Re: [Histonet] Cytology (Special stains & IHC)

[Beth Cox via Histonet]

Hi Karen,

The best resource for Cyto questions would probably be a recent edition 
of the Carson Histotechnology book.  The Fourth edition has some info on 
special stains for Cyto, but the new Fifth edition is expanded on that 
topic and has more info on IHCs for Cyto (Chapter 17).


For _special stains_, you would definitely want them fixed in 95% 
alcohol.  The only exception to that would be fat stains which would of 
course require air-dried slides.  It would likely be advantageous to use 
charged slides, but that is not required. Remember when special staining 
Cyto slides fixed in 95% alcohol, that the deparaffinization steps at 
the beginning of the procedure must be skipped.  You are normally okay 
with using regular FFPE controls with your Cytospins or ThinPrep special 
stains.


Doing _IHCs_ on Cytology preps (Cytospin or ThinPrep) gets a little 
trickier. _IF_ you are going to do them, you would want them to be 95% 
alcohol fixed and on charged slides.  However, you would be required to 
do a validation for Cytology for any/all antibodies you were going to 
run.  (Your CAP or CLIA inspector will eat you alive if you don't!).  
Alcohol fixed specimens will require a new optimization since the 
pretreatments will be different than those for FFPE.   Many antibodies, 
particularly nuclear ones, may be difficult or impossible to stain on 
the intact cells in a Cyto prep.


Also, you would not be able to use FFPE controls for Cyto IHC since they 
are 'fixed and processed differently" than the Cyto slide and therefor 
don't properly control the process.  AND, alcohol fixed smears begin to 
lose their antigenicity in about 24 hours, so they must be stained the 
same day the slides are made, so having a control bank of alcohol fixed 
slides isn't feasible.


Doing IHC on Cyto smears using FFPE protocols and FFPE controls is very 
likely to give you false negative results.  Unless you are doing large 
volume Cyto, it is very strongly recommended that you do your IHCs for 
Cyto on the FFPE cell block.


Beth Cox, HTL/SCT(ASCP)QIHC

--

Message: 5
Date: Wed, 5 Aug 2020 15:34:28 +
From: "Heckford, Karen - SMMC-SF"
To:"histonet@lists.utsouthwestern.edu"

Subject: [Histonet] Cytology
Message-ID:<903be99abb344593a73079761d762...@phx-exch-013.chw.edu>
Content-Type: text/plain; charset="us-ascii"

Good Morning,
I rarely do Special stains or IHC's on Cytology cytospins or thin prep type 
slides.Should these slides be charged, air dried or fixed in 95% alcohol 
before doing Special stains or IHC's on them?

Is there a good book or some sort of publication on cytology procedures 
regarding the above mentioned.   I have tried and look it up and it seems all 
over the place on what to do.

Any help would be greatly appreciated,

Karen Heckford HT ASCP CE
Lead Histology Technician
St. Mary's Medical Center
450 Stanyan St.
San Francisco, Ca. 94117
415-668-1000 ext. 6167
karen.heckf...@dignityhealth.org

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Re: [Histonet] Cytology

[Joe Myers via Histonet]


Ms. Heckford:
As a cytotechnologist with just a little more than average experience with 
immunostaining procedures, I’d like to offer my input.   To my knowledge, there 
are very few publications that address your concerns.  I have, however, 
collected/prepared a significant amount of information on this topic, and I’m 
happy to share it with you.  To address your specific questions: 1) When 
performing immunocytochemical (ICC) staining procedures, it’s always a good 
idea to use charged slides, since that will help with cellular retention; 2) 
specimen material should always be fixed, preferably in 95% ethanol (i.e. 
air-drying should be avoided); and 3) if you plan on routinely performing ICC, 
you’ll need to fully validate such procedures, particularly since antibodies 
that are characterized for IHC will not work in the same manner as they would 
in ICC.  If you’d like, I’d be happy to share with you a PowerPoint 
presentation that I’ve given on several occasions relating to this topic.
Cheers,
Joe Myers, M.S., CT/QIHC(ASCP)

- - - - - - - 


Date: Wed, 5 Aug 2020 15:34:28 +
From: "Heckford, Karen - SMMC-SF" 
To: "histonet@lists.utsouthwestern.edu"
   
Subject: [Histonet] Cytology

Good Morning,
I rarely do Special stains or IHC's on Cytology cytospins or thin prep type 
slides.Should these slides be charged, air dried or fixed in 95% alcohol 
before doing Special stains or IHC's on them?

