Steven R. DeGoey

A Comparison of Plasmapheresis Versus High-Dose IVIG Desensitization in Renal Allograft Recipients with High Levels of Donor Specific Alloantibody

Several protocols allow for the successful transplantation of sensitized renal allograft recipients, yet no one best method has emerged. The aim of the current study was to compare the efficacy of high‐dose IVIG with two different plasmapheresis (PP)‐based regimens in kidney transplant recipients with high levels of donor specific alloantibody (DSA) defined as a positive T‐cell cytotoxicity crossmatch. With the primary goal of achieving a negative crossmatch, we employed three protocols sequentially between April 2000 and May 2005: (i) PP, low‐dose IVIG, anti‐CD20 antibody (n = 32); (ii) high‐dose IVIG (n = 13); and (iii) PP, low‐dose IVIG, anti‐CD20 antibody and pre‐transplant Thymoglobulin combined with post‐transplant DSA monitoring (n = 16). IVIG decreased DSA activity in all treated patient, yet only 38% (5/13) achieved a negative crossmatch. In contrast, a negative crossmatch was achieved in 84% in PP group and 88% in the PP/monitoring group (p < 0.01 vs. IVIG). Even with a negative crossmatch, the rejection rates were 80% (IVIG), 37% (PP) and 29% (PP/monitoring), respectively, (p < 0.05 IVIG vs. PP). We conclude that multiple PP treatments leads to more reproducible desensitization and lower humoral rejection rates than a single high‐dose of IVIG, but that no regimen was completely effective in preventing humoral rejection.

Transplant Glomerulopathy: Subclinical Incidence and Association with Alloantibody

Transplant glomerulopathy (TG) usually has been described as part of a constellation of late chronic histologic abnormalities associated with proteinuria and declining function. The current study used both protocol and clinically‐indicated biopsies to investigate clinical and subclinical TG, their prognosis and possible association with alloantibody. We retrospectively studied 582 renal transplants with a negative pre‐transplant T‐cell complement dependent cytotoxicity crossmatch. TG was diagnosed in 55 patients, 27 (49%) based on protocol biopsy in well‐functioning grafts. The cumulative incidence of TG increased over time to 20% at 5 years. The prognosis of subclinical TG was equally as poor as TG diagnosed with graft dysfunction, with progressive worsening of histopathologic changes and function. Although TG was associated with both acute and chronic histologic abnormalities, 14.5% of TG biopsies showed no interstitial fibrosis or tubular atrophy, while 58% (7/12) of biopsies with severe TG showed only minimal abnormalities. TG was associated with acute rejection, pretransplant hepatitis C antibody positivity and anti‐HLA antibodies (especially anti‐Class II), with the risk increasing if the antibodies were donor specific. We suggest that subclinical TG is an under‐recognized cause of antibody‐mediated, chronic renal allograft injury which may be mechanistically distinct from other causes of nephropathy.

Overcoming a Positive Crossmatch in Living‐Donor Kidney Transplantation

Many patients who have an otherwise acceptable living‐kidney donor do not undergo transplantation because of the presence of antibodies against the donor cells resulting in a positive crossmatch. In the current study, 14 patients with a positive cytotoxic crossmatch (titer ≤ 1 : 16) against their living donor underwent a regimen including pretransplant plasmapheresis, intravenous immunoglobulin, rituximab and splenectomy. Eleven of 14 grafts (79%) are functioning well 30–600 days after transplantation. Two grafts were lost to accelerated vasculopathy and one was lost to death with good function. No hyperacute or cellular rejections occurred. Antibody‐mediated rejection occurred in six patients [two clinical (14%) and four subclinical (29%)] and was reversible with plasmapheresis and steroids. Our results suggest that selected crossmatch‐positive patients can be transplanted successfully with living‐donor kidney allografts, using a protocol of pretransplant plasmapheresis, intravenous immunoglobulin, rituximab and splenectomy. Longer follow‐up will be needed, but the absence of anti‐donor antibody and good early outcomes are encouraging.

