[Histonet] PAS/Decal Question

[Gudrun Lang]

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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