Development and Preliminary Clinical Validation of a High Sensitivity Assay for Cardiac Troponin Using a Capillary Flow (Single Molecule) Fluorescence Detector

(CV) of <10%. As none of the commercial assays can detect cTnI in healthy subjects with the requisite precision, many have advocated a cTnI cutoff at the 10% CV value. We developed and evaluated a high-sensitivity cTnI assay and determined its analytical and preliminary clinical performance. Methods: The Singulex cTnI assay utilizes a 384-well ELISA plate, and the ZeptX™ Digital Molecule Counting (DMC) System. This assay uses a monoclonal capture antibody and a fluorescently-tagged affinity-purified goat detection antibody. After washing, the fluorescently- tagged antibody is chemically released into each well. An aliquot is pumped into the analyzer. Individually-labeled antibodies are measured during capillary flow by setting the interrogation volume such that the emission of single fluorescent molecules is detected in a defined space following laser excitation. With each signal representing a digital event, this configuration enables extremely high analytical sensitivities, with total fluorescent signal determined as a sum of the individual digital events. Each molecule counted is a positive data point with hundreds to thousands of DMC events/sample. The limit of detection the Singulex cTnI assay was determined by the mean plus 3 SD method. The normal range was determined on a population of 148 apparently healthy subjects. This assay was correlated to the Bayer Centaur on 130 samples from patients admitted with chest pain. We also examined 47 serial samples from 15 patients who presented to the ED with a diagnosis of AMI. In this latter group, all had initial Centaur cTnI results that were <0.35 ng/mL (10% CV cutpoint), and 12 were <0.1 ng/mL (99 th %). The cTnI concentration was positive on all subsequent serial samples from these patients on the Centaur, establishing the diagnosis of AMI. Results: The analytical sensitivity of the Singulex assay was 1 pg/mL. The precision was 10% at 4 and 12 pg/mL. The reference population exhibited a normal distribution. The 99 th percentile was determined to be <8 pg/mL. The linear regression between Singulex (y) and Centaur (x) was: y= 0.113x + 0.048, r=0.937. In the 3 cases that had initial Centaur cTnI value between 0.1 and 0.35 ng/mL, all were positive for Singulex. In the 12 cases that had initial Centaur cTnI value <0.1 ng/mL, 5 of 12 cases were positive. The prospective use of the Singulex assay would have detected 53% more AMI cases than the Centaur when the admission sample was tested. Conclusion: At a precision of 10% and correcting for the difference in standardization, the sensitivity of the Singulex cTnI assay at least 10-fold higher than the Centuar assay. This enabled earlier detection of AMI. It is also probably that a higher number of patients at risk for adverse cardiac events will be detected. The increased sensitivity was achieved by counting individual fluorescent emission events.