Study population
The present study is a subanalysis of a previous investigation of the performance of different biomarkers in a well-established HF cohort [10–13]. From May 2006 to 2013, ambulatory patients treated at a multidisciplinary HF clinic were consecutively included in the study. Referral inclusion criteria and blood sample collection are described elsewhere [4–7]. All analyses of biomarkers were performed on the same blood sample, which had been stored at −80° without prior freeze–thaw cycles. All samples were obtained between 9:00 a.m. and 12:00 p.m. Serum concentrations of NT-proBNP (N = 1030), hs-TnT (N = 803), ST2 (N = 814), galectin-3 (N = 811), hs-CRP (N = 773 after we excluded 16 patients with levels ≥100 mg/dL), cystatin-C (N = 804), STfR (N = 794), and neprilysin (N = 1069) were measured.
All of the participants provided written informed consent, and the local ethics committee approved the study. All of the study procedures were in accordance with the ethical standards outlined in the Helsinki Declaration of 1975 (revised in 1983).
Follow-up and outcomes
All patients made follow-up visits at regular, predefined intervals, and they made additional visits as required in cases of decompensation. The regular visitation schedule included a minimum of quarterly visits with nurses; biannual visits with physicians; and elective visits with geriatricians, psychiatrists, and rehabilitation physicians. Patients who did not attend the regular visits were contacted by telephone.
The primary outcomes of the present study were all-cause and cardiovascular death. A death was considered cardiovascular in origin if it was caused by HF (decompensated HF or treatment-resistant HF in the absence of another cause), sudden death (unexpected death, witnessed or not, of a previously stable patient with no evidence of worsening HF or any other cause of death), acute myocardial infarction (due to mechanical, haemodynamic, or arrhythmic complications), stroke (in association with recent acute neurological deficits), procedural (death after diagnostic or therapeutic procedures), and other cardiovascular causes (e.g., rupture of an aneurysm, peripheral ischaemia, or aortic dissection). Fatal events were identified from paper and electronic clinical records, from general practitioners, and by contacting the patients’ relatives. Data were verified by comparisons with the databases of the Catalan and Spanish health systems. Events were adjudicated by two of the authors (MD and JL). Follow-up was closed on 30 September 2015. Five patients were lost during follow-up and appropriately censored.
DM diagnosis
A diagnosis of DM was made when one of the following criteria were met: (1) a diagnosis of DM was previously established and recorded in the patient’s electronic history, (2) fasting plasma glucose ≥126 mg/dL or HbA1c ≥6.5% identified by laboratory testing [18], or (3) the patient had a current prescription for oral hypoglycaemic medication or insulin. All the included patients in this study had type 2 diabetes.
NTproBNP assay
NTproBNP levels were determined by an immunoelectrochemiluminescence method (Elecsys®; Roche Diagnostics, Switzerland). The assay had inter-run CVs of 0.9–5.5%.
Hs-TnT assay
Hs-TnT levels were measured by electrochemiluminescence immunoassay on the Modular Analytics E 170 (Roche Diagnostics). The hs-TnT assay had an analytic range of 3–10,000 ng/L. At the 99th percentile value of 13 ng/L, the CV was 9%.
ST2 assay
ST2 levels were measured from plasma samples with a high-sensitivity sandwich monoclonal immunoassay (Presage® ST2 assay; Critical Diagnostics, San Diego, CA, USA). The ST2 assay had a within-run coefficient of <2.5% and total CV of 4%.
hsCRP assay
hsCRP concentrations were measured by particle-enhanced turbidimetry (CRPHS, ref. 04628918 190; Roche Diagnostics) on the automatic analyser Cobas 6000 (Roche Diagnostics). The linearity of the method was 0.15–20.0 mg/L, the detection limit was 0.15 mg/L, the functional sensitivity was 0.3 mg/L, and the interserial CV was <8.4%.
Galectin-3 assay
For galectin-3 measurements, we used an enzyme-linked fluorescent assay (BioMerieux ref. 411191) on a mini-VIDAS® analyser (BioMerieux, France). The CV for the assay was <10%, the linearity was 3.3–100.0 ng/mL, and the limit of detection was 2.4 ng/mL.
Cystatin-C assay
Cystatin C was measured by a nephelometric technique that assesses immune complex formation between cystatin and antiserum anticystatin-C attached to latex particles (Cystatin C Radim, ref. NPP42; Radim Group, Pomezia, Italy). The CV between assays was 2.9%.
STfR assay
Soluble transferrin receptor concentrations were measured with a particle-enhanced turbidimetric immunoassay (Tina-quant Soluble Receptor Transferrin STFR, Roche Diagnostics) on the automatic analyser Cobas 6000 (Roche Diagnostics). The linearity of the method was 0.5–40.0 mg/L, the detection limit was 0.50 mg/L, and the interserial CV was <3.8%.
Neprilysin assay
Human neprilysin (NEP) was measured with a modified sandwich immunoassay (HUMAN NEP/CD10 ELISA kit, ref. SK00724-01, lot no. 20111893; Aviscera Biosciences, Santa Clara, CA, USA). At a positive control value of 1.4 ng/mL, the intra- and interassay CVs were 3.7 and 8.9%, respectively.
Statistical analysis
Categorical variables are expressed as percentages. Continuous variables are expressed as means (SD) or medians (quartiles Q1–Q3) for normal and non-normal distributions, respectively. Normal distribution was assessed with normal Q–Q plots. Differences between nondiabetic and diabetic patients were assessed by Chi-squared test, Student’s t test, and Mann–Whitney U test, as required. We also assessed differences in the levels of the different biomarkers between nondiabetic and diabetic patients (subsequent to log transformation) after adjustments for age, sex, body mass index, and estimated glomerular filtration rate.
We performed two Cox regression analyses, with all-cause or cardiovascular death as the dependent variable and the selected biomarker plus DM plus the interaction between the covariate biomarker and DM as independent covariables. In the Cox models, to fulfill the assumption of linearity of the covariables NT-proBNP, hs-TnT, ST2, hs-CRP, galectin-3, and sNEP, we used the logarithmic functions of NT-proBNP, hs-TnT, hs-CRP, galectin-3, and sNEP; the quadratic term of ST2; and log(hs-TnT). Afterwards, Cox regression analyses were performed separately for diabetic and nondiabetic patients for each biomarker. We used a 1SD increase, calculated jointly for the whole cohort, for HR calculations in the five logarithm-transformed variables. ST2 analyses were performed for every 10 ng/mL change. In patients with sNEP levels below the lower range of detection (0.250 ng/mL), a concentration of 0.249 ng/mL was introduced as a continuous variable. Survival curves for all-cause death were plotted for nondiabetic and diabetic patients based on the best cut-off points of ST2, obtained from AUC. As a sensitivity analysis of the 265 diabetic patients with all the biomarkers collected, we performed two comprehensive multivariable Cox regression analyses with all the biomarkers and also clinical variables, again for all-cause and cardiovascular death. Finally, a simple clinical model (age, sex, New York Association functional class, left ventricular ejection fraction and estimated glomerular filtration rate (CKD-EPI equation) was developed and discrimination evaluated (AUC) adding each one of the biomarkers and the best short combinations. Statistical analyses were performed with SPSS 15 (SPSS Inc., Chicago, IL, USA) and version 2.11.1 of the R statistical package (Foundation for Statistical Computing, Vienna, Austria). Two-sided p < 0.05 was considered significant.