ENUMERATION OF ERYTHROCYTES
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ENUMERATION OF ERYTHROCYTES (R.B.C. COUNT)
AIM To find out the number of red blood cells in one cubic millimeter of blood
PRINCIPLE: The number of RBC in a known volume of diluted blood is counted and the number of cells in one cmm of undiluted blood is calculated from this.
APPARATUS: Hemocytometer, RBC diluting fluid, compound microscope, sterile lancet, watchglass, cotton, rectified spirit
HAEMOCYTOMETER: This includes a counting chamber, a special cover slip, and RBC diluting pipette and a WBC diluting pipette.
The improved Neu-Bauer’s double counting chamber: This is a thick rectanguar glass with a polished transverse bar in the centre, separated from the rest of the slide by two parallel grooves on either side. The polished bar is divided into two equal platforms by a groove in the middle resulting in ‘H’ shaped depression (moats). The surface of the platforms is 1/10 mm below the surface of the rest of the slide. So if a cover glass is placed over the surface of the counting chamber, the under surface of the coverglass remains 1/10 mm above the polished surface of the platform.
The counting area is in the form of a central ruled area on the polished surface of each platform. It is a square of 3 mm side, divided into 9 equal squares of 1 mm side. Of these, the four corner squares are used for WBC counting. Each WBC square of 1 mm side is again divided into sixteen smaller squares each of 1/4mm side. The central 1 mm square is divided into 25 equal small squares of 1/5mm side, by means of triple lines of which the 4 corner ones and the central one are used for RBC counting. Each of these squares is subdivided into 16 smallest squares each of 1/20mm side.
R.B.C. Pipette (Thoma glass pipette)
This is a bulb pipette having a long stem with a capillary bore and a pointed tip. The bulb contains a red bead inside. A small rubber tube provided with a mouth piece is connected to the small narrow portion above the bulb for sucking blood and fluid into the pipette. The pipette has three markings on in it, 0.5 mark in the middle of the stem, 1 mark at the junction between stem and bulb, and 101 mark above the bulb. The total volume of the pipette is 101 parts, of which one part is in the stem and 100 parts in the bulb. Other uses of R.B.C. pipette:
1. WBC count in leukemias 2. Platelet counting Uses of the bead in the bulb 1. For proper mixing 2. To know whether the pipette is dry 3. To identify the pipette R.B.C diluting fluid: Hayem’s fluid is the commonly used diluting fluid.
Composition:
Sodium chloride 0.5 Gm Sodium sulphate 2.5 Gm Mercuric perchloride 0.25 Gm Distilled water 100 mlSodium chloride and sodium sulphate together keeps the isotonicity of fluid. Sodium sulphate also prevents clumping of red cells. Mercuric perchloride fixes the cells and acts as a preservative. Other diluting fluids: Gower’s fluid Toison’s fluid Formol Citrate solution
Procedure:
Clean and dry the counting chamber and put on the special cover slip provided. Focus under the high power objective and identify the RBC counting area. Clean the RBC pipette first with distilled water, then with absolute alcohol and finally with ether and keep it dry. Take a small quantity of diluting fluid in a watch glass and keep aside. Clean the finger tip using rectified spirit and make a deep prick with a sterile lancet,so that blood comes out freely without squeezing. Wipe off the first drop which may contain tissue fluid also. Allow a good sized blood drop to form hanging drop and keep the pointed tip of the pipette touching the drop. Suck in blood upto the 0.5 mark carefully, without any air bubble. Excess blood at the tip of the pipette is removed using a bloating paper or piece of cotton. Immediately, diluting fluid from the watch glass is sucked in upto the 101 mark with out any air bubble by keeping the pipette in vertical position.
Then thoroughly mix the blood and diluting fluid in the pipette by gently rolling the pipette held horizontally between the palms and keep aside. Mixing takes place only in the bulb of the pipette. The column of diluting fluid contained in the stem of the pipette does not enter into the dilution (i.e. 101-1 = 100). So that the blood sucked upto 0.5 mark will have a dilution of 0.5 in 100 or 1 in 200. Now take out the counting chamber for charging discard first few drops from the pipette, as the stem contains only diluting fluid. Bring one small drop of diluted blood at the tip of the pipette, to the edge of the cover slip on the counting chamber at an angle of about 450 The fluid enters by capillary action under the cover slip and fills the counting chamber. Both areas are filled. Focus the RBC counting area under high power. Keep the counting chamber undisturbed about 3 minutes for the cells to settle down in the counting area, and start counting. At least 5 squares, each having 16 smallest squares (preferably 4 corner and 1 central) should be counted to obtain a satisfactory average and a better dispersal value. While counting each small square, cells touching the top and left margin of each square should be omitted and cells touching bottom and right margin of each square should be counted. Draw a chart of the counting squares in the record and enter the number of cells in each square and when counted.
Precautions:
1. Counting chamber and pipette should be clean and dry. 2. Fingertip and pricking lancet should be sterile. 3. Blood should freely come out without squeezing. 4. Be careful to prevent clotting of blood inside the pipette. 5. While filling the pipette and charging the counting chamber, no air bubble should enter. 6. Blood should be taken only upto the 0.5 mark and diluting fluid sucked only upto 101 mark. 7. Blood should be properly mixed with the diluting fluid. 8. Discard first few drops before charging because it will not contain RBC5. 9. While charging the counting chamber, over filling and spilling should be avoided. 10. Cells should be settled down and more or less evenly distributed before counting. 11 Don’t keep the microscope in tilted position. 12. Count from Left to Right and avoid counting of the same cell.
Calculation:
Let the number of cells counted in (5x16) 80 smallest squares be “N” Number of cells in 1 smallest square is N/80 Side of 1 square = 1/20mm Area of 1 square = 1/400mm2Depth of fluid film in counting chamber is 1/10mm Volume of diluted blood in 1 square=1/400x1/10=1/4000mm3 Number of cells in 1/4000mm3 diluted blood = N 80 Numberof cells in 1 mm3 of diluted blood N 80x1/4000 = Nx4000 80 The dilution factor is 1 in 200 (Total diluted volume in bulb of the pipette is 100 parts, out of which 0.5 is blood. So dilution is 0.5 in 100 i.e.1 in 200) So numberof cells in 1 mm3 of undiluted blood =Nx 4000x200 = Nxl0000 80
Discussion: (Expressed in millions/mm3) Normal RBC Count Adult male: 5- 5.5 millions/mm3 Adult female: 4.5 - 5 millions/mm3 Variations in Count: Increase in count Decrease in count
Physiological variations 1.Diurnal - 2.Age - 3.Sex - 4. High altitude - 5. Muscular exercise - - Polycythemia (Erythrocytosis) - Anaemia (Erythropenia) Lessduring night, minimum in early morning, gradually increasesduring day In newborn, high count is seen More in males Count higher due to hypoxia Count increases
Pathological increase in count:
1. Lung diseases like emphysema, pulmonary tuberculosis 2. Congenital heart disease 3. Carbon monoxide poisoning 4. Primary polycythemia (Polycythemia rubra vera) Secondary polycythemis is due to hypoxia resulting from any cause, physiological or pathological. Pathological decrease in count: 1. Increased destruction of RBC 2. Decreased production of RBC
Questions: Result: 1. Enumerate the sources of error in this experiment. 2. Diagrammatically represent various stages of Erythropoiesis 3. Discuss the dietary factors in Erythropoiesis. 4. How can you differentiate between primary and secondary polycythemia?
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