Platelet interaction with erythrocytes and propensity to aggregation in essential thrombocythaemia

Abstract

3 years after diagnosis with essential thrombocythaemia (JAK2 Val617Phe mutation) in January, 2011, a healthy 26-year-old white woman was referred to the Applied Morphology Research Centre at the University of Pretoria for an ultrastructural blood analysis because of a platelet count of 1240 × 109 cells per L (normal range 150–300 × 109 cells per L). Essential thrombocythaemia is a myelo proliferative disorder characterised by hyperplasia of megakaryocytes in the bone marrow and thrombocytosis. The JAK2 Val617Phe mutation is present in about 50% of people with essential thrombocythaemia and leads to megakaryocyte hyperplasia and consequent thrombo cytosis. Although other organs such as the liver can be aff ected, our patient had no organ involvement, and no history of thrombosis. She was not on any regular medication, had no other medical history, and did not smoke. To study her platelet morphology and activation we used light and scanning electron microscopy to study whole blood smears (fi gure). We drew blood in a citrate tube and prepared it on the same day. We did a standard light microscopy smear preparation stained with Giemsa’s azur eosin methylene blue solution (Merck, Darmstadt, Germany). We prepared platelet-rich plasma samples for scanning electron microscopy by centrifuging whole blood for 600 s at 3000 g. We placed the platelet-rich plasma on a coverslip and spread it. Standard scanning electron microscopy fi xation preparation was done (in 2·5% gulteraldehyde/ formaldehyde solution), secondary fi xation in osmium tetroxide, and dehydration. We coated samples with carbon and examined them with a Zeiss ULTRA Plus fi eld emission gun-scanning electron microscope with InLens capabilities (Oberkochen, Germany). We took micrographs at 1 kV. Typically, in a blood smear from a healthy person, platelets appear spherical, with a minimal pseudopodia formation due to contact activation during preparation, and no spreading. In our patient, platelet activity was strikingly increased and we noted pseudopodia formation, spreading, and extreme aggregation. Essential thrombocythaemia is characterised by a prothrombotic tendency, consistent with the platelet hyperactivation seen in these micrographs. Platelet activation can cause interaction with both erythrocytes and leucocytes, which might aff ect haemostasis in these individuals. We saw frequent platelet–erythrocyte interaction in scanning electron microscopy. On light microscopy we saw rouleaux formation with visible platelet aggregates and evidence of platelet–leucocyte interaction on light microscopy, which corresponds with previous studies indicating that activated platelets might adhere to circulating leucocytes, creating heterogenous aggregates that further aff ect haemostasis. The samples from our patient show changes in platelet morphology (indicating activation) and the interaction of the activated platelets with erythrocytes and leucocytes, which has often been described in literature but not visualised in detail before.