Comparison Between Total IgG, C1q, and C3d Single Antigen Bead Assays in Detecting Class I Complement-Binding Anti-HLA Antibodies.

BACKGROUND: Complement-binding donor-specific antibodies (DSAs) are associated with antibody-mediated rejection and allograft loss. Novel single antigen bead (SAB) assays-that is, complement component 1q (C1q) and complement component 3d (C3d) assays-have been developed to specifically detect complement-binding DSA, but it remains unclear whether these assays have an improved ability to detect complement-binding DSA as compared with using the total IgG SAB assay with a high mean fluorescence intensity (MFI) cutoff. The aim of this study was to compare the ability of the total IgG, C1q, and C3d SAB assays in detecting complement-binding anti-HLA antibodies. METHODS: Twenty sera known to have complement-binding anti-HLA antibodies (serologic class I HLA typing by complement-dependent cytotoxicity method) were tested with 3 different SAB assays: total IgG (undiluted and 1:8 dilution), C1q, and C3d. Serologic anti-HLA specificities were compared with those obtained by IgG, C1q, and C3d SAB assays. RESULTS: IgG SAB was more sensitive in detecting complement-binding antibodies (sensitivity 24 of 24 = 1, odds ratio infinity). Pearson correlation showed the association between (1) C1q and IgG SAB assays (cutoff C1q SAB 1000 MFI, cutoff IgG SAB 5000 MFI: r = 0.347, P < .0001) and (2) C3d and IgG SAB assays (cutoff 500 MFI C3d SAB, 5000 MFI for IgG SAB: r = -0.173, P = .279). CONCLUSIONS: For class I anti-HLA antibodies, IgG SAB (cutoff MFI > 5000) was more sensitive in detecting complement-binding antibodies when compared with C1q and C3d SAB assays.

Effect of pretransplant human leukocyte antigen antibodies detected by solid-phase assay on heart transplant outcomes.

INTRODUCTION: The significance of pretransplant human leukocyte antigen antibodies (HLA-Abs), especially donor-specific HLA-Abs (DSA), as detected by single antigen bead assay (SAB), is not well characterized in cardiac transplantation (CTX). We analyzed the significance of DSA detected by SAB in predicting crossmatch (XM) results and post-transplant rejection. MATERIALS AND METHODS: We performed a retrospective study of 85 CTX with negative cytotoxicity XM. We tested pretransplant sera collected within 24 hours of transplantation by flow cytometric XM (FXM) and SAB. DSA identified by SAB were utilized to perform a virtual crossmatch (VXM). Positive VXM was defined as the presence of DSA at mean fluorescence intensity (DMFI)>1500. Additionally, to analyze the significance of low-level DSA weakly positive VXM was DMFI 300 to 1500. We defined a negative VXM as MFI<300. VXM results were correlated with FXM results and with posttransplant rejection. RESULTS: Patients in the weakly positive and negative VXM had similar posttransplant rejections. DMFI>1500 correlates well with FXM results (accuracy=90%). Patients with DMFI>1500 had a higher incidence of antibody-medicated rejection (AMR; P=.0052), AMR grade I (P<.0001), cell-mediated rejection (CMR) grade>1R/1A (P=.018), and CMR grade>2R/3A (P=.057). Similarly patients with positive FXM had a higher incidence of AMR (P=.091), AMR grade 1 (P<.0001), CMR grade>1R/1A (P=.05), and CMR grade>2R/3A (P=.56). CONCLUSIONS: In conclusion, SAB defined DMFI>1500 can be used as a surrogate for FXM. Recipients with DMFI>1500 pretransplant and positive FXM have significantly higher rates of AMR and CMR compared to recipients with DMFI<1500 or negative FXM.

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.

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.

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.

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 < or = 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.

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.

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.

Serum platelet antibody testing: evaluation of solid-phase enzyme immunoassay and comparison with indirect immunofluorescence.

