BLOOD GROUP SUBSTANCES - CHEMICAL BASIS OF BLOOD GROUP ANTIGENS (ABO

SYSTEM)
ABO is the original blood group system and the most important clinically. There are two antigens, A and B,
and four phenotypes: A, B, AB, and O. Almost all adults have antibodies against the ABO antigens they lack.
These antibodies can cause severe haemolytic transfusion reactions and hyperacute rejection of solid organ
grafts. The antigens of the ABO, H, and Lewis systems are oligosaccharides on glycoproteins and glycolipids
and are closely related biochemically. They are often referred to as histo-blood group antigens because they
are present in many tissues. A and B antigens are produced by the addition of N-acetylgalactosamine and
galactose, respectively, to a common acceptor substrate, H antigen. This biosynthesis is catalysed by N-
acetylgalactosaminyltransferase or galactosyltransferase, the products of the A and B alleles of ABO.
The O allele produces no active transferase. H antigen is synthesised by an α-1, 2-fucosyltransferase, the
product of FUT1, and Lewis antigens, which are adsorbed onto red cells from the plasma, are synthesised by
an α-1, 3/4-fucosyltransferase, the product of FUT3.

At the end of the lesson, students should be able to:

1. State the chemical basis of blood group antigens (ABO system).
2. Discuss the structure and role of carbohydrate determinants in blood group antigens.
3. Describe the ABO blood group system antigens and antibodies.
4. Review the blood typing methods and reasons why discrepancies in blood typing can occur.
5. Summarize typical scenarios of hemolysis due to ABO incompatibility.
6. Identify appropriate blood products that should be ordered for patients in the context of their ABO
blood type.

ABO Blood Group System

ABO blood group system, is the classification of human blood based on the inherited properties of red blood
cells (erythrocytes) determined by the presence or absence of the antigens A and B, carried on the surface of
the red cells. Persons may therefore 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.

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-carrying Glycoproteins and glycolipids of unknown function
molecules The ABO blood group antigens are attached to oligosaccharide chains that project 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 group B: 9% Caucasians, 20% Blacks, 27% Asians
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 phenotypes Caucasians: group O, 44%; A1, 33%; A2, 10%; B, 9%; A1B, 3%; A2B, 1%
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, A 1 and A2.

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 Yes — typically causes an acute hemolytic transfusion reaction
Most deaths caused by blood transfusion are the result of transfusing ABO-
incompatible blood.
Hemolytic disease of No or mild disease
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.

The ABO and Rh Groups in Transfusion

*Not if the patient's serum contains anti-A1 (antibody to common type A red cell in subgroup A patients).
**Not if the patient is a female less than 45 years old (childbearing possible), unless life-threatening
hemorrhage is present and transfusion of Rh-positive blood is lifesaving.
***Not if the patient's serum contains anti-D (antibody to positive red cells), except under unusual medical
circumstances.
System Recipient Type Donor Red Cell Type Donor Plasma Type
ABO A A* or O A or AB
ABO B B or O B or AB
ABO O O only O, A, B, or AB
ABO AB AB*, A*, B, or O AB
Rh positive positive or negative positive or negative
Rh negative negative or positive**, *** negative or positive**

Blood group O is the most common blood type throughout the world, particularly among peoples of South
and Central America. Type B is prevalent in Asia, especially in northern India. Type A also is common all
over the world; the highest frequency is among Australian Aboriginal peoples, the Blackfoot Indians of
Montana, and the Sami people of northern Scandinavia.
The ABO antigens are developed well before birth and remain throughout life. Children acquire ABO
antibodies passively from their mother before birth, but by three months of age infants are making their own;
it is believed that the stimulus for such antibody formation is from contact with ABO-like antigenic substances
in nature. ABO incompatibility, in which the antigens of a mother and her fetus are different enough to cause
an immune reaction, occurs in a small number of pregnancies. Rarely, ABO incompatibility may give rise to
erythroblastosis fetalis (hemolytic disease of the newborn), a type of anemia in which the red blood cells of
the fetus are destroyed by the maternal immune system. This situation occurs most often when a mother is
type O and her fetus is either type A or type B.
Blood Typing
Blood typing, is classification of blood in terms of distinctive inherited characteristics that are associated with
the antigens located on the surface of red blood cells (erythrocytes). The ABO and the Rh blood groups are
among those most commonly considered. Identification of these determinants has become indispensable in
connection with blood transfusion, because the recipient and donor must have the same, or compatible, blood
groups. Otherwise, hemolysis (destruction) or coagulation (clotting) results from interaction of an antigen on
the red blood cells of one with an antibody in the serum of the other. In addition, blood typing serves to
promptly identify the cause of such disorders as erythroblastosis fetalis (hemolytic disease of the newborn),
which results from blood group incompatibility between mother and fetus. Since blood group determinants
are inherited according to generally known mechanisms of heredity, blood typing sometimes provides a
method for resolving cases of disputed paternity.

THE IMPORTANCE OF ANTIGENS AND ANTIBODIES

The red cells of an individual contain antigens on their surfaces that correspond to their blood group
and antibodies in the serum that identify and combine with the antigen sites on the surfaces of red cells of
another type. The reaction between red cells and corresponding antibodies usually results in clumping—
agglutination—of the red cells; therefore, antigens on the surfaces of these red cells are often referred to
as agglutinogens.
Antibodies are part of the circulating plasma proteins known as immunoglobulins, which are classified by
molecular size and weight and by several other biochemical properties. Most blood group antibodies are found
either on immunoglobulin G (IgG) or immunoglobulin M (IgM) molecules, but occasionally the
immunoglobulin A (IgA) class may exhibit blood group specificity. Naturally occurring antibodies are the
result of immunization by substances in nature that have structures similar to human blood groups. These
antibodies are present in an individual despite the fact that there has been no previous exposure to the
corresponding red cell antigens—for example, anti-A in the plasma of people of blood group B and anti-B in
the plasma of people of blood group A. Immune antibodies are evoked by exposure to the corresponding red
cell antigen. Immunization (i.e., the production of antibodies in response to antigen) against blood group
antigens in humans can occur as a result of pregnancy, blood transfusion, or deliberate immunization. The
combination of pregnancy and transfusion is a particularly potent stimulus. Individual blood group antigens
vary in their antigenic potential; for example, some of the antigens belonging to the Rh and ABO systems are
strongly immunogenic (i.e., capable of inducing antibody formation), whereas the antigens of the Kidd and
Duffy blood group systems are much weaker immunogens.
The blood group antigens are not restricted solely to red cells or even to hematopoietic tissues. The antigens
of the ABO system are widely distributed throughout the tissues and have been unequivocally identified on
platelets and white cells (both lymphocytes and polymorphonuclear leukocytes) and in skin, the epithelial
(lining) cells of the gastrointestinal tract, the kidney, the urinary tract, and the lining of the blood vessels.
Evidence for the presence of the antigens of other blood group systems on cells other than red cells is less
well substantiated. Among the red cell antigens, only those of the ABO system are regarded as tissue antigens
and therefore need to be considered in organ transplantation.

CHEMISTRY OF THE BLOOD GROUP SUBSTANCES

The exact chemical structure of some blood groups has been identified, as have the gene products (i.e., those
molecules synthesized as a result of an inherited genetic code on a gene of a chromosome) that assist in
synthesizing the antigens on the red cell surface that determine the blood type. Blood group antigens are
present on glycolipid and glycoprotein molecules of the red cell membrane. The carbohydrate chains of the
membrane glycolipids are oriented toward the external surface of the red cell membrane and carry antigens of
the ABO, Hh, Ii, and P systems. Glycoproteins, which traverse the red cell membrane, have a polypeptide
backbone to which carbohydrates are attached. An abundant glycoprotein, band 3, contains ABO, Hh, and Ii
antigens. Another integral membrane glycoprotein, glycophorin A, contains large numbers of sialic acid
molecules and MN blood group structures; another, glycophorin B, contains Ss and U antigens.
The genes responsible for inheritance of ABH and Lewis antigens are glycosyltransferases (a group of
enzymes that catalyze the addition of specific sugar residues to the core precursor substance). For example,
the H gene codes for the production of a specific glycosyltransferase that adds l-fucose to a core precursor
substance, resulting in the H antigen; the Le gene codes for the production of a specific glycosyltransferase
that adds l-fucose to the same core precursor substance, but in a different place, forming the Lewis antigen;
the A gene adds N-acetyl-d-galactosamine (H must be present), forming the A antigen; and the B gene adds d-
galactose (H must be present), forming the B antigen. The P system is analogous to the ABH and Lewis blood
groups in the sense that the P antigens are built by the addition of sugars to precursor globoside and
paragloboside glycolipids, and the genes responsible for these antigens must produce glycosyltransferase
enzymes.
The genes that code for MNSs glycoproteins change two amino acids in the sequence of the glycoprotein to
account for different antigen specificities. Additional analysis of red cell membrane glycoproteins has shown
that in some cases the absence of blood group antigens is associated with an absence of minor membrane
glycoproteins that are present normally in antigen-positive persons.

SUGARS IN BLOOD GROUP PROTEINS

There are three blood group antigens in the human, O, A, B, and AB system. Every individual is able to
synthesize the O antigen. The O antigen is a carbohydrate attached to a lipid, the carbohydrate being the
antigenic region. From the lipid outward, the O antigen is a sequence of glucose, galactose, N-
acetylglucosamine, galactose and fucose. Fucose is a methylated pentose. The blood group antigens are
visualized in the figure below.

Carbohydrate constituents attached to proteins and lipids that form the blood group antigens. All humans can
synthesize the O antigen. Type A antigen is characteristic of an individual expressing an enzyme that adds an N-
acetylgalactosamine to the terminal galactose of the O antigen (lower right). The B antigen occurs in an individual
expressing an enzyme that adds another galactose to the terminal galactose of the O antigen (lower left). The AB type
(mixture of both A and B antigens) expresses both enzymes and the O type lacks expression of these enzymes.

These antigens are found on glycoproteins and glycolipids. When they occur on the cell surface, they are
linked to sphingolipids. In the serum, they represent the secreted forms and they are linked to glycoproteins.
Secreters, constituting about 80% of the people in the United States, secrete their blood antigen into various
body fluids, including saliva and mucus. Nonsecreters have low levels of glycoproteins and secrete little or
no blood antigen into body fluids. This distinguishing feature of a secreter versus a nonsecreter can be used
forensically in the courts, especially in cases of rape.

THE ABO BLOOD GROUP SYSTEM

The ABO blood group system was first discovered in the early 1900s, and since that time, the understanding
of the ABO blood group system has increased significantly. These discoveries have led to safer transfusion
practices. The ABO blood group system spans beyond transfusion medicine and has been used in the study of
populations by anthropologists, police in forensic science, and lawyers in paternity claims.
The ABO blood group system is used to denote the presence of one, both, or neither of the A and
B antigens on erythrocytes (red blood cells). For human blood transfusions, it is the most important of the 44
different blood type (or group) classification systems currently recognized. A mismatch in this serotype (or in
various others) can cause a potentially fatal adverse reaction after a transfusion, or an unwanted immune
response to an organ transplant. Such mismatches are rare in modern medicine. The associated anti-A and
anti-B antibodies are usually IgM antibodies, produced in the first years of life by sensitization to
environmental substances such as food, bacteria, and viruses.
The ABO blood types were discovered by Karl Landsteiner in 1901. ABO blood types are also present in
other primates such as apes and monkeys.

ABO blood group antigens present on red blood cells and IgM antibodies present in the serum

Characteristics of the ABO Blood Group

There are four common blood groups in the ABO system: O, A, B, and AB. The blood groups are defined by
the presence of specific carbohydrate sugars on the surface of red blood cells, N-acetylgalactosamine for the
A antigen, and D-galactose for the B antigen. Both of these sugars are built upon the H antigen—if the H
antigen is left unmodified, the resulting blood group is O because neither the A nor the B antigen can attach
to the red blood cells.
Individuals will naturally develop antibodies against the ABO antigens they do not have. For example,
individuals with blood group A will have anti-B antibodies, and individuals with blood group O will have
both anti-A and anti-B. Before a blood transfusion takes place, routine serological testing checks the
compatibility of the ABO (and Rh) blood groups. An ABO incompatible blood transfusion can be fatal, due
to the highly immunogenic nature of the A and B antigens, and the corresponding strongly hemolytic
antibodies.
Compared to other blood groups, individuals with blood group O may have a lower risk of pancreatic cancer
and thromboembolic disease. In addition, in certain African populations, individuals with the blood group O
may be protected from life-threatening malaria. However, this blood group is not more common in some
regions where malaria is endemic. This might be because individuals with blood group O are at higher risk of
cholera and severe diarrhea due to Vibrio cholerae 01, with individuals with the AB blood group being the
most protected.
Over 80 ABO alleles have been reported. The common alleles include A1, A2, B1, O1, O1v, and O2. Whereas
the A and B alleles each encode a specific glycosyl-transferring enzyme, the O allele appears to have no
function. A single-base deletion in the O allele means that individuals with blood group O do not produce
either the A or B antigens. Blood type frequencies vary in different racial/ethnic groups. In the US, in
Caucasians, the ratio of blood group O, A, B, and AB is 45%, 40%, 11%, and 4% respectively. In Hispanics,
the distribution is 57%, 31%, 10%, and 3%; and in Blacks, 50%, 26%, 20%, and 4%.

FUNCTION OF THE ABO BLOOD GROUP

ABO Antigens and Antibodies
People can be divided into four main groups (A, B, O, and AB) based on the agglutination patterns of their
red blood cells. The ABO blood group system consists of A and B antigens on red blood cells and
their corresponding antibodies in the sera of people who do not express those antigens. ABO antigens exist
on the surface of red blood cells as well as the surfaces of the other tissues and secretions. Anti-A and anti-B
antibodies are naturally-occurring and are made by immunocompetent people starting at approximately six
months of age.
The A and B blood group antigens are oligosaccharide antigens generated by reactions catalyzed by
glycosyltransferases. The expression of these antigens determines the blood type of the patient. The human
ABO gene is found on chromosome 9; it spans over 18 kilobases and is made up of 7 coding exons. Functional
A and B alleles encode the A and B glycosyltransferases, leading to the synthesis of A and B antigens. Due to
nucleotide substitutions leading to amino acid substitutions, A and B genes encode the A and B transferases
with different sugar specificities. The O genes are not active because they cannot produce functional enzymes.
Group O individuals have an H gene found on chromosome 19, forming the H-antigen. This antigen serves as
a precursor oligosaccharide needed to form the A and B antigens. H antigens are abundantly noted in type O
individuals. If the A and B genes are both present, then some of the H becomes A, and some become B,
forming the AB group. If the H gene is defective or not present, then the H antigen cannot be formed, and
therefore A or B cannot be formed. The consequence is a rare phenotype known as the Bombay phenotype.
Patients with the Bombay phenotype have antibodies in the plasma similar to those present in the plasma of
type O individuals, but they also have clinically significant anti-H antibodies.
Based on the pattern and degree of agglutination using reference antibodies and red blood cells, subgroups of
ABO have also been identified. Subgroups are distinguished by decreased amounts of A, B, or O (H) antigens
on the red blood cells. Blood type A has the most variation with different subgroups; type A1 has the standard
amount of antigen A, with decreasing amounts of this antigen noted in the subsequent subtypes.

