In vitro whole blood clot lysis for fibrinolytic activity study using d-dimer and confocal microscopy - PubMed (original) (raw)

In vitro whole blood clot lysis for fibrinolytic activity study using d-dimer and confocal microscopy

Abuzar Elnager et al. Adv Hematol. 2014.

Abstract

This study aimed to evaluate in vitro whole blood (WB) clot lysis method for the assessment of fibrinolytic activity. Standardized unresected (uncut) retracted WB clot was incubated in pool platelet poor plasma (PPP) for varying incubation times and in streptokinase (SK) at different concentrations. The fibrinolytic activity was assessed by D-dimer (DD), confocal microscopy, and clot weight. DD was measured photometrically by immunoturbidimetric method. There was a significant difference in mean DD levels according to SK concentrations (P = 0.007). The mean DD ± SD according to the SK concentrations of 5, 30, 50, and 100 IU/mL was: 0.69 ± 0.12, 0.78 ± 0.14, 1.04 ± 0.14 and 2.40 ± 1.09 μ g/mL. There were no significant changes of clot weight at different SK concentrations. Gradual loss and increased branching of fibrin in both PPP and SK were observed. Quantitation of DD and morphology of fibrin loss as observed by the imaging features are in keeping with fibrinolytic activity. Combination of DD levels and confocal microscopic features was successfully applied to evaluate the in vitro WB clot lysis method described here.

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Figures

Figure 1

Figure 1

Line graph of D-dimer level against time of whole blood clot lysis in plasma.

Figure 2

Figure 2

The difference in clot median weight between before and after pool PPP incubation.

Figure 3

Figure 3

D-dimer level (_μ_g/mL) against various concentrations (IU/mL) of streptokinase using Box-and-Whisker plot.

Figure 4

Figure 4

The difference in clot median weight between before and after SK induced fibrinolysis.

Figure 5

Figure 5

Confocal images for retracted WB clot incubated in pool PPP. (a) showed confocal image of normal retracted WB clot. (b), (c), and (d) showed retracted WB clot after 3, 6, and 9 hrs incubation in pool PPP, respectively. (a) shows confocal images of normal retracted WB clot showing fibrin mass surrounded and covered by RBCs (control untreated). (b) demonstrates a clear separation of fibrin from the RBCs after 3 hrs of WB clot incubation in PPP. (c) shows fibrin separation from the RBCs and a gradual loss of fibrin fibres after 6 hrs of incubation. (d) shows at 9 hrs of incubation in PPP minimal fibrin fibres with some remaining RBCs.

Figure 6

Figure 6

Confocal images for retracted WB clot treated with SK. (a) showed image of normal retracted WB clot. (b), (c), (d), (e) and (f) showed retracted WB clot treated with 5 IU/mL, 30 IU/mL, 50 IU/mL, 100 IU/mL, and 1500,000 IU (stock) of SK concentrations after 1-hour incubation, respectively. (a) demonstrates fibrin integrated with RBCs in retracted WB clot (control untreated). (b) shows 5 IU/mL concentration of SK where the fibrin started to separate from the RBCs. (c) depicts 30 IU/mL concentration of SK showing the fibrin that separated from the RBCs and a gradual thinning of fibrin fibres. (d) shows 50 IU/mL concentration of SK demonstrating fibrin separation from the RBCs and the fibrin fibres became much thinner and increased branching than 30 IU/mL. (e) shows 100 IU/mL concentration of SK where the fibrin almost separated from the RBCs and fibrin fibres became much thinner and increased branching than the 50 IU/mL SK. (f) shows 1500,000 IU (stock) of SK showing complete disappearance of fibrin from the RBCs.

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