The Cord Blood Bank Dilemma

REVIEW Quality rather than quantity: the cord blood bank dilemma

S Querol1,2, SG Gomez1, A Pagliuca3, M Torrabadella2 and JA Madrigal1

1The Anthony Nolan Research Institute, The Anthony Nolan Trust, London, UK; 2Barcelona Cord Blood Bank, Banc de Sang i Teixits, Barcelona, Spain and 3Haematology Department, King’s College Hospital NHS Foundation Trust, London, UK

Growing inventories of cord blood units have facilitated shown when Gluckman et al.2 performed the first cord access to umbilical cord cell transplantation for many blood transplantation (CBT), with subsequent confirma- patients lacking conventional stem cell donors. They are tion over the past 20 years, with 20 000 procedures having in principle ‘off-the-shelf’, ‘fit-for-use’, as well as safe and been conducted.3 In spite of the low blood volume left effective therapy products. Cellular enumeration is used in the placenta and collected in the cord blood unit (CBU), as a surrogate of graft potency, and users rely on the in approximately half of the collections, a single unit may rigorous assessment carried out in banks to avoid poor reconstitute the full haematopoietic system of an adult engraftment after thawing (loss of cells or poor function), patient.4–6 A combination of large-scale banking and when the patient’s situation is critical. However, in transplantation development has made cord blood an practice, when units are selected, initially on the basis of alternative source for BMT.7 HLA matching and cell dose assessment, their absolute Cord blood has a number of advantages compared with quality remains uncertain. Unfortunately, quality-related other stem cell sources. In terms of safety, although a issues (particularly related to viability) are not uncommon waste product, adequately collected, it is not harmful to the in cord blood transplantation. The reasons for potency donor and has a low risk of transmission of communicable failures are diverse, but a lack of thorough validation diseases.8–10 The ability to conduct HLA-incompatible during critical steps of the process and of appropriate use transplants allows for the efficient and affordable design of quality-control tools for timely detection of problematic of national inventories improving access to stem cell units are significant contributors. Moreover, incongruence therapy for almost all patients in need.11 In terms of between different sets of standards and regulations, and logistics, it develops the concept of ‘off-the-shelf’ therapy, lack of common quality systems between banks result in providing near-immediate access for all patients who a highly heterogeneous international inventory. Therefore, require an SCT.12 Furthermore, it also opens new avenues this complicates the matter for the end user of the product. for developing alternative transplantation therapies and To ameliorate this situation, it is essential to improve novel non-haematopoietic applications.13,14 quality at each of the critical manufacturing steps wherein During the past 20 years, outcomes of CBT progressively potency can be threatened, thereby creating homogeneous improved.15–18 This was attributed to a combination of inventories of units with excellent quality and quantity. better clinical management and patient selection, develop- Bone Marrow Transplantation (2010) 45, 970–978; ment of standardized and novel clinical protocols and doi:10.1038/bmt.2010.7; published online 1 March 2010 conditioning regimens, and overwhelmingly, especially in Keywords: cord blood units; umbilical cord cell the adult setting, the increase in the number and quality of transplantation; quality control; viability; international units offered, thus allowing transplantation of more cellular accreditation products with better match.

Pitfalls in cord blood banking

Introduction Despite this experience, many problems remain to be resolved to improve outcomes further. It is not unusual that Cord blood contains naturally mobilized progenitor cells the cell-processing laboratory at the transplant centre that have the capacity to engraft and reconstitute the reports issues on receipt of the product and following haematopoietic system of an individual.1 This ability was thawing. Examples of serious events typically communi- cated are poor cell viability and yield (that is, far below 50%, or failure in colony-forming unit (CFU) growth after thawing);19,20 mislabelling;21 or accidents during transport, Correspondence: Dr S Querol, Cord Blood Bank, The Anthony Nolan including receipt of thawed units, broken bags or impro- Research Institute, Fleet Road, London NW3 2QG, UK. 22,23 E-mail: [email protected] perly labelled products. Received 2 December 2009; accepted 2 December 2009; published online Cord blood is easily collected and manufactured but not 1 March 2010 without risk. Compared with typical adult sources, it The cord blood bank dilemma S Querol et al 971

• Units above 12.5 x108 total nucleated cells (patient body weight >50kg) Suitable for adults

