Selection of unrelated donors and cord blood units for hematopoietic cell transplantation: guidelines from the NMDP/CIBMTR.

Allogeneic hematopoietic cell transplantation involves consideration of both donor and recipient characteristics to guide the selection of a suitable graft. Sufficient high-resolution donor-recipient HLA match is of primary importance in transplantation with adult unrelated donors, using conventional graft-versus-host disease prophylaxis. In cord blood transplantation, optimal unit selection requires consideration of unit quality, cell dose and HLA-match. In this summary, the National Marrow Donor Program (NMDP) and the Center for International Blood and Marrow Transplant Research, jointly with the NMDP Histocompatibility Advisory Group, provide evidence-based guidelines for optimal selection of unrelated donors and cord blood units.

Predictors of Loss to Follow-Up Among Pediatric and Adult Hematopoietic Cell Transplantation Survivors: A Report from the Center for International Blood and Marrow Transplant Research.

Follow-up is integral for hematopoietic cell transplantation (HCT) care to ensure surveillance and intervention for complications. We characterized the incidence of and predictors for being lost to follow-up. Two-year survivors of first allogeneic HCT (10,367 adults and 3865 children) or autologous HCT (7291 adults and 467 children) for malignant/nonmalignant disorders between 2002 and 2013 reported to the Center for International Blood and Marrow Transplant Research were selected. The cumulative incidence of being lost to follow-up (defined as having missed 2 consecutive follow-up reporting periods) was calculated. Marginal Cox models (adjusted for center effect) were fit to evaluate predictors. The 10-year cumulative incidence of being lost to follow-up was 13% (95% confidence interval [CI], 12% to 14%) in adult allogeneic HCT survivors, 15% (95% CI, 14% to 16%) in adult autologous HCT survivors, 25% (95% CI, 24% to 27%) in pediatric allogeneic HCT survivors, and 24% (95% CI, 20% to 29%) in pediatric autologous HCT survivors. Factors associated with being lost to follow-up include younger age, nonmalignant disease, public/no insurance (reference: private), residence farther from the tranplantation center, and being unmarried in adult allogeneic HCT survivors; older age and testicular/germ cell tumor (reference: non-Hodgkin lymphoma) in adult autologous HCT survivors; older age, public/no insurance (reference: private), and nonmalignant disease in pediatric allogeneic HCT survivors; and older age in pediatric autologous HCT survivors. Follow-up focusing on minimizing attrition in high-risk groups is needed to ensure surveillance for late effects.

Clinical risks and healthcare utilization of hematopoietic cell transplantation for sickle cell disease in the USA using merged databases.

Advances in allogeneic hematopoietic cell transplantation for sickle cell disease have improved outcomes, but there is limited analysis of healthcare utilization in this setting. We hypothesized that, compared to late transplantation, early transplantation (at age <10 years) improves outcomes and decreases healthcare utilization. We performed a retrospective study of children transplanted for sickle cell disease in the USA during 2000-2013 using two large databases. Univariate and Cox models were used to estimate associations of demographics, sickle cell disease severity, and transplant-related variables with mortality and chronic graft-versus-host disease, while Wilcoxon, Kruskal-Wallis, or linear trend tests were applied for the estimates of healthcare utilization. Among 161 patients with a 2-year overall survival rate of 90% (95% confidence interval [CI] 85-95%) mortality was significantly higher in those who underwent late transplantation versus early (hazard ratio (HR) 21, 95% CI 2.8-160.8, P=0.003) and unrelated compared to matched sibling donor transplantation (HR 5.9, 95% CI 1.7-20.2, P=0.005). Chronic graftversus host disease was significantly more frequent among those translanted late (HR 1.9, 95% CI 1.0-3.5, P=0.034) and those who received an unrelated graft (HR 2.5, 95% CI 1.2-5.4; P=0.017). Merged data for 176 patients showed that the median total adjusted transplant cost per patient was $467,747 (range: $344,029-$799,219). Healthcare utilization was lower among recipients of matched sibling donor grafts and those with low severity disease compared to those with other types of donor and disease severity types (P<0.001 and P=0.022, respectively); no association was demonstrated with late transplantation (P=0.775). Among patients with 2-year pre- and post-transplant data (n=41), early transplantation was associated with significant reductions in admissions (P<0.001), length of stay (P<0.001), and cost (P=0.008). Early transplant outcomes need to be studied prospectively in young children without severe disease and an available matched sibling to provide conclusive evidence for the superiority of this approach. Reduced post-transplant healthcare utilization inpatient care indicates that transplantation may provide a sustained decrease in healthcare costs over time.

Impact of allele-level HLA matching on outcomes after myeloablative single unit umbilical cord blood transplantation for hematologic malignancy.

We studied the effect of allele-level matching at human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 in 1568 single umbilical cord blood (UCB) transplantations for hematologic malignancy. The primary end point was nonrelapse mortality (NRM). Only 7% of units were allele matched at HLA-A, -B, -C, and -DRB1; 15% were mismatched at 1, 26% at 2, 30% at 3, 16% at 4, and 5% at 5 alleles. In a subset, allele-level HLA match was assigned using imputation; concordance between HLA-match assignment and outcome correlation was confirmed between the actual and imputed HLA-match groups. Compared with HLA-matched units, neutrophil recovery was lower with mismatches at 3, 4, or 5, but not 1 or 2 alleles. NRM was higher with units mismatched at 1, 2, 3, 4, or 5 alleles compared with HLA-matched units. The observed effects are independent of cell dose and patient age. These data support allele-level HLA matching in the selection of single UCB units.

Identification of a permissible HLA mismatch in hematopoietic stem cell transplantation.

In subjects mismatched in the HLA alleles C*03:03/C*03:04 no allogeneic cytotoxic T-lymphocyte responses are detected in vitro. Hematopoietic stem cell transplantation (HSCT) with unrelated donors (UDs) showed no association between the HLA-C allele mismatches (CAMMs) and adverse outcomes; antigen mismatches at this and mismatches other HLA loci are deleterious. The absence of effect of the CAMM may have resulted from the predominance of the mismatch C*03:03/C*03:04. Patients with hematologic malignancies receiving UD HSCT matched in 8/8 and 7/8 HLA alleles were examined. Transplants mismatched in HLA-C antigens or mismatched in HLA-A, -B, or -DRB1 presented significant differences (P < .0001) in mortality (hazard ratio [HR] = 1.37, 1.30), disease-free survival (HR = 1.33, 1.27), treatment-related mortality (HR = 1.54, 1.54), and grade 3-4 acute graft-versus-host disease (HR = 1.49, 1.77) compared with the 8/8 group; transplants mismatched in other CAMMs had similar outcomes with HR ranging from 1.34 to 172 for these endpoints. The C*03:03/C*03:04 mismatched and the 8/8 matched groups had identical outcomes (HR ranging from 0.96-1.05). The previous finding that CAMMs do not associate with adverse outcomes is explained by the predominance (69%) of the mismatch C*03:03/03:04 in this group that is better tolerated than other HLA mismatches.

Impact of KIR and HLA Genotypes on Outcomes after Reduced-Intensity Conditioning Hematopoietic Cell Transplantation.

Natural killer cells are regulated by killer cell immunoglobulin-like receptor (KIR) interactions with HLA class I ligands. Several models of natural killer cell reactivity have been associated with improved outcomes after myeloablative allogeneic hematopoietic cell transplantation (HCT), but this issue has not been rigorously addressed in reduced-intensity conditioning (RIC) unrelated donor (URD) HCT. We studied 909 patients undergoing RIC-URD HCT. Patients with acute myeloid leukemia (AML, n = 612) lacking ≥ 1 KIR ligands experienced higher grade III to IV acute graft-versus-host disease (GVHD) (HR, 1.6; 95% CI, 1.16 to 2.28; P = .005) compared to those with all ligands present. Absence of HLA-C2 for donor KIR2DL1 was associated with higher grade II to IV (HR, 1.4; P = .002) and III to IV acute GVHD (HR, 1.5; P = .01) compared with HLA-C2(+) patients. AML patients with KIR2DS1(+), HLA-C2 homozygous donors had greater treatment-related mortality compared with others (HR, 2.4; 95% CI, 1.4 to 4.2; P = .002) but did not experience lower relapse. There were no significant associations with outcomes for AML when assessing donor-activating KIRs or centromeric KIR content or for any donor-recipient KIR-HLA assessments in patients with myelodysplastic syndrome (n = 297). KIR-HLA combinations in RIC-URD HCT recapitulate some but not all KIR-HLA effects observed in myeloablative HCT.

