Platelets: Overview, Morphology, Quantity, Platelet Function Disorders (Thrombocytopathia or Thrombopathia)

Overview

Function and Physiology

> Platelets are essential for normal hemostasis and perform four distinct functions:

• Maintenance of vascular integrity by sealing minor endothelial deficiencies.
• Helping to arrest bleeding by the formation of platelet plugs following endothelial constriction.
• Contributing membrane lipid procoagulant activity to facilitate secondary hemostasis (coagulation) and fibrin formation.
• Promoting vascular healing through platelet derived growth factor (PDGF).

• Stimulates endothelial cell migration and smooth muscle production.

> Platelets are involved in the initial phase of wound repair through growth factors including PDGF.

• Rapid cell to cell interactions and release of soluble mediators stimulate mitogenesis of smooth muscle cells and fibroblasts.

> Platelets play an essential role in inflammation through cell to cell interaction and the release of soluble mediators.

• Platelets release vasoactive substances such as serotonin and also modulate neutrophil function.

Production

> Platelets are produced in the bone marrow from megakaryocytes.

• Formed by megakaryocytic cytoplasmic demarcation.
• Released directly into the venous blood vessels surrounding the marrow hematopoietic space.

> Thrombopoietin

• Regulates the development of megakaryocytes from hemopoietic stem cells.
• Appears to be involved only in the late development of megakaryocytes and the release of platelets from megakaryocytes.
• Concentration is inversely correlated with platelet numbers.
• The source of thrombopoietin is uncertain but appears to involve liver, endothelial cells, and fibroblasts.

> In the dog, two-thirds to three-quarters of all platelets are in the systemic circulation.

• The remainder exist as a splenic pool.
• This pool exchanges freely with the systemic circulation.

> The presence of a splenic pool in the cat is suspected but not defined.

Destruction

> Platelets have a variable but finite intravascular lifespan in the dog and cat of 3 to 7 days.

> Effete platelets are phagocytized by macrophages of the mononuclear phagocyte system.

• Anti-platelet antibody accelerates the destruction of platelets by macrophages leading to immune-mediated thrombocytopenia (see below).

Laboratory Evaluation of Platelets

Examination of the complete hemogram is essential.

> Must establish whether thrombocytopenia is an isolated finding or associated with anemia or leukopenia

• If thrombocytopenia appears to be an isolated finding, repeat the count to confirm.
• The peripheral smear should be checked for platelet morphologic abnormalities, polychromatophilia, neutropenia, lymphopenia, spherocytosis (not easily recognized in the cat), other red cell shape changes, or abnormal or unidentifiable cells.

Reticulated Platelet Count

> Reticulated platelets are immature with high ribonucleic acid content.

> Counting these platelets requires special stains and instrumentation.

> The number of reticulated platelets is useful in differentiating platelet destruction/consumption from decreased production.

Buccal Mucosal Bleeding Time (BMBT)

> A test to determine adequacy of endothelial function and platelet function.

> More sensitive test of platelet function than endothelial function

> Do not perform on patients with platelet counts < 75,000 per microliter.

Mean Platelet Volume (MPV)

> Estimated platelet size; similar to red cell size and mean cell volume (MCV)

• Inversely proportional to platelet number

> MPV in the dog is 6.1 - 10 femtoliters

> MPV in the cat is 12 - 18 femtoliters

> Increase in MPV is suggestive of responsive thrombopoiesis

• Occurs with secondary platelet destruction, some myeloproliferative diseases, and hyperthyroidism.
• Artifactual increases in MPV arise when platelets are exposed to EDTA, cooled to room temperature or refrigerated, or if there is delayed exposure to anticoagulant.
• Changes in MPV are minimized when blood is collected in citrate anticoagulant and kept at 37°C until analyzed.

> Decreases in MPV have been reported in:

• Dogs with early immune-mediated thrombocytopenia.
• Bone marrow failure

Platelet Distribution Width (PDW)

> A value provided by some automated particle counters.

> Index of variation in platelet size (similar to red cell distribution width  -  RDW)

> PDW is a useful adjunct to smear evaluation of platelets.

> Examination of the PDW histogram may reveal the presence of abnormally sized platelets, either small or large (see morphology).

