Moschowitz ‘ Disease

1. Moschowitz ‘ Disease Galila Zaher MRCPath

2. Case Presentation • 20 years old Saudi patient • Easy fatigability • Easy bruising • Afebrile • Systemic examination normal • Anaemia & thrombocytopenia • Blood film • High urea & creatinin

3. Clinical course • Abdominal US • Auto-immune profile • Seizures • Plasma pharesis • Normal platelets count • Renal dialysis • Relapse

4. Thrombotic Thrombocytopenic Purpura • Thrombotic thrombocytopenic purpura (TTP) is characterized by • Microangiopathic hemolytic anemia • Thrombocytopenia • Microvascular thrombosis causes variable degrees of tissue ischemia and infarction

5. VWF Biochemistry • VWF monomers synthesized in EC • Monomers linked into multimers • Multimers constructed in megacaryocytes & EC • Stored in alpha granules & weibel-palade bodies • ULVWF entangled to sub-endothelial collagen • Bind to platelets GP1b-IX –V and activated plt GPIIb-IIIa  adhesion and aggregation Moake et al 1982

6. VWF Biochemistry • Activation, immobilization and spreading • Recruit more VWF and more platelets • Adisintegrin and metalloprotease with eight thrombospondin -1-like domain ADAMTS • Metalloprotease cleaves ULVWF in A2 domain • Impaired degradation of VWF by deficiency of metalloprotease Tsai 1996 • Characterization of ADAMTS and elicidation of its cDNA and gene structure 2001

8. ADAMTS 13 • Encoded on chromosome 9q34 • Produced mainly in liver • Activator : Zn and Ca requiring protein • Inhibitors : metal chelators & EDTA • Substrate : VWF • Plasma activity 50-178% • Half-life 2-3 days

9. Patho-physiology TTP favored by conditions that combine • Increased VWF level (late pregnancy) • Decreased ADAMTS13 activity • Two-hit model could explain substantial variation in age at which patients with inherited TTP develop symptoms.

10. Platelet-rich Microvascular Thrombi • Wide spread of intravascular thrombosis • Organ ischemia :Renal, Cerebral • Blood flows through turbulent area of partially occluded by platelet aggergates • Schistocytes • High LDH correlates with severity of ischemia

11. Clinical Presentation Pentad • Thrombocytopenia (increased marrow megakaryocytes) • Microangiopathic hemolytic anemia (MAHA) • Renal failure (50%) • Neurologic abnormality (25%) • Fever • Thrombocytopenia , schistocytes & elevated LDH • Nonspecific symptoms: weakness, abdominal pain, nausea, vomiting, and diarrhea • Median duration of symptoms prior to diagnosis was 6 days Oklahoma TTP-HUS Registry

12. Types Of TTP • Congenital TTP • Acquired idiopathic TTP • Secondary TTP

13. Congenital Familial TTP • Moschowitz ‘ Disease • Rare : Autosomal recessive • Homozygous mutations in both ADAMTS13 alleles • Both parents showing 50% of activity • Infancy or childhood • Severe ischemic brain lesions by MRI • ADAMTS13 < 5% of normal plasma • Almost always have ULVWF multimers • Response to FFP infusion is rapid :Prophylactic FFP q2-3 weeks avoid relapses

14. Conditions Associated • Bone marrow transplantation • Pregnancy & Postpartum • Drugs • Autoimmune disorders • TBI • Kidney, liver, heart, or lung transplant

15. Drug-associated TTP • Acute immune-mediated or dose-related toxic • Most common cause of immune-mediated TTP • Quinine ( isolated thrombocytopenia) • Ticlopidine (TTP and HUS) • Clopidogrel (TTP and HUS) • Mitomycin C- Cyclosporine • Tacrolimus (FK506) • Discontinuation or dose adjustment is sufficient • Trial of plasma exchange :efficacy is uncertain

16. Acquired idiopathic • Adults and older children • Sever ADAMTS 13 deficiency :acute episodes • IgG autoantibody produced transiently • Return to normal upon recovery • Mortality rate 13% • Worse prognosis :Prolonged courses & more frequent complications

17. HUS • Children, usually >5 years old • Bloody diarrhea • E.coli O157:H7 • Shiga toxin • Acute renal failure • Thrombocytopenia and MAHA • Mortality is 3-5% • Normal plasma ADAMTS13 activity • Plasma exchange treatment is rarely considered

18. Laboratory Tests • Anemia • Thrombocytopenia • Blood film : (Schistocytes > 1% of total RBCs) • High LDH (hemolysis and leakage from ischemic tissue) • High bilirubin • DCT negative • High urea and ceriatnin • PT , APTT, fibrinogen & D-dimers :normal

20. Diagnosis • No “gold standard” for diagnosis • ADAMTS13 activity • Auto-antibodies against ADAMTS13

21. Clinical Applications • Diagnosis • Discrimination of TTP from HUS • Discrimination of congenital and acquired • Estimating risk of relapse • Monitoring therapeutic efficacy of plasma exchange or plasma infusion.

