For Medical Laboratory Technology

IMMUNOHAEMATOLOGY

Bachelor Student

Chapter one:INTRODUCTION TO

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 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 nonsecretor
- 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 techniques related to modern transfusion therapy. Efforts to save human
lives by transfusing blood have been recorded for several centuries. The area 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 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

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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
1.3 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 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 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 themselves in pairs in the nucleus.

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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.
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.

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- 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 cannot be formed. This
rare group is referred to as Oh (Bombay group).

Fig 1.1 ABO Genetic pathway

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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 bodyNfluids (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.
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.
Review Questions
1. Briefly outline 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.

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

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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 (γ) alpha (α), mu (µ) delta (δ ) , and epsilon(Є) respectively. Each is unique and
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 rol 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.

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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
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

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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.
- Give optimum reaction at a temperature of room or lower, and they are also called cold
agglutinins.
These antibodies do not generally react above 370C 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
- React optimally at a temperature of 370C, 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
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?

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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
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 antiB.
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 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

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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.

To illustrate the mode of inheritance, a particular mating, that in which a group A male
mates with a group B female, is 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 childrenBfrom this particular mating are heterozygous , i.e. AO or BO.
Table 3.2 The ABO mating with possible genotype and phenotype of children.

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 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 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).

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 In group AB, the A gene is normally carried on one chromosome and the B gene on the
other, each being codominant, although rare families have been described in 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.

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 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.
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 groupB.
- 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

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

found in the serum (Table 3.6)

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 - 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

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
Sources of antisera
- Animal inoculation in which animals are deliberately inoculated by known antigen and
the resulting serumcontaining 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

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 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 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 of agglutination which cannot 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.

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 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.
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 differents 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

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 - 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.
- 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 cannot 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.
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 antigenantibody reaction differs for
different antibodies. Most IgG antibodies react best at warm temperature(370C) while IgM
antibodies, cold reacting antibodies react best at room temperature and coldest temperature(4 to
220C).
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.

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 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 aresuspended 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 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 overcome 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 can be overcome 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

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certain impurities; and when necessary you can use this formula to prepare different red cell
concentrations.

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
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 isbperformed 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 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.

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 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 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 welllighted white background.
7. Interpret the results as presented on Table 3.7.

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.

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 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 welllighted white background.
7. Interpretation of results: look at table 3.8
Table 3.8 Reactions of patient serum and reagent erythrocy

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.

24
 Hypogamaglobulininemia: in conditions in which hypogamaglobulininemia may be
demonstrated, these include lymphomas, leukemias, immunodeficiency disorders, use of
immunosuppressive drugs, and following bone marrow transplantation. Resolution: Enhancing
reaction in reverse grouping by incubating of patients serum with the red cells at room
temperature for 15 min or incubation at 160C or 40C 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.
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 red cells coated with sufficient antibody to promote spontaneous agglutination.

25
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:
- Washing the patient red cells in warm (370C) 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.
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.
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

26
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
- Discuss on the common variants of the D antigen (Du), 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 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 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.

27
Later work demonstrated that the animal or rabbit antiRhesus 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 antihesus 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) does not exist, the symbol “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 called R1,
R2 , r, R0, r’,r”,Rz & ry . Comparison of nomenclature is presented in Table 4.1 and 4.2.

28
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

29
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

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 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
Rhindividuals especially to women of child bearing age.
4.4 Variants of antigen Weak antigen D (Du)

30
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 two grades
of Du : High grade Du red cells, which are agglutinated by certain anti-D sera and lower grade
Du red cells, which are agglutinated only by the Indirect Antiglobulin (IAG )test.
In case of blood transfusion, donors with Du + red cells are regarded as Rh+ because, a severe
hemolytic transfusion reaction may result from the transfusion of Du + red cell to a recipient
whose serum contains anti D. As a recipient individuals with Du + red cells regarded as Rh
negative, because of the risk of provoking the formation anti-D in a Du + subject through the
transfusion of D+ blood.
In addition, Du + red cells are clinically important in that, they may be destroyed at a higher rate
by anti-D, and a Du infant can suffer from hemolytic disease of the newborn (HDN) if the
mother possesses anti –D.
- As a donor individuals with Du 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.
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

31
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, 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
3. Place a drop of Rh control (albumin or other control medium) on another labeled slide.
4. Add one drop of a 2-5% cell suspension to each tube
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 a weakly positive (+) and negative sample to perform the Du test
4.6.4 Du Typing Using Indirect Anti- Globulin Test (IAT)
1. Use the intial Rh D typing tube and control in procedure 4.6.3 and incubate the Rh . Negative
or weakly reactive (+) samples and the control at 370C for 30 minutes.

32
2. Wash cells in both test and control tube 3-4 times with normal saline.
3. Add one drop of the poly specific anti- human globulin (coombs) to each tube and mix well.
4. Centrifuge at 2200-2800 rpm for 10 second.
5. Gently re suspended the cell button and observe for agglutination
6. Interpretation: the positive result is agglutination in the tube containing anti–D and the control
is negative. A negative result is absence of agglutination in both the test & control.
Review Questions
1. Discuss the history of the development of the Rh system
2. Contrast the Fisher- Race & 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 do the Rh antibodies are produced?
6. Discuss the clinical significance of the Rh antibodies
7. Out line a brief lab procedure to classify an individual as Rh positive or Rh negative

33
CHAPTER FIVE: THE ANTI- GLOBULIN TEST (COOMB’S TEST)
Learning objective
At the conclusion of the chapter, the student should be able to:
- Describe the purpose of the antihuman globulin (AHG) test.
- Name and contrast the Anti-human globulin (coomb’s) reagents.
- Know the principle and carryout the AHG procedure.
THE ANTI- GLOBULIN TEST is introduced in to clinical medicine by Coomb’s in 1945. It is
a sensitive technique in the detection of incomplete antibodies, anti bodies that can sensitize but
which fail to agglutinate red cells suspended in saline at room temperature, mainly IgG. These
antibodies are agglutinated by the anti- IgG in antiglobulin serum through the linking of the IgG
molecules on neighboring red cells, as shown in Fig 5.1.

Red cells can also be agglutinated by a reaction of complement components on their surface with
anticomplement serum, in the antiglobulin reagent, since the antiC3 in the AHG reagent binds to
the C3 on the sensitized red cells and bridge the gap between the cell- bound human C3 on
adjacent red cells. The anti-complement helps in detecting IgM antibodies, which bind
complement but may elute off the red cells with increase in temperature, leaving complement
components alone on the red blood cells. They also enhance the reactions seen with complement
binding antibodies.
The anti-globulin reagent is prepared by immunizing animals, often rabbits with human gamma
globulin (antibody) and, or beta globulin (components of complement). There are two types of

34
anti-globulin reagents that can be used in laboratory procedure: broad spectrum (polyspecific
sera) and monospecfic sera.
Broad spectrum (polyspecific): prepared by combining antiIgG & anti-complement. The
reagent may also contain antibodies of other specificities such as anti- IgM, anti-IgA anti C-3, or
anti C-4.
Monospecific: contains only a sigle antibody: anti-IgG or only anti-complement. Two kinds of
antiglobulin tests are known: the direct antiglobulin test (DAT) & the indirect antiglobulin test
(IAT).
5.1. The Direct Antiglobulin Test (DAT)
It is used to demonstrate whether red cells have been sensitized (coated) with antibody or
complement in vivo, as in case of HDN, Autoimmune haemolytic anemia, and drug induced
haemolytic anemia, and transfusion reactions.
Principle: Patients erythrocytes are washed to remove free plasma proteins and directly mixed
with Anti human globulin (AHG), and if incomplete antibodies are present, agglutination occurs.
Procedure
1. Place one drop of 5% saline red cell suspension in a test tube.
2. Wash the red cells 3-4 times using normal saline, ensuring adequate removal of the
supernatant after each wash. 3. Add one or two drops of antiglobulin reagent to the tube.
4. Centrifuge at 3,400 rpm, for 15 sec.
5. Gently re suspend the red cells and examine macroscopically and microscopically for
agglutination or hemolysis. 6. Add IgG sensitized red cells as a control, centrifuge and read. If a
negative result is obtained, the test result is invalid if mono anti-complement reagents are used
complement sensitized red cells should be for substituted for IgG sensitized red cells.
5.2 The Indirect Antiglobulin Test (IAT)
It is used for the detection of antibodies that may cause red cells sensitization in vitro. The
sensitizing antibody or complement acts as the antigen for the antiglobulin reagent. IAT used in
cross- matching, to detect antibodies that might reduce the survival of transfused red cells and
Du technique, in the detection of Du antigen.
Principle: The serum containing antibodies is incubated with erythrocytes containing antigens
that adsorb the incomplete antibodies. After washing to dilute the excess antibody in the serum,
the addition anti-globulin serum produces agglutination in the presence of incomplete antibodies.

