When “Neat” Just Isnʼt Enough: Gaining Precision and Depth in HLA Antibody Data Interpretation Utilizing Titers and C1q Antibody Binding Assay.

Abstract

BACKGROUND: Virtual crossmatches (VXM) are routinely performed utilizing solid-phase single antigen (SA) HLA antibody (Ab) assays to determine compatibility and induction and maintenance therapies for transplant recipients. SA tests are also a critical component of post-transplant monitoring for Ab mediated rejection (AMR). Neat, or undiluted, sera samples from highly sensitized patients (pts) are susceptible to prozone in these assays, which masks strong or high titer donor specific Ab (DSA). Titration and/or C1q-binding Ab (C1q) assays could alleviate these issues and add needed data to determine clinical risk from DSA. METHODS: We prospectively tested 47 neat and tittered samples from 34 highly sensitized pts who underwent kidney or heart transplant at our center, using commercially available SA-IgG and C1q kits. SA-IgG data was stratified into Strong (>10,000 MFI), Strong Moderate (5,001-10,000 MFI), Weak Moderate (2,501-5,000 MFI), Weak (1,000-2,500 MFI) and Negative Ab expression (< 1,000 MFI). C1q Ab expression was categorized as Strong (>10,000 MFI), Strong Moderate (5,001-10,000 MFI), Weak Moderate (1001-5,000 MFI), Weak (200-1000 MFI) and Negative (< 200 MFI). RESULTS: Prozone effect, as revealed by titration studies, was observed in 10/34 patients (11/47 tests) using neat sera SA-IgG tests. Prozone was most apparent at a titer of 1:16 on average; 0% of C1q results displayed prozone. Importantly, comparisons between neat MFI values and antibody strength, using titer evaluation, showed disconcordance in 19/34 patients. Specifically, 12/34 patients (15/47 tests) exhibited low neat MFI (1,000-5,000) while antibody titers were high (1:256->1:1024). Furthermore, 6/34 patients (7/47 tests) exhibited high neat MFI (>5,000) but the antibody strength per titer was low (1:1-1:32). CONCLUSION: Prozone effect was observed in 30% of patients in this cohort. Conflicting data between neat IgG MFI values and titers were observed in 56% of patients. While both titration and C1q studies alleviated this issue in 100% of the cohort, only titration studies informed a sense of avidity and affinity of Ab binding while maintaining high sensitivity, as only 9.4% of DSA tittering at or below 1:64 were detected by C1q. We conclude that titration studies provide valuable clinical information in accurately predicting VXM results and effectiveness of desensitization or of therapeutic modalities to resolve AMR. DISCLOSURES:Friedewald, J.: Grant/Research Support, Pfizer, Stockholder, TGI.