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DIFFERENTIAL

LEUKOCYTE COUNT
Group II
Abdullah, Jenny Rose M.
Agleham, Trisha Lyn G.
Becyagen, May Anne M.
Camacho, Khriz Mere Marikit R.
Hipolito, Mac Vince A.
Liban, Jana Marie M.
Orata, Weena D.
Date Performed:
September 20, 2019

Teodoro James B. Mallabo, RN, MSN, MD


Professor
INTRODUCTION
The leukocytes, also called white blood cells are a
central part of the immune system. They help to
protect the body against foreign substances,
microbes, and infectious diseases. These cells are
produced or stored in various locations throughout
the body, including the thymus, spleen, lymph nodes,
and bone marrow.
Leukocytes are subclassified into two general groups
– granulocytes and agranulocytes. Granulocytes
include neutrophils, eosinophils, and basophils.
Agranulocytes include lymphocytes and monocytes.
INTRODUCTION

Neutrophils are the most common granulocyte as


well as the most numerous among the WBCs. They
measure 10 to 12 um in diameter in blood smears
and are obviously larger than erythrocytes. They are
identified by their multilobal nucleus and fine, light,
violet-colored granules.
INTRODUCTION

Eosinophils are about the same size as neutrophils


and their nuclei are typically bilobed connected by a
chromatin strand. Its cytoplasm is course orange to
brick red granules. They are usually associated with
allergic reactions, parasitic infections and chronic
inflammation.
INTRODUCTION

Basophils are about the same size as neutrophils and


are so named because the numerous large granules
in their cytoplasm stain with basic dyes. Its nucleus is
irregular, may be bilobed or S shaped. These cells are
the least numerous accounting for less than 0.5% of
total leukocytes.
INTRODUCTION

Agranular leukocytes include lymphocytes and


monocytes.
Lymphocytes are the main functional cells of the
lymphatic or immune system. There are two main
types of lymphocytes: B cells and T cells.
Monocytes are the precursors of the cells of the
mononuclear phagocytotic system. Monocytes have
large, central, kidney-shaped nucleus and it is known
to be the largest type of WBC.
INTRODUCTION

Blood differential test measures the amount of


each type of WBC in the body. It is used to monitor
the overall health, to keep track of an existing blood
disorder or related condition, and to diagnose a
variety of medical condition. These may include
infections, autoimmune diseases, leukemia and
other types of cancer.
INTRODUCTION

Blood differential test measures the amount of


each type of WBC in the body. It is used to monitor
the overall health, to keep track of an existing blood
disorder or related condition, and to diagnose a
variety of medical condition. These may include
infections, autoimmune diseases, leukemia and
other types of cancer.
INTRODUCTION
Normal count of each type of leukocytes:
Neutrophils: 50-70%
Eosinophils: 1-4%
Basophils: 0-1%
Lymphocytes: 20-40%
Monocytes: 2-8%

The normal count may vary in response to some


conditions such as neutrophilia, neutropenia,
eosinophilia, eosinopenia, basophilia, basopenia,
monocytosis, monocytopenia, lymphocytosis, and
lymphocytopenia.
II. MATERIALS
III. PROCEDURES
PREPARATION OF BLOOD FILM
Glass slides
1. Clean finger and prick with sterile lancet. Discard the first drop of
blood.
2. A clean glass slide is touched to the newly formed drop of blood
1 cm away from the edge.
3. The slide is placed on the table so that the blood drop is on the
right side. It is supported with the left hand and the second slide
(spreader) is held along its long edges with the right hand at an
angle of 45.
4. The smooth narrow edge of the spreader slide is placed in front
of the blood dropped drawn back to touch the drop which then
spreads along the edge of the spreader slide evenly.
5. The spreader slide is pushed towards the left by a quick uniform
motion with a light but even pressure. The blood follows along
the spreader slide to form a blood film.
It is dried quickly by waving in the air.
III. PROCEDURES
III. PROCEDURES
STAINING THE BLOOD FILM
1. The blood smear is placed on the staining rack. Leishman‘s stain
is added drop by drop till the entire film is covered by the stain.
2. The number of drops added is counted (usually8-10drops).
3. The undiluted stain is allowed to act for 2 minutes. It should be
allowed to dry up. The cells are fixed within 2 minutes.
4.Them, double the number of distilled water drop is added to
dilute the stain. It is mixed gently by blowing through a pipette.
5. After 7 minutes, the stain is drained off.
6. The slide is washed in a gentle stream of running tap water until
the film turns pink.
7. The slide is kept in a vertical position to drain and dry.
8. Thick uneven smears should be discarded. It is preferable to
make 3-4 smears.
9. The best stained film should be selected for microscopic
examination.
III. PROCEDURES