35
Procedure (IAT)
1. Put 2-4 drops of serum in a test tube
2. Add a drop of 5% red cell suspension
3. Mix & incubate at 370C for 15-30 minute
4. Centrifuge at 3400 rpm, for 15 sec and examine for agglutination or haemolysis.
5. Wash 3-4 times decanting the supernatant.
6. Add 1 or 2 drops of antiglobulin reagent.
7. Mix and centrifuge at 3400rpm, for 15 sec.
8. Examine for agglutination or haemolysis
Review Questions
1. What is the purpose of the AHG test?
2. How can the AHG reagent make agglutination of sensitized erythrocytes?
3. Write the principle of the DAT and IAT.
4. What conditions can be diagnosed by the DAT

36
CHAPTER SIX: HAEMOLYTIC DISEASES
Learning Objective
At the conclusion of this chapter the student should be able to:
- Understand the cause of and the laboratory method to demonstrate Auto Immune Hemolytic
Anemia (AIHA).
- Briefly explain the cause and consequences of hemolytic disease of the newborn (HDN).
- Name the major immunoglobulin class and sub classes responsible for HDN.
- Describe the laboratory diagnosis & treatment of HDN caused by ABO and Rh incompatibility.
- Compare the relationship of HDN caused by ABO incompatibility to HDN caused by Rh
incompatibility.
- Describe the prenatal treatment that is significant in HDN caused by anti-D.
6.1. Autoimmune hemolytic anemia (AIHA)
AIHA is brought about through the interaction of red cells and autoantibodies. It is classified into
four groups namely: warm- reactive autoantibodies, cold – reactive auto antibodies paroxysmal
cold hemoglobinuria and drug induced hemolysis
The diagnosis of AIHA depends on the demonstration of autoantibodies on the patient’s
red cells using the DAT. The autoimmune antibodies will be either of the ‘warm antibody‘ types
or the ‘cold antibody’ type. The warm types have antibodies active at 370C but no abnormal cold
antibodies. In the cold type the patient’s serum contains high titer cold agglutinins, optimally
active at 20C but with a temperature range which may go as high as 320C.
A positive DAT will be found in both types; in the cold type it is complement which
sensitizes the cells and give rise to the positive DAT rather than specifically bound antibody,
whereas in the warm type it is more usually the attached antibody, although occasionally it may
be due to complement.
6.2. Hemolytic Disease of the New Born (HDN)
HDN, originally known as erythroblastosis fetalis, results from blood group
incompatibility in which maternal antibodies destruct fetal red cells. An infant having inherited
an antigen from the father, which is absent in the mother, causes her to form the corresponding
antibodies. These antibodies pass through the placenta by active transport mechanism, coat the
fetal erythrocytes and cause damage to them.

37
 Every blood group antibody that can occur as IgG can cause HDN. It is only IgG
immunoglobulin that is capable of passing the placental barrier and which is found in cord blood
in a concentration equivalent to that found in maternal blood. IgM agglutinin though produced in
response to fetal red cells in utero, plays no part in the cause of HDN, and are either present in
much lower concentration in the new born than the mother or entirely absent.
Fetal hematopoietic tissue (liver, spleen and bone marrow) respond to hemolysis by
increased production of RBCs , predominantly NRBCs . Increased destruction of red cells leads
the fetus to develop anemia and jaundice from the hemoglobin breakdown product, bilirubin. If
this blirubin reaches excessive levels in the newborn or infants circulation it causes mental
retardation or death.
6.2.1 HDN Due to Rh Blood Group Incompatibility
HDN due to anti- Rh(D) occurs when mother and infant are always incompatible with
respect to the Rh factor: The mother Rh(D) negative, and the infant Rh (D) positive (inherited
the D factor from the father). ABO incompatibility between the mother and fetus reduces the
chance of maternal immunization to the D Ag. This is probably because the fetal cells, which are
incompatible with the maternal ABO antibodies, are destroyed by existing ABO antibodies
before they have a chance to act as an antigenic stimulus.
The first Rh- incompatible infant is usually unaffected because the number of fetal cells
that cross the placenta during pregnancy (after 24 weeks gestation) is small and insufficient to
cause IgG anti D production, unless a prior transfusion of D positive blood has been given.
During transplacental hemorrhage, the amount of fetal blood that enters the maternal
circulation increases, and in 6 months’ time after delivery only 10% of these Rh negative women
could produce detectable antibodies. The actual production of anti-D antibodies depends on the
dosage and antigenecity of the D antigen, and the mother’s ability to respond to these foreign
antigens. About one third of mothers are nonresponders, they fail to form anti- D despite
intentional repeated injections of Rh (D) positive erythrocytes.
During a second pregnancy with a Rh positive fetus, small number of fetal cells cross the
placenta (20 doses of antigen) stimulating the antibody to high concentration, mainly Ig G anti-
D that passes in to the fetal circulation destroying fetal red cells. The severity of the disease
increases with each Rh positive pregnancy. IgG anti-D is found predominantly in sub classes

