Chapter One TO Immunohematology

CHAPTER ONE
INTRODUCTION
TO
IMMUNOHEMATOLOGY
.
1
CONTENTS
1.1 Overview of Immunohematology
1.2 Historical background

1.3 Blood Group Genetics
1.3.1 Inheritance pattern of blood group antigen
1.3.2 Chromosomal assignment
1.3.3 Homozygocity and hetrozygocity
1.3.4 Genetic inheritance
1.4 Blood cell antigen
1.4.1 Blood cell antigen
1.4.2 Human leukocyte antigen (HLA)
1.4.3 Platelet antigen
2
CONTENTS
1.5 Blood group antibodies and their stimulation
1.5.1 Natural (non red cell immune) antibodies
1.5.2 Immune antibodies
1.6 Antigen –antibodies interaction
1.7. Antiserum
1.8. Invitro Antigen antibody interactions---
added
1.8.1The influence of antibody type on
agglutination
1.8.2 Mthods of enhancing agglutination
3
Learning Objectives
At the end of this chapter, the student will be able
to:
- Explain a brief history of Immunohematology.
- Discuss patterns of inheritance of A and B
antigens.
- Describe the synthesis of H, A and B antigens.
- State the genotype of individuals with the
Bombay phenotype.
- State the characteristic genotype of secretors
and non-secretors.
4
1.1 Overview of Immunohematology
• Immunohematology:
– is more commonly known as "blood banking“
– deals with the concepts and clinical techniques

related to modern transfusion therapy.
– collecting, preparing, storing and transfusing blood.
5
Immuno hematology…
– 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.
6
1.2 Historical background
• The era of blood transfusion began when

William Harvey described the circulation of blood
in 1616.
• In 1665, Richard Lower- the first animal-to-

animal blood transfusion.
7
Historical background…
• 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.
8
• 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.
9
• 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.
10
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.
11
1.3 Blood Group Genetics
• 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.
12
Blood Group Genetics…
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.
13
• 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.
14
• 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.
15
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 .
16
Table 1.1. The ABO phenotypes and their
corresponding genotypes
Phenotype Genotype
A AA, AO
B BB,BO
AB AB
O OO
17
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 .
18
Table1.2. 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
19
1.3.1 Inheritance pattern of blood
group antigens
• 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.
20
Inheritance pattern…
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.
21
• 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.
22
Mendelian principles (law of independent
segregation)
• refers to the transmission of a trait in a

predictable fashion from one generation to the
next.
• Independent assortment is demonstrated by the

fact that blood group antigens inherited on
different chromosomes, are expressed
separately and discretely.
23
1.3.2 Chromosomal assignment
Table 1.3. Chromosomal assignment of genes in blood
group system
Blood group system chromosome
• Rh---------------------------------------------------------1
• Duffy------------------------------------------------------1
• Gerbich--------------------------------------------------2
• MNS------------------------------------------------------4
• Kell--------------------------------------------------------7
• ABO-------------------------------------------------------9
• Kidd-------------------------------------------------------18
• Lewis-----------------------------------------------------19
• Landsteiner-Wiener-----------------------------------19
• Lutheran-------------------------------------------------19
• Hh---------------------------------------------------------19
• P-----------------------------------------------------------22
24
1.3.3 Homozygosity & Hetrozygosity
Homozygous-
– Genotype is made up of identical genes, such
as AA, BB, or OO,
• Heterozygous.
– Genotype is made up of different alleles from
each parent, such as AO, AB, or BO,
25
Table 1.3.Dosage effect on antigen
expression
Genotype Dosage effect on antigen
expression
Homozygous :MM Red blood cell tested with

anti-M : +4
Heterozygous :MN Red blood cell tested with

anti-M : +2
26
1.3.4 Genetic inheritance
• Genes can inherit with each other depending on

whether they are inherited on the:
– same chromosome (Cis) or
– opposite chromosome (Trans).
27
Genetic inheritance…
• Trans interaction may weaken the expression of

one of the antigens encoded by the genes,
For example:
– The C and D genes of Rh system are inherited on

different genetic loci .
– When C is inherited in trans to D, it will weaken the D

antigen expression on the red blood cell.
28
Linkage and Haplotypes
• In some blood group systems, the antigens are

encoded by two or more genes on the same
chromosome.
• When genes are very close together, they are

inherited from each parent as a unit and are
known as linked
29
Genetic inheritance….
• Independent assortment does not occur when

genes are linked.
• These gene units are called haplotypes
Silent genes
• In some blood group systems genes do not
produce a detectable antigen product and are
called "silent” genes or amorphs.
30
• Amorphs can result in an unusual phenotype if

passed on by both parents.
• The phenotypes are often called "null" type

because expressions of the blood group system
antigen are not apparent.
31
• Rare gene must be inherited from both parents
(homozygous) to produce a null phenotype.
– null types caused by amorphic genes are

rare.
• Unusual phenotypes may also result from the

action of suppressors, or regulator genes.
32
• These genes (suppressor /regulator)
– act to inhibit the expression of another gene and,

– must be inherited in the homozygous state to create
this effect.
– suppressor genes that affects the blood group antigen
are rare.
• Null phenotype therefore can be resulted of

either an amorphic or a suppressor gene.
33
Table 1.4. Blood group genes that can
result an unusual phenotypes
Blood group Amorph/ Phenotype
system Regulator
gene
H h Bombay
Rh r/x0r Rh null
Kell K0 Kell null
Lutheran Lu/in(lu) Lu(a-b-)
Kidd JK JK(a-b-) 34
1.4 Blood cell antigens
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.
35
Red cell antigens…
• In biochemical terms these antigens may take
the form of:
– proteins,
– Glycoprotein,
– Glycolipids
36
Red cell antigens…
• Some of the red blood cell antigens are more

immunogenic than the others
Example
– The D antigen within the Rh group system.
37
1.4.2 Human leukocyte antigens
(HLA)
• 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.
38
Human Leukocyte antigens…
• The MHC system is important in the:
– recognition of non self ,
– coordination of cellular and humoral immunity , and
– graft rejection .
39
• The MHC region is on chromosome 6 and is

divided in to three categories or classes :
– Class I includes the A, B and C locus,

– Class II includes the DR, DP and DQ
– Class III includes the complement proteins
40
• The MHC region is called polymorphic , because

there are so many possible alleles .
For example :
• At least 49 different alleles or possible genetic

expressions have been identified at the A locus.
• At the B locus 97 alleles are identified.

41
1.4.3 Platelet antigens
• 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.
42
Platelet antigens…
• 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.
43
1.5 Blood group Abs & their stimulation
Blood group antibodies are classified into:
– Natural and
– Immune antibodies.
44
1.5.1. Natural / non red cell immune Abs
• 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.
45
Non-red cell immune antibodies…
Characteristics
• They are mainly IgM type.
• Exhibit optimum in vitro agglutination saline

media
– complete antibodies.
• Optimum reaction at room temperature or lower

– cold agglutinins.
46
Non-red cell immune antibodies…
• 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
47
1.5.2. Immune antibodies
• 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.
48
Immune antibodies…
• Optimally react at 370C

– warm agglutinins.
• Causes more serious transfusion reactions than

the naturally occurring ones.
• Can cross the placental barrier.
49
1.6 Antigen - Antibody interactions
• 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.
50
Ag-Ab interactions…
• 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
51
Ag-Ab interactions…
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
52
1.7.The Anti-serum
• 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.
53
The antiserum…
• 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
54
The antiserum…
• 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
55
The antiserum…
• 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
56
The antiserum…
• Have certain avidity or strength of reaction with

corresponding red cells
For example
Anti-A1should agglutinate:
– A1 cells in 10seconds or less,
– A2 cells in 20sec or less, and
– A2B in 30 sec or less
57
The antiserum…
• Be free from haemolysins, fat and rouleaux
• Be sterile and clear
• Preserved with 1% sodium azide and be stable.
58
The antiserum…
• Have a marked expiration date, and
• Should be stored at 40C
• The manufacturer directions must be followed

carefully.
59
1.8. Invitro detection of Ag and
Ab reaction
• The presence of Invitro antigen and
antibody interaction can be detected by:
– Hemolysis
– Precipitation
– Agglutination (Most commonly used)
60
The mechanism of agglutination
Agglutination:
• Visible clumping of particulate Ags caused by

interaction with a specific Ab
• Occurs in two stages:
– sensitization and
– lattice formation.
61
Stages of Ag-Ab Reaction…
A-Sensitization-the first phase
• represents the physical attachment of Ab

molecules to Ags on the RBC membrane.
62
Factors affecting the sensitization phase
1. The antigen - antibody ratio
For example : pro-zone phenomenon.
(overcome by serial dilution)
2. Physical conditions such as:

– PH
– Temperature
– Time of incubation
– Ionic strength, and
– Steric hindrance. 63
Stages of Ag-Ab Reaction…
B- Lattice formation – the second phase
• Is the establishment of cross links between

sensitized particles and Abs resulting in
clumping
64
Factor affecting the lattice formation phase
• 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.
– d/ce b/n net charge at cell surface & at surrounding
media, e.g cell suspension in NaCl solution
65
1.8.1. The influence of Ab type
on agglutination
• IgM antibodies are more efficient than IgG or IgA
antibodies in exhibiting invitro agglutination
• IgG antibodies are less efficient due to:

• The deep location of the antigen
determinants and
• Restricted movement of the hinge region
causes them to be functionally monovalent.
66
1.8.2.Methods of enhancing
agglutination
• Centrifugation
• Treatment with proteolytic enzyme
• Use of colloids, and
• Addition of anti-human globulin (AHG) reagent.
• Others
– Poly ethylene glycol (PEG)
– Low Ionic strength saline (LISS)
– Polybrene
67
Review Questions
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.
68
CHAPTER TWO
THE ABO BLOOD GROUP SYSTEM
69
CONTENT
2.1 Historical Overview of the ABO system
2.2 General characteristics of the ABO Antigens

2.3.In heritance and development of the A, B and H
Antigens
2.3.1 Common structure for A, B and H antigens
2.3.2 Development of the H antigen
2.3.3 Development of the A and B Antigens

2.3.3.1.Bombay and para-bombay blood groups
70
CONTENT
2.4. Secretors status
2.5. ABO subgroups
2.5.1. A1 and A2 subtype
2.5.2. Other sub groups
2.5.3. subgroups of B
2.6. ABO system antibodies
2.6.1 General characteristics

2.7. Routine ABO phenotyping
2.7.1.Direct(cell grouping)
2.7.1.Indirect(serum grouping)
2.8. ABO discrepancies
2.8.1.Technical error
2.8.2. Sample- Related

71
Learning objectives
At the end of the chapter, the student should be able to:
 Describe the historical overview of the ABO system.
 Describe the general characteristics of the ABO antigens
and antibodies.
 Discuses the pattern of inheritance and development of
the A, B and H antigens
 Differentiate the common structure of A, B and H antigen
 Describe some of the ABO subgroups.
 Explain Bombay phenotype and acquired B phenotype.
 Discuses the general characteristic of the human anti-A
and anti-B antibodies.
 Perform ABO blood grouping using different methods.
72
Learning…
 Describe the three important observations which
indicate the presence of ABO discrepancies.
 List the common technical errors that results
ABO discrepancies.
 List and explain sample related ABO
discrepancies.
 Discus why and how to do saline replacement
technique.
73
2.1 Historical Overview of the ABO system
• 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
74
2.2. General characteristics of the ABO
Ags
• ABO antigens are widely distributed and located

