Standardization of Pre-analytical Conditions for Murine Plasma Thrombin Generation using Calibrated Automated Thrombography

Phenotypic variability exists between individuals with bleeding disorders that is not reflected in the traditional laboratory evaluation of clotting times or factor assays. Global assays of coagulation, such as thrombin generation assays (TGA), have been developed to further define the full process of clot formation and lysis. The Calibrated Automated Thrombography (CAT) assay is a TGA that has been developed for use in human samples and is being adapted for pre-clinical purposes in animals. Pre-analytical conditions influence the measurement of thrombin; there is, however, no consensus in the literature for managing these conditions in murine samples.;The goal of this study is to standardize the pre-analytical conditions for murine plasma collection for CAT assay by evaluating the dilution of anticoagulant in blood utilized, the way to administer that anticoagulant and the blood collection method. CAT assay conditions including the dilution of the plasma in HEPES buffered saline, final in-well volume, tissue factor concentration, phospholipid concentration, measuring interval and temperature were previously optimized. The thrombogram readout of the CAT assay includes the lag time, time to peak, peak thrombin generation and endogenous thrombin potential. Increased sensitivity of the assay due to optimization of pre-analytical conditions was evaluated by comparing the thrombogram components differences between Factor 8 knockout (B6;129S4-FBtmlKaz) (F8K0) and Wild type (B6129SF2/J) (WT) mice. A pooled human plasma sample was added to each plate as an assay control.;Prior to blood collection, various methods of loading anticoagulant into the syringe were tested to reduce contact activation. Drawing anticoagulant into the lumen of the needle was optimal. The concentration of anticoagulant must be sufficient to prevent clotting and allow for evaluable data. Three dilutions (1:5.5, 1:7.75 and 1:10) of 3.2% sodium citrate in blood were suggested in the literature. The results were optimal for the 1:7.75 dilution compared to the 1:10 and 1:5.5 dilutions.;Blood collection was optimized to minimize bleeding, reduce contact activation, reduce tissue factor exposure, and obtain precise volumes of blood to create an accurate dilution with the anticoagulant. Blood was collected from either the inferior vena cava (IVC) or from the heart by cardiac puncture (CP) into lmL syringes using 22 gauge needles containing anticoagulant. IVC collection was optimized by varying the angle of the syringe to obtain precise volumes. The method of anticoagulant delivery was also optimized. Blood was either collected into a syringe containing anticoagulant (ex vivo) or anticoagulant was injected into the mouse intravenously (in vivo). Less clotting and activation was associated with the ex vivo method of anticoagulant delivery and was preferred.;These pre-analytical optimized conditions standardize evaluation of thrombin generation in mouse plasma by the CAT assay. Consensus to utilize these optimized conditions offers an opportunity for discourse about treatment and coagulation profiles in animal research not possible in human trials.