Technical limitations of the C1q single antigen bead assay to detect complement binding HLA-specific antibodies

Background: Solid-phase assays to distinguish complement binding from non-complement binding HLA-specific antibodies have been introduced, but technical limitations may compromise their interpretation. We have examined the extent to which C1q-binding to HLA-class I single-antigen-beads (SAB) is influenced by denatured HLA on SAB, antibody titre, and complement interference that causes a misleading low assessment of HLA-specific antibody levels. Methods: Sera from 25 highly-sensitised patients were tested using Luminex IgG-SAB and C1q-SAB assays. Sera were tested undiluted, at 1:20 dilution to detect high-level IgG, and after EDTA treatment to obviate complement interference. Conformational HLA and denatured HLA protein levels on SAB were determined using W6/32 and HC-10 monoclonal antibodies respectively. Denatured HLA was expressed as HC-10 binding to untreated SAB as a percentage of maximal binding to acid treated SAB. Results: For undiluted sera, Luminex mean fluorescence intensity (MFI) values for IgG-SAB and C1q-SAB correlated poorly (r2 0.42). EDTA and serum dilution improved the correlation (r2 0.57 and 0.77 respectively). Increasing levels of denatured HLA interfered with the detection of C1q binding. Consequently the correlation between IgG-SAB MFI and C1q-SAB MFI was lowest using undiluted sera and SAB with >30% denatured HLA (r2 0.40) and highest using diluted sera and SAB with ≤30% denatured HLA (R2 0.86). Conclusion: Antibody level, complement interference and denatured HLA-class I on SAB may all affect the clinical interpretation of the C1q-SAB assay. The C1q-SAB assay represents a substantial additional cost for routine clinical use and we question its justification given the potential uncertainty about its interpretation.This study was supported by the Cambridge National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre and the NIHR Blood and Transplant Research Unit in Organ Donation and Transplantation at the University of Cambridge in collaboration with Newcastle University and in partnership with NHS Blood and Transplant (NHSBT). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health or NHSBT. VK was supported by the Academy of Medical Sciences and the Evelyn Trust.This is the author accepted manuscript. The final version is available from Wolters Kluwer via http://dx.doi.org/10.1097/TP.0000000000001270