all tests included in liver function test. also some non invasive biomarkers of liver fibrosis

1. LIVER FUNCTION TEST
EKTA JAJODIA

2. SEVERAL BIOCHEMICAL TESTS
CAN BE USED –
•To detect presence of liver disease
•Distinguish among different types of liver disorders
•Gauge the extent of known liver disease
•Follow the response to treatment

3. •Liver tests rarely suggest a specific diagnosis
•They suggest a general category of liver disease,
such as hepatocellular or cholestatic
•This further directs the evaluation

4. Metabolic
function
Excretory
function
Synthetic
function
Detoxification
function
Storage
function
FUNCTIONS OF LIVER

5. CLASSIFICATION OF LFT
According to function of liver
Tests based on
excretory function
Tests based on
metabolic function
Tests based on
detoxification function
Tests based in
storage function
Tests based on
synthetic function

6. 1.Serum bilirubin
2.Urine bilirubin
3.Urine and feacal urobilinogen
4.Urine bile salts
5.Dye excretion tests
TESTS BASED ON EXCRETORY
FUNCTION

7. Hippuric acid
test
Determination
of blood
ammonia
TESTS BASED ON
DETOXIFICATION FUNCTION

8. Plasma
proteins
Prothrombin
time
TESTS BASED ON SYNTHETIC
FUNCTION

9. Tests related to
CARBOHYDRATE
metabolism
Tests related to
LIPID
metabolism
Tests related
to PROTEIN
metabolism
Galactose tolerance
test
Serum cholesterol Serum proteins
Aminoaciduria
TESTS BASED ON METABOLIC
FUNCTION

10. ENZYMES IN DIAGNOSIS OF
LIVER DISEASE
SERUM
TRANSAMINASES
AST
ALT
ALP
GGT
5’NT

11. 1.Serum bilirubin
2.Urine bilirubin
3.Urine and feacal urobilinogen
4.Urine bile salts
5.Dye excretion tests
TESTS BASED ON EXCRETORY
FUNCTION

18. SERUM BILIRUBIN
Total bilirubin (<1mg/dl) is found in blood in 2 fractions
•Conjugated bilirubin
(<0.3mg/dl)
•Soluble in water so
excreted by kidney
•Unconjugated bilirubin
•Insoluble in water so
bound to albumin in
blood

19. Significance of hyperbilirubinemia
•Daily production of bilirubin <500mg
•But normal liver can conjugate upto 1500mg/day
•So plasma bilirubin concentration is an insensitive test
for liver disease - since it begins to rise only after
significant liver damage has occured

20. CAUSES OF HYPERBILIRUBINEMIA
Isolated increase in unconjugated bilirubin is due to –
1. Hemolytic disease
2. Genetic disorders – crigler najjar and gilbert’s syndrome
3. Neonatal jaundice/physiological jaundice
Isolated increase in conjugated bilirubin is due to –
1. Cholestasis
2. Genetic disorders – Dubin johnson syndrome and rotor’s
syndrome
Increase in both conjugated and unconjugated bilirubin is due
to –
1. Intrahepatic /liver disorders

23. Pigment deposition in
hepatocytes in DJ syndrome
• Coarsely- granular brown pigment
• Diffuse but more heavily concentrated in the
perivenular zone
• grey to black colour grossly
• pigment shares some of its physiochemical
properties with lipofuscin and melanin
• Oil Red O-positive (in frozen sections), stains
black with the Fontana stain
• autofluorescent when examined by ultraviolet
microscopy

24. DELTA BILIRUBIN / BILIPROTEIN
•When diseases/cholestasis prevents excretion of
conjugated bilirubin into bile
it enters the plasma
Filtered by the kidneys
Excreted in urine
•Some monoconjugated bilrubin can become
covalently bound to albumin

25. •This protein bound conjugated bilirubin is
known as - biliprotein or delta – bilirubin
•Normally present in very small amount
•Increases in cholestasis
•Half life is longer – 20 days (like albumin)
•Normally half life of conjugated bilirubin is 24
hrs

26. TESTS FOR SERUM BILIRUBIN
• Spectrophotometric method
• Diazo method
• Peroxidase method
• Diazo- peroxidase method
• HPLC
• Transcutaneous bilirubinometer

