Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

The overall goal of this procedure is to examine the morphological differences in abnormal clot structures that occur in diabetes and sickle cell anemia, disease conditions for simulated diabetic clots. This is accomplished by imaging glycated clot structures by confocal microscopy for blood samples from sickle cell patients. Fibrin clots containing red blood cells are imaged real-time confocal.

Microscopy analysis can then be performed to evaluate the fibrinolysis rates on the normal and glycated clot structures. Ultimately, this real-time analysis can be used to evaluate the morphological differences that occur in abnormal clot structures in response to the disease states in which they arise. This method can help answer key questions in the field of thrombosis and hemostasis, such as How are fibrin clot structures different in patients with diabetes or sickle cell disease.

To evaluate simulated diabetic clots by confocal microscopy, first defrost human fibrinogen, and 10%fluorescently labeled human fibrinogen conjugate at 37 degrees Celsius when the solutions of warmed mixed to human fibrinogen and the fibrinogen conjugate in a glucose trics sodium chloride solution and incubate the solution protected from light in a 37 degree Celsius water bath for 48 hours. Nearing the end of the incubation, use a non-liquid based adhesive to affix a thin adhesive on two sides of a glass microscope slide to make a chamber, then mixed a fibrinogen solution with room temperature factor 13 and thrombin to a final volume of 50 microliters, and immediately transfer 30 microliters of the fibrin solution into the microscope slide chamber. Now place a 22 by 22 millimeter glass cover slip with a point 15 millimeter thickness on top of the adhesive and seal the open sides of the chamber with clear adhesive taking care that the adhesive does not interact with the clot.

Allow the fibrin clots to polymerize at 21 to 23 degrees Celsius for two hours. Then acquire confocal microscopy images of the clots using a 40 x objective and a 488 nanometer argon laser to evaluate clots from sickle cell patients. First dilute the red blood cell sample at 1 million cells per milliliter in serum free cell culture medium, and mixed the cells with five microliters of the appropriate cell labeling solution by gentle pipetting.

After 20 minutes at 37 degrees Celsius, wash the cells three times in 37 degrees Celsius medium while the cells are in the centrifuge mix freshly prepared fibrinogen solution factor 13 and thrombin as just demonstrated. Then transfer 10 microliters of the labeled red blood cells into the fibrin solution and immediately dispense 30 microliters of the cell solution into an adhesive chamber. On a glass microscope slide, allow the fibrin to polymerize at 21 to 23 degrees Celsius after two hours, acquire confocal images of the clots using excitation lasers with wavelengths of 488 nanometers and 633 nanometers to excite the fluorescently labeled fibrin and red blood cells respectively.

To evaluate the fibrinolysis rates and the glycated clot structures first prepare glycated clots as just demonstrated this time. However, after dispensing the fibrin clot solution into the adhesive chamber, do not seal the ends of the chamber. Instead, allow the clots to polymerize at 21 to 23 degrees Celsius in a sealed enclosure containing 250 milliliters of water to prevent dehydration.

After one and a half hours, acquire images of the clots by confocal microscopy as just demonstrated. Then pipette 25 microliters of plasmin into the open end of the chamber and allow the plasmin to disperse throughout the clot by capillary action. Finally, open the time series features of the confocal microscope software, click acquisition mode and select time series.

Then select the number of cycles and the recording time interval to capture real-time video footage of the plasmin induced clot lysis analysis of normal and glycated clots reveals that glycated clots are denser with smaller pores than normal clots, both with and without the addition of factor 13 during clot polymerization. Indeed, in the UN glycated clots, there is a lower fibrin concentration, which creates a less dense structure than that observed in the glycated clots, which contain a higher fibrin concentration. Fibrin clots from sickle cell patients contain red blood cells and exhibit denser structures than those of healthy fibrin clots due to an increased fibrinogen concentration in these individuals.

Further, the natural tendency of red blood cells from sickle cell patients to form aggregates and genders, clumps of red blood cells that become interwoven into the fibrin network, resulting in the formation of aberrant clot structures that are markedly different in morphology than those observed in normal patients. Finally, analysis of the fibrinolysis rates of normal versus glycated clots demonstrates a significantly impaired ability of the simulated diabetic clots to ly fibrin in the absence of sufficient plasmin. This research will help to pave the way for researchers in the field of thrombosis and a hemostasis, especially for patients that develop comorbid pathologies as a result of sickle cell disease and diabetes.