S. Scherer

Survival of DNA HLA-DR typed and matched cadaver kidney transplants

The clinical value of serological HLA matching for cadaver kidney transplantation remains uncertain because the success rate for HLA-matched cadaver transplants is lower than that of HLA-matched sibling grafts. Up to 25% of serological HLA-DR typings may be incorrect when compared with a more accurate DNA-RFLP method, and we have now examined whether incorrect HLA-DR typings account for the lower than expected success rates of HLA-matched cadaver transplants. 58 transplant centres took part in this study and DNA was extracted from over 4000 samples of frozen tissue at the study centre. 8 laboratories then completed blind RFLP typing for HLA-DR. Serological typing data were reported by individual transplant laboratories. 29 of 107 transplants (27%) that were reported as HLA A, B, DR compatible and 76 of 273 (28%) transplants that were reported as HLA B, DR compatible according to serological typing were found to be HLA-DR mismatched by DNA typing. The one-year transplant success rate for DNA-matched HLA, A, B, DR grafts was 87% compared with 69% for mismatched grafts (p less than 0.02); the corresponding success rate for DNA-matched HLA B, DR grafts was 85% compared with 72% for mismatched grafts (p less than 0.01). Many transplants that were previously thought to be HLA matched are mismatched, and this finding may account for previously unexplained graft failures.

No Association of Kidney Graft Loss With Human Leukocyte Antigen Antibodies Detected Exclusively by Sensitive Luminex Single-Antigen Testing: A Collaborative Transplant Study Report

Background. It is unclear whether kidney transplant recipients with preformed donor-specific human leukocyte antigen (HLA) antibodies (DSA) detectable only in the highly sensitive Luminex single-antigen (LSA) assay are at an increased risk of graft failure. Methods. We studied 3148 patients who received a deceased donor kidney graft between 1996 and 2008 and were enrolled in the prospective serum project of the Collaborative Transplant Study. There were 118 patients with graft loss during the first 3 years after transplantation on whom recipient and donor DNA was available for complete HLA typing. We compared the incidence of LSA-detected DSA in these patients with graft failure and matched controls with functioning grafts. All patients were found negative in the less-sensitive complement-dependent lymphocytotoxicity and enzyme-linked immunosorbent assays. Results. When mean fluorescence intensity (MFI) of greater than or equal to 1000 was used as a cutoff for Luminex positivity, 118 patients with graft loss did not show a higher incidence of DSA against HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, or -DPB1 antigens than 118 matched controls without graft loss (for all loci P not significant). The incidence of strong DSA (MFI ≥2000 or MFI ≥3000) detected only by LSA was low (for all loci between 0% and 5%) and did not identify unacceptable antigens that were relevant for graft loss within the first 3 years after transplantation. Conclusion. We conclude that, given currently practiced crossmatch procedures and immunosuppressive regimens, exclusion of donor organs carrying “unacceptable” HLA based exclusively on sensitive LSA antibody testing is not justified.

Influence of Test Technique on Sensitization Status of Patients on the Kidney Transplant Waiting List

The exquisitely sensitive single antigen bead (SAB) technique was shown to detect human leukocyte antigen (HLA) antibodies in sera of healthy male blood donors. Such false reactions can have an impact on critical decisions, especially with respect to the determination of unacceptable HLA‐antigen mismatches in patients awaiting a kidney transplant. We tested pretransplant sera of 534 patients on the kidney waiting list using complement‐dependent cytotoxicity (CDC), enzyme‐linked immunosorbent assay (ELISA) and SAB in parallel. Evidence of HLA antibodies was obtained in 5% of patients using CDC, 14% using ELISA, and 81% using SAB. Among patients without history of an immunizing event, 77% showed evidence of HLA antibodies in SAB. In contrast 98% of these patients were negative in ELISA and CDC. In patients without an immunizing event, SAB‐detected antibodies reacted not always weakly but with mean fluorescence intensity (MFI) values as high as 14 440. High‐MFI‐value antibodies were found in some of these patients with HLA specificities that are rather common in general population, consideration of which would lead to unjustified exclusion of potential kidney donors. False SAB reactions can be unveiled by testing with additional antibody assays. Denial of donor kidneys to recipients based on HLA‐antibody specificities detected exclusively in the SAB assay is not advisable.

