Several circulating biomarkers have been found to play a role in the surveillance and risk stratification of heart failure without congenital heart disease, but these have not been widely studied in patients with single ventricles palliated with a Fontan operation. Imaging predictors of worse outcomes in this population include ventricular dilation and dysfunction. Patients who weighed >30 kg with a Fontan circulation referred for cardiac magnetic resonance imaging were invited to participate in the study. Blood and urine samples were obtained at the time of imaging and multiple conventional and novel biomarkers were measured. A total of 82 patients with a median age of 18 years were enrolled. Among the novel biomarkers, N-terminal pro–B-type natriuretic peptide (NT-proBNP) and high-sensitivity troponin T had the strongest correlation with ventricular dilation and dysfunction. NT-ProBNP >100 pg/ml has a sensitivity of 91% for the detection of significant ventricular dilation (end-diastolic volume >120 ml/body surface area 1.3 ) and 82% for detection of ejection fraction <50%. The urinary neutrophil gelatinase-associated lipocalin-2 to creatinine ratio correlated with ejection fraction and estimated glomerular filteration rate. In conclusion, abnormalities in biomarkers of heart failure are common in ambulatory, largely asymptomatic patients with Fontan circulation. NT-ProBNP may serve as a sensitive marker for the identification of patients with significant ventricular dilation or dysfunction. Further work is needed to understand how these easily measured circulating biomarkers may be integrated into clinical care.
Morbidity and premature mortality are common in the Fontan population, with a 15-year survival of around 90% following the Fontan operation and an annualized risk of late mortality about 2.1% per year. Circulating biomarkers are accepted as key metrics in adults with ischemic heart disease and heart failure, but these have not been well studied in the Fontan population. This includes markers of myocardial wall stress, injury, inflammation, myocardial remodeling, and collagen metabolism. Biomarkers such as N-terminal pro–B-type natriuretic peptide (NT-proBNP) or high-sensitivity troponin T (Hs-troponin T) may improve risk stratification and also advance the understanding of pathophysiology, potentially allowing patient-specific therapy. In addition to these circulating biomarkers, cardiac magnetic resonance (CMR)-derived imaging biomarkers such as higher ventricular volumes, depressed function, and myocardial fibrosis have been associated with death and heart transplantation. The present study aims to characterize circulating and urine biomarkers in a cohort of patients with Fontan circulation and explore associations between these biomarkers and contemporaneously derived CMR metrics with known associations with poor clinical outcomes.
Methods
This was a cross-sectional study of patients with Fontan circulation undergoing CMR at Boston Children’s Hospital. The study was approved by the institutional review board, and written informed consent was obtained. Patients who weighed <30 kg were excluded to meet the research blood-draw volume guidelines. Medications, arrhythmia history, and New York Heart Association functional class were extracted from the closest clinical documentation within 1 year of the CMR.
