Symposium on Clinical Laboratory Medicine
The Diagnosis of Hemostatic Disorders Gary J. Kociba, D.V.M.*
The wide variations in etiology, type, and severity of bleeding disorders necessitate a systematic approach to diagnosis. Data obtained from the history, physical examination, and results of selected laboratory tests are vital for evaluation of hemostatic defects. The emphasis in this paper is on a simple, organized approach to bleeding disorders rather than on a detailed discussion of the various defects and the laboratory tests used for diagnosis. A brief summary of the major components of the hemostatic mechanism is followed by a description of the stepwise approach to the diagnosis of bleeding problems.
THE HEMOSTATIC MECHANISM The hemostatic mechanism can be divided into three components to facilitate clinical and laboratory evaluation: blood vessels, platelets, and coagulation mechanism. Normal blood vessels and adjacent connective tissues are essential for protection from blood loss and the arrest of bleeding when injuries occur. Vasoconstriction and diversion of blood flow serve to decrease the blood loss from injured vessels. The platelets play a vital role in the arrest of bleeding from an injured blood vessel by adhering to subendothelial collagen, releasing platelet constituents, and aggregating to form the platelet plug which temporarily fills the vessel defect. The primary role of the coagulation mechanism is the formation of a fibrin network around a platelet plug, thereby anchoring it in position as a stable hemostatic plug. Schemes used to depict the series of reactions of the coagulation mechanism are frequently divided into intrinsic and extrinsic mechanisms (Fig. 1). The intrinsic mechanism is activated by exposure of factor XII to subendothelial collagen or other abnormal *Associate Professor, Ohio State University; Clinical Pathologist, The Ohio State University Veterinary Teaching Hospital, Columbus, Ohio
Veterinary Clinics of North America-Yo!. 6, No.4, November 1976
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610 EXTRINSIC TISSUE THROMBO PLASTIN
XI IX
VII
V Ill
OSPT
PTT APTT ACT COAGULATION TIME
\
PHOSPHOLIPID PROTHROMBIN
RVVT
Figure 1. Simplified scheme of the coagulation mechanism. The parts of the coagulation mechanism evaluated in the various screening tests are indicated.
FIBRINOGEN
Fl BRI N
surfaces. A series of sequential enzyme activations then follows with the eventual conversion of fibrinogen into a network of fibrin strands (clot). The extrinsic mechanism begins with exposure of clotting proteins to tissue thromboplastin which is present in tissue juices. Tissue thromboplastin acts on clotting proteins, thereby contributing to the rapid formation of the fibrin clot. Both the intrinsic and extrinsic mechanisms share the terminal portion of the sequence of clotting enzyme reactions. The dissolution of fibrin clots is accomplished by fibrinolysis. Activation of the fibrinolytic mechanism is effected by conversion of plasminogen, a plasma precursor, to plasmin. Plasmin is a widely active proteolytic enzyme which digests fibrin and a variety of other proteins into fragments. The digestion products of fibrin are referred to as fibrin split products, fibrin degradation products, or fibrinolytic split products.
APPROACH TO THE BLEEDING PATIENT The following outline is recommended for use in the evaluation of patients with suspected hemostatic defects. Each of the components of this approach to the bleeding patient will be discussed in more detail. 1. History 2. Physical examination 3. Collect samples for screening tests a. Whole blood-citrate anticoagulant (1) extrinsic mechanism-one stage prothrombin time (OSPT) (2) intrinsic mechanism-activated partial thromboplastin time (APTT) b. Whole blood-EDT A anticoagulant (1) platelets
DIAGNOSIS OF HEMOSTATIC DISORDERS
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4. Classify defect a. platelet or vessel defect-petechiae, thrombocytopenia, superficial hemorrhage, prolonged bleeding time b. coagulation defect-abnormal OSPT or APTT; deep hemorrhage c. multiple defects 5. Perform specific tests or therapeutic trial
History
Objectives. (1) Determine if hemostatic defect is present. (2) Describe hemorrhages. (3) Describe possible episodes of spontaneous hemorrhage. (4) Describe environmental factors. (5) Describe any previous treatments. (6) Investigate possibility of bleeding in siblings or ancestors. The history is extremely important for diagnosis of bleeding disorders. The first objective should be to determine if the animal has a hemostatic defect. In obtaining a description of all previous hemorrhages, one should determine the time, location, size, and duration of the lesions. If the owner is aware of the cause of the bleeding, a judgment is necessary to decide if the hemorrhage is compatible with and proportionate to the injury. Evidence for earlier hemorrhage or an associated disease may be pursued with questions regarding the animal's condition and behavior prior to any observed hemorrhages. Specific inquiry should be made for evidence of spontaneous hemorrhages including color of the urine and stool, petechiae on the skin or mucous membranes, unexplained lamenesses which could be due to hemarthroses or intramuscular hemorrhages, or periods of weakness and inactivity. Evidence for a longstanding hemostatic defect can be gained by questions regarding elective surgical procedures. It should be noted, however, that some dogs with congenital coagulation defects have survived ear cropping or tail docking procedures with no apparent bleeding problems. If the animal has experienced any lacerations or severe trauma the degree and duration of bleeding should be determined. Environmental factors which should be investigated include the animal's housing, area of confinement, and possibilities for exposure to warfarin or other chemicals and drugs. Any previous treatments, especially blood transfusions, should be recorded along with comments regarding their effectiveness in controlling the bleeding. If a congenital bleeding defect is suspected, the medical history of the littermates is frequently helpful. Since some defects (factor VIII deficiency and factor IX deficiency) are sex-linked recessive characteristics, the sex of possibly affected littermates provides a helpful clue to the diagnosis. Physical Examination The necessity for a thorough physical examination is not only to characterize the hemorrhag~ but also to evaluate the patient for evi-
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dence of other disease processes. Recognition of abnormalities, such as icterus, fluid in body cavities, and the type and location of hemorrhages, will aid in the classification of bleeding defects. Petechial hemorrhages are characteristic for hemostatic defects involving either platelets or blood vessels. Patients with defects of the plasma coagulation mechanism are more likely to suffer large, deep hemorrhages into the body cavities, subcutaneous tissues, or large muscle masses. Gastrointestinal bleeding with dark, tarry stools is a very common cause of blood loss in thrombocytopenic dogs, but is not specific since it is also seen in association with some blood coagulation defects. It is difficult to classify the causes of ecchymoses and large bruises in the skin because they may be associated with defects of any of the components of the hemostatic mechanism. In animals with light skin, evidence for resorbing hemorrhage is provided by the observation of yellow, orange, or brownish areas of discoloration of the skin. Screening Tests
Screening tests are important for the differential classification of hemostatic defects. These tests are most effectively used in combinations. These tests ideally would be selected to evaluate the blood vessels, platelets, and coagulation mechanism. The routine tests screen for defects involving the extrinsic coagulation mechanism, the intrinsic coagulation mechanism, and the platelets. Vascular defects are more difficult to evaluate. Tests for blood vessel functions are therefore not recommended as routine screening procedures. The minimum screening tests should include: 1. One extrinsic mechanism test a. one stage prothrombin time 2. One intrinsic mechanism test a. partial thromboplastin time b. activated partial thromboplastin time c. activated coagulation test d. Lee-White clotting time 3. Platelet count
For the classification of the hemostatic defects, the author has selected the OSPT, APTT, and platelet count. A brief discussion of the principles of these tests and related screening tests is provided. Sample Collection and Preparation. Samples should be collected from the patient prior to the administration of any treatment whenever possible. Careful collection techniques are vital for hemostatic tests. The introduction of small amounts of tissue juices during problem venipunctures can lead to erroneous coagulation results and platelet clumping in a sample. Samples should be collected from a large vein with a plastic syringe. If the venipuncture is not achieved on the first attempt a new syringe and needle should be used for the subsequent attempt. A
DIAGNOSIS OF HEMOSTATIC DISORDERS
613
desirable alternative procedure is to use one syringe for positioning the needle in the vein and aspirating a small amount of blood prior to substituting a second syringe for sample collection. If vacuum tubes are used for sample collection, a small amount of blood should be allowed to flow through the needle prior to attachment of the tube with anticoagulant. Blood for platelet counts should be added to EDT A, because this anticoagulant is a good inhibitor of platelet aggregation, thereby preventing platelet clumping. Platelet counts should be performed within four hours after sample collection. For coagulation studies, nine volumes of blood are mixed with one volume of 3.8 per cent (0.13 m) sodium citrate in a plastic or siliconized glass tube. The plasma should be separated by high speed centrifugation (3000 rpm for 15 minutes) directly after sample collection and used for coagulation tests, or frozen at -20°C. for testing later. Since marked variations in normal clotting test values may occur in different laboratories, and occasionally in the same laboratory, it is extremely important that blood samples be collected from at least one normal animal of the same species and submitted along with the patient samples. Clotting test results for the patient should always be compared with those of the normal control animals. Blood Vessels. Bleeding time and biopsy. Defects of blood vessels are difficult to evaluate. The majority of these defects are associated with other systemic disease processes. An understanding of the pathogenesis of the primary disease process is helpful for explaining the vessel lesions. Although the bleeding time is easy to perform, it is difficult to obtain consistent results from this test in most animals. Variations in the size, number, and location of injured vessels will affect the results. This test is not recommended as a routine screening test. Prolonged oozing hemorrhage from venipuncture sites may be an indication of a long bleeding time. Biopsy of affected tissues should be restricted to special instances where unusual vascular lesions are suspected. Platelets. Platelet count, clot retraction, platelet retention, platelet aggregation, and bleeding time. Platelets may be evaluated in a variety of tests. For routine screening of patients with hemostatic defects, only the platelet count and platelet evaluation on a blood smear are recommended. Platelet defects are more extensively discussed under "Defects Involving Blood Vessels or Platelets." Extrinsic Clotting Mechanism. One stage prothrombin time (OSPT) and Russell's viper venom test (RVVT). The one stage prothrombin time is performed by measuring the time to clot formation in a plasma sample after tissue thromboplastin and calcium are added. This test is used routinely for evaluation of the extrinsic mechanism (Fig. 1). An extrinsic mechanism defect should be' suspected if the OSPT of the patient is prolonged longer than 5 seconds compared with the control.
