Hemostasis and Diagnosis of Disorders

The objective of hemostasis is to maintain a balance of coagulation and fibrinolysis to preserve proper vascular structure and function and blood fluidity. When a vascular structure is damaged, it is necessary to have adequate platelet numbers and function (primary hemostasis), as well as sufficient factors and cofactors of the coagulation system (secondary hemostasis), to stop bleeding. An intact fibrinolytic system localizes clotting to the area of vascular damage and acts to reopen blood vessels while healing occurs. An imbalance of these factors leads either to hypercoagulation and thromboembolic disorders or to hypocoagulation and hemorrhage or both in DIC.

This chapter has three sections. The first section introduces the components of normal hemostasis, including vascular endothelium, platelets, coagulation factors, and fibrinolysis. The second section describes selected hemostatic tests and factors that affect how they may be used in diagnosis. The third section briefly describes selected inherited and acquired disorders of hemostasis and their laboratory diagnosis.

Evaluation of the cause of bleeding varies from handling of overt problems (e.g., severe epistaxis), to detection of suspected problems (e.g., von Willebrand’s disease [vWD] in a Doberman pinscher), to investigation of unexpected problems found during laboratory examination (e.g., mild to moderate thrombocytopenia, evidence of hepatic disease). Signs of bleeding problems may include prolonged bleeding after parturition, estrus, or minor trauma (e.g., venipuncture, loss of deciduous teeth), or spontaneous hemorrhages (i.e., petechiae, ecchymoses, hematomas). Gastrointestinal tract bleeding may appear as fresh red blood or dark tarry stools and is often occult. Bleeding into joints and muscle may cause lameness. Bleeding into body cavities may be diagnosed from fluid cytology. Small hemorrhages such as petechiae and ecchymoses suggest a platelet or vascular defect (Figure 5-1). Epistaxis is often associated with platelet defects, perhaps because of the paucity of tissue between the vessels and the nasal mucosa. In contrast, coagulation defects are characterized by large, deep hemorrhages (e.g., hematomas, hemarthroses).

FIGURE 5-1 Physical evidence of thrombocytopenia or coagulopathy. The difference in the size of hemorrhage in thrombocytopenia versus coagulopathy is illustrated. A, A break in a vessel is normally repaired quickly when platelets and coagulation factors are stimulated by exposure to collagen to form a platelet plug that is rapidly stabilized by fibrin strands. B, A proper platelet plug is not formed in thrombocytopenia, but only a little blood leaks into the collagen before a fibrin clot is formed. C, A coagulation factor defect prevents rapid or firm fibrin stabilization of the platelet plug, and the platelet plug breaks, allowing abundant hemorrhage.

Evaluation of a patient with a suspected bleeding disorder requires not only selection of appropriate tests but, for the best diagnosis, requires a complete history and physical examination because of the high association of hemostatic disorders with breed, age, treatments, and poisonings. Some screening tests should be run in private practice. Point-of-care analyzers such as the VetScan VSpro Coagulation Analyzer (Abaxis North America, Union City, CA) are available for in-clinic analysis of aPTT and prothrombin time (PT). However, hemostasis testing by reference laboratories and especially hemostasis centers should have better control of methods and reagents and a more complete selection of tests that may be necessary to identify mild or uncommon problems (see later discussion of methodology).

A simple approach for test selection is shown in Figure 5-2. If physical examination shows no cause of the bleeding or bleeding seems disproportionate to the injury, the expense of hemostatic tests (relatively expensive testing) is warranted to document and localize the defect. Some situations require only a single test (e.g., von Willebrand’s factor [vWF] assay or capillary bleeding time as a presurgical screen for elective surgery on a Doberman due to their high incidence of having vWD). Similarly, PT is the preferred single test for suspected ingestion of warfarin-type rodenticide. However, a profile of tests performed the same day is recommended to localize an unknown bleeding defect (Table 5-1; Table 5-2). Some hemostatic disorders affect multiple areas of hemostasis, so evaluation of only one or two tests may lead to an incomplete or erroneous conclusion. Variation in production and inactivation of coagulation and anticoagulation factors and variation in production and removal of platelets and effects of treatment confuse diagnosis; therefore, performing a complete profile of tests concurrently at initial presentation permits a more likely correct diagnosis. Normal results in a profile exclude some diseases from consideration.

FIGURE 5-2 Selection of laboratory tests. No hemostatic tests are necessary if an adequate explanation for a bleeding process exists. If a hemostatic defect is likely and either several causes are possible or the cause is uncertain, one should screen all parts of the hemostatic mechanism for a defect with a profile of laboratory tests. When the clinical evidence suggests only one probable cause, one should select the most appropriate test to evaluate that defect.

TABLE 5-1 HEMOSTATIC SCREENING PROFILE

TABLE 5-2 HEMOSTATIC MECHANISMS: FUNCTIONS AND APPROPRIATE LABORATORY TESTS

A reasonable profile includes a platelet count, a test of platelet function, the aPTT to assess intrinsic and common coagulation pathways, the PT to assess extrinsic and common pathways, and a test of fibrinolytic activity such as D-dimer (see Table 5-1). Alternative and additional tests are used in certain situations (see section on Laboratory Tests).

Hemostatic defects should initially be localized to a general area of the hemostatic mechanism, which has four main components: the blood vessels, the platelets, coagulation factors, and the fibrinolytic system (Table 5-2). A plug of aggregated platelets initially seals damaged vessels after circulating platelets come into contact with exposed subendothelial collagen (primary hemostasis). Platelets bind to collagen and each other with the help of vWF (Figure 5-3; see Figure 5-1). The coagulation factors stimulated by the collagen, tissue thromboplastin, and platelets form fibrin strands to stabilize the platelet plug (secondary hemostasis). The fibrinolytic system degrades the clot to reopen the vascular lumen to blood flow while the vessel heals. A platelet plug forms in coagulopathies, but easily breaks down and allows rebleeding because it is not stabilized by fibrin strands. A severe deficiency of one or more coagulation factors slows the formation of a clot, which should form rapidly after contact with the subendothelial collagen. During this time, larger sized hemorrhages can occur before a fibrin clot is finally formed or pressure of adjacent tissues stops the bleeding (see Figure 5-1).

FIGURE 5-3 Depiction of the factor VIII molecular complex. von Willebrand’s factor (vWF) is a long multimer of many vWF units. Factor VIII coagulant activity (fVIIIc) is a smaller molecule linked to vWF in the complex that protects fVIIIc. To facilitate platelet adhesion to sites of vascular injury, vWF binds to collagen at the sites; vWF also facilitates receptor-specific platelet aggregation. In the intrinsic system, fVIIIc acts to activate the common pathway at factor X, which ultimately results in fibrin formation to stabilize the platelet plug.

(Modified from Cotran RS, Kumar V, Robbins SL: Robbins pathologic basis of disease, ed 5, Philadelphia, 1994, WB Saunders, p 621.)

Vascular Endothelium and Wall

Endothelial cells secrete substances such as prostacyclin and nitric oxide that act as inhibitors of platelet adhesion. However, when the vascular endothelium is disrupted because of trauma, inflammation, neoplasia, or toxicity, coagulation is initiated by platelet adherence to exposed subendothelial collagen (primary hemostasis), which initiates the coagulation cascade and the formation of a fibrin mesh that subsequently stabilizes the clot (secondary hemostasis). Clot retraction and vasoconstriction also prevent continued blood loss.