– on red blood cells
– lymphocytes
– platelets
– tissue cells
– bone marrow, and
– organs such as the kidneys.
75
Characteristics of the ABO Ags…
• 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
76
Characteristics of the ABO Ags…
• 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 in utero.
– 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.
77
Table 2.1. Frequency of ABO blood groups in
different population
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
78
2.3. In heritance and development of the
A, B and H Antigens
• 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.
79
Inheritance and development of…
• 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.
80
Inheritance and development of…
• ABO antigens are assembled on a common

carbohydrate structure that also serves as the
base for the formation of the H, Lewis I/i and P,
antigens.
• This common carbohydrate structure(antigen

building block) is capable of antigen expression
for more than one blood group system
81
2.3.1.Common structure for A, B and H
antigens
• 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.
82
Structure…
• The two terminal sugars, D-galactose and N-

acetylglucosamine, may be linked together in
two different configurations.
– β13 and
– β1 4 linkage
83
Structure…
• When C1 of D-galactose is linked with C3 of

N-acetylglucosamine, the linkage is
described as β13.
84
85
Structure…
• When the C1of D-galactose is linked with C4 of

N- acetyl glcuosamine, the linkage is described
as β1 4.
• Then type 2 oligosaccharide chains are formed
86
87
Structure…
• Type 2 structures are primarily associated with

Glycolipids and Glycoproteins on the RBC
membrane
• Type 1 structures are associated primarily with

body fluid.
• Some type 2 Glyco protein structures are

located in body fluids and secretions.
88
2.3.2.Development of H antigen
• 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 (amorph), frequency rare.

89
Development of H antigen…
• 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.
90
In the formation of H antigen :

• L-fucosyl transferase transfers L-fucose, to
either type 1 or type 2 common oligosaccharide
chains.
• The added L-fucose is called the

immunodominant sugar for H antigens
– It confers H specificity.
91
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.
92
93
2.3.3. Development of the A and B
Ags
• 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
94
95
Development of….
• The B-allele codes for D-galactosyl transferase

which transfers D-galactose (Immunodominant
sugar) to the H-antigen.
96
97
Development of Ags…
• 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.
98
Development of Ags…
• Other ABO phenotypes have fewer copies of H

Ags, since the H Ags is the acceptor molecule
for the A and B enzymes.
• Group A1B phenotype possesses the lowest

number of unconverted H sites.
99
100
2.3.3.1 Bombay and para-bombay groups
• Bombay phenotype (hh) individuals
– do not inherit the H gene and do not possess

the enzyme H transferase
– are unable to produce H substance.
101
Bombay….
– 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.
102
Para Bombay
• 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.
103
2.4.Secretor status
• 80% of individuals inherit the Se ( Secretor)

gene
• Are termed as Secretors
• Secretor A and B antigens are found:
– in secretions and all body fluids except CSF
– in a soluble form
• They are glycoproteins and has no clinical
significance
104
2.5. ABO sub groups
• Differ in the amount of antigen expressed on the

red blood cell membrane.
• Some sub groups possess more highly

branched, complex Antigenic structures
• Others have simplified linear forms of antigen.
105
106
2.5.1. A1 and A2
• Are major subgroups of A phenotype
• Are genetically controlled by the A1 and A2

genes, respectively and
• Differ slightly in their ability to convert H Ag to

A Ag.
107
A1 and A2…
A1 phenotype
– Is encoded by the A1 gene
– exists in approximately 80% of group A individuals
– A antigens are highly concentrated on branched and

linear oligosaccharide chains.
– A1 gene effectively acts on the H antigens in the

production of A Antigen.
108
A1 and A2…
A2 phenotype
– encoded by the A2 gene,
– constitutes approximately 20% of group A individuals.
– A antigen copies are fewer in number than A1

phenotype.
– is assembled on the simplified linear forms of the

oligosaccharide chains.
109
A1 and A2…
• Both A1 and A2 RBCs agglutinate with

commercially available anti- A reagents.
• Dolichos biflorus lectin (anti- A1 lectin)
– Is extracted from the seeds of the plant Dolichos

biflorous ,
110
Dolichos biflorus lectin…
– possesses anti- A1 specificity.
i.e. Agglutinates A1, but not A2 red blood

cells.
– is not used in the routine ABO testing of donors and

recipients
– useful in resolving ABO typing problems and

identifying infrequent sub groups of A.
111
2.5.2 Other sub groups of A
These are:
• Aint, A3, Ax, Am, Ael, and A bantu,
• More infrequent than the A1 and A2
• Have reduced expression of A antigens.
112
2.5.3. Sub group of B
• Are less common and include B3, Bx ,Bm and Bel.
• React weakly with or not at all with anti-B, anti -

AB and strongly with anti H.
113
Acquired B phenomena
• 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 to Galactose
• restored when infection is controlled
114
2.6. ABO system antibodies
• 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
115
2.6.1.General characteristics of Human
anti- A and anti - B
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.
116
General characteristics…
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.
117
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.
118
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.
119
Anti- A1
• Sera from group O and B individuals contain
anti- A antibodies.
• can be separated into: anti- A and Anti- A1.
• A1 antibody is specific for the A1 antigen and
does not agglutinate A2 RBCs.
• Optimally react at room temperature or below.
120
Anti- A1…
• Is not clinically significant for transfusion

purposes.
• becomes a concern when it causes

problems with ABO pheno typing and
incompatible cross matches on immediate
spin.
121
2.7. Routine ABO phenotyping
ABO phenotyping has two components:
1. Forward grouping
2. Reverse grouping
122
Routine ABO phenotyping…
2.7.1.Direct (cell grouping)

Testing of the red blood cells for the presence of
ABO Antigens (or forward grouping).
2.7.2. Indirect (serum grouping)

Testing of serum or plasma for the expected
ABO Antibodies (or reverse grouping).
123
Principle
• When an antigen is mixed with its corresponding
antibody under the right conditions it causes
agglutination or haemolysis of the red cells.
124
Clinical significance
– For safe blood transfusion

– Prevention of Hemolytic Disease of the Fetus
and Newborn (HDFN)
125
Points to be considered in routine
phenotyping
• 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 .
126
Points to be considered …
• 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.
127
Points to be considered…
• ABO discrepancy
– occurs when ABO phenotyping of red blood cells
does not agree with expected serum testing results
for the particular ABO phenotype.
128
The methods for grouping

1. Tube method
2. Emergency tile or slide grouping
3. Tile or slide grouping of blood donors
129
1.Tube grouping method
Specimens
-Patient’s serum .
-Patient’s cells.
130
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
131
Preparation of Red Cell Suspension:

% required= PCV x100
Volume of suspension
132
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.
133
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.
134
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.
135
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.
136
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.
137
Table 2.2. ABO phenotyping Reaction
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
138
Grading of Antigen-antibody
agglutination reaction in ABO grouping
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.
139
Grading of …
+ 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
140
2. Emergency tile or slide grouping
Emergency tile grouping is carried out using a
white glassed porcelain tile, Perspex tray or
slides.
141
Emergency tile or slide grouping…
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
142
• 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
143
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
C.1 volume of group O serum
144
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.
145
Illustration Of The Forward And Reverse Grouping
Reaction Patterns Of the ABO groups
146
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 .
147
Tile or slide grouping of blood donors…
Specimen
– Whole blood
Reagents and equipments:
– Anti- A and anti-B serum
– Group O serum (anti-AB)
– Tile or slide
– Applicator stick
148
Method
1. Take two slides or a white tile and mark as
follows
Anti-A Anti-B Anti-AB
149
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.
150
Other methods of Detection of Ag-Ab
Reaction
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.
151
Other methods ….
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.
152
2.8. ABO discrepancies
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+.
153
ABO discrepancies…
• 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.

154
ABO discrepancies…
Can be technical or sample –related problems.
2.8.1.Technical error
• Occur more in student laboratory
• Identification or documentation error.
• Reagent or equipment errors.
• Standard operating procedure errors
155
Technical error cont’d
Clerical error
• Improper identification of patient sample
• Improper recording of reactions
Technical error
• Failure to follow manufacture’s directions
• Contaminated reagents
• Improper concentration of subject red blood cell
• Failure to add reagents or improper amounts
• Improper centrifugation and warming of test
156
2.8.2. Sample- Related ABO Discrepancies
• ABO discrepancies that affect the ABO red

blood cell testing.
– Extra antigens present
– Missing antigens
– Mixed field reaction
157
Sample- Related…
• 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
158
Review Question
1. Briefly discus the historical overview of
ABO system.
2. Describe the general characteristics of
human anti-A and anti-B antibodies.
3. Draw and label the structure of ABH
antigen.
4. Describe the general characteristics of
ABO antigen.
5. Discuss the inheritance and development
of the ABO antigens.
159
6. Discuss how to perform direct and indirect method
of ABO blood grouping.
7. Explain the inheritance and synthesis of ABH
antigens.
8. What is acquired B phenotype?
9. Compare and contrast A1 and A2 subgroups of A.
10.What are the three major indicators of ABO
discrepancies?
11. List the basic technical errors during ABO
grouping.
160
Chapter Three
The RH-Hr blood group system
161
CONTENT
3.1 Historical Background of Rh-Hr Blood Grouping
3.2.Genetics and Biochemistry of the Rh antigen-modi
3.2.1 Rh Nomenclature
3.2.1.1Fisher- Race: CDE Terminology
3.2.1.2. Wiener: Rh- Hr Terminology
3.2.1.3 . International society of Blood transfusion (ISBT)

standardized numeric Terminology
3.3 Determining the Genotype from the phenotype--added
162
Content…
3.4 Antigens of the Rh system

3.4.1. D-antigen concentration
3.4.2. Weak D(Du)
3.5 Rh- antibodies
3.6. The Rh-hr blood grouping technique
3.6.1 Slide Test Method
3.6.2 Modified Tube Test Method
3.6.3 DU Typing
163
Learning objectives
At the end of the chapter the student should be

able to:
• Describe the historical background of the Rh
system.
• Compare the genetic inheritance and
nomenclature of the Rh antigen in the Wiener
and Fisher –Race theories
• List the most common Rh antigens and their
characteristics.
164
Learning…
• Discuss the common variant of D antigen (DU)

including its clinical significance.
• Discuss the characteristics and the clinical

significance of Rh antibodies.
• Describe the methods of Rh phenol typing.
165
3.1 Historical Background of Rh-Hr Blood
Grouping
• 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.
166
Historical Background…
• 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.
167
• 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.
168
• 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.
169
• 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).
170
• 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.
171
• 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.
172
3.2.Genetics and Biochemistry of the Rh
antigen
• 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.
173
Genetics and Biochemistry…
174
Genetics and Biochemistry…
• 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
175
Table 3.1 the different Rh blood group
amino acids and their sequence
Antigen Amino acid N0
C Serine 103
c Proline 103
E Proline 226
E Alanine 226
176
3.2.1 Rh Nomenclature
3.2.1.1. Fisher-Race:CDE terminology

