Flow cytometry based detection of HLA alloantibody mediated classical complement activation

 

M. Wahrmann1, M. Exner2, H. Regele3, K. Derfler1, G. Körmözci4, K. Lhotta5 G.J. Zlabinger6, G.A. Böhmig1

Dept. of Internal Medicine III1, Dept. of Lab. Medicine2, Inst. of Pathology3, Dept. of Blood Group Serology4, Inst. of Immunology6, University of Vienna, and Dept. of Internal Medicine5, University of Innsbruck, Austria

 

Background: Complement-dependent cytotoxicity (CDC)-PRA testing is used to assess recipient presensitization and posttransplant alloantibody formation in kidney transplantation. Lymphocytotoxicity testing, however, can be affected by false positive results (e.g. autoantibodies, anti-lymphocyte reagents). As an alternative to CDC-PRA testing, detection of HLA antigen-specific PRA reactivity using HLA-class I or HLA-class II coated fluorescent microparticles was recently established. FlowPRAâ testing, however, does not distinguish between (presumably more harmful) complement-fixing and non-fixing alloantibodies. In this study, we established a novel assay allowing flow cytometric detection of alloantibody-dependent complement activation using the FlowPRAâ test.

 

Methods: CDC-PRA and FlowPRAâ testing (HLA-class-I and II) were performed according to standard protocols. For detection of alloantibody-dependent complement activation, FlowPRAâ beads were incubated with sera from highly sensitized dialysis patients and stained for C4 and C3 fragment, as well as C1q deposition using indirect immunofluorescence. To exclude preformed Ig-complement complexes and to demonstrate integrity of C4, Western blot analysis was performed.

 

Results: Testing sera from highly sensitized patients (PRA reactivity >60%) we demonstrate alloantibody-induced induction of C4 fragment (C4b/C4d) and, in parallel, C1 and C3 fragment deposition to FlowPRAâ beads. As shown by Western blot analysis C4 staining is not due to the presence of preformed C4 fragment-IgG/IgM complexes. Indeed, C4 deposition in our in vitro system was demonstrated to result from de-novo C4 activation: (I) Cleavage of the internal thioester of C4 by treatment of sera with the nucleophile methylamine completely abolished C4 fragment deposition. (II) C4 fragment deposition was not observed when beads were incubated with C4-free immunoadsorption eluates obtained from highly sensitized dialysis patients subjected to preemptive immunoadsorption therapy. C4 fragment deposition was partially restored by addition of normal serum but not C1q-depleted serum. As observed for alloantibody-binding to FlowPRAâ beads, deposition of C4 fragment binding was temperature- and time-dependent with maximal binding after 60 min incubation at 4°C. Similarly maximum C1q deposition was observed at 4°C. In contrast, maximum C3 fragment deposition was found at 37°C. At this temperature, C3 fragment deposition occurred in an alloantibody-independent fashion presumably as a result of alternative complement activation.

 

Conclusions: In summary, we herein present a novel cell-independent and easy to perform PRA test which enables flow cytometry-based detection of alloantibody-induced classical complement activation. Future studies testing larger patient cohorts will have to elucidate its relevance as an alternative to CDC-PRA testing and, moreover, as a non-invasive test to evaluate the presence of complement-activating alloreactivity in allograft rejection.

 

 

 

Contact regarding acceptance of the abstract:

 

“Flow cytometry based detection of HLA alloantibody mediated classical complement activation”

 

Georg A. Boehmig, M.D.

Div. of Nephrology and Dialysis

Dept. of Internal Medicine III

Währinger Gürtel 18-20

A-1090 Vienna, Austria

Tel.: +43-1-40400-4390

Fax: +43-1-40400-4499

Email: georg.boehmig@nephro.imed3.akh-wien.ac.at