Blood grouping

AGA KHAN UNIVERSITY HOSPITAL

1. Blood Grouping

2. History - Karl Landsteiner Discovered the ABO Blood Group System in 1901 He and his five co-workers began mixing each others red cells and serum together and inadvertently performed the first forward and reverse ABO groupings

3. Landsteiners Rule If an antigen (Ag) is present on a patients red blood cells the corresponding antibody ( Ab ) will NOT be present in the patients plasma, under ‘normal conditions’.

4. Major ABO Blood Group ABO Group Antigen Present Antigen Missing Antibody Present A A B Anti-B B B A Anti-A O None A and B Anti-A&B AB A and B None None

5. ABO Basics Blood group antigens are actually sugars attached to the red blood cell. Antigens are “built” onto the red cell. Individuals inherit a gene which codes for specific sugar(s) to be added to the red cell. The type of sugar added determines the blood group

6. Principle of blood grouping There are two principles 1-almost all normal healthy individuals above 3-6 months of age have “ naturally occurring Abs ” to the ABO Ags that they lack These Abs termed naturally occurring because they were thought to arise without antigenic stimulation

7. Principle of blood grouping 2- These “ naturally occurring ” Abs are mostly IgM class . That means that, they are Abs capable of agglutinating saline/ low protein suspended red cell without enhancement and may activate complement cascade .

8. ABO and H Antigen Genetics Genes at three separate loci control the occurrence and location of ABO antigens The presence or absence of the A, B, and H antigens is controlled by the H and ABO genes

9. 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 gene

10. H Antigen The H gene codes for an enzyme that adds the sugar fucose to the terminal sugar of a precursor substance (PS) The precursor substance (proteins and lipids) is formed on an oligosaccharide chain (the basic structure)

11. RBC Precursor Structure Glucose Galactose N-acetylglucosamine Galactose Precursor Substance (stays the same) RBC

12. Formation of the H antigen Glucose Galactose N-acetylglucosamine Galactose Precursor Substance (stays the same) RBC H antigen Fucose

13. H antigen The 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 the H antigen Immunodominant sugars are present at the terminal ends of the chains and confer the ABO antigen specificity

14. A and B Antigen The “ A ” gene codes for an enzyme ( transferase ) that adds N- acetylgalactosamine to the terminal sugar of the H antigen N- acetylgalactosaminyltransferase The “ B ” gene codes for an enzyme that adds D- galactose to the terminal sugar of the H antigen D- galactosyltransferase

15. Formation of the A antigen Glucose Galactose N-acetylglucosamine Galactose RBC Fucose N-acetylgalactosamine A antigen

16. Formation of the B antigen Glucose Galactose N-acetylglucosamine Galactose RBC Fucose Galactose B antigen

17. H antigen Certain blood types possess more H antigen than others: O>A 2 >B>A 2 B>A 1 >A 1 B

18. Why do Group O individuals have more H antigen than the other groups? Group O individuals have no A or B genes to convert the H antigen to A or B antigens….that means more H antigen sites

19. Group O Group A Many H antigen sites Fewer H antigen sites A A A A A Most of the H antigen sites in a Group A individual have been converted to the A antigen

20. Genetics The H antigen is found on the RBC when you have the Hh or HH genotype, but NOT from 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

21. Bombay Phenotype (O h ) Inheritance of hh The h gene is an amorph and results in little or no production of L- fucosyltransferase Originally found in Bombay Very rare (130 worldwide)

22. Bombay Phenotype (O h ) The hh causes NO H antigen to be produced Results in RBCs with no H, A, or B antigen (patient types as O) Bombay RBCs are NOT agglutinated with anti-A, anti-B, or anti-H (no antigens present) Bombay serum has strong anti-A, anti-B and anti-H, agglutinating ALL ABO blood groups What blood ABO blood group would you use to transfuse this patient?? Another Bombay Group O RBCs cannot be given because they still have the H antigen You have to transfuse the patient with blood that contains NO H antigen

23. ABO antibodies Group A serum contains anti-B Group B serum contains anti-A Group AB serum contains no antibodies Group O serum contains anti-A, anti-B, and anti-A,B