• Postulated that the Rh system antigens were
inherited as a gene complex or haplotype that
codes for three closely linked sets of alleles.
– D is inherited at one locus,
– C or c at the second locus, and
– E or e at the third locus.
Each parent contributes one haplotype or set of Rh
genes.
177
Fisher-Race:CDE terminology…
• Each gene expresses an Antigen that is given

the same letter as the gene except that when
referring to the gene the letter is italicized.
For example:
The gene that produces the “C” antigen is C.
178
• The original theory assumed the d allele was

present when the D allele was absent.
• The “d” is still some times written to denote the

absence of the D antigen.
179
• The order of the genes on the chromosome,

according to the Fisher- Race theory, is DCE;
however, it is often written alphabetically as
CDE.
180
181
3.2.1.2. Wiener: Rh- Hr Terminology
• Alexander Wiener postulated that alleles at one

gene locus were responsible for the expression
of the Rh system antigens on the RBCs.
• Each parent contributes one Rh gene.
• The inherited form of the gene may be identical

(homozygous) or different (heterozygous) from
each parent.
182
Wiener: Rh- Hr Terminology…
Allele: short hand

RO, R1,R2, Rz
r, rI, rII, ry
Antigenic specificities: Dce, DCe,
DcE, DCE, dce, dCe, dcE, dCE
Fig.3.3 Wiener genetic theory: 1

locus
183
• According to Wiener, eight alleles exist at the Rh

gene locus: RO, R1 R2, Rz, r, rI rII and ry.
• The gene encodes a structure on the RBC called

an agglutinogen which are detected by their
factors
• These factors are identified with the same

antisera that agglutinates the D, C, c, E and e,
antigens.
184
• The difference between the wiener and Fisher-

Race theories is the inheritance of the Rh
system is on a single gene locus rather than 3
separate genes.
185
Table 3.2 Wiener Theory: Genes and the
factors or antigens they encode
Gene Antigens Gene Antigens
(Fisher- Race) (Fisher- Race )
RO cDe r Ce
R1 CDe rI Ce
R2 cDE rII cE
Rz CDE ry CE
Factors in wiener terminology:

D =Rho, C= rhI, E= rhII e= hrII, c=hrI
186
Table 3.3 Converting Fisher- Race
terminology to Wiener`s
Fisher- Race Antigen R (D+) r (D-)
C 1 '
E 2 ''
CE z Y
Ce O
* Upper- case R indicates the D antigen is present,

lower- case r indicates the D antigen is absent, 1 ‘ , 2 “ ,
O, z, and y refer to the presence or absence of the C,E,
c, and e Antigens. 187
3.2.1.3. International society of Blood transfusion
(ISBT) standardized numeric Terminology

• ISBT assigned a six- digit number to each blood
group specificity.
• The first three numbers represent the system,

and the remaining three represent the
specificity.
188
ISBT…
• The Rh system assigned number is 004, and the

remaining three numbers corresponds to the
Rosenfield system.
For example
The C Antigen’s ISBT number is 004002.
189
ISBT…
• An ISBT “symbol” or alpha numeric designation

similar to the Rosen field terminology is used to
refer to a specific antigen.
• The term Rh is written in upper- case letters, and

the antigen number immediately follows the
system designation
190
3.3 Determining the Genotype from the
Phenotype
• 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.
191
Genotype from the Phenotype…
• Five anti sera are used to determine the Rh

system phenotypes - anti- D, anti- C, anti- c,
anti- E and anti- e.
• Once the phenotype is known, the most

probable genotype can be determined by
knowing the most common Rh system genes for
the race of the person being tested.
192
Table 3.4. Order of frequency of the common
Rh system Haplo type Rh Gene frequency
Whites Blacks
Highest CDe(R1) cDe (R0)
cde (r) cde (r)
cDE (R2) CDe (R1)
cDe (R0) cDE (R2)
Lowest
Cde(r’) is 2% or less, cdE(r”), CDE(R2), CdE(Ry)

are less than 1%
193
3.4 Antigens of the Rh system
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.
194
3.4.1.D-antigen concentration
• Varies depending on the antigen inherited at the

RHCE gene
• The D deletion phenotype has the most D- Ag
sites.
• The C gene weakens the D expression if
inherited on the opposite chromosome.
• R2R2 cells show stronger D expression than R1R1
cells
• D> R2R2 > R1R1 > R1r or Ror>R1r1 or Ror1

195
3.4.2.Weak D (Du)
• Most RBC can be typed for the D Ag directly with

anti-D reagents, but when the D-Ag is weakly
expressed on the RBC, its detection requires the
indirect antiglobuline test (IAT).
• Red blood cells that are positive for D only by the

IAT are referred to as weak D.
• Older terminology classified weak D-Ag as Du and

the test is still sometimes referred to as the Du test.
196
Weak D (Du)….
• Newer monoclonal typing reagents for Rh

system antigens have enhanced the ability to
detect the weaker D-antigens without additional
testing.
• Weaker D expression can result from several
different genetic circumstances, such as
– Genetic
– Position effect and
– Partial D.
197
3.5 Rh- antibodies
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.
198
Rh- antibodies…
• Some Rh Abs may be IgM (anti-E) or found in

individuals never transfused or pregnant (anti-
Cw)
• Anti- D is typically stronger with cDE/cDE (R2R2),

since these cells have more D-Ag sites.
• Are not associated with complement activation

which would be detected by hemolysis in tube
testing or the use of poly-specific AHG reagent.
•
199
Rh- antibodies…
• 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).
200
3.6 The Rh-Hr Blood grouping Technique
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.
201
Rh grouping…
– For safe blood transfusion

– Prevention of Hemolytic Disease of the Fetus
and Newborn (HDFN)
202
Rh grouping…
Principle
• When an antigen is mixed with its corresponding
antibody under the right conditions it causes
agglutination or haemolysis of the red cells.
203
3.6.1.Slide Test
Specimen
• Washed RBC(40-50%)
• Anti- D serum
– Microscopic slide
– Microscope
– Applicator stick
– Albumin (Control)
204
Slide Test….
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.
205
Slide Test Method…
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.
206
3.6.2.Modified Tube Test
Specimen
• Anti- D serum
– Test tube
– Centrifuge
– Microscope
207
Modified Tube Test…
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.
208
Modified Tube Test…
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.
209
3.6.3 DU Typing Using Indirect Anti-
Globulin Test (IAT)
Specimen
• Anti- D serum
– Test tube
– Centrifuge
– Microscope
– AHG serum
210
Du Typing…
1. Use the initial Rh D typing tube and control in the

above procedure. Incubate the Rh negative or weakly
reactive (+/_) samples and the control at 370c for 30
minutes.
2. Wash cells in both the test and the control tubes 3-4
times with normal saline.
3. Add one drop of the poly specific anti-human globulin

(coomb`s) to each tube and mix well.
211
Du typing…
4. Centrifuge at 2200-2800 rpm for 10 seconds.

5. Gently re suspend the cell button and look for
agglutination.
Interpretation
Positive -shows agglutination in the tube
containing anti-D while the control is negative .
Negative-absence of agglutination in both the test
and control tubes
212
Du typing…
• Potentiators (reagents added to the serum- cell

mixture to enhance antibody up take during the
incubation phase of the IAT) includes:
– high – protein
– low- ionic strength solution (LISS),
– proteolytic enzymes, and
– poly ethylene Glycol (PEG) is useful in identification
procedures.
213
Review Questions
1. Discuss the development of the Rh system.

2. Contrast the Fisher-Race and Wiener’s genetic theories
on the Rh antigens.
3. List the common Rh antigens and their characteristics.
4. Discuss the clinical significance of the Du antigen.
5. How are the Rh antibodies produced?
6. Discuss the clinical significance of the Rh antibodies
7. Outline a brief laboratory procedure to classify an
individual as Rh positive or Rh negative.
214
CHAPTER FOUR
OTHER MINOR BLOOD GROUP
SYSTEMS
215
Content
4.1.Kell Blood Group system
4.2 .Duffy Blood Group system
4.3 .Lutheran blood group system
4.4 .Lewis Blood group system

4.5. I blood group system
4.6. P blood group system
4.7.MNSs blood group system
4.8. Kidd (JK) blood group system
216
Learning objectives
At the end of this chapter, the student should be

able to:
• Briefly explain the characteristics and
biochemistry of other blood group antigen.
• Name the type of immunoglobulin class
produced in other blood group system and their
reactivity.
• Compare and contrast the characteristics of
antibodies in other blood group system.
217
4.1.The kell Blood Group System
• Discovered in 1946
• Includes 21 high and low frequency antigens
well developed at birth
• Kell (KEL) locus is found on chromosome 7 and
has 4 sub locus – Kpa/Kpb, K/k, Jsa/Jsb,
KELL11/KELL17
– High frequency antigens – k, Kpb, Jsb, KELL11
– Lower frequency antigens – K, Kpa , Jsa, KELL17
218
The kell Blood Group ….
• The K antigen is a powerful immunogen

• Anti-K is
– the kell system antibody most commonly
encountered in routine blood bank practice.
– an immune mediated IgG
– can cause both HDN and HTR
219
Kell…
ISBT ISBT Clinical Antibody Optimal Reactive Enzyme

system system significance class To phase phase
symbol No
Kell 006 Yes IgG At 370C AHG No

effect
220
4.2.The Duffy blood group system
• discovered in 1950
• is a single locus system with two antigens, Fya
and Fyb
• Possible phenotypes includes Fy (a+b-), Fy(a-
b+) Fy(a+b+), Fy(a-b-)
• The only rare phenotype is Fy (a-b-).
– Protects from P.falciparum infection.
221
Duffy…
• Anti-Fya and anti-Fyb are the antibodies

encountered in this Fy sytem with anti-Fya being
seen much more commonly.
• These antibodies usually are IgG
• Even though rare antibodies of this system are

associated with mild HDN and HTR
222
Duffy…

system system significan class TO phase phase
symbol No ce
Fy 008 Yes IgG 370C AHG
223
4.3.The Lutheran Blood Group
system
• Identified in 1945
• Single locus (on chromosome 19) system with

antigens Lua and Lub
• Antibodies (anti-Lua and anti-Lub) are IgG

– Are the mild causes of HDN even not common.
– They are not associated with HTR.
224
system system significance class TO phase phase
symbol No
Lu 005 Yes (Lub) IgG/IgM Room Room E

To (RT) temp. 
37oC AHG
E
Key:  No significant change is observed after the
addition of enzyme
225
4.4.The Lewis blood group
• Identified in 1946
• Lele locus is on chromosome 19.
• Lea and Leb are the major antigens.
• Others include Lec, Led, and Lex (LE3)
226
Difference of Lewis blood group from the
others
• Lewis antigens are not intrinsic to the RBC
membrane produced during cell development
• An individual’s Lewis phenotype is not

determined solely by genes at the Lele locus but
also by the action of genes at the Hh and Sese
loci.
• The amount of Lewis antigen expressed on the

RBC varies according to the cell’s ABO
phenotype.
227
Lewis…
• Phenotypes commonly seen in the Lewis blood

group system are:
• Le (a- b+)
• Le (a + b-) and
• Le (a+b+) ---- rare in adults, more frequent in
young children.
228
Acquisition of Lewis Ags by RBCs
• The Lewis phenotype of RBC’s

– depends on the phenotype of the plasma in which
they are suspended
– can be changed by incubating the cells in plasma
containing different Lewis active glycolipids.
Eg. If Le (a – b-) cells incubated with plasma

containing Lea or Leb glycolipid,
– they adsorb the available antigen from the plasma
and
– subsequently type as Le (a+ b-) or Le (a-b+)
229
Lewis system Abs
Two main types: anti-Lea and anti-Leb

• Anti-Lea
– Very common and naturally accuring
– Produced almost exclusively by the Le (a-b-)
phenotype
– Usually IgM, react best at room temp and rarely at
370C
– Can bind complement
– Not associated with HDN because the antigen is not
present on fetal RBCs
230
Lewis..
• Anti Leb
– usually IgM,
– binds complement
– produced by Le(a-b-) individuals and rarely by
Le (a+b-)
– Two subgroups of anti-Leb
• Anti- LebL reacts with all Le (a-b+) cells regardless
of ABO type
• Anti-LebH reacts with Le (a-b+) cells of O or A2
type
231
system system significance class TO phase phase
symbol No
Lu 007 No IgM room To IS

37oC RT
370C E
Key:E :No agglutination seen on the addition of enzyme, rather

agglutination reaction will disappear
IS: agglutination reaction on immediate spin
232
4.5.The I Blood group system
• Discovered around 1956 by Winner and his

associates
• The I Ag is found on almost all adults
• Is part of the precursor component of the

oligosaccharide that forms the A, B and H Ags.
233
I…
• Red cells that posses this Ag are labeled as I
and those that lack as i.
• The anti-I antibodies are naturally occruing, of

IgM type with their optimal temp at 40C
• The antibodies of the I blood group system are

not associated with HDN and HTR
234
4.6.The P Blood group system
• Discovered in 1927
• The most common phenotype are P1 and P2

which are analogous to the A1 and A2
phenotypes seen in the ABO system.
• P1 individuals have two antigens on their RBCs:

P1 and P.
235
P…
• P2 individuals have only the P Ag and can

produce anti-P1.
• Anti-P1 is frequently encountered antibody

– It is naturally occurring
– IgM, cold – reactive agglutinin, and does not react
above RT
– Not associated with HDN or HTR
236
4.7.The MNSs Blood group system
• Discovered in 1927 by Land Steiner and Levine

• There are two loci on chromo. 4 M/N and S/S.
• The Antigens are M, N, S and s
• Anti-M, Anti-N and Anti-S Anti-s are the major
Abs of the system
Anti-M
– Naturally occurring reacting at RT or below
– May be either IgM or IgG
– Rarely causes HDN or HTR
237
MNSs…
Anti-N
– Usually weak cold-reactive,
– naturally occurring IgM Ab produced by individuals
who are M+N- and who are positive for S or s
– Clinically not significant
Anti-S and Anti-s
– Usually present as immune Abs.
– Usually IgG but rare IgM types present
– Anti-M and anti-N are not associated with HDN or
HTR but Anti-s and anti-s
238
4.8.The KIDD (JK) Blood group system
• was discovered in 1951

• is a single locus system with two antigens
Jka and Jkb
• Phenotypes
– Jk (a-b-)-------rare
– Jk(a-b+)
– Jk (a+b+)
– JK (a+b-).
239
KIDD…
• The primary antibodies of the kidd system,

– are anti-Jka and anti-JKb
– are usually immune mediated ( IgG)
– mild HDN and delayed HTR
240
Review questions
1.Explain why blood groups like Lewis, Kidd… are

called minor blood groups?
2. Explain the relationship and difference of ABO
and Lewis system antigens development.
2. Compare and contrast the different other blood
group antibodies.
3. List the different other blood group antibodies
responsible for hemolytic reactions.
241
CHAPTER FIVE
THE ANTI-GLOBULIN (COOMB`S)
TEST
242
Content
5.1 The anti –human globulin test
5.1.1 Direct antiglobulin test (DAT)
5.1.2 Indirect antiglobulin test (IAT)
243
Learning objectives
At the end of the chapter, the student will be able

to:
• Define coomb`s test and its purpose.

• Explain the different principle of anti-human
globulin test.
• Describe how the anti-human globulin reagent is
prepared
• Describe the purpose of antihuman globulin test
244
5.1.THE ANTI-GLOBULIN (COOMB`S) TEST
• It was introduced by Coomb’s in 1945.

• Is a sensitive technique to detect incomplete Abs
– Abs that are sensitized but which fail to agglutinate

RBCs suspended in saline at room temperature, mainly
IgG.
– are agglutinated by the anti-IgG in antiglobulin serum

through the linking of the IgG molecules on neighboring
red cells.
245
AHG…
• Red cells can also be agglutinated by a

reaction of complement components on
their surface with anti-complement serum.
246
AHG…
Types of AHG:
1. Broad spectrum (polyspecific):
• prepared by combining anti-IgG and anti-

complement.
• The reagent may also contain antibodies of

other specificities such anti-IgM, anti-IgA, anti
C3, or anti C4.
247
AHG…
• The anti-C3 in the antiglobulin reagent

(AHG)
– binds to the C3 on the sensitized red cells;
– bridges the gap between the cell – bound
human C3 on adjacent red cells.
– helps in detecting IgM antibodies,
– enhance the reactions seen with complement
binding antibodies.
248
AHG…
2. Monospecific:
• contains only a single antibody:
– anti-IgG or
– only anti-complement.
• It is advantageous to detect RBC bound IgA,

IgM and/or complement components by this test.
• The antiglobulin test can be used to detect

invivo or invitro red blood cell sensitization
249
AHG…
• Types of AHG tests
– the direct antiglobulin test (DAT)
– the indirect antiglobulin test (IAT).
250
5.1.1 Direct antiglobulin test (DAT)
It is the detection of in vivo sensitized RBC.
Clinical significance:
• Diagnosis of HDN
• Diagnosis of autoimmune hemolytic anemia
• Investigation of drug sensitized RBCs
• Investigation of transfusion reactions.
251
DAT…
Principle
• Washed red blood cells from the patient are
directly tested with the antiglobulin serum
Specimen
• 2-5% RBC suspension
252
DAT…

• Test tubes
• Centrifuge
• Microscope
• Microscopic slides
• AHG
• Physiological saline
253
DAT…
Procedure
1. Place one drop of a 2% to 5% saline suspension of
cells to be tested in a labeled tube.
2. Wash 3 or 4 times with saline. After last wash decant
completely. Add one or two drops of antiglobulin serum
and mix.
3. Centrifuge: examine for agglutination with an optical
aid; record results. (The tube should be held at an
angle and shaken gently until all cells are dislodged.
Then it should be tilted gently back and forth until an
even suspension of cells or agglutinates is observed.
254
DAT…
Control
• To control for inadvertent contamination of the
AHG,
– add one drop of IgG- sensitized red blood cells to
any tubes that have been recorded as negative and
re-centrifuge
– If the patient’s cells were washed adequately in the
first stage of the test, the control cells should be
agglutinated and the negative result on the patient
is valid.
255
5.1.2 Indirect antiglobulin test (IAT)
It is the detection of Ab that may cause RBC

sensitization invitro.
Clinical Significance:
• Detection and identification of unexpected

Antibodies.
• Cross matching
• Detecting RBC Antigens not demonstrable by
other techniques etc….
256
IAT…
Principle
• The Antibody-containing serum is incubated with
specific RBCs which, following washing, are
reacted with antiglobulin serum to see whether
RBC sensitization has occurred.
257
IAT…
Equipments and Reagents

• Test tubes
• Centrifuge
• Microscope
• Microscopic slides
• AHG
• Physiological saline
• Anti-D
258
IAT…
Procedure
1. Place two to six drops of the serum under test
(patient’s serum) in a test tube.
2. Add one drop of washed 5% suspension of the
test cells (donor’s RBC, screening RBC, etc)
optional: Two drops of 20-22% bovine albumin
may be added to the mixture
3. Mix well
4. Incubate at 370C for 15 to 30 minutes.
259
IAT…
5. Centrifuge immediately on removal from the

incubator for 15 seconds at 3400 rpm:
examine for hemolysis and agglutination using
an optical aid, and record results
6. Wash 3 or 4 times in large amounts of saline.
Decant each wash as completely as possible.
7. Add one or two drops of antiglobulin reagent.
8. Mix well.
260
IAT…
9. Centrifuge at 3400 rpm for 15 sec.

10. Examine for agglutination using an optical aid.
11. Record results
12. Add one drop of known sensitized red cells to
all negative test. Centrifuge at 3400 rpm for 15
sec: examine for agglutination. If no
agglutination is seen, the test result is invalid
and must be repeated.
261
Review Questions
1. What is the purpose of the AHG test?

2. How can the AHG reagent cause agglutination
of sensitized red cells?
3. Write the principle of the DAT and IAT.
4. What conditions can be diagnosed by DAT.
262
CHAPTER SIX
HEMOLYTIC DISEASES
263
Content
6.1.Hemolytic trasnfusion reaction

6.2.Autoimmune Hemolytic Anemia (AIHA)
6.3. Hemolytic Disease of the Fetus and New born (HDFN)
6.3.1 overview of HDFN------added

6.3.2.HDFN due to Rh
6.3.3.HDFN due to ABO
6.3.4 Assessment of HDFN -------added
6.4 Rho (D) Immune Globulin (Human) – RhIG -mod
6.5.Treatment of Infants Suffering from HDFN
264
Learning objectives
At the end of this chapter, the student will be able to:
• Explain the cause and laboratory diagnosis of the

different hemolytic diseases .
• Explain the cause and consequence of HDN due to ABO

and Rh incompatibility.
• Name the major immunoglobulin class responsible for

hemolytic disease of the new born
265
Learning..
• Explain the different etiology HDN
• Describe the prenatal treatments that are

significant in HDN caused by anti-D.
• Compare and contrast the HDN caused due to

ABO and Rh incompatibility.
• Describe how to prevent Rh-alloimmunization.
266
6.1 Hemolytic Transfusion reaction (HTR)
• Is a condition in which there is intravascular

destruction of transfused RBCs
• Is mainly caused by antibodies of the ABO and
Rh system.
• Clinical consequences include
– Dissiminated intravascular hemolysis(DIC)
– Hypotension
– Irreversible shock and renal failure
267
6.2 Autoimmune Hemolytic Anemia (AIHA)
• Brought about through the interaction of red

cells and auto antibodies.
• Classified into four groups

– warm-reactive auto antibodies
– cold- reactive auto antibodies
– paroxysmal cold hemoglobinuria and
– drug induced hemolysis.
268
AIHA…
• The diagnosis depends on the demonstration of

auto antibodies on the patient’s red cells using
the DAT.
• The autoimmune antibodies are either

– ‘warm antibody’ type or
– ‘cold antibody’ type.
269
AIHA…
• The warm antibodies are active at 370C
• The cold types are optimally active at 20C but

with a temperature range which may go as high
as 320C.
270
AIHA…
• A positive DAT will be found in both types;
– In the cold type , complements rather than the bound

antibody sensitizes the cells and give rise to the
positive DAT
– In the warm type, the attached antibody sensitizes the

cells, although occasionally it may be complement.
271
6.3 Hemolytic Disease of the Fetus and
New born (HDFN)
• Originally known as erythroblastosis fetalis
• Initially observed in babies from D- negative

women with D-positive mates.
• Initial pregnancies were not usually affected.
• Infants from subsequent pregnancies were often

still born or severely anemic and jaundiced.
272
HDFN…
Rh immune globulin (RhIG)

