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Analytical performance of the Sanofi Access[R] cardiac troponin-I procedure.

Troponin-I (TnI), a regulatory protein of the thin filaments of striated muscle, is part of the troponin complex (I, T, C) involved in the calcium-sensitive switch that regulates the interaction of actin and myosin in these tissues [1]. TnI exists in three isoforms: one for slow-twitch, one for fast-twitch, and one for cardiac muscle. Cardiac TnI (cTnI) is differentiated from the skeletal muscle isoform by a significant difference in amino acid sequence [2]. cTnI is not expressed in human fetal skeletal muscle or in adult human skeletal muscle in response to any pathological stimuli but is specific to cardiac muscle. Consequently, an increasing interest has been paid to this protein as a marker of myocardial injury [3, 4].

Several different immunoassays have been reported for quantification of cTnI in serum. They use two cTnI-specific monoclonal antibodies, do not show cross-reactivity with skeletal muscle TnI or other cardiac proteins, and are not subject to nonspecific binding effects from the sample matrix [5,6]. The Access [R] cTnI is an automated procedure performed with the Access Immunoassay Analyzer (Sanofi Diagnostics Pasteur, Madrid, Spain), a random and continuous-access instrument that performs various immunoenzymatic assays. Paramagnetic particles coated with monoclonal antibodies against the analyte constitute the solid phase, and chemiluminescence is used to detect antigen-antibody binding. The protocol is initiated by adding the sample and anti-cTnI antibody labeled with alkaline phosphatase to a reaction vessel containing the paramagnetic particles in a buffered solution. After incubation, the unbound material is separated and removed by applying a magnetic field and washing. Finally, a chemiluminescent substrate is added to the reaction vessel. The signal is directly proportional to the amount of the antigen, which is determined by comparison with the stored calibration curve. The test takes ~15 min. The Access cTnI procedure resembles the previous manual assay designed by Pasteur [6] only through use of the same monoclonal antibodies.

To evaluate the analytical characteristics of the Access cTnI assay, we followed the protocol proposed by the Sociedad Espanola de Quimica Clinica (SEQC) [7]. Access reagents, calibrators, and controls were provided by the manufacturer. The control materials and calibrators were prepared and stored according to the manufacturer's instructions. Imprecision was calculated by assaying in triplicate, in 2 runs per day for 20 days, cTnI controls at three concentrations covering the analytical range. The linearity study was carried out by diluting a specimen pool of high concentration with various volumes of the SO control (zero cTnI concentration) and analyzing the diluted solutions in duplicate. The detection limit of the method was calculated by determining 5 times the SO control and a sample with low cTnI concentration (0.1 [micro]g/L calibrator) and establishing a calibration curve from these two points. The detection limit was taken as 2 SD above the mean measured for the SO control. We repeated this process on 3 days in a 1-month period. To determine the amount of carryover, we analyzed in quadruplicate the sequence AABBB, where A is a specimen pool with high cTnI concentration (40 [micro]g/L) and B is a specimen pool with low concentration (0.1 [micro]g/L). The percentage of carryover was calculated with the formula proposed by Broughton [8]:

carryover, % = 100 X (B1 - B3)/(A2 - B3)

The imprecision of the method (CV) ranged from 4.8% to 7.8% (Table 1)--values comparable with the published data for the automated analyzers Stratus [R] (Dade) [9] and Opus Plus [R] (Behring Diagnostics) [10] and better than those of the manual procedures (CVs between 2.1% and 26.7% [5, 61). The detection limit was determined as 0.037 [micro]g/L, lower than the reported minimum detectable concentrations for other assays: Stratus assay, 0.5 [micro]g/L; Bodor et al. [5], 1.9 [micro]g/L; Larue et al. [6], 0.2 [micro]g/L. The manufacturer's recommended cutoff for myocardial injury (cTnI 0.1 [micro]g/L) is also lower than the decision points recommended for the Stratus analyzer--0.6 [micro]g/L [11], 0.8 [micro]g/L [121, and 1.5 [micro]g/L [13,141--and the Opus Plus analyzer--2 [micro]g/L [15,161. The method showed excellent linearity over the range of concentrations studied (1-50 [micro]g/L), yielding a linear regression equation of [y.sub.measured] = [0.95x.sub.expected] - 0.68 (r = 0.999), and the percentage difference between the expected and measured data was 8694%. The amount of sample-related carryover was insignificant, i.e., 0.008%.

We conclude that the Sanofi Pasteur Access assay of cTnI is a linear and precise method.

We thank Sanofi Diagnostics Pasteur, Madrid, Spain, for loan of equipment and gifts of reagent.


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[3.] Bodor GS, Porterfield D, Voss EM, Smith S, Apple FS. Cardiac troponin-I is not expressed in fetal and healthy or diseased adult human skeletal muscle tissue. Clin Chem 1995;41:1710-5.

[4.] Adams JE III, Bodor GS, Davila-Roman VG, Delmez JA, Apple FS, Ladenson JH, Jaffe AS. Cardiac troponin-I: a marker with high specificity for cardiac injury. Circulation 1993;88:101-6.

[5.] Bodor GS, Porter S, Landt Y, Ladenson JH. Development of monoclonal antibodies for an assay of cardiac troponin-I and preliminary results in suspected cases of myocardial infarction. Clin Chem 1992;38:2203-14.

[6.] Larue C, Calzolari C, Berinchart JP, Leclerq F, Grolleau R, Pau B. Cardiac- specific immunoenzymometric assay of troponin-I in the early phase of acute myocardial infarction. Clin Chem 1993;39:972-9.

[7.] Aluma A, Alsina MJ, Armenter C, Bertran N, Biosca C, Dolade M, et al. Protocolo de Evaluacion de Analizadores Automaticos por el Usuario. In: Seleccion y Evaluacion de Sistemas Analiticos. Barcelona: SEQC, 1994:4656.

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[9.] Flaa C, Sabucedo A, Geist W, DeMarco C, Troy S, Chavaillaz P, Bauer R. Development of a rapid, automated procedure for the determination of troponin-I on the Stratus[R] immunochemistry analyzer [Abstract]. Clin Chem 1993;39:1273.

[10.] Panteghini M, Bonora R, Pagani F. An automated immunoassay for determination of cardiac troponin-I in serum evaluated [Abstract]. Clin Chem 1996;42:S186-7.

[11.] Russel A, Loonan M, Lester D, Henke A, Feld R. Divergence between cardiac troponin-I (cTnI) and creatine kinase MB (CKMB) in the diagnosis of acute myocardial infarction (AMI) [Abstract]. Clin Chem 1996;42:S99.

[12.] Apple F, Sharkey S, Falahati A, Christensen D, Miller E, McCoy M, Murakami MA. A prospective study for detection of myocardial infarction [Abstract]. Clin Chem 1996;42:S97.

[13.] Omand K, Leung P, Lopez H, Johnson T, Almazan C, Kirk D, Kost G. Early diagnosis of acute myocardial infarction using troponin I & T, CK-MB isoforms, myoglobin and CK-MB [Abstract]. Clin Chem 1996;42:S102-3.

[14.] Dohnal J, Bowie L, Kouzov E. Troponin I in the evaluation of patients with normal total CK and elevated CK-MB serum results [Abstract]. Clin Chem 1996;42:S163.

[15.] Thomas L, Yvonne L. Specificity of Behring troponin I serum assay compared to CKMB in emergency department patients with possible acute myocardial infarction (AMI) [Abstract]. Clin Chem 1996;42:S107-8.

[16.] Anderson FP, Jesse RL, Roberts SC, Lawson CJ. Early diagnosis of myocardial infarction with troponin I [Abstract]. Clin Chem 1996;42:S157.

Diego Lozano, * Pino Carreno, Inmaculada Moreno, and Manuel Mendez (Servicio de Bioquim., Hosp. La Paz, Paseo de la Castellana 261, 28046 Madrid, Spain; * present address and address for correspondence: c/Ferrocarril del Puerto 16 5-C, 29002 Malaga, Spain)
Table 1. Imprecision of the Access cTnI assay.

 Within-run Between runs

Mean cTnI, Mean cTnI,
 [micro]g/L CV, % [micro]g/L CV, %

 0.183 5.68 0.209 7.75
 6.180 4.78 5.920 7.87
 22.420 5.10 26.810 6.32
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Title Annotation:Technical Briefs
Author:Lozano, Diego; Carreno, Pino; Moreno, Inmaculada; Mendez, Manuel
Publication:Clinical Chemistry
Date:Jun 1, 1997
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