2Hemoglobin 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
3The absorbance is measured using spectrophotometer of 540 nm wave length. Then it is the concentration determined using standard curve.
4Principle 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
5Methods 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.
6Results 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
7Standard curve A Test Absorbance Hgb concentration glL Test concentration
8Linearity 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