27. DIAZO METHOD
•Sample – Serum or plasma EDTA or heparin
•Note – protect from light
•Exposure to direct sunlight can decrease bilirubin
in samples by 50% within one hour

28. Van Den Bergh reaction
Serum bilirubin Diazotized sulphanilic acid
(Ehrlich diazo reagent)
Azobilirubin( red)
Direct bilirubin – reading is taken at one minute
Add activator /accelerator
2nd reading at 30 minutes – Total bilirubin
Measure absorbance at 600nm

29. Test Principle Comments
Evelyn Malloy method Diazo method – product is
azobilirubin
Activator – ethanol
Readings- at one minute and 30
minutes
Sensitive to pH changes and
serum protein changes
Overestimates dB
Jendrassik-Groff Diazo method
Product- azobilirubin
Activator – caffeine sodium
benzoate
Insensitive to pH changes
And serum protein changes
Direct
Spectrophotometric
Method
Absorbance of bilirubin in serum at
455 nm
Abs Hb at 455 nm is corrected by
subtracting the
abs at 575 nm.
Insensitive to hemolysis
Affected by the presence of
lipochromes and turbidity
Only used in new born infants
HPLC Alkaline methanolysis method
Conjugated forms are methylated
Unconjugated forms remain
unchanged
Extracted into chloroform

30. URINE BILIRUBIN
•Any bilirubin found in urine is conjugated bilirubin
•Presence of bilirubinuria is s/o liver disease
•Can be tested by dipstick test
•When dipstick test show presence of urine bilirubin
– confirmatory test to be done
•Ictotest tablet – based on diazotization reaction –
coupling of solid diazonium salt with bilirubin in
an acidic medium gives a
blue/purple color

31. •Ictotest can be used to rule out presence of any
interfering substance that give false positive reaction
with dipstick test
• While recovering from jaundice urine bilirubin clears
before serum bilirubin
•Presence of bilirubin in urine impart it dark brown
color
• Also fouchet’s test done – if bilirubin is
present a green color develops
due to formation of biliverdin

32. Colour Cause
Orange Conc. Urine , urobilin , drugs
Orange-reddish
brown
Drugs , rhubarb ingestion
Dark brown Altered blood , myoglobin, porphyrins , phenolic drugs
Red Blood, beetroot or blackberry ingestion , food dyes
Purple-red Phenolphthalein laxative
Brown black Altered blood, melanin, homogentisic acid

33. URINE UROBILINOGEN
• Increase in urine is sensitive indicator of
hepatocellular disease
• It is markedly increased in hemolysis
• In cholestatic jaundice urobilinogen disappears
from urine
• Urine strips are available
• Fresh urine should be used
• Ehrlich’s test – gives
pink-red color

34. BILE SALTS
•Products of cholesterol metabolism
•Facilitate absorption of fat from intestine
•Constitute a substantial amount of bile in bilirubin
excretion and can be used in diagnosing cholestasis
•Primary bile salts – cholate and
chenodeoxycholate are produced in liver
Metabolised by bacteria in intestine

35. Produces secondary bile salts – lithocholate,
deoxycholate and ursodeoxycholate
•In cirrhosis – reduced ratio of primary to secondary
bile salts
•In cholestasis – as secondary bile salts are not
formed – so increased ratio of primary to secondary
bile salts

36. •In normal condition – renal excretion of bile salts is
negligible
•In cholestasis – increased renal excretion of bile salts
•
•For measuremnet – chromatography (HPLC)
•Hay’s test – bile salts when present lower the surface
tension of urine
• When sulphur powder is added to the urine, sulphur
particles sink to the bottom of the tube
• In case of normal urine,
it will float on the surface

37. DYE EXCRETION
TEST
3 synthetic dyes are commonly employed to test liver
function
1. Bromosulphthalein (BSP)
2. Indocyanine green
3. Rose bengal
Bromosulphthalein excretion test –
5ml/kg weight i.v is given
BSP is taken up by hepatocytes, conjugated and
excreted in bile

