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  • BILIRUBIN CC Lab

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    Maw Berry
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    This document describes a quantitative colorimetric method for determining total and direct bilirubin levels in serum or plasma. The method involves reacting bilirubin with diazotized sulfanilic acid to produce azobilirubin, which is measured colorimetrically. Several reagents are required, including sulfanilic acid, a bilirubin oxidant containing sodium nitrite, and a bilirubin calibrator. Serum or plasma samples should be collected from a fasting patient and stored between 2-8 degrees C for up to 7 days to measure bilirubin levels accurately.

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    BILIRUBIN-CC-Lab

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    This document describes a quantitative colorimetric method for determining total and direct bilirubin levels in serum or plasma. The method involves reacting bilirubin with diazotized sulfanilic acid to produce azobilirubin, which is measured colorimetrically. Several reagents are required, including sulfanilic acid, a bilirubin oxidant containing sodium nitrite, and a bilirubin calibrator. Serum or plasma samples should be collected from a fasting patient and stored between 2-8 degrees C for up to 7 days to measure bilirubin levels accurately.

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    52 views2 pages

    BILIRUBIN CC Lab

    Uploaded by

    Maw Berry
    This document describes a quantitative colorimetric method for determining total and direct bilirubin levels in serum or plasma. The method involves reacting bilirubin with diazotized sulfanilic acid to produce azobilirubin, which is measured colorimetrically. Several reagents are required, including sulfanilic acid, a bilirubin oxidant containing sodium nitrite, and a bilirubin calibrator. Serum or plasma samples should be collected from a fasting patient and stored between 2-8 degrees C for up to 7 days to measure bilirubin levels accurately.

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    © All Rights Reserved
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    of 2

    BILIRUBIN: QUANTITAIVE COLORIMETRIC REAGENT STORAGE AND STABILITY:

    DETERMINATIONNOF TOTAL AND DIRECT BILIRUBIN IN


    - store at 15-30 C
    SERUM OR PLASMA
    - Direct bilirubin reagent (15-30 C)
    SUMMARY AND PRINCIPLE: - Oxidant discard if a dark yellow color
    develops
     Most routine clinical procedures for the
    determination of serum bilirubin are based on SPECIMEN COLLECTION AND PREPARATION:
    the classical Diazo reaction of Ehrlich
    - Serum or plasma collected in the
    - was first applied to the estimation of bile
    fasting state
    pigment in serum in 1913 by Van den Berg
    - sample should be protected from
    and Snapper
    sunlight and white artificial light
     Winkelman and Co-workers – has an excellent
    (50% of bilirubin can be lost to
    review of many bilirubin methodologies
    sunlight for 1 hour)
    - avoid anticoagulants containing
    1. Nitrous acid: formed when a solution of sulfanilic
    ammonium or fluoride salts
    acid in dilute hydrochloric acid is combined with
    sodium nitrite SAMPLE STABILITY
    - this unstable acid reacts to form diazotized
    - bilirubin is stable in serum or plasma
    sulfanilic acid
    4-7 days (2-8 C), 3 months (-20 C)
    2. Diazotized sulfanilic acid ( p-benzenediazonium
    sulfonate) couples with bilirubin to produce: INTERFERING SUBSTANCES: Gross hemolysis ( can cause
    azobilirubin falsely low bilirubin values due to inhibition of diazo
    - color intensity is equal to bilirubin reaction by oxyhemoglobin) Turbid or lipemic serum
    concentration (may cause falsely elevated bilirubin values)
    3. Calibrator – a aqueous solution of N-1-
    naphthylethylenediamine dihydrochloride
    (Bilissis and Speer)
    - this coupled with diazotized sulfanilic acid to
    give an azo dye
    - color equivalent at 540 nm

    REAGENTS:

    Total Bilirubin reagent

     Sulfanilic acid, 32 mmol/L in dilute hydrochloric


    acid, Accelerator and stabilizer added

    Direct Bilirubin Reagent

     Sulfanilic acid, 32 mmol/L in dilute hydrochloric


    acid and stabilizer added

    Bilirubin Oxidant

     Sodium nitrite, 20mmol/L, aqueous, stabilizer


    added

    Bilirubin Calibrator (10mmol/L)

     N-naphthylethylenediamine dihydrochloride,
    0.346 mmol/L, stabilizer added. Equivalent to
    10mg/dL Bilirubin in method described

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