Diagnostic Tests for Thrombocytopenic Patients
ACVIM 2008
Margi Sirois, EdD, MS, RVT
Scottsdale, AZ, USA

Introduction

Hemostasis involves a number of complex pathways, platelets and coagulation factors. Any alteration in these parameters can result in a bleeding disorder. Primary hemostasis, can be initiated when a blood vessel is ruptured or torn. The exposed blood vessel endothelium is a charged surface and platelets are attracted to this surface. As platelets congregate at the site, they undergo morphologic and physiologic changes. These changes cause the platelets to adhere to each other as well as the blood vessel. This also causes platelets to release the initiating factor for the chemical phase of hemostasis. Secondary hemostasis (the coagulation cascade) involves a number of coagulation factors. The end result of the coagulation cascade is the formation of a mesh of fibrin strands that forms the clot. The final phase, tertiary hemostasis, involves degradation of the fibrin clot.

Laboratory Evaluation

Laboratory evaluation of the thrombocytopenic patient must begin by ruling out sample collection and processing errors. Platelets adhere to charged surfaces such as uncoated glass and this will manifest as a decrease in platelet count. Platelets that are activated during the sample collection and processing procedures will also falsely decrease the platelet count. Common causes of platelet activation during sample collection include prolonged venous stasis and patient excitement. EDTA can trigger platelet activation so samples should be evaluated as soon as possible after collection. Use of automated analyzers for a cell count can also mask abnormalities in both numbers and function of platelets. Lysing reagents used in cell counting do not affect platelet aggregates. The presence of aggregated platelets often results in falsely decreased platelet numbers and falsely increased red or white blood cell counts. Macrothrombocytes in the blood sample can also lead to erroneous results when using automated blood cell counters. A scanning peripheral blood film should be performed to provide an overall indication of platelet size. Some automated analyzers can then be adjusted to count the larger platelets. Since thrombocytopenia can occur in a wide variety of conditions, samples for bacterial culture and immunologic testing may be needed.

Platelet Counting Methods

The preferred method for counting of platelets counts is the manual method performed with the Unopette® 5855 diluting chamber. Always use a freshly collected blood sample to perform manual platelet counts. Platelet counts using automated analyzers with impedance methods are often inaccurate due to platelet clumping and platelet/RBC overlap. Morphologic changes in platelets include aggregation and giant platelets. These abnormalities will not be evident with most automated analyzers and must therefore be detected using the differential blood film. If a platelet histogram is available, this will aid in the overall evaluation. The platelet histogram can identify the presence of macrothrombocytes, aggregated platelets, and alteration in platelet indices. Automated analyzers that utilize flow cytometry, light scatter, or fluorescence methods are more accurate and can usually identify the presence of platelet clumps in the sample. A few analyzers utilize a combination of test principles to obtain hematologic data and some include the ability to perform tests of platelet aggregation and activation.

Platelet Estimates

Examination of the blood film can provide a rough estimate of platelet numbers but should not be used as a primary means of evaluation of adequate platelet numbers in patients suspected of hemostatic problems. Platelet numbers should be evaluated in the monolayer area of the blood film. This is generally done by counting the numbers of platelets in a minimum of ten microscopic fields should be counted. Platelet estimates can be reported as the average number seen in ten microscopic fields or as the range seen in ten fields. The expected number of platelets for normal patients using this method varies with different types of microscopes and can range from three to five in older microscopes to eight to ten with newer microscopes. If platelet clumps are observed, platelets are probably adequate in number.

Another indirect measure of platelet number involves counting the number of platelets seen per 100 white blood cells on the differential blood film. This number is then used to calculate the platelet estimate using the following equation:


 

Platelet Indices

Automated analyzers usually provide additional platelet parameters, referred to as platelet indices. These include mean platelet volume (MPV), platelet distribution width (PDW), and Plateletcrit (PCT). Other platelet indices may also be provided depending on the instrument manufacturer. Different analyzer manufacturers vary in the methods used to obtain these results. Clinical usefulness of platelet indices is under investigation. Research is focusing on utilization of platelet indices in differential diagnosis and as prognostic indicators. Current research indicates that the highest values for the various platelet indices may be most likely in patients with immune-mediated thrombocytopenia.

