LABORATORY DIAGNOSIS OF JAUNDICE by Dr. Varughese George

LABORATORY DIAGNOSIS OF JAUNDICE by Dr. Varughese George

JaundiceYellow 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

Serum bilirubin levelsNormal

◦ 0.2 to 0.8 mg/dLUnconjugated/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

Bilirubin metabolism & elimination

Classification of JaundiceI. 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 includeHemolysisResorption of a large hematoma,Ineffective erythropoiesis (Thalassemia, Megaloblastic anemia)Physiologic jaundice of newborn.CongenitalGilbert’sSyndromeCrigler-Najjar syndrome.

Causes includeHepatitisCirrhosisCholestasisDrug (anabolic steroids, oral contraceptives)ToxinsCongenitalDubin-Johnson SyndromeRotor syndrome.

Causes includeViral HepatitisAlcoholic Hepatitis

Classification of JaundiceII. 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, faeces are clay-colored, and bilirubin (being conjugatedand water-soluble) appears in urine.

Classification of Jaundice

III. According to site of disease:Class of Jaundice Type of

Bilirubin raisedCauses

Pre-hepatic or Hemolytic Unconjugated Hemolysis/Abnormal RBCs

Antibodies drugs and toxins

Thalessemia Hemoglobinopathies

Hepatic or Hepatocellular Unconjugated and conjugated

Viral hepatitisToxic hepatitis Intrahepatic cholestasisGilbert’s SyndromeCrigler-Najjar Syndrome

Post-hepatic or Obstructive Conjugated Extrahepatic cholestasisGallstonesTumors of the bile ductCarcinoma of pancreasCarcinoma of ampulla of Vater

DIFFERENTIAL DIAGNOSIS OF JAUNDICE

Parameter Pre-hepatic Hepatocellular ObstructiveBasic 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 PresentUrine 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)

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

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.

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.

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.

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

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.

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.

Other HyperbilirubinemiasPrimary 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.

Other HyperbilirubinemiasPrimary 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.

Complications – Biliary cirrhosis cholangiocarcinoma

Investigating Clinical Jaundice

Evaluation of acute hepatocellular injury

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 TimeGroup 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)

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.

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

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’.

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.

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.

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.

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 toALP 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.

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.

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.

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

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.

Detection of bile pigments in urine

Foam Test5ml of urine in test-tube is shaken andyellow 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).

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.

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.

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

JaundiceHepatocellular Jaundice

Obstructive Jaundice

Urobilinogen Increased

Increased Absent

UrobilinogenCauses 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 jaundiceIn 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.

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 TestTest 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 urobilinogento urobilin)

•Antibiotic therapy (gut bacteria which•produce urobilinogen are destroyed).