CHAPTER ONE

INTRODUCTION TO
IMMUNOHAEMATOLOGY

Learning Objectives
At the conclusion of the chapter, the student should be able
to:
- Explain a brief history of the science of
Immunohaematology
- Discuss the patterns of inheritance of A and B genes
- Describe the synthesis of H, A and B antigens
- Name the specific transferase for the A, B & H genes
- State the genotype of individuals with the Bombay
phenotype
- State the characteristic genotype of secretor and non-
secretor
- Identify the product or products found in the saliva of
persons of various ABO groups

1.1 Historical Overview of Immunohematology

Immunohematology is one of the specialized branches of

medical science. It deals with the concepts and clinical

1
techniques related to modern transfusion therapy. Efforts to
save human lives by transfusing blood have been recorded for
several centuries. The era of blood transfusion, however,
really began when William Harvey described the circulation of
blood in 1616.

In 1665, an English physiologist, Richard Lower, successfully

performed the first animal-to-animal blood transfusion that
kept ex-sanguinated dogs alive by transfusion of blood from
other dogs.

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. However, after the third transfusion
of lamb’s blood the man suffered a reaction and died. Denys
also performed subsequent transfusions using animal blood,
but most of them were unsuccessful. Later, it was found that it
is impossible to successfully transfuse the blood of one
species of animal into another species.

Due to the many disastrous consequences resulting from

blood transfusion, transfusions were prohibited from 1667 to
1818- when James Blundell of England successfully
transfused human blood to women suffering from hemorrhage
at childbirth. Such species-specific transfusions (within the

2
same species of animal) seemed to work about half the time
but mostly the result was death.

Blood transfusions continued to produce unpredictable

results, until Karl Landsteiner discovered the ABO blood
groups in 1900, which introduced the immunological era of
blood transfusion. It became clear that the incompatibility of
many transfusions was caused by the presence of certain
factors on red cells now known as antigens. Two main
postulates were also drawn by this scientific approach: 1.
Each species of animal or human has certain factor on the red
cell that is unique to that species, and 2, even each species
has some common and some uncommon factor to each other.
This landmark event initiated the era of scientific – based
transfusion therapy and was the foundation of
immunohematology as a science.

1.2 Blood Group Genetics

Blood group genetics are concerned with the way in which the
different blood groups are inherited, that is passed on from
parents to children.

Chromosomes and Genes: In the human body, the nucleus

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

3
chromosome is divided in to many small units called genes,
which are important as they contain the different physical
characteristics, which can be inherited including those of the
blood groups.

Allomorphic genes (Alleles): Each gene has it own place

called its locus along the length of the chromosome. However,
a certain inherited characteristic can be represented by a
group of genes, and the place or locus can be occupied by
only one of these genes. Such genes are called alleles or
allomorphic genes.

For example, every one belongs to one or other of the

following blood groups: group A, group B, group O or group
AB. Therefore, there are three allelomorphic genes which
make up the ABO Blood group system such as gene A, gene
B, and gene O. Only one of these alleles can occupy the
special place or locus along the chromosomes for this blood
group characteristic.

Body cells and mitosis: When body cells multiply they do so

by producing identical new cells with 46 chromosomes. This
process is called mitosis.

Sex cells and meiosis: When sex cells are formed either
male or female the pairs of chromosomes do not multiply but

4
simply separate so that each of the new cells formed contains
only 23 chromosomes not 46 as in the body cells. This
process is called meiosis.

However, during fortification when the egg and sperm unite,

the fertilized 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 again arrange them
selves in pairs in the nucleus.

For example, a child who inherits gene A from its father and
also gene A from its mother would be homozygous, where as
a child who inherits gene A from its father and gene B from its
mother would be heterozygous.

Dominant and recessive genes: A dominant gene will

always show itself if it is present but a recessive gene will only
show itself if there is no dominant one, that is if both genes
are recessive.

For example, in the ABO blood group system the gene A and
B are dominant over gene O. Thus if a child receives from its
parents gene A and O it will belong to group A. In the same
way if a child receives from its parents genes B and O it will
belong to group B only if it receives gene O from both its
parents will it belong to group O.

5
Genotype and phenotype: The genetic composition from a
particular inherited characteristic is called the phenotype and
the way this can be seen is called phenotype. Thus if a person
is group A (phenotype) his phenotype could be either AA or
AO.

1.3 The Role of H-Gene in the Expression of

ABO Genes

Inheritance of A and B genes usually results in the expression

of A and B gene products (antigens) on erythrocytes, but H,A
and B antigens are not the direct products of the H,A, and B
genes, respectively. Each gene codes for the production of a
specific transferase enzyme (Table 1.1), which catalyzes the
transfer of a monosaccharide molecule from a donor
substance to the precursor substance, and enable us to
convert the basic precursor substance to the particular blood
group substance.

Table 1.1 ABH Genes and Their Enzymatic Products

Gene Enzyme
H L- fucosyltransferase
A 3 N-acetyl- D- galactosaminyl transferase
B 3-D- galactosyl transferase
O None

6
- As predicted in Fig 1.1 the H gene (HH/Hh) encodes for
an enzyme, which converts the precursor substance in
red cells in to H substance (H antigen).
- A and B genes encode specific transferase enzymes
which convert H substance in to A and B red cell
antigens. Some H substance remains unconverted (the H
substance is partly converted).
- O gene encodes for an inactive enzyme, which results in
no conversion of the substance in-group O red cells. This
indicates group O individual contains the greatest
concentration of H antigen.
- Persons who do not inherit H gene (very rare hh
genotype) are unable to produce H substance and
therefore even when A and B genes are inherited, A & B
antigens can not be formed . This rare group is referred to
as Oh (Bombay group).

