CLS 3311 Advanced Clinical Immunohematology

1. CLS 3311Advanced Clinical Immunohematology ABO Blood Group System

2. History Carl Landsteiner: • Discovered the ABO Blood Group System in 1901 • He and five co-workers began mixing each others red blood cells and serum together and inadvertently performed the first forward and reverse ABO groupings. • Landsteiners Rule: If an antigen is present on a patients red blood cells the corresponding antibody will NOT be present in the patients plasma, under ‘normal conditions’.

3. The ABO Blood Group System was the first to be identified and is the most significant for transfusion practice. AABBTechnical Manual It is the ONLY system that the reciprocal (antithetical) antibodies are consistently and predictably present in the sera of people who have had no exposure to human red cells. AABB Technical Manual ABO Blood Group System

4. Major ABO Blood Groups

5. Forward GroupingDefinition: Determination of ABO antigens found on patient red blood cells using reagent antisera.

6. Reverse GroupingDefinition: Determination of ABOantibodies found in patient serum using reagent red blood cells.

8. ABO Antibodies • Generally IgM class antibodies • I need you to recall the characteristics of IgM antibodies, because now they apply to the ABO blood group system antibodies. • ABO Antibody Development: Hypothesis • Immune response following exposure to environmental antigens (such as bacterial cell walls) similar to A and B antigens during infancy results in production of ABO antibodies. Remember, babies have a tendency to put EVERYTHING into their mouths…

9. ABO Antibodies • For Group A and Group Bpersonsthepredominant antibody class is IgM • For Group O people the dominant antibody class is IgG(with some IgM) • React best at room temperature (22-24oC) or below in vitro. • Activates complement to completion at 37oC • Can cause acute hemolytic transfusion reactions • RBC Immune form: Predominantly IgG

10. Which ABO blood group presents a higher risk for Hemolytic Disease of the Newborn? Why? • Group O - because the dominant immunoglobulin class is IgG, which crosses the placenta. • Group A and B can but only the immune form. Which means that only after exposure to foreign ABO antigens will the mother make immune anti-A or anti-B that is predominantly IgG.

11. ABO Antibodies Time of appearance: • Generally present within first 4-6 months of life • Do we perform a reverse grouping on newborns (<4-6 months of age) and cord blood? • If there are anti-A or anti-B antibodies in newborn serum where did they most likely originate? What source? • ABO antibody titers with age: • Reach adult level at 5-10 years of age • Level off through adult life • Begin to decrease in later years: >65 years of age

12. ABO AntibodiesGroup O Phenotype • Anti-A,B Antibody • Inseparable anti-A and anti-B antibody. If we add A cells to anti-A,Bserum all of the antibody activity is removed, not just anti-A!! • RBC immune Anti-A,B • When exposed to Group A or B antigens (or both) Group O persons will have an immune response that results in the production of separate immune anti-A and/or anti-B antibodies. This could be seen in a fetomaternal bleed of a Group O mom with a Group A baby. (Hemolytic Disease of the Newborn)

13. ABO Antibodies GroupB or O phenotype • Have bothanti-Aand Anti-A1antibodies Anti-A • Reacts with both A1 and A2 red blood cell antigens Anti-A1 • Reacts only with A1 antigens on red blood cells • A2 and A2B phenotypes can make anti-A1 antibodies. What is clinical significance? Thermal range is up to 25oC - not usually clinically significant. Can cause an ABO discrepancy.

14. ABO Antibodies • Is there a reagent anti-A1 antisera? • NO!! • But there is Dolichos biflorus,a plant lectin that has anti-A1 activity when diluted properly. • This is not an antibody, but a chemical that acts like an antibody in that it specifically agglutinates A1 red blood cells.

15. ABO Genetics • Genes at three separate loci control the OCCURRENCE and LOCATION of A and B antigens • Hh genes– H and h alleles • H allele codes for a fucosyltransferase enzyme that adds a fucose on Type 2 chains (primarily) to form the H antigen onto which A and B antigens are built on red blood cells. • h allele is a silent allele (amorph)

16. ABO Genetics 2. Se genes– Se and se alleles • Se allele codes for a fucosyltransferase enzyme that adds fuscos onto Type 1chains (primarily) in secretory glands. Controls expression of H antigens in secretions (i.e. saliva, body fluids, etc.) • se allele is an amorph 3. ABO genes– A, B and O alleles • A and B alleles code for glycosyltransferase enzymes that add a sugar onto H antigens to produce A and B antigens • O allele does not code a functional enzyme

17. A, B and H antigens are built on oligosaccharide chains of 4 types. The most common forms are Type 1 and Type 2. Type 1: #1 carbon of Gal is attached to the #3 carbon of GlcNAc. Type 2: #1 carbon of Gal is attached to the #4 carbon of GlcNAc.

18. This represents a method to determine possible genotypes and phenotypes. For future reference, if you happen to be asked a question concerning possible ABO genotypes and/or phenotypes of offspring, now you could do it!!

19. Genes at three separate loci control the Occurrence and Locationof ABO antigens • OCCURRENCE • The presence or absenceof the ABH antigens is controlled by the H and ABO genes. • LOCATION • The presence or absence of the ABH antigens on the red blood cell membrane is controlled by the H gene • The presence or absence of the ABH antigens in secretions is indirectly controlled by the Se genes.

