HEMATOLOGY II

By :
Name : Devina Alifah
Student ID : B1B017010
Group : VII
Subgroup :2
Assistant : Siti Masrifah

PRACTICAL REPORT OF ANIMAL PHYSIOLOGY I

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO

2018
 I. INTRODUCTION

A. Background

Blood is a tissue whose blood cells do not stick together in the blood flow
system. Like most body cells, red blood cells have an internal concentration that is
maintained so that red blood cells can function optimally. In different external
environmental conditions blood cells will show cell responses in the form of
shrinkage or swelling. For this reason, the response of red blood cells can be
studied by placing blood in a hypotonic, isotonic, or hypertonic medium
(Anggraini, 2010).
According to Arifin et al (2013), blood is a special organ that is different
from other organs because it is liquid. Blood is a part of the body that is 6-8% in
total body weight. In men this percentage is slightly greater than in women. Forty-
five to 60% of blood consists of blood cells, especially erythrocytes. Leukocytes
and platelets, although functionally essential are only a small part of the blood. The
main function of blood is as a medium of transportation, maintaining temperature
and fluid balance, acids and bases.
The normal coagulation process, termed hemostasis, is the body’s defense
mechanism to prevent severe blood loss due to uncontrolled bleeding. Blood
coagulation involves multiple plasma proteins (coagulation factors), platelets, and
red blood cells (RBCs) that work together through a cascade of biochemical
processes to stop bleeding following injury. Coagulopathy, a condition in which
blood coagulation is impaired, can result from a variety of conditions including
severe trauma, illness or surgery and can cause life-threatening bleeding or
thrombotic disorders. For instance, deficient blood coagulation can lead to
‘hypocoagulable’ states resulting in prolonged or uncontrolled bleeding, which may
cause severe anemia, shock and multiple organ failure (Tripathi et al., 2014).

B. Purpose

The objectives of this laboratory activity are:

1. To know the response of red blood cells to a various medium that have different
osmotic concentration
2. To know the internal concentration of red blood cells
3. To know the cell shape and cell structure and compare the shape and the strcture
of frog blood cell and human blood cells
4. To know the process of blood clotting in human and determine the blood clotting
time in human.
 II. MATERIAL AND METHODS

A. Material

The materials that used in this practice are Human’s blood, paddy frog
(Fejervarya cancrifora) blood, NaCl solution (0,2; 0,4; 0,6; 0,9; 1%), 70% alcohol
solution, EDTA, cotton.
The tools that used in this practice are microscope, object glass, cover glass,
capillary tube, drop pipette, dissection kit, lancet, srynge, beaker glass and
stopwatch.

B. Methods

1. Concentration of Blood Cell

a. Blood from animal are taken, drop it on object glass, NaCl is added, covered
with cover glass, observed under the microscope.
b. The shape of blood cells are observed (min 5 cells).
2. Structure of Red Blood Cell
a. Blood from animal are taken, drop it on object glass, NaCl ( 0,6% for frog;
0,9% for human) are added.
b. The difference of the red blood cell’s structure are observed.
3. Blood Clotting Time
a. The finger is cleaned by alcohol 70%
b. Finger is punctured by using sterile lancet
c. Blood are taken by capillary tube
d. The capillary tube is broken by using a scissor per 1 minute
e. The time when the fibrin are form is noted.
 III. RESULT AND DISCUSSION

A. Result

Table 3.1. The Result of Blood Amount, Hb Level, and Hematocrit Value

Calculation Descriptive
Group Concentration (%)
Frog Human Frog Human
1 13,92 10,56 0,2% L L
2 0,526 0,72 0,4% N L
3 4,204 5,488 0,6% C N
4 13,44 9,6 0,9% N N
5 10,32 9,12 1% C C

Table 3.2. The Result of Human Blood Clotting Time

Group Clotting Time
1 2 minutes
2 6 minutes
3 6 minutes 20 seconds
4 2 minutes
5 9 minutes 9 seconds

Calculation of group 2
𝐷1 𝑥 𝐷2
Frog = x Calibration Human = D x Calibration
2

𝐷1 𝑥 𝐷2
1st cell = x 2,4 = 0,132 1st cell = 0,3 𝑥 2,4 = 0,72
2

𝐷1 𝑥 𝐷2
2nd cell = x 2,4 = 0,144 2nd cell = 0,3 𝑥 2,4 = 0,72
2

𝐷1 𝑥 𝐷2
3rd cell = x 2,4 = 0,156 3rd cell = 0,3 𝑥 2,4 = 0,72
2

𝐷1 𝑥 𝐷2
4th cell = x 2,4 = 2,04 4th cell = 0,2 𝑥 2,4 = 0,48
2

𝐷1 𝑥 𝐷2
5th cell = x 2,4 = 0,156 5th cell = 0,4 𝑥 2,4 = 0,96
2

∑ = 0,526 ∑ = 0,72
Microscopic images Observations on the Blood Cell (Eritrocyte) Concentration

Figure 3.1. Figure 3.2.

