Step-by-step Procedure For Estimation of RBCs Using a haematocytometer

Principle of Estimation of RBCs using a haematocytometer

There is a large numbers of Red Blood Cells in a the Blood Sample. Practically, counting this amount of Red cells directly under the microscope is not possible. therefore, the Red Blood cells are counted by using a special type of chamber, designed for the counting of blood cells in the specimen, known as Hemocytometer or Neubauer’s chamber.

A suitable dilution of blood (1/200) is made in formal citrate solution. This diluent breaks down or lyses the white cells leaving the red cells intact. The number of RBCs in 1/5th of the RBC square is counted using an improved Neubauer counting chamber called a hematocytometer. The hematocytometer consists of 9 counting squares with each square having an area of 1mm2. Every square is filled with 0.1 ml of diluted blood. The result in expressed as RBCs/ lit of blood.

After diluting the specimen, the content is charged on Hemocytometer / Neubauer’s chamber and the cells are counted in the areas specific for RBC count.

Nowadays, two types of RBC Diluting fluid are commonly used, thus

• Hayem’s RBC Diluting fluid
• Formalin Citrate diluting fluid

The Contents of Hayem’s diluting Fluid

• Mercuric Chloride:  0.25 grams
• Sodium sulfate: 2.5 grams
• Sodium chloride: 0.5 grams
• Distilled water: 100 ml
• The Final pH of the solution (at 25°C) varies from 5.8 – 6.0 which depends on the composition and
  companies who manufacture it.

The composition of Formalin Citrate diluting fluid

• Trisodium citrate: 3 grams
• Formalin: 1 ml
• Distilled water: 99 ml

This diluting fluid is commonly used because it is cheaper than the Hayem’s fluid. However, Hayem’s diluting fluid gives the better results.

Hemocytometer / Neubauer’s Chamber

This is a special type of glass chamber that is used for the cell counting, especially for Blood cells.

Of late, Improved Neubauer’s Chamber is used and in some laboratories, other types of chambers are also employed like Burkers chamber, Levy’s chamber and Fusch – Rosenthal chamber etc.

The Neubauer’s Chamber has ruled the area of total 9 square mm and the depth is 0.1 mm as when the coverslip is placed on the surface of the counting chamber, the space between the bottom of the cover glass and the base of grooved area measures 0.1 mm in depth.

The central 1 square is highly ruled which is divided into 25 squares. Each square of the Central square is further subdivided into 16 small squares.
For RBC count the cells are counted in the 5 squares of the Central square as 4 Corner squares of the Central square (divided into 25 squares) and 1 central square of the Larger Central Square (divided into 25 squares).

Methods of  Estimation of RBCs using a haematocytometer

• Macrodilution Method
• Microdilution Method

Macrodilution method of Estimation of RBCs using a haematocytometer

Materials Required for the Estimation of RBCs by Macrodilution Method

• Blood sample (Capillary blood or EDTA anticoagulated specimen)
• RBC diluting fluid (preferably Hayem’s fluid)
• Hb pipette or Micropipette (0.02 ml or 20 µl)
• Hemocytometer / Neubauer’s Chamber
• Gauze piece or Cotton swab
• Graduated Pipette (5 ml)
• Test tubes
• Cover Slip

Procedure of Estimation of RBCs using Macrodilution Method

• Get 3.98 ml of RBC diluting fluid in a Clean, Dry and Grease free Test tube. Note: If there is no variable pipette in the lab which can measure 3.98ml or 3980 µl of Diluting fluid then use 4 ml of Diluting fluid with the help of 5ml Graduated pipette in the test tube and discard 20 µl of fluid using a micropipette or RBC pipette.
• Add 0.02 ml or 20 µl of Blood Specimen to the tube containing diluting fluid with the help of micropipette or RBC pipette.
• Mix well for few minutes and ready your Hemocytometer / Neubauer’s Chamber.
• Get the Neubauer’s chamber / Hemocytometer from its case and clean it using a swab or gauze piece. Similarly, clean out the cover glass and place it over the grooved area of Hemocytometer. Note: Here a special type of cover glass is used which is 0.4 mm thick with very smooth surface and even thickness so that the space between the grooved area of the chamber and cover glass is exactly 0.1 mm.
• Now, take out the RBC pipette and fill it with the Diluted Specimen, mix the solution well and then discard 1-2 drops from the pipette before charging the chamber.
• Gently press the rubber tube of the RBC pipette, so that the next drop of fluid is in hanging position.
• Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately.
• Allow a small amount of fluid from the pipette to fill into the chamber which occurs by the Capillary action. Do not overcharge the chamber and there should be no air bubble in the Chamber

by Use Micropipette instead of RBC pipette for charging the Hemocytometer

• It is also possible to use a micropipette instead of RBC pipette for charging the Hemocytometer. So, with a micropipette, carefully fill around 20 µl of the diluted specimen.
• Press the knob of the pipette to make a hanging drop at the tip of the micropipette.
• Gently place the pipette tip against the edge of the cover glass and if needed slowly expel the more liquid until the counting chamber is full. This process occurs by Capillary action, but care should be\taken not to overfill the chamber.
• A volume of 10 µl is sufficient to fill out the one counting chamber.
• After charging, wait for 3-5 min so that the cells settle down in the chamber.

