GRANULOCYTE AND AGRANULOCYTE IMMUNE CELLS

EXAMINATION

By :
Name : Nisrina Fairuz Salsabila
Student ID : B1B015038
Group : IV
Subgroup : 4
Assistant : Lovendo Ilham Widodo

PRACTICAL REPORT OF IMUNOBIOLOGY

MINISTRY OF RESEARCH TECHNOLOGY AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2017
 I. INTRODUCTION

1.1 Background

Leukocytes are blood cells containing nuclei, also called white blood
cells. In the human blood, normal leukocyte counts are found to average 5000-
9000 cells / mm3, when the number is more than 12000, this is called
leukocytosis, when less than 5000 is called leukopenia. Leucocytes can perform
amuboid movements and through leucocyte diapedesis process can leave the
capillaries by breaking through the endothelial cells and penetrating into the
connective tissue. Seen in a light microscope, the white blood cell has a specific
granule (granulocyte), which is alive in the form of a semi-liquid droplet, in its
cytoplasm and has a varied nucleus form, which has no granules, its
homogeneous cytoplasm with a round or renal form (Effendi, 2003).
According to Bevelander (1988), leukocyte function is as a system of
immunity or immunity, when the body infiltrated foreign objects such as bacteria
or viruses then by neutrophil cells or lymphocytes of foreign bodies will be in
phagocytosis where T cells will kill lymphocytes directly or form a lymphokin
Substances that strengthen the power of phagocytosis while B lymphocytes will
release antibodies that will destroy the foreign object.
Leukocyte cells consist of two categories: granulocytes and
agranulocytes. Granulocytes or also called polymorphonuclear are white blood
cells in which there are granules such as: eosinophils, basophils, neutrophils.
75% of leukocyte components are granulocyte cells and these cells are formed in
the spinal cord. Agranulocytes: are part of a white blood cell that has 1 lobe cell
and the cytoplasm does not have granules such as lymphocytes and monocytes
(Paulsen, 2000).

1.2 Purpose

The purpose of this practicum is to know the types of immune cells

granulocytes and agranulocytes and their function and shape and to know the
percentage of immune cells in various animals.
 II. MATERIALS AND METHODS

2.1 Materials

The materials that used in this practice are blood of chicken, blood of
fish, blood of mice, blood of human, Giemsa solution, methanol, aquades,
and emersi oil.
The tools that used in this practice are syringe injection, object glass,
and light microscope.

2.2 Methods

1. Blood of chicken was taken using an injection syringe on the wings of

the pectoral vein. Blood of mice was taken using an injection syringe at
the lateral vein portion of the tail. Fish blood is taken using an injection
syringe. Human blood is taken using an injection syringe in the median
cubital vein.
2. The tip of a clean glass object and free of fat dripped one drop of blood
one of the preparate.
3. Removed forward at the same time with the other glass object (chosen by
the edges flat) by forming a 45o angle.
4. Preparate are fixed with methanol for 5 minutes.
5. The remaining methanol is removed and the preparate is subjected to a
diluted solution of Giemsa. Note: the making of Giemsa solution is 1 ml
of giemsa dropped with 20 ml of aquadest.
6. Washed with running water and dried.
7. Examined with objective lens magnification 100 times using immersion
oil.
8. Types of leukocytes are examined in separate erythrocytes. Calculated in
100 leukocyte cells and the results are expressed in percent (%).
 III. RESULT AND DISCUSSION

3.1 Result

Table 3.1.1 The result of Granulocyte and Agranulocyte Immune Cells

Observation
%
Neutrophyl
Lymphocyte Monocyte Eosinophyl Basophyl
Rod Segment
Chicken 70% 25% - 5% - -
Fish 70% 10% - 10%/ 10% -
Human 15% 10% 5% 40% 5% 25%
Mice 80% 8% 8% - 4% -

 Calculation of group 4 (Calculation of Chicken’s Blood)

% Type of Cells : ∑ Calculated cells/the amount of cells x 100%
The Amount of Cells : 40
% Lymphocyte : 28/40 x 100% = 70%
% Monocyte : 10/40 x 100% = 25%
% Neutrophyl (Rod) : 0/40 x 100% = 0%
% Neutrophyl (Segment) : 2/40 x 100% = 5%
% Eosinophyl : 0/40 x 100% = 0%
% Basophyll : 0/40 x 100% = 0%
Figure 3.1.1 The Picture of Ten Viewing Field of Granulocyte and
Agranulocyte Immune Cells Observation
3.2 Discussion

