RESUMEN

Using a combination of Cohn ethanol fractionation, virus inactivation, glycine and sodium chloride precipitation, and lysine-Sepharose affinity chromatography, a unique and rapid simplified method was developed to obtain highly purified fibrinogen for diagnostic use with both biological (Clauss method) and immunological (Jacobsson method) activity. Yield was 0.66 g of fibrinogen per liter of starting pooled plasma, and the purified product showed good agreement in activity with the starting material. The purified fibrinogen solution contained over 95% clottable protein and had a clear appearance. No degradation was observed after urokinase treatment and the preparation provided good precision in fibrinogen measurement compared to pooled plasma. The simplified method was, thus, shown to result in a high-purity fibrinogen preparation, suitable for in vitro diagnostic use, as well as for use to prepare a fibrinogen reference material and to perform fibrinogen quality control using an automated coagulation analyzer.

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RESUMEN

A commercially available fibrinogen standard calibrated by a World Health Organization (WHO) reference material is widely used in Japan, and most clinical laboratories use the Clauss method for plasma fibrinogen measurement. However, a current issue in fibrinogen measurement is poor laboratory-to-laboratory variability. To improve the reliability of fibrinogen values and thereby solve the poor precision and accuracy of plasma fibrinogen testing, the present paper develops a simple and large preparation procedure for a suitable fibrinogen standard and quality control material and evaluates their basic performance. With a new procedure getting high purified fibrinogen by glycine precipitation, the calibrator determined by both the Clauss and Jacobson methods produced a fibrinogen concentration of 2.20 g l(-1). The total precision of the calibrator was excellent (coefficient of variation 1.4-2.1%) in comparison with current plasma fibrinogen materials from the WHO (#98/612) and with a commercial standard (CV 1.9-3.9%). The within-run precision of the calibrator on the coagulation analysers was 1.7-2.8%. Within-analyser variability among the five instruments had good consistency (mean 2.20 +/- 0.022 g l(-1); CV 1.0%). The degradation study of the calibrator suggested that storage at 9 degrees C for two years was as predicted. In conclusion, the results show that the calibrator prepared herein can be useful as a candidate Japanese fibrinogen standard and is applicable to automated and semi-automated coagulation analysers. Additionally, it is expected that it will be widely used in Japan by diagnostic manufacturers and clinical laboratories as a recommended secondary standard to estimate a fibrinogen value according to the WHO primary standard.

RESUMEN

Plasma fibrinogen measurement is a routine laboratory procedure commonly performed on automated coagulation analysers. Its determination is quantitative, not quantitative. Yet, a lack of precision has been an issue for fibrinogen measurement. A control material derived from plasma comprises many proteins, inhibitors and fatty acids, any or all of which can interfere in the fibrinogen assay. This study has attempted to develop a quality control material using purified human fibrinogen and has compared measurement precision between both purified and plasma materials. Purified fibrinogen was prepared using Cohn fraction 1 and glycine precipitation. Purified fibrinogen clottability was greater than 95%, with no main plasma proteins, lipids or fibrinogen degradation products observed. Two purified control materials were lyophilized at normal (2.30 g l(-1)) and abnormal (1.20 g l(-1)) levels of fibrinogen concentration. Precision was evaluated using a liquid-type reagent, Thrombocheck Fib(L), on automated coagulation analysers. Coefficient of variation for within-run, intraday and between-day precision of the purified materials was 0.7-3.5%. In comparison, the coefficient of variation for plasma materials ranged from 1.2 to 5.3%. These results suggest that materials prepared from purified fibrinogen can be useful to laboratory quality control by improving overall precision of fibrinogen measurement and are applicable to automated coagulation analysers.

RESUMEN

BACKGROUND: Remnant lipoprotein-cholesterol (RLP-C) concentrations in sera of healthy individuals are very low (0.080-0.437 mmol/L), making conventional cholesterol methods poorly suited to this purpose. We have developed a highly sensitive cholesterol assay (CD method) and applied it to the RLP-C assay. METHODS: The CD shuttled cholesterol reversibly between reduced and oxidized forms in the presence of thio-NAD and NADH. The production rate of thio-NADH correlated with the cholesterol concentration and was measured by the absorbance at 404/500 nm. This CD method was combined with an immunoaffinity separation procedure with specific monoclonal antibodies to apolipoprotein (apo) A1 and apo B-100 and used for RLP-C assay. Results were compared with a RLP-C method that uses cholesterol oxidase, peroxidase, and chromogenic substrate. RESULTS: The CD method could detect 0.10 x 10(-3) mmol/L cholesterol and was at least 5 times more sensitive than the conventional enzymatic method. Within- and between-day imprecision (as CVs) of the RLP-C assay with the CD method was <4%. Regression analysis of RLP-C assays with the new (y) and conventional (x) cholesterol methods yielded: y = 1.02x - 0.008 mmol/L (S(y/x) = 0.0065 mmol/L; r = 0.997; n = 297). CONCLUSIONS: Serum RLP-C can be measured by the CD method. The CD method may be useful for other assays that require sensitive cholesterol measurements in biological materials.

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