The authors have declared that no competing interests exist.
Conceived and designed the experiments: TK MN AO AG HW JGM JM TB MT VK JW. Performed the experiments: TK MN AO AG MT VK JW. Analyzed the data: TK MN AO AG DP HW JGM JM TB MT VK JW. Contributed reagents/materials/analysis tools: TK AO AG HW JGM JM TB VK JW. Wrote the paper: TK MN DP JGM JM TB VK JW. Revision of manuscript: TK MN AO AG DP HW JGM JM TB MT VK JW.
Cystic fibrosis-related liver disease (CFLD) is present in up to 30% of cystic fibrosis patients and can result in progressive liver failure. Diagnosis of CFLD is challenging. Non-invasive methods for staging of liver fibrosis display an interesting diagnostic approach for CFLD detection.
We evaluated transient elastography (TE), acoustic radiation force impulse imaging (ARFI), and fibrosis indices for CFLD detection.
TE and ARFI were performed in 55 adult CF patients. In addition, AST/Platelets-Ratio-Index (APRI), and Forns' score were calculated. Healthy probands and patients with alcoholic liver cirrhosis served as controls.
Fourteen CF patients met CFLD criteria, six had liver cirrhosis. Elastography acquisition was successful in >89% of cases. Non-cirrhotic CFLD individuals showed elastography values similar to CF patients without liver involvement. Cases with liver cirrhosis differed significantly from other CFLD patients (ARFI: 1.49 vs. 1.13 m/s; p = 0.031; TE: 7.95 vs. 4.16 kPa; p = 0.020) and had significantly lower results than individuals with alcoholic liver cirrhosis (ARFI: 1.49 vs. 2.99 m/s; p = 0.002). APRI showed the best diagnostic performance for CFLD detection (AUROC 0.815; sensitivity 85.7%, specificity 70.7%).
ARFI, TE, and laboratory based fibrosis indices correlate with each other and reliably detect CFLD related liver cirrhosis in adult CF patients. CF specific cut-off values for cirrhosis in adults are lower than in alcoholic cirrhosis.
Cystic fibrosis (CF) is the most common lethal genetic disease in populations of European descent. Cystic fibrosis-related liver disease (CFLD) is caused by biliary hyperviscosity which results in bile duct obstruction and is present in up to one third of CF patients. Five to 10% of patients develop liver cirrhosis and may require liver transplantation
In the last decade, ultrasound-based elastography methods for non-invasive assessment of chronic liver diseases have been developed
However, liver elastography has not been systematically evaluated in adult patients, and a direct comparison of TE and ARFI is missing so far. Thus, the aim of the present study was to evaluate both methods simultaneously in a cohort of adult CF patients.
The study was performed in strict accordance with the ethical guidelines of the Helsinki Declaration and was approved by the Leipzig University ethics committee (registration number 091-10-19042010). All study participants provided written informed consent.
Adult CF patients were prospectively investigated at presentation to the pulmonary outpatient clinic for clinical routine examinations. Patients with pregnancy, age <18 years, and liver transplantation were not included. Patients underwent conventional upper abdomen ultrasound evaluation, elastography and blood tests (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), bilirubin, gamma-glutamyltransferase (GGT), blood count, INR, albumin, creatinine, and cholesterol) at the same day. Fasting for at least three hours was required prior to examination, however exceptions were permitted when clinically required.
Previous ultrasound reports, recent pulmonary function tests (time span <6 months), and results of previous routine blood tests were collected from clinical records.
Cystic fibrosis-related liver disease was defined if at least 2 of the following conditions were present on at least 2 consecutive examinations spanning a 1-year period
ARFI results were compared with a control group of 50 healthy volunteers which has already been described in detail before
Liver stiffness was measured by ARFI technology (Acuson S2000; Siemens Medical Solutions, Mountain View, California, USA; Software Version 350.1.050.36) using a convex probe (4C1, Siemens Healthcare).
Patients were examined in a supine position with the right arm elevated above the head in a resting respiratory position if tolerable (short breath-hold without deep inspiration). The measurement depth from the transducer surface was between 20 and 55 mm. The examiner aimed for a measuring angle close to 0° (region of interest position in the center of the transducer surface).
