Thrombotic Thrombocytopenic Purpura and the Role of ADAMTS-13

Transcription

1 Thrombotic Thrombocytopenic Purpura and the Role of ADAMTS-13 Mark Cunningham,MD Director, Hematology Laboratory Department of Pathology University of Kansas Medical Center College of American Pathologists Materials are used with the permission of Mark Cunningham, MD, FCAP. 1

2 Course Objectives Review the definition, diagnosis, classification, pathophysiology, and treatment of TTP Discuss the role of ADAMTS-13 in the pathophysiology, diagnosis, classification, and treatment of TTP 2

3 TTP and ADAMTS-13 Lecture Outline I. Review of TTP II. Role of ADAMTS-13 in TTP A. Laboratory assays B. Diagnosis C. Classification D. Treatment III. Case Studies IV. Summary V. Pearls of Pathology 3

4 TTP: Definition Microvascular occlusive syndrome Systemic platelet aggregation Multiple organ ischemia Severe thrombocytopenia Red cell fragmentation 4

5 TTP: Definition Purpura Schistocytes Thrombocytopenia Microvascular Thrombosis 5

6 TTP: Diagnosis Diagnostic Criteria 1. Microangiopathic hemolysis - elevated LDH, schistocytes on blood smear 2. Thrombocytopenia 3. Fever, renal failure, and neurologic manifestations are NOT required for diagnosis 4. Exclusion of similar disorders - hemolytic uremic syndrome - disseminated intravascular coagulation - malignant hypertension - eclampsia - vasculitis - HEELP syndrome - Evans syndrome 6

7 TTP: Diagnosis Disorders Mimicking TTP Disorder TTP HUS DIC Malignant hypertension Eclampsia Vasculitis Mechanism ADAMTS-13 deficiency Shiga toxin ST1 or ST2 Excess tissue factor High blood pressure Unknown vasoconstrictor Hypersensitivity reaction Treatment Plasma exchange Supportive Antibiotics, chemotherapy, deliver placenta, etc. Anti-hypertensives Deliver placenta Immunosuppressives 7

8 TTP: Classification Classification Frequency Age of Onset Clinical Course Inherited 10% Infancy Recurrent episodes Childhood (regular intervals) Acquired 90% Adult Single episode, or Recurrent episodes (irregular intervals) 8

9 TTP: Classification Acquired TTP Idiopathic Drugs Autoimmune disorders Pregnancy Infection Stem cell transplant Malignancy 9

10 TTP: Classification Drug Associated TTP Cardiovascular - quinine Immunosuppressive - cyclosporine A, tacrolimus (FK506) Chemotherapy - mitomycin C, carmustine, pentostatin Antithrombotic - ticlopidine (Ticlid), clopidogrel (Plavix) 10

11 TTP: Pathophysiology Von Willebrand Factor (VWF) Synthesis - endothelial cells and megakaryocytes Primary structure - single polypeptide (MW = 280 kd) - several repetitive domains (A1-3, B, C1-2, D1-4) Quaternary structure - monomers disulfide linked into multimers - varying multimer size (600 to 20,000 kd) 11

12 TTP: Pathophysiology Von Willebrand Factor (VWF) Function - platelet adhesion (collagen, GP Ib/V/IX) - platelet aggregation (GP IIb/IIIa - high shear) - factor VIII carrier protein Cell Biology - endothelial cells produce unusually large von Willebrand factor multimers (ULVWF) - ULVWF mediate platelet aggregation (high shear stress) - ULVWF are absent from normal plasma 12

13 TTP: Pathophysiology VWF Structure and Function ULVWF Multimers Multimer Structure Platelet Adhesion Factor VIII Carrier Protein 13

14 TTP: Pathophysiology Von Willebrand Factor (VWF) Abnormality in TTP - ULVWF multimers persist in plasma - ULVWF mediate spontaneous platelet aggregation and thrombosis in arterial microcirculation (high shear stress) 14

15 TTP: Pathophysiology ULVWF Multimers Moake JL. Sem Hematol 41: 4-14,

16 TTP: Pathophysiology Acronym ADAMTS-13 - a disintegrin and metalloprotease with thrombospondin - like domains - ADAMTS family (18 members) Synonym - VWF cleaving protease Synthesis - Liver perisinusoidal cells 16

17 TTP: Pathophysiology ADAMTS-13 Primary Structure - Single polypeptide (180kD) - Multiple distinct domains Gene - 9q34 Function - Specific proteolytic activity against VWF Cofactors - Zinc and calcium 17

18 TTP: Pathophysiology ADAMTS-13 Structure and Function Moake JL. Sem Hematol 41: 4-14, 2004 Plaimauer B et al. Sem Hematol 41: 24-33,

