Troponin I Measurement: Evolution of a Biomarker Essential for Assessment of Myocardial Injury

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1 Troponin I Measurement: Evolution of a Biomarker Essential for Assessment of Myocardial Injury Robert H. Christenson, Ph.D., DABCC, FACB Professor of Pathology Professor of Medical and Research Technology University of Maryland School of Medicine

2 Troponin

When troponin is increased think heart Cardiac isoforms in blood = Trauma Contusion Ablation Pacing ICD firing Cardioversion Endomyocardial biopsy Cardiac surgery Hypertension Hypotension Acute

3 When troponin is increased think heart Cardiac isoforms in blood = 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

4 Meta-Analysis: Troponin T or I and NSTE MI JACC 2001;38: Troponin I Troponin T 2.6 ( ) 3.0 ( ) 5.1( ) 8.5 ( )

5 25% ctnt Quartiles: PARAGON B J Am Coll Cardiol 2003;41: Six-month Death/(re)MI Rate 20% 15% 10% 5% Trend P-value P = ng/ml (N=365) (N=113) (N=241) (N=340)

6 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; 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

7 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) 99 th percentile of a reference control population Imprecision (CV) at 99 th percentile should be 10% Thygesen K, et al, JACC 2000;36:

8 age >65 years TIMI Risk Factors JAMA 2000;284: >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

9 (New)Type 1A Recommendations Circulation 2002;106: 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

10 Not all Troponin Methods are Created Equal

11 Systematic Variation Between ctni Assays Clin Chem 2001;47: 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 Mean: 28.1 SD: 36.4 CV: 130% Within assay reproducibility good, but measurement of 10 ng/ml material yielded results that were >30-fold different!

12 Imprecision of Troponin Assays Clin Chem 2004;50: 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.

13 Imprecision Ratio at Low ctni Clin Chem 2004:50;

14 Compliance of Assays High sensitivity designation Analogous to: hs-crp 3 rd generation TSH Manufacturers will have to improve assays Provide data to FDA for new claim

15 ctni and One-year Clinical Events Am J Cardiol 2002;89: Access, 99th <0.02 ng/ml 0.02 ng/ml Access, 20% <0.03 ng/ml 0.03 ng/ml AxSym, 99th <0.6 ng/ml 0.6 ng/ml AxSym, 20% <1.0 ng/ml 1.0 ng/ml Death P-Value Death/MI P-Value < < <0.001 <0.001

16 Is Discordance Important? Am J Cardiol 2002;89: Classification based on equal assay performance (CV) Access, <0.03 Access, 0.03 AxSym AxSym < (TN) 3 (FP) 109 (FN) 595 (TP) Death p=0.01 Death/MI p=0.001 Death p=0.01 Death/MI p=0.001

17 Is Discordance Important? Am J Cardiol 2002;89: Classification based on equal assay performance (CV) ctnt, <0.03 ctnt, 0.03 AxSym AxSym < (TN) 17 (FP) 27 (FN) 581 (TP) Death p=ns Death/MI p=0.05 Death p=ns Death/MI p=0.01

18 Cardiac Troponin I Antibody binding to stable region for stable immunoassay N-terminal C-terminal Proteases Proteases Katrukha et al. Clin. Chem. 1998; 44: Shi et al. Clin. Chem. 1999; 45:

19 ctni Standardization is Desirable But 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

20 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

21 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

22 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.)

