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Seeing Beyond Limits

2020 Webinar Schedule

Link to register: https://immucor.webinato.com/register

Seeing Beyond Limits

2020 “Mini” WebinarsShort,10-minute educational videos with an industry leader.

Link to register: https://immucor.webinato.com/register


learn.immucor.com


Continuing Education

• PACE, ABHI, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:

https://www.surveymonkey.com/r/SolidPhaseAntibodies

• Registration deadline is 28 February, 2020

• Certificates will be sent via email only to those who have registered 6 March, 2020


7

Making “Cents” of

Unexpected Reactions

Michael Spigarelli, MD

VP, Medical Affairs

8

Expectations:

9

Expectations:

“Climate is what you expect, weather is what you get”


The Difficulty with Expectations

• Goal of any testing is to obtain the best answer

• Expectations are a type of bias

• Bias can lead to misinterpretations

• Misinterpretations lead to mistaken conclusions

• Mistaken conclusions can lead to errors in care

• Errors in care are never the goal


Sorting Out the Truth – Easily Done


Simple Sort

???


Simple Sort

???

A


Simple Sort

???

AB


Sorting Out the Truth – Which Truth

What if you had 9 Quarters, 7 Dimes, 3 Euros, 2 Loonies and 1 Washer?


Sorting Out the Truth – Which TruthWhat if you had 9 Quarters, 7 Dimes, 3 Euros, 2 Loonies and 1 Washer?

Based upon the simple sorter we just used:

A Bin 7 Dimes 1 WasherB Bin 9 Quarters 3 Euros??? Bin 2 Loonies





How well did the sorting work?





How well did the sorting work? Depends on the Question!






1. Can we sort money from non-money?






1. Can we sort money from non-money?

A Bin 7/8 (87.5%)B Bin 12/12 (100%)??? Bin 2/2 (100%)Overall 21/22 (95.5%)






1. Can we sort Quarters?






1. Can we sort Quarters?

A Bin 8/8 (100%)B Bin 9/12(75%)??? Bin 2/2 (100%)Overall 19/22 (86.4%)

26

Theoretic Example


Example

• A patient, with a known, historic E antibody, presents for care

• Automated antibody screen is negative

• Interpretation:

• Instrument result is correct OR

• Instrument result is incorrect


Example (continued)

• Decision is made to confirm

• Manual Gel for E antibody is negative

• Interpretation:

• Manual Gel result is correct OR

• Manual Gel result is incorrect


Example (continued)

• Decision is made to be certain

• Tube testing for E antibody is negative

• Interpretation:

• Tube result is correct OR

• Tube result is incorrect


Example (continued)

• Decision is made to be EXTRA certain

• Tube testing repeated with weaker shaking

• Tube testing for E antibody is Positive

• Interpretation:

• Tube result is correct OR

• Tube result is incorrect


Sample Reactivity

• Is the pathway correct?

• Is the result valid?

• Did all the testing need to be done?


Sample Reactivity

• Is the pathway correct?

• Is the result valid?

• Did all the testing need to be done?

• What if there was no known history?

• Why do the paths seem different with or without history?


Example Deux

• A patient, with a known, historic E antibody, presents for care


• Interpretation:




Example Deux

• A patient, presents for care


• Interpretation:




Methods are Method Specific

+ Purple- Red

+ Purple+ Red

- Purple+ Red

- Purple- Red


Methods are Method Specific+ Purple- Red+ Green

+ Purple+ Red+ Green

- Purple+ Red+ Green

- Purple- Red- Green


Methods are Method Specific+ Purple- Red- Green

+ Purple+ Red+ Green

- Purple+ Red- Green

- Purple- Red- Green


Definition of Unexpected Negative

• Expected outcome vs Actual outcome

• Definition of an Unexpected Negative Reaction

“Unexpected Negative (UN) is defined as an actual negative assay

result that was expected to yield a positive assay result. For example,

a sample that contains anti-Fyᵃ that was expected to yield a positive red

blood cell antibody screen, but actually produced a negative result”.

-Immucor Validation Guide










• That means two things:

– There must be an antibody present

– The antibody must be detectable by the methodology


Example: Complaints of Unexpected Negatives:What is the expectation?

