Post-experiment Questions:

1. Calculate the Red Blood Cell count and show your calculation. (6%)
Volume of cell suspension included in each ruled square:
= (0.2 mm)2 X 0.1mm
= 0.004mm3
= 0.004uL
Total volume included in 5 ruled squares:
= 0.004uL x 5
= 0.02uL

Total number of red cells counted in 5 ruled square = R

Red cell count = (number of red cells counted/ volume) X dilution factor
= (R/0.02ul) x 200
= 10000R X 106/L
= R X 1010/L
= 473/100 X 1012/L
= 4.73 X 10 12/L

2. Show the results of WBC differential count. (12%)

Cell Type Count

Total WBC x 103/µL 100

Neutrophils % 67

Lymphocyte % 25

Monocyte % 5

Eosinophil % 2

Basophil % 1

3. State the colour of the following cell and its organelle after dying with Wright stain. (7%)

Cell/Cell organelle Colour

Erythrocytes Reddish pink

Eosinophils: Nuclei purple

Eosinophils: Granules Bright orange granules

Eosinophils: Cytoplasm Bright red

Basophils: Nucleus Dark purple

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 Basophils: Granules Dark purple

Platelets red

4. What are the common causes of a poor blood smear? List FIVE of them. (5%)
During the preparation of blood smear, there are some reasons which will cause the
appearance of poor blood smear.
When the drop of blood is placed on the slide, the smear should be made without any delay as
the WBC will have abnormal distribution such as accumulation of WBC at the thin edge of
smear if it has delay. Moreover, the RBC may form rouleaux and platelets may clump.
Furthermore, if the drop of blood to the slide is too large or too small will cause the film
becomes too thick or too thin.
Sometimes, the failure will happen during the spreader slide pushed across the horizontal slide. For example, in
a jerky manner. Or even cannot push the spreader slide completely across the slide.
Moreover, people may fail to keep the entire edge of the spreader slide against the horizontal slide while
making the smear. Also, the inappropriate angle for the spreader slide will make a poor blood smear. If the
sample has low hemoglobin, the spreader slide should increase the angle. However, if the sample with high
hemoglobin, the spreader slide should decrease the angle.
Sometimes, the smear may have many holes on it which will disturb the observation under a microscope. This
may be due to the contamination of fat or grease to the glass slide.
Finally, the cellular degeneration may change because of the delay in fixing, inadequate fixing time or methanol
contamination with water.

5. Why is it important to smear the blood as soon as the drop is placed on the slide?
(4%)
The reason to smear the blood as soon as the drop is placed on the slide as it can prevent the
blood from drying out or the possibility of clotting in some cold agglutinin disease.

6. What is Macrocytosis and list FIVE cause? (7%)

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 greater than 100 fL. Those enlarged RBC will be called the macrocytes or megalocytes.
There are some reasons for the appearance of macrocytosis. The first reason may be related to
bone marrow dysplasia. The second cause may be due to the alcohol abuse and chronic
alcoholism. Third reason is the absorption deficiency of vitamin B12 in the digestive tract.
Moreover, Crohn's disease and chronic obstructive pulmonary disease (COPD) are the fourth and fifth reasons
for macrocytosis.

7. What is/are the clinical significance(s) if high eosinophil in blood sample

8. What is/are the composition(s) of Wright’s stain and what is/are the function(s) of
individual components? (5%)
The Wright’s stain is a Romanowsky stain. A Romanowsky stain is any stain combination
consisting of eosin Y or eosin B with methylene blue and any of its oxidation products. While
methanol is used as both a solvent and fixative in this procedure.
Eosin is a red color dye. And it has a negative charge and is able to stain positive charge
substances (base), such as granules and hemoglobin. While methylene blue is a blue color
dye which has a negative charge(acidic) and is able to stain the negatively charged
substances, such as granules, DNA and RNA.

9. How to handle the blood sample if the blood smear cannot be stained or examined
within a short period of time? (2%)
To preserve the blood smears for further investigation in the future, there are some method to
achieve it.
The first method is performing the fixation. 100% ethanol can be utilized for fixation after the
smear is made and put it into a dark box for preventing any exposure. Moreover, the
condition of preserve should be at 4 °C and avoid from freezing and thaw.
Except, air dry and heat fix can also assist in preservation of blood smear. This method can
let us fix the smear without the utilizing of solvent such as methanol. First, the slides were placed in a fused

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silica dish and covered by a Pyrex petri dish lid for heating with 5 minutes in an oven at 150°C.This method can
avoid the contact of the cells with solvents.
There are few reasons will lead to the smear cannot be stained including the following. Firstly, the problem of
dye. The dye is already expired or reused so many times. If the dye is being re-use, it will lead to too much
water or dilute the stain and may lose the staining function. Or may be the dye is not suitable being use in that
case, we may use another dye to instead the old one, such as using May-Grunwald-Giemsa stain instead of
using wright stain. Second, there are some problems with the sample. The sample contains too many proteins
or lipids may the dye be hard to stain. This could be solved by using the PBS or saline to dilute the sample.
Another solution may be to ask the patient to redraw.
Another cause leading to poor blood smear staining could be improved by following steps. For example, the
presence of crenated erythrocyte found in blood smear, we could try to dry smear quickly. Another example,
you may find thin smear due to anaemia; then we could increased spreader slide angle and increase push
speed. Or you may find the thick smear due to polycythaemia, we may decrease spreader slide angle and
decrease the push speed. Another problem you may find in blood smear was presence of agglutinated
erythrocytes associated with cold agglutinin disease, we may warm the blood in 37C for 15 minutes before
staining. Moreover, if you find the blood increase the viscosity due to multiple myeloma, we could decrease
spreader slide angle and decrease push spread.

~END of the Laboratory Assignment~

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