Clinical utility of serial serum dilutions for HLA antibody interpretation

Abstract

Luminex single antigen bead (SAB) testing has increased the sensitivity and specificity of accurately identifying HLA antibodies, in support of all organ transplantation. However, as described in manufacturers' recommendation, the output of the assay, using mean fluorescence intensity (MFI) units, is only semi‐quantitative. Therefore, the ability to use MFI values to compare between different assays, to accurately guide clinical practice, or be used as an endpoint measure in clinical trials, is limited. To improve potential quantification, one must circumvent inherent limitations of SAB assays such as interference and saturation phenomena. In this review, we discuss how measurement of pre‐transplant serum dilutions can be used to determine unacceptable antigens for wait‐listing, determine the likelihood for successful HLA antibody reduction with desensitization, and compare degree of HLA (in)compatibility among various living donors. We also discuss how serum dilutions are optimal for measuring and comparing the efficacy of antibody depletion therapies for desensitization or antibody mediated rejection treatment post‐transplant. Historically, one of the main criticisms for the use of serum dilutions and titer has been the potential labor and cost associated with additional testing. Here, we show how only one or two dilutions can add major value in most circumstances. In summary, the practical use of serum dilutions and titer determination are important methods that can be used before and after transplantation of all organs to quantify antibody accurately and reliably in routine practice and in clinical trials.

BSHI and BTS UK guideline on the detection of alloantibodies in solid organ (and islet) transplantation

“…Although the term 'prozone' is often used to describe these inhibitions within the literature, this is an erroneous use of the term in this setting, and its use often causes confusion (Tambur & Schinstock, 2022). Prozone refers to agglutination / precipitation assays to describe a zone in which reactions do not happen.…”

“…The presence of inhibitory factors which negatively impacts the accurate determination of HLA antibody by SAB assays, have been widely reported in the literature (Tambur & Schinstock, 2022). Such inhibitory factors reported include presence of IgM antibodies (Kosmoliaptsis et al., 2009), high concentration HLA IgG (Tambur & Schinstock, 2022) and complement components (Schnaidt et al., 2011; Schwaiger et al., 2014; Weinstock & Schnaidt, 2013), the result of these reported inhibitions being a negative result or falsely low MFI readout. Although the term ‘prozone’ is often used to describe these inhibitions within the literature, this is an erroneous use of the term in this setting, and its use often causes confusion (Tambur & Schinstock, 2022).…”

“…Such inhibitory factors reported include presence of IgM antibodies (Kosmoliaptsis et al., 2009), high concentration HLA IgG (Tambur & Schinstock, 2022) and complement components (Schnaidt et al., 2011; Schwaiger et al., 2014; Weinstock & Schnaidt, 2013), the result of these reported inhibitions being a negative result or falsely low MFI readout. Although the term ‘prozone’ is often used to describe these inhibitions within the literature, this is an erroneous use of the term in this setting, and its use often causes confusion (Tambur & Schinstock, 2022). Prozone refers to agglutination / precipitation assays to describe a zone in which reactions do not happen.…”

“…While the writing committee agrees that this is the most effective mechanism of removing assay inhibition, several studies have shown dilutions may continue to be of benefit. Tambur and colleagues (Tambur & Schinstock, 2022) demonstrate that falsely low MFI values can still be observed in some sera treated with EDTA. Most recently the use of dilutions has been suggested to assist in the investigations of HSP sera, particularly where CDC XM assay may not be available (Daga & Briggs, 2023).…”

Antikörpertestung in der Nierentransplantation

“…Falsch-negative Ergebnisse durch Inhibitoren oder durch das "Prozonenphänomen", die für Seren mit einer hohen Konzentration an HLA-Antikörpern beschrieben sind, lassen sich durch Vorbehandlung der Seren mit Ethylendiamintetraazetat (EDTA) oder Hitzeinaktivierung oder durch die Verdünnung des Serums umgehen [14][15][16]. In hochtitrigen Patientenseren mit abgesättigten Beads kann nur durch Verdünnungsreihen die eigentliche Stärke einzelner Antikörper herausgearbeitet werden, die im unverdünnten Serum vergleichbar hohe Werte für die Fluoreszenzintensität (MFI) haben [17].…”