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Articles |
1
Department of Pathology, University of Maryland School of Medicine, 22 S. Greene St., Baltimore, MD 21201.
2
Department of Laboratory Medicine and Pathology,
Hennepin County Medical Center, and the University of Minnesota School
of Medicine, Minneapolis, MN 55415.
3
Denver Health Medical Center, Denver, CO 80204.
4
Analytical Chemistry Division, National Institute of
Standards and Technology, Gaithersburg, MD 20899.
5
Clinical Chemistry Laboratory, Azienda Ospedaliera
’Spedali Civili, 25125 Brescia, Italy.
6
Department of Pharmacology, University of Miami School
of Medicine, Miami, FL 33101.
7
Department of Pathology and Laboratory Medicine,
Hartford Hospital, Hartford, CT 06102.
8
Departments of Pathology, Cell Biology, Neurobiology,
and Anatomy, Loyola University Medical Center, Maywood, IL 60153.
a Author for correspondence. Fax 410-328-8672; e-mail
rchriste{at}umaryland.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Solutions of 10 cTnI cRMs, each characterized by NIST, were shipped to the manufacturers of 13 cTnI measurement systems. Manufacturers used their respective diluents to prepare each cRM in cTnI concentrations of 1, 10, 25, and 50 µg/L. For the purpose of ranking the cRMs, the deviation of each cTnI measurement from the expected response was assessed after normalization with the 10 µg/L cTnI solution. Normalized deviations were examined in five formats. Parameters from linear regression analysis of the measured cTnI vs expected values were also used to rank performance of the cRMs.
Results: The three cRMs demonstrating the best overall rankings were complexes of troponins C, I, and T. The matrices for these three cRMs values differed; one was reconstituted directly from the lyophilized form submitted by the supplier; one was submitted in liquid form, lyophilized at NIST, and subsequently reconstituted; and the third was evaluated in the liquid form received from the supplier. The cRM demonstrating the fourth best performance was a binary complex of troponins C and I supplied in lyophilized form and reconstituted before distribution.
Conclusions: The cRMs demonstrating the best performance characteristics in 13 cTnI analytical systems will be included in subsequent activities of the cTnI Standardization Committee of the AACC.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The "troponin era" has been based on evidence that cTnI and cTnT are tissue-specific biochemical markers for indicating myocardial injury and that measurements of these proteins have excellent performance characteristics for diagnosis of myocardial infarction (1)(2)(3)(4), risk stratification of acute coronary syndrome patients (5)(6), and guidance of therapeutic intervention (7)(8)(9). For cTnT measurement, there have been three generations of quantitative assays and two generations of whole-blood qualitative tests. Assay harmonization has been achieved among these several cTnT assay formats because all cTnT assays have been produced by a single manufacturer (Roche Diagnostics). As a result, standardization of cTnT measurements is not currently an issue within the laboratory community. cTnI assays, on the other hand, present a somewhat different situation.
There are numerous cTnI assays that have been developed and marketed by various manufacturers. This has led to a situation where cTnI measurements using different methods on identical specimens may differ by 100-fold (10), creating a substantive problem for the clinical and laboratory communities, particularly as the use of cTnI measurements increases. To help address this situation, the AACC formed a cTnI Standardization Committee in cooperation with NIST and in collaboration with the Standardization of Markers of Cardiac Damage Committee of the IFCC. The goal of the cTnI Standardization Committee is the designation of international reference materials for cTnI that will substantially reduce intermethod variation.
The use of candidate reference materials (cRMs) in standardization of cTnI assays may be complicated because release of this protein after myocardial injury is not yet completely understood. cTnI may be released predominantly as a troponin C-troponin I (CI) protein complex (11), in part as free cTnI (12) or as a combination of these forms (13), and as degradation products of the free cTnI subunit (13). In addition, cTnI and the complex may be undergoing posttranslational changes after release, such as oxidation, phosphorylation, and proteolysis (14). Further complicating standardization activities are the effects of lyophilization and reconstitution of reference materials and the need to demonstrate the commutability of measurements using a cRM from an artificial matrix to a physiologic one.
The primary objective of this report is to describe the efforts of the AACC cTnI Committee to select a feasible number of cRMs from among 10 that were characterized by NIST (unpublished data). This selection consisted of ranking the cRMs to identify those showing the best performance (i.e., most consistent and predictable analytic response), using 13 cTnI assay systems as measurement tools. In addition, the activities described here were intended to assist in developing reasonable expectations and directing further phases of cTnI standardization and harmonization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were characterized by NIST for composition, purity, and
approximate cTnI concentration (unpublished data). After
characterization, each cRM was reconstituted or diluted in distilled
water at NIST to contain cTnI in an estimated concentration of 50 mg/L
and was stored at -70 °C. Vials containing each reconstituted cRM
were shipped to manufacturers for dilution, using each respective
recommended diluent, and measurement.
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cTnI METHODS
The cTnI systems used as measurement tools in this study are
listed in Table 2
.
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measurement protocol
Upon receipt, the 10 cTnI cRMs were maintained at -70 °C until
measurement, which occurred within 30 days. For studies in phase I, all
dilutions of the cRMs were prepared in the diluent recommended by the
manufacturer. Details of the cRM dilution scheme may be obtained as a
data supplement to this article at Clinical Chemistry
Online
(http://www.clinchem.org/content/vol47/issue3). Briefly,
aliquots of each of the 10 cRMs were prepared from the 50 mg/L cRM to
produce 1.0-mL solutions with estimated cTnI concentrations of 1, 10,
25, and 50 µg/L, using a 1000 µg/L intermediate working solution.
Manufacturers were directed to analyze these solutions in duplicate
with their respective analytical systems immediately after preparation.
Both replicate measurements were reported to the AACC cTnI Committee.
data analysis
The rationale for assessing consistency and predictability of the
cRMs was to normalize the response of cTnI assays for serial dilutions
of each material. The deviation of the measured normalized response
from the expected response was then calculated. The magnitude of the
deviation (measured vs expected) reflected the consistency and
predictability of each cRM, allowing ranking of the materials. In
addition, cTnI systems use different calibrators; therefore, it was
expected that part of this deviation was attributed to calibration
differences. To take this into account, linear regression was performed
in which the measured results were the dependent variable and the
expected value was the independent variable.
Several formats of absolute deviation analysis (see below) were used. Although the formats were not strictly independent, they provided a means to rank the cRMs under different constraints. Where linear regression analyses were used, random and systemic errors were assessed. The lower the random error for a cRM, the better the ranking. All analyses reported here were performed by individuals who were unaware of the identity of the various cRMs used in the phase I studies.
Absolute deviation.
The intended purpose of this set of
analyses was to evaluate the overall response of the cTnI systems to
each cRM in a linear series of concentrations. For this purpose,
results for the 10 µg/L solution were used to normalize cRM response
for the 1, 25, and 50 µg/L solutions for each of the cTnI systems.
The 10 µg/L solution was used for normalization because this
concentration was within the dynamic range of all assay systems and it
was expected to have the lowest CV across the assay range. This
normalization was done by calculating the mean of the duplicate
measured values for the 10 µg/L solution. The 10 µg/L mean value
was multiplied by 5, 2.5, and 0.1 to calculate the respective values
expected for the 50, 25, and 1 µg/L concentrations. The mean of the
measured replicates for each of the 50, 25, and 1 µg/L concentrations
was then divided by the expected value derived from the 10 µg/L
concentration to calculate recovery, as shown below:
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The ideal recovery of each cRM, i.e., the recovery if there was a
perfect match between expected and measured at each concentration, was
1.00. The absolute deviation of the measured value from the expected
result of 1.00 for each concentration was calculated for each cRM with
each cTnI method, according to the following:
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The mean absolute deviation within a cTnI method was a unitless
quantity that was calculated according to the following:
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Absolute deviation data were examined in five formats in an effort to avoid possible bias. The first format included data for all cTnI systems participating in the study in which results for at least three of the four cTnI concentrations were within the dynamic range of the assay. In the second format, the criteria were the same as the first format, except that data for three cTnI measurement systems, which had data sets for less than eight cRMs (i.e., systems for which three or more of the cRMs did not yield results within the dynamic range for at least three of the four cTnI concentrations), were excluded. This was done in an attempt to eliminate possible bias attributable to measurement systems having a relatively limited analytical range. The third format included only data for cTnI systems yielding valid results for all four cTnI concentrations. The fourth format also included only data for which all four concentrations yielded valid cTnI measurements, except that data for the five cTnI measurement systems having data sets for less than six cRMs (i.e., systems for which five or more of the cRMs did not yield results within the dynamic range for all four cTnI concentrations) were excluded. This was also done in an attempt to eliminate possible bias attributable to measurement systems having a relatively limited analytical range.
