Digestive and Liver Disease 49 (2017) 947–956

Contents lists available at ScienceDirect

Digestive and Liver Disease

journal homepage: www.elsevier.com/locate/dld

Review Article

Clinical usefulness of serum antibodies as biomarkers of

gastrointestinal and liver diseases
Antonio Di Sabatino a,∗ , Federico Biagi a , Marco Lenzi b , Luca Frulloni c ,
Marco Vincenzo Lenti a , Paolo Giuffrida a , Gino Roberto Corazza a
a
First Department of Internal Medicine, San Matteo Hospital Foundation, University of Pavia, Pavia, Italy
b
Department of Medical and Surgical Sciences, Sant’Orsola-Malpighi, University of Bologna, Bologna, Italy
c
Department of Medicine, Pancreas Center, University of Verona, Verona, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The progressively growing knowledge of the pathophysiology of a number of immune-mediated gas-
Received 2 April 2017 trointestinal and liver disorders, including autoimmune atrophic gastritis, coeliac disease, autoimmune
Received in revised form 5 June 2017 enteropathy, inflammatory bowel disease, autoimmune hepatitis, primary sclerosing cholangitis, pri-
Accepted 7 June 2017
mary biliary cholangitis and autoimmune pancreatitis, together with the improvement of their detection
Available online 23 June 2017
methods have increased the diagnostic power of serum antibodies. In some cases – coeliac disease and
autoimmune atrophic gastritis – they have radically changed gastroenterologists’ diagnostic ability, while
Keywords:
in others – autoimmune hepatitis, inflammatory bowel disease and autoimmune pancreatitis – their diag-
Autoimmune atrophic gastritis
Autoimmune liver disease
nostic performance is still inadequate. Of note, serum antibody misuse in clinical practice has raised a
Autoimmune pancreatitis number of controversies, which may generate confusion in the diagnostic management of the aforemen-
Coeliac disease tioned disorders. In this review, we critically re-evaluate the usefulness of serum antibodies as biomarkers
Inflammatory bowel disease of immune-mediated gastrointestinal and liver disorders, and discuss their pitfalls and merits.
© 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

1. Introduction by diseases instead of by biomarkers, even if serum antibodies are

The expansion of the pathogenic knowledge of immune-
mediated gastrointestinal and liver diseases, including autoim-
mune atrophic gastritis (AAG), coeliac disease (CoeD), inflamma-
tory bowel disease (IBD), autoimmune hepatitis (AIH), primary
sclerosing cholangitis (PSC), primary biliary cholangitis (PBC) and
autoimmune pancreatitis (AIP), has led to a deeper awareness of
the clinical management of these conditions [1–4]. In particular,
serum antibodies have increased our diagnostic power in most of
the aforementioned disorders (Tables 1 and 2), although their mis-
use has often led to diagnostic mistakes thus generating a number
of controversies regarding their accuracy and appropriate use in
real life. Fig. 1 shows the accuracy of serum antibodies according
to the figures commonly reported in the literature.
On this basis, we aimed to critically re-evaluate the usefulness
of serum antibodies as biomarkers in immune-mediated gastroin-
testinal and liver disorders, and to discuss both their pitfalls and
merits. For clarity, we decided to divide the review and discussion
∗ Corresponding author at: Clinica Medica I, Fondazione IRCCS Policlinico San Mat-
teo, Università di Pavia, Piazzale Golgi 19, 27100 Pavia, Italy. Fax: +39 0382 502618.
E-mail address: a.disabatino@smatteo.pv.it (A. Di Sabatino).
the core of the review.
2. Autoimmune atrophic gastritis
AAG is an organ-specific disease which affects the corpus-
fundus mucosa of the stomach, causing hypo-achlorhydria,
deficiency of vitamin B12 and iron [5,6], and may predispose to gas-
tric adenocarcinoma or type I neuroendocrine tumour [7,8]. Specific
clinical symptoms may be absent for many years [9], and anti-
parietal cell antibodies (PCA) and anti-intrinsic factor antibodies
(IFA) could be potentially helpful in the diagnosis of this condition
(Table 1) [10,11].
PCA are directed against the ␣ and ␤ subunit of gastric H+ /K+
adenosine triphosphatase [12–14], and they can be identified
by either immunofluorescence or enzyme-linked immunosorbent
assay (ELISA), the latter being more accurate than the former
[15,16]. How to interpret serum PCA positivity in the absence of gas-
tric atrophy is still under debate [17–19]. We recently followed-up
58 patients with serum PCA positivity and normal gastric mucosa,
and we observed that thirteen of them subsequently developed
atrophy over a median 30-month follow-up period [20]. Of course,
further studies are needed to verify whether the isolated PCA pos-

http://dx.doi.org/10.1016/j.dld.2017.06.010
1590-8658/© 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
948 A. Di Sabatino et al. / Digestive and Liver Disease 49 (2017) 947–956

Table 1
Serum antibody biomarkers of immune-mediated gastrointestinal disorders.

