Multicentre standardisation of a clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood

Abstract

Background

In addition to a largely prevalent use for bleeding prophylaxis, platelet concentrates from adult blood have also been used for many years to prepare platelet gels for the repair of topical skin ulcers. Platelet gel can be obtained by activation of fresh, cryopreserved, autologous or allogeneic platelet concentrates with calcium gluconate, thrombin and/or batroxobin. The high content of tissue regenerative factors in cord blood platelets and the widespread availability of allogeneic cord blood units generously donated for haematopoietic transplant but unsuitable for this use solely because of low haematopoietic stem cell content prompted us to develop a national programme to standardise the production of allogeneic cryopreserved cord blood platelet concentrates (CBPC) suitable for later preparation of clinical-grade cord blood platelet gel.

Materials and methods

Cord blood units collected at public banks with total nucleated cell counts <1.5×10⁹, platelet count >150×10⁹/L and volume >50 mL, underwent soft centrifugation within 48 hours of collection. Platelet-rich plasma was centrifuged at high speed to obtain a CBPC with target platelet concentration of 800–1,200×10⁹/L, which was cryopreserved, without cryoprotectant, below −40 °C.

Results

During 14 months, 13 banks produced 1,080 CBPC with mean (± standard deviation) volume of 11.4±4.4 mL and platelet concentration of 1,003±229×10⁹/L. Total platelet count per CBPC was 11.3±4.9×10⁹. Platelet recovery from cord blood was 47.7±17.8%. About one-third of cord blood units donated for haematopoietic transplant could meet the requirements for preparation of CBPC. The cost of preparation was € 160.92/CBPC. About 2 hours were needed for one technician to prepare four CBPC.

Discussion

This study yielded valuable scientific and operational information regarding the development of clinical trials using allogeneic CBPC.

Introduction

Umbilical cord blood (CB) (also termed placental blood) collected by venipuncture of umbilical cord vessels after delivery of healthy term neonates is an established source of haematopoietic stem and progenitor cells that has been used in more than 30,000 haematopoietic stem cell transplants carried out in patients suffering from leukaemia, lymphoma, thalassaemia, immunodeficiencies and metabolic disorders. Since the development of regular CB banking programmes in the USA and in Europe in the early 1990s, about 600,000 CB units voluntarily donated after informed consent by healthy mothers have been cryopreserved for long-term storage in more than 100 public CB banks worldwide¹.

Consolidated clinical experience shows that the clinical outcome of CB transplantation is significantly and positively associated with the number of nucleated cells contained in the CB unit (a proxy of haematopoietic stem cell content)². Moreover, recent reports from organisations in charge of managing CB unit requests by transplant centres show that CB units containing fewer than 1.5×10⁹ nucleated cells are infrequently selected and used for clinical transplants³. Following the publication of the above evidences and in consideration of the high cost of cryogenic space, several public banks, including those forming the Italian Cord Blood Network (ITCBN), elected to exclude from banking for haematopoietic transplant purposes those CB units containing less than 1.5×10⁹ nucleated cells, which represent more than 80% of CB collections^4,5. Although justified on grounds of clinical evidence and economic evaluations, this decision has the potential to discourage mothers in their generous attitude towards altruistic CB donation for public use.

The above considerations and the presence of several potentially valuable biological materials in CB prompted the development of investigations to identify new blood components that could be obtained from routine CB collections. A procedure for the preparation of allogeneic CB platelet gel (CBPG) suitable for tissue regenerative purposes was developed at the Milan Cord Blood Bank and the Blood Transfusion Service of the Policlinico Hospital in Milan, Italy⁶, and a patent for the preparation of a “Platelet Fraction Deriving From Placental Blood” (US patent n. 8,501,170 B2) was granted to the Policlinico Hospital on August 6^th, 2013.

This article describes the outcomes of a national programme developed to standardise the production of cryopreserved CB platelet concentrates (CBPC) suitable for future preparation of clinical grade CBPG by the public CB banks belonging to the ITCBN.

