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TRANSLATIONAL AND CLINICAL RESEARCH |
aStem Cells and Immunotherapies Department,
bNational Health Service Cord Blood Bank,
cBristol Institute of Transfusion Sciences,
dHistocompatibility and Immunogenetics Department,
eTissue Services Department,
fDivision of Diagnostics, Development and Research, National Blood Service, National Health Service Blood and Transplant, United Kingdom;
gDepartment of Immunology and Molecular Pathology, University College London, London, United Kingdom;
hNuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, United Kingdom
Key Words. Directed cord blood banking • Stem cells • Transplantation • Accreditation
Correspondence: Jon Smythe, MPhil, Ph.D., Stem Cells and Immunotherapies Department, National Blood Service, NHS Blood and Transplant, Headington, Oxford OX3 9BQ, U.K. Telephone: 44-0-1865-44-79-67; Fax: 44-0-1865-44-79-77; e-mail: jon.smythe{at}nbs.nhs.uk
Received January 25, 2007;
accepted for publication April 20, 2007.
First published online in STEM CELLS EXPRESS May 17, 2007.
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ABSTRACT |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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Cord blood transplants have the advantage of less graft-versus-host disease (GvHD) than similarly matched non-T-cell-depleted bone marrow (BM) transplants, although time to engraftment for single UCB unit transplants may be delayed [2–5, 9–11]. Factors associated with improved UCB engraftment include lower recipient age and weight, closer human leukocyte antigen (HLA) matching, and cell dose [9]. Low UCB cell doses have often restricted their use for transplantation to children or small adults, where recommended doses range from or exceed 2.5–5 x 107 total nucleated cells (TNC) per kilogram [9]. More recently, the use of multiple UCB units for larger recipients or combinations of UCB with selected bone marrow or mobilized peripheral blood CD34+ cells in a haploidentical setting have proven successful in increasing cell dosages for specific disease indications [2, 3, 9, 11–14].
Approximately 25% of patients requiring an allogeneic stem cell transplant have an HLA-matched sibling, and for the majority of the remainder, if white, an appropriate unrelated BM or peripheral blood stem cell (PBSC) harvest or a stored unrelated UCB donation will probably be found [9]. For some children, especially those from ethnic minority groups and those who suffer from inherited disorders such as hemoglobinopathies or immune deficiency, a directed collection of UCB following the birth of a sibling may be the only opportunity for an HLA-matched transplant until the sibling is able to donate BM. In addition, such directed umbilical cord blood (DCB) donations from matched siblings have generally been shown to give better overall results than matched unrelated BM [15–17]. Preimplantation genetic testing in the U.K. to ensure that a pregnancy will result in a child free from a serious inherited disorder known to exist in the family is now accepted practice regulated through the Human Fertilisation and Embryology Authority (HFEA) [18, 19]. Combined with preimplantation HLA typing, this practice will increase the chances of finding a matched disease-free DCB collection, and so the number of DCB collections is likely to rise [20, 21].
The National Blood Service in England, through its network of accredited stem cell services and histocompatibility laboratories, has taken a leading role in the provision of a national DCB service for high-risk families. This service encompasses liaison with the obstetric delivery unit to arrange collection, processing, testing, storage, and issue for transplantation. This service has been developed to operate in a GMP-compliant environment as prescribed by the U.K. Department of Health Code of Practice for Tissue Banks 2001 [22]. The DCB service was also developed to meet the evolving standards of the U.K. Blood Transfusion Services' "Red Book" quality standards [23], FACT-NETCORD guidelines [24], the requirements of the U.K. Human Tissue Act [25], and the European Union Directive for Tissues and Cells [26].
Here, we review the service we have provided for DCB collections over 10 years, describing the quality of the units, practices adopted, regulatory issues, and the outcome of the resulting transplants. We also recommend best practices, to ensure a system for DCB collection and storage that is ethical, robust, of high quality, and clinically responsive to patient needs.