Measurements were performed at two different sites in an area of homogenous tissue: i) right liver lobe through the intercostal space and ii) left liver lobe in the epigastric region in the median line. Measurements were performed by experienced operators (TK, VK, MN, MT, JW).
10 valid measurements at each site were required. The ARFI success rate was calculated as the ratio between the valid and the total number of measurements. The median value of each measuring site was used for further analysis. In case of a success rate below 60% the result was regarded as invalid and excluded from further analysis
For transient elastography (TE; Fibroscan®, Echosens, Paris, France; Software Version 1.40) all subjects were examined in a supine position immediately after ARFI measurement. TE was performed in a right intercostal space in resting respiratory position. 10 valid measurements were taken according to the manufacturer's recommendation (M probe). Measurements were performed by experienced operators (TK, VK, MN, MT). Patients with an interquartile range (IQR)>median value/3 or a success rate below 60% were considered as invalid and excluded from further analysis.
In addition to ultrasound examination, liver fibrosis was investigated by the two non-invasive fibrosis indices AST/Platelets-Ratio-Index (APRI) and Forns' score
Ordinal and nominal data were collected in a Microsoft® Excel file. Statistical analyses were conducted by using MedCalc® 11.4 (MedCalc Software, Belgium) and PASS 11.0.2 (NCSS, UT; USA). Clinical and laboratory data were expressed as median ± range and mean ± standard deviation (SD) where appropriate. Elastography results are presented as boxplots.
Fisher's exact test and chi-square test were used to test for association of variables. Nonparametric tests were chosen to compare median values of two independent samples (Mann-Whitney U test) or groups (Kruskal-Wallis test). Correlations between variables were examined using the Pearson's correlation coefficient. Diagnostic performance of elastography methods and serologic fibrosis indices were evaluated using receiver operating characteristic curves. The probabilities of a true-positive (sensitivity) and a true-negative (specificity) assessment with selected cut-off values were determined, and the area under the receiver operating characteristics curve (AUROC) was calculated
55 adult CF patients were prospectively included in the study from April to December 2010 (
Cystic fibrosis patients | CFLD subgroups | Healthy controls | Alcoholic cirrhosis | ||||
Total study cohort | Without CFLD | With CFLD | Without cirrhosis | With cirrhosis | |||
Gender (n) | |||||||
Male/female | 31/24 | 24/17 | 7/7 | 4/4 | 3/3 | 21/29 | 8/2 |
Age (years) |
31.9±8.8 | 32.9±9.0 | 29.0±8.0 | 29.6±7.8 | 28.3±8.9 | 27.8±4.0 | 57.5±5.9 |
BMI (kg/m2) |
21.5±3.8 | 21.7±4.1 | 20.8±3.0 | 20.9±3.0 | 20.6±3.2 | 22.1±1.9 | 26.0±5.1 |
Time (years) since initial CF diagnosis |
26.6±10.1 | 26.2±10.9 | 27.8±7.3 | 28.4±7.6 | 27.0±7.5 | / | / |
Age (years) at CF diagnosis |
5.2±10.7 | 6.6±12.0 | 1.1±1.2 | 1.3±1.0 | 1.0±1.5 | / | / |
CFTR-genotype ΔF508/other (n) | 37/18 | 27/12 | 12/4 | 5/3 | 5/1 | ||
Lung transplantation (n) | 8 | 7 | 1 | 1 | 0 | / | / |
Ursodeoxycholic acid treatment (n) | 35 | 22 | 13 | 7 | 6 | / | / |
Diabetes mellitus (n) | 18 | 11 | 7 | 5 | 2 | / | / |
FVC (l) |
3.2±1.2 | 3.2±1.2 | 3.2±1.3 | 3.1±1.3 | 3.3±1.5 | / | / |
FEV1 (l/s) |
2.3±1.0 | 2.3±0.9 | 2.4±1.2 | 2.3±1.1 | 2.4±1.4 | / | / |
FEV1 in % of VC | 71.2±13.1 | 70.6±13.5 | 72.8±12.1 | 74.8±11.2 | 70.5±13.7 | ||
TLC (l) |
6.1±1.2 | 6.2±1.1 | 5.9±1.4 | 6.2±1.2 | 5.7±1.7 | / | / |
RV (l) |
2.8±0.9 | 2.9±0.9 | 2.5±0.9 | 2.8±1.0 | 2.2±0.6 | / | / |
|
|||||||
- FVC (%) | 73.8±23.1 | 72.5±21.6 | 77.4±27.7 | 74.5±30.4 | 80.9±26.6 | ||
- FEV1 (%) | 62.3±24.8 | 60.5±22.3 | 67.5±31.5 | 65.8±32.4 | 69.9±33.3 | ||
- TLC (%) | 102.0±12.2 | 101.0±11.8 | 105.0±13.5 | 105.2±11.9 | 104.9±16.5 | ||
- RV (%) | 172.7±54.3 | 173.8±52.6 | 169.5±61.1 | 178.0±72.6 | 159.7±49.2 | ||
Platelets (109/l) |
308.1±135.9 | 341.0±131.0 | 211.7±102.5 | 269.1±80.0 | 135.2±77.7 | / | 164.9±87.2 |
ALT (µkat/l) |
0.5±0.2 | 0.4±0.2 | 0.6±0.4 | 0.4±0.2 | 0.8±0.4 | / | 0.5±0.2 |
AST (µkat/l) |
0.5±0.2 | 0.5±0.1 | 0.6±0.2 | 0.5±0.1 | 0.7±0.3 | / | 0.9±0.4 |
AP (µkat/l) |
2.00±1.37 | 1.63±0.59 | 3.08±2.25 | 1.98±0.51 | 4.56±2.86 | / | 1.8±0.8 |
GGT (µkat/l) |
0.69±1.11 | 0.55±0.81 | 1.12±1.67 | 0.44±0.18 | 2.04±2.34 | / | 2.7±2.3 |
Bilirubin (µmol/l) |
8.1±7.2 | 6.6±3.0 | 12.5±12.3 | 7.3±4.8 | 19.4±16.2 | / | 23.1±15.0 |
Albumin (g/l) |
43.8±4.3 | 43.9±4.5 | 43.6±4.1 | 43.2±4.3 | 44.0±4.2 | / | 35.1±12.3 |
INR |
1.0±0.1 | 1.0±0.1 | 1.1±0.1 | 1.0±0.1 | 1.1±0.1 | / | 1.3±0.2 |
mean ± standard deviation.
CFLD – cystic fibrosis-related liver disease; FVC – functional vital capacity; FEV1 – forced expiratory volume in 1 second; VC – vital capacity, TLC – total lung volume; RV – residual lung volume.
In the ARFI control group, healthy subjects were significantly younger than the CF individuals (p = 0.012). However, the age difference was moderate (12% of median CF patient age) and there was no correlation between age and liver stiffness.
The group of patients with alcoholic liver cirrhosis was significantly older (p = 0.002) and had a higher body mass index than CF-cases with cirrhosis (p = 0.025). None of the individuals presented with either acute hepatitis (aminotransferase levels >2× upper limit of normal) or ascites.
Six of the CFLD patients had distinct clinical and sonographical signs of advanced liver disease, three of them showed collateral circulation of the portal vein. These six cases were classified as CFLD cirrhosis (
CF patients with cirrhosis showed higher values of alkaline phosphatase (p = 0.028), bilirubin (p = 0.039) and a significantly lower platelet count than CFLD cases without cirrhosis (135 vs. 269×109/l, p = 0.010). There were no significant differences concerning age, body mass index, time since and age at CF diagnosis, and pulmonary function. The distribution of ΔF508 CFTR-genotype, treatment with ursodeoxycholic acid, and the presence of diabetes mellitus did not vary significantly.
ARFI-acquisition was completely successful in 53 cases (96%). In two cases without CFLD the success rate was <60% for one liver lobe.
