ABO Blood Groups System

• A blood group system consists of antigens produced by alleles at a single genetic locus or closely linked loci and can exist as components of cellular membranes and in soluble forms.

ABO Blood Group System

• ABH-soluble antigens exist in all body secretions and depend on inherited ABO and Sese genes.

Landsteiner’s Rule

• Sera from group O and B individuals contain anti-A antibodies.
• Anti-A produced by group O and B individuals contains anti-A and anti-A1.
• Anti-A1 is specific for the A1 antigen and does not agglutinate A2 red cells.

ABO, H, and Se Genes

• ABO antigens are influenced by three genetically independent loci: ABO, H, and Se.

Genetics

• Genes at 3 separate loci influence the occurrence and location of ABO antigens: ABO, H, Se
• H gene has H and h alleles (h is an amorph). The Bombay phenotype lacks the H antigen (hh)
• Se gene has Se and se alleles (se is an amorph)
• ABO genes have A, B, and O alleles (O is an amorph)
• The presence or absence of the ABH antigens on the RBC membrane is controlled by the H gene.
• The presence or absence of the ABH antigens in secretions is influenced by the Se gene.
• ABO antigens can be intrinsic to the RBC membrane or soluble (body fluids).
• ABO antigens are detected in the embryo as early as 5 to 6 weeks’ gestation.
• Newborns’ RBCs have fewer numbers and partially developed antigens; full expression occurs at about 2 to 4 years of age.

Inheritance

• The H gene controls the production of H antigens and is on a different chromosome from the ABO genetic locus, influenced by the secretor gene (Se gene).
• Group A secretors secrete glycoproteins carrying A and H antigens. The Sese genes (SeSe, Sese) regulate their formation.
• Se genes do not affect the formation of A, B, or H antigens on RBCs but determine whether ABH-soluble substances will be secreted.

Oligosaccharide Chains

• Oligosaccharide Chain: A chemical compound formed by a small number of simple carbohydrate molecules linked in simple linear forms or complex branched structures.
• Type 1 structures are associated primarily with body fluids.
• Type 2 structures are associated primarily with glycolipids and glycoproteins on the red cell membrane.

Genetic Basis of H Antigen

• The H antigen is determined by the H locus on chromosome 19, which is closely linked to the Se locus.
• There are two significant alleles at the H locus: H (dominant) and h (recessive).

Enzymatic Process of H Antigen

• The H antigen forms through the action of a glycosyltransferase enzyme, specifically l-fucosyltransferase (FUT-1), which transfers l-fucose to type 1 or type 2 common oligosaccharide chains. The addition of l-fucose to the terminal galactose confers H specificity.

Relationship to ABO Antigen Expression

• The presence of the H antigen is crucial for the expression of A and B antigens because the gene products of the ABO alleles (A and B transferases) require the H antigen as the acceptor molecule to form A and B antigens.
• Without the H antigen, A and B antigens cannot be formed.
• Bombay phenotype individuals appear to be blood type O because they lack A and B antigens on their red blood cells, but they can only safely receive blood from other Bombay phenotype individuals due to the lack of the H antigen.

Red Blood Cell (RBC) Transfusions

• ABO Compatibility: It is determined by the serologic compatibility between the recipient's ABO antibodies and the donor's ABO antigens on red cells.
• A group A recipient with anti-B antibodies can receive group A or group O red cells, as these cells do not express B antigens.
• A group B recipient can receive group B or group O red cells, as these do not express A antigens.
• Incompatibility Consequences: A group A recipient who receives group B or AB red cells will have their anti-B antibodies bind to the B antigens on donor cells, potentially causing an acute hemolytic transfusion reaction through complement activation.

Whole Blood Transfusions

• ABO-Identical Requirement: The donor's blood must be ABO-identical to the recipient’s blood because whole blood contains both plasma and red cells, necessitating compatibility of both components.
• A group A recipient must receive group A whole blood, and a group O recipient must receive group O whole blood.

Plasma Products

• Plasma transfusions are less restrictive because plasma does not contain red cell antigens that could react with recipient antibodies, reducing the risk of adverse reactions. Plasma from any ABO group can be used more flexibly, considering clinical circumstances and plasma availability.

