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Deborah Baudler MS, MT(ASCP) SBBAssistant Professor
University of Illinois-SpringfieldPatchwork Conference
April 15, 2014
Investigative Techniquesin Blood Banking
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University of Illinois-Springfield
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UIS CLS Students
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• Identify common problems that occur in day to day blood banking
• Discuss various techniques for problem-solving
• Apply new knowledge to case studies for resolution
Objectives
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“The science of deduction and analysis is one which can only be acquired by long and patient study...”
Sherlock Holmes
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• ABO Discrepancies
• Weak Positive Antibody Screen……no antibody identified
• Miscellaneous Reactivity showing up on the antibody panel
• Incompatible Crossmatch when antibody screen is negative
Common Problems That Can Occur
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• Cry a little• Start over, hoping the problem will just go
away• Shake the tubes harder• Pretend the weak reactions don’t exist• Call your blood bank supervisor at 2 am to
see if she/he is reading a good book• Leave it for the next shift to resolve!
Common Solutions Available
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ABO Discrepancy
• A discrepancy occurs when the red cell testing does NOT match the serum test results
• In other words, the forward type does NOT match the reverse.• What is the discrepancy here?
Anti-A Anti-B Anti-D A1 cells B cells
2+ 4+ 4+ 1+ 0
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• Recall: the production of ABO antigens is controlled by the genes we inherit
• ABO forward and reverse reactions are typically very strong: 3+ to 4+.
• Where do we start?
What we do know….
Anti-A Anti-B Anti-D A1 cells B cells
2+ 4+ 4+ 1+ 0
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• Most of the time, the problem is technical• Failure to add patient plasma• Reversed the A1 and B cells in the rack• Reagent contamination• Incubation time too short• Clot in specimen• Interpretations not accurately recorded
Discuss the Possibilities
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Forward vs Reverse
Courtesy of School of Health Related Professions University of Mississippi Medical Center
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
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• If you have extra reactivity:• Recent Bone Marrow/ Stem Cell Transplant: check
medical history• Excess protein coating red cells or Rouleaux: Wash red
cells and retest• Strong cold agglutinin coating cells: Treat cells with
0.01 M DTT• Antibody coated red cells causing autoagglutination:
can be seen in HDFN. Perform DAT and Incubate cells and wash several times with 37°C saline
Reasons for Red Cell Discrepancies
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• Acquired B antigen: occurs in Group A individuals with gram neg sepsis. True group A cells will not agglutinate with patient’s own Anti-B in plasma
Reasons for Red Cell Discrepancies
Enzyme cleaves off acetyl group
D-Galactose
Acetyl group
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• If you have missing or weak reactivity:• Subgroup of A: test cells with A1 Lectin, Anti-A,B and Anti-H• Massive red cell transfusion: check transfusion history• Cancer or Chemotherapy: require longer incubation period
Reasons for Red Cell Discrepancies
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• If you have extra reactivity:• Rouleaux: Check for “stack of coins”
and perform Saline Replacement• Cold or RT alloantbody: Antibody ID
and repeat reverse cells with antigen negative cells
• Cold or RT autoantbody: Antibody ID and pre-warm plasma and reverse cells in separate tubes, combine and read
Reasons for Plasma Discrepancies
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• If you have extra reactivity:• Issoagglutinins: Passive ABO
antibodies: check recent transfusion history
• Subgroup of A: A1 Lectin and antibody ID with A1 cells
Reasons for Plasma Discrepancies
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• If you have missing or weak reactivity:• Check age of patient:
• Newborn: no antibody production until 4 mos• Elderly: extend incubation or increase serum/cell ratio
• Hypogammaglobulinemia: extend incubation or increase serum/cell ratio
• Chemotherapy or recent Bone Marrow Transplant: check medical history
Reasons for Plasma Discrepancies
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• We have extra reactivity on the plasma side• Most frequent cause for ABO discrepancy
