[Histonet] decalcification

Joseph Saby saby_joseph_a <@t> yahoo.com
Mon Aug 2 16:49:51 CDT 2010


If your bones are indeed large, 24 hours in 10% NBF will not be sufficient for 
fixation.  If the fixation isn't sufficient, none of the processes following 
that will be as effective as they might be.  Even after trimming, I would 
recommend fixing an additional 24-48 hours for every mm of thickness of your 
bone section, at a minimum.  If you are in a hospital setting and need reults 
quickly, you can use a processor (with heat, pressure and vacuum) to speed up 
the fixation.

I use buffered formic acid to decal.  The final concentration of formic acid is 
about 20-25%, so it is rather aggressive, but it also allows you to read the 
bone marrow after decal.  If you need results in a short time (hospital setting 
again) you can use an aggressive reagent like RDO for decal, but keep in mind 
that nuclear detail will be lacking, but bone structure should still be 
readable.  If you do not have the reagents to determine decal endpoints, you can 
(with training/practice) use a needle to pass through the sections to help 
determine if decal is complete.  The needle should move through the specimen 
smoothly and not get hung up and stiff in the middle of the section.  
Flexibility of the bone section can tell you when you are appoaching the time 
when the needle test will give you useable results.

Please do not read this as an endorsement for RDO or any other super aggressive 
decal solution.  Personally, I always insist on being able to read all tissue 
elements, and these solutions, although fast, give less than optimal staining 

If you have further questions, please ask me off line.

Joe Saby, BA HT

From: "Webb, Dorothy L" <Dorothy.L.Webb <@t> HealthPartners.Com>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Sent: Mon, August 2, 2010 10:14:35 AM
Subject: [Histonet] decalcification

I would appreciate any feedback on what all are using in your decalcification 
process.  We get a lot of large bones in and the past 2-3 months have noticed a 
huge problem in our microtomy process with these samples.  We have been grossing 
the bones in and leaving the sample in the cassette in 10% formalin for 24 hours 
befoere placing in decal for up to 8 hours and still having the inner portion of 
the sample look underprocessed and crunchy!  Any suggestions would be 

Dorothy Webb, HT
Regions Hospital Technical Supervisor

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