Objectives. Thrombotic Thrombocytopenic Purpura (TTP) and ADAMTS13 Testing. Disclosure

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1 Thrombotic Thrombocytopenic Purpura (TTP) and Testing Dong Chen MD PhD Special Coagulation Laboratory Mayo Clinic-Rochester 2018 Disclosure Chair, CAP Coagulation Resource Committee Mayo Medical Laboratory Objectives The pathophysiology of thrombotic thrombocytopenic purpura (TTP) Laboratory characteristics of various tests Clinical applications of testing in diagnosis and management of patients with TTP 1

2 Thrombotic Microangiopathy (TMA) Clinical Presentation Hemolytic anemia Thrombocytopenia Organ damage Histologic Findings Schistocytosis Microvascular Thrombosis Pathophysiology Endothelial damage Coagulation abnormality deficiency Pathogenesis Compliment Shiga toxin dysfunction Other etiologies Differential Diagnosis of TMA TMA Thrombotic Thrombocytopenic Purpura (TTP) Severe Deficiency Hemolytic Uremic Syndrome Other Congenital TTP Immune mediated TTP (ittp) Infection-HUS E. Coli 0157: H7 Autoimmune diseases (e.g. catastrophic antiphospholipid syndrome) Primary ittp) Secondary (TTP) Complementmediated HUS DIC Autoimmune disorder (e.g., SLE, Sjogren syndrome,ra) Drugs (ticlodipine, quinine, simvastatin, trimethoprim,pegylated interferon ) Disseminated malignancy Infection (HIV) Infections Malignant hypertension Pregnancy (e.g. HELLP) Clinical Features of TTP Presenting features and clinical course ( ) N=65 No. (%) of patients History and physical examination Neurologic abnormalities 43 (66) Major abnormalities (eg. focal, Seizure, stroke and coma) 23 (35) Minor abnormalities (eg, confusion, headache) 20 (31) Fever 15 (23) Laboratory data (range) Hematocrit, percentage 25 (15-43) Platelet count, 10 3 /μl 16 (2-124) LDH, U/L 1613 ( ) Creatinine, μmol/l 97.2 ( ) Patients with the classic pentad (thrombocytopenia, microangiopathic hemolytic anemia, neurologic and renal abnormalities, fever) 3 (5) George, J. N. Blood, 2010, 116(20):

3 Clinical Diagnostic Criteria of TTP 1. Micro-angiopathic hemolytic anemia (MAHA) 2. Thrombocytopenia + With or without neurologic symptoms History of TTP Galbusera, M., M. Noris, et al. (2006). Seminars in thrombosis and hemostasis 32(2): Scully, M., et al. (2017). "Consensus on the standardization of terminology in thrombotic thrombocytopenic purpura and related thrombotic microangiopathies." Journal of thrombosis and haemostasis : JTH 15(2): Biology and Pathophysiology Von Willebrand Factor and J. Evan Sadler, Hematology 2015;2015:

4 Wild Type ADAMTS-13 and Mutations 1427aa Mutations Levy G G et al. Blood 2005;106:11-17 Anti- Autoantibody Epitopes Klaus et al r Patient Number N=25 Luken et al r N=7 Zheng et al r N=67 Johanna A. Kremer Hovinga, and Bernhard Lämmle Hematology 2012;2012: Laboratory Testing Activity Antigen Bethesda Titer Autoantibody Titer 4

5 AdamTS13 Activity Assays Full Native VWF Denature 1.5 M urea AdamTS13 Activation Residual BaCl 2 24 Hours VWF: Collagen Binding Activity VWF: Ristocetin Cofactor Activity VWF: Antibody Binding (IRMA) VWF: Multimer analysis VWF Peptide AdamTS13 Activation VWF-73aa or -78aa BaCl hour Detection FRET ELISA Mass spec. Most Frequently Used Assays Method Substrate, monoclonal or polyclonal antibodies Measurement of cleaved VWF Reference Blotting Purified (plasma-derived) VWF VWF western blot N Engl J Med 1998; 339: CBA Recombinant or plasma-derived VWF Collagen binding Thromb Haemost 1999; 82: FRET Fluorogenic VWF73 peptide (residues of VWF) Fluorescence resonance energy transfer (FRET) assay Br J Haematol 2005; 129: ELISA VWF78 peptide conjugated with HRP (Nterminus) and labelled with biotin (C-terminus) (HRP-VWF78-biotin) Recombinant VWF73 (residues of VWF) tagged with both GST and His (GST- VWF73-His) Streptavidin-agarose absorption J Thromb Haemost 2006; 4: and measurement of HRP activity in solution Anti-N10 MoAb and measurement Transfusion 2006; 46: 1444 of HRP activity 52. Mass Spec. Recombinant VWF73-His Mass spectrometry J Thromb Haemost Feb;4(2): Fluorescence Resonance Energy Transfer FRETS-VWF73 Assay Excitation at 340 nm Emission at 450 nm 1605 Substrate: Fluorescent and quenching agent labeled Peptide of VWF-73aa (A2 domain D 1596 to R 1668 ) Product: cleavage of substrate abolishes quenching effect permitting fluorescence to occur. Kokame, et al. Br J Haematol 2005:129 (1),

6 Limitations of the Activity Assays Difference in low limit of quantification (LLQ) High coefficient variation (CV) at the lower end Discrepant results among different assays Various Interfering substances Static assay, which may be non-biological Discrepancy Among Different Assays Journal of Thrombosis and Haemostasis : The New WHO International Standard Pooled plasma from 38 normal healthy donors. 32 laboratories from 14 countries Using local pooled normal plasma preparations as calibrators. FRET (n = 18) or ELISA (n = 9). Combination of all results for activity gave an overall mean of 0.91 units/ml. The inter-laboratory variability (geometric coefficient of variation, GCV) was 12.4%. For estimates of antigen the combination of all results gave an overall mean of 0.92 units/ml with inter-laboratory variability (GCV) of 16.3%. Hubbard, A. R., A. B. Heath, et al. (2015). "Establishment of the WHO 1st International Standard, plasma (12/252): communication from the SSC of the ISTH." JTH 13(6):

7 Then New International Standard Does not Resolve Method/Laboratory Discrepancy L: Local Standard IS: International Standard Activity Assay Interference Free Hemoglobin Falsely low activity Falsely positive inhibitor Severe Hemolysis: Free hemoglobin inhibits activity. Studt JD, et al: Blood Free Hemoglobin Inhibitor Titer mg/dl BU 200 < >2 7

8 Bilirubin Meyer SC, et al: JTH 2007 Meyer, S. C., I. Sulzer, et al. (2007). JTH 5(4): EDTA AdamTS13 activity <5% Inhibitor screen (mixing study): Positive NPP expected= 53% Patient + NPP= <5% Titer: >16 BU EDTA negative control EDTA positive control Patient s sample Assays of Inhibitor Inhibitor Screen Assay: When activity <30%. Heat inactivation of residual activity Mix patient plasma with normal pooled plasma. Assess activity. Inhibitor Bethesda Titer Assay: Semiquantitative similar to FVIII inhibitor assay. 1 BU inhibits 50% activity in a 1:1 mix with normal pooled plasma. 8

9 The Antibody (e.g. IgG) Present in most patients with activity < 10% (90%) Mostly IgG antibody (Reiger M, et al: Blood 2005) High titer antibody is associated with worse prognosis (Ferrari S: Blood 2007) Low specificity: Positive in patients with autoimmune disorders. Up to 5-10% positivity in normal donors. Activity In Normal Adult and Pediatric Population Baseline AdamTS13 Levels in General Population Kokame, K. et al. J Thromb Haemost, (8): activity in Pediatric population is about 80% of adults Nguyen TC. et al. Haematologica

10 Activity Clinical Utility at the Initial Diagnosis of TTP Prevalence of Severe Deficiency (<10%) in Patients with an Acute On-set of TTP Author Design of study Positive Cases/Total (%) Furlan et al Retrospective 26/30 86* Tsai and Lian 1998 Retrospective 37/37 100* Veyradier et al Prospective, multicenter 47/66 71 Mori et al Retrospective? 12/18 66 Vesely et al Inception cohort, single center 16/48 33 Zhou et al Retrospective 34/34 100* Zheng et al Single center, prospective 16/20 80 Peyvandi et al Multicenter 48/ Matsumoto et al Multicenter 56/ Kremer Hovinga Multicenter 56/93 60 Matsumoto, et al Based on initial assessment 56/ Groot et al Retrospective 24/27 89* Kremer-Hovinga 2010 Inception cohort, single center 46/98 48 George et al Retrospective 51/ * Retrospective study with exclusion of cases of renal failure or other secondary TMA. Severe Deficiency Using VWF Collagen Binding Method is Highly Associated with TTP TMA Etiology activity < 5% activity 5%-50% activity >50% + inhibitor Total Idiopathic TTP HSCT and cyclosporine Cancer and chemotherapy Clopidogrel Pregnancy Other Total Zheng, X. L., et al. Blood, 2004, 103(11):

