How reliable are HLA antibody detection assays under immunosuppressive regimen?

B. Duygu, M.H.v.a.n Tuijl, C.v.a.n. Groesen, L. Wieten, C.E.M. Voorter

Thursday 14 march 2019

13:23 - 13:25h at Tropentheater

Categories: Klinisch/Basaal, Parallelsessie

Parallel session: Parallelsessie XV – Basaal / Klinisch 2

Background: Precise and accurate assessment of donor-specific HLA antibodies (DSA) is a crucial step prior to transplantations to reduce the risk for antibody mediated rejection (AMR), as well as to screen for the development of de novoDSA post-transplantation. However, immunosuppressive reagents used for desensitization of highly immunized patients or for the treatment of AMR can interfere with antibody detection assays leading to misinterpretation of the test results. Therefore, we examined the effect of two commonly used agents, Intravenous immunoglobulin (IVIG) and Rituximab, on Luminex mixed screen and single antigen (SA) assays and on flow- and CDC- crossmatches.

Methods: IVIG and Rituximab were added to patients’ sera or negative and positive control sera. Also, sera from patients treated with either agents were included. Sera were tested with Luminex mixed and SA, flow and CDC crossmatch assays.

Results: In vitro treatment of negative sera with IVIG (dilution 1:10) resulted in increased MFI value of certain beads in both Luminex SA and mixed assays, creating a broad pattern of false positive specificities. This pronounced pattern of reactivity was reproducible with four different IVIG batches. Similar patterns were observed when patient’s sera were analyzed shortly after IVIG treatment although sera before IVIG infusion were negative. Spiking IVIG in the positive control serum did not affect the Luminex assay results. Further, testing the IVIG effect on CDC crossmatches using 3 different cells, did not show any difference with untreated sera. On the other hand, Rituximab treatment of negative serum had no effect on the Luminex assay results, but gave false positive reactions in unseparated CDC and B cell flow crossmatches, again using three different samples of cells.

Conclusions: Our results indicate the presence of HLA antibody specificities in negative sera treated with IVIG when tested with Luminex assays, but no induction of false positive crossmatches, probably either because antibodies are not complement-fixing or the titer is not high enough for CDC reaction. Furthermore, in line with previous findings, our data revealed the interference of Rituximab with crossmatch assays, but not with Luminex bead assays, so the latter ones can be safely used to monitor the effect of Rituximab therapy. Further investigation is required to test for other medications and to develop strategies to eliminate the interference of each medication or therapy on antibody detection assays.