Flow cytometric method for detecting thiazole orange-positive (reticulated) platelets in thrombocytopenic horses

Abstract

Abstract Objective To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry. Animals 16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, ElAV-positive horses and ponies; and 2 thrombocytopenic, ElAV-negative horses. Procedure Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample of blood or platelet-rich plasma was incubated with thiazole orange, and flow cytometric analysis was performed. Percentage of circulating TO+ platelets was determined from fluorescence (FL-1) logarithmic histograms. Results Healthy ponies (n = 9) had 1.28 to 2.83% (mean ± SD, 2.03 ± 0.50%) and horses (n = 7) had 0.9 to 3.44% (2.12 ± 1.14%) TO+ platelets in circulation. Thrombocytopenic ponies (n = 7) had 11.14 to 48.41 % (26.51 ± 11.99%) and thrombocytopenic horses (n = 4) had 2.33 to 8.52% (6.19 ± 2.68%) TO+ platelets in circulation. Mean platelet counts for the thrombocytopenic ponies and horses were 24,400 ± 20,500 and 39,300 ± 13,500 platelets/μl, respectively (reference range, 94,000 to 232,000 platelets/μl). Conclusion Thiazole orange-positive platelets can be detected in equine blood and percentages of TO+ platelets are increased in thrombocytopenic horses. Clinical Relevance Enumeration of TO+ platelets may prove to be a helpful noninvasive clinical measurement of bone marrow platelet production and aid in the assessment of platelet kinetics in thrombocytopenic horses. (Am J Vet Res 1997;58:1092–1096)