Flavia Sequeira

C1q-fixing human leukocyte antigen antibodies are specific for predicting transplant glomerulopathy and late graft failure after kidney transplantation.

Background. Human leukocyte antigen (HLA) antibodies, especially those that fix complement, are associated with antibody-mediated rejection and graft failure. The C1q assay on single antigen beads detects a subset of HLA antibodies that can fix complement and precede C4d deposition. The aim of this study was to determine whether C1q-fixing antibodies distinguish de novo donor-specific antibodies (DSA) that are clinically relevant and harmful. Methods. We retrospectively studied 31 of 274 kidney transplant recipients who had pretransplant and concurrent biopsy and serum specimens, 13 with C4d-positive and 18 with C4d-negative staining. We measured IgG and C1q DSA pretransplant and at the time of biopsy using single antigen bead assays. We identified 13 recipients who developed de novo DSA by IgG or C1q and examined associations with C4d deposition, transplant glomerulopathy, and graft failure. Results. Testing for DSA by IgG is more sensitive for C4d deposition (IgG: 100%, 95% confidence interval [CI] 0.60–1; C1q: 75%, 95% CI 0.36–0.96). Testing for DSA by C1q is more specific for transplant glomerulopathy (C1q: 81%, 95% CI 0.57–0.94; IgG: 67%, 95% CI 0.43–0.85) and graft loss (C1q: 79%, 95% CI 0.54–0.93; IgG: 63%, 95% CI 0.39–0.83). Absence of de novo DSA by IgG and C1q has a high negative predictive value for the absence of C4d deposition (IgG: 100%, 95% CI 0.73–1; C1q: 88%, 95% CI 0.62–0.98), transplant glomerulopathy (IgG: 100%, 95% CI 0.73–1; C1q: 100%, 95% CI 0.77–1), and graft failure (IgG: 86%, 95% CI 0.56–0.97; C1q: 88%, 95% CI 0.62–0.98). Conclusion. Monitoring patients with the C1q assay, which detects antibodies that fix complement, offers a minimally invasive means of identifying patients at risk for transplant glomerulopathy and graft loss.

Novel C1q assay reveals a clinically relevant subset of human leukocyte antigen antibodies independent of immunoglobulin G strength on single antigen beads.

It has been known for 40 years that cytotoxic human leukocyte antigen (HLA) antibodies are associated with graft rejection. However, the complement-dependent cytotoxicity assay (CDC) used to define these clinically deleterious antibodies suffers from a lack of sensitivity and specificity. Recently, methods exploiting immunoglobulin G (IgG) antibody binding to HLA single antigen beads (SAB) have overcome sensitivity and specificity drawbacks but introduced a new dilemma: which of the much broader set of antibodies defined by these methods are clinically relevant. To address this, we developed a complement-fixing C1q assay on the HLA SAB that combines sensitivity, specificity, and functional potential into one assay. We compared the CDC, IgG, and C1q assays on 96 sera having 2,118 defined antibodies and determined that CDC detects only 19% of complement-fixing antibodies detected by C1q, whereas C1q detects only 47% of antibodies detected by IgG. In the same patient, there is no predictability by IgG mean fluorescence intensity (MFI) as to which of the antibodies will bind C1q because fixation is independent of MFI values. In 3 clinical studies, C1q(+) antibodies appear to be more highly correlated than those detected by IgG alone for antibody-mediated rejection in hearts as well as for kidney transplant glomerulopathy and graft failure.

Complement‐fixing donor‐specific antibodies identified by a novel C1q assay are associated with allograft loss

Sutherland SM, Chen G, Sequeira FA, Lou CD, Alexander SR, Tyan DB. Complement‐fixing donor‐specific antibodies identified by a novel C1q assay are associated with allograft loss. 
Pediatr Transplantation 2012: 16: 12–17.

Antibody-mediated rejection despite inhibition of terminal complement

Terminal complement blockade has been shown to decrease the incidence of early acute antibody‐mediated rejection (eAMR) in the first month after positive cross‐match kidney transplant recipients, yet some patients still develop eAMR. The current study investigated possible mechanisms of eAMR despite eculizumab treatment. Of the 26 patients treated with eculizumab, two developed clinical eAMR and another patient developed histologic signs of eAMR without graft dysfunction (‘subclinical eAMR’). Twenty‐three did not have histologic injury on early surveillance biopsies. All 26 patients had therapeutic levels of eculizumab and showed complete blockade of complement in hemolytic assays. High levels of donor‐specific alloantibody (DSA) including total IgG, IgG3, and C1q+ DSA were present in patients with and without eAMR, and none correlated well with eAMR. In contrast, IgM DSA was present in only four patients after transplantation: the two patients with clinical eAMR, one patient with subclinical AMR, and one patient without eAMR (P = 0.006 correlation with eAMR). Both clinical eAMR episodes were easily treated with plasma exchange which removed IgM more completely and rapidly than IgG, resulting in normalization of function and histology. These data suggest a possible role of antidonor IgM DSA in the pathogenesis of eAMR in patients treated with terminal complement blockade (ClinicalTrials.gov Identifier: NCT00670774).

Complement (C1q) fixing solid-phase screening for HLA antibodies increases the availability of compatible platelet components for refractory patients.

BACKGROUND: Immune refractoriness to platelet (PLT) transfusion is primarily due to HLA antibody. Patients at our institution are identified as refractory due to HLA by a Luminex‐based immunoglobulin (Ig)G–single‐antigen‐bead (SAB) assay, but in highly sensitized patients, antigen‐negative compatible donors cannot be found due to the high sensitivity of the IgG‐SAB method. We developed an assay that detects only HLA antibodies binding the first complement component (C1q). We hypothesized that the C1q‐SAB method might be more relevant than the IgG‐SAB method because the antibodies identified may activate the complement cascade causing PLT destruction.