CROSS TESTS: WHAT ARE THEY FOR, TECHNIQUE, RATIONALE, PHASES - MEDICINE

The Crossmatch are a number of laboratory studies are conducted to determine whether blood products from a donor (mainly whole blood and packed globular) are compatible with the blood of the recipient.

It is an additional complementary test to ABO compatibility and Rh factor. The reason for crossmatching is that sometimes two individuals (donor-recipient) may have the same ABO and Rh group but their blood is still incompatible.

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Such incompatibility is due to the presence of antibodies against a series of red blood cell proteins known as minor antigens. These antigens are not routinely tested as they are for blood group (ABO) and Rh factor.

This is because the minor antigens are much less frequent and have a variable expression in each individual, therefore it is virtually impossible to group them into categories as it is done with the group and Rh factor.

Instead, donor red cells are mixed with patient serum (major match test) and patient red cells with donor serum (minor match test) to detect the presence of antigen-antibody reactions.

When there are antibodies to minor antigens, either in the patient's or donor serum, the test is said to be positive, so in most cases that particular unit of blood cannot be transfused.

ABO Group

To fully understand what cross-reactions are about, you first need to know the basics of blood groups.

In this sense, the most important thing is to know that blood can be classified into four groups: A, B, AB and O.

Each of these groups expresses on the surface of the red blood cells a particular protein (antigen), which is identified as a foreign element by the antibodies of a potential receptor of a different group.

The most striking thing about antigen-antibody reactions in blood matching is that no prior exposure to the antigen is required for antibodies to exist. This is known as natural antibodies.

Natural antibodies

Generally, for antibodies to exist in an individual's body, it is necessary that the white blood cells of the individual have been previously exposed to the antigen.

This means that in the first contact between the foreign antigen and the organism, there are no antibodies, since these are generated later, after the initial contact. Therefore, it is impossible for the immune system to have antibodies against for example a particular virus, if it has not been exposed to it in the past.

The only exception to the above are anti-AB antigens. In these cases, the person has antibodies against the antigen that their red blood cells do not have, even though they have never been in contact with another person's red blood cells. This is known as natural antibodies.

Antigens and antibodies related to blood group

Blood groups are determined in the case of the ABO system by the presence of specific antigens (A or B) on the red blood cell membrane and, in contrast, antibodies against the absent antigen on the erythrocyte membrane.

Thus, a person with blood group A expresses antigen A on the surface of his red blood cells, while there are anti-B antibodies in the serum.

On the contrary, in group B patients the B antigen is found while the antibodies are anti-A.

However, patients with AB blood have both A and B antigens. Therefore, there are no antibodies since doing so would destroy that person's red blood cells.

Quite the opposite occurs in group O, where the erythrocyte membrane does not present any of the two antigens (neither A nor B), while in the serum there are anti-A and anti-B antibodies.

ABO group incompatibility

From the above, the compatibility of ABO blood groups can be easily deduced, since knowing the antigen of the erythrocyte membrane automatically knows the antibodies in the serum. So that:

- Blood A is compatible with group A and group O.

- Blood group B is compatible with blood B and O.

- People with group O can only receive O blood (since they have anti-A and anti-B antibodies), although their blood is received by all other groups without problems since it lacks antigens.

- Finally. those with blood group AB can receive blood from all other groups (A, B, O and of course AB), since they do not have antibodies against any of the antigens. However, only people in group AB can receive AB blood, as all other groups have antibodies that would destroy these red blood cells.

Minor antigens

As with ABO groups, a series of proteins can be found on the surface of erythrocytes that function as antigens in the same way as ABO group antigens.

However, these antigens are not present in all individuals. Their combination is heterogeneous and the penetrance (level of protein expression) is variable, therefore a classification into groups like the one that exists for ABO and Rh is impossible. This is where it derives its name from "minor antigens", also known as "low incidence antigens."

Although they are not frequent, there may be natural antibodies against minor antigens. Among them the most common are Lewis, MNSs, anti N, Kell, Duffy, anti Fyb and Kidd. All of them responsible for very serious hemolytic and post-transfusion reactions.

In addition, there may be the case of sensitization against minor antigens by previous contact, either with said antigenic proteins due to previous transfusions or due to cross immunity.

