Triglycerides, but not bilirubin or hemoglobin, can interfere with the modified ham (mHam) functional complement assay

Abstract

Abstract Complement-mediated thrombotic microangiopathy (CM-TMA)/atypical hemolytic uremic syndrome (aHUS) is a rare, potentially lethal TMA resulting from uncontrolled complement activation, often due to genetic mutations or acquired complement dysregulation. Previously a diagnosis of exclusion, the modified Ham (mHam) test was developed to directly diagnose aHUS. In the mHam, cell-killing of a complement-susceptible bioluminescent reporter cell line by patient serum is measured. In serum from a patient with uncontrolled complement activation due to aHUS, reporter cells are killed, measured as a loss of bioluminescence. The mHam became available clinically only recently; however, the assay performance in samples with potentially-interfering substances (hemoglobin, bilirubin, and lipids) has not been fully characterized. For this study, interfering substances were assessed by spiking hemoglobin, bilirubin, and triglycerides into positive and negative serum samples. Our findings indicate that triglycerides significantly impact the assay readout but not hemoglobin or bilirubin. Specifically, addition of increasing amounts of triglycerides increased the measured viability for the positive sample (19.05% for unspiked, 42.35% for the sample spiked with 125 mg/dL, 53.31% for the sample spiked with 250 mg/dL, 101.26% for the sample spiked with 1000 mg/dL triglyceride). Student T-tests on log-transformed viability ratios for triglyceride-spiked vs. unspiked positive controls showed a significant difference in viability measurements for positive controls at all triglyceride levels. Student T-tests on log-transformed viability ratios for triglyceride-spiked vs. unspiked negative controls showed no significant differences in viability measurements for negative controls at all triglyceride levels.