Systematic Comparison of Four Cell- and Luminex-Based Methods for Assessment of Complement-Activating HLA Antibodies

Abstract

Efforts to increase the specificity and sensitivity of human leukocyte antigen (HLA) antibody detection assays recently led to the establishment of two novel Luminex bead-based assays to detect complement-activating antibodies by the assessment of complement products C1q or C4d. Here, we present a systematic comparison of the four methods, complement-dependent lymphocytotoxicity (CDC) and C1q-, C4d-, and IgG-Luminex, to assess or predict the complement-binding capability of HLA IgG antibodies. Forty-five sera of highly immunized patients have been assessed by in-house modified C1q- and C4d-Luminex assays and compared with standard CDC and IgG-Luminex. Antibody specificities assigned by the C1q- and C4d-Luminex assay revealed an excellent concordance of 94% and 97% for HLA class I and II, respectively. Complement-fixing HLA class II antibodies were found less frequently among IgG antibodies compared with class I. Both C1q- and C4d-Luminex detected, on average, three times more specificities than CDC. Although we found a high correlation of mean fluorescence intensity values between C1q- and C4d-Luminex assays, IgG mean fluorescence intensity was not a suitable surrogate marker for the prediction of complement binding. C1q- and C4d-Luminex assays are characterized by an increased sensitivity and specificity compared with CDC, the current standard in detecting complement-fixing HLA antibodies. Pretransplantation risk assessment for transplantation but also posttransplantation monitoring are important applications for both assays to improve overall allograft survival.