Reticulated Platelets Counts in the Assessment of Bone Marrow Thrombopoiesis in Dogs--Preliminary Data
World Small Animal Veterinary Association World Congress Proceedings, 2008
Regina Kiomi Takahira, PhD; Luis Fernando Negro Silva; Marjorie de Assis Golim
São Paulo State University, Distrito de Rubião Junior
São Paolo, Brazil

Thrombocytopenia is one of the most common hematological disturbances in dogs and may occur in several diseases. Reticulate platelets are young platelets that are usually evaluated by flow cytometry and may be useful in determining the etiology of multifactorial thrombocytopenia. The aim of this study was to evaluate the reticulated platelet importance as a thrombopoiesis marker in regenerative and nonregenerative thrombocytopenic conditions. These preliminary data were obtained from 15 healthy dogs (control group), 10 thrombocytopenic dogs (< 100,000 platelets/μL) without megakaryocytic hypoplasia (group A) and 10 thrombocytopenic dogs (< 100,000 platelets /μL) with megakaryocytic hypoplasia (group B).

Blood samples were collected and the platelet rich plasma (PRP) was extracted. Thiazole orange (TO) was used to stain RNA content in reticulated platelets. A negative control (without staining) and TO fixed PRP were incubated in the dark during one hour and the fluorescence was quantified by flow cytometry. CBCs were obtained using a Cell-Dyn 3500 hematology analyzer. Megakaryocytes were quantified in marrow particles, collected from aspiration cytology and correlated to reticulated platelets counts.

The results are shown in the table:

 

Mean ± SD

Mk/Particle

Reticulated platelet
(%)

Reticulated platelet
(/μL)

Control

4.1 ± 1.4

1.4 ± 0.9a

3,857 ± 2,971 a,b

Regenerative

5.6 ± 2.4

14.0 ± 8.1a

6,540 ± 4,125 b

Nongenerative

0.8 ± 1.0

10.7 ± 8.5 a

2,694 ± 2,309 a

No significant difference was observed in the percentages of reticulated platelets between the two groups (A and B), but when the absolute values were analyzed, higher values (p<0.05) were observed in the regenerative group. The relative and absolute values of reticulated platelets observed in control group were in agreement with a report for non thrombocytopenic dogs. There was no correlation between megakaryocytes count and absolute reticulated platelets.

The absence of correlation with the number of megakaryocytes indicates that even in a condition of apparent megakaryocytic hypoplasia thrombopoiesis may be not impaired. Although the marrow cytology is usually utilized to verify the thrombopoietic activity, it may be not representative of the real platelet production, once megakaryocytes may show an irregular distribution in bone marrow aspirates.

Nevertheless, reticulated platelets demonstrate that they are peripheral markers of marrow thrombopoiesis in dogs, with the advantage of being a less invasive technique than bone marrow cytology.

Speaker Information
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Regina Kiomi Takahira , PhD
São Paulo State University
Distrito de Rubião Junior, São Paolo, Brazil


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