Determination of the percentage of thiazole orange (TO)‐positive, “reticulated” platelets using autologous erythrocyte TO fluorescence as an internal standard

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Abstract
Determination of the percentage of thiazole orange (TO)‐positive or “reticulated” platelets by flow cytometry has been advocated as an aid in the diagnosis of thrombocytopenic disorders. However, a reproducible method for determining control fluorescence and setting threshold values on a routine clinical basis has not been described. We used erythrocyte TO fluorescence in whole blood as an internal standard to set threshold markers for TO fluorescence of autologous, purified platelets. Since platelets have approximately a threefold higher TO fluorescence than erythrocytes, multiplying the erythrocyte threshold marker by 3 allowed a determination of the percentage of “reticulated” platelets in 29 normal controls. This value was 3.64% ± 2.10% (S.D.) in men and 5.79% ± 2.22% (S.D.) in women; there was a significant (P < 0.05) difference between normal men and women for this measurement. Applying this method to our earlier studies of normals and patients with immune thrombocytopenic purpura confirmed its ability to discriminate between these populations with similar sensitivity and specificity as previously reported. This method controls for fluctuations in TO labeling and fluorescence and does not require daily determination of a new normal control value.