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Haemocytometery

Haemocytometery • Various counting chambers • Methods of counting RBC, WBC, and platelets, their calculation, and reference values. and Errors involved in haemocytometery and means to minimize them. Unit II

HAEMATOLOGY

Dr Pramila Singh

3/3/202414 min read

HSBTE, DMLT IInd Semester. Haemocytometery: Various counting chambers, Methods of counting RBC, WBC, and platelets, their calculation, and reference values.  Errors involved in a hemocytometer and means to minimize them. Unit II.

The process of counting blood cells manually or by automated technique is called haemocytometry. An apparatus to count blood cells visually in a blood sample or other fluid under the microscope is called a hemocytometer. The counting chamber, Thoma pipette (diluting pipette), and cover glass for the counting chamber are parts of the hemocytometer. Red blood cells (RBCs) or Erythrocytes, white blood cells (WBCs) or Leucocytes, and platelets or Thrombocytes number are counted by hemocytometer.

VARIOUS COUNTING CHAMBERS:

The two most commonly used counting chambers are

A. Improved Neubauer counting chamber and

B. Fuchs-Rosenthal counting chamber.

Improved Neubauer Counting Chamber:

  • 1. Improved Neubauer Counting Chamber is made of thick rectangular glass slides. The Glass slide has an H-shaped trough (gutter). The horizontal line of the ‘H’ shaped trough forms two counting chambers. One above the horizontal line of H shaped trough and the other below the horizontal line of the H-shaped trough.

  • 2. The left of the ‘H’ shaped trough has one raised vertical ridge line. Similarly right of the ‘H’ shaped trough has one raised vertical ridge line. These two raised vertical ridges hold coverslips (cover glass). The depth between the cover slip and the counting area is 0.1 mm.

  • 3. Cover slip has a very smooth and even surface, made of thick optically flat glass. It is available in two sizes: 16 x22 mm and 22 x23 mm.

  • 4. Improved Neubauer Counting Chamber has a 9 sq. mm area and depth of 0.1 mm.

  • 5. The 9 sq. mm area of the counting chamber has 9 squares. Each square has a 1 sq mm. area.

  • 6. Out of 9 squares, there are four corner squares. Each has 1 sq. mm. area. Each corner square has 16 small squares. Corner squares are used to count WBCs (White Blood Cells).

  • 7. Out of 9 squares, there is one center square. The center square has 25 small squares. Each small square has 16 squares. Out of these 25 small squares, 5 squares are used to count RBCs (Red Blood Cells). Four corner squares of this center square and one in the center of the center square. It means a total of 80 squares (16x5) are used to count RBCs.

Fuchs-Rosenthal counting chamber

The Fuchs-Rosenthal counting chamber consists of a thick glass slide with a rectangular groove in the center. This groove forms a counting area with a known volume and surface area. The counting area is typically divided into a grid of smaller squares. This facilitates the accurate counting of cells or particles. There are two types of Fuchs-Rosenthal counting chambers.

  • 1. Type I: It consists of 16 squares each has 1 square mm area. Each square is separated by three lines. Each square is further divided into 16 squares. Each with0.068 (1/16) square mm area. The depth of the chamber is 0.2 mm.

  • 2. Type II: It consists of 9 squares each has 1 square mm area. Each square is separated by three lines. Each square is further divided into 9 squares. Each with 0.12 (1/9) square mm area. The depth of the chamber is 0.2 mm.

Working: A small volume of the liquid sample containing the cells or particles to be counted is placed on the counting area of the chamber. Capillary action draws the sample into the space. A coverslip is placed on top of it, creating a thin, uniform layer of the sample within the counting area.

The cells in the sample are then visually counted under a microscope. The grid lines on the counting area help to organize the counting process and ensure accuracy. The concentration of cells in the original sample can be calculated by counting the number of cells within a defined area of the grid and knowing the volume of the counting chamber.

Uses: The Fuchs-Rosenthal counting chamber is widely used in medical laboratories for the following:

  • 1. Counting blood cells, such as red blood cells, white blood cells, and platelets.

  • 2. Determining the concentration of cells in cell culture samples.