7
 A & H antigens

H B & H antigens
HH or Substance
Hh
Genes
AB & H antigens

Precursor
H antigens
Substance

ABO Genes
hh
genes
No ABO or H antigens
(Bombay)
Unchanged
Precursor

Fig 1.1 ABO Genetic pathway

1.4 Secretors and Non-Secretors

The term secretor and non-secretor only refer to the presence

or absence of water- soluble ABH antigen substances in body
fluids (saliva, semen, urine, sweat, tears, etc). Every individual
contains alcohol soluble antigens in body tissues and on the
red cells, whether secretor or non-secretor, but secretors, in
addition to this, possess the water soluble (glycoprotein) form
of antigen, which appears in most body fluids.

8
Majority of the population secrete water- soluble substances
in saliva and most other body fluids that have the same
specificity as the antigens on their red cells.

The production of A, B & H antigens in saliva is controlled by

a secretor gene, which is in herited independently of the ABO
and H genes. The relevant gene is called Se, and its allele
which amorphic is se. At least one Se gene (genotype SeSe
or Sese) is essential for the expression of the ABH antigens in
secretors. Individual who are homozygous for se (sese) do not
secrete H,A, or B antigens regardless of the presence of H,A
or B genes.

The Se gene does not affect the formation of A,B or H

antigens on the red cells or in hematopoietic tissue, which are
alcohol soluble and which are not present in body secretions.
Oh (Bombay) individuals do not secrete A, B or H substance,
even when the Se gene is present.

9
Review Questions

1. Briefly out line the historical background of blood

transfusion.
2. What was the reason for the failure of attempted intra
and inter species blood transfusions (relate this with the
discovery of blood group by Karl Landsteiner).
3. Define the following terms:
A. Chromosome
B. Gene
C. Dominant gene
D. Phenotype
E. Secretors
4. Explain why group O individuals contain the greatest
concentration of H antigen.

10
 CHAPTER TWO

PRINCIPLES OF ANTIGENS AND

ANTIBODIES

Learning Objectives

At the conclusion of the chapter, the student should be able

to:
- Define an antigen
- Explain the basic essential for antigenic substances
- Define an antibody
- List the classes of immunoglobulin
- Compare the characteristics of IgG, IgM and IgA
- Contrast between the natural and immune antibodies
- Explain the non- red cell- immune antibodies

2.1 Antigens

An antigen can be defined as any substance which, when

introduced in to an individual who himself lacks the substance,
stimulates the production of an antibody, and which, when
mixed with the antibody, reacts with it in some observable
way.

11
Foreign substances, such as erythrocytes, can be
immunogenic or antigenic (capable of provoking an immune
response) if their membrane contains a number of areas
recognized as foreign. These are called antigenic
determinants or epitopes.

The immunogenicity of a substance (relative ability of a

substance to stimulate, the production of antibodies when
introduced in to a subject lacking the substance) is influenced
by a number of characteristics:

Foreignness: The substance should present, at least in part,

a configuration that is unfamiliar to the organism. The greater
the degree the antigenic determinant is recognized as non-
self by an individual’s immune system, the more antigenic it is.

Molecular weight: The antigen molecule must have a

sufficiently high molecular weight. The larger the molecule,
the greater is its likelihood of possessing unfamiliar antigenic
determinant on its surface, and hence the better the molecule
functions as an antigen.

Molecules with a molecular weight of less than 5000 fail to act

as antigen, with 14,000 are poor antigens unless conjugated
with adjuvant and with 40,000 or more are good antigens.
High MW molecules of 500,000 or more are the best antigens.

12
However, physical size of the molecule is not a controlling
factor. Since dextran (a carbohydrate) with a MW of 100,000
is not antigenic.

Structural stability: Structural stability is essential

characteristic; structurally instable molecules are poor
antigens, eg. Gelatin.

Structural complexity: The more complex an antigen is, the

more effective it will be complex proteins are better antigens
than large repeating polymers such as lipids, carbohydrates,
and nucleic acid, which are relatively poor antigens.

Route of administration: In general, intravenous (in to the

vein) and intraperitoneal (into the peritoneal cavity) routes
offer a stronger stimulus than subcutaneous (beneath the
skin) or intramuscular (in to the muscle) routes.

2.2 Antibodies

Antibodies are serum proteins produced in response to

stimulation by a foreign antigen that is capable of reacting
specifically with that antigen in an observable way. Five major
immunoglobulin (Ig) classes exist; which are called IgG, IgA,
IgM, IgD and IgE, with heavy chains gamma ( ) alpha ( ), mu
(µ) delta( ) , and epsilon( ) respectively. Each is unique and

13
possesses its own characteristic. Blood group antibodies are
almost exclusively IgG, IgM and IgA.

Characteristics of immunoglobulin

IgG:
- Is the predominant immunoglobulin in normal serum,
accounting for about 85% of the total immunoglobulin
- Is the only immunoglobulin to be transferred from mother
to fetus, through the placenta, a fact that explains its role
in the etiology of hemolytic disease of the new born
(HDN)
- Is the smallest antibody which has a MW of 150,000
- Is capable of binding complement
- Is predominantly produced during the secondary immune
response.

Sub classes of IgG: within the major immunoglobulin classes

are variants known as sub classes. Four sub classes of IgG
have been recognized on the basis of structural and
serological differences and are known as IgG1, IgG2, IgG3 and
IgG4. They also have different characteristics as shown in
Table 2.1.

14
Table 2.1. IgG subtype characteristics

Characteristic IgG1 IgG2 IgG3 IgG4

% of total lgG in
65 25 6 4
serum
Complement
4+ 2+ 4+ +/-
fixation
Half-life in days 22 22 8 22
Placental
Yes Yes Yes Yes
passage
Some Immune Immune
specificities Anti-Rh Anti-A Anti-Rh Anti-A
Anti-B Anti-B

IgM:
- Accounts for about 10% of the immunoglobulin pool, with
a concentration of about 1.0 g/l in normal serum.
- Is the predominant antibody produced in a primary
immune response
- Is structurally composed of five basic subunit
(pentameric), and has the largest MW of 900,000.
Because of its large size IgM cannot pass the placental
barrier to the fetus
- Is complement binding

15
IgA:
- Ig A with a MW of 160,000 constitutes 10 to 15 % of the
total circulatory immunoglobulin pool.
- Is the predominant immunoglobulin in secretions such as,
tears, saliva, colostrum, breast milk, and intestinal
secretions.
- Does not fix complement and is not transported across
the human placenta.

