SUMMARY OF FORWARD & REVERSE GROUPINGS

ABO BLOOD GROUP SYSTEM

FORWARD REVERSE
Blood Group
Antigen (RBC) Antibody (Serum)
O No A or B antigen Anti-A, Anti-B
INTRODUCTION:
A A Anti-B
B B Anti-A
• ISBT Number: 001
AB A and B No A or B antibodies
• Number of antigens: 4
o ABO1: A ○ ABO3: A,B
o ABO2: B ○ ABO4: A1 GRADING OF TUBE AGGLUTINATION REACTIONS:
• Most important of all blood groups in both transfusion
and transplant medicine. Macroscopic (Visual) Readings:
Grade Description of Reaction
• The only blood group with naturally occurring
antibodies (found in the serum) against the antigens 4+ One solid agglutinate; clear background
that are absent from their red blood cells. 3+ Several large agglutinates; clear background
2+ Medium-sized agglutinates; clear background
o NOTE: ABO antibodies are primarily IgM-type and 1+ Many small agglutinates; turbid background
naturally occurring, which means they are present 0 No agglutination; turbid background
in the serum without any prior exposure to foreign Microscopic Readings:
RBCs through blood transfusion, organ donation, or
Grade Description of Reaction
pregnancy (in contrast to alloimmunization).
W+ Barely visible agglutination
• When a donated blood unit is given to a patient with 0 No agglutination; cells float freely
an incompatible blood type, this will result in the W+, weakly positive
“tagging” of donor RBCs for destruction.
o ABO incompatibility reactions are the most feared • The contents of each tube are examined after gentle
immune-mediated hemolytic transfusion reactions rotation and inclination using the naked eye.
due to their ability to cause rapid intravascular • Weaker grades of agglutination are verified by
hemolysis, potentially resulting in patient death. microscopic examination.
o Shortened survival of the donor RBCs (in-vivo or • Reaction gradings vary from patient to patient.
post-transfusion).
ABO Forward Grouping:

HISTORICAL PERSPECTIVE: Donor/Patient Donor/Patient Interpretation of

RBCs with RBCs with Patient/Donor
• Karl Landsteiner (1901) discovered the ABO blood Anti-A Anti-B Blood Group
group system by mixing the red blood cells and serum
0 0 O
of each of his staff. He demonstrated that the serum of
some people agglutinated the red cells of others. 4+ 0 A
• Blood grouping: or blood typing; is the most frequently 0 4+ B
performed laboratory test in the blood bank. Ideally, 4+ 4+ AB
both FORWARD and REVERSE typing must be
performed on all donors and patients. ABO Reverse Grouping:

