Estimation of
Hemoglobin
Concentration
Reem Alsolami
Hb concentration is estimated by:
Colorimetric techniques or spectrophotometer, based on
light intensity principle
Methods
1. Oxyhemoglobin method
2. Hemoglobincyanide method ( HICN)
Reference method
nternationally recommended
Principle
 Blood (from EDTA) is diluted in a solution called
(Drabkin’s solution) :
 It contains potassium cyanide + potassium
ferricyanide.
 As a result:
 RBCs are hemolyzed and the hemoglobin is
released.
 And the released hemoglobin is oxidized as the
following reaction is occurred:
Cont. Principle
• 1.Hemoglobin (Hgb)+ ferricynide  methemoglobin
• 2. methemoglobin + cyanide  cyanmethemoglobin ( or
also called stable) (HiCN).
• Absorbance of the HiCN solution is read in a
spectrophotometer at ( 540 nm).
• The absorbance of the HiCN solution is compared with
of a reference HiCN standard solution.
• Hemoglobin values are obtained form tables prepared
from calibration graph or by using hemoglobin meter.
Objective
• Hemoglobin is measured to detect anemia and its
severity and to monitor anemic patient under the
treatment.
• It is also used to check the Hb level of potential donor’s
blood, prior of donation.
Methodology
•
The blood is diluted (EDTA) in Drabkin’s solution by 1:201
 20µL of blood in 4000 µL (equal to 4 mL).
•
The tube is then covered and inverted several times.
•
The tube is left to stand at R.T for 5-10 minutes to insure complete
conversion.
•
The HiCN solution is poured into cuvette .
•
The spectrophotometer is set to 100% transmittance at 540nm,
using Drabkin’s solution as blank.
•
The Hgb value is then determined by using, Beers law and STD
curve.
 HiCN is used as standerd
 To calculate the Hgb concentration use the
following equation:
 Hgb concentration=
 Abs of test sample x concentration of STD (in mg/l) x dilution factor
Abs of Standard
 = result g/l --- g/dl ÷ 10
 1000 = factor to convert mg/dl to gm/dl
1000
Abs
.70
.60
.50
.40
.30
.20
.10
0
20
40
60
80 100 120 140
160
180
200 Hgb
Concentration
g/l
• For home work:
• Calculate the Hgb concentration, by Beer’s Law, write
the calculation and draw the graph for your standard and
test sample.
• Give attention to the units.
• Don’t write the method or the procedure
• Write it in two separate papers, graph paper and A4
paper.
• Thanks for listening
• Time for practical work