troponin i measurement: evolution of a biomarker essential for … · 2005. 2. 1. · round robin...

Troponin I Measurement: Evolution of a Biomarker Essential

for Assessment of Myocardial Injury

Robert H. Christenson, Ph.D., DABCC, FACBProfessor of PathologyProfessor of Medical and Research TechnologyUniversity of Maryland School of Medicine

Troponin

When troponin is increased think heart

• Trauma– Contusion– Ablation– Pacing– ICD firing– Cardioversion– Endomyocardial biopsy– Cardiac surgery

• Hypertension• Hypotension• Acute neurologic disease, CVA• Rhabdomyolysis, cardiac injury

• Post-op non-cardiac surgery• Renal failure• Critically ill, esp. DM• Hypothyroidism• Myocarditis• Post PCI• Pulmonary embolism• Sepsis• Amyloidosis• Exhaustion• Heart Failure, Acute and Chronic

=

Cardiac isoforms in blood

Meta-Analysis: Troponin T or I and NSTE MIJACC 2001;38:478-85.

3.0 (1.6-5.5)

5.1(3.2-8.4)

2.6 (1.8-3.6)

8.5 (3.5-21.1)

Troponin I Troponin T

cTnT Quartiles: PARAGON BJ Am Coll Cardiol 2003;41:371-80.

0.0 ng/mL(N=365)

0.0- 0.047(N=113)

0.048-0.277(N=241)

0.278-8.37(N=340)

5%

10%

15%

20%

25%

Six-

mon

th D

eath

/(re)

MI R

ate

Trend PTrend P--value = 0.001value = 0.001

0

Myocardial Infarction Redefined-A Consensus Document of the Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of

Myocardial Infarction J Am Coll Cardiol 2000;36;959-969.

“Any amount of myocardial necrosis caused by ischemia should be labeled as myocardial

infarction.”

Troponin I or T are a surrogate for myocardial necrosis (in the setting of cardiac ischemia).

Whole New Way of Doing Business

“Definition of MI. Criteria for acute, evolving or recent MI.”

• Typical rise and gradual fall (troponin) or more rapid rise and fall (CK-MB) of biochemical markers for myocardial necrosis with at least one of the following:• Ischemic symptoms• Development of pathologic Q waves on ECG• ECG changes indicative of ischemia (ST↑ or ST↓)• Coronary artery intervention (e.g. coronary angioplasty)

• 99th percentile of a reference control population

• Imprecision (CV) at 99th percentile should be 10%

Thygesen K, et al, JACC 2000;36:959-969.

TIMI Risk FactorsJAMA 2000;284:835-42

• age >65 years• >3 risk factors for coronary artery disease• prior coronary stenosis > 50%• ST deviation on the ECG• > 2 anginal events in previous 24 hours• aspirin use in last 7 days

Elevated Cardiac Markers

(New)Type 1A RecommendationsCirculation 2002;106:1893-1900

• An early invasive strategy in patients with UA/NSTEMI without serious comorbidity and who have any of the following high-risk indicators:– Recurrent angina/ischemia at rest or with low level

activities despite intensive anti-ischemic therapy

– Elevated cTnI or cTnT Elevations in cTnI (0.1 ng/mL) and cTnT (0.01 ng/mL) provides superior predictive capability.

– New or presumably new ST-segment depression

Not all Troponin Methods are Created Equal

Systematic Variation Between cTnI AssaysClin Chem 2001;47:431-437

• 10 ng/mL troponin CIT standard material, value-assigned by NIST, was measured by 13 participating cTnI assays, in duplicate: Assay Value 1 Value 2 Mean

1 143.7 135.7 139.72 46.9 47.9 47.43 19.9 19.9 19.94 49.2 50.1 49.75 4.3 4.1 4.26 12.5 12.6 12.67 7.4 8.9 8.28 6.0 6.1 6.09 16.5 17.4 17.0

10 12.6 13.2 12.911 12.6 12.4 12.512 18.3 18.9 18.613 17.6 16.9 17.3

• Within assay reproducibility good, but measurement of 10 ng/mL material yielded results that were >30-fold different!

Mean: 28.1 SD: 36.4CV: 130%

Imprecision of Troponin AssaysClin Chem 2004;50:327-332

• Construct clinically relevant imprecision profiles to identify 10% CV values for each cTnI assay.

• Eight pools were shipped to manufacturers and measurements were made according to NCCLS EP5-A.

• Data sent to centralized lab for data analysis.

Imprecision Ratio at Low cTnIClin Chem 2004:50;327-333.

Compliance of Assays

• High sensitivity designation• Analogous to:

– hs-CRP– 3rd generation TSH

• Manufacturers will have to improve assays• Provide data to FDA for new claim

cTnI and One-year Clinical EventsAm J Cardiol 2002;89:1035-41.

Access, 99th<0.02 ng/mL≥0.02 ng/mLAccess, ≤20%<0.03 ng/mL≥0.03 ng/mLAxSym, 99th<0.6 ng/mL≥0.6 ng/mLAxSym, ≤ 20%<1.0 ng/mL≥1.0 ng/mL

DeathP-Value0.024

0.037

0.54

0.28

Death/MIP-Value<0.0001

<0.0001

<0.001

<0.001

Is Discordance Important?Am J Cardiol 2002;89:1035-41.

Classification based on equal assay performance (CV)

Access, <0.03Access, ≥0.03

AxSym AxSym<1.0 ≥1.0

172 (TN) 3 (FP)109 (FN) 595 (TP)

Death p=0.01Death/MI p=0.001

Death p=0.01Death/MI p=0.001

Is Discordance Important?Am J Cardiol 2002;89:1035-41.

Classification based on equal assay performance (CV)

cTnT, <0.03cTnT, ≥0.03

AxSym AxSym<1.0 ≥1.0

254 (TN) 17 (FP)27 (FN) 581 (TP)

Death p=NSDeath/MI p=0.01

Death p=NSDeath/MI p=0.05

Cardiac Troponin I

Antibody binding to stable region for stable immunoassay

1 30 110 209

N-terminal C-terminal

Proteases Proteases

Katrukha et al. Clin. Chem. 1998; 44: 2433-40Shi et al. Clin. Chem. 1999; 45:1018-25

cTnI Standardization is DesirableBut there are a host of issues

Blood cTnI in patients with disease includes CIT and CI complexes, free cTnI

cTnI may be oxidized, reduced or phosphorylated

Different assays use different anti-cTnI MAb’s directed to different protein epitopes

Certain antigenic regions may be metabolized first

Susceptible areas may show altered immunoreactivity

Preanalytical variation a concern - e.g., fibrin, heparin

Analytical variation a concern - e.g., HAMA, RF

AACC cTnI Standardization Committee

• Desired endpoint is to reduce inter-method variability

• Possible strategies– Define common reference material– Establish consensus values for cTnI in “real”

patient specimens by a normalization process to minimized inter-method variability

– Combination of RM and patient specimens

AACC’s cTnI Standardization Committee*

• Fred S. Apple, Ph.D.• Geza S. Bodor, M.D.• Robert H. Christenson, Ph.D.• Stephen E. Kahn, Ph.D. (Chair)• Mauro Panteghini, M.D. • Michael J. Welch, Ph.D.• Alan H.B.Wu, Ph.D.

* Most committee members affiliated with medical centers have been consultants or research grantees of companies that produce cTnI assays

AACC’s cTnI Standardization Committee Acknowledges

• David M. Bunk, Ph.D. (NIST)• Show Hong Duh, Ph.D. (Univ. Maryland)• Joseph Dalluge, Ph.D. (NIST)• James D. Potter, Ph.D. (Univ. Miami)• Aleksei G. Katrukha, Ph.D. (HyTest, Inc.)• Joseph Keffer, M.D. (Spectral Diag., Inc.)

Strategies• Phase 1

– Obtain wide variety of candidate RMs– Evaluate with measurement methods (commercial)– Narrow field of candidate RMs

• Phase 2– Full characterization of remaining candidate RMs– Evaluate commutability– Investigate harmonization strategies– Select final Reference Material

• Phase 3– Obtain large amount of RM– Characterize RM– Validate performance of RM

Phase I: Round Robin 1Results of AACC cTnI Standardization Committee

Evaluation of 10 cRM’s which varied with respect to -

Human or recombinant material

CIT complex, CI complex or free cTnI

Liquid or lyophilized and reconstituted

Provided from Univ.Miami, HyTest or Spectral Diag.

13 different cTnI assays, 8 different manufacturers

Assessed consistency and predictability of analytical response using a cRM through normalizing the response of each cTnI assay over serial dilutions of each cRM

cRM’s ranked in 10 statistical categories

Two cRM’s selected for further study

Round Robin 1Candidate Reference Materials (cRMs)

Evaluation of 10 candidate reference materials: Human or recombinant material CIT complex, CI complex or free cTnI

Clin Chem 2001;47:431-437.

