Flow cytometry based chronologic analysis of surface glycoproteins of resting and stimulated platelets in normal individuals
Abstract
Objective: To evaluate the effect of timed incubation on the activation dependent glycoproteins on the surface of unfixed platelets after stimulation with adenosine diphosphate (ADP).
Methodology: A total of 32 normal subjects were recruited in this study. CBC, Hb A1C, fasting and random blood sugar, urea, creatinine, cholesterol, triglycerides, high density as well as low density lipoproteins were determined. Flow cytometric analysis of platelets was done using FACSCalibur (BD Bioscience). Two sets of CD markers i.e. platelets CD41& CD61 and CD63 & CD 62p were studied during resting state and after stimulation with ADP.
Results: Mean± SD values of CD41 and CD61 surface markers of resting and stimulated platelets showed no statistical difference; p value being 0.198 and 0.486 respectively. Comparison of CD41 and CD61 markers at 20 minutes Vs 2 hrs, 20 minutes Vs 3 hrs and 2 hrs Vs 3 hrs didn’t show any significant difference after stimulation with ADP. This indicates that these markers remain stable and they are not affected by incubation for up to 3 hours. Activation of platelets with ADP resulted in an increase in the number of CD63 and CD62p positive platelets with a p value of 0.001. Activity of CD63 remained high while that of CD62p progressively decreased after incubation for 2 and 3 hours.
Conclusion: CD63 is an ideal marker to evaluate functional status of the platelets while CD62p positivity, though it increases after activation, cannot be used alone as a marker of platelet activation because its positivity decreases with the passage of time. Changes in CD62p positivity when considered in conjunction with increased CD63 positivity lend support to the presence of increased number of activated platelets in the peripheral blood.
Methodology: A total of 32 normal subjects were recruited in this study. CBC, Hb A1C, fasting and random blood sugar, urea, creatinine, cholesterol, triglycerides, high density as well as low density lipoproteins were determined. Flow cytometric analysis of platelets was done using FACSCalibur (BD Bioscience). Two sets of CD markers i.e. platelets CD41& CD61 and CD63 & CD 62p were studied during resting state and after stimulation with ADP.
Results: Mean± SD values of CD41 and CD61 surface markers of resting and stimulated platelets showed no statistical difference; p value being 0.198 and 0.486 respectively. Comparison of CD41 and CD61 markers at 20 minutes Vs 2 hrs, 20 minutes Vs 3 hrs and 2 hrs Vs 3 hrs didn’t show any significant difference after stimulation with ADP. This indicates that these markers remain stable and they are not affected by incubation for up to 3 hours. Activation of platelets with ADP resulted in an increase in the number of CD63 and CD62p positive platelets with a p value of 0.001. Activity of CD63 remained high while that of CD62p progressively decreased after incubation for 2 and 3 hours.
Conclusion: CD63 is an ideal marker to evaluate functional status of the platelets while CD62p positivity, though it increases after activation, cannot be used alone as a marker of platelet activation because its positivity decreases with the passage of time. Changes in CD62p positivity when considered in conjunction with increased CD63 positivity lend support to the presence of increased number of activated platelets in the peripheral blood.
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