VPB0904 - Flow cytometry test with ADP stimulation for platelet functional activity assessment

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: Virtual

Presenting Author(s)

Evgeniya A. Ponomarenko

Junior researcher
Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology
Moscow, Moskva, Russia

Co-Author(s)

AI

Anastasia A. Ignatova

Researcher
Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology
Moscow, Moskva, Russia
AF

Alexandra Filkova

Junior researcher
National Medical Research Center of Pediatric Hematology, Oncology and Immunology Named after Dmitry Rogachev, Russian Ministry of Healthcare
Moscow, Moskva, Russia
AP

Alexandra Pisaryuk

MD
City Clinical Hospital named after V.V. Vinogradov, Russian Ministry of Healthcare
Moscow, Moskva, Russia
MT

Marina Teterina

MD
City Clinical Hospital named after V.V. Vinogradov, Russian Ministry of Healthcare
Moscow, Moskva, Russia
IM

Imad Meray

MD
City Clinical Hospital named after V.V. Vinogradov, Russian Ministry of Healthcare
Moscow, Moskva, Russia
ZK

Zhanna Kobalava

MD
City Clinical Hospital named after V.V. Vinogradov, Russian Ministry of Healthcare
Moscow, Moskva, Russia
AS

Anastasia N. Sveshnikova

Principal Investigator
Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology
Moscow, Moskva, Russia
GN

Galina Novichkova

Director
National Medical Research Center of Pediatric Hematology, Oncology and Immunology Named after Dmitry Rogachev, Russian Ministry of Healthcare
Moscow, Moskva, Russia
MP

Mikhail A. Panteleev

Director
Center for Theoretical Problems of Physico-Chemical Pharmacology
Moscow, Moskva, Russia

Background: Adenosine diphosphate (ADP) potentiates platelet activation caused by other agonists as well as stimulates platelets on its own. There are studies on platelet functional activity after activation with ADP using flow cytometry, however, no standard protocol is developed. It’s important in monitoring antiplatelet therapy in cardio patients with the acute coronary syndrome, which includes ADP receptors’ inhibitors.

Aims: The aim of our study was to develop a flow cytometry protocol for platelet functional activity assessment after stimulation with ADP and to test it on cardio patients on dual antiplatelet therapy.

Methods: Venous blood was collected into vacuum plastic tubes with sodium citrate, final concentration 3.8%. 20 µL of blood was diluted 1:20 in HEPES-buffered Tyrode buffer. For platelet activation, a mixture of ADP (5μM) and 2.5 mM CaCl2 was used. We determined forward scatter, side scatter, CD42bPE, CD61-PE, CD62P-Alexa Fluor 647, PAC-1-FITC, annexin V-Alexa Fluor 647 binding and mepacrine release levels.

Results: We found that blood should not be transported, while time after blood collection and inter-operator variability didn’t influence the results. Platelets should be activated and dyed simultaneously for 15 minutes with the addition of CaCl2. We used our protocol on children aged 2 months-3 years old (n=5) and healthy adult donors aged 18-45 years old (n=22) and found that after stimulation both groups similarly had 5-fold increase in CD62p and PAC-1 binding levels, while no dense granules release was observed. We then used our protocol on cardio patients on dual antiplatelet therapy (acetylsalicylic acid + ticagrelor or prasugrel, n=9) and found that after a month of therapy stimulated platelets of cardio patients had 2.7-fold lower CD62p and CD61 and 1.2-fold lower PAC-1 binding levels compared to healthy donors.

Conclusion(s): We developed a flow cytometry protocol for comprehensive platelet functional activity assessment after stimulation with ADP, which is sensitive to dual antiplatelet therapy.