Journal of Autoimmunity 95 (2018) 144–158

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Journal of Autoimmunity
journal homepage: www.elsevier.com/locate/jautimm

The clinical usage and definition of autoantibodies in immune-mediated T

liver disease: A comprehensive overview
Benedetta Terziroli Beretta-Piccolia, Giorgina Mieli-Verganib, Diego Verganic,∗
a
Epatocentro Ticino, Lugano, Switzerland
b
Paediatric Liver, GI and Nutrition Centre, MowatLabs, King's College Hospital, Denmark Hill, London, SE5 9RS, UK
c
Institute of Liver Studies, MowatLabs, King's College Hospital, Denmark Hill, London, SE5 9RS, UK

A R T I C LE I N FO A B S T R A C T

Keywords: Autoimmune serology is key to the diagnosis and management of autoimmune liver diseases. Its correct use in
Anti-nuclear antibody clinical practice requires a basic knowledge of the laboratory techniques used for autoantibody detection.
Anti-smooth muscle antibody Indirect immunofluorescence (IIF) on triple rodent tissue is still the gold standard screening procedure for liver-
Anti-liver kidney microsomal antibody relevant autoantibodies, while HEp2 cells and human ethanol-fixed neutrophils are used as substrates to char-
Anti-liver soluble antibody
acterize nuclear reactivities and to detect anti-neutrophil cytoplasm antibody, respectively. Assays based on
Anti-mitochondrial antibody
Perinuclear anti-neutrophil nuclear antibody
purified or recombinant antigens are increasingly used, having the main advantage of being observer-in-
dependent and the disadvantage of detecting only autoantibodies whose antigenic target has been identified. The
AIH-specific anti-soluble liver antigen antibody cannot be detected by IIF and a molecular-based assay should be
used at the screening level. Since autoantibodies may be present in the context of viral hepatitides and other
inflammatory liver diseases it is important to exclude these conditions before diagnosing autoimmune liver
disease. Anti-nuclear antibody (ANA), most often with a homogeneous IIF pattern on HEp2 cells, characterizes
type 1 autoimmune hepatitis (AIH), and is found in association with anti-smooth muscle antibody in about half
of the cases. Two IIF ANA patterns are specific for primary biliary cholangitis, namely the rim-like/membranous
pattern, and the multiple nuclear dots pattern. Anti-liver kidney microsomal antibody type 1 is the serological
hallmark of type 2 AIH, often in association with anti-liver cytosol type 1 antibody. Atypical perinuclear anti-
neutrophil antibody, referred to as perinuclear anti-neutrophil nuclear antibody, is frequently detected in pri-
mary sclerosing cholangitis, in AIH type 1 and in inflammatory bowel diseases. The anti-asiaglycoprotein re-
ceptor antibody is liver-specific but not disease-specific, and reliable commercial assays for its detection are
lacking. Anti-mitochondrial antibody is the hallmark of primary biliary cholangitis (PBC), being disease-specific
and present in about 95% of the PBC patients. Its incidental detection presages the future development of PBC.

1. Introduction
Autoantibodies are an essential tool in diagnosis and management
of autoimmune liver diseases, particularly in autoimmune hepatitis
(AIH) and primary biliary cholangitis (PBC).
According to the simplified diagnostic criteria for AIH [1], a definite
AIH diagnosis is not possible in absence of autoantibodies. The ser-
ological profile also allows classification of AIH into type 1 AIH,
characterized by anti-nuclear antibody (ANA) and/or anti-smooth
muscle (SMA) antibody, and type 2 AIH, associated with anti-liver
kidney microsomal type 1 (LKM1) and/or anti-liver cytosol 1 (LC1)
antibody [2]. The two types are clinically different, as type 2 AIH af-
fects mainly children and adolescents, is much rarer and more ag-
gressive than type 1 AIH [2]: therefore distinction has relevant clinical
consequences.
Anti-mitochondrial antibody (AMA), the serological hallmark of
primary biliary cholangitis (PBC), is considered the most specific au-
toantibody in human immunology [3] and is detected in about 95% of
PBC patients, being part, together with a specific subset of ANA, of the
PBC diagnostic criteria [4].
Anti-neutrophil cytoplasmic antibody (ANCA) is detected in up to
94% of the cases of primary sclerosing cholangitis (PSC) [5]. The pe-
diatric condition known as autoimmune sclerosing cholangitis (ASC)
has a serological profile overlapping with type 1 AIH except for a higher
frequency of ANCA positivity [6].
This paper aims at offering a comprehensive review of the clinical
significance of the autoantibodies associated to AIH, PBC and PSC and
ASC, including the history of their discovery and the appropriate

∗
Corresponding author.
E-mail address: diego.vergani@kcl.ac.uk (D. Vergani).

https://doi.org/10.1016/j.jaut.2018.10.004
Received 11 September 2018; Received in revised form 9 October 2018; Accepted 11 October 2018
Available online 23 October 2018
0896-8411/ © 2018 Elsevier Ltd. All rights reserved.
B. Terziroli Beretta-Piccoli et al.
laboratory techniques for their detection, whose basic knowledge is a
prerequisite for the correct interpretation of the laboratory reports in
the clinical context.
2. Laboratory techniques
Indirect immunofluorescence (IIF) on triple rodent tissue remains
the gold standard methodology to detect liver-relevant autoantibodies
[7], though observer-independent immunochemical techniques are ei-
ther already available or under development. IIF nuclear reactivities
should be further characterized on HEp2 cells, which have a prominent
nucleus allowing the detection of different patterns [7]. The main ad-
vantages of IIF on rodent tissue are the simultaneous detection of vir-
tually all autoimmune liver disease relevant autoantibodies, the re-
cognition of patterns characteristic of a specific disease, and, most
importantly, the detection of autoantibodies directed against still un-
known targets, in contrast to assays based on purified or recombinant
antigens. However, this procedure is operator-dependent, requiring
experienced laboratory personnel and high quality rodent substrates:
these hurdles have led to screening of autoantibodies by solid-phase
immunoassays in many extra-European countries [8,9], with the risk of
missing key positivities in clinical practice. Indeed, identification of the
molecular targets of some autoantibodies giving a characteristic IIF
pattern has led to the establishment of immuno-assays for the detection
of AMA, anti-LKM1, anti- LC1 and partially ANA and anti-neutrophil
cytosolic antibody (ANCA) [10], the IIF substrate of the latter being
ethanol-fixed human neutrophils [7]. These assays are based either on
recombinant or purified antigens, and still lack standardization [9,11].
In this context, it is important to note that the AIH-specific anti-soluble
liver antigen (SLA) antibody is not detected by IIF [12], and, according
to recent guidelines [13], an appropriate molecular test should be
employed for its detection at the initial diagnostic work-up of suspected
AIH.
Autoantibodies are considered positive in IIF when present at a di-
lution titer ≥1:40 in adults, while in children the cut-off for positivity is
lower, being ≥1:20 for ANA and SMA and ≥1:10 for anti-LKM1 and
LC1 [7,14]. The ANCA positivity cut-off on ethanol-fixed human neu-
trophils is 1:20 in both adults and children [15].
3. Anti-nuclear antibody
ANA was the first antibody to be associated with AIH [15]. It is
responsible for the lupus erythematosus (LE) cells, i.e. neutrophils en-
gulfed with denatured nuclei of damaged cells, whose phagocytosis is
mediated by ANA (originally named anti-nuclear factor) [16,17]. LE
cells were first detected in the blood of LE patients [18], and, seven
years later, also in the ascites of patients with “chronic hy-
pergammaglobulinemic hepatitis” [19]. This observation led to the
original label for AIH, namely “lupoid hepatitis” [19]. It was later re-
cognized that LE and what at that time was called “chronic hy-
pergammaglobulinemic hepatitis” or “lupoid hepatitis”, are different
entities, only rarely coexisting in the same patient [20].
IIF on triple rodent tissue is recommended as screening test for ANA
in the context of liver autoimmunity [7] (Fig. 1), and any reactivity
should be further characterized on HEp2 cells. The use of HEp2 cells as
a substrate for screening is not recommended, since by this metho-
dology low titer (< 1:40) ANA is frequently detected in healthy in-
dividuals [15]. Some three quarters of AIH patients display a homo-
geneous IIF pattern on HEp2 cells, the remainder showing a speckled or
nucleolar pattern (Fig. 2) [15,21]. Commercial kits are available for a
wide range of identified nuclear antigens, but they are of limited help as
a screening tool in AIH, since the molecular targets of ANA in AIH are
only partially known and ANA positivity detectable by IIF may be
missed by molecular-based assays [22,23], with potentially serious
consequences for patients. Reported ANA reactivities in AIH include
anti-double- and single-stranded DNA, anti-histones, anti-centromere,
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Journal of Autoimmunity 95 (2018) 144–158
anti-chromatin, anti-ribonucleoproteins and anti-cyclin A [10,24–29].
About one third of type 1 AIH patients do not react with any of the
known nuclear antigens [21]. Neither the IIF pattern on HEp2cells nor
any of the target antigens are associated with specific clinical pheno-
types of AIH [15]. However, a homogeneous IIF pattern on HEp2 cells
(Fig. 2, panel A) is the most commonly found in AIH.
ANA, together with SMA, defines type 1 AIH [12] (Table 1). If au-
toantibodies are tested according to recommendations [7], > 95% of
type 1 AIH patients are seropositive for ANA, SMA or both [15]. ANA is
detected as the only serological marker in some 10–15% of type 1 AIH
patients, and in association with SMA in half of the cases [15], SMA
being the only autoantibody in the remainders [28,29]. ANA is not
specific for AIH, since it is found in a broad spectrum of diseases, in-
cluding autoimmune conditions, infections, malignancies and drug-in-
duced damages [21]. In the context of liver diseases, ANA may be
present in a wide array of acute and chronic conditions such as viral
hepatitis B, C, D and E, acute liver failure, non-alcoholic fatty liver
disease, alcohol-induced liver disease, hepatocellular carcinoma and
drug-induced liver damage [7,8,15,31,32]. Moreover, as mentioned
above, ANA may be present in healthy individuals, both in children and
in adults [33,34], the frequency and the titer increasing with age [21].
AIH-like drug-induced hepatitis is a particularly challenging differential
diagnosis, as it may be clinically, serologically and histologically in-
distinguishable from genuine AIH, and often only long-term follow up
can help the clinician arriving at a definite diagnosis [35,36]. This
highlights the concept that autoantibodies, though an important piece
in the diagnostic jigsaw puzzle of AIH, are not diagnostic on their own.
In PBC, ANA is detected by IIF on HEp2 cells in 30–50% of patients,
with specific and non-specific subtypes [4,37,38]. Non-specific sub-
types include anti-centromere (ACA), anti-Ro/SSA, anti-La/SSB, anti-
Scl-70 and anti-histones, which give a centromere, speckled (SSA and
SSB), nucleolar or homogeneous IIF pattern on HEp2 cells [39]. Anti-
double stranded DNA, characteristic of systemic lupus erythematosus,
has also been detected in PBC, some data suggesting that this reactivity
is particularly common in PBC patients with AIH overlapping features
[40,41]. ACA, which is characteristic of systemic sclerosis with a pre-
valence of about 90%, is identified on HEp2 cells, as this cell line
contains a high number of mitotic figures, especially in its Hep-20-10
form, which has more than ten times mitotic cells than HEp-2 cells
[42,43]. The ACA pattern shows multiple discrete dots distributed
throughout the entire nucleus. Mitotic cells also exhibit this speckled/
dot pattern in a typical alignment within the condensed chromosomal
material. ACA targets proteins localize to the kinetochores of chromo-
somes. Commercially available ELISA kits have been developed, mainly
using the immunodominant autoantigen CENP-B [44]. ACA is found in
9–30% of PBC patients, the frequency being higher in those with con-
comitant systemic sclerosis [43,45]. A large Italian study reports that
ACA positivity predicts systemic rheumatic diseases, particularly sys-
temic sclerosis, in PBC patients [46]. ACA-positivity in PBC has been
associated also with a higher risk of developing portal hypertension
[47,48]. Of note, a study investigating the frequency and significance of
rheumatologic serology in PBC patients found that anti-Ro/SSA and
ACA are specific PBC markers in AMA-negative patients [49]. Two PBC
specific ANA subtypes, i.e. only very rarely detected in subjects without
PBC, have been reported, which display a multiple nuclear dots (MND)
and a nuclear-rim/membranous IIF patterns on HEp2 cells, respectively
(Fig. 3) [8]. They are particularly useful as a diagnostic tool in AMA-
negative PBC, [37,42]. Both have a prognostic value in PBC, since they
have been demonstrated to be associated with a more severe disease
course, the evidence being stronger for rim-like/membranous ANA
[38,42,50]. In this context, it is of interest to note that the association
with disease severity is particularly strong for the IgG3 subtype of anti-
MND [51–53], possibly due to its ability to activate complement. The
same observation has been made for IgG3 AMA (see below) [54]. Anti-
MND stains the nuclear bodies of interphase cells, appearing at IIF on
HEp2 cells as 3–20 nuclear dots 0.2–1 μm in size (Fig. 3, panel C). The
B. Terziroli Beretta-Piccoli et al.
chromosomal plates of mitotic cells do not stain. Anti-MND is mainly
directed against the nuclear proteins called sp-100 and promyelocytic
leukemia (PML) protein. More recently reported antigenic targets are
sp140 and small ubiquitin-related modifiers [42]. ANA giving a nu-
clear-rim/membranous IIF pattern (Fig. 3, panel B) is directed against
multiple nuclear envelope proteins, including gp210, nucleoporin p62
and lamin B receptor, the two latter giving a slightly different IIF pat-
tern, at times referred to as smooth rim-like pattern [38,42]. The nu-
clear envelope is a complex structure, consisting of a double bilayer
membrane, the nuclear pore complex and the nuclear lamina layer, and
thus includes a variety of proteins, explaining the relative high number
of antigenic targets corresponding to the nuclear-rim/membranous IIF
Fig. 2. Anti-nuclear immunofluorescence pattern on the large nuclei of HEp2 cells, showing the homogeneous pattern (panel A) and the speckled pattern (panel B).
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Journal of Autoimmunity 95 (2018) 144–158
Fig. 1. Indirect immunofluorescence. Panel A: anti-nu-
clear antibody (ANA) pattern on rodent liver tissue. Panel B:
anti-liver-kidney microsomal type 1 (LKM-1) antibody pat-
tern on rodent kidney tissue (left hand side) showing
staining of the larger, proximal tubules, and on rodent liver
tissue (right hand side) showing bright staining of the he-
patocyte cytoplasm, sparing the nuclei. Panel C: anti-
smooth muscle antibody (SMA) pattern on rodent kidney
tissue showing staining of vessels (V) and glomeruli (G).
Panel D: anti-liver-cytosol type 1 (LC-1) antibody pattern on
rodent liver tissue showing hepatocyte cytoplasm staining
weakening around the central vein.
pattern. Anti-sp100 antibodies are found in 20–40% of PBC patients,
while anti-PML in some 15–20%, often coexisting in the same patient
[38,51]. Interestingly, anti-sp100 has been found to be associated with
AMA in women with recurrent urinary tract infections, with or without
PBC, suggesting a role of bacteria in inducing autoantibodies in PBC
[55]. Anti-MND serum titers remain unchanged during the disease
course, mirroring the findings of a Japanese study on anti-gp210
[52,56] [57]. ELISA and immunoblotting assays based on recombinant
gp210 and sp100 have been developed, and can be used to confirm IIF
results, and to detect anti-gp210 and anti-sp100, as they are more
sensitive than IIF, partially because at IIF concomitant AMA may mask
ANA [42]. However, these assays do not include all antigenic targets
 Table 1
Clinical significance of autoantibodies in autoimmune hepatitis.
B. Terziroli Beretta-Piccoli et al.

Antibody Method of Antigenic target Frequency Other hepatic diseases Extrahepatic diseases Clinical significance
detection

ANA IIF Unknown in 30% 75% in AIH-1 Primary sclerosing cholangitis Autoimmune conditions Typical of type 1 AIH, most often with
Chromatin Autoimmune sclerosing Infections homogeneous pattern on HEp2 cells
Histones cholangitis Malignancies Coexisting with anti-SMA in 50% of type 1 AIH
Centromeres, Cyclin A, Ribonucleoproteins, Double Primary biliary cholangitis Drug-induced disorders
stranded DNA, Single stranded DNA Chronic hepatitis B and C
Hepatitis E
Non-alcoholic fatty liver disease
Drug-induced liver injury
SMA IIF Unknown in 20% 85% in AIH-1 Primary sclerosing cholangitis Autoimmune conditions Typical of type 1 AIH, most often with VG/VGT
Filamentous actin Autoimmune sclerosing Infections patterns on rodent kidney tissue
Vimentin cholangitis Malignancies Titer correlates with disease activity in AIH
Desmin Primary biliary cholangitis Drug-induced disorders
Chronic hepatitis B and C
Hepatitis E
Non-alcoholic fatty liver disease
Drug-induced liver injury
Anti-LKM1 IIF Cytochrome P4502D6 At least 70% in AIH-2 Chronic hepatitis C None reported Diagnostic of type 2 AIH in absence of HCV

147
ELISA, IB, LIA, RIA Rarely in autoimmune sclerosing infection
cholangitis Titer correlates with disease activity
Anti-LC1 IIF, DID, CIE Formimino-transferase cyclodeaminase 30% in AIH-2 Chronic hepatitis C None reported Diagnostic of type 2 AIH in absence of HCV
ELISA, LIA, RIA Autoimmune sclerosing infection
cholangitis Often associated with anti-LKM1
Rarely in pediatric type 1 AIH Toter correlates with disease activity
Anti-SLA/LP ELISA, IB, RIA tRNP(Ser)sec 10–50% in AIH-1 and Chronic hepatitis C and B None reported Specific for type 1 AIH
AIH-2 Autoimmune sclerosing Associated with a more severe disease course
cholangitis
pANNA IIF Beta-tubulin isotype 5 and other yet unknown 40–96% in AIH-1 Autoimmune sclerosing Inflammatory bowel Absent in type 2 AIH
autoantigens cholangitis disease Rarely detected as the only serological marker in
Primary sclerosing cholangitis type 1 AIH
Anti-ASGPR IIF on human ASGPR 24–82% in AIH-1; Primary biliary cholangitis None reported Not used in diagnostic practice because of
substrate 16–57% in AIH-2 Drug-induced liver injury cumbersome technique
Chronic HCV, HBV and HDV
infection
Non-alcoholic steatohepatitis
Alcoholic hepatitis

ANA, anti-nuclear antibody; IIF, indirect immunofluorescence; AIH, autoimmune hepatitis; SMA, smooth muscle antibody; LKM1, liver kidney microsomal type 1; LC1, liver cytosol type 1; SLA, soluble liver antigen; LP,
liver pancreas; pANNA, perinuclear anti-neutrophil nuclear antibody; ELISA, enzyme-linked immunosorbent assay; IB, immunoblot; LIA, line-immuno-assay; RIA, radio-immune-precipitation assay; DID, double-di-
mension immune-diffusion; CIE, counter-immune-electrophoresis; HCV, hepatitis C virus; ASGPR, asialoglycoprotein receptor; HBV, hepatitis B virus; HDV, hepatitis D virus.
Journal of Autoimmunity 95 (2018) 144–158
B. Terziroli Beretta-Piccoli et al.
Fig. 3. Panel A: anti-mitochondrial antibody (AMA) immunofluorescence
pattern on rodent gastric parietal cells (top), and on rodent kidney tissue
(bottom) showing staining of the distal, smaller and mitochondria-rich tubules.
Panel B and C: immunofluorescence pattern of anti-nuclear antibodies diag-
nostic of primary biliary cholangitis: rim-like/membranous pattern (RML, panel
B); multiple-dots pattern (MND, panel C).
and therefore their use should be complementary to IIF on HEp2 cells.
In PSC, reported ANA frequencies range between 7.4 and 77%,
without predominant IIF pattern or antigenic target, and without as-
sociations with clinical phenotypes [58,59]. Reported.
4. Anti-smooth muscle and anti-actin antibodies
SMA was first reported in the context of lupoid hepatitis in 1965
[60], and, shortly afterwards, was detected also in “chronic active he-
patitis”, but not in patients with systemic lupus erythematosus [61,62],
leading to further characterization of the clinical entity later named
AIH type 1 [63]. In the same paper, it was reported that SMA stains
renal tissue in addition to the smooth muscle of muscularis mucosa of
the gastric wall [62]. Three different IIF staining patterns on rodent
renal tissue were recognized by Bottazzo in 1976 [64], namely staining
of arterial vessels (V), glomerular mesangium (G) or kidney tubules (T):
while the V pattern was found also in PBC, malignancies and viral in-
fections, the VG (Fig. 1, panel C) and VGT patterns were detected only
in patients with an aggressive form of chronic hepatitis, nowadays
known to be type 1 AIH [31]. Therefore, the IIF pattern of SMA should
always be reported by the laboratory, since this information is very
valuable for the clinician [21], although the VG and VGT patterns are
not entirely specific for AIH [20,65], and, conversely, 20% of SMA-
positive AIH patients do not have these patterns [66]. Interestingly, a
recent report confirms that the GT SMA pattern is associated with the
development of AIH in patients with abnormal transaminase levels
[67]. If cultured fibroblasts or vascular smooth muscle (VSM) 47 cells
from rat embryonic thoracic aorta are used as a substrate, the VGT
pattern generally corresponds to the anti-actin or anti-microfilament
pattern [15]. This observation led to the introduction of commercial
kits using filamentous actin as an antigen. These molecular tests have
been shown to be less specific for AIH than the VGT pattern on rodent
kidney tissue, particularly if SMA is present at low titers [68–71]. In
addition, molecular tests are less sensitive than IIF [68]. For these
reasons, it is recommended that they are used as complementary to IIF
[21].
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Journal of Autoimmunity 95 (2018) 144–158
The molecular target antigen of SMA remains to be defined. Early
studies suggest that AIH-1-specific VGT-positive sera target different
antigens as compared to non-AIH-1-specific V-positive SMA sera
[72–75]. This is also suggested by the different staining pattern at IIF.
The target antigen of the VGT sera is probably part of actin [76], in its
filamentous form [72,75]. However, this is challenged by the ob-
servation that some 20% of AIH-1-specific VGT SMA do not react with
purified actin [68,69,75,77,78]. It can be speculated that this is due to
loss of some antigens during protein purification.
Like ANA, SMA is not disease-specific, and can be found in a variety
of conditions, including viral hepatitis, malignancies, rheumatic dis-
eases, drug-induced liver injury, non-alcoholic fatty liver disease and
alcohol-induced liver disease [10]. As mentioned above, IIF on kidney
tissue is helpful in distinguishing the more AIH-specific VG and VGT
SMA patterns. In addition, titers are usually higher in AIH than in other
conditions [31,65]. In children with AIH, SMA titers correlate with
disease activity [79]. This has been suggested in adults as well [80].
In pediatrics, the same serological profile of type 1 AIH char-
acterizes also ASC [6], a condition affecting about half of children
presenting with autoimmune liver disease. These patients have an ab-
normal cholangiogram already at presentation and often have asso-
ciated inflammatory bowel disease [81]. The high frequency, up to 83%
[58], of SMA positivity reported in PSC may point toward a link with
ASC, but whether PSC is an advanced/burnt out form of ASC is still
debated.
5. Anti-liver kidney microsomal antibody
Anti-LKM antibody was identified in 1973 by Mario Rizzetto in
Deborah Doniach's laboratory in London [82]: he detected in the serum
of 16 patients (12 with liver disease) an antibody that stained brightly
hepatocyte cytoplasm and proximal renal tubuli, the reactivity being
abolished by incubation of the serum with a “microsomal fraction”
obtained by ultracentrifugation of a liver homogenate [82,83]. The
rarity of this reactivity was already noted in Rizzetto's landmark paper,
being detected only in a small fraction (0.12%) of sera form patients
with liver diseases [82,84]. The liver disease associated with anti-LKM
has been fully characterized by Alagille's group in France in 1987 [85],
following the publication of smaller and less well characterized case
series [83,84,86]. The French series included 65 patients, 89% females,
56% aged less than 20 years, and reported a 14-year survival rate of
only 51% despite treatment with prednisone and azathioprine [85],
leading to the proposal of naming this aggressive and juvenile form of
AIH, AIH type 2. Anti-LKM1, as opposed to anti-LKM2 and anti-LKM3
described below, is the serological hallmark of type 2 AIH, which is a
particularly aggressive condition affecting mainly children and ado-
lescents, much rarer than type 1 AIH, and accounting for approxima-
tively one third of juvenile AIH, and only 10% of adult AIHs [87–89].
Anti-LKM1 is often found in association with anti-LC1 antibody (see
below), which rarely is the only serological marker of type 2 AIH [10].
As stated above, the recommended screening test for anti-LKM1 is
IIF on triple rodent tissue [7], whereby it stains brightly the hepatocyte
cytoplasm and the proximal renal tubules, sparing stomach tissue
(Fig. 1, panel B) [31], and thus allowing distinction of anti-LKM1 from
AMA, which stains stomach tissue as well (Fig. 3, panel A) [7]. The IIF
pattern on kidney and liver tissue displays subtle differences between
AMA and anti-LKM1: on kidney tissue sections, anti-LKM1 stains the
proximal, larger tubules, whereas AMA stains the small mitochondria-
rich distal tubules; on liver tissue, anti-LKM1 stains the hepatocyte
cytoplasm more brightly [10]. Only an experienced observer may be
able to appreciate these differences, therefore screening uniquely on
kidney tissue should be avoided [7]. The use of triple rodent tissue
sections allows also for the detection of anti-LC1, which only stains liver
(see below) [21]. Molecular-based assays have been established after
the identification of the target antigens of anti-LKM1, anti-LC1 and
AMA: they have high specificity and sensitivity and can be used to
B. Terziroli Beretta-Piccoli et al.
clarify any doubtful case, but they still lack standardization [90].
The target antigen of anti-LKM1 has been identified as cytochrome
P4502D6 (CYP2D6) [91–93]. Subsequent studies were able to identify
different linear epitopic regions within this large protein hierarchically
recognized by anti-LKM1 [94–97], indicating that anti-LKM1 is poly-
clonal, as reported for other autoimmune conditions [98]. A possible
pathogenic role for anti-LKM1 is suggested by the presence of its target
not only in the endoplasmic reticulum, but also on the external aspect
of the hepatocyte membrane rendering the hepatocyte vulnerable to the
autoimmune attack, though this needs to be confirmed [99]. In children
with type 2 AIH, anti-LKM1 titers correlate with disease activity and
should be used to monitor treatment response [79].
Anti-LKM antibodies with IIF staining patterns different from anti-
LKM1 have been described. Anti-LKM2 was detected in sera of patients
with ticrynafen-induced hepatitis, also referred to as tielinic acid, an
uricosuric anti-hypertensive drug withdrawn from the market in the
‘80s owing to its hepatotoxicity [84,100]. On kidney tissue sections,
anti-LKM2 stains more intensely the first and second portion (P1 and
P2) of the proximal tubule, while on liver tissue sections it stains more
intensely the centrilobular hepatocytes [101]. The target antigen is
CYP2C9 [102,103]. Anti-LKM3 was first reported by Rizzetto's group in
association with chronic hepatitis delta infection, where it is found in
some 13% of patients [104]. It stains hepatocyte cytoplasm and prox-
imal renal tubules of human and primate tissue sections, thus ham-
pering its testing in clinical practice, although it has been described in
up to 19% of AIH type 2 patients, at times as the only serological
marker [105–107]. The target antigen is family 1 uridine 5′-dipho-
sphate glucuronosyltransferase (UGT-1) [106].
Anti-LKM1 is detected also in a proportion ranging from 0 to 13% of
patients with chronic hepatitis C (HCV) infection [108,109]. An asso-
ciation with female gender, higher serum transaminase levels and,
histologically, more severe necroinflammatory activity in HCV-infected
patients has been reported [65]. The main target epitope of CYP2D6 in
type 2 AIH, namely CYP2D6193-212, is targeted by 93% of type 2 AIH
patients, and by 50% of anti-LKM1 positive HCV patients [94]; the
second most commonly targeted epitope in AIH, CYP2D6254-271 is also
more frequently targeted in type 2 AIH than in HCV patients [110]:
these observations show that the targets of anti-LKM1 in HCV and type
2 AIH are partially overlapping, suggesting that anti-HCV antibodies
may cross-react with self-epitopes, and thus lead to autoimmunity [15].
Homologous regions of CYP2D6 with Herpes virus and Cytomegalo-
virus have also been identified [96,111], leading to the “multiple-hits
hypothesis”, stating that exposure to multiple common viruses may
induce loss of tolerance in auto-reactive lymphocytes. In this context,
exposure to HCV and Herpes viruses may lead to type 2 AIH in ge-
netically predisposed patients [111,112]. Anti-LKM1-positive HCV-in-
fected patients were a challenging clinical problem in the interferon
treatment era, since interferon may trigger autoimmunity [113], but
this is no longer the case in the direct-antiviral agents era, where HCV
infection can be effectively and safely cured [21].
Lastly, anti-LKM1 has been detected in patients suffering from he-
patitis in the context of autoimmune polyendocrinopathy-candidiasis-
ectodermal dystrophy (APECED), a rare autosomal recessive disorder
linked to homozygous mutations in the autoimmune regulator gene
(AIRE). Anti-LKM1 in this condition are indistinguishable in IIF from
anti-LKM1 in AIH type 2, but the molecular target differs, being
CYP2A6 in APECED-linked hepatitis [114].
6. Anti-liver cytosol antibody
Anti-LC1 antibody was reported first by Martini in 1988 in juvenile
AIH, who detected it in the serum of 21 patients with “chronic active
hepatitis”, coexisting in two thirds of them with anti-LKM1 [115]. No
clinical differences were observed in LKM1-positive patients positive or
negative for anti-LC1 [115]. Anti-LC1 was not detected in the serum of
1392 patients with various liver diseases, of 1200 patients with
149
Journal of Autoimmunity 95 (2018) 144–158
extrahepatic diseases and of 100 healthy controls [115].
Anti-LC1 is an organ-specific antibody, and, when tested by IIF on
triple rodent tissue, it stains brightly the hepatocyte cytoplasm sparing
the centro-lobular cell layers (Fig. 1, panel D) [31]. No zonality of
hepatocyte staining is seen when using human liver as a substrate
[115]. Importantly, it can be masked on liver tissue by the concomitant
presence of anti-LKM1 antibody, as both antibodies frequently occur
together. The target antigen of anti-LC1 is formiminotransferase cy-
clodeaminase, a liver enzyme that catalyzes the conversion of histidine
to glutamic acid [116,117], leading to the establishment of molecular-
based assays, which are particularly useful when anti-LC1 and anti-
LKM1 coexist, making anti-LC1 difficult to visualize on IIF. Anti-LC1 is,
together with anti-LKM1, the serological hallmark of type 2 AIH. It is
found in about two thirds of type 2 AIH patients, the frequency of type 2
AIH patients having anti-LC1 on its own is unknown but probably is low
[21]. In one recent Italian study on a cohort of 63 pediatric type 2 AIH,
anti-LC1 was detected in two thirds of the patients, being the only
serological marker in one third [118]. Anti-LC1 disease-specificity was
already apparent in the first report, since it was not found in the serum
of a large number of patients with other hepatic or extra-hepatic dis-
eases [115]. One may argue that in 1988 HCV had not yet been dis-
covered and some HCV-positive patients could have been included in
the 21 patients with chronic active hepatitis: indeed, it was later shown
that anti-LC1 can rarely be detected in HCV infection [119], most fre-
quently in association with anti-LKM1 [120]. Anti-LC1 has been re-
ported in a small proportion of pediatric AIH type 1 and ASC patients
[121]. It can be concluded that it is not entirely disease-specific, and its
detection must be interpreted in the clinical context. Anti-LC1 titers
correlate with disease activity in children with type 2 AIH [122].
7. Anti-soluble liver antigen/liver-pancreas antibody
Anti-liver-pancreas (LP) antibody was first reported in 1981 by
Peter Berg in Germany, who detected an antibody reacting with the
supernatant of rodent liver and pancreas homogenates in the serum of
20 (18 females) patients with liver disease [123]. All treated patients
(14/20) responded well to prednisone with or without azathioprine.
Anti-LP antibody was detected by complement-fixation test, being un-
detectable by IIF. The same group suggested that anti-LP antibody
characterizes a distinct subgroup of AIH, which they proposed to call
AIH type 3 [124,125]. Six years after Berg's report, a German group led
by Michael Manns reported an antibody directed against an antigen
contained in the supernatant of liver homogenate, which they called
anti-soluble liver antigen (SLA), identified by immunoassays based on
the use of cytosolic liver cell fractions [126]. The antibody was detected
in the serum of 23 patients, mostly young women, with chronic active
hepatitis, hypergammaglobulinemia, good response to im-
munosuppressive treatment, and was the only antibody detected in six
patients, leading again to the proposal of a third type of AIH [126]. This
proposal was later declined by the IAIHG, since the conventional cut-off
for ANA positivity at that time was a titer exceeding 1:80, higher than
the cut-off of 1:40 on which the international AIH community later
agreed [7]; therefore some anti-SLA positive patients were likely to be
positive also for ANA. It was later recognized that anti-LP and anti-SLA
are the same antibody, thus termed anti-SLA/LP [127]. Both early re-
ports by Berg and Manns underscored the high specificity of this anti-
body for AIH, which was extremely rarely found in large control groups
including healthy blood donors, patients with extrahepatic diseases or
other liver diseases [124,126]. Indeed, anti-SLA/LP is the most specific
serological marker of AIH [8], as confirmed by later studies
[111,127–129], though its disease sensitivity is low [130]. In view of its
specificity, being as high as 98.9%, anti –SLA/LP has a high value in the
simplified AIH diagnostic scoring system [1] [131]. The specificity of
this autoantibody was challenged by one study reporting anti-SLA/LP
positivity in 10% of HCV-infected patients, often in association with
anti-LKM1, but these results have not been replicated [120,132]. In the
B. Terziroli Beretta-Piccoli et al. Journal of Autoimmunity 95 (2018) 144–158