Antibody Acronym Disease Target Detection method

Anti-gastric parietal cell PCA Autoimmune atrophic gastritis H+/K+ ATPase ELISA
Anti-intrinsic factor IFA Intrinsic factor ELISA, IB

Anti-tissue transglutaminase TTA Coeliac disease Tissue transglutaminase ELISA, CLIA

IgA
Anti-endomysial IgA EMA Tissue transglutaminase IF
Anti-deamidated gliadin DGP Deamidated gliadin-related peptide ELISA
peptide IgA

Anti-enterocyte EA Autoimmune enteropathy Enterocyte IF

Anti-Saccharomyces cerevisiae ASCA Crohn’s disease Yeast mannan IF, ELISA

Perinuclear anti-neutrophil pANCA Ulcerative colitis Neutrophil cytoplasm IF

cytoplasmic

Abbreviations: CLIA, chemiluminescence immunoassay; ELISA, enzyme linked immunosorbent assay; IB, immunoblot immunoassay; IF, immunofluorescence; Ig, immunoglob-
ulin; tRNA, transfer ribonucleic acid; UGA, uracil–guanine–adenine.

Table 2
Serum antibody biomarkers of immune-mediated liver disorders.

Antibody Acronym Disease Target Detection method

Anti-nuclear ANA Autoimmune hepatitis Heterogeneous IF

Anti-smooth muscle SMA Filamentous actin IF
Anti-liver kidney microsomal LKM Cytochrome P450 IF
Anti-soluble liver SLA/LP UGA serine tRNA-associated ELISA
antigen/liver-pancreas protein complex
Anti-liver cytosol 1 LC1 Formimino-transferase IF
cyclodeaminase (FTCD)

Perinuclear anti-neutrophil pANCA Primary sclerosing cholangitis Neutrophil cytoplasm IF

cytoplasmic

Anti-mitochondrial AMA Primary biliary cholangitis Mitochondria IF

Immunoglobulin G4 IgG4 Autoimmune pancreatitis Pleiotropic Nephelometry

Abbreviations: ELISA, enzyme linked immunosorbent assay; IF, immunofluorescence; Ig, immunoglobulin; tRNA, transfer ribonucleic acid; UGA,uracil–guanine–adenine.

Fig. 1. Schematic representation of the accuracy of the most commonly used serum antibodies in the diagnosis of immune-mediated gastrointestinal and liver diseases.
Accuracy is calculated as the average of sensitivity and specificity by using the best available tests in the literature. AEA, anti-enterocyte antibody; AGA, anti-gliadin antibody;
AMA, anti-mitochondrial antibody; ANA, anti-nuclear antibody; ASCA, anti-Saccharomyces cerevisiae antibody; DGP, anti-deamidated gliadin peptide antibody; EMA, anti-
endomysial antibody; GCA, anti-goblet cell antibody; IFA, anti-intrinsic factor antibody; Ig, immunoglobulin; LC1, anti-liver cytosol 1 antibody; LKM1, anti-liver kidney
microsomal antibody 1; pANCA, perinuclear anti-neutrophil cytoplasmic antibody; PCA, anti-gastric parietal cell antibody; SLA/LP, anti-soluble liver antigen/liver-pancreas
antibody; TTA, anti-tissue transglutaminase antibody.
 A. Di Sabatino et al. / Digestive and Liver Disease 49 (2017) 947–956
itivity could be considered a hallmark of “potential AAG”. A few
considerations regarding the value of PCA in clinical practice should
be made. Firstly, there is evidence from a single study [17] that
serum PCA titres may fluctuate along the natural course of AAG,
becoming negative in patients with longstanding disease, thus
affecting PCA accuracy in late AAG. In these cases, if the clinical sus-
picion is high, other laboratory parameters and histology should
be taken into account before ruling out AAG diagnosis. In partic-
ular, in a previous study we designed and validated a laboratory
score model that evaluates serum basal 17-gastrin, haemoglobin
and mean cell volume, able to detect AAG with a high accuracy
[21]. Furthermore, the concern that Helicobacter pylori infection
might affect PCA accuracy [22,23] was not confirmed by a recent
large study [24]. Although there are no studies assessing the perfor-
mance of PCA in a case-finding strategy, the relevant association of
AAG with other autoimmune disorders [9,17,18,25,26] might make
it worth using serology to screen patients with autoimmune thy-
roiditis, type I diabetes, Addison’s disease and vitiligo. In addition,
the value of PCA in patients with megaloblastic anaemia has been
widely investigated and confirmed [27,28].
With regard to IFA, they can be classified in two types, i.e. type
I preventing vitamin B12 from binding to intrinsic factor [29], and
type II preventing the ileal absorption of vitamin B12 –intrinsic fac-
tor complex [30]. IFA, even detected by ELISA, due to their low
sensitivity (<30%) have limited clinical usefulness. However, in
patients presenting with pernicious anaemia, a late finding in AAG
[27], IFA may have a role in increasing diagnostic accuracy when
combined with PCA [15].
3. Coeliac disease
The discovery of serum antibodies specific for CoeD is certainly
the key factor that revolutionized our knowledge of this condition
(Table 1). Formerly, CoeD was considered to be a rare disease, affect-
ing almost exclusively children, and to be taken into account only
in the case of severe malabsorption symptoms. Thanks to “coeliac
serology” we know that CoeD is very frequent, it affects adults as
much as children, and its clinical presentation is extremely variable
[4,31].
The first serum antibodies discovered to be specific for CoeD
were R1 reticulin antibodies, identified in 1971 [32]. They were
detected by standard indirect immunofluorescence on cryostat sec-
tion of rodent tissues, stomach, liver and kidney. Although some
authors reported remarkably good levels of sensitivity and speci-
ficity [33], in everyday clinical practice the results were more
disappointing and they never became a test used world-wide. The
era of coeliac serology kicked off in the early 1980s with the discov-
ery of ELISA anti-gliadin antibodies (AGA) and immunofluorescent
anti-endomysial antibodies (EMA) [34,35]. In 1997, the discovery
of the role of tissue transglutaminase 2 (TG2) in the pathogene-
sis of CoeD made it possible not only to detect EMA by means
of an ELISA technique, i.e. anti-tissue transglutaminase antibod-
ies (TTA), but also to detect deamidated gliadin peptide antibodies
(DGP) [36,37]. Immunofluorescence IgA anti-actin and ELISA anti-
glutenin antibodies were also investigated [38,39]. A total of seven
different types of coeliac antibodies have been investigated so far.
However, it must be stressed that nowadays only EMA, TTA and DGP
are the antibodies that need to be taken into account. Anti-reticulin
and AGA are now obsolete, have exhausted their role and should be
abandoned. Anti-actin and anti-glutenin antibodies never managed
to get beyond the experimental level and enter into clinical prac-
tice, although IgA anti-actin antibodies were shown to correlate
with histological damage.
EMA are directed against TG2 in soft connective tissues sur-
rounding smooth muscle fibres, the so-called endomysium. They
949
are detected with traditional immunofluorescence on cryostat sec-
tions of monkey oesophagus, monkey jejunum or human umbilical
cord. When detected on monkey jejunum they are defined jejunum
antibodies. Although the very first studies clearly showed that EMA
of IgA class are the relevant ones and IgG EMA should be taken into
account only in patients with IgA deficiency [35,40], it is nowadays
quite common to have patients tested for both IgA and IgG EMA “in
case there is an unknown IgA deficiency”. The problem with this
strategy is that the sensitivity and specificity of IgG EMA in subjects
with normal IgA levels are rather low. So, immunocompetent sub-
jects with false positive IgG EMA are more common than patients
with unknown IgA deficiency and true positive IgG EMA. There-
fore, testing for total IgA should be part of the serological search
for CoeD, and only when total IgA deficiency is detected should
IgG EMA be performed [41,42]. The sensitivity of EMA has been
reported to be around 95%, and since EMA specificity is close to
100%, a positive patient with a normal duodenal biopsy implies a
diagnosis of potential CoeD [43].
As stated before, TG2 is the antigen of EMA and this allowed TTA
detection by means of an ELISA technique [36]. Therefore, although
they are defined with two different names, EMA and TTA are exactly
the same antibodies. Again, only TTA of IgA class have a diagnostic
role and TTA of IgG class should be considered only in the case of
IgA deficiency. An ELISA technique is obviously cheaper to perform
and does not require specific training for the operator. However, we
do not agree with those authors who suggest that EMA detection
is subjective and operator-dependent, and that TTA are more reli-
able because they provide a numerical result [48]. An EMA positive
pattern is not just “stronger” than a negative one but it is different.
We could say that to distinguish positive EMA from negative ones
it is like distinguishing the Eiffel tower from the leaning tower of
Pisa: a glance is enough and we do not need any measurement, any
number to distinguish between them!
The sensitivity of TTA is maybe even higher than that of EMA
but the specificity is not 100%. The main problem is represented by
“low positive” TTA that are often unrelated with CoeD. We showed
a few years ago that there is a very good relationship between TTA
optical density and EMA titres, though this relationship tends to
be weaker at low EMA titres and at low TTA optical density [44].
Since, in our experience, low positive TTA are among the causes of
misdiagnosing CoeD [45], we strongly recommend that weak TTA
should always be confirmed by EMA. In children, even strong TTA
need to be confirmed by EMA according to the latest ESPGHAN cri-
teria, which allow the diagnosis of CoeD without duodenal biopsy.
This can be done providing that TTA are higher than 10 times the
normal value, EMA are found positive in a second serum sample,
HLA-DQ2 and/or DQ8 are positive, clear-cut symptoms consistent
with CD are present, and there is a response to a gluten-free diet
[46]. Finally, the observation that during an infectious disease TTA
can be produced temporarily and independently of gluten further
supports the importance of confirming TTA-positivity with EMA
[47].
As regards DGP, similarly to old AGA, both DGP of IgG and IgA
class should be taken into account. Since the sensitivity of EMA/TTA
tends to be lower in children below 3 years of age, DGP have been
proposed as the serological tool of choice in this age group [48].
The proper use of coeliac antibodies in adulthood should be
based on the pre-test probability of CoeD. Screening of the gen-
eral population, i.e. mass screening, should be performed with TTA
first, followed by EMA on TTA-positive samples and then duodenal
biopsy. In at risk groups with an expected prevalence of CoeD rang-
ing from 5 to 10% (case-finding strategy), patients should undergo
either EMA or TTA determination followed by duodenal biopsy
in those who test positive. Finally, patients with frank symptoms
of malabsorption and unexplained malnutrition should undergo
950 A. Di Sabatino et al. / Digestive and Liver Disease 49 (2017) 947–956
Fig. 2. Strong positivity of anti-enterocyte antibodies (white arrows) detected by
indirect immunofluorescence on cryostat sections of monkey jejunum (brush border
and enterocyte cytoplasm). Original magnification: ×250.
upper endoscopy with duodenal biopsies in spite of the EMA/TTA
result [41,49].
Tons of papers have been written on the use of coeliac anti-
bodies in the follow-up of CoeD and for checking gluten-free diet
adherence. Although they are reported as a satisfactory tool in some
papers [50,51], they are not as reliable as they are for diagnosis in
monitoring dietary adherence or histologic response [52–55]. Con-
versely, we showed that coeliac antibodies can persist despite strict
dietary adherence and good histological response. More precisely,
when histological response was used as the gold standard, the sen-
sitivity and specificity of the serological response was 48% and 71%,
respectively [56].
Common variable immunodeficiency is a condition that can
present subtotal villous atrophy that does not always respond to a
gluten-free diet. So, proving that these patients are also affected by
CoeD can be a real challenge. We have recently shown that CoeD
serology has no role in these patients and can even be mislead-
ing [57]. Therefore, the histological response to a gluten-free diet,
together with the presence of DQ2 or DQ8 molecules, remains the
only diagnostic criterion for CoeD in these patients.
4. Autoimmune enteropathy
Autoimmune enteropathy (AIE) is a chronic enteropathy first
described in children [58] and then in adults [59]. According to
Unsworth & Walker-Smith [58], the diagnosis had to be based
on subtotal villous atrophy refractory to any dietary exclusion,
anti-enterocyte antibodies (EA) and/or associated autoimmune
conditions, and absence of significant immunodeficiency. EA are
therefore the serological marker of this condition (Table 1). They are
detected by indirect immunofluorescence on cryostat sections of
monkey or human jejunum where they bind to the cytoplasm and,
to a lesser extent, the brush border of villi and crypt enterocytes
(Fig. 2).
AIE is really very rare. To figure out how rare it is, it should
be sufficient to say that we described the first two adult cases in
1997 [59]. Since then we have been actively looking for an AIE that
represents an important differential diagnosis with CoeD and its
complications. Although we are a referral centre for CoeD and we
use monkey jejunum on a regular basis to look for EMA, only three
more patients have been diagnosed in the last 20 years. The rarity
of the condition is therefore the first obstacle to estimate the accu-
racy of EA for AIE, but it is not the only one. A second problem is
linked to the diagnostic criteria themselves. Although those origi-
nally proposed by Unsworth and Walker-Smith [58] work perfectly
well for children, we are not sure that this is also true for adults.
In children, refractory CoeD simply does not exist, and so there
is no problem of differential diagnosis with AIE. This is obviously
not the case in adults where refractoriness and other complica-
tions of CoeD are a major problem. Since associated autoimmune
conditions are very frequent in CoeD, EA are the only tool that can
be used to discriminate between adult AIE and refractory CoeD.
So, sensitivity of EA would inevitably be 100%. After having tested
serum samples from more than 2200 patients undergoing duode-
nal biopsy, we think that the specificity of EA for AIE is virtually