11 Severe Deficiency Using the New FRET method is Highly Associated with TTP 10% Kokame, K. et al. Br J Haematol 2005;129: Hassan, S. et al. Br J Haematol 2015; 171: Deficiency in Non-TTP Conditions Mild deficiency of (>10%): Uremia, Sepsis, chronic inflammation, DIC, pregnancy, post-operatively, liver disease (Mannucci PM, et al. Blood, 2001). Rare Non-TTP conditions with severe deficiency (<10%): Liver disease and cirrhosis Levels as low as 6% (Mannucci PM, et al. Blood, 2001). Alcoholic hepatitis < 5% (Uemura M, et al. Alcohol Lin Exp Res, 2005) Sepsis-induced DIC or severe sepsis < 5% (Ono T, et al. Blood 2006) Disseminated malignancy Mean was about 5% in 10 cases (Oleksowicz, et al. J Thrombo Haemost 1999) Timing of Activity Testing 11

12 Activity Before Each Plasma Exchange % of severe deficiency (<10%) 100% 89% 83% 78% 59% Pre-PE1 Wu, N., J. Liu, et al. (2015). Transfusion 55(1): TMA Diagnostic Algorithm If PLASMIC Score is >4, proceed with testing Modified from J. Evan Sadler Hematology 2015;2015: Association Between Patient Characteristics and Deficiency Using Multivariate Analysis. Coppo P, et al. (2010). PLoS ONE 5(4): e doi: /journal.pone

13 The Impact of Timely Results on Clinical Diagnosis and Management of Patients with TMA Suspected TMA pts Initial clinical and laboratory evaluation with available results TTP (7pts) Possible TTP (21 pts) Non-TTP (81) No PLEX Re-examine when results were available PLEX (7 pts) PLEX (21 pts) Continued PLEX Continued PLEX (7 pts) Continued PLEX (6 pts) Discontinued PLEX (15 pts) Mohamed Alsammak et al ISTH Abstract Benefit of Rapid Activity Turnaround Time on Plasma Utilization for Suspected TTP Outcome parameter Slow-TAT (n = 32) Rapid-TAT (n = 28) p value TAT (days), median (range) 9 (5-51) 1(<1-1) < Exchanged Total exchanges Exchanges per patient, mean (range) 14.1 (1-38) 5.3 (0-35) Total plasma units utilized Number of units of plasma per patient, mean (range) Plasma volume exchanged (ml) per patient per kg of body weight, mean (range) (13-386) ( ) 63.3 (0-425) ( ) day mortality 7 (21.9) 6 (21.4) Cost (18 months) $75/ unit of plasma and $2400/plasma exchange treatment Nathan Baseline T. Connell et al. $715,000 Transfusion 2015, , Connell, N.T., Transfusion, Utility of Testing in Assessing Prognosis of Congenital and Acquired TTP 13

14 Residual Plasmatic Activity of and Phenotype Severity in Congenital TTP Lotta LA, et al. Blood Jul 12;120(2): Studies activity Predictors of Outcome During the Acute Phase of Acquired TTP Design Outcomes (% of patients) Remission Activity Death Activity <10% 10% <10% 10% Vesely et al Prospective Zheng et al Prospective Mori et al Retrospective Coppo et al Retrospective NA 13 0 Raife et al Retrospective NA 8 18 Utility of Monitoring During Treatment 14

15 Poor Responder to Plasma Exchange (PE) due to Inhibitor Boosting Isonishi A, et al. Transfusion. 2015; 55: Shaded areas show normal ranges, and the vertical bars indicate values of mean ± SD. Utility of Testing in Assessing Risk of Relapse of Acquired TTP Activity at Time of Initial Diagnosis and Risk of Relapse N=136 N=47 The median follow-up= 7.5 years Johanna A. Kremer Hovinga et al. Blood 2010;115:

16 Results at Remission and TTP Relapse Flora Peyvandi et al. Haematologica 2008;93:232-9 When to Order Testing and Result Interpretation TMA is suspected : Activity Inhibitor Screen Bethesda Titer During treatment Pre-PE testing may help to confirm TTP or a TTP that is refractory to PE. During remission activity or inhibitor titer to assess risk of relapse or verify the onset of a relapse. <10% is highly suggestive for TTP But higher activity does not exclude TTP Improvement of platelet count and activity indicate treatment response and approaching to remission. However, some patients could continue to have severe deficiency despite clinical remission. Persistent deficiency and positive titer may explain refractory TTP. Severe deficiency and positive inhibitor titer during remission indicate a risk of relapse. A sudden platelet count decline and activity <10%, in conjunction with other TMA features suggest TTP relapse. Summary The pathophysiology of in thrombotic thrombocytopenic purpura (TTP) Laboratory characteristics of various assays Clinical applications of testing in diagnosis and management of patients with TTP 16

17 Thank You 17