The Fibrinogen Antigenic Turbidimetric Assay (FIATA): The X 2x Test—The Corrected Chi-Square Comparison Against the Control-mean

Abstract

Vancomycin precipitates fibrinogen. The turbidity induced by this vancomycin-fibrinogen interaction is used to establish a simple standardized antigenic assay for plasmatic fibrinogen, the FIATA. 1 mM vancomycin or 2 mM chloramine-T inactivates 50% of fibrinogen in human plasma. In contrast to chloramine-T, vancomycin does not react in NaJ-based photometric assay for chloramines, vancomycin does not inactivate the singlet oxygen-sensible antithrombin III, and the vancomycin action against fibrinogen is not changed in spite of the presence of the ¹O₂ quenchers methionine or ascorbic acid. The FIATA is performed as follows: to 25 μL plasma 50 μL PBS are added and the absorbance (A) at 405 nm is read. Then 50 μL FIATA-reagent, consisting of 4.4 mM vancomycin in PBS, are added. After 2 minutes (RT) δA is determined and standardized against a plasma pool of 100% of norm (2.8 g/L) fibrinogen. The FIATA is nearly linear up to a fibrinogen concentration of about 150% of norm (4.2 g/L), resulting in a δA of about 600 mA. The lower detection limit is 4% of norm (0.1 g/L). The intra-assay and interassay CV values are < 4%. The normal range of FIATA is 100% ± 20% ([ILLEGIBLE] ± 1 SD). In = 321 or 344 unselected patient plasmas the FIATA ([ILLEGIBLE] = 130%; SD = 52% or 43%) correlated with the functional fibrinogen assays a) modified Clauss-Method ([ILLEGIBLE] = 4.1 g/L; SD = 1.7 g/L) with r = 0.755 and b) FIFTA ([ILLEGIBLE] = 124%; SD = 40%) with r = 0.813. The vancomycin/fibrinogen interaction (binding of about 16 molecules of vancomycin/molecule of fibrinogen) can be used to purify fibrinogen out of plasma. Vancomycin also clouds dysfunctional fibrinogen (fibrinogen in presence of EDTA or chloramine-T) or soluble fibrin. Vancomycin-reacted fibrinogen stimulates tissue-type plasminogen activator (t-PA) up to about 20-fold. The experimental data are analyzed by a new significance test: the twofold Yates-corrected chi-square comparison against the mean value of the control-collective, called the [ILLEGIBLE] Test. The P < .05 barrier calculated with the [ILLEGIBLE] significance - Test is equivalent to that calculated with the Fisher’s Exact Test. The FIATA might be considered an interesting screening test for inactive fibrinogen forms or soluble fibrin, as eg in disseminated intravascular coagulation. Fibrinogen precipitation by vancomycin within the blood vessel might explain why vancomycin has to be infused slowly (< 10 mg/min) to prevent nephrotoxicity. The FIATA is of such a simplicity that the determination of fibrinogen antigen in plasma can be performed anywhere—even outside a hospital—within seconds. Thus, the presented FIATA might contribute to extrahospital testing of patients for assessing their risk for myocardial or cerebral ischemia/infarction.