100%. In children, in whom the diagnosis does not require posi-
tive EA, the highest sensitivity (91%) was obtained in 12 children
affected by immune dysregulation, polyendocrinopathy, enteropa-
thy, or X-linked disease, which is a syndromic form of AIE [60].
However, EA specificity in AIE is still under debate, EA being said to
be positive in more than two-thirds of human immunodeficiency
virus-infected patients with chronic diarrhoea [61]. We think that
these discrepant results are due to the lack of immunofluorescent
diagnostic criteria for EA. To look for EA is difficult. We would say
that it is like distinguishing a black cat from a white one. It seems to
be easy and indeed it is easy in the most obvious cases (Fig. 2), but
sooner or later we will come across a grey cat, i.e. an intermediate
pattern difficult to define as either black or white. So, on the basis of
our experience, we recommend that only very bright patterns like
those in Fig. 2 should be considered to be positive EA. Weak fluo-
rescent staining of enterocytes, especially in the crypts, are on the
other hand very common and totally non-specific. High EA titres
do not correlate with histological severity and they might appear
late during AIE natural history [62–65]. Finally, whether EA have a
pathogenic role or they represent only a secondary phenomenon is
still under debate.
Other authors based the diagnosis of AIE on positive anti-goblet
cell antibodies (GCA) [66]. We have clearly shown that GCA are
totally non-specific, and are very frequent even in controls with
normal villi [67]. Their use for the diagnosis of AIE should be dis-
couraged.
5. Inflammatory bowel disease
Serum antibodies detectable in IBD patients can be classified
into three main groups, namely autoantibodies directed against
autoantigens [1], anti-microbial antibodies against the luminal
microbiota [68], and anti-drug antibodies (ADA), which target
anti-tumour necrosis factor (TNF) antibodies or anti-␣4 ␤7 anti-
body vedolizumab [69,70]. The first class includes perinuclear
anti-neutrophil cytoplasmic antibodies (pANCA), which exhibit a
non-granular distribution [71] and have a sensitivity of 52% and
a specificity of 91% in differentiating ulcerative colitis (UC) from
Crohn’s disease (CrD) [72]. Moreover, the small group of pANCA-
positive CrD patients have endoscopic, pathological and clinical
features of left-sided colitis, thus indicating a predominant UC phe-
notype [73]. Among the subgroup of pANCA-positive IBD patients,
the anti-bacterial flagellin CBir1 antibodies are expressed in 44% of
CrD patients and only in 4% in UC cases, and this may help in dis-
criminating UC from CrD [74]. Negativity for pANCA in UC predicts
an early response to the anti-TNF monoclonal antibody infliximab
[75], while its positivity predicts the development of chronic pou-
chitis after ileal pouch-anal anastomosis [76].
In a meta-analysis on 14 studies performed in Europe, Israel and
Canada [77], serum levels of the anti-glycan anti-Saccharomyces
cerevisiae antibodies (ASCA) have a sensitivity of 56% and a speci-
ficity of 88% in discriminating CrD from UC. ASCA-positive CrD
patients have a higher risk of disabling disease, including strictur-
ing and penetrating behaviour, earlier onset and perianal disease,
resulting in an increased need for surgery [77,78]. A recent study
 A. Di Sabatino et al. / Digestive and Liver Disease 49 (2017) 947–956
Fig. 3. Algorithm for optimization of anti-tumour necrosis factor (TNF) therapy in
inflammatory bowel disease patients depending on serum anti-TNF antibody levels
(therapeutic range: 3–7 ␮g/ml) and the presence or absence of anti-drug antibodies
(ADA).
showed that faecal ASCA have the same sensitivity (52%) but
a lower specificity (71%) compared to serum ASCA (87%) in a
paediatric cohort of 83 CrD patients [79]. Apart from ASCA, five
further serum anti-glycan antibodies, i.e. anti-chitobioside carbo-
hydrate IgA (ACCA), anti-mannobioside carbohydrate IgG (AMCA),
anti-laminaribioside IgG (ALCA), anti-laminarin carbohydrate anti-
bodies (anti-L), and anti-chitin carbohydrate (anti-C), have been
detected in the serum of IBD patients. It has been shown that
serum levels of all the anti-glycan antibodies remain stable over
six years of follow-up in most patients [80]. In general, each anti-
glycan antibody has a good specificity and positive predictive value
in discriminating CrD from UC [80]. CrD patients positive for ASCA,
AMCA and anti-L have a higher risk of complications, whereas anti-
C is strongly associated with IBD-related surgery [80]. ALCA or
ACCA were detected in 44% of a small cohort of ASCA-negative CrD
patients from Israel, increasing the sensitivity and specificity up to
77% and 90%, respectively, in CrD patients positive for at least one
out of the three antibodies, ALCA, ACCA and ASCA [77]. On the other
hand, positivity for anti-Escherichia coli outer membrane protein
C antibodies (anti-OmpC), which are frequently associated with a
disabling disease course and surgery in CrD [81], are not associ-
ated with IBD in ASCA-negative individuals [82]. A recent study on
twins with CrD showed a high degree of similarity in anti-OmpC and
in anti-Pseudomonas fluorescens-associated peptide I2 antibodies
(anti-I2) in discordant monozygotic twin pairs, but not in discor-
dant dizygotic twin pairs, suggesting that both anti-OmpC and
anti-I2 stand for a genetically determined loss of tolerance [83]. In
addition, over six years before the diagnosis in 65% of CrD patients,
at least one of ASCA IgA, ASCA IgG, anti-OmpC, anti-CBir1, and two
other anti-flagellin antibodies, anti-Fla2 and anti-FlaX, is positive
[84]. Similarly, a panel of serum antibodies, including pANCA, ASCA
IgA, ASCA IgG, anti-OmpC, anti-CBir1, has been shown to predict the
diagnosis of both CrD and UC in the previous four years [85]. Anti-
microbial antibodies are clinically useful in predicting a disabling
disease course in CrD, but not in discriminating between stricturing
and non-stricturing phenotypes [86].
Due to its chimeric nature and consequent high immunogenic-
ity, the monoclonal anti-TNF antibody infliximab may induce the
development of ADA, generally within the first 12 months of
therapy [87]. ADA, whose appearance may be delayed by concomi-
