IMMUNO HEMA CHROMOSOMAL LOCATION OF MAJOR

module3-lecturenotes BLOOD GROUP SYSTEMS

(Kell, Kid – Most dangerous blood groups)
THE ABO BLOOD GROUP SYSTEM CHROMOSOMAL LOCUS BLOOD GROUPS

● Most important system in transfusion and 1 Rh, Duffy

transplantation therapy 4 MNSs

● Discovered by Karl Landsteiner in 1901 In
7 Kell
1902, AB blood type was discovered by Von
Decastello and Sturli 9 ABO
● Relation of genetics to blood banking: Any
18 Kidd
trait produced by the body is due to genes
including blood types. 19 H, Lewis Lutheran
Genes
22 P1
● Any trait produced by the body DNA
contains the genetic information Specific
position called Locus (Chromosome Locus) LESSON 1: Discovery, Laws, Properties,
Genetics Characteristics, and Inheritance
● the study of transmission of inherited traits
or characteristics Normal number of DISCOVERIES
chromosomes (46 in each nucleated cells) – Karl Landsteiner
23 pairs
● discovered the first human blood group
Autosomal pairs
system (ABO)
● Chromosome 1-22
● In 1901: He is the first individual to perform
● Sex Chromosome: Chromosome 23
FORWARD and REVERSE typing from
Homozygous (Double dose)
drawing blood from himself and five
● expresses more antigen than heterozygous.
associates.
Dosage effect (Ex. AA, BB, OO)
Heterozygous (Single dose) Forward grouping (Front type) – determine antigen
● single dose (AO, BO)
Genotype ● defined as using known sources of
● It is the total genetic composition of an commercial antisera, anti-A & anti-B, to
individual. Maternally and paternally derived detect antigens on an individual’s RBC.
genes. AA gene, AO gene = A antigen
Reverse grouping (Back type)
Phenotype
● It is the detectable or expressed ● defined as detecting ABO antibodies in
characteristics of genes. The resulting the patient’s serum by using known reagent
product or the trait itself. (A antigen) RBCs, namely A1and B cells.
Laws
MAJOR ABO GENOTYPES AND PHENOTYPES
A1,A2 more common ● The frequency of the ABO blood groups
differs among selected populations and
PHENOTYPE/ GENOTYPE
BLOODTYPE ethnic groups (e.g. group B is found twice
as frequently in blacks and Asians as in
A1 A1A1, A1O, A1A2 whites or subgroup A2 is rarely found in
A2 A2A2, A2O Asians)
● There is always an inverse reciprocal
A1B A1B relationship between the forward and
A2B A2B reverse type. For example, if the individual
has A antigens only on their red blood cells,
B BB, BO
there will be an “expected” naturally
O OO occurring anti-B antibody in their serum
since they lack the B antigen.
 Blood Anti- Anti-B Anti-A A B O ● Basic precursor is named as
type A Anti-B cells cells cells paragloboside/glycan Precursor substances
A 4+ 0 4+ 0 4+ 0 in RBCs are termed as type 2 = terminal
galactose is attached to
B 0 4+ 4+ 4+ 0 0 Nacetylglucosamine in beta 1 > 4 linkage.
AB 4+ 4+ 4+ 0 0 0 ● On the 37th day of fetal life,attachment of
immunodominant sugars occur on the RBC
O 0 0 0 4+ 4+ 0 membrane and it is dependent of ABH
genes inherited
INHERITANCE

ABH genes Punnet square – Reginald C. Punnet

● In 1924, the Theory of inheritance for ABO

blood groups was first described by
indicating that an individual inherits one
ABO gene from each parent and that these
two genes determine which ABO antigens
are present on the RBC membrane.
● This follows the simple Mendelian genetics.
● The O gene is considered an amorph.
The group O phenotype is an autosomal
recessive trait with the inheritance of two
nonfunctional O genes.
● A & B: Inherited as autosomal codominant
● 50% haplotype from mother, 50% haplotype
from father
● It is inherited in a codominant manner
● Dominant (A, B) – Traits or characteristics
that are always expressed whenever
present. Ex. AO= A ag BO= B ag
ABO ANTIGENS
● Recessive (O) Traits or characteristics that
are not express whenever the dominant ● Develop early in fetal life and do not
gene is present increase much in strength during the
● Silent/Amorph gene: a gene with no gestational period
observable effect, manifestation or product ● RBCs of the newborn have been estimated
to carry anywhere from 25% to 50% of the
PARENT A B O number of antigenic sites found on the
ALLELES
adult RBC
A AA AB AO ● Formation of ABH antigens results from the
interaction of genes at three separate loci
B AB BB BO (ABO, Hh, and Se). these genes produce
O AO BO OO glycosyltransferases that add sugars to a
basic precursor substance
Frequency of ABO blood group in Asian ● ABH antigens on the RBC are constructed
Population: O (40%) > A (28%) > B (25%) > AB on oligosaccharide chains of a type 2
(7%) precursor substance
● Antigens can be glycolipids, glycoproteins,
FORMATION OF ABH ANTIGENS or glycosphingolipids
● It has been postulated that bacteria, pollen
● ABO genes do not code for the antigen
particles, and other substances present in
themselves but for the production of
nature are chemically similar to A and B
glycosyltransferases that add
antigens
immunodominant sugars to a basic
precursor substance.
 ● The formation of ABH antigens results from
AB AB None
the interaction of genes at three separate
loci (ABO, Hh, and Se). O None/H antigen Anti-A, Anti-B,
● These do not produce antigens but produce Anti AB
specific glycosyltransferases that add sugar O – most common
to basic precursor substances. BLOOD TYPING
● Basic precursor is named as ● Routine blood bank procedure performed to
paragloboside/ceramide. detect unknown antigens or antibodies
● When the terminal galactose on the using known reagents.
precursor substance is attached to the N- ● Types:
acetylglucosamine in a beta 1 → 4 linkage, 1. Forward Typing/ direct/ cell typing
the precursor substance on erythrocytes is 2. Reverse Typing/ indirect/ backward/
referred to as type 2. ABH antigens on the serum
RBC are constructed on oligosaccharide FORWARD REVERSE
chains of a type 2 precursor substance. (screening (Confirmatory)
● A type 1 precursor substance refers to a PRIMAR To find out the To counter check
beta 1 → 3 linkage between galactose and Y blood type of a the result of
PURPOS person forward blood
N-acetylglucosamine. E typing

ABO ANTIBODIES SECONDARY To detect antigen To detect antibody

PURPOSE

● Naturally occurring because they are SPX Patient’s Red Cell Patient’s serum
produced without any exposure to RBCs Suspension (2-5%
RCS), (tomato red)
● Predominantly IgM, activate complement,
Whole blood or
and react at room temperature or colder anticoagulated
● ABO Antibodies includes: AntiA, AntiB, blood WB+ NSS

AntiAB REAGENTS Antisera (typing Known cells (A,

● Produce strong direct agglutination sera): AntiA B, O Known
reactions during ABO testing TS(Blue), AntiB TS cells)
(Yellow), AntiAB TS
● ABO antibody production is initiated at birth, (Colorless)
but titers are generally too low for detection RESULTS (+) (+) Agglutination, ()
until infants are 3 to 6 months old Agglutination, () No No Agglutination
● Antibody production peaks between 5 and Agglutination

10 years of age and declines later in life slide screened @ 2mins / TS - sodium azide
● ABO antibodies can cause rapid (preservative)
intravascular hemolysis if the wrong ABO
group is transfused, potentially resulting in
patient death

General Characteristics Of ABO Antibodies

● Not normally present at birth
● They develop 3-6 months after birth (3-6
mos.)
● They react better at RT They are present in
some plants and animals They are present
in low titer or even absent in some
conditions