Back to Our Case
Anti-A Anti-B Anti-D A1 cells B cells
2+ 4+ 4+ 1+ 0
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In This Case
Anti-A Anti-B Anti-D A1 cells B cells
2+ 4+ 4+ 1+ 0
1. Patient has never been transfused and is not pregnant
2. Patient is here for elective surgery
3. Antibody Screen is negative, Auto control = 0
4. Checked under the scope, no Rouleaux
5. What’s left to do?
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1. Recall: 20% of group AB individuals are actually A2B. 25% of A2B will make an alloantibody called Anti-A1
2. Test patient’s red cells with A1Lectin. A2B will not agglutinate with A1Lectin
3. Test patient’s plasma with several lots of A1 cells to confirm that the antibody is Anti-A1
Solution
Agglutination
No Agglutination
A1 cells
A2 cells
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• Patient is an A2B Pos with Anti-A1
• Solution: Use A2 reverse cells to eliminate extra reactivity and resolve discrepancy
In This Case
Anti-A Anti-B Anti-D A2 cells B cells
2+ 4+ 4+ 0 0
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• 2. Weak Positive Screen: Negative Antibody ID
• Get the Patient’s Medical History• Possible Solutions:
• Repeat antibody screen and ID by a second method• Check expiration dates of reagents• Increase serum/cell ratio• Increase incubation time• Contact the manufacturer
• How should this be reported?
Weak Antibody Activity
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• 3. Positive Screen: No specific antibody identified• All alloantibodies have been ruled out!
Miscellaneous Antibody Activity
D C E c e K Fya Fyb
+ + 0 + + 0 + 0
+ 0 0 + + 0 0 +
+ + 0 0 + + + +
0 + + + + 0 + +
0 + 0 0 + 0 + 0
0 0 + + 0 + 0 0
0 0 0 + + 0 + +
Jka Jkb M N S s Lua
0 + + + + + 0
+ 0 + 0 + 0 0
0 + 0 + 0 + +
+ 0 0 + 0 + 0
+ 0 0 + 0 + 0
0 + + 0 + 0 0
+ + + + + + 0
Lub IS AHG cc
+ 0 + nt
+ 0 0 √
+ 0 0 √
+ 0 + nt
+ 0 0 √
+ 0 0 √
+ 0 + nt
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What Should You Do?
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• 3. Positive Screen: No specific antibody identified
• Possible Solutions• Check lot number of antigrams!• Check expiration dates of reagents • Repeat antibody screen and ID by a second method • Increase serum/cell ratio• Increase incubation time
Miscellaneous Antibody Activity
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• Highlight positive reactions• Check for Dosage
Miscellaneous Antibody Activity
D C E c e K Fya Fyb
+ + 0 + + 0 + 0
+ 0 0 + + 0 0 +
+ + 0 0 + + + +
0 + + + + 0 + +
0 + 0 0 + 0 + 0
0 0 + + 0 + 0 0
0 0 0 + + 0 + +
Jka Jkb M N S s Lua
0 + + + + + 0
+ 0 + 0 + 0 0
0 + 0 + 0 + +
+ 0 0 + 0 + 0
+ 0 0 + 0 + 0
0 + + 0 + 0 0
+ + + + + + 0
Lub IS AHG cc
+ 0 + nt
+ 0 0 √
+ 0 0 √
+ 0 + nt
+ 0 0 √
+ 0 0 √
+ 0 + nt
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• Additional Suggestions: • Get the Patient’s Medical History• Enzyme panel• Check Direct Coombs• Perform an Eluate
Miscellaneous Antibody Activity
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• For a DAT to become positive: > 200 molecules of IgG on red cell
• Purpose of an eluate:• Removes an antibody that’s coating the red cell• Concentrates antibody• Allows identification of newly forming or weak antibodies• Can be positive even when DAT is negative
Benefits of an Eluate
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Miscellaneous Antibody Activity
• What antibody is detected?
