Reticulocyte count using thiazole orange. A flow cytometry method

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.