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The Russell's viper venom test (Stypven test) is performed by adding snake venom to a plasma sample and timing to clot formation. This extrinsic mechanism screening test involves the same clotting factors as the OSPT except for factor VII (Fig. 1). Intrinsic Clotting Mechanism. Partial thromboplastin time (PTT), activated partial thromboplastin time (APTT), Lee-White clotting time, and activated coagulation time. The partial thromboplastin time and the activated partial thromboplastin time are widely available and reliable tests for evaluating the intrinsic clotting mechanism (Fig. 1). In these tests, platelet poor plasma is incubated with a phospholipid (partial thromboplastin) which substitutes for the phospholipid that is derived in vivo from platelets. The sample is recalcified and timed to clot formation. The APTT is a modification of the PTT, in which a surface activator is incorporated to ensure maximal factor XII activation (in the PTT factor XII is activated only by exposure to the glass wall of the test tube). The clotting times in the APTT are therefore usually shorter than those of the PTT, but either test is effective in screening for intrinsic mechanism defects. In our laboratory the APTT is considered to be abnormal when it is 7 seconds longer than the control in small animals and 15 seconds longer than the control in cattle and horses. Smaller changes may be significant in some of the less severe defects but are more difficult to interpret. The Lee-White clotting test is performed by measuring the time required for clotting of a whole blood sample under controlled conditions in glass tubes. The test is affected by variations in temperature which must be minimized. The test screens for defects in the intrinsic mechanism (Fig. 1), but is not as sensitive or precise as the PTT or APTT. Severe thrombocytopenia can lead to a mildly prolonged LeeWhite clotting time since platelet phospholipid is needed for the intrinsic mechanism. The test, however, is not sensitive for most platelet deficiencies, and a normal Lee-White clotting time does not eliminate the possibility of thrombocytopenia. The activated coagulation time (ACT)* is a relatively new screening test for intrinsic mechanism defects. In this test a blood sample is collected into a vacuumized glass tube containing a surface activator substance to rapidly activate factor XII in the sample. The tube is incubated at a constant temperature in a heat block or water bath and timed to clot formation. This test combines the advantages of using whole blood rather than plasma with the contact activator to improve the precision of the procedure. Preliminary work in our laboratory suggests that this test is superior to the Lee-White coagulation time and is easier to perform than the PTT or APTT. This test may be very useful for routinely *Activated Coagulation Time, Vacutainer 3206XF534, Becton, Dickenson and Company, Rutherford, New Jersey.
DIAGNOSIS OF HEMOSTATIC DISORDERS
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screening for the more severe intrinsic mechanism defects in domestic animals. Fibrinolytic Split Products. Latex agglutination test. Increased levels of circulating fibrinolytic split products are usually evidence of enhanced fibrinolysis. The Thrombo-Wellcotest* is a latex agglutination test that is simple and reliable. In this test latex particles are used which are coated with antibody directed toward human fibrinogen degradation products (FDP; fragments from the proteolytic digestion of fibrinogen by plasmin). The antibody appears to cross react with FDP's of most domestic animals and can be used to screen for enhanced fibrinolysis. Because the affinity of the antibody for FDP's of different species probably varies, the results should be reported as positive or negative at various dilutions and not in ~-tg/ml. Fibrinolytic split products are present in only very low concentration in the blood of normal animals. In our laboratory a positive Thrombo-Wellcotest at a serum dilution of 1 /zs or greater is evidence of enhanced fibrinolysis in the dog, horse, and cat. Samples for fibrinolytic split products assays must be preserved with a proteolytic enzyme inhibitor to block in vitro fibrinolysis which would lead to increased levels of fibrinolytic split products. Classification of Hemostatic Defects Using the data from the history, physical examination, and screening tests one should attempt to classify the defect. The following outline is provided as a guideline. The identification of any of the abnormalities listed warrants tentative assignment of the defect to that category and further evaluation. A. Vessel or Platelet Defect 1. Physical findings a. petechial hemorrhages b. superficial hemorrhages (purpura) 2. Laboratory findings a. thrombocytopenia b. prolonged bleeding time B. Coagulation Defect 1. Physical findings a. large, deep hemorrhages disproportionate to injury (1) subcutaneous (2) intramuscular (3) intraarticular (4) body cavities 2. Laboratory findings a. prolonged OSPT b. prolonged APTT C. Combined Defects of Vessels, Platelets, and Coagulation
*Thrombo-Wellcotest, Burroughs-Wellcome Company, Wellcome Research Division, Research Triangle Park, North Carolina.
GARY J. KocmA
616 PIATELET COUNT
~ NORMAL PLATELET NUMBER
THROMBOCYTOPENIA
~
~
APTT
PLATELET FUNCTION TESTS
/~ PROLONGED
NORMAL
i
~
l
BONE MARROW
DIG
/
~
BONE MARROW HYPOPLASIA MYROPROLIFERATIVE DISORDERS
Figure 2. blood vessels.
i
VASCULAR DEFECTS
~
THROMBOCYTOPATHY DRUGS DYSPROTEINEMIA
vWD
UREMIA FIBRINOLYSIS
MEGAKARYOCYTE$
DECREASED
/~ ABNORMAL
NORMAL
~ INCREASED ~
ITP INFECTIOUS DISEASES MYELOPROLIFERATIVE DISORDERS
Flow chart for the evaluation of patients with defects involving platelets or
After classification of a hemostatic defect in one of the above groups the appropriate steps toward definitive diagnosis can be selected. The schemes for the evaluation of defects involving blood vessels or platelets and the coagulation defects are designed to quickly narrow the differential diagnoses to a few diseases on the basis of the history, physical examination, and a few laboratory tests. With this information it is usually possible to decide on probable prognosis and treatment, select special tests, and decide if referral to a specialized center is appropriate.
