An automated method for fibrin clot permeability assessment

Abstract

The fibrin clot permeability coefficient (Ks) is a useful measure of porosity of the fibrin network, which is determined by a number of genetic and environmental factors. Currently available methods to evaluate Ks are time-consuming, require constant supervision and provide only one parameter. We present an automated method in which drops are weighed individually, buffer is dosed by the pump and well defined clot washing is controlled by the software. The presence of a straight association between drop mass and their dripping time allows to shorten the measurement time twice. In 40 healthy individuals, Ks, the number of drops required to reach the plateau (DTP), the time to achieve the plateau (TTP) and the DTP/TTP ratio (DTR) were calculated. There was a positive association between Ks (r = 0.69, P < 0.0001) evaluated by using the manual [median of 4.17 (3.60-5.18) ·10⁻⁹ cm²) and the automated method [median of 4.35 (3.74-5.38) ·10⁻⁹ cm²]. The correlation was stronger (r = 0.85, P < 0.001) in clots with DTP of 7 or less (n = 12). DTP was associated with total homocysteine (tHcy) (r = 0.35, P < 0.05) and activated partial thromboplastin time (APTT) (r = -0.34, P < 0.05), TTP with Ks (r = -0.55, P < 0.01 for the manual method and r = -0.44, P < 0.01 for the automated method) and DTP (r = 0.75, P < 0.0001), and DTR with Ks (r = 0.70, P < 0.0001 for the manual method and r = 0.76, P < 0.0001 for the automated method), fibrinogen (r = -0.58, P < 0.0001) and C-reactive protein (CRP) (r = -0.47, P < 0.01). The automated method might be a suitable tool for research and clinical use and may offer more additional parameters describing fibrin clot structure.

How to Assess Fibrinogen Levels and Fibrin Clot Properties in Clinical Practice?

“…32 Recently, we have developed and reported on an automated method for K s assessment, in which drops are weighed individually, buffer is dosed by a peristaltic pump, and welldefined clot washing is controlled by dedicated software. 33 A close association between drop mass and dripping time after proper clot washing allowed to shorten the measurement time. 33 Collet et al investigated clots generated from platelet-rich and platelet-poor plasma in thin glass microchambers (250 µm).…”

“…33 A close association between drop mass and dripping time after proper clot washing allowed to shorten the measurement time. 33 Collet et al investigated clots generated from platelet-rich and platelet-poor plasma in thin glass microchambers (250 µm). 34 Using this approach, they showed that platelets modify fibrin clot structure leading to a 3.5-fold increased K s .…”

“…31 In that study, clotting of 110 µL of plasma was activated by adding 55 µL mixture containing 1.5 IU/mL thrombin and 75 mM CaCl 2 in a Tris-buffered solution in the presence of aprotinin to inhibit spontaneous fibrinolysis during an overnight incubation. 33 Over the first 20 minutes of measurement, pressure drop increased from 2 to 4 cm H 2 O, and then fibrin clot was permeated (equilibrated) for the next 100 minutes before analysis. 31 In 2012, the Factor XIII and Fibrinogen Subcommittee of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis proposed and tested in healthy individuals a novel approach to K s assessment, in which plasma is minimally diluted by clotting activator (100 µL of plasma and 10 µL of activator containing 1 U/mL thrombin and 20 mmol/L CaCl 2 ); clotting mixture is transferred into mechanically scratched tubes, and clot washing is defined as time to percolate six drops of buffer through the clot.…”

“…34 Using this approach, they showed that platelets modify fibrin clot structure leading to a 3.5-fold increased K s . 33 Until now, little is known about the practical use of K s measurement using this technique.…”

Importance of Endogenous Fibrinolysis in Platelet Thrombus Formation

“…The lack of standardised approach to fibrin clot structure assessment and permeability studies limits reproducibility and usefulness [ 43 ]. However, the possibility of an automated system might allow this technique to be used more widely [ 115 ].…”