Role of Immunological Markers in Diagnosis of
Autoimmune Liver Disease
Lusiana Lim Product Manager
HASLD
Hanoi 16th December 2018
EUROIMMUN (South East Asia) Pte Ltd
Autoimmunity
Agenda
1. Types of ALD
2. EASLD Serological Guidelines for ALD Diagnosis
3. EUROIMMUN’s Serological Diagnostic Tests
4. Conclusion
Types of
Autoimmune Liver Diseases
Autoimmune Liver Diseases
Autoimmune hepatitis (AIH) Primary biliary cirrhosis
(PBC)
Primary sclerosing
cholangitis (PSC)
Types of Autoimmune Liver Disease
AIH-PBC
AIH-PSC
8~9%
6~8% AIH-cholestatic syndrome
Overlap Syndrome
> occurs in 3% to 17% of patients with ALD
Albert J. Czaja M.D Clinical Liver Disease, Vol 3, No 1, January 2014
International Guidelines
American Association for The Study of Liver Diseases
June 2010
European Association for the Study of
Liver
EASL Clinical Practice Guidelines:
1. Autoimmune hepatitis (June 2015)
2. Primary Biliary Cholangisit (July 2017)
June 2015
The identification of AIH as the aetiology of acute hepatitis
and/or fulminant hepatic failure is very important because a
delay in diagnosis and thus delay of initiation of therapy results
in poorer prognosis of AIH, whereas administration of
immunosuppression with steroids might avoid the need for liver
transplantation.
EASL CPG AIH. J Hepatol 2015;63:971–1004
Diagnosis of AIH
*Possibly more severe
EASL CPG AIH. J Hepatol 2015;63:971–1004
Sub-type Features
AIH-1 • Almost 90% of AIH cases
• Detection of ANAs, SMAs or anti-SLA/LP
• Association with HLA DR3, DR4 and DR13
• Any age at onset
• Variable clinical and histopathological severity
• Usually excellent treatment response,
but variable relapse rates after drug
withdrawal and need for long-term
maintenance therapy
AIH-2 • Up to 10% of AIH cases
• Anti-LKM1, anti-LC1 and rarely anti-LKM3
• Association with HLA DR3 and DR7
• Onset usually in childhood/young adulthood
• Clinical and histopathological severity
commonly acute and advanced
• Sometimes failure of treatment and
frequent relapse rates after drug
withdrawal; need for
long-term maintenance therapy
very common
AIH-3 • Up to 10% of cases
• Only SLA/LP positive
• Otherwise very similar to AIH-1*
• Often Ro52-antibody positive
• Lifelong immuno-suppression in
most, if not all patients
New Sub-classification of AIH
Suggested diagnostic algorithm for AIH
*Test also for elevated IgG levels; †These antibodies are highly specific for PBC diagnosis EASL CPG AIH. J Hepatol 2015;63:971–1004
Positive
Negative
Liver disease of unknown origin
IFL autoantibody test on
rodent tissue sections
+ SLA/LP (ELISA or blot)
ANA+ SMA+ LKM1/
LC1+
Test
Negative
SLA/
LP+
Consider AIH* Clinical suspicion*
remains
Consider alternate
diagnoses or
Autoantibody-
negative AIH
Repeat testing in
specialty lab
(including pANCAs and specific
immunoassays for LKM1, LKM3,
LC1, SLA/LP, F-actin, Ro52,
gp210†, sp100†)
Consider AIH
Liver
biopsy
Recommended to use:
Rat kidney stomach for SMA
Rat liver kidney stomach or
blot for LKM1 & LC1
IAIHG simplified scoring system (2008)
Hennes EM, et al. Hepatology 2008;48:169–76; EASL CPG AIH. J Hepatol 2015;63:971–1004
Feature/parameter Discriminator Score
Antibodies (max 2 points)
ANA or SMA+
ANA or SMA+
or LKM+ or
SLA/LP+
≥1:40
≥1:80
≥1:40
Any titre
(02 points total)
+1
+2
+2
+2
IgG or γ-globulins level >ULN
>1.1x ULN
+1
+2
Liver histology
(evidence of hepatitis is required)
Compatible with AIH
Typical of AIH Atypical
+1
+2
0
Absence of viral hepatitis No
Yes
0
+2
Score ≥7 = Definite AIH
Score ≥6 = Probable AIH
Diagnostic criteria for routine clinical use
Guideline statements*
AIH is a clinical diagnosis. Diagnosis of AIH relies particularly on the presence of
autoantibodies, hypergammaglobulinaemia and typical or compatible histology II-2
Elevated IgG levels, especially in the absence of cirrhosis, are a distinctive
feature of AIH. A selectively elevated IgG in the absence of IgA and IgM elevation is II-3
particularly suggestive of AIH
Normal IgG or γ-globulin levels do not preclude the diagnosis of AIH.
