A non-clotting and non-instrumental method for sensing heparin and monitoring of extracorporeal anticoagulation.

Abstract

A non-clotting and non-instrumentally based heparin assay was developed. This was achieved by studying the immobilization of protamine (a heparin antidote) on a porous filter paper strip; and the subsequent migration of a heparin sample through the paper in a descending manner. The area of paper to which heparin was adsorbed would be proportional to the heparin level in the sample. The bound heparin was visualized by spraying Methylene Blue NNX (a dye that interacts with heparin causing a metachromatic shift of the dye's absorption maximum from blue to purple) solution onto the paper strip upon the exhaustion of the sample reservoir. Heparin levels in the samples could be estimated according to the length of the purple region on the paper strip. The sensitivity, resolution and time requirement could be properly adjusted by varying the amount of protamine immobilized on paper, the sample volume applied and the type of paper used. This system was shown to be capable of accurately detecting heparin, at all relevant levels in aqueous sample in a simple and time efficient manner. Interference and long assay times were encountered when this system was applied to untreated plasma heparin samples; the accuracy of the assay was also significantly decreased. Subsequent investigations indicated that the plasma components sodium chloride and globulin were primarily responsible for the interference; whereas the high viscosity albumin imparted to plasma was accountable for the long assay time needed. The types of filter papers used (Whatman grade 541 and 113) contributed to difficulties in applying the sensing system to detect heparin in plasma, particularly at low levels. A modified "standard addition" method of sample treatment was devised in which plasma specimens were diluted with aqueous heparin solution of known concentration in order to lessen the interferences of plasma components. The heparin levels determined by applying treated plasma samples to the grade 31ET Chr paper strips were in agreement with their counterparts derived by plasma APTT clotting assay.Ph.D.PharmaceuticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105502/1/9135567.pdfDescription of 9135567.pdf : Restricted to UM users only