Laboratory Investigations in a case of Jaundice

Tapeshwar Yadav(Lecturer)BMLT, DNHE, M.Sc. Medical Biochemistry

BILE PIGMENTS Daily bilirubin production - 250-300mg% 85% heme moiety of aged RBC 5% RBC precursors destroyed in bone marrow ( ineffective erythropoiesis),Catabolism of some heme proteins – myoglobin, cytochrome, peroxidase

BILIRUBIN LEVELS: Conjugated : 0.0 – 0.2 mg / dl

Unconjugated : 0.2 – 0.8 mg / dl

Total : 0.2 - 1.0 mg / dl – normal

1.0- 2.0 mg /dl – latent jaundice

> 2.0 mg /dl – jaundice

> 3.0 mg /dl – clinical jaundice

Bilirubin-types 1. Unconjugated bilirubin ( - bilirubin )-27%

2. Mono- conjugated bilirubin (-bilirubin )- 24%

3. Di-conjugated bilirubin (- bilirubin ) -13%

4. Protein-bound bilirubin ( - bilirubin ) -37% ( irreversible )

PROEPRTIES Un conjugated Conjugated

1. Solubility in water in alcohol2. In bile3. Absorption from GIT4. Diffusion into tissues5. In urine6. Vandenberg’s test

Insoluble Soluble Absent

Absorbed Diffuses Always absent Indirect + ve

Soluble Soluble Present

Not absorbed Not diffused Normally absent Indirect + ve

PROPERTIES OF CONJUUGATED & UNCONJUGATED BILIRUBIN

Properties of bilirubin products No. of Hatoms colour Bilirubin (BR) 36 Orange yellow Mesobilinogen (MB) 40 yellow Urobilinogen (UBG) 44 Colour less Stercobilinogen (SBG) 48 Colour less Urobilin (UB) 42 Orange brown Stercobilin (SB) 46 Dark brown

Tests for bile pigments

Bilirubin (BR) Fouchet’s test, Gmelin’s test, Vandenberg’s test

Urobilimogen (UBG) Ehrlich’s test Urobilin (UB) Schlesinger’s test Stercobilin (SB) Schlesinger’s test

Bile salts

helps in digetion and absorption of lipids by forming molecular aggregates called “ micelle “ cholesterol (27C)

(+3) OH groups ( +2) OH groups Cholic acid chemodeoxy cholic (24C) acid(24C)

Primary Glycocholic acid Taurochenodeoxy cholic acid

Secondary deoxy cholicacid Litho cholicacid Test for bile salts - Hay’s test .

Rolleston & Mc nee (1929) classification1. Haemolytic Jaundice (Pre hepatic)2. Infective Jaundice (Hepatic)3. Obstructive Jaundice (Post hepatic)

Rich’s classification1. Retention Jaundice2. Regurgitation Jaundice

Determination Haemolytic Obstructive Hepatic

(Prehepatic) (Post hepatic) Infective Cholestasis

toxic

1. Serum Total bilirubin ++++ ++++ +++ +++ Direct bilirubin N +++ + ++ Indirect bilirubin +++ N/+ ++ +

2. Feces Colour Dark Pale dark Pale Urobilin ++++ -/Absent N / + - / N

Stercobilin ++++ -/Absent N / + - / N 3. Urine Color dark dark moderate Dark Bilirubin Absent +++ + ++ Urobilin +++ Absent + + - / N bile salts ++ Absent + Absent

GROUP II TESTSLiver enzyme panel

Serum enzymes (Routinely done)

1. Transaminases ( ALT and AST)2* Alkaline phosphatase (ALP)

Serum enzymes (Research purposes )

1* 5!- nucleotidase 2* - glutamyl transferase (-GT)3. Lactate dehydrogenase (LDH) 4. Iso citrate dehydrogenase (ICD)5. Ornithine trans carbamoylase

(OTC) 6* Leucine aminopeptidase (LAP)7. Cholinesterases 8. Sorbitol dehydrogenase (SDH)

* Biliary tract enzymes

Serum enzymes- routinely done in the lab

1. Transaminases. SGOT(AST) 8-40 IU/L

SDPT(ALT) 13-40 IU/L AST- heart muscle. ALT- Liver Liver diseases – ALT >> AST

Alcoholic hepatitis – AST >> ALT Infectious hepatitis Pre icteric phase – elevations are noticed

Icteric phase – peak 12-14 hrs (10-20 times) Recovery phase – normal with in 2-5 weeks Biliary obstruction elevated 3-4 times.

Decreased shortly after relief of obstruction. ALT- cytosol. AST- mitochondria Normally ALT- AST ratio is 1 or <1.

acute hepatitis > 1. chronic hepatitis <<1

2. Alkaline phosphatase 3-13 KA units/ 100ml (40-125 IU/L).

Rich in intestine epithelium, kidney , liver, bones, placenta.

Site of production from liver –hepatocyte adjacent to biliarycanaliculi.

ALP- elevated in obstructive jaundice (3 folds)

(Extra hepatic > intra hepatic)

ALP- elevated in infective hepatitis and obstructive jaundice , but elevation is more in obstructive jaundice dividing line 35 KA unit/100ml

> 35 KA unit /100ml suggestive of obstructive jaundice

< 35 KA unit /100ml infective hepatitis.

Markedly elevated in biliary cirrhosis Space occupying lesions of liver –abscess, carcinomas metastasis lymphoma and granuloma.

