[C4d]FlowPRA screening--a specific assay for selective detection of complement-activating anti-HLA alloantibodies

Hum Immunol. 2005 May;66(5):526-34. doi: 10.1016/j.humimm.2004.12.007. Epub 2005 Feb 12.


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.

Publication types

  • Comparative Study

MeSH terms

  • B-Lymphocytes / immunology
  • Complement Activation / immunology*
  • Complement C4 / immunology*
  • Complement C4 / metabolism
  • Cytotoxicity Tests, Immunologic
  • Flow Cytometry / methods
  • HLA Antigens / immunology*
  • Histocompatibility Antigens Class I / immunology
  • Histocompatibility Antigens Class II / immunology
  • Humans
  • Immunoassay / methods
  • Isoantibodies / immunology*
  • Leukocytes, Mononuclear / immunology
  • Microspheres
  • Reproducibility of Results
  • T-Lymphocytes / immunology


  • Complement C4
  • HLA Antigens
  • Histocompatibility Antigens Class I
  • Histocompatibility Antigens Class II
  • Isoantibodies