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EXPERIMENT TOTAL LEUKOCYTE

NUMBER 8 COUNT
ARUGAY BAYONGASAN CAUILAN JOSE LIZARDO PAGADDU
TAGUINOD
INTRODUCTION
White blood cells or leukocytes -- spherical, motile, and
nucleated cells of varying sizes

Two types according to the presence of granules:


1.Agranular : Lymphocytes and the Monocytes
2.Granular WBCs : Basophils, Neutrophils, and Eosinophils.
INTRODUCTION

Total leukocyte count -- measures the total number of


leukocytes in a given volume of blood.

Normal leukocytes count : 4000-11000 cells/cu mm.


APPARATUS
Microscope
Hemocytometer
WBC pipette
Cover slip
Disposable lancet
Cotton and spirit
Turks fluid
PROCDURE
1. Filling the pipette and diluting the blood
sample:

The finger tip was cleaned with spirit and then


pricked with a sterile lancet(the first drop of blood
was discarded)

Blood was drawn up to the 0.5 in the WBC Pipette


then Turks fluid was sucked up to mark 11.

The blood sample was mixed with the diluting


fluid thoroughly by rolling the pipette into the
palm horizontally.
2. Charging the Chamber

The cover slip was placed over the chamber and the
chamber was placed on the microscope stage, and
the pipette was rolled again before charging. The
first 2-3drops were discarded.

A moderate sized drop was allowed to form at tip


of pipette and the tip was made to touch the slide
and coverslip. The drop was made to run into the
capillary space and was not made to run into the
trenches.

The fluid was then allowed to settle for 2-3


minutes
3. Counting the Cells

The WBC area was first focused under low power


objective and it was made sure that there were no
air bubbles

The cells lying on the upper horizontal and left


vertical lines were counted along with those lying
within the square

The cells in 4 large corner squares were counted


and entered in the observation table
RESULTS & DISCUSSION
WBC count =
Number of WBCs counted x Correction for dilution x
Correction for volume

Number of white blood cells counted:


Sum of WBC counted in the four large squares of the counting chamber.
Square 1 = 25
Square 2 =31
Square 3 =30
Square 4 =24
TOTAL= 110
Correction for volume: 2.5 Correction factor for dilution: 20
Obtain the WBC as the number of white
Since the blood was initially diluted 1:20,
blood cells in 1ul of blood.
the correction factor for dilution is 20.
Therefore if the cells are counted in four
large squares, the total volume counted is
4(1.0 x 1.0 x 0.1) or 0.4 ul.

To obtain the volume of 0.1ul, Therefore,


0.4 is multiplied by 2.5(1.4/0.4) the WBC/ul= 110 x 2.5 x 20
correction factor for volume then is 2.5. =5.5 x 103 /ul or 5.5 x 109 /L
Normal Range : 4.3-10.8 x 103/ ul

The procedures were properly executed and it was made sure


that the blood sample was not over or under diluted. The patient can
be assumed to be normal since the obtained WBC count which 5.5 x
103 /ul, is within the normal range.
ANSWERS TO QUESTIONS
1. Is this a routine test? What is the usefulness of this test?

Total leukocyte count (TLC) is a routine test of CBC.

As it can serve as part of a routine health examination, this test can


specifically help diagnose infection or inflammatory processes. This
differentiates acute and chronic infection.

It may be used to determine presence of other diseases that affect WBCs such
as allergies, leukemia or immune disorders. It is also done as a follow up test
on patients on chemotherapy or to monitor bone marrow function.
2. What other tests has to be done with total count to make
it more meaningful?

CBC includes all kinds of cell count especially with RBCs, WBCs and
platelets. Along with total leukocyte count, other tests come with to
make sense of some cases.

The closest one is called the differential leukocyte count (DLC) which
accounts for the individual amount of major types of WBC. Tests like
RBC, PCV, Hgb, red blood cell indices, thrombocyte, and mean platelet
volume count may be added with total leukocyte count as components
of a complete blood count (CBC).
3. Why should you destroy the RBCs while doing WBC count?

The destruction of RBC will allow a better examination under the


microscope.

The RBCs are hemolyzed by a glacial acetic acid present in Turks fluid while
it does not affect WBCs. A dye stains the nuclei of leukocytes to be able to
recognize the minute WBCs.
4. What is the normal range of total WBC count? List three
important physiological variations?

