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The impact of activating KIR (aKIR) and inhibitory KIR (iKIR) on OS, relapse-related mortality (RRM) and acute GVHD (aGVHD) was prospectively studied in 84 adults with high-risk hematologic malignancies receiving reduced intensity conditioning (RIC) T-cell depleted hematopoietic SCT (HSCT) from haploidentical related donors. In this clinical model, freedom from RRM is dependent on GVL effect. Patients were divided into myeloid (n=49) and lymphoid (n=35) malignancy groups. KIR-ligand and ligand-ligand models were studied in both GVH and rejection directions and statistically correlated with outcome measures. In the myeloid group, OS was higher (P=0.009) and RRM was lower (P=0.036) in patients missing HLA-C group2 ligand to donor iKIR. OS was higher if patients had >1 missing ligand (P=0.018). In lymphoid malignancy, missing ligand to donor KIR had no impact on OS or RRM. However, OS was better with donor aKIR 2DS2 (P=0.028). There was a trend towards shorter OS in recipient with KIR 2DS1, 2DS5 and 3DS1, although sample sizes were too small to provide inferential statistics. Findings in lymphoid malignancy patients should be further studied. These results suggest that the absence of appropriate HLA ligands in the recipient to donor iKIR may induce GVL without aGVHD in myeloid malignancy patients undergoing TCD-RIC transplants.

Aim The linkage between DPB1 and DPA1 was extensively studied in 5,944 European American stem cell donors (Hollenbach et al., 2012). Thirty-three DPB1 alleles and associated DPA1 were reported. In 2014, Voorter et al. reported the special association of DPB1∗105:01 with DPA1∗03:01 in contrast to DPB1∗04:02-DPA1∗01:03 association although both DPB1∗105:01 and DPB1∗04:02 belong to the same G group. Here we retrospectively reviewed 1938 renal recipients with DP typing and report new DPB1/DPA1 associations frequently seen in our cohort. Methods HLA-DPB1 and DPA1 typing was performed by rSSO (One Lambda, Thermo-Fisher). Haplotypes were inferred by Clark’s method published in 1990. Results We identified 11 DPB1 alleles whose association with DPA1 has never been reported previously (Table 1). Five DPB1 alleles, DPB1∗124:01, 126:01, 131:01, 350:01 and 352:01, are not in the 2013 CWD list. Of note, DPB1∗131:01 was detected at high frequency (2.9%) in our cohort. The association of these five DPB1s with DPA1 was the same as the most frequent allele in the same G group, e.g. DPB1∗126:01 and DPB1∗350:01 are both associated with DPA1∗01:03 the same as DPB1∗04:01. Six DPB1 CWD alleles observed in our cohort were not reported in Hollenbach’s study. In 2015, Aaron et al. reported significant variation of DPB1 frequency in four major ethnic groups. In their study, DPB1∗29:01, 39:01, 40:01, and 85:01 were much more frequent in African American than other three ethnic groups. High percentage of African American in our renal recipients (44%) may explain the detection of these alleles in our cohort. Interestingly DPB1∗106:01 was associated with DPA1∗02:01, unlike PB1∗19:01 which is in the same G group however is associated with DPA1∗02:02. Conclusions We reported 11 new DPB1/DPA1 haplotypes frequently seen in DP low resolution typing. Five non-CWD DPB1 alleles were identified frequently. The enrichment of some alleles in our cohort could be attributed to high incidence of African American in our patients. Download : Download high-res image (190KB) Download : Download full-size image

