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CHAPTER ONE
Definition
Blood transfusion Science is a Science that deals with the study of
the various Blood Group antigens, their Medico- legal significance and
reactions. The Science stems from the importance of Blood as a
tissue fluid.
History of Blood Transfusion
The transfer of blood from a healthy individual to a sick one is an
ancient idea. Drinking of animal blood was practiced during
medieval times, but this did not produce the desired results.
In 1916 William Harvey advanced the story of blood circulation.
Within that same period Christopher Wren attempted the first
transfusion. Using an intravenous needle he transfused blood from
one dog to another.
In 1667 , a French Physician had performed a transfusion of
Lamb’s blood to a human patient , but after the death of the
second patient no further transfusion were attempted.
A scientist by name James Blundell showed that blood from one
specie could not successfully be transfused to another. This
Scientist was the person to perform a successful blood transfusion
between members of the same species. His exercise also resulted
into some problems such a clotting of Blood after transfusion.
This problem got a break through in 1900 when Karl Land
Steiner demonstrated the ABO Blood grouping system. Land Steiner
and Levine went ahead to discover another blood group system in
1927 known as the MN and P system. They also in 1939 discovered
the Rhesus System. Since then a lot of different Blood Group
Systems have been discovered e.g the Lutheran , Kell , Lewis ,
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Duffy and Kidd Systems. This scientific advancement continues
today.
BLOOD CENTRES
The Blood transfusion Science has explored the Biochemistry
nature of Blood groups antigens. The Science has greatly increased
our knowledge about Blood group systems.
A Blood transfusion Laboratory is thus a Laboratory mainly
concerned with the Blood Grouping of patients, Provision of
suitable Blood for Transfusion and detection of a typical antibodies
which may complicate blood transfusion and for Pregnancy.
Blood transfusion centers are concerned with the bleeding and
testing of blood donors and provisions of suitable blood , blood
products and reagents to Hospital Laboratories. These Centres may
also act as reference and confirmatory centres for Hospital
Laboratory findings and to perform test and solve problems
beyong the scope of a Hospital Laboratory.
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CHAPTER TWO
Blood Group Immunoglobulins
Man lives in an environment in which he is surrounded by
potentially dangerous bacteria and Viruses. It is necessary that , the
body has suitable defense in order to remove foreign organism,
which enter it. This is carried out in several ways . One of which
is the production of an antibody ( Immunogloblins) which reacts
with the foreign organism/s or antigens.
Blood group Immunoglobulins are
high molecular weight
proteins produced as a defense to remove foreign substances in
circulation. Their Production is govern by the presences of an
antigen/s. These Immunoglobulins are referred to as Antibodies.
Definition of Antigen:
An Antigen is a Substance which stimulates the production of
antibodies , and when missed with the antibody reacts in some
observable way.
Definition of Antibodies :
An Antibody is a protein formed in the spleen or lymph nodes in
response to the Presences of an antigen, and reacts specifically
with that particular antigen in some observable way.
Basic Structure of Immunoglobulins
All type of Immunoglobulins have the same basic structure . This
consist of four polypeptide chains , two long heavy chains and two
short chains which exist in two forms ( Kappa or lambda).
The heavy and light chains are joined to each other by
disulphide bonds. The disulphide bonds confer the molecule
strength but with flexibility.
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The variable region occurs at the end of both light and heavy
chains, made of a sequence of amino acids that gives the antibody ,
the antigen specificity. The more the amino acids the more the
antibody specificity.
Classes of Immunoglobulins
Immunoglobulins are classified base on the Structure of the heavy
Chain. The Classes include : IgG, IgA, IgM, IgE, IgD. Blood group
antibodies exclusively are made of IgG , IgM and IgA.
Structure of Immunoglobulin Molecule
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Basic Differences Between the Immunoglobuins
Designation
Heavy Chain
Light Chain
Molecular Wt
Sedimentation
Coef
Content in
Normal
Serum per
100ml
Percent of
Normal Igm
Placental
Crossing
IgG
y
K-λ
150.000
7s
IgA
α
K-λ
160.000
7s -gs
IgM
µ
K-λ
900.000
19s
8001600mg
140400mg
50 -200mg 0 – 40mg
10 -70mg
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6
1
0.002
None
None
80
Free
None
None
IgD
∂
K-λ
180.000
7s
IgE
€
K-λ
200.000
8s
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CHAPTER THREE
The Immune Response
When an antigen is introduced into an individual, antibodies are
produced in response to the stimulation. The production and increase
in antibody is known as the Immune response. The Peak of this
reaction usually occurs at an interval of 10- 20 days after the initial
stimulation . The response to the antigen depends on whether the
individual has previously been exposed to that particular antigen.
