Normal physiology

LABORATORY WORK 1
BLOOD PHYSIOLOGY

UNIT 2

Determination of WBCs and platelets in peripheral blood:

4.1 White blood cell count (TLC)
4.2 Preparation and staining of thin blood smear
4.3 Determination of leukocyte count (DLC)
4.4 Determination of the platelet count

Name_________________________________________________________________________

Group________________________________________________________________________

Teacher_________________________Signature_________________Date_________________
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Plan of class
Lab. w: Detection of WBCs number concentration (TLC - total leucocyte count)
Lab. w: Preparation and staining of thin blood film
Lab. w: Determination of leucocytes type number fraction (DLC – differential leucocyte count)
Lab. w: Determination of platelet count

LAB. W: DETECTION OF WBCS NUMBER CONCENTRATION (TLC - TOTAL

LEUCOCYTE COUNT)
Principle: The blood is diluted in a leukocyte diluting fluid which hemolysis the erythrocytes, but
leaves the leukocytes intact. The leukocytes are then counted in a counting chamber under the
microscope, and the number of cells per liter of blood is calculated. The blood specimen diluted in 20
times in 3% Acetic acid solution, calculate leukocytes in 12.5 biggest squares of Gorayev chamber
which consist of 50 big squares (by four in one biggest).
Materials and reagents
1. Gorayev chamber (counter) with cover glass
2. 0.4 ml of 3% Acetic acid solution (colored with methylene blue)
3. Test-tubes
4. Capillary tube (Sahle pipette 0.02 ml)
5. Researching specimen of blood
6. Microscope
7. Scarifier
8. Methanol
9. Iodine
10. Cotton

Procedure
Fill the test-tube till 0.4 ml of 3% CH3COOH solution
Take capillary blood in capillary tube till 0.02 ml
Mixed blood specimen with CH3COOH solution and shake
One drop of preparing solution distributes in Gorayev chamber under the cover glass
Use x10 objective lenses for calculation
Calculate the leukocytes in placed in 12.5 biggest squares

Normal rates of human leukocytes is 4-9 x 109/l or 4000-9000 mm3

 Normal physiology
LAB. W: PREPARATION AND STAINING OF THIN BLOOD FILM
Principle: A thin blood film is prepared by spreading a small drop of blood evenly on a slide so that
there is only one layer of cells. Thin blood films are stained with Romanowsky stains, which contain
essential azure and eosin dyes. The Romanowsky stains prepared in methanol can used to fix thin
films before being diluted on the slides to stain the films. Better results are obtained by fixing first
with methanol, then staining with pre-prepared diluted stains, as described below.
- After staining, blood films are used for:
- determining leukocyte type number fractions
- detecting abnormal erythrocytes
- identifying certain parasites
- estimating the number of thrombocytes
Materials and reagents
Microscope
Microscope slides (should be well washed and, if necessary, cleaned with ethanol or ether using a
piece of soft cloth)
Glass spreader
Blood lancet
Bottles containing clean tap water
Rack for drying slides
Methanol
Diluted Romanowsky stain

Procedure
Collection of specimens
Take the blood from the side of the third or fourth finger.
Let the blood flow freely. First take the samples for determining the erythrocyte or leukocyte
number concentrations, if possible.
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NOTE:
Do not take blood from:
the index finger or thumb
an infected finger
the ear (too many monocytes).
If it is possible to prepare the film within 1-2 hours of collection of the blood specimen, EDTA
dipotassium salt solution should be added. Other anticoagulants such as heparin alter the appearance
of leukocytes and thrombocytes and should not be used.
Preparation of the film

Figure. Place a drop of blood on the slide

Collect a drop of blood of about 4mm diameter by touching it lightly with one end of the slide
Hold the slide with one hand. Using the other hand, place the edge of the spreader just in front of the
drop of blood.

Figure. Using a second slide, pull the drop of blood across the slide surface, leaving a thin layer of
blood on the slide. After the blood dries, apply a stain for contrast. Place a coverslip on top.
Draw the spreader back until it touches the drop of blood
Let the blood run along the edge of the spreader.
Push the spreader to the end of the slide with a smooth movement (all the blood should be used up
before you reach the end). Blood from patients with anemia should be spread more rapidly.
Check that the film is satisfactory as shown in fig.
There should be no lines extending across or down through the film
The film must be smooth at the end, not ragged and lined as shown in fig.
 Normal physiology
The film must not be too long
The film must not be too thick
The film must not contain holes because a greasy slide has been used

Figure. Blood film

A well-spread film is of great importance. A badly spread film will give the wrong leukocyte type
number fractions and make it impossible to report erythrocyte morphology.
III. Drying the film
Adequate drying is essential to preserve the quality of the film, especially in humid climates. The
film can be left to air-dry in dry climates. Mark the dry film with the patient’s name or number.
Write with a lead pencil on the thick part of the film not used for examination.
IV. Fixation of the film
If the film is intended for determining leukocyte type number fractions, it should be fixed with
methanol before staining with Romanowsky stain.
V. Staining of the film
1. Fix the thin blood film with methanol for 2-3 minutes
2. Prepare the stains as follow
Dilute Romanowsky stain 1 in 10 using one volume of stain and nine volumes of distil water. Mix
gently.
Example: use 1 ml of stain and 9 ml of distil water
Note: Prepare sufficient stain for 1 day’s use, as the diluted stains do not keep well.
3. Cover the slide with diluted Romanowsky stain for 15-20 minutes
4. Wash the stain off in a stream of distils water. Do not tip the stain off as this will leave a deposit
of stain on the film
5. Place the slide in a draining rack to dry.
VI. Microscope examination
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Figure. When view under the microscope, blood smear reveals the components of the formed
elements.

LAB. W: DETERMINATION OF LEUCOCYTES TYPE NUMBER FRACTION (DLC –

DIFFERENTIAL LEUCOCYTE COUNT)

Principle: A total of 100 leukocytes are counted and the number of each type seen is recorded. The
proportion of each type of leukocyte is reported as a decimal fraction1.

Materials
Microscope
Immersion oil
Well-spread thin blood films stained with a Romanowsky stain
Counter (as a table)
WBC, %
Neutrophils Eosinophils Basophils Monocytes Lymphocytes
Band Segmental

Procedure
Using the x90 oil immersion objective, check that the leukocytes are evenly distributed till 1001.
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1
In the Traditional system, the leukocyte type number fractions are called the “differential leukocyte
count”, and the proportion of each type is reported as a percentage (e.g. Neutrophils 56%,
lymphocytes 25%, eosinophiles 12%, monocytes 6% and basophiles 1%).
 Normal physiology

Age___________ Weight___________ Height___________

a) Male b) Female
Your How much should be normal

1 Detection of WBCs number

concentration (TLC - total
leucocyte count)
2 Preparation and staining of thin
blood film

3 Determination of leucocytes type

number fraction (DLC – differential
leucocyte count)
4 Determination of platelet count

Explain your choice: