Transcript ABO Discrepancies & other problems

Practical
Blood Bank
ABO Discrepancies
ABO Discrepancy

Definition: When the results of the forward
grouping (patient cells) do not correspond to the
results of the reverse grouping (patient serum) or
abnormal reactivity is present (i.e. Mixed Field):
1. Strength of reaction

Weak or missing
2. Additional reactions
3. Abnormal reactions
HINT

ABO forward and reverse reactions are typically
very strong: 3+ to 4+. Weaker reactions should
immediately send up red flags indicating that
something is wrong.

Since production of ABO antigens is genetically
controlled they are less vulnerable to problems
than does the production of ABO antibodies.
Therefore we see more problems in which grouping:
Forward or Reverse?
Patient Anti-A Anti-B A1-Cells B-Cells
A
4+
1+
0
4+
B
0
4+
1+
0
C
4+
4+
1+
0
D
0
3+
0
0
Patient A: Additional reaction with anti-B and patients cells.
Patient B: Weak reaction with patients serum and A1-cells.
Patient C: Additional reaction with patients serum and A1-cells.
Patient D: Missing reactions with patients serum A1-cells
Red Cell Problems

Affect the forward grouping results
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Missing or weak antigens
Extra antigens
Mixed field reactions
Mixed Field reactions
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Chimera: Two cell populations
Causes: Recent transfusion (O cells to an A
patient), Bone marrow transplant
(Testing using Serum or plasma , suspended
Patient RBCs)
Can cause non specific aggregation of RBC’s
1. Increased serum proteins: Multiple Myeloma patient
2. Contamination in cord blood sample: Wharton’s jelly
3. Infusion of macromolecular solutions: Dextran, etc.
Mixed Field Agglutination (Post transfusion)
~ (ABO Testing) Can be seen in A, B and AB individuals who have received O units. The
antisera reacts with the patient’s RBCs, but not with the transfused O cells.
~ (Antibody screen) Can also be seen post transfusion if a person makes an antibody to
antigen on donor cells; antibody agglutinates with donor cell, but not their on cells.
Weakened Antigen Expression
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Potent cold reacting autoagglutinins
Antibody coats patient RBC and agglutinate
spontaneously in the diluent
Subgroups of A or B
Some leukemia’s, Hodgkin's disease
Excess soluble A and B blood group substances
 Carcinoma of the stomach and/or carcinoma of
the pancreas
Missing or Weak antigens
Anti-A
Anti-B
A1 Cells
B Cells
0
0
0
4+
Group O
Group A
• Since the forward and reverse don’t match, there must be a
discrepancy (in this case, a missing antigen in the forward grouping)
Subgroups of A (or B)
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Subgroups of A account for a small portion of
the A population (B subgroups rarer)
These subgroups have less antigen sites on
the surface of the red blood cell
As a result, they show weakened (or missing)
reactions when tested with commercial
antisera
Resolution: test with Anti-A1, Anti-H, and
anti-A,B for A subgroups
Extra ABO antigens
1.
Acquired ‘B’ Antigen
a) Microbial deacetylating enzymes such as E.
coli cleave off the N-Acetyl of the Group A Nacetyl-D-galactosamine immunodominant
sugar. The remaining D-galactosamine
becomes similar enough to the Group B Dgalactose immunodominant sugar that it does
react with reagent anti-B.
2. Secondary to bowel obstruction or carcinoma
of the bowel
3. Polyagglutinable state


Exposure of ‘crypt’ or buried antigens (T, Tk, etc.) by
inheritance or bacterial enzymes – RBC’s agglutinate
with most sera.
Exposure of T, Tn and Tk (etc.) antigens. Antibodies to
these antigens are present in virtually all human
antisera. If using human source anti-A and anti-B these
cells will agglutinate.
Forward Grouping: Extra Antigens
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Acquired B
B(A) phenotype
Rouleaux
Polyagglutination
Wharton’s Jelly
Anti-A Anti-B
4+
1+
A1
Cells
0
EXAMPLE
B
Cells
4+
Acquired B Phenotype
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Limited mainly to
Group A1 individuals
with:
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Lower GI tract disease
Cancer of colon/rectum
Intestinal obstruction
Gram negative
septicemia (i.e. E. coli)
Acquired B

Bacteria (E. coli) have a deacetylating
enzyme that effects the A sugar….
Resolving Acquired B
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Check patient diagnosis: Infection?
Some manufacturers produce anti-B reagent
that does not react with acquired B
Test patients serum with their own RBCs

The patients own anti-B will not react with the
acquired B antigen on their red cell (autologous
testing)
B(A) phenotype
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Similar to acquired B
Patient is Group B with an apparent extra A
antigen
The B gene transfers small amounts of the A
sugar to the H antigen
Sometimes certain anti-A reagents will detect
these trace amount of A antigen
Resolution: test with another anti-A reagent
from another manufacturer
Other reasons for “extra” antigens

