A Novel Rapid Method of Red Blood Cell and Platelet Permeabilization and Staining for Flow Cytometry Analysis.

BACKGROUND: Flow cytometry essentially focuses on surface-expressed proteins, with few protocols being devoted to intracellular components. We evaluated a two-step procedure using new formaldehyde-free permeabilization and staining reagents that allow the staining of platelets and red blood cells (RBCs) from whole blood. METHODS: Citrated blood was treated with the new staining protocol (NSP) or control reagent (phosphate-buffered solution bovine serum albumin) and stained with antibodies against surface or intracellular markers. The effects of the NSP on cell integrity, morphology, and content were evaluated. RESULTS: The NSP slightly reduced the cell count (~20%) and changed the RBC morphology with a 42% mean diameter reduction. Conversely, the NSP did not affect platelet discoid morphology and led to a minor size decrease (11%). These morphological changes neither impelled a gating strategy modification nor interfered with the discrimination among populations based on surface markers. The NSP provided intracellular access to all the tested antigens: CD62P, FXIII, and CD63 in platelets and glycated and fetal hemoglobin (HbA1c and HbF) and nucleic acid in RBCs. The NSP gave excellent intra-assay precision with minimal impact on cell morphology and fluorescence labelling over time (up to 24 h). CONCLUSIONS: With the ability to detect surface and intracellular antigens through a rapid preparation protocol without washing steps or toxic formaldehyde treatment, this NSP designed for research offers a marked improvement in the analysis of platelets and RBCs isolated directly from whole blood. Consequently, the NSP opens new avenues to investigate platelet degranulation and erythrocyte subpopulations. © 2019 International Clinical Cytometry Society.

Macrothrombocytopenia and dense granule deficiency associated with FLI1 variants: ultrastructural and pathogenic features.

Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 variant carriers' platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers' platelets, presumably due to a biogenesis defect. Additionally, 25-29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7-9%) and autophagosome-like structures (0-3%). In vitro study of megakaryocytes derived from circulating CD34+ cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the FLI1 variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in FLI1 variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of FLI1 variant-associated thrombocytopenia.