Other sources of error include the "weak D" phenomenon, in which people who are positive for the RhD antigen show weak or negative reactions when tested for RhD, and the presence of immunoglobulin G antibodies on red blood cells, which can interfere with antibody screening, crossmatching, and typing for some blood group antigens.

[4][5] It is also used before hematopoietic stem cell transplantation, because blood group incompatibility can be responsible for some cases of acute graft-versus-host disease.

The most important of these in medicine are the ABO and RhD antigens[7]: 585  but many other blood group systems exist and may be clinically relevant in some situations.

[1]: 62  Some of these antibodies can bind to incompatible red blood cells and cause them to be destroyed, resulting in transfusion reactions and other complications.

If the relevant antigen is present, the antibodies in the reagent will cause the red blood cells to agglutinate (clump together), which can be identified visually.

[1]: 104 In the indirect antiglobulin test, the mixture of antiserum or plasma and red blood cells is incubated at 37 °C (99 °F), the ideal temperature for reactivity of IgG antibodies.

After incubation, the red blood cells are washed with saline to remove unbound antibodies, and anti-human globulin reagent is added.

If the test result is indeed negative, the check cells should react with the unbound anti-human globulin and demonstrate agglutination.

[3]: 716–9 In ABO and Rh typing, reagents containing antibodies against the A, B, and RhD antigens are added to suspensions of blood cells.

[3]: 722–4 Agglutination of the screening cells by the plasma, with or without the addition of anti-human globulin, indicates that an unexpected blood group antibody is present.

By examining the antigen profiles of the red blood cells the person's plasma reacts with, it is possible to determine the antibody's identity.

Each row represents "reference" or "control" red blood cells of donors which have known antigen compositions and are ABO group O.

The "result" column to the right displays reactivity when mixing reference red blood cells with plasma from the patient in 3 different phases: room temperature, 37°C and AHG (with anti-human globulin, by the indirect antiglobulin test).

Still, if a subsequent cross-matching shows reactivity, additional testing should be done against previously discounted antigens (in this case potentially E, K, Kpa and/or Lua).

If the antigen of interest is present, the red blood cells agglutinate, forming a solid clump in the tube.

In this procedure, the plasma and red cells are mixed together in a tube containing a medium that enhances agglutination reactions, such as low ionic strength saline (LISS).

The test sample is added and the microplate is centrifuged; in a positive reaction, the red blood cells adhere to the surface of the well.

[11] The AABB recommends RhD antigen genotyping for women with serologic weak D phenotypes who have the potential to bear children.

This is because some people with weak D phenotypes can produce antibodies against the RhD antigen, which can cause hemolytic disease of the newborn, while others cannot.

Genotyping can identify the specific type of weak D antigen, which determines the potential for the person to produce antibodies, thus avoiding unnecessary treatment with Rho(D) immune globulin.

When a pregnant woman has a blood group antibody that can cause HDN, the fetus can be typed for the relevant antigen to determine if it is at risk of developing the disease.

An unexpected difference between the two results is termed an ABO discrepancy, and must be resolved before the person's blood type is reported.

[1]: 136 Weak reactions in the forward grouping may occur in people who belong to certain ABO subgroups—variant blood types characterized by decreased expression of the A or B antigens or changes in their structure.

[1]: 138 [24]: 314 People with cold agglutinin disease produce antibodies against their own red blood cells that cause them to spontaneously agglutinate at room temperature, leading to false positive reactions in forward grouping.

[1]: 141 In a rare phenomenon known as "acquired B antigen", a patient whose true blood type is A may show a weak positive result for B in the forward grouping.

[1]: 139 Infants under 3 to 6 months of age exhibit missing or weak reactions in reverse grouping because they produce very low levels of ABO antibodies.

The discrepancy can be resolved by testing the person's red blood cells with an anti-A1 reagent, which will give a negative result if the patient belongs to the A2 subgroup.

[10]: 477–8  A direct antiglobulin test can be performed to demonstrate that the positive reaction is due to sensitization of red cells.

[3]: 749 [27] Those who are at risk of developing graft-versus-host disease, such as bone marrow transplant recipients, receive blood that has been irradiated to inactivate the T lymphocytes that are responsible for this reaction.

[29] In 1901, Karl Landsteiner published the results of an experiment in which he mixed the serum and red blood cells of five different human donors.