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Red Blood Cell Count Manual Method

In this red blood cell count manual method post we briefly summarise about: principle, reagents requirements, red blood cell count practical procedure, result, application and limitations of red blood cell count manual method.

Red Blood Cell Count Manual Method

Red blood cell count practical is part of haemocytometry, which measures the amount of RBCs in a blood sample quantitatively. RBCs are counted manually using a haemocytometer or Neubauer’s chamber slide. More accurate or automated instruments, such as electrometric and photometric counters that can count the cell elements of a blood sample, have been developed in recent years.

It is impossible to count RBCs in a blood sample directly. As a result, one of the RBC diluting fluids (hayem’s diluting fluid or formalin citrate diluting fluid) must be used to dilute the blood sample or blood specimen. In this content, we will discuss the requirements, preparations and red blood cell count practical procedure of the RBC count through Neubauer’s chamber. You will also get to know the formula for red blood cell count practical.

Red Blood Cell

Red blood cells (RBCs) are biconcave discs with a round shape that are found in the blood and contribute in the transfer of gases throughout the body. The biconcave form of RBCs aids in the flexibility of red cells, allowing them to flow through capillaries with ease. The size of Red Blood Cells (RBCs) is 7.2–7.4 mm on average in red blood cell count practical analysis.

RBCs are non-nucleated cells with Hemoglobin, an iron-containing pigment that helps in the transport of oxygen from the lungs to tissues and carbon dioxide from tissues back to the lungs for excretion. Red Blood Cells have a lifespan of 100 to 120 days.

Requirements

The haemocytometer is a micro-slide that may be used to count the quantity of erythrocytes or RBCs using two methods: microdilution and macrodilution red blood cell count practical method. The red blood cell count practical method has the following requirements:

Neubauer’s Chamber

This is a specific type of glass chamber that is used to count cells, particularly blood cells. Nowadays, the Improved Neubauer’s Chamber is the most often used chamber, however other types of chambers such as Burkers chamber, Levy’s chamber, and Fusch Rosenthal chamber are also employed in some red blood cell count practical laboratories.

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.

RBC Count Method

Red Blood Cell Count Manual Method: Red blood cell count practical

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). Each square of the Central Square (divided into 25 squares) contains 16 small squares so the total no. of the area to be counted for RBC Count – 16 × 5 = 80 small squares.

RBC Pipette

The RBC pipette is a graduated pipette that allows for 1:100 and 1:200 dilutions. The bottom of the pipette has two markings: 0.5 and 1, while the top of the pipette has the number 101. It has a circular bulb that houses the Red bead that is used to mix the blood samples with the diluting solution. A rubber tube is linked to the pipette at the top for sucking blood samples and diluting liquids.

Red Blood Cell Count Practical: RBC Pipette

When blood is sucked up to 0.5 mark and diluting fluid is sucked up to 101 mark, the blood is diluted to 1:200. The 1:100 dilutions of Blood: Diluting fluid is often employed in anaemic patients when the blood is sucked up to 1 mark and the diluting fluid is sucked up to 101. After sucking the specimen and diluting fluid, the contents are gently combined by twisting the pipette on its long axis to ensure that the blood and diluting fluid are well mixed.

Micropipette

Dr.Heinrich Schnitger invented the device. In practical or research labs, the micropipette is widely used to aspirate or dispense liquid in the necessary volume. The micropipette comes in a variety of sizes. The sample of interest is loaded or dispensed using disposable pipette tips. A diagram of the pieces or components of a micropipette can be found below.

RBC Count Method

Red Blood Cell Count Practical: Micropipette

RBC-Diluting Fluid

To dilute the RBC samples, Hayem’s fluid and formalin citrate diluting fluids are commonly utilised. Both are isotonic solutions that do not cause haemolysis or the crenation of RBCs. The following is a list of the ingredients in Hayem’s and formalin citrate diluting fluid.

Hayem's diluting fluid

Formalin citrate diluting fluid

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 for red blood cell count practical purposes.

