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.

中文翻译：

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A-171 用于儿科抗 Xa 因子测试的新型荧光测定法：最大限度地减少全血中的胆红素和溶血干扰


背景 高胆红素水平在儿科患者中很常见，并且在接受体外膜肺氧合 (ECMO) 的患者中尤为明显。急性抗凝治疗需要频繁监测抗 Xa 因子活性 (aFXa)，高胆红素水平会干扰显色测定。此外，大量血液会导致儿科和新生儿患者出现医源性贫血。我们开发了一种荧光测定法，使用低容量全血样本（< 50 μL）在患者附近的数字微流体（DMF）平台上测量 aFXa 活性，并呈现胆红素和溶血对 aFXa 活性的干扰结果。方法在DMF盒上进行aFXa测定，其中将50μL全血加载到盒中，通过盒内的凝集分离血浆液滴，然后与含有外源FXa和荧光底物的液滴一起孵育。荧光与样品中肝素的浓度成反比。 aFXa 测定所需的所有试剂均在盒内干燥，从而可以实现集成的样品制备和测定自动化，以供护理点使用。为了测试胆红素的干扰，我们在全血中加入了不同浓度的非结合胆红素，范围为 0-40 mg/dL。我们还通过在 aFXa 测定中测试 4 种不同浓度的溶血液 (0-1,000 mg/dL) 来测试血红蛋白干扰。所有实验均以每个浓度重复 6 次进行。结果 在每个胆红素浓度中，我们看到 aFXa 活性的测量值一致（见表）。我们在 0、13.33 和 26.67 mg/dL 胆红素水平下观察到 aFXa 活性的 < 5% CV。所有测得的普通肝素溶血物干扰值均为 <5% CV。 结论 我们的干扰结果表明，高胆红素和溶血产物水平不会干扰我们的新型 aFXa 荧光测定。进一步的临床研究正在进行中以确定临床表现。