Transcript ABO Discrepancies & other problems

Practical
Blood Bank
ABO Discrepancies
ABO Discrepancy


When the results of the forward grouping (patient
cells) is not matching the results of the reverse
grouping (patient serum) or abnormal reactivity is
present (i.e. Mixed Field) then we called this ABO
discrepancy.
The Discrepancy will be noticed by:
 Strength of reaction



Weak or missing.
Additional reactions
Abnormal reactions
HINT
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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
Missing or Weak antigens

Subgroups of A and B.
 Solution: test with Anti-A1, Anti-H, and anti-A,B for A
subgroups
• Since the forward and reverse don’t match, there must be a
discrepancy (in this case, a missing antigen in the 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+
Solutions:

Acquired B


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
B(A) phenotype

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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)
Test with another anti-A reagent from another manufacturer
Polyagglutination, Rouleaux,
Wharton’s Jelly
 Wash red cells or request new sample from heel,
etc
Mixed Field agglutination


Can be seen in A, B and AB individuals who have received O
units.
Can also be seen post transfusion if a person makes an
antibody to antigen on donor cells.
Mixed Field Agglutination (Post transfusion)
Unexpectedly Weakened Antibodies
Immunodeficient due to therapy or disease


Immunosuppressive drugs
Certain leukemia’s (CLL) or lymphoma’s (malignant
lymphomas) have hypogammaglobulinemia (Little or
no antibody production)
Age related


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
Extra Antibodies



Cold antibodies (allo- or auto-)
 Cold antibodies may include anti-I, H, M, N, P, Lewis
 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
 Stronger at IS and weak reaction at 37° C and no
agglutination at AHG phase
 Solutions
Anti-A1 in an A2 or A2B individual
Resolving Rouleaux

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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)
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

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, anti-A,B, and antiH
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:
re-label tubes, re-drop
patient and reagent
drops, etc.
1.
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?
5.
Test patient cells with
anti-A1 (Dolichos
biflorus), anti-A,B or
anti-H (Ulex
europaeus)
5.
For suspected
subgroups of A
6.
Test patient serum with
A1 or A2 cells
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: