Analytical and between-subject variation of thrombin generation measured by calibrated automated thrombography on plasma samples*

Abstract

Background: The Calibrated Automated Thrombography (CAT) is an in vitro thrombin generation (TG) assay that holds promise as a valuable tool within clinical diagnostics. However, the technique has a considerable analytical variation, and we therefore, investigated the analytical and between-subject variation of CAT systematically. Moreover, we assess the application of an internal standard for normalization to diminish variation.

Methods: 20 healthy volunteers donated one blood sample which was subsequently centrifuged, aliquoted and stored at −80 °C prior to analysis. The analytical variation was determined on eight runs, where plasma from the same seven volunteers was processed in triplicates, and for the between-subject variation, TG analysis was performed on plasma from all 20 volunteers. The trigger reagents used for the TG assays included both PPP reagent containing 5 pM tissue factor (TF) and PPPlow with 1 pM TF. Plasma, drawn from a single donor, was applied to all plates as an internal standard for each TG analysis, which subsequently was used for normalization.

Results: The total analytical variation for TG analysis performed with PPPlow reagent is 3–14% and 9–13% for PPP reagent. This variation can be minimally reduced by using an internal standard but mainly for ETP (endogenous thrombin potential). The between-subject variation is higher when using PPPlow than PPP and this variation is considerable higher than the analytical variation.

Conclusion: TG has a rather high inherent analytical variation but considerable lower than the between-subject variation when using PPPlow as reagent.

Keywords:

• Analytical variation
• between-subject variation
• blood coagulation
• blood coagulation test
• thrombin
• thrombin generation

Acknowledgements

The present study (N-20110068) was approved by the local ethical committee and informed consent was obtained from all healthy donors prior to collection of blood samples. All healthy donors furthermore consented to publication of their anonymized data. The work was performed in compliance with relevant legislation and mandatory laboratory safety procedures.

Disclosure statement

The authors declare that they have no conflicts of interest to declare.

Additional information

Funding

This research has been funded by the Danish Council for Independent Research and the Obel Family Foundation.