[Histonet] RE: Histonet Digest, Vol 96, Issue 24 reprocessing

Steve McClain SteveM <@t> mcclainlab.com
Thu Nov 17 05:17:38 CST 2011

This sounds like a complicated problem- Generally fixed tissues left in the processor overnight can just be re-hydrated in formalin for a few hours and then run (we've made that mistake)
Exceptions abound in histology methods, but Unless the tissue was well-fixed to begin with, Re-processing generally stinks.
Given that this is research and a one of a kind, it may still be worth trying.
I would not expect Immunostains and other high-end work  to be reliable even if you do get decent sections.

First INVESTIGATE and determine what likely happened- Study the slides and see what is wrong -are they sunken and shriveled as if paraffin did not infiltrate?
Second Study the cut surface of the blocks with a 10x lens or dissecting scope and determine if there was a processing failure also, any reason to suspect reagents needed changing?
Your tissues sound maybe under-fixed (shrinkage described), probably dehydrated and then fixed by coagulation in the alcohols on the processor.
Is it possible that the cleaning cycle was run before you started processing the next day?
Third try Luna's method given by Shelly and do it manually and at room temp
Fourth buy lunch for the investigator and meet to apologize and work out the details of fixation and any other issues discovered from your 'NTSB Accident Investigation'.
Who knows, it may work.
Fifth, assign one person to supervise (be in charge of) processing and have them keep statistics ( I have a large  graph outside my door with 1 months data on each processor, graphing # of blocks for each run, when reagents were replenished/changed, noting every failure)- even in a small lab like ours, once we got to 6 processors running 1-4 times a day, someone needs to be in charge and held responsible.
Six, hang a sign on the exit door (like the one on my back door) "DID YOU START THE TISSUE PROCESSOR?"
Some may think that is over-reacting to a one-time error; when it happens a second time you earned and deserve the reputation.  
Be thankful it did not harm any patients- in the accident world that is known as a near-miss.
Good luck

Steve A. McClain, MD
631 361 4000
Message: 9
Date: Wed, 16 Nov 2011 15:05:38 +0000
From: Shelly Christenson <christen <@t> vet.k-state.edu>
Subject: RE: [Histonet] is there a way to recover ruined tissues
To: Patsy Ruegg <pruegg <@t> ihctech.net>, 'histo net'
	<histonet <@t> lists.utsouthwestern.edu>
	<06E342B6098ED9478347E1407764C80404B13411 <@t> VETMXHT.ads.vet.k-state.edu>
Content-Type: text/plain; charset="us-ascii"

I have used this reprocessing schedule from Lee Luna's book "Histopathologic Methods and Color Atlas of Specials Stains and Tissue Artifact". This procedure has worked for me on a couple of occasions, You may need to change the times a little since your tissue samples are so small. Luna's book has a lot of information that is very helpful, and would be a good reference book to have in the lab. Hope this helps you out, give it a try.

Tissue Softening Solution
The following solution is used for softening dried tissue specimens prior to reprocessing or processing

      Formol-Sodium Acetate (Stock) Solution
Concentrated formalin (37%) solution    10 ml
Sodium acetate                                             2 gm
Tap water                                                     90 ml

     Formol-Glycerol (Working) Solution
Formalin-sodium acetate (stock)              90 ml
Glycerol (glycerin)                                      10 ml
       Reprocessing Schedule
Melt the paraffin blocks down
Place the tissue in 3 changes of xylene for 1 hour each
Place in 100% alcohol, 2 changes for 1 hour each
Place in 95% alcohol, 2 changes for 1 hour each
Place under running tap water for 20 minutes
Place in formol-glycerol working solution until tissue becomes soft (In most instances this will occur within 5-8 hours: extended exposure to the formol-gycerol will not harm tissue specimens)
Place the tissue in the process and proceed with routine Processing schedule. How ever it is not necessary for the tissue to go through the fixative stations on the processor

Best of Luck,

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