Transcript ABO Discrepancies & other problems

Practical
Blood Bank
ABO Discrepancies
Importance
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It is important to recognize discrepant results
and how to (basically) resolve them
Remember, the ABO system is the most
important blood group system in relation to
transfusions
Misinterpreting ABO discrepancies could be
life threatening to patients
Discrepancies
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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
Why?
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Reaction strengths could be weaker than
expected
Some reactions may be missing in the
reverse or forward typings
Extra reactions may occur
Patient
Anti-A
Anti-B A1 Cells B Cells
1
4+
1+
0
4+
2
0
4+
1+
0
3
4+
4+
1+
0
4
0
3+
0
0
What do you do?
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Identify the problem
Most of the time, the problem is technical
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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
Discrepancy ?
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If a real discrepancy is encountered, the
results must be recorded
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However, the interpretation is delayed until
the discrepancy is RESOLVED
Technical Errors
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Clerical errors
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Reagent or equipment problems
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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
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Reagents not added
Manufacturer’s directions not followed
RBC suspensions incorrect concentration
Cell buttons not resuspended before grading agglutination
Clotting deficiencies
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Serum that does not clot may be due to:
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Low platelet counts
Anticoagulant therapy (Heparin, Aspirin, etc)
Factor deficiencies
Serum that does not clot completely before
testing is prone to developing fibrin clots that
may mimic agglutination
Thrombin can be added to serum to activate
clot formation
OR, tubes containing EDTA can be used
Contaminated samples or reagents
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Sample contamination
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Microbial growth in tube
Reagent contamination
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Bacterial growth causes cloudy or discolored
appearance…do not use if you see this!
Reagents contaminated with other reagents (don’t
touch side of tube when dispensing)
Saline should be changed regularly
Equipment problems
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Routine maintenance should be performed on
a regular basis (daily, weekly, etc)
Keep instruments like centrifuges,
thermometers, and timers calibrated
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Uncalibrated serofuges can cause false results
Hemolysis
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Detected in serum after centrifugation (red)
Important if not documented
Can result from:
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Complement binding
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Anti-A, anti-B, anti-H, and anti-Lea
Bacterial contamination
ABO Discrepancies
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Problems with RBCs
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Weak-reacting/Missing antigens
Extra antigens
Mixed field reactions
Problems with SERUM
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Weak-reacting/Missing antibodies
Extra antibodies
Red Cell Problems
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Affect the forward grouping results
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Missing or weak antigens
Extra antigens
Mixed field reactions
Forward Grouping:
Missing or Weak antigens
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ABO Subgroups
Disease (leukemia, Hodgkin’s disease)
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
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
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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
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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
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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)
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Wash red cells or request new sample from heel, etc
Forward Grouping:
Mixed Field Agglutination
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Results from two different cell populations
Agglutinates are seen with a background
of unagglutinated cells
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All groups transfused with Group O cells
Bone marrow/stem cell recipients
A3 phenotype (sometimes B3)
Anti-A
Anti-B
0
2+ mf
A1 Cells B Cells
4+
0
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.
Reverse Grouping Problems
Reverse Grouping
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Affect the reverse grouping results
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Missing or weak antibodies
Extra antibodies
Reverse Grouping:
Missing or Weak antibodies
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Newborns
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Elderly
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Weakened antibody activity
Hypogammaglobulinemia
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Do not form antibodies until later
Little or no antibody production (i.e.
immunocompromised)
Often shows NO agglutination on reverse
groupings
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
Cold antibodies
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Sometimes a patient will develop cold-reacting
allo- or auto-antibodies that appear as “extra”
antibodies on reverse typing
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.
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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)
Finding the problem…
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Forward type tests for the
antigen (red cell)
Reverse type tests for the
antibody (serum)
Identify what the patient
types as in both Forward
& Reverse Groupings
Is there a weaker than
usual reaction?
Is it a missing, weak, or
extra reaction??
Resolving ABO Discrepancies
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Get the patient’s history:
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age
Recent transplant
Recent transfusion
Patient medications
The list goes on….
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
1+
• Probably a subgroup of A (Ax)
• if the result was negative (0), adsorption or elution studies
with anti-A could be performed (these will help determine what
A antigens)
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:
Example 6
Screening Cells
(I and II)
Autocontrol
(AC)
Conclusion
Patient Serum
1
Pos
Neg
Cold alloantibody
Patient Serum
2
Pos
Pos
Cold autoantibody
• if alloantibody – antibody ID techniques
• if autoantibody – special procedures (minicold panel, prewarming
techniques); no prior transfusions. If they have had a recent
transfusion, then it could be an alloantibody.