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Reticulocyte count using thiazole orange. A flow cytometry method.

L Van Hove·W Goossens·V Van Duppen·R L Verwilghen
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Abstract

Recently flow cytometry techniques have been developed to replace the microscope reticulocyte count. We used thiazole orange, a RNA binding fluorochrome, to discriminate reticulocytes from mature erythrocytes. Thiazole orange and the Retic-COUNT software package were evaluated for performance of routine analysis on different flow instruments. The applied methodology analysed 10(4) cells semi-automatically in an easily performed manner. Consistent results were obtained with dipotassium EDTA anticoagulated blood (stable for 30 h after venesection), with incubation times in thiazole orange solution ranging from 2 to 7 h at 25 degrees C. This allowed flexibility in specimen collection and storage and assay performance with no change in results. Changes of incubation temperature up to 30 degrees C had no measurable effect. The values obtained showed good linearity, precision and accuracy for normal, low and high reticulocyte counts. However interferences were observed: RBC autofluorescence, nucleated RBC, Howell-Jolly bodies, high leucocyte count, high platelet count and giant platelets, all falsely increased the number of reticulocytes. These artifacts were eliminated by software gate corrections, thus leaving less than 5% of the specimen to be reanalysed by the microscopic method. The thiazole orange flow cytometric method was determined to be a fast, reliable method for the routine clinical quantitation of reticulocytes.

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BenzothiazolesErythrocyte CountFlow CytometryFluorescent DyesHumansMicroscopyQuinolinesReproducibility of ResultsReticulocytesSensitivity and SpecificitySoftwareThiazolesComparative StudyJournal ArticleResearch Support, Non-U.S. Gov't

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L Van Hove, W Goossens, V Van Duppen, R L Verwilghen. 1990. Reticulocyte count using thiazole orange. A flow cytometry method.. https://doi.org/10.1111/j.1365-2257.1990.tb00039.x

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