1._BLOOD_Donation_and_Testing_Blood_Transfusion.pptx_5.pptx

BLOOD TRANSFUSION
Blood Donation and Testing
JB 2020

BLOOD Donation and Testing (Blood
Transfusion
Session 1. Selection of suitable blood donors
• By the end of this session, students are expected to be able to:
• Define terminologies used in blood donation (blood donor, blood
recipient ).
• Explain types of blood donors.
• Explain Historical background of blood donation.
• Describe criteria for suitable blood donors (Hb, pre-counselling:
age, weight, history of travel, medical history, history of
transfusions and last donation date).
• Describe specimen collection procedure for selection of suitable
blood donors for Blood Transfusion tests (for Hb, ABO and Rh, type,
and transfusion transmissible infections(TTIs).
• Describe the method of haemoglobin (Hb) estimation in Blood
Transfusion for blood donor selection.

• Blood transfusion: The transfer of blood or
blood components from one person (the
donor) into the blood stream of another
person (the recipient).
• Blood donation is a process by which a blood
donor voluntarily has blood drawn for storage
in a blood bank for subsequent use in a blood
transfusion

Types of blood donors
• There are four types of blood donors
1. Voluntary non remunerated (un paid donors)
– It is advisable to group donors into two main groups, namely
autologous and allogenic Voluntary non-remunerated, replacement or
paid and commercial donors are all allogenic blood donors
2. Replacement or family unpaid donors
3. Autologous donations
4. Paid commercial or professional donors
Definition of autologous: involving one individual as both donor and
recipient; the patient donates blood for himself and it is saved for
use within one month.

Historical background of blood transfusion
The idea of transfer of blood from a healthy person
to a sick one is an ancient idea.
Attempts were made but failed because of two
problems:
• Blood to be transfused clotted during the
procedure
• Patients suffered severely and sometimes fatal
reactions to donated blood

• In 1900 Karl Landsteiner discovered the ABO
blood group system and its importance in the
safe transfusions of blood was recognized.
• It was also suggested that blood groups were
inherited characters.
• Landsteiner and Wiener also discovered Rhesus
blood group system in 1940. This discovery led to
recognition of Hemolytic disease of the newborn.
• Other blood groups were subsequently
discovered which include MN, P, Lutheran, Kelly,
Duffy, Kid and Lewis blood Group Systems.

Criteria for suitable blood donors
This criteria is in place to protect both the donor and the
potential recipient(s).
• Curious about whether you can donate blood? Learn the
age, weight, and other considerations for donating blood
• There are four criteria to be considered in selection of
suitable donor. These are:-
I. Pre donation counseling
II. Medical History
III. Health check criteria
IV. Post donation counseling.

1.Pre donation counseling
• The donor must be informed about the
diseases that could be transmitted through
blood transfusion such as HIV, HBV,HCV,
malaria, syphilis, etc
• Provide the knowledge about the
consequences of the laboratory results

2.Risk behavior for Transfusion Transmissible
Infections
• Having more than one sexual partner
• Prostitution/commercial sex work
• Homosexual (man having sex with other Man)
• Bisexual (having both sex with man and woman)
• Injecting drug use
• Skin scarification and tattooing
• Window period for HIV infection should be
explained well to the donor

3.Medical history:
• Before donating blood, the donor should
always be asked whether he/she is suffering
from heart diseases, kidney problems, Ulcers,
Asthma, Diabetes, high blood pressure,
Rheumatic fever, Malaria, Tuberculosis,
Hepatitis, HIV/AIDs, STI, Menstrual problems
or under breast feeding, Pregnant or any drug
use.

4. Health check criteria
• Blood pressure should be normal: up to
150/60 (accept)
• Body weight should be 50kg and above
• Age should range from 18- 65 years old
• Haemoglobin should be normal 12.5g/dl –
18.5g/dl

Health check criteria..
• Physical assessment of the donor for symptoms such as skin rashes
and swollen glands or needle marks might indicate injection drugs
use.
• The time of the donors last meal to avoid fainting attack during
donation
• Last donation date
• Clean, safe, pleasant and convent environment
• Screening tests should be performed on all donated blood in
accordance with the National policy and strategy on blood
screening. Quality measures should be in place for specimen
collection and handling, analysis and reporting and record keeping.
Instruments and equipment should be well maintained, reagents
should be fresh and not expired and quality control should be
performed to check the reliability of results before patient testing is
performed.

Post donation counselling
• Post donation counselling is a process where a
donor gets back a feedback of his test results
voluntarily. Donors should be advised of the
need to contact the BTS if they become
unwell, particularly with an illness that they
may have incubating at the time of donation
(usually within 14 days of donation)

• Post donation counselling should be conducted as soon as practical
after test results are available. It should be provided by a trained
health care professional who is able to explain the results, elicit the
donors history and risk for the TTIs and counsel the donor with
understanding and empathy. Post donation counselling should
always be conducted privately in a safe and conducive environment
that protects the donors confidentiality.
• Post donation counselling enables the BTS to collect demographic
and risk exposure information about TTI positive donors as part of
its haemovigilance programme. This information can be valuable in
making future decisions about donor selection and the usefulness
of questions in the donor questionnaire.
• Note: Observe professional ethics in patient care and
communications, maintaining confidentiality.

Record Management
• Donated units of blood are labelled and tracked
carefully so that they can be taken out of the
blood donation supply if tested positive for any of
the Transfusion Transmissible Infections.
Confidentiality of donated blood results is strictly
maintained but is used for ensuring safety of the
blood supply and for collecting data for the
haemovigilance programme

• Specimen collection procedure for selection of
suitable blood donors for Blood Transfusion tests
For the blood transfusion screening test, the blood should
be collected in vacutainer ® system (for
Haemoglobin, ABO and Rhesus, type, transfusion
transmittable infections (TTI,S) according to SOPs.
• Refer to Specimen Management Module 7 MLT 04207
for specific details of blood collection and to
Laboratory Safety and Waste Management Module 3
MLT 04103

• Method of Haemoglobin estimation in Blood Transfusion
for blood donor selection
for blood donor selection is by use of copper sulphate.(and
other methods available)
for blood donor selection is by use of copper sulphate or
other rapid method. (The principle of copper sulphate will
be discussed in future sessions in this module. Rapid
haemoglobin estimation by modified Drab kin's using the
Hemocue ® was discussed and demonstrated in MLT 04205
Basic Laboratory Investigations.

• Before being eligible to donate blood or blood components,
prospective donors must be tested to determine if they have
adequate amounts of hemoglobin for their safety.
• The minimum qualification is 12.5 grams per deciliter (g/dL)
(Female) and 13.5g,dl (Male) and causes the majority of donor
deferrals.
• One of the standard methods involves placing a drop of blood into a
test tube/ universal bottle of copper sulfate solution: if the drop of
blood sinks to the bottom in an acceptable amount of time,(30 sec)
the donor qualifies.
• If the drop of blood floats or takes too long to sink the donor is
deferred. This article describes, for educational purposes, the steps
involved in qualitatively testing blood for hemoglobin level using
copper sulphate.

• The copper sulphate solution is placed into a clear, clear brown
container that is kept at room temperature and covered to prevent
evaporation. A new tube/bottle is used daily or after 20-25 tests.
• A small amount of blood is produced from the side of an alcohol
swabbed finger using a lancet followed by pressure at the stick site.
The blood drop is then drawn into a small capillary tube by capillary
action. The finger prick site has gauze applied to it to stop any
bleeding.
• A latex dropper bulb is then attached to the capillary tube containing
the blood. The dropper bulb is squeezed slightly to expel a blood drop
half an inch above the now opened copper sulfate test tube. The
blood drop automatically forms a pellet upon contact with the copper
sulfate. The used capillary tube is disposed of as biohazardous waste.

• The blood drop is observed for a short time to determine whether it
sinks (donor haemoglobin above 12.5 g/dL cut-off) or swims (donor
hemoglobin MAY be below 12.5 g/dL cut-off). Since the test is just an
estimate, many false-negatives tests (hemoglobin is not <12.5 g/dL)
are produced and hemoglobin may be checked in another more
accurate manner.
• Principle of the method (How the test works): the specific gravity of
blood is directly proportional to the amount of hemoglobin it
contains.
• Blood with a hemoglobin level of 12.5 g/dL has a specific gravity of
1.053 g/mL. Hence when a drop of blood is placed in a solution having
specific gravity of 1.053 g/mL, it will sink if it has a higher specific
gravity and thus greater than 12.5 g/dL hemoglobin and vice versa.
• A haemoglobin of 12.5 g/dL corresponds to a 38% hematocrit (percent
blood volume occupied by the red cells).

