Im1759u

Haematocrit samples of a few of our patients in the cohort were exposed to the two fluorescent markers, CD62P (PE-conjugated) (platelet surface P-selectin) (IM1759U, Beckman Coulter, Brea, CA, USA) and PAC-1 (FITC-conjugated) (340507, BD Biosciences, San Jose, CA, USA) (17). CD62P is a marker for P-selectin that is either found on the membrane of platelets or inside them. PAC-1 identifies platelets through marking the glycoprotein IIb/IIIa (gpIIb/IIIa) on the platelet membrane. To study platelet pathology, 4 µL CD62P and 4 µL PAC-1 was added to 20 µL haematocrit, followed by incubation for 30 min and protected from light at room temperature. The excitation wavelength band for PAC-1 was set at 450 to 488 nm and the emission at 499 to 529 nm and for the CD62P marker it was 540 to 570 nm and the emission 577 to 607 nm [17 (link)]. Samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).

The whole blood (WB) (haematocrit) samples of healthy volunteers, COVID-19 and Long COVID/PASC patients were exposed to the two fluorescent markers, CD62P (PE-conjugated) (platelet surface P-selectin) (IM1759U, Beckman Coulter, Brea, CA, USA) and PAC-1 (FITC-conjugated) (340507, BD Biosciences, San Jose, CA, USA). CD62P is found in the granules  of platelets and then translocate to the platelet membrane surface. The translocation occurs after the platelet P-selectin is released from the cellular granules during platelet activation [6 (link), 9 (link)]. 4 µL CD62P and PAC-1 was added to 20 µL haematocrit. The haematocrit exposed to the markers was incubated for 30 min (protected from light) at room temperature. The excitation wavelength for PAC-1 was set at 450 to 488 nm and the emission at 499 to 529 nm and for the CD62P marker it was 540 nm to 570 nm and the emission 577 nm to 607 nm. Processed samples were viewed using the Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).

Haematocrit samples of all 80 patients in the cohort were exposed to the two fluorescent markers, CD62P (PE-conjugated) (platelet surface P-selectin) (IM1759U, Beckman Coulter, Brea, CA, USA) and PAC-1 (FITC-conjugated) (340507, BD Biosciences, San Jose, CA, USA). CD62P is a marker for P-selectin that is either on the membrane of platelets or found inside them [13 (link), 33 (link)]. PAC-1 identifies platelets through marking the glycoprotein IIb/IIIa (gpIIb/IIIa) on the platelet membrane. To study platelet pathology, 4 µL CD62P and 4 µL PAC-1 was added to 20 µL haematocrit, followed by incubation for 30 min (protected from light) at room temperature. The excitation wavelength band for PAC-1 was set at 450 to 488 nm and the emission at 499 to 529 nm and for the CD62P marker it was 540 nm to 570 nm and the emission 577 nm to 607 nm. Samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).