Interference in the anti-Xa heparin activity assay due to hemolysis and icterus during pediatric extracorporeal life support

Chromogenic anti-Xa assays for unfractionated heparin monitoring (heparin activity) are susceptible to interference from hemolysis and icterus. The purpose of this study was to better understand the effect of hemolysis and icterus on anti-Xa heparin activity and to predict the magnitude of the error. Increasing levels of hemoglobin and unconjugated bilirubin were added to pooled normal plasma or buffer containing known levels of heparin. Increased plasma hemoglobin or bilirubin produced falsely increased residual factor Xa activity as measured by the absorbance change (OD/min) in the Stago heparin activity assay. This increased absorbance change slope resulted in falsely lower estimates of heparin activity. The falsely lower heparin activity measurement occurred even when heparin was not present, indicating it was not due to heparin neutralization. In a sample containing 0.62 +/- 0.06 U/mL heparin and 228 mg/dL hemoglobin, the measured heparin activity was 0.41 +/- 0.03 U/mL, underestimating heparin activity by 0.21 +/- 0.07 U/mL. Interference occurred if plasma hemoglobin was above 70 mg/dL or bilirubin was above 16 mg/dL, which happened in 16%-26% of samples from pediatric patients on extracorporeal life support (ECLS). In conclusion, hemolysis and icterus were common in ECLS patients, leading to underestimates of unfractionated heparin activity and potentially higher doses of heparin than intended. The magnitude of the heparin activity measurement error could be predicted based on plasma hemoglobin and bilirubin levels until these levels exceeded the technical limits of the assay, ~230 mg/dL hemoglobin and 55 mg/dL bilirubin.