Materials and methods

The Italian Cord Blood Network

The ITCBN comprises 19 public CB banks accredited by the health authorities of the Italian Regions. Among them, the CB banks located in Bologna, Milan, Pavia and Treviso have also been accredited by the Foundation for the Accreditation of Cellular Therapy (FACT). A detailed report of activities of the ITCBN is available at the website of the Italian National Blood Centre⁵. Briefly, during 2014 the 19 CB banks belonging to the ITCBN collected 19,459 CB units for allogeneic non-family-related use in 320 delivery rooms. Of them, 1,738 (8.9%) were added to the national inventory of cryopreserved CB units stored for allogeneic haematopoietic transplantation, which totalled 38,437 CB units on December 31, 2014. Starting from 1993 (date of the onset of the first public CB banking programme in Italy) until December 31, 2014, 1,352 CB units from the ITCBN inventory have been distributed for allogeneic non-family-related haematopoietic transplants worldwide.

The Italian Cord Blood Platelet Gel project

In 2011, the public CB banks located in Milan and Pavia developed a research project aimed at standardising the routine production of cryopreserved CBPC from CB units not fulfilling the criteria for banking for haematopoietic transplant purposes but otherwise potentially usable for other therapeutic applications as compliant with negative donor medical history, infectious markers screening and additional criteria (see below). Eleven additional ITCBN banks later agreed to participate in a national project. After a pilot exercise carried out during January-October 2013, routine CBPC production was started on November 1^st, 2013. Table I reports the criteria adopted for the selection of CB units to be processed into CBPC. Meanwhile, an inquiry into the appropriate classification of CBPC and CBPG (as blood components or as blood derivatives) was submitted to the Committee for Experimentation in Phase I Clinical Trials of the Italian National Institute of Health (Istituto Superiore di Sanità) which determined that allogeneic CBPC and CBPG, in analogy to similar products obtained from adult peripheral blood, belong to the category of blood components. The Italian National Health Council, which is the scientific counselling board of the Minister of Health (Consiglio Superiore di Sanità), released the same opinion under the regulatory point of view.

Table I.

Criteria adopted for the selection of cord blood (CB) units to be processed into CB platelet concentrates (CBPC).

- Maternal informed consent to CB use for the preparation of CBPC and CBPG if CB unit does not match the criteria for inclusion in the cryopreserved inventory for haematopoietic transplant. - Negative medical history of the CB donor. - Total nucleated cell count <1.5×109/CB unit. - CB unit volume including anticoagulant >50 mL. - Platelet count >150×109/L. - Negative CB donor screening for blood transmissible infections (hepatitis B virus, hepatitis C virus, human immunodeficiency virus, syphilis, human T-lymphotropic virus I/II).

Preparation and quality control of cord blood platelet concentrates

CB units were collected after the mothers’ informed consent into plastic bags containing 25–30 mL of citrate-phosphate-dextrose anticoagulant by trained midwives, before and after placental delivery in natural deliveries and in Caesarean sections respectively, according to locally validated standard operating procedures. After storage and transportation at monitored room temperature to the CB banks, the units were processed within 48 hours of collection.

The participating banks were allowed to use locally available centrifuges and bags of convenient size and nominal volume that they considered appropriate for this protocol. Moreover, in agreement with the platelet concentration defined by the Italian Society of Transfusion Medicine for platelet gel obtained from adult peripheral blood⁷, it was decided to define a mean target platelet concentration in the CBPC of 1,000×10⁹/L (range, 800–1,200×10⁹/L).

During pilot optimisation studies at each bank, a common protocol was defined which included an initial centrifugation of CB at 200–210 g×10–15 minutes, followed by transfer of the platelet-rich plasma (PRP) into a secondary bag, centrifugation of the PRP at 1,800–2,600 g×15 minutes and removal of the supernatant platelet-poor plasma (PPP) in excess of the final target volume of the CBPC. The latter was defined by an automated algorithm performed by an Excel spreadsheet used for data collection (Microsoft Corp., Redmond, WA, USA), which takes into account the platelet concentration in the PRP and the minimum (800×10⁹/L) and maximum (1,200×10⁹/L) values of the platelet concentration expected in the final CBPC. The platelet concentrations in the PRP divided by 800×10⁹/L and by 1,200×10⁹/L provided the upper and lower bounds of the target CBPC volume, respectively. The final volume was set as (upper + lower bounds)/2, by determining the net weight of the CBPC on an electronic scale.

