T cell receptor (TCR) T cell therapies target tumor antigens in a human leukocyte antigen (HLA)-restricted manner. Biomarker-defined therapies require validation of assays suitable for determination of patient eligibility. For clinical trials evaluating TCR T cell therapies targeting melanoma-associated antigen A4 (MAGE-A4), screening in studies NCT02636855 and NCT04044768 assesses patient eligibility based on: (1) high-resolution HLA typing and (2) tumor MAGE-A4 testing via an immunohistochemical assay in HLA-eligible patients. The HLA/MAGE-A4 assays validation, biomarker data, and their relationship to covariates (demographics, cancer type, histopathology, tissue location) are reported here. HLA-A∗02 eligibility was 44.8% (2,959/6,606) in patients from 43 sites across North America and Europe. While HLA-A∗02:01 was the most frequent HLA-A∗02 allele, others (A∗02:02, A∗02:03, A∗02:06) considerably increased HLA eligibility in Hispanic, Black, and Asian populations. Overall, MAGE-A4 prevalence based on clinical trial enrollment was 26% (447/1,750) across 10 solid tumor types, and was highest in synovial sarcoma (70%) and lowest in gastric cancer (9%). The covariates were generally not associated with MAGE-A4 expression, except for patient age in ovarian cancer and histology in non-small cell lung cancer. This report shows the eligibility rate from biomarker screening for TCR T cell therapies and provides epidemiological data for future clinical development of MAGE-A4-targeted therapies.

The impact of donor-host chimerism in post-hematopoietic stem cell transplantation (HSCT) outcomes is poorly understood. We were interested in studying whether pre-HSCT variables influenced lineage-specific donor-host chimerism and how lineage-specific chimerism impacts post-HSCT outcomes. Our main objective was to study pre-HSCT variables as predictors of lineage-specific donor-host chimerism patterns and to better characterize the relationship between post-HSCT lineage-specific chimerism and adverse outcomes, including graft failure and disease relapse. We conducted a retrospective data analysis of all patients who underwent allogeneic HSCT at the Pediatric Transplantation and Cellular Therapy service at Memorial Sloan Kettering Cancer Center between January 2010 and June 2015 and had at least 2 measurements of split-lineage chimerism. The trend of lineage-specific donor-host chimerism post-HSCT and the impact of age, disease, graft type, and pretransplantation conditioning regimen on chimerism at 3 months and 12 months post-HSCT were studied. The Wilcoxon signed-rank test, Mann-Whitney-Wilcoxon test, and Cox proportional hazard models were used for statistical analyses. A total of 137 patients were included (median age, 11.3 years). Most patients had a hematologic malignancy (n = 95), and fewer had a nonmalignant disorder (n = 27) or primary immune deficiency (n = 15). Myeloablative conditioning regimens (n = 126) followed by T cell-depleted (TCD) peripheral blood stem cell or bone marrow grafts (n = 101) were most commonly used. Mixed chimerism (MC) of total peripheral blood leukocytes (PBLs) did not predict loss of donor chimerism in all lineages and when stable was not associated with graft failure or rejection in this analyses. Split chimerism with complete donor chimerism (CC) of myeloid, B, and natural killer cells, but not T cells, occurred early post-HSCT, but full donor T cell chimerism was achieved at 12 months post-HSCT by most patients. MC within the T cell lineage was the major contributor to PBL MC, with lower median donor T cell chimerism at 3 months than at 12 months (91%) post-HSCT (51% versus 91%; P < .0001). Predictors of MC at 3 and 12 months were (1) age <3 years (P = .01 for PBLs and P = .003 for myeloid lineage); (2) nonmalignant disorder (P = .007 for PBLs); and (3) the use of reduced-intensity conditioning regimens. TCD grafts produced lower donor T cell chimerism at 3 months post-HSCT compared with unmodified grafts (P < .0001), where T cell lineage CC was achieved early post-HSCT. The donor T cell chimerism was similar at 12 months in the 2 types of grafts. Umbilical cord blood grafts had CC in all lineages at all time points post-HSCT. Loss of donor B cell chimerism was associated with increased risk of relapse in hematologic malignancies (hazard ratio, 1.33; P = .05). Age, underlying disease, conditioning regimen, and graft manipulation can impact post-HSCT donor-host chimerism and be predictors for early MC. MC in total PBLs and T cells was not related to graft failure or disease relapse. Whole-blood PBL chimerism analysis is not sufficient to assess the significance of post-HSCT donor-host status; rather, lineage-specific chimerism, particularly formyeloid, T, and B cells, should be analyzed to guide interventions and inform outcomes. (C) 2021 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Single antigen bead (SAB) assays are used to identify human leukocyte antigen (HLA) antibodies in patients with platelet refractoriness due to HLA Class I alloimmunization. Some laboratories use serum pretreatment regimens to eliminate interference from immunoglobulin M antibodies and complement. These modifications may contribute to interlaboratory variability, which is a recognized problem with the SAB assay.

BackgroundIsolated mixed T-cell chimerism in otherwise fully engrafted recipients of TCDHCT has been associated, in some series, with risk of graft rejection or relapses and an indication for DLI. Thereto, we have analyzed the impact of donor chimerism on outcomes in a prospective clinical trial of conditioning regimens used with CD34+ selected TCDHCT.MethodsThe trial (NCT0119066) compares A) HFTBI, Thiotepa and cyclophosphamide with B) Busulfex, melphalan and fludarabine or C) Clofarabine, melphalan and thiotepa as conditioning for CD34+ TCDHCT used to treat high risk hematologic malignancies. As part of the trial, we sequentially assessed lineage-specific donor chimerism in the marrow and the myeloid cells and T-cells in blood by analysis of donor and host-specific STRs in cell fractions isolated by immunotype. In the landmark analysis, quartiles of donor chimerism were compared for T-cell levels, and clinical outcomes.Results283 consecutive patients, transplanted between 5/13/2010 and 12/31/2016 (106 in Arm A, 144 in Arm B, 33 in Arm C). All engrafted by day 10-11. While 3 pts in Arm B and 1 in C had late graft failure, all others from 3 mos on had full donor chimerism in marrow and myeloid blood cells. However, at 3 mos median donor CD3+ T-cell chimerism (MDTC), was mixed in each arm but significantly lower in Arm B (10%) vs A (50%) and C (63%) (p<0.001). While MDTC for Arms A and C rose to 80% and 85% by 6 mos and ≥95% by 1yr, but in Arm B was 25% at 6 mos, 55% at 1 yr and 65% at 2 yrs.To assess the impact of this sustained mixed chimerism, we did a landmark analysis at 3 months, comparing subsequent T-cell recovery and clinical outcomes for pts in the first (lowest) and fourth (highest) quartile of T-cell chimerism in Arms A and B. As shown in Table 1, CD3+ and CD8+ T-cells in both arms were significantly higher in Q1, as were CD3+CD4+ cells in Arm B. Non-relapse mortality (NRM) at 2 yrs was also significantly lower for Q1 pts in both arms but relapse risks were not. In Arm B, but not A, 2yr OS and DFS were also significantly better in Q1 vs Q4.ConclusionsConditioning with Bu/Mel/Flu (Arm B) secured durable engraftment and hematopoietic reconstitution but a high proportion of pts have isolated and persistent mixed T-cell chimerism. However, surviving host T-cells can expand, persist and contribute significantly to earlier T-cell recovery and long-term reduction of NRM without increasing risk of graft rejection, relapse or need for DLI. Isolated mixed T-cell chimerism in otherwise fully engrafted recipients of TCDHCT has been associated, in some series, with risk of graft rejection or relapses and an indication for DLI. Thereto, we have analyzed the impact of donor chimerism on outcomes in a prospective clinical trial of conditioning regimens used with CD34+ selected TCDHCT. The trial (NCT0119066) compares A) HFTBI, Thiotepa and cyclophosphamide with B) Busulfex, melphalan and fludarabine or C) Clofarabine, melphalan and thiotepa as conditioning for CD34+ TCDHCT used to treat high risk hematologic malignancies. As part of the trial, we sequentially assessed lineage-specific donor chimerism in the marrow and the myeloid cells and T-cells in blood by analysis of donor and host-specific STRs in cell fractions isolated by immunotype. In the landmark analysis, quartiles of donor chimerism were compared for T-cell levels, and clinical outcomes. 283 consecutive patients, transplanted between 5/13/2010 and 12/31/2016 (106 in Arm A, 144 in Arm B, 33 in Arm C). All engrafted by day 10-11. While 3 pts in Arm B and 1 in C had late graft failure, all others from 3 mos on had full donor chimerism in marrow and myeloid blood cells. However, at 3 mos median donor CD3+ T-cell chimerism (MDTC), was mixed in each arm but significantly lower in Arm B (10%) vs A (50%) and C (63%) (p<0.001). While MDTC for Arms A and C rose to 80% and 85% by 6 mos and ≥95% by 1yr, but in Arm B was 25% at 6 mos, 55% at 1 yr and 65% at 2 yrs. To assess the impact of this sustained mixed chimerism, we did a landmark analysis at 3 months, comparing subsequent T-cell recovery and clinical outcomes for pts in the first (lowest) and fourth (highest) quartile of T-cell chimerism in Arms A and B. As shown in Table 1, CD3+ and CD8+ T-cells in both arms were significantly higher in Q1, as were CD3+CD4+ cells in Arm B. Non-relapse mortality (NRM) at 2 yrs was also significantly lower for Q1 pts in both arms but relapse risks were not. In Arm B, but not A, 2yr OS and DFS were also significantly better in Q1 vs Q4. Conditioning with Bu/Mel/Flu (Arm B) secured durable engraftment and hematopoietic reconstitution but a high proportion of pts have isolated and persistent mixed T-cell chimerism. However, surviving host T-cells can expand, persist and contribute significantly to earlier T-cell recovery and long-term reduction of NRM without increasing risk of graft rejection, relapse or need for DLI.

BACKGROUND Adoptive transfer of donor-derived EBV-specific T-cells (EBV-CTLs) can eradicate EBV associated lymphomas post hematopoietic cell (HCT) or solid organ (SOT) transplants but is not available for most patients. METHODS We developed a 3rd-party, allogeneic, off-the-shelf bank of 330 GMP grade EBV-CTL lines from specifically consented healthy HCT donors. We treated 46 recipients of HCT (N=33) or SOT (N=13) with established EBV associated lymphomas, who failed rituximab therapy, with 3rd-party EBV-CTLs. Treatment cycles consisted of 3 weekly infusions of EBV-CTLs and 3 weeks of observation. RESULTS The EBV-CTLs did not induce significant toxicities or graft injury. One patient developed grade I skin GVHD requiring topical therapy. Complete and sustained partial remissions were achieved in 68% of HCT recipients and 54% of SOT recipients. For patients who achieved CR/PR or stable disease after cycle 1, overall survival was 88.9% and 81.8% respectively at 1 year. Although only 1/11 patients (9.1%) with progression of disease (POD) after cycle 1 who received additional EBV-CTLs from the same donor survived, 3 of 5 with POD subsequently treated with EBV-CTLs from a different donor achieved CR or durable PR (60%) and survive > 1 year. Maximal responses were achieved after a median of 2 cycles. CONCLUSIONS Third party EBV-CTLs of defined HLA restriction provide safe, immediately accessible treatment for EBV PTLD. Secondary treatment with EBV-CTLs restricted by a different HLA allele (switch therapy) can also induce remissions if initial EBV-CTLs are ineffective. These results suggest a promising potential therapy for patients with rituximab refractory EBV-associated lymphoma post transplant.Phase II protocols (NCT01498484 and NCT00002663) were approved by the Institutional Review Board at Memorial Sloan Kettering Cancer Center, Food and Drug Administration and National Marrow Donor Program.This work was supported through NIH grants CA23766, NIH R21CA162002, Aubrey Fund, The Claire Tow Foundation, Major Family Foundation, Max Cure Foundation, Richard "Rick" J. EIsemann Pediatric Research Fund, Banbury Foundation, Edith Robertson Foundation, Larry Smead Foundation. In June 2015 Atara Biotherapeutics licensed the EBV-CTL bank and is developing this as ATA-129.