Platelets and Primary Hemostasis

Platelets are small cytoplasmic fragments shed from megakaryocytes in the bone marrow. They circulate for approximately 5 days in dogs and about 30 hours in cats. Increased thrombopoiesis is simulated by decreased total circulating platelet mass (measured by plateletcrit) and not necessarily reduced platelet count (thrombocytopenia). For example, Cavalier King Charles spaniels with a hereditary dysplasia with fewer but larger platelets have a normal platelecrit.^11,54 Plateletcrit, like hematocrit, is the percentage of blood volume composed of platelets. Thus the platelet count, though universally used to judge the adequacy of platelets for hemostasis, is not as physiologic a test as the plateletcrit.

Platelets adhere to a defect in a vascular wall and clump together as a platelet plug. Platelets then enhance and accelerate the coagulation cascade and formation of stabilizing clots. vWF is needed for primary hemostasis and can affect some tests of platelet function. The binding of platelets results in a conformational change to expose previously internalized phosphatidylserine (formerly known as PF-3, a potent receptor for factor X and prothrombin; Figure 5-4), which causes platelet activation, release of platelet granule contents, aggregation of more platelets, and initiation of secondary hemostasis by formation of a procoagulant surface of fibrinogen receptors on the primary plug. Release of adenosine diphosphate (ADP) from the dense granules causes synthesis of thromboxane A₂, a prostaglandin that causes irreversible platelet aggregation and viscous metamorphosis and local vasoconstriction. Primary hemostasis requires adequate numbers of platelets that function adequately.

FIGURE 5-4 A simplified coagulation cascade. One may consider the cascade as a letter Y with three portions (or pathways): The intrinsic pathway includes factors XII, XI, IX, and VIII; the extrinsic pathway contains factor VII; and the common pathway includes factors X, V, prothrombin, and fibrinogen. Phosphatidylserine (i.e., platelet factor 3 [PF-3]) on the surface of activated platelets speeds the coagulation process. The intrinsic pathway is initiated by contacting abnormal surfaces (e.g., collagen) not covered by endothelium. The intrinsic system activates the common pathway at factor X by a complex of IX, VIII, and phosphatidylserine on the activated platelet surface. The end point of the common pathway (and thus the intrinsic or extrinsic pathways) is the fibrin clot. Tissue thromboplastin from damaged cells and factor VII in the extrinsic pathway also activates the common pathway.

Coagulation Factors

Stabilization of the initial platelet hemostatic plug is accomplished by a mesh of fibrin formed by the coagulation cascade. The primary objective of the coagulation system is the formation of thrombin, which converts circulating fibrinogen to insoluble fibrin. These generated fibrin strands then cross-link to form a mesh, which traps more blood cells and stabilizes the platelet plug, which is stronger than the initial plug and is resistant to proteolytic degradation by plasmin, a fibrinolytic enzyme.

Coagulation factor interactions are much more complex than this description, but may be divided into three parts—(1) intrinsic pathway, (2) extrinsic pathway, and (3) common pathway—for diagnostic purposes. These three parts are diagrammed as a letter Y (see Figure 5-4). The intrinsic pathway includes factors XII, XI, IX, and VIII and is initiated by contact activation. Factor XII is activated when it contacts a nonendothelial surface such as collagen outside a blood vessel.

The extrinsic pathway is initiated by interaction of tissue factor and activated factor VII. The extrinsic system initiates the coagulation response, whereas the intrinsic system sustains the generation of fibrin. These two pathways converge into the common pathway at the point where factor X is activated, which then leads to formation of a fibrin clot. Most coagulation tests have fibrin formation as the end point of the test. The common pathway consists of factors X, V, prothrombin, and fibrinogen.

In the case of a difficult venipuncture, tissue thromboplastin may be released from injured cells and initiate the clotting cascade ex vivo. Deficiencies of tissue thromboplastin and ionized calcium levels are never low enough in a patient to cause bleeding. Activated platelets are important in clot formation, because they provide a negatively charged phospholipid surface on which coagulation factors assemble. In severe thrombocytopenia, the coagulation process is slower (e.g., slight prolongation of the activated clotting time). The negatively charged surface of platelets reacts with factors IX and VIII as a complex in the intrinsic pathway. Activated factor IX from this complex interacts with another complex of factors X and V of the common pathway.

