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“What is ADAMTS13 Anyway?”

“What is ADAMTS13 Anyway?”. Maxwell Smith, MD August 19th, 2005. Objectives. Review the pathology, epidemiology, differential diagnosis, and treatment of Thrombotic Thrombocytopenic Purpura (TTP) using a case presentation

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“What is ADAMTS13 Anyway?”

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  1. “What is ADAMTS13 Anyway?” Maxwell Smith, MD August 19th, 2005

  2. Objectives • Review the pathology, epidemiology, differential diagnosis, and treatment of Thrombotic Thrombocytopenic Purpura (TTP) using a case presentation • Describe the discovery of ADAMTS13 (von Willebrand Factor Cleaving Protease) and its role in TTP • Introduce selected testing methods for vWF-CP and evaluate their use in the diagnosis and management of TTP

  3. 35 year-old Hispanic Female • Presents with easy bruising and abdominal pain • Multiple bruises had developed “all over” without traumatic history • Multiple small red spots as well • No neurologic complaints • 1 week prior had URI symptoms and diarrhea

  4. Physical Exam • Vitals: Temp 38.5, HR 94, BP 121/75 • Active gingival and mucosal bleeding • Innumerable ecchymoses over all extremities and trunk • No neurologic abnormalities • Scattered petechiae on BUE and BLE • Cervical lymphadenopathy

  5. Lab Studies • CBC: WBC 12.8, HCT 30, PLTC 12 • BUN/Cr: 14 / 0.9 • CoAg: PT 13.2, PTT 40.9, d-dimer 3.91 • T-bili: 2.5 (2.4 unconjugated) • LDH 1522 • Peripheral smear • Thrombocytopenia • 1+ schistocytes • Direct Coombs neg. • Drug screen neg.

  6. Brief Differential Diagnosis • TTP vs. Hemolytic uremic syndrome vs. ITP • TTP • Classic pentad • Patients often have only 3/5 • HUS • Lack of neurologic defects • “Prominence” of renal compromise • ITP • Isolated thrombocytopenia with no other clinical findings - A diagnosis of exclusion • No anemia, fever, neuro, or renal signs/symptoms

  7. TTP - Classic pentad • Fever • Thrombocytopenia • Transient neurologic dysfunction • Microangiopathic hemolytic anemia • Renal failure YES YES NO YES NO

  8. TTP Pathology • Unique composition of microvascular thrombi • Platelet rich • vWF rich • Fibrin poor (lack of involvement of the traditional clotting cascade) • Sheer forces lead to fragmentation of the RBC - hemolytic anemia with schistocytes • Vascular compromise leads to end organ dysfunction (kidneys, brain, etc.)

  9. TTP - Epidemiology • Classic history is sudden onset of symptoms in an otherwise health adult • Propensity for females of child bearing age

  10. TTP - Treatment • Plasma exchange • Started in 1970’s • Changed prognosis from >90% mortality to <25% mortality • Effectiveness related to the proposed etiology of TTP

  11. “What is ADAMTS13 Anyway?” • Moake et al., 1982 • In 4 patients with chronic TTP, large multimers of vWF were identified in their serum • Multimers were similar to those found in media surrounding in vitro endothelial cells suggesting vascular cells as a possible source • Possible failure of cleavage of the vWF multimer was causing TTP

  12. ADAMTS13 discovery • 1996 - 2 groups identified a 300 kD metalloprotease which cleaved the vWF multimers at a Tyr-Met bond • Required divalent cations (inhibited by calcium chelating agents) • Kinetics were slow in undisturbed plasma • Mild denaturation of the vWF protein or fluid shear stress accelerated the reaction

  13. ADAMTS13 discovery cont. • Levy et al.,2001 • Mapped the gene for the metalloprotease to chromosome 9q34 with linkage analysis • Identified a new member of the ADAMTS family of zinc metalloproteinases, ADAMTS13 • Identified 12 mutations in patients with hereditary TTP clinical picture

  14. Von Willebrand Factor Cleaving Protease, vWF, and Platelets Under Normal Conditions vWF-cleaving protease (ADAMTS13) Tyr-Met AA bond in vWF Receptor for GP Ib on the platelets Platelet

  15. vWF and Platelets When Von Willebrand Factor Cleaving Protease is Absent or Deficient

  16. vWF and Platelets When Von Willebrand Factor Cleaving Protease is Absent or Deficient, Cont.

  17. Back to Our Patient • Heme/Onc diagnosed TTP clinically • Sample drawn for vWF-CP testing and sent to the Blood Center of Wisconsin • 7 rounds of plasma exchange over 4-5 days • Patient discharged home with normal platelet count • Lab test pending

  18. Laboratory Testing for vWF-CP • Genomic Studies • Limited use unless documented family history of TTP like illness (FISH analysis for multiple known genetic mutations in the ADAMTS13 gene) • Activity & Inhibitor Studies • Wide variety of methods currently in use • Initial methods required laboratory and personnel expertise • Subsequent methods have decreased turnaround time and complexity

  19. Flaws in current vWF-CP Testing • The test result is invariably compared to the current gold standard for TTP diagnosis - clinical (universally accepted to be difficult and often incorrect) - misclassification bias • Most measure enzyme activity indirectly • Most carry out enzymatic reaction in non physiologic conditions • No standardization of methods

  20. Questions to be Answered from the Literature • 1. What test is our send out lab using and does it work? • 2. How well do the various testing methods compare with each other? • 3. How has this specific test (or similar tests) been evaluated and how well has it performed?

  21. 1. What test is our send out lab using and does it work?