2.2.1 Types of Antibodies

Based on their development, blood group antibodies are

classified into Natural and Immune antibodies.

Natural antibodies: are red cell antibodies in the serum of an

individual that are not provoked by previous red cell
sensitization. But, it is believed that these antibodies must be
the result of some kind of outside stimulus and the term
naturally occurring gives an inaccurate connotation, so they
are called non- red cell or non- red cell immune antibodies.

Characteristics
- Exhibit optimum in vitro agglutination when the antigen
bearing erythrocytes are suspended in physiologic saline
(0.85%) sodium chloride, sometimes referred to as
complete antibodies.

16
- Give optimum reaction at a temperature of room or lower,
and they are also called cold agglutinins.
0
These antibodies do not generally react above 37 C that
is at body
temperature, for this reason most of these do not
generally give rise
to transfusion reactions.
These antibodies are of high MW that they can’t cross the
placental barrier, eg. IgM.

Immune antibodies: are antibodies evoked by previous

antigenic stimulation either by transfusion or pregnancy, i.e.
as a result of immunization by red cells.

Characteristics
- Do not exhibit visible agglutination of saline- suspended
erythrocytes, and called incomplete antibodies
0
- React optimally at a temperature of 37 C, and are so
called warm agglutinins.
These antibodies obviously have more serious transfusion
implications than the naturally occurring ones.
- These antibodies are so small that they can cross the
placental barrier, e.g. IgG

17
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
A. Primary immune response?
B. Secondary immune response?

18
 CHAPTER THREE

THE ABO BLOOD GROUP SYSTEM

Learning Objectives

At the end of the chapter the student should be able to:

- Describe the history of the discovery of the ABO system
- Discuss the patterns of inheritance of A and B genes
- Contrast the antigens & antibodies found in the blood in
the ABO system
- Define antiserum and its acceptance criteria for laboratory
work
-
Explain the method of grading the strength of
agglutination reactions
- Name the methods commonly used in routine blood
banking to enhance the agglutination of erythrocytes
- Prepare different percentage of red blood cells
suspensions
- Perform ABO blood grouping using different methods
- Discuss some of the result discrepancies that can be
encountered in ABO grouping

19
3.1 The Discovery of ABO Blood Group

In the 1900, a German Scientist Karl Landsteiner established

the existence of the first known blood group system, the ABO
system. Classification of the blood group was based on his
observation of the agglutination reaction between an antigen
on erythrocytes and antibodies present in the serum of
individuals directed against these antigens. Where no
agglutination had occurred, either the antigen or the antibody
was missing from the mixture.

Landsteiner recognized the presence of two separate

antigens, the A & B antigens. The antibody that reacted with
the A antigens was known as anti A, and the antibody that
reacted with the B antigen was known as anti B. Based on the
antigen present on the red cells, he proposed three separate
groups A, B & O. Shortly hereafter, von Decastello and Sturli
identified a fourth blood group AB, by demonstrating
agglutination of individuals red cells with both anti-A and anti-
B.

3.2 Inheritance of the ABO Groups

In 1908, Epstein and Ottenberg suggested that the ABO blood

groups were inherited characters. In 1924 Bernstein
postulated the existence of three allelic genes. According to

20
the theory of Bernstein the characters A,B and O are inherited
by means of three allelic genes, also called A,B and O . He
also proposed that an individual inherited two genes, one from
each parent, and that these genes determine which ABO
antigen would be present on a person’s erythrocytes. The O
gene is considered to be silent (amorphic) since it does not
appear to control the development of an antigen on the red
cell. Every individual has two chromosomes each carrying
either A, B or O, one from each parent, thus the possible ABO
genotypes are AA, AO, BB, BO, AB and OO. ABO typing
divides the population in to the four groups, group A, B, O
and, AB, where the phenotype and the genotype are both AB
(heterozygous), see Table 3.1.

Table 3.1 The ABO phenotypes and their corresponding

genotypes
Phenotypes Genotypes
A AA
AO
B BB
BO
O OO
AB AB

To illustrate the mode of inheritance, a particular mating, that

in which a group A male mates with a group B female, is

21
considered. The group A male may be of genotype AA or AO
and similarly the group B female may be of the genotype BB
or BO; therefore within this one mating four possibilities exist,
namely (a) AA with BB, (b) AA with BO, (c) AO with BB and
(d) AO with BO, see Table 3.2.

- This mating can result in children of all four ABO groups

or phenotypes although it is only in mating AO with BO
that children of all four ABO groups can occur in the same
family.
- This mating also shows that a knowledge of the groups of
relatives will sometimes disclose the genotype of group A
or group B individuals, eg. the finding of a group O child in
an AxB mating demonstrates the presence of the O gene
in both parents, and it follows that any A or B children
from this particular mating are heterozygous , i.e. AO or
BO.