To keep coagulation localized to the defect, once procoagulant factors have been activated, they are inhibited by anticoagulant factors such as antithrombin III (AT III, complexed with heparin) and removed by the phagocytic system. Those that escape AT III will bind with thrombomodulin on endothelial cells, which activates protein C. Protein C is an anticoagulant and profibrinolytic factor that, with protein S as a cofactor, specifically hydrolyzes factors V and VIII and inhibits thrombin formation.³⁷ Several factors (II, VII, IX, X and protein C) are dependent on vitamin K. Vitamin K antagonists (i.e., warfarin) interfere with hemostasis by interfering with these factors. These factors have different half-lives, which affects which of them become deficient earliest during warfarin poisoning. Factor VII and protein C become deficient earliest, and lack of protein C can lead to often unexpected hypercoagulability early in warfarin poisoning or high-dose warfarin treatment.

Heparin inhibits thrombin and other procoagulant factors indirectly by activating AT III, a circulating natural anticoagulant that is the main physiologic inhibitor of thrombin. Because AT III is usually decreased in DIC, heparin therapy may be ineffective. Heparin has been used clinically to treat hemostatic abnormalities associated with severe gastrointestinal disease, septicemia, and endotoxemia.³⁶ Decreased AT III occurs in hepatic insufficiency, owing to decreased synthesis, and in glomerular disease because of increased loss through the glomerulus. Availability of the AT III assay varies. Automated assays with chromogenic substrates are used by some veterinary laboratories and allow routine testing. However, tests of AT III function, protein C, and protein S are expensive to maintain in terms of time, quality control, and reagents, so they are available only at a few hemostasis reference laboratories. Lack of information about the activity of these anticoagulant factors impedes ability to fully understand the balance of hemostasis in a patient.

Fibrinolysis

Fibrinolysis is the process of fibrin clot dissolution once the damaged blood vessel is repaired, which reestablishes normal vascular patency. Plasmin digests fibrin clots (and even circulating fibrinogen) to produce fibrin degradation products (FDPs) or fibrinogen degradation products (FgDPs). FDPs have anticoagulant effects, which helps explain bleeding signs during DIC. Fibrinolysis may be detected by quantification of products of fibrinolysis such as D-dimer, FDPs, or alteration in TEG curves 30 to 60 minutes after clot formation.

Blood Collection for Hemostatic Testing

Proper sample collection and management avoid preanalytical errors in test results. A clean venipuncture is essential to minimize introduction of tissue factors, which could falsely activate platelets and clotting factors. One should avoid “raising” a vein by hitting it. A sharp 20-gauge (1.1-mm) or larger needle should be used. A two-syringe technique is preferred for platelet counting and coagulation testing. The first tube receiving blood with any tissue thromboplastin from the venipuncture is used for another purpose (e.g., serum clot tube for chemistry) or is discarded. Allowing blood to flow from a bare needle also flushes out tissue thromboplastins. Blood should be mixed well with the anticoagulant by inverting the tube several times without violent shaking. The collection tube may be swayed during collection to aid mixing. Additionally, turbulence, clotting, or extremes in temperature may invalidate hemostatic testing. Ethylenediaminetetraacetic acid (EDTA) is an excellent anticoagulant for canine platelet counts and routine hematology, though exceptions occur in which EDTA (and even heparin and citrate to a lesser effect) seems to promote clumping of platelets to cause pseudothrombocytopenia in certain patients. A venipuncture adequate for a routine complete blood count (CBC) is often inadequate for an accurate platelet count. (See later comments on feline platelet counting and prostaglandin E₁.)

The citrated plasma-clotting tests (i.e., aPTT, PT, fibrinogen, thrombin time [TT], FDP) require nine parts fresh whole blood and one part 3.8% trisodium citrate anticoagulant to separate plasma from cells and platelets, which must be done within 30 minutes of sample collection. Citrate or oxalate blood collection tubes must be filled completely to a mark on the tube’s label to avoid errors in dilution. Plasma can be stored at 4° C for 48 hours or 20° C for 6 hours, or frozen for transport, to preserve factors VII and VIII. Furlanello has stated that samples were reasonably stable for 48 hours at room temperature and so may be sent to a laboratory.¹⁵ Certain tests require use of special tubes containing various reagents, so one should ask or read information from the laboratory before collecting the sample. Plastic tubes should be used because routine glass surfaces activate coagulation.

Blood Vessel Evaluation

Vascular disease is rarely diagnosed as the cause of bleeding in dogs and cats. Blood vessels can be evaluated histologically. Skin biopsy may document vasculitis in bleeding animals and is indicated in the presence of edema, petechiae, and a normal platelet count, or edema plus DIC. Immune-mediated vasculitis in dogs is uncommon, but antigen-antibody complexes occasionally deposit in vessels, causing an Arthus reaction and nonthrombocytopenic purpura with petechiae and edema. Widespread petechial hemorrhages seen at necropsy are a sign of septicemia-induced DIC. In rare instances, cutaneous hemorrhages are noted in dogs with hyperadrenocorticism, in which bleeding is secondary to catabolism of supporting collagen around vessels.

Platelet Enumeration

Hematology instrument platelet counts are accurate, sensitive, and precise methods for detecting and evaluating thrombocytopenia and should be used when available. However, blood smear estimates are often adequate for initial quick diagnosis of severe thrombocytopenia. Severe thrombocytopenia is a very common cause of bleeding, and an estimate may be used during off hours or when instruments are not available. After routine preparation and staining of a blood smear, one should determine the average number of platelets in 5 to 10 microscope fields using the 100× oil objective (i.e., 1000× magnification) to estimate platelet numbers.⁵² Dogs normally have about 8 to 29 platelets/field, and cats should have 10 to 29/field. If the thrombocytopenia is severe enough to cause bleeding, only 0 to 3 platelets/field are expected. Platelets should be counted in the thin “monolayer” area. On most smears, this is where the red blood cells (RBCs) infrequently touch each other, and central pallor in canine RBCs is prominent. Anemic, bleeding animals have fewer RBCs per field in the proper area, so it is easy to look too deeply into the thicker areas of a smear. An alternative is to consider each platelet/100× oil immersion field as approximately equal to 15,000 to 20,000 platelets/µl. There is variation to the number of platelets seen per field of view depending on the microscope objective. Better objectives have a larger field of view.

One must ensure uniform platelet distribution in the sample. Blood smears should be screened for platelet aggregates to assure accuracy of any platelet count or estimate. Platelet clumps that are large enough to be seen at scanning magnification (4× or 10× objective) are usually pulled to the distal end of the smear. If many or large platelet clumps are present, all platelet methods, such as estimated, manual, and instrument platelet counts, will be inaccurate. If an accurate platelet result is needed for the patient, then a new sample should be taken in such a manner as to avoid aggregation of platelets. Aggregation of erythrocytes or leukocytes also causes laboratory error. All tubes of anticoagulated blood should be examined for grossly visible clots and not submitted for hematologic testing.

Thrombocytopenia is a common initial clue to the presence of many diseases. Instrument platelet counts are accurate and precise and are the preferred method to detect mild to moderate thrombocytopenia. Instrument counts are preferred to monitor the course of a thrombocytopenia and response to treatment. Reference values vary with the instrument, method, and breed; thus specific reference values improve sensitivity in detection of mild thrombocytopenia.