24. ABO antibodies IgM is the predominant antibody in Group A and Group B individuals Anti-A Anti-B IgG (with some IgM ) is the predominant antibody in Group O individuals Anti-A,B (with some anti-A and anti-B)

25. ABO antibodies Reactions phase: Room temperature Complement can be activated with ABO antibodies (mostly IgM , some IgG ) High titer: react strongly (4+) Usually present within the first 3-6 months of life Stable by ages 5-6 years Decline in older age Newborns may passively acquire maternal antibodies ( IgG crosses placenta) Reverse grouping (with serum) should not be performed on newborns or cord blood

26. ABO routine testing Several methods for testing the ABO group of an individual exist. The most common method is: Serology: This is a direct detection of the ABO antigens. It is the main method used in blood transfusion centres and hospital blood banks. This form of testing involves two components: a) Antibodies that are specific at detecting a particular ABO antigen on RBCs.        b) Cells that are of a known ABO group that are agglutinated by the naturally occurring antibodies in the person's serum.

27. ABO ROUTINE TESTING DIRECT OR FORWARD GROUPING Test for antigens Patient’s cells containing unknown antigens tested with known antisera Antisera manufactured from human sera Aantisera used: Antisera Color Source Anti-A Blue Group B donor Anti-B Yellow Group A donor Anti-A,B Red Group O donor

28. Forward Grouping Reaction of patient red blood cells tested with Reagent anti-A and anti-B antisera Slide: 20-40% RBC suspension + anti-serum Tube (12x75mm): 2-5% RBC suspension + anti-serum (centrifuge before read)

29. Forward Grouping Reaction Patterns for ABO Groups Blood group Agglutination with Anti-A Agglutination with Anti-B A + - B - + AB + + O - -

30. Reverse grouping serum is combined with cells having known Ag content in a 2:1 ratio uses commercially prepared reagents containing saline-suspended A 1 and B cells

31. Reverse grouping Reaction Patterns for ABO Groups Blood Group Agglutination with A cells Agglutination with B cells A - + B + - AB - - O + +

32. Grading of Agglutination : Negative (0) No clumps or aggregates Weak (+/-) Tiny clumps or aggregates barely visible macroscopically or to the naked eye 1+ Few small aggregates visible macroscopically 2+ Medium-sized aggregates 3+ Several large aggregates 4+ One solid aggregate

33. ABO blood group (forward blood grouping) Patient Red Cells Tested With Interpretation Anti-B Anti-A Patient 1 4+ 2 4+ 3 4+ 4+ 4

34. ABO blood group (forward blood grouping) Patient Red Cells Tested With Interpretation Anti-B Anti-A Patient O 1 A 4+ 2 B 4+ 3 AB 4+ 4+ 4

35. Reverse Grouping (Confirmatory grouping Patient SERUM Tested With Interpretation B Cells A 1 Cells Patient 4+ 4+ 1 4+ 2 4+ 3 4

36. Reverse Grouping (Confirmatory grouping Patient SERUMTested With Interpretation B Cells A 1 Cells Patient O 4+ 4+ 1 A 4+ 2 B 4+ 3 AB 4

37. Forward & reverse ABO blood grouping Reaction of Cells Tested With Reaction of Serum Tested Against ABO Group Anti-A Anti-B A 1 Cells B Cells 1 + + O 2 + + A 3 + + B 4 + + AB

38. Forward & reverse ABO blood grouping Reaction of Cells Tested With Reaction of Serum Tested Against ABO Group Anti-A Anti-B A 1 Cells B Cells 1 + + 2 + + 3 + + 4 + +

39. ID card system This ID-Card contains a mixture of human polyclonal and monoclonal anti-A, human polyclonal anti-B and human polyclonal anti-D antibodies. The microtube ctl is the negative control. Two microtubes with neutral gel serve for reverse grouping with A1 and B cells.

40. Fully-automatic walk-away for ID-Cards Stand alone instrument Continuous sample loading Continuous reagent loading Priority samples (STAT) Reagent stock High throughput

41. Optimized for small blood volumes Dispense verification Easy-to-use Full test menu Wi-Fi Touchscreen 17" Host connectivity Internal & external validation Capacity 180 samples 240 ID-Cards 28 reagent vials

42. Thank you….