Finding Strength in Weak Reactions

November 30, 2016

Raeann Thomas, MLS(ASCP)CM

Blood Bank Supervisor

Hospital of the University of Pennsylvania

2

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 “non-

specific”

Illustrate situations in which weak Capture results resulted in

the discovery of clinically significant antibodies through case

studies.

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Background Information

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)

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

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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

5

In first 3 months of 2015-

7 of 16 JK required solid

phase to identify

Anti-Jkb is identified in solid

phase only

6

Reference Testing Flow Chart Positive Ab

Screen

Full Gel Panel

Additional methods

if necessary

New Antibody

Identified

Select cells in

appropriate method

Manual Solid

Phase panel

New

positive?

History of

Antibodies?

Any Nonspecific

reactivity?

Historical

antibodies

demonstrating

Work flow prior to

Echo installation

Primary method for

identification was

gel

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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

8

New Reference Testing Flow Chart Positive Ab

Screen

Echo Panel

Additional methods

if necessary

New Antibody

Identified

Select cells in

appropriate method

New

positive?

History of

Antibodies?

Historical

antibodies

demonstrating

Work flow after

Echo installation

(September 2016)

Primary method for

newly positive

antibody screens is

solid phase

Primary method for

select cells is gel

Additional rule

outs in PEG

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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

15

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

18

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/non-

specific 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?