Autoimmune liver diseases

Serological diagnostics of autoimmune hepatitis, primary biliary

cholangitis and primary sclerosing cholangitis

 IIFT Liver Mosaics

 Anti-M2-3E ELISA, Anti-LKM-1 ELISA, Anti-LC-1 ELISA, Anti-SLA / LP ELISA
 EUROLINE Autoimmune Liver Diseases
 EUROASSAY Liver Profile (Anti-M2, -LKM-1, -LC-1, -SLA / LP)
Autoimmune liver diseases
Autoimmune liver diseases mainly include three different disease images: autoimmune hepatitis (AIH), primary biliary cholangi-
tis (PBC) and primary sclerosing cholangitis (PSC). Out of these three diseases, AIH is the most frequent, with a prevalence of 17
cases per 100,000 persons, followed by PBC and PSC. AIH and PBC mainly occur in women during menopause, AIH can also
affect children and young adults. PSC mainly affects men from 20 to 40 years of age. In most cases, the three diseases can be
reliably distinguished by serological analysis. However, overlap syndromes may occur in which patients present symptoms of
two autoimmune liver diseases.

The detection of specific autoantibodies allows precise differentiation between autoimmune liver diseases and infectious, toxic
and other forms of hepatitis. AIH is often associated with chronic inflammatory rheumatic systemic diseases such as rheumatoid
arthritis, Sjögren’s syndrome and systemic lupus erythematosus. The majority of patients respond excellently to anti-inflamma-
tory or immunosuppressive treatment, which should be initiated promptly after the diagnosis is made.

Autoimmune hepatitis (AIH)

AIH is a progressive chronic form of hepatitis which occurs
Autoimmune hepatitis
in phases and leads to destruction of the hepatocytes. The
aetiology of the disease has not yet been clarified. However, Associated autoantibodies Prevalence
a connection with infections with hepatitis, measles, cyto- Type 1 AIH
megalo- or Epstein-Barr viruses have been discussed. While
ASMA type F-actin 70 – 80 %
some patients show no to only mild symptoms, fulminant
liver failure can also occur. The most frequent complaints ANA 70 – 80 %
encompass stomach ache, pruritus, nausea, anorexia and SLA / LP ~ 20 %
general malaise. Type 2 AIH
LKM-1 1– 3 %
In laboratory diagnostic analyses, AIH patients show ele-
vated levels of transaminases and bilirubin as well as an LC-1 1–3%
increase in the total IgG titer. In at least 80 % of AIH patients, SLA / LP ~ 20 %
autoantibodies are also detected. These allow distinction of
two AIH types (see table). Delimitation from other chronic
forms of hepatitis and consequently a clear diagnosis of AIH
is essential since untreated AIH is associated with a five-year
mortality rate of 50 %.

Primary biliary cholangitis (PBC)

PBC is characterised by progressive inflammation-mediated
Primary biliary cholangitis
destruction of the small intrahepatic bile ducts which leads
to cholestasis and fibrosis. Liver cirrhosis and liver failure are Associated autoantibodies Prevalence
possible consequences of the disease. The symptoms are AMA-M2 90 – 95 %
very similar to those of AIH. With PBC, the laboratory analy-
ANA (nuclear dots): PML, Sp100 25 – 40 %
ses yield an increase in cholestatic enzymes, elevated immu-
noglobulin values (mainly IgM) and the presence of different ANA (nuclear envelope): gp210, p62 25 – 40 %
autoantibodies.

Primary sclerosing cholangitis (PSC)

PSC is a chronic fibrosing inflammation of the intra- and
Primary sclerosing cholangitis
extrahepatic bile ducts. Over the course of disease, liver cir-
rhosis develops, requiring liver transplant within 7 to 12 years. Associated autoantibodies Prevalence
Around 60 to 80 % of PSC patients also suffer from the Atypical pANCA (DNA ANCA) 42 %
chronic inflammatory bowel disease ulcerative colitis. Atypi-
cal (p)ANCA (DNA ANCA) are characteristic markers of both diseases, but do not represent a diagnostic proof. Approximately
5% of the patients show clinical, laboratory and histological signs which can be assigned to PSC, but have an inconspicuous
cholangiogram (small-duct PSC).

