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

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.”

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

Blood ◽

10.1182/blood.v75.1.116.116 ◽

1990 ◽

Vol 75 (1) ◽

pp. 116-121 ◽

Cited By ~ 174

Author(s):

J Kienast ◽

G Schmitz

Keyword(s):

Fluorescence Enhancement ◽

Flow Cytometric Analysis ◽

Normal Subjects ◽

Thiazole Orange ◽

High Quantum Yield ◽

Specific Test ◽

Blood Samples ◽

Flow Cytometric ◽

Patient Groups ◽

The Absolute

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.”

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Flow Cytometric Analysis of Feline Reticulocytes

Veterinary Pathology ◽

10.1177/030098589202900603 ◽

1992 ◽

Vol 29 (6) ◽

pp. 503-508 ◽

Cited By ~ 8

Author(s):

W. J. Reagan ◽

L. M. Vap ◽

M. G. Weiser

Keyword(s):

Flow Cytometry ◽

Flow Cytometric Analysis ◽

State University ◽

Thiazole Orange ◽

Adult Males ◽

Blood Samples ◽

Colorado State University ◽

Flow Cytometric ◽

Manual Counting ◽

Two Samples

Hemolytic anemia was induced in five Domestic Shorthair cats (four adult males and one spayed female obtained from a breeding colony at Colorado State University, CO), and blood samples were analyzed from five other cats (two castrated male Domestic Shorthairs, one castrated male Domestic Longhair, one castrated male Persian, and one spayed female Siamese presented to the Veterinary Teaching Hospital at Colorado State University for miscellaneous problems). Blood samples taken from these cats had percentages of aggregate reticulocytes that ranged from 0% to 14.5% as determined by manual counting and were used to identify the best technique for staining cat reticulocytes for flow cytometric analysis. The best technique was mixing a blood sample (1/2,000 dilution) with 0.2 μg thiazole orange in 1 ml of diluent and incubating the mixture in the dark at room temperature for 30 to 60 minutes. The percentage of reticulocytes determined by flow cytometry correlated well ( r = 0.88) with manually determined aggregate reticulocyte percentages; no significant differences were observed between the two techniques ( P > 0.05). For the conditions used, punctate reticulocytes were not detected by flow cytometry. Samples with very high platelet numbers and very low packed cell volumes may show falsely elevated percentages of reticulocytes as determined by flow cytometry. The reproducibility of the flow cytometric technique was good; the coefficient of variation ranged from 4.8% to 17.9% in two samples with two different times of incubation. Staining of cat aggregate reticulocytes with thiazole orange and use of flow cytometric quantification is a reproducible technique that has a good correlation with the manual reticulocyte counting method.

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Aspirin Does Not Affect the Flow Cytometric Detection of Fibrinogen Binding to, or Release of α-Granules or Lysosomes from, Human Platelets

Clinical Science ◽

10.1042/cs0870575 ◽

1994 ◽

Vol 87 (5) ◽

pp. 575-580 ◽

Cited By ~ 70

Author(s):

Nicolas A. F. Chronos ◽

Darren J. Wilson ◽

Sarah L. Janes ◽

Ronald A. Hutton ◽

Nigel P. Buller ◽

...

Keyword(s):

Flow Cytometry ◽

Arachidonic Acid ◽

Thromboxane A2 ◽

Normal Subjects ◽

Human Platelets ◽

Aspirin Therapy ◽

Flow Cytometric Assay ◽

Blood Samples ◽

Flow Cytometric ◽

Fibrinogen Binding

1. Aspirin inhibits the conversion of arachidonic acid to thromboxane A2 which reinforces the effects of weak agonists such as ADP in platelets. 2. In this study the effect of aspirin (300 mg/day) on platelet agonist response was measured by whole blood flow cytometry of unfixed blood samples from normal subjects (n = 10), an assay that investigates aggregation-independent changes in the platelet. 3. Fibrinogen binding to unstimulated platelets or to platelets stimulated with ADP or thrombin was unaffected by aspirin. 4. Under the conditions of this assay, platelets undergo a partial degranulation of α-granules and lysosomes (evidenced by expression of P-selectin and CD63, respectively) in response to ADP, and full degranulation in response to thrombin. P-selectin expression was paralleled by release of β-thromboglobulin. None of these events was affected by aspirin. 5. Thromboxane formation was totally prevented by the aspirin treatment, as shown by Born aggregometry in which the platelet aggregatory response to arachidonic acid was abolished and secondary aggregation by ADP was inhibited. 6. The flow cytometric assay can therefore be used to investigate platelets in patients, regardless of aspirin therapy. 7. These findings suggest that platelet fibrinogen binding and the release of platelet α-granule and lysosomal contents, in response to stimulation with physiological agonists, can continue in patients despite aspirin therapy. This may help to explain why aspirin is only partially effective in preventing thrombotic events.

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High stability of blood samples for flow cytometric analysis of VASP phosphorylation to measure the clopidogrel responsiveness in patients with coronary artery disease

Thrombosis and Haemostasis ◽

10.1160/th05-04-0886 ◽

2009 ◽

Author(s):

Christian Gachet

Keyword(s):

Coronary Artery Disease ◽

Coronary Artery ◽

High Stability ◽

Flow Cytometric Analysis ◽

Blood Samples ◽

Flow Cytometric ◽

Vasp Phosphorylation ◽

Artery Disease

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P0934THE IMPACT OF HAEMODIALYSIS INITIATION ON CKD-ASSOCIATED MYELOID CELL DYSREGULATION

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0934 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Sarah Cormican ◽

Serika Diane Naicker ◽

Denise Connolly ◽

Gemma Prendergast ◽

M Conall Dennedy ◽

...

Keyword(s):

Flow Cytometric Analysis ◽

Myeloid Cell ◽

Absolute Number ◽

Flow Cytometer ◽

Dialysis Initiation ◽

Monocyte Subset ◽

Cell Lineages ◽

Flow Cytometric ◽

The Absolute ◽

Population Numbers

Abstract Background and Aims Abnormalities of the myeloid immune cell lineages are well described in people with both Chronic Kidney Disease (CKD) and End Stage Renal Disease (ESRD). Increased numbers and proportions of the intermediate (CD14++CD16+) and nonclassical (CD14+CD16+) monocyte subsets and increased neutrophil numbers have been reported in both settings. These changes are one aspect of the chronic inflammatory state which exists in people with CKD and ESRD and which contributes to complications including cardiovascular disease and protein-energy malnutrition. The effects of transition from advanced CKD to dialysis on myeloid cell populations has not been well studied. We investigated these changes by measuring major leucocyte population numbers and monocyte subset numbers and proportions in adults with Stage 5 CKD prior to and after haemodialysis initiation. Method Adults with stage 5 CKD and a non-emergent indication for dialysis initiation (in-centre haemodialysis) were recruited after informed consent. Blood samples were collected within the week prior to dialysis initiation and follow-up samples were collected prior to a dialysis session at one week and one month after haemodialysis initiation. Major leucocyte population numbers were determined by two-colour flow cytometric analysis of whole blood on an Accuri™ Flow Cytometer. Peripheral blood mononuclear cells were isolated and stained for multi-colour flow cytometric analysis on a Canto II™ flow cytometer. The absolute number of each monocyte subset/ml blood was calculated based on the above values. Results Ten individuals (six male, four female) with a mean age of 78 ± 6.4years were enrolled and completed all follow-ups. The mean eGFR was 9 ± 1.9ml/min at the time of haemodialysis initiation. Total monocyte numbers had not changed after one month of haemodialysis (Figure 1A) (3.6x105 ± 1.6x105cells/ml) compared to initiation (5.2x105 ± 3.2x105, p=0.11). However, the proportion of nonclassical monocytes was markedly increased after one week of haemodialysis (16.8% (IQR 12.8-21.0%) compared to 11.2% (IQR 9.3-12.3%) at initiation, p=0.007) (Figure 1B). At one month the proportion of nonclassical monocytes was maintained (17.1% (IQR 14.5-20.5%)) but had not increased further compared to the one week timepoint (p=0.97). This proportionate change was not reflected in the absolute nonclassical count. There were no significant changes in the proportion of classical or intermediate monocytes. Neutrophil numbers were reduced at one month (3.6x106 ± 1.3x106cells/ml) compared to initiation (4.8X106 ± 1.6x106cells/ml, p=0.04) (Figure 1A). Total lymphocyte numbers did not change significantly after dialysis initiation. Conclusion Haemodialysis initiation is associated with an increase in the proportion of nonclassical monocytes without significant increases in the absolute number of any monocyte subset. A reduction in total neutrophil number also occurs one month after dialysis initiation. It has been previously been shown that progression of CKD results in increasing abnormalities of the myeloid cell lineages. Here we have demonstrated that transition from advanced CKD to haemodialysis results in further modulation of myeloid cell numbers and myeloid cell subset proportions.

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Flow Cytometric Detection and Phenotype of Circulating CD34+ Cells in Patients Affected by Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v106.11.4329.4329 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4329-4329

Author(s):

Fabio Stagno ◽

Nunziata Laura Parrinello ◽

Giovannella Fargione ◽

Anna Triolo ◽

Antonella Privitera ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Absolute Number ◽

Lymphocytic Leukemia ◽

Unexpected Finding ◽

Advanced Stage ◽

Flow Cytometric ◽

Cd34 Cells ◽

The Absolute

Abstract Flow cytometric determination of peripheral blood CD34+ cells provides reliable measurements of circulating hemopoietic progenitors. Since the detection of the absolute number of circulating CD34+ cells has been found of clinical utility in the setting of chronic myeloproliferative disorders, we investigated whether peripheral CD34+ cells could play any role in the clinical work-up of B-cell chronic lymphocytic leukemia (B-CLL). In this view, we determined by flow cytometry the absolute number of circulating CD34+ cells in the peripheral blood of 28 patients (16 males and 12 females, median age 67 years) affected by typical B-CLL (Matutes score 5,4,3) and in different Rai stages of the disease (19 early stage: Rai 0, I, II; 9 advanced stage: Rai III, IV). Conventional and multiparameter flow cytometric analysis was performed utilizing a FACSCalibur cytometer (Becton Dickinson). Our data showed a significant increase in the number of circulating CD34+ cells in the peripheral blood of patients with B-CLL (median CD34+ cells:7.8mL) as compared to controls (median CD34+ cells 0.1mL) (p=0.008). No statistical difference between B-CLL patients in early versus advanced stage (p=0.5) and between untreated versus treated (p=0.7) was found, as well as there was no correlation with some of the clinical characteristics of B-CLL (WBC-count, LDH levels, Beta-2M). In 10 out of 28 B-CLL affected patients, circulating CD34+ cells were correlated with ZAP-70 and CD38 antigen but no correlation was found. In addition, we detected in the peripheral blood of 22 out of 28 patients small numbers of circulating CD34+ cells displaying the CD19+/CD5+ phenotype (median CD34+/CD19+/CD5+ cells:5.7mL) whereas these cells were absent in normal controls. This unexpected finding, whose significance remains to be clarified and still restricted to a small number of cases, could be directly correlated to the underlying lymphproliferative disease and might represent a pool of leukemic stem cells. However, further studies are warranted.

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