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MATERIALS AND METHODS |
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Written Informed Consent
Written informed consent was obtained from the donor mother for screening of both the mother and cord blood for mandatory microbiological markers, for tissue typing, and for storage of samples and confidential information. Consent was taken with reference to the U.K. Department of Health's Reference Guide to Consent for Examination or Treatment [27]. An information sheet was latterly provided by the NBS as an aid to those undertaking consent. Consent was also obtained by the NBS from the obstetrician for the delivery hospital to help with the collection.
Mandatory Screening
Samples from mothers were tested in advance for hepatitis B surface antigen, anti-hepatitis C virus (HCV), HIV 1 and 2, syphilis, and, more recently, human T-lymphotropic virus I and II, HCV, and HIV by polymerase chain reaction (PCR), by the NBS. Potential collections were not necessarily excluded on the basis of positive maternal virology results.
Directed Cord Blood Collection and Transport to the NBS
Collections were organized and processed by four NBS centers located across England to limit transit times; collection could therefore be performed in the hospital selected by the mother for birth. Initially, NBS staff collected the DCB, but later, for logistical reasons, midwives collected the DCB using detailed instructions provided by the NBS. The instructions covered collection both in utero and ex utero, with additional advice from the NBS provided by telephone when necessary. NBS collection kits were sent to a named individual in the delivery hospital in an insulated box, validated for transport of the DCB at 2°C–8°C using cool pack inserts. The kit included a spare collection pack (Macopharma, Twickenham, U.K., http://www.macopharma.com) and alcohol and iodine sterile swabs for cleaning the cord. A partially completed label was sent, along with line clips, a sealer, and a labeled bottle for a segment of the umbilical cord as a backup source of DNA for HLA typing. Where the mother's history indicated that the fetus/infant was at risk for an inherited disorder, the referring hospital liaised directly with the delivery hospital to request neonatal samples for genetic testing. The kit included details of a courier able to deliver at short notice, and shipping records were maintained. The NBS centers were staffed 24 hours a day, and collections were stored at 4°C before cryopreservation within 24 hours of collection.
HLA Typing, Including Preimplantation Studies
A blood sample from the potential recipient (and family members if required) was requested for HLA typing. Alternatively, a copy of the HLA typing results was provided by the referring hospital. HLA typing by the NBS for HLA-A, B, Cw, DRB1, and DQB1 antigens was carried out by polymerase chain reaction with sequence-specific primers (PCR-SSP), as detailed previously [28]. Before 1997, some HLA typing was done by serology. DNA was stored for any confirmatory HLA typing required subsequently. Prenatal HLA typing from chorionic villus sampling was as described [29]. Preimplantation screening [30], introduced during this study, was the responsibility of the referring clinician and followed U.K. guidelines issued by the HFEA [31].
Directed Cord Blood Processing and Storage
If the DCB was badly clotted or of negligible volume, the laboratory head called the referring clinician to discuss whether to proceed. The default position, however, was to proceed, with a final decision on storage being made when all results were available. A unique 12-digit barcode number was used for labeling, paperwork, and files, in addition to other details. A bleedline sample was removed for TNC and CD34+ counts, cell viability, and blood grouping. CD34 analysis was performed using a single-platform lyse no-wash flow cytometric protocol [32, 33] that incorporated calibrated beads to quantitate CD34 cell concentration directly and 7-actin-actinomycin to determine viability.
Processing was carried out according to European Union GMP standards [34] under grade A air quality conditions in a clean room. A sample was taken prior to processing for tissue typing and virology screening. Cryoprotectant solution was prepared from dimethyl sulfoxide (DMSO) and 10% (wt/vol) dextran-40/saline or 4.5% (wt/vol) human albumin solution [32]. An equal volume of cooled 20% (vol/vol) DMSO solution was added to the cells in a controlled manner. To maximize the cell dose, stored volume reduction was not carried out [23]. A small volume was taken postprocessing for bacteriological screening and to prepare frozen reference samples before the cells were frozen in cryocyte bags (Baxter Healthcare, Newbury, U.K., http://www.baxterhealthcare.co.uk). DCB collections were processed individually to avoid inadvertent mixing of bags or samples. A contiguous line sample was left attached to the frozen bags for future confirmatory HLA typing, although this practice varied in the early part of this study. The collections were frozen double-wrapped using a controlled-rate freezer and then transferred to liquid nitrogen-based storage tanks, maintained at less than –150°C and continuously monitored [33]. DCB units from mothers known to be positive for microbiology markers or awaiting results were stored in quarantine tanks. Reference samples were cryopreserved with the DCB unit and stored in the same tank [33].
Reporting to the Referring Clinicians
A preliminary report was sent indicating volume, cell counts, and any adverse events, with an additional report sent once all test results were available. Where HLA typing of the potential recipient and family members was done by the NBS, the report included information on the degree of match. NBS histocompatibility staff were available to discuss the HLA typing with the referring hospital's histocompatibility laboratory or transplant medical consultant.
Storage Policy
The NBS advice on long-term storage of the DCB depended on HLA compatibility, the potential recipient weight, disease progression, and the likelihood and timing of using the DCB unit. Latterly, the NBS policy has been to store units for patients with at least one haplotype match and cell counts greater than 0.3 x 109 TNC. A written statement from the referring clinician was requested to confirm whether or not long-term storage was required. NBS costs (on a not-for-profit basis) were recovered from the referring hospital.
Directed Cord Blood Issue for Transplantation
All final decisions on transplantation remained the responsibility of the transplant unit. All requests to issue the DCB were confirmed in writing. Frozen reference samples were thawed by the NBS for cell viability, colony-forming unit (CFU) testing, confirmatory HLA typing, and short tandem repeat analysis [33] to confirm identity with the sample that had been HLA typed at the time of banking. Where the reference sample was not the unit bleedline, the sample had been given the same unique barcode number as the unit to avoid incorrect identification. The NBS has shown that viability testing on reference samples from both the bleedline and vials can be used to predict viability in the whole units. CFU assays were carried out latterly using a methylcellulose-based medium according to the manufacturer's instructions (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) [34]. DCB units were transported in temperature-monitored dry shipper containers at less than –150°C and were thawed by NBS or hospital staff, with instructions provided by the NBS.
Transplant Outcome
A form requesting information on adverse reactions, primary engraftment data, and transplant outcome was issued with the DCB unit, and additional information was requested by telephone. Each transplant center determined engraftment using its usual protocols. Neutrophil engraftment was defined as the first of three consecutive days to reach 0.5 x 109 neutrophils per liter and platelet engraftment as the first of three consecutive days to reach 20 x 109 platelets per liter, with at least 7 days since the last platelet transfusion.
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RESULTS |
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Transplant Characteristics
Of the 233 units collected for 217 existing siblings, 65 (28.0%) were a full 10/10 antigen match at HLA A, B, Cw, DR and DQ loci. Of the matched units, 13 were issued for transplantation (Table 3), with all but 3 being used for nonmalignant disorders. The median age of the recipients at transplantation was 5.8 years. This represented median and mean ages of 5.0 years for recipients with malignant disease (n = 3) and 6.0 and 6.5 years, respectively, for recipients with nonmalignant disorders (n = 10). Cord blood was stored, on average, for 17 months (median, 13; range, 4–47) before transplant. Three units were transplanted for acute lymphoblastic leukemia (ALL), seven for ß-thalassemia major, one for Fanconi anemia, one for Diamond-Blackfan Anemia (DBA), and one for neutrophil dysplasia. One of the transplanted cords was collected following prenatal HLA typing, and another was collected following preimplantation HLA typing of the embryo prior to pregnancy. A second matched donation was stored following preimplantation typing. None of the units collected from mothers with a family history of inherited disease were transplanted; however, the mothers may yet give birth to affected children who are HLA matches.
