Autoimmune liver disease is a heterogenous group of disorders. Laboratory diagnosis plays an important role in early diagnosis. Availability of transfected cells(F-Actin HEK cells) & cell based assays have increased the test specificity significantly.

1. Autoimmune Liver
Disease - Laboratory
Diagnosis
Dr RAJESH V BENDRE
Chief Pathologist
MD(Path), DNB(Path), DPB

2. Organ-Specific Systemic
Hashimoto thyroiditis Systemic lupus
erythematosus
Autoimmune hemolytic anemia Rheumatoid arthritis
Autoimmune atrophic gastritis
of pernicious anemia
Sjögren syndrome
Multiple sclerosis Reiter syndrome
Autoimmune orchitis Inflammatory
myopathies
Goodpasture syndrome Systemic sclerosis
(scleroderma)
Autoimmune thrombocytopenia Polyarteritis nodosa
Insulin-dependent diabetes
mellitus
Ulcerative colitis
Myasthenia gravis
Graves' disease
Autoimmune liver diseases
AUTOIMMUNE DISEASES

3.  Autoimmune liver diseases (ALD) are
characterized by immune-mediated injury
of bile ducts or hepatocytes, thus including
cholangiopathies such as primary biliary
cirrhosis (PBC), primary sclerosing
cholangitis, and autoimmune hepatitis.
 Further, ALD variants manifesting with
both hepatocellular and cholangiocellular
damage are becoming more common.
 In contrast to Western studies where the
estimated prevalence for autoimmune liver
diseases is 11–20% of all cases of chronic
liver diseases; Indian studies have reported
a low prevalence of 3.5–6.1%.
 Majority of the patients are in the third and
fourth decades with a female
preponderance.
Autoimmune Liver Disease
Autoimmune hepatitis in India: profile of an uncommon disease
G Choudhuri, S K Somani. BMC Gastroenterology 2005, 5:27

4. Autoimmune Liver Disease

5. Autoimmune Liver Disease

6. Autoimmune Liver Disease

7. Primary Biliary Cirrhosis
PSC-
In early stages, changes associated with
PSC can be focal and may be non-
specific. The characteristic finding is of
concentric "onion-skin" fibrosis
surrounding the bile ducts . Other bile
duct abnormalities may include necrosis
of epithelial cells, inflammatory infiltrates
and fibrosis. There may be intrahepatic
bile duct proliferation with ductopaenia or
oedema in some portal tracts
PBC –
is an inflammatory autoimmune
disorder causing damage usually to
medium-sized intrahepatic bile ducts
eventually leading to cirrhosis. In the
early pre-cirrhotic stages, however, the
disease is marked by chronic
inflammation in portal tracts only,
“florid duct lesion”.
Autoimmune Liver Disease

8. Autoantibodies in
Primary Biliary Cirrhosis
Mitochondrial and Nuclear Autoantigens
Nine AMA types react with antigens which are
associated either with inner (M1, M2, M7) or
outer mitochondrial membranes (M3, M4, M5,
M6, M8, M9) derived from rat liver or beef heart
mitochondria & can be related to distinct clinical
entities:
-Anti-M1, anti-M5, and anti-M7 are found in
nonhepatic disorders, such as syphilis ,
undefined collagen diseases , and some
forms of cardiac diseases.
- Anti-M3 and anti-M6 are detected in drug-
induced autoimmune disorders.
- Anti-M2, anti-M4, anti-M8, and anti-M9
are confined to primary biliary cirrhosis
(PBC). Anti-M2 is a specific marker for the
diagnosis of PBC; 96% of PBC patients.
Anti-M4 and anti-M8 seem to reflect disease
activity. Anti-M9 antibodies occur
preferentially in early PBC.
AMA Native Antigen Associated diseases
M2 Pyruvate
dehydrogenase
complex
Primary biliary cirrhosis,
other chronic liver
diseases
M4 Sulfite oxidase Primary biliary cirrhosis
M9 Glycogen
phosphorylase
Early phase of primary
biliary cirrhosis

