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Platelet activation and platelet–leukocyte interaction in β-thalassemia/hemoglobin E patients with marked nucleated erythrocytosis

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Abstract

Patients with thalassemia, an inherited hemolytic anemia, have increased risk of hypercoagulable complications. A whole blood flow cytometric (FCM) method has been used for studies of platelet activation and platelet–leukocyte aggregation in these patients. However, this FCM method presents technical difficulties because of the high proportion of immature red blood cells (RBCs) in these patients. A protocol for the simultaneous measurement of platelet activation and their aggregation with leukocyte populations in whole blood using four-color FCM which excluded immature RBC was devised, and evaluated for the evaluation of platelet function in patients with β-thalassemia/hemoglobin E (HbE). Whole blood from these patients and from healthy volunteers was stained for platelet activation and platelet–leukocyte aggregates using anti-CD42a, anti-CD62P, anti-CD45 and glycophorin A (GPA) conjugated with different fluorochromes. Our FCM method is simple, effective and based on the assumption that GPA is present on all immature RBCs, but is not expressed on CD45+ leukocytes. Results from the studies showed that blood samples from these patients contained a high frequency of circulating activated platelets (CD42a+/CD62P+) when compared to samples from healthy individuals. The percentage of platelet–neutrophil, platelet–monocyte—but not platelet–lymphocyte—aggregates were also elevated in both thalassemia genotypes with marked increase in patients who had undergone splenectomy. These findings suggest that platelets adhere to neutrophils and monocytes are activated which support the clinical observation that splenectomized thalassemia patients have an increased risk of arterial or venous thrombotic manifestations.

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Acknowledgments

We sincerely thank Professor Aftab A. Ansari of the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, USA, for valuable discussions. Support for this study was provided by the Thailand Research Fund – Senior Research Scholar Award, Chalermprakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University, and Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative. S.F. and K.P. are presently Thailand Research Fund – Senior Research Scholar.

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Authors and Affiliations

  1. Center of Excellence for Flow Cytometry, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Rassamon Keawvichit, Ladawan Khowawisetsut, Porntip Chaichompoo, Korakot Polsrila, Suchana Sukklad, Kasama Sukapirom & Kovit Pattanapanyasat

  2. Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Rassamon Keawvichit, Ladawan Khowawisetsut & Suchana Sukklad

  3. Thalassemia Research Center, Institute of Molecular Biosciences, Salaya Campus, Mahidol University, Nakhon Pathom, Thailand

    Porntip Chaichompoo & Suthat Fucharoen

  4. Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Archrob Khuhapinant

  5. Center for Emerging and Neglected Infectious Diseases, Mahidol University, Nakhon Pathom, Thailand

    Kovit Pattanapanyasat

  6. Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand

    Kovit Pattanapanyasat

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  1. Rassamon Keawvichit
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  2. Ladawan Khowawisetsut
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Correspondence to Kovit Pattanapanyasat.

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Keawvichit, R., Khowawisetsut, L., Chaichompoo, P. et al. Platelet activation and platelet–leukocyte interaction in β-thalassemia/hemoglobin E patients with marked nucleated erythrocytosis. Ann Hematol 91, 1685–1694 (2012). https://doi.org/10.1007/s00277-012-1522-2

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  • DOI: https://doi.org/10.1007/s00277-012-1522-2

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