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DISCUSSION |
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We recommend that the decision to retain the DCB is best made once the cell counts and HLA typing results have been obtained. Other studies using unrelated UCB units indicate that a single HLA antigen mismatch can be compensated for by higher cell doses and recommend that only HLA-A-, -B-, and -DR-matched units be used when cell doses <2.5 x 107 TNC per kilogram are available [9]. However, even for low volume collections, there is the possibility of using the DCB unit in conjunction with small volumes of BM from the same donor once the child has reached sufficient age for this to be an option [13, 14, 16]. There is also the possibility of using donor lymphocytes to counter relapse or infection post-transplantation [9, 42, 43]. Donor lymphocytes were used successfully in this study to combat a rejection episode following one transplant for thalassemia.
Those collections that were less than 40 ml were not associated with one hospital or NBS center but were slightly more likely to have been collected ex utero (57%). We and Wall et al. [44] reported little difference in the volume of UCB units collected using either in utero or ex vivo techniques, whereas others [45] have observed higher CD34+ cell counts and lower rates of contamination with in utero collections. However, the safety of mother and child is paramount, and the U.K. Royal College of Obstetricians and Gynaecologists [38] has recently suggested all collections should be made ex utero.
The median TNC viability prior to cryopreservation was 99%, with only four samples scoring lower than 90%. This suggests that a validated means of transport with a reliable courier is an effective way to avoid loss of viability in transit. Wada et al. [46] have noted lower viabilities of UCB units with increased transit times and lower volumes. However, there was no clear reason (as noted by others [47, 48]) to explain the few low viabilities in our studies. We recommend using a single-platform CD34 assay that includes viability assessment on fresh samples and combining this with the CFU assay on frozen samples to measure cell numbers accurately and determine viability of the CD34+ cell population.
UCB banks typically volume-reduce to 21 ml [7, 8, 33, 49], whereas we did not reduce the volume of DCB units. The numbers of DCB units are relatively low, and so the benefit from not losing as much as 20% of cells through volume reduction [49] or even total loss during processing becomes more important than the larger storage space required. Improving umbilical cord cleaning by using alcohol wipes followed by iodine swabs dropped bacterial contamination to 2.4%, which compares very well with other DCB programs [16] (3.3%) and is probably at a minimum [50, 51]. Bacterial contamination can be addressed at the time of transplant with antibiotics, but units contaminated with dangerous organisms are discarded. Units from an HCV-positive mother could be considered where follow-up testing of the donor child indicates there was no vertical HCV transmission.
All units are now typed at the time of collection using PCR-SSP for HLA-A, -B, -Cw, -DRB1, and -DQB1 to ensure that the degree of match can be determined fully and so best inform the decision to store the cord blood. NBS Histocompatibility laboratories are all accredited by the European Federation for Immunogenetics. The NBS recommends high-resolution (allele) typing for all potential transplant donors and recipients where any uncertainty regarding HLA compatibility is identified. We found that 28% of collections for a sibling were an HLA 10/10 match, in line with Mendelian segregation. An identical matched sibling donor is preferred as the standard of care to an unrelated donor as a clinical option in many transplant settings, as indicated recently by the fourth European Bone Marrow Transplantation Society (EBMT) report on current practice for transplantation in Europe [52]. Where stem cells from related and unrelated donors are both listed as standard of care options, an unrelated UCB unit may be preferred to a related UCB unit to provide a sufficient cell dose. As the practice of preimplantation HLA testing becomes more widely accepted, it is likely that the proportion of related matched units and transplants will increase [53–55].
UCB collections that are not a full match may be used successfully, as shown for unrelated UCB transplants, where 80% of transplants have one or two mismatches. Indeed, a limited mismatch may be preferable for some malignancies, bearing in mind that cell doses of 3–5 x 107 TNC per kilogram have been recommended for transplants with a one- or two-HLA disparity [9]. Where there is a family history of an inherited disorder, the DCB collections are almost always stored for potential future use. Most of the collections for nonmalignant disorders were for severe combined immunodeficiency, and matched UCB units have been successfully used for transplant in immunodeficiency from both related and unrelated donors [9, 56]. Cord blood units have been transplanted after many years [9, 11, 57], so long-term storage may be appropriate. Continued storage should, however, be reviewed at least annually to ensure that units are not stored unnecessarily. A unit with one HLA haplotype match or none is unlikely to be used, and so storage of such a unit over the longer term is most likely not required.
The final decision to store or otherwise is the responsibility of the referring clinician, but where this ignores current acceptable practice for transplantation [52], the NBS may request that the unit be transferred to a private bank. For nonidentical units, the decision should be made with regard to the availability of better-matched unrelated UCB units of sufficient dose or BM/PBSC being available from UCB banks and BM registries. The initial search against HLA -A, -B, and -DRB1 does not attract a charge, but a number of factors should be taken into account when considering the potential availability of unrelated UCB or BM [9, 11, 58]. First, the majority of potential unrelated donors will be BM or PBSC donors, and so the advantages of cord blood over these two will not be provided. Second, at least 30% of identified registry donors will not be available [59]. Third, the match will only be for HLA -A, -B, and -DRB1, and the DRB1 typing may have only been of low resolution, leading to a reduction in the number and degree of matches once full high-resolution allele-level typing has been performed. Fourth, the availability of an unrelated unit or donor cannot be guaranteed if the transplant is likely to take place some time later. Fifth, a UCB unit may not contain sufficient cells for the heavier prospective patient, but there is no opportunity to "top up" the cell dose with a BM collection from the same donor.
In our study, 13 collections have been transplanted to date, and all were matched for HLA-A, -B, -Cw, -DRB1, and -DQB1 loci. This represents a 4.9% take-up rate from all collections, a 5.5% transplant rate where a sibling is alive, and a 20% transplant rate for matched units. The figures compare very well with DCB programs in several countries [15–17]. Twelve patients engrafted, with 11 alive for a median of 49 months (range, 13–116) post-transplant. Of the three ALL patients, all survived 2 years post-transplant, but two subsequently died following leukemic relapse. Nine patients transplanted for nonmalignant diseases engrafted, and all are alive and well 1–9 years post-transplant. Although these figures are from low numbers, they compare very well with those published for unrelated and related cord blood and BM transplants for malignant and nonmalignant disorders [9, 10, 15, 16, 60, 61]. One patient with thalassemia relapsed within 4 months but survived following a BM transplant from the same sibling. This patient was conditioned with busulfan and cyclophosphamide only, whereas more recently, similar patients have been conditioned with fludarabine, busulfan, and cyclophosphamide. Notably, fludarabine-based conditioning for Fanconi anemia patients receiving matched cord blood transplants has been suggested as an essential component of choice [62]. Four thalassemic patients and 1 DBA patient suffered minimal GvHD and other adverse reactions. One rejection episode in a thalassemic patient was countered with a donor lymphocyte infusion, and this patient survives with 70% donor chimerism. Our results demonstrate that DCB transplantation is as effective as unrelated UCB transplantation, albeit in the limited number of cases where a DCB collection is possible.
We are able to offer a cost-effective national service for DCB banking by using the NBS's network of stem cell and immunotherapies laboratories that already have the appropriate facilities, staff, and equipment in place for handling PBSC and BM. The costs for DCB cryopreservation are therefore similar to those for PBSC. The laboratories have been accredited by the U.K. Medicines and Healthcare Products Regulatory Authority and The Joint Accreditation Committee for ISCT-Europe and EBMT (JACIE) or FACT-NETCORD. Our laboratories have recently also been given licenses by the U.K. Human Tissue Authority to operate under the European Union Directive for Tissues and Cells, which became mandatory in April 2006.
Recently, large investments in cord blood banking were announced by the U.S. Congress, and recommendations were published by the Institute of Medicine on establishing a national cord blood bank program in the U.S. [9]. The approach that we have taken within the NBS encompasses many of these recommendations and demonstrates that a national high-quality program can be maintained despite the logistical difficulties of organizing collections from many hospitals and issuing units for transplantation to diversely situated transplant units.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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