role of immunological markers in diagnosis of autoimmune liver...

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 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


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


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

https://doi.org/10.1016/S0140-6736(87)92024-1

https://doi.org/10.1016/S0140-6736(87)92024-1

https://doi.org/10.1016/S0140-6736(87)92024-1

https://doi.org/10.1016/S0140-6736(87)92024-1

https://doi.org/10.1016/S0140-6736(87)92024-1

Special patient populations: AIH and pregnancy

*MMF is contraindicated in pregnancy


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

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



(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


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


Hep-2 cells Liver (primate)

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



(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


Hep-2 cells

Kidney (rat)

Stomach (rat)

Liver (primate)

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


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



(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



(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


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


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


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)

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

role of immunological markers in diagnosis of autoimmune liver...

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