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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. ispartof: BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION vol:26 issue:3 pages:553-561 ispartof: location:United States status: published

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.

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.

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. (c) 2021 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

The 17(th) 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 1,017 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, and confirming them using HLA allele segregation at the DNA sequence level.

A limitation of solid-phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high-titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.

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.

Introduction HLA matching is not current practice in heart transplant (HT) donor allocation due to scarcity of donor organs and ischemic time. However HLA mismatch might adversely affect transplant outcomes. Even if HLA matching is not possible the degree of HLA mismatch might help inform clinicians on a patients risk for long term transplant complications. Hypothesis We hypothesize that the degree of HLA mismatch affects long-term complications in a stable HT cohort. Methods 65 clinically stable HT recipients (no cellular (ACR) or antibody mediated rejection (AMR) in the preceding 6 months) ≥ 2 years post transplant were enrolled. Serologic HLA typing was obtained for donor and recipients (D/R) pairs and the number of matches was analyzed combined for HLA class I and HLA II. Study endpoints were donor specific antibody (DSA) formation and significant cardiac allograft vasculopathy (CAV). Results HLA-A,-B,-C, -DR and-DQ (ABCDRQ) typing were available for 37 D/R pairs and were analyzed for matches. Additionally, 53 D/R pairs had HLA-A,-B, -DR (ABDR) typing. Meantime post HT was 8.5±5.4 years. A total of 28 patients had CAV and 9 patients had DSA. Among the ABCDRDQ cohort the number of matches was 1.54 ± 1.17 and 1.00 (0.00-1.00) in the ABDR cohort. There were no differences in the number of HLA matches for both cohorts among those with and without DSA and those with and without CAV (Table).However among the ABCDRDQ cohort there was a trend towards higher number of HLA matches in those without DSA 1.63 ± 1.19 versus 1.14 ± 1.07 in the DSA group (p 0.32), this was likely limited by the small sample size.There was no significant relationship between total HLA matches and number of ACR episodes (p 0.89). The incidence of AMR was too low for analysis. Conclusion In this highly selected cohort of clinically stable long time transplant survivors the degree of serological HLA matching was not associated with the development of CAV or ACR. However, there was a trend towards less DSA formation in those with more D/R HLA matches. Future longitudinal studies should evaluate the development of long-term complications as a function of HLA matching and potentially incorporate HLA mismatch as an indicator for the need of enhanced clinical surveillance.

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.

HLA-DP antigens are beta-alpha heterodimers encoded by polymorphic HLA-DPB1 and -DPA1 alleles, respectively, in strong linkage disequilibrium (LD) with each other. Non-permissive unrelated donor (UD)-recipient HLA-DPB1 mismatches across three different T-cell epitope (TCE) groups are associated with increased mortality after hematopoietic SCT (HCT), but the role of HLA-DPA1 is unclear. We studied 1281 onco-hematologic patients after 10/10 HLA-matched UD-HCT facilitated by the National Marrow Donor Program. Non-permissive mismatches defined solely by HLA-DPB1 TCE groups were associated with significantly higher risks of TRM compared to permissive mismatches (hazard ratio (HR) 1.30, confidence interval (CI) 1.06-1.53; P = 0.009) or allele matches. Moreover, non-permissive HLA-DPB1 TCE group mismatches in the graft versus host (GvH) direction significantly decreased the risk of relapse compared to permissive mismatches (HR 0.55, CI 0.37-0.80; P = 0.002) or allele matches. Splitting each group into HLA-DPA1*02:01 positive or negative, in frequent LD with HLA-DPB1 alleles from two of the three TCE groups, or into HLA-DPA1 matched or mismatched, did not significantly alter the observed risk associations. Our findings suggest that the effects of clinically non-permissive HLA-DPB1 TCE group mismatches are independent of HLA-DPA1, and that selection of donors with non-permissive DPB1 TCE mismatches in GvH direction might provide some protection from disease recurrence. OI Heemskerk, Martin/0000-0002-9432-7623

Role of donor specific antibodies (DSAs) in liver allograft function has not been fully defined. We report an ABO compatible orthotopic liver transplant case with DSAs to donor HLA, where the patient developed immediate antibody-mediated rejection (AMR).The patient, a 43-year-old female with cirrhosis, underwent ABO-compatible living-donor liver transplant from her husband. On post-operative day (POD)1, serum transaminases were sharply elevated. Retrospective testing of pre-transplant serum demonstrated presence of strong class I and class II anti-HLA antibodies and positive T- and B-cell flow-cytometric crossmatches (FCXM). Transaminase levels improved with plasmapheresis and thymoglobulin. On POD7, her liver enzymes became elevated again and allograft biopsy stained positive for C4d. Patient was treated with intravenous immunoglobulin and rituximab and recovered over time. Pre-transplant sera of patient were retrospectively tested by C1q assay to determine the cytotoxic function of DSAs; DSAs were positive for C1q binding. Our results suggest that pre-liver transplant antibody testing may be helpful in identifying patients at risk for development of AMR.

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.

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.

Background Intestinal tissues of patients with Crohn's disease (CD) contain expanded populations of T cells which are believed to mediate inflammation. We performed a detailed characterization of these T-cell repertoires. Methods We obtained biopsies from the neoterminal ileum of 12 patients undergoing evaluation for postoperative recurrent CD and 4 individuals with normal terminal ileum and no history of inflammatory bowel disease (controls). Samples of diseased terminal ileum were obtained from 5 patients undergoing surgery for stricturing or penetrating CD. Total RNA was extracted from tissues and peripheral blood mononuclear cells, and cDNAs were generated. We used next-generation sequencing to characterize T-cell receptor (TCR)-α and TCR-β cDNAs in ileal mucosal tissue and matched peripheral blood mononuclear cells of 17 patients with CD to identify oligoclonal expansions of T-cell populations associated with CD. Results TCR diversity in mucosal tissue was significantly lower than that of matched peripheral blood mononuclear cells, indicating expansion of certain T-cell populations in inflamed intestinal tissue. A single TCR-β clonotype, CASSWTNGEQYF (TRBV10-1-TRBJ2-7), was enriched at a frequency of 7.0% to 28.9% in the neoterminal ileum of 4 of 12 patients with recurrent CD. The abundance of this clonotype significantly correlated with the severity of disease recurrence, based on Rutgeerts score (P = 0.015). Conclusions Specific populations of T cells are expanded in the inflamed intestinal mucosa of patients with CD; their abundance correlates with severity of disease recurrence. Studies of these T cells could provide information about mechanisms of CD pathogenesis. Deep TCR sequencing is a powerful tool that rapidly provides in-depth, real-time assessment of the T-cell repertoire.

