ABO Subgroups and Discrepancies
Nada Jamjoom
Faculty of Applied Medical Sciences BLOOD BANK
MEDICAL TECHNOLOGY
KAU
ABH Antigens
H antigen
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.
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
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.
Difference between A
1and A
2• 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.
Difference between A
1and A
2Qualitative differences
•1 to 8% of A2 individuals produce anti-A1 in their serum
•25% of A2B individuals produce anti-A1.
Difference between A
1and A
2Reagent 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.
A
1versus A
2Phenotype
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
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 .
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.
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.
Technical Errors
• 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
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.
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)
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
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.
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)
ABO Discrepancies
Acquired B
• Bacteria (E. coli) have a deacetylating enzyme that modify the immunodominant BG A sugar….
Group A individual
N-acetyl galactosamine
Acquired B Phenotype
Bacterial enzyme removes acetyl group
Galactosamine now resembles D-galactose (found
in Group B)
ABO Discrepancies
Resolution of Group II Discrepancies
ABO 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.
ABO 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).
Group III Discrepancies
ABO 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.
Resolution of Group III Discrepancies
ABO 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.
Group IV discrepancies
Common sources of technical errors resulting in ABO discrepancies
False Negative False Positive
Over centrifugation and over reading
Use of incorrect reagents or samples
Failure to add serum or reagents
Cell suspention is too heavy or too light
Inadequate identification of
samples or test tubes
Contaminated reagents Dirty tubes or
glassware 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 +++ Negative Negative
Possible discrepancy: Group I discrepancy; because of weakly reacting or missing antibodies.
Patient’s probable group: B ABO Discrepancies
Forward Grouping Reaction of Patient’s
Cells with
Reverse Grouping Reaction Patient’s
Serum with
Anti-A Anti-B A1 cells B cells
Patient ++++ ++ ++ ++++
Example II
Possible discrepancy: Group III discrepancy; caused by Rouleaux formation Patient’s probable group: A
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 ++ ++++
Possible discrepancy: Group II discrepancy; because of weakly reacting or missing antigens
Patient’s probable group: A (subgroup A2) 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 ++++
Possible discrepancy: Group II discrepancy; caused by an acquired B Ag Patient’s probable group: A
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 ++ + ++++ ++++
Possible discrepancy: Group IV discrepancy; caused by T activation.
Patient’s probable group: O ABO Discrepancies
