Clinical impact of KIR haplotypes in 10/10 HLA-matched unrelated donor-recipient pairs undergoing allogeneic hematopoietic stem cell transplantation.

To evaluate the impact of killer immunoglobulin-like receptor (KIR) genotyping in allogeneic hematopoietic stem cell transplantation for myeloid disorders at our institution, retrospective KIR genotyping was performed on 77 patients and their 10/10 matched unrelated donors. In a multivariate model including donor age, HLA-DPB1 permissiveness, and presence of donor KIR B/x, an association with overall survival was observed (p = .047). Within the model, increasing donor age increased risk (RR 1.03 [1.00-1.06]/year, p = .046), while donor KIR and HLA-DPB1 permissiveness were not associated with risk (RR 0.51 [0.26-1.03] and RR 0.68 [0.34-1.36]). Grouping recipients by conditioning regimen or limiting the analysis to recipients of peripheral blood stem cells, no association between donor KIR and survival or relapse was identified. No significant associations were observed between overall survival, relapse, grade III-IV acute, or chronic graft versus host disease and presence of KIR B (B/x), quantity of donor KIR B haplotype motifs, or centromeric KIR type (all p > .05).

Prozone rates in the solid-phase platelet crossmatch assay and correlation with class I HLA antibody levels.

BACKGROUND: Solid-phase platelet crossmatch (PXM) testing is used to help manage patients with platelet transfusion-refractoriness. Recently, we published the first report of false-negative PXM results from prozone effect that was mitigated using sample dilution. This study aimed to describe the prevalence of PXM prozone effect and the levels of class I HLA antibodies (HLA-Abs) associated with positive PXM results and with false-negative PXM results from prozone effect. STUDY DESIGN AND METHODS: A cross-sectional study of patients undergoing PXM testing from July 2019 through December 2020 was performed. All PXM tests were run simultaneously using undiluted and 1:4 diluted patient plasma. Prozone effect was defined as a negative PXM result using undiluted patient plasma but a positive PXM result using 1:4 diluted patient plasma. RESULTS: Among 59 patients, 830 individual ABO-compatible PXM results yielded an overall positivity rate of 25.8% (214/830) and a false-negative rate from prozone effect of 4.7% (10/214). Among the 28 patients with class I HLA-Ab testing and no other anti-platelet antibodies, maximum HLA-Ab mean fluorescence intensity (MFI) was significantly associated with a positive PXM result (p < .0001; AUC approx. 0.9) and categorized into negative (<3700), indeterminate (3700-10300), and positive (>10300) maximum HLA-Ab MFI zones. Maximum HLA-Ab MFI, however, was not associated with prozone effect (p = .17; AUC approx. 0.6). DISCUSSION: While there is a strong predictive association between class I HLA-Ab levels and positive PXM results, PXM prozone effect is a common occurrence not associated with class I HLA-Ab levels, so additional testing with diluted samples should be considered.

Daratumumab interference in flow cytometric anti-granulocyte antibody testing can be overcome using non-human blocking antibodies.

BACKGROUND: Daratumumab (DARA), a human IgG1K monoclonal antibody targeting CD38, is used to treat refractory multiple myeloma patients. CD38 is expressed on many cell types (RBCs, granulocytes, lymphocytes, etc.), and thus, DARA can interfere with serological tests. Information regarding how DARA affects anti-granulocyte antibody (AGA) testing and optimal neutralization of DARA will help laboratories perform accurate testing. METHODS: Screening of AGA was performed by the granulocyte agglutination test (GAT) and the flow cytometric granulocyte immunofluorescence test (Flow-GIFT). Samples were tested from patients on DARA (n = 7), non-transfused blood donors (healthy controls, n = 7) and AGA reactive samples (positive controls, n = 5). Two neutralization experiments, CD38 removal with DTT and DARA epitope blockage with mouse anti-CD38, were evaluated. RESULTS: Positive reactivity of human IgG binding was observed in 5/7 DARA cases when tested by Flow-GIFT; however, all 7 cases had negative GAT agglutination results. Further studies by Flow-GIFT revealed DARA concentrations >0·63 µg/ml bound to granulocytes. DARA binding was negated by DTT though a reduced Flow-GIFT sensitivity was observed in positive control samples due to increased background detection of human IgG. Mouse anti-CD38 neutralized the detection of human IgG observed in DARA-treated patient serum without effecting controls. CONCLUSION: We established that DARA can interfere with AGA testing, leading to false positive Flow-GIFT results without causing GAT agglutination. DTT treatment increased background binding of secondary antibodies causing a decrease in Flow-GIFT sensitivity. In comparison, blockage of the DARA binding epitope using mouse anti-CD38 antibody was effective in neutralizing DARA interference while maintaining Flow-GIFT sensitivity.

False-negative solid-phase platelet crossmatch results due to prozone phenomenon.

