Confirmatory Typing Research Articles

Biological and Genetic Aspects of Donor-Recipient Matching in HSCT

Bone marrow transplantation is a routine clinical activity offering salvage therapy in a number of hematological diseases and inborn errors. There are two obstacles that may delay or even postpone this curable treatment approach. The first is a lack of matched family donors, which affects up to 75% of patients. In this situation a search for unrelated donors, if successfully completed, makes this approach feasible. HLA genes of five loci (A, B, C, DR, and DQ) are currently considered as a basis for matching. Each day brings information of new alleles. Genetic typing can lead to detection of diversity at the single nucleotide level. It ensures that a level of matching is achieved resulting in transplant success rates similar to those seen among siblings sharing the same HLA genotype. While we wish to have a perfect match, also important is elapsing time during the search process, which is related to the presence in the patient of rare alleles and unusual B-C, DR-DQ associations. Having a primary typing of a patient we can predict the chance for a proper match. Each day new donors are recruited worldwide. Iterative searching must be applied in the latter situation. In some cases 6 or more potential donors are required to have a donor accepted by a clinician. Finally, a compromise must be reached between the aspiration of matching at the level of 10 alleles and the urgency of transplantation in patients suffering from relapsing disease. To facilitate the decision-making process, modern information technology must be at hand. The search process includes the complete donor pool which is screened for potential donors. The chosen potential donors must be activated for confirmatory typing which includes 5 loci specificities typed at the high resolution level with exchange of information between registries and the hospital iteratively coming to the optimal decision. The process must be reliable, safe, and transparent, and must operate efficiently in real time. The European Marrow Donor Information System (EMDIS), used in many countries worldwide, ensures fulfillment of the above requirements. The present volume illustrates the above points, supporting the rational basis for the decision-making process.

The influence of HLA polymorphism and recombination events on the selection of a donor for hematopoietic stem cell transplantation

Background: HSC transplantation outcome is associated with the level of HLA matching between donor and recipient. A search for HSC donor starts in the patient’s family. In the absence of related donor, HLA matched unrelated donors are searched in registries. In the present study we sought to determine the number of donors found in Slovenian families and the influence of initial typing and HLA polymorphism on the selection of unrelated donors. Patients and methods: We set up genotypes in 744 families that were typed for HLA-A, B, DR with PCR-SSP. Recombination events were located in 17 of them. Average number of children and percentages of possible related donors per year were calculated. SBT of HLA-A, B, C, DR, DQ alleles was performed for 162 patient/unrelated donor pairs. An influence of initial typing level on the selection of the donor was defined using Fischer’s exact test. A Slovenian population sample consisting of 77 unrelated individuals was typed for HLA-C using PCR-SSO and PCR-SSP methods. Phenotype frequencies were determined by direct counting. Results: Recombination was observed in 2.28 % of families. Among all patient/donor pairs matched after the initial phase, only 49.4 % remained HLA compatible after confirmatory typing. In particular, 38 % of the pairs were mismatched only owing to disparity at HLA-C. Conclusions: Lower percentage of related HLA identical donors is a consequence of decreasing number of children and occurrence of recombination events in families. Disparities at HLA-C were observed as a result of high level of polymorphism and initial typing not including this locus. This leads to the conclusion that the addition of HLA-C to the initial typing would contribute to faster and more efficient identification of well-matched stem cell donors, which is in most cases crucial for patient survival.

Both Optimal Matching and Procedure Duration Influence Survival of Patients after Unrelated Donor Hematopoietic Stem Cell Transplantation

Eighty-six patients suffering from hematological malignancies, immunodeficiencies, and aplastic anemias received alloHSCT from unrelated donors. Donors were selected from the BMDW files and further matching was performed according to the confirmatory typing procedure with the use of PCR SSP and that based on sequencing. The time from the clinical request of the donor search to the final decision of clinicians accepting the donor was from 0.3 to 17.8 months (median 1.6). Matching was analyzed at the allele level, and 50, 27, and 9 donor-recipient pairs were 10/10 matched, mismatched in one or more alleles, respectively. In an univariate analysis we found better survival if patients were transplanted: (i) from donors matched 10/10 (P = 0.025), (ii) not from female donor to male recipient (P = 0.037), (iii) in female donation from those with ≤1 pregnancy than multiparous (P = 0.075). Notably, it became apparent that duration of the confirmatory typing process affected the survival (HR = 1.138, P = 0.013). In multivariate analysis only the level of matching and the duration of the matching procedure significantly affected the survival. In conclusion, the duration of the matching procedure in addition to the level of matching should be considered as an independent risk factor of survival.

