Clinical Chemistry 2

BS Medical Laboratory Science

Professor: Ms. Alliah Dela Cruz, RMT

LIVER FUNCTION TEST TEST MEASURING HEPATIC SYNTHETIC ABILITY:

• Chief metabolic organ of the body! A. TOTAL PROTEIN METHODOLOGIES
• MACROSCOPIC VIEW: Bilobed organ richly vascularized
with two main supply vessels: the hepatic artery (supply 1. KJELDAHL METHOD:
oxygenated blood - 25% total of blood supply) and the PRINCIPLE REAG QUANTIFICAT INTERFERENC
portal vein ENT ION OF E
• MICROSCOPIC COMPONENT: AMMONIA
o LOBULE - structural functional unit of the liver 1. Digestion H2SO 1. Alkali Ammonia
▪ CORDS - ‘’hepatocytes”; surrounds the of protein 4 Titration Contamination
central vein using strong 2. Nesslerization
▪ SINUSOIDS - Blood spaces lined with acid 3. Berthelot
endothelial cells and Kupffer's cells that followed by Reaction
surrounds the cords measureme
▪ BILE CANALICULI - Small channels nt of
between hepatocytes that carry bile nitrogen
formed by the hepatocytes to the bile content
duct 2.
NOTES: Reference
Method
• Hepatic to portal vein: 75%
but not
• 80% damage of liver it will only shows you have a
recommen
liver disease or illness
ded

FUNCTIONS OF LIVER
2. BIURET REACTION:
➢ Synthetic
Principle Reagent Notes Interference
➢ Detoxification
1. Based on Alkaline Recommended Ammonia
TEST MEASURING HEPATIC SYNTHETIC ABILITY: the ability of Copper for measuring Contamination
TPAG TEST: peptide Sulfate total protein
➢ TPAG bonds to
o Useful for quantitating the severity of hepatic react with Rochelle Absorbance of
dysfunction copper ions Salt the resultant
o Serum albumin and Vitamin K dependent to form a complex is
coagulation factors provide the most useful biuret/purple NaOH read at 545
indices for assessing severity of liver disease complex Potassium nm which is
o TPAG – most important liver functions test 2. Routine Iodine proportional to
➢ PROTHROMBIN TIME Method protein
concentration
TEST MEASURING HEPATIC SYNTHETIC ABILITY:
A. TOTAL PROTEIN AND ALBUMIN NOTE:
Cupric ions + di peptide + NaK tartrate (Rochelle salt) + K
Test Specimen Specimen Reference iodine + NaOH = Violet – 545 nm (UV length)
Collection Range
Total SERUM or 1. Prevent Total protein: 3. FOLIN CIOCALTEU (LOWRY) METHOD:
protein PLASMA overapplication Principle Reagent Notes Interference
and (higher and tourniquet A. Ambulatory: 1. Based on Phosphotungstic Highest Other
albumin total 2. Avoid 6.5-8.5 g/dl oxidation of -molybdic acid analytical oxidizable
protein due Hemolysis B. Recumbent: phenolic Biuret Reagent sensitivity substances
to the 6.0-7.8 g/dl compounds
presence of such as
fibrinogen) Albumin: 3.5- tyrosine,
5.0 g/dl tryptophan
and histidine
to give color

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 Clinical Chemistry 2
BS Medical Laboratory Science
Professor: Ms. Alliah Dela Cruz, RMT

SERUM ELECTROPHORESIS: ABNORMAL SPE PATTERN

2. Biuret SPE PATTERN ASSOCIATED
reaction CONDITION
followed by Gamma Spike Multiple Myeloma
Oxidation of Beta Gamma Bridging Hepatic Cirrhosis
Phenolic Flat cure in A1 band AAT deficiency
compounds Flat curve in Gamma Hypogammaglobulinemia
Globulin band
4. SERUM ELECTROPHORESIS A1-Globulin Band Spike Nephrotic Syndrome
Principle Reagent Notes A1, A2 and Beta Band Spike Inflammation
Separation based on Cellulose After separation
electrical charges of Acetate or protein fractions • Ionto electrophoresis and zone electrophoresis -
protein fraction Agarose Gel - are immersed in two UV light
COMPONENTS OF support media acidic solution
• Specimen to be used in electrophoresis are serum,
ELECTROPHORESIS followed by
CSF, and urine
SYSTEM: quantification by
• Serum electrophoresis pattern can be seen at gamma
• Support medium Densitometry,
spike
• Buffer UV Light
• Sample Visualization or
5. REFRACTOMETRY:
• Driving force by used of
Principle Interferences
• Detecting System specific dyes.
Based on measurement of Hyperlipidemia
refractive index of the Hemoglobinemia
Common dyes:
sample due to the presence Bilirubinemia
• Commasie
of protein solutes in the
Brilliant Blue
sample.
• Ponceau S
• Amido Black
Capacity of the solutes to
• Sudan Black
alter light transmitted
• Periodic Acid
through a solvent
Schiff

TEST MEASURING HEPATIC SYNTHETIC ABILITY:

1. ALBUMIN METHODOLOGIES
4. SERUM ELECTROPHORESIS: COMMON SPE PATTERN

1. SALT PRECIPITATION
Principle Reagent Notes
Based on the Sodium Sulfate Albumin in the
Protein Fraction Mobility separation of Ammonium Sulfate supernatant after
ALBUMIN 1st band, fastest migration Albumin and Methanol precipitation is
A1-Globulins 2nd fastest band globulin through measured using
A-2 Globulins Located at the middle section of the precipitation Biuret method
SPE pattern using high
B-Globulins 4th band concentration of
G-Globulins Slowest band in term in migration Salt

SERUM ELECTROPHORESIS: NORMAL SPE PATTERN 2. DYE BINDING TECHNIQUE

Principle Dye Notes
Based on Methyl Non specific dye for albumin
the ability Orange
of
Albumin
the

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 Clinical Chemistry 2
BS Medical Laboratory Science
Professor: Ms. Alliah Dela Cruz, RMT

absorb
dyes
Bromcresol Most commonly used;
Green Advantage: absorb strongly
below 500 with maximum
absorbance at 630 nm
HABA Affected by interferences due
to salicylates and bilirubin
Bromcresol Most specific dye; High
Purple precision and sensitivity to
albumin

TEST MEASURING HEPATIC SYNTHETIC ABILITY:

TPAG GENERAL CLINICAL SIGNIFICANCE
CLINICAL SIGNIFICANCE

Causes of Causes of
Increased Value Decreased Value
Total protein Cancer, Multiple LIVER DAMAGE, TEST MEASURING CONJUGATION AND EXCRETION:
Myeloma, Gastrointestinal TWO TYPES OF BILIRUBIN
Dehydration, cancers,
Infection Malnutrition,
BILIRUBIN 1 BILIRUBIN 2
Glomerulonephritis
Other name Unconjugated Conjugated
Bilirubin, Bilirubin, Direct
Albumin Dehydration, LIVER DAMAGE,
Nonpolar Bilirubin, Polar
Sunstroke, Multiple Burns, Kidney
Bilirubin, Indirect Bilirubin,
Sclerosis, Diseases,
Bilirubin, Cholebilirubin
Hypothyroidism Malnutrition and
Hemobilirubin
Malabsorption,
Solubility in No Yes
Pregnancy
H2O
Solubility in Yes Yes
TEST MEASURING HEPATIC SYNTHETIC ABILITY:
Alcohol
B. Prothrombin time
Bound to Yes No
• Useful for differentiating intrahepatic disorder Protein
(ABNORMAL PROTIME) from extrahepatic (NORMAL
Affinity for High Low
PROTIME)
Brain Tissue
• Prolonged protime despite Vitamin K Present in Urine No Yes
administration indicates loss of hepatic or Stool
capacity to synthesize proteins (specifically
Reaction with Indirect Direct
clotting factors)
Diazo Reagent

TEST MEASURING CONJUGATION AND EXCRETION:

TEST MEASURING CONJUGATION AND EXCRETION:
I. BILIRUBIN
DELTA BILIRUBIN
• Principal pigment in bile that is derived from
DELTA BILIRUBIN:
hemoglobin breakdown
• Conjugated Bilirubin tightly bound to Albumin
• Major heme waste product
• Has a longer half life compared to B1 and B2
• Formed due to prolonged increased of B2 in biliary
TEST MEASURING CONJUGATION AND EXCRETION:
obstruction
BILIRUBIN FORMATION

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 Clinical Chemistry 2
BS Medical Laboratory Science
Professor: Ms. Alliah Dela Cruz, RMT

TEST MEASURING CONJUGATION AND EXCRETION: TEST MEASURING CONJUGATION AND EXCRETION:
BILIRUBIN METHODOLOGY PRE-HEPATIC JAUNDICE
• Avoid Hemolysis (hemolysis will cause falsely elevated Other name Causes Lab. Findings
Bilirubin measurement) Hemolytic Excessive B1: INCREASED
• Lipemic samples will cause falsely decreased Bilirubin jaundice destruction of B2: NORMAL
Measurement Retention RBC UROBILINOGEN:
• Bilirubin is a PHOTOSENSITIVE analyte jaundice Decreased NORMAL
Hepatic Uptake
A. BILIRUBIN ASSAY Gilbert’s Disease
PRINCIPLE: VAN DEN BERGH REACTION – Diazotization Criggler-Najjar
of Bilirubin to produce azobilirubin SYndrome
REACTION: BILIRUBIN + DIAZOTIZATION SULFANILIC
ACID → AZOBILIRUBIN TEST MEASURING CONJUGATION AND EXCRETION:
POST HEPATIC JAUNDICE
Assay Evelyn and Jendrassik and Grof Other name Causes Lab. Findings
Malloy Regurgitative Obstruction of the B1: INCREASED
pH Acid Alkaline jaundice biliary duct B2: INCREASED
Dissociating Methanol Caffeine Sodium UROBILINOGEN:
Agent/Coupling Benzoate Obstructive Increased ALP INCREASED
Accelerator jaundice and 5’N activity URINE
Additional Diazo Reagent Diazo Reagent BILIRUBIN:
Reagent (Sulfanilic Acid) Sodium Acetate Cholestatic Impairment of POSITIVE
Ascorbic Acid jaundice Hepatic Section
Diazo Product Red or Reddish Blue or Blue-Purple
Purple Azobilirubin Intrahepatic
Azobilirubin Cholestasis
Notes Maximum Maximum absorbance:
absorbance: 600nm Cholelithiasis or
560nm Candidate Gallstones
Reference Method
Most Sensitive Strictures,
Preferred Method Spasms or Atresia
by automated
analyzers Cancer of the
Pancreatic Head
TEST MEASURING CONJUGATION AND EXCRETION:
BILIRUBIN REFERENCE VALUES Ascaris
CONJUGATED BILIRUBIN 0-0.2 mg/dl Lumbricoides
UNCONJUGATED BILIRUBIN 0.2-0.8 mg/dl
TOTAL BILIRUBIN 0.2-1.0 mg/dl Dubin-Johnson

TEST MEASURING CONJUGATION AND EXCRETION: Rotor Syndrome

JAUNDICE
• Yellowish pigmentation of the skin, mucous membrane TEST MEASURING CONJUGATION AND EXCRETION:
and sclera of the eyes due to hyperbilirubinemia OTHER DERANGEMENTS IN BILIRUBIN
• Not apparent until the bilirubin exceeds 2-3 mg/dl (3.0-5.0 Dubin Rotor Lucey
mg/dl BISHOP) Johnson Syndrome Driscoll
• CAUSES: Pathophysiolog Bilirubin Bilirubin Inhibition
o Accumulation of abnormal amounts of either free y excretion excretion of Bilirubin
or conjugated Bilirubin or both deficit deficit Conjugatio
o Excessively high rate of red cell destruction n
(Erythroblastosis Fetalis) Analytes Increased Increased Increased
B2 B2 B1

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 Clinical Chemistry 2
BS Medical Laboratory Science
Professor: Ms. Alliah Dela Cruz, RMT

Other Liver No TEST MEASURING DETOXIFICATION FUNCTION:

Pigmentatio pigmentatio A. REYE SYNDROME
n n REYE SYNDROME
Pathophysiology Ingestion of aspirin during a viral
syndrome
Clinical Finding Non-inflammatory encephalopathy
Fatty Degeneration of Liver
Profuse Vomiting
Personality Changes
Laboratory Finding Increased in total bilirubin
Three fold increase in Ammonia
Increase ALT and AST

TEST MEASURING DETOXIFICATION FUNCTION:

B. ENZYMES

ENZYMES:
TEST MEASURING DETOXIFICATION FUNCTION: • Used to assess the extent of liver damage and to
A. AMMONIA differentiate hepatocellular from obstructive disease
AMMONIA: • Damage to liver cells will allow the release of various
• Least NPN in the body enzymes found in the hepatocytes
• Only NPN substance not used to assess kidney • ENZYMES secreted by liver:
function but a liver test o ALP
• MARKER FOR DETOXIFICATION o Aminotransferase
• In liver disease: Increase AMMONIA, Decrease UREA o 5’ Nucleotidase
o GGT
TEST MEASURING DETOXIFICATION FUNCTION: o LAP
A. AMMONIA METHODOLOGY o LD
AMMONIA ASSAY (SPECIMEN CONSIDERATION)
• SPECIMEN: Arterial Blood w/o Tourniquet Markers for Markers that reflect
• TRANSPORT: Transported in Ice Water (Chilled hepatocellular necrosis cholestasis
Water) a. ALT: Most specific for a. Alkaline phosphatase
• Avoid Hemolysis, Smoking (Falsely Increased hepatocyte injury b. Gamma-glutamyl
Ammonia) b. AST: Less specific than transferase
ALT; significant presence in
1. KJELDAHL METHOD: other tissues
Principle Reagent Quantification of c. LD: Least specific;
Ammonia significant presence in other
1. Digestion of H2SO4 1. Alkali Titration tissues
protein using strong 2. Nesslerization
acid followed by 3. Berthelot Reaction
measurement of
nitrogen content

2. Reference Method
but not
recommended

2. GLUTAMATE DEHYDROGENASE METHOD

• NH4+ + 2-oxaloglutarate + NADPH – (GLDH) →
Glutamate + NADP

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