Sensitivity EstimationSituation: What if there was large hospital system with 30 sites filed complaints

of 22 “Unexpected Negatives” on Echo Lumena/v2.0 since conversion

Assumptions (conservative):

• Small Hospital ~ 5 blood bank samples/day

• Large Hospital ~ 35 blood bank samples/day

• “Unexpected Negatives” are all actual negatives (likely not true)• Average ~ 20 blood bank samples/day

• 20 samples x 30 days/month x 11 months x 30 hospitals =

198,000 total samples

• True Positive = 198,000 x 5% = 9,900

Calculation: Sensitivity = TP/(TP+FN)

9,900 / (9,900 + 22) * 100 = 99.8%


Relative Comparisons

• In the absence of an absolute standard all assignments are relative

• Detection of an antibody is based upon numerous factors

• If one method detects an antibody and another does not, then depending upon which one is considered the gold standard

• A positive result could be considered• correct and called a true positive – should be expected positive

• Incorrect and thus a false positive – should be unexpected positive

• A negative result could be considered• correct and a true negative – should be expected negative

• Incorrect and an Unexpected Negative – should be unexpected negative


Relative Comparisons

• When compared to each other the relative assignments become part of a continuum,

• If one method detects an antibody and another does not then the results are based on relative sensitivity

• A positive result on the more sensitive method and negative on the less sensitive method

• Is correct unless definitive analysis proves it wrong

• It represents an Unexpected Negative for the less sensitive method

• It is NOT a false positive

• A negative result on the more sensitive method • Is correct unless definitive analysis proves it wrong

• It likely represents a true negative for the less sensitive method

• It is NOT necessarily a Unexpected Negative


Why are unexpected negatives reported

• Comparison with known history• Was positive on a previous test, now negative

• Antibodies are known and expected to diminished with time

• At a minimum, transfusion would be based upon prior history

• Further work up may reveal antibody or not

• Harm to patient should be zero

• Discrepancy with another test• Frequently multiple tests are run on the same sample

• At a minimum, transfusion is based preponderance of tests

• Harm to patient should be zero

Capture Proof Sources


Capture Proof Source

Anti-Jka that are detected by solid-phase red blood cell adherence but missed by gel testing can cause hemolytic transfusion reactions, B Kay et al.

• The goal of the study was to define the clinical significance of anti-Jka antibodies undetected by gel, but detected by Capture.

• Anti-Jka was detected in 105 of the 88,478 Capture screens performed during the period evaluated.

• “The use of [Capture] testing significantly increased the detection and identification of patients with clinically important anti-Jka. This reduced the risk of delayed hemolytic transfusion reactions due to anti-Jka”.

TRANSFUSION, 2016; 56;2973-2979

32 of 105 Jka antibodies were initially discovered by Capture but undetectable (n = 26) /inconclusive (n = 6) in gel.

• 17 of these 32 patients were recently transfused• 6 met the criteria for delayed hemolytic transfusion reaction• 3 had possible delayed hemolytic transfusion reactions• 8 had delayed serologic reactions


Capture Proof Source

• A COMPARATIVE EVALUATION OF TWO AUTOMATED SYSTEMS (SOLID PHASE AND GEL COLUMN AGGLUTINATION) VS TUBE METHODS FOR DETECTION OF IRREGULAR ANTIBODIES , Vox Sanguinis (2014) 107 (Suppl. 1), 57-248

• Capture-R sensitivity was higher than DG Gel in this study

• Capture only missed 2 clinically significant antibodies that PeGdetected, both were weakly reactive in PeG.

• DG Gel missed 5 clinically significant antibodies that PeG detected.


Capture Proof SourceAABB 2018: IGT106 Antibody Detection Using Solid Phase Technology: A Three Year Retrospective Analysis of Reference Laboratory Samples That Were Non-Reactive in Tube and Gel Testing and Reactive in Solid Phase Testing Marianne F. Leininger, Mollie Bell, David Oh, Matthew Montgomery, Jeffrey Papiernik and Gregory Halverson. Mercy Health - West Hospital, Hoxworth Blood Center

• Table 1. Shows 16 cases where reactivity was detected only with SP

• In 8 of the 16 cases the patient was pre-natal with identified antibody specificities of anti-C, -E, -Jk(a) or passive anti-D

49

Capture Sensitivity


Importance of Sensitivity

• Immucor’s design goal is to maintain the highest level of sensitivity for clinically significant antibodies that our customer’s have come to expect from our platform

• New design is always compared to previous design, so we have plenty of current data to understand cross method sensitivity


Why is Capture sensitive?

