Abstract
Ultrastructure analysis of immature platelets is difficult because of the lack of a suitable marker and their relatively low concentration in total platelets. We investigated the morphological and optical properties of human immature platelets produced and enriched in immunodeficient mice via human CD34-positive cell administration. Immunodeficient mice were injected with human CD34-positive cells and administered eltrombopag orally for 14days (eltro-mice). Some of these mice were maintained for 2-3months (steady-state-mice). Platelets were double-stained with a human CD41 antibody and a nuclear staining dye (Sysmex hematology analyzer XN series reagent), and then analyzed by flowcytometry FCM to identify human immature platelets. Human CD41-positive cells were isolated from citrated blood by magnetic cell sorting with human CD41 antibody, and examined using electron microscopy. Flow cytometric analysis with the XN reagent demonstrated that peripheral blood from eltro-mice had a higher percentage of immature platelet fraction in human platelets than that from steady-state-mice. The geometric mean of XN reagent fluorescence for human platelets, divided with that for mouse platelets, revealed that the ratios in eltro-mice were significantly higher than those in steady-state-mice, thus indicating that immature platelets were highly enriched in eltro-mice. Scanning and transmission electron microscopy revealed that human citrated platelets isolated from eltro-mice tended to be larger (n=15, p=0.276) and contained more mitochondria than those isolated from steady-state-mice (n=10, p=0.0002). Therefore, an increased number of mitochondria, rather than platelet size, is a distinctive feature of immature platelets.
Highlights
Platelets are enucleated cells which are derived from megakaryocytes in the bone marrow and are present in the blood at concentrations ranging from 150,000 to 400,000 cells/μL [1]
Analysis for immature platelet fraction (IPF)% in eltro-mice and steady-state-mice The intensity of peripheral blood samples double-stained with a FITC-conjugated human CD41 antibody (Figures 2(a) and 2(c) right) or the negative control antibody (Figures 2(a) and 2(c) left), along with the XN series reagent specific for nucleic acids, was measured using flow cytometry (FCM)
IPF% values are useful biomarkers to distinguish platelet consumptive disorder, such as primary immune thrombocytopenia (ITP), from hypoplastic thrombocytopenia, such as aplastic anemia (AA). They are useful for predicting thrombopoietic recovery after stem cell transplantation and/or chemotherapy [8,9,18,19]
Summary
Platelets are enucleated cells which are derived from megakaryocytes in the bone marrow and are present in the blood at concentrations ranging from 150,000 to 400,000 cells/μL [1]. When blood vessels are damaged, platelets immediately adhere to the damaged endothelial cells to form a platelet plug and stop bleeding. Owing to their highly polyploid nuclei, megakaryocytes in the bone marrow are large cells (~ 60 μm), with one megakaryocyte releasing approximately 1000 platelets, which are derived from its proplatelets and pass through holes in the bone marrow blood vessels, into the peripheral blood [2]. The Sysmex hematology analyzer (XN-series) and Abbott CELLDYN Sapphire can measure RP as an immature platelet fraction [3]. Platelets that display higher intensity of both FSC and SFL are identified as being in the immature platelet fraction (IPF) [6,7]. It has been demonstrated that IPF% values, especially when measured using the XN series, as well as RP% values, are useful to determine platelet turnover in thrombocytopenic disorders, and for predicting platelet recovery after stem cell transplantation [8,9,10,11,12,13,14]
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