Based on the result of practical activity of IV group, in chicken blood

preparate obtained percentage of lymphocyte cell equal to 70%, monocyte cell
equal to 25%, and neutrophil segment 5%. In mice blood preparate obtained the
number of percentage of lymphocyte cells by 80%, monocytes 8%, neutrophil
rods by 8% and eosinophils by 4%. In the human blood preparate obtained
percentage of lymphocyte cells 15%, monocytes 10%, neutrophil rod 5%, 40%
neutrophil segment, 5% eosinophils, and 25% basofil. In the preparate of fish
blood obtained percentage of lymphocytes by 70%, monocytes by 10%, 10%
neutrophil segment, and 10% eosinophils. The results obtained from practicum
performed by group IV showed that the value of the results of examination of
immune system cells in this lab when compared with the standard values there
are some quite significant differences that the average value in the lymphocytes
58.75%, which should amount 20-40% only. The mean value of 3.25% in stem
neutrophils. The average value on the neutrophil segment is 13.75%, which
should be 50-70%. The mean value of basophils with a standard value of 0-1%,
in practice is obtained 6.25%. Eosinophils are 1-3% standard, at 4.75%
practicum. The average monocyte in practice 13.25% while the standard value is
only 2-8%. This percentage difference is possible due to the inaccuracy of
praktikan in observing and counting immune system cells because the difference
of leukocyte cells with each other is not clearly visible. It could also be because
people who observed their blood cells in abnormal circumstances or are infected
with the disease so that the number of blood cells can be reduced or more
(Widmann, 1995).
The number of immune system cells in the blood is different from each other,
this is due to several factors such as the presence of the disease, allergy to a
certain substance, genetic factors, and age. Leukemia sufferers such as the
number of leukocytes in the blood will be very excessive and this will be very
dangerous. In addition, the number of eosinophils in people affected by allergies
will be more than the healthy people. A person with an infection has many
neutrophils to kill germs. This can be demonstrated by incubating the blood
smear that is not fixed with NBT. The activated neutrophils contain large black
formazan grains in the cytoplasm. A normal person of less than 10% neutrophils
contains formazan. Patients with an infection of the number of neutrophils in the
blood containing formazan can reach 45% (Widmann, 1995).
The immune system of a mammal consists of a specific immune system
(adaptive) and a nonspecific immune system (natural). Neutrophil, eosinophil,
basophil and monocyte cells are included in non-specific immune systems,
whereas lymphocyte cells belong to specific immune systems (Astuti et al.,
2009). Specific and non-specific immune systems interact in the face of
infection. Non-specific immune systems work quickly and are often required to
stimulate specific immune systems. Neutrophil cells play a role in the early
defense of non-specific immunity against bacterial infections. Eosinophils cells
play a role in response to parasitic and allergic diseases (Primawati & Khalid,
2015).
According to Bevelander (1988), the content of leukocytes in peripheral
blood is 2-5%. In contrast to the nucleus of a neutrophil, the nucleus of an
acidophyl usually consists of two intercellular lobes interconnected with
chromatin strands, except for the central region occupied by the cytosentrum, the
cytoplasm containing a large number of coarse granules in a spherical human
shape. Basophil cells are not seen in the observation because they are only seen
in the aftermath. Types of leucocyte cells according to Baratawidjaja (2004),
including:
A. Neutrophils
Neutrophil cells are part of the leukocytes that act as the front line in the
immune system, neutrophils will phagocytosis bacteria and dilute them with the
enzyme amino acid D oxidase in azurophilic granules. Mielo peroxidase present
in neutrophils binds to peroxides and halides acts on the molecular biosin of
bacterial cell walls and destroys them. Neutrophils are formed in the bone
marrow and are removed in the circulation, the number of leukocytes is 60
-70%. The midline neutrophil cell is about 12 μm, has one nucleus and consists
of 2-5 lobes. The cytoplasm is heavily filled by specific granules (0; 3-0,8μm)
close to the optical resolution limit, with the coloring of the giemsa appearing
purplish.
B. Eosinophils
Eosinophils are part of the immobilized cell leukocytes to phagocytosis
bacteria or foreign bodies that enter the body even though their movements are
not as fast as neutrophils. The number of eosinophils is slightly only 1-4% of
blood leukocytes, having a median line of 9 μm (slightly smaller than
neutrophils). Having a nucleus usually has two inlets, having an ovoid granule
with an acidophilic eosin that looks red, granules are lysosomes containing
acidic phosphatase, katepsin, ribonuclides, but do not contain lysosimes.
B. Basophils
Basophils account for 0-1% of blood leukocytes, 12 μm midline size, nucleus
one, large irregular choice of forms, generally S shape, larger granular cytoplasm
filled basophils, and often granules covering the core, blue irregularly shaped
granules.
C. Lymphocytes
Lymphocytes are spherical cells, midline 6-8 μm, amounts in leukocytes
about 20-30%. Normal nucleated cells are relatively large, rounded slightly
hollow on one side, solid core chromatin, very small cytoplasm, slightly
basophilic, containing azurophilic granules. Lymphocyte cells are formed in the
lymph nodes and bone marrow. It has no amuboid movement and can not
phagocytosis bacteria but lymphocyte cells play a role in forming antibodies to
boost immunity against infection. The increased number of lymphocytes in the
body is called lymphocytosis. The number of lymphocyte cells will decrease
with age, at birth about 5% but in the elderly the body's ability will be reduced in
producing lymphocytes so that the immune system will decrease as well.
 D. Monocyte
Monocytes are large leukocyte cells 3-8% of normal leukocytes, 9-10 μm in
diameter but in dry blood preparate diameters reaching 20 μm, or more. The core
is usually eccentric, the indentation in the shape of a horseshoe. Chromatin is
less dense, more fibrillar arrangement, azurophile granula, is the primary
lysosome, more but smaller. Monocytes are present in the blood, connective
tissue, and body cavities. The number of leukocytes per microliter of blood, in
normal adults is 4000-11000, birth time 15000-25000, and by the fourth day
down to 12000, at the age of 4 years according to normal amount. Quantitative
variation in white blood cells depends on age. Birth time, 4 years and at the age
of 14 -15 years a typical adult percentage is reached (Effendi, 2003).
IFN-γ plays an important role because: 1) potential activators of
mononuclear phagocyte cells, such as MAF (macrophages activating factors), 2)
increase the expression of MHC I or II molecules; 3) because they directly
differentiate against T cells and cells; 4) cell activation NK, neutrophils, and
endothelial vascular cells are therefore adeshi energies on the increase of CD4 +
T cells (Ismawati et al., 2015). Lymphocytes in humans account for 20-25% of
all white blood cells. Lymphocytes are relatively small in number and an
increase in size is the result of a large number of cytoplasm. The cytoplasm
usually contains some dispersed mitochondria and granules. According to
Underwood (1999), the normal leukocyte counts in men and women are:
Basophils (0 - 1%), eosinophils (1 - 3%), neutrophil stems (2 - 6%), neutrophil
segments (50-70%), lymphocytes (20-40%), and monocytes (2 - 8%). Diseases -
diseases associated with immune system disorders according Underwood (1999),
including:
A. Deficiency of B cells or T cells, ie the immune system fails to keep the body
from attacking bacteria or viruses
B. Autoimmune diseases, the immune system wrongly attacks the body's own
tissues that are no longer recognized and tolerated
C. Complex disease - immune, antigen complex - excessive antibodies and
resulting in lethal complement components that damage normal surrounding
cells
D. Allergies, the immune system inappropriately cause symptoms and damage
the body against allergens. Cell diagnostic diagnostic techniques can be
performed using blood smears, antigen detection, and PCR.
According to Ndao et al., (2004), the diagnostic technique of cell
examination that is mostly done is to use a blood smear with Giemsa solution.
The use of this blood smear is observed with a microscope. According Jawetz et
al., (1974) blood smear is one way of observing the material - matter in the
blood either solid or liquid material. Solid material consists of red blood cells,
white blood cells, and blood clots. Making a blood smear using Giemsa staining.
When staining preparates using Giemsa solution must be waited until dry first
washed with running water because if not yet dried but was washed then when
observed using a microscope then the blood will look clot. Blood type of blood
thin and thick blood preparate. The process of blood formation is called
hemopoiesis (or hematopoiesis), and it occurs within the hemopoietic tissues.
The shaped blood element can be divided into two groups according to where it
develops and differentiates in adults. Lymphocytes and monocytes mainly
develop in lymphoid tissue and are called lymphoid elements. Based on the
dualistic or difletic theory that monocytes and lymphocytes originate from a
single stem cell (lymphobas) and granular leukocytes and erythrocytes derived
from myeloblasts (Ganong, 1989).
 IV. CONCLUSION

Based on the result can be concluded that:

1. Immune cells are divided into agranulocyte cells and granulocytes. Agranulocyte
cells consist of monocyte and macrophage cells while granulocyte cells consist
of neutrophils, eosinophils and basophils.
2. Calculation results of immune system cells in the laboratory obtained the
following results: 0% Basofil, Eosinophils in chickens 2.6%, fish 5.88%, 72.5%,
Neutrophils 32.2%, 25%, an 23 %, Lymphocytes 52.2%, 88.23% (fish), 2.7%
(Humans), 65% (Mice) and Monocytes 13% (Chicken), 5.88% (Fish), and 12%
(Mice ).
3. The number of immune system cells in the blood is different from each other,
this is due to several factors such as the presence of the disease, allergic to a
certain material, genetic factors, and age.
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