Internal Medicine 1. Diagnostic Approach to the Bleeding Patient
Urs Giger, PD, Dr. med. vet., MS, FVH, DACVIM, DECVIM
University of Pennsylvania
Philadelphia, PA, USA

Bleeding is a common clinical presentation in small animal practice. Bleeding disorders occur more commonly in dogs than in cats. Clinical manifestations of bleeding disorders may range from mild and self-limited to life-threatening hemorrhage requiring immediate medical attention. Animals may bleed due to vascular injury caused by any trauma, surgery, ulcer, and tumor. In case of hemostatic disturbances, the hemorrhagic tendency is exaggerated as exemplified by spontaneous, multifocal, and unexpected severe bleeding.

Hemostasis is the complex physiologic response to bleeding and can be divided into primary and secondary hemostasis. Vascular endothelium, platelets, and vonWillebrand factor (vWF), are required for primary hemostasis or the formation of the unstable platelet plug, which is sufficient to stop capillary bleeding. Von Willebrand factor, a large plasma protein synthesized by endothelium facilitates adhesion between platelets and subendothelium. With larger vessel injury, coagulation factors are also needed to form a stable fibrin clot known as secondary hemostasis. Hemostatic disorders can be conveniently classified into vasculopathies, thrombocytopenias, thrombopathias, vonWillebrand disease (vWD), and coagulopathies realizing that some disease processes cause combined hemostatic defects (e.g., disseminated intravascular coagulopathy [DIC]). Hemorrhagic presentations may suggest certain hemostatic disorders and practical tests are available to characterize the bleeding tendency. An accurate diagnosis of the cause of bleeding will influence the treatment and outcome of a patient.

Coagulation Cascade

The coagulation cascade is a series of enzymatic reactions involving coagulation factors. This complex biochemical pathway plays three pivotal roles in the formation of fibrin from fibrinogen:

1.  Acceleration of fibrin generation >10 million-fold

2.  Regulation of fibrin plug size appropriate for injury

3.  Localization of fibrin clot formation to site of injury

The coagulation cascade can be divided into an extrinsic and intrinsic system which merge into the common pathway. When blood vessel walls are injured, the extrinsic coagulation system is initially activated by tissue factor (thromboplastin produced in the subendothelium), which combines with factor VII. The small amount of thrombin produced through Factor VIIa appears sufficient to activate factor XI to factor XIa and also to activate other cofactors (factors V, VIII, XIII). Through the action of factor XIa on factor IX, thrombin formation is maintained. The other contact phase coagulation factors (e.g., factor XII) are in vivo of lesser importance in the activation of the intrinsic system. The above described coagulation process explains why hemophilic (Factor VIII and IX deficient) or Factor VII deficient animals bleed, whereas Factor XII deficient animals do not.

Coagulation factors are enzymes, cofactors, or substrates of particular reactions of the coagulation cascade. Most factors are given roman numerals from I-XIII, but the numbering is not sequential and there is no factor VI. Calcium (factor IV) is required for most reactions and is the reason why chelators, e.g., citrate and EDTA, are used for blood collection and processing, where plasma or blood cells are analyzed. All coagulation factors are synthesized in the liver and circulate as inactive precursors in the plasma. They need to be activated at the site of vessel injury. Vitamin K is needed for the functional synthesis of the coagulation factor II, VII, IX, and X. The half-lives of the coagulation factors vary from hours (Factor VII) to a few days (Fibrinogen). Following a fibrin plug formation, plasminogen will be activated and plasmin, an unspecific protease will commence breaking down fibrinogen as well as fibrin which results in fibrinogen lysis and fibrin (-ogen) split product (FSP) as well as D-dimer formation.

Diagnostic Approach

Signalment & Family History--Although hereditary coagulopathies may occur in any breed, each coagulopathy has thus far only been reported in certain breeds. Hemophilia A and B occur in many different breeds and are X-chromosomal recessively inherited; thus, only males are generally affected and females are asymptomatic carriers. Signs of bleeding typically occur at an early age and are often recurring, but may not be recognized until adulthood.

History--Bleeding may be induced (trauma, surgery) or appear spontaneous. Careful history taking may reveal exposure to toxins (rodenticides, mushrooms) and drugs (warfarin, heparin). It is important to identify the specific product, as e.g., different rodenticides have quite varied potency. Any evidence of other diseases, e.g., hepatopathies and cancer, may be responsible for the hemorrhage.