The response after the first dose known as the Primary or
sensitizing dose , is usually slow and weak , but subsequent exposure
to the same antigen produces a strong response with large amounts
of antibody being produced quickly. These may remain in the
Circulation for many years. The antibodies initiated by the Primary
response are Predominantly IgM. While subsequent exposure to the
antigen results in the production of IgG antibodies.
The Immune Response Curve.
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CHAPTER FOUR
ANTIGEN- ANTIBODY REACTIONS
The Forces holding an antigen- antibody complex together are :
 Ionic bonds
 Hydrogen bonds
 Vander waal forces
 Hydrophobic bonds
In Blood grouping the two most commonly observed results of
antigen- antibody reactions are :
1) Agglutination or Clumping of Red Cells caused by cross
linking of a multivalent antibody.
2) Haemolysis , where antigen antibody reaction results in
the breakdown of break down of cells.
Red cells when suspended in Saline, although appearing to touch
each other when viewed under the microscope , in fact do not as
they are surrounded by what is referred to as an ionic cloud.
Red Cells have a net negative electrostatic charge on their surface
due to the ionization of the carboxyl group of sialic acid present at
the cell membrane which attracts positive ions from the
surrounding medium. This positive layer subsequently attracts
negatively charged ions to its surface . This process continues in
layers until there is insufficient force to attract more ions and the
outer edge is called the surface of shear. These electrostatic charge
is referred to as the Zeta Potentials.
The relative size of the Immunoglobulin and the techniques for
demonstrating specific red cell antigens and antibodies takes into
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account the surrounding ionic cloud. The IgG antibodies has two
binding sites which are not enough to bind with the corresponding
antigen in saline. While the IgM antibodies has five binding sites
which make it easily reactive to its corresponding Antigen.
Fig : Illustrated Diagram
Application of Zeta Potentials
The relative size of Immunoglobulins and the techniques for
demonstrating Specific Red Cell Antigens and antibodies takes into
account the surrounding Ionic Cloud. The IgM or Complete
antibodies are large enough to bridge the ionic cloud and therefore
agglutination can occur in a saline medium.
The IgG or incomplete antibodies are much smaller and because
of this , the majority are unable to straddle the ionic cloud ,
producing blocking of the receptor site , but no agglutination in
saline. These Red Cells can be said to be Sensitized . Each
molecule of IgG and IgA has two antigen binding sites, whereas
IgM has ten . This may facilitate agglutination in saline by IgM
antibodies. In order for an IgG antibody to agglutinate two red cells
, it would have to bind each each of its two binding sites onto
separate Red cells. An IgM antibody having many more antigen
binding sites , would be able to bind more than one antigen
binding site to each red cell.
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Fig : Ilustration.
CHAPTER FIVE
THE ABO BLOOD GROUP SYSTEM
Land Steiner observed that the Red Cells of some individuals were
agglutinated by the serum of other individuals. He demonstrated
that these people could be classified into four groups according to
which of the two antigens were detectable A , B, AB and O. He also
showed that an individual possesses antibodies against the antigen
or antigens that he lacks on his Red cells.
For Example :
Antigen on Red Cell
Antibody in Serum
A
Anti- B
B
Anti-A
AB
None
O
Anti- A +B
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Because of the presence of these antibodies , the ABO system is of
major importance when transfusing blood in an individual. And
whenever possible blood should not be transfused d if it carries an
ABO antigen which the recipient lacks.
The presences of these anti A and anti B agglutins means that
ABO grouping can be performed on both cells and serum. This
acts as a double check to ensure that the Correct ABO group has
been determined.
ABO Antigens
The A and B antigens can be detected at an early stage in the
foetus, but are still not fully developed at birth. There is no problem
grouping cord blood cells with potent anti-sera , but weaker sub
groups may be difficult to detect.
There are many antigens on a single red cell which express the
presence or absence of the various blood group.
For example
Antigen A on Red cell has approximately 1.000.000 antigen sites
Antigen B on Red Cell has approximately 700.000 antigen sites
Antigen AB on Red cell has approximately 500.000 antigen sites.
These ABO antigens are also present in White Blood cells , Platelets
and Tissue cells.
GROUP SPECIFIC SUBSTANCES
Blood group Substances are carbohydrate substances called hapten
.They are non antigenic and are found in tissue cells and body
fluids. These substances are present in the saliva of certain
individuals. Individuals whose saliva contains appropriate ABO
substances are called Secretors. Secretion of these Substances are
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determined by a pair of allelomorphic genes, Se and se giving rise
to three genotypes.
Se
Se
Se
se
80%
se
se ----20%
These blood groups specifc Substances appear in two forms :
1) Water Soluble form present in body fluids and tissues
2) Alcohol –Soluble forms present in Red Cells , but absent in
body fluids.
ABO SUB- GROUPS
In 1911 , Van Dungern and Hirszfeld described two different types
of A antigens known as A1 and A2. Nearly all anti-A produced by
group B individuals contain two anti A antibodies i.e Anti A and
Anti A 1. Anti A agglutinates red cells of the group A1 , A2 , A1B
and A2B1. But anti-A1 agglutinates only the red cells of group A
and A1B.
The A2 antigens react more weakly than the A1 antigen when
mixed with anti A. This is because there are fewer antigen sites
of which the anti A can become attached. Another Subgroup of A
cells include A3, Ax , Aint , Am and Aend.
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 groups.
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ABO ANTIBODIES
They are also referred to as naturally occurring antibodies or allo
-antibodies. They are said to be formed naturally circulation
following stimulation from ABO antigens. The production of ABO
antibodies in infants does not begins until about 4 months of age.
Which means that it may only be possible to determine an infants
ABO type from a red cell group. However antibodies may be
detected in the cord blood which has been transferred via the
placenta from the mother.
Characteristics of Natural Antibodies
They have the following properties.
 React maximally at 4.0C , but the thermal range of activity
includes 37.0c
 Agglutinate cells suspended in Saline
 Are Absorbable
 The agglutinated Cells adhere very strongly and agglutinates
are difficult to break.
Anti- A and Anti- B levels are highest between the ages of 5 – 10
, after which the decrease and are sometimes difficult to detect in
elderly patients.
ABO antibodies like other antibodies are found in the globulin
fraction of plasma. They can be demonstrated in other body
fluids which contain plasma globulins such as lymph , exudates and
milk.
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ABO GENES
The presences of antigens on the RBC is determined by genes. The
genes are carried on Chromosomes which are present in the
nucleus of all Red Cells of the body. There are 46 Chromosomes
arranged in 23 pairs in each nucleus , with exception of the sex
chromosomes which has only 23 chromosomes. So that only one
chromosome from each pair is presence in the ovum and spleen.
The fusion of the ovum and sperm brings the total number back
again to 23 pairs.
The two genes ( one from each Parent ) which control the ABO
Group can be the same or different . if the genes are the same ,
the person is called Homozygous that character and if different is
called heterozygous . The gene which can occupy the same site or
locus on the chromosomes are called Allemorphy or alleles.
Ignoring the question of subgroups , the inheritance of the ABO
Group depend upon their genes A, B and O. The O gene is codominant while the AB are dominant. The O gene is an amorph
which is a recessive gene , showing no observable change when
in the homozygous form. An individual inherits the A,B and O
genes from one parent and the A, B or O gene from the other
thereby making a pair of gene called the genotype. Therefore
six genotype can occur Viz : AA , AB, BB, AO, BO and OO.
It is not possible to differentiate Red cells of genotype AA or
AO, BB or BO and so the term Phenotype is used to describe the
observed reaction . AB Red cells and OO red cells are both a
phenotype and the genotype.
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Genotype of Parents
Permutation
A
O
B
AB
BO
O
AO
OO
Possible genotype
AB , AO BO and OO
Possible Phenotype AB , A O and B
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CHAPTER SIX
ABO GROUPING SERUM
These serum are usually obtained from selected donors whose
antibody levels are suitable for us as Laboratory Reagent.
