Hemoglobin concentration

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Hemoglobin concentration

• Hematology Lab

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Hemoglobin concentration

• Hgb concentration estimated by
• light intensity, or color matching technique
• Spectrophotometer
  
• Methods used are
• Hemoglobin cyanide method
• Reference method
• Internationally recommended for Hgb concentration
  measurement
• Oxyhemoglobin method

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• The absorbance is measured using
  spectrophotometer of 540 nm wave length.
• Then it is the concentration determined using
  standard curve.

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Principle of Hgb cyanide method

• Blood is diluted in a solution
• contains potassium cyanide potassium
  ferricyanide (Drabkin solution)
• As a result this reaction will occure
• Hgb, HbCO ? methemoglobin? HiCN
  (cyanmethemoglobin)
• HbS will not be converted using this method

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Methods

• 1201 dilution (blood diluents)
• 20µL of blood in 4 mL
• Cover and invert the tube several times
• Let it stand at R.T for 5-10 minutes to insure
  complete conversion
• Pour into cuvette and read absorbance at 540nm
  wave length spectrophotometer.
• Set spectrophotometer to 100 transmittance at
  540 wave length, using cyanmethemoglobin as
  blank.
• Continue reading patient samples and record
  transmittance or absorbance.
• Determin Hgb value of sample using, Beers law and
  STD curve.

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Results and Calculations

• HiCN is used as standerd
• To calculate the Hgb concentration use the
  following equation
• A of test sample x concentration of STD x
  dilution factor
• A of Standard
  1000

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Standard curve

• A

Test Absorbance
Hgb concentration glL
Test concentration
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Linearity of standard curve

• To ensure linearity of standard curve
• 3 STD are used
• 11 dilution 20µL blood 20µL diluent
• 12 dilution 20µL blood 40µL diluent
• 13 dilution 20µL blood 60 µL diluent