Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders

Abstract

Thiazole orange (TO), a fluorescent dye originally synthesized for reticulocyte analysis, is characterized by a large fluorescence enhancement and high quantum yield on binding to nucleic acids, particularly RNA. In addition, the dye readily permeates live cell membranes. We applied TO staining, followed by fluorescence-activated flow cytometric analysis, to platelets in whole blood samples from hematologically normal subjects and patients with various quantitative platelet disorders. The percentage of TO-positive platelets in 50 control subjects was 8.6 +/- 2.8% (mean +/- SD) ranging from 2.8% to 15.8%. In 21 thrombocytopenic patients whose bone marrow contained normal to increased numbers of megakaryocytes, the percentage of fluorescently labeled platelets was significantly elevated (P less than .0001) to 26.9 +/- 10.9% (range, 13.3% to 57.1%). In contrast, the proportion of positively stained platelets in 23 patients with thrombocytopenia due to impaired platelet production (various conditions with reduced marrow megakaryocytes) did not significantly differ from the controls, whereas the absolute counts of TO-positive platelets were significantly lowered (P less than .0001). Differences in the distributions of the percentage values as well as of the absolute counts for TO-positive platelets between the two patient groups were again highly significant (P less than .0001). Both the sensitivity and the specificity of this method in distinguishing between these categories of thrombocytopenia were greater than or equal to 95%. We conclude that flow cytometric analysis of platelets after staining with TO is a sensitive and specific test that rapidly provides information on the thrombopoietic activity in thrombocytopenic disorders. Our data further suggest that increased amounts of residual RNA characterize platelets released under conditions of “stress thrombopoiesis.”

Lipidomic analysis of platelet senescence

“…4C) during PLT storage. Although PLTs are anucleate cells and do not show cell growth, reticulated PLTs seem to undergo terminal differentiation 42 and programmed cell death during senescence, 43,44 biologic processes in which sphingolipids play an important role 45 . The sphingolipid metabolism in PLTs shown by Tani and coworkers 46 has special features: PLTs are deficient in de novo sphingolipid biosynthesis 46 .…”

Immature platelet fraction measurement: a future guide to platelet transfusion requirement after haematopoietic stem cell transplantation

“…During the past decade, an increasing body of evidence has suggested that the reticulated platelet count may be a useful indicator of thrombopoiesis in the bone marrow (Kienast & Schmitz, 1990; Ault et al ., 1992; Richards & Baglin, 1995; O'Malley et al ., 1996; Robinson et al ., 2000). However, measurement by flow cytometry is expensive, time consuming and requires expertise not usually available on a daily basis in the routine laboratory.…”

Measurements of immature platelets with haematology analysers are of limited value to separate immune thrombocytopenia from bone marrow failure

“…They are assumed to be a surrogate marker of thrombopoietic activity in analogy to reticulocytes. To use this fact for clinical decision‐making, since the 1990s, several studies using flow cytometry with different fluorescent dyes have shown that the amount of reticulated platelets reflects the rate of thrombopoiesis, but standardization remained difficult (Kienast & Schmitz, ; Ault et al , ; Richards & Baglin, ; Matic et al , ). Later on, Sysmex (Kobe, Japan) introduced the immature platelet fraction (IPF), measured by a haematology analyser based on RNA staining as a routine parameter within the automated blood count (Briggs et al , ).…”