  • 3. Measuring the density of microbial cells in environmental samples.

  • 4. Assessing sperm concentration in semen samples for fertility testing.

METHODS OF COUNTING OF RBCs, WBCs, AND PLATELETS, THEIR CALCULATION AND REFERENCE VALUES.

METHODS OF COUNTING OF RBCs:

A Haemocytometer, dilution fluid, microscope, and blood specimen shall be required to count RBC (Red blood cells or Red blood corpuscles). The hemocytometer includes an improved Neubauer Counting Chamber, RBC pipette, and WBC pipette. RBC pipette and Improved Neubauer Counting Chamber are used in RBC counting (RBC determination or RBC estimation) in a given sample of blood.

The RBC pipette (Thoma RBC pipette) is a part of the Haemocytometer. It is a graduated micropipette to hold blood for RBC counting in a Haemocytometer. It is made of thick solid glass. Glass is resistant to acid and alkali. It is attached to a large bulb. The large bulb has a red glass bead. The graduated micropipette has 10 marking lines that divide the capillary into 10 divisions. There are two additional markings i.e.0.5 at the 5th line and 1.0 at the 10th marking line. This capillary opens into a large mixing bulb. This mixing bulb opens into another glass capillary that has mark 101.

The red glass bead mixes whole blood and dilutes fluid inside the mixing bulb. Free movement of glass bead in empty glass bulb ensures RBC pipette dryness.

RBC pipette holds 20 microliters or 0.02ml blood. The whole blood dilution inside the RBC pipette is 1:200. The RBC pipette is connected to an aspirating tube made of rubber. It has a mouthpiece.

Dilution fluid formula

  • 1. Sodium citrate: 3.00gm

  • 2. Formaldehyde: 1.00 mL

  • 3. D. Water quantity sufficient to produce; 100mL

Sodium citrate acts as an anticoagulant and maintains the isotonicity of diluted fluid. Formaldehyde acts as a preservative.

Hayem’s diluting fluid formula (Composition)

  • 1. Mercuric chloride 0.25 gm

  • 2. Sodium sulphate 2.5 gm

  • 3. Sodium chloride 0.5 gm

  • 4. D Water 100 ml.

  • Adjust pH between 5.8 and 6.0.

Sodium sulfate and sodium chloride maintain the isotonicity of Hayem’s diluting fluid. Mercuric chloride acts as a preservative. Hayem’s diluting fluid is toxic in nature due to presence of mercuric chloride. Thus handle it with care and dispose off properly.

Specimen: Whole blood containing EDTA or double oxalate as an anticoagulant. Capillary blood is immediately diluted and used to count RBC.

  • Procedure: Rinse RBC pipette capillary and bulb with alcohol, spirit, or ether. Dry it thoroughly. Suck blood into the RBC pipette up to 0.5 markings. Suck dilution fluid carefully to make volume up to the 101 mark. Hold the pipette horizontally and rotate several times by using both palm surfaces. The red bead in the bulb mixes blood and dilutes the solution properly. This dilution of blood is 1: 200 (200 times the dilution of blood).

Place cover slip on improved Neubauer Counting Chamber. Reject the first four drops of dilute blood from the RBC pipette. Put a few drops of dilute fluid under the cover slip. Avoid entry of air with diluted blood under the cover slip.

Set aside the improved Neubauer Counting Chamber for a few minutes. This allows settling RBCs on the bottom of the improved Neubauer Counting Chamber. Count the number of RBCs present in the center square of the improved Neubauer Counting Chamber under a microscope 40X objective. Select the 1st 5th 13th 21st and 25th smaller squares of the center square to count RBCs. Each smaller square has also 16 small squares. It means 80 squares (16 X 5) are used to count RBC. The total area of five smaller squares shall be 0.2 square mm.

Calculation:

  • Total Number of RBC per cc. mm = i.e. Area x depth

  • Area=Central 1 sq. mm (25 sq.)

  • Out of which, 5 are counted.=

  • 25= 1 Sq. mm.

  • 5=?

