BILIRUBIN DETERMINATION.pdf

CLINICAL CHEMISTRY 2 Bilirubin Determination Prepared by: Mike Lenard M. Leocadio, RMT Functions of the Liver Synthetic function Detoxification function Excretory function Detoxification and removal of Bilirubin Bilirubin – Product of hemoglobin determination B1...

CLINICAL CHEMISTRY 2 Bilirubin Determination Prepared by: Mike Lenard M. Leocadio, RMT Functions of the Liver Synthetic function Detoxification function Excretory function Detoxification and removal of Bilirubin Bilirubin – Product of hemoglobin determination B1 B2 Unconjugated Conjugated bilirubin bilirubin Polar bilirubin Non-polar bilirubin Water soluble Water insoluble Direct Bilirubin Indirect bilirubin Cholebilirubin Hemobilirubin Methods for Bilirubin Analysis Van den Bergh / Diazo reaction - Reagent: Diazotized Sulfonic Acid = Only Detects B2 Modified Van den Bergh Jendrassik-Grof – Reagent: Caffeine Na Benzoate Evelyn-Malloy - Reagent: 50% Methyl Alcohol/ Methanol Bilirubin determination : Evelyn-Malloy Modified Principle: The procedure is based on the reaction of Diazo Reagent which reacts with bilirubin to produce a colored chromophore. Reagents: Bilirubin Reagent, Absolute Methyl alcohol and Bilirubin accelerator Specimen: Strictly non-hemolyzed serum Procedure: Direct Bilirubin – Mix 0.1 mL of the Test/Control, 1.4 mL Bilirubin reagent and 1 drop of Bilirubin accelerator. Stand for exactly one minute at room temperature then read %T and Absorbance against water blank at 550 nm. Total Bilirubin – use the same test tube just add 1.5 mL of the absolute methyl alcohol stand for exactly 5 minutes at room temperature then read %T and Absorbance against water blank at 550 nm. Calibration / Standardization Transfer the bilirubin accelerator directly into the cuvette and read against water blank. Read %T and Absorbance against water blank at 550 nm. Calculation of the Results: Value of the test: Absorbance of the test / absorbance of the standard X Value of the standard X 17.10 = umol/L. Calibrator: 2 mg/dL (B1), 4 mg/dL (Total bilirubin) Indirect bilirubin = TB – B1 = B2 Bilirubin Conventional Factor SI Unit B1