Cross immunity

It is said that there is cross immunity when two antigens from two different sources (for example a red blood cell and a bacterium) are very similar, to the point that the antibodies against one of these antigenic proteins also react with the other because they are almost identical.

To understand this better, take the previous hypothetical example (antigens from a red blood cell and a bacterium). In neither case are there natural antibodies, but if a person is exposed to the bacteria, they will generate antibodies against it.

Such antibodies will later react against a red blood cell if its antigens are very similar to those of the bacteria that induced the antibodies to form.

If this happens, the red blood cells with that particular antigenic protein cannot be given to the person who has the antibodies, as there would be rejection. Herein lies the importance of cross-reactions.

What is crossmatching for?

Since it is impossible to group the blood of different individuals based on the minor antigens, the only way to know if there are antibodies against the minor antigens of the red blood cells of another person in the blood of one person is through crossmatching..

In those cases where antibodies are present, a hemolysis or agglutination reaction is triggered, which is why it is concluded that the reaction was positive; that is, there are antibodies against minor antigens (although it is not known exactly which one). Otherwise the test is negative.

Basis

Crossmatches are based on the antigen-antibody reaction. Therefore, with them it is possible to detect if there are antibodies in the serum of a recipient against the antigens of the donor's red blood cells (or vice versa), inducing an antigen-antibody reaction.

If there are no antibodies, no reaction occurs and the test is reported negative. On the contrary, if the reaction is positive (there is hemolysis or agglutination during the test) it can be concluded that the antibodies are present.

In this sense, it is important to note that there may be antibodies against red blood cells in both the donor's and the recipient's serum. This is why there are two types of cross reactions.

Types of cross reactions

Antibodies against donor erythrocytes may exist in the patient's serum; but the opposite can also occur, that is, antibodies in the donor's serum against the patient's red blood cells.

That is why there are two types of crossmatch:

- Major crossmatch.

- Minor crossmatch.

Both types are performed routinely in the blood bank before transfusing blood products, since if any of the tests are positive there is a high risk of transfusion reactions that can endanger the life of the patient.

Major crossmatch

This test assesses whether the recipient's serum contains antibodies to the donor's red blood cells.

If this occurs, blood products cannot be administered, as a large amount of antibodies present in the patient's plasma will destroy the donor's red blood cells very quickly, generating catastrophic reactions in the recipient's body in the process. These reactions are so severe that they can be life threatening.

Minor crossmatch

In this case, it is determined whether there are antibodies against the recipient's red blood cells in the donor's serum.

If so, the antibodies will begin to destroy the recipient's erythrocytes. However, given that the amount of antibodies is limited, the reaction is less intense; although it is still dangerous.

Phases

Both the major and minor crossmatch are divided into three phases:

- Saline.

- Thermal or incubation.

- Coombs.

In the first phase the red blood cells and serum are mixed in saline solution. Subsequently, albumin is added, and the sample is incubated at 37ºC for 30 minutes to finally proceed with the coombs phase.

Technique

The crossmatch technique is relatively straightforward, as it involves adding donor red blood cells to the patient's serum (major crossmatch) as well as recipient erythrocytes to donor serum (minor crossmatch).

In order to induce the antigen-antibody reaction in a relatively short time, a series of standardized steps must be followed. These steps are summarized in a simplified way below.

It is important to note that the next section describes the major compatibility test, although the steps are the same for the minor compatibility test, but exchanging the origin of red blood cells and serum.

Saline phase

- Add to a test tube 2 drops of serum from the recipient (from the donor if it is the minor crossmatch).

- Take a sample of red blood cells from the donor (from the recipient if it is the minor crossmatch).

- Wash and centrifuge the red blood cells.

- Resuspend in a solution between 3% and 5%.

- Place a drop of this solution in the tube containing the recipient's serum.

- Mix gently.

- Centrifuge.

- Read the result in display lamp.

Thermal phase

- Add 2 drops of 22% albumin to the tube where the saline phase was completed.

- Incubate at 37ºC for 30 minutes.

- Centrifuge for 15 seconds.

- Read the result in display lamp.

Coombs phase

- Take the cells from the tube and wash them with saline solution.

- Eliminate the supernatant.

- Add two drops of Coombs reagent.