Standard Anti A serum should have a titre of 1 in 512 and anti
B a titre of I in 256, when titrated against A and B cells. Several
dilution of the serum are made in Saline and Red cells of the
appropriate group added.
The titre is the reciprocal of the highest dilution at which
agglutination occurs . Although some sera may confirm to these
requirements , the avidity of the antibody may not be suitable.
Avidity is the power of the antibody to agglutinate quickly and
strongly.
LECTINS
These are plant extracts that can react with red cells and antigens
to form agglutination .Their use can be extended to the
identification of Blood Types . For Examples :
1. Dolichos biflorus : This can react specifically with A1
antigen. It can be used to differentiate A1 and A1B red cells
from A2 and AB Red cells.
2. Ulex Europaeus : This has anti A Specificity and
agglutination of A2 , A2B cells are more strongly than A1B1
or B red cells.
General Characteristics of Antibody and Antigen Reactions
 Potency of antibody
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 Titre ( Level of Antigen and Antibody Binding )
 Specificity ( Exact Antigen binding Sites )
 Detection of Subgroups
 No Rouleaux Formation
CHAPTER SEVEN
LAW OF HEREDITY
Two Laws of inheritance have been proved in accordance with
Bernstein theory.
1. The Off- Spring cannot possess the antigen A or B alone or in
Combination except that it has been inherited from one or both
parents.
2. The Parents of group AB cannot produce an offspring of group
O , nor can parents of group O give rise to a child of group AB.
This is because the group AB is heterozygous , so that the A gene
must come from one parent and the B gene from the other.
Phenotypes of Parents
Possible Phenotype of
Offspring
O X O --------------------------------------------------------------------O
O X A ------------------------------------------------------------------O or A
O X B------------------------------------------------------------------O or B
O X AB ----------------------------------------------------------------A or B
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CHAPTER EIGHT
THE RHESUS SYSTEM
In 1940 Landsterier and Wiener injected the red cells of the Rhesus
Monkey into rabbits thereby producing an antibody which not only
agglutinates Rhesus Monkey red cells but also the red cells of some
human beings. As these people apparently possessed an antigen similar
to the Rhesus Monkey , these individuals were called Rhesus Positive or
Rhesus Negative. The antigen was called D and the antibody anti- D
The Rhesus system is very complex and contain more than 40antibodies.
At the level of general use, six common Rhesus genes exist C, D and E
with their allelomorphs c d and e.
THE Du ANTIGENS
It has been postulated that the D antigen is in fact made up of a mosaic
of 4 parts, Rh A, Rh B, Rh C and Rh D. An individual who is Rh D
positive possesses all 4 parts of the mosaic whereas none of these parts
is present in Rh D negative individuals,
The D u antigen is the weaker form of the D antigen. The D u antigen
does not usually react with complete anti D but will react with varying
amount of different incomplete anti D sera depending on whether it is
high grade or low grade D u antigen. When the D u antigen is present ,
any anti D serum which does not agglutinates the red 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 anti human
globulin test.
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Diagram of D Antigen
A Du person if given RhD positive blood may although rarely ,
form an anti-D and similarly Du Blood given to a Rhesus
Negative person may well stimulate the formation of Anti-D. It
is therefore accepted that a Du individual is considered as
Rhesus Positive as a donor and Rhesus negative as a recipient .
HAEMOLYTIC DISEASE OF THE NEW BORN ( HND)
In 1939 , it was demonstrated by Levine and Stetson that
maternal antibodies crossing the placenta could damage foetal red
cells possessing the antigen specific for the maternal antibody.
Since only IgG antibodies are able to cross the placenta . IgM
antibodies being too large . It is these IgG antibodies when
active at 37.0c can cause HND.
In the majority of cases the causative antibody is anti-D.
The mother being RhD negative and the foetus having inherited
the D antigen from the father.
The first child is seldom affected by HND , since the
stimulation of the antibody is frequently due to as transplacental
hemorrhage from the foetus to the mothers during delivery.
If a Pregnancy with a second Rh Positive foetus occurs , then
small bleeds from foetus to mother may further stimulate
antibody production. The antibody produced as a result of the
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primary dose of the antigen will be mainly IgM ,but on
subsequent exposure to the antigen, IgG Production replaces IgM. It
is the IgG antibodies which are able to cross the placenta, enter
the foetal circulation and destroy red cells.
