MLS Review - CC - Liver

MEDICAL LABORATORY SCIENCE REVIEW TESTS FOR LIVER SYNTHETIC FUNCTION CLINICAL CHEMISTRY: LIVER FUNCTION TESTS OUTLINE • Liver • Tests for Liver Synthetic Function o Total Protein o Albumin o Prothrombin Time • Tests for Conjugation and Excretion Function o Bilirubin o Urobilinogen o BSP Bye Extraction Test • Tests for Detoxification Function o Enzyme Tests o Ammonia Legend:  Mnemonic/Tip to Remember • Quantitates severity of hepatic dysfunction TOTAL PROTEIN • • • • • Measures totality of protein fractions in the serum o Albumin + globulins Assessment of nutritional status and presence of severe disease involving the liver, kidney, and bone marrow Sample: serum o Plasma is not used as it contains Fibrinogen ▪ False increase in protein by 0.2-0.4 g/dL Interference: hemolysis (reddish coloration), ictericia (yellowish coloration due to ↑ bilirubin) Reference value (serum): 6.5-8.3 g/dL (CF to SI g/L: 10) METHODS FOR TOTAL PROTEIN LIVER 1. • • Liver o The chief metabolic organ ▪ Most metabolic and biochemical processes occur in the liver ▪ Synthesizes majority of biochemical substances o A large organ divided unequally into two lobes by a falciform ligament o Lobules – functional unit of the liver ▪ Responsible for all metabolic and excretory functions of the liver o Two major cell types ▪ Hepatocytes (80%) - functional hepatic cells • Performs major functions of the liver • Responsible for liver’s regenerative properties ▪ Kupffer cells (20%) - hepatic macrophages • Act as phagocytes against infections/pathogens • Lines the sinusoid of the liver o 2 blood supply: supplied by 1500 mL blood per min ▪ Hepatic artery - 25% of total supply ▪ Portal vein - 75% of total supply Functions of the Liver o Synthetic Function ▪ Ability to synthesize substance ▪ Liver synthesizes all proteins except immunoglobulins (from lymphocytes) o Conjugation Function ▪ Conjugation - involved in bilirubin metabolism o Detoxification and Drug Metabolism ▪ Ability to protect from potentially toxin substances absorbed by GI and other metabolic by products o Excretory and Secretory Function ▪ Excretion of bile • Bile - eliminates cholesterol and bile salts o Storage Function ▪ Storage site for fat-soluble (A, D, E, K) and watersoluble (B, C) vitamins ▪ Storage site for glycogen 2. Kjeldahl Method o Classical method for protein quantitation o Reference method but not routinely used ▪ The process is time consuming ▪ The test is based on assumption N content • Contributes to the inaccuracy of the test o Principle: Measures the amount of nitrogen in specimen (N in the CHON structure) o Assumes that nitrogen content of protein is 16% (15.1-16.8%) o Assumes that 1 g of nitrogen = 6.54 g of protein o 3 Steps: ▪ Digestion ▪ Distillation ▪ Titration Biuret Method o Colorimetric non-enzymatic method ▪ End-product is deep purple ▪ The reagent do not utilize enzymes o Most widely used method for total protein determination o Measures based on the number of peptide bonds present in the protein ▪ More peptide bonds = deeper color o Absorbance of color is read at 540 nm o Principle: peptide bonds present in protein forms a bond with cupric ions forming a violet-colored chelate.  Biuret = sounds like violet o Interference: lipemic sample o Biuret reagents consist of: ▪ Alk. CuSO4 ▪ NaK Tartrate (Rochelle salt) - prevent precipitation of Copper ▪ NaOH ▪ KI - stabilizer 1 3. ▪ Folin-Ciocalteu (Lowry) Method o Principle: Oxidation of phenolic compounds such as tyrosine, tryptophan, & histidine to give a deep blue color o Has the highest analytical sensitivity ▪ Can measure minute concentration of proteins o Main reagent: Phosphotungstic-molybidic acid or phenol reagent o Color enhancer: biuret reagent o End-product: deep blue color UV Absorption Method o Principle: The absorbance of proteins at 210nm is due to the absorbance of the peptide bonds at specific wavelength. ▪ Majority of proteins are measured at 210 nm except aromatic AA o Absorption @ 280nm: tryptophan, tyrosine, and phenylalanine ▪ Aromatic amino acids are measured at 280 nm 5. Refractometry - based on measurement of refractive index of serum total proteins o Based