Leukemia specific loss of heterozygosity of MHC in a CLL patient: Disease state impacts timing of confirmatory typing

A 63 year old white male with refractory B-CLL presented for allogeneic HSCT evaluation; HLA typing was performed on PBL at time of WBC = 53K, ALC = 47K and revealed homozygosity at Class I locus and heterozygosity at Class II locus. Two siblings were full mismatches with the recipient and an unrelated search initiated. The patient was treated with Fludaribine and Rituxan complicated by aplastic anemia and bacteremia. Prior to transplant, confirmatory typing performed on PB revealed two full haplotypes at Class I and II. Sample identification error and the presence of third party lymphocyte engraftment as a result of prior red cell or granulocyte transfusion(s) were ruled out by STR analysis of 8 loci of all samples, T and B cells from cryopreserved PB at blast crisis were HLA typed independently. T cell typing yielded both complete haplotypes (genotype verified by offspring HLA typing); B cells typed for homozygous haplotype indicating loss of heterozygosity of MHC locus. Microarray based comparative genomic hybridization of tumor cells confirmed LOH at 6p.

96-P

Human Leukocyte Antigens (HLA), which is extremely polymorphic, plays an important role in stem cell transplantation. The Chinese Han comprise a large population of approximate 1.3 billion with diverse HLA alleles need to be characterized. Data from 3296 independent, unrelated Chinese Han individuals (1457 recipients and 1839 donors) were provided by China Marrow Donor Program (CMDP) for donor-recipient confirmatory typing. SBT, SSOP/HD-SSOP and SSP methods were used to obtain 4-digit alleles. A total of 49, 86, 50, 63 and 24 HLA-A, B, C, DRB1, and DQB1 alleles were observed. Following ASHI CWD criteria, CWD alleles for Chinese Han in our laboratory test and other laboratory reports do not quite correlate with the ASHI CWD alleles: A ∗ 11:53, A ∗ 02:34, A ∗ 02:53N, B ∗ 27:24, B ∗ 46:02, B ∗ 55:12, C ∗ 01:06, C ∗ 03:17, C ∗ 06:06, C ∗ 07:66, C ∗ 07:67, C ∗ 08:22, DRB1 ∗ 12:10, DQB1 ∗ 03:13 and DQB1 ∗ 06:05 are CWD, but are not included in the ASHI CWD list. A series of alleles are Well-Documented alleles and listed in the ASHI CWD list. Conversely, A ∗ 26:03, B ∗ 51:03, C ∗ 12:05, C ∗ 15:09, C ∗ 15:11, C ∗ 17:03, DRB1 ∗ 11:07, DRB1 ∗ 11:11, DRB1 ∗ 13:05, DRB1 ∗ 13:13, DRB1 ∗ 14:06, DRB1 ∗ 14:12, DRB1 ∗ 14:22, DRB1 ∗ 14:25 and DQB1 ∗ 06:11 are rare alleles, but are included in the ASHI CWD list. HLA Ethnic diversity is the main reason for the differences in HLA alleles worldwide. The ASHI HLA CWD alleles help reduce the workload and expenses in high-resolution donor registries and the HLA allele frequencies provide a basis from which to predict the chances of finding HLA matching donors. Our data will be meaningful for the CMDP, for other worldwide donor registries, and for an updated ASHI CWD alleles list.

