Hemoglobin is the main
intracellular conjugated protein present in the red blood cells which gives red
color to the blood. It is made up of large protein molecule globulin and four
ring like structure each containing one atom of iron. Its main function is to
transport oxygen to the tissues and to remove the carbon dioxide from the
tissues by the lungs.
METHOD OF ESTIMATION
1. Colorimetric Method
Ø
Tallquist Method
Ø
Sahli's Acid Hematin Method
Ø
Haldane Method
Ø
Dare Method
Ø
Spencer Method
Ø
Photoelectric Method
§ Oxyhemoglobin
Method
§ Cyanmethemoglobin
Method
2. Specific Gravity Method
(Physical Method)
3. Iron Content Method
(Chemical Method)
4. Oxygen Combining Capacity
Method (Gasometric Method)
MANUAL METHOD
Sahli's Acid Hematin Method
Principle
Hemoglobin is converted to acid
hematin by the addition of N/10 or 0.1N hydrochloric acid. It gives the brown
color, which is compared with standard glass reference blocks. The intensity of
brown color depends upon the amount of acid hematin, which is directly
proportional to the concentration of hemoglobin present in the specimen.
Requirements
1.Sahli's Hemoglobinometer
It consists of
- Standard brown glass mounted on a comparator.
- Graduated tube
- Hb pipette.
- Stirrer
2. 0.1N HCl and dropper
3. Distilled water
4. Pasteur pipette.
Procedure
Ø
By using a Pasteur pipette, add 0.1 N HCl in the
tube up to the lowest mark.(20% mark)
Ø
Draw blood up to 20μL mark in the pipette.
Ø
Adjust the blood column, carefully without
bubbles.
Ø
Wipe excess of the blood on the sides of the
pipettes by using a day piece of cotton.
Ø
Transfer blood to the acid in the graduated
tube.
Ø
Rinse the pipette well mix well the reaction mixture
and allow the tube to stand at least 10 minutes.
Ø
Dilute the solution with distilled water by hemoglobin
estimation.
Ø
By adding few drops at a time carefully and by
mixing the reaction mixture until the color matches with the glass plate in the
comparator.
Ø
The matching should be done only against natural
light and the reading of the fluid is noted at its lower meniscus and the
reading corresponding to this level on the scale is recorded in gm/dl.
Note
Precautions
·
Avoid squeezing the finger tip while drawing
blood.
·
Avoid air bubbles in the pipette.
·
Wipe the tip of pipette before transferring
blood into the hemoglobinometer tube.
·
Blow the blood directly into the HCl into the
hemoglobinometer tube quickly and rinse it with the HCl
·
While matching the color:
§
Hold the hemoglobinometer against bright light
§ Graduations on the tube should not be visible.
§ Don’t block the passage of light through tube with the fingers.
§ Raise the stirrer above the fluid level while matching the color.
§ Don’t remove the stirrer out of the tube once it is introduced into it.
§ Graduations on the tube should not be visible.
§ Don’t block the passage of light through tube with the fingers.
§ Raise the stirrer above the fluid level while matching the color.
§ Don’t remove the stirrer out of the tube once it is introduced into it.
Cynmethhemoglobin method
Principle
Drabkin's reagents contain
potassium ferricyanide and potassium or sodium cyanide. When whole blood is
mixed with Drabkin's reagent, the hemoglobin present in whole blood first
converted to the methemoglobin (unstable) by the action of potassium
ferricyanide and this methemoglobin is further converted to stable
cyanmethemoglobin by the action of potassium or sodium cyanide. The resultant
color complex formed is directly proportional to the hemoglobin concentration,
which is directly measured spectrophotometrically at 540nm.
Requirements
1. Cyanmethemoglobin solution
(Drabkin's reagent)
Sodium Bicarbonate
|
1gm
|
Potassium Ferricyanide
|
200mg
|
Potassium Cyanide
|
50mg
|
Nonionic Detergent
|
0.5ml
|
Make up to 1000ml.with distilled water. Store
in brown color bottle in refrigerator.
2. Test tubes (15 x 12.5mm)
3. Calorimeter
4. Hemoglobin pipette
5. Standard of cyanmethemoglobin
Procedure
Ø
Pipette 5 ml of cyanmethemoglobin reagent in
test and blank.
Ø
Add 0.02ml of whole blood in test.
Ø
Wait for 5 mins.
Ø
Read the absorbance of blank
Ø
Read the test value at 540 nm.
Ø
Read the absorbance of standard (15gm/dl) by
pipettes it directly in a cuvette
Calculation
Hb concentration = OD of test/OD of standard x
15
v
In the blood bank during the screening the test
of donor the specific gravity hemoglobin method is done for the hemoglobin
estimation by using coppersulphate solution.
Principle
The hemoglobin concentration of
the given sample depends upon its specific gravity which is calculated by using
copper sulphate solution.
Procedure
Ø
A drop of blood is allowed to fall into CuSO4
solution of varying specific gravity
§ If the drop of blood sinks specific gravity of
blood is greater than the solution
§ If the drop of blood floats specific gravity of the blood is lesser than the solution
§ If the drop of blood floats specific gravity of the blood is lesser than the solution
Ø
It does not exact estimate the concentration. It
only determines whether the person’s hemoglobin is above the specified level or
not. In blood bank a solution of specific gravity 1.047 is used and is
equivalent to the hemoglobin level of 10.5gm%
Normal value
Men
15.5±2.5gm%
Women 14±2.5gm%
New born 16.5±3gm%
Children 12-13gm%
Clinical Significance
A. Increased
·
In polycythemia
·
After vigorous exercise
·
Excitement
·
Hemoconcentration like dehydration, burns,
severe vomiting, and intestinal obstruction
·
Congenital heart disease
B. Decreased
·
In anemia
·
Drugs that cause aplastic anemia then cause
hemolysis
·
Pregnancy
·
Premature infants
Note
Ø
Protein, lipid bilirubin, methemoglobin,
carboxyhemoglobin, sulfhemoglobin, influence the depth of the color and cannot
be converted to acid hematin by HCL.
Ø
Cyanmethemoglobin estimate all the hemoglobin
except sulfhemoglobin.
Ø
In Drabkin's solution instead of sodium
bicarbonate potassium dihydrogen phosphate buffer can also be used which
shortens the time needed for complete conversion of hemoglobin to
cyanmethemoglobin .The non ionic detergent enhances the lysis of erythrocytes
and decreases turbidity from protein precipitation.
Ø
Since the potassium cyanide present in Drabkin’s
solution is poisonous it should be disposed in running tap water.
Ø
The mean hemoglobin in the dark persons is lower
than in whitish.
Ø
Falsely high results may occur due to prolonged
venous stasis during venipuncture
The
rough value for Hb estimation can be
done by
PCV/3
Or
RBC count x 3