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Liver functrion tests
- 1. Liver Function Tests CaviteState University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 2. • Storage • Metabolism –Carbohydrates, Lipids, Proteins • Synthetic – Synthetic site of plasma proteins • Detoxification – Ammonia • Excretory and Secretory – Biliary system Biochemical Functions Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 3. • Tests forExcretory and Secretory Function – Bilirubin – Urobilinogen, Bile Acids • Tests for Detoxification Function – Liver Enzymes – Ammonia • Tests Measuring Hepatic SyntheticAbility – Total Proteins, Albumin, Gamma Globulins – PT/ PTT • Autoimmune Markers • OtherTests Assessment of Liver Function Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 4. • Serum Bilirubin:Spectrophotometry – Van den Berg Reaction (Diazotization) Evelyn-Malloy Jendrassik-Grof – Enzymatic Method Bilirubin Oxidase • Urine Bilirubin – Bilirubinuria - Conjugated Bilirubin Bile duct obstruction, Liver damage – Ehrlich’s Diazo Reaction, Ictotest • Icterus Index • Direct Spectrophotometry Bilirubin Determination Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 5. RBCs breakdown: 120days Degraded by macrophages releasing hemoglobin molecules HEMOGLOBIN HEME (Porphyrin ring) GLOBIN BROKEN DOWN TO AMINO ACIDS RE-ENTERS BLOOD FOR ERYTHROPOIESIS UNCONJUGATED BILIRUBIN IRON ALBUMIN RE-ENTERS BLOOD FOR ERYTHROPOIESIS NOT RECYCLED YELLOWISH ORANGE COLOR LIPID SOLUBLE BILIVERDIN
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- 7. CONJUGATED BILIRUBIN (WATER SOLUBLE) GLUCORONIC ACID EXCRETED BY THELIVER IN BILE CB CB BACTERIA UROBILINOGEN 10-15% RE-ABSORB BY THE BLOOD BOUND TO ALBUMIN BACTERIA 85-90% OXIDIZE BY BACTERIA FORMING STERCOBILIN UROBILINOGEN 5% WILL PARTICIPATE IN ENTEROHEPATIC UROBILINOGEN CYCLE UROBILINOGEN 5% TRANSPORTED BY THE BLOOD TO THE KIDNEYS UROBILIN UROBILINOGEN
- 8. • Specimen Collection –Serum – Fasting ? - Lipemia – Hemolysis – Avoid specimen exposure from light Bilirubin Determination Cavite State University – Main Campus College of Nursing, Department of Medical Technology Clinical Chemistry 2 Laboratory Liver Function Tests
- 9. • Van denBerg Reaction (Diazotization) • Diazo Reagent: – Diazo A = 0.1 % Sulfanilic Acid + HCl – Diazo B = 0.5 % Sodium Nitrite • Read at 560 nm • Direct Bilirubin – Bilirubin + Diazo Reagent → Azobilirubin (Pink to Purple) (Acidic) • Total Bilirubin – Bilirubin + Methanol+ Diazo Reagent → Azobilirubin (Pink to Purple) (Acidic) Solubilizer/ Coupling Accelerator : Methanol • Indirect Bilirubin – Total Bilirubin – Direct Bilirubin = Indirect Bilirubin (Unconjugated) Evelyn-Malloy Method Cavite State University – Main Campus College of Nursing, Department of Medical Technology Clinical Chemistry 2 Laboratory Liver Function Tests
- 10. • Van denBerg Reaction (Diazotization) • Buffer: Sodium Acetate • Ascorbic Acid – stop the reaction • Alkaline tartrate - to make the solution alkaline • Read at 600 nm • Direct Bilirubin – Bilirubin + Diazo Reagent + Ascorbic + Alkaline Tartrate → end product (Blue) (Alkaline) • Total Bilirubin Bilirubin + Diazo Reagent + Caffeine–Benzoate + Ascorbic + Alkaline Tartrate →(Blue) end prod Coupling Accelerator : Caffeine – Benzoate ( preferred over methanol because methanol promotes protein precipitation and increases turbidity) • Indirect Bilirubin – Total Bilirubin – Direct Bilirubin = Indirect Bilirubin (Unconjugated) Jendrassik-Grof Method Cavite State University – Main Campus College of Nursing, Department of Medical Technology Clinical Chemistry 2 Laboratory Liver Function Tests
- 11. • Enzymatic Method –pH 8.0: Total Bilirubin Conjugated, Unconjugated, Delta Bilirubin Bilirubin Oxidase Biliverdin – pH 4.5: Direct Bilirubin Conjugated Bilirubin Bilirubin Oxidase Biliverdin – Decrease in absorbance at 460 nm Bilirubin Determination Cavite State University – Main Campus College of Nursing, Department of Medical Technology Clinical Chemistry 2 Laboratory Liver Function Tests
- 12. • Icterus Index –Hyperbilirubinemia – Standard Solution (0.01% Potassium dichromate) – Dilute the serum sample with NSS • Direct Spectrophotometry – Neonatal Sample (First 24 Hours) – No reagent – 455 nm • Bilirubinometer – Transcutaneous and Non-invasive – To remove possible interferences: 1st wavelength: 455 nm 2nd wavelength: 575 nm Bilirubin Determination Cavite State University – Main Campus College of Nursing, Department of Medical Technology Clinical Chemistry 2 Laboratory Liver Function Tests
- 13. • Reference value: •Conversion Factor : 17.1 Bilirubin Determination Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 14. • Urine andFeces • Urobilinogen (colorless) Bacterial Enzymes Urobilin (Brown Pigment) • Increased urinary urobilinogen – hemolytic disease – defective liver cell function • Decreased fecal urobilinogen – biliary obstruction Urobilinogen Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 15. • Ehrlich’s Reagent:p-dimethylaminobenzaldehyde (p-DAB) • Urine Urobilinogen – hour urine sample – urobilinogen in urine + Ehrlich’s reagent red color (spectrophotometer) – Reference value: 0.1 to 1.0 Ehrlich unit every 2 hours 0.5 to 4.0 Ehrlich units per day • Fecal Urobilinogen – Semi-quantitative determination – aqueous extract of fresh feces – Urobilin alkaline ferrous hydroxide urobilinogen + Ehrlich reagent red color – Reference value: 75 to 275 Ehrlich units per 100 grams of fresh feces 75 to 400 Ehrlich units per 24-hour specimen Ehrlich’s Test Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 16. • Increased SerumBile Acids = Liver Disease • Not Specific • Ratio: Primary Bile Acids/Secondary BileAcids – Cholic: Chenodeoxycholic acids High Ratio – Biliary Obstruction Low Ratio – Primary BiliaryCirrhosis Serum Bile Acids Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 17. • AST/ALT (Aminotransferase) –damaged or necrotic hepatocytes – High concentration-drug hepatotoxicity • ALP (Phosphatase) – extrahepatic biliary obstruction • 5’-nucleotidase – differentiates ALP elevations • GGT – differentiates ALP elevations – chronic alcohol and drug ingestion • LD – LD 4 and 5 (Skeletal Muscle and Liver) Acute Hepatitis and Cirrhosis – Alcohol dehydrogenase Drug Hepatotoxicity and obstructive jaundice Liver Enzymes Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests Condition ALP 5’N GGT Not Liver Disease ↑ Normal Normal Biliary Obstruction ↑ ↑ ↑ Chronic Alcohol/ Drug intake Normal or Slight ↑ Normal ↑ Differentiate hepatocellular (functional) from obstructive (mechanical) disease
- 18. • ↑Ammonia =HepaticComa and advance liver disease – For diagnosis of hepatic failure and Reye’s Syndrome • Liver converts ammonia to urea • Pre-Analytical: – Plasma (EDTA or Heparin) – Fasting Sample – Avoid Hemolysis – AvoidTourniquet, Fist Clenching Arterial is preferred – No Exercise and Smoking – Separate plasma and red cells –Transport on Ice Ammonia Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 19. • Berthelot Reaction: –NH3 + Phenol + Hypochlorite Indophenol Blue • EnzymaticAssay: – Glutamate Dehydrogenase (GD) – Decrease in absorbance at 340 – NH4 + α- ketoglutaric acid + NADH GD Glutamic Acid + NAD+ • ReferenceValue: Ammonia Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 20. • TPAG –Cirrhosis (Decrease) • Albumin • A/G ratio • PT/ PTT • Gamma globulins – IgG and IgM Chronic active hepatitis – IgM Biliary cirrhosis – IgA Alcoholic Cirrhosis Tests Measuring Hepatic Synthetic Activity Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 21. • Anti-mitochondrialAntibody – PrimaryBiliary Cirrhosis (PBC) – Method: ELISA AMA with Anti-M2 specificity (AMA-M2) is 100% specific for PBC • Anti-neutrophil cytoplasmicAntibodies (ANCA) – Primary Sclerosing Cholangitis Autoimmune disease associated with destruction of extrahepatic and intrahepatic bile ducts • Anti-nuclearAntibody and Anti-smooth muscle Antibody – greater than 1:80 titer – Type 1 Autoimmune Hepatitis • Antibodies to Liver-Kidney Microsomal Antigens – Type 2 Autoimmune Hepatitis Autoimmune Markers Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 22. • Hepatitis Profile •MELD Score (Model for End Stage Renal Disease) – Bilirubin, INR (PT), Serum Creatinine – Probability of survival of patients with liver disease TIPS –Transjugular Intrahepatic Post-systemic Shunt > 24 MELD Score =TIPS not recommended • PGA Index – ProthrombinTime (PT) - Prolonged – Gamma glutamyl transferase (GGT) - Elevated – Apo A1 Increased –Alcoholic liver Hepatitis /Liver Injury Decreased - Cirrhosis • A1 Anti-trypsin, Ceruloplasmin – Excretory function of the liver – Ceruloplasmin – Removed by the liver – AAT- hereditary deficiency; associated with early cirrhosis Other Tests Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 23. • Vitamin KResponseTest – Supplement –Vit. K – Normal 2nd Test =Vit. K deficiency • Hippuric Acid SynthesisTest – Assess the detoxification function of the liver – Normal liver = benzoate is detoxified into hippuric acid – Normal Result: Hippuric Acid in the Urine Other Tests Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 24. • Bishop, Fody,and Schoeff. ClinicalChemistry Principles, Techniques, Correlations, Seventh Edition. 2013. • McPherson, RichardA. and Pincus, Matthew. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 23rd Edition. Philadelphia: Elsevier Inc. 2017. • Burtis, Ashwood, Bruns.Tietz Fundamentals of Clinical Chemistry 6th edition. 2008 • Anderson and Cockayne. ClinicalChemistry Concepts and Applications. 2003. References Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests
- 25. End of Presentation Thankyou and God Bless Cavite State University – Main Campus College of Nursing, Department of MedicalTechnology Clinical Chemistry 2 Laboratory Liver FunctionTests