Knut Bartl

A functional photometric assay for plasma fibrinogen.

We have developed a photometric assay for fibrinogen concentration. The sample is mixed with a snake venom enzyme (Batroxobin) and fibrin formation is recorded turbidimetrically at 334 nm. Reaction conditions are such that a linear increase in absorbance is obtained over a concentration range of fibrinogen from 80-700 mg/dl. Higher or lower ranges can be measured by adjusting the sample volume. Calibration is performed with a single standard. Precision and accuracy are comparable with the usual clinical chemical methods. Experiments with plasma samples digested with streptokinase showed that interference of fibrinolytic split products (FSP) is less than using the Clauss method. Large amounts of FSP in the sample, however, delay the increase in absorbance. This phenomenon can be utilized for the additional estimation of the FSP-content of the sample. Results obtained with the present method corresponded well with those obtained using the method of Clauss.

Development of a photometric assay for activated partial thromboplastin time and its application to the cobas(R) biocentrifugal analyzer

We describe a two-step procedure for APTT that can be performed on photometric devices. It includes preincubation of diluted plasma with ellagic acid and phospholipids and a starting reagent that contains calcium and a chromogenic peptide substrate for thrombin, Tos-Gly-Pro-Arg-pNA. Reaction time is recorded from addition of the starting reagent until thrombin formation occurs, and a prefixed amount of substrate is cleaved. The pattern of sensitivity to clotting factors and heparin was similar to clotting assays and the substrate used did not interfere with the activity of factor Xa. An application of the method was made for the Cobas(R) Bio centrifugal analyzer. Absorbance readings were sent to an external computer and were transformed into reaction times by a computer program. Although the results are independent on fibrinogen concentrations, from kinetic data of the reaction curve fibrinogen concentrations can be estimated. Correlation studies showed good correspondence to clotting methods (r = 0.92, n = 53) as well as an excellent precision (CV 3% for inter-assays, n = 15) and high throughput of samples (greater than 100/h) in the automated assay.

Turbidimetric kinetic method for serum low density lipoprotein quantitation.

A turbidimetric kinetic fixed-time method for the rapid determination of serum low density lipoprotein (LDL) has been developed. It is based on specifically precipitating this lipoprotein with the use of a combination of heparin, Ca2+, EDTA and lipase. The method has been adapted to the Eppendorf spectrum line photometer and to the ENI Gemsaec centrifugal analyzer. It yielded satisfactory results with regard to linearity and precision. The values agreed closely to those obtained by conventional procedures for LDL-cholesterol and LDL-apolipoprotein B. The predictive value of this new method for the assessment of the individual risk of vascular disease remains to be proven by clinical and epidemiological studies.