PB0126 - A Novel Flow Cytometric Method for Studying Murine Reticulated Platelets Using SYTO 13 Green

Sunday, June 25, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Muataz Ali Hamad

PhD Student
Department of Cardiology and Angiology I, Heart Center, University of Freiburg, Freiburg, Germany
Freiburg, Baden-Wurttemberg, Germany

Co-Author(s)

NS

Nancy Schanze

Research Group Leader
Department of Cardiology, Angiology, Haemostaseology and Medical Intensive Care, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
Mannheim, Baden-Wurttemberg, Germany
KK

Krystin Krauel

Research Group Leader
Department of Cardiology, Angiology, Haemostaseology and Medical Intensive Care, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
Mannheim, Baden-Wurttemberg, Germany
MZ

Mark Zurek

MD
Department of Cardiology and Angiology II, Heart Center, Faculty of Medicine, University of Freiburg, Bad Krozingen, Germany.
Freiburg, Baden-Wurttemberg, Germany
TN

Thomas Nührenberg

MD
Department of Cardiology and Angiology II, Heart Center, Faculty of Medicine, University of Freiburg, Bad Krozingen, Germany.
Freiburg, Baden-Wurttemberg, Germany
DD

Daniel Duerschmied

Director of Department of Medicine I
Department of Cardiology, Angiology, Haemostaseology and Medical Intensive Care, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
Mannheim, Baden-Wurttemberg, Germany

Background: Reticulated platelets (RP) are immature platelets with increased RNA content that circulate in the blood and represent the youngest platelet subpopulation. RP are associated with various pathological conditions and their detection is crucial for diagnosis and management.

Aims: To evaluate the effectiveness of the fluorescent nucleic acid dye SYTO 13 Green in detecting murine RP using flow cytometry.

Methods: Diluted anticoagulated blood (1:10) from C57BL/6J mice was lysed/fixed, incubated with SYTO 13 Green in combination with a platelet-specific marker (anti-mouse CD42b-DyLight 649), washed, and analyzed by flow cytometry. For certain samples, the blood was first incubated with a murine PAR-4 agonist (400 µM) or PBS, followed by incubation with a surface platelet activation marker (anti-CD62P-PE Cy7). Several staining times (15, 30, 60, 90 min) and concentrations of SYTO 13 Green (1.5-100 µM) were tested to find the optimal staining conditions. As a proof-of-principle, RP were also evaluated in platelet-depleted mice during the recovery phase (D4 after anti-CD42b treatment).

Results: SYTO 13 staining was successful under various conditions (n=3). The mean baseline level of circulating RP in the untreated group was 9% (SD: 0.99, n=3). Four days after inducing thrombocytopenia, the circulating RP levels increased to 60% (SD: 21, n=3) (Figure 1). Platelets did not show any surface positivity for P-selectin at baseline, but treatment with the agonist resulted in most platelets becoming positive for P-selectin (mean: 1% vs. 68%, SD: 0.4 vs. 11, PBS vs. PAR-4 agonist, n=4).

Conclusion(s): The use of SYTO 13 Green in combination with platelet (activation) markers enables the identification of RP in both resting and activated states. This method is potentially useful to simultaneously monitor RP levels and platelet activation in murine models of human disease.