Glass Cover

It’s a square-shaped coverslip with a 20 mm width. Keep the glass cover over the centre portion or the ruled region of Neubauer’s chamber to count the number of eukaryotic cells. The glass cover and the core portion of the haemocytometer normally have a 0.1 mm gap.

Blood sample

Blood is usually collected from capillaries or anticoagulated blood. Capillary blood can be collected immediately by pricking the tip of a ring finger, as capillaries are the tiniest blood arteries near the skin surface.

Red Blood Cell Count Practical

For red blood cell count practical, you can perform microdilution and macrodilution quantitative red blood cell count practical by using Neubauer’s chamber.

Microdilution Method

1. Blood sample

2. RBC diluting fluid

3. Gauze piece or Cotton

4. RBC pipette

5. Neubauer’s Chamber

6. Coverslip

7. Microscope

1. Fill the RBC pipette up to the 0.5 mark with the blood specimen and wipe out the pipette externally to avoid false high results.

2. Fill the same pipette with the RBC diluting fluid (preferably Hayem’s Fluid) up to the mark 101. Be cautious that there should be no air bubble in the pipette bulb.

3. Mix the Blood and Diluting fluid in the pipette by rotating the pipette (horizontally) between your palms.

4. 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.

5. Now, put the RBC pipette, mix the solution present in it again and then discard 1-2 drops from the pipette before charging the chamber.

6. Gently press the rubber tube of the RBC pipette, so that the next drop of fluid is in hanging position.

7. Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately.

8. 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.

9. 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.

Macrodilution Method

1. Blood sample

2. RBC diluting fluid

3. Hb pipette or Micropipette (0.02 ml or 20 µl)

4. Neubauer’s Chamber

5. Gauze piece or Cotton swab

6. Graduated Pipette

7. Test tubes

8. Cover Slip

1. Take 3.98 ml of RBC diluting fluid in a Clean, Dry and Grease free Test tube. Now add 0.02 ml or 20 µl of Blood Specimen to the tube containing diluting fluid with the help of micropipette or RBC pipette.

2. Mix well for few minutes and ready your Hemocytometer / Neubauer’s Chamber. 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.

3. 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.

4. Gently press the rubber tube of the RBC pipette, so that the next drop of fluid is in hanging position.

5. Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately.

6. 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.

7. You can also use a micropipette instead of RBC pipette for charging the Hemocytometer. So, with a micropipette, carefully draw up around 20 µl of the diluted specimen. Press the knob of the pipette to make a hanging drop at the tip of the micropipette.

8. Now gently place the pipette tip against the edge of the cover glass and if required 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.

9. 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.

Count under Microscope

First, focus the rulings of the haemocytometer slide using a 10X objective lens. Using coarse and fine adjustment knobs, focus on the five squares of the large central square to count the number of red blood cells under the 40X objective.

A diagram below represents the pattern to count RBCs in all the five medium squares of a large central square. As already discussed, each medium square possesses 16 small squares. We need to manually count the number of RBCs in five medium squares via hand tally.

In each square, you need to count the red blood cells located within the square. The red lines in the upper and right corners indicate the areas not to count RBCs, whereas green lines indicate the areas to count the RBCs.

Calculation

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.

Total RBCs/µL = Number of RBCs counted X Dilution factor / Area X Depth

The number of red blood cells (N) =?

Dilution factor = 1:200 or 200

A large central square is subdivided into 25 medium squares or sub squares. So, the area will be one sq. mm. The number of RBCs is enumerated in 5 squares out of 25 squares. So, the area of 5 small squares will be 5/25 or 1/5 sq. mm.

Depth of the sample = 0.1 mm

Total RBCs = N X 200 / 1/5 X 0.1 = N X 200 X 50 = N X 10,000 cells/µL

Suppose, N or number of RBCs in the five squares is 486, then the equation will be represented as:

Total RBCs = 486 X 10,000 = 48, 60, 000 cells/µL

Further Readings

Reference

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