2 Hemoglobin concentration Hgb concentration estimated by:light intensity, or color matching technique*Spectrophotometer*Methods used are:Hemoglobin cyanide method :Reference methodInternationally recommended for Hgb concentration measurementOxyhemoglobin 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 timesLet it stand at R.T for 5-10 minutes to insure complete conversionPour 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 standerdTo calculate the Hgb concentration use the following equation:A of test sample x concentration of STD x dilution factorA of Standard
7 Standard curve A Test Absorbance Hgb concentration glL Test concentration
8 Linearity of standard curve To ensure linearity of standard curve:3 STD are used1:1 dilution 20µL blood : 20µL diluent1:2 dilution 20µL blood : 40µL diluent1:3 dilution 20µL blood : 60 µL diluent