1. ABO Subgroups and Discrepancies
Nada Jamjoom
Faculty of Applied Medical Sciences
BLOOD BANK
MEDICAL TECHNOLOGY
KAU

2. ABH Antigens
H antigen

3. ABO Antibodies ABO Abs are generally IgM class antibodies.
For Group A and Group B individuals the predominant antibody class is IgM.
Serum from Group O individuals contains not only anti-A and anti-B but also anti-A,B.
Anti-A,B is a mixture of class IgG and IgM.
The anti-A,B “immune” antibodies are predominantly IgG.

4. ABO Antibodies Time of appearance:
Generally present within first 4-6 months of life
Reach adult level at 5-10 years of age Level off through adult life
Begin to decrease in later years: >65 years of age

5. ABO Subgroups
Von Dungern in1911 described 2 different A Ag based on reactions between group A RBCs and anti-A and anti-A1 antisera.
Group A red cells that react with both anti-A & anti-A1 are classified as A1.
Group A red cells that react with anti-A and not anti-A1 are classified as A2.
The majority (80%) of the A and AB population are A1 or A1B; the remaining 20% are A2 or A2B.
The production of both types of antigens (A1 and A2) is a result of an inherited gene at the ABO locus & the immunodominant sugar on both A1 and A2 RBCs is N-acetyl-D-galactosamine.

6. Difference between A1 and A2
The difference between A1 and A2 is both quantitative and qualitative.
Quantitative difference
Inheritance of an A1 gene elicits production of high concentrations of the enzyme α-3-N-acetylgalactosaminyltranferase, which converts almost all of the H precursor structure to A1 antigens on the red cells.
The number of A1 antigen sites is higher than the number of A2 antigens sites on the red cell because A1 gene is a very potent gene that creates large numbers of A1 antigens sites on RBC.

7. Difference between A1 and A2
Qualitative differences
1 to 8% of A2 individuals produce anti-A1 in their serum
25% of A2B individuals produce anti-A1.

8. Difference between A1 and A2
Reagent anti-A is a mixture of two Abs ;
Anti-A which react with both A1 and A2 cells.
Anti-A1 which reacts with A cells but not with A2 cells in simple testing .
The seeds of the plant Dolichos biflorus serve as a source of anti-A1 which is known as anti-A1 lectin.
This reagent agglutinates A1 or A1B but does not agglutinate A2 or A2B cells.

9. A1 versus A2 Phenotype Blood Group A1 + A2 Negative
Reactions of patient’s red cells with
Blood Group
Anti-A (from B Sera)
Anti-A1 Lectin A1 + A2
Negative
A1 has 2 antigens A and A1
A2 has only one, A antigen

10. Weak A and B Subgroups
Other A subgroups: RBC of the A end, A3, Ax, Ay or A el. are only rarely seen in transfusion practice.
Subgroup of B: infrequent than the weaker subgroup of A, identified by anti-B and anti-A,B. Subgroups B3 , Bx , Bm and Bel .

11. ABO Discrepancies
A discrepancy occurs when the red cell testing does NOT match the serum testing results
In other words, the forward does NOT match the reverse
ABO discrepancies are usually technical in nature and can be simply resolved by correctly reporting the testing and carefully checking reagents with meticulous reading and recording of results.

12. ABO Discrepancies Most of the time, the problem is technical
Mislabeled tube
Failure to add reagent
Either repeat test on same sample, request a new sample, or wash cells
Other times, there is a real discrepancy due to problems with the patient’s red cells or serum.

13. Technical Errors ABO Discrepancies Clerical errors
Mislabeled tubes
Patient misidentification
Inaccurate interpretations recorded
Transcription error
Computer entry error
Reagent or equipment problems
Using expired reagents
Using an uncalibrated centrifuge
Contaminated or hemolyzed reagents
Incorrect storage temperatures
Procedural errors
Reagents not added
Manufacturer’s directions not followed
RBC suspensions incorrect concentration
Cell buttons not resuspended before grading agglutination

14. ABO Discrepancies
Group I Discrepancies
Group I discrepancies are between forward and reverse groupings because of weekly reacting or missing antibodies.
Group I discrepancies are the most common type.
If a reaction in the reverse grouping is weak or missing  Group I discrepancy.
It means that the patient has depressed antibody production or cannot produce the ABO antibodies.

15. Examples of Group I Discrepancies
ABO Discrepancies
Examples of Group I Discrepancies
Newborns
Do not form antibodies until they 3-6 months
Elderly patients
Weakened antibody activity
CLL, malignant lymphoma, using immunosuppressive drugs can cause hypogammaglobulinemia (little or no antibody production)

16. Resolution of Group I Discrepancies
ABO Discrepancies
Resolution of Group I Discrepancies
Eliminate all technical errors
Determine patients age, diagnosis
Incubate patient’s serum with reagent A1 and B cells for 15 to 30 minutes (RT) to enhance antibody reactions
If negative, place serum-cell mixture at 4°C for 15 to 30 minutes

17. Group II Discrepancies
ABO Discrepancies
Group II Discrepancies
These discrepancies are between forward and reverse groupings because of weakly reacting or missing antigens.
Group II discrepancies is the least common type.

