Resolution of ABO Discrepancies

ABO discrepancies are recognized when the reactions obtained in the forward type do
not "match" the reactions obtained in the reverse type. For example,
Forward type is group O and reverse type is group B.
If an individual is group B they MUST have anti-A present in their serum or
plasma. Failure to agglutinate the reagent A1 cells will cause additional
investigation to be performed.
When a discrepancy is observed one MUST determine if the problem is associated
with the forward type, reverse type or both.
FALSE POSITIVE means the reaction should have been NEGATIVE but
agglutination occurred. FALSE NEGATIVE means the reactions should have
been POSITIVE but no agglutination occurred. The key is to recognize which type of
problem is occurring so that appropriate testing can be performed.
When evaluating an ABO discrepancy it needs to be determined whether there is a
problem with the FORWARD type (antigens on cells), the REVERSE type
(antibodies in serum/plasma) or BOTH.
Problems With the Forward Type
The forward type detects antigens present on the RED BLOOD CELLS.
False negative reactions in the forward type may occur due to:
Weak subgroups of A or B.
A, B or AB individual transfused with massive quantities of group O
blood.
ABO non-identical bone marrow or stem cell transplant.
Inhibitor substances which neutralize anti-A or anti-B.
False positive reactions may occur due to:
heavy protein coating of the red blood cells.

 coating of cord blood cells with Wharton's jelly.

antibodies to dyes used to color anti-A or anti-B
Problems with the Reverse Type
The reverse type detects antibodies present in the SERUM or PLASMA.
False negative reactions in the reverse type may be due to weak or missing antibodies
due to a variety of factors which influence the production of antibodies.
Patient is an infant.
Patient is elderly.
Seriously immunocompromised due to disease, therapy or depressed
immunoglobulin levels.
Large amounts of IV fluids present due to treatment or drawing blood
above an IV.
Antibodies passively transfused in non-ABO identical products
containing excessive plasma, usually platelets.
False positive reactions may be due to "unexpected antibodies", those antibodies
other than anti-A or anti-B.
False positive reactions occur in situations such as a group A individual
reacting with BOTH the A1 and B reagent cells. In this situation one
must think of the most frequent cause of the problem. Anti-A1 is not
uncommon in A2 or A2B individuals.
Cold agglutinins are another cause of an unexpected positive in the
reverse type. It is important that red blood cell reagents be allowed
to warm to room temperature prior to use to prevent false positives
due to cold agglutinins.
ABO Discrepancies - Example 1
Anti-A

Anti-B Anti-D

D ctrl A1 cell B cell

4+

4+

In this example the forward type, which detects antigens on red cells, appears to be
valid and the patient appears to be B pos. The reverse type appears to be group AB.
In looking at these results the discrepancy appears to be with the reverse type
which detects ANTIBODIES in the patient serum or plasma which has caused a false
negative in the reverse type. The forward type has 4+ reactions which are to be
expected. The patient should have reacted with the A1 cells but has not. This is most
frequently due to the absence or extremely low levels of antibodies. The patient may
be an infant, elderly or have some condition which has caused significantly decreased
antibody levels. This type of discrepancy is relatively easy to resolve by allowing the
reverse type to incubate at RT for 10-15 minutes. One may also add 1-2 more drops of
serum/plasma. This adds more antibody to the test system. After incubation the tubes
are respun. Most patients will react with the appropriate reverse cell after this
procedure. If the reactions are still negative then incubation at 4C may be necessary.
A patient control (patient red blood cells plus patient serum or plasma) must be run to
ensure that a false positive due to the presence of cold autoagglutinins does not occur.
After incubation at RT for 15 minutes the following reactions were obtained:
Anti-A
0

Anti-B Anti-D
4+

4+

D ctrl A1 cell B cell

2+

The forward and reverse now match, the reaction with the A1 cells is 2+ and the
patient can now be reported out as Bpos.
NOTE: Most transfusion services require that the appropriate reverse cell react 2+ or
stronger to be valid.
Continue

ABO Discrepancies - Example 2

Anti-A
4+

Anti-B Anti-D
0

D ctrl A1 cell B cell

4+

1+

4+

In this example the forward type, which detects antigens on red cells, appears to be
valid and the patient appears to be A positive. The reverse type appears to be group
O. But look at the STRENGTH of the reverse cells. A 1+ reaction should raise a red
flag. One of the most commonly encountered ABO discrepancies are with
individuals who are A2 and have a naturally occuring anti-A1 in their serum or
plasma. Both A1 and A2 individuals will react 4+ with the anti-A typing serum. The
discrepancy illustrated in this example is detected when an unexpected reaction with
the A1 cells is obtained. Group A people should not have anti-A in their serum.
There are TWO components to resolving this discrepancy:
1. Test the patient red blood cells with anti-A 1 lectin, a negative reaction will be
obtained if the individual is A2. Anti-seras always detect antigens on the red
blood cells. A positive reaction indicates presence of the antigen on the cell, a
negative reaction indicates the absence of the antigen on the cell. A negative
reaction indicates that the patient lacks the A1 antigen and is, therefore, an A2.
2. Test the patient serum or plasma agains A2 reagent red blood cells,
a negative reaction will be obtained if the individual is A2. Patient serum
contains antibodies. If a patient serum reacts with a red blood cell it indicates
the presence of an antibody in the patient's serum against an antigen on the cell.
No agglutination indicates no antibodies present in the patient's serum against
this particular cell.
The following results were obtained on this sample:
Results of patient Results of patient
RBCs tested with serum tested against
Anti-A1
A2 Cell
0
0
Once recognized, this type of discrepancy is relatively easy to resolve. YOU MUST
MEMORIZE THE TESTING REQUIRED FOR THIS TYPE OF
DISCREPANCY.