Laboratory Diagnosis of Jaundice

1. LABORATORY DIAGNOSIS OF
JAUNDICE
by
Dr. Varughese George
LABORATORY DIAGNOSIS OF JAUNDICE
by
Dr. Varughese George

2. Jaundice
 Yellow discoloration of skin, sclera, and
mucous membranes due to increased level
of serum bilirubin.
 Jaundice becomes clinically evident when
serum bilirubin level exceeds 2.0 mg/dl. (N-
1.2mg/dl)
 Causes
 Overproduction of bilirubin
 Defective hepatic bilirubin uptake
 Defective conjugation
 Defective excretion

3. Serum bilirubin levels
 Normal
◦ 0.2 to 0.8 mg/dL
 Unconjugated/free/indirect (bilirubin-albumin
complex)
◦ 0.2 to 0.7 mg/dL
 Conjugated/direct:
◦ 0.1 to 0.4 mg/dL
 Latent jaundice:
◦ Above 1 mg/dL
 Clinical Jaundice:
◦ Above 2 mg/dL

4. Bilirubin metabolism &
elimination

5. Classification of Jaundice
I. According to the main type of bilirubin increased in plasma:
Predominantly
unconjugated
hyperbilirubinemia
Predominantly
conjugated
hyperbilirubinemia
Mixed (conjugated +
unconjugated)
hyperbilirubinemia
Indirect or
unconjugated bilirubin
is > 85% of total;
Direct or conjugated
bilirubin is >50% of
total
Conjugated bilirubin is
20-50% of total
Causes include
Hemolysis
Resorption of a large
hematoma,
Ineffective erythropoiesis
(Thalassemia, Megaloblastic
anemia)
Physiologic jaundice of
newborn.
Congenital
Gilbert’sSyndrome
Crigler-Najjar syndrome.
Causes include
Hepatitis
Cirrhosis
Cholestasis
Drug (anabolic steroids, oral
contraceptives)
Toxins
Congenital
Dubin-Johnson Syndrome
Rotor syndrome.
Causes include
Viral Hepatitis
Alcoholic Hepatitis

6. Classification of Jaundice
II. According to etiology:
Hemolytic Increased rate of RBC destruction
Increased Hb breakdown to bilirubin in RES
cells
This exceeds the capacity of conjugation in
liver.
Hepatocellular Inability of hepatocytes to conjugate and/or
excrete bilirubin.
Obstructive Failure of excretion of conjugated bilirubin into
the intestine, causing its regurgitation in
circulation.
Urinary and fecal urobilinogen are decreased,

7. Classification of Jaundice
III. According to site of disease:
Class of Jaundice Type of Bilirubin
raised
Causes
Pre-hepatic or Hemolytic Unconjugated  Hemolysis/Abnormal
RBCs
 Antibodies
 drugs and toxins
Thalessemia
 Hemoglobinopathies
Hepatic or Hepatocellular Unconjugated and
conjugated
Viral hepatitis
Toxic hepatitis
 Intrahepatic cholestasis
Gilbert’s Syndrome
Crigler-Najjar
Syndrome
Post-hepatic or Obstructive Conjugated Extrahepatic cholestasis
Gallstones
Tumors of the bile duct
Carcinoma of pancreas
Carcinoma of ampulla of
Vater

8. DIFFERENTIAL DIAGNOSIS OF JAUNDICE
Parameter Pre-hepatic Hepatocellular Obstructive
Basic mechanism
of raised bilirubin
Hemolysis leading
to excess
production
Deficient uptake,
conjugation, or
excretion by
hepatocytes
Deficient excretion
due to obstruction of
biliary tract
Type of serum
bilirubin increased
Mainly
unconjugated
Unconjugated +
Conjugated
Mainly conjugated
(>50%)
Urine Bilirubin Absent Present Present
Urine urobilinogen Increased Variable Decreased/Absent
Prototype Hemolytic anemia Viral hepatitis Common duct stone
Prothrombin time Normal Abnormal that isnt
corrected with
Vitamin K
Abnormal that is
corrected with
Vitamin K
Additional features Features of
hemolysis on blood
smear
(reticulocytosis, low
haptoglobin, low
Hb
Marked rise of
serum ALT and
AST
Marked rise of serum
ALP (>3 times
normal)

9. Increased RBC turnover
 RBCs are the major source of bilirubin.
 Etiology
 Hemolytic anemia
 Ineffective erythropoiesis (thalassemia, megaloblastic anemia).
 Lab : increased unconjugated bilirubin.
 Chronic hemolytic anemia patients often develop pigmented
bilirubinate gallstones

10. Physiological jaundice of the newborn
 Transient unconjugated hyperbilirubinemia
observed in almost all newborns due to
immaturity of the liver i.e deficiency of
glucuronyl transferase leading to impaired
conjugation during the first few days of life.
 First appears between 24-72 hrs of age
 Maximum intensity seen on 4-5th day in term
& 7th day in preterm neonates.
 Doesn’t exceed 15 mg/dl
 Clinically undetectable after 14 days.
 Risk factors –
 Prematurity
 Hemolytic disease of the newborn
(Erythroblastosis foetalis)
 Major complication is kernicterus especially in
infants, in which increased levels of
unconjugated lipid-soluble bilirubiin may cross
the blood brain barrier and deposit in the
basal gangllia, thus causing irreversible brain
damage.

11. Congenital
Hyperbilirubinemias
Gilbert’s Syndrome
 Most common inherited cause of unconjugated
hyperbilirubinemia.
 It is a familial, benign disease with autosomal dominant mode
of inheritance.
 Affects 5% of general population.
 Mild deficiency or decreased hepatic levels of Bilirubin
glucoronosyltransferase attributed to mutation in the encoding
gene; polymorphisms in the gene may play a role in the
variable expression of this disease
 Jaundice is mild and fluctuating, goes unnoticed for years;
often related to stress/fasting/infection.
 Mildly elevated serum bilirubin is the sole abnormality; other
LFTs are normal.
 Not associated with any morbidity.

12. Congenital
Hyperbilirubinemias
Crigler Najjar Syndromes
 Autosomal recessive disorder.
 Unconjugated hyperbilirubinemia.
 Type I -
 Serum bilirubin > 20 mg/dl
 Complete absence of Bilirubin UDP-glucoronosyltransferase1-A1
expression detected in liver tissue.
 Fatal due to kernicterus – causes rapid death in neonate.
• Type II (Arias Syndrome) –
• Serum bilirubin < 20mg/dl.
• Reduced levels of detectable Bilirubin UDP-glucoronosyltransferase1-
A1 expression in liver tissue owing to single base pair mutations.
• Bile is pigmented instead of pale or dark as normal.
• No risk of kernicterus.

13. Congenital
Hyperbilirubinemias
Dubin Johnson Syndrome
• Benign autosomal recessive disorder.
• Conjugated hyperbilirubinemia
• Decreased hepatocellular bilirubin excretion due to a defect in the
canalicular cationic transport protein.
 Gross : black pigmentation of the liver due to accumulation of
polymerized epinephrine metabolites
 Lab - Bromosulphthalein excretion test is abnormal.
• No clinical consequences

14. Congenital
Hyperbilirubinemias
Rotor Syndrome
 Autosomal recessive disorder.
 Conjugated hyperbilirubinemia.
 Decreased intraheptic binding.
 Similar to Dubin-Johnson but without liver pigmentation.
 Bromosulphthalein test is normal.
 No clinical consequences.

15. Other Hyperbilirubinemias
Biliary Tract Obstruction
Etiology -
 Gall stones
 Tumors (pancreatic, gall bladder and bile duct)
 Strictures
 Parasites (Liver flukes – Clonorchis sinensis)
Presentation –
 Jaundice & icterus.
 Pruritis due to raised plasma levels of bile acids.
 Abdominal pain, fever and chills
 Dark urine (bilirubinuria)
 Pale clay colored stools
Laboratory Diagnosis –
 Increased conjugated bilirubin.
 Increased alkaline phosphatase.
 Increased 5’ nucleotidase.

16. Other Hyperbilirubinemias
Primary biliary cirrhosis
 Chronic liver disease of unknown etiology
probably autoimmune characterised by
inflammation & granulomatous destruction of
intrahepatic bile ducts.
 Males: Females = 1:10, age – 30-65 yrs.
 Presentation : middle-aged women with
obstructive jaundice: xanthomas,
xanthelasmas, increased serum cholesterol,
fatigue, cirrhosis.
 Laboratory Diagnosis -
 Increased conjugated bilirubin
 Increased alkaline phosphatase
 Increased 5’ nucleotidase.
 Antimitochondrial Antibody
 Most people have another autoimmune
disease.
 Microscopy : Lymphocytic & granulomatous
destruction of interlobular bile ducts.

17. Other Hyperbilirubinemias
Primary sclerosing cholangitis
 Chronic liver disease of unknown etiology
characterized by segmental inflammation &
fibrosing destruction of intra-& extrahepatic
bile ducts.
 Males: Females = 2:1, age – 20-40yrs; most
associated with ulcerative colitis.

 Presentation – similar to Primary Biliary
Cirrhosis.
 Microscopy –
 Periductal chronic inflammation.
 Concentric fibrosis around bile ducts.
 Segmental stenosis of the bile ducts.
 Beaded appearance of bile ducts on
cholangiogram.
 ANCA is a Marker for Primary Sclerosing
Cholangitis.

18. Investigating Clinical Jaundice

19. Evaluation of acute hepatocellular injury

20. Classification of Liver Function
Tests
Group I:
Markers of liver dysfunction
 Serum bilirubin: total,
conjugated and
unconjugated
 Urine: bile pigments, bile
salts and urobilinogen
 Total protein, serum albumin
and albumin/globulin ratio
 Prothrombin Time
Group II:
Markers of hepatocellular injury
 Alanine aminotransferase
(ALT)
 Aspartate aminotransferase
(AST)
Group III:
Markers of cholestasis
 Alkaline phosphatase (AKP)
 γ- glutamyl transferase (GGT)
 5’-nucleotidase (5’-NT)

21. Hepatocellular Locations of
Enzymes
 Alanine aminotransferase (ALT) and
the cytoplasmic isoenzyme of aspartate
aminotransferase (ASTc) are found
primarily in the cytosol. With membrane
injury as in viral or chemically-induced
hepatitis, these enzymes are released
and enter the sinusoids, raising plasma
AST and ALT activities.
 Mitochondrial aspartate
aminotransferase (ASTm) is released
primarily with mitochondrial injury, as
caused by ethanol as in alcoholic
hepatitis.
 Alkaline phosphatase (ALP) and
Gamma-glutamyltransferase (GGT) are
found primarily on the canalicular
surface of the hepatocyte. Bile acids
accumulate in cholestasis and dissolve
membrane fragments, releasing bound
enzymes into plasma. GGT is also
found in the microsomes, represented
as rings in the figure; microsomal
enzyme-inducing drugs, like
phenobarbital and dilantin, can also
increase GGT synthesis and raise
plasma GGT activity.

22. INTERPRETATION OF LIVER FUNCTION TESTS
Typical LFT profile in
hepatocellular disease
 Marked elevation of AST
and ALT (usually >500 IU)
 Mild increase of ALP (<3
times normal)
 Hyperbilirubinemia, if
present, is of both
conjugated and
unconjugated type
Typical LFT profile in
cholestatic jaundice
 Marked elevation of ALP
(>3 times normal)
 Elevation of GGT and 5’-
NT.
 Mild or no increase of ALT
and AST (usually <200 IU).
 Elevation of conjugated
bilirubin

23. Estimation of serum bilirubin
Spectrophotometry
 used for measurement of total
serum bilirubin in newborns
and infants (<3 months of
age).
 Concentration of serum
bilirubin is directly proportional
to its absorbance in a
spectrophotometer at 454 nm.
 This method cannot be used
in older children and adults
because their sera may also
contain carotene and other
pigments, which absorb light
at the same wavelength. In
newborns, other pigments are
absent.
Diazo Method
 Direct bilirubin (Conjugated bilirubin):
It reacts directly with diazo reagent. It consists of
monoconjugated bilirubin, diconjugated bilirubin, and
bilirubin tightly bound to albumin (delta bilirubin).
 Indirect bilirubin (Unconjugated bilirubin):
It reacts with diazo reagent in the presence of alcohol. It
consists of bilirubin bound to albumin. It is calculated
as ‘total bilirubin minus direct bilirubin’.

