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\begin{document}

\title{Early Detection of Hepatobiliary Involvement in Cystic Fibrosis:
Biomarkers, Elastography and Genetic Influences}



\author[1]{Senthilkumar Sankararaman}%
\author[2]{A. Jay Freeman}%
\affil[1]{University Hospitals Rainbow Babies \& Children's Hospital}%
\affil[2]{Nationwide Children's Hospital}%


\vspace{-1em}



  
  \date{June 06, 2024}


\begingroup
\let\center\flushleft
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\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Cystic fibrosis-related hepatobiliary involvement (CFHBI) is a term used
to describe a spectrum of hepatobiliary involvement ranging from benign
elevation of transaminase levels to advanced cystic fibrosis associated
liver disease (aCFLD). While CFHBI is common among people with cystic
fibrosis (PwCF), aCFLD is rare impacting only approximately 5-10\% of
the CF population. Importantly though, aCFLD is the third leading cause
of death among PwCF, is an independent predictor of all-cause mortality
and is associated with significant morbidity. Despite this recognition,
our ability to predict those patients at greatest risk for aCFLD,
identify early aCFLD and monitor incremental progression of CFHBI is
lacking. Here we review the strengths and weaknesses of the common
biomarkers and imaging modalities used in the evaluation and monitoring
of CFHBI, as well as the current understanding of genetic modifiers
related to aCFLD.%
\end{abstract}%



\sloppy


\textbf{Early Detection of Hepatobiliary Involvement in Cystic Fibrosis:
Biomarkers, Elastography and Genetic Influences}

Senthilkumar Sankararaman, MD

Division of Pediatric Gastroenterology, Hepatology \& Nutrition

UH Rainbow Babies \& Children's Hospital, Cleveland Associate Professor,
Case Western Reserve University SOM, Cleveland, OH Email -
S.SankaraRaman@UHhospitals.org

A. Jay Freeman, MD, MSc

Division of Pediatric Gastroenterology, Hepatology \& Nutrition

The Ohio State University College of Medicine

Director of Pancreatic Care for the Pancreas and Liver Care Center

Nationwide Children's Hospital, Columbus, OH

Email - Alvin.Freeman@nationwidechildrens.org

\textbf{Corresponding author}

A. Jay Freeman, MD, MSc

Professor of Pediatrics, The Ohio State University College of Medicine

Director of Pancreatic Care for the Pancreas and Liver Care Center

Nationwide Children's Hospital

700 Children's Drive

J West 4\textsuperscript{th}~Floor

Columbus, OH 43205

Senthilkumar Sankararaman has no conflicts to disclose

A. Jay Freeman has received grant funding from National Institute of
Health and the Cystic Fibrosis Foundation. Has done advisory work Abbvie
and Takeda.

\textbf{Early Detection of Hepatobiliary Involvement in Cystic Fibrosis:
Biomarkers, Elastography and Genetic Influences}

\textbf{Abstract:}

Cystic fibrosis-related hepatobiliary involvement (CFHBI) is a term used
to describe a spectrum of hepatobiliary involvement ranging from benign
elevation of transaminase levels to advanced cystic fibrosis associated
liver disease (aCFLD). While CFHBI is common among people with cystic
fibrosis (PwCF), aCFLD is rare impacting only approximately 5-10\% of
the CF population. Importantly though, aCFLD is the third leading cause
of death among PwCF, is an independent predictor of all-cause mortality
and is associated with significant morbidity. Despite this recognition,
our ability to predict those patients at greatest risk for aCFLD,
identify early aCFLD and monitor incremental progression of CFHBI is
lacking. Here we review the strengths and weaknesses of the common
biomarkers and imaging modalities used in the evaluation and monitoring
of CFHBI, as well as the current understanding of genetic modifiers
related to aCFLD.