GROUP ANTIGEN ANTIBODY

A A Anti-B

B B Anti-A

LESSON 2: ABO Blood Group System
BLOOD GROUP
ABO SUBGROUPS
● The antigens under the ABO system that
reacts less strongly with commercially
prepared typing agents.
● The original reports of most ABO subgroups
were made before the availability of the
monoclonal typing reagents currently used
in routine ABO grouping.
● ABO subgroups represent phenotypes
showing weaker and variable serologic
reactivity with the commonly used human
polyclonal anti-A, anti-B, and anti-A, B
reagents.
Anti-A1 Lectin - Differentiate A1 from A2
Anti-H Lectin differentiate O and Bombay
A Subgroups
● In 1911, Von Dungern described two
different A antigens based on reactions
between group A RBCs and anti-A and anti-
A1.
● Group A RBCs that react with both anti-A
and anti-A1 are classified as A1, whereas
those that react with anti-A and not anti-
A1 are classified as A2.
● RBCs from A1 and A2 individuals react
equally strongly with current reagent
monoclonal anti-A in ABO forward typing
tests. The A subgroups are more
common than B subgroups.
● The weaker serologic reactivity of ABO
subgroups is attributed to the decreased
number of A and B antigen sites on their red
blood cells
● The cells of approximately 80% of all
group A (or AB) individuals are A1 (or
A1B), and the remaining 20% are A2 (or
A2B) or weaker subgroups.
A1 VS. A2 PHENOTYPES
ANTI-A Anti-A1 LECTIN
REAGENT REAGENT
(Anti-A plus
Anti-A1)
A1 + +
A2 + 0
● The production of both types of antigens is
a result of an inherited gene at the ABO
locus. Inheritance of an A1 gene elicits
production of high concentrations of the
enzyme
α-3-N-acetylgalactosaminyltransferase,
which converts almost all of the H precursor
structure to A1 antigens on the RBCs.
● The very potent gene A1 creates between
810,000 and 1,170,000 antigen sites on the
adult A1 RBC, whereas inheriting an A2
gene results in production of only 240,000
to 290,000 antigen sites on the adult A2
RBC.
● The immunodominant sugar on both A1 and
A2 RBCs is N-acetylgalactosamine.
● This antibody can cause discrepancies
between forward and reverse ABO testing
and incompatibilities in crossmatches with
A1 or A1B cells.
● Anti-A1 is a naturally occurring IgM cold-
reacting antibody and is unlikely to cause
a transfusion reaction because it usually
reacts only at temperatures well below
37°C.
● In routine forward grouping, reagent anti-A
strongly agglutinates both A1 and A2
phenotypes.
● Differentiation of A1 and A2 phenotypes is
determined serologically using anti-A1
lectin, a reagent made from the seeds of
the plant Dolichos biflorus.
● Lectins: are seed extracts that agglutinate
human cells with some degree of specificity.
1. Dolichos biflorus - agglutinates A1 or
A1B (light yellow)
2. Bandeiraea simplicifolia -
agglutinates B cells (Anti-B lectin)
3. Ulex europaeus - agglutinin OF cells
(H specificity) and other ABO blood
groups depending on the amount of
H antigen available
CHARACTERISTICS OF A1 AND A2
PHENOTYPES
A. Reagents (A1 has more A antigenic sites)
B. Antibodies in serum
C. Others (A1, A2)
(Greatest amount of H) O > A2 > B > A2B > A1 >
A1B (Least amount of H)
● Due to the presence of so many A1
antigens, the H antigen on A1 and A1B
RBCs may be hidden and therefore may not
be available to react with anti-H antisera.
● In the presence of an A2 gene, only some
of the H antigen is converted to A antigens,
and the remaining H antigen is detectable
on the cell.
● This anti-H is a naturally occurring IgM cold
agglutinin that reacts best below room
temperature.
WEAK A SUBGROUPS
● Subgroups weaker than A2 occur
infrequently and are most often recognized
through an ABO discrepancy (unexpected
reactions in the forward and reverse
grouping).
● These subgroups of A make up 1% of
those encountered in the laboratory and
therefore are mainly of academic interest.
Characteristics of weak A subgroups include the
following:
● Decreased number of A antigen sites per
RBC (resulting in weak or no agglutination
with human polyclonal anti-A)
● Varying degrees of agglutination by human
anti-A
● Increased variability in the detectability of H
antigen, resulting in strong reactions with
anti-H
● Presence or absence of anti-A1 in the
serum
Weak A phenotypes can be serologically
differentiated using the following techniques:
● Forward grouping of A and H antigens with
anti-A, anti-A,B, and anti-H
● Reverse grouping of ABO isoagglutinins
and the presence of anti-A1
● Adsorption-elution tests with anti-A
- Confirmatory for weak blood subgroups
● Saliva studies to detect the presence of A
and H substances
● Molecular testing
Weak Subgroups Of A
● A3: Demonstrate mf reaction with anti-A
and most anti-AB (mixed field)
● Ax: RBC do not agglutinate with Anti-A but
do not agglutinate with Anti-AB
● Aend: Demonstrate mf reaction but only
small amounts of rbc agglutinate less than
or equal 10 red cells
● Ay3: RBCs are not agglutinated or weakly
agglutinated by either Anti-A or Anti-B
● Ay: RBCs are not agglutinated by either
Anti-A and Anti-B – small a substance
● Ael: Typically no agglutination by Anti-A or
Anti-AB (only H substance)
A3, Ax, Aend - blood
Ay3, Ay, Ael - saliva

B SUBGROUPS
Subgroups of B are very rare and much less
frequent than A subgroups.
● Subgroups of B are usually recognized by
variations in reaction strength using anti-B
and anti-A,B.
● Inheritance of B subgroups, similar to that of
the majority of A subgroups, is considered
to be a result of alternate alleles at the B
locus.
Criteria used for differentiation of weak B
phenotypes include the following techniques:
● Strength and type of agglutination with anti-
B, anti-A,B, and anti-H
● Presence or absence of ABO isoagglutinins
in the serum
● Adsorption-elution studies with anti-B
● Presence of B substance in saliva
● Molecular testing
Weak B Subgroups
● RBCs demonstrating serologic activity that
are weaker than normal are designated
weak B phenotypes or B subgroups.
● A classification system similar to A
subgroups has been used because of
common serologic characteristics.
● Serologic techniques can be used to
characterize B subgroups in the following
categories: B3, Bx, Bm, and Bel.
Weak Subgroups Of B ( no Bend, By)
B3, Bx - Blood / Bel, Bm - Saliva
● B3: Demonstrate with anti-B and most anti-
AB ( Mixed field)
● Bx: RBC do not agglutinate with Anti-A but
do not agglutinate with anti-AB
● Bel: Extremely rare subgroup
unagglutinated by Anti-B or Anti-AB – only
H substance
● Bm: RBCs are not agglutinated or weakly
agglutinated by either anti-A or anti-B
LESSON 3: Bombay Phenotype
THE BOMBAY PHENOTYPE (Oh)
● The Bombay phenotype was first reported
by Bhende in 1952 in Bombay, India.
● The phenotype results from the inheritance
of a double dose of the h gene, producing
the very rare genotype hh or Hnull.
● 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 (Oh)
Bombay Phenotype
● hh or Hnull 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 the serum
● Can only be transfused with blood from
another Bombay (Oh)
● The (Oh) Bombay phenotype is inherited as
an autosomal recessive trait.
● The underlying molecular defect is most
often a mutation in the gene FUT1 (H
gene), producing a silenced gene
incapable of coding for the enzyme, L-
fucosyltransferase (H transferase).
● This mutation underlying the Bombay
phenotype is also associated with a
silenced FUT2 gene (Se gene).
GENERAL CHARACTERISTICS OF BOMBAY Oh
(H null) PHENOTYPES
● Absence of H, A, and B antigens;
● Presence of anti-A, anti-B, anti-A,B, and a
potent wide thermal range of anti-H in the
serum
● A, B, H nonsecretor
● Absence of L-fucosyltransferase (H
enzyme) in serum and H antigen on red
blood cells
● Presence of A or B enzymes in serum
(depending on ABO genotype)
● A recessive mode of inheritance (identical
phenotypes in children but not in parents)
● RBCs of the Bombay phenotype (Oh) will
not react with the anti-H lectin (Ulex
europaeus)
● RBCs of the Bombay phenotype (Oh) are
compatible only with the serum from
another Bombay individual
● Since these RBCs lack normal ABH
antigens, they fail to react with anti-A, anti-
B, and anti-H antisera. The Bombay would
phenotype as an O blood group
● However, RBCs of the Bombay phenotype
(Oh) do not react with the anti-H lectin (Ulex
europaeus)
● Bombay serum contains anti-A, anti-B, anti-
A,B, and anti-H
 ● The Bombay anti-H can often be potent and
reacts strongly at 37°C
● It is an IgM antibody capable of binding
complement and causing RBC lysis
● Transfusing normal group O blood (with the
highest concentration of H antigen) to a
Bombay recipient (anti-H in the serum)
would cause immediate cell lysis
● Only blood from another Bombay individual
