Diagnosis of ETV6-mutation Related Thrombocytopenia by Immunofluorescence Microscopy

Background: Inherited thrombocytopenias are rare but important differential diagnoses of low platelet counts. Many hereditary thrombocytopenias and platelet function disorders have been determined. Autosomal dominant mutations in ETV6 are one cause of inherited thrombocytopenia. ETV6 mutations are associated with mild to moderate thrombocytopenia, delta storage pool deficiency, increased bleeding tendency and increased risk for hematological malignancies. Until now, no specific clinical or laboratory features of the platelet phenotype in ETV6-mutation related thrombocytopenia have been identified. ETV6 is a nuclear protein, which is also expressed in the cytoplasm of megakaryocytes of patients with ETV6 mutations. We have identified a specific platelet morphologic phenotype allowing identification of patients with ETV6-mutation by immunofluorescence microscopy on a blood smear.

Methods: Platelets from peripheral blood smears, sent by regular mail, had been stained with monoclonal and polyclonal antibodies for the glycoprotein complexes: IbIX and IIbIIIa; the cytoskeleton proteins: non-muscular myosin IIa, α-, β1-tubulin, and filamin; α-granule markers: von-Willebrand factor, thrombospondin and P-selectin; lysosomal and delta-granule markers: Lamp1, Lamp2 and CD63; ETV6; and CD34.

Results: In eight of nine patients from six families with ETV6-related thrombocytopenia, ETV6 was detectable in the platelet cytoplasm. In eight of nine patients, the markers of the delta granules Lamp 2 and/or CD 63 were diminished and diffusely distributed in the platelet cytoplasm. All other assessed markers (see methods) were normally expressed, except thrombospondin, which was additionally diminished in two patients. In none of the controls, including a non-affected member of a concerned family, platelets were stained for ETV6.

Conclusion: Immunofluorescence microscopy of platelets on a blood smear is a powerful tool to characterize the phenotype of hereditary thrombocytopenias and to guide genetic diagnostic. We are now able to identify by immuno fluorescence analysis of blood smears patients with Bernard-Soulier-syndrome, Glanzmann thrombasthenia, MYH9-associated thrombocytopenias, α and β1 tubulin disorders and filamin-defects, α and delta storage pool disorders, Gray platelet syndrome, Wiskott-Aldrich Syndrome, GFI1B-mutation, and ETV6 mutation. ADDIN EN.REFLIST

No conflict of interest has been declared by the author(s).