Hemoglobin concentration Hematology Lab Hemoglobin concentration Hgb concentration

Hemoglobin concentration Hematology Lab

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

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

Principle of Hgb cyanide method n Blood is diluted in a solution: n contains potassium cyanide + potassium ferricyanide (Drabkin solution) n As a result this reaction will occure: n Hgb, Hb. CO methemoglobin Hi. CN (cyanmethemoglobin) n Hb. S will not be converted using this method

Methods 1: 201 dilution (blood: diluents) n 20µL of blood in 4 m. L Cover and invert the tube several times n Let it stand at R. T for 5 -10 minutes to insure complete conversion n Pour into cuvette and read absorbance at 540 nm wave length spectrophotometer. n Set spectrophotometer to 100% transmittance at 540 wave length, using cyanmethemoglobin as blank. n Continue reading patient samples and record % transmittance or absorbance. n Determin Hgb value of sample using, Beers law and STD curve.

Results and Calculations n Hi. CN is used as standerd n To calculate the Hgb concentration use the following equation: n A of test sample x concentration of STD x dilution factor A of Standard 1000

Standard curve A Test Absorbance Hgb concentration Test concentration gl. L

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