• Protects D- negative mothers against the
production of anti- D following delivery.
• Anti-C, anti-c, anti-E, anti-e are not protected by
RhIG and can cause HDN.
273
6.3.1 Overview of HDFN
HDFN is a condition in which the red blood cells

of a fetus or neonate are destroyed by
Immunoglobulin G (IgG) antibodies produced by
the mother.
274
Overview of HDFN…
Factors that must be present for HDFN to occur:
1. The mother must lack the Ag, and, following

exposure to the Ag should produce Abs of the
IgG class.
2. The Fetus must possess the Antigens
3. The Antigen must be well developed at birth
275
Etiology of HDFN
• The placental barrier limits the number of fetal

RBCs entering the maternal circulation during
pregnancy and thus reduces the chances of Ab
production during pregnancy.
276
Etiology of HDFN……
• At the time of delivery , a fetal RBCs escape into

the maternal circulation (known as feto maternal
hemorrhage (FMH).
• Immunization can result from fetal RBC

exposure following:
• Abortion
• Ectopic pregnancy, or
• Abdominal trauma.
277
• Ags on the fetal RBC can stimulate the maternal

immune system which results in the production
of IgG antibodies .
• In a subsequent pregnancy the IgG antibodies

cross the placental barrier by active transport
mechanism.
278
• The antibodies bind to the fetal antigens which

results in RBC destruction by macrophages in
fetal liver and spleen.
• Hemoglobin liberated from the damaged RBCs

metabolized to indirect bilirubin.
279
Etiology of HDFN….
• Bilirubin is harmlessly excreted by the mother.
• If the RBC destruction continues, the fetus

becomes increasingly anemic
• Fetal liver and spleen enlarge as erythropoiesis

increases in an effort to compensate for the RBC
distruction.
280
• Erythroblasts are released into the fetal
circulation
– Erythroblastosis Fetalis
• If this condition is left untreated, cardiac failure

can occur accompanied by hydrops fetalis, or
edema and fluid accumulation in fetal peritoneal
and pleural cavities.
281
• Thus the greatest threat to the fetus is cardiac

failure resulting from uncompensated anemia.
• Following delivery, red blood cell destruction

continues with the release of indirect bilirubin
• The new born liver is deficient in glucuronyl

transferase
282
• The indirect bilirubin binds to tissues results in

jaundice.
• Bind with tissues of the CNS and cause

permanent damage (kernicterus)
– resulting in deafness, mental retardation, or death.
283
Etiology of HDFN….
• HDN is often classified into three categories

based on Antibody specificity:
– Rh
– ABO and
– Other (non-Rh) Antibodies.
284
Fig.6-1 Development and prevention of HDN
285
6.3.2.HDFN due to RH
• Anti- D is responsible for the most severe cases

of HDFN.
• In most cases, D-Negative women become

alloimmunized (produce anti- D) after the first D-
Positive pregnancy.
286
HDFN due to RH…
• In rare cases, alloimmunization occurs during

the first pregnancy but rarely results in clinical
signs of HDFN.
• In some cases, the maternal anti-D binds to fetal

D-positive red blood cells and causes a positive
direct antiglobulin test (DAT).
287
HDFN due to RH…
• Moderately affected infants develop signs of

– jaundice, and
– corresponding elevations in bilirubin levels, during the
first few days of life.
• Severely affected D- positive infants,

– experience anemia in utero and develop jaundice
within hours of delivery.
288
6.3.3.HDFN due to ABO
• Occurs most frequently in group A or B babies

born to group O mothers.
• Is due to the increased incidence of IgG ABO

Abs in group O individuals compared to other
ABO groups.
• ABO incompatibility often affects the first

pregnancy because of the presence of non-RBC
stimulated ABO Abs
289
HDFN due to ABO…
• Red blood cell destruction by ABO Abs is more

common than by anti-D.
• Fortunately, most cases are sub clinical and do

not necessitate treatment.
290
HDFN due to ABO…
• Some infants may experience mildly elevated

bilirubin levels and some degree of jaundice with
in the first few days of life.
• These cases can usually be treated with

phototherapy.
291
HDFN due to ABO…
• Mild red blood cell destruction, despite high

levels of maternal antibody occurs because:
– A or B substances are present in the fetal tissues

and secretions and bind or neutralize ABO
antibodies, which reduces the amount of ABO
antibody available to destroy fetal red blood cells.
292
HDFN due to ABO…
– Poor development of ABO Antigens on fetal or infant

red blood cells.
– Presence of reduced number of A and B antigen sites
on fetal or infant red blood cells.
• This also explains why the DAT is only weakly

positive in most cases of ABO HDN.
293
Alloantibodies causing HDN other than anti-D
• Other Rh-system antibodies are known to cause

HDN alone or in combination with anti D.
• Anti-c is the second most common cause of

HDN, followed by anti-K.
294
6.3.4 Assessment of HDFN
Post partum testing

• It is advantageous to collect a sample of cord
blood from every new born.
• The specimen should be properly labeled and

stored for up to 7 days
– for testing if the mother is D Negative or if the new
born develops signs or symptoms of HDFN.
• Cord blood should be washed free of wharton’s

jelly.
295
Assessment…
Post partum testing of infants Born to D-

Negative Mothers
• Infants should be tested for the D antigen,

including a test for weak D.
296
Assessment…
Postportum Testing of infants with suspected

HDFN
• Is based on medical history, physical

examination of the new born, and results of
Laboratory testing on both mother and child
297
Assessment…
ABO Testing
• In cases of HDFN the DAT may be positive,
which can lead to false positive or false negative
Rh- testing results.
• False Rh- negative results may be due to a

blocking phenomenon requiring gentle elution
to resolve.
298
Assessment…
DAT
• Its result may be weak especially in cases of
ABO HDFN.
• When positive, performing an elution is optional

if Ab identification test were performed on the
mother at the time of admission.
• If a maternal sample is unavailable, testing the

elute may be useful to confirm HDFN.
299
Assessment…
• If the maternal Antibody screen is negative and

the DAT is positive, ABO HDN should be
suspected.
• ABO HDN can be confirmed by performing

either a heat or freeze thaw elutes.
• The elute should be tested against A1, B and O

cells using an antiglobulin technique.
300
Assessment…
• Positive results with A1 and/or B cells and

negative results with the O cells is indicative and
/or ABO HDN.
• If the elute is negative with all cells, an antibody

to a low frequency antigen should be suspected
and maternal serum tested against the paternal
cells.
301
6.4. Rho (D) Immune Globulin (Human)-
RhIG
• Administered
– to non immunized Rho- negative mothers
– who deliver Rho- positive babies
• Use
– prevent Rh- alloimmunization.
302
RhIG…
• Candidates for this prophylaxis are:

– mothers who are Rh-negative and
– Du - negative, and
– have an Rh-positive or Du positive new born.
• All Rh-negative women who have abortions are

candidates unless the father or fetus is known to
be Rh-negative.
303
RhIG…
The following are not RhIG candidates:

• Rho-negative women
– Who deliver Rh-negative babies.
– Whose serum contains anti-Rho (D).
– Who are Rho- positive or Du- positive
304
RhIG….
• Is a concentrate of IgG anti-D prepared

from pools of human plasma.
• Is given intramuscularly
– to non-sensitized D-negative women at 28
weeks of gestation (ante partum) and
– again within 72 hours of delivery (post
partum) of a D positive infant.
305
RhIG…
Mechanism of action
• Suppresses the immune response following
exposure to D positive fetal red blood cells and
prevents the mother from producing anti-D.
306
RhIG …
Rosette Test-
Purpose
• To detect the presence of Rh positive
RBCs in the circulation of Rh negative
person
307
Rosette Test - Procedure
• Use EDTA mother’s sample drawn after delivery

• Wash cells, add chemically modified anti-D and
enhancement
• Incubate 37oC
• Wash times four with normal saline
• Add R2R2 cells
• Centrifuge, mix, read microscopically
308
Acid Elution Stain- Purpose
• To detect the presence of Hgb F
309
Acid Elution Stain Procedure
• EDTA sample
• Make a slide
• Fix smear
• Treat with acid
• Stain with Eosin
• Count number of stained HbF cells within 2000
HbA cells
310
RhIG…
Acid Elution Stain Calculations
• # fetal cells/2000 adult cells x 100 = %of

fetal cells
• % of fetal cells X 50 = number of mLs of
fetal bleed
311
RhIG…
Determine # vials RhIG

– FB/30 = # vials needed
– 300 ug RhIG will neutralize 30 mL of D+
whole blood
– If # calculated is <0.5 round down, > or =0.5
round up
• 1.4 round down to 1 and add 1 vial=give 2 vials
• 1.6 round up to 2 and add 1 vial – give 3 vial
312
RhIG Dose
• Recommended dose (contained in one vial) is

about 300 µg
– offers protection against a feto-maternal hemorrhage
(FMH) of 30 ml (15 ml packed cells) or less.
• If a massive FMH has occurred, the volume of

the hemorrhage must be determined to calculate
the number of vials to administer.
313
6.5 Treatment of Infants Suffering from
HDFN
• For infants who develop hyperbilirubinemia

and/or anemia due to HDFN, exchange
transfusion is usually carried out.
314
Treatment…
Exchange transfusion:
– is a continuous removal of small amounts of blood
from the neonate with simultaneous continuous
infusion of donor blood until a one or two-volume
exchange is accomplished.
– Helps to reduce the concentration of bilirubin and

incomplete antibodies
– Provides the infant is with compatible donor red cells.
315
Treatment…
• To give exchange transfusion to an infant clinical

and laboratory findings must be considered.
– Cord Haemoglobin(<10g/dl) and
– raised serum bilirubin are strong indicators for

treatment.
316
Treatment…
• For compatible exchange transfusion, donor’s

blood should be:
– cross-matched with the maternal serum and

– lack the RBC Ag corresponding to the maternal Abs
– ABO group and Rh type compatible with the infant’s
blood group.
• If the mother’s antibody is not available, group

O Rh negative red blood cells must be selected.
317
Phototherapy
• The use of light to degrade bilirubin in mildly

jaundiced infants
• Usually ABO incompatibility
318
Review Questions
1. Compare the causes and laboratory

demonstrating methods, of AIHA with HDN.
2. What is the cause of HDN? What
consequences could result from this
condition?
3. What is exchange transfusion?
319
Review…
4. Discuss the dose of IgG anti-D given to Rh-

pregnant women to prevent HGN?
5. When Rh HDN does occur?
6. List and discuss the postnatal laboratory
investigations to be carried out to know the
presence and extent of HDN.
320
CHAPTER SEVEN
THE CROSS-MATCH
(COMPATIBILITY TESTING)
321
Content
7.1. Cross matching
7.2. Types of Cross Match
7.3. Steps for compatibility testing

7.4. Choice of Blood for cross-match
7.5. Procedure for cross-match

7.5.1.Standard cross matching
7.5.2. Emergency cross matching
7.5.3. Rapid slide crossmatching
322
Learning Objectives

able to:
• Understand the cross-match and its primary
purpose.
• Explain the constituents of the major and minor
cross match.
• Select appropriate blood for cross-match.
• Describe the types of antibodies that can be
encountered at various phases of a cross-match.
323
7.1 Cross-Matching
• 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
324
Cross-Matching…
• Will not:
– prevent immunization of the patient
– guarantee normal survival of transfused
erythrocytes
– detect all unexpected antibodies in a patient’s
serum.
325
7.2 Types of Cross Match
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
326
Types….
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
327
7.3. Steps for compatibility testing
• 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).
328
Steps for…
• 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
329
Steps for….
• Finally, during the actual transfusion :
– careful observation of the recipient’s vital

signs and
– post transfusion hematocrit and Heamoglobin

levels must be considered.
330
7.4.Choice of Blood for cross-match
• 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.
331
Choice of…
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
332
Choice of…
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.
333
Choice of…
• 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.
334
Choice of…
• 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..
335
Choice of…
• 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
336
1. Saline tube
• 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.
337
2. Albumin tube
• 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
338
Albumin tube…
• 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.
339
3. Anti-human globulin tube
• 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

340
7.5. Procedure for cross-match
7.5.1.Standard cross-match
• Is cross-match that is performed in three tubes

(Saline, albumin and AHG) within 45 to 60
minutes
• detects unexpected (irregular) antibodies in the
recipient/ donor serum
341
Cross match (Standard)…
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
342
Cross match….
Type of specimen
– Serum (plasma) not older than 48 hrs

– Washed cells (20-30% and 2-5%)
343
• Test tubes
• Centrifuge
• Microscope
• Microscopic slide
• Normal saline
• 20% albumin
• AHG (Coombs reagents)
344
Procedure
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
Tube 3 3 volume of patent’s serum
1volume of 20-30% suspension of
donor’s cells
345
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.
346
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.
347
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.
348
Incompatibility investigation
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
349
Cross match (Incomp. Invest.)…
Anti- Duffy and anti- kidd incompatibilities

• Saline tube …… Does not usually show agglutination
• Albumin tube … does not usually show agglutination
Anti- Lewis incompatibility

• Saline tube …………………….. Shows agglutination
• Albumin tube ………………….. Shows agglutination
350
7.5.2 Emergency cross match
• 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.
351
Cross match (Emergency)…
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
352
Type of specimen
– Serum (plasma) not older than 48 hrs

– Washed cells (2-5%)
353
• Test tubes
• Centrifuge
• Microscope
• Microscopic slide
• Normal saline
• 20% albumin
354
Procedure:
1. Take 2 small tubes, mark them 1 and 2 and

add to each the following

1volume of 3-5% donor’s red cells.
1volume of 20% bovine albumin
355
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.
356
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.
357
7.5.3. Rapid direct slide cross match
(Request for un cross matched blood )
• 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.
358
Procedure
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
359
Cross match (Rapid)…
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.
360
Cross match (Rapid)…
Sources of errors in cross-matching

• Rouleaux
• Auto agglutinins
• Infected donor cells
• Anti- A1
• Over centrifugation
• Dirty glass wares etc..
361
Review Questions
1. What is cross-matching?
2. What is the purpose of cross-matching?
3. List the types of cross-match with their

constituents
4. List the stages of cross-match and their

respective importance in antibody detection.
362
CHAPTER EIGHT
THE DONATION OF BLOOD
363
Content
8.1 Selection of blood donors
8.1.1 Selection Criteria
8.2 Collection of Blood

8.2.1 Blood collecting bottles and bags--------added
8.2.2. Preservative solution
8.2.3. Danger in taking blood ----------------------------added
8.2.4. Donor reactions --------added

8.3.The cold chain ---------added
8.4Transporting blood
8.4.1Transport within the blood bank or hospital

8.5.Storage of blood
8.5.1. Importance of storage
8.5.2.Temperature and storage time for blood products
8.6. Preservative solution
364
Learning objectives

able to:
• Discuss the medical and physical requirements
that would exclude an allogenic donor
• Describe the proper procedure for collecting
blood from donors.
• Name the commonly used anticoagulants for
donated blood and their respective approved
maximum storage time.
• Explain the possible donor reactions.
365
Learning…
• Describe the cold chain and its use.

• Explain the different storage and
transportation temperatures of blood and
blood products.
• Discuss the different preservative
solutions and the purpose of their
components.
366
8.1 Selection of blood donors
Aim
• to prepare safe blood from a safe donor to give
a recipient
– by identifying conditions which could harm both the

donor and the recipient
367
8.1.1 Selection Criteria
Age
• Between 17 – 65 years
Hemoglobin
• Females -not less than 12.5 g/dl (PCV 38 %)
• Males -not less than 13.5 g/dl (PCV 41%)
• In both sexes Hgb above 19g% (Hct above 57%) are not
acceptable.
368
Selection Criteria…
Pulse, Blood pressure and Temperature
• Pulse - between 60 – 100 per minute.

• B.P
– Systolic between 90 and 180 mmHg.
– Diastolic between 50 and 100 mmHg.
• Temperature should not exceed 370C.
369
Weight
• A person
– between 45-50 kgs can donate 350 ml of blood.
– above 50 kg can donate 450ml of blood.
• Obese donors who are unable to climb onto the

couch are not acceptable.
• Donors with unexplained weight loss of a
significant degree are not acceptable to donate.
370
Pregnancy
• Are excluded from donating for 1 year after the
conclusion of their pregnancy.
Medication
• Deferral of donors depends on the nature of the
disease for which the drug was ordered.
• Consult a medical doctor about a donor’s long
term treatment.
371
Illness
Prospective donors with:

– disease of the heart, liver, lungs, or
– history of cancer, or
– bleeding problems should be excluded, subject upon
evaluation by a physician.
372
Selection (illness)…
• Donors who have had leukemia must never be

accepted.
• Donors with a previous history of tuberculosis

are acceptable after completion of therapy and if
the disease is no longer active.
373
Selection…
Infectious diseases
• A donor must be free from transfusion
transmissible infections
• Recipients of blood or blood products known to

be possible sources of hepatitis and donors
having had close contact with an individual with
viral hepatitis must be deferred for 1 year.
374
Selection (infectious)…
• who have a history of malaria, or were Persons

at high risk for acquiring or transmitting AIDS
should not donate blood.
• Donors previously resident in an endemic area,

should be deferred for 3 years after becoming
symptomatic or after leaving the endemic area.
375
Selection…
Previous donation
• An interval of at least
– four months for men and
– six months for women, is required before the next
donation.
– The recipient can also donate a blood after 12 months
of recovery
376
Selection…
Surgery
• If the surgery is minor (such as tooth extraction)

a donor is excluded until healing is complete and
full activity has been resumed.
377
Selection…
Vaccinations
• Persons recently immunized with
– toxoids and
– killed viral, bacterial and rickettsial vaccines are
acceptable, if they are symptom free and not febrile.
• After small pox vaccination, a donor is

acceptable when the scab has fallen off, or 2
weeks after an immune reaction.
378
Selection…
• A donor who has received an attenuated live

virus vaccine such as mumps or yellow fever is
deferred for 2 weeks after the last immunization..
• If rabies vaccination has been given following a

bite by a rabid animal, the donor must be
deferred for 1 year after the bite.
379
8.2.Collection of Blood
Before blood collection Basic information about the

donor like
– date of donation
– full name,
– Address
– Sex
– age and
– the ABO and Rh blood group
– donor’s medical history, must be obtained and signed
by the phlebotomist who performs the procedure.
380
Collection…
• Patient identification also is an important step in

blood collection.
• Blood collection should be carried out with the

blood donor lying on a simple bed with a support
for his head.
• The donor must not be left alone while donating

blood.
381
8.2.1 Blood collecting bottles and bags
• A standard unit of blood contains 450 cc blood

and 63cc of preservative solution.
• If lesser amounts of blood is to be collected the

amount of the preservative should be reduced
proportionally.
– 350 ml blood bag with 49 ml of anti coagulant.
382
8.2.2. Preservative solution
• The common anticoagulant preservative

solutions are:-
– CPD (citrate, phosphate & dextrose)- For 21 days/at

2-80C
– CPDA- 1 (CPD with adenine)----For 35 days at 2-80C
– ACD (Acid Citrate Dextrose)------For 28 days /at
2-80C
383
Method for blood collection
1. Apply a pressure cuff to the upper arm a few inches

above the elbow, and raise the pressure to between 80
and 100mmHg.
2. And select a large deeply situate vein for the vein

puncture, usually near the bend of the elbow.
3. Clean very well the required part of the arm with cotton
wool and 70% methylated spirits.
384
Collection (Methods)…
4. Make the vein puncture with the needle directed up

wards in the line of the vein. If necessary secure the
needle in place with a small strip of adhesive tape.
5. When the blood begins to flow, reduce the pressure of

the cuff to 40-60 mmHg, and ask the donor to squeeze
a small object.
6. When the blood has almost reached the 450 ml mark,
reduce the pressure to zero and remove the object
from the donor’s hand.
7. Clamp off the tubing and remove the cuff.
385
8. Take the needle out of the vein, applying

pressure with cotton wool.
9. Ask the donor to bend his arm up wards, with

the cotton wool over the puncture wound.
10. Empty the blood in the tubing of the taking set
in to – a clean dry test tube
- for grouping and screening the blood.
386
11. Mix the blood well with the anti coagulant by inverting
the bottle at least four times, but not shake it as this
could damage the cells.
12. Seal the tops of the collecting and pilot bottle with
adhesive tape on which is written the group and the
bottle number.
13. Label the sample with- the blood group, the bottle
number and the date of collection.
387
13. Place a small pad of cotton wool and an adhesive

plaster on the donor’s arm as a dressing, making sure
that the bleeding has stopped
14. See that the donor is given a rest and a drink, perhaps
of tea or orange juice, but not alcohol to make up his
fluid lose.
15. Thank the donor and give him a certificate of blood

donation
388
8.2.3.Danger in taking blood
• The greatest danger in taking blood is an air

embolism
– can be caused by the air out let needle becoming
blocked, either before or during the taking of blood.
• If the air out let is blocked, the following can
happen.
– The flow of blood can slow down and even stop
– The blood may move in the opposite direction,
returning to the arm
389
Danger…
• Slowing down of the flow of blood can also be

occurred due to:
– A fall of the pressure cuff
– A bend in the tubing of the taking set
– The needle in the arm requiring adjusting
390
8.2.4.Donor reactions
Include:
• syncope (fainting),
• weakness,
• excessive perspiration,
• dizziness,
• pallor, and nausea.
• Occasionally convulsions, loss of consciousness
or involuntary bowl or urinary passage.
391
Donor reactions…
Note:
• At the first sign of the reaction, the phlebotomist

should stop the phlebotomy
• Preliminary measures taken when donor

reactions occur……
392
Donor reactions…
Syncope
- Place the donor on his or her back and raise
the feet above the level of the bend.
- Loosen tight clothing, and ensure that the
donor has an adequate air way.
- Apply cold compress to the donor’s forehead
or back of the neck.
- Refer if the condition is not improved
393
Donor reactions…
• Nausea
– instruct the donor to breath slowly and deeply,
– Have emergency basin and damp towel
available in case the donor vomits.
• Convulsions
– the convulsions are rare, when they occur
help should be summoned immediately.
394
Donor reactions…
• Weakness, excessive perspiration, Dizziness,

pallor
– Apply cold compress to the donor’s forehead
395
Donor reactions…
• Hematoma
– remove the tourniquet and the needle from

the donor’s arm ,
– place gauze and apply pressure for 7-10
minutes with the donor’s arm held above the
level of the heart.
396
Donor reactions…
• Air-embolism
– this is very dangerous and always take care.
– If it happens, immediately call for help.
Because, it cause rapid fall of systolic blood
pressure, death can occur.
397
8.3.The cold chain
• Is a system for storing and transporting blood

and blood products in a safe a way to maintain
all their function
• The two essential parts of the cold chain are:
– People to organize and manage the storage and
transportation of blood and blood products.
– Equipment to store and transport the blood and the

products safely.
398
8.4 Transporting blood
• During transportation the temperature of:
– Red cell products must be kept between 2-80C
– Platelet products must be kept between 22-240C
– Frozen plasma in a frozen state at < -180C
399
Transport…
• When blood is to be transported using cold

boxes for long distances in hot weather
– there should be as much ice as there is do blood and
– if above 100C it should be discarded.
400
Transport…
• Containers for transporting red cell products

should be pre- cooled to 40C and,
• For transporting platelet should be kept open at

room temperature for 30 minute before use.
401
Transport…
• When blood is collected outside the blood bank-

at a mobile session, cold boxes should again be
used.
– If possible a maximum and minimum thermometer

should be put inside the box without touching the ice
box.
402
Transport…
• On arrival at the blood bank the blood must be

put into the refrigerator as soon as possible.
• Periodic temperature checks of received

components under all encountered weather
conditions should be performed & documented
to ensure shipping methods are adequate to
meet these criteria.
403
Transport…
• Returned blood and blood products should not
be re- issued for transfusion if:
– the bag has been penetrated or entered
– there is leakage
– visual change or
– it has not been maintained continuously within the

approved temperature range.
404
8.4.1 Transport within the blood bank or
hospital
• The blood should be issued in a cold box (80C) if
– the temperature inside the blood bank is greater than
250C or
– the blood will not be transfused immediately
– stored away from the main blood bank, such as in

operating theater or hospital wards
• The blood must be allowed to “warm” before

transfusion and this will take time.
405
8.5 Storage of blood
Changes occurring in stored blood
Viability
• It is the capacity of the transfused cells to
survive in the recipient’s circulation after 24
hours
• Progressive loss of viability is the most
important change occurring in the RBC during
storage.
• the lower viability limit for successful transfusion.
– 70% survival of transfused red cells after 24 hrs.
406
Storage….
• Viability depends on preservative solution.

– Viability in CPD (which contain less citric acid than
ACD) appears to be lost as little more slowly.
– After 21 days of storage in
• CPD 79% of the cells were viable
• ACD 74%,
– After 28 days
• CPD 77%
• ACD 71%
407
Storage…..
• The progressive loss of viability during storage is

related to:
– Depletion of ATP
– Depletion of 2-3 DPG
– Decrease in PH of the solution
408
Storage….
• Changes taking place during liquid storage of

blood at 40C:-
– Bilirubin and potassium levels increase in weeks
– ATP, 2 3 – DPG, PH decrease in weeks
– Factors V,VII,VIII, VWF decreased in days
– Function of white cells and platelet Decrease in hours.
• Factors I,II,VI,IX,X,XI,XII levels are not affected
409
8.5.1 Importance of storage
• The purpose of storing blood is to:-
– Maintain viability and function of each relevant

constituent.
– To prevent physiological changes harmful to the
constituent.
– To minimize bacterial multiplication.
410
8.5.2 Temperature and storage time for
blood products
• Whole blood, 2-80C for 21-42 days
• Packed cells up to 42 days at 2-80C
• Red cells frozen in glycerol -650c up to 10 years
• Fresh frozen plasma -180 C or lower for 1 year
411
Temp. and storage time….
• Platelets 20-240C (Room temp) with rotation for

5 days.
• Before blood is issued, every pint must be

examined visually for hemolysis and any
abnormal discoloration
412
8.6 Preservative solution
• The survival of blood cells depends during

storage on the delicate biochemical balance of
nutrients specially glucose, PH, and ATP.
• The preservatives contain citrate, phosphate,

Dextrose and Adenine
• The common anti coagulants are ACD, CPD and

CPD-1
413
Preservative solution…
• The purpose of the solution during storage

are:
– Citrate binds calcium and prevents clotting of
the blood
– Citric acid is added to the anticoagulant in
order to obtain a hydrogen ion concentration
414
Preservative…..
– Dextrose is used by the red cells during storage and

each dextrose (glucose) gives 2 molecules of ATP.
– Adenine helps the red cells to synthesis new ATP.
Note: Low temperature slows glycolysis, and

reduce bacteria proliferation.
415
Preservative…..
Other anticoagulants
• SAGM- contain saline, Adenine, Glucose, Mannitol (it is
RBC’ additive solution)
• CPDA-1- contains CPD+125% Glucose +0.25 mmol

adenine
• CPDA-2 – contains CPD+ 175% Glucose + 0.25 mmol

more adenine
416
Review questions
1. List some clinical conditions that exclude a

donor
2. What are the steps in performing a
venipuncture?
3. Where is the most common venipuncture site?
4. List some potential hazards that occur during
or after blood donation.
417
Review…
5. What it the transportation temperatures of red

cell products, platelet products and fresh
frozen plasma?
6. What are the different components of blood
preservative solutions and their use?
7. How does the viability of red cells can be
affected during storage
418
CHAPTER NINE
WHOLE BLOOD,BLOOD
COMPONENTS AND DERIVATIVES
419
Content
9.1 . Whole blood, blood components ad

derivatives
9.2. Whole Blood
9.3. Red cell preparation

9.4. Plasma product
9.5 Platelet concentrates (PC)
9.5.1.ABO and RH compatibility in platelet
transfusion
420
Learning objectives
At the end of this chapter, the student will be able

to:
• Identify the different blood components and their
preparation
• Mention the clinical indications for blood
component transfusion.
421
9.1 WHOLE BLOOD,BLOOD COMPONENTS
AND DERIVATIVES
• 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.
422
Whole blood….
• 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
423
Whole blood….
• 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
424
9.2 Whole blood (w/b)
• 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.
425
Indications for whole blood
• 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
426
Blood components
427
Blood components..
• 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
428
9.3.Red cell preparation
• 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
429
Red cell preparation….
• 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
430
Red cell preparation…
• 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.
431
9.4. Plasma products
• 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
432
Plasma products….
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.
433
Plasma products….
• 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)
434
Plasma products….
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.
435
Single donor plasma (SDP)/Ordinary plasma
(OP)
• 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.
436
plasma….
• 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.
437
plasma…..
• Indications:
– In the treatment of stable coagulation factors
deficiencies
– For volume and protein replacement under
special circumstances
438
plasma…..
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.
439
plasma…..
• CDP is indicated for patients requiring volume

expansion or protein replacement when labile
clotting factors are not required
440
9.5.Platelet concentrates (PC)
• 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)
441
Platelet …….
• 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
442
Platelet …….
• 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.
443
Platelet …….
Indications
Patients who are actively bleeding from the

thrombocytopenia
– due to drugs, toxins, infections and bone marrow

failure
• Bleeding caused from qualitatively abnormal

platelet.
444
9.5.1.ABO and Rh compatibility in platelet
transfusions
• 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
445
9.6.Cryoprecipitate (CRyo)
• 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
446
(CRyo)…
• 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.
447
(CRyo)……..
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.
448
Review questions
1. Name the common blood components that can

be prepared from a unit of donated blood.
2. Describe the clinical indications for transfusion
of the different blood components.
3. What is OP? Describe its use.
4. What are the optimum storage temperatures of
plasma products?
449
CHAPTER TEN
TRANSFUISON REACTIONS
450
Content
10.1. Types of transfusion reaction

10.1.1.Laboratory tests to be done when
transfusion reaction occurs
451
Learning Objectives
At the conclusion of this chapter, the student

should be able to:
• Define the transfusion reaction
• Classify the transfusion reactions
• Carryout laboratory tests during transfusion
reactions
452
10.1 Transfusion reactions
• Also called adverse reactions to transfusions

• Are any unfavorable responses by a patient
following transfusion of blood, blood components
or derivatives.
Example:
– Facial flushing
– Chest/back pain
– Chills
– Fever
– Cyanosis, Dyspnea
453
10.1.1. Types of Transfusion Reaction
Divided into:
A .Hemolytic reactions-
-abnormal destruction of RBCs of either the donor or recipient
B. Non hemolytic reactions-

-not usually associated with RBC hemolysis
a. Febrile reactions (pyogenic reactions)
b. Allergic reactions
c. Bacteriogenic reactions
d .Circulatory overload
454
Types of Transfusion…..
Characteristics non hemolytic reactions:
• shortened post transfusion survival of RBCs

• febrile reactions,
• allergic response and
• disease transmission.
455
Cause hemolytic transfusion reactions (HTR)

– incompatible blood
– blood under great pressure
– transfusion of hemolyzed, overheated or frozen
blood,
• HTR can be
1. immediate or
2. delayed.
456
1. Immediate Type
• hemolysis evidence is obtained during or
immediately after blood is infused due to
destruction of donor cells by the recipient’s Abs.
• Two types:
a. intravascular
b. extravascular
457
a. Intravascular-
It is due to:
– ABO incompatibilities due to clerical errors (Usually)
– Antibodies to other blood group antigen systems
may also be involved in immediate HTR (Rarely)
458
b. Extravascular-
It is due to:
-Coating of red cell antigen by incompatible Abs which
results in the removal of the red cells in liver &
spleen.
-Any IgG, non agglutinating, non-complement binding

form of antibody
459
Causes of immediate hemolytic transfusion

reactions
1.Failure to recognize antibodies present recipient
serum/plasma prior to transfusion
2.Failure to recognize donor antibodies before
transfusion
3.Administration of blood considered compatible
on the basis of an incompatible cross match
4.Erroneous identification of recipient, donor or
both etc...
460
2. Delayed HTR
• Are not so dramatic and are usually noticed a
week or two after a transfusion.
• Abs to Ags other than ABO,( Rh, Kell, Duffy,

Kidd) Ag systems are commonly responsible.
• occur because of an anamnestic response

(previously encountered Ags)
-transfusions or
-pregnancies.
461
• The Abs screen on the patient's serum and the

major cross-match may well have been normal
during the initial testing.
• They are subtle in their manifestations and
present
– only as a mild jaundice or
– as a failure of the Hb to rise as expected.
• Are not preventable ,b/c
Abs -are not present or
-below the detectable level
462
2.Non-Hemolytic transfusion reactions
a. Febrile reactions (pyogenic reactions)

– The patient develops fever and chills during or after
transfusion
– The rise in body temperature may be due to leukocyte

Abs ,platelet Abs or Pyrogens
– Prevented by giving leukocyte and platelet poor RBCs
463
b. Allergic reactions
– Most common type is urticaria (an itchy rash)
– Characterized by flushing of the skin &
dyspenia
– Commonly caused by the interaction b/n
transfused IgA and class specific anti-IgA in
the recipients plasma
– controlled by giving anti-histamine
464
c. Bacteriogenic reactions
• Caused by bacteria that may contaminate
solutions or equipments before sterilization
– Transfusion of bacterially contaminated blood

components
• Common problem for platelet concentrates

stored at room temperature
465
Signs of bacteriogenic reactions

– Rapid onset of chills & fever
– Vomiting
– Diarrhea
– Profound hypotension
– Shock
466
To minimize the risk of infection of blood

– Blood should be maintained at refrigerator
temperature at all times during storage
– the container should not be opened or punctured to
obtain a sample
– the recommended storage time must not be
exceeded
– Blood should be examined routinely for
• unusual color or discoloration
• the presence of hemolysis, both of which may suggest
bacterial contamination.
467
d. Circulatory overload
• Results from the transfusion of a patient with a
normal blood volume but a reduced RBCs
volume
• The total circulatory volume becomes too great
for the pumping action of the heart
– Cause congestive heart failure manifested by:
• Coughing
• Cyanosis, and
• Difficulty in breathing, dyspenia
468
10.1.1 Laboratory tests to be done when
transfusion reaction occurs

1. Check the identification of the patient and transfused
unit
2. Obtain a post transfusion specimen from the patient and
visually examine it for hemolysis
3. Perform direct Antiglobulin test from post transfusion
sample, taken as soon as possible after the reaction
has taken place.
4. Re-type the RBCs of both donor and recipient for ABO
and Rh grouping.
5. Re-cross-match blood from each unit transfused using
serum from both pre-and post-transfusion specimens
from the patient.
469
Review questions
1. What is transfusion reaction?