Physical Examination--Careful clinical evaluation may differentiate between primary and secondary hemostatic defects. Surface bleeding is typically seen with primary hemostatic disorders. Petechia and ecchymosis are hallmark features of thrombocytopenias and thrombopathias. However, von Willebrand disease (vWD) is causing bleeding at sites of injury (trauma, dental disease, estrus, gastrointestinal) rather than petechia or ecchymosis. Coagulopathies may be associated with single or multiple sites of bleeding characterized by cavity bleeding such as hematoma, hemarthrosis, hemomediastinum, hemoperitoneum, and hemothorax, but gastrointestinal hemorrhage and bruising may also occur. Signs of other underlying diseases may be recognized.

Hemostatic Tests--Hemostatic tests are indicated: 1) whenever an animal is bleeding excessively 2) prior to surgery when an increased bleeding tendency is suspected 3) to monitor therapeutic interventions 4) for genetic screening in certain breeds or families with a known bleeding disorder.

Hemostatic abnormalities should be assessed prior to instituting therapy whenever possible or at least appropriate blood samples should be collected pretreatment. Excellent venipuncture with discarding of the first few drops of blood (to avoid platelet activation and tissue factor) and extended compression over jugular, saphenous or femoral vein is required. The cuticle bleeding time crudely assesses overall hemostasis, but is not standardized and painful and is, therefore, not recommended. A minimal database includes a packed cell volume and total protein evaluation. Evaluation of a blood smear can provide a platelet estimate and identify platelet size and clumping as well as schistocytes. The results can provide some measure of the extent of blood loss and red blood cell transfusion requirement.

Primary Hemostasis: Platelets & vWF

Since 8-15 platelets are normally found per high power oil emersion microscopic field, an absence to low number of platelets suggests a severe thrombocytopenia. Hemorrhage is generally not observed unless the platelet count is <40,000/µl (normal 150-500,000/µl). Detection of platelet-associated antibodies further supports an immune-mediated thrombocytopenia, but this test is rarely available. Serum titers or PCR tests for tick-born and other infectious diseases are indicated in certain countries or areas. The presence of schistocytes and thrombocytopenia suggests intravascular disseminated coagulation, where intravascular fibrin strands fragment erythrocytes. Because von Willebrand disease is such a common mild primary hemostatic defect in dogs, plasma vWF measurements by ELISA are indicated. Alternatively, DNA testing is available in some canine breeds for breeding purposes. Finally, in light of normal platelet count and plasma vWF values, a prolonged buccal mucosal bleeding time (BMBT) indicates a thrombopathia. Disposable devices are available that facilitate making 1-2 standard 1mm deep mucosal incisions. The platelet function analyzer (PFA100) is a simple tool to functionally assess primary hemostasis. Electron microscopic and platelet aggregation and nucleotide studies allow further characterization of platelet dysfunctions in specialized laboratories.

Secondary Hemostatic Tests: Coagulation Tests

Whereas the whole blood clotting time test is insensitive and inaccurate, there are several standardized coagulation screening tests that are useful to define coagulopathies in clinical practice. The screening tests assess coagulation in vitro, which is helpful, but is now known not to be identical with the in vivo coagulation process. Nearly all coagulation tests assess the function of certain parts of the coagulation system in fresh whole blood or fresh (frozen) plasma to generate fibrin in a fibrometer; recalcified citrated plasma is used and many tests are comparing a patient sample directly with a simultaneously obtained control or pool plasma (plasma from 10 animals). Generally coagulation times, the time to clotting (fibrin formation), are much shorter in small animals than in humans; thus, tests need to be validated for the animal species.

The intrinsic and common pathways are assessed by either the activated coagulation time (ACT) or activated partial thromboplastin time (PTT). Factor XII of the intrinsic cascade is activated by diatomaceous earth (celite) in the ACT test and by kaolin or other contact phase substrates in the PTT test.

The extrinsic and common pathways can be assessed by either the prothrombin time (PT) or the protein induced by vitamin K antagonism or absence (PIVKA) test. Different tissue factors (thromboplastins) are activating factor VII, which in turn will lead to fibrin formation. It should be noted that the PIVKA test is not specific for the detection of anticoagulant rodenticide poisoning, but detects any coagulation factor deficiency of the extrinsic and common pathway and does not add information to the generally run PT test.