Introduction Solid phase HLA antibody detection is more sensitive and specific compared to traditional CDC tests. Different solid phase methods use different platforms. FlowPRA uses 30 beads coated with HLA antigens of 30 individuals representing the most common HLA antigens. Luminex single antigen bead assay (LSA) uses up to 100 beads, each of which is coated with antigen molecules of a single specificity, and has much better coverage of the HLA antigens. Aims This study was to determine a correlation between FlowPRA and LSA in regarding if a negative FlowPRA would correlated to a negative LSA assay. Methods We use FlowPRA for screening and LSA for identification. Every new heart and lung transplant candidate is evaluated for presence of HLA antibodies by performing both FlowPRA and LSA up front. The routine cutoff of LSA is MFI = 1000. For this study we retrospectively reviewed records of FlowPRA and LSA performed in 100 new heart transplant candidates evaluated between April – December 2013. Results Of these 100 heart transplant candidates, 37 had either class I and/or class II LSA detectable antibodies. Among these 37 patients, 32 had LSA class I antibodies, 14 had LSA class II antibodies, and 9 had both class I and II antibodies. Among these 32 patients with positive LSA class I antibodies, only 20 demonstrated a clear positive FlowPRA. Class I FlowPRA of the other 12 patients with positive LSA appeared more likely to be negative. Among the 14 class II LSA positive patients, 7 patients’ FlowPRA appeared more likely to be negative. The majority of these HLA antibodies detected by LSA, but that did not cause a clear positive FlowPRA, had an MFI under 2000. A few of these antibody bindings were confirmed to be not reactive to antigens on cell surface in crossmatch. Conclusion The antigen density on LSA beads may be higher than on the FlowPRA beads which may explain that some antibodies may be detected by LSA but not FlowPRA. It could also not be excluded that some of the antibodies detected only by LSA might be non-specific which need further clarification. The clinical relevant of the weak antibodies detected only by LSA warrantees further studies. It is meaningful to test the HLA antibody using a combination of different detection platforms, especially for patients who will be transplanted using donors selected based on a virtual crossmatch.

Aim DQB1∗03:19 allele has been reported to be associated with DRB1∗11:01 or DRB1∗11:02 allele. Here, we report its extended association with DRB1 and DQA1 in our local population. Materials and methods Randomly selected 1425 reports from our routine HLA typing were analyzed for DQB1∗03:19 allele association with DRB1 and DQA1 alleles. High and intermediate resolution of HLA typing was performed by SBT, SSP and rSSOP methodology. The most common alleles were selected if intermediate resolution typing results had ambiguities in DRB1 locus. Results DQB1∗03:19 allele has been detected in 86 out of 1425 individuals, which consists 6% of the population analyzed. Among the DQB1∗03:19 individuals, the most frequent DRB1 alleles were DRB1∗08:04 (42 out of 86, 49%) and DRB1∗11:02 (32 out of 86, 37%). Among the DRB1∗08:04-DQB1∗03:19 individuals, 37 had been DQA1 typed, of which 36 carried a DQA1∗04:01. Among the DRB1∗11:02-DQB1∗03:19 individuals, 29 had been DQA1 typed, of which 28 carried a DQA1∗05:CMM as shown in Table 1. 73 out of 84 individuals with known ethnicity among DQB1∗03:19 population were identified as African American descendants and only 10 and 1 of them were Caucasians and Native American, respectively. No ethnicity was reported in 2 individuals (Table 2). Conclusion Based on our observations, we can demonstrate the presence of the following two most likely haplotypes DRB1∗08:04-DQB1∗03:19-DQA1∗04:01 and DRB1∗11:02-DQB1∗03:19-DQA1∗05:CMM. Both of these two most likely haplotypes were mainly observed among African Americans in North Carolina.

Aim Major ABO incompatible bone marrow transplantation can cause Pure Red Cell Aplasia (PRCA), which can evolve into AML. We will present here incomplete reactions of missmatched donor allele(s) in relapsed AML patient with a history of ABO incompatible, haplo-identical HSCT. Methods High and low resolution HLA typing was performed by SBT and rSSO, respectively. Post-transplant engraftment analyses were conducted with STR. Results Patient is a 35 y/o, “O” blood group female with AML, who had HSCT from her “A” blood group sibling in Oct 2009. Patient’s initial HLA class I and II typing was homozygous. Her sibling was haplo-identical with 6/12 mismatch in host vs graft direction. Engraftment testing performed in Jan 2010 revealed more than 98% donor allele composition in un-fractioned whole blood, as well as in CD3+ and CD15+ populations, which was then decreased in Feb 2010 and recovered again after receiving donor lymphocyte infusion (DLI) in Aug 2010. BM biopsy of Feb 2010 revealed that 30–40% cellularity with M:E ratio greater than 10:1. Sustained erythroid hypoplasia with intact myelopoiesis and megakaryopoiesis raised a possibility of acquired PRCA. HLA typing in Jan 2010 showed full engraftment. However, following low resolution HLA verification typing performed by rSSO in Apr 2010 using buccal sample could not call mismatched alleles in HLA-A, B and DQB1 loci. In HLA-A and HLA-B loci, multiple beads presented weaker reactions than positive cut off value. To be heterozygous in DQA1 and DQB1 loci, 2 beads should be positive, however, they were too weak to be adjusted as positive. When HLA typing was tested by SBT in parallel, one DQB1 mismatched allele was still missing. Despite of DLI and other treatments, patient expired in a year after HSCT. Conclusions Summary: Both low and high resolution HLA typing methodologies revealed incomplete reactions of donor alleles (s) in relapsed AML patient, which supports other clinical findings of disease relapse.

The anti-Factor VIII (F.VIII) antibodies produced by hemophilic patients have the potential to be directed against many different epitopes on the F.VIII molecule. Although first recognized as inhibitors of coagulation factor activity these antibodies may or may not interfere with F.VIII bioactivity.1,2 Indeed, as has been suggested by Nilsson and co-workers, antibodies without inhibitory reactivity may play a critical role in the generation and/or maintenance of tolerance in hemophiliacs.3 At the same time, many individuals with severe hemophilia A may never develop inhibiting antibodies (inhibitors), even after hundreds of days of replacement therapy. What factors are important in determining which individuals with hemophilia A will have an immune response to F.VIII and become refractory to continued replacement therapy? To date, there are no clear answers. It is clear that inherited factors both at the F.VIII locus and at other loci, possibly associated with the immune response, play a role in inhibitor production. This brief review will try to summarize some of the more recent information on how 1) the structure of the F.VIII protein, 2) the nature of F.VIII mutation that results in hemophilia A, 3) additional genetic factors (at the HLA complex locus) may affect the probability of an individual with severe hemophilia (F.VIII:C >1%) developing antibodies to F.VIII, and 4) how the clonality of the immune response to F.VIII varies with time.

Aim Single antigen bead (SAB) assay allows in-depth characterization of HLA antibody specificities. In addition to detection of antibodies against natural HLA antigens, the SAB may also detect antibodies against denatured HLA antigens. Recently, we observed antibody reactivity to all DR antigens (pan-DR) in serum samples of two transplant candidates using the Luminex SAB assay. This study was to characterize the pan-DR antibodies detected by Luminex SAB on two serum samples. Methods Serum samples received from two transplant candidates were analyzed in the study. Initial antibody screening and specificity tests were performed using Flow PRA and Luminex SAB. Additional tests using Flow SAB, Flow phenotype bead assay and Flow T and B cell crossmatch were performed to confirm the presence of HLA antibodies. Results Flow PRA was clearly negative for both samples, while Luminex SAB surprisingly revealed antibody reactivity to all DR antigens. No antibodies to DQ and DP were detected. However, no HLA antibodies were detected by Flow phenotype bead assay. In the Flow SAB assay, patient #1 showed reactivity with majority of the DR antigen beads, while patient #2 showed no any binding reactivity. The crossmatch with a donor cell carrying DR4, DR15, DR51, and DR53 was completely negative for both serum samples. No specific antibody removals were observed on serum samples adsorbed using the donor cells in a Luminex SAB assay. Conclusions Flow PRA and Flow phenotype beads are coated with natural HLA antigens extracted from cell lines, while SABs are coated with recombinant HLA antigens. The antibody binding detected by SAB, but not by FlowPRA and Flow phenotype, may indicate the presence of antibodies against denatured antigens. Our results suggest that the pan-DR antibodies detected in these patients were possibly antibodies against denatured HLA antigens, more likely to a shared epitope uncovered on denatured DR antigens. Antibodies to denatured antigens would not result in crossmatch reactivity.

Women who conceive as a result of rape often elect to abort the fetus. We describe twelve cases where genetic markers were tested on the aborted fetal material to provide evidence of the genetic constitution of the rapist. Two cases are presented in detail, and the problems encountered with the testing are discussed.