[Histonet] PAS/Decal Question

[Rene J Buesa]

My impression is that your problem is during the decalcification step. It cannot be hurried and has to be in EDTA at pH 7All reagents have to be prepared in pH7 phosphate buffer.The inconsistency resides in the fact that not all core Bx are the same regarding thickness, tissue condition or size.Besides you are hurrying too much. As yourself (and your pathologists) the following question: what good you take out of your protocol if the "failure" rate is as big as you describe?Change to EDTA and process more time.René 

    On Tuesday, May 3, 2016 1:01 PM, "Marcum, Pamela A via Histonet" <histonet at lists.utsouthwestern.edu> wrote:
 

 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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