PB1205 - In vitro analysis of plasma clots with 0.7 to 10.4 g/L fibrinogen concentration by turbidity, permeation and laser scanning electron microscopy

Background: Fibrinogen concentration [Fg] can vary tremendously in certain diseases and during acute phase reactions. The analyses of the clot structure by different techniques in extremes [Fg] of a commercial control plasma could be extrapolated to pathological situations.

Aims: To analyze the outputs and limitations of techniques used to characterize clot structure from plasma clots formed with a broad range of [Fg].

Methods: Siemens Healthineers Fibrinogen Calibrator was used as plasma source with six different [Fg] (0.7 to 10.4 g/L). Human thrombin was used as clotting reagent. Fibrin structure was analyzed by turbidity (405 nm), measuring the lag time (min), the slope (OD/s)x10-4, the MaxAbs (mOD) and the T50% (min), and by permeation (Ks, cm2). Fibrin network was visualized by laser scanning confocal microscopy clotting the plasma with 0.1, 0.5 and 1 units/mL thrombin. Clots were labeled with Alexa Fluor 488-Fg and pores size and fiber diameter measured with a software written at the Bioimaging facility.

Results: Results are summarized in Table 1. Increasing fibrinogen concentration decreased the lag time, increased the slope and MaxAbs, while lengthening fibrinolysis. The flow through the clots decreased exponentially as [Fg] increased (Ks = 10-7 x [Fg]-1.617). Pore size decreased as [Fg] increased, although from 3.9 to 10.4 g/L Fg no changes were detected by the software. The fiber size almost did not change from 0.7 to 10.4 g/L. At 0.7 g/L Fg clotting with 0.1 units/mL thrombin did not form a 3D network.

Conclusion(s): The turbidity and permeation techniques were suitable for studying the clot structure at all [Fg], while LSCM seemed insensitive to fiber diameter, probably due to the fact that a fixed amount of Alexa Fluor 488-Fg is used lowering its concentration from 30% at 0.7 g/L Fg to 3% at 10.4 g/L, and to the resolution of the optical system.