Investigation of an anti-activated factor X (anti-Xa) assay for the quantification of enoxaparin in human plasma

Abstract

Objectives Enoxaparin is a low-molecular weight heparin (LMWH) widely used for the treatment of thrombosis and measured through a chromogenic assay (anti-Xa). The aim of this project was to investigate the impact of patient and sampling sources of variation on an anti-Xa assay for enoxaparin. Methods COATEST was used in accordance with manufacturer's instructions. A standard curve of LMWH concentration (0.1-1.0-IU/ml) was prepared. The shelf-life and freeze-thaw stability of Xa over a 6-month period were investigated. The effects of blood sample haemolysis and plasma antithrombin-III (AT) concentration were examined. Key findings The standard curve performed well with high accuracy (average bias of 8.1%) and precision (average CV of 2.8%). The shelf-life of Xa once reconstituted could be extended from 1 month to 4 months if aliquots were frozen at -20°C. Some loss of anti-Xa activity was observed on freeze-thawing (bias up to 25%). Haemolysis of 2.5% was found to interfere with the assay. AT level as low as 50% did not affect assay accuracy. Conclusions Xa standards when frozen immediately after reconstitution could be used for the following 4 months. Haemolysis interferes with the assay, but a 50% reduction in AT does not significantly affect the assay result.