o Forward grouping: Donor/Patient Donor/Patient Interpretation of

▪ Also called front typing Serum with Serum with Patient/Donor
▪ Uses known sources of commercial antisera to A1-cells B-cells Blood Group
detect ABO antigens on an individual’s RBCs. 4+ 4+ O
✓ Anti-A: Monoclonal IgM; clear; blue color
0 3+ A
✓ Anti-B: Monoclonal IgM; clear; yellow color
✓ Anti-A,B: Monoclonal IgM; clear; colorless, 3+ 0 B
not routinely used; used to confirm group O 0 0 AB
donor units; it is not a mixture of anti-A and
anti-B blood grouping reagents.
▪ Methods: slide, tube, and gel methods
ABO ANTIBODIES (ISOAGGLUTININS):
o Reverse grouping:
▪ Also called back typing • Isoagglutinins are antibodies produced by an
▪ Using commercial reagent or manually prepared individual that cause agglutination of RBCs in other
reagent RBCs of known blood groups. individuals. People possess isoagglutinins directed
✓ Reagent A1 cells: 4%–5%; human source toward the A or B antigen absent from their own RBCs.
✓ Reagent B cells: 4%–5%; human source For example, type B or O individuals will usually
▪ It is used to detect ABO antibodies in the possess anti-A.
patient’s serum using the known reagent RBCs. • ABO antibodies have been described as naturally
▪ Confirmatory testing for the forward grouping. occurring because they are produced without any
▪ Methods: tube and gel methods exposure to RBCs.
• Predominantly IgM antibodies (small quantities of IgG
• There is always an inverse reciprocal relationship may also be present), which can activate complement
between the forward and reverse type; thus, one and react at room temperature or colder.
serves as a check on the other. • Produce strong direct agglutination reactions, but
reaction gradings vary from patient to patient.
Example: If the individual has A antigens only on their • ABO antibody production is initiated at birth, but titers
red blood cells, there will be an “expected” naturally are generally too low for detection until infants are 3 to
occurring anti-B antibody in their serum since they 6 months old.
lack the B antigen.
 o Results of serum ABO testing before 3 to 6 months
of age cannot be considered valid.
o Perform only FORWARD grouping on cord blood
from newborn infants (some or all of the antibodies
present are mostly IgG maternal antibodies).
• Antibody production peaks between 5 and 10 years
of age and declines later in life.
o Elderly people usually have lower levels of anti-A
and anti-B; therefore, antibodies may be
undetectable in the reverse grouping.
INHERITANCE OF ABO BLOOD GROUPS:
• Inheritance of the ABO blood group is codominant in
expression.
o Codominance means that neither allele can
mask the expression of the other allele; both
alleles will be expressed by the offspring.
o An offspring inherits one ABO gene from each
parent.
• The ABO gene is located on chromosome 9 (locus:
9q34.1-q34.2.) and has three alleles—A, B, and O.
• A and B genes: autosomal dominant
• O gene: considered as amorph (no detectable
antigen is produced in response to the inheritance of
this gene); autosomal recessive
• ABO designations:
o A, B, AB, and O are phenotypes.
o AA, AO, BB, BO, AB, and OO are genotypes.
• The ABO blood group gene codes for a specific
carbohydrate transferase enzyme that adds an
immunodominant carbohydrate to the H antigen.
• Group A phenotype:
o The A allele encodes for the α-3-N-
acetylgalactosaminyltransferase enzyme that
adds N-acetyl-D-galactosamine (GalNAc) to
the glycoprotein H antigen that is expressed on
all normal red cells.
• Group B phenotype:
o The B allele encodes for the α-3-D-
galactosyltransferase enzyme that adds D-
galactose to the glycoprotein H antigen that is
expressed on all normal red cells.
• Group O phenotype:
o The group O phenotype is an autosomal
recessive trait with the inheritance of two
nonfunctional O genes.
FORMATION AND INTERACTION OF ABH ANTIGENS:
H and Se Genes:
• The H antigen (a glycoprotein) is a basic precursor
substance to the ABO blood group antigens. The H
antigen is expressed on all normal red cells.
o Immunodominant sugar: L-fucose
o The H antigen is not part of the ABO blood group
system, but its inheritance influences the
expression of the ABO antigens.
o Chromosome 19: location of FUT 1 (H) and FUT
2 (Se) genes.
o Individuals not expressing the H antigen belong
to the Bombay (h/h or Oh) phenotype (H antigen
deficiency).
o NOTE: Bombay phenotype individuals lack the H
antigen on their RBCs; hence, when erroneously
transfused with H-antigen-containing RBCs, anti-
H alloantibodies will be formed – a severe or
lethal hemolytic transfusion reaction will result.
o H gene: elicits the production of an enzyme
called α-2-L-fucosyltransferase that transfers
the sugar L-fucose to an oligosaccharide chain on
the terminal galactose of type 2 chains.
▪ H genotypes: HH, Hh, and hh (Bombay)
▪ H gene is present in more than 99.99% of the
random population.
▪ The hh genotype does not elicit the
production of α-2-L-fucosyltransferase. This
genotype is extremely rare.
Type 1 and Type 2 Precursor Substance:
• Type 1: Beta 1→3 linkage between galactose and
N-acetylglucosamine
• Type 2: When the terminal galactose on the
precursor substance is attached to the N-
acetylglucosamine in a beta 1→4 linkage.
Sugars occupying the terminal positions of this precursor
chain and conferring blood group specificity are called the
immunodominant sugars:
• H antigen: L-fucose
• A antigen: N-acetyl-D-galactosamine
• B antigen: D-galactose
Formation of the Blood Group O:
• Historically designated as “C” (was later renamed “O”)
• Inheritance:
o Individuals who are blood group O inherit at least
one FUT 1 (H) gene (genotype HH or Hh) and
two O genes.
o O gene (genotype OO) is an amorph and does
not elicit the production of a catalytically active
polypeptide transferase; therefore, the H
substance remains unmodified.
o The O blood group has the highest concentration
of the H antigen.
o Anti-H lectin blood grouping reagent is prepared
from an extract of Ulex europaeus seeds.
Formation of the Blood Group A:
• The A gene (genotypes AA or AO) codes for the
production of α-3-N-acetylgalactosaminyltransferase,
which transfers an N-acetyl-D-galactosamine
(GalNAc) sugar to the H substance.
• The A gene tends to elicit higher concentrations of
transferase than the B gene, leading to conversion of
nearly all of the H antigen on the RBCs to A antigen
sites.
• As many as 810,000 to 1,170,000 antigen sites exist
on an A1 adult RBC in response to inherited genes.
• Group A genotypes and phenotypes:
Genotype Phenotype
A1A1 A1
A1A2 A1
A1O A1
A2A2 A2
A2O A2
Formation of the Blood Group B:
• The B gene (genotypes BB or BO) codes for the
production of α-3-D-galactosyltransferase, which
attaches D-galactose (Gal) sugar to the H substance
previously placed on the type 2 precursor substance
through the action of the H gene.
• Anywhere from 610,000 to 830,000 B antigen sites
exist on an adult group B RBC.
• Group B genotypes and phenotypes:
Genotype Phenotype
BB B
BO B
Formation of the Blood Group AB:
• This blood group results when both the A and B genes
are inherited.
• Genotype: AB
• The A and B enzymes compete for the binding to the
H substance.
o B enzyme seems to compete more efficiently for
the H substance than the A enzyme.
• Group AB genotypes and phenotypes:
Genotype Phenotype
A1B AB
A2B AB
FORMATION OF ABH SOLUBLE ANTIGENS:
• Their presence is dependent on the ABO genes
inherited and on the inheritance of another set of
genes called secretor genes (genotypes SeSe or
Sese) that regulate their formation.
• The inheritance of a FUT 2 (Se) gene codes for the
production of the α-2-L-fucosyltransferase that
modifies the type 1 precursor substance in secretions
to form an H substance (L-fucose).
• It is the presence of the Se gene–specified α-2-L-
fucosyltransferase that determines whether ABH-
soluble substances will be secreted.
• People who inherit the sese (take note of the
lowercase letter “s”) genotype are termed non-
secretors.
Body fluids in which ABH substances can be detected
in secretors (people who inherited the secretor genes
[SeSe or Sese]):
1. Saliva
2. Tears
3. Urine
4. Digestive juices
5. Bile
6. Milk
7. Amniotic fluid
8. Pathological fluids: pleural, peritoneal, pericardial,
ovarian cyst
Comparison of ABH Antigens on RBCs with A, B, and
H Soluble Substances:
ABH Antigens on RBC Membrane:
• RBC antigens can be glycolipids, glycoproteins, or
glycosphingolipids.
• RBC antigens are synthesized only on type 2
precursor chains.
• Type 2 chain refers to a beta 1→4 linkage in which
the number one carbon of the galactose is attached
to carbon number four of the N-acetylglucosamine
sugar of the precursor substance.
• The enzyme produced by the H (FUT 1) gene (α-2-L-
fucosyltransferase) acts primarily on type 2 chains,
which are prevalent on the RBC membrane.
A, B, and H Soluble Substances:
• Secreted substances are glycoproteins.
• Secreted substances are primarily synthesized on
type 1 precursor chains.
• Type 1 chain refers to a beta-1→3 linkage in which
the number one carbon of the galactose is attached
to carbon number 3 of the N-acetylglucosamine
sugar of the precursor substance.
• The enzyme produced by the Se (FUT 2) gene (α-2-
L-fucosyltransferase) preferentially acts on type 1
chains in secretory tissues.
ABH Substance in the Saliva of Secretors (Genotypes
SeSe or Sese):
Substances in the Saliva of Secretors*
ABO Group A B H
O None None ↑↑
↑
A ↑↑ None
B None ↑↑ ↑
AB ↑↑ ↑↑ ↑
*Non-secretors (genotype: sese) have no ABH
substances in saliva. ↑↑ and ↑, respectively, represent the
approximate concentration of ABH substances in the
saliva of secretors (genotypes: SeSe or Sese).
Lectins Used in Blood Banking:
• Lectins: they are phytohemagglutinins (plant-
derived cell-agglutinating proteins) prepared from
seed extracts that agglutinate human red cells with
some degree of specificity to certain carbohydrates.
• Dolichos biflorus: agglutinates group A1 or A1B cells
(binds to N-acetyl-D-galactosamine residues).
• Griffonia (Bandeiraea) simplicifolia: agglutinates
group B cells (binds to D-galactose residues).
• Ulex europaeus: preferentially agglutinates group O
cells (H antigen specificity; binds to L-fucose
residues) and other ABO blood groups (e.g., group A2
red blood cells) depending on the amount of H
antigen available.
ABO SUBGROUPS:
GROUP A SUBGROUPS:
• First described by Emil von Dungern (1911).
• The differentiation between A1 and A2 phenotypes is
determined serologically using the anti-A1 lectin,
which is obtained from an extract of the Dolichos
biflorus seeds.
Anti-A Anti-A1
A Subgroups
(Anti-A + Anti-A1) Lectin
A1 + +
A2 + 0
+, positive (agglutination); 0, negative (no reaction)
• The immunodominant sugar on both A1 and A2 RBCs
is N-acetyl-D-galactosamine.
• Weak subgroups of the A antigen will often have an
inverse reciprocal relationship between the
amount of H antigen on the RBC and the amount of A
antigens formed (i.e., the more A antigen is formed,
the less H antigen is expressed on the RBC).
o O > A2 > B > A2B > A1 > A1B
(Arranged from greatest to least amount of H
antigen)
WEAK GROUP A SUBGROUPS:
• Weak A subgroups are often mistyped as group O
and may be potentially dangerous.
• They occur infrequently and are most often
recognized through an ABO discrepancy.
• Weak A phenotypes can be serologically
differentiated using the following techniques:
o Forward grouping of A and H antigens with anti-
A, anti-A,B, and anti-H
o Reverse grouping of ABO isoagglutinins and the
presence of anti-A1
o Adsorption-elution tests with anti-A (Am, Ay, Ael)
o Saliva (secretor) studies to detect the presence of
A and H substances
• Characteristics of weak A subgroups include the • Agglutination reactions (forward grouping):
following: o Anti-A: negative to weak
o Decreased number of A antigen sites per RBC o Anti-B: negative (no reaction)
(resulting in weak or no agglutination with human o Anti-A,B: negative to weak
polyclonal anti-A) o Anti-H: strongly positive
o Varying degrees of agglutination by the human • Antibodies in serum (reverse grouping):
anti-A,B reagent. o Anti-A: none
o Anti-B: present
Characteristics of the Weak A Subgroups: o Anti-A1: none

