ABO Discrepancies

Dr. Nada Hosny

 • Introduction
Agenda
• ABO discrepancies rules
• Some bla bla bla
• Examples
Discrepancies
A discrepancy occurs when the red cell testing does NOT match the serum testing results
In other words, the forward does NOT match the reverse
Why?
• Reaction strengths could be weaker than expected
• Some reactions may be missing in the reverse or forward typing
• Extra reactions may occur
Patient Anti-A Anti-B A1 Cells B Cells

1 4+ 1+ 0 4+

2 0 4+ 1+ 0

3 4+ 4+ 1+ 0

4 0 3+ 0 0
What do you do?
Identify the problem
Most of the time, the problem is technical
• Mislabeled tube
• Failure to add reagent
• Either repeat test on same sample, request a new sample, or wash cells
Other times, there is a real discrepancy due to problems with the patient’s red
cells or serum
Discrepancy ?
If a real discrepancy is encountered, the results must be recorded

However, the interpretation is delayed until the discrepancy is RESOLVED

Errors
 Technical Errors
Clerical errors Reagent or equipment problems Procedural errors
• Mislabeled tubes Using expired reagents Reagents not added
• Patient misidentification Using an uncalibrated centrifuge Manufacturer’s directions not
• Inaccurate interpretations
Contaminated or hemolyzed followed
recorded reagents RBC suspensions incorrect
Incorrect storage temperatures concentration
• Transcription error Cell buttons not resuspended
• Computer entry error before grading agglutination
 Clotting deficiencies
Serum that does not clot may be due to:
• Low platelet counts
• Anticoagulant therapy (Heparin, Aspirin, etc)
• Factor deficiencies
Serum that does not clot completely before testing is prone to developing fibrin clots that may
mimic agglutination
Thrombin can be added to serum to activate clot formation
OR, tubes containing EDTA can be used
 Contaminated samples or reagents

Sample contamination
• Microbial growth in tube
Reagent contamination
• Bacterialgrowth causes cloudy or discolored appearance…do not use if you see this!
• Reagents contaminated with other reagents (don’t touch side of tube when dispensing)
• Saline should be changed regularly
Equipment problems
Routine maintenance should be performed on a regular basis (daily,
weekly, etc)
Keep instruments like centrifuges, thermometers, and timers calibrated
• Uncalibrated serofuges can cause false results
Hemolysis
Detected in serum after centrifugation (red)
Important if not documented
Can result from:
• Complement binding
Anti-A, anti-B, anti-H, and anti-Lea
• Bacterial contamination
Red supernatant
ABO discrepancies
ABO Discrepancies' rules
ABO Discrepancies
Problems with RBCs
• Weak-reacting/Missing antigens
• Extraantigens
• Mixed field reactions

Problems with serum

• Weak-reacting/Missing antibodies
• Extra antibodies
 Grouping

Forward Reverse

Missing/Weak Extra Mixed Field Missing/Weak Extra

Young
Elderly Cold
A/B Subgroup Acquired B O Transfusion
Immunocompromi Autoantibody
sed

Disease Bone Marrow Cold

B(A) Phenotype
(cancer) Transplant Alloantibody

Rouleaux May cause all + reactions Rouleaux

Anti-A1
 RBCs problem
“Forward grouping problem”
Red Cell Problems
Affect the forward grouping results
• Missing or weak antigens
• Extra antigens
• Mixed field reactions
 Forward Grouping:
Missing or Weak antigens
ABO Subgroups
Disease (leukemia, Hodgkin’s disease)
Anti-A Anti-B A1 Cells B Cells

0 0 0 4+

Group O Group A
• Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)
Subgroups of A (or B)
Subgroups of A account for a small portion of the A population (B subgroups
rarer)
These subgroups have less antigen sites on the surface of the red blood cell
As a result, they show weakened (or missing) reactions when tested with
commercial antisera
Resolution: test with Anti-A1, Anti-H, and anti-A,B for A subgroups
Forward Grouping:
Extra Antigens
Acquired B
B(A) phenotype
Rouleaux Anti-A Anti-B A1 B
Polyagglutination Cells Cells
4+ 1+ 0 4+
Wharton’s Jelly

EXAMPLE
 Acquired B Phenomenon
Cause: there are two causes of acquired B phenomenon:
In vivo, patients with bacterial infections and often cancer of the colon or rectum may develop a false B-like antigen.

