Our research mainly focuses on cerebral thrombosis. Many studies have shown that cerebral blood clot cell components correlate with the diagnosis, treatment, and prognosis of cerebral thrombosis. In this study, we established a novel method for the analysis of cell components of cerebral blood clots.
This challenge in analyzing cell components of cerebral blood clots is the release of intact cells from the clots. This is because the cross-linked fibrin tightly wraps the blood cells in the clot. Current approaches to studying the cell components of the clots are based on in-situ staining, which is unsuitable for the comprehensive study of the cell components.
On the contrary, our method enables both quantitative and qualitative analysis of the cerebral blood clot cell components. Our method utilizes a fibrinolytic enzyme to degrade the fibrin in blood clots, releasing the intact cells. The data obtained from the analysis of the cell components will facilitate the study of the mechanisms of cerebral thrombosis at a molecular level.
Our team will continue to focus on the study of fibrinolysis and the coagulation system. To begin, place the collected cerebral blood clot samples on a clean dish using tweezers. Then, using a pipette, add five milliliters of physiological saline to it and gently shake the dish.
Remove the saline using a pipette. Using a pair of scissors, chop the clots into small pieces. Once again, add five milliliters of physiological saline to the clots and shake the dish gently before aspirating the saline with a pipette.
Then using tweezers, transfer the pieces of clots to a new clean U-plate. Prepare the sFE working solution by adjusting the concentration of sFE to 2000 units per milliliter using physiological saline. Add 300 microliters of the sFE working solution to the pretreated clots.
To initiate the first round of degradation, incubate the mixture of sFE and clots at 37 degrees Celsius for half an hour. After that, gently shake to mix the sFE-treated sample and using a clean pipette, transfer the liquid portion to a sterile tube. Set aside the remaining clot for another round of degradation.
Centrifuge the collected liquid at 200 G for five minutes at four degrees Celsius. Next, using a pipette, transfer the supernatant or the recovered sFE solution to another clean tube. Resuspend the resultant cell pellet in 50 microliters of physiological saline by gentle mixing.
After this, perform subsequent rounds of degradation on the remaining clots using the recovered SFE solution as demonstrated previously. Gather the cell mixture from each round of degradation to prepare the blood cell sample. Add five microliters of the obtained cell sample to the poly-L-lysine-coated glass slide and using a cover slip, smear the cells.
Allow the liquid to evaporate at room temperature. To perform cell staining, gently add 100 microliters of Wright's dye to the cell smear. Allow the cells to stain for 30 minutes at room temperature before gently rinsing the dye off with clean water.
Finally, examine the stained cells using a light microscope. Compact red blood clots with a colorless working solution were observed during the early stage of degradation. Clot dissolution was observed after a 30-minute incubation and a prolonged incubation of up to five hours dissolved most clots, while there was no significant change in the negative control group, even after a 10-hour incubation.
After a Wright's staining, mature red blood cells, platelets, and various granulocytes were clearly identifiable under a light microscope. The cell components could be quantitatively analyzed with the aid of auto hematology successfully.