The CBPC units were finally transferred into a storage bag and cryopreserved without cryoprotectant in a mechanical freezer at a temperature below −40 °C in view of future clinical use of the CBPG, which requires thawing at 37 °C in a water-bath before activation. The main characteristics of the disposable bags used for the preparation of CBPC by the 13 CB banks are shown in Table II.

Table II.

Number of CBPC prepared from November 2013 to December 2014 by each CB bank participating in this study (total 1,080), bag manufacturer, code and volume of anticoagulant in the primary bags used for CB collection, manufacturer and code of the bags used for the production of CBPC.

Bank (n. of CBPC)	Type of bag used for
	CB collection (mL of anticoagulant)	CBPC preparation	CBPC cryopreservation
A (180)	J: 811–1010 (30, CPDA1)	FK: P4208	FK: A3AB0010 B: PRPS
B (152)	MA: MSC1202PU (29, CPD)	MA: VQX0001XU FK: A3AB0010	B: PRPS
C (129)	MA: MSC1201PU (29, CPD)	FE: R4R2004	B: PRPS
D (112)	MA: MSC1205DU (29, CPD)	FK: A3AB0020 FK: A3AB0010	B: PRPS
E (89)	FK: T4029 (30, CPD) J: 811–1010 (30, CPDA1)	MA: VRT0000XU	B: PRPS
F (71)	J: 811–1010 (30, CPD)	FK: P4208	B: PRPS
G (70)	FK: T4029 (30, CPD)	J: 814–0132	B: PRPS
H (64)	MA: MSC1201DU (29, CPD)	FK: A3CA0060	MA: GSR 1000 AU B: PRPS
I (61)	G: 722270 (25, CPD) MA: MSC1206DU (29, CPD) FK: T4029 (30, CPD)	T: BBT015CM FK: A3AB0030	B: PRPS
L (58)	FK: T4029 (30, CPD) MA: MSC1201DU (29, CPD)	B: CBB150	B: PRPS
M (47)	MA: MSC1201DU (29, CPD)	MA: VSE2001XR	B: PRPS
N (30)	FK: T4029 (30, CPD)	FK: P4208	B: PRPS
O (17)	J: 811–1010 (30, CPDA1)	T: BBT015CM	B: PRPS

CB: cord blood; CBPC: CB platelet concentrates; B: Biomed Device, Modena, Italy; FK: Fresenius Kabi, Bad Homburg, Germany; FE: Fenwal, Mont Saint Guibert, Belgium; G: Grifols, Sant Cugat del Vallès, Spain; J: JMS, Singapore; MA: MacoPharma, Turcoing, France; T: TerumoBCT, Lakewood, CO, USA.

PPP was used for the detection of bacteria and fungi, according to standard blood component culture procedures.

Quality assurance

A common Excel database was developed and distributed to the banks to collect detailed information (weight and complete blood counts) on each CB unit before processing, the PRP, the PPP and the CBPC before cryopreservation. Automated internal controls checked the coherence, expressed as percent recovery, between the sums of cell counts (platelets, white blood cells, red blood cells, haemoglobin) and volumes in the recovered fractions and the total cell counts and volume in the original CB unit.

A common bleeding list was used by all the participating banks to report information relevant for the validation of clinical grade CBPC and CBPG units and for national haemovigilance purposes to the Italian National Blood Centre, including manufacturer, code and lot number of kits used for microbiological screening tests carried out on blood samples collected from the mothers of neonates from whom the CB units were collected, according to standard CB banking procedures.

Statistical analysis

Data showing a normal distribution are presented as mean±SD, those diverting from a normal distribution as median, 5^th and 95^th percentiles. The statistical significance of differences in volume and platelet content in CB units and in CBPC prepared by the different banks was evaluated by analysis of variance (ANOVA).

Results

Between November 2013 and December 2014, the 13 banks produced 1,080 CBPC. The mean±SD time from CB collection to the start of processing into CBPC was 31±12 hours.

