[C4d]FlowPRA screening--a specific assay for selective detection of complement-activating anti-HLA alloantibodies.
On waiting lists, transplant candidates are routinely screened for potentially harmful complement-fixing alloantibodies using complement-dependent cytotoxicity (CDC) panel-reactive antibody (PRA) testing. We have recently developed a novel cell-independent assay for assessment of complement-activating panel reactivity ([C4d]FlowPRA), which is based on selective flow-cytometric detection of alloantibody-triggered C4 complement split product deposition to human leukocyte antigen (HLA)-coated FlowPRA beads. Serum specimens selected from 120 transplant candidates were evaluated by [C4d]FlowPRA (HLA class I vs II) in comparison with FlowPRA IgG alloantibody screening (HLA class I vs II), a method detecting both complement- and noncomplement-activating alloantibodies, and with CDC-PRA on separated T (T-CDC) or B cells (B-CDC, evaluation on platelet-absorbed sera). For each assay, >/=10% PRA reactivity was considered positive. Comparing complement-dependent PRA assays with standard FlowPRA, the specificity calculated for [C4d]FlowPRA (HLA class I: 92%; class II: 100%) was found to be superior to that of CDC testing (T-CDC-PRA: 79%; B-CDC-PRA: 86%). Because noncomplement-activating alloreactivities were not detected for both techniques, low sensitivities were calculated ([C4d]FlowPRA HLA class I: 61%; class II: 31%; T-CDC-PRA: 70%; B-CDC-PRA: 55%). Compared with CDC-PRA, [C4d]FlowPRA gave a high specificity (HLA class I compared with T-CDC: 89%, HLA class II compared with B-CDC: 95%) but, at least in part because of false-positive CDC results, a modest sensitivity (66% and 38%, respectively). For both HLA classes, we found a highly significant association between absolute [C4d]FlowPRA and CDC-PRA levels (p < 0.0001). Our results suggest that detection of C4 split product deposition to FlowPRA beads may represent an attractive HLA-specific and time-effective alternative to CDC-PRA screening.
Wahrmann
Markus
M
Department of Medicine III, Medical University of Vienna, Vienna, Austria.
Exner
Markus
M
Haidbauer
Bettina
B
Schillinger
Martin
M
Regele
Heinz
H
Körmöczi
Günther
G
Böhmig
Georg A
GA
D001402:B-Lymphocytes / Q000276:immunology; D003167:Complement Activation / Q000276:immunology*; D003181:Complement C4 / Q000276:immunology* / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic; D005434:Flow Cytometry / Q000379:methods; D006680:HLA Antigens / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007518:Isoantibodies / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008863:Microspheres; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000276:immunology
D003181:Complement C4; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D007518:Isoantibodies