• References.
• P.L. Mollison, C.P. Engelfriet Marcela Contreras
(1993): Blood Transfusion in Clinical Medicine
9th Edition Blackwell Scientific Publications.
• Medline Plus online medical dictionary:
http://www.nlm.nih.gov/medlineplus/mplusdi
ctionary.html

Session 2. Collection Of Suitable Blood Donors and Practical
Material and reagents required .
• Blood bags, syringe, prickers,
• Sphygonomanometer
• Capillary tubes,
• Copper sulphate solution
• 70% methanol,
• sterile swab,
• donation bed,
• ABO and Rhesus antisera,
• water bath,
• Syphilis, Hepatitis and HIV testing kits,
• weighing scale and spring balance

Specimen collection procedure for selection of suitable
blood donors for Blood Transfusion tests10 minutes
For the blood transfusion screening test, the blood should
be collected in vacutainer ® system, or other evacuated
tubes and holder. The procedure for venipuncture that
was learned in MLT 04207 Basic Laboratory Specimen
Management should apply. Universal precautions
should be practiced including the use of personal
protective equipment and safe disposal of sharps as
waste as learned in MLT 04103 Laboratory Safety and
Waste Management. For (ABO and Rh, type,
transfusion transmissible infections)

Method of Hb estimation in Blood Transfusion for blood donor selection 4
• Method of Haemoglobin estimation in Blood Transfusion for blood donor
selection is by use of copper sulphate
• Before being eligible to donate blood or blood components, prospective
donors must be tested to determine if they have adequate amounts of
hemoglobin for their safety.
• The minimum qualification is 12.5 grams per deciliter (g/dL)(female) and (
13,5 grams per deciliter (g/dL) (male), and causes the majority of donor
deferrals.
• One of the standard methods involves placing a drop of blood into a test
tube/universal container of copper sulphate solution: if the drop of blood
sinks to the bottom in an acceptable amount of time, the donor qualifies.
• If the drop of blood floats or takes too long to sink the donor is deferred.
This article describes, for educational purposes, the steps involved in
qualitatively testing blood for hemoglobin level using copper sulphate.

• References:
• P.L. Mollison, C.P. Engelfriet Marcela Contreras (1993): Blood Transfusion
in Clinical Medicine 9th Edition Blackwell Scientific Publications.
• Dacie and Lewis (2001): Practical Haematology, 9th Edition (SM Lewis, B.J.
Bain, I Bates, Churchill Livingstone Inc.;
• R.D Eastham (1984): Clinical Haematology 6th Edition ELBS, John Wright &
Sons Ltd;
• Mary .L. Turgeon (1999): Clinical Haematology; Theory and Procedures 3rd
Edition Lippincott & Wilkins (A Wolters Kluwer Company);
• Radak (1995): Haematology Clinical Principles and Applications 2nd Edit.
Sounders and Imprint of Elsevier; and
• S.M. Lewis (2001): Decie and Lewis Practical Haematology 9th Edit.
Harcourt Publishers Limited.
•

• Session 3. Maintenance of cold chain process for
blood and blood specimens
• Learning Objectives
• By the end of this session, students are expected to
be able to:
• Define cold chain and an unbroken cold chain
• Explain the importance of the cold chain process
• Describe how to maintain the cold chain process for
blood, blood d product and blood specimens
according to SOPs (ice container during transportation

Definition of cold chain
• A cold chain is the systematic process for the safe storage
and transportation of blood from its collection from the
donor to its administration to a patient who requires
transfusion. It is referred to as cold because blood and
blood and blood products must be kept cold as they are
moved from one storage facility to another until they are
required for transfusion. The blood cold chain fragile, one
weak link and the chain may break. Always remember that
the failure in quality in any part of the chain can have a very
serious even fatal consequences for the recipient
• An unbroken cold chain is an uninterrupted series of
storage and distribution activities which maintain a given
temperature range

Explain the importance of the cold chain
process.
• It help to extend and ensure the shelf life of
products such as fresh frozen plasma,
cryoprecipitate, whole blood, blood grouping
sera and blood specimens according to SOPs.
Shelf-life is how long the blood product is safe
to be transfused into a patient

• Describe how to maintain the cold chain process
for blood and blood specimens according to
SOPs (ice container during transportation)
• The blood cold chain is often thought to be
simply a collection of refrigerators, freezers and
cool boxes but the importance of people like you
cannot be stressed enough. Even if the finest and
most modern equipment and vehicles are
available, the blood cold chain will not be
effective if people do not handle properly.

• Cold chain is also important during transportation from mobile
collection sites to the blood transfusion centre. It is very important
to consider good manufacturing practice (GMP) when choosing
refrigerators, freezers and cool boxes. Transportation containers
need to validated by continuously monitoring their temperature
before they are used to transport products. Special thermometers
can be used which monitor minimum and maximum temperature.
Special tape that changes colour when temperature falls outside of
expected range can also be used to monitor temperature during
transport.
• Cool box should be used when transporting blood, and blood
products from the National Blood Transfusion Services to the
Individual Hospital Blood Bank.
• These insulated cool boxes are strongly built and are designed to
take an ice in the centre such that the blood and blood products are
maintained at 4-60c for at least six hours

• Use of Blood transfusion refrigerator: These
refrigerators are known as blood bank refrigerators. It
should have an excellent thermal insulation properties
and ability to maintain a temperature of 40c plus or
minus 20c
The Blood transfusion refrigerator should have the
following properties
• A continuous temp recorder.
• Must be connected to an audible alarm system which is
triggered if the temp rises or falls outside the
prescribed limit. The alarm should sound in place
where staff are always on duty.

• Temp control chat should be displayed on the wall of
the Blood transfusion refrigerator
• Thermometer should be placed inside the refrigerator
as a control.
• Note. There should be an Auto-alternative source of
power.
• A cold chain can be managed by a quality management
system. It should be analyzed, measured, controlled,
documented, and validated. Record keeping is
important so that blood transportation conditions can
be monitored over time as part of haemovigilance and
other quality management measures

REFERENCES
• Dacie and Lewis (2001): Practical Haematology, 9th Edition
(SM Lewis, B.J. Bain, I Bates, Churchill Livingstone Inc.;
• R.D Eastham (1984): Clinical Haematology 6th Edition ELBS,
John Wright & Sons Ltd;
• Mary .L. Turgeon (1999): Clinical Haematology; Theory and
Procedures 3rd Edition Lippincott & Wilkins (A Wolters
Kluwer Company);
• Radak (1995): Haematology Clinical Principles and
Applications 2nd Edit. Sounders and Imprint of Elsevier; and
• S.M. Lewis (2001): Decie and Lewis Practical Haematology
9th Edit. Harcourt Publishers Limited.
•

• Session 4. Maintenance of Cold Chain Process For
Blood and Blood Specimen Practical
Identify the materials used to maintain or control cold
chain in blood donation.
• Blood transfusion refrigerator,
• Temperature control chat
• Thermometer
• Alarm system
• Ice packs
• Cool box

Apply the cold chain process for blood specimens storage according to SOPs .
Demonstrate the Process of cold chain for blood specimens.
• Visit Blood transfusion refrigerator (Blood bank) and have students observe the following
properties and note in their laboratory logbook:
• A continuous temp recorder.
• Whether the refrigerator is connected to an audible alarm system which is triggered if the
temp rises or falls outside the prescribed limit. The alarm should sound in place where staff
are always on duty.
• Observe range of temperature and fill Temp control chat which is displayed on the wall of the
refrigerator.
• Use the Thermometer control the temperature inside the refrigerator and record your
findings.
• Apply Cool box when transporting blood, see how is insulated strongly built and are designed
to take an ice in the centre such that the blood and blood products are maintained at 4-6 for
at least six hours. Record your findings how temperature is maintained in the cool box.
• Observe the current records and previous months records for documentation of cold chain of
refrigerators and cool boxes.

