Toward development of a point-of-care assay of enoxaparin anticoagulant activity in whole blood

There is need for a rapid assay to determine the efficacy of low-molecular-weight-heparin (LMWH) in whole blood. Heparinase was used to eliminate, and thereby quantify, the anticoagulant activity of the low-molecular-weight-heparin, enoxaparin. The percent change in the clotting time of whole blood in the presence of heparinase yielded the anticoagulant contribution of enoxaparin. A minimally activated assay (MAA) of whole blood clotting time was evaluated for the detection and relative quantification of enoxaparin. The assay cartridge consisted of a plain glass tube and detection magnet, with no additional sources of activation. Comparisons were also made with a point-of-care, activated partial thromboplastin time (aPTT) assay. Plasma or whole blood was spiked with enoxaparin at concentrations ranging from 0.1 to 1.0 anti-factor Xa IU/ml. A commercial preparation of heparinase I was used to digest enoxaparin, and clotting times were determined with and without heparinase incubation. Heparinase digestion caused an average shortening of clotting time of 21.1% (47.3 s) in blood spiked with 0.4 anti-Xa IU/ml enoxaparin, an amount expected in the therapeutic range; also, 0.1 anti-Xa IU/ml of enoxaparin could be reliably detected. The assay performed comparably when transferred to a point-of-care setting with heparinase being added directly to citrated blood collection tubes, followed by either MAA or aPTT assay. Strong correlations were obtained with both assays between the percent change in clotting time (after heparinase) and the added concentration of enoxaparin, or in comparison with the chromogenically measured concentration of enoxaparin. The assays for an individual blood sample can be completed within 10 min.