Module 10

Cardiovascular Physiology:
Differential Leucocyte Count

At the end of this module, you are expected to:

1. to be able to identify different types of leukocytes
2. to be able to do differential leukocyte count
3. to compute and know the significance of absolute and relative leukocyte count

Introduction
Differential Leukocyte count can be expressed as relative or absolute count.
Relative LC is simply the percentage of each classification of WBC whereas absolute LC
is the percentage of each classification multiplied by the total WBC count/µl. Fresh blood
without anticoagulants is preferred, however oxalated blood is commonly used. In the
case of the latter, smears should be prepared within 20 minutes from the time the
samples are collected because leukocytes may show changes in morphology due to
aging in the presence of oxalate.

Materials

Fresh blood – one EDTA tube per group*

Slides – one box per group*
Microscope
Diff Quick Stain
* - must be purchased/prepared
Methods

A. Smear Preparation
1. Select several clean and grease-free slides with smooth, unbroken ends.
2. Place the slide on a horizontal surface
3. Transfer a small amount of blood with a dropper or applicator stick (a toothpick
will do) from the container to the slide one inch from the right and midway
between the edges of the slide
4. Secure the slide by holding the left end with the left forefinger and thumb
5. Get another slide (spreader) holding one end and placing the other end on the
center of the horizontal slide forming an acute angle.
6. Draw the spreader to the right until it touches the drop of the blood. Stop at this
point and allow the blood to spread to the edge of the horizontal slide, push the
spreader forward in a smooth continuous glide. Thickness is determined by the
size of the drop and the angle of the spreader
7. Allow the smear to dry by waving in the air. Do not blow the slide
8. Identify the slide with a marker.

B. Staining the Smear

1. Dip the blood smear in the fixer approximately 5 times.
2. Remove excess fixer by tapping the glass slide.
3. This is followed by dipping again the smear into the container this time with the
red stain.
4. Rinse with tap water and remove excess water before doing the same
procedure this time using a blue stain.
5. Rinse again and let dry.

C. Examination of the smear

1. First scan with 10x objective, note abnormal cells or structures, particularly in the
feather edge of the smear
 2. Next, scan the smear with 40x objective in an area where RBCs are not touching
each other and note the distribution of WBC. Distribution is uniform in properly
made smears.
3. Place a drop of oil on the smear in a selected area and examine it with oil
objective. Note the distribution and morphology of the palettes.
4. Move to the edge of the smear and start the count, moving across the blood
smear counting each WBC as it is encountered.
5. Count and classify at least 100 WBC.

Results

1. Familiarize yourself with the appearance of different leucocytes. Find pictures online
and place them in the spaces provided below.

1. Neutrophil
Count (%) - ____________
 2. Basophil
Count (%) - ____________

3. Eosinophil
Count (%) - ____________

4. Lymphocyte
Count (%) - ____________
 5. Monocyte
Count (%) - ____________

Discussion
1. Is it important to differentiate absolute from relative leukocyte count? Why?
It's critical to understand the difference between absolute and relative leukocyte counts
since they provide different insights into an individual's white blood cell counts. The actual
quantity of white blood cells in a person's blood is measured by the absolute leukocyte
count, which is essential for assessing the severity of an infection, inflammation, or other
medical issues. Conversely, the percentages of various white blood cell types in relation
to the total white blood cell count are expressed by the relative leukocyte count. This can
aid in the diagnosis of some medical diseases and provide information about the
particular immune response. As a result, leukocyte counts that are both absolute and
relative are useful and offer complementary data that can support medical diagnosis and
treatment.

2. What will be the effect of inflammation, parasitism, and other disease conditions
on the different types of WBCs?
Neutrophils are the most abundant type of WBCs and are often the first to respond to
inflammation. Inflammatory conditions can cause an increase in the number of
neutrophils in the blood. Neutrophils are responsible for phagocytosis, the process of
engulfing and destroying pathogens. In parasitic infections, neutrophils may be less
involved compared to other WBCs. Eosinophils are involved in allergic reactions and
defense against parasitic infections. In inflammatory conditions, the number of
eosinophils may increase. In parasitic infections, eosinophils play a crucial role in
combating the parasites. They release toxic substances to kill the parasites. Basophils
are involved in allergic reactions and release histamine, which contributes to
inflammation. Inflammatory conditions can lead to an increase in basophil numbers.
However, their specific role in parasitic infections is not well-defined. Lymphocytes are
a key component of the immune system. There are different types of lymphocytes,
including T cells, B cells, and natural killer (NK) cells. Inflammation can stimulate the
production and activation of lymphocytes. T cells play a crucial role in orchestrating the
immune response, while B cells produce antibodies. NK cells are involved in the
destruction of infected and cancerous cells. The specific effect on lymphocytes may vary
depending on the disease condition. Monocytes are precursors to macrophages, which
are phagocytic cells involved in engulfing and destroying pathogens. Inflammatory
conditions can lead to an increase in monocyte numbers. Monocytes migrate to the site
of inflammation and differentiate into macrophages to eliminate pathogens.

References
https://flebo.in/health/neutrophils-count-what-is-it-diagnosis-treatment/
https://stock.adobe.com/ph/search?k=eosinophil
https://stock.adobe.com/images/lymphocyte-cells-white-blood-cell-in-blood-smear-
analyze-by-microscope/199157149
https://www.britannica.com/science/neutrophil
https://en.wikipedia.org/wiki/Basophil
https://novaappai.page.link/TDZmq26bwuFJbBoT9
https://quillbot.com/paraphrasing-tool