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.

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 towards 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.

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.

The identification of important amino acid substitutions associated with low survival in hematopoietic cell transplantation (HCT) is hampered by the large number of observed substitutions compared with the small number of patients available for analysis. Random forest analysis is designed to address these limitations. We studied 2107 HCT recipients with good or intermediate risk hematological malignancies to identify HLA class I amino acid substitutions associated with reduced survival at day 100 post transplant. Random forest analysis and traditional univariate and multivariate analyses were used. Random forest analysis identified amino acid substitutions in 33 positions that were associated with reduced 100 day survival, including HLA-A 9, 43, 62, 63, 76, 77, 95, 97, 114, 116, 152, 156, 166 and 167; HLA-B 97, 109, 116 and 156; and HLA-C 6, 9, 11, 14, 21, 66, 77, 80, 95, 97, 99, 116, 156, 163 and 173. In all 13 had been previously reported by other investigators using classical biostatistical approaches. Using the same data set, traditional multivariate logistic regression identified only five amino acid substitutions associated with lower day 100 survival. Random forest analysis is a novel statistical methodology for analysis of HLA mismatching and outcome studies, capable of identifying important amino acid substitutions missed by other methods.

Aim Turnaround time (TAT) for HLA typing is critical for transplantation. We sought to establish a faster protocol for low-resolution HLA typing using the rSSOP methodology. Methods The LABType™ rSSOP method (One Lambda) was altered to reduce hybridization time. Modifications included centrifugation and incubation duration reductions, and reagent volume adjustments. Single parameters were first changed and tested. Successful modifications were used in the next round of tests. All modifications were tested with a set of PCR amplicons prepared from three samples, which contained previously HLA-typed high quality DNA. Following a series of rSSOP typings for these samples at HLA-A, B, C, DRB1, and DQB1/DQA1 loci, an accelerated protocol was established. Discovery phase concluded with protocol validation at all loci including HLA-DRB3/4/5 and DPB1/DPA1. Full validation confirmed intra-operator, inter- and intra-assay, as well as multiple DNA source reproducibility. DNA was extracted from different samples including ACD blood (n = 4), EDTA blood (n = 4), cord segment (n = 5), buccal swab (n = 5) and mouth-wash samples (n = 4). Results The original centrifugation of 3000 rpm for 2 min (total 2 min 22 s) was changed to a quick spin (up to 3000 rpm; 33 s), reducing each centrifugation step by 1 min 50 s. Additionally, one centrifugation and one transfer of plate step were eliminated. The duration of incubation time was changed for denaturation (from 10 min to 2.5 min), hybridization (from 15 min to 5 min), and streptavidin-PE (SAPE, from 5 min to 2.5 min). The reagent volume modifications includes the reduction of washing buffer (from 100 μl to 50 μl per well) and increase in SAPE (from 25 μl to 50 μl per well). Compared to the original protocol, the accelerated protocol reduced the hybridization time approximately 30 min. This method allowed us to obtain consistent and accurate typing results for all tested HLA loci among different operators, intra- and inter-assays, and a variety of DNA samples. Conclusions Fulfilling requests for faster TAT may be achieved by adaptation of current methodologies, which may help improving patient management. The accelerated rSSOP protocol is an effective method for reducing testing time, while still giving robust results for HLA class I and class II typing. (RLU and JJX contributed equally).

A female patient presented for follow-up care approximately ten years post-cardiac transplantation. HLA antibody testing revealed multiple class I and class II donor specific antibodies (DSA), including anti-HLA A1 (MFI > 2500), DQ6, and DQ7/DQ8/DQ9 antibodies (DQ antibodies have MFIs >15,000). Per our current testing protocols, the sample was reflexed to C1q to determine if any DSAs were complement- fixing. As expected, based on antibody strength, the class II DSAs were C1q positive, while anti-HLA A1 was C1q negative. Interestingly, C1q testing revealed reactivity for anti-HLA B8 which was not previously detected by our standard IgG testing methodology. While anti-HLA B8 was not a DSA to the previous transplant, we wanted to determine the underlying cause for the positive reactivity seen with C1q testing: prozone effect or IgM? Since our laboratory routinely treats all sera tested for HLA antibodies with EDTA prior to testing, we thought prozone was unlikely as previous studies performed in our lab demonstrated the removal of this effect. Nonetheless, to rule out prozone with another method, IgG testing was repeated using a series of dilutions (Neat, 1:4, 1:16, 1:64, and 1:256). This testing did not yield positive reactivity for anti-HLA B8, and therefore prozone effect was excluded, indicating that EDTA methodology is robust to overcome prozone effect. To confirm the isotype of this antibody, IgM testing was conducted in three sera using the same assays as the IgG testing which produced positive results. In conclusion, anti-HLA B8 was determined to be complement-fixing and of the IgM isotype. Unexpectedly, our results supported the absence of an IgM-IgG isotype class switch for the period of testing performed in our lab spanning 17 months. *RLU and JJX contributed equally to this work.

Allogeneic hematopoietic cell transplantation (HCT) offers the potential to cure hematologic malignancies. Absent an HLA matched donor, HLA mismatched unrelated donors may be used although risks of graft-versus-host disease (GvHD) and treatment related mortality (TRM) are higher. Identification and avoidance of amino acid substitutions and position types (AASPT) conferring higher risks of TRM and GvHD would potentially improve the success of transplantation from single HLA mismatched unrelated donors. Using random forest and logistic regression analyses, we identified 19 AASPT associated with greater risks for at least one adverse transplant outcome: grade III-IV acute GvHD, TRM, lower disease free survival or worse overall survival relative to HLA matched unrelated donors and to other AASPT. When tested in an independent validation cohort of 3,530 patients, none of the AASPT from the training set were validated as high-risk, however. Review of the literature shows that failure to validate original observations is the rule and not the exception in immunobiology and emphasizes the importance of independent validation prior to clinical application. Our current data do not support avoiding any specific class I AASPT for unrelated donors. Additional studies should be performed to fully understand the role of AASPT in HCT outcomes.