38
IgG1 & IgG3 and these subclasses possess properties that play and effective role in
erythrocytolysis in vivo.
6.2.2 HDN due to ABO Blood Group Incompatibility
HDN due to ABO blood grouping usually occurs when the mother is invariably group O
(posses Ig G anti- A&B), the infant: group A or B (usually group A) and when the mother &
infant are Rh compatible.
The fetal red cells cross the placenta in to the maternal circulation stimulating the existing
anti A& B to high titers; the “immune” anti A& B stimulated is largely IgG. ABO HDN occurs
in the first pregnancy because anti A & anti- B are always present (naturally occurring) and
therefore readily stimulated. Although ABO incompatibilities between mother and baby occur
frequently and represent a common form of HDN, the clinical course of ABO HDN is relatively
mild, probably because of the antigenic development of the fetal red cells and may also be due to
the presence of A&B substances in the fetal tissues and fluids that will neutralize the anti A &
anti – B antibodies before they can attack the fetal red cells.
6.2.3 Assessment of HDN
Prenatal
Some investigations are carried out on blood of the mother to identify women at risk of
having a child affected with HDN. It is recommended that all pregnant women at their first
attendance at a clinic need to have ABO grouping, Rh typing for D & Du, alloantibody screening
test and amniocentesis.
Postnatal
After birth different laboratory procedures are helpful in determining the presence and assessing
the severity of HDN.
1. ABO & Rh Typing: the ABO group of the infant is based on forward (cell) grouping as
anti- A and anti B agglutinins do not develop until a few months after birth.
- ABO grouping most commonly reveals the mother to be group O and the baby to be
group A or possible group B.
- Rh typing shows the baby to be D or DU positive and the mother D or DU negative
2. DAT on cord or infants blood:

39
 - In ABO HDN, DAT is usually negative or weakly positive , as weak Ag-Ab interaction
which cause the antigen to be removed during the washing phase of the DAT. Also, due to too
low antibody titer to be detected.
- In Rh HDN, DAT gives positive result.
3. Antibody elution test of cord blood: done if DAT is positive, may reveal the presence
of immune anti- A or anti-B in ABO HDN and anti-D in Rh HDN.
4. Hgb level of cord blood: may be slightly to severely decreased
5. Serum bilirubin level on cord serum: may exceed the normal values of cord total serum
bilirubin of 1 to 3 mg/ml.
6. Peripheral blood smears on cord blood: blood smear evaluation shows anemia with
RBC morphology abnormalities: hypochromia, microspherocytosis with the demonstration of
reticulocytes and immature nucleated RBCs.
7. Kleihauer- Betke acid elution test: is a test to be performed for quantitating the extent
of fetal maternal hemorrhage (number of fetal cells in the maternal circulation). It is an indicator
for treatment of the mother with anti-D immunoglobulin, more importantly used to determine the
size of dose to be given.
Reagents: 80% ethanol
Solution A: 0.75 haematoxylin in 96% ethanol
Solution B : 2.4 g FeCl3 & 2 ml of 25% HCl in 100 ml distilled water
Elution solution: 2 parts of A mixed with 1 part of B & 9 part of 80% ethanol
Counter stain: 0.1% erythrosin or 0.5% aqueous eosin.
Method: Mother’s whole blood is diluted with an equal quantity of saline and used for
making films. The slides are fixed in 80% ethanol for 5 minutes at RT. They are thoroughly
dried. They are then placed for 20 seconds in the elution solution, rinsed in distilled water and
counterstained for 2 minutes. At least 50 fields are examined using a magnification x 400. The
white cells, including lymphocytes, stain gray, the adult red cells, which have soluble
hemoglobin appear as ‘ghost’ cells, while the fetal cells stain bright red. Report the percent &
fetal cells after counting 2000 cells in the acid elution smear.
6.2.4 Prevention of HDN
Fetal red cells in the maternal circulation might be destroyed by administration of
suitable quantity of IgG anti- D to prevent Rh immunization of the mother, given to Rh-negative