\textbf{Keyworlds -- CF, cystic fibrosis, CFTR, liver disease, portal
hypertension}

\textbf{Introduction:}

Cystic fibrosis-related hepatobiliary involvement (CFHBI) is common in
people with cystic fibrosis (PwCF) with a varying severity. A wide
spectrum of hepatic manifestations are noted in CF including but not
limited to steatosis, intermittent or persistent elevated liver enzymes,
abnormalities noted on radiological imaging and end-stage liver disease.
The pathophysiology of CFHBI is poorly understood but likely
multifactorial including non-specific and related extrahepatic causes
such as malnutrition, drug-related toxicities, viral/bacterial
infections, and hepatic congestion, but also could be due to the effects
of CFTR dysfunction in the biliary epithelia \textsuperscript{1} or
vascular abnormalities including obliterative portal venopathy or
non-cirrhotic portal hypertension \textsuperscript{2,3}. The clinically
significant pathognomonic finding of focal biliary fibrosis is noted in
approximately 30\% of PwCF with a varying degree of
severity\textsuperscript{4-9}. In autopsy studies, focal biliary
fibrosis has been documented up to 70\% of PwCF \textsuperscript{10}.
However, only about 5-10\% of PwCF progress to the severe form of
fibrosis namely the multilobular cirrhosis usually by the end of the
first decade of life\textsuperscript{4-9}. Along with multilobular
cirrhosis (with or without portal hypertension), the other phenotypes of
advanced CF-related liver disease (aCFLD) include one (or more) of the
following: nodular liver, advanced hepatic fibrosis (F4 of the METAVIR
staging) and non-cirrhotic portal hypertension (obliterative portal
venopathy).

The prevalence of clinically defined CFHBI varies but is generally felt
to occur in 30-40\% of PwCF in most studies depending on the definition
utilized \textsuperscript{7,11,12}. To minimize confusion, experts
proposed an unified approach for the classification of
CFHBI\textsuperscript{11,13}. According to the Cystic Fibrosis
Foundation 2021 Annual Registry, aCFLD accounts for the third leading
cause of mortality in PwCF accounting for 2.6\% of the deaths after
respiratory and transplant-related complications \textsuperscript{14}.
Even though this is relatively small portion of the population, aCFLD
may adversely impact lung function and nutritional status
\textsuperscript{7,15}, and is strongly associated with CF related
diabetes (CFRD)\textsuperscript{16}, all of which may further impact
morbidity and mortality. This is supported by a review of the French CF
Registry finding that liver involvement at baseline was an independent
risk factor for lung transplant or death \textsuperscript{17} and a
review of the United Kingdom CF registry concluding that aCFLD also has
significantly increased all-cause mortality with a hazard ratio (HR) of
1.54 \textsuperscript{18}.

Historically, CFHBI was viewed as a complication impacting PwCF almost
exclusively prior to 20 years of age \textsuperscript{5}. However, as
survival for PwCF improves, liver involvement among adults with CF is
increasingly recognized. A report by the National Institutes of Health
(NIH) proposed a modified criteria to evaluate CFHBI among adults that
revealed 47\% of their population had some degree of hepatic involvement
and that CHHBI was more common among adults than previously
reported\textsuperscript{19}. Importantly though, aCFLD remains a rare
finding among adults with single-center studies concluding that adult
CFHBI is a largely a benign condition \textsuperscript{20-23} and
well-illustrated by the French CF Modifier Gene Study
\textsuperscript{7} (see\textbf{Figure 1} )\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

The primary challenge facing the CF clinician is early identification of
those patients that will progress to aCFLD compared to those with more
benign forms of CFHBI. Initial strategies focused on early screening for
hepatic fibrosis. Liver fibrosis is often utilized as the surrogate
endpoint for clinical trials and considered an early marker of liver
disease progression, yet this strategy has been largely underwhelming to
date likely due to a number of a factors. Although histological
assessment is considered the gold standard method in the evaluation of
liver fibrosis, it has a number of limitations in evaluating CFHBI. It
is both invasive and expensive which precludes its use as a screening
test, and requires general anesthesia/sedation in the pediatric
population. Despite double-pass liver sampling, liver biopsy is still
prone to sampling errors (both underestimation or overestimation are
possible based on the site of sampling) as CFHBI is felt to have a
patchy distribution \textsuperscript{24,25}. Liver sampling may not
predict the exact severity of the disease process based on the area
sampled \textsuperscript{4,26}. Also, the pathologists'
histopathological assessment is prone to intra- and interobserver
variabilities, which may be further complicated by unusual findings such
as non-obliterative portal venopathy or non-cirrhotic portal
hypertension.

