ABO Discrepancies &

other problems

Importance
It

is important for students to recognize

discrepant results and how to (basically)
resolve them
Remember, the ABO system is the most
important blood group system in relation
to transfusions
Misinterpreting ABO discrepancies could
be life threatening to patients

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

?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 patients 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

Mislabeled tubes
Patient misidentification
Inaccurate interpretations recorded
Transcription error
Computer entry error
Using expired reagents
Using an uncalibrated centrifuge
Contaminated or hemolyzed reagents
Incorrect storage temperatures

Procedural errors

Reagents not added

Manufacturers directions not followed
RBC suspensions incorrect concentration
Cell buttons not resuspended 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

Bacterial growth causes cloudy or discolored

appearancedo not use if you see this!
Reagents contaminated with other reagents
(dont 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
Problems

Weak-reacting/Missing antigens
Extra antigens
Mixed field reactions

Problems

with RBCs

with serum

Weak-reacting/Missing antibodies
Extra antibodies

Grouping

Forward

Missing/Weak

Extra

Reverse

Mixed Field

Missing/Weak

A/B Subgroup

Acquired B

O Transfusion

Disease
(cancer)

B(A) Phenotype

Bone Marrow
Transplant

Young
Elderly
Immunocompromised

Rouleaux

Extra

Cold
Autoantibody

Cold
Alloantibody

Rouleaux

May cause all +

reactions
Anti-A1

Forward Grouping
Problems

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, Hodgkins disease)
Anti-A

Anti-B

A1 Cells

B Cells

+4

Group O

Group A

Since the forward and reverse dont 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
Polyagglutination
Whartons Jelly
Anti-A Anti-B
+4

+1

A1
Cells
0

EXAMPLE

B
Cells
+4

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 (Nacetyl-D-galactosamine) into D-galactosamine.

Because the terminal sugar of the B antigen is galactose, antiB 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

N-acetyl galactosamine

Bacterial enzyme
removes acetyl group

Acquired
B
Phenotype
Galactosamine
now resembles
D-galactose (found
in Group B)

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

A1 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 patients serum with their own RBCs

The patients 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

Whartons 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

+2

A1 Cells B Cells
+4

Mixed Field Agglutination

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
#2

4+
-

4+

A
B or AB

#3

#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.); Missing antigen
#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 4C [anti-A and anti-B
react best at 4C].
QC required: because all persons have a
harmless auto-anti-I reactive at 4C, include an
autocontrol. (Auto-anti-I may agglutinate the A1
cells, the B cells, and the patient's own cells at
4C.)
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 4C 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 37C and no
agglutination at AHG phase
Associated with:

Multiple myeloma
Waldenstroms 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+

#2

4+

4+

2+

AB

#3

4+

4+

1+

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+

#2

4+

4+

2+

AB

#3

4+

4+

1+

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 4C. The harmless autoanti-I of most people will not react above 1015C, 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 A1 individuals

The

antibody is a naturally occurring IgM

Reacts with A1 Cells, but not A2 Cells
+ A1 cells
Anti-A1 from
patient

+ A2 cells

AGGLUTINATION
NO AGGLUTINATION

Resolving anti-A1 discrepancy

2

steps:
Typing patient RBCs with Anti-A1 lectin
Repeat reverse grouping with A2 Cells instead
of A1 Cells
Both results should yield NO agglutination
Anti-A Anti-B
+4

A1
Cells

B
Cells

+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 antiP1 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 (Bombays dont 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 patients history:

age
Recent transplant
Recent transfusion
Patient medications
The list goes on.

!Lets practice

Example 1
Anti-A

Anti-B

A1 Cells

B Cells

+3

+1

Problem:
Causes:
Resolution:

Example 2
Anti-A

Anti-B

A1 Cells

B Cells

+3

+1

+4

Problem:
Causes:
Resolution:

Example 3
Anti-A

Anti-B

A1 Cells

B Cells

+2

+0

+1

+4

Problem:
Causes:
Resolution:

Example 4
Anti-A

Anti-B

A1 Cells

B Cells

+3

Problem:
Causes:
Resolution:

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

mf+2

+3

Problem:
Causes:
Resolution:

Example 6
Anti-A

Anti-B

A1 Cells

B Cells

+4

+4

+1

Problem:
Causes:
Resolution:

Example 7
Anti-A

Anti-B

A1 Cells

B Cells

Problem:
Causes:
Resolution:

Example 8
Screening Autocontrol
Cells
(AC)
(I and II)

Conclusion

Patient
Serum 1

Pos

Neg

Cold
alloantibody

Patient
Serum 2

Pos

Pos

Cold
autoantibody

if alloantibody antibody ID techniques

if autoantibody special procedures (minicold panel, prewarming
techniques

References

Rudmann, S. V. (2005). Textbook of Blood Banking and Transfusion

Medicine (2nd Ed.). Philadelphia, PA: Elsevier Saunders.
Blaney, K. D. and Howard, P. R. (2000). Basic & Applied Concepts
of Immunohematology. St. Louis, MO: Mosby, Inc.