40
women within 72 hours of delivery. This dramatically decreases the incidence of anti-D HDN.
Combined prenatal-postnatal treatment is more effective than postnatal treatment alone in
suppressing Rh immunization. All pregnant Rh- women should receive Rh IG even if the Rh
status of the fetus is unknown because fetal D antigen is present in fetal erythrocytes as early as
38 days of conception.
Dose: The usual recommended dose (contained in one vial) is about 300 µg which is
believed to offer protection against a fetomaternal hemorrhage of 30 ml (15 ml packed cells) or
less.
If a massive fetomaternal hemorrhage has occurred, the volume of the hemorrhege must
be determined to calculate the number of vials of Rh (D) immune globulin to administer.
Calculated as follows:
Volume of fetomaternal Percentage of fetal cells hemorrhage = (seen in the acid elution
stain) x*50

NB: Factor of *50 = 5000 ml (estimated maternal volume) x 1/100%

*2 is a common factor, because the actual fetomaternal hemorrhage may be twice the
estimate.
Example:

Candidates for the administration of RhIG:

Anti- D prophylaxis should be given as soon as possible If the women is Rh D negative
and has not already developed anti D within 72 hrs after delivery of a D positive infant, or after
obstetric intervention such as amniocentesis, abortion, miscarriage, or pregnancy.
The laboratory criteria are:
- The mother is D and DU negative.
- The screening test for alloantibodies is negative for anti D antibody.
- The infant is D or Du positive.

41
 - DAT on cord or infant’s cells is negative. If DAT is positive perform elution test to
establish anti
– D is not the coating antibody.
6.2.5 Treatment of Infants Suffering from HDN
For infants who develop hyperbilirubinemia and/or anemia due to HDN, exchange transfusion is
usually carried out.
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. By exchange transfusion the concentration of bilirubin and incomplete antibodies
decrease, simultaneously the infant is provided with compatible donor red cells. To give
exchange transfusion for an infant clinical & laboratory findings must be considered. Cord Hgb
(<10g/dl) and raised serum bilirubin are strong indicator for treatment.
For compatible exchange transfusion, donor’s blood should be cross- matched with the maternal
serum and this blood should lack the red cell antigen corresponding to the maternal antibodies. It
must also be ABO group & 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.
Review Questions
1. Compare the causes and lab 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?
4. Discuss on the dose of IgG anti-D given to Rh- pregnant women to prevent HDN?
5. When does Rh HDN occurs?
6. List & discuss the postnatal lab investigations to be carried out to know the presence & extent
of HDN.

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 CHAPTER SEVEN: THE CROSS- MATCH
(COMPATIBILITY TESTING)
Learning Objective
At the conclusion of this chapter, the student should be 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.
7.1. Purpose of Cross-Match
The cross- match (compatibility testing) is a procedure performed before transfusion to select donor’s blood that will
not cause any adverse reaction like hemolysis or agglutination in the recipient. In addition it helps the patient to
receive maximum benefit from transfusion of red cells, which will survive maximum in his circulation. This is done
by ensuring the ABO and Rh group of the blood to be transfused is compatible with patient’s ABO and Rh group
and by detecting most unexpected (irregular) antibodies in the patient’s serum that will react with the donor’s red
cells causing their destruction or reducing their normal survival.
However, a cross match will not prevent immunization of the patient, and will not guarantee normal survival of
transfused erythrocytes or detect all unexpected antibodies in a patient’s serum.
7.2. Types of Cross Match
There are two types of cross- matches major & minor curse match.
Major cross- match: includes mixing recipient’s serum with the donor’s red cells. It is much more critical for
assuring safe transfusion than the minor compatibility test. It is called major because the antibody with the
recipient’s serum is most likely to destroy the donor’s red cells and that is why it is called major cross match.
Minor cross match: involves mixing the donor’s serum with patient’s red cells. It is usually thought that any
antibody in the donor’s serum will be diluted by the large volume of the recipient’s blood, so it causes relatively less
problem and so called minor cross match.
7.3 Selection of Blood for Cross Match
Generally, when whole blood is to be transfused, 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.
Table 7.1 Selection of ABO blood for cross-match

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7.4 Procedure for Cross-Match
For a full cross match for non- emergency transfusions the following procedure which include different phases (the
saline, protein, AHG and enzyme) is recommended:
1. Saline tube technique at RT: provides the optimum temperature and medium for the detection of IgM antibodies
of ABO system and other potent cold agglutinins.
2. Saline 37oC: is the optimum for the detection of warm agglutinin, of which are saline reactive IgG antibodies of
the Rh/ Hr system.
3. AHG: is highly efficient for the detection of most kinds of incomplete antibodies.
4. Enzyme technique- is a very sensitive one for the detection of some low affinity Rh antibodies, which are not
detected by other methods including the anti-globulin technique.
Procedure:
1. Put 3 drops of patient’s serum in to a test tube.
2. Put one drop of donor’s 3% red cells suspension.
3. Mix and centrifuge at 3400 rpm for 15 seconds.
4. Examine for agglutination or haemolysis, if compatible proceed with the next phase.
5. Mix the contents of the tube and incubate at 370C for 20-30 min.
Note: potentiators such as a drop of 22% albumin may be added at this phase to increase the sensitivity of the test.
6. Centrifuge at 3400 rpm for 15 seconds and examine for agglutination or hemolysis. If there is no hemolysis or
agglutination proceed with the next phase.
7. Wash the contents of the tube 3-4 times with normal saline.
8. After the last wash, decant all saline and add two drops of AHG reagent and mix.
9. Centrifuge at 3400 rpm for 15 seconds.

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10. Gently re suspend the cells button and examine macroscopically and microscopically for agglutination
orhemolysis .
Enzyme cross match can be performed by using different enzymes: bromelin, ficin, papain & trypsin. Two methods
are available to carry out enzyme cross mactch - One stage & two stage methods. The one-stege technique involves
enzyme, patient’s serum and donor’s red cell incubated together. The two-stage technique involves red cells
pretreated with enzyme and then tested with the patient’s serum.
Review Questions
1. What is cross match?
2. What is the purpose of cross-match? 3. List the types of cross-match with their constituents.
4. List the phases of cross- match and their respective importance in antibody detection.

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CHAPTER EIGHT: THE DONATION OF BLOD
Learning objective:
At the end of this chapter the student should be able to:
- Discuss the medical and physical requirements that would exclude an allogeneic 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
- Name the common blood components with their storage temperature and shelf life
- Explain the possible donor reactions
8.1 Selection of blood donors
A blood transfusion service aims to prepare safe blood from a safe donor to a recipient who needs blood. The
medical person who screens donors should identify conditions which can harm both the donor who gives his blood
on one hand and the recipient who receives blood and blood products on the other hand. Therefore, to ensure the
wellbeing of both donors and patients the screening person should understand the requirements that make a donor
acceptable and not acceptable to donate blood.
8.1.1 Selection Criteria:
Age:
If between 17-65 years acceptable.
If less than 17 years after guardian’s consent or depending on the local law If more than 65 years after consulting a
medical doctor.
Hemoglobin:
Females should not be less than 12.5 g/dl (PCV 38%)
Males should not be less than 13.5 g/dl (PCV 41%)
In both sexes Hgb above 19g% (Hct above 57%) are not acceptable.
Pulse, Blood pressure & Temperature:
Pulse between 60-100 per minute acceptable.
Systolic pressure between 90 and 180 mmHg acceptable
Diastolic pressure between 50 and 100 mmHg acceptable
A donor’s temperature must not exceed 37.5oC.
Weight:
If between 45-50 kgs can donate 350 ml of blood If above 50 kg can donate 450 ml of blood
- Obese donors who are unable to climb the coach are not acceptable.
- If weight is very low compared to the height of the donor do not accept.
- Donors with unexplained weight loss of a significant degree (more than kg) are not acceptable to donate.
If a prospective donor weighs less than 50 kg, a lesser amount of blood may be collected, and the amount of
anticoagulant in the collecting bag must be reduced proportionally, calculated as follows:

