The technique of measuring thrombin generation with fluorescent substrates: 4. The H-transform, a mathematical procedure to obtain thrombin concentrations without external calibration.

Synapse BV, Cardiovascular Research Institute CARIM, P. O. Box 616, 6200 MD, Maastricht, The Netherlands.
Thrombosis and Haemostasis (Impact Factor: 5.76). 02/2009; 101(1):171-7. DOI: 10.1160/TH08-09-0562
Source: PubMed

ABSTRACT In fluorogenic thrombin generation (TG) experiments, thrombin concentrations cannot be easily calculated from the rate of the fluorescent signal increase, because the calibration coefficient increases during the experiment, due to substrate consumption and quenching of the fluorescent signal by the product. Continuous, external calibration via an in a parallel sample therefore was hitherto required for an accurate calculation of the TG curve. A technique is presented that allows mathematical transformation of experimental fluorescence intensities into "ideal" data, i.e. in the data that would have been obtained if substrate consumption and quenching by the product would not play a role. The method applies to fluorescence intensities up to 90% of the maximal fluorescent signal corresponding to total substrate conversion and thereby covers the entire region of interest encountered in practice. The first derivative of the transformed signal can then be converted into thrombin concentrations via a conventional, fixed calibration factor. This calibration factor can be obtained from a separate experiment but also by measuring the amidolytic activity of the alpha(2)macroglobulin-thrombin complex present in the reaction mixture ("serum") after thrombin generation is over. This method halves the amount of sample required per experiment.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Despite increasing recognition as a more precise test of in vivo hemostatic conditions, standardization of the thrombin generation test (TGT) continues to hinder its development as routine clinical practice. Prior efforts largely focused on comparing the effects of experimental conditions and different reagents. Commercialized kits, instruments and software have been introduced to calculate the TG curve and its parameters. However, modified versions of the TGT continue to be used worldwide on a variety of microplate reader instruments and processed using individualized algorithms. No prior study has compared the effect of instrument choice and its inherent noise profile on the processing of the TG curve and its common endpoint parameters. Hemophilia A plasma supplemented with buffer or Factor VIII, mimicking hemophilic or normalized samples respectively, was monitored for thrombin generation after activation with TF on six different fluorescent microplate readers. Each instrument was optimized for TGT signal recording prior to testing. An automated software package containing various mathematical algorithms was utilized to compute the TG curves and parameters, and compare different TG processing approaches. Instruments produced unique noise profiles and end-point parameters that were incomparable in absolute signal terms. Similar relative hemophilic responses were obtained across various instruments when the normalized plasma sample was used as an internal standard. Smoothing algorithms corrected destructive instrument noise. Instrument-induced errors from numerical differentiation during TG curve processing cannot be eliminated by external calibrators, and require careful qualification of the instrument and implementation of noise-reducing software algorithms.
    Thrombosis Research 07/2013; · 2.43 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To obtain a thrombin generation (TG) curve from the conversion of added fluorogenic substrate, thrombin concentrations are to be derived from the observed velocity of increase of fluorescence (dF/dt). The relation between velocity and thrombin concentration varies during the experiment because substrate is consumed and because fluorescence is not linear with the concentration of product. Here we review the techniques that we developed to:
    Thrombosis Research 11/2012; · 2.43 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel supramolecular complex formed by N,N′-diethyl viologen (EV2+) and symmetrical tetramethylcucurbit[6]uril (TMeQ[6]) was described both in solution and in the solid state by 1H NMR, UV spectroscopy and Single-crystal structure analysis. In the solid state, EV2+ and TMeQ[6] formed two types of inclusion models in the self-assembled complex, a pseudorotaxane of a N,N′-diethyl viologen threading into the cavity of TMeQ[6] and a dumbbell-like structure of a N,N′-diethyl viologen shouldering two TMeQ[6] molecules, and the two types of complexes assembled to 1D supramolecular chain through hydrogen bonding.
    Chemical Physics Letters 10/2011; 514(4):317-320. · 1.99 Impact Factor