Clinical laboratory methods lecture notes

for midwifery students

Section IV Immunohematology

By Henok.B (MLT,HI)

January 2018
DDU

1
1. Understand Explain the basics of
immunohematology
2. Understand and explain ABO blood grouping
system
3. Explain the concepts regarding Cross-match
4. Perform and interpret laboratory tests for cross-
match

2
 Immunohematology:
◦ is more commonly known as "blood banking“

◦ deals with the concepts and clinical techniques

related to modern transfusion therapy.

◦ Is the area of laboratory medicine dealing with

the general procedures involved in collecting,
preparing, storing and transfusing blood.

3
◦ refers to immunologic reactions involving blood
components

◦ an application of the principles of immunology to

the study of
 red cell antigens and
 their corresponding antibodies on blood for resolving
the problems of blood transfusions.

4
 The era of blood transfusion began when
William Harvey described the circulation of
blood in 1616.

 In 1665, Richard Lower, successfully

performed the first animal-to-animal blood
transfusion.

5
 In 1667, jean Bapiste Denys transfused,

◦ blood from the carotid artery of a lamb into the

vein of a young man, which at first seemed
successful.

◦ using animal blood, but they were unsuccessful.

 Later, it was found that it is impossible to

successfully transfuse the blood of one
species of animal into another species.

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 Transfusions were prohibited from 1667 to
1818
◦ Due to the disastrous consequences resulting.

 In 1818, James Blundell of England successfully

transfused human blood to women suffering
from hemorrhage at childbirth.

 Such species-specific transfusions seemed to

work sometimes but mostly the result was
death.

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 Karl Landsteiner

◦ discovered the ABO blood groups in 1900,

◦ introduced the immunological era of blood
transfusion.

 It became clear that the incompatibility of

many transfusion was caused by the
presence of certain factors on red (blood)
cells now known as antigens.

8
Two main postulates were drawn:
1.Each species of animal or human have certain
factors on the red cells that are unique to that
species, and
2.Each species have some common and some
uncommon factors to each other.
 This landmark event initiated the era of
science based transfusion therapy and was
the foundation of immunohematology as a
science.

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 Concerned with the way in which the
different blood groups are inherited

Chromosomes and Genes:

 The nucleus of each human body cell
contains 46 small thread-like structures
called chromosomes, arranged in 23 pairs.

 The length of each chromosome is divided

into many small units called genes.

 Genes code for different inherited physical

characteristics, including blood groups.

10
Allomorphic genes (Alleles),and
Polymorphism
 Each gene has its own locus, along the
length of the chromosome.
 Certain inherited characteristic can be
represented by a group of genes, and the
locus can be occupied by only one of these
genes.
 Such genes are called alleles or allomorphic
genes.

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 Mitosis: While body cells multiply they do so
by producing identical new cells with 46
chromosomes.

 Meiosis: When sex cells are formed either

male or female, the pairs of chromosomes
do not multiply but simply separate so that
each of the new cells formed contains only
23 chromosomes.

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 During fertilization when the egg and
sperm unite the fertilizer ovum receives 23
chromosomes from each sex cell.

◦ Half of these from the male and

◦ half from the female and thus will contain 46
chromosomes which arrange themselves in pairs
in the nucleus.

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Genotype versus phenotype
 Phenotype
◦ Physical expression of inherited traits,
◦ Determined by reacting red cells with known
antisera
 Genotype
◦ Actual genes inherited from each parent
◦ Can only be inferred from the phenotype .
◦ Family studies are required to determine the
actual genotype .

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Phenotype Genotype

A AA, AO

B BB,BO

AB AB

O OO

15
Punnet square
 Illustrates the probabilities of phenotypes
from known or inferred genotypes.

 Visually portrays the potential offspring`s

genotypes or the probable genotypes of
the parents .

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Table4.1.2. Punnet squares showing ABO inheritance

• Two group A parents can have a group O child.

• The parents of an AB child can be A, B or AB,
but not group O.

A O

A AA AO

O AO OO

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 In most cases blood group antigens are
inherited with co dominant expression.

 The product of each allele can be identified

when inherited as a co dominant trait.

◦ If one parent passed on an A gene the other

parent passed on a B gene, both the A and B
antigens would be expressed equally on the red
blood cells.

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Recessive or dominant inheritance patterns

◦ recessive
 inheritance would require that the same alleles from
both parents be inherited to demonstrate the trait

◦ Dominant
 expression would require only one form of the allele to
express the trait.

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 O gene is recessive, since it is expressed
only when both parents contribute the O
allele.

 The product of an O gene however, does

not affect the membrane proteins.

◦ Its expression is termed as amorphic (a gene that

does not express a detectable product) rather
than recessive.

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1.4.1 Red blood cell antigens

 A unique set of red blood cell Ag is

determined through genetic inheritance.

 These antigens protrude from the surface of

the RBC in three dimensional
configurations.

◦ As a result, they are accessible to Ab molecules

for agglutination reaction.

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 In biochemical terms these antigens may
take the form of:
◦ proteins,
◦ Glycoprotein,
◦ Glycolipids

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 Some of the red blood cell antigens are
more immunogenic than the others
Example

◦ The D antigen within the Rh group system.

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 Is possessed by nucleated cells such as
leukocytes and tissues

 Can readily provoke an immune response if

transferred in to a allogenic individual.

 Encoded by genes which are parts of Major

Histocompatibility Complex (MHC) gene
system.

