ABO Blood Group System

• ABO Blood Group System is the most important of all blood groups in transfusion practice.
• It is the only blood group system in which individuals have antibodies in their serum to antigens that are absent from their RBCs, naturally occurring due to exposure to RBCs through transfusion or pregnancy, for example, blood group A has anti-B.
• Transfusion of an incompatible ABO type may result in immediate lysis of donor RBCs, producing a very severe, if unresolved, fatal transfusion reaction.
• ABO incompatibility detection testing between a donor and a potential transfusion recipient is the foundation on which all other pre-transfusion testing is based.

Historical Perspective

• 1901 – ABO was discovered.
• Karl Landsteiner discovered the first human blood group system and first conducted reverse and forward typing.
• A, B, and O blood groups were the first to be identified.
• Sturli and Von Decastello discovered the AB blood group.
• The I gene codes for the ABO allele's: IA, IB, and i.

The Landsteiner Law

• The antigen is present on the RBC surface and determines the blood group/type.
• The corresponding antibody is never found in an individual's serum.
• The OPPOSITE naturally occurring antibody is always present in the individual's serum.
• Blood group percentages in the American population:
  • A: 40%
  • B: 10%
  • AB: 5%
  • O: 45%

Importance of ABO

• Almost all normal healthy individuals above 3-6 months of age have naturally occurring antibodies to the ABO antigens that they lack.
• Antibodies are termed naturally occurring because they were thought to arise without antigenic stimulation.
• These "naturally occurring" antibodies are mostly IgM class.
• They are capable of agglutinating saline/ low protein suspended red cells without enhancement and may activate complement cascade, causing strong rapid intravascular hemolysis.

Inheritance of the ABO blood group

• Inheritance theory of the ABO blood group was first described by Bernstein in 1924.
• An individual inherits one ABO gene from each parent (CODOMINANCE), and these two genes determine which ABO antigens are present on the RBC membrane.
• One position, or locus, on each chromosome 9 is occupied by an A, B, or O gene.
• The O gene is considered an amorph, with no detectable antigen being produced in response to the inheritance of the genes AA, BO and OO, which denotes genotype.
• A and B denotes phenotype.
• In the case of a blood type O individual, they possess a homozygous OO genotype.

Formation of A, B and H Red cell antigen

• Formation of ABH antigens results from the interaction of genes of three separate loci: ABO, Hh, and Se.
• These genes do not code for the production of antigens but rather produce specific enzymes (glycosyltransferase) that add sugars to a basic precursor/precursor substance called paragloboside or glycan.
• GENE → ENZYME → SUGAR (PS) → ANTIGEN
• H and Se genes are located on chromosome 19 and are not part of the ABO blood group system, but their inheritance affects the ABO blood group.
• The H gene must be inherited to form ABH antigens on the RBCs and codes for the H antigen:
  • Enzyme produced acts on Type II precursor substance.
  • Most individuals are homozygous HH.
  • The hh genotype is extremely rare and referred to as the Bombay phenotype.
• The Se gene must be inherited to form ABH soluble antigens in secretions and codes for the H substance:
  • The enzyme produced acts on Type I precursor substance.
• ABO genes are located on chromosome 9.
• The H antigen precursor structure on which A and B antigens are made.

Precursor Substances

• The precursor substance on erythrocytes is referred to as Type 2 precursor substance.
  • It means that the terminal galactose on the precursor substance is attached to the N-acetylglucosamine in a beta 1-4 linkage.
• Type 1 precursor substance means that the terminal galactose on the precursor substance is attached to the N-acetylglucosamine in beta 1-3 linkage.
• Type I and Type II precursor substances are both composed of 4 sugars:
  • 2 molecules of D-galactose
  • 1 molecule of glucose
  • 1 molecule of N-acetylglucosamine
• They differ only in the linkage of the terminal sugars (terminal D-galactose is attached to N-acetylglucosamine).
• Type 1 precursor substance is water-soluble and is found in plasma and tissues and body secretions (glycoproteins).
• Type 2 precursor substance is fat-soluble and is found as an integral part of the red cell membrane (glycolipids).

Glycosyltransferases and Immunodominant Sugars

• Glycosyltransferases and Immunodominant Sugars Responsible for H, A, and B Antigen Specificities:
  • H gene: a-2-l-fucosyltransferase and L-fucose.
  • A gene: a-3-w-acetylgalactosaminyltransferase and N-acetyl-D-galactosamine.
  • B gene: a-3-D-galactosyltransferase and D-galactose.
• L-fructose is attached to TYPE 2 PS.
• The A gene codes for the production of a-3-N-Acetylgalactosaminyltransferase. N-Acetylgalactosamine attaches to the H antigen to expressed the A antigen.
• B genes code for the production of a-3-D-galactosyltransferase. D-galactose is attached to the H antigen, expressing the B antigen.
• Type O blood has a red blood cell with Galactose, Fructose, and N-Acetyl-glucosamine.
• Type A blood has a red blood cell with Galactose, Fructose, N-Acetyl-glucosamine, and N-Acetyl-galactosamine.
• Type B blood has a red blood cell with Galactose, Fructose, N-Acetyl-glucosamine, and N-Acetyl-galactosamine.
• The occupied H antigen sites by blood type:
  • A: 810,000 to 1,170,000
  • B: 610,000 to 830,000
  • AB: A: 600,000 and B: 720,000
  • None: O
• Amount of H-antigen / reaction with anti-H is O > A2 > B > A2B > A₁ > A₁B.