DEFECTS INVOLVING BLOOD VESSELS OR PLATELETS These defects are usually recognized by the observation of characteristic superficial hemorrhages in the skin or mucous membranes. The hemorrhages are frequently noted as purple discolorations which may be referred to as purpura. The hemorrhages usually are petechiae or ecchymoses. Petechiae are characteristic of hemostatic defects involving platelets or blood vessels. The laboratory tests that are helpful in evaluating patients with petechial hemorrhages include hemogram, one stage prothrombin test (OSPT), activated partial thromboplastin time (APTT), platelet count, bone marrow examination, and platelet function tests (optional). Figure 2 is a flow chart that incorporates the results of the laboratory tests in the classification of purpuric diseases. Using the stepwise approach that is outlined one can quickly classify the defect. After clas-
DIAGNOSIS OF HEMOSTATIC DISORDERS
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sification of the defect, decisions are made relative to specific diagnosis, prognosis, and treatment. Several clues may be gained from the hemogram and examination of the peripheral blood smear. Platelet numbers can be estimated by determining the number per field. If less than three platelets are observed per oil immersion field the possibility of thrombocytopenia should be considered. When estimating platelet numbers from a blood smear it is important to check for irregularities in platelet distribution due to clumping of platelets which is most obvious near the distal (feathered edge) end of the smear. A direct platelet count on a carefully collected, fresh, EDT Aanticoagulated sample is the best way to determine platelet number. Even with this technique a blood smear should be examined to corroborate the results of the platelet count. If a thrombocytopenia is recognized it may be due to either decreased production or increased destruction of platelets. Concurrent nonregenerative anemia and leukopenia would suggest that bone marrow hypoplasia may be the cause of the thrombocytopenia. Large platelets (macroplatelets) are evidence for diseases with increased platelet turnover rate. These platelets (up to 10 JLm diameter) are produced and released during periods of rapid platelet production. Syndromes of increased platelet consumption are usually associated with immunologic thrombocytopenic purpura (ITP), disseminated intravascular coagulation (DIC), or infectious diseases. The APTT is used to screen for the coagulation defects that are associated with DIC since marked prolongation of the APTT is usually evident in animals with clinically significant thrombocytopenia related to DIC. If the APTT is normal in a thrombocytopenic patient, bone marrow aspiration biopsy is indicated. A bone marrow sample can be aspirated from the iliac crest of a dog using a local anesthetic. Abnormal bleeding from the biopsy site is generally not a significant problem even in severe thrombocytopenias. Bone marrow smears can be stained with either new methylene blue stain or Wright's stain and examined for the number of megakaryocytes. If few or no megakaryocytes are found, syndromes of bone marrow hypoplasia, such as estrogen toxicity in the dog, must be considered. If normal or increased number of megakaryocytes are seen in a dog without evidence of other disease processes, a tentative diagnosis of ITP is suggested and corticosteroid therapy can be initiated. It should be noted that ITP is usually diagnosed by evidence of increased platelet turnover rate and exclusion of other diseases. Tests for anti-platelet antibody, such as platelet factor III release test, are available from some specialized laboratories and could be used to substantiate a diagnosis of ITP. Direct effects of drugs or drug-induced, immune-mediated thrombocytopenia are distinct possibilities if the thrombocytopenia developed during drug therapy. The suspected drug must be incorporated into the test system if
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tests for demonstration of anti-platelet antibodies induced by drug exposure are attempted. It should be kept in mind that some infectious diseases can lead to enhanced platelet destruction or megakaryocyte damage. A transient thrombocytopenia and/or platelet function defect has been reported in dogs following vaccination with either measles or some canine distemper vaccines. The possibility of myeloproliferative diseases causing a defect in platelet production must also be considered. This may be recognized when the bone marrow smears are examined. Platelet function defects should be considered in purpuric animals with normal or increased numbers of platelets. Tests for these defects include the simple clot retraction test, glass bead column platelet retention test, and platelet aggregation tests. The more complicated platelet function tests are most effectively handled by specialized laboratories experienced with them. Acquired platelet function defects are more common than congenital defects and may be related to disease processes such as uremia, dysproteinemia, or exposure to drugs. A wide variety of drugs, including aspirin and many of the phenothiazine tranquilizers, cause platelet function defects. Hereditary platelet function defects have been described in dogs, especially otterhounds and basset hounds, and fawn hooded rats. Vascular defects are less common causes of purpuric lesions. Scurvy in guinea pigs or primates is accompanied by increased vascular fragility and defective collagen synthesis. Some infectious diseases and some immune-mediated diseases are characterized by vasculitis and purpuric lesions. Dogs with hyperadrenocorticism rarely have petechial or ecchymotic hemorrhages. These hemorrhages are probably related to abnormal collagen metabolism. COAGULATION DEFECTS The hemostatic defects involving the blood coagulation mechanism can usually be subdivided using the results of three screening tests as indicated in Table 1. After classification of the defect in one of the four major groups one can utilize the following information to proceed to a specific diagnosis. Defects in the Early Stages of the Intrinsic Clotting Mechanism
Screening Test Results.
OSPT (normal), APTT (prolonged), and
platelets (normal).
Differential Diagnoses. Factors VIII, IX, XI, and XII deficiencies, and von Willebrand's disease. Factor VIII deficiency (classic hemophilia) and factor IX deficiency are both transmitted as sex-linked recessive defects. The bleeding tendencies are therefore limited to males except for rare instances involving intensive inbreeding. Factor VIII deficiency has been recognized in most breeds of dogs and mongrels, cats, and horses. Factor
619
DIAGNOSIS OF HEMOSTATIC DISORDERS
of Coagulation Defects According to Screening Test Results
Table 1. Classification SCREENING TEST
COAGULATION DEFECTS
OSPT*
APTT*
Platelets
Normal
Prolonged
Normal
Factor VIII deficiency Factor IX deficiency Factor XI deficiency Factor XII deficiency von Willebrand's disease
Prolonged
Normal
Normal
Factor VII deficiency
Prolonged
Prolonged
Normal
Fibrinogen deficiency Factor X deficiency Liver disease Warfarin poisoning Vitamin K deficiency
Prolonged
Prolonged
Decreased
DIC
*OSPT, one stage prothrombin time; APTT, activated partial thromboplastin.
IX deficiency is a much less common defect than classic hemophilia and has been recognized only in Cairn terriers, black and tan coonhounds, and St. Bernards. Diagnosis of these defects is made with specific factor VIII and factor IX assays. Animals that are bleeding as a result of factor VIII deficiency usually have less than 20 per cent of normal factor VIII activitity, with severely deficient patients having less than 1 per cent activity. Dogs that have been detected with factor IX deficiency have had defects with less than 1 per cent of normal factor IX activity. Female carriers for either factor VIII or factor IX deficiency can usually be identified in the laboratory using specific assays, since these animals usually have 40 to 60 per cent of normal factor VIII or factor IX activity, respectively. Factor XI deficiency has been recognized in Holstein cattle and springer spaniel dogs. The bleeding defect is usually less severe than that of factor VIII or factor IX deficiency. Spontaneous hemorrhages have been detected in dogs with factor XI deficiency, but the bleeding defect is usually recognized as abnormal bleeding which starts within 24 hours after surgery. The disease is transmitted as an autosomal characteristic with the homozygotes having a bleeding tendency and with less than 15 per cent factor XI activity, whereas the clinically normal heterozygotes have 25 to 70 per cent normal factor XI activity. Factor XII deficiency has not yet been reported in domestic animals. In man the defect is not associated with a clinical bleeding defect. Therefore, if an animal has a bleeding problem, the chances that a factor XII
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deficiency is causing the defect are remote. Factor XII assays are available for specific diagnosis of factor XII deficiency. Marine mammals and birds do not have detectable factor XII activity. Von Willebrand's disease has been recognized in pigs and appears to be one of the more common congenital hemostatic defects in dogs. The disease ranges from that of a moderately severe bleeding defect to a mild defect in different animals. The majority of these animals have a mild clinical bleeding tendency which is recognized following surgical procedures. The hemorrhages may be severe and life-threatening. The results of the APTT in von Willebrand's disease usually range from normal to moderately prolonged. Unlike the congenital deficiencies of most other coagulation factors, the bleeding time may be prolonged in von Willebrand's disease. This is related to a defect in platelet function. The disease has an associated mild to moderate ( 10 to 60 per cent) deficiency of factor VIII activity. A unique feature of this disease that is sometimes used for differential diagnosis is the paradoxical increase in factor VIII activity that peaks about 24 hours after transfusion with either factor VIII deficient or normal plasma. A recent finding that is useful for the diagnosis of von Wille brand's disease is that patients with the disease have low levels of factor VIII related antigen that correlate with low factor VIII activity in clotting tests. In contrast, dogs with classic hemophilia have normal or increased quantities of factor VIII related antigen (apparently nonfunctional molecules) with low factor VIII clotting factor activity. Von Willebrand's disease is transmitted as an autosomal characteristic and therefore is seen in both males and females. Defects Restricted to the Extrinsic Clotting Mechanism
Screening Test Results. OSPT (prolonged), APTT (normal), and platelets (normal). Disease. Factor VII deficiency. Factor VII deficiency is a defect which is usually discovered incidentally in beagle dogs being screened with OS PT. One malemute with factor VII deficiency has been reported. The autosomal defect causes only a very mild clinical bleeding tendency with easy bruising and possible increased bleeding after surgery. Since factor VII is not a part of the intrinsic clotting mechanism, the APTT and whole blood clotting tests will be normal. Specific diagnosis is most effectively accomplished with a factor VII assay. Since the Stypven test bypasses factor VII of the extrinsic clotting mechanism, the finding of a normal Stypven test in a dog with a prolonged OSPT should be conclusive for factor VII deficiency. Defects Involving Both Intrinsic and Extrinsic Clotting Mechanisms; Normal Platelets
Screening Test Results. platelets (normal).
OSPT (prolonged), APTT (prolonged),
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Differential Diagnoses. Warfarin toxicity, liver disease, vitamin K deficiency, factor X deficiency, fibrinogen deficiency, and anticoagulants. Warfarin poisoning is the most common coagulation defect in this group of diseases. Nevertheless, the disease is incriminated in animals more often than it actually occurs. The diagnosis of warfarin poisoning in an untreated animal with a normal one stage prothrombin time is unwarranted. The majority of bleeding episodes in dogs with warfarin poisoning are massive hemorrhages into the body cavities, gastrointestinal tract, musculature, or subcutaneous tissues. Petechial hemorrhages are not common and the platelet count is normal or on the low side of the normal range. The administration of natural vitamin K quickly corrects the defect with shortening of the OSPT detectable six hours after injection. If specific clotting factor activities are assayed, low activity is detected for factors II (prothrombin), VII, IX, and X. The diagnosis is based on the history, compatible laboratory findings, and rapid response to vitamin K therapy. Since the majority of clotting factors are synthesized in the liver, diseases which cause liver dysfunction can lead to clotting defects owing to decreased synthesis of certain clotting factors. The severity of the defect is generally proportionate to the liver disease. Clinical hemostatic defects are seen only in animals with very severe liver diseases. Animals with coagulation defects due to liver disease should have decreased ability to excrete BSP. Vitamin K deficiency is rarely the result of a dietary deficiency. Since vitamin K is a fat soluble vitamin, defects in fat absorption related to defects such as bile salt deficiency can lead to vitamin K deficiency. Other possible causes include gut "sterilization" with long-term antibiotic therapy, germ free animals, and some specific pathogen free animals especially if coprophagy is not permitted. The defect of vitamin K deficiency mimics that of warfarin poisoning . . Factor X deficiency has been recognized as an inherited defect in cocker spaniel dogs. The disease is transmitted as an autosomal trait. The homozygotes for factor X deficiency usually die in the early neonatal period or are stillborn with hemorrhage from the umbilicus or into the body cavities. In adults the defect is relatively mild since the dogs are usually heterozygotes with 18 to 65 per cent of normal factor X activity. Hemorrhage in adult dogs with factor X deficiency usually occurs post-surgically. The results of screening tests may be normal in the heterozygotes for factor X deficiency. Specific diagnosis is made with factor X assays. Fibrinogen deficiency is usually an acquired defect related to disseminated intravascular coagulation or decreased synthesis related to liver disease. Congenital deficiency of fibrinogen has been recognized in dogs and Saanen goats, but is rare. Hemorrhages include hemarthroses, umbilical bleeding, subcutaneous bleeding, and bleeding from mucous
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membranes. Fibrinogen deficiency is an autosomal trait with heterozygotes having about 50 per cent of normal fibrinogen levels. Fibrinogen assays are readily available to facilitate diagnosis. Exposure to some anticoagulants, especially heparin, may lead to erroneous low results in some fibrinogen assays. This possibility can be evaluated by anticoagulant screenings tests or by observing for the normal precipitation of fibrinogen which occurs when plasma is heated to 58°C. Circulating anticoagulants are not commonly encountered in domestic animals. When they are detected in a sample, heparinization related to use of concentrated heparin solutions to maintain patency of an indwelling catheter, or collection of sample through a heparinized catheter should be eliminated first. Circulating anticoagulants can be demonstrated in some patients with disseminated intravascular coagulation. The author has detected high levels of circulating heparin-like activity in one cat with mastocytosis. Defects Involving the Coagulation Mechanism and Platelets
Screening Test Results. OSPT (normal---+prolonged), APTT (normal---+prolonged), platelets (normal--'>decreased). Differential Diagnoses. Disseminated intravascular coagulation (DIC) and dysproteinemias. Disseminated intravascular coagulation is an acquired syndrome that is characterized by activation of both the hemostatic mechanism and fibrinolytic mechanism. This syndrome is always secondary to another disease process which can be neoplastic, infectious, or metabolic. DIC is usually induced by diseases with a combination of a number of the following changes: exposure of blood to abnormal surfaces (activates intrinsic mechanism), release of tissue thromboplastin from damaged tissue or neoplasms, hypotension, stasis of blood flow, vasculitis, and endotoxin exposure. DIC may range in severity from a mild disease to a severe lifethreatening syndrome with severe bleeding and organ dysfunction related to capillary obstruction by fibrin thrombi. Thrombocytopenia is usually present in the more acute forms of DIC since platelets are consumed in the overactive hemostatic process. Platelets that are present are usually large reflecting rapid platelet turnover. The OSPT and even more constantly the APTT are markedly prolonged due to consumption of clotting factors in the coagulation process and/or anticoagulant effects of fibrinolytic split products. Fibrinolytic split products are present in increased concentration in the bloodstream reflecting active fibrinolysis. Animals with DIC frequently have petechial hemorrhages and prolonged bleeding times, which often is suggested by prolonged oozing of blood from venipuncture sites. The bleeding defects are related to thrombocytopenia and platelet function defects induced by exposure of platelets to fibrinolytic split products. Since DIC is a complex syndrome, the changes that are associated with the process are not always spectacu-
DIAGNOSIS OF HEMOSTATIC DISORDERS
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lar nor are all present simultaneously. The changes that are helpful for the diagnosis of DIC include: (1) OSPT-prolonged, (2) APTTprolonged, (3) thrombocytopenia, (4) fibrinolytic split productsincreased, (5) petechial hemorrhages, and (6) abnormal bleeding from venipunctures. Dysproteinemias are rare diseases and hence are seldom recognized as causes of hemostatic defects. The abnormal quantities of protein that are produced in animals with plasma cell myeloma or macroglobulinemia may cause hemostatic defects. The defects apparently are related to interference with some coagulation enzyme reactions and platelet function defects associated with the paraproteins. Since plasma hyperviscosity and stasis of blood flow may occur in macroglobulinemia, some of the bleeding defects may be related to changes associated with DIC. REFERENCES 1. Biggs, R. (ed.): Human Blood Coagulation, Haemostasis, and Thrombosis. Philadelphia, F. A. Davis Co., 1972. 2. Bowie, E.]. W., Thompson,]. H., Didisheim, P., and Owen, C. A.: Laboratory Manual of Hemostasis. Philadelphia, W. B. Saunders Co., 1971. 3. Dodds, W. ].: Bleeding Disorders. In Ettinger, S. (ed.): Textbook of Veterinary Internal Medicine, Vol. 2. Philadelphia, W. B. Saunders Co., 1975. 4. Dodds, W. ]., and Kaneko, J. ].: Hemostasis and Blood Coagulation. In Kaneko, J.J., and Cornelius, C. E. (eds.): Clinical Biochemistry of Domestic Animals, Vol 2. New York, Academic Press, 1971. 5. Rowsell, H. C.: Blood Coagulation and Hemorrhagic Disorders. In Medway, W., Prier, ]., and Wilkinson,]. (eds.): Textbook of Veterinary Clinical Pathology. Baltimore, The Williams and Wilkins Co., 1969. College of Veterinary Medicine Ohio State University 1935 Coffey Road Columbus, Ohio 43210
The Diagnosis of Hemostatic Disorders Gary J. Kociba, D.V.M.*
The wide variations in etiology, type, and severity of bleeding disorders necessitate a systematic approach to diagnosis. Data obtained from the history, physical examination, and results of selected laboratory tests are vital for evaluation of hemostatic defects. The emphasis in this paper is on a simple, organized approach to bleeding disorders rather than on a detailed discussion of the various defects and the laboratory tests used for diagnosis. A brief summary of the major components of the hemostatic mechanism is followed by a description of the stepwise approach to the diagnosis of bleeding problems.