22
Table 3.2 The ABO mating with possible genotype and
phenotype of children.

Mating Children
Phenotypes Genotypes Genotypes Phenotypes
AxA (1)AAxAA (1)AA A and O
(2)AAxAO (2)AA and AO
(3) AOxAO (3)AA,AO and OO
AxB (1)AAxBB (1)AB A,B AB, and O
(2)AAxBO (2)AB and AO
(3)AOxBB (3)AB and BO
(4)AOxBO (4)AB,BO, AO, and
OO
AxAB (1)AAxAB (1)AA and AB A,B and AB
(2)AOxAB (2)AB, AO,BO and
OO
AxO (1)AAxOO (1)AO A and O
(2)AOxOO (2)AO and OO
BxB (1)BBxBB (1)BB B and O
(2)BBxBO (2)BB and BO
(3)BOxBO (3)BB,BO, and BO
BxAB (1)BBxAB (1)AB and BB A,B, and AB
(2)BOxAB (2)AB,BB, AO, and
BO
BxO (1)BBxOO (1)BO B and O
(2)BOxOO (2)BO and OO
ABxAB (1)ABxAB (1)AA,AB and BB A,B, and AB
ABxO (1)ABxOO (1)AO and BO A and B
OxO (1)OOxOO (1)OO O

In1930 Thompson proposed a four allele theory of inheritance

based on the discovery of von Dungern and Hirszfeld in 1911,
which demonstrated that the A antigen could be divided in to

23
A1 and A2 sub groups. Thompson’s four-allele theory
encompassed the four allelic genes, A1, A2, B and O. This four
allelic genes give rise to six phenotypes: A1, A2, B, O, A1B and
A2B and because each individual inherits one chromosome
from each parent, two genes are inherited for each
characteristic and these four allelic gene give rise to ten
possible genotypes (table 3.3).

Table 3.3 ABO phenotypes and genotypes, including A1 and

A2

Phenotypes Genotypes
A1 A1A1
A1A2
A1O
A2 A2A2
A2O
B BB
BO
A1B A1B(or A1B/O)
A2B A2B(orA2B/O)
O OO

In group AB, the A gene is normally carried on one

chromosome and the B gene on the other, each being co-
dominant, although rare families have been described in

24
which both A and B have been shown to be inherited from one
parent, this condition is called Cis- AB . In serological testing,
individuals of this type have a weaker B antigen and possess
some kind of anti- B in the serum.

Table 3.4 shows the six possible genotype mating included in

the one phenotype mating A1 x B together with the
phenotypes which can be found among the offspring of each
mating.

Table 3.4 The mating A1xB.

Mating possible Possible

Genotypes phenotypes of
children
A1A1xBB A1B
A1A1xBO A1B,A1
A1OxBB A1B,B
A1OxBO A1,B,A1B,O
A1A2xBB A1B,A2B
A1A2xBO A1,A2,A1B,A2B

Sometimes by studying the phenotypes of the children it is

possible to say which genotype the parents belong. For
example, it can be seen that for the matings A1xB, A2 and A2 B
children never occur in the same family as B or O children.

25
This follows that taking all A1xB mating together, all six
phenotypes can occur. However, the finding of, for instance, a
group O child in a family where other children are A2 and A2 B
would not be possible if they all had the same parents.

3.3 The ABO Blood Group

A person’s ABO blood group depends on the antigen present

on the red cells.
- Individuals who express the A antigen on their red cell i.e.
their red cells agglutinate with anti - A belong to group A.
- Individuals who express the B antigen on their red cells
i.e. their red cells agglutinate with anti-B belong to group-
B.
- Individuals who lack both the A and B antigen on their red
cells that is their red cell show no agglutination either with
anti- A or anti- B belong to group O.
- Individuals who express both A and B antigens on their
red cells that is their red cells show agglutination with both
anti- A and anti –B belong to group AB.

The distribution of ABO blood groups differ for various

population groups, different studies have provided statistics as
given in table 3.5

26
Table 3.5 Frequency of ABO blood groups in different
population

Examples A% B% AB% O%
Asian 28 27 5 40
African 26 21 4 49
Nepalese 33 27 12 28
Caucasian 40 11 4 45

Ethiopians 31 23 6 40
(Blood donors)

Whenever an antigen A and, or B is absent on the red cells,

the corresponding antibody is found in the serum (Table 3.6)
- Individuals who possess the A antigen on their red cells
possess anti- B in their serum.
- Individuals who possess the B antigen on their red cells
possess anti A in their serum.
- Individuals who possess neither A nor B antigen have
both anti A and anti- B in their serum.
- Individuals with both A and B antigens have neither anti A
nor anti B in their serum.

27
Table 3.6. Classification of the ABO blood groups

Antigen on Red Antibodies in Serum Blood Group

Cells
A Anti-B A
B Anti-A B
Neither A nor B Anti-A and Anti-B O
A and B Neither anti-A nor AB
anti-B

3.4 Antiserum

An antiserum is a purified, diluted and standardized solution

containing known antibody, which is used to know the
presence or absence of antigen on cells and to phenotype
once blood group.

Antiserum is named on the basis of the antibody it contains:

- Anti- A antiserum which contains anti- A antibody
- Anti- B antiserum which contains anti- B antibody
- Anti- AB antiserum, which contain both anti A and B
antibodies.
- Anti –D antiserum which contains anti- D antibody

28
Sources of antisera
- Animal inoculation in which animals are deliberately
inoculated by known antigen and the resulting serum
containing known antibody is standardized for use as
antiserum.
- Serum is collected from an individual who has been
synthesized to the antigen through transfusion, pregnancy
or injection.
- Serum collected from known blood groups

Antisera requirements: Antiserum must meet certain

requirements to be acceptable for use. In using antisera the
manufacturer’s instruction should always be followed. The
antiserum has two be specific: does not cross react, and only
reacts with its own corresponding antigen, avid: the ability to
agglutinate red cells quickly and strongly, stable: maintains it
specificity and avidity till the expiry date. It should also be
clear, as turbidity may indicate bacterial contamination and
free of precipitate and particles. It should be labeled and
stored properly.

3.5 Manifestation and Interpretation of

Antigen- Antibody reactions

The observable reactions resulting from the combination of a

red cell antigen with its corresponding antibody are

29
agglutination and/ or haemolysis. Agglutination is the widely
observed phenomenon in blood grouping.

Agglutination: is the clumping of particles with antigens on

their surface, such as erythrocytes by antibody molecules that
form bridges between the antigenic determinants. When
antigens are situated on the red cell membrane, mixture with
their specific antibodies causes clumping or agglutination of
the red cells.

An agglutination in which the cells are red cells synonymously

called hemagglutination. In hemagglutination the antigen is
referred to as agglutinogen and the antibody is referred to as
agglutinin.

