pathology

1. RECENT ADVANCES IN
DIAGNOSIS OF
CHRONIC HEPATITIS
DR.RASHMI HANDE

2. CHRONIC HEPATITIS REPRESENT A SERIES OF
LIVER DISORDER OF VARYING CAUSES AND
SEVIRITY IN WHICH HEPATIC INFLAMMATION
AND NECROSIS CONTINUE FOR MORE THAN AT
LEAST 6 MONTH.
DEFINITION

3. •Chronic alcoholism
•Wilson disease • α1AT deficiency
•Drugs( isoniazide, methyldopa,
methotrexate
•Autoimmunity
•Viral hepatitis
CHRONIC
HEPATITIS
ETIOLOGY

4.  HBV virus
HBV + HDV virus
HCV virus

5. Double stranded circular DNA virus.
3200 nucleotides
42nm sperical double layered Dane
particle that has outer surface envelop
of protein, lipid, carbohydrate
enclosing an 28nm hexagonal core.
Belongs to family of hepadnaviridae
TRANSMITTED
 Transfusion , blood product ,
dialysis , intravenous drug
abuse
 Homosexual activity , needle
stick injury in health workers.

6. GENOME OF HBV VIRUS
HBV DNA code for four set
of viral protein
S gene- envelop glycoprotein
C gene – core protein
P gene-DNA polymerase
X gene- HBx Ag necessary for
virus replication – which can
activate transcription of viral
gene

7. Several mutant strains – identified
Impact on clinical outcome with increased likelihood
of chronicity & severity of disease
Mutants arise from exogenous factors
 Nucleoside & nucleotide analogue
 Hepatitis B immunoglobulin
 Vaccination
Mutation of pre-S region
 Escape from vaccine induced viral clearance in
already infected patients
 Ability to infect despite prior vaccination
 Change in antigenic properties of HBsAg - decrease
their affinity to neutralizing antibodies
MUTANTS

8. Outcome of Hepatitis B
infection

10.  called as hepatitis delta virus
Unique RNA virus having defective replication
Infect only when it is encapsulated by HBs Ag.
Dependent on genetic information provided by HBV for
multiplication
35 nm double shelled particle resemble Dane particle
Externally coated with HBs Ag enclose internal
polypeptide designated delta antigen HDV Ag
Causes two types of infection

11. OUTCOME OF HDV
INFECTION

12. Small envelop single stranded
Positive sense 9600-nucleotide
RNA virus
flaviviridae family
Transmitted
Blood transfusion
Sexual transmission
Vertical transmission

13. Genome of HCV

14. Outcome of Hepatitis C
infection

15. Cytomegalovirus
Epstein-Barr virus
Yellow fever virus
In immunosupressed and childrens
hepatitis caused by
 rubella virus
 Herpes virus
 Adenovirus
 enterovirus
Other viruses causing
chronic hepatitis

16. AUTOIMMUNE CHRONIC
HEPATITIS
Female predominance (young and menopausal)
Absence of viral serological markers
Raised serum IgG and gamma globulin
level(>1.5 times normal)
High serum antibody level (ANA, antismooth
muscle antibody, anti LKM)
Associated with other autoimmune diseases

17. DRUG INDUCED
CHRONIC HEPATITIS
Results from
 direct toxicity
 from hepatic conversion of xenobiotics to active
metabolite
 Through immune mediated
 two types of hepatic injury
 predictable- overdose of acetaminophen , CCL4,
amanita phalloides toxin
 unpredictable/ idiosyncratic-Alfa methyldopa
,Allopurinol , sulfonamides

18. ALCOHOL INDUCED
CHRONIC HEPATITIS
Quantity and duration play most important role
> 60 to 80 g/day of alcohol for 10 years
Females are more susceptible to alcoholic liver
injury when compare to men
Concurrent hepatitis c infection - important
comorbidity in progression of alcoholic liver disease
Malnutrition
Gene polymorphism – alcohol dehydrogenase ,
Cytochrome P-450

19. Non alcoholic steatohepatitis
Hepatic manifestations' of metabolic syndrome –
 Obesity
 Insulin resistance(DM)
 Hyperlipidemia
 Hypertension
histological similarity to alcoholic liver disease
Risk factors for NASH
 Obesity BMI>30kg/m2,waist : hip ration > 0.9(male) ,>o.85
(female)
 Hypertension BP > 140/90
 Dyslipidemia -low HDL<0.9mmole/l,
 Hypertriglyceridemia > 1.7mmole/l
 microalbuminuria , urinary albumin excretion >20 ug /min

