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Flow Cytometry Analysis of Tissue Factor Expression in Human Platelets
Published on: November 22, 2024
Rapid flow cytometric quantitation of reticulated platelets in whole blood
N Chavda1, I J Mackie, J B Porter
1Department of Haematology, University College Hospital, London, UK.
Abstract:
Young or reticulated platelets contain some residual mRNA, which is rapidly degraded after platelet release into the circulation. In order to minimize platelet activation and possible loss of large platelets during sample handling a whole blood method has been developed utilising the RNA fluorochrome thiazole orange (TO) in combination with an antibody to anti-glycoprotein Ib (GpIb) directly conjugated to phycoerythrin (PE), to specifically stain reticulated platelets via flow cytometric analysis. In this study whole blood analysis of platelet mRNA was undertaken in healthy normal subjects and a variety of patients with haematological abnormalities. The percentage of Gp Ib positive platelets containing mRNA in normals (n = 22) was 11.61% with a two SD range of 3.19-20.01%. The percentage of reticulated platelets was significantly increased (mean mRNA content ± one SD) in sickle cell disorders (n = 22) 38.12% ± 18.42 (P < 0.001); thalassaemia (major, intermedia and trait) (n = 24) 29.76 ± 19.15 (P < 0.001); ITP (N = 20) 23.53% ± 13.04 (P < 0.02) and essentialthrombocythemia (N = 32) 37.12 ± 19.84 (P < 0.001). Platelets from patients with reactive thrombocytosis (N = 15) were only 12.23% positive (±6.95) and not significantly different from the normal range (P = 0.95). This method offers a rapid and simple procedure for assessment of reticulated platelets in whole blood and suggests that there may be an increased platelet turnover in certain haemoglobinopathies.
Insights
A new whole blood method accurately measures reticulated platelets, which contain mRNA. This research reveals increased reticulated platelets in various blood disorders, suggesting higher platelet turnover.
Area of Science:
- Hematology
- Molecular Biology
- Flow Cytometry
Background:
- Young platelets, termed reticulated platelets, contain residual mRNA that degrades post-release.
- Platelet activation and loss of large platelets during sample handling necessitate optimized analysis methods.
Purpose of the Study:
- To develop and validate a whole blood method for assessing reticulated platelets using flow cytometry.
- To investigate reticulated platelet levels in healthy individuals and patients with hematological abnormalities.
Main Methods:
- Utilized thiazole orange (TO) RNA fluorochrome and anti-glycoprotein Ib (GpIb)-phycoerythrin (PE) antibody for direct staining.
- Employed flow cytometry for whole blood analysis of platelet mRNA content.
- Analyzed samples from healthy controls and patients with sickle cell disorders, thalassaemia, ITP, essential thrombocythemia, and reactive thrombocytosis.
Main Results:
- Established normal range for reticulated platelets in healthy subjects (11.61% ± 2 SD range 3.19-20.01%).
- Significantly elevated reticulated platelet percentages observed in sickle cell disorders (38.12%), thalassaemia (29.76%), ITP (23.53%), and essential thrombocythemia (37.12%).
- Reactive thrombocytosis patients showed no significant difference from normals (12.23%).
Conclusions:
- The developed whole blood method is rapid and simple for reticulated platelet assessment.
- Increased platelet turnover is indicated in certain hemoglobinopathies and other hematological conditions.
- This technique aids in understanding platelet kinetics in various disease states.
