MODULE 03- ABO BLOOD GROUP SYSTEM

Learning Outcomes:
1. To explain the Landsteiner’s Rule.
2. To compare and contrast forward and reverse typing.
3. To identify the frequencies of the four major blood types in white, black, Hispanic and
Asian populations.
4. To describe the immunoglobulin classes of ABO antibodies in group O, A and B
individuals.
5. To predict the ABO phenotypes and genotypes of offspring from various ABO matings.
6. To explain the formation of H, A and B antigens on the blood cells (RBCs) from precursor
substance to immunodominant sugars.
7. To describe the characteristics of Bombay phenotype.
8. To discuss ABO incompatible pregnancy.
9. To describe the common ABO discrepancies.

Introduction:
The discovery of the ABO blood group system by Landsteiner in 1900 marked the beginning of
the modern blood banking and transfusion medicine. In a series of experiments designed to
show serologic incompatibilities between humans, Landsteiner recognized different patterns of
agglutination when human blood samples were mixed in random pairings. He described the
blood groups as A, B and O. Several years later, Landsteiner’s associates, von Decastello and
Sturli, added group AB to the original observations. In his investigations, Landsteiner noted the
presence of agglutinating antibodies in the serum of individuals who lacked corresponding ABO
antigen. He observed that group A red cells agglutinated with the serum from group B
individuals. This observation has been termed Landsteiner’s rule (or Landsteiner’s Law).

Landsteiner’s Rule:
A normal, healthy individuals possess ABO antibodies to the ABO blood group
antigens absent from their red cells. (Figure 3.1)
Individuals with group A red cells possess the A antigen lack the B antigen. Therefore, these
individuals possess anti- B antibodies. Individuals with group B red cells possess the B antigen
and lack the A antigen. Therefore, these individuals also possess anti-A antibodies.
The first blood group system to be described, the ABO blood group system, remains the most
important blood group system for transfusion purposes. Accurate donor and recipient ABO
types are fundamental to transfusion safety because of the presence ABO antibodies in
individuals with no previous exposure to human red cells. The transfusion of ABO- incompatible
blood to a recipient can result in intravascular hemolysis and other serious consequences of an
acute hemolytic transfusion reaction.
Testing to detect ABO incompatibility between a donor and potential transfusion recipient is
the foundation in which all other pretransfusion testing is based.
Even today, transfusion of wrong ABO group remains the leading cause of death in hemolytic
transfusion reaction fatalities reported to the FDA; however, transfusion related acute lung
injury (TRALI) was the most frequent cause of death in the fiscal year 2009 (refer to your book
table 6-1).

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Forward and Reverse Grouping
Forward grouping (front type) is defined as using known sources of commercial antisera (anti-
A and anti- B) to detect antigens on an individual’s RBCs. (Figure 3.2)
Reverse grouping (back type) is defined as detecting ABO antibodies in the patient’s serum by
using known reagent RBCs, namely A1 and B cells. (Figure 3.3)
ABO forward and reverse grouping tests must be performed on all donors and patients. ABO
grouping is the most frequently performed test in the blood bank. There is always an inverse
reciprocal relationship between the forward and reverse type; thus, one serves as a check on
the other. Example, if an individual has A antigens only on their red cells, there will be an
expected naturally occurring anti-B antibody in their serum since they lack the B antigen.

Figure 3.1 Landsteiner Law

Figure 3.2 Forward Typing (screening test); slide or tube method

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Figure 3.3 Reverse Typing (confirmatory testing; not applicable to babies and elderly); tube
method (sample: serum, reagent: known A cells and known B cells)

