DELHI PUBLIC SCHOOL, KALINGA

AN INVESTIGATORY PROJECT OF BIOLOGY

“ABO BLOOD GROUPING & CLOTTING TIME”

As per the partial fulfilment of AISSCE, 2023-24
(CENTRAL BOARD OF SECONDARY EDUCATION, NEW DELHI)

SUBMITTED BY: MAITREYEE DAS GUIDED BY:

AISSCE ROLL NO.: 1. MR. SANJAY JENA
2. MR. V VEERA
BHADRA RAO

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 CERTIFICATE

This is to certify that MAITREYEE DAS of class XII E Science bearing

the CBSE roll no. _________ has prepared and submitted the
investigatory project entitled, “ABO BLOOD GROUPING AND
CLOTTING TIME” in partial fulfillment of AISSCE 2023-24 of CBSEW,
New Delhi under my personal guidance and supervision.
I wish her all success in life
Dt: ________

Counter signature of Principal Signature of guide

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 ACKNOWLEDGEMENT
I feel proud to present my investigatory project in biology on the
topic “ABO BLOOD GROUPING AND CLOTTING TIME” for the partial
fulfillment of “All India Secondary School Examination 2023-24”.
I am extremely thankful to the principal Mrs. Anuradha Rakshit for
her help and guidance.
This project wouldn’t have been feasible without the proper and
rigorous guidance of my biology teachers Mr. V. Veerabhadra Rao,
Mr. Sanjay Jena. I am extremely thankful to them for their guidance
in every possible way throughout the project.
An investigatory project involves various difficult experiments which
have to be carried out by the student to obtain the observations and
conclude the report on a meaningful note.
Thereby, I would like to thank our lab assistant Mr. Bidhu Ranjan
Panda for his patience and help.
I would like to thank my family members and friends for their
constant support.
At last but not the least I would like to thank CBSE for giving the
opportunity to undertake this project.

Submitted by – MAITREYEE DAS

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 CONTENTS

TOPIC PAGE NO.

1. Introduction 4-16

2. Experiment-1 17-18

3. Blood Clotting Time 19-20

4. Experiment-2 20-22

5. Bibliography 23

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Introduction
Blood may be described as a specialized connective tissue in which there is
liquid intercellular substance known as plasma formed elements, the RBCs, the
WBCs and the platelets suspended in the plasma.

Landstiener in 1900 demonstrated that the human beings can be

divided into several groups depending upon the antigens and
antibodies present in their blood

i. A, B and O groups
ii. Rh factor
iii. M and N factors
iv. Others

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Blood group system is the most important blood type system in
human blood transfusion. The ABO is the most common blood type
throughout the world, particularly among peoples of South and
Central America. Type B is prevalent in Asia, especially in northern
India. Type A also is common all over the world: the highest
frequency is among the Blackfoot Indians of Montana and in the
Sami people of northern Scandinavia.

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DISCOVERY:
It was not until the year 1900, (1) when Karl Landsteiner at the
University of Vienna, discovered why some blood transfusions were
successful while others could be deadly. Landsteiner discovered the
ABO blood group system by mixing the red cells and serum of each of
his staff. He demonstrated that the serum of some people
agglutinated the red cells of other. From these early experiments, he
identified three types, called A, B and C (C was later to be re-named
O for the German “Ohne”, meaning “without”, or “Zero”, “null” in
English). The fourth less frequent blood group AB, was discovered a
year later. In 1930, Landsteiner received the Nobel Prize in
physiology and medicine for his work.

The gene that determines human ABO blood type is located on

chromosome 9and is called ABO glycosyltransferase. The ABO locus
has three main allelic forms: A, B, and O, as mentioned above and
each of them is responsible for the production of its glycoprotein. It
is therefore the combination of alleles that are inherited from
parents that determines which glycoproteins (antigens) are found on
persons’ blood cells and thereby their ABO blood type.
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ANTIGENS: (2)
The ABO blood group antigens remain of prime importance in
transfusion medicine—they are the most immunogenic of all the
blood group antigens. The most common cause of death from a
blood transfusion is a clerical error in which an incompatible type of
ABO blood is transfused. The ABO blood group antigens also appear
to have been important throughout our evolution because the
frequencies of different ABO blood types vary among different
populations, suggesting that a particular blood type conferred a
selection advantage (e.g., resistance against an infectious disease.)
However, despite their obvious clinical importance, the physiological
functions of ABO blood group antigens remain a mystery. People
with the common blood type O express neither the A nor B antigen,
and they are perfectly healthy. Numerous associations have been
made between particular ABO phenotypes and an increased
susceptibility to disease. For example, the ABO phenotype has been
linked with stomach ulcers (more common in group O individuals)
and gastric cancer (more common in group A individuals). Another

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observation is that individuals with blood type O tend to have lower
levels of the von Willebrand Factor (vWF), which is a protein involved
in blood clotting.