Microdilution method of Estimation of RBCs using a haematocytometer

Materials Required for Estimation of RBCs by Microdilution Method:

• Hemocytometer/Neubauer’s Chamber
• Coverslip
• Microscope
• Blood sample (Capillary blood or EDTA anticoagulated specimen)
• RBC diluting fluid (preferably Hayem’s fluid)
• Gauze piece or Cotton
• RBC pipette: This is a graduated pipette that gives the dilution of 1:100 and 1:200. It is got two markings at the bottom as 0.5 and 1 and the top of the pipette is marked 101. It is got a round shape bulb which has the Red bead to mix the blood specimen and the diluting fluid. On the top, a rubber tube is attached to the pipette for sucking the blood specimen and dilution fluid. When blood is sucked up to 0.5 mark and the diluting fluid up to 101 marks, gives the 1:200 dilution of Blood: Diluting fluid and When the Blood is sucked up to 1 mark and the diluting fluid up to 101, gives the 1:100 dilution of Blood: Diluting fluid which is commonly used in anemic patients. After sucking the Specimen & Diluting fluid, the content is gently mixed by rotating the pipette on its long axis to ensure thorough mixing of blood and diluting fluid.

Procedure of Estimation of RBCs using Microdilution Method

• Fill the RBC pipette up to the 0.5 mark with the blood specimen and clean the pipette externally to avoid erroneous high results.
• Fill the same pipette with the RBC diluting fluid (can be Hayem’s Fluid) up to the mark 101.
• Careful making sure there are no air bubbles in the pipette bulb.
• Mix the Blood and Diluting fluid in the pipette by rotating the pipette (horizontally) between the palms.
• Take out the Neubauer’s chamber / Hemocytometer from its case and clean it using a swab or gauze piece. Similarly, clean out the cover glass and place it over the grooved area of Hemocytometer. Note: Here a special type of cover glass is used which is 0.4 mm thick with very smooth surface and even thickness so that the space between the grooved area of the chamber and cover glass is exactly 0.1 mm.
• Put the RBC pipette, mix the solution present in it again and then discard 1-2 drops from the pipette before charging the chamber.
• Gently press the rubber tube of the RBC pipette, so that the next drop of fluid is in hanging position.
• The Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately.
• Let a small amount of fluid from the pipette fill into the chamber which occurs by the Capillary action. Do not overcharge the chamber and there should be no air bubble in the Chamber.
• After charging, wait for 3-5 min so that the cells settle down in the chamber & then focus the chamber under the microscope to calculate Red Cells.

Cell counting

• Focus the ruling using the 10x Objective lens and then Count the RBCs in 5 small squares of the central square as described above, using the 40x Objective lens.
• Count the cells which are lying on the right and lower lines of the 5 small squares but not the opposite line. In case of marginal cells, count the cells on ‘L’ line that is either on Right and Lower lines or Left and Upper lines.

Calculations

• After counting the cells under the microscope, we know the No. of RBC in 5 squares of the central square. Let’s consider it as ‘N’ no. of cells.
• Now, the volume of the fluid inside the chamber is the product of Area and depth of the Hemocytometer / Neubauer’s chamber.
• The central area is the 1 sq. mm which is divided into 25 parts so the area is 25 squares = 1 sq. mm
• Out of these 25 squares, the RBCs are counted in 5 squares. So the Area of 5 small squares is 5/25 i.e. 1/5
• The depth of the Hemocytometer is 0.1 mm as described above in a short description of Hemocytometer.
• Now Apply the Following formula to get the Total Red Blood Cell Count –
  Total RBC Count = N × Dilution / Area × Depth
  N × 200 (or 100 as the dilution is made) / (1/5 × 0.1)
• Total RBC count = N × 10,000 / mm cubed

Normal Ranges

• Males: 4.8-5.5 million/mm cubed
• Females: 4.5-5 million/mm cubed

Note

Red cells and other blood cells can be counted in systems based on either aperture impedance or light scattering technology. Because large numbers of cells can be counted rapidly, there is a high level of precision. Consequently, electronic counts have rendered the RBC of much greater clinical relevance than was possible when only slow and imprecise manual RBC count was available.

Also, check out How to calculate Red Blood Cell Indices