Finding Strength in Weak Reactions
Raeann Thomas, MLS(ASCP)CM
Blood Bank Supervisor
Hospital of the University of Pennsylvania
November 30, 2016
Objectives
 Describe how antibody resolution work-flow at the Hospital of
the University of PA has evolved over time with solid phase
 Explain how to look at serological results in depth to help
resolve antibody problems that may otherwise be called “nonspecific”
 Illustrate situations in which weak Capture results resulted in
the discovery of clinically significant antibodies through case
studies.
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Background Information
 Hospital of the University of PA
• University of Pennsylvania Health
System
• >750 beds
• Large Heme/Onc outpatient population
• All types of transplants performed
• No longer a Trauma Center as of 2014
• Transfused 83,883 blood products in
2015
 Blood Bank
• Receive approximately 250 samples a day for testing
• Instrumentation:
– 2 Neos for performing all ABO/Rh types and antibody screens (2014)
– Echo for performing antibody panels on all new positive screens and for
additional testing as needed (2016)
• Also use gel and tube methods
• All reference testing is performed in-house (except molecular)
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Background Information
 In our lab, we have been gradually relying more and more on solid
phase- WHY?
• Periodically, we would see patients with strong positive antibody screens in
solid phase (Galileo) and negative testing in other methods (gel, tube, ficin)
• Not only 1 sample, but consecutive samples on the same patient
• We sent a few patients to another hospital to run panels on their Echo
• Nearly all came back with JK identifications!
– Bhoj V. Detection of Kidd Antibodies With Unclear Serology. Poster
Presented at: AABB Annual Meeting; October 2012; Boston, MA
• Through additional studies, found that PEG was closest in sensitivity to solid
phase for JK
 Early 2012- installed a manual solid phase station for additional
testing of solid phase panels
• All patients with newly identified inconclusive reactivity MUST have solid phase
panel before releasing products
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 Anti-Jkb is identified in solid
phase only
 In first 3 months of 20157 of 16 JK required solid
phase to identify
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Reference Testing Flow Chart
Positive Ab
Screen
History of
Antibodies?
Select cells in
appropriate method
Historical
antibodies
demonstrating
 Work flow prior to
Echo installation
New
positive?
Full Gel Panel
 Primary method for
identification was
gel
Additional methods
if necessary
Any Nonspecific
reactivity?
Manual Solid
Phase panel
New Antibody
Identified
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Background Information
 After manual solid phase was introduced:
•
•
•
•
Gel remained our primary testing method for reference testing
Our reference testing could more accurately reflect our screening methods
Began to pick up more antibodies than we had originally expected, not just JK
But manual reading is difficult and subjective.
 2014- Began running all antibody screens in solid phase (Neo)
• Unless an antibody to the test method was detected
• Found patients that had JK antibodies in samples that were repeatedly missed
due to gel antibody screens, causing some patients to have severe delayed
transfusion reactions
• However- our reference testing work flow still remained the same
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New Reference Testing Flow Chart
Positive Ab
Screen
History of
Antibodies?
New
positive?
Select cells in
appropriate method
Echo Panel
Additional rule
outs in PEG
Historical
antibodies
demonstrating
 Work flow after
Echo installation
(September 2016)
Additional methods
if necessary
 Primary method for
newly positive
antibody screens is
solid phase
 Primary method for
select cells is gel
New Antibody
Identified
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Background Information
 September 2016- Echo is live
•
•
•
•
All first time positive antibody screens have a panel run on the Echo
Additional testing methods vary depending on Echo results
BIG adjustment for the staff; still fine-tuning our work flow
Additional unexpected process changes:
– Due to increase in unexpected weak/equivocal reactions observed during
validation, plasma is placed in a clean tube and spun again for 8 minutes
before loading on the Echo for a panel.
• Also validated our Neos for panels in case the Echo is out of service
 Problem remains that no one method is perfect for picking up all
antibodies all the time
 Weak reactions continue to be a source of frustration
 However, it is important not to become complacent with our testing
and review of work ups
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Why are weak reactions important?