Crossmatching and Other Compatibility Tests

• Crossmatching: Donor red cells are mixed with recipient serum to detect any agglutination or hemolysis, indicating incompatibility.
• Antibody Screening: Tests are performed to detect unexpected antibodies in the recipient's serum that may react with donor red cells, identifying the presence of clinically significant antibodies that could cause transfusion reactions.

Risks and Management

• Acute Hemolytic Transfusion Reactions: Severe reactions are caused by ABO incompatibility, leading to the rapid destruction of transfused red cells. Symptoms include fever, chills, back pain, hemoglobinuria, and potential renal failure, and management includes immediate cessation of transfusion, supportive care, and monitoring of renal function.
• Delayed Hemolytic Transfusion Reactions: Reactions occur days to weeks after transfusion, often due to minor antigen incompatibilities, with symptoms including mild jaundice, decreased hemoglobin levels, and a positive direct antiglobulin test (DAT).

Glycosyltransferases and Immunodominant Sugars

• A glycosyltransferase enzyme catalyzes the transfer of glycosyl groups (simple carbohydrate units) in biochemical reactions.

H Allele

• L-fucosyltransferase (FUT-1) is the glycosyltransferase, and L-fucose is the immunodominant sugar. The H antigen is a precursor structure for the ABO blood group antigens, formed when fucosyltransferase transfers L-fucose to a type 1 or type 2 common oligosaccharide chain on red blood cells and other tissues.

A Allele

• N-acetylgalactosaminyltransferase is the glycosyltransferase, and N-acetylgalactosamine is the immunodominant sugar. The A allele encodes N-acetylgalactosaminyltransferase, which transfers N-acetylgalactosamine to the H antigen, converting it to the A antigen.

B Allele

• D-galactosyltransferase is the glycosyltransferase, and D-galactose is the immunodominant sugar. The B allele encodes galactosyltransferase, which transfers D-galactose to the H antigen, converting it to the B antigen.

O Allele

• O allele has an enzymatically inactive protein (nonfunctional) glycosyltransferase and no immunodominant sugar because the H antigens remain unconverted.

A1 Phenotype

• Approximately 80% of group A individuals have a high concentration of A antigens present on both branched and linear oligosaccharide chains.
• The A1 gene product effectively converts H antigen to A antigen, resulting in a highly concentrated presence of A antigens on the red cell membrane.
• Both A1 and A2 red cells agglutinate with commercially available anti-A reagents.
• A1 red cells can be distinguished from A2 red cells using Dolichos biflorus lectin, a reagent extracted from the seeds of the plant Dolichos biflorus, which agglutinates specifically with A1 red cells.

A2 Phenotype

• About 20% of group A individuals have fewer A antigens assembled on simplified linear oligosaccharide chains.
• The A2 gene product is less efficient at converting H antigen to A antigen, leading to a lower number of A antigen copies on the red cell membrane.
• A2 red cells do not agglutinate with Dolichos biflorus lectin.
• An alloantibody, anti-A1, can be detected in 1% to 8% of A2 individuals and 22% to 35% of A2B individuals, causing agglutination with A1 red cells but not A2 red cells.

A3 Subgroup

• The A3 subgroup characteristically demonstrates mixed-field agglutination patterns, where not all red cells agglutinate uniformly, and individuals in the A3 subgroup may possess anti-A1 antibodies in their serum.
• A3 red cells may react weakly or not at all with anti-A monoclonal antibodies but react better with anti-A,B reagents.

Ax Subgroup

• The Ax subgroup typically shows weak or no agglutination with commercial anti-A monoclonal antibody reagents, and individuals in the Ax subgroup may have anti-A1 antibodies in their serum.
• Ax red cells have enhanced detection when using anti-A,B reagents due to the blended specificity toward both A and B antigens.

Ael Subgroup

• Ael red cells exhibit weak or no agglutination with anti-A monoclonal antibody reagents, and individuals in the Ael subgroup may possess anti-A1 antibodies in their serum.
• Saliva studies for the detection of soluble forms of A and H antigens, and testing with anti-H lectin (Ulex europaeus), can provide additional information.
• The amount of H antigen present in weak subgroups of A (like Ael) is equivalent to group O red cells, showing strong (3+ to 4+) reactions.