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 The MHC system is important in the:

◦ recognition of non self ,

◦ coordination of cellular and humoral immunity ,

and

◦ graft rejection .

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 Platelet possesses inherited membrane
proteins that can also elicit an immune
response.

 Platelet antibodies are less frequently

found, because there is less antigen
variability in the population.

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 Antibodies to platelet antigens are the
major cause of :
◦ neonatal alloimmune thrombocytopenia,

◦ Post transfusion purpura ,

◦ It can also decrease the expected increment of

platelet transfusion.

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Blood group antibodies are classified into:

◦ Natural and

◦ Immune antibodies.

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 Are RBC Abs in the serum of an individual
that are not provoked by previous RBC
sensitization.

 The term non red cell immune have crept in

to modern use.

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Characteristics
 They are mainly IgM type.

 Exhibit optimum in vitro agglutination

saline media
◦ complete antibodies.

 Optimum reaction at room temperature or

lower
◦ cold agglutinins.

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 Do not react above the body temperature

◦ most of these do not give rise to transfusion

reactions.

 They are of high molecular weight

◦ cannot cross the placenta

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 Produced due to previous antigenic
stimulation either by transfusion or
pregnancy

Characteristics
 Mainly IgG type
 Do not exhibit visible agglutination in
saline, but in albumin medium .
◦ Incomplete antibodies.

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 Optimally react at 370C
◦ warm agglutinins.

 Causes more serious transfusion reactions

than the naturally occurring ones.

 Can cross the placental barrier.

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 The binding follow the law of mass action
and is a reversible process.

 This union complies with the principles of a

chemical reaction that has reached
equilibrium.

 When Ag and Ab combines, an immune

complex is produced.

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 The amount of Ag - Ab complex formation
is determined by the association constant of
the reaction .

 When the forward reaction rate is faster

than the reverse reaction rate Antigen-
Antibody complex formation is favored.

 Therefore a higher association constant

influences greater immune complex
formation at equilibrium

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Properties that can influence the binding of Ag
and Ab
 The goodness of fit (as a lock and key fit)

 complementary nature of the antibody

 size, shape, and charge of antigen

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 To determine a person’s blood type, some
sort of substance must be available to show
what antigens are present on the red cell.

 The substance used for this purpose is

referred to as anti serum.

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 Is highly purified solution of antibody.

 named on the basis of the antibody it

contains
For Example:
◦ Solution of Anti-B antibodies is called
anti –B antiserum

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 The anti-sera used in Immuno hematology
are prepared in one of the two ways:

◦ By deliberately inoculating animals with an

antigen

◦ By collecting serum from humans who have been

sensitized with corresponding antigens

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 Anti-serum must:
◦ Be specific for the antigen to be detected
◦ Have sufficient titer to detect antigen
For Example
Anti-A should have a titer of at least
 1/128 against A1 cells,
 1/64 againstA2 cells, and
 1/16 against AB cells
Anti-B should have a titer of at least 1/64
against B
cells

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 Be free from haemolysins, fat and rouleaux

 Be sterile and clear

 Preserved with 1% sodium azide and be

stable.

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 Have a marked expiration date, and

 Should be stored at 40C

 The manufacturer directions must be

followed carefully.

42
 The presence of Invitro antigen and antibody
interaction can be detected by:

◦ Hemolysis

◦ Precipitation

◦ Agglutination (Most commonly used)

43
Agglutination:

 Visible clumping of particulate Ags caused

by interaction with a specific Ab

 Occurs in two stages:

◦ sensitization and

◦ lattice formation.

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A-Sensitization-the first phase

 represents the physical attachment of Ab

molecules to Ags on the RBC membrane.

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1. The antigen - antibody ratio

For example : pro-zone phenomenon.

2. Physical conditions such as:

◦ PH,
◦ Temperature
◦ Time of incubation
◦ Ionic strength, and
◦ Steric hindrance.

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B- Lattice formation – the second phase

 Is the establishment of cross links between

sensitized particles and Abs resulting in
clumping

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 Cross linking is influenced by Zeta potential

 Zeta potential

◦ is the difference in electrostatic potential between

the net charge at the cell membrane and the
charge at the surface of shear.

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 Centrifugation
 Treatment with proteolytic enzyme,
 Others
◦ Low Ionic strength saline (LISS)

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1. Define:
A. Antigen
B. Antibody
C. Immunogenicity
2. Identify some characteristics of the IgG subtypes
3. What are the characteristic differences between
Natural and Immune antibodies?
4. Which classes of antibodies predominate during
the primary immune response and secondary
immune response?
5. List the factors that affect antigen and antibody
interaction
6. List the methods that are routinely used in the
blood banking laboratory to enhance
agglutination reaction.

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4.2 THE ABO BLOOD GROUP
SYSTEM

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 Karl Landsteiner in 1900

◦ the beginning of modern blood banking and

transfusion medicine.
◦ described the blood groups as A, B and O.
◦ Stated rule- Land steiner’s rule
“normal, healthy individuals possess ABO
antibodies to the ABO Antigens lacking on their
RBCs.”

 Von Decastello and Sturli- group AB

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 ABO antigens are widely distributed and
located
◦ on red blood cells
◦ lymphocytes
◦ platelets
◦ tissue cells
◦ bone marrow, and
◦ organs such as the kidneys.

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 Soluble forms of the antigens can be

◦ synthesized and secreted by tissue cells

◦ found in association with cellular membranes and
◦ in all body fluids except CSF
◦ are glycoproteins

54
 ABO system Ags, which are intrinsic to the
RBC membrane

◦ exist as either Glycoprotein or Glycolipid

molecules,
◦ are detectable as early as 5 to 6 weeks invitero.
◦ are weaker on cord blood and result in weaker
ABO phenotype reaction.
◦ develop slowly and reach the full expression of
adult levels at approximately 2 to 4 year of age.

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 A% B% AB% O%

Asian 28 27 5 40

African 26 21 4 49

Nepalese 33 27 12 28

Caucasian 40 11 4 45

Ethiopian 31 23 6 40

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 The production of H antigen is genetically
controlled by the H gene, located on a
different chromosome from the ABO genetic
locus.

 The expression of soluble ABO antigen is

influenced by inheritance of the Se
(secretion) gene.

57
 The Se gene genetically influences the
formation of ABO antigens in saliva,tears,
and other body fluids.

 The occurrence and location of the ABO

antigens are influenced by three genetically
independent loci ABO, H, and Se.

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 Ag building block for A, B, and H Ag is
oligosaccharide chain attached to either a
protein or lipid carrier molecule.

 The oligosaccharide chain comprises four

sugar molecules linked in simple linear
forms or in more complex structures with
a high degree of branching.

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 The H antigen is the only antigen in the H
blood group system .
 The H-gene is assigned to H -locus on
chro.19
 The H-locus has two significant alleles: H &
h.
◦ The H allele is a dominant allele,
 frequency greater than 99.99%

◦ h allele frequency rare.

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 Each gene (H, A and B genes) codes for the
production of a specific transferase
enzyme.

 Transferase enzyme promote the transfer of

a biochemical group from one molecule to
another.

 A glycosyl transferase enzyme catalyzes the

transfer of glycosyl groups (simple
carbohydrate units) in biochemical
reactions.

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Note:
 The formation of H antigen is critical to the
expression of A and B antigens

◦ the gene products of the ABO alleles require the

H antigen to be the acceptor molecule.

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63
 The gene for A and B Ags are located on
Chr.9.

 Three major alleles on ABO locus: A,B, and

O.

 A-allele codes for N-acetylgalactosaminyl

transferase, which transfers N- acetyl
galactosamine (immunodominant sugar) to
H-Ags

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65
 The B-allele codes for D-galactosyl
transferase which transfers D-galactose
(Immunodominant sugar) to the H-antigen.

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67
 The O allele is considered non functional,
since the resulting gene product is an
enzymatically inactive protein.

 Group O RBCs carry no A or B Ags but are

rich in unconverted H Ags.

 Adult group O RBCs possess the greatest

concentration of H Ags per RBC.

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 Bombay phenotype (hh) individuals

◦ do not inherit the H gene and do not possess the

enzyme H transferase
◦ are unable to produce H substance.

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◦ Can inherit the A and B genes, as they do not
make the H substance the A and B gene is not
expressed and so typed as group “O” unlike
group O individuals they lack H.

◦ Cause- mutation in the H gene on chromosome

19 that causes a non functional H glycosyl
transferase to be produced.

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 Do not have H antigen but possess trace
amounts of A or B antigen depending on the
ABO gene on chromosome 9.
◦ Small amount of H antigen is produced and almost
completely converted to A or B antigen if these
enzymes are present.
 These individuals are termed Ah or
Bh respectively.
 Cause -production of very weakly acting H
glycosyl transferase.

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 It is a temporary replacement of Blood
group A by group B due to infection with a
gram negative bacteria especially by
P.vulgaris

 Deacetylation of the N-acetylgalactosamine

sugar

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 Present in individuals with no known
exposure to blood or blood products

 Are not detected in the serum of new born

until 3 to 6 months of age.

 With advanced age the ABO titers tend to

decrease

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Immunoglobulin class

 Anti- A and anti- B found

◦ In group B and group A individuals are primarily

IgM with small amounts of IgG.

◦ In group O individuals are composed primarily of

IgG class.

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Hemolytic properties and clinical significance

 are capable of activation and binding of

complement and
 eventual hemolysis of the red blood cells in
vivo or invitro.

◦ Are thus considered of clinical significance in

transfusion medicine.

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In vitro serologic reaction
 directly agglutinate a suspension of RBC in
a physiologic saline
 Optimally react in immediate spin phases at
room temperature (250C).
 Agglutination reactions do not require an
incubation period
 React without delay up on centrifugation.

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Human anti- A, B from Group O individuals
 possesses unique activities beyond mixtures
of anti- A and anti- B antibodies.

 is cross – reactive with both A and B antigens.

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ABO phenotyping has two components:

1. Forward grouping

2. Reverse grouping

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4.3.2. 1.Direct (cell grouping)
Testing of the red blood cells for the
presence of ABO Antigens (or forward
grouping).

4.3.2. 2. Indirect (serum grouping)

Testing of serum or plasma for the
expected ABO Antibodies (or reverse
grouping).

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Principle
 When an antigen is mixed with its
corresponding antibody under the right
conditions it causes agglutination or
haemolysis of the red cells.

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Clinical significance

◦ For safe blood transfusion

◦ Prevention of Hemolytic Disease of the Fetus and
Newborn (HDFN)

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 Donor and recipient red blood cells must
be tested using anti- A and anti-B.

 Donor and recipient serum or plasma must

be tested for the expected ABO
antibodies using reagent A1 and B red
blood cells.

 Cord blood and samples from infants less

than 4 months old should be tested by
forward ABO grouping .

82
 The ABO phenotype is determined when the
red blood cells are directly tested for the
presence or absence of either A or B
antigen.

◦ Serum testing provides a control for red blood

cells testing, since ABO antibodies would reflect
Landsteiner’s rule.

83
 ABO discrepancy
◦ occurs when ABO phenotyping of red blood cells
does not agree with expected serum testing
results for the particular ABO phenotype.

84
The methods for grouping
1. Tube method
2. Emergency tile or slide grouping
3. Tile or slide grouping of blood donors

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1.Tube grouping method

Specimens
-Patient’s serum .
-Patient’s cells.

86
 Tube grouping method….

Equipments and reagents

◦ Anti- A serum and anti- B serum
◦ Antigen A and antigen B ( Red cell suspension)
◦ test tube
◦ Centrifuge
◦ wash bottles
◦ normal saline

87
 Tube grouping method….

Preparation of Red Cell Suspension:

% required= PCV x100
Volume of suspension

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Procedure: (e.g. preparation of a 2% red blood cell
suspension of 10 ml volume)

1. Place 1 to 2ml of anticoagulated blood in a test

tube
2. Fill the tube with saline (0.85%) and centrifuge
3. Aspirate or decant the supernatant saline.
4. Repeat (steps 2 and 3) until the supernatant saline
is clear.
5. Pipette 10 ml of saline into another clean test tube
6. Add 0.2 ml of the packed cell button to the 10ml
of saline
7. Cover the tube. Immediately before use, mix the
suspension by inverting the tube several times until
the cells are in suspension.

89
Method
1. Take 4 tubes and label them 1-4.
2. Place each of the following in its
numbered tube.
Tube 1: 1 volume of anti- A sera.
1 volume of 3-5% suspension of
patient’s washed cells.
Tube 2: 1 volume of anti- B serum.
1 volume of 3-5% suspension of
patient’s
washed cells.

90
Tube 3: 1 volume of patient’s serum.
1 volume of 3-5% suspension of
washed
RBC (reagent RBC) containing
antigen A.

Tube 4: 1 volume of patient’s serum.

1 volume of 3-5% suspension of
washed
RBC (reagent RBC) containing
antigen
B.

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3.Mix the contents of each by gently taping
the tube with the finger and leave for five
minutes at room temp.
4. Centrifuge the tube for one minute at
1000 RPM for one minute.
5. Replace the tube in the rack in the same
position as before centrifugation, and read
the result by gently tapping each tube,
looking for agglutination or heamolysis.

92
6. Check the tube which shows no visible
agglutination by examining the contents
microscopically on a slide using a 10x
objective.
7. Decide what a blood group of a patient
is and record the result in the book
provided and one the patient’s form.
Controls
Anti- A and Anti- B should be controlled
using known A (preferably – A2) cells and B
cells.

93
Phenotype RBC reaction Serum or plasma reaction

Anti-A Anti-B A-cells B-cells

A + O O +
B O + + O

AB + + O O
O O O + +

+= agglutination, O= no agglutination

94
4+ A single agglutinate with no free cell,
clear
supernatants
3+ Several large aggregates, some free
erythrocytes, clear supernatants.
2+ medium sized aggregates and some free
cells,
clear supernatant.
1+ A few small aggregates just visible
macroscopically, many free erythrocytes,
turbid
and reddish supernatant many.

95
+ Weak granularity in the cell suspension. A
few
macroscopic agglutinates but
numerous agglutinates microscopically.
Weak (+/_) Tiny aggregates that are barely
visible
macroscopically, many free erythrocytes,
turbid
and reddish supernatant
Mixed field Few isolated aggregates, mostly free
floating cells, supernatants appear red.
Negative No aggregates

96
2. Emergency tile or slide grouping
Emergency tile grouping is carried out using
a white glassed porcelain tile, Perspex tray
or slides.

97
Specimen
◦ Patient’s serum
◦ Patient’s cells
Reagents and equipments:
◦ Anti- A and anti-B serum
◦ Group O serum (anti-AB)
◦ Antigen A and antigen B (20% known RBC
suspension)
◦ Group AB serum( Control)
◦ Tile or slide

98
 Method
 1. Divide and mark a white tile as follows

a b c d e f
Anti- A Anti- B Anti- AB A - cell B- cell Control

99
Then add the following
a. 1 volume of anti- A serum
1 volume of 20% suspension of patient cell
b. 1 volume of anti- B serum
1 volume of 20% suspension of patient cell
C.1 volume of group O serum
1 volume of 20% suspension of patient cell

10
0
d. 1 volume of patient serum
1 volume of 20% suspension of A cell
e. 1 volume of patient serum
1 volume of 20% suspension of B cell
f. control
1 volume of group AB serum
1 volume of 20% suspension of patient washed
cells (there should not be agglutination here).
3. Mix the contents of each division, using a small
clean applicator stick for each.
4. After 2-3 minutes read the results and decide the
blood group.

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1
10
2
3. Tile or slide grouping of blood donors

 This is a rapid method used to group

donors , using a white tile or two glass
slides

 It consist only cell grouping using anti-A

serum and anti-B serum .

10
3
Specimen
◦ Whole blood
Reagents and equipments:
◦ Anti- A and anti-B serum
◦ Group O serum (anti-AB)
◦ Tile or slide
◦ Applicator stick

10
4
Method
1. Take two slides or a white tile and mark as
follows

Anti-AB
Anti-A Anti-B

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5
2. Place in to each marked area (anti-A, anti-
B, anti-AB) one drop of donor capillary
blood.
3. In to the division marked
- Anti-A, place one drop of anti-A serum,
- Anti-B, place one drop of anti-B serum,
- Anti-AB, place one drop of anti-AB serum.
4. Mix the content of each division with small
clean applicator stick.
5. After two or three minute read the results
by naked eye and decide the blood group
the donor.