Amino acid substitution at peptide-binding pockets of HLA class I molecules increases risk of severe acute GVHD and mortality.

HLA disparity has a negative impact on the outcomes of hematopoietic cell transplantation (HCT). We studied the independent impact of amino acid substitution (AAS) at peptide-binding positions 9, 99, 116, and 156, and killer immunoglobulin-like receptor binding position 77 of HLA-A, B, or C, on the risks for grade 3-4 acute graft-versus-host disease (GVHD), chronic GVHD, treatment-related mortality (TRM), relapse, and overall survival. In multivariate analysis, a mismatch at HLA-C position 116 was associated with increased risk for severe acute GVHD (hazard ratio [HR] = 1.45, 95% confidence interval [CI] = 1.15-1.82, P = .0016). Mismatch at HLA-C position 99 was associated with increased transplant-related mortality (HR = 1.37, 95% CI = 1.1-1.69, P = .0038). Mismatch at HLA-B position 9 was associated with increased chronic GVHD (HR = 2.28, 95% CI = 1.36-3.82, P = .0018). No AAS were significantly associated with outcome at HLA-A. Specific AAS pair combinations with a frequency >30 were tested for association with HCT outcomes. Cysteine to tyrosine substitution at position 99 of HLA-C was associated with increased TRM (HR = 1.78, 95% = CI 1.27-2.51, P = .0009). These results demonstrate that donor-recipient mismatch for certain peptide-binding residues of the HLA class I molecule is associated with increased risk for acute and chronic GVHD and death.

Evaluation of HLA matching in unrelated hematopoietic stem cell transplantation for nonmalignant disorders.

The importance of human leukocyte antigen (HLA) matching in unrelated donor transplantation for nonmalignant diseases (NMD) has yet to be defined. We analyzed data from 663 unrelated marrow and peripheral blood stem cell transplants performed from 1995 to 2007 for treatment of NMD. Transplantation from a donor mismatched at the HLA-A, -B, -C, or -DRB1, but not -DQB1 or -DPB1, loci was associated with higher mortality in multivariate analyses (P = .002). The hazard ratio for mortality for single (7/8) and double mismatched (6/8) transplants was 1.29 (0.97-1.72; P = .079) and 1.82 (1.30-2.55; P = .0004), respectively, compared with 8/8 matched transplants. HLA mismatches were not associated with acute or chronic GVHD, but were strongly associated with graft failure. After adjustment for other factors, the odds ratio for graft failure for 7/8 and 6/8 (allele and/or antigen) matched pairs compared with 8/8 matched transplants was 2.81 (1.74-4.54; P < .0001) and 2.22 (1.26-3.97; P = .006), respectively. Patients with NMD should receive transplants from allele matched (8/8) donors if possible. Unlike the case with malignancies, HLA mismatching in NMD is associated with graft failure rather than GVHD.

Next-generation sequencing identifies two novel HLA-DPB1 alleles: HLA-DPB1*1069:01 and DPB1*1072:01.

Characterization of two novel HLA-DPB1 alleles: HLA-DPB1*1069:01, and DPB1*1072:01 containing non-synonymous nucleotide substitutions.

Reduced-intensity conditioning with combined haploidentical and cord blood transplantation results in rapid engraftment, low GVHD, and durable remissions.

We conducted a 45 patient prospective study of reduced-intensity conditioning (RIC) and transplantation of unrelated umbilical cord blood (UCB) and CD34(+) stem cells from a haploidentical family member. Median age was 50 years; weight was 80 kg. Fifty-eight percent had active disease. Neutrophil engraftment occurred at 11 days (interquartile range [IQR], 9-15) and platelet engraftment at 19 days (IQR, 15-33). In the majority of patients, early haploidentical engraftment was replaced by durable engraftment of UCB by 100 days, with regular persistence of minor host and/or haplo-hematopoiesis. Percentage of haplochimerism at day 100 correlated with the haplo-CD34 dose (P = .003). Cumulative incidence of acute GVHD (aGVHD) was 25% and chronic GVHD (cGVHD) was 5%. Actuarial survival at 1 year was 55%, progression-free survival (PFS) was 42%, nonrelapse mortality (NRM) was 28%, and relapse was 30%. RIC and haplo-cord transplantation results in fast engraftment of neutrophils and platelets, low incidences of aGVHD and cGVHD, low frequency of delayed opportunistic infections, reduced transfusion requirements, shortened length of hospital stay, and promising long-term outcomes. UCB cell dose had no impact on time to hematopoietic recovery. Therefore, UCB selection can prioritize matching, and better matched donors can be identified rapidly for most patients. This study is registered at http://clinicaltrials.gov as NCI clinical trial no. NCT00943800.

Spherotech-EDTA combined serum treatment reduces background more effectively as compared to One Lambda Adsorb Out™ and LIFECODES Serum Cleaner in Luminex-based solid-phase immunoassays for HLA antibody detection.

Spherotech (SPT) microparticles capture non-specific binding materials present in test serum, and EDTA removes the so called" prozone effect". This study presents a novel approach of combined SPT-EDTA serum treatment prior to Luminex HLA antibody testing to remove high background, and prozone effect in a single step process, and compared the efficacy of SPT-EDTA serum pre-treatment with AdsorbOut (ADS) and Serum Cleaner (SC) to reduce background in solid phase immunoassays (SPI). A total of 21 serum samples with a history of elevated negative control (NC) values ≥500, and 20 samples with normal NC values were included to assess the potential adverse effects. A problem of high background was noted in 25% of our samples in SPI. We observed 80% effectiveness in reducing NC values <500 with SPT-EDTA serum pre-treatment compared to 72%, and 67% for ADS and SC-treated sera, respectively. Interestingly, we found a strong correlation in antibody-binding levels between SPT versus ADS; and ADS versus SC-treated sera for both phenotype and single antigen bead assays (p < 0.001). No adverse effect was noted on NC, positive control (PC) values, PC/NC ratios in the upfront use of SPT-EDTA as compared to EDTA alone. Our data revealed that combined SPT-EDTA treated sera is more effective than ADS, and SC in reducing high background in SPI. Taken together, SPT-EDTA serum treatment prior to Luminex HLA Ab testing is cost-effective, our laboratory saves nearly 30% of the annual total cost for Ab testing and improved test turnaround time by two business days.

Plasma cell densities and glomerular filtration rates predict renal allograft outcomes following acute rejection.

The contribution of T cells and graft-reactive antibodies to acute allograft rejection is widely accepted, but the role of graft-infiltrating B and plasma cells is controversial. We examined 56 consecutive human renal transplant biopsies classified by Banff schema into T-cell-mediated (N = 21), antibody-mediated (N = 18), and mixed (N = 17) acute rejection, using standard immunohistochemistry for CD3, CD20, CD138, and CD45. In a predominantly African-American population (75%), neither Banff classification nor C4d deposition predicted the return to dialysis. Immunohistochemical analysis revealed CD3(+) T cells as the dominant cell type, followed by CD20(+) B cells and CD138(+) plasma cells in all acute rejection types. Using univariate Cox Proportional Hazard analysis, plasma cell density significantly predicted graft failure while B-cell density trended toward significance. Surprisingly T-cell density did not predict graft failure. The estimated glomerular filtration rate (eGFR) at diagnosis of acute rejection also predicted graft failure, while baseline eGFR ≥6 months prior to biopsy did not. Using multivariate analysis, a model including eGFR at biopsy and plasma cell density was most predictive of graft loss. These observations suggest that plasma cells may be a critical mediator and/or an independently sensitive marker of steroid-resistant acute rejection.

HLA-C antigen mismatch is associated with worse outcome in unrelated donor peripheral blood stem cell transplantation.

The association between HLA matching and outcome in unrelated-donor peripheral blood stem cell (PBSC) transplantation has not yet been established. In the present study, a total of 1933 unrelated donor-recipient pairs who underwent PBSC transplantation between 1999 and 2006 for acute myelogenous leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, or chronic myelogenous leukemia and received high-resolution HLA typing for HLA-A, -B, -C, -DRB1, -DQA1, and -DQB1 were included in the analysis. Outcomes were compared between HLA-matched and HLA-mismatched pairs, adjusting for patient and transplant characteristics. Matching for HLA-A, -B, -C, and -DRB1 alleles (8/8 match) was associated with better survival at 1 year compared with 7/8 HLA-matched pairs (56% vs 47%). Using 8/8 HLA-matched patients as the baseline (n = 1243), HLA-C antigen mismatches (n = 189) were statistically significantly associated with lower leukemia-free survival (relative risk [RR], 1.36; 95% confidence interval [CI], 1.13-1.64; P = .0010), and increased risk for mortality (RR, 1.41; 95% CI, 1.16-1.70; P = .0005), treatment-related mortality (RR, 1.61; 95% CI, 1.25-2.08; P = .0002), and grade III-IV graft-versus-host disease (RR, 1.98; 95% CI, 1.50-2.62; P < .0001). HLA-B antigen or allele mismatching was associated with an increased risk for acute GVHD grade III-IV. No statistically significant differences in outcome were observed for HLA-C allele (n = 61), HLA-A antigen/allele (n = 136), HLA-DRB1 allele (n = 39), or HLA-DQ antigen/allele (n = 114) mismatches compared with 8/8 HLA-matched pairs. HLA mismatch was not associated with relapse or chronic GVHD. HLA-C antigen-mismatched unrelated PBSC donors were associated with worse outcomes compared with 8/8 HLA-matched donors. The study's limited power due to small sample size precludes conclusions about other mismatches.

One-antigen mismatched related versus HLA-matched unrelated donor hematopoietic stem cell transplantation in adults with acute leukemia: Center for International Blood and Marrow Transplant Research results in the era of molecular HLA typing.

Approximately 13% of patients lacking an HLA-identical sibling have a one-antigen-mismatched related donor (MMRD). Historically, outcomes from the use of a one-antigen MMRD were considered equivalent to those from the use of a matched unrelated donor (UD). Recent improvements in UD stem cell transplantation (SCT) resulting from better molecular HLA matching justifies investigating whether UD should be preferred over MMRD in adult patients with acute leukemia. Here, we compared the outcomes of MMRD (n = 89) and HLA-A, -B, -C, and -DRB1 allele-matched UD (n = 700) SCT reported to the Center for International Blood and Marrow Transplant Research between 1995 and 2005. The patients underwent transplantation for acute myelogenous leukemia or acute lymphoblastic leukemia in first or second complete remission. Donor type was not associated with hematologic recovery. Univariate and multivariate comparisons of MMRD versus HLA-matched UD transplants showed no statistically significant differences in overall survival, disease-free survival, treatment-related mortality, relapse, or 100-day grade III-IV acute graft-versus-host disease (GVHD). MMRD SCT was associated with a lower rate of chronic GVHD at 1 year (35% vs 47%; P = .03), which was confirmed by multivariate analysis (relative risk, 0.58; 95% confidence interval, 0.39-0.85; P < .01). According to our data, HLA-matched UD and MMRD SCT are associated with comparable survival. Given that less chronic GVHD was observed in the MMRD transplantations, this option, when available, remains the first choice in patients with acute leukemia without an HLA-identical sibling in need of allogeneic SCT.

High-resolution HLA allele and haplotype frequencies in several unrelated populations determined by next generation sequencing: 17th International HLA and Immunogenetics Workshop joint report.

The primary goal of the unrelated population HLA diversity (UPHD) component of the 17th International HLA and Immunogenetics Workshop was to characterize HLA alleles at maximum allelic-resolution in worldwide populations and re-evaluate patterns of HLA diversity across populations. The UPHD project included HLA genotype and sequence data, generated by various next-generation sequencing methods, from 4,240 individuals collated from 12 different countries. Population data included well-defined large datasets from the USA and smaller samples from Europe, Australia, and Western Asia. Allele and haplotype frequencies varied across populations from distant geographical regions. HLA genetic diversity estimated at 2- and 4-field allelic resolution revealed that diversity at the majority of loci, particularly for European-descent populations, was lower at the 2-field resolution. Several common alleles with identical protein sequences differing only by intronic substitutions were found in distinct haplotypes, revealing a more detailed characterization of linkage between variants within the HLA region. The examination of coding and non-coding nucleotide variation revealed many examples in which almost complete biunivocal relations between common alleles at different loci were observed resulting in higher linkage disequilibrium. Our reference data of HLA profiles characterized at maximum resolution from many populations is useful for anthropological studies, unrelated donor searches, transplantation, and disease association studies.

Effective desensitization for a strong donor-specific HLA antibody in a case of HLA-mismatched allogeneic hematopoietic cell transplantation.

We describe a unique ABO compatible and 9/10 HLA-matched case of successful allogeneic hematopoietic cell transplantation (HCT) after effective desensitization of a strong anti-HLA-A24 donor-specific antibody (DSA) with mean fluorescence intensity of approximately 18 000. Due to absence of a suitable matched unrelated donor the patient sibling was considered the best available donor, and it was decided to desensitize patient prior to transplant. The strength of HLA-A24 DSA slowly decreased over the course of treatment, necessitating a total of 23 sessions of therapeutic plasma exchange in order to bring the DSA strength to undetectable levels, followed by a successful transplant. In summary, the outcome of this case shows effective application of desensitization treatment to remove strong DSA in HCT patients.

HLA alleles and haplotypes observed in 263 US families.

The 17th International HLA and Immunogenetics Workshop (IHIW) conducted a project entitled "The Study of Haplotypes in Families by NGS HLA". We investigated the HLA haplotypes of 1017 subjects in 263 nuclear families sourced from five US clinical immunogenetics laboratories, primarily as part of the evaluation of related donor candidates for hematopoietic stem cell and solid organ transplantation. The parents in these families belonged to five broad groups - African (72 parents), Asian (115), European (210), Hispanic (118) and "Other" (11). High-resolution HLA genotypes were generated for each subject using next-generation sequencing (NGS) HLA typing systems. We identified the HLA haplotypes in each family using HaplObserve, software that builds haplotypes in families by reviewing HLA allele segregation from parents to children. We calculated haplotype frequencies within each broad group, by treating the parents in each family as unrelated individuals. We also calculated standard measures of global linkage disequilibrium (LD) and conditional asymmetric LD for each ethnic group, and used untruncated and two-field allele names to investigate LD patterns. Finally we demonstrated the utility of consensus DNA sequences in identifying novel variants, confirming them using HLA allele segregation at the DNA sequence level.

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.

Reanalysis of the role of pronase treatment of B cells in the flow cytometric crossmatch assay: Fc receptor is not the primary target.

Pronase, a mixture of nonspecific bacterial proteases, is used to pretreat human lymphocytes to prevent false-positive B cell results in the flow cytometric crossmatch (FCXM) assay. The target of pronase has been reported to be B cell-expressed Fc receptors, which nonspecifically bind IgG. As pronase use in FCXM can induce other complications, including degradation of HLA leading to inappropriate FCXM results, and false-positive T cell results when testing serum from HIV-positive patients, we tested whether specifically blocking Fc receptor CD32 could replace pronase. Anti-CD32 mAb 6C4 was superior to pronase for blocking binding of aggregated IgG to B cells. However, 6C4 was unable to replace pronase in clinical FCXM, as it did not prevent false-positive B cell FCXM results, or enhance sensitivity of the assay. We conclude that the functional targets of pronase in the FCXM assay are poorly understood, and that B cell-expressed Fc receptor plays an insignificant role.

Identification and characterization of novel HLA alleles: Utility of next-generation sequencing methods.

The HLA genes are the most polymorphic of the human genome, and novel HLA alleles are continuously identified, often by clinical Sanger sequencing-based typing (SBT) assays. Introduction of next-generation sequencing (NGS) technologies for clinical HLA typing may significantly improve this process. Here we compare four cases of novel HLA alleles identified and characterized by both SBT and NGS. The tested NGS system sequenced broader regions of the HLA loci, and identified novel polymorphisms undetected by SBT. Subsequent characterization of the novel alleles in isolation of coencoded alleles by SBT required custom-designed primers, while the NGS system was able to sequence both alleles in phase. However, the tested assay was unable to amplify buccal cell DNA for subsequent NGS sequencing, presumably due to the lower quality of these samples. While NGS assays will undoubtedly increase novel allele identification, more stringent DNA sample requirements may be necessary for this new technology.