Until recently the ACT tube test was the only point of care test available for clinical practice, whereas PTT and PT tests were performed in reference laboratories. There are now new point of care coagulation instruments (e.g., SCA2000) introduced that are capable of determining without delay on small amounts (50µl) of fresh citrated whole blood the PTT and PT, thereby making the chilling, rapid separation of citrated plasma and shipment of frozen plasma on dry ice to the laboratory for initial coagulation screening unnecessary.

In fact, a reasonable and simple approach for a bleeding animal to be screened for a coagulopathy would be to measure the ACT or PTT first as either test detects all coagulopathies (except for hereditary factor VII deficiency in Beagles). If the PTT (or ACT) is prolonged, a PT test would be indicated to differentiate between an intrinsic and common pathway defect or a combined coagulopathy involving several coagulation factors.

Although hereditary coagulopathies can be suspected based upon the pattern of coagulation test abnormalities, specific factor analyses are needed to confirm a diagnosis. A bleeding male animal with a prolonged PTT (or ACT) and normal PT likely has hemophilia A or B (factor VIII or IX deficiency), an X chromosomal recessive disorder. However, factor XI deficiency is associated with the same test abnormalities and is inherited by an autosomal recessive trait (e.g., Kerry blue terriers). Finally, factor XII deficiency, particularly common in domestic shorthair cats, and prekallikrein deficiency causes marked PTT (ACT) prolongations but no excessive bleeding tendency.

Rodenticide poisoned animals that are bleeding or are at risk for bleeding will have prolongations in all of the above coagulation tests, but would have a normal thrombin time (TT). The thrombin time is independent of vitamin K-dependent coagulation factors and is a functional assay for fibrinogen to form fibrin. The PIVKA test is not diagnostic, but a toxicological investigation (product identification, blood toxicology analysis) may confirm the rodenticide poisoning. Moderate thrombocytopenia may be associated with rodenticide poisoning.

All liver diseases may result in varied coagulopathies due to impaired coagulation factor synthesis and vitamin K malabsorption. Similarly, disseminated intravascular coagulopathies (DIC), due to many different disorders is associated with variably prolonged coagulation times. More helpful to the diagnosis of DIC are the recognition of schistocytes, thrombocytopenia, low antithrombin III levels, and increased D-dimers and fibrin split (degradation) products.

Hemostatic Tests in Clinical Practice·

Test

Normal Dog

Disorder

PCV %

37-55%

Anemias; May not be evident in first few hours

Total Protein

5.5-7.5 g/dl

Hypoproteinemias with external blood loss

Platelet estimate
Platelet count

8-15 per oil field
(1/15,000 per µl)
150-400,000/µl

Thrombocytopenias also schistocytes

Buccal mucosal
bleeding time (BMBT)

< 4 minutes

Thrombopathias vWD

von Willebrand factor (vWF)

65-150% also mutation tests for breeding

von Willebrand disease

Activated clotting
time (ACT)

<110 seconds (tube assay)

Intrinsic and common coagulopathies

Partial Thromboplastin Time (PTT)

12-16 seconds (Lab*)
54-94 seconds (SCA 2000)

Intrinsic and common coagulopathies

Prothrombin time (PT)

10-14 seconds (Lab*)
12-16 seconds (SCA 2000)

Extrinsic and common coagulopathy

Protein Induced by Vitamin K Antagonism/Absence (PIVKA)

<25 seconds

Like PT, not specific for warfarin

Thrombin time (TT)

10-12 seconds (Lab*)

Hypofibrinogenemia functional

Fibrinogen

100-300 mg/dl
(precipitated)

Hypofibrinogenemia

Fibrin split products
(FSP/FDP)

<1:5 (Lab*); <5µg/dl (Lab*)

Fibrin(-ogen) degradation in DIC

D-dimers

<250µg/dl;
-/+ with kit

Fibrin degradation in DIC

Antithrombin III

90-120% (Lab*)

Low levels with thrombosis, DIC

*Use veterinary laboratories' established reference range.
SCA 2000 instrument using fresh citrated whole blood.
Giger, U: Practical Guide to the Diagnosis of Coagulopathies, Synbiotics, 2000.

Speaker Information
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Urs Giger, PD, Dr. med. vet., MS, FVH, DACVIM, DECVIM
University of Pennsylvania
Philadelphia, PA, USA


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