The agglutination of red cells takes place in two stages. In the

first stage- sensitization, antibodies present in the serum
become attached to the corresponding antigen on the red cell
surface. A red cell, which has thus coated by antibodies is
said to be sensitized. In the second stage, the physical
agglutination or clumping of the sensitized red cells takes
place, which is caused by an antibody attaching to antigen on
more than one red cell producing a net or lattice that holds the
cells together. The cells form aggregates, which if large
enough, are visible to the naked eye. There are also degrees

30
of agglutination which can not be seen without the aid of a
microscope.

The strength of an agglutination reaction can be indicated by

the following grading system (Fig. 3.1 a-f), as recommended
by the American Association of Blood Banks.

(4+) one solid aggregate;

With no free cells
clear supernatant
Fig. 3.1a

(3+) several large aggregates;

Few free cells
Clear supernatant
Fig 3.1b

(2+) Medium sized aggregate

Some free cells
Clear supernatant
Fig 3.1 c

31
(1+) Small aggregates
Many free cells
Turbid reddish supernatant
Fig 3.1 d

(Weak +) Tiny aggregate

many free cells
turbid reddish supernatant
Fig 3.1e

(Negative) No aggregates,
red blood cell all intact.
Fig3.1f

Hemolysis: is the break down or rupture of the red cell

membrane by specific antibody (hemolysin) through the
activation of complement with the release of hemoglobin, and
the librated hemoglobin can easily be observed staining the
supernatant fluid.

32
3.6 Techniques:

Determination of ABO grouping is important in pretrarsfusion

studies of patients and donors as well as in cases of obstetric
patients. There are different technique to determine ABO
grouping in the laboratory: slide, test tube & microplate. In
each technique results are interpreted based on the presence
or absence of agglutination reaction. Agglutination reaction is
interpreted as a positive (+) test result and indicates, based
on the method used, the presence of specific antigen on
erythrocytes or antibody in the serum of an individual. No
agglutination reaction produces a negative (-) test indicating
the absence of specific antigens on erythrocytes or antibody
in the serum of an individual.

3.6.1 Rules for Practical Work

- Perform all tests according to the manufacturer’s direction

- Always label tubes and slides fully and clearly.
- Do not perform tests at temperature higher than room
temperature.
- Reagent antisera should be tested daily with erythrocytes
if known antigenicity. This eliminates the need to run
individual controls each time the reagents are used.
- Do not rely on colored dyes to identify reagent antisera.
- Always add serum before adding cells.

33
- Perform observations of agglutination against a well –
lighted background, and record results immediately after
observation.
- Use an optical aid to examine reactions that appear to the
naked eye to be negative.

3.6.2 The Right Conditions for RBCs to Agglutinate

The correct conditions must exist for an antibody to react with

its corresponding red cell antigen to produce sensitization and
agglutination of the red cells, or hemolysis. The following
factors affect the agglutination of RBCs:

Antibody size: normally, the forces of mutual repulsion keep

the red cells approximately 25 nanometer apart. The
maximum span of IgG molecules is 14 nanometer that they
could only attach the antigens, coating or sensitizing the red
cells and agglutination can not be effected in saline media. On
the other hand, IgM molecules are bigger and because of their
pentameric arrangement can bridge a wider gap and
overcome the repulsive forces, causing cells to agglutinate
directly in saline.

34
pH: the optimum PH for routine laboratory testing is 7.0.
Reactions are inhibited when the PH is too acid or too
alkaline.

Temperature: The optimum temperature for an antigen-

antibody reaction differs for different antibodies. Most IgG
0
antibodies react best at warm temperature(37 C) while IgM
antibodies, cold reacting antibodies react best at room
0
temperature and coldest temperature(4 to 22 C).

Ionic strength: lowering the ionic strength of the medium

increases the rate of agglutination of antibody with antigen.
Low ionic strength saline (LISS) containing 0.2% NaCl in 7%
glucose is used for this purpose rather than normal saline.

Antibody type: Antibodies differ in their ability to agglutinate.

IgM antibodies, referred to as complete antibodies, are more
efficient than IgG or IgA antibodies in exhibiting in vitro
agglutination when the antigen - bearing erythrocytes are
suspended in physiologic saline.

Number of antigen sites: Many IgG antibodies of the Rh

system fail to agglutinate red cells suspended in saline,
however IgG antibodies of the ABO system (anti-A & anti-B)
agglutinate these red cells, because there are many A&B

35
antigen sites (100 times more than the number of Rh sites)
than the D site on the cell membrane of erythrocytes.

Centrifugation: centrifugation at high speed attempts to over

come the problem of distance in sensitized cells by physically
forcing the cells together.

Enzyme treatment: treatment with a weak proteolytic

enzymes (eg. Trypsin, ficin, bromelin, papain) removes
surface sialic acid residue- by which red cells exert surface
negative charge, thereby reducing the net negative charge of
the cells, thus lowering the zeta potential, and allowing the
cells to come together for chemical linking by specific antibody
molecules. However, enzyme treatment has got a
disadvantage in that it destroys some blood group antigens.

Colloidal media: certain anti-D sera especially some IgG

antibodies of the Rh system would agglutinate Rh positive
erythrocytes suspended in colloid (bovine albumin) if the zeta
potential is carefully adjusted by the addition of the colloid.

Ratio of antibody to antigen: There must be an optimum

ratio of antibody to antigen sites for agglutination of red cells
to occur. In prozone phenomena (antibody excess), a surplus
of antigens combining site which are not bound to antigenic
determinants exist, producing false- negative reactions. These

36
can be over come by serially diluting the anti body containing
serum. It is also important to ensure that the red cell
suspension used in agglutination test must not be too week or
too strong, as heavy suspension might mask the presence of
a weak antibody.

