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Updated: Apr 26, 2026
Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry
Published on: May 19, 2020
Deciphering complement interference in anti-human leukocyte antigen antibody detection with flow beads assays
Jonathan Visentin1, Margaux Vigata, Sophie Daburon
11 Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France. 2 UMR CNRS 5164, Université de Bordeaux, Talence, France. 3 Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Laboratoire d'immunologie biologique, Paris, France. 4 Service des Maladies Respiratoires, Hôpital Haut-Lévêque, CHU de Bordeaux, Pessac, France. 5 Service de Chirurgie Cardiaque, Hôpital Haut-Lévêque, CHU de Bordeaux, Pessac, France. 6 Service de Transplantation Hépatique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France. 7 Address correspondence to: Jean-Luc Taupin, Pharm. D., Ph.D., Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Place Amélie Raba Léon, 33076 Bordeaux Cedex, France.
Background:
Anti-human leukocyte antigen (HLA) antibody detection in solid-phase flow beads assays can be quenched by complement activation, but the precise mechanism of this interference is not fully elucidated yet.
Methods:
Using the Luminex flow beads screening assay for detection of anti-HLA antibodies, we analyzed the binding of high concentrations of the pan class I anti-HLA monoclonal antibody W6/32 in neat normal, ethylenediaminetetraacetic acid-treated normal and complement factors C1q, C4/C3, C2, C3, factor B or C5-depleted human sera, using anti-mouse immunoglobulin G as the detection antibody. Complement activation and binding to beads were revealed using anti-human C1q, C4d, and C3d antibodies. To translate our findings to the human setting, we used the class I and class II HLA single-antigen flow beads assays and sera from four patients with high titers of antibodies.
Results:
Detection of W6/32 did not suffer any interference with C1q and C4/C3-depleted sera. A partial quenching was observed with C2, C3, and factor B-depleted sera, but was more pronounced with the factor B-depleted serum. W6/32 was undetectable in presence of C5-depleted serum. The binding of activation products derived from C3 principally, and also from C4, impaired immunoglobulin G and C1q detection. Accordingly, C4d detection was hindered by deposition of activated C3. Similar findings were obtained with patients' sera.
Conclusion:
Binding of C4 and C3 activation products is the main responsible for complement interference in flow beads assays. A complete quenching requires complement activation through C3 cleavage and its amplification by the alternative pathway.
