The ABO Blood Group System

• ABO blood group system is crucial in transfusion as it is the only system with antibodies in serum against absent RBC antigens.

ABO System

• Involves forward grouping (detecting antigens on RBCs with known antisera) and reverse grouping (detecting antibodies in serum with known RBCs) as checks.
• Exposure to A-like and B-like antigens from bacteria can stimulate anti-A & anti-B production.

ABO Antibodies

• ABO antibodies can cause rapid, intravascular hemolysis.
• Anti-A, B detects weakly expressed A & B antigens more effectively than Anti-A or anti-B reagents.
• IgG and IgM ABO antibodies react at room temperature (20-24°C) or below and activate the complement system at 37°C.

Inheritance of ABO Blood Group

• ABO is codominant.
• The ABO gene is present on chromosome 9.

Formation of A, B, and H RBC Antigens

• ABH antigen formation results from gene interactions at three loci: ABO, Hh, and Se.
• These genes produce glycosyltransferases that add sugars to a basic precursor substance.
• Paragloboside is a basic precursor that forms A, B, and H antigens.
• Precursor I has a β-1-3 linkage between galactose and N-acetylglucosamine.
• Precursor II has a B-1-4 linkage between galactose and N-acetylglucosamine.
• ABH antigens develop as early as the 37th day of fetal life on RBCs.
• The A gene elicits a higher concentration of transferase than the B gene.
• When both A & B genes are inherited, the B-enzyme works more efficiently on the H substance than the A enzyme.

Molecular Genetics of ABO

• The A & B gene is on chromosome 9 and has seven exons.
• Exons 6 & 7 encode the catalytic domain of ABO glycosyltransferases.
• The O gene is identical to the A gene except for a deletion in the coding region at a nucleotide position.
• A secretor gene (SeSe or Sese) is responsible for producing a transferase (α-2-L-fucosyltransferase).
• It modifies the type 1 precursor substance in secretion to express the H substance.
• A, B, and H substances in saliva are evidence of A, B, and H Se gene inheritance.
• Fluids where A, B, and H substances can be found in secretors include saliva, tears, urine, digestive juices, bile, milk, amniotic fluid, and certain pathologic fluids.

ABH Antigens Comparison

• RBC antigens can be glycolipids, glycoproteins, or glycosphingolipids and are only synthesized on type 2 precursor chains.
• Type 2 chain has a beta 1→4 linkage.
• The H gene produces an enzyme (α-2-L-fucosyltransferase) that acts primarily on type 2 chains on the RBC membrane.
• Secreted substances are glycoproteins, primarily synthesized on type 1 precursor chains.
• Type 1 chain has a beta-1-3 linkage.
• The Se gene produces an enzyme (α-2-L-fucosyltransferase) that acts preferentially on type 1 chains in secretory tissues.

ABO Subgroups

• A subgroups are characterized by a decreased number of A antigen sites per RBC.
• They have varying degrees of agglutination by human Anti-A,B.
• RBCs react well with Anti-H.
• Presence or absence of Anti – A₁ can occur in the serum.
• A enzyme transferase added sugar (NALNAC) on four forms of H antigens
• Two unbranched straight chains - H1, H2
• Two branched chain - H3, H4
• Secretor studies and adsorption-elution tests can subdivide A individuals to A3, Ax, Aend, Am, Ay and Ael.

A1 blood group

• The A1 enzyme transferase can convert all H antigens (H₁ to Aª, H₂ to Ab, H3 to Aº, H4 to Ad).

A2 blood group

• A glycosyltransferase convert only H₁ and H2
• Some A2 individuals produce Anti – A₁ and can cause a transfusion reaction.
• Characterized by mixed-field agglutination with Anti-A and Anti – A,B reagent occurs in A3 blood groups.
• Anti – A₁ may be present in the serum.
• Weak α-3-N-acetylgalactosaminyltransferase activity is detectable in the serum.
• Group 1: enzyme is reactive at pH 7 and has very low activity.
• Group 2, enzyme is undetectable.
• Group 3, enzyme is reactive at pH 6 and has weak activity.

Ax blood group

• RBCs react with Anti – A,B but not Anti - A.
• Anti – A can be adsorbed to RBCs and then eluted from Ax RBCs.
• Transferase is undetectable.
• Anti A₁ is present in the serum.
• Ax secretors contain A substance detectable only by agglutination/inhibition studies using Ax RBCs as indicators.
• An Ax donor mistyped as blood group O can cause rapid intravascular hemolysis.