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Pregnancy:
pregnant women excluded from donating for 1 year after the conclusion of their pregnancy.
Medication:
In general, medications taken by a donor are not harmful to a recipient. Deferral of a donor because of drug depends
on the nature of the disease for which the drug was ordered. Consult medical doctor for donor’s on long term
treatment.
Illness: prospective donors with disease of the heart, liver, lungs, or individuals with a history of cancer, or those
with bleeding problems should be excluded subject to evaluation by a physician.
- Donors who have had leukemia must be permanently deferred.
- Donors with previous history of tuberculosis are acceptable after completion of therapy and if no longer active.
Infectious diseases: A donor must be free from infectious diseases that can be transmitted by blood such as
hepatitis, HIV & malaria.
- 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.
- Persons at high risk for acquiring or transmitting AIDS should not donate blood.
- Donors who have a history of malaria, or were previously resident in an endemic area, should be deferred for 3
years after becoming symptomatic or after leaving the endemic area.
Previous donation: If a person has donated blood, an interval of at least four months for men and six months for
women is required before the next donation.
Surgery: If the surgery is minor (such as tooth extraction) a donor is excluded until healing is complete and full
activity has been resumed.
Vaccinations: Persons recently immunized with toxoids and killed viral, bacterial and rickettsial vaccines (such as
for anthrax, cholera, diphtheria, influenza, polio, tetanus, typhoid, typhus) are acceptable, if they are symptom free
and a febrile.
- After small pox vaccination, a donor is acceptable when the scab has fallen off, or 2 weeks after an immune
reaction.
- 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.
8.2 Collection of Blood

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Trained personnel must collect donation of blood. Basic information from the donor: date of donation, full name,
address, sex, age and the ABO & Rh blood group including the prospective donor’s medical history must be
obtained and signed by the phlebotomist who perform the procedure. A form similar to Figure 8.1, which is taken
from Ethiopian Red Cross Society, is the basic guideline for donor registration and the medical history interview.
Patient identification also is an important step in blood collection. Forms accompanying blood samples from the
recipient must contain sufficient information: full name, identification number of patient, sex, age, clinical diagnosis
and the like for identification of the recipient.
8.2.1 Micro Sampling
Micro sampling is the collecting of small quantity of blood from capillaries of the fingertip. Capillary blood
collection is performed using a sterile, disposable lancet. In immunohematology laboratory, this blood is used for
blood grouping, hemoglobin or hematocrit determination.
Copper sulphate method of hemoglobin determination:
CuSO4 method is based on the relationship of specific gravity to hemoglobin concentration. It is used to check that a
donor has sufficiently high hemoglobin level to be eligible to give blood. Two strengths of CuSO4 solution are
normally used, each of which has a different specific gravity: one for male donor with a sp.gr. of 1.055 (equivalent
to 13.5g/dl of hemoglobin)and one for female donors with a sp.gr of 1.053(equivalent to 12.5g/dl of hemoglobin ).
In this method, a drop of blood is allowed to fell gently at a height of about 1 cm above the surface of the CuSO 4
solution. If the drop of blood has a satisfactory hemologin concentration, it will sink in the solution within 15
seconds. An unacceptable specimen will either remain suspended or will sink slightly and then rise to the top of the
solution within 15 seconds.
8.2.2 Macro Sampling
Macro sampling is the collection of large volume of blood from the veins by venipuncture. In Blood Bank
laboratory, this blood is usually collected for transfusion purpose from volunteer blood donors, and for serologic
tests of diseases that are transmittable by blood transfusion: hepatitis, HIV and syphilis. - Before starting the
phlebotomy, the phlebotomist should introduce himself/ herself pleasantly to the patient and briefly explain the
phlebotomy procedure in early- tounderstand terms.
1. Visually inspect both arms, and choose the arm that is free of bruises and brasions. In the arm, three veins can be
used for venepuncture the cephalic, basilic, and median cubital veins. (Fig. 8.2).

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 Fig. 8.2 Veins commonly used for venipuncture (left arm shown)
2. Apply the tourniquet, and ask the patient to make a fist (sometimes a roll of gauze is placed is the
patient’s hand).
This usually makes the veins more prominent. Using the left index finger, palpate for an appropriate vein. The ideal
vein is generally near, or slightly below, the bend in the arm. Do not leave the tourniquet on for more than 2
minutes (After an appropriate site has been chosen,
release the tourniquet).
Using 70% alcohol swab cleanse the intended site of venepuncture in a circular motion from the center outward.
Allow the site to dry.
The phlebotomy procedure
1. Inspect the anticoagulant donor bag for leaks, and make sure that the anticoagulant solution is clear.
2. Position the bag below the level of the donor- arm balance system, making sure that the counterbalance is level
and adjusted for the amount of blood to be drawn. Make a loose knot of the blood bag tubing.
3. Reapply the tourniquet or blood- pressure cuff (inflated to 40-60 mm Hg) and have the donor open and close the
hand until the selected vein is again prominent.
4. Apply the hemostat clamp to the tubing atleast 5 cm above the needle, uncover the sterile needle, and perform he
venipuncture immediately. A clean venepuncture will guarantee a full, clot free unit
5. Open the hemostat clamp and check that the blood flow is adequate. Carefully tape the tubing to hold the needle
in place and cover the venipuncture site with a sterile gauze pad. Have the donor squeeze a rubber ball or other soft
object every 10 to 12 seconds during collection.
6. Keep the donor under observation throughout the phlebotomy. A person should never be left unattended during
or immediately after donation. 7. Mix the unit of blood periodically (every 30 seconds). Time limits for collecting a
unit are not fixed, so long as the blood flow is continuous However, it usually takes 8-10 minutes. A unit
containing 450-495 mL should weigh 425-520 g plus the weight of the container with its anticoagulant.

