Evaluation of flow cytometric counting procedure for canine reticulocytes by use of thiazole orange.
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 less than or equal to 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.
Abbott
D L
DL
Toxicology Division, Division of Eli Lilly & Co, Greenfield, IN 46140.
McGrath
J P
JP
D000818:Animals; D052160:Benzothiazoles; D004285:Dogs / Q000097:blood*; D004906:Erythrocyte Count / Q000662:veterinary; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D011237:Predictive Value of Tests; D011804:Quinolines; D012016:Reference Values; D015203:Reproducibility of Results; D012156:Reticulocytes; D013844:Thiazoles
D052160:Benzothiazoles; D005456:Fluorescent Dyes; D011804:Quinolines; D013844:Thiazoles; C051000:thiazole orange