• Session 5 Describe Blood Transfusion terms and the
principles of Immunology related to Blood Transfusion
• Learning Objectives
• By the end of this session, students are expected to be
able to:
• Define the terms relevant in blood transfusion-
(compatibility, rouleaux formation, cold agglutinin,
agglutination. Autoimmune haemolytic anaemia, fresh
frozen plasma , packed red cells, frozen plasma, platelets
haemolytic disease of the new born and Immunoglobulin)
• Explain types and role of immunoglobulins: IgG G, IgM M,
Ig A, Ig D, Ig E
• Explain the basic immune function

• Compatibility test:
– Is the test used to detect or to demonstrate ABO grouping
and clinical significant antibodies to red cell antigens. This
test is important for blood transfusion services unless
there is an urgent need for blood, Compatibility test must
be done in order to avoid transfusion reaction.
• Rouleaux formation
– Is a pseudo form of agglutination which can be easily
misinterpreted as a positive reaction, particularly by the
less experienced.
– are stacks of red blood cells (RBCs) which form because of
the unique discoid shape of the cells in vertebrate body.

• The flat surface of the discoid RBCs give them a large
surface area to make contact and stick to each other
like a pile of coins; thus, forming a rouleaux. They
occur when the plasmaprotein concentration is high,
and because of them, the ESR
(erythrocytesedimentation rate) is also increased. ESR
is a non-specific indicator of the presence of
inflammatory disease, often when plasma protein
concentation is high.
• Conditions which cause rouleaux formation include
infections, Multiple myeloma, inflammatoryand
connective tissue disorders, and cancers. It also occurs
in diabetes mellitus.

Cold agglutinin.
• Antibodies that cause clumping of red blood
cells when the blood temperature falls below
normal body temperature (98.6°F/37°C) or
antibody which aggregates a particulate
antigen, e.g., bacteria, following combination
with the homologous antigen

Agglutination :
• Is the binding together of antigens and antibodies which results in
the formation of visible clumps,
• Clumping of red blood cells is caused by multivalent antibody which
cross-link cells.
• The clumps fall to the bottom of a tube. Multivalent antibodies are
effective against, and combine with, more than one antigen on the
red blood cells.
• Laboratory agglutination reactions may be performed as slide and
tube tests.
• The slide test is usually a screening test while the tube test is a
confirmatory test.
• Refer to MLT 04207 Basic Investigations where students first
learned about tile testing for ABO and Rh blood grouping

• Autoimmune haemolytic anaemia occurs
when antibodies directed against the person's
own red blood cells (RBCs) cause the RBCs to
burst (Lyse), leading to insufficient
haemoglobin and red blood cell count. The
lifetime of the RBCs is reduced from the
normal100–120 days to just a few days in
serious cases. Half and the intracellular
components of the RBCs are released into the
circulation causing more complications

Packed red cells:
• These are cells whose plasma has been removed
almost completely. This is commonly the blood product
given to patients.
• Whole blood:Complex fluid consisting of different
blood cells suspended in a yellowish liquid called
plasma. The blood cells comprise a mixture of red cells
(erythrocytes),white cells (leucocytes and platelets
(thrombocytes).
• Frozen plasma blood: Refers to the liquid portion of
human blood that has been frozen and preserved 24
hours a blood donation.

Fresh frozen plasma.
• The term fresh frozen plasma (FFP) refers to the
liquid portion of human blood that has been
frozen and preserved few hours (6 to 8) after a
blood donation which preserves the coagulation
factors. It is used in patient who have severe
bleeding like post partum haemorrhage due to
dysfunction of clotting mechanism, severe burn
due to reduction of plasma.

Haemolytic disease of the new born (HDFD)
• Is a diseases of newborns and foetuses which is
usually caused by Rhesus D or ABO
incompatibility due to destruction of red cells.
• Haemolytic Disease of the Newborn is caused by
maternal IgG antibodies crossing the placenta
into foetal circulation where it causes damage to
foetal red cells if they possess the antigen that is
specific for the maternal antibody. Levine and
Stetson discovered this condition in 1939. It can
be fatal to the foetus or newborn

Immunoglobulin(Antibodies)
• Are proteins which are produced from the
globulin part of plasma proteins by B
lymphocytes (specialized WBCs)
• They are termed immunoglobulin and are
subdivided into five subclasses i.e. IgG, IgA,
IgM, IgE and IgD.
• All the immunoglobulins have a similar basic
structure

Types and role of immunoglobulin 50 minutes
• Types of immunoglobulin:
• Five subclasses of Immunoglobulin. The immunoglobulins
can be divided into five different classes, based on
differences in the amino acid sequences in the constant
region of the heavy chains. Note the diagram of IgG which
shows the heavy chains and light chains. All
immunoglobulins within a given class will have very similar
heavy chain constant regions. These differences can be
detected by sequence studies or more commonly by
serological means (i.e. by the use of antibodies directed to
these differences).

• IgG - Gamma heavy chain
• IgM - Mu heavy chains
• IgA - Alpha heavy chains
• IgD - Delta heavy chains
• IgE - Epsilon heavy chains

General Functions Of Immunoglobulins
• A. Antigen binding
Immunoglobulins bind specifically to one or a few closely
related antigens. Each immunoglobulin actually binds to a
specific antigenic determinant. Antigen binding by
antibodies is the primary function of antibodies and can
result in protection of the host.
• The valency of antibody refers to the number of antigenic
determinants that an individual antibody molecule can
bind. The valency of all antibodies is at least two (divalent)
and in some instances more (multivalent or polyvalent).
These antigens could be found on incompatible blood and
the patient recipient has antibodies that bind to the blood
cell antigens and allow them to be destroyed.

B. Effector Functions
Frequently the binding of an antibody to an antigen has no direct biological effect.
Rather, the significant biological effects are a consequence of secondary "effector
functions" of antibodies. The immunoglobulins mediate a variety of these effector
functions. Usually the ability to carry out a particular effector function requires
that the antibody bind to its antigen. Not every immunoglobulin will mediate all
effector functions. Such effector functions include:
• Fixation of complement - This results in lysis of cells and release of biologically
active molecules
• Binding to various cell types - Phagocytic cells, lymphocytes, platelets, mast cells,
and basophils have receptors that bind immunoglobulins. This binding can activate
the cells to perform some functions. Some immunoglobulins also bind to receptors
on placental trophoblasts, which results in transfer of the immunoglobulin across
the placenta to the foetus.

• As a result, the transferred maternal
antibodies provide immunity to the foetus but
in the case of haemolytic disease of the
newborn, it can harm the infant when
maternal antibodies attack RBCs with Rh
antigens. Due to thei large size, IgM
antibodies can not cross the placenta to the
foetus so only IgG antibodies are associated
with HDFN

Major Functions of the Immune 45 minutes
• The immune system that protects the body
from foreign substances, cells, and tissues by
producing the immune response. It includes
especially the thymus, spleen, lymph nodes,
special deposits of lymphoid tissue (as in the
gastrointestinal tract and bone marrow),
lymphocytes including the B cells and T cells,
and antibodies

Organs of the Immune System Cells of the Immune System
www.niaid.nih.gov people.eku.edu

Natural Immunity
• The first set of the main function of the immune
system is defense against microbial infection. The other
major functions of the immune system depend on a
number of major actions that defend us against threats
to our health. These actions help our immune system
sort out a system of complicated reactions that our
body uses every day to defend us from infection and
disease. When these properties are not functioning,
they often spell disaster if not watched carefully

• Functions is usually called "natural" or
"innate" immunity since it does not have to
be turned on and is always (under normal
circumstances) present. This set of functions
is non-specific, which means that it does not
single out a specific microbe when defending
the body and does not require the body to
identify the potential pathogen before it acts.
This includes barriers, complement,
phagocytosis and inflammation

• Barriers
– Natural immunity includes things like our skin or
other organs that create a barrier between our
insides and the infectious outside world. Another
type of barrier is a secretion like a tear or mucous
that is sloughed off and takes the infectious agent
with it.