18. Examples of Group II Discrepancies
ABO Discrepancies
Examples of Group II Discrepancies
Subgroups of A and or subgroups of B may be present.
Leukemias may yield weakened A or B antigens.
Hodgkin’s disease
“Acquired B” phenomenon is most often associated with lower GI tract disease
Cancer of colon/rectum
Intestinal obstruction
Gram negative septicemia (i.e. E. coli)

19. Acquired B
ABO Discrepancies
Bacteria (E. coli) have a deacetylating enzyme that modify the immunodominant BG A sugar….
Acquired B Phenotype
Group A individual
N-acetyl galactosamine
Galactosamine now resembles D-galactose (found in Group B)
Bacterial enzyme removes acetyl group

20. Resolution of Group II Discrepancies
ABO Discrepancies
Resolution of Group II Discrepancies
Eliminate all technical errors
Wash patient’s cells with saline
The reaction can be enhanced by incubating the test mixture at RT for up to 30 minutes to increase the association of Ab with Ag.
If negative, reduce the temperature to 4°C.
Testing the patient’s serum against autologous RBCs gives a negative reaction because the anti-B in the serum does not agglutinate autologous RBCs with the acquired B Ag.

21. Group III Discrepancies
ABO Discrepancies
Group III Discrepancies
These discrepancies are between forward and reverse grouping due to protein or plasma abnormalities and result in Rouleaux formation.
These can be caused by elevated levels of globulin from certain diseases such as multiple myeloma and Hodgekin’s lymphoma.
Wharton’s jelly ( a viscous mucopolysaccharide material present on cord bloods).

22. Resolution of Group III Discrepancies
ABO Discrepancies
Resolution of Group III Discrepancies
Rouleaux or red cells result from a stacking of erythrocytes that adhere in a coin-link fashion giving the appearance of agglutination.
To resolve this kind of problem, washing the patient’s red cells with saline or adding a drop or two of saline to the tube in case of rouleaux formation.
If the agglutination is true red cell clumping will remain.
Cord blood must be washed 6-8 times in forward grouping ONLY.

23. Group IV discrepancies
ABO Discrepancies
Group IV discrepancies
These kind of discrepancies are between forward and reverse groping due to miscellaneous problems.
Polyagglutination can occur due to exposure of hidden erythrocyte Ag (T antigen) in patients with bacterial or viral infection.
Bacterial contamination in vitro or vivo produces an enzyme that alters and exposes the hidden Ag on red cell leading to T activation.
This problem can be resolved by using monoclonal antibody typing reagent.

24. Common sources of technical errors resulting in ABO discrepancies
Dirty tubes or glassware
Common sources of technical errors resulting in ABO discrepancies
False Negative
False Positive
Inadequate identification of samples or test tubes
Over centrifugation and over reading
Failure to add serum or reagents
Use of incorrect reagents or samples
Contaminated reagents
Cell suspention is too heavy or too light

25. Example I Forward Grouping Reaction of Patient’s Cells with
ABO Discrepancies
Example I
Forward Grouping
Reaction of Patient’s Cells with
Reverse Grouping Reaction Patient’s Serum with
Anti-A
Anti-B
A1 cells
B cells
Patient
Negative
+++
Patient’s probable group: B
Possible discrepancy: Group I discrepancy; because of weakly reacting or missing antibodies.

26. Example II Forward Grouping Reaction of Patient’s Cells with
ABO Discrepancies
Example II
Forward Grouping
Reaction of Patient’s Cells with
Reverse Grouping Reaction Patient’s Serum with
Anti-A
Anti-B
A1 cells
B cells
Patient
++++ ++
Patient’s probable group: A
Possible discrepancy: Group III discrepancy; caused by Rouleaux formation

27. Example III Forward Grouping Reaction of Patient’s Cells with
ABO Discrepancies
Example III
Forward Grouping
Reaction of Patient’s Cells with
Reverse Grouping Reaction Patient’s Serum with
Anti-A
Anti-B
A1 cells
B cells
Patient
++++
negative ++
Patient’s probable group: A (subgroup A2)
Possible discrepancy: Group II discrepancy; because of weakly reacting or missing antigens

28. Example IV Forward Grouping Reaction of Patient’s Cells with
ABO Discrepancies
Example IV
Forward Grouping
Reaction of Patient’s Cells with
Reverse Grouping Reaction Patient’s Serum with
Anti-A
Anti-B
A1 cells
B cells
Patient
++++ ++
Negative
Patient’s probable group: A
Possible discrepancy: Group II discrepancy; caused by an acquired B Ag

29. Example V Forward Grouping Reaction of Patient’s Cells with
ABO Discrepancies
Example V
Forward Grouping
Reaction of Patient’s Cells with
Reverse Grouping Reaction Patient’s Serum with
Anti-A
Anti-B
A1 cells
B cells
Patient ++ +
++++
Patient’s probable group: O
Possible discrepancy: Group IV discrepancy; caused by T activation.