  • 5/25=1/5

  • Total RBC count= N x Dilution/Area x Depth

  • =N x 200/ 1/5x0.1

  • Total RBC count =N x 10000

Normal values of RBCs:

  • 1. In male adults: 4.5 to 6 X1012 per litre of blood. (4.5 to 6 million/cu mm)

  • 2. In female adults: 4.0 to 5.1 X1012 per liter of blood (4.0 to 5.1 million/cu. mm).

Interpretation

  • · Increased: Polycythemia, Hemoconcentration (Example: Cholera), Congestive heart failure (CHF) and in infants.

  • Decreased: Anaemia

METHODS OF COUNTING OF WBC:

A hemocytometer, glacial acetic acid, gentian violet solution, distilled water, microscope, and blood specimen shall be required to count WBC (White blood cells or White blood corpuscles). The hemocytometer includes an improved Neubauer Counting Chamber, RBC pipette, and WBC pipette.

WBC pipette and Improved Neubauer Counting Chamber are used in WBC counting (WBC determination or WBC estimation) in a given blood sample.

Glacial acetic acid is used to haemolyse the RBCs present in blood. Gentian violet is used to stain nuclei of WBCs.

WBC pipette (Thoma WBC pipette) is a part of Haemocytometer. .It is a graduated micropipette to hold blood for WBC counting in a Haemocytometer. It is made of thick solid glass. Glass is resistant to acid and alkali. It is attached to a large bulb. The large bulb has a white glass bead. The graduated micropipette has 10 marking lines that divide the capillary into 10 divisions. There are two additional markings i.e.0.5 at the 5th line and 1.0 at the 0th marking line. This capillary opens into a large mixing bulb. This mixing bulb opens into another glass capillary that has mark 11. White glass bead mixes whole blood and dilutes fluid inside the mixing bulb. Free movement of glass bead in empty glass bulb ensures WBC pipette dryness.

Whole blood dilution inside WBC pipette is 1:20. WBC pipette is connected with aspirating tube made of rubber. It has mouth piece.

Diluting fluid: Turk’s fluid is used as diluting fluid in WBC counting.

  • 1. Glacial acetic acid: 4ml.

  • 2. Methylene blue: 10 drops.

  • 3. D Water: 200 ml.

  • 4. Glacial acetic acid lyses the RBC. Methylene blue stains WBC.

Procedure WBC Counting:

Rinse the WBC pipette capillary and bulb with alcohol spirit or ether. spirit or ether. Dry it thoroughly. Suck blood into WBC pipette up to 0.5 markings. Suck diluting fluid carefully to make volume up to the 101 mark. Hold the pipette horizontally and rotate it several times by using both palm surfaces. The white bead in the bulb mixes blood and dilutes fluid properly. This dilution of blood is 1: 20 (20 times dilution of blood)

Place cover slip on improved Neubauer Counting Chamber. Reject the first four drops of dilute blood from the WBC pipette. Put a few drops of dilute fluid under the cover slip. Avoid entry of air with diluted blood under the cover slip.

Set a side-improved Neubauer Counting Chamber for a few minutes. This allows the settling of RBCs on the bottom of the improved Neubauer Counting Chamber. Count WBCs number in all four corner squares under a microscope using suitable objectives.

Calculation:

Total WBC count

Dilution-1/20

Depth - 1/10

No. of WBCs in 4 large corner squares=N

Vol. of 4 large squares=1x1x4/10 cumm

=2/5 cumm.

2/5 cumm contains =N x Dilution

.. 1 cumm contains=Nx20x5/2 WBCs

=Nx50.

Normal values of WBC:

1. In male and female adults: 4.5 to 11 X 109 per liter of blood

Interpretation/Significance

  • · Increased: Leucocytosis. at birth, pregnancy, muscular exercise, high temperature, severe pain, pneumonia, asthma.

  • · Decreased: Leukopenia. Anaemia, dengue, malaria.

Difference between RBC Pipette and WBC pipette

RBC and WBC pipettes are used for diluting blood for cell counting in a laboratory setting.

  1. Function: The RBC pipette is used for counting RBC.

  • A WBC pipette is used for counting WBC.

  1. Identification:

  • · Color-coded bead: An RBC pipette has a red bead.