20. • Wilson disease
• Alfa 1 antitrypsin deficiency
• Hemochromatosis
• Cryptogenic
• Primary billiary cirrhosis
Other causes of chronic
hepatitis

21. History
Thorough examination
Laboratory diagnosis
 Liver function test
 Serology
 Immunoassay
 RNA detection by PCR
 Non-invasive techniques
 Invasive - Biopsy
 Proteomics
 Imaging modalities
APPROACH TO PATIENT

22. DISEASES
ENZYMES
CHRONIC
HEPATITIS B
CHRONIC
HEPATITIS C
CHRONIC
HEPATITIS D
ALCOHOL
INDUCE
CHRONIC
HEPATITIS
AUTOIMMUNE
HEPATITIS
SERUM BILIRUBIN
3 TO 10mg/dl
Normal
SERUM ALBUMIN Normal
AST
ALT
ALT>AST AST>ALT
In bet 100 to 1000
units
ALKALINE
PHOSPHATASE
Marginally
elevated
Marginally
elevated
Marginally
elevated
normal
PROTHROMBINE
TIME
prolonged Prolonged prolonged Prolonged prolonged
HYPGAMMAs
GLOBULINEMIAMIA
Not
present
Present Present Not
present
Present
>2.5g/dl
AUTOANTIBODIES Not raised AntiLKM1 Anti LKM3 Not raised ANA ,anti
LKM1

23. SEROLOGY OF
CHRONIC HEPATITIS B
Markers
Disease
HBs Ag IgM Anti
HBc
IgG Anti
HBc
HBe Ag Anti HBe
Acute
hepatitis B + + +
_
+ +
Chronic
hepatitis B + _ + + _
 HBV DNA –PCR , direct blot or liquid hybridization
 HBs Ag- RIA, EIA,
 HBs Ag in liver tissue-immunofluroscence ,immunoperoxidase

24. ACUTE HEPATITIS B CHRONIC HEPATITIS B

25. Serology of HCV
MARKERS
DISEASE
HCV RNA IgM Anti HCV IgG Anti HCV
Acute HCV + + +
Chronic HCV + _ +

26. Serology of HCV

27. MARKERS HBs
Ag
HBV
DNA
HBe
Ag
HDV
RNA
HDV
Ag
IgM
Anti
HBc
Ag
IgM
Anti
HD Ag
IgG
Anti
HBs
Ag
IgG
Anti
HDV
Coinfection
_ + + + + + + _ _
superinfection
+ + _ + + _ + + _
Serology of HDV

28.  Autoimmune hepatitis
Type 1 autoimmune hepatitis-anti LKM1, ANA
Type2a autoimmune hepatitis-high anti LKM1
Type2b autoimmune hepatitis-low anti LKM1
Type 3 autoimmune hepatitis- lack of ANA & Anti
LKM ,& presence of antibody against
cytoplasmic cytokeratin 8 and 18

29. Technique to detect
serological markers
Antigen (HBs Ag, HCV Ag, anti HCV, HDV Ag)
 Radioimmunoassay
 Enzyme immunoassay
 Immunofluroscence
 Immunoblot assay
 Immunostaining
 Immunoperoxidase method
Viral DNA and RNA detection by
 Hybridization method
 PCR
 TMA( transcriptional mediated amplification)

30. Enzyme immunoassay
and radioimmunoassay
 RIA and EIA are direct binding assay and work on same
principle
 Based on the competitive binding reaction to antibodies
between labeled antigen and non labeled antigen
 two important aspect
 should be one reagent in pure and detectable form
 Separating mean –to separate bound fraction of labeled
antigen from unbound

31. RIA
.
.

32. ENZYME IMMUNO ASSAY
Radioactive label enzyme
EIA require secondary process to obtained signal –
catalytic reaction of enzyme
Commonly used enzymes
 Horseradish peroxidase
 Alkaline phosphtase
 Beta-galactosidase
 Glucose oxidase
antigen coupled to solid surface-bind antibody present
in sample –enzyme conjugated anti Ig antibody added
- product measured by spectrometry

33. HCV RNA –RT-PCR , TMA
RT-PCR
 Based on enzymatic amplification of DNA fragments flanked by primer
(short oligonucleotides fragments complimentary to DNA.
 convert RNA to c DNA –template for PCR
 Primer whose sequences correspond to the 5UTR (most conserved
region of genome)
TMA(transcriptional mediated amplification)
 More complex reaction with T7 RNA polymerase and RT under isothermal
condition for detectable level of RNA
 TMA uses primer containing T7RNA polymerase binding site , RT
synthesize C DNA – template from which T7 RNA polymerase
synthesize numerous copies of RNA.

