If your controls always work, at least you can be sure that your Perl's reagents are in good condition.
Your method differs from what I used in the following:
1- the aspirate and the core were placed immediately into NBF that was pH corrected to 7.0 exactly. When received in the laboratory the core Bx was placed in EDTA to decalcify. You decalcify first and that is not recommendable because fixation should precede decalcification, perhaps here you have a source of inconsistency depending on the characteristics of the core Bx.
2- the smears were allowed to air dry and fixed with methanol and stained afterward.
3- processing was similar to what you describe (overnight) to be sectioned early morning next day to stain, etc.
Decalcifying before fixing could be a cause of problem.
--- On Mon, 11/24/08, Tom McNemar wrote:
From: Tom McNemar
Subject: [Histonet] Iron stains
Date: Monday, November 24, 2008, 12:06 PM
We may be having a problem with our iron stains. I say that we 'may' be
having a problem because our positive control always works.
For bone marrows, we stain the core, aspirate, a smear, and a purchased
positive control. The stain is all over the place. We may have a loaded smear
with a negative core and aspirate. We may have a positive aspirate with a
negative core and smear. We may have all negatives even though clinically,
there should be iron. Our control always works. The pathologists do not trust
the stain and really do not believe the results. I have stood by the stain due
to the positive control but I find it increasingly difficult.
I would appreciate any thoughts. Our procedures are outlined below...
The aspirate is allowed to clot before placing it into 10%NBF.
The core is placed briefly into DecalStat (until it floats) then placed into
Both are then processed with our regular tissues. The spend anywhere from 4
to 10 hours in formalin.
The following day, they are cut and along with one of the purchased controls,
run down to water on the stainer. (8 mins onboard oven, americlear, alcohols,
The smear is placed into 95% alcohol then rinsed in distilled.
All slides are then stained using Perl's Method for iron pigment.
We have already explored the way the specimens are collected during the BM
procedure, and decal solution. All reagents are good. I'm wondering about
the formalin fixation. I know that threre are other probably better fixatives.
Does anybody use another fixative prior to formalin? For those using formalin
only, do you limit the fixation time in any way?
I really would appreciate any comments. Such a common and simple stain. We
should be able to trust it.
Tom McNemar, HT(ASCP)
Licking Memorial Health Systems
Histonet mailing list
Histonet mailing list