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

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 used
1:1 dilution 20µL blood : 20µL diluent
1:2 dilution 20µL blood : 40µL diluent
1:3 dilution 20µL blood : 60 µL diluent