Counting reticulocytes by flow cytometry: use of thiazole orange

Thiazole orange is a new fluorescent dye which will bind to the residual RNA in the cytoplasm of reticulocytes and allow their enumeration by FACS analysis. We have evaluated the use of this dye in the routine haematology laboratory. There is an excellent correlation between manual and FACS reticulocyte counts (r = 0.98) but FACS counting showed significantly higher precision (CV = 3.1) than the manual method (CV = 11.9) for single observer, 20.8% for multiple observers). Clinical specimens showed stable reticulocyte counts for 6 h if stored at 4 degrees C allowing efficient batching of samples. There was a significant fall in reticulocyte counts stored for 24 h at both 4 degrees C and 21 degrees C. Evaluation of 78 male and 76 female blood donors by FACS analysis gave normal ranges (mean % +/- 2 SD) of 0.74 +/- 0.48 and 0.84 +/- 0.56 respectively (P less than 0.005). When corrected to absolute values there was no sex difference (36 +/- 24 x 10(9)/l). Thiazole orange is an effective stain for the automated counting of reticulocytes by FACS analysis.