Morphology

Normal Morphology: Dog

Shape:

> Discoid, oval, slightly elongated, slightly biconvex or flat with an even contour (Fig. 10-1).

Figure 10-1. Canine platelets. Small round to oval, light blue, anucleated cells with a central cluster of purple granules (50x).

Romanowsky (Wright's or modified Wright's) stain:

• Central cluster of fine azurophilic granules surrounded by a pale blue matrix enclosed in a delicate membrane.
• Some platelets appear agranular or have only a few granules.

Size:

• 2.2 - 3.7 microns in diameter and 0.5 microns thick (approximately 1/10th the size of an erythrocyte).
• Young platelets are often large macroplatelets (megathrombocytes) (Fig. 10-2).

Single platelets are most common. Small groups may be observed.

Figure 10-2. Canine platelets exhibiting platelet anisocytosis. Large purple platelet in center of field (arrow) is a young platelet. These are occasionally observed in normal canine blood smears (50x).

Normal Morphology: Cat

Feline platelets are morphologically similar to those of other domestic animals.

Shape:

> Small spherical bodies (Fig. 10-3)

• Sometimes appear as elongated structures

> Tend to clump, often resulting in amorphous masses (Fig. 10-4)

Romanowsky stain:

> Central cluster of purple azurophilic granules surrounded by a pale blue background and enclosed in a delicate membrane.

Size:

> Variable

> Giant forms equal to the size of red blood cells are often observed in normal animals.

Figure 10-3. Normal feline platelets and red blood cells. Platelet size can be normally variable (anisocytosis) in feline blood smears (50x).

Figure 10-4. Large clump of feline platelets (center). Note size of the platelet clump as compared to the relatively tiny dark white cells at the periphery of the clump. Also note red cell clumps. These are typical of platelet and red cell appearance at the feathered edge of feline blood smears. Platelet clumping in cats could affect validity of both platelet and white cell counts (10x).

Abnormal Morphology: Dog

Activated canine platelets have a spider-like appearance with small cytoplasmic pseudopodia.

> May form small clumps or an agglutinated mass (Fig. 10-5). Large platelet masses are generally found on the feathered edge of push preparations (Fig. 10-4).

Figure 10-5. Clumped canine platelets (center) generally indicative of poor ex vivo anticoagulation (50x).

Microplatelets

> When they predominate, they suggest an early immune-mediated event (immune-mediated thrombocytopenia) (Fig. 10-6)

Figure 10-6. Canine blood smear from immune-mediated hemolytic anemia patient. Platelet numbers appear increased and platelet anisocytosis is evident. The tiny platelets are microplatelets suggesting the immune activity also involves the platelets. Large blue-red blood cells are immature red cells (polychromatophils). The small red cells without obvious central pallor are spherocytes (50x).

Platelet anisocytosis and numerous macroplatelets suggest marrow release of young platelets.

> Often observed in responsive thrombocytopenias (Fig. 10-7, Fig. 10-8 and Fig. 10-9).

Figure 10-7. Canine blood smear. Platelet anisocytosis is evident. Large macroplatelet is in upper left quadrant. Large oval platelet is in lower right quadrant. These findings are associated with both increased platelet turnover and probable microhemorrhage (50x).

Figure 10-8. Canine blood smear in responsive anemia. Do not confuse the immature blue red cells (polychromatophils) with the large blue granular platelet in the center of the field (arrow). Platelet numbers appear reduced and the large platelet (macroplatelet) could be indicative of increased platelet turnover in this situation (50x).

Figure 10-9. Canine blood smear in immune-mediated hemolytic anemia and thrombocytopenia. Platelet numbers are reduced and a large blue granular cell (arrow) is a macroplatelet suggesting increased platelet turnover in this thrombocytopenic situation. Small dark red blood cells without central pallor throughout field are spherocytes (50x).

Notable numbers of elongated, cigar-shaped platelets suggest focal or generalized systemic hemorrhage.

> Further clinical investigation is warranted, such as determination of occult fecal or urinary blood or examination of body cavities, mucous membranes and hairless areas for evidence of hemorrhage.

Poorly granular platelets or platelets with few large granules suggest developmental abnormalities.

> Bone marrow examination is recommended.

Large granules must be examined as potential inclusions such as those associated with Ehrlichia platys (Fig. 10-10).