Aim Our laboratory has recently reported that serum treatment with EDTA is sufficient to overcome the prozone effect. However, it has been reported that in rare cases, specimens are partially resistant to EDTA treatment, and serum dilutions are still necessary. Although we have not encountered such case in our laboratory, we sought to determine if differences in the technical aspects of the assay may contribute to EDTA’s effectiveness. Methods We screened samples sent to our laboratory for HLA antibody testing for suspected prozone effect, based on MFI values of >15,000. Selected samples were tested in three protocols. Protocol #1: to determine the presence of prozone effect, serum samples were frozen, thawed, centrifuged (14,000 rpm for 10 min), and tested at neat, 1:4, 1:16, 1:64, and 1:264 by single antigen (SA) bead assays (One Lambda). Protocol #2: to test the clinically-validated method used by our lab, samples were frozen, thawed, centrifuged, treated with EDTA (4 mM), and tested at neat only; incubation and washing was performed in a 1 μM filter plate on a vacuum plate-washing system (Biotek ELx50). Protocol #3: to test the effect of EDTA in fresh samples with the manual washing method, fresh serum samples were centrifuged, treated with EDTA, and then tested at neat only; incubation and washing was performed in a traditional round-bottom plate. Data were acquired on a Luminex-200 and analyzed with HLAFusion software (One Lambda). Results We identified prozone effects in 18 patients’ samples: 8 patients’ class I antibodies, 8 patients’ class II antibodies, and 2 patients’ class I and II antibodies. Following EDTA treatment, fresh and frozen samples showed similar MFI values for the corresponding assignments, including 138 class I and 103 class II specificities. In addition, all determined specificities showed higher MFI values in EDTA-treated samples than the peak MFI value in sample with dilution only, regardless of which technical protocol was used. Conclusions Samples with prozone effect resistant to EDTA treatment were not detected in our study, independent of technical variability for sample processing. EDTA treatment fully overcomes prozone effect in our testing system. Factors causing low efficacy of EDTA in rare cases remain unknown.

Due to the expanded genomic coverage of many next-generation sequencing (NGS) assays for HLA typing, and their recent introduction into immunogenetics laboratories, there has been an increase in the identification of novel alleles. While the majority of novel alleles are simple, single nucleotide polymorphisms (SNP), others require an extensive knowledge of the NGS analysis software in order to be suitably identified. Herein, we describe a case of gene conversion, a primary way in which HLA diversity is maintained, in a novel DPB1 allele. An optimized NGSgo protocol (GenDx) was used for sequencing the DPB1 locus, with analysis by NGSengine (GenDx). Initially analyzed using default parameters, including both coding and non-coding regions of the amplicon, a typing assignment of DPB1∗02:01:19, and a novel allele, similar to DPB1∗512:01, with an SNP in exon 2 was obtained. Using a lab-developed process, the data was reanalyzed using exon 2 only, with a resulting assignment of DPB1∗40:01, without mismatches. Utilizing the full amplicon and manually aligned to DPB1∗40:01, the sample was reanalyzed, revealing that the novel allele matched DPB1∗40:01 in exons 1 and 2. However, multiple mismatches were present in the remaining sequence. This result indicated that, while the 5′ end of the novel allele likely derived from DPB1∗40:01, the 3′ end may have been derived from another allele. In order to determine which DPB1 allele may have served as the template for this gene conversion event, known DPB1 alleles were manually aligned to the sample, with the finding that the sequence for DPB1∗06:01:01 was identical to the sample sequence at exons 3–5. We concluded that the novel allele likely resulted from a gene conversion event between the DPB1∗40:01 and 06:01:01 alleles. With the increased adoption of NGS assays for HLA typing, the detection of novel alleles due to gene conversion will also likely increase. With the aid of a skilled analyst, and using the comprehensive process described here, the proper identification of such alleles may be simplified and reported appropriately to the clinician.

Aim DNA sequencing has long been the gold standard for HLA typing, offering the ability to interrogate each base pair of a targeted region. Sanger sequencing-based typing assays are known to suffer from allele dropout, whereby one of two encoded alleles is less represented in the results, due to poor amplification. Next-generation sequencing (NGS) includes several distinct technologies, defined by their ability to sequence clonal DNA fragments in a high-throughput manner. One such technology, provided by Illumina, sequences thousands of short DNA fragments, with read depth orders of magnitude higher than Sanger sequencing, which may allow for the identification of poorly-amplified alleles. Here we sought to determine the rate of allele dropout in an Illumina-based HLA typing assay. Methods NGSgo (GenDx) was used to produce libraries for the HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 loci for sequencing on a MiSeq (Illumina). NGSengine (GenDx) was used to analyze the data and type the loci. 85 unique samples were run in parallel by a combination of previously-validated assays (SBT, SSP, rSSOP) and by NGS. Results The NGS assay resulted in very high read depths (>100 for our clinical results) and a very low error rate. Use of NGSengine software allowed detection of both alleles at heterozygous loci when the alleles were unevenly represented (imbalanced). In cases of extreme allele imbalance, data visualization by NGSengine allowed detection of the minor allele during manual review. Of the 811 patient loci tested by the previously-validated assays, 584 were heterozygous, 108 were homozygous and 119 were absent; the NGS results were 100% concordant. Conclusions In our experience with a clinical NGS typing assay, we found the Illumina sequencing technology was able to successfully identify all alleles present in tested samples, including poorly-amplified alleles. While the possibility of rare allele dropout still exists with this technology, due to alleles with novel polymorphisms at a primer-binding site, we found that with an appropriate combination of NGS library construction, sequencing technology, and analysis software, allele dropout is a low frequency, to non-existent, event. This reduces the need for extra testing to confirm homozygous loci during routine clinical HLA typing.

A 71 year old female was initially diagnosed with myelodysplastic syndrome (del 5q) which progressed to AML one year later. Patient had pancytopenia, experienced bruising, and needed blood transfusions. Allogeneic stem cell transplant was recommended and she presented to the University of Chicago Medicine for allo-stem cell transplant work up. This patient had one potential related donor (sibling), who was a 9/10 match (mismatch at HLA-A). When examining the patient’s HLA antibodies, it was found that she was highly sensitized, and anti-HLA-A24 was a donor specific antibody (DSA). A search for matched unrelated donors was conducted but no suitable donors were found. It was decided to accept her sibling and desensitize the patient prior to transplant. The desensitization protocol consisted of plasmapheresis (PP), with IVIG (100 mg/kg) given after each PP. The initial MFI of the DSA was >18,000 and showed evidence of rebound after each PP. As a result, PP was performed daily or every other day, with A24 MFI measurement before and after each PP (Figure). While the strength of A24 decreased over the course, a total of 18 sessions of PP were needed to decrease the strength to Download : Download high-res image (161KB) Download : Download full-size image

