Platelets from thrombocytopenic ponies acutely infected with equine infectious anemia virus are activated in vivo and hypofunctional.

Thrombocytopenia is a consistent finding and one of the earliest hematological abnormalities in horses acutely infected with equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus. Multifactorial mechanisms, including immune-mediated platelet destruction and impaired platelet production, are implicated in the pathogenesis of EIAV-associated thrombocytopenia. This study was undertaken to investigate whether regenerative thrombopoiesis and platelet destruction occurred in ponies acutely infected with EIAV. Circulating large, immature platelets were increased in ponies acutely infected with EIAV late in the infection when platelet count was at a nadir. Morphometric analysis of bone marrow from acutely infected ponies revealed significant increased in megakaryocyte area and megakaryocyte nuclear area. A trend toward increased numbers of megakaryocytes was also observed. Platelets from acutely infected ponies had increased surface-bound fibrinogen and ultrastructural changes consistent with in vivo platelet activation. Platelets also had hypofunctional aggregation responses to three agonists in vitro. We conclude that thrombocytopenia in ponies acutely infected with EIAV is regenerative and suggest that bone marrow platelet production is not severely compromised in these ponies. Our findings reveal that in vivo platelet activation occurs in ponies acutely infected with EIAV, and as a result platelets are hypofunctional in vitro. Activation of platelets in vivo may cause platelet degranulation or formation of platelet aggregates, which would result in removal of these damages platelets from circulation. This may represent a form of nonimmune-mediated platelet destruction in ponies acutely infected with EIAV.

Platelet-associated immunoglobulin (antiplatelet antibody) in canine Rocky Mountain spotted fever and ehrlichiosis.

Antiplatelet antibodies were detected in the sera of dogs with naturally occurring and experimentally induced Rickettsia rickettsii and Ehrlichia canis infections. This is the first known report documenting elevated platelet-associated immunoglobulin (PAIg) titers in Rocky Mountain spotted fever (RMSF) infections. In the naturally occurring RMSF infections and ehrlichiosis, the antibodies persisted for weeks or months, even when the platelet counts had normalized. Results of this study indicate an immunological component for rickettsial thrombocytopenia. Therefore, current therapeutic recommendations, especially regarding avoiding the use of immunosuppressive drugs in patients with rickettsial diseases, need to be critically reviewed.

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

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, EIAV-positive horses and ponies; and 2 thrombocytopenic, EIAV-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/microliters, respectively (reference range, 94,000 to 232,000 platelets/ microliters). 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.

Flow cytometric analysis of punctate and aggregate reticulocyte responses in phlebotomized cats.

Five cats were made anemic by one-time phlebotomy, and their reticulocyte responses were monitored daily for 20 days, using manual enumeration and a standardized feline reticulocyte protocol developed and validated in our laboratory. The reticulocyte responses of 38 clinically normal client-owned cats also were analyzed manually and cytometrically to determine clinical reference ranges. Increases in the percentage of aggregate reticulocytes over the reference range were detected in 5 of 5 phlebotomized cats, using the cytometric protocol. Only 4 of the 5 cats had an increase by results of manual enumeration. Manual aggregate counts had considerable daily variation and often fluctuated in and out of reference range, whereas cytometric aggregate counts remained consistently increased for distinct periods. Increased numbers of aggregate cells could also be detected for longer periods when evaluated by flow cytometry. Increased numbers of punctate reticulocytes were detected in 4 of 5 cats, using the cytometric protocol. None of the cats had increased numbers of punctate cells when evaluated by use of the manual technique. Aggregate reticulocytes in the 38 clinically normal cats ranged from 0.1 to 0.5%, which corresponded to 8,487 to 42,120 cells/microliter. Punctate reticulocytes ranged from 2 to 17%, which corresponded to 225,400 to 1,268,584 cells/microliter. Flow cytometry, using a standardized analysis protocol, was a more reliable and sensitive technique for detection and evaluation of feline reticulocytosis than was manual enumeration. The sensitivity of the flow cytometer to small amounts of intracellular nucleoprotein makes this assay especially valuable for detection of punctate reticulocytosis and low degrees of aggregate reticulocytosis in cats.