Figure 10-10. Canine blood smear. The platelet located at lower center contains Ehrlichia platys morula (100x).

Abnormal Morphology: Cat

Poorly granulated platelets or platelets with large granules suggest developmental defects (Fig. 10-11).

> Bone marrow examination is recommended.

Elongated forms or macroplatelets may be encountered in nonthrombocytopenic cats.

Figure 10-11. Feline blood smear in anemic patient. Note large blue-red cells (polychromatophils) throughout the field. Platelet anisocytosis is extreme with a bizarre macroplatelet in lower left quadrant (arrow) suggesting developmental defect and need for bone marrow examination (50x).

Quantity

Normal

For both dogs and cats, 10 - 12 platelet per 1000x (oil immersion) field are typical of a normal count.

In the normal dog, platelets circulate at a concentration of approximately 200,000/μl to 600,000/μl.

In the cat, platelet numbers are 300,000/μl to 800,000/μl.

> Excitement in cats results in sudden increases in platelet counts with normal morphology.

> A decrease in platelet counts can be seen in splenectomized cats. Morphology remains normal.

Feline platelets are prone to clumping.

> Produces inaccurately low platelet counts

> Automated cell counters that rely on impedance may count platelets as WBCs resulting in artifactually increased WBC counts (see Hematology In Practice).

Decreased Platelet Count (Thrombocytopenia)

Clinical Signs of Thrombocytopenia

The clinical hallmark of thrombocytopenia is the occurrence of petechiae.

Petechiae

> Reflect capillary or postcapillary venule bleeding.

> Usually occur at sites of increased intravascular pressure.

• Lower abdomen
• Oral or genital mucosae
• Axillary or inguinal friction sites

Large coalescing petechiae are called purpura.

> Purpura are not usually palpable (skin texture, thickness are normal).

> Palpable purpura suggest an underlying systemic vasculitis.

Ecchymosis, or bruising, is also associated with primary hemostatic defects.

Clinical signs of bleeding can be seen in any thrombocytopenic patient.

> The rapidity with which thrombocytopenia occurs affects the platelet number at which clinical signs occur.

• Rapid platelet destruction leads to hemorrhagic tendencies at much higher platelet counts.

> If platelets are also dysfunctional, clinical signs occur with only modest reductions in numbers.

• Often occurs with rickettsial diseases. Patients with concomitant illnesses and thrombocytopenia bleed more.

Causes of Thrombocytopenia

There are four major mechanisms which result in thrombocytopenia (Table 10-1).

> Abnormal platelet production

> Accelerated platelet removal

> Abnormal distribution of platelets

> Some combination of the above

Abnormal Platelet Production (Rare)

Virtually always accompanied by another cytopenia such as anemia and/or neutropenia.

Platelet production defects include:

 > Pure megakaryocytic hypoplasia

• Immune or infectious etiologies

> Marrow panhypoplasia

• Drug, infectious, or toxic etiologies

> Dysthrombopoiesis (myelodysplasia or megakaryocytic leukemia)

• Diagnosis requires bone marrow aspirate at least; marrow core biopsy is recommended.
• Multiple aspirates on different days may be required.

Infectious causes include:

> Ehrlichiosis

> Feline leukemia virus (FeLV) infection

> Feline immunodeficiency virus (FIV) infection

Vaccination-induced interference with platelet production has been reported.

> In dogs: measles, distemper, parvoviral vaccinations

> In cats: feline panleukopenia vaccination

Drug-induced suppression of thrombopoiesis

> Usually affects other cell lines also.

> Neutropenia usually occurs at day 5 and thrombocytopenia at days 8 - 10 after exposure to drug.

> Anemia is not usually seen because of longer red cell lifespan.

> Commonly implicated drugs are estrogen, sulfadiazine, and nonsteroidal anti-inflammatory drugs.

Accelerated Platelet Removal (Common)

Causes of accelerated platelet removal include immune mediated thrombocytopenia, alloimmune thrombocytopenia, and secondary non-immune thrombocytopenia (See Fig. 10-8 and Fig. 10-9).