The affected infant usually presents with anaemia and jaundice.
The Anaemia usually have an increase in reticulocytes and a high
nucleated red cell count.
DANGERS OF HND
 Massive infant anaemia
 Massive Jaundice that can lead to irreversible brain damage.
 Still birth in High sensitization
SOLUTIONS
 Treat for Anaemia
 Removal of sensitized Red Cells in Circulation
 Infusion of Compatible ABO Rh – ve Blood
 Antenatal Screening of antibody levels
 Exchange transfusion
 Injection of anti- D Immunoglobulins to Rh negative woman
bearing a Rh D positive Children.
OTHER BLOOD GROUP SYSTEMS
Over 100 blood group antigens maybe demonstrated using the
specific antisera and these have been classified into blood group
systems. Many of these antigens fortunately have no clinical
significance. But as the blood group systems are inherited quite
independently from each other, they are of immene value as genetic
19
markers. Some of the other blood group systems apart from ABO
are : MNs, P , Kell , Lewis , Lutheran , Duffy , Kid and I.
MEDICO-LEGAL ASPECT OF BLOOD GROUP
In paternity dispute , the blood grouping of all parties concerned
can do no more than exclude one of the parents. Usually it is the
father who is a dispute and he is excluded if antigen which he
genetically must pass are not present in the child. Also if the child
possesses an antigen which both he and the mother lack , the
disputed father must be excluded. This type of work is not usually
carried out in the Hospital Laboratory because of the legal
implication.
FORENSIC ASPECTS
The determination of the Blood group antigen of an individual are
nearly as exclusive as the finger print. This fact is used frequently
by Police Departments to investigate criminal cases. Blood groups
antigens can be detected from dried stain saliva and other body
fluids inclusive.
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CHAPTER NINE
COLLECTION AND STORAGE OF BLOOD.
BLOOD DONATION CRITERIA
 Donor must be within the ages of 18 and 65 of both sexes
 Females Donors must have HB values greater than 12.5 g/dl
 Male Donors must have HB Values greater than 13.5 g/dl
 Donors should be absolutely well devoid of malaria, Hepatitis
B, Hepatitis C , HIV , Syphilis and other diseases.
 Donors should not have any history of jaundice or allergy
BLOOD COLLECTION
Blood is collected in plastic bags or Medical Research Council
(MRC) Bottles. The said container contain the relevant
anticoagulant. The anticoagulant commonly used are :
 Acid Citrate Dextrose ( ACD)
 Citrate Phosphate Dextrose Adenine ( CPDA)
The CPD Adenine anticoagulant has a better preservation of red
cells enzymes, Oxygen carrying capacity than ACD. Blood stored
at 4.0c is well preserved in CPD- adenine and maybe used for up
to 35 days after the date of collection.
FUNCTION OF THE COMPONENTS OF THE
ANTICOAGULANT
Citrate
- Prevents the Blood Clotting by acting on Prothrombin
preventing its conversion to thrombin.
Phosphate – Act a buffer , thus buffering the Blood Electrolytes
Dextrose – Provides the energy needed by the Blood Cells
Adenine - Amino acids helping the maintaining the internal
structure of the Hb
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BLOOD BANK
Since Blood deteriorates rapidly if not kept under ideal condition,
it is very necessary to store blood in a specially constructed
refrigerator which have a high insulator. Most maintain a
temperature of 4.0c with a Maximum range of 2 – 6.0C. It is
essential that the temperature does not exceed 6.0c or fall below
2.0c. If this occurs damage to the red cell may occur.
To prevent this, the Blood bank must have a temperature
recorder so that it is possible to tell the temperature at a glance
and also to be able to keep a record of the stability of
temperature. The Blood Bank must also be connected to an alarm
system. Preferably a laud bell or buzzer which will give an audible
signal, If the temperature rises or falls outside the prescribed
units. The alarm must also sound in a place where the staff are
always on study thereby ensuring that a responsible person will
take the predetermined action even when the Laboratory is
closed.
The alarm should also sound if the electricity supply fails. It
therefore follows that the alarm system must be battery
operated. On no account must blood be stored in a domestic
refrigerator because of its temperature fluctuation.
If there is any doubt regarding the storage of blood, once it has
left the Laboratory blood blank, it must be discarded and not
returned to the stock to be re-cross matched for another patient.
TRANSPORTATION OF BLOOD
Blood should be transported in an insulated box which keeps at a
temperature of 4-6.0c for at least 6 hours.
STORAGE OF BLOOD
22
Apart from a 21 -35 days storage duration at 4.0c. Blood can also
be used in frozen form. For example Red Cells can be frozen
for 2 years at - 80.0c in the present of glycerol. Blood can also
be stored in the presences of liquid nitrogen at - 196.0c, for 10
years. The Blood cells can be recovered by thawing and washing
of the cells.
BLOOD PRODUCTS AND SUBSTITUTES
With the advent of more intensive and sophisticated methods of
Clinical management, the demand for blood have greatly increased.
In order to keep up with these demands , it has been necessary
to split blood into various components such as red cells , Platelets ,
white cells etc. In this way the same amount of blood may be used
to treat many more patients.
1. Fresh Frozen Plasma
This is plasma that has been separated from red cells on
collection and stored at – 20. 0C. The plasma contain clotting
factors and hence can be used for the treatment of multiple
clotting factor deficiencies.
2.Plasma Protein Fraction
This is a solution of the protein of human plasma and is a clear
amber fluids. It is prepared from pooled plasma which has been
precipitated with suitable organic solvents and re-dissolved in
water. This may be used to replace depleted plasma volume and
may be used in place of blood in an emergency while awaiting
issues of blood. It is also called Human Albumin solution 4.5%.
3. Dried Human Plasma
This is plasma that has been freezed dried and can be reconstituted with sterile isotonic saline for usage.
4. Albumin Solution
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These are used to give colloid. They are pasteurized at 60.0C for
one hour and have no risk of transmitting Hepatitis B or HIV.
4% and 2% solution are the most common.
5. Cryoprecipitate
This is prepared from fresh Plasma which is frozen solid in and
a mixture of solid CO2 ( DRY ICE) and ethanol is then thawed
slowly at 4.0c for 24 hours. This contains clotting factors
including factor V111. It is indicted in coagulation failure.
Treatment of haemophilia and other bleeding disorders.
BLOOD SUBSTITUTES
These are solutions which are used in the absence of blood to
replace blood volumes.
1. Dextran 6 %
2. Gelatin 3 -5 %
3. 4% Glucose Saline
4. Hartmann’s Lactate Solution and Isotonic Saline.
All these solutions are predominantly used to maintain the
blood volume and prevent dehydration after surgery. The
development of synthetic compounds called
perfluorocarbons are being developed . These are somehow
capable of carrying Oxygen to the tissues.
CHANGES TO BLOOD DUE STORAGE
1. Oxygen carrying capacity is reduced due to
deterioration of red cell function, during storage. Fresh blood
has the best Oxygen carrying capacity.
2. Platelets function declines rapidly so that after 48 - 72
hours of storage the platelets will not assist in Coagulation.
3. HyperKalaemia
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During Storage potassium is released from damaged red
cells. Red cells membrane become less efficient with
increasing length of storage.
5. Deterioration of Coagulation factors
6. Acidosis from stored blood
7. Citrate toxicity above 35 days storage.
8. Hypothermia : Since blood is stored at 4.0C , large
transfusion may produce hypothermia. Blood can be
warmed by placing a sealed bag or bottle in water at 35 40.0c for 7 minutes. If temperature exceeds 45.0C
heamolysis will occur.
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CHAPTER TEN
FACTORS AFFECTING ANTIGEN/ANTIBODY REACTIONS
The speed at which Antigen -Antibody reaction takes lace and the
strength of the reaction are affected by many factors.
1) Concentration : The sensitivity of blood grouping tests is
dependent on the use of Optimum Antigen-Antibody
concentrations. An increase in the number of antibodies that
are bound to each red cells results in an increase in the
strength of the reaction.
2) PH : Most blood group antibodies show optimum activity
between PH 6.5 and 7.5.
3) Temperature : The Maximum rate of formation of AntigenAntibody complex is at 37.0c, with a reduction in this rate at
lower temperatures.
