Liver Function Tests

CLINICAL CHEMISTRY 2 LABORATORY

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
Biochemical Functions
• Storage
• Metabolism
– Carbohydrates, Lipids, Proteins
• Synthetic
– Synthetic site of plasma proteins
• Detoxification
– Ammonia
• Excretory and Secretory
– Biliary system

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
Assessment of Liver Function
• Tests for Excretory and Secretory Function
– Bilirubin
– Urobilinogen, Bile Acids
• Tests for Detoxification Function
– Liver Enzymes
– Ammonia
• Tests Measuring Hepatic Synthetic Ability
– Total Proteins, Albumin, Gamma Globulins
– PT/ PTT
• Autoimmune Markers
• Other Tests
Cavite State University – Main Campus Clinical Chemistry 2 Laboratory
College of Nursing, Department of Medical Technology Liver Function Tests
Bilirubin Determination
• 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
Cavite State University – Main Campus Clinical Chemistry 2 Laboratory
College of Nursing, Department of Medical Technology Liver Function Tests
 RBCs breakdown: 120 days

PRE-HEPATIC Degraded by macrophages

PHASE releasing hemoglobin
molecules
HEMOGLOBIN

BILIVERDI BROKEN DOWN TO AMINO

N HEME ACIDS RE-ENTERS BLOOD FOR

ERYTHROPOIESIS
(Porphyrin GLOBIN
ring)

RE-ENTERS BLOOD FOR

ERYTHROPOIESIS
ALBUMIN
IRON

UNCONJUGATED
BILIRUBIN

 NOT RECYCLED
 YELLOWISH ORANGE
COLOR
 LIPID SOLUBLE
 UNCONJUGATED
BILIRUBIN

HEPATIC PHASE GLUCORONIC

ACID

UDP-GLUCORONYL
TRANSFERASE

GLUCORONIC
ACID

CONJUGATED
BILIRUBIN
 GLUCORONI
CONJUGATED C ACID
BILIRUBIN
(WATER
EXCRETED BY SOLUBLE)
POST-HEPATIC THE LIVER IN
BILE

PHASE
UROBILINOGEN CB
5% TRANSPORTED BY
THE BLOOD TO THE
KIDNEYS
UROBILIN
UROBILINOGEN CB
5% WILL PARTICIPATE
IN ENTEROHEPATIC BACTERIA
UROBILINOGEN
CYCLE UROBILINOGEN

UROBILINOGEN
BACTERIA
10-15% RE-ABSORB 85-90% OXIDIZE
BY THE BLOOD BY BACTERIA
BOUND TO FORMING
ALBUMIN STERCOBILIN
BROWN
PIGMENT IN
FECES
Bilirubin Determination
• Specimen Collection
– Serum
– Fasting ? - Lipemia
– Hemolysis
– Avoid specimen exposure from light

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
Evelyn-Malloy Method
• Van den Berg 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)
Cavite State University – Main Campus Clinical Chemistry 2 Laboratory
College of Nursing, Department of Medical Technology Liver Function Tests
Jendrassik-Grof Method
• Van den Berg 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)
Cavite State University – Main Campus Clinical Chemistry 2 Laboratory
College of Nursing, Department of Medical Technology Liver Function Tests
Bilirubin Determination
• 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

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
Bilirubin Determination
• 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
Cavite State University – Main Campus Clinical Chemistry 2 Laboratory
College of Nursing, Department of Medical Technology Liver Function Tests
Bilirubin Determination
• Reference value:
• Conversion Factor : 17.1

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
Urobilinogen
• Urine and Feces
• Urobilinogen (colorless) Urobilin (Brown Pigment)
Bacterial Enzymes

• Increased urinary urobilinogen

– hemolytic disease
– defective liver cell function
• Decreased fecal urobilinogen
– biliary obstruction

