DETERMINATION OF SOLUBLE FIBRIN IN PLASMA WITH A CHR0M0GENIC KIT METHOD UTILIZING THE HIGH AFFINITY PLASMIN SUBSTRATE S-2390

Abstract

A sensitive and quantitative assay of soluble fibrin is of clinically diagnostic relevance in an early thrombotic state where there is a risk for development of DIC. Recently Wiman and Ranby (Thromb. Haemostas 55, 189-193 (1986)) published a spectro-photometric assay which met these criterions. The single-stage assay procedure is based upon activation of Glu-Plasminogen to Plasmin by t-PA in the presence of soluble fibrin and hydrolysis of the chromogenic plasmin substrate S-2390, H-D-Val-Phe-Lys-pNA, which has a high affinity for plasmin. The rate of plasmin generation is correlated to the amount of soluble fibrin monomers present in the sample.A complete kit containing optimized, stable reagents has now been developed which allows a quantitative determination of soluble fibrin in the range 30-200 nmol/1 within 30 min. at room temperature (20-25°C). The assay procedure is straightforward involving addition of 200 pi diluted plasma sample to 200 pi Glu-Plasminogen and 100 ul of a t-PA/S-2390-reagent.The results show a high resolution of the standard curve as illustrated by a AA405 amounting to about one absorbance unit between a 200 nmol/1 sample of soluble fibrin and the reagent blank, some variation, ±0.1 absorbance unit, being caused mainly by differences in temperature. In combination with an intra-assay variation coefficient = 6.3% and 5.0% at 150 and 50 nmol/1, respectively, this will allow safe and reliable differentiation of pathological levels of soluble fibrin from levels found in healthy subjects (below 10 nmol/1). A similar precision is also obtained when the assay is performed in microplates.In the original procedure fresh frozen human plasma was utilized as a dilution medium for soluble fibrin. Comparisons with carefully collected bovine plasma proved this source to be a convenient substitute. Furthermore, lyophilization of the bovine plasma did not produce any significant degradation of fibrinogen which otherwise might interfere in the assay. This simple kit procedure should make it a suitable tool in early determinations of soluble fibrin in a number of pathological states which may result in severe haemostatic disturbances.