*A good smear should be:


1. Be buff colored.
2. Be uniform.
3. Be broader at the head and taper off into a trail.
4. Occupy the middle-third of the slide leaving a
margin of about 5mm along the edges.
5. Have no longitudinal or transverse striations or
windows.
6. Have no stained granules or precipitates.
7. Have discrete red cells without overlapping each
other.
III. PROCEDURES
MICROSCOPIC EXAMINATION
1. The microscope is adjusted for oil immersion lens.
2. The condenser is raised, the diaphragm is completely opened,
and the plane mirror is used.
3. Two drops of cedar wood oil are placed near the head end.
4. The oil immersion objective lens is made to touch the oil by
viewing from the side.
5.The fine adjustment screw is adjusted till the cells are brought
into focus
6. The slide is moved towards the tail end and the cells are counted.
7. The slide is then shifted up and moved in the opposite direction.
This pattern of movement of the side (zigzag pattern) takes into
consideration all the parts of the film and ensures that a cell is
not counted more than once.
Hundred white cells are identified and entered. The number of
each type of WBC is counted and expressed in percentage.
III. RESULTS AND DISCUSSIONS
The percentage of the different types of white blood cells is called
the differential count. It is done to find out if there is an increase or
decrease in a particular type of WBC.
III. RESULTS AND DISCUSSIONS
III. RESULTS AND DISCUSSIONS
• Normal values (according to Chandrasekar & Mishra, 2014):
Neutrophils: 50-70%
Eosinophils: 1-4%
Basophils: 0-1%
Lymphocytes: 20-40%
Monocytes: 2-8%

• Table 1 reveals that all members had their leukocyte


differential count within or close to the normal reference
ranges. It shows the different quantities of the various white
blood cell types per one hundred white blood cells. Different
laboratories provide different normal ranges for blood tests
so it is appropriate to assert that a 6% eosinophil count
similar to May‘s is still a normal value.
III. RESULTS AND DISCUSSIONS

• Depending on the type of cell, high or low levels can


indicate different health issues.

• Neutrophils, which are responsible for destroying


bacteria in injured or infected tissue, when present in
high amounts indicates an acute bacterial infection,
inflammation, tissue death, stress on the body, or
chronic leukemia. On the other hand, if they are present
in low amounts it indicates the presence of illnesses
such as myelodysplastic syndrome, autoimmune
disorders, bone marrow cancers, and aplastic anemia.
III. RESULTS AND DISCUSSIONS
• Eosinophils primarily serve as our defenders in the
presence of infections caused by parasites. When these
parasite-fighting cells grow in number higher than
normal, The patient must be suffering from an allergic
reaction, Inflammatory disorders, or inflammatory
bowel disease. Low eosinophil count does not usually
indicate any issue.
• Basophils, which play a role in allergic reactions, are the
least common type of leukocyte. When present in high
quantities, it could be a result of certain types of
leukemia including chronic myeloid leukemia. severe
allergy reactions are also the cause of high basophil
count. Low basophil count, on the other hand, does not
typically suggest a medical condition.
III. RESULTS AND DISCUSSIONS

