3.5 ABO typing : front and reverse

ABO typing has two stages: one direct red blood cells -RBCs- typing using commercial antisera (anti-A, anti-B) and one indirect typing utilizing plasma/serum and known - A and B - RBCs suspensions.

1. Direct ABO blood typing (or front grouping)

This test detects the presence of A and B antigens on RBCs surface by means of agglutination utilizing commercial antisera. It is a basic and simple test, it can be performed on few drops of venous or capillary blood (i.e. obtained by finger pricks) .

Commercial monoclonal anti-A and anti-B antisera , (anti A,B is also available*)

* Note: Anti-A,B antiserum agglutinates both A and B expressing RBCs, it is useful as a confirmation of anti-A and anti-B reagents


Anticoagulated blood (with EDTA or citrate) is preferrable for RBCs typing. In alternative fresh capillary blood or RBCs obtained from dissolved blood clots can be used..

Two techniques are possible: on a tile and in tube.

On a tile (or on a glass slide):

• Dispense 2 separate drops of whole blood on a tile (or on a slide)

Note: a third drop is usually added for Rh-D typing, another one can be added if anti A,B antiserum is available

• Dispense 1-2 drops of each antiserum next to each blood drop

• Mix separately the reagents with each blood drop with a clean plastic or wooden stick

Note: take care not to use the same stick tip to mix the different drops in order not to drag the antisera from one drop to another causing so erroneous positive results

• Tilt and rock gently the tile to facilitate reaction for 30-60 seconds

• Observe for agglutination

In tube:

• Mix together one drop of whole blood with about 15 drops of saline in order to obtain a 3-5% suspension of RBCs:

• Dispense 1 drop of RBCs suspension in each of the test tubes (one for A, one for B plus eventually the one for anti A,B and the one for Rh-D)

• add in each tube 1-2 drops of antiserum and mix by gently shaking the tube

• spin at low speed (1000 rpm) for about 30 seconds

• gently tip and roll the tubes on order to show possible agglutination

A video tube test typing (italian language) is available at :



• Agglutination = the antigen is present

• No agglutination: the antigen is absent


• Anti-A + RBCs suspension = agglutination
• Anti-B + RBCs suspension = no agglutination
• Anti A,B + RBCs suspension = agglutination

Interpretation: group A

False positives or artifacts are rarely seen and they are essentially caused by the plasma proteins when whole blood is used as specimen. These plasma-related agglutinations may be false, like in the case of ruleaux formation (see the page "agglutination tests") or they may be real agglutinations caused by auto-antibodies. These apparently positive results turn generally negative when saline-suspended RBCs are tested instead of whole blood.
In case high titer cold anti-antibodies are present repeated washing of RBCs (dilution with saline> centrifugation> supernatant removal) with warm – at 37°C – normal saline may be needed in order to eliminate the  the auto-agglutination of RBCs. Remember that auto-antibodies usually cause agglutination of all tested blood drops so beware of AB positive results ! In these cases the reverse typing will help to clarify the situation (see below).

False negatives or weak reactions: they are in theory possible if antisera are no longer working. So antisera should be quality checked regularly with RBCs samples of known A and B type. If reagents are working properly the only “false” negatives may be due to the presence of very rare group A or B variants. These may be also suspected because of discrepancies between the direct typing and the reverse – or indirect – serum grouping.

2. Reverse “serum” grouping

This test is used to confirm and must not substitute the front grouping that remains the pillar of ABO typing. Reverse grouping is useful in resolving the infrequent but possible dubious or weak results in the front typing, and it helps to identify the rare cases of false negative and false positive results.

The test puts in evidence the presence of “naturally” occurring Anti-A and Anti-B that are present after 6 months of life in all subjects that lack the reciprocal A/B antigen.

In order to perform the reverse test we need a saline 3-5% suspension of “fresh” RBCs of group A, B and O. The O type RBCs suspension represents a negative control. The suspensions can be stored refrigerated for few days.


• Mix 2 drops of serum or plasma under investigation with one drop of each 3-5% RBCs suspension (group A, B and O)

• Read for agglutination

Again the test can be performed in tubes or on a tile. With the tube test reading is immediate after low speed centrifugation for 30-60 seconds. In case of tile (or slide) method agglutination may require some minutes and low power microscope magnification may be needed in order to detect weak agglutinations.

Expected results:

• Group O serum/plasma will agglutinate A and B cells

• Group A serum/plasma will agglutinate only B cells

• Group B serum/plasma will agglutinate only A cells

• Group AB serum/plasma will not agglutinate any cells suspension

Group O RBCs suspension is expected not to be agglutinated by any serum/plasma sample representing a kind of “negative” control.

In case reverse results do not match the front typing we are in front of a “discrepancy” that must be resolved before transfusion is issued. Agglutination of O group RBCs is another unexpected result that must be further investigated.

It must be remembered that natural anti-A/anti-B may be absent in some physiological or pathological situations:
- newborns up to 4-8 months don’t have anti-A or anti-B antibodies
- old persons may have a very low titer or absence of detectable anti-A and/or anti-B antibodies
- persons that are deeply immunosuppressed because of drugs or of disease may not have detectable Anti-A and/or anti-B

No comments: