Measurement of hemoglobin

1. ESTIMATION OF
HEMOGLOBIN

2. Why do we estimate haemoglobin?
• To detect the oxygen carrying capacity of blood.
• Disease detection, which causes a deficiency or excess of
haemoglobin.
• Studying changes in haemoglobin concentration before or after
operations and blood transfusions.
• To detect anaemia and its severity and monitor an anaemic patient's
response to treatment.
• To check haemoglobin level of blood prior to blood donation.
• To calculate red cell indices.

3. Required samples for Hb estimation
• Capillary blood from finger prick.
• Intravenous sample: Should be well anticoagulated,
preferably in EDTA.
Liquid anticoagulants should not be used as they dilute and
decrease Hb concentration.

4. Methods of Haemoglobin Estimation
These are divided into 4 groups;
1. Colorimetric method: This is based on colorimetric
measurement of the intensity of color developed on
addition of some substance to the blood. They include:
- Visual Methods:
- WHO Hb Color Scale,
- Sahli’s method.
- Photoelectric Methods:
- Oxyhaemoglobin method & alkaline Haematin methods,
- Cyanmethaemoglobin method,
- Electronic counter method,
- Direct reading electronic haemoglobinometer.

5. Methods of Haemoglobin Estimation
2. Gasometric Method or Measurement of O2 carrying
capacity of Hb: It measures the O2-carrying capacity of Hb and
can not be used for mass screening but used in referral or
research laboratories only.
3. Chemical Method or Measurement of iron content of Hb:
This measures the iron content of Hb and used only for research
purpose
4. Specific gravity method: It is a very rapid method and is
useful for screening blood donors for aneamia in blood donation
program especially in resource-limited settings. The normal
specific gravity of blood ranges from 1.048-1.066.

6. Commonly Used Visual
Haemoglobin Estimation Methods
i. The WHO Haemoglobin Color Scale: This technique of
estimating haemoglobin involves placing a drop of blood on a
particular type of chromatography paper, and the color
developed after absorption compared against a printed scale
of colour.
• This color corresponds to different levels of haemoglobin
ranging from 4-14 g/dl which forms a color scale.
• It is rapid, simple, inexpensive and reliable.

7. The WHO haemoglobin color scale

8. Commonly Used Visual Haemoglobin
Estimation Methods
ii. Sahli’s Method or Acid Hematin method
Principle: Haemoglobin is converted to acid haematin by N/10
HCl and the resulting brown colour is compared with standard
brown glass reference blocks.
The intensity of the brown colour depends on the amount of
acid haematin produced, and this is directly proportional to
the amount of haemoglobin in the blood sample.

9. Sahli’s Method Procedure
• Place N/10 HCl into the Sahli’s Hb tube up to the lowest mark.
• Deliver (0.02 ml) of blood from a Hb pipette into the tube
thereafter.
• Stir with a glass rod/stirrer and wait for 10 minutes to allow
color development.
• Add distilled water drop by drop and stir till color matches with
the comparator because 95% of Hb is converted at the end of
10mins and others much later.
• Take the reading at upper meniscus.

10. Salhi’s Haemoglobinometer

11. Sahli’s Method
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.

12. Commonly Used Spectrophotometric
Haemoglobin Estimation Methods
i. Oxyhaemoglobin method
• Principle: Blood is diluted in weak alkali (0.04% ammoniun
hydroxide,sp gravity: 0.88) which lyses the red blood cells
and release oxyhaemoglobin into the solution. This
conversion is complete and immediate with a resulting
stable colour.
• In this method blood sample is mixed with a weak
ammonia solution and the absorbance of this solution is
measured in a spectrophotometer at 540nm.

13. Commonly Used Spectrophotometric
Haemoglobin Estimation Methods
• Absorbance of this solution is compared with that of the standard
solution.
• It is a rapid & simple method, however derivatives other than oxyHb
are not measured.
Advantages
- It is fast and no time is required for colour development.
- It is a simple, economical and accurate method than visual
comparative method (error of 2-3%).
Disadvantages
- Standard solution not easily available and unstable.
- Methhaemoglobin and carboxyhaemoglobin are not accurately
detected.

14. Commonly Used Spectrophotometric
Haemoglobin Estimation Methods
• Cyanmethaemoglobin method: It is the preferred and most
accurate method of determining the haemoglobin
concentration in the haematology laboratory.
• Principle: Blood is diluted in a solution of potassium
ferricyanide and potassium cyanide. The ferricyanide oxidizes
haemoglobin to methaemoglobin and potassium cyanide
provides cyanide ions (CN–) to form Cyanmethaemoglobin.
• The absorbance of the solution is then measured in a
spectrophotometer at a wavelength of 540 nm or in a
colorimeter using a yellow-green filter.

15. Specific gravity method (Physical method)
• Haemoglobin as the largest single constituent, affects the
specific gravity of blood more than other substances.
• This procedure does not give the exact Hb value, as it is not
accurate. It uses the principle that when a drop of whole
blood is dropped into a solution of copper sulphate (CuSo4),
which has a given specific gravity, the blood will maintain its
own density for approximately 15 seconds.
• The density of the drop is directly proportional to the
amount of haemoglobin in that drop. If the blood is denser
than the specific gravity of the solution,the drop sinks to the
bottom,if not it will float.

16. Specific gravity method procedure
• A drop of blood is allowed to fall in copper sulphate (CuSo4)
solution of specific gravity 1.053 from a height of 1cm and 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 but if
it floats, the specific gravity is low hence low Hb.
• Since the exact Hb value of the donor is not needed, the blood
bank sets a cut-off value for men and women using CuSo4
solutions with corresponding specific gravity.