24. Serum Aminotransferases
 Serum aminotransferases (AST/ALT) are the sensitive markers of acute hepatocellular injury.
 Serum alanine aminotransferase or ALT (formerly called serum glutamic-pyruvic transaminase
or SGPT) is a cytosolic enzyme.
 Serum aspartate aminotransferase or AST (formerly called serum glutamic-oxaloacetic
transaminase or SGOT) is both cytosolic and mitochondrial.
 When necrosis or death of cells containing these enzymes occurs, aminotransferases are
released into the blood and their concentration in blood increases whose level correlates with
extent of tissue damage.
 Most marked elevations of ALT and AST (>15 times normal) are seen in acute viral hepatitis,
toxin-induced hepatocellular damage (e.g. carbon tetrachloride), and centrilobular necrosis due
to ischemia (congestive cardiac failure).
 Moderate elevations (5-15 times) occur in chronic hepatitis, autoimmune hepatitis, alcoholic
hepatitis, acute biliary tract obstruction, and drug-induced hepatitis.
 Mild elevations (1-3 times) are seen in cirrhosis, nonalcoholic steatosis, and cholestasis.
 Increase of AST and ALT is much more in hepatocellular jaundice (>500 units/ml) than in
cholestatic jaundice (<200 units/ml).
 ALT and AST are elevated in acute viral hepatitis even before the appearance of jaundice.
 Persistence of elevated ALT and AST beyond 6 months in a case of hepatitis indicates
development of chronic hepatitis
 In massive liver necrosis, aminotransferase levels gradually decrease.
 Normal ALT:AST ratio is 0.7 to 1.4.

25. Serum Alkaline Phosphatase
(ALP)
 In the liver, ALP, GGT, and 5’-NT are
located normally on canalicular surface of
hepatocytes.
 Serum ALP levels are increased in most
cases of cholestatic type of jaundice and
hence helps in differentiating it from
hepatocellular jaundice.
 Hepatobiliary causes of increased alkaline
phosphatase include –
 Bile duct obstruction (cancer of head of
pancreas/stone in common bile
duct/stricture of bile duct/ biliary atresia)
 Primary biliary cirrhosis
 Primary sclerosing cholangitis
 Infiltrative diseases of liver
- Granulomatous diseases like
tuberculosis/sarcoidosis
- Amyloidosis
- Cysts
- Primary/secondary cancer.
 Serum ALP levels are also raised in
diseases of bone and pregnancy.

26. Serum γ-glutamyl Transferase
(GGT)
Relatively high levels of this enzyme are present in liver, pancreas,
kidney & prostate.
Estimation of this enzyme is particularly useful in following liver
diseases:
 Alcoholism - Increased enzyme activity is present in alcoholism, and
is a helpful clue in suspected cases of occult alcoholism.
 Cholestasis: - Elevation of GGT generally parallels that of ALP and
5’-NT in liver diseases like primary biliary cirrhosis or sclerosing
cholangitis.
 Recovery in acute hepatitis: - Serum GGT is the last enzyme to
return to normal following acute hepatitis and its normalization is
indicative of a favourable outcome.

27. 5’-nucleotidase (5’-NT)
5’-NT is present in liver as well as in various other tissues.
It is located mainly along the cell membrane, similar to
ALP and GGT.
Estimation of 5’-NT is helpful in deciding whether increased ALP
is due to liver disease or due to increased osteoblastic activity
in growing children.

28. Serum Albumin
 Serum albumin is a sensitive but nonspecific test for liver disease
 Albumin is synthesized exclusively in liver and constitutes about 60% of total
proteins in serum  important investigation in liver disease
 Half-life of albumin is about 20 days.
 In acute liver disease (e.g. viral hepatitis), there is little change in albumin level.,
whereas it is low in chronic liver disease (cirrhosis) and correlates with synthetic
capacity of hepatocytes  it is helpful in following progression of cirrhosis.
 Fall in serum albumin level correlates with severity of ascites.
 Serum albumin is estimated by bromocresol green which binds selectively and
tightly to it thus imparting a blue color  Absorbance (in a spectrophotometer at
632 nm) is directly proportional to concentration of albumin.
 Causes of decreased serum albumin:
 Decreased intake: malnutrition.
 Decreased absorption: malabsorption syndromes.
 Decreased synthesis: liver disease, chronic infections.
 Increased catabolism: thyrotoxicosis, fever, malignancy, infections.
 Increased loss: nephrotic syndrome, severe burns, protein-losing enteropathies,
ascites
 Increased blood volume: pregnancy, congestive cardiac failure.

29. Prothrombin Time (PT)
 Prothrombin Time(PT) measures three out of four vitamin K-dependent
factors (II, VII, and X) and is prolonged in hepatocellular disease and in
obstructive jaundice.
 Deficient synthesis of Vitamin K dependent factors occurs in hepatocelllular
jaundice.
 In obstructive jaundice, vitamin K (a fat-soluble vitamin) cannot be absorbed
due to the absence of bile in the intestine.
 Intramuscular injection of vitamin K corrects prolonged PT in obstructive
jaundice but not in hepatocellular jaundice.
 In acute fulminant liver failure, marked prolongation of PT is an unfavourable
prognostic sign.

30. Bile pigments in Urine
 The main bile pigments are bilirubin and biliverdin.
 Bilirubin is converted to non-reactive biliverdin on exposure to light
(daylight or fluorescent light) and on standing at room temperature.
Biliverdin cannot be detected by tests that detect bilirubin. Therefore
fresh sample that is kept protected from light is required.
 Presence of bilirubin in urine indicates conjugated hyperbilirubinemia
(obstructive or hepatocellular jaundice) as only conjugated bilirubin
is
 water-soluble.
 Bilirubin in urine is absent in hemolytic Jaundice because
unconjugated bilirubin is water-insoluble.
Urine Test Hemolytic
Jaundice
Hepatocellular
Jaundice
Obstructive
Jaundice
Bile pigments Absent Present Present

31. Detection of bile pigments in
urine
 Fouchet’s test
2.5 ml of 10% of barium chloride is mixed with 5 ml of fresh urine in a
test tube. A precipitate of sulphates appears to which bilirubin is
bound (barium sulphate-bilirubin complex).
Filter to obtain the precipitate on a filter paper followed by an addition
of
1 drop of Fouchet’s reagent(25g of CCl3COOH, 10ml of 10%
FeCl3 & distilled water 100ml) to it.
Immediate development of blue-green color around the drop =>
presence of bilirubin.

32. Detection of bile pigments in
urine
 Foam Test
5ml of urine in test-tube is shaken and
yellow foam => presence of bilirubin.
 Gmelin’s test:
3 ml of concentrated HNO3 is added to an equal quantity
of urine in a test tube and shaken gently. Play of
colors(yellow, red, violet, blue, and green) => +ve
test.
 Lugol iodine test:
4 ml of Lugol iodine solution (I 1gm, KI 2 gm, and
distilled water to make 100 ml) is added to 4 drops
of urine in a test tube and mixed by shaking and
green color => +ve test.
 Reagent strips or tablets impregnated with diazo
reagent:
Tests based on reaction of bilirubin with Diazo reagent.
Color change is proportional to the concentration of
bilirubin.
Tablets (Ictotest) detect 0.05-0.1 mg of bilirubin/dl of
urine.
Reagent strip tests are less sensitive (0.5 mg/dl).

33. Bile salts in urine
 Bile salts are salts of four different types of bile acids:
 cholic
 deoxycholic
 chenodeoxycholic
 lithocholic.
 Bile acids combine with glycine or taurine to form complex salts
or acids.
 Bile salts enter the small intestine through the bile and act as
detergents to emulsify fat and reduce the surface tension on fat
droplets so that enzymes (lipases) can breakdown the fat.
 In the terminal ileum, bile salts are absorbed and enter in the
bloodstream from where they are taken up by the liver and re-
excreted in bile (enterohepatic circulation).
 Bile salts along with bilirubin can be detected in urine in cases of
obstructive jaundice in which bile salts and conjugated bilirubin
regurgitate into blood from biliary canaliculi (due to increased
intrabiliary pressure) and are excreted in urine.

34. Detection of bile salts in urine
 Hay’s surface tension test
 The property of bile salts to lower the surface tension is
utilized in this test .
 Fresh urine at room temperature taken in a conical glass tube
is sprinkled on the surface particles of sulphur.
 If bile salts are present, sulphur particles sink to the bottom
because of lowering of surface tension by bile salts.
 If sulphur particles remain on the surface of urine, bile salts
are absent.
 Thymol (used as a preservative) gives false positive test.

35. Urobilinogen
 Conjugated bilirubin excreted into the duodenum through bile
is converted by bacterial action to urobilinogen in the
intestine.
 Major part is eliminated in the feces.
 A portion of urobilinogen is absorbed in blood, which
undergoes recycling (enterohepatic circulation);
 A small amount, which is not taken up by the liver, is excreted
in urine.
 Urobilinogen is colorless; upon oxidation it is converted to
urobilin, which is orange-yellow in color.
 Normally about 0.5-4 mg of urobilinogen is excreted in urine
in 24 hours. Therefore, a small amount of urobilinogen is
normally detectable in urine.
 A 2-hour post-meal sample is often preferred as urinary
excretion of urobilinogen shows diurnal variation with highest
levels in afternoon.
Urine Test Hemolytic
Jaundice
Hepatocellular
Jaundice
Obstructive
Jaundice
Urobilinogen Increased Increased Absent

36. Urobilinogen
Causes of Increased
Urobilinogen in Urine
 Hemolysis:
Excessive destruction of red cells leads
to hyperbilirubinemia and therefore
increased formation of urobilinogen
in the gut.
 Hemorrhage in
tissues:
There is increased formation of bilirubin
from destruction of red cells.
 Causes of Decreased
Urobilinogen in Urine
 Obstructive jaundice
In biliary tract obstruction,delivery of
bilirubin to the intestine is restricted
and very little or no urobilinogen is
formed. This causes stools to become
clay-colored.
 Reduction of intestinal
bacterial flora:
 This prevents conversion of bilirubin to
urobilinogen in the
 Intestine; observed in neonates and
following antibiotic treatment.

37. Detection of Urobilinogen in urine
 Ehrlich’s aldehyde test:
0.5 ml Ehrlich’s reagent (pdimethylaminobenzaldehyde) is added to a 5ml
fresh 2 hr postprandial sample of urine.
If urobilinogen present, it ll reach with Ehrlich’s reagent and produce a
pink color.
Intensity of color developed depends on the amount of urobilinogen
present.
Development of pink color  normal amount of urobilinogen.
Development of dark red color  increased amount of urobilinogen.
 Watson-Schwartz’s Test
Test distinguishes between urobilinogen and porphobilinogen which
gives similar results in Ehrlich’s test.
1-2ml chloroform is added to the sample, shaken for 2 mins & allowed to
stand.
Pink color in the chloroform layer indicates presence of urobilinogen,
while pink coloration of aqueous portion indicates presence of
porphobilinogen
 Reagent strip method:
 Method is specific for urobilinogen.
 Test area is impregnated with either
p-dimethylaminobenzaldehyde or 4-methoxybenzene
diazonium tetrafluoroborate
False -ve reaction
• UTI (nitrites oxidize
urobilinogen
to urobilin)
•Antibiotic therapy
(gut bacteria which
•produce
urobilinogen are