Blood grouping in compliance with the guidelines includes the following:

  1. identification of the antigens on the red blood cells
  2. identification of isoagglutinins (natural antibodies against blood group antigens) in plasma
  3. the determination of the rhesus factor
  4. ruling out irregular AK



A tube with EDTA blood is suitable for blood grouping.

In most immunohaematology laboratories, the blood group is determined on an automated system.


In the first step, the patient's erythrocytes are placed in gel cards containing antisera (anti_A, anti_B, anti_AB, anti_D, anti_CDE).










Principle of the gel test:


These wells are approx. 1.5 cm high and 6 of each are joined together to form a card. 


A drop of the diluted patient's blood is placed on top and incubated for approx. 20-30 min. at 37°C or RT, depending on the test setting. Afterwards, the cards are centrifuged for 15 min. and analysed.


The higher the number of cells agglutinated by the antibodies present in the wells during incubation, the more difficult it is for them to migrate down the column during centrifugation.


If all cells form a line at the top of the gel, it means that all cells are agglutinated and there is a maximum antibody-antigen reaction.


If all cells are at the bottom of the well, there was no reaction at all and the free erythrocytes were able to migrate down without any problems.

And this is how it looks in practice:

After the cards have been centrifuged, the results can now be read.

In the well with anti-A, the antibodies were not interested in the erythrocytes at all. These went down to the bottom unhindered.

On the other hand, a strong reaction can be seen in the well with anti-B and anti-AB: all the cells were captured and agglutinated by the antibodies and remained on top of the gel.

The same reaction is seen with anti-D and anti-CDE - our patient has the blood group B Rhesus D positive.








Although we now technically already know the patient's blood group, this is not sufficient to exclude even rare combinations. We already know that everyone has antibodies against these blood group antigens that they do not express themselves. In the case of blood group B, it would therefore need to be isoagglutinins against the A antigen. In order to determine these, a serum reverse typing is carried out: erythrocytes of the blood groups A1, A2, B and O (previously typed donor cells) are incubated with one drop each of the patient's serum and then centrifuged.


And the result then looks like this:

The cells reacted where an A antigen was present.

(I will not explain the terms A1 and A2 in more detail - that already goes into too much detail - both are blood group A and that is all you need to know - unless you are a transfusion specialist).

We don't see any reaction with the blood group B cells and obviously we don't see any reaction with blood group O cells.

The O blood group cells are actually meant to be the perfect negative control.

An additional task of the O cells is to reveal the Bombay type. Even though most of us do not ever actually get to see the Bombay type in our whole career - it is important to recognise it immediately.




More about the Rhesus factor can be found on the separate Rhesus website.

In most cases, the Rhesus factor can already be determined from the antigen identification card.

In our case, it is blood group B and because the well with anti-D also reacted very strongly, there are also D antigens on top of the cells - i.e. Rhesus D positive. The well with the anti-CDE antibody only provides additional information if the anti-D is negative. This is because it is used to check whether there is perhaps a rhesus C or rhesus E in the CDE rhesus formula (for Rh neg ccddee).




This test is usually carried out with purchased erythrocytes of blood group O. On these cells, all serologically and molecularly detectable blood group antigens are already determined, but the package insert mainly lists antigens that are mandated by law.


Excerpt from the regulations:

"... The test cells shall have the following characteristics: C, Cw, c, D, E, e, K, k, Fya, Fyb, Jka, Jkb, S, s, M, N, P1, Lea, Leb. It is recommended that the following traits are present in high antigenic density (homozygous inheritance for the allele) on the test cells: D, c, Fya, Fyb, Jka, Jkb, S, s."


Either two or three cells are used and the same procedure is followed as for the reverse typing. First, test cells are placed in the well, then the patient's serum (or plasma) - incubate - centrifuge - interpret.

It is common to do the reverse typing together with the antibody screening test. In both our examples, two cells are used for the screening. In the left picture, the screening is positive (all test cells have reacted with an antibody and remained on the gel during centrifugation). In the right picture, on the other hand, the two test cells (I, II) are all at the bottom of the well - so there were no antibodies to react with the cells.


If the serum examined in the picture on the right belongs to our patient, then we have now determined the complete blood group. And it is:

B Rh pos, no irregular antibodies present


In the left case, the screening is positive. Here, one would now have to carry out an antibody identification. It is particularly interesting that the O cell is also 4x positive. Without this cell, it would be a simple blood group O - due to the strongly reacting O cell in the reverse typing, the Bombay type would also be possible. However, this is very unlikely. It is much more likely to be one or more irregular antibodies caused by previous immunisation (transfusion, pregnancy). The two cells of the antibody screening test also react 4x strongly - in order to be able to release the blood group here, the specificity of the antibody or antibodies must be identified - an antibody identification.

Updated on 02.06.2023.