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Technical Briefs |
Department of Medical Sciences,
1
Clinical Chemistry and
2
Internal Medicine, University of Uppsala, SE-751 85 Uppsala, Sweden
aaddress correspondence to this author at: Department of Clinical Chemistry, University Hospital, SE-751 85 Uppsala, Sweden; fax 46-186113703, e-mail Per.Venge{at}clm.uas.lul.se
The measurement of troponins in blood has rapidly become an alternative to conventional methods of detecting myocardial damage (1)(2)(3)(4)(5)(6)(7)(8), particularly in unstable angina, and several studies have indicated the prognostic importance of increased troponins in various clinical settings (9)(10)(11)(12)(13)(14). These studies, however, have also pointed out the need for more sensitive methods because patients with even small increases of troponin seem to be at increased risk of cardiac events. Currently, cardiac troponin I (cTnI) can be quantified by assays from several manufacturers (15)(16)(17)(18)(19), whereas only one company currently commercializes a cardiac troponin T assay (2)(20)(21). The aim of this work was to evaluate the analytical performance of a new generation of the Access cTnI assay. We also provide data on values in apparently healthy subjects.
Venous blood was drawn from 70 patients admitted to our Coronary Care Unit because of suspicion of an acute coronary syndrome. Only patients found to have increased myocardial markers such as creatine kinase-MB and troponin I were included. The study was approved by the ethics committee of the Medical Faculty of Uppsala University. Serum samples were also obtained from 122 apparently healthy subjects (70 women and 52 men; median age, 41 years; range, 26–73 years) as part of a health-screening program.
The new ACCESS cTnI assay (Beckman Coulter, Inc., Chaska, MN) is a two-site immunoenzymatic (sandwich) immunoassay. Paramagnetic particles coated with mouse monoclonal anti-cTnI, mouse monoclonal anti-cTnI-alkaline phosphatase conjugate, and sample are added to a reaction vessel to form a particle-cTnI-conjugate sandwich. The cTnI in the sample binds to the immobilized anti-cTnI on the solid phase. The mouse anti-cTnI conjugate reacts with a different antigenic site on the cTnI molecule. Separation in a magnetic field and washing remove materials not bound to the solid phase. A chemiluminescent substrate (dioxetane Lumigen PPD) is added to the reaction vessel, and light generated by the reaction is measured with a luminometer. The photon production is proportional to the quantity of cTnI in the sample. The amount of analyte is determined by means of a stored multipoint calibration curve.
We defined the detection limit as the concentration of cTnI at a signal
2 SD above the mean signal of 10 replicates of the zero calibrator, as
calculated from the calibration curve. Three studies were performed on
one lot of reagents. The mean detection limit was 0.0036 µg/L (range,
0.0024–0.005 µg/L). The lower limit of the reporting range was
defined as the concentration at which the variation in duplicate
samples was 
20% (CV) and was calculated by means of the computer
software Multicalc® (Wallac Oy). It was
estimated by assaying serial dilutions of five different patient
LiHeparin (cat. no. 367993; 3 mL PET tube; 72 IU of lithium heparinate;
final concentration in filled tube, 48 IU/mL of blood; BD Vacutainer
Systems) plasma samples with increased cTnI (range, 0.21–0.81 µg/L).
The lower limit was 0.0085 µg/L. In the same experiment, the cTnI at
a CV of 10% was 0.03 µg/L.
To determine assay imprecision, in one set of experiments trilevel controls provided by Beckman Coulter (range, 0.582–29.2 µg/L) were analyzed in triplicate in a total of 36 assays representing 6 tests per day during 6 different days. The model used to estimate imprecision was a one-way ANOVA, assuming random effect. Estimates were calculated for intraassay, interassay, and total imprecision for each control level and showed intraassay variations (CVs) of 2.2–3.2%, interassay variations of 1.2–4.4%, and total imprecision of 2.5–5.4%. In an additional study, three patient samples (LiHeparin) with mean cTnI values (range) of 0.098 µg/L (0.09–0.11µg/L), 0.069 µg/L (0.06–0.07 µg/L), and 0.034 µg/L (0.03–0.04 µg/L) were assayed six times on each of 2 separate days, and the total imprecision was calculated. The CVs were 6.4%, 4.3%, and 15%, respectively.
A linearity study was performed with LiHeparin-plasma samples from six
subjects with increased cTnI (0.86–75 µg/L). The samples were
measured in quadruplicate at five different dilutions (0.8, 0.6, 0.4,
0.2, and 0.1). The mean apparent recovery was 98.5% (95% confidence
interval, 95.9–101.0%). To analyze the linearity at lower
concentrations, we used the results for the estimation of the
functional sensitivity as above. As shown in Fig. 1A
, dilutions of the five patient samples were linear down to
cTnI concentrations <0.03 µg/L (r = 0.9991).
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In vitro sample stability was tested using matched serum and
LiHeparin-plasma samples obtained from eight patients with cTnI
concentrations ranging from 0.81 to 52.0 µg/L. Accepting a ±
10% deviation from the values at 0 h, cTnI was stable in
LiHeparin-plasma samples left at room temperature for 48 h after
blood sampling, although the values at 48 h showed increased
variation (median, 91%; range, 85–111%). After 
72 h at room
temperature, mean results were 80% of the initial concentration.
Values in LiHeparin-plasma samples stored at 4 °C also changed
<10% at 48 h, and median values were 89% of control (range,
85–98%) at 72 h or more. The in vitro stability of cTnI in serum
was similar to that in LiHeparin plasma at both temperatures. In all
sample types, the changes after storage at either room temperature or
4 oC were independent of the initial cTnI
concentration. Five freeze-thaw cycles with four samples at different
concentrations had no significant effect on cTnI recovery (median,
100%; range, 92–107%).
A sample type comparison was performed in matched serum and
LiHeparin-plasma samples (n = 54) collected at the same time from
the same subject (Fig. 1B
). Results in LiHeparin plasma and serum were
not significantly different [2.4% lower in serum (95% confidence
interval, -1.3% to 6.1%)]. Plasma and serum showed a correlation
coefficient of 0.9975.
In 122 sera from apparently healthy subjects, the cTnI concentrations of all except 2 subjects were <0.01 µg/L. Thus, the 95th and 99th percentiles were 0.01 and 0.02 µg/L, respectively, in this group.
We conclude that the new generation of Access cTnI assay has several desirable features. The lower limit of the reported range is below the 95th percentile of our preliminary reference range for a healthy population. Both serum and LiHeparin plasma may be used for measurement of cTnI, although there was a tendency to lower values in LiHeparin plasma at lower cTnI concentrations. [Part of this difference might be explained by the loss in cTnI that has been shown to occur for both cTnI and troponin T during the early phase of acute myocardial infarction (22)(23). The data presented in this study, however, do not allow conclusions on this point.] When measured by this assay, cTnI is stable in vitro at both room temperature and 4 °C and can be stored up to 48 h under these conditions without any major effect on recovery. It seems, however, reasonable to recommend storage for <24 h. [The in vitro stability observed with the new generation cTnI assay contrasts with our experience with the Access first-generation cTnI assay. Indeed, storage of samples at room temperature for 1–2 h produced decreases of 20–30% of the measured cTnI concentration (unpublished observations and Ref. (15).]
The improved in vitro stability may be explained by the selection of different monoclonal antibodies to develop the new generation cTnI assay. According to Beckman Coulter, Inc., the monoclonal antibodies used in the new generation cTnI assay were selected based on published information that suggested that the antibodies recognize epitopes located within a region encompassing amino acids 30–110 in the N-terminal half of the cTnI molecule. In recent studies, the region encompassing amino acids 30–110 was shown to be more resistant to proteolytic cleavage (15)(24).
Acknowledgments
This study was supported by grants from Beckman Coulter Inc. The technical expertise of Ing-Britt Persson is gratefully appreciated.
References
The following articles in journals at HighWire Press have cited this article:
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  P. A. Kavsak, X. Wang, D. T. Ko, A. R. MacRae, and A. S. Jaffe Short- and Long-Term Risk Stratification Using a Next-Generation, High-Sensitivity Research Cardiac Troponin I (hs-cTnI) Assay in an Emergency Department Chest Pain Population Clin. Chem., October 1, 2009; 55(10): 1809 - 1815. [Abstract] [Full Text] [PDF]
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 J. Sundstrom, E. Ingelsson, L. Berglund, B. Zethelius, L. Lind, P. Venge, and J. Arnlov Cardiac troponin-I and risk of heart failure: a community-based cohort study Eur. Heart J., April 1, 2009; 30(7): 773 - 781. [Abstract] [Full Text] [PDF]
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P. Venge, S. James, L. Jansson, and B. Lindahl Clinical Performance of Two Highly Sensitive Cardiac Troponin I Assays Clin. Chem., January 1, 2009; 55(1): 109 - 116. [Abstract] [Full Text] [PDF]
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 P. Bahrmann, G. S. Werner, G. Heusch, M. Ferrari, T. C. Poerner, A. Voss, and H. R. Figulla Detection of Coronary Microembolization by Doppler Ultrasound in Patients With Stable Angina Pectoris Undergoing Elective Percutaneous Coronary Interventions Circulation, February 6, 2007; 115(5): 600 - 608. [Abstract] [Full Text] [PDF]
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P. A. Kavsak, A. M. Newman, V. Lustig, A. R. MacRae, G. E. Palomaki, D. T. Ko, J. V. Tu, and A. S. Jaffe Long-Term Health Outcomes Associated with Detectable Troponin I Concentrations Clin. Chem., February 1, 2007; 53(2): 220 - 227. [Abstract] [Full Text] [PDF]
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P. A. Kavsak, A. R. MacRae, G. E. Palomaki, A. M. Newman, D. T. Ko, V. Lustig, J. V. Tu, and A. S. Jaffe Health Outcomes Categorized by Current and Previous Definitions of Acute Myocardial Infarction in an Unselected Cohort of Troponin-Naive Emergency Department Patients Clin. Chem., November 1, 2006; 52(11): 2028 - 2035. [Abstract] [Full Text] [PDF]
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A. R. MacRae, P. A. Kavsak, V. Lustig, R. Bhargava, R. Vandersluis, G. E. Palomaki, M.-J. Yerna, and A. S. Jaffe Assessing the Requirement for the 6-Hour Interval between Specimens in the American Heart Association Classification of Myocardial Infarction in Epidemiology and Clinical Research Studies Clin. Chem., May 1, 2006; 52(5): 812 - 818. [Abstract] [Full Text] [PDF]
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S. James, M. Flodin, N. Johnston, B. Lindahl, and P. Venge The Antibody Configurations of Cardiac Troponin I Assays May Determine Their Clinical Performance Clin. Chem., May 1, 2006; 52(5): 832 - 837. [Abstract] [Full Text] [PDF]
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B. Zethelius, N. Johnston, and P. Venge Troponin I as a Predictor of Coronary Heart Disease and Mortality in 70-Year-Old Men: A Community-Based Cohort Study Circulation, February 28, 2006; 113(8): 1071 - 1078. [Abstract] [Full Text] [PDF]
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D. A. Morrow, N. Rifai, M. S. Sabatine, S. Ayanian, S. A. Murphy, J. A. de Lemos, E. Braunwald, and C. P. Cannon Evaluation of the AccuTnI Cardiac Troponin I Assay for Risk Assessment in Acute Coronary Syndromes Clin. Chem., August 1, 2003; 49(8): 1396 - 1398. [Full Text] [PDF]
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P. Venge, N. Johnston, B. Lagerqvist, L. Wallentin, and B. Lindahl Clinical and Analytical Performance of the Liaison Cardiac Troponin I Assay in Unstable Coronary Artery Disease, and the Impact of Age on the Definition of Reference Limits. A FRISC-II Substudy Clin. Chem., June 1, 2003; 49(6): 880 - 886. [Abstract] [Full Text] [PDF]
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D. Uettwiller-Geiger, A. H.B. Wu, F. S. Apple, A. W. Jevans, P. Venge, M. D. Olson, C. Darte, D. L. Woodrum, S. Roberts, and S. Chan Multicenter Evaluation of an Automated Assay for Troponin I Clin. Chem., June 1, 2002; 48(6): 869 - 876. [Abstract] [Full Text] [PDF]
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