Thrombotic Thrombocytopenic Purpura (TTP) and ADAMTS13 … Program Handouts/TTP and... · Suspected...
Transcript of Thrombotic Thrombocytopenic Purpura (TTP) and ADAMTS13 … Program Handouts/TTP and... · Suspected...
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Thrombotic Thrombocytopenic Purpura (TTP) and ADAMTS13 Testing
Dong Chen MD PhDSpecial Coagulation Laboratory
Mayo Clinic-Rochester2018
Disclosure
• Chair, CAP Coagulation Resource Committee
• Mayo Medical Laboratory
Objectives
• The pathophysiology of thrombotic thrombocytopenic purpura (TTP)
• Laboratory characteristics of various ADAMTS13 tests
• Clinical applications of ADAMTS13 testing in diagnosis and management of patients with TTP
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Thrombotic Microangiopathy (TMA)
Pathogenesis
ADAMTS13 deficiency
Compliment dysfunction Shiga toxin Other etiologies
Pathophysiology
Endothelial damage Coagulation abnormality
Histologic Findings
Schistocytosis Microvascular Thrombosis
Clinical Presentation
Hemolytic anemia Thrombocytopenia Organ damage
Differential Diagnosis of TMA
TMA
Thrombotic Thrombocytopenic Purpura (TTP)
Severe ADAMTS13 Deficiency
Congenital TTP
Immune mediated TTP (iTTP)
Primary iTTP) Secondary (TTP)
Autoimmune disorder (e.g., SLE, Sjogren
syndrome,RA)
Drugs (ticlodipine, quinine, simvastatin, trimethoprim,pegylated
interferon )
Infection (HIV)
Hemolytic Uremic Syndrome
Infection-HUS
E. Coli 0157: H7
Complement-mediated HUS
Other
Autoimmune diseases (e.g.
catastrophic antiphospholipid
syndrome)
DIC
Disseminated malignancy
Infections
Malignant hypertension
Pregnancy (e.g. HELLP)
Presenting features and clinical course (1995-2009) N=65No. (%) of patients
History and physical examinationNeurologic 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, ×103/μL 16 (2-124)
LDH, U/L1613 (256-3783)
Creatinine, μmol/L97.2 (61.9-406.6)
Patients with the “classic pentad” (thrombocytopenia, microangiopathichemolytic anemia, neurologic and renal abnormalities, fever)
3 (5)
George, J. N. Blood, 2010, 116(20): 4060-69.
Clinical Features of TTP
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1. Micro-angiopathic hemolytic anemia (MAHA)2. Thrombocytopenia
+With or without neurologic symptomsHistory of TTP
Clinical Diagnostic Criteria of TTP
Galbusera, M., M. Noris, et al. (2006). Seminars in thrombosis and hemostasis 32(2): 81-89.
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): 312-322.
ADAMTS13
Biology and Pathophysiology
Von Willebrand Factor and ADAMTS13
J. Evan Sadler, Hematology 2015;2015:631-636
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Levy G G et al. Blood 2005;106:11-17
Wild Type ADAMTS-13 and Mutations
ADAMTS13Mutations
ADAMTS13
1427aa
Anti-ADAMTS13 Autoantibody Epitopes
Johanna A. Kremer Hovinga, and Bernhard Lämmle Hematology 2012;2012:610-616
Klaus et al
Luken et al
Zheng et al
N=25
N=7
N=67N=67
Patient NumberrADAMTS13
rADAMTS13
rADAMTS13
ADAMTS13
ADAMTS13
Laboratory TestingActivity
Antigen
Bethesda Titer
Autoantibody Titer
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AdamTS13 Activation
BaCl224 Hours
Denature
1.5 M ureaResidual
VWF: Collagen Binding ActivityVWF: Ristocetin Cofactor ActivityVWF: Antibody Binding (IRMA)VWF: Multimer analysis
Full Native VWF
VWF Peptide
VWF-73aa or -78aa BaCl20.5-1 hour
DetectionFRETELISAMass spec.