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6
A. Gel Technology method
-It is a new and unique method

-uses dextran acryl amide gel particles

combined with diluents or reagent in pre
filled plastic cards

-used for AHG test, cross match procedure,

ABO and Rh typing.

10
7
B. Micro plate testing methods
-A micro titer plate with 96 wells serves as
the substituted tube test to which the
principles of blood banking are applied.

-It can be adapted to red blood cell antigen

testing or serum testing for antibody
detection.

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8
May be indicated by the following
observations

 Agglutination strengths of the typing

reactions are weaker than expected

◦ typically the reactions in red blood cell testing

with reagents anti- A and anti- B are 3+ to 4+
agglutinations;

◦ the results of serum testing with reagent A1 and

B cells 2+ to 4+.

10
9
 Expected reactions in ABO red blood cell
testing and serum testing are missing

For example
◦ a group O is missing one or both reactions in
the serum testing with reagent A1 and B cells).

 Extra reactions are noted in either the ABO

red blood cell or serum tests.

11
0
Can be technical or sample –related
problems.

2.8.1.Technical error
 Identification or documentation error.
 Reagent or equipment errors.
 Standard operating procedure errors

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1
 ABO discrepancies that affect the ABO red
blood cell testing.

◦ Extra antigens present

◦ Missing antigens

◦ Mixed field reaction

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2
 ABO discrepancies that affect the ABO
serum testing.

◦ Presence of additional antibodies other than anti–

A and anti-B or

◦ The absence of expected antibody reactions

11
3
 In 1940 Landsteiner & Wiener discovered a
human blood factor, which they called
Rhesus factor.

 They immunized guinea pigs and rabbits

with blood from the Macacus rhesus
monkey, and the antiserum obtained
agglutinated not only the red cells of the
rhesus monkey but also 85% of human.

11
4
 This discovery followed the detection of an
antibody occurred in the serum of a woman
who delivered a stillborn fetus by Levine &
Stetson in 1939.

11
5
 They also postulated that the antibody had
arisen as the result of immunization of the
mother by a fetal antigen which had been
inherited from the father.

11
6
 In 1940, Wiener & Peters showed that the
antibody (anti- Rh) could be found in the
serum of individuals who had transfusion
reactions following ABO group – compatible
transfusions.

11
7
 In 1941, Levine & his co- workers showed
that not only could Rh negative mother
become immunized to an Rh positive fetus
in utero but also that the antibody could
then traverse the placenta and give rise to
erythroblastosis fetalis or Hemolytic Disease
of the New born (HDN).

11
8
 Later work demonstrated that the animal or
rabbit anti-Rhesus and human anti-Rh are
not detecting the same antigen but the
system had already named the human
antibody anti-Rh.

11
9
 The animal anti-rhesus was detecting
another antigen possessed by Rh positive &
Rh negative persons but in much greater
amount in Rh positives.

 Therefore the animal antibody was renamed

anti- LW after Landsteiner and Wiener who
discovered it, and the human antibody
retained the title anti-Rh.

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0
 A theory originally postulated by Tippett, describes
two closely linked genes- RHD and RHCE- on
chromosome 1.
 RHD determines the D Ag expression on the
surface of RBCs. D-negative individuals have no
genetic material at this site. An antithetical d allele
does not exist.
 Adjacent to the RHD locus, the gene RHCE
determines the C,c,E and e antigens. The alleles at
this locus occupy the gene RHCE, RHCe, RHcE, and
RHce. These genes encode the red blood cell
Antigens CE,Ce, cE, and ce.
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1
12
2
 The assortment of other antigens in the Rh
system occurs as a result of variation
of these polypeptides embedded in the
cell membrane bilayer in unique
configurations.

 A model of the difference in the amino acid

sequence for the antigens produced by the
RHCE gene.
 An identical basic structure differs in the
amino acid at the residue number indicated

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3
Antigen Amino acid N0

C Serine 103
c Proline 103
E Proline 226
E Alanine 226

124
 Phenotype- refers to the test results
obtained with specific antisera

 Genotype- refers to the genetic makeup of

an individual.

 The genotype cannot be absolutely

determined without family studies but can
be inferred from the phenotype based on
the frequency of genes in a population.

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5
D antigen
 Is the most immunogenic antigen in the Rh
system.

 More than 80% of D-negative people

receiving a D-positive red blood cell
transfusion produce an antibody with anti-
D specificity.

 For that reason ,a D-negative patient

should receive D- negative red blood cells.

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6
General characteristics

 Are usually made by exposure to Rh

antigens through transfusion or through
pregnancy and show similar serologic
characteristics.

 Most are IgG (IgG1) and bind with the

corresponding antigen at 370C
◦ Agglutination is observed by the IAT.

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7
 Can cause a severe hemolytic transfusion
reaction in a recipient if transfused with
blood possessing the offending antigen .

 Being IgG, are capable of crossing the

placenta and are associated with hemolytic
disease of the new born (HDN).

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8
Methods of Rh Grouping Technique
◦ Direct slide and
◦ Direct tube

 Reverse grouping cannot be done due to

the absence of naturally occurring Rh
antibodies in the serum of persons lacking
the corresponding Rh antigen.

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9
Clinical significance

◦ For safe blood transfusion

◦ Prevention of Hemolytic Disease of the Fetus and
Newborn (HDFN)

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Principle
 When an antigen is mixed with its
corresponding antibody under the right
conditions it causes agglutination or
haemolysis of the red cells.

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1
Specimen
 Washed RBC(40-50%)
Equipments and reagents
 Anti- D serum
◦ Microscopic slide
◦ Microscope
◦ Applicator stick
◦ Albumin (Control)

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2
1. Place a drop of anti-D on a labeled slide.

2. Place a drop of Rh control (albumin or other

control medium) on another labeled slide.

3. Add two drops of 40-50% Red cell suspension to

each slide.

4. Mix the mixture on each slide using an applicator

stick, spreading the mixture evenly over on most
of the slide.

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Interpretation of the test result

-Agglutination of red cells ----------Rh

positive.
-No agglutination of red cells------Rh
negative.
A smooth suspension of cell must be
observed in the control.
Note: Check negative reactions
microscopically.

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Specimen
 Washed RBC(2-5%)
Equipments and reagents
 Anti- D serum
◦ Test tube
◦ Centrifuge
◦ Microscope
◦ Albumin (Control)

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5
1. Make a 2-5% red cell suspension.

2. Prepare two test tubes and mark “D” on the first

tube and add two drops of anti-D

3. Mark “A” on the second tube and Place a drop of Rh

control (albumin).

4. Add one drop of a 2-5% cell suspension to each

tube.

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6
5. Mix well and centrifuge at 2200-2800 rpm for 60
seconds.

6. Gently re suspend the cell button and look for

agglutination and grade the results (a reaction of
any grade is interpreted as Rh positive) a smooth
suspension of cells must be observed in the
control.

7. Collect all weakly positive (+/-) and negative

sample to perform the Du test.

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7
 It is a procedure performed before
transfusion to select donor’s blood that will
not cause any adverse reaction, (hemolysis
/agglutination)

 It helps the patient to receive maximum

benefit from the transfusion of red cells

 This is done by ensuring compatibility

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8
 Will not:
◦ prevent immunization of the patient
◦ guarantee normal survival of transfused
erythrocytes
◦ detect all unexpected antibodies in a patient’s
serum.

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9
Two types
Major cross-match:
 involves mixing recipient’s serum with the
donor’s red cells.

 is much more critical for assuring safe

transfusion than the minor compatibility
test.

 called major b/c the Abs in the recipient’s

serum are most likely to destroy the
donor’s RBC

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Minor cross match:
 Involves mixing the donor’s serum with
patient’s red cells

 Called minor because

◦ any Ab in the donor’s serum will be diluted by the
large volume of the recipient’s blood

◦ the destructed RBCs of the patient may be

compensated by the transfused RBC of the
donors

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1
 Accurate Patient Identification
 Proper sample collection and handling

 Review of the recipient’s past blood bank

records
 Careful ABO/Rh determination

 Antibody screening of the recipient (cross

matching of the donor unit).

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 In cases when the recipient possess a
clinically significant Antibody, donor units
must be:

◦ Screened for the corresponding Ag and should

be negative

◦ Cross- matched

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3
 Finally, during the actual transfusion :

◦ careful observation of the recipient’s vital signs and

◦ post transfusion hematocrit and Heamoglobin

levels must be considered.

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4
 The blood selected for cross-match should
be of the same ABO and Rh (D) group as
that of the recipient.

 However, Rh positive recipients may receive

either Rh positive or Rh negative blood.

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5
Whenever possible blood of the patients own
blood group should be given.

Otherwise the following rules should be

applied.
Group A patient.
- Should receive group A blood, if not available
group O
Group B patient.
- Should receive group B blood, if not available
group O

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6
Group O patient.
 Can only receive group O blood

Group AB patient.
 Should receive from group AB, if not
possible can receive blood from group A,B,
and O.

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7
 When cross-matching is carried out, the
serum is tested against the cells.

 The serum should be fresh, that is not more

than 48hours old, to make sure that it
contains complement.

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8
 When deciding on methods for cross-
matching, the following conditions are
required for Ag-Ab reactions.

◦ The right Temperature.

◦ Suitable surrounding medium
◦ Antigen-Antibody ratio etc..

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9
 The safe cross-matching of blood requires
that the donor’s cells be mixed with the
patient’s serum in three separate tubes,
using :

1. Saline
2. Albumin
3. Anti-human globulin reagents

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 The red cells from the donor are suspended
in saline and mixed with the patient’s
serum .
 show the presence of any complete
antibodies
 Agglutination in the saline tube is usually
caused by:
◦ anti-A or anti-B antibodies and
◦ Occasionally by Lewis, MNSs, Lutheran and kell
antibodies.

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1
 The red cells from the donor’s suspended in
saline, are mixed with the patient’s serum,
and albumin is added.
 The tube is incubated at 370C
 shows the presence of any incomplete
antibodies
 the antibodies react in albumin or any other
protein medium

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2
 Agglutination in the albumin tube is often
caused by:

◦ the rhesus antibodies,

◦ Lewis, MNSs, Lutheran and P antibodies, and
◦ occasionally by anti-kell.

 Reaction caused by anti- A or anti- B

antibodies usually occur in albumin as well
as in saline.

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 A more concentrated suspension of red
cells is mixed with the patient’s serum and
incubated at 370C and then AHG is added.

