ESTIMATION OF %AGE OF HEMOGLOBIN IN HUMAN BLOOD

Hemoglobin is a combination of haem and globulin. One molecule of hemoglobin contains four subunits. Various methods can be used to determine percentage of hemoglobin in blood.
APPARATUS:
1. Sahle’s hemoglobinometer, which consists of graduated capillary pipette.
2. Two standard colored tubes.
3. Graduated mixing tube.
4. Stirrer, sterilized pricking needles, alcohol, cotton, etc, etc.
PRINCIPLE:
When HCl is added to blood, hemoglobin comes out of red blood cells and reacts with HCl to form hematin, which dark brown in color. Then dilution of the resultant solution is continued by drop wise addition of distilled water till the color matches with the color of the standard tubes.
PROCEDURE:
1. Clean and dry a capillary pipette and mixing tubes.
2. Take 0.1N HCl in the graduated mixing tubes up to the bottom line, which means up to the mark 2g%.
3. Clean the fingertip of the patient with alcohol and prick with sharp sterilized pricking needle. Blood will appear on the fingertip.
4. Suck the blood into capillary pipette up to the mark 2mm3.
5. Immediately add this blood into mixing tubes. The mixture looks dark brown in color.
6. Wait for 3-4 minutes.
7. Start adding distilled water drop by drop to the graduated mixing tube till the color the color of the solution matches with the color of the standard tubes.
8. Take two readings; first, when color is slightly darker than the color of standard tubes and second, when color is slightly lighter than the color of standard tubes.
9. Take the mean of the two readings as result.
PRECAUTIONS:
1. The blood should be added immediately to the mixing tube after taking in the capillary pipette.
2. Apparatus must be clean and dry.
3. Addition of water should be in drop wise pattern.
4. The blood should be taken in the capillary pipette up to the mark and there should be no air bubbles in the capillary pipette.
5. Stirring should be done after every addition of distilled water.


DETERMINATION OF RED BLOOD CELLS PER CUBIC MILLIMETER OF HUMAN BLOOD
PRINCIPLE:
The blood is diluted to 1:200 with a specific pipette using an isotonic fluid for the preservation of RBCs. The diluted blood is then placed in a special counting chamber and the cells in a measured volume are counted. This figure is multiplied to appropriate factor to obtain the number of RBCs in 1mm3 of blood.
APPARATUS:
1. Hemocytometer, consisting of counting chamber.
2. RBCs pipette, containing red bead in the bulb.
3. A special thick cover slip.
4. Microscope, pricking needle and cotton.
CHEMICALS:
1. Alcohol
2. Hayem’s fluid, which is prepared as: Na2SO4 5grams + NaCl 1gram + HgCl2 0.5grams and add quantity sufficient of distilled water to produce 100ml.
PROCEDURE:
1. Clean the fingertip of hand with alcohol and dry it.
2. Prick the fingertip with a sterilized pricking needle and let a drop of blood to appear on fingertip.
3. Suck the blood into RBCs pipette up to the mark 0.5ml.
4. Immediately draw up the hayem’s fluid up to mark 101.
5. In this way a dilution of 1:200 is prepared.
6. Hold the pipette between thumb and forefingers and rotate it gently to mix the blood thoroughly with the diluted fluid.
7. Clean the Neubauer scale’s area of the chamber and cover slip so that these must be free from dust.
8. Focus the Neubauer scale under microscope.
9. Place cover slip on the area of counting chamber. Drop 3-4 drops of clear fluid from capillary stem of pipette.
10. Add a drop of diluted blood to the edge of cover slip under which it automatically spread.
11. Wait for 3-4 minutes for settling of RBCs.
12. Observe RBCs under high power in 80 small squares designated as E1, E2, E3, E4 and E5.
13. Calculate the number of RBCs per cubic millimeter of undiluted blood.
PRECAUTIONS:
1. Apparatus must be clean and dry.
2. Blood and fluid must be taken exactly up to the mark.
3. Blood should be diluted immediately otherwise it will block capillary pipette.
4. 3-4 drops of clear solution must be dropped from the capillary stem.


DETERMINATION OF WHITE BLOOD CELLS PER CUBIC MILLIMETER OF HUMAN BLOOD
PRINCIPLE:
The blood is diluted accurately to 1:20 with a diluting fluid, which causes complete hemolysis but do not affects leukocytes. WBCs are then counted in special counting chambers. The normal number of leukocytes is usually from 4000 to 11000 per cubic millimeter of blood. However normal count may be lower or higher according to age of individual. There are great fluctuations in the number of WBCs as compared to RBCs.
APPARATUS:
1. Hemocytometer, consisting of counting chamber.
2. WBCs pipette, containing white bead in the bulb.
3. A special thick cover slip.
4. Microscope, pricking needle and cotton.
5. Diluting fluid (Turk’s fluid, which is prepared as: Acetic acid 3ml +distilled water 97ml + few crystals of methylene blue.)
PROCEDURE:
1. Clean the fingertip of hand with alcohol and dry it.
2. Prick the fingertip with a sterilized pricking needle and let a drop of blood to appear on fingertip.
3. Suck the blood into WBCs pipette up to the mark 0.5ml.
4. Immediately draw up the Turk’s fluid up to mark 110.
5. In this way a dilution of 1:20 is prepared.
6. Hold the pipette between thumb and forefingers and rotate it gently to mix the blood thoroughly with the diluted fluid.
7. Clean the Neubauer scale’s area of the chamber and cover slip so that these must be free from dust.
8. Focus the Neubauer scale under microscope.
9. Place cover slip on the area of counting chamber. Drop 3-4 drops of clear fluid from capillary stem of pipette.
10. Add a drop of diluted blood to the edge of cover slip under which it automatically spread.
11. Wait for 3-4 minutes for settling of WBCs
12. Observe WBCs under low power in 64 medium size squares designated as L1, L2, L3, and L4.
13. Calculate the number of WBCs per cubic millimeter of undiluted blood.
PRECAUTIONS:
1. Apparatus must be clean and dry.
2. Blood and fluid must be taken exactly up to the mark.
3. Blood should be diluted immediately otherwise it will block capillary pipette.
4. 3-4 drops of clear solution must be dropped from the capillary stem.