Evaluation of flow cytometric counting procedure for canine reticulocytes by use of thiazole orange
1991
Abbott, D.L. | McGrath, J.P.
An automated reticulocyte counting method that used a flow cytometer and the nucleic acid staining dye, thiazole orange, was developed. Anticoagulated (EDTA) blood specimens were suitable for flow cytometric reticulocyte counting when stored at 4 C for 96 hours after collection. Thiazole orange-stained samples were stable for 5.5 hours after staining when stored capped at 20 C and protected from light. Flow cytometric and manual microscopic reticulocyte counts were compared for counts in the 0.27 to 5.32% range (as determined by flow cytometry) and 0.10 to 4.90% range (as determined by 1 technician). Although the results of flow cytometric analysis generally correlated well (r = 0. 821) with manual counts, there was poor correlation between the procedures for counts less than or equal to 2.0% (r < 0.272). Linearity of flow cytometric counts over the range 0.27 to 14.46% was excellent (r = 0.999). Within-run precision of flow cytometric counts (% coefficient of variation [CV] = 3 to 5) was superior to manual microscopic counts obtained by one technician (% CV = 19 to 23) and to manual microscopic counts, which were an average of counts done by 3 technicians (% CV = 8 to 18). Comparable flow cytometric counts were obtained by counting 50,000 or 100,000 blood cells in the flow cytometer.
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