Liquid Heparin Assay: A Straightforward Approach for Monitoring the Clinical Use of Heparins.

There are number of commercial heparins available in the market. The choice of heparin may depend on the clinical condition of the patient. For example, patients with deep-vein thrombosis (DVT), low molecular weight heparins (LMWH) (such as enoxaparin) may be used; whereas for pulmonary embolism (PE) cases, unfractionated heparins (UFH) may be the heparin of choice. In clinical studies, the synthetic heparin pentasaccharide, Fondaparinux (commercially known as Arixtra) has been shown to be efficacious in the treatment of both types of patients. Monitoring heparin activity in patients under treatment is critical in reducing the patient risk of bleeding or thromboembolism. The majority of current available tests allow measurement of heparins in the range of 0.0 – 1.0 U/mL. In many clinical laboratories, the analysis of each type of heparin requires a separate calibration curve, which makes the assay more complex and time consuming. In most cases these calibrators are “home made” and are not related to a standard, which may contribute to variability in testing. The reagents are usually provided in lyophilized form and require prior reconstitution which can introduce additional variation and increase handling time. An assay developed here, based on ready to use liquid reagents and designed for use on ACL TOP™, ACL Futura®/, ACL Advance™ ACL Elite™/ACL Elite Pro™ and ACL™ 9000/10000, addresses these issues and minimizes testing time. The Liquid Heparin Assay is built on the one stage principle of heparin detection, utilizing endogenous antithrombin III. The assay kit consists of two reagents, a liquid Factor Xa and a chromogenic substrate for Factor Xa, S2732. The composition of the kit and assay parameters are optimized to achieve overlay of the calibration curves for UFH and LMWH in the range from 0.0 to 2.0 U/mL of heparin activity. Furthermore, the assay is evaluated with respect to the various commercial heparins including Enoxaparin, Fragmin and Arixtra. The calibration curves for WHO UFH standard, WHO LMWH standard, Enoxaparin, and Fragmin are observed to overlap, with a maximum difference of 10% at each calibration point. The recoveries of heparin activities in samples spiked with various heparins are also within 10% of the expected values. The weight concentration (μg/mL) of Arixtra in plasma samples and its corresponding heparin activity (U/mL) can also be measured using this assay. The potential utility of the Universal Calibrator, composed of the mixture of UFH and LMWH is also evaluated and shown to be promising. Thus, the Liquid Heparin Assay permits simultaneous determination of various commonly used heparins in patient samples using a single calibration curve, and ready to use reagents. This can simplify analysis, reduce time and improve assay reliability. Since one assay is sufficient to measure different heparins, this assay could also reduce the overall costs of heparin analysis in the clinical laboratory.