COAGULATION OF HUMAN AND CHICKEN BLOOD SAMPLES AND AGGLUTINATION

REACTION OF DIFFERENT HUMAN BLOOD TYPES

Carandang | Cruz | Pasumbal | Salem | Tolentino
Institute of Biology, University of the Philippines Diliman, Diliman, Quezon City

ABSTRACT

Blood samples from human and chicken were used for observing different properties of blood. A
human blood sample was obtained and placed on a slide and the fibrin formation was observed every
minute to determine the clotting time of the blood sample. A survey was also conducted for the blood
types of a sample of students. Coagulation of chicken blood was observed under varying conditions
including different surfaces (paraffin, cotton fibers), different temperatures, additions of different
anticoagulants (0.1% heparin, 1% sodium oxalate, 1% sodium citrate), and under continuous mechanical
stirring. Blood pigmentation was also observed through the use of citrated blood. The human blood
sample coagulated after five minutes, and the chicken blood sample coagulated after two minutes.
Chicken blood contained in test tubes with paraffin and cotton fiber surfaces both coagulated after two
minutes. Samples in lowered temperatures displyed a marked increase in coagulation time of over 20
minutes, while those in elevated temperatures displayed 5 minutes. Continuous stirring resulted in a
coagulation time of 4 minutes. Sodium oxalate displayed signs of coagulation after 5 drops of calcium
chloride, while sodium citrate did the same after 40 drops. Heparin showed no signs of coagulation after
100 drops of the calcium chloride solution. The citrated blood turned bright red when aerated, and dark
red when bubbled with carbon dioxide.
INTRODUCTION leading to the formation of a mesh which entraps
red blood cells and plasma (Randall et al., 2002).
Blood is a liquid tissue which flows through a
network of closed circulating channels (blood Agglutination is a physiological response
vessels) whose liquid component is chiefly resulting in blood clumping that occurs in the
plasma which contain the blood cells. It presence of antibodies which physically hinder
performs a multitude of transport and regulatory some antigens from exerting their effects.
functions and is essential in vital life processes Multiple antibody molecules cross-link
(Rastogi, 2007). As such, systems are in place to numerous antigen molecules into chains or
prevent excessive blood loss or degradation. lattices of antigen–antibody complexes. Through
this means foreign cells, such as bacteria or
Clotting occurs only when there is damaged mismatched transfused red blood cells, bind
tissue or exposed collagen, which occurs when together in a nonmotile clump (Sherwood et al.,
the endothelium of a vessel is damaged and 2013). This property of blood is used in blood
exposes connective tissue in the vessel wall to type determination. Blood samples of an
blood. This causes a cascade of events in which unknown type are exposed to known antibodies,
many factors normally found in the blood are Anti-A, Anti-B, and Anti-D (also known as anti-
activated in sequence and ultimately leading to sera). Blood type is determined based on which
the formation of thrombin. Thombrin converts antibody the unknown blood shows
fibrinogen to fibrin which then polymerizes into agglutination (“Blood Typing,” n.d.).
an insoluble fibrin clot and adheres to platelets,
In the experiment, coagulation was observed Blood coagulation for the human sample
under varying substrate/surface conditions, occurred around five minutes as indicated by the
temperature, mechanical stress, and anti- formation of fibrin threads as it was parted every
coagulating chemical substances. The 1 min. However, the chicken blood sample
mechanism behind these anticoagulatory coagulated faster wherein a semi solid gelatin-
substances was also determined. Blood types of like consistency was observed after two minutes
participants were also taken to observe the (Table 1).
distribution within the group.
Table 1. Different factors affecting clotting time
MATERIALS AND METHODS of human and chicken blood samples.
Time of coagulation
Setup
To observe the coagulation of human blood, a (minutes)
blood sample was obtained by pricking the Empty test tube 2
finger with a blood lancet. The pricked finger Paraffin lining 2
was placed near a glass slide and collected a few Cotton fibers 2
drops of blood. The coagulation time of the Ice bath >20
blood was monitored every minute by lifting the Hot bath 5
Continuously stirred 4
blood with a needle to observe fibrin formation.

It can be observed from the experiment that the

The coagulation of chicken blood was also
factor of paraffin lining and cotton fibers did not
observed in different conditions. Different
affect the clotting of blood with respect to the
surfaces were prepared by covering the second
control, the empty test tube. The ice bath,
test tube with paraffin wax and cotton fibers
however increased the time it took to clot the
were added on the third test tube. The chicken
blood to 20 mins while the hot bath has a much
sample was held in place and the area around the
faster clotting time of 5 mins. Continuous
neck was defeathered. Using a clean blade, an
stirring of blood acquired 4 mins of clotting
incision was made on the neck, and about two
among the samples.
mL of fresh chicken blood were placed in 11
tubes. The fourth tube was placed in an ice bath,
Table 2. Number of CaCl2 added in the anti-
while the fifth tube was placed in a warm water
coagulant before clotting occurs
bath. The sixth tube was continuously stirred.
Anticoagulant # of CaCl2 Drops
The seventh to ninth tubes are added with 0.1%
100
heparin, 1% sodium oxalate, and 1% sodium 0.1% Heparin
(No coagulation)
citrate, respectively. The last two tubes were 1% Sodium
added with 1% sodium citrate and aerated 5
oxalate
initially with oxygen and then with carbon 1% Sodium
40
dioxide conversely to observe blood citrate
pigmentation of oxygenated and deoxygenated
blood. Lastly, a survey was conducted on a From the results obtained, heparin is the best
sample of students to determine the number of anti-coagulant from the sample because no
students sharing the same blood typing. coagulation occurred regardless of the number
of CaCl2 added. This was followed by Sodium
RESULTS Citrate with accumulated 40 drops before
clotting occurs. Sodium oxalate happens to be
the weakest anti-coagulant in the group which oxygen affinity of fetal hemoglobin, the oxygen
clotted after 5 drops of CaCl2. unloading capacity of hemoglobin in neonates is
about 50% less than in adults (Linderkamp et al.,
Blood pigments of the citrated blood was also 1994).
observed with respect to color change. Initially
aerated blood which is known to be oxygenated Deviations from these values are likely caused
has a native bright red solution while by pathological disorders such as polycythemia,
deoxygenated blood has a deep red appearance. and oligocythemia. Polycythemia vera is a
chronic myeloproliferative disorder
Survey of the majority of the blood type the characterized by increased red blood cell mass.
students share obtained a result of almost all The resultant hyperviscosity of the blood
blood type O and the students were not sure of predisposes such patients to thrombosis. It
their Rh factor also. should be suspected in patients with elevated
hemoglobin or hematocrit levels, splenomegaly,
DISCUSSION or portal venous thrombosis. It can be due to an
increase in the number of red blood cells,
Blood is a liquid tissue which flows through a absolute polycythemia, or to a decrease in the
network of blood vessels where blood cells are volume of plasma, relative polycythemia (Stuart,
suspended in a liquid component called plasma. 2004). Oligocythemia is a reduction in red cells
It performs a multitude of transport and (erythrocytes) in the peripheral circulation,
regulatory functions and is essential in vital life anaemia. It is also a reduction in all cells in the
processes (Rastogi, 2007). The red blood cell peripheral circulation or pancytopenia
number in males are more than in females (“Oligocythemia,” n.d.) However, there are no
because adult females maintain their venous differences between the white blood cell number
haemoglobin levels at a lower level than adult in males and females. The differences in the
males as a physiological steady state which they parameters were found insignificant
do not try to maintain the same levels as adult (Matyushichev et al., 2001).
males. This physiological process is controlled
by the modulation of the juxtaglomerular An increase in white blood cells is called
apparatus (JGA) through regulating renal leukemia which is the is cancer of the body's
filtration and the amount of oxygen that goes to blood-forming tissues, including the bone
the erythropoietin producing peritubular cells. marrow and the lymphatic system. It is an
(Murphy, 2014). However, the reference values abnormal growth of the white blood cells. A
for red blood cell counts are identical in male decrease in white blood cells is called
and female children and as well in elderly men leukopenia which is a decrease in disease-
and women. The differences in these levels fighting cells. It is almost always related to a
between fertile women and men are caused by decrease in a certain type of white blood cell
repetitive loss of blood and iron especially in (neutrophil) (Kasper et al., n.d.).
menstruating women (Cheong et al., 1991).
Blood Types
Infants have a higher red blood cell number than
adults because there is a high oxygen The ABO blood groups in humans may be A, B,
consumption and cardiac output in neonates AB, or O. These groupings are caused by three
three times those of adults. Due to the high alleles of a single gene: I A, IB, and i. The letters
refer to two carbohydrates A and B that may be O contains anti-A and anti-B antigens in the
found on the surface of red blood cells. A plasma, only the RBC which do not contain
person’s blood cells may have carbohydrate A antigens is transfused to a receiver. If the plasma
(type A blood), carbohydrate B (type B), both of the donor is also transfused, only a relatively
(type AB), or neither (type O). few cells of the receiver will clump because
plasma contains a relatively low amount of
antibodies and blood transfused are usually
diluted. The antibodies in plasma can be
neutralized by intravenous immunoglobulins or
can be removed from plasma by plasmapheresis
(Bethesda, 2005).