Baseline Donor‐Specific Antibody Levels and Outcomes in Positive Crossmatch Kidney Transplantation

Renal transplant candidates with donor‐specific alloantibody (DSA) have increased risk of antibody‐mediated allograft injury. The goal of this study was to correlate the risk of antibody‐mediated rejection (AMR), transplant glomerulopathy (TG) and graft survival with the baseline DSA level (prior to initiation of pretransplant conditioning). These analyses include 119 positive crossmatch (+XM) compared to 70 negative crossmatch (−XM) transplants performed between April 2000 and July 2007. Using a combination of cell‐based crossmatch tests, DSA level was stratified into very high +XM, high +XM, low +XM and −XM groups. In +XM transplants, increasing DSA level was associated with increased risk for AMR (HR = 1.76 [1.51, 2.07], p = 0.0001) but not TG (p = 0.18). We found an increased risk for both early and late allograft loss associated with very high DSA (HR = 7.71 [2.95, 20.1], p = 0.0001). Although lower DSA recipients commonly developed AMR and TG, allograft survival was similar to that of −XM patients (p = 0.31). We conclude that the baseline DSA level correlates with risk of early and late alloantibody‐mediated allograft injury. With current protocols, very high baseline DSA patients have high rates of AMR and poor long‐term allograft survival highlighting the need for improved therapy for these candidates.

Histologic findings one year after positive crossmatch or ABO blood group incompatible living donor kidney transplantation.

Recent protocols have allowed successful positive crossmatch (+XM) and ABO incompatible (ABOI) kidney transplantation, although their long‐term outcome is not clear. To begin to assess this issue we compared protocol biopsies performed 12 months posttransplant in 37 +XM, 24 ABOI and 198 conventional allografts. Although the majority in all three groups had only minimal histologic changes, transplant glomerulopathy (TG) was significantly increased in +XM (22% vs. 13% ABOI vs. 8% conventional, p = 0.015), and correlated with prior humoral rejection (HR) by multivariate analysis (odds ratio 17.5, p ≤ 0.0001). Patients with a prior history of HR also had a significant increase in interstitial fibrosis (No HR 54% vs. HR 86%, p = 0.045). In the absence of HR no difference in histologic changes was seen between groups, although all three groups had a demonstrable mild increase in interstitial fibrosis from biopsies performed at the time of transplant. Thus, although HR is associated with an increase in TG, in its absence allograft histology is similar in +XM, ABOI and conventional allografts 1 year posttransplant.

Persistence of low levels of alloantibody after desensitization in crossmatch-positive living-donor kidney transplantation.

Background: Desensitization protocols have been developed to allow successful kidney transplantation in sensitized recipients. However, a detailed analysis of the impact of these protocols on alloantibody has not been performed. Methods: We studied 12 living-donor kidney-transplant recipients with positive antihuman globulin-enhanced complement dependent cytotoxicity (AHG-CDC) crossmatches against their donors. Using a variety of crossmatch techniques and single-antigen flowbeads (SAFBs), we characterized the specificity and amount of alloantibody at baseline before desensitization, after desensitization (using plasmapheresis followed by 100 mg/kg intravenous immunoglobulin, and anti-CD20 antibody), and 4 months after transplantation (after splenectomy and on maintenance immunosuppression). Results: All 12 patients with a positive baseline AHG-CDC crossmatch were AHG-CDC crossmatch negative at the time of transplant (after desensitization). However, despite desensitization, the majority of patients had low-level donor-specific alloantibodies demonstrable on the day of transplantation by both flow crossmatch (FXM 8/12) and SAFBs (10/11). Four months after transplantation, no patient had a positive AHG-CDC crossmatch, but again the majority had persistent low levels of donor-specific alloantibodies by FXM (6/12) and SAFBs (9/11). No patient experienced hyperacute rejection, and the persistence of low levels of donor-specific alloantibodies did not correlate with the development of humoral rejection in the early posttransplant period. Conclusions: Despite desensitization, a majority of positive crossmatch transplant recipients demonstrate low levels of donor-specific alloantibodies both on the day of transplant and 4 months after transplantation. The impact of these antibodies appears to be minimal early after transplant, but their long-term significance bears further study.