9. Autoantibodies in Primary
Biliary Cirrhosis
Antimitochondrial antibodies ~ 95 %
-Antibodies against oxo-acid dehydrogenase
E2 subunits are nearly 100% specific
Antinuclear antibodies ~ 50%- nuclear dots &
cytoplasmic speckled patterns
Antibodies against gp210 and Sp100 are
nearly 100% specific but present in only 20%
to 30% of cases
Anti-Mitochondrial Antibodies (M2) on
Kidney/Stomach Substrate

10. Revised Scoring System for the Diagnosis of Autoimmune Hepatitis (Revised 1999)
The overall sensitivity of the score to establish a diagnosis of definite or probable AIH was 89.8%,
however, the specificity for discriminating AIH from overlapping syndrome such as PSC or PBC was low.
Autoimmune Hepatitis

11.  Simplified score of International Autoimmune Hepatitis Group (IAIHG) 2008
The score was found to have 88% sensitivity and 97% specificity (cutoff > or =6) and
81% sensitivity and 99% specificity (cutoff > or =7) in the validation set
Autoimmune Hepatitis

12.  Timely diagnosis and immunosuppressive
therapy contain disease activity in majority
patients & with near normal life expectancy
Untreated AIH, however, has a 5-year
mortality above 50%.
 In order to establish a diagnostic scoring
system for AIH, in 1993, the International
Autoimmune Hepatitis Group (IAIHG)
proposed diagnostic criteria, which were
revised in 1999.
 Further improvised as Simplified score by
Hennes et al 2008 included autoantibodies,
immunoglobulin G, histology and exclusion
of viral hepatitis..
 Simplified IAIHG score identifies the
presence of AIH with high degree of
specificity but with poor sensitivity in
comparison with 1999 criteria.
Autoimmune Hepatitis

13. Autoimmune hepatitis is unresolving inflammation of the liver of unknown cause
associated with interface hepatitis on histological examination,
hypergammaglobulinemia, and autoantibodies.
Autoimmune hepatitis - Interface hepatitis – portal
& periportal inflammation with abundant
lymphoplasma cell infiltrate, Rosette formation
Autoimmune hepatitis

14. Autoimmune Hepatitis
Autoimmune hepatitis and Autoantibodies :-

15. Autoimmune Hepatitis
Autoimmune hepatitis and Autoantibodies :-
It has been described that about 70 to 80 % patients of AIH present with significant titers of
ANA or SMA (or both) and about 3 to 4 % have anti-LKM-1 antibodies, while up to 20 %
have none of these antibodies
AIH Type 1 Type 2 Type 3
Age
Sex
Gamma globulin
Steroid responsive
Progression to Cirrhosis
HLA association
Common Associated
Autoimmune diseases
AUTOANTIBODY
Bimodal (10-20 & 45-75)
F (78%)
+++
+++
45%
B8, DR3, DR4
Autoimmune thyroiditis,
ulcerative colitis, synovitis
ANA , ASMA
Pediatric (2-14)
F (89%)
+
++
82%
B14, DR3,C4AQ0
Vitiligo, type 1diabetes,
autoimmune thyroiditis
LKM1
Adults
F (80%)
++
+++
50%
DRB1, DQA1
Same as
Type 1
LA/SLP

16. Autoantibody test by
Indirect Immunofluorescence
Uses Hep2 cells & tissue substrates–
Detects ANA of clinical relevance
Optimized sample screening dilution &
Titers association with clinical relevance
Reduce fading, special Mounting Medium
Optimum Antigen Expression
Significant Mitotic Cells
Large Nuclear size
Pattern correlates with the antibody
specificity

17. Antismooth muscle antibodies (ASMA) - are believed to be directed against a range
of cellular autoantigens in the cytoskeleton of smooth muscle and other cells.
The cellular cytoskeleton consists of a family of filaments that are broadly
classified by size into microfilaments (actin and vinculin), intermediate filaments
(vimentin and desmin), and microtubules (tubulin).
• Antibodies to tubulin and intermediate filaments are commonly encountered
in viral infections, including viral hepatitis and other autoimmune and
nonautoimmune diseases.
• Further research has shown that Actin: a globular protein and exists as either
G-actin (monomeric) 46kD or F-actin (polymerized into filaments) 34kD
• Anti-F-actin antibody is highly predictive of autoimmune hepatitis, with a
sensitivity of approximately 80%, and a specificity of about 90%.
In order to increase the specificity in the Immunofluorescence assays following
improvements have been developed –
-Sample screening dilution
-Use of polyvalent(GAM) conjugate with FITC label
-Substrate used
Anti Smooth Muscle Antibodies

18. Mechanisms of smooth muscle antibody production A clinical
study in children with infections, haemolytic syndromes, and
idiopathic thrombocytopenic purpura . f. kanakoudi-tsakalidis,
c.cassimos Journal of Clinical Pathology, 1979, 32, 1257-1263
• Autoantibodies are seen in low titres in
the general population and are termed
‘natural autoantibodies’ . It is known that
the appearance of serum autoantibodies
may precede the onset of clinical disease
by several years in conditions such as
rheumatoid arthritis and thyroid disease.
•The prevalence of autoantibodies in any
population depends on both the laboratory
techniques used and the initial screening
dilution.
• Screening of asymptomatic blood donors
detected antinuclear antibodies of 1:10 or
more in 45% of subjects, but titres of at
least 1:80 in only 5.6%.
• The incidence of SMA in the normal
population has been reported to be in the
range of 1.5-7.5%
Anti Smooth Muscle Antibodies

19. Anti Smooth Muscle Antibodies
The BIOCHIP Mosaic consists of 6 substrates:
human epithelial cells (HEp-2), primate liver, rat kidney,
rat liver, rat stomach, VSM47.
Thus, a broad spectrum of antigens is present, allowing not
only targeted serological diagnoses, but also frequently
yielding additional results with clinical relevance.
In the case of ASMA - the tunica muscularis, the lamina
muscularis mucosa as well as the interglandular contractile
fibrils fluoresce on the rat stomach. ASMA directed
against the target antigen F-actin furthermore react with
the cytoskeleton of HEp-2 cells and the bile canaliculi of
primate liver. The substrate VSM47 reacts very
specifically, showing a filamentous, needle-like
fluorescence

20. Types of LKM Antibodies
Type Immunofluorescence Pattern Antigen Specificity Disease Association
LKM1 Liver; evenly staining cytoplasmic P4502D6, 50kD Autoimmune hepatitis
fluorescence kidney; proximal tubules
LKM2 Liver; Kidney; same as LKM 1 P4502C9, 50kD TienilicAcid
associated hepatitis
LKM3 Liver; Kidney; primate-specificUridine diphosphate Hepatitis D
glucuronosyltranferases
Kidney Liver

21. Autoimmune Liver Disease

22. • Diagnosis of AIH should be made when compatible clinical signs & symptoms,
laboratory abnormalities, serological and histological findings are present and
other conditions that can cause chronic hepatitis including viral, hereditary,
metabolic, and cholestatic & drug induced disease have been excluded.
• Incorporation of clinical, laboratory, serological & histological findings using
the
diagnostic scoring system for accurate diagnosis.
• Diagnosis of AIH should be considered in all pts with acute or chronic hepatitis
of undermined cause including pts with acute severe hepatitis.
• The aetiology of AIH is unclear, though both genetic and environmental factors
are nvolved in its expression. Immune reactions against host liver antigens are
believed to be the major pathogenic mechanism.
• Autoimmune hepatitis is one of the few liver diseases with excellent response
to immunotherapy achieving remission in 65% and 80% patients at 18 month
and
Conclusion -

23. THANK YOU