INVESTIGATION OF A QUANTITATIVE ANOMALY ENCOUNTERED IN ASSAY OF FIBRINOGEN BY IMMUNO-DIFFUSION

Abstract

When diluted platelet-poor normal plasma is allowed to react with a specific antifibrinogen antiserum by double diffusion in agar the fibrinogen content of the plasma, as estimated immunologically, is less than would be expected from chemical estimation. This discrepancy is even more marked in plasma exposed to procedures bringing about increased coagulability such as storage of platelet-rich plasma, glass contact or pre-treatment of the blood with traces of thrombin insufficient to cause coagulation. On the other hand, full development of immune precipitation occurs with purified fibrinogen and is often almost complete in plasmas with severe coagulation defects. Full development of immune precipitation is restored when chelating agents such as sodium citrate or sequestrene are added to both the agar medium and the diluting fluid in re-testing plasmas initially showing the anomaly in saline-agar. Investigation of this phenomenon suggests that it is not mediated by any one of the plasma factors concerned in thromboplastin generation, but that the anomaly is closely concerned with thrombin evolution. Evidence is presented that the quantitatively anomalous immune behaviour of plasma fibrinogen in saline-agar may be due to fibrin deposition in the agar consequent upon lowered citrate concentration in the plasma and the formation of traces of thrombin during diffusion. Antifibrinogen-antibody interaction with the products of plasm in lysis of fibrinogen also shows a quantitative anomaly in saline-agar, but this is only demonstrable early in the digestion process. In conformity with other workers it is suggested that the early digestion products (fibrino-peptides) formed as a result of the proteolytic action of thrombin complete with the digestion products of plasmin for interaction with antibody. The practical implications of this anomaly are discussed in relation to immunological estimation of the fibrinogen content of plasma and the development of a screening test for plasma coagulation defects.