Shared Molecular Eplet Stimulates Acute Antibody-Mediated Rejection in a Kidney Transplant Recipient With Low-Level Donor-Specific Antibodies: A Case Report

HLA antibodies usually recognize epitopes rather than antigens. This case report reveals that acute antibody-mediated rejection (AMR) that occurred in a kidney transplant recipient with low-level donor-specific antibodies (DSAs) could be explained by shared epitope. A 39-year-old woman received a first kidney transplant from a deceased donor (HLA-DRB1*11:06, *12:02, DRB3*02:02, *03:01). She developed acute AMR confirmed by kidney biopsy on day 4 after transplantation. Antibody testing with pretransplant serum showed anti-DR11 DSA below cutoff level (mean fluorescence intensity [MFI], 702; cutoff >1,000). However, high-level DSAs were detected on day 5 after transplantation (anti-DR11 MFI, 8,531; anti-DR12 MFI, 3,146). We hypothesized that the sharp rise in DSA levels was a result of anamnestic response with donor-antigen sensitization that occurred during pregnancy. High-resolution HLA-DR typing of her husband showed HLA-DRB1*03:01, *15:02:01, DRB3*02:02, DRB5*01:02. No sharing between donor HLAs eliciting reactive antibodies and her husband's HLAs was detected. Nevertheless, we speculated that shared epitope, not antigen, was the cause of allosensitization. To identify the shared epitope recognized by patient's antibodies, we used HLAmatchmaker, a computer algorithm that considers small configurations of polymorphic residues referred to as eplets as essential components of HLA epitopes for analysis. The results showed that 149H, which was the eplet shared by HLA-DRB1*03:01 (from her husband) and DRB1*11:06, DRB1*12:02, DRB3*03:01 (from donor), was the most prevalent eplet on DRB1 reactive alleles in Luminex assay. In conclusion, pretransplant low-level DSAs can induce AMR early after transplantation as a result of shared epitopes with a previous immunizer.     

Cytotoxic flow cytometric crossmatch in renal transplantation: a single assay to simultaneously detect antibody binding and cytotoxicity

Objectives

The complement-dependent lymphocytotoxicity crossmatch (CDC-XM) detects cytotoxic parameters of preformed antibodies. The flow cytometric crossmatch (FCXM) is used to detect the binding of recipient antibodies to donor cells. Because these two assays provide different information, both methods are often performed to assess the compatibility of donor-recipient pairs. The aim of this study was to develop a single assay that can simultaneously detect antibody binding and cytotoxicity.

Methods

A procedure called cytotoxic flow cytometric crossmatch (cFCXM) that determines cell death and antibody binding simultaneously was developed. The assay was validated in parallel with extended incubation CDC-XM. Receiver operating characteristic analysis was used to determine the cut-off level. Furthermore, pretransplantation sera from seven recipients with pretransplantation donor-specific antibodies (DSA) and negative CDC-XM were retrospectively tested for cFCXM (4 without antibody-mediated rejection (AMR) and three with AMR).

Results

The optimal method for the simultaneous detection of antibody binding and cytotoxicity in a single assay has been determined. Four of four patients (100%) with pretransplantation DSA and without AMR had negative cFCXM in both parameters. Of three patients with pretransplantation DSA who developed AMR, two patients (66.7%) had positive B-cell cFCXM in both parameters, and 1 patient (33.3%) had positive T-cell cFCXM in a binding parameter only. The first patient had anti-DR9, DR53, DQ9, the second patient had anti-A11, DR12 and the last one had an anti-B46 in their pretransplantation sera. These 3 cases experienced biopsy-proven AMR after living-donor kidney transplantation.

Conclusion

The newly developed assay, cFCXM, can simultaneously determine cytoxicity and antibody binding using a single platform. Furthermore, this assay can detect clinically significant HLA alloantibodies undetectable by conventional crossmatches. The cFCXM could serve as a new tool for the detection of a recipient's alloantibodies.     

Outcomes of transplantation with related- and unrelated-donor stem cells in children with severe thalassemia.

Recently published reports indicate that the outcome of unrelated donor transplantations in patients with leukemia is currently comparable to that of transplantation from identical family donors. We investigated the possibly favorable outcomes of related and unrelated transplantation in children with severe thalassemia. We reviewed transplantation outcome in 49 consecutive children with severe thalassemia who underwent allogeneic stem cell transplantation with related-donor (n=28) and unrelated-donor (n=21) stem cells between September 1992 and May 2005 at the Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Bangkok, Thailand). Analysis of engraftment, frequency of procedure-related complications, and thalassemia-free survival showed no advantage from use of related-donor stem cells. The 2-year thalassemia-free survival estimate for recipients of related-donor stem cells was 82% compared with 71% in the unrelated-donor stem cell group (P=.42). The present study provides evidence to support the view that it is quite reasonable to consider unrelated-donor stem cell transplantation an acceptable therapeutic approach in severe thalassemia, at least for patients who are not fully compliant with conventional treatment and do not yet show irreversible severe complications of iron overload.     

One-year experience of nucleic acid technology testing for human immunodeficiency virus Type 1, hepatitis C virus, and hepatitis B virus in Thai blood donations

BACKGROUND: Blood donations collected at the National Blood Center, the Thai Red Cross Society, Bangkok, in 2007 were tested by nucleic acid amplification technology (NAT) using the Chiron TIGRIS/Procleix Ultrio test and the Roche cobas s 201/cobas TaqScreen multiplex (MPX) test.
STUDY DESIGN AND METHODS: The sensitivity, specificity, and robustness were determined by testing 486,676 seronegative blood donations. Samples from each day of collection were divided into two sets; the odd-numbered samples were tested individually on the TIGRIS and the even-numbered samples were tested in pools of 6 on the cobas s 201. The status of reactive samples was confirmed by duplicate testing of samples from the plasma bag to calculate the test specificity. Reactive samples were tested on the alternate system and followed up.
RESULTS: The analytical sensitivity of both systems met the 95% limits of detection claimed by the respective package inserts. No cross contamination was seen with either system. Test specificity was 99.93 and 99.90% for the Procleix Ultrio and cobas TaqScreen tests, respectively. The NAT yield rates for human immunodeficiency virus Type 1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) were 1:97,000, 1:490,000, and 1:2800, respectively. Several occult HBV donors, the majority of whom were detected by both tests, were also identified. The HIV-1 and HCV window cases were detected with both tests.
CONCLUSION: The performances of the systems and tests indicated that both were acceptable for routine NAT by the National Blood Center, the Thai Red Cross Society. However, the Procleix Ultrio test appeared to be less sensitive than the cobas TaqScreen test for HBV.     

Vibrio cholerae O139 in Thailand in 1994.

Vibrio cholerae O139 first appeared in India and Bangladesh in 1992. Surveillance for O139 was started at three hospitals in Thailand in 1993. By 1994 all three hospitals surveyed in Thailand had experienced an increase in Vibrio cholerae O139 infections.     

Influence of HLA-DQ Matching on Allograft Outcomes in Deceased Donor Kidney Transplantation

Background and Objectives

HLA matching at the A, B, and DR loci influences the graft survival rate of deceased donor kidney transplants. The effect of HLA-DQB1 matching on transplant outcomes is still controversial. The aim of this study was to investigate the association of HLA-DQB1 matching with allograft outcomes in deceased donor kidney transplant recipients.

Occult hepatitis B virus infection in Thai blood donors

BACKGROUND: An evaluation by the National Blood Center, the Thai Red Cross Society, of two commercial multiplex nucleic acid tests (NATs; the Chiron PROCLEIX ULTRIO test and the Roche Cobas TaqScreen MPX test) for screening Thai blood donors for hepatitis B virus (HBV), hepatitis C virus, and human immunodeficiency virus Type 1 identified 175 HBV NAT–reactive/hepatitis B surface antigen (HBsAg)‐negative donors. The classification of the HBV infection of these donors was confirmed by follow‐up testing. STUDY DESIGN AND METHODS: Index samples were tested for HBV serologic markers and HBV viral loads were determined. Donors were followed for up to 13 months and samples were tested with both NAT assays and for all HBV serological markers. RESULTS: Of 175 HBV NAT–yield donors, 72 (41%) were followed. Based on the follow‐up results, the majority of donors who were followed had an occult HBV infection (66.7%), followed by donors with a primary, acute infection (26.4%). The majority of donors in this latter group (20.8%) were in the window period. Three donors (4.2%), who were anti‐HBs positive, had a reinfection or breakthrough infection. CONCLUSION: The majority of donors detected during routine screening, who were HBsAg negative and NAT reactive, had an occult HBV infection, thus validating the decision to introduce NAT for blood donations in Thailand.     