8 Suitable • Units below 12.5 x10 total nucleated cells, for children (patient body weight <50kg)

• No attached segment • No viability test ever performed • No CD34 enumeration Non standard units • Mislabelling risk • Counting discrepancies

Non accredited banks • Only 19 CBB accredited by FACT-Netcord worldwide

Figure 1 Figurative representation of the quality of the international cord blood inventory (400 000 Units from 107 cord blood banks according to www.bmdw.org) and suitability for adult and paediatric transplantation on the basis of accreditation, administrative and quantitative factors—The Iceberg effect. includes a cryopreservation step in samples that have that a small percentage of them are really acceptable usually aged 424 h. Processing automation facilitated products. banking growth but undermined the capacity to perform Promotion of congruent international standards is exhaustive in-process quality control. In addition, general relevant because as shown in the last update of WMDA knowledge on quality assessment has not spread at the (World Marrow Donor Association),27 42% of the cord same speed. Warning reports have been published describ- blood activity resulted from imported units. Communica- ing severe losses of viable stem cells after thawing.24,25 tion and harmonization between the Netcord Foundation, Worryingly, many CBUs were stored without an attached FACT, WMDA and other stakeholders with public health segment, making it impossible to confirm the cellular representatives to gain consensus on releasing test criteria content and viability later on. As we mentioned, the fast and searching policies is required, with the intention of development of worldwide inventories with limited valida- achieving an international harmonization on accreditation tion (that is, banks without transplantation data on and policy. A FACT-Netcord joint venture pioneered this engraftment and follow-up), the lack of ‘fit-for-purpose’ initiative and became the first true international body to integrated quality management systems and the delayed conduct the same strategy of inspection worldwide for all acquisition of common international standards (first set cord blood bank modalities.28 This process validates the developed in 2000,26 but at present, only 19 have been quality management system in these centres, affirming the granted Foundation for Accreditation on Cell Therapy storage of reliable units and should constitute an important (FACT)-Netcord accreditation worldwide) has contributed requirement in the future to regain confidence in the to an ambience of distrust between the banks and end users. international CBUs available for public use. After growing to a reasonable size, the banks need to now focus on improving quality. In spite of the 400 000 Units available worldwide, it is clear that many of these units Quality as a tool to improve outcomes need to be assessed and judged ‘fit for purpose’ on the basis of current recommended practice, thereby defining the Every pharmaceutical product has to establish identity, actual number of reliable quality units. Figure 1 simulates a strength, purity and other quality characteristics designed distribution of the current worldwide inventory stressing to ensure the required levels of safety and effectiveness. the qualitative problems discussed above, and suggesting Cord blood banks, as tissue therapy centres, must ensure

Bone Marrow Transplantation The cord blood bank dilemma S Querol et al 972 300% 20% NC yield CD34 yield 18% 250% 16% 200% 14% 12% 150% 10% 100% 8%

50% 6% 4% 0% 1234567891011121314151716 2% Consecutive units 0% Figure 2 Experience of the ﬁrst 17 cord blood units received at King’s <10 40 70 100 130 160 190 College Hospital and their cell yields after thawing. CD34/microlitre

Figure 3 Distribution of cord blood according to CD34 per microlitre at collection (results from 1383 samples recovered at the Barcelona CBB). that the product is disease free, and that its manufacturing process prevents contamination and preserves integrity and function. Existing and new cord blood bank establish- assess the stem cell function. New methods, especially the ments need to design and develop quality management so-called ‘functional flow cytometry’ that assess early systems that ensure that the product consistently attains its apoptosis are required to increase sensitivity and reduce predefined quality at the end of the process.29,30 Below, our the time it takes to obtain culture results.24 We are review will concentrate on determining which are the validating a method using the early apoptosis marker, critical steps of the cord blood process, especially those that Annexin-V, and the ISHAGE gating strategy38 to improve can affect final cell viability, and will discuss which the functional assessment of cord blood grafts. From these releasing tests need to be conducted to reassure that the data, we observed that a substantial number of CD34 þ unit selected is ‘fit for purpose’. events were 7-Actinomycin D negative but stained for Annexin-V (25% more on average), indicating further functional impairment. In our hands, this approach better Building quality: validation predicts CFU than 7-Actinomycin D alone, especially in Discussion on traceability and safety can be found thawed samples, and might become an ideal releasing test. elsewhere.20,21 However, there are no reports ascertaining how many engraftment failures are due to low graft Procurement. Cells are obtained after cord clamping. viability. In Figure 2, we show the results of the first CBUs Collection techniques may influence the volume harvested used at the King’s College Hospital cord blood programme, but should not influence cell viability. In contrast, and it is clear that there is marked variability in cell conditions during delivery influence the number of pro- recovery and viability observed. In our experience, up to genitors and cells in the graft, especially high in stressful 9 out of 17 thawed units (53%) had problems in labours.39,40 Figure 3 shows the distribution of CD34 þ cell enumeration. These accounted for cell yields o50% cells per microlitre of cord blood collected. The majority of in many cases, and exceptionally big discrepancies in CD34 products range from 20 to 100 with some significant enumeration, perhaps reflecting the difficulties in the outliers above these figures. Interestingly, as transplant standardization of these particular tests when using thawed centres request high cell dose, these outliers are the grafts samples. preferentiality selected for transplantation. Other authors have reported the same issues regarding Cells are drained into a conventional blood donation bag graft potency,19 and remarkably Barker et al. presented with anticoagulant (citrate–phosphate–dextrose in most of data suggesting that in double CBT, the unit that the cases) adjusted to 25–35 ml. This introduces variability preferentially engrafts is that having better viability.31 as units collected with little volume might have excess Worryingly, 430% of the units used for transplantation anticoagulant that might interfere with cellular perfor- in their series had CD34 viability o75%. mance. Another theoretical issue is that the plastic In principle, transplant data suggest that the graft composite of the collection bag was originally designed to content of CD34 þ progenitors, and especially, that of contain red cells and may prove suboptimal for cord blood CFUs correlates with the speed of engraftment and long- storage akin to what happened with platelet function.41 term survival.32–36 These data suggest that a simple assess- These aspects have never been analysed in the context of ment of viable CD34 can be used as a surrogate of cord cord blood banking, and only indirect evidence suggests blood potency. At the moment, the best practice is to that this type of collection bag is not critical, at least if the follow the international ISHAGE (International Society of process is conducted in the first 24 h after collection. Hematotherapy and Graft Engineering) recommendations Longer contact times are still a matter of scientific debate, to enumerate CD34 þ events and using 7-Actinomycin D in spite of the standards allowing processing up to 72 h as a viability marker,37 but maintaining CFU assay to after collection in private banking.

