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Analytical and between-subject variation of thrombin generation measured by calibrated automated thrombography on plasma samples*
Authors:Anne F. Kristensen  Søren R. Kristensen  Ursula Falkmer  Anna-Marie B. Münster  Shona Pedersen
Affiliation:1. Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark;2. Department of Clinical Medicine, Aalborg University, Aalborg, Denmark;3. anflk@rn.dk;5. Department of Clinical Medicine, Aalborg University, Aalborg, Denmark;6. Department of Oncology, Aalborg University Hospital, Aalborg, Denmark;7. Department of Clinical Biochemistry, Hospital Unit West, Holstebro, Denmark
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
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