Determination of Soluble Fibrin in Plasma by a Rapid and Quantitative Spectrophotometric Assay

Abstract

A rapid, sensitive and quantitative spectrophotometric assay of soluble fibrin in plasma samples has been developed. The method is based on the principle that fibrin stimulates the activation of plasminogen by tissue plasminogen activator (t-PA). A sample containing fibrin is incubated with t-PA, plasminogen and a plasmin-sensitive chromogenic substrate. An increase in absorbance which is dependent on the fibrin concentration in the sample is obtained. In plasma samples an initial lag-phase, due to the presence of alpha₂-antiplasmin is observed. However, the change in A₄₀₅ per square minute during the later part of the reaction was found to be proportional to the fibrin content and to generate linear standard curve intercepts at the origin. The detection limit of bathroxobin-digested fibrinogen added to plasma was about 5 nmol/L. Recovery experiments at 20 and 75 nmol/L levels in plasma samples from healthy individuals were excellent. The “within-run” variation (CV) was determined as 3.7%. The formation of fibrin after addition of minute amounts of thrombin to plasma could be monitored with the method. Plasma from 8 healthy individuals was found to contain about 9.2 ± 1.9 nmol/L fibrin. Plasma samples from 46 patients with a suspected haemostatic disturbance had higher levels of soluble fibrin (53 ± 62 nmol/L). Seven of the 46 samples had concentrations above 150 nmol/L.