Methods

Forty-nine consecutive patients admitted to the hospital with acute HF (AHF) of ischemic origin were recruited and compared with 37 patients with stable chronic HF (SHF), 43 patients with stable coronary artery disease (CAD) free of HF (“disease controls”), and 37 healthy controls. AHF was defined in accordance with the European Society of Cardiology guidelines as the rapid onset or progression of HF symptoms and signs related to reduced cardiac contractility requiring hospital admission (New York Heart Association [NYHA] class IV symptoms). All patients had left ventricular ejection fraction (LVEFs) ≤40% on echocardiography or left ventriculography. Patients were excluded if the hospital admission was caused by acute coronary syndromes (chest pain with ST/T-wave changes on electrocardiography with or without positive troponin) or hemodynamically compromising arrhythmia or valvular pathology.

The SHF group included patients with LVEFs ≤40% and no deterioration in clinical condition, hospital admission, or change in medication for the preceding 6 months. All patients with SHF had ischemic causes of HF. In this group, 10 patients (27%) had NYHA class I symptoms, 24 patients (65%) had NYHA class II symptoms, and 3 patients (8%) had NYHA class III symptoms. For CAD disease control group eligibility, criteria included a history of myocardial infarction >6 months previously and/or angiographically documented stenosis >50% in ≥1 coronary artery and LVEF >50%. Patients with SHF and CAD were recruited from outpatient clinics at Sandwell and West Birmingham Hospitals NHS Trust. The study was performed in accordance with the Declaration of Helsinki and was approved by the Warwickshire Research Ethics Committee. All participants provided written informed consent.

To minimize potential confounders, CAD was the cause of HF in all patients. This inclusion criterion enabled AHF and SHF patients to be compared with the CAD group as disease controls with similar profiles of cardiovascular risk factors and co-morbidities (e.g., diabetes, hypertension) and medications used. For all study groups, exclusion criteria included infectious and inflammatory disorders, cancer, hemodynamically significant valvular heart disease, creatinine >200 μmol/L, steroids, and hormone replacement therapy.

At baseline, all participants underwent full medical histories and clinical examinations, performed by a cardiologist. Nonfasting peripheral venous blood samples were analyzed by flow cytometry <60 minutes after collection, and plasma was stored at −70°C for batch analysis of biomarkers, including brain natriuretic peptide (BNP), high-sensitivity C-reactive protein (hsCRP), cFLCs, and cystatin C.

To assess the dynamics of cFLCs in patients with AHF over time, blood samples were analyzed at the following time points: (1) during the first 24 hours after admission, (2) on the day of hospital discharge, and (3) 3 months after hospital admission. Recruitment began on October 30, 2009, and all patients were followed until July 30, 2011, to register any cases of death or rehospitalization. The primary end point was defined as a combination of the first occurrence of rehospitalization or death; the secondary end point was any cause of death alone.

Cystatin C and cFLCs (Combylite assay; The Binding Site Group Ltd., Birmingham, United Kingdom) were measured on the SPAPLUS turbidimeter (The Binding Site Group Ltd.) following the manufacturer’s recommendations. The reference ranges were 0.56 to 0.99 mg/L and 9.3 to 43.3 mg/L (determined in serum samples), respectively. Combylite quantifies the combined FLC κ and FLC λ concentrations in a single assay. There was no evidence of monoclonal expansions, which could potentially influence FLC concentrations, in any sample either by positive serum protein electrophoresis or abnormal FLC κ/λ ratio (Freelite; The Binding Site Group Ltd.). hsCRP concentrations were measured on the BNII nephelometer (Siemens Healthcare, Erlangen, Germany).

Plasma levels of interleukin-6 were measured by cytometric bead array technology. The BD FACSCalibur flow cytometer was used for data acquisition, with FCAP Array version 2.0.2 software (Burnsville, Minnesota) for data analysis. Commercially available Human interleukin-6 Flex (Becton Dickinson, Oxford, United Kingdom) was used according to the manufacturer’s recommendations. The lower limit of detection of interleukin-6 was 1.0 pg/ml. BNP was measured using a commercially available enzyme immunoassay set (human BNP-32; Peninsula Laboratories, LLC, Torrance, California) according to the manufacturer’s specifications. The inter- and intra-assay coefficients of variation for interleukin-6 and BNP assays were <5%.