Is there a good book or some sort of publication on cytology procedures 
regarding the above mentioned.   I have tried and look it up and it seems all 
over the place on what to do.

Any help would be greatly appreciated,

Karen Heckford HT ASCP CE
Lead Histology Technician
St. Mary's Medical Center
450 Stanyan St.
San Francisco, Ca. 94117
415-668-1000 ext. 6167
karen.heckf...@dignityhealth.org

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[Histonet] Protocol for cutting frozen sections for RNA Sequencing

[Kristyn Ferber via Histonet]

Hello Histonet!

Can anyone share their protocol for cutting frozen section (rolls) that will be 
used for RNA sequencing downstream much like this nature article: 
https://www.nature.com/articles/s41591-020-0844-1

The only direction we have been given is to cut the sections at 100 microns. 
Knowing RNA sequencing can be finicky, we want to ensure they have the best 
yield and we have a protocol that enables that.

Best regards,

Kristyn

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[Histonet] slide scanning and glass vs plastic RE: Histonet Digest, Vol 201,

[Morken, Timothy via Histonet]

Steve, I agree with your whole thing. And our pathologist fought against 
plastic for years simply due to the fact it scratches easily and we are 
constantly pulling and filing slides for various needs. However, when we 
decided to go to scanning slides wet mounting media was never going to work - 
takes too long to dry and any gloppiness on the slide gums up the scanner. And 
no one is going to wait a day or more for their slides to be scanned when the 
purpose is to do remote diagnostics in real time. 

We started with frozens several years ago and worked out a lot of details 
(hand- applied). Then started scanning control slides in histology a couple 
years ago to allow remote sign off of those. This year we started slowly moving 
various types of specimens to plastic and scanning. Then when Covid hit and 
everyone had to work remotely we went in for scanning everything. Luckily we 
had enough scanning capacity to do that. 

So far it has worked well. For diagnostics the scanned slide is fine 99 or more 
percent of the time. Occasionally we get problem scans and a slide has to be 
viewed manually. Actually the biggest problem has been network bandwidth to 
review images from home - often too slow. 

Currently we are still sending the slides to the pathologists office, but most 
of the time they are not looked at and end up going to storage. We expect to 
move to simply scanning and storing and may never touch the slide again - the 
images can be used for all teaching, conferences, etc. So the scratch problem 
is solved in that way. 

Glass will probably always be better than plastic for photography, but the vast 
majority of diagnostic slides in practice are never photographed. We'll see 
within the year if various pathologists want recut with glass coverslipping 
expressly for photography for articles and books. 

Tim Morken
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center

-Original Message-
From: Steve McClain via Histonet  
Sent: Wednesday, August 05, 2020 8:41 PM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Histonet Digest, Vol 201, Issue 3 Update info. on old 
topic?

Tim, Paula and Jeanine
I have little experience w slide scanning, yet I have captured nearly 1.8M 
slide images, mostly on film coverslipped slides. One year ago we switched back 
to glass.
1) I suspect what Tim describes may be due to the fact that the refractive 
index of film differs from glass.
We used film for nearly 20 years and for 4  years I could never get a sharp 
high power image. This was most noticeable when using 40x lens designed for 
glass cover slips.
About 16 years ago, we found an Olympus 40x lens w a correction collar allowing 
for adjustment/ focusing suitable for coverslips of different thickness but 
useful also with either film or glass. The lens was expensive ($3600 if memory 
serves me).