Blood was obtained from a peripheral vein, collected in ethylenediaminetetraacetic acid plasma, lithium heparin, and serum separating tube tubes on the same day as the CMR (typically immediately before or after the CMR). Samples were sent to a commercial laboratory (LabCorp, Burlington, NC) for the following tests: comprehensive metabolic profile, uric acid, high-sensitivity C-reactive protein (Hs-CRP), and a complete blood count with differential cell count. In addition, samples were divided into aliquots, and stored at −80°C for future use. A portion of the urine sample was tested for microalbumin and creatinine, and the remainder was stored at −80°C after centrifuging at 3,000 rotations per minute for 10 minutes. The stored blood and urine samples underwent testing by the Department of Laboratory Medicine at Boston Children’s Hospital for the following tests: NT-ProBNP, Hs-Troponin T, growth differentiation Factor 15, soluble suppression of tumorigenicity 2, transforming growth factor beta-1, insulin-like growth factor binding Protein 7, C-terminal telopeptides (Beta-CTx), Procollagen Type I C-terminal peptide (PIP), urinary neutrophil gelatinase-associated lipocalin 2 (NGAL), and urinary C-type natriuretic peptide (CNP). Supplement 1 contains assay details for these measurements. Model for end-stage liver disease excluding international normalized ratio or MELD-XI score and estimated glomerular filtration rate (eGFR) was calculated as previously described. ,
CMR studies were performed and analyzed as previously described. The following indices were recorded: indexed end-diastolic volume (EDV i ), indexed end-systolic volume (ESV i ), ejection fraction (EF), indexed ventricular mass. Indexing was performed for mass and volume measurements to body surface area (BSA) 1.3 .. Feature tracking analysis was performed in mid-ventricular short-axis and 4-chamber views as previously described, and global circumferential strain (GCS) and global longitudinal strain (GLS) were recorded. Late gadolinium enhancement (LGE) imaging was qualitatively graded as major, minor, or absent. The minor category was defined as minimal isolated involvement of septal/free wall junctions and/or involvement of known areas of previous ventriculotomy or surgical patch. The degree of semilunar valve regurgitation and atrioventricular valve regurgitation (AVVR) was categorized as moderate or worse if the regurgitation fraction was ≥20%. When CMR-derived grading was unavailable, the degree of regurgitation was recorded from the closest available echocardiogram.
Continuous data are presented as medians with interquartile range (IQR) unless otherwise specified and categorical data are presented as percentages. The lower limit of detection for Hs-Troponin T by the assay was <6.0 pg/ml. For analysis as a continuous variable, values <6.0 pg/ml were considered equal to 3.0 pg/ml. Urine NGAL and CNP levels were analyzed as ratios to urine creatinine. Natural log-transformed values were used to assess the correlation between circulating and urine biomarkers and CMR measurements; the strength of the association is presented as Pearson’s r . The lowest significance level was set at 0.05. Cut-off values for Hs-Troponin T (>14 pg/ml) and Gal-3 (>14.3 ng/ml) were based on published values in patients with Fontan circulation, whereas that for ST2 (>35 ng/ml) was derived from adults with heart failure. Cut-off values for Hs-CRP levels (<1 mg/L, 1 to 3 mg/L, and >3 mg/L) were based on published literature on patients with Fontan circulation and those without congenital heart disease. , Receiver operating characteristic curve analysis was performed to establish cut-off values for NT-proBNP in detecting the presence of significant ventricular dilation (EDV i >120 ml/BSA , ) or decreased ventricular function (EF <50%). Linear regression analysis was performed to assess the relation of eGFR and log-transformed novel biomarker levels with age as a covariate. Analysis was performed using IBM SPSS Statistics v24 (IBM Corp, Armonk, NY) and JASP v0.14.1 (University of Amsterdam, The Netherlands).
Results
The study cohort included 82 patients whose demographic, clinical, and imaging characteristics are summarized in Table 1 . New York Heart Association functional class was 1 for 89% and 2 for 11% of these participants. The results of complete blood count, comprehensive metabolic profile, and urinary microalbumin levels are summarized in Table 2 . Hematocrit was elevated (>0.50 in men, >0.44 in women) in 16%, red-cell distribution width was elevated (>15.5%) in 10%, mean corpuscular volume was low (<80%) in 10%, and lymphocytopenia (absolute lymphocyte count <0.9 × 10 9 /L) was present in 12%. Two patients had a serum albumin level <3.5 mg/dL, one of whom had protein losing enteropathy. A total bilirubin >1.2 mg/dL was present in 15%. A MELD-XI score >11 was present in 11%; the highest score was 15. Uric acid was slightly elevated (>8.6 mg/dL) in 4 men (range 9-9.6 mg/dL) and normal in all women. AST >40 IU/L was present in 20% and ALT >55 IU/L was present in 5%. No participant had AST>80 IU/L or ALT >110 IU/L. Urine microalbumin/creatinine ratio >30 mcg/mg was present in 25%, but only 1 patient had a value >300 mcg/mg. Blood urea nitrogen and creatinine were normal in all participants. An eGFR <90 ml/min/1.73 m 2 was present in 1 patient.
| Age at cardiac magnetic resonance imaging (years) | 17.8 (13.5, 22.8) |
| Body surface area (m 2 ) | 1.59 (1.33, 1.84) |
| Heart rate (beats per minute) | 78 (69, 88) |
| Male | 51 (62%) |
| New York Heart Association functional class | 1(1,1) |
| Medications | |
| Angiotensin converting enzyme inhibitor or angiotensin II receptior blocker | 46 (56%) |
| Aspirin | 62 (76%) |
| Warfarin | 16 (20%) |
| Atrial arrhythmia | 24 (29%) |
| Ventricular arrhythmia | 3 (3%) |
| Ventricular morphology | |
| Right ventricle | 37 (45%) |
| Left ventricle | 25 (31%) |
| Mixed | 20 (24%) |
| Primary anatomic diagnosis | |
| Hypoplastic left heart syndrome | 23 (28%) |
| Tricuspid atresia | 15 (18%) |
| Double outlet right ventricle | 14 (17%) |
| Unbalanced atrioventricular `canal defect | 9 (11%) |
| Double inlet left ventricle | 7 (9%) |
| Pulmonary atresia with intact ventricular septum | 4 (5%) |
| Other | 10 (12%) |
| Fontan type | |
| Lateral tunnel | 56 (68%) |
| Extracardiac | 16 (20%) |
| Right atrium-pulmonary artery | 5 (6%) |
| Other | 5 (6%) |
| End-diastolic volume (ml/BSA , ) | 99.5 (84.8, 122.2) |
| End-systolic volume (ml/BSA , ) | 46.8 (37.7, 70.6) |
| Mass i (gm/BSA , ) | 50.3 (43.0, 61.2) |
| Ejection fraction (%) | 52.0 (42.8, 57.9) |
| Global circumferential strain (%) | -12.3 (-14.0, -10.6) |
| Global longitudinal strain (%) | -13.4 (-15.9, -10.9) |
| Variable | n | Median (IQR) |
|---|---|---|
| White blood cell (x 10 3 /mm 3 ) | 78 | 6 (4.9, 7.2) |
| Lymphocyte (%) | 73 | 23 (19, 33) |
| Absolute lymphocyte count (x 10 3 /mm 3 ) | 73 | 1.39 (1, 1.81) |
| Hematocrit | 78 | 0.45 (0.42, 0.47) |
| Mean corpuscular volume (fl) | 77 | 87 (83, 90) |
| Red cell distribution width (%) | 71 | 13.9 (13.6, 14.5) |
| Platelets (10 3 /mm 3 ) | 78 | 193 (147, 251) |
| Blood urea nitrogen (mg/dl) | 78 | 13 (11, 16) |
| Creatinine (mg/dl) | 78 | 0.76 (0.6, 0.86) |
| Estimated glomerular filtration rate (ml/min/1.73 m 2 ) | 78 | 126 (113, 145) |
| Total protein (g/dl) | 77 | 7.1 (6.7, 7.3) |
| Albumin (g/dl) | 75 | 4.6 (4..4, 4.8) |
| Total bilirubin (mg/dl) | 76 | 0.7 (0.5, 1) |
| Alkaline phosphatase (IU/L) | 77 | 103 (70, 211) |
| Aspartate transaminase (IU/L) | 77 | 30 (19, 39) |
| Alanine transaminase (IU/L) | 77 | 27 (19, 38) |
| Uric acid (mg/dl) | 75 | 5.5 (4.4, 7.1) |
| Urine creatinine (mg/dl) | 76 | 71 (42, 134) |
| Urine microalbumin (mcg/ml) | 76 | 12.5 (3.5, 32.4) |
| Microalbumin/creatinine ratio (mg/g) | 76 | 16.6 (8.6, 29.6) |
| Model for end-stage liver disease excluding international normalized ratio score | 75 | 9.4 (9.4, 9.9) |
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