Fibrofast and Fibrosis-4 versus Fibroscan as Indicators of Hepatic Fibrosis in Non-Alcoholic Fatty Liver Disease Patients: A Cross-Sectional Study

: Non-alcoholic fatty liver disease (NAFLD) is a broad category for a disease spectrum that includes simple steatosis, which can proceed to non-alcoholic steatohepatitis, cirrhosis, and, finally, hepatocellular carcinoma. Owing to the invasive nature of liver biopsy, the need for non-invasive tools were required for diagnosis. Objective: To compare the performance of simple biochemical scores (fibroblast) FIB-5 and (fibrosis-4) FIB-4 with fibroscan to differentiate mild to moderate fibrosis (MF; F0 to F2) from advanced fibrosis (AF; F3 to F4) in patients with NAFLD.


Introduction:
Non-alcoholic fatty liver disease (NAFLD) was identified as a pathological entity in 1980 as a disorder that resembles alcoholic fatty liver disease with significant fat infiltration to the liver but without excessive alcohol use or other causes of liver disease [1].
With an estimated prevalence of twenty to forty percent, NAFLD is one of the most frequent liver disorders in the developed and developing world [2]. Our expertise in NAFLD has progressed over the last forty years to broadly establish a relationship to metabolic dysregulation as a significant factor in the disease's pathophysiology [3][4][5][6].
NAFLD is a broad category for a disease spectrum that includes non-alcoholic fatty liver (NAFL) or simple steatosis, which can proceed to non-alcoholic steatohepatitis (NASH), and cirrhosis. Finally, hepatocellular carcinoma (HCC) and end-stage liver disease (ESLD), in the end, severely impaired liver function has occurred [7]. Even without cirrhosis, advanced cases of NAFLD can develop into HCC [8].
The liver biopsy is the gold standard for diagnosing NAFLD patients. However, due to its intrusive nature and related complications (e.g., hemorrhage), non-invasive techniques for evaluating liver fibrosis and steatosis have been developed in recent years, such as transient elastography, controlled attenuation parameter, or magnetic resonance depending. As a result, emphasis was placed on non-invasive imaging modalities, particularly transient elastography. For example, vibration-controlled transient elastography (VCTE) is a novel technology for measuring mean liver stiffness in a non-invasive way. In addition, some devices offer a controlled attenuation parameter (CAP) that may measure hepatic steatosis, allowing for the assessment of both hepatic fibrosis and steatosis in the same situation without any side consequences [9,10].
The FIB-5 score was developed by Attallah et al. and is based on three biochemical markers (AST/ALT ratio, albumin, alkaline phosphatase (ALP), and one hematological marker (platelet count). The score was verified on 604 chronic HCV patients [11].

Original research
Fibrosis-4 (FIB-4) is a scoring system to estimate the grade of liver fibrosis using a combination of the patient's age, platelet count, aspartate transaminase (AST), and alanine transaminase (ALT), all readily available to a primary care physician, besides being inexpensive [12].
The objective of this study was to compare the performance of simple biochemical scores; FIB-5 and FIB-4, with fibroscan to differentiate mild to moderate fibrosis (MF; F0 to F2) and advanced fibrosis (AF; F3 to F4) in patients with NAFLD.

Patients and methods:
The site, Type, and Study Period This cross-sectional study was done on 116 patients who presented to Tanta University The diagnosis of hepatic steatosis in NAFLD patients was confirmed by a CAP score of more than 237 dB/ m. Of these, 116 male or female patients older than 18 with CAP score More than 237 dB/ m are included in the study (figure 1). All patients who fulfilled the inclusion criteria were encouraged to get a detailed history. Therefore, they were subjected to thorough clinical examination, including height, weight, body mass index, waist-hip ratio (WHR), history of other metabolic diseases, e.g., diabetes mellitus and hypertension, as well as basic laboratory tests such as complete blood count (CBC), blood urea, serum creatine, ALT, AST, international normalization ratio (INR), total bilirubin, serum albumin, alkaline phosphatase, and total lipid profile.