In a fifth analysis format, the absolute deviation was used to rank the performance of each cRM within each cTnI system (as first, second, third, and so forth), according to the sum of absolute deviations from each set of cRMs. The criterion for incrementing the rank of a cRM within a cTnI system (i.e., from 1 to 2) was an increase in the sum of absolute deviation by either 0.1 or 1 SD, whichever was smaller. The cRMs were then ranked according to the means of their ranks across all cTnI systems. In addition, the number of times each material was ranked either first or second was recorded.
Linear regression analysis of cRM data.
Least-squares linear
regression analysis of cTnI results for each measurement system was
carried out for each cRM according to the following:
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This linear regression method was used because it assigns all of
the error to the dependent variable and, therefore, provides the most
conservative estimate of error for the observed values. Regression
parameters calculated included the slope, y-intercept,
Sy|x, and correlation coefficient. Because of the
large differences in the measured values among the cTnI systems, a
relative Sy|x
(SRy|x) was calculated
by dividing the Sy|x by the mean of the measured
values for that set of cRMs, and is expressed as a percentage as
follows:
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 4
. As seen from Tables 3
and 4
, the cRMs designated E, B, G, and
I (see Table 1
for identity) consistently showed favorable performance
across the cTnI systems used as measurement tools, except I, which was
the lyophilized version of the CI material from Spectral Diagnostics.
The other three are all CTI complexes. The full sets of data used to
develop Tables 3
and 4
are available as a data supplement to this
article at Clinical Chemistry Online
(http://www.clinchem.org/content/vol47/issue3).
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Table 5
displays the performance of the cRMs within each cTnI
measurement system as described for analysis format 5 in
Materials and Methods. Materials E, B, D, and G showed the
highest frequency of scoring top ranks among the various systems. (For
the full data set used to develop Table 5
, see data supplement to this
article at Clinical Chemistry Online,
http://www.clinchem.org/content/vol47/issue3).
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The linear regression results for the cRMs are summarized in Tables 6
and 7
. Except for the slopes, materials B, G, E, and I showed overall
satisfactory performance. Of note, the overall slopes for the cRMs
showed a rather narrow range of 0.9342–1.0639, suggesting that
recalibration with a common standard material could harmonize
methods. The SRy|x data
displayed in Table 7
are particularly representative of overall
performance because this term normalizes for differences between the
measurement values for the cTnI systems. The full data set used to
develop Tables 6
and 7
is available as a data supplement to this
article at Clinical Chemistry Online
(http://www.clinchem.org/content/vol47/issue3).
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Table 8
is a summary of the overall performance of the cRMs included in
this analysis. The overall analysis indicated that cRM E showed the
best overall performance, followed by cRMs B, G, and I.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, the 10 µg/L cTnI concentration for each cRM was used for normalization with the reasoning that imprecision of cTnI systems would most likely be satisfactory at this concentration. The 10 µg/L concentration yielded results within the measurable ranges of all the cTnI methods for all cRMs. Use of the 10 µg/L concentration allowed calculation of the absolute deviations used in formats 1–5 of the analyses. Different formats (2 and 4) were used to avoid bias that may have been present because of missing data. These analyses yielded similar results, indicating that the missing data had little impact on the final conclusions. Format 5 was performed to rank the consistency of each cRM within the cTnI measurement tools. Format 5 gave each cRM an ordinal rank within each cTnI method to avoid bias attributable to large differences between some of the assays. The mean rank of each cRM across the methods was used as rating criteria. As expected, the number of times the cRMs ranked first or second were closely paralleled with the mean rank.