The mechanism: The bacterial produce a deacetylase (enzyme) which chemically alters the terminal sugar of A antigens (N -
acetyl-D-galactosamine) into D-galactosamine.

Because the terminal sugar of the B antigen is galactose, anti-B antisera will cross react with the B-like D-galactosamine
antigen. Because of this, in vivo, only group A people can develop an acquired B-like antigen. The condition is transient and
disappears when the infection is cured.
Acquired B
Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar….

Group A individual Acquired B

Phenotype

N-acetyl galactosamine Galactosamine now resembles D-

galactose (found in Group B)
Bacterial enzyme removes
acetyl group
 Another mechanism
In vitro, blood specimens can get an acquired B-like antigen if they are bacterially contaminated. This is
because the membranes of some bacteria (e.g., E. coli and P. vulgaris ) have determinants which are
chemically similar to the B antigen.

In this case, anti-B antisera is actually reacting with the bacterial antigens which have attached to the
red cells.

In vitro, both group O and group A cells can acquire the B-like antigen. Note: most examples of acquired
B phenomenon detected in the blood bank happen in vivo to group A people only.
 ES4 Anti-B antisera
The use of monoclonal ABO typing antisera (specifically an anti-B clone designated "ES4")
initially caused an increase in acquired B phenomenon.
because

The ES4 monoclonals can detect even a small number of galactosamine molecules on red
cells. However, the reactions are particularly sensitive to pH and can be reduced (not
eliminated totally) if the pH is lowered, something that the manufacturers have done.
Typical reaction pattern

The reactions with anti-B are weaker than expected (e.g., 1+ or 2+). The
patient's autocontrol is negative even though anti-B is present (patient is group
A).

The patient's own anti-B will not recognize and agglutinate the B-like antigen,
but everyone else's anti-B (including the typing sera) will.
 Resolution of acquired B
Check the past records in case the patient is a known group A.
Check the diagnosis for bacterial infection (with or without Cancer of the colon or rectum).
Test the red cells with anti-B reagent acidified to pH 6.0
If using human polyclonal reagents, redo the ABO group using monoclonal anti-A and anti-B typing sera,
which may resolve the problem.
If using monoclonal reagents, redo the ABO group using human polyclonal anti-A and anti-B typing sera.
Do autologous control it should give negative result.
Try secretor status studies (usually not necessary). If the patient is group A and a secretor, he will secrete A
and H antigens only.
 Implication in blood transfusion

Group A people (especially children with a small blood volume) who have acquired B
phenomenon should receive group A washed red cells (or group O washed red cells).

The red cells should be washed to remove all traces of donor anti-B which can react
with the patient's B-like antigens.
Acquired B Phenotype

Limited mainly to Group A 1 individuals

with:
• Lower GI tract disease
• Cancer of colon/rectum
• Intestinal obstruction
• Gram negative septicemia (i.e. E. coli)
Resolving Acquired B
Check patient diagnosis: Infection?
Some manufacturers produce anti-B reagent that does not react with
acquired B
Test patient’s serum with their own RBCs
• Thepatients own anti-B will not react with the acquired B antigen on their
red cell (autologous testing)
B(A) phenotype
Similar to acquired B
Patient is Group B with an apparent extra A antigen
The B gene transfers small amounts of the A sugar to the H antigen
Sometimes certain anti-A reagents will detect these trace amount of A antigen
Resolution: test with another anti-A reagent from another manufacturer
Other reasons for “extra” antigens
Polyagglutination – agglutination of RBCs with human antisera no matter what blood type
• Due to bacterial infections
• Expression of hidden T antigens react with antisera

Rouleaux – extra serum proteins

Wharton’s Jelly – gelatinous substance derived from connective tissue that is found in
cord blood and may cause false agglutination (Remember: only forward typing is
performed on cord blood)
• Wash red cells or request new sample from heel, etc
Forward Grouping: Mixed Field Agglutination

Results from two different cell populations

Agglutinates are seen with a background of unagglutinated cells
• All groups transfused with Group O cells
• Bone marrow/stem cell recipients
• A3 phenotype

Anti-A Anti-B A1 Cells B Cells

0 2+ 4+ 0
Mixed Field Agglutination
Serum promlems “Reverse Grouping
Problems”
Reverse Grouping
Affect the reverse grouping results
• Missing or weak antibodies
• Extra antibodies
Reverse Grouping:
Missing or Weak antibodies
Newborns
• Do not form antibodies until later
Elderly
• Weakened antibody activity
Hypogammaglobulinemia
• Little or no antibody production (i.e. immunocompromised)
Often shows NO agglutination on reverse groupings
Missing or Weak antibodies
Example Anti-A Anti-B A1 cells B cells Tentative group

#1 +4 - - - A
#2 - +4 - - B or AB
#3 - - - - O
#1: Patient is a newborn: Anti-A and anti-B are not present at birth and develop about 3-6 months of age. (Usually the reverse
group is not done when grouping newborns.)
#2: Patient is very elderly: Anti-A and anti-B levels decrease in old age because levels of immunoglobulins decrease. Because the
levels may only be decreased and not totally missing, further investigation can be done. (Note: It would be unusual for an elderly
person to totally lack ABO antibodies in the absence of an immune disorder.)
#3: Patient has a- or hypogammaglobulinemia: Anti-A and anti-B will be weak or missing in patients with a gammaglobulinemia
or hypogammaglobulinemia.
 Resolution
Check the age of the patient
Repeat the ABO group at 4°C [anti-A and anti-B react best at 4°C].
QC required: because all persons have a harmless auto-anti-I reactive at 4°C, include an
autocontrol. (Auto-anti-I may agglutinate the A1 cells, the B cells, and the patient's own cells
at 4°C.)
Check the diagnosis.
If undiagnosed, have gammaglobulin levels tested
Resolving Weak or Missing antibodies
Determine patients age, diagnosis
Incubate serum testing for 15 minutes (RT) to enhance antibody reactions
If negative, place serum testing at 4°C for 5 minutes with autologous control (a.k.a.
Autocontrol, AC)
This is called a “mini-cold” panel and should enhance the reactivity of the antibodies
Reverse Grouping:
Extra Antibodies

Cold antibodies (allo- or auto-)

• Cold antibodies may include anti-I, H, M, N, P, Lewis
Rouleaux
Anti-A1 in an A2 or A2B individual
Cold antibodies
Sometimes a patient will develop cold-reacting allo- or auto-antibodies that appear as “extra”
antibodies on reverse typing
Alloantibodies are made against foreign red cells
Autoantibodies are made against ones own red cells. Cold reacting antibodies cause agglutination
with red cells at room temperature and below. The autocontrol will be positive.
• Resolution: warming tube to 37° and washing red cells can disperse agglutination; breaking the IgM
bonds.
Rouleaux
Can cause both extra antigens and extra antibodies
“stack of coins” appearance
May falsely appear as agglutination due to the increase of serum proteins (globulins)
Stronger at IS and weak reaction at 37°C and no agglutination at AHG phase
Associated with:
• Multiple meloma
• Waldenstrom’s macroglobulinemia (WM)
• Hydroxyethyl starch (HES), dextran, etc
Resolving Rouleaux
Remove proteins!
If the forward grouping is affected, wash cells to remove protein and repeat test
If the reverse grouping is affected, perform saline replacement technique (more
common)
• Cells(reagent) and serum (patient) centrifuged to allow antigen and antibody to react (if
present)
• Serum is removed and replaced by an equal volume of saline (saline disperses cells)*
• Tube is mixed, centrifuged, and reexamined for agglutination (macro and micro)

*some procedures suggest only 2 drops of saline.

