Rapid serum tube technology overcomes problems associated with use of anticoagulants

Abstract

Introduction: Failure to obtain complete blood clotting in serum is a common laboratory problem. Our aim was to determine whether snake prothrombin activators are effective in clotting blood and producing quality serum for analyte measurement in anticoagulated patients. Materials and methods: Whole blood clotting was studied in a total of 64 blood samples (41 controls, 20 Warfarin patients, 3 anticoagulated patients using snake venom prothrombin activator (OsPA)) with plain tubes. Coagulation was analysed using a visual assay, Hyland-Clotek and thromboelastography. Healthy control blood was spiked with a range of anticoagulants to determine the effectiveness of OsPa-induced clotting. A paired analysis of a Dabigatran patient and a control investigated the effectiveness of the OsPA clotting tubes. Biochemical analytes (N = 31) were determined for 7 samples on chemistry and immunoassay analysers and compared with commercial tubes. Results: Snake venom prothrombin activators efficiently coagulated blood and plasma spiked with heparin and commonly used anticoagulants. Clotting was observed in the presence of anticoagulants whereas no clotting was observed in BDRST tubes containing 3 U/mL of heparin. Snake venom prothrombin activator enhanced heparinised blood clotting by shortening substantially the clotting time and improving significantly the strength of the clot. Comparison of 31 analytes from the blood of five healthy and two anticoagulated participants gave very good agreement between the analyte concentrations determined. Conclusions: Our results showed that the snake venom prothrombin activators OsPA and PtPA efficiently coagulated recalcified and fresh bloods with or without added anticoagulants. These procoagulants produced high quality serum for accurate analyte measurement.

PAR2 Activation on Human Kidney Tubular Epithelial Cells Induces Tissue Factor Synthesis, That Enhances Blood Clotting

“…In initial experiments, a simple tube mixing clotting assay was used with plain collection tubes (Greiner Bio One, Kremsmünster, Austria) pre-coated with hydrophilic surfactant (Dow Corning, Midland, MI, United States). This has been previously reported and shown to be a reliable estimate of blood clotting times (Zhao et al, 2019). To each tube, 20 µL of the 10-fold concentrated cell conditioned medium, 50 µL of 1 M CaCl 2 and 3.95 mL of citrated normal whole blood were added.…”

Potential Application of Recombinant Snake Prothrombin Activator Ecarin in Blood Diagnostics

“…These PAs can clot blood with high efficiency, without being subject to the regulation observed with human Factors X and V [ 1 , 2 ]. Snake prothrombin activator sources include those from Oxyuranus scutellatus (Australian Coastal Taipan (OsPA) that from Pseudonaja textilis (Australian common brown snake) (PtPA) and ecarin from Echis carinatus [ 3 , 4 , 5 , 6 , 7 ]. Recently, we employed OsPA for the rapid preparation of consistently high-quality serum using blood from both normal individuals and anticoagulated patients [ 5 , 6 ].…”

“…Snake prothrombin activator sources include those from Oxyuranus scutellatus (Australian Coastal Taipan (OsPA) that from Pseudonaja textilis (Australian common brown snake) (PtPA) and ecarin from Echis carinatus [ 3 , 4 , 5 , 6 , 7 ]. Recently, we employed OsPA for the rapid preparation of consistently high-quality serum using blood from both normal individuals and anticoagulated patients [ 5 , 6 ]. We demonstrated that OSPA converted prothrombin to thrombin in the blood sample, generating [ 5 , 6 ] a bolus of thrombin significantly greater than the amount used in commercially available thrombin tubes [ 5 , 6 ].…”

“…Recently, we employed OsPA for the rapid preparation of consistently high-quality serum using blood from both normal individuals and anticoagulated patients [ 5 , 6 ]. We demonstrated that OSPA converted prothrombin to thrombin in the blood sample, generating [ 5 , 6 ] a bolus of thrombin significantly greater than the amount used in commercially available thrombin tubes [ 5 , 6 ]. However, the use of snake prothrombin activators, such as OsPA and PtPA, in commercial blood collection tubes is problematic due to the supply considerations associated with procuring venom from snakes in quantity, quality and continuity.…”

“…This single chain metalloproteinase has an advantage over prothrombin activators in that it does not require other cofactors, Ca 2+ , or phospholipids [ 14 ]. As with other snake venom prothrombin activators, it is minimally or not affected by any regulatory components of the blood coagulation-fibrinolysis system in humans, e.g., Activated Protein C [ 5 , 6 , 15 ]. N-Ecarin has been used for measuring the total prothrombin in plasma.…”