Thrombin generation in whole blood – response to Al Dieri & Hemker

Abstract

We read with interest the comments by Al Dieri and Hemker on our recent article on thrombin generation (TG) in plateletpoor plasma (PPP), platelet-rich plasma (PRP) and whole blood (WB) (Tappenden et al, 2007).Al Dieri and Hemker report that they have tried unsuccessfully to reproduce our method for determining TG in WB. They suggest that, because of this, we must have omitted one or more details of the experimental procedures in our paper. They base their argument on two major points.Their first point deals with the fluorescent signals obtained in WB. They state that they found these to be 30· weaker in WB than those in plasma and that the signal to noise ratio was so poor in WB that they were unable to obtain useful first derivatives.The results described in our paper follow over 2 years development work on the plasma-based and WB TG methods including comparisons of different microtitre plates (clear or black and shape), studies on a variety of fluorogenic peptide substrates, the determination of the actual amounts of tissue factor added in the assays and excitation and emission wavelength scans to find optimal assay conditions. These studies were performed using a Gemini XS fluorimeter (Molecular Devices, West Sussex, UK), which is equipped with a monochromator rather than a filter wheel and has the ability to mix the incubating material between readings. In our method the samples were read every 30 s and continually mixed during the incubation period between readings. A major influence on obtaining results in WB was the use of different excitation and emission wavelengths for WB compared to those used for PPP and PRP. Using the wavelengths specified the signal obtained for WB was two to three times weaker than that seen in plasma samples. We were able to obtain relative fluorescence units (RFUs) in excess of 3500 in WB. This produced a signal to noise ratio (at an RFU of 2600) some 3· poorer than that obtained in PPP. This did not however prevent the calculation of the first derivative.The second point in the letter by Hemker and Al Dieri deals with erratic TG curves obtained with WB and the conversion of the fluorescence data. While the curves obtained in the TG assay are more erratic in WB than those in PPP (Fig 1A and B) they still allow for conversion to the first derivative and thus the calculation of the thrombin concentration. In those samples where the fluorescence is higher than that of the calibrator, the effect may be an underestimation of the inner filter effect; this may reduce the final thrombin concentration calculated by the mathematical model. It is worth noting that in comments made by AlDieri and Hemker they fail to state the type of fluorimeter used in their experiments. We believe the reason for our success in measuring TG in WB is not only because of the optimized assay conditions and wavelengths chosen, but also to the type of fluorimeter used. We surmise that this is probably as a result of the fluorimeter using a monochromator rather than a filter wheel but there may be other propert...