25-OR

Aim Chromosomal abnormality which may down regulate the human leukocyte antigen (HLA) on chromosome 6 may result in inaccurate homozygous typing of a patient and thus adversely affect donor and recipient matching and transplant outcome. We present a case of a 60 year old female with AML who had chromosomal abnormality in tumor cells. Methods HLA typing was performed by SSP, rSSO microarray and SBT. Short Tandem Repeat (STR) was performed using primers targeting 10 genetic loci. Results Initial typing on patient’s blood revealed the following: SSP method: A∗32, weak bands for A∗2; B∗51, weak bands of B∗44; C∗01, weak band of C∗12, DRB1∗1301, weak band of 07:01; DRB3, weak band of DRB4; DQB1∗06:03, weak band of 02:02. rSSO (class I only): A∗02:XX, 32:XX; B∗51:XX, C∗01:XX. SBT (class I only): A∗32:01, 32:01; B∗51:01, 51:01; C∗01:01, 01:01. Since SSP showed presence of a second haplotype with weak bands, and rSSO detected two A locus antigens, HLA typing was questionable and thought that could be due to transfusion or chromosomal abnormality. HLA typing was repeated using patient’s buccal swab. Results of both SSP and SBT confirmed heterozygosity (mentioned above) with strong bands in all loci in SSP and amplification of both haplotypes in SBT. rSSO typing was not performed on buccal swab. Short tandem repeat (STR) profiles of blood and buccal swabs samples showed identical amplification for 10 primers used. Conclusions The above results show the loss of HLA genes on chromosome 6 in an AML patient. This abnormality was not included in 10 short tandem repeat we tested for. In this case, SSP method showed some weak amplification and helped us to address the possibility of chromosomal abnormality. HLA typing of buccal swab, blood at the time of remission or confirmatory typing must be considered to accurately type leukemia patients with possible chromosomal abnormality.

176-P

All stem cell recipients have their own story, each one being more complicated than others. We report here the case of an 18 year old female who developed an acute lymphoblastic leukemia 10 years ago and went through many stem cell grafts. Complications in HLA typing due to chimerisms had to be addressed in an unconventional way. The HLA-A, -B, -C and -DRB1 were analysed in 2002 using serological methods and SSO low resolution and DRB1 and DQB1 were also analysed in high resolution by SSP. More than nine years after, the HLA-A, -B, -C, -DRB1 and -DQB1 were performed by sequencing, using PCR reactions of exons 2, 3 for loci A, B and DQB1, exon 2 for DRB1, exons 2, 3 and 4 for C and SSP. Blood sample, mouth swabs and hair follicles were used for DNA extraction and HLA typing. The patient was diagnosed with acute leukemia and Ph(+) in 2002. HLA typing was performed at low resolution for the related donor search. No sibling was compatible and the transplant team had to rapidly graft a 4/6 compatible cord blood unit. In 2008, the patient relapsed and received a second 4/6 compatible cord blood graft, using once again the HLA low resolution typing. Again, by the end of 2011, the patient relapsed and blood samples were sent to Hema-Quebec HLA lab to perform high resolution typing and start an unrelated donor search. The level of chimerism was interfering with the HLA typing of loci B, C, DRB1 and DQB1. The DNA extracted from mouth swabs was also chimeric. The unrelated donor search had to be initiated with high resolution HLA typing deduced from the parent’s inherited haplotypes. The patient’s typing still had to be confirmed and the only material left to do it was the hair follicles. Because this material is not routinely used in our lab, DNA extraction was performed by the Quebec forensic laboratory, allowing us to perform the high resolution confirmatory typing. An unrelated donor could be identified and the patient is now being prepared for her third stem cell graft.

27-OR

Case: 36 year old female with AML diagnosed in Dec 2011 and persistent disease after 3 rounds of chemotherapy. She received 7 granulocyte transfusions for neutropenic sepsis in Dec 2011. Single antigen bead (SAB) testing in Jan 2012 demonstrated alloimmunization to HLA-DPB1. Patient has one mismatched sibling and unrelated search was initiated. She demonstrated donor specific antibodies (DSA) to all donors. We present here the progression of DSA over a 5 month period and the clinical care based on these DSA. Confirmatory typing of 5 donors found that 2 were 10/10 match with the recipient, while 3 were mismatched at DQB1. Since the recipient demonstrated DP antibodies with MFI >1000, DPB1 typing was performed on the recipient and donors. Recipient and donor DPB1 typing and DSA are summarized [ Table 1 ]. Serial SAB demonstrate decreasing MFI with no specific DSA reducing intervention and reflected in B-FXM with donor-2 cells [ Table 2 ]. Based on the high DSA levels, an autologous bone marrow harvest was performed as backup to allotransplant. As the DSA MFI was <1000 with negative B-FXM on 4/23/12, transplant was performed with donor 2 cells using reduced intensity conditioning on 5/2/2012. DPB1 antibodies are known to play a role in graft rejection. We present a case of alloimmunization to HLA-DPB1 antibodies following granulocyte transfusion and with decreasing MFI after discontinuation of transfusion. Post transplant DPB1 antibody levels and clinical outcome will be presented at the meeting.

122-P

Aim Current practice for identifying a suitable 4/6 matched CBU is antigen level matching at HLA-A and B and allele level matching at HLA-DRB1, while matching at HLA-C is not typically considered. Recent studies have suggested that matching at HLA-C may improve transplant related mortality (Eapen et al., 2011). Determining the availability of a suitably matched (x/8) CBU is important for potentially improving transplant related outcomes for patients. Methods We examined a cohort of single CBU transplants from December to February 2012 to determine if a better matched (antigen level at HLA-A, B, C and allele level at HLA-DRB1, x/8) unit was available in the Be The Match Registry®. The better matched unit had to retain the traditional 4/6 or better match category (MC) and a minimum cell dose of 2.5 × 107 TNC/kg or no lower than the selected unit TNC. CBU without HLA-C typing were considered matched if algorithm predictions were ⩾50%. Results 119 single CBU transplant cases were assessed. 79 (66%) of these cases selected the best matched unit. 40 (34%) had a better matched x/8 unit available, where 36 (90%) improved one MC (x/8) and four cases (10%) improved two MCs. The cell dose of the better matched unit was lower in 57.5% of cases, with the median change in cell dose of −9.3%. Conclusions The benefit of HLA-C matching for CBU transplant has not been fully elucidated. This analysis highlights that although current practice may not always consider HLA-C matching, often a similar or better matched unit is available including HLA-C. This study cannot account for the myriad of factors considered in CBU selection such as cell dose, mismatching at a particular locus (e.g. HLA-DRB1), previous confirmatory typing, patient antibodies, CBU protocols or physician preference. These data indicate that when solely considering HLA matching in CBU selection, units that also matched at the C locus while maintaining similar unit cell dose were available.

Measuring Ambiguity in HLA Typing Methods

In hematopoietic stem cell transplantation, donor selection is based primarily on matching donor and patient HLA genes. These genes are highly polymorphic and their typing can result in exact allele assignment at each gene (the resolution at which patients and donors are matched), but it can also result in a set of ambiguous assignments, depending on the typing methodology used. To facilitate rapid identification of matched donors, registries employ statistical algorithms to infer HLA alleles from ambiguous genotypes. Linkage disequilibrium information encapsulated in haplotype frequencies is used to facilitate prediction of the most likely haplotype assignment. An HLA typing with less ambiguity produces fewer high-probability haplotypes and a more reliable prediction. We estimated ambiguity for several HLA typing methods across four continental populations using an information theory-based measure, Shannon's entropy. We used allele and haplotype frequencies to calculate entropy for different sets of 1,000 subjects with simulated HLA typing. Using allele frequencies we calculated an average entropy in Caucasians of 1.65 for serology, 1.06 for allele family level, 0.49 for a 2002-era SSO kit, and 0.076 for single-pass SBT. When using haplotype frequencies in entropy calculations, we found average entropies of 0.72 for serology, 0.73 for allele family level, 0.05 for SSO, and 0.002 for single-pass SBT. Application of haplotype frequencies further reduces HLA typing ambiguity. We also estimated expected confirmatory typing mismatch rates for simulated subjects. In a hypothetical registry with all donors typed using the same method, the entropy values based on haplotype frequencies correspond to confirmatory typing mismatch rates of 1.31% for SSO versus only 0.08% for SBT. Intermediate-resolution single-pass SBT contains the least ambiguity of the methods we evaluated and therefore the most certainty in allele prediction. The presented measure objectively evaluates HLA typing methods and can help define acceptable HLA typing for donor recruitment.

