Standarization of the method to determine the expression of P-selectin on platelets by means of the flow cytometer and comparison with impedance aggregometry method in acute myeloid leukemia patients
Journal Title: Postępy Nauk Medycznych - Year 2015, Vol 28, Issue 6
Abstract
Introduction. Damage to vascular endothelium, inflammation or thrombotic disorders result in platelet activation. P-selectin is expressed on the surface of the activated platelets. Examination of P-selectin expression on platelets using the flow cytometer allows for simultaneous determination of platelet activation as well as their reactivity (function).Aim. Determining both optimal reaction conditions for ADP-induced platelet activation and maximum storage time of the samples after labeling and fixation. Comparative analysis of the platelet function using flow cytometry and impedance aggregometry.Material and methods. 33 patients with acute myeloid leukemia (AML) together with 26 healthy blood donors were studied. Blood samples collected from healthy blood donors were incubated with 10 μM/l and 20 μM/l ADP for 2, 5, 10 and 15 minutes. Platelets were labeled using CD61-FITC, CD45-PerCP, and CD62P-PE antibodies. Stability of the samples was determined after 2, 3, 4, 5 and 6 hours of storage. Platelet function in patients with AML was determined using the cytometric method and the impedance aggregometry. The results were then compared.Results. Expression of P-selectin after application of 20 μM/l ADP for 2 and 5 minutes was at 67.7 ± 3.0% and 67.6 ± 2.3%. Longer activation was connected with reduction in the number of CD62P cells: 60.5 ± 3% and 56.4 ± 4.1% (p < 0.05). Platelets activated with 10 μM/l ADP showed the lowest amount of CD62P (p < 0.05). Longer incubation time entailed growth in the number of aggregates (p < 0.05). Expression of P-selectin at the five points in time was compared V = 2.21% (p < 0.01). In patients with AML, expression of P-selectin was at 42.6 ± 13.2% (compared with the control group 64.0 ± 7.4%) (p < 0.01).Correlation between the results of the platelet function examination conducted by means of the two methods was R = 0.66 (p < 0.01).Conclusions. The most effective conditions for platelet activation were obtained by incubating the platelets with 20 μM/l ADP for 2 minutes. It was proved that after fixation with paraformaldehyde, the samples remain stable for up to 6 hours. The flow cytometry method as well as the impedance aggregometry proved the platelet function in patients with AML to be lower in comparison with the control group.
Authors and Affiliations
Joanna Bienias, Krystyna Jagoda, Mirosław Markiewicz, Agata Wieczorkiewicz-Kabut, Sławomira Kyrcz-Krzemień
Keywords
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