Most of these patients demonstrate a fall of IgG levels upon treatment III
Circulating non-organ-specific antibodies are present in the vast majority of AIH
patients. Autoantibody profiles have been used for sub-classification of AIH.† The II-2
clinical implications arising from this sub-classification are uncertain
Indirect immunofluorescence is the test of choice for the detection of ANA, SMA,
LKM and LC-1 autoantibodies. Immunoassays‡ are the tests of choice for the III
detection of SLA/LP autoantibodies. Methods and cut-off values should be reported
by the laboratory
•
•
•
•
*Statement numbers: 10–14 † AIH-1 (ANA and/or SMA positive); AIH-2 (LKM1, LKM3 and/or LC-1 positive); AIH-3 (SLA/LP positive); ‡ELISA/Western blot
EASL CPG AIH. J Hepatol 2015;63:971–1004
Diagnosis Guidelines Statement
Grade of recommendation
Indirect immunofluorescence is the test of choice for the detection
of ANA, SMA, LKM and LC-1 autoantibodies. Immunoassays‡ are
the tests of choice for the detection of SLA/LP autoantibodies.
Methods and cut-off values should be reported by the laboratory
Summary for AIH Serology Testing
1. Immunofluorescence is the recommended gold standard
for detection of:
ANA
SMA (Rat kidney, Rat stomach)
Anti-LKM and Anti-LC1 (Rat liver, kidney, stomach)
2. Detection of Anti-SLA/LP by solid immunoassay
ANA
HEp-2
LC-1 Rat liver
AMA
Rat kidney
Miniaturization and standardization of IFA (high lot consistency)
Combination of different or differently fixed substrates in one test field
BIOCHIP Mosaics for analysis of antibody profiles or screening and
confirmation in one step
More than 300 substrates already available
BIOCHIP technology
Single Slide Screening of AIH Type I & II Liver Mosaic
Liver Mosaic Profile 8
Hep-2 cells
Kidney (rat)
Stomach (rat)
Liver (primate)
Liver (rat)
VSM47 (cells) HEp-2 / Liver primate
Kidney rat / Liver rat
Stomach rat / VSM47
FA 1800-####-8
VSM47 (cells)
Anti-F-actin
Takashi Himoto • Mikio Nishioka Autoantibodies in liver disease: important clues for the diagnosis, disease activity and
prognosis. 2013. Autoimmune Highlights DOI 10.1007/s13317-013-0046-7
Autoantibodies for ALD
ANA
HEp-2
Homogenous,
granular pattern
Antibodies against F-actin (VSM47 cells)
Anti-F-actin
Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International
Autoimmune Hepatitis Group Vergani, Diego et al. Journal of Hepatology , Volume 41 , Issue 4 , 677 - 683
Summary for AIH Serology Testing
1. Immunofluorescence is the recommended gold standard
for detection of:
ANA
SMA (Rat kidney, Rat stomach)
Anti-LKM and Anti-LC1 (Rat liver, kidney, stomach)
2. Detection of Anti-SLA/LP by solid immunoassay Sub-type Features
AIH-3 • Up to 10% of cases
• Only SLA/LP positive
• Otherwise very similar to AIH-1*
• Often Ro52-antibody positive
• Lifelong immuno-suppression
in most, if not all patients
SLA/LP cannot be detected on IIFT
The Lancet (1987)
Baeres, Herkel, Czaj et.al 2002. Establishment of standardised SLA/LP immunoassays: specificity for autoimmune hepatitis, worldwide occurrence, and
clinical characteristic. Gut 2002;51:259–264
Manns et. al The Lancet. 1987 Feb 7;1(8528):292-4. Characterisation of a new subgroup of autoimmune chronic active hepatitis by autoantibodies against a
soluble liver antigen. DOI:https://doi.org/10.1016/S0140-6736(87)92024-1
Detection of SLA/LP
should be via solid
phase immunoassay
Baeres et. Al (2002) Gut Journal
Special patient populations: AIH and pregnancy
*MMF is contraindicated in pregnancy
EASL CPG AIH. J Hepatol 2015;63:971–1004
Characteristics
Manifestation • AIH can manifest within a few months after pregnancy/delivery
• AIH can also manifest during pregnancy, though rarely
Management • Maternal and foetal complications are similar to general population
• Anti-SLA/LP antibodies and anti-Ro52 antibodies seem to be associated with a higher rate
of spontaneous abortion/congenital heart block
• Immunosuppressive therapy with azathioprine plus/minus predniso(lo)ne is not a
contraindication for pregnancy,* and patients should be counselled accordingly
• The risks for mother and baby are higher when treatment is stopped and a disease flare
occurs, than on continued treatment
• Disease activity is usually milder during pregnancy (as in other immune-mediated
diseases), and may thus allow a dose reduction of immunosuppression
• As flares after delivery are common, transient increase in
immunosuppression after delivery may be considered
Anti-SLA/LP specific for AIH
ANA or
ASMA
Anti-SLA/LP
none ASMA
Anti-
LKM
none Anti-LKM
AIH Hepatitis B
ANA or
none ASMA
Hepatitis C
ANA or
Anti-SLA antibodies have high specifity (98.9%) for AIH!
Wen-Chao et al. Meta-Analysis: Diagnostic Accuracy of Antinuclear Antibodies, smooth Muscle Antibodies and Antibodies to a Soluble Liver Antigen/Liver Pancreas in
Autoimmune Hepatitis. PLOS. 2014 https://doi.org/10.1371/journal.pone.0092267
IAIHG simplified scoring system (2008)
Score
Antibodies (max 2 points) (02 points total)
ANA or SMA+ ≥1:40 +1
ANA or SMA+ ≥1:80 +2
or LKM+ ≥1:40 +2
or SLA/LP+ Any titre +2 IgG or γ-globulins level
>ULN +1
>1.1x ULN +2
Liver histology Compatible with AIH +1
(evidence of hepatitis is required) Typical of AIH +2
Atypical 0 Absence of viral hepatitis No
Yes
0
+2
Score ≥7 = Definite AIH Score ≥6 = Probable AIH
Hennes EM, et al. Hepatology 2008;48:169–76; EASL CPG AIH. J Hepatol 2015;63:971–1004
• Diagnostic criteria for routine clinical use
Feature/parameter Discriminator
Detection ELISA for anti-SLA/LP
EA 1302-9601 G
EUROLINE for ALD
DL 1300-1601-2 G DL 1300-1601-4 G DL 1300-1601-5 G
Liver Profile 2 EUROLINE Profile ALD EUROLINE ALD 14 Ag
Autoimmune Liver Diseases
Autoimmune hepatitis (AIH) Primary biliary cirrhosis
(PBC)
Primary sclerosing
cholangitis (PSC)
Type 1 Type 2
ANA
ASMA
F-actin
LKM-1
LC-1
Type 3
SLA/LP
Autoantibodies in ALD
OR
July 2017
2017 EASL Guidelines for PBC Diagnosis
Recommendations*
Perform serological screening for AMA and PBC-specific ANA by
immunofluorescence in all patients with unexplained cholestasis III 1
In adults with cholestasis and no likelihood of systemic disease, an elevated
ALP plus AMA at a titre >1:40 is diagnostic III 1
In the correct context, a diagnosis of AMA-negative PBC can be
made in patients with cholestasis and specific ANA immunofluorescence
(nuclear dots or perinuclear rims) or ELISA results (sp100, gp210)
III
1
Liver biopsy not required for diagnosis of PBC, unless PBC- specific
antibodies absent, coexistent AIH or NASH suspected, or other (usually
systemic) comorbidities are present
III
1
PBC should be suspected in patients with persistent cholestatic serum liver tests or symptoms
Including pruritus and fatigue
PBC and PSC Diagnostic Guidelines
Structured algorithm to diagnose chronic* cholestasis
•
•
*Lasting for >6 months
EASL CPG PBC. J Hepatol 2017;67:145–72
Elevated serum ALP/GGT
and/or conjugated bilirubin
(HBsAg and anti-HCV negative)
History, physical examination,
abdominal US
AMA, ANA
(anti-sp100, anti-gp210)
Extended imaging
MRCP (± EUS)
Liver biopsy
Genetics
Observation/re-evaluation
No abnormalities
No abnormalities
No abnormalities
Negative and no specific drug history
DIAGNOSIS
ESTABLISHED
(with/without additional
specific diagnostic
measures)
Suspicion of DILI
Focal lesions; dilated bile ducts
Serum antibodies
Stenoses (sclerosing cholangitis)
Parenchymal damage
Biliary lesions
Gene mutations
Autoantibodies of PBC
AMA reactivity is only sufficient to diagnose PBC when combined with abnormal serum liver
tests. Only one out of six with AMA positivity and normal ALP develops PBC within 5 years.
