Impact of blood centrifugation on the parameters of thrombin generation assay revisited to look for possible revision of the current guidance

Blood centrifugation affects thrombin generation assays (TGA). Current guidance recommends double-centrifugation, which is uncommon in clinical laboratories. We evaluated the impact of 4 centrifugation speeds on TGA performed with low-triggers (1pM tissue-factor/1.0 μM phospholipids) or high-triggers (5pM tissue-factor/5.0 μM phospholipids). TGA parameters were evaluated in the presence/absence of thrombomodulin. We included 20 healthy subjects. Centrifugation speeds were: (i)Double-centrifugation: blood at 2,500g(15min) and plasma at 2500(15min) (reference method). (ii)Single-centrifugation at 3,000g(20min). (iii)Single-centrifugation of blood at 3,000g(20min), plasma freezing, then centrifugation of thawed plasma at 10,000g(5min). (iv)Single-centrifugation at 1,700g(10min). Results were also expressed as percentage difference relative to reference centrifugation. Lag-time was affected when centrifugation speed was relatively slow (1,700g), regardless of low- or high-triggers, presence or absence of thrombomodulin, whereas it was scarcely affected by centrifugation at 3,000g. Peak-thrombin was marginally affected at relatively low-speed (1,700g). ETP was marginally affected at relatively low-speed (1,700g), except when TGA was performed in the presence of thrombomodulin. Peak-thrombin and ETP were not or were poorly affected by centrifugation at 3,000g or 10,000g after thawing, respectively. In conclusion, slow-centrifugation (1,700g) had a considerable impact on lag-time. This centrifugation speed represents common practice in clinical laboratories and should not be used for TGA, unless controls samples centrifuged at the same speed are used for comparison. Single-centrifugation at 3,000g may be a suitable alternative, which would allow TGA testing without the complex and time-consuming double-centrifugation as recommended by current guidance. We propose that current guidance on plasma preparation for TGA be switched from double-to a more intense single-centrifugation.