Engraftment kinetics after transplantation of double unit cord blood grafts combined with haplo-identical CD34+ cells without antithymocyte globulin.

Double unit cord blood (dCB) transplantation (dCBT) is associated with high engraftment rates but delayed myeloid recovery. We investigated adding haplo-identical CD34+ cells to dCB grafts to facilitate early haplo-identical donor-derived neutrophil recovery (optimal bridging) prior to CB engraftment. Seventy-eight adults underwent myeloablation with cyclosporine-A/mycophenolate mofetil immunoprophylaxis (no antithymocyte globulin, ATG). CB units (median CD34+ dose 1.1 × 105/kg/unit) had a median 5/8 unit-recipient human leukocyte antigen (HLA)-match. Haplo-identical grafts had a median CD34+ dose of 5.2 × 106/kg. Of 77 evaluable patients, 75 had sustained CB engraftment that was mediated by a dominant unit and heralded by dominant unit-derived T cells. Optimal haplo-identical donor-derived myeloid bridging was observed in 34/77 (44%) patients (median recovery 12 days). Other engrafting patients had transient bridging with second nadir preceding CB engraftment (20/77 (26%), median first recovery 12 and second 26.5 days) or no bridge (21/77 (27%), median recovery 25 days). The 2 (3%) remaining patients had graft failure. Higher haplo-CD34+ dose and better dominant unit-haplo-CD34+ HLA-match significantly improved the likelihood of optimal bridging. Optimally bridged patients were discharged earlier (median 28 versus 36 days). ATG-free haplo-dCBT can speed neutrophil recovery but successful bridging is not guaranteed due to rapid haplo-identical graft rejection.

Quality control project of NGS HLA genotyping for the 17th International HLA and Immunogenetics Workshop.

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.

HLA-genotyping of clinical specimens using Ion Torrent-based NGS.

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.

Sustained donor engraftment in recipients of double-unit cord blood transplantation is possible despite donor-specific human leukoctye antigen antibodies.

The impact of human leukocyte antigen (HLA) donor-specific antibodies (DSA) on cord blood (CB) engraftment is controversial. We evaluated the influence of pre-existing HLA-antibodies (HLA-Abs) on engraftment in 82 double-unit CB recipients (median age, 48 years) who underwent transplantation for hematologic malignancies. Of 28 patients (34%) with HLA-Abs, 12 had DSA (median mean fluorescence intensity 5255; range, 1057 to 9453). DSA patients had acute leukemia (n = 11) or myelodysplasia (n = 1) and all received either high-dose or reduced-intensity (but myeloablative) conditioning. After myeloablative CB transplantation (CBT) (n = 67), sustained donor engraftment was observed in 95% without HLA-Abs (median, 23 days), 100% with nonspecific HLA-Abs (median, 23 days), and 92% with DSA (median, 31 days, P = .48). Of 6 patients with HLA-Abs to 1 unit, 3 engrafted with that unit and 3 with the other. Of 6 patients with HLA-Abs against both units, 1 had graft failure despite being 100% donor, and 5 engrafted with 1 unit. Successful donor engraftment is possible in patients with DSA after myeloablative double-unit CBT. Our data suggest potential deleterious effects of DSA can be abrogated in patients with hematologic malignancies.

A comparison of two robotic platforms to screen plateletpheresis donors for HLA antibodies as part of a transfusion-related acute lung injury mitigation strategy.

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.