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Hemoglobin concentration Hematology Lab. Hemoglobin concentration Hgb concentration estimated by: light intensity, or color matching technique *Spectrophotometer*

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Presentation on theme: "Hemoglobin concentration Hematology Lab. Hemoglobin concentration Hgb concentration estimated by: light intensity, or color matching technique *Spectrophotometer*"— Presentation transcript:

1 Hemoglobin concentration Hematology Lab

2 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

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

4 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

5 Methods 1:201 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.

6 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

7 Standard curve A Hgb concentration glL Test concentration Test Absorbance

8 Linearity of standard curve To ensure linearity of standard curve: 3 STD are used 1:1 dilution 20µL blood : 20µL diluent 1:2 dilution 20µL blood : 40µL diluent 1:3 dilution 20µL blood : 60 µL diluent


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