Normally distributed data are presented as mean ± SD and non-normally distributed data as median (interquartile range [IQR]). Cross-sectional comparisons among the 4 study populations were made using a chi-square test (for categorical variables), 1-way analysis of variance with Tukey’s post hoc test (for normal data), or the Kruskal-Wallis test with Dunn’s post hoc test (for non-normal data). Longitudinal analysis was performed using repeated-measures analysis of variance with Bonferroni’s adjustment (normal data) or the Friedman test with Dunn’s post hoc test (non-normal data). Only patients with AHF who completed 3-month follow-up were included in the longitudinal analysis. For patients with AHF, correlation coefficients were calculated by Pearson’s and Spearman’s tests for normal and non-normal data, respectively. Cox regression analysis was used to establish predictive value of the study for parameters for the study outcome.

Kaplan-Meier estimates for the distribution of time from index admission to the primary end point were computed, and log-rank analysis was performed to compare event-free survival for patients with values of cFLCs, BNP, hsCRP, and estimated glomerular filtration rate (eGFR) higher and lower than the median values at admission. Data analysis was carried out using SPSS version 18.0 (SPSS, Inc., Chicago, Illinois), and a 2-sided p value <0.05 was considered statistically significant.

Results

The 3 patient groups (AHF, SHF, and CAD) were comparable for age, gender, blood pressure, and body mass index ( Table 1 ). Patients with AHF had higher BNP and creatinine levels and lower eGFR compared with those with SHF. As expected, patients with AHF more often received loop diuretics and less often received β blockers than participants from all other groups. Patients with AHF had increased counts of monocytes and neutrophils and lower hemoglobin concentrations compared with other groups (p <0.001).

Patients with AHF had highly significant increases in levels of cFLCs, cystatin C, hsCRP, interleukin-6, monocytes, and neutrophils compared with other groups (p <0.001; Table 1 , Figure 1 for cFLCs). Also patients with SHF showed higher levels of cFLCs and cystatin C compared with healthy controls (p <0.05 and p = 0.008, respectively). There were no significant differences in hsCRP, interleukin-6, and leukocyte counts between patients with SHF and control groups free from HF.

cFLCs in the study groups. Medians and IQRs are indicated by the solid lines .

In patients with AHF, cFLCs were correlated with cystatin C (Spearman’s r = 0.63, p = 0.000001), creatinine (Spearman’s r = 0.47, p = 0.002), and inversely with eGFR (Spearman’s r = −0.39, p = 0.01). Cystatin C was correlated with creatinine level (Pearson’s r = 0.70, p = 0.0000004) and inversely with eGFR (Pearson’s r = −0.64, p = 0.00001). hsCRP was correlated with interleukin-6 level (Spearman’s r = 0.69, p = 0.0000001) and neutrophil count (Spearman’s r = 0.51, p = 0.004). There was no significant association between concentrations of cFLCs, cystatin C, and hsCRP and LVEF or BNP value.

Thirty-two patients with AHF (65%) completed all 3 blood-test time points. The median length of hospital stay was 9 days (IQR 5 to 13). Nineteen patients (39%) reached the primary end point of death or rehospitalization, with a median duration of follow-up of 221 days (IQR 140 to 453). Fourteen patients (29%) reached the secondary end point of death. Twelve patients died of HF, 1 died of a ruptured abdominal aortic aneurysm, and the cause of death in 1 patient was unknown.

During the 3-month follow-up period, there were significant reductions in BNP concentrations (p = 0.017) but no significant changes in cFLC or hsCRP levels ( Table 2 ). A nonsignificant trend was observed toward a further increase in cystatin C (p = 0.072).

Table 2

Longitudinal analysis of the study parameters

N = 32	Admission	Discharge	Follow Up	p
Combined free light chains (mg/L)	61 (45–77)	62 (48–83)	63 (48–86)	0.14
Cystatin C (mg/L)	1.90 ± 0.49	1.95 ± 0.53	2.04 ± 0.56	0.072
High sensitivity C-reactive protein (mg/L)	13.1 (5.0–29.9)	9.4 (2.9–27.1)	6.9 (2.7–17.5)	0.42
Brain natriuretic peptide (pg/L)	480 (230–900) ∗	253 (183–581)	218 (74–342)	0.017
Interleukin-6 (pg/mL)	12 (6–16)	8 (4–18)	6 (3–14)	0.60

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