ISSUES OF CONCERN
Discrepancies in Blood Typing
Before a transfusion, ABO typing is routinely performed to provide the safest transfusion possible. Forward
typing (antigen typing for A and B on red cells) and reverse typing (testing for anti-A and anti-B in the patient’s
plasma) are performed. The forward and reverse typing are performed to confirm the blood type. However,
discrepancies between forward and reverse typing may occur. The discordance can be due to a lack or excess
of antigens or antibodies. If a discrepancy is found, the first step is to perform confirmatory assessments to
evaluate for any errors in testing or technical problems. Various clinical scenarios can also explain these
discrepancies.


Diagram showing the carbohydrate chains that determine the ABO blood group


Student blood test. Three drops of blood are mixed with anti-B (left) and anti-A (right) serum. Agglutination
with anti-A suggests this individual is type A.
 
There are three basic variants of immunoglobulin antigens in humans that share a very similar chemical structure but
are distinctly different. Red circles show where there are differences in chemical structure in the antigen-binding site
(sometimes called the antibody-combining site) of human immunoglobulin. Notice the O-type antigen does not have a
binding site.

In forward typing, the patient’s red blood cells are combined with commercial antisera. Weaker RBC
agglutination than expected (weak or missing reactivity) can occur in cases of A or B subgroups. Additional
RBC reactivity on forward typing can occur in cases of acquired B phenomenon. The acquired B phenotype
occurs in cases where bacterial enzymatic changes lead to the conversion of N-acetylgalactosamine (A
antigen) into galactose (B antigen); this phenomenon has been documented in cases of bacterial infections and
colorectal malignancy.
Reverse ABO typing combines a patient’s plasma with commercial red blood cells. Possible causes of weaker
than expected plasma reactivity include immunosuppression, very young or old age,
hypogammaglobulinemia, and prior treatment with rituximab. Unexpected or “extra” reactivity detected in
reverse typing can be seen in patients who have received intravenous immune globulin (IVIG) or non-ABO-
matched plasma products.

Choosing the Safest Blood Products

In the simplest terms, individuals with type O blood are considered “universal donors” for red blood cells, and
type AB patients are “universal recipients” for red cells from patients with any ABO blood type. Type AB
plasma is compatible with all other ABO blood types. There are, however, many caveats and clinical scenarios
to consider when choosing the safest and most appropriate blood products for each patient.
The ABO antigen is fully developed at birth; however, newborns do not start making antibodies until 3 to 6
months of age. The antibodies present in the serum of newborns under four months of age are passively
transferred from the mother. Therefore, when a red cell transfusion is ordered for an infant under four months
of age, the mother’s blood type must be considered. Forward typing is performed to determine a newborn’s
blood type, but reverse typing is not performed in infants in the first few months of life.
Platelets have ABO antigens, but the expression is variable; these antigens are only strongly expressed in a
minority of patients. Platelets are, however, suspended in plasma containing ABO antibodies. The plasma
accompanying the platelets may cause hemolysis if the plasma is not compatible with the recipient’s red blood
cells. In the emergency setting for the bleeding patient requiring blood products, group AB plasma, and group
O-negative red blood cells have been traditionally transfused to avoid ABO incompatibility and avoid delays
while blood typing is being performed. Group AB plasma does not contain Anti-A or Anti-B antibodies and
is therefore compatible with all ABO blood groups. The supply of AB plasma is, however, limited. Newer
approaches in the emergency treatment of patients without a known ABO blood type include transfusion of
type A plasma and low-titer group O whole blood. While transfusion of even a small amount of ABO-
incompatible red blood cells can lead to severe hemolysis and morbidity, clinically significant hemolysis has
not been described in cases of type A plasma or low-titer group O whole blood transfusions into patients for
whom these products are ABO-incompatible.
Clinical Significance
While there is variability in the ABO phenotype frequencies among different ethnic and racial groups, blood
type O is generally the most prevalent worldwide, followed by types A and B, respectively. Type B is more
common in the Asian population. Blood type AB is the rarest of the ABO phenotypes. ABO antigens are not
only found on red blood cells but are also expressed on the surface of many different types of human cells.
The importance of ABO blood type antigens stretches beyond transfusion medicine, as many reports suggest
the involvement of the ABO system in several disease processes. ABO blood groups have been linked with
the susceptibility to various diseases, including hematologic disorders, cancer, infections, and cardiovascular
diseases.
Clinically significant antibodies can lead to adverse events during transfusion and can also lead to hemolytic
disease of the fetus and newborn after placental transmission from mother to fetus. Hemolytic disease of the
newborn (HDN) occurs due to incompatibility between the blood of the mother and the baby. Antibodies from
the mother cross the placenta during pregnancy and attack the baby’s red blood cells. ABO incompatibility
between mother and infant can cause hemolysis and hyperbilirubinemia in the infant. HDN due to ABO
incompatibility tends to be less severe than that caused by Rh compatibility because fetal red cells tend to
express less of the ABO blood group antigens than adults. Also, the ABO blood group antigens are present on
various tissues, which decreases the likelihood that ABO antibodies will bind to their targets on fetal red cells.
The degree of severity of hemolysis varies greatly. HDN can occur with the first pregnancy and has a high
recurrence rate.
A hemolytic transfusion reaction is one type of reaction that can occur with the transfusion of blood products.
Acute hemolytic transfusion reactions most commonly occur with transfusion of red blood cells, although
they can develop with transfusions of other blood products. An acute hemolytic transfusion reaction occurs
within 24 hours of the transfusion. Acute hemolytic reactions are most often caused by incompatibility
between the donor product and recipient blood group system, most commonly the ABO blood group system.
The classic presentation of acute hemolytic transfusion reaction includes fever, red/brown urine, and
back/flank pain. However, not all patients will present in this way; other symptoms that may be noted include
hypotension, chills, renal failure, and disseminated intravascular coagulation.
Once an acute hemolytic transfusion is suspected, the transfusion should be immediately stopped. The most
common cause of death during a transfusion is a clerical error, where an incompatible unit of blood was
transfused, and so clerical error should be ruled-out and ensure the correct blood product was given to the
intended patient. The remaining blood product, along with a sample of blood from the patient, should be sent
to the blood bank for repeat ABO testing on the patient after the reaction. Repeat cross-matching is then
performed, and patient samples should be sent to evaluate for renal failure, hemoglobinemia, hemoglobinuria,
disseminated intravascular coagulation, and hemolysis. Treatment for an acute hemolytic transfusion reaction
is mainly supportive care, and specific treatments are determined by the specific complications noted.

Enhancing Healthcare Team Outcomes

The discovery of the ABO system has led to increased safety in the transfusion of blood products.
Understanding the ABO blood group system is essential to ensure appropriate blood products are administered
to patients. Practitioners should know the appropriate blood products to transfuse based on ABO typing and
recognize typical presentations of ABO incompatibility. Knowledge of the ABO blood system, prevention of
complications with appropriate blood product administration, and prompt identification of signs/symptoms of
ABO incompatibility will improve patient care and decrease morbidity.
From the bedside to the blood bank, the medical team must collaborate to ensure that transfusions are
administered safely to minimize complications. The blood bank team must determine the most appropriate
blood product to release. Communication from the clinical team is necessary to provide clinical background
and information regarding adverse transfusion-related events to the blood bank team. Administering a
transfusion safely can only occur with the teamwork of physicians, advanced care practitioners, nurses, and
technologists in the blood bank.