Figure 3.4 Forward and Reverse Typing

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Formation of A, B and H Red Cell Antigens
The formation of ABH antigens results from the interaction of genes at three separate loci
(ABO, Hh, and Se). these genes do not actually code for the production of antigens but rather
produce specific glycosyltransferases that add sugars to a basic precursor substance (refer
Table 6-9 on your book). A, B and H antigens are formed from the same basic precursor
material (called a paragloboside or glycan) to which sugars are attached in response to specific
enzyme transferases elicited by an inherited gene.
The H antigen is actually the precursor structure on which A and B antigens are made.
Inheritance of the H gene results in the formation of H antigen. The H and Se genes are closely
linked and located on chromosome 19, in contrast to ABO genes, which are located on
chromosome 09. The H and Se genes are not part of the ABO system; however, their
inheritance does influence A and B antigen expression. The H gene must be inherited to form
the ABO antigens on the RBCs, and the Se gene must be inherited to form the ABO antigens in
the secretions.
Individuals who are blood group O inherit at least one H gene (genotpe HH or Hh) and two O
genes. The H gene elicits the production of an enzyme called a-2-L-fucosyltransferase, which
transfers the sugar L-fucose to an oligosaccharide chain on the terminal galactose of 2 chains.
The sugars that occupy the terminal positions of this precursor chain and confer blood group
specificity are called immunodominant sugars. Therefore, L-fucose is the sugar responsible for
the H specificity (blood group O). The O blood group has the highest concentration of H antigen.
The H substance (L-fucose) must be formed for the other sugars to be attached in response to
inherited A and/or B gene.
The H gene is present in more than 99.9% of the random population; its allele. “h” is quite rare,
and the genotype hh is extremely rare. The term Bombay has been used to refer to the
phenotype that lacks normal expression of ABH antigens because of the inheritance of the hh
genotype.

Gene Glycosyltransferase Immunodominant Antigen

sugar
H a-2-t-fucosyltransferase L- fucose H
A a-3-N- N-acetyl-D- A
acetylgalactosaminyltransferase galactosamine
B a-3-D-galactosyltransferase D- galactose B
Table 3.1 Glcosytransferase and Immunodominant Sugars Responsible for H,A and B Antigens

Comparison of A, B and H antigens on RBCs with A, B and H Soluble Substances

The formation of soluble A, B and H substances is the same as that described for the formation
of A, B and H antigens on the RBCs.
In the past, tests for ABH secretion has been used to establish the true ABO group of an
individual whose RBC antigens are poorly developed. The demonstration of A, B, and H
substances in saliva is evidence for the inheritance of A gene, B gene and H gene and Se gene.
The term secretor refers only to the secretion of A, B and H soluble antigens in the body fluids.
(Refer to table 6-11 in your book.)
People who inherit the sese genotype are termed nonsecretors.

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Table 3.2 Frequency of ABO blood types

Table 3.3 Distribution of Blood Groups in the U.S According to Race

ABO Antibodies
Individuals normally produce antibodies directed against the A and/or B antigen (s) absent from
their RBCs. These antibodies have been described as naturally occurring because they are
produced without any exposure to RBCs. The ABO antibodies are predominantly IgM, and they
activate complement and react at room temperature or colder. ABO antibodies produce strong
direct agglutination reactions during ABO testing. The production of ABO antibodies is initiated
at birth, but titers are generally too low for the detection until the individual is 3 to 6 months of
age. Therefore, most antibodies found in the cord blood serum are of maternal origin. Results
of serum ABO testing before 3 to 6 months of age cannot be considered valid because some or
all of the antibodies present maybe IgG maternal antibodies that have crossed the placenta. It is
therefore logical to perform only forward grouping on cord blood from newborn infants.

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Antibody production peaks when an individual is between 5 and 10 years of age and declines
later in life. Elderly people usually have lower levels of anti-A and anti-B; therefore, antibodies
may be undetectable in the reverse grouping. ABO antibodies can cause rapid intravascular
hemolysis if the wrong blood ABO group is transfused; this can result in the patient dying.
Although anti-A (from group B individual) and anti-B (from group A individual) contains
predominantly IgM antibody, there may be small quantities of IgG present. Serum from group O
individuals contains not only anti-A and anti-B but also, anti-AB which reacts with A and B cells.
Knowing the amount of IgG anti-A, anti-B and anti-A,B in a woman’s serum sometimes allows
prediction or diagnosis of hemolytic disease of the newborn (HDFN) caused by ABO
incompatibility. Often cord blood samples from babies of group O mothers are examined for
possible ABO HDFN. Both immunoglobulin classes of ABO antibodies react preferentially at
room temperature, or below and efficiently activate complement at 37 0C.
LECTINS USED IN BLOOD BANKING
Lectin is a seed extract that has antibody specificity. Lectins do not contain antibodies, instead
they contain proteins that react similarly to antibodies. They are used to identify certain types
of blood group antigens by binding to the carbohydrate determinant of the antigen, resulting in
agglutination. Other use of lectin is the investigation of red cell polyagglutination.
Dolichos biflorus- agglutinates A1 or A1B
Bandeiraea simplicifolia- agglutinates B cell
Ulex europaeus- agglutinates O cells (H specificity) and other ABO blood groups depending on
the amount of H antigen available.
Table 3.4 Subgroups of A