Rh factor

The Rh blood group system refers

to the most immunogenic D antigen
of the Rh blood group system, or
the Rh− blood group system. The
status is usually indicated by Rh
positive (Rh+ does have the D
Figure 1. Alleles, genotypes and phenotypes of
antigen) or Rh negative (Rh− does
the ABO blood groups (Reece et al., 2014).
not have the D antigen) suffix to
the ABO blood type. This blood
Matching compatible blood groups is critical for
group system consists of 50
safe blood transfusions because certain bacteria
defined blood group antigens,
normally present in the body have epitopes very
among which the five antigens D, C,
similar to the A and B carbohydrates.
c, E, and e are the most important.
Responding to the bacterial epitope similar to
The commonly used terms Rh
the B carbohydrate, a person with type A blood
factor, Rh positive and Rh negative
makes antibodies that will react with the B
refer to the D antigen only.
carbohydrate. If a person with type A blood
Besides its role in blood
receives a transfusion of type B blood, the anti-
transfusion, the D antigen is used
B antibodies in the type A blood will cause an
to determine the risk of hemolytic
immediate and devastating transfusion reaction.
disease of the newborn (or
The transfused red blood cells undergo lysis,
erythroblastosis fetalis) for Rh
which can lead to chills, fever, shock, and
disease management. A pregnant
kidney malfunction. The anti-A antibodies in the
mother who is Rh negative can give
donated type B blood will act against the
birth to a normal Rh negative baby
recipient’s type A red blood cells. (Reece et al.,
with no complications but during
2014). Hence, a type O blood can be transfused
the birth. If the same mother has a
to a type B because type O does not have any
pregnancy in which the offspring
carbohydrate that type B will react. Whole blood
is Rh positive, the Rh positive
is normally not transfused. The components of
antigens will enter the mother’s
blood samples were were separated by the use of
blood causing the mother's Rh
a centrifuge. Therefore, even though blood type
negative antibodies to respond and being foreign to the coagulation factors in the
will begin to lyse the offspring's plasma. This is why theoretically, paraffin wax
red blood cells and may even cause would have the highest coagulation time because
the death of the offspring. unlike the two, it is non-polar and smooth which
However, there is no risk if the makes it the closest match to the surface of a
mother is Rh positive carrying a blood vessel (Lozner & Taylor, 1941) Cotton
Rh positive fetus (Echavez, n.d.). fibers should have the shortest time since it is
charged when in contact with the water in the
Coagulation and Agglutination plasma, resulting to a polar surface foreign to the
clotting components. Its rough surface also adds
Coagulation is the is the process by which blood to it being classified as foreign since blood
changes from a liquid to a gel, forming a blood vessels are usually smooth when unharmed.
clot while agglutination is another process that Glass should also hasten the reaction since it
occurs if an antigen is mixed with its becomes polar when in contact with water but
corresponding antibody called isoagglutinin not faster than cotton because of its smooth
(Coelho, 2014). surface (Margolis, 1957). However, the
experimental data gathered did not coincide with
The coagulation of blood is divided into two the theoretical observations, all three of them
parts: primary hemostasis and secondary coagulating after 2 minutes leading to
hemostasis. The former accounts for the insignificant differences. This might have been
constriction of blood vessels to reduce blood caused by other factors such as temperature.
flow as well as the formation of a plug made
from platelets present in the plasma. The latter Sodium oxalates and citrates are known
encompasses the formation of fibrin from a anticoagulants because it only causes partial
cascade of reactions, starting from the activation inhibition and its effect can be easily countered
of coagulation factors which would convert by addition of calcium ions. Sodium oxalates act
prothrombin to thrombin which in turn would as anticoagulant by forming insoluble
cleave fibrinogen into fibrin. Fibrin is insoluble precipitates with calcium while citrates only
in plasma and would form threads to would bind to calcium to inhibit its activity and form
entangle platelets, forming a clot (Lotha, 2017). soluble compounds with it (Heisermann, 2004).
This cascade could be activated by two Citrates are generally preferred over heparin and
pathways: intrinsic and extrinsic. The intrinsic oxalates (Mann et.al., 2007). Excess in heparin
pathway would promote clotting when blood could cause distortion in the shape of the
comes into contact with a foreign surface inside erythrocytes and even hemolysis. Oxalates, on
the blood vessel, while the extrinsic pathway the other hand, could cause a more severe
would be activated by presence of damaged damage in that it can alter blood plasma
tissue that is exposed to the external concentrations. This happens under extreme
environment. In order to observe the effect of temperature conditions, where oxalates can be