Bone Marrow Transplantation The cord blood bank dilemma S Querol et al 973 Table 1 Characteristics of a cord blood unit at reception (table cell yield for quality control (that is, we use 50% as represent data from first 172 Units recovered at the Anthony Nolan reference). To minimize variability on yield, automation is Cord Blood Bank) beneficial, especially in cord blood banks with a high turn n ¼ 172 Median Maximum Minimum over. Different systems are commercially available, including Sepax, AXP and others.47 These systems have the Volume+25 ml CPD (ml) 89 210 31 ability to register critical parameters during the process, 9 Nucleated cells per l ( Â 10 ) 11.4 29.0 2.8 including volume, spinning speed and time. Total nucleated cells ( Â 108)a 11.0 29.8 0.9 % Lymphocytesb 39 75 19 Finally, another parameter that can affect viability is the % Monocytesb 11 29 5 final haematocrit, and this should be reported to the % Granulocytesb 46 69 12 transplant centre.48 Platelets per l ( Â 109) 218 415 57 Haematocrit 38 49 9 12 Cryopreservation and long-term storage. It is important to RBCs per l ( Â 10 ) 3.54 4.90 0.08 Median corpuscle volume 107 124 88 ensure that the processing of each single unit is performed CD34+ cell total ( Â 106) 3.00 14.87 0.22 by a single technician to minimize the risk of mislabelling. Colony-forming units ( Â 106) 0.32 1.41 0.06 Cells can be potentially damaged depending on time and 7-AAD viability (CD45+) 98% 100% 40% temperature during the cryoprotectant addition.47 We use 7-AAD viability (CD34+) 100% 100% 51% the Coolmix Device (Biosafe, Eysins, Switzerland) that Abbreviation: 7-AAD ¼ 7-Actynomycin D. registers temperature at the time of DMSO mixing, aPercentage of units eligible for different cellular thresholds (higher than increasing the repeatability of the process. After freezing, 5 Â 108 or higher than 12.5 Â 108) are 90 and 40%, respectively. units need to be sealed and packed within the canisters. b Impedance counts using ACT-5-Diff, Beckman Coulter (UK) Ltd, High This is a risky manoeuvre again but does not directly affect Wycombe, UK. potency, unless warming occurs due to excessive handling. Other major risks of this step include tube sealing that can After collection, there is a preference to use cool put the bag integrity at risk. Particular attention needs to be environments for transit times longer than 24 h.42–44 It is paid to the type of bags used, the canister adaptability and very important that each bank generates its own validation the overwraps added to prevent cross-contamination in on the basis of CD34 þ viability or CFU assessment before case of bag damage. Cryovials should be avoided as sample selecting time and temperature parameters for non-frozen handling, especially thawing, is complex and may generate storage and transport. Furthermore, these studies should unexpected variables. also analyse the product after thawing as the viability After freezing, the unit needs to be placed in the problem might be magnified after cryopreservation.45 quarantine space before finally being transferred to the Transport containers need to minimize temperature long-term storage tank. Any event that results in an changes and include temperature logs to register tempera- increase of temperature above À130 1C is considered a ture variations. As a reference, Table 1 shows the data on significant warming event. Their accumulation during the the procurement validation at the Anthony Nolan Cord storage can theoretically affect cell viability after thawing Blood Bank. due to unstable glass transition. This individual thermal history of the sample adds additional certainty that the Processing. At reception, many cord blood banks have integrity and viability of the samples is maintained. It is threshold policies, which remove units that are 448 h old necessary to note that any event at this stage cannot be from the collection and units below a defined volume or cell quality tested, unless a representative segment linked to the content. According to the US Food and Drug Adminis- bag is present. We have demonstrated that the segment tration recommendations,46 values that help to make this linked to the bag and inside the overwrap correlates well decision are suggested in Table 2. Conservation in the with transplant bag viability,49 but this needs to be processing laboratory is sometimes poorly defined. There validated individually by each cord blood bank. are two periods wherein units can be at risk: one, The duration of storage for cryopreserved CBU, includ- immediately after reception and before volume reduction ing assigning an expiration date to CBUs where appro- (if this occurs), and second, after volume reduction and priate, is still not defined. This time should be determined before starting cryoprotectant addition. In particular, this on the basis of the medical literature and/or on the banks’ second period can be critical if the bank uses colloids to own experimental data. However, if no expiration date has improve yield after red cell depletion (that is, starch) as the been established, this should be documented by a con- cells conserved in these solutions sediment, generating a current/retrospective validation of storage to generate data high concentrated pellet if the bag is kept statically on the to use for decision making in the future. The ongoing policy bench or in the fridge. of banks to investigate the effects of long-term storage on Another potential interference in potency is the techni- CBU includes testing for viability, function and/or stability que used for volume reduction and the protocol used for along time. In future, if this is determined and bags are that. Time and temperature of this process, as well as speed beyond this time point, the CBU should be discarded. of centrifugation can be detrimental to final viability, and again this needs to be carefully tested in each cord blood Distribution and transplantation. Finally, after shipment bank programme. In general, standards only recommend and before use, there are three potential points wherein plasma and red cell depletion to reduce the cord blood product quality can be compromised: (1) in the vapour volume. It is helpful to establish a minimum total nucleated shipper used for transportation, if the intended temperature

Bone Marrow Transplantation The cord blood bank dilemma S Querol et al 974 Table 2 Required and recommended tests and test results according the US FDA for cord blood and HPC-C (ﬁnal cord blood product)46

Product Testing Sample (type and timing) Results of product testing characteristics

