DETERMINATION of BLOOD TYPE by AGGLUTINATION

Aim of this practical hour

To understand the basic concept of Blood Grouping. A simple testing that
provides everyone with general knowledge and practice will be instructed to evaluated

Principle:
It was in 1901, that Austrian-American immunologist and pathologist Karl Landsteiner
discovered human blood groups. Karl Landsteiner's work helps to determine blood
groups and thus opened a way for blood transfusions, which can be carried out
safely. He was awarded the Nobel Prize in Physiology or Medicine in 1930 for this
discovery.

Death of the patient was the result in most cases before 1900, when blood
transfusion was attempted. Blood transfusion was made much safer by the discovery
of blood groups, as blood of the same ABO group could be chosen for each patient.
However, there were still many cases of unexplained blood transfusion reactions.
Biologists still went in search of these unexplained questions.

In 1902, the fourth main type, AB was found by Decastrello and Sturli.It was the
observations of Levine and Stetson in 1939, and Landsteiner and Weiner in 1940,
that laid the foundations of our knowledge about the remaining major blood group-
the Rhesus system. Once reliable tests for Rhesus grouping had been established,
transfusion reactions became rare! For this discovery Landsteiner was awarded the
Nobel Prize in Physiology or Medicine in 1930.

Karl Landsteiner

The Components of Blood

The circulatory system distributes about 4-6 liters of blood to the adult human body.
The blood mainly has 2 portions: the Plasma and the Blood Cells. Plasma is mainly
composed of water, but contains different types of proteins and other chemicals
such as: enzymes, glucose, fat particles1 salts, hormones, antibodies etc.It
constitutes about 60% of the blood. Blood cells can be observed under a microscope
on staining. The formation of blood cells occurs in the bone marrow by the
 'Hematopoietic stem cells'. They can be divided into 3 basic cell types:

Erythrocytes Red Blood Cells {RBC):

As the name suggests, these red coloured cells give blood its red colour. (The
word erythrocyte is from erythro-Gk.meaning red and Latin-cytos meaning cell.) 1
ml of blood contains approximately 5 million RBCs! The proportion of blood occupied
by red blood cells is referred to as the hematocrit, and is normally about 45%.
Mature RBCs are biconcave in shape; lack a Nucleus and many other
organelles. They circulate in the system for about 120 days, carrying out their job,
i.e., to supply oxygen.
They carry out this function most efficiently. Since they are rich in an Iiron-containing
biomolecule called haemoglobin. Haemoglobin has high affinity for oxygen, thus
binds to it and is transported from the alveoli (in lungs) to every part of the body.
There is constant replenishment of RBCs in order to remove old cells that break
down. (This process is carried out in organs like the liver, also producing by-products
like bile pigments.) Millions of eels are released into the bloodstream from the bone
marrow each day.

Leukocytes- White Blood Cells:

These cells are key players in our immune system. They are of different types such
as neutrophils, lymphocytes, eosinophils, monocytes, basophils. Each of them
have a variety of functions in our immunity. 1 ml of blood of an adult human
contains about 4,000-11,000 leukocytes. Basically, they destroy and remove old or
aberrant cells and clear cellular debris, as well as attack foreign substances and
infectious agents (pathogenic entities).

Thrombocytes- Platelets:

The coagulation or blood clotting process is taken care of by them. They act on
clotting proteins like Fibrinogen, converting it into Fibrin. They create a mesh onto
which RBCs collect and form a clot. This prevents excessive blood loss and also
checks the entry of pathogens into the body. 1 ml of blood of an adult human
contains about 200,000-500,000 platelets.
The observations that led to the discovery of blood groups:
At times, it wa s observed t h a t m i x i n g b l o o d from two individuals led to blood
clumping or, agglutination. Later it was understood that the agglutinated red cells
can clog blood vessels and stop the circulation of the blood to various parts of the
body. The agglutinated red blood cells also crack and their contents leak out in the
body.

The RBCs contain haemoglobin, which becomes toxic when outside the cell. This
must have been the phenomena that occurred in the blood transfusion cases that
ended up with fatality of the patient at the receiving end. Karl Landsteiner
discovered that blood clumping was an immunological reaction. Which occurs when
the receiver of a blood transfusion has antibodies against the donor blood cells!
People learned that, compatibility of blood groups needed to be checked before
anything else was done. If they are not, the red blood cells from the donated blood
will agglutinate. This can have fatal consequences for the patient

ABO blood grouping system:

According to the ABO blood group system. There are four different kinds of
blood groups: A, B, AB and 0 (null).

Blood group A: If you belong to the blood gr oup A, you have A antigen o n the
surface of red blood cells and B antibodies in your blood plasma.
Blood group B: If you belong to the blood gr oup B, you have B antigen o n the
surface of red blood cells and A antibodies in your blood plasma.
Blood group AB: If you belong to the blood gr oup AB, you have A and B antigen o n
the surface of red blood cells but, neither A nor B antibodies in your blood plasma.
Blood group O: If you belong to the blood gr oup 0 (NULL), you have neither A nor B
antigen o n the surface of red blood cells but you have both A and B antibodies in
your blood plasma.
Rh (Rhesus) factor is found on the RBC's surface in most people. Like A and B,
this is also an antigen and those who have it are called Rh +. Those who lack the
antigen on the surface of RBC are called Rh-. A person with Rh- blood does not have
Rh antibodies naturally in the blood plasma. But a person with Rh- blood can develop
Rh antibodies in the blood plasma if he or she receives blood from a person with Rh+
blood, whose Rh antigens can trigger the production of Rh antibodies (as the immune
system is triggered by the presence of an unknown antigen in the system). A person
with Rh+ blood can receive blood from a person, with Rh­ blood without any problems.

Inheritance of Blood Groups:

Blood groups for each individual are determined by genes or alleles (small packets of
information in cells contained in the DNA) which are inherited from both parents. Genes
for the Rh- and O groups from one parent are masked (i.e., they, are recessive) by the
presence of Rh positive and A or B genes from the other parent. That is, O and Rh
negative genes only produce an effect when there is a "double dose" of such genes,
i.e., one from each parent (homozygous condition). Thus, people who are apparently A
or B Rh+ may also carry genes 0 and Rh negative blood groups which can be inherited
by their children.

Principle behind blood tests: Blood clumping or Agglutination observation

Compatibility b e t w e e n t h e blood groups of donor and recipient determines
the success of a blood transfusion. The ABO and Rh blood groups are determined
in regular blood typing test. In a diagnostic lab, Monoclonal antibodies are available
for A, B and Rh antigen. Monoclonal antibody against Antigen A (also called Anti-A)
comes in a small bottles with droppers; the monoclonal suspension being BLUE in
colour. Anti-B comes in YELLOW colour Anti-D. (monoclonal antibody against Rh) is
colourless. The entire colour codes are universal standards. When the monoclonal
antibodies are added one by one to wells that contain the test sample (blood from
patient), if the RBCs in that particular sample carry the corresponding Antigen, clumps
can be observed in the corresponding wells. A drop of blood is left without adding any
of the antibodies; it is used as a control in the experiment. The monoclonal antibody
bottles should be stored in a refrigerator. It is recommended to tilt the bottle a couple
of times before use in order to resuspend the antibodies that have settled at the
bottom of the bottle.
DETERMINATION of BLOOD TYPE by AGGLUTINATION

gu1e1eren.yaem1rel/UAU/lmmuno 1ogy/Uece1'11ber2Ul!:l
 Eastern Mediterranean University
BIOL 412 - Immunology
Faculty of Arts and Sciences
Department of Biological Sciences

DETERMINATION of BLOOD TYPE by AGGLUTINATION

LAB REPORT

Student no:
Name: Submission Date:
Group: Experiment Date:

Heading: Exercise # and Title of the lab

Purpose: A short paragraph describing the objectives of the lab.

Please write a short summary about principle of this test.
1. Explain the mechanism of agglutination you have observed in this test. Explain
with an immunological approach.
2. What is the purpose of blood group typing?

Procedure : Brief outline.

• Materials include: items and the chemicals used in the experiment.

• Methods are usually presented chronologically. Schematically explain the

method you have followed.

Results :
1. Make a table of your result evaluation, explaining how you have decided on
the specific blood type, who can donate blood for you and to whom you may
donate?

2. Name one disease that mismatch of blood groups of parents will lead to fetal
death; Explain the disease shortly.

Conclusions/Discussion:
What is your interpretation of the results you have obtained?
• “What does it mean?”

– summarise your findings

– Try to explain your results

 “What are the errors (possible errors)? How to eliminate them?

Conclusion: In a single paragraph and Sum up what happened in the experiment.

Eastern Mediterranean University
BIOL 412 - Immunology
Faculty of Arts and Sciences
Department of Biological Sciences