Blood Bank 3

Diagnostic Hematology and Blood Banking
LM )453(
Lab No.3, ABO Discrepancy
TA.shefaa aleslam almasri
First semester, 2024
Definition: Cell grouping and Serum grouping do not agree.
Discrepancies may be signaled either because negative results
are obtained when positive results are expected, or positive
results are found when tests should have been negative.
When a discrepancy is encountered:

1. Record ABO discrepancy.
2. ABO interpretation should be delayed until the discrepancy is
resolved.
3. Discrepancy in donor unit makes it unsuitable for transfusion.
4. Discrepancy in patient (recipient) sample: if urgent = O RBCs
of appropriate Rh should be released for transfusion before
investigation is completed.
• The most common cause of discrepancies:
Technical and
Personal errors Red cell mediated Serum mediated
1.Technical errors:
False Negative results False Positive results

1) Failure to add reagent or serum 1) Over centrifugation
2) Failure to identify hemolysis as 2) Contaminated reagent, RBCs
a positive reaction or saline
3) Failure to add appropriate ratio 3) Dirty tubes
of serum or cells or reagents. 4) Incorrect interpretation or
4) Under centrifugation recording of test results
5) Failure to recognize the
temperature of reaction
6) Bad or inactive reagents
7) Error in results interpretation
2. Red cell mediated:
A) Acquired B antigen (Extra reactivity in cell grouping tests

("Extra antigens"):
Tentative
Example Anti-A Anti-B A1 Cells B Cells
group
#1 4+ 1+ -- 4+ A
The forward grouping resembles group AB.

The reverse grouping resembles group A.
A serum containing strong anti-B and red cells agglutinated strongly

by anti-A and weakly by anti-B suggests the acquired B state.
• The acquired B phenotype arises when microbial (bacterial
infections and often cancer of the colon or rectum)
deacetylating enzymes modify the A antigen by altering the
A-determining sugar (N-acetylgalactosamine) so that it
resembles the B-determining galactose. Anti-B antisera will cross
react with the B-like D-galactosamine antigen.
• Only A1 red cells exhibit acquired B activity in vivo. Most red
cells with acquired B antigens react weakly with anti-B, but
occasional examples are agglutinated quite strongly. The
condition is transient and disappears when the infection is cured.
Resolutions:
a. Check the past records in case the patient is a

known group A.
b. Check the diagnosis for bacterial infection (of the
colon or rectum).
c. If using human polyclonal reagents, redo the ABO
group using monoclonal anti-A and anti-B typing sera,
which may resolve the problem.
d. Treat the red cells with acetic anhydride, which
reacetylates the surface molecules and markedly
diminishes the reactivity of acquired B red cells.
• Reactivity of normal group B antigens is not
affected by acetic anhydride.
3) Serum mediated:
A) Weak or missing reactivity in serum grouping

tests:
All three of these examples in table1 could
represent cases of missing anti-A or anti-B due to
one of three causes:
1- Patient is new-born.
2- Patient is an elderly.
3- Patient has agammaglobulinenia or
hypogammaglobulinemia.
Table 1 "Weak or missing antibodies”
Tentative
Examples Anti-A Anti-B A1 Cells B Cells
group
#1 4+ -- -- -- A
#2 -- 4+ -- -- B
#3 -- -- -- -- O
Resolutions:
a. Check age of patient.
b. Look to level of gammaglobulins.
c. Repeat reverse grouping by incubating patient's serum and
known cells for 30 minutes at room temperature with the addition
of extra serum drop, then centrifuge and check results, if still
negative incubate another 30 minutes at 4°C.
B) Subgroups of A (Extra antibody):
Anti-A Anti-B A1 Cells B Cells
4+ 0 1+ 4+
The forward grouping resembles A group

The reverse grouping resembles O group
• Many subgroups for A-group have been identified, but most are rare. The
most important are A1 and A2, these subtypes difference qualitatively and
quantitatively (the number of antigens expressed per erythrocytes surface).
A1 cells carry about 1 million copies of A1 antigen, whereas A2 carry
250,000 copies.
• In a similar way: AB blood is either an A1B or A2B. About 80% of
A-grouped are A1 or A1B and about 20% are of A2 and A2B subgroups.
Anti-A1 may occur in 1-8% of A2 persons and 22-35% of A2B persons.
• The serological discrimination of A-subgroups is based on an agglutination
reaction of red cells with anti-A1 reagent prepared from Lectin of Dolichos
Biflorus seeds.
• Resolutions:
1. Test the patient red blood cells with anti-A1 lectin, a negative
reaction will be obtained if the individual is A2. A negative
reaction indicates that the patient lacks the A1 antigen and
is,therefore, an A2.
2. Test the patient serum or plasma against A2 reagent red blood
cells, a negative reaction will be obtained if the individual is A2.
Results of patient RBCs Results of patient serum

tested with Anti-A1 tested against A2 Cell
0 0
C) Rouleaux (Extra reactivity in serum grouping tests):
Tentative
Examples Anti-A Anti-B A1 Cells B Cells
group
#1 4+ -- 1+ 4+ A
#2 -- 4+ 4+ 1+ B
Rouleaux is a type of false agglutination caused by an increase

in serum globulins. This can occur in diseases such as multiple
myeloma or macroglobulinemia.
• Resolutions:
a. Check that all serum tests are "positive" (2+ or less).
b. Check microscopically for Rouleaux formation "coin stacks".
c. Check the diagnosis for diseases such as multiple myeloma.
d. Try to disperse the rouleaux using the serum replacement
method: Centrifuge the test tube, remove the serum and replace
it with an equivalent amount of saline. Mix and centrifuge then
re-read the tube:
• If no agglutination is present after the addition of saline, the

agglutination is due to rouleaux.
• If agglutination persists after the addition of saline, an antigen-
antibody reaction has taken place and true agglutination will
occur.

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Diagnostic Hematology and Blood Banking

When a discrepancy is encountered:

False Negative results False Positive results

A) Acquired B antigen (Extra reactivity in cell grouping tests

The forward grouping resembles group AB.

A serum containing strong anti-B and red cells agglutinated strongly

a. Check the past records in case the patient is a

A) Weak or missing reactivity in serum grouping

Anti-A Anti-B A1 Cells B Cells

The forward grouping resembles A group

Results of patient RBCs Results of patient serum

Rouleaux is a type of false agglutination caused by an increase

• If no agglutination is present after the addition of saline, the

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