Kidney Transplantation in Patients with Antibodies against Donor HLA Class II

The immunologic risk associated with donor‐specific antibodies (DSA) against Class II human leukocyte antigens (HLA) in kidney transplant (KTx) recipients is unclear. The aim of this study was to determine the outcome of KTx when DSA was detected only against HLA Class II. To isolate the impact of anti‐Class II DSA, we retrospectively analyzed 12 KTx recipients who at baseline had a positive B‐cell flow cytometric crossmatch (FXM) and a negative T‐cell FXM. Using alloantibody specification analysis, 58.3% (7/12) had DSA against donor Class II and 41.7% had no demonstrable DSA. Biopsy‐proven AMR occurred in 57% (4/7) in the Class II+ group and 0% in the Class II− group (p > 0.05). Peritubular capillaries stained positive for C4d in 86% (6/7) of the Class II+ patients and in 40% (2/5) of the Class II− patients (p > 0.05). One patient in the Class II+ group lost their graft at 3 months to accelerated transplant glomerulopathy, while all other grafts were functioning 3–37 months posttransplant despite the persistence of anti‐Class II DSA. We conclude that KTx recipients with clearly defined anti‐Class II DSA are at risk for humoral rejection suggesting that desensitization and/or close posttransplant monitoring may be needed to prevent AMR.

The effect of antithymocyte globulin on anti-human leukocyte antigen antibody detection assays.

Background. We evaluated the effect of antithymocyte globulin (ATG) on anti-human leukocyte antigen (HLA) antibody assays. Methods. We tested sera from six in vivo ATG-treated kidney transplant patients after measuring serum concentrations, as well as six nonsensitized sera with ATG added in vitro. T- and B-cell complement-dependent cytotoxicity (CDC), flow cytometric (FXM), and solid-phase HLA class I and II assays based on antigen-coated microspheres and enzyme-linked immunosorbent assay (ELISA) were studied. Sera were then retested after treatment to remove ATG. Results. We found that ATG affects test results differently depending on whether sera is obtained from in vivo treated patients or added in vitro. In vitro treated sera produced ATG concentration-dependent positive results for T/B CDC, FXM, and flow bead testing for HLA I/II, while the ELISA-based assay was unaffected. In vivo treated sera from ATG-treated patients produced positive test results for T CDC and T/B FXM, while the B-cell CDC crossmatch remained negative. Solid phase assays were minimally affected using in vivo treated sera. After ATG extraction, all tests became negative. Conclusion. We conclude that ATG produces positive results in anti-HLA antibody testing, and treatment to remove ATG abolishes this effect. This treatment allows ATG-treated patients to be monitored for anti-HLA antibodies.

Inter and intra laboratory concordance of HLA antibody results obtained by single antigen bead based assay.

Single antigen bead based assays (SAB) to identify antibodies to HLA are marketed as a qualitative test, however often used as a quantitative test as the results provide mean fluorescence intensity (MFI) which is found to correlate with the strength/avidity of the antibody. We studied the between and within laboratory variability in performing the SAB from one manufacturer. Ten samples were tested at four laboratories according to the manufacturers suggested protocol. Additionally same samples were tested on four consecutive days in one laboratory. All tests were performed using the same lot of beads and secondary antibody. Results were classified as positive at four different MFI cutoffs: 1000, 5000, 8000 and 10,000. MFI values across and within the laboratory were compared in a pair-wise fashion with Pearsons correlations. Overall concordance for Class-I was 97% between laboratories and 98% within laboratory at all cutoffs. Pair-wise Pearson correlation between laboratories was 0.989-0.99, while within laboratory it was 0.998-0.999. For Class-II, overall concordance between and within laboratory was 98%. Pair-wise Pearson correlation between laboratories was 0.991-0.997, while within laboratory it was 0.997-0.999. There is good correlation between laboratories and within laboratory using the same manufacturer, same lot and same protocol while performing SAB.

High frequency of HLA-A*0103 allele in a Somali population

We report the existence of class I HLA allele A*0103 in an ethnic group (Somali) where this allele has not been reported. This allele was discovered in a study to examine the relationship between HLA alleles and humoral antibody response to measles vaccine among recent immigrants from Somalia to Olmsted County, Minnesota. We initially used polymerase chain reaction-sequence-specific primers (PCR-SSP) to carry out HLA class I typing. Based on PCR-SSP, 55 subjects were assigned the allele HLA-A*0101. Following direct DNA sequencing of the PCR products, 37 of the 55 subjects (67.3%) that were initially assigned the A*0101 allele were found to actually be A*0103. Our data are significant because it demonstrates that many of the previously typed A*0101 individuals are actually A*0103 as the SSP or sequence-specific oligonucleotide probes method cannot distinguish between the two alleles. Lastly, this is the first identification of this allele in the homozygous state.