Establishing a reference range for thrombin generation using a standard plasma significantly improves assay precision.

BACKGROUND: Thrombin generation is a global coagulation assay which appears to be an effective method for assessing the potential for an individual's plasma to coagulate. However for this assay to become accepted in routine clinical practice it requires standardization. OBJECTIVES: To establish a reference range for the NIBSC reference plasma (TGT-RP) which can then become an internal quality control (IQC) for thrombin generation assays. PATIENTS/METHODS: Thrombin generation was measured in TGT-RP in 153 independent experiments using 4 assay conditions; 1 pM tissue factor (TF) or 5 pM TF +/- thrombomodulin (TM). A target value +/- 2 SD was calculated to provide an acceptance range under the 4 conditions. A plasma sample from a healthy volunteer was subsequently tested in 11 separate experiments using the TGT-RP for (i) normalisation and (ii) exclusion of experimental results when the TGT-RP results did not fall within the established acceptance range. RESULTS: An acceptance range was established for TGT-RP for the 4 assay conditions. Normalisation of all results from a healthy volunteer reduced inter-assay variability significantly (ETP: p=0.0003; Peak: p=0.001). Exclusion of results from the volunteer when concurrently run TGT-RP results fell outside the acceptance range reduced inter-assay variability significantly when reporting raw data (ETP: p=0.001; Peak: p=0.004). However normalisation of this data had no beneficial effect (ETP: p=0.126; Peak: p=0.232). CONCLUSIONS: Our work represents further progress in the standardization of thrombin generation techniques with the establishment of an IQC reference range. Using an IQC reduces inter-assay variability, whilst allowing reporting of raw data and ensures production of accurate and reproducible data.