A standard curve for hemoglobin is created by plotting optical density on the y-axis and concentration of hemoglobin standards on the x-axis. Cyanmethemoglobin standard solutions of known concentrations are used to generate a straight line. The unknown hemoglobin concentration of a sample can then be determined using the standard curve by measuring its optical density. To generate the standard curve, various dilutions of a cyanmethemoglobin standard are prepared and their optical densities measured and plotted against concentration to form the calibration line.

1. Standard Curve of Haemoglobin :
When Plotting a graph of optical density
(O.D.) of the haemoglobin standard
solutions on the vertical axis (y-axis) and
the concentration of standard on the
horizontal axis (x-axis), a straight line is
passes through the origin is called the
Standard Curve of Haemoglobin.

2. A standard curve is prepared by
using cyanmethemoglobin standard
solutions of known hemoglobin
concentrations

3. An unknown hemoglobin concentration
may be calculated from the measured
optical density (O.D.) and can be read
from a standard calibration curve
directly.

4. Reagents and Equipment:
1. Cyanmethemoglobin standard 60 mg/dl available commercially.
Concentration of Haemoglobin standard in gm%
= 60 mg/dl x Dilution Factor
60
5020 µl
=
----- gm/dl X
--------1000
20 µl

5. = 0.06 gm% X 251
Hb standard concentration =15.06 gm%
(Since the Hb Cyanmethaemoglobin procedure
uses 20 µl of whole blood + 5ml Drabkin's
solution)

6. 2. Test tubes
3. Micropipette, 20 μl (0.020 ml)
4. Micropipette tips
5. Volumetric pipettes: 1 ml, 2 ml, 3 ml, 4 ml, 5
ml
6. Colorimeter
7. Graph paper/pencil/scale etc.

7. Preparation of a Standard
Haemoglobin Curve:
1.
Prepare
dilutions
of
the
cyanmethemoglobin
standard
representing
3 gm%, 6 gm%, 9
gm%, 12gm%, and 15 gm%.
2. Take 6 clean, dry test tubes. Label test
tubes.

8. 3. Using the appropriate volumetric
pipettes pipette the solutions as
described below:

9. Tube Cyanmethae Drabkin’s
moglobin
Standard
solution
Concentration
standard
of
1.
0 ml
5 ml
Blank
2.
1 ml
4 ml
1/5 x 15 gm%= 3 gm%
3.
2 ml
3 ml
2/5 x 15 gm% = 6 gm%
4.
3 ml
2 ml
3/5 x 15 gm%= 9 gm%
5.
4 ml
1 ml
4/5x15 gm% = 12 gm%
6.
5 ml
0 ml
5/5 x 15gm%= 15 gm%

10. 4. Mix and stand for 5 minutes.
5. Read the dilutions in the colorimeter:
6. Place the appropriate filter in the
colorimeter or set to 540nm.
7. Zero the colorimeter using tube 1, of
Drabkin’s solution.

11. 8. Read optical density (O.D.) of the
contents of tubes 2-6 relatively.
9. Plot a graph of optical density (O.D.) of
the haemoglobin standard on the
vertical
axis
(y-axis)
and
the
concentration of standard with gm% on
the horizontal axis (x-axis) .

12. Below is an example of a calibration
curve:
TUBE
Concentration of standard ABSORBANCE (O.D.)
suppose
1.
0 gm%
.00
2.
3 gm%
.07
3.
6 gm%
.14
4.
9 gm%
.21
5.
12 gm%
.28
6.
15 gm%
.35

13. 0.4
0.35
0.3
O.D. of standard
0.25
0.2
0.15
0.1
0.05
0
0
2
4
6
8
Concentration of standard
10
12
14
16

14. A new calibration graph must be
prepared
whenever
the
colorimeter, cuvette type or the test
method is changed.
• Always allow the colorimeter to warm up
before measuring the test samples.

15. • Ensure that the correct filter (540nm
wavelength—yellow/green filter) is in
place.
• To test for accuracy use a control sample
of known value.

16. • A new stock of Drabkin’s solution must
be checked against the old solution with
samples of known value before it is used
for patients
• Drabkin’s solution should be clear and
pale yellow in colour. If it is turbid or
loses its
colour it must be discarded.

17. • Handle Drabkin’s solution with care. It is
very poisonous. Do not mouth pipette.
• Leave the blood mixed with Drabkin’s
solution for 10 minutes before reading
the optical density so that the
haemoglobin can convert to
Cyanmethaemoglobin.

18. • Be careful to avoid air bubbles in the
cuvette.

19. Thank you