Blood biomarkers were measured in all participants using morning samples collected after an overnight fast. Participants were supine for approximately five to ten minutes prior to phlebotomy. Blood samples were immediately centrifuged, and plasma and serum were stored at −70°C. The samples did not undergo any freeze-thaw cycles prior to the performance of the assays below.
The concentration of sST2 was determined using a high-sensitivity, second-generation enzyme-linked immunosorbent assay with a detection limit of 2 ng/mL (Presage®ST2, Critical Diagnostics).28 (link) sST2 values above 35 ng/mL have been linked to adverse outcomes in the setting of overt heart failure.18 (link),29 (link) Quantification of hsTnI was performed with an ultra-sensitive immunoassay for cardiac troponin I that utilizes a novel, single-molecule counting technology (Erenna hsTnI, Singulex).30 (link) The limit of detection is 0.2 pg/mL, with an assay range of 0.5 to 70 pg/mL. The 99th percentile value for this assay has not been well-established, but in small studies of normal subjects ranges from approximately 7 pg/mL to 10 pg/mL.30 (link)–32 (link) hsCRP and BNP were measured as previously described.2 (link)GDF-15 levels were measured with a pre-commercial, automated electrochemiluminescent immunoassay on a Cobas e 411 analyzer (Roche Diagnostics). The assay has a limit of detection below 10 ng/L, a linear measuring range up to 20,000 ng/L, and an inter-assay imprecision of 2.3% and 1.8% at GDF-15 concentrations of 1,100 ng/L and 17,200 ng/L, respectively (Roche Diagnostics, data on file). GDF-15 values obtained with the electrochemiluminescent assay correlate closely with the values measured with our previously described immunoradiometric assay33 (link) (r = 0.980, slope 1.049, intercept −136 ng/L, n = 45 samples with GDF-15 concentrations ranging from 567 to 13,334 ng/L). Using the immunoradiometric assay, 1200 ng/L was previously proposed as the upper limit of the reference interval in apparently healthy elderly individuals.33 (link)
The concentration of sST2 was determined using a high-sensitivity, second-generation enzyme-linked immunosorbent assay with a detection limit of 2 ng/mL (Presage®ST2, Critical Diagnostics).28 (link) sST2 values above 35 ng/mL have been linked to adverse outcomes in the setting of overt heart failure.18 (link),29 (link) Quantification of hsTnI was performed with an ultra-sensitive immunoassay for cardiac troponin I that utilizes a novel, single-molecule counting technology (Erenna hsTnI, Singulex).30 (link) The limit of detection is 0.2 pg/mL, with an assay range of 0.5 to 70 pg/mL. The 99th percentile value for this assay has not been well-established, but in small studies of normal subjects ranges from approximately 7 pg/mL to 10 pg/mL.30 (link)–32 (link) hsCRP and BNP were measured as previously described.2 (link)GDF-15 levels were measured with a pre-commercial, automated electrochemiluminescent immunoassay on a Cobas e 411 analyzer (Roche Diagnostics). The assay has a limit of detection below 10 ng/L, a linear measuring range up to 20,000 ng/L, and an inter-assay imprecision of 2.3% and 1.8% at GDF-15 concentrations of 1,100 ng/L and 17,200 ng/L, respectively (Roche Diagnostics, data on file). GDF-15 values obtained with the electrochemiluminescent assay correlate closely with the values measured with our previously described immunoradiometric assay33 (link) (r = 0.980, slope 1.049, intercept −136 ng/L, n = 45 samples with GDF-15 concentrations ranging from 567 to 13,334 ng/L). Using the immunoradiometric assay, 1200 ng/L was previously proposed as the upper limit of the reference interval in apparently healthy elderly individuals.33 (link)