3.6.3 Preparation of Red Cell Suspension

The concentration of erythrocytes in a saline suspension is

important to the accuracy of testing in the blood bank. Red
cell suspension can be prepared directly from anticoagulated
blood or from packed red cell (after separating the serum or
plasma). Proper concentration of suspensions can be
prepared visually as experience allows; however, as a student
you should follow the following procedures. The procedures
include a red blood cell washing step to remove certain
impurities; and when necessary you can use this formula to
prepare different red cell concentrations.

% Required = Packed cell volume x 100

Volume of suspension required

Procedure: (as an example preparation of 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 and centrifuge the tube

37
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 in to another clean test tube.
6. Add 0.2 ml of the packed cell button to the 10 ml of saline
7. Cover the tube until time of use. Immediately before use,
mix the suspension by inverting the tube several times
until the cells are in suspension.

3.6.4 The ABO Blood Grouping

The ABO blood groups (A, B, AB, &O) represent the antigen
expressed on the erythrocytes of each group, and whenever
an antigen (A and / or B) is absent on the red cells, the
corresponding antibody is found in the serum. Based on the
above facts, an individual’s unknown ABO blood group is
performed by two methods in the laboratory: Forward (Direct)
and Reverse (Indirect) grouping. Reverse grouping is a cross
check for forward typing. Both tests should be done side by
side and the results of both methods should agree.

3.6.4.1 The Direct ABO Blood Grouping

The direct blood grouping also called cell grouping employs

known reagent anti sera to identify the antigen present or their

38
absence on an individual’s red cell.It can be performed by the
slide or test tube method.

Slide method
1. Make a ceramic ring on the slide.
2. Label one ring as anti- A and the other ring as anti-B
3. Add anti- serum to the ring labeled anti-A
4. Add anti-B serum to the ring labeled anti-B
5. Add 10% unknown cell suspension to both rings
6. Mix using a separate applicator stick.
7. Observe the reaction within 2 minutes by rotating the slide
back and forth
8. Interpreter the results: Look at Table 3.7

Test tube method:

1. Take two tubes, label one tube ‘anti- A’ and the second ‘
anti -B’
2. Add one drop of anti- A serum to the tube labeled ‘anti-A’
and one drop of anti- B to the tube labeled anti- B’
3. Put one drop of the 2-5% cell suspension to both tubes
4. Mix the antiserum and cells by gently tapping the base of
each tube with the finger or by gently shaking
5. Leave the tubes at RT for 5- minutes. Centrifuge at low
speed (2200-2800 rpm)for 30 seconds

39
6. Read the results by tapping gently the base of each tube
looking for agglutination or haemolysis against a well-
lighted white background.
7. Interpret the results as presented on Table 3.7.

Table 3.7 Reactions of patient Erythrocytes and known

Antisera
RED CELLS TESTED BLOOD GROUP
WITH INTERPRETATION
ANTI- A ANTI- B
Positive Negative A
Negative Positive B
Positive Positive AB
Negative Negative O

3.6.4.2 The Indirect ABO Blood Grouping

The indirect blood grouping, also called serum grouping

employs red cells possessing known antigen to see the type
of antibodies (anti A & -B) present, or absence of these
antibodies in serum. It usually is performed by test tube
method alone. Slide reverse grouping is not reliable as serum
antibodies agglutinate most cell samples when centrifuged,
and use of test tube enhances the agglutinated reaction.

40
Test tube method
1. Take two tubes, label one tube A- Cells’ and the second
‘B cells’
2. Put one drop of the serum to be tested each tube.
3. Add one drop of 2-5% A cells to the tube labeled ‘A cells’
and one drop of 2-5% B cells to the tube labeled ‘B cells’.
4. Mix the contents of the tubes.
5. Leave the tubes at RT for 5- minutes. Centrifuge at low
speed (2200-2800 rpm) for 30 seconds.
6. Read the results by tapping gently the base of each tube
looking for agglutination or haemolysis against a well-
lighted white background.
7. Interpretation of results: look at table 3.8

Table 3.8 Reactions of patient serum and reagent

erythrocytes

SERUM TESTED WITH BLOOD GROUP

A cell B cell INTERPRETATION
Negative Positive A
Positive Negative B
Negative Negative AB
Positive Positive O

41
3.6.5 Anomalous Results in ABO Testing

Technical errors and various clinical conditions can contribute

to a discrepancy between erythrocyte and serum results in
ABO grouping. Most ABO discrepancy’s however, are
technical in nature, and can be resolved by careful repeating
of the test procedure. These include: contaminated reagents
or dirty glass ware, over centrifugation, incorrect serum: cell
ratio, under centrifugation or incorrect incubation temperature,
failure to add test specimen or reagents, and the like. If
carefully controlled repeat testing yields the same
agglutination patterns, the variation can be assigned to one of
the following four categories.

1. Missing or weak reacting antibodies

Age: testing of infants who have not begun to produce their

own antibodies, or who possess antibodies that have been
passively acquired from the mother, or during testing of
elderly persons whose antibody levels have declined.

Hypogamaglobulininemia: in conditions in which

hypogamaglobulininemia may be demonstrated, these include
lymphomas, leukemias, immunodeficiency disorders, use of

42
immunosuppressive drugs, and following bone marrow
transplantation.

Resolution: Enhancing reaction in reverse grouping by

incubating of patients serum with the red cells at room
0 0
temperature for 15 min or incubation at 16 C or 4 C for 15
min.

2. Missing weak antigens

Sub groups of A or B antigens: The A or B antigens may

be weakly expressed because of an unusual genotype (i.e,
sub groups of A&B).

Disease: In some conditions like acute leukemias, the red cell

antigens in the ABO system may be greatly depressed that
they give weak reactions.
Blood group specific substances: in conditions like ovarian
cyst & carcinomas, blood group specific substance may be of
such high concentration is that anti-A & and – B are
neutralized when unwashed cells are used.

Acquired B antigen: effect of bacterial enzymes & absorption

of bacterial polysaccharide on to the red cells of group A or O
patients results in B specificity which involve weak B antigen
reaction in the forward grouping.

43
Additives to sera: acriflavin, the yellow dye used in some
commercial anti B reagents, can produce false agglutination in
some persons, which results from antibodies against acriflavin
in the serum combining with the dye and attaching to the
erythrocytes of the individual.

Mixtures of blood: Mixture of cell types in recently transfused

patients or recipients of bone marrow transplants can produce
unexpected reactions in forward typing.

Resolution:
- Investigating the possibility of sub groups of A&B
- Investigating the diagnosis
- Washing the patient’s red cells in saline to eliminate the
problem with blood group specific substances.
- Acidifying the anti- B reagent to PH 6.0 to rule out
acquired B and then determining secretor status
- Washing the patient’s cells three times and then
regrouping if dye is suspected as the problem or using
reagents that do not contain dye.

3. Additional antibody

Autoantibody: cold autoantibodies can cause spontaneous

agglutination of the A and B cells used in reverse grouping.
Patients with warm autoimmune hemolytic anemia may have

44
red cells coated with sufficient antibody to promote
spontaneous agglutination.

Anti A1: A2 & A2 B individuals may produce naturally occurring

anti-A1, which cause discrepant ABO typing.

Irregular antibodies: Irregular antibodies in some other blood

group system may be present that react with antigens on the
A or B cells used in reverse grouping.

Resolution:
0
- Washing the patient red cells in warm (37 C) saline to
establish cold autoantibodies as the cause.
- Treating cells with chloroquire diphosphate to eliminate
bound antibodies if warm autoantibody is suspected.
- Identifying the irregular antibody, and using A & B cells,
which are negative for the corresponding antigen.

4. Plasma Abnormalities

Increased gamma globulin: elevated levels of globulin from

certain disease states such as multiple myeloma result in
rouleaux formation.

45
Abnormal proteins: Abnormal proteins, altered proportions
of globulins, and high concentration of fibirogen may cause
rouleaux formation, which could be mistaken for agglutination.

Wharton’s jelly: when cord blood is used, reverse grouping

may be affected by wharton’s jelly which causes rouleaux.

Resolution: wash the patients cells with saline or to add a

drop of saline to the test tube is sufficient to remove proteins
that cause rouleaux

46
Review Questions

1 Briefly discuss on the discovery of the ABO blood group

system
2. Classify the ABO blood group system based on the
antigen and antibodies present in an individual
3. Give a description for grade of agglutination reaction as
recommended by the America blood bank society.
4. List conditions that influence agglutination of red cells
5. Describe how to prepare a 10 ml volume of 5% red cell
suspension
6. Discuss how to perform direct & indirect method of ABO
blood typing
7. Discuss conditions that lead to anomalous results in ABO
testing

47
 CHAPTER FOUR

THE Rh-Hr BLOOD GROUP

SYSTEM

Learning Objectives

At the conclusion 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 antigens in Wiener and Fisher- Race theories
- List the most common Rh antigens including their
characteristics
u
- Discuss on the common variants of the D antigen (D ),
including the clinical significance.
- Discuss the characteristics and clinical significance of Rh
antibodies.
- Describe and perform the techniques used in Rh antigen
detection in routine laboratory

4.1 Historical Background of Rh-Hr Blood

Grouping

In 1940 Landsteiner & Wiener reported the discovery of a

human blood factor, which they called rhesus. They

48
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 humans. They realized that this serum which they
called anti-Rh was about detecting an unknown human blood
group antigen which, independent of all other blood groups
discovered before that time. They used it to type as Rh
positive those donors whose red cells were agglutinated by
the new antibody and as Rh negative to those whose red cells
were not so agglutinated.

This discovery followed the detection of an antibody by Levine

& Stetson in 1939. This antibody occurred in the serum of a
woman delivered a stillborn fetus, who suffered a hemolytic
reaction to her husband’s ABO compatible blood transfused
shortly after delivery. The antibody was found to agglutinate
approximately 80% of randomly selected ABO compatible
donor’s and latter was shown to be anti-Rh in specificity.
Levine and Stetson 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.

In 1940, Wiener & Peters showed that the antibody anti- Rh

could be found in the serum of certain individuals who had
had transfusion reaction following ABO group- compatible
transfusions. In 1941 Levine & his Co- workers showed that

49
not only could an 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 (HDN).

Later work demonstrated that the animal or rabbit anti-

Rhesus and human anti-Rh are not the same, and were not
detecting the same antigen but the system had already
named the human antibody as anti-Rh. The animal anti-
hesus 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 & Wiener who discovered it, and
the human antibody retained the title anti Rh.

4.2. Nomenclature & Genetic Theories

Fisher- Rase Nomenclature

The Fisher- Race theory states that there are three closely
linked loci, each with one of the set of allelic gene (D& d, C &
c, E &e) and these three genes are inherited as a complex.
These three loci are believed to be so closely linked that
crossing over occurs only very rarely.

Complex Rh genes control the Rh antigens, these genes are

C, D, E, c, d & e. The Rh antigens are therefore named C, D,
E, c, d &e. The antigen d (and anti- d) do not exist, the symbol

50
“d” is used to express the absence of D.The Rh gene complex
possesses closely linked genes (antigens) which could be
assembled in eight different ways: CDe, cDE, cde, cDe, cdE,
Cde, CDE & CdE. Because of the strong antigenic characters
of D, all individuals who lack the D antigen are said to be Rh
negative regardless of whether the C or E antigen or both are
present.

Wiener nomenclature
Wiener’s theory states one gene instead of three closely
linked ones, produces one complex antigen which is made up
of three factors found on the red cells. There are eight genes
1 2 0 z y
called R ,R ,r,R ,r’,r”,R &r . Comparison of nomenclature is
presented in Table 4.1 and 4.2.