The ABO antigens exist in two groups-

a) Water-soluble, present in most body fluids.
b) Alcohol-soluble present in RBCs
Transfusion reactions
The routine practice of blood typing and cross matching blood
products should prevent adverse transfusion reactions caused by
ABO antibodies. However, clerical error can result in "the wrong
blood" being transfused into a patient, an error which can result in
the death of the patient.
If a recipient who has blood group O is transfused with non-group O
RBCs, the naturally occurring anti-A and anti-B in the recipient's
serum binds to their corresponding antigens on the transfused RBCs.
These antibodies fix complement and cause rapid intravascular
hemolysis, triggering an acute hemolytic transfusion reaction that
can cause disseminated intravascular coagulation, shock, acute renal
failure, and death.
Hemolytic disease of the newborn
For hematopoietic diseases, the loss of expression results
predominantly from a mutation affecting antigen production in the
stem cell. Complete or partial loss of antigen expression is seen
among the progenitors of RBC arising from this affected stem cell,
whereas the RBCs arising from unaffected stem cells usually express
normal RBC antigens.
Most cases of hemolytic disease of the newborn (HDN) that arise
from an ABO incompatibility require no treatment. Cases of severe

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hemolysis that require exchange transfusions are less common, and
fetal hydrops is rare.
HDN caused by ABO antibodies occurs almost exclusively in infants of
blood group A or B who are born to group O mothers. This is because
the anti-A and anti-B formed in group O individuals tend to be of the
IgG type (and therefore can cross the placenta), whereas the anti-A
and anti-B found in the serum of group B and A individuals,
respectively, tends to be of the IgM type. Although uncommon, cases
of HDN have been reported in infants born to mothers with blood
group A2and blood group B.
HDN tends to be relatively mild in nature mainly because fetal RBCs
don't express adult levels of A and B antigens. However, the strength
of fetal ABO blood group antigens can vary, and therefore the degree
of hemolysis and hence the severity of HDN can be unpredictable.
Early studies suggested that the race of a neonate was a risk factor
for developing ABO HDN.However, later studies showed that the
prevalence of disease that required treatment did not differ
significantly among Asian, Black, Hispanic, and Caucasian infants.

Alteration of ABO antigens for transfusion

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In April 2007, an international team of researchers announced in the
journal Nature Biotechnology an inexpensive and efficient way to
convert types A, B, and AB blood into type O. This is done by using
glycosidase enzymes from specific bacteria to strip the blood group
antigens from red blood cells. The removal of A and B antigens still
does not address the problem of the Rh blood group antigen on the
blood cells of Rh-positive individuals, and so blood from Rh negative
donors must be used. The sort of blood is named "enzyme converted
to O" (ECO) blood. Patient trials will be conducted before the
method can be relied on in live situations. One such Phase II trial was
done on B-to-O blood in 2002.

Another approach to the blood antigen problem is the manufacture

of artificial blood, which could act as a substitute in emergencies.

Genetics:
Blood groups are inherited from both parents. The ABO blood
type is controlled by a single gene (the ABO gene) with three types
of alleles inferred from classical genetics: i, IA, and IB.
The I designation stands for isoagglutinogen, another term
for antigen.[3] The gene encodes a glycosyltransferase—that is,
an enzyme that modifies the carbohydrate content of the red blood
cell antigens. The gene is located on the long arm of the ninth
chromosome (9q34).

The IA allele gives type A, IB gives type B, and i gives type O.

As both IA and IB are dominant over i, only ii people have type O
blood. Individuals with IAIA or IAi have type A blood, and individuals
with IBIB or IBi have type B. IAIB people have both phenotypes, because
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A and B express a special dominance relationship: codominance,
which means that type A and B parents can have an AB child. A
couple with type A and type B can also have a type O child if they are
both heterozygous (IBi,IAi). The cis-AB phenotype has a single enzyme
that creates both A and B antigens. The resulting red blood cells do
not usually express A or B antigen at the same level that would be
expected on common group A1 or B red blood cells, which can help
solve the problem of an apparently genetically impossible blood
group.

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 The table above summarizes the various blood groups that
children may inherit from their parents. Genotypes are shown in the
second column and in small print for the offspring: AO and AA both
test as type A; BO and BB test as type B. The four possibilities
represent the combinations obtained when one allele is taken from
each parent; each has a 25% chance, but some occur more than once.
The text above them summarizes the outcomes.

ROLE IN THE BODY: (4)

The carbohydrate molecules on the surfaces of red blood cells have
roles in cell membrane integrity, cell adhesion, membrane
transportation of molecules, and acting as receptors for extracellular
ligands, and enzymes. ABO antigens are found having similar roles on
epithelial cells as well as red blood cells.

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Bleeding and thrombosis (von Willebrand factor)

The ABO antigen is also expressed on the von Willebrand factor

(vWF) glycoprotein, which participates in hemostasis (control of
bleeding). In fact, having type O blood predisposes to bleeding, as
30% of the total genetic variation observed in plasma vWF is
explained by the effect of the ABO blood group, and individuals with
group O blood normally have significantly lower plasma levels of vWF
(and Factor VIII) than do non-O individuals. In addition, vWF is
degraded more rapidly due to the higher prevalence of blood group
O with the Cys1584 variant of vWF (an amino acid polymorphism in
VWF): the gene for ADAMTS13 (vWF-cleaving protease) maps to
human chromosome 9 band q34.2, the same locus as ABO blood
type. Higher levels of vWF are more common amongst people who
have had ischemic stroke (from blood clotting) for the first time. The
results of this study found that the occurrence was not affected by
ADAMTS13 polymorphism, and the only significant genetic factor
was the person's blood group.

Disease risks

Compared to O group individuals, non-O group (A, AB, and B)

individuals have a 14% reduced risk of squamous cell carcinoma and
4% reduced risk of basal cell carcinoma. Conversely, type O blood is

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associated with a reduced risk of pancreatic cancer. The B antigen
links with increased risk of ovarian cancer. Gastric cancer has
reported to be more common in blood group A and least in group O.

According to Glass, Holmgren, et al., those in the O blood group have

an increased risk of infection with cholera, and those O-group
individuals who are infected have more severe infections. The
mechanisms behind this association with cholera are unclear in the
literature.

COVID-19 AND BLOOD GROUP:

The ABO blood group
system is known to be
associated with several
parameters of healthy
aging and disease
development.[5]

Recently a study was conducted to explore any relationship between

the ABO blood group and the coronavirus disease 2019 (COVID-19)
susceptibility, we compared ABO blood group distributions in 2173
COVID-19 patients with local control populations, and found that
blood group A was associated with an increased risk of infection,
whereas group O was associated with a decreased risk.[6]

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There is one more study that showed a relationship between the
ABO blood group and Covid 19.[7]

However, the study did not address other factors, such as chronic
pre-existing medical conditions that could potentially affect the
chance and severity of SARS-CoV-2 infectionThe 2019 coronavirus
disease (COVID-19) has become a global pandemic. Several studies
report that ABO blood group polymorphism may be related to
COVID-19 susceptibility and clinical outcomes; however, the results
are controversial.People with B and/or O blood groups were less
represented among COVID-19 patients, thereby highlighting the
possible beneficial role of anti-A antibodies in COVID-19
susceptibility. In fact, it was known that anti-A antibodies can block
the adhesion of SARS-CoV S-protein to ACE2 expressing cell lines.

Pseudoscience: -

1.Blood groups and personality traits

The idea of linking blood groups to personality traits arose in the
early 20th century. The personalities ascribed to the several blood
types carry forth stereotypes that ultimately backdate themselves to
the abiding influence of Japanese feudalism. Japan saw itself as a
society of samurai (type O) and peasants (type A), the most frequent
blood types in Japan; with a smaller number of merchants and artisans
(type B) and intellectuals (type AB).

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There are studies that show a correlation between blood groups and
personality [8]. However, they significantly differ from the Japanese
idea.

Though the results are significant, there is little consensus as to the

personality characteristics of members of any one blood type.

2.Blood groups and IQ

There are no known studies which show a relation between iq and
blood group, but there are some which discuss the methods that could
be used.

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 PROCEDURE TO DETERMINE ABO BLOOD
GROUPING

FOR EXPERIMENT

Material required:
 Antigen – A
 Antigen – B
 Ten pieces of sterile syringes
 150 ml of spirit
 One bundle of cotton
 Antigen – D
 A number of volunteers are selected by random selection from different
classes.
 Respective measures were taken to have the laboratory set up for the
experiments with the necessary materials required.
Steps taken to carry out a successful test on an individual:
o Volunteer right ring finger was washed and cleaned with spirit using
cotton.
o The finger was pricked using sterilized needle.
o The blood sample was collected on three different places on he slide.
o Antigen – A, antigen – B and antigen -D were added to the three
collected blood samples respectively.
o The slides were observed and the conclusions were recorded.

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RECORD SHEET
Name Age Gender Blood Rh Factor Signature
Group

Conclusion:

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The above experiment concludes that out of every 10 people,__________are
O, _________are Ab, _________are A and ___________are B blood groups.

BLOOD CLOTTING TIME

To determine the clotting time of a blood sample by capillary glass method.

