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DOI: 10.1055/s-0038-1651291
Properties of Thrombin and Fibrinogen
The Comparison of Clotting Time Averages Supported by research grants from the Molecular Biology Program of the National Science Foundation and the Institute of General Medical Sciences of the National Institutes of Health.Publication History
Publication Date:
27 June 2018 (online)
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Summary
A standard clotting system has been analysed according to the average clotting time and variations due to thrombin, fibrinogen, day-to-day preparation of reagents and the clotting assay itself. The variations, εT, εF and ετ are reproducible and normally distributed with zero mean.
Transfer of thrombin into a vessel produces adsorption losses at the solution-air (S/A) and solution-wall (S/W) interfaces. Specific loss at τ = 30 sec = (% loss in τ) (thrombin volume)/surface area. For clean non-polar surfaces such as polyethylene or paraffin the average S/W loss of 3.8%-cm (0.77 mg/m2) has the large coefficient of variation of 3.2%. Vessel surfaces may be saturated by prior conditioning using a thrombin concentration tenfold that required to give τ = 50 sec. Emptying and immediately refilling now gives an S/W loss of 0.19%-cm (0.04 mg/m2). The specific S/A loss, always present, is 2.7%-cm (0.54 mg/m2). The combined average loss for conditioned vessels is 1.0% at τ = 30 sec and is reproducible within sτ 2 for single vessels. A set of vessels gives a coefficient of variation of 0.53%.
Stock fibrinogen aliquots are stored frozen at —95° and are thawed and diluted under standard conditions. If a syringe is used to withdraw clotting tube aliquots, SF2 is significantly increased over that obtained by using pipettes. Since pipettes do not contribute, the observed coefficient of variation of 1.7% then applies to the variation between tubes of stock-F. Six different lots of Fraction I gave a coefficient of variation of 12% between lot numbers.
Experiments revealed a variance between equivalent experiments performed on different days (coefficient of variation 1.9%). Within this variance pairs of stock reagents were stable within the maximum limits of ± 0.12% of τ per day. There is no reason to suspect that either stock reagent has an intrinsic instability. The day-to-day variance given above arises from non-reproducibilities in the preparation of buffer diluents. Variances were reduced when buffers were prepared in deaerated water at room temperature.
The total day-to-day coefficient of variation of fibrinogen of 1.5% and the variation between fibrinogen lots effectively eliminates this reagent as a reference standard. Thrombin appeared otherwise : by selecting and reusing reservoirs the maximum total thrombin day-to-day coefficient of variance is 0.36%. Thus, 95% of the thrombin concentrations prepared on different days will lie within 0.72% of the true value. By making use of known purification procedures it is possible to reproduce a stock thrombin reagent. This would serve as a universal standard.
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References
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