Cord blood banking for clinical transplantation - Nature

Bone Marrow Transplantation (2009) 44, 635–642 & 2009 Macmillan Publishers Limited All rights reserved 0268-3369/09 $32.00

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REVIEW

Cord blood banking for clinical transplantation P Rubinstein National Cord Blood Program, New York Blood Center, New York, NY, USA

Cord blood (CB) stem and progenitor cells from related donors have been transplanted for past 20 years and from unrelated donors issued by public CB banks for 16 years. This brief look at public CB banking highlights aspects of its current status to suggest that accomplishing the currently required tasks, though no small undertaking, is not enough: much remains to be contributed. CB banking started in the 1930s, collecting blood for transfusion and showed that CB could be effectively collected, stored and administered intravenously without negative consequences. The realization that it contains hematopoietic ‘stem’ cells (actually, colony-forming units) followed discoveries elsewhere in hematopoiesis research, while HLA and unrelated BMT were being investigated. Progress in the exploration of ethnically stratified HLA allele frequencies, together with plausible neonatal (partial) immunological tolerance, seemed to predict initially frequent, unavoidable, but sufficiently tolerable HLA mismatching with CB grafts. Gluckman et al. and Boyse et al. proved that HLA-identical sibling CB grafts led to definitive engraftment. Technical developments in processing and freezing enabled public banks to accumulate large inventories and to supply grafts that could succeed despite major HLA incompatibility and low cell doses and provide hope for universal access to unrelated-donor transplantation. Public CB banking has thrived worldwide. Regulation and accreditation defined Good Tissue Practice in the CB banking environment and provided accepted do’s, don’t’s and how to’s. Startling advances continue to be made, not only technical, but including the description of molecular regulation in the function of natural killer and other cells involved in allogeneic recognition that will have dramatic effects and will permit further improvement in CB selection and use. Bone Marrow Transplantation (2009) 44, 635–642; doi:10.1038/bmt.2009.281; published online 5 October 2009 Keywords: public cord blood banking; colony-forming units; hematopoiesis; allogeneic recognition; technical developments

Correspondence: Dr P Rubinstein, National Cord Blood Program, New York Blood Center, 310 East 67th Street, New York, NY 10065, USA. E-mail: [email protected] Received 2 September 2009; accepted 2 September 2009; published online 5 October 2009

Introduction ‘Banking’ is the systematic procurement, testing, storage and organization of cord blood (CB) donations and data, with the aim of providing tissue for hematopoietic transplantation and transplant outcome data for analysis. A source of hematopoietic stem (HSC) and progenitor cells for transplantation, the procurement and distribution of CB differs in important ways from adult donation of BM or mobilized stem cells harvested from the peripheral blood (PBSC), until recently the main clinical sources of these cells. Perhaps the most important practical difference is concerned with the selection of donors, which is not limited to HLA match criteria, as in the case of adult donors, but which also matches patient weight and CB cell dose. In addition, because the adult grafts are patient specific and are infused fresh after collection (typically less than 3 days), adult donations usually do not require cryopreservation and frozen storage. Prolonged storage, often for several years, which is an obligatory step for the unrelated CB source, constitutes a logistical advantage (off-the-shelf grafts). It is also a hurdle: CB must be continuously frozen below 150 1C and shipped, transported and received still frozen below 150 1C at the transplant center (TC), if at all possible before patient conditioning starts. Freezing of CB grafts also applied to the sibling donors in all cases of ‘directed CB donor transplantation’ since the publication of the first CB transplantation, in 19891 (we now celebrate its twentieth anniversary) and to the situation in which cord blood units (CBUs) are stored speculatively by private CB banks (CBBs). The latter situations, which resemble ‘deposits’ in financial banking operations more than ‘donations’, are the subject of some controversy because of the incompletely supportable claims often made in advertising, the profit mode of operation typical of such banks and the relatively very low frequency with which such ‘deposits’ have been actually withdrawn and used for transplantation. Thus, private banking will not be included in this review. An interesting third (hybrid) model for CBBs makes private ‘deposits’ potential public ‘donations’ in case they offer a better-matched transplant to patients for whom sufficiently ‘close’ donations cannot be found in the public domain. Different variants of this model exist, from an outright displacement from ‘private’ to ‘public’ status pursuant to an unrelated recipient’s requirement, to having the ‘deposit’ split in two, up-front, one part of each deposit being allowed to become ‘donation’ on need. Given that the

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cell dose is an important predictor of successful transplant outcomes,2–4 such ‘split CB units’ might place their potential recipients at a disadvantage and their application, therefore, unlikely, unless successful stem cell expansion becomes practical in this setting. Transplants of (public) CB donations, in contrast, are performed frequently and the large number of successful outcomes, throughout the world, attests to their clinical usefulness. This success is, however, dependent on patient- as well as CBU-dependent issues and very likely also, on others that have escaped identification thus far. Among the latter are technical difficulties, such as delay in the processing and freezing of grafts and ‘transient warming events (TWE)’ of frozen units from below 150 1C to above 130 1C, which, depending on the extent of warming and number of occurrences, has been demonstrated to reduce overall CD34 þ cell viability and colony-forming unit (CFU) count.5 This review will include some of the unpublished reports and current practices of the first public CBB, the National Cord Blood Program (NCBP) of the New York Blood Center (NYBC), in CB collection, processing, testing/ typing and distribution of clinical CBUs.

Historical notes The fetal/neonatal blood left in the placental and umbilical vessels after birth was first used clinically as a source of whole citrated blood for blood transfusion, before the Second World War.6 Placental/Umbilical cord blood (CBU) was still used for routine blood transfusions in some hospitals in New York and other places at least in the 1960s.7 Transfusion of an acute leukemia patient with CBU unrelated donors was found to have led to the transitory change of the patient’s MN blood group, claimed to be caused by temporary engraftment of stem and progenitor cells of one of the transfused CBUs.8 That report attributed the observed engraftment and described the features of such engraftment in relation to that reported from conventional BMTs. Although this paper was disbelieved, human placental/umbilical CB does contain abundant ‘hematopoietic and progenitor stem cells’, as shown by the development of hematopoietic colonies in CB culture assays by several investigators.9–11 Knudtzon9 had postulated that because the concentration of these cells in CBU is similar to that in BM, CB might be used ‘as a source of hematopoietic stem cells for the restoration of bone marrow function in humans’. Improved techniques for functional characterization and enumeration of these cells took place during the 1980s.12–14 Nakahata and Ogawa12 showed that individual cells taken from GEMM colonies could be re-plated and give rise to colonies of diverse lineages, that is, displayed a degree of stemness, and Koike13 showed that colony-forming cells from CBU remain viable after cryopreservation and suggested storing frozen CB for eventual transplantation, particularly, for genetic disease. The same suggestion was made independently by Besalduch14 who also raised the possibility that easier tolerance induction in newborns might permit transplanting CB from HLA-mismatched donors. Subsequently, Boyse and colleagues15–17 anticipated that the Bone Marrow Transplantation

concentration of HSC in cryopreserved placental blood collections was sufficient for the clinical reconstitution of ablated marrow, including in adults. In 1989, Gluckman et al. and Boyse et al. reported the first successful marrow reconstitution by transplanting CB from an HLA-identical sibling to a child with Fanconi anemia.1 A number of other CBU transplants were performed quickly afterwards from related donors, both HLA identical and non-identical.18–21 The Placental Blood Program at the New York Blood Center, designed to support marrow reconstitution by CBU transplantation from unrelated donors,22,23 was initiated in September 1992 and within 1 year had supplied grafts for the first two unrelated-donor CBU transplants in August and September 1993, to children with acute leukemia at Duke University.24,25 As of April 2009, this Program has banked more than 50 000 CBUs and released grafts for more than 3000 patients, to over 100 TCs in four continents.

Cord blood harvesting technique The earliest reports of CB collection for clinical applications in blood transfusion6 describe the trans-section of the doubly ligated umbilical cord during the third stage of labor, after the baby’s delivery, followed by the squirting and dropping of blood from the sectioned umbilical vein into a graduated glass beaker or bottle containing sodium citrate anticoagulant. This, however, has been replaced in modern practice by venipuncture and CB collection into an integral plastic blood collection bag. There are two approaches about the timing of the CB collection, with the venipuncture in the third stage of labor, while the placenta is in utero, being probably the most frequently used. The alternative method bleeds the umbilical vein in the (ligated) cord attached to the delivered placenta by gravity into a blood bag for transfusion.25 The latter method avoids interference with the conduction of the delivery itself but still presents the risk of blood clotting within the vein, especially when the cord has been previously clamped or otherwise traumatized. Although many workers in the field believe the latter method to result in lower volume and hence lower total nucleated cell (TNC) numbers in CB collections than the first, only early comparisons and no definitive analyses have been performed, although several CBBs reported comparisons (with relatively few collections). These were reviewed by Lasky et al.26 who reported that the results were similar with either method. Some individual banks (ours included) have found marked improvement with the use of adequate placental support. The position of the newborn relative to its mother after the delivery and before severing the cord influences the volume of blood that can be collected: if it is placed above the mother, the volume of the placental transfusion would be smaller, at the expense of the infant’s volemia, than if it is at the same level. Conversely, placing the infant below the mother results in a larger placental blood flow to the infant increasing its blood volume and eventually, its hematocrit and decreasing the amount of the blood lost in the umbilical vessels. The result will be a larger CB


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collection in the first mode and a smaller one if the infant is kept below the level of the placenta in the uterus. Additional aspects of obstetrical management may affect the volume of blood that can be collected, such as the size of the conceptus, the time between the infant’s delivery and the ligation of the cord, the length and condition of the cord attached to the placenta (mechanical attrition of which releases tissue factor, causing blood clots) and bleeding from the umbilical vein after the donor’s birth.27

Mother’s informed consent General consensus holds that a valid informed consent must be obtained before the onset of labor when the placental blood is to be obtained during its third phase (see above). Informed consent may be obtained after delivery when blood is collected from the delivered placenta, but, in this case, the mother must be informed of her right to direct the program not to collect, retain or test the unit and/or to discard it without further ado. For the consent to be valid, the mother must be able to agree freely, after receiving all germane information available, to: donate the CBU unconditionally as a graft for anyone who might need it; grant an interview focusing on her and the father’s ethnicity, family antecedents, general health history, and risks for infectious and genetic disease including travel to regions with high endemicity of malarial and Chagas disease; allow a review of the hospital charts of the mother and newborn, for additional information on such risks; allow collection of a blood specimen from herself, taken during 7 days before and 7 days after delivery for infectious disease marker testing, HLA typing, confirmatory testing and for future reference and improved testing techniques and allow the program to report the results of all testing on hers and the infant’s blood to her and/or her physicians.

mainly according to their TNC content. Not surprisingly, the volume of CB collected correlates with its total cellular content, but the TNC concentration can affect markedly that number and an eventual patient’s cell dose. A threshold TNC dose has been identified as 2.5 107/kg patient weight.4 Nucleated red blood cells add to that variation, as their numbers, usually low as a fraction of the RBC count, are more important relative to leukocyte numbers and can also be elevated by obstetrical causes that interfere with blood oxygenation, including the anemia of erythroblastosis fetalis due to maternal–fetal blood group incompatibility (caused by, for example, maternal anti-RhD and anti-A or B blood group antibody). Obviously, erythroblasts, as differentiated cells, are not capable of mediating engraftment, but their increase in fetal/neonatal blood usually portends the presence of higher concentrations of HSCs and progenitor cells. Thus, nucleated erythroid cell numbers correlate in multivariate analyses with the probability of engraftment and its speed27,29 as do monocyte numbers, but not lymphocyte or neutrophil counts (as measured by clinical hematology analyzers).27 In a multivariate analysis of 1213 single-unit grafts (NCBP, unpublished data, 2004), including the transplant’s neutrophils, lymphocytes, monocytes and nucleated erythrocytes, these cell types were numerically associated with the following relative risks of engraftment (and corresponding probabilities) respectively, 0.998 (P ¼ 0.3), 0.994 (P ¼ 0.08), 1.047 (Po0.001) and 1.015 (Po0.001) Thus, only the monocytes (as defined) and nucleated erythrocytes (of the classical cell types in human CB) are positively associated with engraftment. Consequently, TNC numbers that include nucleated erythroid cells are better indicators of hematopoietic potency than TNC numbers that exclude them. Most important for the transplant recipient, however, is the presence of sufficient numbers of viable stem and progenitor cells in CBUs, more closely reflected by the counts of mononuclear cell (MNC) and CFUs30 and CD34 þ cells.31 Other tests of hematopoietic potency have been proposed, the expression of aldehyde dehydrogenase by MNCs32 and the levels of ATP after culture in the presence of selective cytokine cocktails.33 Information on the clinical relevance of these reasonably putative markers is still preliminary.

Collected cord blood: cells and hematopoietic potency Clearly, standardized, reliable and reproducible tests, predictive of the engraftment capacity of HSCs and progenitor cells in CB cell suspensions, are necessary for appropriate pretransplantation evaluation of the hematopoietic potency and thus, clinical utility of CB transplants, as well as for studies of their complex physiology and regulation of their differentiation. Despite the availability of some useful assays, current assays are based mainly on enumeration of cells carrying the CD34 marker and/or of cells capable of giving rise to hematopoietic colonies in vitro and a straightforward potency test is still not available. Just as is the case with BMTs,28 CBUs may provide insufficient doses of the stem and progenitor cells responsible for engraftment in allogeneic recipients, particularly in adult recipients. Thus, the utility of CBUs a priori is judged

Testing and typing Assays that must be performed on each banked unit are now specified by both NETCORD-FACT and AABB accreditation standards. These reflect FDA’s guidance documents and final rules and also consider HRSA requirements for participation in the US National Cord Blood Inventory. They include the following: (a) Cellular content. These indices are fundamental for the determination of suitability and hematopoietic potential of the CBU. 1. Enumeration of the nucleated cells, leukocytes and immature erythrocytes, at the end of processing— before cryopreservation (TNC) and recovery rates for Bone Marrow Transplantation


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viable and total TNC as well as MNC, CD45 þ cells (pan-leukocyte marker), CD34 þ cells and CFU (also see below). 2. Viability of nucleated and progenitor cells at the end of processing—before cryopreservation. For this evaluation, the techniques must be documented to provide reliable and stable dye-exclusion estimates. (b) Microbiological contamination should be examined by validated culture methods for the detection of bacterial and fungal agents. Current Standards require identification and documentation of the contaminant(s) in all cases of directed CB donation, whereas in conventional donation for unrelated recipients, the presence of contamination is exclusionary. (c) Immunogenetic markers, including the main ABO and Rh erythrocyte markers and the HLA-A, B and DRB1 types (typing for HLA-C and DQB is currently recommended). Typing must be performed at the DNA level and at least DRB1 should be typed at the highest resolution level available at the time. (d) Testing for hematopoietic CFU should be carried out before release for transplantation. This in vitro culture test adds to the enumeration of viable CD34 þ cells, a functional evaluation of the progenitors’ proliferation capability. (e) Hemoglobinopathy screening is necessary to avoid the transmission of Hb defects by CB hematopoietic transplantation detecting and identifying genetic variants of Hb in the population. (f) Markers of communicable viral disease agents should be used to screen CB samples before release for transplantation (and must be used when tests approved for use on CB samples are available), including, as of April 2009, 1. HIV viruses, types 1 and 2. 2. Hepatitis B and C viruses. 3. Human T-cell lymphotrophic viruses types I and II. 4. Trypanosoma cruzi, the causative agent of Chagas disease. 5. West Nile virus. 6. Treponema pallidum (syphilis). 7. Other agents, as required by FDA and other regulatory agencies at the time of release for transplantation. 8. (Information on requirements for blood and tissue donation is frequently updated in the CBER (FDA) website www.fda.gov/CBER) (g) Mothers. The mothers of the donors also contribute exclusionary information on units potentially carrying communicable disease agents both through history and blood sample analysis. We must inquire specifically about communicable disease history and about genetic traits segregating in their families and the fathers’. Maternal blood specimens collected between 7 days before or following the donor’s birth must be tested for markers of infection by the agents listed above (f1–f7) and for CMV. The presence of serum antibodies to CMV is not exclusionary and neither is antibody to hepatitis B core if they are negative for hepatitis B antigen by DNA tests. Mothers found positive by Bone Marrow Transplantation

T. pallidum screening can be accepted if they are negative in specific confirmatory tests.

CBU processing for banking Cord blood preparation for cryopreservation and freezing into storage requires no more than minimal manipulation. In its simplest form, a cryoprotective solution containing a cell-permeating agent (DMSO is used essentially exclusively with nucleated cells) is added to a full unit of CB and mixed slowly and thoroughly with an orbital rotator, as the DMSO concentration increases to a final 10%. Because the dilution of DMSO is strongly exergonic, some means to absorb the heat released as it mixes with the CBU are advised, at least, simple ‘cold packs’ optimally with eutectic points between 0 and 4 1C, which rotate in direct contact with the CBU as DMSO is being admixed. Controlled freezing procedures are required to chill and freeze the blood more or less slowly (freezing speeds close to 1–2 1C/ min are considered optimal). Two methods have been used for this purpose, bulk freezing, by depositing the unit inside a plastic foam container inside the hold of a 80 1C mechanical freezer and controlled-rate freezing, by vaporizing liquid nitrogen in the chamber of a computerized cooling container, usually set at the 1–2 1C/min rate. The freezing of unmodified CBUs is simple to perform and obviates the TNC losses that accompany red cell elimination to reduce the CBU volume, a problem reported very early in the clinical use of CB: attempts to reduce the volume always recover less than 100% of the TNC. Such losses have been estimated as at least in the order of 50%.17 The main problems of storing cryopreserved whole CBU is the large volume, which occupies much cryogenic storage room and can, upon administration, cause hemodynamic stress in small recipients, as well as potential toxicity, due to the large amount of DMSO used for whole CBU cryopreservation. To ameliorate these problems, we reduce the volume of CBUs, by removing most of the erythrocyte bulk (99.9% of the total cellular content) and some plasma, to provide a final volume of 25 ml after 5 ml of 50% DMSO cryoprotectant, yielding nucleated cell concentrations safe for cryogenic freezing (o2 108/ml), with hematocrit below 30% and unchanging volume (for reproducible freezing rates). Erythrocytes were initially removed by adding hydroxyethyl starch (to 1%) and centrifuging the CBU at low acceleration and then sedimenting the nucleated cells off the (separated) supernatant.34 The latter were suspended in 20 ml of supernatant plasma and cryopreserved by adding 5 ml of cryoprotectant containing 55% (w/v) DMSO to 10% (also containing 5% Dextran 40) for a 1% final concentration. Freezing and storage was, as usual, in conventional liquid nitrogen freezers. To infuse, after quickly thawing the frozen unit and removing the supernatant, we restored the volume with a saline solution containing 1% dextran and 5% human albumin before infusion. This simple wash, recommended to reduce the quantity of DMSO that is injected into the patient, can be obviated by simply diluting the unit threefold with the 1% dextran, 5% human albumin solution that reduces the


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potential cell loss. This method allowed banking many 25 ml CBUs in relatively small freezer rooms and is being successfully used by many banks worldwide, albeit with a slightly lower cell recovery than after freezing whole-blood CBU (unpublished NCBP data, 2004). A new, computerized device, the BioArchive System (Thermogenesis, Rancho Cordova, CA, USA), which combined the controlled-rate freezing with storage capacity for 3626 frozen units in the same device, was developed to prevent the brief exposure of the frozen CBU to over 200 1C temperature gradients as it travels from a freezer to another, thus, from 196 1C after controlled-rate freezing, to þ 22 1C room air and back into a different 196 1C storage freezer) (TWE).5 The BioArchive allowed automated freezing and individually controlled storage of each CBU and improved recovery and viability of cells after thawing, before transplantation. In a 2004 retrospective validation study of post-thaw cell viability, we compared 1032 CBU that were processed and frozen (a) without volume reduction (whole, cryopreserved units slowly frozen in conventional liquid nitrogen dewars) with (b) volumereduced units slowly frozen in similar dewars and with (c) volume-reduced units frozen and stored in BioArchive systems. When these units were thawed in the respective TCs the viable TNC recoveries were (a) 84.5±0.8% (s.e.m.) for whole blood, (b) 81.8±0.7% (s.e.m.) for volume-reduced CBU in conventional freezers, (c) 88.0±1.0% (s.e.m.) for volume-reduced frozen and stored in BioArchive. Although the losses were all small, they were significantly larger for (b) than for (a) or for (c). The improvement in (c) underlies the usefulness of avoiding TWEs. More recently, instrumented volume reduction techniques have been introduced. Two such technologies are now in use by CBBs, Sepax (Biosafe, Eysins, Switzerland) and AutoExpress (AXP) (Thermogenesis). Sepax consists of a centrifugal device and disposable built to allow spinning the CB around a vertical axis. The disposable has rigid walls and a computer-controlled piston, which, when lowered, aspirates the CB containing 1% Hespan filling the device; after centrifugation, the erythrocytes settle next to the walls and the leukocytes into a concentric layer slightly closer to the axis of rotation. Elevating the piston under light centrifugation while opening the exit valve (top center) forces a fluid stream into a valve-connected sterile distribution tubing. Through the valve exit, sequentially, acellular plasma, leukocyte suspension and finally, erythrocyte mass. Average CB TNC recoveries reported with Sepax volume reduction are in the range of 80–87% (mean ¼ 84.2) with CD34 recoveries being slightly higher 86%. Hematocrits are in the 36–45% range (data from Rodrı´ guez et al.35 and Lapierre et al.36 and Sepax website). These results compare well with those achievable by manual techniques using Hespan to accelerate red cell settling and top-and-bottom bag centrifugation. The other automated method (AXP) consists of a device that fits in the carrier of a blood bank centrifuge and uses a circuit board to drive a stopcock valve, part of the

disposable. As with Sepax, the CB collection bag is steriledocked to the disposable main bag and the CBU is transferred before centrifugation. AXP processes CB without Hespan, an advantage with respect to the manual procedure.34 After the cellular components have been sedimented, the valve opens to let the erythrocytes migrate under low centrifugation into a red cell bag; under optical sensor control, the valve then switches connecting the flow path to the inlet of a Thermogenesis freezing bag placed on a gyroscopically weight-controlled section and allows the transfer of the separated light fraction from the main bag. When the target weight is reached, the valve closes and the CBU product in the freezing bag is ready for cryoprotection. The bag set includes in line ‘pillows’ that permit the collection of pre- and post-centrifugation test samples. AXP and Sepax have been granted FDA 510(k) clearance and AXP has been used by the NCBP since August 2006. In our opinion, AXP without Hespan recovers better than 95% MNC and averages 76–82% of the TNC with very high viabilities (above 95 and 90%, respectively). Viable CD34 þ cell recovery is regularly 497%, higher than Sepax’s albeit with a more laborious procedure. Hematocrit is consistently o30% and post-processing volume is remarkably constant with the target volume. The loss of up to 30% of the granulocytes, with consequent decrease of the TNC, reduces the appeal of CBUs prepared with AXP technology because the TNC criterion has been broadly adopted. Because the cells thus lost are mature, their loss is not expected to reduce the hematopoietic potency of the units. Evaluation of the cell counts and flow cytometric data (Table 1) and examination of the results of transplants with NCBP AXP-volume-reduced units bear this out, although it is still early to quantify any potential transplant outcome differences. Because threshold TNC doses are part of the criteria for CBU selection, CBUs manufactured with AXP technology are proportionally richer in the MNCs, thought to include the stem and progenitor cells. Hence, they should also be more potent than units conventionally processed and having similar TNC.

