Detection of reticulated platelets: estimating the degree of fluorescence of platelets stained with thiazole orange.

The primary problem in the measurement of reticulated platelets (RP) stained with thiazole orange (TO) by flow cytometry is the definition of a threshold limit for fluorescence positivity. We evaluated settings for the threshold gate for TO positivity based on two principles: a fluorescence histogram (median FL1, Relative FL1) or a plot of forward light scatter (FSC; reflecting the distribution of the platelet size) versus fluorescence intensity (% RP). These methods were applied prospectively in examination of 54 healthy blood donors (16 females) and a total of 50 blinded patient samples: pregnant women with thrombocytopenia (Group 1A, n = 11), thrombocytopenic women after delivery (Group 1B, n = 9) and healthy women with a thrombocytopenic newborn (Group 2, n = 30). Group 1A displayed higher median FL1 (mean 306, CI 279-332) as compared to that of Group 2 (mean 266, CI 255-277; p = 0.0038) or to that of the female controls (mean 249, CI 231-268; p < 0.001). Relative FL1 was also higher in the patients of Group 1A than those of Group 2 (p = 0.037). When analysing the % RP, the difference between these groups was not significant. In the patients (n = 50), the median FSC (mean 407, SD 40, CI 395-418) was also higher than that of the controls (n = 54; mean 383, SD 25, CI 376-390; Mann-Whitney U-test, p = 0.0015). In Group 1A, a significant correlation was observed between the Patient median FL1 and Patient median FSC (r = 0.62, p = 0.043). When developing methods for the measurement of RP, it seems to be useful to analyse the data with more than one principle to define the threshold limit for TO positivity.