TE measurements were invalid in six cases without CFLD (one patient: success rate <60%; five patients: IQR exceeding the third of the median value) resulting in a rate of valid measurements of 89. Rates of valid results between ARFI and TE were not significantly different (p = 0.271).
APRI score and Forns index could be calculated in all CF patients.
Results of elastography measurements and serologic fibrosis indices are shown in
Results of shear-wave velocity measurement in the liver tissue are shown as boxplots (median, 25%- and 75% quartile, maximum and minimum, outliers) according to the site of measurement. Shear-wave velocity is not increased in CF-related liver disease (CFLD) patients compared to healthy controls and CF patients without liver involvement. ARFI measurement in the right liver lobe (A) can detect patients with cirrhosis in the CFLD group. Patients with alcoholic liver cirrhosis have significantly higher shear-wave velocity values than CF patients with cirrhosis (p = 0.002).
Results of liver stiffness measurement are shown as boxplots (median, 25%- and 75% quartile, maximum and minimum, outliers). Liver stiffness is not increased in CF-related liver disease (CFLD) patients compared to CF patients without liver involvement. CFLD-patients with liver cirrhosis show a significant increase of liver stiffness.
Cystic fibrosis patients | CFLD subgroups | Healthy controls | Alcoholic cirrhosis | ||||
Total study cohort | Without CFLD | With CFLD | Without cirrhosis | With cirrhosis | |||
n = 55 | n = 41 | n = 14 | n = 8 | n = 6 | n = 50 | n = 10 | |
|
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R-ARFI (m/s) | 1.16±0.21 | 1.12±0.15 | 1.28±0.31 | 1.13±0.15 |
1.49±0.36 |
1.15±0.17 | 2.99±0.77 |
Success rate | 0.95±0.13 | 0.94±0.14 | 0.96±0.09 | 0.94±0.11 | 0.98±0.04 | 0.95±0.09 | 0.91±0.09 |
valid/invalid (n) | 54/1 | 40/1 | 14/0 | 8/0 | 6/0 | 50/0 | 10/0 |
L-ARFI (m/s) | 1.29±0.21 | 1.25±0.18 | 1.41±0.25 | 1.35±0.29 | 1.48±0.20 |
1.28±0.19 | 3.05±0.54 |
Success rate | 0.92±0.16 | 0.91±0.17 | 0.95±0.09 | 0.96±0.06 | 0.95±0.12 | 0.93±0.09 | 0.88±0.20 |
valid/invalid (n) | 54/1 | 40/1 | 14/0 | 8/0 | 6/0 | 50/0 | 9/1 |
TE (kPa) | 4.5±2.5 | 4.0±1.1 | 5.8±4.2 | 4.2±1.3 |
8.0±5.9 |
/ | / |
Success rate | 0.94±0.10 | 0.94±0.10 | 0.96±0.08 | 92.8±8.9 | 100 | / | / |
valid/invalid (n) | 49/6 | 35/6 | 14/0 | 8/0 | 6/0 | / | / |
|
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APRI | 0.314±0.395 | 0.207±0.099 | 0.630±0.687 | 0.28±0.12 |
1.09±0.87 |
/ | / |
Forns | 2.471±2.064 | 1.933±1.627 | 4.045±2.444 | 2.66±1.17 |
5.89±2.54 |
/ | / |
results shown as mean ± standard deviation.
significant difference compared to other subgroup (p-value<0.03).
significant difference compared to positive control group (p-value = 0.002).
CFLD – cystic fibrosis-related liver disease; TE – transient elastography, R-ARFI – right liver lobe ARFI; L-ARFI – left liver lobe ARFI; APRI – AST/Platelets-Ratio-Index.
Both ARFI and TE did not reveal a significant difference between CF patients without liver involvement and non-cirrhotic CFLD cases (n = 8): 1.12±0.15 vs. 1.13±0.15 m/s (right liver lobe ARFI, p = 0.83) and 4.0±1.1 vs. 4.2±1.3 kPa (TE, p = 0.94), respectively (
TE results were highly correlated with median ARFI values of the right liver lobe in all patients (rho = 0.762; p<0.001). ARFI values of the left liver lobe showed good correlation with right liver lobe ARFI (rho = 0.311; p<0.001) and with TE (rho = 0.333; p<0.021). All elastography methods displayed a significant correlation with APRI and Forns indices (rho>0.4; p<0.01).
