Haemocytometer: Principle & How To Collect Blood Sample:

Haemocytometer is an apparatus used to count various blood cells (RBC , WBC and a easinophll and platelets). It consists of RBC and WBC pipette (Thoma diluting pipettes) thick slide (Neubauer’s chamber).

REAGENTS:

Hayem’s fluid for RBC counting and its composition:

• NaCl 0.5 g for isotonicity.
• Na2SO4- 2.5 g Breaks R.B.Cs and prevents their Rouleaux formation.
• HgCl2-  2.5 g Antibacterial / antifungal and as preservative.
• Distilled H’O upto 100 ml for dilution.

Diluents for leukocyte Counting:

Turk’s solution is used for WBC’s counting. Its composition is as follows:

1. 1.5ml glacial acetic acid for destruction of RBC’s and platelets.
2. 1 ml aqueous gentian violet to stain nuclei of WBC’s
3. 100 ml distal water.

Diluents for platelet counting (Rees Ecker’s solution) and its composition:

• 3.8g Na citrate as anticoagulant.
• 0.2 ml neutral formaldehyde (38%) as Antifungal and fixative for the cells.
• 0.5 g brilliant cresyl blue in 50 ml distal water for the staining of platelets.
• Distal water upto 100 ml as diluent. RBC Pipette.

RBC  PIPETTE:

1. This consist of glass stem having capillary tube in it which opens in a bulb containing red bead and Opposite to the bulb again there is a small stem. This small stem is connected to the red coloured mouth piece with the help of a rubber tube
2. The stem has three markings, 0.5, 1.0 and 101. From the tip of the pipette to the marking 1.0 there are 10 equal divisions. These are simple divisions not of any specific unit system like mm, ml, and cu mm.
3. The stem has the capacity of one part and bulb has 100 parts. IV. Bead of pipette serves two purposes First mixing of the blood with diluting fluid and other acts as an identification mark of RBC pipette.

 

WBC Pipette:

This pipette is similar in shape except size of bulb is smaller, marking are 0.5, 1.0 and 11, and bulb contains white bead and colour of the mouth piece is white .

 (Levy Counting Chamber with Improved Neubauer Ruling):

This is a thick glass slide having central platform divided into two positions with the help of H shape groove or trench .

1. On both the sides of lateral groove there are raised ridges of a height of 0.1 mm (1/10 mm) from the central-platform. When a coverslip is placed on the ridges, a space of 0.1 mm height is created below the coverslip on the central platform.
2. Counting chamber (Grid) is made up of ruled area of 3mm x3 mm size on each central platform (Figure 8). Each central area is further divided by triple lines into 9
3. squares of equal size (1 square mm each).
4. Four corner squares are further divided into 16 squares of equal size. These four corner squares are used to do total leucocytic count (TLC)
5. Volume of each big square is 0.1 cu mm (1 mm x 0.1mm).
6. Central big square is divided into 25 (medium size) squares each having arm 1/5 mm. Area of each medium size square is 1/25 sq.mm (1/5×1/5) and the volume.
7.  Further these medium size square are divided into 16 small squares of equal size. Area of each small square is 1/20 mm . Area Of each square is 1/400 mm squares(1/20 x 1/20) and volume is 1/4000 mm3 (1/400 x 1/10). Total number of smallest squares in big central square are 400 (25 x 16).
8.  RBCs are counted in 5 medium size square R2, R3, R4, four corners and one central.

COLLECTION OF BLOOD SAMPLE :

Blood is collected for various haematological investigations:

Capillary Blood:

• It can be collected from fingertip and ear lobule in adult and heal in case of newborn or infant.
• Take 20-24 gauge sterilized disposable hypodermic needle for this purpose.
• Clean ring finger of left hand (preferably ring finger) with spirit swab and let it dry. Sterilization is effective when spirit dries up and spirit causes haemolysis when it comes in contact with blood.
• After collection of blood sample put a spirit swab at pricking site and hold it therefor 2-3 minutes (till bleeding stops).
• Do not squeeze the finger for collection of blood, it will cause dilution of blood because tissue fluid mixes
• Give 2-3 mm deep prick at centre of the tip of ring fingers so that free flow of blood will be there.

VENOUS BLOOD :

• Take Sml disposable syringe with 1920 gauge needle.
• Support the arm of the subject on the edge of the table and locate the vein in antecubital fossa (area).
• Clean the area with spirits swab and let it dry. Do not touch the area again with finger once it is cleaned.
• Apply rubber or cloth tourniquet around the arm to occlude venous return. Ask the subject to close and open the fist repeatedly so as vein gets engorged with blood.
• Place the thumb of your left hand on the skin about 4-5 cm distal to the vein to be pricked so that vein will not slip.
• Introduce the needle into the skin and push the needle on the side of vein, when needle enters the vein resistance feels to cease.
• Draw the blood in syringe slowly to the required quantity (slowly means not faster than the filling of vein).
• Release the tourniquet and withdraw the needle after putting the fresh spirit swab at the site of the puncture of skin.
• Ask the subject to press the swab at the site of the puncture of skin.
• Eject the blood from the syringe Anticoagulants  after removing needle in the vial having anticoagulant. For serum no anticoagulant is required in the vital.

Anticoagulants Used:

• Ethylene diamine tetracetic acid (EDTA): –Calcium chelating agent

• No effect on blood cells
• 2.4 mg dry powder in a vial for 2 ml of blood (1.2mg/ml of bloo )
• Used for ESR and PCV measurement.
• Sodium citrate:
• Calcium chelating agent
• 0.4 ml of 3.8% solution for 1.6 ml of blood
• used for ESR and collection of blood from donors
• Double oxalate mixture:
• Potassium oxalate and ammonium oxalate in the ratio of 2:3.
• Single oxalate is not used mainly to measure PCV. Because potassium oxalate causes shrink-age of cells and ammonium oxalate causes increase in volume of the cells.
• Heparin
• Solution or powder may be used according to need.
• Does not affect cell volume