Complement
– Natural immunity includes proteins called complement that make it more
attractive for a microbe to undergo phagocytosis. These complement proteins
attach themselves to the target microbe to mark it for death in the spleen and
lymph nodes by labeling it an unauthorized microbe. They also stimulate
inflammation and final bacteria cell destruction by a series of reactions that
destroys cell membranes. Complement can play a harmful role in
autoimmune haemolyticanaemia which destroys the patient’s red blood cells
from their own antibodies and complement.
Phagocytosis
• Natural immunity includes phagocytes that "eat" microbes appearing to
be a threat within the body. Phagocytes are any cell that eats another. The
most common of these is the white blood cell. If the phagocyte cannot
identify the microbe, it eats it by surrounding it and dumping digestive
enzymes to break it down into digestible pieces

Inflammation
– Natural immunity includes inflammation. Inflammation is also referred
to as flare, flame and wheal in medical schools. The "flare" is the
redness. The "flame" is the heat of inflammation and itch. Fever is
also a part of this reaction. "Wheal" is the swelling of the area caused
by fluid getting under the area and phagocytes migrating into the area
which results in pus formation.
Aquired Immunity
• Acquired immunity includes all of the reactions in the body that
need lymph node involvement. Acquired immunity is also known as
adaptive immunity or specific immunity. This process is broken into
two parts that include what happens in the fluids (humoral
immunity) and what happens in the rest of the body (including
cellular immunity

Humoral vs Cellular Immunity
• Humoral immunity involves making antibodies by
B lymphocytes when they are educated by pieces
of antigens processed by macrophages, mast cells
and monocytes about the threat. The B
lymphocytes and plasma cells (mature B
lymphocytes) then make antibodies that bind to
either a microbe or a toxin to mark it so that it is
seen as a good candidate for elimination by the
spleen and lymph tissue and speeds up the
process.

• Cellular immunity involves T lymphocytes which
do two things: They change so that they activate
B cells to "raise the alarm" and start
inflammation. T cells also undergo changes to
allow them to become a cell that kills other cells
(T cytotoxic lymphocytes and Natural killer cells)
T suppressor lymphocytes help to turn off the
inflammatory response when the foreign
substance has been destroyed. They suppress
the function of other cells including T
lymphocytes

References:
• Dacie and Lewis (2001): Practical Haematology, 9th Edition
(SM Lewis, B.J. Bain, I Bates, Churchill Livingstone Inc.;
• R.D Eastham (1984): Clinical Haematology 6th Edition ELBS,
John Wright & Sons Ltd;
• Mary .L. Turgeon (1999): Clinical Haematology; Theory and
Procedures 3rd Edition Lippincott & Wilkins (A Wolters
Kluwer Company);
• Radak (1995): Haematology Clinical Principles and
Applications 2nd Edit. Sounders and Imprint of Elsevier; and
• S.M. Lewis (2001): Dacie and Lewis Practical Haematology
9th Edit. Harcourt Publishers Limited.

• Session 6 . Describe principles of ABO blood group
antigens and antibodies related to Blood Transfusion
able to:
• Describe the ABO and blood group system (antigen and
antibodies and inheritance) purpose, principle, results.
• Describe the purpose, principle and expected results of
ABO blood grouping.
• List the clinical significance of mistyping ABO blood
group.

• Inheritance is passing on of genes from each
parent to the offspring.
• Genotype is a specific gene combination
inherited from both parents.
• Phenotype is the physical characteristics that can
be observed directly to reflect the genetic
makeup.
• Recessive genes do not provide the phenotype
and are silent genes but will be passed to
offspring.
• Dominant genes do provide a phenotype

• The ABO blood group system (antigen and antibodies and
inheritance)
INHERITANCE OF ABO BLOOD GROUPS:
• Blood groups are inherited from our biological parents,
• Inheritance of ABO groups depend on three genes A, B and O.
• A and B genes express themselves dominantly to O gene.
• The A and B genes express themselves co-dominantly.
• The O gene is called an amorph, which is a recessive gene that does
not show observable change when present in the homozygous
form.
• Individual inherit the A, B, or O gene from one parent and the A, B
or O gene from the other parent making a pair of genes called
genotype

• The genes direct production of an enzyme which places certain antigens
on the red blood cells. The antigens are unique polysaccharide (sugar)
structures.
• Six genotypes are possible: AA, AB, AO, BB, BO and OO.
• Phenotypes are the physical expression of the genotypes and are easily
measured by ABO blood grouping.
• There are four phenotypes from these genotypes: A, AB, B and O.
• Red cells of genotype AA, AO phenotype as A, so can not be differentiated
by type.
• Likewise, BB and BO phenotype as B and can not be differentiated by type.
• These cells however have observable reactions (phenotype) that can be
differentiated.
• AB and OO cells are both phenotypes and genotypes.
• The Rh D group is also dominant and will be expressed if inherited from
either parents, but is made up of several combinations of genotypes

• LAWS OF HEREDITY
• Heterozygous: two different genes are inherited; eg. AB or AO
• Homozygous: the same gene is inherited from each parent; eg. AA
or OO
• Two laws of heredity have been proved
• Offspring cannot possess antigen A or B antigens alone or in
combination except that it is inherited from one or both parents.
• The parent of group AB cannot produce an offspring of groups O
nor can a parent of group O give rise to a child of groups AB.
• This is because the AB is a heterozygous so that A gene must come
from one parent and the B gene from the other. A rare form called
cis AB exists

• INCIDENCE OF THE ABO GROUPS.
• The incidence of the ABO groups varies strikingly in
different parts of the world and certain races have a
predominance of different groups to others; for
example, Negroes have a higher percentage of group B
within their ethnic group. The approximate incidence
of the ABO groups in Britain is given below, but even in
this small part of the world there is a difference
between the north and south of the country.
• Group O = 47% Group B = 8%
• Group A = 42% Group AB = 3%

• ABO ANTIGENS.
• The Genetic pathway
• The pathway begins with precursor substance
which is common to all human RBC
• The precursor is converted into H-substance in
the presence of H-gene three
• There are common alleles at the ABO locus on
the chromosome of A,B and O gene.

• The A and B alleles encode the glycosyltransferases,
enzyme that produce the A and B antigens respectively.
• O alleles does not contain a functional enzyme therefore
the red cells from group O individual lack A and B antigen.
•
• The ABO antigens are not fully developed at birth but cord
blood cells can be grouped without a problem except for
weaker subgroups, which may be difficult to detect.
• There are many antigen sites on a single red cell, which
express presence or absence of various blood groups.
• It is estimated that the A antigen is represented by 1million
times and the B antigen 700,000 times.

• In AB individual A sites are reduced to 500,000 sites.
• ABO antigens are also present in WBC, platelets and tissue cells.
• Less common ABO group: Bombay Oh
• Especially found in Asian or people whose origins are from India
– Lack H antigen & ABO Ag expression
– Both RBCs & secretions lacked H & ABO expression
– Forward & reverse typing typical for group 0 –but strong antibody
called anti-H is found in addition to other A, B or AB antibodies.
– Anti-H is clinically significant because it is capable of strong reactions
with all blood groups except another Oh. It is very difficult to find
compatible blood for these people except for another Bombay type.

• Purpose, principle and expected ABO blood grouping
• This means that:
• Blood should not be transfused to people if it carries an
ABO antigen, which the recipient is lacking. For example,
type B blood group should not be given to type A or type O
recipients.
• ABO grouping can be performed on both cells and serum.
• Serum and cell grouping acts as a double check to ensure
that correct ABO group has been determined. The cell
grouping is often called the type or “front” type and the
serum grouping is often called the “reverse” type or group

• ABO ANTIBODIES.
• ABO antibodies are usually called “naturally Occurring” or non-red-
cell-stimulated antibodies.
• ABO antibodies are not present at birth and they start to appear at
the age of 4 months.
• It is suggested that these antibodies are stimulated by the
inhalation or ingestion of bacteria, seeds or foodstuffs, which have
similar chemical structure to ABO antigens (which are large sugars)
• ABO antibodies are also called” Cold” antibodies because they have
maximum reactions at 5oc. They usually are IgM type.
• Anti. A/Anti B levels are highest at the ages of 5 and 10 years.
• The levels of these antibodies decrease with age and may be
difficult to detect in elderly patients

• ABO antibodies are found in the globulin fraction
of plasma
• Are demonstrated in body fluids e.g. lymph,
exudates and milk, are not present in tears, saliva
or urine
• There are two Antigen and two Antibodies in ABO
blood group system.
• These are Antigen A and Antigen B, Antibody A
and Antibody B and anti-A,B.
• Antibodies to A and B can be tested by a reverse
or indirect type.

GROUP RBC ANTIGEN ATIBODY
A A ANT B
B B ANT A
AB A AND B NONE
O NONE ANT A AND B
•Expected Type and Reverse Type

Clinical Significance of ABO Antibodies
• Due to the non-red cell stimulated or so-called naturally occurring
antibodies in the ABO system, it is important to provide donated packed
red blood cells with minimal plasma. Type specific and type compatible
blood can be provided to the recipient, as long as the compatibility test is
not reactive. Group O used to be considered the “universal donor” since
all other blood groups are expected to be compatible with donor Blood
Group O. Group AB is sometimes considered the “universal recipient”
because all other blood groups are expected to be compatible when the
recipient is Blood Group AB.
• For example a Blood Group B recipient can receive compatible Blood
Group B and O packed cells. Since O blood lacks both A and B antigens it
is expected to be compatible.
• The following are expected compatible types of donor blood for recipients

• When selecting donor Blood groups, Rhesus (Rh) type is
also considered. This will be discussed in another lesson.
• Since AB type is rare, it is often suggested to select another
Blood group for the donor blood, such as A or O.
• It is important to not provide incompatible donor blood to
a recipient because an immediate life-threatening
transfusion reaction is likely to occur. For example, Blood
Group O should not be given A, B or AB donor blood cells
because of “naturally occurring anti-A, anti-B which will
immediately react with the donated blood and start an
immune response

Patient Recipient Group Compatible Donor Group
A A, O
B B,O
AB AB, A,B,O
O O

• Session 7. Describe principles Rhesus and other blood
group antigens and antibodies related to Blood
Transfusion
able to:
• Describe the Rhesus blood group system.
• Describe the principle and clinical significance of testing for
Rh blood groups and antibodies.
• List other blood group system of clinical significance
(Lutheran, Kell, Duffy, MNS)
• Describe compatibility testing for blood transfusion
(immediate spin, room temperature incubation

• Antigenic: a substance strongly able to
stimulate an immune response such as a
foreign protein.
• Mosaic: a mixture such as a mosaic gene is a
mixture of several
• Du: a lesser common form of Rhesus antigen
that is weakly reactive so may be typed as Rh
negative.

Description of the Rhesus blood group system (30 minutes)
The Rhesus Blood Group System
• Rhesus blood group system was discovered by Karl Lansteiner and Wiener in 1940.
• They injected red cells of the Rhesus monkey into rabbits.
• The rabbits produced an antibody, which agglutinated Rhesus monkey red blood
cells.
• However this antibody also agglutinated red blood cells of approximately 85% of
people of the white races (Caucasians).
• It means therefore that these people possessed an antigen similar to the Rhesus
monkey.
• The red blood cells, which were agglutinated by the antibody, were called Rhesus
positive.
• The remainder whose cells did not agglutinate was called Rhesus negative.
• The antigen on the red cells of these individuals was called D and the antibody was
called anti-D.

• Further investigations discovered other Rhesus antigens and
antibodies and Rhesus blood group system was established.
• More than 40 antibodies have been described in the Rhesus blood
group system.
• At the level of general use there are six common Rhesus genes,
which give rise to the Rhesus antigens i.e. - C, D and E and their
allomorphs c, d and e.
• A chromosome can carry C or c, E or e and D or d but not both.
• Each chromosome can carry the genes in only eight possible
combinations that is CDe, cDE, cDe, CDE, Cde, cdE, CdE or cde.
• These combinations are difficult to say without causing some
confusion, therefore a shorthand system has devised for easy
identification.

Shorthand system for Rhesus antigens.
• Rhesus antigens Shorthand
• Cde R1
• cDE R2
• cDe Ro
• CDE Rz
• Cde r’
• cdE r”
• CdE ry
• cde r

• INHERITANCE OF RHESUS ANTIGENS
CLOSE LINKAGE
• The three genes C or c, D or d and E or e are carried on the
same chromosome positioned close together.
• We know they are close together because an individual
receiving, for example, cde from one parent and Cde from
another, passes on to his offspring either cde or Cde.
• If the genes were positioned at some distance apart on the
chromosome the genetic frequencies would differ widely
from those observed because of ‘crossing over’ etc.
• The genes are therefore said to be closely linked

• GENOTYPE OF THE RHESUS ANTIGENS
• Since there will be two Rh chromosomes found in the red cell, eight combinations
can then be paired in 36 different ways and result in 36 possible Rhesus
genotypes.
• RHESUS ANTIGENS
• Rhesus antigens are found only on the red cells as structural part of the cell
membrane.
• Rhesus antigens are well developed before birth and can be demonstrated on red
blood cells of early fetuses
• IMPORTANCE OF THE RHESUS ANTIGEN
• All the Rhesus antigens are capable of stimulating the formation of a specific
antibody except the Rhesus d antigen.
• Studies have failed to find an antibody anti-d, which indicates that there is no
antigen

• Rh Incompatibility
• The D antigen is the most antigenic after the A and B antigens of the ABO
system.
• This means that when the D antigen is introduced into an individual who is
lacking the D antigen he will more likely form the anti- D antibody than
other Rhesus antigens.
• It is usual therefore to determine only the presence or absence of the D
antigen in the routine hospital laboratory. The second time that
individual is given D antigen (Rh positive blood), the existing anti-D will
react with the blood and have an immune response. Foetuses who have D
antigen on their red blood cells can cause the mothers who lack the D
antigen to cause an immune response and result in haemolytic disease of
the foetus and newborn.
• It is important however to note that any Rhesus antigen (except d)
transfused, which is lacking in the recipient may well give rise to the
production of the specific antibody. In clinical practice the problem is not
common with the exception of the D antigen, as stated above.

• THE DU ANTIGEN
• Rare genes exist in the Rhesus blood group system.
• The most important of these genes for the routine laboratory
worker is the Du antigen. The Du antigen appears to be a weaker
form of the D antigen.
• It has been postulated that the D antigen is in fact made up of a
mosaic A,B,C and D . An individual who is Rh D positive possesses all
four parts of the mosaic, whereas none of these parts is present in
the Rh D negative individual. In some people, however, one or more
of the parts of the mosaic may be missing and their red cells are
agglutinated by some, but not all, anti-D sera. The anti-D serum
should react with all four parts of the mosaic, but some may fail to
react with one part, hence variable reactions may occur

• The Du antigen does not usually react with complete anti-D, but will
react with varying numbers of different incomplete anti-D sera
depending on whether it is a high grade or a low grade Du antigen.
• When the Du antigen is present, any anti-D serum which does not
agglutinate the cells will have sensitized the cells, that is to say the
red cells will have been coated with the antibody and this may be
demonstrated using the antiglobulin test
• A Du person if given RhD positive blood may, although rarely, form
an anti-D, and similarly Du blood given to aRhd, stimulate the
formation of an anti-D. It is therefore accepted that a Du individual
is considered as Rhesus positive as a donor and Rhesus negative as
a recipient.
• Du antigen is highest among the Negro races but also occurs in
about 1% of the population in the UK.
•

• APPARENT DU.
• An apparent Du can occur in some people with the genotype R1r’
(CDe/Cde). The C on the other chromosome (in the trans-position)
suppresses the D antigen so those cells will give similar reactions to Du
cells. However, when the R1 gene is passed on to the next generation the d
antigen will appear normal.
• RHESUS ANTIBODIES
• The following rhesus antibodies have been described, Anti D, C, c, E, e.
• Anti D is the most important clinically because it can cause haemolytic
transfusion reactions and frequently cause haemolytic disease of the
newborn.
• The other Rhesus antibodies are less antigenic and rarely cause
haemolytic transfusion reactions and haemolytic disease of the newborn

• CHARACTERISTICS:
• Immune antibodies- Rhesus antibodies are always produced due to immunization by previous
transfusion or pregnancy. However there are some naturally occurring forms of anti E
• IgG – Rhesus antibodies are IgGimmunoglobulins
• Optimum temperature – They react favourably at 370C
• Rhesus antibodies don’t fix complement and do not therefore cause in vitro haemolysis
• Step 4 Other Blood Group Systems (15 minutes)
• List of other blood group system of clinical significance
• Some of the other blood group systems apart from the ABO and Rhesus are called as follows:
• MNSs
• P Kell
• Lewis
• Lutheran
• Duffy
• I,

• These blood group antigens may also be found on
red blood cells and are inherited in certain
patterns. They may stimulate production of
antibody when blood is transfused into a
recipient who is lacking the antigen. The type of
antibody formed may be IgM or IgG. Antibodies
to these blood group antigens may also be
associated with certain infections. For example,
patients who have been infected with
mycoplasma pneumonia may develop anti-I
which reacts in cold temperature phase of
compatibility testing.

• Compatibility testing (Cross-Matching) for blood transfusion 45 minutes
• Definition= Compatibility test is a test used to detect or demonstrate ABO
incompatibly and clinically significant antibodies to red cell antigens
• After determining the ABO and Rhesus D groups of the patient, Donor blood of
appropriate ABO and Rhesus D groups is also selected for compatibility tests.
• The principle of compatibility testing is to mix wash donor cells with patient serum
to see if the blood cell antigens react or agglutinate with any antibody in the
patient serum. Since anti-A and anti-B are naturally occurring, if the incompatible
blood is mixed with the patient serum containing these antibodies, agglutination
will occur. Most other antibodies will not be visible until centrifugation is applied.
Centrifugation increases the force between red blood cell antigens and serum
antibodies to enhance agglutination. Other reagents can be used in compatibility
testing to enhance detection of warm IgG antibody-RBC reactions.
• It is essential to carry out compatibility tests between the serum of recipient and
the red cells of the donor before blood transfusion because of the following
reasons

– To detect antibodies in the recipient’s serum, which
are active against antigens in the donor’s red cells.
– To detect mistakes in ABO and sometimes Rhesus
grouping.
– Compatibility is signified by no agglutination or
haemolysis reaction.
– Incompatibility is signified by agglutination or
haemolysis reaction
– Strength of incompatibility may be graded by weak,
1+. 2+, 3+ 4+

Compatibility testing for blood transfusion by immediate spin technique
• Wash donor cells three times by using physiological saline
• Make 3% cells suspension of donor cells, positive and negative control
cells and put into the tube.
• Put two drops of patients serum into the tube marked T
• Add two drops of cells suspension and mix well.
• Centrifuge the tube immediately at 3000 rpm for 30 seconds.
• Re-suspend the cells bottoms and examine for haemolysis or
agglutination.
• Quality control of reagents and charting of refrigerator and cold box
temperatures must be performed routinely with results recorded on
proper forms and properly stored for retrieval. Periodic preventative
maintenance and calibration of the centrifuge timer and speed should be
followed, documented and records kept

• RESULTS: -
• No agglutination – Compatible
• Agglutination – Incompatible
• If negative ,continue with IAT, If positive find
out the course according to SOP

• Some of the other blood group systems apart from the ABO and
Rhesus are known as follows:
• MNSs
• P Kell
• Lewis
• Lutheran
• Duffy
• I,
• Compatibility testing is performed by mixing donor cells with
recipient patient serum to see if any reaction occurs though several
phases. The most important phase is Immediate spin because it
helps to identify ABO incompatibility of the donor blood when
given to the recipient

• Session 8. Describe principles of Immunology
and blood group antigens and antibodies related
to Blood Transfusion PRACTICAL
• Identify equipment and material for ABO and Rh
blood group and compatibility testing
• Perform ABO and Rhesus blood group by tube
method
• Perform compatibility testing for blood
transfusion (immediate spin, room temperature
incubation) and IAT

• Material and Equipment for blood grouping and compatibility
Materials needed are:
• Tiles
• beakers,
• pipettes,
• normal saline,
• centrifuge,
• marker pen,
• test tubes,
• test tube rack,
• patient serum,
• blood cells,
• antisera.

• ABO BLOOD GROUPING USING TILE METHOD. 50 minutes
• ABO Blood grouping is performed to classify an individual, or a patient into one of the four ABO classifications. It is
important to know the ABO blood group of a donor and that of a patient before any step can be made in the
blood transfusion process
• CELL GROUPING BY TILE
• MATERIALS:
• Tile (opal glass), grease pencil/mark pen, Pasteur pipette, tubes, beakers physiological saline,. Anti A, Anti B, and
Anti A+B, A cells, B cells and O cells.
• PROCEDURE:
• Wash patient and the control A, B and O cells in large amount of normal saline.
• Make a 2 -5 % suspension of all cells in normal saline.
• Make squares on an opal tile using grease pencil as shown in the diagram below.
• Place one drop (volume) of each of the grouping sera in respective squares in columns marked anti A, anti B, anti
AB (O serum) using Pasteur pipette.
• Rinse Pasteur pipette three times in normal saline from one antisera to another, to avoid contamination.
• Add one drop of 2-5 % washed A, B, O control cells in the respective squares.
• Mix the serum and red cells by gently rotating the tile Read results macroscopically within 5 minutes.
• Read controls first and if they work as expected, proceed to read the tests.
• View suspension under low power microscope to check doubtful agglutinations

SERUM GROUPING USING TILE METHOD.
• PROCEDURE:
• Make2 -5 % suspension of control A cells, B cells, O cells.
• Make squares on an opal tile as shown in diagram below.
• Place one drop of each of the anti A anti B and anti AB(for controls)
• Rinse the Pasteur pipette in normal saline
• Place one drop of each of the patient serum marked T 1-T4 in respective rows.
• Rinse Pasteur pipette in saline after each addition.
• Add one drop of 2-5% washed A, B and O cells in respective columns.
• Mix the serum and cells
• Rock the tile gently as in cell grouping and read results within 5 minutes.
• View doubtful reactions under low power of Microscope
• NB: it is important to use reagents that have been checked with positive and
negative control cells to verify they are reliable for patient blood grouping.

PROBLEMS IN (ABO) BLOOD GROUPING
• CAUSES OF FALSE POSITIVE RESULTS IN ABO BLOOD GROUPING
ROULEAUX FORMATION
• Rouleaux formation is sometimes called ‘pseudo-agglutination. Rouleaux
formation is not true agglutination, because it is not caused by an
absorbable agglutinin. It does not usually give trouble to an experienced
worker.
• When rouleaux formation is marked, the auto-agglutination control tube
will be positive and any cells added to the patient’s serum will give a
similar reaction. However if the serum is diluted 1 in 2 or 1 in 3 with
physiological saline, the rouleaux formation should disappear, but true
agglutination will persist.
• Rouleaux formation may cause a mistake when reading results in tile
method if reading is delayed. A ‘graininess’ appears which may be read as
a weak positive. The graininess will disappear when a drop of saline or
albumin is added to the mixture

INFECTED RED CELLS (THE THOMSEN PHENOMENON)
• Several types of bacteria are capable of causing red cells to
agglutinate by any normal serum, except young infants’
serum. This phenomenon is called polyagglutination and
the cells are called polyagglutinable. The bacteria expose
the T antigen in the cells and most sera contain the T
antibody and therefore agglutination occurs.
• The reaction may appear within 18 h at 4oc or within
shorter periods at room temperature.
• It is important to use fresh cells or cells which have been
stored for the shortest time in the refrigerator when doing
blood grouping. In blood stored in the form of a clot, the
phenomenon is rarely found.

INFECTED SERUM
• Occasionally this gives a false positive reaction, but in most cases it gives a false
negative reaction.
• COLD AGGLUTININS
• Cold agglutinins will usually react at temperatures not higher than 250c. If they are
active at room temperature the auto-agglutination control will be positive as well
as the A, B and O cells. This type of reaction is termed pan-agglutination.
• High titre cold antibodies are sometimes formed during viral or atypical
pneumonia e.g. infection by Mycoplasma pneumonia, infectious mononucleosis or
in lymphomas.
•
• When this type of reaction is encountered, the patient’s red cells should be
washed in warm saline at 37oc to remove the cold antibody, and the test repeated.
• The serum grouping must be repeated at 37oc and the auto-agglutination control
should be negative, although at this temperature any anti-A or Anti-B present will
still react

• CAUSES OF FALSE NEGATIVE RESULTS IN ABO
GROUPING
• Impotent sera which have deteriorated being
stored incorrectly or repeatedly frozen and
thawed.
• Failure to recognise the time factor in the tube
method may cause false negative results to be
reported.
• Failure to recognisehaemolysis and report it as
such and not recorded as negative may cause
false negative results to be reported.