tant administration of immunosuppressant drugs (i.e. thiopurines),
induce and frequently precede the loss of response to infliximab
[87]. Conversely, transient ADA may appear randomly during inflix-
imab therapy, but have little clinical impact [87]. Although ADA
against the fully human anti-TNF antibody adalimumab are not
951
so frequent in IBD patients (from 2 to 44%) [88,89], their titres
inversely correlate with adalimumab concentration and, hence,
high amounts of ADA are associated with disease activity due to
their drug inactivation and clearance in CrD [90]. In the case of
loss of response, a number of different strategies may be adopted
on the basis of the presence or absence of ADA positivity and
according to drug trough levels (Fig. 3) [91]. However, it has been
proposed that adequate serum trough levels (3–7 ␮g/ml) do not
necessarily reflect translated amounts of anti-TNF agents sufficient
to neutralize all the TNF present in IBD mucosa [92]. Until now,
no study has been conducted on ADA directed against golimumab
in UC patients. Similarly, treatment with the humanized mono-
clonal antibody vedolizumab is associated with the development
of ADA in 4% of patients [70]. According to our recent demonstra-
tion that increased amounts of matrix metalloprotease-3 and -12
in active IBD mucosa may cleave not only anti-TNF agents but also
endogenous IgG at hinge region level, raised serum levels of matrix
metalloproteinase-cleaved endogenous IgG and anti-hinge autoan-
tibodies against neo-epitopes of cleaved IgG may be considered as
predictors of poor response to anti-TNF therapy [93].
In conclusion, none of the aforementioned autoantibodies cur-
rently have enough accuracy to justify their use in day-to-day
clinical practice, whereas ADA appear to be a useful tool in the
clinical management of IBD patients exposed to biologic agents.
6. Autoimmune liver disease
Autoimmune serology has a central role in the diagnosis and
classification of autoimmune liver disease (Table 2). However,
there are a number of controversial issues as most autoantibodies
characteristically associated with autoimmune liver disease lack
specificity and pathogenic significance. Indirect immunofluores-
cence is the main technique for routine autoantibody testing [94].
It is based on the use of a freshly frozen rodent substrate that usu-
ally includes kidney, liver and stomach, a combination that allows
the simultaneous detection of anti-nuclear antibodies (ANA), anti-
smooth muscle antibodies (SMA), anti-liver kidney microsomal
antibody (LKM) 1, anti-mitochondrial antibody (AMA), and anti-
liver cytosol 1 (LC1), if LKM1 is absent. In adults, significant titres are
≥1:40 dilution by indirect immunofluorescence. In children, titres
of 1:20 for ANA or SMA and 1:10 for LKM1 are supportive of the
diagnosis of AIH when used in combination with other laboratory
tests and clinical features suggestive of the disease. However, the
indirect immunofluorescence technique has many drawbacks, as it
is time consuming, requires an experienced observer, and is insuf-
ficiently standardized. Commercially available substrates are used
in routine laboratory practice, but their quality varies. These sub-
strates are treated with fixatives in order to lengthen their shelf life,
but this also causes enhanced background staining, which might
cause difficulties in the interpretation of fluorescence patterns and
can explain the low reproducibility of the test. Methods other than
indirect immunofluorescence, such as ELISA or immunoblotting,
are gaining popularity. This shift has been supported by the intro-
duction of assays based on recombinant/purified target antigens,
such as cytochrome P 450 2D6-CYP2D6, formimino-transferase
cyclo deaminase (FTCD), soluble liver antigen/liver pancreas, and
filamentous actin-F-actin. However, the use of ELISA as the sole
primary screening test is inappropriate because there is no useful
combination of molecular specificities for a dependable detection
of ANA and SMA, while the results are interchangeable with indi-
rect immunofluorescence for those autoantibodies (AMA, LKM1
and LC1) whose target antigen has been identified at molecular
level [95,96].
Autoantibody titres and specificity may vary during the course of
the disease, and seronegative individuals at diagnosis may express
952 A. Di Sabatino et al. / Digestive and Liver Disease 49 (2017) 947–956
the conventional autoantibodies later in the disease course [97,98].
In these patients, repeated testing allows autoantibody detec-
tion, thus correct disease diagnosis and classification [94,96,99]. In
adulthood, autoantibody titres correlate poorly with disease activ-
ity, clinical course and treatment response [100], and do not need
to be monitored regularly, unless a significant change in the clini-
cal phenotype appears. On the contrary, in childhood autoantibody
titres may be useful biomarkers of disease activity, and they can be
used to monitor treatment response [101]. In particular, LC1 have
been demonstrated to correlate well with disease activity show-
ing a significant decrease in titre (>50%) or disappearance during
remission and flare-up during relapse [102].
6.1. Autoimmune hepatitis
ANA and SMA, markers of type 1 AIH, which account for about
75% of patients [103,104], are not disease-specific and show a wide
range of heterogeneity in terms of antigenic specificity. The fluores-
cence pattern of ANA in AIH is usually homogeneous using HEp-2
cells, but a speckled pattern is rather frequent. ANA-positivity
is found in 43% of type 1 AIH patients [105], and is associated
with a variety of antigenic specificities including histones, double-
stranded DNA (15%), chromatin and ribonucleoprotein complexes.
However, no single pattern or combination is pathognomonic of
AIH. Thus, the investigation of different ANA staining patterns
has no clinical or diagnostic relevance in routine practice, and
the use of HEp2 cells at AIH screening stage is not recommended
[106–108]. SMA react to several cytoskeletal elements including
F-actin with a reported prevalence of anti-actin antibodies in 41%
of patients. When kidney sections are used as a substrate for indi-
rect immunofluorescence, SMAvg (vessel/glomerulus) and SMAvgt
(vessel/glomerulus/tubule) patterns can be identified, which are
frequently associated with AIH, though not pathognomonic. SMAgt
correlate with F-actin antigenicity [106]. In the diagnostic work-up
for AIH, SMA/anti-actin antibody testing is appropriate and may
also be done by ELISA [107,108]. However, indirect immunofluo-
rescence remains superior to ELISA and provides the best accuracy.
Indeed, actin is not the only target antigen of AIH-specific SMA reac-
tivity, and thus ELISA can miss the diagnosis in about 20% of cases
[109–111]. ANA and SMA reactivity frequently coexist in the same
serum, thus improving the strength of the diagnosis.
LKM1 and/or LC1 are the serologic markers of type 2 AIH. The
two antibodies often coexist in the same serum, but in some cases
LC1 is present alone and is the only marker for the diagnosis of
type 2 AIH. The major target autoantigen of LKM1 has been clearly
identified as the cytochrome P4502D6 (CYP2D6) and the FTCD for
LC1. However, neither LKM1 nor LC1 are highly disease-specific, as
they have been described in a small proportion (5–10%) of adult
and paediatric patients with chronic HCV infection [97,112–115].
Anti-soluble liver antigen/liver-pancreas antibodies (SLA/LP) are
the only disease-specific biomarker. The target antigen has been
identified as a synthase (S) converting O-phosphoseryl-tRNA (Sep)
to selenocysteinyl-tRNA (Sec), named as SepSecS [116,117]. This
has led to the development of reliable commercial assays for SLA/LP
detection (ELISA and dot-blot) [118]. SLA/LP is detected in approx-
imately 30% of patients with AIH (particularly in type 1 AIH) and is
often associated with anti-Ro52 antibodies [116–119], but some-
times it is the only detectable autoantibody reactivity. SLA/LP has
never been described in association with LKM1 and LC1 in type
2 AIH. The positivity of pANCA, originally considered specific for
PSC and IBD, is also frequently present in patients with type 1 AIH
[120,121], and it can be used as an additional element support-
ing the diagnosis of AIH, particularly if other autoantibodies are
negative [94,99]. A recent meta-analysis indicated that ANA have
moderate sensitivity and specificity, SMA have moderate sensitiv-
ity and high specificity, and SLA/LP have low sensitivity and high
specificity [122].
6.2. Primary biliary cholangitis
AMA at titres higher than 1:40 have a high sensitivity and speci-
ficity for the diagnosis of PBC, being present in 90–95% of patient
sera. AMA are in fact one of the diagnostic criteria of PBC along with
elevated alkaline phosphatase and a compatible liver histology
[122,123]. AMA are typically detected by indirect immunofluores-
cence, characterized by the staining of all three substrates, namely
smaller distal renal tubules, gastric parietal cells and liver cell
cytoplasm. AMA target the 2-oxoacid dehydrogenase complex fam-
ily. The major epitope (AMA-M2) is located on the subunit of the
pyruvate dehydrogenase complex-E2, but AMA also react with the
other two components of the pyruvate dehydrogenase complex.
Immunoblotting and ELISA tests are now available, and these assays
have an increased sensitivity and specificity (greater than 95%)
[124], being positive in nearly 20% of individuals originally consid-
ered as AMA negative by indirect immunofluoresence [125]. The
role of AMA in the pathogenesis of PBC is under debate because of
the accessibility of the autoantibody to an antigen located in the
inner membrane of mitochondria. Recent evidence indicates that
AMA recognize pyruvate dehydrogenase complex-E2 in apoptotic
bodies resulting in a complex that stimulates innate immune sys-
tems in genetically susceptible individuals [126]. AMA reactivity
can be detected decades before the clinical onset of PBC; how-
ever, 76% of asymptomatic patients with incidental AMA serum
reactivity eventually develop PBC over more than 10 years of obser-
vation [127]. AMA titre is not associated with disease severity or
rate of progression [128], can occasionally be detected (8–12%)
[129] in patients with the classic phenotype of AIH without any
other evidence of PBC, and may hint at co-existent or underlying
PBC. Nonetheless, these patients should be classified and treated
according to their clinical phenotype.
Non-specific ANA are found in at least 30% of PBC sera [130]
without an apparent correlation with diagnosis or disease pheno-
type. However, some ANA reactivity with rim-like/membranous or
multiple nuclear dot immunofluorescence patterns are highly spe-
cific to PBC. The identified targets are the nuclear body 100 kDa
(sp100), promyelocitic leukaemia and small ubiquitin-like modi-
fiers corresponding to the multiple nuclear dot-ANA, and proteins
within the nuclear pore complex, including the 210 kDa glycopro-
tein (gp210) and the 62 kDa nucleoprotein corresponding to the
rim-like/membranous pattern [131]. Anti-sp100 and anti-gp210
are highly specific for PBC and can be used as markers of disease
when AMA are absent [123]. Patients positive for anti-nuclear pore
complex have a more severe disease course [131].
6.3. Primary sclerosing cholangitis
Unlike AIH and PBC, autoantibody testing is not included in the
diagnostic work-up of PSC. The most frequent autoantibody found
in patients with PSC is pANCA, which is routinely detected by an
indirect immunofluorescence assay. The target antigen is located
in the nuclear membrane [132,133], but the antigens targeted by
pANCA in PSC are still unknown. Several cytoplasmic proteins have
been proposed, including lactoferrin, myeloperoxidase, cathepsin
G, proteinase 3 and catalase [134]. The presence of pANCA in PSC
has been described in up to 94% of patients with a mean prevalence
of 63% [135]. Their specificity for the diagnosis of PSC is low [96]
because the prevalence can be found at equal rates in AIH and PBC.
No association links pANCA to the genetic susceptibility of PBC in
terms of a particular HLA haplotype [136], nor does pANCA repre-
sent a marker of association between PSC and IBD. In fact, only one
small study [137] has reported a higher prevalence of pANCA in
 A. Di Sabatino et al. / Digestive and Liver Disease 49 (2017) 947–956
patients with PSC and IBD than in patients without IBD. The prog-
nostic role of pANCA in PSC has also been investigated, but, even
though studies seem to suggest an association between pANCA and
end-stage liver disease [138], most studies reported no differences