Human Leukocyte Antigen Class II Allele Frequencies and Haplotype Association in Iranian Normal Population

The polymorphism of the HLA class II genes DRB1, DQA1, and DQB1 was investigated in 100 unrelated Iranian individuals from Fars province in Southern Iran, using the restriction fragment length polymorphism (RFLP) method. Subtyping of DRB1*04, *15, and *16 alleles was performed using PCR amplification with sequence specific primes (PCR-SSP). The allele and the haplotype frequencies were calculated. The most common DRB1 alleles were DRB1*11, DRB1*15, and DRB1*04 with a frequency of 25.0%, 14.5%, and 10.5%, respectively. In contrast, the allelic frequency of DRB1*12 and DRB1*08 was very low (1.5% for each). In the DR15 group DRB1*1501 was the most prevalent variant (6.0%). Concerning DR4, the most common alleles were DRB1*0405 and DRB1*0402 (3.5% for each). Interestingly, DRB1*0402 was associated with DQB1*0302 and DRB1*0405 was associated with DQB1*0302 and DQB1*02, the latter being a rare DRB1/DQB1 haplotype in Caucasian individuals. The most frequent DQB1 alleles were DQB1*0301 (31.0%), and DQB1*05 (22.0%). The most frequent DQA1 variants were DQA1*0501 (39.0%) and DQA1*0102 (14.5%). The most common haplotype was DRB1*11-DQB1*0301-DQA1*0501 (25.0%) followed by DRB1*0301-DQB1*02-DQA1*0501 (10%) and DRB1*0701- DQB1*02-DQA1*0201 (6.5%). Data presented in this study suggest that the Iranian population shares some HLA components with populations resident in eastern and southern European countries.

An integrative approach for the transplantation of high-risk sensitized patients.

Background. Sensitized patients have a lower chance of receiving a crossmatch-negative kidney and, if transplanted, are at risk of antibody-mediated allograft rejection. Methods. For safe and timely transplantation of sensitized patients at our center, we developed an integrative algorithm that includes identification of high-risk patients, good human leukocyte antigen match, inclusion in the Eurotransplant Acceptable Mismatch Program when applicable, apheresis, anti-CD20 therapy, posttransplant antibody monitoring, and protocol biopsies. Thirty-four high-risk recipients of a deceased donor kidney (DDK: n=28) or living donor kidney (LDK: n=6) were transplanted using this algorithm. Results. One-year graft survival, death-censored graft survival, and patient survival rates in DDK recipients were 92.4%, 96.4%, and 95.8%, respectively. No graft loss or patient death was observed in the six LDK patients. Median serum creatinine at 1 year in DDK and LDK recipients was 1.2 and 1.4 mg/dL, respectively. Eleven DDK and three LDK patients experienced at least one biopsy-proven acute rejection episode, mostly showing borderline changes. Antibody-mediated rejection without graft loss was diagnosed in two DDK and one LDK patients. Delayed graft function was observed in 13 DDK and 1 LDK patients. Infectious complications were infrequent. Conclusions. We describe an algorithm for the categorization and treatment of presensitized high-risk patients. This protocol provides effective prevention of antibody-mediated rejection and is associated with a low rate of side effects and good graft outcome.

Analysis of KIR ligand incompatibility in human renal transplantation

Natural killer cells express killer immunoglobulin-like receptors (KIR) that bind to MHC class I antigens. Lack of self-MHC on the target cell can cause NK-cell mediated killing. Here, we analyzed the effect of KIR ligand incompatiblity on renal allograft survival in humans. Kidney recipient/donor pairs were separated according to their HLA-Cw alleles and HLA-Bw4 specificity which are considered epitopes for KIR. A total of 2,757 renal transplants were examined. Graft survival rates were computed according to the Kaplan-Meier method. No effect of KIR ligand matching on graft survival was observed in cadaver kidney transplants. Our results indicate that KIR ligand matching cannot be recommended as a strategy for improving renal allograft survival.

Living donor kidney transplantation in crossmatch-positive patients enabled by peritransplant immunoadsorption and anti-CD20 therapy

Living donor kidney transplantation in crossmatch‐positive patients is a challenge that requires specific measures. Ten patients with positive crossmatch results (n = 9) or negative crossmatch results but strong donor‐specific antibodies (DSA; n = 1) were desensitized using immunoadsorption (IA) and anti‐CD20 antibody induction. IA was continued after transplantation and accompanied by HLA antibody monitoring and protocol biopsies. After a median of 10 IA treatments, all patients were desensitized successfully and transplanted. Median levels of mean fluorescence intensity (MFI) of Luminex‐DSA before desensitization were 6203 and decreased after desensitization and immediately before transplantation to 891. Patients received a median of seven post‐transplant IA treatments. At last visit, after a median follow‐up of 19 months, 9 of 10 patients had a functioning allograft and a median Luminex‐DSA of 149 MFI; serum creatinine was 1.6 mg/dl, and protein to creatinine ratio 0.1. Reversible acute antibody‐mediated rejection was diagnosed in three patients. One allograft was lost after the second post‐transplant year in a patient with catastrophic antiphospholipid syndrome. We describe a treatment algorithm for desensitization of living donor kidney transplant recipients that allows the rapid elimination of DSA with a low rate of side effects and results in good graft outcome.

Association of Kidney Graft Loss With De Novo Produced Donor-Specific and Non-Donor-Specific HLA Antibodies Detected by Single Antigen Testing

Background The association of donor-specific HLA antibodies (DSA) with kidney graft failure has been addressed previously; however, the majority of studies were based on small numbers of patients with graft failure. Methods We investigated 83 patients with failed kidney transplants for a possible association of de novo development and persistence or loss of pre-existing DSA with graft failure. Single Antigen Bead assay-detected DSA and non-DSA antibodies were compared between patients with graft loss and matched controls with functioning grafts. Results The incidence of weak de novo DSA or non-DSA at a mean fluorescence intensity of 500 or higher was higher in the graft loss than in the nonrejector group (76% vs 40%, P < 0.001). Because of the low number of patients developing de novo DSA, the DSA results did not reach statistical significance (only 22% of patients with graft loss developed de novo DSA). However, at all cutoffs, there was a significantly higher rate of graft loss in patients with de novo non-DSA. The incidence of strong pretransplant DSA that persist after transplantation was higher in the graft loss group (10% vs 1%, P = 0.034). When C1q-binding ability in sera of rejectors and nonrejectors with posttransplant de novo or persistent DSA was compared, none of the nonrejectors demonstrated C1q positivity, whereas 43% of patients with graft loss showed C1q-positive antibodies, although not necessarily donor-specific (P < 0.001). Conclusions Our data show that the posttransplant presence of persisting or de novo HLA antibodies, especially if C1q binding, is associated with graft loss, even if the antibodies are not specific for mismatched donor HLA.

Comparison of serological and DNA PCR-SSP typing results for HLA-A and HLA-B in 421 Black individuals: a Collaborative Transplant Study report.

In a recent study, we observed a discrepancy rate of 8.5% between the results of molecular and serological HLA class I typing in Caucasian kidney donors and recipients. In the present study we addressed the question how often black individuals are mistyped using the serological technique. 421 Blacks whose HLA typing results were reported to the Collaborative Transplant Study (CTS) were typed retrospectively for HLA-A and -B using a PCR-SSP method. 78 of the 421 individuals (18.5%) showed a discrepancy for HLA-A and 107 individuals (25.4%) for HLA-B. 36.3% of all individuals tested showed either an HLA-A or an HLA-B discrepancy. 13.1% of the discrepancies at the HLA-A locus were due to antigen misassignments and 4.8% were due to missed antigens. HLA-B discrepancies were caused in 15.7% by antigen misassignments and in 10.5% by missed antigens. These results demonstrate an impressive advantage of the PCR-SSP method for HLA-A and HLA-B locus typing over serological typing in black individuals. The high typing discrepancy rate observed in Blacks provides a strong argument for replacing serological typing by the DNA method. It is likely that this will improve the HLA matching correlation in clinical transplantation in Blacks.

The collaborative transplant study registry.

The Collaborative Transplant Study (CTS) was initiated in 1982. Over the last 30 years, it has collected information on over half a million kidney, liver, heart, lung, and pancreas transplant procedures. Participation is voluntary and the study has strictly scientific objectives. Analyses of the CTS database serve as an international reference source in the field of solid organ transplantation.