Advanced Clinical

Immunohematology

ABO Discrepancies
 ABO Discrepancy
• Definition: When the results of the
forward grouping (patient cells) do not
correspond to the results of the reverse
grouping (patient serum) or abnormal
reactivity is present (i.e. Mixed Field):
1. Strength of reaction
• Weak or missing
2. Additional reactions
3. Abnormal reactions
 HINT
• ABO forward and reverse reactions are
typically very strong: 3+ to 4+. Weaker
reactions should immediately send up red
flags indicating that something is wrong.

HINT
• Since production of ABO antigens is
genetically controlled they are less
vulnerable to problems than does the
production of ABO antibodies. Therefore
we see more problems in which grouping:
Forward or Reverse?
 Patient Anti-A Anti-B A1-Cells B-Cells
A 4+ 1+ 0 4+
B 0 4+ 1+ 0
C 4+ 4+ 1+ 0
D 0 3+ 0 0
Patient A: Additional reaction with anti-B and patients cells.
Patient B: Weak reaction with patients serum and A1-cells.
Patient C: Additional reaction with patients serum and
A1-cells.
Patient D: Missing reactions with patients serum A1-cells
 Algorithm for resolving ABO
discrepancies
• See Figure 6-14 of Harmening p.137
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 Categories of ABO Discrepancies
Group I: unexpected reactions in the reverse grouping due to weakly reacting or
missing antibodies
Causes Notes Resolution
1. Age • Production of ABO • Review patient's profile
• Newborns antibodies is not detectable • Review patient's history
• Elderly until 4 to 6 months of age • Enhance reaction by
• ABO titer decreases with (1) incubating the patient serum
age with reagent A1 and B cells at
room temperature for
2. Patients with
approximately 15 to 30 minutes.
leukemia or
If there's still no reaction,
lymphoma
incubate further at 4°C for 15
demonstrating
minutes. Autocontrol and O cells
hypogammaglobuli
must be used to rule out
nemia
naturally occuring cold
3. Patients using autoantibodies
immunosuppressiv (2) by adding one or two drops
e drugs more plasma or serum to the
test
Group I: unexpected reactions in the reverse grouping due to weakly reacting or
missing antibodies

Causes Notes Resolution

4. Patients with congenital • Enhance reaction by
or acquired (1) incubating the patient
agammaglobulinemia or serum with reagent A1 and B
immunodeficiency diseases cells at room temperature for
approximately 15 to 30
minutes. If there's still no
5. Patients with bone reaction, incubate further at
marrow or stem cell 4°C for 15 minutes. Autocontrol
transplantations and O cells must be used to
rule out naturally occuring cold
6. Dilution of ABO autoantibodies
antibodies due to massive (2) by adding one or two drops
transfusion or exchange more plasma or serum to the
transfusion test
7. ABO subgroups • Very weak (1) Use Anti-A1 lectin, test
phenotypes (e.g. serum against additional A1,
Ael, Bel) may A2 and O cells
produce unexpected (2) Perform saliva studies or
ABO isoagglutinins adsorption-elution
Group II: Associated with unexpected reactions in the forward grouping due to
weakly reacting or missing antigens

Causes Notes Resolution

1. Subgroups of A or B Enhance reaction by

incubating the patient serum
with reagent A1 and B cells
at room temperature for
approximately 15 to 30
minutes. If there's still no
reaction, incubate further at
4°C for 15 minutes.
Autocontrol and O cells
2. Weakening of ABO
must be used to rule out
antigens due to leukemia or
naturally occuring cold
Hodgkin's disease
autoantibodies
Group II: Associated with unexpected reactions in the forward grouping due to
weakly reacting or missing antigens
Causes Notes Resolution
3. Acquired B phenomenon Bacterial enzymes modify (1) Do not use monoclonal
the immunodominant blood anti-B clone (ES4)
group A sugar (N-acetyl-D- (2) Autoincubation: patien'ts
galactosamine) into D- anti-B will NOT agglutinate
galactosamine, which is acquired B cells
sufficiently similar to the (3) Modify pH of antiserum.
group B sugar (D- Acquired B antigen is not
galactose) and cross- agglutinated at pH >8.5 or
reacts with anti-B antisera. <6
This pseudo-B antigen is (4) Perform secretor studies
formed at the expense of (5) Treat red cells acetic
the A1 antigen and anhydride (reacetylates
disappears after recover acquired B cells)
(6) Check patient history

