raptor analysis guide - storyblok · 2020-01-20 · 2. ccd inhibition 3. spot morphology 1.dirt,...

Raptor Analysis GuideFor tricky results

For Raptor version 1.3.0.34 and higher

Guide Version 1.1

Authors: Martina Gunacker & Peter Forstenlechner

www.macroarraydx.com 1

Content

1. Grid Alignment

2. CCD Inhibition

3. Spot Morphology1.Dirt, Dust, Hair & Scratches

2.Spot diameter

3.Spot bleeding

4. Components & Extracts

5. High Background


1. Grid Alignment


In almost all cases Raptor is able to position the grid correctly, as shown below.

In those cases move ahead according to the manual

1. Grid Alignment


In rare cases the grid can become misaligned, see below

The QC will be registered as failed (see right)

You will still be able to approve the

misaligned results, but you will be warned

(see below).

1. Grid Alignment


If you choose to approve and export misaligned results, your patient

will get completely false results, which could lead to incorrect and

possibly dangerous treatment. As a further hurdle to exporting faulty

results the report will contain a warning in the header (see below).

To remedy the situation, press CTRL+R, adjust the grid manually and press F5

1. Grid Alignment

• Circle overlap – misalignment

• Can happen to circles where the result is negative and the neighbour is positive or where very few results are positive

• In almost all cases this does not affect the result enough to lead to falsely elevated results.

• To correct the misalignment press F, click on the circle and move it to its position, then press Q


1. Grid Alignment


TAKE HOME MESSAGE

1. If you are not sure about the grid alignment press CTRL+R, adjust the

grid manually and press F5

2. If you are still not sure of the grid alignment, or if many grids are

misaligned, rerun the TEST ARRAY

3. Do not approve misaligned & QC failed ALEX

2. CCD Inhibition


1. The automatic blocking of CCD specific antibodies is one of the key

advantages of ALEX above all other IgE in vitro test systems

2. Incomplete CCD inhibition occurs rarely: in 6 out of 263 samples Hom s LF

was positive (done according to standard assay protocol, internal data).

An incomplete

CCD inhibition will

display a positive

result for Hom s LF

and many other

allergens.

To increase the

CCD blocking

efficiency please

consult ALEX

instruction for use

page 10-11:

sample

incubations/CCD

inhibiton

Press N to annotate allergen features

2. CCD Inhibition


1. Incomplete CCD blocking will lead to a lot of positive allergen extracts

(plant- and venom allergens, some seafood and mite allergens) and also

naturally purified components.

a. This leads to a very high interpretative burden for clinically irrelevant

IgE results

2. Incomplete CCD blocking is a very rare occurrence

3. Rerun the samples according to the optional CCD Inhibition protocol (ALEX IFU)

TAKE HOME MESSAGE

3. Spot Morphology


To check the spot morphology is of vital

importance to get a correct result

Dust, hairs, scratches and dirt can affect

results i.e. lead to falsely elevated

results (in very rare cases)

Acceptable

morphology

unacceptable

morphology

3. Spot morphology

• If a spot is smaller than 70% of its designated size set signal to negative (press F, click spot, press B, press Q)

• Dust or dirt on the array. Set affected spots negative (press F, click spot, press B, press Q)


acceptable

spot

unacceptable

spot

3. Spot morphology


• Scratch or hair on the array

can lead to falsely elevated

results

• Set spots negative, see

manual or previous slide on

how to

Hairy artefact

Scratchy

artefactBoth kind of artefacts will in most

cases not push the result above the

cut off of 0.3

3. Spot morphology

• Spot bleeding: in rare cases a highly positive spot can bleed over to its neighbour and simulate a falsely elevated signal – check spot morphology (clear circle yes or no).

For better spot visibility press CTRL + G


Doubtful positive result for Cyp c 1

Spot morphology bad! – not a

clear circle, spot bleeding from

neighbouring spot likely reason for

pos. result

Correct setting for Cyp c 1

(in this case)

CTRL + F, click spot, press

B, press Q

Spot morphology – Allergen family

• An alternative way to judge the validity of results is the analysis of signals according to their allergen family. Within allergen families there can be a high or a limited form of cross reactivity.

• E.g. Cyp c 1 (Parvalbumin from Carp) shows a low positive and Gad m 1 (Parvalbumin from Cod) a negative result.

• the result for Cyp c 1 is most likely falsely elevated (provided the spot morphology of Cypc 1 is bad)


Gad m and Gad m 1 spot

negative

Doubtful positive result for Cyp

c 1

Spot morphology bad! – not a

clear circle, spot bleeding from

neighbouring spot likely

reason for pos. result

3. Protein families• Highly cross reactive protein(allergen) families are

• Profilin• PR-10• nsLTP• Serumalbumin• Parvalbumin • Tropomyosins• NPC2

• Protein families with limited cross-reactivity are• 2S Albumins• 7&11S Globulins• Ole e 1 family• Pectate Lyase• Lipocalin

• Judging results according to protein families requires experience and literature knowledge, if you are not sure contact [email protected]


mailto:[email protected]

3. Spot Morphology


• Check spots carefully

• In most cases artefacts don’t push results above cut off

• Correct falsely elevated results, if they are near or above the cut off (0.3)

• Knowledge on protein (allergen) families useful to judge validity of pos./neg. signals

• If you are not sure about a signal, contact [email protected]

TAKE HOME MESSAGE

mailto:[email protected]


• It can happen that components are positive i.e. Ara h 9 positive and Ara h Extract negative.

• This occurs mostly when components are slightly positive

• Reason: in a component spot the whole surface is dedicated to one component, in an extract the surface has to be shared with many allergens


Surface of component

spot: 100% of surface

dedicated to one molecule

Surface of extract spot:

x% of surface dedicated

to one molecule

Ara h 9

Ara h 6

Ara h 1

Ara h 2

Ara h 3/4Ara h 7

Ara h 8

Ara h 9

Ara h 5


• When analysing messy results (bad spot morphology, possible influence of artefacts) the constellation of extract and component can give a clue to the right result

• Component result is low positive and the corresponding extract is negative

• Points to a falsely elevated component result

• If spot morphology is clear than approve positive result for component

• See slide 13

• Extract positive and component negative• The component that is causing the positive extract result could not be spotted on

ALEX



• Wheat component Tri a Gliadin positive, Tri a Extract negative• Tri a Gliadin not well represented in Tri a Extract (extract is watery, Gliadin is

insoluble in water)• Tri a Gliadin result valid!



• Look at component and corresponding extract results if spot morphology is bad

• Check for possible artefacts

• Negative extract and positive component result ok, if spot morphology is ok

• Positive extract and negative component result ok, if spot morphology is ok


TAKE HOME MESSAGE

5. Background: Edge effect


• Edges gives high

background (precipitated

substrate)

• Check spots close to the

edge carefully

• Repeat the assay and pay

careful attention to

knocking out the washing

solution after the substrate

and before drying.

• For optimal knocking out

technique visit:

https://www.macroarraydx.co

m/downloads-and-clips

→Videos

https://www.macroarraydx.com/downloads-and-clips

5. Background


• Guide Dots and Standards are too low

• QC fail

• Repeat the assay procedure and follow the instructions of the manual verycarefully, especially during the washing steps (forcefully knock out).

Array with very

high

background

TAKE HOME MESSAGE

raptor analysis guide - storyblok · 2020-01-20 · 2. ccd inhibition 3. spot morphology 1.dirt,...

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