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

1. Laboratory Investigations in a case
of Jaundice
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

2. 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

3. Bilirubin-types
1. Unconjugated bilirubin ( α- bilirubin )-27%
2. Mono- conjugated bilirubin (β-bilirubin )- 24%
3. Di-conjugated bilirubin (γ- bilirubin ) -13%
4. Protein-bound bilirubin ( δ- bilirubin ) -37%
( irreversible )

4. PROEPRTIES Un conjugated Conjugated
1. Solubility
in water
in alcohol
2. In bile
3. Absorption from
GIT
4. Diffusion into tissues
5. In urine
6. 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

5.  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

6. 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 .

7.  Rolleston & Mc nee (1929) classification
1. Haemolytic Jaundice (Pre hepatic)
2. Infective Jaundice (Hepatic)
3. Obstructive Jaundice (Post hepatic)
 Rich’s classification
1. Retention Jaundice
2. Regurgitation Jaundice

10. Determination Haemolytic Obstructive Hepatic
(Prehepatic) (Post hepatic) Infective Cholestasis
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

11. GROUP II TESTS
Liver enzyme panel
Serum enzymes
(Routinely done)
1. Transaminases
( ALT and AST)
2* Alkaline phosphatase
(ALP)
Serum enzymes
(Research purposes )
1* 5!
- nucleotidase
2* γ- glutamyl transferase (γ-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

12. 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

13. 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

14. 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. γ-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.

15. 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.

16. 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.

17. Determina- pre-hepatic Hepatic post-
tions (haemolytic)
SGPT(ALT) N ++++ +++ + ++
SGOT(AST) N ++ +++ + ++
ALP N + + ++ ++++
viral toxic cholestatic
hepatic
(obstructive)
GROUP II TEST
liver enzyme panel - interpretationinterpretation
differentiation between hepatic and obstructive jaundice.differentiation between hepatic and obstructive jaundice.

18. Groups III tests
plasma protein panel – differentiation between acute, chronic
hepatites and cirrhosis liver
Determinatios Pre-
haepatic
Hepatic
Viral hepatitis
Acute chronic
cirrhosis
Post
hepatic
Total proteins N N / – N / – – N
Albumin N N / – – – – N
Globulin N N + + + N
A/G ratio N N / – N / – – – N
(reverse)

19. Other TestsOther Tests
Thymol turbidity test
Prothrombin test
BSP ( Bromo Sulphthalin) retention test
Rose bengal dye test
MEGX (Mono ethyl glycine xylidine) test
Anti pyrine breath test

20. How do you investigate a case ofHow do you investigate a case of
JaundiceJaundice

21.  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

22. Hyper BilirubinemiaHyper Bilirubinemia
Unconjugated
(Haemolysis)
Conjugated
(Hepatobiliary
damage)
Urine - Hb
Haemosederine
+ ve - ve
Intra vascular
haemolysis
Extra vascular
haemolysis
(Resorption of
large haemotoma)
Auto immune,
hemoglobino pathies,
Micro angio pathic

23. Conjugated hyper bilirubinemiaConjugated hyper bilirubinemia
Liver 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

24. Conjugated hyper bilirubinemiaConjugated hyper bilirubinemia
Alkaline phosphatase (Hepato biliary obstruction)
5’ NT, γ 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 - Ve
Primary Sclerosing Cholengitis Primary biliary cirrhosis

25. 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

26. 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

27. 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.