Prozone is a known phenomenon affecting immunoassays causing falsely low or negative results when excess target is present in the test system. For assays used to evaluate immune-mediated platelet (PLT) transfusion refractoriness, prozone-like phenomenon has been described in solid-phase human leukocyte antigen (HLA) antibody testing and can be mitigated by diluting samples or pretreating samples with ethylenediaminetetraacetic acid (EDTA) or dithiothreitol. Prozone phenomenon has not yet been described in solid-phase red blood cell (RBC) adherence PLT crossmatch assays. CASE REPORT: A 40-year-old female with myeloid sarcoma and PLT transfusion refractoriness underwent repeated solid-phase PLT crossmatches; however, crossmatch-compatible PLTs units did not yield adequate PLT count responses. Class I HLA antibody testing with neat, diluted, and EDTA-pretreated serum demonstrated significant prozone-like effect and the presence of numerous high strength HLA antibodies. Based on this HLA antibody profile, HLA antigen-negative PLTs gave an adequate PLT count response. It was noted that the HLA types of her crossmatch-compatible PLTs were incompatible with her HLA antibody profile (eg, HLA-A2). With ABO-identical, HLA-A2-positive PLT units, a solid-phase PLT crossmatch was repeated using undiluted and diluted EDTA plasma. Undiluted EDTA plasma demonstrated negative or weakly positive PLT crossmatches while the diluted EDTA plasma demonstrated strongly positive PLT crossmatches. CONCLUSION: The prozone phenomenon can cause false-negative results in solid-phase RBC adherence PLT crossmatch assays, which can be mitigated with sample dilution. In immune-mediated PLT transfusion-refractory patients with high-strength HLA antibodies, sample dilution should be considered to correctly identify compatible PLT inventory.

Concordance between predicted HLA type using next generation sequencing data generated for non-HLA purposes and clinical HLA type.

We explored the feasibility of obtaining accurate HLA type using pre-existing NGS data not generated for HLA purposes. 83 exomes and 500 targeted NGS pharmacogenomic panels were analyzed using Omixon HLA Explore, OptiType, and/or HLA-Genotyper software. Results were compared against clinical HLA genotyping. 765 (94.2%) Omixon and 769 (94.7%) HLA-Genotyper of 812 germline allele calls across class I/II loci and 402 (99.5%) of 404 OptiType class I calls were concordant to the second field (i.e. HLA-A*02:01). An additional 19 (2.3%) Omixon, 39 (4.8%) HLA-Genotyper, and 2 (0.5%) OptiType allele calls were first field concordant (i.e. HLA-A*02). Using Omixon, four alleles (0.4%) were discordant and 24 (3.0%) failed to call, while 4 alleles (0.4%) were discordant using HLA-Genotyper. Tumor exomes were also evaluated and were 85.4%, 91.6%, and 100% concordant (Omixon and HLA-Genotyper with 96 alleles tested, and Optitype with 48 class I alleles, respectively). The 15 exomes and 500 pharmacogenomic panels were 100% concordant for each pharmacogenomic allele tested. This work has broad implications spanning future clinical care (pharmacogenomics, tumor response to immunotherapy, autoimmunity, etc.) and research applications.

Upregulation of HLA-Class I and II in Placentas Diagnosed with Villitis of Unknown Etiology.

The placenta utilizes many mechanisms to protect the haploidentical fetus from recognition by the maternal immune system. However, in cases of villitis of unknown etiology (VUE), maternal lymphocytes gain access into the placenta, causing significant health risks for the fetus. Evidence suggests that VUE is a rejection response between the mother and the haploidentical fetus. Therefore, we profiled human leukocyte antigen (HLA), an important predictor of transplant rejection, in VUE using placental tissue from ten patients with VUE and ten gestational age matched controls. Placentas were stained using novel multiplexed immunofluorescence (MxIF) to investigate morphology and HLA classes I and II. Gene expression was evaluated by microarray, and where available, tissue typing of mother/baby pairs was completed to determine HLA type. MxIF demonstrated strong CD8+ T cell infiltration and HLA class I staining both the distal and stem villi of VUE placentas. Compared to controls, VUE cases had significantly higher expression of HLA class II mRNA and pathway analysis demonstrated that 40% of the differentially expressed genes in VUE are related to tissue rejection. The data suggest that VUE resembles a rejection response between the mother and the fetus. It remains unknown what initiates immune recognition and why some mothers appear to be at higher risk for developing this condition than others. Understanding this etiology will be critical for developing effective interventions or prevention strategies during pregnancy.

HLA-B*45:22: a novel allele that differs from HLA-B*45:01:01 at several locations.

Discovery of a novel HLA-B*45:22 allele in a Middle Eastern heart and lung transplant candidate.

The association of de novo anti-HLA-DPB1 donor-specific antibody formation and primary graft failure after allogeneic hematopoietic cell transplantation.

