Search

Background Human immunoglobulins (H-Ig) are widely used in solid organ transplantation for immunoglobulin G (IgG) replacement and for desensitization and treatment of antibody-mediated rejection. They are obtained from plasma pools and may contain HLA antibodies that can be detrimental to transplant recipients. The goal of this study was to evaluate HLA antibodies in multiple lots of 2 commercial H-Ig preparations by Luminex single-antigen bead (SAB) and cell-based crossmatch assays. Methods Thirty lots of 2 commercial H-Ig products (CSL Behring, King of Prussia, PA) were evaluated: 6 Hizentra and 24 Privigen. All were adsorbed and diluted 1:10 before testing. HLA IgG antibodies were determined by 2 Luminex SAB kits and C1q screen for complement-binding capability. Lots were tested for the presence of antibody to denatured vs. intact class I HLA alleles using acid-treated SAB. Surrogate T and B-cell flow cytometry crossmatches (FCXM) were performed with peripheral blood lymphocytes from 2 healthy donors. Results Twenty-two (73%) lots at 1:10 showed SAB reactivity with mean fluorescent intensity of 2000 or greater for HLA class I, 67% (20/30 lots) for class II. The reactivity pattern was similar using both SAB kits. Acid treatment revealed antibodies to denatured class I: the majority of HLA-C, half of HLA-B and few HLA-A alleles. No C1q reactivity was observed. Surrogate flow cytometry crossmatch results were positive (>150 median channel shift), but were fourfold to eightfold lower than expected. Conclusions The H-Ig products tested consisted of low titer, non-complement-binding HLA class I and class II antibodies; most of the observed class I HLA reactivity was toward denatured HLA antigens.

Background The presence of antibodies to angiotensin type 1 receptor (AT1R) and endothelin type A receptor (ETAR) is associated with allograft rejection in kidney and heart transplantation. The aim of our study was to determine the impact of AT1R and ETAR antibodies on graft outcome in lung transplantation. Methods Pretransplant and posttransplant sera from 162 lung recipients transplanted at 3 centers between 2011 and 2013 were tested for antibodies to AT1R and ETAR by the enzyme-linked immunosorbent assay (ELISA) assay. Clinical parameters analyzed were: HLA antibodies at transplant, de novo donor-specific antibodies (DSA), antibody-mediated rejection (AMR), acute cellular rejection, and graft status. Results Late AMR (median posttransplant day 323) was diagnosed in 5 of 36 recipients with de novo DSA. Freedom from AMR significantly decreased for those recipients with strong/intermediate binding antibodies to AT1R (P = 0.014) and ETAR (P = 0.005). Trends for lower freedom from acute cellular rejection were observed for recipients with pretransplant antibodies to AT1R (P = 0.19) and ETAR (P = 0.32), but did not reach statistical significance. Lower freedom from the development of de novo DSA was observed for recipients with antibodies detected pretransplant to AT1R (P = 0.054), ETAR (P = 0.012), and HLA-specific antibodies (P = 0.063). When the pretransplant antibody status of HLA-specific antibody (hazard ratio [HR], 1.69) was considered together with either strong binding to AT1R or ETAR, an increased negative impact on the freedom from the development of de novo DSA was observed (HR, 2.26 for HLA antibodies and ETAR; HR, 2.38 for HLA antibodies and ETAR). Conclusions These results illustrate the increased negative impact when antibodies to both HLA and non-HLA antigens are present pretransplant.

Aim. Humoral sensitization affects transplant outcome, and it is now apparent that human leukocyte antigen (HLA) antibodies are specific for epitopes rather than antigens. Such epitopes can be structurally defined by HLAMatchmaker, an algorithm that considers eplets as critical elements of epitopes recognized by alloantibodies. This study addressed the question how mismatched HLA antigens induce specific antibodies in context with eplet differences with the antibody producer. Methods. HLA class I-specific human monoclonal antibodies derived from women sensitized during pregnancy were tested in Luminex assays with single allele panels. Their epitope specificity was determined from reactivity patterns and eplet differences between immunizing antigen and the antibody producer. Results. This study focuses on the reactivity patterns of 10 monoclonal antibodies specific for epitopes defined by a mismatched eplet paired with a self-eplet shared between immunizing HLA antigens and HLA antigens of the antibody producer. The eplets in these pairs are between 7 and 16 angstrom apart, a sufficient distance for contact by two separate complementarity-determining regions of antibody. Conclusions. These findings demonstrate that immunizing antigens have mismatched eplets that can form antibody-reactive epitopes with self-configurations on the molecular surface. They seem to suggest that HLA antibodies can be produced by autoreactive B cells that have undergone receptor editing to accommodate the recognition of nonself-eplets, the driving force of the humoral alloresponse. This concept enhances our understanding of structural epitope immunogenicity and the interpretation of antibody reactivity patterns with HLA panels.

Background During the 12th International Histocompatibility Workshop, a collaborative study between 35 laboratories was conducted on a group of highly allosensitized patients who had received a kidney transplant from 1981 to 1995. The major goal of the study was to assess how serum screening against a large cell panel could determine donor HLA mismatch acceptability in relation to graft outcome. Methods Twenty laboratories participated in an extensive screening of 92 high panel-reactive antibody (PRA) sera from patients at 29 transplant centers worldwide; each patient had received a kidney allograft from an HLA-A,B mismatched unrelated donor. Screening was done by complement-dependent lymphocytotoxicity and antihuman globulin augmentation techniques using a common protocol and shared standardized reagents. After an extensive quality-control assessment, we selected data from 14 participants who had screened the sera against a combined panel of 535 HLA-typed cells. Results With the 2x2 table-based Multiscreen computer program, we could readily determine for virtually every patient the significant correlations between serum reactivity and the presence of panel cell markers, including private and public HLA-A,B epitopes and amino acid residues assigned from published sequencing data. Donor mismatch acceptability was assessed at the amino acid residue level. In the complement-dependent lymphocytotoxicity (n=49; PRA=84.1+/-12.1%) and antihuman globulin (n=60; PRA=92.5+/-5.8%) groups, the 3-month graft survivals were 28% and 30% lower for unacceptable residue mismatches. Conclusions These studies underscore the importance of a comprehensive serum screening analysis in the selection of appropriately mismatched donors for highly sensitized transplant patients.

Background. The performance characteristics and interlaboratory comparisons of the T-cell flow cytometry crossmatch remain largely unknown. Methods. This study was performed using data from the ASHI-CAP proficiency testing program. Four unknown sera and two unknown cells were sent to participating laboratories twice a year for 4 years. Results. In one survey in which different crossmatch techniques were compared, flow cytometry was slightly more sensitive than the antiglobulin method and considerably more sensitive than direct cytotoxicity. However, the proportion of participants in any given survey detecting antibodies in all sera expected to be positive was 50-60% and has not changed over the years. Failure to detect antibodies correlated with low antibody concentration, diluting the unknown serum by the testing laboratory, and with the instrument used. False positive results with normal sera were infrequent. Fluorescence intensity values were not standardized and were highly variable, but when fluorescence units reported by individual laboratories were divided by their own positive-negative cutoff values, results from different centers were more comparable. In general, fluorescence-to-cutoff ratios >5 correlated with complement binding activity, whereas values

During the 12th International Histocompatibility Workshop, a collaborative study between 35 laboratories was conducted on a group of highly allosensitized patients who had received a kidney transplant from 1981 to 1995. The major goal of the study was to assess how serum screening against a large cell panel could determine donor HLA mismatch acceptability in relation to graft outcome.Twenty laboratories participated in an extensive screening of 92 high panel-reactive antibody (PRA) sera from patients at 29 transplant centers worldwide; each patient had received a kidney allograft from an HLA-A,B mismatched unrelated donor. Screening was done by complement-dependent lymphocytotoxicity and antihuman globulin augmentation techniques using a common protocol and shared standardized reagents. After an extensive quality-control assessment, we selected data from 14 participants who had screened the sera against a combined panel of 535 HLA-typed cells.With the 2x2 table-based Multiscreen computer program, we could readily determine for virtually every patient the significant correlations between serum reactivity and the presence of panel cell markers, including private and public HLA-A,B epitopes and amino acid residues assigned from published sequencing data. Donor mismatch acceptability was assessed at the amino acid residue level. In the complement-dependent lymphocytotoxicity (n=49; PRA=84.1+/-12.1%) and antihuman globulin (n=60; PRA=92.5+/-5.8%) groups, the 3-month graft survivals were 28% and 30% lower for unacceptable residue mismatches.These studies underscore the importance of a comprehensive serum screening analysis in the selection of appropriately mismatched donors for highly sensitized transplant patients.

The performance characteristics and interlaboratory comparisons of the T-cell flow cytometry crossmatch remain largely unknown.This study was performed using data from the ASHI-CAP proficiency testing program. Four unknown sera and two unknown cells were sent to participating laboratories twice a year for 4 years.In one survey in which different crossmatch techniques were compared, flow cytometry was slightly more sensitive than the antiglobulin method and considerably more sensitive than direct cytotoxicity. However, the proportion of participants in any given survey detecting antibodies in all sera expected to be positive was 50-60% and has not changed over the years. Failure to detect antibodies correlated with low antibody concentration, diluting the unknown serum by the testing laboratory, and with the instrument used. False positive results with normal sera were infrequent. Fluorescence intensity values were not standardized and were highly variable, but when fluorescence units reported by individual laboratories were divided by their own positive-negative cutoff values, results from different centers were more comparable. In general, fluorescence-to-cutoff ratios5 correlated with complement binding activity, whereas values5 denoted concentrations below those required to fix complement.Flow cytometry, as used by most centers, is highly sensitive and allows relative antibody quantitation. Furthermore, the data define objective parameters that may help to standardize the test and improve its predictive value in clinical transplantation.

A three-year-old boy with severe aplastic anemia (HLA-A1,B8(Bw6), Cw7,DR3, MB2, MT2, SB4/A1,B8 (Bw6), Cw7,DR3,MB2,MT2,SB-) received a bone marrow transplant from a phenotypically HLA-identical, SB-compatible female unrelated donor. This donor was selected from eighteen HLA-A1,-B8,-blood donors after extended serotyping, mixed leukocyte culture testing and secondary proliferation assays with primed lymphocyte typing reagents specific for SB. Although patient cells proliferated well as responders in MLR, their stimulatory capability was greatly impaired. Because the patient had inherited the same serological HLA-D haplotype from each parent, it was concluded that a compatible unrelated donor must be homozygous for the same HLA-D antigens as the patient. This HLA-D homozygosity was demonstrated by the lack of MLR responses of both parents to stimulators from the donor. The SB typing results suggested SB compatibility because both the patient and the donor typed as SB4,-. Following bone marrow transplantation, there was rapid hematopoietic engraftment. The patient developed severe diarrhea caused by graft-versus-host disease of the gastrointestinal tract, which necessitated hyperalimentation. He is currently eighteen months posttransplant with full hematopoietic reconstitution and moderate chronic skin graft-versus-host disease.