Duration of Unrelated Donor Search for Hematopoietic Stem Cell Transplantation

Abstract 2046▪▪This icon denotes a clinically relevant abstract Objective:Donor search is a major challenge once the indication for allogeneic hematopoietic stem cell transplantation (HCT) has been established. For those patients who lack an HLA-identical sibling, unrelated donor search (UDS) needs to be initiated. Only if UDS is not successful, alternative stem cell sources like cord blood (CB) or alternative concepts like haploidentical HCT have to be considered. Since UDS is a time consuming process it is important to assess the probability to find a matched unrelated donor (UD) during prolonged UDS. Yet, very few data on the required times and the success rates of unrelated donor searches are available. Furthermore, information on the probability to find a donor within short time is crucial in the context of early allogeneic transplantation including HCT in aplasia after induction chemotherapy in AML or for the interpretation and planning of clinical trials employing donor versus no donor comparisons. Therefore, we have retrospectively analyzed the duration of UDS for a large cohort of patients. Methods:All unrelated donor searches of one large search unit, which were initiated between January 2004 and July 2010 were analyzed retrospectively. Throughout that period blood from all donor-recipient pairs has been typed for HLA-A, -B, -C, -DRB1 and –DQB1 at the allele level (4 digits). A full match was defined by HLA-identity for HLA-A, -B, -C and DRB1 (8/8), while a partial match was defined as a single mismatch in the HLA-A, -B, -C or DRB1 locus without additional DQB1 mismatch (9/10). The duration of UDS was calculated from the date of data entry to the date of receipt of confirmatory typing (CT) of the first 8/8 HLA-matched donor or the first 9/10 HLA-matched donor. When given as reasons for the termination of the search, poor clinical condition or death of a patient were considered as competing events. Unsuccessful searches were censored at the day of the receipt of the last typing request or when UDS was stopped for other reasons. Cumulative incidences of successful UDS were calculated with competing event statistics. Approximative 95% confidence intervals are provided for point estimates. Results:In the respective time period 852 UDS comprising 8477 donor requests were performed. In the Caucasian population, an HLA compatible UD (8/8) has been found for 29% (95% CI, 26% to 32%) of the patients within two weeks, 54% (95% CI, 51% to 58%) of patients within 4 weeks, and 61% (95% CI, 57% to 64%) of patients within 6 weeks (Figure 1). In high risk diseases and high urgent indications for allogeneic transplantation one antigen or allele mismatch may be acceptable. Therefore, we calculated the probabilities to find an HLA matched UD (8/8) or a partially matched UD (9/10) within defined times. The resulting probabilities of finding an acceptable donor who met these criteria for Caucasian patients were 35% (95% CI, 32% to 39%), 66% (95% CI, 63% to 70%) and 75% (95% CI, 72% to 78%) at two, four, and six weeks respectively. After six weeks of UDS the probability of finding a donor declined. Between the six week landmark and the 12 week landmark 8/8 HLA matched unrelated donors have been identified only for additional 4% of the patients. Extending UDS up to one year resulted in additional 3% HLA matched donors (8/8). The incidence of the competing events (death or deteriorated condition of the patient) was 10% (95% CI, 6.6% to 13.8%) at one year when the aim was to find an 8/8 HLA matched UD. Eight UDS (1%) were started for non-Caucasian patients. Only for one of the eight non-Caucasian patients an HLA-compatible donor (8/8) was found. Conclusions:For Caucasian patients HLA compatible UDs (8/8) can be identified in our population for 61% of the patients within six weeks. If donors with one mismatch (9/10) are accepted the probability increases to 75% at that time. Beyond that landmark, the probability of finding donors for the remaining patients declines steeply and alternatives like haploidentical or cord blood transplantation should be considered. Since an HLA matched donor can be identified for 29% of the patients within two weeks, UDS is a valid option even for very high urgent searches like in the context of allogeneic HCT in aplasia. Unrelated donor searches for patients with non-Caucasian ancestry are much less successful. Further analyses are planned in order to define the probability to find a donor related to the haplotype frequency of the patient. [Display omitted] Disclosures:Off Label Use: pentostatin is not licensed for use in acute GvHD.