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Diagnostics of autoimmune liver diseases
In suspected cases, abdominal ultrasound is indicated for initial diagnostics.1 Increased alanine aminotransferase (ALT),
aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), alkaline phosphatase (AP), glutamate dehydrogenase
(GLDH), bilirubin or albumin values can also indicate liver disease.1 If the clinical suspicion of autoimmune liver disease is
substantiated, specific autoantibodies should be determined. Serological diagnostics of AIH, PBC and PSC encompasses:

 Quantification of the total immunoglobulins of classes IgG, IgM and IgA for assessment of the selective increase 1,2

 Detection of autoantibodies in serum by means of IIFT: 1,2

 ANA, ASMA, AMA and LKM-1 on a combination of three rat tissue sections: stomach, liver and kidney
 PBC-associated ANA against Sp100, gp210 and centromeres by means of HEp-2 cells
 ASMA against F-actin using VSM47 cells
 Anti-SLA / LP antibodies using transfected cells

 Monospecific antibody detection in serum by ELISA or immunoblot

Positive serological findings indicating the diagnosis AIH should be verified by liver biopsy. In PBC diagnostics, biopsy is only
recommended with unclear findings.1 The International AIH Group (www.iaihg.org) has developed simplified diagnostic crite-
ria for AIH based on antibody titers, histology and the exclusion of viral hepatitides.3 For diagnostics of paediatric AIH, a
modified IAIHG scoring system should be applied. 1 The AIH-PCB overlap syndrome lacks generally accepted diagnostic crite-
ria.1

Serological test systems for diagnostics of autoimmune liver diseases

Indirect immunofluorescence Monospecific tests, e.g. ELISA or blot

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Serological diagnostics by means of immunofluorescence
IIFT Liver Mosaics
The combination of the IIFT substrates HEp-2, kidney, liver and stomach on one BIOCHIP allows simultaneous detection of
the antibodies relevant for diagnostics of autoimmune liver diseases. Positive samples show specific fluorescence patterns,
e.g. of the cell nuclei and tissue structures.

Detection of ASMA type F-actin by means of the IIFT Liver Mosaic 8: The IIFT substrates stomach and VSM47 cells ena-
ble simultaneous detection and differentiation of ASMA type F-actin and type non-actin. With frozen sections of rat stomach
ASMA show a distinct cytoplasmic fluorescence of the tunica muscularis as well as the lamina muscularis mucosae and the
interglandular contractile fibrils of the tunica mucosa. If specific antibodies against F-actin are present, there is a fibrillar flu-
orescence of the cytoskeleton of the VSM47 cells.

Primate liver Rat liver VSM47 HEp-2 Rat kidney Rat stomach

Detection of AMA and ANA using the IIFT Liver Mosaic 8: For the detection of AMA, rat kidney is used as the standard
substrate. The cytoplasm of the proximal and distal tubule cells shows a granular, basally emphasised fluorescence. The glo-
meruli are only weakly stained by AMA. AMA show a weaker reaction with liver tissue and lead to a fluorescence of the
proximal and distal renal tubules and the parietal cells. Liver tissue and especially HEp-2 cells, however, are particularly suited
for ANA detection and differentiation.

Primate liver Rat liver VSM47 HEp-2 Rat kidney Rat stomach

Suitability of IIFT substrates for the determination of autoantibodies

Substrate
Antibodies SLA / LP Primate
HEp-2 VSM47 Rat kidney Rat stomach Rat liver Primate liver
transf. cells heart
AMA M2 ++ + + ++ + + + +
AMA-M7 – – – – – – – ++
AMA-M9 + – – – – – – –
Nuclear dots ++ + + + + + ++ +
Nuclear envelope ++ + + + + + ++ +
Centromeres ++ + + – – – + –
SS-A / SS-B ++ + + – – – + –
Scl-70 ++ + + + – – + –
Nuclear homogeneous ++ + + + + + ++ +
ASMA type non-actin – – – + ++ + – –
ASMA type F-actin – – ++ – – – – –
LKM – – – ++ – ++ + –
LC-1 – – – – – ++ – –
SLA / LP – ++ – – – – – –
++ well suited + suited with limitations – not suited