• CROSS MATCHING BY TUBE METHOD (40 minutes)
REQUIREMENTS: -
• Donor cells, recipient’s serum, test tubes, 30% bovine albumin, normal
saline, Pasteur pipette, water baths 300c and 370c, microscope, slides,
sensitized cells, anti-human globulin (AHG or AGS). Incomplete anti-D, O
cells
• PROCEDURE Part 1 Compatibility testing for blood transfusionby spin
technique
• Wash donor cells, positive and negative control cells three times by using
physiological saline
• Make 3% cells suspension of donor cells.
• Put two drops of patient’s serum into the tube marked T.
• Add two drops of cells suspension and mix well.
• Centrifuge the tube according to SOP (30 seconds and 3000 RPM) and
then immediately gently re-suspend the cells bottoms and examine for
haemolysis or agglutination.

Compatibility testing for blood transfusionby incubation and enhancement technique -
• Wash the donor cells three times with large volume of saline.
• Arrange four test tubes on a rack for each donor.
• Label the tubes 1, 2, 3 and 4.
• Place one drop of patient’s serum in tubes 1-3.
• Place 4 drops of patient’s serum in tube marked 4
• Add one drop of 2-5% donor cells in tubes 1-3.
• Add one drop of 30% bovine albumin in tube marked 3.
• Add one drop of 50% donor cells in the tube marked 4.
• Mix by shaking the test tube rack.
• Incubate for 30- 60 minutes as follows: -
• Tube marked 1, at room temperature (to detect cold antibodies and to detect errors)
• Tube marked 2, at 300c water bath (to detect significant cold antibodies with wide range of
temperature).
• Tubes marked 3 and 4, at 370c water bath to detect incomplete antibodies.

• Centrifuge the tubes according to SOP (minutes and RPM)
and then immediately gently re-suspend the cells bottoms
and examine for haemolysis or agglutination.
• Read results of tubes marked 1-3 using low power objective
of microscope by spreading a drop of the serum/cell
suspension on microscope slide.
• Perform Indirect Coomb’s test (ICT, IAT) on the tube marked
4 according to SOP including the use of control cells.
• Record the results of your 4 tubes for positive or negative
agglutination according to instructions from the tutor and
the SOP. An example of results are shown below

• Session 9. Describe principle for collection and safety of blood for
transfusion
• By the end of this session, students are expected to be able to:
• Define anticoagulant, TTIs, Transfusion reaction
• Explain anticoagulant used for collection of blood for transfusion
(acid citrate dextrose, citrate phosphate dextrose, and citrate
phosphate dextrose adenine
• List advantages and disadvantages of each type of anticoagulant
used in blood transfusion (storage duration)
• List common transfusion transmissible infections (TTIs): (HIV,
Hepatitis B,HCV, Syphilis)
• List causes of transfusion reactions (allergic, anaphylactic,
incompatibility / haemolysis, circulatory overload)

• Anticoagulant is any agent used to prevent the formation of blood clots.
• Anticoagulants have various uses. Some are used for the prophylaxis
(prevention) or the treatment of thromboembolic disorders
• Transfusion Transmissible Infection (TTIs) Are the infection that are
transmitted through blood transfusion example HIV, Hepatitis B, Syphilis,
Malaria, Trapanasomiasis,
• Transfusion reaction,
• a systemic response by the body to the administration of blood
incompatible with that of the recipient.
• The causes include red blood cell incompatibility; allergic sensitivity to the
leukocytes, platelets, plasma protein components of the transfused blood;
or potassium or citrate preservatives in the banked blood. See also
haemolysis.
• Adverse: bad outcome or serious and harmful consequence

• Explain anticoagulant used for collection of blood transfusion 35 minutes
• Acid Citrate Dextrose Solution (sometimes called Anticoagulant Citrate Dextrose
Solution) is a solution of citric acid, sodium citrate and dextrose in water.
• It is mainly used as an anticoagulant to preserve blood specimens required for
tissue typing
• Ingredient of Acid Citrate Dextrose (ACD)
• Dextrose (glucose)......................3g
• Tri-sodium hydrogen citrate.......2g
• Distilled water............................120mls
• Advantages ofAcid Citrate Dextrose (ACD)
• It preserve blood better.
• It preserve blood for 21 days.
• It is wide used anticoagulant.
• Disadvantages ofAcid Citrate Dextrose (ACD)
• It preserve blood for short duration

• Citrate phosphate dextrose,
• The effect of varying adenine concentrations in citrate-phosphate-
dextrose (CPD) blood was studied in an attempt to optimize the storage
conditions for human erythrocytes with regard to post transfusion viability
and oxygen release function. The maintenance of diphosphoglycerate
(DPG) was impaired by adenine supplementation; this effect was closely
related to the adenine concentration. A 0.25 mM adenine concentration in
CPD blood improved the adenosine triphosphate (ATP) levels and the post
transfusion viability markedly, without appreciably impairing the DPG
maintenance. The results suggest that CPD solution supplemented with
adenine to give a 0.25 mM concentration in the blood is a better
preservative for human erythrocytes than the commonly used acid-citrate-
dextrose (ACD), CPD, and ACD-adenine solutions with regard to
posttransfusion viability and oxygen release function. Adenine addition to
this low concentration is not expected to cause renal damage even after
massive transfusion.

Ingredient of Citrate phosphate dextrose (CPD)
• Sodium citrate......................2.63g
• Dextrose...............................2.9g
• Citric acid...............................0.327g
• Sodium acid phosphate.........0.251g
• Adenine................................0.0275g
• Distilled water............................100mls
•

• Advantages ofAcid Citrate Dextrose (APD)
• Is an anticoagulant of choice for storage of blood for
transfusion.
• It preserve red cells metabolism and hence oxygen carrying
capacity.
• After collection it preserve blood for 35 days under 4oC.
• Below 28oC out of direct sunlight APDA has half life of 2-3
years when silted with foil envelope.
• Disadvantages of Acid Citrate Dextrose (APDA)
• Beyond 35 days there is a considerable loss of viability of
blood

• List common transfusion transmissible infectious
(TTIs)
• This organisms are transmitted through blood
and body fluids
• HIV
• Hepatitis B
• Syphilis)
• Malaria
• Trypanasomiasis

List causes of transfusion reactions
• Allergic
• Anaphylactic
• Incompatibility / haemolysis
• Circulatory overload
• All of these adverse reactions can be harmful to the patient.
Incompatibility due to ABO is usually the most severe and can threaten
the life of the patient immediately and within minutes of starting the
transfusion.
• Allergic reaction may be most common and often mild. It results in a
slight fever or hives.
• Anaphylactic is less common and can be due to something in the donated
blood such as a medication that the patient has a strong allergy with.
• Circulatory overload happens in older individuals with failing heart when
large amounts of packed cells are given. It was more common when
whole blood was transfused but can still be a problem.

• Donor Haemovigliance
• Haemovigilance is a set of surveillance procedures for the
monitoring, investigation and reporting of adverse or unexpected
events or reactions in donors and transfusion recipients. It includes
epidemiological follow up of donors. The goal of Haemovigilance is
to prevent the occurrence of transfusion reactions and adverse
events and improve the outcomes for donors and patients.
• Blood donation may produce undesireable effects in donors with or
without consequence for the quality and safety of blood products.
• All adverse reactions in donors and recipients should be fully
documented and data should be regularly analysed in order to
generate possible corrective and preventative actions.
•

• Session 10. Describe the principles of tests used in
Blood TransfusionTheory
able to:
• Define Haemagglutination, Vitro antigen- antibody
reaction, Vivo antigen – Antibody reaction
• Describe type of in-vitro antigen antibody reactions
(haemagglutination)
• Explain the principle of compatibility by the tube
method in Blood Transfusion
• Explain the principle of ABO and Rhesus grouping by
the tube method in Blood Transfusion

• Haemagglutination,
• Is a specific form of agglutination that involves red blood cells (RBCs). It has two common uses in
the laboratory: blood typing and the quantification of virus dilutions.
• Blood Typing
• Blood type can be determined by using antibodies that bind to the A or B blood group in a sample
of blood.
• For example, if antibodies that bind the A blood group are added and agglutination occurs, the
blood is either type A or type AB. To determine between type A or type AB, antibodies that bind the
B group are added and if agglutination does not occur, the blood is type A.
• In blood grouping the patient's serum is tested against RBCs of known blood groups and also the
patient's RBCs are tested against known serum types. In this way the patient's blood group is
confirmed from both RBCs and serum. A direct Coombs test is also done on the patient's blood
sample in case there are any confounding antibodies.
• In-Vitro antigen- antibody reaction is a type of antigen antibody reaction which take place outside
the blood circulation
• In-Vivo antigen – Antibody reaction is a type of antigen antibody reaction which takes place inside
the circulation .

