Detection of Activated Feline Platelets in Platelet-rich Plasma by Use of Fluorescein-labeled Antibodies and Flow Cytometry

Abstract

Platelets contribute to prethrombotic or thrombotic states; however, accepted evaluation methods (i.e., in vitro testing by use of an aggregometer) of platelet function in cats can be difficult because of the large volume of blood required from which platelets are isolated and the potential for platelet activation due to difficult venipunctures in sometimes uncooperative or excited animals. The activation problem also contributes to errors in platelet counts. Platelets from four domestic short haired cats (two males, two females, 2–3 years old) minimally restrained without sedation or anesthesia were evaluated. Blood (5 ml) was collected by jugular venipuncture directly into syringes containing 3.8% trisodium citrate (nine parts blood to one part anticoagulant) plus prostaglandin E1 (3 μM; 0.25, 0.5, 1, or 2 μl/500 μl citrate) or 3.8% trisodium citrate alone. Prostaglandin E1, which is a stable metabolite of arachidonic acid with platelet inhibitory properties similar to those of prostaglandin I2 was added to the anticoagulant to prevent activation of platelets during the collection process. Feline platelets exposed to prostaglandin E1 became immediately and persistently nonreactive to agonists, which negated their use in functional studies (aggregation, 14C-serotonin release, binding of fluorescein-conjugated antifibrinogen) but improved platelet counting accuracy. Detection of in vivo activation of platelets in prethrombotic and thrombotic states in humans has been done by identification of activation-dependent molecules on platelet surfaces by use of specific antibody recognition and detection by flow cytometric analysis. Many activation-dependent platelet surface receptor changes are species specific; however, fibrinogen appears to be conserved across species. Results of flow cytometric analysis by forward and side angle light scatter easily identified feline platelets in 10 μl of platelet-rich plasma but were less definitive in identification of platelets in whole blood. Feline platelets in platelet-rich plasma activated by ADP (5 or 50 μM), which allowed binding of fibrinogen to exposed fibrinogen receptors, were labeled with fluorescein-conjugated anti-human fibrinogen. Labeled platelets were identified as a separate population by flow cytometric staining pattern and fluorescence intensity from nonlabeled platelets or nonspecifically-labeled resting platelets. This technique should allow detection of activated feline platelets from extremely small quantities of blood, which could facilitate repetitive and serial platelet testing in diseases thought to be accompanied, caused, or augmented by thrombotic conditions.