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Monospecific antibody detection by ELISA or EUROLINE
Anti-M2-3E ELISA (IgG)
The detection of AMA is of great importance in the diagnosis
Designer antigen BPO
of PBC. Antibodies against the M2 antigen represent the
most sensitive and specific diagnostic marker. The Anti-
M2-3E ELISA uses the native PDH complex and the designer
(His)8 BCOADH-E2 PDH-E2 OGDH-E2
antigen BPO as antigen substrate. The combination of artifi-
cial polypeptide PBO and native PDH increases the sensitivity
by 14 % compared to the classic Anti-M2 ELISA. The AMA
target antigens belong to the enzyme family of ketoacid
dehydrogenase complexes from the mitochondrial respiratory chain. The lipoyl-binding regions (E2) of these enzyme com-
plexes are the main autoantigens in PBC. The recombinant polypeptide His-BPO consists of the E2 subunits of the branched
chain keto acid dehydrogenase complex (BCOADH), the pyruvate dehydrogenase (PDH), and the ketoglutarate dehydroge-
nase (OGDH) and an N-terminal His-tag.

Sera from 251 PBC patients, 15 patients with PBC / AIH overlap syndrome and 1,129 control individuals were investigated with
the EUROIMMUN Anti-M2-3E ELISA (IgG). The sensitivity of the Anti-M2-3E ELISA (IgG) for PBC was 93.2 % with a specificity
of 97.9 %.4

Anti-SLA / LP ELISA (IgG)

The ELISA test kit provides a semiquantitative or quantitative in vitro detection of human antibodies of the IgG class against
SLA / LP in serum or plasma. Autoantibodies against SLA / LP have the highest diagnostic relevance for AIH.5 The presence of
anti-SLA autoantibodies in AIH patients is of prognostic relevance since they seem to be associated with greater occurrence
of relapses, poor prognoses, relapses after liver transplantation and pregnancy complications.6 In AIH, anti-SLA / LP antibodies
usually occur as the only specific antibodies. While their prevalence is only between 10 and 30 %, their predictive value is
nearly 100 %. Basically every positive anti-SLA / LP result confirms an AIH as long as the corresponding clinical symptoms are
present. Anti-SLA / LP autoantibodies cannot be reliably detected by IIFT on tissue sections, but only by means of monospe-
cific tests such as ELISA, immunoblot or IIFT with transfected cells.

Sera from 454 AIH patients, 147 patients with other liver diseases and 200 blood donors were investigated using the EURO-
IMMUN Anti-SLA / LP ELISA (IgG). The specificity of the ELISA was 100 %.

Anti-LC-1 ELISA (IgG)

The Anti-LC-1 ELISA allows semiquantitative determination of anti-LC-1 antibodies. Anti-LC-1 antibodies in AIH can occur
independently of or together with other autoantibodies. Especially in cases of anti-LKM-1 negative serology, anti-LC-1 anti-
bodies indicate type 2 AIH.

Sera from 93 AIH patients, 183 patients with other liver diseases including PBC, and 200 blood donors were investigated
using the EUROIMMUN Anti-LC-1 ELISA. The prevalence of antibodies against LC-1 in the panel of autoimmune hepatitis
amounted to 5.4 %, at a specificity of 100 %.

Anti-LKM-1 ELISA (IgG)

Detection of autoantibodies against LKM is part of routine AIH diagnostics. Antibodies against LKM-1 (target antigen:
cytochrome P450 IID6) occur especially in children, while they are only found in 1 % of adult AIH patients. Antibodies against
LKM-1 are also detectable in 1 to 2 % of patients with positive hepatitis C serology. In patients with AIH, autoantibodies
against LKM-1 typically do not occur together with antibodies against SLA / LP. Testing both parameters can increase the
serological hit rate for AIH. The parallel determination of other AIH-associated autoantibodies, such as ANA, pANCA, ASMA
and antibodies against LC-1 and SLA / LP is recommended for differentiation from viral hepatitis. 5

18 sera from AIH patients and 489 serum samples from a reference laboratory were investigated using the EUROIMMUN
Anti-LKM-1 ELISA (IgG) and an IIFT Mosaic with the substrates rat liver and kidney (IgG) as a reference method. The specific-
ity of the ELISA amounted to 99.4 % with a sensitivity of 100 % with respect to the IIFT.

