Case challenges in hemostasis (Proceedings)

Bleeding disorders can pose diagnostic challenges, but a logical approach to testing for bleeding disorders can simplify the process. We'll go through some cases we've encountered and how the laboratory tests have helped determine the cause of the bleeding disorder.

1.) Jake

     • 9 year old neutered male Labrador

     • Presented for epistaxis and hemoptysis

2.) Sarah

     • 5 month old female Great Pyrenees

     • Presented for anorexia, lethargy, gingival bleeing and epistaxis

3.) Ozzy

     • 9 year old Jack Russell Terrier

     • Presented for 3 days history of wheezing, coughing and decreased eating and drinking.

4. )Bigby

     • 10 year old neutered male Welsh corgi

     • Presented with dyspnea, lethargy, anorexia

5.) Moe

     • 9 year old spayed female Doberman Pinscher

     • Presented with epistaxis for 1 day. Had been given one aspirin that morning.

6. )Tsunami

     • 4 year old spayed female Great Dane

     • Presented with bleeding gums, hyphema in left eye

7.) Marley

     • 9 year old spayed female golden retriever

     • Presented for lethargy, anorexia and a mass at the elbow.

Approach to diagnosis of a bleeding disorder

History

A pertinent history should include

     • Frequency, duration, severity of progression

     • Age at onset, breed, inherited/acquired

     • Type of hemorrhage and location

     • Environmental factors

     • Drug therapy

     • Presence of underlying disease that could compromise hemostasis

Physical examination

     • Type and sites of bleeding should be considered carefully

     • Small, focal hemorrhage (petechiae) in the skin and mucous membranes suggest vessel wall defect, platelet defects or von Willebrand disease. Ecchymosis can be observed in both platelet and coagulation defects.

     • Deep hemorrhages in body cavities or muscle (hematoma) suggests coagulation factor deficiency.

     • Massive bleeding from a single site without a history of a previous bleeding episode or purpura is more suggestive of a surgical or anatomic defect rather than a coagulation disorder.

Laboratory evaluation of a bleeding disorder

Sampling

Sodium citrate (light blue top tube) is the anticoagulant of choice for PT and PTT

     • Use 9 parts whole blood: 1 part sodium citrate. Tubes must be filled to the fill line or results will be artifactually altered

     • Samples should be kept refrigerated and centrifuged within 60 minutes, plasma collected and tested within 2 hours or frozen.

     • EDTA is the anticoagulant of choice for platelet counts.

Evaluation of platelet numbers

Doing a platelet count is probably the most logical first step in the work-up of any clinically bleeding patient.

Platelet estimate

An adequate platelet concentration corresponds to a minimum of 8 to 10 platelets per oil immersion field in a monolayer area of a well-made, stained blood smear. If platelets appear to be decreased, the smear should be scanned at 100 X magnification to search for clumps at the feathered edge of the smear

Platelet count by unopette and hemocytometer

The same basic Unopette used for counting white blood cells can be used for platelets. This should be done within 5 hours of drawing the blood.

Automated platelet counts

The newer hematology cell counters use whole blood or platelet rich plasma as a sample and can give the mean platelet volume, platelet histogram, and platelet count.

Interpretation

Although there are species differences, most animals have 200,000 - 500,000 platelets/µl. Less than 100,000 platelets/µl in all species constitutes a thrombocytopenia; clinical signs of hemorrhage can be seen at platelet counts <20,000/µl.

Tests of primary hemostasis

Buccal mucosal bleeding time (BMBT)

This is a measurement of the time it takes for a platelet plug to form after a superficial vessel is cut. An automated incision-making device or lancet is used to make a small cut and filter paper is used to wick away the blood without disturbing the platelet plug..

A normal bleeding time is 1-3 min in dogs. Prolonged bleeding times are seen in animals with severe thrombocytopenias, in von Willebrand's disease, and in patients with platelet function defects. This test isn't indicated when there's a thrombocytopenia, as it will be prolonged- and doesn't give you any new information.

Von willebrand's factor (VWF) assay

If BMBT is prolonged, the platelet count is within reference ranges and there is no history of use of drugs that may affect platelet function, vWF deficiency is a logical differential. vWF can be measured by activity assays or plasma concentrations by ELISA. These are usually reported as a percent of the activity or concentration in a pool of normal plasma.

Tests of secondary hemostasis

Activated coagulation time (ACT)

This is a rough evaluation of the function of the intrinsic pathway that can easily be done in a general practice. All that is required is a gray-topped tube that contains sterile diatomaceous earth (a contact activator of the intrinsic pathway). The tube needs to be at body temperature when the blood hits the diatomaceous earth. The easiest way to do this is to hold it in your armpit. Excellent venipuncture technique is required to avoid introducing tissue factor. Blood is drawn into the tube and mixed well- at the same time a stopwatch is started. After one minute of incubation, the tube is checked every 5-10 seconds for the first formation of fibrin strands. At the first sign of fibrin, the stopwatch is stopped. Factors must be fairly markedly decreased to prolong the ACT.

Normals: Dogs = 60-110 sec; Cats, = 50-75sec; horses 120-190 sec.

Partial Thromboplastin Time (PTT)

This is a test of the intrinsic and common pathway function. The instruments and reagents used in human laboratories are the same as those used in veterinary laboratories, but there can be variation in reference ranges among labs. It's a good idea to submit a sample from a "normal" animal from the same species as your patient if you send a sample to a predominantly human laboratory. Reference intervals from one lab: Dogs = 8-13 seconds, Cats =10-15 seconds, Horses = 27-39 seconds.

Prolonged PTT usually reflects decreased production or increased consumption of factors in the intrinsic and common pathway. Hereditary defects, such as hemophilia A (Factor VIII deficiency) may also occur.

Prothrombin time (PT)

This test is often performed along with the PTT as it assesses the extrinsic and common pathway. Sample and laboratory considerations are the same as for the PTT.

Again- reference ranges may vary from lab to lab. Here's one: Dogs = 6-8 seconds, Cats = 4-10 seconds, Horses = 8-10 seconds.

The PT is the test most often used to assess therapy for warfarin toxicity

Interpretation

Defect in intrinsic pathway - normal PT and prolonged PTT

Defect in extrinsic pathway - normal PTT and prolonged PT

Multiple defects or defect in common pathway - prolonged PT and PTT

Evaluation of fibrinolysis

Fibrin/fibrinogen degradation products (FDPs)

These are produced by proteolytic action of plasmin on fibrinogen and fibrin and thus reflect the activity of the fibrinolytic system. Samples must be collected in specialized tubes that contain either thrombin or snake venom (the venom produces hemolysis but does not alter FDP results). The serum has to be collected promptly (in 30 minutes) and should be tested right away or frozen.

Normal FDP <10 µg/ml. In DIC, the concentration is usually > 40 µg/ml. Severe internal bleeding or thrombosis can increase FDPs- but usually only to > 10 µg/ml but < 40 µg/ml.

D-dimer assay

The D fragment (two combine to form the D-dimer) is a product of plasmin degradation of cross-lined fibrin. The D-dimer assay is therefore more specific assay for fibrin formation and breakdown than is the FDP assay. As there is lots of species variation in how the various test kits available work, be sure your laboratory is using a kit that's validated for use in the given species.