See 
https://urldefense.proofpoint.com/v2/url?u=https-3A__www.edmundoptics.com_p_olympus-2Duplxapo-2D40x-2Dobjective-2Dwith-2Dcorrection-2Dcollar_43026_=DwIF-g=iORugZls2LlYyCAZRB3XLg=7cy9qXFa73jDX2Iixpjkq1XlWAfHgLLHm33agI_sCKA=IZq7sglnD7BZh6tQuwuXoW8Xdke6Vv0QQ-hNSDBkFzk=B_LEPrG8dgAhhmRiptOVT5tukWNA5pNLY1iJ7AYW5do=
 

Perhaps the scanner manufacturer has a similar solution?

2) we were especially diligent (mono-maniacal) about using fresh reagents to 
keep water out of the last clearing reagent step-film has a nasty reputation 
for delaminating over time and pulling the sections off the glass. Several 
major academic centers have years and hundreds of thousands of slides now 
completely useless due to delamination.

3) film has problems with certain specimens routinely (bone and toenails and 
the thick cornified layer on volar skin) not cover slipping well leaving 
bubbles and 'cornflakes' or brown spots over the tissue. Cornflake artifact 
also occurs w water in the last reagent and may be seen on obscenely humid days.

4) film scratches readily when you stack slides or file them too tightly. Film 
Slides can be filed immediately after exam.  Glass generally needs to wait 2 
days before filing or one can glue up a brick of slides by filing too soon.

5) Film is faster and dries faster but glass is sharper and produces better 
images for my purposes (we switched back to glass 1 year ago- and the Leica 
model works perfectly well)

6) our old SCA film coverlipper generated more fumes than the new Leica. 
However all new instruments produce less fumes than older models.

7) glass coverslips are better stored in a desiccator/dry environment, before 
use.

8) This will sound stupid, yet not all coverslips of clean.- most (3/4) cheap 
coverslip glass is dirty and cannot be used in our lab where most/every slide 
is imaged. To determine this pick up an entire stack of coverslips and look 
through them. If they look clear-good to go; if they appear cloudy, find a new 
supplier.

9) glass coverslippers are finickyabout viscosity and volume 

Re: [Histonet] Film coverslip vs glass coverslip

[Joe W. Walker, Jr. via Histonet]

I respect your view point but as someone who has trained histotechs and 
cytotechs, the film leaves residual dots from dotting pens making it a 
challenge to test individual's ability to locate and identify tissues and cells 
of interest.  That is the only draw back that I have seen in my years with 
using both film and glass.

Joe W. Walker, Jr. MS, SCT(ASCP)
Anatomical Pathology and Interim Phlebotomy Manager
Rutland Regional Medical Center
160 Allen Street, Rutland, VT 05701
P 802.747.1790  F 802.747.6525
joewal...@rrmc.org, www.rrmc.org

-Original Message-
From: Anne van Binsbergen via Histonet 
Sent: Wednesday, August 5, 2020 2:58 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Film coverslip vs glass coverslip

[External Email] This email originated from outside of the organization. Think 
before you click: Don’t click on links, open attachments or respond to requests 
for sensitive information if the email looks suspicious or you don’t recognize 
the sender.


Hi all
I have used Sakura film coverslippers in several different facilities since 
1997.
In the ‘early days’ there were some issues with slip detachment but those were 
smartly sorted out by the manufacturers.

1) Film coverslips dry really fast - no clumps of stuck together slides to soak 
for sometimes days to separate.
2) Film is easy to remove - 3 mins in Acetone and a few dips in ethanol and 
xylene and you are good to go.
3) Drop a slide with a glass slip and it shatters. Drop a slide with a film 
slip and the glass breaks but the film slip holds it all together preserving 
the section.
4) Eager pathologists in a hurry to view the section, cannot ‘smear’ the film 
slip off the slide.
5) No pesky glass shards to deal with.

Over 40 years in the saddle, in my opinion it’s no contest. Film is the best.

Annie in Africa
(aka annieinarabia)
Recently retired Artist and Artisan

Sent from my iPhone
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Re: [Histonet] process formalin-fixed tissues from animals infected with a virus

[Greg Dobbin via Histonet]

Very interesting paper John! Thank you. I wish the authors had also
experimented with higher concentrations of formaldehyde (eg 10% formalin).
Might one infer that 10% would be even more efficient in inactivating viral
infectivity than 2 and 4%? 樂
Cheers
Greg

On Thu, Aug 6, 2020 at 11:02 AM John Garratt  wrote:

> Evaluation of Virus Inactivation by Formaldehyde to Enhance Biosafety of
> Diagnostic Electron Microscopy
>
>
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4353909/
>
>
> It is nice to have a reference.
>
>
>
> John
>
> On Thu, Aug 6, 2020 at 4:10 AM, Greg Dobbin via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
> Hi Amy,
> Formalin fixed tissue is no longer infectious...unless you are talking
> about prions (eg scrapie, BSE, etc). So there should otherwise be no
> concerns or additional precautions required.
> Cheers,
> Greg
>
> --
> *Greg Dobbin*
> 1205 Pleasant Grove Rd
> 
> RR#2 York,
> PE C0A 1P0
>
>
> *Everything in moderation...even moderation itself**!*
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
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>
>
> --
*Greg Dobbin*
1205 Pleasant Grove Rd
RR#2 York,
PE  C0A 1P0


*Everything in moderation...even moderation itself**!*
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Re: [Histonet] process formalin-fixed tissues from animals infected with a virus

[John Garratt via Histonet]

Evaluation of Virus Inactivation by Formaldehyde to Enhance Biosafety of 
Diagnostic Electron Microscopy

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4353909/

It is nice to have a reference.

John

On Thu, Aug 6, 2020 at 4:10 AM, Greg Dobbin via Histonet 
 wrote:

> Hi Amy,
> Formalin fixed tissue is no longer infectious...unless you are talking
> about prions (eg scrapie, BSE, etc). So there should otherwise be no
> concerns or additional precautions required.
> Cheers,
> Greg
>
> --
> *Greg Dobbin*
> 1205 Pleasant Grove Rd
> RR#2 York,
> PE C0A 1P0
>
> *Everything in moderation...even moderation itself**!*
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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Re: [Histonet] process formalin-fixed tissues from animals infected with a virus

[Greg Dobbin via Histonet]

Hi Amy,
Formalin fixed tissue is no longer infectious...unless you are talking
about prions (eg scrapie, BSE, etc). So there should otherwise be no
concerns or additional precautions required.
Cheers,
Greg

-- 
*Greg Dobbin*
1205 Pleasant Grove Rd
RR#2 York,
PE  C0A 1P0


*Everything in moderation...even moderation itself**!*
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Re: [Histonet] Update info. on old topic?

[MARR, Penelope (EAST SUSSEX HEALTHCARE NHS TRUST) via Histonet]

Hi Jeanine,
- Film coverslips scratch very easily.  Any dust in the system will scratch the 
film.
- Staining is often compromised if the film coverslip needs to be removed.
- Specific storage conditions are required to validate the adhesive on the 
film.  If the storage conditions are not met the longevity of the tape is not 
guaranteed.

- Glass is better for photography
- Glass does not scratch as easily
- Less fussy with storage requirements
- Removal does not compromise staining quality.

I hope this helps.

Penny Marr
Senior BMS

C/- Histology
Conquest Hospital
St Leonards-on-Sea TN37 7RD

penny.m...@nhs.net

(01424) 758023 ext 734914
0300 133 4914


-Original Message-
From: Sanders, Jeanine (CDC/DDID/NCEZID/DHCPP) [mailto:j...@cdc.gov]
Sent: 05 August 2020 13:40
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Update info. on old topic?

Good morning!

I need to provide some information to my team about film vs. glass 
coverslipping. I have a lot of positive info. regarding film but are any cons? 
And I need to come up wit the pros of glass over film. Anyone with extended 
experience have some current information for me?

Thanks very much,

Jeanine Sanders, BS, HT(ASCP), QIHCCM(ASCP)
Centers for Diseases Control and Prevention
1600 Clifton Road NE
MS H18-SB
Bldg. 18, Rm SB-114
Atlanta, GA 30329
404-639-3590







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