ANATOMY

AND
PHYSIOLOGY
MODULE 17
(ABO Blood Grouping)

Prepared by:

MARICEL L. DATOY, RN, MAN, LPT

Instructor
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Introduction:

ABO blood group system, the classification of human blood based on the inherited properties of
red blood cells (erythrocytes) as determined by the presence or absence of the antigens A and B,
which are carried on the surface of the red cells. Persons may thus have type A, type B, type O,
or type AB blood. The A, B, and O blood groups were first identified by Austrian
immunologist Karl Landsteiner in 1901.
Blood containing red cells with type A antigen on their surface has in its serum (fluid) antibodies
against type B red cells. If, in transfusion, type B blood is injected into persons with type A
blood, the red cells in the injected blood will be destroyed by the antibodies in the recipient’s
blood. In the same way, type A red cells will be destroyed by anti-A antibodies in type B blood.
Type O blood can be injected into persons with type A, B, or O blood unless there is
incompatibility with respect to some other blood group system also present. Persons with type
AB blood can receive type A, B, or O blood.

Objectives:
At the end of this lesson, you should be able to:
1. Discuss the ABO Blood System
2. Understand the antibodies of the ABO Blood System
3. Discuss the Inheritance of the ABO Blood Group
4. Discuss the practical significance of the ABO blood group in blood transfusion
5. Discuss the Rh and Bombay blood group
6. Narrate the history of blood transfusion
7. Explain Landsteiner’s Law
8. Discuss the procedures and the principles of reverse and forward Blood typing.
9. Discuss the agglutination process in transfusion reactions
10. Discuss the medico-legal importance of the ABO blood group

Definition of Terms:
Blood Type- The specific reaction pattern of red blood cells of an individual to the antisera of
one blood group as, for example, of the ABO blood group, which consists of four major
blood types, O, A, B, and AB.
Antigen- any substance that can stimulate the production of antibodies and combine specifically
with them.
Antibody- a blood protein produced in response to and counteracting a specific antigen,
chemically combine with substances which the body recognizes as alien, such as
bacteria, viruses, and foreign substances in the blood.
Blood Typing- the testing of a sample of blood to determine an individual's blood group.
Cross-Matching- test the compatibility of a donor's and a recipient's blood.
Blood Transfusion- the process of transferring the blood of a person into the veins of another
person.
Agglutination- is the clumping of particles, the process that occurs if an antigen is mixed
with its corresponding antibody.
Medico-legal- relating to, or concerned with both medicine and law, as when medical testing
or examination is undertaken for a legal purpose.

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BLOOD TYPE
The surfaces of erythrocytes contain a genetically determined assortment of antigens
composed of glycoproteins and glycolipids. These antigens, called agglutinogens, occur in
characteristic combinations. Based on the presence or absence of various antigens, blood is
categorized into different blood groups. Within a given blood group, there may be two or more
different blood types. There are at least 29 blood groups and more than 100 antigens that can be
detected on the surface of red blood cells. Here in this module we discuss major blood groups—
ABO and other blood groups.

ABO Blood Group

The ABO blood group is based on two glycolipid antigens called A and B (Figure 19.12).
 People whose RBCs display only antigen A have type A blood.
 Those who have only antigen B are type B.
 Individuals who have both A and B antigens are type AB;
 Those who have neither antigen A nor B are type O.

Blood plasma usually contains antibodies called agglutinins that react with the A or B
antigens if the two are mixed. These are the anti-A antibody, which reacts with antigen A, and
the anti-B antibody, which reacts with antigen B. The antibodies present in each of the four
blood types are shown in Figure 19.12.

You do not have antibodies that react with the antigens of your own RBCs, but you do
have antibodies for any antigens that your RBCs lack. For example, if your blood type is B, you
have B antigens on your red blood cells, and you have anti-A antibodies in your blood plasma.
Although agglutinins start to appear in the blood within a few months after birth, the reason for
their presence is not clear. Perhaps they are formed in response to bacteria that normally inhabit
the gastrointestinal tract. Because the antibodies are large IgM-type antibodies (see Table 22.3)
that do not cross the placenta, ABO incompatibility

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Antigens of the ABO blood group
Number of 4: A, B, AB, and A1
antigens

Antigen Carbohydrate
specificity The sequence of oligosaccharides determines whether the antigen is A, B, or A1.
Antigen- Glycoproteins and glycolipids of unknown function
carrying The ABO blood group antigens are attached to oligosaccharide chains that project
molecules above the RBC surface. These chains are attached to proteins and lipids that lie in
the RBC membrane.
Molecular basis The ABO gene indirectly encodes the ABO blood group antigens.
The ABO locus has three main allelic forms: A, B, and O. The A and B alleles each
encode a glycosyltransferase that catalyzes the final step in the synthesis of the A
and B antigen, respectively. The A/B polymorphism arises from several SNPs in
the ABO gene, which result in A and B transferases that differ by four amino acids.
The O allele encodes an inactive glycosyltransferase that leaves the ABO antigen
precursor (the H antigen) unmodified.
Frequency of A: 43% Caucasians, 27% Blacks, 28% Asians
ABO blood B: 9% Caucasians, 20% Blacks, 27% Asians
group antigens A1: 34% Caucasians, 19% Blacks, 27% Asians
Note: Does not include AB blood groups
Frequency of Blood group O is the most common phenotype in most populations.
ABO Caucasians: group O, 44%; A1, 33%; A2, 10%; B, 9%; A1B, 3%; A2B, 1%
phenotypes Blacks: group O, 49%; A1, 19%; A2, 8%; B, 20%; A1B, 3%; A2B, 1%
Asians: group O, 43%; A1, 27%; A2, rare; B, 25%; A1B, 5%; A2B, rare
Note: Blood group A is divided into two main phenotypes, A1 and A2 (1).

Antibodies produced against ABO blood group antigens

Antibody type IgG and IgM
Naturally occurring. Anti-A is found in the serum of people with
blood groups O and B. Anti-B is found in the serum of people with
blood groups O and A.
Antibody reactivity Capable of hemolysis
Anti-A and anti-B bind to RBCs and activate the complement cascade,
which lyses the RBCs while they are still in the circulation (intravascular
hemolysis).
Transfusion reaction Typically causes an acute hemolytic transfusion reaction
Most deaths caused by blood transfusion are the result of transfusing
ABO-incompatible blood.
Hemolytic disease No or mild disease
of the newborn HDN may occur if a group O mother has more than one pregnancy with a
child with blood group A, B, or AB. Most cases are mild and do not
require treatment.

