Inter-Laboratory Variation and the Effect of the Type of Fluorimeter on the Results of the Technothrombin® Thrombin Generation Assay.

Thrombin generation is an essential step in the clotting system. Therefore the measurement of thrombin generation is a desirable parameter to elucidate deviations in the clotting system. However till recently the measurement of thrombin generation was a difficult and laborious laboratory method. Nowadays commercial tests are available at the market. We evaluated in a multicenter trial the Technothrombin® TGA assay from Technoclone GmbH Vienna, Austria with a focus on inter-laboratory variability. The Technothrombin® TGA assay is a fluorogenic method. To our knowledge this was the first large-scale inter-laboratory comparison of a thrombin generation test. A set of 8 different samples were distributed to 45 clinical centers in Europe. Thirty-six returned results (80%). Samples were selected on a different level of thrombin generation as indicated by the thrombin concentration at peak height (PTC). The PTC in the samples used varied from 4 nM to 335 nM. The inter-laboratory variability varied from 16% for high PTC (~335 nM) to about 100% for low PTC (~4 - 15 nM).

Using a normal citrated pooled plasma as a reference the other samples can be significantly discriminated by all participants (P < 0.03) using an activator with a low phospholipid concentration (0.32 μM) in the test. With a high phospholipid concentration (3.2 μM) two of the samples can not be significantly discriminated from normal pooled plasma.

Furthermore, we evaluated the effect of the type of fluorimeter used by the clinical laboratories. The most frequently used fluorimeters in this study were the Biotek FLx 800 (n=11), Thermo Fluoroskan (n=8), Tecan Genios (n=5) and the BMG Fluostar (n=4). Overall there are no significant differences between the measurement of the PTC by the different fluorimeters. However remarkable differences were observed for the inter-laboratory variability between the different fluorimeters. The average coefficient of variation of all samples increased from BMG (19.3% ; range 11.1 - 26.9%) < Biotek (28.8% ; range 15.9 - 59.3%) < Tecan (38.5% ; range 12.6 - 81.5%) < Thermo (49.7% ; 19.1 - 96.6%).

This data shows the impact the selection of a type of fluorimeter may have on the comparability of test results of clinical samples. Selection of a measurement system with the lowest inter-laboratory variation in clinical studies shows a benefit for the comparability of study results.