Original research
The laboratory investigations were carried out in the clinical pathology department, faculty of medicine, Tanta University. CBC was performed on K3 EDTA blood using an automated cell coulter (ERMA PCE 210, Tokyo, Japan). Serum levels of urea, creatinine, total bilirubin, albumin, and complete lipid profile, as well as ALT, AST, and ALP enzymes activity were measured using a fully automated chemistry analyzer (Konelab Prime 60i, Konelab, Helsinki, Finland) with the compatible chemicals supplied from ThermoFisher scientific ™.
The score was calculated using the following equation:

Fibroscan; Transient Elastography
All patients were scanned with the EchosensTM FibroScan. A 3.5 MHz ultrasonic transducer is installed on the axis of a low amplitude vibrator in the Fibroscan® probe (frequency of 50 Hz and amplitude of 2 mm peak-to-peak).
An experienced operator blinded to the patient's diagnosis and data performed the liver stiffness measurement (LSM) and Controlled Attenuation Parameter (CAP). Only findings with ten accurate shots and an interquartile range IQR/median liver stiffness ratio of 30% were considered credible. Both liver stiffness measurement (LSM) and CAP were collected from the same region of the liver parenchyma (between 25 and 65mm in depth) [14].
The final LSM and CAP values were presented in Kpa and dB/ m, respectively [15].
According to the METAVIR scoring system, significant fibrosis was classified as fibrosis stage ≥ F2, severe fibrosis was defined as fibrosis stage ≥ F3, and cirrhosis was defined as fibrosis stage = F4. These categories constituted at least significant fibrosis and impacted patient management in therapy indications [16,17].
The following CAP cut-off values were adopted from another investigation to indicate liver steatosis (S): S0 denotes no steatosis (237 dB/m), S1 represents mild steatosis (that range from 237.0 to 259.0 dB/m), S2 represents moderate steatosis (that range from

Results
The present study included 116 patients. The mean age of patients was 45.47 ± 9.01 years, with no significant age difference between the advanced fibrosis group (group II) and mild to moderate fibrosis group (group I). Seventy patients were females (60.34%), while 46 patients were males (39.65%) (Tab 1).

LSM: liver stiffness measurements
The mean liver stiffness measurement (LSM) score of group II patients was (9.53 ± 1.05 kPa) which was significantly higher than group I (5.18 ± 0.99 kPa) (p-value <0.001) ( Table 3). The mean steatosis score of groups II (319.4± 43.49 dB/m) is higher than group I (284.5± 38.85 dB/m) but with no significant difference between the two groups (pvalue = 0.640). The frequency of liver steatosis according to ultrasound criteria and fibroscan CAP examination was shown in (Tab. 3).
In an attempt to compare the laboratory data of both groups; the patients with advanced fibrosis (group II) had statistically significant higher Bilirubin (p value = 0.001), waist hip ratio (p value =0.002), BMI (p value = 0.001), VLDL (p value =0.001), TG (p value = 0.008) as compared with mild to moderate fibrosis patients (group I) respectively. While, significantly low Hb (p value = 0.001), LDL (p value =0.001) and ALT (p value = 0.005) were noted among group II as compared with group I respectively ( Table 4).

Discussion
In our study, we compared the performance of biochemical scores, including FIB-4 and FIB-5, with fibroscan to rule out the advanced stages of liver fibrosis in patients with NAFLD.
Our study revealed no significant age difference between AF and MF, in contrast to the previous research, which found a high prevalence of fibrosis (40%) and cirrhosis (14%) in the liver biopsies of these older individuals [18]. This may be attributable to the inclusion of older patients in this study.
Our results showed that the patients with AF had higher BMI, VLDL, and TG than those with MF. This is consistent with other studies [19,20] that concluded that fibrosis frequently occurs in overweight and obese patients. showing higher scores among significant or advanced fibrosis compared to mild to moderate fibrosis. Also agree with previous studies [22] that found FIB-4 has great potential in diagnosing liver fibrosis caused by viral hepatitis and NAFLD in patients with advanced fibrosis . This was in agreement with Kumari et al. [23], who concluded that FIB-4 is one of the best indices to assess liver fibrosis in NAFLD patients [23].
Also, Amernia et al. supported this finding; their study showed that FIB-4 is the best index to assess liver fibrosis in NAFLD patients [24]. Another study is consistent with our results; this study showed that The FIB4 index was superior to other tested noninvasive markers of fibrosis in Japanese patients with NAFLD [25].
We also found in our study; that the FIB-5 score has no significant difference between groups. This was in agreement with Kolhe et al., who stated that FIB 5 could not be used to rule out advanced fibrosis [28], but this is in contrast to the previous study [23] that reported the FIB-5 score of the group with advanced fibrosis was significantly lower as compared with patients with mild to moderate fibrosis in NAFLD patients.
Also, other studies showed FIB-5 score was more specific than FIB-4 for diagnosing significant from non-significant hepatic fibrosis in patients with chronic HBV infection [26,27].

Conclusion: FIB-4 is considered a diagnostic tool for fibrosis in NAFLD patients
and can be used for differentiation between advanced fibrosis and mild to moderate fibrosis in NAFLD patients.

Funding
This research received no specific grant from public, commercial, or not-forprofit funding agencies.

Declaration of competing interest
There are no conflicts of interest related to this study.
Authors contributions: