ABO Phenotyping
ABO Blood Group: A Review
ABO blood group antigens
ANTIGENS A and B
distribution blood cells, tissues, body fluids, secretions
biochemical glycolipid/glycoprotein
composition
expression on cord weak
blood
genetic loci ABO: chromosome 9
gene products glycosyltransferases
immunodominant A antigen: N-acetylgalactosamine
sugars B antigen: D-galactose
secretor status Se: chromosome 19
Formation of A and B antigens
In RBCs:
A/B
H A/B/O
PS II H gene Antigen
Antigens
genes in RBCs
In secretions:
A/B
H A/B/O Antigens
PS I Se gene Antigen in
genes
secretions
Formation of H antigen
H antigen
Gal PS
Gal GlcNac
fucosyl
H gene Fuc fucose
transferase
Formation of A antigen
A antigen
H
Gal PS
Gal GlcNac
Fuc
H gene
N-acetyl-
A gene galactosaminyl GalNac N-acetyl-galactosamine
transferase
Formation of B antigen
H
B antigen
Gal PS
Gal GlcNac
Fuc
H gene
galactosyl
B gene
transferase
Gal galactose
Formation of O antigen???
H antigen
Gal PS
Gal GlcNac
Fuc
H gene
O gene NO GENE PRODUCT
ABO Phenotypes and Genotypes
Phenotype Genotype
AB AB
A AO/AA
B BO/BB
O OO
ABO antibodies
ANTIBODIES anti-A, anti-B, anti-A,B
Ig class IgM, IgG and IgA
In vitro reactions at or below room temperature
complement binding Yes
Clinically significant Yes
Antibody production NATURALLY OCCURING and immune;
absent in first few months of life;
decreased in the elderly
Landsteiner’s rule serum possesses the ABO antibody
directed toward the A or B antigen that is
absent from red cells
Other ABO phenotypes… Subgroups
ABO subgroups represent phenotypes that show weaker
variable serologic reactivity with the commonly used human
polyclonal anti-A, anti-B, and anti-A,B reagents.
due to the amount of ABO antigens on the RBC (quantitative)
and the type of precursor substance used (qualitative)
Often recognized in ABO discrepancies as having weaker than
expected reactions with routine ABO typing sera
ABO Phenotyping
Forward and Reverse Grouping
FORWARD
GROUPING Anti-A reagent Anti-B reagent
-Monoclonal -Monoclonal
antibody antibody
-IgM -IgM
-Clear blue -Clear yellow
-Expected 3+ to 4+ -Expected 3+ to 4+
reaction reaction
-Usually use 1-2 -Usually use 1-2
drops drops
REVERSE
GROUPING Reagent A1 and B cells
-Human source
-4%-5% RBC suspension
-Expected 2+ to 4+ reaction
-Usually use 1 drop
FORWARD GROUPING REVERSE GROUPING
Direct hemagglutination Direct hemagglutination
Unknown ABO antigens react Unknown ABO antibodies react
with known antisera with known cells
May use whole blood, washed May use plasma or serum
RBC or RBC suspension
Performed at room Performed at room
temperature/immediate spin temperature/immediate spin
phase phase
Positive reactions Positive reactions
characteristically show 3+ to 4+ characteristically show 2+ to 4+
agglutination agglutination
ABO Phenotyping Reactions
PHENOTYPE FORWARD REVERSE
(Red cell reactions) (Serum or plasma
reactions)
anti-A anti-B A1 cells B cells
A 3-4+ θ θ 2-4+
B θ 3-4+ 2-4+ θ
O θ θ 2-4+ 2-4+
Oh* θ θ 2-4+ 2-4+
AB 3-4+ 3-4+ θ θ
* Oh = Bombay phenotype; differentiate with O phenotype using anti-H lectin
from Ulex europaeus
ABO Phenotyping
ABO Discrepancies and their resolution
ABO Discrepancies
ABO discrepancy – occurs when red cell testing does not agree
with the expected serum testing
It is suspected when:
1. agglutination strengths of the typing reactions are weaker
than expected
2. expected reactions in ABO red cell testing and serum testing
are missing
3. extra reactions are noted in either the ABO red cell or serum
tests
Problems with red cell testing
1. Weak ABO Ag expression resulting from
(a) inheritance of ABO subgroups,
(b) leukemias or other malignancies
2. Mixed-field agglutination seen in
(a) out-of-group red cell transfusion
(b) hematopoietic progenitor cell (HPC) transplantation
(c) mosaicism in fraternal (dizygotic) twins
(d) mosaicism from dispermy
3. Neutralization of typing sera by high concentrations of A or B
blood group substance.
4. Spontaneous agglutination or autoagglutination of serum- or
plasma-suspended red cells caused by heavy coating of RBCs
by potent autoagglutinins
Problems with red cell testing
5. Non-specific aggregation of serum- or plasma-suspended RBCs
caused by abnormal concentrations of serum proteins or
infused macromolecular solutions.
6. Anomalous red cell grouping resulting from acquired B, B(A),
or A(B) phenotypes.
7. Polyagglutination (e.g. T activation) resulting from inherited or
acquired abnormalities of the red cell membrane, with
exposure of “cryptic autoantigens”.
Problems with Serum/plasma testing
1. Small fibrin clots in plasma or incompletely clotted serum that can be
mistaken for red cell agglutinates.
2. Lack of detectable isoagglutinins in infants less than 4 to 6 months of
age.
3. Unexpected absence of ABO agglutinins in children resulting from long-
term parenteral and enteral nutrition, that are sterile and free of
bacteria.
4. ABO-incompatible HPC transplantation with induction of tolerance.
5. Severe hypogammaglobulinemia:
(a) inherited immunodeficiency
(b) disease therapy
(c) dilution of isoagglutinins seen in plasma exchange with albumin
replacement
Problems with Serum/plasma testing
6. Cold alloantibodies (e.g., anti-M) or autoantibodies (e.g. anti-I)
reacting with reverse grouping cells, leading to unexpected
positive reactions.
7. Antibodies directed against constituents in the diluents used
to preserve reagent A1 and B red cells.
8. Nonspecific aggregation or agglutination caused by high-
molecular-weight plasma expanders, or high serum protein
concentrations.
Unexpected Positive Reaction Unexpected Negative Reaction
Problems with RBC testing Problems with RBC testing
1. Acquired “B” Ag
2. Contamination w/ Wharton’s Jelly
3. Autoagglutination by cold autoAb
1. A or B subroups
4. Polyagglutination
2. Antigen depression due to leukemia or
5. Acriflavine Ab
other disease states
6. Genetic Chimerism*
3. High levels of soluble blood-group
7. Administration of RBCs outside ABO
substances (BGSS)
group*
8. B(A) phenotype
9. fetomaternal hemorrhage*
Problems with serum testing Problems with serum testing
1. Rouleaux-forming proteins
2. Cold-reactive alloAb 1. Elderly or newborn
3. Cold autoagglutinins 2. Hypogammaglobulinemia
4. Passively acquired ABO Abs 3. Immunosuppression
5. Subgroup of A with anti-A1
☼ All discrepant results must be recorded, and ABO group
interpretation delayed until resolved.
☼ Blood units with unresolved ABO discrepancy should NOT be
labeled nor transfused.
☼ If the recipient is involved, it may be necessary to transfuse
group O, Rh-compatible red cells pending investigation.
Resolving ABO discrepancies
A. Technical error
1. review sample identification
2. review manufacturer’s inserts in using their
reagents
3. retest using the same sample, a newly
collected sample, or washed red cells
Resolving ABO discrepancies
B. Intrinsic problems with red cells or plasma/serum
1. review patient medical history
2. for weak or missing Ag or Abs,
(a) prolonged incubation @ rm temp for 15- 30mins
(b) cold enhancement
(b) enzyme treatment of red cells
(c) adsorption and elution studies
(d) testing for soluble antigens in saliva
3. retest using different typing sera from another
manufacturer
Resolving ABO discrepancies
4. Use of lectins for suspected ABO subgroups
5. Saline replacement or saline dilution to
distinguish rouleaux from true agglutination
ABO discrepancy
Identified
Was initial testing Do the reagents
Problem with spx
done on whole still perform
ID and manner of
blood or within
collection?
unwashed RBCs? specifications?
Request a new Recheck QC of
Wash patient’s
sample to be reagents used,
RBCs with saline
drawn from the get a new batch
and repeat
patient, and and repeat
testing
repeat testing testing
No discrepancy?
Still with
REPORT OUT ABO
discrepancy
GROUP
Still with
discrepancy
Look up px information: Age, diagnosis,
medications, transfusion, and pregnancy
history
Is the discrepancy observed in the Forward
OR Reverse grouping?
Forward only Reverse only Both
What is the nature of the discrepancy? Are
there unexpected weak/missing reactions
OR unexpected positive/additional
reactions?
Sample problem #1
ABO Typing Result
Patient RBCs with Patient serum with reagent RBCs
Anti-A Anti-B A1 B
4+ 4+ 2+ 2+
Evaluation:
Which reaction/s is/are strong reaction/s?
FORWARD: Anti-A and anti-B
What is the most likely blood group of the patient?
AB
Are there discrepant results, if any? Which are they?
REVERSE: unexpected positive reactions in A1 and B cells
Probable cause/s: (1) rouleaux (2)cold-reacting alloAb (3) cold-
reacting auto-Ab (4) passively acquired Ab
Resolution
1. Rouleaux – Saline replacement or dilution
2. Cold reacting alloAb – run an O-cell control/
run an Ab screen
3. Cold reacting autoAb – run an auto control
4. Passively acquired Ab – check Px history
Sample problem #2
ABO Typing Result
Patient RBCs with Patient serum with reagent RBCs
Anti-A Anti-B A1 B
θ 2+mf 4+ θ
Evaluation:
Which reaction/s is/are strong reaction/s?
REVERSE: A1 cells
What is the most likely blood group of the patient?
B
Are there discrepant results, if any? Which are they?
FORWARD: unexpected weak with mixed-field reaction in Anti-B
Probable cause/s: (1) Out-of-group transfusion (2) Out-of-group
bone marrow/HPC transplant (3) Feto-maternal bleed
Resolution
(1), (2), (3) – check Px history
(3) - Acid elution test
Sample problem #3
ABO Typing Result
Patient RBCs with Patient serum with reagent RBCs
Anti-A Anti-B A1 B
4+ 4+ θ 1+
Evaluation:
1. Strong agglutination reactions are observed in the red cell
testing and are consistent with a group AB individual
2. The results of serum testing with reagent B demonstrate a
weaker extra reaction. This serum testing appears to be
consistent with group A.
Probable cause: cold-reacting allo- or autoantibody
Antibodies to non-ABO antigens
• patients may posses antibodies to other blood group systems
• reagent A and B cells used in the ABO serum grouping may
posses antigens to these antibodies in addition to the A and B
antigens
• common cold alloAbs: anti-P1, anti-M, anti-N, anti-Lea, anti-Leb
• common cold autoAbs: anti-I, anti-IH
Antibodies to non-ABO antigens
How to resolve:
1. determine the patient’s diagnosis and transfusion history
2. test patient’s serum with screening cells and an autocontrol
at room temperature:
screening cells autologous cells conclusion
patient’s serum pos neg cold alloAb
patient’s serum pos pos cold autoAb
3. allo- or autoadsorption techniques may be used to remove
interfering Abs.
4. if alloAb, antibody identification techniques can be performed.
