ABO Grouping

The ABO system is regarded as the most important blood group system in transfusion medicine because of severe hemolytic Transfusion Reactions and, to a lesser degree, Hemolytic Disease of the Newborn.

ABO grouping is a test performed to determine an individual's blood type. It is based on the premise that individuals have specific oligosaccharide structures in their red blood cell (RBC) antigens that correspond to the four main blood groups: A, B, O, and AB. ^[1] Antibodies (isohemagglutinins) in an individual's plasma are directed against blood group antigens that their own RBCs lack (see Table 1). These antibodies (isohemagglutinins) form early in the first 3 months of life after exposure of the immune system to bacterial antigens in the gut that resemble human blood group antigens. ^[2] ABO antigens are expressed on RBCs, platelets, leukocytes, plasma proteins, and endothelial cells and on certain tissues. Blood group antigens are also present in body fluids such as sweat, saliva, urine, breast milk, amniotic fluid, seminal fluid, and gastric secretions in soluble form. ^[1] ABO testing is performed to prevent an adverse transfusion reaction that could be caused by ABO incompatibility between the blood of a patient (recipient) and that of a donor.

Table 1. ABO Genotyping

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ABO blood group antigens are genetically determined glycoconjugates expressed on the membranes of RBCs, surfaces of other cells, body fluids, and secretions. The H antigen on RBCs and in secretions is encoded by the H (FUT1) gene and Se (FUT2), respectively. ^[3] Both FUT1 and FUT2 genes are located on chromosome 19q13.3, 35 kb apart. ^[4]

• The FUT1 gene encodes H-type α(1,2) fucosyltransferase (H enzyme) to create H antigen in the erythroid lineage, a key precursor for producing A and B antigens in the presence of their respective transferases. ^[4] Chromosome 9q34 encodes for A and/or B glycosyltransferases, which are necessary to produce the various ABO antigens (mainly glycolipoproteins) on blood components.
• The FUT2 or "secretor"/"se" gene encodes for the secretor-type α(1,2) fucosyltransferase (Se enzyme) that produces H antigen in the saliva and various secretory epithelia. ^[4] Most of the population (approximately 80%) expresses the secretor gene. ^[5]

The precursor glycoprotein/glycolipoprotein that allows the expression of all ABO antigens is composed of oligosaccharide chains with the essential addition of L-fucose. α-1,2-L-fucosyltransferase is the enzyme responsible for adding L-fucose to the primary galactose of the oligosaccharide chain. This is the foundation "H" antigen. The gene for type "O" is silent and thus maintains the original formation of the H antigen.

The A and B antigens are carbohydrate epitopes built upon the H antigen with the help of their respective glycosyltransferase enzymes which attach sugar moieties to the oligosaccharide chains. ^{[3, 1]} For type A, an N-acetyl-D-galactose is attached at the end of H antigen to the primary, initial galactose by α-1,3-N-acetyl-D-galactosyltransferase. For type B, an additional D-galactose is attached to the primary galactose by α-1,3-D-galactosyltransferase on the H antigen. ^[3] For type AB, both these transferases would add both the sugars, whereas in individuals with blood group O, the transferases lack enzymatic activity. ^[3]

The O allele is an autosomal recessive trait, and the A and B alleles are codominant traits. Each parent contributes an A, B, or O allele to their offspring, depending on the ABO type of the parents (see Table 1).

The frequency of ABO blood group antigens varies in different populations. Most people have the antigen "H" encoded because this is the precursor for antigen A or B. Thus, depending on whether the other genes are encoded, it will then be determined if they will remain a type O or change to type A, B, or AB. Type O blood is the most frequent, and type B and AB are the least frequent (see Table 2).

Table 2. ABO Phenotype Frequencies Among Different Ethnic Groups

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In rare cases, even the initial precursor H antigen is not genetically encoded. These individuals are known as Bombay or para-Bombay phenotypes and show absent or reduced H antigen expression on RBCs due to deficient or minimal activity of the H-type α(1,2)fucosyltransferase enzyme encoded by the FUT1 gene. ^[4] They can only receive the Bombay type blood because they make antibodies to not only type A, B, or AB donor RBCs but also to type O donor red cells (anti-H), causing hemolysis of the transfused donor RBCs.

The ABO test may be part of a group of tests performed in various clinical settings.

Type and screen

A type and screen includes ABO, Rh, and atypical, non-ABO antibody detection/identification.