An ideal screening test for evaluating CFHBI should be noninvasive,
broadly available, reliable, reproducible, and cost-effective for both
evaluating and follow-up of CFHBI. Specifically for CF, screening
modalities should be able to capture various forms of hepatic
involvement. The currently available screening tests can be broadly
classified as laboratory biomarkers (primarily from blood) and
radiological methods utilizing greyscale ultrasound and/or elastography
technologies. Often these tests are utilized together for diagnostic
precision of liver fibrosis \textsuperscript{13,27,28}. Unfortunately,
current screening methods are best at identifying those without aCFLD,
or those unlikely to advance to aCFLD. While more work is required to
improve early detection of aCFLD, here we review the widely available
screening methods currently employed.

\textbf{BioMarkers:}

\textbf{Indirect laboratory biomarkers for screening hepatobiliary
involvement in PwCF:}

Conventional markers of liver disease, such as aspartate transferase
(AST), alanine aminotransferase (ALT) and gamma-glutamyl transferase
(GGT) along with platelet levels are obtained annually for PwCF. These
commonly employed indirect blood tests used independently or in
combination (AST to platelet ratio index (APRI), fibrosis index (FIB-4)
based on four factors (age, AST, ALT, and platelets), GGT to platelet
ratio (GPR), and AST/ALT ratio) have been widely
studied.\textsuperscript{29-34}. These tests have been extensively
utilized and validated in other chronic liver diseases such as viral
hepatitis (hepatitis B and hepatitis C), and nonalcoholic fatty liver
disease in both children and adults \textsuperscript{28,35,36}. The
tests are based on the general principle that AST remains stable or
increases, ALT typically increases with worsening fibrosis, and platelet
levels decrease with the development of portal hypertension, with or
without cirrhosis \textsuperscript{37,38}.

A number of studies have reported statistically different values among
these conventional markers of liver disease among PwCF with aCFLD
compared to those without liver disease \textsuperscript{39-44}. Among
these, GPR ([?]0.28), APRI ([?]0.425) have the diagnostic precision with the
highest AURUC's, generally between 0.75 to
0.9\textsuperscript{29,31,32}. Importantly though, these tests have
shown differences between disease classifications (e.g. normal and
advanced liver disease) but their utility in detecting early disease,
identifying small, incremental disease progression, or predicting the
development of aCFLD are not clear.

Even though these tests are simple, noninvasive, and cost-effective,
they have a number of major drawbacks. Non-specific elevation of liver
enzymes in PwCF is not uncommon due to reasons such as intercurrent
infections and medications (CFTR modulator therapies, and
antibiotics)\textsuperscript{45,46}. In a prospective CF study, by age
21 years, at least one increased liver enzyme (ALT, AST, and GGT) was
noted in all participants, and most elevations were noted in the early
years of life \textsuperscript{4,45}. Therefore, persistently elevated
liver enzymes are generally more valuable in the predicting liver
disease \textsuperscript{4,45}. Also, in people with aCFLD, liver
enzymes can be near normal with advanced stages of
fibrosis\textsuperscript{5}. Standalone, they demonstrate poor
sensitivity and specificity but can be effectively incorporated into the
algorithmic approach of liver involvement evaluation
\textsuperscript{27}. Another challenge in utilizing these tests is the
wide range of values that extend between disease category and
determining the definition of the upper limit of normal
\textsuperscript{32}. Additionally, the lack of adjustment for
confounding variables such as age, sex, and modulator status may further
complicate the interpretation of these markers among PwCF. All
non-invasive tests utilized in CFHBI are summarized in