Quality control data from the 1,080 CBPC are shown in Table III. It can be seen that the CB units used for the production of CBPC had a mean±SD volume (including anticoagulant) of 97.6±20.2 mL. The PPP fractions obtained after centrifugation of the PRP had a mean±SD volume of 19.6±9.8 mL and median total platelet count of 0.13×10⁹ platelets (5^th–95^th percentiles, 0–1.54).

Table III.

Volume, total platelet count, total white blood cell (WBC) count, total red blood cell (RBC) count, and haemoglobin (Hb) content of cord blood (CB) units before processing, of platelet rich plasma (PRP) obtained after the first centrifugation and of the final CBPC before cryopreservation.

Unit/Fraction	Volume (mL)	Platelets (109)	WBC (106)	RBC (109)	Hb (g)
CB unit	97.6±20.2	23.4±6.8	956.8 (535.9–1430.2)	305.5 (195.1–463.5)	7.4±2.1
PRP	34.7±9.8	14.3±5.8	12.7 (2.7–151.5)	0.8 (0–3.0)	n.d.
% recovery from CB	n.d.	59.7±19.3	1.4 (0.3–13.2)	0.3 (0–0.9)
CBPC	11.4±4.4	11.3±4.9	7.2 (1.0–105.7)	0.6 (0.2–2.4)	n.d.
% recovery from PRP	n.d.	79.2±21.7	67.5 (10.6–144.8)	n.d.
% recovery from CB	n.d.	47.7±17.8	0.8 (0.1–12.0)	0.2 (0.1–0.7)

Data are given as mean ± standard deviation (volume, platelets, Hb) or median (5^th–95^th percentiles) (WBC, RBC).

CBPC: CB platelet concentrates; n.d.: not determined.

The 1,080 CBPC had a mean ± SD volume of 11.4±4.4 mL, platelet concentration of 1,003±229×10⁹/L and total platelet count of 11.3±4.9×10⁹ platelets. Platelet recovery from CB was 47.7±17.8%. The median white cell count per CBPC was 7.2×10⁶ (5^th–95^th percentiles, 1.0–105.7).

The ANOVA showed highly statistically significant differences (p<0.001 in both cases) in volume and platelet counts both in the initial CB units and in the final CBPC processed at the individual banks (data not shown). However, differences in platelet concentration, absolute platelet count and volume of CBPC produced by the different banks were relatively small. In particular, the coefficient of variation of platelet concentration in the CBPC ranged between 8% and 22% in ten banks and was 35%, 42% and 45% in three banks (Table IV).

Table IV.

Volume and platelet concentration (mean±SD) and coefficient of variation (CV) of platelet concentration of the CBPC prepared at the different banks participating in the Italian Cord Blood Platelet Gel project.

Bank (n. of CBPC)	Volume (mL)	Platelet concentration (×109/L)	CV of platelet concentration (%)
A (180)	10.0±3.7	991±83	8
B (152)	11.6±4.8	1,016±216	21
C (129)	11.8±4.7	939±156	17
D (112)	13.4±5.0	1,041±133	13
E (89)	10.8±3.6	944±327	35
F (71)	10.1±3.4	1,066±225	21
G (70)	13.6±4.2	895±374	42
H (64)	15.5±4.5	1,031±225	22
I (61)	11.1±2.7	1,020±195	19
L (58)	8.4±2.1	1,066±185	17
M (47)	11.0±4.4	983±138	14
N (30)	10.3±3.3	1,174±530	45

SD: standard deviation; CBPC: cord blood platelet concentrates.

An evaluation carried out in one bank over the period from January to June 2014 showed that about one third of CB units originally donated for haematopoietic transplant purposes complied with the selection criteria listed in Table I for the production of CBPC.

Table V reports costs incurred at one bank for the production of one CBPC (€ 160.92). On average, banks reported that about 2 hours were needed for one technician to prepare four units of CBPC.

Table V.

Costs (in Euro) incurred at one bank for the production of one CBPC.

Item	Cost/item	N.	Cost/CBPC
Materials
Wafers for sterile docking	3.29	3,00	9.87
Multiple bag kit for 1st centrifugation	6.10	0.25	1.53
Multiple bag kit for 2nd centrifugation	17.69	0.50	8.85
Multiple bag kit for CBPC cryopreservation	25.25	0.33	8.33
Subtotal			28.58
Laboratory tests and labour
CB group (ABO/Rh)	7.90	1,00	7.90
Complete blood count	4.05	5,00	20.25
HBsAg	10.00	1,00	10.00
Anti-HCV	8.95	1,00	8.95
Anti-TPHA	3.70	1,00	3.70
Anti-HIV1/2	8.95	1,00	8.95
HTLV-I/II	13.15	1,00	13.15
Tri-NAT (HCV, HIV, HBV)	18.00	1,00	18.00
PPP culture	26.44	1,00	26.44
Technician’s time (Euro/hour)	30.00	0.5,	15.00
Subtotal			132.34
Total			160.92

CBPC: cord blood platelet concentrates; HBsAg: hepatitis B virus surface antigen; HCV: hepatitis C virus; TPHA: Treponema pallidum hemagglutination assay; HIV: human immunodeficiency virus; HTLV: human T-lymphotropic virus I/II; NAT: nucleic acid testing; HBV: hepatitis B virus.

Discussion

Platelet gel from adult peripheral blood is a standard blood component that has been used for therapeutic purposes for many years, primarily as an autologous biological product for the treatment of skin ulcers and bedsores^8–11 and other conditions^12,13. Autologous platelet gel is generally considered microbiologically safer than allogeneic donor blood with regard to the risk of acquiring microbial and viral transmissible infections. However, we are unaware of comparative studies of appropriate size supporting or contrasting this hypothesis. Moreover, autologous platelet gel has significant practical limitations which may prevent its clinical use in different categories of patients, for example elderly hypo-mobile patients, neonates and children for whom repeated blood collections for multiple platelet gel applications may be difficult or clinically inappropriate.

These limitations prompted several groups to standardise platelet gel as an allogeneic blood component obtained from healthy adult blood donors, to be routinely offered to clinicians for the treatment of patients suffering from different conditions, thus avoiding the inconvenience of autologous blood collection^12,14–16. This approach also reduces potential negative effects of pathological biological effectors possibly present in the patient’s blood in relation to his or her morbid conditions.

The positive clinical outcomes of allogeneic platelet gel from adult blood in our setting¹⁷ and the very high local rate of discarded CB units donated to our public CB bank for haematopoietic transplant purposes because of low cell dose prompted us to develop a national cooperative effort within the ITCBN starting from the standardisation of the routine production of cryopreserved CBPC.

The results of this study suggest that CBPC can be easily standardised at the national level through a simple programme of institutional cooperation among delivery rooms, CB banks and blood transfusion services. In our programme, we deliberately chose not to select a unique set of bags for the production of CBPC for this initial national exercise, thereby facilitating participation by institutions using different disposables and equipment conveniently available at the local level. However, we recognise that the selection of a unique, optimised kit of bags specifically designed for the preparation of CBPC could further decrease variability and improve standardisation in the future. An optimised processing kit could also improve the platelet yield in the CBPC from the current levels slightly below 50% to higher values typically found in the routine preparation of platelets for transfusion purposes.

Not unexpectedly, ANOVA carried out on a relatively large dataset from 13 institutions detected statistically significant differences in the CBPC produced at the different banks. However, the coefficient of variation of the platelet concentration in the final CBPC was below 23% in ten of the 13 banks, a promising result in view of improved standardisation that could be achieved in future, larger studies carried out with a unique, optimised processing kit. With regard to the most appropriate primary parameter for the standardisation of CBPC, we preferred to aim at the standardisation of platelet concentration rather than to select a more easily achievable fixed volume for the CBPC, which would have necessarily increased the variability of platelet concentration. In fact, a highly variable platelet concentration (i.e. the platelet count per unit volume) in CBPC could represent an important confounding variable for the evaluation of outcomes in clinical trials developed to test the therapeutic efficacy of CBPG.