4. Excess amounts of BGSS formed secondary to • Wash patient red cells

blood group–specific gastric carcinoma
soluble (BGSS) substances neutralize reagent antisera
present in the plasma
Group II: Associated with unexpected reactions in the forward grouping due to
weakly reacting or missing antigens

Causes Notes Resolution

5. Antibodies to low- • Repeat forward type
incidence antigens in using antisera with a
reagent anti-A or anti-B different lot number

6. Chimerism
Acquired B Antigen
 Blood Group A

Bae‹arial

Free
Acecyl

Acquired B
 Example of ABO Discrepancy Caused by an Acquired B
Antigen
Forward Grouping Reverse Grouping

Anti-A Anti-B A1 cells B cells

Patient 4+ 2+ 0 4+

Patient’s probable group: A

Note: Patient RBCs have acquired a B-like antigen that reacts

with reagent anti-B and is associated with cancer of the colon
or other diseases of the digestive tract.
Group III: Caused by protein or plasma abnormalities resulting in rouleaux
formation or pseudoagglutination
Causes Resolution
1. Elevated levels of globulin from certain • Saline replacement technique
disease states, such as multiple myeloma,
Waldenström’s macroglobulinemia, other
plasma cell dyscrasias, and certain
moderately advanced cases of Hodgkin’s
lymphomas

2. Elevated levels of fibrinogen

3. Plasma expanders, such as dextran and

polyvinylpyrrolidone

4. Wharton’s jelly in cord blood samples • Wash cord cells six to eight times
Group IV: Miscellaneous problems

Causes Notes Resolution

1. Cold reactive RBCs are so heavily coated (1) Incubate red cells at
autoantibodies with antibody that they 37°C , then washed three
spontaneously agglutinate times with saline at 37°C
regarless of antibody and retype
specificity (2) Treat red cells with 0.01
M DTT
(3) Cold autoadsorption

2. Chimerism

3. Unexpected ABO (1) A2 and A2N phenotypes (1) Repeat serum grouping
isoagglutinins may produce Anti-A1 with at least three examples
(2) A1 and A1B phenotypes of A1, A2 and O cells and
may produce Anti-H autologous control
(3) Weak ABO phenotypes (2) Test patient rbcs with
may form ABO antibodies Dolichos biflorus
Group IV: Miscellaneous problems

Causes Notes Resolution

4. Unexpected non-ABO Perform antibody screening
alloantibodies and identification. Once
antibodiy is identified, use
A1 and B cells negative for
the corresponding antigen

5. Presence of antibody to Acriflavine-anti-acriflavine Wash the red cells three

acriflavine complex attaches to red times with saline
cells causing spontaneous
agglutination
6. Cis-AB Inheritance of three ABO
genes instead of two. A
and B alleles are found on
the same/”cis”
chromosome.
Cis-AB individuals have
weakened ABO antigen
expression and some
produce Anti-B
Group IV: Miscellaneous problems

Causes Notes Resolution

7. Polyagglutination Exposure of crypt antigens • Use lectins to
due microbial infections or characterize the crypt
in association with inherited antigen present
disorders • Review patient history
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with The Forward Grouping: Extra AB
Polyagglutinable state

• Exposure of ‘crypt’ or buried antigens

(T, Tk, etc.) by inheritance or bacterial
enzymes
– RBC’s agglutinate with most ABO-
compatible sera.
• Exposure of T, Tn and Tk (etc.) antigens.
Antibodies to these antigens are present in
virtually all human antisera. If using human
source anti-A and anti-B these cells will
agglutinate.
 Polyagglutination
Acquired
A. Microbially associated
• T (Hubener-Thomsen-Friedenreich phenomenon), Th,
Tx, Acquired B, VA
B. Non-microbially associated
• Tn