 A postive test detects the presence of

antibodies of:
◦ rhesus, kell, kidd, S and Lewis

 Anti globulin is essential for detection anti-

Duffy

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4
4.6. 5.1.Standard cross-match

 Is cross-match that is performed in three

tubes (Saline, albumin and AHG) within 45
to 60 minutes

Clinical significance
 detects unexpected (irregular) antibodies in
the recipient/ donor serum

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5
Principle

 Serum of the recipient / donor is tested

against the red cells of the donor/ recipient
under different conditions in order to
establish their compatibility

15
6
Type of specimen

◦ Serum (plasma) not older than 48 hrs

◦ Washed cells (20-30% and 2-5%)

15
7
Equipments and reagents

 Test tubes
 Centrifuge
 Microscope
 Microscopic slide
 Normal saline
 20% albumin
 AHG (Coombs reagents)

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8
1. Take 3 small tubes mark them 1,2 and 3, and add
to each the following

Tube 1 1volume of patient’s serum

1volume of 3-5% donor’s red cells
Tube 2 1 volume of 20% bovine albumin
1volume of patient’s serum
1volume of 3-5% donor’s red cells
Tube 3 3 volume of patent’s serum
1volume of 20-30% suspension of
donor’s cells

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9
2. Incubate tube 1 and 2 for 30 minutes at
370C. Incubate tube 3 for 15 minutes at
370C.

3. After incubation, remove tube 3 and wash

the cells three times with clean saline to
make sure that all the globulins are
removed from the cells.

4. And make a 3% saline suspension of the

washed cells in a tube.
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0
5. To one volume of red cell deposit add 2
volumes of fresh diluted antiglobulin
(coombs) Reagent.

6. Remove tube 1 and 2 and centrifuge with

tube 3 for one minute at 1000 rpm

7. Examine the tube for heamolysis

macroscopically and microscopically for
agglutination.

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1
Results
 No hemolysis or agglutination is seen in
tube 1, 2 or 3
◦ the blood is compatible and can be issued with
the completed cross-match label.
 If there is agglutination or hemolysis in any
of the tubes
◦ the blood is incompatible, and must not be
issued for the patient.

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2
ABO incompatibility (anti A and Anti-B.)
 Saline tube ………………….Shows strong
agglutination
 Albumin tube ………………. Shows agglutination
 Anti- globulin tube ………… show no agglutination

Rhesus incompatibility (anti-D ,c, e)

 Saline tube …………………….does not usually show
agglutination
 Albumin tube …………………. Shows agglutination
 Anti- globulin tube ……………. Shows agglutination

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3
 Performed when there is no enough time to
perform the standard cross match

 Takes about 25 to 30 minutes and

 Does not include antiglobulin test.

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4
Principle
 Serum of the recipient / donor is tested
against the red cells of the donor/ recipient
in saline and albumin medium in order to
establish their compatibility

16
5
Type of specimen

◦ Serum (plasma) not older than 48 hrs

◦ Washed cells (2-5%)

16
6
Equipments and reagents

 Test tubes
 Centrifuge
 Microscope
 Microscopic slide
 Normal saline
 20% albumin

16
7
1. Take 2 small tubes, mark them 1 and 2
and add to each the following

Tube 1 1volume of patient’s serum

1volume of 3-5% donor’s red cells
Tube 2 1volume of patient’s serum
1volume of 3-5% donor’s red cells.
1volume of 20% bovine albumin

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8
2. Leave tube 1 at room temp for 15 minutes
incubate tube 2 for 15 minutes at 370C.
3. Centrifuge both tubes for one minute at
1000
rpm/min
4. Examine the tubes macroscopically for
hemolysis and microscopically for
agglutination.

16
9
Results
 If no hemolysis or agglutination is seen in
either tube 1 or 2
◦ the blood is compatible and can be issued with
the emergency cross match.

 If agglutination or hemolysis is seen in

either of the tubes
◦ the blood is incompatible and must not be issued
for the patient.

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0
 Takes only 3 or 4 minutes

 Plasma is used instead or serum.

 Not safe and must only used in extreme

emergencies

 Standard cross match should be carried out

while the transfusion is in progress.

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1
1. Take 2 volume of patient’s plasma on a
slide

2. Add 1 volume of donor’s whole blood of

the same group as the patient and mix.

3. Leave for 2 minutes and examine

microscopically for agglutination

17
2
Results

 If the cells show agglutination the blood

must not be given and will usually indicate
that the wrong ABO group blood is being
cross marched.

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3
Sources of errors in cross-matching
 Rouleaux
 Auto agglutinins
 Infected donor cells
 Anti- A1
 Over centrifugation
 Dirty glass wares etc..

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4
 Only human blood and its components are
used for transfusion into humans
 Transfusions are the introduction of either
◦ Whole blood
◦ blood components (RBCs, Plts, plasma or WBCs )
or
◦ blood derivatives (albumin, gamma globulin,
Factors VII,VIII,Von willebrand,or Immune
globulins and prothrombin) directly into the
blood stream.

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5
 Whole blood- complex tissue ,composed of
cells and plasma

 Blood components-prepared from blood by

mechanical method especially by
centrifugation

 Blood derivatives-separated by more

complex process

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6
 The use of component therapy:

◦ Gives a better form of treatment

◦ Often permits considerable economy in the use of

blood

◦ Minimizes the risk of immune reactions

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7
 Contains all cellular elements and
coagulation factors
 Freshly drawn w/b maintains all its
properties for a limited time.
 Upon storage a number of changes occur,

Example:
◦ increase in O2 affinity and
◦ loss of viability of RBC.