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History
At the beginning of the 20th century an Austrian scientist, Karl Landsteiner,
noted that the RBCs of some individuals were agglutinated by the serum from
other individuals. He made a note of the patterns of agglutination and showed
that blood could be divided into groups. This marked the discovery of the first
blood group system, ABO, and earned Landsteiner a Nobel Prize.
Landsteiner explained that the reactions between the RBCs and serum were
related to the presence of markers (antigens) on the RBCs and antibodies in the
serum. Agglutination occurred when the RBC antigens were bound by the
antibodies in the serum. He called the antigens A and B, and depending upon which antigen the
RBC expressed, blood either belonged to blood group A or blood group B. A third blood group
contained RBCs that reacted as if they lacked the properties of A and B, and this group was later
called "O" after the German word "Ohne", which means "without". The following year the fourth
blood group, AB, was added to the ABO blood group system. These RBCs expressed both A and
B antigens.
In 1910, scientists proved that the RBCs antigens were inherited, and that the A and B
antigens were inherited codominantly over O. There was initially some confusion over how a
person's blood type was determined, but the puzzle was solved in 1924 by Bernstein's "three
allele model".
The ABO blood group antigens are encoded by one genetic locus, the ABO locus, which
has three alternative (allelic) forms—A, B, and O. A child receives one of the three alleles from
each parent, giving rise to six possible genotypes and four possible blood types (phenotypes).

Nomenclature
 Number of ABO blood group antigens: 4
 ISBT symbol: ABO
 ISBT number: 001
 Gene symbol: ABO
 Gene name: ABO blood group (A transferase, α1,3-N-acetylgalactosaminyltransferase;
B transferase, α1,3-galactosyltransferase)

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Basic biochemistry
ABO phenotypes
The four basic ABO phenotypes are O, A, B, and AB. After it was found that blood
group A RBCs reacted differently to a particular antibody (later called anti-A1), the blood group
was divided into two phenotypes, A1 and A2. RBCs with the A1 phenotype react with anti-A1
and make up about 80% of blood type A. RBCs with the A2 phenotype do not react with anti-A1
and they make up about 20% of blood type A. A1 red cells express about 5 times more A antigen
than A2 red cells, but both types of red cell react with anti-A, and as far as transfusion purposes
are concerned, the A1 and A2 blood groups are interchangeable.
There are many other subgroups of blood group A in which RBCs tend to weakly express
the A antigen, whereas weak variants of the blood group B phenotype are rare
The immune system forms antibodies against whichever ABO blood group antigens
are not found on the individual's RBCs. Thus, a group A individual will have anti-B antibodies
and a group B individual will have anti-A antibodies. Blood group O is common, and individuals
with this blood type will have both anti-A and anti-B in their serum. Blood group AB is the least
common, and these individuals will have neither anti-A nor anti-B in their serum.

ABO antibodies in the serum are

formed naturally. Their production is
stimulated when the immune system
encounters the "missing" ABO blood group
antigens in foods or in micro-organisms. This
happens at an early age because sugars that
are identical to, or very similar to, the ABO
blood group antigens are found throughout
nature.
The ABO locus has three main alleleic
forms: A, B, and O. The A allele encodes a
glycosyltransferase that produces the A
antigen (N-acetylgalactosamine is its
immunodominant sugar), and the B allele
encodes a glycosyltransferase that creates
the B antigen (D-galactose is its
immunodominant sugar). Structures of the A, B, and O antigens

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 The O allele encodes an enzyme with no function, and therefore neither A or B
antigen is produced, leaving the underlying precursor (the H antigen) unchanged. These
antigens are incorporated into one of four types of oligosaccharide chain, type 2 being the most
common in the antigen-carrying molecules in RBC membranes. Some of the other enzymes
involved in the earlier stages of ABO antigen synthesis are also involved in producing antigens
of the Hh blood group and the Lewis blood group.
Expression
Although the ABO blood group antigens are regarded as RBC antigens, they are actually
expressed on a wide variety of human tissues and are present on most epithelial and endothelial
cells.
Each human RBC expresses about 2 million ABO blood group antigens. Other blood
cells, such as T cells, B cells, and platelets, have ABO blood group antigens that have been
adsorbed from the plasma. In individuals who are "secretors", a soluble form of the ABO blood
group antigens is found in saliva and in all bodily fluids except for the cerebrospinal fluid.
A number of illnesses may alter a person's ABO phenotype. Patients can "acquire" the B
antigen during a necrotizing infection during which bacteria release an enzyme into the
circulation that converts the A1 antigen into a B-like antigen. During this time, patients should
not receive blood products that contain the B antigen because their sera will still contain anti-B.
Once the underlying infection is treated, the patients' blood groups return to normal.
Illness can also cause patients to "lose" ABO blood group antigens. Any disease that
increases the body's demand for RBCs may weaken the expression of ABO blood group
antigens, e.g., thalassemia. In addition, ABO blood group antigens can be altered by
hematological cancers that can modify the sugar chains that bear the ABO blood group antigens,
lending to the use of the A and B antigens as tumor markers for acute leukemia,
myeloproliferative disorders, and myelodysplasia.

Function of the A and B antigens

The functions of the ABO blood group antigens are not known. Individuals who lack the
A and B antigens are healthy, suggesting that any function the antigens have is not important, at
least not in modern times.

Diseases associated with ABO blood group antigens

No diseases are known to result from the lack of expression of ABO blood group
antigens, but the susceptibility to a number of diseases has been linked with a person's ABO
phenotype. Such correlations remain controversial and include the observation that gastric cancer
appears to be more common in group A individuals whereas gastric and duodenal ulcers occur
more often in group O individuals.
A clear correlation has been established between the ABO phenotype and the level of two
proteins involved in blood clotting; factor VII (FVIII) and von Willebrand factor (vWF). Blood
group O individuals have about 25% less FVIII and vWF in their plasma. It is well established
that low levels of FVIII and vWF are a cause of excess bleeding, and therefore it may also be the
case that increased levels make clotting more likely, increasing the risk of both arterial (ischemic
heart disease) and venous (thromboembolic disease) problems. Indeed, non-group O individuals
have been shown to be at an increased risk of both arterial and venous disease.

Clinical significance of ABO antibodies

ABO antibodies are of major clinical significance for two reasons: they are naturally
occurring and are found universally, and, they are highly reactive.

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Transfusion reactions
The routine practice of blood typing and cross matching blood products should prevent
adverse transfusion reactions caused by ABO antibodies. However, clerical error can result in
"the wrong blood" being transfused into a patient, an error which can result in the death of the
patient .
If a recipient who has blood group O is transfused with non-group O RBCs, the naturally
occurring anti-A and anti-B in the recipient's serum binds to their corresponding antigens on the
transfused RBCs. These antibodies fix complement and cause rapid intravascular hemolysis,
triggering an acute hemolytic transfusion reaction that can cause disseminated intravascular
coagulation, shock, acute renal failure, and death.
Anti-A1 is a less significant cause of transfusion reactions and does not appear to fix
complement.

Hemolytic disease of the newborn

Most cases of hemolytic disease of the newborn (HDN) that arise from an ABO
incompatibility require no treatment. Cases of severe hemolysis that require exchange
transfusions are less common, and fetal hydrops is rare.
HDN caused by ABO antibodies occurs almost exclusively in infants of blood group A or
B who are born to group O mothers. This is because the anti-A and anti-B formed in group O
individuals tend to be of the IgG type (and therefore can cross the placenta), whereas the anti-A
and anti-B found in the serum of group B and A individuals, respectively, tends to be of the IgM
type. Although uncommon, cases of HDN have been reported in infants born to mothers with
blood group A2 and blood group B.
HDN tends to be relatively mild in nature mainly because fetal RBCs don't express adult
levels of A and B antigens. However, the strength of fetal ABO blood group antigens can vary,
and therefore the degree of hemolysis and hence the severity of HDN can be unpredictable. Early
studies suggested that the race of a neonate was a risk factor for developing ABO HDN.
However, later studies showed that the prevalence of disease that required treatment did not
differ significantly among Asian, Black, Hispanic, and Caucasian infants.

Molecular information
Gene
The ABO locus encodes specific glycosyltransferases that synthesize A and B antigens on RBCs.
For A/B antigen synthesis to occur, a precursor called the H antigen must be present. In RBCs,
the enzyme that synthesizes the H antigen is encoded by the H locus (FUT1). In saliva and other
bodily secretions, the enzyme that synthesizes the H antigen is encoded by the Se locus (FUT2).
The ABO locus
The ABO locus is located on chromosome 9 at 9q34.1-q34.2. It contains 7 exons that span more
than 18 kb of genomic DNA. Exon 7 is the largest and contains most of the coding sequence.
Exon 6 contains the deletion that is found in most O alleles and results in a loss of enzymatic
activity.
The A and B alleles differ from each other by seven nucleotide substitutions, four of which
translate into different amino acids in the gene product (R176G, G235S, L266M, G268A). The
residues at positions 266 and 268 determine the A or B specificity of the glycosyltransferase they
encode.
The O allele differs from the A allele by deletion of guanine at position 261. The deletion
causes a frameshift and results in translation of an almost entirely different protein that lacks
enzymatic activity.

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 There are many variant ABO alleles that encode a number of variant ABO phenotypes,
but they do not encode specific antigens other than the A and B antigens. For example, weak A
subgroups, such as A3, Ax, and Ael, express the A antigen, and weak B subgroups, such as B3 and
Bx, express the B antigen.

The H locus (FUT1)

The H locus is located on chromosome 19 at 19q13.3. It contains three exons that span
more than 5 kb of genomic DNA, and it encodes a fucosyltransferase that produces the H antigen
on RBCs.
Individuals who are homozygous for null alleles at this locus (h/h) do not produce H
antigen, and because the H antigen is an essential precursor to the ABO blood group antigens,
they cannot produce A and B antigens. Therefore, their serum contains anti-A and anti-B, in
addition to potent anti-H. This rare phenotype of H-deficient RBCs is called the "Bombay
phenotype" (Oh) after the city in which it was first discovered. Individuals with the Bombay
phenotype are healthy, but if they ever needed a blood transfusion, the antibodies in their serum
would place them at a high risk of having an acute hemolytic transfusion reaction. This can be
avoided by using only blood products from a donor who also has the Bombay phenotype (usually
a relative).

The Se locus (FUT2)

The Se locus is located on chromosome 19 at 19q13.3. It contains two exons that span
about 25 kb of genomic DNA.
The Se locus encodes a specific fucosyltransferase that is expressed in the epithelia of
secretory tissues, such as salivary glands, the gastrointestinal tract, and the respiratory tract. The
enzyme it encodes catalyzes the production of H antigen in bodily secretions.
"Secretors" have at least one copy of the Se gene that encodes a functional enzyme—their
genotype is Se/Se or Se/se. They secrete H antigen which, depending on their ABO genotype, is
then processed into A and/or B antigens.
Non-secretors are homozygous for null alleles at this locus (se/se). They are unable to
produce a soluble form of H antigen and hence do not produce A and B antigens.

Paternity testing and Medico Legal Importance of ABO Blood Group

Although blood group studies cannot be used to prove paternity, they can
provide unequivocal evidence that a male is not the father of a particular child. Since the
red cell antigens are inherited as dominant traits, a child cannot have a blood group antigen that
is not present in one or both parents.

For example, if the child in question belongs to group A and both the mother and
the putative father are group O, the man is excluded from paternity. The table shows the
phenotypes (observed characters) of the offspring that can and cannot be produced in the matings
on the ABO system, considering only the three alleles (alternative genes) A, B, and O. Similar
inheritance patterns are seen in all blood group systems. Furthermore, if one parent is genetically
homozygous for a particular antigen—that is, has inherited the gene for it from both the
grandfather and grandmother of the child—then that antigen must appear in the blood of the
child. For example, on the MN system, a father whose phenotype is M and
whose genotype is MM (in other words, a man who is of blood type M and has inherited the
characteristic from both parents) will transmit an M allele to all his progeny.