Mean Platelet Volume (MPV)

Even in healthy dogs and cats, individual platelets can vary markedly in size. Increased MPV might be expected in situations where increased loss, destruction, or consumption of platelets is accompanied by megakaryocytic hyperplasia. Accelerated thrombopoiesis tends to result in the release of larger platelets. A. high MPV in dogs indicates adequate bone marrow response; however a normal or low MPV in thrombocytopenic dogs does not predict an inadequate bone marrow response. Healthy cats generally have variably sized platelets and some may be quite large. Some breeds of dogs, e.g., Cavalier King Charles Spaniels, have larger platelets than other breeds. These may be missed with some automated counters because the platelets fall outside the size threshold the instrument uses for counting platelets.

Platelet Distribution Width (PDW)

This measurement is also referred to as platelet size deviation width, platelet component distribution width (PCDW) and platelet dry mass distribution width (PMDW). Increases in PDW are associated with bone marrow hyperplasia.

Plateletcrit (PCT)

This measurement is a calculated ratio of the platelet volume to the whole blood volume. Its usefulness in diagnosis and prognosis of hemostatic disorders is not well characterized.

Clot Retraction Test

This procedure provided a crude evaluation of platelet number and function and intrinsic and extrinsic pathways. Blood is drawn into plain sterile tube and incubated at 37 degrees. The tube is examined at 60 minutes and re-examine periodically over a 24-hour period. A clot should be evident in 60 minutes, retracted in about 4 hours, and markedly compact at 24 hours.

Bone Marrow Biopsy

A bone marrow biopsy can be used to determine whether thrombocytopenia is accompanied by a concurrent pancytopenia. Mature megakaryocytes are large cells containing 32 to 64 fused nuclei. Shedding of platelets by the bone marrow megakaryocyte is stimulated by thrombopoietin. Thrombopoietin is produced by the liver. Megakaryocytes are not evenly distributed in a bone marrow aspirate. They are often seen in clusters, particularly at the edges of the slide. Megakaryocytes may number as much as 8-10 per low-power field, although 2-3 per low power field is more common. Dysmegakaryopoiesis (Dysthrombopoiesis) is characterized by the presence of megakaryocytes with separate nuclear lobes or dwarf megakaryocytes.

D-Dimer and FDP's

Both of these tests are used to evaluate tertiary hemostasis. D-dimers and fibrin degradation products (or fibrin split products) are formed as a clot is degraded. These tests are therefore useful in identifying the presence of disseminated intravascular coagulation (DIC) and will also provide diagnostic information in cases of liver failure, trauma, and hemangiosarcoma. Patients with DIC show evidence of thrombocytopenia as platelet consumption increases. Agglutination assays are available for evaluation of d-dimers and FDP's. A canine in-house test is available for d-Dimer analysis.

Disorders of Platelet Numbers

Thrombocytopenia is defined as insufficiency of platelets and is the most common disorder of hemostasis observed in dogs and cats. The normal ranges of platelets in healthy animals tend to be relatively stable. Causes of thrombocytopenia include a variety of conditions that decrease platelet production, increase platelet destruction, increase platelet consumption, or alter platelet distribution. The clinician often diagnoses the cause of thrombocytopenia by first ruling-out infectious causes. Platelet underproduction usually occurs with underproduction of other blood cell lines, and is therefore often accompanied by pancytopenia. Thrombocytopenia caused by increased platelet destruction develops when the rate of platelet destruction surpasses the ability of the bone marrow to produce platelets, and can be caused by immune or non-immune mechanisms. Altered platelet distribution usually involves sequestration of platelets in the spleen. Thrombocytopenia can result from congenital or acquired conditions. Whether or not bleeding occurs with thrombocytopenia depends on additional factors such as platelet size and function.

Congenital Thrombocytopenia

Congenital thrombocytopenia can occur when maternal antibodies are produced against fetal platelets. This condition is more common in large animals than in dogs and cats. Some dogs, particularly collies, can be affected by congenital cyclic hematopoietic disorders that depress platelet production. The disorder is autosomal recessive and affected animals usually die by six months of age. Certain dog breeds, such as Cavalier King Charles Spaniels and Greyhounds, have platelet counts lower than the reference interval. In these breeds, a slightly low platelet count is a normal finding and should not be regarded as pathological. In the Cavalier King Charles Spaniels, platelet counts may seem unusually low due to the presence of macrothrombocytes.

Acquired Thrombocytopenia

Generalized septicemia, bacteremia, and a variety of systemic diseases can all cause thrombocytopenia. A summary of some of the infectious causes of thrombocytopenia can be found in Table 1. Immune-mediated destruction of platelets is one of several possible mechanisms by which infectious diseases can cause thrombocytopenia.