Subgroup A3: Subgroup Ay:

• Characteristically demonstrate a mixed-field pattern • Not agglutinated by anti-A or anti-A,B reagents.
of agglutination with anti-A and most anti-A,B • Adsorption and elution of anti-A is the method used
reagents. to confirm the presence of A antigens.
• Anti-A1 may be present in the serum of A3 individuals. • Normal quantities of A and H substances are found in
• A and H substances are detected in the saliva of A3 the saliva of Ay secretors.
secretors. • Serologically, ‘Ay’ is almost similar to ‘Am’ and even
• Number of antigen sites: 35,000 per RBC the adsorption–elution test fails to differentiate the two
o Mixed-field: can be defined as small agglutinates phenotypes.
within predominantly non-agglutinated RBCs. • Agglutination reactions (forward grouping):
• Agglutination reactions (forward grouping): o Anti-A: negative (no reaction)
o Anti-A: positive (mixed-field) o Anti-B: negative (no reaction)
o Anti-B: negative (no reaction) o Anti-A,B: negative (no reaction)
o Anti-A,B: positive (mixed-field) o Anti-H: strongly positive
o Anti-H: moderately positive • Antibodies in serum (reverse grouping):
• Antibodies in serum (reverse grouping): o Anti-A: none
o Anti-A: none o Anti-B: present
o Anti-B: present o Anti-A1: none
o Anti-A1: sometimes present Subgroup Ael:
• Ael RBCs typically are not agglutinated by anti-A or
Subgroup AX: anti-A,B reagents; however, adsorption and elution
• Characteristically demonstrate a negative reaction can be used to demonstrate the presence of the A
(no agglutination) by anti-A reagent but do antigen.
demonstrate agglutination with anti-A,B. • Ael individuals usually produce an anti-A1 that is
• Number of antigen sites: 4,000 per RBC reactive with A1 cells and sometimes produce anti-A,
• Secretor studies detect the presence of only H which agglutinates A2 RBCs.
substance in the saliva of Ax secretors. • Secretor studies demonstrate the presence of only H
• Agglutination reactions (forward grouping): substance in the saliva of Ael secretors.
o Anti-A: negative to weak • No detectable A transferase enzyme activity can be
o Anti-B: negative (no reaction) demonstrated.
o Anti-A,B: positive • Agglutination reactions (forward grouping):
o Anti-H: strongly positive o Anti-A: negative (no reaction)
• Antibodies in serum (reverse grouping): o Anti-B: negative (no reaction)
o Anti-A: none to weak o Anti-A,B: negative (no reaction)
o Anti-B: present o Anti-H: strongly positive
o Anti-A1: almost always present • Antibodies in serum (reverse grouping):
o Anti-A: sometimes present
Subgroup Aend: o Anti-B: present
• Characteristically demonstrate very weak mixed-field o Anti-A1: present
agglutination with some anti-A and anti-A,B reagents.
• Number of antigen sites: 3,500 per RBC Summary of the weak A subgroups:
• Secretor studies detect the presence of only H ABO Forward Grouping
Weak A
substance in the saliva of Aend secretors.
Subgroup Anti-A Anti-B Anti-A,B Anti-H
• No detectable A transferase enzyme activity can be
demonstrated. A3 2+ (mf) 0 2+ (mf) 3+
Ax wk/0 0 2+ 4+
• Agglutination reactions (forward grouping):
o Anti-A: weak (mixed-field) Aend wk (mf) 0 wk (mf) 4+
o Anti-B: negative (no reaction) *Am 0/wk 0 0/1+ 4+
o Anti-A,B: weak (mixed-field) *Ay 0 0 0 4+
o Anti-H: strongly positive *Ael 0 0 0 4+
• Antibodies in serum (reverse grouping): +, positive (agglutination); 0, negative (no reaction);
o Anti-A: none mf, mixed-field; wk, weak reaction
o Anti-B: present
o Anti-A1: sometimes present *A subgroup specificity can be demonstrated only by
adsorption–elution procedures –– Am, Ay, and Ael.
Subgroup Am:
• Characteristically not agglutinated, or are Weak A Reverse Grouping / Secretor Study
agglutinated only weakly, by anti-A or anti-A,B Subgroup Anti-A Anti-B Anti-A1 Sec.
reagents. A3 0 + 0/+ A, H
• Number of antigen sites: 200 to 1,900 per RBC Ax 0/wk + + H
• These individuals usually do not produce anti-A1 in Aend 0 + 0/+ H
their sera. Am 0 + 0 A, H
• Normal quantities of A and H substances are found in Ay 0 + 0 A, H
the saliva of Am secretors. Ael 0/+ + + H
WEAK GROUP B SUBGROUPS: Subgroup Bel:
• Subgroups of B are very rare and much less • Bel RBCs are not agglutinated by anti-B or anti-A,B
frequent than A subgroups. antisera.
• Subgroups of B are usually recognized by variations • This extremely rare phenotype must be determined
in reaction strength using anti-B and anti-A,B. by adsorption and elution of anti-B.
• Criteria used for differentiation of weak B phenotypes • No B glycosyltransferase has been identified in the
include the following techniques: serum or RBC membrane of Bel individuals.
o Strength and type of agglutination with anti-B, • A weak anti-B may be present in the serum of this
anti-A,B, and anti-H subgroup.
o Presence or absence of ABO isoagglutinins in the • Only the H substance is demonstrated in the saliva of
serum Bel secretors.
o Adsorption-elution studies with anti-B • Agglutination reactions (forward grouping):
o Presence of B substance in saliva o Anti-A: negative (no reaction)
o Molecular testing o Anti-B: negative (no reaction)
o Anti-A,B: negative (no reaction)
Characteristics of the Weak B Subgroups: o Anti-H: moderately positive
• Antibodies in serum (reverse grouping):
Subgroup B3: o Anti-A: Present
• Characterized by a mixed-field pattern of o Unexpected antibodies: weak anti-B
agglutination with anti-B and anti-A,B antisera.
• B glycosyltransferase is present in the serum but not
in the RBC membranes of these individuals. Summary of the weak B subgroups:
• Anti-B is absent in the serum of B3 phenotypes, yet B
substance is present in normal amounts in the saliva Weak B ABO Forward Grouping
of secretors. Subgroup Anti-A Anti-B Anti-A,B Anti-H
• The B3 subgroup is the most frequent weak B B3 0 2+ (mf) 2+ (mf) 3+
phenotype. Bx 0 wk wk 3+
• Agglutination reactions (forward grouping): *Bm 0 0/wk 0/wk 3+
o Anti-A: negative (no reaction) *Bel 0 0 0 3+
o Anti-B: moderately positive (mixed-field)
o Anti-A,B: moderately positive (mixed-field) +, positive (agglutination); 0, negative (no reaction);
o Anti-H: moderately positive mf, mixed-field; wk, weak reaction
• Antibodies in serum (reverse grouping):
o Anti-A: Present *B subgroup specificity can be demonstrated only by
o Unexpected antibodies: none adsorption–elution procedures –– Bm and Bel.