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8
 To provide both O2 carrying capacity and
blood volume expansion
Example In the treatment of massive hemorrhage.
 Used as a source material for blood
component preparation.
 For exchange transfusion in new born.
◦ Whole blood less than 4-5 days old is often the
component of choice

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9
 Component make blood use more
economical by using one unit one can
treat:-

 Anemia with the packed cells

 Plate late deficiency with plate late preparations

 Clotting factor and other plasma deficiencies

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0
 Is the product remaining after the removal
of most of the plasma from freshly drawn
whole blood by centrifugation.

 The commonly available RBC preparations

are
◦ Concentrated red cell suspension
◦ Frozen/ Deglycerolized red cells
◦ Leukocyte poor red cell suspension

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1
 The red cell prepared in this form contain
the same:
◦ red cell mass and
◦ oxygen carrying capacity as whole blood with
approximately one half of the volume.

 The final Hematocrit of the product should

be between 70-80% in 250-300 ml of
volume

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2
 Concentrated red cell (CRC) is used to treat
patients with symptomatic anemia

 Transfusion of 1 unit red cell increases the

HcT by 3% and the hemoglobin by 1 gm.

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3
 Plasma is a fluid portion of one unit of blood collected and
separated in a closed system and intended for intra venous
use.

 The therapeutically useful plasma products are:

◦ Fresh Frozen plasma (FFP)

◦ Ordinary plasma (OP)/ single donor plasma
◦ Cryo depleted plasma

 All plasma products have to be stored at -180C or colder

18
4
Fresh Frozen plasma (FFP)
 Is the plasma obtained from a unit of whole
blood after centrifugation

 It is then separated and frozen solid at a

temperature that will adequately maintain
the labile coagulation factors in a functional
state.

18
5
 The indications for FFP are for:

◦ replacement of labile and stable coagulation

factors
◦ deficiencies of antithrombin III
◦ deficiencies of protein C and protein S
◦ treatment of thrombotic thrombocytopenic
purpura (TTP)

18
6
FFP
 Should be stored frozen at –300C or colder
for 12 months
◦ beyond this period the factors VII may have
decreased
◦ To maintain adequate levels of coagulation
factors
 If not used in 12 months time may be re
designated and relabeled ”Plasma” and has
4 more years of shelf life at –180C or colder.

18
7
 is deficient in labile coagulation factors.

 It is separated from single whole blood unit

at any time during its storage and up to 5
days after the expiration of the original
unit.

18
8
 OP is different from FFP in that it contains
high levels of K and ammonia since it is
prepared after long contact with red cells

 is stable up to 5 years after preparation if

kept frozen at-180C or colder.

18
9
 Indications:
◦ In the treatment of stable coagulation factors
deficiencies
◦ For volume and protein replacement under special
circumstances

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0
Cryo depleted plasma (CDP)
 CDP is the supernatant plasma following
removal of cryo precipitate.
- albumin
◦ immunoglobulin and coagulation factors are the
same as that of FFP,
◦ fibrinogen concentration and levels of the labile
coagulation factors V and VIII are markedly
reduced.

19
1
 CDP is indicated for patients requiring
volume expansion or protein replacement
when labile clotting factors are not required

19
2
 Is conversion of whole blood into
concentrated red cells with in the first 6
hours of collection.

 PC from random donations are the most

frequently used form of platelet product.
(“random” platelet)

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3
 Each unit of PC
◦ contains 5.5x1010 platelet in 50-65ml of plasma
◦ represents 60-80% of the plate late present in a
unit of whole blood.
◦ increases the platelet count by approximately
5,000-10,000 / ml in an average adult.

 The normal adult dose is 6-10 units, and

for child 1 unit/ 10kg

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4
 Platelet should be stored at
◦ 1-60C with agitation for 72 hours if prepared in a
close system
◦ 20-240C for

 24 hours if prepared in an open system

 5 days if prepared in closed system

 Refrigerated PC don’t maintain function or

viability as well as the PC stored at room
temperature.

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5
Indications

Patients who are actively bleeding from the

thrombocytopenia

◦ due to drugs, toxins, infections and bone

marrow failure

 Bleeding caused from qualitatively abnormal

platelet.

19
6
 PC of the same ABO group should be used

 Rh (D) incompatibility has no effect on

platelet survival
◦ but the extent of RBC contaminating in PC may
be sufficient to immunize Rh (D) negative women
in reproductive age group.

 In such cases Rh (D) negative Pc should be given

19
7
 Is a cold- precipitated concentration of
factor VII, anti hemophilic factor (AHF),
obtained after the plasma has been thawed.
 Contains:
◦ Factor VIII: C (pro coagulation factor)
◦ Factor VIII: VWF (Von-Willebrand’s factors)
◦ Fibrinogen factor XIII
◦ Fibronectin factor

19
8
 Each unit has at least 80μl of factor VIII,
250 mg of fibrinogen in a volume of 15 to
20 ml.

 Cryo:

◦ has a shelf life of 12 months at a temperature

of180C or colder (preferably-300C) and
◦ it must be transfused within 6 hours after
thawing at 370C.

19
9
Indications
 In the management of classic hemophilia
(factor VIII deficiency)
 In the management of factor XIII deficiency
 In the management of von willebrand’s
disease and
 source of fibrinogen for the treatment of
hypo fibrinogenemia.

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