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.

The 17th International HLA and Immunogenetics Workshop (IHIW) organizers conducted a Pilot Study (PS) in which 13 laboratories (15 groups) participated to assess the performance of the various sequencing library preparation protocols, NGS platforms and software in use prior to the workshop. The organizers sent 50 cell lines to each of the 15 groups, scored the 15 independently generated sets of NGS HLA genotyping data, and generated "consensus" HLA genotypes for each of the 50 cell lines. Proficiency Testing (PT) was subsequently organized using four sets of 24 cell lines, selected from 48 of 50 PS cell lines, to validate the quality of NGS HLA typing data from the 34 participating IHIW laboratories. Completion of the PT program with a minimum score of 95% concordance at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci satisfied the requirements to submit NGS HLA typing data for the 17th IHIW projects. Together, these PS and PT efforts constituted the 17th IHIW Quality Control project. Overall PT concordance rates for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 were 98.1%, 97.0% and 98.1%, 99.0%, 98.6%, 98.8%, 97.6%, 96.0%, 99.1%, 90.0% and 91.7%, respectively. Across all loci, the majority of the discordance was due to allele dropout. The high cost of NGS HLA genotyping per experiment likely prevented the retyping of initially failed HLA loci. Despite the high HLA genotype concordance rates of the software, there remains room for improvement in the assembly of more accurate consensus DNA sequences by NGS HLA genotyping software.

The outcome of allogeneic HCT is dependent on several pre-transplant variables. We hypothesize that these pre-transplant factors may influence the donor/host chimerism post HCT, which in its turn may affect the outcome. We performed an analysis to study lineage specific D/H chimerism patterns of total blood leukocytes, myeloid cells/neutrophils, B-cells, NK-cells and T-cells.All consecutive patients who underwent an allogeneic HCT between 01/2010 to 06/2015 on the Pediatric BMT Service at MSKCC were reviewed. Chimerism analyses were performed by short tandem repeat (STR) polymorphism analysis at the American Red Cross Blood Services (Philadelphia, PA). Lineage specific donor chimerism post HCT was studied including donor chimerism trend, and factors with potential impact on chimerism including: age, disease, graft source, and T-cell depletion (TCD). Preliminary analyzes performed on this cohort included Wilcoxon Rank Test and cox proportional hazard analyses.140 patients with a median age of 11.5 years were included. They included 97 patients with hematologic malignancies, 27 patients with non-malignant hematologic disorders and 16 patients with immunologic disorders. Cytoreduction included TBI in 48 patients or chemotherapy only in 98 patients. 105 patients received T-cell depleted grafts, 28 patients unmodified marrow or peripheral blood grafts, and 7 patients unrelated cord blood grafts. No patients received donor leukocyte infusions (DLI).Observational studies revealed that full donor chimerism of myeloid cells, B-cells and NK-cells, but not T-cells occurred early post-transplant. In Wilcoxon Rank Test, there was no difference in the percentage of total donor leukocytes at 3 months vs 12 months post HSCT (n=30), while the median of donor T-cell chimerism was 51% at 3 months and 91% at 12 months post HSCT (p<0.0001, n=42). For most grafts, full donor chimerism of T-cells occurred early, while for T-cell depleted transplants, it took up to one year to complete. Cord blood grafts were associated with high T-cell donor chimerism throughout the post-transplant period.The studies of the impact of different factors on chimerism were performed, including age, disease, and type of graft. Loss of donor total leukocytes chimerism between 3 and 12 months post-transplant was significantly more pronounced for patients < 3 years of age (p=0.01) and for patients with nonmalignant disorders (p=0.007, n=30).This preliminary analysis of lineage specific chimerism post-transplant showed that donor T-cells may take one year to fully recover post-transplant, mostly following T-cell depleted grafts, without intervention. Cord blood grafts were associated with high donor chimerism throughout the post-transplant period. Lastly, factors associated with loss of donor chimerism post-transplant were younger age and non-malignant disorders. More in-depth analyses are being performed.

