Preanalytic processing of rat plasma influences thrombin generation and fibrinolysis assays

Thrombin generation assays (TGA) have potential applications as measures of hemostatic balance in animal models. However, variations in plasma processing greatly influence human TGA, and may also impact on the translational value of TGA in animal studies.

The purpose of the study was to compare the performance characteristics of Sprague-Dawley rat plasma prepared by single vs double centrifugation protocols in TGA and fibrinolysis assays.

Platelet-poor plasma (PPP) from adult rats (n = 20 males; 20 females) was prepared by centrifugation at 1200g × 12 min, or 2 sequential centrifugations of 2500g × 15 min. Plasma aliquots were assayed fresh and after freeze-thaw in a commercial fluorogenic TGA (Technothrombin TGA, Technoclone) using 2 different trigger reagents containing approximately 7 pM human tissue factor. In addition to TGA variables (lag time, peak thrombin, endogenous thrombin potential), we compared clotting time test and fibrinogen concentration, residual platelet and platelet-derived microparticle (PMP) counts measured by flow cytometry, and variables of fibrin clot formation and lysis measured in turbidimetric assays.

Single-centrifugation PPP demonstrated significantly greater thrombin-generating potential regardless of trigger reagent, yielded higher residual platelet and procoagulant PMP counts, and more stable fibrin clot profiles. The influence of a freeze-thaw cycle on TGA varied depending on trigger reagent, and male sex was associated with an overall "procoagulant" phenotype.

Preanalytic processing and sex have significant effects on many functional measures of hemostasis in rats. A standardized double centrifugation protocol to prepare PPP is recommended for future studies.