BILIRUBIN (TOTAL AND DIRECT)

BILIRUBIN (TOTAL AND DIRECT) PRINCIPLE TOTAL and DIRECT Colorimetric method END POINT Bilirubin is converted to coloured azobilirubin by diazotized sulfanilic acid and is measured photometrically. Of the two bilirubin fractions in serum ?bilirubin-glucuronide and free bilirubin which is bound to albumin? only the former reacts directly, while free albumin reacts after being displaced from protein by an accelerator. The difference of two measurements total bilirubin (with accelerator) and direct bilirubin (without accelerator) enables the calculation of indirect bilirubin. The terms ?direct? and ?indirect? bilirubin refers exclusively to the reaction characteristics in the presence or absence of an accelerator or solubilizer and are only approximate equivalents of the two bilirubin fractions. REAGENT COMPOSITION 2 x 100 ml RT Sulfanilic acid 29 mmol/L, hydrochloric acid 0.24 mol/L, Duposol? 3% (w/v). 2 x 80 ml. RD Sulfanilic acid 29 mmol/L, hydrochloric acid 0.24 mol/L. RN Sodium nitrite 11.6 mmol/L. 1 x 20 ml. Ancillary kit: CAL Bilirubin Calibrator. REF. 1912005 Bilirubin freeze-dried into a protein matrix. Concentration value is traceable to Standard Reference Material 916a. The concentration of total T and direct D Bilirubin is stated on the label and is lot-specific. The target values are derived using LINEAR reagents on the Cobas Mira. STORAGE AND STABILITY Store at 2-30?C. REAGENT PREPARATION Working reagents. Mix 1 mL RN + 4 mL RT (Total) or 1 mL RN + 4 mL RD (Direct). Stable for 8 days at 2-8?C. Calibrator. Reconstitute the vial by adding exactly 1.0 mL of distilled water. Mix carefully and let stand for 5-10 minutes before use. The stability of bilirubin in the dark is: 8 hours 16-25?C, 2 days 2-8?C and 28 days ?20?C frozen once. SAMPLES Fresh hemolysis-free serum. Store in the dark until use. Samples can be frozen at ?15?C or below in which case bilirubin is stable for 2 months. MATERIALS REQUIRED Photometer or colorimeter capable of measuring absorbance at 540 ? 20 nm. Constant temperature incubator set at 37?C. Use the same temperature for assay of calibrator, controls and samples. Pipettes to measure reagent and samples. BILIRUBIN (TOTAL AND DIRECT) Reagents per box