What is the clinical relevance of HLA-C antibodies detected by luminex single antigen bead assays?

G.E. Karahan, K.H. Bakker, S. Brand-Schaaf, D.L. Roelen, F.H.J. Claas, S. Heidt

Wednesday 4 march 2020

16:40 - 16:50h at Joep Nicolas zaal

Parallel session: Parallel sessie VII – Basale- en Klinische abstracts

Background: HLA antibody detection using luminex single antigen beads (SAB) aids in defining acceptable and unacceptable HLA antigens for patients awaiting a transplant. While being highly sensitive and specific, not all HLA antibodies detected by SAB assay appear to be clinically relevant, possibly resulting from differences in the quality and quantity of HLA molecules on the beads compared to natively expressed HLA antigens. Considering the instability of the HLA molecules and low HLA-C expression on the cell surface, we investigated whether HLA-C antibodies with high MFI values in SAB assay bind to cells natively expressing the corresponding HLA-C antigen.

Methods: Eight serum samples containing HLA-C antibody specificities with MFI values >5000 as detected by SAB assay (One Lambda, USA) were included. Complement dependent cytotoxicity (CDC; n=28) and/or flow cytometric crossmatches (FC-XM; n=33) were performed using peripheral blood mononuclear cells (PBMC) from 38 healthy donors. Each serum sample was crossmatched with 1-8 different donor PBMC targeting only one HLA-C antigen in each crossmatch (XM).

Results: Three sera harboured only HLA-C antibodies while in 5 sera HLA-C antibodies were coinciding with HLA-A and HLA-B antibodies. All CDC-XM were negative in samples with isolated HLA-C antibodies (median MFI: 8520; range: 5624-13990). Only 3 (11%) CDC-XM were positive in samples with multi-loci class I antibodies. Overall, only 36% of FC-XM were positive and MFI values were higher (median: 19803; 8520-23612) for FC-XM positive specificities than for negative ones (median: 10659; 5624-22215). Subsequently, when we absorbed a serum sample positive in FC-XM for HLA-C*03:04 with cells bearing HLA-C*03:04, MFI value for C*03:04 decreased from 23612 to 8622. FC-XM became negative when absorbed serum was re-crossmatched against the C*03:04, indicating that C*03:04 antibody can be absorbed and can bind to intact C*03:04 on cells. In contrast, when we absorbed the same serum sample negative in FC-XM for HLA-C*01:02 with cells bearing HLA-C*01:02, there was only a slight decrease in MFI values from 11590 to 6900, suggesting that reactivity was due to denatured HLA-C molecule on the beads.

Conclusions: HLA-C antibodies may be binding epitopes on native and denatured HLA-C antigens coated on beads affecting MFI values. Results from SAB assays must be assessed in combination with cellular assays in order not to preclude a possible transplantation for a particular patient.