A-171 Novel Fluorometric Assay for Pediatric Anti-Factor Xa Testing: Minimizing Bilirubin and Hemolysis Interference in Whole Blood

Background High bilirubin levels are common in pediatric patients and are noted in patients undergoing extracorporeal membrane oxygenation (ECMO). Acute anticoagulation therapy necessitates frequent monitoring of anti-Factor Xa activity (aFXa), and high bilirubin levels cause interference in chromogenic assays. Moreover, large volumes of blood lead to iatrogenic anemia in pediatric and neonatal patients. We developed a fluorescence assay to measure aFXa activity on a near-patient digital microfluidic (DMF) platform using low volume whole blood samples (< 50 μL) and present the results of interference of bilirubin and hemolysis on aFXa activity. Methods aFXa assay was performed on a DMF cartridge wherein 50 μL whole blood was loaded into the cartridge which separates plasma droplets through agglutination within the cartridge followed by incubation with a droplet containing exogenous FXa and a fluorogenic substrate. The fluorescence is inversely proportional to the concentration of heparin in the sample. All required reagents for the aFXa assay are dried within the cartridge, allowing for integrated sample preparation and assay automation for point of care use. To test the interference of bilirubin, we spiked into whole blood different concentrations of unconjugated bilirubin, ranging from 0-40 mg/dL. We also tested hemoglobin interference by testing 4 different concentrations of hemolysate (0-1,000 mg/dL) on the aFXa assay. All experiments were performed with 6 replicates of each concentration. Results Across each bilirubin concentration, we saw uniform measurements of aFXa activity (see Table). We observed < 5% CV in aFXa activity with 0, 13.33 and 26.67 mg/dL bilirubin levels. All measured unfractionated heparin values for hemolysate interference were <5% CV. Conclusions Our interference results suggest that high bilirubin and hemolysate levels do not interfere with our novel aFXa fluorescence assay. Further clinical studies are underway to establish clinical performance.