Interference of erythrocytes on the measurement of thrombin generation, a universal test of hypercoagulability

The Thrombin Generation Assay (TGA) is used to measure TG in PPP and PRP . Thrombin is central to process of coagulation and monitoring its activity is a reliable indicator of rate and extent of coagulation. Previous studies suggest microparticles and pre-analytic preparation vary TG in PPP. Using WB would prevent the need for centrifugation removing these varying factors producing a more universally applicable method, however, it has not been studied.
We aim to assess the reproducibility of the TGA using WB or reconstituted WB (PPP-RBC) and determine if erythrocytes are responsible for any interference observed. Activation of platelets by collagen addition to the WB and application of stirring will also be tested.
TG was measured with Technoclone TGA kit by the use of fluorescent substrate: Z-Gly-Gly-Arg-AMC. Excitation and emission wavelengths of 365 and 455nm were used. Erythrocytes were isolated and fixed before PP addition to produce a HCT of 0.4 in the case of PPP-RBC. Collagen addition (5µg/mL WB) and stirring was applied for 2 or 10 minutes.
PPP-RBC samples had 78 and 79%; SD of 4.3 and 4.2% lower peak thrombin (PT) with or without RCL addition respectively relative to PPP-buffer samples. The PT of PFP was 70%; SD of 25% lower than PPP. Stirred PPP samples showed increased PT by 43%; SD of 25% and reduced lag time (LT) by 7%; SD of 1%. 10 minute stirring and addition of collagen to WB resulted in a 32%; SD of 25% decrease in PT and the addition of collagen reduced LT by 47% and 43%, SD’s of 20% and 26% after 10 and 2 minute stir respectively relative to NS WB.
The results confirm erythrocytes interfere with the measurement of TG, however the role of their interference needs to be tested further. Absence of microparticles in PFP reduces TG compared with PPP. Stirring PPP resulted in higher PT and shorter LT which can be explained by the activation of microparticles. Collagen and stirring decreased LT and PT and longer stirring time was correlated with lower LT and PT. Shortening of LT may mean that there is fast thrombin formation in WB that involves platelets, with rapid clot formation, which entraps thrombin in a time dependent manner.