AdamTS13 Activation
AdamTS13 Activity Assays
MethodSubstrate, monoclonal or polyclonal
antibodiesMeasurement of cleaved
VWFReference
Blotting Purified (plasma-derived) VWF VWF western blotN Engl J Med 1998; 339: 1578–84.
CBA Recombinant or plasma-derived VWF Collagen bindingThromb Haemost 1999; 82: 1386–9.
FRETFluorogenic VWF73 peptide (residues 1596–1668 of VWF)
Fluorescence resonance energy transfer (FRET) assay
Br J Haematol 2005; 129: 93–100.
ELISA
VWF78 peptide conjugated with HRP (N-terminus) and labelled with biotin (C-terminus) (HRP-VWF78-biotin)
Streptavidin-agarose absorption and measurement of HRP activity in solution
J Thromb Haemost 2006; 4: 129–36.
Recombinant VWF73 (residues 1596–1668 of VWF) tagged with both GST and His (GST-VWF73-His)
Anti-N10 MoAb and measurement of HRP activity
Transfusion 2006; 46: 1444–52.
Mass Spec.
Recombinant VWF73-His Mass spectrometryJ Thromb Haemost. 2006 Feb;4(2):333-8.
Most Frequently Used ADAMTS13 Assays
Fluorescence Resonance Energy TransferFRETS-VWF73 Assay
Kokame, et al. Br J Haematol 2005:129 (1), 93-100.
• Substrate: Fluorescent and quenching agent labeled Peptide of VWF-73aa (A2 domain D1596 to R1668)
• Product: ADAMTS13 cleavage of substrate abolishes quenching effect permitting fluorescence to occur.
Excitation at 340 nm Emission at 450 nm
1605
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Limitations of the ADAMTS13 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 ADAMTS13 Assays
Journal of Thrombosis and Haemostasis. 2006 4:1707-17
The New ADAMTS13 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 ADAMTS13 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 ADAMTS13 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 ADAMTS13, plasma (12/252): communication from the SSC of the ISTH." JTH 13(6): 1151-53.
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L: Local StandardIS: International Standard
Then New ADAMTS13 International Standard Does not Resolve Method/Laboratory Discrepancy
ADAMTS13 Activity Assay
Interference
Free Hemoglobin
• Severe Hemolysis:Free hemoglobin inhibits ADAMTS13 activity.
Free Hemoglobin Inhibitor Titer
mg/dL BU
200 <1
500 1-2
1000 >2Studt JD, et al: Blood 2005.
Falsely low ADAMTS13 activityFalsely positive inhibitor
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Bilirubin
Meyer SC, et al: JTH 2007
Meyer, S. C., I. Sulzer, et al. (2007). JTH 5(4): 866-867
EDTA
AdamTS13 activity <5%
Inhibitor screen (mixing study): PositiveNPP expected= 53%Patient + NPP= <5%
Titer: >16 BU
EDTA negative control
EDTA positive control Patient’s sample
Assays of ADAMTS13 “Inhibitor”
• Inhibitor Screen Assay:
• When ADAMTS13 activity <30%.
• Heat inactivation of residual ADAMTS13 activity• Mix patient plasma with normal pooled plasma.• Assess ADAMTS13 activity.
• Inhibitor Bethesda Titer Assay:• Semiquantitative similar to FVIII inhibitor assay.• 1 BU inhibits 50% ADAMTS13 activity in a 1:1 mix with
normal pooled plasma.
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The ADAMTS13 Antibody (e.g. IgG)
• Present in most patients with ADAMTS13 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.
ADAMTS13 Activity
In Normal Adult and Pediatric Population
Baseline AdamTS13 Levels in General Population
Kokame, K. et al. J Thromb Haemost, 2011. 9(8):1654-6.
ADAMTS13 activity in Pediatric population is about 80% of adults’ Nguyen TC. et al. Haematologica 2007.