Safety Infectious diseases—testing required Maternal peripheral blood obtained All tests negative except non-treponemal test (21 CFR 1271.45 through 1271.90) within 7 days of cord blood for syphilis when confirmatory test is negative. collection—type and timing required. CMV results are recorded). CMV report (21 CFR 1271.80(a) and (b)) Sterility—bacterial and fungal HPC-C a(pre- cryopreservation) No growth cultures—testing required. (21 CFR 211.165(b) and 21 CFR 610.12) Haemoglobin Cord bloodb or appropriate donor No homozygous haemoglobinopathy sample obtained at time of cord blood recovery Purity and Total nucleated cells (TNCs) HPC-C (pre-cryopreservation) X5.0 Â 108 TNCc per unit HPC-C potency Viable nucleated cells HPC-C (pre- cryopreservation) X85% viable nucleated cells Viable CD34+ cells (flow HPC-C (pre-cryopreservation) X1.25 Â 106 viable CD34+ cellsd per unit cytometry) HPC-C Identity HLA typing Cord blood Report Confirmatory HLA typing Attached segment of HPC-C Confirms initial typing Blood group and Rh type Cord blood Report

Abbreviation: FDA ¼ Food and Drug Administration. aSample may be obtained before or after addition of the cryoprotectant. bCord blood ¼ cord blood before undergoing volume reduction. cBased on 20 kg recipient, a target dose of X2.5 Â 107 nucleated cells per kg and 70% post-thaw recovery ¼ 1.7 Â 107 nucleated cells per kg. dBased on CD34+ cells X0.25% of TNC before freezing.

is not properly achieved; (2) during the transient storage including small segments that are unmistakably linked to at the transplant centre, because usually there is no the principal transplant product split into two detachable appropriate racking to store the canister received; (3) and compartments for cell therapy applications, and developed finally, the thawing method used. by the New York Blood Center group.47 These segments Unfortunately, accidents have been reported at this stage can be used for specific tests with one still remaining, once mainly due to excessive transit time or failure in the cryo- shipped for quality control at the transplant centre. Such shipper (the product was received thawed at the transplant tests may include assays for safety (infectious or genetic centre), or specifically, problems during the handling in disease markers), identity (HLA, ABO, gender) and the transplant centre especially accidents during thawing potency (CFU, viability, functional flow cytometry).54–57 (for example, broken cryobags). Table 3 shows the guidelines However, in our opinion, the responsibility to verify graft we use to check suitability of units upon arrival. At that time, potency remains the duty of the cord blood banks (in if major issues, regarding identity, safety or potency, are addition to safety and identity) and not the individual detected, the unit needs to be substituted immediately by transplant centre. They should expect to purchase a high- an alternative one. Therefore, it is recommended that all quality product just as they do for any other clinical transplant centres have a back-up CBU reserved that can product, especially when a poor product may interfere with be immediately shipped. This will avoid significant delay in the outcome of transplantation. Proactive decisions in the obtaining new cells for infusion. cord blood bank discarding non-suitable or undetermined The thawing technique remains controversial, despite units are expected. significant data supporting methods that prevent osmotic At present, the best test to demonstrate haematopoietic shock by DMSO dilution and/or washing.50–53 Interest- potency is assessing the clonogenic potential. In our ingly, recent data suggest that, especially in adult patients particular programmes, we use a parameter that is helpful for whom the volume infused can be bigger, an alternative in identifying problems. That is, the so-called clonogenic option is dilution (without washing), thereby reducing efficiency (CLONE), a parameter based on the correlation complexity and validation requirements at transplant between CFU and CD34 þ cells (Figure 4). It means that centres. for a given amount of CD34 þ cells, a defined number of CFUs are expected. We hypothesize that a decrease in the CLONE can predict relevant losses in functionality. As a Confirming quality: verification or quality control reference and according our experience, mean clonogenic Certainly, there is controversy regarding how to perform efficiency (CLONE) corresponding to CB CD34 þ cells is batch quality control; however, a sample from a contiguous 36±25%. As a safety threshold, we arbitrarily chose 10% segment attached to the transplant bag seems the better or more (equivalent to the mean minus one standard method to test the final function of cells present in the deviation) to qualify a unit as optimal and therefore product. Special cord blood bag design and adequate establishing this correlation as a releasing criterion. This releasing tests may become critical to confirm the quality of value, if useful, need to be calculated by each cord blood cord blood products. We comment on a model of cryobag, bank and used as an internal reference. This simple

