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Abstract Objective Cigarette smoking is associated with immune-mediated disorders. We explored the contribution of smoking to polymyositis (PM) and dermatomyositis (DM) phenotypes and attempted to determine whether cigarette smoking effects differ by race and genotype. Methods Associations of tobacco smoking with disease features, autoantibodies, HLA types, and race were evaluated using multiple logistic regressions in 465 patients. Results Caucasian ever-smokers (n = 140) were more likely to have PM (adjusted OR = 2.24, 95% CI: 1.41\x963.57), anti-synthetase (adjusted OR = 1.93, 95% CI: 1.12\x963.34) and anti-Jo-1 autoantibodies (adjusted OR = 1.94, 95% CI: 1.08\x963.46) and less likely to have anti-p155/140 autoantibodies (adjusted OR = 0.36, 95% CI: 0.14\x960.92). In Caucasians, ever-smokers had a greater interstitial lung disease (ILD) frequency than never-smokers, while in African-Americans this relationship was inverted, but neither trend reached statistical significance. Pack-years of cigarette smoking showed significant positive associations with PM (adjusted OR = 1.02, 95% CI: 1.002\x961.04) and ILD (adjusted OR = 1.02, 95% CI: 1.001\x961.03) and was inversely associated with anti-p155/140 autoantibodies (adjusted OR = 0.93, 95% CI: 0.87\x960.99) in Caucasians. Caucasian heavy smokers (=20 pack-years) were more likely to have PM (adjusted OR = 2.52, 95% CI: 1.25\x965.09), ILD (adjusted OR = 2.48, 95% CI: 1.23\x965.00) and anti-Jo-1 autoantibodies (adjusted OR = 2.65, 95% CI: 1.16\x966.08) than never-smokers. In Caucasians, compared to never-smokers without HLA-DRB1*03:01 allele, ever-smokers with HLA-DRB1*03:01 allele had the highest odds of PM, ILD, ASA, and anti-Jo-1 autoantibodies. Risks for those with only one of these two factors were intermediate. An inverse pattern was observed regarding anti-p155/140 autoantibodies. Conclusion Tobacco smoking was associated with clinical and autoantibody phenotypes in Caucasians. Our findings also suggest possible interactions among HLA-DRB1*03:01 and smoking on the risk of PM and ILD, as well as, anti-synthetase, anti-Jo-1, and anti-p155/140 autoantibodies in Caucasians. [ABSTRACT FROM AUTHOR]

Stem cell transplantation (SCT) from a healthy donor can be curative for patients with hematologic malignancies resistant to other treatments. Elimination of malignant cells through a graft-versus-leukemia (GVL) effect involves donor T and natural killer (NK) cells, but their relative contribution to this process is poorly defined. NK cell alloreactivity and GVL effects are controlled by the nature of the interaction of NK activation receptors and killer-immunoglobulin-like-receptors (KIR) with major histocompatibility locus class I antigens on the target cell. We performed KIR-genotyping of HLA-identical sibling donors in 246 T cell-depleted SCTs to identify genetic factors affecting transplant outcome (treatment-related mortality [TRM], leukemic relapse, and survival). Univariate and multivariate analysis of transplant-related risk factors and KIR genotyping was performed to identify independent variables predictive of outcome for different forms of leukemia. Further to confirming known predictive factors for TRM and survival (CD34 cell dose, patient age, disease stage), statistical analysis revealed that 3 donor B haplotype KIR genes, 2DL5A, 2DS1, and 3DS1, were associated with significantly less relapse in patients with acute myelogenous leukemia (AML) (13% versus 57%) but not in patients with other myelogenous or lymphoid malignancies. AML patients receiving SCT from donors with these KIR genes relapsed 4 times less frequently than patients transplanted from donors with other KIR genotypes. These findings suggest specific, genetically determined, interactions between NK cells and AML cells that facilitate the GVL effect, and have implications for donor selection for AML patients. [ABSTRACT FROM AUTHOR]

Antibodies to neutrophil and HLA antigens can cause pulmonary transfusion reactions, and in some cases acute lung injury. When evaluating cases of pulmonary transfusion reactions, it is often necessary to test donors for neutrophil and HLA antibodies and also type the recipient for neutrophil and HLA antigens. A variety of enzyme-linked immunosorbent assay (ELISA) and flow cytometry–based solid phase assays are available to test for HLA class I and class II antibodies, but not neutrophil antibodies. Screening for neutrophil antibodies requires the preparation of panels of fresh neutrophils and testing in agglutination, immunofluorescence, or flow cytometry assays. Genotyping of HLA class I and II antigens is performed with a variety of sequence-specific primers, sequenced-specific oligonucleotide probe, and sequence-based typing assays. Neutrophil-specific antigens HNA-1a, -1b, -1c, -4a, and -5a can be genotyped, but not HNA-2a or -3a. Phenotyping of HNA-2a can be performed with CD177 monoclonal antibodies, but the gene encoding HNA-3a has not been identified, and the genomic basis for the HNA-2a–negative phenotype is not known. In conclusion, patients and donors involved with pulmonary transfusion reactions can be quickly typed for HLA antigens and tested for HLA antibodies, but testing for neutrophil antibodies and antigens requires the use of a reference laboratory. [Copyright &y& Elsevier]

• The best donor options for patients with primary immunodeficiencies (PIDs) are often unrelated or older, HLA-haploidentical donors • Inherited diseases, particularly autosomal dominant, can diminish related donor options • Additional evaluations, such as genetic testing, add complexity to donor searches for patients with PIDs. Patients with primary immunodeficiencies (PIDs) are potentially cured by allogeneic hematopoietic cell transplantation (HCT). The spectrum of PIDs has expanded greatly beyond those that present in infancy or are diagnosed on newborn screening and require urgent, preemptive HCT. Many PID diagnoses are now made later in life, and the role of HCT is only considered for severe disease manifestations; in these cases, the kinetics and goals of a donor search may be different than for severe combined immunodeficiency. Across all PIDs, related donor searches have the additional selection factor of the inherited disease, and such searches may yield more limited options than searches for patients with hematologic malignancies; thus, unrelated donor options often become more critical in these patients. We retrospectively evaluated the outcomes of donor searches among patents with PIDs referred for HCT at the National Institutes of Health, where the minimum patient age for evaluation is 3 years and where donor options include matched sibling donors or matched related donors, HLA-haploidentical (haplo), or 7-8/8 HLA matched unrelated donors (mMUDs/MUDs). Patient (n = 161) and donor demographics, MUD search results, HLA typing, pedigrees, mutation testing, and donor selection data were collected. The National Marrow Donor Program HapLogic 8/8 HLA match algorithm was used to predict the likelihood of a successful MUD search and categorized as very good, good, fair, poor, very poor, or futile per the Memorial Sloan Kettering Cancer Center (MSKCC) Search Prognosis method. There were significant differences by PID mode of inheritance in patient age, disposition (receipt of HCT or not), donor source, and donor relatedness. A related or unrelated donor option could be identified for 94% of patients. Of living first-degree relatives (median, 3; range, 0 to 12 per patient), a median of 1 donor remained for autosomal dominant and X-linked (XL) diseases after HLA typing, mutation testing, and other exclusions, and a median of 2 donors remained for autosomal recessive (AR) diseases. Among patients with a PID of known mode of inheritance (n = 142), the best related donor was haplo for 99 (70%) patients, with 56 (39%) haplos age 40 years or older and 5 (4%) second-degree haplos; 13 (9%) had no family donor options. The best related donor was a heterozygote/asymptomatic carrier of the PID mutation in 36 (49%) patients with AR or XL disease (n = 73). Among patients with MUD search performed (n = 139), 53 (38%) had very poor/futile 8/8 MUD searches, including 6 (32%) of those with unknown PID mutation and therefore no family donor options. The MSKCC Search Prognosis was less favorable for those of non-European ancestry compared with European ancestry (P =.002). Most patients of Hispanic or African ancestry had very poor/futile MUD searches, 71% and 63%, respectively. No HCT recipients with very poor/futile MUD searches (n = 38) received 8/8 MUD grafts. Alternative donor options, including haplo and unrelated donors, are critical to enable HCT for patients with PIDs. MUD search success remains low for those of non-European ancestry, and this is of particular concern for patients with PIDs caused by an unknown genetic defect. Among patients with PIDs, related donor options are reduced and haplos age 40 years and older and/or mutation carriers are often the best family option. [ABSTRACT FROM AUTHOR]

Abstract The CYP21A2 gene encoding 21‑hydroxylase is on chromosome 6p21.3 within the human leukocyte antigen (HLA) class III major histocompatibility complex and an association between congenital adrenal hyperplasia (CAH) due to 21‑hydroxylase deficiency and HLA class I and II alleles has been shown in genetically isolated populations. One-third of CAH causing alleles are 30-kb deletions due to homologous recombination events between active and pseudogenes resulting in chimeric genes. The aim of this study was to re-visit the association between the CYP21A2 variants and HLA polymorphisms in a large ethnically diverse cohort of patients with CAH who underwent comprehensive CYP21A2 genotyping, including specification of chimeric gene subtypes (CAH CH-1 through CH-9 of CYP21A1P/CYP21A2 chimeras; CAH-X CH-1 through CH-3 of TNXA/TNXB chimeras) in alleles with 30-kb deletions. The study population included 201 patients (86 males, 115 females, age 3–75 years) with CAH due to 21‑hydroxylase deficiency (159 classic, 42 nonclassic) and 194 parents. Based on the availability of parental genotype, we determined the haplotypes of CYP21A2 mutations and HLA types in 95 probands (190 alleles). Five prevalent haplotype associations were found: p.V281L and B*14-C*08 (P < 0.0001); p.I172N and DQB1*03 (P = 0.035); and of the chimeric genes caused by 30-kb deletions: CH-1 and A*03 (P = 0.033); CH-5 and C*06-DRB1*07 (P < 0.0001); and CAH-X CH-1 and DQB1*03 (P = 0.004). Our findings show that a number of associations between HLA alleles and haplotypes and CYP21A2 mutations, including large 30-kb deletions, exist commonly across ethnicities. These HLA associations may have clinical implications for patients with CAH and may provide insight into the genetics of this highly complex region of the human genome. Graphical abstract Unlabelled Image Highlights • CYP21A2 mutations are associated with HLA haplotypes, regardless of ethnicity. • p.V281L-HLA B*14-C*08 accounts for the majority of nonclassic alleles. • p.I172N-HLA DQB1*03 accounts for over one-third of simple virilizing alleles. • CYP21A1P/CYP21A2 and TNXA/TNXB chimeras are HLA linked. [ABSTRACT FROM AUTHOR]

Aim To develop assays to quantify the mRNA transcribed from 15 KIR functional genes utilizing a quantitative real-time PCR (qPCR) method. To study the KIR mRNA expression patterns in NK cells and peripheral blood mononuclear cells (PBMC) among 50 healthy volunteers. To correlate the qPCR results from NK cells and PBMC. Methods PBMC were obtained from freshly drawn whole blood (maximum 48 h) from 50 healthy volunteers using a Ficoll method. NK cell enrichment from PBMC was performed by negative selection using a magnetic bead kit. mRNA extraction and cDNA synthesis was followed by qPCR using EvaGreen dye. Relative quantification (RQ) of KIR mRNA expression was quantified by 2-ΔΔCt algorithm with GAPDH as the housekeeping gene and a commercial human cDNA as the universal reference. Results 16 mRNA expression assays for 15 KIR genes, including assays for both the full-length and truncated variants for 2DS4, have been developed and validated. Among the 50 individuals, KIR mRNAs were detected in all samples carrying respective KIR genes and were negative for the KIR gene negative samples tested. In particular, 2DL1, 2DL4 and 3DL2 were positive for all 50 individuals at both genomic DNA and mRNA levels, whereas mRNA was undetected for all 2DL5B and 3DL3 gene positive samples. Among 50 individuals, the most variably expressed KIR mRNA was 2DL1 in NK cells and 3DL1 in PBMC. The strongest correlation between NK cells and PBMC was found for 2DS5 ( R = 0.9582, n = 9, p < 0.001, one-way ANOVA). Conclusions We established qPCR assays to quantify the mRNA expression of 15 KIR genes. KIR mRNA expression varied among healthy individuals carrying the same KIR genotype. Analyzing NK cells and PBMC will allow establishing NK cell specific and overall KIR mRNA expression signatures from an individual. Results from NK cells and PBMC are correlated to a different degree for the 15 KIR genes, confirming that NK cells are the main predictor for KIR mRNA expression in PBMC. However, any KIR mRNA expression in PBMC is only partially reflective of the KIR mRNA expression in NK cells. [ABSTRACT FROM AUTHOR]

Aim: To develop assays to quantify the mRNA transcribed from KIR genes utilizing a quantitative real-time PCR (qPCR) method. To study the KIR mRNA expression patterns in PBMC and enriched NK cells among healthy volunteers. Methods: PBMCs were obtained from freshly drawn whole blood (maximum 48 hours) from 14 healthy volunteers using a Ficoll method. NK cell enrichment from PBMC was performed by negative selection using a magnetic bead kit. mRNA extraction and cDNA synthesis was followed by EvaGreen based qPCR. Relative KIR mRNA expression was quantified by 2-ΔΔCt algorithm with GAPDH as reference. qPCR with a Cq ⩽ 38.0 was considered positive. Results: 10 mRNA expression assays for the 9 KIR genes 2DL1, 2DL5A, 2DL5B, 2DS1, 2DS2, 2DS3, 2DS4 (2DS4-full length and 2DS4-truncated), 2DS5 and 3DS1 have been developed and validated. Among the 14 individuals, KIR mRNAs were detected in all samples carrying respective KIR genes and were negative for KIR gene negative samples. The mRNA expression varied among individuals. In PBMC, the relative quantification (RQ) of cDNA showed the largest variation for 2DL1 (14-fold, n = 14) and the smallest for 3DS1 (1.8-fold, n = 3) among volunteers who were positive for the respective genes. 2DL5B cDNA was not detected, as expected for this gene. More KIR cDNA was detected in the enriched NK cell fraction when compared to PBMC, with the largest difference observed for 2DL1 (NK : PBMC ratio, mean ± SD: 37.8 ± 25.0, n = 14). Conclusions: Development of qPCR assays with high specificity and easy applicability will be useful, because all known 15 KIR genes share > 90% identity at the genomic level. We established 10 assays for the mRNA expression of 9 KIR genes. KIR mRNA expression varied among healthy individuals carrying the same KIR genotype. Because NK cells are the predominant, but not the only source of KIR mRNA, analyzing PBMC and NK cells will allow establishing an overall and NK cell specific KIR mRNA expression signature from an individual. [ABSTRACT FROM AUTHOR]

Patients with PIDs are potentially cured by allogeneic HCT, although related donor searches for PID patients have the additional selection factor of the inherited disease and therefore may yield more limited options than searches for hematologic malignancy patients. Unrelated donor options are thus critical in these patients. We evaluated the outcomes of donor searches among PID patients referred to the National Institutes of Health (NIH) for HCT, where donor options included matched related donors (MRD), HLA-haploidentical (haplo), or 7-8/8 HLA-matched unrelated donors (mMUD/MUD). After IRB approval (NCT03188419, clinicaltrials.gov), retrospective chart review of PID patients referred to NIH for HCT between 3/30/12-2/27/18 was performed. Patient and donor demographics, MUD search results, HLA typing, pedigrees, mutation testing, and donor selection data were collected. For the purposes of this study, patients with unknown PID mutation were considered to have no related donor options. The HapLogic 8/8 HLA-match algorithm was used to predict the likelihood of a successful MUD search and categorized as very good, good, fair, poor, very poor, or futile per the MSKCC Search Prognosis method. Patient (n = 161), donor, and HCT (n = 109) characteristics are shown in Fig 1, with significant differences by PID mode of inheritance in patient age, disposition (receipt of HCT or not), donor source, and donor relatedness. Of living 1st degree relatives (median 3 (range 0-12) per patient), a median of 1 donor remained for autosomal dominant and X-linked (XL) diseases after HLA typing, mutation testing, and other exclusions, and a median of 2 donors remained for autosomal recessive (AR) diseases. Among patients with a PID of known mode of inheritance (n = 142), the best related donor was haplo for 100 (70%) patients, with 56 (39%) haplos age 40+ years and 4 (3%) 2nd degree haplos; 13 (9%) had no family donor options. The best related donor was a heterozygote/asymptomatic carrier of the PID mutation in 36 (49%) patients with AR or XL disease (n = 73). Among patients with MUD search performed (n = 139), 53 (38%) had very poor/futile 8/8 MUD searches, including 6 (31%) of those with unknown PID mutation and therefore no family donor options. The predicted likelihood of a successful MUD search was lower for those of non-European ancestry, p = 0.01. The majority of patients of Hispanic or African ancestry had very poor/futile MUD searches, 71% and 63%, respectively. No HCT recipients with very poor/futile MUD searches (n = 38) received 8/8 MUD grafts. Median time from HLA typing to HCT was 7 months. Haplo and MUD options are critical to enable HCT for patients with PID. Related donor options are reduced and haplos age 40 years+ and/or mutation carriers are often the best family option. MUD search success remains low for those of non-European ancestry and this is of particular concern for patients with PID due to an unknown genetic defect. [ABSTRACT FROM AUTHOR]

Aim High resolution HLA typing with clinical applications is preferred in some applications of transfusion and transplantation medicine. Concordance of results was determined between new reverse sequence specific oligonucleotide assays (rSSO; LABType CWD and LABType XR, One Lambda, Canoga Park, CA) and our current assays, rSSO (LABType SSO) and sequence-based typing (SBT; SeCore). Methods Forty-eight patient samples previously tested by our current rSSO and SBT assays were selected randomly. The 2 new assays and a new multiplex flow analyzer (LABScan3D TM ) were utilized to detect HLA-A common and well-defined alleles (LABType CWD) and HLA-B and DRB1 alleles at high resolution (LABType XR). DNA was amplified with group specific primers targeting exons 2, 3, 4, and 5 for HLA-A and HLA-B, and exon 2 for HLA-DRB1. Results We determined the concordance between the 2 new and our current assays (Table 1). The LABType CWD identified 6 out of 96 HLA-A alleles (6.25%) at the allele level (4-digit resolution) and was also able to resolve 1 ambiguous allele combination, exceeding the resolution obtained by our current LABType SSO. The LABType XR assay provided results with shorter allele strings in 59 out of 96 HLA-B alleles (61.5%) and 22 out of 96 HLA-DRB1 alleles (22.9%), respectively, exceeding the resolution obtained by our current SeCore SBT without reflux testing. Discordances were observed resulting from false bead reactions or ambiguous allele combinations. Conclusions The new LABType CWD and XR assays are suitable platforms for high throughput testing. Utilization of the LABType XR kits potentially could yield better results than SBT thus making them a consideration for clinical applications requiring quicker turn around time. However, even with LABType XR, additional testing may still be necessary to resolve any remaining discrepancies and ambiguities. [ABSTRACT FROM AUTHOR]

Introduction Allogeneic hematopoietic stem cell transplantation using UCB (UCBT) is an alternative approach for severe aplastic anemia (SAA) patients (pts) lacking an HLA matched donor. However, UCBT is associated with delayed engraftment, high graft failure and mortality rates. Ex-vivo expanded UCB using nicotinamide (NAM) can engraft in NOD/SCID mice, and in pts with hematological malignancies in pilot studies. Here we investigated whether NAM-expanded UCBT would accelerate engraftment and immune reconstitution in refractory SAA pts. Methods Eligible SAA pts with severe neutropenia who failed immunosuppressive therapy underwent a NAM-expanded UCBT at a single center in a phase II trial. Pts were conditioned with cyclophosphamide (60 mg/kg × 2), horse ATG (40 mg/kg × 4), fludarabine (25 mg/m2 × 5) and 200cGy of TBI. GVHD prophylaxis included tacrolimus and MMF. Cohort 1 is designed to transplant six pts with a single NAM-expanded unit combined with 3 × 106 CD34+ cells/kg from a haploidentical donor as a stem cell backup. Once adequate cord engraftment is established, Cohort 2 will transplant a NAM-expanded unit alone. Engraftment and immune recovery were assessed and compared with a conventional UCBT combined with haploCD34+ cells using the same conditioning and GVHD prophylaxis. Results From 2017 to 2018, two SAA pts (22 years male and 45 years female, pre-transplant ANC ≤300/uL, failed ATG/CSA/Eltrombopag) successfully underwent a single 6/8 HLA-matched NAM-expanded UCBT combined with haplo CD34+ cells. UCB units before expansion contained a median total nucleated cell (TNC) dose of 2.8 × 107/kg and 1.7 × 105 CD34+ cells /kg. At transplant, NAM-expanded units contained a median 6.0 × 107 TNCs/kg and 96.4 × 105 CD34+ cells /kg, representing a median TNC and CD34+ cell expansion of 2-fold and 52-fold, respectively. At 14 and 7 months post-transplant, both pts were GVHD-free and survived with stable engraftment and transfusion independence (Figure 1). The median time to neutrophil and platelet recovery was only 6.5 days (range 6-7) and 35.5 days (31-40), with >95% cord donor myeloid and T-cell chimerism occurring at days 6.5 and 23.5, respectively. Immune recovery was brisk (Figure 2): absolute CD4+ > 200 cells/μL occurred at 17 and 60 days; at day 100, mean CD4+ cells were 382/μL and IGA 92 mg/dL. Compared to 16 SAA pts who received a single unexpanded UCBT with haplo CD34+ cells at our institute from 2013 to 2016, engraftment and immune recovery were superior in SAA pts with NAM-expanded UCBT (P <0.05, Figures 1 and 3). Conclusion These results show for the first time that NAM-expanded UCB results in rapid cord engraftment, sustained hematopoiesis and accelerated immune recovery in treatment refractory, neutropenic SAA pts. The high numbers of CD34+ progenitor cells in NAM-expanded grafts could potentially overcome graft failure which limits conventional UCBT for SAA. [ABSTRACT FROM AUTHOR]

Aim Holotype HLA is a powerful NGS-based method for HLA genotyping being adopted by HLA laboratories over legacy technologies such as SSO and Sanger SBT for its increased accuracy, resolution, repeatability and high-throughput nature. The elimination of human and random error is a desirable attribute of any diagnostic test and the implementation of a liquid handling system can significantly reduce both sources of error in an NGS workflow. Development of a protocol that streamlines multiple modules of the NGS process was the goal of this multi-center study. Methods The Beckman Coulter Biomek 4000 automated liquid handler is a small footprint solution for pre- and post-PCR processes in NGS-based HLA typing. During development, the protocol was optimized through comparison of multiple data points at measurable stopping points to provide a customized modular liquid handling solution for any HLA lab. Results Technologists and scientists from three sites performed multiple runs of 24 samples for 11 HLA loci using the Holotype HLA 24/11 configuration automated on the Biomek 4000 to generate data on the accuracy, specificity, repeatability and reproducibility of an automated Holotype HLA solution. Conclusion Data obtained from the three centers were analyzed using Omixon HLA Twin and assessed for accuracy, reproducibility, and allele assignment ambiguities to demonstrate that high-throughput NGS-based HLA typing is achievable in under 48 h from gDNA to genotype using the Beckman Coulter Biomek 4000 modular based NGS protocol for Holotype HLA 24/11. [ABSTRACT FROM AUTHOR]

Aim: The goal of the study was to evaluate a correlation of an HLA restricted minor histocompatibility antigen (mHAg) with the development of graft versus host disease (GVHD). The objective was to assess whether testing for mHAgs should be done on a prospective basis, to guide donor selection and reduce the high incidence of GVHD in post-transplant allografts when treating hematologic malignancies. Methods: A cohort of 45 patient/unrelated donor pairs, 10/10 HLA match, in the NCI protocol 07-C-0195 were typed for 18 mHAgs using an SSP-PCR typing kit (Minor Histocompatibility Antigen Typing Kit; Life Technologies, Carlsbad, CA). GVHD data was obtained from the clinical protocol database. Patients were determined not evaluable if they relapsed or had residual disease requiring a donor lymphocyte infusion or additional chemotherapy. Due to clinical status, one patient could not be evaluated for acute or chronic GVHD. In addition, 4 patients could not be evaluated for chronic GVHD. Therefore, 44 out of 45 patients were evaluated for acute GVHD and 40 out of 45 patients for chronic GVHD. Statistical analysis was performed using the Fisher exact test. Results: Patients with mHAg mismatches were analyzed for both acute and chronic GVHD. Preliminary analysis demonstrates that out of the 45 patient/donor pairs, 30 pairs had mHAgs mismatched in the GVHD direction. 19 of the 30 patients (63%) with mismatches developed acute GVHD (P=0.062) and 15 of the 30 patients (50%) with mismatches developed chronic GVHD (P=0.061). Nine of these patients developed both acute and chronic GVHD. Conclusions: Data suggests some correlation regarding mHAg mismatches and the development of GVHD. Further study and a larger cohort may allow a gauge for the efficacy of testing for the mHAgs on a prospective basis. Testing on a prospective basis could give clinicians more knowledge to select donors with a favorable mHAg constellation to reduce the risk of their transplant patients developing GVHD. [Copyright &y& Elsevier]