Transcript Immunological Testing in Liver and Gastrointestinal Disease

Immunological Testing in Liver and
Gastrointestinal Disease
Dr David Sinclair
Department of Clinical Biochemistry,
Queen Alexandra Hospital,
Portsmouth
PO6 3LY
Liver related antibodies





Anti-nuclear antibodies
Anti-mitochondrial antibodies
Anti-smooth muscle antibodies
Anti-liver kidney microsomal antibodies
Gastric parietal/ intrinsic factor antibodies
Anti-nuclear antibodies

Homogenous



Most common and least
disease specific
Found in most SLE cases
and 50% liver disease and a
multitude of other conditions
Speckled

Found in SLE, liver disease,
Sjogren’s etc.
Mitochondrial antibodies


Traditionally detected by
immunofluorescence on rodent
tissue
Seen in primary biliary cirrhosis
(90%) but also in 30% Type I AIH
and non-specifically.
Mitochondrial antibodies

9 Different subtypes; most distinguishable
by microscopy.





M2 (pyruvate dehydrogenase complex), M3
and M4 associated with PBC (for the most part)
M1: Probably an anti-phospholipid antibody:
Syphilis, SLE, scleroderma, Sjogren’s etc
M6: Iproniazide induced hepatitis.
M7: Acute myocarditis.
M5: SLE, connective tissue disorders.
Mitochondrial antibodies

PBC accompanied by raised ALP and/or IgM



ALP activity increased in PBC as a marker of
intrahepatic cholestasis
IgM because the isotype switching mechanism
from IgM to IgG and IgA is impaired.
Can predate PBC by months or years.

M2 ELISA or Western blot assay can be useful.
Mildred


Born 1926
Presented 2001 to her GP.



TATT, ?polymyalgia rheumatica
No other significant symptoms
U/Es, glucose, Ig profile, FBC/ESR requested



U/Es normal
Glucose 6.5mmol/L
MCV 100; ESR 29 otherwise normal FBC
Mildred



IgG = 12.9g/L (ref 6-15)
IgA = 1.37g/L (ref 0.8-4.0)
IgM = 2.91g/L (ref 0.5-2.0)

Slightly raised polyclonal IgM



? acute infection
? 7s monomeric IgM secondary to early lymphoma
? Primary Biliary Cirrhosis
Mildred

We added LFTs on to original request




Bilirubin = 6 umol/L (ref 3-20)
Alkaline phosphatase = 180 IU/L (ref 30-95)
AST = 35 IU/L (ref 12-40)
We then added a liver autoimmune profile



Smooth muscle antibodies = Negative
Mitochondrial antibodies= Moderate positive
Anti-nuclear antibodies = Negative
Mildred

Comment added to the raised IgM, ALP and
mitochondrial antibodies for the GP

“On the basis of the raised IgM, we added LFTs
and liver antibodies which showed raised ALP
and mitochondrial antibodies. These may be a
“red herring” but PBC cannot be discounted
and I’d rather you were aware of this
possibility. ”
Mildred

Referred to Hepatologists for liver biopsy


Mononuclear inflammatory infiltrate at the
portal tracts-mainly lymphocytes, plasma cell
and eosinophils with some areas showing
spillage into surrounding hepatic parenchyma;
Occasional granulomata seen; appearances in
keeping with early primary biliary cirrhosis.
Treated with steroids rather than
ursodeoxycholic acid, later developed CCF
and died in 2003
Mildred

Practice points


If you see an unexplained polyclonal IgM
increase or raised ALP activity or positive
mitochondrial antibodies or any combination of
these three, in someone of this age and sex :
Don’t forget Primary Biliary Cirrhosis.
The role of the laboratory?


9 years retrospective study (J Clin Pathol, in press)
22 PBC cases reported and the lab had a role in all
of them:

11 had high ALP; 7 had abnormal liver antibodies; 1
had a raised IgM




All these diagnoses were based on the lab following up a
biochemical or immunological abnormality.
One had no liver related antibodies.
One had a request for follow on samples which were
not received.
One had a previous diagnosis made on clinical and
serological grounds and the biopsy was confirmatory.
Smooth muscle antibodies

Seen in chronic active hepatitis
 Directed against cytoskeletal
proteins
 actin – autoimmune liver
disease.
 tubulin – infectious
mononucleosis
 vimentin- infection,
rheumatic disease etc.
 desmin – myocarditis and
CHD
Smooth muscle antibodies




Present in 70-90% patients; often accompanied by ANA.
 Type 1 autoimmune hepatitis
Not specific for autoimmune hepatitis and found in
infectious and rheumatologic diseases as well as 50% of
PBC cases.
Antibody titres are not predictive and large variations can
be seen in individual cases
Clearly AST/ALT will be more of a help here but look also
for a raised IgG.
 Combination of all three is suspicious.
Anti-liver kidney/microsomal antibodies

Characterises Type 2
autoimmune hepatitis



i.e. LKM positive but no
nuclear or smooth muscle
antibodies.
The LKM-1 antigen is
cytochrome P450 II D6
Also described in chronic
hepatitis C infection,
cryptogenic cirrhosis and
drug induced hepatitis.
“Stomach” related antibodies

Gastric parietal cell antibodies


Autoimmune gastritis, pernicious
anaemia (90%), PBC (10%),
autoimmune hepatitis (75%); chronic
liver disease (30%) ; autoimmune
thyroiditis (30%) otherwise well elderly
(10-15%)
Intrinsic factor antibodies

Done by ELISA: Specific and virtually
“diagnostic” for PA but only present in
40-50% of PA cases.
Gut related antibodies



Anti- reticulin and gliadin antibodies
Anti- endomysium antibodies
Anti-tissue transglutaminase (TTG) antibodies



Coeliac Disease with its incidence of 1/100-1/300
Anti- Saccharomyces cerevisae antibodies
Anti-neutrophil cytoplamsic antibodies (ANCA)

Crohn’s and Ulcerative Colitis
Reticulin antibodies



Usually detected on rodent tissue
Poorly sensitive and specific for
coeliac disease and not worth
performing in their own right.
Very often a chance finding as
part of an ANA screen

Best to add other tests if one of
these
IgA and IgG Gliadin antibodies

Sensitive but poorly specific markers for
coeliac disease.


15-20% of children with gliadin antibodies will
not have coeliac disease - inappropriate
biopsies?
Superceded by other tests with better
specifity
Endomysium antibodies


Endomysium is the
connective tissue
covering individual
smooth muscle fibres
IgA antibodies usually
detected using primate
oesophagus or human
umbilical cord tissue.
Endomysium antibodies

IgA antibodies have >90% sensitivity and
specificity for coeliac disease and dermatitis
herpetiformis.


Titres correlate with intestinal damage.


Excellent figures for an antibody test!
Expect antibodies to disappear within 6-9
months on gluten free diet.
Monitoring dietary compliance is possible
Tissue transglutaminase antibodies




Replacing endomysium antibodies as the method
of choice for coeliac screening.
TTG is an intracellular enzyme and is the major
auto-antigen involved in the EMA response.
ELISA: Similar sensitivity and specificity to EMA
Small bowel biopsy still the gold standard but
does this removes the need for more invasive
tests?
IgA deficiency in coeliac disease



Significant proportion (~3-5%)
of coeliac disease patients are
IgA deficient (IgA <0.05g/L)
Need to exclude IgA deficiency
Measure total IgA on every TTG
antibody request?
Follow on testing with total IgA
and if below age related range
proceed to IgG antibodies
Figure 1 IgA concentration vs TTg assay OD
4
3.5
3
2.5
IgA g/L

2
1.5
1
0.5
0
-0.5 0
0.02
0.04
TTg OD units
0.06
0.08
IgA deficiency in coeliac disease





SC; 4 year old girl presented with failure to thrive
and abdominal pain
TTG negative; TTG ELISA OD = 0.02
Total IgA = 0.25g/L i.e. low for age but not absent
Moderate positive IgG endomysium antibody
Biopsy: villous flattening: Appearances consistent
with coeliac disease.
When should these be tests be performed?







Family history of coeliac disease
Type I diabetes
Irritable bowel
Fe deficiency anaemia
Chronic diarrhoea, chronic fatigue
Weight loss, short stature, FTT
Unexplained transaminase enzyme increases
Anti-Saccharomyces cerevisiae
Antibodies (ASCA)


Found in Crohn’s
Disease
Saccharomyces cerevisiae
stained sequentially with the red
fluorescent Rhodamine B hexyl
ester, which selectively labels
yeast mitochondria and the
green fluorescent yeast vacuole
membrane marker
Anti-Saccharomyces cerevisiae
Antibodies (ASCA)





Proabably a marker of an immune response to an
envirnmental antigen in early stage Crohn’s Disease.
40-70% of Crohn’s patients and 6-12% of patients with
ulcerative colitis; also found in autoimmune liver disease
and gluten sensitive enteropathy.
Considered too insensitive to diagnose Crohn’s or
ulcerative colitis.
No evidence that it is useful in selecting therapeutic
interventions
No correlation with disease activity, duration of illness,
extent of disease, extraintestinal complications.
ANCA


Atypical p-ANCA
found in 50-80 % of
ulcerative colitis and
10-40% of Crohn’s
diseases
Insufficient evidence
to include ANCA as a
front line test for gut
related pathologies.
What happens to patients with positive
gut antibodies in General Practice?




In 2002, we published an audit that showed:
9 month period in 2000-2001
67 of 1450 patients had positive EMA
55 of these had no biopsy done


82% of patients with positive antibodies had no
biopsy done.
47 (69.9%) patients were not referred for
Gastroenterology review.
Background


Clearly referral and biopsy rates were much too
low and something had to be done to improve this.
“Referral for further gastroenterological
investigations, including duodenal biopsy, is
suggested as per current BSG guidelines for the
management of suspected coeliac disease.”

Comment added to each positive gut antibody result
to General Practice
Materials and Methods





5632 patients screened for coeliac antibodies
April 2001 to May 2003
Screened for IgA (Behring BN2) and TTG
antibodies (Celikey: Sweden Diagnostics)
Positive TTGs are backed up by EMA on
monkey oesophagus
Patients under 16 years, those under Gastro
care and known CD/dermatitis herpetiformis
excluded
Results



89 patients tested positive and had the
comment added to their results
71 were subsequently referred and biopsy
samples were taken from all of them
i.e Vast majority 79.5% now reviewed by
Gastroenterology c.f. 18% biopsy rate
before.
Results

18 patients were not referred


9 not on GFD, not referred and no biopsy taken.






Letter to GPs about current management/referral.
One GP had no recollection of the result, patient had moved away and
he was writing to the new GP with the good news
2 on GFD but not referred to Gastro.
1 on GFD and referred to a Physician.
1 no longer registered, unable to track but no referral
or biopsy data found.
4: No reply to letter; no referral or biopsy data found
1 On GFD and referred to Gastro but did not attend
Discussion



To ensure the BSG guidelines are followed,
there is a much improved referral and
biopsy rate after adding an “instructive“
comment.
If this is common to other areas, this
comment works.
Still of concern are the 15 patients who
have not been referred and only two of them
are known to be on GFD