As with all methods of analysis, there are strengths and caveats for assessing the various parameters from linear regression analysis. The slope and y-intercept clearly described the proportionality and limits between cRMs; however, the slope and y-intercept depend, in part, on calibration and are therefore not intrinsic characteristics of the antibodies used as reagents. The correlation coefficient showed a highly significant relationship between the cRMs and the calibrators of the manufacturers. However, all of the correlation coefficients were high, and therefore provided little discrimination. The Sy|x is representative of the SE at the mean of measurements, which can be difficult to interpret when comparing measurements that vary greatly in value. The relative Sy|x (SRy|x) corrects for the wide differences and was, therefore, better suited for this analysis. In this way, SRy|x represents the normalized dispersion of measured values over the regression line, with smaller values suggesting better performance.
The analytical response for each cRM varied >40-fold for the 13
participating cTnI systems, underscoring the need for standardization.
The differences in analytic response among cTnI systems, reflected by
the mean absolute deviation values, were only 30–50% for most of the
cRMs (see Tables 3
and 4
). Furthermore, regression parameters and
SRy|x data from the
linear regression analysis also indicated that better agreement between
methods can be achieved via recalibration with a common standard. The
slope and y-intercept data are not as informative because
these two analytical parameters are associated with calibration and
presumably can be adjusted or compensated for when an assay is
recalibrated with a new reference material. This preliminary
information offers the promise of producing reasonable harmony among
cTnI measurement systems through the use of one of the cRMs as a
reference material.
The overall outcome in this study, as indicated in Table 8
, is that the
three cRMs composed of the CTI complex demonstrated the best
performance. This finding is consistent with a recent study that showed
a reduction in between-assay variability by calibration with a material
composed of the CTI complex (15). Of interest, two of the
three cRMs were the same material with the only difference being
lyophilization.
The cTnI Standardization Committee plans to use both serum pools and individual patient specimens to evaluate the ability of cRMs to reduce cTnI result variation across analytical systems. Evaluations of commutability of cRMs and other important characteristics are under consideration.
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Acknowledgments |
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Footnotes |
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3 Chair, AACC Cardiac Troponin I Standardization Committee.
1 Nonstandard abbreviations: CTI, troponin C-troponin T-troponin I protein complex; cTnI and cTnT, cardiac troponin I and T; cRM, candidate reference material; and CI, troponin C-troponin I protein complex.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  E. Kuhn, T. Addona, H. Keshishian, M. Burgess, D.R. Mani, R. T. Lee, M. S. Sabatine, R. E. Gerszten, and S. A. Carr Developing Multiplexed Assays for Troponin I and Interleukin-33 in Plasma by Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Clin. Chem., June 1, 2009; 55(6): 1108 - 1117. [Abstract] [Full Text] [PDF]
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F. S. Apple, M. M. Murakami, R. Ler, D. Walker, M. York, and for the HESI Technical Committee of Biomarkers Wor Analytical Characteristics of Commercial Cardiac Troponin I and T Immunoassays in Serum from Rats, Dogs, and Monkeys with Induced Acute Myocardial Injury Clin. Chem., December 1, 2008; 54(12): 1982 - 1989. [Abstract] [Full Text] [PDF]
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 D. C Gaze and P. O Collinson Multiple molecular forms of circulating cardiac troponin: analytical and clinical significance Ann Clin Biochem, July 1, 2008; 45(4): 349 - 355. [Abstract] [Full Text] [PDF]