AMA, anti-mitochondrial antibody; IIF, indirect immunofluorescence; ELISA, enzyme-linked immunosorbent assay; IB, immunoblot; LIA, line-immuno-assay; PDC, pyruvate dehydrogenase; PBC, primary biliary cho-
original report by Berg's group anti-LP positivity was detected in 4/128

Associated with more severe liver disease

Associated with more severe liver disease

Virtually diagnostic of PBC, predicts the

More frequent in patients with PBC and

Lower frequency in the Mediterranean
patients with chronic HBV or HCV infection [124].

May be the only serological marker in

Associated with portal hypertension

Anti-SLA/LP target antigen, originally reported to be a cytosolic
protein [126], was later identified by Wies et al. as a 50 kDa enzyme, by

AMA-negative PBC patients

using human lymphoma cDNA libraries [127]. A 35 kDa polypeptide

Virtually diagnostic of PBC

Virtually diagnostic of PBC

fragment of the same enzymatic protein was identified as an anti-SLA
development of PBC

target shortly thereafter using human liver cDNA library screening

Clinical significance

systemic sclerosis
[133]. In both studies, the protein was procariotically cloned. However,
nearly a decade before these reports, Gelpí reported an autoantibody
targeting the UGA serine tRNA protein complex (tRNP(Ser)sec) in the sera
of patients with a particularly aggressive form of ANA/SMA positive-
area

AIH [134]. The same group subsequently demonstrated that tRNP(Ser)sec

is the same protein identified by Wies et al. as the molecular target of
Recurrent bacteriuria
Extrahepatic diseases

Sjögren's syndrome

Sjögren's syndrome

Sjögren's syndrome

anti-SLA [127], allowing the establishment of molecular-based immune

Systemic sclerosis

Systemic sclerosis
assays [128]. However, such assays only detect antibody reacting with
tuberculosis

linear epitopes, with suboptimal sensitivity [15]. Assays using eu-

Pulmonary

kariotically expressed antigens, thus maintaining conformational epi-

Leprosy

topes, are cumbersome and remain research tools, therefore not used in
clinical practice [135]. Fine specificity studies of anti-SLA/LP using the
procariotically expressed protein, have identified tRNP(Ser)sec 395-414 as
Other hepatic diseases

Rarely in chronic HCV

Autoimmune hepatitis

the immunodominant epitope [136]. Of note, this serologically defined

Acute liver failure

epitope overlaps with one epitope recognized by CD4+ T cells in a

mouse model of AIH and in anti-SLA/LP-positive AIH patients [137].
The reported frequency of anti-SLA/LP antibody in AIH is highly
None

None

variable, ranging from 10 to 50% [8,15]: this variability is due to dif-

ferent sensitivities of the assays used. If tested by high-sensitivity
Up to 95%
Frequency

radioligand assays, up to 58% of type 1 and type 2 AIH patients are

10–40%

20–40%

9–30%

anti-SLA/LP seropositive, as well as 41% of ASC pediatric patients

[135].
Anti-SLA/LP is strongly associated with a subtype of antibodies to
branched-chain 2-oxo acid dehydrogenase complex; E3 binding protein of

Sjögren's syndrome type A autoantigen (anti-SSA), namely anti-Ro52

Sp100, promyelocytic leukemia protein, Sp140, small ubiquitin-related
gp210, nucleoporin p62, lamin B receptor, nuclear membrane proteins
E2 subunits of PDC, of 2-oxoglutarate dehydrogenase complex and of

[138,139], possibly explaining the high rate of fetal loss in pregnant

women seropositive for anti-SLA/LP and anti-Ro52 [140]. Transpla-
cental passage of anti-SSA and SSB antibodies causes neonatal lupus,
whose most severe manifestation is congenital heart block, which may
be fatal [141].

