Presence Of HLA Abs Research Articles

P148 Uncovering antibodies to cryptic epitopes: Use of flow cytometry crossmatch to differentiate between functional and denatured epitopes

HLA antibody (Ab) detection and characterization is a critical step in transplantation. With high sensitivity and allele level Ab identification, the Luminex-based single beads assay is an essential tool in all HLA labs. One of the challenges with the current testing is the potential detection of cryptic or not-physiologically expressed epitopes. Here we report an interesting case of cryptic epitopes and describe the decision making process in preparation for virtual crossmatch requests. A 2 y/o female patient being evaluated for small bowel transplant was tested for presence of HLA Ab. Her class II profile was positive for all DR except DR10, with strongest reactions to alleles with epitopes 98E (DR4,7,9) and 48YQ6 (DR53). The patient typed as DR7,10,53 and the sample was immediately flagged as a possible false pattern of reactivity. Single antigen beads from 2 different vendors showed similar profiles; the same reactivity pattern was also detected by multi-antigen beads. Upon dilution the Ab profile was weaker but similar to the undiluted sample (figure). The pattern “all DR but DR10” could be explained by the patient carrying a rare DR7 allele or a non-expressed DR7 variant. However, no patient cells were available for serological DR7 detection. Molecular typing by SSO and/or SSP may detect a rare DR7 but could not exclude the possibility of a SNP or frameshift mutations creating a premature stop codon behind exon 2. Therefore, the sample was tested by NGS. By sequencing past exon 2 we confirmed that the recipient expresses DRB1∗07:01:01 and no premature stop codons were present in exons 3 or 4. To determine the functional characteristics of the Ab, surrogate donors were tested by flow cytometry crossmatch (FCXM). One donor was DR7 homozygous and the other was DR4, DR7. No reactivity was detected against these donors’ DR antigens, despite the presence of 4 potential targets (figure). This case demonstrates the usefulness of additional testing by FCXM to confirm the presence of false pan-DR reactivity, probably due to a denatured epitopes. Download : Download high-res image (533KB) Download : Download full-size image

Potential hyperacute renal rejection due to non-hla antibody in a pediatric patient

Non-HLA antigen targets have been associated with hyperacute, acute, and chronic kidney allograft rejection. Although there is controversy regarding the predictive role of non-HLA antibodies (Abs) in transplant (Txp) outcome - there are cases that indicate causality between the presence of such Abs and graft loss. A 17 month old boy with end-stage kidney disease, secondary to kidney dysplasia, received a living donor Txp from his uncle in April 2014. The presence of HLA abs was tested in 5 sera samples prior to Txp with a PRA of 7% and 0% for class I and II respectively (flow PRA). Single antigen testing confirmed the absence of donor specific HLA Abs (DSA). Initial and final flow cross matches were negative, as expected. During the Txp surgery, it was noted that the kidney became firm, urine output was below expectation and the child became increasingly pressor-dependent. Subsequently, the patient had an episode of ventricular tachycardia and an exploratory laparotomy demonstrated that the Txp kidney became tense and enlarged, with thrombosis of the renal vein. The kidney was nephrectomized within 48 hours and the patient’s status improved significantly. To investigate a potential role for HLA DSA, we tested a 2 days post-Txp serum sample, confirming the absence of HLA-DSA. We also repeated Abs testing on the pre-Txp serum sample ruling out HLA-DSA as the cause of rejection. To assess potential causes for this apparently hyperacute Ab mediated rejection, pre-Txp (pre-14 days) and post-Txp (post-24 days) samples were sent out for AT1R Abs screening and donor specific endothelial cell crossmatch (XM-One). Retrospective testing of a pre- and post-Txp sera for AT1R antibody was out of range for pre-Txp sample and the post-Txp sample was positive ( > 30 Units/ml). The XM-One assay using endothelial precursors isolated from the donor as targets was strongly positive using a pre-Txp serum but negative using post-Txp serum. Approximately two month’s post-Txp, the patient developed HLA Abs, on top of the AT1R antibodies. In conclusion, this patient still requires a kidney Txp and the question is how to proceed – shall we consider only obtaining an organ from a living donor? This would allow for the necessary time to run XM-One and potentially inhibit AT1R pathway with an AT 1 receptor antagonist. What to do if no living donor becomes available?

Poor HLA Matching and Post-Operative Blood Transfusions Are Associated With the Development of De Novo DSA Post Renal Transplantation.

Introduction. De novo donor specific antibodies [dnDSA] are associated with antibody mediated rejection and allograft loss. In the first study of its kind, we have undertaken a detailed multivariate analysis of pre- and post- transplant factors associated with the development of dnDSA. Methods. We retrospectively analysed 871 ABO/HLA compatible renal transplant recipients who received a tacrolimus based immunosuppressive protocol with monoclonal antibody induction. Results. 159/871[18.3%] patients developed dnDSA. Pre-transplant factors associated with dnDSA on univariate analysis were the presence of HLA Abs [OR: 1.48(1.0-2.17), p=0.049], being mismatched (MM) at both the class I and II HLA loci [OR: 3.84(2.52-6.36), p<0.01] and having a DQ HLA MM [OR: 2.96(1.92-4.54), p<0.01]. Post-transplant factors analysed included blood transfusion [OR: 2.15(1.33-3.49),p=0.002], delayed graft function [OR: 1.25(0.8-1.95),p=0.33], culture positive infection [OR: 0.95(0.63-1.42),p=0.80], non-compliance (assessed by the coefficient of variance of tacrolimus levels) [OR: 1.29(0.9-1.84), p=0.17] and biopsy proven rejection [OR: 1.81(1.16-2.84),p=0.01]. On multivariate analysis, factors found to be independently associated with dnDSA are shown below.Table: No Caption available.Conclusion. Poor HLA mismatching and post-operative blood transfusions are strongly associated with dnDSA and are therefore potentially avoidable.

No Effect of HLA Antibodies Detected by Luminex Based Single Antigen Tests Only, on Kidney Survival, Graft Function or Acute Rejection

Introduction: There are conflicting reports about the risk of kidney transplants performed in the presence of donor specific HLA Antibodies (Abs) that are only identified with Luminex single antigen testing. Furthermore there are reports that other non-DSA HLA Abs might also carry some additional risk. Our experience has shown in the past that such transplants can be performed safely. Aim and Methods: This was a prospective study to investigate in a series of consecutive kidney transplant patients performed in the knowledge of a negative crossmatch if the presence of HLA Abs that were Donor specific or otherwise resulted to an increased incidence of graft failure, acute rejection or to a worse kidney function as measured by creatinine and eGFR. Pre-transplant sera from 343 adult renal transplant recipients transplanted during a 45 month period were investigated for the presence of antibodies to HLA using Luminex LABScreen Mixed. Mixed positive sera were further tested by LABScreen Single Antigen for determination of HLA specificity. There was a minimum follow up of three years. A significant number of those patients did not receive any antibody induction. Results: There were 213 patients with Luminex only Abs with a level over 500 out of which 85 had DSAs. There were 28 patients with DSA level over 2000 (9 class I and 19 class II) and 9 with level over 5000 all class II. There was no impact of the Ab presence (DSA or not) or the antibody level (as measured by the Single Antigen beads) on the kidney graft survival up to 3 years. Furthermore the rejection rate including borderline rejection was no different between the positive and the negative DSA groups either class I (22% in patients with DSA class I over 2000 vs 28% on those without) or class II (36% in patients with DSA class II over 2000 vs 27% on those without) and at any antibody level. The 6 months, 1 year and 2 years creatinine and creatinine clearance were similar between the positive and negative DSA groups again at any level of sensitisation. Conclusion:Transplantation in the presence of Luminexonly HLA Abs, in the absence of a positive crossmatch, is safe, resulting in equivalent graft survival, kidney function and even rejection rates up to 3 years post transplant. This is the largest prospective study of single antigen testing in kidney transplantation with a complete 3 year follow up.

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Aim The presence of HLA Ab in the allograft is associated with transplant vasculopathy (TV). Ligation of HLA class I elicits signaling events and induces the remodeling of actin cytoskeleton leading to cell survival, migration and proliferation in EC. However, the mechanism by which anti-HLA class II Ab contribute to TV development is not clear. We hypothesize that HLA II ligation induces signaling cascades mediating cytoskeleton reorganization. Methods To express HLA II, human aortic EC were infected with adenoviruses encoding Class II transactivator (Ad-CIITA). Expression of HLA II was determined by flow cytometric analysis using Ab against a monomorphic epitope on HLA II or locus specific Ab against HLA-DR, DQ and DP. Ad-CIITA infected EC were stimulated with anti-HLA II Ab and protein phosphorylation was examined by Western blot. Stress fiber formation was evaluated by fluorescence microscopy in EC stained with Texas Red-phalloidin. The FAK siRNA was utilized to deplete FAK expression. Results Infection with Ad-CIITA for 2d induced expression of HLA II in 80% of EC. Staining with locus specific Ab showed HLA-DR was expressed at the highest level with comparable levels of DP and DQ. Treatment of EC with HLA II Ab significantly increased the phosphorylation of FAK, Src, ERK and p70 S6 kinase (S6K). Ligation of HLA II also significantly augmented formation of stress fibers in EC compared to isotype control IgG [Fig. 1] Knockdown of FAK attenuated phosphorylation of Src, ERK and S6K and also abolished class II-mediated stress fiber formation. Download : Download full-size image Conclusions Ab ligation of HLA II molecules stimulates signal transduction and induces actin cytoskeleton remodeling in EC in a FAK dependent manner.

Impact of HLA Antibodies on Transplant Glomerulopathy

The influence of humoral rejection on the development of chronic allograft nephropathy (CAN) is controversial, especially in relation to transplant glomerulopathy. The aim of our study was to analyse the influence of anti-HLA antibodies on the development of transplant glomerulopathy ( cg0, cg1, cg2, and cg3; Banff’97). We selected all renal transplants patients from 1975 to 2003 who had a functioning graft for at least 6 months and a clinically indicated graft biopsy with CAN and chronic glomerular changes (case group). We studied the presence of anti-HLA antibodies (Ab) in the last serum taken while the graft was functioning and divided them into three groups according to the severity of glomerular lesions. We also selected 52 contemporary and comparable cases without transplant glomerulopathy (control group). A total of 77 case had transplant glomerulopathy: 39 cg1, 29 cg2, and 9 cg3. Pretransplant Ab titers and number of previous blood transfusions were higher among the subgroup with the most severe glomerulopathy. Patients who developed posttransplant anti-HLA Ab more frequently showed transplant glomerulopathy. Serum creatinine and proteinuria were higher among cases with chronic glomerulopathy, and more grafts were lost in that group. Thus, the presence of HLA-Ab is a key factor in the development of transplant glomerulopathy and chronic allograft rejection.

Vascular endothelial growth factor gene polymorphism are associated with acute rejection after heart transplantation

Lambda) for the detection of HLAab in serum/plasma of 54 patients. 10/54 patients exhibited a positive ELISA post-LTx (ELISA ). 9 ELISA patients were in the ACSAgroup and one patient with preformed donor-specific HLAab received ACSA for 4 months. The HLAab pattern was: 2 patients had both class I and II HLAab, 3 class II and 5 class I. 2/10 of these patients tested ELISA pre-LTx. In 6/9 patients we were able to identify donor-specific HLAab. De novo HLAab were developed in 5/10 ELISA patients. In 9/10 of ELISA patients, the presence of HLAab was associated with a P/R during the first 18 months, comparatively with only 2/14 in the ELISA-/ACSA group (p 0.0003) and with 13/30 in ELISA-/ACSA(p 0.01). There was a tendency for fewer P/R in ACSA /ELISArelative to ACSA-/ ELISApatients (p 0.08). The frequency of HACR episodes in ELISA group was significantly higher than in ELISAgroup (28/83 versus 36/340, 2 27.84, p 0.00001). Furthermore, the ELISA patients required increased immunosuppression ( 2 course of bolus steroids and/or antilymphocytic agents-p 0.005). Conclusions: The presence of ELISA-detected HLAab positively correlated with increased risk of severe acute rejection. These LTx patients may benefit from an altered strategy of immunosuppression. LTx recipients in the ACSA protocol exhibited the lowest prevalence of P/R.