23 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

24 Phase I: Round Robin 1 Results 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

25 Round Robin 1 Candidate Reference Materials (crms) Evaluation of 10 candidate reference materials: Human or recombinant material CIT complex, CI complex or free ctni Clin Chem 2001;47:

26 Round Robin 1 ctni Measurement Methods Evaluated 13 troponin I assays from US, Japan, Germany, the UK, and France Clin Chem 2001;47:

27 Round Robin 1 crm Ranking Within Each Measurement System CTI CTI CI CTI Free I Clin Chem 2001;47:

28 Conclusion: Round Robin 1 Clin Chem 2001;47: 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: ).

29 Phase 2: Round Robin 2 Results 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

30 Round Robin #2 Candidate Reference Materials (crms) TIC complex, crm TIC IC complex, crm IC Characterized at NIST for purity and concentration. ctni-negative Serum Pool

31 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

32 ctni 2nd Round Robin Participants and Measurement Systems ctni Measurement System (15) Manufacturer (10) Access Beckman Coulter Instruments ACS 180 Bayer Corporation AIA TOSOH Alpha Dx First Medical Inc AxSYM Abbott Diagnostics Centaur Bayer Corporation Dimension RxL Dade Behring Immulite 1000 Diagnostic Products Corporation Immulite 2000 Diagnostic Products Corporation Immuno 1 Bayer Corporation Liaison Byk-Sangtec Diagnostica Opus, 2 nd Generation Dade Behring Stratus CS Dade Behring Vidas BioMerieux Vitros ECi Ortho Clinical Diagnostics

33 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

34 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 ISO Central Secretariat, Brussels, Belgium, 2000.

35 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:

36 Round Robin #2 Commutability: Step 1 What 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 intersystem variation

37 Commutability: Step 2 Does 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

38 crm CIT crm CI Serum Pools ctninegative Serum Diluent b ctninegative Serum Diluent b SP SP SP SP SP SP Sum 2 (2.2%) 1 (1.1%) 1 (1.1%) 12 (13.3%)

39 Round Robin #2 Commutability: Step 3 What 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

40 Commutability of (crm CIT & crm CI ) The numbers in each cell indicate how many of the 15 analytical systems had relative residual values that were outside the +3/-3 residual criteria. crm CIT crm CI Nominal ctni Concentration ctni-negative ctni-negative Serum Diluent a Serum Diluent a 5.0 μg/l 5/15 2/15 3/15 5/15 a = Manufacturers specified diluent

41 ctni Standard Material Does Not Improve Measurement Harmony ctni ( g/l) crm CIT in Serum crm CI in Serum Manufacturer's Calib SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 Serum Pools

42 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.

43 Harmonization: Present State Systems calibrated with Manufacturers calibrators and instructions 6 serum pools measured with each system Determine inter-method variability

44 Measurements, Field Calibrations Pool Usual Measurement ctni, µg/l 1 Mean SD (CV) (93%) 2 Mean SD (CV) (82%) 3 Mean SD (CV) (86%) 4 Mean SD (CV) (90%) l 5 Mean SD (CV) (97%) 6 Mean SD (CV) (91%)

45 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

46 Variability of ctni Measurement through Harmonization Process 200% 180% 160% 140% CV 120% 100% 80% 60% Manufacturer Alignment Only ctni Serum CIT Serum Aligned 40% 20% 0% Serum Pools

47 Phase 3: Next Steps One Reference Material selected Human CIT complex Large batch prepared Analyzed by NIST Standard Reference Material (SRM) 2921 by NIST

48 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

49 LC/MS of Cardiac Troponin Complex used in SRM 2921 Troponin T Troponin I Troponin C Extract from human heart tissue

50 ASSIGNED VALUES FOR SRM 2921 CARDIAC TROPONIN COMPLEX ctni 31.0 mg/l ± 3.4 mg/l Certified ctnt ctnc 37.2 mg/l ± 7.9 mg/l 24.1 mg/l ± 4.8 mg/l Reference Reference

52 Phase 3: Round Robin 3 AACC 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

53 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)

54 Thank you!

Cardiac Troponin Testing and Chest Pain Patients: Exploring the Shades of Gray

Cardiac Troponin Testing and Chest Pain Patients: Exploring the Shades of Gray Nichole Korpi-Steiner, PhD, DABCC, FACB University of North Carolina Chapel Hill, NC Learning Objectives Describe the acute