[Histonet] paraffin sectioning-dry tissue? - long response - image analysis related

Elizabeth Chlipala liz <@t> premierlab.com
Thu Feb 5 11:48:54 CST 2015


You are not overthinking this at all if you are utilizing your sections for any image analysis applications.  You need to be able to standardize as much of the histology process as possible.  There are so many other parameters that can cause section thickness to fluctuate.  Such as the sharpness of your knife, how fast you turn the hand wheel.  Blowing on the block is not acceptable in our lab that will create thicker sections since you are warming up the block.   Great care is taken to standardize what we do, from soaking blocks to how we collect the sections to placement on the slide, and how often we move our knife blade.  We routinely soak all of our blocks but we keep in mind so many other factors when we are providing histology for image analysis.  It starts at the beginning with fixation, it all has to be standardized, our goal is to decrease the potential for variability.  That is on our minds at every step through the histology process.

The other thing to consider is how well the algorithm functions - you need to determine the limits of the algorithm and when it will stop functioning properly, which is usually due to staining issues (over or under staining or inconsistent staining), section thickness and overall section and stain quality which is so important.  As a part of algorithm validation we test for these parameters we want to understand where we lose functionality, accuracy and precision of the algorithm.  So we look at different section thicknesses and how that impacts analysis, we look at over and under staining parameters to see how that affects the algorithm, etc.  

Just my two cents.


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz <@t> premierlab.com

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lucie Guernsey
Sent: Thursday, February 05, 2015 10:22 AM
To: James Watson
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] paraffin sectioning-dry tissue?

By adding water, ice, or warm humidity (through exhalations) to the mix, though, doesn't the block contract/expand? Wouldn't that change the ultimate thickness of the section? I've always wondered how much it affects things.

Could your sections be 1 um (or multiple um!) thicker/thinner that you expect? What happens if individual blocks contract/expand differently (due to the amount of time left soaking, how far into the block you've cut since you last soaked, etc.)? I feel like you couldn't properly compare quantifications across a study if the thickness of your tissue is an unknown variable.

Am I overthinking this?

Lucie Guernsey
UC San Diego
(858) 822-5797
lguernsey <@t> ucsd.edu

On Thu, Feb 5, 2015 at 7:35 AM, James Watson <JWatson <@t> gnf.org> wrote:

> We use a 5% glycerin in denatured alcohol for our 100% alcohol on our 
> tissue processor for routine animal tissues, this reduces the over 
> dehydration of the animal tissue and greatly reduces the time required 
> to soak the blocks.  Warning, if processing fat or cell pellets do not 
> use this, we switch the containers to straight 100% reagent alcohol for them.
> For fat we have a longer processing schedule and for cell pellets we 
> have a short processing cycle.
> James Watson HT  ASCP
> GNF  Genomics Institute of the Novartis Research Foundation Scientific 
> Technical Leader II, Histology
> Tel    858-332-4647
> Fax   858-812-1915
> jwatson <@t> gnf.org
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:
> histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Caroline 
> Miller
> Sent: Thursday, February 05, 2015 6:41 AM
> To: Geoff
> Cc: histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] paraffin sectioning-dry tissue?
> Yes, exactly what Mike and Geoff said. All mouse tissue, especially 
> liver, can be really dry and needs a 'soak'. I have left them for an 
> hour before now but don't leave it for longer than 4 hours though 
> because it can start to swell and de-process!
> You will still only get a few non-chattery sections so be gentle. 
> Thinner sections also help too (3-4.5). Plus low um polishing after 
> you trim
> Good luck! It is weird at first but you will get used to it!
> Caroline
> Sent from my iPhone
> > On Feb 5, 2015, at 6:29 AM, Geoff <mcauliff <@t> rwjms.rutgers.edu> wrote:
> >
> > This is common with mouse and rat tissues, they get "over-dried" 
> > with a
> typical processing schedule.
> > Soaking the face of the block with a kimwipe wet with ice water for 
> > 60
> -120 seconds will enable you to cut 10 nice sections, maybe more.
> >
> > Geoff
> >
> >> On 2/5/2015 6:23 AM, Emily Brown wrote:
> >> Hello all!
> >>
> >> I just started sectioning mouse liver in paraffin and the tissue is 
> >> very dry.  I know it's not supposed to have water due to the 
> >> processing, but the weird thing is that one tech's solution is to 
> >> put a wet kimwipe on the block for a while.
> >> It seems to me that there is a larger processing issue if this is 
> >> happening, am I correct? And why add water when you've already 
> >> dehydrated it?
> >> Unfortunately, we do not have the set up to embed them ourselves, 
> >> so we have to send them to a histology lab.  They were sectioning 
> >> for us, but they are backlogged, so my boss wants me to do it.
> >> Therefore, I can't tell you how they were processed, but I think 
> >> usually the histology lab manages to get good sections.
> >> Is putting a wet kimwipe (using distilled water) the best way to 
> >> get rid of chatter that's only in the tissue? The surrounding 
> >> paraffin sections excellent.
> >> This may have been answered already, but a very quick google search 
> >> didn't help.  My googlefu is probably erratic as it's still early.
> >>
> >> Emily
> >>
> >>
> >> "By bitching and bitching and bitching, they could exhaust the 
> >> drama of their own horror stories. Grow bored. Only then could they 
> >> accept a new story for their lives. Move forward."
> >>
> >> -Chuck Palahniuk, "Haunted"
> >> _______________________________________________
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> >
> >
> > --
> > --
> > **********************************************
> > Geoff McAuliffe, Ph.D.
> > Neuroscience and Cell Biology
> > Robert Wood Johnson Medical School
> > 675 Hoes Lane, Piscataway, NJ 08854
> > voice: (732) 235-4583; fax: -4029
> > mcauliff <@t> rwjms.rutgers.edu
> > **********************************************
> >
> >
> >
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