8. Anti-asiaglycoprotein receptor antibody

Asiaglycoprotein receptor (ASGPR) is a liver-specific lectin con-

stitutively expressed on the sinusoidal and basolateral membrane of
hepatocytes [142]. This receptor binds circulating altered glycopro-
teins, i.e. whose sialic acid moieties has been enzymatically removed
leading to exposure of the galactose residues, which are the ASGPR
ligands [142]. The existence of a liver receptor clearing asiaglycopro-
Clinical significance of autoantibodies in primary biliary cholangitis.

teins form the circulation was first reported by Ashwell and Morell in
Antigenic target

1974 [143] and was identified soon thereafter as the ASGPR [144]. It
modifiers

was subsequently shown that ASGPR has a variety of functions, in-

CENP-B

cluding mediating cell entry of hepatitis A and B virus [145,146] and

PDC

elimination from the circulation of activated lymphocytes [147]. Prior

to the identification of their main target antigen, autoantibodies to
ELISA, IB, RIA

ELISA, IB, LIA

ELISA, IB, LIA

ASGPR identified by IIF were known as anti-liver specific protein an-

tibody, whose presence in AIH patients had been shown to correlate
Method of

ELISA, IB
detection

with disease activity [148,149]. There is evidence that ASPGR is a

pathogenic autoantigenic target: anti-ASGPR antibody has been shown
IIF

IIF

IIF

IIF

to be able of inducing immune-mediated hepatocyte injury, ASGPR-

ANA with multiple nuclear dots IIF

langitis; HCV, hepatitis C virus.

specific T-cell clones inducing anti-ASGPR antibody have been re-

ANA with anti-centromere IIF

ported, and ASGPR-specific T cells infiltrating the liver of AIH patients

membranous IIF pattern

have been detected in AIH patients [150,151] [152]. The reported

ANA with nuclear-rim/

prevalence of anti-ASGPR antibody depends on the employed assays.

Purified or recombinant ASGPR is needed to detect anti-ASGPR. Pur-
ified ASGPR from different species has been used as an autoantigenic
pattern

pattern

source in a variety of methodologically different assays, whereby pur-

Antibody

ification procedures may not preserve conformational epitopes, leading

Table 2

AMA

to unsatisfactory quality of the assays [59,142]. Attempts to produce

recombinant ASGPR have been unsuccessful [59]. Reported prevalence

150
B. Terziroli Beretta-Piccoli et al. Journal of Autoimmunity 95 (2018) 144–158

Table 3
Clinical significance of autoantibodies in primary sclerosing cholangitis.
Antibody Method of Antigenic targets Frequency Other hepatic diseases Extrahepatic diseases Clinical significance
detection

pANNA IIF Beta-tubulin isotype 5 and yet 26–94% Autoimmune hepatitis Inflammatory bowel Bile ANCA correlate with disease severity
unknown autoantigens Autoimmune sclerosing disease PSC ANCA-positive patients are younger
cholangitis and have less frequently biliary cancer
ANA IIF dsDNA 8–77% Autoimmune hepatitis Autoimmune conditions AIH overlap to be excluded
SSA/B Autoimmune sclerosing Infections
RNP cholangitis Malignancies
SCL70 Drug-induced disorders
Sm ssDNA
SMA IIF Not studied in PSC 0–83% Autoimmune hepatitis Autoimmune conditions AIH overlap to be excluded
Autoimmune sclerosing Infections
cholangitis Malignancies
Primary biliary Drug-induced disorders
cholangitis
Chronic hepatitis B and C
Acute hepatitis E
Non-alcoholic fatty liver
disease
Drug-induced liver injury

pANNA, perinuclear anti-neutrophil nuclear antibody; ANA, anti-nuclear antibody; SMA, smooth muscle antibody; IIF, indirect immunofluorescence; ANCA, anti-
neutrophil cytoplasm antibody; PSC, primary sclerosing cholangitis; AIH, autoimmune hepatitis.

Table 4
Novel autoantibodies in autoimmune liver diseases.
Antibody Method of Antigenic target Frequency Other epatic diseases Extrahepatic diseases Comments
detection

Anti-GP2 [225] IIF Glycoprotein 2 47–71% in PSC None Crohn's disease Large bile-duct involvement
Ulcerative colitis Increased mortality
Increased cholangiocarcinoma risk
Anti-GW bodies [226] IIF, ALBIA, IB RAP55, GW182, GW2, 2–28% in PBC Not investigated Scleroderma No association with clinical
GRASP phenotypes
Anti-Kelch-like 12 IB, ELISA Kelch-like 12 40% in PBC by Acute liver failure Scleroderma Highly PBC specific
[227] ELISA, 16% by IB PSC Lupus erythematosus
AIH Sjögren's syndrome
Anti-hexokinase 1 IB, ELISA Hexokinase 1 45% in PBC by Acute liver failure Scleroderma Highly PBC specific
[227] ELISA, 16% by IB PSC
AIH
Anti- AHPA9419 [228] DELFIA, IB AHPA9419 45% in AIH Rarely detected in Not investigated Combined sensitivity: 95%
viral hepatitis Combined specificity: 76.2%
Anti-CHAD [228] DELFIA, IB CHAD 52% in AIH Rarely detected in Not investigated
viral hepatitis

IIF, indirect immunofluorescence; IB, immunoblot; ALBIA, addressable laser bead immunoassay; ELISA, enzyme-linked immunosorbent assay; DELFIA, dissociation-
enhanced lanthanide fluorescence immunoassay; PBC, primary biliary cholangitis; AIH, autoimmune hepatitis; PSC, primary sclerosing cholangitis, CHAD, con-
droadherin precursor.

in type 1 AIH ranges from 24 to 82% and in type 2 AIH from 16 to 57%
[59]. Anti-ASGPR may disappear when disease control is achieved with
immunosuppressive treatment [79,142]. Anti-ASGPR is organ-specific
but not disease-specific for AIH, having been reported in a variety of
hepatic diseases, including PBC, drug-induced liver injury, chronic
HCV, HBV and HDV infection, non-alcoholic steatohepatitis and alco-
holic hepatitis [131]. In a study by Lohse et al., eight of 10 AIH patients
seronegative for ANA, SMA, anti-SLA and anti-LKM1 were anti-ASPGR
positive [153]. Probably, anti-ASGPR may play a role in patients with
suspected AIH who are seronegative for conventional AIH auto-
antibodies [13,154]. However, difficulties in establishing reliable as-
says coupled with the absence of disease specificity have hindered their
clinical use.
In PSC, one study reported a prevalence of anti-ASGPR of 33.3%
[59], but other studies reported lower prevalences [155,156]. In PBC,
reported prevalence of anti-ASGPR ranges from 6.2 to 100%
[59,155,156], depending on the employed assays.
9. Anti-neutrophil cytoplasmic antibody
Anti-neutrophil cytoplasmic antibody (ANCA) was first reported in
patients with small and medium-sized vessel vasculitis [157–159].
ANCA is detected by IIF using ethanol fixed human neutrophilic gran-
ulocytes as a substrate, where they have different staining patterns,
namely a diffuse cytoplasmic granular fluorescence (cANCA) or a
perinuclear, often with nuclear extension, fluorescence (pANCA) [160].
While cANCA is found in the majority of patients with granulomatosis
with polyangiitis, pANCA is associated with microscopic polyangiitis
and eosinophilic granulomatosis with polyangiitis [160]. The target
antigen of cANCA is mainly the cytoplasmic protein leukocyte protei-
nase 3, whereas pANCA is directed against myeloperoxidase, which is
an also cytoplasmic protein: the pANCA staining pattern is an artefact
due to the ethanol fixation which leads to migration of positively
charged cytoplasmic proteins to the negatively charged nuclear mem-
brane [31]. If the staining pattern is unaffected by ethanol fixation, the
detected antibody is referred to as atypical pANCA, which is directed
against components of the nuclear envelope, leading to the designation
as perinuclear anti-neutrophil nuclear antibody (p-ANNA)
[31,58,161,162], also referred to as nuclear anti-neutrophil antibodies
(NANA) [163]. It is important to remember that presence of ANA may
interfere with pANCA detection by IIF, since neutrophil nuclei are
stained by ANA, masking the perinuclear staining by pANCA [164].

151
B. Terziroli Beretta-Piccoli et al.
Atypical p-ANCA have been found in patients with PSC, ASC, PBC, in-
flammatory bowel disease and AIH type 1, being absent in type 2 AIH
[165,166,166–170]. An antigenic target has been identified as beta-
tubulin isotype 5, cross-reacting with the bacterial cell division protein
FtsZ [171], suggesting a possible role for bacteria in PSC and AIH pa-
thogenesis [172]. In AIH and ulcerative colitis, other nuclear antigenic
targets of atypical pANCA have been described, namely the high-mo-
bility group non-histone chromosomal proteins HMG1 and HMG2, and
histone H1 [58].
ANCAs are found in up to 94% of PSC patients, no data existing on
ANCA-frequency in small duct PSC [5,173]. A large study in PSC found
that 80% of patients are ANCA-positive, 70% showing pANCA staining
pattern, 5% cANCA and 5% other patterns [5]. The only phenotypic
difference between ANCA-positive and ANCA-negative patients was
diagnosis at a younger age and lower cholangiocarcinoma frequency in
ANCA-positive patients; no differences in clinical phenotypes were
found according to ANCA-subtypes [5]. However, an earlier study with
low patient numbers suggested a more severe disease course in pANCA
positive PSC patients [174]. In addition to the above mentioned mye-
loperoxidase and proteinase 3, which are rarely targeted in PSC, and to
the targets reported in AIH and ulcerative colitis, a variety of antigenic
targets have been reported in ANCA-positive PSC patients, including
bactericidal/permeability increasing protein, lactoferrin, elastase, ca-
thepsin G, catalase, and human lysosomal-associated membrane protein
2 [58]. Of note, one recent study reported a prevalence as high as 38%
of anti-proteinase 3 ANCA in PSC patients, detected by a new chemi-
luminescence test, which correlated with higher serum transaminase
levels [175]. Previously, the frequency of anti-proteinase 3 ANCA was
reported to be low [58]. The same study, reported anti-proteinase 3
ANCA positivity in 11.1% of PBC patients, 21.5% of AIH patients, and
54.5% of AIH-PSC overlap and 30% of AIH/PBC overlap patients [175].
The reported frequency of pANNA in AIH ranges from 40 to 96%,
whereas cANCA is rarely detected [108]. pANNA can occasionally be
the only serological marker in AIH type 1, making them useful in
clinical practice [21]. According to current guidelines, ANCA should be
tested by IIF in cases of suspected AIH who are negative for ANA, SMA,
anti-LKM1, anti-LC1 and anti-SLA/LP antibodies [13,154].
ANCA has been reported in PBC patients, with a prevalence ranging
from 11 to 33%, some studies suggesting a more severe diseases in PBC-
ANCA-positive patients [58,170,175,176].
10. Anti-mitochondrial antibody
The presence of high-titers autoantibodies to tissue homogenates in
the serum of a PBC patient has been first reported in 1958 by Ian
Mackay [177]. Subsequently, it was demonstrated that the reactivity
was abolished by a mitochondrial fraction of rodent hepatic tissue
[178]. In a landmark paper by Walker, Doniach, Roitt and Sherlock
published in 1965 it was shown that sera from 32 PBC patients, tested
by IIF on human tissue sections rich in mitochondria, gave a char-
acteristic staining pattern, while sera from patients with extra-hepatic
bile duct obstruction, drug-induced cholestasis, viral hepatitis and
cholestatic disease associated with ulcerative colitis did not [179].
Based on this strong association, AMA detection has rapidly become
essential in the diagnostic workup of cholestasis, which included in
those early days surgical exploration of the extrahepatic biliary tree.
The IIF technique first described in this paper is still used for AMA
detection, although rat instead of human tissue sections are employed
nowadays [7]. The association of AMA with PBC was confirmed in
subsequent studies, using either complement-fixation or IIF [180,181].
In 1967, it was demonstrated that antibodies in the serum of PBC pa-
tients react with the mitochondrial fraction obtained by differential
centrifugation of liver homogenates [182]. The same group demon-
strated that, in a variety of species, the main target antigen is a 74 kDa
enzymatic protein located on the inner surface of the mitochondrial
inner membrane [183–185]. It was named M2 antigen, this designation
152
Journal of Autoimmunity 95 (2018) 144–158
including other, less frequently detected mitochondrial antigens
[186,187], as opposed to M1, which is the target of anti-cardiolipin
antibody in syphilis [188]. A nomenclature spanning M2-M9 was de-
veloped based on the different anti-mitochondrial reactivities [189],
which is no longer used [37]. The M2 antigens have been identified as
components of the 2-oxo acid dehydrogenase complexes, which are key
enzymes in the mitochondrial respiratory chain, namely the pyruvate
dehydrogenase complex (PDC), the E3 binding protein of PDC, the 2-
oxoglutarate dehydrogenase complex (OGDC) and the branched-chain
2-oxo acid dehydrogenase complex (BCOADC) [37,190]. All of them are
multienzyme complexes consisting of a minimum of three enzymes,
namely E1, E2 and E3, which have a common structure [37]. The
74 kDa target enzymatic protein was cloned by Eric Gershwin in 1987,
using rat liver cDNA libraries, as the E2 subunit of the pyruvate de-
hydrogenase complex (PDC-E2) located on the inner mitochondrial
membrane [191]. This was a major advance in the study of PBC,
leading to the establishment of immune-assays based on the use of re-
combinant or purified antigens. Sera from 80 to 95% of PBC patients
recognize PDC-E2, while reactivity against the E2 subunit of OGDC and
BCOADC is detected in 30–80% and 50–70% of the cases, respectively
[37,192]. Isolated reactivity to one of the latter target antigens is rare,
the majority of patients being positive for anti-PDC-E2 and at least one
of the two minor antigenic targets [37]. Interestingly, all autoantigenic
proteins share similar conformational epitopes, the inner lipoylated
domains [193], which represent the specific epitopic region targeted by
AMA [194]. Recent data suggest that AMA arises, in genetically pre-
disposed individuals, via molecular mimicry between self and en-
vironmental xenobiotics [195].
In IIF on kidney tissue sections, AMA stains preferentially the distal
tubules, which are smaller and mitochondria-richer than the proximal
tubules, whereas on gastric tissue sections, it gives a bright granular
pattern and on liver tissue a faint cytoplasmic stain (Fig. 3, panel A) [7].
This IIF pattern on triple rodent tissue is unique to AMA [37]. AMA
stains the cytoplasm of HEp2 cells by IIF, with a diffuse, granular cy-
toplasmic pattern. However, this pattern may not be consistent with
AMA detected on triple rodent tissue or by molecular based assays, and
therefore the sole use of HEp2 cells for AMA detection is not re-
commended [7]. Immunoblotting, also referred to as Western blot,
identifies AMA by using mitochondrial preparations from different
tissues and species as an antigen source or, more recently, recombinant
antigens [37]. This technique is highly sensitive and specific, but also
very demanding for clinical laboratories [194]. In contrast, ELISA based
on purified or recombinant antigens, is a much more practical assay,
since it is automated, expeditious and allows testing a large number of
samples at once. In 1996, a recombinant antigen called MIT3 co-ex-
pressing the three immunodominant epitopes of PDC-E2, BCOADC-E2
and OGDC-E2 has been developed by Eric Gershwin's group [192].
MIT3 is used as antigen source in Western blot as well as in ELISA, and
shows higher sensitivity than conventional M2-antigens and IIF
[196,197]. This improved sensitivity leads to diminished specificity: on
the one hand, this has the advantage of reducing the number of AMA-
negative PBC patients, but on the other hand may lead to AMA detec-
tion at low titers in conditions such as chronic infections in patients
without PBC [198–201]. Recently, an ELISA based on MIT3 and pur-
ified sp100 and gp210 has been established (PBC screen), and proved to
be highly sensitive and specific (area under the curve of 0.92), in-
cluding positive results in 44% of patients who were AMA-negative by
IIF [202].
AMA is the serological hallmark of PBC, and is considered as the
most specific autoantibody in human pathology [3]. However, the
clinician must be aware that using the particularly sensitive MIT3 assay
AMA is transiently positive in up to 40% of patients with acute liver
failure, probably as a result of oxidative stress [203]. AMA may precede
disease onset by decades, as demonstrated by an early study from the
Newcastle's group, reporting PBC-typical or PBC-compatible liver his-
tology in 24 of 29 AMA-positive subjects despite normal cholestasis
B. Terziroli Beretta-Piccoli et al.
indices [204]. On a median follow-up of 17.8 years, 83% developed
overt PBC [205]. These results were confirmed by a study from Estonia,
reporting that three of eight subjects with isolated AMA positivity de-
veloped PBC over a nine-year observation period [206]. More recently,
a large, nationwide study from France based on positive AMA in la-
boratory reports, found a 5-year PBC-incidence of 16% in asymptomatic
AMA-positive subjects with normal alkaline phosphatase [207]. Taken
together, these data indicate that AMA-positive subjects without PBC
deserve long-term follow-up, since they are at risk of developing PBC.
Data on correlation of AMA with disease activity have been con-
flicting. A study published in 1990 reported a correlation of AMA titers,
measured by an ELISA using purified PDC-E2 and PDC-E3 binding
protein, with histological activity, as well as with albumin and bilirubin
serum levels [208], in line with previous data reporting an increase of
AMA titers with disease progression [209,210]. A Greek study found a
correlation between IgG3 subclass AMA and the PBC Mayo score
[54,211], whereas another study from the same group demonstrated a
correlation between IgG and IgA AMA titers, assessed by MIT3-ELISA,
and the PBC Mayo score [196]. A study in AMA-positive subjects with
and without PBC, found that patients with well-defined PBC had higher
avidity anti-PDC-E2 and higher IIF-AMA titers [212]. Interestingly, the
trial first describing the beneficial effect of ursodeoxycholic acid
(UDCA) in PBC, published in 1991, reported a significant decrease in
AMA titers in patients treated with UDCA [213]. These data have not
been confirmed by other reports, which were unable to demonstrate a
correlation between AMA titers and disease progression/prognosis
[214–216]. A recent study analyzing serial serum samples of 27 PBC
patients did not find a correlation between AMA titers, assessed with a
MIT3-ELISA, and histological features and clinical outcomes [217].
11. Clinical significance of autoantibodies in autoimmune liver
diseases
The clinical significance of autoimmune serology is presented in
Tables 1–3. Table 4 summarizes recently reported autoantibodies which
are not yet part of the recommended serological work up in patients
with suspected autoimmune liver disease.
The importance of ruling out viral hepatitis in patients presenting
with acute or chronic hepatitis cannot be stressed enough. Besides acute
hepatitis A, acute and chronic HBV, including delta superinfection, and
HCV infection, an emerging cause of acute hepatitis in Western coun-
tries is hepatitis E virus (HEV) infection. Acute HEV infection is asso-
ciated with a high frequency of autoantibodies [32], leading to AIH
misdiagnosis in a number of case reports [218–222], with inappropriate
immunosuppressive treatment.
If AIH is suspected, autoimmune serology should be investigated by
IIF on triple rodent tissue and by a molecular based assay for anti-SLA
[13,154]. In some cases, particularly in patients presenting with acute
hepatitis, the initial serological work up may be negative and may turn
positive during follow up [20]. It is thus advisable to retest seronegative
patients on a later time. Positive ANA sera should be further char-
acterized on HEp2 cells: rim-like/membranous or multiple nuclear dots
IIF patterns are diagnostic for PBC, whereas homogeneous or speckled
patterns are indicative, but not diagnostic, of AIH [21]. These two PBC-
specific ANA subtypes, particularly ANA giving a rim-like/membranous
pattern on HEp2 cells, are associated with an unfavorable clinical
course [50,52,56,223]. The recent EASL guidelines recommend solely
to use anti-gp210 as a prognostic marker [4]. Detection of AMA is
highly suggestive of PBC or, in absence of established liver disease,
identify patients at high risk of developing PBC in the long term. AMA
coexisting with typical AIH positivities suggests AIH/PBC variant syn-
drome [224]. If SMA is detected, it is advisable that the laboratory
reports include the IIF pattern on kidney tissue, which is a very im-
portant information for the clinician, allowing to support the AIH di-
agnosis if AIH-specific patterns are detected. The presence of anti-LKM1
and/or anti-LC1 is diagnostic of type 2 AIH, provided that HCV
153
Journal of Autoimmunity 95 (2018) 144–158
infection has been ruled out. Anti-SLA is diagnostic for AIH, although
rare cases of HCV seropositive for anti-SLA have been reported [132].
Detection of pANNA in a patient with acute hepatitis of unknown origin
points toward the diagnosis of AIH, and should prompt a work up to
rule out ASC, PSC and IBD due to its strong associations with these
conditions.
12. Concluding remarks
Autoimmune liver serology is an important ally to the clinician in
diagnosing and managing patients with autoimmune liver disease.
Within the clinical context, a basic knowledge of the laboratory meth-
odologies and a close interaction between physician and laboratory are
essential to maximize the value of this powerful tool.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jaut.2018.10.004.
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