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    Estimation of Bilirubin

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    Nihal
    This document discusses the estimation of bilirubin levels using a quantitative, colorimetric method. Total and direct bilirubin are estimated by reacting serum samples with reagents to produce an azobilirubin complex, which is measured photometrically. The absorbance readings are used to calculate bilirubin levels against a standard calibration curve. Normal bilirubin ranges are provided as well as clinical interpretations and symptoms of hyperbilirubinemia.

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    Estimation of Bilirubin

    Uploaded by

    Nihal
    363 views36 pages
    This document discusses the estimation of bilirubin levels using a quantitative, colorimetric method. Total and direct bilirubin are estimated by reacting serum samples with reagents to produce an azobilirubin complex, which is measured photometrically. The absorbance readings are used to calculate bilirubin levels against a standard calibration curve. Normal bilirubin ranges are provided as well as clinical interpretations and symptoms of hyperbilirubinemia.

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    This document discusses the estimation of bilirubin levels using a quantitative, colorimetric method. Total and direct bilirubin are estimated by reacting serum samples with reagents to produce an azobilirubin complex, which is measured photometrically. The absorbance readings are used to calculate bilirubin levels against a standard calibration curve. Normal bilirubin ranges are provided as well as clinical interpretations and symptoms of hyperbilirubinemia.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    363 views36 pages

    Estimation of Bilirubin

    Uploaded by

    Nihal
    This document discusses the estimation of bilirubin levels using a quantitative, colorimetric method. Total and direct bilirubin are estimated by reacting serum samples with reagents to produce an azobilirubin complex, which is measured photometrically. The absorbance readings are used to calculate bilirubin levels against a standard calibration curve. Normal bilirubin ranges are provided as well as clinical interpretations and symptoms of hyperbilirubinemia.

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    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 36

    Estimation of Bilirubin

    Structure of Bilirubin
    Types of Bilirubin

    • Conjugated
    • Un-conjugated
    • Total
    Bilirubin Metabolism
    Clinical Significance
    Jaundice
    Causes for Jaundice
    Estimation of Bilirubin

    • By general colourimetric analysis


    • By photometric analyzer
    Estimation of Bilirubin: History

    • Biliverdin measurement
    • Icteric index
    • Van den Bergh and Muller method
    • Jendrassik and Grof
    • Diazyme assay
    By Photometric Analyzer
    Absorption of Bilirubin
    Total and Direct Bilirubin Estimation
    by
    Quantitative, Colourimetric Method
    Principle
     Direct bilirubin (conjugated)
    +
    diazotized sulfanilic acid ---- > azobilirubin (blue at
    alkaline pH)

     Total bilirubin (conjugated +unconjugated0


    +
    caffeine-benzoate-acetate mixture + diazotized
    sulfanilic acid ------→ azobilirubin (blue at alkaline
    pH)
    Requirements

    • Apparatus
    • Reagents
    • Serum samples
    Apparatus
    Reagent: Bilirubin Estimation Kit
    Reagent Kit: Components

    Caffeine Reagent
    Alkaline Tartrate
    HCl
    Diazo Reagent
    Cysteine Reagent
    Standard Bilirubin
    Preparation of Kit Reagents

    • Diazo reagent--13 mg. of sulfanilate and


    0.457 mg. of sodium nitrite in 6ml HCl.
    • Caffeine reagent--25 g./liter of caffeine and
    38 g./liter of sodium benzoate in sodium
    acetate solution.
    • Cysteine reagent--Prepared by adding 10.5 ml
    water
    • Sodium potassium tartrate (350 g./liter) in
    2.5 N sodium hydroxide
    • HCl--200 ml. of 0.05 N
    Preparation of Standard Bilirubin
    Serum
    Method

    • Preparation of reaction assay systems


    • Incubation
    • Measurement
    • Preparation of calibration curve
    Working Table
    Parameters

    Wave length : 540-600nm


    Optical pathway : 1 cm
    Temperature : 37°C
    Measurement : Against reagent blank
    Method : End point analysis
    Result

    • Prepared calibration curve is used to


    determine the concentration of unknown
    samples.
    • Total and direct bilirubin levels are
    determined from the curve.
    • The indirect bilirubin is the difference
    between the total and the direct.
    • All results are tabulated in worksheet.
    Calculation by Standard Formula
    Example

    • Absorbance of Unknown = 0.052


    • Absorbance of Calibrator = 0.180
    • Calibrator Value = 5.2 mg/dL
    0.052
    • Total Bilirubin = ———— x 5.2 = 1.5 mg/dL
    0.180
    Calculation—Calibration Curve
    Analytical Reliabilities

    • Precession
    • Accuracy
    Precaution

    • Blood should be drawn with aseptic precaution.


    Pouring of blood from syringe to cuvette, niddle must
    be avoided.
    • Hazardous materials: Alkaline tartrate is strong base
    so caution should be taken (for eyes and clothes),
    This method uses sulphanilic acid and sodium
    hydroxide. Avoid contact with eyes, skin and mucous
    membranes.
    • During incubation appropriate temperature and
    time should be maintain.
    • Appropriate filter should be used.
    Advantages
    • Specific
    • Sensitive
    Limitation
    • Dilution
    • Interference
    Specific Performance Characteristics

    • Reproducibility studies
    • Recovery studies
    • Numerous variations
    Standard Reference Range
    Clinical Interpretation

    • Adult- Total: 0.3-1.3 mg/dl.


    Direct: Adults 0.1-0.4 mg/dl.
    Indirect: 0.2 – 0.9mg/dl.
    • Infants- Total: Infants 1.0-12.0 mg/dl.
    Clinical Symptoms

    •Plasma bilirubin exceeds 1mg/dl – hyperbilirubinemia


    •B/w 1-2 mg/dl – latent jaundice
    •>2 mg/dl – yellowish discolouration of sclera, conjunctiva, skin , mucous
    membrane resulting in jaundice.
    Diagnosis and Therapy (Infants)
    Diagnosis and Therapy (Adults)

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