36. Fibrotest is noninvasive alternative technique to liver biopsy in
diagnosis of chronic hepatitis and other liver disorders
Fibrotest uses algorithm to combine the results of five serum
markers
 alpha 2 macroglobulin
 Heptaglobulin
 Apo lipoprotein A1
 Total bilirubin
 Gamma glutmyltranspeptidase
To asses level of fibrosis and necroinflammatory activity
Helpful in diagnosis of
 Chronic hepatitis B and C
 Alcoholic hepatitis
 Non alcoholic hepatitis

37. Fibro test scoring
Fibro test score is calculated from above parameter blood
test and combine this serum marker with age and gender
of patient
 fibro test score derived from equation
f=4.467xlog 10(alpha-2 macroglobulin in g/l)-
1.357xlog10(haptoglobulin g/l)+ 1.017xlog10(GGT
IU/L)+0.028x(age in years)
Fibrosis staging done by METAVIR system

38. F0
NO FIBROSIS
0.00TO
0.21
F1 PORTAL FIBROSIS
WITHOUT SEPTA
O.28 to
0.31
F2
PORTAL FIBROSIS
WITH SEPTA
0.49 to
0.58
F3
NUMEROUS SEPTA
0.59 to
0.74
F4
CIRROSIS
o.75 to
1.00

39. Advantages of fibro test
over biopsy
Liver biopsy is gold standard till now to asses histological
features of liver and estimate liver damage
 fibro test having some advantages over biopsy
 is noninvasive
 not Prone to complication
 not having Sampling variability
 no high intra and inter pathologist variability
Fibro test can be repeated easily
 validated in those
• over 65 yrs & children
• Renal insufficiency, transplant
• Hemophiliacs
• Pt with chronic inflammatory ds.

41. Acti- test
 Acti test asses viral
necroinflammatory activity
 Parameters of Fibrotest +
ALT = Acti test
 It gives grading as
 A0-no histologic activity
 A1-minimal activity
 A2-moderate activity
 A3- severe activity

42. NASH test
Alternative non invasive diagnostic technique for inflammatory
steatosis of metabolic origin (Obesity, DM, Hyperlipidemia)
Use combinations of 10 highly concentrated biochemical
markers in blood.
• Fasting glucose
• Triglyceride
• Cholesterol
• ALT and AST adjusted on age, gender, wt of patient .
• It gives grading as
• N0 – no NASH
• N1- borderline NASH
• N2-severe NASH

43. Full set of protein encoded by DNA –proteomes
Study of proteomes- proteomics
Describe basic structure and function of DNA , protein ,or antibodies
produce against viral antigen
 analyses the difference between gene expression of healthy and
diseased tissue.
basis of use of Proteomics technology is to detect distribution , and
characterization of protein in body fluids and tissue in diseases
Studies translation process of RNA into protein

44. Analysis involve disease and control sample – 2D gel
electrophoresis, protein are separated by molecular wt,
shape and charges, expression level and amino acid
sequences are then determined.

45. Doppler sonography
fibro scan

46. High diagnostic accuracy in cirrhosis and in follow up of
progression of chronic hepatitis
sensitive to hemodynamic alteration resulting from inflammation
and fibrosis
Useful in differentiation of cirrhosis and chronic viral hepatitis
A series of Doppler indices of hepatic vasculature
Portal vein velocity
Portal vein pulsatility score ( reduced)
Flow volume of portal vein
Waveform of hepatic vein( loss of sinusoidal wave form )
Focal acceleration of flow

47. ultrasonic transient elastography
Noninvasive diagnostic method alternative to biopsy for
liver fibrosis
Elasticity of hepatic parenchyma is assessed by USG
study of liver wherein tissue compression and lateral
displacement of tissue is asses which is produce by
fibrosis.
Gives curve of fibrosis
 F2( mild fibrosis )—0.69 TO 0.77
 F3(mod fibrosis) –0.75 TO 0.82
 F4(severe fibrosis) –O.76 TO 0.83
FIBROSCAN