 Extra Antibodies
Example Anti-A Anti-B A1 cells B cells Tentative group

#1 +4 - +1 +4 A
#2 +4 +4 +2 - AB
#3 - +4 +4 +1 B
Anti-A1 in A2 or A2B people: examples #1 and #2 illustrate the presence of anti-A1. The autocontrol (not shown) would be
negative.
Irregular IgM Alloantibodies: All three examples could represent the presence of irregular IgM alloantibodies such as anti-M, -N, -
Lea, -Leb, or -P1. The A1 cells (or B cells) may be agglutinating because they are positive for the corresponding antigen. The
autocontrol (not shown) would be negative.
 Example Anti-A Anti-B A1 cells B cells Tentative group

#1 +4 - +1 +4 A
#2 +4 +4 +2 - AB

#3 - +4 +4 +1 B

Rouleaux: providing both cells in the reverse grouping show agglutination (examples #1 and #3), the discrepancy could be due to Rouleaux. The
autocontrol (not shown) would be positive.
Causes: 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 or can be caused by infusion of macromolecular substances such as dextran or polyvinyl pyrollidone (PVP), which are used as blood
volume expanders.
Autoanti-I: Many people have a harmless autoanti-I that is IgM and reacts best at 4°C. The harmless autoanti-I of most people will not react above
10°-15°C, but some people have an autoanti-I that can react at RT and cause unexpected agglutination in both cells of the reverse serum group
(examples #1 and #3).
Anti-A1
• Sometimes A2 (or A2B) individuals will develop an anti-A1 antibody
• A2 (or A2B) individuals have less antigen sites than A 1 individuals
• The antibody is a naturally occurring IgM
• Reacts with A1 Cells, but not A 2 Cells

+ A1 cells
AGGLUTINATION
Anti-A1 from patient
+ A2 cells
NO AGGLUTINATION
Resolving anti-A1 discrepancy
2 steps:
• Typing patient RBCs with Anti-A1 lectin
• Repeat reverse grouping with A 2 Cells instead of A 1 Cells
• Both results should yield NO agglutination

Anti-A Anti-B A1 B
Cells Cells
4+ 0 2+ 4+
Resolution of discrepancies caused by anti-A1
First step:
We must determine if the person is group A1 or group A2. (If group A1, the
discrepancy with the A1 cells is NOT due to anti-A1).

To do this, we antigen type the person's red cells with the anti-A1 lectin which is
Dolichos biflorus . If the red cells agglutinate, the person is group A1. If the red cells
do not agglutinate, the person is not group A1, and probably is group A2 assuming the
red cells reacted strongly (3+ or 4+ with anti-A).
Resolution of discrepancies caused by anti-A1

Second step:
If the person appears to be group A2, we must prove that the extra antibody is anti-A1, and not
some other IgM irregular antibody.

To do this we test the person's serum against a panel of 3 A1 cells and 3 A2 cells. If anti-A1 is
present, only the A1 cells should agglutinate.
 Resolution of discrepancies caused by anti-A1
NOTE:
3 A1 and 3 A2 cells are required in order to ensure that the antibody reacting is anti-A1 and not some other
antibody.

With 3 cells of each group we can achieve a statistical probability of 95% that the right antibody has been
identified.

For example, if only one A1 cell and one A2 cell were tested, by chance, another antibody like anti-M or anti-
P1 could react with the A1 cells (if they were M+ or P1+), but not the A2 cells (if they were M- or P1-).
Others…
The Bombay phenotype (extremely RARE) results when hh is inherited

These individuals do not have any antigens and naturally produce, anti-A, anti-B, anti-A,B, and anti-H

Basically, NO forward reaction and POSITIVE reverse

Resolution: test with anti-H lectin (Bombay’s don’t have H and will not react)
Finding the problem…
Forward type tests for the antigen (red cell)
Reverse type tests for the antibody (serum)
Identify what the patient types as in both Forward
& Reverse Groupings
Is there a weaker than usual reaction?
Is it a missing, weak, or extra reaction??
Resolving ABO Discrepancies
Get the patient’s history:
• age
• Recent transplant
• Recent transfusion
• Patient medications
• The list goes on….
Let’s practice!
ABO Discrepancies' rules
Examples
Example 1
Anti-A Anti-B A1 Cells B Cells

3+ 0 0 1+

Problem: Reverse grouping, weakened patient antibody

Causes: Age related or weakened immune system

Resolution: Incubate at Room Temperature 15-30 minutes and respin. Check patient history.
Example 2
Anti-A Anti-B A1 Cells B Cells

3+ 1+ 0 4+

Problem: 1+ reaction with anti-B. Appears to have additional antigens.

Causes: Acquired B antigen
Resolution: Patient history – bowel obstruction, carcinoma of colon/rectum. (E. coli)
Example 3
Anti-A Anti-B A1 Cells B Cells

2+ 0+ 1+ 4+

Problem: Weak forward with anti-A and 1+ reaction with A1 cells

Causes: 1) Subgroup of A (A2 with anti-A1)
2) unexpected cold reacting antibody to antigen on reagent A1 cells
Resolution: 1) test patient cells with anti-A1 lectin and with patient serum test with A2 cells
2) an unexpected cold antibody would be detected in the antibody screen
Example 4
Anti-A Anti-B A1 Cells B Cells

0 0 0 3+

Problem: missing antigen in forward grouping. Patient appears as group A in reverse grouping
Causes: A subgroup
Resolution: extend incubation time because this may enhance the reaction. Test with a polyclonal or monoclonal blend of anti-
A,B (may contain subgroup antigens)…..
Example 4
Anti-A,B

Patient RBC 1+

• Probably a subgroup of A (Ax)

• if the result was negative (0), adsorption or elution studies
with anti-A could be performed (these will help determine
what A antigens)
Example 5
Anti-A Anti-B A1 Cells B Cells

0 2+mf 3+ 0

Problem: strength of anti-B is weaker than expected; reverse indicates a group B individual
Causes: Group B individual transfused with group O cells
Resolution: recent transfusion? Bone marrow/stem cell transplant? Find what ABO type the patient was prior to
transfusion
Example 6
Anti-A Anti-B A1 Cells B Cells

4+ 4+ 0 1+

Problem: Forward shows AB individual, Reverse shows weaker “extra” reaction with B cells (looks like a group A)
Causes: Possible cold allo- or autoantibody (patient may have an antibody to another blood group system; A1 and B cells
may have the antigens to these antibodies) (allo: P, M, N, Lewis) (auto: I or IH)
Resolution: screen for antibodies using Screening Cells and an autocontrol
Example 7
Anti-A Anti-B A1 Cells B Cells

0 0 0 0

Problem: Reverse grouping, missing patient antibody (probably group O with no antibodies)
Causes: Age related or weakened immune system
Resolution: Incubate at Room Temperature 15-30 minutes and respin. Check patient history.
Example 8
Screening Autocontrol Conclusion
Cells (AC)
(I and II)
Patient Pos Neg Cold
Serum 1 alloantibody
Patient Pos Pos Cold
Serum 2 autoantibody
• if alloantibody – antibody ID techniques
• if autoantibody – special procedures (minicold panel, prewarming techniques
Thanks