22. Furlan • NP VWF as substrate • Test plasma is activated by barium chloride • Mix over night in presence of 1.5M urea • Separated by SDS agarose gel electophoresis • Followed by immunoblotting. • Excellent resolution leader • Very sensitive • Reproducible • Requires several days

23. Bohm et al • Test plasma is incubated with recombinant substrate • Separated by SDS polyacrelamide gel • Quantitate residual VWF using RIPA • Short incubation

24. Immunoassay Assay • Residual VWF binding to collagen after its degradation • ELISA simple • Fast few hours • Less sensitive and less precise • Problems with reproducibility

25. Low Levels Of ADAMTS • PLiver disease • DIC • Chronic metabolic conditions & inflammatory conditions • Uremia • HIT • Pregnancy third trimesters • Newborn • Healthy controls :levels 20-50% and none < 10%

26. Management • Untreated almost always fatal (90%) • FFP Byrnes and Khurana 1977 • Cryo-supernatant contain ADAMTS Tsai 1998 • Solvent/detergent-treated plasma contain ADAMTS Tsai and Lian and Furlan 1998

27. Plasma pheresis • Plasma exchange reduced MR from 90%- 25% • Remove circulating ULVWF multimer-platelet strings • Remove circulating auto-antibodies against ADAMTS13 • Infusion of FFP or cryosupernatant , SD or methylene blue/light-treated plasma • Response rate 80%–90% • T1/2 of infused ADAMTS13 activity is 2 days • Prompt and complete response : no further therapy

28. Risk For Relapse • Severe ADAMTS13 deficiency • Idiopathic TTP auto-Abs • Non following SCT • No relapse following drug toxicity • No relapse who had a prodrome of bloody diarrhea Oklahoma TTP-HUS Registry

29. Management Of Relapse • Most relapses occur within the first year • Suboptimal response or relapse :steroids • Sever neurologic defect :immunosuppressive treatment • Spleenectomy: eliminates autoantibody-producing B cells

30. Acquired idiopathic TTP • Lower titers better responses • High titer inhibitor wore prognosis • Inconsistent response to steroids and other immunosuppressive agents • Rituximab or cyclophosphamide • Removal of autoantibody-producing cells by splenectomy.

31. Pregnancy And TTP • During pregnancy ,postpartum 70% around time of delivery • Pre-eclampsia/HELLP :3rd trimester or following delivery, HT , protinurea & Spontaneous postpartum recovery • Observation for several days after delivery • Acute, severe multi-organ failure :prompt plasma exchange

32. TTP Following BMT • Post allogeneic : 2-76% • Post autologous : 0-27% • Mortality rate : 0-93% • DD : Renal and neurotoxicities of GVHD • Cyclosporine and Tacrolimus • Clinically suspected TTP: efforts to diagnose & treat GVHD and sepsis & delay a decision for plasma exchange • ADAMTS13 activity :normal • No response to plasma exchange

33. HIV • Typical TTP • Acquired autoantibody to ADAMTS13 • Rapidly fatal course • Plasma exchange :one plasma volume once daily • Glucocorticoids auto-antibodies to ADAMTS13

34. Thank you

35. Central Venous Catheter Insertion • Deaths • Cardiac arrest with near-death • Hemorrhage • Pneumothrax • Pericardial tamponade • Allergic reactions • Severe hypotension and hypoxia • Fatal sepsis

36. ADAMTS13 Absent Clinical Presentation

37. Tsai • Plasma samples incubated with guanidine HCl-treated VWF X1hr • Products separated by SDS–polyacrylamide gel electrophoresis • Immunoblotting • Obert et al • Plasma samples incubated with recombinant VWF overnight • Degraded VWF fragments detected by two-site immunoradiometric assay • Performed in hospital laboratory • High-throughput method • Gerritsen et al Functional assay • Preferential binding of HMWt forms of VWF to collagen. • Plasma treated with EDTA to abolishes the VWF-cleaving activity. • Dialyzed against the buffer and used as substrate.

38. Recombinant protein as ADAMTS13 substrate E coli culture • Direct assay for measuring ADAMTS13 product generation • More accurate • Protease-free VWF. • Substrate tagged with two different molecules makes it easy to modify the detection of product. • One potential disadvantage : GST-VWF73-H is not a natural substrate

39. Sensitivity And Specificity • Specificity: Severe deficiency (<5% ) is specific • Sensitivity remains questionable 66%- 100% Tsai and Lian. • Levy et al identified 12 gene mutations

41. Differential Diagnosis • Thrombotic thrombocytopenic purpura (TTP) • Immune thrombocytopenia purpura (ITP) • Autoimmune hemolytic anemia • Hemolytic uremic syndrome (HUS) • Pregnancy, eclampsia • Disseminated intravascular coagulation (DIC) • Septicemia with DIC • Systemic lupus erythematosus (SLE) • Scleroderma • Paroxysmal nocturnal hemoglobinuria (PNH)

42. Comparison of the features of TTP and hemolytic uremic syndrome (HUS)