Round Robin 1cTnI Measurement Methods

–Evaluated 13 troponin I assays from US, Japan, Germany, the UK, and France

Clin Chem 2001;47:431-437.

Round Robin 1cRM Ranking Within Each Measurement System

CTICTICICTIFree I

Clin Chem 2001;47:431-437.

Conclusion: Round Robin 1Clin Chem 2001;47:431-437.

The outcome of this study is that the three cRMs composed of the CTI complex demonstrated the best

performance. This finding is consistent with a separate study that showed a

reduction in between-assay variability by calibration with a CTI complex

material (Katruka et al. Scand J Clin Lab Invest 1999;59:124-127).

Phase 2: Round Robin 2Results of AACC cTnI Standardization Committee

• Evaluate two candidate RMs from Phase 1– TIC complex (Human)– IC complex (Recombinant)

• Full characterization of remaining cRMs• 15 different cTnI assays

– Evaluate commutability– Investigate harmonization strategies

• Select final Reference Material

Round Robin #2

Candidate Reference Materials (cRMs)• TIC complex, cRMTIC

• IC complex, cRMIC

Characterized at NIST for purity and concentration.

cTnI-Negative Serum Pool

Human Serum Pools

• Pool 1, <24 hrs symptoms onset, ~0.4 ng/mL• Pool 2, <24 hrs symptoms onset, ~1.5 ng/mL• Pool 3, <24 hr symptoms onset, ~15.0 ng/mL

• Pool 4, >48 hrs symptoms onset, ~0.4 ng/mL• Pool 5, >48 hrs symptoms onset, ~1.5 ng/mL• Pool 6, >48 hrs symptoms onset, ~15.0 ng/mL

cTnI 2nd Round Robin Participants and Measurement Systems

cTnI Measurement System (15) Manufacturer (10)Access Beckman Coulter InstrumentsACS 180 Bayer CorporationAIA TOSOHAlpha Dx First Medical IncAxSYM Abbott DiagnosticsCentaur Bayer CorporationDimension RxL Dade BehringImmulite 1000 Diagnostic Products CorporationImmulite 2000 Diagnostic Products CorporationImmuno 1 Bayer CorporationLiaison Byk-Sangtec DiagnosticaOpus, 2nd Generation Dade BehringStratus CS Dade BehringVidas BioMerieuxVitros ECi Ortho Clinical Diagnostics

Round Robin #2

Protocol Overview

• Calibrate “field” cTnI methods according to manufacturers’ instructions– Measure six (6) serum pools– Measure dilutions of the cRMs

• Calibrate methods with the cRMs– Measure six (6) serum pool

• All measurements made in triplicate• Report all data to the committee

Commutability Definition:

“degree to which a material yields the same numerical relationships

between results of measurements by a given set of measurement procedures applied to those types of material for which the procedures are intended.”

European Committee for Standardization (CEN). Draft International Standard ISO/DIS 17511. ISO Central

Secretariat, Brussels, Belgium, 2000.

Are the materials commutable?Commutability represents comparison of inter-system variation of cTnI measurements in the biological matrix of interest (patient serum) with measurements of the cRM in biological matrix (cTnI negative serum).

Adapted from Clin Chem Lab Med 2002;40:167-173.

Round Robin #2

Commutability: Step 1What is the variability of the human serum material?

• Calibrate systems with field cTnI methods• Measure serum pools with each cTnI system.• Designate “common comparison” method• Deming’s regression method to yield parameters• Recalculate cTnI values for each serum pool

using regression parameters• Determine mean, SD, and CV for recalculated

values for each serum pool• The CV represented the characteristic inter-

system variation

Commutability: Step 2Does calibration with cRMs cause variability to exceed the characteristic

variability of genuine (human serum) material with field calibration?