  22. Gerritsen et al., 1999 • Assay of von Willebrand Factor (vWF)-cleaving Protease Based on Decreased Collagen Binding Affinity of Degraded vWF • Developed a simple activity and inhibitor assay for vWF-CP • Evaluated the test in 40 patients

  23. Gerritsen et al., 1999 • Activity Assay • Deactivate donor plasma vWF-CP with EDTA • Mix donor and patient plasma • If vWF-CP is present and functional in the patient sample, it will cleave vWF-multimers in the donor plasma (more vWF-CP, more vWF monomers, less vWF multimers) • Add solution to plates coated with human type III collagen (preferentially binds vWF multimers) • Quantify the collagen bound vWF multimers with a anti-vWF multimer peroxidase labeled antibody • Measure absorbance at 492nm • Calibration curve done with serially diluted donor plasma samples

  24. Gerritsen et al., 1999 • Inhibitor assay • Add non-deactivated donor plasma with patient sample in 1:1 ratio • If antibodies to vWF-CP are present in the patient sample, they will bind and deactivate vWF-CP from the donor plasma • Perform same test as previously described

  25. Gerritsen et al., 1999 • Plasma sample selection • Based on clinical findings (classic pentad) and a history of relatives with a similar condition • 10 “normal” control patients • 10 with familial TTP • 11 with acquired TTP • 9 with HUS

  26. Gerritsen et al., 1999 N 10 10 11 9 10 11

  27. Gerritsen et al., 1999 • Conclusions • vWF-CP activity can be used to distinguish TTP from HUS • The presence of a vWF-CP inhibitor can further differentiate acquired from hereditary TTP • The collagen binding assay is sensitive and specific

  28. Gerritsen et al., 1999 • Study deficiencies • Low sample number (40) • Misclassification bias • Diagnosis of TTP • Definition of familial

  29. 2. How well do the various testing methods compare with each other?

  30. Studt et al., 2003 • Measurement of von Willebrand factor-cleaving protease (ADAMTS-13) activity in plasma: a multicenter comparison of different assay methods

  31. Studt et al., 2003 • Methods • Identical aliquots from 30 different patients with acquired TTP, hereditary TTP, and “other” conditions were sent to 5 different laboratories • Each lab used its standard testing method for activity and presence of inhibitor • (1) Immunoblot assay • (2) Residual collagen binding assays • (1) Residual ristocetin cofactor activity assay • (1) Immunoradiometric assay

  32. Studt et al., 2003 • Results

  33. Studt et al., 2003 • Results Cont.

  34. Studt et al., 2003 • Conclusion • In general, correlation fairly good [Spearman rank order correlation coefficient = 0.89 - 0.97 (p<0.001)] • Deviations were more common in the collagen binding assay suggesting it is more delicate

  35. Studt et al., 2003 • Problems • Dose not address the accuracy or reproducibility of the labs (each sample was only tested once) • Poor correlation of data at the higher activity level compensated for correlation at the lower activity levels

  36. Tripodi et al., 2004 • Measurement of von Willebrand factor cleaving protease (ADAMTS13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas

  37. Tripodi et al., 2004 • Method • Normal plasma (100% activity) and plasma from a patient with familial TTP (0% activity) were mixed to have ADAMTS activity, by volume, of 0%, 10%, 20%, 40%, 80%, and 100%

  38. Tripodi et al., 2004 • Results • Linearity (expected vs. observed) = from 0.98 to 0.39 (1 = perfect linearity) • Reproducibility = from <10% to 83% (coefficient of variation) • Better correlation between the very low and high ADAMTS13 levels

  39. Tripodi et al., 2004 • Conclusion • Best methods included measuring vWFCP by ristocetin cofactor, residual collagen binding, and immunoblotting • Varied inter-laboratory agreement

  40. 3. How has this specific test (or similar tests) been evaluated and how well has it performed?

  41. Furlan et al., 1998 • Von Willebrand Factor-Cleaving Protease in Thrombotic Thrombocytopenic Purpura and the Hemolytic -Uremic Syndrome

  42. Furlan et al., 1998 • Methods • Plasma from patients with a clinical diagnosis of TTP or HUS along with a worksheet containing clinical and laboratory data was sent for study (selection bias) • Patients were classified as TTP, acute or in remission and as HUS, acute or in remission by the PI without knowledge of the vWFCP testing (based on the work-sheet) • vWFCP testing using an immunoabsorbent assay

  43. Furlan et al., 1998 • Results

  44. Furlan et al., 1998 • Results Cont.

  45. Furlan et al., 1998 • Conclusion • Nearly absent levels of vWFCP is a sensitive test for acute TTP (low false negative) • Plasma from patients with non-familial TTP tends to have a vWFCP inhibitor while plasma from those with familial TTP does not

  46. Furlan et al., 1998 • Problems • Test bias • Selection bias • No “normal” patients included • No referred patients were excluded • Gold standard (PI interpretation of work sheet) - misclassification bias

  47. Bianchi et al., 2002 • Von Willebrand factor-cleaving protease (ADAMTS13) in thrombocytopenic disorders: a severely deficient activity is specific for thrombotic thrombocytopenic purpura

  48. Bianchi et al., 2002 • Methods • 68 patients with thrombocytopenia (<140 K) recruited for study with the following clinical diagnoses • Sepsis (17) • HIT (16) • Osteomyelofibrosis (3) • Myelodysplastic syndrome (4) • ITP(10) • Acute leukemia (6) • Severe aplastic anemia (2) • Miscellaneous (10) • ADAMTS13 activity and inhibitor measurement with immunoblotting method

  49. Bianchi et al., 2002 • Results Only 18% had levels <30% 10% is lowest level

  50. Bianchi et al., 2002 • Conclusion • These results along with prior publications indicate that very low levels (<5% activity) of ADAMT13 activity is very specific for TTP

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