2. RBCs last 120 days, degraded by reticuloendothelial(RE) system  Under physiologicconditions in the human adult, 1~2×10 8 erythrocytes are destroyed per hour. Thus, in 1 day, a 70kg human turns over approximately 6g of hemoglobin.  When hemoglobin is destroyed in the body, globin is degraded to its constituent amino acids, which are reused, and the iron of heme enters the iron pool, also for reuse. most heme from RBCs (85%) - rest from turnover of cytochromes, p450s, immature erythrocytes globin HemeHeme Amino acids iron+ Bile pigments

3. Bile pigments Any of several coloured compounds derivedfrom porphyrin that are found in bile; principally bilirubinand biliverdin.

4. Unconjugated bilirubin: Bilirubin that are not conjugated with gluconic acid, also called hemobilirubin, indirected bilirubin. conjugated bilirubin: Bilirubin that are conjugated with gluconic acid, also called hepatic bilirubin, directed bilirubin.

5. BLOOD CELLS LIVER Bilirubin diglucuronide (water-soluble) via bile duct to intestines Stercobilin excreted in feces Urobilinogen formed by bacteria KIDNEY Urobilin excreted in urine CO Biliverdin IX  Heme oxygenase O 2 NADP + NADPH Biliverdin reductase HemeHeme Globin Hemoglobin reabsorbed into blood 2 UDP-glucuronic acid Bilirubin (water-insoluble) via blood to the liver INTESTINE Catabolism of hemoglobin Bilirubin (water-insoluble) unconjugated

6. METABOLISM Senescent redcells are major source of hemeproteins Breakdown of heme to bilirubin occur in macrophage of reticuloendithelial system ( tissue macrophages, spleen and liver). Unconjugated bilirubin is transported through blood ( complex to albumin) to liver. Bilirubin is taken into liver and conjugate with glucuronic acid. Bile is secreted into intestine where glucuronic acid is removed and the resulting bilirubin is converted to urobilinogen. A portion of urobilinogen is reabsorbed into blood, where it is converted to the yellow urobilin and excreted by kidneys. Urobilinogen is oxidized by intestinal bacteria to the brown stercobilin.

7. Occult: the concentrationof blood bilirubinare increased, but have no clinic sympotom, normally 1-2mg/dl. Jaundice : ( also called icterus) refers to the yellow color of the skin and scleare caused by deposition of bilirubin, secondry to increased bilirubin levels in the blood. Although not a disease itself, jaundice is usually a symptom of an underlying disorder. Hyperbilirubinemia:the concentrationof blood bilirubin are more than 1mg/dl.

9. Based onpathophysiology, jaundice may result from one or more of the following mechanism: 1.Increased bilirubin production ( excessive red cell destruction) 2.Decreased hepatic uptake ( ligandin, drug, prolonged starvation, and sepsis) Decreased hepatic conjugation (enzyme,drugs, cirrhossis) 3.Decreeased excretion of bilirubin into bile( gallstone, tumour)

10. SIMPLECLASSIFICATION OF JAUNDICE Accordingly, a simple classification of jaundice is to divided into 3 predominant type: ① Pre-hepatic (hemolytic jaundice) ② Hepatic jaundice ③ Post – hepatic cholestatic (obstructive jaundice)

11. HEMOLYTIC JAUNDICE massive lysis of red blood cells (for example, in patients with sickel cell anemia or malaria) may produce bilirubin faster than the liver can conjuagte it. More bilirubin is excreted into the bile, the amount of the urobilinogen entering the enterohepatic circulation is increased, and urinary urobilinogen is increased. Unconjugated bilirubin is elevated in blood.

12. CAUSES OF HEMOLYTIC JAUNDICE  Malaria  Side effects of certain drugs :antibiotic and anti-tuberculosis medicines, levodopa,  Certain drugs in combination with a hereditary enzyme deficiency known as glucose-6-phosphate dehydrogenase (G6PD)  Poisons Snake and spider venom, certain bacterial toxins, copper, and some organic industrial chemicals directly attack the membranes of red blood cells  Artificial heart valves  Hereditary RBC disorders sickle cell disease  Enlargement of the spleen  Diseases of the small blood vessels  Immune reactions to RBCs cancer  Transfusions  Kidney failure and other serious diseases  Erythroblastosis fetalis

13. HEPATOCELLULAR JAUNDICE Damage to liver cells( for example in patient with cirrhosis or hepatitis) causes a decrease in both bilirubin uptake and production of conjuagted bilirubin. Unconjugated bilirubin occur in the blood and increased urobilinogen in the urine. The urine is dark in color and stool are pale, clay color. Plasma level of AST and ALT are elevated and the patient experience nausea and anorexia.