20. Hh gene – H and h alleles (h is an amorph) Se gene – Se and se alleles (se is an amorph) ABO genes– A, B and O alleles Controls presence of H, A, and B antigens on both RBCs and in Secretions Controls presence of H antigen in the secretions Inherit 1 gene from each parent that codes for an enzyme that adds a sugar totheH antigen ABO Antigen Genetics

21. H Antigen The H gene codes for an enzyme (fucosylytranferase) that adds a Fucose to the terminal sugar of a Precursor Substance (PS*). The biochemical structure below constitutes the H Antigen. (h gene is an amorph.) H gene acts on a Precursor substance(PS)* by adding Fucose *PS = oligosaccharide chain attached to eitherglycosphingo-lipid, Type 2 chain (on RBC)or glycoprotein, Type 1 chain (in secretions)

22. H antigen is the foundation upon which A and B antigens are built. A and B genes code for enzymes that add an immunodominant sugar to theH antigen.

23. Formation of theA Antigen TheA gene codes for an enzyme that adds GalNAc (N-Acetyl-D galactosamine) to the terminal sugar of theH Antigen. This biochemical structure constitutes theA antigen.

24. Formation of theB Antigen Bgene codes for an enzyme that adds D-Galactoseto the terminal sugar of the H Antigen. This biochemical structure constitutes theB Antigen.

25. The H antigen is found on the rbc when you have the Hh or HH genotypes but NOT with the hh genotype. The A antigen is found on the rbc when you have the Hh, HH, and A/A,A/O or A/B genotypes. The B antigen is found on the rbc when you have the Hh, HH, and B/B,B/O or A/B genotypes.

26. Amount of H Antigen According to Blood Group • Blood Group O people have red blood cells rich in H antigen. Why? Neither the A or B genes have converted the H antigens to A or B antigens - just a whole bunch of H! LeastAmount of H Greatest Amount of H O > A2 > B > A2B > A1 > A1B

27. Bombay (Oh) Phenotype • Homozygous inheritance of the h gene (hh) results in the inability to form the H antigen and subsequently the A or B antigens. • This is referred as the Bombay or Oh phenotype due to the location of its discovery. • This phenotype has no H, A or B antigens on the red blood cell membrane, only an abundant amount of precursor substance. • They also have anti-H, anti-A and Anti-B. What blood type can we safely transfuse?

28. ABO Antigens in Secretions • Secretions: • Body fluids including plasma, saliva, synovial fluid, etc. • Blood Group Substance:Soluble antigen • Soluble antigen found in the secretions not bound to a membrane such as a rbc or epithelial cell. Soluble blood group substances (A, B and H) can be found in the secretions. This is controlled by the H and Se genes.

29. FORMATION OF ABO ANTIGENS IN SECRETIONS Se/seH/HA/O PS1 PS2 H Ag A,HAg genesgenesgenes From left to right is the gene interactions necessary for the production of ABH antigens in secretions. Must have Se gene (78% of population) for ABO Ag’s to be in secretions.

30. FORMATION OF ABO ANTIGENS IN SECRETIONS Se/seH/HO/O PS1 PS2 H Ag H Ag genesgenesgenes Inheritance of the O/O genotype results in the presence of only H antigen in the secretions.

31. LACK OF ABO ANTIGENS IN SECRETIONS Se/seh/hA/O PS1 PS2 PS2 PS2 genesgenesgenes Two mechanisms exist that account for a LACK of ABO antigens in secretions: Either se/se or h/h genotypes.

32. LACK OF ABO ANTIGENS IN SECRETIONS se/seH/hA/O PS1 PS1 PS1 PS1 genesgenesgenes Two mechanisms exist that account for a LACK of ABO antigens in secretions: Either se/se or h/h genotypes.

33. Relationship of ABO Blood Group with Le, I and P • As we continue to study the Other Blood Groups we will learn that there is a genetic and biochemical relationship between the ABO blood group and the Lewis, I and P systems. • The following table gives us a taste of that relationship. Don’t worry about it now, just know that it exists.

35. ABO Subgroups • ABO Phenotypes that differ in the amount of antigen carried on red cell and saliva, for secretors: There are fewer Ag sites! • Subgroups are the results of less effective glycosyltransferase enzymes – just not as good at attaching the immunodominant sugar to the H antigen. • Subgroups of A are more common than Subgroups of B.

36. ABO Subgroups • 80% of all Group A’s are A1 and about 19% are A2. • A1’s have 4-6 times the # of antigen sites on the RBC surface than A2’s. • Both react strongly with reagent Anti-A but… • Only A1 cells are agglutinated with Dolichos biflorus plant lectin and not A2 cells. • The remainder of the Subgroups of A have even weaker expression of A antigen.

37. ABO Subgroups • You will be responsible for knowing the reaction patterns of the A1, A2, A3 and Ax subgroups of A. The table on the next slide if a very good reference. • Knowing these reactions will help you resolve weak reaction strengths and ABO discrepancies.

39. Subgroups of B • The same explanation of Subgroups of A applies to Subgroups of B. • Subgroups of B are less common than Subgroups of A and thus encountered less often. • You will be responsible for knowing the reaction patterns of the B, B3 and Bx subgroups of B.