BOMBAY PHENOTYPE
The Bombay phenotype was first reported by Bhende in 1952 Bombay, India. It represents the
inheritance of a double dose of the h gene, producing very rare genotype hh. As a result, the
ABO genes cannot be expressed, and ABH antigens cannot be formed, since there is no H
antigen made in the Bombay phenotype.
The Bombay Phenotype (Oh)

 hh genotype
 no H antigens formed; therefore, no A or B antigens formed
 phenotypes as blood group O
 anti-A, anti-B, anti-A,B and anti-H present in serum
 can only be transfused with blood from another Bombay (Oh)

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ABO DISCREPANCIES
ABO discrepancies occur when unexpected reactions occur in the forward and reverse
grouping. These can be due to problems with the patient’s serum (reverse grouping), problems
with the patient’s red cells (forward grouping), or problems with both the serum and cells. The
unexpected reaction can be due to an extra positive reaction or a weak or missing reaction in
the forward and reverse grouping. All ABO discrepancies must be resolved prior to reporting a
patient or donor ABO group.
Technical Errors
Technical errors can also cause ABO discrepancies. This includes errors in labeling of blood
sample at the patient’s bedside or in the laboratory; therefore, patient and sample
identification are essential. Other errors include the failure to add reagents or the addition of
incorrect reagents or sample. Therefore, it is recommended that serum and antiserum be
added first, then the patient reagent red cells. It is also recommended that results be recorded
immediately to avoid transcription errors.
In addition, contaminated reagents can cause errors in testing. Therefore, looking at all the
reagent vials when performing ABO testing and during quality control testing is extremely
important. Refer to Box 6-8 of your textbook for the table on common sources of technical
errors resulting in ABO discrepancies.
Resolution
If the initial test was performed using RBCs suspended in serum or plasma, repeat testing the
same sample using a saline suspension of RBCs can usually resolve the ABO discrepancy. It is
important to make sure that any and all technical factors may have given rise to the ABO
discrepancy are reviewed and corrected. It is also essential to acquire information regarding the
patient’s age, diagnosis, transfusion history, medications, and history of pregnancy. If the
discrepancy persists and appears to be due to an error in specimen collection or identification,
a new sample should be drawn from the patient and the RBC and serum testing repeated.
When a discrepancy is encountered, results must be recorded, but interpretation of the ABO
type must be delayed until the discrepancy is resolved. If blood is from a potential transfusion
recipient, it may be necessary to administer group O- compatible RBCs before the discrepancy
is resolved. In general, when investigating ABO discrepancies, it should be noted that RBC and
serum grouping reactions are very strong (3+ to 4+); therefore weaker reactions usually
represent the discrepancy.
Refer the algorithm of resolving ABO discrepancies in your text book (Figure 6-14) and the
different categories of ABO discrepancies .

Change in ABH types with Various Diseases

Weakening of the A antigen may occur in some persons with acute leukemia or in chronic
myeloproliferative diseases with leukemic evolution. Certain cancers, particularly cancer of the
colon, maybe associated with the acquisition of a B antigen termed the acquired B antigen.
Acquired B can also occur with certain gram- negative infections and intestinal obstruction.
Thus, occasionally with these diseases an individual of Group o phenotype may acquire a B and
apparently type as B, or a Group A person may acquire a B and type as AB.

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Table 3.6 ABO Compatibility for Whole Blood, Red blood cells, Platelet and
Plasma transfusions

Universal donors: Group O donors for RBC transfusions: these RBC’s may be transfused to any
ABO phenotype because the cells lack both A and B antigens.
Universal recipients: Group AB recipients may receive transfusions of RBCs from any ABO
phenotype; these recipients lack circulating ABO antibodies in plasma.
Universal donor for RBC transfusion is group O; universal donor for plasma transfusion is
group AB.
Universal recipient for RBC transfusion is group AB; universal recipient for plasma transfusion
is group O.

Assignment:
A. Illustrate (draw) ABO and Rh Pregnancy incompatibility.
B. Compare and contrast in table form.