• Capture-R is an antiglobulin test that has two stages that should be considered separately

Sensitization

The binding of

antibodies in a sample

to red blood cell

antigens on the

membranes

immobilized on the

microwell surface

Adherence

Indicator Red Cells

that are impeded from

migrating to the

bottom of a well in

where anti-IgG-IgG

complexes are formed

resulting in antibody

bridging which forms a

second immobilized

layer


Sensitization

• Classical textbooks such as Issitt & Anstee’s Applied Blood Group Serology tell us that several factors affect the binding of antibodies from the sample to corresponding antigens

• Incubation conditions

• Liquid medium: (LISS, PEG etc.)

• The ratio of test sample (antibodies) to cell surface area (antigens) influences the sensitivity of the test

• Same sample volume to RBC surface area


Sample to cell ratio: 3 Methods

MethodSample Volume

(µL)

Red Cell Surface Area

(mm2)

Sample to Cell Ratio

(µL/mm2)

Capture 25* 66.4 .38

Gel 25 598 .04

Tube 100 2244 .04

•The ratio of sample volume to antigen-bearing surface is .38 in a Capture reaction vs .04 in an agglutination reaction such as tube or gel methods

•This difference means that a higher proportion of the antigens in a Capture well are likely to have antibody attached to them vs other methods

*Automated use


Agglutination & Adherence

• Tube IAT and gel methods are all based on agglutination - cells that are cross-linked by antigen-antibody-AHG complexes

• In Capture tests, binding of antibody to antigens on the surface of the well is visualized by adherence - binding of the AHG on the Indicator Red Cells to antibodies bound to antigens immobilized on the microwellsurface


Solid Phase Adherence

• During centrifugation, Indicator Red Cells begin to migrate along the sides of microwells towards the bottom of the well unless impeded by a sensitized reagent cell

• This process results in multiple contacts providing increased opportunity for the Indicator Red Cells to be immobilized as the anti-IgG-IgG complexes form


Sensitivity Summary

• The discrimination by centrifugation in the curved surface of the microwell combined with the migration effect of the Indicator Red Cells provides many binding opportunities

• This coupled with the ratio of sample volume to antigen-bearing surface makes Capture very sensitive


• Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations.

• The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor.


Continuing Education

• PACE, ABHI, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at: https://www.surveymonkey.com/r/SolidPhaseAntibodies

• Registration deadline is 28 February, 2020

• Certificates will be sent via email only to those who have registered 6 March, 2020


62

Thank you!

AABB 2017: CP208 Frequencies and Specificities of “Solid-Phase Only” Detected Erythrocyte Antibodies: Is Solid Phase Testing Worth the Headache? Karen Finegan, Karen Gray, Jill Adamski, Theresa Kinard and Qun Lu. Mayo Clinic, Arizona

Background/Case Studies: An effort to re-evaluate automated testing platforms (Automated Solid-phase red blood cell adherence vs automated Gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in “unnecessary” workup at one site of a hospital system. The data available from parallel testing on Solid-phase, Gel and PEG performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of “solid-phase only” detected erythrocyte antibodies and to see if solid-phase antibodies workup is necessary for patient care.

Study Design/Methods: Throughout 2016, the transfusion service used automated Solid-phase red blood cell (RBC) adherence as the primary method for antibody screening and identification. All Solid-phase antibody screen positive samples were re-tested using both Gel column agglutination and PEG method manually in order to determine which method should be used for antiglobulin phase crossmatch of RBC products. All antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed.

Results/Findings: A total of 398 patients were positive on Solid-phase antibody screen and re-tested on Gel and PEG antibody screen. In 12% (n=49) patients antibody reactivity observed in solid phase only and the concurrent Gel and PEG testing were completely negative. Of them clinically significant RBC alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in 22% (n=11), 4% (n=2), and 74% (n-36) of the cases, respectively. RBC alloantibodies identified in solid-phase only included: anti-E (n=4), anti-Jka (n=3), anti-K (n=2), and anti-Jka (n=1), both anti-E and Anti-C (n=1) (See Table 1).

Conclusion: Solid-phase only RBC antibodies are clinically important in a significant portion of cases (roughly 1 in 4 cases). Workup for Solid-phase only antibodies is not “unnecessary” workload. Transfusion of corresponding antigen negative RBCs to these patients prevent possible hemolytic transfusion reactions.

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