Subgroup BX:
• Typically demonstrate weak agglutination with anti-B
and anti-A,B antisera. BOMBAY PHENOTYPE (Oh):
• B glycosyltransferase has not been detected in the
serum or in the RBC membranes of Bx phenotypes, • Absence of the H antigen (Hnull phenotype)
but a weakly reactive anti-B usually is produced. • First reported by Bhende in 1952 in Bombay, India.
• Bx RBCs readily adsorb and elute anti-B. • Bombay phenotype results from the inheritance of a
• Secretor studies demonstrate large amounts of H double dose of the h gene (note the lowercase h),
substance. producing the very rare genotype hh.
• Agglutination reactions (forward grouping): • Phenotypes as blood group O in forward-typing.
o Anti-A: negative (no reaction)
o Anti-B: weak General Characteristics of the Bombay Phenotype:
o Anti-A,B: weak o Absence of H, A, and B antigens; no agglutination
o Anti-H: moderately positive with anti-A, anti-B, or anti-H lectin.
• Antibodies in serum (reverse grouping): o Presence of anti-A, anti-B, anti-A,B, and a potent
o Anti-A: Present wide thermal range of anti-H in the serum.
o Unexpected antibodies: none o A, B, H non-secretor (no A, B, or H substances
present in saliva).
Subgroup Bm: o Absence of α-2-L-fucosyltransferase (H enzyme)
• Bm RBCs are characteristically not agglutinated by in serum and H antigen on red blood cells.
anti-B or anti-A,B antisera. o Presence of A or B enzymes in serum (depending
• The Bm RBCs easily adsorb and elute anti-B. on ABO genotype).
• B glycosyltransferase is present in the serum of Bm o A recessive mode of inheritance (identical
phenotypes but is usually lower in enzymatic activity. phenotypes in children but not in parents).
• Anti-B is not characteristically present in the serum of o RBCs of the Bombay phenotype (Oh) will not
Bm individuals. react with the anti-H lectin (Ulex europaeus)
• Normal quantities of H and B substance are found in o RBCs of the Bombay phenotype (Oh) are
the saliva of Bm secretors. compatible only with the serum from another
• The Bm subgroup is reported to be more frequent in Bombay (Oh) individual.
Japan.
• Agglutination reactions (forward grouping): Blood ABO Forward Grouping
o Anti-A: negative (no reaction) Group Anti-A Anti-B Anti-A,B Anti-H
o Anti-B: negative (no reaction) A + 0 + +
o Anti-A,B: negative (no reaction) B 0 + + +
o Anti-H: moderately positive AB + + + +
• Antibodies in serum (reverse grouping): O 0 0 0 +
o Anti-A: Present Bombay 0 0 0 0
o Unexpected antibodies: weak anti-B
+, positive (agglutination); 0, negative (no reaction)
ABO DISCREPANCIES:
• They occur when unexpected reactions are
obtained in the forward and/or reverse grouping.
• These can be due to problems with:
o The patient’s serum (reverse grouping);
o Problems with the patient’s RBCs (forward
grouping); or
o Problems with both the serum and cells.
• The unexpected reaction(s) may 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.
o Enhance the weak or missing reaction by
extending the incubation time of the serum–red
cell mixture at room temperature for
approximately 15 to 30 minutes and then
centrifuge. Observe for agglutination.
o If there is still no reaction after centrifugation, the
serum-red cell mixture can be incubated at 4°C
for 15 to 30 minutes.
o An auto-control and O cell control must always
be tested concurrently with the reverse typing.
▪ The lower temperature of testing will most
likely enhance the reactivity of other
commonly occurring cold agglutinins, such as
anti-I, that react with all adult RBCs.

Categories of ABO Discrepancies: Group II Discrepancies:

• Associated with unexpected reactions in the forward
Group I Discrepancies: grouping due to weakly reacting or missing antigens
• Associated with unexpected reactions in the reverse in the patient’s red blood cells.
grouping due to weakly reacting or missing antibodies
in the patient’s serum. For example: For example:

Forward Typing Reverse Typing Patient’s Forward Typing Reverse Typing Patient’s
(Patient RBCs) (Patient Serum) ABO (Patient RBCs) (Patient Serum) ABO
Patient Patient
Blood A B Blood
A B Anti-A Anti-B
Anti-A Anti-B Group (A1 cells) (B cells) Group
(A1 cells) (B cells)
1 0 0 3+ 4+ O✔️ 1 4+ 0 0 4+ A✔️
2 4+ 0 0 4+ A✔️ 2 3+ 4+ 0 0 AB✔️
3 3+ 4+ 0 0 AB✔️ 3 1+ (mf) 0 0 3+ A?
4 0 3+ 4+ 0 B✔️ 4 0 ±/+ 4+ 0 B?
5 0 0 0 0 O? 5 4+ 2+ 0 4+ A?

+, positive (agglutination); 0, negative (no reaction) +, positive (agglutination); 0, negative (no reaction)

• These discrepancies are more common than the • This group of discrepancies is probably the least
group II, III, and IV discrepancies. frequently encountered.

• One of the reasons for the missing or weak
isoagglutinins is that the patient has depressed
antibody production or cannot produce the ABO
antibodies.
• Common populations associated with this category:
o Newborn––antibody production is not yet
detectable until 3-6 months of age.
o Elderly—depressed antibody production.
o Hypogammaglobulinemia:
▪ Leukemia patients;
▪ Patients taking immunosuppressive drugs;
▪ Patients with congenital or acquired
agammaglobulinemia or immune-deficiency
diseases; and
▪ Patients with bone marrow or hematopoietic
stem cell transplants.
o Iatrogenic causes (caused by medical
examination or treatment): patient’s existing ABO
antibodies may have been diluted by plasma
transfusion of exchange transfusions.
o Presence of ABO subgroups.
• The following are some of the causes of
discrepancies in this group:
o Subgroups of A or B may be present
o Leukemias—may yield weakened expression of
the corresponding A and B antigens (see
reactions for patients 3 and 4)
o The “acquired B” phenomenon will show weak
reactions with anti-B antisera and is most often
associated with diseases of the digestive tract
(e.g., cancer of the colon) (see reactions for
patient 5).
• Resolution of Group II ABO Discrepancies:
o Enhance the weak or missing reaction by
extending the incubation time of the serum–red
cell mixture at room temperature for
approximately 15 to 30 minutes and then
centrifuge. Observe for agglutination.
o If there is still no reaction after centrifugation, the
serum-red cell mixture can be incubated at 4°C
for 15 to 30 minutes.

• Resolution of Group I ABO Discrepancies:
o Obtain and examine the patient’s medical history.
▪ Check the patient’s age;
▪ Check the patient’s transfusion history;
▪ Check the patient’s medications (e.g.,
immunosuppressive drugs);
▪ Check for diagnosis of congenital or
acquired causes of agammaglobulinemia or
hypogammaglobulinemia.
o Include group O and autologous cells as controls.
RBCs can also be pretreated with enzymes and
retested with reagent antisera.
o Testing the patient’s serum or plasma against
autologous (their own) RBCs gives a negative
reaction because the anti-B in the serum does
not agglutinate the patient’s RBCs with the
acquired B antigen.
Group III Discrepancies: • Resolution of Group IV ABO Discrepancies:
• These discrepancies between forward and reverse
groupings are caused by protein or plasma COLD AUTOANTIBODIES:
abnormalities and result in rouleaux formation or
o FORWARD TYPING (Patient’s RBCs):
pseudo-agglutination, attributable to the following:
1. To resolve this discrepancy, the patient’s
o Elevated levels of globulin from certain disease
RBCs could be incubated or warmed at
states, such as multiple myeloma, Waldenström’s
37°C for 10 to 15 minutes, then washed with
macroglobulinemia, other plasma cell dyscrasias,
NSS three times, and then retyped.
and certain moderately advanced cases of
• If not successful in resolving the forward
Hodgkin’s lymphomas
type, the patient’s RBCs can be treated
o Elevated levels of fibrinogen
with 0.01M dithiothreitol (DTT) to
o Plasma expanders, such as dextran and
disperse IgM-related agglutination.
polyvinylpyrrolidone
2. Run a direct antihuman globulin test (DAT)
o Wharton’s jelly in cord blood samples
and autocontrol.
3. Run an indirect antihuman globulin test (IAT)
• Rouleaux [roo-low] is a stacking of erythrocytes that
or antibody screen.
adhere in a coinlike fashion, giving the appearance of
agglutination.
o REVERSE TYPING (Patient’s Serum):
• The reagent RBCs and patient’s serum can
be warmed to 37°C for 10 to 15 minutes,
mixed, tested, and read at 37°C.

• The test can be converted to the antihuman

globulin phase if necessary.

• If the reverse typing is still negative (and a

positive result was expected), a cold
autoabsorption (patient cells with patient
serum) could be performed to remove the
cold autoantibody from the serum. The
Rouleaux formation absorbed serum can then be used to repeat
the serum typing at room temperature.
• Resolution of Group III ABO Discrepancies:
o Cell grouping can usually be accomplished by UNEXPECTED ABO ISOAGGLUTININS:
washing the patient’s RBCs several times with
plain normal saline solution (NSS). o Examples of this type of ABO discrepancy include
A2 and A2B individuals, who can produce
o Performing a saline replacement technique will naturally occurring anti-A1, or A1 and A1B,
free the cells in the case of rouleaux formation in individuals who may produce naturally-occurring
the reverse type. The serum is removed and anti-H.
replaced by an equal volume of saline (1:1
mixture). o Serum grouping can be repeated using at least
three examples of A1, A2, B cells; O cells; and an
o Cord blood samples contain a viscous autologous control (patient’s serum mixed with
mucopolysaccharide called Wharton’s jelly. patient’s RBCs). The specificity of the antibody
Thoroughly washing cord cells 4 to 6 up to eight can be determined by examining the pattern of
times with NSS should alleviate spontaneous reactivity.
rouleaux due to Wharton’s jelly and result in an
accurate ABO grouping. o The patient’s RBCs can be tested with A1 lectin
(Dolichos biflorus) to confirm the presence of the
ABO subgroup. Dolichos biflorus will agglutinate
• Testing is usually not performed on cord
cells of the A1 but not the A2 phenotype.
serum because the antibodies detected are
usually of maternal origin, reverse grouping
may still not correlate with the RBC forward UNEXPECTED ALLOANTIBODIES:
grouping.
o Unexpected alloantibodies in the patient’s serum
Group IV Discrepancies: other than ABO isoagglutinins (e.g., anti-M) may
• These discrepancies between forward and reverse cause a discrepancy in the reverse grouping.
groupings are due to miscellaneous problems that
have the following causes: o In this situation, an antibody identification panel
o Cold-reacting alloantibody (e.g., Anti-M, Anti-P1 should be performed with the patient’s serum.
most common) Once the unexpected alloantibodies are
o Cold-reacting autoantibody (e.g., Anti-I, Anti-H, identified, reagent A1 and B cells negative for the
Anti-IH) corresponding antigen should be used in the
o Passively acquired RBCs—circulating RBCs of reverse grouping.
more than one ABO group due to RBC
transfusion or marrow/stem cell transplant
Forward Typing Reverse Typing Patient’s
o Unexpected ABO isoagglutinins (Patient RBCs) (Patient Serum) ABO
o Unexpected non-ABO alloantibodies Patient
A B Blood
Anti-A Anti-B Group
(A1 cells) (B cells)
• Potent cold-reacting antibodies can cause 1 4+ 4+ 1+ 1+ AB ?
spontaneous agglutination of the patient’s cells.
2 4+ 4+ 1+ 0 A2B ?
These cells often yield a positive direct Coombs’ or
antiglobulin test. 3 2+ 4+ 4+ 2+ B?