 A clinically significant antibody could be newly developing or
could be waning
• JK
 Could be signs of an antibody that may be better detected in a
different test method
• K and tube with LISS
• JK and solid phase
 May require enhancement to aid in identification
• Lewis and ficin
 The patient’s condition/treatment may be causing weakened
expression
• Patient’s age
• Immunosuppression
• Apheresis
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What can we do?
 Look for patterns
• Are there any antigens that the weak reactions have in common?
• Dosage?
 Enhancement media
• Ficin or PEG
 Changes test methods
• Solid phase vs gel vs tube
 Antigen type the patient
• Serology or molecular
 Try to find any history from an outside hospital
 Weak/Equivocal reactions in solid phase
• Equivocal reactions on antibody screens- always interpret as positive!
• Weak/equivocal reactions in panel– Ask a coworker for a second opinion
– Start by verifying reactions you are confident are pos/neg, then go
back to the reactions you are having difficulty grading
– Refer to the references provided by Immucor
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 Guidance for Interpreting Galileo
EchoTM Images
 Guidance for Troubleshooting
Atypical Echo Images (Table-2:
Atypical Capture-R Reactions
Interpreted as Negative on the Echo)
 Great references for
grading questionable
reactions!
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Case 1
 46 year old male admitted for a possible liver transplant
 Historically O pos and antibody screen negative May 2016
• Patient received several transfusions during the previous 6 months
 Currently O pos and antibody screen positive
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 Cell 3 and 6 were graded at 1+ by the technologist
•
Adherence is seen in the background of the well. Cell button is smaller and lighter in
color when compared to a negative reaction
 Cell 1 was graded as 1+ by the technologist
 All other cells were verified as negative
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Auto Control performed
in tube with LISS
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Interpretation




Anti-E identified. Rh phenotype is R1r
Patient received the transplant
Received 16 RBCs during surgery without complication
Patient was discharged a week later
 Important to review and grade equivocals according to
Immucor’s guide for interpreting weak/equivocal reactions
 Important to review and verify all negative reactions
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Case 2
 44 year old male with history of mechanical aortic valve repair
(2009) transferred from an outside hospital with possible
endocarditis
 Historically O pos and antibody screen negative in 2010
• Received several products during admission
 Currently O pos and antibody screen positive
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 Cell 12 is the only positive reaction report by the Echo
 Cells 5 and 6 were graded as 1+ by the technologist
 All other cells were verified as negative
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Auto Control performed
in tube with LISS
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What can we do next?
 Look for patterns
• Are there any antigens that the weak reactions have in common?
• Which antigens correspond to the reactions on the antibody screen?
• Dosage?
 Enhancement media
• Ficin or PEG?
 Antigen type the patient?
• At this point- we are unsure if the patient has been transfused recently
 Try to find any history from an outside hospital
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 Little c, Fya, and Leb
are positive on all 4
cells
 Cob is positive on
cell 12
 Kell is homozygous
on cell 6
 Possibly multiple
antibodies
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



Select cells were run in tube with PEG
Little c, Leb and Cob are ruled out on the first cell.
Kell is ruled out on homozygous cell
Fya is ruled in with 2 positive reactions
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Interpretation
 Anti-Fya identified. Patient typed Fya negative
• Able to obtain history from an outside hospital
• No antibodies, no recent transfusions
 Important to verify all negative reactions!
 Just because all clinically significant antibodies appear to be
ruled out, don’t be quick to interpret as inconclusive/nonspecific reactivity!
 Look for any pattern in the reactions
 Try different test methods to rule in/out
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Conclusion
 Antibodies do not read textbooks!
• Don’t expect perfect “textbook” demonstration when performing panels
 Investigation of weak reactions can be critical to identifying a
clinically significant alloantibody
• Especially when is has been years since their last exposure
 Review of positive reactions is also necessary to ensure
antibodies to low frequency antigens are not missed
 Use additional test methods to rule in/out when necessary or
to enhance weak reactivity
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THANK YOU!!
QUESTIONS?
COMMENTS?
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