Hemoglobin contains two pairs of polypeptide chains and four haem groups with ferrous iron. It is the main component of red blood cells, accounting for 34% of their weight. Methods for estimating hemoglobin include colorimetric methods and photoelectric methods. The commonly used Sahli's method is a visual colorimetric method where blood is mixed with hydrochloric acid to form acid hematin, and the color is matched to a comparator. The WHO Hemoglobin Color Scale is a simple, rapid and inexpensive alternative that matches blood to a printed color scale.

1. Hemoglobin
(Hb) contains two pairs of polypeptide chains α2 β2
and four haem groups each having an atom of ferrous
iron(Fe+2).
Hb is the main component of RBCs and accounts for
approximately 34% of the RBCs by weight.
Iron content of Hb is 0.347 gm/100 g

2. Samples used for
Hb estimation
i. Capillary blood from finger prick.
ii. Intravenous sample—It should be well
anticoagulated, preferably in EDTA. Liquid
anticoagulants should not be used at all as these
dilute and decrease Hb concentration.

3. METHODS FOR ESTIMATION OF
HAEMOGLOBIN
By: M. Ishaq (A.P.)
Nims University
Rajasthan
-are divided into 4 groups as under:
1. Colorimetric method:
Colorimetric method is based on colorimetric measurement
of the intensity of color developed on addition of some
substance to the blood.
Include the following:
A.Visual Methods
B.Photoelectric Methods

4. METHODS FOR ESTIMATION OF
HAEMOGLOBIN
By: M. Ishaq (A.P.)
Nims University
Rajasthan
A. Visual Methods
a. Sahli’s method
b. Tallquist chart (obsolete)
c. WHO Hb Color Scale
B. Photoelectric Method
a. Cyanmethemoglobin method
b. Oxyhaemoglobin method & Alkaline Hematin method
c. Electronic counter method
d. Direct reading electronic haemoglobinometer

5. Commonly used methods
By: M. Ishaq (A.P.)
Nims University
Rajasthan
Sahli’s Method – A Color Based Method
Principle :
 Hb is converted into acid hematin with the action of dilute
hydrochloric acid (N/10 HCl).
 The acid hematin is brown in color and its intensity is matched
with a standard brown glass comparator in a visual colorimeter
called Sahli’s colorimeter.

6. Sahli’s Method
By: M. Ishaq (A.P.)
Nims University
Rajasthan
Procedure
 „FillSahli’s Hb tube upto mark 2 with N/10 HCl.
 Deliver 20 μl (0.02 ml) of blood from a Hb pipette into it. „
 Stir with a stirrer and wait for 10 minutes. „
 Add distilled water drop by drop and stir till color matches
with the comparator. „
 Take the reading at upper meniscus.

7. ADVANTAGES:
 Simple bedside test.
 Reagents and apparatus are cheap.
DISADVANTAGES
 There can be visual error.
 Carboxy, met and sulfhaemoglobins
cannot be converted to acid hematin.
 Comparator can fade over the years.
 Color attainment of acid hematin takes long
time and also fades quickly.
 Source of light (day light or artificial) influences
the color comparison.
By: M. Ishaq (A.P.)
Nims University
Rajasthan

8. Normal Values of Hb
By: M. Ishaq (A.P.)
Nims University
Rajasthan
Men
Women
Infants
15 ± 2 g/dl
13.5 ± 1.5 g/dl
16.5 ± 3 g/dl
CriticalV
a
l
u
e
s
Less than 7 gm/dl-
More than 20 gm/dl-
SevereAnemia
Hperviscosity

9. W
H
OHemoglobinColorScale
This is rapid, simple, inexpensive and reliable.
P
r
o
c
e
d
u
r
e
:
A drop of blood is placed on strip of chromatography
paper and the color developed is matched visually against
the printed color scale.
Color scale consists of printed set of colors
corresponding to Hb values.
By: M. Ishaq (A.P.)
Nims University
Rajasthan

10. By: M. Ishaq (A.P.)
Nims University
Rajasthan