Type and cross

ABO, Rh, atypical, non-ABO antibody detection/identification, and compatible matching with a donor unit are included in a type and cross. ^[8]

Transfusion recipient testing

A type and cross needs to be completed before blood is issued to prevent ABO incompatibility. ^[8]

Recipient organ/hematopoietic stem cell testing

A type and screen of the recipient is performed to assess ABO compatibility between the recipient and potential donor of organ and hematopoietic stem cells. In hematopoietic stem cell transplantation, progenitor cells engraft into the recipient's chemotherapy-induced "empty" marrow and replace the recipient's blood ABO type and Rh type with that of the donor's if the engraftment is successful.

Blood donor testing

A type and screen of the donor is completed as one of the required tests for blood donation. This allows blood collection facilities to quickly assess the inventory status of ABO/Rh type by label identification. Before a donor unit is transfused, additional compatibility testing with the intended recipient's plasma/serum is completed. This will also act as a second validation step of the initial donor ABO/Rh typing.

Donor organ/hematopoietic stem cell testing

A type and screen of the donor is completed as one of the required tests for organ or hematopoietic stem cell donation to assess the level of compatibility of the donor's and intended recipient's ABO/Rh types, which will dictate the subsequent type of blood components transfused. Sometimes, donor lymphocytes present in the transfused blood can engraft and proliferate in the recipient, which may lead to graft vs host disease. ^[2]

Evaluation of hemolytic disease of the fetus and newborn associated with ABO incompatibility

Hemolytic disease of the fetus and newborn (HDFN) occurs when a fetus inherits paternal RBC antigens that the maternal immune system does not recognize as her own. A small percentage of fetal blood may come into direct contact with maternal blood circulation through fetal maternal hemorrhage (e.g., amniocentesis, trauma, miscarriage/abortion, placental abnormalities).

The ABO system of fetal RBC antigens is not as fully developed in utero, and RBC antigens are lesser in number. Anti-ABO antibodies form naturally at an early age in response to ubiquitous environmental antigens, without requiring prior sensitization. ^[9] Mild ABO HDFN may result if there is ABO incompatibility between the baby and the mother due to the following reasons ^[9] :

• Naturally occurring anti-A and anti-B antibodies are predominantly IgM, which does not cross the placenta. Only the IgG component (which may be produced in group O individuals or after transfusion exposure) can cross and cause hemolysis. ^[9]  
• Reduced antigen density on fetal RBCs — fetal erythrocytes express lower quantities of A and B surface antigens, limiting antibody binding. ^[10]
• Widespread tissue expression of A/B antigens in the fetus — the fetus expresses A/B antigens on many non-erythrocyte tissues, effectively "diluting" or adsorbing the antibodies so that fewer target the RBCs specifically. ^[11]  

The scenario that is indicted more often for an increased severity of hemolysis is when the mother is type O and the fetus has either type A, B, or AB.

Type O mothers produce both IgM and IgG anti-A/B, but they produce more IgG  anti-A/B than type A or B mothers — this is why ABO HDFN is predominantly a type O mother phenomenon. ^[12]

The maternal IgG crosses the placenta via active FcRn-mediated transport, not passive diffusion based on size ^[13] and, thus more likely to expose fetal RBCs to the mother's antibody in the right clinical setting (see also Rh Incompatibility for information on hemolytic disease of the fetus and newborn) (Rh Typing is discussed in a separate article).

Platelet refractory evaluation

A platelet refractory evaluation attempts to evaluate why a patient's platelet count did not increase as expected after a platelet transfusion; it includes evaluating immune-mediated vs nonimmune-mediated causes. ABO incompatibility is an example of an immune-mediated cause. Platelets with incompatible ABO antigens undergo faster clearance from circulation, leading to reduced post-transfusion counts. ^[14]

Because ABO antigens are minimally expressed on platelets and less than 2 mL of RBCs are left in a unit of platelets, it is often unnecessary to give ABO-compatible platelets. However, donor plasma containing ABO antibodies may cause hemolysis with incompatible recipient RBCs. Thus, if an individual requires frequent platelet transfusions, the amount of RBC exposure and platelet ABO antigens may increase enough that an individual's antibodies will attack and cause hemolysis. Although platelets lack the Rh antigen, residual intact RBCs or fragments present in the donor plasma may lead to RhD alloimmunization when platelets obtained from RhD-positive donors are given to RhD-negative recipients. ^[14]