in pANCA-positivity between early and advanced PSC, and a signif-
icant correlation between titres and disease activity has not been
clearly demonstrated [134,139]. ANA and SMA have been detected
with variable prevalence in patients with PSC, i.e. 8–77% for ANA
and 0–83% for SMA [135]. No particular ANA reactivity seems to
predominate [140]. None of the autoantibodies described in PSC
have sufficient specificity and sensitivity to be used for screening
or diagnosis.
In conclusion, although autoimmune liver disease encompasses
a wide – and sometimes overlapping – range of immune-mediated
disorders, AMA and SLA/LP are the only disease-specific antibodies
for these conditions. Therefore, in the case of overlapping autoim-
mune characteristics, making an appropriate diagnosis might still
be tricky and it should rely on clinical, serological, radiological and
histological features.
7. Autoimmune pancreatitis
AIP is a fibro-inflammatory condition involving the pancreas
and, sometimes, extra-pancreatic organs. There are two distinct
subtypes of the disease (type 1 and type 2), which clearly differ
in histological, clinical and epidemiological features. Currently, the
International Consensus Diagnostic Criteria are widely accepted for
the diagnosis of AIP subtypes, and they are based on five cardinal
features: (i) pancreatic parenchymal and ductal imaging, (ii) serol-
ogy, (iii) other organ involvement, (iv) pancreatic histology, and (v)
response to steroid therapy [141]. In the absence of a definitive his-
tological diagnosis, which generally allows the distinction between
type 1 and type 2 AIP, a combination of several criteria is needed
for the diagnosis of type 1 AIP. Serological abnormalities and other
organ involvement are consistent with type 1 AIP [141].
There is increasing interest in identifying specific biomarkers
for AIP since a differential diagnosis with pancreatic cancer is
frequently required [142]. The only biomarker for AIP in clinical
practice is serum IgG4, an antibody that accounts for less than 5%
of the total IgG in healthy subjects [141,143]. Between 75 and 85%
of patients with type 1 AIP and only a minority (less than 10%)
of patients with type 2 AIP have elevated serum IgG4 [144]. Since
type 1 accounts for 80–90% of AIP and type 2 for only 10–20%,
increased serum levels of IgG4 are observed only in up to 70% of
AIP patients. Furthermore, IgG4 elevation may be observed in up
to 10% of patients suffering from other diseases, such as pancreatic
cancer, cholangiocarcinoma and PSC [144]. Indeed, the specificity
of serum IgG4 in the diagnosis of AIP is higher if the elevation is
more than twice the upper limit of normal, and it increases propor-
tionally with the serum IgG4 level. Considering the low prevalence
of the disease (4.6/100.000) [145] and the difficult differential diag-
nosis with cancer [144], serum IgG4 should be managed carefully
in clinical practice, in order to avoid misdiagnoses, and should only
be considered as a part of the diagnostic algorithm [141].
Many studies have proposed other biomarkers for the diagnosis
of AIP, but none have reported satisfactory specificity and sensi-
tivity levels, and their use is still limited to research protocols.
Up to 40% of AIP patients have ANA [146]. Autoantibodies against
carbonic anhydrase II and alfa2-amylase [147], lactoferrin [148],
and pancreatic secretory trypsin inhibitor [149] were identified in
55%, 75% and 33% of patients, respectively. Other autoantibodies
described in AIP patients are against trypsinogens PRSS1 and PRSS2
[144]. Antibodies against the H. pylori plasminogen-binding protein
have been proposed as a sensitive marker of AIP (sensitivity 94%,
953
specificity 95%) [150], but a recent study has not confirmed these
data [151].
In conclusion, the only useful biomarker in clinical practice for
AIP is serum IgG4. Considering its low specificity and sensitivity,
other criteria are needed for the diagnosis of this disorder. New
serological markers are expected in order to improve, alone or in
combination with serum IgG4, the diagnosis of a disease that still
remains difficult to recognize.
8. Concluding remarks
Based on recent advances in most fields of medicine we can
expect increasing progress in the future in the development of
more reliable serum antibody biomarkers for diagnosing immune-
mediated gastrointestinal and liver diseases. These new tests
should make it possible to predict a patient’s risk of developing a
disease, facilitate early diagnosis, promote case-finding strategies,
allow assessment of patient prognosis, and prove the efficacy of
therapeutic strategies. This is particularly the case in those condi-
tions, such as AIE, IBD, AIH, PSC and AIP, in which serum antibodies
are of little use because of their poor diagnostic accuracy.
Key messages
– Case-finding is a good strategy in coeliac disease and autoim-
mune atrophic gastritis based on the measurement of their
specific serum antibodies – anti-tissue transglutaminase and
anti-endomysial antibodies for the former and anti-gastric pari-
etal cell antibodies for the latter condition – followed by
histologic confirmation on duodenal and gastric biopsy, respec-
tively.
– Anti-enterocyte antibodies are virtually 100% sensitive, but the
lack of immunofluorescent diagnostic criteria may decrease their
accuracy for the diagnosis of autoimmune enteropathy.
– Optimization of anti-tumour necrosis factor therapy in patients
with inflammatory bowel disease relies on serum drug levels and
the presence or absence of anti-drug antibodies.
– Unlike autoimmune hepatitis and primary biliary cholangitis, the
diagnostic work-up of primary sclerosing cholangitis does not
encompass autoantibody testing, and only anti-mitochondrial
antibodies and anti-soluble liver antigen/liver-pancreas anti-
bodies are disease-specific (for primary biliary cholangitis and
autoimmune hepatitis, respectively).
– The only available marker for autoimmune pancreatitis is serum
IgG4, and considering the low prevalence of the disease and the
difficult differential diagnosis with cancer, this marker should be
managed carefully in clinical practice in order to avoid misdiag-
noses.
Conflict of interest
None declared.
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