2. On what basis are the transfusion
reactions classified?
3. List laboratory investigations to be
carried out when incompatible transfused
reactions are suspected?
470
CHAPTER- ELEVEN
TRANSFUSION TRANSMITTED
DISEASES AND SCREENING
ASSAYS
471
Content
11.1. Transfusion transmission diseases

11.1.1. Viral
11.2.2. Parasitic
11.1.3.Bacterial
11.2. Screening assays
472
11.1 TRANSFUSION TRANSMITTED DISEASES
AND SCREENING ASSAYS
• There are three main types of infectious agents

known to be transmitted by transfusion.
They are:
– Viruses
– Bacteria
– Protozoa
473
11.1.1.Viral Infections
– Hepatitis A,B,C
– HTLV
– HIV
– West nile virus
– Herpes and parvovirus
474
11.1.2. Parasitic infections
– Malaria
– Chagas
– Toxoplasmosis
– Babesiosis
– Leishmaniasis
475
11.1.3. Bacterial infections
– Syphilis
– Lyme disease (Borreliosis)
476
11.2. SCREENING ASSAYS
Types of screening assays

• The choice of markers to screen for depends on
the infectious agents that are screened.
• Screening for specific antibody is usually most
appropriate for HIV,HCV and Syphilis.
• Screening for specific antigens (HBsAg) is
necessary for HBV.
477
assays …..
• The three main kinds of primary
screening assay are available to detect
infectious disease markers in donated
blood:
1. ELISA/EIA
2. Particle agglutination assay
3. Simple rapid assay
478
assays …..
• Criteria for selecting the most appropriate
assays for screening of blood:
– Principle of the assay
– Complexity of the assay
– Incubation time
– Sensitivity
– Specificity
– Suitability for different situation
– Availability and
– Cost
479
Review questions
1. Define TTD
2. List viral, bacterial and parasitic TTD
480
CHAPTER TWELVE
QUALITY ASSURANCE
IN
IMMUNOHEMATOLOGY
481
Content
12.1.Quality assurance
12.2. Achieving total quality
12.3.Frequency and specificity of control
material
12.4.Quality requirements for safe
transfusion practice
482
Content..
12.5.Checklist/critical control points

12.5.1. Sample reciept/accepatnce
12.5.2. patient detail
12.5.3. laboratory test
12.5.4.unit for transfusion
12.5.5.Blood collection
12.5.6.Blood administration
12.5.7.Blood return
483
Learning objectives
• Define Quality assurance

• List the steps important in achieving total
quality
• Discuss quality requirements for safe
• List the critical control points in transfusion
• Explain the importance of QA in
Immunohematology
484
12.1.Quality assurance
Quality - ‘degree of excellence’

Assurance - ‘positive assertion’.
Quality assurance (QA) :

-Of a process ; confidence in the outcome can be
guaranteed at a pre-defined level of quality.
-In transfusion; the ideal to be achieved is zero
defects throughout the transfusion chain.
– is of prime importance
485
Quality assurance (QA) ….
• Total quality management (TQM) stresses the

importance of collecting data as a -
-basis for -decision-making and
-problem solving.
• In this way, the whole transfusion process can

be -analyzed and
-improved in the long term.
486
487
12.2. Achieving total quality
1.Set appropriate standards.

2.Ensure good communication.
3.Full documentation and document control.
– Traceability of all laboratory procedures, including
-Quality Control (QC) results,
-batch numbers,
-expiry dates of reagents, and
-staff identification.
488
total quality…..
4.Training: external as appropriate;

– in-house, relevant, and continuous.
5.Standard operating procedures (SOPs), which
must be complied with.
6.QC for individual procedures, which must reflect
the technology used and be appropriate.
7.External assessment by participating in QA
schemes to monitor standards, spot trends, and
help to reduce errors
489
12.3. Frequency and specificity of control
material
• Controls must be carried out for all test
procedures.
– generally at least daily and when new batches of

reagents are introduced.
– Single tests, for example in an emergency situation,

require that controls are included at the same time.
490
Frequency and specificity….
• In order to ensure best practice, QC, QA, and

continuous quality improvement in the laboratory
should include the review and approval of:
– procedures and policies;
– training/education;
– validation of protocols and results;
– calibration of equipment;
– document control/record keeping;
491
Frequency and specificity..
– product specifications;
– suppliers;
– corrective action -planning
-monitoring;
– error reports;
– health and safety issues (including waste
management);
492
Frequency and specificity….
– audits;
– SOPs;
– analysis of trends;
– problem solving;
– external QA results;
– accreditation issues
493
12.4.Quality requirements for safe
• The responsibilities of all personnel involved in the
transfusion process should be clear and each individual
notified.
• Pre-labelled tubes for patients’ samples should not be

used.
• Samples for pre-transfusion compatibility procedures

should be
– stored at 4–6°C and
– taken not more than 24 hours before transfusion if a previous
transfusion has been administered within the previous 3–14
days.
494
Quality requirements….
• A sample not older than 72 hours may be used if the last

transfusion was between 14 and 28 days previously.
• Positive patient identification at sampling and at the time

of transfusion is essential for both inpatients and
outpatients.
• The request system should ensure prescription issue of

blood/components and needs to include procedures to
ensure the correct administration of the component.
495
• Written policies for the collection of components

are essential.
• Emergency issue, special requirements, or

telephone requests should also be covered in
the regulations.
• Laboratories should have access to previous

transfusion records including historical grouping
and screening results.
496
• Transfusion committees can help to monitor the

efficiency of the procedures and recommend
changes where necessary.
• Multidisciplinary audits of the transfusion

process on a regular basis.
• Continuous training of all staff involved in the

transfusion chain to ensure compliance with
procedures.
497
12.5.Checklist of critical control
points
12.5.1.Sample receipt/ Acceptance
• Patient details match on transfusion request

form and sample
• Any doubts (e.g. illegible handwriting), new
sample to be requested
498
Checklist….
12.5.2.Patient details entered

• Check the information is correct (Spelling,
hospital no.)
• Take care about patients with common or similar
names
– minimum 2-point match
Example.- Full name and
-unique number or according to local /national
guidelines
499
Checklist….
12.5.3.Laboratory tests
• Perform in appropriate SOPs, Enter details
• If previously tested check that results match
exactly.
• Check history of- previous
-transfusions
-preganancies,
-previous antibodies
500
Checklist….
12.5.4.Units for transfusion

• Check ABO group/blood group label
• Check component is correct
Example CMV negative…
• Compatibility label after negative cross match
• Check and enter details.
• Transfer to storage( Laboratory/OR refrigerator
etc.)
501
Checklist….
12.5.5.Blood collection
• Follow established SOP.
– Recipient identity signature of individual taking units
etc.
• Retain samples post-transfusion, sample from

unit after transfusion
502
Checklist….
12.5.6.Blood administration
• Beside check as per SOP
• Monitor patient
503
Checklist….
12.5.7.Blood return
• Establish the units’s suitability to be returned to
stock
• If accepted, remove compatibility label and
register return details
504
Review questions
1. What is quality assurance?

2. List the steps important in achieving total
quality.
3. Discuss quality requirements for safe
transfusion practice.
4. List down the critical control points in
transfusion
5. Explain the importance of QA in
Immunohematology.
505
Glossary
-Added on the notes
506

Chapter One TO Immunohematology

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Chapter One TO Immunohematology

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CHAPTER ONE

1.2 Historical background

– is more commonly known as "blood banking“

– deals with the concepts and clinical techniques

– collecting, preparing, storing and transfusing blood.

– refers to immunologic reactions involving

– an application of the principles of immunology

• The era of blood transfusion began when

• In 1665, Richard Lower- the first animal-to-

– blood from the carotid artery of a lamb into the vein of

– using animal blood, but they were unsuccessful.

• Later, it was found that it is impossible to

• Transfusions were prohibited from 1667 to 1818

• In 1818, James Blundell of England successfully

• Such species-specific transfusions seemed to

– discovered the ABO blood groups in 1900,

• It became clear that the incompatibility of many

Two main postulates were drawn:

Chromosomes and Genes:

• The length of each chromosome is divided into

• Genes code for different inherited physical

• Each gene has its own locus, along the length

• Certain inherited characteristic can be

• Such genes are called alleles or allomorphic

• Mitosis: While body cells multiply they do so by

• Meiosis: When sex cells are formed either male

• During fertilization when the egg and sperm

– Half of these from the male and

Genotype versus phenotype

• Visually portrays the potential offspring`s

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

• The product of each allele can be identified

– If one parent passed on an A gene the other parent

Recessive or dominant inheritance patterns

• O gene is recessive, since it is expressed only

• The product of an O gene however, does not

– Its expression is termed as amorphic (a gene that

• refers to the transmission of a trait in a

• Independent assortment is demonstrated by the

Homozygous :MM Red blood cell tested with

Heterozygous :MN Red blood cell tested with

• Genes can inherit with each other depending on

– same chromosome (Cis) or

– opposite chromosome (Trans).

• Trans interaction may weaken the expression of

– The C and D genes of Rh system are inherited on

– When C is inherited in trans to D, it will weaken the D

• In some blood group systems, the antigens are

• When genes are very close together, they are

• Independent assortment does not occur when

• Amorphs can result in an unusual phenotype if

• The phenotypes are often called "null" type

– null types caused by amorphic genes are

• Unusual phenotypes may also result from the

• These genes (suppressor /regulator)

– act to inhibit the expression of another gene and,

• Null phenotype therefore can be resulted of

• A unique set of red blood cell Ag is determined

• These antigens protrude from the surface of the

– As a result, they are accessible to Ab molecules for

• Some of the red blood cell antigens are more

– The D antigen within the Rh group system.

• Is possessed by nucleated cells such as