\textbf{Table 1.}

\textbf{Direct serum biomarkers of fibrosis:}

In hepatic fibrosis, there is excessive extracellular matrix deposition
which occurs due to a homeostatic imbalance between matrix deposition
and removal \textsuperscript{47}. Several biomarkers involved either in
fibrogenesis or fibrolysis have been utilized in screening fibrosis.
Tissue inhibitors of metalloproteinase (TIMPs), collagen-IV, matrix
metalloproteinase (MMP)-2,~hyaluronic acid, and~prolyl hydroxylase~have
been explored in CFHBI \textsuperscript{48-50}. MicroRNAs (miRNAs) are
short RNA molecules that modulate gene expression at the
post-transcriptional level which are increased in chronic liver diseases
and have also been investigated in CFHBI \textsuperscript{24,51,52}.
However, these tests are expensive, not widely available for clinical
settings, results can also be confounded by other fibrotic processes
seen CF \textsuperscript{53-55}, and have not resulted in improved
diagnostic accuracy compared to conventional markers outlined
above\textsuperscript{56,57}.

\textbf{Biomarker conclusions:}

Taken together, direct and indirect serum biomarkers have yet to show
the ability to predict the progression to aCFLD. Normal liver indices
are very sensitive (i.e. exclude aCFLD) but specificity and
predictability remain poor. Clinically, intraindividual changes over
time are likely more significant and should be followed closely rather
adhering to strict cut-offs for any individual marker.

\textbf{Radiological methods:}

\textbf{Ultrasound in evaluation of CFHBI:}

Ultrasound (US) is a relatively inexpensive and widely available tool to
assess the liver. The initial role of US was established in a
single-center study involving a cohort of 106 children with CF followed
for approximately 10 years. In this study, a heterogeneous echogenic
pattern on liver US was utilized to evaluate children at risk for aCFLD
(defined as a nodular liver in US) with or without portal
hypertension\textsuperscript{58}. The authors demonstrated that children
with CF with a heterogeneous echogenic appearance had a 5.2-fold
increased incidence of a nodular appearance and a 6.1-fold increased
incidence of portal hypertension compared with participants with a
normal echogenic pattern \textsuperscript{58}. These results were
confirmed by the Prospective Study of Ultrasound to Predict Hepatic
Cirrhosis in CF (PUSH study) which showed the heterogenous pattern had
9.5 times increased incidence of the nodular pattern when compared to
participants with normal liver appearance over a six-year period
\textsuperscript{59}.

In addition to showing an association between a heterogenous US pattern
and the development of a nodular pattern on US, the PUSH study also
demonstrated significant differences between US groups (normal,
heterogenous, homogenous and nodular) for spleen size, and non-invasive
hepatic markers depicting the ability of a research ultrasound in the
evaluation of CFHBI and aCFLD \textsuperscript{44}. FIB-4, GPR, and APRI
were significantly increased, and platelets decreased in participants
with a heterogeneous pattern when compared with PwCF with a normal liver
appearance \textsuperscript{60}. In another study with longitudinal
follow up, the same group of PUSH study investigators noted that in
approximately 6.3 years of average follow-up, six participants with
nodular liver appearance developed esophageal varices, two had bleeding
related to varices, and two had liver transplants \textsuperscript{61}.
The control population had no liver-related adverse events highlighting
the feasibility of research US in discriminating the progression of
aCFLD from the individuals with normal liver appearance.