38. •A blood specimen is taken after 45 mins and 2 hrs
•After 45 mins – if >50% dye is retained in blood –
abnormal Liver function is present
• This test is useful in differentiating dubin johnson
from rotor syndrome
•In DJS – At 45 mins – normal blood levels of BSP
• At 2 hrs – higher level of BSP
•In rotor syndrome – at 45 mins – higher levels
• at 2 hrs – lower levels

39. Hippuric acid
test
Determination
of blood
ammonia
TESTS BASED ON
DETOXIFICATION FUNCTION

40. BLOOD AMMONIA
•Produced in body by normal protein metabolism
and by intestinal bacteria
•For detoxification of ammonia
In liver
converted to urea
Excreted by kidneys
In striated muscles
Combines with glutamic acid
Forms glutamine

41. Plasma
proteins
Prothrombin
time
TESTS BASED ON SYNTHETIC
FUNCTION

42. PROTEIN
•Liver is the sole site for synthesis of most plasma proteins
except immunoglobulins (gamma globulins)
•Serum albumin comprises 60% of all plasma proteins
TESTS FOR PROTEINS INCLUDE-
1. Total serum proteins
2. Serum albumin
3. Sr albumin/globulin ratio
4. Serum protein electrophoresis
5. Prealbumin
6. Procollagen III peptide
7. Ceruloplasmin
8. Alpha fetoprotein
9. Alpha antitrypsin

43. TOTAL SERUM PROTEIN
•2 methods of estimation – 1. Refractometer method
2. Biuret method
Biuret method -
• Principle: Cu2 ions present in biuret reagent complex
with peptide bonds in proteins in alkaline medium and
give violet colour
Absorbance is measured at 540 nm

44. ALBUMIN
•Synthesized exclusively by liver
•Its half life is 18-20 days
•Due to its slow turn over – not a good indicator of
acute or mild hepatic dysfunction
• In hepatitis - <3g/dl of albumin – possibility of
chronic liver disease
•Non hepatic causes of Hypoalbuminemia -
• Protein losing enteropathy
• Nephrotic syndrome

45. Dye binding method
Immunoassay
Chromatography
Salt -fractionation
Methods of estimation

46. BROMOCRESYL GREEN METHOD
•Most common method
•The complex becomes blue in color
•Absorbance at 632 nm is directly proportional to
the concentration of albumin

47. GLOBULINS
•Made up of – alpha, beta and gamma globulins
•Gamma globulin is produced by plasma cells
• alpha and beta globulins are synthesized in liver
• In cirrhosis – gamma globulin is increased
•Cirrhotic liver fails to clear bacterial antigens that
normally reach the liver – Abs are formed against such Ags
– so , increased gamma globulin
• Polyclonal gamma globulin if increases by 100% -
autoimmune hepatitis
•Increased IgM – primary biliary cirrhosis
•Increased IgA – alcoholic liver disease

48. PREALBUMIN /TRANSTHYRETIN
• Levels fall in liver disease
• Half life – 2 days
• Sensitive indicator of any changes affecting its
synthesis and catabolism
• PAB is a negative acute phase reactant
• Particularly useful in drug-induced
hepatotoxicity

49. • Immunoturbidimetric procedure
• Sample is incubated with antibodies specific
to prealbumin
• Insoluble complexes are formed

50. • Normal plasma levels - 0.2-0.4g/L
• Acute phase protein
• Decreased in multiple conditions –
1. Wilson’s disease
2. Menkes disease
3. Aceruloplasminemia
4. Copper deficiency
CERULOPLASMIN

51. Increased in –
1. Copper toxicity
2. Pregnancy
3. OCPs

52. PROCOLLAGEN III PEPTIDE
• Cleavage product of the type III procollagen
molecule
• Radioimmunoassay
• Elevated Conc. Of PIIIP- the transformation of
viable hepatic tissue into connective
tissue/fibrosis

53. • AFP is a gp and MW – 70,000 daltons
• Normally present in fetus
• Liver, yolk sac and small intestine
• AFP- ELISA
HCC
Non seminomatous testicular cancer
Ataxia telangiectasia
Hereditary tyrosinemia
Neonatal hyperbilirubinemia
Chronic active hepatitis
α- FETO PROTEIN