Aend blood group

• Characterized by mixed field agglutination with Anti – A and Anti – A,B reagent with weak agglutination.
• Transferase is undetectable; Anti – A₁is present in some.

Am blood group

• RBCs show very weak or no agglutination with Anti – A or Anti – AB.
• Anti – A can be adsorbed to RBCs and then eluted from Ax RBCs.
• Transferase is detectable in the serum, but Anti –A₁ is not produced.

Ay blood group

• Ay RBCs are unagglutinated with Anti – A or Anti – A,B.
• Anti A can be adsorbed to RBCs and then eluted from Ay RBCs.
• A little amount of transferase is detectable and Anti – A₁ is usually not produced.

Ael blood group

• RBCs unagglutinated with Anti – A or Anti – A,B.
• Anti – A can be adsorbed to RBCs and then eluted from A1 RBCs.
• The enzyme transferase is not detectable, and Anti –A₁ is not produced.

Weak B Subgroups

• B3, Bx, Bm, Bel are weak B subgroup.
• Use the agglutination strength with Anti-B, Anti-A,B, and Anti-H to differentiate weak B phenotypes
• Determine ABO isoagglutinins presence/absence in the serum
• Perform adsorption-elution studies with anti-B.
• Determine B substance precense in the saliva.

B3 blood group

• Characterized by mixed-field agglutination with anti-B and anti-A,B.
• B transferase is present, while anti-B is absent in the serum.

Bx blood group

• Gives a weak agglutination with anti-B and anti-A,B.
• B transferase can't be detected, and weak anti-B is produced.

Bm blood group

• RBCs are not agglutinated by anti-B or anti-A,B.
• B transferase is detected in the serum and anti-B is absent.

Bel blood group

• RBCs are not agglutinated by Anti-B or Anti-A,B.
• B transferase is detected in the serum and weak Anti-B is produced.

The Bombay Phenotypes (Oh)

• The H gene is necessary for forming A and B antigens (99.9% of individuals have HH or Hh genotype).
• The h is rare allele which do not produce the L-Fucose transferase required to form H-Structure.
• The (hh) or H null genotype is extremely rare and known as the Bombay Phenotype or Oh.
• The H gene codes for (α-2-L-fucosyltransferase) that adds a Fucose to the terminal sugar of a Precursor Substance (PS*).
• H antigen structure is made of a precursor substance and fucose while A and B genes encode that adds immunodominant sugar to the H antigen.
• The O allele does not code for a functional enzyme.
• The H antigen amount varies among blood types in the order O > A2 > B > A2B > A1 > A1B.
• First reported in Bombay, India in 1952.
• Bombay cells cannot be converted to group A or B due to lack of the H structure (acceptor molecule for the A or B gene transferase.
• Therefore devoid of normal A, B, H antigens failing to react with anti-A, anti-B, anti-A,B, and anti-H.
• Contain anti-A, -B & anti-H which is effective during a wide thermal range is an IgM antibody.
• Bombay blood is incompatible with all ABO donors due to the ubiquitous expression of the H antigen. In routine forward grouping, using anti-A, anti-B, and anti-AB will display as group O.
• Transfusion of normal group O would cause immediate cell lysis due to the potent anti-H in Bombay individuals.
• Only Bombay blood can be transfused to a Bombay recipient. Oh (hh) individuals are non-secretors of ABH substances (both the H and Se gene are inherited for the ABH antigens to be in secretions).
• Lack of the H antigen can be confirmed by testing red cells with Ulex Europaeus or sera containing Anti-H activity, pre-adsorbed with anti-A and anti-AB activity.
• Ulex Europaeus serves as readily available source of anti-H like.
• Should be employed to identify the presence/ absence of H antigen on the RBC surface.

General Characteristics of Bombay Oh (Hnull Phenotypes)

• Absence of H, A, and B antigens, which cause NO agglutination with anti-A, anti-B, or anti-H Lectin.
• The presence of anti-A, anti-B, anti-A,B, and potent anti-H active over a wide thermal range in the serum.
• A, B, H non-secretor and no A, B, or H substances are present in saliva
• Absence of α-2-L-fucosyltransferase (H enzyme) in serum & H antigen on red cells
• Red cells of Bombay phenotype (Oh will not react with anti-H Lectin (Ulex europaeus).& are only compatible with the serum from another Bombay individual.