Polyagglutination – agglutination of RBCs with
human antisera no matter what blood type
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Due to bacterial infections
Expression of hidden T antigens react with antisera
Rouleaux – extra serum proteins
Wharton’s Jelly – gelatinous substance derived
from connective tissue that is found in cord blood
and may cause false agglutination (Remember:
only forward typing is performed on cord blood)

Wash red cells or request new sample from heel, etc
Reverse Grouping

Affect the reverse grouping results

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Missing or weak antibodies
Extra antibodies
Unexpectedly Weakened Antibodies
Immunodeficient due to therapy or disease

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Immunosuppressive drugs
Certain leukemia’s (CLL) or lymphoma’s (malignant
lymphomas) have hypogammaglobulinemia (Little or
no antibody production)
Age related
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Very young: <6 months of age (Newborns)
Very old: >65 years of age (Weakened Abs Activity)
Dilutional Effect

Plasma Exchange, Transfusion, etc. dilutes out
patient antibodies
Resolving Weak or Missing antibodies
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Determine patients age, diagnosis
Incubate serum testing for 15 minutes (RT) to
enhance antibody reactions
If negative, place serum testing at 4°C for 5
minutes with autologous control (a.k.a.
Autocontrol, AC)
This is called a “mini-cold” panel and should
enhance the reactivity of the antibodies
Reverse Grouping:
Extra Antibodies
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Cold antibodies (allo- or auto-)
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Cold antibodies may include anti-I, H, M, N, P,
Lewis
Rouleaux
Anti-A1 in an A2 or A2B individual
Extra Antibodies :Cold antibodies
Sometimes a patient will develop cold-reacting
allo- or auto-antibodies that appear as “extra”
antibodies on reverse typing
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Alloantibodies are made against foreign red cells
Autoantibodies are made against ones own red
cells.
Cold reacting antibodies cause agglutination
with red cells at room temperature and below.
The autocontrol will be positive.

Resolution: warming tube to 37° and washing red
cells can disperse agglutination; breaking the IgM
bonds with 2-ME will also disperse cells
Rouleaux
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Can cause both extra antigens and extra
antibodies
“stack of coins” appearance
May falsely appear as agglutination due to the
increase of serum proteins (globulins)
Stronger at IS and weak reaction at 37°C and no
agglutination at AHG phase
Associated with:
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Multiple meloma
Waldenstrom’s macroglobulinemia (WM)
Hydroxyethyl starch (HES), dextran, etc
Resolving Rouleaux
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Remove proteins!
If the forward grouping is affected, wash cells
to remove protein and repeat test
If the reverse grouping is affected, perform
saline replacement technique (more common)
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Cells (reagent) and serum (patient) centrifuged to
allow antigen and antibody to react (if present)
Serum is removed and replaced by an equal volume
of saline (saline disperses cells)*
Tube is mixed, centrifuged, and reexamined for
agglutination (macro and micro)
*some procedures suggest only 2 drops of saline (UMMC)
Anti-A1
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Sometimes A2 (or A2B) individuals will
develop an anti-A1 antibody
A2 (or A2B) individuals have less antigen sites
than A1 individuals
The antibody is a naturally occurring IgM
Reacts with A1 Cells, but not A2 Cells
Resolving anti-A1 discrepancy
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2 steps:

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Typing patient RBCs with Anti-A1 lectin
Repeat reverse grouping with A2 Cells instead
of A1 Cells
Both results should yield NO agglutination
Anti-A Anti-B
4+
0
A1
Cells
2+
B
Cells
4+
Others…
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The Bombay phenotype (extremely RARE)
results when hh is inherited
These individuals do not have any antigens
and naturally produce, anti-A, anti-B, antiA,B, and anti-H
Basically, NO forward reaction and
POSITIVE reverse
Resolution: test with anti-H lectin
(Bombay’s don’t have H and will not react)
Popular LAB CAUSES Of ABO Discrepancies
1.
2.
3.
4.
5.
6.
7.
Poorly labeled specimen OR test tubes
Patient RBC suspension too heavy or light
Wrong specimen put in Patient’s labeled test
tubes
Oh? Is hemolysis really a Pos. Rx’n?
Wrong results recorded on Pt. Form
Didn’t follow manufacturer’s instructions
Poor centrifugation: over or under!
Popular LAB CAUSES Of ABO Discrepancies
Didn’t add:
1.
2.
3.
Patient Serum
Reagents
Correct Reagent
Reaction Reading:
1.
2.
3.
Shaking tubes while looking elsewhere
Shaking tubes too hard
Shaking tubes too gently or not completely resuspending cell button
ABO Discrepancy
When an ABO Discrepancy is encountered:
1.
2.
3.
Results must be recorded, but interpretation of
the ABO group must be delayed until the
discrepancy is resolved…by you!
Begin follow up by getting an accurate patient
history – age, medications, diagnosis, etc.
Repeat testing to rule out tech errors such as
mislabeling, adding reagents, wrong patient
sample, etc.
Resolving ABO Discrepancies
1.
Repeat testing on the
same sample…
2.
Repeat testing using
saline suspended
and/or washed patient
red blood cell’s.
Saline Replacement.
1.
From the beginning: relabel tubes, re-drop
patient and reagent
drops, etc.
2.
Many labs make the
patients red blood cell
suspension with the
patient’s serum/plasma. If
the patient has increased
plasma proteins it can
cause non-specific red
cell aggregation.
3.
Weak or missing
reactions?
3.
Incubate test system at
room temperature for
15-30 minutes! Get
patient history.
4.
Mislabeled or
contaminated
specimen:
4.
Redraw Patient!!
a)
ALL of the above: any
labeling error may
account for the problem
and needs to be redrawn.
b)
Drawn above an IV?
Resolving ABO Discrepancies
Call the floor!!!
1.
Get patient history.
a)
b)
c)
d)
Recent transplant: two cell populations
dilutional effect
Patient medication
etc.
5.
6.
Test patient cells with
anti-A1 (Dolichos
biflorus), anti-A,B or
anti-H (Ulex
europaeus)
Test patient serum with
A1 or A2 cells
5.
For suspected
subgroups of A
6.
Ditto!
7.
Review Antibody
Screening tests
1.
8.
7.
Can react with
reagent A1 and B
cells
8.
Should strengthen
weakened ABO
antibody reactivity!
WHY?
Allo antibody or cold
reactive allo or auto
Ab
Incubate tests and
controls for 10-30
minutes room
temperature
Anti-A
3+
Anti-B
0
A1-Cells
0
B-Cells
1+
Problem: Reverse grouping - weakened patient
antibody
Causes: Age related (>65, infant),
immunosuppressed or immunocompromised,
Resolution: Incubate Room Temperature 1530 minutes and respin. Check Patient history.
Anti-A
3+
Anti-B
1+
A1-Cells
0
B-Cells
4+
Problem: 1+ Reaction with Anti-B. Appears
to have additional antigens.
Causes: Acquired ‘B’ antigen.
Resolution: Patient history – bowel obstruction,
carcinoma of the bowel. (E. coli deacetylation of
the Group A antigen.)
Anti-A
2+
Anti-B
0
A1-Cells
1+
B-Cells
4+
Problem: Weak forward anti-A and 1+ reaction with A1
Cells.
Causes:
1. Subgroup of A – A2 with anti-A1.
2. Unexpected cold reacting antibody to antigen on
reagent A1 cells.
Resolution:
1. Test patient cells with anti-A1 lectin and with patient
serum test A2 cells
2. Antibody screen should demonstrate unexpected cold
reacting antibody.
EXAMPLES of ABO Discrepancies
and Possible Resolution
Forward:
Reverse:
Screening
Anti-A
1
2
3
4
5
6
0
4+
4+
3+
0
4+
Anti-B
0
4+
0
4+
0
2+
A1 Cells
0
2+
1+
1+
4+
0
B Cells
0
2+
4+
0
4+
4+
Cells
0
2+
0
0
4+
0
Autocontrol:
Possible Causes
Possible Resolutions
0
Group O newborn; elderly
patient; low
immunoglobulin levels
Incubate tests at 4°C,
check age of patient
Rouleaux; cold
autoantibody
0
Probable A2 subgroup
with anti-A1
Wash RBCs and repeat
testing; test for cold
antibodies
Test with anti-A1 and antiH lectins and A2 cells
0
Probable A2B subgroup
with anti-A1
Test with anti-A1 and antiH lectins and A2 cells
Probable Oh (Bombay)
Test with anti-H lectin;
may sent to reference lab
for confirmation
Investigate patient history;
test with anti-B lectin if
available
Perform antibody
identification (antibody
panel)
Test for cold antibodies
and identify if appropriate
2+
0
0
Probable acquired B
phenotype
Probable alloantibody
7
8
4+
0
4+
4+
2+
4+
0
1+
2+
1+
0
1+
Probable group B with
cold autoantibody
Adapted from Table 3-11: Flynn, J. C. (1998). Essentials of Immunohematology. Philadelphia: W.B. Saunders Company.
Example 1
Anti-A
Anti-B
A1 Cells
B Cells
3+
0
0
1+
Problem:
Causes:
Resolution:
Example 2
Anti-A
Anti-B
A1 Cells
B Cells
3+
1+
0
4+
Problem:
Causes:
Resolution:
Example 3
Anti-A
Anti-B
A1 Cells
B Cells
2+
0+
1+
4+
Problem:
Causes:
Resolution:
Example 4
Anti-A
Anti-B
A1 Cells
B Cells
0
0
0
3+
Problem:
Causes:
Resolution:
Example 4
Anti-A,B
Patient RBC
Problem:
Causes:
Resolution:
1+
Example 5
Anti-A
Anti-B
A1 Cells
B Cells
0
2+mf
3+
0
Problem:
Causes:
Resolution:
Example 6
Anti-A
Anti-B
A1 Cells
B Cells
4+
4+
0
1+
Problem:
Causes:
Resolution:
Example 7
Anti-A
Anti-B
A1 Cells
B Cells
0
0
0
0
Problem:
Causes:
Resolution: