Abstracts of the 44th Annual Congress of the German Society for Transfusion Medicine and Immunohematology (DGTI)

Transfus Med Hemother 2011;38(suppl 1):1–72

Abstract Session 01 – Cell Therapy: Immunotherapy

OS 1.01

HSP70/peptide complexes: potent mediators for the generation of antiviral T cells with low precursor frequencies

Tischer S.¹, Basila M.¹, Kramer M.¹, Maecker—Kolhoff B.², Figueiredo C.¹, Immenschuh S.¹, Oelke M.³, Eiz—Vesper B.¹

¹Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany, ²Hannover Medical School, Pediatric Hematology/Oncology, Hannover, Germany, ³Department of Pathology and Medicine, Johns Hopkins School of Medicine, Baltimore, USA

Background: Viral infections resulting from reactivation of latent viruses (e.g. CMV, ADV, EBV) are associated with high morbidity and mortality after transplantation. Low precursor frequencies often hamper the application of virus—specific T cells. In this study the ability of HSP70/peptide complexes (HSP70/PCs) to elicit antiviral T cells even with low precursor frequencies through cross—presentation was evaluated. Patients and Methods: We examined the role of HSP70—chaperoned viral peptides (CMVpp65: A∗02:01, ADVhexon: A∗01:01, A∗24:02, B∗07:02) in HLA class I—restricted cross—presentation for ex vivo expansion of antiviral CTLs. T cells generated from PBMCs of healthy donors by HSP70/PCs were analyzed for phenotype and function. Results: About 90% of T cells generated with HSP70/CMV—PC were CMV—specific as compared to about 69% stimulated alone with the CMVpp65 peptide. In response to HSP70/ADV—PCs an up to 210fold increase of ADV—specific T cells was achieved as compared to the corresponding peptides. HSP70/PCs—induced T cells exhibited a significantly higher IFN—γ secretion and cytotoxic activity compared to those stimulated with the viral peptides. The enhancement of antiviral T—cell responses by HSP70—chaperoned peptides was most significant in donors with low memory precursor frequencies. Blockage of CD91 by α2—macroglobulin substantially reduced the T—cell proliferation suggesting a major role of this receptor in the HSP70/PC uptake. Conclusions: This study demonstrates that HSP70/PCs are stronger mediators for inducing antiviral T cells than soluble peptides, thus offering new approaches to generate relevant quantities of antiviral CTLs. HSP70—mediated cross—presentation may dispense with the need of dendritic cells or enriching CD8+ T cells. In particular, HSP70/PCs may even be able to overcome limitations related to low precursor frequencies and broaden the application of antigen—specific T cells to much more targets than currently possible.

OS 1.02

MHC histamer technology: a new tool for visualization, characterization, isolation and clinical application of virus—specific T cells

Tischer S.¹, Kaireit T.¹, Rokittta D.¹, Figueiredo C.¹, Maecker—Kolhoff B.², Geyeregger R.³, Hiller O.¹, Immenschuh S.¹, Blasczyk R.¹, Eiz—Vesper B.¹

¹Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany, ²Hannover Medical School, Pediatric Hematology/Oncology, Hannover, Germany, ³Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria

Background: Multimers of soluble peptide—major histocompatibilty complex (pMHC) molecules are used in both basic and clinic immunology. They allow a specific visualization, phenotype characterization and isolation of antigen—specific T cells from ex vivo samples. For treatment of patients with malignancies or infectious diseases after transplantation, the adoptive transfer of antigen—specific T cells sorted by pMHC multimers is an effective therapeutic strategy. Patients and Methods: A new reversible pMHC multimer, called pMHC Histamer, was developed enabling a specific detection and isolation of antiviral T cells from peripheral blood mononuclear cells. The HLA—A02/ CMVpp65 Histamer was generated by coupling 6xHis—tagged pMHC molecules onto cobalt—magnetic beads. The specificity and sensitivity of the magnetic bead—based Histamer was evaluated by flow cytometry. Sorting of antiviral CD8+ cytotoxic T cells (CTLs) was performed by magnetic cell separation, followed by the monomerization of the pMHC Histamer in the presence of L—histidine. Sorted T cells were analyzed in phenotypical and functional assays. Results: The reversible Histamer showed high specificity and sensitivity (up to 99.5%). CMV—specific T cells were isolated by the Histamer technology with a high purity of up to 99.6%. A rapid and complete reversibility of A02/CMV Histamer from the T—cell receptor (TCR) of CD8+ CTLs using 100mM L—histidine was accomplished. CMV—specific T cells enriched by Histamer staining did not differ in function with regard to cyto—toxicity and IFN—γ production in comparison to CD8+ CTLs induced by standard T—cell assays. Conclusions: This reversible T—cell staining procedure preserves the functionality of antigen specific T cells and can easily be adapted to GMP conditions. The pMHC Histamer technology offers full flexibility and meets the all requirements to generate clinical grade T lymphocytes.

OS 1.03

Automated generation of antigen—specific T cells for adoptive T cell therapy directly from cryopreserved material

Fahrendorff M., Stuth J., Essl M., Rettmer I., Brandt M., Miltenyi S., Hattenhorst U.E., Biehl M.

Miltenyi Biotec GmbH, Bergisch—Gladbach, Deutschland

The adoptive transfer of antigen—specific T cells can be a powerful tool for immunotherapy of malignant diseases or infectious complications after allogeneic stem cell transplantation. Human adenovirus, Epstein—Barr virus or cytomegalovirus infections are frequent and often life—threatening complications post allogeneic stem cell transplantation. Virus specific CD4+ and CD8+ T—cells can rapidly be isolated after a short—term antigen—specific restimulation of peripheral blood cells with pools of peptides covering complete viral antigens using the Cytokine Capture System IFN—gamma (CCS). A novel cell processing device was developed (CliniMACS Prodigy), which performs all steps of the CCS procedure, i.e. restimulation, magnetic enrichment, and potentially in vitro expansion fully automated under sterile conditions. All components for the generation of the cellular product including the cellular starting material (e.g. leukapheresis, fresh or thawed after cryopreservation in CryoMACS Freezing bags, antigen(s), reagents, buffer, and media are connected to a sterile single—use functionally closed tubing set via sterile filters or docking technique. Cell processing can run overnight and the isolated cells might be used directly after magnetic enrichment or after an additional phase of in vitro expansion. Using this cell processing device, IFN—gamma secreting virus—specific T—cells were enriched to the same purity as with the semi—automated procedure. Cell loss is markedly reduced, leading to an increased yield of IFN—gamma positive cells. An improved viability was observed resulting in better expansion rates. The final cellular product can be obtained in the medium/buffer of choice, with the desired cell concentration and volume. In conclusion, automating the generation of antigen—specific T—cells for adoptive therapy with the new device is easy, safe, fast, robust, and reduces workload, manual intervention and clean room requirements.

OS 1.04

HLA—C encoded KIR ligands in matched unrelated hematopoietic peripheral progenitor cell (PBPC) transplantation impact influence of HLA—C allele matching on survival

Fischer J.C.¹ Bruns I.², Kobbe G.², Haas R.², Enczmann J.¹, Uhrberg M.¹

¹Institut für Transplantationsdiagnostik und Zelltherapeutika, Universitätsklinik Düsseldorf, Düsseldorf, Deutschland,

²Klinik für Hämatologie, Onkologie und Immunologie, Universitätsklinikum Düsseldorf, Düsseldorf, Deutschland

HLA—C—dependent KIR ligand mismatch improves EFS survival in AML patients. In CML patients clinical outcome was significantly influenced by specific C1/C2 donor recipient constellations due to the presence of C1but not C2 ligand immunocompetent NK cells early after transplantation. We investigate whether this holds true for other cohorts than CML. 97 patients (meanOS 618d) with MDS (n = 24), AML/CML (n = 39), ALL/NHL (n = 34) received unmanipulated PBPC after myeloablative (n = 72) or non—myeloablative conditioning. HLA was determined by sequencing. Patients and donors were further classified to their KIR ligand (C1C1, C1C2, C2C2). 42 recipients received grafts from fully MUD (10/10), 37 recipients received a graft with HLA—C allele mismatch. 20 of those were mismatched for the C1/C2 KIR ligand. Results: Both, HLA—C dependent KIR ligand and allele match influenced OS rates (p < 0.02), independent from other factors. Especially in AML/ CML/MDS patients C1C1 patients profited from an HLA C allele match (meanOS 542d vs. 268d, p > 0.05), fitting to the hypothesis that early NK cell reconstitution is dominated by the presence of C1 specific NK cells. C1C1 patients did not profit from further HLA—C mismatch. In contrast C1C2 or C2C2 patients profited from HLA—C allele mismatch (980d vs. 245d, p < 0.01). Only AML/CML patients benefited from C1/C2 dependent KIR ligand mismatch (n = 6, donors KIR haplotype all patients alive, median OS 575d vs. 254d, p > 0.02). Several effects contributed to survival rates: in patients bearing the C2 KIR—ligand significant lower aGVHD (p < 0.02), but higher incidence of death due to infection (p < 0.04) or relapse (p < 0.02) could be seen. MDS/AML/CML C1C1 patients showed lower TRM and relapse rates when matched on the HLA—C allele level (p < 0.06 resp.). Based on this cohort, patients with C1C1 KIR—ligand phenotype should be matched at the HLA—C allele level, whereas patients with C1C2 or C2C2 phenotype should be mismatched at the HLA—C allele level.

OS 1.05

KIR receptor dependent NK cell alloreactivtiy in hapoidentical CD19/CD3 depleted stem cell transplantation (SCT)

Becker P.¹, Susan A.¹, Richter R.¹, Schulz M.¹, Klingebiel T.², Seifried E.¹, Kohl U.², Seidl C.¹, Bader P.²

¹DRK—Blutspendedienst Baden Württemberg – Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Klinikum der Johann Wolfgang Goethe—Universität, Frankfurt, Frankfurt, Deutschland

²Department of Paediatric Stem Cell Transplantation, Johann Wolfgang Goethe University Hospital, Frankfurt/Main, Germany

Donor—vs—recipient NK cell alloreactivity has been demonstrated to occur when donor—NK cells express inhibitory killer cell immunoglobulin—like receptors (iKIR) missing the HLA ligand in the patient (KIR—HLA MM). For a better prediction of GvL effects mediated by the alloreactive NK cell subset, we phenotypically and functionally characterized the donor— and the patient—NK cell population after haploidentical CD19/CD3 depleted SCT with a mean follow—up of one year. KIR genotyping was performed by PCR—SSO and HLA typing by SBT. KIR phenotyping of NK cells was performed by flow cytometry using antibodies against 2DL1, 2DL2, L3, 3DL1 and NKG2A. To determine the frequency of alloreactive NK cells, combinations of these antibodies were used depending of the specific KIR—HLA MM between donor and patient. Activity of NK cells was evaluated by CD 107 degranulation assay against 721.221 target cells and leukaemic blasts. Phenotypic analysis of NK cells showed a variation between 0.9% and 43.3% (n = 14) in the frequency of potentially KIR dependent alloreactive donor—NK—cell subpopulations. The lowest frequencies were observed in the group missing the C2 ligand (mean 4.6%). Highest frequencies were determined in the group missing the C1/Bw4 ligand (mean 23.5%). Early after SCT, NK cells dominate the lymphocyte recovery (mean 75% at week +2, compared to mean 21.5% at week +14). Importantly, KIR dependent alloreactive NK—cells are detectable (range: 0.7% —9.3% at week +7). These NK cells show clear cytotoxic activity against target cells missing the corresponding ligand (L721.221) and blast cells. Although NK cells seem to vary in their alloreactive potential, individual donor— patient combinations carry cytotoxic NK cell clones that could assist the GvL effect. Thus, phenotypic and functional characterization of NK cells in addition to genotyping should be considered in the finding of a donor with an optimal NK cell repertoire to improve the outcome of SCT.

OS 1.06

NK—cell activity against K562 target cells is associated with donor KIR genotype and allelic HLA class I background

Duske H., Binder T.C.M., Eiermann T.H.

Institute for Transfusion Medicine, University Hospital Hamburg Eppendorf, Hamburg, Germany

In myeloid leukemia, stem cell grafts from donors with low number of stimulating killer cell immunoglobulin—like receptors (KIRs) seem to be beneficial. The relapse rate was reduced when patients received T—depleted grafts from donors with homozygous KIR haplotype A. We speculated that the combination of stimulating and inhibitory KIRs influence the NK cell activity in individuals. To address this hypothesis we collected PBMC of HLA identical healthy donors and measured the cytotoxic activity against K562 cells. We identified native frozen PBMC (in contrast to IL—2 stimulated PBMC or isolated NK cells) as the optimal source to assess physiological NK cell activity in a standardized chromium release assay. The potential of NK cells to lyse K562 cells increased with the number of licensing relevant HLA ligands expressed in the donor. To separate the KIR effect from the HLA background, cells were grouped according to their HLA type. In the HLA identical donors with ligand group C1 the beneficial effect of donor KIR AA could be affirmed: the mean specific lysis was 14,8% in KIR AA (n = 9), 12,9% in KIR AB (n = 15) and 5,3% in KIR BB (n = 4) individuals. Preliminary data of the smaller HLA—C1/C2/Bw4 ligand group suggested a contrary impact of KIR genotypes on lytic capacity. Contrary to the HLA class I identical groups, the lytic activity of NK cells derived from HLA non—identical donors was not associated with the KIR genotype, suggesting a differential triggering of NK cell education via different HLA—A, —B and/or —Cw alleles. Our results are in line with the arguing results of clinical studies that investigate the influence of donor KIR genotype on the outcome of HSCT in leukaemia patients: they indicate the relevance of the HLA class I polymorphism on NK cell licensing success, which rules out the possibility to determine alloreactive NK cell properties as a function of KIR genotype only.

OS 1.07

POCKETCHECK: A comprehensive contact calculation for distinct peptide—HLA structures

Huyton T., Ladas N., Blasczyk R., Bade—Döding C.

Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany

Background: There have been several attempts over the years to define which positions in the HLA binding groove (pockets) influence the specificity of bound amino acids at each position in the peptide. Structural determination of the HLA molecule by X—ray crystallography provided valuable information for understanding how peptides bind to HLA. Originally, six pockets (A—F) were defined by calculating the surface of the binding groove based upon the crystal structure of HLA—A2 (Saper et al., 1991). Since then, x—ray crystallography has been performed for a variety of HLA alleles bound to a range of peptides, which has lead to broader pocket definitions (Chelvanayagam, 1996). Several studies performed peptide sequencing for allelic variants to understand magnitude of mismatches. We have previously described the ability of distinct HLA variants to present peptides of >10 amino acids in length. Here it is especially important to define which positions within the HLA binding cleft are in contact with the peptide. The knowledge about individual peptide features allowed for the crystallographic analysis of selected pMHC complexes. Methods: The Protein data bank was searched for all deposited structures of peptide: HLA—complexes http://rcsb.org/pdb and those were submitted to the contact map analysis webserver http://ligin.weizmann.ac.il/cma. The output html was piped into an InterSystems Cach post—relational database allowing object—oriented data storage. Tabular data with residue position, amino acid and buried surface that contact a particular peptide position were compared for >100 HLA class I structures. Results: We here describe a new dynamic definition that will increase the precision of peptide prediction and support the characterization of individual weights for individual amino acids. Conclusions: This knowledge facilitates a rating of the allogenicity of mismatches and will be a further step towards intelligent HLA mismatching.

OS 1.08

Allele frequency distribution of minor histocompatibility antigens (mHags) in 174 African Americans

Bakker G.R., Uribe M.R., Adams S.D., Flegel W.A. National Insitute of Health, Bethesda, USA

In hematopoietic stem cell transplantation (HSCT) the nature and extent of the genetic disparity between donor and recipient determines the ability to obtain engraftment, eradication of abnormal host cells, graft—vs—leukemia (GVL), and control of graft—vs—host disease (GVHD). In HLA identical HSCT, polymorphic peptides foreign to recipient or donor and recognized by selected T—cell clones are known as minor histocompatibility antigens (mHags). These are encoded by polymorphic genes located throughout the genome, the origin of their polymorphism are single nucleotide polymorphisms, or gene deletions. Eighteen mHags were tested to establish their distribution in African Americans, specifically 8 of them for which no frequency data has been published. The method utilized was an SSP—PCR typing kit, visualization with gel electrophoresis, to detect mHag alleles in 174 African Americans. Utilizing this SSP method, genotype and allele frequency in 18 mHags in the African American population was determined. Frequency data for the 9 mHags HA—1, HA—2, HA—3, HA—8, HB—1, ACC—1, ACC—2, HwA—10, and UGT2B17 were in accordance with previous publications. Further analysis of primer pairs for HwA—9 is needed. HY was found in males only, as expected. The frequency data for the 8 mHags LB—ECGF—1, HwA—11, CTSH, LRH1, LB—ADIR, and CD31 exons 1, 3, and 8 were novel (Table 1). Utilization of this frequency information may prove useful in the transplant setting. Frequencies of the mHag alleles provides information regarding the chance of having mHag mismatches in a HLA—matched donor/recipient pair. This may help estimate the GVHD risk or the possibility of the GVL effect. Hence, mHag genotyping may provide useful information in donor selection, if the mHag contribution to GVHD and GVL is known.

Table 1.

Minor Histocompatibility Antigens (mHags) in 174 African Americans

	Genotype		Frequency
Gene	Allele Combination	n	Alleles	%
LB-ECGF-1	HH	0	H	1
	HR	4
	RR	170	R	99
HwA-11	SS	3	S	17
	ST	55
	TT	116	T	83
CTSH	RR	0	R	5
	RG	17
	GG	157	G	95
CD31 Exon 3∗	VV	29	80V-125V	42
	VL	87
	LL	58	80V-125L	58
CD31 Exon 8	NN	6	N	22
	NS	65
	SS	103	S	78
CD31 Exon 1	RR	117	R	83
	RG	54
	GG	3	G	17
LRH1	5C/5C	74	5C	70
	5C/4C	93
	4C/4C	7	4C	30
LB-ADIR	FF	0	F	6
	FS	21
	SS	153	S	94

^∗80M-125V and 80M-125L are two additional alleles assayed in CD31 Exon 3, but no donors were found to be positive for these alleles.

Abstract Session 02 – Blood Safety: Pathogen Transmission

OS 2.01

Prevalence of cytomegalovirus DNA in whole blood samples from 10,000 donations

Ziemann M., Grève S., Juhl D., Hennig H.

Institut für Transfusionsmedizin, Universitätsklinikum Schleswig—Holstein, Lübeck, Deutschland

Background: It is unknown, whether the risk of transfusion—transmitted CMV—infections can be decreased by selection of leukoreduced blood products from subgroups of donors (e. g. seronegative donors). In most studies, CMV DNA is determined only in plasma samples, and potentially infected white blood cells are ignored. Methods: Whole blood samples from 10,000 blood donations are screened for CMV DNA. For reactive samples, CMV PCR is repeated both from a second whole blood sample and from a plasma sample from the same donation. Repeatedly positive donations are presumed to be potentially infectious. Results: In an interims analysis after about 5000 tested donations, there were four CMV DNA—positive samples (0.1%). Presumably, all four samples were from donors with primary CMV infection: One sample from a seronegative donor in the window period, two samples from primarily seropositive donors, and one sample from a donor with presumed CMV seroconversion within the last vear. Reactivations of latent CMV infection could not be detected. One donor was positive for CMV DNA in the whole blood samples only, but not in plasma. During the DGTI meeting, results of the completed study will be shown. Conclusions: Some donors with active CMV infection might be detectable only by CMV PCR from whole blood samples, but not from plasma samples. As shown earlier for plasma samples, the majority of potentially infectious donors is detected in connection with seroconversion. Therefore, blood products from newly seroconverted donors should not be used for patients at risk for CMV infection. After completion of the study, it could perhaps be shown, whether the prevalence of CMV DNA is higher in seronegative donors or in seropositive donors with remote seroconversion.

OS 2.02

CMV—seroconversion in blood donors

Heuft H.—G.¹ Heim A.², Blasczyk R.¹ Ziemann M.³

¹Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany, institute for Virology, Hannover Medical School, Hannover, Germany, institute for Transfusion Medicine, University Hospital of Schleswig—Holstein, Lübeck, Germany

Background: To evaluate the rate of Cytomegalovirus (CMV) seroconversions in blood donors, in particular the rate of seroconversions (SC) with short donation intervals. Patients and Methods: We analyzed the CMV status among Hannover Medical School blood donors from 2006 to 2010 and identified all short term CMV—SC (≤35 days between two do—nations) since 2001. CMV screening was performed using a Behring IgG/IgM—Elisa test (2001–2005), thereafter with the Abbott Architect CMV IgG—Elisa test system (since 2006). Apparent SC with short donation intervals underwent confirmation procedures (repeated Architect testing, western blot tests) and additionally real—time PCR testings. Results. In addition, since 2001 fourty donors with an apparent CMV—SC and an interdonation time ≤35 days were detected. Of these, only 2 (5%) could be confirmed by repeated Elisa and WB testing (one of them with low titer CMV—DNA); 3 further samples remained doubtful. Conclusion: The 2006–2010 apparent CMV—SC rate fluctuated around 0.5%. The low number of confirmed short—term CMV—SC require further investigations (e.g. testing of repeat samples) and raise concerns to the specificity of CMV Eli sa—screening.

OS 2.03

Risk of transfusion transmitted CMV—infection after use of leukocyte reduced blood products not tested for anti— CMV—antibodies: a prospective study in high risk patients undergoing allogeneic hematopoietic stem cell transplantation

Thiele T.¹, Krüger W.², Zimmermann K.³, Wessel A.¹, Dölken G.²,

Greinacher A.¹

¹Institute for Immunology and Transfusion Medicine, Greifswald, Germany,

²Department of Hematology and Oncology, Ernst—Moritz—Arndt University Hospital Greifswald, Greifswald, Germany, ³Institut für Medizinische Mikrobiologie, Ernst—Moritz—Arndt Universitätsklinik Greifswald, Greifswald, Deutschland

Background: Strategies for prevention of transfusion transmitted CMV infection (TT—CMV) after hematopoietic stem cell transplantation (HSCT) include transfusion of CMV seronegative blood units, and/or transfusion of leukocyte reduced cellular blood products. We assessed the incidence of TT—CMV in CMV seronegative patients receiving CMV seronegative HSC—transplants, who were transfused with leukocyte reduced cellular blood products not tested for anti—CMV antibodies. Methods: In a prospective observational study between 1999 and 2009 all HSCT—patients received leukocyte reduced cellular blood products not tested for anti—CMV antibodies. Patients were screened for CMV—serostatus and CMV negative recipients of CMV negative transplants were monitored for TT—CMV clinically and by CMV—NAT. Anti—CMV—IgG and —IgM antibodies were assessed after three time intervals (interval I: study inclusion – day +30 after HSCT; interval II: day +30 – day +100; interval III: after day +100). Results: Among 142 patients treated with allogeneic HSCT, 23 CMV negative donor/patient pairs were identified. These 23 patients received 1,847 blood products from 3,180 donors. All patients remained negative for CMV—DNA and none developed CMV—associated clinical complications. The risk for TT—CMV per donor exposure was 0% (95% confidence interval 0.0–0.12%). 17/23 patients seroconverted for anti—CMV—IgG, but none for anti—CMV—IgM. CMV—IgG seroconverters received significantly more transfusions per week than non—converters. These anti—CMV—IgG titers decreased if no further transfusions were administered. Conclusion: The risk of TT—CMV is low in high risk CMVneg/neg HSCT—patients transfused with leukocyte reduced blood products not tested for anti—CMV antibodies. The cause of anti—CMV—IgG seroconversion is most likely passive antibody transmission by blood products.

Table for abstract OS 2.02

Year	Donors, n	Donors, CMV neg (n/%)	Apparent CMV-SC (n/%)	Time neg/pos result (days)
2006	8159	5590/68.5	33/0,59	186 (43–1534)
2007	9361	6508/69.5	44/0,68	226 (63–2220)
2008	9500	6622/69.7	29/0,44	252 (66–2380)
2009	10175	7322/72.0	46/0,63	260 (43–1794)
2010	9888	7224/73.0	39/0,54	175 (70–1933)
Total			191/0.57%	199 (43–2380)

OS 2.04

Results of anti—HBc surveillance in Germany

Kang M.U.—Z.

Robert Koch—lnstitut, Berlin, Deutschland

Since September 2006, testing for antibodies against hepatitis B core—antigen (anti—HBc) is mandatory in Germany. Subsequently the National Advisory Committee ‘Blood’ recommended reporting of anti—HBc positive donations which were tested negative for hepatitis B surface—antigen (HBs—Ag) to the Robert Koch Institute additionally to the mandatory epidemiological reporting according to §22 of the Transfusion Act. 24 million donations were tested between 2006 and 2009. 113.201 (0.46%) of these were anti—HBc positive and HBs—Ag negative. 70.398 (62%) were further tested individually for HBV—DNA with a required analytic sensitivity of ≤12 IU/ml. 148 (0.21%) anti—HBc positive and HBs—Ag negative donations tested NAT—positive. Most of the NAT—positive donations were observed among first—time (n = 79) and repeat donors (n = 42) who were tested for anti—HBc for the first time. While the total number of all donations tested increased annually the proportion of anti—HBc positive and HBs—Ag negative donations declined by more than 30% from 2006 until 2009, mainly due to decreasing number of first time tested repeat donors. Donors who tested anti—HBc—positive were deferred from don—tating by the majority of the blood establishments. 3377 repeat donors with preceding negative tested donations had reactive anti—HBc—results during the observation period. It is unclear whether these reflect true seroconversions, because only four of these donations were NAT—positive. 27396 donations (39%) of NAT—negative donors had to be deferred, due to a low (n = 23455) or not specified (n = 3941) antibody titer against hepatitis B surface—antigen (anti—HBs). The additional testing for anti—HBc has lead to the identification of 148 HBV—DNA positive donations that would have been missed by HBs—Ag—screening only. This reduces the residual risk of transfusion transmitted HBV—infections. In order not to loose too many donors the non—specificity of reactive anti—HBc tests has to be evaluated further.

OS 2.05

Rapid detection of microbial contamination of cell based products by fluorescence staining of microcolonies

Störmer M., Brachen J., Schurig U., Becker B., Löschner B., Montag T.

Paul—Ehrlich—Institut, Fachgebiet Mikrobiologische Sicherheit, Langen, Deutschland

Background: Cell based products represent new dimensions in microbial safety of drugs. The international regulated methods for sterility testing require 1–2 weeks up to result. Considering the extremely short shelf life of much cell therapeutics, the results from these established methods are often available too late. Sterility testing can be improved if the detection takes place in less than 14 days with a rapid detection method. This study evaluates the use of the Milliflex Quantum System (Millipore, Molsheim, France) by using selected PEI Bacteria References to demonstrate the suitability for bacterial monitoring of cell based products. Methods: Ten PEI Bacteria References which are defined in identity, count and ability to grow in platelet concentrates, were involved in the study. Artificially contaminated chínese hamster ovary cells (CHO, 4 × 10⁶ cells/ml) and samples of physiological saline, both containing 20–80 CFU per sample, were filtered through a Milliflex funnel and incubated onto agar media at 32.5°C. Afterwards the samples were applicated with reagents and microcolonies were enumerated by using the Milliflex Quantum machine. CHO—samples were pre—treated with a selective mammalian cell lysis solution. Results: Nine PEI Bacteria References (Klebsiella pneumoniae, Yersinia enterocolitica, Escherichia coli, Pseudomonas fluorescens, Bacillus cereus, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans) could be detected between 7 and 20 hours. The anaerobic slow—grower Propionibacterium acnes was detectable between 66–72 hours. The loss of CFU on the membrane was depended on the bacterial species. Conclusion: The Milliflex Quantum System is a unique system to detect and enumerate viable microorganisms in one—fourth to one—fifth the time of traditional microbiology methods. The results demonstrated the suitability of the Milliflex Quantum for rapid bacterial monitoring of cell based products.

OS 2.06

A new bacterial screening strategy to extend platelet shelf life from 4 days to 5 days in Germany

Sireis W., Rüster B., Diass C., Gubbe K., Karl A., Hourfar M.K, Capalbo G., Pfeiffer H.U., Janetzko K., Goebel M., Kempf V.A.J., Tonn T., Klüter H., Seifried E., Schmidt M.

German Red Cross Blood Service Baden—Württemberg – Hessen, Institute of Transfusion Medicine and Immunohaematology, Medical Faculty Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany

Background: The Paul—Ehrlich—Institute analysed all fatalities caused by bacterial infections between 1997 and 2007. After this, platelet shelf life was reduced to a maximum of 4 days after blood donation because the majority of cases that involved severe transfusion—transmitted bacterial infections used 5—day—old platelets. Aims: The current study compares the analytical sensitivity and the diagnostic specificity of four rapid bacterial detection procedures. Methods: Nine transfusion—relevant bacterial strains were spiked in pooled platelets or apheresis platelets at a low concentration (10 CFU/bag). Samples were collected after day 3, day 4 and day 5 and investigated by four rapid bacterial detection methods (modified BacT/ALERT with a reduced incubation period, Bactiflow, in—house FACS method and in—house 16s DNA PCR method). Results: Seven out of nine bacterial strains were adequately detected by BacT/ALERT, Bactiflow and PCR in apheresis platelets, and pooled platelets after samples were collected on day 3, day 4 and day 5. For three bacterial strains, the analytical sensitivity was reduced for the FACS method. The diagnostic specificity for BacT/ALERT and Bactiflow was higher than 99% due to more than 400 investigations. Conclusions: A late sample collection on day 3, day 4 or day 5 after blood donation in combination with a rapid bacterial detection method offers a new opportunity to improve blood safety and reduce sampling errors. Therefore, BacT/ALERT, Bactiflow and 16s ID—NAT are feasible for late bacterial screening in platelets and may provide data that support the extension of platelet shelf life in Germany to 5 days. Clinical field trial data are now eagerly awaited in the next step.

OS 2.07

First interlaboratory comparison of different rapid methods for the detection of bacterial contamination in platelet concentrates

Vollmer T.¹, Hinse D.¹, Schottstedt V.², Bux J.², Tapernon K.³, Sibrowski W.³, Kleesiek K.¹, Knabbe C.¹, Dreier J.¹

¹Institut für Laboratoriums— und Transfusionsmedizin, Herz— und Diabeteszentrum NRW, Bad Oeynhausen, Deutschland, ²DRK Blutspendedienst West Zentrallabor, Hagen, Deutschland, ³Institut für Transfusionsmedizin und Transplantationsimmunologie, Universitätsklinikum Münster, Münster, Deutschland

The shelf life of PCs has been reduced to four days for non pathogen—inactivated PCs (vote 38, German Blood working party). Bacterial screening methods were initially not recommended due to the absence of an adequate test system. In the present study, the analytical quality of three different rapid screening methods (BactiFlow flow cytometry (BF), Pan Genera Detection Assay (PGD), 23S rRNA RT—PCR (NAT)) was assessed in an interlaboratory comparison. The performance was evaluated in three different German blood services. Per sample set, 20 samples were randomly analyzed with three different rapid screening methods while the BacT/Alert automated culture system served as reference method. Twelve samples were inoculated with different bacteria (gram—positive: S. aureus, S. epidermidis; gram—negative: E. coli (2 strains), K. pneumoniae (2 strains), E. aerogenes (1 strain), S. marcescens (1 strains)) at different concentrations (4.5 × 10E+03 to 4.5 × 10E+08 CFU/mL). Stability of bacterial concentration during transportation of samples was secured by temperature control (16 to 20°C) and a stated maximum transportation period. Results of the interlaboratory comparison revealed that the BF assay and NAT—screening detected all samples correctly (positive: 12/12, negative: 8/8). The PGD test detected only four of the positive samples. Four of the non—detected positive samples were below the PGD detection limit (E. coli < 2.4xE+04 CFU/mL (2 strains), S. marcescens < 8.6 × 10E+05 CFU/mL, S. aureus < 8.2 × 10E+03 CFU/mL). Another four inoculated samples with relatively high bacterial concentrations were detected false—negative (E. coli: 2.1 × 10E+07 CFU/mL, K. pneumoniae: 4.8 × 10E+06 CFU/mL, S. auxeus: 6.1 × 10E+05 CFU/mL). All rapid screening methods revealed no false positive results. Both, BF and NAT, demonstrated a high sensitivity to detect bacterial contamination in PCs. The PGD test had some shortcomings regarding the sensitivity especially for the detection of gram—negative strains.

OS 2.08

Growth kinetics of various bacteria species in red blood cell units after spiking with high and low bacterial titers

Gravemann U., Lambrecht B., Seltsam A.

DRK—Blutspendedienst NSTOB Institut Springe, Springe, Deutschland

Background: Bacterial contamination of blood products still remains one of the major risks of transfusion. Platelet concentrates are known to have a high risk of bacterial growth due to storage at room temperature. Taking into account the significantly higher number of red blood cell (RBC) units transfused, however, the incidence of transfusion—associated bacterial contamination is similar for RBC. Aim of the present study was to characterize the growth kinetics in RBC units of a series of different transfusion—relevant bacteria species. Methods: RBC units in additive solution SAG—M were produced from whole blood using standard procedures. Bacteria species typically found in blood products were used for spiking: E.cloacae, E.coli, K.pneumoniae, Pacnes, Paeruginosa, S.aureus, S.epidermidis, S.liquefaciens, S.marcescens, S.pyogenes, Y.enterocolitica. RBC units (n = 3) were spiked with an inoculum concentration of 100 colony forming units (CFU)/mL on the day of production. Samples for bacterial testing were taken on days 1, 14, 28 and 42 of storage. Those strains that multiplied in RBC units at high spiking concentrations were further used to contaminate RBC units with a lower spiking concentration of 1 CFU/mL. Results: Bacterial strains could be divided into three groups according to their growth behaviour. E.cloacae, E.coli, K.pneumoniae, Paeruginosa, S.epidermidis and S. marcescens did not show any growth in RBC units; their titers significantly decreased or became even no more detectable during storage. Pacnes, S.aureus and S.pyogenes did not grow up in RBC units; their titers remained almost constant during storage. Y.enterocolitica and S.liquefaciens rapidly grew to high titers of up to 1 × 10⁹ CFU/mL after 28 days. Conclusion: Although most bacteria do not survive or amplify in RBC units at 4°C, psychrophilic bacteria such as Y.enterocolitica and S.liquefaciens can proliferate in RBC units from a very low level of contamination to clinically significant levels.

Abstract Session 03 – Hemostasis: Platelets

05 3.01

IgM memory B—cells specific for PF4/heparin are present in the normal population

Hietkamp B.¹, Krauel K.², Fürll B.¹, GreinacherA.³

¹Institut für Immunologie und Transfusionsmedizin, Abteilung Transfusionsmedizin; Universitätsmedizin Greifswald, Greifswald, Deutschland, ²Institut für Immunologie und Transfusionsmedizin, Greifswald; Zentrum für Innovationskompetenz „Humorale Immunreaktionen bei kardiovaskulären Erkrankungen”, Greifswald, Deutschland, ³Institut für Immunologie und Transfusionsmedizin, Greifswald, Deutschland

Background: Heparin—induced thrombocytopenia (HIT) is an adverse drug reaction mediated by antibodies against complexes of platelet factor 4 (PF4) and heparin that does not follow the typical immune response pattern. Even heparin—naïve patients can generate specific IgG as early as day 4 of heparin exposure and there is no memory in case of reexposure. Recently we have shown that anti—PF4/heparin IgM and IgG are present in the normal population. Therefore we assessed whether PF4/heparin—specific memory B—cells circulate in the blood of healthy individuals. Patients and Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from hirudinized blood of healthy donors (n = 15) by density gradient centrifugation and cultured for 6 days with mitogens (Lectin, Phytolacca americana; Protein A, S. aureus: CpG2006) stimulating memory B—cells, i.e. cells secreting anti—PF4/heparin IgM and IgG. Memory B—cells were quantified by a modified enzyme—linked immuno spot technique (ELISPOT) using PF4/heparin—coated plates (PF4—only coated plates were used as control for specificity). Positive controls were total IgM and IgG producing B—cells. Results: Titration dependent total IgM and total IgG producing B—cells were present in all individuals, showing validity of the method. Anti—PF4/heparin IgM producing B—cells were present in 10/15 individuals (not reacting with PF4 alone in 6/6 tested), no anti—PF4/ heparin IgG producing B—cells were found. Conclusions: The high frequency of PF4/heparin—IgM memory B—cells in healthy individuals is consistent with the occurrence of these antibodies in ∼70% of patients after heart surgery and with the concept that these antibodies are a bacterial defense mechanism. We provide further evidence that PF4/heparin—IgG memory B—cells are absent, which is consistent with a lack of IgG—memory during reexposure with heparin. Anti—PF4/heparin antibody producing B—cells seem to be different from memory—B—cells in other immune reactions.

OS 3.02

Development of a rapid nano particle based immunoassay for the exclusion of heparin induced thrombocytopenia

(HIT)

Kolde H.—J.¹, Mauracher S.², Dostatni R.²

¹Consulting Diagnostics Dr. H.—J. Kolde, Ottobrunn, Deutschland, ²Milenia Biotec, Deutschland, Giessen, Deutschland

Background: HIT patients require alternative anticoagulants, but such drugs are associated with more bleeding and much higher costs. A reliable detection of IgG—antibodies (AB) against complexes of platelet factor 4 (PF4) and polyanions (PA) can reliably exclude HIT; but ELISAs are laborious and time consuming. We developed a rapid lateral flow HIT assay based on nano particles (NP). Methods: The assay is based on PF4 in complex with a proprietary PA bound to gold NPs (patent pending). Simply, 5 μl serum and 2 drops of the NP reagent are added to a test strip. Human ABs against PF4/PA in the sample bind to PF4/PA on the NP. During lateral flow, NPs with bound IgG against PF4/PA are retained by an immobilized goat anti—human IgG AB capture line on the membrane. In positive HIT cases the NPs form a red line while free NPs or NPs with bound IgA or IgM pass the capture line. A control line is integrated. Test time is 10 minutes. Results can either be read visually with the help of a color card or the line is quantitated with a POCScan reader. Results: The test was developed with a panel of sera from pat. with suspected HIT confirrmed with HIPA. After optimization, an internal validation with 60 sera agreed with HIPA, with a tendency of less falsely positive results than ELISA. A clinical trial confirmed these results (Sachs U, et al., in prep.). Preliminary tests with whole blood and plasma demonstrated principal feasibility for these specimens, a validation is in progress. Discussion: The new rapid assay QuickLine HIT detects or excludes reliably the presence of AB of the IgG type directed against PF4/PA AB within 10 minutes with minimum handling. Quantitative results can be obtained as well. The format of a single HIT test unit fits well into any lab; such tests are ordered infrequently. Together with clinical data this assay could be used for deciding on the anticoagulation strategy more evidence based and may reduce risks and costs in patients with HIT.

OS 3.03

The assessment of autoantibodies from patients with primary immune thrombocytopenia using platelet phagocytosis assay

Walek K.M.¹, Krautwurst A.¹, Santoso S.², Sachs U.¹, Bein G.¹, Bakchoul T.¹

¹Institut für Tranfusionsmedizin und Klinische Immunologie am Universitätsklinikum Gießen/ Marburg GmbH, Deutschland, ²Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Germany

Background: Primary Immune thrombocytopenia (ITP) is a bleeding disorder caused by platelet specific autoantibodies (PLT—AAbs). Although PLT—Abs are frequently detected using serological laboratory investigations, little is know about the effector function of these antibodies. In this study, the phagocytic activity of PLT—AAbs was analyzed in vitro using phagocytosis assay. Methods: Monocytes were isolated from healthy donors using CD 14—microbeads. Autologous PLTs were isolated by centrifugation and labeled using an intra—cellular FITC—dye. Labeled PLTs were then opsonized with purified IgG from ITP— and non—immune thrombocytopenic patients and introduced to isolated monocytes. After incubation, antibody phagocytic activity was determined using flow cytometry as the percentage of FITC—positive monocytes out of all gated monocytes. To exclude non—specific positive signal from antibody—independent PLT—monocytes interactions, investigations using confocal microscope were performed in parallel. Results: By testing IgG—fraction from non—immune thrombocytopenic patients, no phagocytic activity was observed (median: 3.1%) indicating the high specificity of the phagocytosis assay. The phagocytosis induced by AAbs was significantly higher using platelets obtained from citrated than from EDTA— blood samples (21,33% vs. 10,33%, p = 0.0013). The phagocytic activity of AAbs was largely, but not completely, inhibited by adding ivIgG at high concentration (660 μg/ml), suggesting that other mechanisms than engaging Fc gamma receptors may contribute to platelet destruction in ITP—patients. Conclusion: Better conditions for the effector function of PLT—AAbs can be provided using citrated blood samples. This finding could have a huge impact on laboratory investigations of ITP where a better sensitivity may be obtained using citrated blood samples. The phagocytosis assay allows better understanding of the pathophysiology of ITP and may be used to investigate new therapies.

OS 3.04

Platelet kinetics in ITP patients treated with thrombopoietin receptor agonists

Meyer O.¹, Herzig E.², Salama A¹

¹Institute of Transfusion Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany, ²Ambulantes Gesundheitszentrum der Charité GmbH, Nuklearmedizin mit PET/CT, Germany

Background: ITP is characterized by a reduced platelet count, antibody—mediated platelet destruction, and a significant decrease in platelet survival (PS). Impaired platelet production and T—cell mediated platelet destruction has been implicated as playing a potential role. Approximately 40% of patients with ITP display a reduced platelet turnover, and megakaryocytes are often decreased and/or damaged. It has also been demonstrated that ITP patients have normal or only slightly increased levels of thrombopoietin (Tpo). Furthermore, studies with thrombopoietin receptor agonists (Tpo RA) have demonstrated a dose—dependent response with a significant increase in platelets in almost 80% of patients treated for ITP. To further elucidate this, platelet kinetics were measured in six ITP patients treated with Tpo RA. Methods: In order to determine PS, autologous platelets were labeled with ¹¹¹In oxine and retransfiised in six patients undergoing treatment with Tpo RA (romiplostim n = 3; eltrombopag n = 3). Results: Stable platelet counts of greater than 100 × 109/μE were observed in all six patients. Platelet survival was decreased in all cases (mean 2.10 d; range 0.13 − 3.73 d). No correlation was found between platelet count and PS. Similarly, there was no significant relationship either between platelet turnover and platelet counts or PS. However, a high platelet turnover, exceeding 25 and three times the norm, respectively, was observed in two patients who presented the lowest PS (0.13 and 0.83 d, respectively). Two patients had a moderately shortened PS (1.91 d and 2.42 d), and, correspondingly, a moderately increased platelet turnover rate (63,072 and 72,872 plt/μL/d), respectively. Conclusion: These results indicate that Tpo RAmay not only overcompensate platelet destruction in ITP, but may interfere with other mechanisms, which, in some cases, results in a reduced platelet destruction rate.

OS 3.05

Pitfalls in the diagnosis of acute thrombocytopenia leading to the misdiagnosis of heparin—induced thrombocytopenia – case report

Knutson F.¹, Althaus K.², Greinacher A.³, Lubenow N.¹

¹Uppsala University Hospital, Dept. Clinical Immunology and Transfusion Medicine, Uppsala, Sweden, ²Institut für Immunologie und Transfusionsmedizin, Abt. Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Deutschland, ³Institut für Immunologie und Transfusionsmedizin, Greifswald, Deutschland

Background: Patients developing thrombocytopenia while treated with heparin as well as other drugs can pose a diagnostic challenge. The correct differential diagnosis between the procoagulatory syndrome of heparin—induced thrombocytopenia (HIT) and other drug—induced thrombocytopenias associated with an increased bleeding risk is important as management strategies are diametrically opposed. Case report: A 63 year old female presented with a traumatic fractured neck of femur (day 1). On day 3 an endoprothesis was inserted. She received low molecular weight heparin from day 3. On day 10 piperacillin/tazobactam (Tazocin) was commenced because of suspected pneumonia. On day 19 platelet counts fell from 208 to 41 × 10⁹/1, on day 20 they were 5 × 10⁹/1 and petechiae developed, there were no signs of thrombosis. Heparin and piperacillin/tazobactam were stopped. A particle—gel immu—noassay (PaGIA, DiaMed GmbH, Cressier, Switzerland) for anti—PF4/ heparin—antibodies was positive and danaparoid 2 × 750 units s.c. was started. Platelets rose to a stable plateau until day 30. At that time piperacillin/tazobactam was restarted because of relapsing fever. Platelet count decreased within 1 day from 446 to 8 × 10⁹/1. Further laboratory testing revealed strongly reactive piperacillin—dependent platelet antibodies, whereas an anti—PF4/heparinantibody ELISA showed HIT antibodies of the IgA class only, but no anti—PF4/ heparin IgG antibodies and a negative HIPA test. Comment: The PaGIA has high sensitivity, but low specificity for clinically relevant PF4/heparin antibodies. Reliance on a positive antigen assay alone can lead to wrong conclusions – in this case the diagnosis of HIT. Only reexposure to piperacillin revealed drug dependent thrombocytopenia. The case underscores that a positive antigen—assay in patients with suspicion of HIT needs to be further investigated by a functional assay (HIPA), especially if there is a low pre—test probability of HIT.

OS 3.06

Flow cytometric multi parameter acquisition improves analysis of megakaryopoiesis

Lorenz F., Grassl M., Ecker C., Bieback K., Klueter H., Bugert P., Schedel A.

Institut of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Germany

Background: Different in vitro cell systems are available to analyze megakaryopoiesis. A well—established model is the use of the human megakaryoblastic leukemia cell line MEG—01. A more physiological approach represents the use of ex—vivo generated megakaryoblastic cells isolated from human CD34⁺—progenitors. Because a detailed investigation of CD34⁺—generated progenitors is limited by low cell numbers, a technique is needed to gain as much information as possible using small cell numbers. In this study, we used the in vitro MEG—01 cell model to establish a flow cytometric panel for multiple cell differentiation marker analysis and applied it to the ex—vivo CD34⁺—progenitor model. Methods: MEG—01 cells and cord blood generated CD34⁺ progenitors (CB—MKs) were cultured and megakaryocytic differentiation was stimulated with (TPA) for 4 days and thrombopoietin (TPO) for three weeks, respectively. Data acquisition was performed daily for MEG—01 cells and twice a week for CB—MK cells on a FACS Canto II. Cells were simultaneously stained for the stem cell marker CD34, the leukocyte marker CD45, the megakaryocytic differentiation markers CD61 and CD41, as well as the viability dye 7—Aminoactinomycin D (7—AAD). Results: The multi parameter panel could be well applied to both MEG—01 cells and CB—MKs. The numbers of CB—MK cells for one measure could be reduced down to 10⁵ cells. As expected, a steady increase of CD61 and CD41 expression and a decrease of

CD34 expression were determined during megakaryocytic differentiation in both cell systems, being more clearly for CB—MKs. Conclusion: The simultaneous acquisition of five differentiation markers by flow cytometry from less than 10⁵ cells can be applied to analyze in vitro megakaryopoiesis on clinical samples with small cell numbers. By extending the FACS panel with further markers of interest new insights into the differentiation process could be achieved.

OS 3.07

Low—avidity anti—HPA—1a alloantibodies are capable of antigen—positive platelet destruction in the NOD/scid mouse model of alloimmune thrombocytopenia

Bakchoul T., Kubiak S., Krautwurst A., Bein G., Sachs U., Santoso S.

Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Germany,

Background: Neonatal alloimmune thrombocytopenia (NAIT) is mostly caused by maternal antibodies against human platelet antigen la (HPA—1a) expressed on glycoprotein (GP) IIb/IIIa. Accumulated evidence indicated that anti—HPA—1a antibodies could be overlooked by standard methods due to low avidity. Low—avidity HPA—la antibodies were shown to be detectable by surface plasmon resonance (SPR). We sought to investigate the frequency and in vivo relevance of low—avidity anti—HPA—1a. Study design and Methods: A retrospective cohort consisting of 82 HPA—lbb—mothers of thrombocytopenic HPA—lab newborns was analyzed using standard serological methods. Maternal IgG fractions were investigated for low avidity antibodies in SPR using purified GPIIb/IIIa (HPA—la or —lb). The capability of HPA—1a antibodies to clear platelets in vivo was analysed using the NOD/SCID mouse model of alloimmune thrombocytopenia. Results: HPA—antibodies were detectable in sera from 68/82 (83%) mothers using standard serological methods and undetectable in 14/82 sera. In SPR, IgG fractions of sera reacting positive in MAIPA showed specific binding to an HPA—la flow cell (mean: 87±21 resonance units (RU)). When MAIPA—negative sera were tested in SPR, binding with low—avidity was observed in 7/14 to HPA—1a— (mean: 31±5 RU), but not to HPA—1b—flow cell (mean: 5±2 RU). In vivo, low—avidity antibodies were capable to clear HPA—lab platelets but not HPA—1bb platelets in a NOD/SCID mouse model. Elimination kinetics was slower than observed with MAIPA positive antibodies. Conclusions: Low—avidity HPA—la antibodies are present in a significant number of NAIT cases and, although they can escape detection by standard serology, they harbour the capability of platelet destruction in vivo.

OS 3.08

Polymorphic variants of platelet integrin allbβ3: evidence of different receptor activation

El—Khattouti A., Stoldt V.R., Chahem B.C., Scharf R.E. Department of Hemostasis, Hemotherapy and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf, Germany

Background: The HPA—1 polymorphism of allbβ3 is due to a Leu→Pro exchange resulting in HPA—la (Leu33) or HPA—lb (Pro33). We have shown that patients with coronary artery disease who are carriers of HPA—lb experience their myocardial infarction 5.2 yrs earlier than HPA—la patients (JTH 2005; 3: 1522–1529). To explore the prothrombotic phenotype of the HPA—lb (Pro33) variant, we have generated transfected HEK293 cells expressing either isoform of allbβ3 fused to fluorescent proteins (CFP or YFP). Methods: Conformational changes of the integrin were analyzed by fluorescence resonance energy transfer (FRET) to assess the special separation between allb—CFP and β3—YFP upon integrin activation. Results: Specific activation of the phosphotyrosine motif of Src kinase (pY418) associated with allbβ3 was higher in HPA—lb than HPA—la adherent cells upon outside—in signaling. Likewise, the relative decrease in FRET signal, indicating spatial separation of the cytoplasmic tail as a consequence of integrin activation, was more pronounced in HPA—lb than in HPA—la cells. Upon exposure of adherent cells to increasing shear rates, the spatial separation of the integrin subunits occurred significantly faster in HPA—lb than HPA—la cells (p < 0.05). Under same conditions, the percentage of HPA—1b cells still adherent onto immobilized fibrinogen was 78%, while the relative number of residual HPA—1a cells decreased to 16% upon exposure to 1600 s—1 (p < 0.05). By contrast, HPA—1a cells displayed a stable FRET signal indicative of a rather resting or less activated state of αIIbβ3. Conclusions: Our findings suggest that HPA—1b (Pro33) has a significant impact on the activation of αIIbβ3. This is evident from a higher phosphorylation (pY418) of Src and higher resistance to biome—chanical stress upon exposure to increasing shear rates. Thus, the difference in allosteric changes of αIIbβ3 can contribute to the prothrombotic character of the HPA—1b (Pro33) variant.

Abstract Session 04 – Cell Therapy: Hematopoietic Stem Cells

OS 4.01

Establishment of an extended in vitro assay to detect the most primitive human hematopoietic cells at a clonal level

Radtke S.¹, Görgens A.¹, Punzel M.², Horn P.A.¹, Giebel B.¹

¹Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, ²Center for Bone Marrow and Stem Cell Donation, Cologne, Germany

Hematopoietic stem cells (HSC) can either self renew or give rise to multipotent hematopoietic progenitor cells (HPCs). Accordig to the classical model of hematopoiesis, these HPCs become either restricted to the lymphoid or to the myeloid lineage, e.g. to the common lymphoid (CLP) or common myeloid progenitor (CMP) cells. Via more restricted HPCs, CLPs then give rise to T, B and NK cells as well as to dendritic cells (DCs), while CMPs differentiate to macrophages, granulocytes, megakaryocytes and erythrocytes as well as a second subtype of DCs. Due to the recent characterization of HPCs containing partial myeloid and partial lymphoid developmental potentials this classical model has been challenged. New data suggests the existence of additional or alternative developmental pathways. Aiming to set up a functional in vitro read out system for the most primitive human hematopoietic cells, we originally developed a so called myeloid—lymphoid initiating—cell (ML—IC) assay. Within this assay individual HSC/HPC candidates are expanded on a murine stromal feeder cell layer. To test for their lymphoid or myeloid developmental capacities, the daughter cells are either transferred into an assay allowing NK cell or granulocyte and macrophage development. Before discovering the novel HPC types, deposited primitive hematopoietic cells whose offspring gave rise to NK cells as well as granulocytes and macrophages were retrospectively claimed as cells containing the potential to develop into all hematopoietic cell types. However taking the new findings into account, this conclusion cannot be justified anymore. Since we are still interested in an in vitro read out system allowing the functional detection of the most primitive human hematopoietic cells and also to define novel hematopoietic routes, we decided to extend the ML—IC assay for additional lineage read outs including T cell, B cell, megakaryocyte and erythrocyte development. Our experimental strategy will be presented.

OS 4.02

Endosomal machinery in hematopoietic stem and progenitor cells (HSPCs)

Ludwig A.—K.¹, Horn P.A.², Giebel B.²

¹Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, ¹Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany

One important aspect in the field of stem cell biology is to elucidate the mechanisms which control the decision whether progenies of somatic stem cells are maintained as stem cells or whether they become committed to differentiate. In principal two different settings have been identified that can control such decisions, the process of asymmetric cell division and the stem cell niches. For the most investigated mammalian stem cells, the hematopoietic stem and progenitor cells (HSPCs), it has been elaborated that they reside in special niches, which are required for their maintenance. In addition, due to the identification of asymmetrically segregating proteins, we have previously proven for the first time that HSPCs also have the ability to divide asymmetrically. Remarkably, three of the four identified, asymmetrically segregating proteins are associated with the endosomal machinery. According to recent findings that endosomes also can segregate asymmetrically in model organisms and that they participate in the regulation of cell fate decisions, we have started to elvaluate impacts of the endosomal machinery on cell fate decisions within the human primitive hematopoietic cell compartment. Since small GTPases play key roles in regulating fission, fusion and trafficking of endosomal vesicles, we decided to set up a technical platform to genetically manipulate human HSPCs with constructs expressing normal as well as constitutive active and dominant negative isoforms of a variety of different Rab—GTPases. After cloning corresponding coding regions into lentiviral vectors allowing to stably transduce human somatic stem cells, we are now exploring their functional impacts on the decision self—renewal versus differentiation of human HSPCs in different in vitro assays.

OS 4.03

Interleukin 32 promotes hematopoietic progenitor expansion and attenuates bone marrow cytotoxicity

Moldenhauer A.¹, Futschik M.², Lu H.¹, Helmig M.¹, Götze P.¹, Bal G.¹, Zenke M.³, Han W.⁴, Salama A.¹

¹Institute of Transfusion Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany, ²institute of Theoretical Biology, Humboldt—University, Berlin, Germany, ³Helmholtz Institute for Biomedical Engineering, RWTH, Aachen, Germany, ⁴Institute for Regeneromics, Jiaotong—University, Shanghai, China

The identification of soluble factors involved in stem cell renewal is a major goal in the assessment of the BM niche. We have previously shown that human endothelial cell (EC) supernatants can induce the proliferation of hematopoietic progenitor cells (HPCs), especially after stimulation with IL—1β. To identify new potential growth factors, we compared the expression profile of IL—1β— stimulated ECs over 4, 8 and 16h with non—stimulated ECs using oligonucleotide microarrays covering more than 46000 transcripts. Most significant changes were detected after 4h. Utilization of Gene Ontology annotation for the stimulated EC transcriptome indicated multiple upregulated genes encoding extracellular proteins with a cell—cell signaling function. Using flow cytometry, delta, colony and cobblestone assays, we assessed the proliferative capacities of 11 gene products, i.e. IL—8, IL—32, FGF—18, osteoprotegerin, Gro 1–3, ENA78, GCP—2, CCL2 and CCL20, which are not known to induce HPC expansion. Notably, IL—32 and to a lesser degree osteoprotegerin and Gro 3 significantly induced the proliferation of HPCs. Furthermore, IL—32 attenuated chemotherapy—related BM cytotoxicities by increasing the number of HPCs in mice. Our findings confirm that the combination of microarrays and gene annotation helps to identify new hematopoietic growth factors.

OS 4.04

Haematopoietic stem cell mobilization with Plerixafor in patients with non—haematological diseases

Worel N.¹, Apperley J.F.², Basak G.W.³, Douglas K.W.⁴, Gabriel I.H.², Geraldes C.⁵, H übel K.⁶, Jaksic O.⁷, Koristek Z.⁸, Lanza F.⁹, Lemoli R.M.¹⁰, Mikala G.¹¹, Selleslag D.¹², Duarte R.F¹³, Monty M.¹⁴

¹Dept for Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria, ²Department of Haematology Imperial College, London, UK, ³Department of Haematology, Oncology and Internal Diseases, Medical University of Warsaw, Warsaw, Poland, ⁴HPC Transplant Programme, Beatson West of Scotland Cancer Centre, Glaskow, UK, ⁵Department of Haematology, University of Coimbra, Coimbra, Portugal, ⁶Klinik I für Innere Medizin, Universitätsklinikum Köln, Cologne, Germany, ⁷Department of Haematology, University Hospital Dubrava, Zagreb, Croatia, ⁸Department of Internal Medicine, Haemato—Oncology, Masaryk University Hospital, Brno, Czech Republic, ⁹Section of Hematology & BMT Unit Hospital of Cremona, Cremona, Italy ¹⁰Department of Hematology and Oncological Sciences L and A Seràgnoli, Institute of Hematology, University of Bologna and Stem Cell Research Center, Bologna, Italy, ¹¹Department of Haematology and Stem Cell Transplantation, St Laszlo Hospital of Budapest, Budapest, Hungary, ¹²Algemeen Ziekenhuis Sint—jan, Brügge, Belgien, ¹³Department of Haematology, Catalan Institute of Oncology, Barcelona, Spain, ¹⁴Centre Hospitalier et Universitaire de Nantes, Hématologie Clinique, Nantes, France

Autologous stem cell transplantation (ASCT) can be a curative procedure for a variety of malignancies. Successful ASCT is dependent on transplantation of sufficient CD34+ cells to ensure prompt and durable engraftment. However, a number of patients fail to mobilize the threshold of >2 × 10⁶/kg CD34+ cells. Recently, plerixafor was shown to be safe and effective for stem cell (SC) mobilization from patients with myeloma and lymphoma. This report aimed to evaluate the effect of plerixafor on SC mobilization in patients with non—haematological malignancies in whom data regarding pretreatment, mobilization and CD34+ cell yield were available. Thirty—two patients who previously failed HSC mobilization were included in this study. Patients were diagnosed with germ cell (11), Ewing sarcoma (5), Wiscott Aldrich disease (5), neuroblastoma (3), medulloblastoma (2), and others (6). Median age was 26 (2–70) years, and 78% were males. The median number of prior chemotherapy regimens was 2 (0–5), with 22% of patients receiving prior radiotherapy. In the majority of patients the mobilization consisted of G—CSF + plerixafor, whereas a few patients received chemotherapy followed by G—CSF + plerixafor. On the first apheresis day (about 10–11 hrs. after plerixafor), the peripheral blood CD34+ cells count reached a median of 34 (2–250) CD34+ cells/μl. Twenty—five of 32 (78%) patients achieved the target cell dose of 2 × 10⁶ CD34+ cells/ kg (median 5.2, range 2–29.53 × 10⁶/kg CD34+ cells), while 7 patients failed to collect a sufficient cell dose (median 1.5, range 0.33–1.8 × 10⁶/kg CD34+ cells). At last follow—up, 16 patients (50%) underwent ASCT and received a median of 3.6 (2.6–6.7) × 10⁶/kg CD34+ cells. In all, these data suggest that plerixafor + G—CSF ± chemotherapy is effective in patients with non—haematological diseases. In patients eligible for ASCT who have failed prior mobilization attempts, plerixafor can provide an opportunity to still pursue a potentially curative procedure.

OS 4.05

Analysis of intraapheresis recuitment of hematopoietic progenitor cells during mobilization with Plerixafor and comparison to controls

Humpe A.¹, Renders U.¹, Jung N.¹, Buwitt—Beckmann U.¹, Günther A.¹, Horst H.², Schrauder A.³, Biersack H.⁴, Gramatzki M.¹

¹Division of Stem Cell and Immunotherapy, Second Department of Medicine, University of Kiel, Kiel, Germany, ²Second Department of Medicine, University of Kiel, Kiel, Germany, ³Department of Pediatrics, University Kiel, FRG, Kiel, Germany, ⁴First Department of Medicine, Hematology and Oncology, University of Lübeck, FRG, Lübeck, Germany

Background: Successful mobilization of sufficient or even optimal amounts of hematopoietic progenitor cells (HPC) is necessary for transplantation after high—dose therapy. In patients who do not mobilize sufficient numbers of HPC, the addition of Plerixafor offers a new therapeutic option. We analyzed a series of patient undergoing mobilization with Plerixafor and compared the data to a control group. Patients and Methods: Data from 19 aphereses of 18 patients being mobilized with chemotherapy, G—CSF, and Plerixafor were retrospectively analyzed regarding number of CD34+ cells in peripheral blood (PB) before apheresis, after apheresis and in the graft. These data were compared with retrospective data from 34 aphereses of 27 patients being mobilized in the same time period and exhibiting a maximum CD34+ cell count in PB before apheresis not higher than the maximum count in the Plerixafor group. Intraapheresis recruitment (IR1) is defined as followed: (yield of CD34+ cells in the graft) – (number of CD34+ cells in PB before apheresis). In addition, an IR2 was calculated by the following difference: (yield of CD34+ cells in the graft) – ((total number of CD34+ cells in PB before apheresis) – (total number of CD34+ cells in PB after apheresis)). Results: The median number of CD34+ cells before apheresis was significantly (p < 0.05) higher in the control group than in the Plerixafor group (59 and 25, respectively). In addition, the median IR1 as well as the median IR2 were significantly (p < 0.05) higher in the control group (IR1: 2.04 × 10E+5/kg; IR2: 3.77 × 10E+5/kg) compared with the Plerixafor group (IR1: 8.33 × 10E+4/kg; IR2: 1.37 × 10E+5/kg). In the Plerixafor group the median number of processed peripheral blood volumes during apheresis was significantly (p < 0.05) higher (4.25 times versus 3.46 times) compared to the control group. Conclusions: In poorly mobilizing patients Plerixafor can induce an IR of HPC but at a significantly lower extent compared with controls.

OS 4.06

Clonogenicity, viability and cytochemical quality of blood progenitor cell products

Gebauer W., Barklage I., Kirchberg S.

German Red Cross Blood Transfusion Service N.S.T.O.B., Oldenburg, Germany

Progenitor cells of malignant and benign origin are known to express increased activity of aldehyde dehydrogenase (ALDH) making them more resistant to alkylating agents. Immunological characterization of blood progenitor cells (bpc) was combined with flow cytochemical determination of ALDH expression and with viability determination by 7—Aminoactinomycin D (7AAD). Furthermore clonogenic potential was determined. After freezing and thawing of progenitor cell products analysis of twenty five consecutive transplants was performed. An aminoacetaldehyde was converted to the corresponding aminoacetate by the cytosolic enzyme ALDH and was detected by flow cytometry. By confocal microscopy ALDH expression was localized in the cytosol, qdot 605nm marked secondary antibodies showed CD34 related to the cell surface. Clonogenic potential of blood progenitor cells was determined by short term culture assays. Enumeration of CD34 and 7—AAD expressing events by multicolour flow cytometry was performed using commercially available assays. ALDH expressing cells with low orthogonal light scatter can be characterized by gating strategies without physical enrichment procedures or depletion of cellular fractions. ALDH activity as nonimmunologic marker led to the identification of a cellular population overlapping but not identical to the population of CD34 expressing events. To describe quality and viability of cryopreserved and then thawed blood progenitor cell transplants 7—AAD, ALDH expression and clonogenic potential in short term culture assays was performed. Preliminary results demonstrated a weak relation of ALDH expression and a stronger relation of 7—AAD expression of CD34 positive cells to clonogenic potential. A more close relation of ALDH activity and 7—AAD defined viability of CD34 expressing events was observed. Cells with high ALDH activity will be useful in the fields of regenerative medicine as the cells after staining procedures are viable.

OS 4.07

Platelet differentiation from CD34+ progenitor cells is inhibited by Semaphorin 7A

Jaimes Y.¹, Immenschuh S.¹, Eiz—Vesper B.¹, Seltsam A.², Blasczyk R.¹, Figue i red o C.¹

¹Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany, ²DRK—Blutspendedienst NSTOB Institut Springe, Springe, Deutschland

Background: Thrombocytopenia is a common side effect in patients after high dose chemotherapy. New treatments for thrombocytopenia focus on the transfusion of ex vivo CD34+ cell derived—megakaryocytes (MKs). We observed that 50% of the patients that underwent chemotherapy upregulated the expression of Semaphorin7A (Sema7A), a signalling molecule involved in immunomodulation. We studied the effect of Sema7A in the differentiation and function of platelets (PLTs) derived from CD34+ progenitor cells. Patients and Methods: Recombinant Sema7A was used to stimulate CD34+ cell—derived MKs or PLTs. For PLT differentiation, CD34+ cells were isolated from G—CSF mobilized patients and incubated with thrombopoietin and inter—leukin—3 during 25 days in presence or absence of Sema7A. Expression of CD41, CD61 and CD42a was used to assess the MK and PLT differentiation via flow cytometry. The effect of the Sema7A cytokine secretion profile of MKs was measured using multiplex bead technology. Cell density and morphology during MK differentiation was assessed by fluorescence microscopy. Results: In presence of Sema7A, we observed that only 30% of the cell populations expressed the MK and PLT markers CD41, CD61 and CD42a in comparison to 70% of the cell populations when incubated in absence of the molecule. Moreover, a significant higher quantity of the pro—inflammatory cytokines IL—6 (245±67pg/ml, p < 0.05) and IL—8 (10925±2254pg/ml, p < 0.05) was secreted by MKs and PLTs exposed to Sema7A in comparison to the concentrations detected when the cells were differentiated in the absence of Sema7A (59±35pg/ml and 801±117pg/ml, respectively). Also, the MK cell density was up to 40% lower in presence of Sema7A compared to the cell density obtained in the absence of Sema7A. Conclusions: This data shows that Sema7A is a strong inhibitor of MK and PLT differentiation from CD34+ cells and offers new therapeutic approaches to face thrombocytopenia.

OS 4.08

Ex vivo improvement of defective red blood cell function in sickle cell disease

Dorn l.¹, Mazurier C.², El Nemer W.³, Picot J.³, Giarratana M.C.², Kobari L.², Douay L.²

¹Pediatric Hematology and Oncology, University Hospital Münster, Münster, Germany, ²INSERM, UMR_S938, Faculté de Médecine Pierre et Marie Curie 27, Paris, France, ³UMRS 665, Institut National de la Transfusion Sanguin, France

Background: In sickle cell disease (SCD), pathological hemoglobin S (HbS) induces sickling of red blood cells (RBC), their reduced deformation capacity and increased adherence to the endothelium.¹‘^2,3 Resulting vaso—occlusive events are responsible for high morbidity and mortality. Fetal hemoglobin (HbF) is known as the most potent modifier in disease severity, although mechanisms by which HbF ameliorates RBC physiology remain unclear.²‘⁴ To investigate the effect of HbF on RBC physiology, we applied our protocol for the ex vivo generation of RBC on hematopoietic stem cells (HSC) from patients with SCD⁵. Patients and Methods: CD34+ HSC from SCD patients were cultured over 25 days in an ex—vivo model for erythropoiesis. Cultured RBC (cRBC) were compared with native SCD—RBC from the same patient for their level of HbF (HPLC, flow cytometry), the expression of different adhesion molecules (flow cytometry), their deformation capacity (LORCA) and adhesion to human laminin and primary endothelial cells (flow adhesion assay). Results: The applied ex—vivo model was able to generate >90% enucleated RBC with an ∼100.000fold amplification. Compared to native SCD—RBC, cRBC produced a higher level of HbF (26.5% vs. 2.3%, p < 0.01). Whereas native SCD—RBC showed strong adhesion to laminin and endothelial cells, this was reduced or absent in cRBC. In line with this, cRBC showed a modified expression of adhesion molecules. In contrast to the reduced deformation capacity of native SCD—RBC, the deformation capacity of cRBC was also normalized. Conclusion: RBC generation under ex vivo conditions allows for the improvement of RBC physiology in SCD. Besides an increased level of HbF, cRBC cells show a normalized deformation capacity and reduced adhesion to elements of the endothelial wall. As the most interesting clinical application, such ex vivo generated RBC might be suitable as an autologous transfusion product, able to to circumvent shortages of compatible RBC units.

Table for abstract OS 5.01

Year Pat., PC (n)	CI (×109/L)	CCI (×109/L)	PC transfusion intervals (days)	Bleedings (pat., n)
2007, 36, 334	7 (0–33)∗	4 (0–20)∗	1.0	20 (56%)
2008, 41, 375	14 (0–106)∗∗	10 (0–68)∗∗	1.9	9 (22%)
p-values	≤ 0.0001	≤ 0.0001	≤ 0.0001	≤ 0.01

^∗17 and

^∗∗18 hours after transfusion (median values)

Abstract Session 05 – Hemotherapy

OS 5.01

Clinical effect of platelet concentrate storage time reduction

Heuft H.—G.¹, Tiede A.², Goudeva L.¹, Buchholz S.², Blasczyk R¹

¹Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany, ²Department for Hematology, Hemostaseology, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany

Background: To evaluate the effect of apheresis platelet concentrate (PC) transfusion after im—plementation of the 4—days PC storage period. Patients and Methods: We compared the platelet count increment (CI), the corrected plate—let count increment (CCI), red cell (RC) and PC transfusion needs or intervals, and bleeding events in 77 haematological patients transplanted from August to November, 2007 (n = 36, 5 days routine PC storage, median storage time 79 [11–136] hours) versus August to Novem—ber, 2008 (n = 41, 4 days routine PC storage, median time 55 [11–112] hours, p≤0.001). Patients were comparable for demographic data (age, weight, body surface area [BSA]), me—dian PC transfusion dose (2007, 1.38 × 101 l/m2 BSA/PC as compared to 1.41 × 1011/m2 BSA 2008, p = 0.84), and allogeneic transplantations (2007, 23/36 patients, 64%; 2008, 26/41 patients, 63%). Underlying diseases (2007/2008) were: acute leukaemia 12 (33%)/20 (49%); lymphoma 4 (11%)/5 (12%); multiple myeloma 10 (28%)/9 (22%), myelodysplastic syndrome and other diseases 10 (28%)/7 (17%). Bleeding evaluation comprehended petechiae, haematomas, nose and mouse bleeds, bloody stools and haematuria. Results: Within 5 days after PC transfusion, in 74% of the observation points the patients required ≥2 RC in 2007 as compared to 58% in 2008 (p ≤0.002). Conclusion: PC storage time shortening was associated with highly significant CI/CCI in—crease, reduced PC/RC needs and fewer patients with significant bleedings.

OS 5.02

Magnetically labelled platelets for recovery and survival studies

Aurich K.¹, Riepenhausen S.¹, Spoerl M.—Ch.², Fuerll B.¹, Sietmann R.³, Weitschies W.⁴, Greinacher A.¹

¹Institut für Immunologie und Transfusionsmedizin, Greifswald, Deutschland, ²Institut für Diagnostische Radiologie, Universitätsmedizin Greifswald, Greifswald, Deutschland, ³Institut für Mikrobiologie, Universität Greifswald, Greifswald, Deutschland, ⁴Institut für Pharmazie, Universität Greifswald, Deutschland

Background: Platelets play a dominant role in the pathogenesis of bleeding disorders and cardiovascular pathology. Non—radioactive labelling of platelets may offer several clinical applications ranging from survival studies of transfused platelet concentrates to the pathogenesis of stroke. We established a protocol using superparamagnetic iron oxide nanoparticles (SPIO; Resovist^®, Bayer Schering AG, Germany) to label platelets. Additionally, we used magnetically labelled platelets for visualization of platelets by magnetic resonance imaging (MRI). Methods: SPIOs were fractionated by magnetic fractionation and incubated with human platelets in Tyrodes buffer (37°C; 30 min). Particle location within cells was determined by transmission electron microscopy (TEM) and fluorescence microscopy (FM). Iron content per cell was determined by atomic absorption spectroscopy (AAS). To reduce interference with membrane proteins, SPIO at the exterior cell membrane were eliminated by enzymatic digestion. MRI settings for in vitro MR analysis of labelled platelets were established using an artificial circular tube blood flow system and a 7 Tesla scanner. Results: Platelets internalized SPIOs. Incubation time of 30 min with SPIO suspension of 10 mM iron concentration sufficed for adequate labelling of 2 pg iron/platelet as determined by AAS. This will enable sensitive ex vivo determination of transfused labelled platelets (sensitivity ∼2 platelets/μL), allowing survival studies. In vitro, labelled platelets gave a clear signal by 7 Tesla magnetic resonance imaging. Thereby, the largest SPIO fraction (95 nm) exhibit highest contrasts. Conclusion: Magnetic labelling of platelets may offer a new tool for diagnosis and research in transfusion medicine and cardiovascular medicine. It will allow tracking of their biodistribution in vivo and opens the perspective to study platelet distribution and platelet kinetics in humans without the need of radioactivity.

OS 5.03

Hemostasis endpoints (EP) in platelet transfusion clinical trials with differential follow—up

Sherman C., Reed W., Corash L. Cerus Corporation, Concord, CA, USA

Background: Platelet transfusion (PCTXN) is vital to prevent bleeding (B) in patients (PTS) with thrombocytopenia. Identification of relevant EP to assess interventions to improve PCTXN outcomes is challenging. Many EP to assess PCTXN efficacy combine different grades (G) of B—WHO G2 through G4 into a composite EP. Severe B may not be affected by PCTXN, but requires plasma TXN. Prior assessments of B used dichotomized B EP (+/—) during PC TXN. This analysis does not consider recurrent B during PCTXN. Methods: In the SPRINT trial efficacy of conventional PC (C—PC) was compared to pathogen inactivated PC (PI—PC) for up to 28 days(d) of PCTXN. This large database was used to identify recurrent B, confounding factors, and propensity for G2B to precede G3B or G4B bleeding. Data were evaluated for key confounders: HSCT—allo/auto, total body irradiation (TBI), transplant GVHD, and G2B prior to study PCTXN. For days with G2B, the duration of PCTXN was considered since with longer PCTXN G2B is more likely. Results: The distribution of d with G2B was positively skewed and best fit a negative binomial regression model. Mean d of observation (PCTXN) ranged from 10 (auto HSCT) to 25 d (allo HSCT with TBI + GVHD). When the regression model for d with G2B was adjusted for d of observation and confounders, the rate of G2B did not differ significantly between treatments (C—PC vs PI—PC). 6 of 33 PTS (18%) with G3B or G4B had G2B at study entry; but after study entry, only 2 of 27 PTS (7%) had G2B preceding G3B or G4B. 23 of 33 PTS (70%) with G3B or G4B had plasma phase coagulopathy at entry or during PC TXN. Conclusions: D with G2B characterized recurrent bleeding, was positively skewed, was impacted by confounders, and d of observation. G3B or G4B occurred frequently before G2B and was commonly associated with coagulopathy. G2B did not predict G3B or G4B, and should not be pooled in a composite EP.

OS 5.04

Platelet transfusion alters plasma CD40L and CD40L release capacity in the peripheral blood

Wenzel F.¹, Günther W.², Haas R.³, Giers G.²

¹Institut für Transplantationsdiagnostik und Zelltherapeutika, Düsseldorf, ²Dept. of Experimental and Clinical Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf, Germany, ³Klinik für Hämatologie, Onkologie und Klinische Immunologie, Düsseldorf, Deutschland

Background: Soluble CD40L (sCD40L) is proven to have various effects on the adaptive immune system. Platelet derived cytokines, like sCD40L, play an important role in the development of adverse transfusion reactions associated with the administration of platelet products. In the present study we determined sCD40L concentration and release capacity in thrombocytopenic patients before and after receiving a platelet transfusion. Study Design and Methods: The study included nine patients suffering from chemotherapy—induced thrombocytopenia. sCD40L levels were measured in plasma and in serum samples of the patients before and after platelet administration as well as in the respective platelet apheresis concentrates. sCD40L concentrations were determined by an ELISA—Kit. Sixteen healthy blood donors served as a control group. Results: In platelet apheresis concentrates, elevated sCD40L levels (2,791 ± 131 pg/ml) were observed in comparison to plasma sCD40L levels in controls (235.2 ± 34.7 pg/ml). sCD40L plasma concentration of thrombocytopenic patients was lowered to 91.9 ± 18.6 pg/ml before transfusion and increased to 210.2 ± 26.7 pg/ml after platelet administration. In parallel, the total sCD40L release capacity raised from 240.1 ± 63.9 pg/ml before to 908.6 ± 188.9 pg/ml after platelet transfusion. Conclusions: In thrombocytopenic patients, sCD40L levels were clearly influenced by platelet transfusions: In the stored platelet apheresis concentrates, an accumulation of sCD40L was found leading to a normalization of sCD40L plasma concentration in the patients immediately after transfusion. Additionally, the sCD40L release capacity was enhanced by platelet administration dependent on the increase in platelet count.

OS 5.05

Preoperative autologous deposit – rather illusion but potential resolution to shortage of allogeneic RBC to come?

Singbartl G.¹, Held A.—L², Singbartl K.³

¹Si_AIT, Soltau, Deutschland, ²Medizinische Klinik 2 Schwerpunkt Geriatrie, Klinikum Nuernberg Nord, Nürnberg, Deutschland, ³Department of Critical Care Medicine University of Pittsburgh, USA

Background: Due to increasing safety of allogeneic blood, relevance of autologous alternatives has strongly declined; especially of preop. autolog. deposit (PAD). However, demographic changes probably give PAD a new chance to substitute in part for shortage of allogeneic RBC associated w/ decrease of volunteer donors. Methods: Search of original PAD—data in English/German literature from 01/1985 to 12/2009 allowing mathematical modeling on efficacy (increase in RBC—mass: +RBC) and effectiveness (max. blood loss compensated autologously, only: MABL) of PAD, and ntra—individually comparing it to intraop. cell salvage (ICS; RBC—recovery rate 30,50,70%, resp.), and acute normovolaemic haemodilution (ANH); either w/o preceding PAD. ANH was applied until reaching hct min 24,21,18%, resp. (intensified ANH: iANH); then starting surgery/retransfu—sion with commencing BL. Overall, when reaching hct min, BL was replaced both by autologous +RBC and colloid to maintain hct min and normovolaemia despite ongoing BL. Formulae have been published elsewhere. Statistical analysis by H—/U—test; p≤0.005 w/ Bonferroni correction. Ranking on effectiveness of either autologous alternative (PAD, ICS30, ICS50, ICS70, iANH); rank 1 (most effective), rank 5 (least effective). Results: 21/957 papers w/ a total of 3.926 pats, have been eligible for analysis. Ranking of effectiveness has demonstrated consistently ICS70 and 50 the most effective measures (rank 1 and 2, resp.), ICS30 and PAD have been shown moderate effective, and iANH has been the least effective autologous alternative; it has gained relevance with decreasing hct min, only; w/ hct min of 18%, it has reached rank 3 ahead of ICS30 and PAD. Conclusion: ICS has been demonstrated the superior autologous alternative when compared to both PAD and iANH. PAD may be in part a resolution to shortage of allogeneic RBC to come, only, if the PAD—concept applied is adapted to the physiological principles of erythropoi—esis (2,3).

OS 5.06

Improving the safety of granulocyte transfusion by introducing the flow cytometric granulocyte immunofluorescence test (Flow—GIFT) for donor and patient screening

Schulze T.J., Huetter G., Stoetzer F., Klüter H., Nguyen D.

Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Germany

Background: Granulocyte transfusions have been effective in treating neutro—penic patients with life—threatening infections. Leukocyte—associated antibodies can cause transfusion—related acute lung injury (TRALI). According to the national guidelines only rbc crossmatch is mandatory prior to granulocyte transfusion. Here we describe the procedures for rapid testing of leukocyte—associated antibodies in donor and patients prior to granulocyte apheresis and transfusion using the novel flow cytometric granulocyte immunofluorescence test (Flow—GIFT). Patients and Methods: Within 2 years we provided 112 granulocyte preparations for 31 patients. Before a donor was declared a match, screening for antibodies against leukocytes in both patient and donor as well as crossmatches between patients’ serum and donors’ leukocytes were performed using Flow—GIFT. This procedure was repeated every two weeks to exclude de—novo alloimmunization. Positive Flow—GIFT results were differentiated by simultaneous analysis of specific granulocyte antibodies assay (SASGA). Results: All patients tolerated the crossmatch negative granulocyte transfusions well. In one patient, anti—HLA class II and —granulocyte antibodies with unknown specificity, but reactivity to all selected donors, were found. In this case, all TRALI—relevant antibodies against HNA—1a, —1b, —2a and —3a as well as lymphocyte—reactive antibodies including specific anti—HLA—class II were excluded. In a further patient, Flow—GIFT results turned positive against one donor's granulocytes after two weeks of granulocyte administration. The antibody specificity could not be determined. This donor was not further permitted to donate granulocytes for this patient. Conclusions: Flow—GIFT is a reliable tool for the investigation of leukocyte Abs in the supply of granulocyte preparations. Screening for leukocyte Abs as well as individual patient donor leukocyte crossmatches should be performed regularly to sustain safety of granulocyte transfusion

Table for abstract OS 5.05

	PAD		ICS30%		ICS50%		ICS70%		ANH
	MABL (L)/Ranking of measures
	Hct min 24%
1 PAD	3.58	5	4.47	3	5.34	2	6.21	1	4.15	4
2 PAD	4.13	5	4.76	3	5.70	2	6.64	1	4.51	4
3 PAD	5.33	3	5.31	4	6.36	2	7.40	1	5.02	5
4+ PAD	4.59	2	3.68	4	4.40	3	5.11	1	3.38	5
Single RBC-Apher.	1.77	4	1.82	3	2.13	2	2.45	1	1.37	5
Double RBC-Apher.	5.13	5	5.37	3	6.45	2	7.53	1	5.19	4
Σ of Ranks	24		20		13		6		27
Rank	4		3		2		1		5
	Hct min 21%
1 PAD	4.41	5	5.57	3	6.72	2	7.86	1	5.51	4
2 PAD	5.07	5	5.86	4	7.08	2	8.31	1	5.90	3
3 PAD	6.45	5	6.59	4	7.95	2	9.32	1	6,60	3
4+ PAD	5.68	2	4.59	4	5.54	3	6.48	1	4.51	5
Single RBC-Apher.	2.47	4	2.64	3	3.13	2	3.61	1	2.21	5
Double RBC-Apher.	6.10	5	6.56	4	7.95	2	9.34	1	6.73	3
Σ of Ranks	26		22		13		6		23
Rank	5		3		2		1		4
	Hct min 18%
1 PAD	5.38	5	6.92	4	8.43	2	9.94	1	7.34	3
2 PAD	6.19	5	7.20	4	8.80	2	10.40	1	7.78	3
3 PAD	7.81	4	8.15	3	9.94	2	11.73	1	7.81	4
4+ PAD	7.02	2	5.71	5	6.95	3	8.19	1	6.00	4
Single RBC-Apher.	3.30	5	3.64	3	4.35	2	5.06	1	3.31	4
Double RBC-Apher.	7.26	5	8.03	4	9.83	2	11.63	1	8.80	3
Σ of Ranks	26		23		13		6		21
Rank	5		4		2		1		3

OS 5.07

Routine use of minor blood group antigen prediction by high throughput methods

Wagner F.F.¹, Doescher A.², Bittner R.¹, Mueller T.H.¹

¹DRK—Blutspendedienst NSTOB Institut Springe, Springe, Deutschland, ²German Red Cross Blood Transfusion Service N.S.T.O.B., Institute Bremen—Oldenburg, Oldenburg, Germany

Background: A crucial factor for the utility of molecular antigen prediction of blood donors is the probability that the obtained typing information will be used for unit selection. We traced typed units to estimate this probability. Methods: Antigens of frequent blood donors were predicted by pooled capillary electrophoresis. Donations from typed donors with useful antigen patterns were guided to laboratory inventories. For one month, release of typed units for patients or other blood services was collated for the “main” deposit A. For each unit, it was checked whether the antigen—negative status was utilized and how many units would have been needed to screened by serology to find a unit with a similar antigen status. Results: In January 2010, 6150 donation were from typed donors with successful predictions for Fy and Jk antigens (mean 298 units per day, range per day 244 to 425). Of these units, 919 (15%) were guided to laboratory inventories. Inventory A received 585 typed units and released 352 units with at least one negative typing result demanded for Jka, Jkb, Fya, Fyb, M, N, S, s, Lub, Kpb, Yta or Coa. Of these units, 315 (89% of released units, 54% of pretyped units) had been identified by molecular donor typing. Considering multiple negative units, 373 negative antigen predictions could be exploited. In a conventional approach, an estimated number of 1723 units would have had to be screened to find the units. Conclusion: In our setting, one out of 12.5 units from typed donors was used in a situation in which the antigen negative status was useful. In the case of searching only serologically, 5.5 units would have to be tested to find a useful unit. Assuming that molecular typing for all relevant antigens costs 4 times that of sérologie screening for a single antigen, molecular prediction would become cost efficient, if typed donors are retained for at least 9 donations (about 4 years).

OS 5.08

High risk of red cell alloimmunization in patients with inflammatory bowel disease

Körmöczi G.F.¹, Hackner K.¹, Vogelsang H.², Mayr W.R.¹, Primas C.², Eser A.², Mikulits A.², Dejaco C.², Reinisch W.², Novacek G.², Papay P.²

¹Dept. of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria, ²Medical University of Vienna, Vienna, Austria

Background: Anemia is frequently found in patients with inflammatory bowel disease (IBD), at times necessitating red blood cell transfusion (RBCT). However, allogenic red cell exposure such as RBCT and pregnancy may induce red blood cell alloantibodies (RBCA), with the potential of complicating further RBCTs and pregnancies. Recent studies indicate that acute inflammation may trigger RBCA formation. Hence, the prevalence and clinical modulators of RBCA acquisition were investigated in IBD patients with chronic or relapsing inflammation. Patients and Methods: In 193 consecutive IBD patients with a history of RBCT and/or pregnancy, RBCA screening and analysis of further clinical data was performed. Transfused sex—matched controls (n = 357) suffering from other diseases served as control cohort.

Results: A RBCA prevalence of 8.4% was observed in IBD patients with a history of ABT, which is about 2.5 times higher than in transfused controls with different diseases (3.4%; p = 0.023). In IBD patients with a history of pregnancy but not RBCT, the RBCA prevalence was 1.4%. Overall, RBCA specificities of clinical significance were detected in 5.7% of all studied IBD patients. The risk of RBCA acquisition increased with more RBCT (OR 1.57 [1.03–2.39]), whereas immunomodulatory therapy during RBCT appeared to be protective (OR 0.12 [0.02–0.61]), as judged by multivariate analysis. Conclusions: These results indicate a considerably increased risk of transfusion—induced red cell alloimmunization in IBD patients, possibly triggered by inflammation. A restrictive transfusion policy and extended red cell phenotype matching for IBD patients could prevent RBCA induction and associated complications, which may be of long—term clinical significance in this particular patient cohort.

Abstract Session 06 – Immunoregulation

OS 6.01

Class II Transactivator (CIITA) A is a Key Regulator of the Semaphorin—receptor System

Schlahsa L., Battermann A., Vahlsing S., Eiz—Vesper B.,

Blasczyk R., Figueiredo C.

Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany

Background: The semaphorin—plexin receptor system was shown to modulate the immune response. This complex system demonstrated to play an important role in several diseases. However, the regulation of the expression of semaphorins and their receptors is still unknown. Patients and Methods: Here, we have investigated the role of CIITA in the regulation of semaphorin receptors. CIITA expression was induced on human cornea endothelial cell line (HCEC) by IFN—γ stimulation or by transfection with a CIITA encoding vector. The mRNA levels and protein levels of the semaphorin receptors Neuropilin—1 (NP—1), vascular endothelial growth factor receptor—2 (VEGFR—2), Plexin—A1, Plexin—A4, triggering receptor expressed on myeloid cells (TREM)—l and TREM—2 were detected by real time PCR, flow cytometry and fluorescence microscopy. To confirm that the upregulation of the semaphorin receptors was directly controlled by CIITA, siRNAs were used to silence the CIITA expression in HCEC. Recombinant Semaphorin3 A was used to stimulate HCEC and its effect on CIITA levels was assessed. Results: IFN—7 induction or CIITA overexpression caused an upregulation of Plexin—Al mRNA levels by up to 37%. A significant upregulation of NP—1 and VEGFR—2 mRNA levels by up to 45% (p < 0.05) and 91% (p < 0.001) was detected upon CIITA expression, respectively. Furthermore, a significant increase in mRNA levels of Plexin—A4 (2.2 fold, p < 0.05), TREM—1 (2.3 fold, p < 0.05) and TREM—2 (2.4 fold, p < 0.001) was detected. siRNA—mediated knockdown of CIITA resulted in the downregulation of the expression of NP—1 (35.4%), Plexin—A4 (59.3%), TREM—1 (30.6%), and TREM—2 (41.5%) after two days. Semaphorin3A stimulation caused an increase by up to 2—fold of CIITA levels. Conclusion: This data suggest that a comprehensive understanding of the regulation of the semaphorin—receptor expression may open new avenues in the treatment of cancer or prevention of auto— or allo—immune diseases.

OS 6.02

Semaphorin 5A activates NK and T cell responses in an antigen—independent manner

Gras C, Jaimes Y., Eiz—Vesper B., Immenschuh S., Blasczyk R., Figueiredo C.

Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany

Background: Semaphorin 5A (Sema5A) was shown to be upregulated in cancer, however its function in the immune system is still unknown. We investigated the effect of Sema5A in the regulation of NK and T cell immune responses. Patients and Methods: Recombinant Sema5A was used for the stimulation of Natural Killer (NK) and T cells. Intracellular signal transduction was assessed by Western Blot. Sema5A stimulated and non—stimulated NK or T cells were analyzed for receptor phenotype using flow cytometry. NK and T cell proliferation assays were performed in presence and absence of Sema5A protein. Cytokine secretion of NK cell populations in response to Sema5A was analyzed using Luminex technology. The capacity of Sema5A to modulate the cytotoxic potential of NK cells was measured in cytotoxic assays using K562 cells as targets. Results: Sema5A showed to induce the phosphorylation of ERK1/2 and Akt. Sema5A stimulation caused an increase of NKp44+ NK cells frequency by up to 94%. NKG2A+ and NKG2D+ NK cell population increased by up to 44% and 7% respectively. Upon Sema5A stimulation, the frequencies of CD25 and CD69 increased by up to 2.4 fold and 4.3 fold in NK cells, and by 2 fold and 9.5 fold in T cells, respectively. Stimulation with a combination of Sema5A, IL—2 plus IL—15 induced a significant increase by up to 67% and 59% in the proliferative capacity of NK cells and T cells, respectively. In NK cells, Sema5A was able to induce de novo production of IL—1β and GM—CSF, and to increase the secretion levels of IL—6, IL—8 and TNF—α by 45—fold, 80—fold, and 1.8—fold, respectively. Furthermore, preincubation of NK cells with Sema5A increased their cytotoxic activity against K562 cells by up to 32%. Conclusions: These results show for the first time that Sema5A is a potent activator of NK cells and T cells. Thus, Sema5A and its pathway might be promising targets for the development of new therapeutic approaches against cancer.

OS 6.03

Activation of acute lymphocytic leukemia blasts with CpG ODN, IL—4 and CD40 ligand facilitates enhanced anti—leukemic CTL responses

Jahrsdörfer B.¹, Breckerbohm L.², Vollmer A.², Queudeville M.², Eckhoff S.², Fulda S.², Strauss G.², Schrezenmeier H.¹, Debatin K.—M.², Meyer L.², Fabricius D.³

¹Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden—Württemberg—Hessen, and University of Ulm, Ulm, Germany, ²Department of Pediatrics, University of Ulm, Ulm, Germany, ³Department of Pediatrics, University of Ulm, Ulm, Germany

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the majority of patients initially respond to up—front chemotherapy, relapses with poor prognosis occur in approximately 20% of cases. Thus, novel therapeutic strategies are required to improve long—term survival. B cell precursor (BCP)—ALL cells express low levels of immunogenic molecules and therefore are poorly recognized by the immune system. In the present study we investigated the effect of various combinations of potent B cell stimulators including CpG, Interleukin (IL)—2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of primary BCP—ALL cells and a series of BCP—ALL cell lines. The combination of CpG, IL—4 and CD40L was identified as most effective to enhance expression of immunogenic molecules on BCP—ALL cells, resulting in an increased capacity to induce both allogeneic and autologous cytotoxic T lymphocytes (CTL). Importantly, such CTL exhibited significant anti—leukemic cytotoxicity not only towards treated but also towards untreated BCP—ALL cells. Our results demonstrate that the combination of CpG with other B cell stimulators is more efficient than CpG alone in generating immunogenic BCP—ALL cells and anti—leukemic CTL. Our results may stimulate the development of novel adoptive T cell transfer approaches for the management of BCP—ALL.

OS 6.04

MHC—II+ cells are essential to promote expansion of human peripheral blood gamma—delta T cells in response to phosphoantigens

Sambia N.S.¹, Jomaa H.¹, Bein G.², Hackstein H.¹

¹Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Germany, ²Zentrum für Transfusionsmedizin und Hämotherapie, Universitätsklinikum Giessen und Marburg GmbH, Giessen, Deutschland

Background: γδT cells represent an unique subset of effector lymphocytes attracting increasing interest for cellular immunotherapy due to their cytotoxicity and capacity to promote adaptive immunity. Vγ2δ2 (V52) cells representing the principle γδT cell subset in human peripheral blood recognize small nonpeptid phosphoantigens, such as E—4—hydroxy—3—methyl—but—2—enyl pyrophosphate (HMBPP) or isopentenyl pyrophosphate (IPP). Little is known about the specific cellular requirements for γδT cell expansion. It has been suggested that various cell types including cell lines or T cells can promote γδT cell expansion. Methods: High purity fluorescent activated cell sorting was used to systematically dissect the cellular requirements for phosphoantigen—driven V52 cell expansion. Results: We show for the first time that MHC—11+ cells are indispensable for phosphoantigen—driven human V52 cell expansion. In contrast, cultures with highly purified (>99.8%) MHC—II— cells were unable to promote phosphoantigen—driven V52 cell expansion. Purified αβT cells, CD4+, or CD8+ T cells (purity >99%) also did not promote HMBPP—mediated V52 cell expansion. Interestingly, depletion of CD4+CD25+ T regulatory cells demonstrated that the inability of αβTCR T cells to promote Vδ2 cell expansion was not related to the presence of regulatory T cells. Furthermore, sorting of MHC—II+ subsets into >99% pure dendritic cells, mono—cytes and B cells indicated that dendritic cells were the most potent MHC—II+ subset promoting Vδ2 cell expansion. Additionally, pulsing experiments with purified populations demonstrated that short—time HMBPP pulsing transformed MHC—II+ but not MHC—II— cells into Vδ2 expanders and eliminated the need for HMBPP to be present during Vδ2 expansion. Conclusion: This is the first study that has systematically analyzed the specific cellular requirements for the expansion of primary human Vδ2 cells. The results may be useful for the development of cellular therapy protocols.

OS 6.05

Reduced alloreactivity of NK cells after G—CSF mobilization in stem cell donors

Kordelas L.¹, Ottinger H.¹, Beelen D.W.¹, Heinemann F.M.², Rebmann V.³, Horn P.A.³

¹Department of Bone Marrow Transplantation, University Hospital Essen, Essen, Germany, ²Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, ³Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany

Granulocyte colony—stimulating factor (G—CSF) is commonly used for stem cell mobilization in the context of autologous or allogeneic stem cell transplantation. A limited number of studies elucidating the effect of G—CSF on the specific leukocyte subtypes of the donor have been published so far, especially for NK cells. Therefore we studied the cytotoxic function of NK cells derived from 13 peripheral stem cell donors before and 5 days after G—CSF administration. NK cell cytotoxicity was determined by flow cytometry using the HLA—E transfected K562 cells and the parental HLA class I negative chronic myelog—enous leukemia K562 cell line as target cells in an effector/target ratio of 6:1. Lysed target cells were detected by propidium iodide. The expression of HLA—E specific receptors was analysed on NK cells from 10 donors before and after G—CSF treatment by flow cytometry. The NK cells procured after G—CSF administration showed a reduced cytotoxicity (mean +/— SEM %) towards HLA class I negative K562 target cells (10.4 +/— 3.3) compared to NK cells before treatment (5.3 +/— 1.6, p = 0.06). This effect was found to be more prominent in HLA—E transfected K562 target cells (10.9 +/— 3.7 vs. 2.8 +/—1.3; p = 0.03). The reduced capability to lyse HLA—E transfected K562 target cells might be explained by the fact that the proportion of NK cells bearing HLA—E specific receptors (CD94, the inhibitory receptor NKG2A and the activating receptor NKG2C on NK cells) were significantly decreased after G—CSF administration (CD94: 66.4 +/— 4.3 vs. 57.7 +/— 4.9; NKG2A: 46.7 +/— 4.9 vs. 43.7 +/— 6.1; NKG2C: 8.1 +/—4.5 vs. 3.4 +/—1.3). To the best of our knowledge this study is the first one showing that G—CSF decreases the proportion of NK cells with HLA—E specific receptors, which in turn results in a reduced lytic function towards HLA—E positive target cells. Further studies are needed to elucidate whether the expression of other NK cell receptors is influenced by G—CSF.

OS 6.06

The TLR7/8 ligand resiquimod targets monocyte—derived dendritic cell differentiation via TLR8 and augments functional dendritic cell generation

Hackstein H.¹, Knoche A.¹, Jurk M.², Vollmer J.², Bein G.³

¹Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany, ²Pfizer Oligonucleotide Therapeutics Unit, Coley Pharmaceuticals, Düsseldorf, Germany, ³Zentrum für Transfusionsmedizin und Hämotherapie, Universitätsklinikum Giessen und Marburg GmbH, Giessen, Deutschland

Background: Dendritic cells (DC) represent powerful antigen presenting cells modulating innate and adaptive immunity. Imidazoquinolone compounds, such as resiquimod are TLR7/8 ligands representing novel immune response modifiers and co—compounds for DC vaccination trials. Resiquimod has been reported to inhibit conventional human monocyte—derived DC (moDC) differentiation and maturation, but the role of TLR7 and TLR8 is unclear. Methods: To dissect the contribution of TLR7 and TLR8 on moDC differentiation we performed experiments with selective TLR7 ligands and directly dissected the TLR7— and TLR8—dependency by resiquimod—coculture experiments with S—Class inhibitory oligonucleotides (iODN) suppressing TLR7, TLR7+8 or TLR7+8+9. Results: In contrast to resiquimod, selective TLR7 ligands did not affect conventional moDC differentiation as analyzed by CD14/CD1a expression. iODN experiments confirmed that resiquimod's effects during DC differentiation were antagonized only in the presence of TLR8 iODNs. Direct comparison of resiquimod DC with TLR7—monostimulated DC and control DC revealed significantly higher T—cell costimulatory molecule CD40, CD83, CD86, MHC class II expression. Resiquimod DC promoted significantly stronger allogeneic CD4+ and CD8+ T—cell proliferation and stronger proliferation of sorted naïve CD4+ T cells. Conclusion: These results revealed the relevance of TLR8 for human DC differentiation and activation. Resiquimod efficiently promoted human monocyte—derived DC differentiation and activation. Triggering of TLR8 may be relevant for successful DC vaccination trials employing novel immune response modifiers.

OS 6.07

DAMPs – including S100A4 – used to pulse dendritic cells (DCs) are capable of inducing the proliferative response of autologous lymphocytes

Wiegmann D.S., Nienhaus C, Schrezenmeier H., Lotfi R.

Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden—Württemberg—Hessen, and University of Ulm, Ulm, Germany

Background: Necrotic cell death with subsequent release of damage associated molecular pattern molecules (DAMPs) is a characteristic feature of advanced solid tumor. S100A4 is a DAMP family member associated with clinical outcome of patients with colorectal and breast cancer. Materials and Methods: Immature DCs (iDCs) were generated from human monocytes in the presence of IL—4 and GM—CSF. In the presence or absence of anti—S100A4 antibodies iDCs were pulsed for 3 days with DAMPs obtained by repeated freeze—thaw lysis of HCT—116 cells. Alternatively, DCs were pulsed with S100A4 protein. Pulsed DCs were cocultured with autologous lymphocytes for 5 days and lymphocyte proliferation was assessed by WST—1 Assay as well as by flow cytometric analysis of CFSE labeled lymphocytes. Results: We could demonstrate a dose—dependent proliferative response of lymphocytes co—cultured with autologous DCs which were pulsed with necrotic material (DAMPs) from lysed colorectal tumor cells at a concentration of 105, 104,103 lysed HCT cells/ml, the relative stimulation index of lymphocytes was up to 2.1, 2.6, and 3.9 respectively, compared to the lymphocyte response to non—pulsed DCs. The observed proliferation—inducing effect of necrotic material on lymphocytes could be specifically inhibited with antibodies to the DAMP—member S100A4. Using recombinant S100A4 protein, we could confirm our results with necrotic material (DAMPs) and obtained a relative stimulation index of up to 4.7 for lymphocytes co—cultured with autologous DCs which were pulsed with 1μg S100A4 protein/ml culturing media. Conclusions: Given that necrotic material is generally found within advanced tumor tissue, there is an urgent need to characterize specific members of DAMPs and their impact within tumor microenvironment. By describing the effect of S100A4 protein our results shed some light into the underlying mechanisms playing a crucial role in adaptive immune response to tumor.

OS 6.08

TLR—4—dependent down—regulation of human heme Oxygenase—1 by lipopolysaccharide via a Bach 1—regulated pathway in macrophages

Paine A.¹, Eiz—Vesper B.¹, Figueiredo C.¹, Theilmeier G.², Blasczyk R.¹, Immenschuh S.¹

¹Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany, ²Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany

Background: Heme oxygenase—1 (HO—1) is the inducible isoform of the first and rate—limiting enzyme of heme degradation. In animal models of genetic HO—1 deficiency this enzyme has been shown to exhibit potent anti—inflammatory and immunomodulatory functions and to play a major beneficial role in organ transplantation. In particular, the cell type—specific functions of HO—1 in myeloid cells such as macrophages and monocytes have been shown to be protect against ischemia/reperfusion injury and adoptive transfer of ex vivo HO—1 modified macrophages have salutary effects in experimental mouse models of kidney and liver transplantation. In contrast to mouse, the species—specific regulation of HO—1 gene expression in human macrophages is largely unknown. Methods and Results: Here, it is demonstrated that activation of toll—like receptor (TLR)—4 by lipopolysaccharide (LPS) markedly down—regulated HO—1 gene expression in cell cultures of primary human macrophages, but caused an up—regulation in murine macrophages as determined by realtime RT—PCR and immunoblot studies. This LPS—dependent down—regulation of HO—1 gene expression was largely attenuated by the protein synthesis inhibitor cycloheximide, but only to a minor extent by the transcription inhibitor actinomycin D. Blockage of proteasomal degradation by various compounds such as MG132 attenuated LPS—mediated decrease of HO—1 gene expression. Finally, studies with siRNA—dependent knock—down of BTB and CNC homologue 1 (Bachl), which is a key repressor of HO—1 gene transcription, suggested a major role for this nuclear factor for the TLR—4—dependent decrease of HO—1 gene expression in human macrophages. Conclusions: Taken together, LPS mediates a species—specific down—regulation of HO—1 gene expression in human macrophages. These findings may have major implications for targeted therapeutic approaches in organ transplantation.

Abstract Session 07 – Cell Therapy: Non—Hematopoietic Stem Cells

OS 7.01

Ex vivo generation of red blood cells from human pluripotent stem cells

Dorn l.¹, Schlenke P.², Bleidissel M.³, Sibrowski W.², Schöler H.R.³, Zaehres H.³

¹Pediatric Hematology and Oncology, University Hospital Münster, Münster,

Germany, ²Institut für Transfusionsmedizin und Transplantationsimmunologie, Universitätsklinikum Münster, Münster, Deutschland, ³Max Planck Institut für Molekulare Biomedizin, Münster, Deutschland

Background: Generating red blood cells (RBC) from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPS) offers the potential to generate large quantities of patients’ specific RBC for transfusion purposes from an unlimited source. However, recapitulation of the different physiological steps of RBC maturation from ESC/iPS including mesoderm induction, generation of hematopoietic stem cells (HSC), which undergo erythroid maturation, hemoglobin switching and enucleation remains a challenge. Materials and Methods: We used human ESC (H1) and iPS cells from different human tissue for the ex vivo generation of RBC. Somatic cells were reprogrammed by retroviral expression of the transcription factors OCT4, SOX2, KLF4 and C—MYC. For the generation of HSC, ESC/iPS cells were allowed to form embryoid bodies (EB) under cytokine stimulation for >18 days. Thereafter, EB were dissociated and single cells were applied to a three step protocol for erythropoiesis over 25 days. For morphological analysis, cells were stained with May—Grünwald—Giemsa and evaluated by microscopy. Furthermore, cells were analysed by flow cytometry (CD34, CD45, CD36, CD71, glycophorin A (GPA). Results: The used EB protocol allows for the generation of 3%—15% CD34+ and CD45+ cells from human ESC/iPS cells, able to form erythroid and myeloid colonies in semisolid cultures. These hematopoietic cells further developed into erythroid precursors as determined by >80% expression of CD36, CD71 and GPA, followed by maturation into normoblasts and partielly enucleated RBC. Compared to ESC, first results indicate a lower proliferation capacity of iPS cells, whereas maturation into RBC seems to be equal. Conclusions: We were able recapitulate the development of erythroid cells from human ESC and iPS cells under ex vivo conditions. This might contribute as a first step towards the large scale ex vivo generation of patients specific RBC from human pluripotent stem cells.

OS 7.02

Evaluation of basal membrane—derived and biomaterial matrices for endothelial cell expansion and maintenance of their primitive potential

Horsch L.¹, Denecke B.², Fischer J.³, Horn P.A.¹, Giebel B.¹

¹Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, ²Interdisciplinary Center for Clinical Research, RWTH Aachen, Aachen, Germany, ³Institute of Transplantation Diagnostics and Cellular Therapeutics, University Hospital Düsseldorf, Düsseldorf, Germany

The discovery of so—called circulating endothelial progenitor cells (EPCs) has highly stimulated the field of vascular biology. During recent years different protocols have been applied to raise and expand such cells. However, a recent comprehensive comparison of obtained EPCs revealed that many of them are of hematopoietic origin and just mimic an endothelial cell surface phenotype. According to the published studies only the endothelial colony forming cells (ECFCs) seem to represent true circulating endothelial progenitor cells. Of note, ECFCs raised from human umbilical cord blood (UCB) are phenotypically almost identical to human umbilical cord vein endothelial cells (HUVECs). Furthermore, similar to HUVECs their proliferation capacity is limited to a few passages. In this context it has been suggested that both cell types become senescent and stop to proliferate. Provided that both endothelial cell types are organized in a hierarchical manner similar to primitive hematopoietic cells, such a limited expansion would also be expected if the culture conditions would not allow self—renewal of the more primitive cells. Favoring the latter hypothesis we aim to optimize the culture conditions for these endothelial cells. For a number of different cell systems cell fate modulating features of matrices and biomaterials have been reported. To this end we decided to compare the impact of basal membrane—derived matrices as well as distinct biomaterials on the adherence capacity, the metabolic activity, the necrosis and apoptosis rate and the expansion rate of primary human endothelial cells. So far, we compared the impact of collagen IV, fibrin, gelatin, heparin, hyaluronic acid, laminin, platelet lysate coated plastic dishes and advanced plastic with conventional plastic ware. In addition, we studied biological features of ECFCs expanded on different biomaterials used in tissue engineering. Obtained results will be presented.

OS 7.03

The capacity of peripheral tissue precursor cells to form new vessels in primary avascular three dimensional (3D) fibrin matrices in dependence of hypoxia

Rüger B.M., Yanagida G., Fischer M.B.

Dept. of Blood Group Serology and Transfusion Medicine, Medical

University of Vienna, Austria, Vienna

New vessels can be generated by sprouting of new capillaries from the preexisting vascular network and subsequent stabilization of the newly formed immature vessel by the coordinate growth of endothelial cells, pericytes, smooth muscle cells and fibroblasts. In addition, endothelial precursor cells can be found in areas of active angiogenesis in peripheral tissues. The sequence of events leading to the formation of new vessel takes place in a microenvironment that is determined by chronic inflammation, hypoxia and tissue destruction and regeneration. In this study we aimed to establish an in vitro culture system to investigate the mechanisms of neo—vascularization in the context of chronic inflammatory processes and hypoxia that occurs in RA tissue. We used fibrin matrices for a three—dimensional (3D) angiogenesis assay because fibrin plays an important role in tissue damage induced by inflammation. Dependent on infiltration of the synovial tissue with inflammatory cells we found new vessels growing into primary avascular 3D fibrin matrices without exogenous growth factors. Vascular structures could originate from cell clusters containing CD34 positive precursor cells, co—expressing CD133 and CXCR4 with various intensity. These cells are surrounded by STRO—1 positive mesenchymal precursor cells. The newly formed vessel can develop in the 3D fibrin gel to a state where newly formed vessels showed a lumen and were stabilized by α—smooth muscle actin (SMA) positive pericyte. Remarkably, these newly formed vessels contained CD45 positive lymphocytes and erythrocytes that migrated from the embedded synovial tissue in the absence of a functional circulation most likely due to hypoxia. Hypoxia was confirmed in embedded tissue by the expression of HIF—1 alpha and HIF—2alpha.

OS 7.04

Cord blood derived endothelial colony forming cells emerge from a CD45dim circulating precursor

Elvers—Hornung S.¹, Vinci M.², Gloe T.³, Klüter H.¹, Bieback K.¹

¹Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Donor Service Baden—Württemberg – Hessen, Mannheim, Germany, ²The Institute of Cancer Research; Cancer Research UK Cancer Therapeutics Unit Tumour Biology and Metastasis Team McElwain Laboratories, London, UK, ³Cardiovascular Physiology; Centre for Biomedicine and Medical Technology, Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Germany

Background: Within the last decade numerous studies have been published dealing with endothelial progenitor cells (EPC). Concomitant it became apparent that a variety of different cell types have been subsumed under the term EPC. Depending on the protocol culture adapted EPC have now been classified as i) colony forming unit endothelial cells (CFU—EC), ii) early outgrowth/ proangiogenic (CAC) or iii) late outgrowth/endothelial colony forming cells (ECFC). For circulating EPC the precise phenotypic definition is still heavily discussed and different authors propose different combinations of the markers CD34, CD 133, VEGFR—2/KDR, CD31 and CD45 debating whether EPC are of hematopoietic origin. Methods and Results: CD34+ mononuclear cells (CD34+ MNC) were isolated from fresh cord blood and cultured to derive endothelial colony forming cells (ECFC). These were compared to mature endothelial cells from the umbilical vein (HUVEC). We succeeded to analyse two ECFC batches already in primary passage (p0). Interestingly, despite a homogenous appearance as cobblestone cells one batch represented a very early phenotype with 99.41% of cells stained dimly for CD45 and 91.43% CD31. The other batch comprised two populations, one with a higher proportion of cells positive for endothelial markers (66.17 − 82.88%) and a subpopulation expressing CD45dim. Functionally ECFC were compared to HUVEC in terms of adhesion, migration and transmigration. ECFC demonstrated significant higher adhesion, both under static and flow conditions. Migration and transmigration exceeded that of HUVEC. Conclusions: Our study supports the notion that ECFC emerge from a CD45dim progenitor and very rapidly mature in culture. Our data strengthen the necessity to identify a set of markers capable of prospectively discriminating endothelial from hematopoietic cells as well as progenitor from mature cells.

OS 7.05

Assessment of cornerstones during HOXB4—assisted hematopoietic development of pluripotent stem cells, in vitro

Teichweyde N., Hinrichs C, Kleinbielen A., Horn P.A., Klump H. Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany

In hematopoietic stem cells (HSCs), many mutations can lead to diseases associated with blood formation and effector cell function. However, isolation, gene targeting and selective expansion of HSCs is currently not feasible. An alternative could be the utilization of pluripotent embryonic stem cells (ESCs) because they allow the efficient application of the aforementioned procedures and can, in principle, be differentiated towards HSCs, in vitro. An attractive candidate to support their hematopoietic differentiation is the homeodomain transcription factor HOXB4. Our group and others have repeatedly shown that ectopic expression of this protein enhances formation of hematopoietic stem and progenitor cells (HSPCs) from mouse ESCs and mediates expansion of HSCs, in vitro and in vivo. To explore the influence of HOXB4 during the development of HSPCs in more detail, we differentiated ESCs as embryoid bodies (EBs) for 6 days and cultured the dissociated cells on OP9 stroma with appropriate cytokine support. Clusters of round, semiat—tached cells first appeared after 5 days with dramatically increasing cell numbers during the following days. These putative hematopoietic cells emerged from islands presumably containing hemogenic endothel cells being positive for CD201 (endothelial Protein C Receptor). The ESC—derived hematopoietic cells (ES—HCs) were characterized by FACS analyses using the cell surface markers CD117, CD41, CD45, CD201, VE—Cadherin and CD34. Cells with the phenotype c—kit⁺/CD41⁺/CD34” have been shown to be capable of engraftment after transplantation into lethally irradiated recipient mice. CD41 marks the earliest known hematopoietic cells during embryonic development. In this study CD41+ as well as CD41⁺/CD201⁺ cells were detectable only in HOXB4 expressing ES—HC cultures. To this end, our results suggest that HOXB4 influences hematopoietic development at the stage of hemogenic endothelium generation during ES—cell differentiation, in vitro.

OS 7.06

Induced pluripotent stem cells of the common marmoset monkey

Wiedemann A.¹, Schwamborn J.², Bernemann I.³, Figureido C.¹, Honndorf S.¹, Schambach A.⁴, Blasczyk R¹— Müller T.¹

¹Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany, ²Center for Molecular Biology and Inflammation ZMBE, Institute for Cell Biology, University Muenster, Muenster, Germany, ³Institute for Multiphase Processes, Leibniz University Hannover, Hannover, Germany, ⁴lnstitute for Experimental Hematology, Hannover Medical School, Hannover, Germany

Background: Regenerative medicine is in need of a solid animal model as a link between rodents and human to evaluate functionality, immunogenicity and clinical safety of embryonic stem cells (ESCs) or reprogrammed cell types. The common marmoset monkey (Callithrix jacchus) is an excellent large animal model, genetically closly related to the human and readily used worldwide in clinical research. Besides the studies on Marmoset ESCs, our group is focusing on reprogramming somatic cells of the common marmoset to explore their potential for preclinical studies. Material and Methods: For reprogramming bone—marrow derived mesenchymal cells of the common marmoset were used. In the presence of TAV, SB431542, PD0325901 and ascorbic acid, the cells were reprogrammed via a lentiviral SFFV driven quad—cistronic vector system in mTESR medium and transferred onto mouse embryonic feeder cells at day 21. Results: The cells obtained showed typical ESC—like morphology and were positive for alkaline phosphatase and the endogenous pluripotency markers Oct3/4, Nanog, Sox2, KLF4 and MYC while exogenous genes were downregulated. From passage 19 on cells were also differentiated successfully to embryoid bodies. Marker genes and morphology of early neuronal progenitors, adipocytes, chondrocytes, osteogenic cells and megakaryocytes could be observed so far. Conclusions: These cells appear to be fully reprogrammed iPS cells of the common marmoset which display pluripotency markers and can be differentiated into various cell types. They are a promising tool for animal models close to human for preclinical studies in the field of regenerative medicine.

OS 7.07

Human mesenchymal stem cells can promote phenotypic maturation of human plasmacytoid dendritic cells via Tolllike receptor 7/8

Hackstein H.¹, Neubauer A.², Bein G.³, Brendel C.²

¹Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany, ²Department of Hematology, Oncology and Immunology, Philipps—University Marburg, Marburg, Germany, ³Zentrum für Transfusionsmedizin und Hämotherapie, Universitätsklinikum Giessen und Marburg GmbH, Giessen, Germany

Background: Human mesenchymal stem cells (hMSC) represent important multipotent cells hitherto believed to exert primarily immunosuppressive effects on human dendritic cells. We show here that this view is incomplete by providing evidence that hMSC can efficiently promote phenotypic maturation of purified plasmacytoid dendritic cells (pDC). Methods: hMSC were generated from bone marrow employing standard procedures and human BDCA—4+ pDC precursors were microbead—sorted according from human buffy coat samples. Cocultures of purified pDC and hMSCs were stimulated through different toll—like receptor (TLR) ligands and pDC activation was analyzed by flow cytometry and ELISA. Results: Triggering of hMSC pDC cocultures through TLR4 (LPS) or TLR9 (CpG ODN) did not affect upregulation of T cell costimulatory m,olecule expression CD40, CD80, CD86 and HLA—DR on pDCs. In contrast TLR7/8 activation of hMSC pDC cocultures resulted in marked upregulation of CD80 and CD86 surface expression on pDC when compared to pDC—only controls. Moreover, pDC maturation by hMSC was dose—dependent and a hMSC—pDC ration of 1:10 was already sufficient to increase pDC CD80 and CD86 surface expression (mean fluorescence intensity; MFI) by >75% and 190%, respectively. Increasing the hMSC concentration to 20% resulted in further stimulation of pDC CD80 and CD86 MFI after TLR7/8 activation by 331% and 669%, again compared to pDC—only controls. Furthermore, whereas presence of hMSC resulted in moderate reduction of TLR9—triggered interferon—alpha production by pDC, it was not inhibited after TLR7/8—mediated stimulation. Conclusion: These data indicate that hMSC are not per se immunosuppressive. The results identify TLR7/8 as a candidate pathway circumventing the immunoregulatory function of hMSC. Given the fact that TLR7/8 activation plays a critical role in the development of autoimmune diseases such as lupus erythematodes and during viral infection, our results may add to the understanding of basic principles of autoimmunity.

OS 7.08

Electrochemical Impedance Sensing to non—invasively monitor mesenchymal stromal cell differentiation potential

Brinkmann l.¹, Angstmann M.², Maercker C.³, Breitkreutz D.⁴, Bieback K.¹

¹Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Donor Service Baden—Württemberg – Hessen, Mannheim, Germany, ²Mannheim University of Applied Sciences, Biotechnology, Mannheim, Germany; German Cancer Research Center (DKFZ), Genetics of Skin Carcinogenesis, Heidelberg, Germany, ³Mannheim University of Applied Sciences, Biotechnology, Mannheim; German Cancer Research Center (DKFZ), Genomics and Proteomics Core Facilities, Heidelberg, Germany ⁴Mannheim University of Applied Sciences, Biotechnology, Heidelberg, Germany

Background: Because of their mesodermal differentiation potential mesenchymal stromal cells (MSCs) are attractive candidates for tissue engineering. Up—to—date, MSC differentiation potential is evaluated by exposure to inducing factors or media usually for three weeks, followed by staining for distinct markers, proteomic analysis or gene expression profiling. These invasive methods do not allow monitoring cultures continuously and can only analyse single time points. Thus, standardized and non—invasive quality control assays monitoring differentiation over time are lacking. Methods: By monitoring impedance profiles during adhesion and cell growth, we have employed a non—invasive assay to discriminate osteogenic and adipogenic differentiation potential of MSCs. Impedance profiles were recorded with MSCs derived from bone marrow and adipose tissue, either non—induced or induced for osteogenesis or adipogenesis. These were correlated to differentiation markers assessed by RT—qPCR and western blot. Results: The impedance profiles of osteogenic induced MSCs revealed an initially rapid and continuous rise corresponding to the formation of mineralised matrix. Conversely, adipogenic induction caused shallower initial slopes and eventually declining profiles, corresponding to more compact, adipocyte—like cells with numerous lipid vacuoles. Importantly, impedance recordings correlated well to the extent of differentiation evaluated by histochemical staining, protein and RNA analysis and were capable of discriminating well differentiating from poor differentiating MSCs. Conclusions: Impedance profiling offers a basis for standardized real time, non—invasive high—throughput screening of MSC properties. The fact that impedance measurements indicate already early changes which can neither be addressed reliably by histochemical stainings nor by changes in gene expression, further recommends this method as suitable quality control also for larger sample cohorts.

Abstract Session 08 – Blood Safety: Pathogen Reduction

OS 8.01

Inactivation of Hepatitis C Virus (HCV) by UVC treatment of platelet concentrates as determined by an in vitro HCV infection system

Steinmann E.¹, Gravemann U.², Friesland M.¹, Doerrbecker J.¹, Pietschmann T.¹, Seltsam A.²

¹TWINCORE, Center for Experimental and Clinical Infection Research GmbH, Hannover, Germany, ²DRK—Blutspendedienst NSTOB Institut Springe, Springe, Germany

Background: A novel pathogen reduction procedure, called THERAFLEX UV—Platelets system, has been developed to reduce the infectious risk associated with transfusion of platelet concentrates (PCs). The process is based on the application of UVC light combined with intense agitation of the blood units to ensure a uniform treatment of the entire product. Although HCV is known since 1989 only recently a HCV infection system allowing the propagation of infectious HCV in cell culture has been developed. Aim of the current investigation was to investigate the inactivation capacity of the THERAFLEX UV—Platelets system by using this in vitro HCV infection system. Methods: HCV Jc1 virus was generated by electroporation of 10 μg RNA into Huh7.5 cells. Cell culture supernatants were harvested 48, 72 and 96h post transfection. Plasma reduced PC were prepared from 5 buffy coats in additive solution SSP+ (MacoPharma). PCs (N = 3, 315 mL) were spiked with cell—culture grown HCV (35 mL). PCs were irradiated on the MacoTronic UV device and samples were taken at different dose steps. Infectivity was determined by immunohistochemistry—based endpoint titration and large volume plating on Huh7.5 cells. The titer which causes a positive result in 50% of all infected cultures (TCID50) was determined according to the method of Spearmann and Kaerber. Results: The virus titer was determined at a dilution that was neither cytotoxic nor did interfere with the viral titer. Spiking of the PC resulted in a titer of 5.61 ± 0.54 log10 TCID₅₀. HCV was rapidly inactivated in PCs by UVC treatment below the limit of detection with a UVC dose of 0.2 J/cm². Conclusions: UVC treatment of PCs using the THERAFLEX UV—Platelets system efficiently inactivates HCV with a reduction factor ≥ 4.99 log steps. The procedure therefore has the potential to significantly reduce transfusion transmitted HCV infections.

OS 8.02

Hepatitis C is highly sensitive to MB/light treatment of plasma as shown by a novel in vitro HCV infection system

Gravemann U.¹, Friesland M.², Doerrbecker J.², Pietschmann T.², Steinmann E.², Seltsam A.¹

¹DRK—Blutspendedienst NSTOB Institut Springe, Springe, Deutschland, ²TWINCORE, Center for Experimental and Clinical Infection Research GmbH, Hannover, Germany

Background: Photodynamic treatment using methylene blue (MB) and visible light is a well established method for virus inactivation of human plasma. It has been shown to inactivate a broad range of different DNA and RNA viruses. Although HCV is known since 1989 only recently a HCV infection system allowing the propagation of infectious HCV in cell culture has been developed. Aim of the current investigation was the investigation of the inactivation capacity of the THERAFLEX MB—Plasma system by using this in vitro HCV infection system. Methods: HCV Jc1 virus was generated by elec—troporation of 10 μg RNA into Huh7.5 cells. Cell culture supernatants were harvested 48, 72 and 96h post transfection. Leukodepleted plasma was prepared from whole blood using standard blood banking technology. Plasma (n = 3, 285 mL) was spiked with cell—culture grown HCV (30 mL). MB/light treatment was done using the MacoPharma Theraflex MB—Plasma bag system and the MacoTronic B2 illumination device. Samples were taken at different steps of the process. Infectivity was determined by immunohistochemistry—based endpoint titration and large volume plating on Huh7.5 cells. The titer which causes a positive result in 50% of all infected cultures (TCID₅₀) was determined according to the method of Spearmann and Kaerber. Results: The virus titer was determined at a dilution that was neither cytotoxic nor did interfere with the viral titer. Results Spiking of the PC resulted in a titer of 5.41 ± 0.22 log₁₀ TCID50. HCV in plasma was efficiently inactivated by the MB/ light treatment below the limit of detection already with a light dose of 10 J/ cm². After 40 J/cm² no infectivity was detectable by large volume plating. Conclusions: MB/light treatment of plasma using the THERAFLEX MB—plasma system (MacoPharma) efficiently inactivates HCV with a reduction factor ≥ 3.83 log steps. The procedure therefore has the potential to significantly reduce transfusion transmitted HCV infections.

OS 8.03

Liquid plasma storage under different conditions and after Methylen Blue pathogen inactivation

Selleng K.¹, Kellner S.¹, Hron G.¹, Wasner C.², Greinacher A.¹, Thiele A.¹,

¹Institut für Immunologie und Transfusionsmedizin, Abt. Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Deutschland, ²Institut für klinische Chemie und Labormedizin, Universitätsmedizin, Greifswald, Deutschland

Background: Plasma (FFP) is stored at —30°C and thawing/ issuing require 40–50 min, which is too long in bleeding emergencies. Liquid plasma storage would allow rapid delivery, but had been considered problematic due to anticipated loss of clotting factor activities and the risk of bacterial growth. We assessed different storage conditions and the effect of pathogen inactivation on clotting factor stability and clotting assays in thawed FFP over time. Patients and Methods: 50 apheresis FFP (≥750 ml each) were divided into 3 biologic aliquots (250 ml each). After thawing, aliquot A was stored at 4°C, aliquot B at 20–24°C (RT) and aliquot C was treated with méthylène blue (MB) and UV light (Macotronic; Macopharma) and stored at 4°C. After thawing, clotting factor activities (FII, V, VII—XIII, fibrinogen, AT, vWF—Ag, PrC, PrS) were assessed on day 0,3,5,7 by standard methods. Functional assays (INR, aPTT, ProC—Global, thrombin generation) were performed. Microbial growth was tested on day 7. Statistical analysis was performed using the Wilcoxon signed rank test. Results: No microbial growth was detected in any aliquot. At RT clotting factor activities decreased more strongly than at 4°C At 4°C storage with or w/o MB treatment only FVIII (105%±25% vs. 56%±16% w/o MB, 47%±5% after MB; medianiSD) and PrS (78%±20% vs. 50%±15% w/o MB, 48%±15% after MB) fell below the reference range (FVIII < 70%, PrS < 52%)(day 0 vs. 7, p < 0.0001 for both conditions). INR, aPTT, ProCGlobal kept stable within the reference range for 4°C—storage w/o MB. Conclusions: Only FVIII and PrS fell below the reference range during 7 days storage of liquid plasma at 4°C with and w/o MB. The results are encouraging to use liquid plasma for patient treatment in emergency bleeding situations. Further studies are needed to assess if these changes in clotting factor activities are clinically relevant.

OS 8.04

Efficacy and safety of quarantine plasma compared to pathogen inactivated plasma (Intercept) for support of hepatic transplantation

Kientz D.¹, Rémy E.², Cinqualbre J.², Cazenave J.—P.¹

¹EFS—Alsace, Hôpitaux Universitaires de Strasbourg, Strasbourg, France, ²Pôle des pathologies digestives hépatiques et transplantation. Hôpitaux Universitaires de Strasbourg, Strasbourg, France

Background: Transfusion of plasma(P) is an important supportive therapy for hepatic transplantation (HTP). During HTP, patients (PTS) require large P volumes with potential exposure to transfusion—transmitted pathogens. Ineffective support of hemostatic and anti—thrombotic coagulation factors may impact graft and patient survival. Therapeutic P treated with pathogen inactivation by amotosalen + UVA light (IP, INTERCEPT, Cerus BV, Amersfoort, Netherlands) was implemented into routine use by a regional blood center (EFS—Alsace, Strasbourg, France) in place of quarantine plasma (QP). Study Aim: To compare efficacy and safety of IP and QP for support of HTP. Methods: A retrospective analysis was conducted for 2 years with QP (2005–2007) and 3 years with IP (2008–2010). All PTS undergoing HTP were included except PTS with simultaneous multiple organ transplant. The primary efficacy measure was volume (L) of P transfused during and 7 days post HTP. Secondary endpoints included: Day 7— Fibrinogen(g/L), Factor V(IU/dL), hemoglobin(g/L), platelet count(109/L), and hepatic enzymes (TGO and TGP, IU/L). The primary safety endpoint was mortality (day 0–7 post HTP). Proportions were analyzed by Wilcoxon rank sum test with a significance level of p = 0.05. Results: During the QP and IP periods 215 and 214 HTP PTS, respectively, were identified for analysis. Mean age was 50 years in both periods, but mean days awaiting HTP was less for QP than IP PTS (79 vs 143 days, p < 0.05). Indications for HTP (% PTS: QP/IP) were similar: carcinoma(33/29), ETOH cirrhosis(23/24), hepatitis cirrhosis(9/15), acute hepatitis(4/4), repeat HTP(13/9), and other diverse pathology(18/19). Efficacy endpoints were not different for QP and IP PTS; and mortality was not different (Table). Conclusion: Replacement of QP with IP did not impact safety and efficacy for HTP.

Table 1.

More relevant results in both study groups (for Abstract OS 8.04)

Group	P Volume (L/pt)	Fibrinogen (g/L)	F-V (%)	Hb (g/dL)	PLT CT (G/L)	TGO (UI/mL)	TGP (UI/mL)	Mortality (% at day 7)
QP	2.27	4.2	106	10.8	122	85	222	2.8
IP	2.27	4.9	134	10.9	129	61	202	3.3

OS 8.05

Safety of platelet components prepared with Amotosalen—UVA pathogen inactivation treatment (INTERCEPT™) transfused in routine practice

Corash L, Elliott A., Sherman C, Stassinopoulos A. Cerus Corporation, United States, Concord, CA, USA

Background: Platelet components (PC) are transfused to prevent or treat bleeding. Many PC recipients require repeated transfusion, and are at risk for transfusion—transmitted disease (TTD), the most important pathogen including bacteria. To reduce the risk of TTD pathogen inactivation (PI) of PC (INTERCEPT Blood System, Cerus BV, Amersfoort, Netherlands) was developed. Randomized clinical trials supported Class III CE Mark and national registrations (Afssaps, PEI, Swissmedic). Active hemovigilance programs (AHVP) post—marketing are important to extend the safety profile of PI—PC. Study Aim: An AHVP was continued to extend the safety profile of prior AHV for 12,543 PI—PC transfused to 2,051 patients. Methods: Transfusion centers using PI—PC (N = 14) completed a web—based report for each transfusion of PI—PC. Patient demographics, adverse events (AE) 24 hr after PI—PC transfusion, and Serious Adverse Events (SAE) 7 days after transfusion were reported. AE and SAE were assessed for severity and relationship to PC transfusion by physicians using standardized criteria. Transfusion—related AE and SAE were classified as Acute Transfusion Reactions(ATR) or Serious Acute Reactions(SAR). The proportion of transfusions and patients with ATR and SAR and the respective 95% confidence intervals (95%CI) were tabulated. Results: 6632 PI—PC were transfused to 2016 patients (50 infants < 1 year, 133 children age range 1–18 years, and 1833 adults). Indications for transfusion were: hematologic disease(46%), surgery(14%), and medical(40%). 50.7% of patients received more than 1 PI—PC (mean 3.3±5.5, range 1–66). ATR were observed for 0.4% of transfusions and 23/2016 (1.1%) of patients. 1 SAR was reported. For 22/23 patients the ATR was Grade 0–1, and 1 patient had a Grade 2 ATR. The sole SAR was a Grade 3 anaphylactic reaction which resolved. Conclusions: PI—PCs transfused to a diverse patient population were well tolerated without unexpected ATR or SAR.

OS 8.06

Flexible organization of the Intercept pathogen inactivation process for platelets omits extension of product shelf—life

Valek A., Goslings D., Rothlisberger A., Yavuzcan G., Frey B. Blutspende Zürich, Schweiz

Background: In 2011 the blood transfusion service Zurich was one of the first center in Switzerland which introduced the INTERCEPT™ pathogen inactivation (PI) technology for platelet concentrates (PC) into routine. PCs are produced by apheresis (PCA) or by pooling 5 of buffy—coats (PCBC). UVA illumination is performed on 4 days/week (Tue—Fri) during 2 shifts/d. Infectious markers are available at 2pm of dl, while drawing day being dO. Due to quality concerns, the shelf—life for PI treated PCs was kept at max. 5 days. Therefore several process adaptations were necessary to avoid weekend shifts and stockouts. Methods: PCAs were scheduled for illumination in the morning of dl followed by CAD—incubation and release in the afternoon. During PCA illumination PCBCs were produced using OrbiSac System (Caridian—BCT), illuminated immediately after PCAs and released in the evening (1st run of PCBCs). A 2nd run of PCBCs was started in the afternoon and products were released on d2. A 2nd run of PCAs was scheduled between 1 st and 2nd run of PCBCs, if required. 1–2 operators using 2 Illuminators managed 100% of PCs. Throughput, cycle time of PI (CT), CAD incubation time (CAD—t) and product defects during PI as well as PC purchasing due to shortage were assessed. Data were collected by in—house adapted IT—system. Results: During the first 16 weeks (11/1 − 29/04/2011) 1850 PCAs and 1449 PCBCs were worked up. In average 52 PCs/d with a throughput peak of 108 PCs/d were processed. 81% of PCs were released on dl. Mean CT for PCAs and PCBCs were 6.4h (PCAs), 7.3h (PCBCs, 1st run) and 15.3h (PCBCs, 2nd run), respectively. CAD—t was approx. 1h shorter than CT. No product defects due to PI occurred. 10 PCs (0.3%) needed to be purchased to fulfill hospital requests. Conclusions: The designed PI work flow runs well and allows to adapt on increasing product needs. No extension of product shelf—life was necessary to comply with irregular hospital demand on products.

Table 1. for Abstract OS 8.06.

Standard Organization of Pathogen Inactivation and Production of PCBCs

CAD Incubation	All process steps from starting CAD incubation until removing PC from the incubation-shaker
Illumination	All process steps from connecting the intercept-set to the PC until start of CAD Inkubation
PC	Platelet concentrate (origin not defined)
PCA	Platelet concentrate from apheresis
PCBC	Platelet concentrate from 5 pooled buffy-coats
Processing	All process steps from loading the centrifuges with WB until end of separation with blood press
Release	All process steps from removing PC from the incubation-shaker until final labeling
WB	Whole blood donation

OS 8.07

Pathogen inactivation of erythrocyte concentrates using the S—303 Intercept system

Leibacher J.¹, Erickson A.², Giese M.¹, Mufti N.², Seifried E.¹, Henschler R.¹

¹DRK—Blutspendedienst Baden Württemberg – Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Klinikum der Johann Wolfgang Goethe—Universität, Frankfurt, Deutschland, ²Cerus Corporation, Concord, CA, USA

Background: A safe and pharmaceutically acknowledged GMP process is not yet in place for pathogen—inactivated erythrocyte concentrates (RCCs). Aim: For filing a “Herstellungserlaubnis”/Manufacturing License (ML) for PI—RCCs, we adapted the RCC inactivation process for the buffy coat method and using Saline—Adenine—Glucose—Mannitol (SAGM). Method: Leukodepleted RCCs were incubated with S—303 and Glutathione (GSH) overnight at room temperature. During processing, RCCs were held at 4°C+/—2°C. Results: We found nearly identical values of MCV (86.8+2.1 g/dL vs 89.0+3.9 g/dL; means+SD), MCHC (32.6+2.4 vs 31.1+1.8 fl) and HCT (57.4+0.4 vs 59.9+1.8%) between test and control groups after 42 days of storage, which were not statistically significant (n.s.). Similarly, hemolysis rate (0.6+0.1 vs 0.7+0.2%) and free hemoglobin values (2.8+0.6 vs 3.2+0.7 g/1) were comparable between both arms. In contrast, PI treated products revealed higher ATP (36.4+8.0 vs 26.3+4.4 μη??ι/dl), higher glucose (585+43 vs 324+22 mg/dl) and lower lactate (17.7+3.2 vs 31.8+2.7 mmol/1) concentrations than control products, Altering the sequence of addition of S—303 and GSH resulted in a reduced hemolysis rate (0.6 — > 0.4%; control values, 0.3%). Analysis of immune reactivities against Pi—treated erythrocytes revealed 0 positive reactions in 1500 samples from healthy volunteers, and only very few (3/1000) reactivies with sera form chronically transfused patients which were both not hemolytically active. Pathogen reduction was found to be 5.9–4.8 log for 4 model viruses, and >5.1–6.8 log for 4 model bacteria. Discussion: Together, we found that the modified PI process for RCCs fulfils all criteria for the pharmaceutical quality and points to a reduced storage lesion in erythrocytes. The GMP realization of the PI process for erythrocytes is feasible. These data confirm a process that can be further validated to support phase III clinical trials using PI—RCCs with S—303 and GSH in SAGM.

OS 8.08

Implementation of the first international repository for transfusion relevant bacteria reference strains (ISBT working party TTID, subgroup on bacteria)

Melanie Stoermer¹, Antonio Arroyo², Julia Brachert¹, Hector Carrero³, Dana Devine⁴, Jay S. Epstein⁵, Christian Gabriel⁶, Cohava Gelber⁷, Ray Goodrich⁸, Kay—Martin Hanschmann¹, David G. Heath³, Michael R. Jacobs⁹, Shawn Keil⁸, Dirk de Korte¹⁰, Bernd Lambrecht¹¹, Cheuk—Kwong Lee¹², Jan Marcelis¹⁰, Susanne Marschner⁸, Carl McDonald¹³, Siobhan McGuane¹³, Marian McKee⁶, Thomas H. Mueller¹¹, Tshilidzi Muthivhi¹⁴, Annika Pettersson¹⁵, Piotr Radziwon¹⁶, Sandra Ramirez—Arcos⁴, Henk W. Reesink¹⁷, Julieta Rojo², Ineke Rood¹⁰, Michael Schmidt¹⁸, Christian K Schneiden, Utta Schurig¹, Erhard Seifried¹⁸, Ute Sicker¹, Silvano Wendel¹⁹, Erica M. Wood²⁰, Roslyn A. Yomtovian²¹ and Thomas Montag¹

¹Paul Ehrlich Institute, Langen, Germany, ²Centro Nacional de la Transfusion Sanguínea, México, ³Walter Reed Army Medical Center, Washington DC, USA, ⁴Canadian Blood Service, Ottawa, Canada, ⁵Food and Drug Administration, Rockville, Maryland, USA, ⁶Austrian Red Cross, Blutzentrale Linz, Austria, ⁷American Type Culture Collection, Manassas, Virginia, USA, ⁸CaridianBCT Biotechnologies, Lakewood, Colorado, USA, ⁹Case Western Reserve University, Cleveland, Ohio, USA, ¹⁰Sanquin Blood Supply Foundation, Amsterdam, The Netherlands, ¹¹German Red Cross, NSTOB, Springe, Germany, ¹²Hong Kong Red Cross Blood Transfusion Service, Hong Kong, China, ¹³NHS Blood and Transplant, London, United Kingdom, ¹⁴South African National Blood Service, Weltevreden Park, South Africa, ¹⁵VU University Medical Centre, Amsterdam, The Netherlands, ¹⁶Regional Centre for Transfusion Medicine, Bialystok, Poland, ¹⁷Academic Medical Center, Amsterdam, The Netherlands, ¹⁸German Red Cross, Frankfurt/Main, Germany, ¹⁹Hospital Sirio Libanês, São Paulo, Brazil,²⁰Australian Red Cross Blood Service, Melbourne, Australia, ²¹Louis Stokes Veterans Administration Medical Center, Cleveland, Ohio, USA

Background: Methods for bacterial detection or reduction in platelet concentrates (PCs) are important tools for improving the safety of blood components. No international transfusion relevant bacterial reference material existed for investigation of these methods. The ISBT Working Party on Transfusion—Transmitted Infectious Diseases (WP—TTID), Subgroup on Bacteria, organised an international study to establish the first international Repository for Transfusion Relevant Bacteria Reference Strains (TRBR) as a tool for validation and comparison of bacteria screening and pathogen reduction methods. Methods: Four TRBR (Staphylococcus epidermidis PEI—B—06, Streptococcus pyogenes PEI—B—20, Klebsiella pneumoniae PEI—B—08, E. coli PEI—B—19) were distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and proliferation analyses in PCs after low spiking (0.3 and 0.03 CFU/ml). Results were submitted to WHO Expert Committee Biological Standardisation (ECBS). Results: The TRBR were correctly identified in 98%. Str. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, K. pneumoniae and St. epidermidis replicated in all laboratories. The 95% confidence intervals [value × 10⁷ CFU/ml] were for St. epidermidis 1.19–1.32, Str. pyogenes 0.58–0.69, K. pneumoniae 18.71–20.26, and E. coli 1.78–2.10. During the annual meetings of ECBS in 2009 and 2010 the potential advancements in this field aiming to establish a recognized standard for worldwide use were discussed. Conclusion: The study was undertaken as a proof of principle with the aim to demonstrate a) the quality and suitability of TRBR for low—titre spiking, b) the property of donor—independent proliferation in PCs, and c) their suitability for worldwide shipping. The WHO ECBS approved the adoption of these four TRBR as the first WHO Repository for Transfusion Relevant Bacteria Reference Strains, and also endorsed as a project the addition of 9 further strains.

Abstract Session 09 – Hemostasis: Coagulation and von Willebrand Factor

OS 9.01

A novel factor VIM bypass strategy based on FIX variants is efficacious and does not increase the thromboembolic risk

Milanov P.¹, Abriss D.¹, Quade—Lyssy P.C.¹, Tonn T.², Seifried E.¹, Schüttrumpf J.¹

¹DRK—Blutspendedienst Baden Württemberg – Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Klinikum der Johann Wolfgang Goethe—Universität, Frankfurt, Deutschland, ²German Red Cross Blood Donation Service East, Institute Dresden, Dresden, Germany

Therapy of patients with haemophilia A and inhibitory antibodies against factor VIII (FVIII) is a major clinical Background. Acute bleedings can be controlled by infusion of activated coagulation proteases, so called bypass agents. However, substances available have short half—lives and raise concerns about the thromboembolic risk. FVIII is the obligate co—factor of the serine protease factor IX (FIX). Previously, we identified FIX variants with up to 22% FVIII independent clotting activity at physiological FIX levels. Furthermore we could demonstrate that these proteins correct the bleeding phenotype of FVIII K.O. mice. We now expressed FIX variants with bypass activity (V181I/ K265T/I383V, ITV) in FVIII K.O. mice using non—viral minicircle based delivery and demonstrated reconstitution of clot formation following laser induced injury in the microvasculature. Therapeutic efficacy was present even at low levels (around 1000 ng/ml) of expression, 21/21 stable clots formed, while only 2/23 small clots formed for FIX wild type (WT) controls. In order to examine vascular risks involved, we expressed FIX variants at different levels in hemostatic normal C57B1/6 mice. We compared FIX variant ITV with a variant ITAWV with bypass and additionally high FIX specific activity (1600% of FIX WT). Below circulating FIX of 5 μg/ml (physiological FIX plasma level) no change in thrombin anti—thrombin complex (TAT) levels was observed for the wild—type and the triple variant groups (n = 6 mice/group). Only at highest plasma levels (5 to >50 μg), elevated TAT levels were measured. In contrast, the variant ITAWV increased TAT levels even at expression levels below 5 μg/ml. None of the mice showed signs of DIC or thrombosis (drop in platelet counts, high D—dimers). The experiments in these mice suggest that the bypass activity of FIX variants alone does not increase the thromboembolic risk.

OS 9.02

Factor VIM locus itself explains all cases of hemophilia A cases without mutations in the F8 cDNA

Pezeshkpoor B.¹, Vidovic N.¹, Nüsgen N.¹, Nanda I.², Haaf T.², Budde U.³, Oldenburg J.¹, El—maarri O.¹

¹Institut für Experimentelle Hämatologie und Transfusionsmedizin; Universitätsklinikum Bonn, Bonn, Deutschland, ²Institut of Human Genetics, Würzburg, Germany, ³Hemostaseology Department Medilys, Hamburg, Germany

In a small group of typical hemeophilia A patients with reduced F8 activity no mutations in the F8 coding sequence could be found. To investigate possible molecular causes we addressed some open questions. The first: is the phenotype in all affected members within one family segregate with the same F 8 allele, which would indicate the localization of the defect to the F8 locus? The second: is the mRNA level from these patients reduced and is it correlating with the F8 activity and antigen levels? The third: is there a correlation between the F8 activity and F8 antigen levels in these patients, which would suggest a reduced secretion of rather normally functional protein? To answer the above question we have analyzed 11 such patients. We excluded the involvement of vWF, MCFD2 and LMAN1 by direct sequencing and by the vWF collagen binding, vWF multimer analysis and vWF—F8 binding assays; all of which were normal. Gross rearrangement or translocation in the F8 locus was excluded by long range PCRs, that cover 190 Kb spanning all the genomic F8 locus, and by FISH analysis. Abnormalities in mRNA splicing were excluded by sequencing all exons junctions from RT—PCR products. Our analysis revealed that all affected members, within one family share the same F8 allele. The F8 antigen levels are considerably reduced and correlate well with both the severity and the activity levels, in contrast to the F8 mRNA expression levels. The specific activity of FVIII didn't show a significant difference to the healthy individuals suggesting that the lack of activity is not due to loss of functionality of the protein, rather than to un—effective expression or secretion. Our data indicate that the disease at least in these families segregate with the F8 locus itself. Based on these data we could conclude that the defect is with high probability localized to the F8 gene itself or it has high linkage disequilibrium to F8 gene; however, the molecular mechanism remains elusive.

OS 9.03

Von Willebrand disease: genotype—phenotype correlation

Yadegari H., Driesen J., Pavlova A., Dermer H., Schuering I.K., Hertfelder H.—J., Oldenburg J.

Institut für Experimentelle Hämatologie und Transfusionsmedizin; Universitätsklinikum Bonn, Bonn, Deutschland

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of the von Willebrand factor (VWF). VWD is classified into three subtypes – type 1 (partial quantitative deficiencies), type 2 (qualitative defects) and type 3 (complete deficiency of VWF). In this study we explored genotype and phenotype correlation in patients with VWD. Materials and Methods: One hundred seventeen patients belonging to 81 families diagnosed to have VWD were studied. Mutation analysis was performed by direct sequencing of the VWF gene. Large deletions were investigated by multiplex ligation—dependent probe amplification (MLPA). Result and Discussion: We identified putative mutations in 68 index patients (IP) (84%). In total, 68 different mutations were detected comprising of 36 missense mutations (53%) and 32 null mutations (47%) including 11 small deletions (16.2%), 2 small insertions (2.9%), 7 nonsense mutations (10.3%), 5 splice—site mutations (7.4%), 1 silent mutation (1.4), and 6 large deletions (8.8%). Novel mutations comprise 47% (32). The majority of identified mutations in type 1 and type 2 VWD (73% vs 84.5%) were missense, while the type 3 VWD was mostly caused by null alleles mutations (81%). The mutations were found in all regions of the VWF gene. However, a substantial number of the candidate mutations found for type 1 and type 2 VWD were clustered in exon 28. Conclusion: Our data support a complex spectrum of molecular pathology resulting in VWD. In most cases, changes within the VWF gene were identified and correlated with VWD phenotype although for type 1 VWD this correlation was less pronounced. Moreover, our results indicate that large deletions should be taken into consideration in the diagnosis of VWD especially in patients without clear causative mutations.

OS 9.04

Von Willebrand factor binding factor VIII: a new method to determine relative stoichiometry and affinity

Stein E.—L.¹, Pachmann U.²

¹SIMFO Spezielle Immunologie Forschung + Entwicklung GmbH, Bayreuth, Deutschland, ²Labor Dr. U. Pachmann, Bayreuth, Deutschland

Backgruond: If factor VIII (FVIII) is low, compared to von Willebrand factor (vWF), this may have different reasons, including Normandy type (2N) of von Willebrand Disease (vWD). However, testing hitherto does not comply with GLP criteria. Neither frequency nor strength of vWD type 2N can be determined, implying relevant diagnostical, organisational and therapeutic consequences. Therefore we have developed a simple new method to determine the relative stoichiometry and affinity of vWF interaction with FVIII. Methods: The binding of FVIII to vWF was determined in a solid phase assay under standardized conditions and independent of the ligand concentration. Standard deviations and coefficients of variation were determined. Results: 15 duplicates of a normal plasma pool ranged from 0,8 to 1,3 for the relative number of the binding sites and from 0,7 to 1,5 for the relative binding affinity. The reference range (CI 95%) as derived from 30 nonrelated healthy donors was 0,34 − 1,98 for the relative affinity and 0,27 − 1,47 for the relative stoichiometry. The interaction of Wilate^® with Recombinate^® was found within the reference range. Even after 4 days plasma storage at 4+/— 2°C the results did not differ significantly. Conclusions: Whenever percent FVIII and vWF differ by more than a factor of two, the relative stoichiometry and affinity can help to distinguish between vWD 2N and other causes of the irregularity.

OS 9.05

Direct measurement of plasma thrombin levels

Müller J.¹, Becher T.¹, Berdel P.², Gravius S.², Mayer G.³, Oldenburg J.¹, Pötzsch B.¹

¹Institut für Experimentelle Hämatologie und Transfusionsmedizin; Universitätsklinikum Bonn, Bonn, Deutschland, ²Clinic for Orthopedic Surgery, University of Bonn, Bonn, Germany, ³Life and Medical Sciences (LIMES)— Institute, University of Bonn, Bonn, Germany

Background: The serine protease thrombin is the key—player within the coagulation system. Thus, levels of circulating thrombin might represent a promising biomarker reflecting a patient's individual hemostatic status. However, no clinically applicable test system for quantification of plasma thrombin levels is available to date. To close this gap, we developed an oligonucleotide—based enzyme—capture—assay (OECA) for the direct measurement of plasma thrombin levels. Patients and Methods: Plasma thrombin levels were measured in healthy volunteers and patients undergoing total hip arthroplasty (THA). Blood was drawn into tubes containing citrate and the reversible active site inhibitor argatroban to prevent complex—formation between thrombin its plasma inhibitors ex vivo. The bivalent DNA—aptamer HD 1—22 was immobilized in the wells of MTPs to capture thrombin from processed plasma samples. The amount of captured thrombin was assessed using plasma—based calibrators and an appropriate fluorogenic peptid substrate. Results: Evaluation of pre—analytical handling conditions revealed that argatroban completely preserves thrombin activity in taken whole blood for a time period of least 8 hours. The applied thrombin—OECA showed a limit of quantification (LLOQ) of 0.039 + 0.019 ng/ml while the limit of detection (LOD) was calculated as 0.017 + 0.004 ng/ml. In healthy normals, plasma levels of thrombin were found to be low and therefore fell short of either the determined LLOQ or LOD of the assay. During THA, increased plasma thrombin levels were detectable reaching peak values above 100 pM. Correlation of data with that found for TAT—complexes revealed high inter—individual variability of proco—agulant responses. Conclusions: We propose that the direct determination of the active reaction partners of the coagulation cascade more accurately reflects disease—relevant intermediate phenotypes. The OECA platform presented herein demonstrates a possible route for such measurements.

OS 9.06

Thrombin inactivation kinetics are not influenced by antiphospholipid antibodies

Rühl H., Müller J., Harbrecht U., Oldenburg J., Pötzsch B. Institut für Experimentelle Hämatologie und Transfusionsmedizin; Universitätsklinikum Bonn, Bonn, Deutschland

Background: Impaired thrombin (FIIa) inactivation generates a prothrombotic state. This is demonstrated by the high thrombotic risk of patients with antithrombin—(AT)—deficiencies. It has been suggested that antibodies against prothrombin/phospholipid complexes generate a thrombophilic phenotype through interference with AT—dependent FIIa inactivation. To study thrombin inactivation in plasma we developed a thrombin inhibition assay (TIA). Methods: The TIA is based on an oligonucleotide enzyme capture assay (OECA) platform that uses a DNA—aptamer to catch Fila. FIIa is added to citrate plasma at a final concentration of 2 ng/ml (54.5 nM). The FIIa—inhibitor argatroban is added at intervals from 0 − 360s to stop FIIa inactivation by endogenous inhibitors. Subsequently the amount of argatroban—protected FIIa is quantified by OECA. To control inter—assay variation, FIIa half—life of individual samples is divided by half—life of an internal reference to calculate a “θ—ratio”. Using this approach, FIIa inactivation rates were determined in AT—deficient plasma, in plasma samples of healthy probands (n = 16), samples of patients with antibodies against prothrombin (n = 16), and samples of 24 other thrombophilic patients. Results: The TIA demonstrated coefficients of intra—and inter—assay variation of 7.8% and 19.2%, respectively. Mean half—life of FIIa in normal human plasma was 57.6 ± 5.4s (mean ± SD). We observed a strong correlation (r = —0.94) between AT activity and FIIa half—life which ranged between 168s (0% AT) and 57.6s (94% AT). The mean θ—ratio was 0.90 ±0.18 in healthy probands, 0.81 ±0.20 in patients with prothrombin/phospholipid antibodies, and 0.90 ± 0.24 in the whole patient population. Conclusion: The TIA allows monitoring of FIIa inhibition in plasma which strongly depends on AT activity. FIIa inactivation does not seem to be impaired in patients with a history of thrombosis and in the presence of antibodies against prothrombin/phospholipid complexes.

OS 9.07

Exploration of fibronectin (Fn) unfolding by β3 integrins during platelets adhesion and aggregation in vitro using fluorescence resonance energy transfer (FRET)

Huynh K.C, Stoldt V.R., Scharf R.E.

Department of Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf, Germany

Background: Unfolding of Fn when interacting with platelets may modulate platelet adhesion and aggregation. We have used fluorescent Fn and FRET (1) to examine kinetic differences in Fn unfolding caused by activated platelets in suspension or adherent platelets and (2) to assess Fn unfolding when interacting with distinct β3 integrins (allbβ3 or α?β3) upon platelet adhesion. Methods: To assess FRET signals in response to conformational changes, fluorescent Fn was exposed to guanidine (Gdn) HC1. Fluorescent Fn was mixed with a 10 fold excess of unlabeled Fn to prevent energy transfer between adjacent molecules. Fn mixtures (20 μg/ml) with or without 20 μg/ml 10E5 or LM609 antibodies were incubated for 3 h at 22°C with suspended platelets or platelets adherent onto immobilized Fn (50 μg/ml). For control, FRET signals of Fn mixtures without platelets were recorded by fluorescence spectrometry. Results: Upon incubation of fluorescent Fn with GdnHCl, FRET decreased by 60% from 0.46±0.08 to 0.20±0.05 (mean ratio of FRET+SD, p < 0.001) indicating a maximal conformational change of Fn from its compact to the unfolded state. Addition of fluorescent Fn to platelets adherent onto immobilized unlabeled Fn caused a progressive decrease by 6% in FRET after 3 h of incubation. The decrease in FRET was slower when LM609 (blocking α?β3) was added. By contrast, FRET remained unchanged in control experiments without platelets. The same was true with suspended platelets or in the presence of 10E5 (blocking allbβ3). Conclusion: Our findings suggest that activated adherent but not suspended platelets can progressively unfold fibronectin, thereby inducing profound conformational changes in the ligand. Its different conformational states may also explain the oppositional effects in platelet adhesion and aggregation. Unfolding of fibronectin caused by platelet adhesion onto immobilized fibronectin is governed by β3 integrins in which allbβ3 plays a predominant role in comparison with α?β3.

OS 9.08

Orally administered non—viral vectors for gene therapy of hemophilia B

Quade—Lyssy PC, Milanov P., Abriss D., lingerer C, Seifried E., Schüttrumpf J.

DRK—Blutspendedienst Baden Württemberg – Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Klinikum der Johann Wolfgang Goethe—Universität, Frankfurt, Deutschland

Non—viral vectors are becoming attractive gene delivery systems due to their excellent safety profile. Although gene transfer is less efficient compared to viral vectors, non—viral vectors allow repeated administration over long periods of time. Hemophilia B, a bleeding disorder caused by deficiency of coagulation factor IX (FIX), is especially well suited for a gene therapy approach since plasma levels above 1% can already prevent major long—term complications. In this study we are developing an oral delivery system for the treatment of hemophilia B based on the cationic polymer chitosan. Protection of vector DNA in nanoparticle constructs was confirmed by DNase I digest and subsequent gel electrophoresis. In cell culture experiments transfection with nanoparticles resulted in expression of up to 100 ng/ml (2% of normal plasma concentration). Next, we treated groups of mice (n = 4–5/group) with naked DNA or nanoparticle complexes containing FIX or mock DNA. Circulating plasma levels in all groups remained below the therapeutic range (< 1%). To improve therapeutic efficacy, different amino acid substitutions know to increase FIX clotting activity were combined and introduced in the expression cassette by site—directed mutagenesis. One variant G4Y/V86A/R338L/S377W (YALW) was identified with 1500% FIX specific clotting activity compared to the wild—type protein. Following oral gene transfer in mice, FIX antigen expression levels remained low. However, the FIX variant YALW shortened the aPTT—based one stage clotting assay with FIX activity in the therapeutic range (1–4% of normal). Immune histochemical staining confirmed FIX accumulation in the extracellular matrix of gut, liver, and spleen. In conclusion, gene transfer strategies based on non—viral vectors are still hampered by their low efficacy. Protein engineering, in our case for FIX, might provide a possibility to overcome this shortcoming without compromising the excellent safety profile of simple DNA vectors.

Abstract Session 10 – Blood Donation

OS 10.01

Trends in the supply of blood products in the period 2000 to 2010 – data pursuant to section 21 German Transfusion Act (TFG)

Henseler O., Heiden M., Haschberger B., Hesse J., Seitz R. Paul—Ehrlich—Institut, Division Haematology/Transfusion Medicine, Langen, Germany

Background: The German Transfusion Act obligates the Paul—Ehrlich—Institut to provide data on blood supply in Germany. Thus, data on collection, manufacture, import, export and use are collected and published as annual reports. Methods: Data are submitted annually via web—application. Results: Between 2000 and 2010, whole blood donations steadily increased from 4.4 to 4.9 million per year. Concordantly, annually distributed red blood cell concentrates increased to 4.8 million, a plus of 11.8%. Over the same time period the number of apheresis donations varied between 0.7 and 2.6 million due to variable amounts of collected source plasma. Collection of source plasma intended for further processing into plasma derived medical products account for about 90% of all apheresis donations. 1.8 million litres source plasma and 1.1 million litres recovered plasma were collected in 2010 which corresponds to almost the biggest per capita amount collected in Europe. Furthermore, 16,316 autologous hematopoietic stem cell preparations were manufactured in 2010, 50% thereof from cord blood. A total of 3,255 autologous transplantations were reported but none of them were cord blood derived ever. The number of allogeneic peripheral blood stem cells increased by 67% to 6,054 since 2004. 51% thereof were exported and 36% transplanted. In contrast the number of allogeneic cord blood preparations nearly doubled to 3,261 in the last six years but only 0.34% was reported as transplanted in 2010. Conclusions: With a mean of 58 whole blood donations per 1,000 inhabitants and year a sufficient blood supply is assured. In Germany a huge amount of source plasma is collected which exceeds the local fractionation capacity. Due to imports of plasma derivatives in sufficient quantities no shortage is to expect. The number of hematopoietic stem cell preparations and the portion exported increased, indicating that stem cell donations are well organized in Germany.

Figures to blood donations and supply with blood components and source plasma

OS 10.02

Is the deferral of men who have sex with men (MSM) from donating still appropriate?

Offergeld R., Kang M.U.—Z., Ritter S., Marcus U., Hamouda O. Robert Koch—lnstitut, Berlin, Deutschland

The permanent deferral of men who have sex with men (MSM) from donating blood is claimed to be discriminatory by some advocacy groups. Before changes in deferral policy in this complex area are considered a careful analysis of the epidemiological situation especially with respect to HIV infections has to be made. Data from the national HIV surveillance and epidemiological data from all blood establishments on confirmed HIV, HCV, HBV and syphilis donors are collected and evaluated at the RKI. Special attention is given to the most likely modes of transmission. The number of newly diagnosed HIV infections in the general population in Germany increased from 2001 to 2007 has been stable since with close to 3000 HIV infections/year. Approximately 2/3 of all infections are acquired through sexual contacts between men. Among blood donors HIV prevalence in new donors was 6.7/100,000 and 2.2/100,000 in repeat donors in 2009. An increasing trend among whole blood repeat donors was observed since 2001. Information from post donation interviews was available for roughly 40% of HIV positive donors between 2006 and 2010. 45.7% of donors declared a heterosexual risk, 41.4% were MSM and 11.7% stated other risks like occupational exposure. Due to the possibility of window phase donations or test failures individuals with a high risk of acquiring transfusion relevant infections are permanently deferred from donating blood including MSM. Still, more than 40% of the HIV positive donors are MSM. The incompliance with donor selection criteria may be due to HIV test seeking behaviour or opposition to the regulation. In order to increase blood safety it is necessary to inform target groups, improve donor educational material, make a direct assessment of sexual risk behaviour in the donor questionnaire and offer anonymous testing free of charge. The possibility of a temporal rather than lifetime deferral is currently under investigation on a national and international level.

OS 10.03

Motivation of blood donors in West Pomerania – a cross—sectional study

Sumnig A.¹, Fendrich K.², Hoffmann W.², Greinacher A.³

¹Institut für Immunologie und Transfusionsmedizin, Abt. Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Deutschland, ²Institut für Community Medicine, Universitätsmedizin Greifswald, Germany, ³Institut für Immunologie und Transfusionsmedizin, Greifswald, Deutschland

Background: This cross—sectional study aimed to analyze motivational factors for blood donation in first—time compared to regular donors. Methods: During a 12 months period a sample of every 6th whole blood donor (age—range 18–68 years) of the blood donation centre of the University hospital of Greifswald was drawn. 2,570 donors (response rate: 74.1%) participated in the study. The donors were informed about the study and after giving informed consent asked to fill in a self—completion questionnaire. The standardized questionnaire contained modules about socio—demographic characteristics, donor status, adverse reactions after blood donation, information sources about blood donation, satisfaction with blood donation centre, travel time and distance to the donation centre, reasons/motivational factors for blood donation, current health status. Differences in prevalence between age groups, gender and donor status were tested by nonparametric test for trend (Cuziak) or Chi—Square—Test. Results: 12.9% of the 2,570 participating donors were first time donors in the donation centre Greifswald. The strongest motivational factor for blood donation were “help other people” (94.6%), “medical assessment of blood values” (67.8%), and “feeling satisfied after donation” (48.4%). The most pronounced differences between first time and regular donors occurred in the factors “are taken here by a friend/relative” (first time donors: 48.2% vs. regular donors: 15.9%; p < 0.05) and “getting a remuneration” (first time donors: 38.6% vs. regular donors: 49.2%; p < 0.05). Discussion: Obtaining more knowledge about the motivation to donate blood is important in order to recruit more new and regular donors. In the light of the demographic change yielding a potential blood shortage in the near future the design of specific campaigns based on the different motivational factors for younger or older, female or male potential donors in the population is of high importance.

OS 10.04

A statistical analysis of blood donors on medication

Hartmann L., Zeiler T.

German Red Cross Blood Service West, Center of Transfusion Medicine Breitscheid, Ratingen, Germany

Background: The consequences of medications taken by blood donors on donor deferral or product quality have been discussed previously. However there are no data about the extent and kind of medication in our donor population. To assess the effects of a modification of donor deferrals due to certain medications we collected and analyzed corresponding data. Methods: In a spot check of blood donors any medication taken within the previous 4 weeks before blood donation, time of taking the last medication, sex, age and demographic data were recorded. Using a “Prolog” based programme we assessed the effect of various rules on donor deferral and use of blood components due to different assessments of medication. Results: 1,299 donors were entered in the study. The study population was representative for our donor population (age, sex, donation frequency). For every 100 donors 64 drug takings had to be evaluated. 0.95% of the donors were deferred due to medication that prohibited blood donation. 29.9% of the donors took drugs that allowed blood donation but were considered relevant for certain blood components (e.g. ASS). 13.2% of the donors took two or more different drugs. Drug intake increased with age from 22% (18–45 years) to 62% (older than 65 years). We could identify the “top ten” of drugs that constituted 95% of all drug intakes. A simulation of different rules for assessment of medication provided only minimal effect on donor deferrals (due to polymedication) but 3.2% less restrictions for the use of blood components. Conclusion: Medication is frequently seen in blood donors especially in elderly donors. Sophisticated rules for assessment of medication considering pharmacodynamics may result in less donor deferrals or restrictions for the use of donated blood. Our database provides an instrument for developing rules and estimating the effects of these rules on eligibility of blood donors and blood products.

OS 10.05

Results of additional laboratory tests for blood donors as part of a donor relationship program

Rothe R.¹, Galle G.², Tonn T.³, Knels R.⁴

¹German Red Cross Blood Donation Service East, Institute Cottbus, Cottbus, Germany, ²German Red Cross Blood Donation Service East, Institute Chemnitz, Chemnitz, Germany, ³German Red Cross Blood Donation Service East, Institute Dresden, Dresden, Germany, ⁴Eurocode IBLS e.V., Dresden, Germany

Higher levels of cholesterol, LDL and lower levels of HDL are strongly associated with cardiovascular diseases. Thus, today hypercholesterolemia is an important life style parameter. To stimulate active donors for higher intensity and to honor very active donors the German Red Cross Blood Donation Service East decided to measure cholesterol starting from April 2008 and if cholesterol is >240 mg/dl additionally LDL/HDL from July 2009. Till July 2010 188,432 cholesterol and 17,638 LDL/HDL—values were measured from donors which donated whole blood three times in 12 months. The first analysis was started to evaluate the results from a supposed healthy population and to answer the question whether the additional checks stimulated the donors for a higher blood donation frequency. Cholesterol levels >240 mg/dl were found in 21% of the donors resulting in higher LDL—levels in 95.7%. Only 4.5% of the HDL values were lower the minimum standard of 40 mg/dl, the mean value was 61 mg/dl, possibly a compensative effect to high LDL and cholesterol levels. Sex, age and living area show in result of the high number of values significant differences, but the absolute values vary only by 1–2 mg/dl. Exception was the higher HDL—values by women versus men. A surprise was the increase of the cholesterol mean values by follow up the donors. 22,892 donors were three time qualified for additional testing in the evaluation time—frame. Mean values increased from 214.3 up to 218.1 respectively 219.4 mg/ dl. Today the reason for this increase is unknown, but because most of these donors were very active before starting the program, the high frequency of donation could be excluded. Investigating the intensity of blood donation we found that in result of the health check for very active donors the number of donors with three or more donation per year increased from 16.6% in 2007 up to 18.7% in 2010. Thus we decided to continue the program and to implement further parameters into the program.

OS 10.06

Safety and frequency of whole blood donations from elderly donors

Müller—Steinhardt M.¹, Müller—Kuller T.², Menzel D.³, Wiesneth M.⁴, Seifried E.², Klüter H.¹

¹Institut of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Germany, ²German Red Cross Blood Service Baden—Württemberg – Hessen, Institute of Transfusion Medicine and Immunohaematology, Medical Faculty Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany, ³Institute of Clinical and Experimental Transfusion Medicine, Medical Faculty University of Tübingen, Tübingen, Germany, ⁴Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden—Württemberg—Hessen, and University of Ulm, Ulm, Germany

Background: Within the coming decades a steadily growing demand for cellular blood products will face a shrinking eligible blood donor population in many countries. After increasing the donor age of repeat donors for whole blood donation (WB) from 68 to 70 years in 2009 in our Blood Service, we investigated whether this is sufficient as a safe and effective strategy to sustain future blood supply. Materials and Methods: Between March 1, 2009 and February 28, 2011 WB donations from donors aged between 69–70 and their proportion of total WB donations in 2010 were determined. We analyzed local and systemic adverse reactions (overall versus systemic reaction rate) in 710,048 WB donations and determined annual donation frequencies with respect to sex and age. Results: 4,157/12,799 (32.5%) of all invited donors responded and contributed 0.98% (males) and 0.56% (females) to all WB units (723,606) collected in 2010. The overall and systemic adverse reaction rate declined progressively by age for both sexes (males: p < 0.0001, p < 0.0001, females: p < 0.0001, p = 0.0004). On the other hand mean annual donation frequencies were positively correlated with increasing age (males: r = 0.953, p < 0.0001; females: r = 0.913, p < 0.0001) with peak values for 70 year old male: 2.53 ± 1.37 vs. 1.79 ± 1.05, p < 0.0001 and female donors: 2.15 ± 1.06 vs. 1.52 ± 0.78; p < 0.0001. Conclusions: We conclude that elderly WB donors have a declining risk of adverse reactions with 69–70 year old donors having the lowest frequency of any adverse reactions of all. In parallel they are highly committed to donate blood. Thus we consider WB donations from repeat donors aged 69–70 safe and sustainable and suggest it a very powerful short to mid—term strategy to, at least in part, overcome the challenges of the demographic change.

OS 10.07

Prospective paired study on gamma—irradiation of fresh red blood cells (RBC) in SAG—M: evidence for a significant storage lesion within 24 hours

Mansouri B.¹, Vogt M.¹, Brüllhardt R.¹, Schulzki T.²

¹University Clinic of Hematology and Central Hematological Laboratory Inselspital, University Hospital, Bern, Switzerland, ²Regionaler Bluspendedienst SRK Graubünden, Chur, Schweiz

Background: The negative impact of gamma—irradiation on RBC storage variables is well known. But there are rare data about possible early alterations during the first week of storage. We evaluated the in—vitro quality of gamma—irradiated RBC units during their early storage time. Methods: Twenty fresh (< = 48 hours), prestorage leukoreduced RBCs in additive solution saline—adenine—glucose—mannitol (SAG—M) were split in two equal halves. One of each pair was irradiated (IR) with 25 Gray (Gammacell 3000 Elan, Canada), the other served as control (CO). Blood samples were taken from the original products (day 0) and from their splits (days 1, 3, 5, 7 and 9). In—vitro analyses included K+ (mmol/L), Na+ (mmol/L), free haemoglobin (FHB, g/L), adenosintriphosphate (ATP, μmol/g HB) and 2,3—diphosphoglycerate (DPG, mg/g HB). Three further RBC units were gamma—irradiated only and K+ and Na+ were measured before and directly after gamma—irradiation, and after 5', 10', 15', 30', 45', lh, 2h, 4h, 8h, 12h, and 24h. Results: Results are summarised in the table. Significant differences between IR and CO (p < 0.01) are marked (∗). Particularly, IR splits showed an immediate increase of K+ on day 1 with levels being comparable to day 9 of CO units. In addition, IR splits disclosed an accelerated increase of Na+ with levels at day 9 being comparable to those of regularly stored RBC at the end of their shelf life (= 42 days). IR units further disclosed a significant higher FHB on days 7 and 9. Conclusion: Gamma irradiation of RBC units induces an early and accelerated storage lesion with a particularly desperate leakage of potassium and corresponding influx of sodium. Therefore we recommend to irradiate RBC preferably straight before transfusion to avoid potassium exposure of the recipient. This applies particularly for vulnerable patient groups like neonates or intra—uterine transfusions.

Table for abstract OS 10.07.

	Day 0	Day1, CO/IR	Day 3, CO/IR	Day 5, CO/IR	Day 7, CO/IR	Day 9, CO/IR
K	4.75	6.45/19.10∗	10.55/34.85∗	14.25/44.40∗	17.40/50.30∗	20.35/54.30∗
Na	147	146/135∗	143/119.5∗	140/112∗	140/106.5∗	136.5/101.5∗
FHB	0.41	0.33/0.36	0.32/0.37	0.40/0.46	0.34/0.46∗	0.41/0.60∗
ATP	4.96	5.15/5.10	4.99/4.95	4.86/4.85	4.88/5.07	5.01/5.20
DPG	2.27	2.03/1.70	1.18/1.09	1.00/0.87	0.60/0.37	0.02/0.05

OS 10.08

Gamma ray irradiation of red blood cells does not impair mechanical stability during centrifugation

Weiss D.R., Goehring J., Weisbach V, Strasser E.R, Ringwald J., Zimmermann R., Eckstein R.

Transfusionsmedizinische und Hämostaseologische Abteilung, Universitätsklinikum Erlangen, Erlangen, Deutschland

Background: No data were available about the question whether irradiation of erythrocytes might impair the mechanical stability during centrifugation, possibly leading to elevated haemolysis. For preparing RBCs for intrauterine transfusion, for example, irradiation and centrifugation of red blood cell concentrates (RBCs) is necessary. We performed the study to find the optimal sequence of the preparing steps. Methods: 20 RBC units not older than 9 days were divided into two subunits. These subunits were washed by diluting with 0.9% NaCl, centrifuging and discarding the supernatant. Subsequently, hae—matocrit was adjusted to 75% with 0.9% NaCl. One of the 2 subunits was γ—irradiated with 30 Gy before these preparing steps, the other subunit was irradiated afterwards. The units were evaluated in vitro before preparation and at days 1 and 7. Results: Haemolysis rate, extracellular LDH and a—HBDH in the two groups of RBCs were similar. We conclude that centrifugation after

Irradiation of RBCs does not accelerate haemolysis. There was no difference in the ATP content in the two subunits indicating no difference in energy metabolism. However, the extracellular potassium concentration was significantly lower in the subunits washed after irradiation (19.1 mmol/L ± 2.3 vs. 21.9 mmol/L ± 2.7 after 24 hours). Conclusions: Regarding haemolysis, it is equal if RBCs are irradiated before or after centrifuging. Because the extracellular potassium is about 8% lower in units washed after irradiation, it might be favourable to wash RBC units after irradiation.

OS 10.09

Increasing donor deferral rates after the implementation of the new German blood donor questionnaire

Michael Müller—Steinhardt^1,2, Christian Weidmann³, Markus Wiesneth^1,4, Eberhard Weck^1,5, Erhard Seifried^1,5, Joachim Brade⁸, Harald Klüter^1,2

¹German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Germany; ²Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; ³institute of Public Health, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; ⁴Institute of Clinical Transfusion Medicine and Immunogenetics, Medical Faculty University of Ulm, Ulm, Germany; ⁵Institute of Transfusion Medicine and Immunohaematology, Medical Faculty Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany; ⁶Department of Medical Statistics and Biomathematics, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany

Background: The implementation of a new national German blood donor questionnaire (DQ) was proposed to improve donor and recipient safety. Materials and Methods: We compared deferral/exclusion rates of whole blood (WB) donors before (May 2010, n=64.735) and after (May 2011, n=71.687) the implementation of a new DQ. Considering seasonal variations, analysis was performed with respect to collection—site (mobile vs. fixed), sex, donor—status (first—time (FT) vs. repeat), age and the frequency of specific medical reasons for deferral.

Results: We observed an increase of the overall deferral/exclusion rate from 6.2 vs. 8.1%, irrespective of: type of collection site (fixed: 6.0 vs. 8.5%; mobile: 6.2 vs. 8.0), sex (females: 7.5 vs. 9.9%; males: 5.1 vs. 6.6%), donor—status (FT: 19.7 vs. 24.7%; repeat donors: 4.6 vs. 6.3%) or age (18–29: 9.1 vs. 11.7%; 60–71 years: 5.1 vs. 6.6%) (p < 0.001 for all respectively). Confidential self—exclusion increased from 0.08 to 0.14% (p < 0.001). Besides risk behaviour, various medical reasons were identified to explain this increase. Conclusions: The new DQ resulted in an increased deferral/exclusion of all donor groups. As FT, young or female donors are overproportionally affected, its possible impact on future blood supply must be considered. Long—term studies and investigation of donor acceptance will be needed.

Abstract Session 11 – Interventional Therapy

OS 11.01

Extracorporeal photochemotherapy in small children with acute and chronic GvHD

Witt V.

St. Anna Kinderspital, Austria, Vienna

Background: ECP is meanwhile a recommended treatment modality in acute and chronic GVHD after BMT. In children data are very rare. Moreover due to the restriction of the available systems to patient weighing more than 40 kg (Therakos system) the feasability in especially small children is not widely shown. Patients and Methods: From 1996 to 2010 35patients, 11 with a.GVHD and 24 with c.GVHD were treated with ECR Patients weighing less than 40 kg were available for the offline system consisting of one leukapheresis on dl and 2 reinfusions on d 1+2 separately (n = 15) after 8MOP and UVA irradiation with the PUVA CombilightTM or MacogenixTM system or in very small children or critical ill children with a MINI ECP (n = 2)consisting of the preparation of MNC from 50 to 100 ml peripheral blood and consecutively the irradiation with UVA after adding 8—MOP, and finally the closed system (UVAR XTS, Therakos Corp., n = 15). Results: In a.GVHD 10/11 patients responded with improvement after 2 or 3 ECP cycles. In median 10 ECP cycles were necessary before stopping the treatment. In c.GVHD 16 patients improved and 8 patients did not improve. In median 27 ECPs were necessary per patient. In chronic GVHD the overall survival was determinated by the response to the ECP treatment There was no difference between the methods used for ECP. In total 712 procedures were performed (227 (acute GVHD 78) apheresis with 454 reinfusions with the offline method, 16 MINI ECP, and 489 closed ECP with the UVAR XTS) (Neither with the offline nor with the closed system any very severe adverse events were registered. Conclusion: We used different methods for ECP in pediatric patients and could demonstrate in acute GVHD an overal repsonse of 90%, in chronic GVHD of 74%. This was not depended on the method of ECP used. ECP could be used safely in pediatric patients with all available methods.

OS 11.02

Successful use of mini buffy coat photopheresis in the treatment of graft—versus host disease and severe heart allograft rejection

Hackstein H.¹, Misterek J.¹, Woessmann W.², Reiter A.³, Bein G.^{4 1}Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany, ²Department of Pediatric Hematology and Oncology, Justus—Liebig—University Giessen, Giessen, Germany, ³Department of Pediatric Hematology and Oncology, Justus—Liebig—University Giessen, Giessen, Germany, ⁴Zentrum für Transfusionsmedizin und Hämotherapie, Universitätsklinikum Giessen und Marburg GmbH, Giessen, Deutschland

Background: Classical extracorporeal photopheresis has proven efficacy in the treatment of graft versus host disease (GVHD) and heart allograft rejection but is limited to patients with sufficient body weight. We have developed a novel simplified mini buffy coat photopheresis technique that allows treatment of small children and patients with apheresis contraindications. We present 15 children after allogeneic stem cell and heart allograft transplantation. Methods: White—blood cell rich buffy coat fractions were prepared from 100–200 ml whole blood in a closed system, diluted and UVA—irradiated after addition of 8—Methoxypsoralen. Results: A total of 429 mini photopheresis treatments (median mini photopheresis treatments per patient n = 21) were performed in 12 patients with steroid—refractory acute or chronic GVHD and in three heart allograft recipients suffering from refractory rejection or steroid—refractory sarcoidosis. The median age of the patients was 6 years (range 1–15 years) and the median body weight 20 kg (range 7–48 kg). Most of the patients with steroid—refractory skin GVHD and all heart allograft recipients responded to mini photopheresis. Mini photopheresis was tolerated well without severe side effects including the heart allograft patients suffering from heart failure. Conclusion: To our knowledge this is the largest patient cohort treated with mini photopheresis to date. Our findings suggest that mini photopheresis is a safe and effective alternative to conventional photopheresis for therapy of GVHD and heart allograft rejection in small patients with apheresis contraindications.

OS 11.03

Isoagglutinin apheresis for ABO—incompatible renal transplantation

Black A.¹, Mack M.², Dullinger K.¹, Schmitz G.¹, Ahrens N.¹

¹Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Regensburg, Regensburg, Deutschland, ²Klinik und Poliklinik für Innere Medizin II, Universitätsklinikum Regensburg, Regensburg, Deutschland

Blood group antigens of the ABO—type are expressed with broad tissue distribution, including endothelial, epithelial and red blood cells. This has traditionally restricted solid organ transplantation to major ABO compatibility. We therefore asked, if this barrier could be overcome by isoagglutinin apheresis before transplantation. In four patients on dialysis with ABO incompatible living related donors, apheresis was performed daily until transplantation with Cobe Spectra and Adasorb using Glycosorb columns. In addition, patients received Rituximab (Anti—CD20) in standard dosage for B cell depletion before transplantation. Isoagglutinin titers were determined at baseline before Rituximab application, before and after apheresis, and after transplantation. The isoagglutinin titers at baseline were 8 to 128 (IgG), and 1 to 32 (IgM). Apheresis resulted in a median reduction of 3 titer steps, and a median of 5 procedures were required prior to transplantation. In all patients titers could be reduced to 4 or less before transplantation, and all grafts could be transplanted successfully with excellent function. Isoagglutinin titers remained low (4 or less) after transplantation under standard immunosuppression. ABO—incom—patible living donor renal transplantation may be facilitated by prior isoagglutinin apheresis.

OS 11.04

Donor— and recipient—derived immunity in ABO incompatible living donor liver transplantation

Lindemann M.¹, Schumann A.², Fiedler M.³, Beckebaum S.⁴, Cicinnati V.R.⁴, Witzke O.⁵, Lenz V.¹, Roggendorf M.³, Horn P.A.¹

¹Institute for Transfusion Medicine, University Hospital Essen, Essen, Hufelandstr. 55, ²Institute for Transfusion Medicine; Institute of Virology, University Hospital Essen, Essen, Germany, ³Institute of Virology, University Hospital Essen, Essen, Germany, ⁴Department of Gastroenterology and Hepatology & Department of General, Visceral and Transplantation Surgery, University Hospital Essen, Essen, Germany, ⁵Department of Nephrology, University Hospital Essen, Essen, Germany

Previously, we have shown that donor—derived T and B cell immunity can be transferred with a liver transplant. We now addressed the question how donor—and recipient—derived immunity is influenced by immunosuppressive treatment of ABO incompatible transplantations in the long—term. We describe a 4—year course of Hepatitis B virus (HBV) immunity, isohemagglutinins, and B cell numbers in an ABO incompatible donor/recipient pair receiving rituximab treatment and immunoadsorption/plasmapheresis. The 48—year old female donor (blood group A) was successfully immunized against HBV; the 49—year old recipient (blood group 0) suffered from multiple liver abscesses and was HBV naive. Whereas cellular HBV immunity was stably transferred from donor to recipient, humoral HBV specific immune transfer was lacking. Starting at month 17 after transplantation, the recipient was vaccinated six times against HBV (months 17–44). Interestingly, anti—HBs did not appear until the sixth vaccination. Antibodies against the isohemagglutinin Al could be reduced prior to transplantation from a titer of 320 to 2 (IgG). Within the first month post transplantation low titers (4–64) were reached by immunoadsorp—tion/ plasmapheresis. From month 6 onwards anti—Al IgG titers remained low (8–16). CD19 and CD20 B cell numbers were below normal levels up to month 26 post transplantation. Due to treatment by rituximab and immunoad—sorption/ plasmapheresis donor—derived B cell immunity against HBV had been completely abrogated and recipient—derived B cell immunity against isohemagglutinins severely impaired. The treatment inhibited de novo synthesis of anti—HBs antibodies until month 44, but not the pre—existing production of isohemagglutinins.

∗Funded by the DFG (A—III, KFO 117)

OS 11.05

Influence of immunoadsorption with Protein A (PA—IA) on vaccination status

Rummler S., Beier J., Ban. D.

Institut für Transfusionsmedizin Universitätklinikum der Friedrich Schiller Universität Jena, Jena, Deutschland

Background: PA—IA is an extracorporeal, immunosuppressive procedure for elimination of alio— and autoantibodies in patients’ blood. Although, in addition to eliminating immunopathologic antibodies, specifically acquired antibodies are also removed. Material and Methods: We examined 20 patients, 8 male and 12 female between 26 and 65 years of age before, right after resp. in follow up examination after PA—IA with respect to immunity to Pertussis, Tetanus and Diphtheria antitoxins. 9 of 20 patients [cohort 1] (1) received PA—IA because of kidney rejection, 2 patients with antibody—complicated pregnancy [cohort 2] (2) and 9 patients with progressive autoimmune diseases (2). Results: Pertussis Leerzeichen 4 patients of (1) and 2 patients of (2) showed long time immunity before PA—IA changing to no vaccination protection after treatment. IgG serum concentration was reduced to 16% (1) and 28% (2) of baseline level per PA—IA. By the time of follow up examination IgG recoveries were 66% (1), 99% (2) compared to baseline values. Diphtheria: 3 of 9 patients of (1) showed antitoxin >0.1 IU/ml before PA—IA. Regarding (2) 8 of 11 patients displayed immunisation protection. After PA—IA none of the patients of (1) and (2) presented protecting antitoxin titre >0.1 IU/ml. Antitoxin concentration was reduced to 9% (1) and 14% (2) of baseline level per PA—IA. By the time of follow up examination antitoxin recoveries were 36% (1), 86% (2) compared to baseline values. Tetanus: 7 of 9 patients of (1) displayed antitoxin >0.11 IU/ml before PA—IA, reducing to 4 patients after. All patients of (2) were sufficiently vaccinated with >0.11 IU antitoxin/ml before PA—IA, leaving only 5 of 11 patients after PA—IA. At follow up examination 18 of all patients showed immunisation protection with antitoxin recoveries of 71% (1), 62% (2) compared to baseline values. Conclusion: Patients with immunosuppression undergoing PA—IA lose vaccination protection status temporarily and should therefore be informed.

OS 11.06

Dependency of the collection efficiency of CD14+ monocytes to gender and pre—treatments of the patients with malignant melanoma during leukapheresis prior to DC—vaccination

Strasser E.F.¹, Born L.¹, Erdmann M.², Weiss D.R.¹, Zimmermann R.¹, Eckstein R.¹

¹Transfusionsmedizinische und Hämostaseologische Abteilung, Universitätsklinikum Erlangen, Erlangen, Deutschland, ²Department of Dermatology, Erlangen, Germany

Background: This study analyses product quality and specific characteristics of patients with advanced malignant melanoma due to the production of Mo—DCs. Methods: Blood samples were taken from the patients before and after each leukapheresis and from the product. Blood cell counts were analysed by a blood cell counter and by flow cytometer. For leukapheresis the COBE Spectra machine (Caridian BCT, Denver Colorado) was used. Results: 116 patients with malignant melanoma, 48 women and 68 men underwent up to seven leukaphereses, all in all 249 procedures. The average age was 53 years (range, 16 to 80 years). 105 patients had a melanoma stage IV and 11 patients stage III. The monocyte yield of products was 2,75+/—1,31 ∗10e9 cells (range, 0,73 to 8,61 ∗10e9 cells), whereas the monocyte yield in men was higher (2,95+/—1,39 ∗10e9 cells) compared to women (2,49+/—1,16 ∗10e9 cells) (p = 0,004). The collection rate of monocytes in men (1,07+/—0,47 ∗10e7 cells/ min.) differed significantly (p = 0,004) from women (0,92+/—0,39 ∗10e7 cells/ min.). The collection efficiency was on average 84,2+/—43,9%. The collection efficiency was significantly lower in men with side effects compared to male patients without problems during apheresis (79+/—45,3% vs. 92,6+/—48,l%, p = 0,037). Good correlations were found between monocyte counts in blood of patients prior to apheresis and the monocyte concentration of products, in men (r = 0,604, p < 0,001) and women (r = 0,545, p < 0,001). Male patients, who had a chemotherapy six month before apheresis procedure, had significant higher collection rates (male: 1,21+/—0,51 ∗10e7 Zellen/Min, n = 50) than the patients without treatment (male: 0,99+/—0,43, n = 88). Conclusion: Leukapheresis, the gold standard for the collections of MNCs to produce Mo—DC—vaccines depend on the gender, side effects during apheresis and pre—treatments of the patients due to the collection rate and the collection efficiency of monocytes.

OS 11.07

Hereditary hemochromatosis patients/carriers as blood donors – a single center experience

Radojska S., Hengeöz Ö., Gathof B.

Department of Transfusionmedicine, University of Cologne, Cologne, Germany

Background: Hemochromatosis is one of the most common genetic diseases in white persons of western European descent. The disorder is characterized by an increase in iron absorption from the gastrointestinal tract, which leads to an accumulation of iron in many organs. The preferred therapy of iron overload in hemochromatosis patients/carriers is regular, if clinically possible weekly, phlebotomy, until the serum ferritin level decreases to low normal range (≈ 50 μl/l). This preventive/therapeutic ferritin level can be maintained in hemochromatrosis patients/carriers by 3 to 4 phlebotomies per year. These patients/carriers can become voluntary blood donors, if there are no medical contraindications. European countries have varying regulations for the use of blood of Hemochromatosis patients: in some countries the use is explicitly forbidden. In Germany like in the US and Canada, regulations permit donations. Admission to donation may depend on each blood service. Methods: Evaluation of phlebotomies and blood donations of hemochromatosis patients/carriers (n = 11) at our blood center. Results: The mean age of hemochromatosis probands was 47±17 (range 21 to 81 and 91% were men. 54% were blood donors since their first contact with us. 45% had first therapeutic phlebotomy and after iron reduction they became regular blood donors. Figure 1 shows the long time follow up of our oldest hemochromatosis patient and effectiveness of phlebotomy/blood donation. Hemochromatosis carriers/patients donated 3 to 4 times a year, twice as much as the average other blood donors. Conclusions: Our experience suggests that if there are no medical contraindications hemochromatosis patients are a potential resource as regular and motivated blood donors. This option may help to compensate for donor losses reported by many blood centers.

Figure for Abstract OS 11.07

OS 11.08

Comparative evaluation of a heparin—citrate—anticoagulation for LDL—apheresis with the primary apheresis systems COBE Spectra and Spectra Optia

Handschel D.

DHZ, Deutsches Hämapherese Zentrum gGmbH, Köln, Deutschland

For decades, we have successfully conducted LDL—apheresis procedures using the COBE^® Spectra system for primary plasma separation in conjunction with the secondary systems ADA/ADA Sorb using a mixed heparin/citrate anticoagulation protocol. With the introduction of the new Spectra Optia^® apheresis system a re—evaluation of our established mixed anticoag. protocol became opportune. In a pilot study,8 patients of whom five were medicated with 100 mg/day ASS, underwent 2 treatments with Spectra using an inlet blood to AC (I:A) ratio of 25:l,and 2 treatments with Optia using LA split ratios of 16:1/18:1 or 20:1/25:1 per treatment. In a follow—up study, 9 patients underwent 20 Optia procedures with an LA ratio of 20:1. The primary endpoint was the highest LA ratio that assured efficient anticoag. in a particular procedure and patient. The anticoag. state was recorded by various coagulation parameters(platelet counts, P—selectin, INR, aPTT). Max. platelet activation was measured in the return line 5 minutes after heparin bolus independent of LA ratios. In procedures for ASS(+) patients the average increase of P—selectin is similar in Spectra(to 26.8%) and in Optia 16/18:1 (to 29%) runs and higher for Optia 20/25:1 (to 42%). Towards the end of procedure, P—selectin returned to almost starting levels. Anticoagulation was sufficient throughout each procedure (therap. values for INR and aPTT). In ASS(+) patients the average platelet loss measured as drop in circulating platelets was similar in all Spectra and Optia runs. In ASS(—) patients platelet loss varied more in Optia runs independent of the LA ratio with an average of 0%—ll%. Transient platelet—microaggregates were observed in six Optia runs (by Optia run data file images) with unstable blood flow rates at LA ratio higher than 20:1. Apheresis procedures on the Optia system with low—dose heparin—citrate anticoagulation are feasible and safe. At LA ratios of 20:1 and below, anticoagulation is sufficient and in spite of increased platelet activation platelet aggregation is low.

Abstract Session 12 – Immunohematology

OS 12.01

Leukocyte—associated antibodies in exposed and non—exposed female blood donors

Nguyen D.¹, Lauber S.¹, Schulz—Linkholt M.¹, Dengler T.², Klueter H.¹

¹Institut of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Mannheim, Germany, ²Institute of Transfusion Medicine, Baden—Baden, German Red—Cross Blood Service of Baden—Württemberg – Hessen, Baden—Baden, Germany

Background: Leukocyte—associated antibodies may cause severe pulmonary transfusion reactions (TRALI). As transfusion of plasma from female donors with history of pregnancy might be associated with an increased TRALI risk, they are excluded from plasma donation. Thus, the use of Fresh Frozen Plasma from male and never pregnant female is the standard. Here, we determine the frequency of granulocyte antibodies in exposed and non—exposed female whole—blood donors using an automated high—throughput flow—cytometric granulocyte—immunofluorescence test (Flow—GIFT). Methods: We investigated 6977 female whole blood donors (60.4% reported previous pregnancies). Leucocytes from two HNA—typed donors were used as test cells. For testing, sera were incubated with isolated test cells in 96—deep well plates. Antibody binding was detected using FITC—conjugated antibodies and analysed by Flow—GIFT. All pipetting steps were automated using Biomek NXp Workstation. For the detection of HLA—antibodies the ELISA assay was used. Results: In 924 (21.9%) of 4212 exposed females, granulocyte antibodies (n = 62; 1.5%), HLA class I and/or class II (n = 862; 20.5%) antibodies were detected. In 96 (3.5%) of 2765 non—exposed females, antibodies against granulocyte—antigens (n = 13; 0.47%), HLA class I and/or class II (n = 83; 3.0%) were found. To investigate the possible decay of the respective antibodies, each of the donor groups was split into four age groups: < 35/35–44/45–55 and >55 years. The presence of detectable antibodies in exposed women declined with advancing age (p < 0.0001). This was not seen in non—exposed women (p > 0.05). Conclusion: The automated high—throughput Flow—GIFT allows effective anti—granulocyte antibody detection in a large donor population to prevent TRALI due to transfusion of blood products. The presence of antibodies in exposed females declines with age. Thus, testing of exposed female donors or the re—entry of exposed females for plasma donation should be considered.

OS 12.02

Comparative leukocyte antibody testing in fluorescence conjugated bead assays and established techniques

Reil A.¹, Flesch B.², Bux J.¹

¹DRK—Blutspendedienst West, Hagen, Deutschland, ²DRK—Blutspendedienst West, Bad Kreuznach, Deutschland

Background: Detection of HNA and HLA antibodies is an important tool in TRALI diagnostics. Established and validated techniques now are supplemented by fluorescence conjugated bead—based assays. Methods: 68 sera with HNA antibodies previously confirmed by the granulocyte agglutination test (GAT), indirect granulocyte immunofluorescence test (GIFT) or the monoclonal antibody immobilization of granulocyte antigens (MAIGA) were tested by the LabScreen Multi assay (BMT OneLambda) for the presence of antibodies to HNA—1a, —1b, —1c, —2, —3a, —3b, —4a and HLA class I and II. Twelve sera were additionally tested in the HLA class I Single Antigen assay (LSA, Tepnel Lifecodes) and results were compared to those obtained by the indirect lymphocyte immunofluorescence test (LIFT) and complement—dependent cytotoxicity (CDC). Results: HNA—2 antibodies were reliably detectable with a cut—off value of 5.0 NBG (negative background ratio) while HNA—1a and —1b IgG antibodies were detected at a cut—off of 3.0 with a sensitivity and specificity between 88 and 100%. Only 10 out of 29 HNA—3a sera proved to be positive (>3.0 NBG) in the LabScreen Multi assay, but additional 6 sera were false positive. Testing 7 HNA—3b antibodies resulted in 39% sensitivity. 62 of 68 sera were positive for HLA class I in the LabScreen Multi assay at a cut—off of 1.5 NBG and even 22 remained positive at a cut—off of 3.0. Twelve of these (NBG 1.7 to 76.2) were additionally tested in the LSA HLA class I assay, LIFT and CDC and reported concordant positive results for 8 sera in each assay. Three sera were reactive only in LIFT and LSA and one only in LSA but the total number of observed specificities was far higher in LSA assay. Conclusions: The LabScreen Multi assay produces reliable results for HNA—2—anti—bodies, while the detection of HNA—1 and HNA—3 antibodies requires improvement. The clinical significance of numerous HLA specificities detected in the LabScreen and LSA at low detection levels remains open.

OS 12.03

Implication of transfected cell lines for the detection of HNA—3 alloantibodies

Bayat B.¹, Tjahjono Y.¹, Werth S.¹, Reil A.², Kroll H.³, Sanioso S.¹

¹Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany, ²DRK—Blutspendedienst West, Deutschland, Hagen, ³German Red Cross Blood Transfusion SAervice NSTOB, Dessau, Germany

Antibodies against human neutrophil antigen—3 in blood products are responsible for the fatal reaction of transfusion related acute lung injury (TRALI). Consequently, reliable detection of anti—HNA—3 alloantibodies is mandatory to improve blood transfusion safety. Recently, synthetic peptides have been introduced for the detection of HNA—3a antibodies in ELISA. The results in regards to sensitivity and specificity, however, are not satisfactory. In this study, we developed transfected cell lines expressing HNA—3 for the detection of HNA—3 antibodies. Mammalian cell lines (HEK) were transfected with HNA—3a or HNA—3b constructs and sorted by flow cytometry according to high and homogenous surface expression. Cell lines were tested with sera containing HNA—3 antibodies in flow cytometry. The results were compared with current serological methods. In flow cytometry, 11/14 HNA—3a sera reacted specifically with HNA—3aa cells, but not with HNA—3bb cells. Two HNA—3a sera showed moderate reaction. One serum reacted with both HNA—3aa and HNA—3bb cells. All HNA—3b sera recognized HNA—3bb cells. No reaction was observed with broad reactive antibodies against HLA class I and II. Serial testing of transfected cells over period of 3 months showed stable expression of HNA—3a on the cell surface. In addition, we found by genotyp—ing of 249 blood donors, a new HNA—3 allele (HNA—3a∗) caused by a nucleo—tide substitution C > T at position 457 leading to L153F mutation. This mutation may impair PCR—SSP based genotyping of HNA—3a and HNA—3b. Analysis with transfected HEK cells expressing HNA—3a∗, showed that LI 53F mutation did not alter the binding of HNA—3 antibodies. This study demonstrated that HEK cells expressing stable recombinant HNA—3 are suitable for the detection of HNA—3 alloantibodies in flow cytometry allowing reliable screening of blood products. In addition, such cells are useful for accurate analysis of HNA—3 antibody binding.

OS 12.04

Molecular and serologie characterization of a HNA—3a variant

Grabowski C.¹, Borowsky A.¹, Neumann K.², Jorks S.¹, Winterstein K.¹, Hoyer P.¹ Reil A.³, Santoso S.⁴, Kroll H.¹

¹Red Cross Blood Transfusion Service NSTOB, Institute for Transfusion Medicine Dessau, Dessau, ²Institute of Pathology, City Hospital Dessau, Dessau, Germany, ³Red Cross Blood Transfusion Service West, Centre for Transfusion Medicine Hagen, Hagen, Germany, ⁴University Clinics Giessen and Marburg, Centre for Transfusion Medicine and Hemotherapy, Giessen, Germany

Background: Granulocyte—reactive alloantibodies in blood products, in particular against antigens of the HNA—3 and HNA—1 systems, can cause transfusion—related acute lung injury (TRALI). Recently, the molecular background of the HNA—3 alloantigen system was elucidated. In this study, we analyzed the gene frequencies and the sérologie reaction patterns of the HNA—3a and —3b antigens as well as a new variant of the HNA—3a allele, characterized by a single nucleotide substitution 4bp upstream of the HNA—3 polymorphism. Methods: A total of 345 unrelated healthy blood donors were included into the study. DNA was extracted from EDTA—anticoagulated blood using chemagic

Magnetic Separation Module I (Chemagen AG, Baesweiler, Germany) or GenoM6 workstation (GenoVision Vienna, Austria). Genotyping was performed by a newly developed PCR—SSP. For primer design, the additional polymorphism of the HNA—3a variant allele (HNA—3a∗) was taken into account. For sérologie analysis granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) were used. Results: Genotyping resulted in the following gene frequencies: HNA—3a: 220/230 (95.7%), HNA—3b: 77/230 (33.5%), HNA—3a∗C: 248/248 (100%), HNA—3a∗T: 1/345 (0.3%). Serologie HNA—3a and —3b typing was performed in 178 donors. The serology matched completely with molecular testing. Granulocytes carrying the HNA—3a∗T variant were extensively analyzed with ten well defined HNA—3a anti—sera. Two of the HNA—3a antibodies did not react with the HNA—3a variant in GAT whereas no differences in the reaction patterns were detectable in GIFT (microscopic or flow cytometry). Conclusions: Amissense mutation close to the HNA—3a polymorphism was shown to interfere with the binding of selected HNA—3a antibodies. Genotyping of the HNA—3 alleles can be hampered by the mutation and may lead to false negative allele assignment. This can be of special impact if TRALI prevention is based on the exclusion of HNA—3a negative blood donors.

OS 12.05

Neutrophil activation by HNA—2a antibodies mediated by Mac—1 integrin

Bayat B.¹, Jerke U.², Ditimar G.³, Sanioso S.¹, Kettritz R.²

¹Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany, ²Medical Faculty of the Charité', Experimental and Clinical Research Center, Berlin, Germany ³Max—Delbruck—Center for Molecular Medicine, Berlin, Germany

Antibodies against human specific neutrophil antigen—2a (HNA—2a; also known as NB1) play a role in immune mediated neutropenia and transfusion—related acute lung injury (TRALI). In our previous studies, we found that exposure with anti—NB 1 antibodies can cause neutrophil activation and super—oxide production depending on the NB1 expression. Since NB1 is a GPI—linked protein lacking a cytoplasmic domain, signal transduction could not be directly ascribed by this molecule. In this study, we searched for integral membrane protein on neutrophils, which may act as NB1 co—receptor for the transduction of outside—in signalling. For this study, digitonin lysates of intact neutrophil plasma membrane fractions were immunoprecipitated by anti—NB 1 and analysed by tandem mass spectrometry analysis (MS/MS). A major protein, Mac—1 integrin, which forms a complex with NB1 protein, could be identified by this approach. This result was confirmed by immunoblotting analysis using a panel of mabs against NB1 and Mac—1 integrin as well as by real—time analysis of protein—protein interaction using surface plasmon resonance technology (SPR). Additionally, confocal microscopy showed strong HNA—2a co—localization with Mac—1 on neutrophil surface. Moreover, NB1 stably transfected cells bound significantly to Mac—1 protein coated surface in compared to non—transfected cells, and this interaction could be inhibited by mabs against NB1 as well as antibodies specific for Mac—1 integrin. Furthermore, the involvement of NBl/Mac—1 complex in signalling process could be identified by the presence of both molecules in lipid raft fraction. Inhibition studies using selected mab against Mac—1 blocked HNA—2a positive neutrophil activation and Superoxide production induced by HNA—2a antibodies. This study demonstrated for the first time the mechanism underlying NB 1 —antibody mediated activation in neutrophils, which may add our understanding in the pathomechanism of TRALI.

OS 12.06

Rapid molecular blood group prediction allowing “point of serology” molecular testing

Wagner F.F.¹, Doescher A.², Bittner R.¹, Mueller T.H.¹

¹DRK—Blutspendedienst NSTOB Institut Springe, Springe, Deutschland, ²German Red Cross Blood Transfusion Service N.S.T.O.B., Institute Bremen—Oldenburg, Oldenburg, Germany

Background: Current molecular methods for blood group antigen prediction involve a delay of several hours until the results are available, which limits their value in cases of urgent blood need. We wanted to develop a rapid typing method that yields the result within 40 min from the decision to type. Methods: A LightCycler DNA typing method with direct amplification from plasma and ultra—rapid cycling was developed based on the LightCycler typing method of Polin H et al1. Major modifications were an accelerated cycling scheme and the use of Finnzymes Phusion BloodDirect PCR mix to eliminate DNA isolation. Multiplex detection was accomplished using LC 705 for FY. One μl of serum or plasma was added to 9 μl of PCR mix in a light cycler capillary. Melting curve analysis was performed between 45 to 80°C at 0.1°C/s. Results were evaluated by two investigators. One hundred blood donor samples were typed for Fy and Jk in multiplex and compared with capillary electrophoresis and serology. Results from serum and plasma were compared in 20 samples. Results: Total analysis time from the first pipetting step to the result was 37 min. Among 100 samples, 99 were predicted correctly. The Jk(a) antigen of a single Jk(a+b+)Fy(a+b+) sample was missed in 1 of 5 repeats. Frozen pre—pipetted capillaries were successfully used up to 67 days after freezing. Conclusion: Rapid LightCycler PCR directly from plasma or serum allows the molecular prediction of several transfusion relevant antigens within 40 min from the decision to type and is therefore more than twice as fast as “conventional” LightCycler PCR using isolated DNA. The new technology involves only 1 pipetting step, takes not much longer than serologic antigen determination and therefore has the potential to leave specialized molecular laboratories and complement serology as “point of serology” molecular typing.

OS 12.07

Allele specific PCR and microbead based genotyping for the Wright blood group alleles DI3 and DI4

Strathmann K., Nzinga K., Hägerbäumer M., Stenzel A., Bringmann G., Zeiler T.

German Red Cross Blood Service West, Center of Transfusion Medicine Breitscheid, Ratingen, Germany

Background: We developed an allele—specific PCR and a microbead—based genotyping test for the Wright blood group antigens Wr^a and Wr^b wich are determined at the DI3/DI4 alleles of the Diego blood group system. The alleles are differentiated by a single nucleotide polymorphism A/G at bp 1972 in exon 16 at chromosome 17. The bead based test was developed in addition to an inhouse multiplex application for the typing of 18 blood group alleles based on the data of Karparsitou K et al. Methods: The allele—specific primers and the DI3 and DI4 probes are designed with open source “primer3” software. The detection of allele—specific PCR products has been done by sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS—PAGE). The primer for the microbead test based on the data of Huang CH et al. The detection of genotypes with the microbead—based method works in a flow cytometry method with the direct hybridisation of PCR products of target sequences to allele—specific oligonucleotides coupled with colour—coded microbeads. Results: The specific PCR products in the allele—specific PCR of DI3 and DI4 have 338 bp. For cut—off determination of the bead based test we used 50 blood samples typed for Wr^a and Wr^b. The allelic ratio for the bead based test was calculated as MFIa/MFIa+MFIb for each sample. The cut—off ranges are: DI3/ DI4 0,6989 − 0,7728 and for DI4/DI4 lower than 0,2622. To accept test results allelic ratio had to be within the cut—off range. Both tests were validated with additional 25 samples. Conclusion: The DI3/DI4 allele—specific PCR is a good tool for single sample test for patients with warm autoimmune hemolytic anemia and additional anti—Wra antibody to determine the DI3/DI4 genotype. On the other hand the microbead based genotyping test for the alleles DI3 and DI4 is well suited for routine use as addition for a multiplex application. The test has a great potential for patient and blood donor screening, especially for determination of rare blood groups.

OS 12.08

A set of 52 single nucleotide polymorphisms as individual internal controls in prenatal blood group typing

Doescher A.¹, Wagner F.F.²

¹German Red Cross Blood Transfusion Service N.S.T.O.B., Institute Bremen—Oldenburg, Oldenburg, Deutschland, ²German Red Cross Blood Transfusion Service N.S.T.O.B., Institute Springe, Springe, Germany

Background: Determination of fetal blood groups in plasma of pregnant women is increasingly utilized as an additional diagnostic method. However, test interpretation can fail, if insufficient amplification of fetal DNA occurs (a negative test result is expected, if the fetus is negative but may also occur if the amplification of the fetal DNA fails). To discriminate fetal and maternal DNA we evaluated the use of 52 SNPs as individual internal positive controls in our PCR settings. Methods: DNA from 224 plasma samples was screened for 52 SNPs divided into four primer pools in a multiplex PCR setting. Detection of amplicons was done using SBE (single base extension) and the GeneS—can Method in an ABI310. SNP frequencies were calculated and compared to ALFRED (allele frequency database). A ranking of the most informative SNPs calculated from 224 tested samples were done and modified primer pools were generated for use in prenatal typing of RH, KEL and HPA. Results: 97.8% of all samples showed differences in maternal and fetal SNP patterns when tested with four primer pools, 2.2% lacked these differences because of identical genotype or due to failure of fetal DNA extraction. In each primer pool a set of about 5–6 SNPs with frequently observed differences between maternal and fetal DNA were detected and rearranged in three new pools. The first set of the new SNP combination (15 SNP) was usuable as internal control in 59% of samples (original set 39%), the addition of a second set of 15 SNPs increases the positive results to 76%. Co—amplification with sequences for RHD, RHc, RHE and HPA 1 was successful, the other systems are still in progress. Conclusion: Negative results in fetal blood group determination can be confirmed with SNPs as a positive control for the presence of fetal DNA, even if a paternal blood sample is not available. Detection of amplicons with single base extension gives also the opportunity to include several blood group specific sequences.

Session 01 – Blood Donation

P1.01

Evaluation of a new in—line leukocyte reduction filter system for red blood cells

Moog R.¹, Mönninghoff J.²

¹Hospital Laboratory Network Brandenburg—Berlin, Bernau, Ladeburger Street 17, ²University Clinics Essen, Essen, Germany

Background: White blood cells (WBC) may cause both immunogenic and infectious adverse events in the transfusion recipient. To avoid these side effects universal WBC reduction was introduced in many countries. Currently flexible soft shell leukocyte reduction filters made of polymeric materials such as polyester or polyamide are used. New filter innovations have to prove their effectiveness and quality under routine conditions. Materials and Methods: The flexible in—line red blood cell filtration system Leucoflex LCR Diamond (MacoPharma, Tourcoing, France) was evaluated. The system has a smaller volume than the previous filter LCR—5 and a rhomboid design. The filter tube connections are not at the level of the centre edge, but at two opposite corners. 25 RBC concentrates were produced of which 7 were irradiated with 30 Gy. The packed RBCs were produced under Good Manufacturing Practice conditions. To ensure the quality of the filter system every 7 days metabolic parameters such as WBC count, haemoglobin level, haemolysis rate, potassium, pH and ATP were analysed. The parameters were measured for a storage period of 49 days for non—irradiated packed RBCs and 35 days for irradiated RBCs. Results: The average product volume was 260 mL after filtration. Mean WBC contamination was 0.02 × 10⁶/unit and average haemoglobin content was 51.8 g/unit. Haemolysis rate was 0.05% directly after filtration and 0.35% at the end of the storage time period for irradiated RBCs and 0.20% for non—irradiated RBCs, resp. Mean potassium level was 1.3 mmol/L and the pH was 7.37 after filtration. ATP level remained over 2.0 μmol/g Hb for the whole storage time. Conclusion: Effective WBC reduction was observed with the new inline leukocyte reduction filter system. All quality control parameters met national and international guidelines. Packed RBCs also showed good quality of metabolic parameters within the target range over the whole storage period.

P1.02

Routine determination of reticulocyte hemoglobin content in non—anemic whole blood donors to detect impaired hemoglobinisation caused by functional iron deficiency

Semmelrock M.J.¹, Raggam R.B.², Semmelrock H.J.², Prüller F.², Amrein K.³, Schallmoser K.¹, Lanzer G.¹, Rohde E.⁴

¹Department of Blood Group Serology and Transfusion Medicine, Medical University, Graz, Austria, ²Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University, Graz, Austria, ³Department of Internal Medicine, Division of Endocrinology and Metabolism, Medical University, Graz, Austria, ⁴Department of Blood Group Serology and Transfusion Medicine, Paracelsus Medical University, Salzburg, Austria

Background: Frequent blood donations may lead to depletion of body iron stores resulting in development of anemia. In Austria, donor screening is done by determination of hemoglobin concentration (Hb) prior to each donation, but Hb measurement has limitations due to the late response in case of a present iron deficiency (ID). Several methods to detect ID are available but lack practicability. Using the ‘Thomas’ Plot', we evaluated the association of reticulocyte hemoglobin content (CHr) – a marker for impaired hemoglobinisation (IH) – with iron status in regular whole blood donors. Methods: In a prospective study, 918 frequent and 689 first—time or reactivated whole blood donors were included after written informed consent; 35.3% of the study participants were female. The iron status including serum ferritin and soluble transferrin receptor (sTfR) was determined and the ferritin index (sTfR—F), representing the most relevant marker for ID, was calculated as the ratio of sTfR to the logarithm of ferritin. Red blood cell count including CHr was measured. Results: ID is defined as being present at a sTfR—F ratio above 1.5 and IH is defined as being present at a CHr value below 28.0 pg. Among regular whole blood donors, 8.9% of male and 23.9% of female subjects were diagnosed as being iron deficient, while only 4.2% of male and 11.8% of female donors had signs of a present IH. In 72.1% of cases IH was associated with ID, while only 1.84% of subjects without ID had a CHr below 28.0 pg (specificity 98.16%). Conclusion: There is a high degree of individuality for iron stores and measurement of iron status is expensive, but routine CHr determination in blood donors seems to be a sensitive and specific tool to detect IH which is mostly associated with verifiable functional ID. The results allow for a decision whether iron substitution has to be recommended. Testing of CHr in frequent blood donors can probably prevent development of ID based anemia at a very early stage.

P1.03

Evaluation of low red blood cell mean corpuscular volume is useful in the detection of iron deficiency in a whole blood donor population

Just I.—M., Süßner S., Schaumberger R., Renke C., Gabriel C. Red Cross Transfusion Service for Upper Austria, Linz, Austria

Background: Regular blood donation can lead to iron deficiency. Low red blood cell mean corpuscular volume (MCV) in the presence of normal haemoglobin can indicate iron deficiency. Unlike low haemoglobin level MCV is not a deferral criteria for blood donation. We assessed the correlation between microcytosis and iron deficiency to detect donors with incipient iron deficiency in time. Patients and Methods: Over a period of one year 59651 predonation samples were evaluated by complete blood count (CBC). Samples with a MCV equal or less than 80 fl were further investigated (serum iron, ferritin, transferrin and percentage of transferrin saturation). Iron deficiency was defined as a ferritin level below 20 μg/L in males and 15 μg/L in females or a transferrin saturation level of less than 15%. Results: 1028 (1,72%) donors had low MCV levels. Within this group 55,06% presented with iron deficiency compared to 19,34% in 946 donors with normal MCV values. In the iron—deficient group mean MCV values were 77,9 fl. Conclusions: The results show that MCV is useful in the detection of iron deficiency. Therefore in regular donors low MCV values should be investigated to advise iron replacement therapy or to reduce the frequency of donations if indicated.

P1.04

Analysis of donor adverse reactions after whole blood donation reported in the years 2005 to 2010

Koppen A., Nowak K., Alt T., Bux J. DRK—Blutspendedienst West, Hagen, Deutschland

Background: Aspects of safety in blood transfusion services are no more only focused on prevention from transmission of infectious diseases or adverse transfusion events. Safety of the donor is essential part of the hemovigilance system. Monitoring, analyzing and researching the risks during and after donation will help to understand donor's behaviour. Donors feeling good treated in the rare case of an adverse event will probably return as active blood donor. The quality assurance department of the DRK—Blutspendedienst West gather and evaluates information and trends related to donor reactions since 2005. Methods: Retrospective analysis of the adverse reactions during whole blood donation in the years 2005–2010. Standardized protocols for documentation of the complications were used. Data were transferred into the blood bank software and evaluated by specific reports and statistics. Results: In the reviewed period 5.7 million venepunctures for whole blood collection from voluntary non—remunerated donors were performed. 15,491 donors sustain an adverse event. The overall rate of complications is at a low level (≈0.27%). 80% suffered from vasovagal reactions, respectively 5% from convulsions and hematomas. Differences are observed by first—time—/repeat—donors, gender and age. When evaluating the return rates of donors with adverse events in the years 2007–2010, we could follow up 13,810 donors until the end of 2010. 4,622 (33%) of them returned to blood donation. Return rates can be estimated three years after occurrence of the complication. We observed the highest return rates within three years after the adverse event in the group of 60–68 year—old donors (60%) and the lowest rates in the group of 20–29 year old donors (35%). Conclusion: The large number of donations and observations during blood donation, which are systematically analyzed, allow very reliable descriptions of our donor population and is an opportunity to extent our knowledge about donor reactions.

P 1.05

Save transport of red blood cell concentrates via a pneumatic tube system

Hecker J.¹, Burkart l.¹, Faustmann J.¹, Halm—Heinrich l.¹, Parkner A.¹, Kropf S.², Heim M.U:¹

¹Institute of Transfusion Medicine, Magdeburg, Germany,

²Institute of Biometrics, Magdeburg, Germany

Background: For a fast transport of red blood cell concentrates (RCC) an integrated pneumatic tube system is necessary. To find out wether a RCC can be delivered by such a tube system without being destroyed and getting unusable we measured haemolysis. Methods: We tested RCCs of different ages (3–5 days, 14 days, 35 days) and blood groups (0, A, B, AB) from healthy blood donors. Overall we sent 54 units across about 500 meters from our institute to the emergency room. The RCCs were sent back and forth 3 times via a “normal” pipeline in order to simulate routine use with a mean sending time of about 7 minutes (one way). The tube containers traveled within the tubes with an average speed of 7m/sec. Before and after the transport with the pneumatic tube system we drew blood samples of the concentrates. From each sample we analysed a blood count, measured potassium, LDH and extracellular haemoglobin to find out haemolysis rate and index. Moreover, we measured temperature within the containers filled with a cool RCC while being sent there and back. Results: In our studies we found that no RCC was damaged. The samples taken before transport had significantly lower rates of haemolysis than those taken after transport. Also depending on the age of RCCs the parameters of haemolysis except for potassium increased significantly from younger to older concentrates. But all collected data of haemolysis in the RCCs were considerably lower than those approved by law as a maximum limit. A mean temperature of about 10 degrees Celsius could be measured within the containers during transport. Conclusion: RCCs can be delivered by a pneumatic tube system safely and fast without being afraid of losing stability in shape, quality or usefulness. Nevertheness, a validation of each newly installed tube system is still mandatory.

P1.06

Does frequent plateletpheresis donation influence donor platelet count?

Schmidt V.¹, Starck R.¹ Alt T.², Markovic A.¹

¹DRK—Blutspendedienst Rheinland—Pfalz und Saarland, Bad Kreuznach, Deutschland, ²DRK—Blutspendedienst West, Hagen, Deutschland

Background: A retrospective study was performed to evaluate the effects of long—term regular plateletpheresis donation on donor platelet counts. Even after multiple donations over an extended period, the platelets from a donor should not exceed or fall below a pathological value. Methods: 144 high—frequency donors, who donated a minimum of 20 times from January 1, 2000 through January 31, 2011, were chosen. Donor pre apheresis platelet counts were collected and the average rate of change in donor platelet count for each group was calculated by regression analysis. Donors were grouped based on their number of donations in the observed period (20–10, 41–80, 81–120 and >120) (table). Results: The mean change in platelet counts clustered around an increment of 15.98 × 10³/μ1 in the observed period, which is a clinically insignificant change. 12.59 percent of donors platelet count increased more than 2 standard deviations from their respective group mean. Correlations were noticeable in each group, but they are all weak (r is about 0.2). No comparable control data were available. Conclusion: There was a consistent relation between the changes in platelet counts and the number of products donated in the observed period. A small number of donors may be predisposed to progressive increases in platelet count with donation, but the underlying variables responsible for the changes and long—term clinical significance in this group of donors is not well—known. This is similar to those reported in earlier studies by Richa and coworkers.

Donation Frequency Category	Number of Donors	Mean Change in Platelet Count × 103/μl	Median Change in Platelet Count × 103/μl
20–40	50	+8.60	+8.5
41–80	50	+14.40	+12.5
81–120	35	+23.80∗	+14.0
> 120	9	+35.30	+36.0
Overall	144	+15.98∗∗	+14.0

^∗p < 0.05

^∗∗p < 0.01

P1.07

Consecutive triple platelet apheresis donations have no impact on serum Thrombopoietin (TPO) levels in donors

Gross J., Stetzenbach M., Kekukh E., Eichler H.

Institut für Klinische Hämostaseologie und Transfusionsmedizin, Universität des Saarlandes, Homburg, Deutschland

Background: Compared to double apheresis platelet donations (DAD), triple donations (TAD) might further enhance cost effectiveness in blood services. Because a possible impact of long—term TAD on the hematopoietic system could be crucial regarding donor's safety, a prospective study was performed to compare serum TPO levels and other standard safety parameters in donors for consecutive DAD and TAD procedures. Methods: Two groups of 13 donors with mean pit levels of 267 and 323/nl were recruited for the DAD and TAD cohorts, and each of six consecutive collections were performed with the Trima Accel™ device with an interval of 16 and 23 days and a target dose of 5 and 8×10ell pits, respectively. Pre—donation serum TPO levels were measured by Quantikine™ sandwich ELISA (R&D Systems). Results: Prior to the 156 donations of the 26 donors, we measured mean serum TPO levels of 37.7 ± 29.2 pg/ml with significant inter—donor variability. Neither within the DAD/TAD cohorts nor between the cohorts nor for a single donor, significant changes in TPO levels could be observed during the study. All procedures were well tolerated and no major side effects were observed. Conclusion: Both DAD and TAD have no impact on donor's TPO levels over the course of at least six consecutive donations.

P1.08

Impact of immediate or delayed hold time prior to platelet additive solution addition on the in—vitro quality of single donor platelets

Ringwald J.¹, Tully S.¹, Geier C.¹, Weiss D.R.¹, Callaert M.², Eckstein R.¹

¹Transfusionsmedizinische und Hämostaseologische Abteilung, Universitätsklinikum Erlangen, Erlangen, Deutschland, ²CaridianBCT Europe, Belgium

Background: There is still little knowledge on whether different hold times as hyperconcentrated single donor platelets (SDPs) before adding of platelet (PLT) additive solution (PAS) might affect PLT quality. We compared the in—vitro quality of SDPs with immediate or delayed addition of PAS—IIIM and studied the quality of collected by—plasma. Methods: 31 blood donors underwent apheresis on Trima Accel^® (V6.0) collecting a hyperconcentrated SDP (6 × 1011 PLTs/175 mL plasma) with a maximum amount of by—plasma. The SDP was split into 2 similar products. The system added 162 mL of PAS to one product (0h—SDP). The same volume of PAS was added to the second product after 2 hours (2h—SDP) resulting in SDPs containing 1.2 × 106 PLTs/ μL and 65% PAS. Lactate, glucose, mean PLT volume (MPV), pH (22°C), CD62P—expression, hypotonic shock response (HSR), and extent of shape change (ESC) were tested on days 1,5, and 7. Activity of coagulation factors FV and FVIII and concentration of D—dimers were determined in by—plasma and in the donor. Results: Mean volume of the 0h— and 2h—SDPs was 248.7+5.0 and 243.4+5.5 mL with 1.05+0.14 and 1.07+0.16 xl06 PLTs/μL resulting in a PLT content of 2.62+0.35 and 2.60+0.42×1011 PLTs, respectively. Maximum content of residual WBCs was 0.12 × 106 WBCs/unit. Glucose utilisation, lactate increase, and pH were similar throughout storage for both kinds of products. Similarly low values for CD62P—expression indicated no platelet activation during storage. HSR and ESC were well preserved during storage with similar values for 0h— and 2h—SDPs (Table). The recovery of FV and FVIII was 100.0+14.0 and 98.6+14.9%, respectively. Concentration of D—dimers in the donor and the by—plasma was 173.7+90.1 and 177.6+91.2 ng/ dL, respectively. Conclusions: Adding PAS immediately or 2h after collection does not result in a different in—vitro quality of PLTs stored up to 7 days. The good recovery of CF with no signs of activation indicates a good quality of by—plasma.

In-vitro parameters (n=31, ∗n=30)	Oh-SDP Mean±SD	2h-SDP Mean±SD	P-value
HSR day 5 (%)	64.8±10.0	64.7±12.0	0.969
HSR day 7 (%)	59.9±10.7	60.2±10.6	0.883
ESC day 5 (%)	24.4±4.8	25.2±4.8	0.334
ESC day 7 (%)	20.3±4.4	19.6±4.9	0.377
CD62P day 5 (%)∗	2.03±1.02	2.02±0.88	0.938
CD62P day 7 (%)	3.03±1.26	2.99±1.23	0.844
MPV day 5 (fl)	7.83±0.51	7.84±0.54	0.904
MPV day 7 (fl)	7.98±0.50	7.99±0.53	0.742
pH at 22°C day 5	7.47±0.07	7.47±0.07	0.685
pH at 22°C day 7	7.49±0.09	7.48±0.09	0.511
Glucose utilization until day 7 (mmol/day/1011 PLTs)	0.31±0.07	0.31±0.08	0.648
Lactate increase until day 7 (mmol/day/1011 PLTs)	0.63±0.11	0.62±0.13	0.582

P1.09

Influence of storage conditions on thrombocyte concentrates

Plöderl K., Fenz M., Kemptner J., Hennerbichler S., Peterbauer—Scherb A., Gabriel C.

Red Cross Transfusion Service for Upper Austria, Linz, Austria

Background: Pools made from thrombocyte concentrates are usually produced for transfusion purposes and stored at room temperature under permanent agitation. These pools have to be used within five days after production or otherwise have to be discarded. The aim of this study was to determine whether outdated pools, pools with shorter storage period or pools with different storage conditions would be ideal for further processing into platelet lysate. Methods: 144 whole blood donations were further processed into thrombocyte concentrates. In order to minimize product variation 36 concentrates were pooled. From each pool a sample was drawn to obtain starting values. Each pool was then split into six bags. These represent the normal size of thrombocyte pools used for transfusion purposes. Twelve of them were stored at room temperature and 12 at +4°C. Samples were drawn on day 3, 5 and 7. The samples were used for pH measurement and gas analysis, determination of the hypotonic shock reaction as well as platelet activation and platelet aggregation. Additionally, LDH, glucose and total protein concentrations were determined. For the detection of potential changes in the concentrates’ compositions 2—D gel electrophoresis was performed. STATISTICA 9.1 was used for statistical evaluation of the results. Results: Thrombocyte concentrates stored for three days would be best for further production of platelet lysate. Concentrates from day 5 stored at room temperature representing outdated products also obtained good results for hypotonic shock reaction and platelet activation. However, concerning platelet aggregation storage at +4°C was superior. Other parameters did not show any significant differences. Conclusion: Storage conditions definitely influence the quality of thrombocyte concentrates. Concentrates from day 5 may be used as starting material for the manufacturing of platelet lysate, which would be cost effective by extensively exploiting natural resources.

P 1.10

In vitro quality of pooled platelets prepared using a combined centrifugation/separation method and treated with the Theraflex UV—System

Larrea L.¹, Ortiz de Salazar M.—l.¹, Guinot M.¹, Carbonell F.¹, Solves P.¹, Mirabet V.¹, Tolksdorf F.², Alvarez I.³, Roig R.¹

¹Centro De Transfusion De La Comunidad Valenciana, Valencia, Spain, ²Maco Pharma International GmbH, Langen, Germany, ³Macopharma, Madrid, Spain

Background: THERAFLEX UV—Platelets (Macopharma, France) uses short wave UV light (254 nm) for pathogen reduction in platelet concentrates (PC), without the need of any photoactive chemicals. We evaluated the in vitro quality of UVC—treated BC—derived platelets prepared using an automated technique for centrifugation and separation. Materials and Methods: A pool made out of 8 BCs and suspended in 600mL SSP+ (Macopharma) was split into two aliquots, which were further processed using the TACSI system (Terumo Automated Centrifuge & Separator Integration, Terumo, Japan). The average volume of the two final PC was 340mL with a plasma ratio of 35%. Both UVC—treated (test, n = 11), as well as the untreated units (control, n = 11) were stored on a flat bed agitator at 22°C for in vitro testing at day 1, 2, 5, and 7. Results: There was no significant difference in the platelet concentration between the test group and the control group along the week. Between day 5 and day 7, glucose and HCO₃^– decreased more in the test group (9.8 ± 2.5% and 11.7 ± 2.6%, respectively). Lactate increased more in the test units throughout storage, but without significant difference at any time. pH remained above >7.0 until day 7 in all units. Visual inspection did not reveal any abnormality and swirling remained at the highest level (score = 3) for all units throughout storage. The differences for Annexin V and CD62P were not statistically significant between the groups. Conclusions: The in vitro quality of UVC—treated TACSI platelets correspond to current in vitro standards and storage stability of PC is well maintained until day 7. The results indicate that TACSI platelets are compatible with the incoming product requirements of the THERAFLEX UV—system, particularly regarding the range of plasma substitution by SSP+. Moreover, the simple and rapid THERAFLEX UV—Platelets process can be easily incorporated into established preparation procedures for BC—derived PC.

P1.11

Impact of plasma—reduction of platelet concentrates stored for seven days on platelet activation and platelet function in vitro

Macher S.¹, Rosskopf K.¹, Kozma N.¹, Klinnik V.², Lanzer G.¹, Schallmoser K.¹

¹Clinic for Blood Group Serology and Transfusion Medicine. Medical University of Graz, Graz, Austria, ²MacoPharma, LAB. Pharmaceutiques, Tourcoing, France

Reducing the plasma fraction in platelet (pit) concentrates (PCs) is an option to save resources and to decrease transfusion reactions. Thus plasma—reduced buffy coat—derived PCs (BC—PCs) suspended in pit additive solution (SSP+, MacoPharma) were prepared and compared to standard BC—PCs. Pit function, metabolic parameters and pit activation state in vitro were analyzed during 7 days (d). Three groups of PCs were prepared: Group 1 (Gl; standard method, n = 12) with a plasma content of 30% and group 2 (G2; n = 12) with a plasma content of 20%. For decreasing the plasma content to < 5% (G3; washed pits; n = 12) 15 vol% ACD—A was added and the pits were centrifuged at 2000 × g for 10 minutes. After discarding the supernatant pits were resuspended in 250 ml SSP+. BC—PCs were tested for blood cell counts, pH, glucose, potassium, lactate, P—selectin (CD62P) and LAMP—3 (CD63) expression, aggregometry and hypotonie shock response (HSR). Pit yields (x 10ⁿ; mean ± SD) were 2.39 ± 0.24 (Gl), 2.32 ± 0.34 (G2) and 2.31 ± 0.51 (G3) on d2. pH values always met the required range of 6.4 − 7.4 and metabolic parameters were well maintained during 7 days. Activation markers in Gl and G2 did not show significant differences, whereas pits in G3 expressed significantly higher CD62P levels (d2, d5, d7) and CD63 levels (d2) compared to Gl. The in vitro capacity for pit aggregation was significantly reduced in G2 on d5 and d7 compared to Gl. Moreover, pits with < 5% residual plasma volume (G3) did not show any collagen—induced aggregation in vitro. HSR was significantly diminished in G2 and G3 compared to Gl during the whole storage period. The preparation of plasma—reduced BC—PCs is feasible and metabolic parameters were well maintained in all three study groups. Plasma—reduction has an impact on pit function in vitro and BC—PCs with a residual plasma volume of < 5% (G3) have to be considered for special and immediate application only.

P 1.12

Stability studies on fresh frozen plasma, thawed

Madia W.¹, Alt T.¹, Jungk H.², Markovic A.²

¹DRK—BlutspendedienstWest, Hagen, Deutschland, ²DRK—Blutspendedienst Rheinland—Pfalz und Saarland, Bad Kreuznach, Deutschland

Background: Provisions for the storage and transport conditions of blood products are found in the German Haemotherapy Guidelines, amongst others. Fresh frozen plasma (FFP), thawed, is recommended “for immediate transfusion”; distribution resp. transportation should take place at “room temperature”. For this study thawed FFP was temporarily stored for up to 24h at room temperature (RT = 15 − 25°C). We subsequently examined the influence this had on various activators and inhibitors of coagulation and fibrinolysis, on the development of activation markers of coagulation and on the complement system. Methods: 18 leucocyte—depleted FFPs (blood group AB) after 12 months of storage at < —30°C have been thawed and studied for following parameters after Oh, 8h, 12h, 16h and 24h: PT, aPTT, factors I, II, V, VII, VIII, IX, X, XI, XII, XIII, vWF, antithrombin, protein S, protein C, plasminogen, alpha 2—antiplasmin, TAT, D—dimer, CH50 and C3a. Results: After 8h of storage at RT, none of the parameters revealed any significant difference to the initial contents level (value at Oh). After 12h only protein S exhibited a significant decrease. This tendency continued after 16h and 24h. After 24h some factors exhibited a slight, though insignificant decrease (F I, II, VII, VIII, IX, XII, protein C, alpha 2—antiplasmin, CH50) or an increase (PT, C3a) in activity. The decrease, if any, was most noticeable in F VII (p = 0.057) and F VIII (p = 0.087). The other parameters (aPTT, F V, F X, F XI; F XIII, vWF, antithrombin, plasminogen, TAT, D—dimer) were unremarkable. Conclusion: According to the data available, storing (or distribution) of thawed FFP at RT for 8h is considered safe in respect of all the parameters tested. Although a longer interim storage period (12h to 24h) has a detrimental effect on protein S only, it is not recommended because the activities of other factors — coagulation factors VII and VIII in particular — revealed a downward trend.

P 1.13

Homeopathic arnica as supportive therapy in bone

marrow donation

Wenzel F.¹, Hoegn C.¹, Rox J.¹, Trapp T.¹, Giers G.², Fischer J.^{1 1}Institut für Transplantationsdiagnostik und Zelltherapeutika, Düsseldorf, Deutschland, ²Dept. of Experimental and Clinical Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Duesseldorf, Germany

Background: Arnica montana is widely used as homeopathic dilution for treating wounds on account of its supposed abilities. It is one of the widely used homeopathic preparations and is popular with patients undergoing surgery. In bone marrow (BM) donation, there is a well defined osseous lesion in healthy donors. In this report we could observe the effects of arnica on the postoperative physical health in five allogenic BM donors. Methods: Five healthy BM donors conducting a self—medication with Arnica for BM donation could be evaluated and compared to a respective matched control group (n = 8) without homeopathic medication. BM was collected by repeated puncture of the iliac crest under general anesthesia. The physical health status was examined by a standardized SF—36 questionnaire before, immediately and four weeks after BM donation. Results: In the arnica group, the amount of CD34+ cells was found to be 3.9 ± 1.1 ∗ 10⁁6 cells/kg BW recipient in comparison 4.1 ± 1.8 ∗ 10⁁6 CD34+ cells/kg BW recipient in the matched control group. Preoperatively, the SF—36 scores for pain, physical function and general health were within a normal range (arnica: 99 ± 5, 98 ± 2, 84 ± 10, respectively; controls: 90 ± 10, 99 ± 2, 83 ± 11, respectively). Immediately after surgery, the SF—36 scores were significantly altered in both groups, and especially the pain score showed a tendency to lower values in the controls (arnica: 78 ± 31 vs. controls: 60 ± 14). After four weeks the SF—36 scores reached a normal range. Conclusions: In BM donation, immediately after surgery the SF—36 scores of the arnica group as well of the controls showed significant alterations. However, donors receiving homeopathic arnica showed a trend towards less postoperative pain. Because arnica is a safe and easy to use alternative compared to other treatments it seems helpful to consider the use of homeopathic arnica in BM donation.

P1.14

Verlauf von Hämoglobin— und Ferritinwerten nach Knochenmarkentnahme bzw. Mehrfachstammzeilspenden unter Eisensubstitution mittels Eisen(ll)—Gluconat Saftes

Völker G., Fischer I., Kreissig C.

DRK Blutspendedienst West – ZBST, Ratingen, Deutschland

Fragestellung: Pro Jahr erfolgen bei uns etwa 150 Stammzellentnahmen von Fremdspendern, circa 15 bis 20 Prozent als Knochenmarkentnahmen und 80 bis 85 Prozent als periphere Blutstammzellapheresen. Bei den Nachuntersuchungen fielen erhebliche Hb— und Ferritinabsenkungen auf, die besonders bei den Knochenmarkentnahmen relativ lange anhalten. Wir wollten wissen, ob durch die regelmäßige Einnahme eines Multivitaminsaftes – Rabenhorst Eisenblutsaft, versetzt mit Eisen (II)—Gluconat – eine schnellere Erholung der entsprechenden Blutwerte zu erzielen ist. Gleichzeitig erhofften wir, die bei einer Eisensubstitution mittels Arzneimittel zu erwartenden Verdauungsprobleme durch die Einnahme des Saftes zu umgehen. Spender und Methode: Wir haben 14 Knochenmark— bzw. 27 Mehrfachblutstammzellspender gebeten, 12 Wochen lang täglich 70 ml (50 mg Eisen (II)—Gluconat) des Saftes zu trinken. Eine Blutbild— und Ferritinkontrolle wurde nach 1 Woche, nach 1 Monat und nach 3 Monaten durchgeführt. Parallel zu den Laboruntersuchungen erfassten wir mittels Fragebögen Informationen über die Verträglichkeit und die korrekte Einnahme des Saftes. Die Daten werden verglichen mit einer Kontrollgruppe mit 28 Spendern. Ergebnisse: Es ist ersichtlich, dass die Einnahme des mit Eisen (II)—Gluconat versetzten Multivitaminsaftes keine schnellere Regeneration der entsprechenden Blutwerte zur Folge hat. Die Compliance bezüglich Einnahme ist aufgrund der guten Verträglichkeit im Vergleich zu apothekenpflichtigen Eisenpräparaten sehr gut. Schlussfolgerung: Im Gegensatz zur medikamentösen Behandlung wurde der Multivitaminsaft gerne von den Spendern angenommen. Auch der Geschmack wurde von den Meisten als angenehm beurteilt. Eine bessere bzw. schnellere Regeneration der Erythropoese konnte jedoch trotzt regelmäßiger Einnahme nicht beobachtet werden. Insgesamt war das Feedback der Spender durchaus positiv, die sich gewertschätzt fühlten durch unsere Bemühungen um eine schnelle Erholung nach der Spende.

P 1.15

Development and clinical application of the first functionally closed collection and application system for autologous serum eye drops

Hackstein H.¹, Misterek J.¹, Bein G.²

¹Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany, ²Zentrum für Transfusionsmedizin und Hämotherapie, Universitätsklinikum Giessen und Marburg GmbH, Giessen, Germany

Background: Eye drops produced from autologous serum are used for the treatment of ocular surface disorders. Several controlled clinical studies have proved the effectiveness of autologous serum eye drops in the treatement of persistent epithelial defects, drye eye syndrome (keratokonjunctivitis sicca) and during ocular surface reconstruction. Currently, all procedures for the preparation of serum eye drops include the opening and aliquotation of the serum collection bag and depend therefore on cleanroom facilities. Results: We have developed a continuous tubing system with pouches and branches including sterile opening adapter. This serum eye drop tubing system can be connected to a serum collection bag via TSCD—coupling while maintaining a functionally closed system. Subsequently, serum eye drop bottles are simply created by sealing off the tubing system between the branches. The remaining serum eye drop bottles each contain a closed sterile opening device allowing easy dosing and instilling of sterile serum to ocular surfaces. Clinical application of the system was approved by local authorities and facilitated the production of autologous erum eye drops for patient treatment. The novel serum eye drop tubing system has been introduced into the clinical routine. Conclusion: The closed serum eye drop tubing system represents a novel alternative for clinical production of autologous serum eye drops.

P 1.16

Why it is useful to provide mobile collection teams with automatic external defibrillators (AED)

Deitenbeck R.

German Red Cross Blood Service West; Center of Transfusion Medicine,

Hagen, Germany

Background: With the opening of the upper age limit for blood donors in 2005, there is a rise in the contribution of elderly donors to the total amount of blood donations in Germany. According to an analysis of the Federal Statistical Office, the proportion of donors older than 65 years will increase by 5.5 million between 2010 and 2030. Therefore it can be expected that the proportion of donors with unknown cardiac diseases will increase simultaneously. We report on our first successful use of an AED on a mobile collection campaign. Patient and Methods: A 62—year—old volunteer of a local Red Cross association assisted in the organization of a mobile collection campaign. Just before starting the campaign at 7.45 pm, the collection team was called for help by the volunteer's colleague since he collapsed. The collection team found the man without any vital signs. The AED has been brought in position immediately and the result of the analysis required an immediate shock due to an asystole state of the heart. The subsequent CPR merged in a stabile cardiac and circulatory condition within 2 minutes until the patient could be released into the care of an external emergency team. Given artificial respiration and sedation, the patient was hospitalized for further diagnostics and treatment. Diagnostics revealed a previously unknown coronary heart disease. Within the first cardiac catheterization two stents have been implanted. The patient could be discharged a few days later without any sequelae. Conclusions: There is a change in the demographic structure of both donor population and staff/volunteers, each associated with an increased risk of previously unknown but relevant cardiac diseases. As mobile collection campaigns are carried out under the responsibility of medical organizations such as the Red Cross and in public facilities it is recommendable to provide mobile collection teams with AEDs to be able to intervene immediately and effectively as described above.

P 1.17

Seasonal fluctuations of vitamin D3 in a cohort of blood donors in upper Austria

Süßner S., Falkinger A., Schreiberhuber S., Frühwirt K., Ecklbauer D., Gabriel C.

Red Cross Transfusion Service for Upper Austria, Linz, Austria

Background: Vitamin D3 (calcediol, 25—OH—vitamin D3) is generated by UV—irradiation and stored in the human body, in summer more than in winter. Reduced sun exposure in winter can result in a decrease of vitamin D3. We decided to detect seasonal variations of vitamin D3 in voluntary blood donors to evaluate the impact of this parameter for our donors. Patients and Methods: Samples were taken from 105 blood donors (18 to 65 years) before and after winter and stored immediately without light exposure. After centrifuga—tion, freezing and thawing the vitamin D content was tested with the testing kit “25—OH—Vitamin D3/D2” (Chromsystems, Germany) according to manufacturer's instructions on a HPLC instrument (LC20, Shimadzu, Japan). The results of vitamin D3 were categorized in normal range (20 − 70 μg/L), under—supply (10 − 20 μg/L) and deficiency (< 10 μg/L). Vitamin D2 was not interpreted because the tested cohort did not supplement vitamin D in this period. Results: The difference of vitamin D3 concentration before and after winter was significant (p = 0.000). The mean reduction was above 40% and does not depend on gender. Vitamin D3 deficiency was present in 1.9% of the samples before winter and in 21.9% afterwards. 17.1% of the cohort had an undersupply at the first point of testing. This group increased to 50.5% after the winter. Only 27.6% of the donors were in the normal range at the first as well as at the second sampling. Conclusions: The prevalence of an undersup—ply/deficiency of vitamin D3 of 0.190 before winter and 0.724 afterwards implicate that reservoirs of vitamin D3 are emptied in winter due to reduced solar irradiation. Our results suggest that the parameter vitamin D3 could be a gratification for our voluntary blood donors in and after wintertime. In case of undersupply/deficiency a supplement therapy could be recommended.

P 1.18

Spender—Nebenwirkungen: eine prospektive Beobachtungsstudie

Infanti John L, Sigle J.P., Job S., Keshelashvili K., Buser A.

Blutspendezentrum SRK beider Basel, Basel, Switzerland

Einleitung: Seit 2007 werden im Blutspendezentrum Basel die Neben—wirkunegn der Blutspende systematisch erfasst. Methoden: NW (ohne Anämie, Eisenmangel und leichte Zitrattoxizität) wurden erfasst und gemäss den Kriterien der ISBT Haemovigilance, EHN 2008, klassifiziert. NW wurden eingeteilt in akut (innerhalb 24 Stunden) und verzögert (24 Stunden bis 7 Tage nach Blutspende). Schwere NW (SNW) benötigten ärztliche Interventionen oder zeigten eine verzögerte Erholung. Erfasst wurden NW der Vollblutspenden (VB), Eigenblutspenden (EB), Thrombapheresen (TA), Doppeleryth—rocyten—apheresen (DEA) und Plasmapheresen (PA). Resultate: Januar 2007— Dezember 2010 wurden 47I74 Entnahmen durchgeführt (38'796 VB, 5'864 TA, 419 PA, 2′O95 DEA, 168 EB). Bei 500 Entnahmen (1%) durch 455 Spender wurden insgesamt 519 NW dokumentiert; 45 (0,1%) waren SNW bei 44 Spendern. In 19 Fällen traten 2 NW gleichzeitig auf. Der 455 betroffenen Spender 55% waren weiblich; 334 (39 SNW) waren Wiederholspendern, 118 Erstspender (6 SNW) und 3 (keine SNW) EB—Spender. Spender mit Komplikationen gehörten meistens zu der Altersgruppe 18—25 und 46—55 Jahre (jeweils 24% und 23% aller Spender mit NW). Insgesamt 0,8% der VB, 2,8% der TA, 1,9% der DEA und < 1% der PA wurden von NW kompliziert, insbesondere von vasovagalen Reaktionen von Sofortyp (51% aller NW) und lokalen Hämatome (31%). SNW waren vorwiegend akut (31 Fälle); 21/45 Fälle waren Nervenverletzungen. Die Rate der Spender, die nach einer NW und SNW erneut Blut spendeten war 63% bzw. 59%; 38/118 Erstspender mit NW (32%) und 1/6 Erstspender mit SNW spendeten Blut wieder. Schlussfolgerungen: Spender—NW sind insgesamt selten, häufiger bei Apheresen. Die systematische Erfassung der NW ermöglicht eine optimierte Beratung der Blutspender, sowie das Umsetzen von individuellen Massnahmen. Eine aufmerksame Betreuung der Spender mit NW ist eine wichtige Aufgabe der Blutspendezentren und verbessert möglicherweise die Rate an Wiederholungsspenden bei Blutspendern.

P 1.19

New blood donor ID—card of the German Red Cross Blood Donation Services (GRC—BDS) – Results of the acceptance analysis and the pilot project

Knels R.¹, Graeulich N.², Rau T.², Gillert F.³, Stangenberg W.⁴

¹Eurocode IBLS e.V., Dresden, Germany, ²Technische Hochschule Wildau [FH], Wildau, Deutschland, ³UbiConsult, Berlin, Germany, ⁴German Red Cross Blood Service Mecklenburg—Vorpommern, Germany

The 7 GRC—BDS established a working group with experts for donor recruitment, data management and with physicians to create a new common donor ID—card which allows donors to give blood in all GRC—BDS with this card. After technical evaluation the usage of 13.56 MHz RFID chips for data storage embedded in plastic cards (EC—card format) was decided. This allows the storage of the different donor numbers of the GRC—BDS as well as number of donations, date of last donation, personal data and blood group on the RFID chip. Questions about data protection, donor and staff acceptance and the cost/ benefit relation had to be addressed. Thus, an acceptance analysis and a pilot project were performed in cooperation with the University of Applied Sciences in Wildau and Ubiconsult. For the acceptance analysis 1,853 blood donors and non—donors from urban and rural areas were asked to fill in a questionnaire. All asked topics have a high acceptance: change of format in 86%, special appraisal of jubilee donors with different card layouts in 93% and using RFID in 95%. The high acceptance of RFID was especially surprising because 34% of the interviewees would appreciate to get more information about the storage of personal data in general. Another surprise was that donors and non—donors in rural areas showed a higher acceptance for all four topics than the urban population. Comparable analyses for using personal documents with RFID are unavailable. The pilot project with 2,000 donors was performed in GRC—BDS East and West with the new RFID cards. The new ID—cards and the reader technology were well accepted by both, donors and staff. Besides gauging the acceptance the pilot project was very helpful identifying corrective actions in data management and calculating the implementation costs. In result of the successful acceptance analysis, pilot project and positive cost/ benefit—relation the GRC—BDS decided to start the roll—out of the new donor ID—card in autumn 2011.

P1.20

Secular trends in German blood donor rates between 1994 and 2009

Weidmann C.¹, Schneider S.¹, Litaker D.¹, Weck E.², Klueter H.²

¹Mannheimer Institut für Public Health, Mannheim, Deutschland, ²Institut of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Germany

Background: The demand for blood has increased significantly over the last two decades due to the development of novel treatment options and to the increasing prevalence of chronic disease in an ageing population. As a consequence, blood establishments have been required to intensify recruitment efforts. Have these efforts resulted in increased blood donor rates between 1994 and 2009? Methods: Three representative cross—sectional waves of the Eurobarometer survey (1994, 2002, and 2009) were used to compare ageadjusted percentages of German adults ever having donated blood.

Respondents aged 18 to 60 in each population—based sample were included in the analysis (n = 1490, n = 1287, n = 893). A logistic regression was applied to the pooled data to assess temporal trends. The dependent variable in this model was donor status (having donated or not) and the study period was the main predictor. We adjusted for age, sex, and education of the respondents and included interaction terms between study period and the sociodemographic characteristics. Results: Although the percentage of respondents donating at least once was similar between 1994 (31.8 percent) and 2002 (32.2 percent), report of donation increased to 41.4 in 2009. Increases between 1994 and 2009 were found consistently for both men and women and in all age and education groups. The logistic regression model showed that the odds of reporting donation in 2009 were 1.57 (CI 1.31 − 1.88) times higher than in 1994. In all waves the likelihood to report donating increased with age and education and was higher for men. Conclusion: In the face of increasing demand, results from this study suggest that supply, in the form of blood donation rates, has increased as well. The reasons for these changes is unclear, but may be related to intensified recruitment efforts of the blood establishments, an increased sense of altruism, and a raised awareness for the need for blood.

P1.21

Motivation for blood donation: results of a representative survey of a university blood donation service – Identification of factors improving the willingness to donate blood

Wienzek—Lischka S.¹, Toepel G.², Hackstein H.¹, Bein G.¹

¹Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, dessen, Germany, ²Rehazentrum Bad Nauheim, Dt. Rentenversicherung Bund, Klinik Taunus, Bad Nauheim, Deutschland

The need for transfusion of blood components is increasing steadily for the past few years. Medical advances of surgical and conservative therapy as well as the demographic development of the population are the reasons for the increased demand for blood components. The number of blood donations, however, remains almost constant or decreases depending on region and age structure of the blood donors. Often long—time donors withdraw due to their age. On the other hand, the willingness for blood donation of young people is often not sufficient or a donation is not possible, for example by long—distance travel activities. To continue to guarantee the supply of the population with blood components and to motivate people for long—term blood donation, a survey was conducted among 1000 volunteers of our blood donation service. The participation rate of the voluntary survey was excellent and carried out with a standardised and anonymous questionnaire. The survey included the interrogation of age, gender and employment. Furthermore, the questions differentiate in first—time and repeat donors. Analyzed were their own motivation for blood donation (for example medical check—up, to do a good deed, remuneration for their efforts) and how the donors were made aware to donate blood. Furthermore, proposals should be referred how their own donation rate could be increased and how new blood donors could be obtained at best. The analysis of the survey is not only useful for our blood transfusion service as a marketing tool to attract new blood donors, but could be also a valuable stimulus for transfusion medicine in Germany.

P1.22

Demographic analysis of first time donors versus elderly donors in a single blood center

Röhrig O., Nowak—Harnau S., Luz B.

Zentralinstitut für Transfusionsmedizin und Blutspendedienst, Klinikum Stuttgart, Stuttgart, Deutschland

Background: In Aug. 2008, at Katharinenhospital Stuttgart, we started to allow first time donors to donate blood at the day of their first contact. Before that, we performed only medical and laboratory examinations. Donors were then ivited to donate by letter. Regular blood donors may donate blood until their 69th birthday. Method: Data were analyzed, retrospectively, from aug. 2008 to feb. 2010. During that time, 7.325 first time donors registered. Demographics, donation frequencies and outcomes were analyzed. A comparative analysis was performed on elderly, regular blood donors, aged ≥65 years. Results: During the time studied 20.475 donors came to our blood center for donation. 35,8% (n = 7.325) were first time donors, 2,2% (n = 450) were 65yrs or older at the time of their donation. 66.360 whole blood donations were performed during that period. First time donors contributed to 20,3%(13.481) donations. 2,8% (1.845) donations were given at an age of 65 or higher. 83,3% of the registered first time donors (n = 6.098), gave blood at least once. In 7,0% (428) of these donations no product could be obtained. 5.256 first time donors gave blood already at their first encounter. 3297 donors came at least twice. The majority, of the first time donors, 61,1%, were at 18 to 25 yrs. 22,4% were ≤35yrs. Sex distribution was almost equal among first time donors, F: 49,2%, M: 50,8%. In contrast to that, among the elderly donors there were 31,3% female vs. 68,7% male donors. Overall ratio was 43,2% female vs. 56,8% male donors during that time. Conclusion: Our analysis showed a high contribution of first time donors to the blood supply. First time donors are a very important group with a high percentage of young donors. Emphasis should be put on finding ways to increase recurrence rates after the first and second successful donation. Elderly donors are already used to give blood regularly. Legislative rules should be fixed to allow blood donation until the age of 71years.

P 1.23

Loss of donors – loss of blood components – what are the reasons? Analysis of the 10—year period 2001–2010

Kühnel D., Spier B., Walther—Wenke G.

German Red Cross Blood Service West, Center for Transfusion Medicine Münster, Münster, Germany

Background: Efficient blood supply relies on successful donor recruitment. Our aim is to provide a sufficient quantity of blood components for transfusion. Not every prospective donor and not every blood donation leads to the production of releasable blood components. In our blood donation service with nearly 2.9 Mio. donors in the observation period, mainly on mobile donation sites, we analysed the causes for donor retention and loss of blood resulting from various steps in the production processes. Methods: A retrospective analysis of data about donors, donations and production steps was performed for a ten—year production period. Results: Between 2001 and 2010 2.897.377 donors appeared of whom 301.108 (10.39%) were deferred due to their medical history or examination (e. g. low Hb 2.14%, surgery 1.18%, infection diseases 1.17%, blood pressure 0.98%). During the donation process we detected a loss of 1.92% of whole blood donations, among these are under—or over—filled blood bags (1.27%), incomplete or missing laboratory samples (0.34%) and technical faults (0.16%). Control of actual and former donor data showed unsuitable whole blood donations which were excluded from further processing (confidential self exclusion 0.31%, medical reasons 0.06%). During component preparation technical problems were followed by a loss of RBCs (0.24%). 0.50% of RBCs where discarded due to laboratory donor screening results, 0.05% by post donation informations and 1.25% by product quality control. The loss of RBCs during blood processing and laboratory examination amounts to a total of 3.31%. Conclusions: To summarise, the willingness to donate blood does not regularly lead to a blood donation and consequently to the production of applicable RBCs. 100 prospective donors are needed to generate 84 RBCs. The implementation of medical, technical and organizational measures should be monitored critically to prevent loss of donors and donations.

P1.24

The impact of immediate versus delayed donation on return behavior of first time donors

Röhrig O., Nowak—Harnau S., Luz B.

Zentralinstitut für Transfusionsmedizin und Blutspendedienst, Klinikum Stuttgart, Stuttgart, Deutschland

Background: In August 2008, at the Katharinen Hospital Stuttgart, the enrollment procedure for First Time Donors (FTD) was changed. Previously only medical and laboratory examinations were performed at the first appointment and donations were delayed until these results were checked. In our new procedure eligible FTD following interview and physical examination, were encouraged to donate blood on the same day. The impact of this change on donation procedures for FTD was studied. Method: Starting on August 11th, 2008, for 6 months, all FTD were included in the study. Follow up—time was one year, individually calculated for each donor. The study was finished on February 5th, 2010, when a new donor management system was introduced. FDT who came in between August 13th 2007 and February 11th, 2008 were taken as a control. Demographics, donation frequency and outcome were studied. Results: During the time studied there were a total of 3512 FTD, 1496 before and 2016 after the change. In the control period there were 2453 whole blood donations taken from FTD, however after we changed our procedure, we were able to collect 4938 donations. Before, only 62,2% of the First time Donors donated at least once. 45,1% donated two times, but only one third, 30,1% performed three or more donations, and became regular donors later. After we had changed our procedure, independently of the absolute increase of first time donors, the proportion of those who actually donated blood at least once, rose to 84,8%. 62,4% donated two times, and almost half of them performed three or more donations. Conclusion: The Change in our enrollment procedure for first time donors had a tremendous impact on the overall donations and we were able to collect blood from these donors. Additionally, there was a much higher proportion of FTD who came to donate at least three times with potential for donation on a regular basis.

Session 02 – Blood Safety

P 2.01

Epidemilogy of CMV infection in patients

Seferi I.

National Blood Transfusion Centre, Tirana, Albania

Aim: To evaluate the epidemiology of CMV in immunocompromised and multitransfused patients. Materials and Methods: 372 patients belonging to the risk groups for CMV, and 76 patients of the control group were tested for IgG and IgM anti—CMV antibodies. Patients that were IgG and IgM negative in the risk group and in the control group were monitored over a period of one year in order to identify an anti—CMV antibody seroconversion. They were tested every month for anti—CMV IgG and IgM; negative results were confirmed by PCR. Results: Anti—CMV IgG was found in 96.3% of thalassemia patients, 100% of haematologic patients, 82% of pregnant women, 100% of patients with HIV/AIDS and 85.5% of patients in the control group. The prevalence of anti—CMV IgG in haematologic patients and in HIV/AIDS patients was significantly higher than in patients of the control group. Anti—CMV IgM was found in 11.1% of thalassemia patients, 18.6% of haematologic patients, 8% of pregnant women, 65.4% of HIV/AIDS patients and 6.6% of the control group patients. The analysis showed that thalassemia patients have a 2.6 times, haematologic patients of 5.4 times and HIV/AIDS patients of 44.7 times higher propability of being IgM positive than the conrol group patients. 20% of patients with thalassemia and none of the control group seroconverted. Conclusion: Immunocompromised and multitransfused patients have a very high prevalence of CMV infection (96–100%). In addition, a high seroconversion rate has been observed in multitransfused patients. A high prevalence of CMV is also found in other groups of not transfused individuals such as pregnant women, blood donors or control patients. This hampers the supply of CMV—negative blood components for “at risk” patients and supports the policy of universal leukodepletion to diminish the risk of a transfusion mediated transmission of CMV.

P2.02

Assessment of the blood cell count of blood donors who tested positive for Parvovirus B 19 DNA.

Juhl D.¹, Steppat D.², Glessing P.², Gorg S.³, Hennig H.³

¹Institut für Transfusionsmedizin, Universitätsklinikum Schleswig—Holstein, Lübeck, Deutschland, ²Institut für Transfusionsmedizin, Universitätsklinikum Schleswig—Holstein, Kiel, Deutschland

Background: Parvovirus B19 (PVB19) infections result in a decline of the blood cell counts in the acute viremic phase and PVB19 DNA can persist thereafter at lower levels for a long time. Whether PVB19 viremia affects the blood cell count in blood donors is unknown. Patients and Methods: All donors, who donated whole blood or platelets at the institute of transfusion medicine of the university hospital of Schleswig—Holstein, either at the location in Kiel or in Lübeck, were screened routinely for PVB19 DNA using the cobas TaqScreen DPX test (Roche diagnostics; 95% detection limit 12 IU PVB19 DNA/ml pool plasma) in minipools containing up to 96 samples 6 weeks after their donation. Blood cell counts of those donors tested positive for PVB19 DNA were compared to a matched control group who tested negative for PVB19 DNA. Results: Overall, 8922 blood donors were tested for PVB19 DNA between January 4th and March 4th 2011. Of those, 40 (0.45%) tested positive for PVB19 DNA with a median viral load of 1045 IU/ml (range: < 75 − 34013 IU/ml). The median blood cell count of those donors (Leukocyte count [L]: 6.600/ul, Erythrocyte count [E]: 4.66 Mio/μl, Hemoglobine [Hb]: 14.45 g/dl, Hematocrit [Hkt]: 40.5%, platelet count [P]: 209.000/μl) did not differ significantly compared to the results of the PVB19 DNA negative control group (L: 6.450/μl, E: 4.72 Mio/μl, Hb: 14.85 g/dl, Hkt: 41.55%, P: 221.000/μl). Conclusions: The prevalence of PVB19 DNA in our study is comparable to those described in other reports. The viral load of the donors tested positive for PVB19 DNA is relative low, compatible rather with long—lasting, not acute PVB19 infections, as in acute infections high viral loads, up to 10¹⁴ IU/ml, are measurable. A low PVB19 viral load seems not to affect the donor's blood cell count. To clarify whether those donors are at risk to transmit PVB19 to susceptible patients, look—back investigations are intended.

P2.03

Blood donor screening for HAV, HBV, HCV, HIV—1 (dual targeting), HIV—2, PB19 and for bacterial pathogens by the mini—pool NAT extraction system Zelos x100

Hourfar M.K., Capalbo G., Rüster B., Sireis W., Gubbe K., Karl A., Tonn T., Seifried E., Schmidt M.

German Red Cross Blood Service Baden—Württemberg – Hessen, Institute of Transfusion Medicine and Immunohaematology, Medical Faculty Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany

Background: The German Red Cross Blood Donor Service Baden—Württemberg – Hessen introduced the mini—pool NAT testing on a voluntary basis in 1997, before it was mandated by the German Authority, the Paul—Ehrrich—Institute, for hepatitis C in 1999 and for HIV—1 in 2004. The current study presents clinical experience with an automated mini—pool NAT system named Zelos x 100. In 2011 the DRK HIV—1 PCR kit was improved by adding a dual target strategy. Additional parameters (HIV—2 and a generic bacterial detection system) were implemented this year. Methods: The Zelos xlOO system is based on a high—volume bead extraction process with a 95% level of detection (LOD) of 0.6 IU/ml, 6.8 IU/ml and 8.9 IU/ml for HBV, HCV and HIV—1, respectively. Results: Four HCV and one HIV pre—seroconversion samples were detected by the mini—pool NAT system (Hepatitis C virus load range: 1.4 × 104 IU/ml − 2.5 × 105 IU/ml; HIV—1 virus load was 3 × 104 IU/ml). Based on window period models, the residual transfusion transmitted infection (TTI) risk was recalculated to 1:458,082, 1:10.88 million and 1:4.3 million for HBV, HCV and HIV—1, respectively. Additional all platelets were screened on day 3 after donation in mini—pools of 10 samples per pool. Shelf life of platelet components with negative MP—NAT results can be extended to five days after donation. Conclusion: The introduction of a complete barcode—controlled automated mini—pool NAT system was successful. Based on the high amount of pre—seroconversion samples detected by the automated system for hepatitis C and the change from an enrichment centrifugation to a bead—based extraction process, Zelos xlOO represents a state—of—the—art procedure comparable to several other commercially available mini—pool NAT systems (like Roche s201 or Novartis Tigris). The Zelos xlOO system convinced with one extraction for transfusion relevant viruses as well as for bacteria.

P2.04

First data on HTLV—I/II seroprevalence in Tyrolean blood donors

Mühlbacher A., Mayersbach P., Grabmer C, Schennach H.

Central Institute for Blood Transfusion and Immunology, University Clinics, Innsbruck, Austria

Background: The aim of the study was to obtain epidemiological data for HTLVI/II seroprevalence in the Austrian federal state of Tyrol and to evaluate the specificity of the ARCHITECT rHTLV—I/II assay. Methods: A total of 4811 randomly selected serum samples of volunteer blood donors from Tyrol was investigated with the fully automated Architect rHTLV—I/II assay. Repeatedly reactive samples were confirmed in the Austrian reference laboratory (Institute for Hygiene, Vienna) using Western blot assays and/or PCR. Results: Out of the 4811 samples 10 had repeatedly reactive results, none of them was confirmed positive. 9 were negative in Western blot and 1 showed a border result, but all 10 samples were negative in PCR. Therefore specificity was 99,79%. Conclusions: Specificity of ARCHITECT rHTLV—I/II assay meets the criteria for blood donor screening. There was no evidence for HTLV—I/II infections in Tyrol, but of course for final proof a higher number of donors must be investigated.

P2.05

CMV—NAT screening of blood donations

Weber—Schehl M.¹, Hedges D.¹, Weinauer F.²

¹Blutspendedienst des BRK gGmbH, Wiesentheid, Deutschland, ²Blutspendedienst des BRK gGmbH, München, Deutschland

Background: According to German guidelines, the use of leukocyte—depleted blood components is an effective strategy of reducing transfusion transmitted human Cytomegalovirus infection. A remaining risk still exists due to non cell—associated Cytomegalovirus (CMV). Therefore, we developed our own in—house CMV—NAT for the detection of free virus in blood plasma. Methods: CMV detection by NAT was performed in minipools (up to 96 samples) from EDTA—plasma. Screening whole blood donations 0.1 ml plasma of each sample was pooled. Testing aphaeresis or stem cell donations, a larger input volume of 1 ml plasma was used. Sample preparation was performed by autoX—System (GFE Blut mbH). Detection of viral genome was done by in—house real—time PCR, using published oligonucleotide sequences. Detection limit of CMV—NAT, with 0.1 ml sample volume of each tested sample, was determined with a sensitivity of 1,000 IU/ml (95% hit rate). IgM and IgG antibodies to CMV were determined by ELISA. Results: 123,765 whole blood donations, 6.636 aphaeresis and 72 stem cell donations were screened for CMV by NAT (11/2010–04/2011), resulting in the identification of 12 CMV—NAT positive donations. One of 13.752 tested whole blood donations was tested PCR positive. CMV infection was confirmed by screening for antibodies to CMV. All 12 DNA positive donations showed detectable levels of antibodies. 2 DNA positive donations were only positive for IgG and 1 only positive for IgM antibodies to CMV. CMV contamination of leukocyte—reduced blood components from CMV—DNA positive donors could be proved by PCR analysis. Conclusion: CMV—NAT screening identified CMV DNA positive donors. Despite leukocyte—reduction Cytomegalovirus could be detected in blood products.

P2.06

Comparison between two pooling robot systems

Hourfar M.K., Capalbo G., Rüster B., Sireis W., GubbeK., Karl A., Tonn T., Seifried E., Schmidt M.

German Red Cross Blood Service Baden—Württemberg – Hessen, Institute of Transfusion Medicine and Immunohaematology, Medical Faculty Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany

Background: Blood safety was improved by the introduction of the mini—pool NAT screening procedure within the last decade. The pooling procedure represents a critical step within the mini—pool NAT screening system and should be optimised by modern robot technologies regarding aspiration and dispense monitoring. The current study examined two modern pipetting robot systems, which included an aspirating and dispensing monitoring system. Methods: In the current study, the Tecan Evo Clinical 2 and the Hamilton MicroLab Star were compared in a direct study with each other regarding precision, accuracy, handling of pre—frozen plasma, handling of lipemic plasma and handling of plasma contaminated with a high virus load (up to 1012 IU/ml of B19 viruses). Results: The overall precision for both systems yielded a VC (variation coefficient) of below 2%, and the accuracy was close to 100% with a range of 97.8% to 100%. Both systems were able to handle pre—frozen plasma and lipemic plasma without any significant differences. The handling of plasma with a high viraemic concentration (contaminated with Parvovirus B19 from 10⁹ to 10¹² IU/ml) was completed without cross contamination. Work flow analysis demonstrated that the Tecan Evo Clinical 2 was able to build 3 minipools per hour with 96 samples per mini—pool, whereas the Hamilton Micro—Lab Star was able to prepare 4 mini—pools per hour. No differences were recognised between the data from the aspirating and dispensing monitoring data and the data regarding the weighing. Summary: Both modern pipetting robot systems were feasible for routine preparations of mini—pools for blood donor screening, and they improved the whole process by including an aspirating and dispensing monitoring system and a weighing of the pipetting steps. Our Blood Donor Service decided to implement the Hamilton pipetting system based on the slight improvement that four pools can be built within one hour per system.

P2.07

Proliferation of bacteria in platelets: a requisite property of international bacteria references – ISBT WP—TTID subgroup on bacteria

Melanie Stoermer¹ Antonio Arroyo², Julia Brachert¹, Hector Carrero³, Dana Devine⁴, Jay S. Epstein⁵, Christian Gabriel⁶, Cohava Gelber⁷, Ray Goodrich⁸, Kay—Martin Hanschmann¹, David G. Heath³, Michael R. Jacobs⁹, Shawn Keil⁸, Dirk deKorte¹⁰, Bernd Lambrecht¹¹, Cheuk—Kwong Lee¹², Jan Marcelis¹⁰, Susanne Marschner⁸, Carl McDonald¹³, Siobhan McGuane¹³, Marian McKee⁶, Thomas H. Mueller¹¹, Tshilidzi Muthivhi¹⁴, Annika Pettersson¹⁵, Piotr Radziwon¹⁶, Sandra Ramirez—Arcos⁴, Henk W. Reesink¹⁷, Julieta Rojo², Ineke Rood¹⁰, Michael Schmidt¹⁸, Christian K Schneider¹, Utta Schurig¹ Erhard Seifried¹⁸, Ute Sicker¹ Silvano Wendel¹⁹, Erica M. Wood²⁰, RoslynA. Yomtovian²¹, Thomas Montag¹¹

¹Paul Ehrlich Institute, Langen, Germany, ²Centro Nacional de la Transfusion Sanguínea, México, ³Walter Reed Army Medical Center, Washington DC, USA, ⁴Canadian Blood Service, Ottawa, Canada, ⁵Food and Drug Administration, Rockville, Maryland, USA, ⁶Austrian Red Cross, Blutzentrale Linz, Austria, ⁷American Type Culture Collection, Manassas, Virginia, USA, ⁸CaridianBCT Biotechnologies, Lakewood, Colorado, USA, ⁹Case Western Reserve University, Cleveland, Ohio, USA, ¹⁰Sanquin Blood Supply Foundation, Amsterdam, The Netherlands, ¹¹German Red Cross, NSTOB, Springe, Germany, ¹²Hong Kong Red Cross Blood Transfusion Service, Hong Kong, China, ¹³NHS Blood and Transplant, London, United Kingdom, ¹⁴South African National Blood Service, Weltevreden Park, South Africa, ¹⁵VU University Medical Centre, Amsterdam, The Netherlands, ¹⁶Regional Centre for Transfusion Medicine, Bialystok, Poland, ¹⁷Academic Medical Center, Amsterdam, The Netherlands, ¹⁸German Red Cross, Frankfurt/Main, Germany, ¹⁹Hospital Sirio Libanês, São Paulo, Brazil, ²⁰Australian Red Cross Blood Service, Melbourne, Australia, ²¹Louis Stokes Veterans Administration Medical Center, Cleveland, Ohio, USA

Background: In order to validate and assess methods for bacterial screening and pathogen reduction, it is crucial to apply bacterial strains which are able to grow in blood components. Aiming to establish the first international Repository for Transfusion Relevant Bacteria Reference Strains (TRBR), the ISBT Working Party on Transfusion—Transmitted Infectious Diseases (WP—TTID), Subgroup on Bacteria, organised an international study on PEI Bacteria References to inter alia demonstrate the ability to proliferate in platelet concentrates (PCs) worldwide. Method: Four TRBR (Staphylococcus epider—midis PEI—B—06; Streptococcus pyogenes PEI—B—20; Klebsiella pneumoniae PEI—B—08; E. coli PEI—B—19) were prepared by the Paul—Ehrlich—Institute (PEI) as deep—frozen suspensions which are stable, shippable on dry ice, defined in count of living bacterial cells and able to grow in PCs. The TRBR were distributed to 14 laboratories from 10 different countries that were asked to inter alia determine their ability to grow in PCs after low spiking (∼ 0.3 and 0.03 CFU/ml). Results: All data received included the growth results of 12 of 14 laboratories. In general K. pneumoniae, St. epidermidis, and E. coli replicated in all participating laboratories worldwide (Austria, Canada, China, Germany, Mexico, Poland, South Africa, The Netherlands, UK, USA). Str. pyogenes grew in all regions with the exception of Mexico. Conclusion: The international study was organised to demonstrate the proliferation in PCs from donors of different regions of the world, which could be confirmed in general. On account of this property these TRBR can be used as a worldwide tool for development, validation and comparison of bacteria screening and pathogen reduction methods. The WHO Expert Committee Biological Standardisation (ECBS) has approved the adoption of these four PEI Bacteria References as the first Repository for Transfusion Relevant Bacteria Reference Strains.

Actually the author list included 38 authors in total because everyone was involved in the study and publication.

P2.08

Seroprevalence of antibodies to Borrelia burgdorferi in a healthy Swiss blood donor population

Tinguely C.H.¹, Engler O.², Niederhauser C.¹, Fontana S.¹, Tschaggelar A.¹, Strasser M.², Mütsch M.³

¹Blood Transfusion Service SRC Berne, Bern, Switzerland, ²Spiez Laboratory, Federal Office for Civil Protection, Spiez, Switzerland, ³Institute of Social and Preventive Medicine, University of Zurich, Zurich, Switzerland

Background: Lyme borreliosis is the most common tick—borne infectious disease and is endemic in nearly the whole of Switzerland, the only exception being higher regions of the Alps. At present, little is known on the seroprevalence of antibodies to Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, in the Swiss population. Older studies have indicated a seroprevalence range between 0.6% − 10.7%. The aim of this investigation was to evaluate current seroprevalence of antibodies to Borrelia burgdorferi within healthy blood donors from rural regions of Switzerland. All the donors were informed and gave their consent. Methods: 4580 blood donors were screened by the recomWell Borrelia IgG ELISA (Microgen Diagnostics). Those IgG positive donors who reported knowledge of a previous tick bite, or symptoms related to an active Lyme borreliosis during the last 6 months, or had a Borrelia diagnosis confirmed by their physician, were further confirmed by the recomLine Borrelia IgG Immunoblot. Results: From the initially screened 4580 donors 551 (12.03%) were found to be positive for anti—Borrelia IgG antibodies, 36 (0.79%) had a result near the cut—off. 238 were further analyzed by Immunoblot. From these donors 188 (79%) were confirmed positive, 17 were borderline positive and 33 were negative. The immunoblot profiles indicated the contact with Borrelia burgdorferi had occurred previously as there was an existing IgG immune response. Conclusion: The high seroprevalence (12.03%) in this population is surprising. As 188 (79%) of these blood donors were confirmed positive without current disease and only 22 (9%) had a borreliosis, this shows the course of most Borrelia infections is subclinical. The detection of anti—Borrelia IgG antibodies must be interpreted with caution and always in a clinical context with symptoms. As the donors are in a recovered, not bacteremic phase, they should therefore be allowed to continue to donate their blood.

P 2.09

Routine screening of platelet concentrates for bacterial contamination using the BactiFlow flow cytometry: results of a multicenter study

Vollmer T.¹, Schottstedt V.², Bux J.², Tapernon K.³, Sibrowski W.³, Kleesiek K.¹, Knabbe C.¹, Dreier J.¹

¹Institut für Laboratoriums— und Transfusionsmedizin, Herz— und Diabeteszentrum NRW, Bad Oeynhausen, Deutschland, ²DRK Blutspendedienst West Zentrallabor, Hagen, Deutschland, ³Institut fuer Transfusionsmedizin und Transplantationsimmunologie, Universitätsklinikum Münster, Münster, Domagkstr. 11

Background: Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk. As a result of septic complications, particularly observed with older PCs, the shelf life has been reduced to four days in Germany. In the present study, a novel flow cytometry—based rapid bacterial screening method (BactiFlow, BF) was implemented as a routine in—process control to extend the shelf life of PCs. The robustness and applicability of the assay was further evaluated in two other German blood services. Methods: 2939 PCs (apheresis and pooled PCs) were tested using the BF to detect and count bacteria based on esterase activity in viable cells. The BacT/Alert culture system served as reference method. PCs were tested on day 2, 4 and 6 after PC production. Results: Six PCs were tested confirmed positive by culture only (aerob and anaerob) and were identified as Propionibacterium species (n = 1) or Staphylococcus species (n = 5). Additionally, six PCs were culture positive only in one culture bottle (aerob n = 1, anaerob n = 5). Bacteria were identified as Streptococcus mitis (n = 1), P. acnes (n = 1) or Staphylococcus species (n = 4). In these PCs, corresponding bacterial titers were most likely below the BF detection limit (< 150 CFU/mL) and had probably no transfusion relevance. Two PCs were positive for S. mitis by BF and culture. All remaining specimens were tested negative. Conclusions: The BactiFlow flow cytometric assay is a sensitive method for bacterial screening of PCs and is most applicable as a routine in—process control.

P2.10

Flow cytometric detection of bacteria in stem cell products

Rehfeld I.S.¹, Kreissig C.¹, Bux J.²

¹DRK Blutspendedienst West – ZBST, Ratingen, Deutschland, ²DRK—BlutspendedienstWest, Hagen, Deutschland

The traditional system for detection of microorganism in stem cell products is Bact/Alert system. This system provides many advantages and two disadvantages: First products with a high count of white cells may cause false— positive results. Second seven days of incubation are needed, so results are available long time after transplantation of allogeneic products. In this study we compared Bact/Alert with Bactiflow—system, a flow cytometry—based screening method for bacterial contamination. We examined 43 peripheral blood stem cell product (27 allogeneic, 16 autologous) and 10 bone marrow products (allogeneic). We got equal results in 51 cases. No bacterial contamination was detected in these samples. Two samples were contaminated with germs. We observed a positive result in Bact/Alert system after 96h in the aerobic bottle of autologue PBSC aliquot, differentiated as Staphylococcus epidermidis. We got a second positive result in Bact/Alert system after 27h in the aerobic bottle and 32h in the anaerobic bottle of allogeneic PBSC aliquot, differentiated as Staphylococcus auricularis. We did not detect these germs in Bactiflow system after 24h. In 3 cases Bactiflow system showed false—positive results. 5 anaerobic bottles were flagged as unspecific positive by the Bact/alert system in whereas Bactiflow results were negative. Colony forming assays of these samples were negative. The Bact/Alert system shows ongoing issues over 7 days. Bactiflow testing is finished after 24 hours. Lower bacteria growth after 24 h of incubation could not be detected using Bactiflow system. Because of lower series of samples per day the test is labour and cost intensive. But if you want to differentiate positive Bact/Alert results in specific and unspecific positive result the additional testing with Bactiflow system may be a valuable tool to get rapide analysis results.

P2.11

Challenges in the microbial safety of cell therapeutics

Montag T., Störmer M., Spreitzer I., Schneider C.K., Schurig U.

Paul—Ehrlich—Institut, Fachgebiet Mikrobiologische Sicherheit, Langen, Deutschland

Problems regarding microbial safety of cell therapeutics are not more than partially solved. The sterility of source for cell based medicinal products cannot be guaranteed in many cases. Cells and tissues would not survive a heat treatment, and sterile filtration would hold back the cells. As a rule, these source materials cannot be sterilised, as it holds true for the final products. Furthermore, the established methods for sterility testing are not applicable for cell preparations. Since there are some parallels in the warranty of microbial safety of cellular blood components and cell therapeutics, the experiences collected in transfusion medicine in the past decades can be successfully used in a safe production of cell based medicinal products. Comparably to the situation regarding cellular blood components, there is a need for new principles in rapid bacteria detection considering the extremely short shelf life of much cell therapeutics. Relevant limitations regarding microbial safety of cell therapeutics and pyrogenicity are demonstrated. Current opportunities are discussed how to handle the problems. Additionally, some novel solutions for the rapid sterility testing of cell products are proposed in comparison to classical sterility testing. The established methods for sterility testing are not applicable for cell preparations. Therefore the warranty of microbial safety of cell based medicinal products is a challenge for industry and authorities. A new understanding is required as there are fundamental differences to the approved and well defined manufacturing processes of conventional drugs. On the other hand, there are many similarities between cell based medicinal products and cellular blood components regarding potential bacterial contamination. Thus, the experiences of transfusion medicine collected mainly in the past decade can be successfully used in improvement of microbial safety of cell based medicinal products.

P 2.12

UVC treatment of platelet concentrates in the Theraflex UV—Platelets System only slightly affects disulfide bonds on platelet proteins

Knüver—Hopf J., Gravemann U., Lambrecht B., Mueller T.H., Seltsam A.

DRK—Blutspendedienst NSTOB Institut Springe, Springe, Deutschland

Background: Irradiation with short ultraviolet light (UVC) using the THERAFLEX UV—Platelets system results in effective pathogen inactivation of platelet concentrates (PCs). The UVC light directly interacts with nucleic acids mainly causing dimerization of thymins. However, UVC may also induce alterations of platelet (PLT) proteins. In the present study, the impact of the Theraflex UV—Platelets procedure on the integrity of disulfide bonds of PLT membrane proteins was investigated. Methods: Plasma—reduced, pooled PCs derived from buffy coats were treated with the Theraflex UV—Platelets system. Besides the standard UVC dose of 0.2 J/cm², higher UVC doses of 0.4 and 0.6 J/cm² were used. Untreated PCs and gamma—irradiated PCs were used as controls. A set of in vitro parameters was used to assess the PLT quality. The amount of free thiol groups on the PLT surface as a parameter for disrupted disulfide bonds was measured using maleimide which has a high binding affinity to free thiol groups. Results: UVC irradiation caused a dose—dependent increase in free thiol groups on the surface of PLTs. More specifically, a UVC—induced increase in free thiol groups could also be detected for the fibrinogen receptor. However, in contrast to the higher UVC doses, the amount of free thiol groups on PLTs and on the fibrinogen receptor was only slightly increased when using the standard UVC dose of 0.2 J/cm² of the THERAFLEX UV—Platelets procedure. Conclusions: The results of this study showed that UVC—irradiation of PLTs causes disruption of disulfide bonds of proteins on the cell surface. Expression of free thiol groups on the fibrinogen receptor may be responsible for an increased activation of UVC—treated PLTs. However, data obtained for the UVC dose of 0.2 J/cm² suggest that treatment of PCs with the THERAFLEX UV—Platelets procedure may only slightly affect PLT quality as it seems to activate only a minor part of the GPIIb/IIIa complexes.

P 2.13

Polymerase chain reaction inhibition assay documenting the pathogen reduction process of platelet concentrates treated by UVC in the Theraflex UV—Platelets system

Knüver—Hopf J., Gravemann U., Mueller T.H., Seltsam A.

DRK—Blutspendedienst NSTOB Institut Springe, Springe, Deutschland

Background: The pathogen reduction procedure, called THERAFLEX UV—Platelets system, is based on application of UVC light of a specific wavelength (254 nm) combined with intense agitation of the platelet concentrates (PCs). UVC inactivates pathogens mainly by its direct interaction with nucleic acids. This results predominantly in formation of cyclobutane pyrimidine and pyrimidine pyrimidone dimers, which block the elongation of nucleic acid transcripts. Platelets contain viable mitochondria and mitochondrial DNA (mtDNA) and this study aimed at evaluating UVC—induced mtDNA alterations in platelets (PLTs) derived from PCs treated by the THERAFLEX UV—Platelets system. Methods: Plasma—reduced PCs prepared from pools of buffy coats and stored in additive solution (SSP+, MacoPharma, Langen, Germany) were treated in the THERAFLEX UV—Platelets system using the standard UVC dose of 0.2 J/cm² and higher doses of 0.4 and 0.6 J/cm². A mtDNA—specific long—range real—time polymerase chain reaction (PCR) assay was performed one day after irradiation using primers selected to amplify a 1059 base—pair fragment of the 16S rDNA region. Results: Long—range real—time PCR revealed that mtDNA in PLTs was dose—dependently degraded after treatment in the THERAFLEX UV—Platelets system. Compared to the results obtained with mtDNA from untreated and gamma—treated PCs which served as controls, the mtDNA—specific PCR signal was reduced by 2 log₁₀ steps after irradiation with the UVC standard dose of 0.2 J/cm² and further inhibited each by 1 log₁₀ step when UVC doses of 0.4 and 0.6 J/cm² had been applied. Conclusions: A PCR inhibition assay was established with a large amplicon demonstrating the influence of UVC—irradiation on mtDNA in PLTs. Due to its susceptibility to UVC—induced mtDNA modification this assay can be used to document the success of pathogen reduction treatment in the THERAFLEX UV—Platelets system.

P 2.14

Critical evaluation of available pathogen inactivated plasma products in France

Kientz D.

EFS—Alsace, Hopitaux Universitaires de Strasbourg, Strasbourg, France

Background: Currently three licensed plasma products constitute the 370,000 therapeutic plasma units transfused annually in France: Solvent Detergent plasma (SD FFP; 38%), Methylene Blue/VIS light plasma (MB FFP; 55%) and Amotosalen/UVA light plasma (IBS FFP; 6%). Methods: We compared the efficacy and safety data of the products, and evaluated hemovigilance data obtained after routine use in France. Results: Comparative studies of the three products demonstrated that the coagulation factors meet EU standards. However, MB FFP Fibrinogen and SD FFP Protein S are reduced. Modification of HIS residues in Fibrinogen in MB FFP may cause this reduction. The ability to inactivate pathogens met basic requirements for all products and IBS—FFP had the broadest spectrum of inactivation for viruses, bacteria and parasites. Randomized clinical trial data prior to regulatory approval are only available for IBS FFP based on 3 trials demonstrating equivalence to control plasma for the support of congenital and acquired clotting factor deficiencies and TTP. In prior studies SD FFP was equivalent to control FFP for patient support, whereas MB FFP was found to require more exchanges, or a larger volume to support patient needs in TTP or surgery (liver or CV). Concerns over allergic reactions with MB FFP arose from Hemovigilance reports of a higher rate per 104 products for Allergies (5.56 vs 3.05 (SD FFP) and 3.36 (PCT FFP)) and a significant number of severe allergic reactions (0.58 vs. 0.28 for SD FFP and 0 for PCT FFP1). Three severe reactions were also reported separately and in follow up investigations proven to be connected to MB. Conclusions: SD FFP, MB FFP and IBS FFP have been implemented to reduce the risk of transfusion—transmitted infection. Some differences have been found for the coagulation factors after MB treatment, while significant concerns remain with regard to severe anaphylactic reactions strongly associated with transfusion of MB FFP.

Session 03 – Cell Therapy: Hematopoietic Stem Cells

P 3.01

Influence of the cryo—protectant DMSO on the detectability of bacteria in sterility testing of cell therapeutics

Schurig U., Störmer M., Brachert J., Sicker U., Schneider C.K., Montag T.

Paul—Ehrlich—Institut, Fachgebiet Mikrobiologische Sicherheit, Langen, Deutschland

Background: DMSO (dimethyl—sufoxide) is widely used as a protecting agent in cryo—preservation of cell therapeutics. Data on its influence on bacterial growth and on the outcome of testing for sterility are rare in literature. This study shows whether DMSO in concentrations as usually applied in cell therapeutics are able to inhibit bacterial growth and may influence the detection of microorganisms in sterility testing using automated culture systems. Methods: A) Klebsiella pneumoniae PEI—B—08, B) Staphylococcus epidermidis PEI—B—06, C) Escherichia coli PEI—B—19, D) Staphylococcus aureus PEI—B—23, E) Propionibacterium acnes DSM 1897, F) Micrococcus species ATCC 700405 were used for low count spiking. At first A, B, and C were cultivated in the BacTrac in the presence of 0%, 0.5%, 1%, 2%, 4%, 6%, 8%, and 10% DMSO. Secondly, supposed samples containing 2–10% DMSO were spiked with strains A—F and analyzed in the BACTEC and BacT/Alert. A worst—case scenario was disposed applying final concentrations of 10% DMSO. Results: Up to a final DMSO concentration of 4% in the cultivation medium no impairment of bacterial growth was observed for all bacterial strains. Starting with 6% DMSO, a slight delay in growth kinetics was observed becoming more obvious with 8% and 10% DMSO. Only strain C showed significant delay in growing at 8% and no growth in the presence of 10% DMSO. No obvious difference when samples containing 10% DMSO (medium concentration of 0.5%) were compared with control samples without DMSO. Conclusion: Concentrations of DMSO in cell and tissue preparations are usually specified between 5% and 10%. Addition of test sample volumes between 1 and 10 ml into blood culture bottles for automated systems will result in final DMSO concentrations between 0.1% and 2% in the culture. For these conditions, the study results give no indication for an inhibition of bacterial growth or false negative results, respectively, in sterility testing using automated culture systems.

P3.02

Long—term storage of human peripheral peripheral blood stem cells (PBSC) in a mechanical (—150°C) freezer compared to storage over liquid nitrogen

Sputtek A.¹, Peine S.², Rowe A.W.³

¹University Medical Center Hamburg—Eppendorf, Dept. of Transfusion Medicine, Hamburg, Germany, ²Institute for Transfusion Medicine, University Hospital Hamburg Eppendorf, Hamburg, Germany, ³New York University School of Medicine, New York, USA

The aim of this study was to investigate the equivalence of 2 different cryogenic storage conditions for PBSC, namely storage in a −150°C mechanical freezer vs. storage in the vapor phase (below −170°C) over liquid nitrogen (LN2). Long—term storage stability is required for preservation of autologous PBSC in high—dose chemotherapy protocols for patients with various malignancies. The study material consisted of 24 frozen autologous transplant units (= 12 pairs of identical units) which were no longer needed. All units (volume 105 ± 5 ml) containing 10% (v/v) dimethyl sulfoxide had been cooled down by means of a controlled rate LN2 operated freezer to −40°C at approx. 1°C/ min, followed by cooling at 10°C/min down to −100°C. Thereafter the units were immediately transferred to the vapor phase of a LN2 storage tank for 4 to 127 months (average 73 months). The pairs were split and one unit of each pair was transferred into a −150°C mechanical freezer (MDF—1156, SANYO), the other unit remained in the vapor phase over LN2. The following storage period at each temperature amounted to 16.4 months (range 15.7 to 16.7). The highest temperature measured inside the bag stored in the mechanical freezer did not exceed −143°C, the lowest was not below −150°C, and the “baseline” temperature for the mechanical freezer was at −148°C. The “baseline” temperature inside the control bag stored over LN2 was −193°C with a range of −196°C and −177°C. After thawing we found no significant differences (student's t—test) and high correlation coefficients (r > 0.90) between both groups for WBC concentration, membrane integrity under the microscope (trypan blue exclusion), by FACS as 7—AAD exclusion and CD34+ recovery. Clono—genicities (e.g. CFU—C, BFU—E) measured in a semi—solid cell culture assay did not differ as well. These data support the conclusion that a reliable −150°C freezer is as suitable for long—term storage of PBSC as storage in the vapor phase over LN2.

P3.03

MHC resequencing highlights potential transplantation determinants in HLA identical haematopoietic stem cell transplantation

Pröll J., Damer M., Niklas N., Stabentheiner S., Hofer K., Gabriel C.

Red Cross Transfusion Service for Upper Austria, Linz, Austria

In haematopoietic stem cell transplantation adverse effects like graft—versus—leukaemia (GVL) and graft—versus—host—disease (GVHD) are observed despite full human leukocyte antigen (HLA) matched donor—recipient pairings. In addition to HLA major histocompatibility complex (MHC) resident immuno—regulatory genes and other unidentified transplantation determinants have been proposed to contribute to GVHD. In a clinical transplant setting with 10/10 HLA—loci matched and a patient died of acute GVHD we performed MHC resequencing, genome wide Single Nucleotide Polymorphism (SNP) analysis and compared data. Microarray sequence capture and targeted enrichment combined with next generation pyrosequencing (Roche/454Life Sciences) are used for analysing the 3.5 million base pair human MHC. We show that direct resequencing provides the near—complete information with 87% of the MHC at median coverage of 16 in a single experiment. Analyzing donor and recipient sequences directly to each other at the nucleotide resolution level reveals 4,554 genotypic differences. These variant positions locate 53% to intergenic, 8% to exonic and 9% to intronic regions. 551,954 SNPs (62%) among a total of 885,576 are concordant between donor and recipient genome wide. Whereas, concerning SNPs inside MHC 1,365 (87%) match. Using this approach a total of 270 genes can be analysed regarding potentially important aminoacid exchanges or other structural discrepancies. Taken together, the presented data show, that sequence capture and massively parallel pyrosequencing can be used as a new practical tool for extended MHC analysis.

P3.04

Impact of KIR ligand mismatches in hematopoetic stem cell transplantation – Antithymocyte globulin therapy matters

Richter R.¹, Serve H.², Seifried E.¹, Bug G.², Martin H.², Seidl C.¹

¹DRK—Blutspendedienst Baden Württemberg – Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Klinikum der Johann Wolfgang Goethe—Universität, Frankfurt, Deutschland, ²Department of Internal Medicine II – Hematology and Oncology, Stem Cell Transplant Unit, Johann Wolfgang Goethe University, Frankfurt am Main, Germany

Natural killer (NK) cells play a major role in rejection of tumors and cells infected by viruses. In SCT, inhibitory KIR receptors – regulating the activity of NK cells – are suggested to be a predictor for the success of SCT. In addition, in vivo T—cell depletion by antithymocyte globulin (ATG) treatment prior to SCT is suggested to be critical for the HLA—KIR ligand—mismatched SCT. To evaluate the impact of inhibitory KIR receptors in allogeneic SCT and the impact of ATG pre—treatment on the KIR mediated effect we performed a study with 243 patients with haematological malignancies (137 AML, 29 CML, 13 MDS, 38 ALL, 4 CLL, 6 NHL, others). Patients were transplanted with HLA matched unrelated— or family related donors. Clinical analysis included relapse, transplant—related—mortality, event free survival, and overall survival (OAS) with a follow—up of 11 years. Our results confirm that a donor KIR2DL – recipient HLA—C ligand mismatch (MM) is associated with an improved clinical outcome (Log—rang test: p = 0.13). Contrary, a lack of KIR2DL1 or KIR2DL3 genes in the donor is associated with a significantly reduced OAS (Log—rang test: p = 0.02). Considering the donor KIR2DL —recipient HLA—C—MM, the benifical effect was exclusively found in transplants without ATG treatment (5 year OAS; no ATG treatment: KIR2DL —HLA—C—mismatch group − 71%; KIR2DL —HLA—C—match group − 37%) whereas in the patients with ATG pre—treatment the OAS in the KIR2DL —HLA—C—mismatched and matched group was equal (5 year OAS; ATG treatment: KIR2DL —HLA—C—mismatch group − 50%; KIR2DL —HLA—C—match group − 54%). In conclusion, our results support the significance of NK cells and their KIR2DL repertoire in the immune control of haematological malignancies. ATG treatment might abrogate the beneficial impact of the KIR —HLA—C MM and the negative impact of a KIR – HLA—C match constellation. Therefore, patients with SCT would benefit from KIR typing and evaluation of the KIR – HLA MM constellation.

P3.05

Analysis of transcription factors differentially expressed in the primitive human hematopoietic compartment

Jansen S.¹, von Levetzow G.², Görgens A.¹, Hanenberg H.³, Horn P.A.¹, Giebel B.¹

¹Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, ²University of Southern California, Children's Hospital Los Angeles, Los Angeles, USA, ³Department for Pediatric—Oncology, —Hematology and Clinical Immunology Heinrich—Heine—University Düsseldorf, Düsseldorf, Germany

Somatic stem cells are required to maintain homeostasis in different tissues. In this context stem cells give rise to differentiating cells which replace cells getting lost in the lifetime of a multi—cellular organism. To fulfil this function over time, the pool of stem cells needs to remain relatively constant. Since both, abnormal loss as well as uncontrolled expansion of stem cells is fatal for organisms, the decision of self—renewal versus differentiation needs to be tightly regulated. A number of transcription factors have been identified to take part in the decision process self—renewal versus differentiation of primitive hematopoietic stem cells, including HoxB4, AML1/Runx1, SCL/Tal1, Meis1. While loss of function of these transcription factors is generally associated with defects in the development of the hematopoietic system, the aberrant expression often results in an expansion of primitive hematopoietic cells and seems to be connected to different forms of leukemia. With the aim to identify additional transcription factors required for the self—renewal process of primitive human hematopoietic cells, we have performed genome wide GeneChipTM analyses of different cell fractions, containing either primitive or more mature hematopoietic cells. We identified a number of transcription factors encoding genes which are specifically expressed in the most primitive hematopoietic cell fractions, whose function has not yet been associated with hematopoiesis. In order to characterize the early hematopoietic function of some of these candidate genes, we decided to perform over expression as well as RNAi mediated knock down experiments. We are using a lentiviral transduction to genetically manipulate primary human umbilical cord blood derived CD34+ cells and analyze effects on the cell fates of transduced cells in different functional read out systems.

P 3.06

Functional analyses of cell polarity organization in human hematopoietic stem and progenitor cells

Görgens A.¹, Beckmann J.², Horn P.A.¹, Rajendran L.³, Giebel B.¹

¹Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, ²Institut für Transplantationsdiagnostik und Zelltherapeutika, Düsseldorf, Germany, ³Department of Psychiatry Research, University of Zurich, Zurich, Switzerland

Hematopoietic stem and progenitor cells (HSPC) contain capabilities to home to specific bone marrow niches. Providing a basis for their clinical use, several studies have analyzed mechanisms involved in HSPC homing. Many molecules, including the chemokine receptor CXCR4, phosphoinositol—3—kinase (PI3K), atypical protein kinase C (aPKC) and Rho—GTPases have been associated with HSPC homing and control sub—cellular processes allowing cellular migration in vivo and in vitro. Since cell polarization is a prerequisite for cellular migration and we aim to unravel mechanisms controlling establishment and maintenance of human HSPC polarity, we investigated impacts of pharmaceutical substances on this process that are known to interfere with HSPC homing. To complete our studies we also applied drugs which affect protein synthesis, the integrity of the cytoskeleton or that of lipid rafts. By analyzing sub—cellular distributions of lipid raft—associated proteins in HSPCs, we were able to distinguish two levels of cellular polarization, a molecular and a morphological cell polarization level. Our data suggest that PI3K, aPKC activities, protein synthesis and lipid raft integrity are required to organize the molecular cell polarity. The morphological cell polarization process additionally depends on actin polymerization and rho—GTPase activities. Pertussis Toxin, an inhibitor of stromal derived factor—1 (SDF—1)—mediated chemokine signaling, did not affect cell polarity but inhibited in vitro migration of investigated HSPCs. Since all drugs affecting the morphological cell polarity also reduced in vitro migratory capabilities of treated cells, our data qualify HSPC polarization processes as new pharmaceutical targets to interfere with migratory and possibly homing capabilities of HSPCs. Currently, we investigate whether components of the evolutionary conserved cell polarity organizing Par/aPKC complex control HSPC polarization and hematopoietic cell fate specification processes.

P3.07

Enumeration of CD34+ HPC and lymphocyte subsets in CD34+ selection of HPSC—A for transplantation: comparison of a dual— (DPM) with a single—platform method (SPM)

Wagner B.¹, Oduncu F.², Wittmann G.¹, Grützner S.¹, Schramm W.¹, Albert M.H.³

¹Abteilung Transfusionsmedizin und Hämostaseologie, Klinikum der Universität München – Campus Großhadern, München, Deutschland, ²Medizinische Klinik Schwerpunkt Haematoonkologie, Klinikum der Universität München – Campus Innenstadt, München, Deutschland, ³Dr von Haunersches Kinderspital, Zentrum für Pädiatrische Hämatologie und Onkologie, Klinikum der Universität München, München, Deutschland

Background: The recent 6th edition of the European Pharmacopoeia describes a SPM for the enumeration of CD34+ hematopoietic progenitor cells (HPCs) in cell products thus causing manufacturers to change their protocols. CD34 selection of PBSC—Anot only enriches CD34+ HPCs, but also depletes other cells. The dose of CD34+ HPCs and residual T cells is highly critical in the transplantation of CD34 selected grafts thus requiring validation of their enumeration. Patients and Methods: 5 PBSC—A from 4 patients with high risk neuroblastoma were subjected to 4 CD34+ selection procedures (Clini—MACS, Miltenyi Biotec). The starting material (SM) and the fractions preselection, positive (PF), negative and buffer waste were analysed in parallel by SPM and DPM on a blood counter (KX—21, Sysmex) and a flow cytometer (Calibur, BD) for the determination of NCs, CD34+ HPCs and lymphocyte subsets. For SPM TruCOUNT tubes and the Stem Cell Enumeration Kit (BD) were used. Accordingly lymphocyte subsets were determined applying CD3, CD19 and CD16/56 fluorescence conjugated antibodies (BD). Viability was determined using 7—AAD. Results were analysed for agreement by Bland—Altman plot, Spearman's rank correlation and Wilcoxon—Test. Results: The mean counts in SM measured by DPM and SPM were 93 NC/nl vs. 94/nl, CD34+ HPCs 1.6/nl and 1.7/nl, T, B, NK cells 8.2/nl, 1.1/nl, 1.3/nl vs. 8.3/nl, 1.1/nl, and 1.4, respectively. The graft (PF) contained 3.7 NC/nl vs. 4.1/nl, CD34+ HPCs 3.4/nl and 3.8/nl, T, B, NK cells 0.6/μ1, 0.9/μι, l.l/μl vs. 0.7/ μl, 1.1/μ1, 1.2/μ1, respectively. HPC doses were 22.2 vs. 22.6∗10e6/kg in SM and 15.7 vs. 17.3 in PF. In spite of a 2—4 logs’ range the results including viability showed an excellent agreement according to the tests described above. Conclusions: The results demonstrate the equivalence of DPM and SPM. Further data revealed a higher consistency of SPM at the lower end of the measuring range. Thus SPM should be preferred for the analysis of CD34+ selected grafts.

As to my opinion your policy not to consider tables for printing is unfavourable for abstracts like mine on the agreement of methods. Otherwise you would have been provided a table with the summary of all results which I would prefer as an interested reader of your abstract book.

P3.08

Matched—pair analysis of peripheral blood progenitor cell apheresis: Spectra Optia vs. COBE Spectra

Rox J.M.¹, Wenzel F.¹, Kobbe G.², Fischer J.C.³

¹Institute of Transplantation Diagnostics and Cellular Therapeutics, University Hospital Düsseldorf, Düsseldorf, Germany, ²Klinik für Hämatologie, Onkologie und Klinische Immunologie, Düsseldorf, Deutschland, ³Institut für Transplantationsdiagnostik und Zelltherapeutika, Düsseldorf, Deutschland

Peripheral blood progenitor cells (PBPC) are the most common stem cell source for autologous or allogeneic transplantation. Recently a novel automated apheresis system (Spectra Optia, Caridian BCT) was introduced. The Spectra Optia system provides an automated buffy coat interface control and combines continuous centrifugation with subsequent cellular collection into an elutriation chamber. PBPCs are harvested intermittently from this chamber. In contrast, its predecessor the COBE Spectra MNC apheresis system permits manually controlled continuous PBPC harvesting. 50 consecutive apheresis procedures using the Spectra Optia were analyzed. These donations were matched by type of donation, gender, vein access, weight, total blood volume (TBV), and pre collection WBC count to a database of historical donations using the COBE Spectra. Apheresis procedures were performed according to the manufacturer's recommendations with exception of anticoagulation (using ACDA with Heparin to allow as fast as possible inlet flow rates). In all procedures, collection efficiency (CE2, calculated by CD34+ cells collected/CD34+ cells processed: 52% ±17 vs 46% ±10) and collection ratio (CR) per processed TBV (calculated by CD34+ cells collected/kgBW/CD34+ cells in TBV: 0.034 ±0.011 vs 0.030 ±0.007) were better for the Spectra Optia than for the COBE Spectra. Throughput (TP, calculated by CD34+ cells collected/kgBW/minute/ CD34+ cells in circulation), which takes apheresis time into account, was better for the COBE Spectra than for the Spectra Optia (540 ±195 vs 373 ±132). Subgrouping allogeneic procedures there were no differences in CE2 and CR per TBV between the two devices. In autologous procedures CE2 and CR per TBV were better for the Spectra Optia. Compared to the COBE Spectra the Spectra Optia harvests PBPC with better (autologous patients) or equal (allogeneic donors) collection efficiency. However, due to its discontinuous mode of PBPC harvesting apheresis time is longer.

P 3.09

In vitro expansion of hematopoetic stem— and progenitor cells from cord blood in co—culture with MSC from different placenta tissues, cord blood and bone marrow

Klein C., Strobel J., Zingsem J., Zimmermann R., Eckstein R., Weisbach V.

Transfusionsmedizinische und Hämostaseologische Abteilung, Universitätsklinikum Erlangen, Erlangen, Deutschland

Background: The therapeutic efficacy of cord blood (CB) hematopoietic progenitor cell (HPC) transplantation is limited by the low cell dose of CB. It has been shown that cell dose can be increased by ex vivo CB expansion in co—culture with bone marrow—derived mesenchymal stroma cells (MSC). We investigated whether bone marrow—derived MSC can be replaced by MSC from the placenta, which are readily available. Patients and Methods: MSC were prepared from amnion, chorion, wharton's yelly, amniotic fluid, cord blood and bone marrow. Characterisation of MSC was done by classic means: plastic adherence, flow cytometry and adipogenic, chondrogenic and osteogenic differentiation. 14 day dual stage ex vivo cultures of mononuclear cells (MNC) containing 5 × 10³ to 1 × 10⁵ CD34+ cells from cryopreserved cord blood were performed in 9,6 cm² wells in medium supplemented with G—CSF, SCF and TPO in co—culture with different MSC. Expansion was followed by measuring total nucleated cell (TNC), CD34+ cell, colony—forming unit (CFU) and LTC—IC output. Results: MSC of all origins showed a unique pattern in multi parameter flow cytometry (positive for CD29, CD90, CD73, CD44 and HLA class I, negative for CD14, CD34, CD45 and HLA class II) and differentiation experiments. Expansion was higher when a smaller initial number of MNC containing a lower count of CD34+ cells was cultured (1 × 10⁴ versus 1 × 10⁵ CD34+ cells). We observed no expansion of LTC—IC. The expansion of CD34+ cells/CFU in co—culture with MSC from different origins was: Amnion 4962/12357; Chorion 2960/2259; Wharton's Jelly 12217/6553; cord blood 12486/25400; amniotic fluid 36246/23547; bone marrow 97376/121919. Conclusions: There was no expansion of hematopoetic stem cells, no expansion of LTC—IC was observed. The expansion of hematopoietic progenitor cells from cord blood was successfull in co—culture with MSC from the placenta but less effective compared to co—culture with bone marrowderived MSC.

P3.10

Successful autologous stem cell mobilisation after chemotherapy with G—CSF and Plerixafor (Mozobil^®) in a 2 9/12 year old child

Hölig K.¹, Zimmer K.¹, Hermanns V.², Bornhäuser M.³, Knöfler R.², Suttorp M.²

¹Medical Clinic I, Department of Transfusion Medicine, University Hospital Dresden, Dresden, Germany, ²Department of Pediatrics, University Hospital Carl Gustav Cams, TU—Dresden, Dresden, Germany, ³Department of Internal Medicine I, University Hospital Carl Gustav Cams, TU—Dresden, Dresden, Germany

Background: Plerixafor (Mozobil^®) has been approved for the mobilisation of autologous peripheral blood stem cells (PBSC) since 2010. There is still limited experience with this mobilisation regimen in children with solid tumors. We report on the successful mobilisation of autologous PBSC in a 2 9/12 year old girl with neuroblastoma. Case report: A girl was diagnosed with neuroblastoma (stage IV with 10—20% bone marrow infiltration) at the age of 2 5/12 years. In accordance with the NB—2004 protocol of the Society of Pediatric Oncology and Hematology (GPOH) she was treated in the high risk group with 6 cycles of chemotherapy. Following the 4th cycle PBSC mobilisation was attempted with G—CSF (10μg/kg). During the 1st autologous stem cell apheresis (6.0 × blood volume, Cobe Spectra, Caridian BCT) 1.2 × 106/ kg CD 34+ cells could be collected. A 2nd PBSC—mobilisation was undertaken after the next cycle of chemotherapy with G—CSF (10μg/kg) and Plerixafor (240μg/kg). The 2nd collection revealed 1.7 × 106/kg CD 34+ cells. Both apheresis products underwent CD 34+ selection with Clinimacs® (Miltenyi) according to the treatment protocol. Results: Leucocyte counts (WBC), peripheral CD 34+ counts and apheresis yields with G—CSF alone and after administration of G—CSF plus Plerixafor are shown in table 1. The patient underwent high dose chemotherapy (Melphalan, Etoposide, Carboplatin) and was transplanted with 2.0 × 106 CD34+cells/kg. Engraftment was delayed (need for platelet and red cell transfusion for about 3 months). The patient is alive, well and in complete remission of her underlying disease 6 months after autologous PBSC transplantation. Conclusion: Administration of Plerixafor enabled mobilisation and collection of a sufficient amount of CD 34+ cells for autologous transplantation in a heavily pretreated 2 9/12 years old child with neuroblastoma. Plerixafor had no side effects and mobilised peripheral blood stem cells effectively in this toddler.

Table 1 for abstract P 3.10

Mobilisation	WBC [×109/l]	Peripheral CD34 count [/μl]	CD 34 yield [×106/kg]	CD 34 yield after CD 34+ selection [x106/kg]	CFU-GM+CFU-MM [×104/kg]
Chemo+G-CSF	3.2	5.0	1.2	0.9	10
Chemo+G-CSF+Plerixafor	5.8	12.0	1.7	1.1	10

P3.11

RBC & HLA—alloantibodies influence indication for allogeneic hematopoietic cell transplantation in patients with haematological malignancies

Slonka J.¹, Sohlbach K.², Alrifai M.¹, Bein G.³, Sachs U.J.⁴

¹Zentrum für Transfusionsmedizin und Hämotherapie, Universitätsklinikum Giessen und Marburg GmbH, Marburg, Deutschland, ²Klinik für Innere Medizin, Schwerpunkt Hämatologie, Onkologie und Immunologie, Marburg, Deutschland, ³Zentrum für Transfusionsmedizin und Hämotherapie, Universitätsklinikum Giessen und Marburg GmbH, Giessen, Deutschland, ⁴Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany

Background: Alloantibodies against red blood cell— and HLA—antigens in patients with hematological malignancies can significantly impede blood supply. This can affect the course of treatment during chemotherapy, particularly in patients with rare blood groups. Allogeneic hematopoietic cell transplantation (HCT) is a curative treatment in hematological malignancies. The indication has also been extended to some benign hematological and non hematological diseases. We report on a patient with acute myeloid leukemia (AML) who presented with multiple alloantibodies. Case report: A 52 y old female patient presented with a diagnosis of AML. She was 0 Rhesus (D) positive, ccD.EE, K negative and red cell—alloantibodies anti—C, —e, —K, —Fya plus HLA—antibodies were detected. Cytological examination confirmed the diagnosis of AML without maturation, classified as subtype M1 according to FAB, karyotype 46, XX, NPM1 pos, FLT3 ITD neg, MLL neg. In this subtype conventional chemotherapy containing induction and consolidation therapy is usually indicated as the first line therapy, allogeneic HCT is only recommended when a related donor is available or in case of relapse. However, because of the detected alloantibodies, blood supply problems, platelet transfusion refractoriness and recurrent bleeding complications seen already in first chemotherapy course, decision to allogeneic HCT has been made. The patient was successfully transplanted in first complete remission after one induction therapy to reduce further cycles of chemotherapy. Conclusions: We conclude that a combination of multiple red cell alloantibodies, HLA antibodies, platelet transfusion refractoriness and bleeding complications in patients with hematological malignancies can influence the indication for allogeneic HCT.

P 3.12

How healthy are matched unrelated stem cell donors?

Leitner G.C., Pelzmann B., List J., Tosenmayr A.

University Clinic for Blood Group Serology and Transfusion Medicine, Vienna, Austria

Background: The use of matched unrelated stem cell grafts (MUD) for allogeneic peripheral stem cell transplantation (PBSCT) has become common practice. A prerequisite (beside the HLA match) is a good health status of the donor. In this evaluation we tried to figure out health impairments which were inconsistent with an allogeneic donation due to a health risk for either donors or recipients. Materials and Methods: Between 2007 and 2010 eighty three healthy volunteer donors (30, females and 53 males, median age 41 yrs (20 − 55) were examined. We routinely performed physical and explorative examinations, virus serological testing including Hbc antibodies, EKG, blood chemistry including renal, liver and coagulation parameters. Additionally, chest and abdominal x—rays were done. When necessary, the investigations were extended to include computertomography or ergomertry. Results: Thirty nine donors showed pathological blood parameters (for example elevated bilirubin or triglycerides), in 12 cases a fatty liver was diagnosed, 7 donors had liver hemangiomas, hepatic cysts or spleen cysts. In 4 donors we diagnosed severe hypertonia, unknown up to examination. A right bundle branch block was found in 1 donor and a WPW syndrome in another one. Three donors were rejected from donation because of a.) primary biliary cirrhosis, b.) cholecystolithiasis and nephrolithiasis and c.) status post unknown hepatitis B infection. One donor was postponed because of a 4 cm spleen enlargement due to a flu like infection. Conclusion: Most of the diagnosed health problems were easy to handle and there was no need to reject the donor. In several cases this intensive exploration led to first time diagnosis and in 3 presumptive to prevention of potential harm. In one case the risk of transplant related HBV infection could be avoided. In summery, a thorough pre—donation examination is a valuable contribution to health care.

P3.13

Severe pulmonary inflammation after allogeneic unrelated peripheral stem cell donation – a case report

Blechschmidt M.¹, Hölig K.¹, Cotta L.², Wetzko K.³, Schuler M.³, Kroschinsky F.³, Ehninger G.³

¹Medical Clinic I, Department of Transfusion Medicine, University Hospital Dresden, Dresden, Germany, ²DKMS, Deutsche Knochenmarkspenderdatei gGmbh, Tübingen, Deutschland, ³Department of Internal Medicine I, University Hospital Carl Gustav Carus, TU—Dresden, Dresden, Germany

Background: We report on a case of a healthy allogeneic unrelated stem cell donor with severe pulmonary disease occurring shortly after PBSC donation. Case report: A 31 years old female healthy donor underwent G—CSF mobilisation and PBSC collection without any unexpected side effects. Three days after the donation treatment with amoxicillin was started because the donor suffered from an otitis media. 1 day later she was admitted to hospital due to fever, thoracic pain and pronounced respiratory insufficiency. (First) CT scan of the chest demonstrated disseminated cloudy shadows on both middle and lower lung fields. Furthermore, the donor developed papular skin lesions, muscle pain and impaired renal function. A non—infectious, serum—sicknesslike disorder was assumed and treatment with high—dose steroids was started (Prednisolon 3.3 mg/kg KG). Exanthema and muscle pain disappeared. Bronchoscopy demonstrated normal respiratory mucosa; microbiologic analyses did not detect any microbes. Biopsies from lung tissue and skin lesions showed infiltration by neutrophiles. All tests for auto—antibodies and virus—specific PCRs remained negative. Maximum CRP and procalcitonin were 477 mg/L and 17 μg/L, respectively. Antibiotics were initiated (Piperacillin/Tazobactam and Clarithromycin). During the following days the donor recovered and could be discharged from hospital on day 21 after the PBSC donation. Steroid treatment was tapered and stopped. Discussion/Conclusion: Etiology and pathogenesis of this disorder after PBSC donation remain unclear. Although microbes could not be detected, we hypothesize an acute respiratory tract infection. Transient bacteraemia, complicated by persisting neutrophilia could have induced disseminated infiltrates in skin, lungs and consecutively impaired organ functions. This case might indicate on a different clinical picture of infectious diseases in the course of G—CSF—administration in allogeneic donors.

P 3.14

Factors influencing search duration in allogenic stem cell donor search

Fürst D., Recker K., Schrezenmeier H., Mytilineos J.

Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden—Württemberg—Hessen, and University of Ulm, Ulm, Germany

In patients who need to undergo hematopoietic stem cell transplantation, treatment scheduling must consider the time required for the identification of a suitable donor. This time is highly variable and may lead to adverse outcome if no donor can be found in a given time frame. We analysed 4208 searches completed between 2004 and 2009 in our institute aiming at identification of factors influencing search duration. This was defined as days from initiation to identification of the first donor who fulfilled the transplant centre's compatibility criteria. Urgency was classified by the transplant clinic as urgent or standard. International search was defined as search which involved a confirmatory typing of at least one foreign donor. Phenotype frequency estimation was defined as number of potentially HLA—A—B—DRB1 matched donors divided by the total number of donors available at that time. For comparison patients were categorized in phenotype frequency quartiles. The median search duration for the periods 2004, 2005/2006, and 2007–2009 was 20d, 22d, and 18d (p < 0.001) respectively. Median search time for international and national searches was 26d, and 16d (p < 0.001), respectively. Urgent searches were accomplished in a median time of 19d vs 20d in “standard” searches (p = 0.0096). Phenotype frequency also correlated with search duration: Decreasing frequencies resulted in increasing median search durations of 15d, 18d, 21d and 31d, respectively (p < 0.001). Search duration most likely increased between 2004 and 2005 due to the switch to matching at high resolution level for HLA—class I alleles. The decrease after 2006 is due to faster turnaround times in our laboratory (automation) and due to an increasing donor pool worldwide. The difference between standard and urgent searches was marginal. Low frequency phenotypes were associated with long search duration, highlighting the difficulties in donor search for these patients.

Session 04 – Cell Therapy: Non—Hematopoietic Stem Cells

P4.01

Identification of the active components within human platelet lysate enhancing proliferation of mesenchymal stromal cells (MSC)

Fekete N., Fürst D., Lotfi R., Wiesneth M., Rojewski M., Schrezenmeier H.

Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden—Württemberg—Hessen, and University of Ulm, Ulm, Germany

Background: Clinical use of human MSC requires ex—vivo expansion. We have processed clinical grade pooled platelet concentrates as a safe source rich of human growth factors to obtain a GMP compliant supplement for large—scale expansion of MSC. Methods: Platelets were frozen, quarantine stored, thawed and sterile filtered to obtain platelet lysate (PL). PL was used in a single—step isolation and expansion protocol for BM—derived MSC. variability of growth factors within PL was analyzed by custom—designed Multiplex assay. MSC proliferation was analyzed via CyQuant. Results: Low batch—to—batch variability was observed in regard to content of growth factors and MSC proliferation. PL contains high levels of sCD40L, VCAM—1, ICAM—1, PDGF—AA, PDGF—AB/BB, RANTES, and GRO. Neutralizing antibodies to PDGF—AA, RANTES, VEGF, GRO, MIP—la, MIP—lb, VCAM—1, ICAM—1, sCD40L had no inhibitory effect on PL—induced proliferation. Inhibition of PDGF—BB or bFGF alone decreased MSC proliferation by about 20% and 50%, respectively. Strongest inhibition (about 75%) was observed with a combination of either anti—bFGF + anti—PDGF—BB or anti—bFGF + anti—TGF—ßl + anti—PDGF—BB. Conversely, addition of recombinant PDGF—BB did not promote cell proliferation neither by itself nor in addition to 10% PL. Cocktails of recombinant PDGF—BB, TGF—ßl or ActivinA, bFGF and other components were not sufficient to stimulate MSC proliferation. Conclusions: PL enhances MSC proliferation and can be regarded as a safe tool for expansion of primary cells for clinical purposes. Neutralizing PDGF—BB, bFGF and TGF—ßl in various combinations resulted in substantial reduction of PL—induced MSC proliferation to 75% suggesting a synergistic effect. Failure to enhance MSC proliferation with a combination of respective recombinant proteins suggests that they are necessary but not sufficient for MSC proliferation, and that PL contains other additional growth supporting factors.

P4.02

Effect of radiation on human bone marrow—derived mesenchymal stem cells (MSC)

Fekete N.¹, Sensebé L.², Langonné A.³, Fürst D.¹, Rojewski M.¹, Schrezenmeier H.¹, Schmidtke—Schrezenmeier G.¹

¹Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden—Württemberg—Hessen, and University of Ulm, Ulm, Germany, ²Etablissement Français du Sang Pyrénées—Méditerranée, Toulouse, France, ³Etablissement Français du Sang—Centre—Atlantique, Tours, France

Background: Cell therapy with MSC is of high interest in varying indications. However, long—term persistence of ex—vivo expanded cells in the recipient may not always be desirable. Irradiation is a routine procedure in transfusion medicine to prevent long term persistence of nucleated cells and could therefore be applied to MSC. Methods: MSC were irradiated with either 30 or 60Gy. Proliferation, clonogenicity, differentiation and expression of hTERT, p 16, p21, p53 and c—myc were evaluated. Cytokine levels of cell culture supernatant on day 0,7,14,21 and 28 were analyzed via custom—designed Multiplex assay. FACS analysis was performed on day 0, 3, 56 and 112 post irradiation. Results: Irradiated MSC show a significant decrease in proliferation and CFU—F. However, a subpopulation of surviving cells remains able to differentiate and form colonies even 28 days after irradiation. Irradiated MSC showed stable expression of CD90, CD105, absence of CD3, CD34, CD45, decrease of CD9 and CD49c, and an increase of CD200 compared to controls. During culture of non—irradiated MSC we observed a consistent change of cytokine levels, i.e. increased VEGF concentration and decrease of PDGF—AA, PDGF—AB/BB, RANTES. Irradiated MSC showed an inverse pattern, i.e. no increase of VEGF, but also no consumption of PDGF—AA and PDGF—AB/BB. Interestingly, IL—6 levels increased during culture regardless of irradiation. After irradiation expression of p21 andhTERT was significantly higher on day 56 and 112 compared to non—irradiated controls and pi 6, p56 and c—myc varied in their expression. Conclusions: A subgroup of MSC shows radiation resistance with survival of clonogenic cells. Irradiation substantially changes the pattern of cytokine production/consumption and —given the importance of paracrine effects —might substantially change MSC activity in vivo. MSC surviving high—dose irradiation are characterized by expression of hTERT and p21 and may comprise a separate functional subgroup.

P4.03

Differential proteomic analysis to identify mesenchymal stromal cell stimulating factors in platelet lysate and platelet releasate

Kinzebach S.¹, Thierse H.—J.², Klüter H.¹, Bieback K:¹

¹Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Donor Service Baden—Württemberg – Hessen, Mannheim, Germany, ²Laboratory of Immunology & Proteomics, Department of Dermatology, University Medical Center Mannheim, Ruprecht—Karls University of Heidelberg, Mannheim, Germany

Background: Mesenchymal stromal cells (MSC) are promising candidates for widespread clinical applications. For clinical—grade expansion fetal bovine serum – commonly used in the expansion medium – shall be replaced by suitable non—xenogenic supplements. We already demonstrated that pooled human platelet lysate (pHPL) and thrombin activated platelet releasate in plasma (tPRP) can support the expansion of MSC. Whereas pHPL strongly accelerated the proliferation of MSC from bone marrow (BM), the effect on MSC from lipoaspirate (LA) was less intensive compared to tPRP indicating that MSC from different tissue sources respond differentially to these supplements. Methods: While tPRP contains those growth factors released from platelets after thrombin activation, pHPL is a whole cell extract obtained by freeze/thaw lysis. Since we hypothesize different stimulating factors in pHPL and tPRP, a differential differential proteomics approach was performed: differential gel electrophoresis (DIGE) followed by matrix—assisted laser desorption ionization mass spectrom—etry (MALDI). Identified proteins were verified by western blot analysis. Selected proteins were assayed for their effects on MSC proliferation. Results: We were able to identify 28 proteins which were higher expressed in pHPL and 8 proteins higher expressed in tPRP. Identified proteins have been validated by western blot analyses. Fibrinogen was the protein highest expressed in pHPL compared to tPRP Accordingly, we analysed whether external fibrinogen can further accelerate the proliferation of LA—MSC and BM—MSC. The current data suggest a tissue specific and supplement related response to fibrinogen. Conclusion: The higher abundance of proteins in pHPL and the effects of fibrinogen support the observation of accelerated proliferation. The identification of growth factors relevant for MSC expansion may pave the way towards a chemically defined medium which is yet missing for clinical—grade MSC manufacturing.

P4.04

Human serum sustains the effects of long—term expansion of adipose—tissue derived stromal cells

Hecker A.¹, Becker M.², Hofmann I.³, Klüter H.¹, Bieback K.¹

¹Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Donor Service Baden—Württemberg – Hessen, Mannheim, Germany, ²lnstitut für Medizinische Strahlenkunde und Zellforschung der Universität Würzburg, Würzburg, Germany, ³Joint Research Division Vascular Biology of the Medical Faculty Mannheim, Heidelberg University, and the German Cancer Research Center (DKFZ—ZMBH—Alliance), Center for Biomedicine and Medical Technology Mannheim (CBTM), Mannheim, Germany

Background: Mesenchymal stromal cells (MSCs) are highly interesting candidates for novel cell therapeutic applications. To replace fetal bovine serum (FBS) commonly used in MSC expansion media human blood derived alternatives have been evaluated. We previously demonstrated that pooled blood group AB human serum (HS) accelerates the expansion of adipose tissue—derived MSCs (ASCs) while maintaining key functions. Albeit cell expansion is required to obtain clinically relevant cell numbers, this may alter cellular qualities. Methods: We compared the effect of long—term culture on ASCs cultivated in either FBS or HS with respect to growth and cell cycle prolife, telomerase activity and telomere length, senescence marker expression, anchorage independent growth in soft agar assays, differentiation potential, immunosuppressive capacity and marker expression. Results: Compared to FBS HS seems to prolong the replicative life span of ASCS, however no differences regarding the clinically important features differentiation potential and immune suppression were apparent. Adipogenic differentiation potential was rapidly impaired, whereas the osteogenic potential appeared less affected by the culture age of ASCs. Senescence was paralled by the lack of telomerase expression along with progressive telomere shortening. Cell cycle regulator expression appeared stronger regulated in HS. Anchorage—independent growth indicative of tumorigenic properties was not observed in either culture. Regarding marker expression, ASC cultures were heterogeneous and stayed heterogeneous through—out long—term expansion. Conclusions: Our data demonstrates that HS is a reliable surrogate for FBS as key cellular features in long—term culture are maintained and cellular aging is unaffected. Especially the rapidly impaired differentiation potential further denotes to carefully assess the stage of replicative aging with respect to the intended cell therapeutic application.

P4.05

Preprogramming cells of the nonhuman primate common marmoset towards pluripotency

Zuk M., Stolp K., Kleinbielen A., Horn P.A., Klump H.

Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany

Treatment of monogenetic diseases of the hematopoietic system by targeted gene correction of its stem cells (HSCs) is not feasible yet since prospective isolation, homologous recombination and selective expansion of gene—corrected HSCs remains a largely unsolved challenge. In contrast, pluripotent stem cells may represent an attractive alternative source for HSC—based therapies as they offer all required prerequisites and can in principle be differentiated towards somatic hematopoietic stem and progenitor cells (HSPCs), in vitro. To test the medical relevance of this notion in the long run we sought to employ an animal model system more closely related to humans than the mouse model and, thus, initiated work on the non—human primate common marmoset (Callithrix jacchus). As a starting point for the generation of transplantable HSPCs, in vitro, we wished to generate induced pluripotent stem (iPS—) cells. Because the reprogramming conditions for cells from this primate are ill—defined, we performed a side—by—side comparison of marmoset skin fibroblasts and human umbilical vein endothelial cells (HUVECs) using a lentiviral and transposon—based system for expression of the human or mouse pluripotency factors Oct4, Klf4, Sox—2, c—Myc and +/— Lin28. Formation of colonies with ES—cell like morphology was observed approximately 15 days after gene transfer using either approach, both with human and marmoset cells. Whereas human colonies could be well propagated and showed hallmarks of pluripotency, marmoset colonies ceased further proliferation under the employed culture conditions, suggesting only partial reprogramming of the primate cells so far. Such partially reprogrammed cells may already represent a useful source for generating autologous HSPCs in vitro. Nevertheless, to support the identification and isolation of fully reprogrammed iPS cells, a pluripotency reporter system driven by the Oct4 core enhancer from the conserved region 4 (CR4) is currently being employed.

Session 05 – Cell Therapy: Immunotherapy and Immunoregulation

P5.01

EBV—specific T cell immunity in pediatrie solid organ graft recipients with lymphoprolitérative disease

Wilsdorf N.¹, Eiz—Vesper B.², Henke—Gendo C.³, Klein C.¹, Maecker—Koihoff B.¹

¹Hannover Medical School, Pediatric Hematology/Oncology, Hannover, Germany, ²Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany, ³Hannover Medical School, Institute of Virology, Hannover, Germany

Background: Post—transplant lymphoproliferative disease (PTLD) is a severe complication after organ transplantation. In children, most PTLDs are Epstein—Barr virus (EBV) associated and impaired EBV—specific immunity is a risk factor for PTLD development. Here we present data on EBV—specific T cells in patients treated with Rituximab +/— chemotherapy on the Ped—PTLD Pilot 2005 protocol. Patients and Methods: PBMCs were isolated from 15 pédiatrie patients with PTLD and 18 healthy controls. PBMC were stimulated with autologous EBV—transformed LCL, stained with extracellular antibodies for CD3, CD4, and CD8, followed by intracellular staining of interferon—g and detection by flow cytometry. EBV load was analyzed by quantitative PCR of whole blood. Results were correlated with clinical data from the Ped—PTLD registry. Results: At diagnosis, PTLD patients had similar numbers of EBV—specific CD4+ (mean 0.16%) and CD8+ (0.59%) T cells as healthy controls (0.18% and 0.49%). Numbers were lower in early compared to late PTLD. In 11/12 patients, a modest increase in CD4+ and/or CD8+ EBV—specific T cells was noticed during treatment with Rituximab, possibly reflecting increased immunocompetence after reduction of immunosuppressive therapy and anti—genic stimulation after liberation of EBV antigen from destroyed B cells. However, this increase did not predict response to Rituximab or chemotherapy. EBV load and circulating B cells decreased to zero in most patients during Rituximab treatment. Recurrence of B cells was accompanied by re—detection of EBV in peripheral blood, which was controlled by strong T cell responses in 12/14 cases. Conclusions: In pédiatrie PTLD patients EBV—specific T cells increase moderately upon reduction of immunosuppression and treatment with Rituximab. In contrast, recurrence of EBV during complete remission is paralleled by strong T cell responses. EBV—specific T cell therapy may augment endogenous T cell responses to secure successful control of PTLD.

P5.02

Profiling of self peptides derived from lymphoblastic cells selected by B∗44 variants

Bade—Döding C, Huyton T., Blasczyk R.

Institute for Transfusion Medicine, Hannover Medical School, Hannover, Hannover, Germany

Background: HLA class I molecules are mediators of the adaptive immune response including rejection of organs and HSC transplants. From the HLA—perspective the way to analyse the impact of mismatches is a comprehensive analysis of the peptide binding spectra of key alíeles that exhibit critical polymorphisms at controversial pocket positions. However little is known about the peptide—perspective and which peptides of different origin and/or different length would be selected for presentation by different alíeles. The aim is to provide a comprehensive ligand dataset selected by allelic variants using a lymphoblastic cell line, thereby providing each individual alíele with the same host proteome. Methods: LCL 721.221 cells were lentivirally transduced with truncated HLA—B∗44 constructs. The alíeles 4402, 4403, 4404, 4405, 4406, 4407, 4408, 4409, 4410, 4427, 4428 and 4435 were chosen for fishing of host proteome derived ligands. All these alleles feature polymorphism located in the alpha 1 domain (positions 24, 32, 41, 45, 46, 63, 67, 77, 80, 81, 82, 83), alpha 2 domain (positions 99, 103, 113, 114, 116, 156, 163) or the alpha 3 domain (position 199). From the host proteome a representative pool of ligands (>1500 peptides in total) were thought to be fished. Eluted peptides from soluble B∗44 products were sequenced by LC—ESI—MSMS (LTQ—Orbi—trap) technology. Results: Throughout the whole genome peptides encoded by all chromosomes could be sequenced. Major categories of peptides featured proteins involved in cell metabolism, cell maintenance and catalytic proteins. The length of the derived peptides varies from 8–19 amino acids in length. Conclusions: The results suggest that for comprehensive prediction tools ligands of non canonical length need to be considered. Furthermore these data represent a comprehensive overview of the proteomic content of lymphoblastic B—cells and provide knowledge about self and non—self ligands for vaccination purposes, immunotherapy and bioinformatic prediction algorithms.

P 5.03

Differential regulation of the effector cell function by Semaphorin 7a variants explains their disease association

Figueiredo C.¹, Jaimes Y.¹, Gras C.¹, Eiz—Vesper B.¹, Seltsam A.², Blasczyk R.¹

¹Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany, ²DRK—Blutspendedienst NSTOB Institut Springe, Springe, Germany

Background: Semaphorin 7A (Sema7A) is a membrane—bound member of the conserved Semaphorin family. Although initially described as axon pathfinder, Sema7A has been shown to modulate the immune response. Only very few polymorphic variations of Sema7A sequence were described. However, those have been associated with autoimmune and bone diseases. Patients and Methods: The wild—type Sema7A protein (Sema7A_wt) and a natural variant (Sema7A_R461C) were produced in human embryonic kidney (HEK) cells. The effect of Sema7A molecules in the regulation of NK and activity was evaluated in phenotypic, cytotoxic and proliferation assays. Sema7A_wt caused a significant reduction in the NK cell proliferation rates, whereas the natural variant Sema7A_R461C protein strongly induced NK cell proliferation. Results: Only the Sema7A_wt led to a decrease in expression of activating receptors and demonstrated to inhibit NK cell cytotoxicity against K562 cells. In contrast, NK cell stimulation with Sema7A_R461C protein showed to significantly increase K562 cell lyses rates. RNAi—mediated knockdown of the transmembrane Sema7A_wt or Sema7A_R461C proteins demonstrated that membrane bound and soluble Sema7A proteins have similar effects on the regulation of NK cell cytotoxicity. Antibody blocking studies showed that the inhibitory effect of Sema7A_wt on NK cells is mediated by plexin C1, whereas the activation effect of the natural variant is β1 integrin dependent. This study demonstrates for the first time that wild type Sema7A and its polymorphic variation Sema7A_R461C regulate NK cell function in a completely opposite manner. Conclusion: This unexpected effect of Sema7A polymorphic variations causing a differential regulation of cell function might explain why exclusively Sema7A variants are associated with autoimmune diseases.

P5.04

Skin triggering of TLR7 modulates respiratory dendritic cells and natural killer cells – identification of a novel mode for manipulation of respiratory leukocytes

Hackstein H.¹, Baal N.¹, Hagel N.¹, von Wulffen W.², Bein G.³

¹Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany, ²Klinikum Universität München Medizinische Klinik I, München, Deutschland, ³Zentrum für Transfusionsmedizin und Hämotherapie, Universitätsklinikum Giessen und Marburg GmbH, Giessen, Germany

Background: Complete dendritic cell (DC) maturation and activation in situ is a critical element of successful DC vaccination protocols whereas suppression of functional DC activation represents an important step in the development of immunoregulatory cellular therapy protocols. The TLR7 agonist imiquimod has been used successfully as novel topical immune adjuvant but the systemic use has been limited due to significant toxicity. We hypothesized that skin application of a synthetic TLR7 ligand may represent a novel means to modulate respiratory leukocytes. Methods: In a placebo—controlled experimental animal study we have used 7—colour flow cytometry to systematically analyze the modulation of murine respiratory leukocyte subsets after skin administration of the synthetic TLR7 ligand imiquimod. Results: Compared to placebo, skin administration of imiquimod markedly increased respiratory dendritic cells and innate lymphocytes within 24h, whereas total respiratory leukocytes, alveolar macrophages, T helper and killer lymphocyte numbers were not affected or reduced. DC subpopulation analyses revealed that elevation of respiratory DC was related to an increase of monocytic DC (CD11c+CD11b+MHC—classIIdimSiglecFneg, CD45+). Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a reduction of B cells. In contrast, bronchoalveolar cell counts and histopathology were not affected suggesting that skin TLR7 stimulation markedly modulated respiratory leukocyte composition without inducing a respiratory inflammatory response. Conclusion: These data suggest for the first time the possibility to modulate respiratory leukocyte subsets after skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel and safe means to modulate respiratory immunity. DC vaccination trials targeting the respiratory system may be modulated by skin TLR7 triggering.

P 5.05

Intracellular concentrations of angiogenic cytokines in human platelets change after surgery for colorectal cancer

Zimmermann R.¹, Schellerer V.², Schmieger T.¹, Stuetzer T.¹, Weiss D.R.¹, Hauck B.¹, Eckstein R.¹

¹Transfusionsmedizinische und Hämostaseologische Abteilung, Universitätsklinikum Erlangen, Erlangen, Deutschland, ²Chirurgische Klinik, Universitätsklinikum Erlangen, Deutschland

Background: Previous work on circulating growth factors in patients with colorectal cancer (CRC) is significantly biased from severe pre—analytical errors. The questions, in what concentrations the growth factors vascular endothelial growth factor (VEGF), platelet—derived growth factor AB (PDGF—AB) and transforming growth factor beta 1 (TGF—ß1), which circulate freely or stored in platelets, can be found in the blood of patients with CRC and to what extent the measured values are changed by the surgical removal of the tumor, have therefore not yet been clarified. Methods: In 20 patients, blood was taken on admission before surgical tunor removal and before discharge after surgery. Extracellularly circulating levels of VEGF, PDGF—AB, and TGF—ß1 were determined from CTAD plasma. Total intra— and extracellularly circulating amounts of these growth factors were measured from a lysate of blood in 0.5 percent Triton X—100. Results: The extracellularly circulating cytokine levels remained unchanged after surgery. Surprisingly, however, there was a significant increase of the VEGF concentration in the lysate (0,14 ± 0,09 vs. 0,45 ± 0,17 pg/10e5 PLTs; p < 0.05) and a significant decrease of the PDGF—AB concentration in the lysate (6,7 ± 7,4 vs. 2,0 ± 0,7 pg/10e5 PLTs; p < 0.05) after tumor removal. Conclusions: The significant change of the levels of the cytokines VEGF and PDGF—AB 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 the resection of a colorectal tumor or at least of a tumor resection in itself, or whether we discovered a general effect of major surgery. These issues must be clarified by further studies. Their first objective must be the reproduction of the most interesting finding of this study in a second, different group of patients.

P5.06

Differentiation and maturation of CD14+ monocytes in leucocyte concentrates during short—term liquid storage

Steininger P., Weiss D.R., Zimmermann R., Eckstein R., Strasser E.R

Transfusionsmedizinische und Hämostaseologische Abteilung, Universitätsklinikum Erlangen, Erlangen, Deutschland

Background: Adoptive immune therapy with dendritic cells (DCs) has achieved great importance. Thereby the development of monocyte subpopulations and the impact of proinflammatory monocytes on DC differentiation still needs clarification. Methods: The leucoreduction system chamber of the Trima Accel cell separator (CaridianBCT^®) was used as a source of high quality leucocytes. Five different leucocytes products were stored in polytetraflu—orethylene—bags at room temperature before analysis. By staining for CD 14 and CD 16 in flow cytometry (FACS Calibur, BD) we could divide the blood monocytes into the three most established subsets of classical CD 14highCD 16—, intermediate CD14highCD16+ and non—classical CD141owCD16+ monocytes, the latter two known as proinflammatory monocytes. We measured the change of monocyte subpopulation in course of time and compared it with their particular rate of apoptosis and necrosis by staining with Annexin V and 7—Aminoactinomycin D. The DCs were selected as lineage negative (CD3, CD14, CD16, CD19, CD20, CD56) and HLA—DR+ cells and were further distinguished by labelling with anti—CDllc. Finally the maturation of DCs should be observed by loss of CDla and up—regulation of CD86. Results: We found the CD14highCD16— monocytes with a proportion of 86,5% at the beginning and an increase to 92,0% at 48 h, vs. approximately constant levels of CD14highCD16+ monocytes (4,1% to 3%) and decreasing levels of CD141owCD16+ monocytes (9,5% to 4,9%). In this period the corresponding apoptotic rates decreased, 24,9% to 11,7% vs. 37% to 15,7% vs 21,7% vs. 11,4%, respectively. DCs revealed a lack of CD la—expression at any time, whereas the CDllc+ were entire CD86+ and the fraction of CD lie— augmented from 55,7% to 81,8%. Conclusions: The existing storage conditions did not elicit a de novo generation of CD11c+DCs. Further approaches must identify micro—environmental factors, which control the fate of monocytes in order to guide their differentiation into DCs.

P5.07

Peripheral blood mononuclear cells obtained from leukoreduction system chambers show better viability than those from leukapheresis

Strasser E.F.¹, Weidinger T.², Keller A.³, Weiss D.R.¹, Zimmermann R.¹, Eckstein R¹

¹Transfusionsmedizinische und Hämostaseologische Abteilung, Universitätsklinikum Erlangen, Erlangen, Deutschland, ²Department of Surgery, University Hospital Erlangen, Erlangen, Germany, ³Department of Medical Informatics, Biometry and Epidemiology, University of Erlangen—Nuremberg, Erlangen, Germany

Background: PBMCs obtained from leukoreduction system chambers show excellent quality. We compared viability of stored PBMCs obtained from LRSCs and leukapheresis products. Study Design and Methods: In this randomized prospective pilot study we stored six samples each taken from LRSCs of SPUs or DPUs or taken from leukapheresis products. Within one hour after production and after storage for six, 24 and 48 hours in PVC—bags we analysed pH, pO2 and PCO2, as well as blood cell counts. PBMCs were analysed by flow cytometry with regard to Annexin—V+ and 7—AAD+ events. Statistical analysis was performed by use of a multivariate general linear model for measurements followed by post hoc tests adjusted by the method of Sidák. Results: Within one hour after production in samples obtained from leukapheresis the fraction of Annexin—V+/7—AAD– lymphocytes was double compared to the lymphocytes of LRSC (SPUs, p = 0.005, DPUs, p = 0.001). In stored samples obtained from leukapheresis products we recognized a significant increased of Annexin—V+/7—AAD+ PBMCs after 48 hours (lymphocytes, p < 0.001, monocytes, p < 0.001). In contrast, we could not observe any significant increase of Annexin—V+/7—AAD+ PBMCs obtained from LRSCs. PBMCs obtained from leukoapheresis products, thus, showed more apoptotic events compared to PBMCs of LRSCs during storage. PH measured in storage bags obtained from PBMCs of leukapheresis decreased to a higher extent suggesting a higher level of anaerobic metabolism compared to LRSCs. Conclusion: Viability of mononuclear cells derived from LRSCs was better to those from leukapheresis. Differences in PBMC—viability could be caused by methodical specifics of both apheresis procedures. Reason for that may be different ways to collect PBSCs by both apheresis procedures. We suggest that leukocyte reduction system chambers could be an interesting new method for gently collecting PBMCs in future that should be subject to further studies.

P 5.08

Influence of the secreted fraction from bone marrowderived stroma cell lines on NK—mediated lysis

Rebmann V.¹, Veit T.D.¹, May T.², Opalka B.³, Dührsen U.³, Horn P.A.¹

¹Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, ²Helmholtz Centre for Infection Research, Department of Gene Regulation and Differentiation, Braunschweig, Germany, ³Department of Hematology, University Hospital Essen, Essen, Germany

The impairment of the immunologic response against tumors is a known issue in cancer biology. Besides alteration of the tumor cells themselves components of the tumor microenvironment might modulate the immune response. Tumor—derived stromal cells (TStrC) were previously shown to impair NK cell function. In this work we investigate the potential influence of the secreted fraction from five different TStrC lines as well as a stromal cell line derived from healthy subjects in NK cell function. The cell lines were derived from bone marrow stroma by immortalization with different transgenes. Primary plastic—adherent stroma cells prior to immortalization were cultured from patients suffering from acute myeloid leukemia, from lymphoma patients without malignant infiltration in the bone marrow, and from a healthy donor. All stromal cell lines expressed ligands for the activating NKG2D receptor i.e. MICA, MICB, ULBP2, which have been described to have activating properties when in membrane form and, conversely, inhibitory features when in soluble form, as well as cytokines. These molecules were also present on microvesicles or exosomes. Such vesicles displayed significant amounts of cytokines: IL—8 was always high whereas variable levels were found for IL—1b and IL—6, and low amounts of other cytokines. Analyzing NK92 cell—mediated lysis we observed a strong inhibition of NK92 killing by stromal cell supernatants. However, TStrC derived vesicles did not inhibit NK cell lysis, probable due to the high amount of pro—inflammatory cytokines. Taken together, our results support the use of cell lines derived from bone marrow stroma as an interesting model to study the interaction between tumor—derived stromal cells and the immune system in patients with malignancies.

P5.09

Human B cells differentiate into granzyme B—secreting cytotoxic B lymphocytes upon incomplete T cell help

Jahrsdörfer B.¹, Hagn M.², Hofmann S.¹, Hofmann S.¹, Beyer T.¹, Schrezenmeier H.¹

Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden—Württemberg—Hessen, and University of Ulm, Ulm, Germany, ²Peter MacCallum Cancer Centre, Melbourne, Australia

Recently, CD4+ T helper cells were shown to induce differentiation of human B cells into plasma cells by expressing interleukin (IL—)21 and CD40 ligand (CD40L). In the present study we show, that in the absence of CD40L, CD4+ T cell—derived IL—21 induces differentiation of B cells into granzyme B (GzmB)—secreting cytotoxic cells. Using FACS analysis, ELISpot and confo—cal microscopy, we demonstrate that CD4+ T cells, activated via their T cell receptor without co—stimulation, express IL—21, but not CD40 ligand and rapidly induce GzmB in co—cultured B cells in an IL—21 receptor—dependent manner. Of note, we confirmed these results with recombinant reagents, highlighting that CD40 ligand suppresses IL—21—induced GzmB induction in B cells in a dose—dependent fashion. Surprisingly, although GzmB—secreting B cells did not express perforin, they were able to transfer active GzmB to tumor cell lines, thereby effectively inducing apoptosis. In contrast, no cytotoxic effects were found when effector B cells were activated with IL—2 instead of IL—21 or when target cells were cultured with IL—21 alone. Our findings suggest GzmB+ cytotoxic B cells may play a role in early cellular immune responses including tumor immunosurveillance, before fully activated, antigen—specific cytotoxic T cells are on the spot.

P5.10

Optimization of cell type specific gene transfer into human lymphocytes

Wu S.¹, Jonas S.², Lang C.³, Lorenzen T.¹, Barz D.¹, Geßner R.²

¹Institut für Transfusionsmedizin Universitätklinikum der Friedrich Schiller Universität Jena, Jena, Deutschland, ²Department of Visceral, Transplant, Thoracic and Vascular Surgery, Leipzig University medical school, Leipzig, Germany, ³Institute of Microbiology and Genetics, Technical University Berlin, Berlin, Germany

Background: Using ex—vivo genetically modified T lymphocytes has been considered to be particularly potent method for immunotherapy against various types of diseases. However, the development of genetically modified T cells still remains a major challenge because transfection efficiency is low in non—viral DNA—uptake approaches. Recently, we have developed a novel two—stage screening strategy for optimizing transfection procedures and have identified a new class of lysosomotropic drugs that are capable of enhancing an endoctosis—based transfection protocol of mammalian adherent cells significantly (Neukamm et al. J Gene Med 8, 745—753, 2006). Methods: In our current study we analyzed the effect of twelve previously identified compounds on the transfection efficiency of several human B and T cell lines using an non—specific endocytosis—based DEAE—dextran transfection assay and a luciferase—based read—out system. Cytotoxicity was determined in parallel by a MTT assay. Results: Phenotiazines and related drugs enhance the transfection efficiency of human B and T cells up to 10—fold in a dose— and cell type—dependent manner. Chloropromazine enhances specifically the transfection of T cells. Phenotiazines appear to interact with both, DEAE—dextran and the purified plasmid DNA that was used to quantify the transfection efficiency. Conclusions: The optimization of B and T cell transfection will facilitate ex—vivo gene transfer for a number of medical applications. Furthermore, our transfection enhancers seem also be suitable to enhance the uptake of other therapeutic macromolecules into human B and T cells.

P5.11

An unusual HLA—DRB1∗11:03 – DQB1∗02:01 association in a Caucasian family from north—eastern Germany

Binder T.C.M.¹, Dittmer C.¹, Kisselmann L.¹, Schmidt C. A.², Krüger W.H.², Eiermann T.H.¹

¹HLA Laboratory, Transfusion Medicine, University Hospital Hamburg— Eppendorf (O38), Hamburg, Germany, ²Department of Hematology and Oncology, Ernst—Moritz—Arndt University Hospital Greifswald, Greifswald, Germany

The limited diversity of HLA class II haplotypes encompassing DRB1 and DQB1 indicates a low recombination rate between these genes. On the occasion of HLA—typing of a female patient with Myelodysplastic Syndrome (MDS), we found the combination from DRB1∗11:01 with DQB1∗02:01. Normally in German Caucasians DRB1∗11:01 is associated with DQB1 ∗03:01, or in rare cases with DQB1∗05:02. To verify this unusual DRB1—DQB1 HLA haplotype and to find an applicable donor, the patient, her brother, her sister, her mother and her three children were typed for HLA class I and class II using the LCT (for HLA class I only), PCR—SSOP, PCR—SSP and SBT. We have identified segregation of the extraordinary HLA haplotype A∗24:02—C∗03:03—B∗15:01—DRB3∗02:02—DRB1∗11:01—DQA1∗05—DQB1∗02:01 in this Caucasian family from the North—Eastern of Germany. Her mother, her son and one of her two daughters are owner of this haplotype. No one of her relatives could be her stem cell donor, because all are HLA haploidentical, only. We were not able to find an HLA identical allogeneic stem cell donor, too. For that reason we had to select an allogeneic stem cell donor with a “normal” DRB1∗11:01/DQB1∗03:01 haplotype association. After two independent transplantations with stem cells from the same donor, we found no ‘Graft versus Host’ (GvH) or ‘Host versus Graft’ (HvG) reactions, but CMV reactivations, sepsis and BKV infections after both transplantations. A prolonged time of neutropenia after the stem cell transplantations was observed. The HLA DR/DQ haplotype we found, is very rare and most likely reflects an ancestral recombinatorial event between the DRB1 and the DQB1 loci in the past.

P 5.12

DNA—alignment driven JAK2 exon 12 mutation analysis defines 2 broad genetic mutant—“clusters” and simplifies genetic testing by PCR—SSP

Gassner C., Sigurdardottir S., Meyer E., Frey B.M.

Blutspende Zürich, Zurich, Switzerland

Background: Somatic V617F mutation located in exon 14 of the JAK2 gene, is known to be present in approximately 90–95% of Polycythemia Vera patients. In V617F negative patients a variety of different mutations in JAK2 exon 12 has been identified with comparable gain of function effects of the Janus Kinase as reported for V617F. Methods: In order to design a practicable genotyping approach, 25 different JAK2 exon 12 mutant alleles known so far, were analysed using a DNA alignment software tool.[1–5] Following this analysis, artificial plasmid DNA for the wildtype and the two clusters was used as positive control DNA. Applying PCR Using Sequence Specific Priming (PCR—SSP), the two major allelic groups (clusters) of JAK2 exon 12 mutant alleles were made detectable. Results: Cluster 1 alleles were characterized by a A1616T, A1617T substitution, (n = 4 alleles, 16 observations) and cluster 2 by a 6 bp deletion, which may be positioned after T1619 for all of them (n = 5 alleles, 31 observations). Representative alleles are F537—K539de—linsL and N542—E543del for cluster 1 and 2, respectively. Using artificial DNAs, PCR—SSPs were proven to work correctly and within a wide range of emulated DNA concentrations. The method should be capable of detecting 23.9% (cluster 1) and 46,3% (cluster 2), or 70.2% in total of all previously observed JAK2 exon 12 mutant alleles (total n = 67). Together with our in—house adapted V617F PCR—SSP, most of the pathogenic JAK2 mutations are assessed “all—in—one” using genomic DNA. Conclusion: JAK2 genotyping is an important criterion for the diagnosis of PV and other myeloproliferative disorders according to WHO. Using the PCR—SSP method presented, JAK2 exon 12/exon 14 mutation detection is simplified drastically and provides a pragmatic approach to assess V617F negative cases, which normally present with isolated polyglobulia. Identified positive samples for exon 12 mutations may be DNA sequenced for final alíele definition.

Session 06— Hemostasis: Coagulation

P6.01

Novel mutation in the gamma carboxylase gene resulting in VKCFD1

Watzka M.¹, Khaniyev S.², Unal S.², Gumruk F.², Oldenburg J.³

¹Institut für Experimentelle Hämatologie und Transfusionsmedizin; Universitätsklinikum Bonn, Bonn, Deutschland, ²Hacettepe University Division of Pedriatric Hematology, Ankara, Turkey, ³lnstitut für Experimentelle Hämatologie und Transfusionsmedizin, Bonn, Deutschland

Background: Vitamin K functions as a cofactor for the endoplasmatic enzyme gamma—carboxylase (GGCX), which catalyses the posttranslational modification of glutamate residues into gamma—carboxy glutamate residues (Gla). Gla residues are essential for the function of coagulation factor II, VII, IX, and X. Combined deficiency of these factors (VKCFD) is caused by defects in either GGCX or VKORC1 and represents a very rare autosomal recessive disorder. Methods: Direct sequencing of VKORC1 and GGCX genes was applied for mutation detection a two year old girl originating from Turkey, which is offspring of presumptive consanguineous parents. Vitamin K dependant coagulation factor activity was significantly dimishid. Furthermore, the child was presented with hypertelorism, short—neck, flat nasal bridge and underdeveloped ear helix. Treatment with 3 μg vitamin K/day results in approximately 50% VKD coagulation factor activity. Results: In our patient a homozygous missense mutation in GGCX gene was detected (Arg83Pro) whereas VKORC1 gene showed wild type sequence. Thus, VKCFD 1 can be diagnosed as cause of combined vitamin K dependant coagulation factor deficiency. The mutation Arg83 Pro lies at the interface of the first transmenbrane helix and the first ER lumenal loop of GGCX. Conclusions: The amino acid sequences around Arg83 are highly conserved throughout all GGCX enzymes known in vertebrates and therefore functional relevance is likely. Indeed, this region forms part of the interface of the first transmembrane helix and the first extra—membranous loop. As the resulting proline due to its structure is known as a “helix—breaker”, the observed mutation might result in a disturbed topology of this enzyme. The observed defects in bone/cartilage development most probably are result of missing gamma—carboxalation of MGP and BGP. In conclusion, further investigation of this mutation might give new insights into structure – function relationship of GGCX.

P6.02

Mutations in VKORC1 : a new assay for functional investigation

Czogalla K.¹, Blaurock N.¹, Westhofen P.¹, Wendeln A.—C.¹, Watzka M.², Oldenburg J.¹

¹Institut für Experimentelle Hämatologie und Transfusionsmedizin, Bonn, Deutschland, ²Institut für Experimentelle Hämatologie und Transfusionsmedizin; Universitätsklinikum Bonn, Bonn, Deutschland

Background: For therapy and prevention of thromboembolic conditions, coumarins have been the most often prescribed drugs globally for more than four decades. However, clinical use of coumarins is complicated by their narrow therapeutic window and the broad variation of individual drug requirement ranging from very low doses up to partial or total coumarin resistance. In few cases, resistance phenotype originates from mutations in VKORC1, the molecular target of coumarins. To gain further insight in this supposed mechanism we investigated human mutations in VKORC1 dependend on different Warfarin—concentrations in a cell—based system. Methods: After co—transfec—tion of VKORC 1 and F9 into HEK 293 cells, FIX activity served as a surrogate marker for VKORC 1—activity. After introduction of defined VKORC 1 mutations (H28Q, mild phenotype; V29L, strong phenotype; Y139H, total resistant phenotype), resistance to coumarins was measured dependent on different Warfarin concentrations (0.001 μmol, 0.003 μmol, 0.01 μmol, 0.03 μmol). Results: The mutation H28Q showed low to abscent FIX— activity after treatment with low concentrations of Warfarin. Co—transfected cells with the V29L—mutation needed higher concentrations of Warfarin for blocking VKORC1—activity indicated by low residual FIX—activity at high Warfarin concentrations. VKOR—activity of Y139H—mutation was not affected by Warfarin at each concentration applied. Discussion: The described phenotypes of the human VKORC1—mutations Y139H, H28Q and V29L were reproducible in our cell—test system. All mutations resulted in different strength of Warfarin resistance, ranging from total (Y139H) to mild resistance (H28Q) as observed in the corresponding patients.