THE HEMOSTATIC MECHANISM The hemostatic mechanism can be divided into three components to facilitate clinical and laboratory evaluation: blood vessels, platelets, and coagulation mechanism. Normal blood vessels and adjacent connective tissues are essential for protection from blood loss and the arrest of bleeding when injuries occur. Vasoconstriction and diversion of blood flow serve to decrease the blood loss from injured vessels. The platelets play a vital role in the arrest of bleeding from an injured blood vessel by adhering to subendothelial collagen, releasing platelet constituents, and aggregating to form the platelet plug which temporarily fills the vessel defect. The primary role of the coagulation mechanism is the formation of a fibrin network around a platelet plug, thereby anchoring it in position as a stable hemostatic plug. Schemes used to depict the series of reactions of the coagulation mechanism are frequently divided into intrinsic and extrinsic mechanisms (Fig. 1). The intrinsic mechanism is activated by exposure of factor XII to subendothelial collagen or other abnormal *Associate Professor, Ohio State University; Clinical Pathologist, The Ohio State University Veterinary Teaching Hospital, Columbus, Ohio
Veterinary Clinics of North America-Yo!. 6, No.4, November 1976
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610 EXTRINSIC TISSUE THROMBO PLASTIN
XI IX
VII
V Ill
OSPT
PTT APTT ACT COAGULATION TIME
\
PHOSPHOLIPID PROTHROMBIN
RVVT
Figure 1. Simplified scheme of the coagulation mechanism. The parts of the coagulation mechanism evaluated in the various screening tests are indicated.
FIBRINOGEN
Fl BRI N
surfaces. A series of sequential enzyme activations then follows with the eventual conversion of fibrinogen into a network of fibrin strands (clot). The extrinsic mechanism begins with exposure of clotting proteins to tissue thromboplastin which is present in tissue juices. Tissue thromboplastin acts on clotting proteins, thereby contributing to the rapid formation of the fibrin clot. Both the intrinsic and extrinsic mechanisms share the terminal portion of the sequence of clotting enzyme reactions. The dissolution of fibrin clots is accomplished by fibrinolysis. Activation of the fibrinolytic mechanism is effected by conversion of plasminogen, a plasma precursor, to plasmin. Plasmin is a widely active proteolytic enzyme which digests fibrin and a variety of other proteins into fragments. The digestion products of fibrin are referred to as fibrin split products, fibrin degradation products, or fibrinolytic split products.
APPROACH TO THE BLEEDING PATIENT The following outline is recommended for use in the evaluation of patients with suspected hemostatic defects. Each of the components of this approach to the bleeding patient will be discussed in more detail. 1. History 2. Physical examination 3. Collect samples for screening tests a. Whole blood-citrate anticoagulant (1) extrinsic mechanism-one stage prothrombin time (OSPT) (2) intrinsic mechanism-activated partial thromboplastin time (APTT) b. Whole blood-EDT A anticoagulant (1) platelets
DIAGNOSIS OF HEMOSTATIC DISORDERS
611
4. Classify defect a. platelet or vessel defect-petechiae, thrombocytopenia, superficial hemorrhage, prolonged bleeding time b. coagulation defect-abnormal OSPT or APTT; deep hemorrhage c. multiple defects 5. Perform specific tests or therapeutic trial
History
Objectives. (1) Determine if hemostatic defect is present. (2) Describe hemorrhages. (3) Describe possible episodes of spontaneous hemorrhage. (4) Describe environmental factors. (5) Describe any previous treatments. (6) Investigate possibility of bleeding in siblings or ancestors. The history is extremely important for diagnosis of bleeding disorders. The first objective should be to determine if the animal has a hemostatic defect. In obtaining a description of all previous hemorrhages, one should determine the time, location, size, and duration of the lesions. If the owner is aware of the cause of the bleeding, a judgment is necessary to decide if the hemorrhage is compatible with and proportionate to the injury. Evidence for earlier hemorrhage or an associated disease may be pursued with questions regarding the animal's condition and behavior prior to any observed hemorrhages. Specific inquiry should be made for evidence of spontaneous hemorrhages including color of the urine and stool, petechiae on the skin or mucous membranes, unexplained lamenesses which could be due to hemarthroses or intramuscular hemorrhages, or periods of weakness and inactivity. Evidence for a longstanding hemostatic defect can be gained by questions regarding elective surgical procedures. It should be noted, however, that some dogs with congenital coagulation defects have survived ear cropping or tail docking procedures with no apparent bleeding problems. If the animal has experienced any lacerations or severe trauma the degree and duration of bleeding should be determined. Environmental factors which should be investigated include the animal's housing, area of confinement, and possibilities for exposure to warfarin or other chemicals and drugs. Any previous treatments, especially blood transfusions, should be recorded along with comments regarding their effectiveness in controlling the bleeding. If a congenital bleeding defect is suspected, the medical history of the littermates is frequently helpful. Since some defects (factor VIII deficiency and factor IX deficiency) are sex-linked recessive characteristics, the sex of possibly affected littermates provides a helpful clue to the diagnosis. Physical Examination The necessity for a thorough physical examination is not only to characterize the hemorrhag~ but also to evaluate the patient for evi-
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dence of other disease processes. Recognition of abnormalities, such as icterus, fluid in body cavities, and the type and location of hemorrhages, will aid in the classification of bleeding defects. Petechial hemorrhages are characteristic for hemostatic defects involving either platelets or blood vessels. Patients with defects of the plasma coagulation mechanism are more likely to suffer large, deep hemorrhages into the body cavities, subcutaneous tissues, or large muscle masses. Gastrointestinal bleeding with dark, tarry stools is a very common cause of blood loss in thrombocytopenic dogs, but is not specific since it is also seen in association with some blood coagulation defects. It is difficult to classify the causes of ecchymoses and large bruises in the skin because they may be associated with defects of any of the components of the hemostatic mechanism. In animals with light skin, evidence for resorbing hemorrhage is provided by the observation of yellow, orange, or brownish areas of discoloration of the skin. Screening Tests
Screening tests are important for the differential classification of hemostatic defects. These tests are most effectively used in combinations. These tests ideally would be selected to evaluate the blood vessels, platelets, and coagulation mechanism. The routine tests screen for defects involving the extrinsic coagulation mechanism, the intrinsic coagulation mechanism, and the platelets. Vascular defects are more difficult to evaluate. Tests for blood vessel functions are therefore not recommended as routine screening procedures. The minimum screening tests should include: 1. One extrinsic mechanism test a. one stage prothrombin time 2. One intrinsic mechanism test a. partial thromboplastin time b. activated partial thromboplastin time c. activated coagulation test d. Lee-White clotting time 3. Platelet count
For the classification of the hemostatic defects, the author has selected the OSPT, APTT, and platelet count. A brief discussion of the principles of these tests and related screening tests is provided. Sample Collection and Preparation. Samples should be collected from the patient prior to the administration of any treatment whenever possible. Careful collection techniques are vital for hemostatic tests. The introduction of small amounts of tissue juices during problem venipunctures can lead to erroneous coagulation results and platelet clumping in a sample. Samples should be collected from a large vein with a plastic syringe. If the venipuncture is not achieved on the first attempt a new syringe and needle should be used for the subsequent attempt. A
DIAGNOSIS OF HEMOSTATIC DISORDERS
613
desirable alternative procedure is to use one syringe for positioning the needle in the vein and aspirating a small amount of blood prior to substituting a second syringe for sample collection. If vacuum tubes are used for sample collection, a small amount of blood should be allowed to flow through the needle prior to attachment of the tube with anticoagulant. Blood for platelet counts should be added to EDT A, because this anticoagulant is a good inhibitor of platelet aggregation, thereby preventing platelet clumping. Platelet counts should be performed within four hours after sample collection. For coagulation studies, nine volumes of blood are mixed with one volume of 3.8 per cent (0.13 m) sodium citrate in a plastic or siliconized glass tube. The plasma should be separated by high speed centrifugation (3000 rpm for 15 minutes) directly after sample collection and used for coagulation tests, or frozen at -20°C. for testing later. Since marked variations in normal clotting test values may occur in different laboratories, and occasionally in the same laboratory, it is extremely important that blood samples be collected from at least one normal animal of the same species and submitted along with the patient samples. Clotting test results for the patient should always be compared with those of the normal control animals. Blood Vessels. Bleeding time and biopsy. Defects of blood vessels are difficult to evaluate. The majority of these defects are associated with other systemic disease processes. An understanding of the pathogenesis of the primary disease process is helpful for explaining the vessel lesions. Although the bleeding time is easy to perform, it is difficult to obtain consistent results from this test in most animals. Variations in the size, number, and location of injured vessels will affect the results. This test is not recommended as a routine screening test. Prolonged oozing hemorrhage from venipuncture sites may be an indication of a long bleeding time. Biopsy of affected tissues should be restricted to special instances where unusual vascular lesions are suspected. Platelets. Platelet count, clot retraction, platelet retention, platelet aggregation, and bleeding time. Platelets may be evaluated in a variety of tests. For routine screening of patients with hemostatic defects, only the platelet count and platelet evaluation on a blood smear are recommended. Platelet defects are more extensively discussed under "Defects Involving Blood Vessels or Platelets." Extrinsic Clotting Mechanism. One stage prothrombin time (OSPT) and Russell's viper venom test (RVVT). The one stage prothrombin time is performed by measuring the time to clot formation in a plasma sample after tissue thromboplastin and calcium are added. This test is used routinely for evaluation of the extrinsic mechanism (Fig. 1). An extrinsic mechanism defect should be' suspected if the OSPT of the patient is prolonged longer than 5 seconds compared with the control.
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The Russell's viper venom test (Stypven test) is performed by adding snake venom to a plasma sample and timing to clot formation. This extrinsic mechanism screening test involves the same clotting factors as the OSPT except for factor VII (Fig. 1). Intrinsic Clotting Mechanism. Partial thromboplastin time (PTT), activated partial thromboplastin time (APTT), Lee-White clotting time, and activated coagulation time. The partial thromboplastin time and the activated partial thromboplastin time are widely available and reliable tests for evaluating the intrinsic clotting mechanism (Fig. 1). In these tests, platelet poor plasma is incubated with a phospholipid (partial thromboplastin) which substitutes for the phospholipid that is derived in vivo from platelets. The sample is recalcified and timed to clot formation. The APTT is a modification of the PTT, in which a surface activator is incorporated to ensure maximal factor XII activation (in the PTT factor XII is activated only by exposure to the glass wall of the test tube). The clotting times in the APTT are therefore usually shorter than those of the PTT, but either test is effective in screening for intrinsic mechanism defects. In our laboratory the APTT is considered to be abnormal when it is 7 seconds longer than the control in small animals and 15 seconds longer than the control in cattle and horses. Smaller changes may be significant in some of the less severe defects but are more difficult to interpret. The Lee-White clotting test is performed by measuring the time required for clotting of a whole blood sample under controlled conditions in glass tubes. The test is affected by variations in temperature which must be minimized. The test screens for defects in the intrinsic mechanism (Fig. 1), but is not as sensitive or precise as the PTT or APTT. Severe thrombocytopenia can lead to a mildly prolonged LeeWhite clotting time since platelet phospholipid is needed for the intrinsic mechanism. The test, however, is not sensitive for most platelet deficiencies, and a normal Lee-White clotting time does not eliminate the possibility of thrombocytopenia. The activated coagulation time (ACT)* is a relatively new screening test for intrinsic mechanism defects. In this test a blood sample is collected into a vacuumized glass tube containing a surface activator substance to rapidly activate factor XII in the sample. The tube is incubated at a constant temperature in a heat block or water bath and timed to clot formation. This test combines the advantages of using whole blood rather than plasma with the contact activator to improve the precision of the procedure. Preliminary work in our laboratory suggests that this test is superior to the Lee-White coagulation time and is easier to perform than the PTT or APTT. This test may be very useful for routinely *Activated Coagulation Time, Vacutainer 3206XF534, Becton, Dickenson and Company, Rutherford, New Jersey.
DIAGNOSIS OF HEMOSTATIC DISORDERS
615
screening for the more severe intrinsic mechanism defects in domestic animals. Fibrinolytic Split Products. Latex agglutination test. Increased levels of circulating fibrinolytic split products are usually evidence of enhanced fibrinolysis. The Thrombo-Wellcotest* is a latex agglutination test that is simple and reliable. In this test latex particles are used which are coated with antibody directed toward human fibrinogen degradation products (FDP; fragments from the proteolytic digestion of fibrinogen by plasmin). The antibody appears to cross react with FDP's of most domestic animals and can be used to screen for enhanced fibrinolysis. Because the affinity of the antibody for FDP's of different species probably varies, the results should be reported as positive or negative at various dilutions and not in ~-tg/ml. Fibrinolytic split products are present in only very low concentration in the blood of normal animals. In our laboratory a positive Thrombo-Wellcotest at a serum dilution of 1 /zs or greater is evidence of enhanced fibrinolysis in the dog, horse, and cat. Samples for fibrinolytic split products assays must be preserved with a proteolytic enzyme inhibitor to block in vitro fibrinolysis which would lead to increased levels of fibrinolytic split products. Classification of Hemostatic Defects Using the data from the history, physical examination, and screening tests one should attempt to classify the defect. The following outline is provided as a guideline. The identification of any of the abnormalities listed warrants tentative assignment of the defect to that category and further evaluation. A. Vessel or Platelet Defect 1. Physical findings a. petechial hemorrhages b. superficial hemorrhages (purpura) 2. Laboratory findings a. thrombocytopenia b. prolonged bleeding time B. Coagulation Defect 1. Physical findings a. large, deep hemorrhages disproportionate to injury (1) subcutaneous (2) intramuscular (3) intraarticular (4) body cavities 2. Laboratory findings a. prolonged OSPT b. prolonged APTT C. Combined Defects of Vessels, Platelets, and Coagulation
*Thrombo-Wellcotest, Burroughs-Wellcome Company, Wellcome Research Division, Research Triangle Park, North Carolina.
GARY J. KocmA
616 PIATELET COUNT
~ NORMAL PLATELET NUMBER
THROMBOCYTOPENIA
~
~
APTT
PLATELET FUNCTION TESTS
/~ PROLONGED
NORMAL
i
~
l
BONE MARROW
DIG
/
~
BONE MARROW HYPOPLASIA MYROPROLIFERATIVE DISORDERS
Figure 2. blood vessels.
i
VASCULAR DEFECTS
~
THROMBOCYTOPATHY DRUGS DYSPROTEINEMIA
vWD
UREMIA FIBRINOLYSIS
MEGAKARYOCYTE$
DECREASED
/~ ABNORMAL
NORMAL
~ INCREASED ~
ITP INFECTIOUS DISEASES MYELOPROLIFERATIVE DISORDERS
Flow chart for the evaluation of patients with defects involving platelets or
After classification of a hemostatic defect in one of the above groups the appropriate steps toward definitive diagnosis can be selected. The schemes for the evaluation of defects involving blood vessels or platelets and the coagulation defects are designed to quickly narrow the differential diagnoses to a few diseases on the basis of the history, physical examination, and a few laboratory tests. With this information it is usually possible to decide on probable prognosis and treatment, select special tests, and decide if referral to a specialized center is appropriate.
