Download Group I discrepancy

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 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.
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.
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.
A1 versus A2 Phenotype
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.
ABO Discrepancies
Technical Errors
•
Clerical errors
–
–
–
–
–
•
Reagent or equipment problems
–
–
–
–
•
Mislabeled tubes
Patient misidentification
Inaccurate interpretations recorded
Transcription error
Computer entry error
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
Bacterial enzyme
removes acetyl group
Acquired
B
Phenotype
Galactosamine
now resembles
D-galactose (found
in Group B)
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.
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).
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.
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.
ABO Discrepancies
Dirty tubes or
glassware
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
Common sources of technical errors resulting in
ABO discrepancies
False Negative
False Positive
ABO Discrepancies
Example I
Forward Grouping
Reaction of Patient’s
Cells with
Anti-A
Anti-B
Patient
Negative
+++
Reverse Grouping
Reaction Patient’s
Serum with
A1 cells
B cells
Negative
Negative
Patient’s probable group: B
Possible discrepancy: Group I discrepancy; because of weakly reacting or
missing antibodies.
ABO Discrepancies
Example II
Forward Grouping
Reaction of Patient’s
Cells with
Anti-A
Anti-B
Patient
++++
++
Reverse Grouping
Reaction Patient’s
Serum with
A1 cells
B cells
++
++++
Patient’s probable group: A
Possible discrepancy: Group III discrepancy; caused by Rouleaux formation
ABO Discrepancies
Example III
Forward Grouping
Reaction of Patient’s
Cells with
Anti-A
Anti-B
Patient
++++
negative
Reverse Grouping
Reaction Patient’s
Serum with
A1 cells
B cells
++
++++
Patient’s probable group: A (subgroup A2)
Possible discrepancy: Group II discrepancy; because of weakly reacting or
missing antigens
ABO Discrepancies
Example IV
Forward Grouping
Reaction of Patient’s
Cells with
Anti-A
Anti-B
Patient
++++
++
Reverse Grouping
Reaction Patient’s
Serum with
A1 cells
B cells
Negative
++++
Patient’s probable group: A
Possible discrepancy: Group II discrepancy; caused by an acquired B Ag
ABO Discrepancies
Example V
Forward Grouping
Reaction of Patient’s
Cells with
Anti-A
Anti-B
Patient
++
+
Reverse Grouping
Reaction Patient’s
Serum with
A1 cells
B cells
++++
++++
Patient’s probable group: O
Possible discrepancy: Group IV discrepancy; caused by T activation.