Association studies between the ABO group and infectious/noninfectious diseases

Studies have been conducted to elucidate the correlations between ABO blood types and the susceptibility to various infectious and noninfectious diseases, including cancer, cardiovascular diseases, and hematologic disorders. ^[1] For example, blood group A is strongly linked to a higher risk for stomach cancer, possibly due to a higher susceptibility to Helicobacter pylori, which may interact with A antigens to promote tumor development. Several studies have reported loss of expression of blood group antigens in the primary breast tumors, metastases, and about 50% of proximal colon cancers, suggesting its role as a marker of tumor invasion. ^[15]

ABO typing is performed by taking a sample of blood, placing it in a centrifuge, and separating RBCs from the serum/plasma. A "front," or forward type, and "back," or reverse type, are then performed.

The front/forward type detects antigens present on the individual's RBCs based on RBC agglutination. It takes an individual's RBCs and mixes them with commercially prepared reagents of anti-A antibody and with separate anti-B antibody. The test measures visual agglutination or lack of agglutination. The back/reverse type detects anti-A, anti-b, and anti-AB antibodies in the plasma or serum sample. ^[3] It mixes an individual's plasma with reagent RBCs positive for antigen A and separate reagent RBCs positive for B antigen. The test also measures visual agglutination or lack of agglutination.

Most ABO antibodies are Ig belonging to class M. ^[3] When IgM comes into contact with a foreign antigen, it attaches or "coats" the antigen. Once attached, IgM can come into contact with other antigen-binding sites and antibodies, which will bring them closer together, thus causing visual agglutination (see the following image). In the tube agglutination test, consistent RBC clumping after centrifugation and gentle agitation indicates complete agglutination, whereas uniformly distributed RBC suspension indicates nonagglutination reaction. ^[3] The presence of this reaction is designated with a positive symbol (+) and the absence with the number zero, as shown in Table 3, further down in this section.

Manual tube testing. (Image created by Jaye Parsley)

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ABO grouping, common test reactions, and interpretations. (Image created by Jaye Parsley)

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The amount of sample can limit ABO typing. For standard tube testing, column agglutination "gel card" methodology (see the image below), and solid phase test systems, at least 1 mL of blood is required.

Column agglutination, "gel card". (Image created by Jaye Parsley)

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Factors that can cause interference of ABO testing

They include mixed field reactions and bacterial infections and malignancies.

1. Mixed field reaction: In a mixed field reaction, two different ABO groups are present in the same sample, causing discordant ABO typing. The front and back type results are not as predicted. Examples are discussed below.
   • Chimerism or mosaicism - It occurs when two or more genetically distinct cell populations coexist within the same individual. Congenital chimerism arises from either embryonic fusion or dizygotic twin-twin vascular anastomoses between two dichorionic placentas, resulting in hematopoietic cell exchange. Partial hematopoietic chimerism is seen as a result of transfusion or stem cell transplantation and is restricted to hematopoietic cells. ^[16]
   • Massive transfusion - Massive transfusion of type O–negative RBCs to a non-O–type individual may cause a mixed field reaction or discordant ABO typing. The front and back type will not be concordant.
   • Hematopoietic stem cell recipients - Hematopoietic stem cell recipients may receive a transplant from a donor who has a different ABO type. Initial engraftment of the donor stem cells into the recipient's marrow may cause mixed typing because there are two different ABO types within the recipient until full engraftment occurs.
2. ABO subtypes: ABO subtypes are defined as a blood type that has most of the chemical characteristics of type A, B, or O but that also has a slight variation in a portion of the structure that can be recognized through testing. These changes may occur through various mutations, such as frameshift mutations, amino acid substitutions, single point mutations, single missense mutations, and deletions. The amount of variation determines if it is clinically significant enough to cause hemolysis. The two most common subtypes of A are A1 (approximately 80% of all type A individuals) and A2 (approximately 20% of all type A individuals) (see Table 3). A1 and A2 subtypes have distinct qualitative characteristics, namely A1 RBCs have 8.1-11.7 × 10⁵ antigenic sites, while A2 RBCs possess 2.4-2.9 × 10⁵ antigenic sites. Both A1 and A2 show strong agglutination by anti-A antiserum. ^[17] Most commercial testing reagents use A1 RBC antigen. Although there will be discrepancies between the front and back typing between an A2 individual's cells and A1 reagent cells, this usually does not cause clinically significant hemolysis. If a concern exists, a blood bank research laboratory can use the chemical Dolichos biflorus to confirm the A1 status of a patient: Only A1 will react with D. biflorus. Similarly, the A2 phenotype exhibits enhanced reactivity with the anti-H lectin of Ulex europaeus. Other weaker and rarer subtypes of blood group A include A₃, A_end, A_x, A_m, A_y, and A_el , which are seen in < 1% of cases. ^[17] Similar subtypes of B occur, but they are much rarer. ^[18]

Table 3. Subgroups of A Identification

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      3. Bacterial infections and malignancies: Although uncommon, another discrepancy in ABO testing can result from certain bacterial infections and malignancies that may cause an acquired "B" typing from an A type individual. The patient's underlying disorder can cause enzymatic deacetylation of group A antigen, thus forming a B-like antigen, "acquired" B phenomenon. Although this will cause some weak discrepancies in the laboratory, the patient will still receive type A blood products.

Limitations

As noted earlier, the amount of sample can limit ABO typing. For standard tube testing, column agglutination "gel card" methodology (see the image below), and solid phase test systems, at least 1 mL of blood is required.

Column agglutination, "gel card". (Image created by Jaye Parsley)

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If a newborn has received a substantial amount of type O RBCs, the baby may be typed as O even if the baby is non-O.

For many years, manual agglutination testing using a test tube was the main methodology for ABO/Rh typing. With the advent of automated testing, new methodology has been introduced, including column agglutination and solid phase test systems.

Manual tube testing

Testing forward typing, an individual's red cells (in a 2-5% saline suspension) are mixed in a test tube with separate reagents anti-A and anti-B at room temperature. After centrifugation, the pelleted cell button is gently resuspended and examined for agglutination. If there is agglutination, this will indicate a positive reaction. The amount of agglutination is graded on a scale of 0 to 4+, as shown in the following image.

Testing back typing, an individual's plasma is mixed in a test tube with separate reagent A and B RBCs. The remainder of the testing and interpretation is completed as stated above for forward typing.

Manual tube testing. (Image created by Jaye Parsley)

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Limitations: This method is unreliable for blood group determination in infants (due to limited reverse typing), patients with leukemia having weak antigen expression, and individuals with low antibody titers. Accurate results require meticulous tube cleaning, standardized centrifugation parameters, and observation under controlled ambient conditions. ^[3]

Column agglutination ("gel," "gel card")

Testing forward typing, an individual's RBCs are initially mixed at room temperature in small, gel-filled tubes containing separate reagent anti-A and anti-B. The specimen is centrifuged and assessed for agglutination. Agglutination seen at the top of the tube column indicates a strong positive reaction; agglutination seen at the bottom signifies no reaction. The amount of agglutination is also graded on a scale of 0 to 4+, as depicted in the image below.

Testing back typing, an individual's plasma is initially mixed at room temperature in small, gel-filled tubes that contain separate reagent A and B RBCs. The remainder of the testing and interpretation is completed as stated above for forward typing.

Column agglutination, "gel card". (Image created by Jaye Parsley)

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Solid phase test systems ("microplate test")

Separate anti-A and anti-B reagent is embedded in the bottom of a 96- or 120-well trapezoidal or V-shaped ladder microplate with stepped wells, which are designed in such a way that the width of each step is approximately equal to the diameter of one RBC, while the height exceeds the dimeter of the RBC. ^[3] An individual's RBCs (in a 2-5% saline suspension) are mixed together with an enhancing reagent and centrifuged. The plates are then assessed. Following antigen-antibody reaction, if there is a covering of RBCs throughout the bottom of the microplate, this would indicate agglutination, a positive reaction. Conversely, if there is just one tightly packed RBC pellet at the bottom of the microplate, it is considered to be no reaction. As with the previous two tests, the amount of agglutination is graded on a scale of 0 to 4+.

Testing back typing, separate portions of reagent A and B RBCs are embedded in the bottom of the microplate wells. An individual's plasma is mixed together with an enhancing reagent and centrifuged. The plates are interpreted as stated above for forward typing.

Table 4 provides further description of conventional agglutination grading.

Table 4. Standard Agglutination Grading

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The following are common laboratory resources:

• ARUP Laboratories
• John Hopkins Clinical Tests and Labs
• Massachusetts General Hospital: Laboratory Handbook
• Mayo Medical Laboratories
• Labcorp
• Quest Diagnostics
• Stanford Anatomic Pathology & Clinical Laboratories