H deficient phenotype categories

• (1) RBC H deficient, non secretor, Bombay phenotype (hh sese) called Classic Bombay (inheritance of hh genotypes, non secretor sese). These cells lack H, A and B antigen in RBCs and secretion. Anti – H is clinically significant can cause HTRs.
• (2) Para-Bombay phenotype (mutant alleles at the H genes but normal Se genes). Demonstrate minute quantities of ABH antigen on RBCs, or fail to demonstrate ABH antigen on RBC but expressed in the secretion based on Se inheritance. Adsorption and elution of anti – H may show the present of H antigen. Anti – H react at 25C.
• (3) RBC H – Partially deficient, Nonsecretor. Weak expression of antigens A or/and B on RBC, notation Ah, Bh and AB have been used to describe these individual. No A,B or H antigens in the saliva, Anti – H is weaker than Bombay phenotype/may react at 37C.

Discrepancy

• Exists when result of red cells and serum tests do not align.
• The discrepancy must be resolved.
• Donor samples with discrepancy must not be released until it is resolved (similar recipient discrepancy).
• Discrepancies can stem from lab errors or test problems resulting in false positives/negatives.

Error Causes

• Not adding reagents or test serum to the tube.
• Incorrectly identifying hemolysis as a positive result
• An inappropriate serum/ reagent ratio.
• Not centrifuging sufficiently.
• Temperature control.
• Faulty result interpretation.
• Incorrect reagent/sample us
• Inactive contaminated expired reagent use

Process to resolve discrepancy

• Check the age, diagnosis, transfusion and pregnancy history, medication, immunoglobulin levels.

Causes of Discrepancies

• Type 1 caused due to depressed antibody production/ inability to produce ABO antibodies - New-born in infant 4-6 mth/ Elderly patient/Leukemia(CLL)/Lymphoma/ Patient use immunosuppressive Drug/ Immunodeficiency disease/ transplant/ chimersim.
• Two cell population arise -Two fertilisation of the ova (artificial chimears or sperm fertilizing one egg
• T-activation of transplanted cells/ Exchange BM .Fetal maternal bleeding/Utero exchange.
• Type 2: Weakness of A or B antigen expression or grouping of A or B or both/ the hodgkin disease.
• Type 3: Excessive amount of soluble group - B.G.S .S enzymes of P vulgaris neutralise the reagent anti a and anti B.

Further Discrepancies

• B phenonmenon/ transferases which yield immuno dominance / intestinal obstruction
• The elevation of globulin or Wharton’s Jelly increases false positives
• Reverse ABO can also be caused/ elevated global levels
• polyagluniation leads to spontaneous cell agglutination by most human serum.
• Drug/H DNA transfusion reaction can be reversed via polyagglutination

Steps for Resolving discrepancies

• 1st remove any technical error
• Repeat serum test/wash cells
• Check sample
• Obtain relevant patient history
• Read AB screenings test.

Steps for resolving

• Enhance reverse sample through incubation at room temp/ 4 degrees, and ensure use of autologous controls.
• Confirm any immunoglobulin by running immunoassay, and repeating all forward sample testing
• Wash cells again
• Treat cells via enzyme such as ficin
• Run controls with those tests

Steps for resolving weak reactions

• Wash those cells
• Get a new diagnosis and history
• check to ensure there were no contaminations from anti-B antibodies or anti-saliva
• Use antibody blood test with different lot numbers
• Wash the cells in order/dilute reagent.

Steps for confirming other blood tests

• Monoclonal check via performing lectin studies
• Prewarm and incubate for warm and cold reactors
• Treat with DTT /perform auto adsoption to ensure all cells and plasma test correctly.

Addressing allo and antibodies

• Raise the temperatures to room temp levels before mixing the substance.
• A new blood test/ serum test for checking AB like antigen/enzyme treatments with ficin or saline wash after retyping to clear and isolate.

Key Facts

• Blood O is about 45% of the population (A:40%; B:11%; AB:%)
• ABO created with antibodies via the IGM
• ABO is dominant genes
• Soluble tissue antigens found in the body depend on AB blood group
• Reverse grouping is not performed on new borns

Further information

• O>2.B>A ( H amounts by dominance
• 80% chance of inheriting gene by generation
• Glycoproteins made of type one precusers are common with strong reactions .
• The last 22% inharot the code so