Among the limitations of this initial standardisation study, we acknowledge that microbiological testing of PPP rather than the final product may generate some false negative results due to lack of sensitivity at low bioburden and cause difficulties in the regulatory pathway and in the performance of some clinical trials. To overcome this limitation, the Emilia Romagna Cord Blood Bank recently completed local validation studies based on requirements of the European Pharmacopoeia, General Part “Biological tests”, chapter 2.6.27 “Microbiological control of cellular products” and aimed at the miniaturisation of testing, which use 100 μL of CBPC (Marina Buzzi and Alessandra Maso, written communication on unpublished observations, 10 May 2015). Plans are in progress to implement this method within the ITCBN.

Although the cost of € 160.92 for the production of one CBPC reported by one bank participating in this study cannot accurately represent costs in all banks, it is encouraging to note that the outcome of a parallel national study yielded a mean cost per CBPC of € 164, a very similar value (Simonetta Pupella, written communication, 12 May 2015). These cost data could be used as a reference for local estimations and comparisons with the cost of standard and advanced skin ulcers medications, which need to be accurately carried out in view of clinical uses. In this regard, total costs of CBPG should also take into account the cost of reagents used for CBPC activation. The product can be activated inexpensively by the addition of one volume of 10% calcium gluconate (at a cost of about € 0.2/vial) to four volumes of CBPC or by the use of a commercial batroxobin kit. Advantages and disadvantages of the above methods of platelet activation and their effect on growth factor release have been extensively discussed in published studies^18–21.

Conclusions

In conclusion, our study suggests that CBPC can be easily standardised at the national level. This evidence could facilitate the development of clinical trials aimed at the full evaluation of the therapeutic value of this new blood component in different conditions. Based on the promising outcomes of two recently published pilot clinical studies carried out in Milan with CBPG in children suffering from epidermolysis bullosa^22,23, an international, multicentre, randomised clinical trial using CBPG obtained from the CBPC units prepared in this study for the treatment of diabetic foot ulcers has been started.

Finally, we believe that the identification of novel, clinical applications for allogeneic biological products obtained from CB, the “youngest” donated blood^24–26, could further increase the ethical motivation of families that generously donate CB for public benefit.

Appendix 1. Italian Cord Blood Platelet Gel Study Group

• - Marino Argiolas (Cagliari);

• - Paola Bergamaschi (Pavia);

• - Maria Bianchi (Rome, Catholic University);

• - Tiziana Bonfini (Pescara);

• - Daniela Bovo (Padua);

• - Marina Buzzi (Bologna);

• - Mauro Carta (Cagliari);

• - Pier Luigi Cocco (Cagliari);

• - Lidia De Felice (Rome, La Sapienza University);

• - Lazzaro Di Mauro (San Giovanni Rotondo);

• - Giovanni Foti (Reggio Calabria);

• - Noemi Greppi (Milan);

• - Mariacarla Iorio (Pisa);

• - Elisabetta Liberatore (Pescara);

• - Maurizio Marconi (Milan);

• - Benedetta Mazzanti (Florence);

• - Laura Mazzucco (Alessandria);

• - Mario Pagano (Naples);

• - Pasqualepaolo Pagliaro (Bologna);

• - Vincenzo Poggi (Naples);

• - Giulia Pucci (Reggio Calabria);

• - Simonetta Pupella (Rome, Italian National Blood Centre);

• - Paolo Rebulla (Milan);

• - Sara Rinalducci (Tuscia University, Viterbo).

• - Bina Romano (Pavia);

• - Riccardo Saccardi (Florence);

• - Laura Salvaneschi (Pavia);

• - Michele Santodirocco (San Giovanni Rotondo);

• - Ilaria Sbarsi (Pavia);

• - Valentina Schiavo (Rome, La Sapienza University);

• - Maria Screnci (Rome, La Sapienza University);

• - Serena Spartano (Rome, Catholic University);

• - Angela Totaro (San Giovanni Rotondo);

• - Patrizia Urciuoli (Pisa);

• - Stefania Vaglio (Rome, Italian National Blood Centre);

• - Claudio Velati (Bologna);

• - Ida Villanova (Pescara);

• - Lello Zolla (Tuscia University, Viterbo).