Blood compatibility testing

Blood compatibility testing is conducted in a

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.^[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.^[6]

Principles

Blood types are defined according to the presence or absence of specific

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 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.[3]^: 716–9

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 (clumping).^[1]: 65 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,^[1]: 120 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.^[7]: 595 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.^[1]: 236

Other blood group systems

Prior to receiving a blood transfusion, individuals are screened for the presence of antibodies against antigens of non-ABO

Antibodies to most blood group antigens besides those of the ABO system develop after exposure to incompatible blood.[1]^: 62 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(D) immune globulin (Rhogam).^[1]: 233

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.^{[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. If this occurs, further testing using more cells (usually 10–11) 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

The image above shows the interpretation of an antibody panel used in

• 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 (0 in column at right, in this case cell donor 2), and excluding (here marked by X) each present antigen where the other pair is either practically non-existent (such as for DT) or 0 (presence is homozygous, in this case homozygous c).
  When both pairs are + (heterozygous cases), they are both excluded (here marked by X), except for C/c, E/e, Duffy, Kidd and MNS antigens (where antibodies of the patient may still react towards blood cells with homozygous antigen expression, because homozygous expression results in a higher dosage of the antigen).[17] Thus, in this case, E/e is not excluded in this row, while K/k is, as well as Js^b (regardless of what Js^a would have shown).^{[note 2]}
• Step 2: Annotated in brown: Going to the next reference cell row with a negative reaction (in this case cell donor 4), 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 (here marked with \). 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 (in this case Fy^a), 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 (in this case potentially E, K, Kp^a and/or Lu^a).^[16]

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.

• Enhanced:
  P1, Ii
• Destroyed: Duffy (Fy^a and Fy^b), 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.^[20]

Crossmatching

Crossmatching, which is routinely performed before a blood transfusion, involves adding the recipient's blood plasma to a sample of the donor's red blood cells. If the blood is incompatible, the antibodies in the recipient's plasma will bind to antigens on the donor red blood cells. This antibody-antigen reaction can be detected through visible clumping or destruction of the red blood cells, or by reaction with anti-human globulin, after centrifugation.^{[7]: 600–3}

If the transfusion recipient has a negative antibody screen and no history of antibodies, an "immediate spin" crossmatch is often performed: the red blood cells and plasma are centrifuged immediately after mixing as a final check for incompatibility between ABO blood types. If a clinically significant antibody is detected (or was in the past), or if the immediate spin crossmatch demonstrates incompatibility, a "full" or "IgG crossmatch" is performed, which uses the indirect antiglobulin test to detect blood group incompatibility caused by IgG antibodies. The IgG crossmatching procedure is more lengthy than the immediate spin crossmatch, and in some cases may take more than two hours.^[20]

Individuals who have a negative antibody screen and no history of antibodies may also undergo an "electronic crossmatch", provided that their ABO and Rh type has been determined from the current blood sample and that the results of another ABO/Rh type are on record. In this case, the recipient's blood type is simply compared against that of the donor blood, without any need for serologic testing.^{[7]: 600–3} In emergencies, blood may be issued before crossmatching is complete.^{[1]: 262–3}

Methods

Tube and slide methods

Blood typing by slide method: type A positive

Blood typing can be performed using test tubes, microplates, or blood typing slides. The tube method involves mixing a suspension of red blood cells with antisera (or plasma, for reverse grouping) in a test tube. The mixture is centrifuged to separate the cells from the reagent, and then resuspended by gently agitating the tube. If the antigen of interest is present, the red blood cells agglutinate, forming a solid clump in the tube. If it is absent, the red blood cells go back into suspension when mixed.^{[7]: 611–12 [9]: 214} The microplate method is similar to the tube method, except rather than using individual test tubes, blood typing is carried out in a plate containing dozens of wells, allowing multiple tests to be performed at the same time. The agglutination reactions are read after the plate is centrifuged.^[21]: 201

Antibody screening and identification can also be carried out by the tube method. In this procedure, the plasma and red cells are mixed together in a tube containing a medium that enhances agglutination reactions, such as

The slide method for blood typing involves mixing a drop of blood with a drop of antisera on a slide. The slide is tilted to mix the cells and reagents together and then observed for agglutination, which indicates a positive result. This method is typically used in under-resourced areas or emergency situations; otherwise, alternative methods are preferred.[9]^{: 214 [10]: 476}

Column agglutination

Column agglutination techniques for blood compatibility testing (sometimes called the "gel test") use cards containing columns of

Solid-phase assays (sometimes called the "antigen capture" method) use reagent antigens or antibodies affixed to a surface (usually a microplate).[9]^: 214 Microplate wells coated with anti-A, -B and -D reagents are used for forward grouping. 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.^{[7]: 590 [10]: 477} Some automated analyzers use solid phase assays for blood typing.^{[1]: 275–6}

Genotyping

Genetic testing can be used to determine a person's blood type in certain situations where serologic testing is insufficient. For example, if a person has been transfused with large volumes of donor blood, the results of serologic testing will reflect the antigens on the donor cells and not the person's actual blood type.

In ABO typing, the results of the forward and reverse grouping should always correspond with each other. 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

Forward grouping

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. Weakened expression of ABO antigens may also occur in

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. Cold agglutinins can usually be deactivated by warming the sample to 37 °C (99 °F) and washing the red blood cells with saline. If this is not effective, dithiothreitol can be used to destroy the antibodies.^[1]: 141

Cord blood samples may be contaminated with Wharton's jelly, a viscous substance that can cause red blood cells to stick together, mimicking agglutination. Wharton's jelly can be removed by thoroughly washing the red blood cells.^[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. This condition, which is associated with gastrointestinal diseases such as

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.[1]^: 136 Therefore, reverse grouping is generally not performed for this age group.^[10]: 486 Elderly people may also exhibit decreased antibody production, as may people with hypogammaglobulinemia. Weak reactions can be strengthened by allowing the plasma and red cells to incubate at room temperature for 15 to 30 minutes, and if this is not effective, they can be incubated at 4 °C (39 °F).^{[1]: 137–8}

Approximately 20% of individuals with the blood type A or AB belong to a subgroup of A, termed A₂, while the more common subgroup, encompassing approximately 80% of individuals, is termed A₁. Because of small differences in the structure of the A₁ and A₂ antigens, some individuals in the A₂ subgroup can produce an antibody against A₁. Therefore, these individuals will type as A or AB in the forward grouping, but will exhibit an unexpected positive reaction with the type A₁ red cells in the reverse grouping. 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. Anti-A₁ antibodies are considered clinically insignificant unless they react at 37 °C (99 °F). Other subgroups of A exist, as well as subgroups of B, but they are rarely encountered.^{[1]: 127–32}

If high levels of protein are present in a person's plasma, a phenomenon known as rouleaux may occur when their plasma is added to the reagent cells. Rouleaux causes red blood cells to stack together, which can mimic agglutination, causing a false positive result in the reverse grouping. This can be avoided by removing the plasma, replacing it with saline, and re-centrifuging the tube. Rouleaux will disappear once the plasma is replaced with saline, but true agglutination will persist.^{[1]: 140–1}

Antibodies to blood group antigens other than A and B may react with the reagent cells used in reverse grouping. If a

Approximately 0.2 to 1% of people have a "weak D" phenotype,[25] meaning that they are positive for the RhD antigen, but exhibit weak or negative reactions with some anti-RhD reagents due to decreased antigen expression or atypical variants of antigen structure. If routine serologic testing for RhD results in a score of 2+ or less, the antiglobulin test can be used to demonstrate the presence of RhD.^[1]: 159 Weak D testing is also performed on blood donors who initially type as RhD negative.^[22] Historically, blood donors with weak D were treated as Rh positive and patients with weak D were treated as Rh negative in order to avoid potential exposure to incompatible blood. Genotyping is increasingly used to determine the molecular basis of weak D phenotypes, as this determines whether or not individuals with weak D can produce antibodies against RhD or sensitize others to the RhD antigen.^[25]

Red cell antibody sensitization

The

Some groups of people have specialized transfusion requirements. Fetuses,

A direct antiglobulin test (Coombs test) is also performed as part of the antibody investigation.^[28]

Donor blood is generally screened for

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. He observed that a person's serum never agglutinated their own red blood cells, but it could agglutinate others', and based on the agglutination reactions the red cells could be sorted into three groups: group A, group B, and group C. Group C, which consisted of red blood cells that did not react with any person's plasma, would later be known as group O.^[30]: 5 A fourth group, now known as AB, was described by Landsteiner's colleagues in 1902.^[30]: 7 This experiment was the first example of blood typing.^[1]: 120

In 1945,