Detailed Manual White blood cell count (WBC count)-The method only


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Detailed Manual White blood cell count (WBC count)-The method only

Here we will talk about the method only and we don’t mention the condition in which the WBCs increased or decreased but we will mention it in anther posts

The aim and the Value of test:

The number of leukocytes contained in 1 liter of blood is called the leukocyte number concentration or leukocyte count.


A white blood cell (WBC) count is used to investigate HIV/AIDS, infections and unexplained fever, and to monitor treatments which can cause leukopenia. In most situations when a total WBC count is requested it is usual to perform also a differential WBC count (coming soon).

Principle of  the test

Whole blood is diluted 1 in 20 in an acid reagent which haemolyzes the red cells (not the nucleus of nucleated red cells which is called reticulocyte), leaving the white cells to be counted.

White cells are counted microscopically using an Improved Neubauer ruled counting chamber (haemocytometer) and the number of WBCs per litre of blood calculated.

Note: When after examining a stained blood film, many nucleated red cells are present (more than 10%), the WBC count should be corrected (see end of Test Method).

Blood sample:

EDTA anticoagulated blood or capillary blood can be used for counting white cells. Heparin or sodium citrate anticoagulated blood must not be used. The count should be performed within 6 hours (blood should not be refrigerated).

Tools:

1) Counting chamber (hemocytometer)

The counting chamber recommended for cell counts is

-       A metallized surface (‘Bright-line’)

-       Double cell Improved Neubauer ruled chamber.

Non-metallized haemocytometers: While these are less expensive, they are not recommended because the background rulings and cells are not as easily seen which makes it is more difficult to count WBCs reliably using this type of chamber . Non-metallized chambers are also more difficult to fill.

Improved-Neubauer-ruled-counting-chamber-non-metalized2

Improved-Neubauer-ruled-counting-chamber-metalized3

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cover glass for hemocytometer2) Counting chamber cover glasses

Special optically plane cover glasses of defined thickness (designed for use with hemocytometers) are required.

Other cover glasses must not be used.

Manufacturers of counting chambers provide two cover glasses with each chamber.

The laboratory should always keep in stock spare cover glasses.

220px-Pasteur_PipetsPasteur-Pipette3) Pipettes/calibrated capillaries and safe filling device

We need 3 types of Pipettes

First: Graduated Pipette of 1 ml volume to get the volume needed from the diluting fluid

Second: Pasteur pipette of a capillary pipette to mix the blood sample and with diluting fluid

Third: Calibrated pipette of 20 µl (0.02 ml, 20 cmm) to aspirate 20 µl accurately

 Sahli PipetteIn case of Calibrated Pipette:

==> You may buy a Calibrated pipette of 20 µl (0.02 ml, 20 cmm) e.g. Sahli is required to measure blood samples.

==> Of It may be supplied with hemocytometers by some manufacturers and it is called a Bulb Pipette. and there is 2 Bulb pipettes supplied; one for Red blood cell count and one for WBCs count,>>>>>>  

BUT:

  • Bulb Pipettes should not be used for counting WBCs because it is not possible to obtain reliable mixing of the blood and diluting fluid inside the bulb of the pipette.

  • Bulb pipettes are also difficult to clean and expensive to replace

A safe pipette/capillary filler should be used to aspirate and dispense the blood. This can be a simple bulb filler or device.  (CLIKC HERE TO SEE THE Pipette filter)

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blood-diluting-pipettes-for-white-corpuscles blood-diluting-pipettes-for-red-corpuscles

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They are differ in the volume

You can use Variable Micropipette instead of all the THREE types

 micropipettes-a-volume-variable

4) Hand counter

To count white cells accurately, a simple inexpensive mechanical hand tally counter or bead counter

Turning the knob on the side returns the counter to zero after each count.

hand-tally-counter

Bead-Score-Counter

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Reagent

CLICK HERE to see how you can prepared it manually in the lab

It is WBC diluting fluid

This is a weak acid solution to which gentian violet is added which stains the nucleus of white cells.

 See dilution step in Page 2

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