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

Abstract

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.

Monitoring of Unfractionated Heparin Therapy in the Intensive Care Unit Using a Point-of-Care aPTT: A Comparative, Longitudinal Observational Study with Laboratory-Based aPTT and Anti-Xa Activity Measurement

“…In addition, analytical interferences on anti-Xa activity, including antithrombin deficiency (for the kits that do not add exogenous antithrombin to the reagents), hemolysis or hyperbilirubinemia due to the photo-optical detection method used, may also occur [ 9 , 28 ]. According to two previous studies, increased plasma-free hemoglobin concentrations resulted in a concentration-dependent underestimation of heparin activity [ 29 , 30 ]. As a consequence of shear stress and the exposure of blood to non-biological substances in ECMO patients, thrombocytopenia and altered platelet function may occur due to decreased levels of adhesion receptors, activation markers and surface expression of CD62/CD63 [ 9 , 31 ].…”

“…Some patients may also experience high levels of fibrinogen during their ECMO course, to which anti-Xa assays are not sensitive. All of these conditions frequently encountered in patients on extracorporeal devices may therefore lead to inadequate UFH dosing [ 29 , 30 , 31 , 32 ]. Since 48.6% of the patients included in this study were on ECMO (8.6%) or CVVH (40.0%), this led to a poorer correlation between POCT-APTT and anti-Xa pairs.…”

Anti-Factor Xa–Based Anticoagulation during Extracorporeal Membrane Oxygenation: Potential Problems and Possible Solutions

“…50 mg/dL) had significantly lower anti-FXa activity despite an identical UFH infusion rate, with a median difference approaching 0.1 IU/mL. Khan and Chandler, in a very recent study, 25 found that samples with large free hemoglobin concentration (228 mg/dL) showed an underestimation of the UFH concentration by 0.21 IU/mL; interference between free hemoglobin, bilirubin, and anti-FXa activity was noticed for free hemoglobin values greater than 70 mg/dL or bilirubin greater than 16 mg/dL, conditions that were found in 16 and 26% of pediatric patients under extracorporeal life support. In both these studies, the anti-FXa activity was measured using the Stago liquid anti-FXa assay (Diagnostica Stago).…”

Artificial Organs 2019: A year in review

“…Jenna Khan and Wayne L. Chandler of the University of Washington, Seattle, WA, USA investigated the interferences in the anti‐Xa heparin activity assay due to hemolysis and icterus during pediatric ECLS. Increasing levels of hemoglobin and unconjugated bilirubin were added to pooled normal plasma or buffer containing known levels of heparin.…”