on how protein bends light o Changes is proportional to concentration of protein ▪ High refractive index = more protein 6. Turbidimetry and Nephelometry - Measurement depends on formation of a uniform fine precipitate which scatters incident light in suspension (nephelometry) or block light (turbidimetry) o Turbidimetry - amount of light blocked by proteins o Nephelopmetry - amount of light scattered by proteins 7. Salt Fractionation 8. Coomasie Brilliant Blue Dye - A dibinding technique used for detection of proteins as little as 1 ug 9. Ninhydrin Test - develops violet color by reacting with primary amines widely used for detection of peptides and amino acids after paper chromatography 10. Serum Protein Electrophoresis (SPE) o Principle: separation technique based on migration of charged particles in an electric field o Component of SPE ▪ Cathode - negative electrode ▪ Anode - positive electrode o 4. The acidic and basic amino acids content of proteins determines its net charge. (positive and negative components: amine & carboxylic acids) ▪ Buffer: Barbital (Veronal) - pH 8.6 • Renders proteins with negative charge • Proteins migrate toward the anode Normal SPE Pattern ▪ Separates proteins into 5 fractions 1. Albumin band (1st band) • Fastest band and most anodal • Most abundant band • Reference value: 3.5-5.0 g/dL (53.65%) 2. α1 Globulin band • 2nd fastest band and 2nd most anodal • α1-antitrypsin - 90% of α1 Globulins o Deficiency cause flat band in α1 • Reference value: 0.1-0.3 g/dL (2.5-5%) 3. α2 Globulin band • Reference value: 0.6-1.0 g/dL (7-13%) 4. β Globulin band • Reference value: 0.7-1.1 g/dL (8-14%) 5. γ Globulin band • Includes immunoglobulins • Reference value: 0.8-1.6 g/dL (12-22%) ALBUMIN • • • • • • Most abundant among all proteins Synthesized by the liver o Liver synthesizes 9-12 g of albumin per day Concentration is directly proportional to the severity of hepatic disease. Low albumin due to decreased liver synthesis Reference value: 3.5-5.0 g/dL Methods: o Salt precipitation o Dye-binding ALBUMIN MEASUREMENT • • o Isoelectric Property of Proteins ▪ Amphoteric – either positive or negative depending on pH condition • Basic (↑ pH) = negative charge • Acid (↓ pH) = positive charge ▪ No charge at isoelectric point Salt Precipitation o Globulins are precipitated ▪ Removes globulins from the total protein o Albumin is quantitated using biuret reaction Dye Binding o Used in SPE, charges protein positively to bind dyes (anionic dyes are used) o Bromcresol Green (BCG) – most commonly used, sensitive dye for albumin o Bromcresol purple (BCP) – most specific dye o Methyl Orange – not specific for albumin o 2,4’-hydroxyazobenzene-benzoic acid (HABA) – more specific to albumin than methyl orange ▪ Interferences: conjugated bilirubin, aspirin, penicillin, sulfonamides 2 o ALBUMIN/GLOBULIN RATIO • • • • To determine if globulin is higher than albumin Inverted A/G ratio = globulin > albumin o Present in cirrhosis, multiple myeloma, Waldenström macroglobulinemia Reference value: 1.3-3.1 g/dL (CF to g/L: 10) Formula: AG ratio = Albumin Globulin Globulin = Total protein - Albumin PROTHROMBIN TIME • • • • • • Aka in CC as Vitamin K Response Test o Patient should be administered with Vit. K (10 mg for 1-3 days) prior to testing Based on the clotting factors that are Vitamin-K dependent o Prothrombin group (II, VIII, IX, X) - vit. K dependent Differentiates intrahepatic disorder from extrahepatic disorder Acute/Chronic Hepatitis – prolonged prothrombin time signifies massive cellular damage. o No CF production after vit. K administration Intrahepatic - prolonged PT after vit. K administration Extrahepatic - normal PT after vit. K administration • TESTS FOR CONJUGATION AND EXCRETION FUNCTION • Involves measurement of Bilirubin BILIRUBIN • • • • • • End product of hemoglobin metabolism o Has no physiologic function; a waste product o Must be excreted Conjugated and excreted through the liver Principal pigment in bile Also formed from destruction of heme-containing proteins such as myoglobin, catalase, and cytochrome oxidase Distribution: o 85% - heme portion of the hemoglobin o 15% - other heme-containing proteins Synthesis and Excretion • • • • Hemoglobin components ▪ Iron - transported by transferrin to storage ▪ Heme - converted to bilirubin ▪ Globin - degraded to amino acids o Albumin detaches B1 in the liver and B1 attaches with ligandins o UDPGT ▪ uridine diphosphate glucoronyl/glucoronosyl transferase ▪ responsible for conjugation of bilirubin o B2 → hepatic duct (combine B2 + gallbladder secretions) → common bile duct → intestine o Stercobilin - orange colored-substance responsible for giving the brown color of the stool o Urobilin - one of urochromes responsible for urine color Bilirubin 1 & 2 Bilirubin 1 (IB) Bilirubin 2 (DB) • Unconjugated bilirubin • Conjugated bilirubin • Indirect bilirubin • Direct bilirubin • Water insoluble • Water soluble • Non-polar bilirubin • Polar bilirubin • Hemobilirubin (found • Cholebilirubin and synthesized in the • One-minute/prompt circulation) bilirubin • Pre-hepatic bilirubin • Post-hepatic/ Hepatic (formed in the bilirubin (formed in the circulation before liver) reaching the liver) • Obstructive/ • Slow-reacting Regurgitative bilirubin • Normally present in the • Normally not present in circulation the circulation • Small amounts may leak in the circulation (0 to minute amounts) o Bilirubin should be conjugated to be excreted in body Plasma concentration of bilirubin increases upon birth and reaches its peak on the 5th day. o Newborns are normally slightly jaundiced after birth due to high bilirubin levels If bilirubin synthesis > liver clearance rate = ↑↑ bilirubin o Increased bilirubin but no conjugation for excretion Reference value: o Conjugated bilirubin: 0-0.2 mg/dL (0-3 µmol/L) o Unconjugated bilirubin: 0.2-0.8 mg/dL (3-14 µmol/L) o Total bilirubin: 0.2-1.0 mg/dL (3-17 µmol/L) Delta Bilirubin o An abnormal form of bilirubin o Conjugated bilirubin bounded to albumin o Have longer half-life than other bilirubin forms o Formed in prolonged elevation of conjugated bilirubin in the circulation o Helps in monitoring the decline of serum bilirubin following cholecystectomy o It reacts with diazo reagent ▪ Measured with total bilirubin (B1 + B2) ▪ ↑ Delta bil = ↑ Total bil ▪ Delta bil = TB - (B1 + B2) o Measurement should not be performed to neonates (≤14 days old) = false ↑↑ ▪ Neonates normally have increased delta bilirubin o Reference value: <0.2 mg/dL 3 JAUNDICE INHERITED DISORDERS OF BILIRUBIN METABOLISM • • • • • • • Hyperbilirubinemia (↑ bilirubin) characterized by yellow discoloration of the skin, sclera, and mucus membranes. o Visible jaundice = Total Bilirubin = 3.0-5.0 mg/dL or ↑ ▪ Visible discoloration is observed within this levels o Overt Jaundice: 1.0-1.5 mg/dL ▪ Hyperbilirubinemia but no observable yellowish discoloration Icterus/Ictericia – more commonly termed for serum or plasma with yellow discoloration due to hyperbilirubinemia. o In the past, term is used interchangeable with jaundice Categorized based on the site of disorder Pre-Hepatic Jaundice o Hemolytic jaundice, unconjugated hyperbilirubinemia o Bilirubin increased in the pre-hepatic stage (B1) o Caused by too much red blood cell destruction ▪ Hemolytic disease of the newborn (HDN) • Fetus do not develop jaundice because the placenta can remove bilirubin • After birth, neonates develop jaundice ▪ Hemolytic anemia ▪ Malaria o Laboratory findings: ▪ Indirect Bilirubin: increased • Toxic as it can cross BBB (Blood Brain Barrier) • Cause Kernicterus o Bilirubin deposition in the brain o Can lead to severe motor dysfunction and retardation ▪ Direct Bilirubin: normal ▪ Urobilinogen: normal ▪ Urine bilirubin: negative Post-Hepatic Jaundice o Obstructive jaundice o Involves stages after the liver; excretion of bilirubin ▪ B1 was formed and conjugated to B2 ▪ Issue with excretion of bilirubin o Caused by failure of bile to flow to the intestine or impaired bilirubin excretion o Conjugated bilirubin increases o Laboratory diagnosis: ▪ Indirect bilirubin: normal ▪ Direct bilirubin: increased ▪ Urobilinogen: decreased ▪ Urine bilirubin: positive • Bilirubin is excreted in the urine ▪ Alkaline phosphatase: increased • Primary marker for obstructive jaundice • Increases with increased excretion rate due to obstruction Hepatic Jaundice o Hepatocellular combined jaundice o Involves conjugation of bilirubin o Caused by disorders of bilirubin metabolism, transport defects, hepatocellular injury or destruction. o Most common causes are inherited disorders o Laboratory findings: ▪ Indirect bilirubin: increased ▪ Direct bilirubin: increased ▪ Urobilinogen: decreased ▪ Urine bilirubin: positive • • • Gilbert’s Syndrome o Bilirubin transport deficit o Characterized by impaired cellular uptake of bilirubin due to genetic mutation in UGT1A1 gene (chromosome #2) o Impaired ability to transport bilirubin in the liver and impaired hepatocyte uptake of bilirubin o Affected individuals may have no symptoms but may have mild icterus & predisposed acetaminophen toxicity o Laboratory findings: ▪ Indirect Bilirubin (↑): 1.5-3.0 mg/dL Crigler-Najjar Syndrome o Conjugation deficit (deficiency in UDPGT) o Chronic non-hemolytic unconjugated hyperbilirubinemia o Laboratory result: elevated indirect bilirubin o Crigler-Najjar Syndrome Type I ▪ Complete deficiency of UDGPT ▪ Laboratory result: • Indirect bilirubin (↑) : >25 mg/dL • Direct bilirubin: none ▪ Symptoms: kernicterus, colorless bile o Crigler-Najjar Syndrome Type II ▪ Partial deficiency of UDGPT ▪ Laboratory results: • Indirect bilirubin: 5-20 mg/dL o Treatment: phototherapy ▪ Indirect bilirubin is photosensitive Lucey-Driscoll Syndrome o Familial form of unconjugated hyperbilirubinemia caused by circulating inhibitor of bilirubin conjugation ▪ Inhibits B2 production o Laboratory findings: ▪ Indirect bilirubin: increased (2-3 weeks of life) Dubin-Johnson Syndrome and Rotor Syndrome o Bilirubin excretion deficit o Conjugated hyperbilirubinemia o Defective excretion of direct bilirubin into the canaliculi caused by hepatocyte membrane defect. o Characterized of an intense dark pigmentation of liver (“black liver”) due to accumulation of lipofuscin pigment o Rotor Syndrome ▪ Similar with Dubin-Johnson Syndrome without the “black liver” ▪ Cause is unknown o Laboratory findings: ▪ Elevation in direct bilirubin and total bilirubin (2-5 mg/dL) • Defective excretion increases bilirubin conc. ▪ Delta Bilirubin: increased • Formation of delta bil due to accumulation of B2 BILIRUBIN METHODOLOGY • Sample: serum o Stored immediately in a dark storage space and protected from light (using carbon paper/aluminum foil) o If sample is not protected from light, concentration decreases at 30-50% per hour o If sample is prepared and stored properly: ▪ Room temperature: 2 days ▪ Refrigerator (4˚C): 1 week ▪ Freezer (-20˚C): indefinite o Fasting sample is preferred but not required 4 • Interferences: o Lipemia: increased bilirubin concentration; false ↑ o Hemolysis: decrease reaction of bilirubin with diazo reagent (decreased bilirubin); false decrease BILIRUBIN ASSAY • • • Principle: Van den Berg Reaction – diazotization of bilirubin to form azobilirubin Only measure conjugated bilirubin and total bilirubin. o Specifically measures conjugated bilirubin (direct bil) o Conjugated bilirubin directly reacts with Diazo reagent o Total bilirubin is measured by adding accelerants to hasten conjugation of unconjugated bilirubin ▪ B1 (indirect bil) are slow-reacting bilirubin Two Methods: o Evelyn and Malloy Method ▪ Principle: Van den berg reaction ▪ Reagents: • Coupling accelerator: methanol o Allows measurement of TB and B1 • Diazo reagents: only measure B2/DB o Diazo A – 0.1% sulfanilic acid + HCl o Diazo B – 0.5% sodium nitrite o Diazo blank – 1.5% HCl ▪ Performed at pH near 1.0 ▪ Final reaction: pink to purple azobilirubin (with maximal absorption at 560 nm) ▪ Disadvantage: accelerator (methanol) can cause protein precipitation causing turbidity o Jendrassik and Grof Method ▪ Popular technique for discreet analyzers (automated) ▪ More commonly used • Increased sensitivity • Do not cause turbidity due to ppt of proteins • Safer technique as more neutral pH is used ▪ Main reagent: Diazo reagent ▪ Accelerator: caffeine sodium benzoate ▪ Buffer: sodium acetate ▪ Ascorbic acid: terminates the accelerator and destroys excess diazo reagent ▪ Alkaline tartrate solution: provides alkaline pH after addition of ascorbic acid ▪ Final reaction: blue azobilirubin (measured at 600nm) CLINICAL SIGNIFICANCE • •  Increased Indirect Bilirubin o Hemolytic anemia o G-6-PD deficiency o Gilbert’s Syndrome (bilirubin transport deficit) o Criggler-Najjar Syndrome (conjugation deficit) o Lucey-Driscoll Syndrome (conjugation inhibition) o Hepatocellular disease Increased Direct Bilirubin o Dubin-Johnson Syndrome o Biliary obstruction (gall stones) o Pancreatic (head) cancer o Alcoholic and viral hepatitis o Biliary atresia o Hepatocellular disease Remember: All present ↑ TB o Pre-Hepatic = ↑ IB o Hepatic = ↑ IB, DB o Post-hepatic = ↑ DB UROBILINOGEN • • • • • • Colorless end product of bilirubin metabolism that is oxidized by intestinal flora to urobilin or stercobilin Collection of sample: avoid light exposure (light-sensitive) Specimen: 2-hr freshly collected urine or freshly collected stool Method: Ehrlich’s method o Uses p-DABA (p-dimethylaminobenzaldehyde) Reference value: o Urine: 0.1-1.0 Ehrlich Unit/2 hour or 0.54 Ehrlich Unit/day urine o Stool: 75-275 Ehrlich Unit/100g of feces o 1 Ehrlich unit = 1 mg urobilinogen For complete biliary obstruction, it may cause decreased urobilinogen o Biliary obstruction prevents B2 from reaching intestine thus B2 won’t be oxidized to urobilinogen o Complete biliary obstruction = absent uro BROMSULFONTHALEIN (BSP) BYE EXTRACTION TEST • • • Test for hepatocellular function and potency of bile duct The test is rarely used as it involves the use of bromsulfonthalein dye o Bromsulfonthalein - has no physiologic function, only functions as a stain and is excreted as waste 2 Methods of Administration: o Rosenthal White Method ▪ Double collection method ▪ BSP Dose: 2 mg/kg body weight ▪ Specimen collection: blood samples • 5 minutes after BSP administration • 30 minutes after BSP administration ▪ Reference value: • After 5 minutes: 50% dye retention • After 30 minutes: 0% dye retention o Mac Donald Method ▪ Single collection method ▪ BSP Dose: 5 mg/kg body weight ▪ Specimen collection: after 45 minutes ▪ Reference value: • After 45 minutes: +/- 5% dye retention TESTS FOR DETOXIFICATION FUNCTION • Involves enzyme and ammonia tests ENZYME TESTS • • • It is used to assess the extent of liver damage. o Enzymes are located intracellularly o Enzymes within hepatocytes are liberate in the event of liver damage o Increased enzyme activity suggests liver damage Indicator of cell injury in early or localized liver disease (hepatocellular/functional disease) Hepatic enzymes: o Alanine aminotransferase (ALT) o Aspartate aminotransferase (AST) o 5’ nucleotidase (5’NT) o Gamma glutamyl transferase (GGT) o Lactate dehydrogenase (LDH) o Ornithine carbamoyltransferase (OCT) 5 AMMONIA • • Diagnostic marker of hepatic failure Detoxified into urea (liver), then eliminated by the kidneys (urine) o Hepatic failure/issues prevent urea cycle and prevents conversion of ammonia into urea o Ammonia - neurotoxin ▪ Converted to glutamate when not converted to urea ▪ Increased glutamate may cause comatose o Urea is eliminated in the urine o • • Ammonia must be converted to urea through the urea cycle or Krebs henseleit cycle which occur in the liver Reference value: 19-60 ug dL Methodology o Preferred specimen: Venous blood ▪ Anticoagulants: Heparin, EDTA, Potassium oxalate ▪ Immediately kept in ice water during transport • Ammonia - converted to urea by bacteria (urease positive bacteria) • Prolonged standing in RT will allow bacteria to convert ammonia to urea = false decrease ▪ During collection, minimal tourniquet use must be observed and fist clenching must be avoided (false increase) o Specimen precautions: ▪ Smoking (false ↑ by 100-200 ug/L per cigar) • The patient and the phlebotomist should not be exposed to smoke as it sticks with the clothing ▪ Exercise ▪ Probing of vein ▪ Under filing of tube ▪ Hemolysis • Ammonia within RBC is 2-3x more concentrated than ammonia in plasma o Storage: frozen GLUTAMATE DEHYDROGENASE • • • Most commonly used ammonia methodology utilized in automated analyzers Inverse colorimetric enzymatic reaction Decrease in absorbance at 340nm as NADPH is consumed (oxidized) • 6