Hemoglobin contains iron and transports oxygen in red blood cells. It can be measured through hemoglobinometry to diagnose anemia, screen for polycythemia, and assess treatment responses. Methods for estimating hemoglobin include colorimetric, gasometric, chemical, and specific gravity tests. The most common colorimetric method is the cyanmethemoglobin process, which uses reagents to convert hemoglobin to cyanmethemoglobin and takes absorbance readings to calculate concentration. Estimation helps evaluate anemia severity, monitor therapy effectiveness, and select blood donors.

1. Hemoglobin
Estimation
By: M. Ishaq
Nims University Rajasthan

2. 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
By: M. Ishaq (A.P.)
Nims University Rajasthan

3. By: M. Ishaq (A.P.)
Nims University Rajasthan
Various forms of Hb
i. Oxyhemoglobin (Hb O2)
ii. Carboxy hemoglobin (Hb CO)
iii. Sulfhemoglobin (SHb)
iv. Methaemoglobin
Variants of Hb
Normal: Hb A, Hb F- gama, Hb A2-delta
Abnormal: HbS, HbC, HbD & HbE
The measurement of concentration of Hb in the blood is
known as HEMOGLOBINOMETRY.

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

5. By: M. Ishaq (A.P.)
Nims University Rajasthan
Indications for Hb Estimation
 To determine presence and severity of Anemia.
 Screening for polycythemia.
 To assess the response to a specific therapy in anemia.
 Estimation of Red Cell Indices
 Selection of blood donor

6. By: M. Ishaq (A.P.)
Nims University Rajasthan
METHODS FOR ESTIMATION OF
HAEMOGLOBIN
-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

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

8. By: M. Ishaq (A.P.)
Nims University Rajasthan
2. Gasometric Method
or Measurement of O2 carrying capacity of Hb:
 Measurement of O2 carrying capacity of Hb can not be used for mass
screening but is used in referral or research laboratories only.
3. Chemical Method
or Measurement of iron content of Hb:
 Measurement of iron content of Hb is used only for research purpose
4. Specific gravity method:
 It is a very rapid method and is useful for screening blood
donors for anemia in blood donation program.
 Normal specific gravity of blood ranges from 1.048-1.066.

9. By: M. Ishaq (A.P.)
Nims University Rajasthan
Commonly used methods
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.

10. By: M. Ishaq (A.P.)
Nims University Rajasthan
Sahli’s Method
Procedure
 „Fill Sahli’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.

11. By: M. Ishaq (A.P.)
Nims University Rajasthan
Equipment:
a. Sahli’s Hemaglobinometer consisting of :
1. Graduated hemoglobin tube
2. Comparator with a brown glass standard
b. Hb Pipette
c. Stirrer
d. Dropper (dropping pipette)
REAGENTS:
a. N/10 HCl b. Distilled Water
Specimen:
a. EDTA anticoagulated venous blood.
b. Blood obtained by skin puncture

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

13. By: M. Ishaq (A.P.)
Nims University Rajasthan
Normal Values of Hb
Men 15 ± 2 g/dl
Women 13.5 ± 1.5 g/dl
Infants 16.5 ± 3 g/dl
Critical Values
Less than 7 gm/dl- Severe Anemia
More than 20 gm/dl- Hperviscosity

14. By: M. Ishaq (A.P.)
Nims University Rajasthan
WHO Hemoglobin Color Scale
This is rapid, simple, inexpensive and reliable.
Procedure:
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.

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

16. By: M. Ishaq (A.P.)
Nims University Rajasthan
Tallquist Hb Chart
This chart consists of a series of lithographic colors said to
correspond to Hb values ranging from 10% to 100%. It is
obsolete.
B. Photoelectric Method
Cyanmethemoglobin method
Oxyhaemoglobin method

17. By: M. Ishaq (A.P.)
Nims University Rajasthan
Cyanmethemoglobin method:
This is method of choice for estimation of Hb,
recommended by ICSH.
EQUIPMENTS & REAGENTS
-Colorimeter/ Spectrometer
-Sahli’s Pipette marked at 20 microns
-5 ml pipette
-Drabkins’s Solution
-Standard soln. with known Hb Value
Pot. Ferricyanide
Pot. Cyanide
Pot. Di-hydrogen
Phosp.
Non-ionic detergent
Water

18. By: M. Ishaq (A.P.)
Nims University Rajasthan
Principle & method:
- Erythrocytes are lysed by non-ionic detergent and Hb released
evenly.
- Pot. Ferricyanide converts Hb to methamoglobin.
- Methamoglobin reacts with Pot. Cyanide to form cyanmetHb.
- All forms are converted except SULF-Hb.

19. By: M. Ishaq (A.P.)
Nims University Rajasthan
Sample: Blood from Skin Puncture or EDTA anti-coagulated blood
Take 5 ml of Drabkin solution in two test tubes each.
Add 20 micro liter of blood and mix well
Allow the tube to stand for at least 5 min. or more.
Transfer the sample to cuvette and read the absorbance in
Spectrometer at 540 nm.
Hb is derived from the formula:
Hb in grams/dl =Absorbance of test / Absorbance Of
Standard X Conc.of standard X Dilution Factor / 100.

20. By: M. Ishaq (A.P.)
Nims University Rajasthan
OXYHEMOGLOBIN METHOD
* In this method blood sample is mixed with a weak ammonia
solution.
* Absorbance of this solution is measured in a
spectrophotometer at 540.
* Absorbance of this solution is compared with that of the
standard solution.
* It is a rapid & simple, however derivatives other than oxyHb
are not measured.

21. By: M. Ishaq (A.P.)
Nims University Rajasthan
SpecificGravityMethod
This method gives approximate value of Hb.
Method
 A drop of blood is allowed to fall in copper sulphate solution of specific
gravity 1.053 from a height of 1 cm.
 Specific gravity of 1.053 is equivalent to Hb of 12.5 grams.
 This drop of blood is covered by CuSo4 and remains discrete for 15-20
seconds.
 If drop sinks within this time its specific gravity is higher.
 If it floats- specific gravity is low hence low Hb.

22. By: M. Ishaq (A.P.)
Nims University Rajasthan
THANK
YOU . . .