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Liver function tests
Liver function tests can detect, distinguish, and monitor various types of liver disease. Tests are grouped based on the liver's excretory, metabolic, detoxification, storage and synthetic functions. Enzyme tests like ALT and AST indicate hepatocyte damage, while elevated ALP and GGT suggest cholestasis. Protein tests such as albumin, PT and AFP evaluate synthetic function. Bilirubin, bile salts and dye excretion tests examine excretory function. Together these tests provide insight into liver health and disease categories.
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Liver function tests
- 1. LIVER FUNCTION TEST EKTAJAJODIA
- 2. SEVERAL BIOCHEMICAL TESTS CANBE 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 rarelysuggest a specific diagnosis •They suggest a general category of liver disease, such as hepatocellular or cholestatic •This further directs the evaluation
- 4.
- 5. CLASSIFICATION OF LFT Accordingto 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.Urineand feacal urobilinogen 4.Urine bile salts 5.Dye excretion tests TESTS BASED ON EXCRETORY FUNCTION
- 7. Hippuric acid test Determination of blood ammonia TESTSBASED ON DETOXIFICATION FUNCTION
- 8. Plasma proteins Prothrombin time TESTS BASED ONSYNTHETIC FUNCTION
- 9. Tests related to CARBOHYDRATE metabolism Testsrelated to LIPID metabolism Tests related to PROTEIN metabolism Galactose tolerance test Serum cholesterol Serum proteins Aminoaciduria TESTS BASED ON METABOLIC FUNCTION
- 10. ENZYMES IN DIAGNOSISOF LIVER DISEASE SERUM TRANSAMINASES AST ALT ALP GGT 5’NT
- 11. 1.Serum bilirubin 2.Urine bilirubin 3.Urineand 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 •Dailyproduction 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 Isolatedincrease 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 hepatocytesin 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 boundconjugated 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 SERUMBILIRUBIN • 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 Berghreaction 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 EvelynMalloy 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 bilirubinfound 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 beused 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 • Increasein 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 ofcholesterol 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 bilesalts – 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 syntheticdyes 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 specimenis 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 TESTSBASED ON DETOXIFICATION FUNCTION
- 40. BLOOD AMMONIA •Produced inbody 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 ONSYNTHETIC FUNCTION
- 42. PROTEIN •Liver is thesole 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 •2methods 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 byliver •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 •Mostcommon 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 • Levelsfall 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 plasmalevels - 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 isa 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 theexception 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 DIAGNOSISOF LIVER DISEASE SERUM TRANSAMINASES AST ALT ALP GGT 5’NT
- 57. 1.Enzymes whose elevationreflects 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 >1000IU/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 ratiois 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 isusually 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 formsof 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 ASTratio – 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 riseof 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 theseenzymes 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 procedureutilizes 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 inabsorbance 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 procedureis 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 inabsorbance due to the consumption of NADH is measured at 340nm and is proportional to the ALT activity in the sample
- 71. 3 enzyme activitiesare 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 groupsO 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 anisolated 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 GGTis 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 ANTITB 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 groupsare – 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 – •Theseare 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 ANDINDIRECT 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 fibrosisin HIV/HBV coinfected patients – 1. Fibrometer 2. Hepascore 3. Zeng’s score
- 88. REFERENCES 1.Harrison’s – textbook of medicine 2.Kawthalkar clinical pathology 3.Henry’s textbook of clinical pathology
- 89.