Bone Marrow Transplantation The cord blood bank dilemma S Querol et al 975 Table 3 Checklist for each unit upon arrival at the transplant Table 4 How to choose a reliable cord blood bank? (Items to centre consider)

Related quality Issue Check Aspect Topic Item area Availability Accreditation FACT-Netcord Safety/identity Traceability Identifiers of external audits HLA (EFI-ASHI) Labels IDM Cryoshipper seal Proficiency testing CD34 (that is, UK-NEQAS) Leakage Bag integrity CFU (that is, stem cell Confirmatory testing Cord blood HLA technology) on attached segments Maternal blood HLA Availability of Procurement Donor inclusion criteria ABO relevant data on Collection technique Gender the unit reports Manufacturing Conservation temperature SOPs Instruction upon arrival Volume reduction technique Freezing technique Potency Transport risks Schedule fulfilment Long-term storage In-house transference times Timing Collection to freezing Cryoshipper weight Long-term storage Cryoshipper position Qualification Nucleated cells per ml at Significant warming Data logger collection events (4À150 1C) Actual temperature reading Erythroblasts Release test on Viable nucleated cell yields CD34 enumeration by single attached segments (450%) platform Viable CD34+ yields Flow cytometry viability on (450%) CD45 and CD34 events CFU growth CFU growth CLONE410% Release checks One attached segment left SOPs Validated thawing procedure Additional reference samples left Traceability Abbreviations: CFU ¼ colony-forming unit; CLONE ¼ clonogenic effi- Disclosing NC yield o50% a ciency of CD34+ cells, namely percentage of CFU scored/CD34+ cells discrepancies Cell counts out of range seeded. Final haematocrit 450% CLONE o10% Administration Accessibility Immediate contact with key individuals 60 Advice Help in decision-making process Disclosure policy Transparency Speed Fast answers 50 Cost Proportionate Per procedure not per unit 40 Scientific profile Reports Activity Quality indicators Peer-review papers Banking 30 Engraftment and outcomes

Abbreviations: CFU ¼ colony-forming unit; CLONE ¼ clonogenic effi- 20 2 ciency; FACT-Netcord ¼ Foundation for Accreditation on Cell Therapy- Total CFU (x10e6) R = 0.54 Netcord. aWe consider a range of normality an initial nucleated cell concentration 10 between 5–30 Â 106 per ml and a %CD34 per CD45+ events between 0.1 and 1%. 0 0 10 20 30 40 50 60 Total CD34 (x10e6) reliable cord blood banks. In Table 4, we propose checklist Figure 4 Linear correlation CFU/CD34 (results from 1383 samples features that may help transplant centres to identify such recovered at the Barcelona CBB). establishments. approach is used in our cord blood banks to discard units Proposal for CBU categorization in public inventories that probably lost potency during manufacturing, thereby increasing the likelihood of high-quality effective units. Although HLA matching remains important in CBT, cell Finally, the most important quality check is the feedback dose dependency on outcomes suggests that the cell dose the bank requires from the transplant centre regarding data should be used as a major quality criterion. In Figure 5, we on cell yields and engraftment. Every deviation would show how the likelihood of finding a donor for a given require a thorough analysis of potency data to identify body weight varies within two cord blood banks having potential problems in manufacturing and prevent the future different thresholds of acceptance. To increase the effec- release of unreliable products. An active communication tiveness of a cord blood inventory, a adequately high policy on engraftment outcomes and other quality indica- threshold need to be establish in order they can benefit as tors will help transplant centres in identifying the most much patients as possible. As shown in Table 5, units

Bone Marrow Transplantation The cord blood bank dilemma S Querol et al 976 120% transplanted by the Barcelona Cord Blood Programme ANCBB have progressively increased in size over time. This thresh- bcB 100% old is especially important in the design of national cord blood inventories. The size of the inventory is calculated on 80% the basis of match categories; however, the units stored should fulfil minimum criteria to make the investment cost- 11 60% effective. In our recently published study, we proposed a minimum of 9 Â 108 total nucleated cells as the cost 40% effective threshold. Finally, to facilitate user decisions around the quality of the cord, we propose to categorize the CBUs into three Percentage of suitable units 20% categories. These categories need to distinguish clearly units with potential issues (category C), from reliable units that 0% 20 30 40 50 60 70 80 90 100 can be further classified according to their cellular content Body weight as suitable for children (category B) or suitable for adults Figure 5 Effectiveness of finding a donor by two cord blood banks with (category A) (see Table 6). Assessment of the inventories different acceptance thresholds based on patient body weight. Anthony Nolan based on these categories can give us a real picture of the cord blood bank (ANCBB): current acceptance thresholds are 6 Â 108 or 9 Â 108 quality of the current international inventories and the need total nucleated cells for non-predominant and predominant ethnic groups to replace or discard unreliable units. (resulting in a clinical inventory suitable for patients of 33 kg median body weight). Barcelona cord blood bank (bcB): Current acceptance thresholds are 20 Â 108 total nucleated cells or 4 Â 106 CD34+ cells for units between 11– 20 Â 108 (clinical inventory suitable for patients of 63 kg of median body weight). Summary

Table 5 Cell dose of units selected for transplantation over time In summary, cord blood banking has developed extensively within the Barcelona Cord Blood Programme since the pioneering work by Rubinstein et al.58 in New Year Number of Total nucleated Total CD34+ York in 1993. Certainly, there has been an impressive units cells cells increase in activity over the last few years; however, provided (median, 108) (median, 106) signiﬁcant quality issues remain. In this paper, we emphasize the need to reinforce validation, in-process 1996–1998 26 11 3.8 1999 26 14 4.3 quality control and common international accreditation 2000 17 18 5.2 strategies, such as those promoted by FACT-Netcord. 2001 25 17 4.4 Safety and identity issues remain the most critical factors of 2002 17 15 6.0 the cord blood bank responsibility. Extensive manipulation 2003 21 18 6.1 2004 36 15 6.2 with signiﬁcant variables such as transit time, storage 2005 44 16 6.1 temperature and cell composition result in unexpected 2006 52 17 6.8 viability problems. In this review, we identify many critical 2007 68 17 6.8 points in the production chain in which the process can 2008 94 17 7.5 damage cells if a proper validation and quality control are 2009 until October 114 21 9.3 not well designed. We support a model of cryobag that

Table 6 Proposal for the categorization of cord blood units within the cord blood bank inventories and simulation using the Barcelona Cord Blood Bank

Category Code Description bcB Unitsa

A. Standard, reliable units A1 Suitable adultsb 4096 (32%) A2 Suitable childrenb 4906 (38%)

B. Discrepancies affecting potency B1 Viable TNC yield after processing or thawing o50% 824 (6%) B2 Low cell countsc B3 %CD34 out of ranged B4 CLONE out of rangee

C. Non-standard units C1 No contiguous segment left 2992 (23%) C2 No reference samples left C3 Not fulﬁlling other standards

Abbreviations: bcB ¼ Barcelona Cord Blood Bank; CBU ¼ cord blood unit; CLONE ¼ clonogenic efﬁciency; FDA ¼ Food and Drug Administration; TNC ¼ total nucleated cell. aCategory distribution of 12 818 CBUs available from bcB. bBased on a target dose of X2.5 Â 107 NC per kg and X0.25% CD34 for adult (X50 kg, corresponding to 1.25 Â 108 TNC and 3.12 Â 106 TCD34) or children (o50 kg, 0.5–1.25 Â 108 or 1.25–3.12 Â 106) according US FDA recommendations (see Table 2). cTNC o5 Â 108 or CD34 o1.25 Â 106. dNormal range: TCD34 cells/TNC before freezing 0.1–1%. eNormal range: CLONE (total CFU/total CD34+ cells ) before freezing 10–100%.

Bone Marrow Transplantation The cord blood bank dilemma S Querol et al 977 allows the verification of not only safety and identity but 15 Kurtzberg J, Prasad VK, Carter SL, Wagner JE, Baxter-Lowe also potency, and encourages researchers to develop LA, Wall D, et al., COBLT Steering Committee. Results of appropriate tests to measure these parameters from the the Cord Blood Transplantation Study (COBLT). Clinical small volume of blood kept in them. Finally, we encourage outcomes of unrelated donor umbilical cord blood transplan- a revision of the available CBU inventories based on tation in pediatric patients with hematologic malignancies. Blood quality categories to help users to identify the most 2008; 112: 4318–4327. 16 Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, appropriate products. 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