Inventory, search and release To be most useful, as a whole, the CBU inventory should approximate the requirements of patients in need of grafts for hematopoietic reconstitution. Because clinical outcome

Table 1

AXP processing: cell recoveries

Cell type

Pre-AXP processing Total no.

TNC MNC Granulocytes CD34+ Volume (ml)

7

137 10 55 107 82 107 37 105 108

TNC (%) 100 39 61

Post-processing Total no. 7

101 10 51 107 50 107 36 105 25

TNC (%) 100 51 49

Abbreviations: MNC ¼ mononuclear cell; TNC ¼ total nucleated cell. NCBP unpublished data. N ¼ 6336 consecutive CBUs processed during 2007–2008. Bone Marrow Transplantation


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depends in part on the sharing of genetic histocompatibility factors (mostly of the HLA system, whose alleles reach different frequencies in different populations) the chance to find an unrelated donor who matches the histocompatibility antigens of the patient in a genetically heterogeneous population, such as the US’, will be maximal among donors of the same genetic subset. For this reason, the inventory of CBBs should be carefully constructed to provide similar probabilities of matching for patients of the diverse racial/ ethnic groups in the population. With CB inventories, this can be approximated by establishing collection centers in birthing hospitals where mothers’ ethnicities occur in known proportions, collectively suitable to achieve balance with respect to the patient population. The prior probability of finding matches is lower for people of genetically distinct subsets who comprise a minority within the overall population. Therefore, the distribution of ethnic groups among CB donors should not merely reflect the population proportions but should favor donors of the minority subgroups.37 Only having disproportionately more donors can make the probabilities of matching approximately similar. We anticipate the correct solution to the genetic heterogeneity problem (obtaining well-matched CB grafts for people of all ethnicities) to be the sharing of search requests with CBBs around the world, including those in places where our ‘minority’ constitutes a large population. Technical and regulatory homogeneity will be essential for accrediting CBB and sustaining the necessary excellence. The ample diversity of HLA phenotypes observed in the population as a whole influences the outcome of clinical transplantations by causing both allograft rejection, when the graft expresses an antigen absent from the recipient, and GVHD, when there is rejection of the recipient by an allograft lacking one of its HLA antigens. Better HLA matches associate with improved transplantation outcomes4 despite difficulties in demonstrating this clinical correlation in all data sets. Hopes of overcoming the odds against adequate HLA matching, at least for patients with some diseases, may come from the more recently uncovered family of receptors expressed in natural killer cells (killer immunoglobulin-like receptors, KIR). KIR receptors interact with ligands in HLA class I molecules to prompt/ prevent natural killing. The involvement of natural killing modulated by KIR–HLA interactions in combating leukemic relapse has described38 to follow transplantation with adult marrow, with evidence of reduced relapse incidence and increased disease-free survival. Similar data have been recently gathered in CB transplantation by Willemze et al.39 As mentioned above, the cell dose is the other CBU characteristic that strongly affects the probability of a successful outcome. It is now well understood that the probability and speed of engraftment correlate with the cell dose and that a sharp decrease in both accompanies the evolution of grafts with TNC doses below 2.5 107/kg whereas improvements result from progressively higher TNC doses. Clear indications exist that the negative effects of a low cell dose can be countered by a well-matched graft and vice versa, a poor match can have better prognosis if it occurs with a high cell dose CBU (Table 2). The reciprocally compensating relationship of HLA match and Bone Marrow Transplantation

Table 2 Number of mismatches 2 1 2 1 2 1

HLA mismatches and cell dose interaction (TNC dose) range 107

Transplant-related mortality at 12 months (%)

o2.5 o2.5 2.5–4.9 2.5–4.9 45 45

74 65 58 48 41 34

RR

P

1.9 o0.001 1.7 0.002 1.4 0.009 0.96 0.8 Reference 0.8 0.091

Abbreviation: TNC ¼ total nucleated cell. Single CB transplants with outcome data, N ¼ 1667; NCBP 2007.

cell dose on transplant outcome is potentially useful in selecting the best among potential donors for a patient and should be investigated systematically before focusing attention on a CBU donor because of match or dose. Although great attention has been devoted to the attempts to improve the cell dose by in vitro cell amplification, no clear demonstration of clinical improvement has been obtained with amplification strategies thus far used. Still, more innovative methods are being tried including the blocking of differentiation in stem and progenitor cells being grown in vitro by modulating the concentration of copper ions40 and with specific enzymes that methylate DNA and others that acetylate histones.41 In preparation for the eventual development of a useful expansion technique, however, we have developed a patented, two-compartment, 25 ml freezing bag, to allow culture of the small compartment (5 ml) without disrupting the continuous storage of the stem cells in the large compartment (20 ml) and there are many thousands of CBUs stored in such containers. Obviously, if the small cell doses that limit the number of clinical level CBUs could be solved by in vitro amplification, the speed with which inventories could be increased would accelerate vastly. In particular, the problem of the smaller volume and lower cellularity of the CB that can be detected from some minority ethnic groups, especially African Americans, could be corrected by in vitro expansion, helping to finally achieve balance between the numbers of CBUs in storage and those required. A different solution to the problem of low CB cell doses has been sought through the transplantation of more than a single CBU. A similar solution was attempted in the early years of BMT, by pooling the marrow aspirated from several members of the patient’s family, with improvement in the outcomes. The largest experience relies on the infusion of two clinical units, because initial trials using several units proved to incur excessive mortality. From the CB banking viewpoint, using two CBUs in a patient increases the burden on the inventory and the costs, but will provide solutions for patients whose size and HLA types do not allow finding an appropriate single CBU graft. Considerable work on this approach has been already carried out by Dr John Wagner and Dr Juliet Barker in the use of double CB graft transplantation, to obviate the insufficiency of available cell doses.42 Matching under these conditions becomes an issue that, currently, is not fully solved. Typically, only one of the two units survives after


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an initial period after engraftment, which, surprisingly is not necessarily the largest or the better matched of the two transplanted CBUs. Although a number of hypotheses about the mechanism that guides the in vivo selection of the ‘winner’ have been advanced, there are no definitive answers. Research under way is attempting to define whether there is immunological interaction between the two grafts, in which one may reject the other. HLA matching between the units is being attempted, for example, in addition to each unit with the host. An intriguing observation from the outcome data analysis of NCBP is that one-way HLA-mismatched grafts, resulting only in graft-vs-host reactivity, are associated with a very good outcome, approximately equal to that of fully matched transplantations. This is true for one or two antigen-mismatched grafts, provided that no mismatches in the rejection direction are detectable at the HLA-A, -B, -DRB1 loci (A and B at the serological antigen level, DRB1 at the high-resolution level). A few three-antigen mismatched grafts of this type have been performed with similar results. These observations, if confirmed, might indicate that in the absence of host alloimmune HLA activation, rejection reactivity may be truly minimal. In this case, CBUs homozygous for one or more HLA alleles (say, at HLA-A11) would be extremely valuable as sources equivalent to full 6/6 grafts for transplants to patients that are matched for A 11 and for the HLA: B and DR loci, irrespective of whether the other HLA-A allele is mismatched (that is, 5/6 matches).

Conflict of interest The authors declare no conflict of interest.

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