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Liver function tests

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

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Liver function tests

  1. 1. LIVER FUNCTION TEST EKTA JAJODIA
  2. 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. 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. 4. Metabolic function Excretory function Synthetic function Detoxification function Storage function FUNCTIONS OF LIVER
  5. 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. 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. 7. Hippuric acid test Determination of blood ammonia TESTS BASED ON DETOXIFICATION FUNCTION
  8. 8. Plasma proteins Prothrombin time TESTS BASED ON SYNTHETIC FUNCTION
  9. 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. 10. ENZYMES IN DIAGNOSIS OF LIVER DISEASE SERUM TRANSAMINASES AST ALT ALP GGT 5’NT
  11. 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
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15. 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
  16. 16. 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
  17. 17. •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
  18. 18. TESTS FOR SERUM BILIRUBIN • Spectrophotometric method • Diazo method • Peroxidase method • Diazo- peroxidase method • HPLC • Transcutaneous bilirubinometer
  19. 19. 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
  20. 20. 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
  21. 21. 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
  22. 22. 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
  23. 23. •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
  24. 24. 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
  25. 25. 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
  26. 26. 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
  27. 27. 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
  28. 28. •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
  29. 29. 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
  30. 30. •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
  31. 31. Hippuric acid test Determination of blood ammonia TESTS BASED ON DETOXIFICATION FUNCTION
  32. 32. 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
  33. 33. Plasma proteins Prothrombin time TESTS BASED ON SYNTHETIC FUNCTION
  34. 34. 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
  35. 35. 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
  36. 36. 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
  37. 37. Dye binding method Immunoassay Chromatography Salt -fractionation Methods of estimation
  38. 38. BROMOCRESYL GREEN METHOD •Most common method •The complex becomes blue in color •Absorbance at 632 nm is directly proportional to the concentration of albumin
  39. 39. 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
  40. 40. 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
  41. 41. • Immunoturbidimetric procedure • Sample is incubated with antibodies specific to prealbumin • Insoluble complexes are formed
  42. 42. • 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
  43. 43. Increased in – 1. Copper toxicity 2. Pregnancy 3. OCPs
  44. 44. 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
  45. 45. • 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
  46. 46. 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
  47. 47. ENZYMES IN DIAGNOSIS OF LIVER DISEASE SERUM TRANSAMINASES AST ALT ALP GGT 5’NT
  48. 48. 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
  49. 49. • 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
  50. 50. •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
  51. 51. • 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
  52. 52. • 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
  53. 53. • 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
  54. 54. • 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
  55. 55. • 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
  56. 56. •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
  57. 57. •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+
  58. 58. •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+
  59. 59. • 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+
  60. 60. The decrease in absorbance due to the consumption of NADH is measured at 340nm and is proportional to the ALT activity in the sample
  61. 61. 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
  62. 62. 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
  63. 63. • 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
  64. 64. • 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
  65. 65. • 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
  66. 66. 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
  67. 67. 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
  68. 68. 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
  69. 69. 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)
  70. 70. 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)
  71. 71. 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
  72. 72. 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)
  73. 73. For assessing fibrosis in HIV/HBV coinfected patients – 1. Fibrometer 2. Hepascore 3. Zeng’s score
  74. 74. REFERENCES 1.Harrison’s – text book of medicine 2.Kawthalkar clinical pathology 3.Henry’s textbook of clinical pathology
  75. 75. THANK YOU

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