BACKGROUND:A simplified protocol for HLA-typing -by NGS, developed for use with the Illumina MiSeq, was performed by technologists with varying NGS experience to assess accuracy and reproducibility. METHODS:Technologists from six laboratories typed the same 16 samples at HLA-A, B, C, DRB1, and DQB1. The protocol includes long range PCR, library preparation, and paired-end 250bp sequencing. Two indexing strategies were employed: locus-specific indexing whereby each locus was tagged uniquely and sample-specific indexing whereby all 5 loci for a sample were pooled prior to library preparation. Sequence analysis was performed with two software packages, Target HLA (Omixon) and NGSengine (GenDx). RESULTS:The average number of sequence reads per library was 387,813; however, analysis was limited to 40,000 reads for locus-indexed libraries and 200,000 reads for sample-indexed libraries resulting in an average depth of coverage of 1444 reads per locus. Sufficient reads for genotype analysis were obtained for 98.4% of libraries. Genotype accuracy was >97% in pooled amplicons and >99% in individual amplicons by both software analysis. Inter-laboratory reproducibility was 99.7% and only cause of discordance was cross-contamination of a single amplicon. CONCLUSIONS:This NGS HLA-typing protocol is simple, reproducible, scalable, highly accurate and amenable to clinical testing.

To validate the BLDType Multiplex Typing Kit (ThermoFisher) for genotyping hematopoietic stem cell (HSC) registry donors for ABO, RHD and CCR5 using Fragment Analysis (FA). Ninety-one archived genomic DNA samples (gDNAs) that had prior serology and/or ABO, RHD or CCR5 genotyping were typed using BLDType which employs fragment analysis using ABI 3130XL and Genemapper software 5 to distinguish A1, A2, B, O1, O2 and O3 alleles, detects deletion of RHD and CCR5Δ32. Fifty were typed for ABO and CCR5 alleles by restriction fragment length polymorphism (RFLP) and sequence-specific primer (SSP) PCR assays respectively. Twelve were genotyped for CCR5 by SSP and standard tube or automated red blood cell agglutination methods were used for ABO and RhD typing. Concordance rates were calculated for each sample subset as follows: 79 specimens with ABO and RhD phenotyping and 62 specimens with ABO by RFLP. Concordance between RhD serology and FA was 100% (79/79). Concordance between ABO serology and FA was 96% (76/79). The concordance between ABO RFLP and FA was 97% (60/62). The concordance between CCR5 genotyping by FA and SSP methods was 100% (91/91). The discrepancies encountered in this study were limited to ABO and were due to lack of serologic subtyping to distinguish A1 from A2 (N = 3) and a limitation of the RFLP which does not detect O3 alleles (N = 2) or cannot resolve unusual banding patterns. The BLDType technique is user friendly and easy to implement. FA of 96 gDNAs can be completed in 8 h, and if automated and performed on a 3730XL, more than 1000 gDNAs can be tested per day. The validation data presented here demonstrates that BLDType is a highly sensitive and specific typing tool. Incorporating BLDType along with HLA typing for registry donors would expedite the selection processes for both HLA-matched and ABO-compatible donors simultaneously.

Using high resolution HLA typing, crossing over (CO) between HLA-A/B, B/DRB1, or DRB1/DPB1 loci can be identified among siblings from families with both parents or with large sibship. Locating the breaking points along the C I and II genomic regions can be estimated via SNP genotyping of the MHC region. We report the rare occurrence of two recombinant children in a single family and the application of SNP genotyping to pinpoint the CO in each. Blood specimens of 6 siblings from a HSCT family (F) were HLA-A/B/C/DRB1/DQB1/DPB1 typed by LABType (OLI), SBT (SeCore, Life Tech) and NGS (NXType on the Ion Torrent platform, OLI) methods. SNP genotyping for the six siblings was performed using the OMNIExpress-24 v1.1 to query over 713K markers throughout the genome. Within the MHC there were 3754 SNPs covering the region between HLA-A position 29913067 and DQB1 position 32628428 a distance of 2.7Mb. 100% concordant results were obtained from all three methods (Table 1). The 4 parental haplotypes segregated appropriately among siblings #3–6. This fact helps to determine sibling #1 and sibling #2 are paternal HLA-A & B and maternal HLA-B & DRB1 recombinants, respectively. The derivation of SNP haplotypes for the 6 siblings revealed the approximate breaking points where CO most likely have occurred for siblings 1 and 2. The actual breaking points were identified to have occurred within 700bp in two respective regions, one centromerically to HLA-A for sibling 1 and the other centromerically to HLA-B for sibling 2. Precise break points will be discussed when analysis has been completed.

We have evaluated and validated the NXType™ workflow (One Lambda, Inc.) and the accompanying TypeStream™ software on the Ion Torrent Next Generation Sequencing (NGS) platform using a comprehensive testing panel. The panel consisted of 285 genomic DNA (gDNA) samples derived from four major ethnic populations and contained 59 PT samples and 226 clinical specimens. The total number of alleles from the six loci interrogated by NGS was 3420. This validation panel provided a wide range of HLA sequence variations including many rare alleles, new variants and homozygous alleles. The NXType™ system (reagents and software) was able to correctly genotype the vast majority of these specimens. The concordance rate between SBT-derived genotypes and those generated by TypeStream™ auto-analysis ranged from 99.5% to 99.8% for the HLA-A, B, C, DRB1 and DQB1 loci, and was 98.9% for HLA-DPB1. A strategy for data review was developed that would allow correction of most of the few remaining typing errors. The entire NGS workflow from gDNA amplification to genotype assignment could be completed within 3 working days. Through this validation study, the limitations and shortcomings of the platform, specific assay system, and software algorithm were also revealed for further evaluation and improvement.

Abstract 960 Background: Pre-transplant human leukocyte antigen (HLA)-antibodies have an established role in failed engraftment after single-unit cord blood (CB) transplantation (CBT). Their role in double-unit CBT (DCBT) engraftment, however, is more controversial. Some transplant centers advocate pre-transplant screening of the recipient for HLA-antibodies, and avoidance of units with the corresponding antigens if antibodies are detected. Methods: We evaluated the influence of pre-transplant HLA-antibodies on sustained donor neutrophil engraftment and unit dominance in 82 recipients (median 48 years, range 2–69) of 4–6/6 HLA-A, B antigen, DRB1 allele matched double-unit CB grafts, and transplanted for hematologic malignancies at MSKCC from July 2008 to July 2012. HLA-antibodies were measured by the HLA Laboratory of the American Red Cross Blood Service using Single Antigen Luminex beads and analyzed by HLA Fusion software. The cut-off for positive results was a normalized mean fluorescence intensity value > 1000. Results: Overall, 28/82 (34%) patients were positive for HLA-antibodies [16 (19.5%) had antibodies without graft specificity, and 12 (14.5%) had antibodies with graft specificity]. These patients were more likely to be female with acute leukemia and cytomegalovirus seropositive. All patients with graft-specific antibodies received myeloablative conditioning; 5 had antibodies against class I HLA, 6 against class II, and one patient had antibodies to both Class I/II. Moreover, of these 12 patients, 6 had antibodies against one unit, and 6 had antibodies against both units. Neutrophil engraftment according to antibody presence is summarized in the Table. 64 of 66 (97%) evaluable myeloablative DCBT recipients (Table 1A) engrafted at a median of 24 days (range 12–40); one patient without antibodies and one with antibodies against both units had primary graft failure, both in the setting of early onset multi-organ failure. Both patients were 100% donor with one unit but did not recover counts. Of the 6 patients with antibodies to one unit, 3 engrafted with that unit and 3 with the opposite unit. Of the 6 patients with antibodies against both units, one had clinical graft failure as described above, and the 5 others had sustained donor engraftment (4 with one unit and one with both). In engrafting myeloablative recipients, the median time to neutrophil recovery was 8 days slower in patients with graft-specific antibodies, but the engrafting unit in these patients also had the lowest infused dose: median CD34+ cell dose/kg if no antibodies 0.83 × 105/kg, non-specific antibodies 1.09 × 105/kg, and antibodies specific to graft 0.65 × 105/kg. In non-myeloablative recipients, 15/16 (94%) engrafted (Table 1B). The single patient with graft rejection and autologous recovery had HLA-antibodies that were not graft specific. Conclusions: 11 of 12 double-unit CBT recipients with graft-specific antibodies engrafted successfully. While myeloablative recipients with graft-specific antibodies engrafted more slowly, this may be explained by a lower infused CD34+ cell dose of their engrafting units. Multivariate analysis of larger patient numbers will be required to further evaluate the effect of graft-specific antibodies on engraftment speed. At this time, however, the presence of graft-specific antibodies should not preclude DCBT, and whether their presence should influence graft selection is not clear. Furthermore, there is no suggestion that the presence of HLA-antibodies influences unit dominance after DCBT. Disclosures: Giralt: Celgene: Honoraria, Research Funding.

BACKGROUND: Efforts to minimize white blood cell alloantibodies, responsible for transfusion-related acute lung injury (TRALI) in components with high-volume single-donor plasma include consideration of plateletpheresis donor screening for human leukocyte antigen (HLA) antibodies. High-throughput screening platforms make this feasible for large blood centers. Which platform to use, donor subgroups to screen, characteristics of detected antibodies, and operational impact of deferring reactive donors are important questions.STUDY DESIGN AND METHODS: We screened 2462 plateletpheresis donor sera for HLA antibodies on automated instruments using HLA Class I and II enzyme-linked immunosorbent assays (ELISA) or a mixed Class I/II Luminex flow analyzer. Screen-reactive samples were further tested by manual Luminex single-antigen assay to determine antibody specificity, estimated corresponding antigen frequency, and signal strength.RESULTS: Alloexposed females had the highest reactivity rate on both platforms (21.0%), with much lower rates for nonexposed individuals or transfused males (1.4%-5.4%). Increasing parity and more recent pregnancy increased their likelihood of screen reactivity. Deferring screen-reactive parous females would result in at least a 4.8% plateletpheresis donor base decrement. Supplemental testing showed higher rates of nonspecific or natural antibodies in ELISA screen-reactive alloexposed females (2.5%) than Luminex (0%). Both assays were more likely to identify antibodies directed against a larger number of HLA antigens and/or of presumed higher titer in alloexposed donors.CONCLUSION: A strategy screening only parous female donors is reasonable. Both automated HLA antibody detection platforms are easy to use and preferentially identify alloexposed individuals with antibodies of presumed higher titer directed against more recipient HLA antigens.