• Lymphocytes have a wide range of functions. these


include the production of antibodies that attack specific
viruses, bacteria, and foreign invaders. They also help
identify cells that require an immune response and
destroy cancer cells and viruses. When lymphocyte
count is high it can indicate an acute viral infection such
as chicken pox, herpes, or hepatitis or it may be due to
bacterial infection and conditions like lymphocytic
leukemia or lymphoma. A low lymphocyte level can
point to an autoimmune disorder like lupus or
rheumatoid arthritis.
III. RESULTS AND DISCUSSIONS
• Lastly, monocytes which also defend the body from
bacterial infection and help repair damaged tissues
must be present in the body in low amounts. Too high
monocyte count can result from a chronic infection or
the presence of conditions like monocytic leukemia,
juvenile myelomonocytic leukemia, or rheumatoid
arthritis. However if monocytes are present in numbers
that are too low, this may indicate bone marrow
damage.

• Based on the above-data, no one among the 7 members


of the group tested too high or too low in their
leukocyte differential counts relative to their WBC
Counts.
• Differential Count help determine the cause of
abnormal results on a white blood cell (WBC) count; to
help diagnose and/or monitor an illness affecting your
immune system, such as an infection or inflammatory
CONCLUSION

condition, or cancers that affect your white blood cells,


such as leukemia.
• This determines the percentage of each type of WBC
present in the blood.
• Leishman stain is commonly used when there is need to
examine the Blood smear for the various blood cells,
Differential Leukocyte count, Type of Anemia, Toxic
Granules & Platelet count etc.
• The leukocyte differential counts of the seven members
of the group are well within the normal range indicating
that they have no health/medical issues.
QUESTIONS
1. What is the purpose of methyl alcohol in
Leishman’s stain?
The purpose of methyl alcohol is fixative and must be free
from acetone and water. It serves two functions: It fixes the
blood smear to the glass slide. The alcohol precipitates the
plasma proteins, which then act as a “glue” which attaches
(fixes) the blood cells to the slide so that they are not
washed away during staining and the alcohol preserves the
morphology and chemical status of the cells.
QUESTIONS
2. What is the pH of the buffered water that is
used?
Neutral distilled water or buffered distilled water of pH
6.8 was for diluting the stain. Buffered distilled water tends
to keep original pattern on addition of small amount of
alkali or acid.
3. Give the normal
differential count. What
QUESTIONS

are the physiologic


variations?
NORMAL COUNT PHYSIOLOGIC VARIATIONS
-Neutrophils 50-70% -Exercise
-Eosinophils 1-4% -Stress
-Basophils 0-1% -After meals
-Lymphocytes 20-40% -Pregnancy & parturition
-Monocytes 2-8%
4. Explain the terms (a) neutrophilia and (b) neutropenia
and list conditions where it occurs.
QUESTIONS
Neutrophilia-Increase in the number of circulating neutrophils
caused by bacterial infections and products of inflammation that
enter the bloodstream
Conditions resulting to neutrophilia:
1. Acute pyogenic infection such as tonsillitis, appendicitis, pneumonia
2. Tissue necrosis as in Myocardial Infarction(MI)
3. Following Hemorrhage
4. Trauma, postoperative burns
5. Hemolysis
6. Metabolic disorders like gout, diabetic acidosis, uremia
7. Drugs such as glucocorticoids, adrenaline, digitalis, phenaticin
8. Poisoning with lead, mercury, insect venom
9. Physiological: Exercise, stress, after meals, pregnancy, and parturition
Neutropenia- decrease in the number of circulating
QUESTIONS

neutrophils primarily caused by infections or other


underlying factors

Conditions resulting to neutropenia:


1. Typhoid and Paratyphoid fever, kala-azar(Visceral
leishmaniasis)
2. Viral Infection
3. Depression of bone marrow due to irradiation
4. Drugs such as chloramphenicol
5. Autoimmune diseases
5. What are the terms which refer to (a) increase and (b)
QUESTIONS

decrease in the lymphocyte count?

