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Technical Briefs |
1
Bayer Diagnostics, 333 Conney St., E. Walpole, MA 02032;
2
University of Texas-Houston, Houston, TX 77030;
a author for correspondence: fax 508-660-4591, e-mail pradip.datta{at}chirondiag.com
Troponin, consisting of three components, troponin C (TnC), troponin I (TnI), and troponin T (TnT), is a major component of the structural proteins involved in striated and cardiac muscle contraction (1)(2). TnI and TnC bind tightly to each other in the presence of Ca2+ with an association constant, Ka, of ~108-109 L/mol (1)(2)(3). TnT binds to both TnC and TnI, although less weakly than the binding between TnC and TnI. The cardiac isoforms of TnI (cTnI) and TnT are structurally different from the corresponding skeletal isoforms, and therefore they have recently established themselves as biochemical markers of myocardial damage (4)(5)(6)(7).
The currently available cTnI assays produce differing results (8)(9)(10). One important reason is that the assays may differ in their responses to the various isoforms of cTnI present in circulation or in biochemical preparations. In addition to "free" cTnI (I) and its binary and ternary complexes with TnC and TnT (IC or ICT) (8)(11)(12), cTnI may exist in phosphorylated (13), oxidized (14), and proteolytically degraded (8)(11)(12) forms. The latter modified forms of cTnI also may exist as binary or ternary complexes. All of these forms may have different recognition patterns in different immunoassays. The predominant form of cTnI in acute myocardial infarction (AMI) patients is the binary complex IC (8)(11). Samples treated with a Ca2+ chelator such as EDTA would contain mostly free cTnI because chelation of Ca2+ disrupts IC and ICT complexes.
We explored the possibility that the sera from patients with different pathological conditions may contain different isoform distributions of cTnI, thus generating discordant results among assays that recognize the isoforms differently. Here we report the immunoreactivity of five commonly used commercial cTnI immunoassays (Bayer ACS:180®, Dade Stratus®, Beckman Access®, Behring OPUS®, and Abbott AxSYM®) toward the two major forms of the analyte: free cTnI and its binary complex, IC.
Free cTnI (I) and its binary complex (IC), both isolated from human heart, were obtained from Scripps Laboratories. Their concentrations, determined by protein assay (Bio-Rad), were provided by the vendor and then converted to molar cTnI concentrations, using molecular weights of 21 000 and 35 000 for I and IC, respectively. Normal human serum, obtained from Scantibodies, tested negative for cTnI in all five cTnI assays.
Buffered stock solutions of both I and IC, as obtained from the vendor,
and the serum were mixed to produce 0, 5, 10, and 20 µg/L solutions
of cTnI, which were analyzed by five cTnI immunoassays: ACS:180,
Stratus, AxSYM, OPUS, and ACCESS. Manufacturer-suggested assay
protocols and platforms were used for all assays. All five assays use a
"sandwich" method, where the analyte is sandwiched between capture
and label antibodies, and thus the generated signal is directly
proportional to the concentration of TnI in the sample. Details of the
assays are in listed in Table 1
.
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When the results of the methods listed in Table 1
(y) were
compared with the Stratus (x), the linear correlation slopes
were 0.14 for ACCESS, 1.13 for ACS:180, 1.43 for OPUS, and 3.42–5.14
for AxSYM. We calculated the OPUS (y)/Stratus (x)
slope as the ratio of slopes for (ACS:180/Stratus) and (ACS:180/OPUS).
The two different slopes of AxSYM (y) vs Stratus
(x) were obtained from different studies: 3.42 from the
package insert of the AxSYM kit, and 5.14, from our own studies
(15). The AMI cutoff values varied in slightly different
order: ACCESS (0.15 µg/L) < ACS:180 ~ Stratus (1.5
µg/L) < AxSYM ~ OPUS (2.0 µg/L). The ACCESS and AxSYM
assays differed by a factor of as much as 36-fold (5.14/0.14). The
other three assays agreed more closely (± 25%). Various survey data
for cTnI also indicate similar differences among the assay results
(16). Many of the difference are probably contributed by
standardization. As noted by other researchers (8)(9)(10), such
interassay differences underscore the urgent need for universal
standardization for this important analyte.
Fig. 1
presents the comparison of immunoreactivity of the five assays
to IC (Fig. 1A
) and I (Fig. 1B
). The assays have different orders of
immunoreactivity toward free cTnI and IC complex: ACCESS <
Stratus < ACS:180 < OPUS < AxSYM for IC; and
Stratus < ACCESS < OPUS < ACS:180 < AxSYM for
I. The order of immunoreactivity for IC was similar to the order of
their responses in the method comparison study (Table 1
). However,
whereas the ACS:180, ACCESS, and AxSYM cTnI assay responses to I
remained similar to their responses to IC, the Stratus and OPUS assays
behaved quite differently to I than to IC, producing responses to I
that were 6.41 and 3.19 times less than IC (as determined
the ratio of slopes, IC/I; Fig. 1
). The ACS:180 cTnI assay showed the
best "equimolarity" (i.e., the ability to recognize I and IC
equally well), with a IC/I slope ratio of 1.07. The slope ratios for
the ACCESS and AxSYM assays were 1.23 and 1.4, respectively.
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Our observations agree well with those of Wu et al. (8) who compared nine different immunoassays for cTnI toward synthetically prepared oxidized or reduced forms of I or IC. When they compared the more common oxidized forms of IC and I, six assays in their study were approximately equimolar, two others responded to IC better than to I by ~1.5-fold, and one assay responded to IC ~3.2-fold more than to I.
Because the binary IC complexed form of cTnI is believed to account for
90% of cTnI in the serum of most AMI patients
(8)(11)(12), the ACS:180 and Stratus
assays show close agreement among most samples (Table 1
). However, if
the complexed form of cTnI is released into the circulation only after
extensive damage to myocytesand the subsequent necrosis of cardiac
muscle, the appearance of IC or ICT complexes in the circulation could
take several hours to days, thus delaying the confirmation of AMI
diagnosis. On the other hand, 5–6% of the total cardiac cTnI exists
as a free form in the cytoplasm (17). If the free cTnI is
released faster than IC complex from damaged myocytes, initial cTnI
concentrations may contain a higher percentage of free cTnI. These
samples may show a higher frequency of discordance between the
equimolar and nonequimolar cTnI assays.
We think that the differential immunoreactivity of cTnI assays to its isoforms may explain some cases of assay discordance. A possible example can be found in a method comparison study with samples from many categories of cardiac patients (18). When the Stratus, OPUS, and ACCESS assay results for 138 such samples were compared, a poorer linear regression coefficient (r = 0.774) was found between Stratus and ACCESS than between Stratus and OPUS (r = 0.92) or OPUS and ACCESS (r = 0.90) (18). Our finding that the order of equimolarity between these three assays is ACCESS (most equimolar) > OPUS > Stratus (least equimolar) may explain those data. An assay that detects both forms of cTnI equally well could be an advantage over a method that detects only one form of cTnI because the presence of any form of cTnI in serum is indicative of cardiac damage.
References
The following articles in journals at HighWire Press have cited this article:
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  F. Pagani, F. Stefini, G. Micca, M. Toppino, F. Manoni, L. Romano, P. Hoffer, A. Iervasi, M. Caputo, R. Dorizzi, et al. Multicenter Evaluation of the TOSOH AIA-Pack Second-Generation Cardiac Troponin I Assay Clin. Chem., September 1, 2004; 50(9): 1707 - 1709. [Full Text] [PDF]
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R. Labugger, J. A. Simpson, M. Quick, H. A. Brown, C. E. Collier, I. Neverova, and J. E. Van Eyk Strategy for Analysis of Cardiac Troponins in Biological Samples with a Combination of Affinity Chromatography and Mass Spectrometry Clin. Chem., June 1, 2003; 49(6): 873 - 879. [Abstract] [Full Text] [PDF]
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A. Lavoinne, B. Cauliez, H. Eltchaninoff, C. Tron, and A. Cribier Release of Macromolecular Cardiac Troponin I Complex after Successful Percutaneous Transluminal Coronary Angioplasty in Acute Myocardial Infarction Clin. Chem., March 1, 2003; 49(3): 505 - 507. [Full Text] [PDF]
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 T. M. Welsh, G. D. Kukes, and L. M. Sandweiss Differences of Creatine Kinase MB and Cardiac Troponin I Concentrations in Normal and Diseased Human Myocardium Ann. Clin. Lab. Sci., January 1, 2002; 32(1): 44 - 49. [Abstract] [Full Text] [PDF]
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 M. J. Quinn and D. J. Moliterno Troponins in Acute Coronary Syndromes: More TACTICS for an Early Invasive Strategy JAMA, November 21, 2001; 286(19): 2461 - 2462. [Full Text] [PDF]
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B. Cassinat, D. Darsin, P. Guardiola, M.-E. Toubert, J.-D. Rain, E. Gluckman, and M.-H. Schlageter Intermethod Discordance for {alpha}-Fetoprotein Measurements in Fanconi Anemia Clin. Chem., August 1, 2001; 47(8): 1405 - 1409. [Abstract] [Full Text] [PDF]
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 R. C. Payne, B. I. Bluestein, D. L. Morris, R. Labugger, L. Organ, C. Collier, J. E. Van Eyk, and D. Atar Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction Response Circulation, July 31, 2001; 104 (5): e26 - e27. [Full Text] [PDF]
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R. Siebers How Accurate Are References in Clinical Chemistry? Clin. Chem., March 1, 2001; 47(3): 606 - 607. [Full Text] [PDF]
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A. Lavoinne, B. Cauliez, H. Eltchaninoff, R. Koning, and A. Cribier Analytical and Clinical Performance of the Immulite Cardiac Troponin I Assay Clin. Chem., December 1, 2000; 46(12): 1989 - 1990. [Full Text] [PDF]
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K. M. ver Elst, H. D. Spapen, D. N. Nguyen, C. Garbar, L. P. Huyghens, and F. K. Gorus Cardiac Troponins I and T Are Biological Markers of Left Ventricular Dysfunction in Septic Shock Clin. Chem., May 1, 2000; 46(5): 650 - 657. [Abstract] [Full Text] [PDF]
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