  • The WBC pipette has a white bead.

  • · Markings: An RBC pipette has markings of 0.5, 1, and 101.

  • WBC pipette has markings of 0.5, 1, and 11.

  • · Dilution Ratio: The RBC pipette is designed for a 1:200 dilution ratio. The 101 marking indicates the total volume the pipette can hold, and the 0.5 marking indicates the amount of blood to be drawn.

  • WBC pipette is designed for a 1:20 dilution ratio. The 11 marking indicates the total volume and the 0.5 marking indicates the amount of blood to be drawn in.

  1. Size: The RBC pipette bulb is larger than the WBC pipette bulb. This reflects the difference in dilution ratios.

  2. Diluting fluid: Hayem’s diluting fluid is used as diluting fluid in the RBC pipette.

  • Turk’s fluid is used as diluting in the WBC pipette.

METHODS OF COUNTING OF PLATELETS:

Improved Neubauer Counting Chamber, RBC pipette, microscope, and platelets diluting fluids are required to count platelets in blood specimens. Composition of diluting fluid

  • 1. Procaine hydrochloride: 3.0 gm

  • 2. Sodium chloride: 10 gm

  • 3. Distilled water quantity sufficient to produce 100mL.

Prepare a solution. Filter it by using Whatman filter paper No. 44. Store it in a well-closed plastic container at 2 to 8 degrees C.

Other diluting fluid:

  • Ammonium oxalate diluting fluid: 10 gm per liter (1% solution) C.

Precautions:

  • 1. 3% w/v Procaine hydrochloride in diluting solution is used to haemolyses the RBC. Intact RBC may be mistaken as platelets during platelet counting. 1% w/v Ammonium oxalate can also be used to haemolyse RBC.

  • 2. 3%w/v sodium citrate and !% w/v formalin can also dilute fluid. But this does not haemolyse RBC.

  • 3. Diluting fluid should be free from bacteria and dust. Bacteria and dust may look like platelets. They may be counted as platelets during platelet counting.

  • 4. Siliconised, clean, and dry blood collection bottles and syringes should be used. They prevent adherence of platelets with the wall of the syringe and blood collection bottle.

  • 5. Proper care should be taken to avoid breakage and agglutination of platelets during blood collection.

Procedure:

Rinse the RBC pipette capillary and bulb with alcohol, spirit, or ether. Dry it thoroughly. Suck blood into the RBC pipette up to 0.5 markings. Suck dilution fluid carefully to make volume up to the 101 mark. Hold the pipette horizontally and rotate several times by using both palm surfaces. The red bead in the bulb mixes blood and dilutes the solution properly. This dilution of blood is 1: 200 (200 times the dilution of blood).

  • Place cover slip on improved Neubauer Counting Chamber. Reject the first four drops of dilute blood from the RBC pipette. Put a few drops of dilute fluid under the cover slip. Avoid entry of air with diluted blood under the cover slip.

  • Set aside the improved Neubauer Counting Chamber for a few minutes. This allows the settling of RBCs on the bottom of the improved Neubauer Counting Chamber.

  • Set aside the improved Neubauer Counting Chamber for 15 minutes. This allows the settling of platelets on the bottom of the improved Neubauer Counting Chamber. Count the number of platelets present in the center square (RBC counting area) of the improved Neubauer Counting Chamber under microscopes. Select one corner of small squares of center square to count platelets. It means a total of 25 squares are used to count platelets. Total area to count platelets shall be 0.1 square mm.

Calculation:

  • Total Platelets count=

    • Dilution-1/20

    • Depth - 1/10mm

  • No.of Platelets in 5 medium square in  central large sq.=N

  • Vol..of 5 medium sq.=1/5x1/5x1/10x5 cu mm =1/50cu mm

  • 1/50cu mm =N x Dilution

  • 1cu mm contains=Nx20x50

  •  =Nx1000 Normal values of platelets: 250,000 to 500,000/ cu. mm.

Interpretation

  • · Increased: Thrombocytosis. It occurs during burn injury, pregnancy, cancer, immediately after severe bleeding, etc.