Lymphocytosis- Increase in the number of lymphocytes


Lymphocytopenia-Decrease in the number of
lymphocytes
6. Name two conditions in which each occurs?
QUESTIONS

Lymphocytosis
1. Whooping cough, diphtheria
2. Chronic infections like TB, Syphilis and malaria

Lymphocytopenia
1. Steroid Therapy
2. Acute infections and illnesses
3. Bone marrow failure
7. What is monocytosis? When does it occur?
Monocytosis is an increase in the number of monocytes
circulating in the blood.It is not a representative of a specific
QUESTIONS

rather it is often a marker of chronic inflammation, either as a


result of infection, auto immune disease, and blood born
malignancy
Conditions resulting to monocytosis:
1. Chronic Infection like TB, Syphilis, sub-acute bacterial
endocarditis, brucellosis
2. Protozoal infections like malaria, kala azar(visceral
leishmaniasis)
3. Infectious mononucleosis
4. Monocytic leukemia and multiple myeloma
5. Collagen diseases
6. Granulomatous diseases like sarcoidosis, ulcerative colitis
8. What is the composition of Leishman’s stain? What are
the functions of each component?
QUESTIONS

Eosin- An acidic dye which stains the basic protoplasmic


materials(acidophilic material).
-It stains red/pink

Methylene blue- A basic dye, which stains the acidic


nuclear chromatin(basohilic material).
-It stains blue.

Acetone-free methyl alcohol-To fix the smear to the slide


9. Name some conditions where eosinophils and
neutrophils increase in number.
In underdeveloped areas of the world, increased peripheral
QUESTIONS
blood eosinophils are seen in patients with parasite infestation,
especially helminthes and protozoa. A major function of eosinophils
is degranulation, where substances are released that damages an
offending organism (i.e., parasites) or target cell. In developed
countries eosinophilia is most often associated with allergic
conditions, including asthma, hay fever, urticarial, and atopic
dermatitis. Eosinophilia is also seen in scarlet fever, HIV, fungal
infections, autoimmune disorders, and hypersensitivity to antibiotics
and antiseizure medications.
An increase in neutrophils can be related to several factors, this
can occur from strenuous exercise, emotional stress, shock, burns,
trauma, labor, or an increase in epinephrine. Neutrophilia also
occurs in conditions that result in an increase in bone marrow
production.
10. Which WBC has got the phagocytic function?
QUESTIONS

Neutrophils,Monocytes/Macrophage
11. What is the function of lymphocytes in the body?
QUESTIONS

Lymphocytes. They play a fundamental role in immune


responses of the body.
Types of Lymphocytes. There are 3 primary lymphocyte
populations:
a. T- lymphocytes
b. B-lymphocytes
c. Third party or nil (non-T, non-B) lymphocytes. The
function of 3rd party cells is not clear, and unlike T
and B cells, they do not contain antigen receptors in
their plasma membranes.
• Innate or Inborn Immunity. This immunity is present
from birth and does not require first exposure to foreign
QUESTIONS
agents (antigens). The cells involved include: neutrophils,
macrophages, natural killer (NK) cells, and large
lymphocytes that are cytotoxic.

• Acquired Immunity. (“My”body contains a huge variety


of chemical substances, especially proteins, which are
part of me and constitute my “self”‖ material. Material
which is not part of me, such as another person‘s cells, or
microbes, etc. which invade the body, is “nonself‖
material”).
• The key to acquired immunity is the inborn ability (without
first exposure) of lymphocytes to recognize a billion or more
QUESTIONS
nonself antigens. Before T cells and B cells leave thymus and
bone marrow, they start to form a number of distinct proteins
that are inserted into their plasma membranes. Many of them
function as antigen receptors (“T cell markers” or “T cell
receptors”, TCRs) in T cells and “surface antibody receptors”
in B cells. In addition to antigen receptors, they also contain
other markers, which have been given CD (clusters of
differentiation) numbers. Most cytotoxic T cells have
glycoprotein CD8 and helper T cells have glycoprotein CD4.
These proteins are closely associated with T cell receptors and
may function as coreceptors.
Types of Acquired Immunity. There are 2 types of acquired
immunity: humoral and cellular.
QUESTIONS