The mechanism behind blood clotting:

Coagulation (Thrombogenesis) is a process by which blood changes from a
liquid to a gel, forming a clot. It potentially results in hemostasis, the cessation
of blood loss from a damaged vessel, followed by repair. The mechanism of
coagulation involves activation, adhesion and aggregation of platelets along
with deposition and maturation of fibrin. Disorders of coagulation are disease
states which can result in bleeding (hemorrhage or bruising) or obstructive
clotting(thrombosis).
Coagulation is highly conserved throughout biology; in all mammals,
coagulation involves both a cellular (platelet) and a protein (coagulation factor)
component. The system in humans has been the most extensively researched
and is the best understood.
Coagulation begins almost instantly after an injury to the blood vessel has
damaged the endothelium lining the vessel. Exposure of blood to the space
under the endothelium initiates two processes: change in platelets, and the
exposure of subendothelial tissue factor VII, which ultimately leads to fibrin
formation. Platelets immediately form a plug at the site of injury: this is called
primary hemostasis. Secondary hemostasis occurs simultaneously: additional
coagulation factors or clotting factors beyond factor VII (listed below) respond
in a complex cascade to form fibrin strands, which strengthen the platelet plug.

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 Injury

Thtromboplastin release

Prothrombin Thrombin

Fibrinogen Fibrin

Polymerisation Fibrin threads form mesh

Cells clogged in Mesh

Semisolid clot

Bleeding stops

PROCEDURE TO DETERMINE THE CLOTTING TIME OF AN

INDIVIDUAL
Materials Required:
 Sterile disposable pricking needle or lancet
 Dry glass capillary tube (Narrow diameter, top two mm, 10 cm long)
 Cotton swab of absorbent cotton
 Spirit wetted cotton swab
 Seventy percent volume / volume ethyl alcohol
 Stopwatch
Method:

o Apply alcohol 70% v/v to the clean finger by cotton swab

o Prick the finger with usual aseptic precautions. Immediately stop watch
is started.
o Dip one end of the capillary into the blood gently without any pressure.
o Allow the capillary to fill by lowering the end of fitted capillary. Fill
around 3/4th of the length of the capillary.

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 o After every 30 seconds, using stopwatch, break a small piece of the
capillary.
o Repeat breaking at regular time intervals, till fibrin threads appear at the
broken end of the capillary tube. Do not pull away the cut pieces apart
briskly.
o Record the time interval between pricking finger and first appearance of
fibrin thread at the broken ends of capillary tube. That is clotting time of
blood.
RECORD SHEET:
NAME AGE GENDER CLOTTING TIME

Ronak Mishra 17 Male 6.57 mins

Shreema Das 16 Female 5.51 mins

Ankita Mishra 18 Female 4 mins

Shaswat Rath 17 Male 5.48 mins

Manasi Sahu 17 Female 8.12 mins

Ayush Mohanty 16 Male 3.58 mins

Aniket Mahapatra 18 Male 4.55 mins

Shweta Rath 17 Female 7.49 mins

Result:
The normal clotting time is 4 to 9 minutes.
The blood taking shortest time for clotting is __3.58 mins______ and longest
time for clotting is __8.12 mins________.

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OBSERVATION:

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BIBLIOGRAPHY :
1. Chatterjee CC, Blood Transfusion: Blood Groups, In Human
Physiology,11th ed, Medical Allied Agency, Kolkatta, pg 181-2
2. Dean L. Blood Groups and Red Cell Antigens [Internet].
Bethesda (MD): National Center for Biotechnology Information
(US); 2005. Chapter 5, The ABO blood group. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK2267/
3. Klug, William S.; Cummings, Michael R. (1997). Concepts of
Genetics (5th ed.). Upper Saddle River, NJ: Prentice Hall.
p. 83. ISBN 978-0135310625.
4. Wikipedia contributors, "ABO blood group system," Wikipedia,
TheFreeEncyclopedia, https://en.wikipedia.org/w/index.php?
title=ABO_blood_group_system&oldid=1008261349 (accessed
March 7, 2021).
5.Genetically determined abo blood group and its association with
health and disease (Hilde e Groot et al) Arteriosclerosis,
Thrombosis, and Vascular Biology. 2020; 40:830–838.
6.Relationship Between the ABO Blood Group and the Coronavirus
Disease 2019 (COVID-19) Susceptibility, Clinical Infectious Diseases,
2020;, ciaa1150, https://doi.org/10.1093/cid/ciaa1150, (Jiao Zhao et
al)
7. Zietz M, Tatonetti NP. Testing the association between blood type
and Covid 19 infection, intubation, and death. Medrxiv 2020
doi:10.1101/2020.04.08.20058073
8. Lester, d & Gatto (1987) personality and blood group,
Personality and individual differences

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