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Hydrophobic Slides??!

09/13/2008 12:27 PM

Does anyone know of a way to measure and verify the charge strength on a histology slide? We believe we are seeing a slide issue that is preventing an automated oncology screening device from staining occassionally. We see the problem randomly, but always, and only happens on tissue samples placed in the bottom half of the slide.

We have seen the phenomenon before (always at this time of year, almost to the week! However we haven't been able to validate any environmental causes and are looking elsewhere.) We have also observed it when it happened, but are looking for a quicker test to show that the slide is hydrophobic.

Also, if anyone knows how a glass slide is charged, this would help. Previous testing lab results stated that there was NO detectable coating on the glass from a frosted, positively charged slide.

Thank you.

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#1

Re: Hydrophobic Slides??!

09/14/2008 7:04 PM

There are static detecting devices available on the market that "sense" the charge and can give you a value in apparent volts as to what charge is present. Contact a local painting or electronics specialist. They should be readily able to lend you their hand held unit for a while.

Note however that local experience is that charge does not make a surface hydrophobic.

Try the opposite experiment. Intentionaly charge (as best you can) a slide by rubbing with cloth and see if you can replicate the problem.

You mention that the phenomenon ONLY happens at the bottom of the slide. This again indicates a handling concern or contamination that may be happening during the slide preparation.

What else is "special" about this time of year? Is a key technician on leave? Are you using recycled slides that have not been cleaned properly? Is there a trainee in the department that is following a different process? Has someone changed brand of gloves used and so is contaminating the slides at the bottom where they hold it?

Might also suggest getting a corrupted slide checked (Spectroscopic analysis) for contaminants that are not representative of normal samples. You may have perfectly clean slides being supplied and there may be an intermitant contaminant happening that is seasonal in type.

If you need details of types of static sensing devices, I can find the brand of what we use here and let you know.

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#2
In reply to #1

Re: Hydrophobic Slides??!

09/14/2008 11:18 PM

I would like to know if you think a hand held device will work.

We tried one that is used for ESD monitoring that had an Ion Bond band that went down to the single volt range, and we saw nothing different on well stained slides or these outliers. Typically, the surface IB was non-detectable.

I think I can get a blank slide, so I'll try to over charge one. Our problem was not knowing how they are charged in the first place either. In addition, when we find out about the problem, the slide already has tissue on it, and was not stained properly. The tissue samples are centered in top and bottom halves, to prove the machine can stain a whole slide, but the bottom half sees nothing. We have been through the instrument again to try to see if something wasn't set up properly, but all tests proved to be perfect with respect to specifications. This means, that if the stains are dispensed on the slide, and only the top half accepted the stain, yet the tissue was de-parafinized and prepped properly, that the stain "liquid" didn't get onto the lower half tissue for some reason. I'll look for a cover slip leak, but I've been working on this instrument with our number one technician all last week.

Nobody wants to blame the slides, but if we see this happen and then we change the slide lot, and it goes away. We don't make our own slides, either, they are purchased.

Thanks for any more ideas you may provide, they are jogging the thought circuits!

Don

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#3

Re: Hydrophobic Slides??!

09/15/2008 1:51 AM

I just lost about 200 typed words in a reply, so if that comes through, then it was incomplete. Hopefully I can remember what was in it for this repeat.

I presume that when you say the bottom half of the slide that you mean the lower portion on the same surface and not the opposite surface.

Some additional questions to help me understand what's happening.

How large are the slides?

Is the indicator applied to the whole slide or locally?

If it is applied locally, then are the two nozzles both working? Is there a minor air lock in one line for your indicator?

Is there a previous process that may not be working for the bottom portion and thus not removing fatty residue from that portion of the sample.

Has the reagent from the previous process been properly removed/dried? If it is only the lower portion of the sample, then maybe there's a drop of the rinse/de-fatting reagent still on that portion of the slide and at best it's diluting the indicator.

Is the sample contacting the nozzle for the indicator solution and obstructing/blocking it and thus not getting indicator applied?

Is the previous process applicator nozzle blocked/air locked/....????

Are the slides/samples thicker causing it to block dispensing nozzle?

Is there an air blast or something that is stopping the indicator from reaching the sample?

Is there a contaminant inside the indicaor dispenser? (We once got a spray gun with a small piece of plastic film inside it. Brand new from the supplier.)

Hope I'm not sending you on wild goose chases, just questions from someone who is "outside the box".

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#4
In reply to #3

Re: Hydrophobic Slides??!

09/15/2008 8:22 AM

All good questions. Sorry you encountered difficulty with it, and thanks for trying again, despite the frustration.

Slides are regular sized microscope-type slides.

Indicator is applied to the top (on the front side, nothing going on the back side) half & mixed across whole slide. All reagents were primed before starting run. Both tissue samples were preppred properly (rendering the one on the bottom half of the slide almost invisible after it was finished. All fat was removed.

Subsequent slides were stained fine.