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ADAMTS13 Activity
Clinical Utility at the Initial Diagnosis of TTP
Prevalence of Severe ADAMTS13 Deficiency (<10%) in Patients with an Acute On-set of TTP
Author Design of study Positive Cases/Total (%)
Furlan et al. 1998 Retrospective 26/30 86*
Tsai and Lian 1998 Retrospective 37/37 100*
Veyradier et al. 2001 Prospective, multicenter 47/66 71
Mori et al. 2002 Retrospective? 12/18 66
Vesely et al. 2003 Inception cohort, single center 16/48 33
Zhou et al. 2004 Retrospective 34/34 100*
Zheng et al. 2004 Single center, prospective 16/20 80
Peyvandi et al. 2004 Multicenter 48/100 48
Matsumoto et al. 2004 Multicenter 56/108 52
Kremer Hovinga. 2004 Multicenter 56/93 60
Matsumoto, et al. 2005 Based on initial assessment 56/108 52
Groot et al. 2006 Retrospective 24/27 89*
Kremer-Hovinga 2010 Inception cohort, single center 46/98 48
George et al. 2010 Retrospective 51/107 48
* Retrospective study with exclusion of cases of renal failure or other secondary TMA.
TMAEtiology
ADAMTS13 activity
< 5%
ADAMTS13 activity 5%-50%
ADAMTS13 activity >50%
+ADAMTS13 inhibitor
Total
Idiopathic TTP 16 4 0 7 20 HSCT and cyclosporine
0 4 4 0 8
Cancer and chemotherapy
0 2 1 0 3
Clopidogrel 0 0 1 0 1 Pregnancy 0 1 1 0 2 Other 0 1 2 0 3 Total 16 12 9 7 37
Zheng, X. L., et al. Blood, 2004, 103(11): 4043-4049
Severe ADAMTS13 Deficiency Using VWF Collagen Binding Method is Highly Associated with TTP
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Kokame, K. et al. Br J Haematol 2005;129: 93-100.
10%
Severe ADAMTS13 Deficiency Using the “New” FRET method is Highly Associated with TTP
Hassan, S. et al. Br J Haematol 2015; 171:830-5.
ADAMTS13 Deficiency in Non-TTP Conditions
• Mild deficiency of ADAMTS13 (>10%): Uremia, Sepsis, chronic inflammation, DIC, pregnancy, post-operatively, liver disease (MannucciPM, et al. Blood, 2001).
• Rare Non-TTP conditions with severe ADAMTS13 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• ADAMTS13 < 5% (Ono T, et al. Blood 2006)
• Disseminated malignancy• Mean ADAMTS13 was about 5% in 10 cases (Oleksowicz, et al. J
Thrombo Haemost 1999)
Timing of ADAMTS13 Activity Testing
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Wu, N., J. Liu, et al. (2015). Transfusion 55(1): 18-24.
ADAMTS13 Activity Before Each Plasma Exchange
% of severe ADAMTS13 deficiency (<10%)
100% 89% 83% 78% 59%
Pre-PE1
Modified from J. Evan Sadler Hematology 2015;2015:631-636
TMA Diagnostic Algorithm
If PLASMIC Score is >4, proceed with ADAMTS13 testing
Association Between Patient Characteristics and ADAMTS13 Deficiency Using Multivariate Analysis.
Coppo P, et al. (2010). PLoS ONE 5(4): e10208. doi:10.1371/journal.pone.0010208
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Continued PLEX
Re-examine when ADAMTS13 results were
available
Initial clinical and laboratory evaluation with
available ADAMTS13 results
Suspected TMA2012-2014
109 pts
TTP
(7pts)
PLEX
(7 pts)
Continued PLEX
(7 pts)
PossibleTTP
(21 pts)
PLEX
(21 pts)
Continued PLEX
(6 pts)
Discontinued PLEX
(15 pts)
Non-TTP
(81)
No PLEX
The Impact of Timely ADAMTS13 Results on Clinical Diagnosis and Management of Patients with TMA
Mohamed Alsammak et al. 2015 ISTH Abstract
Benefit of Rapid ADAMTS13 Activity Turnaround Time on Plasma Utilization for Suspected TTP
Nathan T. Connell et al. Transfusion 2015, 354-359,
Outcome parameterSlow-TAT(n = 32)
Rapid-TAT(n = 28)
pvalue
TAT (days), median (range) 9 (5-51) 1 (<1-1) <0.0001
Exchanged 32 12
Total exchanges 451 148
Exchanges per patient, mean (range) 14.1 (1-38) 5.3 (0-35) 0.0008
Total plasma units utilized 6242 1771
Number of units of plasma per patient, mean(range)
144.5(13-386)
63.3(0-425)
0.0002
Plasma volume exchanged (mL) per patient per kg of body weight, mean (range)
634.4(21.5-2093.9)
263.5(0-1762.5)
0.0045
30-day mortality 7 (21.9) 6 (21.4) 0.9659
Cost (18 months) $75/ unit of plasma and $2400/plasma exchange treatment
Baseline –$715,000
Connell, N.T., Transfusion, 2015.
Utility of ADAMTS13 Testing in
Assessing Prognosis of
Congenital and Acquired TTP
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Residual Plasmatic Activity of ADAMTS13 and Phenotype Severity in Congenital TTP
Lotta LA, et al. Blood. 2012 Jul 12;120(2):440-8.
ADAMTS13 activity Predictors of Outcome During the Acute Phase of Acquired TTP
Studies Design
Outcomes (% of patients)
RemissionADAMTS13 Activity
DeathADAMTS13 Activity
<10% ≥10% <10% ≥10%
Vesely et al Prospective 84 55 16 45
Zheng et al Prospective 82 49 18 51
Mori et al Retrospective 85 20 15 80
Coppo et al Retrospective NA 13 0
Raife et al Retrospective NA 8 18
Utility of Monitoring ADAMTS13 During Treatment
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Poor Responder to Plasma Exchange (PE)
due to ADAMTS13 Inhibitor Boosting
Isonishi A, et al. Transfusion. 2015; 55: 2321-30.
Shaded areas show normal ranges,and the vertical bars indicate valuesof mean ± SD.
Utility of ADAMTS13 Testing in Assessing Risk of Relapse of Acquired TTP
ADAMTS13 Activity at Time of Initial Diagnosis and Risk of Relapse
Johanna A. Kremer Hovinga et al. Blood 2010;115:1500-1511
The median follow-up= 7.5 years
N=47
N=136
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ADAMTS13 Results at Remission and TTP Relapse
Flora Peyvandi et al. Haematologica 2008;93:232-9
TMA is suspected
During treatment
During remission
ADAMTS13:•Activity•Inhibitor Screen•Bethesda Titer
Pre-PE ADAMTS13 testing may help to confirm TTP or a TTP that is refractory to PE.
ADAMTS13 activity or inhibitor titer to assess risk of relapse or verify the onset of a relapse.
When to Order ADAMTS13 Testing and Result Interpretation
ADAMTS13 <10% is highly suggestive for TTP
But higher ADAMTS13 activity does not exclude TTP
Improvement of platelet count and ADAMTS13 activity indicate treatment response and approaching to remission. However, some patients could continue to have severe ADAMTS13 deficiency despite clinical remission.
Persistent ADAMTS13 deficiency and positive titer may explain refractory TTP.
Severe ADAMTS13 deficiency and positive inhibitor titer during remission indicate a risk of relapse.
A sudden platelet count decline and ADAMTS13 activity <10%, in conjunction with other TMA features suggest TTP relapse.
• The pathophysiology of ADAMTS13 in thrombotic thrombocytopenic purpura (TTP)
• Laboratory characteristics of various ADAMTS13 assays
• Clinical applications of ADAMTS13 testing in diagnosis and management of patients with TTP
Summary
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Thank You