Effect of Pre-Analytical Conditions On the Results of Thrombodynamics Assay
Authors: Dashkevich Natalia M. , Vuimo Tatiana V. , Balandina Anna N. , Ovsepyan Ruzanna A. , Soshitova Natalia P. , Seregina Elena A. , Surov Stepan S. , Lipets Elena N. , Panteleev Mikhail A. , Ataullakhanov Fazoil I , Negrier Claude 
Published: October 2019

Abstract
Abstract 4393

Introduction
Thrombodynamics is a novel global coagulation assay based on spatial separation of clotting activation and propagation. In this assay fibrin clot grows from the TF-coated surface in a thin layer of non-stirred plasma. Clot formation is monitored via light scattering (Fig.1). The main parameters measured in the assay are: lag-time (Tlag), time between bringing plasma in contact with TF and actual start of clot growth: initial rate of clot growth (Vin), rate of clot growth during first 5 minutes; stationary rate of clot growth (Vst), rate of clot formation in 20 minutes after the beginning. (Fig.2)
In this study we analyzed effect of the pre-analytic conditions (blood collection and plasma preparation) on the results of Thrombodynamics assay.

Materials and Methods
Blood was collected into sodium citrate tubes with 9:1 volume ratio. It was usually processed by two centrifugations to obtain PPP (1 600g, 15 min) and then PFP (10 000g, 5 min). 20 μg/ml of Corn Tripsin Inhibitor (CTI) was used to prevent contact activation. 20 mM of CaCl2 was added to plasma prior to the experiment. Coagulation was activated by tissue factor immobilized to plastic surface (100 pmol/m2).

Results
Sodium citrate venous blood collection tubes of three major manufacturers (BD Vacutainer, Greiner Vacuette and Sarstedt Monovette) and citrate concentrations 3.2% and 3.8% for Greiner Vacuette tubes were compared. Blood from 12 healthy volunteers was used for that purpose. All experiments were performed in duplicates. BD Vacutainer tubes showed significant hypercoagulation compared to the other tubes tested. Stationary rate was significantly higher (Mann-Whitney test p=0.05) in these tubes while three other types of tubes showed similar results (Table 1).
There was no significant difference between 3.2% and 3.8% of sodium citrate for Vacuette tubes in all parameters measured. Sarstedt Monovette and BD Vacutainer tubes revealed an increased initial rate of clot growth compared to both Vacuette tubes (p=0.05). Lag times were similar for all the tubes tested.
To estimate the possible error introduced by blood collection procedure, 3 independent collections from different veins were performed for 3 healthy volunteers. Standard deviation for the Thrombodynamics parameters were 12% for Tlag, 7% for Vin, 8% for Vst.
Two centrifugation protocols were studied: PPP was centrifuged for 5 min at 10 000 g or for 20 min at 1600 g. No significant difference was observed for these methods (n=7, p=0.05).
PPP can potentially be used in the Thrombodynamics assay as well as PFP with adjusted range of normal values. Clot growth rates increase in PPP compared to PFP (n=50, Vin =48.8±7.4μm/min and 45.6±12.8 μm/min for PPP and PFP respectively; Vin= 28.6±4.2 μm/min and 24.1±3.0 μm/min for PPP and PFP respectively).
Inhibition of contact activation is important as thin layer of plasma is used and surface to volume ratio in experimental chamber is high. Addition of CTI to PPP with subsequent centrifugation induced significant decrease in initial rate (44.8±6.7 μm/min) compared to 51.1±7.6 μm/min when CTI was added to PFP directly, n=12, p=0.05. Stationary rate did not change significantly (25.8±3.6 μm/min and 28.2±4 μm/min respectively, p=0.05).
Finally we studied the effects of sample storage. For healthy donors no significant change in Thrombodynamics parameters was observed during 3 hours (n=8) and 24 hours (n=3) of storage of PFP at RT.
50 samples of fresh and frozen plasma from healthy volunteers were compared. Clot growth rates increased for frozen plasma (Vst = 28.4±4.0μm/min) compared to non-frozen samples (24.1±3.0μm/min). Therefore frozen plasma can be used in the assay but normal ranges for frozen and non-frozen samples should be defined separately.
These data represent a first step for standardization of Thrombodynamics assay and decrease of variability due to pre-analytic conditions.

Disclosures:
Dashkevich: HemaCore LLC: Employment. Vuimo:HemaCore LLC: Employment. Balandina:HemaCore LLC: Employment. Ovsepyan:HemaCore LLC: Employment. Soshitova:HemaCore LLC: Employment. Seregina:HemaCore LLC: Employment. Surov:HemaCore LLC: Employment. Lipets:HemaCore LLC: Employment. Panteleev:HemaCore LLC: Employment. Ataullakhanov:HemaCore LLC: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.