Transient Elastographic Values of Healthy Volunteers in a Tertiary care Hospital

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INtrODUctION
Chronic liver diseases often result in fibrosis that may eventually lead to cirrhosis, a state that carries a risk of lethal complications, including hepatocellular carcinoma. 1,2The prognosis and management of chronic liver diseases often depend strongly on the degree of liver fibrosis. 3ese facts point out the clinical interest in quantifying hepatic fibrosis and detecting patients with cirrhosis.At present, liver biopsy is the "gold standard" method to assess the grade of liver fibrosis.5][6] Liver biopsy examination is difficult to repeat in generally asymptomatic patients.Furthermore, due to the limited size of liver samples and the subjective assessment made by pathologists, accuracy and reproducibility of histological grading has been questioned.Therefore, there is an increasing need for alternative noninvasive methods to estimate the grade of liver fibrosis. 7Among the other potentially interesting approaches, elastography seems to be one of the most promising.Indeed, it is well known that liver stiffness is related to the degree of hepatic fibrosis, and palpation has been used from decades to establish a clinical diagnosis of hepatic fibrosis and cirrhosis.
Transient elastography (FibroScanR, Echosens, and Paris, France) is a recently described non-invasive procedure which consists of a 5-MHz ultrasound transducer probe mounted on the axis of a vibrator.The vibrator generates a completely painless vibration (with a frequency of 50 Hz and amplitude of 2 mm) which generates an elastic share wave propagating through the skin and the subcutaneous tissue to the liver.The velocity of the wave is directly related to the tissue stiffness which allows evaluating liver stiffness. 8he basic principle of transient elastography is that the propagation velocity of a wave through a homogenous tissue is proportional to its elasticity, which is correlated with the amount of fibrosis in the liver.The FibroscanR test has the advantage of being pain-free, easy-to-learn and fast 9 and give result immediately.
In the context of growing burden of chronic liver disease in our country, we need a novel, noninvasive and bedside method like transient elastography to assess liver fibrosis by measuring liver stiffness.Unlike liver biopsy, transient elastography can be repeated without risk to patients.Since, transient elastography is a relatively new diagnostic tool and it has been tested in different chronic liver disease but there are few data regarding healthy population.There are no published studies of transient elastography on either healthy or people with chronic liver diseases in Nepalese population.So, we aimed in this study to see the transient elastographic values in apparently healthy Nepalese population.

MEtHODs
This was a cross sectional descriptive study done in Liver Unit, National Academy of Medical Sciences, Bir Hospital on July-August 2013.
Healthy volunteers were taken for study after explaining about study purpose and methods.Volunteers were Resident Doctors and Nursing Staffs working in Bir Hospial at the study time.Healthy Volunteers aged 18 years and above were included and those volunteers who had any liver disease or suspected of having systemic disease affecting liver like raised transaminases, fatty liver in ultrasonography and those not willing to participate in study were excluded.All volunteers gave informed consent and the study protocol was approved by the institutional review board (IRB), National Academy of Medical Sciences.Those who agreed to participate had a complete medical examination with laboratory tests on the same day.Data were collected for each volunteer: age, gender, ethnicity, occupation and vital signs.Similarly, history of liver diseases, diabetes, arterial hypertension, smoking and alcohol use were also recorded.Routine laboratory tests including complete haemogram, random blood glucose, renal function test, liver biochemistries, serum total protein, albumin and total cholesterol, serological tests for hepatitis B&C virus was also done.Likewise, ulrasonography of the abdomen done where size of liver and spleen was recorded and chest x-ray also carried out.Body mass index was calculated and classified according south Asian BMI value (Normal BMI: 18.0-22.9kg/m 2 , Overweight: 23.0-24.9kg/m 2 , Obesity: >25 kg/m2). 10en FibroscanR of all volunteers were done in Centre for Liver Disease next day by the same clinician who was blinded to clinical and biochemical profiles.FibroscanR was performed on the right lobe of the liver, through intercostals spaces, with the patient lying in dorsal decubitus with the right arm in maximal abduction.The operator placed the fibroscanR transducer perpendicularly to the intercostals space, assisted by time motion ultrasound images, located the probe in a liver portion at least 6cm thick and free of large vascular structures and gallbladders and presses the probe button to commence the measurement.Ten valid measurements were performed to examine a patient with transient elastography.The median value of the ten valid measurements was considered representative of liver elasticity.The success rate was calculated as the number of valid measurements divided by the total number of measurements.Examinations with a success rate higher than 60% and interquartile range (IQR), which represents the intrinsic variability of TE, <30% of the median were considered reliable.The results were immediately obtained after performance of TE and expressed as the median value of 10 validated measurements in kilopascals (kPa), values ranging from 2.5 to 75 kPa.Only procedures with at least ten successful acquisitions and a success rate of at least 60% were considered reliable and recorded.
Descriptive statistical measures such as mean, standard deviation, median, range, etc for continuous variables and percentages for categorical variables had been reported to summarize the data distribution.Continuous variables between two groups were compared by independent t-test/ Mann-Whitney U test wherever applicable and the three groups were compared by using one way ANOVA.The associations between two independent categorical variables were assessed through the use of Chi-square test/ Fisher's exact test wherever applicable.
All statistical analyses were carried out with statistical software SPSS version 16.0.Results were considered statistically significant with p value less than 0.05.

rEsULts
A total of 51 apparently healthy volunteers were enrolled in this study.Six of them were excluded in which two volunteers had raised transaminases, two had fatty liver in ultrasonography and two had both raised transaminases and fatty liver.Of the remaining 45 volunteer subjects with normal liver biochemistries and normal liver ultrasonography were taken for subsequent analysis.Demographic profiles and vital parameters of the study subjects are given in Table 1 and laboratory parameters in Table 2. Similarly, ultrasonographic measurements of spleen and liver and fibroscan values are presented in Table 3.Among these volunteers percentage of male was higher than female (66.66% vs 33.33%) and percentage of resident doctors was higher than nursing staff (80% vs 20%).The mean age of our study subjects was 30.13±4.96 years and according to gender was comparable (31.70 ± 3.80 vs 27.00 ± 5.62 years for men and women ,respectively, p = 0.002).  2 and spleen size (cm) in Figure 3 are shown.

DIscUssION
Transient elastography (TE) is a recently described noninvasive procedure, which measures the transmission of an elastic share wave generated by vibration.This technique allows to evaluate liver stiffness. 8Briefly, TE consists of an ultrasound transducer mounted on the axis of the vibrator, which produces vibration of a mild amplitude and low frequency (50 Hz), consequently inducing an elastic shear wave that propagates through the liver.Pulse-echo ultrasound follows the propagation of the shear wave and measures its velocity, which is related to liver tissue stiffness.It is reported that the velocity of elastic waves is faster in fibrotic liver than normal liver.Performance of TE takes only a few minutes, and it is well tolerated by most patients.TE is performed on the right lobe of the liver with the patient lying in dorsal decubitus with the right arm in maximal abduction.Usually, 10 valid measurements are performed and median value of the ten valid measurements is considered representative of liver elasticity.Examinations with a success rate higher than 60% are considered reliable and results expressed in kilopascals (range 2.5-75 kPa).The interquartile range (IQR), which represents the intrinsic variability of TE, <30% of the median indicates a high-quality result. 8ransient elastography has already been validated in different liver diseases like chronic viral C hepatitis, [11][12][13][14] chronic viral B hepatitis, 1516 HCV-HIV co-infection, 17,18 liver transplant recipients, [19][20][21][22] cholestatic conditions 23 and hemochromatosis. 24broscanR is gaining popularity in various chronic liver diseases to determine status of liver in term of stiffness with different cut-off values.But data of normal values of liver stiffness is still few.We studied 45 apparently healthy volunteers with normal liver chemistries and normal liver ultrasound finding.The study subjects are few due to limitation of available resources.FibroscanR is available in single center (Center for liver diseases) and relatively inexpensive, easier to perform and safer than liver biopsy.It can also be repeated, in order to monitor changes in fibrosis during the course of treatment and can yield an immediate result after procedure.It measures liver stiffness of a value of parenchyma approximately 100 times bigger than a needle biopsy specimen so it is more representative of liver parenchyma.In this study, we found mean liver stiffness value (4.24 ± 0.70 kPa) in apparently healthy volunteers.Liver stiffness values were found higher in males than in females (4.32±0.74vs 4.07±0.61kPa, respectively, P =0.26) but not statistically significant which is similar to Kim SU et al 25 where they found the mean liver stiffness value of 4.6±0.5 kPa (range 3.3-5.6kPa) in their study, which was calculated from 69 strictly selected living liver and kidney donors and liver stiffness values were not significantly different between men (4.6±0.6 kPa) and women (4.5±0.This study is the first of its kind in Nepalese population, so is expected to serve a benchmark for future research in this field.The major limitation of our study is its small sample size in comparison to different studies discussed here but still this gave us a baseline value of liver stiffness in our population.It is recommended that future studies are required with larger sample size and in subjects of various demographic backgrounds.

cONcLUsIONs
We examined liver stiffness value of relatively small group of healthy subjects and found mean liver stiffness value of 4.24 ± 0.70 kPa {male(4.32±0.74 kPa ) and female (4.07±0.61kPa)}.The influence of age, gender and body mass index on liver stiffness value was not significant.

AcKNOWLEDGEMENts
We would like to thank the center for liver disease (Bijulibazar, Kathmandu) for support by performing fibroscanR of study subjects free of cost.We would also like to thank Dr. Shankar Khanal (Statistician) as well as Mr. Prakash Kumar Nepal for support with the laboratory tests.Finally, we would like thanks to all volunteers who consented and participated in this study. rEFErENcEs

Figure 1 .Figure 2 .
Figure 1.Scatter diagram between liver stiffness and age of the study subjects.

Figure 3 .
Figure 3. Scatter diagram between liver stiffness and spleen size of study subjects.

table 4 . Association between liver stiffness and different group of BMI. bMI n Liver stiffness Mean ± s.D.
l in validation set ), spleen size (13.1±3.0 cm in training set and 15.5 ± 2.9 in validation set) and platelets to spleen ratio (1156±704 in training set and 817±443 in validation set) which correlate with liver stiffness values 25.2±16.6kPa in training set and 27.4±12.1 kPa in validation set.They concluded these values from patients with compensated cirrhosis in contrast to our values.In our study where we found platelet count (244000±59295/cumm) and spleen size (9.50 ± 1.12 cm), platelets to spleen ratio (2618.63±726.67) and liver stiffness value 4.24±0.70kPa which does not correlate with it, as these values are from apparently healthy volunteers.