No air blasts interrupt the indicators, but small flow rates used to mix the indicator throughout the liquid puddle on the slide. Indicator dispensers were disproven, as they were changed several times, and someone else used them on an adjacent instrument and got all good staining.

Any other ideas for charge or what else could cause hydrophobicity?

Thanks again.

Don

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#5

Re: Hydrophobic Slides??!

09/15/2008 5:41 PM

Hi Don, I'm an EE so the ideas are coming from distant past exposure in medical instruments and processes. (Have you been able to try the same slide "upside down" through the process, to see whether it takes stain on the second pass.) Why is it only the bottom????

Your observation is that there is no staining.

We use a problem solving tool that suggests the following possible causes.

a) There was no stain applied, so nothing got stained. (Sounds like you've chased that one almost to the end.)

b) There was nothing for the stain to react with, like the sample was completely negative for the test. (Have you re-tried the tissue sample manually and found that stain did take.)

c) Something is contaminating the stain during application. (Like lubricant/oil either on an applicator surface, in the dosing equipment and so on. Or maybe some reside from a previous step in the process.)

d) Something is intervening before the stain can take effect. Like some other material diluting the stain prematurely.

The question that we ask is "What would I have to do to make this problem happen?" or "How could I make this effect?" Dont' think about how the equipment is supposed to work, just look at the slides and try to imagine how you could make that effect happen, then look for that deviation inside the process.

You also have some strong clues since the problem is happening on some slides and not others. Use a spreadsheet and plot EVERY detail of the preparation of EVERY slide and see if there is a pattern as to when the problems happen. (For instance it might be the last slide in each packet and the paper interleaf may have some oiliness on it. It might be that a pack of slides sits on a shelf and contaminant is absorbed through the pack into the bottom portions of some slides. It may be that your "parafin removal" process is the culprit. Imagine a small floating piece of plastic film, any slide that is dipped with that in the way would have the bottom portion of the slide shielded from the reagent/solvent and would then still be full of parafin during later stages.

I'm glad that my ideas seem to be helpful. Problem solving in complex assembly lines is my major focus where I work. Sometimes get sent overseas on very short notice (like 2 hours notice of a 3 week trip to India some years ago.) to help other parts of our company with issues.

The feeling when the final solution is dicsovered is breathtaking. Often though, the challenge is how to prove that the answer you've discovered is the actual root cause.

I'll end this note here and send another soon.

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#6

Re: Hydrophobic Slides??!

09/15/2008 5:46 PM

Yes, it's me again.

When you do discover the suspected cause, there's another step I'd suggest.

Use that knowledge to make the fault happen (EXACTLY as it is on the problem slides) and then correct it and make it not happen. Repeat this a few times to prove to yourself that you have found the cause.

If you can turn it "on" and turn it "off", then you have probably identified the root cause. If they are not EXACTLY as the current failures, then you have found what we would call a "likely" cause and the proof is not definitive.

All too often in problem solving we identify a possible cause, fix that, give ourselves a pat on the back and walk away without confirming that we have solved the actual problem.

Again, good luck and happy hunting.

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#7

Re: Hydrophobic Slides??!

09/15/2008 10:51 PM

One of our probglems is that the slides are not of our making.

If the problem is the odd slide randomly chasing away the aqueous puddle the stain is mixed within. We changed slide lots, and got 2 full successful runs, so the instrument seems sound. We found no leaks or overspray problems that could explain the successful deparafinization, but no staining except the odd lucky splash that landed right on the tissue. The tissue itself is readily stainable.

We keep meticulous records, so we'll have to see if the data can tell us anything. We're trying to get it to happen again now.

Cheers,

Don

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#8
In reply to #7

Re: Hydrophobic Slides??!

09/16/2008 5:58 PM

It's good to hear that you have a way to maintain your process with the new batches of slides.

That would also appear to confirm that the machine/equipment itself is not the root cause.

Some additional ideas came to mind overnight, but I'm running out of suggestions.

Firstly, is it possible that the samples that are not stained have raised edges on the specimen, effectively turning the specimen into a "lilly pad". (If that was the case, then you'd see staining at the perimeter but not the centre.)

Secondly, is it possible that the specimen has been desicated to a point where it's "water repellig?" (Sounds like that's not the case from your last note.)

My final thought is regading the dimensional repeatability of the slides. I'm thinking that if the slides work through some slide/guide mechanism to receive the stain, then if they were either "thick" or "wide" and could not fully travel into the dosing position, then there would only be stain on part of the slide.

The following experiment could then confirm or rule out that cause.

Do you have access to measuring equipment (micrometer or similar) to check the size of the differet lots of slides? Measure EVERY failed slide that you can get your hands on. (Width at "OK" end, width at "not OK" end, thickness at both ends etc.) also measure a sample of around 30 pieces from your "good" slides using the same measuring tool and if possible the same person doing the measurements.

For the good slides, use EXCEL and get mean and standard deviation for each parameter. Then "test" the bad slides against that population predictor. If you want to send me the data, I can do the tests here, but I'm guessing that statistical testing is in your zone of confidence.

Hope I'm helping you get closer to the solution.

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