Does high-resolution donor typing of HLA-C or other loci upon registration confer advantages to patients?

Our study compared all requests for confirmatory typing (CT requests) received in our center between May 2007 and December 2009 (n = 134) for donors issued from 3 groups defined by different human leukocyte antigen (HLA) loci typed at different levels of resolution. We observed a significant advantage for volunteers when HLA-C 2-digit typing was available or with HLA-A, -B, -C, -DRB1 4-digit typing compared with generic HLA-A, -B, -DRB1, -DQB1 DNA typing: increased percentage of CT requests (p < 0.001), increased rate of donor selection for donation (p < 0.001), and decreased time frame for donor search (p = 0.025). The time frame for a successful search (donation) is similar among the 3 groups, indicating that the search might be concluded more rapidly when the pathology is clinically active or when the patient is at a high risk of relapse (76% of our cases) or for pediatric patients (24% of our cases), regardless of HLA typing resolution. Improvement of HLA typing for volunteers could be a great advantage for first selection in the absence of emergency or high-risk disease. Knowledge of HLA-C should be used to prioritize the selection of donors for further testing and could allow a better donor selection process, reducing search duration and increasing efficiency. In most cases, 2-digit typing for HLA-C associated with specific tools to estimate the probability of finding a matched donor could be sufficient.

Common and well-documented (CWD) alleles of human leukocyte antigen-A, -B, -C, -DRB1, and -DQB1 loci for the Chinese Han population do not quite correlate with the ASHI CWD alleles

Human leukocyte antigen (HLA), which is extremely polymorphic, plays an important role in stem cell transplantation. The Chinese Han comprise a large population of approximately 1.3 billion with diverse HLA alleles that need to be characterized. Data from 3,296 independent, unrelated Chinese Han individuals (1,457 recipients and 1,839 donors) were provided by the China Marrow Donor Program (CMDP) for donor-recipient confirmatory typing. Sequence-based typing, sequence-specific oligonucleotide probe (SSOP)/High Definition-SSOP, and sequence-specific primer methods were used to obtain 4-digit alleles. A total of 49, 86, 50, 63, and 24 HLA-A, -B, -C, -DRB1, and -DQB1 alleles were observed. Following American Society for Histocompatibility and Immunogenetics (ASHI) common and well-documented (CWD) criteria, CWD alleles for Chinese Han in our laboratory test and other laboratory reports do not quite correlate with the ASHI CWD alleles: A*11:53, A*02:34, A*02:53N, B*27:24, B*46:02, B*55:12, C*01:06, C*03:17, C*06:06, C*07:66, C*07:67, C*08:22, DRB1*12:10, DQB1*03:13, and DQB1*06:05 are CWD, but are not included in the ASHI CWD list. A series of alleles are well-documented alleles and are listed in the ASHI CWD list. Conversely, A*26:03, B*51:03, C*12:05, C*15:09, C*15:11, C*17:03, DRB1*11:07, DRB1*11:11, DRB1*13:05, DRB1*13:13, DRB1*14:06, DRB1*14:12, DRB1*14:22, DRB1*14:25, and DQB1*06:11 are rare alleles, but are included in the ASHI CWD list. HLA ethnic diversity is the main reason for the differences in HLA alleles worldwide. The ASHI HLA CWD alleles help reduce the workload and expenses in high-resolution donor registries and the HLA allele frequencies provide a basis from which to predict the chances of finding HLA matching donors. Our data will be meaningful for the CMDP, for other worldwide donor registries, and for an updated ASHI CWD allele list.