D C E c e K Fya Fyb
+ + 0 + + 0 + 0
+ 0 0 + + 0 0 +
+ + 0 0 + + + +
0 + + + + 0 + +
0 + 0 0 + 0 + 0
0 0 + + 0 + 0 0
0 0 0 + + 0 + +
Jka Jkb M N S s Lua
0 + + + + + 0
+ 0 + 0 + 0 0
0 + 0 + 0 + +
+ + + + + + 0
+ 0 0 + 0 + 0
0 + + 0 + 0 0
+ + + + + + 0
Lub AHG ELU cc
+ + + nt
+ 0 0 √
+ 0 + nt
+ + + nt
+ 0 + nt
+ 0 0 √
+ + + nt
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• Possibilities:• Perform clerical check on specimen• Check agglutination under scope if <2+• Specimen at room temp or out of
refrigerator• Age of specimen: protein precipitation
• If Reactivity is 3-4+• Repeat patient’s blood type • Strong Cold Agglutinin
Incompatible Crossmatch When Antibody Screen is Negative
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• Other Possibilities:• Patient has an antibody to a Low
Incidence antigen on unit• Unit has positive DAT
• Most likely• Return unit to blood center
• Solution: Try another unit
Incompatible Crossmatch When Antibody Screen is Negative
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Let’s do some Investigating
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Case 1
• 71 yr. old woman comes through the ER on a Friday night with a 6 g Hb. While her antibody screen is incubating, you get the following blood type:
Anti-A Anti-B Anti-D A1 cells B cells
0 4+ 0 4+ 2+
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• ABO discrepancy present• Most probably blood type?• What should we do next?
Thoughts?
Anti-A Anti-B Anti-D A1 cells B cells
0 4+ 0 4+ 2+
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Oh No!
• Next thing you know, her antibody screen comes up positive, YIKES!
D C c E e K Fya Fyb Jka Jkb M N S s IS AHG cc
1 + 0 + 0 + 0 + + + 0 0 + + 0 0 0 √
2 + 0 + + 0 + 0 + 0 + + + 0 + 0 0 √
3 0 + + 0 + 0 + 0 + + + 0 + + + + nt
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Let’s Do the Cross-out Technique
Which Antibodies Could Possibly be Present?
D C c E e K Fya
Fyb Jka
Jkb
M N S s IS AHG cc
1 + 0 + 0 + 0 + + + 0 0 + + 0 0 0 √
2 + 0 + + 0 + 0 + 0 + + + 0 + 0 0 √
3 0 + + 0 + 0 + 0 + + + 0 + + + + nt
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Which Antibody is Present?
Antibody ID Results
D C E c e K Fya Fyb
+ + 0 0 + 0 + +
+ 0 0 + + 0 0 0
+ + 0 0 + + + 0
+ 0 + + 0 0 0 +
0 + 0 0 + + + +
0 0 + + 0 0 0 +
0 0 0 + + 0 + 0
Jka Jkb M N S s Lua
0 + + 0 + 0 0
+ 0 + 0 + + 0
0 + 0 + 0 + +
0 + + + + 0 0
+ 0 0 + 0 + 0
0 + + 0 + + 0
+ 0 + + 0 + 0
Lub IS AHG cc
+ + + nt
+ + + nt
+ 0 0 √
+ 0 0 √
+ 0 0 √
+ + + nt
+ 0 0 √
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•Antibody identified as Anti-M
•Anti-M can possess both IgM and IgG components
•Phenotype patient for M if not recently transfused
•Test B Neg, M Neg cells with patient plasma
Are We Done?
Anti-A Anti-B Anti-D A1 cells B cells
0 4+ 0 4+ 0
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• 62 yr. old man comes through the ER on a Saturday night with abdominal pain. He is rushed to surgery for a possible bowel obstruction
• While his antibody screen is incubating, you get the following blood type:
Case 2
Anti-A Anti-B Anti-D A1 cells B cells
3+ 0 4+ 0 4+
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• Patient’s medical history indicates he had cardiac by-pass surgery 4 weeks ago and received 3 units of prbcs
• Perform cross-out technique• Which antibodies can not be ruled out?