Diagnosis/testing
Serological testing is sufficient to determine an individual’s blood type (e.g., blood group A) for the purposes
of blood donation and transfusion. Molecular genetic testing can be used to determine an
individual’s ABO genotype (e.g., genotype AO or AA). This may be useful in the research setting, for example,
to investigate the link between ABO blood groups and particular diseases, and also in the forensic setting.

Management
Determining an individual’s blood group is important prior to blood transfusion and prior to the donation or
receiving of a kidney transplant.
Occasionally, a person’s blood type may appear to change. For example, the ABO antigens can act as tumour
markers. Their presence may be decreased in particular diseases, such as acute myeloid leukemia, AML. In
contrast, occasionally the B antigen may be acquired in certain infectious diseases. A bacterial infection with
specific strains of E. coli or Clostridium tertium can generate a B-like antigen from an individual who has
the A1 allele.

Genetic counseling
The ABO blood type is inherited in an autosomal codominant fashion. The A and B alleles are codominant,
and the O allele is recessive.

BLOOD GROUP
Blood Group, classification of blood based on inherited differences (polymorphisms) in antigens on the
surfaces of the red blood cells (erythrocytes). Inherited differences of white blood cells
(leukocytes), platelets (thrombocytes), and plasma proteins also constitute blood groups, but they are not
included in this discussion.
Blood groups are inherited from both parents. The ABO blood type is controlled by a single gene (the ABO
gene) with three types of alleles inferred from classical genetics: i, IA, and IB. The I designation stands
for isoagglutinogen, another term for antigen. The gene encodes a glycosyltransferase, an enzyme that
modifies the carbohydrate content of the red blood cell antigens. The gene is located on the long arm of
the ninth chromosome (9q34).
The IA allele gives type A, IB gives type B, and i gives type O. As both IA and IB are dominant over i,
only ii people have type O blood. Individuals with IAIA or IAi have type A blood, and individuals
with IBIB or IBi have type B. IAIB people have both phenotypes, because A and B express a special dominance
relationship: codominance, which means that type A and B parents can have an AB child. A couple with type
A and type B can also have a type O child if they are both heterozygous (IBi and IAi). The cis-AB phenotype
has a single enzyme that creates both A and B antigens. The resulting red blood cells do not usually express
A or B antigen at the same level that would be expected on common group A 1 or B red blood cells, which can
help solve the problem of an apparently genetically impossible blood group.

Blood group inheritance

Blood
O A B AB
type
Genotype ii (OO) IAi (AO) IAIA (AA) IBi (BO) IBIB (BB) IAIB (AB)
O O or A A O or B B A or B
O ii (OO) OO OO AO OO AO AO AO BO OO BO BO BO AO BO AO
OO OO AO OO AO BO OO BO BO
O, A, B or
O or A O or A A B or AB A, B or AB
AB
I Ai (AO) AO AO AA AO AA AA AO AB AB BO AA AB AO
AB AO
OO OO AO OO AO BO BO
A BO OO
A A A A or AB AB A or AB
IAIA (AA) AO AO AA AO AA AA AA AB AO AB AB AB AA AB AA
AO AO AA AO AA AB AO AB AB
O, A, B or
O or B A or AB O or B B A, B or AB
AB
B I Bi (BO) BO BO AB AB AO BB BO BO BB BB BO AB BB AO
AB BO
OO OO AO OO BO BO
AO OO
 B B or AB AB B B B or AB
IBIB (BB) BO BO AB BO AB AB AB AB BB BO BB BB BB BB AB BB AB
BO BO BO AB BO BB BB
A or B A, B or AB A or AB A, B or AB B or AB A, B, or AB
AB IAIB (AB) AO AO AA AO AA AA AB AB AO AB AB BB AA AB AB
BO BO AB BO AB BB BO BB BB

Individuals with the rare Bombay phenotype (hh) produce antibodies against the A, B, and O groups and can
only receive transfusions from other hh individuals. The table above summarizes the various blood groups
that children may inherit from their parents. Genotypes are shown in the second column and in small print for
the offspring: AO and AA both test as type A; BO and BB test as type B. The four possibilities represent the
combinations obtained when one allele is taken from each parent; each has a 25% chance, but some occur
more than once. The text above them summarizes the outcomes.

Human Blood Group Antigens and Antibodies

There are over 300 blood group antigens, most of which are included in 30 different blood group
systems, Table 1. Many of the proteins carrying blood group antigens reside in the erythrocyte membrane as
complexes with other proteins (Figure 1).
Table 1. Human blood group systems
No. System name System Gene name(s)a Chromosomal CD
symbol location numbers
001 ABO ABO ABO 9q34.2
002 MNS MNS GYPA, GYPB, GYPE 4q31.21 CD235
003 P1PK P1PK A4GALT 22q13.2
004 Rh RH RHD, RHCE 1p36.11 CD240
005 Lutheran LU LU 19q13.32 CD239
006 Kell KEL KEL 7q34 CD238
007 Lewis LE FUT3 19p13.3
008 Duffy FY DARC 1q23.2 CD234
009 Kidd JK SLC14A1 18q12.3
010 Diego DI SLC4A1 17q21.31 CD233
011 Yt YT ACHE 7q22.1
012 Xg XG XG, MIC2 Xp22.33 CD99+
013 Scianna SC ERAMP 1p34.2
014 Dombrock Do ART4 12p12.3 CD97
015 Colton CO AQP1 7p14.3
016 Landsteiner–Wiener LW ICAM4 19p13.2 CD242
017 Chido/Rodgers CH/RG C4A, C4B 6p21.3
018 H H FUT1 19q13.33 CD173
019 Kx XK XK Xp21.1
020 Gerbich GE GYPC 2q14.3 CD236
021 Cromer CROM CD55 1q32.2 CD55
No. System name System Gene name(s)a Chromosomal CD
symbol location numbers
022 Knops KN CR1 1q32.2 CD35
023 Indian IN CD44 11p13 CD44
024 Ok OK GSG 19p13.3 CD147
025 Raph RAPH CD151 11p15.5 CD151
026 John Milton Hagen JMH SEMA7A 15q24.1 CD108
027 I I GCNT2 6p24.2
028 Globoside GLOB B3GALT3 3q26.1
029 Gill GIL AQP3 9p13.3
030 Rh-associated RHAG RHAG 6p21-qter CD241
glycoprotein
031 FORS FORS GBGT1 9q34.13
032 JR JR ABCG2 4q22
033 LAN LAN ABCB6 2q36

Figure 1. Graphic representation of the structures that carry different

blood group antigens in the red blood cell membrane

Structure of Blood Group Antigens

Some blood groups are carbohydrate structures attached to glycoproteins or glycolipids. These include the
antigens of the histo-blood group systems, ABO, H, and Lewis. The genes controlling expression of
the carbohydrate antigens do not encode the antigen directly, but produce enzymes, called transferases, which
catalyze biosynthesis of the antigens by stepwise addition of monosaccharide residues to
an oligosaccharide chain. Most blood group antigens are proteins or glycoproteins in which the main factor
determining the blood group polymorphism is the amino acid sequence, encoded directly by the blood group
gene. With these glycoproteins, the presence of carbohydrate may still play a role in expression of the antigen.
Many blood group polymorphisms represent single amino acid substitutions, but some, in the more complex
MNS and Rh systems, involve a variety of different genetic mechanisms that include intergenic recombination
and splice site mutations.

