Preanalytic variables of thrombin generation: towards a standard procedure and validation of the method
Article first published online: 12 DEC 2012
© 2012 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 10, Issue 12, pages 2544–2554, December 2012
How to Cite
LOEFFEN, R., KLEINEGRIS, M.-C. F., LOUBELE, S. T. B. G., PLUIJMEN, P. H. M., FENS, D., van OERLE, R., ten CATE, H. and SPRONK, H. M. H. (2012), Preanalytic variables of thrombin generation: towards a standard procedure and validation of the method. Journal of Thrombosis and Haemostasis, 10: 2544–2554. doi: 10.1111/jth.12012
- Issue published online: 12 DEC 2012
- Article first published online: 12 DEC 2012
- Accepted manuscript online: 30 SEP 2012 03:46AM EST
- Received 3 May 2012, accepted 13 September 2012
- collection tube;
- preanalytic variables;
- thrombin generation;
Summary. Background: Thrombin generation assays are sensitive methods for assessment of the overall clotting potential of plasma, but, despite their common use in thrombosis research, standardization of preanalytic conditions is lacking. In order to set up a standardized protocol, we analyzed different preanalytic variables and validated the calibrated automated thrombogram method.
Methods and Results: Thrombin generation was assessed with 0, 1 and 5 pm tissue factor (TF). Variations in thrombin generation were mostly attributable to the type of collection tube, mainly because of variations in contact activation. The collection tube also determined the influence of other preanalytic variables on thrombin generation, e.g. the need for a discard tube, the storage of whole blood, and the centrifugation method. Regarding the collection system, blood drawn through intravenous catheters or butterfly needles showed significantly more hemolysis than blood obtained with venipuncture using conventional needles. The results showed that a discard tube is still needed for thrombin generation measurements. After blood collection, whole blood is best centrifuged immediately, to prevent activation or degradation of coagulation proteins, and a second centrifugation step at 10 000 × g is recommended. After thawing, plasma is best analyzed immediately, as storage resulted in thrombin generation results outside the 10% range of the reference sample. On the basis of these results, we set up an in-house standardized protocol, which was used for validation, resulting in coefficients of variations of < 15% for all derived parameters with both the 1 and 5 pm TF triggers.
Conclusion: Thrombin generation was greatly influenced by preanalytic conditions, demonstrating the need for an international standardized protocol.