ABO Antibodies

• ABO antibodies are initiated at birth, but titers are generally too low.
• ABO testing before 3-6 months of age cannot be considered valid because some or all antibodies present may be IgG maternal antibodies.
• More logical is performing forward typing on cord blood from newborns.
• Antibody production peaks between 5-10 years of age and declines later in life.
• Elderly people usually have lower levels of Anti-A and Anti-B; therefore, antibodies may be undetectable in Reverse grouping.
• They are present in diluted amounts in some animals and plants as lectins of plant or seed extracts to agglutinate specific human blood group antigens.
• Dolichos biflorus agglutinates Al or AlB cells(anti-A₁ lectin).
• Bandeiraea simplicifolia agglutinates B cells (anti-B lectin).
• Ulex europaeus agglutinates 0 cells (H specificity) and other ABO blood groups depending on the amount of H antigen (anti-H lectin).

ABO Antibodies in Type O

• Type O individuals contain Anti-A, Anti-B, and Anti-A,B.
• Anti-A,B reacts with A and B cells and was originally thought to be just a mixture of anti-A and anti-B.
• Anti-A, B is not part of ABO testing, but some believe that anti-A,B is more effective at detecting weakly expressed A and B antigens than Anti-A and Anti-B.

Two Methods in determining blood type

• Defined as using known sources, forward/direct/cell blood typing's commercial anti-sera (anti-A and anti-B) detects antigens on an individual RBC, such as the patient's red cells with an antisera reagent.
• Defined as detecting ABO antibodies in the patient's serum using known reagent RBC's, reverse/indirect/back blood typing is unique to the ABO blood group system and checks the forward typing results where the specimen is a patient's serum/plasma and the reagent is known cells.

Reagents for Forward Typing

• Anti A
• Anti B
• Anti A,B

Reagents for Reverse Typing

• Al cells
• B cells
• O cells

Interpreting Forward/Direct Typing

• Patient/Anti-A/Anti-B/Interpretation
  • 1/0/0/O
  • 2/4+/0/A
  • 3/0/4+/B
  • 4/4+/4+/AB

Interpreting Reverse/Indirect Typing

• Patient/A₁ Cells/B Cells/Interpretation
  • 1/4+/4+/O
  • 2/0/4+/A
  • 3/4+/0/B
  • 4/0/0/AB

Forward/Direct Typing Vs. ABO Cell Grouping

• ABO cell grouping detects the recipient's blood specimen with Antibody (Anti-A) that agglutinates a present A Antigen on the individual's RBC, as opposed to non-agglutination where A Antigen is not present in the individuals RBC.

Reverse/Indirect Typing Vs. ABO Serum Grouping

• ABO serum grouping detects a recipient with Group A Red Cells in their blood specimens, where agglutination means anti-A is present in the individual's serum, and non-agglutination means anti-A is not present.

Interactions of the Sese, Zz and ABH GENES

• The Sese system regulates the formation of H antigen and, subsequently, of A and B antigens in secretory cells (ABH soluble substances).
• ABH soluble substances exist and are found in the following genotypes: SeSe, Sese, sese, Secretors: SeSe, Sese, and Non-secretors: sese.
• The Zz system regulates the production of H antigens on erythrocytes.

Distinction of ABH Antigens and ABH Soluble Substances

• ABH Antigens exist in: RBC, epithelial cells, platelets, lymphocytes, and endothelial cells.
• ABH Soluble Substances exist in all body secretions except CSF.
• ABH Antigens are secreted substances of glycolipids.
• ABH Soluble Substances are secreted substances of glycoprotein.
• The 1st sugar in the precursor substance for ABH Antigens is glucose.
• The 1st sugar in the precursor substance for ABH Soluble Substances is N-acetylgalactosamine.
• ABH Antigen precursor chains are Type 2. ABH Soluble Substances precursor chains are Type 1.
• ABH Antigens possess beta 1-4 linkage.
• ABH Soluble Substances possess C1-C3 (Beta 1-3 linkage).
• Zz gene is regulating ABH Antigens.
• Se gene is regulating ABH Soluble Substances.

Test for ABH soluble substances/Determination of secretor status

• The SALIVA NEUTRALIZATION TEST is based on the principle of Hemagglutination Inhibition. (+) reaction: without agglutination, signifying the soluble substance is present.
• (-) reaction: with agglutination, signifying the soluble substance is absent:

1. Saliva should be heated to boil at 56°C for 10 minutes to denature salivary amylase to prevent interference.
2. Saliva should be diluted with a buffer such as NSS (1:2) to reduce non-ABH soluble substances present in saliva.
3. Antisera should be diluted (1:4) to reduce potency to avoid a false positive reaction.

Interpreting Steps

• B is present because it has been neutralized first by anti sera and is not reacting to the B cell without agglutination.
• A is not present because anti A was not neutralized, so anti A reacts with a cell, resulting in agglutination and an individual who is a B secretor.