Prevalence, distribution and amplitude of the complement interference phenomenon in single antigen flow beads assays

Abstract

HLA antibody detection with single antigen flow beads (SAFB) assays is impaired by complement interference whose frequency, predictability and distribution among HLA antigens have not been analyzed in large cohorts. We compared in two patients' cohorts the routine follow-up SAFB profiles obtained in class I (n = 129) and class II (n = 85) with and without ethylenediaminetetraacetic acid (EDTA)-treatment. The presence of complement interference was defined according to the reproducibility of the SAFB assays evaluated with our class I and II routine positive control sera. Interference occurred in 29.5% and 45.9% of patients in class I and II, respectively. In the untreated condition, at serum level, neither the number of positive beads, the highest bead mean fluorescence intensity (MFI) nor MFI at bead level, satisfactorily predicted interference. HLA-C were the least affected among class I beads. HLA-DQ beads were the most affected in class II. At least one antibody specificity was falsely negative without EDTA for about 3% of sera in class I and 9% in class II. EDTA-treatment did not significantly modify the low-MFI strengths (500-3000 range). This study emphasizes the high frequency of complement interference and the importance and advantages of systematically pretreating sera with EDTA before performing SAFB assays.

Caveats of HLA antibody detection by solid‐phase assays

“…The formation of the C1 complex can be inhibited by pretreatment of the blood sample with heat, dithiothreitol (DTT), or ethylenediaminetetraacetic acid (EDTA) . Using plasma samples or adding EDTA to the patient serum sample has meanwhile become the preferred option of many HLA laboratories to prevent this so‐called “complement interference” , which often is still erroneously termed “prozone effect” as pointed out by Berth et al . .…”

Reassessment of the clinical impact of preformed donor-specific anti-HLA-Cw antibodies in kidney transplantation

“…However, these studies were conducted before the discovery of 2 major interferences with SAFB results. The first is the complement interference phenomenon, which prevents the detection, or leads to the underestimation, of high‐titer antibodies . The second is the detection of antibodies against denatured HLA (anti‐dHLA), particularly class I HLA, which are strongly suspected to be clinically irrelevant .…”

Technical challenges and clinical relevance of single antigen bead C1q/C3d testing and IgG subclass analysis of human leukocyte antigen antibodies

“…Importantly, luminex SAB assays are semiquantitative at best, since MFI values do not reflect antibody titers . Furthermore, false‐positive reactions are observed because of antibody reactivity to denatured HLA molecules on the beads while false‐negative results are because of the so‐called prozone effect or interference of serum substances other than IgG . Although not part of the commercial protocol provided by the manufacturers of the IgG SAB assays, serial dilutions, heat inactivation or hypotonic dialysis of the serum samples as well as pretreatment with dithiothreitol (DTT) or ethylenediaminetetraacetic acid (EDTA) are means to circumvent the prozone effect or interference of serum components such as IgM and complement in the detection of circulating IgG antibodies .…”