4) Ionic Strength : An increase in the rate of association
between Antigen-Antibody can be observed if the ionic
strength of the medium is decreased. This results in an
increase in Zeta potential, causing an increased attraction
between the negatively charged cells and Antibody molecules,
most of which are positively charged.
Since the titre of most antibodies can be increased by diluting
the serum in low ionic strength saline, and because of the
increased rate of Antigen –Antibody association, it is possible to
reduce incubation time of blood grouping and cross matching
tests without any loss of sensitivity.
This fact has led to the widespread routine use of low ionic
strength saline in Hospital Laboratories. A marked reduction in
26
ionic strength has been associated with non specific antibody
uptake onto red cells. A low ionic strength of saline with
glycine with a molarity of 0.03 has been found to combine
maximum with increase sensitivity without large false positive
results. Physiological Saline has a molarity of 0.17.
27
CHAPTER ELEVEN
COMPLEMENT
Complement is a complex group of serum globulins which are
present in fresh normal serum and are able to lyse red cells
and destroy certain bacteria.
Eleven components of complements are recognized C1-C9, C1
having three subunits C1q , C1r and C1s. Complement activation
can occur in at least two ways.
a) The Classical Pathway : In this pathway antibody binding
leads to haemolysis of the Cell by the later stages of the
sequence.
b) The Alternate Pathway : In this pathway Antibodies are not
essential for activation to take place.
The activation of the Classical Complement pathway can be divided
into three main steps.
1) The Recognition Stage where the first component of the
Complement C1 is activated by the exposed complement
binding site on the FC portion of the Immunoglobulin
molecule.
2) The activation stage which leads to the formation of a C2C4 complex which acts on C3. Parts of C3 and its activating
enzyme forms another enzyme which , in turn bring about
formation of C5.
3) The final stage or membrane attack sequence involves
component C6-9 which interact to produce cell lysis by means
of circular holes in the red cell membrane.
It is possible to detect the complement bound onto the red cell
by using anti-complement antibodies.
28
When Antigen-Antibody reactions occur on the cell surface, some
are capable of binding complement to the red cell. During binding
the Antibody is thought to undergo configurational change ,
exposing a complement activation site on the Antibody molecule.
The complement activation site is located on the FC position of
an Immunoglobulin molecule. IgM antibodies are therefore better
activating complement because of their many bind sites than IgG
molecules which have only two binding sites.
CHAPTER TWELVE
TECHNIQUES USED IN BLOOD TRANSFUSION
Although IgG Antibodies are mostly unable to cause
agglutination of red cells in a saline medium they are of
significance , when grouping and cross matching. Several
techniques may be used to demonstrate these antibodies.
1) Use of Bovine Albumin
The use of a medium which is able to dissipate electric charge(
and therefore disperse the ionic cloud surrounding the Red Cell)
allows the cell to become more closely associated . Agglutination of
the cells by IgG or incomplete Antibodies is then Possible.
2) Use of Proteolytic Enzymes
The net negative charge which the red cell carries is due to the
ionization of carboxyl group of Sialic acid present at the cell
surface. As a result of Proteolytic action, some enzymes are able to
librate Sialic acid residues from the cell membrane. This has the
effect of decreasing the negative charge at the red cell surface, thus
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allowing cells to approach one another more closely. IgG antibodies
are then able to bring about agglutination of the cells.
Type of Enzymes
Papain extracted from paw-paw fruits
Bromelin extracted from Pineapples
Ficin extracted from Figs
Trypsin extracted from Pancreas.
TWO TYPES OF ENZYME TECHNIQUES
a) Two Stage Technique
In this technique the cells are pre-treated with enzymes before
setting up the test. This method is more sensitive and very ideal tests
where the cells are tested against a number of sera ( Antibody
Screening and Antibody Identification ).
b) One Stage Technique
In this technique the serum , enzymes and cells are layered in the
tube and the cells become enzyme treated as they fall through the
enzyme layer into the serum. This stage is frequently used because
of less consuming time.
NB. Using enzyme techniques , by Proteolytic action on the Sialic
acid ,more of the Red Cell antigen can be destroyed e.g M , N, and
Fya antigen.
