3-differential Wbc Count

Practical Physiology 3rd Lab.

Differential White Blood Cell Count (Differential WBC count) [email protected]

Lab. no. 3: Differentail WBC Count Preparation of a Blood film and staining it with Leishman’s stain: 4.Clean thoroughly three microscopic slides, two to be covered with the blood film and one to be used as a spreader. 5.Clean the finger with spirit, allow it to dry and then prick (puncture) it with a sterile lancet to obtain a drop of blood. 6.Touch one end of the two slides to the drop of the blood, only a small quantity is required. Lay the slide on a flat surface. 7.Place the edge of the spreader on the surface of the slide just in front of the drop of blood, and at an angle of 45º. [email protected]

Lab. no. 3: Differentail WBC Count

2.Draw

the spreader back until it makes contact with the drop. The blood will now run along the full width of the spreader at the line of junction. 3.When this has happened, push the spreader slowly and smoothly to the other end of the slide. -The faster the spreader is moved, the thicker the film; a properly made film should be only one cell thick throughout. • Allow the film to dry in air. You can fasten this by waving it. (Repeat with the second slide). • Carefully drop Leishman’s stain∗ on to the blood film until the film is covered. Allow the stain to act for one minute. *Leishman’s stain: is a mixture of methylene blue and eosin in methyl alcohol.

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Lab. no. 3: Differentail WBC Count

3.Then add as much distilled water to the stain as will stay on the slide without spilling over. -This gives a dilution of (1:1 or 1:2). With a teat pipette suck the diluted stain up and down and mix it thoroughly. If the dilution of the stain is correct, the fluid will be covered by a thin greenish scum. 5.The diluted stain should be allowed to act for 7-10 min., then wash it off with distilled water. Make sure that none of the greenish scum settles on the surface of the blood film. 6.Continue washing with D.W to differentiate until the film has a salmon pink color. -Shake off excess water and allow it to dry in air without heat. [email protected]

Lab. no. 3: Differentail WBC Count

spreader

1

A drop of Blood

2

45º(

-Blood film preparation; arrows indicate the sequence of movements of the spreader.

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Lab. no. 3: Differentail WBC Count Examination of the Blood smear: Fix the stained slide on the mechanical stage of the microscope. Examine the blood film first by the low power objective (X10), then by the high power objective (X40). Before getting the oil immersion objective into position add on to the blood film a drop of cedar wood oil. Dip the oil immersion objective into the drop of oil and focus using only the fine adjustment. Numerous Red Blood cells are seen as circular discs stained deep pink at the periphery, and light pink in the center (due to the biconcavity). [email protected]

Lab. no. 3: Differentail WBC Count In iron deficiency anaemia the central light pink area is increased in size. Normal RBCs have about 7 microns diameter and hence are used by comparison to judge the size of the white cells. Scattered, in groups, here and there are platelets seen as small purplish─pink bodies (2─4 microns) in size. Use the mechanical stage of the microscope to traverse the full length of the film. Move the slide along its width by (2mm) and again traverse the full length of the film in the reverse direction. Never stop a count in the middle of the length of the scan, otherwise the result will not be accurate. [email protected]

Lab. no. 3: Differentail WBC Count Identify each white cell in a series of successive microscopic fields; count a sample of (100) white cells and determine the number (percentage) of each cell type. - A table shows the normal ranges, mean values, and normal size of each WBC type. Types of WBCs

Normal range

Mean Value

Normal size

Neutrophils

35-85 %

60 %

10 –14 μ

Lymphocytes

20-50 %

30 %

6 –12 μ

Monocytes

2-6 %

4%

12 –18 μ

Eosinophils

0-4 %

2 %

10 –14 μ

Basophils

0-1 %

0.5 %

10 –14 μ [email protected]

Lab. no. 3: Differentail WBC Count

Granular Leukocytes Neutrophil

Eosinophil

Basophil

Agranular Leukocytes Small Large Lymphocyte

Monocyte

-A figure showing different types of WBCs.

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Lab. no. 3: Differentail WBC Count -A table shows how to determine the number of WBC types (%). WBCs

Neutro.

№

//// //// //// //// ////….

Eosno. Baso.

//..

/

Lympho.

//// //// //// //// ….

Mono.

///…

Percent %

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