The antibodies in ABO are usually naturally occurring and
are mostly IgM. However, IgG classes are also present. IgG
anti-A and IgG anti-B are found more commonly in group
Anti-A and anti-B are usually not produced in infants
up to age of 3–6 months. However, they reach a maximum
titer by 5–10 years and then gradually become weaker as
the individual ages. The antibodies found in the serum of
infants at birth are almost of maternal origin. The serum
grouping of a newborn is, therefore, not recommended.
Anti-H very rarely occurs as cold reactive agglutinin in
individuals with very low levels of H antigens on their
cells and has little clinical significance. However, anti-H
found in Bombay blood group (Oh) is an all antibody
and is clinically significant. It occurs as a hemolysin and
FIG. 11.1: The possible phenotypes and genotypes
FIG. 11.2: Stages in production of blood group
TABLE 11.2: The possible phenotypes and genotypes in ABO group
Blood Banking (Immunohematology) 319
¾ For demonstration of true ABO group of an individual,
it is important to do both cell grouping (forward typing)
and serum grouping (reverse typing). Both forward and
reverse typing must match to confirm the true ABO
¾ Serum should always be added before adding the cells
and examine each tube after serum has been added to
ensure that none has been left.
¾ ABO grouping test should be done at room temperature,
or below. Testing at 37°C weakens reaction.
¾ Tubes, slides and microplates should be labeled
The development of monoclonal antibodies obtained from
cultures of cells secreting antibodies called hybridomas
has made available a new source of ABO typing reagent.
Before the advent of hybridoma technology, the ABO
grouping reagent was derived from human donors with or
without immunization and are called polyclonal reagent.
The monoclonal reagents anti-A, anti-B and anti-AB have
significant advantage over earlier traditional polyclonal
reagent in terms of specificity, potency, consistency and
should be free from virus such as HIV and hepatitis.
The red cells used in ABO grouping are pooled A cells,
B cells and O cells. The cells should be washed in saline
to remove serum or plasma. The supernatant of last
wash should be clear. Use 2–4% cell suspension for tube,
microplate typing and 30–40% for slide typing.
Group ‘O’ cells are used in serum grouping to detect
antibodies other than anti-A or anti-B in some donors.
These antibodies are not naturally occurring and are called
irregular antibodies. They occur due to immunization
Supplementary reagent used are:
¾ Anti-A lectin, which reacts strongly with A individuals
¾ Anti-H reacts selectively with ABO group according to
H substance. Group O individuals which contain only H
react strongly with anti-H while A, B contains very little
H and thus, reacts very weakly or negatively with anti-H.
Other supplementary reagent that may be used to
resolve the discrepancies of ABO grouping may include
¾ Group O reagent screen cells.
Group O reagent screen cells contain red cells with
various antigen specificities. These 2 or 3 group O cells
are complimentary to each other to provide antigens for
detection of most of clinically significant antibodies. They
are used to rule out ABO typing discrepancy caused by
Preparation of Red Cell Suspensions
Depending upon the specific technique employed, 2, 5,
10 or 50% red cell suspensions are required. These can
be prepared by transferring freshly obtained blood from
a skin puncture into saline or suspending in saline the
packed red cells obtained from citrated or oxalated blood.
Preservative anticoagulant solutions are also available
that permit preservation of red cells for one month or
longer. This is most useful for controls and panel cells of
known antigenic composition (reagent red cells). Most
frequently, suspensions are made by gently breaking up
blood clots with an applicator stick and transferring the
red cell aggregates into saline or other suspending media.
1. To about 5 mL of normal saline, add several drops of
whole blood (fresh, citrated, oxalated or fragments of
2. Centrifuge, in order to pack the red cells.
3. Withdraw the supernatant fluid as completely as
4. Add 0.1 mL of packed red cells to a test tube containing
4.9 mL of normal saline and mix well. This represents
a 2% suspension of red cells in saline.
All red cell suspensions must be refrigerated when not
in use. They are unsuitable if they show hemolysis and
should be used within 12 hours of preparation.
Blood Grouping Sera should Meet the following
1. It should have titer of recommended potency.
2. It should be free of cold agglutinins.
3. It should be free of so-called irregular agglutinins.
4. It should not form rouleaux when mixed with red
5. It should be clear, of normal color (except when a dye is
added for identification), and free of cells or any other
particles, not hemolyzed, icteric or chylous.
6. It should be free of complement.
• Anti-A serum (minimum titer with A1 cells 256)
• Anti-B serum (minimum titer with B cells 256)
• Anti-A1 reagent (absorbed anti-A serum, plant
• Anti-Rh (D) serum (minimum titer 32)
• Anti-A serum is colored blue
• Anti-B serum is colored yellow.
(Courtesy: Tulip’s Erybank Range)
Human red blood cell antigens can be divided into four
groups A, B, AB and O depending on the presence or
absence of the corresponding antigens on the red blood
cells. Approximately, 41% of the Caucasian population
have the A-antigen, 9% have the B antigen, 4% have both
A and B antigens, while the remaining have neither A nor
Erybank anti-A, anti-B and anti-A/B are ready to use
reagent prepared from human serum. These reagents of
the immunoglobulin class IgM are a pool of specific human
serum obtained from certified selected donors who are
found to be negative for HBsAg and anti-HIV antibody.
Each batch of reagent undergoes rigorous quality control
at various stages of manufacture for its specificity, avidity
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