmol/dl [d1] to 8.78 ± 1.65 µmol/dl [d7] and control 11.43 ± 1.43 µmol/dl [d1] to 10.70 ± 0.82 µmol/dl [d7]). There was no difference in response to hypotonic stress. Treated platelet units showed a slightly increased expression of activation marker CD62 (47% versus 36% for control at Day 7). Platelets maintained their ability to be activated by ADP and TRAP agonists. After 7 days of storage, all tested units were negative in bacterial cultures.

Conclusion

Pathogen inactivated platelets processed using the TS set retained adequate in vitro function for up to 7 days and met the criteria of the “Council of Europe Recommendation N°.R(95) 15” as well as the German guidelines.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):47.

P03-4 First experiences with a new Waferless Sterile Tube Welding Device (WSTW)

H Jungk 1, R Tryankowski 1, U Heil 1, W Madla 2, A Opitz 1

Introduction

Sterile tube connection procedures are a well-established technique for manufacturing tube connections without compromising the sterility of the product. The Genesis RapidWeld™ STW Device, is a waferless sterile tube welding device that utilizes a non-contact infrared heating source to yield accurate welds without the requirement of a heated consumable copper wafer. The GMP compliant qualification procedures and the first experiences with the new welding device are being demonstrated under operative manufacturing conditions.

Methods

The qualification of the WSTW followed the valid Annex 15 of the EU-GMP-guidelines including the risk analysis assessment according FMEA. The qualification was subdivided into the phases DQ (design qualification), IQ (installation qualification), OQ (operating qualification), and PQ (processing qualification). The OQ was split into 3 phases. Following a successful education and training phase, the staff of the preparation department performed 100 wet to wet, 100 wet to dry and 100 dry to dry tube welds with the Genesis RapidWeld. The tensile strength of 90 dry to dry tube connections were measured by an independent laboratory according to ISO3826. Secondly the manual handling and performance of the WSTW device was rated by the staff in the form of a questionnaire. The final phase of the OQ was defined by evaluating the handling and performance of production of pooled platelet concentrates under routine operating conditions.

Results

The leak test was performed efficiently on the entire count of welds. The total number of tube connections met the requirements for the tensile strength (> 20 N for > 15 seconds) according to ISO3826. The usability, manual handling and performance of the WSTW device was rated very good by all staff members. The pooled platelet concentrates produced fulfilled all required specification criteria regarding platelet count, purity and sterility. The testing for sterility was negative for all pooled platelet concentrates.

Conclusion

All tube connections were tested and were 100% leak proof. The WSTW, Genesis RapidWeld Device obtains analogous solid results in welding tube connections compared to conventional tube welding devices. The advantage of the WSTW lies in the savings of huge amounts of consumable supplies.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):47.

P03-5 When platelets become sour … Unusual big pH drop at the end of storage time in pathogen reduced platelet concentrates of several consecutive donations from the same donor

T Humbel 1, T Weingand 1

Introduction

Platelets are metabolically active. Due to the isolated environment in a platelet concentrate (PC), pH-value drops during storage time. This decrease is more intense in pathogen reduced (amotosalen/UVA) platelet concentrates (pPC) than in native, non treated platelet concentrates (nPC) [Picker et al., Transfusion, 2004; Moog et al., J Clin Apher 2004]. Pathogen reduction in PC is mandatory in Switzerland, for now, there is only one approved method (Cerus Intercept, amotosalen/UVA). According to the applicable regulations in quality control, the pH-value of at least one percent of all produced PC has to be measured at or after the end of maximal storage time of 7 days (acceptance limit pH > 6.4).

Case presentation: We report about three consecutive apheresis donations from the same donor (donation 1: 29.12.2014, donation 2: 09.02.2015, donation 3: 23.03.2015). Two units of pPC were produced from each donation of the donations 1 and 2. All four units dropped < pH 6.3 on day 8 after donation. Donation 3 was taken solely for research purposes (in consent with the donor). Three products were made from donation 3: one pPC, one nPC and one plasma unit. All three products have been stored at 22°C, the PC were stored on the platelet agitator. pH-values from all three products were taken daily except on day 6 after donation. pH from pPC was massively below acceptance limit for PC at the end of maximal storage time (day 7: pH 6.1, day 8: pH 6.1) while pH-values from nPC and plasma remained > 6.4. Since the introduction of the pathogen reduction procedure in our blood donation service, a shortfall of the pH acceptance limit never occurred except in this mentioned case (total number of pH measurements in pPC until December 2015: 90). Our donor was healthy, fully suitable for donation and took no medication. There were no known metabolic diseases, platelet pathologies or coagulation disorders. pH measurement in pPC from his daughter showed normal values (pH > 6.57 on days 5 to 8 after donation).

Discussion and Conclusion

The reason for this multiple pH drops in pPC of consecutive donations from the same donor remains unclear, but it seems reasonable to assume that it is connected to the pathogen reduction procedure since pH-values of nPC and plasma were above pH 6.4. Regular pH measurements in PC at the end of storage time are essential to indentify similar cases and possibly get to the bottom of the phenomenon that occurred in the pPC of our donor.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):47–48.

P03-6 Apheresis procedures can be reflected by the platelet volume

A Bramhoff 1, G Giers 1, J Fischer 2, F Blessing 3, F Wenzel 4

Introduction

Referring to current standards the quality of an apheresis procedure is estimated by the quantity of collected cells. Nowadays a new kind of quality measurement could be found in the detection of cell volumina. Recent diagnostics have shown that stem cells and platelets - when separated - are likely to appear in a higher volume inside the cell product. Therefore, in this study the question should be discussed wether platelets of higher volume are more likely to be separated than platelets showing a lesser volume.

Methods

Blood samples of three different apheresis procedures could be observed: allogenic platelet donations (n = 8) (Trima, Terumo), autologous (n = 6) and allogenic stem cell donations (n = 6) (Cobe Spectra, Terumo). To examine the blood samples the Sysmex hematology analyser (XT-2000) has been used.

Results

During stem cell apheresis, the volume of the separated platelets was 1.2fold increased compared to the platelet volume in the peripheral blood before separation. Before apheresis the mean platelet volume in the peripheral blood was found to be 6,32 fl, after apheresis 6,14 fl and inside the platelet concentrate 7,54 fl. The platelet number in the peripheral blood was also significantly decreased (before separation 180.8/nl and after separation 134.9/nl). In the blood products the concentration of platelets was 7,86fold higher than in the peripheral blood before separation.

Conclusion

Overall, the observed apheresis procedures are more likely to separate platelets showing a higher voulme than common in the peripheral blood. This might indicate that not only the amount of separated cells reflects the quality of the apheresis procedure but also that the volume of the separated cells can be used as a parameter for quality assessment.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):48.

P03-7 First comparative analysis concerning the plasma platelet contamination during MNC collection using three different cell separators

H Pfeiffer 1, J Strobel 1, R Zimmermann 1, R Eckstein 1, EF Strasser 1

Background and objectives

Monocytes can be cultured into dendritic cells with addition of autologous plasma, which is highly prone to platelet contamination due to the apheresis process. Our goals were to analyze whether the type of machine or the set used had an effect on the number of contaminating platelets in the concurrently collected plasma and if a high platelet pre-donation count in the donor led to a high platelet yield in the donated product.

Materials and Methods

The MNC program was compared using three different apheresis devices with different sets: COM.TEC with either the P1Y- or C4Y-set, Cobe Spectra with the P1Y-set, Spectra Optia with either the IDL- or MNC-set. A total of 103 MNC collections from random blood donors were analyzed and 41 single-donor pairs were formed for statistical analysis. A prospectively-paired statistical analysis was performed with the new machine (Spectra Optia, n = 9 single-donor pairs). Concurrent plasma collected during leukapheresis was analyzed for residual cell contamination. Donor pre-donation counts of platelets were analyzed for their predictive value of contaminating platelets in plasma harvests.

Results

The Spectra Optia showed the lowest residual platelet count of the collected concurrent plasma (mean 8.50 × 103/μl) independent of pre-donation counts. The Cobe Spectra showed a 27.6 times higher platelet contamination, and the COM.TEC showed a 23.2 to 26.4 times higher platelet contamination, depending on the set. The plasma contamination with other residual cells was very low (only 2.0% had leukocytes higher than 0.5 × 103/μl).

Conclusions

The study showed that plasma collected by long-established machines during leukapheresis procedures was prone to PLT contamination. The newest device showed the lowest platelet contamination. Because platelets affect the maturation process of monocytes into dendritic and might even lead to a diminished harvest of dendritic cells, it is crucial to reduce the platelet contamination.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):48.

P03-8 Influence of irradiation on leukodepleted small unit red blood cell (RBC) bags for infant transfusion in additive solution SAG-M

B Hauck-Dlimi 1, K Schiffer 1, R Eckstein 1, J Strobel 1, R Zimmermann 1

Aim

There are sparse data about the effect of irradiation on leukoreduced RBCs stored in SAGM that are subdivided to use them for the transfusion to preterm infants and small children.

Method

We studied 30 leukoreduced SAGM-preserved RBCs. The units were divided in 2 groups. Every unit was divided into 3 bags. One of these bags served as unirradiated control, the other 2 were irradiated at different times. In vitro evaluation of irradiated and non-irradiated units was performed on the Days +3, +7, +14, +21, and +28 from the collection.

Result

Gamma irradiation induced a higher increase of extracellular hemoglobin, LDH and potassium than non-irradiated storage over the time. Potassium (as an early marker of quality loss) is shown in the figure. No irradiated or non-irradiated unit showed a hemolysis rate over the recommended limit of 0.8% until day +28.

Conclusion

Our findings show, that subdivision of RBCs does not have an appreciable influence on the storageability of leukoreduced, irradiated RBCs in AS SAGM. Our “worst case scenario” was irradiation on day +3 after donation and subsequent storage until day +28. Especially for infant use it is important to have the possibility to irradiate also late after donation, because this procedure offers the possibility to use one RBC over a longer period of time and to reduce the donor exposition for infants. Therefore subdivided leukoreduced RBCs can be safely irradiated until day +14 and subsequently stored until day +28 after donation.

Fig. 1.

Fig. 1

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shows potassium levels from day +3 to day +28 in all groups.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):48–49.

P03-9 First-time determination of hemoglobin content in packed red blood cells non-invasively in the blood bank

U Netz 1

Introduction

Currently no method exists to non-invasively determine the hemoglobin (Hb) content in red blood cell concentrates (RCC). LMTB has developed a new optical method with which the hemoglobin content of a unit can be determined. After the successful tests with the new demonstrator under laboratory conditions [1], the tests were extended to measurements of RCC at the blood bank.

Method

The tests were performed at expired RCC in the routine quality control process at the blood bank of Charité Campus Virchow-Klinikum. In total, 75 measurements of 45 RCC have been performed at 9 days, 32 in bags from Macopharma and 13 in bags from Fresenius. A number of 30 RCC were measured a second time in smaller bags from Fresenius intended for microbiological testing. The RCC is irradiated through the bag film in the lower label-free area of the bag with light from selected wavelengths. For technical reasons, only one of two wavelengths could be used. For quality control and determination of the Hb value as a reference, a sample was taken from the bag content and the standard blood count (ABX pentra XL80) was performed. From the measured light attenuation and the reference Hb value an algorithm is derived to calculate the Hb value from the measurements. The optical measurement in no way affects the quality control of the blood products.

Results

The mean value of the Hb concentration was 19.0 (16.5 – 21.8) g/dl, of the volume was 310 (238 – 345) ml and of the calculated Hb content was 59 (39.3 – 72.6) g. The determined accuracy (1.96 × σ) is ± 2.4 g/dl for all measurements. We found that in some cases for RCC with low Hb reference the calculated Hb was abnormally high. These measurements have been taken at only one out of 9 days. Assuming a systematic error, the removal of these measurements leads to ± 1.8 g/dl or ± 9% regarding the mean of 19.0 g/dl. No significant dependency on the manufacturer of the bag was found.

Conclusion

With the new technology it was possible to non-invasively determine the Hb content of expired RCC in a blood bank for the first time. The achieved accuracy is promising in respect to the planned improvement of the device with regard to the evaluation in the production process of blood products. This method will open up new possibilities in the quality control of RBC and facilitate the dosimetry of hemoglobin administration for transfusion.

Reference

  • 1.DGTI Kongress Basel, 15.-18.9.2015, Poster P-08-1.
Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):49.

P03-10 Aggregates in apheresis platelet concentrates can be removed by filtration while preserving quality

R Offner 1, AM Brosig 1, V Hähnel 1, F Dormann 1, N Ahrens 1

Introduction

Platelet aggregates are known to occur in 1–2% of apheresis platelet concentrates (PC) and cause specification failure. We asked therefore, if inline-filtration could remove aggregates without compromising platelet quality.

Study design and Methods

We developed a filtration method in a closed system with sterile welding using a 40 µm filter (Cell-Max) and an additional PC storage bag (Terumo BCT). Platelet concentration and content, pH, sterility, CD62P expression, CD61-positive extracellular vesicles (EV) as well as platelet quality (ThromboLux, LightIntegra Technology) were investigated in filtered PC in parallel with non-filtered twin products from double PC donation immediately after filtration, after two hours agitated storage, and at the end of product shelf life.

Results

Filtration removed aggregates completely without significantly effecting PC quality. Median platelet concentration, CD62P expression, EV, and ThromboLux (TL) scores were: 1111/nL, 12.8%, 3.7%, and 22, respectively, two hours after filtration, and 987/nL, 11.4%, 4.3%, and 20 in the non-filtrated twin products (each n = 2). Post-filtration PC met all specification criteria and can be released for clinical use.

Conclusion

Filtration of PC with 40 µm filters proved to be feasible without loss in product quality. The filter also removes aggregates, which would pass through the usual transfusion equipment (200 µm). Filtration could be performed fast (10 min) and cost-effective, thus enabling release of aggregate-containing apheresates that otherwise would have to be discarded and which should be replaced through purchase from another blood suppliers.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):49.

P03-11 Evaluation of the efficiency of the Sepacell™ PLX-5 in-line filter regarding leucocyte removal and platelet recovery in buffy coat derived platelet concentrates

M Störmer 1, S Radojska 1, B Gathof 1

Introduction

Leukoreduction of platelet concentrates (PCs) prior to storage is performed by filtration to achieve a reduction of white blood cells (WBC) to a certain threshold with minimal platelet loss. In Germany the limit of residual WBCs in PCs is < 1 × 106/Unit. Here we present the evaluation of the Kanbarrier Pooling Set (Kansuk Laboratuari) including the hard housing Sepacell™ PLX-5 leukocyte removal filter (Asahi Kasai Medical) for routine production and storage according to the German authorities’ requirements.

Methods

In total, 75 Buffy Coats (BC) were needed to produce 15 BC derived PCs (PPCs) made from 5 BCs in additive solution (InterSol, Fresenius Kabi). A hemogram was performed on the Sysmex (Sysmex Europe GmbH) before BC Pool filtration to obtain WBC and PLT counts. Quality parameters including volume, residual WBCs and red blood cells, platelets (PLT), haematocrit, platelet aggregation and sterility were assessed after production and at the end of storage.

Results

In total 15 PPCs with a mean volume of 342.33 ± 10.18 ml and yield of 3.71 ± 0.62 x1011 PLT/Unit were produced. The mean filtration time was 7.05 ± 0.60min. The WBC removal and PLT recovery results are shown in the table. During storage a loss in platelet aggregation of 67% after activation with TRAP-6, a fall in pH from 7.09 ± 0.06 on day 1 down to 6.49 ± 0.10 on day 5, and decreased swirling scores were observed. All PCs were tested negative for microbial contamination at the end of storage.

Conclusion

The Sepacell™ PLX-5 filter proved to be suitable for filtration of PCs due to efficient WBC depletion without loss of PLTs. The low pH of stored PCs might be associated with high metabolism or inadequate oxygenation. Therefore special focus should be taken on the relationship between the chosen container, the composition and the amount of the additive solution, platelet yield and volume, and the positioning of the bag in the incubator in further studies.

Tab. 1.

WBC Removal and Platelet Recovery

BC Pool before filtration PPC after filtration
Haematocrit [%] 22.49 ± 1.38 0,10 ± 0,00
WBC/μl 15346.67 ± 2263.02 0,01 ± 0,03
PLT x103/μl 1018.80 ± 106.54 1085.47 ± 198.78
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Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):49–50.

P03-12 Collection of fresh frozen plasma (FFP) with plasmapheresis device AURORA

F Kahlenberg 1, B Brys 1, A Dürrenfeld 2, N Kelle 2, E Ulrich 1, B Baumann-Baretti 1

Introduction

The preservation of the coagulation factors and functioning are mandatory. This study aims to demonstrate that fresh frozen plasma (FFP) obtained by plasmapheresis at the Aurora corresponds specification. We investigated the clotting factor stability according to the varying time of storage and in comparison with individual citrate blood tube of donors.

Methods

9 plasmapheresis at the Aurora, Fresenius (version 1.3.), using the collection set S4R2252C were performed. For each apheresis 3 FFP (n = 9) were produced. Additionally citrate blood was removed from donation system and donor blood samples were taken in separate tubes. Tubes were frozen within 2 hours. The coagulation factor activity of FVIII, INR and aPTT were analyzed in FFP and citrate tubes according to production and storage. Additional coagulation factors (FV, FXI and Fibrinogen) were measured just in FFP using the BCS (Siemens). Residual cell content was analyzed by Flow Cytometry (Facs Calibur, BD).

Results

The mean residual cell count (RBC, WBC and Plt) was well below the required limit within the specification. The mean FVIII activity of FFP after preparation (1.53 ± 0.24 IU/ml) and after storage for 1 month (1.52 ± 0.20 IU/ml) is provided in normal range; after storage of 1 month the mean recovery of FVIII showed 99% of the initial value. The mean FVIII activity measured in donor single citrate blood tubes revealed a considerably higher activity with 1,77 ± 0,34 IU/ml. During storage aPTT and INR slightly increased within the norm: the mean aPTT from 28.68 ± 1.04 s to 31.30 ± 1.07 s and the mean INR from 0.94 ± 0.02 (0.91 to 0.99) to 0.97 ± 0.02 (0.95 to 1.01) in FFP. In individual citrate blood tube aPTT of 29.58 ± 3.60 s and INR 0.93 ± 0.04 were measured. All tested units were negative in bacterial cultures.

Conclusion

The residual cell concentrations are in accordance with the specification and were met in all tested samples.

The comparison of FVIII, aPTT and INR between donor samples and FFP demonstrated that the actual coagulation activity of the donor is well kept in FFP and no activation of coagulation parameters is seen. Although compared with previous studies, there is a slightly increased F VIII activity within specification. Overall, the results of tests on quality control samples of frozen plasmapheresis fresh plasma using the Aurora device met the criteria of the “Council of Europe Recommendation N°.R (95) 15” as well as the German guidelines.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):50.

P03-13 App to optimise platelet donor approval

R Offner 1, M Mohrez 1, I Pamler 1, N Ahrens 1

Introduction

Platelet donors may not undershoot post-donation counts of 100.000/µL according to German regulations. Donor approval for double platelet concentrate donation is therefore commonly restricted to pre-donation counts of 220.000/µL or above and disregards the donor's total platelet count. We asked therefore, if additional donors could be approved by taking height and weight into calculation additionally.

Study design and Methods

Height, weight, gender and the predonation blood count was used to calculate predicted donation time with a software tool Trima Prediction of Donation (T-POD, Terumo BCT) that was set up to allow a maximum of a 90 min. donation time with Trima Accel or Amicus as apheresis devices. Platelet target yields were 5.4 × 10^11.

Results

T-POD software provides identical apheresis duration time predictions as Trima with its 6.0 software and Amicus with its 3.2 software. In this study, 12 donors were included with median predonation platelet counts of 210.400/µL (range 197.000/µL to 218.000/µL). None of these donors would be eligible for double platelet concentrate donation based on simple platelet concentration as approval parameter. The donors’ (all 12 men) weight was 86,5kg and their size 183,2cm. All donors (men) gave double platelet concentrates with median platelets of 2,52*10^11/unit (range 2,35 to 2,70). All post-donation counts were in the allowed range (median 140.200/µL; range 101.000/µL to 172.000/µL).

Conclusion

Donor approval with platelet calculation based on blood count, height and weight allowed successful double platelet concentrate donation in all tested donors, provided predonation platelet counts were 200.000/µL or above. This translates to a predicted estimated 2.5% increase in our production.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):50.

P04-1 Characterisation of Parvovirus B19-positive Swiss blood donors

P Gowland 1, N Widmer 1, M Stolz 1, C Niederhauser 1

Introduction

Human parvovirus B19 (B19V) a common human pathogen that causes a variety of diseases with outcomes ranging from an asymptomatic or mild childhood disease to severe symptoms, especially in immunocompromised patients. Though transmitted mainly by a respiratory route, B19V transmission via blood and blood products has been reported. Molecular screening of blood for B19V (NAT) is not required for the release of labile blood products but is performed to prevent a high B19V contamination of plasma manufacturing pools. Our routine B19V NAT of Swiss blood donations began in 2008. Here we present an extensive characterisation of B19V identified between 2008–2015.

Methods

B19V NAT is performed in pools of up to 480 donations on a twice weekly basis with an in-house quantitative PCR assay. Donations with viral loads >1E+6 IU/ml B19 DNA are removed from the transfusion process if not yet transfused. Look-back is conducted when necessary. B19V serology (IgG and IgM) was performed with commercial assays. Viruses were characterized by sequencing and phylogenetic analysis revealing genotypes and novel virus variants.

Results

A total of 70 B19V positive blood donations from 2'119'520 were identified exceeding >1E+6 IU/ml cut-off in the pool analysed. The overall frequency was 1:30'278 donations; in 2013 this increased to 1:6'368 donations. The majority had viral loads exceeding 1.0E+08 IU/ml (median: 2.49E+10 IU/ml; range: 5.86E+03 - 3.43E+12 IU/ml) and >50% were neither IgM nor IgG positive, indicative of a very recent infection. Most (68) belonged to Genotype 1a, but an equal distribution of variant 1a1 and 1a2 was observed. Two related Genotype 2 viruses were identified in 2013 and 2015.

Conclusion

B19V NAT has been successful in preventing B19V entering the blood supply in Switzerland. The 1:30'278 frequency is comparable with that known in other European countries. Seasonal epidemics (e.g. 2013) occurred as reported by others and 2 rare variants were identified.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):50.

P04-2 Update of the evaluation of the Bac-Detect System for rapid bacterial screening of platelet concentrates

M Störmer 1, J Gielen 2, B Gathof 1

Background

Since bacterial screening has been recommended by the Paul-Ehrlich-Institut for prolonging the shelf-life of PCs back to 5 days, blood centers implemented strategies with focus on rapid detection on day 2, 3 or 4 of shelf-life depending on the blood transfusion service structure and logistics. Here we present the update on the results of the evaluation of the Bac-Detect System (Blood Analysis) for bacterial screening of PCs.

Methods

WHO International Repository Bacteria Reference Strains (S. pyogenes, SP; K. pneumonia, KP; E. coli, EC; S. epidermidis, SE) and spores of B. thuringiensis (BT) were inoculated separately in six replicates with 1–18 CFU/bag into buffy coat derived pooled PCs (PPCs, three expired and three in shelf-live) in additive solution and apheresis PCs (APCs, three expired and three in shelf-live) in plasma and stored under routine conditions. Bacterial detection was performed during storage using the Bac-Detect in comparison to the BactiFlow ALS (Biomerieux).

Results

In total, 35 PPCs and 32 APCs were artificially contaminated with a very low bacterial load. Bacterial growth was monitored during storage predominantly up to day 3 after contamination. Actually it was planned to contaminate 12 PCs (6 APCs and 6 PPCs) per bacterial strain but in some cases the contamination was repeated due to no growth during storage. Finally bacteria grew in 59 of 67 PCs (88%). While KP and SE grew in 12 of 12 contaminated PCs (100%), BT grew in 12 of 13 PCs (92%), EC in 12 of 14 PCs (86%), and SP in 11 of 16 PCs (69%) up to day 4 of storage. With the exception of EC all bacterial strains were detectable on day 3 of storage using both technologies in 100%. The Bac-Detect missed two EC contaminated PCs on day 3.

Conclusions

In conclusion the Bac-Detect represents a great alternative to the BactiFlow system for platelet bacteria screening at the end of storage, especially regarding handling, reagents and costs. But there are some aspects that need to be optimized which are specified below. Currently the Bac-Detect system seems to be less sensitive in comparison to the BactiFlow. But optimization of the gating strategy and maybe increasing the sample volume may result in a higher sensitivity. Therefore further testing regarding sensitivity is needed to fully evaluate the acceptance for routine bacterial screening of PCs.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):51.

P04-3 Treatment of Red Blood Cells with the INTERCEPT Blood System for pathogen inactivation for use in a double blinded phase III study preserves physiological erythrocyte morphology

P Paranikulangara 1, V Brixner 1, S Dombos 1, I Weber 1, J Leibacher 1, S Heldke 2, S Graminske 2, C Ravanat 3, A Erickson 4, A North 4, N Mufti 4, E Seifried 1

Background

Prevention of transfusion transmitted infection and preservation of cell vitality are core objectives for pathogen inactivation methods for blood products. The INTERCEPT Blood System for RBC aims to inactivate pathogens and leukocytes through interference of the nucleic acid replication cycle combined with a self-limiting neutralization reaction.

Method

We compare the effect of INTERCEPT treatment, using amustaline (S-303) and glutathione, on erythrocyte morphology in INTERCEPT treated (TEST) and untreated (CONTROL) leukocyte depleted erythrocyte concentrates. Erythrocyte viability characteristics include erythrocyte morphology and CD47 expression. Expression of CD47 on the erythrocyte surface is correlated with viability. INTERCEPT treated TEST and CONTROL RBC of healthy blood donors were analyzed 1–2 days post production (PP) and 35–38 days after donation (end of storage (EOS)) for CD47 expression using flow cytometry. Erythrocyte morphology was determined through light microscopy and the erythrocyte morphology was assessed using the Blasi score. The Blasi score determines the morphologic changes of erythrocytes. Discocytes get a score of 1.0, discoechinocytes get a score of 0.8, echinocytes of 0.5, spheroechinocytes 0.2 and spherocytes are scored as 0. Statistical analysis was done using an unpaired t-test with for CONTROL and TEST groups.

Result

CD47 expression measured by mean fluorescence intensity (MFI) of TEST (n = 242) and CONTROL group (n = 230) shows no difference at time points PP (treatment 42,400 MFI ±8,600 control 44,500 MFI ±8,700) and EOS (treatment 42,800 ± 9,100; control 44,200 ± 9,200). Mean morphology score analyzed on day 38 post donation for treated RBC (n = 108) was 71.8%±10.1 and for untreated (n = 93) 68.20%±10.5 (p < 0.05).

Conclusion

Compared to control group, INTERCEPT treated test erythrocytes showed no significant difference in CD47 expression. INTERCEPT treatment preserves erythrocyte morphology significantly better towards physiological discoid appearance. These findings support INTERCEPT treatment as a potential candidate for routine pathogen inactivation of RBC in clinical practice.

The INTERCEPT Blood System for Red Blood Cells is in development and not approved for use.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):51.

P04-4 Design and validation of a novel set to pool, filter and split 6 units or 1500mL of recovered plasma prior to pathogen Inactivation with Intercept

D Goslings 1, A Valek 1, D Reinert 2, BM Frey 1

Introduction

INTERCEPT pathogen inactivation (PI) technology is used for source and recovered plasma. To treat recovered plasma, approx. 2.5 units should be pooled to use full capacity of the INTERCEPT processing set (650mL). Hence, 5 units are pooled and separated into 2 splits of 650mL each with a commercially available set. Since the set has no filter, filtered plasma must be used to meet Swiss specifications, which leads to tradeoffs. For example, if whole blood filtration is used, buffy-coats lack platelets (plts). If component filtration is applied, often expensive blood collection sets have to be used. The latter approach is particularly uneconomic when most plasma is not used for transfusion but for fractionation not requiring filtered plasma. To solve this issue, we developed a set with filter.

Methods

We previously showed that whole blood filter RZ-2000 can remove white blood cells (WBC) from plasma without getting clogged (Goslings et al., Vox Sang 2012;103; suppl.1). Therefore, we designed a set based on this filter (Fig. 1). Blood was collected with set NGR6428 (Fenwal) and separated into erythrocytes, buffy-coat and unfiltered plasma. Pools of 6 or 7 of these plasma units were processed with the set (26 pools, 8 spiked with WBC).

Fig. 1.

Fig. 1

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Plasma pooling set with filter.

Results

Pools contained 1500–1899mL plasma with WBC conc. up to 0.5970 × 10e3/µL and plts up to 33.6 × 10e9/L. After filtration, WBC were below detection limit of FACS and plts ≤4.1 × 10e9/L while FVIII and fibrinogen conc. did not change significantly (p > 0.05, n = 18). Av. total volume loss was 40mL (n = 22) and filtration times were < 9min.

Conclusion

Our set efficiently filters up to 1500 mL plasma for PI without affecting FVIII and fibrinogen.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):51–52.

P04-5 Introduction of the Cobas 6800/ Cobas 8800 into routine MP-NAT blood donor screening and first experience on a routine basis

K Gubbe 1, K Hourfar 2, K Frank 1, A Karl 1, U Mayr-Wohlfart 3, T Tonn 1, H Schrezenmeier 3, E Seifried 2, M Schmidt 2

Background

Mini-pool NAT was introduced into routine blood donor screening in 1997 at the German Red Cross Baden-Württemberg - Hesse and Saxony for HBV, HCV and HIV-1 by an inhouse PCR system. The inhouse method was CE certified in 2006. Due to increased future requirements to CE certification the blood donor services introduced the Roche Cobas NAT system on the Cobas 6800 and Cobas 8800 platform in 2015.

Aim

The aim of this study was the qualification of the Roche Cobas NAT system at the test laboratories in Frankfurt, Ulm and Plauen and the subsequent introduction of the new NAT system into routine in 2015.

Methods

The analytical sensitivity was evaluated by probit analysis with WHO standards for HAV, HBV, HCV, HIV-1, B19, HEV and WNV (PEI standard) tested in serial dilution steps of at least 144 samples in six different concentrations. The diagnostic specificity was tested by negative blood samples in parallel to the test of record.

Results

The analytical sensitivities were 98.6 IU/ml, 170 IU/ml, 1,487 IU/ml for HBV, HCV and HIV-1, respectively related to the individual donation within a mini-pool screening of 96 samples per pool. The new NAT system was introduced in December 2015 in Frankfurt and in January in Plauen and Ulm. Up to now 12,358 pools were screened for the MPX and DPXand for limited sample pools in addition for HEV and WNV. Inhibited pools were observed in 0.44%. The percentage of false positive test results was 0.06%.

Conclusions

The validation data were slightly better than the data from the manufacturer. The system convinced by an easy handling, a reduced hands on time and an extended walk away time. Results were given as negative or positive for qualitative parameter or with a concentration for B19. Access to amplification curves would be desirable in order to improve the interpretation of reactive test results. The utility channel enables blood donor services the integration of new parameters for emerging pathogens and is a topic for further studies.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):52.

P04-6 RESEP a new medical device for microbiological testing of corneal organ culture medium

Z Skenderi 1, L Giurgola 2, C Gatto 2, J D'Amato Tóthová 2, A Pruß 1, J Schroeter 1

Background

The aim of this study was to validate the microbiological testing of cornea organ culture medium according to the European Pharmacopoeia (chapter 2.6.1.) using RESEP (ALCHIMIA) a new medical device for the removal of antibiotics and blood culture bottles with the automatic BACTEC system (Becton Dickinson, Franklin Lakes, New Jersey, USA).

Methods

For the validation 10–100 colony forming units of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis (ATCC 12228) were inoculated in 9 ml of cornea organ culture medium (MEM with 2% fetal australian calf serum and Streptomycin (130 µg/ml), Penicillin (60 µg/ml) and Amphotericin B (2,5 µg/ml); Merck, Biochrom GmbH, Germany). The medium was treated with RESEP for 20 minutes, inoculated in BACTEC™ Plus Aerobic/F and BACTEC™ Plus Anaerobic/F blood culture bottles and incubated until a positive reading was shown or for at least 14 days at 36 ±1°C. Medium control samples, without RESEP treatment, containing the same microorganisms, were inoculated in BACTEC vials directly. Microbial growth was controlled by direct inoculation of BACTEC flasks (growth control). The decrease of antibiotics from the medium by RESEP was determined by high performance liquid chromatography (HPLC).

Results

After 20 minutes of treatment at room temperature with RESEP (confirmed by HPLC) the antibiotics were completely eliminated. In samples treated with RESEP, the growth of all microorganisms could be detected within 3 days of incubation in the BACTEC system and showed no significant delay compared to the growth controls. In contrast, untreated medium samples showed no reproducibility of Staphylococcus aureus, Enterobacter cloacae, Candida albicans and Bacillus subtilis.

Conclusions

The microbiological testing of cornea organ culture medium with the automatic BACTEC blood culture system using RESEP for the removal of antibiotics has been successfully validated according to the European Pharmacopoeia (Chapter 2.6.1.)

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):52.

P04-7 Change of infectious disease testing and impact on reactivity rate and donor deferral

G Hersmann 1, H Thomas 1, K Linke 1, T Großgebauer 1, C Helbig 1, M Koburger 1, H Kroll 1

Introduction

The Blood Donation Service in Suhl processes more than 170.000 samples annually. For 16 years, serology testing was peformed by Abbott PRISM system, but since July 2013 by the Abbott ARCHITECT system. The introduction of new screening assays may be accompanied with a drop in specificity and may cause non-specific results for donors previously tested negative.

Methods

Repeat reactive rate and specificity of the assays run by either PRISM or ARCHITECT (HIV Ag/Ab Combo, anti-HCV, HBsAg) were calculated for the years 2006 - when anti-HBc testing became mandatory in Germany until end of 2015, more than two years after the switch to ARCHITECT. Due to the lack of a true confirmatory method for anti-HBc we only analysed the number of reactive samples for anti-HBc.

Results

Over the years with PRISM, the rate of nonspecific results (excluding anti-HBc) varied between 0,07 and 0,16%. In the year of the introduction of the new system the rate was slightly elevated to 0,20%, but reached 0,16% again in the second year after introduction. While the specificity for the HIV Combo assay remained at the same level or better, a higher rate of non-specific results was observed for anti-HCV and HBsAg. The number of donors found with anti-HBc reactivity clearly declined over the first four years after introduction of the test from 0,37% to < 0,1% due to deferral of donors that are anti-HBc positive and have anti-HBs levels below 100 IU/L. The introduction of a different anti-HBc assay did not lead to a significant increase. For plasma donations, the change of assays did not have any negative impact, no matter which parameter. The decreased rate of specificity for anti-HCV was leading to a temporary deferral of products and donors only. The impact of donor loss was minimal and within the ranges observed before. The rate of specificity for HBsAG has not reached the level before.

Conclusion

The introduction of the new screening tests was accompanied by an increase temporally in the overall rate of non-specific test results for whole blood donations in the year of the change. This demonstrates that each immunoassay has its own set of non-specific results even when the two systems are from the same manufacturer.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):52–53.

P05-1 Impact of red blood cell alloimmunization on survival of patients with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) treated with azacytidine

M Leisch 1,2,3, L Weiss 1,2,3, N Lindlbauer 4, A Egle 1,2,3, G Mayer 4, E Rohde 4, R Greil 1,2,3, C Grabmer 4, L Pleyer 1,2,3

Introduction

Red blood cell (RBC) alloimmunization frequently occurs in chronically transfused patients patients and poses the patient at risk for delayed hemolytic transfusion reactions and limited blood product supply.

However, the impact of RBC alloimmunization on overall survival (OS) in patients with AML, MDS and CMML is not known.

Methods

We retrospectively analyzed the transfusion and clinical history of patients in the Austrian Azacytidine (AZA) Registry (AAR, NCT01595295; Ethics Committee approval 06.02.2009) treated at the Paracelsus Medical University Salzburg from February 2009 to June 2015. All patients requiring RBC transfusions received extended phenotype matching (D, C, c, E, e and Kell).

Results

A total of 184 patients treated with AZA within the AAR had received RBC-transfusions, and were included in our analysis. 20 patients (10.8%) formed at least one alloantibody (“allo-group”), whereas 164 patients (89.2%) did not (“non-allo-group”). The most common antibody specificities were anti-E (n = 10), anti-Wra (n = 4), anti-Lua (n = 3), anti-D (n = 3), anti-C (n = 3) and anti-Jka (n = 3). Median time from diagnosis to first alloantibody development was 16.3 months (1.2 −86.4 months).

Patients in the allo-group received significantly more RBC-products (68 vs. 38 transfused RBC-products; p = 0.001) and had a significantly longer time under transfusion (16.7 vs. 9.4 months; p = 0.014) than patients in the non-allo group. Patients who developed rhesus alloantibodies received significantly more platelet (PLT) products than patients who did not (40 vs. 8 transfused PLT-products; p = 0.03).

Median OS from initial diagnosis (20.5 vs. 19.4 months; p = 0.296) and from initiation of azacytidine treatment (15.2 vs 9.1 months; p = 0.752) did not differ significantly for patients who developed an alloantibody versus those that did not.

Median survival after detection of the first alloantibody was short (2.8 months, range 1.7 - 3.9 months).

Conclusion

RBC alloimmunization was associated with an increased number of transfused RBCs and time under transfusion, but did not have a significant impact on patient survival. Taking into account extended phenotype matching, formation of rhesus alloantibodies might be a result of platelet transfusions.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):53.

P05-2 Follow up of antibody screen in Rhesus D negative patients transfused with Rhesus D positive red blood cell units

S Flommersfeld 1, R Marschall 1, G Bein 2, UJ Sachs 2

Background

Stock shortage of Rh D negative red blood cell units (RBCU) regularly results in transfusions of Rh D negative patients with Rh D positive RBCU. Since alloimmunization rates are high in contrast to other antigen-incompatible transfusions, antibody screening (ABS) should be performed 2–4 months after exposure (German Guidelines Hemotherapy). This study aimed to assess the adherence to this recommendation.

Material and Methods

From 2009–2015 data from all Rh D- patients exposed to Rh D+ RBCU were collected. All patients without follow-up ABS initiated by the clinicians were invited for an ABS within 6 months after Rh-D-incompatible exposure. Follow up data were evaluated.

Results

302 patients were exposed to D+ RBCU in 7 years (28–59 patients/year). 83 patients (27%) died during their hospital stay. 11 patients (4%) developed Anti-D 10 to 65 days after the first exposure while still in hospital. In 20 cases (7%) patient data were not available for a follow-up. Out of the 188 remaining patients, 85/120 invited to our department and 42/68 patients invited by their general practitioner participated. 61/188 (32.5%) could not be traced. Of the remaining patients 15/127 (12%) had died, 49/127 (39%) developed anti-D whereas 63/127 (49%) patients did not (non-responder).

Mean follow-up of ABS of inpatients after exposure was 21 days (0 – 71), and mean follow-up of ABS after invitation was 256 days after exposure (120–3600).

Conclusions

In total 81/302 patients (27%) could not be assessed. A personal invitation to see the general practitioner or a blood bank physician was followed by 71% and was somewhat higher than through initiation by the clinicians (61%). Follow-up of ABS after Rh D + exposure is time consuming and shows a limited compliance.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):53.

P05-3 Anti-D-alloimmunisation in D-negative recipients of D-positive Red Blood Cell transfusions

R Eichner 1, A Rosner 1, M Kramer 2, K Hölig 1

Introduction

According to the German hemotherapy guidelines, anti-D-alloimmunisation is to be avoided at all costs in female D-negative (D-) patients who might still become pregnant and in patients who depend chronically on transfusions. To guarantee this, transfusions of D-positive (D+) RBC to D- emergency patients with very high RBC demands cannot always be avoided. If needed, D+ RBC were continued for the following 14 days if there was a negative antibody screen. We reviewed our follow-up data of D- recipients after D+ transfusions regarding the frequency of Anti-D alloimmunisation.

Methods

This retrospective study includes 45 D- patients for whom D+ transfusions were provided by our blood bank. In addition to male sex or age > 50 and exclusion of hematologic disorders, a negative antibody screen with and without enzymatic enhancement was required. From January 2013 to January 2016, all D-incompatible transfusions were recorded and both the physician and the patient received a note asking for an antibody screening 2 to 4 months later. If the patient had survived but no blood sample was received, the patient's general practitioner was contacted. The antibody screen was performed by manual gel method using Bio-Rad RBCs with and without enzymatic enhancement.

Results

4 patients were excluded from further analysis because they did not receive D+ transfusions and 4 because no follow-up samples were obtained. 20 of 37 patients (54%) died within 2 months (median 7,5 days, range 0 - 52) after the transfusion, one having an Anti-D antibody detected starting day +41. Diagnoses were emergencies primarily associated with a poor outcome such as gastrointestinal bleeding, polytrauma or aortic dissection. These patients had received a total of 232 RCB concentrates, 109 of which were given after the first day. In 11 of the 17 surviving patients (67%), Anti-D antibodies were detected, often combined with other antibodies. The 6 non-immunized patients (33%) had received a total of 27 RBC concentrates (median 3 per patient, range 1 to 10), the 11 surviving immunized patients a total of 101 RBC concentrates (median 9 per patient, range 2 to 19).

Conclusions

Compared to other groups, we found a rather high immunisation rate among D- patients receiving D+ RBC. There was a trend to a higher number of RBC units in the immunised group. Nevertheless we conclude that our policy of D+ RBC transfusions to D-patients remains still justified.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):53–54.

P05-4 Changing patterns of red blood cell usage in a tertiary-care university hospital

R Zimmermann 1, J Strobel 1, J Zingsem 1, R Eckstein 1, T Ganslandt 2

Introduction

Since five years, the transfusion medicine community, donation services, and authorities in Germany observe a surprisingly pronounced decline in the usage of red blood cell concentrates (RBCs). Although ideas about the cause of this process are present, there are no accurate data to explain the phenomenon.

Material and Methods

The University Hospital Erlangen is a 1400-bed tertiary care hospital. Here, all relevant data for interpreting changes in the usage of RBCs during the years 2010 to 2015 were collected and analyzed.

We integrated the clinical data warehouse components of RBC recipients’ personal data, German Diagnosis Related Group (G-DRG) system codes of inpatients, and RBC component data from the blood bank IT system. The obtained DRGs were associated with the data of the blood component consumption on an individual basis. The results were grouped by Major Diagnostic Categories (MDCs) and by basic DRGs.

Results

Between 2010 and 2015, we observed a 20-percent decline of the number of annually transfused RBCs. This decrease was almost exclusively restricted to inpatients whose diagnoses and procedures were grouped to the MDC 0 (i.e. the so-called Pre-MDC category). This MDC comprises cases with long-term mechanical ventilation and solid organ or stem cell transplant recipients.

Conclusions

We could demonstrate that the pronounced decline in the usage of RBCs is almost exclusively restricted to intensive care patients and to a lesser extent to stem cell transplant recipients. Recent studies on the results of restrictive versus liberal RBC transfusion strategies and patient blood management projects are likely to be responsible for the changes in RBC usage.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):54.

P05-5 Patient blood management und bleeding management guidelines in cardiac surgery – the last Mohawk

D Bujnoch 1, M Weyand 1, E Strasser 2

Background

Cardiac surgery is aware about specific negative effects of CPB on cellular and plasmatic blood coagulation, such as dilution coagulopathy. Furthermore, preoperative administrated platelet inhibitors or coagulopathies by the underlying cardiac disease (e.g. acquired vWS in aortic valve stenosis, VAD) have to be considered. Coagulation disorders thus represent a group of patients with special risks due to bleeding or thrombosis. Preparation of the patient in terms of PBM is essential in cardiac surgery. According to coagulation history, it is helpful to evaluate the patient prior to surgery but it reveals not in every case the coagulation disorder, especially rare coagulopathies, such as thrombocytopenia of unknown origin. ITP is rare and needs special consideration due to planning the perioperative treatment.

Methods

We analyzed ITP patients (n = 11) in a single-center study who underwent cardiac surgery within the last 12 years (7 combined procedures, 1 valve surgery, 2 VAD, 1 TAH). We focused on the pre-operative preparation and diagnostic during the intra-, and postoperative course. Blood loss, units of blood and clotting factor products, the relationship between ITP and its influence on surgery were recorded (2 emergency cases, 1 redo, 1 viral infection, 1 tumor). Furthermore, literature research was performed with view of complications, death, operation planning and the need of blood and coagulation products.

Results

5 patients died due to bleeding. Cumulative blood loss of all patients: 60,090ml (range: 360 – 15,600 ml). On average, each patient needed blood products of 4,943€, clotting products of 4,860€ at a total amount of 107,837€. We found 63 cases and 6 reviews over 42 years, mainly from Asian area, only 17 full text reports in English. 28 cases were written in Japanese whereas no full text was available. Amazingly 3% patients died. Bleeding complications were described in 1.4%. Unfortunately, many cases have incomplete data about the clinical course and treatment algorithm.

Conclusion

ITP patients are at high risk for bleeding complications. There are no treatment algorithms for elective, emergent or special cases (VAD, endocarditis). We need to develop and evaluate diagnostic and therapeutic algorithms and guidelines about it.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):54.

P06-1 Identification of eight novel mutations and one reccurent mutation in F13B gene leading to inherited, heterozygous FXIII deficiency

V Ivaskevicius 1, A Biswas 1, S Gupta 1, G Kappert 2, S Halimeh 2, H Rott 2, S Flommersfeld 3, H Trobisch 4, U Kreibich 5, M Olivieri 6, K Kurnik 6, IC Frers 7, D Graf 8, J Oldenburg 1

Background

Inherited severe factor XIII (FXIII) deficiency is a rare, autosomal recessive bleeding disorder usually caused by mutations in the F13A1 gene and affecting approximately one in 1–3 million people. On the other hand the heterozygous (only one affected allele) FXIII deficiency is expected to be more common affecting approximately one out of 1000 inhabitants with a reduction of FXIII activity by 50% in most cases. Nevertheless, heterozygous FXIII deficiency belongs to one of the most underdiagnosed bleeding disorders so far. This is, because most patients do not develop bleeding complications in daily life.

Aim

Characterization of F13B gene mutations in patients with heterozygous FXIII deficiency.

Materials and Methods

In this work we report the results of the occurrence of F13B gene mutations in patients with mild (FXIII activity < 65% of normal) FXIII deficiency. Genomic DNA of the patients has been analyzed by direct sequencing in the molecular haemostasis unit in Bonn between 2009 and 2015. In total, 16 of more than 200 German patients with reduced FXIII levels had demonstrated mutations in F13B gene.

Results

Eight novel mutations and one reccurent F13B gene mutation have been identified. The majority (12/16) of patients carried missense mutations, one patient had small deletion (c.1958delT), one patient had small insertion (c.365insA) and one patient had splice site (IVS5–1G>A) mutation. All investigated persons were heterozygous for corresponding F13B gene mutation and no causative mutations have been identified in the F13A1 gene. Seven individuals from five unrelated families were heterozygous for a previously by our group described missense mutation in the 6th Sushi Domain of FXIII-B Subunit. Five novel missense mutations (p.Phe42Val, p.Tyr163His, p.Cys213Trp, p.Val266Pro, p.Ala416Glu) affect highly conserved regions of the FXIII-B Subunit.

Conclusions

All identified F13B mutations in this study seem to be causative for FXIII deficiency. F13B gene mutations leading to FXIII (B-Sub-unit) deficiency may not be as rare as reported in the literature.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):54–55.

P06-2 Chloride intracellular channel 1 supports cell adhesive processes in endothelial cells and platelets

LM Knowles 1, I Müller 1, A Drawz 1, E Ampofo 2, H Eichler 1, J Pilch 1

Introduction

Chloride intracellular channel 1 (CLIC1) is involved in vascular biological processes such as angiogenesis and platelet activation but the context of CLIC1 function remains largely unexplored. Our hypothesis is that CLIC1 functions in the context of cell adhesive processes and that this is relevant for thrombus formation, angiogenesis and vascular repair.

Methods

Endothelial cells were treated with CLIC1 siRNA or the small molecule inhibitor IAA94 compared to appropriate controls and probed for cell proliferation on plastic (2D) or cell invasion/survival after embedding in fibrin gel (3D). Platelets were treated with IAA94 to assess the effect of CLIC1 on spreading and aggregation. Flow cytometry was used to determine integrin αIIbβ3 activation as well as CLIC1 cell surface expression on platelets. In addition, we analyzed the subcellular localization of CLIC1 in endothelial cells/platelets using fluorescence or confocal microscopy. The effect of CLIC1 on thrombus formation in vivo was assessed by intravital fluorescence microscopy in a mouse dorsal skin fold chamber model.

Results

Treatment with CLIC1 siRNA or the small molecule inhibitor IAA94 in endothelial cells had a strong anti-proliferative effect in 2D that was matched by a significant reduction of invasion and survival in 3D fibrin-embedded endothelial cells in response to CLIC1 knockdown. Fluorescence and confocal microscopy revealed extensive relocation of CLIC1 into lamellipodia in 2D and invadopodia in 3D suggesting that CLIC1 function is controlled by adhesive interactions with the extracellular matrix. Paralleling these results, we detected co-localization of CLIC1 with F-actin in lamellipodia of platelets, which expressed CLIC1 on the cell surface in an RGD-dependent manner. This mechanism appears to be important for platelet function as CLIC1 relocation to the platelet membrane is reduced after treatment with IAA94, which also reduced platelet spreading, aggregation and integrin αIIbβ3 activation in vitro as well as vaso-occlusion in a mouse model of photo-chemical thrombus formation in vivo.

Conclusion

Our results show that CLIC1 is regulated by adhesive interactions with integrin ligands and that these interactions correlate with prothrombotic functions on platelets. Relocation of CLIC1 into lamellipodia or invadopodia of endothelial cells is associated with relevant pro-angiogenic functions such as cell invasion, survival and proliferation.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):55.

P06-3 Protein S activity measuring is influenced by Factor V Leiden mutation

D Weiss 1, E Strasser 1, J Ringwald 1, R Eckstein 1

Background

For diagnosis of Protein S (PS) deficiency, PS activity (PS:Act) is the clinically most relevant parameter. However, this parameter is preanalytically labile and analysis may be influenced by other parameters. Factor V Leiden mutation (FVL) leads to resistance of factor Va to activated protein C and its cofactor protein S. Thus an influence of FVL to measurement of PS activity by clotting tests might be possible.

Methods

PS:Act was measured by STA protein S clotting test in the STA-R compact system (DiagnosticaStago S.A.S. Asnières sur Seine, France). Furthermore, we measured free PS antigen (PS:AGf) and in the most cases total PS antigen (PS:AGt) by a immunological method. We analyzed 4,882 PS measurements of patients tested for FVL and prothrombin mutation G20210A.

Results

3787, 1033 and 62 samples were negative, heterozygous positive or homozygous positive for FVL with PS:Act of 87.5 ± 26.1, 78.8 ± 24.2 or 66.1 ± 18.4, respectively. The differences between all groups were highly significant in Man-Whitney-test (p < 0,001). There was no significant difference for PS:AGf (87.7 ± 23.8, 85.2 ± 23.9, 83.9 ± 21.8) resp. PS:AGt (80.8 ± 18.2, 89.0 ± 18.8, 88.8 ± 19.0) in relation to FVL. In contrast, there was no dependence of PS:Act and the genetic status of prothrombin mutation G20210A.

Conclusions

Dependent on genetic status of FVL, PS:Act was different. Further studies should investigate if PS:Act is really diminished in patients with FVL or if interactions with the measurement may lead to false-low results in these patients. The latter explanation may be more likely as the first explanation would imply that PS is structurally damaged in persons with FVL. We assume that - despite of added bovine FVa - the test is interfered by mutated endogenous FV. Thus, in samples of patients with FVL, a false-low measuring of PS:Act should be considered. We recommend to compare PS:Act with PS:AGf in patients with FVL.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):55.

P06-4 New aspects on i. v. immunoglobulins in treatment of patients with autoimmune thrombocytopenia

B Mayer 1, F Depre 1, F Ringel 1, A Salama 1

Background

Except from massive platelet transfusion, the most rapidly effective treatment in autoimmune thrombocytopenia (ITP) is the administration of high dose intravenous immunoglobulins (IVIgG). However, there is little information regarding the true onset of treatment effect of IVIgG.

Patients and Methods

Nineteen patients (females, males, age 14–84 years) with chronic ITP required emergency treatment due to significant bleeding and / or an increased bleeding risk.

Immunoglobulin was infused intravenously in doses of 0,8–1,2 g/kg body weight. Platelet count was measured before, directly after and, if possible, 1, 2, 3, 4, 12 and 24 hours after IVIG infusion.

Results

Although the response to IVIgG varied, platelet count was observed to increase in 10 of the treated patients within the first and second hour after IVIgG, respectively. Cessation of bleeding was observed within 12 h in affected cases (n = 7).

Conclusions

The results of our study indicate that the onset of therapy effect of IVIgG in most patients with ITP might be earlier and higher than has yet been thought.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):55.

P06-5 Dysfibrinogenemia during pregnancy treated successfully with low-molecular-weight heparin and fibrinogen

R Zimmermann 1, J Peisl 2, C Rauh 3, MW Beckmann 3, S Achenbach 1, R Eckstein 1

Introduction

Inherited dysfibrinogenemia may be a rare cause of recurrent pregnancy loss. Furthermore, it may be associated with bleeding as well as with thromboembolism. In half of all cases, dysfibrinogenemia remains clinically silent.

Case report

We report on a 31-year-old woman who had experienced four times an early abortion between 2013 and 2015. Presenting in our outpatient clinic the patient reported bruising, but on no other bleeding events and no thromboembolic events. A grandfather had died because of pulmonary embolism while suffering from lung cancer. Laboratory examinations revealed an INR of 1.40, activated partial thromboplastin time of 34.1 s, thrombin time of 35.6 s, and fibrinogen concentrations of 0.67 g/l (Clauss method) and 3.87 g/l (immunoturbidimetry). The patient was diagnosed with dysfibrinogenemia. Shortly later, the fifth pregnancy was diagnosed. Immediately, the patient was treated with a low-molecular heparin using the recommended prophylaxis dose for enhanced thromboembolic risk. Concurrently, the patient received a substitution of fibrinogen concentrate twice weekly (2 × 2 g per week at the beginning, dose adjustment to achieve a fibrinogen concentration of 1.00 g/l). While this was performed, the fifth pregnancy remained uneventful. The child was born six weeks before the calculated delivery time. Since delivery, mother and child are doing well.

Conclusions

Reported cases of dysfibrinogenemia in pregnancy with a history of recurrent early abortion that were successfully treated with fibrinogen replacement from the early stage of gestation are rare. Poor pregnancy outcomes were reported in many cases if functionally active fibrinogen levels were less than 0.6 g/L (Clauss method). After first-time identification of a woman suffering from dysfibrinogenemia without an obvious bleeding tendency and without thromboembolic events in her history, it seems to be best-practice to substitute fibrinogen and to applicate low-molecular-weight heparins simultaneously to prevent bleeding, thrombosis, and pregnancy loss as well.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):56.

P06-6 Our results from ITP patients treated with Eltrombopag and/or Romiplostim

B Mayer 1, F Depré 1, A Salama 1

Background and objectives

The thrombopoietin receptor agonists (TPOs) eltrombopag and romiplostim have revolutionized treatment options in ITP. Both drugs are increasingly used in treatment of patients with ITP. However, there is evidence that these drugs may cause severe side effects.

Patients and Methods

Between 2007 and 2015 a total of 79 patients (31 males and 48 females, age 18 - 87 years) were treated with TPOs at our institution. In addition eltrombopag was switched to romiplostim and/or vice versa in 28 of these patients.

Results

In total, less than 50% of all treated patients have responded and tolerated long-term treatment with TPOs until now. In 25 of our 79 patients, eltrombopag was replaced by romiplostim and in 7 cases romiplostim was replaced by eltrombopag. Switching was indicated in 17 cases (1 romiplostim, 16 eltrombopag) due to resistance, in 14 cases (5 romiplostim, 9 eltrombopag) due to adverse reactions, and due to patient's preference in one case (romiplostim). Until now, most severe side effects included arthritis, vasculitis, maculopathia and in one case multiple organ failure.

Conclusions

Our results indicate that most patients with ITP do not respond and / or develop intolerability to the available TPOs.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):56.

P06-7 Diagnostic work-up of a suspected platelet glycoprotein VI defect

V Klümpers 1, I Müller 2, P Hellstern 3, C Mannhalter 4, P Bugert 1, H Eichler 2

Introduction

Patients with inherited or acquired glycoprotein VI (GPVI) defects often develop mild bleeding diathesis. This case report of a female patient suffering from hypermenorrhea and skin hematoma and a suspected GPVI defect describes our diagnostic work-up. It included different platelet function tests and immunologic as well as molecular assays.

Methods

Fresh anticoagulated blood samples (EDTA, citrated, hirudin) were subjected to platelet function testing including Born aggregometry, whole blood aggregometry, flow cytometry, PFA-200, ATP release, PIFT and MAIPA. Convulxin (CVX) was used as GPVI-specific platelet agonist. Total RNA and protein were extracted from leukocyte-depleted platelets and subjected to GPVI-specific RT-PCR and immunoblotting, respectively. Genomic DNA was prepared from leukocytes and used for GPVI exon re-sequencing.

Results

Aggregation, degranulation and ATP release of the platelets were significantly impaired upon stimulation with low concentration of CVX (5 ng/ml). At higher CVX concentration (>10 ng/ml) or upon stimulation by ADP, platelet function was normal. PFA-200 closure times (ADP and epinephrine) were in the normal range. Anti-platelet or anti-GPVI antibodies could not be detected. Exon re-sequencing of the entire GPVI gene revealed homozygosity for the major allele (SKTQH) without any mutation in the coding region or the flanking intron regions. A heterozygous mutation (1038C>T) was identified in the 3'-untranslated region (UTR). However, expression of GPVI was normal by flow cytometry, qRT-PCR and immunoblotting.

Conclusion

The results indicate a reduced GPVI function as possible cause of the mild bleeding disorder. By immunologic analyses no component causing the reduced platelet activation by GPVI could be identified. A heterozygous mutation was found in the 3'-UTR of the GPVI gene but RNA and protein expression seemed normal. Thus, the cause of reduced GPVI function in our female patient still remains unclear.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):56.