DEFECTS INVOLVING BLOOD VESSELS OR PLATELETS These defects are usually recognized by the observation of characteristic superficial hemorrhages in the skin or mucous membranes. The hemorrhages are frequently noted as purple discolorations which may be referred to as purpura. The hemorrhages usually are petechiae or ecchymoses. Petechiae are characteristic of hemostatic defects involving platelets or blood vessels. The laboratory tests that are helpful in evaluating patients with petechial hemorrhages include hemogram, one stage prothrombin test (OSPT), activated partial thromboplastin time (APTT), platelet count, bone marrow examination, and platelet function tests (optional). Figure 2 is a flow chart that incorporates the results of the laboratory tests in the classification of purpuric diseases. Using the stepwise approach that is outlined one can quickly classify the defect. After clas-
DIAGNOSIS OF HEMOSTATIC DISORDERS
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sification of the defect, decisions are made relative to specific diagnosis, prognosis, and treatment. Several clues may be gained from the hemogram and examination of the peripheral blood smear. Platelet numbers can be estimated by determining the number per field. If less than three platelets are observed per oil immersion field the possibility of thrombocytopenia should be considered. When estimating platelet numbers from a blood smear it is important to check for irregularities in platelet distribution due to clumping of platelets which is most obvious near the distal (feathered edge) end of the smear. A direct platelet count on a carefully collected, fresh, EDT Aanticoagulated sample is the best way to determine platelet number. Even with this technique a blood smear should be examined to corroborate the results of the platelet count. If a thrombocytopenia is recognized it may be due to either decreased production or increased destruction of platelets. Concurrent nonregenerative anemia and leukopenia would suggest that bone marrow hypoplasia may be the cause of the thrombocytopenia. Large platelets (macroplatelets) are evidence for diseases with increased platelet turnover rate. These platelets (up to 10 JLm diameter) are produced and released during periods of rapid platelet production. Syndromes of increased platelet consumption are usually associated with immunologic thrombocytopenic purpura (ITP), disseminated intravascular coagulation (DIC), or infectious diseases. The APTT is used to screen for the coagulation defects that are associated with DIC since marked prolongation of the APTT is usually evident in animals with clinically significant thrombocytopenia related to DIC. If the APTT is normal in a thrombocytopenic patient, bone marrow aspiration biopsy is indicated. A bone marrow sample can be aspirated from the iliac crest of a dog using a local anesthetic. Abnormal bleeding from the biopsy site is generally not a significant problem even in severe thrombocytopenias. Bone marrow smears can be stained with either new methylene blue stain or Wright's stain and examined for the number of megakaryocytes. If few or no megakaryocytes are found, syndromes of bone marrow hypoplasia, such as estrogen toxicity in the dog, must be considered. If normal or increased number of megakaryocytes are seen in a dog without evidence of other disease processes, a tentative diagnosis of ITP is suggested and corticosteroid therapy can be initiated. It should be noted that ITP is usually diagnosed by evidence of increased platelet turnover rate and exclusion of other diseases. Tests for anti-platelet antibody, such as platelet factor III release test, are available from some specialized laboratories and could be used to substantiate a diagnosis of ITP. Direct effects of drugs or drug-induced, immune-mediated thrombocytopenia are distinct possibilities if the thrombocytopenia developed during drug therapy. The suspected drug must be incorporated into the test system if
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tests for demonstration of anti-platelet antibodies induced by drug exposure are attempted. It should be kept in mind that some infectious diseases can lead to enhanced platelet destruction or megakaryocyte damage. A transient thrombocytopenia and/or platelet function defect has been reported in dogs following vaccination with either measles or some canine distemper vaccines. The possibility of myeloproliferative diseases causing a defect in platelet production must also be considered. This may be recognized when the bone marrow smears are examined. Platelet function defects should be considered in purpuric animals with normal or increased numbers of platelets. Tests for these defects include the simple clot retraction test, glass bead column platelet retention test, and platelet aggregation tests. The more complicated platelet function tests are most effectively handled by specialized laboratories experienced with them. Acquired platelet function defects are more common than congenital defects and may be related to disease processes such as uremia, dysproteinemia, or exposure to drugs. A wide variety of drugs, including aspirin and many of the phenothiazine tranquilizers, cause platelet function defects. Hereditary platelet function defects have been described in dogs, especially otterhounds and basset hounds, and fawn hooded rats. Vascular defects are less common causes of purpuric lesions. Scurvy in guinea pigs or primates is accompanied by increased vascular fragility and defective collagen synthesis. Some infectious diseases and some immune-mediated diseases are characterized by vasculitis and purpuric lesions. Dogs with hyperadrenocorticism rarely have petechial or ecchymotic hemorrhages. These hemorrhages are probably related to abnormal collagen metabolism. COAGULATION DEFECTS The hemostatic defects involving the blood coagulation mechanism can usually be subdivided using the results of three screening tests as indicated in Table 1. After classification of the defect in one of the four major groups one can utilize the following information to proceed to a specific diagnosis. Defects in the Early Stages of the Intrinsic Clotting Mechanism
Screening Test Results.
OSPT (normal), APTT (prolonged), and
platelets (normal).
Differential Diagnoses. Factors VIII, IX, XI, and XII deficiencies, and von Willebrand's disease. Factor VIII deficiency (classic hemophilia) and factor IX deficiency are both transmitted as sex-linked recessive defects. The bleeding tendencies are therefore limited to males except for rare instances involving intensive inbreeding. Factor VIII deficiency has been recognized in most breeds of dogs and mongrels, cats, and horses. Factor
619
DIAGNOSIS OF HEMOSTATIC DISORDERS
of Coagulation Defects According to Screening Test Results
Table 1. Classification SCREENING TEST
COAGULATION DEFECTS
OSPT*
APTT*
Platelets
Normal
Prolonged
Normal
Factor VIII deficiency Factor IX deficiency Factor XI deficiency Factor XII deficiency von Willebrand's disease
Prolonged
Normal
Normal
Factor VII deficiency
Prolonged
Prolonged
Normal
Fibrinogen deficiency Factor X deficiency Liver disease Warfarin poisoning Vitamin K deficiency
Prolonged
Prolonged
Decreased
DIC
*OSPT, one stage prothrombin time; APTT, activated partial thromboplastin.
IX deficiency is a much less common defect than classic hemophilia and has been recognized only in Cairn terriers, black and tan coonhounds, and St. Bernards. Diagnosis of these defects is made with specific factor VIII and factor IX assays. Animals that are bleeding as a result of factor VIII deficiency usually have less than 20 per cent of normal factor VIII activitity, with severely deficient patients having less than 1 per cent activity. Dogs that have been detected with factor IX deficiency have had defects with less than 1 per cent of normal factor IX activity. Female carriers for either factor VIII or factor IX deficiency can usually be identified in the laboratory using specific assays, since these animals usually have 40 to 60 per cent of normal factor VIII or factor IX activity, respectively. Factor XI deficiency has been recognized in Holstein cattle and springer spaniel dogs. The bleeding defect is usually less severe than that of factor VIII or factor IX deficiency. Spontaneous hemorrhages have been detected in dogs with factor XI deficiency, but the bleeding defect is usually recognized as abnormal bleeding which starts within 24 hours after surgery. The disease is transmitted as an autosomal characteristic with the homozygotes having a bleeding tendency and with less than 15 per cent factor XI activity, whereas the clinically normal heterozygotes have 25 to 70 per cent normal factor XI activity. Factor XII deficiency has not yet been reported in domestic animals. In man the defect is not associated with a clinical bleeding defect. Therefore, if an animal has a bleeding problem, the chances that a factor XII
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deficiency is causing the defect are remote. Factor XII assays are available for specific diagnosis of factor XII deficiency. Marine mammals and birds do not have detectable factor XII activity. Von Willebrand's disease has been recognized in pigs and appears to be one of the more common congenital hemostatic defects in dogs. The disease ranges from that of a moderately severe bleeding defect to a mild defect in different animals. The majority of these animals have a mild clinical bleeding tendency which is recognized following surgical procedures. The hemorrhages may be severe and life-threatening. The results of the APTT in von Willebrand's disease usually range from normal to moderately prolonged. Unlike the congenital deficiencies of most other coagulation factors, the bleeding time may be prolonged in von Willebrand's disease. This is related to a defect in platelet function. The disease has an associated mild to moderate ( 10 to 60 per cent) deficiency of factor VIII activity. A unique feature of this disease that is sometimes used for differential diagnosis is the paradoxical increase in factor VIII activity that peaks about 24 hours after transfusion with either factor VIII deficient or normal plasma. A recent finding that is useful for the diagnosis of von Wille brand's disease is that patients with the disease have low levels of factor VIII related antigen that correlate with low factor VIII activity in clotting tests. In contrast, dogs with classic hemophilia have normal or increased quantities of factor VIII related antigen (apparently nonfunctional molecules) with low factor VIII clotting factor activity. Von Willebrand's disease is transmitted as an autosomal characteristic and therefore is seen in both males and females. Defects Restricted to the Extrinsic Clotting Mechanism
Screening Test Results. OSPT (prolonged), APTT (normal), and platelets (normal). Disease. Factor VII deficiency. Factor VII deficiency is a defect which is usually discovered incidentally in beagle dogs being screened with OS PT. One malemute with factor VII deficiency has been reported. The autosomal defect causes only a very mild clinical bleeding tendency with easy bruising and possible increased bleeding after surgery. Since factor VII is not a part of the intrinsic clotting mechanism, the APTT and whole blood clotting tests will be normal. Specific diagnosis is most effectively accomplished with a factor VII assay. Since the Stypven test bypasses factor VII of the extrinsic clotting mechanism, the finding of a normal Stypven test in a dog with a prolonged OSPT should be conclusive for factor VII deficiency. Defects Involving Both Intrinsic and Extrinsic Clotting Mechanisms; Normal Platelets
Screening Test Results. platelets (normal).