BLOOD GROUP ANTIBODIES

An antigen can be defined as a substance that will induce the production of antibodies, although there are
certain properties of a molecule, such as foreignness, size, and chemical complexity, that are associated with
increased immunogenicity. Proteins usually induce the most vigorous immune responses, followed by
carbohydrates, whereas lipids and nucleic acids are usually not strong immunogens, although clinically
significant antibodies specific for these types of molecules do exist (e.g., antiphospholipid antibodies and anti-
DNA antibodies found in some autoimmune diseases). Most antigens require helper activity, usually in the
form of cytokines, from helper T cells to induce strong antibody responses, and this helper activity is required
for production of antibody classes other than IgM. Some antigens, usually carbohydrate in nature, can induce
antibodies in the absence of helper T-cell activity, but these responses are primarily IgM, with little, if any,
antibodies of other classes produced.
Antibodies are produced by B lymphocytes, also known as B cells. The basic antibody molecule consists of
four polypeptide chains—two heavy chains of 50 to 70 kilodaltons, depending on the antibody class, and two
light chains of approximately 25 kilodaltons. The two heavy chains produced by a B cell are identical, as are
the two light chains. In each polypeptide chain, whether it is a heavy chain or a light chain, approximately the
first 100 amino acids are known as the variable region, and the remainder of the polypeptide chain constitutes
the constant region, which is identical in all antibodies of the same class. The antigen-binding site is formed
by association of the variable regions of one heavy chain and one light chain, which means that each four-
chain unit has two identical antigen-binding sites. However, in most immune responses, a large number of B
cells are stimulated by an antigen, and each antigen-specific B cell will produce a unique antibody, resulting
in a heterogeneous response consisting of many different antibody molecules directed toward multiple sites
on the antigen.

Immunogenicity
Several factors influence the ability to stimulate antibody production, including antigen size, complexity, and
dose as well as host human leukocyte antigen genotype and other, as yet unidentified, susceptibility factors.
Most carbohydrate-based RBC antigens are T independent and therefore tend to elicit an IgM response. The
protein-based antigens usually are T dependent and induce an IgM primary response that progresses
to IgG. Antigen exposure usually occurs by transfusion of products containing RBCs or during pregnancy
(immune antibodies) or by exposure to microbes (apparently, naturally occurring antibodies). Table 5-
5 summarizes the usual type of immunoglobulin response and the potential clinical significance, in
transfusion or in HDN, of selected blood group antibodies, which are listed in order of clinical significance. 36

Clinical Significance
Antibodies recognizing antigens in the ABO blood group system are by far the most clinically significant.
This is because they occur naturally in people whose RBCs lack the corresponding antigen. Other clinically
significant antibodies occur in the following order, from the most commonly to the least commonly
encountered in transfusion practice: anti-D, anti-K, anti-E, anti-c, anti-Fy a, anti-C, anti-Jka, anti-S, anti-Jkb.
All other clinically significant antibodies occur with an incidence of less than 1% of immunized patients. 57–
59
Antibodies that are considered clinically insignificant unless the antibody reacts in tests performed strictly
at 37°C are anti-P1, anti-M, anti-N, anti-Lua, anti-Lea, anti-Leb, and anti-Sda. Other clinically insignificant
antibodies that react at 37°C in the indirect antiglobulin test (IAT) are those of the Knops and Chido-Rodgers
systems and anti-JMH (Table 5-6).
The incidence of a blood group antibody depends on both the prevalence in the population and the
immunogenicity of the antigen. Immunized patients frequently produce multiple antibodies, and the more
antibodies present, the more difficult they are to identify.

Detection and Identification

Compatibility testing (testing patient's serum against donor's RBCs) still uses techniques that were described
100 years ago for direct agglutination and 50 years ago for indirect agglutination. Even today, with our
detailed understanding of blood group antigens, we have no single technical procedure able to detect all known
blood group antibodies. The hemagglutination technique is simple and inexpensive, does not require
sophisticated equipment, and when done correctly is sensitive and specific in terms of clinical relevance.
Agglutination should be graded according to the strength of reaction, and an evaluation of the reaction strength
can aid in identification of antibodies, especially when multiple antibodies are present in a serum. 33
The first blood group antigens to be identified were those that could be agglutinated by
the alloantibodies when antigen-positive RBCs were suspended in a saline medium (direct agglutination). This
direct agglutination reflects the fact that these antibodies are usually IgM and detect carbohydrate
antigens (ABO, P1, Le, and H antigens). Although anti-A and anti-B are highly clinically significant,
antibodies to the other carbohydrate antigens generally are not.
Most of the other antigens (e.g., Rh, Kell, Duffy, and Kidd) are proteins and are detected by antibodies that
react in the IAT. This test detects IgG antibodies, complement attached to RBCs, or both. The sensitized RBCs
are washed to remove unattached immunoglobulin (which would inhibit the antiglobulin reagent), and anti-
human immunoglobulin is added. The specimen is centrifuged and examined for agglutination. The
antiglobulin test can be direct or indirect. The direct antiglobulin test is used to detect RBCs sensitized in vivo,
such as alloantibodies causing transfusion reactions or HDN and autoantibodies in autoimmune hemolytic
anemia or cold agglutinin disease. The IAT is used to detect RBCs sensitized in vitro; for instance, antigen
typing, antibody detection and identification, and compatibility testing.
To identify antibodies, serum is tested against RBCs of known phenotype by various techniques. It is
sometimes helpful to treat antigen-positive test RBCs with proteolytic enzymes and chemical agents and to
compare the antibody reactivity in tests against treated and untreated RBCs to aid antibody identification
(Table 5-7). Brief, but informative, descriptions of the technical and clinical aspects of most blood group
antibodies can be found elsewhere.

Locating Antigen-Negative Blood

Once a patient is actively immunized to an RBC antigen and produces a clinically significant alloantibody,
the patient is considered immunized for life and must be transfused with antigen-negative RBCs, even if the
antibody is no longer detectable. Patients with passively acquired antibody (e.g., neonates and recipients of
plasma products, Rh immune globulin, or IVIgG) are not actively immunized and may be transfused with
antigen-negative RBCs only while the passive antibody is still present. Selection of blood for transfusion of a
patient with blood group alloantibodies is the joint responsibility of the staff at the transfusion service, the
donor center, and the patient's physician. Thus, it is very important that there be communication regarding the
number of units of RBC products required and the time frame involved. Figure 5-2 is a flow chart that outlines
the process for locating blood. Table 5-8 lists the prevalence of donors whose RBCs lack selected antigens.
To locate antigen-negative blood for transfusion, it is not necessary to identify an antibody to a low-prevalence
antigen detected in the recipient's blood during compatibility testing, because another unit of donor blood is
highly unlikely to be positive for the same uncommon antigen. In contrast, if an antibody to a low-prevalence
antigen is detected in the serum of a pregnant woman, identification of the antibody or determination of its
reactivity with paternal RBCs is required to predict the likelihood and severity of HDN in the baby.
If a patient's serum contains alloantibodies to a high-prevalence antigen, blood for transfusion may be very
difficult to locate. Whether the investigation is for transfusion purposes or for prediction of HDN, the antibody
should be identified. The identification aids in both the assessment of its clinical significance and the location
of appropriate blood for transfusion. Family members, in particular siblings, are the first source to investigate
for antigen-negative units. Most national blood donor centers can often assess national and international Rare
Donor Registries and can help provide the appropriate antigen-negative blood.

Blood group antigens

Antigenic determinants, including both oligosaccharides (e.g., A, B, and O; Figure 8.7) and proteins (e.g.,
Rh), are known as blood group substances. Oligosaccharides are present on the surface and the proteins span
the RBC membrane. The blood group substances are inherited according to Mendel’s law. Identification of
blood group substances is essential for blood transfusions and is valuable in forensic medicine and
anthropological studies. The antigens are recognized by specific antigen–antibody interactions that
produce agglutination. For example, when RBCs containing a specific antigenic determinant are mixed with
plasma containing specific antibodies to that antigen, cells will agglutinate through the formation of a network
of antigen–antibody linkages. More than 100 different blood group antigens have been categorized on the
basis of their structural relationships into 15 independent blood group systems. The blood group substances
are found on RBC membranes and in a variety of tissue cells. Soluble blood group substances
are glycoproteins in saliva, gastric juice, milk, seminal fluid, urine, fluids produced in ovarian cysts,
and amniotic fluid.
 Figure 8.7. The carbohydrate antigenic determinants of the ABO blood group system and the agglutination
reaction.

ABO blood group antigens

Blood group antigens are observed on O-glycoproteins, N-glycoproteins, and glycolipids, both on red blood
cells and various other cells of the body. Blood group antigens are synthesized on type 1, 2, 3, or 4 structures.
Type 1 and 2 structures are Galβ1–3GlcNAc-R and Galβ1–4GlcNAc-R, respectively (Fig. 3). Both are present
on O- and N-glycoproteins as well as on glycolipids. Type 3 and 4 structures are both Galβ1–3GalNAc-R,
however the R group for types 3 and 4 differs. R for type 3 is Ser/Thr of an O-glycopeptide, and R for type 4
is a glycolipid moiety. Type 2 structures are ubiquitous, while type 1 structures are found in the GI tract. Types
1 and 2 can both be found in polymers of (Type 1)n and (Type 2)n, with the latter forming polyLacNAc chains,
also called i blood group. In addition to forming a linear chain, i blood group can also be branched by various
β1–6GnTs to form I blood group. I blood group predominates after embryonic development, increasing
through adulthood.
Synthesis of blood group antigens requires at least two steps. The first is synthesis of H antigen, the structure
corresponding to O blood type. The second is synthesis of either A or B structure. The H antigen is generated
by addition of fucose in α1,2 linkage to a terminal galactose on a type 1–4 chain. Two genetic loci encode the
H transferase. The H loci is functional in red blood cells and the secretor loci is functional in GI epithelia,
getting its name for the secreted blood group antigens produced from secreted glycoconjugates.
These transferases are also important in synthesizing some Lewis antigen as discussed below.
After synthesis of the H structure, the A and B transferases, which differ by four amino acids, utilize the H
structure to synthesize A and B structures on type 1–4 chains. The A transferase transfers GalNAc from UDP-
GalNAc via α3 linkage to the terminal Gal of the H structure, while the B transferase transfers Gal from UDP-
Gal via α3 linkage also to the terminal Gal of the H structure. Individuals carrying mutated A/B transferases
encode neither functional A or B transferase, making them O blood group; only one functional A or B
transferase, making them AA/AO or BB/BO; or both functional transferases, making them AB +. More rarely,
individuals can be H-, Se-, or H-/Se-, making them unable to synthesize AB/H or other blood group structures
such as Lewis antigens.
Susceptibility or protection from various diseases, such as certain infections, has been associated with the
presence of different blood group antigens. Pathogens contain GBPs that may recognize cells of an individual
with one blood type but not another. Alternatively, individuals with a given blood type cannot mount an
adaptive immune response to pathogens expressing the same blood group or blood group-like structures.
Galectins appear to be able to fill this immunologic gap by recognizing and killing ABO-expressing bacteria
(Stowell et al., 2010). In addition to infections, AB/H structures and changes in these structures are observed
in cancers and contribute to the tumor phenotype.
Historical background
The human ABO blood groups were discovered by Austrian-born American biologist Karl Landsteiner in
1901. Landsteiner found that there are substances in the blood, antigens and antibodies, that induce clumping
of red cells when red cells of one type are added to those of a second type. He recognized three groups—A,
B, and O—based on their reactions to each other. A fourth group, AB, was identified a year later by another
research team. Red cells of the A group clump with donor blood of the B group; those of the B group clump
with blood of the A group; those of the AB group clump with those of the A or the B group because AB cells
contain both A and B antigens; and those of the O group do not generally clump with any group, because they
do not contain either A or B antigens. The application of knowledge of the ABO system in blood transfusion
practice is of enormous importance, since mistakes can have fatal consequences.

The discovery of the Rh system by Landsteiner and Alexander Wiener in 1940 was made because they tested
human red cells with antisera developed in rabbits and guinea pigs by immunization of the animals with the
red cells of the rhesus monkey Macaca mulatta.
Other blood groups were identified later, such as Kell, Diego, Lutheran, Duffy, and Kidd. The remaining
blood group systems were first described after antibodies were identified in patients. Frequently, such
discoveries resulted from the search for the explanation of an unexpected unfavourable reaction in a recipient
after a transfusion with formerly compatible blood. In such cases the antibodies in the recipient were produced
against previously unidentified antigens in the donor’s blood. In the case of the Rh system, for example, the
presence of antibodies in the maternal serum directed against antigens present on the child’s red cells can have
serious consequences because of antigen-antibody reactions that produce erythroblastosis fetalis, or hemolytic
disease of the newborn. Some of the other blood group systems—for example, the Kell and Kidd systems—
were discovered because an infant was found to have erythroblastosis fetalis even though mother and child
were compatible as far as the Rh system was concerned. In the table the well-established human blood group
systems are listed in the order of discovery.
Major human blood group systems

system date of discovery main antigens

ABO 1901 A1, A2, B, H

MNSs 1927 M, N, S, s

P 1927 P1, P2

Rh 1940 D, C, c, E, e

Lutheran 1945 Lua, Lub

Kell 1946 K, k

Lewis 1946 Lea, Leb

Duffy 1950 Fya, Fyb

Kidd 1951 Jka, Jkb

Diego 1955 Dia, Dib

Yt 1956 Yta, Ytb

I 1956 I, i

Xg 1962 Xga
 Major human blood group systems

system date of discovery main antigens

Dombrock 1965 Doa

Origin Theories
It is possible that food and environmental antigens (bacterial, viral, or plant antigens) have epitopes similar
enough to A and B glycoprotein antigens. The antibodies created against these environmental antigens in the
first years of life can cross-react with ABO-incompatible red blood cells that it comes in contact with during
blood transfusion later in life. Anti-A antibodies are hypothesized to originate from immune response
towards influenza virus, whose epitopes are similar enough to the α-D-N-galactosamine on the A glycoprotein
to be able to elicit a cross-reaction. Anti-B antibodies are hypothesized to originate from antibodies produced
against Gram-negative bacteria, such as E. coli, cross-reacting with the α-D-galactose on the B glycoprotein.
However, it is more likely that the force driving evolution of allele diversity is simply negative frequency-
dependent selection; cells with rare variants of membrane antigens are more easily distinguished by the
immune system from pathogens carrying antigens from other hosts. Thus, individuals possessing rare types
are better equipped to detect pathogens. The high within-population diversity observed in human populations
would, then, be a consequence of natural selection on individuals.

Clinical relevance
The carbohydrate molecules on the surfaces of red blood cells have roles in cell membrane integrity, cell
adhesion, membrane transportation of molecules, and acting as receptors for extracellular ligands, and
enzymes. ABO antigens are found having similar roles on epithelial cells as well as red blood cells.

Bleeding and Thrombosis (von Willebrand factor)

The ABO antigen is also expressed on the von Willebrand factor (vWF) glycoprotein, which participates
in hemostasis (control of bleeding). In fact, having type O blood predisposes to bleeding, as 30% of the total
genetic variation observed in plasma vWF is explained by the effect of the ABO blood group, and individuals
with group O blood normally have significantly lower plasma levels of vWF (and Factor VIII) than do non-O
individuals. In addition, vWF is degraded more rapidly due to the higher prevalence of blood group O with
the Cys1584 variant of vWF (an amino acid polymorphism in VWF): the gene for ADAMTS13 (vWF-
cleaving protease) maps to human chromosome 9 band q34.2, the same locus as ABO blood type. Higher
levels of vWF are more common amongst people who have had ischemic stroke (from blood clotting) for the
first time. The results of this study found that the occurrence was not affected by ADAMTS13 polymorphism,
and the only significant genetic factor was the person's blood group.
ABO(H) blood group antigens are also carried by other hemostatically relevant glycoproteins, such as platelet
glycoprotein Ibα, which is a ligand for vWF on platelets.[57] The significance of ABO(H) antigen expression
on these other hemostatic glycoproteins is not fully defined, but may also be relevant for bleeding and
thrombosis.

ABO hemolytic disease of the newborn

ABO blood group incompatibilities between the mother and child do not usually cause hemolytic disease of
the newborn (HDN) because antibodies to the ABO blood groups are usually of the IgM type, which do not
cross the placenta. However, in an O-type mother, IgG ABO antibodies are produced and the baby can
potentially develop ABO hemolytic disease of the newborn.

Clinical applications
In human cells, the ABO alleles and their encoded glycosyltransferases have been described in several
oncologic conditions. Using anti-GTA/GTB monoclonal antibodies, it was demonstrated that a loss of these
enzymes was correlated to malignant bladder and oral epithelia. Furthermore, the expression of ABO blood
group antigens in normal human tissues is dependent the type of differentiation of the epithelium. In most
human carcinomas, including oral carcinoma, a significant event as part of the underlying mechanism is
decreased expression of the A and B antigens.[62] Several studies have observed that a relative down-regulation
of GTA and GTB occurs in oral carcinomas in association with tumor development. [62][63] More recently, a
genome wide association study (GWAS) has identified variants in the ABO locus associated with
susceptibility to pancreatic cancer.[64] In addition, another large GWAS study has associated ABO-histo blood
groups as well as FUT2 secretor status with the presence in the intestinal microbiome of specific bacterial
species. In this case the association was with Bacteroides and Faecalibacterium spp. Bacteroides of the same
OTU (operational taxonomic unit) have been shown to be associated with inflammatory bowel
disease,[65][66] thus the study suggests an important role for the ABO histo-blood group antigens as candidates
for direct modulation of the human microbiome in health and disease. [67]

METHODS OF BLOOD GROUPING

Identification of blood groups
The basic technique in identification of the antigens and antibodies of blood groups is the agglutination
test. Agglutination of red cells results from antibody cross-linkages established when different specific
combining sites of one antibody react with antigen on two different red cells. By mixing red cells (antigen)
and serum (antibody), either the type of antigen or the type of antibody can be determined depending on
whether a cell of known antigen composition or a serum with known antibody specificity is used.
In its simplest form, a volume of serum containing antibody is added to a thin suspension (2–5 percent) of red
cells suspended in physiological saline solution in a small tube with a narrow diameter. After incubation at
the appropriate temperature, the red cells will have settled to the bottom of the tube. These sedimented red
cells are examined macroscopically (with the naked eye) for agglutination, or they may be spread on a slide
and viewed through a low-power microscope.
An antibody that agglutinates red cells when they are suspended in saline solution is called a complete
antibody. With powerful complete antibodies, such as anti-A and anti-B, agglutination reactions visible to the
naked eye take place when a drop of antibody is placed on a slide together with a drop containing red cells
in suspension. After stirring, the slide is rocked, and agglutination is visible in a few minutes. It is always
necessary in blood grouping to include a positive and a negative control for each test.
An antibody that does not clump red cells when they are suspended in saline solution is called incomplete.
Such antibodies block the antigenic sites of the red cells so that subsequent addition of complete antibody of
the same antigenic specificity does not result in agglutination. Incomplete antibodies will agglutinate red cells
carrying the appropriate antigen, however, when the cells are suspended in media containing protein. Serum
albumin from the blood of cattle is a substance that is frequently used for this purpose. Red cells may also be
rendered specifically agglutinable by incomplete antibodies after treatment with such protease enzymes as
trypsin, papain, ficin, or bromelain.
After such infections as pneumonia, red cells may become agglutinable by almost all normal sera because of
exposure of a hidden antigenic site (T) as a result of the action of bacterial enzymes. When the patient recovers,
the blood also returns to normal with respect to agglutination. It is unusual for the red cells to reflect
antigenicity other than that determined by the individual’s genetic makeup. The presence of an acquired B
antigen on the red cells has been described occasionally in diseases of the colon, thus allowing the red cell to
express an antigenicity other than that genetically determined. Other diseases may alter immunoglobulins; for
example, some may induce the production of antibodies directed against the person’s own blood groups
(autoimmune hemolytic anemia) and thus may interfere with blood grouping. In other diseases a defect in
antibody synthesis may cause the absence of anti-A and anti-B antibody.

Coombs test
When an incomplete antibody reacts with the red cells in saline solution, the antigenic sites become coated
with antibody globulin (gamma globulin), and no visible agglutination reaction takes place. The presence of
gamma globulin on cells can be detected by the Coombs test, named for its inventor, English
immunologist Robert Coombs. Coombs serum (also called antihuman globulin) is made by immunizing
rabbits with human gamma globulin. The rabbits respond by making antihuman globulin (i.e., antibodies
against human gamma globulin and complement) that is then purified before use. The antihuman globulin
usually contains antibodies against IgG and complement. Coombs serum is added to the washed cells; the tube
is centrifuged; and, if the cells are coated by gamma globulin or complement, agglutinates will form. Newer
antiglobulin reagents (made by immunizing with purified protein) can detect either globulin or complement.
Depending on how it is performed, the Coombs test can detect incomplete antibody in the serum or antibody
bound to the red cell membrane. In certain diseases, anemia may be caused by the coating of red cells with
gamma globulin. This can happen when a mother has made antibodies against the red cells of her newborn
child or if a person makes an autoantibody against his own red cells.

Adsorption, elution, and titration

If a serum contains a mixture of antibodies, it is possible to prepare pure samples of each by a technique called
adsorption. In this technique an unwanted antibody is removed by mixing it with red cells carrying the
appropriate antigen. The antigen interacts with the antibody and binds it to the cell surface. These red cells are
washed thoroughly and spun down tightly by centrifugation, all the fluid above the cells is removed, and the
cells are then said to be packed. The cells are packed to avoid dilution of the antibody being prepared.
Adsorption, then, is a method of separating mixtures of antibodies by removing some and leaving others. It is
used to identify antibody mixtures and to purify reagents. The purification of the Coombs serum (see above)
is done in the same way.
If red cells have adsorbed gamma globulin onto their surfaces, the antibody can sometimes be recovered by a
process known as elution. One simple way of eluting (dissociating) antibody from washed red cells is to heat
them at 56 °C (133 °F) in a small volume of saline solution. Other methods include use of acid or ether. This
technique is sometimes useful in the identification of antibodies.
Titration is used to determine the strength of an antibody. Doubling dilutions of the antibody are made in a
suitable medium in a series of tubes. Cells carrying the appropriate antigen are added, and the agglutination
reactions are read and scored for the degree of positivity. The actual concentration of the antibody is given by
the dilution at which some degree of agglutination, however weak, can still be seen. This would not be a safe
dilution to use for blood-grouping purposes. If an antiserum can be diluted, the dilution chosen must be such
that strong positive reactions occur with selected positive control cells. Titration is helpful when preparing
reagents and comparing antibody concentrations at different time intervals.