• Prepare dilutions of cRMs in cTnI-negative serum or diluent • Recalibrate each method with cRM dilutions.• Measure each serum pool with the recalibrated methods.• Designate “common comparison” method• Deming’s regression to yield parameters• Recalculate values for each serum pool, for each method• Determine relative residual for each serum pool, for each assay• For each cRM: SDI = Relative Residual/CV from serum pool• Beyond +/- 3 SDI defined as non-commutable• Compare each SDI to +3/-3 criteria to manufacturer calibration

results

cRMCIT cRMCI

SerumPools

cTnI-negative Serum Diluentb

cTnI-negative Serum Diluentb

SP1 0 0 0 2

SP2 0 0 0 4

SP3 0 0 0 0

SP4 0 0 1 3

SP5 1 0 0 2

SP6 1 1 0 1

Sum 2 (2.2%) 1 (1.1%) 1 (1.1%) 12 (13.3%)

Round Robin #2

Commutability: Step 3What is the variability of the cRMs when treated like patient samples?• Calibrate systems with field cTnI methods• Measure 5 ng/mL cRMs with each cTnI system.• Designate “common comparison” method• Deming’s regression method to yield parameters• Recalculate cTnI values for each cRM using regression

parameters• Determine mean, SD, and CV for recalculated values for each

cRM• The CV represented the characteristic inter-system variation

• Compare each SDI to +3/-3 criteria to manufacturer calibration results

Commutability of (cRMCIT & cRMCI)The numbers in each cell indicate how many of the 15 analytical systemshad relative residual values that were outside the +3/-3 residual criteria.

cRMCIT cRMCI

Nominal cTnI Concentration

cTnI-negative Serum Diluenta

cTnI-negative Serum Diluenta

5.0 μg/L 5/15 2/15 3/15 5/15

a = Manufacturers’ specified diluent

cTnI Standard Material Does Not Improve Measurement Harmony

0.01

0.10

1.00

10.00

100.00

1000.00

Serum Pools

cTnI

(g/

L)

cRM CIT in Serum

cRM CI in Serum

Manufacturer'sCalib

SP 1 SP 2 SP 3 SP 4 SP 5 SP 6

Harmonization

• Harmonization represents congruency between cTnI systems of measurements performed in the biological matrix (patient serum) such that results are coincident throughout the measurement range.

Harmonization: Present State

• Systems calibrated with Manufacturers’ calibrators and instructions

• 6 serum pools measured with each system• Determine inter-method variability

Measurements, Field Calibrations

Pool Usual Measurement

cTnI, µg/L 1 Mean SD (CV) 0.608 0.562 (93%) 2 Mean SD (CV) 1.280 1.049 (82%) 3 Mean SD (CV) 29.965 25.819 (86%) 4 Mean SD (CV) 0.380 0.341 (90%) l 5 Mean SD (CV) 1.722 1.662 (97%) 6 Mean SD (CV) 22.598 20.498 (91%)

Harmonization by Alignment

•Calibrate methods (field and cRMs)•Measure 6 serum pools with each system•Designate “common comparison” system •Regression analysis to yield parameters •Align each serum pool with regression parameters•Determine inter-method variability of aligned results for each pool

0%

20%

40%

60%

80%

100%

120%

140%

160%

180%

200%

1 2 3 4 5 6

Serum Pools

CV

ManufacturerAlignment OnlycTnI SerumCIT Serum Aligned

Variability of cTnI Measurement through Harmonization Process

Phase 3: Next Steps

• One Reference Material selected– Human CIT complex

• Large “batch” prepared– Analyzed by NIST– Standard Reference Material (SRM) 2921 by NIST

Certification of SRM 2921• Quantification of troponin I in the troponin TIC complex

performed using two methods, LC coupled to amino acid analysis (calibrated using SRM 2389 for SI-traceability) and LC/UV using a purified troponin I primary standard.

• Extensive structural characterization performed using LC/MS for molecular mass and structural heterogeneity evaluation of the intact protein molecules and MALDI/MS on peptide digests of the troponin T, troponin I, and troponin C subunits

LC/MS of Cardiac Troponin Complex used in SRM 2921

Troponin TTroponin I

Troponin C

Extract from humanheart tissue

cTnI 31.0 mg/L ± 3.4 mg/L Certified

cTnT 37.2 mg/L ± 7.9 mg/L

cTnC 24.1 mg/L ± 4.8 mg/L

Reference

Reference

ASSIGNED VALUES FOR SRM 2921 CARDIAC TROPONIN COMPLEX

Phase 3: Round Robin 3AACC cTnI Standardization Committee

• Evaluate RM 2921– TIC complex (Human)

• 10 serum pools– Derive harmonization parameters for

manufacturers

• 19 different cTnI assays– Evaluate commutability

Add to List of Higher-Order Reference Materials

Joint Committee for Traceability in Laboratory Medicine (JCTLM)

Bureau International des Poids et Mesures (BIPM)

International Federation of Clinical Chemistry and Lab Medicine (IFCC)

International Laboratory Accreditation Cooperation (ILAC)

Thank you!rchristenson@umm.edu