M2, M4, M8 and M9 are detected in PBC patients.
Anti-gp210 and anti-sp100 are specific to PBC in the 5-10% of patients with PBC who are
AMA negative.
2017 EASL Clinical Practice Guidelines: Primary biliary cholangitis
Autoantibodies Prevalence
Anti-mitochondrial antibodies (AMA) 35-95%
- AMA M2 95%
- AMA M4 (sulfit oxidase) Up to 55%
- AMA M8 Up to 55%
- AMA M9 (glycogen phosphorylase) 35-85%
Anti-nuclear dots (Sp100, SUMO1/2, PML) 30%
Anti-nuclear membrane (gp210, p62) 20-30%
Anti-nuclear membrane (lamin-B receptor) 2%
(Moteki et al., Hepatology, 24, 1996)
AMA M2-3E ELISA is coated with native PDH + all relevant M2
subunits (designer antigen)! → increased sensitivity
AMA M2-3E (BPO) Our Innovation - Designer antigen 3E
Anti-M2-3E ELISA vs. Anti-M2 ELISA Sensitivity
12
23 Neg.
0 135 Pos. Conventional
Anti-M2 ELISA
negative positive
Anti-M2-3E ELISA
Patients with PBC
(n = 170)
79,4%
92.9%
Higher sensitivity compared to conventional Anti-M2 ELISA
AMA M2-3E (BPO) Our Innovation - Designer antigen 3E
Available in 2 formats:
1. ELISA
2. Immunoblot
ELISA for ALD
EA 1622-9601 G AMA-M2-3E
EA 1302-9601 G SLA/LP
EA 1321-9601 G LKM-1
EA 1307-9601 G LC-1
EUROLINE for ALD
DL 1300-1601-2 G DL 1300-1601-4 G DL 1300-1601-5 G
Liver Profile 2 EUROLINE Profile ALD EUROLINE ALD 14 Ag
Overview of recommended tests for PBC
Test Finding Suspicion Diagnosis Prognosis Notes
ALP
Values associated with disease
progression
AST/ALT
May be suggestive of PBC with
features of AIH
GGT Reflects cholestatic liver injury
IgM Elevated values associated with disease
AMA (>1/40) +
Diagnostic in >90% of cases in correct
clinical context
Specific ANA +
Specific immunofluorescence patterns*
present in 30%
Anti-gp210 + Specific immunoassays available
Anti-sp100 + Specific immunoassays available
Anti-centromere +
Associated with portal hypertensive
phenotype
Bilirubin
Elevation at late stages frequently
indicative of cirrhosis†
Platelets Indicative of cirrhosis
INR Indicative of cirrhosis
Albumin Indicative of cirrhosis
*Perinuclear rims, nuclear dot, centromere; † Except in patients with ductopenic non-cirrhotic variant
EASL CPG PBC. J Hepatol 2017;67:145–72
Summary for PBC Diagnosis
1. Immunofluorescence is used for detection of:
AMA
PBC Specific ANA: Nuclear dots (Target Antigens: Sp100, SUMO1/2, PML)
Nuclear membrane (Target Antigen gp210, p62, lamin-B receptor)
M2-3E
EUROPLUS
AMA
rat kidney
ANA
HEp-2
Nuclear dots
(Primate liver)
Nuclear membrane
(Primate liver)
Single Slide Screening of AIH & PBC Liver Mosaic
Liver Mosaic Profile 8
Hep-2 cells Liver (primate)
Liver (rat)
VSM47 (cells) HEp-2 / Liver primate
Kidney rat / Liver rat
Stomach rat / VSM47
FA 1800-####-8
AMA
rat kidney Nuclear dots
(Primate liver)
Nuclear membrane
(Primate liver)
Kidney (rat)
Stoa ch(rat) HEp-2 / Liver primate Kidney rat / Liver rat Stomach rat / VSM47
