Deciphering Complement Interference in Anti–Human Leukocyte Antigen Antibody Detection With Flow Beads Assays

Visentin, Jonathan; Vigata, Margaux; Daburon, Sophie; Contin‐Bordes, Cécile; Frémeaux‐Bacchi, Véronique; Dromer, C.; Billes, Marc-Alain; Néau-Cransac, Martine; Guidicelli, Gwendaline; Taupin, Jean‐Luc

doi:10.1097/tp.0000000000000315

Cited by 86 publications

(75 citation statements)

References 15 publications

(7 reference statements)

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“…This phenomenon has been recently attributed to a complement inhibitory interference in the SAB assay, likely due to a quenching effect exerted on the bead surfaces by C4 and C3 activation products [8,9]. The presence of a complement dependent hook effect in our SAB assays is supported by the demonstration that DSA detected after heat inactivation, with the exception of DQB1*0201 antibody, were endowed with C1q binding capability.…”

supporting

confidence: 60%

Serum complement inactivation unveiled prepregnancy donor-specific HLA antibodies leading to postpartum kidney graft loss

et al. 2015

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supporting

confidence: 60%

Serum complement inactivation unveiled prepregnancy donor-specific HLA antibodies leading to postpartum kidney graft loss

et al. 2015

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“…For these four patients, the explanation was a complement interference phenomenon due to the accumulation on the beads of activated C3 and C4, dose-dependently hindering the binding of the fluorescent anti-IgG conjugate to the serum HLA antibody. 23 Chelating bivalent cations with EDTA to block complement activation restored the detection of anti-HLA antibodies. 24,25 Therefore, in the absence of EDTA, the SAFB assay underestimated the MFI strength of these C1q+ dnDSAs in these four patients.…”

Section: Discussionmentioning

confidence: 99%

Non-Complement–Binding De Novo Donor-Specific Anti-HLA Antibodies and Kidney Allograft Survival

Guidicelli¹,

Guerville²,

Lepreux³

et al. 2016

Journal of the American Society of Nephrology

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121

112

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C1q-binding ability may indicate the clinical relevance of de novo donor-specific anti-HLA antibodies (DSA). This study investigated the incidence and risk factors for the appearance of C1q-binding de novo DSA and their long-term impact. Using Luminex Single Antigen Flow Bead assays, 346 pretransplant nonsensitized kidney recipients were screened at 2 and 5 years after transplantation for de novo DSA, which was followed when positive by a C1q Luminex assay. At 2 and 5 years, 12 (3.5%) and eight (2.5%) patients, respectively, had C1q-binding de novo DSA. De novo DSA mean fluorescence intensity .6237 and .10,000 at 2 and 5 years, respectively, predicted C1q binding. HLA mismatches and cyclosporine A were independently associated with increased risk of C1q-binding de novo DSA. When de novo DSA were analyzed at 2 years, the 5-year death-censored graft survival was similar between patients with C1q-nonbinding de novo DSA and those without de novo DSA, but was lower for patients with C1q-binding de novo DSA (P=0.003). When de novo DSA were analyzed at 2 and 5 years, the 10-year death-censored graft survival was lower for patients with C1q-nonbinding de novo DSA detected at both 2 and 5 years (P,0.001) and for patients with C1q-binding de novo DSA (P=0.002) than for patients without de novo DSA. These results were partially confirmed in two validation cohorts. In conclusion, C1q-binding de novo DSA are associated with graft loss occurring quickly after their appearance. However, the long-term persistence of C1q-nonbinding de novo DSA could lead to lower graft survival.

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“…In the current study, the vast majority of the patients (71%) exhibited prozone that affected at least one antibody specificity. As we were writing this manuscript, two additional publications using EDTA to uncover prozone were reported (19,20). Those studies used higher concentrations of EDTA and claim to uncover all prozone present.…”

Section: Discussionmentioning

confidence: 99%

Assessing Antibody Strength: Comparison of MFI, C1q, and Titer Information

Tambur

Herrera

Haarberg

et al. 2015

American Journal of Transplantation

225

217

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The presence of donor-specific HLA antibodies before or after transplantation may have different implications based on the antibody strength. Yet, current approaches do not provide information regarding the true antibody strength as defined by antigen-antibody dissociation rate. To assess currently available methods, we compared between neat mean fluorescence intensity (MFI) values, C1q MFI values, ethylenediaminetetraacetic acid (EDTA)-treated samples, as well as titration studies and peak MFI values of over 7000 Luminex-based single-antigen HLA antibody data points. Our results indicate that neat MFI values do not always accurately depict antibody strength. We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with C1q or titration studies. In this study of patients presenting with multiple antibody specificities, a prozone effect was observed in 71% of the cohort (usually not affecting all antibody specificities within a single serum sample, though). Similar to titration studies, the C1q assay was able to address the issue of potential inhibition; however, its limitation is its low sensitivity and inability to detect the presence of weak antibodies. Titration studies are the only method among the approaches used in this study to provide information suggesting antigen-antibody dissociation rates and are, therefore, likely to provide better indication of true antibody strength.Abbreviations: AMR, antibody-mediated rejection; DSA, donor-specific antibody; EDTA, ethylenediaminetetraacetic acid; MFI, mean fluorescence intensity; SAB, single antigen beads; SPA, solid phase assays

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Deciphering Complement Interference in Anti–Human Leukocyte Antigen Antibody Detection With Flow Beads Assays

Abstract: 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.

Cited by 86 publications

References 15 publications

Serum complement inactivation unveiled prepregnancy donor-specific HLA antibodies leading to postpartum kidney graft loss

Serum complement inactivation unveiled prepregnancy donor-specific HLA antibodies leading to postpartum kidney graft loss

Non-Complement–Binding De Novo Donor-Specific Anti-HLA Antibodies and Kidney Allograft Survival

Assessing Antibody Strength: Comparison of MFI, C1q, and Titer Information

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