We compared the ability of solid-phase enzyme immunoassay (SPEIA) with that of indirect immunofluorescence to detect serum platelet antibodies by parallel testing five sets of serum specimens: (1) 12 monospecific HLA class I specimens with defined specificities, (2) 4 HPA-1a specimens, (3) 164 sequential unselected specimens sent to our laboratory for serum platelet antibody testing, (4) specimens from 15 consecutive patients sent for indirect immunofluorescence testing alone, and (5) specimens from 19 consecutive patients sent for both indirect and direct immunofluorescence testing. In addition, specimens of HLA sera were tested by standard complement-dependent lymphocytotoxicity. Solid-phase enzyme immunoassay was consistently more sensitive than indirect immunofluorescence for all 12 HLA specimens and more sensitive than complement-dependent lymphocytotoxicity for 11 of 12 specimens. Similarly, SPEIA was more sensitive than indirect immunofluorescence for all four HPA-1a serum specimens by one to three dilutions. In the nine presumed autoimmune cases (group 5), results were positive with SPEIA in one case; no positive results were noted with indirect immunofluorescence. The solid-phase enzyme immunoassay test permits ready differentiation between alloantibodies directed to HLA and those directed to platelet-specific glycoproteins.

Antagonists of cyclic nucleotide phosphodiesterase (PDE) isozymes PDE 3 and PDE 4 suppress lymphoblastic response to HLA class II alloantigens: a potential novel approach to preventing allograft rejection?

As a potential novel approach to preventing renal allograft rejection, we investigated whether the proliferative response of lymphocytes to mismatched HLA class II antigens in mixed lymphocytic culture (MLC) can be suppressed by antagonists of cyclic nucleotide phosphodiesterase (PDE) isozymes. Cilostamide, an antagonist of isozyme PDE3 and, to an even greater extent, in combination with rolipram, an antagonist of isozyme PDE4, markedly suppressed (delta = -60%; p < 0.01) the mitogenic proliferative response of lymphocytes in MLC to HLA-DR alloantigens from unrelated donors. These observations suggest that the selective PDE isozyme antagonists might have potential as novel drugs in "signal transduction-targeted" pharmacotherapy of renal transplant rejection.

HLA antibody screening: comparison of a solid phase enzyme-linked immunoassay with antiglobulin-augmented lymphocytotoxicity.

BACKGROUND: IgG antibodies to HLA class I antigens can cause hyperacute rejection of renal allografts. Screening of sera from such transplant candidates is laborious, time-consuming, and expensive when performed by sensitive antihuman globulin-augmented lymphocytotoxicity (AHG-CDC). METHODS: Because 60-70% of our transplant screens are negative, we evaluated a solid phase enzyme-linked method (EIA) as a potential prescreen by parallel testing 215 sera by AHG-CDC and by EIA. This EIA method is designed to detect only IgG antibodies, and all positive AHG-CDC sera were retested after dithiothreitol treatment. RESULTS: There was 96.2% concordance between the tests for IgG antibodies. Seven sera (3.25%) were positive by EIA alone, and one (0.46%) was negative by EIA alone. The EIA method was also less costly ($15.00 versus $105.00) and less time consuming (hours versus days) than AHG-CDC panel testing for large numbers of sera. CONCLUSIONS: We conclude that this EIA method is simple, sensitive, objective, and cost effective as a prescreen for HLA class I antibodies.

Platelet crossmatch evaluation in refractory hematologic patients.

Although previous investigators have attempted to identify platelet crossmatching methods that could be used routinely for pretransfusion testing, such studies have excluded patients with underlying clinical conditions inherently associated with low corrected platelet increments. Because many refractory hematologic patients have such underlying conditions, we decided to assess the usefulness of platelet crossmatching (in addition to HLA matching) in determining the posttransfusion corrected platelet count increment in eight medically complicated patients who received a total of 35 single-donor platelet transfusions. In this limited study, the predictive value of a negative crossmatch was only 55%, whereas that of a positive crossmatch was 88%. The test used had a sensitivity of 65% and a specificity of 83%. The results of our study suggest that platelet crossmatching may be a useful additional study for predicting the outcome of transfusion--even in medically complex cases--if it can be done rapidly and relatively inexpensively.

The use of frozen-thawed lymphocytes for DRw typing.

Results of DRw typing using frozen-thawed plastic "adhered" cell preparations were comparable to those obtained using fresh sheep E-rosetted lymphocytes. Estimates of B-cell content of such preparations may be erroneously low if surface immunoglobulin fluorescence is the only method used.

Heterogeneity of HLA-BW35 based on the diallelic BW4-BW6 system.

Utilizing the diallelic BW4-BW6 system of antigens and antibodies, we divided subjects possessing HLA-BW35 into two groups, depending on the association of their BW35 antigens with BW4 or BW6, and demonstrated in family studies that the associated antigens appeared to be inherited together. Absorption experiments were performed to establish the validity of the typings. Anti-human beta2-microglobulin was used in an effort to "map" the location of the BW4 and BW6 antigenic determinants on the HLA molecule.