AMA
ANA
HEp-2
AMA Nuclear dots Nuclear membrane
Summary for PBC Diagnosis
1. Immunofluorescence is used for detection of:
AMA
PBC Specific ANA: Nuclear dots (Target Antigens: Sp100, SUMO1/2, PML)
Nuclear membrane (Target Antigen gp210, p62, lamin-B receptor)
2. Biopsy is not required for diagnosis unless PBC specific
antibodies were not detected
Autoimmune Liver Diseases
Autoimmune hepatitis (AIH) Primary biliary cirrhosis
(PBC)
Primary sclerosing
cholangitis (PSC)
AMA
PBC-specific
ANA
Autoantibodies in ALD
Can we detect for PBC and AIH simultaneously?
Single Slide Screening of AIH Type I & II Liver Mosaic
Liver Mosaic Profile 8
Hep-2 cells
Kidney (rat)
Stomach (rat)
Liver (primate)
Liver (rat)
VSM47 (cells) HEp-2 / Liver primate
Kidney rat / Liver rat
Stomach rat / VSM47
FA 1800-####-8
Simultaneous detection of PBC and AIH Type I & II in
one slide
EUROLINE for AIH & PBC Differentiation
DL 1300-1601-2 G DL 1300-1601-4 G DL 1300-1601-5 G
Liver Profile 2 EUROLINE Profile ALD EUROLINE ALD 14 Ag
AMA-M2 3E (BPO)
Sp100*
PML* gp210**
LKM-1
LC-1
SLA/LP
Ro-52***
AIH
PBC
EA 1622-9601 G 1. AMA-M2-3E
EA 1302-9601 G 2. SLA/LP
EA 1321-9601 G 3. LKM-1
EA 1307-9601 G 4. LC-1
ELISA for ALD
Autoimmune Liver Diseases
Autoimmune hepatitis (AIH) Primary biliary cirrhosis
(PBC)
Primary sclerosing
cholangitis (PSC)
Type 1 Type 2 Type 3
? ANA
ASMA
F-actin
LKM-1
LC-1 SLA/LP
AMA
PBC-specific
ANA
Does it tell a complete story?
Non-conventional findings
Recommends the use of Indirect
Immunofluorescence for
patients without conventional
serological findings:
1. Atypical pANCA
Autoantibodies of PSC
Autoantibodies Prevalence
Atypical pANCA 70-80%
Do take note! • pANCA (atypical) is not specific for PSC
• also found in Ulcerative colitis, PBC, AIH, Hepatitis C,
Hepatitis
• It is however a possible indication of autoimmune liver
disease
Granulocytes (EOH)
Granulocytes (HCHO)
granulo (EOH)
granulo (HCOH)
HEp-2
+ granulo
(EOH)
Description • pANCA gives a characteristic perinuclear fluorescence pattern on ethanol-fixed granulocytes.
• A granular cytoplasmic pattern is seen on formalin-fixed granulocytes.
• No fluorescence is seen on the HEp-2 cells. On the liver fluorescence is seen in the sinusoids where the granulocytes are localized.
Target Antigens • In addition to MPO other proteins have been identified as target antigens of pANCA:
lactoferrin, elastase, BPI, cathepsin G, lysozyme and ß-glucuronidase
Anti Neutrophil Cytoplasmic Antibodies (ANCA) Granulocyte
Autoimmune Liver Diseases
Autoimmune hepatitis (AIH) Primary biliary cirrhosis
(PBC)
Primary sclerosing
cholangitis (PSC)
Type 1 Type 2
ANA
ASMA
F-actin
SLA/ LP
Atypical pANCA LKM-1
LC-1
AMA
PBC-specific
ANA
Type 3
SLA/LP
Complete solution for ALD Serological Diagnosis
Is there a need to detect so many markers at a
time?