The reference range for male and female adults is:


4.1-10.9 x 103/ L
Leukopenia - A decreased WBC count
- WBCs are depleted due to infection, hematopoietic stem cell
abnormality (leukemia), chemotherapeutic or radiation therapy, and collagen-
vascular diseases (such as lupus erythematosus).
Leukocytosis - An elevated WBC count
- seen in response to infection, stress inflammatory disorders
(reactive leukocytosis) or abnormal production. It may be due to anemia, infectious
and inflammatory diseases, leukemia, severe emotional or physical stress and
tissue damage.
- Differential leukocyte count is as follows:
Neutrophils: 2.5-7.5 x 10 9/L
Lymphocytes: 1.5-3.5 x 10 9/L
Monocytes: 0.2-0.8 x 10 9/L
Eosinophils: 0.04-0.4 x 10 9/L
Basophils: 0.01-0.1 x 10 9/l
Neutrophilic leukocytosis is caused by acute bacterial infection and tissue necrosis due to
myocardial infarction, burns and crush injuries.
Eosinophilic leukocytosis is caused by allergy, parasitic infections and drug interactions.
Monocytosis is caused by chronic infections, malaria, bacterial endocarditis and
inflammatory bowel disease. Lastly, lymphocytosis is caused by viral infections (Hep A,
cytomegalovirus, Epstein-Barr virus) and may also accompany monocytosis.
- Biological reference for TLC and DLC depends on factors like age, sex, sample population,
and method of testing.

- Physiological variations can also be called as diurnal variations that affect WBC count:
1. A pregnant woman in labor at the last month of pregnancy may have increased WBC levels.
2. Smoking may also cause increased WBC count.
3. Newborns with normal health status have higher WBC count than adults.
4. Cases such as morning pseudoneutropenia wherein WBC count tends to be lower in the
morning and higher in the late afternoon. This is noticed in some patients who are taking
antipsychotic medications.
EXPERIMENT DIFFERENTIAL COUNT
NUMBER 9 OF WBCs
ARUGAY BAYONGASAN CAUILAN JOSE LIZARDO PAGADDU
TAGUINOD
INTRODUCTION
Differential Count of RBCs - white blood cell types are
separately counted from each other manually in the Neubauer
chamber

-identification of cells involves the observation of the difference


in cell size, characteristics of nucleus, and cytoplasm (granules)
INTRODUCTION
Normal Count:

Neutrophils - 50-70%
Eosinophils - 1-4%
Basophils - 0-1%
Lymphocytes - 20-40%
Monocytes - 2-8%
APPARATUS
Microscope
Clean dry grease free glass slides with even edges
Cedar wood oil
Staining rack
Lancet, cotton and spirit
A drop battle containing distilled water
A drop battle containing Leishmans stain
Eosin
Methylene blue
Acetone-free methyl alcohol
PROCEDURE
Preparation of blood film

1. The finger was cleaned and pricked with sterile


lancet (the first drop of blood was discarded)
2. The newly formed drop of blood was touched
to the glass slide and then spread using
another slide.
3. The spreader slide was pushed towards the left
by a quick uniform motion with a light but
even pressure.
4. The blood followed along the spreader and
formed a blood film then was made to dry.
Staining the Blood

1. The blood smear was placed on the staining rack then Leishmans stain
was added drop by drop until the entire film was covered. The number of
drops added were counted.
2. The undiluted stain was allowed to act for 2 minutes but wasnt allowed to
dry up. The cells were fixed during these 2 minutes.
3. Double the number of distilled water drops were added to dilute the stain
and then it was mixed gently.
4. The stain was not allowed to dry and after 7
minutes the stain was drained off.
5. The slide was washed in running tap water until
the film turned pink and was kept in vertical
position to drain and dry.

6. The uneven smears were discarded and the best


stained film was selected for microscopic
examination.
3. Microscopic examination

The microscope was adjusted for oil


immersion lens. The condenser was raised,
the diaphragm was completely opened and
the plane mirror was used.

Two drops of cedar wood oil were placed


near the head end. The oil immersion
objective lens was made to touch the oil by
viewing from the side. The fine adjustment
screw was adjusted till the cells are brought
into focus.
Hundred squares were drawn for recording the cell
count. The WBC were identified and entered using the
letter N for neutrophil, E for eosinophil, B for
basophil, L for lymphocyte and M for monocyte.

The slide is slowly moved towards the tail end and the
cells are counted. The slides is then shifted up and
moved in opposite direction. This pattern of movement
of slides (zigzag pattern) takes into consideration all
the parts of the film and ensures that a cell is not
counted more than once.

Hundred of cells are identified and entered. The


number of each type of WBC is counted and expressed
in percentage
RESULTS & DISCUSSION
Differential Count of WBCs
Granulocyte Normal value Differential counted

Neutrophil 50-70 % 44 %

Eosinophil 1-4 % 4%

Basophils 0-1 % 0%

Agranulocytes

Lymphocytes 20-40 % 51 %

Monocyte 2-8 % 1 %
The experiment was done by identifying each white blood cell seen and
was recorded until 100 WBCs were counted.
When reporting a manual differential count, the results are reported as
the percentage of each cell type present as seen in the table above.
The patient had a slightly decreased Neutrophil count and a slight
increase in Lymphocyte count.
These results can be correlated to the condition of the patient, since she
is currently having cough and colds, the neutrophils will then slightly
decrease and the lymphocytes will increase due to viral infections. The other
cell types had a normal result.
Other factors can be considered, like for the stain used, the
performers increased the contact time between the stain and the smear.
Since the stain used seems to be contaminated or old, this also gave a
hard time for the performers to identify the cells and they also counted
not only in the thin portion near the feathery edge, but continued to
count in the thick portion of the smear. Nevertheless, they were able to
count 100 WBCs.
ANSWERS TO QUESTIONS
1. What is the purpose of methyl alcohol in Leishmans stain?

It is used to fix the smear to the slide

2. What is the pH of the buffered water that is used?


The pH is 6.8
3. Give the normal differential count. What are the physiological
variations?
- Differential count is as follows:
Neutrophils: 50-70%
Eosinophils: 1-4%
Basophils: 0-1%
Lymphocytes: 20-40%
Monocytes: 2-8%
4. Explain the term (a) neutrophilia and (b) neutropenia and list conditions
where it occurs.

Neutrophilia Increase in the number of Neutrophils


Acute pyogenic infection i.e. Tonsillitis, appendicitis, pneumonia
Tissue necrosis
Following Hemorrhage
Trauma, Postoperative burns
Hemolysis
Malignant Neoplasm
Metabolic Disorder i.e. Gout, Diabetic Acidosis, Uremia
Drugs
Poisoning i.e. Lead, Mercury, Venom
Neutropenia Decrease in the number of Neutrophils
Typhoid and Paratyphoid fever, kala-azar
Viral Infection
Depression of bone marrow due to irradiation
Drugs i.e. chloramphenicol
Autoimmune Disease

5. What are the terms which refer to (a) increase and (b) decrease in the
lymphocyte count?
Lymphocytosis Increase in the number of lymphocyte
Lymphocytopenia Decrease in the number of the lymphocytes
6. Name two conditions in which each occur

a. Lymphocytosis
i. Chronic Infection i.e. TB, Syphilis and
malaria
ii.Viral Infection
b. Lymphocytopenia
i. Acute infection and illnesses
ii.Hodgkins disease
7. What is Monocytosis? When does it occur?

a. Monocytosis increase in the number of


monocytes
i. Chronic infection i.e. tuberculosis,
syphilis, subacute bacterial endocarditis,
brucellosis
ii.Protozoan infection i.e. malaria, kala-
azar
iii.Infectious mononucleosis
iv.Monocytic leukemia and Multiple
Myeloma
v.Collagen Disorder
vi.Granulomatous diseases i.e. sarcoidosis,
ulcerative colitis
8. What is the composition of Leishmans stain? What are the functions of
each components?
The stain is prepared by dissolving 750mg of Leishmans powder in 500ml of
acetone-free methyl alcohol
Components:
i. Eosin Acidic Dye, which stains the basic protoplasmic material
ii.Methylene Blue Basic Dye, which stains the acidic nuclear chromatic
iii.Acetone-free methyl alcohol To fix the smear in the slide;
9. Name some conditions where eosinophils
and neutrophils increase in number.
a. Neutrophilia
I. Acute pyogenic infection i.e. Tonsillitis,
appendicitis, pneumonia
II. Tissue necrosis
III. Following Hemorrhage
IV. Trauma, Postoperative burns
V. Hemolysis
VI. Malignant Neoplasm
VII. Metabolic Disorder i.e. Gout, Diabetic
Acidosis, Uremia
VIII.Drugs
IX. Poisoning i.e. Lead, Mercury, Venom
a. Eosinophilia
i. Allergic conditions i.e. Asthma
ii.Parasitic infection
iii.Skin disease i.e. Eczema
iv.Loefflers Syndrome - can be caused by idiopathic eosinophilia or
eosinophilia in response to parasitic infection
v.Chronic Myeloid Leukemia
vi.Hodgkins Disease
vii.Collagen Disorder
viii.Tropical Eosinophilia is a syndrome of wheezing, fever and eosinophilia
seen predominantly in the Indian subcontinent and other tropical areas. Its
etiological link with Wuchereria bancrofti and Brugia malayi has been well
established
10. Which WBC has got the phagocytic function?
- Basophil , Eosinophil, Neutrophil and Monocyte

