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Blood 2

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BLOOD 2

WHITE BLOOD CORPUSCLES


1. Total white cell count.
2. Differential white cell count.
TOTAL WHITE CELL COUNT
PRINCIPLE:
Whole blood is diluted 1 in 20 using a diluent, it
haemolyses the red cell, leaving all the white
cells to be counted. White cells are counted
microscopically using an Improved Neubauer
counting chamber (hemocytometer).
1. TOTAL WHITE CELL COUNT

APPARATUS:
1. Sterile lancet
2. Methylated spirit
3. Cotton wool
4. Automated micropipette
5. The Improved Neubauer Counting Chamber (Haemocytometer)
6. Cover slip
7. Diluting fluid ( 1% glacial acetic acid in distilled water + Gentian violet)
8. Microscope
Sterile Lancet
Methylated Spirit
Cotton wool
• AUTOMATED MICROPIPETTE
IMPROVED NEUBAUER COUNTING CHAMBER
((HAEMOCYTOMETER)
Cover Slips
DILUTING FLUID (Turk’s Solution)
MICROSCOPE
PROCEDURE
Ø Measure 0.38ml of diluting fluid and dispense into a small test
tube.
Ø Add 20µl (0.02ml) of well mixed EDTA anticoagulated venous
blood or free flowing capillary blood and mix.
Ø Assemble the counting chamber:
Make sure that the central grid area of the chamber and the
special haemocytometer cover glass are completely clean and
dry.
Slide the cover slip into position over the grid areas and press
down on each side until rainbow colours are seen.
ØCharge the hemocytometer with well mixed diluted
blood.
Ø Allow it to settle for 2 minutes.
Ø Using x 10 objectives lens, locate the 4 squares
(1,2,3,4).
Ø Count the total number of white cells in the 4 large
corner squares(4mm²).
Calculation of total WBC/cu.mm
= NO of cell counted × 1/Area counted × 1/depth
× D.F.
=No of cells counted × ¼ × 1/0.1 × 20
=No of cells counted × 50
Normal value:
Adult : 4.0 - 11.0 × 10⁹/L cells/L (4000 – 11,000
cells/mm³).
Neonates = 10.0 – 25.0 × 10⁹ cells/L ( 10,000 ̶
25,000cells/mm³).
CLINICAL SIGNIFICANCE

LEUKOCYTOSIS: Increase in wbc count can be seen in:


1. Infections e.g. pneumonia, meningitis, septicemia.
2. Inflammation e.g. burns, trauma, tumors.
3. Poisoning e.g. chemicals, drugs, snake venoms.
4. Leukemia.
5. Strenuous exercise.
Leukopenia: Main cause of reduced Wbc count are:
1. Viral, bacterial and parasitic infections e.g. Hiv/AIDS,
viral hepatitis.
2. Drugs. 3. Folate and Vit.B₁₂ deficiency.
2. DIFFERENCIAL WHITE BLOOD CELL
COUNT
INTRODUCTION:
This means identification and counting of
various types of white blood cells and expressing
the number of each type per 100 white cells. It
means that the category of the white cells should
be noted.
TYPES OF WHITE BLOOD CELLS:
There are 5 types of white cells observed in a
normal peripheral blood smear. They are grouped
into 2 categories:
1. Granulocytes: includes Neutrophils,
eosinophils, and basophils. It depends on the
staining reaction of the granules.
2. Agranulocytes: lymphocytes and monocytes.
APPARATUS

1. Microscope

2. Microscope Glass Slide

3. Leishman’s stain
4. Electronic blood counter
MICROSCOPE
Microscope Glass Slide
LEISHMAN STAIN
ELECTRONIC BLOOD COUNTER
PROCEDURE
• Using a clean dry slides, drop a drop of blood on
the slide.
• Draw the spreader to touch the drop of blood.
Allow to extend along the edge of the spreader.
• Holding the spreader at the angle of 45°C, spread
the blood to make a film (⅔ of the slide). Not too
thick a film but smooth and not interrupted with
holes and ridges. It should have a tail at the end.
• Dry the slide.
• Stain with Leishman’s stain.
Preparation of slide.
STAINING PROCEDURE

1. Cover the dry film with the stain for 2 minutes.


2. Dilute with distilled water using pipette(twice the
volume of buffer solution).
3. Leave for 8 minutes.
4. Wash the slide with tap water.
5. Drain and dry in air by keeping it in a slanting position.
6. Place a drop of immersion oil on the blood film and
view.
7.Examine the film microscopically. Focus using 100 ×
objectives.
CHARACTERISTICS OF THE WHITE
BLOOD CELLS
1. NEUTROPHILS: This is the most numerous
white cell type in the normal blood smear. The
nuclear chromatin stains deep reddish purple.
They have multi-segmented nucleus with 3- 5
lobes separated by chromatin threads. The
cytoplasm is abundant, light pink in colour.
Nucleoli is absent. The normal range is 40 –
72%. They capture and destroy invading
organisms and other foreign toxic materials as
soon as they enter the body.
2. EOSINOPHILS
• Bilobed nucleus.
• Large, numerous and conspicuous granules
stained orange red with eosin. Normal range is
1- 6 %.
3. BASOPHILS
• Bilobed nucleus
• Few large dark blue granules. Normal range is
0 -0.5 %.
4. Lymphocytes
• They are of 2 sizes: small and large
• Scanty cytoplasm in small lymphocyte but abundant in large lymphocyte.
• Round nucleus central and with dense chromatin.
• Normal range: 20 -45 %.

5.MONOCYTES
• Largest white cell
• Nucleus often indented/ kidney shaped
• Cytoplasm is abundant, and has blue-gray appearance
• Vacuoles may be seen
• Phagocytic in function and are capable of injecting a number of bacteria.
• Normal range: 2 - 8 %.

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