Background: Catheter-based ventricular assist devices are increasingly used to support patients with end stage heart failure.  Anticoagulation to prevent device thrombosis often involves heparin infusions monitored by chromogenic anti-Xa assays.  These devices produce hemolysis due to mechanical shearing of red blood cells, and hemolyzed specimens submitted for anti-Xa assays may be rejected due to anticipated test interference by free hemoglobin.  In fact, the presence of free hemoglobin was shown to underestimate anti-Xa activity using the Stago liquid anti-Xa assay on Stago Compact analyzers with a concentration-dependent effect observed even in the absence of added heparin, suggesting that the mechanism of interference may be inherent in the assay.  The effect of free hemoglobin on other chromogenic anti-Xa assay systems has not been reported.

Aims: The purpose of this study was to investigate the effect of free hemoglobin on anti-Xa activity measurements using the HemosIL Liquid anti-Xa assay system.

Methods: Free hemoglobin was prepared by osmotic lysis of whole blood followed by centrifugation and dilution with OK buffer. Buffered hemoglobin was suspended in pooled normal plasma containing 0.0, 0.3 or 0.6 U/mL unfractionated heparin to final hemoglobin concentrations of 0 – 400 mg/dL.  The absorbance change (mAbs/min) and residual factor Xa activity were determined in duplicate using the HemosIL Liquid anti-Xa assay on the ACL TOP 550 CTS Analyzer.  

Results: Increasing concentrations of plasma hemoglobin produced no significant increase in the absorbance change slope (Figure 1) or decrease in anti-Xa activity (Figure 2) at heparin concentrations of 0.0, 0.3 and 0.6 U/mL.

Effect of Plasma Hemoglobin on Average Absorbance Change
Effect of Plasma Hemoglobin on Anti-Xa Heparin Activity

Conclusions: In contrast to the Stago liquid anti-Xa assay, the presence of free hemoglobin did not result in a clinically significant underestimation of unfractionated heparin activity in the HemosIL Liquid anti-Xa assay. Evaluation of other marketed chromogenic anti-Xa assays is important, as interference from hemolysis may vary across different assay platforms.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/hemolysis-interference-in-the-anti-xa-heparin-activity-assay/