54. COAGULATION FACTORS
•With the exception of F-VIII , all other factors are
synthesized in liver
•Half life ranges from 6hrs for F-VII to 5 days for fibrinogen
• So their measurement is the single best measure of
hepatic synthetic function
• Tests – Serum prothrombin time
•Marked increase in PT >5secs above the control and not
corrected by Vit K administration – is a poor prognostic
sign in acute viral hepatitis and other acute and
chronic liver disease

55. ENZYMES IN DIAGNOSIS OF
LIVER DISEASE
SERUM
TRANSAMINASES
AST
ALT
ALP
GGT
5’NT

57. 1.Enzymes whose elevation reflects damage to
hepatocytes
2. Enzymes whose elevation reflects cholestasis
3. Enzyme test that do not fit into either pattern
• ENZYMES WHOSE ELEVATION REFLECTS DAMAGE TO
HEPATOCYTES
• AMINOTRANSFERASES (transaminases) –
They include AST and ALT
SERUM EMZYME TESTS ARE
GROUPED IN 3 CATEGORIES

58. • AST(SGOT) – found in liver> cardiac muscle > skeletal
muscle> kidneys >brain
• ALT(SGPT) – found primarily in liver
• Normally present in serum in low concentration (0-40
IU/L)
• When there is damage to liver cell membrane –
increased permeability and so increased serum
concentration
• Liver cell necrosis is not required for release of these
enzymes

59. •Levels of >1000 IU/L occurs in –
• Acute viral hepatitis
• Toxin and drug induced hepatitis
• Ischaemic liver injury
• In most acute hepatocellular disorders ALT is higher or
equal to AST

60. • Normal ratio is 0.7 to 1.4
• Useful in Wilson disease, chronic liver disease
and alcoholic liver disease
• AST/ALT ratio of > 2:1 is suggestive of and >3:1
is highly suggestive of ALCOHOLIC liver disease
• AST in Alcoholic live disease is rarely >300 IU/L
AST/ALT RATIO

61. • ALT is usually normal in alcoholic liver disease ; can
be sometimes low due to an alcohol induced
deficiency of pyridoxal phosphate
• AST/ALT <1 is seen in NASH and viral hepatitis

62. • 2 forms of AST are known-
1. Cytosolic
2. Mitochondrial (mAST) – it is synthesized in
precursor form (pre-mAST)
converted to mature mAST
• Accounts for 80% of total AST activity within liver
cells

63. • mAST/total AST ratio – marker of chronic alcohol
consumption
• This distinguishes those who consume excess alcohol
from normal subjects irrespective of the presence or
absence of liver disease

64. • Isolated rise of ALT is seen in
1. Chronic HepC infection
2. Fatty liver
• Isolated AST elevation
1. Alcohol -related
2. Drug -induced liver injury,
3. Hemolysis
4. Myopathic processes

65. •Determination of these enzymes are helpful in
distinguishing hepatocellular from cholestatic
jaundice
•Increase in AST and ALT is much more ( >500 IU/L)in
hepatocellular jaundice than in cholestatic jaundice
(>200 IU/L)
•Persistence of elevated ALT and AST beyond 6
months in a case of hepatitis indicates development
of chronic hepatitis

66. •This AST procedure utilizes a modification of the
methodology recommended by the IFCC(International
Federation of Clinical Chemistry)
• In this method, aspartate aminotransferase (AST)
catalyzes the transamination of aspartate and α-
oxoglutarate, forming L-glutamate and oxalacetate
• The oxalacetate is then reduced to L-malate by malate
dehydrogenase, while NADH is simultaneously
converted to NAD+

67. •The decrease in absorbance due to the consumption
of NADH is measured at 340 nm and is proportional to
the AST activity in the sample
• L-Aspartate + α-Oxoglutarate ---------AST----------------
L-Glutamate + Oxalacetate
• Oxalacetate + NADH + H+ ------------MDH---------------
L-Malate + NAD+

68. • ALT procedure is based on a modification of the
methodology recommended by the International
Federation of Clinical Chemistry (IFCC)
• ALT transfers the amino group from alanine to α-
oxoglutarate to form pyruvate and glutamate
•The pyruvate enters a lactate dehydrogenase (LD)
catalyzed reaction with NADH to produce lactate and
NAD+

69. The decrease in absorbance due to the consumption of
NADH is measured at 340nm and is proportional to the
ALT activity in the sample