Structure of Frog’s Red Blood Cell Structure of Human’s Red Blood Cell

Figure 3.3. Figure 3.4.

Frog’s Blood 0.2% NaCl Solution
Human’s Blood 0.2% NaCl
Solution

Figure 3.5. Figure 3.6.

Frog’s Blood 0.4% NaCl Solution Human’s Blood 0.4% NaCl Solution

Figure 3.7. Figure 3.8.

Frog’s Blood 0.6% NaCl Solution Human’s Blood 0.6% NaCl Solution
 Figure 3.9. Figure 3.10.
Frog’s Blood 0.9% NaCl Solution Human’s Blood 0.9% NaCl Solution

Figure 3.11. Figure 3.12.

Frog’s Blood Cell 1% NaCl Solution Human’s Blood 1% NaCl Solution
B. Discussion
Based on practical observations that have been made, the observed
concentration, structure and timing of blood congealed animal blood test using
frog (Fejervarya cancrivora) and human blood. The results obtained are on blood
concentrations obtained diameter blood cells different after being given different
concentrations of NaCl. Human red blood cells are placed in a solution of NaCl
0.2% we got diameter average of 10,56 μm, a concentration of 0.4% = 0.72 μm,
0.6% = 5.488 μm, 0.9% = 9.6 μm and at concentrations of 1, 0% = 9.12 μm. Red
blood cells of frogs were placed in a 0.2% NaCl solution of 13,92 μm in diameter, a
concentration of 0.4% = 0.526 μm, 0.6% = 4.204 μm, 0.9% = 13.44 μm and at a
concentration of 1.0% = 10,32 μm. In accordance with the statement of Pearce
(2002), that the red blood cell responses differently to different solutions. Thus,
determination of the nature of a solution or liquid hipotonis, hypertonic or isotonic
completely determined by the responses generated cells (Kay, 1998).
According to Jain et al. (2015),the isotonic solutions may or may not be
isoionic. If Erythrocytes are incubated in solutions of a substance with high
membrane permeability, the solute will diffuse into the cells because of the
concentration gradient. This process is followed by an influx of water to maintain
osmotic equilibrium, the concentration of NaCl physiological (isotonic) has no
effect on the concentration of red blood cells. Low concentrations of NaCl
(hypotonis) makes the cells swell and the high NaCl concentration (hypertonic) can
make the cells shrink. The observation of blood concentrations of the two groups
were almost consistent with the theory. Only in 0.1% saline (hypertonic) swelling,
which should undergo shrinkage (Wulangi, 1993).
The observation of the structure that is in human blood cells have no
nucleus and a rounded shape, while the red blood cells of frogs (Fejervarya
cancrivora) are round and nucleated. Nucleated red blood cells are larger than red
blood cells that are not core (Evelyn, 2006). This is consistent with the literature
that says that human blood cells are not cored, rounded shape, discs, and biconcave,
which is concave on both sides and when viewed from the side like two crescent
opposite (Wulangi, 1993). Pieces of human erythrocytes has a diameter of about 6-
8 micrometers and a thickness of 2 micrometers, smaller than the other cells found
in the human body.Frogs (Fejervarya cancrivora) as well as other vertebrates have
red blood cells (erythrocytes) and yellowish red core with the shape and size varies
between one species and another. Sometimes found that the shape of a frog
erythrocytes in contrast to erythrocytes in human form. Mature erythrocytes oval,
small, and a diameter of 7-36 micrometers depending on the species. Red blood
cells of most other vertebrates such as frog oval, cored and biconvex (Evelyn,
2006).
Blood coagulation or formation of blood is the blood transformation of a
liquid into a solid gel. Platelets or clotting of blood cells or pieces of blood are tiny
cells about one-third the size of a red blood cell, irregularly shaped, fragile, and
have no core (nucleus). Every 1 mm of blood there are approximately 300,000
platelets. Clotting of blood cells formed in the red bone marrow and each of the
cells have the enzyme tissue factor. Blood will go out when the skin is injured,
causing clotting cells come out and make contact with rough surfaces. Blood
clotting cells will rupture and out of a substance that is prothrombin. This substance
will turn into thrombin under the influence of salts of calcium (Ca), vitamin K, and
thromboplastin. Thrombin is an enzyme that can change fibrinogen into fibrin
(Irianto, 2004). According to Byrnes et al. (2015), fibrinogen is a 340-kDa plasma
glycoprotein composed of 2 sets each of 3 chains (Aa, Bb, and g) that circulates at 2
to 4 mg/mL. During coagulation, thrombin cleaves N-terminal peptides from the
Aa- and Bb-chains, producing fibrin monomers that polymerize into protofibrils
and subsequently fibers.1 Fibrinogen deficiency is associated with bleeding and/or
thrombosis, whereas elevated fibrinogen (hyperfibrinogenemia) is associated with
thrombosis). Changes into fibrin is catalyzed by the enzyme tombin in the vessel
injury. According to Widada et al. (2016), coagulation converts all fibrinogen to
fibrin by spending factor VIII, V and prothrombin. Other clotting factors and
proteins that have nothing to do with hemostasis remain in the serum at the same
level as plasma. If the abnormal coagulation process of the serum may still contain
residual fibrinogen, the product of fibrinogen or prothrombin by remodeling is not
changed. The formation of a clot at the top of a platelet plug strengthen and support
the stopper, additional strengthening covering the hole in the vessel. Along with
clotting of blood vessels around the defect, blood can no longer flow.
According to Aida (2005), the 12 factors that involved in coagulation are factor
I (fibrinogen), fibrinogen is a soluble protein with a molecular weight of
330,000. With the influence of thrombin, fibrinogen is converted to fibrin. If no
fibrinogen, the blood clots will not happen (afibrinogenemia). Factor II
(prothrombin): Prothrombin is an inactive form of thrombin that is made in the
liver. Thrombin formation is influenced by vitamin K. Changes in prothrombin into
thrombin influenced by prothrombin activator. Factor III (tissue factor, tissue
extracts, thromboplastin): tissue factor convert prothrombin into thrombin, but it is
also influenced by factors V, factor VII, factor X, calcium ions and phospholipids.
Factor IV (calcium ion = Ca ++): needed for the formation of prothrombin activator
and fibrin. Factor V (labile factor): required to convert prothrombin into thrombin
by the influence of plasma tissue factor or factors. Factor V continues to be
consumed during the process of blood clotting.
Then factor VI (stable factor, otoprotrombin I) required for the formation of
prothrombin activator by tissue extracts. During freezing is not consumed, because
it is always present in the blood plasma. Factor VII (factor antihemofilia, globulin
antihemofilia): required for formation of prothrombin activator of the components
of blood. Hemophilia is generally caused because there is no factor VII in the
blood. Factor VIII (factor christmas, otoprotrombin II): required for formation of
prothrombin activator of the components of blood. Factor VIII present in the
plasma and activated during freezing so that its activity in serum was higher than in
plasma. Lack of factor VIII cause bleeding that same situation with hemophilia.
Factor IX (Stuart-Prower factor): present in blood plasma and serum. If you lack
the factor IX will result in bleeding. X factor: lack of factor X will cause bleeding
(Aida, 2005).
The last two factors are factor XI (Hageman factor): has a role in formation of
prothrombin activator of blood components. Factor XI deficiency can cause blood
clots in progress is slow, but did not show any bleeding, and factor XII (fibrin
stabilizing factor): a plasma protein that can cause polymerization of soluble fibrin
into insoluble fibrin. X factor II deficiency will cause bleeding (Aida, 2005).
 IV. CONCLUSION

Based on observations and the previous discussion it can be concluded that:

1. Red blood cell response is different for different solutions. So determining the
nature of a solution or liquid hipotonis, hipertosis or isotonic completely
determined by the responses generated cells.
2. Red blood cells will undergo hemolysis when placed in a solution of NaCl 0.4%
and 0.6% since the solution is hipotonis. Concentration of 1% in the red blood cells
will shrink because the solution is hypertonic. At a concentration of 0.9% blood
cells will not change because the solution is isotonic.
3. Shape of the red blood cells of frogs with humans is different where the results of
observation the human red blood cells biconcave disc-shaped, red because it
contains hemoglobin, but at the frog blood cells are oval with a clear core.
4. The length of time of normal blood clotting in humans ranges from 1–7 minutes.
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