  • · Decreased: Thrombocytopenia. It occurs during aplastic anaemia, myelofibrosis, radiation. hyperplenism, etc.

ERRORS INVOLVED IN HEMOCYTOMETER AND MEANS TO MINIMIZE THEM

A. Errors involved in Thoma pipettes (RBC thoma pipette and WBC thoma pipette) and means to minimize them.

  • 1. Only diluting fluid is present up to 1 mark in the Thoma pipette. Thus first 3 to 5 drops should be discarded from the Thoma pipette.

  • 2. Blood is sucked into the Thoma pipette up to 0.5 mark. Blood may be present inside the Thoma pipette above the 0.5 mark. Extra blood should be removed from the Thoma pipette by tapping the tip of the pipette with a gloved finger. The tip of the Thoma pipette should not be touched by absorbent material such as gauze. Absorbent material shall absorb the liquid portion of blood. This will increase blood cell concentration in blood.

  • 3. Assure proper dilution of blood in the Thoma pipette. Dilution above 11.0 mark in Thoma WBC pipette. or above the 101.0 mark in the Thoma RBC pipette shall disturb blood cell concentration. That will give false results. To avoid this, hold the Thoma pipette horizontally. Draw diluting fluid into the Thoma pipette by using constant suction and turning the Thoma pipette between fingers.

  • 4. Leakage by dripping may occur after the completion of blood dilution in the Thoma pipette. To avoid it, hold the Thoma pipette horizontally after the completion of blood dilution. Rotate it several times by using both palm surfaces. The bead in the bulb mixes blood and dilutes the solution properly.

  • 5. Air bubbles may enter into the Thoma pipette while diluting fluid suction. Avoid it by placing the Thoma pipette tip into dilution fluid properly.

B. Errors involved in counting chamber and means to minimize them:

The following errors may occur related to the counting chamber.

  • 1. Incomplete filling of counting chamber,

  • 2. Overflow of fluid over counting chamber and coverslip,

  • 3. Presence of air bubble under cover slip in counting chamber area,

  • 4. Presence of debris in the counting chamber area.

All these errors can be minimized by following precautions

  • 1. Using clean and dry counting chamber area,

  • 2. Placing cover slip on counting chamber correctly. Correctly placed cover slip shall not fall off even after inverting the counting chamber. There will be rainbow colors over the coverslip if it is correctly placed.

  • 3. Fill the counter chamber with controlled pipetting under the cover slip present on the counting chamber.

  • 4. Despite all precautions, if any one of the above-said errors happens, discard it and repeat the procedure.

C. Errors involved in counting blood cells and means to minimize them:

Errors may occur during the visual counting of blood cells under a microscope. It may be due to improper technique in using a microscope and uneven distribution of blood cells in the counting chamber. These errors can be reduced by following precautions

  • 1. Blood cells collide during the filling of the counting chamber. This causes uneven distribution of blood cells. Thus, after filling the counting chambers, set aside the counting chamber for 3 to 4 minutes to settle down blood cells.

  • 2. To avoid blood cell distribution error, count more number of blood cells.

  • 3. Do not allow to set aside filled counting chamber for a longer duration. It will cause drying of fluid that will disturb blood cells concentration.

  • 4. Examine the counter chamber by using low power objective (10X), then under high power objective (40X).

  • 5. Blood cells touching lines of squares in the counting chamber shall also be counted.

D. Sources of errors in blood counting:

  • 1. False high count of blood cells

    • · Blood specimen collection from body parts with excess blood flow,

    • · Improper wiping of pipette,

    • · Improper pipetting of blood specimen and dilution fluid into Thoma pipette.

    • · Improper mixing of blood specimen and dilution fluid inside Thoma pipette,

    • · Uneven distribution of blood cells in the counting chamber,

    • · Bad placement of coverslip on the counting chamber,

    • · Calculation error

  • 2. False low count of blood cells

    • · Blood specimen collection from body parts with edema

    • · Excess dilution fluid in Thoma pipette

    • · Entry of saliva into Thoma pipette,

    • · Uneven distribution of blood cells in a counting chamber

    • · Calculation error.

Dr Pramila Singh