P06-8 High fibronectin expression levels in invasive brain tumors

LM Knowles 1, I Müller 1, S Urbschat 2, R Ketter 2, D Lessig 1, C Wolter 1, H Eichler 1, J Pilch 1

Introduction

High grade astrocytoma represents a group of highly invasive brain tumors that are very difficult to treat. We previously established that invasion and proliferation in a 3-dimensional environment is supported by the ability of tumor cells to express fibronectin and generate fibronectin fibrils. Considering the relevance of invasion for aggressive astrocytoma we hypothesize that fibronectin expression represents a critical property for the malignancy of this tumor type.

Methods

Fibronectin mRNA expression was analyzed from extracts of primary astrocytoma and meningioma cells using quantitative PCR. In addition we assessed expression of fibronectin and the human embryonic protein Slug/Snail2 by western blot. To determine tumor cell invasion, astrocytoma and meningioma cells were embedded in a 3-dimensional matrix of fibrin or matrigel and scored for invadopodia formation. Fibronectin matrix formation in fibrin was analyzed by fluorescence microscopy following immunocytochemistry for fibronectin.

Results

Analyzing an array of primary astrocytoma cells (grade II-IV) for fibronectin mRNA, we detected markedly increased levels of fibronectin in tumor tissue with the highest expression in glioblastoma (grade IV), intermediate expression in grade III and the lowest expression in grade II astrocytomas while normal brain tissue expressed barely any fibronectin. Strong fibronectin expression in glioblastoma was associated with a highly invasive cell phenotype that readily infiltrated fibrin clots and matrigel. Primary meningioma cells (grade I+II) that are considered to be benign expressed only little fibronectin, which correlated with poor fibrin and matrigel invasion. GBM invasion in fibrin was accompanied by the generation of an elaborate fibronectin network that serves to stabilize the adhesive interactions in the 3D environment. This in turn is relevant for the expression of the EMT transcription factor and tumor stem cell marker Slug/Snail 2, which we found to be upregulated in cultured GBM cells.

Conclusion

Together our results demonstrate a positive relationship between astrocytoma grade, fibronectin expression and tumor cell invasion. Mechanistically, we established that fibronectin matrix formation supports expression of Slug/Snail2 suggesting that adhesive interactions with specific extracellular matrix proteins are able to induce a mesenchymal, stem-like tumor cell phenotype.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):57.

P07-1 NGS-based assessment of T cell receptor repertoire in long-term platelet donors

CS Link 1, K Hölig 2, E Rücker-Braun 1, M Kuhn 3, K Lang 4, A Eugster 5, C Klesse 6, M Schmiedgen 1, F Heidenreich 7, J Schetelig 1, E Bonifacio 8

Introduction

Current protocols of platelet donation can go along with a variable degree of lymphocyte loss. Multiple platelet donations within short intervals have been reported to decrease lymphocyte count. While this lymphopenia does not appear to be immunologically relevant from clinical experience, the immunological repertoire of platelet donors has not been measured directly. It remains unknown if the T cell receptor (TCR) repertoire is restricted after multiple platelet donations. In fact, currently little is known about the TCR-repertoire of healthy long-term donors at all.

Methods

Using next generation sequencing (NGS) we analyzed the TCRα repertoire of naive CD8+ T cells which had not been involved in an immune response and antigen experienced memory CD8+ T cells from five long-term platelet donors and five healthy controls. Diversity was estimated using Simpson's diversity index (Ds).

Results

A mean of 5,942,248 and 6,089,385 TCRα reads were found resulting in 60,082 and 12,058 clonotypes (mean) for the naive and memory compartment, respectively. Within all 10 samples we found a mean of 3,684 shared clonotypes (range 1,289 to 5,345) that were present in the naive and the memory repertoire, representing 11.35% (range 5.86% to 15.06%) of all TCRα reads of the naive cells and 75.13% (range 62.35% to 88.48%) of the reads in memory cells. Within the naive compartment none of the clonotypes was found with read frequencies >1% of reads. Diversity of the naive TCRα repertoire was 665fold higher than the diversity of the memory repertoire (p = 0.002). While the lymphocyte count was slightly lower for platelet donors compared to control samples(1.440 ± 0.178 *106/ml vs. 1.834 ± 0.174 *106/ml, respectively) there were no significant differences in T cell subsets. Furthermore, comparison of the TCRα repertoires of long-term platelet donors and controls showed no differences in TCR diversity for the naive and memory compartment.

Conclusion

To our knowledge this is the first study assessing the TCR-repertoire in a healthy population by NGS. This dataset may help to define future reference criteria. The TCR-repertoire of regular platelet donors was not restricted compared to the repertoire of healthy control subjects who did not donate platelets despite somewhat lower lymphocyte counts. This finding supports the clinical assumption that lymphopenia in regular platelet donors does not implicate restricted immunity.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):57.

P07-2 Iron deficiency: perceptions and management among physicians in Styria

M Schröck 1, C Drexler 1, M Moritz 1, P Schlenke 1, K Amrein 2, S Macher 1

Background

Iron deficiency (ID) is one of the most common nutritional deficiencies worldwide. It is highly prevalent in premenopausal women and also frequently found among regular - especially female but also male - blood donors. Besides anaemia, iron deficiency may lead to more subtle symptoms including fatigue and impaired cognitive and physical performance. We were interested in routine procedures of physicians to handle ID in the outpatient setting with a special focus on blood donors.

Methods

By email, we invited 363 physicians working in general practice (GP) or internal medicine (IM) to answer a total of 15 questions on ID using the free online platform soscisurvey.de. Physicians’ email-adresses were found by online query at www.google.at using the keywords: “Allgemeinmediziner, Hausarzt, Internist, Steiermark”.

Results

50/363 (13.8%) physicians completed the questionnaire (39 GP, 11 IM). Blood loss (93%), malnutrition (64%) and malabsorption (60%) were the most frequently indicated causes for ID. Only 8% (4/50) considered regular blood donation as a potential cause for ID. The count of blood donors among their ID patients was estimated to be low, and only 28% (14/50) reported to usually have knowledge if a patient is a regular blood donor. Generally, the compliance for oral iron therapy was stated to be acceptable for the majority. 70% are content with treatment efficacy. 60% indicated that they administer intravenous iron, especially to patients with poor iron resorption and chronic diseases. 56% would like to applicate intravenous iron more often if the costs were lower, while 44% would not because they fear drug-related adverse events (18/50) or find oral iron therapy sufficiently efficient (4/50).

Conclusion

Blood donors are generally considered to be healthy and are therefore not typical patients in doctors’ practices. Our online survey among physicians showed that there is room to improve the awareness of blood donation as primary cause for ID and iron deficient anemia. We therefore suggest that blood donor status should be recorded in patients’ files.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):57–58.

P07-3 Impact of demographic changes on blood donation numbers and first time donors

L Schönborn 1, V Kiefel 2, W Stangenberg 3, D Gloger 4, K Weitmann 5, W Hoffmann 5, A Greinacher 1

Introduction

The German reunification in 1989 was followed by a remarkable decline in birth rate, especially in the eastern parts of Germany of almost 50%. Here, we describe the consequences of these demographic changes on blood donation numbers using the example of the federal state Mecklenburg-West-Pomerania.

Methods

The data of all blood donations in 2008–2014 were acquired by the four blood donation services in Mecklenburg-West-Pomerania: German Red Cross Blood Donation Service, Haema Donation Service and the donation centres of the university hospitals in Rostock and Greifswald. The census of the Federal Statistical Office in 2011 was used to predict the prospective age structure.

Results

Since 2008 the number of blood donations constantly declined by 25.3% from 136,670 to 102,119 in 2014. Even more impressive is the reduction of the number of first time donors. This decline amounts to 58.3% (absolute reduction of 12,745 donations) between 2008 and 2014. By 2020 the population of Mecklenburg-West-Pomerania older than 65 years increases to 26.3%. A similar situation will be reached in the rest of Germany with a ten years delay, in 2030.

Conclusion

The demographic change is associated with a decreasing number of blood donations. These effects are most pronounced for the numbers of first time donors. While the Eastern states of Germany experience a major demographic shift since 2008 due to the decline in birth rates after 1990, the Western states of Germany will be faced with the same trend approximately 10 years later. Close monitoring of first time donor numbers is strongly recommended to counteract potential shortfalls in time.

Fig. 1.

Fig. 1

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(A) Absolute number of donations 2008–2014 (B) Absolute number of first-time-donors 2008–2014 (HRO = Donation Centre University Rostock; Haema = Haema Blood Donation Centre; HGW = Donation Centre University Greifswald; GRC = German Red Cross Blood Donor Service Mecklenburg-West-Pomerania).

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):58.

P07-4 Does RBC stock optimization influence donor motivation? An anticipatory view in times of demographic changes in the model region Tyrol

P Decristoforo 1, H Schennach 2

Background

Considering the international decrease in blood donations, Tyrol still represents an outlier with its high number of donation rates. The Tyrolean Red Cross Blood Donor Service even decreases for efficiency reasons since 2010 actively the number of donations, to reduce the throw-away (target < 5%) of expired red blood cells (RBCs. Although the fulfilment of demand is momentarily ensured, the question arises, whether the comprehensive self-supply of blood components for Tyrolean citizens can be secured respectively to future demographic changes. The detection of motivational reasons yields information for blood donor services to prospectively apply appropriate strategies, to maintain the willingness of donors as well as regulate the possibility of donations, while being adapted to the need. Socio-demographic data, motives, barriers and attitudes of voluntary, non-remunerated Tyrolean first time and regular donors (FD/RD) as well as the outcomes of blood donation limitations were investigated on an again increasing demand.

Study design and Methods

Blood supply and demand data from 2005–2014 were used for a prediction until 2030. Additionally a standardized, anonymous questionnaire (115 items), was distributed and completed, in the catchment area of Tyrol (36 donation sessions). Retrieved data were analysed via descriptive statistics and inductive factor analysis.

Results

The survey results of 430 questionnaires showed that FDs (n = 193) and RDs (n = 237), regardless of the difference in median age (FD 19 years, RD 39 years) share a similar motivational structure and act for the same reasons. Both donor groups are motivated mainly altruistically, are curious, have a positive approach to life and perceive a feeling of warm glow after donation. Financial incentives do not increase donor motivation.

Conclusion

Altruistically motivated blood donors react sensitively to limitations of donation possibilities. Low-threshold access and a frequent donation possibility do influence donors positively and ensure a reliable donor return. Monetary incentives are unrewarding and might even lead to the contrary. The upcoming shift of demographic populations in Tyrol does not exclude, but rather indicates a future increase of blood requirement. Hence, provident behaviour is needed to avoid conflicts between increasing blood demand, ruined blood acquisition structures and mobilization deficits, by earlier induced blood donation limitations.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):58.

P07-5 Non-invasive hemoglobin screening of blood donors

M Störmer 1, S Radojska 1, K Arenskrieger 1, A Rotem 1, K Verdonck 1, L Oustianskaia 1, B Gathof 1

Background

In December 2015 the Paul-Ehrlich-Institut informed blood centers about the non-effectiveness of a non-invasive method (NIM) for hemoglobin (Hb) screening in excluding ineligible donors which may pose a threat to donors’ health. Blood centers were requested to initiate self-dependent arrangements. Consequently the suitability of the Haemospect (MBR Optical Systems) was re-assessed in our blood donation center in comparison to the invasive (IM, CompoLab Hb, Fresenius Kabi) and venous (VM, Sysmex, Sysmex Europe) measurement.

Methods

The suitability of the Haemospect was assessed in three studies including the VM as reference. Study 1 Evaluation of influences on NIM: NIM during reception (sitting on a bar stool), NIM immediately before blood donation (sitting on a stool), VM during blood donation. Study 2 Evaluation after software update: NIM during reception, VM during blood donation. Study 3 Comparison of NIM and IM: NIM and IM during reception, VM during blood donation as reference.

Results

The table includes the percentage of donors that would have been allowed for blood donation due to a falsely too high Hb-value obtained by the non-invasive method. In these cases the venous values were below the required minimal Hb-values. Differences in gender sensitivity as well as the importance of posture could be demonstrated. After the software update the rate could be slightly decreased. But in comparison to the IM the rate was still too high.

Conclusion

NIM represents an attractive alternative in the blood donation admission process due to the elimination of pain and contamination risk. But as shown in several studies the main problem represents the generation of reliable results, especially regarding female donors with low Hb values. Currently the software is under further development and will be tested in the near future. Meanwhile the IM is used again for Hb-screening of blood donors.

Tab. 1.

Study Results

Study Hb Measurement Donors [N] (female/male) Hb falsely too high [%] total Hb falsely too high [%] female Hb falsely too high [%] male
1 NIM at reception 999 (460/539) 12.21 16.09 8.91
1 NIM before donation 997 (458/539) 10.93 16.59 6.12
2 NIM at reception 313 (142/171) 11.50 12.68 10.53
3 NIM at reception 313 (142/171) 10.31 14.13 7.45
3 IM at reception 659 (283/376) 4.86 5.30 4.52
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Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):59.

P08-1 Relevance of donor-specific anti-HLA-antibody-mediated rejection in liver transplant recipients

N Lachmann 1, T Dziodzio 2, S Ünlü 1,2, S Weiss 2, S Gül 2, C Schönemann 1, J Pratschke 2, R Öllinger 2

Introduction

The clinical role of donor-specific anti-HLA antibodies (DSA) in liver transplantation (LT) has not been clearly established yet. Here, we investigated the impact of antibody-mediated rejection (AMR) on the clinical course, morbidity and mortality of LT recipients.

Methods

A total of 649 LT were performed at our institution in 2008 through 2015. Patients with sufficient pre- and posttransplant immunological and clinical follow-up were included in this retrospective study (N = 250). DSA were determined pre- and posttransplant by the Luminex® single antigen assay. The normalized mean fluorescence intensity (MFI) was used to quantify DSA levels, and was correlated with the patients’ clinical course and success of AMR therapy.

Results

Thirty-three patients with preformed or de novo class I and/or class II DSA were identified. Of those, 19 patients (58%) showed clinical signs of AMR. In 17 patients (52%) an additional cellular rejection was observed. Sixteen patients (49%) were treated with steroids, 11 (33%) with plasmapheresis and immunoglobulins and 6 patients (18%) received anti-thymocyte globuline. Six patients (18%) showed a decay of rejection (MFI value reduction of >84%) and improvement of clinical parameters. Six patients needed a re-transplantation. Overall mortality was 21% with sepsis being the leading cause of death (5 patients). Fourteen patients (12%) with persistent elevated MFI values showed no clinical signs of AMR.

Conclusion

AMR after LT is relatively rare, however DSA can cause fulminant rejections with fatal courses. Current therapeutic concepts were only successful in 18% of patients. Interestingly, sepsis was the leading cause of death. Notably, almost half of the patients with DSA showed no signs of AMR. Thus, future investigations need to focus on the balancing of AMR treatment and its side-effects, as well as the causes of heterogeneous susceptibility of LT recipients to AMR.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):59.

P08-3 Developing a donorspecific B- cell ELISpot assay for immunomonitoring after transplantation by means of Hep B vaccination response

H Kripp 1, J Bucher 2, M Guba 2, S Wolf 1, A Bazhin 2, J Werner 2, T Kauke 1

Introduction

Short term survival rate after solid organ transplantation is well, but long term outcome is still insufficient. Donor specific memory-B cells seem to play an important role in acute and chronic allograft rejection. However to this date no assay is available to identify and quantify these donor-specific memory B-cells in the recipient. The aim of our research is to establish a donor specific total IgG B-cell ELISpot to meet this requirement. To proof the concept of detecting multiple antigen-specific memory B-cells by total IgG-ELISpot we chose the model of Hepatitis-B (Hep-B) immunity in vaccinated and unvaccinated blood donors.

Material and Method: PBMCs of vaccinated and unvaccinated healthy individuals were isolated and incubated with Hepatitis-B-vaccine containing HBs-antigen for three days. Cells were washed and put on goat-anti-human-IgG coated ELISpot plates overnight. After rinsing, biotinylated anti IgG antibody was added, left for 2 hours on the plate, washed away and streptavidin-ALP was added. Spots appeared after a short incubation with BCIP/NBT.

Results

There was a statistically significant difference in total spot-number and Δ-spot-number between vaccinated and unvaccinated individuals with p-values < 0.05. We could successfully distinguish vaccinated from unvaccinated individuals. Due to this we assume, that it is possible to detect and quantify antigen-specific memory-B-cell reactivity in vaccinated and unvaccinated individuals using a total IgG-ELISpot.

Conclusion

The validation of our total IgG ELISpot assay to detect Hep-B immunity was successful. The assay is suitable to quantify the immune response of Hep-B vaccinated individuals in vitro as expected. The validation of this concept in transplant-recipients by measuring the donor specific memory-B-cell response after incubating recipient-PBMCs with irradiated donor-cells still in progress.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):59–60.

P08-5 Humoral and cellular responses to a single dose of Fendrix in renal transplant recipients with non-response to conventional hepatitis B vaccination

M Zaslavskaya 1,2,3, M Fiedler 4, B Wilde 2, FM Heinemann 1, A Heinold 1, PA Horn 1, O Witzke 2,3, M Lindemann 1

Background

In kidney transplant recipients the risk for non-responsiveness to conventional hepatitis B virus (HBV) vaccines is increased to approximately 60%, pointing to the need for better vaccines. Highly potent third generation HBV vaccines, which contain the PreS1 and PreS2 antigens in addition to the small S antigen (e.g., Sci-B-Vac™ or Hepimmune™), are not licensed in Germany. But there could be an alternative, the second generation HBV vaccine Fendrix™, containing 3-O-desacyl-4'-mono-phosphoryl lipid A (MPL) as adjuvant.

Aims

It was examined in kidney transplant recipients whether Fendrix™ could induce humoral and cellular HBV immunity. Their vaccination efficacy was compared with the efficacy in other cohorts tested by the same assays (non-responders receiving Sci-B-Vac™ and non-vaccinated young, healthy controls receiving either Hepimmune™ or HBVaxPro™, respectively).

Methods

We selected clinically stable kidney transplant recipients who had been vaccinated at least three times against HBV but had never displayed anti-HBs antibodies. Twenty-nine transplant recipients fulfilled these criteria and we re-assessed anti-HBs antibody titers and further determined cellular HBV immunity by proliferation assay and interferon (IFN)-γ ELISpot.

Results

Eleven of the 29 recipients did neither display humoral nor cellular immunity and could be tested prior to and at month 1 after vaccination. In four out of them we detected anti-HBs antibodies ≥ 10 IU/L (21 - 264), in three HBV specific lymphocyte proliferation (stimulation index of 2.6 –5.5) and in one specific IFN-γ responses (12 spots increment) at month 1 (Figure). This vaccination response was even higher than the response after a single vaccination with HBVaxPro™ in young healthy controls.

Conclusion

The results show that a single vaccination with Fendrix™ can already induce HBV specific humoral and/or cellular responses in six out of eleven kidney transplant patients

Response to a single dose of Fendrix™ in eleven renal transplant recipients with non-response to conventional hepatitis B vaccination

Fig. 1.

Fig. 1

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Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):60.

P08-6 Luminex technology – for research only?

M-L Arnold 1, M Geithner 1, M Herber 1, S Klöcker 1, B Lauer 1, D Roppelt 1, M Steffen 1, H Wiederschein 1, C Bach 2

Introduction

With implementation of bead based (Luminex) techniques for antibody (Ab) detection in patients awaiting transplantation, the complexity of anti-HLA Ab identification and specification has been increasing sharply. The aim of our study was to investigate Ab-reaction patterns in Luminex analysis with a focus on the various HLA antigen (Ag) targets against HLA-A-B-C-DR-DQ-DP. Two questions occured: Do we need a calculation factor in order to compare different Ab results against various HLA-Ag targets within an Ab status of a singular patient? How to interprete the process of Ab testing during plasmapheresis and immunabsorbtion of a patient.

Methods

The status from 651 Ab positive sera from different patients on the wait list Erlangen/Nürnberg were tested for HLA class I and -class II Ab specificity using Luminex Single Antigen (LSA) technique. MFI-values of Ab specificities were collected and analysed.

Results

Discrepancies are detectable regarding the strength of reaction values (MFI) between the different HLA Ag allels and the corresponding Abs, e.g. the MFI of Abs against HLA DQ are meanly about ten fold higher than those against HLA DR. The “Heidelberger Curve”, which demonstrates the relationship on Ag-Ab reaction, shows clearly, that it is not possible to evaluate a reliable standard curve in order to calculate Luminex assays. For patients, habouring anti-donor Abs, a therapie with plasmapheresis and/or immunadsorbtion might be necessary. It is of high interest to calculate the MFI decrease during this treatment. The MFI values of the control sera are extremely importand to take place in this calculation.

Conclusion

We assume, that the Luminex assay is a very sensitive technique which is therefore going to replace the less sensitive complement dependent tests in the future. The analysis of sera from patients, suffered plasmapheresis and/or immunadsorbtion can exclusively be done by using Luminex technology. It is necessary to mention that the MFI value does not mirror a predictable strength of the anti-HLA-Ab in patient's serum. Based on our data, we can summarise that an overall MFI-cutoff for all anti HLA-Abs - independent of their anti-HLA-Ag-target - can not be determined.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):60.

P09-1 Chip-based digital PCR: an accurate and sensitive method for routine chimerism monitoring after hematopoietic stem cell transplantation

E Gourri 1, U Schanz 2, BM Frey 1, C Gassner 1

Background

After hematopoietic stem cell transplantation (HSCT) recipient and donor populations of leukocytes cohabit in the peripheral blood of the patient, resulting in “chimerism”. Monitoring chimerism is crucial to follow the outcome of the disease and to make informed clinical decision concerning further therapeutic interventions. Current chimerism monitoring is performed using Short-Tandem-Repeats analysis (STR) and quantitative real-time PCR (RT-PCR). However, both methods suffer from limited sensitivity and reproducibility. Digital PCR (dPCR) represents a new alternative for accurate and reproducible chimerism monitoring.

Methods

Chip-based Quantstudio 3D chip-based dPCR (Applied Biosystems, Reinach, Switzerland) allows for SNP detection in approximately 20'000 separate RT-PCRs per sample, with statistical presence of only 0, 1, or 2 DNA molecules per each 865 pL reaction well. Fluorescence of each well is separately measured and delivers absolute counts of the two different alleles. Original samples were analyzed for their SNP genotypes by PCR using Sequence Specific Priming (PCR-SSP).

Results

Using artificial DNA mixes, we estimated our limit of detection to range at approximately 0.5% for the minor DNA. Results within the same samples were highly homogeneous using six different SNP-Taqman assays for evaluation. Typing samples of Instand's external proficiency testing (EPT) for post-HSCT chimerism, was performed on one pre-HSCT patient and donor sample each and on five post-HSCT samples, using only two different SNP-Taqman assaysInstand honored our result quality with 20/20 points. On 15 DNA sample triplets, consisting of one pre-HSCT, one donor and one post-HSCT specimen each, dPCR results were consistent with previous STR results in 11 out 15 triplets. Discrepancies were only observed for 4 triplets, where chimerism was estimated to range below 5% by STR analysis, a method with known limited sensitivity (Stahl et al., Exp Hematol, 2015 Jun).

Conclusion

According to the high performance of our method in the Instand EPT and based on the results of 15 patient/donor/post-HSCT sample triplets, chip-based dPCR appears as an accurate and routinely applicable technique for exact chimerism determination with excellent sensitivity.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):60–61.

P09-2 Hematopoietic cell-derived CXCL12 contributes to G-CSF mediated stem/progenitor cell mobilization by auto-/paracrine signaling

D Karpova 1,2, J Miettinen 1, B Tast 1, E Wiercinska 1, G Spohn 1, H Boenig 1,3,4

Introduction

The critical role of the CXCR4/CXCL12 axis for immature hematopoiesis is well established. Current dogma implicates bone marrow stoma as the main source of CXCL12 in this equation. As we are showing, abundant CXCL12 expression is also found on hematopoietic cells of mature and immature lineage, and the role of paracrine or autocrine, hematopoietic-intrinsic effects of CXCL12 were considered.

Material and Methods

Constitutive CXCL12 deletion being lethal, its hematopoietic cell-intrinsic role was explored by analyzing 14.5 DPC fetal liver (FL) hematopoiesis in offspring of CXCL12+/- matings, as well as by establishing CXCL12-deficient (or WT control) hematopoiesis in a CXCL12 competent stem cell niche by transplanting lethally irradiated WT hosts with FL cells. Standard assays of mature and immature hematopoiesis, including phenotypic, in-vitro functional and in-vivo functional assays were used throughout.

Results

Except for mildly increased cell cycle activity in LSK cells, albeit without effects on HSC or WBC numbers, CXCL12-deficient immature hematopoiesis in FL was quantitatively and qualitatively largely normal in phenotypic and functional analyses, including repopulation assays in lethally irradiated hosts. CXCL12-/- FL established numerically and functionally normal adult hematopoiesis with regards to mature and immature cells during the engraftment period as well as later during homeostasis, including in competitive transplant experiments. Circulating CFU-C during homeostasis were the same in WT and CXCL12-/- engrafted hosts. Also, stress hematopoiesis (5-fluorouracil) and enforced mobilization with the CXCR4 antagonist Plerixafor were quite normal. When stably engrafted mice received a 5-day-course of G-CSF, however, the mobilization response was attenuated by one third in CXCL12-/- repopulated vs. WT mice.

Conclusions

On the weight of the evidence, we propose that hematopoietic cell-derived CXCL12 released under the influence of G-CSF triggers a positive feedback loop re-enforcing mobilization. No role of hematopoietic cell-intrinsic CXCL12 was apparent during homeostasis or other forms of hematopoietic stress than pharmaceutical doses of G-CSF, likely due to relatively overwhelming quantities of CXCL12 from other reservoirs.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):61.

P09-3 Generation of an immortalised erythroid precursor cellline by inducible overexpression of c-myc and BCL-XL as potential source for blood farming

R Kronstein-Wiedemann 1, N Kronstein 1, E Pasini 2, T Tonn 1,3

Background

Blood pharming using embryonic, bone marrow or induced stem cells represents a new and fascinating option to warrant the blood supply in the future. However, little is known about the mechanisms and factors that would allow the most efficient in vitro generation of enucleated blood cells. Since the differentiation from stem cells and/or IPS cells into the erythroid lineage would involve considerable amount of cytokines and purification steps, we aimed to immortalized cells already determined to the erythroid lineage.

Materials and Methods

Using an antibiotic inducible lentiviral vector system we transduced erythroid precursor cells derived from CD34+ cells from mobilized peripheral blood with five different oncogenes (c-myc, BCL-XL, SV40 LargeT, Bmi-1, LhX-2) and combinations thereof. 48h after transduction oncogene expression was induced by the addition of doxycycline (dox). Single cell clones were obtained by cloning in semi solid culture medium, further expanded and analysed for CD235a expression. For differentiation of mature red blood cells dox was withdrawn from the culture to turn off oncogene expression and to allow further erythroid maturation in the presence of (SCF and EPO).

Results

The transduction of any oncogene alone did not lead to immortalization. Among all combinations tested, only a combination of c-myc (proto-oncogene) and BCL-XL (anti-apoptotic protein) overexpression lead to immortalization of erythroid precursor cells. Furthermore sub-cloning revealed a heterogeneous expression pattern for erythroid markers and different morphology. In order to obtain a homogeneous CD235a positive cell population we selected a single cell colony (C35) with the highest expression of CD235a (~40%) and sorted erythroid cells based on CD235a expression. The so derived proerythroblastic cell population shows stable growth with a doubling time of 34 hours (expansion > 9 month). After removal of dox and submission to a 7 day differentiation culture in the presence of EPO, the cells readily matured into normoblasts and reticulocytes.

Conclusions

Immortalization of erythroid precursor cells is feasible using stable inducible lentiviral gene transfer for c-myc and BCL-XL. Withdrawal of dox allows differentiation in to further differentiated red blood cells. The conditions to obtain high yields of enucleated red blood cells, which then would also not harbour the risk attributed to the proto-oncogenes, has to be further optimized.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):61.

P09-4 Cytoskeletal structures of Mesenchymal Stem Cells (MSCs) on the road to fibrosis

D Malakhova 1, M Fischer 1

The most prominent cytoskeletal structures are stress fibers that appear when cells are grown ex vivo in tissue culture adherent to plastic surfaces or extracellular matrix components bound to plastic or glass. They show mechanosensitive properties and comprise of large bundles of actin filaments and associated proteins, typically located at the ventral surface of the cell and are frequently several micrometers long extending most of the length of the cell. Tension forces generated by stress fibers are transmitted to the cell surrounding matrix via focal adhesion. Here we can show that profound differences in the actin cytoskeleton structure were observed by fluorescence microscopy or LSM in adherent P1 or P3 MSCs compared to MSCs of spheric aggregates. More precisely, P1 MSCs showed long, thin stress fibers running in parallel according to the orientation of the MSCs, while P3 MSCs showed robust, thick, crisscrossed pattern of actin cytoskeleton emerging and a disappearance of delicate parallel fibers. In contrast, MSCs grown in spheric cultures and then adhered to plastic surfaces for less than 16 hours, showed a smaller and more rounded cell phenotype with very discrete and thin f-actin filaments. To further investigate stress fiber appearance in MSCs according to different culture conditions, we scanned large flattened MSCs of P1 and P3 by atomic force microscopy (AFM) in deflection mode and found thick and prominent actin stress fibers. MSCs grown in spheric cultures, in contrast, showed basically no stress fiber profiling that can be scanned and the cell membrane appeared smooth and homogeneous. The majority of stress fibers were ventral stress fibers commonly extending most of the length of the MSCs anchored at each end by focal adhesion. Mitochondrial appearance correlated with stress fiber formation, as mitochondrial fragmentation in small and larger spheroids located around the nucleus were found in MSCs of P3. In conclusion we can say that MSCs remodel and fine tune their cytoskeleton in response to the extracellular environment and next to chemical signals these physical signal determine the fate of MSCs on their way to fibrosis.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):61–62.

P09-5 Transfusion strategy in Rhesus-mismatched stem cell transplantations in times of scarce D-negative red blood cell supply

A Rosner 1, J Schetelig 2, M Bornhäuser 2, K Hölig 1

Introduction

D-negative red blood cell (RBC) transfusions are recommended for patients undergoing Rhesus-mismatched hematopoietic stem cell transplantations (HSCT) until complete donor-type erythropoiesis is confirmed. Recently, this has become more and more difficult to achieve without depleting the D-negative RBC stock needed for D-negative women of child-bearing age. HSCT recipients tend to require numerous RBC transfusions. The risk of alloantibody formation is low during HSCT. The main challenge comes from pre-existing recipient antibodies until erythroid engraftment, antibodies arising only after the transplantation are generally not directed against donor-type RBC. Based on these observations, it was decided to transfuse D-positive RBC in D-negative recipients with D-positive HSCT donors starting from day 0. Here, we present our first data with respect to the risk of anti-D alloimmunisation.

Methods

For this retrospective study, records of all HSCT at the Dresden University Hospital in the years 2014 and 2015 were reviewed. Antibody screens had been performed by gel microcolumn method with and without enzymatic enhancement every two weeks during hospitalization and at the hematologist's discretion after the hospital stay. In addition, routine antibody screens (gel microcolumn method without enzymatic enhancement or automated solid-phase test) were conducted according to the German hemotherapy guidelines whenever pre-transfusion RBC cross matches were requested.

Results

29 of the 236 HSCT recipients were D-negative and had a D-positive HSCT donor. Four of them received only D-negative RBC because of pre-existing antibodies (2 x anti-D; 1 x anti-Jk(a), and 1 x high-titre anti-A in a major AB0-mismatch situation). The remaining 25 patients had received a total of 380 D-positive RCB transfusions between the HSCT and the diagnosis of complete donor-type erythropoiesis. The median number of D-positive RCB transfusions per patient was 8 (range 0 to 78). Outcome: No newly formed Anti-D antibodies were detected. 8 patients died after the HSCT (median: day +60, range day +15 to +119). For the surviving 17 patients, the median observation time after HSCT was 361 days (range 150 to 787).

Conclusions

We conclude that post-transplant D-positive RBC transfusions appear to be safe with respect to Anti-D alloimmunisation for non-immunised HSCT recipients with D-positive HSCT donors. This transfusion strategy can be applied to reduce the general shortage of D-negative RBC.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):62.

P09-6 Bone marrow substitute for red blood cell depletion validation

I Pamler 1, N Ahrens 2, V Müller 2, K Besler 2, C Heinrich 2, H Schrezenmeier 3

Background

Allogeneic transplantation with bone marrow may require red blood cell (RBC) depletion. Traditional techniques include the addition of sedimentation accelerating agents, more recent techniques employ apheresis devices. This introduction requires validation according to GMP, however, bone marrow without transplantation is not available. We attempted therefore to use substitute material.

Methods

Three expired RBC units (expiring date 1–14days, 600 mL freshly donated plasma (1–3 days old), and 10 buffy coats (1–2 days old) were used to mix one bone marrow harvest in a 2000 mL collector bag. All components were ABO identical. The collector bag was sampled, sterilely welded and transferred into the bone marrow processing bag. This bag was in turn sterilely welded to a Terumo BCT Spectra Optia new intermediate density layer set. An 8-fold total volume was processed, collection preference was adjusted to an hematocrit of 5% in the lower collection tube.

Results

Target bag volumes had 141–161 mL, hematocrit values of 7.0–8.9% and contained 81–97% of the starting bags leukocytes, 84%-94% of the mononuclear cells. All preparations were sterile.

Conclusion

Bone marrow preparation could be simulated with substitute material successfully. In addition, these results indicate suitability and feasibility. Of note, sterile welding ensured that no clean room environment was required.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):62.

P09-7 Monitoring of adenovirus-specific T cells after HSCT in children: equal detection of hexon and penton-specific T cells

S Tischer 1,2, R Schultze-Florey 2,3, A Heim 4, G Picksak 5, M Mynarek 3, M Sauer 3, K-W Sykora 3, B Maecker-Kolhoff 2,3, B Eiz-Vesper 1,2

Introduction

In pediatric patients, adenovirus (ADV) reactivation after HSCT is frequent and associated with high morbidity and mortality. Viral clearance is usually achieved by a functional anti-ADV T-cell response. So far, ADV hexon has been recognized as a major target of anti-ADV response. Here, we present data from a prospective study monitoring hexon- and penton-specific T cells in 33 children with ADV reactivation after HSCT.

Methods

All patients undergoing HSCT were routinely monitored for ADV reactivation in blood and stool by quantitative PCR following institutional guidelines. All patients with quantifiable ADV in stool or blood entered the T-cell monitoring program. After leukocyte engraftment PBMC were collected once per week and ADV-reactive T cells determined by IFN-g ELISPOT using peptide pools derived from ADV proteins hexon and penton. Patients were grouped into strong (>40 spots), intermediate (20–40) and weak (≤20) responders. Control of viral replication was defined as ≤ 1000 copies/ml in blood and/or < 105 copies/ml in stool.

Results

33 patients with ADV reactivation were identified between 10/2011 and 10/2015. In all patients stool PCR was positive (median 7 days after SCT), while 22 patients had quantifiable ADV loads in blood (median 23 days). Most patients received standard treatment with cidofovir (range 2–11 doses). ADV-specific T cells were first detected at a median of 63 days after SCT with no timely difference between hexon- and penton-reactive T cells. The magnitude of T-cell responses against hexon and penton was individually diverse. While all but one patient demonstrated hexon-reactive T cells, 26 of 31 evaluable patients had T-cells reactive to the penton pool. Six of 31 patients showed strong reactions to penton while 11 of 31 showed strong reactions to hexon. There was no correlation between the target of the immune response and the HLA-type detectable in this cohort. Control of ADV replication was achieved in tight association with first detection of ADV-specific T cells (stool +6 days after first detection; blood −1 day) demonstrating the importance of ADV-specific T cells for ADV control.

Conclusion

Hexon- and penton-reactive T cells were equally effective in mediating viral control, suggesting that penton is a second immunodominant target in ADV infection. Extended monitoring of ADV-specific T cells helps tailoring antiviral treatment and identifies patients suitable for adoptive ADV-specific T-cell transfer.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):62–63.

P09-9 Cryopreservation of peripheral hematopoietic stem cells with uncontrolled freezing and 5% DMSO enhance CD34+ and CD45+ cell outcome compared to 10% DMSO

K Aurich 1, T Oergel 1, M Waterstradt 2, K Althaus 1, A Greinacher 1, W Krüger 2

Background

Controlled-rate freezing and storage in the vapour phase of liquid nitrogen is the standard technique for cryopreservation of peripheral hematopoietic progenitor cells (PHSCs). However, it is associated with high costs and dimethyl sulfoxide (DMSO) toxicity. Uncontrolled freezing, starting with 24 h at −80°C and subsequent transfer into the vapour phase of liquid nitrogen with only 5% DMSO instead of 10% DMSO preserves the functional capacities of PHSCs, and reduces toxicity during infusion. Especially in pediatric applications this is of importance, due to the lower bodyweight of the patients. In the present work, we have evaluated the impact of uncontrolled freezing and decreased DMSO concentration of 5% on CD34+ cell count, CD34+ and CD45+ cell viability and colony forming units (CFU)of ten autologous PHSCs compared to 10% DMSO.

Methods

Autologous PHSCs from ten patients median age 59.0 ± 14.3 years, 8 male, 2 female) were produced by apheresis with Spectra Optia (Terumo, Germany) devices following volume reduction (320 g, 6 min) and addition of cryopreservation solution (autologous plasma/DMSO; 5 or 10% DMSO final concentration). Each PHSC was divided into 4 cryopreserved products, quick-freezed on dry ice, stored at −80 °C in a mechanical freezer for 24h before transferring into vp-LN2 for 28 days. Measurements of CD34+ and CD45+ cell counts and viabilities were performed with aliquots and determined by flow cytometry and the viability marker 7-aminoactinomycin D according to the ISHAGE protocol.

Results

The impact of different DMSO concentrations on CD34+ and CD45+ cells during uncontrolled freezing procedure is shown in table 1. In summary, the use of only 5% DMSO was non-inferior to the standard using 10% DMSO.

Tab. 1.

Impact of different DMSO concentrations

5% DMSO 10% DMSO P
relative CD34+ cell count (% of initial cell count before freezing) 86.13 ± 14.3 67.6 ± 13.5 0.00005
CD34+ vitality (%) 99.1 ± 0.3 97.9 ± 1.3 n.s.
CD45+ vitality (%) 65.1 ± 9.7 53.8 ± 14.9 0.0006
CFU (x10^4/kg) 8.5 ± 4.5 9.4 ± 9.6 n.s.
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Conclusion

Uncontrolled freezing in compliance with decreased DMSO concentration of 5% is practicable and results in enhanced CD34+ cell count, CD34+ and CD45+ cell viability as well as reduced DMSO related side effects.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):63.

P09-10 Autologous stem cell apheresis and transplantation +/-Plerixafor

B Fuchs 1, J Auberger 2, C Straka 2, F Weinauer 1, J Burkhart 1

Introduction

For patients suffering from Multiple Myeloma (MM), an Autologous Stem Cell Transplantation (ASCT) can prolong survival. Prerequisite is a successful mobilization of peripheral hematopoietic blood stem cells. In patients with poor mobilizing abilities, CXCR4 antagonist Plerixafor (Mozobil® /P) in addition to granulocyte colony stimulating factor (G-CSF) is used.

Does the additionally use of P has any effect on parameters like age and sex of patients, efficiency of stem cell collection, stem cell dose, number of bags per transplantation and engraftment (number of days until granulocyte count >500/μl and platelet count > 20.000/μl) after transplantation?

Methods

From mid-2013 to 2015 127 apheresis from 106 patients were evaluated for the parameters. Patients were divided in two groups, G-CSF without and with P. In the period of examination the patients received a total of 139 ASCTs.

Results

31% of all apheresis were performed after mobilization with G-CSF + P. Neither the distribution of female (40%) to male (60%) patients nor the average patient age (62 years, range 33–75) showed differences in comparison. Mean CD34 value with G-CSF + P was lower before apheresis (38 CD34+/μl, range 10–107) than without P (144 CD34+/μl, range 10–569), average collection efficiency was higher (0.52%, range 0.36–0.73% versus 0.45%, range 0.29–0.66%) and the accumulated number of CD34+ cells significantly lower (336 × 10^6, range 75–1248) compared to mobilization without P (901.8 × 10^6, range from 77.7–3202.5). Even with a lower stem cell dose transplanted (2.2 instead of 3.2 × 10^6 CD34+ cells/kg body weight) and a higher number of transfused bags relating to a higher amount of DMSO in patients with G-CSF + P (2.1 to 1.2 bag), engraftment of granulocytes showed no significant difference (10.6 and 10.1 days) after transplantation. After mobilization with G-CSF + P, the engraftment of platelets showed an average delay of one day (12.6 instead of 11.7 days).

Conclusion

The addition of P gives patients with poor mobilizing abilities a good chance for a successful ASCT transplantation. Stem cells mobilized by G-CSF + P showed no difference in time for granulocyte engraftment to stem cells conventionally stimulated only with G-CSF. In the engraftment of platelets, there was a slight delay.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):63–64.

P09-11 Potent stem cell mobilization with the novel CXCR4 antagonist POL6326 – Results of a phase IIa dose escalation study in comparison to G-CSF

D Karpova 1,2, S Brauninger 2, E Wiercinska 2, A Kraemer 3, B Stock 3, J Graff 4, C Escot 5, G Douglas 5, B Romagnoli 5, E Chevalier 5, K Dembowsky 5, L Hooftman 5, H Boenig 2,3,6

Background

Stem cell mobilization (SCM) with G-CSF is efficient but – although overall safe – inconvenient because of the five-day injection regime and certain contraindications. Side effects, sometimes severe, are frequent. POL6326, a 2nd generation macrocycle CXCR4 antagonist, has demonstrated rapid mobilization kinetics and efficacy in mice. We report the results of a trial of SCM in response to POL6326.

Methods

In this Phase IIa open label trial, donors with average mobilization after 5d G-CSF, and a wash-out period of >6 weeks, received POL6326 at 500–2500 μg/kg as single 2-h i.v. infusion. Safety, tolerability, PK and PD were assessed. Subgroups received two doses of POL6326 >2 w apart for paired intra-individual analysis. Blood was collected before (0) and at 2, 3, 4, 6, 8 + 24 h after infusion start. CBC, CD34+, CFU-C count, phenotyping of mobilized mature and immature leukocyte subsets and PK were done. Volunteers underwent clinical and laboratory follow-up.

Results

POL6326 was very well tolerated. Some volunteers experienced mild urticarial / itchy macular rash responsive to H1/H2 blockade. Rating of tolerability/adverse events by volunteers (questionnaire) was favorable compared to G-CSF. Exposure was dose-linear. POL6326 mobilized CD34+ cells and CFU-C exceeding reported peak mobilization with plerixafor at all but the lowest dose. In this dataset mobilization after doses of 1500–2500 μg/kg did not differ meaningfully. SCM for CD34+ cells to doses ≥1500 μg/kg was 36.9 ± 2.4/µL (mean±SEM), or 1/3 that of G-CSF (y=0.324x). Good SCM with G-CSF was predictive of good SCM with POL6326 (r=0.63). POL6326 caused mixed leucocytosis peaking in the mid-20K/µL. B-lymphocytosis was more, neutrophilia and monocytosis less pronounced after POL6326 than G-CSF. At the 24 h time point, blood values were well on their way towards normal, and at follow-up all laboratory values had normalized.

Conclusions

POL6326 is safe, well tolerated, and provides efficient HSPC mobilization. Exploration of alternative dosing regimens may provide even higher mobilization responses. POL6326 can be an effective mobilizing agent for allogeneic donors, including subjects with contra-indications to G-CSF.

Fig. 1.

Fig. 1

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POL- CD34 +.

Fig. 2.

Fig. 2

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POL- CFU-C.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):64.

P09-12 Microbial detection of stem cell preparations. A non-trivial comparison

N Arlt 1, R Rothe 1, T Juretzek 2, S Sielaff 1, H Peltroche 2, T Tonn 3,4, R Moog 1

Background

Autologous stem cell grafts often contain antibiotics due to patients’ antibiotic prophylaxis probably resulting in false negative results of sterility tests.

Methods

The comparison was performed with one buffy coat as the control matrix used as the exemplary for tissue preparations and one stem cell preparation (PBSC) using the blood culture device Bact/Alert®3D with low temperature module (bioMérieux Nürtingen Germany). Samples were spiked twofold > 10 colony forming units (CFU) in standard iAST and iNST culture bottles for 14 days with various microbes. The PBSC was also incubated in iFA/iFNplus culture bottles with resorbing polymers. All aerobic bottles were incubated at 22.5°C and anaerobic bottles at 35°C. In a second analysis exemplary microbes were incubated in the same manner, but with an additional aerobic culture bottle at 35°C and measured with a more sensitive analysis algorithm (i-mode).

Results

The Bact/Alert®3D-System detected all microbes appropriate to their growth behavior in buffy coat matrix in iAST and iNST. The PBSC showed a significant difference in comparison to the used culture bottles. No growth was detected in spiked bottles with Staphylococcus aureus (iAST), Bacillus subtilis (iAST/iNST), Clostridium sporogenes (iNST), Propionibacterium acnes (iNST) compared to iFAplus and iFNplus where a growth could be confirmed (Fig. 1). All results could be achieved independent from i-mode. The temperature comparison showed an expected slower growth at 22.5°C.

Fig. 1.

Fig. 1

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Conclusions

Our study showed that spiked microbe's grow in PBSC using iFA/iFNplus in contrast to iAST/iNST. Only iFA/iFNplus are in line with the results of the spiked buffy coat, which is recommended as a matrix by the PEI. Therefore, iFA/iFNplus allow a safe detection of contaminated PBSC and we recommend the incubation at different temperatures for ATMPs for a safer microbial detection.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):64–65.

P09–13 Identification of novel surface markers on naïve, freshly isolated human bone marrow-derived mesenchymal stem cells

S Hemmer 1, N Baal 1, P Nold 2, G Bein 1, C Brendel 2, H Hackstein 1

Background

Mesenchymal stem cells (MSC) are of increasing clinical interest in tissue engineering and cell-based therapies but most information originates from cultured mixtures of heterogeneous progenitor cells after plastic adherence. We have systematically investigated the phenotype of naïve, freshly isolated human bone marrow derived MSC to identify novel surface markers facilitating prospective isolation, characterization and purification of MSC.

Methods

Bone marrow cells were prepared after confirmed consent and the MSC compartment was identified on CD45Neg cells expressing CD90+ and CD271+ according to the literature. We then combined multi-parameter flow cytometry staining of CD45Neg CD90+ CD271+ MSC with 332 different surface monoclonal antibodies to identify novel surface markers. Isotype-specific Fluorescence-minus one controls were used to precisely evaluate the specificity for surface staining and at least 100,000 cellular events were analyzed per sample.

Results

The analysis confirmed the presence of the few already known MSC makers such as e.g. CD49a, CD73 and revealed the presence of seven novel MSC candidate markers being expressed on >90% of freshly isolated MSC. Moreover, in direct comparison to CD45/CD31+ cells we identified highly discriminative markers being positive on naïve MSC but low or absent on contaminating leukocytes and endothelial cells. Additionally, the analysis revealed the presence of novel MSC subset markers allowing the discrimination of primary human MSC subpopulations.

Conclusion

These markers will facilitate human MSC identification, discrimination and purification and additionally provide novel insight into potentially MSC subset-specific functions.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):65.

P09-14 Peripheral blood stem cell donation by good and poor mobilizers

S Körper 1, D Fürst 1, D Hauber 1, A Beer 1, P Reinhardt 1, P Schauwecker 1, K Schwarz 1, D Bunjes 2, A Schulz 3, J Mytilineos 1, M Wiesneth 1, H Schrezenmeier 1

CD34+ cell count in peripheral blood on day of first apheresis is critical for the success of peripheral blood stem cell transplantation. We retrospectively analysed 337 peripheral blood stem cell donors to evaluate the efficacy of peripheral stem cell donation in poor mobilizers. The cohort consisted of related and unrelated donors of the Deutsche Stammzellspenderdatei Süd.

Good and poor mobilizers were statistically defined by the 10. and 90. percentile of peripheral CD34+ cell (CD34+ c) count after 8 dosages of G-CSF (5 μg per kg body weight of the donor) for the total cohort and for the male and female cohort. If not otherwise specified the values were expressed as mean± standard deviation.

The good mobilizers (n = 34) mobilized a minimum of 132 CD34+ c/μl while the poor mobilizers (n = 35) mobilized a maximum of 28/μl. A second apheresis was necessary in 88% of the poor mobilizers and in none of the good mobilizers. The donations of the poor mobilizers resulted in 2 transplants with less than 1 × 10e6 CD34+c per kg body weight of the recipient (BWR) (both donors were women), 6 transplants contained more than 4.0 × 10e6 and 12 transplants between 1 and 2 × 10e6 CD34+c/kg BWR. The values of CD3+c/kg BWR after the first apheresis are shown in the table. After the second apheresis the CD3+c/kg BWR adds to a total of 368.3 ± 225.2 CD3+c/kg BWR.

As expected the platelet counts were lower among the poor mobilizers but seemed to be normalized in both groups after one month of follow up. Adherence to the follow up is worse among the poor mobilizers. Pregnancy in the medical history seems not to have an influence on CD34+ mobilization.

In most cases (65%) a graft with at least 2 × 10e6 CD34+c/kg BWR could be provided even from poor-mobilizers. Apheresis is therefore justified in this group of donors. The CD3+c in the transplants of poor mobilizers are comparable to the good mobilizers. Further evaluation of the outcomes after transplantation from poor mobilizers is needed.

Table 1.

Good mobilizer N = 34 Poor mobilizer N = 35 Male Good mobilizer N = 22 Female Good mobilizer N = 11 Male Poor mobilizer N = 23 Female Poor mobilizer N = 11
Female Sex [%] 6% 18% – – – –
Pregnancies [%] – – – 64% – 64%
CD34 + c in peripheral blood [pl] 170.2 ± 40.0 19.6 ± 5.3 170.7 ± 35.2 170.7 ± 35.2 163.5 ± 55.2 17.6 ± 4.6
CD34+ c in graft [x10e6] 1005.0 ± 277.9 200.9 ± 77.7 984.6 ± 260.0 812.6 ± 344.8 263.9 ± 94.4 188.0 ± 87.0
CD34+c/ kg BW recipient 21.8 ± 34.9 2.7 ± 1.3 25.4 ± 41.9 12.2 ± 3.2 3.6 ± 1.5 2.6 ± 1.3
Platelets before 1. apheresis [/nl] 262.9 ± 51.1 234.4 ± 64.4 253.4 ± 51.1 318.9 ± 48.6 234.4 ± 47.8 238.7 ± 62.5
Platelets after 1. apheresis [/nl] 186.7 ± 38.3 146.9 ± 47.2 177.2 ± 32.3 217.9 ± 52.0 152.2 ± 43.5 144.8 ± 43.9
Platelets after one month [/nl] 247.7 ± 76.9 (N = 20) 235.8 ± 36.1 (N = 16) – – – –
CD3+ c/ kg BW recipient after 1. apheresis ± 360.9 272.2 ± 204.0 ± 426.2 ± 130.5 249.5 ± 162.9 276.4 ± 86.6
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Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):65–66.

P09-15 HOXB4 acts as a hematopoietic fate determinant in hemangioblasts

N Teichweyde 1, PA Horn 1, H Klump 1

Introduction

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for future somatic gene and cell therapy. So far, HOXB4 remains the most promising transcription factor whose ectopic expression in differentiating embryonic stem cells (ESCs) promotes formation of HSCs and confers their long-term multi-lineage reconstitution after transplantation, in vivo. However, the primary “target” cell of HOXB4 during hematopoietic development, in vitro, is not yet known. Its identification is a prerequisite for identifying the molecular circuits driving HSC development. Thus, we aimed to identify the cellular stage at which HOXB4 unfolds its hematopoiesis-promoting activities.

Methods

We retrovirally expressed HOXB4 constitutively or as a Tamoxifen-inducible fusion protein (HOXB4-ERT2) in Runx1 knockout ESCs containing a doxycyclin-inducible Runx1 coding sequence (iRunx) and in ESCs in which a Venus fluorescence reporter gene is expressed under the control of the proximal Runx1 promotor (Runx1:Venus). ESCs were differentiated for 6 days as embryoid bodies (EBs), dissociated, cocultured on OP9 cells and the number of arising Hemogenic Endothelium (HE) colonies counted. Without Runx1 induction, hematopoietic development was blocked immediately at the Endothelial-to-Hematopoietic Transition (EHT) stage allowing for a quantitative acquisition of generated HE-colony numbers. For evaluation whether the observed endothelial colonies were truly hemogenic, Runx1 expression was induced by addition of 0.1µg/ml doxycyclin. For enumeration of Blast-Colony Forming Cells, the in vitro correlates of hemangioblasts, day 3.5 EBs were dissociated, sorted for Flk1 expression and subsequently cultured in appropriate culture conditions.

Results

We observed that HOXB4 promoted the formation of HE-cells, from which the first definitive HSCs arise. Although these cells were expanded by HOXB4, they rapidly lost their ability to respond to the activity of Runx1. In contrast, HOXB4 acted on the immediate precursor of HE-cells, the bipotent hemangioblast by forcing the development of a hemogenic at the cost of structural endothelial development. Moreover, once the HE was formed, our results suggested that Runx1 needed to be expressed within a short window of time for hematopoietic transition to occur.

Conclusion

Our results strongly suggest that HOXB4 promotes the formation of HSCs by acting as a fate determinant in hemangioblasts.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):66.

P09-16 Red cell depletion of bone marrow grafts due to AB0 incompatibility: Significant shorter procedure times and higher red cell depletion efficiency with equal CD34+ cell recovery comparing the new Spectra Optia with the COBE Spectra cell separator

A Humpe 1, A Günther 1, A Wagner 1, B Trost 1, M Gramatzki 1, U Buwitt-Beckmann 1

Introduction

Transplantation of allogeneic hematopoietic progenitor cells from bone marrow (HPC-M) is a therapeutic option after high dose therapy for patients with different malignancies. Due to major or minor AB0 incompatibility between donor and recipient red cell and plasma depletion is necessary for transfusion of the graft without side effects. Here, 23 consecutive red cell depletion procedures of bone marrow (BM) grafts processed either on a new cell separator (Spectra Optia, I) or on the former machine (COBE Spectra, II) are compared retrospectively.

Material and Methods

From January 2011 until March 2016 a total of 23 consecutive bone marrow products were subject to red cell and plasma depletion due to major, minor or a combined major and minor incompatibility between BM donor and graft recipient. Twelve of these procedures were performed with the COBE Spectra cell separator and 11 were done with the new Spectra Optia separator. All bone marrow products contained heparin and additionally ACD-A at a ratio of at least 1:10. Key parameters like the weight of the recipient and the volume of the graft, the number of CD45+ cells, CD34+ cells, and white blood cells (WBC) were compared before and after depletion procedures.

Results

Comparing BM grafts processed by method I or II, neither the median value of the processed total inlet volume nor the volume before or after depletion, the number of CD34+ cells/kg before or after depletion, the number of CD45+ cells/kg before or after depletion, the number of CD3+ cells/kg before or after depletion, the hematocrit before or after, or the number of red cells before or after depletion exhibited differences. Procedures on the Optia machine were significantly (p < 0.01) shorter (median: 80 minutes versus 113.5 minutes) and the median efficiency in red cell depletion was significantly (p = 0.02) higher (98.7% versus 97.9%) without differences in recovery of CD34+ cells/kg.

Discussion

The Spectra Optia cell separator allowed a time saving red cell depletion procedure and showed a higher efficiency. In addition, although not reaching significance the number of CD34+ cells/kg retained was often somewhat elevated.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):66.

P09-18 Anti-proliferative effect of natural killer subsets on hematopoietic stem and progenitor cells

J Riewaldt 1,2, S Sayed 1,3, T Tonn 1,2,3

Introduction

Human hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow niche within a complex cellular network. The innate immune system - with natural killer (NK) cells as one of its major components - is as well present in the bone marrow niche, although it is currently not clear whether and how it may contribute to HSPC homeostasis, also in the context of HSPC transplantation. Here, we will present preliminary data that show negative regulation of hematopoiesis by NK cell subsets in vitro.

Material and Methods

Human mononuclear cells (MNCs) were isolated from peripheral blood of unstimulated or G-CSF-mobilized donors or bone marrow by density gradient centrifugation. CD34+ HSPCs and CD56+ total NK cells were enriched from MNCs using magnetic beads, while CD56bright and CD56dim NK cell subsets were obtained by flow cytometric cell sorting. HSPCs and NK cells were co-cultured in semi-solid medium to assess CFU capacities of HSPCs.

Results

Co-cultivation of HSPCs with CD56+ cells resulted in diminished colony formation after two weeks (minimum 25% less colonies), as compared to cultivation of HSPCs alone. For this effect, a minimum effector : target ratio of 1:1 was required. Although both myeloid and erythroid progenitor numbers were found to be reduced in presence of CD56+ cells, erythroid colonies appeared to be stronger negatively affected than myeloid colonies. Upon co-cultivation of NK cells with HSPCs previously in vitro skewed into the erythroid lineage, strong inhibition of erythroid progenitor growth was observed, indicating that the inhibition of target cells occurs also beyond the LT-/ST-HSC stage in lineage-determined progenitors. The inhibitory effect of NK cells on HSPCs was dependent on pre-activation of effector cells by interleukin-2 and largely mediated by cells with the phenotype CD56dimCD16+CD62L-CD94-CD57+. Of note, assessing co-cultures of NK cells with autologous HSPCs (in contrast to the allogeneic HSPCs used above) we observed increased progenitor colony numbers as compared to the HSPC monocultures.

Conclusion

We conclude that NK cells are clearly able to directly inhibit growth of HSPCs in vitro, which might in the context of HLA-mismatched HSPC transplantation negatively impact on the immune system's reconstitution. Further studies are required to further quantify NK-cell mediated effects on HSPCs and dissect interaction mechanisms (cell-cell contact-dependent vs. paracrine).

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):66–67.

P10-1 Extracorporeal photopheresis with allogenic cells for treatment of acute GvHD in a mouse model

H Budde 1, S Papert 1, H Reichhardt 2, J Riggert 1, TJ Legler 1

Background

Extracorporeal photopheresis (ECP) is an important second line therapy for Graft-versus-Host disease (GvHD). However, patients with acute or severe chronic GvHD often have a poor health status which can be a contraindication for weekly or even more frequent apheresis cycles. We hypothesized that leukocytes from healthy blood donors could be an alternative or additional source of cells for ECP therapy.

Methods

We used a murine model of C57BL/6→BALB/c bone marrow transplantation for induction of acute GvHD. Afterwards we established an ECP treatment in this model according to the clinical procedure in which ECP cells isolated from GvHD mice were used. Subsequently we used alternative allogeneic sources of the ECP cells from healthy donor mice from different MHC backgrounds. Additionally leukocytes were activated by mixed lymphocyte reactions prior to the 8-MOP/UVA treatment and used as ECP cells.

Results

The conventional ECP with cells isolated from GvHD mice had a significant positive effect on GvHD severity and survival. In contrast, ECP cells from healthy C57BL/6 or BALB/c mice showed no therapeutic effects on GvHD outcome. When using ECP cells from the “third party” C3H strain, with MHC I and II mismatch to C57BL/6 and BALB/c strains, we observed significant increased survival times and improved GvHD scores.

The additional activation of cells by mixed lymphocyte reaction before ECP procedure did not result in improved therapeutic effects.

Conclusions

A mouse model for ECP therapy of acute GvHD could be established. The allogeneic ECP procedure is an interesting option for the replacement of the patients’ apheresis step. However, the MHC background of the ECP cells seems to be important, since MHC independent “third party” cells show significant therapeutic effects whereas healthy cells from MHC related sources were less effective. For further clinical evaluation of the allogeneic ECP a clinical phase I-II trial is required.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):67.

P10-2 Mesenchymal stem cells as gene delivery vector for the expression of Db-scTRAIL: a possible application for cell-based therapy

I Marini 1, M Siegemund 2, R Kontermann 2, K Pfizenmaier 2

Introduction

Mesenchymal stem cells (MSCs) are a promising tool for stem cell-based therapies. One of the possible applications is cancer treatment, due to their tumor homing ability and immunosuppressive properties.

Aim

To investigate the potential use of MSCs as cell-based delivery system for the antitumoral agent TRAIL (Tumor Necrosis Factor Related Apoptosis Inducing Ligand), in vitro and in vivo.

Methods

MSCs, isolated from murine bone marrow, were stably transfected with diabody single-chain TRAIL (Db-scTRAIL). The ability of these cells (MSC.TRAIL) to secrete Db-scTRAIL was tested by ELISA assay. The antitumor activity of the secreted protein in combination with the sensitizer bortezomib (BZB) was analyzed using cytotoxicity assay. Propidium iodide staining and caspase-3 activation were determined by flow cytometry. Finally, the antitumoral activity of MSCs.TRAIL was assessed in vivo. NMRI nude mice were subcutaneously injected with colon cancer cells (Colo 205). Tumor volume was compared between mice injected with BZB+MSC.TRAIL and PBS or Mock MSCs (MSC.Mock).

Results

MSCs.TRAIL showed cumulative secretion of Db-scTRAIL in cell medium (productivity: 0.089 pg/cell/day). The bioactivity of the protein was analyzed on Colo 205 cells, revealing a tumor cell viability reduction more than 80% compared to buffer (BZB) (p < 0.001, n = 4). Next, the activation of apoptosis induced by Db-scTRAIL was confirmed by a significant increase of cleaved caspase-3 levels in Colo 205 cells (p < 0.001 MSC.TRAIL+BZB vs MSC.Mock+BZB) when co-cultured with MSC. TRAIL+ BZB. The MSCs properties, like MSCs markers expression and multipotency differentiation potential, were maintained even after stable transfection for 42 passages, suggesting that MSCs are excellent candidate for cell-based therapies. The in vivo study demonstrated a significant reduction in tumor volumes when MSC.TRAIL were peritumorally injected compared to PBS (n = 10 mice for each condition, mean difference in tumor volume 597.5 mm3, p < 0.001). Interestingly, no hepatotoxic effect was observed in the MSCs.TRAIL treated mice (alanine aminotransferase levels< 50U/L for all samples), confirming the safety of MSCs administration.

Conclusions

Our data demonstrate that MSCs are an efficient and safe cell delivery system for tumor therapeutic applications. This innovative clinical approach can overcome critical drawbacks of canonic treatments like protein stability, short serum half-life and fast renal clearance.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):67.

P10-3 Clinical scale expansion of retargeted CAR expressing ErbB2-specific Natural Killer cells (NK-92) using the Xuri Cell Expansion System Wave 25

N Miller 1, S Matko 1, S Tröger 1, WS Wels 2,2, M Odendahl 1, T Tonn 1,4

Introduction

Retargeted chimeric antigen receptor (CAR) expressing ErbB2-specific NK-92 natural killer (NK) cells have been shown to confer anticancer activity to human ErbB2-positive breast and glioblastoma cells in murine models. To prepare for a phase I clinical trial, we have been analyzing the feasibility of the Xuri™ Cell Expansion System Wave 25 for clinical scale expansion of GMP-compliant generation of CAR expressing ErbB2-specific NK-92 cells.

Methods

CAR expressing ErbB2-specific NK-92 cells were cultured in static cell culture (batch culture) using T75 flasks at a cell density of 5 × 104 cells/mL in X-VIVO 10™ (Lonza) supplemented with 5% heat-inactivated human AB plasma and 500 IU/mL IL-2 (Proleukin-S) (Novartis). The cells were then transferred to a 2L Cell Expansion bag (GE Healthcare) with a starting cell density of 5 × 104 cells/mL in 500mL of X-VIVO 10 supplemented with 5% human AB plasma and 500 IU/mL IL-2 on a Xuri Cell Expansion System Wave 25 (GE Healthcare). CAR expressing NK-92 cells were cultured for three days with a rocking rate of 5 rpm (6° angle) at 37°C and 5% CO2. Once a cell density of approximately 3.6 × 105 cells/mL (in 500ml) was obtained, 500mL of fresh medium was added to the bag and cultured for an additional five days before the rocking rate was increased to 10 rpm. NK-92 cells were cultured for another 2 days before the addition of 350mL culture medium containing 5% human plasma and 1000 IU/mL IL-2 with an increase in the rocking rate to 12 rpm. The NK-92 cells were cultured for two more days before the rocking rate was increased to 15 rpm. After three more days of culturing, the final count was obtained and found to be 4.06 × 106/mL. Cell numbers and viability were determined in triplicates using a Neubauer chamber and trypan blue. CAR expression was determined by immunofluorescence staining and flow cytometry (FACS Canto II (BD Biosciences)).

Results

During the initial static phase (days 1–5), the determined NK-92 cell doubling time and cell density within the 2L cell expansion bag using the Xuri™ Cell Expansion System Wave 25 reached numbers comparable to T75 batch cell culturing in T75 flasks (1.45 × 105 viable cells/mL). At the end of the static phase (day 14), the maximal density reached 2.15 × 106 viable cells/mL (viability 85%) using the Xuri Cell Expansion System Wave 25, outperforming the maximal cell density in T75 flasks by the factor of approximately 4.

Conclusion

The use of the Xuri™ Cell Expansion System Wave 25 for the production of relevant therapeutical doses (up to 5 × 109 cells!) of CAR expressing ErbB2-specific NK-92 cells is feasible and will be crucial for up-scaling GMP compliant cell expansion processes which are frequently restricted by lack of space in a clean room environment. Perfusion cultures might allow the yield of significant higher cell densities (>5 × 10^6 cells/ml) with reasonable cell viability.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):67–68.

P10-4 Flow cytometric analysis of hPBMCs in alginate hydrogel

U Höppel 1, R Detsch 2, T Zehnder 2, A Pfeiffer 3, R Eckstein 1, E Strasser 1

Background

This study investigated printing of mononuclear cells (MNCs) into alginate-gelatin hydrogel, which was crosslinked by calcium ions (Ca2+). MNCs packed in hydrogel may be of interest for improvement of cell storage and implantation in different tissues. The aim of the study was to evaluate cell analysis of MNCs packed in hydrogel as well as comparing two cytometric methods of cell counting (beads vs. “VolSens” technique) using a new flow cytometer. Leucocyte subpopulations in hydrogel scaffolds were analyzed.

Methods

Twelve healthy blood donors (11 male and 1 female donors) underwent plateletpheresis. MNCs were isolated from LRS chambers (volume approx. 8 ml) and prepared.

hPBMCs were encapsulated in alginate-gelatin crosslinked hydrogel (ADA-GEL) matrix. Sodium Citrate (4% w/v) was added to dissolute the hydrogel. The cell suspension was filtered with Filcons syringe type 70μm and 100μm. MNCs from ADA-GEL compared to MNCs of the same donor were analyzed by flow cytometry.

Results

The percentage of leucocyte populations was calculated, because each washing step implies cell loss. No difference of results was found due to filter size (70 μm and 100 μm). Significant differences (p = 0.008) were found analyzing NKTCells (3.5 ± 1.9% of lymphocytes) comparing to baseline (untreated leucocytes) regarding HF70 (13.2 ± 8.6%) and HF100 (12.5 ± 6.7%). No significant differences were found between percentage of leucocyte populations regarding additional washing steps with Stain Buffer (SB) and phosphate buffered saline (PBS) comparing printed HSB (5.7 ± 1.9%) to baseline 5.4 ± 0.2% (SB), as well as comparing HPBS (5.8 ± 1.8%) to PBS 3.5 ± 1.0%. No differences were found between the technique of cell count with Beads vs. VolSens for the leucocyte subsets with SB (p = 0.109) vs. PBS (p = 0.285) washing procedures.

Conclusions

The quality between untreated and in ADA-GEL encapsulated MNCs did not differ significantly. By use of washing steps of MNCs the quality of cytometry results was improved reducing background noise but cells are removed with the washing solution and cell quantification is impaired by this method.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):68.

P10-5 The effect of poly(lactic acid)-based scaffolds coated with hyaluronic acid on primary human monocyte-derived macrophages

K Stankevich 1,2,3, A Gudima 3, V Kudryavtseva 2, E Kibler 2, E Bolbasov 2, V Riabov 3,4, M Zhuravlev 5, V Filimonov 1, H Klüter 6,7, S Tverdokhlebov 2, J Kzhyshkowska 3,4,7

Introduction

Tissue engineering scaffold is a major element of regenerative medicine strategies. As it should possess certain physicochemical properties and biological activity the production of such a scaffold is a challenging task. The electrospun scaffolds made from poly(lactic acid) (PLA) meet a biocompatibility requirements, however they have several drawbacks limiting their application. Hyaluronic acid (HA) is naturally derived polymer that besides high hydrophilicity exhibit anti-inflammatory activity in its high-molecular form towards immune cells. Macrophages are key cells mediating inflammatory response and wound healing after implantation. Thus, coating of PLA-based scaffold by HA could improve its physicochemical properties and reduce inflammation as well.

Methods

Non-thermal atmospheric pressure plasma assisted immobilization of hyaluronic acid on the surface of elecrospun PLA-based scaffold was used as a modification method. The obtained materials were investigated by SEM, NMR, and Raman spectroscopy. Human monocytes were isolated from the buffy coats obtained from healthy donors and cultured under stimulation with IFNγ and IL4 to generate M1 and M2 phenotypes. On day 6 cytokines concentrations (TNFα, IL6, IL8, IL1β, IL10, IL1ra, CCL18, and TGFβ) and MMPs concentrations (MMP7, MMP9) were measured by ELISA. YKL40 gene expression was assessed by qPCR.

Results

It was shown that applied method allows for the attachment of different amount of HA and modified scaffolds have improved wettability. Macrophage cytokine responses to investigated materials were donor-specific. Overall, it was found that produced materials do not promote a strong inflammatory response and do not suppress anti-inflammatory cytokine production. At the same time pro-angiogenic factors IL8, YKL40, and MMP9 were upregulated in M0 and M2 macrophages cultured with modified scaffolds.

Conclusion

Scaffolds coated with HA have advanced surface properties and exhibit pro-angiogenic activity.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):68–69.

P10-6 HLA-silenced platelets derived from induced pluripotent stem cells are protected against refractoriness in a platelet transfusion mouse model

D Eicke 1, A-K Börger 1, K Schulze 2, B Eiz-Vesper 1, CA Guzman 2, R Blasczyk 1, C Figueiredo 1

Introduction

The high variability of the human leukocyte Antigen (HLA) remains the main immunological cause for platelet (PLT) transfusion refractoriness. Multiple transfused patients often develop antibodies against HLA class I, which mediate platelet depletion after transfusion. Here, we evaluated the in vitro and in vivo feasibility to use HLA-silenced PLTs differentiated from induced pluripotent stem cells (iPSCs) to escape refractoriness conditions.

Methods

A lentiviral vector encoding for a β2-microglobulin specifc shRNA was used to transduce iPSCs typed as HLA-A*02. HLA-silenced iPSCs were differentiated into megakaryocytes (MKs) and PLTs using VEGF, BMP-4 and TPO. The capacity of anti-HLA antibodies to target HLA-silenced PLTs was evaluated in vitro using antibody-mediated complement-dependent (CDC) and antibody-mediated cellular-dependent (ADCC) cytotoxic assays. PLT production and survival was analysed after MK transfusion of NOD/SCID/IL-2Rγc-/- mice treated or not with an anti-HLA-A*02 antibody to mimic refractoriness conditions.

Results

A HLA silencing effect of 50% was observed in the differentiated PLTs. In CDC assays, lysis rates of HLA-silenced MKs/PLTs were significantly lower (p < 0.05) in comparison to HLA-expressing MK/PLTs. In ADCC assays, MK lysis rates derived from HLA-expressing iPSCs exposed to anti-HLA-A*02 antibodies were significantly increased (p < 0.001) in comparison to a non-specific antibody. In contrast, no significant changes in lysis rates were observed among HLA-silenced MKs incubated with the non-specific antibody and the specific anti-HLA-A*02 antibody. In absence of anti-HLA antibodies, the transfusion of HLA-expressing or HLA-silenced MKs resulted in PLT production in the circulation. However, HLA-silenced MKs showed significantly lower lysis rates in comparison to HLA-expressing MKs under anti-HLA-A*02-mediated refractoriness conditions (p < 0.01). HLA-silenced MKs were also able to produce significantly higher PLT rates in comparison to HLA-expressing MKs under refractoriness. In biodistribution assays, HLA-silenced MKs were not accumulated in organs such as the lung suggesting that PLT production occurs in the peripheral blood circulation.

Conclusion

iPSC-derived HLA silenced PLTs showed to be a promising strategy to treat severe alloimmunised thrombocytopenic patients.

Fig. 1.

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ATIR- OS Probability.

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TRM Probability.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):69.

P10-8 Mannose-6-phosphate receptor may be a novel checkpoint for T cell expansion in HIV+ patients.

CT Kaltenmeier 1, H Schrezenmeier 1,2, B Jahrsdörfer 1,2

In mice, the mannose-6-phosphate receptor (M6PR, CD222) is known to be differentially expressed on activated T cells and is directly linked to their survival and proliferative capacity. One putative mechanism for this phenomenon may be M6PR-mediated uptake of the cytotoxic serine protease granzyme B (GzmB), which is expressed by various cytotoxic and regulatory cell populations. Recently we identified a novel population of regulatory B cells in HIV patients, which strongly express enzymatically active GzmB in the absence of perforin. Importantly, these so-called GraB cells are able to directly regulate proliferation and survival of T cells both in-vitro and in-vivo, comparable to regulatory T cells (Treg). However, the exact mechanism of action remained elusive. Here we demonstrate that in the early phase of HIV infection M6PR expression by T cells is significantly higher than by T cells from healthy control subjects. Moreover, co-culture of GraB cells from HIV patients with autologous or allogeneic T cells results in perforin-independent transfer of GzmB to T cells and GzmB-dependent degradation of their T cell receptor ζ-chain. Our results suggest that active GzmB can be transferred to T cells in the absence of perforin via M6PR-mediated cellular uptake. Our findings support the current view that after infections with intracellular pathogens such as viruses or intracellular bacteria, activated T cells differentially regulate M6PR on their cell surface. This differential expression can explain how Treg initiate the effector T cell contraction phase after an infection. Furthermore, our results may also explain how other regulatory cell populations expressing GzmB in the absence of perforin such as GraB cells or plasmacytoid dendritic cells may directly regulate T cell expansion in an M6PR- and GzmB-dependent fashion. Modulation of M6PR on T cells by pharmacological means may represent a promising novel approach to enhance or suppress T cell-mediated immunity in different infectious diseases including HIV infection.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):69.

P10-9 Development of plasmacytoid dendritic cells as tumor vaccine

T Trzaska 1, T Fecher 1, H Schrezenmeier 1,2, D Fabricius 3, B Jahrsdörfer 1,2

The majority of clinical studies testing the effect of dendritic cells (DCs) for the purpose of tumor vaccination are currently using autologous monocyte-derived DCs (mono-DCs) from tumor patients. Although a series of these studies demonstrated that mono-DCs can cause immunological responses, this approach is associated with two major disadvantages. On the one hand, the ex-vivo maturation of this DC type requires approximately 7 days, which means that the cells have to be cultured outside their physiological environment for quite a long time. On the other hand, both the quantity and the quality of autologous DCs are often limited due to the underlying disease and the therapy of patients. An interesting alternative to mono-DCs are therefore allogeneic plasmacytoid dendritic cells (pDCs) from HLA-matched healthy donors. pDCs are highly active professional antigen-presenting cells, whose precursors are circulating in the peripheral blood. Importantly, after isolation pDCs can be activated and matured within only 24 hours. Recently, their isolation has been made possible according to good manufacturing practice (GMP)-guidelines, so that a clinical application appears within reach. Here, we present our current concept for the development of plasmacytoid dendritic cells as tumor vaccine with a specific focus on their biphasic activation, which involves temporary overexpression of the serine protease granzyme B (GzmB). According to our hypothesis, activation of pDC precursors occurs in a biphasic process. During the first phase, pDCs develop a tolerogenic phenotype in response to different cytokines including IL-3, IL-10 and IL-21. This phenotype involves the production of large amounts of the serine protease GzmB. GzmB is not only able to suppress effector T cell responses, but may also be involved in antigen uptake and processing. In the second phase, pDCs are rapidly (within 24 hours) matured in response to TLR ligands and CD40 ligand. Now GzmB is downmodulated, while co-stimulatory molecules and MHC/peptide complexes are simultaneously upregulated, resulting in a highly immunogenic and mature APC phenotype. On our poster we will present this hypothesis and our current data on a systematic comparison of different maturation stimuli including TLR7 and TLR9 ligands as well as the T cell-dependent stimulus CD40 ligand. Our project is designed to establish the use of allogeneic pDCs as vaccine against certain tumors and infectious diseases at Ulm university.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):71.

P10-10 Dissection of human regulatory T cells by flow cytometry reveals novel surface markers

J Steinhauser 1, N Baal 1, G Michel 1, G Bein 1, H Hackstein 1

Background

Human regulatory T cells (Tregs) suppress immunological inflammation and promote tolerance. Because Tregs exhibit potential to modulate autoimmune diseases and transplant rejection the biomedical community has developed an intense interest in the clinical application of Tregs. We report the results of a systematic prospective study aiming to identifiy novel human Treg cell markers exhibiting potential to facilitate Treg discrimination and isolation and to reveal novel Treg functions.

Methods

Samples from voluntary blood donors were prepared and human Tregs were identified by flow cytometry based on expression of consensus markers CD4+ CD25+ CD127low in combination with > 300 different surface monoclonal antibodies to identify novel markers. The specificity and gating of the Treg staining was validated by intracellular FoxP3 staining. Screening positive markers were validated by repeated stainings of independent blood samples.

Results

This systematic approach proved to be highly specific and reliable since we were able to confirm recently reported Treg markers such as CD39, CD71 and CD101. Moreover our analysis revealed the presence of two novel Treg surface marker candidates being expressed on higher levels on all Tregs in comparison to non-Tregs. Furthermore, our study design allowed the identification of several negative Treg markers allowing the setup of an antibody cocktail for the depletion of non-Tregs and isolation of untouched human Tregs for in vitro characterization or expansion.

Conclusion

We have identified novel human Treg markers facilitating discrimination, characterization and purification of human Tregs.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):71.

P10-11 Efficient expansion of mesenchymal stromal cells by fibrinogen-depleted pooled human platelet lysate

M Öller 1,2, S Laner-Plamberger 1,2, M Feichtner 2, ZA Dunai 2, A Raninger 2,3, M Gimona 2, A Hartl 4, C Hauser-Kronberger 5, E Russe 6, K Bieback 7,8, E Rohde 1,2, G Brachtl 2,3, C Scharler 2,3, D Strunk 2,3, K Schallmoser 1,2

Efficient animal serum-free propagation of mesenchymal stem/progenitor cells (MSPC) could be established by the use of pooled human platelet lysate (pHPL) as alternative for fetal bovine serum (FBS) and HPL manufacturing is widely implemented. In this study we asked whether porcine heparin can be avoided during MSPC propagation under completely xeno-free conditions. As additional technical variations of the procedure depending on fibrinogen content may impede comparative studies, the influence of fibrinogen depleted modifications of pHPL on MSPC biology was analyzed. According to our recently developed protocol (J Transl Med. 2015;13:354) we prepared medium-clotted pHPL (mcpHPL) by adding pHPL to cell culture medium without heparin and pHPL serum (pHPLS) by adding CaCl2 during pHPL preparation (12mM), both enabling porcine heparin avoidance. All pHPL preparations were tested for fibrinogen by ELISA. Concentration of growth factors was analyzed in differentially supplemented media (culture d0 and d5). White adipose tissue (WAT; n = 3) and umbilical cord (UC; n = 2) derived MSPC were isolated and cultured in medium supplemented with pHPL, mcpHPL, pHPLS, or FBS for up to 4 passages. Cell proliferation, clonogenicity and trilineage differentiation capacity were analyzed. MSPC surface markers were tested by flow cytometry.

During 4 passages cumulative population doublings in all media supplemented with pHPL variations (WAT-MSC median 22; range 18–26; UC-MSC 30; 27–32) were significantly higher than in FBS supplemented media (AT-MSC 16; 10–16; UC-MSC 15; 13–16). In FBS medium clonogenicity remained constant until passage 4 whereas in all pHPL media clonogenicity declined continuously.

MSPCs of all medium conditions revealed characteristic cell surface marker patterns and typical trilineage differentiation potential. Fibrinogen decreased from mean 776 μg/ml in pHPL to < 1 μg/ml in mcpHPL and pHPLS. PDGF-BB (median 285; range 217–317 pg/ml), EGF (127; 118–154 pg/ml) and BDNF (828; 640–985 pg/ml) were similar in all pHPL media on d0 and were exhausted until d5. In pHPL media VEGFa increased until d5 up to 18,448 pg/ml compared to 12,741 pg/ml in FBS media.

In conclusion, MSPC culture was feasible in a completely animal-component free system without fibrinogen and heparin. Our results therefore may contribute to the optimization of clinical-grade MSPC expansion enabling applications in advanced somatic cell therapy and tissue engineering.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):71.

P10-12 Ex vivo expansion of T lymphocytes induces substantial alterations in cell size and actin-binding cytoskeletal proteins

I Weber 1, K Kollar 1, D Pollet 2, R Henschler 3

Background

The isolation of immune cells for cellular therapies often involves culture expansion steps, e.g. in the case of selection and amplification of antigen-specific T cell clones or the generation of chimeric antigen receptor-transduced (CAR) T cells. Reports have indicated that during ex vivo expansion, T lymphocytes and other cells can undergo functional impairment, e.g. in migration or antigen recognition which may affect their therapeutic efficacy. The impact of the culture step on actin-binding proteins, which are crucial for these functionalities, is so far not known.

Methods

We analyzed the expression of seven cytoskeletal proteins at the transcriptional and the protein level, including actin modifiers cofilin and profilin, actin branching/bundling partners alpha actinin and filamin A, as well as the linkage proteins between actin and integrin signalling complexes, paxillin, vinculin and talin. Transcription was quantified using qRT-PCR. Protein levels were measured by flow cytometry of permeabilized cells using fluorescence-labelled antibodies titrated to exceed the concentration of the recognized antigens. Cell size was determined by flow cytometry using calibrated microbeads.

Results

Analysis of cell size during anti-CD3/antiCD28-induced ex vivo expansion of freshly isolated murine CD3+ T lymphocytes in RPMI/10% FCS 30U/ml Interleukin-2 indicated a duplication of the average cell diameter from 7 to 14 μm, and a concomitant increase in cell volume from approximately 179 to 1436 μm3 over the period of 7 days. At the same time, protein levels of all seven actin binding molecules remained constant on a per cell basis, indicating a relative decrease in the intracellular concentrations of these proteins. Moreover, mRNA levels relative to the housekeeping gene GAPDH were reduced 5–7 fold in the case of profilin, cofilin, filamin A and alpha actinin, and approximately 10–50 fold for paxillin, talin and vinculin.

Conclusion

Ex vivo expansion protocols can induce major increases in size and intracellular volume of T lymphocytes. In parallel, amounts of functionally relevant actin-binding proteins such as profilin, cofilin, alpha actinin, filamin A, paxillin, vinculin and talin did not increase in parallel. Moreover, their transcription was strongly reduced. Our findings indicate that a cell culture step can induce abnormalities in proteins with key functions in the migration and function of T lymphocytes.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):72.

P10-13 Multicomponent cytokine cocktail for local control of macrophage phenotype: perspectives for immunomodulation in implantation

V Riabov 1,2, A Gudima 1, NE Vrana 3, H Klüter 4,5, J Kzhyshkowska 1,2,5

Introduction

Macrophages can induce adverse reactions towards implanted biomaterials (including titanium implants) resulting in implant failure. Macrophage-mediated production of pro-inflammatory factors upon contact with biomaterials is one of the main reasons leading to implant-associated inflammation. Generation of cytokine cocktails and application of slow release systems that locally re-polarize implant-associated macrophages towards pronounced anti-inflammatory state is a promising strategy to combat excessive inflammation.

Methods

Monocytes were isolated from buffy coats of healthy donors using CD14+ MACS beads and cultured in a serum-free medium supplemented with 10 ng/ml M-CSF and 10–8M dexamethasone in absence or presence of M2-inducing cytokines (IL4, IL10, TGFβ) and their combinations. Re-stimulation was performed with M1 stimuli IFNγ and LPS. Cytokine production was measured in supernatants by ELISA.

Results

Generation of M2 cocktail (M2ct) containing IL4 (3 ng/ml), IL10 (10 ng/ml) and TGFβ (10 ng/ml) resulted in a very strong inhibition of pro-inflammatory cytokine production (TNFα, IL6) after LPS stimulation compared to prototypical M2 stimulation with IL4. At the same time, production of M2-associated chemokine CCL18 was high under M2Ct stimulation. Importantly, pronounced inhibition of TNFα production was observed for 12 days after addition of M2ct. Moreover, withdrawal of M2Ct from the medium only partially restored macrophage response to LPS suggesting temporary fixation of anti-inflammatory phenotype. Complete restoration of LPS responsiveness was achieved only after withdrawal of M2Ct followed by M1 re-polarization with IFNγ.

Conclusions

The multicomponent anti-inflammatory cytokine cocktails appear to be a perspective tool for local immunomodulation around implants. Such cocktails can be used in slow release systems allowing long-term control of macrophage phenotype on implant surface.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):72.

P10-14 Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allogeneic hematopoietic stem cell transplantation: a prospective multicenter trial

M Neuenhahn 1,2, J Albrecht 1, M Odendahl 3, F Schlott 1, G Dössinger 1, M Schiemann 1, S Lakshmipathi 1, L Germeroth 4, H Boenig 5,6, T Tonn 3,7, H Einsele 8, D Busch 1,2, GU Grigoleit 8

Introduction

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT) especially in recipients of T cell-depleted grafts or grafts from CMV-seronegative donors. We assessed safety and efficacy of stem cell- or third-party donor-derived, MHC streptamer-selected CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial.

Methods

Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo MHC-Streptamer-isolated CMV epitope-specific donor T cells. Graft versus host disease (GVHD) induction rates, kinetics of CMV-specific T cell expansion and CMV viremia were monitored.

Results

44 allo-HSCT patients receiving a T cell-replete (D+; n = 28) or T cell-depleted (D+ depl; n = 16) graft from a CMV seropositive donor were screened for CMV-specific T cell immunity. One of 28 D+ and 8 of 16 D+ depl recipients qualified and 8 D+ depl recipients received adoptive T cell therapy from their stem cell donor. CMV epitope-specific T cells became detectable in all treated patients without concomitant side effects. Complete and partial virological response rates were 62·5% and 25%, respectively. Due to longsome third-party donor (TPD) identification, only 8 of 57 CMV-patients transplanted from CMV seronegative donors (D-) received antigen-specific T cells from partially HLA-matched TPDs. In all but one, co-incidentally 8/8 low-resolution HLA-matched, TPD-derived CMV-specific T cells remained undetectable. TPD-derived CMV-specific T cell products did not induce GVHD.

Conclusion

Adoptive T cell transfer correlated with functional virus-specific T cell reconstitution and reduced virus load in D+ depl patients with persistent CMV viremia. Time to TPD identification delayed treatment of D- patients. Suboptimal HLA-match may counteract expansion of TPD-derived virus-specific T cells.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):72–73.

P10-15 Antigen-switch in VZV reactivity of Fingolimod (FTY) treated MS patients

S Matko 1, K Thomas 2, M Odendahl 1, T Ziemssen 2,3, T Tonn 1,3,4

Introduction

Varicella zoster virus (VZV) is one of the most frequent persistent viruses in the adult population. Its reactivation can cause potentially lethal systematic infections in immunocompromised patients. Though it is assumed that VZV specific humoral immunity correlates with a lower rate of VZV relapses in MS patients on Fingolimod (FTY) therapy, little is known about VZV specific cellular immunity. We aimed to characterize VZV specific T cell responses in patients on FTY treatment to assess their potential as an additional prognostic marker.

Methods

18 MS patients before, 6 and 12 months after FTY therapy onset, 12 long term FTY treated patients (>6 years) and one FTY treated patient showing clinical reactivation were analyzed. Anti-VZV IgG and IGM titers were assessed by ELISA. T cell immunity was measured by intracellular IFN-γ staining after stimulation with VZV antigens IE62, IE63, ORF9, ORF26 or gE.

Results

While the relative percentage of the VZV specific cytotoxic T cell (CTL) compartment increased from 0,01 ± 0,01% to 0,06 ± 0,08%, absolute cell numbers declined from 0,3 ± 0,32 Mptl/L to 0,18 ± 0,36 Mptl/L within 1 year of treatment. T helper cell (THC) immunity decreased in both, relative (start 0,01%) and absolute (start 0,08 ± 0,03 Mptl/L) numbers to a non-quantifiable level. Remarkably, a shift from an IE62 antigen dominated CTL response towards ORF26 was observed. CTLs specific for IE62 decreased after 6 months of therapy from 0,1 ± 0,1 Mptl/L to 0,07 ± 0,08 Mptl/L, while ORF26 specific CTL responses rose from 0,05 ± 0,05 Mptl/L to 0,1 ± 0,3 Mptl/L. Also in long term patients, ORF26 induced a stronger CTL response (0,05 ± 0,06 Mptl/L) than IE62 (0,01 ± 0,01 Mptl/L). Analyzing T cell immunity in a patient before and after reactivation, VZV specific CTL responses increased significantly (P = 0,025). Additionally, after reactivation THC responses exceeded the mean value in normal patients by P = 0,032. Comparing cellular and humoral immune responses, the percentage of patients with detectable CTL immunity increased in proportion to their IgG titer.

Conclusion

We observed a switch regarding the predominant antigen response during the course of FTY treatment, implying that an ORF26 dominated CTL response contributes to the control of VZV reactivation. Even though humoral and cellular immunity correlated in our patient group, further longitudinal analyses in a bigger cohort are necessary to elucidate the prognostic value of cellular immunity.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):73.

P10-16 GMP-compliant production of pooled Human Platelet Lysate – Setting standards for Xeno-free cell propagation

M Feichtner 1, M Öller 1,2, S Laner-Plamberger 1,2, H Wiedemann 3, E Rohde 1,2, D Strunk 1,4, K Schallmoser 1,2

Human platelet lysate (HPL) is an efficient alternative for fetal bovine serum for pre-clinical cell propagation. Routine manufacture of pHPL is well established and animal serum-free propagation of mesenchymal stem/progenitor cells (MSPC) enables applications in advanced somatic cell therapy and tissue engineering. Internationally standardized production protocols and defined release criteria are still lacking (Biomaterials 76:371), but are urgently required to increase quality, safety and comparability of this important cell culture supplement.

We produce pooled HPL (pHPL) in a GMP-compliant manufacture process from expired platelet concentrates of healthy blood donors by −30°C freezing and thawing at 37°C to release platelet-stored growth factors. Ten lysates representing 40 buffy coats are pooled for one pHPL batch (mean 2.5L) to minimize donor variations. After centrifugation for platelet fragment depletion the supernatant is stored at −30°C as ready-to-use reagent. All procedure steps are integrated in our quality management system. We use pHPL for efficient culture of endothelial colony-forming progenitor cells and MSPC from human bone marrow, cord and adipose tissue.

In-process controls and product release include negative testing for bacteria, fungi and mycoplasma. Each pHPL batch is tested for isoagglutinin titers because platelets of different ABO blood groups are pooled. Biochemical analyses include pH, osmolality, total protein, fibrinogen, lipids, glucose and electrolytes. Batch functionality is verified by real-time monitoring of cell proliferation.

Since 2013 >100 pHPL batches (>250L) have been produced and released, sufficient for preparation of >2,500L medium (10% v/v). Isoagglutinin titers were constantly below 64. Testing total protein (6 ± 0g/dL), fibrinogen (250 ± 57mg/dL), lipids (triglycerides 79 ± 10mg/dL; cholesterol 162 ± 9mg/dL), glucose (295 ± 13mg/dL), pH (7.45 ± 0), osmolality (311 ± 3mosm/kg) and cell proliferation revealed no significant variations (mean±SD).

We here provide a standardized production process with quality controls resulting in reasonable batch release criteria that meet safety requirements for GMP-compliant manufacture of pHPL.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):73.

P10-17 Establishing a GMP-compliant protocol and quality controls for production of adipose derived mesenchymal stromal cells (ASC)

M Rojewski 1,2, R Lotfi 1,2, P Bugert 3, T Dengler 3,4, K Keller 2,5, P Zeplin 6, W Funk 7, H Schrezenmeier 1,2

Introduction

Clinical use of mesenchymal stromal cells (MSC) has increased during the last decade. We previously demonstrated that bone marrow derived MSC are a powerful tool for in vivo formation of bone and treatment of bone defects. However, ASC seem to be more powerful in treatment of acute or chronic inflammation, especially when degeneration of tissue is involved (e.g. osteoarthritis). We have adapted a GMP-compliant protocol for MSC production that has been validated at five manufacturing centres in Europe and used within three clinical trials for bone regeneration successfully to produce GMP-grade ASC.

Methods

Lipoaspirate was centrifuged and cell fraction was used for collagenase digestion. The stromal vascular fraction (SVF) was seeded at a density of 2000 cells/cm2 on 2 chamber CellSTACKs using TransferPack Kits from MacoPharma. Non-adherent cells were removed after 24h and additional medium exchanges were performed after 3 and 5 days. After 8 ± 1 days ASC of passage 0 (ASCP0) were harvested using TrypZEAN and reseeded at a density of 4000 cells/cm2. After two medium exchanges (day 10 and day 12), ASC of passage 1 (ASCP1) were harvested at day 14 ± 1 and filled into syringes.

Results

From a volume of 639 ± 754 ml of lipoaspirate (n = 10), 140 ± 60 ml of the cell fraction was digested, yielding in 41 ± 34 × 106 SVF cells. After 6.5 ± 0.6 population doublings within 7.3 ± 0.5 days of culture, cell density was 45.2 ± 28.5 × 106 ASCP0/cm2 and after additional 4.5 ± 1.0 population doublings within 6.4 ± 0.5 days of culture, cell density was 53.4 ± 21.0 ASCP1/cm2. Yield was 3.2 ± 2.5 × 106 ASCP0/ml lipoaspirate and 85.8 ± 64.1 × 106 ASCP1/ml lipoaspirate.

For quality control, matrix validations had to be performed according to Ph.Eur. for parameters indicated in table 1. Validation of flow cytometry included markers CD13, CD14, CD34 and CD45 for SVF and CD13, CD14, CD34, CD45, CD73, CD90 and CD105 for ASCP0 and ASCP1.

Table 1.

Matrix Ph.Eur. Lipoaspirate SVF ASCP0 ASCP1
Cell count and viability 2.7.29 – + + +
CFU-F – – + + –
Flow cytometry 2.7.24 – + + +
Endotoxin testing 2.6.14 – – + +
Mycoplasma PCR 2.6.7 and 2.6.21 + + + +
Microbial testing 2.6.27 + + + +
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Conclusion

Using the described protocol, we were able to establish a GMP-compliant protocol for production of > 200 millions of ASCP1 from 100 ml of lipoaspirate within 14 days that passed all required quality controls.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):74.

P10-18 Human Adipose Stromal Cells in contrast to human retinal pericytes resist the detrimental effects of hyperglycaemic modified extracellular matrix

H Kremer 1, S Elvers-Hornung 1, H Klüter 1, H-P Hammes 2, K Bieback 1

Aim

Diabetic retinopathy, an important cause of blindness in the Western world, is characterized by an early loss of pericytes (“pericyte drop out”) followed by qualitative and quantitative changes of the extracellular-matrix (ECM) of connected endothelial cells. Adipose stromal cells (ASC) largely overlap with pericytes regarding the immune phenotype and endothelial stabilization capacity. This in conjunction with their known immunomodulatory and proregenerative features renders them attractive to prevent pericyte dysfunction and loss of early retinopathy. It is, however, unclear whether ASC and pericytes behave similarly in conditions mimicking the diabetic milieu.

Method

ASC and human retinal pericytes (HRP, primary or immortalized) were seeded on ECM produced by human umbilical vein endothelial cells (HUVEC) under varying glucose conditions: normal glucose (NG, 5,6 mmol/L), high glucose (HG, 28 mmol/L) or intermitted phases of hyperglycemia (NG, HG, NG, every 3h during daytime). Adhesion and growth on the ECM was monitored using kinetic live cell imaging for 48 hours. Apoptotic events were assessed using a cell-membrane permeable, fluorescent dye, detecting Caspase-3 activity.

Results

In contrast to HRP, ASC resisted the detrimental effects of constant hyperglycemic modified ECM of endothelial cells. Only intermitted phases of hyperglycemia affected the adhesion and growth of ASC supporting the negative effects of high glucose fluctuations seen in patients. The higher sensitivity of HRP to hyperglycemic-modified ECM was apparent by the occurrence of apoptotic events, rarely seen in ASC.

Conclusion

Our data document that ASC, in contrast to HRP, resist the detrimental effects of constant hyperglycemic modified matrix of endothelial cells. Thus they may serve as corrective against hyperglycemia-induced pericyte death or dysfunction reducing microvascular complications. Whether the immunomodulatory and secretory properties of ASC contribute further to prevent/treat diabetic complications is a matter of current studies.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):74.

P10-19 The CAR2BRAIN study: a monocentric phase I trial with ErbB2-specific NK-92/5.28.z cells in recurrent glioblastoma

M Burger 1, I Mildenberg 1, C Zhang 2, K Ihrig 3, N Gökbuget 3, M Wagner 4, M Mittelbronn 5, C Senft 6, T Tonn 7, W Wels 2, J Steinbach 1

Recent preclinical studies from our institutions and other researchers suggest that natural killer (NK) cells have the potential for adoptive immunotherapy of GBM. Like T cells, NK cells can be genetically modified to express chimeric antigen receptors (CARs) that recognize tumor-associated cell surface antigens and mediate selective recognition and specific lysis of cancer cells, thereby overcoming endogenous resistance mechanisms in tumor cells. In two previous phase I clinical trials, the continuously expanding human NK cell line NK-92 has been safely applied as an allogeneic cell therapeutic with clinical responses observed in some of the cancer patients treated. To enhance efficiency and specificity, the ErbB2-specific cell clone NK-92/5.28.z carries a codon-optimized CAR (CAR 5.28.z) based on ErbB2-specific antibody FRP5 and CD28 and CD3ζ signaling domains. Elevated ErbB2 protein levels have been reported in a significant proportion of GBM tumors and were correlated with impaired survival. We have previously demonstrated potent antitumor activity of NK-92/5.28.z cells in vitro and in orthotopic GBM models in vivo, suggesting adoptive transfer of these cells as a promising new approach for immunotherapy of ErbB2-positive glioblastoma andother ErbB2-expressing cancers. Based on the convincing preclinical data, we consequently designed the CAR2BRAIN trial, a monocentric phase I dose finding trial investigating the safety and tolerability of NK-92/5.28.z cells in patients with recurrent glioblastoma. The NK-92/5.28.z cells will be repeatedly injected through a Rickham reservoir into the resection cavity or the tumor center. In a dose escalation part of the trial the highest cell number which can be applied safely will be established (maximum tolerated dose = MTD). We plan to escalate the dose up to 1 × 108 cells per injection in four injections. After determination of the MTD, up to twelve injections will be carried out to establish safety of prolonged treatment. Distribution of the injected NK-92/5.28.z cells in the brain, the cerebrospinal fluid and the blood will be monitored. Furthermore, the immune reaction triggered against the target antigen ErbB2 as well as ErbB2-independent immune reactions will be characterized.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):74.

P10-20 CliniMACS Prodigy® Concentrate application: Fully automated, continuous, single-use centrifugation technology used for generation of cell products and cell-free supernatants for research and clinical use

A-K Beinhauer 1, A Arendt 1, D Dietrich 1, I Spiegel-Cürten 1, V Huppert 1, B Kauling 1

Cell- and gene-based therapies have great potentials to prevent and treat diseases, where other conventional methods are possibly inadequate. Cell therapy is based on the transplantation of living cells into patients to repair or reconstruct missing or defective functions. Various cell types are expanded in large scale cell cultures e.g. in shaking or stirring bioreactors, cell culture bags or cell stacks before they are administered to the patient. Cell washing, medium change and concentration of cells are often performed steps when cells are cultivated in large scale cultures. Frequent non-automatic processes may affect subsequent functional analyses and may lead to contamination of the cell product.

Therefore, we designed a clinically applicable and highly flexible CliniMACS Prodigy® Concentrate application which can be used to harvest cells from large volume cultures (up to 50 L) to a minimum volume of at least 60 mL in a sterile, closed and fully automated system. The concentrated cells e.g. tumor infiltrating lymphocytes (TILs), CAR-T cells or NK-cells can be used for research, diagnostic or therapeutic purposes.

For the application, the CliniMACS Prodigy® instrument, a single-use tubing set (TS720) and the Concentrate software are required. With this system cells e.g. from a WAVE reactor are continuously loaded into the CentriCult Centrifugation Unit (CCU) of the CliniMACS Prodigy® while the supernatant is collected in a separate bag. Medium change, cell washing and harvesting are done fully automatically. The Concentrate application is a new tool to make the related handling steps more convenient in cell therapies. Interestingly, not only the cells but also the cell culture supernatant can be used for further downstream processing when cell culture supernatant components should be isolated like for instance viral vectors for cell transduction for gene therapies. The CliniMACS Prodigy® Concentrate system can result in over 95% recovery of the cells in the target cell fraction and less than 5% of cells in the supernatant fraction (n>10) depending on the determined parameters of the customer.

In summary, we developed a concentration application in which all steps from the starting material to the final cell product are done fully automated. The automated concentration of large volume cultures using the CliniMACS Prodigy® instrument is a novel strategy to easily generate small volume cell therapy products for clinical use.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):75.

P10-21 GMP-conform matrix validation of a dual temperature sterility release test for ATMPs

N Arlt 1, R Rothe 1, B Ludwig 2, M Odendahl 3, T Juretzek 4, H Peltroche 4, T Tonn 3,5, R Moog 1

Background

In manufacturing ATMP treatments, like natural killer cells (NK) preparations for cancer therapy or pancreatic islet cell transplants for type 1 diabetes mellitus therapy, sterility is an indisputable necessity and need a good manufacturing practice (GMP) admission performance.

Methods

The matrices validations were performed with NK-92-wild-type cell line (in X-Vivo 10 cell culture media supplemented with 5% heat inactivated plasma and 500IE/ Interleukin-2) and fresh isolated rat islet cells using a blood culture device (Bact/Alert®3D with low temperature module, bioMérieux, Nürtingen, Germany). Samples were spiked with microbes recommended by the Paul-Ehrlich-Institute with respect to Ph. Eur., General Chapter 2.6.1/2.6.27. The following strains were spiked twofold > 10 colony forming units (CFU) in iAST and iNST: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans, Aspergillus brasiliensis, Clostridium sporogenes, Propionibacterium acnes and in islet cells Escherichia coli. Aerobic bottles were incubated at 22.5°C in the Bact/Alert®3D low temperature module and anaerobic bottles at 35°C in a Bact/Alert®3D 240 incubator.

Results

The Bact/Alert®3D-System detected all spiked bacteria, yeast and mold appropriate to their aerobic and anaerobic growth behavior in NK-92 wildtype cells (Fig 1A) and islet cells (Fig. 1B) except Clostridium sporogenes. The latter can we explained through the presence of living cells, which expand the optimal culture conditions resulting in an inhibition of growth. Successful media validations were performed. All negative controls and the carried culture media were confirmed as negative. All results were reproducible.

Fig. 1.

Fig. 1

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Conclusions

Our study showed that Bact/Alert®3D-System safely detects spiked bacteria, yeast and mold in NK-92 wildtype cells and rat islet cells and is suitable for sterile release testing of ATMPs. We recommend an incubation time of 7 days for further routine investigations.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):75.

P10-22 Distinct and homogenous surface expression of the transferrin receptor CD71 on murine plasmacytoid dendritic cells in different tissues and lymphatic organs

A Lippitsch 1, N Baal 1, G Bein 1, H Hackstein 1

Background

Plasmacytoid dendritic cells (pDC) are of increasing interest as cellular mediators for therapeutic vaccination of patients with cancer but many functions of these highly specialized antigen presenting cells are purely understood. CD71 is the transferrin receptor and functions as iron transporter into the cell via receptor-mediated endocytosis. Cells with a high metabolism, i.e. proliferating cells like B cells, reveal a higher expression of CD71. Investigating murine respiratory pDC we found a distinct high CD71 surface expression up to 100% in comparison to other leucocyte populations of the lung. In other analyzed murine lymphatic and non-lymphatic tissues this high CD71 expression of pDC was also found except in bone marrow and blood.

Methods

Lymphatic and non-lymphatic tissues of C57BL/6 mice were prepared and immediately analyzed by flow cytometry (BD Aria II) using fluorochrome-conjugated monoclonal antibodies.

Results

Investigating non-lymphatic tissues such as lung, liver and small intestines, and lymphatic tissues such as spleen, thymus, mediastinal lymph nodes and Peyer's patches, we found a unique high CD71 expression on pDC between 90% and 100% in comparison to other representative leucocyte populations in the respective tissue, including other DC subsets. In contrast, the CD71 expression of non-tissue pDC in bone marrow was reduced (around 80%). An even lower CD71 expression, i.e. around 20%, was found in the blood compartment.

Conclusion

Our experiments revealed a unique high level of CD71 surface expression on tissue pDC in comparison to other leukocyte subsets. The cause and function of this tissue-dependent high CD71 expression of pDC remains to be examined.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):75–76.

P10-23 Different platelet concentrations impact on DC maturation

M Six 1, J Strobel 1, R Zimmermann 1, R Eckstein 1, E Strasser 1

Introduction

This study shows the influence of platelets on the differentiation and maturation of dentritic cells (DCs) derived from monocytes, focusing on the influence of different platelet concentrations in the mononuclear cell culture.

Methods

Mononuclear cells (MNCs) were obtained by leukapheresis and CD14 positive monocytes were isolated with CD14 MicroBeads. The MNCs were cultivated in a culture medium with IL-4, GM-CSF and TNF-α (starting on day 4). The following culture preparations were performed supplementing platelets in different concentrations: Control without platelets added (baseline), TH10 with addition of 1.0 × 10e7 platelets, TH50 (5.0 × 10e7 platelets), TH150 (1.5 × 10e8 platelets) and TH250 (2.50 × 10e8 platelets). The different cell culture preparations were analyzed by flow cytometer on days 1 to 4 and on day 7.

Results

On day 7, high platelets concentrations TH150 (24.0 ± 10.9) and TH250 (24.3 ± 14,0) showed a significantly (p = 0.036) lower ratio of CD1a positive cells than in the baseline (51.8 ± 21.3%). The ratio of CD83 positive cells was significantly (p = 0.028) lower on day 1 for TH250 (4.5 ± 4.1%) compared to baseline (9.3 ± 7.1%). No significant difference (p = 0.139) could be found on day 7 between baseline (42.0 ± 17.4) and TH250 (55.4 ± 23.6). The difference between the baseline (42.0 ± 17.4) and TH10 (52.0 ± 12.6) was significant (p = 0.007). Regarding the measurement of cell viability, there was a significant correlation between the ratio of live cells and the platelets concentration analyzed (day 1: p = 0.021; day 2–7: p values < 0.001). On day 7, there was a significantly (p < 0.001) lower ratio of vital cells in the baseline (29.0 ± 24.5) compared to TH250 (73.8 ± 22.6). Negative correlations were seen between the ratio of dead cells and the platelets concentration regarding all days (p values < 0.001).

Conclusion

Platelets show an inhibitory effect on premature DCs (CD1a positive cells) and apparently, in the further process, a boosting effect on the maturing process of DCs (CD83 positive cells) derived from monocytes. In addition, the part of Annexin V and 7AAD positive cells depends on platelet concentration. The higher the platelet concentration, the lower was the ratio of dead cells.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):76.

P10-24 Epigenetic mechanisms as targets for a pharmacological control of hematopoietic lineage differentiation

ON Kuvardina 1, S Herkt 1, H Boenig 1, E Seifried 1, J Lausen 1

Background

During hematopoiesis binary lineage fate decisions are decisive for a balanced supply of differentiated functional blood cells. This equilibrium is deregulated in thrombocytopenia or anemia and proliferative diseases such as myelodysplastic syndrome or leukemia. Lineage differentiation is controlled by transcription factors and their associated epigenetic function. The knowledge of the molecular mechanisms underlying lineage decisions in hematopoiesis can provide means to therapeutically alter differentiation in patients or to produce defined hematopoietic cell types such as thrombocytes in cell culture for cell therapy.

Methods

To unveil molecular targets for a manipulation of hematopoietic lineage fate decision we are examining the influence of hematopoietic transcription factors and their epigenetic interaction partners at the megakaryocytic/erythroid branching point. For this we use genome wide expression analysis in human CD34+ stem cells. We are studying changes in transcription factor occupancy during differentiation using chromatin immunoprecipitation. Furthermore, we utilize cell culture based differentiation assays and the mouse model to define the role of specific transcription factors and epigenetic cofactors.

Results

We found that the transcription factor RUNX1 is repressing the erythroid gene expression program during megakaryocytic differentiation by recruitment of an epigenetic corepressor complex, which includes the protein arginine methyl transferase 6 (PRMT6). PRMT6 triggers the repressive histone modification H3R2me2, which down modulates the active H3K4me3 mark. This way erythroid gene expression is inhibited. Preliminary data show that inhibition of PRMT6 alters the balance between megakaryocytic and erythroid differentiation.

Conclusion

Our data show that manipulation of the transcriptional complexes containing RUNX1/PRMT6 can alter the balance between megakaryocytic and erythroid differentiation. Further experiments are aimed to develop a small molecule inhibitor of PRMT6 as a potential tool to alter lineage differentiation pharmacologically.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):76.

P11-1 Volume flows during preparative plasmapheresis

ST Kiessig 1

Aim

Unexpected events like hypovolemic reactions during plasmapheresis are rear. A broader knowledge about the exchange of volumes between the different compartments is required to adapt guidelines for an evidence based medicine. During plasmapheresis an extracorporeal volume up to 1100mL can be reached (850mL Plasma + 350mL in the set). The exchange of volumes was observed either by impedance spectroscopy or by the dilution of plasma proteins.

Methods

The donation volumes were adjusted according to German guidelines. 1:16 Citrate was added to the whole blood collected, 300mL of NaCl was given.

The different compartments were evaluated in 41 donors before (n = 21) and after plasmapheresis (n = 20) using impedance spectroscopy (BCM, Fresenius Medical Care AG, Hof/Saale, Germany). BCM measurement procedure followed plasmapheresis either at the same day before and 10–20 min after or before and two days after plasmapheresis. In a second study, the serum content of IgM, IgG, IgG subclasses, Albumin and total protein before and immediately after apheresis were determined by standard procedures. The dilution factors were calculated from these values and compared.

Results

An average of 794mL plasma including citrate volume was collected from donors, here average of about 159mL citrate was used. 133mL citrate went into the product and 26mL into the donor. The Intracellular water was nearly uninfluenced (25.05 before vs. 25.01 L after). Extracellular water decreased by 0.25L (19.38 before vs. 19.13 L after). A total loss of intravasal fluids of 130mL (0.47L before NaCl substitution) was found. The exchange between the compartments was already immediately detectable demonstrating highly accessible extracellular water. By the dilution factor method a loss of 334mL was found. Differences between both methods can be explained by the time gap and the fast exchange of volumes already during plasmapheresis.

Conclusions

Both methods are able to detect the rapid balancing of volumes between the highly accessible interstitial compartment and the plasma compartment in the same range. Between 11% and 18% of the plasma volume was quickly replaced by interstitial volume showing that there a no clear physiological reasons for hypovolemic reactions at this stage of investigations.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):76–77.

P11-2 Quality of source plasma collected with the Aurora plasmapheresis system

T Burkhardt 1, R Rothe 2, A Karl 1, T Tonn 3, R Moog 2

Introduction

The quality of plasma for fractionation is of utmost importance. Total Protein (TP), IgG levels and citrate levels were measured as quality indicators before, during and after plasmapheresis. Furthermore, parameters of product quality were analysed.

Material and Methods

54 plasmapheresis donors (39 male, 15 female) fulfilling current national and European eligibility criteria underwent plasmapheresis using the Aurora Plasmapheresis System (Fresenius, Lake Zurich, IL, USA). Donors’ peripheral blood counts were analysed berfore and after plasmapheresis using an electronic counter (Sysmex, Sysmex Corporation, Kobe, Japan). Platelets and red blood cells in the plasma products were determined by means of a Neubauer hemacytometer. Samples during apheresis were drawn every 200 mL of collected plasma. TP an IgG were measured turbidimetrically (AU 640, Beckman Coulter, Krefeld, Germany) and citrate was measured photometrically (Dr. Lange, Berlin, Germany).

Results

An average of 2,751 ± 247 ml blood was processed in 47 ± 6 min. Citrate consumption was 177 ±15 mL and the collected plasma volume was 850 ± 1 mL. Blood counts, total protein und IgG before and after apheresis are shown in table 1. IgG during plasmapheresis is depicted in figure 1 (samling every 200 mL of collected plasma).

Table 1.

TP, Ig and blood counts before and after apheresis

Parameter Before apheresis After apheresis
TP (g/dL) 6.83 ± 0.33 6.03 ± 0.28
IgG (mg/dL) 817.4 ±185.9 720.0 ± 162.4
WBC (103/L) 6.6 ± 1.6 6.6 ± 1.7
RBC (1012/L) 4.92 ± 0.29 5.23 ± 0.27
Hb (g/dL) 14.8 ± 1.0 15.7 ± 1.0
PLT (103/L) 232 ± 55 234 ± 58
MPV (fL) 9.6 ± 1.1 9.7 ± 1.0
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Fig. 1.

Fig. 1

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IgG level during plasmapheresis

Conclusions

IgG levels during apheresis showed only a slight decrease allowing for the collection of a plasma with good quality.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):77.

P11-3 Open systems for extracorporeal photopheresis do not require a cleanroom environment

T Legler 1, H Budde 1, F Streit 2, L Binder 2, J Riggert 1

Background

Extracorporeal photopheresis (ECP) can be performed by a leukapheresis procedure followed by the addition of 8-methoxypsoralen (8-MOP), ultraviolet A (UVA)-irradiation and subsequent transfusion. We investigated the influence of a bacterial filter for the addition of 8-MOP to the leukapheresis product on the 8-MOP concentration in the final cellular therapy product.

Methods

In this study an UV-permeable bag was filled with 160 ml sodium chloride (NaCl). In addition 8-MOP (40 μg) was applied with (n = 9) and without (n = 9) bacterial filter. Subsequently 40 ml NaCl were added. Microbiological tests were performed in cultrue bottles after the final manipulation step. A LC-MS/MS method for determination of 8-MOP in leukapheresis products and EDTA-plasma has been validated. After protein precipitation and centrifugation supernatant was injected into the UPLS-MS/MS system (Waters, Milford, MA, USA). A short gradient (HSST3 100*2.1mm, 1.8μm column) was performed for chromatographic separation before ESI-MS/MS detection. Imprecision was 3.6%, 4.3% and 8.9% at 400.0 μg/L, 100.0 μg/L and 0.5 μg/L, respectively.

Results

No bacterial growth was observed in the microbiological control of each experiment. The mean 8-MOP concentration in products prepared with filter was 140 ± 15 μg/L whereas in products prepared without filter the concentration was slightly higher (154 ± 23 μg/L, p > 0.05).

Conclusions

The whole procedure is completed within less than 6 hours, a period which is associated with a low risk for bacterial contamination even if open systems are used. A new 8-MOP quantification method was developed which allows quality control of routine preparation of cellular products for ECP. The microbiological safety can be increased by the use of a bacterial filter for the addition of 8-MOP to the apheresis product. Cleanroom environment is thus not required for manufacturing ECP products in open systems.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):77.

P11-5 Propionibacterium acnes, a slow-growing bacterium with a long detection time? A validation with 7 matrices of blood components and ATMPs

N Arlt 1, R Rothe 1, T Juretzek 2, H Peltroche 2, T Tonn 3,4, R Moog 1

Background

Slow-growing bacteria like Propionibacterium acnes represent a challenge for quality control investigations in sterility release testing.

Methods

Here a convenient validation with 7 matrices was performed using: buffy coat, stem cells, islet cells, natural killer cells, red blood cells, platelets and plasma in the microbial detection system Bact/Alert®3D incubator with Dual-T- module. All matrices samples were spiked with microbes recommended by the Paul-Ehrlich-Institute with respect to Ph. Eur., General Chapter 2.6.1/2.6.27. Propionibacterium acnes with 55 colonies forming units (CFU) were spiked twofold in iAST and iNST culture bottles for 14 days using multishot bioballs from biomeréux. Additionally the stem cell preparation was also incubated in iFAplus and iFNplus culture bottles, which include neutralizing polymers. Aerobic bottles were incubated at 22.5°C in the Bact/Alert®3D low temperature module and anaerobic bottles at 35°C in a Bact/Alert®3D 240 incubator.

Results

The Bact/Alert®3D-System detected the Propionibacterium acnes shown in Fig. 1 in anaerobic culture bottles in buffy coat [3.3 d (=positive signal day to detection as mean value)], red blood cells [3.21 d], platelet cells [], plasma [3.67 d], natural killer cells [3.255 d] and islet cells [4.845 d]. No growth of the bacterium was found in stem cells using iAST and iNST culure bottles. Compared to iFAplus and iFNplus culture bottles where Propioni could be detected in stem cell matrix [6.34]. A successful media validation was performed. All negative controls were conformed as negative (data not shown). All results were reproducible.

Fig. 1.

Fig. 1

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Conclusions

Our study shows that Bact/Alert®3D-System safely detects the slow-growing bacteria Propionibacterium acnes in different matrices in a practical way except stem cells, which can may be explained by antibiotic prophylaxis and can be circumvented using iFA/iFNplus culture bottles.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):78.

P11-6 Clinical and functional aspects of HLA-G positive extracellular vesicles in breast cancer

L König 1, S Kasimir-Bauer 1, A-K Bittner 1, S Schramm 2, B Wagner 2, Manvailer LF Santos 2, B Giebel 2, R Kimmig 1, PA Horn 2, V Rebmann 2

The non-classical human leukocyte antigen G (HLA-G) molecule and its soluble forms exert multiple immune suppressive functions in malignancy and in stem cells contributing to immune escape mechanisms. Soluble HLA-G (sHLA-G) can be released as free sHLA-G molecules or via extracellular vesicles (EVs). Extracellular vesicles are secreted vesicles representing important elements for intercellular signal transduction often being operative in tumor immune evasion. Our study on EVs harbouring sHLA-G (sHLA-GEV) derived from blood plasma of locally advanced, neoadjuvant chemotherapy treated BC patients (N = 190) revealed (i) elevated sHLAGEV level in BC patients compared to healthy controls (ii) an association between high sHLA-GEV level and disease progression and (iii) the occurrence of stem cell-like circulating tumor cells. For this reason, we investigated the uptake of sHLA-G pos EVs by peripheral blood cells (PBLs), with respect to HLA-G specific receptors ILT2 and ILT4 expression. HLA-G pos EVs were isolated from gynaecological, malignant ascites fluids by non-blocking HLA-G antibody after differential ultracentrifugation and PKH67 staining. The uptake was visualized using AMNIS image Stream technologies. HLA-G-bearing EVs were up-taken by ILT2 and/or ILT4pos monocytes and B-cells in an accumulating fashion, whereas some ILT2pos and ILT2neg T cells seized HLA-Gpos EVs up in a punctual manner. Cytokine array analysis showed that HLA-G pos EVs contained a higher proportion of VEGF, uPAR, TRAIL receptors and others, which are mostly involved in tumor proliferation, metastases and angiogenesis. In contrast, HLA-G neg EVs displayed elevated levels of pro-inflammatory, chemotactically and cell adhesion factors, such as Fas ligand, E-selectin, PECAM and various chemokines. In conclusion, sHLA-GEV are of prognostic relevance in BC, are uptaken by ILT2pos and some ILT2neg PBLs and contain mediators known to promote tumor spread in gynaecological tumor entities.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):78.

P11-7 Filling the dbGaps – Completing the HLA-DQB1*06:37 sequence by combining long range PCR (LR-PCR) with next generation sequencing (NGS)

TMC Binder 1, R Kelsch 2, A-L Hansen 3, U Kordes 4, H Kabisch 4, P Kühnl 1, M Schäfer 3, W Peter 3, TH Eiermann 1

Some years ago, HLA-typing (rSSO & SBT) of a young Turkish patient with Wiskott-Aldrich syndrome (WAS) revealed a DQB1*03:02:01 in combination with a new HLA-DQB1*06:02 variant, later named as DQB1*06:37. The DQB1*06:37 allele differs from DQB1*06:02:01 in two non-synonymous nucleotide positions in exon 2 (codon 70: AAC→AAG, Asn→Lys and codon 78: GGG→AGG, Gly→Arg). The DNA sequences outside exon 2 were not determined initially. Complete sequence data for HLA class II alleles are still limited and difficult to obtain due to special features of the sequences itself. It was our intention to develop a workflow based on LR-PCR and NGS to provide the complete genomic sequence of this allele. Therefore, we designed different DQB1 specific LR-PCRs. After DQB1 amplicon generation, NGS (MiSeq, Illumina), data evaluation with two different HLA software tools (NGSengine, GenDx and Omixon Twin, Omixon) we ended up with allele-specific contigs computed relying on a phasing analysis of the individual single nucleotide variants (SNVs) pattern present. The final alignment of these contigs was performed with the BioEdit (ClustalW) software together with the published genomic sequences of the DQB1*06:02:01, DQB1*06:37 (exon 2 only) and DQB1*03:02:01 alleles (IMGT/HLA database, http://www.ebi.ac.uk/imgt/hla/). With our approach, we were able to determine the complete gene sequence of DQB1*06:37 (from 5'-UTR over all exons and introns to 3'-UTR), which is identical to the published gene sequence of DQB1*06:02:01 differing only in the two above mentioned nucleotide positions within exon 2. The full-length sequence of the second DQB1 allele (DQB1*03:02:01) of the patient is identical to the published gene sequence. Applying LR-PCR and NGS including phased sequence analysis we were able to identify the complete gene sequences of both DQB1 alleles (DQB1*06:37 & DQB1*03:02:01) of this patient separately. We hope, that in the near future it would become easy to identify the complete sequences of HLA-alleles with the combination of these methods. Apart from compatibility testing this would be helpful especially for evolutionary and ancestry studies.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):78.

P11-8 Donor safety in haemapheresis: Implementation of the IHN-standard 2014 into the online system for assessment and evaluation of donor and apheresis complications

H-G Heuft 1, T Burkhardt 2, G Leitner 3, T Weingand 4, EG Fischer 5, B Mansouri Taleghani 6; Haemapheresis Vigilance Working Party

Aims

To assess and evaluate complications related to preparative haemaphereses, we included the IHN-standard 2014 into the recently developed Internet-based Haemapheresis Vigilance system: http://haemapheresisvigilance.eu

Material and Methods

All potential adverse events (AE: venous access problems [VAP], circulation reactions [CR], citrate toxicity [CT], donor compliance [DC], technical events [TE, apheresis machine/disposable related] and combinations of several events) are assessed and evaluated with respect to preparative plasmaphereses, plateletphereses, leukaphereses (blood stem cells, granulocytes, monocytes), red cell and multicomponent aphereses. To avoid operator-specific interindividual variability the grading for mild, moderate or severe reactions is based on the operator's interventions rather than on the operator's subjective estimation of the severity of the complication. An automated evaluation program allows comparing the centre-specific complication rate with corresponding rates of other centres from the same institution as well as with the benchmark of all participating centres.

Results

Currently, 20 centres (Germany, n = 15, Switzerland, n = 4 and Austria, n = 1) provide data. From January, 2012 to January, 2016 a total of 17093 AE out of approximately 145.000 haemaphereses have been recorded to the system. The distribution of AE to the apheresis procedures was 63.0% for plasmaphereses, 34.5% for plateletphereses, 2.2% for stem cell aphereses, 0.3% for the other leukaphereses. The registered AE comprised VAP (n = 9338; 55.9%), CT (n = 1893; 11.3%), CR (n = 1.930; 11.6%), DC problems (n = 2518; 15.1%), TE (n = 1414, 8.5%), and combinations of several AE simultaneously (e. g. VAP plus CR, n = 396, 2.3%). The AE were graded as mild 90.3%, moderate 8.2% or severe 1.5%. The program for the centre-specific AE evaluation including benchmark function was optimized to take place within 15–30 minutes.

Conclusion

The Internet based Haemapheresis vigilance system provides a stable, comprehensive and effective platform to assess and evaluate apheresis related donor complications for regulatory as well as for scientific purposes.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):79.

P11-9 Examinations of platelet function in voluntary first-time plateletpheresis donors

R Zimmermann 1, H Dankerl 2, A Martin 2, DR Weiss 1, J Strobel 1, R Eckstein 1

Introduction

An aspect which is sometimes held against plateletapheresis is the assumption that the apheresis donor sets his own coagulation unnecessarily at risk. There are a few studies reporting frequently detectable impairments of platelet aggregation responses in platelet donors. Since donor safety is one of the most important goals in Transfusion Medicine, it was our aim to investigate if the system of the so-called primary haemostasis is compromised by becoming a plateletapheresis donor.

Material and Methods

We examined several coagulation parameters in 49 first-time donors to get initial values. From these, 20 subjects recurred and became thrombocytapheresis donors.

Before any donation, after a first whole blood or plasma donation but before any plateletpheresis donation, and a third time after a first plateletpheresis donation, the following aspects of primary hemostasis were examined: Platelet count, von Willebrand factor, and factor VIII were measured as plasmatic components of primary hemostasis. Platelet function was studied by whole blood impedance aggregometry using the Multiplate® apparatus. In-vitro bleeding time was examined using the Platelet Function Analyzer PFA-100®.

Results

The platelet count did not significantly change over the three measuring times. In addition, we could not determine a significant influence of becoming an apheresis donor on the examined plasmatic coagulation factors or any significant change of the tested constituents of primary haemostasis in newly gained apheresis donors. At all time points however, we found a surprisingly high number of slightly pathological results of platelet aggregation responses to weak aggregating molecules as well as in the in-vitro bleeding time.

Conclusions

We could demonstrate that the examined parameters of primary hemostasis are not affected by becoming a plateletpheresis donor. Such donors are not exposed to increased risks of bleeding or thrombosis by the donation process itself. With regard to the relatively large number of slightly pathological values of platelet aggregation responses to weaker aggregation triggers as well as in the measurement of the in-vitro bleeding time it is to discuss whether some donors might use drugs or dietary supplements with effect on their platelets’ function. Alternatively, it is also possible that the established reference ranges of certain tests of primary hemostasis are too narrow and require adaptation.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):79.

P11-10 Knowledge, attitude and practice regarding the voluntary blood donation among the young student population of Karachi.

B Nepal, 1

Introduction

Safe blood is a crucial and irreplaceable component in the medical management of many diseases. The Voluntary non-remunerated blood donation is the ideal sources of quality blood, which forms less than 15% of the demand of the blood in Pakistan. Motivation among the youth, particularly students, is essential to make voluntary blood movement more successful.

Aims

To assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of Karachi so that an effective approach can be made regarding motivation enrollment of voluntary non remunerated blood donors in future in Pakistan

Methods

A cross sectional prospective study was conducted among 600 students from different universities and colleges of Karachi. A well-structured and pre-tested questionnaire, in English, was used to access the knowledge, attitudes and practices about voluntary blood donation. A scoring mechanism was used to understand overall knowledge level. The participants were given a briefing about the objectives of the study and confidentiality about the personal data. Obtained data was analyzed by using statistical package of social sciences (SPSS) version 17.0 in computer. Statistical significance level was set at p≤0.05.

Results

The sample population consisted of 54% male and 46% female students in the age group of 18–28 years. Only 65% of the students have heard about voluntary blood donation and 28% of the students have given blood once in their lifetime and among them 19% are blood donors at the moment. 42% of the participants believed that there is a specific reason why they don't donate blood and 59% believed that there is a risk involved for the donors, when donating blood. 80% students wanted to promote voluntary blood donation. Fear and lack of awareness on blood donation are the reasons for not donating blood. Students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends.

Conclusions

This study showed that myths and misconceptions are leading the youngsters not to donate blood. Study also showed how increasing awareness and marketing through different ways can boost the culture of voluntary blood donation in society. Student population can be motivated to participate in different ways. There is a dire need to mobilize the electronic media for educating our youth about voluntary blood donation due to its access to masses.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):79.

P11-11 Five years of hemovigilance reports of complications of the blood donation reported at a tertiary care centre in Karachi

B Nepal 1

Introduction

There is a minor chance of risk among blood donors. Even though blood donors are usually screened for the presence of risk factors, sometimes blood donations can put a person at panic.

Objective

The safety of the blood supply depends on the actions to protect both; blood transfusion recipient and the blood donor. Hemovigilance practice of learning of complications of blood donation and protecting them from such complications is the best way to minimize the risk to blood donor.

Methods

Comprehensive blood donor hemovigilance program was studied at Dr. Ishrat ul Ebad Khan Institute of blood diseases, Karachi from 2010 to 2015. Outlines of reported and communicated complications were collected after whole blood donation. Analysis was done by general logistic regression.

Results

Complications after 30,000 Whole blood donation procedures calculated 1620 total. (54 per 1,000 donations). The majority of the complications were faint and pre-faint reaction with light headedness (58.6%), Sore arm (24%), Bruises and hematoma (14.4%). Minor complications were Agitation/sweating (2%) and arterial puncture (1%). Markers of the complications were age, sex, race, weight, blood pressure and donation status. All associated independently after whole blood donation. Age and first-time status were associated with a significantly higher risk of complications with 18–22 years old at higher risk compared to 23 to 50 years old. First-time donor were at higher risk compared to repeat donor.

Conclusion

The results of this study are helpful in identifying and understanding the promoter to complication of blood donation. Donor age and status were strong predictors of complications. The remedies and specific areas of care should be provided.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):80.

P11-12 Viral inactivation and enrichment of Factor VIII, Factor XIII, Fibrinogen and von Willebrand factor (VWF) multimers from fresh frozen plasma (FFP) using,” VIPS Plasma, Virus Inactivation Treatment System”

B Nepal 1, N Mughal 1, Mukhtar Z Zainab 1, Ebad Khan I Ul 2

Introduction

General use of plasma components includes replacement for multiple coagulation factor deficiencies or for treatment of single coagulation factor deficiencies for which a concentrate is unavailable. Four major products currently transfused are fresh frozen plasma (FFP), plasma frozen with 24 hours of phlebotomy (FP24), cryoprecipitate poor plasma (CPP), and thawed plasma. FP24, CPP and thawed plasma contain decrease amounts of labile coagulation factors. Moreover there is a risk of viral transmission from single-donor blood components such as fresh frozen plasma (FFP) and cryoprecipitate if it is not subjected to a viral inactivation procedure.

Aim

To assess viral inactivation and, Factor VIII, Factor XIII, Fibrinogen and von Willebrand factor (VWF) multimers enrichment capacity of, “VIPS Plasma, Virus Inactivation Treatment System”.

Method

VIPS Plasma, Virus Inactivation Treatment System” comprise of interconnected bag system where the S/D reagents are removed by filtration and the final products subjected to bacterial (0·2 μm) filtration. Cryoprecipitate mini-pools (400 ± 20 mL) were subjected to double-stage S/D viral inactivation, followed by one oil extraction and a filtration on a S/D and phthalate [di(2-ethylhexyl) phthalate (DEHP)] adsorption device and a 0·2 μm filter. The initial and the final products were compared for visual appearance, blood cell count, factor VIII, Factor XIII, Fibrinogen and Von Willebrand factor (VWF) multimers. Initial and final products were also checked for HIV, HBV, HCV, dengue, malaria and bacterial contaminations.

Results

Our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor VIII, Factor XIII, Fibrinogen and von Willebrand factor (VWF) multimers were well conserved (Table 1). Kit ensured bacterial sterility (Table 3) and most importantly, final product was free of HBV, HCV and HIV (Table 2).

Table 1.

Test performed at Aga Khan Labs

Sr. No Factor Start % Final %
1. Von Willebrand factor 418% 435%
2. Factor VIII 183% 251%
3. Fibrinogen Level 754.1mg/dl 672 mg/dl
4. Factor XIII (quantative) 93.9% –
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Table 2.

Molecular Biology: Performed at Molecular lab, DDRRL

Sr. No Test (By PCR) Pre-analyasis Post-analysia
1. Hepatitis B Virus Negative Negative
2. Hepatitis C Virus Negative Negative
3. HIV Virus Negative Negative
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Conclusion

It's the first time, “VIPS Plasma, Virus Inactivation Treatment System”, is used in South Asia for product enrichment and viral inactivation. Results showed effective product enrichment and viral inactivation in our conditions. But further investigation is needed to characterize functional activity of the enrich component. Irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries.

Results

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):80.

P11-13 Five years of hemovigilance reports of complications of the blood donation reported at a tertiary care centre in Karachi

B Nepal 1, N Mughal 1, S Hussain 1, Ebad Khan I Ul 2, R Nazar 1

Introduction

There is a minor chance of risk among blood donors. Even though blood donors are usually screened for the presence of risk factors, sometimes blood donations can put a person at panic.

Objective

The safety of the blood supply depends on the actions to protect both; blood transfusion recipient and the blood donor. Hemovigilance practice of learning of complications of blood donation and protecting them from such complications is the best way to minimize the risk to blood donor.

Methods

Comprehensive blood donor hemovigilance program was studied at Dr. Ishrat ul Ebad Khan Institute of blood diseases, Karachi from 2010 to 2015. Outlines of reported and communicated complications were collected after whole blood donation. Analysis was done by general logistic regression.

Results

Complications after 30,000 Whole blood donation procedures calculated 1620 total (54 per 1,000 donations). The majority of the complications were faint and pre-faint reaction with light headedness (58.6%), Sore arm (24%), Bruises and hematoma (14.4%). Minor complications were Agitation/sweating (2%) and arterial puncture (1%). Markers of the complications were age, sex, race, weight, blood pressure and donation status. All associated independently after whole blood donation. Age and first-time status were associated with a significantly higher risk of complications with 18–22 years old at higher risk compared to 23 to 50 years old. First-time donor were at higher risk compared to repeat donor.

Conclusion

The results of this study are helpful in identifying and understanding the promoter to complication of blood donation. Donor age and status were strong predictors of complications. The remedies and specific areas of care should be provided.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):80–81.

P11-14 Eculizumab treatment in AB0-incompatible red blood cell transfusion: A case report

V Lenz 1, A Röth 2, S Heinrichs 1, N Fuchs 1, M Breyer 1, D Dirkmann 3, U Dührsen 2, J Peters 3, PA Horn 1

Introduction

Eculizumab is a monoclonal IgG 2/4 antibody that specifically binds to the complement protein C5, thereby inhibiting its cleavage to C5a and C5b and the generation of the terminal complement complex C5b-9. In 2015, Weinstock et al. reported its off-label use as a rescue drug in AB0-incompatible transfusion of red blood cells (RBC). We here report the use of eculizumab in a case of AB0-incompatible transfusion with 0/A blood group constellation: One unit of RBC with blood group A Rh pos. was given to a patient with blood group 0 Rh neg. and anti-D.

Methods

AB0-incompatible transfusion occurred in a 22-year-old spina bifida patient during general anesthesia. The mistake was not noticed until after extubation. The patient felt unwell, shivered and had hemoglobinuria but was cardiorespiratory stable. Forced diuresis was induced immediately, 2,5 hours after transfusion 900 mg eculizumab were infused. After transfer to intensive care, forced diuresis was continued for three more days. LDH, haptoglobine and bilirubine were checked twice a day. Immunohematological workup: We performed daily flow cytometric analysis (FCA) of A/0 BG chimerism, ABD blood group and DAT (Grifols DG Gel® cards), monospecific DAT (mDAT, BioRad DC Screening), anti-A1 IgM and IgG titers (tube test) from samples before and after transfusion until day 6. Titers of anti-D were obtained from samples before transfusion and day 5. Additional samples for complement studies (C3, C4, CH50) were obtained from day 1–6.

Results

The patient fully recovered and left intensive care on day 3 with normalized lab parameters. FCA showed a maximum chimerism of 11% at the evening of the event and declined to 5,3% 24 hours later. During the next days, values decrased to <0,05% on day 4. This was consistent with gel card tests: Up to 5% chimerism, blood group testing showed a mixed field agglutination with anti-A and anti-D, and DAT/mDAT were at least positive with IgG and C3d. Gel card testing of samples with FCA values below 5% were negative. Anti-A1 titers IgM/IgG before incompatible transfusion were 8/32, declined to 0/1 after the event and were back to 8/32 on day 3. Day 6 showed boostered titers of 128/1024. Anti-D IgG, before transfusion at a titer of 16, was boostered to 128 at day 5.

Conclusion

While this case presents an AB0-incompatible transfusion in the most feared 0/A setting, the low preexisting anti-A1 titers were obviously fortunate, as the patient remained clinically stable even before eculizumab was administered 2,5 hours later. The transfused cells were completely eliminated within four days, thus preventing repeat administration of eculizumab. As gel card results for ABD and DAT were consistent to FCA down to 5% chimerism, the elimination progress might be assessed with gel card tests alone if FCA is not available.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):81.

P11-15 A meiotic crossing over in the rare haplotype HLA-A*30:02-B*18:01-C*07:01-DRB1*11:04-DQB1*03:01 of a Caucasian patient's sibling leading to a common spread haplotype

P Paranikulangara 1, P Becker 1, P Krenn 1, P Bader 1, E Seifried 1, C Seidl 1

Introduction

Crossing over in chromosome 6p21.3 is a rare recombination mechanism crucial for polymorphic diversity in HLA system. During the search for a family related bone marrow donor for a pediatric patient suffering from acute lymphatic leukemia, we observed interchanged maternal HLA class I and II haplotypes in his sister's HLA pattern. Here we describe a spontaneous maternal meiotic crossing over in a rare haplotype leading to rearrangement of HLA class I and II haplotypes in the female pedigree.

Method

Low resolution HLA class I typing of the patient, mother, father and two siblings were performed with serological testing for HLA-A, -B and -C and by Sequence Specific Priming (SSP) for HLA-DRB1,-DRB3, -DRB4, -DRB5, -DQB1. Results are confirmed by high resolution sequence based typing of exon 2 and 3 for HLA-A,-B,-C, -DQB1 and exon 2 for -DRB1 and -DPB1. Typing of HLA-DRB3, -DRB4 and -DRB5 was performed by high resolution SSP. Sequences were analyzed by the Sequence Pilot software. Segregated haplotypes are compared with the data from Allele Frequency Net Database for worldwide populations.

Result

From family segregation analysis, the patient's mother showed the following haplotypes: A. HLA-A*30:02-B*18:01-C*07:01-DRB1*11:04-DQB1*03:01 and B. HLA-A*24:02-B*18:01-C*12:03-DRB1*01:01-DQB1*05:01. Besides paternal haplotype (HLA-A*02:01-B*44:02-C*05:01-DRB1*04:01-DQB1*03:01) the patient's sister include class I of the maternal haplotype A and class II of maternal haplotype B (HLA-A*24:02-B*18:01-C*12:03-DRB1*11:04-DQB1*03:01).

Conclusion

The maternal chromosome in the patient's sister presents combined maternal haplotypes. Therefore the chromosomal breaking point is suspected between the HLA-B and -DRB1 loci during meiosis of the mother. The maternal haplotype A is rarely prevalent while the crossing over generated a new widespread haplotype. Documentation of rare crossing over cases like this will help to identify potential crossing over hot spots in HLA genes and contributes to modify linkage disequilibrium frequencies of HLA alleles in word wide population.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):81.

P11-16 Impact of “HSC expansion” cocktails on the cell fate of human multipotent hematopoietic cells

SVC Castro 1, A Görgens 1, PA Horn 1, B Giebel 1

Ex vivo expansion of hematopoietic stem cells (HSCs) is a major goal in the field of stem cell transplantation. HSCs reside in specialized niches which contain a heterogeneous population of cells generating signals for controlling the HSC-self renewal, quiescence and differentiation. In the past years, researchers have tried to reproduce the HSC-microenvironment by defining different in vitro culture conditions that comprise cocktails of cytokines and/or small molecules. Since the proof of principle for HSC expansion is the NOD/SCID repopulating cell (SRC) assay, many conditions were reported to allow “HSC expansion”. However, in addition to HSCs and multipotent progenitors (MPPs), so called lympho-myeloid primed progenitors (LMPPs) were recently found to contain SCR activity. We have tested a number of published culture conditions which claim to allow “HSC expansion”. Our aim is to verify whether HSCs/MPPs or LMPPs containing SRC activity are being expanded. The results showed that none of the conditions were able to support maintenance of the phenotypically defined human HSCs/MPPs.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):81.

P11-17 Optimization of the storage conditions for mesenchymal stromal cell-derived extracellular vesicles (EVs)

M Bremer 1, V Börger 1, PA Horn 1, B Giebel 1

Mesenchymal stem cells (MSCs) have been used in over 500 NIH-registered clinical trials to treat various diseases in humans. Novel data imply that MSC-derived EVs exert therapeutic effects comparable to MSCs themselves. Indeed, we have previously successfully treated a steroid-refractory GvHD patient with MSC-EVs.

The aim of the project is to optimize large-scale MSC expansion and the MSC-EVs production for clinical applications.

In this context, we need to qualify a buffer for long-term storage of MSC-EVs which prevents their degradation without affecting their biological features. Our previous experiments showed that the container used for storage can have a huge impact on the particle concentration. Since virus particles and EVs have been discussed to share physical properties, we compared buffers and plastic containers used for the long-term storage of virus with those conventionally being used in the EV-field.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):82.

P11-18 Immunomodulatory impact of mesenchymal stem cell-derived extracellular vesicles on peripheral blood cells in vitro

K de Miroschedji 1, A Görgens 1, M Bremer 1, PA Horn 1, V Börger 1, B Giebel 1

Human mesenchymal stem cells (MSCs) were administered in more than 500 NIH-registered clinical trials to patients suffering from various diseases including myocardial infarction, stroke and graft-versus-host disease (GvHD). Initially, MSCs were thought to replace lost cells in damaged tissues. Despite controversial reports regarding the efficacy of MSC-treatments, MSCs seem to exert their beneficial effects rather by secretion of immunosuppressive factors than by cell replacement. In this context, extracellular vesicles (EVs), such as exosomes and microvesicles, were identified to execute the MSCs’ therapeutic effects. Our recent successful treatment of a steroid-refractory GvHD patient and the demonstration of pro-regenerative effects in a murine stroke model confirmed the MSC-EVs’ therapeutic potential. Due to the fact that MSCs provide a heterogeneous cell entity, we compared the immunomodulatory effects of MSC-EV fractions obtained from MSCs of 20 healthy donors in in vitro assays. Our ongoing results confirm quantitative and qualitative differences within these MSC-EV fractions.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):82.

P11-19 The origin of DC and macrophage subsets in the human hematopoietic tree

F Murke 1, A Görgens 1, PA Horn 1, B Giebel 1

According to the current model of human hematopoiesis, multipotent progenitors (MPPs) create lympho-myeloid primed (LMPP) and erythro-myeloid primed (EMP) progenitor cells. Thus, subsets of myeloid cells derive from both, the lymphomyeloid and the erythromyeloid branches. Within the last few years, several new immune cell subtypes [e.g. dendritic cell (DC) and macrophage (MP) subtypes] have been described. However, their origin has not been uncovered, yet. It remains an open question whether all MP/DC subtypes derive from the same or different hematopoietic lineages.

Here, we aim to unravel the origin of different human macrophage as well as different DC subtypes. To this end, we have established an in vitro differentiation assay that allows for the generation and quantification of all current DC subtypes from hematopoietic stem and progenitor cells (HPSC). Our first results indicate that all DC subtypes derive from the lympho-myeloid branch.

Transfus Med Hemother. 2016 Aug 19;43(Suppl 1):82–87.

Author Index

Transfus Med Hemother 2016;43(suppl 1):1–84

A

Abel F. R. JS-03-4

Abels W. C. SI-09-7

Achenbach S. P06-5, SI-14-4

Adair J. E. JS-09-2

Ahrens N. P01-8, P02-20, P03-10, P03-13, P09-6

Aigner M. SI-14-4

Albert M. H. SI-14-6

Albrecht J. P10-14

Alex J. SI-04-3

Aljabri A. SI-09-1

Altermann W. JS-10-5, P08-4

Althaus K. P09-9, SI-04-3, SI-04-5

Alt-Mayer T. SI-09-5

Amann E. M. JS-03-3

Ampofo E. P06-2

Amrein K. P07-2

Andres O. SI-04-4

Andresen S. P03-3, SI-12-5

Anliker M. SI-06-4, SI-13-5

Arendt A. P10-20

Arenskrieger K. P07-5

Arlt N. P09-12, P10-21, P11-5

Arnold M.-L. P08-6, JS-10-3, JS-10-6

Arnold R. SI-09-9

Auberger J. P09-10

Aurich K. P09-9

Avendańo C. JS-01-2

Axt-Fliedner R. SI-13-3

B

Baade M. JS-11-3

Baal N. P09-13, P10-10, P10-22, JS-02-4

Babikeir Eln Mustafa K. JS-01-2

Bach C. P08-6, JS-08-8, JS-10-3, JS-10-6

Bade-Döding C. SI-09-6

Bade-Doeding C. SI-09-7

Bader P. P11-15

Bakchoul T. P02-25, SI-04-3, SI-04-4, SI-04-5,

SI-13-4, SI-14-5

Balola A. P02-21, P02-30

Bartolmäs T. P02-18, P02-21, P02-30

Bauhaus M. SI-12-5

Baumann-Baretti B. P03-3, P03-12

Bazhin A. P08-3

Becker J. U. SI-09-1

Becker P. P11-15

Beckmann M. W. P06-5

Beer A. P09-14

Bein G. P01-9, P05-2, P09-13, P10-10, P10-22,

JS-02-4, SI-11-3, SI-13-3, JS-11-5, SI-16-5

Beinhauer A.-K. P10-20

Berens C. P09-17

Berger K. SI-07-4

Besler K. P09-6

Beth L. SI-09-6

Beyer S. JS-08-5

Bieback K. JS-01-1, P10-11, P10-18

Bienemann K. P02-2

Binder L. P11-3

Binder T. M. C. SI-09-3, P08-2, P11-7

Biswas A. P06-1

Bittner A.-K. P11-6

Bittner R. P02-7

Blasczyk R. P10-6, P10-26, SI-09-1, SI-09-6,

SI-09-7

Blessing F. P03-6

Blumentritt C. SI-04-5

Böck M. P01-3, P01-5

Boenig H. JS-05-1, P09-2, P09-11, P10-14,

JS-08-5, JS-09-5

Bolbasov E. P10-5

Bonifacio E. P07-1

Boenig H. JS-09-5

Börger A.-K. P10-6

Börger V. P11-17, P11-18

Bornhäuser M. P09-5

Bosser M. P01-6

Brachtl G. P10-11

Bramhoff A. P03-6

Braumüller S. JS-03-3

Braune J. P01-1

Brauninger S. P09-11

Bräuninger A. SI-13-3

Brendel C. P09-13, JS-02-3, JS-02-4

Bremer M. P11-17, P11-18

Brennan M. JS-01-2

Brenner R. JS-03-3

Breyer M. P11-14

Brixner V. P04-3, SI-07-5

Brosig A. M. P03-10

Brys B. P03-3, P03-12

Bucher J. P08-3

Budde H. P10-1, P11-3, JS-08-4

Budde K. SI-09-4

Bugert P. SI-13-2, P02-4, P02-5, P02-8, P02-27, P06-7, P10-17, SI-04-4, JS-11-3

Buhmann R. SI-14-6

Bujnoch D. P05-5

Buldakov M. P02-6

Bunjes D. P09-14, SI-09-9

Bunos M. JS-08-5

Bunse C. P10-26

Burger M. P10-19

Burkhardt T. P11-2, P11-8

Burkhart J. P09-10

Busch D. P10-14

Buwitt-Beckmann U. P09-16

Bux J. P02-2

C

Caesar A. P02-12

Castro S.V.C. P11-16

Celik A. A. SI-09-6

Cherdyntseva N. P02-6

Chevalier E. P09-11

Cichutek K. JS-07-3

Cohen S. P10-7

Colic L. P02-27

Conradi R. P02-13

Cooper N. SI-11-3

D

D'Amato Tóthovį J. P04-6

Dankerl H. P11-9

De Miroschedji K. P11-18

Decristoforo P. P07-4

Degenhardt J. SI-13-3

Dehl J. SI-13-3

Deisting C. SI-13-3

Deitenbeck R. SI-16-4

Deitmer A. SI-16-5

Delcea M. P02-17

Delle-Chiaie L. P02-8

Dembowsky K. P09-11

Dengler T. P10-17

Denker K. SI-11-5

Depre F. P06-4

Depré F. P06-6

Detsch R. P10-4

Dhople V. P01-1

Dick A. JS-10-3

Diekmann J. JS-11-4

Diel A. P02-4

Dieplinger B. P02-11

Dietrich D. P10-20

Dirkmann D. P14-11

Doescher A. P02-1, P02-3, SI-07-3

Dombos S. P04-3, SI-07-5

Dormann F. P01-8, P03-10

Döscher A. P02-13

Dössinger G. P10-14

Douglas G. P09-11

Drawz A. P06-2

Dreier J. JS-11-4

Drexler C. P07-2

Dunai Z. A. P10-11

Duong Y. C. JS-11-5

Dührsen U. P11-14

Dürig J. JS-09-1

Dürrenfeld A. P03-12

Dütsch M. JS-03-4

Dziodzio T. P08-1

E

Ebell W. SI-09-8

Eckstein R. SI-07-1, P01-6, P03-7, P03-8,

P05-4, P06-3, P06-5, P10-4, P10-23,

P10-25, P11-9, SI-06-5, SI-14-4

Egger-Salmhofer M. P02-11

Egle A. P05-1

Ehrhardt H. SI-11-3

Ehrnthaller C. JS-01-2

Eichler H. P06-2, P06-7, P06-8, SI-06-3

Eichner R. P05-3

Eicke D. P10-6

Eiermann T. H. P11-7

Einsele H. P10-14, SI-09-9

Eisenberger U. JS-08-3

Eiz-Vesper B. JS-05-2, P09-7, P10-6, P10-26

Elvers-Hornung S. P10-18

Emmanuel K. P02-11

Engström C. P02-12

Enkel S. P02-25

Erickson A. P04-3

Escot C. P09-11

Eugster A. P07-1

F

Fabricius D. P10-9

Falk J. SI-12-4

Faltus T. JS-09-3

Fecher T. P10-9

Fechner J. SI-02-1

Feichtner M. P10-11, P10-16

Fenchel K. P01-10

Fiedler J. JS-03-3

Fiedler M. P08-5

Figueiredo C. P10-6, P10-26, SI-09-1, JS-09-6,

SI-10-2

Filimonov V. P10-5

Fischer E. G. P11-8

Fischer J. SI-10-3, P01-6, P03-6

Fischer M. P09-4

Flesch B. K. P02-9, P02-10, P02-19, P02-29,

SI-09-5

Fleury-Cappellesso S. JS-01-2

Flommersfeld S. P05-2, P06-1

Foley R. P10-7

Frank K. P04-5, SI-12-3

Frers I. C. P06-1

Frey B. M. SI-13-1, P02-12, P04-4, P09-1

Fritz L. JS-02-3

Fröhner V. SI-13-3

Fuchs B. P09-10

Fuchs N. P11-14

Fuchs T. SI-12-5

Funk M. SI-12-1

Funk W. P10-17

Fürst D. P01-4, P09-14, JS-03-3, SI-09-9

G

Ganslandt T. SI-05-3, P05-4

Gassner C. SI-13-1, P02-12, P09-1

Gathof B. P03-1, P03-11, P04-2, P07-5

Gattenlöhner S. SI-13-3

Gatto C. P04-6

Gebhard F. JS-01-2

Geithner M. P08-6

Gerez L. P10-7

Germeroth L. P10-14

Giebel B. JS-05-3, JS-09-1, P11-6, P11-16,

P11-17, P11-18

Gielen J. P04-2

Giers G. P03-6

Gimona M. P10-11

Giordano R. JS-01-2

Giptner A. JS-11-5

Giurgola L. P04-6

Gjerde C. G. JS-01-2

Gloger D. P07-3

Göbel S. SI-09-5

Gökbuget N. P10-19

Gomez Barrena E. JS-01-2

Gonzalo Daganzo R. M. JS-01-2

Gorelashvili M. SI-09-8

Görgens A. JS-09-1, P11-16, P11-17,

P11-18, P11-19

Goslings D. P04-4

Gourri E. P09-1

Gowland P. P02-16, P04-1

Graber J. P02-16

Grabmer C. P05-1

Grabowski C. P01-10

Graf D. P06-1

Graff J. P09-11

Gramatzki M. JS-07-2, P09-16, SI-09-9

Graminske S. P04-3

Gravemann U. SI-07-5

Grebhardt C. P01-7

Greger N. P02-23

Greil R. P05-1

Greinacher A. SI-16-2, P01-1, P02-17, P07-3,

P09-9, SI-04-3, SI-04-5, SI-11-5, SI-14-5

Greinix H. JS-06-3

Grigoleit G. U. P10-14

Großgebauer T. P04-7

Guba M. P08-3

Gubbe K. P04-5, SI-12-3

Guberina H. JS-08-3

Gudima A. P10-5, P10-13

Gül S. P08-1

Günther A. P09-16

Gupta S. P06-1

Guzman C. A. P10-6

H

Hackstein H. JS-06-1, P09-13, P10-10, P10-22,

JS-02-3, JS-02-4, SI-11-3, SI-13-3, JS-11-5,

SI-16-5

Hähnel V. P01-8, P03-10

Halimeh S. P06-1

Halle K. P02-22

Haltmayer M. P02-11

Hammer E. P01-1

Hammes H.-P. P10-18

Han W. JS-03-4

Handke W. SI-14-4

Hang R. JS-02-5

Hansen A.-L. P11-7

Harder M. SI-06-4

Harmsen M. P02-14

Hartl A. P10-11

Hasenkamp J. JS-08-4

Hatz R. SI-09-2

Hauber D. P09-14

Hauck-Dlimi B. P03-8

Hauser-Kronberger C. P10-11

Heiden M. SI-12-1, SI-16-4

Heidenreich F. P07-1

Heil U. P03-4

Heinrichs S. P11-14

Heim A. P09-7

Heinemann F. M. P08-5, JS-08-3, JS-10-3

Heinold A. P08-5, JS-08-3

Heinrich C. P09-6

Helbig C. P04-7

Heldke S. P04-3

Hellem S. JS-01-2

Hellstern P. P06-7

Hellweg R. JS-03-4

Helmberg W. SI-12-5

Hemmer S. P09-13

Henkenius K. JS-02-3

Hennig H. SI-14-1

Henny C. P02-16

Henschler R. P10-12

Herber M. P08-6

Herbert K. SI-13-4

Herbst L. SI-06-5

Herkt S. P10-24

Hermann S. P01-5

Hermann W. P01-7

Hersmann G. P04-7

Heuft H.-G. P11-8

Hiller O. SI-09-1

Hitzler W. P02-13

Höchsmann B. SI-06-4

Hoffmann T. SI-02-2

Hoffmann W. P07-3

Hölig K. P05-3, P07-1, P09-5

Holland H. JS-03-5

Holloschi A. JS-03-4

Hooftman L. P09-11

Höppel U. P10-4

Horn P. A. JS-09-1, P11-14, P08-5, P09-15,

P11-6, P11-16, P11-17, P11-18,

P11-19, JS-08-3

Houareau C. SI-14-2, SI-16-4

Hourfar K. P04-5, SI-12-3

Hoyer P. P01-10

Huber-Lang M. JS-03-3

Humbel T. P03-5

Humpe A. JS-07-2, P09-16

Huppert V. P10-20

Hussain S. P11-13

Hutschenreuter G. P02-3

Hütter G. JS-03-5

Huyton T. SI-09-6

I

Ignatius A. JS-01-2

Ihrig K. P10-19

Immenschuh S. SI-09-1

Ivaskevicius V. SI-06-2, P06-1

J

Jahrsdörfer B. P10-8, P10-9, JS-02-5

Jänigen B. JS-08-2

Janzen L. SI-04-3, SI-14-5

Jorks S. P01-10

Jouni R. SI-04-3, SI-13-4

Juhl D. SI-14-1

Jungk H. P03-4

Juretzek T. P09-12, P10-21, P11-5

Jurisic M. SI-13-4

Just B. P02-9

K

Kabisch H. P11-7

Kagan K. O. P02-25, SI-13-4

Kahaly G. J. SI-09-5

Kahlenberg F. P03-3, P03-12

Kaltenmeier C. T. P10-8

Kappert G. P06-1

Karagianni M. SI-14-6

Karl A. P04-5, P11-2, SI-12-3

Karnot A. P02-10, P02-19

Karpova D. P09-2, P09-11

Kasimir-Bauer S. P11-6

Kauke T. P08-3, SI-09-2

Kauling B. P10-20

Kelle N. P03-12

Keller K. P10-17

Kellner C. JS-07-2

Kelsch R. SI-09-3, P11-7

Ketter R. P06-8

Kibler E. P10-5

Kiefel V. P02-23, P07-3

Kiem H.-P. JS-09-2

Kiessig S. T. P11-1

Kießig S. SI-16-3

Killer M. JS-02-3

Kimmig R. P11-6

Klesse C. P07-1

Klöcker S. P08-6

Klopocki E. SI-04-4

Klump H. JS-02-1, P09-15

Klümpers V. P02-27, P06-7

Klüter H. P02-5, P02-6, P02-14, P10-5,

P10-13, P10-18, JS-11-3

Knabbe C. JS-11-4

Kneidinger N. SI-09-2

Knöfler R. SI-04-4

Knowles L. M. P06-2, P06-8, SI-06-3

Kobsar A. P01-3, P01-5

Koburger M. P04-7

Kollar K. P10-12

König E.-M. SI-04-4

König I. R. JS-11-5

König L. P11-6

König S. P02-15

Konrad H. JS-10-5, P08-4

Kontermann R. P10-2

Kordes U. P11-7

Körmöczi G. P02-28

Körper S. P09-14

Kößler A. P01-3, P01-5

Kößler J. JS-06-2, P01-3, P01-5

Kraemer A. P09-11

Kramer M. P05-3

Kramer-Zucker A. JS-08-2

Krautwurst A. SI-13-3

Kreibich U. P06-1

Kremer H. P10-18

Krenn P. P11-15

Kribben A. JS-08-3

Kripp H. P08-3

Kroll H. P01-10, P04-7

Kronstein N. P09-3, SI-11-4

Kronstein-Wiedemann R. P09-3, SI-11-4

Krüger W. P09-9

Küchler S. JS-03-5

Kudryavtseva V. P10-5

Kugel T. JS-03-5

Kühl J.-S. SI-09-8

Kuhn M. P07-1

Kühnl P. P11-7

Kunze-Schumacher H. SI-09-6, SI-09-7

Kurnik K. P06-1

Kuvardina O. N. P10-24

Kwoczek J. C. P10-26

Kzhyshkowska J. P02-6, P02-14, P10-5, P10-13

L

Lachance S. P10-7

Lachmann N. P08-1, SI-09-4, JS-08-3

Lahrberg J. P10-26

Lakshmipathi S. P10-14

Lambrecht B. SI-14-4

Laner-Plamberger S. P10-11, P10-16

Lang K. P07-1

Larmann J. SI-09-1

Lauer B. P08-6

Lausen J. P10-24

Laws H.-J. P02-2

Layrolle P. JS-01-2

Legler T. J. SI-11-2, P10-1, P11-3, JS-08-4

Leibacher J. P04-3, SI-07-5

Leisch M. P05-1

Leithäuser M. P02-23

Leitner G. P11-8

Lenz V. JS-08-3, P11-14

Lessig D. P06-8, SI-06-3

Lewalle P. P10-7

Li F. P02-14

Lindemann M. P08-5, JS-08-3

Lindlbauer N. P05-1

Link C. S. P07-1

Linke K. P04-7

Lippitsch A. P10-22

Litviakov N. P02-6

Liu T. P02-6

Lohmann A. SI-12-1

Lotfi R. JS-01-2, P10-17, JS-02-5, SI-12-4

Lotsch M. SI-13-5

Ludwig B. P10-21

Luz B. P02-8

M

Macher S. P07-2

Madla W. P03-4

Maecker-Kolhoff B. P09-7

Maertens J. P10-7

Malakhova D. P09-4

Manadhar T. SI-09-7

Mannhalter C. P06-7

Mansouri Taleghani B. P11-8

Marais I. P02-29

Marandiuc D. P02-13

Marini I. P10-2, SI-04-3

Marschall R. P05-2

Martin A. P11-9

Matheis N. SI-09-5

Matko S. P10-3, P10-15

Mayer B. P02-18, P02-21, P02-22, P02-24, P02-26, P02-30, P06-4, P06-6

Mayer G. P05-1

Mayr-Wohlfart U. P04-5, SI-12-3

Medina Escobar P. P01-7

Medvedev N. P02-17

Meyer S. SI-13-1, P02-12, Meyer zu Bexten E. SI-16-5

Michel G. P10-10

Mielke S. P10-7

Miettinen J. P09-2

Mildenberg I. P10-19

Miller N. P10-3

Mitrofanova I. P02-6

Mittelbronn M. P10-19

Moerman N. SI-12-5

Moganti K. P02-14

Mohrez M. P03-13

Moldenhauer A. JS-03-4

Moldenhauer S. JS-03-4

Möllmann M. JS-09-1

Montemurro T. JS-01-2

Moog R. P01-2, P01-4, P09-12, P10-21, P11-2, P11-5

Moritz M. P07-2

Mufti N. P04-3

Mughal N. P11-12, P11-13

Müller B. SI-14-5

Müller I. P06-2, P06-7, P06-8

Müller J. P09-17

Müller K. JS-11-5

Müller S. SI-12-1

Müller T. P02-1, SI-07-3

Müller V. P09-6

Murke F. P11,19, JS-09-1

Mynarek M. P09-7

Mytilineos J. P01-4, P09-14, SI-09-9

N

Nazar R. P11-13

Nepal B. P11-10, P11-11, P11-12, P11-13

Netz U. P03-9

Neubauer A. JS-02-3

Neuberger P. P02-8

Neuchel C. P01-4, SI-09-9

Neuenhahn M. P10-14

Neurohr C. SI-09-2

Nguyen T.-H. P02-17

Niederacher D. SI-10-3

Niederhauser C. P02-16, P04-1

Niederwieser D. SI-09-9

Niemann M. SI-09-4

Nold P. P09-13, JS-02-3, JS-02-4

North A. P04-3

Northoff H. SI-16-4

Nowak-Harnau S. P02-25

Nyadu B. JS-08-3

Nydegger U. P01-7

O

Odendahl M. P10-3, P10-14, P10-15, P10-21

Oelke M. P10-26

Oergel T. P09-9

Offergeld R. SI-14-2, SI-14-3, SI-16-4

Offner R. P03-10, P03-13

Olavarria E. P10-7

Oldenburg J. P06-1, P09-17

Olivieri M. P06-1

Öller M. P10-11, P10-16

Öllinger R. P08-1

Opitz A. P03-4

Ostermann H. SI-07-4

Oustianskaia L. P03-1, P07-5

P

Palmer A. JS-03-3

Pamler I. P03-13, P09-6

Papenkort E.-M. JS-11-5

Papert S. P10-1, JS-08-4

Paranikulangara P. P04-3, P11-15

Pasini E. P09-3

Paul A. JS-08-3

Paul-Konietzko K. SI-14-5

Payrat J. M. P03-3

Peipp M. JS-07-2

Peisl J. P06-5

Peltroche H. P09-12, P10-21, P11-5

Peter W. P11-7

Peters J. P11-14

Petershofen E. K. SI-07-3

Petrescu-Jipa V.-M. P03-1

Petters O. JS-03-5

Pfeiffer A. P10-4

Pfeiffer H. P03-7, SI-06-5, SI-14-4

Pfizenmaier K. P10-2

Pichl L. SI-14-5

Picksak G. P09-7

Pilch J. P06-2, P06-8, SI-06-3

Pisarski P. JS-08-2

Platz A. P03-2

Pleyer L. P05-1

Pohler P. SI-07-5

Pollet D. P10-12

Poppe C. JS-08-5

Portegys J. P02-27

Portmann C. P02-12

Posset M. P02-20

Pötzsch B. P09-17

Pratschke J. P08-1

Preußel K. SI-14-2, SI-14-3

Pruß A. P02-21, P04-6

Q

Qiu D. SI-11-3

R

Raab S. P03-2

Rabe A. SI-16-3

Radojska S. P03-1, P03-11, P07-5

Radtke S. JS-09-1

Raninger A. P10-11

Rauh C. P06-5

Rauh M. P10-25

Ravanat C. P04-3

Rebmann V. P11-6

Reichenberg S. SI-14-4

Reichhardt H. P10-1

Reil A. P01-2, P02-2

Reinert D. P04-4

Reinhardt P. P09-14

Reinke P. SI-09-4

Reitsma K. P10-7

Riabov V. P10-5, P10-13

Richter E. P02-4, P02-8, P02-27

Rieger C. SI-07-4

Riewaldt J. P09-18

Riggert J. P10-1, P11-3, JS-08-4

Ringel F. P06-4

Ringwald J. P06-3

Rink G. P02-4, P02-5, P02-8, P02-27, JS-11-3

Risch L. P01-7

Risch M. P01-7

Rodi S. JS-03-3

Rohde E. P05-1, P10-11, P10-16

Rojewski M. JS-01-2, P10-17, JS-02-5, JS-03-3

Romagnoli B. P09-11

Roppelt D. P08-6

Rösler W. P09-8

Rosner A. P05-3, P09-5

Rosskopf K. SI-12-5

Röth A P11-14

Rotem A. P07-5

Rothe R. P09-12, P10-21, P11-2, P11-5

Rothenberger S. P02-4, P02-8, P02-27

Rott H. P06-1

Rouard H. JS-01-2

Rovers J. P10-7

Roy D. C. P10-7

Roy J. P10-7

Rücker-Braun E. P07-1

Ruediger M. P10-7

Rühl H. JS-09-4

Russe E. P10-11

Rüster B. SI-12-3

S

Sachs U. J. JS-10-2, P01-9, P05-2

Sachs U. SI-11-3, SI-13-3, JS-11-5

Salama A. P02-18, P02-21, P02-22, P02-24, P02-26, P02-30, P06-4, P06-6

Sallaj B. SI-06-5

Santos Manvailer L. F. P11-6

Santoso S. SI-13-3, JS-11-5

Sanzenbacher R. JS-07-3

Saragih H. SI-09-1

Sauer M. P09-7

Sayed S. P09-18

Schäfer M. P11-7

Schallmoser K. P10-11, P10-16

Schanz U. P09-1

Scharberg E. A. P02-4, P02-8, P02-27

Scharf C. P01-1

Scharler C. P10-11

Schauwecker P. P09-14

Scheffel T. JS-03-4

Scheffler A. SI-14-6

Schellerer V. P01-6

Schennach H. P07-4

Schetelig J. P07-1, P09-5

Schiemann M. P10-14

Schiffer K. P03-8

Schlaf G. JS-10-5

Schlenke P. P07-2, SI-12-5

Schlott F. P10-14

Schmid-Horch B. SI-13-4

Schmidt A. P03-2

Schmidt C. JS-03-5

Schmidt C. Q. SI-06-4

Schmidt M. P04-5, SI-07-5, SI-12-3

Schmiedgen M. P07-1

Schmieger T. P01-6

Schmitzer M. SI-09-2

Schönbacher M. P02-28

Schönborn L. P07-3

Schönefeld S. SI-12-1

Schönemann C.P08-1, SI-09-4, SI-09-8, JS-08-3

Schopohl D. SI-07-4

Schottstedt V. P02-9

Schramm R. SI-09-2

Schramm S. P11-6

Schramm W. SI-07-4

Schranz D. SI-11-3

Schrezenmeier H. JS-01-2, P01-4, P04-5, P09-6, P09-14, P10-8, P10-9, P10-17, JS-02-5, JS-03-3, SI-06-4, SI-09-9, SI-12-3, SI-12-4, SI-13-5

Schröck M. P07-2

Schroeter J. P04-6

Schultze-Florey R. P09-7

Schulz A. P09-14

Schulz R. P11-4, JS-03-5

Schulz U. P01-2, P01-4

Schulze H. SI-04-4, SI-09-8

Schulze K. P10-6

Schulze T. J. P02-5, JS-11-3

Schütte-Nütgen K. SI-09-3, P08-2

Schwarz K. P09-14

Schwarze B. SI-06-5

Schwertz H. SI-04-2

Schwind P. P02-12

Seidl C. P11-15

Seifried E. P04-3, P04-5, P10-24, P11-15, SI-12-3, JS-08-5

Selleng K. SI-11-5

Selleslag D. P10-7

Seltsam A. SI-11-1, SI-07-5, SI-14-4

Senft C. P10-19

Sensebé L. JS-01-2

Seyboth S. P02-8

Siegemund M. P10-2

Sielaff S. P09-12

Sinzger C. SI-12-4

Six M. P10-23

Skenderi Z. P04-6

Smida D. SI-16-5

Solleder M. P02-20

Sommer M. SI-12-5

Song B. P02-6

Song W. P02-5, JS-11-3

Spiegel-Cürten I. P10-20

Spierings E. SI-09-4

Spohn G. P09-2

Spranger R. SI-12-1

Spriewald B. M. P09-8, JS-10-3, JS-10-6

Staeck O. SI-09-4

Stangenberg W. P07-3

Stankevich K. P10-5

Steffen M. P08-6, P09-8

Steil L. P01-1

Steinbach J. P10-19

Steinhauser J. P10-10

Steininger P. A. JS-09-7

Steppe L. JS-02-5

Stock B. P09-11

Stoecklein N. H. SI-10-3

Stöhr D. SI-12-4

Stolz M. P04-1

Störmer M. P03-1, P03-11, P04-2, P07-5

Stötzer F. SI-16-4

Straka C. P09-10

Strasser E. F. P03-7, P10-25, SI-14-4, P05-5, P06-3, P10-4, P10-23, SI-15-3

Strathmann K. P02-3

Strauß G. SI-09-8

Streif W. SI-04-4

Streit F. P11-3

Strobel J. SI-05-3, P03-7, P03-8, P05-4, P10-23, P11-9

Strobel U. SI-04-5

Strunk D. P10-11, P10-16

Stürtzel A. P02-8

Stützer T. P01-6

Sumian C. SI-14-4

Sümnig A. SI-16-4

Suwelack B. SI-09-3, P08-2

Sykora K.-W. P09-7

T

Tast B. P09-2

Teichweyde N. P09-15

Theilmeier G. SI-09-1

Thiele T. P01-1, SI-14-5

Thierbach J. P03-3

Thomas H. P04-7

Thomas K. P10-15

Thomas S. P10-26

Thornton N. M. P02-29

Tischer S. P09-7, JS-09-6

Todorova K. SI-09-8

Tolios A. P02-28

Tolksdorf F. SI-07-5

Tonn T. P01-4, P04-5, P09-3, P09-12, P09-18, P10-3, P10-14, P10-15, P10-19, P10-21, P11-2, P11-5, SI-07-5, SI-10-1, SI-11-4, SI-12-3

Trobisch H. P06-1

Tröger S. P10-3

Trost B. P09-16

Trost N. SI-13-1, P02-12

Tryankowski R. P03-4

Trzaska T. P10-9

Tsamadou C. P01-4, SI-09-9

Tschapikow I. JS-02-4

Türkmen T. SI-11-3

Tverdokhlebov S. P10-5

U

Ul Ebad Khan P11-12, P11-13

Ulrich E. P03-12, SI-16-3

Ünlü S. P08-1

Urban E. P01-4

Urbschat S. P06-8

Url C. SI-12-5

V

Valek A. P04-4

Van Sandt V. P02-15

Vasic B. SI-12-5

Velthuis J. P10-7

Verdonck K. P07-5

Vijayan V. SI-09-1

Vitoriano S. JS-09-1

Völker U. P01-1

Vollmer T. JS-11-4

Von Dossow V. SI-09-2

Von Zabern I. SI-06-4

Vrana N. E. P10-13

W

Wagner A. P09-16

Wagner B. P11-6, SI-14-6

Wagner E. SI-09-9

Wagner F. F. P02-1

Wagner F. P02-7

Wagner M. P10-19

Wahle A. JS-10-5, P08-4

Wahler A. P03-3

Walker I. P10-7

Waterstradt M. P09-9

Weber I. P04-3, P10-12, SI-07-5

Weber K. P01-3, P01-5

Wehrmann K. SI-14-5

Weinauer F. P09-10

Weingand T. P03-5, P11-8

Weinstock C. SI-06-4, SI-13-5

Weisbach V. SI-06-5

Weiss D. SI-06-1, P06-3, P11-9

Weiss L. P05-1

Weiss S. P08-1

Weitmann K. P07-3

Weitzendorfer M. P02-11

Wels W. S. P10-3

Wels W. P10-19

Wenzel F. P03-6

Werner J. P08-3

Wesche J. SI-14-5

Wessiepe M. P02-3

Weyand M. P05-5

Wichmann C. SI-14-6

Widmer N. P04-1

Wieckhusen C. P02-4, P02-8

Wiedemann H. P10-16

Wiederschein H. P08-6

Wienzek-Lischka S. SI-13-3, JS-11-5

Wiercinska E. P09-2, P09-11

Wiesinger K. P02-11

Wiesneth M. P09-14

Wilde B. P08-5

Wingenfeld E. JS-08-5

Winkelmann M. SI-12-4

Winkler J. P09-8

Winter H. SI-09-2

Winterstein K. P01-10

Wittmann G. SI-07-4, SI-14-6

Witzenhausen C. SI-12-1

Witzke O. P08-5, JS-08-3

Woestmann S. J. P02-19, P02-29

Wolf D. P09-17

Wolf S. P08-3

Wolff J.-C. P01-9

Wolter C. P06-8

Worel N. JS-06-3

Wößmann W. SI-11-3

Wu M. JS-03-4

Wulf G. JS-08-4

Y

Yard B. P02-14

Yürek S. P02-18

Z

Zainab Mukhtar Z. P11-12

Zaslavskaya M. P08-5

Zavjalova M. P02-6

Zehnder T. P10-4

Zeiler T. SI-14-5

Zelenski T. JS-02-5

Zeplin P. P10-17

Zhang C. P10-19

Zhuravlev M. P10-5

Ziehe B. P10-25

Ziemann M. SI-12-2

Ziemssen T. P10-15

Zilian E. SI-09-1

Zimmer K.-P. SI-11-3

Zimmermann B. P02-25

Zimmermann R. SI-07-1, SI-05-3, SI-15-4, P01-6, P03-7, P03-8, P05-4, P06-5, P10-23, P11-9, SI-14-4

Zingsem J. SI-07-1, JS-10-1, P05-4

Zschiedrich S. JS-08-2

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