OSPT (prolonged), APTT (prolonged),
DIAGNOSIS OF HEMOSTATIC DISORDERS
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Differential Diagnoses. Warfarin toxicity, liver disease, vitamin K deficiency, factor X deficiency, fibrinogen deficiency, and anticoagulants. Warfarin poisoning is the most common coagulation defect in this group of diseases. Nevertheless, the disease is incriminated in animals more often than it actually occurs. The diagnosis of warfarin poisoning in an untreated animal with a normal one stage prothrombin time is unwarranted. The majority of bleeding episodes in dogs with warfarin poisoning are massive hemorrhages into the body cavities, gastrointestinal tract, musculature, or subcutaneous tissues. Petechial hemorrhages are not common and the platelet count is normal or on the low side of the normal range. The administration of natural vitamin K quickly corrects the defect with shortening of the OSPT detectable six hours after injection. If specific clotting factor activities are assayed, low activity is detected for factors II (prothrombin), VII, IX, and X. The diagnosis is based on the history, compatible laboratory findings, and rapid response to vitamin K therapy. Since the majority of clotting factors are synthesized in the liver, diseases which cause liver dysfunction can lead to clotting defects owing to decreased synthesis of certain clotting factors. The severity of the defect is generally proportionate to the liver disease. Clinical hemostatic defects are seen only in animals with very severe liver diseases. Animals with coagulation defects due to liver disease should have decreased ability to excrete BSP. Vitamin K deficiency is rarely the result of a dietary deficiency. Since vitamin K is a fat soluble vitamin, defects in fat absorption related to defects such as bile salt deficiency can lead to vitamin K deficiency. Other possible causes include gut "sterilization" with long-term antibiotic therapy, germ free animals, and some specific pathogen free animals especially if coprophagy is not permitted. The defect of vitamin K deficiency mimics that of warfarin poisoning . . Factor X deficiency has been recognized as an inherited defect in cocker spaniel dogs. The disease is transmitted as an autosomal trait. The homozygotes for factor X deficiency usually die in the early neonatal period or are stillborn with hemorrhage from the umbilicus or into the body cavities. In adults the defect is relatively mild since the dogs are usually heterozygotes with 18 to 65 per cent of normal factor X activity. Hemorrhage in adult dogs with factor X deficiency usually occurs post-surgically. The results of screening tests may be normal in the heterozygotes for factor X deficiency. Specific diagnosis is made with factor X assays. Fibrinogen deficiency is usually an acquired defect related to disseminated intravascular coagulation or decreased synthesis related to liver disease. Congenital deficiency of fibrinogen has been recognized in dogs and Saanen goats, but is rare. Hemorrhages include hemarthroses, umbilical bleeding, subcutaneous bleeding, and bleeding from mucous
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membranes. Fibrinogen deficiency is an autosomal trait with heterozygotes having about 50 per cent of normal fibrinogen levels. Fibrinogen assays are readily available to facilitate diagnosis. Exposure to some anticoagulants, especially heparin, may lead to erroneous low results in some fibrinogen assays. This possibility can be evaluated by anticoagulant screenings tests or by observing for the normal precipitation of fibrinogen which occurs when plasma is heated to 58°C. Circulating anticoagulants are not commonly encountered in domestic animals. When they are detected in a sample, heparinization related to use of concentrated heparin solutions to maintain patency of an indwelling catheter, or collection of sample through a heparinized catheter should be eliminated first. Circulating anticoagulants can be demonstrated in some patients with disseminated intravascular coagulation. The author has detected high levels of circulating heparin-like activity in one cat with mastocytosis. Defects Involving the Coagulation Mechanism and Platelets
Screening Test Results. OSPT (normal---+prolonged), APTT (normal---+prolonged), platelets (normal--'>decreased). Differential Diagnoses. Disseminated intravascular coagulation (DIC) and dysproteinemias. Disseminated intravascular coagulation is an acquired syndrome that is characterized by activation of both the hemostatic mechanism and fibrinolytic mechanism. This syndrome is always secondary to another disease process which can be neoplastic, infectious, or metabolic. DIC is usually induced by diseases with a combination of a number of the following changes: exposure of blood to abnormal surfaces (activates intrinsic mechanism), release of tissue thromboplastin from damaged tissue or neoplasms, hypotension, stasis of blood flow, vasculitis, and endotoxin exposure. DIC may range in severity from a mild disease to a severe lifethreatening syndrome with severe bleeding and organ dysfunction related to capillary obstruction by fibrin thrombi. Thrombocytopenia is usually present in the more acute forms of DIC since platelets are consumed in the overactive hemostatic process. Platelets that are present are usually large reflecting rapid platelet turnover. The OSPT and even more constantly the APTT are markedly prolonged due to consumption of clotting factors in the coagulation process and/or anticoagulant effects of fibrinolytic split products. Fibrinolytic split products are present in increased concentration in the bloodstream reflecting active fibrinolysis. Animals with DIC frequently have petechial hemorrhages and prolonged bleeding times, which often is suggested by prolonged oozing of blood from venipuncture sites. The bleeding defects are related to thrombocytopenia and platelet function defects induced by exposure of platelets to fibrinolytic split products. Since DIC is a complex syndrome, the changes that are associated with the process are not always spectacu-
DIAGNOSIS OF HEMOSTATIC DISORDERS
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lar nor are all present simultaneously. The changes that are helpful for the diagnosis of DIC include: (1) OSPT-prolonged, (2) APTTprolonged, (3) thrombocytopenia, (4) fibrinolytic split productsincreased, (5) petechial hemorrhages, and (6) abnormal bleeding from venipunctures. Dysproteinemias are rare diseases and hence are seldom recognized as causes of hemostatic defects. The abnormal quantities of protein that are produced in animals with plasma cell myeloma or macroglobulinemia may cause hemostatic defects. The defects apparently are related to interference with some coagulation enzyme reactions and platelet function defects associated with the paraproteins. Since plasma hyperviscosity and stasis of blood flow may occur in macroglobulinemia, some of the bleeding defects may be related to changes associated with DIC. REFERENCES 1. Biggs, R. (ed.): Human Blood Coagulation, Haemostasis, and Thrombosis. Philadelphia, F. A. Davis Co., 1972. 2. Bowie, E.]. W., Thompson,]. H., Didisheim, P., and Owen, C. A.: Laboratory Manual of Hemostasis. Philadelphia, W. B. Saunders Co., 1971. 3. Dodds, W. ].: Bleeding Disorders. In Ettinger, S. (ed.): Textbook of Veterinary Internal Medicine, Vol. 2. Philadelphia, W. B. Saunders Co., 1975. 4. Dodds, W. ]., and Kaneko, J. ].: Hemostasis and Blood Coagulation. In Kaneko, J.J., and Cornelius, C. E. (eds.): Clinical Biochemistry of Domestic Animals, Vol 2. New York, Academic Press, 1971. 5. Rowsell, H. C.: Blood Coagulation and Hemorrhagic Disorders. In Medway, W., Prier, ]., and Wilkinson,]. (eds.): Textbook of Veterinary Clinical Pathology. Baltimore, The Williams and Wilkins Co., 1969. College of Veterinary Medicine Ohio State University 1935 Coffey Road Columbus, Ohio 43210
