Platelet evaluation by manual blood smear review is essential for verifying an automated platelet count, which is particularly relevant when thrombocytopenia is reported. A key initial step for diagnosis and treatment of bleeding disorders is platelet evaluation. Hemostatic disorders are typically caused by insufficient or defective platelets (primary hemostasis) or insufficient or defective clotting factors (secondary hemostasis). Hemostatic disorders are often multifactorial, requiring assessment of numerous aspects of hemostasis, which involves sample collection technique and evaluation of platelet clumping, estimated platelet count, and platelet morphology.<\/p>\n
Take-Home Points<\/strong><\/p>\n <\/div><\/div><\/p>\n Because multiple factors often play a role in hemostatic disorders, components of primary hemostasis (platelet plug formation) and secondary hemostasis (coagulation fortification of platelet plug into fibrin clot) must be evaluated. Hemostatic disorders can include defects in both phases of hemostasis, including insufficient or defective platelets (primary hemostasis) and\/or clotting factors (secondary hemostasis). Primary hemostatic disorders are typically exhibited as surface bleeding; secondary hemostatic disorders may be exhibited as bleeding into body cavities.1<\/sup><\/span><\/p>\n Platelets are essential for the primary phase of hemostasis, during which a platelet plug forms, coagulation factor binding is promoted, and anticoagulant factors are inhibited at the site of vessel injury.1<\/sup> Thus, platelet analysis is helpful for evaluating a patient with a bleeding disorder. Automated hematology analyzers provide a more accurate and precise assessment of platelet count than manual methods; however, various artifacts can reduce their accuracy.2<\/sup> For this reason, a blood smear review is essential for verifying an automated platelet count and detecting any artifacts, especially if thrombocytopenia is reported. If any questions or concerns arise, do not hesitate to reach out to or forward samples (e.g., premade unstained blood smears) to a local reference laboratory.<\/span><\/p>\n Platelets, or thrombocytes, originate from megakaryocytes in the bone marrow, yet most platelets are not in circulation and are stored in the spleen. In most dogs and cats, the lifespan of a platelet is 4 to 6\u00a0days but can vary, especially with splenectomized patients. Because the spleen is responsible for removing aged platelets, platelet lifespan in splenectomized patients can be up to 8 days.3<\/sup><\/span><\/p>\n A platelet count should be performed within 5 hours of collection.2<\/sup> When a clot forms, coagulation factors are activated and platelets are consumed; thus, blood should be collected into a tube with anticoagulant to assess platelet mass, morphology, and function. The preferred anticoagulant for making blood smears and performing automated platelet counts in dogs and cats is EDTA. When the tube is properly filled, EDTA enables optimal sample staining and minimal sample dilution. However, EDTA has been reported to cause platelet clumping; platelet clumping may be reduced by prewarming EDTA samples to 37 \u00baC (98.6 \u00baF).4,5<\/sup> Collecting samples in citrate may decrease platelet clumping in cats but not in dogs.6-8<\/sup> Of note, samples are diluted by 10% when collected in citrate tubes; platelet counts must be corrected for this dilution.3<\/sup> Adding amikacin to blood samples may also disaggregate clumps.9<\/sup><\/span><\/p>\n Platelet clumping can be enhanced by refrigeration, which is recommended if CBC analysis is not performed immediately. For this reason, 2 unstained, air-dried blood smears should be made at the time of sample collection and submitted with the blood sample. The blood sample should come to room temperature and be mixed thoroughly by gentle inversion immediately before being placed in the hematology analyzer.5,6<\/sup><\/span><\/p>\n The most common cause of artifactually low platelet counts (pseudothrombocytopenia) is clumping. Clumping is also among the most common hematology laboratory artifacts, often attributed to improper sample collection (e.g., delayed transfer of blood to anticoagulant tube, inadequate sample mixing, traumatic sample collection).10<\/sup> In vitro platelet clumping can occur during blood collection, especially with cats.3<\/sup> If platelet clumps are present, an accurate platelet count cannot be determined. Typically, an analyzer will not categorize platelet clumps, but smaller clumps may be counted as white blood cells.11<\/sup><\/span><\/p>\n Automated platelet counts by well-maintained hematology analyzers are more accurate than manual platelet counts (in mammals). However, impedance counters can fail to differentiate feline platelets from erythrocytes and other cells, particularly when platelet clumping occurs.12<\/sup> Thus, impedance counters are often inferior to those that use flow cytometry. Regardless, performing a manual blood smear review is advised to complement any automated platelet count, especially when thrombocytopenia is reported below the limit of <\/span>sensitivity (5000 to 20\u2009<\/span>000 platelets\/\u00b5L).2,3<\/sup> A previous article in this cytology series, \u201cBlood Smear Review: A Step-by-Step Guide to Normal Findings for Cats and Dogs<\/a>,\u201d in the Today\u2019s Veterinary Practice <\/i>January\/February 2025 issue <\/span>provides more in-depth information on how to conduct a blood smear review beyond platelet evaluation.<\/p>\n On low magnification (10\u00d7 or 20\u00d7 objective), scan the feathered edge and periphery of the smear for platelet clumps (<\/span>FIGURE 1A<\/b><\/span>). Scan the rest of the smear as platelet clumping can also be found in the monolayer (<\/span>FIGURE 1B<\/b><\/span>). After degranulation, platelets can be difficult to discriminate from basophilic debris (<\/span>FIGURE 1C AND 1D<\/b><\/span>).<\/span><\/p>\n\n
Sample Collection<\/h2>\n
Platelet Evaluation<\/h2>\n
Step 1: Look for Platelet Clumps<\/h3>\n
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