C1q-binding Donor-specific Antibodies Research Articles

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Aim The C1qScreen™ assay detects complement binding HLA antibodies. Recent studies suggest that the presence of C1q positive donor specific antibodies (DSA) is associated with poor post-transplant outcomes. However, in some patients with documented episodes of antibody mediated rejection or graft failure, C1q binding DSA are not detected by the C1qScreen™ suggesting poor assay sensitivity. The goal of this study was to develop an enhanced C1q assay to improve the detection of complement binding HLA DSA. Methods Nine patient sera with well characterized (using IgG single antigen bead (SAB) assay) post-transplant DSA were tested for C1q binding using the standard C1qScreen™ and three modified protocols: 1) extended incubation (EI), 2) wash modified (WM) and 3) anti-human globulin (AHG) C1q enhanced (ACE) protocol. The number of class I/II DSA detected and DSA MFI values obtained with each protocol were compared. Results The C1qScreen™ was positive for only 30% of Class I (9/30) and 52% of Class II (15/29) IgG DSA specificities. The average MFI DSA values were markedly reduced compared with the IgG-SAB assay. The EI and WM protocols exhibited higher average DSA MFI values (2.25 and 3.4 fold higher respectively) compared to the standard protocol but only 2 additional DSA specificities were detected. In contrast, the ACE protocol identified 10 additional Class I (19/30; 63%) and 4 additional Class II (19/29; 66%) DSA specificities with an average increase in DSA MFI of 4.7 fold when compared with the standard C1qScreen™. Conclusions The C1qScreen™ exhibits poor assay sensitivity and fails to adequately identify many post-transplant complement fixing HLA DSA. The sensitivity of the C1qScreen™ to detect DSA can be improved with various protocol modifications. The ACE protocol was the most sensitive of the assays tested and detected the majority of post transplant HLA DSA. Future studies will investigate the clinical significance of HLA DSA detected by the ACE protocol.

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Aim The presence of C1q antibodies may be important for assessing the risk of antibody mediated rejection (AMR) in solid organ transplants. We studied the correlation between the C1q-single antigen bead assay (C1q-SAB) with the long-incubation complement-dependent lymphocytotoxicity assay (CDC) and the IgG-single antigen bead assay (IgG-SAB). Methods 60 reference sera (38 Class I and 22 Class II) from the UCLA serum exchange program were characterized using the CDC, Luminex PRA (Gen-probe Inc.) and IgG-SAB (One Lambda Inc.) assays. We also tested pre- and post-transplant sera from 16 renal allograft recipients. All patients had persistent posttransplant DSA as demonstrated by the IgG-SAB assay. Results The C1q-SAB assay showed 85% concordance with the CDC assay as 15% of the CDC positive antibodies were negative in the C1q test. Correlation between the SAB MFI and C1q positivity showed that 90% of sera with the MFI of >18,000 in the IgG-SAB assay were C1q binding. A retrospective study of 16 renal transplant recipients developing post-transplant donor specific antibodies (DSA) showed 5 of 9 patients diagnosed as C4d+ AMR displayed C1q positive DSA (P = 0.145). One out of 7 patients without AMR displayed C1q binding DSA. Conclusions Our data indicate that antibody strength correlates with the capacity to bind C1q (Fig. 1). However we identified a subset of antibodies that displayed high MFI values and CDC positivity, that failed to bind C1q. The development of C1q positive DSA was not significantly associated with positive C4d staining on the graft. Additional studies are required using a larger cohort of patients to verify these findings [Fig. 1]. Download : Download full-size image

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Aim HLA antibody identification testing has become increasingly sensitive, especially solid-phase assays using fluorescent detection. Recently, a new Luminex single antigen bead (SAB) based assay has been developed that detects HLA antibodies binding C1q, the initial complement protein. This test may help differentiate HLA antibodies with more clinical relevance than others, but the significance of C1q antibodies post-transplant (Tx) is still being determined. We have introduced C1q testing into our renal allograft antibody monitoring. Patients with donor specific antibody (DSA) also undergo C1q testing to determine DSA complement binding ability. To understand the clinical significance of C1q DSA, we determined the associations between C1q binding DSA and serum creatinine (Cr) and pathological C4d staining in a cohort of renal allograft patients. Methods DSA was detected in 72 sera by conventional Luminex SAB and C1q testing in 41 recipients (31 adult, 10 pediatric). Cr was recorded at the time of DSA testing and at the time of last follow-up. 19 adult patients had 24 biopsies at the time of C1q testing and were scored for peritubular capillary C4d immunostaining. Results At the time of DSA testing (mean 1364 days; range 7-8579), Cr did not differ between patients with or without C1q-binding DSA (1.9 ± 1.3 vs 2.1 ± 1.4; p = 0.41). Patients with DSA testing 1 year post-Tx (p = 0.29). At the time of last follow-up (mean 225d after C1q testing), Cr was significantly greater in patients with C1q DSA (2.8 ± 2.8 vs 1.4 ± 0.6; p = 0.05). In 79% of patients with C1q DSA had C4d focal or diffuse on renal biopsy compared to 60% in those without C1q DSA (Chi-Square = 9.35 p = 0.025). However, some patients with C1q DSA had minimal/no C4d, and patients without C1q DSA displayed focal or diffuse C4d. Conclusions In our renal allograft cohort, serum Cr did not different at the time of C1q DSA testing, but patients with C1q DSA had increased incidence of renal biopsy C4d and higher Cr at the time of last follow-up.