> Both primary and secondary immune mediated thrombocytopenias occur

> Primary immune-mediated thrombocytopenia

• Is most common in dogs with some breed predisposition

• Cocker Spaniels
• English Sheepdogs
• German Shepherds
• Poodles

• Is associated with the presence of antiplatelet antibodies which cause accelerated destruction by the mononuclear phagocyte system
• May also be associated with anti-megakaryocyte antibodies which impair thrombopoiesis.
• Is associated with variable clinical signs including hemorrhage from the mucous membranes, skin, genitalia, nose, or gastrointestinal tract.

• Many severely thrombocytopenic patients remain asymptomatic.

• Laboratory findings in primary immune-mediated thrombocytopenia include:

• Severe thrombocytopenia, often less than 30,000/μl.
• The presence of increased proportions of small or large platelets.
• Megakaryocytic hyperplasia in the marrow if antibodies are directed against circulating platelets, reduced numbers of megakaryocytes if the disease is marrow directed.

• Establishing the diagnosis is mainly by exclusion.

• Rule out pseudothrombocytopenia due to platelet clumping or EDTA anticoagulation.
• Splenomegaly suggests a secondary process.
• Anemia suggests a concurrent disease process.
• Drug exposure, infection, recent vaccination neoplasia, or previous transfusion must be considered.

• Special tests include:

• Serology and polymerase chain reaction (PCR) tests for rickettsial agents.
• Tests for systemic immune-mediated disease such as antinu-clear antibodies, rheumatoid factor, lupus erythematosus cells, and red or white cell-associated antibody tests.
• Antiplatelet antibody assays are useful but require fresh platelets and specialized instrumentation.
• The direct megakaryocyte immunofluorescence assay is a good test but requires special technical expertise and air-dried bone marrows.

> Secondary immune-mediated thrombocytopenia

• Is the most common cause of canine thrombocytopenia and the most common canine hemostatic disorder.
• Is associated with underlying conditions including

• Systemic autoimmune disease

• systemic lupus erythematosus (SLE)
• immune-mediated hemolytic anemia
• rheumatoid arthritis
• pemphigus
• juvenile-onset polyarthritis of Akitas

• Neoplasia

• multicentric or metastatic
• hematologic

• Infectious diseases

• FeLV, FIV in cats
• Ehrlichiosis
• Rocky Mountain spotted fever

• Vaccination with modified live virus distemper vaccines (transient)
• Protozoal infections such as leishmaniasis and babesiosis
• Dirofilariasis
• Histoplasmosis
• Immune-complex vasculitis

Alloimmune thrombocytopenia

> Occurs when maternal antibodies to paternal antigens are transferred across the placenta or in colostrum and cause platelet destruction in the neonate.

> Is not reported in dogs and cats.

> Post-transfusion purpura has been described in dogs receiving DEA1 incompatible erythrocytes, or plasma.

• The resultant thrombocytopenia appears to resolve within hours.

Secondary nonimmune thrombocytopenia

> Causes of secondary nonimmune thrombocytopenia include disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.

> Disseminated intravascular coagulation (DIC)

• Occurs secondary to systemic inflammation (acute, subacute, chronic).
• Is associated with vascular damage, sepsis, release of tissue thromboplastin from diseased or neoplastic tissue.

> Thrombotic thrombocytopenic purpura

• Severe thrombocytopenia, intravascular hemolysis with schistocytosis, and neurologic signs.

> Hemolytic uremic syndrome

• Severe thrombocytopenia, intravascular hemolysis with schistocytosis, and renal dysfunction.

Abnormal Platelet Distribution

Abnormal platelet distribution is associated with hypersplenism and endotoxemia.

Hypersplenism

> A pathologic condition in which a large proportion of circulating platelets become sequestered in the spleen.

> One or more cytopenias is usually present.

> Splenomegaly is sometimes present.

Endotoxemia may cause splenic pooling of platelets

Drug-Induced Thrombocytopenia

Decreased platelet production, accelerated platelet removal, and platelet sequestration can all be associated with the use of various pharmaceuticals.

Implicated drugs include

• Antibiotics
• Antimicrobial agents
• Anticonvulsants
• Anti-inflammatory agents
• Chemotherapeutic agents
• Antiviral drugs
• Diuretics

Drug-induced thrombocytopenias may be marrow-mediated or peripherally mediated.

Drug-induced marrow-mediated thrombocytopenia

> Bone marrow suppression

• Most often associated with direct cytotoxic effects on progenitor cells.

> Immune-mediated drug-induced destruction of megakaryocytes has been reported in the dog.

Drug-induced peripherally-mediated thrombocytopenia

> Can be either immune-mediated or non-immune.

> Immune-mediated drug-related thrombocytopenia

• Some drugs provoke an immune response against unadulterated platelets such as methyldopa, levodopa, and gold.

Other drugs provoke antibody formation against drug-platelet antigen complexes such as heparin and quinine.

Still other drugs cause immune-mediated thrombocytopenia after prolonged drug therapy or the reintroduction of a previously used drug.

• Secondary exposure results in antibody formation
• Drug absorbed or adsorbed to the platelet surface
• Platelet destroyed as an innocent bystander by immune activity

Increased Platelet Count (Thrombocytosis)

Mechanisms of Thrombocytosis

There are three categories of thrombocytosis.

• Essential thrombocytosis (primary bone marrow disorder)
• Secondary to disease
• Physiological

Essential Thrombocythemia

> Is a rare myeloproliferative disorder.

> Is characterized by persistent primary thrombocytosis.

> Synonyms

• Idiopathic thrombocythemia
• Primary hemorrhagic thrombocythemia
• Thrombasthenia

> Has been associated with bleeding or thombosis.

> Described in middle-aged to older dogs and in one cat.

• Platelets are often dysfunctional (thrombocytopathic) in aggregation and adhesion studies.
• MPV is usually within reference range.

> Dogs are presented with both nonregenerative and regenerative anemia, occasional hypogranular macroplatelets, basophilia, and spurious hyperkalemia.

Secondary (reactive) Thrombocytosis

> Characterized by transiently increased platelet counts in patients with conditions other than myeloproliferative disorders (Fig. 10-12).

• Neoplasia

• Lymphoma, hematologic neoplasia, melanoma, mast cell neoplasia, adenocarcinoma, mesothelioma, central nervous system neoplasia.

• Gastrointestinal disorders

• Pancreatitis, hepatitis, inflammatory bowel disease, colitis

• Immune-mediated disease
• Blood loss/hemorrhage

• Iron deficiency, trauma, and surgical trauma

• Fractures
• Drug therapy

• Glucocorticoids and antineoplastic drugs

• Postsplenectomy in dogs

Figure 10-12. Feline blood smear exhibiting thrombocytosis and responsive anemia. Platelet clump is at right center. Three bizarre macro platelets are found in the left half of the field from top to bottom indicating platelet developmental abnormalities and the need for bone marrow examination (50x).

Physiologic Thrombocytosis

> Results from increased mobilization of platelets from splenic and nonsplenic (perhaps pulmonary) pools.

> Transient as the result of exercise or epinephrine injection.

Platelet Function Disorders (Thrombocytopathia or Thrombopathia)

Suggested by clinical hemorrhage and prolonged buccal mucosal bleeding time despite a platelet count >100,000 per microliter (Table 10-2).

Clinical signs:
> Bleeding from mucosal surfaces
> Melena
> Hematuria
> Epistaxis
> Cutaneous ecchymoses
> Prolonged or excessive bleeding at venipuncture, trauma, or surgery sites
> Hallmarks of systemic bleeding disorder due to platelet malfunction:

• Bleeding at more than one location
• Multiple ecchymoses
• Previous bleeding history
• Bleeding is persistent or recurring
• The amount of bleeding is much greater than anticipated
• There is no "apparent" history of trauma
• Platelet count is within reference range
• Activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen concentration are appropriate
• Tests to define or diagnose DIC are negative

Buccal Mucosal Bleeding Time (BMBT)

> Utilizes a "bleeding time device", a guillotine-like device that creates an incision standardized in terms of length and depth.

> Preferred to any other "bleeding time test".

> Ideally performed on a nontranquilized or nonanesthetized patient.

• A somewhat loose gauze tie (in preference to fingers) holds the folded back upper lip in place to expose the buccal mucosa.

> As blood seeps from the incision, paper toweling is used to soak up the blood.

• Use care not to touch the incision site.
• The time from incision to cessation of bleeding is the BMBT.
• The reference interval for the dog and cat: 1.8 to 4.2 minutes
• Most patients will have times closer to the lower end of the interval.

