Diagnostic utility of new immunoassays for the cardiac markers cTnI, myoglobin and CK-MB mass

Clinical Biochemistry 38 (2005) 1027 – 1030

Diagnostic utility of new immunoassays for the cardiac markers cTnI, myoglobin and CK-MB mass Michael Weber a,*, Matthias Rau a, Katharina Madlener a, Albrecht Elsaesser a, Dragic Bankovic b, Veselin Mitrovic a, Christian Hamm a a

Kerckhoff Heart Center, Department of Cardiology, Benekestraße 2-8, 61231 Bad Nauheim, Germany b Prirodno Matematicki Fakultet, Kragujevac, Serbia and Montenegro Received 25 May 2004; received in revised form 8 July 2005; accepted 10 July 2005 Available online 24 August 2005

Abstract Objectives: We investigated the diagnostic value of a new system, the Innotrac Aio!i immunoassays for troponin, myoglobin and CKMB, in 270 samples from patients with ACS, after bypass surgery (CABG) or with stable heart failure in comparison to the respective Roche assays. Results: The values of the cardiac markers assessed by the respective assays correlated (cTnT/cTnI Rho = 0.94, myoglobin Rho = 0.87, CK-MB Rho = 0.84). If values were dichotomised, we found a high concordance of test positive and negative classified patients by troponins with the respective assays. Conclusion: There is strong evidence that the Innotrac Aio!i system for cTnI measurement can be used reliably. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Cardiac markers; Troponin; Myoglobin; CK-MB; Acute coronary syndromes

Background Myoglobin, creatine kinase-MB (CK-MB) and cardiac specific troponins have become the cornerstone for risk stratification and diagnosis of patients with an acute coronary syndrome (ACS) [1]. Since cardiac troponin T (cTnT) or I (cTnI) have demonstrated higher sensitivity than CK-MB, current guidelines recommend the use of troponins rather than CK-MB or myoglobin. This requires high precision troponin assays at values near the decision limit. Therefore, the ESC and the AHA/ACC recommend that the reference interval for each assay should be defined as the 99th percentile of a normal population and that the analytical imprecision at this cut-off level should be 10% [2– 4]. Several assays with high precision for the measurement of troponins and other cardiac markers are commercially

* Corresponding author. Fax: +49 6032 9962313. E-mail address: [email protected] (M. Weber).

available [5]. Recently, a new system, the Innotrac Aio!i (Innotrac Diagnostics, Turku Finland), has been introduced [6 –9]. We performed a prospective observational study with the aim of comparing these new immunoassays for cardiac troponin I (cTnI), CK-MB and myoglobin in patients with ACS, after bypass surgery (CABG) and with stable heart failure versus established assays for cTnT, creatine kinase MB activity and myoglobin (Roche, Elecsys).

Methods Patients We included 85 consecutive patients with ACS, 50 patients after CABG and 50 patients with clinically stable chronic heart failure (HF) NYHA-class III. In patients with an ACS, blood was taken at admission, at average 11.4 h after onset of symptoms (1.3 h– 41.7 h; median 10.9 T 1 h) and the day following admission. In patients after CABG,

0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.07.011

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M. Weber et al. / Clinical Biochemistry 38 (2005) 1027 – 1030

blood sampling was performed on the first postoperative day and in HF patients during a regular visit in the heart failure clinic. Clinical data were assessed on admission. STelevation myocardial infarction (STEMI) and non-STelevation myocardial infarction (NSTEMI) were defined according to the recommendations of the Joint European Society of Cardiology/American College of Cardiology committee for the redefinition of myocardial infarction [10]. All patients with an ACS underwent immediate angiography and revascularisation.

mL for men and 58 ng/mL for women. CK-MB activity was measured with the Roche assay with normal reference value of 7 –25 U/L. Cut-off values for myoglobin (myoglobin Innotrac Aio! 135 ng/mL and myoglobin Roche 72 ng/mL) and CK-MB (CK-MB Innotrac Aio! 6 ng/mL and for CK-MB Roche 24 U/L) were chosen as they were recommended according to the manufacturer information.

Laboratory analysis

Values for cardiac markers are given as mean (AM) T standard deviation (SD). For statistical analysis, Mann – Whitney test (2-groups) and the Kruskal –Wallis test (ngroups) were used for non-parametric data and the Student’s t test for parametric data. For the analysis of the baseline characteristics of the patients, the t test (2-groups) or ANOVA (n-groups) was used for continuous variables, and the CHI2 test was used for categorical variables. In order to determine if two tests are significantly different when dichotomous cut points are determined, the McNemar analysis was used. The Spearman correlation coefficient was calculated for the respective markers of the different manufacturers. For all statistical analysis, the statistical software SPSS 10.0 for windows was used.

Blood was sampled in gel tubes containing no anticoagulant. The specimens were centrifuged within 1 h, and serum was frozen at 80-C until analysis. All assays were performed according to the manufacturer’s instructions. Troponin I, creatinine kinase MB, myoglobin (Innotrac Aio!) The Innotrac Aio!i Immunoassays (Innotrac Diagnostics, Turku, Finland) are based on proven dry chemistry TRF (time-resolved-fluorescence) technology. The cut-off value used for troponin I for the diagnosis of MI was 0.06 ng/mL. For myoglobin, the median and the 95th percentile in apparently healthy volunteers were 64 ng/mL and 122 ng/ mL for women and 71 ng/mL and 135 ng/mL for men, for CK-MB, the 95th percentile of healthy individuals is 4.7 ng/ mL in men and 6.0 ng/mL in women according to the manufacturer’s information. Troponin T, CK-MB activity, myoglobin (Elecsys, Roche) Cardiac troponin T (cTnT), CK-MB and myoglobin were measured by an electrochemiluminescence immunoassay (third generation) for cTnT on an Elecsys 2010 analyzer (Roche Diagnostics. Mannheim, Germany). For cTnT, the recommended cut-off level of 0.03 ng/mL was used. For myoglobin, the 97.5th percentile was 72 ng/

Statistics

Results Baseline characteristics, clinical data and values of the cardiac markers for all patients are shown in Table 1. In patients with an ACS, the definite diagnosis was STEMI in 23 patients, NSTEMI in 40 patients and unstable angina pectoris (UAP) in 22 patients. The values of cardiac markers were elevated in ACS and after CABG but were within the normal range in patients with HF. The highest values were found in ACS and lowest in HF patients with a significant difference of all three cardiac markers between HF patients

Table 1 Characteristics and values for cardiac markets of all patients

n Gender (male) Age (mean; min – max) TnT Roche baseline TnT Innotrac Aio! baseline TnT Roche day 1 TnI Innotrac Aio! day 1 Myoglobin Roche baseline Myoglobin Innotrac Aio! baseline Myoglobin Roche day 1 Myoglobin Innotrac Aio! day 1 CK-MB Roche baseline CK-MB Innotrac Aio! baseline CK-MB Roche day 1 CK-MB Innotrac Aio! day 1

(years) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (U/L) (ng/mL) (U/L) (ng/mL)

ACS

CABG

HF

85 59 (69%) 65 (40 – 89) 0.49 T 0.01 1.83 T 0.36 1.74 T 0.3 9.75 T 2.23 282 T 61 317 T 48 119 T 17 219 T 33 30.4 T 4.9 31.3 T 6.5 53.9 T 8.3 65 T 11.6

50 33 (66%) 66 (43 – 86) 0.52 T 0.16 2.01 T 0.72

259 T 0.16 293 T 33

38.7 T 5.2 34.4 T 6.4

P-value ACS-CABG

P-value ACS-HF

P-value CABG-HF

50 42 (84%) 61 (42 – 81) 0.02 T 0.002 0.01 T 0.004

0.68 0.067 0.86 0.74

0.06 0.04 <0.01 <0.01

0.04 0.01 <0.01 <0.01

0.02 T 0.002 95 T 16

0.86 0.98

<0.01 <0.01

<0.01 <0.01

9.8 T 1.2 3.4 T 0.4

0.27 0.75

<0.01 <0.01

<0.01 <0.01

M. Weber et al. / Clinical Biochemistry 38 (2005) 1027 – 1030

and those with ACS or CABG. There was no difference of any of the three cardiac markers between ACS and CABG patients (Table 1). Comparing all 270 samples, we found a significant correlation of cTnI (Innotrac) and cTnT (Roche) (Rho = 0.94, P < 0.01, Sy/x = 10,217), of CK-MB mass (Innotrac) and CK-MB activity (Roche) (Rho = 0.84, P < 0.01) and of myoglobin (Innotrac) and myoglobin (Roche) (Rho = 0.87, P < 0.01; Figs. 1A – C). We used the abovementioned cut-off levels to dichotomise in marker positive and negative patients. Comparing the 270 samples of all patients, we found no significant differences between the two troponin assays in respect to the number of patients who were classified as troponin positive and negative (cTnT+/cTnI+ 166, cTnT+/cTnI 9, cTnT /cTnI+ 7, cTnT /cTnI 88; P = 0.804 by McNemar) with a significant correlation (r = 0.87; P < 0.01). This resulted in a sensitivity of 93% and specificity of 95% for the cTnI Innotrac Aio assay to predict an elevation of cTnT Roche. Almost identical results were found when the 170 samples of 85 patients with an ACS were compared. There was no significant difference in troponin positive and negative classified patients between the two assays (cTnT+/cTnI+ 118, cTnT+/cTnI 2, cTnT /cTnI+ 6, cTnT /cTnI 43; P = 0.289 by McNemar) with a comparable correlation (r = 0.88; P < 0.01).

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In the group of 85 patients with ACS, myocardial infarction was diagnosed in 63 patients. At admission, 55 patients were troponin positive by the Roche assay and 56 by the Innotrac Aio!i assay (cTnT+/cTnI+ 53, cTnT+/cTnI 2, cTnT /cTnI+ 3, cTnT /cTnI 27; P = 1.00 by McNemar) with a significant correlation (r = 0.75; P < 0.01). Myoglobin (myo) was elevated at admission using the Roche (R) assay in 42 patients and using the Innotrac Aio!i system (I) in 33 patients (myo(R)+/myo(I)+ 33, myo(R)+/myo(I) 9, myo(R) /myo(I)+ 0, myo(R) /myo(I) 21; P = 0.004 by McNemar) with a significant correlation (r = 0.74; P < 0.01). CK-MB at admission was elevated in 27 patients analysed with the Roche assay and in 41 patients analysed with the Innotrac Aio!i (I) system (CK-MB(R)+/CK-MB(I)+ 26, CK-MB(R)+/CK-MB(I) 1, CK-MB(R) /CK-MB(I)+ 15, CK-MB(R) /CK-MB(I) 21; P = 0.001 by McNemar) with a significant correlation (r = 0.57; P = 0.001). Out of 63 patients with a myocardial infarction, 17 were admitted to our hospital early after onset of symptoms so that the first blood sample was taken within 4 h. In these patients, the number of troponin positive patients was not significantly different between the two assays (cTnT+/cTnI+ 11, cTnT+/cTnI 2, cTnT /cTnI+ 0, cTnT /cTnI 4; P = 0.5 by McNemar) with a significant correlation (r = 0.71; P < 0.01). However, it must be noted that the number of patients included in this analysis was quite small.

Fig. 1. Correlation of Roche assays and the Innotrac Aio! assays for the 270 samples from all patients. (A) For cardiac specific troponin T and I, (B) for myoglobin and (C) for CK-MB.

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Discussion The Elecsysi (Roche) system for the measurement of cardiac markers troponin T, myoglobin and CK-MB has been in use for several years and has proven its diagnostic utility with high test accuracy. The Innotrac Aio!i system immunoassays for the determination of cardiac troponin I, myoglobin and CK-MB were introduced recently and have not had extensive clinical use. To date, there are limited data available, which primarily demonstrate the good analytical performance of these new assays [6]. Here, we found good correlation between the respective assays. Thus, the Innotrac Aio!i immunoassays were able to reliably detect elevated cardiac markers as compared with an established method and were appropriate for routine clinical use. The cardiac specific troponins are of major interest in these patients since an elevation of these markers allows accurate risk assessment and is an indication for therapeutical options such as antithrombotic agents or early revascularisation. In daily practice, values for cardiac specific troponins are dichotomised by predefined cut-off levels. The ESC and the AHA/ACC recommend that the cut-off level for each assay should be established as the 99th percentile of a normal population and that the analytic imprecision at this cut-off level should be 10% [2,4]. Using cut-off values for the respective markers according to the above mentioned requirements, we found a high correlation and concordance of both assays for cardiac troponin. This supports the diagnostic utility of the Innotrac Aio!i immunoassay for cTnI. There was also no difference between the different assays to detect elevated cardiac specific troponin on admission in patients with myocardial infarction or to detect elevated troponin of patients who were admitted very early (within 4 h after onset of symptoms). Therefore, none of the assays showed a comparatively greater sensitivity for detecting elevated troponins in patients with myocardial infarction at a very early stage. We also found a good correlation between the absolute values of myoglobin and CK-MB activity obtained by the respective assays. However, if dichotomised at the predefined cut-off values, there was a difference between the two tests. Uses must be aware of this discrepancy, and these cutoff values need further evaluation. It must be noted that cTnT was used as a gold standard to establish the diagnosis of myocardial infarction in this study, in accordance to the current guidelines.

Conclusion In this study, we found a close correlation of the values for the biomarkers troponin I, myoglobin and CK-MB assessed by new immunoassays (Innotrac Aio!) as com-

pared to the values obtained with established assays (Roche, Elecsys). However, using the recommended cut-off values, the results by respective assays for cTnT and troponin I were not different, whereas the results obtained by the respective assays for myoglobin and CK-MB were different with regard to patient classification. Therefore, these findings provide evidence that the Innotrac Aio!i system immunoassay is reliable in measuring these markers, especially troponin I in the clinical setting of acute coronary syndrome, after bypass surgery and in patients with heart failure. However, the recommended cut-off values for myoglobin and CK-MB need to be reevaluated. References [1] Hamm CW. Acute coronary syndromes The diagnostic role of troponins. Thromb Res 2001;103(Suppl 1):S63 – 9. [2] Braunwald E, Antman EM, Beasley JW, Califf RM, Cheitlin MD, Hochman JS, et al. ACC/AHA guidelines for the management of patients with unstable angina and non-ST-segment elevation myocardial infarction. A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee on the Management of Patients With Unstable Angina). J Am Coll Cardiol 2000;36(3):970 – 1062. [3] Braunwald E, Antman EM, Beasley JW, Califf RM, Cheitlin MD, Hochman JS, et al. ACC/AHA 2002 guideline update for the management of patients with unstable angina and non-ST-segment elevation myocardial infarction-summary article: a report of the American College of Cardiology/American Heart Association task force on practice guidelines (Committee on the Management of Patients With Unstable Angina). J Am Coll Cardiol 2002;40(7): 1366 – 74. [4] Bertrand ME, Simoons ML, Fox KA, Wallentin LC, Hamm CW, McFadden E, et al. Management of acute coronary syndromes: acute coronary syndromes without persistent ST segment elevation; recommendations of the Task Force of the European Society of Cardiology. Eur Heart J 2000;21(17):1406 – 32. [5] Apple FS, Wu AH, Jaffe AS. European Society of Cardiology and American College of Cardiology guidelines for redefinition of myocardial infarction: how to use existing assays clinically and for clinical trials. Am Heart J 2002;144(6):981 – 6. [6] Hedberg P, Valkama J, Puukka M. Analytical performance of timeresolved fluorometry-based Innotrac Aio! cardiac marker immunoassays. Scand J Clin Lab Invest 2003;63(1):55 – 64. [7] Lovgren T, Merio L, Mitrunen K, Makinen ML, Makela M, Blomberg K, et al. One-step all-in-one dry reagent immunoassays with fluorescent europium chelate label and time-resolved fluorometry. Clin Chem 1996;42(8 Pt 1):1196 – 201. [8] Pettersson K, Katajamaki T, Irjala K, Leppanen V, Majamaa-Voltti K, Laitinen P. Time-resolved fluorometry (TRF)-based immunoassay concept for rapid and quantitative determination of biochemical myocardial infarction markers from whole blood, serum and plasma. Luminescence 2000;15(6):399 – 407. [9] Pagani F, Stefini F, Panteghini M. Innotrac Aio! Second-generation cardiac troponin I assay: imprecision profile and other key characteristics for clinical use. Clin Chem 2004;50(7):1271 – 2. [10] Alpert JS, Thygesen K, Antman E, Bassand JP. 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(3): 959 – 69.