49
8. Remove the tourniquet & hold a sterile gauze lightly over the venipuncture site and remove the needle from the
donor’s arm. Apply pressure on the gauze. Have the donor raise the arm (elbow straight) and hold the gauze firmly
over the phlebotomy site with the opposite hand.
9. Strip the donor tubing from the end of the tube towards the bag as completely as possible in order to mix well
with the anti-coagulant. Invert the donor unit and allow the line to refill. Then strip again. Seal the tubing attached
to bag into segments suitable for subsequent tests with either a heat sealer or metal clips.(see fig 8.3 for
appropriately collected donor blood).
10. Place blood at appropriate temperature.

Note: immediately after collection, blood must be placed in storage at a temperature between 1 oC & 60C.However,
if the blood is to be used as a source of components, up to 8 hours may elapse before storage.
8.3 The Anticoagulants and Storage of Blood and Blood Products
Anticoagulant is a substance that prevents the clotting of blood some anticoagulants contain preservatives that
provide proper nutrients for metabolism in the red cell during storage. Anticoagulants maintain red blood cells
hemoglobin function and viability and the biochemical balance of certain elements: glucose, ATP, 2,3
diphosphoglycerate (2,3DPG) and PH, so that the red cells will maintain the means of delivering oxygen to the
tissues of the recipient.
Anticoagulants and/or anticoagulant preservation for whole blood and red cell concentrate storage include:
ACD(acidcitrate- dextrose),CPD(citrate- phosphate dextrose).CPD-A 1 and CPD-A2(citrate phospate dextrose
adenine) and heparine.

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1. ACD
- Acts as an anticoagulant by binding Ca
- Composition: Trisodium citrate- binds Ca
Citric acid: maintains PH
Dextrose: acts as a nutrient & preservative
- To prevent the clotting of 100 ml of blood 15 ml ACD is required.
- Shelf life: 75% survival after 21 days of storage.
2. CPD
- Acts by binding Ca.
- Composition: In addition to the composition of ACD, CPD contains sodium phosphate, which maintains ATP
levels in the red cells.
- Advantages of CPD over ACD.
• Contains less acid.
• Gives less hemolysis.
• Smaller leak of K from the red cells.
• Prolonged post- transfusion survival of red cells.
- To prevent the clotting of 100 ml blood 14 ml of CPD is required.
- Shelf life: survival of red cells in CPD 24hrs post transfusion is 80 to 85% after 21 days
The composition of two anticoagulant preservative solutions(CPD and CPDA1) is presented comparison purpose in
Table 8.1

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3. CPD-A: It is a combination of CPD and adenine. Adenine provides a substrate from which red cells can
synthesize ATP during storage.- Survival of red cells is 80% after 28 days storage and 75-80% after 35-days
storage.
4. EDTA and Heparin: are not commonly used in Blood Banking. Whole blood or red cell collected and stored in
heparin solution must be used within 48 hours of phlebotomy.
Avariety of blood components can be harvested from a single unit of whole blood (Fig –8.4). Each component can
be collected, processed and stored under conditions, which maximize its storage capacity.

These components can effectively meet patient transfusion needs while keeping the risk of transfusion to a
minimum. By using a single unit one can treat anemia with the packed cells, platelate deficiency with platelate
preparations, clotting factor and other plasma deficiencies with plasma preparation.
8.4 Potential Hazards During & after Blood Collection
Donor reactions though are rare, may include fainting, nausea, vomiting, development of hematoma and
convulsion. At the first sign of reaction, the phlebotomist should stop the phlebotomy, give initial first aid
procedures and call the blood bank physician.

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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. Write the composition of CPDA with their respective importance.
5. Name the common blood components that can be prepared from a unit of donated blood.
6. List some potential hazards that occur during or after blood donation.

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