The Antibody Screen Results
D C c E e K Fya Fyb Jka Jkb M N S s IS AHG cc
1 + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + nt
2 + 0 + + 0 + 0 + 0 + + 0 0 + 0 0 √
3 0 + 0 0 + 0 + + + + + + + + 0 + √
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The Antibody Screen Results
• Anti-C, e, Fya, Jka, N and Anti-S are not ruled out
D C c E e K Fya Fyb Jka Jkb M N S s IS AHG cc
1 + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + nt
2 + 0 + + 0 + 0 + 0 + + 0 0 + 0 0 √
3 0 + 0 0 + 0 + + + + + + + + 0 + √
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Conclusion? What is the next step?
Antibody ID Results
D C E c e K Fya Fyb
+ + 0 0 + 0 + +
+ 0 0 + + 0 0 0
+ + 0 0 + + + 0
+ 0 + + 0 0 0 +
0 0 + + + + + +
0 0 + + 0 0 0 +
0 0 0 + + 0 + 0
Jka Jkb M N S s Lua
0 + + 0 0 + 0
+ 0 + 0 + 0 0
+ + 0 + 0 + +
+ 0 + + + + 0
+ + 0 + 0 + 0
+ 0 + 0 + + 0
+ + 0 + 0 + 0
Lub IS AHG cc
+ 0 + nt
+ 0 0 √
+ 0 + nt
+ 0 + nt
+ 0 0 √
+ 0 + nt
+ 0 0 √0 + + + 0 0 0 +
0 0 0 + + 0 + 0
0 + + 0 + + 0
0 + 0 + 0 + 0
+ 0 + nt
+ 0 0 √
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Repeated panel with PeG. Who is the culprit?
Antibody ID Results
D C E c e K Fya Fyb
+ + 0 0 + 0 + +
+ 0 0 + + 0 0 0
+ + 0 0 + + + 0
+ 0 + + 0 0 0 +
0 0 + + + + + +
0 0 + + 0 0 0 +
0 0 0 + + 0 + 0
Jka Jkb M N S s Lua
0 + + 0 0 + 0
+ 0 + 0 + 0 0
+ + 0 + 0 + +
+ 0 + + + + 0
+ + 0 + 0 + 0
+ 0 + 0 + + 0
+ + 0 + 0 + 0
Lub IS AHG cc
+ 0 + nt
+ 0 + √
+ 0 + nt
+ 0 + nt
+ 0 + √
+ 0 + nt
+ 0 + √
0 + + + 0 0 0 +
0 0 0 + + 0 + 0
0 + + 0 + + 0
0 + 0 + 0 + 0
+ 0 + nt+ 0 0 √
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• 27 yr. old man is medevac to your facility from a small community hospital. The patient has been in a motor vehicle accident and is bleeding internally. He is being prepped for the OR.
Case 3
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• So you perform a STAT Type and Screen• While the antibody screen is incubating, you record the following
results for the blood type:• Any problems?
Case 3
Anti-A Anti-B Anti-D A1 cells B cells
2+mf 2+mf 2+mf 4+ 4+
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• The patient’s antibody screen results are Negative!• Now what?
The Antibody Screen Results
D C c E e K Fya Fyb Jka Jkb M N S s IS AHG cc
1 + 0 + 0 + 0 + 0 + 0 0 + + 0 0 0 √
2 + 0 + + 0 + 0 + 0 + + 0 0 + 0 0 √
3 0 + 0 0 + 0 + + + + + + + + 0 0 √
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• Patient received:• 10 units Group O Negative rbcs• 4 Group O Single Donor Platelets
Check Medical History
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• Patient’s ABO discrepancy was due to massive transfusion of out of group blood products.
• Important Clue: Mixed-field agglutination• Information from transferring hospital confirmed that patient
was AB Positive
• What blood type of rbcs should be transfused?
Resolution
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You did it!