General Notes for Weak A Subgroups

• Weak subgroups of A are characterized by a decreased number of A antigen sites per red cell, resulting in weak or no agglutination with commercial anti-A reagents.
• Special techniques, such as adsorption and elution, may be necessary to demonstrate the presence of the A antigen, but are not routinely performed.
• Current commercial anti-A monoclonal antibody reagents are blended to enhance the detection of weaker subgroups, although some subgroups may still react weakly or not at all.

Weak A Subgroups

• Saliva studies and anti-H lectin testing can help differentiate weak subgroups by detecting soluble antigens and comparing the amount of H antigen present.

Possible Genotypes for Each ABO Phenotype

• Phenotype A: AA, AO
• Phenotype B: BB, BO
• Phenotype O: OO
• Phenotype AB: AB

Group A Individuals

• Produce anti-B antibodies that are primarily IgM class with small amounts of IgG.

Group B Individuals

• Produce anti-A antibodies that are primarily IgM class with small amounts of IgG.

Group O Individuals.

• Produce both anti-A and anti-B antibodies that are primarily IgG class.

ABO Blood Group System Antibodies

• The titers of anti-A and anti-B can vary widely; in whites, the titer of anti-A is generally higher than anti-B, and in blacks, the titers of both anti-A and anti-B are higher than those found in whites.

Hemolytic Properties and Clinical Significance

• Both IgG and IgM forms of anti-A and anti-B can activate and bind complement, leading to hemolysis of red cells in vivo or in vitro.
• ABO antibodies are of clinical significance due to their ability to cause hemolysis, and an antigen-antibody reaction between a recipient’s ABO antibody and transfused red cells can lead to complement activation and destruction of donor red cells, resulting in acute hemolytic transfusion reactions.

In Vitro Serologic Reactions

• ABO antibodies directly agglutinate a suspension of red cells in a physiologic saline environment and do not require any additional potentiators for this reaction. These antibodies are optimally reactive in immediate-spin phases at room temperature (15°C to 25°C), with agglutination reactions occurring immediately upon centrifugation without an incubation period.

RBC Products

• Universal Donor: Group O (no A or B antigens on RBCs).
• Universal Recipient: Group AB (no anti-A or anti-B antibodies).

Plasma Products

• Universal Donor: Group AB (no anti-A or anti-B antibodies in plasma).
• Universal Recipient: Group O (no restriction on receiving plasma from any group).

Practical Application

• Any discrepancy in ABO testing should be resolved before transfusion of recipients or labeling of donor units.

Guidelines for Investigating ABO Technical Errors

• Identification or Documentation Errors: Correct sample identification on all tubes, results are properly recorded, and interpretations are accurate and properly recorded.
• Reagent or Equipment Errors: Daily quality control on ABO typing reagents is satisfactory, reagents are inspected for contamination and hemolysis, and centrifugation time and calibration are confirmed.
• Standard Operating Procedure Errors: Procedures follows manufacturer’s directions, correct reagents were used and added to testing, and red blood cell suspensions are at the correct concentration.

Acquired B Antigen

• It occurs in individuals with conditions such as intestinal obstruction or carcinoma of the colon, allowing bacterial polysaccharides from E. coli to enter the patient's circulatory system.
• In group A1 individuals, the patient's group A red cells absorb the B-like polysaccharide from E. coli, which then reacts with human-source anti-B antibodies.

B(A) Phenotype

• This phenotype is a rare condition where individuals with the B blood group express A-like antigens on their red cells due to a genetic variation that causes the B glycosyltransferase enzyme to add A-like structures to the red cells.

Resolving Discrepancies

• Repeat Testing: Repeat the test to rule out technical errors such as specimen mix-up, incorrect cell suspension, or failure to add reagents, and request a new specimen if the discrepancy persists.
• Extended Testing: Employ anti-A1 and anti-H lectins to differentiate between subgroups and identify specific antigenic structures.Adsorption-Elution Techniques can help demonstrate the presence of weak antigens while performing serum transferase studies and genetic testing to identify rare phenotypes.

Methods for Resolving ABO Discrepancies

• Resolving acquired B antigen discrepancies uses a monoclonal anti-B clone (ES4) reagent to strongly agglutinate cells with the acquired B antigen. The B(A) phenotype involves extended testing with anti-A1 and anti-H lectins, adsorption-elution techniques, and molecular analysis.

Causes for Missing or Weakly Expressed ABO Antigens

• ABO Subgroups: Subgroups of A and B have a reduced number of A or B antigen sites per red cell, resulting in weaker or no agglutination when tested with commercial anti-A or anti-B reagents.
• Weakened Antigen Expression in Diseases: Conditions such as leukemia or Hodgkin’s disease can cause a reduction in the expression of A and B antigens on red cells, leading to weaker-than-expected reactions in serologic testing.

Test Methods to Resolve ABO Discrepancies

• Repeat Testing: Confirms initial results and rules out technical errors by performing repeat testing with fresh reagents and carefully following standard procedures. (Purpose: Detect weaker subgroups more effectively. Method: Utilize human polyclonal or monoclonal anti-A/B reagents, that blend anti-A and anti-B clones for enhanced detection of weak subgroups.)
• Saliva Studies: Detect soluble forms of A and H antigens (adsorption-elution techniques demonstrates the presence of weak antigens) and molecular analysis confirms rare phenotypes of specific alleles.

Additional testing

• Use anti-A1 (Dolichos biflorus lectin) and anti-H (Ulex europaeus lectin) to identify specific antigenic structures. Anti-A1 reacts with A1 cells but not weaker subgroups; anti-H reacts with H antigen, higher in weak subgroups.

Causes of Extra Reactions in Serum Testing

• Serum reactions can occur to A subgroups with anti-A1, cold alloantibodies, rouleaux formation, cold autoantibodies, and intravenous immunoglobulin therapy.

Strategies for Resolving Extra Serum Reactions in ABO Discrepancies

• Review patient history (transfusions, IVIG, cold agglutinin disease, monoclonal gammopathies), and perform additional serologic testing (cold autoadsorption, enzyme treatment, saline replacement).
• Additional options are to repeat testing at warmer temperatures (37°C) to rule out cold-reacting antibodies and use pre-warmed reagent red cells to minimize false-positive agglutination.

Additional methods

• Use anti-A1 lectin to identify and classify weak or variant A subgroups. Another option is to perform a saline replacement test—if rouleaux is present, true agglutination will disappear after washing with saline

Genetic Pathway of the Bombay Phenotype

• The Bombay phenotype occurs in individuals homozygous for the h allele (hh) who lack the enzyme L-fucosyltransferase necessary to add L-fucose, forming the H antigen. Without the H antigen, A and B glycosyltransferases cannot produce the corresponding antigen structures.

Serologic Reactions of the Bombay Phenotype

• In routine ABO testing, the red cells of Bombay phenotype individuals appear similar to the group O phenotype because they lack H and ABO antigens. Serum testing shows reactions similar to group O individuals, with the presence of anti-A, anti-B, and anti-A,B antibodies.

Anti-H Antibody

• A significant feature of the Bombay phenotype is the presence of anti-H antibodies in the serum, which are clinically significant. Anti-H is capable of high thermal activity at 37°C.

Transfusion Implications of the Bombay Phenotype

• Compatibility Issues: Individuals with the Bombay phenotype can only receive blood from other Bombay phenotype donors because of the absence of H and ABO antigens on their red cells and the presence of anti-H antibodies in their serum.
• Potential Solutions: Stored autologous units (self-donated blood), blood from siblings with the same phenotype, or sourcing from rare donor files are potential options for transfusions.

Secretor

• Genetic Basis: Individuals with the Se allele at the secretor locus (SeSe or Sese).
• Function: The Se allele (FUT2) produces L-fucosyltransferase, adding L-fucose to type 1 oligosaccharide chains in secretory glands, which is crucial for the expression of soluble A, B, and H antigens in body fluids.

Nonsecretor

• Genetic Basis: Individuals with the sese genotype (homozygous for the se allele).
• Function: The se allele is an amorph (nonfunctional), and nonsecretors do not produce the enzyme L-fucosyltransferase required to add L-fucose to glycoprotein antigen precursors.
• Antigen Expression: About 20% of the random population are nonsecretors, and they do not have soluble H antigens in their body fluids, resulting in no soluble A or B antigens present in these fluids.