Aim Next-generation sequencing (NGS) assays for HLA typing are being rapidly adopted by clinical laboratories due to their ability to result in unambiguous, high-resolution typing in the vast majority of cases. Analysis of the NGS data involves specialized software algorithms to align hundreds of short oligonucleotide reads to the allele sequences managed by the IMGT/HLA. There is significant variability in the sequence length of alleles in the IMGT/HLA database, as an allele can be named by providing sequence from as little as one exon, while many alleles have full-locus sequences. This variability is problematic for the matching algorithms, which compute scores for alignment to the entire locus. To overcome this limitation, software vendors use imputed sequences, filling in the sequences missing from IMGT/HLA with sequence from a variety of sources. Since the imputed sequence may not match the patient alleles, use of imputed data can lead to mistypings. Here we describe our attempt to overcome the limitation of matching to imputed data. Methods NGSengine (GenDx) was used to analyze Illumina sequencing data from HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 amplicons. Potential mistypings were identified by further analysis of loci for which NGSengine did not report a typing without exonic mismatches. Results 28 samples were identified without typings that matched exonic data. 25 were determined to be novel alleles. 3 samples were mistyped based on mismatches to imputed data in NGSengine. A process was developed to analyze all potential novel alleles by restricting analysis to the peptide binding domains (Exons 2 and 3 for Class I, and 2 for class II). With this method, true novel alleles were successfully discriminated from mistyped alleles. Conclusions Compared to legacy HLA typing assays, NGS assays often sequence larger portions of the HLA loci, with full-locus coverage for many HLA genes. This presents a challenge to the analysis software, which must align patient data to the incomplete IMGT/HLA database. The use of imputed data helps with the computational algorithms, but can introduce its own problems when correctly-typed alleles are excluded due to mismatches to imputed data. The process described here overcomes this limitation, ensuring the correct typing of all HLA alleles present in the IMGT/HLA.

Aim Autoimmunity introduces a variety of difficulties in interpretation of solid phase HLA antibody testing due to autoantibodies present in patient serum. However, because autoantibodies may be present without obvious symptoms or a clear diagnosis, this information is always available when interpreting HLA test results. The current study sought to establish a mechanism for detecting these patients and generate an algorithm for handling samples with autoantibodies. Methods We retrospectively searched the electronic medical record to identify patients with autoimmune disease who were tested by our lab for HLA alloantibodies, and further cataloged any issues encountered during testing of these patients’ samples. This information was subsequently used to formulate an algorithm for testing autoimmune samples. Results A total of 214 patients with diagnosed autoimmunity were identified, including patients with organ-specific autoimmune conditions, most commonly type 1 diabetes mellitus (n = 43, 20%), and systemic autoimmune conditions, most commonly systemic lupus erythematosus (n = 29, 13.6%) and Sarcoidosis (n = 6, 2.8%). The most common issues encountered during testing were found to be high background on solid-phase antibody testing (n = 38, 17.8%), difficulties in assigning Ab specificities (n = 10, 4.7%), many allele specific antibody assignments (n = 5, 2.3%); and antibodies against self HLA antigens (n = 1, 0.5%). A workflow was designed to detect these patients and handle their samples (Fig. 1). Conclusions Using patients with diagnosed autoimmunity, we identified a high rate of interference of autoantibodies with solid phase HLA antibody assays. Extrapolating these results to a testing algorithm, we hope to be able to more readily detect and document patient samples that require special handling for autoantibody interference, even in the absence of an official autoimmunity diagnosis. Download : Download high-res image (170KB) Download : Download full-size image

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.

Aim The development of de novo donor-specific antibodies (DSA) post-transplant is an area of interest for the transplant community. There are several challenges in detecting DSA, including the use of recombinant HLA antigens vs. affinity-purified antigens from a phenotype, and the lack of donor typing at many loci. Herein, we describe a case where surrogate flow-cytometric crossmatch (FCXM) was used in conjunction with solid-phase immunoassays for the detection of DSA in a heart transplant recipient. Methods Post-transplant serum was tested by solid-phase immunoassays for the detection of DSA to the following donor antigens: A2,23; B7,8; Bw6; Cw7; DR15,17; DR52; DQ2,6. Confirmation of antibody specificity was obtained using FCXM using three surrogate donor cells. Results Initial testing by One Lambda LABScreen® PRA and Single Antigen assays provided negative results for HLA class I, and inconclusive results for HLA class II, suggesting possible reactivity to HLA-DQ2, HLA-DQA1∗05:01/DQB1∗02:01, or false-positive reactivity. In order to confirm the possible specificities obtained, surrogate crossmatching was performed utilizing pronased cells from donors with the following HLA types: Donor 1- DQB1∗02:XX, 03:XX (DQ9); DQA1∗02:01; Donor 2- DQB1∗02:01; DQA1∗05:01; Donor 3- DQB1∗02:01, 03:01 (DQ7); DQA1∗05:01, with recipient serum at dilutions of Neat, 1:2, 1:4, and 1:16. T cell crossmatch results at all dilutions were negative for each donor cell tested. Donor 1 tested B cell negative at all dilutions tested, while donor 2 tested positive at serum dilutions of Neat, 1:2, and 1:4, and donor 3 tested positive at serum dilutions of Neat and 1:2. This evidence allowed for us to assign a strong antibody, defined by our laboratory as predicted to yield a positive FCXM, to the DQA1∗05:01/DQB1∗02:01 heterodimer. Since donor HLA-DQA1 typing was not available, it is inconclusive whether the antibody detected should be considered donor-specific. Conclusion We conclude that while solid-phase immunoassays are useful tools for the detection of HLA antibodies, there are several challenges present which could require the use of additional assays for confirmation of test results. In addition, the lack of complete donor typing often makes it difficult for the laboratory to accurately interpret the findings of such results.

Aim Next-generation sequencing (NGS) assays for HLA typing are rapidly being introduced into clinical practice. For common NGS assays, sequenced libraries consist of a mixture of labeled DNA fragments from multiple amplicons and samples. During our validation of a commercially-available NGS assay, we found sample and locus representation varied considerably, compromising data quality. In order to ensure even amplicon and sample loading, we optimized the locus and sample pooling steps. Methods The NGSgo HLA typing kit (GenDx) was used to type the HLA-A, B, C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1 and DPA1 loci, with modifications to the manufacturer’s protocol described below. After PCR amplification, each amplicon was quantified using Quantifluor One dsDNA dye (Promega). Equimolar amounts of each amplicon from a single sample were pooled, and fragmentation, adapter ligation, and indexing steps were performed per the manufacturer’s protocol. Before pooling to a single library, each patient sample was quantified by two Methods KAPA Library Quantification (Kapa Biosystems) for the number of viable DNA libraries, and TapeStation gel electrophoresis (Agilent) for fragment size measurement. These two metrics were used to estimate equimolar pooling of each patient sample into a final library pool before being loaded onto a MiSeq (Ilumina) for sequencing. Results After introduction of quantification of all amplicons and equimolar pooling, locus representation on a per-sample basis was much more even; loci ranged from 0.24 to 3.94 times ideal representation using the manufacture’s protocol, but only 0.70 to 1.5 times ideal representation after equimolar pooling. Likewise, sample-level variation in pooling was extreme using the manufacturer’s protocol (ranging from 0.26 to 3.39 times ideal representation), and was much more even after addition of KAPA and TapeStation quantification of individual samples (ranging from 0.76 to 1.19 times ideal representation). Conclusions Using the modifications described here, we were able to obtain evenly-loaded samples, which allowed for the use of smaller, less expensive flow cells, or the use of a shared portion of a larger flow cell. Together, these modifications enhance the data quality for all patient samples while reducing the overall cost of the NGS assay.

Aim The United Network for Organ Sharing (UNOS), the sole contractor for deceased-donor transplantation in the US, requires HLA type in serologic nomenclature. Currently, however, the vast majority of patients and donors are typed by molecular methods. The 2008 HLA dictionary (hla.alleles.org/dictionary), which lists serologic assignments for many HLA-A, B, C, DRB1, DRB3/4/5 and DQB1 alleles, is used to “translate” molecular typing to serologic equivalents. However, many HLA alleles have multiple or conflicting entries in the HLA dictionary, while many rare or newly-identified alleles are not present. Moreover, UNOS accepts many “serologic” designations that do not appear in the HLA dictionary. To overcome these limitations, we created an algorithm to compile an internal document for UNOS-specific HLA type. Methods The 2008 HLA dictionary and Organ Procurement and Transplantation Network standards were used as primary sources for HLA typing information. Predicted amino acid sequence was used to assign Bw4/w6 (Tissue Antigens 2001: 57: 474). Results The algorithm, summarized below, was used to determine serologic assignments, which we termed “UNOS equivalents.” Expressed DRB3, DRB4 or DRB5 alleles were listed as DR52, DR53, or DR51, respectively, regardless of HLA dictionary assignments. Similarly, HLA-C alleles with first-field numbers greater than 08 were assigned UNOS equivalents corresponding to their first-field numbers, regardless of HLA dictionary assignments. For other alleles, the “Expert” assignment was given precedence over the “WHO” assignment listed in the HLA dictionary. Alleles not appearing in the HLA dictionary were assigned UNOS equivalents based on the first-field number. For alleles with multiple or conflicting entries, an algorithm was written to result in a single UNOS equivalent. For B∗15 alleles with unknown Bw4/w6 assignments, a published algorithm was used. Conclusions The algorithm described here resulted in a document that standardizes the “translation” of HLA alleles to list in UNOS, so that a single UNOS equivalent can be obtained from any HLA-A, B, C, DRB1, DRB3/4/5, or DQB1 type. As subsequent serologic studies are published, this document can serve as a framework for determining typings to enter into UNOS, to facilitate efficient sharing of deceased donor organs.

Umbilical cord blood (UCB) stem cells are frequently employed for allogeneic stem cell transplant, but delayed myeloid and lymphoid immune reconstitution leads to increased risk of infections. We recently reported the clinical results of 45 patients enrolled on a pilot study combining UCB with a human leukocyte antigen (HLA)-haploidentical donor with reduced-intensity conditioning who showed rapid neutrophil and platelet recovery. We report here preliminary immune reconstitution data of these patients. Patients were assessed for lymphocyte subsets, T-cell diversity, Cylex ImmuKnow assay and serological response to pneumococcal vaccination. Natural killer (NK)-cell and B-cell reconstitution were rapid at 1 month and 3 months, respectively. T-cell recovery was delayed, with a gradual increase in the number of T-cells starting around 6 months post-transplant, and was characterized by a diverse polyclonal T-cell repertoire. Overall, immune reconstitution after haplo-cord transplant is similar to that seen after cord blood transplant, despite infusion of much lower cord blood cell dose.

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.

Summary Background and objectives This study characterizes the pathologic and clinical relationships of thrombotic microangiopathy (TMA) to antibody-mediated rejection (AMR) in renal allograft biopsies. Design, setting, participants, & measurements Consecutive renal allograft biopsies, routinely stained for C4d over a period of 51 months (n = 1101), were reviewed. For comparative analysis of histology and clinical features, additional patients with TMA and peritubular capillary (PTC) C4d (n = 5) were combined with those identified in the 51-month period of review (n = 6). Results One hundred eighty-two of 1073 adequate biopsies from 563 allografts had PTC C4d in the study period. Six of 37 biopsies with TMA had PTC C4d (five at ≤90 days and one at 213 days). Early (≤90 days) C4d+ biopsies (n = 5) had more frequent TMA (11.9% C4d+ versus 3.4% C4d−; odds ratio, 3.84; P = 0.03). Graft loss was significantly greater in an early C4d+TMA+ group (n = 5 study + 2 archival patients) than in C4d+ controls without TMA (n = 21) (57% versus 9.5%; P = 0.02). Early TMA+C4d+ biopsies had more severe glomerulopathy and less severe arteriolopathy than TMA+C4d− and had more frequent neutrophilic capillaritis than TMA−C4d+ biopsies. Conclusions TMA was infrequent in this series of unselected, consecutive, renal allograft biopsies (3.4%). PTC C4d may be a significant risk factor for early TMA, and TMA is associated with glomerular thrombi and neutrophilic capillaritis. TMA in allografts with suspected AMR may portend a higher risk of graft loss.

Combined liver-kidney transplantation has become a common practice for the treatment of patients with concurrent end-stage renal disease and end-stage liver disease. Liver transplantation in the setting of multiorgan transplantation is thought to have a protective effect against humoral rejection even when a positive crossmatch is obtained prior to surgery. In most centers, a pre liver-kidney transplant crossmatch is rarely performed because of the known immunoprotective effect of the liver allograft. In this report, a case of acute humoral rejection in the kidney allograft after a combined liver-kidney transplant is described. Although humoral rejection was treated using plasmapheresis, intravenous immunoglobulin and rituximab, the kidney required 3 months to recover function and finally progressed to chronic allograft nephropathy. A heightened index of suspicion for acute humoral rejection of the renal allograft is necessary when performing combined liver-kidney transplants to highly sensitized patients due to previous organ transplants.

Aim Given the high throughput, better coverage, and free of phase ambiguity with HLA typing using next generation sequencing (NGS) technology, we decided to test one of the commercially available kits (Holotype HLA X4 ) offered by Omixon. Methods Total of 32 samples (27 PBMC derived patient DNA samples and 5 UCLA survey DNA samples) were sequenced by NGS for HLA-A, B, C, DRB1 and DQB1 using Holotype HLA X4. All samples were previously typed at high-resolution by combinations of SBT/SSP. This includes 23 HLA-A, 28 B, 21 C, 28 DRB1, 14 DQB1 CWD2.0 alleles and also 2 non-CWD and 2 novel alleles. With Omixon HLA X4 kit, both individual PCR amplicon barcode and pooled amplicon barcode strategies were evaluated on Illumina MiSeq analyzer. Results Long-range PCR gave robust amplifications on all samples for HLA-A, B, C (full coverage) and DRB1 (exon 2–4). DQB1 set 2 PCR (exon 2–5) had a failure rate of 3% (1/32) and DQB1 set 1 PCR (exon 1–4) had a failure rate of 21.9% (7/32, 5 of them from UCLA survey samples). The accuracy of NGS typing was 100% concordant with previous typing for HLA-A, B, C in both PCR amplicon individually barcoded and pooled amplicon barcoded format. Accuracy dropped to 97% for DRB1 and DQB1 assignment in both formats due to technical errors (1/32) or failed DQB1 PCR (1/32). Non-CWD alleles as well as novel alleles were correctly identified. No ambiguities were present in any of the final typing assignments. Using default quality control matrix of 15 parameters from the HLA Twin software, 4/158 resolved HLA alleles gave “red” light in the two traffic light system for amplicon individually barcoded samples, compared to 7/158 alleles in pooled amplicon barcoded samples. Regardless, none of the sample with “red” flagged QC parameter compromised the final HLA typing assignment. Conclusions Our experience with Omixon Holotype HLA X4 kit indicates that HLA-NGS typing indeed has advantages over Sanger SBT method. The Omixon NGS library construction is easy to perform without noticeable bias introduction. The beta version HLA Twin analysis software is user friendly & performs well with both amplicon individually barcoded as well as pooled amplicon barcoded samples with extensive data quality monitoring. However, the robustness of DQB1 PCR requires improvement & the dual algorithm of HLA Twin software needs more transparency.

Aim One of the limitations of solid-phase HLA antibody assays is the false low/negative reading in a subset of samples with high titer/strength HLA antibodies, a phenomenon known as the prozone effect. The goal of this study was to compare the efficacy of ethylenediaminetetraacetic acid (EDTA) vs. dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect in solid-phase HLA antibody assays on the Luminex platform. Methods A total of 21 serum samples were tested by single-antigen bead assays (One Lambda), including 14 previously identified prozone samples (3 class I, 11 class II), 6 previously identified non-prozone samples with HLA antibodies (3 each for class I and II), and 1 negative sample. Serum was treated either by adding EDTA to a final concentration of 5 mM, or by incubating with 5 mM DTT for 30 min at 37 °C. To evaluate the efficacy of prozone effect reversal on HLA single antigen beads assay, fourfold serial dilutions, from neat to 1:256, were tested for each positive sample, using PBS as a diluent. Results Prozone effects were reduced in all 14 samples by either EDTA or DTT treatment. Results revealed sharp increases of MFI values in treated serum samples at neat, compared to non-treated samples. However, EDTA and DTT treatment were not equally efficient in overcoming the prozone effect. EDTA had nearly 100% reversal of prozone effect in all the tested samples, estimated by the ratio of MFI at neat to peak MFI at any given dilution. In contrast, the efficacy by DTT treatment ranged from 47% to 100%. The difference between the treatments was particularly notable in samples with strong prozone effects, defined by peak MFI values beyond a 1:16 dilution with untreated samples. Consequently, EDTA-treated serum samples showed virtually no further MFI increase when diluted, while some DTT-treated serum samples still had MFI increases when diluted, an indicator of residual prozone effect. For the negative control sample as well as non-prozone samples, comparable results were obtained from EDTA, DTT and non-treated serum. Conclusions Our study indicates that EDTA is more efficient in reversing the prozone effect, compared to DTT treatment. Furthermore, the stability of EDTA and the simplicity and robustness of serum treatment provide additional advantages over DTT for routine clinical testing.

Aim Sanger sequencing-based typing (SBT) requires multiple steps to sequence novel HLA alleles, as allele-specific (AS) primers are needed to separate novel from other encoded alleles. Next-generation sequencing (NGS) includes phase information, and has the potential to sequence novel alleles without AS primers. Here we compare the process of novel allele identification and sequencing using SBT and NGS assays. Methods Novel alleles were identified in peripheral blood during routine clinical testing using Atria SBT kits (Abbot Molecular). Germline encoding was verified by typing buccal samples. For Sanger sequencing of novel alleles, AS amplification primers were designed to take advantage of nucleotide polymorphisms between encoded alleles, and patients with homozygous loci were used to verify primer specificity. Amplified products were Sanger sequenced using custom or commercially-available primers. For NGS sequencing, the Holotype X4 (Omixon) kit was used in its default configuration. Results Four novel, germline-encoded HLA alleles were identified in three patients. Novel HLA-A and B alleles were sequenced using AS amplification primers and Sanger sequencing. A novel DQB1 allele previously identified but not resolved by SBT was sequenced using the NGS assay. A novel DQB1 allele not identified by SBT, as the polymorphism was outside the covered region, was identified and sequenced by NGS. The names HLA-A∗66:22, B∗07:238, DQB1∗03:180, and DQB1∗06:01:15 have been officially assigned by the WHO Nomenclature Committee. Conclusions While SBT assays used in the clinical setting are able to identify novel alleles, subsequent sequencing is a cumbersome and time-consuming process. Furthermore, SBT assays may miss novel polymorphisms outside of the exons covered by the assay. NGS systems, on the other hand, can identify and sequence novel alleles over a larger genomic region as part of the normal clinical workflow. However, we experienced issues with the Omixon NGS kit, including the inability to sequence buccal cell DNA, and difficulties amplifying the DQB1 locus, which could have impacted clinical workflow had NGS been the primary method of testing. As the emerging technology of NGS is introduced to complement established SBT assays, it is important to evaluate the strengths and weaknesses of each.

A single nucleotide deletion in codon 12 of the human leukocyte antigen HLA-DRB1*01 results in a novel null allele, DRB1*01:33N.

Background Donors specific anti-HLA antibodies (DSA) in renal transplantation have been associated with higher incidence of graft failure. Therefore, potential living donors cells are tested against recipient serum using flow cytometric crossmatch (FCXM) to determine if the presence of DSA will cause positive FCXM. Antibody determination prior to crossmatching potential donors allows for the determination of the virtual crossmatch (VXM) as either being positive or negative. Herein, we report a case where the FCXM did not correlate at all with the VXM. Methods Pre-transplant serum was tested by solid-phase immunoassays for the presence of unacceptable antibodies. Strength of the antibody is determined from mean fluorescence intensity (MFI) cut-offs established by the laboratory. Results An initial FCXM was performed with potential donor cells and recipient serum. Based on a single DSA with MFI of Conclusion Distortion of antigen integrity may have caused the B62 from not being detected by solid phase assay. Antibody testing using solid phase methods are helpful tests but one cannot completely relay on them when trying to determine antibody status of recipients waiting for transplant.

Background Identification of novel HLA alleles is a common finding in the HLA lab. This finding is particularly challenging in hematopoietic cell transplant (HCT) patients since may prevent them from finding HLA matched donors. In addition, somatic mutations can occur in blast cells of patients with hematological malignancies and are attributed to a mechanism of immune evasion. Herein, we describe the approach used at the University of Chicago Transplant Immunology Laboratory for patients with hematological malignancies who were evaluated for HCT work-up. DNA from recipients who carried novel HLA alleles detected in peripheral blood (PBL) were confirmed in recipient’s germline DNA obtained from buccal cells. Methods DNA was isolated from peripheral blood cells and buccal cells of 5 patients by automated and manual methods. The novel HLA-A, B, C, and DQB1 genes were amplified by polymerase chain reaction (PCR) using locus specific primers. Exons 2 through 4 for HLA-A, B, C, and exons 2 and 3 for HLA-DQB1 were sequenced by Atria sequencing kits, and analyzed by Assign 3.6 SBT Software (Abbot Molecular, Des Plaines, IL). Results Novel alleles were confirmed in buccal cells of patients 1, 2, 3 and 4. Patient 5 only carried the new allele in the blast cells. Patient 1 had a matched related donor (MRD) that carried the same polymorphism at HLA-DQB1 locus. Patients 2 and 3 did not have MRD, and due to presence of novel allele in the HLA-B locus, an 8/8 matched unrelated donor (MUD) could not be found. For patient 4, the polymorphism was located outside of the hypervariable region and was classified as a group of alleles which have the same antigen binding domains (P group). Patient 4 was most likely to find an 8/8 MUD due to this classification. For patient 5 the new allele was only present on DNA from somatic cells, did not affect donor searches, and multiple MUD were available through the National Marrow Donor Registry (NMDP). Conclusions Identification of novel alleles is a common finding that can affect HCT donor searches. Confirmation of genomic versus somatic nature of the polymorphisms through typing related relatives and/or DNA from an alternative source such as buccal cells might be of help for donor selection.

Aim Standardization of test methods helps provide consistent results with assays that have tech to tech, day to day or other variables that may affect results. Our aim was to validate a plate washer for antibody testing to normalize the washing technique used in our laboratory to determine if day to day and tech to tech variability could be prevented. Method The Biotek ELx50 is an automated plate washer that has the ability to perform washing methods using vacuum filtration. An incubation/washing program was created to provide a routine consistent washing technique to use when performing antibody testing. Results An extensive analytical validation took place by testing samples ( n = 20) that were previously tested by manual washing methods for phenotype beads, class I and class II single antigen beads (SAB) using a program created in the Biotek, Elx50. Using the mean florescence intensity (MFI) values we were able to determine specificity and sensitivity by comparing to the manual wash method by calculating the percent difference between the two methods and factoring in the average deviation of the negative control for both methods. The average specificity for phenotype beads, class I SAB and class II SAB was 99% or greater. The average sensitivity for the phenotype beads and class I SAB was 83% or better and class II SAB was 63%. We also looked at day to day variations and technologist to technologist variations. By comparing runs from two consecutive days of testing for the day to day testing variation, we were able to obtain 100% specificity for phenotype beads, class I SAB and class II SAB. The average sensitivity for the phenotype beads was 80%, class I SAB was 67% and class II SAB was 76%. As for technologist to technologist variation, the overall specificity for phenotype beads, class I SAB and class II SAB was 100%. The average sensitivity for phenotype beads, class I SAB class II SAB was 70% or better. Conclusion As seen by the results for both specificity and sensitivity, the Biotek ELx50 can be used to standardize solid phase assays by eliminating the variation seen with manual washing technique to provide a more consistent MFI value. The plate washer is also beneficial in decreasing hands-on time by 75%; therefore reducing turn-around time.

Aim Polymorphism of human leukocyte antigens (HLA) is the major barrier to hematopoietic stem cell transplantation (HCT). Advancement in DNA typing methods has significantly impacted donor selection for HCT recipients. The ImMunoGeneTics (IMGT)/HLA database, 7/25/2013, lists 9719 HLA and related alleles and continues to grow. We describe 3 patients with hematological malignancies who were evaluated for HCT work up. DNA from recipients A and B, carried single non-synonymous mutations that resulted in new alleles in DQB1 and A loci, respectively. In DNA from recipient C, a rare HLA-A allele was identified. Methods DNA was isolated from peripheral blood cells and buccal swabs by automated and manual methods. The HLA-A and -DQB1 genes were amplified by polymerase chain reaction (PCR) using primers specific for each loci and sequenced by Atria sequencing kits (Abbot Molecular, Des Plaines, IL) exons 2 through 4 for HLA-A and exons 2 and 3 for HLA-DQB1. Data was analyzed by Assign 3.6 SBT software (Abbot Molecular). Results DNA from buccal swabs confirmed each suspected mutation suggesting that mutations were carried by recipient’s genomic DNA and were not limited to malignant cells. Recipient A had a related sibling, which carried the same mutation and was 10/10 matched. Recipient B did not have related donors suitable for testing. The National Marrow Donor Registry (NMDP) search was unsuccessful in finding matched unrelated donors (MUD). For recipient C, 4 related donors were evaluated, 2 siblings, did not carry the mutation however, 2 sons, carried the rare allele. A search for MUD was initiated and 3 international donors were identified. Conclusions Identification of novel alleles are a common finding that can affect HCT donor searches. It is important to confirm the discovery by re-typing the loci from an alternative DNA source, especially in cases of patients with hematological malignancies. It is important that the laboratory works with the HCT program to identify best donors for these patients.

