Study population
Our study cohort included 101 ambulatory patients who attended a multidisciplinary HF unit from January 15th 2014 to May 6th 2015 (Table 1). The referral inclusion criteria are described elsewhere [9, 10]. All patients made follow-up visits at regular predefined intervals, and additional visits when 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, nephrologists, and rehabilitation physicians. Upon missing a regular visit, patients were contacted by telephone.
The primary endpoints were all-cause death and the composite of all-cause death or HF-related hospitalization. Fatal events were identified from electronic clinical records, and by contacting the patients’ relatives when necessary. When verification was required, data were compared with records stored in the databases of the Catalan and Spanish health systems. Events were adjudicated by two of the authors (EE and JL), and by clinical and research nurses.
Each subject gave their written informed consent prior to participation. The study protocol was approved by the Clinical Research Ethics Committee of our institution, was designed in accordance with the principles outlined in the 2013 revision of the Declaration of Helsinki of 1975 [11].
Blood extraction and processing
Blood samples of ~ 3 ml were collected into EDTA tubes via standard forearm venipuncture performed between 9:00 a.m. and 11:00 a.m., and were processed within 4 h after collection. Samples were collected at two time-points: at baseline (n = 101) and at the 1-year follow-up (n = 54) (Additional file 1: Table S1). Samples from the 1-year follow-up were unavailable due to death (7 patients), technical issues (10 patients), or patient’s unwillingness to repeat sampling (30 patients). Samples and data from patients included in this study were processed and collected by the IGTP-HUGTP Biobank integrated in the Spanish National Biobanks Network of Instituto de Salud Carlos III (PT13/0010/0009) and Tumour Bank Network of Catalonia. All laboratory measurements were performed by staff blinded to the patients’ clinical characteristics.
Flow–FISH
Blood samples were first lysed by a 10-min incubation with PharmLyse solution (BD Bioscience, San Diego, CA, USA), and then the cell concentration was measured by flow cytometry using Perfect-Count beads (Cytognos, Salamanca, Spain). In a 15-min incubation at room temperature (RT), 1 × 106 cells were stained with titrated amounts of the following antibodies: CD86-BV605, CD14-BV785 (Biolegend, San Diego, CA, USA), CD16-BV421, and CD15-AlexaFluor647 (BD Biosciences). Next, these cells were fixed with 6 mM bis(sulfosuccinimidyl)suberate (Sigma-Aldrich Química SL, Madrid, Spain) for 30 min at 2–8 °C. The reaction was quenched using 1 M Tris buffer (pH 8.0) for 15 min at RT. Then the residual red blood cells were removed by incubation with FACS lysing solution (BD Biosciences) for 7 min at RT.
FISH was performed using the Telomere PNA kit (Dako, Glostrup, Denmark) following the manufacturer’s instructions. The human 4-year old Caucasian female acute lymphoblastic leukemia 1301 cell line from the Health Protection Agency Culture Collections (HPACC) was used with each sample as an internal control. The 1301 cell line was previously cultured according to HPACC recommendations in RPMI 1640 (Gibco, Life Technologies, Grand Island, NY) with 10% fetal bovine serum (Sigma-Aldrich Química SL), penicillin, streptomycin, and glutamine (Gibco, Life Technologies). After purchase of the 1301 cell line, subsequent cells were obtained from four passages after reaching a maximum of 1 × 106 cells viable cells/ml in culture; cells with the same passage number were aliquoted in large numbers and stored at − 196 °C until use. Samples were acquired by flow cytometry, with up to 10,000 monocytes collected per sample. We performed correction for DNA ploidy of the blood sample vs. the internal control as previously described [12].
All samples were acquired on a Fortessa SORP flow cytometer (BD Biosciences) equipped with four lasers (100-mW 488 nm, 150 mW 532 nm, 50 mW 405 nm, and 100 mW 640 nm) using sample acquisition software FACSDiva v6.2 (BD Biosciences) and analyzed with FlowJo vX (Tree Star, Inc, Ashland, OR). We performed routine daily quality control tests with Cytometer Setup & Tracking Beads (BD Biosciences) in accordance with the manufacturer’s instructions. Daily QC control of 6-peak Rainbow Calibration Particles (BD Biosciences) was used for Flow-FISH MFI standardization to reach initial target MFI values. We initially gated for G0/G1 cells of both leukocyte subsets and 1301 cells based on DNA content, and then by scatter properties. Monocytes were sequentially identified using a CD86 vs. CD16 plot, followed by a CD15 vs. CD16 plot to gate out neutrophils. Next, the gated monocytes were analyzed for CD14 and CD16 expression. Clumped cells were excluded using a plot of propidium iodide (PI) area vs. PI width. Finally, each subset of monocytes and internal control cells was displayed on a plot comprising the FITC-labeled PNA probe on PI (B695-A) vs. the PNA probe (B515-A), and the median fluorescence intensity (MFI) of the PNA probe was measured.
The relative TL value for each monocyte subset was calculated as the ratio between the MFI of each subset and the MFI of the control cells. Corrections were made for the DNA index of G0/G1 cells, as previously described [13].
Statistical analysis
Categorical variables are expressed as percentages. For continuous variables, data distributions were assessed using normal Q–Q plots, and data are expressed as mean and (SD) for normally distributed data, or as median and (quartiles Q1–Q3) for non-normally distributed data. Between-group differences were assessed using Student’s t test and ANOVA with post hoc Scheffe analysis. To assess correlations among the TL of the different monocyte subsets, and between the mean TL of all monocytes and clinical variables, we used the Pearson or Spearman rho correlation test, as appropriate. Comparison between mean TL between baseline and 1-year samples were performed with t test for paired data.
We additionally performed univariate Cox regression analyses with all-cause death and the composite endpoint as the dependent variables, and with the mean TL for monocytes as a whole and for each monocyte subset as the independent variables. In the subgroup of patients for whom a 1-year follow-up blood sample was available, we also assessed the relative TL change using the formula [TL at 1 year –baseline TL]/baseline TL) × 100. Statistical analyses were performed using SPSS 15.0 (SPSS Inc., Chicago, IL, USA). A two-sided p value of < 0.05 was considered significant.