19 TTP: Pathophysiology ADAMTS-13 Abnormality in TTP - Decreased activity due to gene mutation (inherited) - Decreased activity due to autoantibody (acquired) 19

20 TTP: Pathophysiology Moake JL. Sem Hematol 41: 4-14,

21 ADAMTS-13: Laboratory Assays General Principle Add patient plasma to source of VWF Use special buffer conditions - Denaturant (urea, GnHCl) - Barium chloride - Alkaline ph - Low ionic strength - Prolonged incubation (2-24 Hrs) Measure decrease in substrate or increase in product: electrophoresis, VWF function, VWF antigen Compare patient data to standard curve prepared using dilutions of pooled normal plasma EDTA irreversibly inhibits ADAMTS-13 21

22 ADAMTS-13: Laboratory Assays NHP ADAMTS-13 Activity (%) Electrophoresis ELISA: Type III Collagen Binding Gerritsen et al. Thromb Haemost 82: ,

23 ADAMTS-13: Laboratory Assays Assay Method Principle Reference Range Literature Citation Electrophoresis ULVWF multimers Decreased substrate > 50% Furlan et al, 1997 Electrophoresis VWF fragmentation dimers Increased product 68% - 126% Tsai et al, 1998 Functional Collagen binding activity Decreased substrate 29% - 119% Gerritsen et al, 1999 Functional Ristocetin cofactor activity Decreased substrate 52% - 134% Bohm et al, 2002 Antigenic ULVWF multimers Decreased substrate 44% - 178% Obert et al,

24 ADAMTS-13: TTP Diagnosis Sensitivity of ADAMTS-13 Deficiency in TTP Highly Variable 15% - 100% (6 studies) Marques MB et al. Am J Clin Pathol 121(Suppl): S89-S96,

25 ADAMTS-13: TTP Diagnosis Sensitivity of ADAMTS-13 Deficiency in TTP TTP Classification Inherited Acquired - Idiopathic Acquired - Autoimmune Acquired - Malignancy Acquired - Stem Cell Tx Acquired - Pregnancy Acquired Ticlopidine/ Sildenafil ADAMTS-13 Deficiency (<5%) 100% (33/33) 52% (56/108) 23% (10/43) 5% (1/21) 5% (1/19) 56% (5/9) 100% (8/8) ADAMTS-13 Inhibitor 0% (0/33) 79% (54/68) 48% (13/27) 5% (1/21) 5% (1/19) 44% (4/9) 100% (8/8) Matsumoto M, et al. Sem. Hematol. 41(1): 68-74,

26 ADAMTS-13: TTP Diagnosis Specificity of ADAMTS-13 Deficiency Clinical Condition No. of Patients ADAMTS-13 Activity, mean + SD ADAMTS-13 Activity, range Metastatic cancer 20 4% + 5% NA Acute inflammation 15 40% + 12% 15% 59% Liver cirrhosis 42 46% + 33% 6% - 146% Newborn 26 52% + 7% 25% - 118% Postoperative 24 58% + 27% 18% - 131% Pregnancy 2 nd trimester 19 64% + 34% 22% - 135% 3 rd trimester 32 64% + 15% 38% - 102% Chronic uremia 63 74% + 30% 15% - 140% Mannucci PM et al. Blood 98: , 2001 Oleksowitcz L et al. Cancer Res 59: ,

27 ADAMTS-13: TTP Diagnosis Specificity of ADAMTS-13 Deficiency Type of HUS Number ADAMTS-13 ADAMTS-13 ADAMTS-13 ADAMTS-13 < 3% 3 25% 26 50% > 50% Idiopathic 34 0% 12% 53% 35% E. Coli 0157:H7 7 0% 0% 71% 29% Mitomycin C 3 0% 33% 33% 33% Matsumoto M, et al. Sem. Hematol. 41(1): 68-74,

28 ADAMTS-13: TTP Classification ADAMTS-13 Activity > 5% ADAMTS-13 Inhibitor ND Interpretation Cannot Rule Out TTP < 5% < 5% Negative Positive Rule In TTP - hereditary if positive family history Acquired TTP - regardless of family history 28

29 ADAMTS-13: TTP Treatment TTP Classification ADAMTS-13 Activity Treatment Response Rate Inherited < 5% Plasma Infusion 100% - Every 3 to 4 weeks Acquired < 5% Plasma Exchange 83% Acquired > 5% Plasma Exchange 33% Matsumoto M, et al. Sem. Hematol. 41(1): 68-74,

30 Case Study 1 History: 4 yo girl presents with 1 week history of skin bruising and fever. Family history: Two cousins died of similar disorder Physical exam: Multiple petechiae and ecchymoses over arms, legs, trunk, and back. 30

31 Case Study 1 (Cont.) Laboratory Test Hemoglobin White blood cells Platelets Blood smear Lactate dehydrogenase Creatinine Prothrombin time Partial thromboplastin time Fibrinogen D-Dimer ADAMTS-13, activity ADAMTS-13 inhibitor Patient Result 7.2 8,000 10, schistocytes s 300 < 0.5 2% Negative Reference Range g/dl ,000 /ul 150, ,000 /ul < 1 schistocyte /HPF U/L mg/dl s s mg/dl < 0.5 mg/l 30% - 120% Negative 31

32 Case Study 1 (Cont.) Question 1. What is the diagnosis? Answer: Inherited TTP (Upshaw-Schulman syndrome) Question 2. What is the most appropriate therapy? Answer: Daily plasma exchange for initial therapy. After ADAMTS-13 result confirms diagnosis of inherited TTP, then plasma infusion (every 3-4 weeks) would be appropriate. 32

33 Case Study 2 History: 8 yo boy presents with 3 day history of bloody diarrhea and fever. Symptoms began 1 day after eating a hamburger at a local restaurant. Family history: Negative Physical exam: Negative for petechiae or bruising 33

34 Case Study 2 (Cont.) Laboratory Test Hemoglobin White blood cells Platelets Blood smear Lactate dehydrogenase Creatinine D-Dimer ADAMTS-13, activity ADAMTS-13 inhibitor E. Coli Shiga-like toxin Patient Result ,000 50,000 8 schistocytes < % Not done Positive Reference Range g/dl ,000 /ul 150, ,000 /ul < 1 schistocyte /HPF U/L mg/dl < 0.5 mg/l 30% - 120% Negative Negative 34

35 Case Study 2 (Cont.) Question 1. What is the diagnosis? Answer: Hemolytic uremic syndrome Question 2. What is the most appropriate therapy? Answer: Supportive therapy for renal failure 35

36 Case Study 3 History: 45 yo woman presents with one week history of headache, disorientation, and lethargy Past Medical History: Negative. Not taking medicines Family history: Negative Physical exam: Petechiae and ecchymoses noted on legs 36

37 Case Study 3 (Cont.) Laboratory Test Hemoglobin White blood cells Platelets Blood smear Lactate dehydrogenase Creatinine Prothrombin time Partial thromboplastin time Fibrinogen D-Dimer ADAMTS-13, activity ADAMTS-13 inhibitor Patient Result ,200 15, schistocytes s 480 < 0.5 1% Positive, 3.0 BU Reference Range g/dl ,000 /ul 150, ,000 /ul < 1 schistocyte /HPF U/L mg/dl s s mg/dl < 0.5 mg/l 30% - 120% Negative 37

38 Case Study 3 (Cont.) Question 1. What is the diagnosis? Answer: Acquired idiopathic TTP (autoimmune) Question 2. What is the most appropriate therapy? Answer: Daily plasma exchange until platelet count >150 K/uL for 2 consecutive days. Consider immunosuppressive therapy if no response or relapse (Rituximab, etc.) 38

39 Summary 1. ADAMTS-13 maintains normal hemostasis by degrading unusually large VWF multimers 2. Severe ADAMTS-13 deficiency (<5% activity) contributes to the pathogenesis of TTP 3. ADAMTS-13 deficiency due to genetic mutation is a common cause of inherited TTP 4. ADAMTS-13 autoantibodies is a common cause of acquired TTP 5. Severe ADAMTS-13 deficiency predicts a good response to plasma exchange 39

40 Pearls of Pathology Diagnosis of TTP Diagnosis of TTP is based on 3 criteria: thrombocytopenia, microangiopathic hemolysis, exclusion of similar diseases ADAMTS-13 specimen should be collected prior to plasma exchange ADAMTS-13 specimen should be collected in citrate anticoagulant, NOT EDTA 40

41 Pearls of Pathology Diagnosis of TTP Severe ADAMTS-13 deficiency (< 5% activity) - confirms diagnosis of TTP - rules out HUS Mild to moderate ADAMTS-13 deficiency (> 5% activity) is very nonspecific Normal ADAMTS-13 level DOES NOT rule out TTP 41

42 Pearls of Pathology Classification of TTP Severe ADAMTS-13 deficiency without an inhibitor, plus a family history of a TTP-like syndrome, is diagnostic of inherited TTP Severe ADAMTS-13 deficiency due to an inhibitor is diagnostic of acquired TTP 42

43 Pearls of Pathology Treatment of TTP ADAMTS-13 levels should NOT be used to determine if plasma exchange should be initiated for acquired TTP ADAMTS-13 levels should NOT be used to determine the duration of plasma exchange therapy ADAMTS-13 levels should NOT be used to predict relapse 43