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 Exclusions of paternity on the ABO system

Matings possible children impossible children

O×O O A, B, AB

O×A O, A B, AB

O×B O, B A, AB

O × AB A, B O, AB

A×A O, A B, AB

A×B O, A, B, AB

A × AB A, B, AB O

B×B O, B A, AB

B × AB A, B, AB O

AB × AB A, B, AB O
In medicolegal work it is important that the blood samples are properly identified. By
using multiple red cell antigen systems and adding additional studies on other blood types (HLA
[human leukocyte antigen], red cell enzymes, and plasma proteins) and DNA testing, it is
possible to state with a high degree of statistical certainty that a particular male is the father.

Blood Groups And Disease

In some cases an increased incidence of a particular antigen seems to be associated with a
certain disease. Stomach cancer is more common in people of group A than in those of groups O
and B. Duodenal ulceration is more common in nonsecretors of ABH substances than in
secretors. For practical purposes, however, these statistical correlations are unimportant. There
are other examples that illustrate the importance of blood groups to the normal functions of red
cells.
In persons who lack all Rh antigens, red cells of altered shape (stomatocytes) and a mild
compensated hemolytic anemia are present. The McLeod phenotype (weak Kell antigens and no
Kx antigen) is associated with acanthocytosis (a condition in which red cells have thorny
projections) and a compensated hemolytic anemia. There is evidence that Duffy-negative human
red cells are resistant to infection by Plasmodium knowlesi, a simian malaria parasite. Other
studies indicate that P. falciparum receptors may reside on glycophorin A and may be related to
the Wrb antigen.
Blood group incompatibility between mother and child can cause erythroblastosis
fetalis (hemolytic disease of the newborn). In this disease IgG blood group antibody molecules
cross the placenta, enter the fetal circulation, react with the fetal red cells, and destroy them.
Only certain blood group systems cause erythroblastosis fetalis, and the severity of the disease in
the fetus varies greatly. ABO incompatibility usually leads to mild disease. Rh, or D antigen,
incompatibility is now largely preventable by treating Rh-negative mothers with Rh
immunoglobulin, which prevents immunization (forming antibodies) to the D antigen. Many
other Rh antigens, as well as other red cell group antigens, cause erythroblastosis fetalis. The
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baby may be anemic at birth, which can be treated by transfusion with antigen-negative red cells.
Even total exchange transfusion may be necessary. In some cases, transfusions may be given
while the fetus is still within the uterus (intrauterine transfusion). Hyperbilirubinemia (an
increased amount of bilirubin, a breakdown product of hemoglobin, in the blood) may lead to
neurological deficits. Exchange transfusion eliminates most of the hemolysis by providing red
cells, which do not react with the antibody. It also decreases the amount of antibody and allows
the child to recover from the disease. Once the antibody disappears, the child’s own red cells
survive normally.

Rh Blood Group
The Rh blood group is so named because the Rh antigen, called Rh factor, was first
found in the blood of the Rhesus monkey. The alleles of three genes may code for the Rh
antigen. People whose RBCs have Rh antigens are designated Rh+ (Rh positive); those who lack
Rh antigens are designated Rh- (Rh negative). Table 19.5 shows the incidence of Rh+ and Rh- in
various populations.
Normally, blood plasma does not contain anti-Rh antibodies. If an Rh- person receives an
Rh+ blood transfusion, however, the immune system starts to make anti-Rh antibodies that will
remain in the blood. If a second transfusion of Rh+ blood is given later, the previously formed
anti-Rh antibodies will cause agglutination and hemolysis of the RBCs

The most common problem with Rh incompatibility, hemolytic disease of the newborn
(HDN), may arise during pregnancy (Figure 19.13). Normally, no direct contact occurs between
maternal and fetal blood while a woman is pregnant. However, if a small amount of Rh_ blood
leaks from the fetus through the placenta into the bloodstream of an Rh_ mother, the mother will
start to make anti-Rh antibodies. Because the greatest possibility of fetal blood leakage into the
maternal circulation occurs at delivery, the firstborn baby usually is not affected. If the mother
becomes pregnant again, however, her anti-Rh antibodies can cross the placenta and enter the
bloodstream of the fetus. If the fetus is Rh_, there is no problem, because Rh_ blood does not
have the Rh antigen. If the fetus is Rh_, however, agglutination and hemolysis brought on by
fetal–maternal incompatibility may occur in the fetal blood.
An injection of anti-Rh antibodies called anti-Rh gamma globulin (RhoGAM®) can be
given to prevent HDN. Rh_ women should receive RhoGAM® before delivery, and soon after
every delivery, miscarriage, or abortion. These antibodies bind to and inactivate the fetal Rh
antigens before the mother’s immune system can respond to the foreign antigens by producing
her own anti-Rh antibodies. •

Hemolytic Disease of Newborn

Figure 19.13 Development of hemolytic disease of the newborn (HDN).
(a) At birth, a small quantity of fetal blood usually leaks across the placenta into the maternal
bloodstream. A problem can arise when the mother is Rh_ and the baby is Rh_, having inherited
an allele for the Rh antigens from the father. (b) On exposure to Rh antigen, the mother’s
immune system responds by making anti-Rh antibodies. (c) During a subsequent pregnancy, the
maternal antibodies cross the placenta into the fetal blood. If the second fetus is Rh_, the ensuing
antigen–antibody reaction causes agglutination and hemolysis of fetal RBCs. The result is HDN.
CLINICAL CONNECTION |

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Background information
In Bombay, India, an individual was discovered to have an interesting blood type that
reacted to other blood types in a way that had not been seen before. Serum from this individual
contained antibodies that reacted with all RBCs from normal ABO phenotypes (i.e., groups O, A,
B, and AB). The individual's RBCs appeared to lack all of the ABO blood group antigens plus an
additional antigen that was previously unknown.
In 1952, a paper about the "new blood group character related to the ABO blood group"
was published (2). This new blood group character is the H antigen and it is the building block
for the antigens of the ABO blood group.
Named for the city in which it was first discovered, the "Bombay phenotype" describes
individuals whose RBCs lack the H antigen. Because the A and B antigens cannot be formed
without the H antigen precursor, their RBCs also lack these antigens. As a result, these
individuals produce anti-H, anti-A, and anti-B and can therefore be transfused only with RBCs
that also lacks the H, A, and B antigens i.e., they can only receive blood from another person
with the Bombay phenotype. Because of the rarity of this blood type, this normally means using
blood donations from a suitable relative.

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Nomenclature
 Number of H antigens: 1
 ISBT symbol: H
 ISBT number: 018
 Gene symbol: FUT1
 Gene name: Fucosyltransferase 1

Basic biochemistry
The biosynthesis of the H antigen and the A and B antigens involves a series of enzymes
(glycosyltransferases) that transfer monosaccharides. The resulting antigens are oligosaccharide
chains, which are attached to lipids and proteins that are anchored in the RBC membrane.
The H antigen is produced by a specific fucosyltransferase. Depending upon a person's ABO
blood type, the H antigen is converted into either the A antigen, B antigen, or both. If a person
has blood group O, the H antigen remains unmodified. Therefore, the H antigen is present in the
highest amounts in blood type O and in the least amounts in blood type AB.
Two regions of the genome encode two enzymes with very similar substrate specificities—the H
locus (FUT1) and the Se locus (FUT2).
The H locus contains the FUT1 gene, which is expressed in RBCs. At least one
functioning copy of FUT1 needs to be present (H/H or H/h) for the H antigen to be produced on
RBCs. If both copies of FUT1 are inactive (h/h), the Bombay phenotype results.
The Se locus contains the FUT2 gene, which is expressed in secretory glands. Individuals who
are "secretors" (Se/Se or Se/se) contain at least one copy of a functioning enzyme. They produce
a soluble form of H antigen that is found in saliva and other bodily fluids. "Non-secretors"
(se/se) do not produce soluble H antigen. The enzyme encoded by FUT2 is also involved in the
synthesis of antigens of the Lewis blood group.

Molecular information
The H blood group locus (containing FUT1) and the secretor locus (containing FUT2) are
located on chromosome 19 at q.13.3. FUT1 and FUT2 are tightly linked, being only 35 kb apart.
Because they are highly homologous, they are likely to have been the result of a gene duplication
of a common gene ancestor.
The H locus contains four exons that span more than 8 kb of genomic DNA. Both the Bombay
and para-Bombay phenotypes are the result of point mutations in the FUT1 gene
The classical Bombay phenotype is caused by a Tyr316Ter mutation in the coding region of
FUT1 The mutation introduces a stop codon, resulting in a truncated enzyme that lacks 50 amino
acids at the C-terminal end, rendering the enzyme inactive. In Caucasians, the Bombay
phenotype may be caused by a number of mutations Likewise, a number of mutations have been
reported to underlie the para-Bombay phenotype

Common H phenotypes
The two common H phenotypes are "secretor" and "non-secretor".
Secretor (common)
 H antigen is expressed on RBCs.
 H antigen is expressed in saliva.
 No anti-H is produced.
 Genotype: H/H or H/h; Se/Se or Se/se
Non secretor (common)
 H antigen is present on RBCs.
 H antigen is absent from saliva.
 No anti-H is produced.
 Genotype: H/H or H/h; se/se

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Uncommon H Phenotypes
The Bombay phenotype and para-Bombay phenotype are relatively rare. In India, where
H deficiency was first discovered, the frequency of both phenotypes combined is 1 in 10,000 . H
deficiency is slightly more common in Taiwan, affecting 1 of 8,000 people A relatively large
number of H-deficient individuals were found on Reunion Island, which is a small French Island
800 km east of Madagascar in the Indian Ocean . Both the classical Bombay phenotype and a
new variant type of partial H deficiency was seen in the islanders In Europe, 1 per million people
are H deficient.

Bombay phenotype
 H antigen is not expressed on RBCs.
 H antigen is not found in saliva.
 Serum contains anti-H.
 Genotype: h/h se/se
Para-Bombay phenotype
 H antigen is weakly expressed on RBCs.
 H antigen may be present or absent in saliva.
 Serum contains anti-H.
 Genotype: (H), Se/Se or Se/se or se/se

Expression of the H antigen

The H antigen shares the same broad tissue distribution as the A and B antigens.
Likewise, in individuals who are "secretors", a soluble form of the H antigen is found in saliva
and all fluids except cerebrospinal fluid.
Function of the H antigen
The function of the H antigen, apart from being an intermediate substrate in the synthesis
of ABO blood group antigens, is not known although it may be involved in cell adhesion (5).
People who lack the H antigen do not suffer any deleterious effects, and being H-deficient is
only an issue if they were to need a blood transfusion because they would require H-deficient
blood.

Clinical significance of H antibodies:

Transfusion reactions
If patients with anti-H in their circulation receive transfusions of blood that contains the
H antigen (e.g., blood group O), they are at risk of suffering an acute hemolytic transfusion
reaction.

Hemolytic disease of the newborn

In theory, the maternal production of anti-H during pregnancy could cause hemolytic disease in a
fetus who did not inherit the mother's Bombay phenotype. In practice, cases of HDN caused in
this way have not been described, possibly because of the rarity of the Bombay phenotype.

History of Blood Transfusion

The history of blood transfusion can be traced back in 1928 that originated with William
Harvey’s discovery of blood circulation. The first and earliest recorded blood transfusion
occurred in 1665, and was performed by Dr. Philip Syng Physick in 1795. The first transfusion
for the treatment of hemorrhage was performed by Dr. James Blundell in London in 1818. The
first Blood bank was established in United States at Chicago’s Cook Country hospital in 1937.

Page 14
The technology making transfusion of allogenic blood products feasible that includes Karl
Landsteiner’s landmark identification of the human blood groups A,B, and O in 1901. Alfred
von Decastello and Adriano Sturii both Landsteiner's students added the fourth group AB in
1902. In 1940 Karl Landsteiner and A.S. Weiner identified Rh antigens and the most common
RhD, causes the most severe immune reaction. The Bombay blood type was first discovered by
Doctor YM Bhende when it was found in some people of Bombay, India. It is also known as HH
blood type or rare ABO blood group.

Transfusions
Blood is the most easily shared of human tissues, saving many thousands of lives every
year through transfusions despite the differences in RBC antigens reflected in the blood group
systems.
A transfusion is the transfer of whole blood or blood components (red blood cells only
or blood plasma only) into the bloodstream or directly into the red bone marrow. A transfusion is
most often given to alleviate anemia, to increase blood volume (for example, after a severe
hemorrhage), or to improve immunity. The normal components of one person’s RBC plasma
membrane can trigger damaging antigen– antibody responses in a transfusion recipient. In an
incompatible blood transfusion, antibodies in the recipient’s plasma bind to the antigens on the
donated RBCs, which causes agglutination or clumping, of the RBCs. Agglutination is an
antigen–antibody response in which RBCs become cross-linked to one another. (agglutination is
not the same as blood clotting.) When these antigen–antibody complexes form, they activate
plasma proteins of the complement family. In essence, complement molecules make the plasma
membrane of the donated RBCs leaky, causing hemolysis or rupture of the RBCs and the release
of hemoglobin into the blood plasma. The liberated hemoglobin may cause kidney damage by
clogging the filtration membranes. It is possible for the viruses that cause AIDS and hepatitis B
and C to be transmitted through transfusion of contaminated blood products although quite rare.
Consider what happens if a person with type A blood receives a transfusion of type B
blood. The recipient’s blood (type A) contains A antigens on the red blood cells and anti-B
antibodies in the plasma. The donor’s blood (type B) contains B antigens and anti-A antibodies.

In this situation, two things can happen.

1. First, the anti-B antibodies in the recipient’s plasma can bind to the B antigens on the
donor’s erythrocytes, causing agglutination and hemolysis of the red blood cells.
2. Second, the anti-A antibodies in the donor’s plasma can bind to the A antigens on the
recipient’s red blood cells, a less serious reaction because the donor’s anti-A antibodies
become so diluted in the recipient’s plasma that they do not cause significant
agglutination and hemolysis of the recipient’s RBCs.

People with type AB blood do not have anti-A or anti-B antibodies in their blood plasma.
They are called universal recipients because theoretically they can receive blood from donors of
all four blood types. They have no antibodies to attack antigens on donated RBCs. People with
type O blood have neither A nor B antigens on their RBCs and are sometimes called universal
donors because theoretically they can donate blood to all four ABO blood types. Type O persons
requiring blood may receive only type O blood (Table 19.6).
In practice, use of the terms universal recipient and universal donor is misleading and
dangerous. Blood contains antigens and antibodies other than those associated with the ABO
system that can cause transfusion problems. Thus, blood should be carefully cross-matched or
screened before transfusion. In about 80% of the population, soluble antigens of the ABO type
appear in saliva and other body fluids, in which case blood type can be identified from a sample
of saliva.
Page 15
 SAQ # 1. What are the main ABO blood types? How does each blood type differ from other blood
type in terms of blood typing? (5 points of class standing)
ASAQ#1________________________________________________________________
SAQ #2. Discuss the basic biochemistry of each blood type. (5 points of class standing)
ASAQ#2________________________________________________________________

Blood Typing and Cross-Matching Blood for Transfusion

Medical laboratory technologist type the patient’s blood and then either cross-match it to
potential donor blood or screen it for the presence of antibodies to avoid blood-type mismatches.
The procedure for ABO blood typing, drops of blood are mixed with different antisera, solutions
that contain antibodies (Figure 19.14). One drop of blood is mixed with anti-A serum, which
contains anti-A antibodies that will agglutinate red blood cells that possess A antigens. Another
drop is mixed with anti-B serum, which contains anti-B antibodies that will agglutinate red blood
cells that possess B antigens.

 If the red blood cells

agglutinate only when mixed
with anti-A serum,
the blood is type A.
 If the red blood cells
agglutinate only when mixed
with anti- B serum,
the blood is type B.
 The blood is type AB if both
drops agglutinate; if neither
drop agglutinates,
the blood is type O.

The procedure for determining Rh factor, a drop of blood is mixed with antiserum
containing antibodies that will agglutinate RBCs displaying Rh antigens. If the blood
agglutinates, it is Rh+; no agglutination indicates Rh_.
Once the patient’s blood type is known, donor blood of the same ABO and Rh type is
selected. In a cross-match, the possible donor RBCs are mixed with the recipient’s serum. If
agglutination does not occur, the recipient does not have antibodies that will attack the donor
RBCs. Alternatively, the recipient’s serum can be screened against a test panel of RBCs having
antigens known to cause blood transfusion reactions to detect any antibodies that may be present.

Blood Type and Cross Match

To avoid a transfusion reaction, donated blood must be compatible with the blood of the
patient who is receiving the transfusion. More specifically, the donated RBCs must lack the same
ABO and Rh D antigens that the patient's RBCs lack. For example, a patient with blood group A
can receive blood from a donor with blood group A (which lacks the B antigen) or blood group

Page 16
O (which lacks all ABO blood group antigens). However, they cannot receive blood from a
donor with blood group B or AB (which both have the B antigen).
Before a blood transfusion, two blood tests known as a "type and cross match" are done.
First, the recipient's blood type is determined, their ABO type and Rh D status. In theory, once
the recipient's blood type is known, a transfusion of compatible blood can be given. However, in
practice, donor blood may still be incompatible because it contains other antigens that are not
routinely typed but may still cause a problem if the recipient's serum contains antibodies that will
target them. Therefore, a "cross match" is done to ensure that the donor RBCs actually do match
against the recipient's serum.
To perform a cross match, a small amount of the recipient's serum is mixed with a small amount
of the donor RBCs. The mixture is then examined under a microscope. If the proposed
transfusion is incompatible, the donor RBCs are agglutinated by antibodies in the recipient's
serum.

Principle involved in Cross -Matching

Landsteiner’s Law
Landsteiner law states that:
1. If a particular agglutinogen (antigen) is present in the RBCs, corresponding agglutinin
(antibody) must be absent in the serum.
2. If a particular agglutinogen is absent in the RBCs, the corresponding agglutinin mustbe
present in the serum.
The second part of Landsteiner law is a fact, it is not applicable to Rh factor.

PRINCIPLE
Testing with both Anti-A and Anti-B is necessary to determine if red blood cells possess
or lack A and/or B blood group antigens.
 Absence of agglutination is a negative test result, which indicates the corresponding
antigen is not demonstrable.
 Agglutination of red blood cells with a given reagent is a positive test result, which
indicates the presence of the corresponding antigen on the red blood cells. (Forward
Type)
 Direct agglutination of A1 or B reagent red cells with the patient serum/plasma indicates
the presence of the appropriate ABO antibody. (Reverse type)
 Forward and reverse typing will be performed on samples for all patients greater than
four months of age to allow for discovery of subtypes as well as to confirm typing.
Individuals less than four months old may not have developed sufficient antibody to
allow for detection.

TEST TUBE METHOD

Recommended method (Gold standard)
– Allows longer incubation of antigen and antibody mixture without drying
– Tubes can be centrifuged to enhance reaction
– Can detect weaker antigen / antibody

Two steps in ABO grouping

1. Cell grouping (Forward grouping)
– Tests the patients red cells with known Anti-A & Anti- B to determine the antigen expressed
2. Serum grouping (Reverse grouping)
– Test the patients serum with known A & B cells to determine the presence of antibody

Page 17
CELL GROUPING ( Forward grouping)
– Prepare 2-5% suspension of test sample in normal saline
– Set three tubes , label them as A,B, D
– Add two drops of anti A , anti-B, anti D in three different tubes
– Add one drop of 2-5% cell suspension (Ratio of 2:1)
– Mix contents well and centrifuge at 1500 rpm for 1 minute
– Observe for hemolysis
– Gently disperse cell button and check for agglutination
– Confirm negative results under microscope

SERUM GROUPING ( REVERSE GROUPING)

– Prepare 2-5% suspension of pooled cells A,B,O
– Label three tubes A cells, B cells and O cells
– Place two drops of serum in each tube
– Add one drop of cell suspension ( A cell to A tube, B cell to B tube and one drop of O cell to O
tube
– Centrifuge tubes at 1500 rpm for 1 minute
– Gently disperse for agglutination
– Negative results check by microscope

Page 18
 Cross Matching is a procedure performed prior to a blood transfusion to determine
whether donor blood is compatible (or incompatible) with recipient blood.
Compatibility is determined through matching of different blood group systems, the most
important of which are the ABO and Rh system, and/or by directly testing for the presence
of antibodies against a sample of donor tissues or blood.

Purpose of Cross Matching

The crossmatch is routinely used as the final step of pre-transfusion compatibility testing. The
purposes of compatibility testing are to detect: irregular antibodies; errors in ABO grouping, and clerical
errors in patient identification and result recording.

The crossmatch will detect the following:

1. Most recipient antibodies directed against antigens on the donor red blood cells.
2. Major errors in ABO grouping, labeling, and identification of donors and recipients.

Principle
Cross-matching will detect incompatibilities between the donor and recipient that will not be
evident on blood typing. There are two types of cross-matches: Major cross-match and Minor cross-
match.
The major crossmatch involves testing the patient’s serum with donor cells to determine
whether the patient has an antibody which may cause a hemolytic transfusion reaction or decreased cell
survival of donor cells. This is the most important cross-match.
The minor crossmatch involves testing the patients cells with donor plasma to determine
whether there is an antibody in the donor’s plasma directed against an antigen on the patient’s cells.
Procedure
1. Prepare donor and recipient blood samples:
For Major crossmatch : Donor’s red cell and recipient serum or plasma
For Minor crossmatch : Recipient red cells and donor’s serum or plasma
2. Prepare 3 – 5% cell suspensions of red cells.
3. Major Crossmatch:
Label a test tube. Add two drops of the
patient serum and one drop of the
appropriate donor cell suspension.
4. Minor Crossmatch:
Label a test tube. Add two drops of the
appropriate donor serum and one drop of
the patient cell suspension.
5. Mix the tubes and incubate at 37°C for
about 45 minutes.
6. Add two drops of AHG (Antihuman
globulin) and mix well.
7. Centrifuge for 1 minute at 1500 rpm
8. Read macroscopically and microscopically
and record the results

Page 19
Blood transfusions and the immune system
The immune system never rests—its cells constantly patrol the circulation. Without the
immune system, the body would be overwhelmed with infections. With it, blood transfusions
must be performed with great care.
If incompatible blood is given in a transfusion, the donor cells are treated as if they were
foreign invaders, and the patient's immune system attacks them accordingly. Not only is the
blood transfusion rendered useless, but a potentially massive activation of the immune system
and clotting system can cause shock, kidney failure, circulatory collapse, and death.
This chapter discusses the causes of transfusion reactions and how the hazards of blood
transfusions are minimized.

Immune response against transfused red blood cells

Many of the adverse effects of blood transfusions are mediated by the recipient's immune
system.
In general, the formation of this and other immune responses occur in three stages:
1. Antigen detection- the immune system detects foreign material (antigen)
2. Antigen processing - the immune system processes the antigen
3. Immune response- the immune system mounts a response to remove the antigen from
the body
The immune response varies tremendously, depending on the individual (the health of his
or her immune system and genetic factors) and the antigen (how common it is and how
"provocative" it is to the immune system).

Antigen detection
The red blood cells (RBCs) from one person may enter into the circulation of another
person in two different ways, either by a blood transfusion or by pregnancy. The RBCs will
appear foreign if they contain antigens that are not found on the patient's own RBCs.

Antigen processing
When the macrophage encounters an antigen, it engulfs it, digests it, and then presents
the antigenic fragments on its cell surface together with MHCII (Major Histocompatibility
Complex II).
A T helper cell binds to the antigen/MHCII on the macrophage, and the two cells interact.
The macrophage secretes cytokines to stimulate the T cell, which in turn secretes cytokines to
stimulate the growth and production of more T cells.
The T helper cell, now activated, leaves to activate a third type of cell, the B cell.
Existing B cells are stimulated by the T cell to grow, divide, and produce genetically identical
daughter cells. Some of the daughter cells become plasma cells that produce antibodies that are
specific for the antigen that stimulated their production. The amount and type of antibody
produced results from the interaction of T helper cells (which stimulate antibody production) and
T suppressor cells (which inhibit antibody production). Other daughter cells remain as B cells in
the circulation for many years. They serve as "memory cells", remembering the encounter with
the antigen that stimulated their production.

Page 20
Immune response
If this is the first time the antigen has been encountered, a primary immune response is
mounted. Usually there is a delay of several days, then IgM antibody is produced, followed by a
switch to IgG antibody production. The initial IgM molecules bind the antigen weakly, but the
subsequent IgG molecules are much better targeted. IgG continues to be produced long after the
encounter with the antigen, providing long-lasting immunity.
If the immune system has encountered the antigen before, it will already be armed with
primed B cells (memory cells) that accelerate the production of larger amounts of IgG (rather
than IgM). This is called the secondary immune response. It is faster, more specific, and the
production of the specific antibody may remain high for years. B cells may also undergo changes
to further improve how the antibodies they produce bind to the antigen.
There are two main arms of immune response( 1) humoral (using antibodies) and (2)
cellular (using immune cells). Severe immune-mediated transfusion reactions usually involve
the humoral arm. In the case of a foreign red blood cell antigen, the patient's pre-existing
antibodies bind to the antigen, coating the donor RBCs.
Some types of antibody may activate the complement cascade, a series of enzyme-driven
reactions involving protein fragments. The cascade ends with the formation of a "membrane
attack complex", a large molecule that punches a hole in the cell membrane. Other antibodies
simply bind to the donor RBCs and cause them to clump together (agglutinate). The agglutinated
cells may survive or may be prematurely removed from the circulation by the macrophages.
Otherwise, the fate of the incompatible RBCs largely rests in the hands of macrophages
in the liver or the spleen. They remove the antibody-coated cells from the circulation and
phagocytose them. Phagocytosis is aided by the macrophages having a receptor that binds to the
antibodies and another receptor that binds to complement fragments. Therefore, incompatible
RBCs are rapidly destroyed after antibody binding. In addition, this antibody response may cause
dangerous hemolytic transfusion reactions some are described below.

Acute Transfusion Reactions

 Mild allergic: Attributed to hypersensitivity to a foreign protein in the donor product.
 Anaphylactic: Similar to a mild allergic reaction, however resulting in a more severe
reaction. Sometimes this can occur in a patient with IgA deficiency who makes
alloantibodies against IgA and then receives blood products containing IgA.
 Febrile non-hemolytic: Generally thought to be caused by cytokines released from blood
donor leukocytes (white blood cells).
 Septic: Caused by bacteria or bacterial byproducts (such as endotoxin) which may
contaminate blood.
 Acute hemolytic transfusion reactions: Can result in intravascular or extravascular
hemolysis, depending on the specific etiology (cause). Immune-mediated reactions are
often a result of recipient antibodies present to blood donor antigens. Non-immune
reactions are possible, and occur when red blood cells are damaged before transfusion
(e.g., by heat or incorrect osmotic conditions).
 Transfusion-associated circulatory overload (TACO): Occurs when the volume of the
transfused component causes hypervolemia (volume overload).

Page 21
  Transfusion-related acute lung injury: Acute lung injury is due to antibodies in the donor
product (human leukocyte antigen or human neutrophil antigen) reacting with antigens in
the recipient. The recipient’s immune system responds and causes the release of
mediators that lead to pulmonary edema. Possibly contributing to this are clinical
conditions that predispose the patient including infection, recent surgery, or
inflammation.
Delayed Transfusion Reactions
 Delayed hemolytic transfusion reaction: Typically caused by an anamnestic response to a
foreign antigen that the patient was previously exposed to (generally by prior transfusion
or pregnancy).
 Transfusion-associated graft-versus-host disease: Results from engraftment of donor
lymphocytes (commonly found in cellular blood products) into an immunocompromised
recipient’s bone marrow. The donor lymphocytes recognize the patient as foreign and
react against the recipient’s body. The patient’s immune system is unable to clear the
foreign lymphocytes. This is rare but often fatal.

SAQ # 3. Differentiate forward and reverse blood grouping? (5 points of class standing)
ASAQ#3________________________________________________________________
SAQ #4. Discuss the significance of cross matching and effects of transfusion incompatibility or
reactions. (5 points of class standing)
ASAQ#4________________________________________________________________

Summary:

 Antigens usually large proteins, which provoke an immune response. In transfusion

reactions, antibodies attach to antigens on the surfaces of erythrocytes and cause
agglutination and hemolysis. ABO blood group antigens are designated A and B. People
with type A blood have A antigens on their erythrocytes, whereas those with type B
blood have B antigens. Those with AB blood have both A and B antigens, and those with
type O blood have neither A nor B antigens. The blood plasma contains preformed
antibodies against the antigens not present on a person’s erythrocytes.
 A second group of blood antigens is the Rh group, the most important of which is Rh D.
People with Rh− blood do not have this antigen on their erythrocytes, whereas those who
are Rh+ do. About 85 percent of Americans are Rh+. When a woman who is Rh− becomes
pregnant with an Rh+ fetus, her body may begin to produce anti-Rh antibodies. If she
subsequently becomes pregnant with a second Rh+ fetus and is not treated preventively
with RhoGAM, the fetus will be at risk for an antigen-antibody reaction, including
agglutination and hemolysis. This is known as hemolytic disease of the newborn.
 Blood typing and Cross matching to determine blood type is necessary before transfusing
blood, to prevent transfusion reaction that may lead to severe reaction or even death.

Page 22
References:

1. Kevin Patton, Essentials of Anatomy & Physiology 5th Edition, McGraw-Hill

Edition
2. Anatomy and Physiology, Open Stax, Rice University, 2013
3. Hole’s Human Anatomy & Physiology 11th Edition, McGraw-Hill Higher Edition
4. Elaine N. Marieb, Essentials of Human Anatomy and Physiology 10th
Edition,Pearson Education, Inc., 2012
5. Elaine N. Marieb, Human Anatomy& Physiology Laboratory Manual 10th Edition,
Pearson International Edition 2014
6. https://www.ncbi.nlm.nih.gov/books/NBK2265

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