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

This study explored a method for detecting a specific type of platelet (thiazole orange-positive or TO+ platelets) in horses that have low blood platelet counts or thrombocytopenia. The findings suggest that enumerating TO+ platelets could serve as a useful noninvasive measurement of bone marrow platelet production in thrombocytopenic horses.

Participants and Methodology

• The study involved 16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies, nine horses and ponies with low blood platelet counts that tested positive for EIAV, and two similar animals who were EIAV-negative.
• Blood samples were collected from the jugular vein of both healthy and thrombocytopenic individuals. The researchers experimented with different sample types – blood anticoagulated with two substances known as EDTA and sodium citrate, and platelet-rich plasma in sodium citrate – in order to determine the optimal sample requirement and incubation time for the assay.

Procedure and Analysis

• The blood samples or platelet-rich plasma were treated with thiazole orange (TO) then went through a flow cytometric analysis.
• The percentage of circulating TO+ platelets – platelets that had bonded with the thiazole orange – was calculated based on fluorescence histograms.

Results

• In healthy ponies and horses, the amount of TO+ platelets was relatively low. Specifically, the proportions ranged from 1.28 to 2.83% in ponies and 0.9 to 3.44% in horses.
• In horses and ponies with thrombocytopenia, the percentage of TO+ platelets greatly increased. Specifically, thrombocytopenic ponies had 11.14 to 48.41% TO+ platelets, while thrombocytopenic horses had 2.33 to 8.52%.
• The study also recorded the mean platelet counts for the thrombocytopenic ponies and horses which were considerably lower than the reference range.

Conclusion

• The researchers concluded that it is possible to detect TO+ platelets in horse blood.
• They observed that the percentage of TO+ platelets increased in thrombocytopenic horses.
• Consequently, they postulated that the count of TO+ platelets could provide a noninvasive way to assess bone marrow platelet production, and help understand platelet metabolism in horses with thrombocytopenia.