will be compatible and can be transfused to
a Bombay recipient
TWO (2) TYPES OF BOMBAY PHENOTYPE:
1. Classical
● Do not inherit the H gene
● They lack the A, B H antigen
● Designated as Oh
● Produces anti-A, anti-B, anti-H
2. Parabombay
● Weak expression of H gene
● With A and B antigens
● No detectable H antigen
● Designated as: Ah, Bh, ABh
● With anti-H
TWO (2) TYPES OF ANTI-H
1. Naturally occurring anti-H
● Produced by individual with little
amount of H antigen
2. Anti-H produced by Bombay individual
● Active over a wide thermal range. It
can cause RBC lysis.
LESSON 4: ABO Discrepancies
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
● Common Sources of Technical Errors
Resulting in ABO Discrepancies
1. Incorrect or inadequate identification
of blood specimens, test tubes, or
slides
2. Cell suspension either too heavy or
too light
3. Clerical errors or incorrect recording
of results
4. A mix-up in samples
5. Missed observation of hemolysis
6. Failure to add reagents
7. Failure to add sample
8. Failure to follow manufacturer’s
instructions
9. Uncalibrated centrifuge
10. Over Centrifugation or under
centrifugation
11. Contaminated reagents
12. Warming during centrifugation
General Rules to Resolve Discrepancies:
● Always re-test first – Detects presence of
procedural errors
● Check for clerical technical errors
● Weakest reaction is usually the one in doubt
● Check results of the screening cells
● Check the patient’s age
● Check the diagnosis
● Check the transfusion history
ABO discrepancies may be arbitrarily
divided into four major categories: group I, group II,
group III, and group IV discrepancies.
GROUP I DISCREPANCIES
● Discrepancies are associated with
unexpected reactions in the reverse
grouping due to weakly reacting or missing
antibodies
● More common than other group
 ● If a reaction in serum grouping is weak or ● Some of the causes of discrepancies in
missing, Group 1 discrepancy should be this group include:
suspected because, normally, RBC and 1. Subgroups of A (or B) may be
serum grouping reactions are very strong present
(4+) 2. Leukemias may yield weakened
● Low antibody production or cannot produce A or B antigens and Hodgkin’s
ABO antibodies disease
● Patients whose existing ABO antibodies 3. Ab to low incidence of antigen
may have been diluted by plasma 4. Excess Blood Group Specific
transfusion Soluble Substances (BGSS)
● Common populations with discrepancies in
this group are:
1. Newborns
2. Elderly patients
3. Patients with a leukemia (e.g.,
chronic lymphocytic leukemia) or Stomach Cancer
lymphoma (e.g., malignant The “acquired B” phenomenon will show
lymphoma) demonstrating weak reactions with anti-B antisera and is most
hypogammaglobulinemia often associated with diseases of the digestive tract
4. Patients using (e.g., cancer of the colon).
immunosuppressive drugs that
● When bacterial enzyme, Deactylase,
yield hypogammaglobulinemia
modify N-acetylgalactosamine into D-
5. Patients with congenital or
galactose
acquired agammaglobulinemia
● Increased permeability of the intestinal wall
or immunodeficiency diseases
causes adsorption of B-like bacterial
6. Patients with bone marrow or
polysaccharide onto red cells of A or B pxs.
stem cell transplantations
What causes acquired B phenomenon?
7. Patients whose existing ABO
antibodies may have been ● Bacteria: Proteus vulgaris, Escherichia
diluted by plasma transfusion or coli type 086
exchange transfusion ● Bacterial enzyme: Deacetylase
8. ABO subgroups ○ Cleaves N-Acetyl