Causes of Platelet Dysfunction (Thrombocytopathy)

Platelet dysfunction can be acquired or inherited.

Acquired Platelet Dysfunction

> May manifest as reduced platelet function (hyporesponsiveness) or enhanced platelet function (hyperresponsiveness).

Acquired Hyporesponsiveness with Bleeding Tendencies

> Causes include:

• Uremia
• Dysproteinemia
• Infectious agents

• Ehrlichia canis
• Feline leukemia virus

• Snake venom
• Hepatic disease
• Neoplasia

• Especially leukemia or myeloproliferative disease

• Drugs (Table 10-3)

• Anti-inflammatory agents including aspirin, ibuprofen, acetaminophen, flunixin, phenylbutazone (butazolidine)
• Antibiotics including penicillin and cephalothin
• Calcium channel blockers  -  diltiazem, nifedipine, verapamil
• Dextrans
• Colloids

Acquired Hyperresponsiveness or Prothrombotic States

> Causes include:

• Diabetes mellitus
• Hyperadrenocorticism  -  Cushing's disease
• Nephrotic syndrome
• Hormone treatment  -  treatment with erythropoietin or patients with severe responsive anemias may have hyperreactive platelets
• Neoplasia  -  both sarcomas and carcinomas have been implicated in enhanced platelet activity
• Infectious agents

• Feline infectious peritonitis
• Heartworm disease

Inherited Platelet Dysfunction

Inherited platelet functional disorders have been described in many breeds of dogs and in cats.

Von Willebrand Disease (vWd)

> The most common canine hereditary bleeding disorder.

> Three subtype classifications which are dependent on the severity of clinical signs, mode of inheritance, and biochemical abnormalities of von Willebrand factor protein (vWf).

• Type 1 vWd has been observed in many canine breeds and in cats. There is a mild bleeding tendency.
• Type 2 vWd is observed in German shorthaired and wirehaired pointers. There is a moderate bleeding tendency.
• Type 3 vWd is observed in the Scottish Terrier, Chesapeake Bay Retriever, and Shetland Sheepdog. There is a moderate bleeding tendency. Von Willebrand factor is usually 0 percent.

> The pathognomonic feature of vWd is lack of functional of von Willebrand factor (vWf).

• Causes abnormal primary hemostasis

> Clinical signs

• Mucosal hemorrhage, cutaneous bruising, and prolonged bleeding from sites of trauma or surgery.
• Bleeding can be mild (common) to severe (relatively uncommon).
• Bleeding is exacerbated by concurrent thrombocytopenia, disease conditions that impair platelet function, and the use of nonsteroidal anti-inflammatory drugs.

> Many breeds of purebred dogs are affected. Affected mixed breed dogs have been observed.

> Diagnosis: The platelet count, APTT, PT, are normal but the BMBT is prolonged.

• Definitive diagnosis requires specific assay of plasma vWf

• Canine and feline vWf are antigenically and functionally distinct from human vWf.
• Each laboratory should provide reference intervals for each species assayed. Quantitative values less than 50% are considered vWf deficient.
• Structural assays are utilized to subtype vWd.

Canine Thrombasthenic Thrombopathia

> Is an autosomal inherited disorder in Great Pyrenees and Otterhounds

> Clinical hemorrhage is associated with mucosal surfaces and is exacerbated by trauma and stress.

> Bleeding episodes may be associated with vaccination, hypothyroidism, or estrus.

Basset Hound Thrombopathia

> Autosomal inheritance

> Often confused with vWd

> BMBT is prolonged

> Bleeding is usually associated with mucosal or cutaneous surfaces

> Similar defects have been observed in the American Foxhound and in cats.

Spitz Thrombopathia

> A variant of canine thrombasthenic thrombopathia

> Chronic epistaxis, gingival bleeding, and gastrointestinal bleeding

Cocker Spaniel Bleeding Disorders

> Delta storage pool disease associated with moderate bleeding and abnormal platelet aggregation

> Cocker Spaniels have numerous other hereditary hemostatic disorders including factor II (prothombin) deficiency and factor X deficiency

> Both are manifest by bleeding and prolonged APTT and PT.