The technique of measuring thrombin generation with fluorogenic substrates: 1. Necessity of adequate calibration

Abstract

In fluorogenic thrombin generation (TG) measurement the concentrations of thrombin are obtained from the course of fluorescence intensity. Because of fluorescence quenching, in one series of normal plasmas (n = 60), the rate of fluorescence increase at fixed thrombin activity was 70 +/- 13% of that in buffer, in another (n = 139) 75 +/- 8%. Using a calibration factor (CF) measured in buffer therefore underestimates thrombin concentrations in plasma and introduces a source of error. A fixed CF also neglects the 25-35% increase of CF during the experiment and thus distorts the form of the TG curve so that the ETP cannot be determined. Continuous individual calibration (CIC), in which CF is determined continuously in a parallel sample, avoids such systematic errors but adds random error because the thrombin course is calculated from two different measurements. We determined the intra-individual coefficients of variation (CV) of the peak-height and ETP as obtained with CIC to those obtained with a fixed CF measured in buffer. With the fixed CF, the CVs varied between 18% and 49%; with CIC they lowered to 4-7% (n = 5 x 12), i.e. in a range allowing clinical application. It is shown that CIC can be discarded for the measurement of peak thrombin values and replaced by comparison to a reference plasma only if quenching is not a systematic confounder. This was shown to be the case in the set of 139 normal plasmas but not in the set used for determining the intraindividual CVs, a difference that may depend upon preanalytical conditions.

Large external quality assessment survey on thrombin generation with CAT: further evidence for the usefulness of normalisation with an external reference plasma

“…On one hand the use of a calibrator allows to reduce some technical aspects like plasma colour and optical factors (filters, lamp…) [26]. On the other hand, normalisation is very likely to correct batch-tobatch variability of reagents; in addition, it may correct some analytical aspects such as measurement temperature, the pre-heating or not of plates, which is critical [27], or the operator-related variability (reconstitution of reagents or dispensing).…”

Protease-Activated Receptor (PAR) 1 and PAR4 Differentially Regulate Factor V Expression from Human Platelets

“…Thrombin generation was monitored in a FlexStation II 384 at 390/460 nm for 4 hours. After correction for substrate depletion and the inner-filter effect, the first-order derivative was taken from raw fluorescent units and converted into concentrations of thrombin (nM) by comparison with a standard curve generated with fixed concentrations of thrombin (Hemker et al, 2003;De Smedt et al, 2008). Concentrations of thrombin were then plotted against time to establish a period of thrombin production.…”

A review of commercially available thrombin generation assays

“…15 For the Technothrombin system there is no correction for this inner filter effect, resulting in a lower overall ETP and shorter peak height versus that of the Thombinoscope which does factor the inner filter effect into the calculations. 16 In the case of the BCS-XP there is no need for this correction as the inner filter effect only applies to fluorescence.…”