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Resolving ABO Discrepancy
Dr. Sheikh Ahmed .W.
Section In charge Blood bankIMC
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Recognition and resolution are two very
important skills that blood bank
technologists must possess.
Of all the blood group systems, ABO is
THE MOST IMPORTANT.
ABO misinterpretation can lead to serious
transfusion complications in
patients.
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ABO Discrepancies MUST be Resolved
In PATIENTS, an ABO discrepancy must be
resolved before any blood component is
transfused.AABB policy 5.12.1 & 5.12.2 says blood shall not be released for a patient until
any discrepancy in patient ABO grouping is resolved.
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In DONORS, the discrepancy must be resolved
before any blood is labeled .
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Landsteiners Rule
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What IS an ABO Discrepancy?
Definition:When the results of the forward grouping
(patients cells) does not correspond to the
reverse grouping (patients plasma/serum
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What CAUSES an ABO
Discrepancy?
Weak or Missing antigens in theFRONT type
Weak or Missing antibodies in the
REVERSE type
Additional antigens in the
FRONT type
Additional antibodies in the
REVERSE type
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Clerical Errors
Mislabeled tubesPatient misidentification errors
Inaccurate interpretations recorded
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Reagent or equipment
problems
Using expired reagents
Using an un-calibrated
centrifugeContaminated or hemolyzed
reagents
Incorrect storage temperatures
Types of Errors
Procedural errors
Reagents not added
Manufacturers
directions not followed
RBC suspensions
incorrect concentration
Cell buttons not re-suspended before
grading agglutination
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Grouping
Forward Reverse
Missing/Weak Extra Mixed Field Missing/Weak Extra
A/B Subgroup
Disease
(cancer)
Acquired B
B(A) Phenotype
O Transfusion
Bone Marrow
Transplant
YoungElderly
Immunocompromised
Cold
Autoantibody
Anti-A1
Rouleaux
Cold
Alloantibody
Rouleaux
May cause all + reactions
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Mixed Field (mf) ReactionsSmall agglutinates with many un-agglutinated cells
Result of:
Mixed cell population from a massive transfusion of another blood
group. non-O individual receiving O red blood cells)
Bone marrow transplants having both the original type and donor
marrow cells.
The inheritance of weak ABO subgroups such as A3, Ax and B3 and
B can traditionally present a mixed field reaction.
Chimerism
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Resolving Mixed Field (mf)ReactionsDetermine the CAUSE of the mixed field
reaction
Checking the patients transfusion history andclinical history
e.g. HPC transplant
If it is determined that it is a weak subgroup,
perform
specialized testse.g. adsorption and elution.
Molecular testing
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Weak or Missing Antigen
Result of:Inheritance of a weak ABO subgroup
Malignancies may result in the loss of ABH
transferases
H d ki di
Forward Grouping Problems
Hodgkins diseaseLymphomas
Leukemias
Massive transfusion with group O blood to a
non-group O
person
e.g. a group A person receiving lots of group O
blood.
Bone marrow transplant and chemotherapy
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Forward Grouping Problems
Resolving Weak or Missing Antigens
Check the patients transfusion and clinical history
Read the forward group microscopically
Use anti-A,B and monoclonal antisera that is known to react with Ax and
A3 weak subgroups.
Perform adsorption and elution studies 17
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Forward Grouping Problems
Additional Antigens
Result of:Bacterial enzymes deacetylate the A antigen to a B
antigen and the patient front types as an AB and reverses as an A.Acquired B antigens are observed in patients with
recurring GI or colon infections with GI bacteria
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Acquired B
Bacteria (E. coli) have a deacetylating enzyme
that effects the 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)
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This sugar cross-reacts with the reagent anti-B, giving a weak reaction (but
still technically it is extra). Patients should receive Group A units. Acquired
B usually goes away when the condition resolves
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Forward Grouping Problems
Resolving Additional Antigens
Check clinical history for evidence of colon
infections with
Gram negative sepsis
Auto control is negative- that proves the blood
group as A.
As the patients own anti-B will not react with
their own AB cells
Acidify the anti-B to a p.H. of 6 and retest
Acquired B antigens will not react in acidified
antiserum, whereas as normal B antigens will
react.
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Forward Grouping Problems
Spontaneous Agglutination
Result of:
Strong potent cold auto antibodies
Forward Grouping ProblemsWould appear as AB in the front type and O in
the reverse type
Strong positive DAT with IgG, C3d and saline
control
Whartons jelly in cord blood
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Resolving Spontaneous
AgglutinationIncubate plasma and cells (separately) at 37C
for 5 to15 minutes
Wash cells 5 to 6 times with warm saline
Forward Grouping Problems
Retest warm washed cells with warm plasma
Retest the DAT and saline control
Treat cells with 0.01M DTTWash cord blood a minimum of 6 times with
saline
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PolyagglutinationResult of:
Inheriting acquired abnormalities of the red
cell membranes with
exposure to crypt antigens
e g T activationBacterially contaminated sample
Resolve by:
Avoid testing with human antisera; use
monoclonal antisera.
Collection of a new sample
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patientserum-plasma
(with anti-B Ab)
AHGreagent
red cells (type
B)
Reverse Grouping
Problems
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Plasma or serum ABO discrepancies are
more common than red cell discrepancies.
Weak/Missing
Additional Antibodies
Rouleaux
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Weak/Missing Antibodies
Newborns
Antibodies are not present at birth and only develop after 3
to 6 months of age.
Elderly
Weakened antibody activity
Hypogammaglobulinemia
Little or no antibody production
(immune-compromised patient)
NO agglutination on reverse grouping
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Reverse Grouping Problems
Resolving Weak/Missing
AntibodiesDetermine patients age and diagnosisIncubate serum testing for 15 minutes at room
temperature or 18 C to enhance antibody
reactions
If negative, incubate serum testing at 4C for
15 minute
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Extra AntibodiesResult of:
Cold antibodies (allo- or auto-)
Cold antibodies may include anti-I, H, M, N, P,Lewis
Rouleaux
Anti-A1 in an A2 or A2B individual
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Extra Antibodies: Rouleaux
Result of:
Abnormal concentrations of serum proteins
Altered serum/protein ratios
High-molecular-weight volume expanders
Associated with:
Multiple myeloma
Waldenstromsmacroglobulinemia (WM)
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Extra Antibodies: Anti-A1Result of:
A2 (or A2B) individuals development of anti-A1
antibody
A2 (or A2B) individuals have less antigen sites
than A1 individuals.
anti-A1 is a naturally occurring IgM antibody
Antibody reacts with A1 cells, but not A2 cells
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A sub-groups
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Reverse Grouping Problems
Resolution Extra Antibodies: Anti-A1
Type patient red blood cells with Anti-A1 lectin
Test patient serum with A1 , A2 and O cells
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CASE 1
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Case Study #1: Patient History
88 year old male
Diagnosis: ImmunocompromisedMedications: Corticosteroids
Transfusion History: Massive plasma infusion
Lab: Hemoglobin: 6.5 g/dL
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What is the problem?
Both a front AND reverse typing issue
Investigation
Patient age: 88 years oldDiagnosis: Hypogammaglobulinemia
Medications: Immunosuppressive drugs
Resolution:
weak front type, weak reverse
type
A B AB AntiD D
CONTROL
A1cells A2cells Bcells
Patient 0 0 0 3+ 0 W+ W+ 0
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A B AB AntiD D
C
A1cells A2 cells Bcells
patient 0 0 0 3+ 0 W+ W+ 030 RT 0 1+ 2+ 1+ 1+ 0
Weak front type and weak reverse type resolution:
Enhance forward typeIncubate patient cells with antisera (per manufacturers directions)
Enhance reverse type
Incubate reverse type cells with patient serum for 15 to 30 minutes
Room temperature or 18C (per manufacturers directions)
4C for 15 minutes
test concurrently with autologous cells and group O screening cells
Enzyme treat reverse type cells with FICIN.
Final diagnosis---B +ve38
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Case Study #2: Patient History
33 year old female
Diagnosis: Anemia
Medications: NoneTransfusion History: 2 units of packed red
blood
cells 5 years ago
Lab: Hemoglobin: 9.1 g/dL
Case 2
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What is the problem?
Reverse typing issue
Additional antibodies: cold
Investigation
Patient age: 33 years oldDiagnosis: Anemia
Medications: None
Resolution:
Additional antibody in reverse
Anti-A Anti-B AntiD D C A1
Cells
A2
cells
B
cells
patient 0 4+ 4+ 0 3+ 3+ 1+
Screening cells IS IAT
1 1+ 0
2 1+ 0
3 1+ 0
DAT Anit-
IgG
Anti C3d
c3b
cont
Patient 0 2+ 0
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Additional antibody in reverse type resolution:
Prewarm Technique
Incubate the serum and red cells separately at 37C before testing
Cold Adsorption
Incubate equal amounts of red cells (adsorbing cells) and patient
serum at 4C for 30 to 60 minutes
Test adsorbed serum against reverse typing cells.
FINAL DIAGNOSISB+ve
Screening cells IS 4c adsorbed
serum
1 0
2 0
3 0
A1
Cells
A2
cells
B cells
Patient 3+ 3+ 1+
4C adsorbed serum 3+ 3+ 0
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Anti-A Anti-
B
Anti-
AB
Anti-
D
D
contro
l
A1
cells
A2
cells
B
cells
Patient 4+ 0 4+ 4+ 0 2+ 0 3+
Screening
cells
IS IAT
1 0 0
2 0 0
3 0 0
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What is the problem?
Reverse typing
issue
DAT -NEGATIVE
Patient
Investigation
Patient age: 36 years old
Diagnosis: PREGNANTMedications: VITAMINS
Resolution:
Additional antibody in reverse type
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Patientserum
A1 cells 3+
AI cells 3+
A2 cells 0
Anti
A1Lecitin(Dolic
hous biflorus
Patient 0
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Resolution: Patient is (probable)type A2 with anti-A1 in her serum
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73 YEAR OLD MALE
DIAGNOSIS: LYMPHOMA AND SEVERE ANEMIA
MEDICATIONS: ASPIRIN
TRANSFUSION HISTORY: 3 UNITS 6 MONTHS AGO
LABORATORY: HEMOGLOBIN: 4.5 G/DL
Case Study #6
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Forward type:
Weak Reactivity
Weak ABO subgroup? Malignancy?
Spontaneous agglutination?.
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Spontaneous agglutination was not resolved
with warm washed cells.
DTT (0.01M) treatment of the red blood cells
should be performed to
resolve the ABO/Rh discrepancy and disperse
the spontaneous
agglutination.
:
Patient is B PositivePositive DAT and autocontrol was further
investigated by a reference
laboratory, and it was discovered the patient
had a warm autoantibody
in his plasma
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Example 7
Anti-A Anti-B A1 Cells B Cells
3+ 1+ 0 4+
Problem:
Causes:
Resolution:
T k H M
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Take Home Message
ABO discrepancy recognition & resolution is imperative in the blood bank
ABO discrepancies can present themselves as a
front type problem, reverse type problem, or
combination of both.
If ABO discrepancy resolution cannot be performed, and blood is
needed immediately, transfusion of type O blood may be necessary
.
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Relation of H substance (antigen)
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Relation of H-substance (antigen)
and ABO groups
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OhPhenotype (Bombay)
Occurs when two hh genes are inherited atthe Hh locus.
Possess normal A or B genes (if they were
inherited) but unable to express. Must have H on red cell membrane
Can transmit A or B gene to offspring
Term Bombay used since first discovered in
Bombay, India
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OhPhenotype (Bombay)
Symbol Oh denotes this phenotype
RBCs not agglutinated by anti-A, -B orA,B
Serum/plasma agglutinates A and B cells
Not recognized until serum tested against group Ocells and causes strong agglutination.
Have anti-A, -B, -A,B andH
Can only be transfused with Bombay blood
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Blood group
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OhPhenotype (Bombay)
Confirmatory testing
Anti-H lectin (Ulex europaeus)negative
Agglutination of A, B, AB and O cells
Serum/plasma will not agglutinate Oh cells.
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Antigen / Antibody
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Reading in tube method
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Principle of Gel Technology
J The sephadex gel matrix acts as asieve.
J Large agglutinates remain on or near
the top of the gel interface.
J Smaller agglutinates pass partway
through the gel, depending on size.
J Unagglutinated cells pass to the base of
the microtube60
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Extra Antibodies: Rouleaux
Result of:
Abnormal concentrations of serum proteins
Altered serum/protein ratios
Reverse Grouping ProblemsHigh-molecular-weight volume expanders
Associated with:
Multiple myeloma
Waldenstromsmacroglobulinemia (WM)
Hydroxyethyl starch (HES), dextran, etc
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Extra Antibodies: Rouleaux
Result of:
Abnormal concentrations of serum proteins
Altered serum/protein ratiosReverse Grouping Problems
High-molecular-weight volume expanders
Associated with:
Multiple myeloma
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Extra Antibodies: Rouleaux
Result of:
Abnormal concentrations of serum proteins
Altered serum/protein ratiosReverse Grouping Problems
High-molecular-weight volume expanders
Associated with:
Multiple myeloma
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Waldenstromsmacroglobulinemia (WM)Hydroxyethyl starch (HES), dextran, etc
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