a. Humoral Immunity. It is mediated by circulating


antibodies (immunoglobulins) that circulate in the
gamma globulin fraction of plasma proteins. Activated B
cells develop into plasma cells and each cell can secrete
up to 2000 specific antibody molecules. Humoral
immunity is the major defense against the common
bacteria.
b. Cellular (“cell mediated”) Immunity. There are 4 main
types of T cells that take part in this type of immunity:
QUESTIONS

helper/inducer (T4 cells), suppressor (Ts or T8) cells,


cytotoxic (Tc, killer cells), and memory T cells. The cellular
immunity is the major defense against viruses, fungi,
some cancer cells, and foreign cells/tissues.
After an antigen (e.g. a microbe) is phagocytosed by
tissue macrophages and other antigen presenting cells
(APCs, such as dendritic and Langerhan cells of the skin)
it is processed. Some of the partly digested products are
presented to T and B cells which get activated.
12. What are the special features of monocytes? How will
you differentiate monocytes from large lymphocytes?
QUESTIONS

Monocytes, (the monocyte-macrophage system, MMS;


formerly called reticuloendothelial system, RES).
The monocytes, after spending a day or two in the
circulation (here they are immature), enter the tissue where
they increase in size and become actively-phagocytic tissue
macrophages. They guard all the possible points of entry of
foreign invaders. They become activated by T lymphocytes.
The activated tissue macrophages migrate in response to
chemotactic stimuli and engulf and kill the bacteria by
QUESTIONS
processes similar to those seen in neutrophils. Over 100
chemicals are secreted by these cells, including most of the
bactericidal agents described for neutrophils. They also
contain lipases which dissolve the lipid coating of bacteria like
tuberculosis and leprosy. Each macrophage can kill up to 100
bacteria before it itself is killed.
The macrophages also play an important role in
immunity. They pass (present) the partly-digested antigens of
the organisms directly to T and B cells, thus activating them to
perform their specific functions in immunity
Monocyte and large lymphocyte. The monocyte, which
is the largest of the blood cells (12–20 μm) and 2–2½ times
QUESTIONS
bigger than a red cell, can be identified by its pale-staining,
oval, kidney or horseshoe-shaped nucleus, which is usually
eccentric. The difficulty in differentiating it from a large
lymphocyte may arise when the cell is seen from the side
when it will appear oval or round. (The kidney shape of the
nucleus cannot appear so from all directions). Then the much
larger amount of pale-blue cytoplasm (about 1–2 times the
size of the nucleus), as compared to a large lymphocyte
(where it forms only a rim or crescent), and its frosty nature
helps to identify it.
13. How do you differentiate a bilobed eosinophil from a
neutrophil?
QUESTIONS

Eosinophil has bilobed nucleus connected by a


chromatin strand(Spectacle shaped) and cytoplasm with
course orange to brick red granules.
Neutrophil, on the other hand, has 2-6 lobes
nucleus and connected by chromatin and cytoplasm with
fine light violet colored granules
14. What are microscopic adjustments you will do before
starting the experiment?
QUESTIONS

Mount the dried smear on the stage of microscope


first under low power objective (LPO). The microscope is
adjusted for oil immersion lens. The condenser is raised,
the diaphragm is completely opened. Two drops of cedar
wood oil are placed near the head end. The OIO lens is
made touch with the oil by viewing from the side. The
fine adjustment screw is adjusted till the cells are
brought into focus.
15. Why methyl alcohol should be acetone free?
QUESTIONS

The alcohol must be free from acetone because


acetone being a very strong lipid solvent, will, if present,
cause crenation, shrinkage, or even destruction of cell
membranes. This will make the identification of the cells
difficult. (If acetone is present, the stain deteriorates
quickly).
THANK YOU FOR YOUR
ATTENTION.