Table 1. Potential infectious causes of thrombocytopenia.

Viruses

Herpesvirus

Adenovirus

Paramyxovirus

Retrovirus

Coronavirus

Parvovirus

Bacteria

Salmonella spp.

Rickettsia rickettsii

Haemobartonella spp.

Leptospira spp.

Ehrlichia spp.

Ehrlichia (Anaplasma) platys

Protozoa

Toxoplasma gondii

Leishmania spp.

Cytauxzoon felis

Babesia spp.

Plasmodium

 

Fungi

Histoplasma capsulatum

Candida albicans

 

A transient vaccine-induced thrombocytopenia has been reported in dogs. Clinical signs are usually not evident unless concurrent hemostatic defects are present. The condition is thought to result from production of antibodies to viral antigens attached to platelet surfaces. Thrombocytopenia in animals with bacteremia or endotoxemia is due in part to sequestration of damaged platelets in organs such as liver and spleen. However, animals with these diseases are likely to show evidence of disseminated intravascular coagulation (DIC) as well.

DIC is always a secondary condition. It occurs when intravascular microthrombus formation ties up platelets and depletes clotting factors. This produces thrombocytopenia, prolonged buccal mucosal bleeding time, increased activated clotting time, and increased prothrombin time. Fibrin-degradations products (FDP's) are also increased due to systemic fibrinolysis. Schistocytes are nearly always evident on the peripheral blood film.

The incidence of hemostatic defects related to neoplasia is unknown. It has been reported that as many as 83% of cancer patients without symptoms of hemostatic problems showed evidence of abnormal coagulation results, including thrombocytopenia. One of the most common tumors associated with bleeding tendencies is hemangiosarcoma in dogs. DIC is a common finding in these patients. Abnormal hemostasis may result from the malignancy itself, chemotherapy or metastatic disease. Thrombocytopenia due to myelosuppression is a frequent complication of chemotherapy. Immune-mediated destruction of platelets and increased consumption in DIC are also common in animals with neoplastic disease.

Immune-mediated thrombocytopenia (ITP) can be a primary clinical syndrome or be secondary to infectious, neoplastic, or systemic immune system disorders. Tests for detecting antiplatelet antibodies in animals are not readily available. Immunofluorescent testing for presence of IgG on megakaryocytes provides supportive evidence of antiplatelet activity. ITP is suspected when thrombocytopenia is present, infectious causes have been ruled out, and other coagulation test results are normal. Favorable response to immunosuppressive therapy confirms the diagnosis. In severe cases, splenectomy may be indicated since the spleen is a major site of platelet destruction.

Thrombocytopenia also occurs in a variety of organ diseases. Patients with liver disease may develop DIC or platelets may be sequestered in the liver. Renal disease is often characterized by altered platelet function which can result in platelet over-consumption should bleeding occur.

Drugs that have been implicated in primary hemostatic defects normally do not cause reduction in platelet numbers but may alter platelet function. Medications that have been implicated in primary hemostatic disorders include acetaminophen, aspirin, diazepam, estrogens, ibuprofen, phenylbutazone, prednisolone, prostaglandins, thiazide diuretics, trimethoprim sulfa, and vinblastine/vincristine.

Summary

A variety of clinical presentations may be seen with thrombocytopenic patients. A thorough understanding of the varied role of platelets in homeostasis is critical to assessing validity of test results. When advanced automated analyzers are not available, manual platelets counts are preferred in evaluating thrombocytopenic patients. Automated analyzers may provide valuable information in the form of a platelet histogram and evaluation of platelet indices. Patients with thrombocytopenia may have no evidence of clinical bleeding. A variety of infectious diseases can cause thrombocytopenia. Some diagnostic procedures may be based on ruling out such causes or by response to therapy.

References

1.  Hoffman, et al. Hematology, 4th ed. Elsevier, 2005;

2.  Sirois M. Hematology and Hemostasis in Principles and Practice of Veterinary Technology, 2nd ed. Mosby, 2004;

3.  Anthony E, Sirois M. Hematolgy and Hemostasis in Laboratory Procedures for Veterinary Technicians, 2nd ed. Elsevier 2006;

4.  Thrall M. Veterinary Hematology and Clinical Chemistry. Lippincott, 2003

Speaker Information
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Margi Sirois, EdD, MS, RVT
Port Richey, FL


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