Feline platelets have a strong tendency to clump, frequently causing inaccurate and lower platelet counts. Prostaglandin E₁ (PGE1) will prevent platelet aggregation.⁵³ If added immediately to a feline blood sample or preferably included already in the tube, PGE1 will allow accurate platelet counts that on average are 60% higher than those without PGE1. Optical counts (in instruments with laser systems) in routine feline patients can be 50% to 62% higher than impedance counts because impedance-type instruments fail to identify large platelets, which are excluded from their platelet counts.²⁹ Thus the combination of missing large platelets with impedance hematology instruments and not using PGE1 as an anticoagulant supplement will combine for substantial errors in feline platelet counts.⁵³ PGE1 is expensive and must be frozen until just before use, but need only be used in cats where thrombocytopenia is likely or other dogs with persistent platelet aggregation of blood samples. Fortunately true and severe thrombocytopenia is rare in cats. In feline myeloproliferative disorders, huge numbers of large and bizarre platelets may not be counted. Other particles (e.g., ghost cells: partially lysed RBCs) may be erroneously counted as platelets. Ghost cells are not uncommon problems in immune-mediated hemolytic anemia (IMHA) samples. An abnormal appearance of platelet dot-plots from the Advia 2120 or Sysmex XT-2000iV suggests this error (Figure 2-6).⁵¹ Small RBCs in iron deficiency anemia can be counted as platelets, especially with impedance-type instruments. This “small red” error may be detected by inspecting the platelet-RBC histogram and noting a failure of separation of the two peaks representing platelets (left peak) and RBCs (right peak) (see Figure 2-3) or by instrument error flags.

Manual counts with a glass counting chamber (hemocytometer) may be used if automated hematology cell counters are not available or, rarely, if the automated count is in question (e.g., if ghost RBCs are counted as platelets). Manual platelet counts are imprecise and labor intensive but include large platelets and are more similar to optical instrument platelet counts than impedance feline platelet counts.²⁹ The hemocytometer must be clean and free of scratches. Because platelets are the size of dust particles, nicks and scratches in the glass may cause misidentification of dust as platelets. One very good hemocytometer should be reserved just for platelet counts. A phase-contrast microscope helps to differentiate platelets from other particles. Lowering the condenser on a regular microscope also aids in visualizing the relatively transparent platelets. A platelet stain improves platelet detection in a hemocytometer but is usually unnecessary. If the blood sample is poor (e.g., has clots or platelet aggregates), it is not appropriate to request a manual platelet count, because it too will be affected by uneven platelet distribution in the blood.

Blood must be diluted such that the number of cells in the hemocytometer is easy to count. A dilution system with plastic containers is easiest. Becton-Dickinson (BD) discontinued their Unopette System and now recommends, as an alternative, similar products of Bioanalytic GmbH (www.bioanalytic.de). Bioanalytic’s Trombo-tic is advertized to lyse RBCs, disaggregate platelet clumps, and make platelets more rounded for better identification.

Delayed analysis favors platelet clumping and can lead to a lower platelet count. Various references suggest 30 minutes to 6 hours as a maximum tolerable limit. This may vary with the method, because an evaluation of 21 dogs with the Bayer H-1 hematology analyzer (precursor to the Advia 2120) suggested that clinically accurate platelet counts could be obtained with analysis of EDTA blood up to 2 days old (Figure 5-5). This suggests that EDTA blood sent to laboratories, remaining in the mail for 1 or 2 days, may still have clinically acceptable platelet count results if platelet clumping is absent or minimal.

FIGURE 5-5 Stability of the platelet count with the H-1 hematology instrument (precursor to the Advia 2120). The mean percentage change in the platelet count with time is compared with the initial platelet count for 21 canine ethylenediaminetetraacetic acid (EDTA) blood samples stored at room temperature for 8 hours and refrigerated for 2 days. Bars indicate the standard deviation (SD). This indicates platelet counts may be performed on blood samples sent by mail.

Platelet Morphology

Platelets may be characterized in different ways by various instruments, including some of those mentioned in this section. Blood smears should be evaluated routinely to ensure that the platelet count appears reasonable. Large platelets are usually seen on blood smears of dogs with thrombocytopenia. However, neither increased numbers of large platelets, increased mean platelet volume (MPV), nor abnormal platelet distribution width (PDW) have been shown in the dog to differentiate among any of the causes of thrombocytopenia or other diseases. Larger platelets and increased MPV consistently are inversely correlated to platelet numbers, which thus may assure the clinician that a thrombocytopenia in the patient is real and not a laboratory error. Large platelets are more functionally active, which may explain why some dogs with platelet counts less than 10,000/µl do not bleed excessively. Cavalier King Charles spaniels often have a hereditary dysplasia with large but fewer platelets. This macrothrombocytopenia is due to a defect in beta1-tubulin.¹¹ They have no signs of bleeding, though impedance platelet counts are often less than 10,000/µl. The Cavalier King Charles spaniels and Norfork terriers have a normal plateletcrit (percentage of blood volume, similar to hematocrit) despite variably low platelet counts (see Figure 2-7).⁵⁴ Plateletcrit (platelet mass) is a more physiologic measurement than platelet count, but nobody can interpret plateletcrit in % blood volume or L/L. Plateletcrit is directly measured by the IDEXX VetAutoread Hematology Analyzer (QBC) but converted to a platelet number, which most veterinarians can interpret. The QBC measures plateletcrit directly by measuring the width of the platelet layer in a centrifuged tube. The Advia 2120 performs a plateletcrit that works fairly well even in Cavalier King Charles spaniels with their very large platelets (unpublished data). Other instruments enumerate platelet numbers well, but most have problems with accurate measurement of MPV, especially when distribution of platelets by size is not typical. Plateletcrit equals platelet count multiplied by MPV multiplied by a factor.

Decreased MPV was consistently reported by the (then Technicon) H-1 Hematology analyzer in thrombocytopenic dogs with immune-mediated thrombocytopenia (ITP), but this was most likely an error in measuring MPV in very thrombocytopenic samples and not reflective of a true microthrombocytosis. Decreased MPV may occur if small particles (e.g., lipid droplets, cell fragments) are counted as platelets. This “background noise” is not noticed in samples with many platelets but has a relatively strong effect in severe thrombocytopenia. Canine MPV may be artificially high in EDTA blood samples held more than 4 hours at 4° C because of platelet swelling.

Large, RNA-rich platelets are named the high fluorescent platelet fraction (HFPF) in the Sysmex XT-2000iV instrument. These HFPF platelets were shown to be equivalent to reticulated platelets.⁴¹ HFPF platelets are consistently increased during active thrombopoiesis due to thrombocytopenia or treatment with thrombopoietin. The HFPF is greater in number in thrombocytopenia due to DIC, IMHA, and ITP than in dogs with thrombocytopenia due to leukemia.^17,41 This suggests that enumerating the absolute number of large RNA-rich platelets may be useful in differentiating causes of thrombocytopenia (“regenerative” thrombocytopenia), pending further studies. However, greater numbers of large, RNA-rich platelets (HFPF) are seen in hereditary macrothrombocytopenia in Cavalier King Charles spaniels and Norfork terriers, which do not have increased thrombocytosis, and these large platelets are large throughout their lifespans.¹⁷

Platelets often have pseudopodia and an irregular shape on blood smears that suggests platelet activation during collection and handling of the blood. Decrease in the Advia 120 and 2120’s mean platelet component (MPC) concentration is an indicator of platelet activation. MPC was more decreased in dogs with IMHA then normal dogs and sick dogs without IMHA, and more decreased in those dogs with IMHA who died.⁶¹ Primary platelet disorders such as thrombasthenic thrombopathia in otter hounds, foxhounds, and Scottish terriers are rare causes of large and morphologically bizarre platelets.^3,5 Ehrlichia platys may be visible in platelets.