Eg. Gene Antigen factor on the red cell

’
R Rh1 rh’, Rho & hr”

Table 4.1 Comparison of Nomenclature of antigens of the Rh

system

Wiener Fisher- Race Rosenfield

Rho D Rh1
’
rh C Rh2
’’
rh E Rh3
’
hr c Rh4
’
hr e Rh5

51
Table 4.2 Comparison of Fisher- Race & Wiener
nomenclature

Fisher- Race Wiener

’, ’’
CDe Rh1 (rh Rho,hr )
c DE Rh2 (hr’,Rho,rh’’)
c de rh (hr’’,hr’’)
1
Cde rh (rh’,hr’’)
’’ ’ ’’
c dE Rh (hr ,rh )
CdE rhy (rh’,rh’’)
z ’ ’’
CDE Rh (rh ,Rho,rh )
’ ’’
c De Rho (hr ,Rho,hr )

The Rh gene that determine the Rh antigens are inherited as

a single gene (wiener) or gene complex (Fisher- Race) from
each parent. According to Fisher – Race, three pairs of allelic
genes on the same chromosome (haplotype) will determine
the production or non- production of D with C or c, E or e. The
inheritance of the Rh genes through haplotype gene is shown
in Fig 4.1

52
 Parents

Offspring

Fig 4.1 Rh Inheritance with Fisher Race nomenclature

4.3 The antigens of the Rh-Hr blood group

system

The Rh antigens can be demonstrated on fetal red cells as

early as 38 days after conception, and are well developed at
birth. There are five rhesus antigens, D, C, c, E &e which are
only expressed on red cells. They are not found in body fluids
(like saliva, amniotic fluid) and not detected on leucocytes or
platelets. The ‘d’ gene is not expressed and there is no ’d’
antigen, it only implies the absence of ’D’. Individuals who lack
any of these antigens may be stimulated to produce the
corresponding antibodies (anti-D, anti- C, ant -c, anti-E, anti-e)
by transfusion or pregnancy.

Antigen D, having antigen site between 110,000 and 202,000

per erythrocyte, is the most important of the rhesus antigens

53
medically, because it is highly antigenic than the other Rhesus
antigens.

A person is grouped as Rhesus (Rh) positive or negative

based on the presence or absence of antigen D:
- Rh positive: a person who inherits gene D and the red
cell express antigen D.
- Rh negative: a person who does not inherit gene D and
the red cells do not express antigen D
For transfusion purpose, Rh positive blood can be given to Rh
positive individuals and Rh negative blood can be given to
+ - + -
both Rh & Rh individuals. Never give Rh blood to Rh
individuals especially to women of child bearing age.

4.4 Variants of antigen

u
Weak antigen D (D )
Weak forms of antigen D where the number of D sites on the
red cells is reduced. Such weak D cells react less strongly
than red cells with normal numbers of D receptors. There are
u
two grades of D : High grade Du red cells, which are
u
agglutinated by certain anti-D sera and lower grade D red
cells, which are agglutinated only by the Indirect Antiglobulin
(IAG )test.

54
In case of blood transfusion, donors with Du + red cells are
regarded as Rh+ because, a severe hemolytic transfusion
u
reaction may result from the transfusion of D + red cell to a
recipient whose serum contains anti D. As a recipient
u
individuals with D + red cells regarded as Rh negative,
u
because of the risk of provoking the formation anti-D in a D +
subject through the transfusion of D+ blood.

In addition, Du + red cells are clinically important in that, they

u
may be destroyed at a higher rate by anti-D, and a D infant
can suffer from HDN if the mother possesses anti –D.
u
- As a donor individuals with D positive antigen regarded
as Rh positive.
- As a recipient individuals with Du positive antigen
regarded as Rh negative.

4.5. Rhesus Antibodies

The common Rh antibodies are anti –E, anti -e, anti -C,anti-c
and anti –D. Rh antibodies occur in individuals who lack the
corresponding antigens, and as a consequence of transfusion
or pregnancy (i.e as a result of immunization by red cells).
However, some exception, in a few percentage found to be
naturally occurring, as example anti- E & anti- C are non red
cell immune antibodies, and agglutinate red cells suspended
in saline at room temperature.

55
Rh antibodies generally develop from 2 to 6 months after the
initial immunization by red cells. Their production is consistent
with the classical immune response in that the earliest
antibody to appear is IgM , followed by IgG, some IgA have
also bean identified. The predominant Rh antibodies however,
are immunoglobulin class IgG, which most of them are IgG1 or
IgG3 subclasses.

Following transfusion of one or more units of Rh positive

blood, 50 to 75% of D negative recipients develop anti- D; but
25 to 30% of D negative individuals are non responders,
unable to produce anti-D inspite of repeated stimulation with
Rh+ blood. Secondary immunization in subjects who are
primarily immunized to Rh (D) may result in maximal increase
in antibody concentration in 3 weeks.

Rh antibodies cause severe hemolytic transfusion reaction in

a recipient if transfused with blood possessing the offending
antigen. In addition, Rh antibodies being IgG, are capable of
crossing the placenta and are associated with HDN.

4.6 The Rh- Hr Blood Grouping Technique

4.6.1 Methods of Rh Technique

Rh grouping can be performed in the routine laboratory by the

direct slide and tube methods. In the Rh blood group system,

56
naturally occurring Rh antibodies are not found in the serum
of persons lacking the corresponding Rh antigens. Therefore
‘reverse grouping’ cannot be done in Rh blood group system.
In performing Rh grouping the number of drops, time and
speed of centrifugation shall be determined by manufactures
directions.

4.6.2 Slide Test Method

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

2. Place a drop of Rh control (albumin or other control
medium) or another labeled slide.
3. Add two drops of 40-50% suspension of cells to each
slides.
4. Mix the mixtures on each slide using an applicators stick,
spreading the mixture evenly over most of the slide.
Interpretation or results:
Agglutination of red cells- Rh positive.
No red cell agglutination- Rh negative.
A smooth suspension of cell must be observed in the control.
Note: Check negative reactions microscopically.

4.6.3 Modified Tube Test Method

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

2. Mark ”D” on a test tube and add two drops of anti-D

57