Inhibition tests
Inhibition tests are used to detect the presence of antigen with blood group specificity in solutions; inhibition
of a known antibody-antigen reaction by a fluid indicates a particular blood group specificity. If an active
substance is added to antibody, neutralization of the antibody’s activity prevents agglutination when red cells
carrying the appropriate antigen are subsequently added to the mixture. A, B, Lewis, Chido, Rogers, and P
antigens are readily available and can be used to facilitate antibody identification. This technique was used to
elucidate the biochemistry of ABH, Ii, and Lewis systems, and it is important in forensic medicine as a means
of identifying antigens in blood stains.

Hemolysis
Laboratory tests in which hemolysis (destruction) of the red cells is the end point are not used frequently in
blood grouping. For hemolysis to take place, a particular component of fresh serum called complement must
be present. Complement must be added to the mixture of antibody and red cells. It may sometimes be desirable
to look for hemolysins that destroy group A red cells in mothers whose group A children are incompatible or
in individuals, not belonging to groups A or AB, who have been immunized with tetanus toxoid that contains
substances with group A specificity.
Hemolytic reactions may occur in patients who have been given transfusions of blood that either is
incompatible or has already hemolyzed. The sera of such patients require special investigations to detect the
presence of hemoglobin that has escaped from red cells destroyed within the body and for the breakdown
products of other red cell constituents.

SOURCES OF ANTIBODIES AND ANTIGENS

Normal donors are used as the source of supply of naturally occurring antibodies, such as those of the ABO,
P, and Lewis systems. These antibodies work best at temperatures below that of the body (37 °C, or 98.6 °F);
in the case of what are known as cold agglutinins, such as anti-P1, the antibody is most active at 4 °C (39 °F).
Most antibodies used in blood grouping must be searched for in immunized donors.
Antibodies for MN typing are usually raised in rabbits—similarly for the Coombs serum. Antibodies prepared
in this way have to be absorbed free of unwanted components and carefully standardized before use.
Additional substances with specific blood group activity have been found in certain plants. Plant agglutinins
are called lectins. Some useful reagents extracted from seeds are anti-H from Ulex europaeus (common
gorse); anti-A1, from another member of the pulse family Fabaceae (Leguminosae), Dolichos biflorus; and
anti-N from the South American plant Vicia graminea. Agglutinins have also been found in animals—for
example, the fluid pressed from the land snail Octala lactea. Additional plant lectins and agglutinins from
animal fluids have been isolated.

Monoclonal antibodies (structurally identical antibodies produced by hybridomas) to blood groups are
replacing some of the human blood grouping reagents. Mouse hybridomas (hybrid cells of a myeloma
tumour cell and lymphocyte merging) produce anti-A and anti-B monoclonal antibodies. The antibodies are
made by immunizing with either red cells or synthetic carbohydrates. In addition to their use in blood
grouping, these monoclonal antibodies can be of use in defining the hereditary background (heterogenicity)
and structure of the red cell antigen.

USES OF BLOOD GROUPING

Transfusion
The blood donated by healthy persons is tested to ensure that the level of hemoglobin is satisfactory and that
there is no risk of transmitting certain diseases, such as AIDS or hepatitis. It is then fractionated (split) into its
component parts, particularly red cells, plasma, and platelets. Correct matching for the ABO system is
vital. Compatible donors on the basis of their possessing A, B, or O blood are shown in the table.
The ABO and Rh groups in transfusion

system recipient type donor red cell type donor plasma type

*Not if the patient's serum contains anti-A1 (antibody to common type A red cell in subgroup A patients).

**Not if the patient is a female less than 45 years old (childbearing possible), unless life-threatening
hemorrhage is present and transfusion of Rh-positive blood is lifesaving.

***Not if the patient's serum contains anti-D (antibody to positive red cells), except under unusual medical
circumstances.

ABO A A* or O A or AB

ABO B B or O B or AB

ABO O O only O, A, B, or AB

ABO AB AB*, A*, B, or O AB

Rh positive positive or negative positive or negative

Rh negative negative or positive**, *** negative or positive**

As explained above, the most important blood group systems for transfusion of red cells are ABO and Rh.
Persons who have either of the red cell antigens (A and B) have antibody present in their serum of the type
that will oppose an antigen of its opposite nature; for example, group A blood contains A antigens on red cell
surfaces and anti-B antibodies in the surrounding serum. On the other hand, O group individuals lack both the
A and the B antigen and thus have both anti-A and anti-B in their serum. If these antibodies combine with the
appropriate antigen, the result is hemolytic transfusion reaction and possibly death. Red cell transfusions must
therefore be ABO compatible. The blood groups A and B have various subgroups (e.g., A 1, A2, A3, and B1,
B2, and B3). The only common subgroups that are likely to affect red cell transfusions are the subgroups of A.
Potential donors are also tested for some of the antigens of the Rh system, since it is essential to know whether
they are Rh-positive or Rh-negative. Rh-negative indicates the absence of the D antigen. Rh-negative persons
transfused with Rh-positive blood will make anti-D antibodies from 50 to 75 percent of the time. Antibody
made in response to a foreign red cell antigen is usually not harmful but does require subsequent transfusions
to be antigen-negative. Rh-positive blood should never be given to Rh-negative females before or during the
childbearing age unless Rh negative blood is not available and the transfusion is lifesaving. If such a woman
subsequently became pregnant with an Rh-positive fetus, she might form anti-Rh antibody, even though
the pregnancy was the first, and the child might develop erythroblastosis fetalis (hemolytic disease of the
newborn).
Care must be taken not to give a transfusion unless the cells of the donor have been tested against the
recipient’s serum. If this compatibility test indicates the presence of antibodies in the recipient’s serum for the
antigens carried by the donor’s cells, the blood is not suitable for transfusion because an unfavourable reaction
might occur. The test for compatibility is called the direct match test. It involves testing the recipient’s serum
with the donor’s cells and by the indirect Coombs test. These are adequate screening tests for most naturally
occurring and immune antibodies.
If, in spite of all the compatibility tests, a reaction does occur after the transfusion is given (the unfavourable
reaction often manifests itself in the form of a fever), an even more careful search must be made for any red
cell antibody that might be the cause. A reaction after transfusion is not necessarily due to red cell antigen-
antibody reactions. It could be caused by the presence of antibodies to the donor’s platelets or white cells.
Transfusion reactions are a particular hazard for persons requiring multiple transfusions.

Organ transplants
The ABO antigens are widely distributed throughout the tissues of the body. Therefore, when organs such as
kidneys are transplanted, most surgeons prefer to use organs that are matched to the recipient’s with respect
to the ABO antigen system, although the occasional survival of a grafted ABO-incompatible kidney has
occurred. The remaining red cell antigen systems are not relevant in organ transplantation.

Paternity testing
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.
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), 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 K x 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 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.