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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