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Abstracts of Oak Ridge Posters |
1 University of California, San Francisco, CA;2 Singulex Inc., Berkeley, CA;
aaddress correspondence to this author at: San Francisco General Hospital, San Francisco, CA 94110; fax 415-206-3045, e-mail wualan{at}labmed2.ucsf.edu)
The European Society of Cardiology and the American College of Cardiology (ESC/ACC) have redefined the criteria for the diagnosis of acute myocardial infarction, requiring an increase in the concentration of cardiac troponin in the clinical context of myocardial ischemia (1). A subgroup of this committee has recommended that the cutoff concentration be established at the 99th percentile of the reference range, with an acceptable interassay imprecision of 
10% (2). The use of a low troponin cutoff concentration enables the detection of more patients who have minor myocardial damage and are at short-term risk for adverse cardiac events (3). In a study conducted by the IFCC, none of the manufacturers of troponin assays cleared by the Food and Drug Administration in 2004 had a sensitivity for detecting troponin at the 99th percentile with the requisite precision (4). Therefore, troponin assays with higher analytical sensitivity and precision are needed to meet the guidelines of The European Society of Cardiology and the American College of Cardiology.
We examined the analytical performance of the ZeptXTM System (Singulex) assay for cardiac troponin I (cTnI), evaluating its sensitivity, precision, and recovery. We assessed the assay’s clinical performance by comparing assay results for 97 specimens from patients with chest pain with results from the 1st-generation Bayer Centaur cTnI assay. With samples from 88 healthy individuals, we determined the 99th percentile cutoff for the ZeptX assay. We also determined the analytical limit of detection (LoD) across 15 sequential assays and calculated the LoD as the mean value of the zero standard plus 3 SDs from quadruplicate intraassay determinations.
The ZeptX System consists of flow immunoassays linked to a digital molecule-counting instrument. Immunoassays are performed with either 96- or 384-well plate formats and a fluorescently labeled tracer. After the final wash step, tracer bound to the plate is eluted and a few microliters are then sipped into the instrument. We used troponin I-specific antibodies (BiosPacific) in a sandwich format with 10 µL of serum specimen to create a prototype assay. Sample was pumped through a glass capillary within the instrument, and a laser beam was directed through the capillary to establish a sample interrogation volume. The flow rate and sample interrogation volume are set such that only 1 fluorescent molecule is present in the interrogation volume during the time interval during which observations are made. As fluorescent molecules pass through the laser beam, fluorescence is detected via an optical system coupled to a photon detector. This approach allows for the binding of molecules into defined spaces so that they can be counted individually with a 5–6 
threshold over the fluorescence background. Thus each count is a digital event that is summed over time to create total signal without background. This instrument reduced sample background significantly and enabled detection of fluorescence from individual molecules with reproducible, signal-to-background activities and easily measurable molecule counts. Whereas the ensemble measurement of fluorescent or colorimetric analog output typical of other instrumentation provides only a single data point per sample, the ZeptX assay enables each molecule counted to be viewed as a positive data point. There is high resolution of a small number of signal events from the sum of background signal.
In a pilot clinical study, we tested 47 serum samples from 15 patients who entered the emergency department at San Francisco General Hospital with disease presentation that led to final diagnosis of non-ST elevation MI (NSTEMI). The diagnosis was predicated on the clinical history, electrocardiogram, echocardiogram (where available), and the results of troponin testing of serial samples by use of the assay currently in use (Centaur) at San Francisco General Hospital. The concentration at 10% CV for the Centaur assay (350 ng/L) was used as the cutoff for acute myocardial infarction (AMI). According to Centaur assay results, the initial samples from 12 NSTEMI patients were below the 99th percentile, and 3 were between the 99th percentile and the 10% CV cutpoint (350 ng/L). For at least 1 of these patients, a subsequent blood sample collected 4–8 h later was positive (>10% CV cutpoint). In addition, we tested blood from 50 patients who presented with chest pain and in whom AMI was ruled out the basis of negative troponin results for serially collected blood samples analyzed by the Centaur assay. The use of deidentified leftover specimens was approved by the University of California Committee on Human Research. As such, informed consent was deemed unnecessary by the committee.
We used the mean (3SD) of troponin-negative sera to calculate an LOD of 1.7 ng/L for the ZeptX assay. The assay had intraassay imprecision values of 8%, 9%, 11%, 7%, and 9% CV at 90, 18, 8, 3.7, and 1.2 ng/L, respectively (n = 20). Calibrator signal imprecision was 5%–6% CV (11–100 ng/L). We obtained cTnI standard from the National Institute on Standards and Technology (SRM 2921, Gaithersburg, MD) and performed serial dilutions into a plasma pool from which cTnI was removed by an affinity column. Assay of these standards as patient samples produced a linear regression of y = 0.78x –0.39, r = 0.99. The recovery of enriched troponin was 91%–114% at troponin concentrations of 5–135 ng/L. The linear regression of the ZeptX (y) vs Centaur (x) was: y = 0.13x + 50 ng/L, r = 0.94, slope = 0.0038, intercept = 0.0172. The distribution of healthy persons was gaussian (Fig. 1A
). The 99th percentile, calculated with the mean (3 SD), was 7 ng/L. For the 3 patients with AMI whose initial cTnI concentrations were between the Centaur 99th percentile and the 10% CV cutpoints, all results were positive on the ZeptX assay at the 99th percentile (cutoff of 7 ng/L). Of the remaining 12 samples that were below the 99th percentile according to the Centaur assay, 5 were positive according to the ZeptX assay. For example, 1 patient had serial Centaur cTnI results that were <100 (negative), 220, 270, and 320 ng/L (positive). The corresponding ZeptX values were 16, 65, 54, and 59 ng/L (all positive). The overall clinical sensitivities of the admission samples were 53% (95% confidence interval, 28%–78%; n = 15) for the ZeptX assay and 0% for the Centaur (a result of the selection criteria used). The distribution of ZeptX results for the 50 non-AMI chest pain cases are shown in Fig. 1B
. Results on the left side of the distribution (<7 ng/L) were within the reference range for this assay and suggested a noncardiac source for the admission. Results on the right side of the distribution (
12 ng/L) were positive for the ZeptX assay and suggested the possibility of minor myocardial injury. Short-term clinical outcome data for these patients were unavailable for this study.
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To assess whether low cTnI values were related to the presence of troponin or a nonspecific interferrent, we performed highly controlled immunodepletion experiments. A cTnI monoclonal antibody, different from those used in the assay, was coupled to agarose and then incubated with serum specimens overnight at 4 °C. We separated agarose beads from the serum, then measured cTnI. cTnI concentrations were 8–14 ng/L and decreased by 60%–80% to 3–6 ng/L in each case,, i.e., below the cutoff concentration of 7 ng/L, indicating that measurements were reasonably specific to cTnI.
The results of this preliminary clinical study show that a high-sensitivity assay can detect troponin concentrations in a gaussian distribution in the sera of healthy individuals. The data also suggest that a high-sensitivity assay can alert physicians to the presence of AMI earlier than a conventional cTnI assay and may also enable more effective identification of individuals at risk for future adverse events. These data must be confirmed by studies with larger healthy and diseased populations with known clinical outcomes.
Acknowledgments
One of the authors (A.H.B.W.) received grant support from Singulex Inc. for conducting these studies.
References
The following articles in journals at HighWire Press have cited this article:
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