COMPATIBILITY TESTING - CROSS MATCH
In most blood transfusion cases even though the blood donor and
the recipient ABO and Rhesus Groups are the same . It is
essential that Cross match techniques be performed. This is to
ensured that the donor blood will not give rise to any reaction
from antibodies the recipient may have formed to any other blood
group systems.
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As it is true to say that no blood given will exactly match the
antigenic structure of the recipients red cells. It is vital that every
precaution is taken to prevent harm to the patient.
The Cross matching is performed by testing the donor red cells
against the recipients serum using several different techniques.
SOME TECHNIQUES INCLUDE :
1. Saline technique at room temperature for detection of cold
antibodies.
2. Saline at 30. 0C or 37.0c detect antibodies with a wide
thermal ranges.
3. Albumin addition Technique
4. Enzyme technique to detect incomplete warm antibodies that
react at high temperature above 6 - 37. 0C
5. Indirect antihuman globulin Technique
IMPORTANT NOTE
1. Blood should ideally never be given to a patient without cross
matching
2. If Blood is required in an Emergency and it is necessary to
issue uncross-matched blood , the requesting clinician then
assumes responsibility.
3. An emergency Cross match can be performed using reduced
incubation time , the majority of Clinically significant
antibodies will be detected by thes methods.
4. Group O Rhesus negative blood should be given in cases of
emergency. This flying squad blood can be issued in cases
where there is insufficient time to perform a rapid group on
the patient.
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ASSESSMENT 1: ON BLOOD BORNE PATHOGENS
All Questions are to be answered:
Answer All Questions Placing an “X” against the correct answers.
Q.1
Bloodborne Pathogens organisms carried in what way?
Q.2
a)
By mosquitoes
b)
By contaminated water
c)
By blood to blood contact
d)
All of the above
Which of the following are not types of bloodborne pathogens?
Q.3
a)
Malaria
b)
Hepatitus B
c)
HIV
Is the following statement true or false? “Hepatitus B is
transmitted primarily through blood to blood contact”
True ………….. False ……………..
Q.4
Is there a cure for Hepatitus B?
Yes …………
No …………………
Q.5 Personnel at risk of exposure can be vaccinated against this
disease. True or False?
True ………….. False ……………..
Q.6
Q.7
Is it true that HIV may take several years to develop into AIDS?
Yes …………
No ………….
AIDS is a fatal disease but it also weakens the body such that it
cannot resist other dangerous diseases. Is this statement true or
false?
True ………..
False …………..
Q.8
Is there a cure for HIV / AIDS?
Q.9
Yes ………….
No ……………
Can the HIV virus last for long outside the body?
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Yes ………..
No ………….
Q.10 “The HIV virus can easily be transmitted through sexual
contact”. True or false?
True ………….
False ………….
Q.11 HIV infected blood can enter the body through which route?
a)
b)
c)
d)
Cuts
Abrasions
Open sores
All of the above
Q.12 Bloodborne pathogens may be transmitted through the mucous
membranes of :
a)
b)
c)
d)
Mouth
Nose
Eyes
All of the above
Q.13 Which of the signs shown below must be displayed on containers
of blood contaminated waste (regulated waste)? Please put an X
against the correct picture.
Danger
Hazardou
s
Substanc
e
DANGER
BLOOD
Q.14 Is it true or false that when dealing with a casualty who is
bleeding you should wear PPE to minimize exposure to
bloodborne pathogens?
Yes …………..
No …………….
Q.15 If your skin is exposed to another persons blood you should wash
your hands with soap and water?
Yes ………….
No ………….
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Q.16 If your eyes should get splashed with another persons blood you
should:
a)
b)
c)
See a doctor as soon as it is convenient.
Do nothing
Flush your eyes out with water for fifteen minutes.
Q.17 If blood is present following an accident you should:
a)
b)
Treat the blood as potentially infectious.
Assume that there is no risk if you know the injured
person.
Q.18 If decontaminating equipment contaminated with blood,
approximately how much household bleach should you mix with a
gallon of water?
a)
b)
c)
One cupful
Four cupful
One quarter cupful
Q.19 Is it ok to pick up blood contaminated glass with your hands if
you are careful?
Yes ………..
No ……….
Q.20 Is it true that needles and broken glass which may be
contaminated with blood must be disposed of in a specially
marked “sharps” container?
Yes ………..
No ………...
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