Significance of C1q-fixing Donor-Specific Antibodies After Kidney Transplantation

Objectives

De novo donor-specific HLA antibodies (DSA) are associated with allograft rejection and allograft loss. However, not all DSA are equally detrimental to allograft function. The ability to activate complement may be an important factor differentiating clinically relevant DSA from nonrelevant DSA. The C1q assay detects a subset of HLA antibodies that can fix complement. This study aimed to investigate the correlation between C1q-fixing de novo DSA (dnDSA) and clinical outcomes posttransplant.

Methods

This retrospective study included 193 sera from kidney transplant recipients who underwent posttransplant DSA testing and/or kidney biopsy for clinical causes. Thirty-five of the 193 (18.1%) had immunoglobulin G DSA. Seventeen of the 35 patients were excluded owing to the presence of pretransplant HLA antibodies. We then analyzed C1q DSA at the time of biopsy in 18 recipients who developed dnDSA. The clinical outcomes of patients with C1q-positive DSA and C1q-negative DSA were compared.

Results

C1q-positive DSA were detected in 10 of 18 patients (55.6%). The incidences of transplant glomerulopathy were significantly higher among patients with C1q-positive DSA than patients with C1q-negative DSA (80% vs 0%; P = .001). Although patients with C1q-positive DSA experienced more chronic antibody-mediated rejection and graft loss (80% vs 37.5% [P = .145]; 60% vs 25% [P = .188]), the differences were not significant. The receiver operating characteristic curve analysis showed that the C1q assay was an excellent predictor of transplant glomerulopathy with area under the curve of 0.9 (95% CI, 0.769–1.000).

Conclusion

The presence of C1q-positive dnDSA was associated with an increased risk of transplant glomerulopathy. The C1q assay is potentially a powerful method for identifying patients at risk for transplant glomerulopathy.     

Acute Antibody-Mediated Rejection by De Novo Anti-HLA-DPβ and -DPα Antibodies After Kidney Transplantation: A Case Report

The presence of isolated de novo anti-DP antibodies is uncommon, making it difficult to determine the impact of anti-DP antibodies on graft outcome. We describe a case of acute antibody-mediated rejection mediated by de novo donor-specific anti-HLA-DP antibodies. Furthermore, the generation of non–donor-specific anti-DP antibodies (NDSAs) detected in the patient's sera was investigated. An 18-year-old woman with pretransplant 0% panel-reactive antibody received kidney transplantation from a living donor. She experienced combined acute T-cell-mediated and antibody-mediated rejection at 15 months after transplantation. High resolution HLA typing of the donor and the patient revealed that they were mismatched at both DPB1 (DPB1*31:01) and DPA1 (DPA1*02:02) loci. The single antigen bead (SAB) testing of patient's sera revealed antibodies against donor's DPB1*31:01 and DPA1*02:02 alleles. Antibodies against several non–donor-specific DP antigens were also detected. No antibodies against other HLA class I and II antigens were detected. In order to explain the reactivity pattern of NDSAs, HLAMatchmaker program was used to identify immunizing eplets shared between donor alleles and reactive beads. The analysis showed 84DEAV, a DPB1 eplet, as a shared eplet found on DPB1*31:01 (mismatched donor allele) and on DPB1-reactive alleles in SAB assay. Additionally, 50RA, a DPA1 eplet, was identified as a shared eplet found on DPA1*02:02 (mismatched donor allele) and on DPA1-reactive alleles in SAB assay. This case highlights the clinical significance of HLA-DP antibodies. Furthermore, the generation of NDSA anti-DP antibodies by epitope sharing underscores the importance of HLA-DP epitope matching in kidney transplantation.