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Total Leucocytes Count (WBCs count) 4

الكلية كلية طب الاسنان     القسم  العلوم الاساسية     المرحلة 2
أستاذ المادة وسن نجم عبد السادة الربيعي       21/02/2019 18:50:55


Experiment (1)
The Total Leukocyte Count
(W.B.Cs Count)

White blood cells are apart of the body’s immune defense system. White blood cells count is important in the diagnosis of disease especially when accompanied by a differential white cells count.

The Principle
The blood is diluted 20 times with the WBCs diluting fluid .The leukocytes are then counted in 4 corners squares of the improved Neubauer chamber.

Purpose
The WBCs count is to determine the number of circulating WBCs in the body.

Apparatus and Reagents
a-Hemocytometer counting chamber (Neubauer,s chamber).
b-WBCs counting pipette.
c-Rubber tube with a plastic mouth piece for drawing the fluid into a pipette.
d-Special hemocytometer coverslip
e-Diluting fluid.
f-Microscope.

Counting chamber
It is a heavy glass slide H-shaped moat, has two central platforms each of which is ruled .When the cover glass is in place there is a space of 0.1 mm depth over the ruled area .Each of the four corner single square divided into 16 smaller square and used for leukocyte count (Fig.1).
















Figure (1): (A), Hemocytometer counting chamber. (B), A side view of a hemocytometer chamber.(C), the ruled area (a,b,c &d the large corner squares for WBCs count ).

Thoma white cell pipette
This pipette is similar in design to RBC pipette, The stem is lined with two lines marked 0.5 and 1.0, another line above the bulb is marked 11.The bulb contain a white bead, which help in mixing the contents of the bulb and for a quick identification of the WBC pipette (Fig.2).

Diluting fluid
Turk’s solution used for diluting WBCs, this diluting fluid contains an acid solution that lyses the RBCs and a stain that stains the nuclei of the WBCs and allows for easy identification and counting; and an appropriate is:
Distilled water 100 ml
Glacial acetic acid 2 ml
1% solution of gentian violet 1 ml
















Figure(2): WBCs counting pipette

Procedure
1.Examine the chamber under the low power objective of microscope, without the coverslip, in order to understand the ruling .
2.Clean the counting chamber and the coverslip, place the chamber on a flat surface ,and then put the coverslip on the surface of the chamber.
3.Obtain a fresh whole blood sample ( usually EDTA anticoagulant ), but the sample can be capillary blood from a finger puncture.
4.Draw blood in a clean dry WBC pipette up to the mark 0.5. Make sure that there are no air-bubbles .
5.Wipe the outside of the pipette tip clean to remove excess blood .
6.Draw the diluting fluid up to mark 11 (dilution 1:20).
7.Withdraw the pipette from the diluting fluid ,and wipe the outside with apiece of clean gauze .Close the tip of the pipette with the thumb, remove the sucker, place the middle finger over the top and mix well by shaking for 2 minute (Fig.3).
8.Discard the first 2 or 3 drops from the pipette to allow the dilution in the glass bulb to reach the tip end of the pipette .
9.Fill the chamber by holding the pipette at an angle of 45° and lightly touching the tip against the edge of the coverslip (Fig.4) ;allow the diluted blood to flow evenly and slowly under the coverslip by capillary action. Overfilling or underfilling the chamber will result in erroneous counts .If this occur , the chamber should be cleaned and refilled .
10.Leave the chamber for 2 minute ,for the white cells to settle down, and place it on the microscope stage .
















Figure (3): (a) Mixing method (b) Delivery of a diluted specimen to the chamber with the WBC pipette.

Procedure for counting WBCs :
(a)Cells are scanned under a 10x objective to determine the distribution.
(b)Use the 40x objective to count WBCs in each of the four of the corner secondary square on both sides of the chamber.
(c)Count cells starting in the upper left corner square, continue counting to the right hand square ,drop down to the next row continue counting from the right square to the left square.
(d)Count all cells that touch any of the upper and left lines .Do not count any cell that touches a lower or right line, and record your results (Fig.4) .

Calculation
The area of each large corner square is 1 mm2, the depth of the chamber 0.1 mm (from chamber surface to coverslip), the volume of blood in one square is 1 mm (length) × 1 mm (width) × 0.1 mm (high) = 0.1 mm3 ,volume of four corner squares = 0.1 mm3 × 4 = 0.4 mm3 .
No. of cells in large corner square = N.


Dilution factor
No. of WBCs/mm3 = Cells counted × ———————
Volume factor

20
No. of WBCs/mm3 = N × _____ = N × 200
(In one square) 0.1


20
No. of WBCs/mm3 = N × _____ = N × 50
(In four squares) 0.4























Figure (4): WBCs counting procedure

Clinical Applications
1.Leukopenia :The WBCs count is below 4,000 cells/mm3. This condition is indicative of depressed bone marrow or a neoplasm of the bone marrow.
2.Leukocytosis :The WBCs count is in excess of 11,000 cells/mm3.This condition is characteristic of most bacterial infections. It may also occur after hemorrhage, in cases of gout ,and in uremia ,a result of kidney disease.
3.Leukemia :Is an enormous increase in the number of white cells due to a cancer of the tissues that produce these cells (myeloid and lymphoid tissues).WBCs count above 50,000 cells/mm3.

Source of Error
1.Sampling error in collection of blood .
2.Equipment error in the pipette and chamber .
3.Technical errors involved in the exercise from the pipette to the final count.
4.Inherent of field errors of the distribution of cells in the chamber .This can be minimized by counting large number of cells.

Normal Value
Infants 15,000-20,000 cell/mm3
Children 4,500-13,500 cell/mm3
Adult 4,000-11,000 cell/mm3


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