Practical Hematology Diagnosing Coagulopathy

1. Practical HematologyDiagnosing Coagulopathy Wendy Blount, DVM

2. Practical Hematology • Determining the cause of anemia • Treating regenerative anemias • Blood loss • Hemolysis • Treating non-regenerative anemias • Blood & plasma transfusions in general practice • Determining the causing of coagulopathies • Treating coagulopathies in general practice • Finding the source of leukocytosis • Bone marrow sampling

3. Hemostasis Primary hemostasis • Platelets plug up damaged blood vessels • Von Willebrand Factor (vWF) • Vasoconstriction Secondary hemostasis • Platelet plug organized by fibrin • Fibrin generated by coagulation cascade (factors) Fibrinolysis

4. Coagulopathies Poor Clotting • Not enough hemostasis • Problem with primary and/or secondary hemostasis so that clots do not form and stabilize normally Thromboembolic Disease • Too much hemostasis • Natural anticoagulants are missing • Or Fibrinolytics are missing

5. Signs of Bleeding Primary hemostasis – small vessel hemorrhage • Immediate bleeding • Petechiae (except vWDz) - <3 mm • Ecchymoses • Bleeding from surfaces • Nose, mouth • Ocular - hyphema • GI – hematemesis, melena, hematochezia, hematuria • repro • Prolonged bleeding after injury or surgery

6. Signs of Bleeding • Secondary hemostasis • Delayed bleeding after injury or surgery • Bleeding into cavities • Joints, pleural space, abdomen • CNS • Muscular hematomas • Hemoptysis – can present as melena • Blood coughed up, swallowed and digested • Both primary and secondary • Epistaxis • Ecchymoses • If severe 1o and 2o bleeding types blend

7. Assessment of Coagulation • Is bleeding appropriate to injury? • Control arterial bleeding with ligation • If not, assess coag status ASAP • Platelet count • PT, PTT/ACT • BMBT • FDPs, D-dimers • Factor assays

8. Coagulation Tests Primary hemostasis • Platelet estimate • Count platelets in 10 HPF (oil) • Divide by 10 to get average • Multiply by 15-20,000 • 8-15 platelets/HPF is adequate • Automated platelet count • Look at the feathered edge for platelet clumping (cats!!) • If clumping, get a new sample • Use citrate (blue top) and count immediately

9. Coagulation Tests Primary hemostasis • MPV – Mean Platelet Volume • Increased in dogs when immature platelets in circulation • Indicates increased platelet pdxn • DIC, vasculitis, chronic hemorrhage • Decreased with IMT - 50% • Large, bizarre platelets • Otter hound thrombocytopathia • Cats with myeloproliferative disease

10. Coagulation Tests Primary hemostasis • Platelet function tests • Academia • Must be performed within 2-3 hours of blood collection

11. Coagulation Tests Primary hemostasis • Buccal mucosal Bleeding Time (BMBT) • Assesses primary hemostasis • Prolonged if platelets <20,000/ul • Rebleeding can indicate problems with secondary hemostasis • The only in clinic test that evaluates vessel and platelet function • Screen for vWDz in likely suspects • Reasonable pre-operative estimate of likelihood of surgical hemorrhage, if you check for rebleeding

12. Coagulation Tests Primary hemostasis • How severe must Tpenia to cause spontaneous bleeding? • Can happen <50,000/ul • More often <20,000/ul • Splenomegaly of any kind can result in thrombocytopenia • Platelets sequestered in the spleen, liver or lymph nodes

13. Coagulation Pathway

14. Coagulation Pathway

15. Coagulation Pathway

16. Coagulation Tests Secondary hemostasis • Partial thromboplastin time (PTT) • Intrinsic and common pathways • Heparin acts on intrinsic pathway • Prothrombin time (PT) • Extrinsic and common pathways • PIVKA is a form of PT test • PIVKA is not specific for rodenticide toxicity • Factor 7 deficiency • Common pathway - DIC, liver failure • No clinical significance when PT/PTT are shorter than normal • PT <3 sec and PTT <5 sec prolonged may not be clinically significant

17. Coagulation Tests Secondary hemostasis • Activated clotting time (ACT) • Less sensitive version of PTT • Intrinsic and common pathways • Platelets as well • Thrombocytopenia can elevate ACT by <10% • No such platelet effect on PTT • Factors must be 5% of normal for ACT to be elevated • If ACT is increased, things are really bad

18. Coagulation Tests Secondary hemostasis • Thrombin time (TT) • Assesses fibrinogen activity • Part of common pathway

19. Coagulation Tests Fibrinolysis • Fibrin degradation products (FDPs) • AKA FSPs – fibrin split products • Measure fibrinolysis • High with clot formation and breakdown over time • DIC • Chronic bleeding • Hypercoagulable state • Neoplasia • Cushings Disease • PLN, PLE • IMHA

20. Coagulation Tests Fibrinolysis • D-dimers • Measure fibrinolysis • More specific for DIC than FDPs • Normal D-dimers exclude DIC as a diagnosis with 99.5% confidence level

21. Anticoagulants • Antithrombin III • Produced by the liver • Activated by heparin • Modulates excessive coagulation • Consumed by coagulation • Lost with albumin – PLN, PLE • Protein C (vitamin K dependent) • Protein S (vitamin K dependent) • TFPI – Tissue Factor Pathway Inhibitor

22. Coagulation Pathway –THAT’S Not ALL!!

23. Fibrinolysis • tPA – tissue Plasminogen activator converts plasminogen within the clot to plasmin • Plasmin breaks down fibrin clot • So tPA promotes fibrinolysis, so that clots are only temporary • Urokinase and streptokinase work similarly • tPA, urokinase and streptokinase are the “clot busters”

24. Excessive Fibrinolysis • Enzymes prevent excessive fibrinolysis which would lead to rebleeding • They break down the clot busters • Alpha2-antiplasmin • inactivates plasmin • PA1-I: tPA-inhibitor1 • Inactivates tPA and uPA • uPA – urokinase plasminogen activator • FDPs and d-Dimers inhibit the coagulation cascade by negative feedback

25. Coags in Practice Why do them at all??? • Patient shows signs of coagulopathy • Excessive bleeding • thrombosis • Presurgical evaluation • Patient predisposed to coagulopathy • Procedure increases risk of bleeding • Rodenticide toxicity suspected • DIC suspected • Genetic screening for breeding • No expensive equipment for platelet count, ACT, BMBT • Can send out the rest

26. Coags in Practice Platelet count Partial thromboplastin time (PTT) Prothrombin time (PT) • Reference lab • Human hospitals often not calibrated for animals (Pluto) • Synbiotics SCA 2000 • $2000-3000 • Idexx Coag Dx

27. Coags in Practice Activated clotting time • Reference labs • Gray top tubes (other colors now) • Diatomaceous earth (DE) or kaolin • http://www.haemtech.com/ACT.htm • Warming block or hand heat • 2 ml whole blood in the tube immediately • Invert once every 15-30 seconds • First clot is the ACT • Normal less than 2 minutes • SCA 2000 • HESKA i-STAT

28. Coags in Practice

29. Coags in Practice

30. Coags in Practice

31. Coags in Practice Buccal Mucosal Bleeding Time (BMBT) • Simplate, Triplett, Surgicutt device • Lift the upper lip (gauze muzzle) • Remove the device safety tab • Place the device on the mucosa • Push the device trigger button • Dab dripping blood every 15 seconds, but don’t touch the clot

32. Coags in Practice Buccal Mucosal Bleeding Time (BMBT) • When bleeding stops, you have BMBT • Normal is 2-4 minutes • 5 minutes isn’t worrisome • Check the patient in 10-15 minutes for rebleeding • DON’T DO BMBT IF: • Platelets <40,000/ul • Petechiae are present • ACT is increased

33. Coags in Practice EVERY PRACTICE can have in house PLATELET COUNT, ACT & BMBT EVERY PRACTICE can use a lab for the rest

34. Coags in Practice Tips for coag test sample handling • Take blood from a peripheral vein • Avoid cystocentesis if coagulopathy • Holding the vein off for too long can lead to platelet & fibrinolysis activation • Multiple sticks can lead to the same – you want a clean first stick. • If you don’t get blood quickly, move to another vein. • Hemolysis and severe lipemia can prevent accurate results

35. Coags in Practice Tips for coag test sample handling • 1 citrate:9 blood • Vacuum tubes should autodraw • Run platelet count immediately • Run tests on whole citrated blood within 2 hours • Centrifuge promptly to harvest plasma for outside lab tests • Freeze immediately in plastic or siliconized glass tubes • Ship frozen on dry ice • May need special blue top clot tubes for FDPs – ask lab

36. Coagulopathies - DDx Disorders of primary hemostasis • thrombocytopenia • thrombocytopathia • Von Willebrand Disease • vasculitis Disorders of secondary hemostasis • Congenital factor deficiencies • Liver disease (poor pdxn) • Vitamin K antagonism • Snake bite envenomation Disorders of both • Consumption – DIC, massive hemorrhage • Paraneoplastic coagulopathy

37. Thrombocytopenia - DDx • Bone marrow disease – lack of production • Consumption • DIC • Massive hemorrhage • Destruction • immune mediated • Sequestration • Splenomegaly • Hepatomegaly • Lymphadenitis, lymphangitis • vasculitis

38. Thrombocytopenia - DDx Infectious Diseases Multiple causes • Viruses • Canine – CDV, CHV, CPV, CAV2 • Feline – FeLV, FIV, panleukopenia, FIP • Arthropod-Borne • Ehrlichia spp. • Babesia spp. • Heamobartonella spp. • Arthropod-Borne • Rickettsia spp. • Leishmania spp. • Cytauxzoon spp. • Borrelia spp. • Bacterial • Sepsis • Borrelia spp. • Leptospira spp. • Fungal • Histoplasma spp. • Candida spp.

39. Thrombocytopenia - DDx Neoplasia Multiple causes • Marrow suppression • Metastatic disease • Hematopoietic neoplasia • Lymphoma • Multiple myeloma • leukemias • DIC • Hemangiosarcoma • Inflammatory mammary carcinoma • Vasculitis • Cytotoxic drug therapy • Azathioprine • Chlorambucil • Cyclophosphamide • Doxorubicin

40. Thrombocytopenia - DDx Drug Therapy Multiple causes • Impaired platelet pdxn • Immune destruction • Platelet dysfunction Antibiotics • Penicillin • Chloramphenicol • Sulfonamides Antifungals NSAIDs • Ibuprofen Cardiopulmonary drugs • Procainamide Cytotoxic drugs • Azathioprine • Chlorambucil • Cyclophosphamide • Doxorubicin Estrogen Methimazole

41. Immune Mediated Thrombocytopenia • Primary – autoimmune • Very rare in cats • Dog breeds predisposed • Same as IMHA • Cocker spaniel • Poodle • Old English Sheepdog • Diagnosis of exclusion • Rule out other causes of Tpenia (normal coags, normal BMBT unless <5K platelets) • Rule out causes of secondary IMT • Platelets <50,000/ul • Increased megakaryocytes in the marrow • Response to immunosuppressive therapy

42. Immune Mediated Thrombocytopenia • Secondary – same as for IMHA • infection • Drug therapy • Paraneoplastic • transfusion • Anti-platelet antibodies • Doesn’t distinguish between 1o & 2o IMT • Sensitive for IMT • But not very specific (many false negatives) • Low MPV – microthrombocytosis • MPV < 5.5 fL + platelets <20,000/ul is almost always IMT (primary or secondary) • But only 50% of dogs with IMT have this combination of abnormalities

43. Breed Specific Thrombocytopenia • Greyhounds • Normal platelet count 150,00/ul • Coags normal • Remember greyhounds also predisposed to von Willebrand Disease and Babesia • Cavalier King Charles Spaniel • Normal coags • Normal platelets 25,000/ul-100,000/ul • Giant platelets on blood smear • CBC machines won’t count them • Platelet mass usually normal • Platelet mass = Platelet count x MPV • Causes no clinical problems

44. Thrombocytopathia • Platelet dysfunction • hereditary • acquired • Acquired Thrombocytopathia • Drugs • Disease • Anemia • Liver failure (plus factor deficiency) • Uremia (plus vasculitis) • DIC (plus consumptive coagulopathy) • Paraproteinemia • Monoclonal gammopathy • Ehrlichia, plasma cell myeloma

45. Thrombocytopathia - DDx Drug Therapy Antibiotics • Beta lactams • Carbenicillin • cephalosporins Antifungals NSAIDs • Aspirin • Phenylbutazone • Ibuprofen • Naproxen Cardiopulmonary drugs • Aminophylline • Verapamil • Diltiazem • Isoproterenol • Propranolol Dextran Phenothiazines CAUTION in pets with thrombocytopenia, undergoing surgery or signs of coagulopathy

46. Thrombocytopathia • Hereditary Thrombocytopathia • Likely underdiagnosed • Otter hound, Great Pyrenees – thrombasthenia • Basset hound, Spitz • Persian cat – Chediak-Higashi • Cocker spaniel • Collie – cyclic neutropenia (stem cell defect) • Boxer • German shepherd • Consider BMBT prior to major surgery

47. von Willebrand Disease Most common canine hereditary coagulopathy in dogs • Does not occur in cats • Clinical signs of primary hemostasis defect • Mucosal hemorrhage, prolonged bleeding after injury • Petechiae are rare • (Indy) • von Willebrand Factor is not a coagulation factor • Made by the endothelium • Acts as carrier protein for factor 8 • Severe vWDz can cause result in bleeding due to lack of secondary hemostasis

48. von Willebrand Disease • Three subtypes of vWDz • Type 1 vWDz – most common – complex genetics • Mild to moderate bleeding • Low plasma vWAg, normal multimer distribution • Type 2 vWDz – simple recessive • Moderate to severe bleeding • Low vWAg, loss of high MW multimers • Type 3 vWDz – simple recessive • Severe bleeding • Total lack of vWF

49. von Willebrand Disease • Diagnosis • Platelet count • Normal (may be mildly low if hypothyroid) • PT, PTT/ACT • Normal (increased of Type 3) • BMBT • Type 1: 5-12 minutes • Type 2&3: >12 minutes • vWFAg assay • carriers: 35-65% • Type 1: 5-30% (clinical <15%) • Type 2: 1-5%, no high MW multimers • Type 3: <0.1%

50. von Willebrand Disease Type 1 - mild Airedale Akita Dachshund Doberman German shepherd Golden retriever Greyhound Irish wolfhound Manchester terrier Pembroke Corgi Poodle Schnauzer Sheltie Type 2 - moderate German shorthair pointer German wirehair pointer Type 3 - severe Chesapeake Bay Retriever Dutch kooiker Scottish terrier Sheltie Less commonly: Cocker spaniel Spitz Labrador retriever Pit bull terrier Rottweiler DNA Tests available for some breeds