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
Ehrlich’s Test
• 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
Cavite State University – Main Campus Clinical Chemistry 2 Laboratory
College of Nursing, Department of Medical Technology Liver Function Tests
Serum Bile Acids
• Increased Serum Bile Acids = Liver Disease
• Not Specific
• Ratio: Primary Bile Acids/Secondary Bile Acids
– Cholic: Chenodeoxycholic acids
 High Ratio – Biliary Obstruction
 Low Ratio – Primary Biliary Cirrhosis

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
 Liver Enzymes
Differentiate hepatocellular (functional) from obstructive (mechanical) disease
• AST/ALT (Aminotransferase)
– damaged or necrotic hepatocytes Condition ALP 5’N GGT
– High concentration-drug hepatotoxicity
Not Liver
• ALP (Phosphatase) Disease
↑ Normal Normal
– extrahepatic biliary obstruction
• 5’-nucleotidase Biliary
↑ ↑ ↑
– differentiates ALP elevations Obstruction
• GGT Chronic Normal
– differentiates ALP elevations Alcohol/ Drug or Normal ↑
– chronic alcohol and drug ingestion intake Slight ↑
• LD
– LD 4 and 5 (Skeletal Muscle and Liver)
 Acute Hepatitis and Cirrhosis
– Alcohol dehydrogenase
 Drug Hepatotoxicity and obstructive jaundice
Cavite State University – Main Campus Clinical Chemistry 2 Laboratory
College of Nursing, Department of Medical Technology Liver Function Tests
Ammonia
• ↑Ammonia = Hepatic Coma 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
– Avoid Tourniquet, Fist Clenching
 Arterial is preferred
– No Exercise and Smoking
– Separate plasma and red cells – Transport on Ice

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
Ammonia
• Berthelot Reaction:
– NH3 + Phenol + Hypochlorite Indophenol Blue
• Enzymatic Assay:
– Glutamate Dehydrogenase (GD)
– Decrease in absorbance at 340
– NH4 + α- ketoglutaric acid + NADH GD
Glutamic Acid + NAD+
• Reference Value:

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
Tests Measuring Hepatic Synthetic Activity
• TPAG – Cirrhosis (Decrease)
• Albumin
• A/G ratio
• PT/ PTT
• Gamma globulins
– IgG and IgM
 Chronic active hepatitis
– IgM
 Biliary cirrhosis
– IgA
 Alcoholic Cirrhosis

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
Autoimmune Markers
• Anti-mitochondrial Antibody
– Primary Biliary Cirrhosis (PBC)
– Method: ELISA
 AMA with Anti-M2 specificity (AMA-M2) is 100% specific for PBC

• Anti-neutrophil cytoplasmic Antibodies (ANCA)

– Primary Sclerosing Cholangitis
 Autoimmune disease associated with destruction of extrahepatic and intrahepatic bile ducts

• Anti-nuclear Antibody and Anti-smooth muscle Antibody

– greater than 1:80 titer
– Type 1 Autoimmune Hepatitis
• Antibodies to Liver-Kidney Microsomal Antigens
– Type 2 Autoimmune Hepatitis
Cavite State University – Main Campus Clinical Chemistry 2 Laboratory
College of Nursing, Department of Medical Technology Liver Function Tests
Other Tests
• 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
– Prothrombin Time (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
Cavite State University – Main Campus Clinical Chemistry 2 Laboratory
College of Nursing, Department of Medical Technology Liver Function Tests
Other Tests
• Vitamin K Response Test
– Supplement – Vit. K
– Normal 2nd Test = Vit. K deficiency
• Hippuric Acid Synthesis Test
– Assess the detoxification function of the liver
– Normal liver = benzoate is detoxified into hippuric acid
– Normal Result: Hippuric Acid in the Urine

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
References
• Bishop, Fody, and Schoeff. Clinical Chemistry Principles,
Techniques, Correlations, Seventh Edition. 2013.
• McPherson, Richard A. 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. Clinical Chemistry Concepts and
Applications. 2003.

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests
 End of Presentation
Thank you and God Bless

Cavite State University – Main Campus Clinical Chemistry 2 Laboratory

College of Nursing, Department of Medical Technology Liver Function Tests