P6.03

Mutagenesis of thrombin cleavage sites in the factor VIII protein and expression in mammalian cells

Herbiniaux U., Driesen J., Pavlova A., Oldenburg J.

¹Institut für Experimentelle Hämatologie und Transfusionsmedizin; Universitätsklinikum Bonn, Bonn, Deutschland

Background: Qualitative and quantitative deficiency of factor VIII (FVIII) is associated with the haemophilia A (HA). FVIII is activated by thrombin cleavage at three sites (Arg372, Arg740, Argl689). The aim of this study is to investigate the functional properties of recombinant FVIII protein variants representing partially or completely mutated thrombin cleavage sites towards the consensus sequence. Materials and Methods: The three, PI (amino acids (aa) 369—374), P2 (aa 737—742), P3 (aa 1686—1691) thrombin cleavage sites fragments were alone or in combination replaced by the consensus sequence fragment by PCR mutagenesis. FVIII activity (FVIIIrC) was measured by a chromogenic assay (FVIII: Cchr) and two aPTT based one—stage clotting assays (FVIIIrClst). Results and Discussion: The FVIIIrC from the constructs PI, P2 and P3 was reduced in comparison to FVIII WT activity. The construct P2 showed FVIIIrC closest to that of the WT. Construct P3 displayed the lowest FVIIIrC compared to FVIIIrC of WT (FVIIIrClst 11 vs 28%; FVIIIrCchr 28 vs 37%; in—house FVIIIrClst 7 vs 34%; p < 0,005). The FVIIIrC of the combined constructs P1+P2+WT and P1+WT+P3 showed dramatical reduction in comparison to the WT. FVIIIrClst below 2% was measured for constructs P1+P2+WT and P1+WT+P3 compared to 25% for the WT (p < 0.005). These results were confirmed by FVIIIrCchr although the measured values were much higher. Due to low (physiological) concentrations of thrombin in FVIIIrClst a decreased affinity of thrombin to FVIII reduces the activation of FVIII. In contrast, the excess of thrombin and a long incubation time in the FVIIIrCchr results in activation of FVIII even if affinity to thrombin is reduced. Conclusion: Our findings show a reduction of FVIIIrC in constructs with thrombin cleavage consensus sequences compared to WT. Thus, the potential benefit from the thrombin cleavage consensus sequence is less significant than the impact of these changes on the overall structure of the FVIII protein.

P6.04

Discrepancy between antithrombin activity assays due to mutations in the antithrombin gene

Luxembourg B.¹, Sittinger K.¹, Körber S.¹, Lindhoff—Last E.², Seifried E.¹, Geisen C.¹

¹DRK—Blutspendedienst Baden Württemberg – Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Klinikum der Johann Wolfgang Goethe—Universität, Frankfurt, Deutschland, ²Department of Internal Medicine, Division of Vascular Medicine and Haemostaseology, University Hospital Frankfurt, Frankfurt, Germany

Background: Antithrombin (AT) is the main blood coagulation inhibitor and AT deficiency is associated with venous thromboembolism. AT activity assays are used to screen for AT deficiency. The aim of our investigation was to detect molecular defects that cause discrepant results in AT activity assays based on factor Xa—inhibition (AT—FXa) and thrombin—inhibition (AT—FIIa) methods. Patients and Methods: In patients with AT deficiency, the AT activity was determined by AT—FXa (Coamatic LR Antithrombin, Haemochrom Diagnostica, Germany; normal range ≥86%) and AT—FIIa (STA Antithrombin III Liatest, Diagnostica Stago, France; normal range >80%). Mutations in the AT gene were detected by direct sequencing. Results: Two mutations were associated with assay discrepancy: 1) c. 133C > T, p.Arg45Trp and 2) c. 1277C > T, p.Ser426Leu. Replacement of arginine by tryptophan at position 45 has been shown to impair the heparin binding capacity of AT (Picard et al., JTH 2003), p.Ser426Leu has been shown to cause a thrombin binding defect of AT (Stephens et al., J Biol Chem 1987). The mutation p.Arg45Trp was identified in three families. The median AT activity in 5 heterozygotes was 88% (range 82–100%) measured with AT—FXa and 69% (range 63–84%) with AT—FIIa, the AT—FIIa/AT—FXa—ratio varied between 0.66 and 0.84. The AT activities of one homozygous male who suffered from progressive bilateral deep vein thrombosis despite heparin therapy were 77% (AT—FXa) and 65% (AT—FIIa, AT—FIIa—/AT—FXa—ratio 0.84). The mutation p.Ser426Leu was identified in a 19—year—old male with recurrent venous thromboembolism. He exhibited an AT activity of 100% measured with AT—FXa and of 55% with AT—FIIa (AT—FIIa/AT—FXa ratio 0.55). Conclusion: Discrepant AT activities in FXa—and thrombin—inhibition assays can be caused by AT mutations that impair the heparin—binding or the thrombin—binding capacity of AT. Discrepant AT results can cause misdiagnosis of AT deficiency.

P 6.05

Establishing causativity for isolated heterozygous mutations in F13A and F13B gene by in vitro expression.

Thomas A., Biswas A., Ivaskevicius V., Oldenburg J.

¹Institut für Experimentelle Hämatologie und Transfusionsmedizin; Universitätsklinikum Bonn, Bonn, Deutschland

Aim: To determine causativity of 12 reported heterozygous mutationsin F13A gene and F13B gene by expression in Cos—1 and HEK293 cells. Material and Methods: The mutations were incorporated in mammalian expression vectors by site directed mutagenesis and transfected onto Cos—1 and HEK293 cell lines. Thirty—six hours post—transfection lysates were assayed for antigen, activity levels/western blots. Results were expressed as percentage of the wild type (WT). Results: The F13A mutations showed similar activity and antigen levels in Cos—1 and HEK293 cells except for one mutation. Three F13A mutations showed mildly reduced activity (47–73%) and antigen levels [39%—75%], one showed moderate (28%activity/23%antigen) and one severe (4.5%activity/2%antigen) reduction. One mutation (Gly592Ser) shows raised activity in Cos—1 cells (173%) but similar antigenic levels (92%). The same mutation however showed similar activity levels in HEK293 cells (95%). The F13B gene mutation, showed mild (75–97%) to a moderate (40%) reduction in antigen levels when expressed in Cos—1 cells. In the HEK293 cells, however, these mutations show moderately reduced levels of antigen (29–69%) except for one mutation (83%; mild reduction). The western blots results for all mutants correlate with the antigenic levels. Conclusion: The F13A mutations do not seem to influence the stability of the protein radically except for Pro289Arg mutation. These mutations may be causative by influencing protein—protein interactions like the hetero—tetramer assembly. Mutations in F13B gene also do not influence antigenic levels drastically, although in HEK293 cell lines they do show comparatively lower antigen values. The causativity of the F13B gene mutations may be a combined influence of low antigenic stability and inability to bind and protect the A subunit. The difference in antigenic levels of the mutants and wild type might not be apparent in Cos—1 cells owing to lower yields than in HEK293 cells.

P 6.06

Impact of laboratory values monitoring Argatroban and Lepirudin in vivo

Seidel H., Buchbinder S., Makhloufi H., Hoffmann T., Bomke B., Morgenrot U., Scharf R.E.

Dept. of Experimental and Clinical Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Duesseldorf, Germany

Background: Monitoring alternative anticoagulation with direct thrombin inhibitors (DTI) argatroban and lepirudin is essential for efficacy and safety. For its therapeutic use 1.5 − 3.0—fold of baseline activated partial thromboplastin time (aPTT) is aspired. However, the validity of aPTT values may be questioned for several reasons, including sensitivity of aPTT reagents. We compared aPTT, thrombin time (TT) and prothrombin time (PT—Quick) with Ecarin Clotting time (ECT) derived DTI concentration (DTI—c). Patients and Methods: aPTT (Pathromtin SL), TT (BC Thrombin), PT (Thromborel S) and DTI—c were measured in 37 patients on argatroban and in 80 patients on lepirudin. n = 98 (52%) and n = 229 (75%) were ordered from intensive care units (ICU). Correlation analysis was performed by Spearman rank correlation with alpha level of 5%. Results: The mean DTI—c was 0,41 ± 0,36 μg/ml for argatroban and 0,20 ± 0,21 μg/ml for lepirudin, respectively. Standard deviation of aPTT was 25 (analyses of the ICU) and 18 seconds (non ICU) for patients on argatroban, resp. 21 and 19 seconds for patients on lepirudin. TT was significantly (p < 0.01) correlated with DTI—c irrespective of the type of DTI (r = 0,820 for argatroban and r = 0,830 for lepirudin). In contrast, aPTT was correlated with DTI—c only weakly in the lepirudin group (r = 0.572 for lepirudin) but not in the argatroban group (r = 0,136). Multiple regression analyses revealed that the TT was predictive for DTI—c in 54% of argatroban cases and 42% of lepirudin cases, respectively, whereas no significance was found for aPTT or PT. Conclusion: According to ECT based DTI—c, argatroban therapy guided exclusively by aPTT results may be deceptive. A combined assessment of aPTT and TT as well as DTI—c, together with attention to pre—therapeutic baseline values and adherence to clinical conditions (e.g. use of extracorporal devices) seems to be essential for laboratory evaluation of the anticoagulant effects of these DTIs.

Figure 1.

P 6.07

Modulation of cell adhesion and Integrin signaling by immobilized plasma ligands

Gyenes M., EI—Khattouti A., Stoldt V.R., Scharf R.E.

Department of Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf, Germany

Background: αIIvβ3, a platelet integrin for fibrinogen, von Willebrand factor, and other adhesive proteins can be activated by immobilized fibrinogen inducing outside—in signaling. The β3 subunit is polymorphic at residue 33 leading to Leu33 or Pro33. Pro33 platelets exhibit a prothrombotic character. In this work, we examined how fibronectin, a RGD—containing adhesive protein, regulates αIIbβ3—mediated processes in αIIbβ3 overexpressing HEK293 cells upon adhesion. Specifically, we assessed cell adhesion rates and outsidein signaling by the αIIbβ3—associated Src kinase upon exposure to fibronectin as compared to fibrinogen. Methods: Adhesion assays with fluorescently labeled HEK293 cells overexpressing either isoform (Leu33 or Pro33) of αIIbβ3 were performed using various concentrations of immobilized fibrinogen or fibronectin. Adherent cells were counted by a microplate fluorometer. Src pY418 kinase activity was determined by Western blotting and quantified by densitometry. Results: After 5 min at ligand concentrations of 20, 50 or 100 μg/ml, both Leu33 and Pro33 cells exhibited a significantly higher adhesion rate on fibrinogen than on fibronectin (p > 0.05, each). Maximal Src activation of adherent Leu33 cells was detected on fibrinogen at 10 μg/ml and on fibronectin at 50 μg/ml, respectively. Adherent Pro33 cells displayed similar Src activities at all concentrations of both adhesive proteins, demonstrating a ligand— and concentration—independent Src activation. At low concentrations of both ligands (2.5 and 5 μg/ml), the Pro33 isoform showed a 3—fold higher Src pY418 hosphorylation than Leu33 cells (p < 0.05). After 45 min, both isoforms displayed ligand—independent adhesion rates and Src activities. Conclusion: Our results demonstrate a higher affinity of both αIIbβ3 isoforms to immobilized fibrinogen than to fibronectin. The faster and greater αIIbβ3—mediated outside—in signaling of Pro33 cells is consistent with the prothrombotic character of this genotype.

P6.08

Variable activation kinetics of different recombinant full length and B domain deleted factor VIII concentrates

Pahl S., Pavlova A., Driesen J., Müller J., Oldenburg J.

Institut für Experimentelle Hämatologie und Transfusionsmedizin; Universitätsklinikum Bonn, Bonn, Deutschland

Background: Recombinant Factor VIII (rFVIII) treatment centrates differ by the presence of the B—Domain, the cell type used for expression and polymorphism inside the FVIII protein. This study analyses the activation kinetics of four rFVIII concentrates. Material/Methods: To analyse the activation profile, the recombinant FVIII concentrates Advate, Helixate, Kogenate and the B—domain deleted (BDD) rFVIII Refacto AF were subjected to Thrombin digestion. Time course of activation was visualised by Western blot using an antibody directed against the A2—domain. Results: Kogenate/Helixate showed the fastest rate of A2—domain—formation for all thrombin concentrations. With 1nM thrombin Refacto AF represents the lowest rate of A2—formation and Advate showed activation profile between Kogenate/ Helixate and Refacto AF. The fastest FXa—generation was obtained for Kogenate and Helixate. Both, Advate and Refacto, showed statistically significant lower values for FXa—generation. No variation of the FXa—generation was seen when the concentrates were adjusted to the FVIII antigen. Conclusion: The in vitro activation profiles of the various recombinant FVIII proteins analysed by thrombin digestion and western blot showed significant differences. The slower activation kinetics in the BDD—rFVIII might be due in part to the absence of the B—domain which may contribute to the structural integrity of the thrombin cleavage site at aa position 740. However, these data represent in vitro data and do not necessarily translate to in vivo characteristics and function of the recombinant FVIII concentrates in the haemophilia A patients.

Session 07 – Hemostasis: Platelets

P 7.01

Mechanisms of action of Acetylsalicylic Acid in human platelets

Geörg K.¹, Schedel A.², Binzen U.¹, Klueter H.², Treede R.—D.¹, Greffrath W.¹, Bugert P.²

¹Department of Neurophysiology, Center for Biomedicine and Medical Technology Mannheim (CBTM), Mannheim, Germany, ²lnstitut of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Germany

Background: Two mechanisms of action were described for acetylsalicylic acid (ASA), widely used in anticoagulation and pain therapy: 1. acetylationdependent antagonism of cyclooxygenase, the key enzyme of thromboxane A2 (TxA2) biosynthesis; 2. acetylation—independent antagonism at the pain receptor TRPV1. Since it was shown that human platelets express functional TRPV1, it is speculated that ASA inhibits platelet function via both pathways. In this study we investigated both mechanisms of ASA effects on platelets. Methods: Cyclooxygenase—acetylation was investigated by the use of ASA, methylsalicylic acid (MSA) and salicylic acid (SA) in platelet aggregometry and quantification of TxA2 formation (TxB2— EIA). The effects of capsaicin—induced TRPV1 activation were measured by platelet aggregation and degranulation (CD62P surface expression by flow cytometry). In addition, TRPV1 expression was investigated by QRT—PCR and Western blot analysis. Results: As expected, ASA dose—dependently inhibited platelet aggregation and TxA2 formation (IC50 = 8.3 μμ). Because no inhibition was observed for MSA and SA – both lacking the acetylic side chain necessary for acetylation – the ASA effect is most likely dependent on acetylation. We previously showed that ASA, MSA and SA significantly block the TRPV1 receptor in HEK cells, suggesting direct acetylation—independent inhibition (IC50 = 0.22 μμ for ASA). However, we were not able to investigate that mechanism in platelets, because capsaicin (up to 100 μμ) had no effect on platelet function. Therefore we proved TRPV1 expression in platelets and were not able to detect TRPV1 neither at the RNAnor at the protein level. Conclusions: ASA caused inhibition of the cyclooxygenase in platelets by an acetylation—dependent mechanism. Based on our data we would raise doubts about the expression of TRPV1 in platelets and conclude that the acetylation—independent TRPVl—inhibition by ASA does not play a role in platelet function.

P7.02

Double—dose clopidogrel can be helpful for patients with high platelet reactivity on normal dose

Makhloufi H.¹, Seidel H.¹, Vorpahl M.², Kirch hoff E.³, Buchbinders.³, Dücker C.³, Hoffmann T.¹, Scharf R.E.¹

¹Dept. of Experimental and Clinical Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Duesseldorf, Germany, ²Department of Cardiology, Pneumology and Angiology, University Hospital Dusseldorf, Dusseldorf, Germany, ³Department of Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf, Germany

Background: High platelet reactivity measured by platelet function tests is associated with adverse cardiovascular events in patients on antiplatelet therapy. Recently, doubling the dose of clopidogrel showed no significant difference in an overall cohort of ACS patients, but selected patients could benefit from double dose clopidogrel. We compared in—vitro efficacy of antiplatelet therapy in patients with double and standard dose of clopidogrel by P2Y12—specific assay and ADP—induced platelet aggregation. Methods: We retrospectively evaluated a cohort of 431 patients by calculating the platelet reactivity index (PRI) based on the phosphorylation status of the vasodilator phospho—protein (P2Y12—specific assay) and by ADP—induced platelet aggregation (ADPmax) with light transmission aggregometry. N = 390 patients were under dual antiplatelet therapy with ASA and standard dose clopidogrel. N = 129 patients (33.1%) showed a PRI >79, indicating a low response to clopidogrel. Results: Overall PRI and ADPmax were 60 +/— 41% and 52 +/— 36%, respectively. There was no significant difference in PRI and ADPmax between standard and double dose clopidogrel (student t—test p > 0.5). Patients with low clopidogrel response under standard dose revealed a PRI and ADPmax of 83 +/— 14% and 61 +/— 24%, respectively. In patients with previously poor clopidogrel response, treatment with double dose clopidogrel results in significantly lower PRI 60 +/— 33% (n = 41, student t—test p < 0.00001), whereas ADPmax showed only a trend to significance (ADPmax 54 +/— 33%, p = 0.06). Conclusions: Only in patients with high platelet reactivity, doubling the dose of clopidogrel results in a significant reduction in platelet reactivity. In accordance with previously data, we observed no significant difference in an overall cohort of patients between standard and double dose clopidogrel. Therefore, doubling the dose of clopidogrel could be beneficial in selected patients with high platelet reactivity.

Patients with poor clopidogrel response

	Baseline clopidogrel 75 mg/d	Clopidogrel 150 mg/d	Reduction
PRI	83±14 %	60±33 %	28 % (p < 0.00001)
ADPmax	61±24 %	54±33 %	(p > 0.05)

Reduction in platelet reactivity after doubling clopidogrel dose

P7.03

The effect of N—Octanoyl—Dopamine for hypothermic organ preservation on platelet function

Schedel A.¹, Ait—Hsiko L.², Kraaij T.¹, Yard B.¹, Klueter H.¹, Bugert P.¹

¹lnstitut of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Germany, ²Department of Nephrology; Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

Background: In an earlier report we could show that dopamine (DA), through D2—like receptors, is a co—agonist for platelets. DA was also shown to have protective effects in kidney transplantation. Recently, N—octanoyl—dopamine (NOD) was developed which is about 40x more effective against cold preservation organ injury compared to DA. Whereas both compounds have comparable redox potentials, only NOD consists of a long hydrophobic tail that enables rapid cell entry. In the presence of esterases NOD is conducting an increase of the intracellular redox potential. Here, we investigated the effects of NOD and the non—redoxactive analog N—octanoyl—tyramine (NOT) on platelet function. We compared the findings to the effects caused by DA. Methods: Effects of DA, NOD and NOT on the platelet response to classical agonists (ADP, U46619) were investigated in 6 healthy individuals applying whole blood aggregometry (Multiplate), flow cytometry (Pac—1, CD62P, CD63) and cAMP quantification. Results: As expected, DA showed significant co—ago—nism in ADP— and U46619—induced platelet activation. In the same experimental setting NOD caused significant inhibition of platelet function, whereas, NOT had no effect. This clearly indicates that the NOD effect is caused by the intracellular increase of the redox potential. DA, NOD or NOT had no significant effects on cAMP levels. Conclusions: While DA increases platelet activation through D2—like receptors, the hydrophobic derivate NOD significantly inhibits platelet function. Through its hydrophobic tail, NOD rapidly enters the plasma membrane and elicits inhibitory effects most likely by intracellular redox—active processes. Platelet activation and thrombus formation is involved in hypothermic organ deterioration. Thus, the inhibitory effect of NOD on platelets might add to the protective effect of NOD in hypothermic organ preservation.

P 7.04

The impact of a variant platelet Integrin: modulation of adhesion underflow and low concentrations of immobilized RGD containing ligands

Stoldt V.R., Schlesinger S., Scharf R.E.

Department of Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf, Germany

The HPA—1 polymorphism of platelet integrin αIIbβ3 is based on an amino acid exchange from Leu33 (HPA—1a) to Pro33 (HPA—1b). Recently, we have demonstrated in CAD patients that HPA—lb alíeles of αIIbβ3 are associated with premature manifestation of myocardial infarction. We hypothesize that HPA—1b is a prothrombotic phenotype due to stronger receptor ligand interaction mediating a firmer adhesion onto low concentrations of ligands under flow conditions. Citrated blood obtained from healthy donors (HPA—1a/1a n = 11, HPA—1b/1b n = 9) and incubated with 10 μμ mepacrine was perfused through a rectangular flow chamber (flow path 80 μm height) coated with fibrinogen (Fg) or fibronectin (Fn), each at concentrations of 100 or 20 μg/ml. The wall—near shear rates were 100 or 1000 s—1. Controls were performed with abciximab or BSA instead of Fg or Fn. Platelet adhesion was detected by laser scanning microscopy over the time, analyzed by ImageJ Software and quantified in arbitrary fluorescence units (FU). HPA—1a/1a and HPA—1b/1b platelets showed similar adhesion rates onto both ligands at a concentration of 100 μg/ ml and shear rates of 100 and 1000 s—1. By contrast, higher platelet adhesion occurred with HPA—1b/1b compared to HPA—1a/1a using Fg and Fn both at a concentration of 20 μg/ml and a shear rate of 1000 s—1 (∗p < 0.05). At a shear rate of 100 s—1, both HPA—1 variants showed a significantly higher adhesion onto Fn than onto Fg at 100 μg/ml. We conclude that low concentrations of Fg or Fn maintain the adhesion of HPA—1b/1b platelets, specifically when higher shear rates are applied. Hence, HPA—1b/1b rather than HPA—1a/1a platelets mediate a firm adhesion under flow conditions, supporting the prothrombotic character of HPA—lb.

P7.05

Cooperative regulation of PKCalpha activity and nAChRalpha7 expression in platelet precursors

Besenfelder S., Klueter H., Bugert P., Schedel A.

Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Mannheim, Germany

Background: We have previously demonstrated that platelets and their precursors express functional nicotinic acetylcholine receptors (nAChR) of the a7—type that form ligand—gated ion channels with high permeability to calcium ions. Through calcium dependent mechanisms, the nAChRa7 is involved in fibrinogen receptor activation and aggregation in platelets. Furthermore, we could show that the nAChRa7—selective antagonist methyllycaconitine (MLA) significantly inhibited in vitro megakaryopoesis, indicating a role of these receptors in the generation of platelets in vivo. In other non—neuronal cells, the nAChRa7 is known to regulate the expression of many genes through protein kinase Ca (PKCα) signaling. In this study, we investigated PKCa phosphorylation and nAChR gene expression in platelet precursors. Methods: MEG—01 cells were treated with the nAChRα7 selective agonist PNU—282,987 (PNU) for 24 hours at 37°C before preparation of cell lysates. Phosphorylation of PKC was investigated by the use of polyclonal antibodies against Ser657 phosphorylated PKCα in Western blot analysis. QRT—PCR was used to investigate nAChR mRNA expression in response to nAChR agonists (nicotine and PNU). Results: The phosphorylated PKCα isoform was significantly increased in MEG—01 cells treated with the nAChRa7—selective agonist PNU, compared to untreated MEG—01 cells. As revealed by QRT—PCR, treatment of MEG—01 cells with nicotine (500 nM) or PNU (10 μμ) induced significant upregulation of nAChRa7 mRNA. Conclusions: Nicotine increases nAChRa7 mRNA expression in platelet precursors, presumably, through a calcium dependent PKCα signalling pathway. These results contribute to the understanding of cholinergic signaling in platelets and their precursors. Chronic exposure to nicotine in smokers might lead to an increased nAChRa7 receptor density in platelets and thus probably contribute to the known enhanced platelet reactivity in smokers.

P7.06

High prognostic value of analysing sequential pre— and postnatal maternal sera using surface plasmon resonance in neonatal alloimmune thrombocytopenia

Bakchoul T.¹, Bertrand G.², Krautwurst A.³, Sachs U.J.¹, Kroll H.¹, Bein G.¹, Santoso S.¹, Kaplan C.²

¹1nstitute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany, ²the Platelet Immunology Unit and the Immunology Transfusion Unit, INT, Paris, France, ³lnstitut für Tranfusionsmedizin und Klinische Immunologie am Universitätsklinikum Gießen/ Marburg GmbH, Gießen, Deutschland, ⁴Red Cross Blood Transfusion Service NSTOB, Institute for Transfusion Medicine, Dessau, Germany

The most severe cases of fetal/neonatal alloimmune thrombocytopenia (FNAIT) are caused by maternal anti—HPA—la antibodies (Ab). Recent data indicated that maternal Ab concentration may be useful to predict the severity of FNAIT in newborn. In comparison to other assays, surface plasmon resonance (SPR) technology allows exact determination of Ab properties (concentration and avidity) in real time. In this study, we evaluated the advantage of SPR in predicting the severity of FNAIT. Two cohorts of mothers with FNAIT were employed; mothers received ivIgG (lg/kg/week) and corticosteroid (0.5 mg/kg/day) (Group 1) or weekly intrauterine platelet transfusion (Group 2). Maternal sera were collected during pregnancy and Abs was quantified in SPR using an internal standard serum (SIS) and mab against HPA—la (MIS). Quantification was performed at two different endpoints; of the association phase (B350) and the dissociation phase (B700). Area under antibody—binding curve (AUC) was calculated to determine the overall Ab binding. In Group 1, 35 serial samples from 7 mothers were analyzed. By quantification of B350 values using SIS and MIS, Abs ranged from 30–1377 AU/mL and 24–402 ng/mL, respectively. A progressive Abs reduction was observed during all pregnancies (4–57 AU and 4–10 ng/Week). In 2/6 cases, considerable reduction (52% and 71%) of Abs avidity was observed. In Group 2, 22 samples from 5 mothers were analyzed and the values were as followed; B350: 200–810 AU/mL (38–184 ng/ml). Interestingly, no significant change was observed in Abs concentration and avidity in this group. In both groups, strong correlation between Abs concentration in the last collected sera and neonatal PLT count was found. Best correlation was found when AUC was determined (spearman r: 0.96). In conclusions, Treatment with ivIgG and cortisone does not only reduce Abs concentration but also decrease binding avidity. Determination of AUC in SPR represent an useful predictor of NAIT severity.

P 7.07

Clinical evaluation of a rapid lateral—flow immunoassay for the diagnosis of heparin—induced thrombocytopenia (HIT)

Sachs U.J., Santoso S., Bein G., Bakchoul T.

Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany

Background: Heparin—induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies (abs) that recognize platelet factor 4—heparin (PF4/hep) complexes. Several laboratory tests for the confirmation and/or refutation of HIT are available, but until recently a reliable and rapid single—sample test was still pending. In our study, we evaluate a new lateral—flow immunoassay based on nanoparticle technology. Patients and Methods: A clinically well—characterized cohort of 452 surgical and medical patients suspected of having HIT was evaluated. All samples were tested in two IgG—specific ELISAs (GTI, Waukesha, USA and Hyphen Biomed, Neu—ville—Sur—Oise, France), in a particle gel immunoassay (PaGIA; BioRad, Munich, Germany) and in a newly developed lateral—flow immunoassay (MQL; Milenia Biotec, Giessen, Germany) as well as by HIPA. Clinical pretest probability was obtained using the Greifswald modification of the 4T's scoring system. Results: Platelet—activating antibodies were present in 34/452 patients, all of whom had intermediate to high clinical probability. PF4/hep abs were detected in 79, 87, 86, and 63 (55) sera using GTI—IgG, Hyphen—IgG, PaGIA, and MQL visually (or applying a scanner). The negative predictive values (NPV) were 100% for both ELISA tests and MQL (read visually), but only 99.7% for MQL read by scanner (1 false negative result) and 99.2% for PaGIA (3 false negative results). There were less false positives (n = 29) in the MQL test compared to any other test (n = 45, 53, and 55, respectively). Conclusion: In this study, a newly developed lateral—flow assay, MQL, was capable of identifying all HIT patients in a cohort in an extremely short period of time. Beside an excellent NPV of 100%, the rate of false—positive signals is significantly lower with MQL than with any other immunoassay. Because this assay is rapid and easy to perform, it does carry the potential to reduce the risk and costs in patients suspected of having HIT.

P 7.08

Small and large thrombocytes, previously separated by size using counter—flow centrifugal elutriation, show different activation potential and distinct functional capabilities

Fischer M.B.¹, Mader C.², Eibl J.², Panzer S.¹, Gruber R.³

¹Dept. of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria, ²Bio—Products & Bio—Engineering AG, Vienna, Austria, ³Department of Oral Surgery, Medical University of Vienna, Vienna, Austria

To separate small from large thrombocytes we successfully used continuous counter—flow centrifugal elutriation (CCE) and the Gambro Elutra device. This physical cell separation method was used to ensure that thrombocytes are not activated during their separation in small and large subpopulations. After separation, small and large thrombocytes were forwarded for functional analysis. ADP and collagen induced platelet aggregation as well as thrombin receptoractivating peptide—6 (TRAP—6) inducible aggregation was found to be stronger in small platelets as compared to larger ones. Platelet—released supernatant of small and large activated platelets can both activate bone marrow stromal cells and dental pulp fibroblasts and induce proliferation in an ex vivo assay. In addition, small thrombocytes were shown to be more potent to generate platelet—derived microparticles (PMPs), small membrane vesicles larger than 100nm in diameter shed from activated platelets, as determined by flow cytometry. Our results demonstrate CCE as a useful tool to separate small from large thrombocytes in order to study their activation potential and functional capability.

Session 08 – Hemotherapy

P 8.01

Management of pregnancy in a patient with Bombay blood group

Fuchs N.¹, Henneberg—Quester K.—B.¹, Holt mann M.¹, Cordeiro C.¹, Elter—Schulz M.¹, Schmidt M.¹, Greiner D.², Horn P.A.¹, Lenz V.¹

¹Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany, ²Department of Obstetrics and Gynecology, University Hospital Essen, Essen, Germany

The Bombay blood group (O_h) is an extremely rare blood group and has previously been described as a cause of hemolytic disease of the newborn (HDN). We here report the case of an 35 year old primigravida from Sicily who was referred to us in the 37th week of gestation. Antibody screening was positive due to anti—H, and subsequently the woman was typed as O_h. Her blood group was typed again and Bombay status was confirmed serologically. Titers of Anti—A, Anti—B and Anti—H were as follows: Anti—A1 IgM/IgG 4/64, Anti—B 16/512, Anti—H 4/16. Titers were determined by DiaMed ID—Card System (IgM NaCl 20°C, IgG LISS/Coombs 37°C). Due to the rarity of donors and possible need of an exchange transfusion, having O_h blood on standby for delivery was recommended in the literature. Medical induction of labour was planned and three units of packed red blood cells (PRBC) were requested. Both parents and two brothers were called in for blood group typing. All of them are BG O and thus could not be considered as donors. The husband was typed BG A1B. Since autologous blood donation was not possible, blood products from O_hdonors were sought. In addition to frozen units identified through the German Rare Donor Program, we recruited one donor from a family with several cases of O_h blood group in Essen. Two additional units of PRBC from another donor were collected by apheresis by the DRK BSD West. On May 12 2011, a female baby was delivered after Misoprostol induction. The infant (3060 g, 52 cm, APGAR score 10/10/10, bilirubin 4,3 mg/dl) had no clinical signs of HDN. Her blood group was B Rh(pos) CcD. Ee with a negative antibody screen. The direct coombs test (DCT) was positive with IgG (subclass 2 or 4) and an Anti—B antibody was eluted. Our case report thus demonstrates the feasibility of organizing O_h blood on standby for such rare cases despite late admission of the patient in adequate amounts, emphasizing the usefulness of the German Rare Donor Program.

P 8.02

Diagnosis and clinical management of β—Thalassemia minor in pregnancy

Scharf M.—A.¹, Balan P.², Scharf R.E.³, Janni W.², Scharf R.E.³

¹Department of Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf and Department of Gynecology and Obstetrics, Albert Ludwigs University Medical Center, Freiburg, Germany, ²Department of Gynecology and Obstetrics, Heinrich Heine University Medical Center, Duesseldorf, Germany, ³Dept. of Experimental and Clinical Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Duesseldorf, Germany

Hypochromic microcytic red blood cells (RBC) are a typical pattern of iron deficiency anemia but may be also indicative of thalassemia. Here we report on a 37—year—old pregnant woman who presented with anemia caused by a thalassemia trait. The index patient is of Mediterranean origin and had given birth to a child in 2007. Following delivery by primary Caesarean section, the patient had experienced a severe hemorrhagic shock (Hb of 2.8 g/dl) due to a major intraabdominal bleeding (caused by insufficiency of a suture). Upon transfusion of RBC concentrates and surgical revision, she rapidly recovered and became pregnant again in 2010. At presentation in the 15th week of gestation, the patient had moderate anemia with microcytosis and hypochromia (Hb 10.6 g/dl, He 32.4%, MCV 69.1 fl, MCH 22.6 pg, MCHC 32.7%), target cells and reticulocytosis of 320/nl but normal ferritin (75.6 ng/ml) and LDH (196 U/ml). Hb electrophoresis revealed elevated HbA2 of 4.9%, reduced HbA of 93.4% and normal HbF of 1.8% compatible with a heterozygous ß—thalassemia minor. A similar pattern was identified in the patient's father and her 4—year—old daughter. The subsequent course of pregnancy was entirely normal with stable Hb > 10 g/dl, however, ferritin decreased to 10 ng/ml. At the end of the 39th week of gestation, the patient gave birth to a healthy boy. Her postpartal Hb was 7.7 g/dl and gradually increased upon alimentary iron supply. No transfusions of RBC were given to prevent from iron overload or alloimmuni—zation. In conclusion, patients with ß—thalassemia should be warned that their blood picture resembles that of iron deficiency and can be misdiagnosed. They should avoid routine use of iron but know that iron deficiency requiring supplementation can develop, as in other individuals, during pregnancy or as consequence of chronic bleeding. Since iron deficiency can aggravate the dysbalance of Hb chains, oral iron supplementation is indicated in thalassemia at Hb levels < 9 g/dl.

P8.03

Supply of a female patient with an anti—Co3 antibody

Oeke B.¹, Stubert D.², Just B.¹, Lander—Kox J.², Deitenbeck R.¹

¹German Red Cross Blood Service West, Center of Transfusion Medicine, Hagen, Germany, ²German Red Cross Blood Service West, Center of Transfusion Medicine Breitscheid, Ratingen, Germany

Background: Antigens (AG) of the Colton blood group are located on the aquaporin—1 and are represented by the AG Co(a) and Co(b). Individuals with Co(a—b—) are able to form antibodies (AB) reacting with almost all tested cells except those of the Co(a—b—) type. The AB is able to activate complement and causes in vitro hemolysis, presumably in vivo as well. Patient and Methods: We report on a female patient with lower gastrointestinal hemorrhage and a Hb of 4.2 g/dl who could be provided with RCC donated by a relative. An anti—Co3—AB was previously documented in 1985. For immunohematological confirmation we applied column and tube agglutination technique as well as absorption/elution methods and SSP—PCR. Results: In the patient's serum we detected an IgG3—specific anti—Co3—AB (confirmed by the IBGRL Bristol, UK) which reacted moderately with all native and papain treated test cells. The same reaction strength became evident with only Co(a—) and Co(b—) test cells. Screening of 6 relatives resulted in only 1 compatible donor. A brother who was among the eligible donors as well as the patient were found Co(a—b—). The result of SSP—PCR was C01+ and C02—, respectively. Sequencing for identification of an inducing mutation has not been made yet. The brother who showed an Hb of 17.2 g/dl was able to make a whole blood donation as well as a double red cell apheresis within 2 days so that the patient could be supplied sufficiently. Throughout Europe no compatible RCC were available in a reasonable time with respect to the patient's clinical situation. Conclusion: We report on a female patient with an AB of anti—Co3 specificity. Both brother and sister showed a discrepancy between the phenotype Co(a)— and the genotype C01+. It poses a big challenge to provide patients with AB against high frequent AG. Searching for donors among relatives may be succesful. New collection techniques (like double red cell apheresis) are indispensable, as well as cryopreservation of rare RCC.

P8.04

Management of anti—U alloimmunization in severe Sickle Cell anaemia: a case report

Korfmacher S.¹, Hoffmann T.¹, Hamberger R.¹, Fox F.², Deitenbeck R.³, Just B.³, Mayer B.⁴, Salama A.⁴, van den Burg P.⁵, Scharf R.E.¹

¹Dept. of Experimental and Clinical Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Duesseldorf, Germany, ²Department of Hematology, Oncology and Clinical Immunology, Heinrich Heine University Medical Center, Duesseldorf, Germany, ³German Red Cross Blood Service West; Center of Transfusion Medicine, Hagen, Germany, ⁴Institute of Transfusion Medicine, Charité – Universitaetsmedizin Berlin, Berlin, Germany, ⁵Sanquin Blood Supply Foundation, The Netherlands

Background: A 60—year—old male of African parentage was admitted with critical anaemia due to a sickle cell crisis. Upon negative compatibility testing, two red blood cell (RBC) concentrates were transfused uneventfully with adequate increase of the Hb concentration from 4.4 to 6.0 g/dl. Nine days later, urgent transfusion need reoccured. This time, antibody screening revealed irregular antibodies with suspected anti—S specificity. During transfusion of two cross—match negative, S negative RBC concentrates, the patient developed tachycardia and dyspnoea. Transfusion was discontinued and complaints remitted upon symptomatic therapy. Hemolytic activity did not exceed preexisting levels. Subsequent antibody differentiation showed a panreactive pattern; a single test cell lacking Fy(a), Fy(b), ‘S’ and ‘s’ antigens displayed a negative reaction. Further investigation with U negative cells allowed the confirmation of an anti—U and exclusion of other antibodies. Subsequent transfusion was effective and uneventful using a cryopreserved U negative RBC concentrate. In addition, a U negative, AB0 identical donor was traced. He underwent double RBC apheresis and one of the components was transfused. The patient stabilized thereafter at Hb levels > 6.0 g/dl and could be transferred to the outpatient clinic. Conclusion: (1) U incompatible RBC transfusions in patients with anti—U not necessarily cause severe hemolytic transfusion reactions. (2) Given the current migration activity, the incidence of relevant antibodies against Caucasian high frequency antigens will increase. (3) Cryopreserved RBC concentrates of rare phenotypes are helpful in urgent treatment but are unfavourable with respect to cost and viral safety, if donated long time ago. Thus, extended collection and registration of blood donors with rare blood groups are required.

P 8.05

Randomized controlled trial (RCT) comparing the transfusion efficacy of triple and double Amicus platelet concentrates collected in plasma or PAS III additive solution

Fontana S.¹, Summermatter R.¹, Vogt M.², Mauchle P.², Lädrach A.¹, Pellegrini A.¹, Mansouri Taleghani B.²

¹Blood Transfusion Service SRC Berne, Switzerland, Bern, ²University Clinic of Hematology and Central Hematological Laboratory, Inselspital, University Hospital, Bern, Switzerland

Background: A previous study at our institution suggest that corrected count increments 1 hour after transfusion (1h—CCI) of platelets (PLT) collected with Amicus by high yield apheresis and/or stored in PLT additive solution (PAS) may be lower. Methods: RCT including 41 regular donors donating 4 different PLT products in a randomized sequence: double dose PLT in plasma (2PL), in InterSol (2IS), triple dose PLT in plasma (3PL), and in InterSol (3IS). Preliminarily available 1h—CCI in thrombocytopenic hemato—oncological patients are compared between the 4 products of donors completing the 4 donations (paired, Wilcoxon signed rank test) and between all currently transfused products (unpaired, 2—sided t—test). A linear model testing other factors influencing 1h—CCI is calculated by F—test. Paired analysis is also performed with percent of PLT recovery (PPR). Results: Mean 1h—CCI in 4 × 14 transfusions from donors completing all 4 donations are 15.2 (2IS), 11.9 (2PL), 12.4 (3PL), and 6.0 (3IS). Paired 1h—CCI comparisons of these transfusions show no differences between 2IS and 3IS (p unadj 0.049, adj >0.1), 2PL and 3PL (p unadj, and adj >0.1), 2IS and 2PL (p unadj and adj >0.1), and a trend toward difference between 3IS and 3PL (p unadj 0.017, adj 0.066). For all 98 transfused products the mean lh—CCI are 14.2 (2IS), 12.6 (2PL), 13.7 (3PL), 8.5 (3IS), and there are no differences (excepted for 2IS vs. 3IS, p unadj = 0.048, adj >0.1). The linear model shows no relevant influence of other variables on CCI differences. The PPR results in the paired analysis are similar. Conclusions: This preliminary evaluation shows no differences between 2 PL, 2IS, and 3PL, and lower lh—CCI after transfusion of 3IS, particularly in comparison with 3PL. These results seem to support the study hypothesis, but a higher number of transfusions are needed to confirm their significance.

P8.06

A case—control study comparing high yield platelet (PLT) apheresis with Amicus V3.11 and Trima V5.1/5.2 cell separators

Siderow A., Lädrach A., Fontana S.

Blood Transfusion Service SRC Berne, Bern, Switzerland

Background: The Fenwal Amicus (A) version 3.11 and the CaridianBCT Trima Accel (T) Versions 5.1 (PLT in Plasma) and 5.2 (PLT in PAS III) cell separators produce up to 3 PLT concentrates containing >2.4 × 1011 PLT/unit within a single donation. In our blood transfusion service A is used for a randomized study comparing double and triple PLT concentrates in plasma or PAS III (V3.ll), and T has been recently switched from PLT in plasma (PL) to PLT in PASIII (InterSol, IS), in order to introduce pathogen inactivation of PLT concentrates. Study design and Methods: Retrospective case—control study comparing the A study donations with a corresponding T donation type of the same donor if available (donation types: triple PLT concentrate in plasma = 3PL, triple PLT concentrate in PASIII = 3IS; double PLT concentrate in plasma = 2PL, double PLT concentrate in PASIII = 2IS). Primary endpoint: collection rate (CR, PLT∗10ell/hour). Secondary endpoints: collection yield (Y, PLT∗10ell), apheresis duration (d, hours), ACD and Calcium (Ca) use, reported side effects. Results: The comparison of the 87 donation available shows a mean CR of 5.30 (SD +/—1.33) in A and of 6.02 (+/—1.48) in T (p < 0.001). The difference is explained by both a higher Y in T (7.63+/—1.24) than in A (7.09+/—1.77; p < 0.001) and a shorter collection time in T (78.11+/—21.44) than in A (82.92+/—15.09, p = 0.017). CR is significantly higher for T in 3IS, 3PL and 2IS collections (all p < 0.01), and not for 2PL (p = 0.364). Donation characteristics and tolerance are comparable between T and A donations, with 9/87 donors needing Ca (10.3%) and without severe adverse events observed. Conclusion: Both cell separators show safe collections of up to 3 PLT units per donation. T produced higher CR, explained by a higher PLT yield despite shorter apheresis duration.

P8.07

Longterm granulocyte transfusion on a patient suffering from CML relapse after second allogeneic BMT – a case report

Fischer I.¹ Kreissig C.¹, Bux J.², Schündein M.M.³

¹DRK Blutspendedienst West – ZBST, Ratingen, Deutschland, ²DRK—Blutspendedienst West, Hagen, Deutschland, ³Päd. Hämatologie, Onkologie und Palliativmedizin, Universitätskinderklinik Essen, Essen, Deutschland

Longterm granulocyte transfusion in a patient suffering from CML relapse after second allogeneic BMT— a case report. We describe the case of a female 10—year—old patient, who suffers from a second relapse after two HLA identic allogeneic bone marrow transplantations in October 2005 and in April 2010. She was treated with Imatinib from April until June 2010 and Dasatinib from June until December 2010, when she developed a lymphatic blast crisis. The patient was treated with a reinduction consisting of Prednisone, Doxorubicin and Vincristin. During this process the patient was admitted to the hospital with neutropenic fever and treated with broad spectrum antibiotics and anti—mycotic drugs. Bone marrow aspirates were aplastic without any signs of granulopoesis. The chemotherapy was reduced to weekly Vincristin and Prednisone. Within two weeks she developed pneumonia, caused by aspergillus fumigatus; she became oxygen dependent, but was never intubated and ventilated. Over a period of six weeks she received 14 granulocyte transfusions from four experienced platelet donors. All donors were stimulated with G—CSF up to 10 microgram per kilogram bodyweight. No corticosteroids were given. After eight weeks of aplasia the patient redeveloped normal granulopoesis. Currently she is being conditioned for retransplantation from a second, unrelated donor. In our paper we describe the development of leucocyte and thrombocyte counts, Hb level, CrP, fever and clinical conditions of the patient in dependence on granulocyte transfusions.

P8.08

First experiences in the application of „Gefrorenes pathogenreduziertes Apheresefrischplasma Th—J” at the University Hospital Jena

Weinigel C, Rummler S., Barz D.

Institut für Transfusionsmedizin Universitätklinikum der Friedrich Schiller Universität Jena, Deutschland

Background: Acute transfusion reactions often result from blood cells and pathogens. We developed a leukocyte and platelet—depleted pathogen—reduced single donor apheresis plasma via filtration with Plasmaflex PLAS4 (pore size 0,65 μm) and Intercept Blood System for Plasma. This is the first pathogen—reduced plasma using Intercept technology to receive marketing authorization from the PEI in Germany (PEI.H. 11468.01.1). We observed the tolerability of this plasma product in clinic routine at the University Hospital Jena. Material and Methods: During the first two months after PEI approval altogether 923 units of plasma were transfused to 97 patients. We examined the indication for transfusion of plasma, the transfusing clinic and if transfusion reactions according to §16 TFG were registered. Results:

Indication	number of units transfused (%)	number of patients treated (%)
Surgical procedures	559 (60.6)	65 (67.0)
Acquired coagulation factor deficiency/Consumptive coagulopathy	131 (14.2)	24 (24.7)
Therapeutic plasma exchange	233 (25.2)	8 (8.2)

A total number of two transfusion reactions according to § 16 TFG were registered (0.2%).

1) Female patient with diagnosis of HUS undergoing plasma exchange therapy. A suspected TRALI was reported. All relevant plasma donors were immunologically characterized. No evidence for TRALI was detected.

2) Male patient with myelitis showed an allergic reaction within therapeutic plasma exchange treatment with severe urticaria to 2 units of plasma. Both units were obtained from the same donor. Characterization of the donor resulted in no immunological background to the observed reaction. Conclusion: With an overall transfusion reaction rate of 0.2% the pathogen reduced plasma has shown a good tolerability in clinic routine. A haemovigi—lance study is already planned for further observation of the clinical effectiveness and tolerability.

P8.09

Comparison of the transfusion of blood products in the hospitals of the Elblandkliniken—Group from 2005 to 2010

Findeisen R., Schmoz B., Guhr A., Weber K., Rolinski B.

ELBLAB GmbH Zentrum für LaborMedizin, Riesa, Deutschland

Background: This research project investigated the transfusion of blood products in the hospitals of the Elblandkliniken—Group from 2005 to 2010. The Elblandkliniken—Group is a clinical complex which consists of four hospitals with about 1.200 beds and includes clinics for Internal Medicine, Surgery, Orthopaedics and Traumatology, Gynaecology and Obstetrics, Paediatrics, Psychiatry, Ophthalmology, Oto—Rhino—Laryngology and Urology. Methods: The clinics of the four hospitals of the Elblandkliniken—Group were combined to the following three medical disciplines: a) Predominantly internal working disciplines [Internal Medicine; Paediatrics, Psychiatry, Ophthalmology, Oto—Rhino—Laryngology]; b) Predominantly surgical working disciplines [Surgery, Orthopaedics and Traumatology, Gynaecology and Obstetrics, Urology] andc) Intensive care. The transfusion of the following blood products was statistically evaluated from 2005 to 2010: heterologous and autologous red blood cells (RBCs); heterologous and autologous fresh frozen plasma (FFP); platelets; prothrombine complex (PPSB), Antithrombin III (ATIII) and Fibrinogen. Results: In all three disciplines an increase in the transfusion of heterologous RBCs was observed from 2005 to 2010. The transfusion of autologous RBCs continuously decreased during that period. In contrast, the transfusion of platelets increased in all three medical disciplines. While transfusions of PPSB and Fibrinogen increased, the number of transfusions of ATIII significantly decreased. Conclusions: On the one hand, the results of this investigation reflect the new guidelines in transfusion medicine. On the other hand, the results also provide information about the modified and new implemented methods of treatment in most disciplines of the hospitals of the Elblandkliniken—Group.

P 8.10

A comment about a decision of the Social Court Saarbruecken, Germany, on hospital billing and revenue of platelet concentrates

Zimmermann R.¹, Bender A.W.², Ringwald J.¹, Zingsem J.¹, Eckstein R.¹

¹Transfusionsmedizinische und Hämostaseologische Abteilung, Universitätsklinikum Erlangen, Erlangen, Deutschland, ²Administrative Management, University Hospital Erlangen, Erlangen, Germany

Background: Currently, the German DRG system offers different payment rates for single—donor (SD) apheresis PLTs and pooled whole—blood derived (PWBD) PLTs with SD PLTs being more expensive. This fact has recently prompted a decision of the Social Court Saarbruecken to refuse reimbursing hospitals for applicated SD PLTs instead of less expensive PWBD PLTs. Methods: Comparison and analysis of the reasons for the 2009 decision of the Social Court Saarbruecken (S 23 KR 530/08) on the prescription of PLTs and of the 1990 decision of the Federal Court (VI ZR 151/90) on the prescription of a not pathogen—reduced (PR) prothrombin complex concentrate (PCC) instead of a PR PCC. Results: The reasons for the 1990 decision of the Federal Court were: Despite the risk of hepatitis infection the prescription of a not PR PCC was no malpractice. However, if the safer PCC would have been available, only the safer PCC should have been taken. An organizational fault may lie in the fact that the safer drug was not available in the hospital. The reasons for the 2009 decision of the Social Court Saarbruecken were: The hospital may invoice the costs for the more expensive SD PLTs only, if the transfusion of the more expensive PLTs was medically required. The hospital is obliged to ensure that even in emergency situations all the necessary resources for treatment of patients are available. This also applies to PWBD PLTs and SD PLTs. Conclusions: The 2009 decision of the Social Court Saarbruecken seems to follow the 1991 decision of the Federal Court. However, in weighting safety and price it is the exact opposite. The Federal Court preferred the safer drug and not the cheaper drug. The 2009 decision interferes with the autonomy of the contractual partner for the regulation of refunding hospitals according to the German DRG system. This system does not include rules restricting the prescription of SD PLTs. For these reasons, the 2009 decision is juridically wrong.

P9.01

Moleculargenetic characterization of blood groups with problems in phenotyping: a retrospective analysis of six years blood group genotyping

Doescher A.¹, Wagner F.F.²

¹German Red Cross Blood Transfusion Service N.S.T.O.B., Institute Bremen—Oldenburg, Oldenburg, Germany, ²German Red Cross Blood Transfusion Service N.S.T.O.B., Institute Springe, Springe, Germany

Background: 2585 samples were routinely send to our laboratory from January 2005 to January 2010, 1803 samples were from blood donors and patients due to problems in serological testing, 782 for prenatal blood group genotyping in pregnancies with known antibodies or RhD negative women. Frequently observed problems in serological testing were weak reactions with certain antisera or inconclusive reaction pattern in samples from polytransfused patients. Methods: Blood samples were genotyped using multiplex—PCR settings to observe the presence of the questionable antigen and determine frequent blood group variants. Detection of rare/new variants was done by sequencing of the corresponding gene. Results: 732/782 DNA samples from amniocentic fluid were investigated for the presence of fetal RHD sequences. 50 samples were send to our laboratory to determine fetal RHC, RHE, KEL1 and FY1 in pregnancies with known antibodies. The molecular background of samples with weak reactions in serological testing was investigated for RHD (n = 729), RHCE (n = 458), ABO (n = 34), KEL1 (n = 44) and other (n = 184). Inconclusive reactions were observed in 299/1803 samples, investigation of non—expressed genes was done in 55 samples. Conclusion: A large number of molecular variants were the reason for problems in serological testing of blood groups. About 31% of the results could only obtained by sequencing of the corresponding gene, the remaining were detectable with commercial kits.

P9.02

DNAmicroarray system BLOODchip: performance in rare RHD allele detection

Rauch S.¹, von Zabern I.¹, Flegel W.A.², García—Crespo D.³, López M.³, Körmöczi G.F.⁴, Schrezenmeier H.¹

¹Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden—Württemberg—Hessen, and University of Ulm, Ulm, Germany, ²Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA, ³Progenika Biopharma S. A., Derio, Spain, ⁴Dept. of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Austria

Background: Blood group genotyping of patients and donors improves transfusion therapy and the safety of pregnancies. We validated a DNA array for routine molecular blood group determination using a large number of different blood group alleles. Methods: RHD, RHCE, KEL, JK, FY, MNS, DI, DO and CO blood group alleles were analyzed using BLOODchip Reference v2.0 (Progenika, distributed by Grifols). The results obtained by microarray analysis and conventional techniques (serology, PCR, sequencing) were compared. Results: The microarray was evaluated with 180 samples comprising 82 RHD alleles, of which 71 BLOODchip claims to recognize. 64 out of 71 RHD alleles were detected correctly. 6 alleles were not assigned (weak D type 29, DIVa, DFR III, RHD(147delA), D III type 4 and 5). One incorrect assignment was encountered: one sample of weak D type 5 was reported as DIIIc while another was typed correctly. One DEL allele was designated as RhD positive. No sample was falsely typed as RhD negative. In addition, we tested 11 RHD alleles that are not claimed to be recognized by BLOODchip. For none of these a specific call was obtained; as expected, several of these alleles were falsely reported as normal RhD positive. No discrepancies were found when 22 different alleles of the Kell, Kidd, Duffy, MNS, Diego, Dombrock and Colton systems were tested. 7% of samples with normal and 20% with rare RHD had to be repeated due to error messages. Conclusion: More than 200 D alleles are known, therefore correct D typing is a great challenge. 90% of tested D alleles were recognized correctly, only one assignment was incorrect. Optimizing software algorithms may reduce the percentage of error messages and alleles not assigned. All tested blood group alleles other than D were recognized correctly. BLOODchip proved to be a valuable tool to solve complex problems in blood group typing not amenable by serology.

P9.03

Dissection of the X—Chromosomal McLeod locus and direct gene sequencing as tools for the analysis of KX gene deletions and mutations

Gassner C.¹, Meyer E.¹, Fragnière M.¹, Bronnimann C.¹, Frey B.M.¹, Jung H.²

¹Blutspende Zürich, Schlieren, Switzerland, ²Department of Neurology, University Hospital Zürich, Zürich, Switzerland

Background: The X—linked McLeod syndrome (MLS) is a rare disease, which leads to pathognomonic deficiencies of the hematological, neurological, and cellular immune system, mainly in male humans. MLS is caused by either direct gene mutations olXK, or different (large) deletions on Xp 21.1, hitting XKs’ and its neighbour genes’ integrity. MLS usually becomes apparent by either neurological symptoms, improperly expressed KEL epitopes, or presence of chronic granulomatous disease (CGD). Methods: Since males only have one X—chromosome, particular genetic presence or absence can easily be tested by positional PCRs. In this way 32 regularly distanced PCRs, organized in 16 biplexes, were used to test a 8.8 mbp region covering the genes from Dystrophin (DMD, telomeric) to Ornithine Carbamoyltransferase (OTC, cen—tromeric). Regions of deletions were narrowed by further PCRs until breakpoints could be “bridged” by one PCR, which was the DNA for breakpoint—sequencing. Undeleted XK genes with assumed mutations were directly sequenced on 3 exon—specific PCR—fragments. Results: Three individual samples with MLS were found to show no (“Bavarian”), a 151.47 kbp (“Turkish”) and a 1.71 mbp (“Finnish”) deletion around the XK gene, respectively. The present “Bavarian” XK gene, showed a XK(W257X) stop mutation. Deletions reached from 48.15 kbp upstream, until 103.31 kbp downstream, and 1.67 mbp upstream, until 0.03 mbp downstream of the “A” of the start codon of XK, in the Turkish and Finnish sample, respectively. Clinically, the Bavarian patient suffered of a complex movement disorder and the Turkish carrier needed treatment for CGD including allogeneic stem cell transplantation. Conclusions: The presented method allows a reliable and robust approach to the genetic analysis of XK gene deletions and mutations in cases with suspected MLS. The precise definition of underlying molecular defect in MLS allows prognostic prediction of disease development and familial carrier assessment.

P9.04

Potential PCR—SSP mistyping of the HNA—3a alíele due to genetic variation of the SLC44A2 gene

Reil A.¹, Flesch B.²,Bux J.¹

¹DRK—Blutspendedienst West, Hagen, Deutschland ²DRK—Blutspendedienst West, Bad Kreuznach, Deutschland

Background: A single nucleotide exchange in the SLC44A2 gene, which is transcribed into two variants (NM020428.3 variant 1 and NM001145056.1 variant 2), is responsible for the HNA—3a/b antigen polymorphism. For TRALI diagnostics pheno— and genotyping methods have been established. Recently, we identified an additional 537C > T SNP variation of the HNA—3a alíele in about 2.4% of German blood donors (transcript variant 2, numbering starting with the 5 ‘UTR, which is consistent with position 457 when numbering begins with the start—ATG of transcript variant 1) . HNA—3a sense primers used for PCR—SSP typing of the HNA—3a alíele include the 537C position which might provoke mistyping by incomplete primer annealing, which should be investigated. Methods: Two in—house PCR—SSP methods with HNA—3—specific sense primers, one PCR—SSP with HNA—3—specific anti—sense primers and one commercial assay were applied on seven DNA samples with a known C537T variation of the HNA—3a alíele. Additional samples without the 537T variation served as controls. Results: Four HNA—3aa samples with the 537T variation and every control sample tested so far were determined correctly by each of the four methods. Both, the commercial assay and one of the in—house methods with HNA—3 specific sense primers failed to detect the HNA—3a alíele in three

HNA—3ab samples with the additional 537T variation and by mistake reported an HNA—3b homozygous result. Using the second in—house method with HNA—3 specific sense primers only weak HNA—3a specific bands appeared with these samples while the method with the anti—sense primers produced clear and correct results. Conclusions: PCR—SSP mistyping of the HNA—3a alíele is possible in samples with the additional 537T variation when the allele—specific primers are constructed as sense—primers. We suggest to either re—type DNA samples with an HNA—3bb result by an HNA—3 537T specific PCR method or to use an assay with HNA—3 specific anti—sense primers.