48. Jaundice chip
Jaundice chip array resequencing provide a rapid and
cost effective analysis of common genetic causes of liver
diseases which can leads to chronic hepatitis
Is diagnostic test in young patient with intrahepatic
cholestasis of unknown etiology.
It test 5 genes which are specific for some inherited
diseases
 ABCB4-
 ABCB11
 ATP8B1
 JAG1 – Alagille syndrome
 SERPINA1 – alpha 1 antitrypsin deficiency

49. Thrombomodulin related to hepatic inflammatory response in
chronic viral hepatitis
Expression of thrombomodulin on sinusoidal endothelial cells
using immunoperoxide method and both light and electron
microscopy in chronic hepatitis
Thrombomodulin was found on sinusoidal endothelial cells in both
type B ,type C chronic hepatitis

53. LABORATORY FEATURES
OF CHRONIC HEPATITIS B
AMINOTRANSFERASE LEVEL WILL FLUCTUATE BETWEEN 100 TO 1000
ALANINE TRANSFERASE RAISED MORE THAN ASPARTATE TRANSFERASE
ALKALINE PHOSPHATASE MARGINALY ELEVATED
SERUM BILIRUBIN ELEVATED 51.3 TO 171mmol (3 TO 10 mg/dl)
HYPOALBUNEMIA( LESS THAN 3mg/dl)
PROLONGATION OF PROTROMBIN TIME
CHRONIC HEPATITIS C
AMINOTRANSFERASE LEVEL FLACTUATE MORE THAN THAT IN HEPATITIS B
HYPERGLOBULINEMIA
PRESENCE OF AUTOANTIBODIES ANTI LKM1
CHRONIC HEPATITIS D
PRESENCE OF ANTI LKM3 ARE DIRECTED AGAINST URIDINE DIPHOSPHATE
GLUCURONOSYLTRANSFERASE

54. Chronic hepatitis
HBV
HCVHBV+HDV
AIH
Wilson’s Disease
PBC
PSC
A1AT
Hemochromatosis
Drugs
Cryptogenic
NASH

55. Hepatitis B Virus (HBV)
Virion
􀀎 Enveloped 42 nm virus
Envelope contains Surface antigen
HBsAg is produced in excess
􀀎 Capsid
Composed of Core antigen (HBcAg)
Contains viral DNA and a polymerase
Genome
􀀎 Partially ds DNA 3200 nt in length
􀀎 Four open reading frames
􀀎 Four genes: Core, Surface, Polymerase and X genes

56. Serum bilirubin normal
Serum globulin normal
Alkaline phosphatase normal
Aminotransferase minimally elevated
Hypergammaglobuline >2.5g/dl
Serum alkaline phosphatase moderately elevated
Circulating autoantibodies will be present

57. Serum level of both AST and ALT will increases two to seven
fold less than 400 unit/lit
AST > ALT
Serum level of GGT will increases
Hyperbilirubinemia (serum bilirubin > 8mg/dl)
Raised alkaline phosphatase level
Hypoalbunemia (serum albumin concentration< 2.5 mg/dl)
Raised circulating polymorphonuclear cells
Raised protrombin time> 5 sec

58. ABOUT HBV
 Hepatitis B Virus (HBV)
 Virion
 􀀎 Enveloped 42 nm virus
 Envelope contains Surface
antigen
 HBsAg is produced in excess
 􀀎 Capsid
 Composed of Core antigen
(HBcAg)
 Contains viral DNA and a
polymerase
 Genome
 􀀎 Four open reading frames
 􀀎 Four genes: Core, Surface,
Polymerase and X genes
 Hepatitis B - Clinical Features
 • Incubation period: Average
60-90 days
 Range 45-180 days
 • Clinical illness (jaundice): <5
yrs, <10%
 ≥5 yrs, 30%-50%
 • Acute case-fatality rate: 0.5%-
1%
 • Chronic infection: <5 yrs,
30%-90%
 ≥5 yrs, 2%-10%
 • Premature mortality from
 chronic liver disease: 15%-25%

60. SERUM MARKERS

61. Serology of HDV

62.  ‘Treat patients with high DNA
 and raised ALT
 and histologically verified liver
 inflammation and/or fibrosis.
 Treatment not necessarily indicated for ALT x1.5-2 normal, low
necroinflammatory
 score on biopsy’
 Biopsy to show that raised ALT is due to chronic viral hepatitis and
assess
 fibrosis/cirrhosis
 In practice:
 Wide variation in blood tests – ALT, antigens, antibodies, DNA
 Clinical uncertainty – experts disagree
 Treatment individually tailored in each patient