While the data regarding US in CF is encouraging, there remain
significant clinical concerns. US lacks the ability to detect early
stages of aCFLD reliably and a normal US does not preclude significant
liver fibrosis \textsuperscript{12}. Another major limitation is a high
percentage of interpretation variabilities (both intra- and
interobserver) between sonologists specifically in clinical
settings\textsuperscript{12}, an important point to note given the PUSH
study was conducted by trained and experienced radiologist within a
research protocol. US is likely best utilized clinically to compliment
serum biomarkers. This is highlighted by the proposed algorithm by
Sellars et al integrating conventional liver indices to guide when to
perform a liver ultrasound (US). They utilized GGT as an initial
screening test and further utilized APRI and GPR to determine when an
abdominal ultrasound is needed \textsuperscript{27}. This is supported
by the final PUSH multivariate analysis showing that the combination of
initial US pattern, age and GPR produced the best AUC in predicting the
development of a Nodular US \textsuperscript{60}.

\textbf{Elastography in evaluating CFHBI:}

With progressive fibrosis, the stiffness of the liver increases with
decreasing elasticity which can be utilized for screening using several
elastography techniques \textsuperscript{38}. A high cutoff for LSM
increases the specificity of diagnosis (ruling in) advanced liver
fibrosis and conversely a low cut-off provides a relatively higher
sensitivity for the evaluation of early stages of (minimal or no)
fibrosis. Common radiological methods include vibration-controlled
transient elastography (TE), ultrasound-guided elastography such as
point shear wave elastography (SWE) or 2D-shear wave elastography, and
magnetic resonance elastography (MRE) have been studied.

TE (FibroScan\selectlanguage{ngerman}®) has been utilized in the early evaluation and monitoring
of liver fibrosis in both children and adults with
CF\textsuperscript{39-42} Lewindon et al demonstrated that liver
stiffness measured by TE was significantly higher in children with CFHBI
(10.7 kPa, SD 2.4) when compared with children without CFHBI (4.6, SD
0.1) and healthy control population (4.1 kPa, SD
0.1)\textsuperscript{41}. Using 5.5 kPa as a screening cut-off for
CFHBI, the liver stiffness measurement (LSM) identified children with
CFHBI with an AUROC of 0.82, 70\% sensitivity and 82\% specificity (P
\textless{}0.0001). A higher cutoff of 8.7 kPa improved the specificity
of the diagnosis and differentiated children with aCFLD (diagnosis of
aCFLD based on ultrasound findings or by liver biopsy, stages F3-F4) vs
patients with early fibrosis (stages F1-F2), with an AUROC, 0.87; 75\%
sensitivity, 100\% specificity \textsuperscript{41}. Also, the combined
utility of APRI and LSM identified children with CFHBI with higher
diagnostic accuracy (AUROC of 0.89, 87\% sensitivity, and 74\%
specificity). Also, the combination of APRI and LSM improved the
differentiation of patients with severe fibrosis vs mild fibrosis
\textsuperscript{41}. Lam and colleagues did a meta-analysis by pooling
results from six studies (605 participants) and demonstrated an ideal
cut-off for LSM by TE and APRI as \selectlanguage{english}[?]5.95 kPa and \selectlanguage{english}[?]0.329 respectively
\textsuperscript{62} The sensitivity, specificity, and AUROC were 55\%,
87\%, 0.76 and 52\%, 93\%, and 0.84 for LSM and APRI respectively. When
both parameters were combined, the sensitivity, specificity, PPV, and
NPV were 43\%, 99\%, 92\%, and 87\% with a diagnostic odds ratio of 75.

Although encouraging, LSM by TE suffers from poor intra- and
interindividual variation. The poor repeatability of TE was highlighted
by Rowland and colleagues in healthy children (normal range of LSM was
as 2.88--6.52\selectlanguage{english} kPa) across various age categories \textsuperscript{63}. A
difference of \textgreater{}1kPa between paired measurements was noted
in approximately 26\% of the participants \textsuperscript{63}.
Therefore well agreed standard cut-off values for TE in the evaluation
of CFHBI are urgent needed.

Calvopina and colleagues demonstrated that LSM by supersonic SWE showed
good diagnostic accuracy in children with CFHBI \textsuperscript{64}.
LSM was significantly higher in CFHBI (8.1 kPa, IQR = 6.7--11.9) versus
without liver involvement (6.2 kPa, IQR = 5.6--7.0; P \textless{}
0.0001) and control population (5.3 kPa, IQR=4.9--5.8; P \textless{}
0.0001) \textsuperscript{64}. The diagnostic accuracy improved when
supersonic SWE was combined with APRI.