SERUM ENZYMES - RESEARCH PURPOSES

1. 5´- Nucleotidase- 2-17 IU/L Specific marker enzyme – Cholestasis Added advantage over ALP – Not elevated in Bone diseases.

2. -GT ( Gamma Glutamyl Transferase)-10-47 IU/L Elevated in Obstructive Jaundice and drug toxicity. Marker/screening enzyme for alcohol abuse.

3. LDH ( Lactate Dehydrogenase)- 70-240 IU/L Elevated in infective hepatitis, Leukemia, hemolytic anemia and

megaloblastic anemia. Less specific than Amino transferase.

4. ICD (Isocitrate Dehydrogenase)- 0.9-4.0 IU/L Markedly elevated in infectious hepatitis and rug toxicity. Normal in obstructive Jaundice. 5. OTC ( Ornithine transcarbomylase)- 8-20 mIU/L Elevated in acute viral hepatitis, obstructive jaundice and

cirrhosis liver. Sensitive and specific measure of Hepato Cellular injury.

6. LAP (Leucine amino peptidase)- 15-56 mIU/L Moderately elevated in Viral hepatitis and Cirrhosis liver. Markedly elevated in malignant obstruction of bile duct and

liver cancer. Added advantage over ALP is not elevated in osseous

involvement.

7. Cholinesterase- 2.17-5.17 IU/L Decrease in liver cell injury, cirrhosis liver Normal in obstructive jaundice.

8. SDH ( Sorbitol Dehydrogenase)- 0.2 mIU/L Strikingly elevated in acute viral hepatitis and CCl4

poisoning. Normal in chronic hepatitis and obstructive jaundice.

Determina- pre-hepatic Hepatic post-tions (haemolytic)

SGPT(ALT) N ++++ +++ + ++

SGOT(AST) N ++ +++ + ++

ALP N + + ++ ++++

viral toxic cholestatichepatic(obstructive)

GROUP II TEST liver enzyme panel - interpretation interpretation

differentiation between hepatic and obstructive jaundice. differentiation between hepatic and obstructive jaundice.

Groups III tests plasma protein panel – differentiation between acute, chronic

hepatites and cirrhosis liver

Determinatios Pre- haepatic

HepaticViral hepatitis

Acute chroniccirrhosis

Post hepatic

Total proteins N N / – N / – – N

Albumin N N / – – – – N

Globulin N N + + + N

A/G ratio N N / – N / – – – N

(reverse)

Other TestsOther Tests

Thymol turbidity testProthrombin testBSP ( Bromo Sulphthalin) retention testRose bengal dye testMEGX (Mono ethyl glycine xylidine) testAnti pyrine breath test

How do you investigate a How do you investigate a case of case of JaundiceJaundice

Patient with history and clinical features suggest jaundice

URINE

Dark color urine, UBG positive with out Bilirubin- Haemolysis

Dark color urine, H/o Pruritis & UBG negative with high bilirubin - Obstruction

Pale color stools

Hyper BilirubinemiaHyper Bilirubinemia

Unconjugated

(Haemolysis)

Conjugated

(Hepatobiliary damage)

Urine - Hb Haemosederine

+ ve - veIntra vascular haemolysis

Extra vascular haemolysis (Resorption of large haemotoma)Auto immune,

hemoglobino pathies,Micro angio pathic

Conjugated hyper bilirubinemiaConjugated hyper bilirubinemiaLiver enzymesLiver enzymes

Elevated Normal

Amino transferases(Hepato cellular damage) (Hepato biliary obstruction)

ALP

Serological tests

Abnormal Normal(Viral hepatitis)

Acute Chronic

Liverbiopsy

•Drug toxicity

•Obstruction of hepatic veins

•Cirrhosis

CT Scan, Doppler study

Hepato biliary image

Liver biopsy

Pregnancy

Sepsis

H/o recent surgery

Dubin-Johnson, Rotor syndromes

Conjugated hyper bilirubinemiaConjugated hyper bilirubinemia

Alkaline phosphatase (Hepato biliary obstruction)5’ NT, GT

Elevated Normal(Bone disease)US/CT scan

Dilated ducts Normal ducts•Gall stones

•Ca. Pancreas

PTC

ERCP

BIOPSY

* Intra hepatic obstruction (Cholestasis, drugs)

* Extra hepatic obstruction

+ Ve - VePrimary Sclerosing Cholengitis Primary biliary cirrhosis

Neonatal Jaundice

Bilirubin levels (mg/ dl)

Premature Full term

Cord < 2.0 < 2.0

0 – 1 day < 8.0 1.4 – 8.7

1 – 2 day < 12.0 3.4 – 11.5

3 – 5 days < 16.0 1.5 – 12.0

•Urobilinogen & Stercobilinogen abscent- Incomplete development of intestinal bacterial flora

•Phototherapy trans - cis bilirubin

Isolated Hyper bilirubinemiaIsolated Hyper bilirubinemia

Conjugated * Dubin –Johnson* Rotor Syndromes

* Gilberts ( < 3 mg/dl)* Crigler _Najjar II ( 5 – 20 mg/dl)* Crigler _Najjar I ( > 25 mg/dl)* Lucey –Driscoll ( ~ 5 mg/dl)

Un conjugated

Bilirubin - analysisBilirubin - analysis

Sample collection – Transport : Precautions Fasting sample is preferred to avoid lipemia Hemolysis – falsely low values ( absorbance) Photo oxidation – exposure to sunlight or UV light Sensitive to high temperature.