raises similar concerns for HLA class II (HLA-II) antigens, where the specific conformational pairing of α- and β-chains may determine potentially pathogenic epitope expression. Antibodies that recognize these disassociated HCs, for example, by binding to epitopes exposed by the loss of β2M, may contribute to clinically irrelevant or “false positive” results. The native cell surface HLA class I molecule (HLA-I), which exists as trimer of HLA heavy chain (HC), β2-microglobulin (β2M), and peptide, may be disrupted during the manufacturing process, resulting in beads coated with HLA HCs (devoid of β2M and/or peptide), considered as “denatured”. Unfortunately, the solid phase assays have significant limitations. False negative results could also result in adverse short- and/or long-term consequences. False positive reactions may result in denial of a potentially compatible transplant or could falsely elevate the degree of sensitization to inappropriately disadvantage a patient. It is imperative that these assays be as accurate as possible. The presence of anti-HLA antibodies reacting with a broad array of HLA antigens can be assessed and compared to the frequency of those antigens in the entire donor pool, the so-called calculated panel reactive antibody (cPRA). The presence of antibodies can be detected by a secondary fluorescent antibody using either flow cytometry or a Luminex platform. HLA antigens are attached to a polystyrene bead, either as a mixture of HLA antigens, a phenotypic panel consisting of the HLA antigens present on a single cell line, or a single HLA antigen per bead. Solid phase assays have now replaced these cell-based assays for the routine detection of anti-HLA antibodies. Cell-based assays such as the CDC crossmatch and flow cytometric crossmatch are still used in histocompatibility laboratories to assess the safety of transplantation. This assay has essentially abrogated hyperacute rejection.

Patel and Terasaki developed the complement-dependent cytotoxicity (CDC) assay, allowing a pretransplantation crossmatch to be performed between a recipient’s serum and donor lymphocytes. They include hyperacute, accelerated acute, or acute antibody-mediated rejection and delayed graft function in the short term, and chronic active antibody mediated rejection with reduced graft survival in the long term. Upon transplantation, patients with anti-HLA alloantibodies are at increased risk for adverse outcomes. The result is increased morbidity and mortality in this population. Many renal transplant candidates have IgG antibodies against HLA antigens which, depending on the degree of HLA sensitization, can restrict their access to transplantation.

The intraindividual variability observed between beadsets with two detection antibodies elucidates that antigens found as acceptable with one beadset may end up unacceptable with the other beadsets, with the possibility of denying potentially compatible transplants to candidates. For HLA-II, the reverse occurred, with significantly higher levels with FcMonoIgG regardless of the beadsets. For HLA-I, MFIs were higher with IgHPolyFab than with FcMonoIgG with the exception of sera with MFIs > 5000 where they were comparable. For both classes, MFIs were also frequently higher with LS than with LC. Consequently, IgG antibodies against both classes of HLA were reactive to more antigens with LS than with LC-beadsets. LS-beadsets are coated with monomeric variants in addition to intact HLA antigens with or without peptides, while LC-beadsets are devoid of monomeric variants and with lesser levels of peptide-free heterodimers. Positive results at mean fluorescence intensity (MFI) > 500 (at serum dilution 1/10) were used to calculate panel reactive antibody (cPRA) levels. Sera antibodies of the end-stage renal disease patients were compared using LIFECODES (LC) and LABScreen (LS) beadsets monitored with polyclonal-Fab ( IgHPolyFab) and monoclonal-IgG ( FcMonoIgG) second antibodies. Both the configuration of the HLA molecules coated on the beads and the nature of detection antibodies may impede assessment of the presence and strength of anti-HLA IgG- with the Luminex single-antigen-bead assay.

The accurate measurement of anti-HLA alloantibodies in transplant candidates is required for determining the degree of sensitization and for the listing of unacceptable antigens for organ allocation.

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