different surfaces to the coagulation time the converted into carbonates (Heisermann, 2004).
intrinsic pathway would be induced. At the same time, this alters the blood pH level.
Citrated blood is preferred to be transfused to
The endothelial lining of blood vessels are non- patients than the two former reagents, although
polar due to their lipid membranes. A polar it is important to note that recalcification of
surface would hasten the clotting time due to it
citrated blood does not coagulate as efficient as hemocyanin. The metallic ion in hemocyanin is
it does before (Mutschler & Derendorf, 1995). copper and the blood turns dark blue when
oxygenated. Otherwise the blood turns colorless
In research studies, clotting time were measured when deoxygenated. Chlorocruorins are blood
at temperatures ranging from +22 degrees to +37 pigments found only in annelid that have heme
degrees C. At +32 degrees C, the bleeding time groups as its central metallic ions. It is green in
was longer and hematocrit was lower. At lower color when oxygenated and turns a shade lighter
temperatures, clotting times were three times when deoxygenated, although in some cases it
longer at +22 degrees C than at +37 degrees C. can turn red when highly concentrated with
Changes in the temperature of the blood, after it oxygen. Hemerythrin are pigments found
is withdrawn from the body, produce a marked specifically in marine invertebrates containing
affect on its coagulation time. From 10° C. to iron pigments that turn violet in its oxygenated
about 40° C. the time is shortened as the state and colorless when deoxygenated (Hill,
temperature rises, and beyond this from 40° C. et.al., 2012). The color of each pigment, whether
upwards, it is lengthened. At 55° C. or 56° C. oxygenated or deoxygenated, can be attributed
the blood does not coagulate at all. This implies to the metallic ion group it contains. For
why dentists advise patients to take something hemoglobin and hemocyanin, each individual
cold after a tooth extraction so that the clotting heme has conjugated molecules causing them to
time will take effect much faster. (Simpson & absorb different wavelengths in the visible light
Rasmusen, 1916). spectrum, depending on the gas molecule it
carries. On the other hand, the reuse the
Blood pigments hemoglobin found in the liver by converting it to
biliverdin, allowing for its green coloration of
Blood pigments are also known as respiratory the chlorocurorins. Lastly, hemerythrin works on
pigments due to its general function of the same principle as the first two pigments,
transporting oxygen and carbon dioxide gases to however, it has a different coloration produced
the different parts of the body (Randall, et.al., due to its relatively weaker affinity for the
2002). There are four classifications of respiratory gases (Bruning, 2015).
respiratory pigments based on their chemical
composition such as hemoglobin, hemocyanin, Buffers
chlorocruorins and hemerythrin. Hemoglobin is
the most common blood pigment and can be The buffer systems functioning in blood plasma
found across almost all kinds of animal species. include plasma proteins, hemoglobin, phosphate,
It consists of a globular protein (globin) with 4 and bicarbonate and carbonic acid buffers.
subunits that varies among species, and a heme In protein buffers, the charged regions of these
structure, an iron (ferrous) porphyrin, in the molecules can bind hydrogen and hydroxyl ions,
middle, identical among species. and thus function as buffers. Buffering by
Oxyhemoglobin is bright red in color while proteins accounts for two-thirds of the buffering
deoxygenated hemoglobin is a darker shade of power of the blood and most of the buffering
red. This color change was observed in the within cells. Secondly, hemoglobin buffers the
experiment. Hemocyanin is the second most hydrogen ions liberated which is reduced by the
common blood pigment found commonly dissociation of oxygen. This buffering helps
among invertebrates and is further classified into maintain normal pH. The process is reversed in
two: arthropod hemocyanin and mollusk the pulmonary capillaries to re-form CO2, which
then can diffuse into the air sacs to be exhaled http://www.compoundchem.com/2014/10/28/col
into the atmosphere. For phosphate buffers, It oursofblood/
has two forms: sodium dihydrogen phosphate
which is a weak acid, and sodium Cheong, R. Kuizon, M. Tajaon, R. (1991).
monohydrogen phosphate, which is a weak base Menstrual blood loss and iron nutrition in
that buffers reactions with strong acids and Filipino women. Southeast Asian J Trop Med
bases. Lastly, the bicarbonate-carbonic acid Public Health. 22(4):595-604.
buffer is regulated in the blood by sodium, as are
the phosphate ions. (Silverthorn, 2007) Coelho, L. C. (2014). Coagulation,
Flocculation, Agglutination and
CONCLUSION Hemaglutination: Similar Properties?. 51-70.

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however the experimental results obtained have Heiserman, D. L. (2004). Methods of
insignificant differences to support this yield. Hematology. Ohio: SweetHaven Publishing
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