Comparison of Functional Methods with Absolute Quantitation of Heparin Levels in Clinical Samples as measured by Heparin Red Assay

Abstract

Background and Objective Activated partial thromboplastin time (aPTT) is a clot based method which is commonly used for the therapeutic monitoring of heparin. This method represents a global test which is also affected by endogenous factors. However, many endogenous factors contribute to observed prolongation of this test. Heparin Red assay utilizes fluorescence for the direct and sensitive detection of the absolute level of heparin in plasma. The purpose of this study was to compare functional activities (anticoagulant and anti-Xa levels) in clinical patient samples to the absolute levels of heparins as measured by a fluorescence quenching based assay to determine endogenous activity upon the therapeutic administration of this agent. Methods Plasma samples from patients treated with therapeutic dosage of heparin (n=100) were collected from Loyola University Medical Center. Citrated blood samples were analyzed using aPTT clotting method, anti-Xa chromogenic assay and Heparin Red (Redprobes UG, Deutschland) assay relative to a commercially used heparin (Medefil) calibration curve. Normal controls were comprised of commercially available 25 male and 25 female citrated plasma samples (George King Biomedical, Overland Park, Kansas). Results were compiled as mean ± SEM and further analyzed to demonstrate correlation between these methods. Results Marked increases were noted in aPTT (81.11±7.37, normal 30.16±0.80 sec.; p<0.001), anti-Xa (43.90±2.83, normal 0±0% Inhibition; p<0.001), and Heparin Red recovered concentration (2.90±0.16, normal 0.07±0.03 ug/ml; p<0.001) in clinical samples compared to normal controls. Although a large scatter in data in all of the assays was noted and shown in Figure 1, significant correlations were observed between Heparin Red and other functional parameters studied. Conclusion These studies demonstrate that Heparin Red method is a reliable assay for the absolute quantitation of circulating heparin level in plasma. Unlike the functional methods, which are also influenced by many endogenous factors, such as AT levels and variations in the clotting proteins. Heparin Red assay measures absolute amounts of circulating heparin in plasma providing accurate circulating amounts of this anticoagulant which are not modulated by endogenous factors.