Inherited
• Cad (Super-Sid/Sd[a++])
• Hemoglobin M-Hyde Park
• HEMPAS/CDA II
• NOR
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 Example of ABO Discrepancy Seen With Weak or
Missing
Antibodies
Forward Grouping Reverse Grouping
Anti-A Anti-B A1 cells B cells
Patient 0 0 0 0
Patient’s probable group: O (elderly patient or newborn)
Note: The absence of agglutination with reagent cells in the
reverse type is because the production of ABO antibodies can be
weak or absent in the elderly.
Resolution:
(1) Check age of the patient.
(2) Increase incubation time to 30 minutes (not appropriate for
newborn sample).
(3) Lower the temperature to 4°C for 15 minutes (include O cells
and an autocontrol).
 Serologic Reactions Typical of Leukemia

Patient Forward Grouping Reverse Grouping

Phenotype
Anti-A Anti-B A1 cells B cells
A +mf 0 0 3+
B 0 +/+ 4+ 0
Note: Weak reactivity with anti-A and anti-B is
because the disease, leukemia, has resulted in
the weakened expression of the corresponding
antigen.
Example of ABO Discrepancy Caused by Low-Incidence
Antibodies in the Reagent Antisera
Forward Grouping Reverse Grouping
Anti-A Anti-B A1 cells B cells
Patient 0 1+ 4+ 4+
Patient’s probable group:O
Note: Reaction with anti-B in the forward type is due to
agglutination between a low-incidence antibody in reagent
anti-B and the corresponding antigen on the patient’s cells.

Resolution: Use a different lot number for reagent Anti-B

 ABO Grouping in Chimera Twins
Patient Anti-A Anti-B Anti- A1 B cells RBC%
A,B cells
Twin 1 0 2+mf 2+mf 4+ 0 70% B;
30% O
Twin 2 0 +wk +wk 4+ 0 30% B;
70% O
Patient’s probable group:O
0 = negative; mf = mixed field; wk = weak
 ABO Discrepancy Caused by Rouleaux Formation
Forward Grouping Reverse Grouping
Anti-A Anti-B A1 cells B cells
Patient 4+ 4+ 2+ 2+
Patient’s probable group:AB
Note: Agglutination with A1 and B cells in reverse type is
due to rouleaux formation as a result of increased serum
protein or plasma abnormalities.
Resolution:
(1) Microscopic examination
(2) Saline replacement technique
(3) Wash cells with saline three times
(4) Run antibody screen
 ABO Discrepancy Caused by Cold Autoantibodies
Forward Grouping Reverse Grouping
Anti-A Anti-B A1 cells B cells
Patient 2+ 4+ 4+ 2+
Patient’s probable group: B
Note: Reaction with anti-A in forward type is due to
spontaneous agglutination of antibody coated cells; reaction
with B cells in reverse type is due to cold autoantibody
(e.g., anti-I) reacting with I antigen on B cells.
Resolution:
(1) Wash patient cells with warm saline and retest;
(2) Run DAT and autocontrol;
(3) Run antibody screen
 ABO Discrepancy Caused by Unexpected ABO
Isoagglutinin
Forward Grouping Reverse Grouping
Anti-A Anti-B A1 cells B cells
Patient 4+ 4+ 1+ 0
Patient’s probable group: A2B
Note: Reactions with patient serum are due to anti-A1
agglutinating A1 reagent red cells.
Resolution:
(1) Test cells with anti-A1 lectin;
(2) Test serum with A1, A2, and O cells;
(3) Run an autocontrol
ABO Discrepancy Caused by an Unexpected Non-ABO
Alloantibody
Forward Grouping Reverse Grouping
Anti-A Anti-B A1 cells B cells
Patient 4+ 4+ 1+ 1+
Patient’s probable group: AB
Note: Reactions with patient serum are due to non-ABO
alloantibody agglutinating an antigen other than A1 and B
on reagent red cells.
Resolution:
(1) Run an antibody screen and panel