*Statement numbers: 15–20
EASL CPG AIH. J Hepatol 2015;63:971–1004
Diagnosis Guidelines Statement
Guideline statements* Grade of recommendation
Histological demonstration of hepatitis is a prerequisite for the diagnosis of AIH
and needs to be part of the initial diagnostic work-up II-2
No morphological features are pathognomonic of AIH.
Suggestive of AIH: interface hepatitis, periportal necrosis, emperipolesis and II-2
rosetting of hepatocytes. These features should be reported by the pathologist in
addition to grading (hepatitis activity index) and staging of disease
Pericentral necrosis may be present in the acute onset of AIH and histologically
indistinguishable from DILI II-3
The simplified scoring system (2008) of the IAIHG is a useful clinical tool.
By considering response to treatment, the revised scoring system (1999) of the II-2
IAIHG can be helpful in diagnosing difficult cases
Coexistence of features of AIH and cholestatic liver diseases can be observed, both
at diagnosis and during follow-up. Diagnostic tests for PBC and PSC should be II-2
performed in patients showing features of cholestasis
In adult patients with AIH and cholestatic lab changes, and in all children with
AIH, (MR) cholangiography should be performed in order to recognize sclerosing II-3
cholangitis
Coexistence of features of AIH and cholestatic liver diseases can
be observed, both at diagnosis and during follow-up. Diagnostic
tests for PBC and PSC should be performed in patients showing
features of cholestasis
Variant syndromes
*Statement number: 50
EASL CPG AIH. J Hepatol 2015;63:971–1004
Guideline statements* Grade of recommendation
In AIH patients with features of PBC (“AIH-PBC variant syndrome”), combined
therapy with UDCA and immunosuppressants is recommended
III
In AIH patients with PSC features (“AIH-PSC variant syndrome”) addition of
UDCA to immunosuppressant can be considered III
In patients with dominant AIH features, an alternative approach is to start with
immunosuppressants only and then add UDCA if response is insufficient
III
Variant Syndrome
Paris criteria 1. ALP > 2x ULN
2. Gamma-GT >5 x ULN
3. Presence of AMA
4. a liver biopsy specimen showing florid bile duct lesions
Diagnosis Sensitivity
Diagnosis Specificity
92%
97%
Clinical Liver Disease, Vol 3, No 1, January 2014
2015 EASL Clinical Practice Guidelines: Autoimmune hepatitis
ULN = upper limit of normal range
EUROLINE for AIH & PBC Differentiation
DL 1300-1601-2 G DL 1300-1601-4 G DL 1300-1601-5 G
Liver Profile 2 EUROLINE Profile ALD EUROLINE ALD 14 Ag
AMA-M2 3E (BPO)
Sp100*
PML* gp210**
LKM-1
LC-1
SLA/LP
Ro-52***
AIH
PBC
DL 1300-1601-5 G
• PBC patients also have other accompanying
autoimmune diseases (ANA overlap)
• Additional diagnostics may be required
EUROLINE for ALD (DL 1300-1601-5 G)
DL 1300-1601-5 G
EUROLINE for ALD (DL 1300-1601-5 G)
DL 1300-1601-5 G
EUROLINE for ALD (DL 1300-1601-5 G)
Recommended diagnostic approach
IIFT EUROLINE
ASMA
F-actin
AMA-M2 3E-BPO
AMA
gp210 Sp100
PML Ro52
CENP B SS-A
CENP A
PDGH Scl-70
CENP
LKM
LC-1 N. Memb
N. Dots SLA/ LP
AIH PBC Overlap
Legend
PSC
ANCA
Conclusion The role of Serological Testing
1. The determination of autoantibodies is a prerequisite to
diagnosis AIH
2. They are components of the diagnostic scoring system
3. It can help to determine the type of AIH
“Testing for liver-related autoantibodies should be included in the workup of
patients with hepatitis or cholestasis of unknown origin.”
Review published: 27 March 2018 doi: 10.3389/fimmu.2018.00609
Thank You
Autoimmunity