The diagnostic performance for CFLD and cirrhosis detection was comparable between elastography methods and the serological fibrosis indices (
Elastography | Fibrosis Indices | ||||
ARFI (right lobe) | ARFI (left lobe) | TE | APRI | FORNS | |
|
40/14 | 40/14 | 35/14 | 41/14 | 41/14 |
AUROC |
0.682 [0.541; 0.802] | 0.672 [0.531; 0.794] | 0.677 [0.528; 0.803] | 0.815 [0.688; 0.907] | 0.786 [0.654; 0.885] |
Cut-Off | 1.28 m/s | 1.43 m/s | 5.9 kPa | 0.231 | 2.154 |
Sensitivity | 42.9% | 50.0% | 42.9% | 85.7% | 92.9% |
Specificity | 92.5% | 90.0% | 97.1% | 70.7% | 61.0% |
|
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CFLD-no cirrhosis vs. CFLD-cirrhosis (n) | 8/6 | 8/6 | 8/6 | 8/6 | 8/6 |
AUROC |
0.854 [0.568; 0.981] | 0.635 [0.345; 0.868] | 0.875 [0.593; 0.988] | 0.875 [0.593; 0.988] | 0.854 [0.568; 0.981] |
Cut-Off | 1.13 m/s | 1.47 m/s | 4.4 kPa | 0.344 | 4.059 |
Sensitivity | 100% | 66.7% | 100% | 83.3% | 66.7% |
Specificity | 62.5% | 75.0% | 75.0% | 87.5% | 100% |
95% confidence interval.
CFLD – cystic fibrosis-related liver disease; AUROC area under receiver operating characteristics curve.
This is the first prospective study evaluating TE and ARFI simultaneously for the detection of CFLD in a cohort of adult CF patients presenting with baseline characteristics comparable to previously reported studies
ARFI and TE could be applied with a success rate>90% and correlated highly between each other. However, TE had a higher rate of invalid measurements than ARFI in patients without CFLD (15% vs. 2%) which is in line with previously reported TE failure rates
Both TE and ARFI could not significantly discriminate between non-cirrhotic CFLD patients and CF cases without liver disease although a gradual increase would have been expected. This observation should be interpreted very cautiously due to the limited case number in the non-cirrhotic CFLD subgroup. However, TE and ARFI (right liver lobe) distinguished between liver cirrhosis and earlier stages of liver disease in CFLD patients.
Previous studies suggested a potential diagnostic benefit of left liver lobe ARFI in healthy controls which seems to diminish in advanced liver disease
Elastography values in CF patients with liver cirrhosis were significantly lower than in alcoholic cirrhosis and differed from reported cut-off values for liver cirrhosis in chronic hepatitis B and C
There is a set of possible explanations for our findings:
Up to two thirds of pediatric and adolescent CF patients show an ultrasound pattern of fatty liver disease
CFLD is a known risk factor for a severe CF course in children. However, the prognostic role of CFLD in adults is less certain, and disease progression after adolescence is rare
The use of different CFLD definitions in previous studies limits their comparability with our results
The number of recruited patients - although comparable to previous studies
Ideally, non-invasive evaluation of liver fibrosis should be compared to adequate results of liver biopsy. However, the focal nature of liver damage in CFLD is the key problem for histological assessment and limits its use as standard classification in CF
In summary, right liver lobe ARFI and TE correlate with each other in CF patients and can reliably detect CFLD related liver cirrhosis. CF specific elastography cut-off values in adults are lower compared to liver diseases of other etiologies. A structured and prospective use of ultrasound- and laboratory based approaches to investigate CF-related liver disease may detect progression of liver fibrosis invisible by ultrasound and allow further risk stratification in adult CF patients.
We thank the patient advocacy group “Mukoviszidose Selbsthilfe Leipzig e.V.” for support of the study project.