How do I approach ABO‐incompatible hematopoietic progenitor cell transplantation? (CME)

H uman leukocyte antigen (HLA) matching is critically important for successful hematopoietic progenitor cell (HPC) transplantation because the rates of engraftment and transplant outcomes are strongly influenced by the degree of match. This is largely because HLA antigens are expressed on immature pluripotent stem cells. In contrast, ABO incompatibility is not a barrier to successful HPC transplantation because ABO blood group antigens are not expressed on pluripotent or early committed HPCs. HLA and ABO antigens are encoded by different genes; thus it is common that potential donors who are fully HLA matched have an ABO incompatibility. Approximately 40 to 50% of HPC transplants are ABO incompatible and are now performed using bone marrow (HPC-M), apheresis-derived peripheral blood progenitor cells (HPCA), and umbilical cord blood (HPC-C) as HPC sources. Three types of ABO incompatibility exist: major, minor, and bidirectional. A total of 20 to 25% of HPC transplants have a major ABO incompatibility, 20 to 25% a minor ABO incompatibility, and up to 5% a bidirectional incompatibility. Each presents a unique set of potential adverse consequences, including early acute hemolysis that can be ameliorated through HPC product processing to remove incompatible red blood cells (RBCs) or plasma (Table 1). In these cases, the recipient must be closely monitored both at the time of the infusion and in the posttransplant period because serious events can occur many days later. Accreditation agencies such as AABB and the Foundation for the Accreditation of Cellular Therapy (FACT) require that all donors be tested for ABO group and D type before the collection of HPC-A or HPC-M and that HPC-C be typed after product processing before cryopreservation. The testing and its required documentation in the medical record need to be done in accordance with institutional standard operating procedures. Current FACT standards also require that “for allogeneic cellular therapy products containing RBCs at the time of administration, a test for ABO group and Rh type shall be performed on the first product collected, or on blood obtained from the donor at the time of the first collection.” At our institution, the donor ABO group and D type is tested as part the donor evaluation process. Additional donor ABO group and D testing is performed on each collection day from blood samples obtained at time of collection. If donor blood sample is not available for a particular collection, confirmatory typing is performed on a product sample on the day of product processing. For all fresh HPC products received from outside facilities, confirmatory ABO group and D testing is performed before further product processing. HPC-C units are received in the frozen state and are not subject to repeat testing.

Discrepancies between Human Leukocyte Antigen Registry Typing and Confirmatory Typing Results of Unrelated Hematopoietic Stem Cell Donors

In unrelated hematopoietic stem cell transplantation, the accuracy of HLA registry typing (RT) of donors is important for timely search and coordination of HLA-matched donors. We analyzed discrepancies between HLA RT and confirmatory typing (CT) results of stem cell donors in Korean and foreign registries. We analyzed the HLA typing results of 834 donors for whom CT was performed at Seoul National University Hospital between April 1997 and March 2010. For CT, DNA typing was used in majority of the cases and HLA-A and HLA-B serological typing was used in some early cases. The discrepancies between the typing results were analyzed at the serological/generic level. The overall discrepancy rate (RT error rate) was 3.2%, and the rate was similar in the Korean and foreign registries. The discrepancy rates in the Korean and foreign registries were more than 10% in the 1997-2001 searches, but decreased to less than 3% in the 2002-2010 searches. Analysis of 19 cases of RT errors in the Korean registry revealed 3 cases of sample switchover errors and 16 cases of typing errors in one of the HLA-A, HLA-B, or HLA-DR loci. The RT error rate in Japan Marrow Donor Program was lower than those in other foreign registries. The error rate of HLA RT results of unrelated stem cell donors in the Korean registry was similar to those in the foreign registries, and has decreased in the recent searches following the change in the typing method from serological to DNA typing.

Activity of the National Polish Bone Marrow Donor Registry—Analysis of the Matching Process Successfully Completed with Hematopoietic Stem Cell Transplantation

The activity of the National Polish Bone Marrow Donor Registry includes: (1) recruitment and typing of volunteer donors for the registry; (2) primary and confirmatory typing of patients and their families for sibling and family alternative donor transplantations; and (3) donor-recipient matching. There has been an improvement regarding the number of procedures (184 vs 201) and the successfully completed matches (102 vs 145) in 2006–2007 vs 2008–2009, respectively. The success rate of conclusive matching depended on the number of donors in a given registry and the incidence of rare HLA B − C or DR − DQ associations and specificities. In 2008–2009, Polish, German and all European registries, finally matched 129 (out of all 145) donors including 3, 18, 4 donors for patients with and 30, 68, 6 donors for patients without difficult HLA specificities and associations, respectively. Among the HLA-difficult patient group those lacking a donor in Europe and Israel were successfully matched in the USA (10/11 patients); the only one other subject was matched with a donor from Australia.

