[Histonet] PAS/Decal Question

[Marcum, Pamela A]

We have no say in the decal procedure and I have been fighting this for 6 years.  It is determined by our HemePath docs and when they want bone marrows completed for clinics the next day.  We have a 24 hour turnaround time, which is ridiculous however; we cannot not change it.  

The feedback we get now is the PAS stain on the bone marrows Levels 2 and 4 are not staining evenly.  They show a smudginess in some cells on the lower or level 4 section/ or they look over processed/cooked.  WE have done manual, instrument and now Dako Artisan (Dako has bend over backward to help) and nothing changes what they see on the slides.  They will not even consider changing the early steps as I have been told decal is fine (no its not) and processing is fine.  Funny thing H&Es are always fine and only the PAS is a problem.  Just tired of it.  

This has been a losing fight for almost 5 months and I reached out to all of you in hopes it would help us make some changes.  Thank you for your suggestions unfortunately no one wants to hear about changes if they mean it is not a 24 hour turnaround time.  I am almost ready to just say "garbage in garbage out".  I feel very bad for the patients at this point.  I have not given up just trying to find a way to get change and better results.  

Thank You All for Your Suggestions!! 

Pam 

-----Original Message-----
From: Gudrun Lang [mailto:gu.lang at gmx.at] 
Sent: Thursday, May 05, 2016 2:19 AM
To: Marcum, Pamela A <PAMarcum at uams.edu>
Cc: histonet at lists.utsouthwestern.edu
Subject: AW: [Histonet] PAS/Decal Question

Hi Pam,
my personal opinion is, that 2 hours fixation is too short for sufficient tissue-protection before acid decalcification. Formic acid at 50°C must have an impact on glycoproteins. Wether it is a kind of solving the "sugars" or beginning oxidation of the OH-groups (like periodic acid does in the PAS-reaction).

In our lab we do also acid decal with formic acid for at least 6 hours at RT, after one day in NBF. Our processing protocol is the routine-protocol over night. How thick are your BMT, also 3-4 mm?
In my opinion 4 hours are a challenge. Are the other stainings of the BMT optimal or show sometimes similar outcome? "Smudginess" reminds me of insufficient infiltration.

I also see that our PAS is not as bright as in the other specimens without decal. Sometimes it gives more the impression of a diastase-PAS. 

Gudrun

-----Ursprüngliche Nachricht-----
Von: Marcum, Pamela A via Histonet
[mailto:histonet at lists.utsouthwestern.edu]
Gesendet: Dienstag, 03. Mai 2016 18:39
An: histonet at lists.utsouthwestern.edu
Betreff: [Histonet] PAS/Decal Question

We are still having issues with our PAS stain on decaled bone marrows.  The Pathologists in HemePath are seeing what they refer to as smudginess in cells on some areas of the completed PAS slides.  We have looked at everything and cannot find where the issue is coming from at this point.  We have done manual staining for PAS, automated on the Leica stainer and on the Dako Artisan.  All methods show the same result for some slides.  We can go for several days to a week or more with no problem and then suddenly it is back and we have changed nothing in the way we do the processing, embedding, sectioning, deparaffinization and coverslipping.  We do as many as 38 bone marrow cores a night or as few as 8 and can find no correlation in the number we have to deal with for a given period.  All bone marrows drawn today must be completed by 8AM tomorrow morning.

Fixation after pulling the bone marrows is a minimum of 2 hours in AZF with a maximum of 7 hours +/-.

Grossed and placed in cassettes for 15 minute rinse in running DI Water

Decal currently in the Milestone Decal Unit for 45 minutes in Immunocal at 50C

Rinsed in running DI water for 15 minutes

Placed in 10% NBF and processed on a 4 hour program with a delay of 4 hours minimum to come off at 4:45AM.

If anyone knows of any literature on decal effects on PAS staining in bone marrows please contact me.  This has been going on for months and no matter what we do manual staining, Leica adaptation for automated or Dako it is not helping.  Dako has been great with sending in technical experts repeatedly and we cannot get this corrected.

Thanks,
Pam

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