14. OBSTRUCTIVE JAUNDICE In this instance jaundice is results from obstruction of the bile duct. For example, the presence ofa hepatic tumor or bile stone may block the bile ducts, preventing passage of bilirubin into the intestine, patients with obstructive jaundice experienceGI pain, nausea and produce stools that are a pale, clay color.

15. Bilirubin + DPDAzobilirubincaffeine surfactant Principle: A stabilized diazonium salt, 3, 5-diclorophenyldiazonium tetrafluoroborate (DPD), reacts with conjugated bilirubin directly and with unconjugated bilirubin in the presence of an accelerator to form azobilirubin. The absorbance at 540 nm is proportional to the total bilirubin concentration. A separate sample blank is performed to reduce endogenous serum interference.

16. Performance specifications: Linearity: The method is linear for up to 10mg/dlserum, plasma. Measurement Range: The method has a measurement range of 0– 10 mg/dlserum, plasma. Sensitivity: The method has a sensitive of 0.1 mg/dl serum, plasma.

17. Primary Sample: Use only serum / plasmaas specimen for the test. a. Collect 2 ml of venous blood in a plain red-topped Vacutainers tube. Allow the tube to stand for 30 minutes at room temperature and separate the serum by centrifugation at 2500- 3000 rpm for 5 – 10 minutes. b. Collect 2 ml of venous blood in a lithium heparin green topped Vacutainers at the room temperature and separate plasma by centrifuging at2500 to 3000 RPM for 5 to 10 min. Do not use lysed serum for testing as it may give very high results Do not use contaminated / turbid samples for testing Process the sample on the same day If analysis is not done on the same day, separate the serum and store it at 2 to 8  C for up to 7 days.

18. Reagent ConstituentsReaction Concentration Caffeine DPD Surfactant Preservative 2.1 mmol/L 0.31 mmol/L Type of container and additive: 1. Use a Plain Red topped Vacutainers tube for collecting venous sample. 2. Use lithium heparin green topped Vacutainers for collection venous sample Reagents / consumables

19. AssayEND POINT Wave LengthPrimary 540 nm Secondary660 nm Temperature37 o C. Reagent VolumeR1 – 30 Diluent120 ul Sample Volume6 μl Measuring Point 1 CyclesFirst – 0 & Last – 10 Measuring Point 2 Cycles Reaction Slopeincreasing OD (+) Instrument: Olympus AU400 Automated Clinical Chemistry Analyzer Step by Step Procedure: Assay Conditions

20. Calibration procedure: Calibrate the instrument for the assay by using the calibrator specified in the kit W henever a new lot of reagent is used. After major equipment breakdown or maintenance. W henever the internal QC is out of range. Record the details of calibration in the register. Quality Control Procedure: Run two levels of QC in the morning and ensure the QC values are with in the range. Run two levels of QC in the evening and ensure the QC values are with in the range.

21. Reference Interval: Adults 0.3-1.2 mg/dL Interpretation of Results: Increased Serum levels were seen in the following conditions: Increased unconjugated billirubin in hemolytic anemia neo-natal jaundice Increased conjugated billirubin Dubin – Johnson and Rotor Syndrome. Liver cirrhosis. Acute and Chronic viral hepatisis. Hepatocellular carcinoma. Critical /Alert level values: > than 15 mg/dl Safety precautions: Handle all samples as potentially infectious Handle all reagents with care and avoid contact with eye mouth and skin Do not perform mouth pipetteing Discard used reagents and sample as per disposal procedure to avoid possible build up of aside compounds flush pipes with adequate water after use after disposal of undiluted reagent.

22. Potential sources of variability: Presence of fibrin in the sample may lead to erroneous results and hence centrifuge the sample only after complete clot formation. Lysed serum specimens may give falsely elevated values. Interferences vary depending upon icterus, haemolysis and lipemia status.