1. Brian Poirier, MD UCDavis Medical Center
ABO Blood Groups
Brian Poirier, MD UCDavis Medical Center

2. Topics Basic genetics of ABO blood groups
Formation of H, A, and B antigens
ABO antigens and antibodies
ABO testing
ABO discrepancies

3. Objectives
Describe the inheritance of the ABO Blood Groups and predict the ABO phenotypes and genotypes of offspring from various ABO matings
Explain the roles of Secretor and H genes in the formation of H, A, and B antigens on the red cells
Describe the reciprocal relationship between ABO antigens and antibodies for blood types O, A, B, and AB.

4. Objectives (Continued)
Describe the procedures on ABO forward and back typings; interpret the results; and resolve any discrepancies if present.
Describe the quantitative and qualitative differences between the A1 and A2 antigens.
Correctly identify all the ABO compatible blood components for each blood type

6. Co-Dominance A “Big” letter doesn’t mean dominant
A “small” letter doesn’t mean recessive
The exceptions to #1 and #2 are:
The Secretor genes: Se and se
The Lewis genes: Le and le
The H genes: H and h

7. Inheritance of the ABO Blood Groups
First described by Bernstein in 1924.
A, B, O genes on chromosome #9
The expression of antigens are based on the combination of three gene alleles: A, B, and O.

8. Phenotypes vs Genotypes
Phenotypes Genotypes Group O OO Group A AA or AO Group B BB or BO Group AB AB

9. ABO Phenotype Frequencies in U.S. Populations
Phenotype White Blk Mexican Asian O A A Rare B A1B A2B 1 1 Rare Rare

10. B Allele World Map

11. Exercises
Mother is type A and father is type O: What are the possible blood types for their offspring?
Mother is type A and father is type B: What are possible blood types for their offspring?

13. Basic Biochemistry Type I and Type II chains Se gene H gene
Formation of the H antigen
Formation of the A and B antigens

15. Type I and Type II chains
Type I: primarily glycoproteins in secretions and plasma
Saliva, clostrum, mothers’ milk, gastric fluid, bile, urine, serum, plasma, ovarian cyst
Type II: primarily glycolipids on RBCs
RBC, WBC, platelets, normoblast, sperm, epidermal and epithelial cells

16. Type 1: Secretor ABH Glycoprotein Substances

17. Se Gene and Formation of the H Antigen
Secretor = SeSe, Sese; Nonsecretor = sese
80% of random population is either SeSe or Sese
Secretor gene codes for fucosyl transferase
Enzyme (FUT2) adds fucose to type I chains at terminal galactose; product is H antigen.

18. Type I chain: 1-3 linkage

19. H Gene and Formation of the H Antigen
Phenotypes: HH, Hh, hh
Virtually 100% of random population is either HH or Hh; hh genotype (lack of H =“Bombay phenotype”) is rare.
H gene also codes for fucosyl transferase (FUT1)
Enzyme (fucosyl transferase) adds fucose to terminal galactose of type II chains
Final product is H antigen

20. Formation of the H Antigen

21. Type 2: H, A, and B Antigens H Ag: Gal–GlcNAc–Gal-X|
Fuc
A Ag: GalNAc–Gal–GlcNAc–Gal-X
B Ag: Gal–Gal–GlcNAc–Gal-X

22. “A” Gene and Formation of the A Antigen
H antigen is required for A antigen formation on RBCs or in secretions/plasma
Formation of A antigen:
N-acetylgalactosamine is added to H antigen to make A antigen.
A Ag

23. Formation of the A Antigen

24. “B” Gene and Formation of the B Antigen
H antigen is required for B antigen formation on RBCs or in secretions/plasma
Formation of B antigen: D-galactose is added to H antigen to make B antigen.

25. Formation of the B Antigen

26. The Residual H Antigen
The more A or B antigen is made, the less H remains
Relative amounts of H by blood group
O>A2>B>A2B>A1>A1B

27. The Use of Lectins for Antigen Confirmation
Dolichos biflorus = anti-A1
Ulex europaeus = anti-H

28. Questions? How is H antigen formed? How are A and B antigens formed?
Relationship of Type I chain and Se gene
Relationship of Type II chain and H gene
How are A and B antigens formed?
What blood type has the highest amount of H antigen? What blood type has the least amount of H antigen? How would you determine that?
4/19/2017

30. ABO Antigens and Antibodies
ABO antigens based on combinations of three genes: A, B, and O
Antibodies are clinically significant and “naturally occurring”
causing most fatal acute HTRs
some causing HDFN
ABO antibodies neutralized with secretor saliva.