Despite advances in HLA matching, graft failure remains a serious complication after allogeneic hematopoietic cell transplantation (HCT). Pre-formed anti-HLA donor-specific antibodies (DSAs) have been associated with graft failure, but the impact of newly developing (de novo) anti-HLA antibodies on HCT outcomes remains unknown. Here, we present the first reported case of graft failure associated with de novo anti-HLA-DPB1 DSA after a 10/10 matched unrelated donor HCT. Based on this observation, testing for DSA should be considered in case of unexplained pancytopenia after allogeneic transplantation, especially after reduced intensity conditioning.

Does matching for SNPs in the MHC gamma block in 10/10 HLA-matched unrelated donor-recipient pairs undergoing allogeneic stem cell transplant improve outcomes?

BACKGROUND: Matching at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci is important in donor selection for patients undergoing unrelated allogeneic hematopoietic stem cell transplantation (ASCT). Additional matching across the MHC gamma region may further improve outcomes. METHODS: The MHC gamma region was retrospectively genotyped in 66 adult recipients of ASCT and their 10/10 matched unrelated donors. A chart review was performed to determine whether MHC gamma matching impacted survival, relapse, or graft-versus-host disease. RESULTS: Of 66 donor-recipient pairs, 26(39.4%) were gamma-type matches, 34(51.5%) were mismatches, and 6(9.1%) were "indeterminate." Matching status was not associated with overall survival (p = 0.43), relapse (p = 0.21), acute GVHD (p = 0.43), severe aGVHD (p = 0.31), or chronic GVHD (p = 0.23) in univariate analyses, nor in multivariate analyses (p = 0.28, 0.13, 0.29, 0.16, and 0.67, respectively), with or without adjusting for HLA-DPB1 matching status. CONCLUSIONS: In our single institution study, gamma-type matching status was not associated with outcomes of adult ASCT recipients.

How do I manage the platelet transfusion-refractory patient?

BACKGROUND: Platelet transfusion-refractoriness is a challenging and expensive clinical scenario seen most often in patients with hematologic malignancies. Although the majority of platelet transfusion-refractory cases are due to nonimmune causes, a significant minority are caused by alloimmunization against Class I human leukocyte antigens (HLAs) or human platelet antigens (HPAs). Such platelet transfusion-refractory patients can be effectively managed with appropriate antigen-negative products. STUDY DESIGN AND METHODS: Our institution has developed a diagnostic and management algorithm for the platelet transfusion-refractory patient with an early focus on identifying those cases caused by immune-mediated factors. Using physical platelet cross-matches to initially classify platelet transfusion-refractory patients as immune-mediated or not, cross-match-compatible inventory is then provided to immune-mediated patients, whereas subsequent HLA (with or without HPA) testing is performed. RESULTS: Our blood donor program performs Class I HLA typing of all repeat platelet donors to facilitate the identification of antigen-negative platelet units (virtual cross-matching) as well as the recruitment of HLA-matched donors. The platelet transfusion-refractoriness algorithm realizes an initial net cost savings once two apheresis platelets are saved from use for each newly identified, immune-mediated platelet transfusion-refractory patient. CONCLUSION: An algorithm utilizing physical platelet cross-matches, Class I HLA and HPA antibody testing, and upfront Class I HLA typing of platelet donors leads to overall resource savings and improved clinical management for platelet transfusion-refractory patients.

Clinical outcomes of HLA-DPB1 mismatches in 10/10 HLA-matched unrelated donor-recipient pairs undergoing allogeneic stem cell transplant.

OBJECTIVE: HLA-DPB1 matching may impact allogeneic hematopoietic stem cell transplantation (ASCT) outcomes; however, this locus is not in linkage disequilibrium with the remainder of the HLA genes. After classifying HLA-DPB1 mismatches based on T-cell epitope, avoiding non-permissive mismatches may impact survival. We tested this hypothesis at a single academic institution. METHODS: Retrospective HLA-DPB1 genotyping was performed on 153 adult patients who underwent ASCT and unrelated donors matched for HLA-A, B, C, DRB1, and DQB1 loci (10/10). Using the ImMunoGeneTics/HLA T-cell epitope matching algorithm, mismatch status was classified as permissive or non-permissive. RESULTS: Of 153 donor-recipient pairs, 22 (14.4%) were HLA-DPB1 matches, 64 (42.8%) permissive mismatches, and 67 (43.8%) non-permissive mismatches. DPB1 mismatch increased risk of chronic graft-versus-host disease (cGVHD; RR 2.89 [1.19-9.53], P=.016) compared with DPB1-matched transplants, but there were no differences in overall mortality, risk of relapse, or acute GVHD (aGVHD). Combining matches and permissive mismatches and comparing to non-permissive mismatches, there was no significant difference in overall survival or relapse; however, patients receiving non-permissive mismatched transplants experienced greater risk of aGVHD overall and severe aGVHD (RR 1.66 [1.13-2.44], P=.010 and RR 1.97 [1.10-3.59], P=.024, respectively). CONCLUSION: In this single-center study, HLA-DPB1 matching influenced outcomes of patients undergoing ASCT for hematologic malignancy.