PRESENTACION TROPONINA. Analytical consideration for high sensitivitty troponin HSTNT

1. Analytical Considerations for High
Sensitivity Troponin
Alan H.B. Wu, PhD
University of California, San Francisco

2. Why troponin is superior to the others
Myoglobin CK-MB Troponin

3. Superiority of troponin vs. CK-MB
Myocardial tissue content
• CK-MB: 1.4 mg/g wet weight
• cTnI: 6.0 mg/g wet weight
• cTnT: 10.8 mg/g wet weight

4. Heart troponin different from skeletal muscle
Fast skeletal
Slow skeletal
Cardiac
NH2
█ █ █ █ █
COOH
COOH
NH2
COOH
NH2
█ █ █

5. CK-MB Troponin-T
0 0
Residual baseline
content
New onset, minor
myocardial injury
2.5
5.0
0.2
0.4
Cutoff value
Concentration
(ug/L)
Effect of specificity on sensitivity

6. Troponin T after angioplasty
Class
-TnT, -MB
-TnT, +MB
+TnT, -MB
+TnT, +MB
Thrombus or
Sidebranch occ.
NA
NA
9
NA
No. Cases
44 (55%)
0 (0%)
13 (16%)
23 (23%)

7. cTnT vs. cTnI?

8. cTnT vs. cTnI
• Equivalent diagnostic utility for AMI and risk stratification
• cTnT slightly larger than cTnI (37 vs. 24 kDa) remains
positive after AMI longer.
• Abnormal cTnT found more frequently in patients with
chronic renal failure than cTnI due to non-ischemic
myocardial damage (although incidence for cTnI in
ESRD increasing with hs assays).

9. Autoantibodies

10. Influence of antibody selection on troponin
detection
N C
N C
Ab1 Ab2 Ab3
Time
degradation
Troponin
conc.
Ab1-3
Ab1-2

11. Epitopes used in commercial cTnI assays
Company/platform/assay (generation) Epitopes recognized by antibodies
Abbott AxSYM ADV (2nd) C 87-91, 41-49; D 24-40
Abbott Architect C 87-91, 24-40: D: 41-49
Abbott i-STAT C: 41-49, 88-91; D: 28-39, 62-78
Beckman Access Accu (2nd) C; 41-49; D: 24-40
bioMerieux Vidas Ultra (2nd) C: 41-49, 22-29; D: 87-91, 7B9
Innotrac Aio! C: 41-49,190-196; D: 137-149
Inverness Biosite Triage C: NA; D: 27-40
Mitsubishi Chemical PATHFAST C: 41-49; D:71-116, 163-209
Ortho Vitros ECi ES (2nd) C 24-40, 41-49; D 87-91
Radiometer AQT90 C; 41-49, 190-196; D: 137-149
Response Biomedical RAMP C: 85-92; D: 26-38
Siemens Centaur Ultra (2nd) C: 41-49, 87-91; D: 27-40
Siemens Dimension RxL (2nd), Stratus, Vista C: 27-32; D: 41-56
Siemens Immulite 2500 and 100 C: 87-91:D: 27-40
Tosoh AIA II (2nd) C: 41-49; D: 87-91

12. Evolution
of troponin cutoff concentrations

13. Stable
angina
no injury no injury
Unstable
angina
Myocardial
infarction
little to
moderate
significant
injury
injury
Concentration of cardiac marker
Normal
individuals
increasing
Receiver operating characteristic curve derived
Initial strategy prior
to ESC/ACC redefinition
Current strategy post
ESC/ACC redefinition

14. NACB Guideline
Morrow et al. Clin Chem 2007;53:552-574
• “In the presence of a clinical history suggestive of ACS,
the following are considered indicative of myocardial
necrosis consistent with MI (Level of Evidence: C):
• Maximal concentration of cardiac troponin exceeding the
99th percentile of values (with optimal precision defined
by total CV <10%) for a reference control group on at
least 1 occasion during the first 24 h after the clinical
event (observation of a rise and/or fall in values is useful
in discriminating the timing of injury).

15. Cutoff at the 10% imprecision limit
(minimizes false positives due to analytic noise)
5
cTnI μg/L
0%
10%
20%
30%
40%
50%
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20
Frequency
of
normal
population
0%
10%
20%
30%
40%
50%
Total
Imprecision
(CV%)
97.5% URL
99% URL

16. 99th percentile for cTnI
Apple et al. Clin Chem
2007,53:1558-60.
Direct comparison on the same population

17. Next generation troponin assays

18. Singulex 99% of the normal range for cTnI
0
5
10
15
20
25
30
35
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
pg/ml
Frequency
99% Cutoff 10 pg/ml
Wu et al. Clin Chem 2005;52

19. hsTnI in ED patients with chest pain
No ischemia
Ischemia?
0
2
4
6
8
10
12
14
16
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 More
pg/ml cTNI
Frequency
Ischemia?
Non-diagnostic

20. Analytical evaluation of hs-cTnT
Giannitsis et al. Clin Chem 2010;55:in press

21. The Apple Scorecard
Hole where Wu gets slammed

22. Scorecard designations of cTn assays.
Apple. Clin Chem 2009;55:1303-6
Acceptance designation Total imprecision at the 99th percentile, CV%
Guideline acceptable 10
Clinically usable >10 to 20
Not acceptable >20
Assay designation Measurable normal values below the 99th
percentile, %
Level 4 (third generation, hs) 95
Level 3 (second generation, hs) 75 to <95
Level 2 (first generation, hs) 50 to <75
Level 1 (contemporary) <50

23. Serial testing

24. Serial testing requirement
• Serial change in cardiac markers may improve specificity by
eliminating patients who have a consistent increase due to
chronic diseases.
• WHO (1979) and ESC/ACCF/AHA/WHF (2007) both
recommended serial testing for serum biomarkers.
• No consensus on the frequency of testing (e.g., q3 or q4 h) or
Δ change considered statistically significant.
• Some investigators suggested use of 3xSD of troponin at the
cutoff (20%)(Clin Chem 2007;53:2086-96).
• Apple et al. (Clin Chem 2009;55:930-7) used 30% and found
improved clinical specificity for AMI and risk stratification.
• Short-term changes of troponin in healthy subjects (biological
variation) is an objective criteria of serial change.

25. Definition of biological variation
• The analytical variation (CVA) is the within-run assay
precision determined on duplicates.
• The intraindividual biological variation (CVI) is the day-to-
day change in analyte values due to normal physiology.
• The interindividual biological variation (CVG) between
subjects.
• Critical difference for serial change:
2.77 x (CVA
2 + CVI
2)1/2
• High sensitivity assays now enable BV measurements for
troponin.

26. 0.0 1.0 2.0 3.0 4.0 5.0 6.0
cTnI, ng/L
Subject
no.
1−
2−
3−
4−
5−
6−
7−
8−
9−
10−
11−
12−
Biological variability: short term
Wu et al. Clin Chem 2009;55:50-5.

27. Marker CVA CVI CVG II RCV Reference
Creatine kinase 14.0 22.0 42.2 0.52 72.2 Ross et al.
CK-MB, activity 29.1 4.9 14.1 0.35 81.8 Ross et al.
CK-MB, mass 6.8 18.4 61.2 0.30 54.4 Ross et al.
Myoglobin 13.4 17.6 46.6 0.38 61.2 Ross et al.
cTnI 8.3 9.7 56.8 0.21 +46,-32 Wu et al. 2008
Biological variability: short term
Wu et al. Clin Chem 2009;55:50-5.

28. Serial testing for cTnI: cases
Wu et al. Clin Chim Acta 2009;401:170-4

29. Serial testing for cTnI: cases
Wu et al. Clin Chim Acta 2009;401:170-4

30. Point-of care testing for
cardiac markers

31. Pros and cons for POCT for cardiac
• Pro: whole blood (no centrifugation), no
sample delivery, on-instrument TAT
shorter
• Con: regulatory compliance, more costly,
and assay performance issues. None are
high sensitivity (at the moment).

32. ACC/AHA Guideline for UA patients
Circulation 2000;102:1193-1209
“When a central laboratory is used to measure
biochemical cardiac markers, results should be available
within 60 minutes and preferably within 30 minutes.”
BNP Consensus Panel (CHF 10(suppl3):1-30, 2004)
TAT for BNP = 60 min.

33. Discordances from POC v central lab testing
Singh et al. Clin Chim Acta 2009;403:359-60

34. 0
3
6
9
12
0
3
6
9
12
0
3
6
9
12
OR 4.55; 2.66-7.78
Rapid Troponin I Assay
Outcomes in relation to troponin: assay sensitivity
James et al. Int J Cardiol 2004;93:13-20.
%
Neg
Death MI Death or MI
56 98 92 130 132 205
Troponin T (0.1 μg/L)
Troponin T (0.01 μg/L)
OR 1.80; 1.30-2.54
1.82; 1.38-2.40
1.64; 1.31-2.06
OR 3.20; 2.22-4.59 2.26; 1.79-2.85
1.47; 1.12-1.93
3.42; 2.57-5.98 4.29; 3.02-6.09
Pos
%
%
41
15
113
139
86
25
136
197
116
36
221
301

35. Summary
• Analytical improvements in the sensitivity of troponin
assays will improve risk stratification (Morrow lecture).
• More than ever before, interpretation of results will
require correlation to clinical findings.
• Serial testing can improve specificity of increased
troponin due to non-ischemic causes
• Point-of-care testing technology has not caught up to
next-generation lab-based troponin for analytic sensitivity