○ Galactosamine + Anti-B = Weak
Resolution of Common Group I Discrepancies
reaction
● Obtaining the patient’s history may resolve
this type of discrepancy, such as a newborn
sample that would not have ABO antibodies
in the serum until the child was 3 to 6
months of age.
● If the history indicates an elderly individual,
or the diagnosis indicates
hypogammaglobulinemia, enhancing the
weak or missing reaction by incubating at
room temperature 15-30 minutes; if no
Resolution of Common Group II Discrepancies
reaction incubate at 4degC 15-30 minutes
● The agglutination of weakly reactive
GROUP II DISCREPANCIES
antigens with the reagent antisera can be
● Discrepancies are associated with enhanced by incubating the test mixture at
unexpected reactions in the forward room temperature 15-30 minutes; if no
grouping due to weakly reacting or reaction incubate at 4degC 15-30 minutes
missing antigens ● BGSS: Washed RBCS
● This group of discrepancies is probably ● Detection of True Antibody with Acquired B
the least frequently encountered. phenomenon
 1. Use of monoclonal Ab
Monoclonal anti-B clone ES4
2. Use of acidified anti-B - Typing
Serum Acidification, 1-2 drops of
HCl to 1 mL Typing Reagent (pH
maintained at 6.0)
3. Addition of acetic anhydride
4. Determination of secretor status
5. Use of anti-B lectin- best method
■ Bandeiraea simplicifolia
■ Griffonia simplicifolia
GROUP III DISCREPANCIES
These discrepancies between forward and
reverse groupings are caused by protein or
plasma abnormalities and result in rouleaux
formation or pseudoagglutination, attributable to:
● Elevated levels of globulin from certain
disease states, such as multiple myeloma,
Waldenström’s macroglobulinemia, other
plasma cell dyscrasias, and certain
moderately advanced cases of Hodgkin’s
lymphomas
● Elevated levels of fibrinogen
● Plasma expanders, such as dextran and
polyvinylpyrrolidone
● Wharton’s jelly in cord blood samples
Resolution of Common Group III Discrepancies
● Rouleaux is a stacking of erythrocytes that
adhere in a coinlike fashion, giving the
appearance of agglutination
● Cell grouping can usually be accomplished
by washing the patient’s RBCs several
times with saline
● Wash cord cells 6-8 times
● Add 1-2 drops of NSS in the test tube then
view again (LPO and HPO) - limit
rouleaux
GROUP IV DISCREPANCIES
These discrepancies between forward and
reverse groupings are due to miscellaneous
problems and have the following causes:
● Cold reactive autoantibodies in which RBCs
are so heavily coated with antibody that
they spontaneously agglutinate,
independent of the specificity of the reagent
antibody
● Patient has circulating RBCs of more than
one ABO group due to RBC transfusion or
marrow/stem cell transplant
● Polyagglutination (Hubener-Thomsen
Friedenreich Phenomenon)
● Unexpected ABO isoagglutinins
● Unexpected non-ABO alloantibodies
● RBCs with cis AB phenotype
○ Polyagglutination – Due to bacterial
or viral infection that produces
enzyme Muraminidase (Clostridium
sp., Vibrio sp., Pneumococci,
Influenza viruses)
○ RBCs with cis AB phenotype – 3
genes are inherited, resulting
antigen is weak
Resolution of Common Group IV Discrepancies
● Incubating at 37degC for 15-30 mins and
wash with saline @ 37degC 3x then retype
● RBCs heat with 0.01M dithiothreitol DTT to
disperse IgM
● 2 TEST FOR AUTOCONTROL:
1. Perform cold ab panel
2. Rabbit erythrocyte Stroma test
Best detects auto antibody P and I
Anti-M
Anti-N
Anti-S
Anti-P
Anti-Lewis
Cold reacting antibodies
Do not water bath