Luminex® C1q

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  • Sarah Peacock, Vasilis Kosmoliaptsis, Andrew J.Bradley and Craig J.Taylor

    Luminex single antigen bead (SAB) technology enables highly sensitive and rapid characterization of immunoglobulin G (IgG) human leukocyte antigen (HLA)-specific antibodies. It is widely used to determine antibody compatibility for renal transplantation and to aid the diagnosis of antibody-mediated rejection and response to therapy. HLA-specific antibodies may contribute to allograft rejection through a variety of mechanisms, including activation of the classical complement pathway, endothelial cell activation, and recruitment of Fc-dependent effector cells. Of these, complement-mediated cytotoxicity has long been associated with hyperacute rejection and, more recently, antibody-mediated rejection for which the deposition of the complement component C4d on peritubular capillaries is a diagnostic marker. In the standard SAB assay, patient serum is incubated with microbeads coated with purified HLA proteins. Human leukocyte antigenYspecific IgG antibody binding is then detected using a fluorescent anti-IgG detection antibody. Increasing levels of donor HLA-specific antibodies detected by this assay predict inferior long-term graft outcome, but not all patients with IgG donor-specific HLA antibodies (DSA) have a poor graft outcome (1,2). This observation is consistent with the notion that HLA antibodies with the same specificity may differ in their ability to cause graft injury, and this variability may be related to differences in complement fixing activity.

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  • D.Thammanichanond, T.Mongkolsuk, S.Rattanasiri, S.Kantachuvesiri, S.Worawichawong, S.Jirasiritham and P.Kitpoka

    Objectives. De novo donor-specific HLA antibodies (DSA) are associated with allograft rejection and allograft loss. However, not all DSA are equally detrimental to allograft function. The ability to activate complement may be an important factor differentiating clinically relevant DSA from nonrelevant DSA. The C1q assay detects a subset of HLA antibodies that can fix complement. This study aimed to investigate the correlation between C1q-fixing de novo DSA (dnDSA) and clinical outcomes posttransplant.
    Methods. This retrospective study included 193 sera from kidney transplant recipients who underwent posttransplant DSA testing and/or kidney biopsy for clinical causes. Thirtyfive of the 193 (18.1%) had immunoglobulin G DSA. Seventeen of the 35 patients were excluded owing to the presence of pretransplant HLA antibodies. We then analyzed C1q DSA at the time of biopsy in 18 recipients who developed dnDSA. The clinical outcomes of patients with C1q-positive DSA and C1q-negative DSA were compared.
    Results. C1q-positive DSA were detected in 10 of 18 patients (55.6%). The incidences of transplant glomerulopathy were significantly higher among patients with C1q-positive DSA than patients with C1q-negative DSA (80% vs 0%; P ¼ .001). Although patients with C1qpositive DSA experienced more chronic antibody-mediated rejection and graft loss (80% vs 37.5% [P ¼ .145]; 60% vs 25% [P ¼ .188]), the differences were not significant. The receiver operating characteristic curve analysis showed that the C1q assay was an excellent predictor of transplant glomerulopathy with area under the curve of 0.9 (95% CI, 0.769e1.000).
    Conclusion. The presence of C1q-positive dnDSA was associated with an increased risk of transplant glomerulopathy.

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  • Marta Crespo, Alberto Torio, Virginia Mas, Dolores Redondo, Maria J.Pérez-Sáez, Marisa Mir, Anna Faura, Rita Guerra, Olga Montes-Ares, Maria D.Checa, Julio Pascual

    Anti-HLA donor-specific antibodies (DSA) identified by single antigen bead array (SAB) are questioned for their excess in sensitivity and lack of event prediction after transplantation. Population and methods: We retrospectively evaluated specific types of preformed DSA (class I, class II or C1qfixing) and their impact on graft survival. Kidney transplantations performed across negative CDC-crossmatch were included (n = 355). Anti-HLA antibodieswere tested using SAB to identify DSA and their capacity to fix C1q.
    Results: Twenty-eight patients with pretransplant DSA+ with MFI N 2000 were selected to assess C1q fixation. DSA were C1q+in 15 patients and C1q- in 13, without significant differences in demographics, acute rejection, graft loss or renal function. The maximum MFI of DSA in patients with C1q-fixing DSA was significantly higher (p = 0.008). Patients with DSA class-I suffered more antibody-mediated rejection (AMR) and had worse graft survival than class-II. The capacity of DSA I to fix C1q did not correlate with rejection, graft function or graft loss.
    Conclusions: C1q testing in pretransplant serawith DSA was unable to predict acute antibody-mediated rejection or early graft loss, but the presence of DSA class I compared to DSA only class II did. Despite non-fixing complement in vitro, pretransplant C1q-negative DSA I can mediate rejection and graft loss.

    Keywords: Antibody-mediated rejection, Complement, Donor-specific antibody, HLA antibody, Kidney transplantation

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  • Ge Chen and Dolly B. Tyan

    Solid phase Luminex® and flow cytometric single antigen bead assays offer exquisite sensitivity and specifi city for HLA antibody detection. Unlike the historical complement-dependent cytotoxicity (CDC) method, these assays do not distinguish complement fixing from non-complement fixing antibody, the former of which are considered the most clinically relevant in the peri-transplant period. This chapter describes a novel solid phase C1q binding assay to distinguish HLA antibodies that can bind the first component of complement (C1q). These antibodies have the capacity to initiate the complement cascade irrespective of whether that actually occurs. The C1q assay detects many more complement fixing antibodies than are observed by the less sensitive and less specific CDC assay.

    Keywords: HLA antibody, Solid phase assay, Single antigen beads, Luminex®, Transplantation, Complement, C1q

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