P9.05

Antibody screening and identification with the Microtube Column Agglutination System DG Gel: validation of a new test cell generation

Schoenfeld H.¹, Berst—Susanto B.¹, Spenke S.¹, Pruß A.²

¹Institute of Transfusion Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany, ²Tissue Bank, Institute of Transfusion Medicine; Charité —Universitätsmedizin Berlin, Berlin, Germany

Background: The routine method for antibody screening in the Charité blood bank is the gel centrifugation technique using the DiaMed gel centrifugation cards (DiaMed, Ottobrunn, Germany). Objective of the study was the evaluation of specificity and sensitivity in antibody screening and antibody differentiation of the “Microtube Column Agglutination System DG Gel” (Diagnostic Grifols S.A., Barcelona, Spain) including a new generation of Grifols test cells compared with our methodically similar routine method. Material and Methods: Atotal number of 359 samples (EDTA—Plasma) of known antibody specificities were tested with the DG gel system in parallel with the DiaMed system. Both methods based on the gel centrifugation technique. The Grifols System contains 3 new developed test cells (Screencyte 0.8%) for antibody screening and 11 new developed test cells (Data—Cyte 0.8%) for antibody identification with the following specificities: D, C, E, c, e, Cw, M, N, S, s, PI, Le(a), Le(b), Lu(b), K, k, Kp(a), Fy(a) (2 cells homocygous), Fy(b), Jk(a) (2 test cells homocygous), Jk(b). Results: The DiaMed system did not detect one anti—E, which was detected by Grifols system. Three anti—Kp(a) were detected by the DG Gel system only, because the DiaMed antibody panel did not include Kp(a)—antigens. The remaining 355 antibodies were detected in both systems. Specificity and sensitivity were comparable in both systems too. The main result of the study was that reaction strengths (—, +/—, +1, +2, +3, +4) were on average 1 reaction step higher with the Grifols System in comparison to the DiaMed system. Conclusion: Both agglutination systems demonstrated a high estimated diagnostic accuracy. The new Grifols system can be recommended thoroughly for the immunohematological diagnosis of irregular red blood cell antibodies.

P9.06

Performance study of Erythrocyte – Magnetized^® technology on the Qwalys^®3 system

Tucek A.

Zentrales Institut des Sanitätsdienstes der Bundeswehr Koblenz, Außenstelle München, Deutschland

Background: Erythrocyte—Magnetized^®Technology (EMT^®) by Diagast is an innovative method for ABO—RH—K and detection of IgG irregular antibodies. The magnetization of red cells avoids centrifugation and washing phases. Aim: Evaluation of EMT^® for antibody screening, blood grouping and RH—K phenotyping on the fully automated Qwalys^®3 system, a dedicated high throughput analyzer. Methods: The EMT^® was evaluated for accuracy of ABO—RH—K against the Beckman Coulter (Olympus) PK7200 system. Antibody detection was compared between EMT^® and Immucor Capture^® method on the Galileo system. In case of discrepancies, samples were tested manually using DiaMed ID System to compare the EMT® ABS sensitivity to microfil—tration technique. Materials: 4670 samples for ABS (ScreenLys, HemaScreen Pool), 520 samples for ABO with forward and reverse typing (Groupa2Lys, HemaLys AI, A2, B, 0) and for RH—K with 2 clones (Phenol,2Lys). Results: In ABS 4629 samples were negative and 32 positive in both techniques. The antibody screening showed a specificity of 99.93% for both, Qwalys^®3 and Galileo(6 unspecific positive samples, 3 in EMT^® and 3 in Capture^®). 4 known weak positive samples showed discrepant results (detected negative in EMT^® with pooled cells/positive with 3cells panel), weak positive in Immucor Capture^® and undetermined positive in DiaMed ID—System Enzyme Identification. These for 4 discrepant results are due to the detection limits. In ABO—RH—K 506 samples showed a complete concordance to PK7200 method. 14 samples showed non—interpretable results (8 in ABO and 6 in RH—K) with PK7200 method and correct results with EMT^®. Conclusion: EMT^® on the Qwalys3 provides a fully and reliable automation without washing and centrifugation phases. EMT^® is reliable and safe for ABO—RH—K and ABS. The results in ABS confirm excellent sensitivity and specificity.

P 9.07

Blood group chimerism detected by flow cytometric analysis for diagnosis of fetomaternal transfusion

Korfmacher S.¹, Hoffmann T.¹, Hamberger R.¹, Kirchhoff E.¹, Pantazis C.², Hoehn T.², Scharf R.E.¹

¹Dept. of Experimental and Clinical Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Duesseldorf, Germany, ²Department of Pediatrics, Divison of General Pediatrics, Duesseldorf, Germany

Background: We present a 38—year—old pregnant woman (gravida 3, para 2) with non—reassuring fetal heart rate pattern at the 37th weeks of gestation. Delivery was performed by Cesarean section without complications. Postnatally, the infant was anemic (Hb 3.4 g/dl). Following a single transfusion of 120 ml packed red blood cells (RBC), subsequent increase of Hb occurred spontaneously. The initial suspicion of hemolytic disease of the newborn was excluded immunohematologically; antibody screening in mother and infant, direct antiglobulin tests and elutable antibodies in the infant were negative. Considering the differential diagnosis of severe fetomaternal blood transfer, HbF analysis by high—performance liquid chromatography (HPLC) of maternal blood was increased to 5.7% (normal < 0.8%). Further quantification of fetomaternal transfusion was performed by flow cytometry on the basis of distinct Rh C/c phenotypes in mother (Rh CC) and infant (Rh Cc). Counting 20.000 RBC from a maternal sample stained with a primary anti—c antibody and a secondary phycoerythrin—stained murine antihuman Ig antibody revealed 4.6% Rh c—positive cells (background 0.9%). Referring to physiologic values in peripartal mothers and perinatal infants, the volume of fetomaternal blood transfer was estimated at 208 ml, equivalent to 71% of the fetal blood volume. The low reticulocyte production index of 2.56 (immediately after birth) which increased to 13.2 within 48 hrs suggested that the fetomaternal transfusion had occurred acutely. Conclusion: Flow cytometry of maternal blood analyzing fetal blood group polymorphisms nonexisting in the mother allows rapid detection and quantification of fetomaternal blood transfer. The high RBC number that can be examined by flow cytometry provides a more accurate estimation of blood loss as compared to the traditional Kleihauer Betke test. In our case, the data obtained by flow cytometry and HPLC were in good accordance.

P9.08

Significance of antibody screening with „ID—DiaScreen Prophylax” after application of rhesus prophylaxis

Stalder—Seeberger S., Näf S., Minneker S., Rüfer A.

Luzerner Kantonsspital, Abteilung Hämatologie, Luzern, Switzerland

Background: At Luzerner Kantonsspital antibodies of all patient samples with known rhesus prophylaxis will be specified if there is no negative antibody screen within the last month. This is done with „ID—DiaPanel (P)” and results in almost all cases in allo—anti—D antibody specification only. A possible alternative is the use of „ID—DiaScreen Prophylax” as a second antibody screen containing one rhesus D (RhD) positive and five RhD negative screening red blood cells. Methods: We compared „ID—DiaPanel (P)” and „ID—DiaScreen Prophylax” regarding their capability of antibody detection in 50 patient samples with known rhesus prophylaxis, 76 samples with a single alio—, cold— or unspecific antibody and 30 samples with multiple antibodies. Results: In all 50 samples with known rhesus prophylaxis identical results were seen regarding detection of allo—anti—D antibodies in all antibody concentrations (±1 level of deviation) despite different phenotypes of screening red blood cells. In samples with a single antibody identical results were seen in 15 of 19 antibodies and this was the case in 80% of samples with multiple antibodies. There is a trend towards weaker reactions with „ID—DiaScreen Prophy—lax” leading to discrepancies between both tests in some samples. Conclusions: In patients with known rhesus prophylaxis „ID—DiaPanel (P)” can safely be replaced by „ID—DiaScreen Prophylax” with consequently reduction of time and effort as well as costs. Whether „ID—DiaScreen Prophylax” will precisely identify antibodies relevant for transfusion in very small concentrations remains an open issue. To identify anti—Kpa—, anti—Lea— and anti—Leb antibodies inspection of antigen profile of screening red blood cells is mandatory.

P9.09

Vergleichende Untersuchung von Gerätesystemen zum Screening auf irreguläre blutgruppenspezifische Antikörper (AKS)

Schottstedt V., Michel A, Cordeiro S., Fischer M., HilkerF. DRK Blutspendedienst West Zentrallabor, Hagen, Deutschland

Fragestellung: Im Zentrallabor Hagen des DRK BSD West werden täglich über 1.000 Spenden im AKS untersucht. Es sollte die Eignung verschiedener Gerätesysteme mit der Option, selektiv aus Masterplatten pipettierte Proben verarbeiten zu können, untersucht werden. Methode: Bei über 25.000 unaus—gewählten Spenden wurde aus Gelbarriereröhrchen 10 ml (Fa. Sarstedt) nach Zentrifugation (15 min., 5500 RCF) EDTA—Plasma, entnommen. Ein AKS wurde mit folgenden Gerätesystemen durchgeführt: Diamed—Gelsystem pooled Cells® (Fa. Biorad) mit Diamed—Zentrifuge und —Reader, Capture®—Ready—Screen pooled Cells mit Gerätesystem Neo (Fa. Immucor) und Screen—Lys® mit Gerätesystem Qwalys 3® (Fa. Diagast/Tcoag). Bei in mindestens einem System reproduzierbar reaktivem Ergebnis erfolgte eine Antiköperdif—feren—zierung. Initiale Prüfkriterien waren: Spezifität, Antikörpererkennung. Vorläufige Ergebnisse: AKS reaktiv: Biorad: 0,22%, Immucor: 0,57%, Diagast: 0,57%. Von insgesamt 46 (0,18%) überwiegend Rh—, Kell, Duffy—Lewis—, S—Antikörpern wurden 32 von allen Systemen im AKS gefunden. Von nur einem System wurden im AKS erkannt: Diamed/Biorad: 1 Anti—e; Neo: 2 Anti—Cw, 1 Anti—D, 1 Anti—S, 1 Anti—Jka. Von nur einem System wurden im AKS nicht detektiert: Diamed/Biorad. 1 Anti—Cw, 1 Anti—Jka, 1 Anti—Lea/Leb; Neo: 2 Anti—E, 1 Anti—Keil, 1 Anti Mca; Qwalys3: 1 Anti—E. Diskussion und Zusammenfassung: Die höhere Rate der im AKS reaktiv befundeten Spenden bei Systemen der Fa. Immucor und Diagast anschließender Antikörperdifferenzierung ist im Vergleich mit dem Diamed—Gelsystem dem Vorteil der Automatisierung und geringeren hands—on time gegenüber zu stel—len. Die Ursachen für die nicht in allen Systemen detektierbaren Antikörper können z.T. an der Antigenzusammensetzung der Poolzelle (fehlendes Anti—Cw bei Biorad, keine EE—Zelle bei Immucor) liegen. Die Entscheidung über das optimalste Gerätesystem muss anhand weiterer Kriterien getroffen werden (in Arbeit).

P9.10

Looking at hemolysins – test one, get one free?

Wreczycki C.A.¹, Peine S.², Henneberg—Quester K.—B.³, Nykamp M.⁴, Horn P.A.¹, Lenz V.¹

¹University Hospital Essen, Essen, Germany, institute for Transfusion Medicine, University Hospital Hamburg Eppendorf, Hamburg, Germany

Background: ABO minor incompatible allocation of platelet concentrates (PC) to adult patients is often unavoidable in large hospitals providing maximum medical care, such as university hospitals. Severe hemolytic transfusion reactions due to existing isoagglutinins as well as isohemolysins have been described previously. Since there is no method internationally agreed upon for testing for isohemolysins, many institutions use the determination of IgM—Isoagglutinin titers (cut off s 128). Our institute routinely determines solely the in vitro hemolysis capacity of platelet donor—serum. In this study, we investigated the correlation between isoagglutinine titers and hemolysis capacity. Method: From 82 donor sera (BG A, B and 0), IgM—isoagglutinine titers against A— and B—erythrocytes were prepared (NaCl, tube test, RT). To the same sera, complement was added and hemolysis of red cells was visually evaluated. Hemolysis was stated in degrees from 1+ (≤50% lysis of erythrocytes) to 3+ (complete lysis). Results: Typically, hemolysis correlated with high isoagglutinine titers. Sera with hemolytic reactions ≤ 50% lysis (1+) did not show isoagglutinine titer higher than 128. The reverse was not the case: Sera with isoagglutinin titer ≤ 128 induced hemolysis up to 3+ (complete lysis). Conclusion: Determination of in vitro hemolysis capacity is a suitable two—in—one—method for detecting strong hemolysins and also avoiding high titre isoagglutinins. ABO minor incompatible allocation of PC up to an in vitro hemolysis capacity of 1+ reduces the potential risk for AB0 minor incompatible reactions. In our institute, we succeed in applying this policy mostly loss—free.

P 9.11

Anti—blood group A and B antibodies in patients with Common Variable Immunodeficiency and their correlation to Pneumococcal Polysaccharide immune response and switched memory B cells

Wolfram W.¹, Binder C.J.², Wolf H.M.³, Eibl M.M.³, Fischer M.B.¹

¹Dept. of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria, ²Center for Molecular Medicine of the Austrian Academy of Science, Vienna, Austria, ³Institute of Immunology, Medical University of Vienna, Vienna, Austria

CVID (common variable immunodeficiency) is the most prevalent human immunodeficiency with an estimated frequency of 1 in 25 000 in Europe and is characterized by markedly reduced serum levels of IgG, IgA and often IgM, and a failure to produce specific antibodies after vaccination or exposure to foreign antigens. This renders patients susceptible to recurrent sinopulmonary, ear, and gastrointestinal infection. Early PAGID and recent ESID definition of CVID mention absence of blood group ABO—isoagglutinins and poor response to polypeptide and polysaccharide vaccines as diagnostic criteria. The aim of the present study was to investigate whether freshly diagnosed CVID patients, who had not hitherto received IVIG treatment, show marked production of blood group ABO isoagglutinins. According to their capability to produce anti—blood group A/B specific antibodies the 32 patients with CVID could be grouped in two subpopulations. One group of patients showed anti—blood group A/B IgM antibodies but no anti—blood group A/B IgG antibodies in a gel based erythrocyte agglutination assay. The anti—blood group A/B specific IgM antibodies showed a high dissociation rate from blood group A/B trisaccharides immobilized on the surface of a dextran hydrogel layer on the CM—5 sensor chip of a Biacore device, indicating low affinity. In group one, patients were found that responded to pneumococcal polysaccharide immunization and showed CD27+IgD— switched memory B cells, while alternatively other patients showed barely any response to pneumococcal polysaccharide immunization and had few CD27+IgD— switched memory B cells. In contrast, the second group of CVID patients that showed no antibody response to blood group A/B antigens, also showed no response to pneumococcal polysaccharide immunization and had few CD27+IgD— switched memory B cells. All patients presented with normal number of CD5 positive B cells.

P 9.12

Prozone phenomenon (high—dose Hook effect) of the HTLA antibody anti—Chido (a)

Bringmann G.¹, Stenzel A.¹, Koch K.¹, Strathmann K.¹, Schulte U.², Zeiler T.¹

¹German Red Cross Blood Service West, Center of Transfusion Medicine Breitscheid, Ratingen, Germany, ²German Red Cross Blood Service West, Center for Transfusion Medicine Münster, Germany

The prozone or high—dose hook effect is a well known problem of serological tests causing false negative results due to massive excess of the analyte to be tested. Here we report a case of previously unknown prozone effect in the plasma inhibition test (PIT) used for detection of the HTLA antibody Anti Ch(a). Anti—Chido antibodies are directed against the C4 component of the human complement system. In the PIT, anti—Chido antibodies reacting with C4—covered RBC are neutralized by incubation with plasma containing C4. In a positive PIT, the antibody screening test (AST) becomes negative, when AB—plasma is added, whereas it remains positive with sodium chloride as supplement. In the negative case the former positive AST is still positive with both supplements. This test is commonly used for differentiation of anti—Chido and anti—Rodgers from all other HTLA antibodies. An 84—year—old male patient with anaemia caused by leukaemia had received multiple transfusions of erythrocyte concentrates and developed the HTLA—antibody Anti—Ch(a), which is not relevant for transfusion of RBC, but causes positive cross—matches. To exclude other important haemolytic antibodies, the PIT was performed as part of the compatibility diagnostics and was found repeatedly positive up to March 2011. One month later, when the patient again needed RBC, the crossmatches with all units still were positive (+), with an extraordinary high titre of the antibody (4,096), whereas PIT now was negative. Due to our previous experiences with other erythrocyte antibodies we assumed the existence of a high—dose hook effect. Therefore we performed the PIT in a dilution of 1:1,024. In this dilution the inhibition test was clearly positive again. The prozone phenomenon was proved. The existence of a high—dose hook effect in the plasma inhibition test used for identifying HTLA—antibodies has to be taken into account once implausible test results or extraordinary high antibody titres occur.

P 9.13

Alloreactive anti—G in a 22 year old mother with additional alloantibodies anti—D, anti—C and anti—Jk(b)

Schulze T.J.¹, Goebel M.¹, Scharberg E.A.², Klüter H.¹, Bugert P.¹, Janetzko K.¹

¹Institut of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Germany, ²Institute of Transfusion Medicine and Immunohematology, German Red Cross Blood Service Baden—Württemberg – Hessen, Baden—Baden, Germany

Background: A 22 year old gravida—3, para—1 woman with blood group A, Rh neg, ccddee and known alloantibodies anti—Jk(b) and anti—D, gave birth to her second child. Before delivery a new alloanti—C could be detected. The newborn was well and did not present any icterus. Routine diagnostic of the newborn included direct antiglobuline test (DAT), in which an alloanti—D and an alloanti—C could be eluted. However the blood group of the child was A, Rh neg, Ccddee and thus did not bear the Rh D antigen. Alloanti—Jk(b) was no longer detectable in the mother's blood and neither could be eluted from the child's Jk(b) positive red blood cells. Methods: Rhesus antigens were detected by tube test with two different clones (Medion—Grifols). Indirect antiglobuline test (IAT) was performed in the gel ID card system (DiaMed—BioRad). Elution was performed with chloroform technique. Rhesus antigens were confirmed by PCR—SSP—CDE—System (innotrain Diagnostik). Anti—G was confirmed by rare G—expressing cells of Rhesus blood group ccddee (rGr). Results: Both anti—D and anti—C could be eluted from D—negative neonatal red blood cells. IAT from the mother's blood confirmed the specificities of the allo—anti—D and —anti—C. The anti—D titre did not match the Rh—prophylaxis applied five months ago. The child's rare Rhesus blood group Ccddee was then confirmed by use of SSP—PCR. No weak D could be found. Eventually, serological testing with D—negative, C—negative and G—positive cells proved the specificity of the anti—G alloantibody. Conclusion: Alloanti—G is a rarely diagnosed antibody that matches epitopes on both Rhesus D and C. It may occur more often in pregnancies due to high frequency of C and D in all populations. Usually, differentiation between anti—G and anti—D, anti—C resp. is performed by absorption from either D and C positive cells and recombination of antibodies with C and D positive cells that lack D and C. In this case absorption was performed on the red blood cells of the newborn.

P9.14

Autoanti—N antibody in pregnancy. A case report

Strathmann K.¹, Stenzel A.¹, Koch K.¹, Bring mann G.¹, Schmolling J.², Zeiler T.¹

¹German Red Cross Blood Service West, Center of Transfusion Medicine Breitscheid, Ratingen, Germany, ²Hospital of the Augustinerinnen, Clinic of Gynaecology and Obstetrics, Cologne, Germany

Background: Apart from few cases of severe haemolytic anaemia caused by autoanti—N this antibody usually is considered innocuous. The case of a strong reactive autoanti—N in a woman and her newborn we present here – to our knowledge – is the first reported case of autoanti—N in materno—fetal constellation. Patients: A 37—year—old woman in first gravidity was admitted to hospital in the 38th week of pregnancy. The child was delivered by caesarean section because of complications in contraction. Antibody screening tests (ABS) performed in the 10th and 27th week of gravidity were documented as negative, however two days before delivery ABS was positive. The mother showed Hb values in the normal range before cesarean section. The newborn was in good health at birth (Hb 19.9 g/dl, Hct 54%) and showed no signs of haemolysis until discharge on day 10. The mother's Hb values were in the normal range. Methods: Blood specimens (EDTA) from mother and child were tested. ABO and Rhesus blood group were determined by tube technique. Antibody screening, antibody identification and direct coombs test were performed by using the gel technique (DiaMed, Creesier sur Morat, Switzerland). RBC—bound antibodies were eluted with an acid elution kit (DiaCidel, DiaMed). MNS genotyping was done with alíele specific PCR (Ready Gene MNS, Innotrain, Kronberg, Germany). Results: In the mother's plasma and in the newborn's plasma and eluate an autoanti—N antibody could be identified. The antibody showed a strong reactivity with ++ in plasma and +++ in eluate. In sérologie typing the mother and the newborn were N+. The mothers genotype with allele—specific polymerase chain reaction (PCR) was GYPA∗N. Conclusions: In this case autoanti—N, although strongly reacting in—vitro, did not cause haemolytic disease of the newborn. However, until more data about autoanti—N are collected we recommend close monitoring of the child in fetal and neonatal stage to exclude the presence of hemolysis.

P9.15

Are antibodies to the blood group antigen M clinically relevant?

BlackA.¹, Ditz D.², Holler E.², Schmitz G.¹, Ahrens N.¹

¹Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Regensburg, Regensburg, Deutschland, ²Abteilung für Hämatologie und internistische Onkologie, Universitätsklinikum Regensburg, Deutschland

Incompatible transfusions in patients with antibodies to red blood cells (RBC) are usually omitted. Anti—M is one of the most frequent irregular antibodies to RBC. The relevance of this cold reactive antibody remains usually obscure. We report on a M and N positive patient, who got transplanted with peripheral blood stem cells containing 319 mL plasma with an antibody to M that was clearly reactive at 4°C in saline milieu and at 37°C by indirect antiglobulin testing. The patient did not show any clinical signs of reaction during or after transplantation, and hemolysis parameters remained largely unsuspicious: LDH was before, 2 h, and 1 day after transplantation 161 U/L, 169 U/L, and 185 U/L, respectively, and haptoglobin before, 2 h, and 1 day after transplantation was 134 mg/dL, 136 mg/dL, and 133 mg/dL, respectively. Engraftment was regular, and no RBC transfusion was required. This case illustrates that an incompatibility due to a limited amount of a cold—reactive anti—M may be tolerated.

P9.16

Difficult diagnosis of a case of pneumococcal hemolyticuremic syndrome with T—antigen activation

Strobe! E.¹, Dreßel P.², Strotmann P.²

¹Department Medizinische Mikrobiologie und Krankenhaushygiene, Medizet, Städtisches Klinikum München GmbH, München, Deutschland, Germany, ²Klinik für Kinder— und Jugendmedizin, Klinikum Schwabing, Städtisches Klinikum München GmbH, München, Deutschland

Background: Hemolytic—uxemic syndrome (HUS) is often caused by entero—hemorrhagic E. coli, but other pathogens like Cl. perfringens or S. pneumoniae also can cause severe hemolysis. In the last—mentioned cases alterations of red blood cell (RBC) antigens may be observed. Case report: A 9 months old boy was admitted to the hospital because of cough and fever. In the chest X—ray the right hemithorax was completely shadowed by infiltrates and atelectasis. Therapy was started with cefuroxime and clindamycin was added later. At day 6 the patient showed a drop of the hemoglobin value to 3.0 g/dL (from 8.5 g/ dL at day 4). Hemolysis was diagnosed by haptoglobin < 8 mg/dL and LDH 3550 U/L. Now the patient was admitted to the ICU for mechanical ventilation and peritoneal dialysis. Immediate transfusion of 1 RBC concentrate increased the Hb to 6.0 g/dL. At day 45 he left the hospital in a good general condition. Methods: At the time of admission to the ICU the direct antiglobulin test (DAT) was performed by the gelcard method. T antigen activation on the RBCs was tested using Arachis hypogea lectin. Swabs from throat and nose as well as trachéal aspirate were cultured. PCR for detection of influença A and B in a throat swab was done. Feces was tested for Shigatoxin by an ELISA. A rapid immunochromatographic membrane assay for detection of soluble pneumococcal antigen in the urine was performed. Results: The monospecific DAT was positive with anti—C3d. T antigen was detected on the RBCs. Pneumococcal antigen was found in the urine at the same time. No pathogenic bacteria could be cultured from the upper and lower respiratory tract. The tests for influença RNA and for Shigatoxin were negative. Conclusions: In the reported case T antigen activation on the RBCs suggested infection by a neur—aminidase producing pathogen. Detection of pneumoccal antigen in the urine led to the correct diagnosis only a few hours after the onset of hemolysis. In contrast to culture techniques that provide results at the earliest after one day of incubation and that can be negative because of antibiotic treatment before specimen collection, immunological proof of pneumococcal antigen can resolve rapidly the reason for T antigen activation in some cases of HUS.

P9.17

PARG: a new low prevalence Rh blood group antigen

Scharberg E.A.¹, Kanbur N.¹, Bieser R.¹, Roth S.¹, Seyboth S.¹, Richter E.¹, Rink G.², Taszig M.³, Gathof B.³, Burkhart J.⁴, Bugert P.⁵

¹1nstitute of Transfusion Medicine and Immunohematology, German Red Cross Blood Service Baden—Württemberg – Hessen, Baden—Baden, Germany, institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service of Baden—Württemberg – Hessen, Mannheim, Germany, department of Transfusionmedicine, University of Cologne, Cologne, Germany, ⁴Blutspendedienst des BRK gGmbH, München, Deutschland, institut of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden—Württemberg – Hessen, Mannheim, Germany

Background: The serum of a 58 years old Caucasian woman contained anti—Wr(a) and an antibody reacting strongly with a single Wr(a) negative test cell of the antibody screening test. Antibody identification and red cells positive for low prevalence antigens showed negative reactions. The results suggested an antibody against a possibly new low prevalence antigen. The donor of the positive test cell was identified and a serological and molecular testing of his family members was performed. Methods: The antibody screening and identification were performed in the indirect antiglobulin test (IAT) in the gel technique. Antibodies against following antigens could be excluded: Vw, Co(b), He, Yt(b), Di(a), Mg, Lu: 14, Mt(a), Js(a), DAK, FPTT, VS, Go(a), Vr, V, St(a), Mit, LW(b), Crawford, K:25, Ls(a), Ul(a), Tc(a), JAHK, Sc:2, Mi(a), Hut, Mur, Hil, Miny, Dantu. The red cells of the donor, his mother, father, brother and son were tested with the patient's serum in the IAT. As the reaction was enhanced by papain treatment of the cells an antibody against an Rh antigen was suspected and the RHCE was analyzed by exon re—sequencing. Results: The serum of the patient reacted positively with the red cells of the donor and his father. It was negative with red cells of his mother, brother and son. The sequencing of the donor's DNA showed a novel 501G > A mutation in exon 4 of the RHCE gen. The father of the donor was positive, his mother, brother and son were negative for the mutation. The mutation obviously defined a new low prevalence Rh antigen, we named PARG. The screening of 10.799 blood donors with the serum of the patient revealed no PARG positive individual. Conclusions: We describe a novel low prevalence Rh antigen, we named PARG, which is characterized by a 501G< A mutation in exon 4 of the RHCE gen and a predicted Met167Ile substitution in the extra cellular part of the third loop of the RHCE protein. The antigen was found only in one family and its frequency is lower than 0.01%.

P9.18

Recurrent Diclofenac—induced immune hemolytic anemia in a patient with multiorgan failure

Lehmann R.¹, Geerdes—Fenge H.², Führer A.², Mitzner S.², Kiefel v.¹

¹Department of Transfusion Medicine, Rostock University, Rostock, Germany, ²Department of Nephrology, Rostock University, Germany

Diclofenac is a widely used non steroidal anti—inflammatory drug and known to induce immune hemolytic anemia (IHA). Different mechanisms of druginduced IHA reported so far are due to occurence of drug—induced autoantibodies (aab), drug—dependent antibodies (ddab) or as a mixture of both and result in mild to life—threatening hemolytic anemia. Case: A 57—year old patient presented with severe backache in June2010 and acute, severe hemolytic anemia. She had taken diclofenac and ibuprofen before. She received eight units of packed rbc. A preliminary diagnosis of severe sepsis and organ failure of kidney and liver were made, diclofenac and ibuprofen were discontinued. A similar hemolytic episode following therapy with diclofenac had occurred in July 2008. Serological findings: In July 2008 serological analysis revealed a strongly positive direct antiglobulin test (DAT) due to rbc—bound IgG and C3d. These serological findings were interpreted as warm type autoantibodies causing autoimmune hemolytic anemia. In June 2010, the patient presented with positive direct antiglobulin test (IgG, C3d) and autoantibodies in serum and eluate from autologous rbc. Moreover, ddab strongly reacting with rbc in the presence of diclofenac were found (gel centrifuagation test: NaCl card). Conclusion: Drug induced hemolytic anemia remains a diagnostic challenge. It is important to obtain a complete list of drugs taken at the onset of immune hemolysis, and to ask/search for similar incidents in the patient's history. Detection of ddab documents the causal relationship between drug and immune hemolysis. The patient should receive written information about the clinical and serological findings in order to prevent a rechallenge with the drug in the future. This patient is another example for the autoimmune nature of drug induced immune hemolytic anemia.

P 9.19

The frequency of CD36 deficiency in Chinese population

Ye X.¹, Fu Y.¹, Xia W.¹, Deng J.¹, Santoso S.²

¹Institute of Clincal Blood Transfusion, Guangzhou Blood Center, Guangzhou, China, ²Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany

Fetal and neonatal alloimmune thrombocytopenia is rare but potentially serious conditions. Incompatibility between the mother and fetus regarding human platelet antigen 1 (HPA—1) is the most frequent cause of FNAIT in the Caucasian population. The mother (HPA—1a negative) may become immunized against fetal platelet (HPA—1a positive) during pregnancy and can develop alloantibodies (anti—HPA—la), which opsonize fetal platelets leading to thrombocytopenia and bleeding risk in fetus. In Asians, however, no homozygous HPA—1a negative mother has been observed so far. Platelets CD36 deficient (Nak antigen) is known to be responsible for the production of antibodies in FNAIT. Currently, no data regarding the frequency of CD36 deficiency is available in China. By analysis of 470 healthy blood donors in

Guangdong province of Southern China using PCR—SBT method, we found 30 individuals (6.38%) with following CD36 gene mutations; C268T (0.64%), 329—330del AC (2.98%), C380T (0.43%), C760T (0.43%), A1237C (1.70%). In one (0.21%) individual, a combined mutation for C268T and 560Tins was detected. In contrast to previous studies, the 329—330del AC appears to be the major mutation type responsible for CD36 deficient, whereas C268T represents the most frequent mutation among other Asian populations. In addition, we found a case of hydrop fetalis associated with the presence of anti—GPIV in a mother with defective GPIV expression. Molecular genetic analysis of the mother showed C380T mutation of GPIV gene. The presence of GPIV antibodies in maternal platelets could be confirmed by crossmatch analysis using ELISA test and immunoprecipitation analysis.

P9.20

A novel ELISA for the detection of human neutrophil antibodies

Werth S., Bayat B., Tjahyono Y, Sanioso S.

Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany

Antibodies to human neutrophil antigens (HNAs) are responsible for a number of immune—mediated neutropenia and transfusion related acute lung injury (TRALI). In this study, a rapid ELISA using biotin labelled recombinant HNA—1 and HNA—2 antigens was developed to detect HNA—la, —lb and −2a antibodies. Soluble HNA—la, —lb and HNA—2a were isolated from supernan—tant of allele—specific transfected insect cells. Purified recombinant antigens (rHNA) were labelled with biotin and immobilized on streptavidin coated microtiter wells to analyze sera containing HNA—la (n = 15), —lb (n = 12) and HNA—2a (n = 10) antibodies. Bound antibodies were detected with enzyme labelled secondary antibodies. In parallel, MAIGA was performed and the results derived from both techniques were compared. Prior to testing, the cutoff of ELISA was determined by analysis of non—selected sera derived from healthy donors (n = 12). Furthermore, no reaction was observed with HLA class I and class II antibodies (n = 15). 12/15 (80%) sera containing HNA—la antibodies reacted in ELISA with rHNA—la, but not with rHNA—lb. 10/12 (83%) sera containing HNA—lb antibodies showed positive reaction only with rHNA—lb in ELISA. 9/10 (90%) sera containing HNA—2a antibodies showed positive reaction in ELISA with immobilized rHNA—2a. It is known that HNA—2 forms a complex with proteinase—3 (PR3) on the cell surface. Indeed, sera from patients with Wegener's granulomatosis with PR3 antibodies reacted in MAIGA. In contrast, this antibody showed no reaction in ELISA using rHNA—2a. In general, this observation indicates false positive reaction in MAIGA due to complex formation between two different molecules, which can be captured by one mab. These results suggested that streptavidin—biotin ELISA using recombinant neutrophil antigens may provide a valuable and specific method for rapid screening of human alloantibodies against HNA—la, —lb and HNA—2a in patients with neutropenia and in blood products to prevent TRALI reaction.

P9.21

Transfusion—related acute lung injury— neutrophil priming enhances their reactivity with HNA—3a antibodies

Berthold T.¹, Muschter S.¹, Schubert N.¹, Wesche J.¹, Fuerll B.¹, Teumer A.², Reil A³, Bux J.³, Greinacher A.¹

¹1nstitut für Immunologie und Transfusionsmedizin, Abteilung Transfusionsmedizin; Universitätsmedizin Greifswald, Greifswald, Deutschland, ²lnterfakultäres Institut für Genetik und Funktionelle Genomforschung, Universität Greifswald, Greifswald, Deutschland, ³DRK—Blutspendedienst West, Hagen, Deutschland

Background: Referring to the “two hit model” of TRALI (Transfusion—related Acute Lung Injury), neutrophils primed by a first hit, e.g. trauma, infection or surgery are expected to be more susceptible to the second hit by HNA antibodies, compared to non—preactivated neutrophils. To determine, whether primed neutrophils are more sensitive to TRALI inducing HNA—3a antibodies, we assessed granulocyte aggregation of primed or non—primed neutrophils by increasingly diluted HNA—3a antibody containing plasma. Methods: Neutrophils were isolated from anticoagulated whole blood obtained from healthy, HNA—3a homozygous donors by dextran sedimentation and density gradient centrifugation. To simulate the first hit, neutrophils were preincubated with various concentrations of either lipopolysaccharide (LPS) or for—mylmethionyl—leucyl—phenylalanine (fMLP) or buffer. The second hit was the addition of two serially diluted HNA—3a plasmas. Neutrophil aggregation was assessed in the granulocyte agglutination test (GAT). Results: HNA—3a antibody plasmas induced aggregation of primed granulocytes (fMLP ≥ 500 nM; LPS ≥ 200 ng/ml) at 1— to 2—fold higher dilutions compared to their effect on non—primed granulocytes (fMLP: n < 11, p < 0.003; LPS: n < 11, p < 0.003). Plasma 1 diluted up to 1:512 caused aggregation of primed granulocytes. LPS and fMLP did not differ in their capacity to prime granulocytes (n = 22; p = 0.84). In none of the experiments, primed neutrophils aggregated after the incubation with control plasma. Conclusion: LPS or fMLP primed neutrophils aggregate at lower concentrations of HNA—3a antibodies than unprimed cells, supporting the “two hit model” of TRALI. Critically ill patients might be more susceptible to develop TRALI as their granulocytes are aggregated at lower concentrations of HNA—3a antibodies. A 1:512 dilution of HNA—3a plasma corresponds to transfusion of ∼10 mL plasma into 5 L whole blood, which may even occur with transfusion of a red blood cell concentrate.

P 9.22

Toxin of Streptococcus pneumonia bacteria induces NB1 gene transcription in HNA—2a negative neutrophil subpopulation

Bayat B., Werth S., Tjahyono Y., Sanioso S.

Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Giessen, Germany

The recruitment of neutrophil to infection site is the first step of host defense system against invasive organisms. Mediator molecules accelerating neutrophils diapedesis toward infection site play therefore an important role in this defense mechanism. Recently, our group has demonstrated that human neutrophil antigen—2a (HNA—2a, also known as NB1) interacts with PECAM—1 on endothelial cells and mediated thereby neutrophil diapedesis toward different chemo attractants. HNA—2a shows bimodal expression pattern (HNA—2a positive and HNA—2a negative populations) on neutrophil surface, which can be upregulated during bacterial infections. However, the mechanism arranged behind is not yet known. In this study, we investigate the impact of pneumolysin (PLY), the most potent bacterial toxin of Streptococcus pneumonia, on the regulation of HNA—2a expression on neutrophil surface. Neutrophil treated with PLY migrated faster in the flow chamber through endothelial cells in comparison to untreated neutrophils. Interestingly, flow cytometry analysis with different mabs against HNA—2a showed significant upregulation of NB1 on HNA—2a (—), but not on HNA—2a (+) neutrophil subpopulations after exposure to PLY. This result could be confirmed by the use of sorted neutrophils. Treatment of HNA—2a (—) sorted neutrophils with PLY caused increase of total NB1 protein as well as NB1 surface expression as shown by immunoblot and flow cytometry, respectively. In the control experiment, neutrophils derived from HNA—2a defective individuals (NB null) did not show any NB1 production after PLY treatment. In conclusions, this result indicates that bacterial toxin is able to activate neutrophil signaling pathway, which initiates the gene transcription in HNA—2a negative neutrophil subpopulation. This mechanism is probably the most important step for the NB1 mediated neutrophil diapedesis during bacterial infections.

P 9.23

HNA—2a mediated TRALI depends on PECAM polymorphism on neutrophils

Bayat B., Wasel W., Sachs U.J., Sanioso S.

Institute for Clinical Immunology and Transfusion Medicine; Justus Liebig University Giessen, Giessen, Germany

Antibodies directed against human neutrophil antigen (HNA—2a, or NB1) play a role in pathomechanism of transfusion—related acute lung injury (TRALI). Recently, our group described endothelial PECAM—1 (CD31) as counterreceptor for NB1 molecule. This interaction triggers neutrophil migration through endothelial cells. Interestingly, neutrophil diapedesis depends on PECAM—1 polymorphism; neutrophils carrying PECAM—1 Ser536 migrate faster through endothelial cells in comparison to Asn536. Besides endothelial cells, PECAM—1 is found in different blood cells including neutrophils. The role of PECAM—1 S536N dimorphism on neutrophil function, however, is not known. In this study, we sought to investigate the effect of the S536N dimorphism on anti—HNA—2a mediated TRALI using ex—vivo rat lung model. Neutrophils from phenotyped individuals homozygous for S536 or N536 were used for this study. Additionally, only neutrophils expressing similar amount of NB1 molecules have been selected to exclude the role of NB1 in hetero—typic cell—cell interaction process. PECAM—1 phenotyped neutrophils (10⁸) primed with purified anti—HNA—2a antibody or control IgG were injected into the explanted rat lung, which has been pre—treated with LPS. The capillary filtration coefficient (Kfc) was quantified to assess capillary permeability changes and was determined gravimetrically from the slope of the lung weight—gain curve induced by a venous pressure elevation of 10 cm H₂0 for 8 minutes. The injection of neutrophils carrying N536 isoform of PECAM—1 showed no change of lung weight—gain curve in this animal model. In contrast, significant increase of lung weight was observed when neutrophils phenotyped for S536 were injected. This result indicates that neutrophils expressing S536 isoform of PECAM—1 are predisposed to induce the endothelial barrier leakage triggered by neutrophil antibodies, therefore the role of PECAM—1 polymorphism as genetic risk factor for TRALI needs to be considered.

P9.24

Transfusion—related acute lung injury (TRALI) due to HLA class II antibodies in a red blood cell concentrate from a male donor without history of transfusion

Winterstein K.¹, Hoyer P.¹, Jorks S.¹, Adamo W.², Wagner F.F.², Borowsky A.¹, Heinemann G.¹, Winterfeld S.¹, Kroll H.¹

¹Red Cross Blood Transfusion Service NSTOB, Institute for Transfusion Medicine, Dessau, Germany, ²German Red Cross Blood Transfusion Service N.S.T.O.B., Institute Springe, Springe, Germany

Background: Immune TRALI is caused by HNA— or HLA—antibodies (abs) usually present in donor plasma. Here we describe a non—fatal case of TRALI most probably induced by strong HLA class II abs. Interestingly, the involved donor was a man without a history of immunisation. Case report: The 76—year—old male patient suffered from stenosis of the cervical spinal canal. During hospital stay after surgery he developed pneumonia. Two red blood cell (RBC) concentrates were transfused, lh after transfusion an abrupt drop of the oxygen saturation was measured and artificial ventilation with 100% oxygen was administered. Because of radiologically documented new pulmonary infiltrations TRALI was suspected. The clinical condition of the patient and chest X—ray findings improved during the two following days. Methods: Sera from the patient and the two involved donors were analyzed in a bead array screening test for HLA class I and II abs and in granulocyte immunofluo—rescence and agglutination tests for HNA—abs. For crossmatch between donor sera and B—lymphocytes of the patient a whole blood flow cytometric immune fluorescence test was used. HLA class II was genotyped by PCR—SSP. Results: One donor serum contained strong HLA class II abs (anti—HLA—DR7). No HNA or HLA class I abs were detected. The donor was a 30—year—old man without history of transfusion. The second donor, a woman with previous pregnancies, and the patient showed completely negative test results. The HLA class II genotype of the patient was HLA—DRB1∗07,∗13. A cross—match between donor serum containing anti—HLA—DR7 and blood cells from the patient was positive with B—cells. Conclusions: HLA—class II abs are frequently found in cases of immune TRALI. The case described here is of special interest since TRALI was induced by an RBC concentrate which contains minor amounts of plasma and the donor was a man without immunizing event. Thus, the “male only” approach for the prevention of TRALI is limited by natural occurring abs.

P9.25

FCGR3B∗04 – A novel allele of the human Fc gamma receptor IIIb gene

Reil A.¹, Flesch B.², Bux J.¹

¹DRK—Blutspendedienst West, Hagen, Deutschland, Hagen, Feithstr. 182, ²DRK—Blutspendedienst West, Bad Kreuznach, Burgweg 5–7

Background: The neutrophil—specific Fc gamma receptor IIIb is a well—known target of allo—and autoantibody formation. Three alleles (FCGR3B∗01, ∗02, ∗03) have been described. Due to gene deletion and duplication, different individuals can have 0 to 4 copies of the FCGR3B gene. In Europeans, most FCGR3B∗03 positive individuals carry three FCGR3B genes and FCGR3B∗03 is inherited together with FCGR3B∗01. To determine the number of genes, typing of only one characteristic residue per allele is not sufficient. During evaluation of FCGR3B sequence—specific primers we got evidence for the existence of a fourth FCGR3B allele in the European population. Methods: To separate FCGR3B alleles from the homologous FCGR3A gene, sequence—specific primers for 6 polymorphic residues (nucleotide positions 141, 147, 227, 266, 277 and 349) of the FCGR3B gene were designed. Primers were combined to 21 primer pairs for FCGR3B allele typing. DNA sequencing was performed by a nested PCR strategy. Results: Except one, all primer pairs revealed the expected results. Combination of a 227A sense primer (FCGR3B∗01) and a 349A antisense primer (FCGR3B∗02, ∗03) gave positive results in some individuals. Interestingly, all these individuals were FCGR3B∗03 positive. 27 FCGR3B∗03 positive individuals typed by the “classical” FCGR3B PCR—SSP method were retyped for nucleotide positions 141, 147, 227, 266 and 349. 23 out of them were 227A positive (FCGR3B∗01 or ∗04). All these 23 individuals were positive with the 227A—349A primer pair (= FCGR3B∗04). Results were confirmed by DNA sequencing in 3 individuals. Typing of 5 families showed combined inheritance of FCGR3B∗03 and FCGR3B∗04. Conclusions: The novel allele FCGR3B∗04 differs from FCGR3B∗01 only at position 349. Its occurrence is closely linked to FCGR3B∗03. Therefore, FCGR3B∗03 is very likely inherited together with the new FCGR3B∗04, and not with the FCGR3B∗01 allele.

P9.26

Clinical relevance of positive HLA—Antibodies in Luminex technology

Volkholz S., Barz D.

Institut für Transfusionsmedizin Universitätklinikum der Friedrich Schiller Universität Jena, Jena, Deutschland

Background: The § 1 of the German Transfusion Act demands high standards of regulation for the transfusion of blood products. Although all cellular blood products in Germany are leucocyte—depleted transfusion reactions (TR) still appear in clinical treatment. Patients and Methods: Bewteen February 2009 and March 2011a total number of 53474 red cell concentrates (RCC), 13,916 platelet concentrates (PC) and 27,675 plasma units were transfused at the University Hospital of Jena and from these 127 TR were registered. Blood samples from patients with TR were analysed for antibodies (Abs) against red blood cells (RBC). HLAAB antibodies were analyzed by leucocytotoxicity test (LCT) and Luminex technology. In parallel we analysed the correlation of clinical symptoms and the type of blood products used. Results: From the registered TR (n = 127) 63 patients (49%) were transfused with RCC, 51 patients (40.1%) with PC and 13 patients (10.2%) with multiple blood products. In 37 patients (29.1%)we could analyse sera both before and after TR. From these patients Abs against erythrocyte specific antigens were shown not to be the cause for their TR. An immunological correlation for the TR could be shown in 15 of 37 cases (40%). Using LCT, in 6 of 37 cases (16%) we found Abs for HLA—AB, while by Luminex technology 15 of 37 cases (40%) were positive for HLA—AB Abs. The TR caused by PC were urticaria and itch, observed in 35 of 51 cases (68%). However, 32 of 63 cases (51%) showed fever and chill after transfusion with RCC. Conclusion: The current study shows that Luminex technology is a more sensitive method for the detection of HLA—AB. Although even today the international standard for the clinical relevance is the LCT, the immunological reason for TR was not shown in a quarter of cases. From an ethical point of view the question arises whether patients should generally be tested for HLA—AB with sensitive methods e.g. Luminex before and after blood transfusions within the meaning of § 1.

Session 10 – Quality Management and Information Technology

P 10.01

Blood bank software

Goebel, St.; Kiese, M.; Hering, J.

Institute of Transfusion Medicine, Martin—Luther—University Halle—Wittenberg, Halle (Saale), Germany

Background: A good blood bank software package is important, so it is for the satisfaction in the daily routine. A good blood bank software package covers nearly all demands. The users have an easier task – without any limitation. Each concentrates on their own rhythm, but in the tune of the blood bank. Methods : Everybody is introduced in an overview of the section „Automation and Data processing” listed suppliers by blood bank software. There are 11 passive and 9 active suppliers. The web page of every supplier serves the mediation of a first impression of the application. Addresses can be used for an establishment of contacts helpfully. Results: The following bidders have remained in the year 2011 in the German market (look at the attached table). Important overviews such as the following are given:

– Requirements for the software

– Legal bases for blood bank software packages

– Questionnaires to a software manufacturer

– Documents what are to be supported by the software

– Training demands by the introduction

– Expenses to be expected

– Licensing

– Tools for developments

The V—model and the phase model as well as the life cycle of software development attract attention. Conclusion: The overview supports potential prospective customer within the decision.

P 10.02

Validation of a data acquisition software (iTrace) in a plasmapheresis operation

Kießig S.T.¹, de Vries l.¹, Pernom C.², Mignon E.², Philipp R.³, Krause K.—P.⁴

¹HaemaAG, Blood Service Center Dortmund, Dortmund, Germany ²Fenwal Europe s.p.r.l., Belgium, Mont—St.—Guibert, 2, rue Edouard Belin, ³Fenwal Germany GmbH, Munich, Germany, ⁴Haema AG, Germany

Background: iTrace is a data acquisition system for Fenwal machines based on a wireless network. Data are automatically transferred from the devices (procedure information including timing of events and alarms), or captured with barcode scanner (traceability of solutions, kits and operators) or manually entered. The software is compatible with A200 and a common PC. Data stored in a SQL database. Data or log files and backups are saved according to the user requirements. Exports allow the data transfer into other databases. Up to 50 A200 and 100 users on different access levels can be handled in a network. Aims: The objective of the validation was to verify the correctness of the data transfer, evaluate user friendliness and suggest improvements. The creation of complete manufacturing printed protocols adjustable to the user requirements was a major goal. Documentation of adverse events should be carried out as well. Methods: 20 A200 were equipped with Wifi bridges and connected with SQL Data Base. 1091 collection procedures were recorded and entries compared with the manual records. Results: Post procedure information was complete. Two procedures were missed in the report but available in the database (disconnection of machines from power supply). There was 1 error in scanning. 12 deviations were found in the volume due to disturbances to the machines caused by interruption of procedures. All side effects were completely documented. iTrace allows replacing manual entries related to apheresis procedures more accurately and with minimal operator interventions. A joint power supply for the instrument and the bridge was set up after the validation in order to avoid missing procedures in the report. Double scan was implemented to avoid error scanning. Therefore a base for the development of the next software generation is evaluated. All occurred errors could be removed either by optimizing the data capture procedures or the work flow. The new version will be ready for routine application after a revalidation.

P 10.03

Improvement and spreading of Eurocode coding system since 1998

Goebel S., Roos D., Kardeous J., Linke T., Lath K.¹, Knels R.

Eurocode IBLS e.V., Dresden, Germany

In the last years numerous attempts have been made to spread and improve the Eurocode coding system. After the registration of the unique primer identifier of the system, the exclamation mark in the German Industry Norm (DIN), the International Standardization Organization (ISO) admitted the identifier to the ISO/IEC 14518: 2009, too. Actually 124 institutes of Red Cross, private, army, community and university based blood services are Eurocode members. Further the DGTI and Zagrab, Croatia are members of the organization. Croatia plans the Eurocode implementation for all 6 institutes in the country in 2011 and 2012. The Technical Committee defined since 1998 altogether 459 products codes for several product groups. Actually the qualifier tables was enlarged and new qualifier was be implemented for tissue and cell coding. In June 2011 the first product codes for tissue and cells will be defined in cooperation with the Charitè, the German Institute of Cell and Tissue Engineering (DIZG) and the DGTI Working Section 7 “Tissue engineering”. Furthermore the Technical Specification was revised last year and can now be downloaded in PDF—format from the website. At the moment the prescription of the product qualifiers are under redefining. The next issue of the Executive Board and the Technical Committee is the implementation of a quality management system resulting in a DIN/ ISO 9001 accreditation of the system management. Today the labeling of blood products with Eurocode is the standard in Germany and thus in Votum 36 (10/2007) the Advisory Board “Blood” at the Robert Koch Institute advises the implementation of Eurocode in Germany.

Table.

(for Abstract P 10.01)

name	legal form	setting up	instal lations	server	client	data base
Becom Blutdepot	AG	1995	10	Win 2008	XP, Win 7	Sybase
Blues	AG	1984	10	Unix	XP, Win 7	Informix
BluWin	GmbH	1978	10	Win 2008	XP, Win 7	Oracle
Edgecare	GmbH	1996	35	any	XP, Win 7	any
eProgesa	GmbH	1984	35	any	XP, Win 7	Oracle
Eurolab	AG	1989	35	Linux	XP, Win 7	Oracle
PC-Blut	GmbH	2004	35	any	XP, Win 7	Pervasive
Swisslab	GmbH	1977	35	any	XP, Win 7	Sybase
WinTransmed	GbR	1986	4	Win 2008	XP, Win 7	SQL

P 10.04

Eurocode ensures coding of tissues and cells in accordance with EU Directives 2004/23/EC and 2006/86/EC

Knels R.¹, Schnurstein K.¹, Redecker—Klein A.¹, HHIer J.¹, Mönig H.—J.², Pruß A.³

¹Eurocode IBLS e.V., Dresden, Germany, ²Deutsches Institut für Zeil— und Gewebeersatz, Berlin, Deutschland, ³Tissue Bank, Institute of Transfusion Medicine; Charité – Universitätsmedizin Berlin, Berlin, Germany

To avoid mix—ups and to ensure traceability EU Directives 2004/23/EC and 2006/86/EC demand the design of a single European coding system. The system shall provide unique donor and product identification as well as information on the main characteristics and properties of tissues and cells. Coevally the DG SANCO Working Group on the European Coding for Human Tissues and Cells worked on the definition of criteria to attain a high extent of consistency for the concomitant use of different national systems. At the last meeting the single Product Code with basic information about the product remained not finally discussed. The Eurocode system with the ISO—registered, unique primer identifier “!” meets all requirements of the EU Directive and the DG SANCO Working Group regarding the donor and the product identification. One major advantage of Eurocode is that the flexible structure following the Key Code allows the integration of alternative data structures currently employed for identification in different countries, thus ensuring their continued use. The data structure of the product code offers the possibility of an easy incorporation of tissue and cell products by defining new qualifier tables and/ or enlarging the existing ones. Furthermore Eurocode can integrate the actually discussed new basic Product Classification in addition to the “comprehensive” Product Code by defining a new secondary identifier. For this reasons the Eurocode system was approved as the single coding system for cells and tissues in Germany by the Working Party “Tissue preparations” of the DGTI. The implementation is supported by the German Ministry of Health. The presentation deals with the international background in this topic and the available data structures, first implemented in Germany by the Charité and the DIZG.

P 10.05

Computer—aided creation of a blood supply guideline

Liebscher K.¹, Huschke K.², Hammer T.³

¹Klinikum St. Georg gGmbH, Institut für Transfusionsmedizin und klinische Hämostaseologie, Leipzig, Deutschland, ²Klinikum St. Georg gGmbH, Klinik für Anästhesiologie, Intensiv— und Schmerztherapie, Leipzig, Deutschland, ³Klinikum St. Georg gGmbH, GB Controlling und Informationswirtschaft, Germany

Background: Efforts to improve and assure quality in the field of hemother—apy have been promoted significantly during the past few years. In accordance with Guidelines for the Preparation of Blood and Blood Components and for the Use of Blood products (Hemotherapy), the patient must be informed about the risk of an allogeneic blood transfusion in the case of planned surgeries where a transfusion may be seriously considered in a regular operative process. For elective interventions with a transfusion probability of >10%, a sufficient amount of erythrocyte concentrates must be provided. Since perioperative transfusion frequency is associated with a high variance, however, application of in—house data is recommended. Creating a supply guideline for elective surgical interventions has been so far a time—consuming procedure. Owing to this, updates were rarely made, which lead to a numerically inadequate demand for erythrocyte concentrates, in its turn causing additional laboratory and provision costs at the blood bank. Methods: We recorded surgeries requiring blood transfusion within 24 hours after the start of the intervention and the number of transfused red cell concentrates per type of procedure. Data were collected by linking different computerized subsystems, with a subsequent calculation of the probability of transfusion. Results: The blood supply guideline resulting from this can then be updated regularly using the implemented computer—aided procedure, and precisely reflect the blood requirement for elective surgeries. Conclusion: The computer—aided creation of a supply guideline for stored blood units is practically feasible, provides valid transfusion data and is thus an essential tool towards quality assurance.

P 10.06

Requirements for a document management system in a blood services environment

Clemm H.¹, Kießig S.T.²

¹Ainea AG, Germany, ²Haema AG, Blood Service Center Dortmund, Germany

The manufacturing of drugs – especially in blood services – requires a quality management considering different regulatory systems at a time. The practical implementation is effected by SOPs, specifications, manufacturing and QC records. Even in hemovigilance systems appropriate documentation is required. All staff members have to be trained and kept constantly informed in order to stay up to date and to assure always the same grade of quality. Therefore various types of documents have to be included in a document management system. As most organisations are decentralised, a web—based system is necessary to grant access to all current documents from everywhere. Thus also decentralised distribution and trainings of instructions prior to the implementation of SOPs are safeguarded. Furthermore the entire process of creation, revision, amendment, authorisation, validation and invalidation (life cycle of a document) has to be reflected in a DMS. The following document types have to be managed by a DMS: —SOPs, manufacturing records, QC records, release records —transport documents —release documentation —hemovigilance documentation (correspondence) —documentation and track recording of adverse incidents —planning and documentation of audits (internal and external audits, supplier audits etc.) —contract management and planning —OOS and deviation management —CAPA management —change control management. A DMS has to be developed and validated in compliance with the regulations of GAMP 5 (versioning, status updates, audit trail etc.). An implemented DMS is part of the manufacturing and release process of drugs and therefore subject to the drug law regulations (AMG). In order to allow an easy and simple handling, a DMS should observe the processes already established in blood services. Present solutions working with MS Office should be maintained. High acceptance can only be reached if a DMS can be used like the existing paper—based documentation.

P 10.07

Introduction of quality management causes mainly internal process optimization

Richter E.¹, Sattler S.¹, Lehn P.¹, Schmitz G.², Ahrens N.²

¹Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Regensburg, Regensburg, Deutschland, ²Institut für Klinische Chemie und Laboratoriumsmedizin, Universitätsklinikum Regensburg, Regensburg, Deutschland

Quality assurance is mandatory in transfusion medicine. Though the resource requirements for quality management are usually obvious, the benefits are sometimes more obscure. We report retrospectively on the first 22 months of experience with the introduction of a quality management program in a laboratory and transfusion medical institute of an university hospital. Among the introduced measures were, apart from qualification, validation and standard operating procedures, the systematic acquisition of deviations, corrective action requests, and out—of—specification (OOS) procedures as well as quality reviews. An average of 3.0% OOS per apheresis and 5.9 corrective action requests were recorded per month since their introduction. Three types of errors became obvious with some of them being particularly trivial. In one instance, a batch—related problem of starting materials was observed. In three instances, deviations were related to the equipment and could be compensated by adjustment settings of the equipment or by renewal of necessary components. The quantification of equipment reliance showed to be advantageous both for patient safety and economically in negotiation settings. In two instances, staff—related errors were identified that could be addressed by cutting unnecessary procedure steps or by targeted training measures. The introduction of a quality management system started to create transparency and resulted in optimizations especially in procedures that involve more than one team.

P 10.08

Quality management system for use of rFVIIa in hospitals

Niemann I.

Klinikum St. Georg gGmbH, Institut für Transfusionsmedizin und klinische Hämostaseologie, Leipzig, Deutschland

Background: recombinant factor VIIa (rFVIIa) is a potent haemostatic agent that is increasingly used in patients with severe bleeding events. It is important to guarantee high quality standards and cost—effectiveness for application of rFVIIa in hospitals. Patients and Methods: presentation of the experiences with use of rFVIIa in an advanced care hospital. Retrospective analysis of patients treated with rFVIIa between 2002 and 2010 in a single centre. Results: within an observation period from 2002 to 2010 29 patients, age 16 − 86 years, 15 male and 14 female, were treated with rFVIIa. Three patients received rFVIIa for labelled indications (congenital FVII deficiency (n = 2) and acquired haemophilia) and 26 patients were treated for off—label indications: life—threatening bleeding after blunt trauma (n = 12), severe intra— or post—operative bleeding (n = 11), and postpartum haemorrhage (n = 3). Marked therapeutic responses were seen after use of rFVIIa in 50% of patients with severe peri—operative bleedings, in 73% of patients with blunt trauma, and in 100% of patients with postpartum haemorrhage. Significant reduction or stop of bleeding and reduced transfusion requirements were observed in these cases. Initial rFVIIa bolus dosages of 80 − 90 μg/kg body weight were administered. Eight patients required a second bolus. No thromboembolic events were observed during hospital stay. Conclusions: our results demonstrate that use of rFVIIa in patients with uncontrollable massive haemorrhage may be an effective therapy to control bleeding and to reduce blood loss. Further studies are needed to optimize rFVIIa treatment regimens (dosage and start of treatment) and to gain more data on safety and cost—effectiveness of its use in off—label indications. The off—label use of rFVIIa in hospitals should be based on interdisciplinary standard operation procedures.