Resolving ABO Discrepancy

Dr. Sheikh Ahmed .W.

Section In charge Blood bank
IMC
1


Correlate ABO/Rh testing with
the expected result and define
various ways to resolve an ABO
discrepancy.
Identify various clerical and
technical errors that can occur inR affect
ABO/Rh interpretation.
Discuss the effects of disease
on the expression of ABH
antigens and antibodies
2
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.
3
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.

4

In DONORS, the discrepancy must be resolved
before any blood is labeled .
5
Landsteiners Rule
6
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
7
What CAUSES an ABO
Discrepancy?
Weak or Missing antigens in the
FRONT type
Weak or Missing antibodies in the

REVERSE type
Additional antigens in the
FRONT type
Additional antibodies in the
REVERSE type
8
Reasons for ABO discrepancy
Types of ERRORS
Clerical Errors
Reagent or equipment
problems
Procedural errors
TRUE DISCREPANCY SITUATION
Problems with
Red Blood Cells
Problems with
Plasma/Serum
9
Clerical Errors
Mislabeled tubes
Patient misidentification errors

Inaccurate interpretations recorded
10
Reagent or equipment
problems
Using expired reagents
Using an un-calibrated
centrifuge
Contaminated 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

11
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
Young
Elderly
Immunocompromised
Cold
Autoantibody
Anti-A
1

Rouleaux
Cold
Alloantibody
Rouleaux
May cause all + reactions
12
patient
red cells
(type A)
FORWARD GROUPING
PROBLEMS
anti-A
antibodies
AHG
13

Mixed field (mf) agglutination
Weak or missing antigens
Additional or unexpected antigens
Polyagglutinable cells
Mixed Field (mf) Reactions
Small 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
14
Resolving Mixed Field (mf)
Reactions
Determine the CAUSE of the mixed field
reaction
Checking the patients transfusion history and
clinical history
e.g. HPC transplant

If it is determined that it is a weak subgroup,
perform
specialized tests
e.g. adsorption and elution.

Molecular testing
15
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 disease
Lymphomas
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
16
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
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
18
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)
19
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
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.


20
Forward Grouping Problems
Spontaneous Agglutination
Result of:
Strong potent cold auto antibodies
Forward Grouping Problems
Would 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
21
Resolving Spontaneous
Agglutination
Incubate plasma and cells (separately) at 37C
for 5 to
15 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 DTT
Wash cord blood a minimum of 6 times with
saline
22
Forward Grouping Problems
Resolving Spontaneous
Agglutination
Incubate plasma and cells (separately) at 37C
for 5 to
15 minutes
Wash cells 5 to 6 times with warm saline
Retest warm washed cells with warm plasma
Retest the DAT and saline control
Treat cells with 0.01M DTT

Wash cord blood a minimum of 6 times with
saline
23
Polyagglutination
Result of:
Inheriting acquired abnormalities of the red
cell membranes with
exposure to crypt antigens
e g T activation
Bacterially contaminated sample

Resolve by:
Avoid testing with human antisera; use
monoclonal antisera.

Collection of a new sample
24
patient
serum-plasma
(with anti-B Ab)
AHG reagent
red cells (type
B)
Reverse Grouping
Problems
25
Plasma or serum ABO discrepancies are
more common than red cell discrepancies.

Weak/Missing
Additional Antibodies
Rouleaux
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
26
Reverse Grouping Problems
Resolving Weak/Missing
Antibodies
Determine patients age and diagnosis
Incubate 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
27
Extra Antibodies
Result 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
28
Extra Antibodies: Rouleaux
Result of:
Abnormal concentrations of serum proteins
Altered serum/protein ratios
High-molecular-weight volume expanders
Associated with:
Multiple myeloma
Waldenstroms macroglobulinemia (WM)
29
30
Resolution of Extra
Antibodies: Rouleaux

REMOVE PROTEINS!!
If the forward grouping is affected, wash
cells to
remove protein and repeat test.

Reverse Grouping Problems
The reverse grouping is affected, perform
salinereplacement technique (more
common)

Reagent cells and patient serum are
centrifuged to allow antibody attachment (if
present)

Serum is removed and replaced by an equal
volume of saline which
disperses cells
Tube is mixed, centrifuged, and re-examined
for agglutination
31
Extra Antibodies: Anti-A1
Result 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
32
A sub-groups
33
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
34
CASE 1
35
Case Study #1: Patient History
88 year old male
Diagnosis: Immunocompromised
Medications: Corticosteroids
Transfusion History: Massive plasma infusion
Lab: Hemoglobin: 6.5 g/dL
36
What is the problem?
Both a front AND reverse typing issue
Investigation
Patient age: 88 years old
Diagnosis: 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
37
A B AB AntiD D
C
A1cells A2 cells Bcells
patient 0 0 0 3+ 0 W+ W+ 0
30 RT
0 1+ 2+ 1+ 1+ 0
Weak front type and weak reverse type resolution:

Enhance forward type
Incubate 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 +ve
38
Case Study #2: Patient History
33 year old female
Diagnosis: Anemia
Medications: None
Transfusion History: 2 units of packed red
blood
cells 5 years ago
Lab: Hemoglobin: 9.1 g/dL
Case 2
39
What is the problem?
Reverse typing issue
Additional antibodies: cold

Investigation
Patient age: 33 years old
Diagnosis: 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
40
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 DIAGNOSIS B+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
41
36 YEAR OLD FEMALE
DIAGNOSIS: PREGNANCY #3
MEDICATIONS: PRE-NATAL VITAMINS
TRANSFUSION HISTORY: NO TRANSFUSIONS
Case Study 3
42
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
43
What is the problem?
Reverse typing
issue

DAT -NEGATIVE

Patient
Investigation
Patient age: 36 years old
Diagnosis: PREGNANT
Medications: VITAMINS
Resolution:
Additional antibody in reverse type
Patient
serum
A1 cells 3+
AI cells 3+
A2 cells 0
Anti A1
Lecitin(Dolic
hous biflorus
Patient 0
44
Resolution: Patient is (probable)
type A2 with anti-A1 in her serum
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
45
Anti A Ant
B
Anti
AB
Anti
D
DC A1 cells B
cells
patient W+ve 4+ 4+ 4+ W+ 4+ 0
Screeni
ng cells
positive
IS IAT
1 0 0
2 0 0
3 0 0
Auto
control
1+ 3+
DAT Anti -IgG Anti-c3d
Ptaient 3+ 2+
What is the problem?

Patient
Patient age: 73 years old
Diagnosis: Lymphoma
Medications: Aspirin
Resolution:
Weak reactive front type,
positive D control, positive
saline control
46
Forward type:
Weak Reactivity
Weak ABO subgroup? Malignancy?
Spontaneous agglutination?.

47
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 Positive
Positive DAT and autocontrol was further
investigated by a reference
laboratory, and it was discovered the patient
had a warm autoantibody
in his plasma
Example 7
Anti-A Anti-B A1 Cells B Cells
3+ 1+ 0 4+
Problem:
Causes:
Resolution:
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
.
49
Relation of H-substance (antigen)
and ABO groups
50
O
h
Phenotype (Bombay)
Occurs when two hh genes are inherited at
the 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
51
O
h
Phenotype (Bombay)
Symbol O
h
denotes this phenotype
RBCs not agglutinated by anti-A, -B or A,B
Serum/plasma agglutinates A and B cells
Not recognized until serum tested against group O
cells and causes strong agglutination.
Have anti-A, -B, -A,B and H
Can only be transfused with Bombay blood <0.01%
52
Grading of Reactions
53
Blood group
54
O
h
Phenotype (Bombay)
Confirmatory testing
Anti-H lectin (Ulex europaeus) negative
Agglutination of A, B, AB and O cells
Serum/plasma will not agglutinate Oh cells.
55
Results in Tube method
56
57
Antigen / Antibody
58
Reading in tube method
59
Principle of Gel Technology
J The sephadex gel matrix acts as a
sieve.

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 microtube
60

61
Extra Antibodies: Rouleaux
Result of:
Abnormal concentrations of serum proteins
Altered serum/protein ratios
Reverse Grouping Problems
High-molecular-weight volume expanders
Associated with:
Multiple myeloma
Waldenstroms macroglobulinemia (WM)
Hydroxyethyl starch (HES), dextran, etc
62
Extra Antibodies: Rouleaux
Result of:
Abnormal concentrations of serum proteins
Altered serum/protein ratios
Reverse Grouping Problems
High-molecular-weight volume expanders
Associated with:
Multiple myeloma
63
Extra Antibodies: Rouleaux
Result of:
Abnormal concentrations of serum proteins
Altered serum/protein ratios
Reverse Grouping Problems
High-molecular-weight volume expanders
Associated with:
Multiple myeloma
64
Waldenstroms macroglobulinemia (WM)
Hydroxyethyl starch (HES), dextran, etc