In a single-center study, both TE using FibroScan\textsuperscript{(r)} and
point SWE (Virtual Touch\textsuperscript{TM} Quantification) were
compared in children with CF for their repeatability and
reproducibility\textsuperscript{34}. The performance of both techniques
was good and investigators concluded that either one of the two methods
can be utilized for evaluating CFHBI \textsuperscript{34}. However, the
intraclass correlation was superior for TE than SWE for both intra- and
interobserver agreements \textsuperscript{28}. Unlike TE, the LSM
measurements between studies may not be comparable in difference studies
involving SWE as the results may vary based on the technology utilized
(point SWE vs 2D-SWE, probe and frequency (6Hz vs 9 Hz) differences, and
availability of different commercial brands \textsuperscript{65}.

MRE is the most accurate elastographic technique but its availability is
limited to research settings in tertiary care centers. MRE is expensive
and also poses technical challenges in young children, people with
severe obesity, and advanced lung disease \textsuperscript{66}. In a
single-center prospective study involving 55 percipients, all three (TE,
SWE, and MRE) elastography modalities were compared\textsuperscript{67}.
PwCF aged 6-18 years were included and based on US findings,
participants were grouped into three groups (aCFLD, heterogenous
increased echogenicity, and normal/homogeneous echogenicity). All tests
were done on the same day. LSM on all elastography methods was
significantly higher compared to the other two groups. TE and SWE were
highly correlated (r=0.9) and concordant in identifying aCFLD (Cohen's
k=0.84) while MRE was moderately correlated and concordant with TE
(r=0.41; k=36) and SWE (r=0.5; k=0.50)\textsuperscript{67}. Even though
these elastography techniques have shown good reliability in the
evaluation of early diagnosis of significant liver fibrosis, their
effect on longitudinal monitoring of disease progression is lacking, and
further studies are awaited\textsuperscript{1}.

\textbf{Spleen elastography:}

Increased spleen stiffness measured by elastography methods has been
utilized in both children and adults with advanced liver disease and may
help to predict the severity of portal
hypertension\textsuperscript{68,69}. In a single-center study involving
children, higher swear wave velocity was noted in CF than in control
population.\textsuperscript{70}. But no differences in swear wave
velocity were noted in CF children with or without liver involvement and
further studies are needed to explore its utilization in the early
stages of aCFLD \textsuperscript{70}.

\textbf{Radiological methods conclusions:}

Greyscale US is often clinically utilized in evaluation of aCFLD due to
wide availability and relatively low cost. However, it lacks the
sensitivity to detect early stages of aCFLD reliably and also has high
percentage of interpretation variabilities (both intra- and
interobserver) among radiologists. US-based elastographies (TE and SWE)
are relatively readily available, technically easier to perform, and
cost effective. LSM obtained by various elastography techniques (TE,
SWE, and MRE) are modality-dependent, and cannot be compared directly to
one another. Also, these radiological tests are best utilized to
complement the serum biomarkers in the evaluation of CFHBI. Finally,
spleen elastography may be advantageous to evaluate portal hypertension,
with or without cirrhosis, but its availability is limited and
additional CF-specific studies are needed.

\textbf{Genetics in the evaluation of CFHBI} :

While CFHBI has been associated with pancreatic insufficiency, history
of meconium ileus and CFRD, no clear genotype association has been
established \textsuperscript{7}. While some studies have shown F508del
homozygosity was associated with aCFLD, it is unclear if that reflects
the frequency of the mutation rather a true risk factor. It is generally
believed that severe mutations (class 1-3) are associated with
aCFLD\textsuperscript{71}, but given the relatively low incidence rate
among patients with two severe mutations, it has long been believed that
additional genetic modifiers contribute to aCFLD.