Abstract 467 Background: While donor-recipient disparity at HLA loci is associated with greater risk for severe acute graft vs. host disease (GVHD) and inferior survival after unrelated donor allogeneic hematopoietic cell transplantation (HCT), the impact of amino acid substitution (AAS) at peptide binding pockets of the HLA molecule is incompletely understood. Methods: Adult and pediatric patients who received myeloablative or reduced intensity/non-myeloablative first unrelated bone marrow or peripheral blood stem cell transplantation for AML, ALL, CML or MDS between 1988 and 2009 were included. Donors and patients were fully high resolution matched for HLA-A, B, C, and DRB1 (8/8) or had single mismatch (7/8) at one HLA class I locus. Among 7/8 donor-recipient pairs, cases were categorized based on the presence or absence of the AAS of interest at positions 9, 77, 99, 116, or 156 of the class I molecule. In multivariable analysis accounting for patient, disease, and transplantation variables, we studied the independent impact of AAS at these residues on risk for grade III-IV acute GVHD, chronic GVHD, treatment-related mortality, primary malignancy relapse, and overall survival. We compared 7/8 donor-recipient pairs with AAS of interest to 7/8 pairs without these AAS in the primary analyses. Additionally, we performed this analysis restricted to each HLA class I locus. Results: Donor-recipient pairs were 8/8 matched (n=5282), 7/8 with AAS of interest (n=1713), or 7/8 without AAS of interest (n=318). In multivariable analysis, AAS at position 116 was associated with increased risk for grade III-IV acute GVHD (HR 1.21, 1.04–1.42, p=0.0165). No other significant association was detected between AAS studied and clinical outcomes. In multivariable analysis restricted to each class I HLA locus, we detected the following: Among 7/8 matched pairs with mismatch at HLA-C, AAS at position 116 was associated with increased risk for severe acute GVHD (HR 1.42, 1.13–1.79, p=0.0031) and inferior OS (HR 1.2, 1.01–1.41, p=0.0343). AAS at position 99 was associated with increased TRM (HR 1.37, 1.11–1.69, p=0.0037). Of 7/8 pairs with mismatch at HLA-B, AAS at position 9 was associated with increased chronic GVHD (HR 2.19, 1.31–3.66, p=0.0029). Specific amino acid substitution pairs with frequency > 30 were tested for association with HCT outcomes. None met the significance level of 0.00125, pre-specified for multiple comparisons. Conclusions: These results support the concept that AAS at key peptide-binding residues in the HLA class I molecule are associated with increased risk for severe acute GVHD and lower survival. Disclosures: No relevant conflicts of interest to declare.

Abstract 3050 Approximately 30% of Caucasian and 70% of African-American hematopoietic stem cell transplant (HCT) patients are unable to find an 8/8 HLA matched unrelated donor. Mismatches at HLA-A, B, C, or DRB1 alleles reduce survival. Therefore, identification and avoidance of high-risk allele combinations and the associated amino acid substitutions (AAS) that negatively impact HCT outcomes may increase access to this treatment option and allow safer utilization of HLA mismatched donors. Using random forest (RF) analysis, our group has previously reported the AAS associated with 100 day survival (D100S) in single HLA class I mismatched, DRB1 matched recipient-donor pairs. We now extend that analysis to one year outcomes of overall survival (1y OS), disease free survival (1y DFS), transplant related mortality (1y TRM), and acute graft-versus-host disease (aGvHD) grades III-IV using the same clinical variables (recipient age, disease type, disease status, and gender match) and 389 AAS position and types (AASPT). The AASPT were defined by the HLA locus, amino acid position in the HLA class I protein and the actual AAS, e.g. locus: HLA-C, position: 97, type: tryptophan to arginine = C97_WR. Patients (n=2107) received myeloablative (99%) HCT as treatment for ALL, AML, CML, and MDS in early and intermediate stage of disease between 1988 and 2003. RF analysis, a tree-based method for classification was used to assign an importance score (IS), reflecting the association of each potential predictor variable with the outcome of interest. Logistic regression analyses were performed to determine the magnitude of the effect of each individual AASPT (n=600) relative to 8/8 matched cases (n=1507), adjusted for the four clinical variables. Using the criteria of n≥10, a relatively high IS (≥5) and a highly statistically significant odds ratio (p The most common alleles associated with all of the AASPT listed above are: HLA-C*01:02/02:02; 01:02/15:02; 02:02/01:02; 03:03/04:01; 04:01/16:01; 14:02/15:02; and HLA-A*02:01/02:05; 24:02/24:03; 29:02/30:01; 30:01/30:02; 30:01/32:01. To understand the potential biological significance of mismatched allele pairs in the molecular context of peptide antigen binding, computational models were constructed for the 10 most important AASPT. In silico screening of ∼500 unique peptides was conducted against each pair to determine differences in the peptide repertoire capable of binding to each mismatched molecule. We found that the mismatched pairs identified by RF as less permissive displayed greater loss in their ability to bind identical peptides compared to other mismatches in the binding groove predicted to be more permissive by RF analysis. Overall, the computational modeling suggests a different affinity and peptide binding repertoire between mismatched HLA molecules. Results from these analyses indicate that only a small number (6.4%) of AASPT clearly confer adverse outcomes in HCT patients with single HLA class I mismatched unrelated donors, and it is likely that these AASPT are responsible for differential binding of immunogenic peptides. Validation studies in an independent dataset are in progress. Additional prospective studies should be performed to refine HLA matching algorithms in the mismatched setting that may increase donor availability and minimize the adverse effects of donor HLA mismatching. Disclosures: No relevant conflicts of interest to declare.

Aim Presence of anti-donor HLA antibodues is a contraindication to transplantation in most solid organs. In contrast, most patients with high titer donor specific antibodies (DSA) and positive flow cytometric crossmatch (FCXM) still undergo liver transplantation. Antibody mediated rejection (AMR) may or may not develop after liver transplantation in such patients. We speculated that despite positive crossmatch results, cytotoxic function of existing and/or de novo DSA is what will ultimately predict development of AMR. Methods In this study we report two conflicting cases of liver transplant patients with high titer pre-transplant DSA and positive FCXM. Pre-transplant sera of patient A and B were retrospectively tested with a C1q assay to determine the cytotoxic function of DSAs. Results One patient (patient A) developed hyperacute AMR shortly after transplantation while the second patient (patient B) did not develop any signs of AMR and her post-transplant period was uneventful. In patient A, shortly after transplantation, serum amino transaminase and bilirubin levels became elevated. Serum HLA antibody testing showed high levels of class I and class II DSAs that resulted in positive T and B cell FCXM. Positive C4d staining of liver allograft biopsy indicated an episode of AMR. Patient was treated with plasmapheresis (days 1-9), Rituximab and IVIG (days 9-13) and Thymoglobulin (days 2-8). Levels of DSA and positive FCXM decreased overtime and 4 years post-transplant DSA antibodies were no longer present. In contrast, patient B did not develop any signs of post-transplant complications related to presence of HLA antibodies. Consistent with our hypothesis at the time of transplant, DSA of patient A were positive for C1q factor. In contrast C1q was negative on DSA of patient B. Conclusions We conclude that pre-transplant antibody testing and C1q assay should be considered as pre-transplant tests to be performed to liver transplant patients. Larger studies should be performed to confirm our findings.