71. 3 enzyme activities are important –
1. Alkaline phosphatase (ALP)
2. 5’nucleotidase (5’NT)
3. Gamma glutamyl transferase (GGT)
ALP – found in liver , bone , placenta and small intestine
Physiological increase in ALP is seen in –
1. >60 yrs
2. Pregnancy
ENZYMES WHOSE ELEVATION
REFLECTS CHOLESTASIS

72. 3. Blood groups O and B – after fatty meal influx of
intestinal ALP into blood
4. In children and adolescent during rapid bone
growth
ALP >4 times the Normal is seen in –
1. Cholestatic liver disease
2. Infiltrative liver disease such as cancer and
amyloidosis
3. Paget’s disease of bone

73. • If an isolated increase in ALP is seen , identification
of the source of elevated isoenzyme is helpful-
1. Fractionation of ALP by electrophoresis
2. Different isoenzymes have diiferent susceptibility
to inactivation by heat
• If increased heat stable fraction is found – MC
from placenta
• Most sensitive to heat inactivation is – bone ALP
3. Measure serum levels of GGT and 5’NT – they are
elevated in only liver disease

75. • Serum GGT is increased in –
1. Alcoholism- Is a helpful clue in suspected cases
of alcoholism ( even in absence of alcoholic liver
disease)
2. Cholestasis
• GGT and 5’NT is especially used to assess the
nature of ALP

76. • Normal range: 10-47 IU/L
Serum γ-Glutamyl Transferase
• Normal range: 2-17 IU/L
Serum 5’-Nucleotidase
• Normal range: 39-117 IU/L
Serum alkaline phosphatase

77. LFT IN ANTI TB TREATMENT
•LFT should be performed before starting anti TB
treatment
•With use of rifampicin and isoniazid , onset of liver
damage may be as soon as 10days after commencing
therapy
•Onset of liver injury may be upto 1 year after starting
therapy – so there is no point at which it is
safe to stop performing routine LFT

78. High risk groups are –
1. Known case of liver disease – such as
alcoholic liver disease or hepatitis B or C
infection
2. Malnourished
3. Children and elderly

80. NON INVASIVE BIOMARKERS(NIBM) FOR
ASSESSING LIVER FIBROSIS
•For assessing live fibrosis – liver biopsy is a
preferred method
•Utilization of NIBM for liver histology can reduce
but not replace the requirement for liver biopsy
•Classification of NIBMs
Class 1 fibrosis
markers/ direct
biomarkers
Class 2 fibrosis
markers/indirect
biomarkers

81. DIRECT BIOMARKERS –
•These are parts of liver matrix produced by
stellate cells during fibrosis process
•These include
Procollagen type 1 and 2
Type IV collagen
Hyaluronic acid
Laminin
MMP-1 (collagenases)
MMP-2 (gelatinase A)
MMP-9 (gelatinase B)
TIMPs (tissue inhibitor of matrix metalloproteinase)

82. INDIRECT MARKERS –
• These are molecules released into blood due to liver
inflammation
• These include
1. Serum ALT
2. Serum AST/ALT ratio (AAR) - >1 predictive of
cirrhosis
BARD score – AAR + BMI + diabetes
3. AST/platelet ratio (APRI)
4. Forn’s index – age + 3 lab tests (platelet count +
cholesterol level + GGT)

83. 5. PGA index – a marker to differentiate alcoholic
liver disease from cirrhosis
Includes – GTT + prothrombin index +
apolipoprotein A
Modified – PGAA – additional alpha2
macroglobulin
6. Fibrotest and fibrosure
7. Fibro index
8. FIB – 4 score
9. Fibro Q test
10. Steato test

84. COMBINED DIRECT AND INDIRECT
MARKERS –
Fibrometer test
Fibrospect II test
SHASTA index – used in case of HIV/HCV coinfected patients
Hepascore model
European liver fibrosis panel test (ELF)

85. For assessing fibrosis in HIV/HBV coinfected
patients –
1. Fibrometer
2. Hepascore
3. Zeng’s score

88. REFERENCES
1.Harrison’s – text book of medicine
2.Kawthalkar clinical pathology
3.Henry’s textbook of clinical
pathology

89. THANK YOU