27-OR: Leukemia Specific Loss of Heterozygosity at MHC Locus Detected at Confirmatory Typing of HSCT Recipient with CLL

27-OR: Leukemia Specific Loss of Heterozygosity at MHC Locus Detected at Confirmatory Typing of HSCT Recipient with CLL

32-OR: Detecting Chimerism from HLA Confirmatory Typing of Cord Blood Unit Samples

32-OR: Detecting Chimerism from HLA Confirmatory Typing of Cord Blood Unit Samples

Creation of a Segment-Based Aldehyde Dehydrogenase Assay as a Biomarker for Cord Blood Potency.

Abstract 3217Poster Board III-154Banked, unrelated umbilical cord blood units (CBU) provide an option for transplantation in patients where matched adult donors are unavailable. Current methods for selecting a suitable CBU for transplantation includes examining the human leukocyte antigen (HLA) matching, total nucleated cell (TNC) count, and viable CD34 counts on the unit prior to cryopreservation. However, up to 20% of patients experience primary graft failure following UCB transplantation (UCBT), suggesting that a rapid assay to assess CBU potency on the thawed unit prior to the release from the cord blood bank is needed. Preliminary clinical studies demonstrated that neutrophil and platelet engraftment as well as overall survival can be predicted with post-thaw colony forming units (CFU). The usefulness of post-thaw CFU as a potency assay is limited both by time (14 days) and variability. However, CFUs in fresh cord blood are highly correlated with the number of cells expressing the enzyme aldehyde dehydrogenase (ALDHbr cells), which is enriched in hematopoietic stem cells. Therefore, an ALDHbr assay for segments attached to CBUs was developed to measure potency for UCBT. Retrospective studies of transplanted CBUs indicated that engraftment success could be accurately predicted by the number of ALDHbr cells infused per kilogram. In order to facilitate prospective studies, the segment assay was modified to allow HLA matching, CFUs and the ALDHbr assay to be done from one CBU segment. Blood was withdrawn from the segment with a needle and 3 drops of blood (∼30 ml) were spotted onto a FTA card for the HLA confirmatory typing. The remainder of the blood was washed with 5% dextran/albumin (DA) to remove the DMSO. Following the removal of 100,000 white blood cells for CFUs, the segments were assessed using flow cytometry for ALDHbr [Aldecount®], CD45 (Phycoerythrin (PE)-labeled), 7-amino-actinomycin D (7-AAD, viability), Glycophorin A (PE-Cy5-labeled) and CD34 (Allophycocyanin (APC)-labeled). Total nucleated cell (TNC) count was obtained [Sysmex] and the total number of each cell population in the segment was calculated. This method reproducibly assays ALDHbr cells (∼85% CD34/45 positive) from a single segment in less than a day while allowing testing of routine parameters of the CBU (HLA matching, TNC, viable CD34) currently used to select units for transplantation. Furthermore, the assay produces reliable results when performed by people from different laboratories (Duke vs. MD Anderson) and on different cytometers (Accuri C6 vs. BD Bioscience Facscalibur). Therefore, this method will be suitable for further prospective studies on the predictive value of the ALDHbr assay to measure cord blood potency as an indicator of its ability to confer neutrophil and platelet engraftment and overall survival after UCBT. DisclosuresGentry:Aldagen, Inc: Employment. Balber:Aldagen, Inc: Employment, Equity Ownership. Kurtzberg:Aldagen, Inc: Research Funding.

168-P: Discrepancy of HLA typing results of unrelated stem cell donors between registry typing and confirmatory typing

168-P: Discrepancy of HLA typing results of unrelated stem cell donors between registry typing and confirmatory typing