WHITE BLOOD CELL COUNT & DIFFERENTIAL COUNT
WHITE BLOOD CELL COUNT & DIFFERENTIAL COUNT
Objectives :-
At the end of the practical the student should be able to,
1. Identify the apparatus used in counting WBCs.
2. Determine the total white cell count using the Neubauer counting chamber.
3. Prepare a peripheral blood film
4. Identify the different types of white blood cells.
5. Describe how to obtain a differential white cell count.
6. List the normal values of,
i. Total white cell count
ii. Neutrophils
iii. Eosinophils
iv. Basophils
v. Lymphocytes &
vi. Monocytes
And the conditions where these values are abnormal.
Calculation of the Total White Cell Count
Apparatus and materials :-
- The haemacytometer set which contains,
▪ Neubauer counting chamber
▪ White cell pipette
▪ Suction apparatus
▪ Cover glasses
- Microscope
- Sterile lancet. Surgical spirit and cotton swabs.
- Diluting fluid Glacial acetic acid (2%) 1.5 ml
C malachite green 0.5 ml
Gentian violet aqueous 0.5 ml
Distilled water 98 ml
(For this, the blood is diluted with a fluid which lyses red blood cells, but spares white blood cells and nucleated red cells.)
Method :-
- Wipe a volunteer's finger with surgical spirit and allow to dry.
- Stab the pulp of the finger with a sterile lancet and wipe away the first drop of blood (do not rub or squeeze finger to assist flow of blood).
- When a big enough drop of blood forms, apply the tip of the pipette to the drop and suck blood upto the 0.5ml mark.
- Draw up the diluting fluid till the 11 mark - till glass bubble is full. Dilution would be 1 : 20.
- Hold the pipette horizontally and rotate it vigorously between finger and thumb to mix.
- Let out the fluid contained within the stem of the pipette.
- Take the Neubauer counting chamber, place it on the stage of the microscope and put the cover glass over it.
- Touch the pipette to the side of the counting chamber, where it projects beyond the cover glass. Known as "charging the chamber".
- The chamber will fill with the diluted blood.
- Now wait two minutes to allow for the cells to settle.
- Observe under low power.
- As WBCs are larger cells and are found in less amounts than RBCs, the larger squares of the counting chamber are used.
Calculation :-
The volume in one large square (eg. A) = 1 x 1 x 1/10 mm3
= 1/10 mm3
The volume in 4 large squares = 4 x 1/10 mm3
= 4/10 mm3
No. of WBCs in one square = Y
Approx. No. of WBC in 4 squares = 4Y = Z
( concentration of WBC = Z / 4/10
Blood was diluted 20 times
( No. of WBC in 1mm3 of undiluted blood = 10Z x 20 / 4
= 50Z
Preparation of a peripheral blood film
- Clean fingertip with surgical spirit and allow to dry
- Stab with a sterile lancet and wait for blood droplet to form.
- Place the drop of blood on a clean slide.
- Take another clean dry slide and place it vertically, touching the drop of blood.
- Drag this slide slightly backwards and then slide it smoothly forward to spread the drop of blood evenly.
- Allow the slide to air dry for around one minute
- When dry, add 8 - 10 drops of Leishmann stain to the slide and allow to stain for 1 minute. (stain is in alcohol medium).
- Next, add double the amount of drops of water to the slide and mix slightly
- Allow to air dry.
- Add a drop of oil and view under oil immersion lens of the microscope
The Differential Leukocyte Count
- The differential count is obtained by counting the number of white cells on the peripheral blood film
- The is done using a chart as follows, this chart has 10 rows.
|field |N |E |B |L |M |
|1 | | | | | |
|2 | | | | | |
|3 | | | | | |
|4 | | | | | |
|5 | | | | | |
|6 | | | | | |
|7 | | | | | |
|8 | | | | | |
|9 | | | | | |
|10 | | | | | |
- A field is chosen under the microscope, in the 'body' area of the blood film
- 10 white cells are counted, in a field, while filling the above chart according to the different numbers of white cells contained in the total 10 cells
eg. 6 neutrophils, 3 lymphocytes, 1 monocyte
- A similar count is done in 9 more fields, bringing the total number of cells counted, to 100.
- The total number of each type of white cell is then taken as the percentage.
- To obtain the absolute count, these percentages are multiplied by the total white cell count
-----------------------
[pic]
Column of blood
0.5 ml
[pic]
Depth = 1/10 mm
[pic]
[pic]
[pic]
Practical No : 04
[pic]
11 ml
1 ml
White Bead
Head
Body
Tail
................
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