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Multiparametric EUROLINE Profiles
The EUROLINE Profiles for autoimmune liver diseases are
EUROIMMUN EUROLINE Profile
qualitative in vitro immunoassays for detection of antibodies
against liver antigens in human serum or plasma. They allow

PM 0 O)
10 BP
simultaneous qualitative determination of several antibodies

2- 2
M -M

SS / L P
Sp E (

CE P A
PG P B

l
L C -1

ro
LK 10

CE 0

H
Sc 2
3
A

l-7
Ro-A
M

-5
gp L

-1

nt
in one incubation.

N
A

N
2
AM

SL

Co
The EUROLINE Profile Autoimmune Liver Diseases encom-
passes not only the AIH- and PBC specific parameters but
also phosphoglycerate dehydrogenase (PGDH). Determination of antibodies against PGDH facilitates the differentiation
between AIH and HBV infection. In a study, 31% of tested AIH and 9 % of tested PBC patients showed autoantibodies against
PGDH in the EUROLINE assay, while none of the HBV patients presented autoantibodies against PGDH.7

Villalta and colleagues conducted a study on PBC-specific autoantibody profiles and tested sera from 58 PBC patients,
144 patients with other autoimmune liver diseases, and 67 patients with chronic, non-autoimmune liver diseases using the
EUROLINE Autoimmune Liver Diseases Profile 2 and an IIFT Mosaic with HEp-2 cells and rat kidney, liver, and stomach.8 Due
to the high sensitivity and specificity of the test, the authors concluded that the EUROLINE was a well suited assay for con-
firmation of IIFT results or for evaluation of cases with unclear clinical image and / or serological finding. In specialised refer-
ence laboratories where PBC-associated autoantibodies are often detected, the EUROLINE is a good option for primary test-
ing since it allows simultaneous determination of all relevant PBC-specific antibodies and improved and faster definition of
AMA subspecificities.

Serodiagnostic guidelines for autoimmune liver diseases

Clinical suspicion of hepatitis Clinical suspicion of cholangitis

Chronic fluctuating increase in ALT and AST Repeatedly increased GGT and / or AP

Inconspicuous bile ducts, Conspicuous bile ducts,

no CIBD known CIBD

Autoimmune serology
Detection of autoantibodies in the IIFT screening test
Confirmation in the monospecific test (e.g. ELISA, immunoblot)

Repeated testing or other

– diagnosis?

MRCP

   – 
ASMA F-actin Anti-LKM -1 AMA ANCA
ANA Anti-LC-1 Nuclear dots
Anti-dsDNA Anti-SLA / LP Nuclear envelope
Anti-SLA / LP ANCA Biopsy

Type 1 AIH Type 2 AIH PBC Small-duct PSC PSC

ALT: alanine aminotransferase, AST: Aspartate aminotransferase, GGT: gamma-glutamyltransferase, AP: alkaline phosphatase,
CIBD: chronic inflammatory bowel disease, MRCP: magnetic resonance cholangiopancreatography

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Helpful information for serological diagnostics
 IgG serum levels and anti-actin autoantibody titers can correlate with the activity of AIH. AMA titers, however, do not
correlate with the disease activity of PBC.

 Acute / fulminant AIH may lack autoantibodies and IgG increase.

 In the IIFT, sera are usually tested in a 1:100 dilution. Antibody titers up to 1: 80 may occur in adults even without pres-
ence of an autoimmune disease. In children and adolescents, however, titers from 1:10 may be clinically relevant.5

 Serological AIH diagnostics should include determination of thyroid stimulating hormone (TSH), since AIH is often asso-
ciated with autoimmune thyroiditis.1

 Moreover, AIH is often associated with chronic inflammatory rheumatic systemic diseases (e.g. systemic lupus erythe-
matosus, rheumatoid arthritis, Sjögren’s syndrome).1

 A positive AMA finding in the ELISA with negative IIFT is rare, but may be explained by greater sensitivity of the ELISA
or detection of infection-associated AMA.

 With suspected PBC, ANCA detection is recommended alongside serological testing for AMA and ANA.

Order information
Test system Test name Antibodies against Substrate Order number
Cell nuclei (ANA)
HEp-2 cells (human)
Liver antigens, cell nuclei (ANA)
Liver (monkey)
LKM + cell nuclei (ANA) FA 1300-####-1 to
Liver (rat)
LKM + mitochondria (AMA) -21
Kidney (rat)
IIFT: Liver Mosaics Smooth muscle FA 1710-####
Stomach (rat)
Striated muscle FA 1651-####
M. iliopsoas (monkey)
F-actin FA 1302-####-50
VSM47 cells
Soluble liver antigen / liver pancreatic
IIFT Transfected cells (EU90)
antigen (SLA / LP)

Cell nuclei (ANA) HEp-2 cells (human)

IIFT: HEp-2 / liver FA 1510-####-1
(ANA global testing) Liver (monkey)

IIFT: Kidney Mitochondria (AMA) Kidney (mouse) FA 1621-####

Anti-M2 IIFT M2 antigen M2 BIOCHIPs (pig) FA 1622-####

Anti-M2-3E ELISA (IgG) AMA M2 EA 1622-9601 G

Anti-LKM-1 ELISA (IgG) LKM-1 EA 1321-9601 G

Antigen-coated microplate
ELISA
wells
Anti-LC-1 ELISA (IgG) LC-1 EA 1307-9601 G

Anti-SLA / LP ELISA (IgG) SLA / LP EA 1302-9601 G

AMA-M2, M2-3E, sp100, PML, gp210,

Autoimmune
LKM-1, LC-1, SLA / LP, SS-A, Ro-52, DL 1300-####-5 G
Liver Diseases 14 Ag
Scl-70, CENP A, CENP B, PGDH

Antigen-coated test strips

EUROLINE EUROLINE Autoimmune AMA-M2, M2-3E, Sp100, PML, DL 1300-####-9 G
Liver Diseases 9 Ag plus gp210, LKM-1, LC-1, SLA/LP, Ro-52,
F-actin F-actin Soon available!

AMA Profile (IgGM) AMA-M2, M2-3E, M4, M9 DL 1620-####-1 O

EUROASSAY Anti-LKM-1, Anti-SLA / LP LKM-1, SLA / LP Antigen-coated test strips DA 1300-####-1 G

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 References
1
Strassburg C et al., Leitlinie Autoimmune Lebererkrankungen, AWMF-Reg. Nr. 021-27, Zeitschrift für Gastroenterologie 55(11):1135-1226 (2017)
2
Vergani D et al., Unusual Suspects in Primary Biliary Cirrhosis, Hepatology 39(1):38-41 (2004)
3
Hennes E et al., Simplified Criteria for the Diagnosis of Autoimmune Hepatitis, Hepatology 48(1):169-176 (2008)
4
Dähnrich C et al., New ELISA for detecting primary biliary cirrhosis-specific antimitochondrial antibodies, Clinical Chemistry 55(5):978–85 (2009
5
Terziroli Beretta-Piccoli B et al., Serology in autoimmune hepatitis: A clinical-practice approach, European Journal of Internal Medicine 48:35-43 (2018)
6
Efe C et al., Antibodies to soluble liver antigen in patients with various liver diseases: A multicentre study, Liver International 33(2):190-196 (2013)
7
Xiang D et al., Detection of D-3-phosphoglycerate dehydrogenase autoantibodies in patients with autoimmune hepatitis: Clinical significance
evaluation, Hepatology Research 41(9):867-876 (2011)
8
Villalta D et al., Autoantibody profiling of patients with primary biliary cirrhosis using a multiplexed line-blot assay, Clinica Chimica Acta 438:135-138
(2015)

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