To date, only the SERPINA1 Z allele has been associated with the
development of aCFLD \textsuperscript{72,73}, but only accounts for a
small portion of those with aCFLD. However, SERPINA1 has since been
shown to be an independent risk factor for advanced liver disease in a
number of chronic liver conditions \textsuperscript{74}, and thus
unlikely to be a CF-specific modifier. In silico modeling has also
supported the idea that additional genetic modifiers beyond SERPINA1 Z
may contribute to the development of aCFLD
\textsuperscript{75}Currently, both the PUSH study team and researchers
for the CF Foundation are conducting studies to better understand the
role of genetic modifiers in CFLD (Clinical Trials NCT01144507 and
NCT00804583).

\textbf{Genetic modifiers conclusion:}

To date, only the SERPINA1 Z allele has been associated with aCFLD but
only accounts for a small portion of the population, and is unlikely to
be CF-specific. Additional work is underway to better understand the
potential genetic modifiers associated with CFHBI and aCFLD.

\textbf{Conclusions:}

Many questions remain as to how best to predict the onset of aCFLD. It
is unlikely a single test will reliably detect or diagnose CFHBI, but
rather a combination of tests with an algorithmic approach (e.g. blood
tests + radiological studies) relying on changes over time rather than
strict cut-offs. Elastography methods are currently gaining rapid
momentum and soon will likely be incorporated in routine clinical
settings. Future methods also include the incorporation of newer
biomarkers and other upcoming modalities such as multi-omics, and
artificial intelligence (AI)-driven protocols\textsuperscript{38,70,76}.

\textbf{Table 1} -- Summary of non-invasive tests utilized in cystic
fibrosis-related hepatobiliary involvement\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Tests & Advantages & Limitations & References\tabularnewline
\midrule
\endhead
Blood tests -- AST, ALT, GGT, AST to platelet ratio index (APRI),
fibrosis index-4 (FIB-4), GGT to platelet ratio, and AST/ALT ratio &
Inexpensive, readily available, no failure rate & Non-specific changes
in liver enzymes or platelets values (hepatic congestion from
cardiorespiratory failure, hepatic inflammation secondary due to drug
induced injury, systemic inflammation) can confound the values. Low
sensitivity and specificity in early stages of aCFLD &
\textsuperscript{1,11,29}\tabularnewline
Direct biomarkers & Noninvasive, no failure rate & Utilized mostly in
research studies and not available widely in clinical settings. Results
can be confounded by advanced lung disease &
\textsuperscript{24,51,52}\tabularnewline
US & Noninvasive, available in most centers & Intra- and interobserver
variabilities exist among radiologists &
\textsuperscript{12,44,58,60}\tabularnewline
TE & Noninvasive, simple, and can be performed by non-radiology
clinicians & Factors such as hepatic inflammation, venous congestion,
ascites, steatosis, and fasting status can interfere with the
interpretation & \textsuperscript{39-42,66}\tabularnewline
SWE & Noninvasive, simple & Similar limitations as TE. Also needs
expertise in radiology & \textsuperscript{28,64}\tabularnewline
MRE & More accurate and reliable than TE or SWE. The entire liver can be
evaluated with lower sampling error. Can be performed even in patients
with ascites & May need sedation in young children, expensive and not
widely available for clinical use. Failure rare \textless{} 5\% due to
inability to breath hold or motion artefacts &
\textsuperscript{28,77}\tabularnewline
Liver biopsy & Considered as the gold standard test for evaluating
hepatic fibrosis & Invasive, the need for sedation, is expensive, have
complications such as pain, bleeding, and tissue sample may
under-represent or over-represent the disease. Intra- and interobserver
variabilities also do exist among pathologists &
\textsuperscript{4,24,26}.\tabularnewline
\bottomrule
\end{longtable}

aCFLD - advanced cystic fibrosis associated liver disease; US --
ultrasound; TE - transient elastography; SWE - shear wave elastography;
MRE - magnetic resonance elastography

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