11. What is the function of lymphocyte in the body?


- Lymphocytes are a type of white blood cell that are responsible for
initiating an immune response when a foreign invader enters the body.
These cells are primarily in the tonsils, lymph nodes and the spleen, but
they also circulate in the blood

12. What are the special features of monocyte? How will you
differentiate monocyte from large lymphocyte?
a. Monocyte
i. Nucleus Large, centrally located and kidney-shaped
ii.Cytoplasm Large amount of pale grayish-blue cytoplasm and
no granules seen
Monocyte versus Large Lymphocyte
i. Size
1.Lymphocyte - cell is twice as big as the Red Cells
2.Monocyte - cells is about 2.1/2 to 3 times
ii.Nucleus
1.Lymphocyte - completely fills the cells
2.Monocyte - big cell with a kidney shaped nucleus
iii.Cytoplasm
1.Lymphocyte - a bigger cell with a rim of cytoplasm all around
2.Monocyte - a big cell with a large amount of clear cytoplasm in relation to the
nucleus
13. How do you differentiate a bi-lobed eosinophil from a neutrophil?
They are differentiated through their granules
i. Neutrophil Fine neutral granules of Light Violet
color
ii.Eosinophil Coarse and Orange or Red color
14. What are the microscopic adjustment you will do before starting the
experiment?
a. The microscope should be adjusted for oil immersion lens
b. The condenser is raised
c. The diaphragm is completely opened and plane mirror is used
d. The oil immersion lens is made to touch the oil by viewing from the side.
e. The fine adjustment screw is adjusted till the cell are brought into focus.

15. Why should methyl alcohol be acetone free?

Acetone is a strong lipid solvent that tends to damage the


cell membrane, thus an acetone-free methyl alcohol is
used.
EXPERIMENT ABSOLUTE
NUMBER 10 EOSINOPHIL COUNT
ARUGAY BAYONGASAN CAUILAN JOSE LIZARDO PAGADDU
TAGUINOD
INTRODUCTION
Absolute Eosinophil Count -- is a blood test that measures the
number of WBCs called eosinophils

Eosinophils -- granulocyte WBCs with a bilobed nucleus and an


immune function specifically in combating parasites.

Normal AEC count : 40-440 cells/cu mm


APPARATUS
Hemocytometer
Microscope
Cotton and spirit
Sterile needle or lancet
Pilots fluid
PROCDURE
1. The finger tip was cleaned with spirit and then
pricked with a sterile lancet(the first drop of
blood was discarded)
2. Blood was drawn up to the 1 mark in the WBC
Pipette then immediately immersed into the
diluting fluid and was drawn until the 11
mark. It was mixed well by rolling the pipette
on the palm.
3. It was kept aside for 15 minutes for adequate
staining of the cells.
4. The fluid in the stem of the pipette was
discarded and the counting chamber was
charged.
5. The eosinophils were counted in the 9 squares
RESULTS & DISCUSSION
Absolute Eosinophil Count:
Number of cells counted x 100/9
= 8 x 100/9
= 88.8 cell/ mm3
Normal value: 40-440 cells /mm3
The procedures was followed and the eosinophils were counted immediately and the
results were recorded.
The patient had of 88.8 cell/ mm3, which is in the normal range(40-440 cells /mm3).
ANSWERS TO QUESTIONS
1. What is the normal total eosinophil count?

0.04-0.4 x 10 9/L (cells per microliter) or 40-440 cells/mm3.

2. Mention the functions of eosinophils.


Eosinophils are component of the innate immune system.
They have a variety of function which includes modulation of immediate hypersensitivity
reactions wherein they play a vital role in allergic inflammatory processes that include
asthma.
Second role of eosinophils is the defense against parasitic infections. With its granule
proteins, they increase resistance against parasites, particularly helminthes.
Lastly, they can be of defense against intracellular bacteria. They can be mediators of
hypersensitivity diseases caused by bacteria, virus, protozoa and pathogens.
3. Name the conditions in which there is an increase in
absolute eosinophil count.
Increased eosinophil count that is higher than normal are linked
to a variety of disorders. Some of these are:
a. Allergic disease
b. Parasite infection (helminthic)
c. Asthma
d. Autoimmune diseases
e. Dermatologic disorders (eczema)
f. Hay fever
g. Blood disorder (leukemia)
h. Side effects of medication
4. Name the hormone which causes a lowering of eosinophil
count.
CORTICOSTEROID
Eosinopenia which accounts for a low level eosinophil count can be
caused by drunkenness or a medical condition that causes the body to over
produce steroids. The overproduction of cortisol can cause depletion or retrain
of the immune system. Corticosteroids are anti-inflammatory drugs that
decrease hyper-sensitivity or hyper-responsiveness. It increases spontaneous
death of eosinophils.
5. What is the appearance of an eosinophil in a stained smear?
What do the granules contain?
Eosinophils are 1-17 m in diameter, larger than neutrophils. Their nucleus
usually has only two lobes. The cytoplasm appears filled with granules stained
with red or pink under eosin stain. These granules contain proteins that are
cytotoxic in nature and are capable of inducing tissue damage and dysfunction.

6. What is the significance of doing absolute eosinophil count?


An absolute eosinophil count is used to measure the eosinophils among other WBC
components that become active during allergic diseases, infections, inflammations and
other condition that warrants the presence of eosinophils. Absolute eosinophil count is
recommended after an abnormal result of differential leukocyte count (DLC). This test may
help diagnose a severity of an allergic reaction, early stages of Cushings disease, and
infection by parasite.
7. What is major basic protein? What is its role?
Major basic proteins are the cytotoxic and proinflammatory mediator found in
eosinophilic granules. It induces non-cytolytic histamine release from basophil and
is involved in anti-parasitic defense and immune hypersensitivity reactions.

8. Mention the composition of Dungers fluid.


- Dungers fluid contains the following:
a. Aqueous eosin 0.1 gram
b. Acetone 10 mL
c. Distilled water up to 100 mL
9. With a diagram, show the morphology of eosinphils.

MYELOBLAST PROMYELOCYTE EOSINOPHILIC MYELOCYTE

EOSINOPHILIC BAND CELL EOSINOPHILIC


METAMYELOCYTE
10. What is the dilution used in eosinophil count?
The dilution used for eosinophil count is Dungers fluid. It is composed of
aqueous eosin (0.01 gram), acetone (10 mL) and distilled water (up to 100 mL).

11. What is the chemotactic factor for eosinophil?


Eosinophil chemotactic factor (ECF), thought to be primarily associated with
basophils and mast cells, causes eosinophils to move towards inflamed tissues.
They are believed to prevent excess spread of local inflammation by detoxification
of inflammation inducing substances (released by basophils and mast cells) and
phagocytosis of allergen-antibody complexes.
12. How does eosinophil combat infestation by worms?
Eosinophil function in host protection against parasitic worms. These parasitic worms
are typically too big to be targeted by CD8 T cells so they are destroyed by
granulated leukocytes (neutrophil, eosinophil, mast cells) that degranulate and
secrete histamine. For eosinophils, they secrete secondary granule proteins (MBD,
EPO, ECP). Eosinophils phagocytose susceptible parasites while bigger and more
resistant parasites can survive the phagocytic activity.
13. Name the chemicals in the eosinophil granules.
Degranulation of eosinophil granules occurs during protection of body against parasitic
infestation, most often. These are called cytotoxic granule cationic proteins which are
capable of phagocytosis and induction of tissue damage. These are:
a. Eosinophil-derived neurotoxin (EDN)
- It is an attractant to immune cells and has cytotoxic properties that may be capable of
reducing activity of a single strand of RNA viruses
a. Eosinophil Peroxidase (EPX)
- Catalyzes formation of hypophalous acids (oxidizing agents) from hydrogen peroxide and
halide ions to mediate lysis of protozoa or parasitic worms
a. Eosinophil cationic protein (ECP)
- Related to inflammation and asthma
a. Major basic protein (MBP)
- involved in anti-parasitic defense mechanism and in immune hypersensitivity reactions
THANK YOU!

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