• 3 Type of antigen antibody reactions (haemagglutination) 25 minutes
• There are two types of antigen antibody reactions (haemagglutination) These are
• Vivo reaction
• Vitro reaction
• Vivo reaction
• Is a type of antigen antibody reaction which takes place inside the circulation .
• Direct Coomb’s test is done to check if there is sensitisation of red cells in vivo (Within blood
circulation).
• Sensitisation of red cells in vivo can be caused by: -
• Haemolytic disease of the new born (HDN).
• Incompatible blood transfusion reactions.
• Auto- immune haemolyticanaemia with warm antibody.
• NB: If no agglutination after addition of sensitised cells means there are sensitisation within the
circulation with weak antibodies which failed to be detected by AGS solution, then test your
evaluate by IAT.
• Control sensitised cells - Agglutination with AGS.
• No agglutination - DAT negative (no sensitisation in vivo).
• Agglutination - DAT positive (there is sensitisation

• In Vitro reaction
• Is a type of antigen antibody reaction which take place outside blood circulation .
• It literally means “in glass” such as in a test tube or laboratory conditions. Indirect
coomb’s test (indirect Antihuman globulin test (IAT)) is used:
• To check if there is sensitisation of red cells in vitro (outside the blood circulation),
• In cell typing or Rhesus D grouping using incomplete anti D
• In compatibility tests or in identification of antibodies in the blood cells
• Control sensitised cells shows agglutination with anti-human globulin (AHG or
AGS).
• No agglutination – ICT or IAT negative (no sensitisation in vitro).
• Agglutination – ICT or IAT positive (Sensitisation in vitro).
• Sensitised cells are used as control cells in Direct or Indirect Ant globulin test
• They are red cells that have been coated with IgG Immunoglobulin, which are
incapable of causing Agglutination.

Principle of ABO and Rhesus grouping
• The principle of test is based upon agglutination test and specific
antigen – antibody reaction.
• The blood group antigens are located on the surface of the red cells
and any antibody produced is found in the serum.
• Known antibody (antisera) are used to detect unknown antigen
(cells)
• If the reaction occurs when the specific antisera is added to a
patient (donor red cells) it indicate that antigen present in the red
cells surface.
• Therefore the blood group of the patient or donor can be
determined.
• Centrifugation will enhance the reaction in vitro by enhancing the
combining of antigen and antibody in a short period of time.

• Step 5 Principle of compatibility test 25 minutes
• The known red cells (antigen) from donor are mixed with known serum (antibody) from the patient
to detect if there is any incompatibility caused by ABO, Rhesus and Other blood group antibody.
• Centrifugation will enhance the reaction in vitro by enhancing the combining of antigen and
antibody in a short period of time.
• Other enhancement such as 37 C, bovine albumin and anti-human globulin are used to allow the in
vitro antibody-antigen reaction to occur more readily, if the antibody is present.
• The most important phase of compatibility testing is the immediate spin reaction will will detect
naturally occurring anti-A, anti-B or anti-A,B which would react with ABO incompatible donor blood
cells.
• Using reagents that have been verified with known positive and negative control cells is important
to provide reliable laboratory results. In addition, the centrifuge, waterbath and refrigerator should
have regularly scheduled maintenance and verify function as part of good laboratory practice.
Temperatures, PPM, QC results should all be properly recorded and records maintained. In
addition, patient and donor results are maintained in order to track the appearance of unexpected
antibodies and to prevent, if possible, adverse transfusion reactions.

• Session 11. Perform Blood Transfusion investigations
Practical
able to:
• List causes of false positive and negative results in routine
blood transfusion investigation
• Perform all tests for routine blood transfusion investigation
( ABO grouping by the tube method, Rhesus grouping by
the tube method and compatibility testing by the tube
method) according to SOP.
• Interpret results of routine blood transfusion investigation
• Suggest causes of incompatible blood transfusion
investigation results

• Material and Equipments blood grouping and compatibility are
• tiles,
• beakers,
• pipettes,
• normal saline,
• centrifuge,
• marker pen,
• test tubes,
• test tube rack,
• patient,s serum,/cells
• blood cells,
• Antisera.
•

• ABO BLOOD GROUPING USING TILE METHOD.
• ABO Blood grouping is performed to classify
an individual, or a patient into one of the four
ABO classifications. It is important to know the
ABO blood group of a donor and that of a
patient before any step can be made in the
blood transfusion process

• MATERIALS AND REAGENTS
• Tile (opal glass)Grease pencil/mark pen
• Pasteur pipette
• Test tubes
• Beakers physiological saline
• Anti A
• Anti B
• Anti A+B
• A cells
• B cells
• O cells.

• PROCEDURE:
• Wash patient,s and the control A, B and O cells in large amount of normal saline.
• Make a 2- 5 % suspension of all cells in normal saline.
• Make squares on an opal tile using grease pencil as shown in the diagram below.
• Place one drop (volume) of each of the grouping sera in respective squares in
columns marked anti A, anti B, anti AB (O serum) using Pasteur pipette.
• Rinse Pasteur pipette three times in normal saline from one antisera to another, to
avoid contamination.
• Add one drop of 20% washed A, B, O control cells in the respective squares.
• Mix the serum and red cells .
• Gently rock the tile and read results macroscopically within 5 minutes.
• Read controls first and if they work as expected, proceed to read the tests.
• View suspension under low power microscope to check doubtful agglutinations

•
• SERUM GROUPING USING TILE METHOD.
• PROCEDURE:
• Make 2..5% suspension of control A cells, B cells, O cells and patients
cells.
• Make squares on an opal tile as shown in diagram below.
• Place one drop of each of the anti A anti B and anti AB
• Rinse the Pasteur pipette in normal saline
• Place one drop of each of the patient serum marked 1-4 in respective
rows.
• Rinse Pasteur pipette in saline after each addition.
• Add one drop of 2,,5%washed A, B and O cells in respective columns.
• Mix the serum and cells as above.
• Rock the tile gently as in cell grouping and read results within 5 minutes.
• View doubtful reactions under low power of Microscope

• COMPATIBILITY TEST/ CROSS MATCHING BY TUBE METHOD 30 minutes
• REQUIREMENTS: -
• Donor cells
• recipient’s serum,
• test tubes
• 30% bovine albumin
• normal saline
• Pasteur pipette
• water baths 300c and 370c,
• microscope
• slides
• sensitized cells
• AGS.
•
• PROCEDURE: -
• Wash the donor cells three times with large volume of saline.
• Arrange four test tubes on a rack for each donor.
• Label the tubes 1, 2, 3 and 4.
• Place one drop of patient’s serum in tubes 1-3.
• Place 4 drops of patient’s serum in tube marked 4
• Add one drop of 2-5% donor cells in tubes 1-3.
• Add one drop of 30% bovine albumin in tube marked 3.
• Add one drop of 50% donor cells in the tube marked 4.
• Mix by shaking the test tube rack.
• Incubate for 30- 60 minutes as follows: -
• Tube marked 1, at room temperature (to detect cold antibodies and to detect errors)
• Tube marked 2, at 300c water bath (to detect significant cold antibodies with wide range of temperature).
• Tubes marked 3 and 4, at 370c water bath to detect incomplete antibodies.
• Read results of tubes marked 1-3 using low power objective of microscope by spreading a drop of the serum/cell suspension on microscope slide.
• Perform Indirect Coomb’s test (ICT, IAT) on the tube marked 4.

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