31. Group O Generally the most common blood group Genotype: OO Antigen: H
Antibodies: anti-A, anti-B, and anti-A,B
Antibodies are naturally occurring and very strong
Anti-A,B (mostly IgG) may cross placenta to cause HDFN

32. Group A Genotype: AA, AO Antigen: A, H
Antibodies: anti-B (primarily IgM)
A subgroups
A1 (80%) and A2 (20%) most important
A1 has more A than A2 (quantitative difference); qualitative differences, too.
~25% of A2B’s form anti-A1
1-8% of A2’s form anti-A1
Lectin of Dolichos biflorus agglutinates A1

33. Group B Genotype: BB, BO Antigen: B, H
Antibodies: anti-A (primarily IgM)
B subgroups: Not important

34. Group AB Genotype: AB Antigen: A, B, very little H Antibodies: None
B subgroups: Not important
A2B: 25% of A2B individuals produce anti-A1
4/19/2017

36. ABO Testing
Cell typing (forward grouping) to determine antigen types on RBCs
Serum/plasma typing (reverse grouping or backtyping) to determine type of antibody in serum:
Note the opposite reactions
If the forward reactions are opposite of reverse, an ABO discrepancy is not present.

37. ABO Grouping Reagents Forward Grouping Reagent
Reverse or Back Tying Cells

38. Forward Grouping Reagent

39. Forward Grouping Reagent: Monoclonal antibody
Highly specific
IgM
Expected 3+- to 4+ reaction
1 drop
Anti-A=Blue; anti-B=Yellow (Acroflavin dye)
A and B antigens on patient red cells are agglutinated by known sera (anti-A, anti-B)

40. Reverse or Back Tying Reagent Cells

41. Reverse or Back Typing Reagent Cells: Human Source
Expected 2+ to 4+ reaction
4-5% cell suspension
1 drop
Anti-A or anti-B antibodies in patient serum (or plasma) agglutinate with A1 and B antigens on Reagent cells

42. Forward Typing Procedures
To determine what antigens are present on RBCs.

43. Step 1. Label test tubes.

44. Step 2: Make a 2-5% patient red cell suspension.

45. Step 3: Add reagent antisera (1 drop).

46. Step 3A: Add reagent Anti-A antisera (1 drop).

47. Step 3B: Add Anti-B reagent antisera (1 drop).

48. Step 4: Add one drop of 2-5% suspension of patient RBC to each tube.

49. Step 5: Mix and centrifuge (approximately 20 seconds).

50. Group A: 4+ Agglutination with Anti-A 0 Agglutination with Anti-B

51. Group B: 4+ Agglutination with Anti-B 0 Agglutination with Anti-A

52. Group AB: 4+ Agglutination with Anti-A and Anti-B

53. Group O: No Agglutination with Anti-A or Anti-B

54. Back Typing
To determine what antibodies are present in patient’s plasma.

55. Step 1: Label Test Tubes

56. Step 2: Add two drops of patient serum to each tube

57. Step 3: Add one drop of reagent cells to each test tube

58. Step 3A: Add one drop of Reagent A1 cells

59. Step 3B: Add one drop of Reagent B cells

60. Step 4: Mix and centrifuge (approximately 20 seconds)

61. Group A: 4+ Agglutination with B Cells 0 Agglutination with A1 Cells

62. Group B: 4+ Agglutination with A1 Cells 0 Agglutination with B Cells

63. Group O: 4+ Agglutination with A1 Cells 3+ Agglutination with B Cells

64. Group AB: No Agglutination with A1 and B Cells

65. Exercises
Interpretation of test results

66. Exercises: Interpretation of ABO Testing Results
Forward Reverse Interpretation anti-A Anti-B A1 cells B cells ABO Group ?? ?? ?? ??

67. Exercises: Interpretation of ABO Testing Results
Forward Reverse Interpretation anti-A Anti-B A1 cells B cells ABO Group A B AB O

69. What can Cause ABO Discrepancies?
Disagreement between the interpretations of forward and reverse grouping
Antigen problems
Antibody problems

70. Antigen Problems Lack of expected antigens
A subgroup
B subgroup
Bombay
Presence of unexpected antigens
Acquired B phenotype
Polyagglutinable RBCs, recent marrow transplant, nonspecific agglutination

71. Antibody problems Lack of expected antibodies
Immunodeficiency, neonates, abnormally high concentrations of Ab (prozone)
Presence of unexpected antibodies
Anti-A1, cold auto-or alloantibodies, rouleaux (false positive)

72. A Subgroups A1 A2 A3 Ax
Aend Am
etc

73. A1 vs A2 Phenotypes Blood Group Anti-A Anti-A1 lectin A1 (80%) + +
A1 & A2 account for 99% of A group

74. A1vs A2 Phenotypes
Quantitative differences: More antigenic sites on A1 than A2.
Qualitative differences between A1 and A2 antigens:
1-8% of A2 individuals produce anti-A1
25% of A2B individuals produce anti-A1.

75. B Subgroups Very rare and are less frequent than A subgroups.
B subgroups demonstrate variations in the strength of the reaction using anti-B and anti-A,B
Examples are: B3, Bx, Bm, Bel

76. Acquired B phenotype Occurs in type A individuals with:
Colon cancer, intestinal obstruction, gram negative sepsis
Bacteria deacetylate group A sugar (GalNAc); remaining galactosamine crossreacts with reagent anti-B.

77. Acquired B phenotype

78. Acquired B phenotype
AB forward (with weak reactions with reagent anti-B)
A reverse
Reaction with anti-B is negative, if:
Acidify serum
Acetic anhydride treatment
Auto incubation

79. Acquired B typing result
Forward Reverse
Anti-A Anti-B Interp A1 cells B cells Interp
AB A”B”

80. Bombay (Oh) Phenotype Total Lack of H, A, and B antigens
Develop strong anti-H, anti-A, and anti-B
“O” forward, “O” reverse; with positive antibody screen
Require other Bombay donors for blood transfusion
(“Para-Bombay” = H antigen in secretions)

81. Reactivity of Anti-H with ABO Blood Groups
O>A2>B>A2B>A1>A1B

82. Blood Type: Antigens vs Antibodies
Blood Type Antigens Antibodies on rbcs in Plasma
A A Anti-B
B B Anti-A
AB A,B None
O None Anti-A, Anti-B

83. Exercise A ___ ___ ___ B ___ ___ ___ AB ___ ___ ___ O ___ ___ ___
Blood Compatible Compatible Compatible
Type RBCs FFPs Whole Blood
A ___ ___ ___
B ___ ___ ___
AB ___ ___ ___
O ___ ___ ___

84. ABO Compatible Blood Components
Blood Compatible Compatible
Type RBCs FFPs
A A, O A, AB
B B, O B, AB
AB AB, A, B, O AB
O O A, B, AB, O

85. ABO Compatible Whole Blood
Blood type Compatible WB A A B B AB AB O O

86. Consequences of ABO incompatibility
Severe acute hemolytic transfusion reactions
One of the most frequent causes of blood bank fatalities
Clerical errors
Most frequent HDFN; usually mild.

87. Sources of Technical Errors Resulting in ABO Discrepancies
Inadequate identification of blood samples
Cell suspension too heavy or too light
Clerical errors
A mix-up in samples
Missed observation of hemolysis
Failure to add reagents
Failure to follow manufacturer’s instructions
Uncalibrated centrifuge
Contaminated reagents
Warming during centrifugation
4/19/2017

88. Resolving ABO Discrepancies
Problems with RBCs Resolution Techniques Rouleaux wash RBCs 4X MF agglutination check tx hx Unusual phenotype (hh) Test with anti-H Disease processes (Acq. B) check patient diagnosis

89. Resolving ABO Discrepancies (Cont’d)
Problems with serum Resolution Techniques Rouleaux Saline replacement Presence of unexpected Ab Do panel to ID Absence of expected Ab Increase incubation time

90. Objectives
Describe the inheritance of the ABO Blood Groups and predict the ABO phenotypes and genotypes of offspring from various ABO matings
Explain the roles of Secretor and H genes in the formation of H, A, and B antigens on the red cells
Describe the reciprocal relationship between ABO antigens and antibodies for blood types O, A, B, and AB.

91. Objectives (Continued)
Describe the procedures on ABO forward and back typings; interpret the results; and resolve any discrepancies if present.
Describe the quantitative and qualitative differences between the A1 and A2 antigens.
Correctly identify all the ABO compatible blood components for each blood type

92. Reference Materials:
Modern Blood Banking And Transfusion Practices, or 5th Edition Denise M. Harmening. March F.A. Davis. Philadelphia PA.
Textbook of Blood Banking and Transfusion Medicine, Sally V. Rudman. February W.B Saunders. Philadelphia PA.
Transfusion Medicine Interactive: A Case Study Approach . Marian Petrides MD, Nora Ratcliffe MD, and Roby Rogers MD AABB Press Bethesda, Maryland.
Transfusion Reactions, 2nd Edition. Mark A. Popovsky (Editor). AABB Press Bethesda, Maryland.
American Association of Blood Banks Technical Manual (AABB) 14th Edition , American Association of Blood Banks, 8101 Glenbrook Road, Bethesda, Maryland.
Standards for Blood Banks and Transfusion Services 23rd Edition, Standards Committee, AABB. Bethesda, Maryland.