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C. Prontera, A. Fortunato, S. Storti, A. Mercuri, G. Longombardo, G. C. Zucchelli, M. Emdin, and A. Clerico Evaluation of Analytical Performance of the Siemens ADVIA TnI Ultra Immunoassay Clin. Chem., September 1, 2007; 53(9): 1722 - 1723. [Full Text] [PDF]
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 NACB Writing Group Members, F. S. Apple, R. L. Jesse, L. K. Newby, A. H.B. Wu, and R. H. Christenson National Academy of Clinical Biochemistry and IFCC Committee for Standardization of Markers of Cardiac Damage Laboratory Medicine Practice Guidelines: Analytical Issues for Biochemical Markers of Acute Coronary Syndromes Circulation, April 3, 2007; 115(13): e352 - e355. [Full Text] [PDF]
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NACB WRITING GROUP MEMBERS, F. S. Apple, R. L. Jesse, L. K. Newby, A. H.B. Wu, R. H. Christenson, NACB COMMITTEE MEMBERS, R. H. Christenson, F. S. Apple, C. P. Cannon, et al. National Academy of Clinical Biochemistry and IFCC Committee for Standardization of Markers of Cardiac Damage Laboratory Medicine Practice Guidelines: Analytical Issues for Biochemical Markers of Acute Coronary Syndromes Clin. Chem., April 1, 2007; 53(4): 547 - 551. [Full Text] [PDF]
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R. H. Christenson, S. H. Duh, F. S. Apple, G. S. Bodor, D. M. Bunk, M. Panteghini, M. J. Welch, A. H.B. Wu, S. E. Kahn, and for the American Association for Clinical Chemistr Toward Standardization of Cardiac Troponin I Measurements Part II: Assessing Commutability of Candidate Reference Materials and Harmonization of Cardiac Troponin I Assays Clin. Chem., September 1, 2006; 52(9): 1685 - 1692. [Abstract] [Full Text] [PDF]
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D. M. Bunk and M. J. Welch Characterization of a New Certified Reference Material for Human Cardiac Troponin I Clin. Chem., February 1, 2006; 52(2): 212 - 219. [Abstract] [Full Text] [PDF]
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 F. S. Apple Clinical Biomarkers of Cardiac Injury: Cardiac Troponins and Natriuretic Peptides Toxicol Pathol, January 1, 2006; 34(1): 91 - 93. [Abstract] [Full Text] [PDF]
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M. Panteghini Standardization of Cardiac Troponin I Measurements: The Way Forward? Clin. Chem., September 1, 2005; 51(9): 1594 - 1597. [Full Text] [PDF]
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D. L. Crimmins Human N-Terminal proBNP Is a Monomer Clin. Chem., June 1, 2005; 51(6): 1035 - 1038. [Full Text] [PDF]
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D. H. Wilson, G. Williams, R. Herrmann, D. Wiesner, and P. Brookhart Issues in Immunoassay Standardization: The ARCHITECT Folate Model for Intermethod Harmonization Clin. Chem., April 1, 2005; 51(4): 684 - 687. [Full Text] [PDF]
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M. Panteghini The Interfering Component in Cardiac Troponin I Immunoassays: Need for Further Experimental Evidence Clin. Chem., March 1, 2004; 50(3): 676 - 677. [Full Text] [PDF]
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K. B. Wallace, E. Hausner, E. Herman, G. D. Holt, J. T. Macgregor, A. L. Metz, E. Murphy, I.Y. Rosenblum, F. D. Sistare, and M. J. York Serum Troponins as Biomarkers of Drug-Induced Cardiac Toxicity Toxicol Pathol, January 1, 2004; 32(1): 106 - 121. [PDF]
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M. Panteghini Performance of Today's Cardiac Troponin Assays and Tomorrow's Clin. Chem., June 1, 2002; 48(6): 809 - 810. [Full Text] [PDF]
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C. Cavalli, R. M. Dorizzi, M. Caputo, and P. Biban Serendipitous Detection of Umbilical Venous Catheter Displacement by Cardiac Troponin I Measurement Clin. Chem., July 1, 2001; 47(7): 1328 - 1329. [Full Text] [PDF]
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