Blood compatibility testing

Blood compatibility testing is conducted in a medical laboratory to identify potential incompatibilities between blood group systems in blood transfusion. It is also used to diagnose and prevent some complications of pregnancy that can occur when the baby has a different blood group from the mother. Blood compatibility testing includes blood typing, which detects the antigens on red blood cells that determine a person's blood type; testing for unexpected antibodies against blood group antigens ; and, in the case of blood transfusions, mixing the recipient's plasma with the donor's red blood cells to detect incompatibilities. Routine blood typing involves determining the ABO and RhD type, and involves both identification of ABO antigens on red blood cells and identification of ABO antibodies in the plasma. Other blood group antigens may be tested for in specific clinical situations.
Blood compatibility testing makes use of reactions between blood group antigens and antibodies—specifically the ability of antibodies to cause red blood cells to clump together when they bind to antigens on the cell surface, a phenomenon called agglutination. Techniques that rely on antigen-antibody reactions are termed serologic methods, and several such methods are available, ranging from manual testing using test tubes or slides to fully automated systems. Blood types can also be determined through genetic testing, which is used when conditions that interfere with serologic testing are present or when a high degree of accuracy in antigen identification is required.
Several conditions can cause false or inconclusive results in blood compatibility testing. When these issues affect ABO typing, they are called ABO discrepancies. ABO discrepancies must be investigated and resolved before the person's blood type is reported. 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.

Medical uses

Blood compatibility testing is routinely performed before a blood transfusion. The full compatibility testing process involves ABO and RhD typing; screening for antibodies against other blood group systems; and crossmatching, which involves testing the recipient's blood plasma against the donor's red blood cells as a final check for incompatibility. If an unexpected blood group antibody is detected, further testing is warranted to identify the antibody and ensure that the donor blood is negative for the relevant antigen. Serologic crossmatching may be omitted if the recipient's antibody screen is negative, there is no history of clinically significant antibodies, and their ABO/Rh type has been confirmed against historical records or against a second blood sample; and in emergencies, blood may be transfused before any compatibility testing results are available.
Blood compatibility testing is often performed on pregnant women and on the cord blood from newborn babies, because incompatibility puts the baby at risk for developing hemolytic disease of the newborn. 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.

Principles

Blood types are defined according to the presence or absence of specific antigens on the surface of red blood cells. The most important of these in medicine are the ABO and RhD antigens but many other blood group systems exist and may be clinically relevant in some situations. As of 2021, 43 blood groups are officially recognized.
People who lack certain blood group antigens on their red cells can form antibodies against these antigens. For example, a person with type A blood will produce antibodies against the B antigen. The ABO blood group antibodies are naturally occurring, meaning that they are found in people who have not been exposed to incompatible blood. Antibodies to most other blood group antigens, including RhD, develop after people are exposed to the antigens through transfusion or pregnancy. Some of these antibodies can bind to incompatible red blood cells and cause them to be destroyed, resulting in transfusion reactions and other complications.
Serologic methods for blood compatibility testing make use of these antibody-antigen reactions. In blood typing, reagents containing blood group antibodies, called antisera, are added to suspensions of blood cells. If the relevant antigen is present, the antibodies in the reagent will cause the red blood cells to agglutinate, which can be identified visually. In antibody screening, the individual's plasma is tested against a set of red blood cells with known antigen profiles; if the plasma agglutinates one of the red blood cells in the panel, this indicates that the individual has an antibody against one of the antigens present on the cells. In crossmatching, a prospective transfusion recipient's plasma is added to the donor red blood cells and observed for agglutination to detect antibodies that could cause transfusion reactions.
Blood group antibodies occur in two major forms: immunoglobulin M and immunoglobulin G. Antibodies that are predominantly IgM, such as the ABO antibodies, typically cause immediate agglutination of red blood cells at room temperature. Therefore, a person's ABO blood type can be determined by simply adding the red blood cells to the reagent and centrifuging or mixing the sample, and in crossmatching, incompatibility between ABO types can be detected immediately after centrifugation. RhD typing also typically uses IgM reagents although anti-RhD usually occurs as IgG in the body. Antibodies that are predominantly IgG, such as those directed towards antigens of the Duffy and Kidd systems, generally do not cause immediate agglutination because the small size of the IgG antibody prevents formation of a lattice structure. Therefore, blood typing using IgG antisera and detection of IgG antibodies requires use of the indirect antiglobulin test to demonstrate IgG bound to red blood cells.
In the indirect antiglobulin test, the mixture of antiserum or plasma and red blood cells is incubated at, 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 IgG antibodies have bound to antigens on the cell surface, anti-human globulin will bind to those antibodies, causing the red blood cells to agglutinate after centrifugation. If the reaction is negative, "check cells"—reagent cells coated with IgG—are added to ensure that the test is working correctly. If the test result is indeed negative, the check cells should react with the unbound anti-human globulin and demonstrate agglutination.

Blood typing

ABO and Rh typing

In ABO and Rh typing, reagents containing antibodies against the A, B, and RhD antigens are added to suspensions of blood cells. If the relevant antigen is present, the red blood cells will demonstrate visible agglutination. In addition to identifying the ABO antigens, which is termed forward grouping, routine ABO blood typing also includes identification of the ABO antibodies in the person's plasma. This is called reverse grouping, and it is done to confirm the ABO blood type. In reverse grouping, the person's plasma is added to type A₁ and type B red blood cells. The plasma should agglutinate the cells that express antigens that the person lacks, while failing to agglutinate cells that express the same antigens as the patient. For example, the plasma of someone with type A blood should react with type B red cells, but not with A₁ cells. If the expected results do not occur, further testing is required. Agglutination is scored from 1+ to 4+ based on the strength of the reaction. In ABO typing, a score of 3+ or 4+ indicates a positive reaction, while a score of 1+ or 2+ is inconclusive and requires further investigation.

Other blood group systems

Prior to receiving a blood transfusion, individuals are screened for the presence of antibodies against antigens of non-ABO blood group systems. Blood group antigens besides ABO and RhD that are significant in transfusion medicine include the RhC/c and E/e antigens and the antigens of the Duffy, Kell, Kidd, and MNS systems. If a clinically significant antibody is identified, the recipient must be transfused with blood that is negative for the corresponding antigen to prevent a transfusion reaction. This requires the donor units to be typed for the relevant antigen. The recipient may also be typed for the antigen to confirm the identity of the antibody, as only individuals who are negative for a blood group antigen should produce antibodies against it.
In Europe, females who require blood transfusions are often typed for the Kell and extended Rh antigens to prevent sensitization to these antigens, which could put them at risk for developing hemolytic disease of the newborn during pregnancy. The American Society of Hematology recommends that people with sickle cell disease have their blood typed for the RhC/c, RhE/e, Kell, Duffy, Kidd, and MNS antigens prior to transfusion, because they often require transfusions and may become sensitized to these antigens if transfused with mismatched blood. Extended red blood cell phenotyping is also recommended for people with beta-thalassemia. Blood group systems other than ABO and Rh have a relatively small risk of complications when blood is mixed, so in emergencies such as major hemorrhage, the urgency of transfusion can exceed the need for compatibility testing against other blood group systems.

Antibody screening and identification

Antibodies to most blood group antigens besides those of the ABO system develop after exposure to incompatible blood. Such "unexpected" blood group antibodies are only found in 0.8–2% of people; however, recipients of blood transfusions must be screened for these antibodies to prevent transfusion reactions. Antibody screening is also performed as part of prenatal care, because antibodies against RhD and other blood group antigens can cause hemolytic disease of the newborn, and because Rh-negative mothers who have developed an anti-RhD antibody are not eligible to receive Rho immune globulin.
In the antibody screening procedure, an individual's plasma is added to a panel of two or three sets of red blood cells which have been chosen to express most clinically significant blood group antigens. Only group O cells are used in antibody screening, as otherwise the cells would react with the naturally occurring ABO blood group antibodies. The mixture of plasma and red cells is incubated at 37 °C and tested via the indirect antiglobulin test. Some antibody screening and identification protocols incorporate a phase of testing after incubation at room temperature, but this is often omitted because most unexpected antibodies that react at room temperature are clinically insignificant.
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. If this occurs, further testing using more cells is necessary to identify the antibody. 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. An "autocontrol", in which the individual's plasma is tested against their own red cells, is included to determine whether the agglutination is due to an alloantibody, an autoantibody, or another interfering substance.
The image above shows the interpretation of an antibody panel used in serology to detect antibodies towards the most relevant blood group antigens. Each row represents "reference" or "control" red blood cells of donors which have known antigen compositions and are ABO group O. The + symbol means that the antigen is present on the reference red blood cells, and 0 means it is absent; nt means "not tested". 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.

Step 1; Annotated in blue: starting to exclude antigens without reaction in all 3 phases; looking at the first reference cell row with no reaction, and excluding each present antigen where the other pair is either practically non-existent or 0.

When both pairs are +, they are both excluded, except for C/c, E/e, Duffy, Kidd and MNS antigens. Thus, in this case, E/e is not excluded in this row, while K/k is, as well as Js^b.

Step 2: Annotated in brown: Going to the next reference cell row with a negative reaction, and repeating for each antigen type that is not already excluded.
Step 3: Annotated in purple. Repeating the same for each reference cell row with negative reaction.
Step 4: Discounting antigens that were absent in all or almost all reactive cases. These are often antigens with low prevalence, and while there is a possibility of such antibodies being produced, they are generally not the type that is responsible for the reactivity at hand.
Step 5: Comparing the remaining possible antigens for a most likely culprit, and selectively ruling out significant differential antigens, such as with the shown additional donor cell type that is known to not contain Fy^a but contains C and Jk^a.

In this case, the antibody panel shows that anti-Fy^a antibodies are present. This indicates that donor blood typed to be negative for the Fy^a antigen must be used. Still, if a subsequent cross-matching shows reactivity, additional testing should be done against previously discounted antigens.

Neutralizing substance	Antigen cancelled
Hydatid cyst fluid Pigeon eggs	P₁
Saliva	H, Le_a
Breast milk	I
Guinea pig urine	Sd^a

When multiple antibodies are present, or when an antibody is directed against a high-frequency antigen, the normal antibody panel procedure may not provide a conclusive identification. In these cases, hemagglutination inhibition can be used, wherein a neutralizing substance cancels out a specific antigen. Alternatively, the plasma may be incubated with cells of known antigen profiles in order to remove a specific antibody ; or the cells can be treated with enzymes such as ficain or papain which inhibit the reactivity of some blood group antibodies and enhance others. The effect of ficain and papain on major blood group systems is as follows:

Enhanced: ABO, Rh, Kidd, Lewis, P1, Ii
Destroyed: Duffy, Lutheran, MNS
Unaffected: Kell

People who have tested positive for an unexpected blood group antibody in the past may not exhibit a positive reaction on subsequent testing; however, if the antibody is clinically significant, they must be transfused with antigen-negative blood regardless.