Blood was drawn from the cubital vein into the
VACUETTE tubes (Greiner bio-one, Austria) containing sodium citrate (3.2%). Immediately after this, sample fixation was performed with
glutaraldehyde (0.1%, 4.5 mL, Sigma). After sedimentation of some RBCs for 20 minutes at room temperature, 20 µL was collected from the upper layer and placed into 2.5%
glutaraldehyde for complete fixation prior to performing scanning electron microscopy. Polycarbonate isopore membrane filters of 0.22 to 0.45 µm diameter were utilized as the matrices for RBC. Then a quarter of the filter was cut out to count the total number of CECs. To control precision of CEC scoring, the cells were recounted by flow cytometry. Specifically, CECs were scored with the method of sequential gaiting, which we modified to count not only intact, but also the damaged endothelial cells (ECs). The cells were examined with a scanning electron microscope (
Inspect F50; FEI Company, Eindhoven, The Netherlands) and
X-Max EDS-Detector (Oxford Instruments, Abingdon, United Kingdom). Since the scatter in this score of desquamated EC was rather large due to the small volume of the specimens, we assessed the accuracy of the electron microscopy data with flow cytometry.
Melkumyants A., Buryachkovskaya L., Lomakin N., Antonova O, & Serebruany V. (2022). Mild COVID-19 and Impaired Blood Cell-Endothelial Crosstalk: Considering Long-Term Use of Antithrombotics?. Thrombosis and haemostasis, 122(1).