Re: [Histonet] Histonet Digest, Vol 208, Issue 14 Best Slides for Leica IP S Slide Printers

[Steve McClain via Histonet]

Be careful.
 Look at the slides before you commit.
The least expensive may also be the dirtiest. So stack up 12 slides and look 
through them. If they look cloudy or dirty.  Nix

Do the drop test. A slide should be able to survive being dropped to the floor 
from waist high.

Choose on price after those two tests.
Just saying,

Steve
Steve A. McClain, MD
631-361-4000  Cell 631-926-3655

On Mar 27, 2021, at 13:11, histonet-requ...@lists.utsouthwestern.edu wrote:

Best Slides for Leica IP S Slide Printers
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Re: [Histonet] Histonet Digest, Vol 207, Issue 3 Bouin's vs Vinegar

[Steve McClain via Histonet]

If the desire is to fix tissue marking dyes on tissue, 5 % acetic acid 
(vinegar) works well and does not stain or alter/fix the tissue like Bouin's 
fixative.

Thanks,
Steve
Steve A. McClain, MD
631 361-4000 cell 631 926-3655


-Original Message-
From: histonet-requ...@lists.utsouthwestern.edu 
[mailto:histonet-requ...@lists.utsouthwestern.edu] 
Sent: Friday, February 5, 2021 1:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 207, Issue 3

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Today's Topics:

   1. Re: Histonet Digest, Vol 207, Issue 2 (Luigi Mascio)


--

Message: 1
Date: Thu, 4 Feb 2021 22:36:26 +
From: Luigi Mascio 
To: "histonet@lists.utsouthwestern.edu"

Subject: Re: [Histonet] Histonet Digest, Vol 207, Issue 2
Message-ID:



Content-Type: text/plain; charset="iso-8859-1"

Buy Bouin's from a consumable company and pour it in your own spray bottle!

Luigi M. Mascio
BS HT (ASCP)
Technical Director
lu...@medequipsource.com
724-687-7342 direct number
724-369-1604 ext. 102
412-216-0496  cell


For a great resource visit medequipsource.com, especially our blog with 
histology tips of the week.

MES?Premium Services:
. New Histology and Pathology Products
. High Quality Remanufactured Equipment
. Signature Preventative Maintenance Programs
. Lab Design & Workflow Layout
. Remanufacture Existing Equipment
. Innovative Financing Programs
. Service Contracts (*Limited Area)
. We carry all major OEM brands
. 
-Original Message-
From: histonet-requ...@lists.utsouthwestern.edu 
 
Sent: Thursday, February 4, 2021 1:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 207, Issue 2

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Re: [Histonet] Histonet Digest, Vol 206, Issue 1 New Tissue Processor? (Sandra Cheasty)

[Steve McClain via Histonet]

While (conceptually) looking forward to the virtues of new and improved modern 
tissue processors w WiFi and SMS messaging capability, I fully expect to be 
operating the 1979 K-series for years to come.

I reckon we may be behind the times, but with regular annual maintenance, our  
VIP5 and even more reliable K-series VIP processors refuse to die. These 
run/process 6 days a week and twice a day, if/when rapid processing is needed.

PS we also have two spare operational (but slightly hacked into) K-series 
processors from when we were contemplating an experiment in cold processing 
using refrigerated alcohols and xylenes to preserve some obscure research 
antigens. In hindsight, it was a goofy academic exercise and soon was abandoned 
and rarely mentioned, after we (meaning Joel) adjusted the antigen retrieval.

Now seriously, has anyone else ever modified their  tissue processor with a 
Saws-all? Please reply confidentially.

Steve
Steve A. McClain, MD
631-361-4000  Cell 631-926-3655

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Re: [Histonet] Histonet Digest, Vol 201, Issue 3 Update info. on old topic?

[Steve McClain via Histonet]

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://www.edmundoptics.com/p/olympus-uplxapo-40x-objective-with-correction-collar/43026/

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 of the mounting 
media. Once dialed in, we didn’t change media and use one brand and only one.

10) Not all film coverslip rolls are satisfactory either. We had the best luck 
w Brand S film and did not switch.

Hope these observations help.
Steve
Steve A. McClain, MD
631-361-4000  Cell 631-926-3655

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?


Message: 3
Date: Wed, 5 Aug 2020 13:00:37
I am interested in this too, with my main question being the quality of digital 
images from film coverslipped slides.
Paula Keene Pierce, BS, HTL
--

Message: 6
Date: Wed, 5 Aug 2020 15:38:14 +
From: "Morken, Timothy" 
To: "Sanders, Jeanine (CDC/DDID/NCEZID/DHCPP)" 
Cc: Histonet 
Subject: Re: [Histonet] Update info. on old topic?
Message-ID:
   


Content-Type: text/plain; charset="us-ascii"

Hi Jeanine!

We are using film coverslipping exclusively now and scanning all slides. We 
moved to film because it dries almost instantly and can be scanned right away. 
It has worked very well. The only issues, and not specific to film 
coverslipping,  are that some slides with low contrast sometimes are out of 
focus. That is primarily IHC slides with low contrast counter stain and low 
contrast specific staining.

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

-Original Message-
From: Sanders, Jeanine (CDC/DDID/NCEZID/DHCPP) via Histonet 

Sent: Wednesday, August 05, 2020 5:40 AM
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,
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Re: [Histonet] Histonet Digest, Vol 197, Issue 23 recovering RNA from FFPE

[Steve McClain via Histonet]

1) Does anyone know of a method of recovering RNA from FFPE?
2) Does anyone know a lab that has done it?

Thanks,
Steve A. McClain, MD
McClain Labs
45 Manor Road Smithtown, NY 11787
631 361-4000 cell 631 926-3655

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Re: [Histonet] Histonet Digest, Vol 197, New Topic Coronavirus testing from tissue sample

[Steve McClain via Histonet]

The CDC website with specimen submission form does not seem to be working.
https://www.cdc.gov/laboratory/specimen-submission/pdf/form-50-34.pdf

Does anyone know who can else can test for Corona virus from FFPE tissue?
I have an unusual skin biopsy from late December (earlier than the first known 
US case)

Thanks,
Steve A. McClain, MD
McClain Labs
45 Manor Road Smithtown, NY 11787
631 361-4000 cell 631 926-3655

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Re: [Histonet] Histonet Digest, Vol 196, Issue 3 Incomplete sectioning

[Steve McClain via Histonet]

Two methods to avoid incomplete sectioning are:
1) ink the specimens well, using acetic acid (vinegar) to fix the ink, thereby 
making the ink easier to see in the block
2) have a microscope or 10 x lens adjacent to the microtome to allow for visual 
inspection. 
One of my histotechs used an old (really old- had a mirror for a light source) 
for years.  Gradually as scopes are upgraded, the 'retired microscopes can be 
put to use in the lab.

It is unfair to the histotechs that their work is judged by pathologists w 
microscopes, when the histotechs do not have access to the same tool, but life 
is not fair.

Thanks,
Steve A. McClain, MD
McClain Labs
45 Manor Road Smithtown, NY 11787
631 361-4000 cell 631 926-3655

-Original Message-
From: histonet-requ...@lists.utsouthwestern.edu 
[mailto:histonet-requ...@lists.utsouthwestern.edu] 
Sent: Wednesday, March 4, 2020 1:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 196, Issue 3

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Today's Topics:

   1. Re: [EXTERNAL] Re: Incomplete cross sections of all tissue in
  blocks (Perl, Alison)
   2. Re: Incomplete sectioning (Terri  Braud)
   3. Cutting benchmarks (Hagon, Christopher (Health))
   4. Looking for a sliding microtome (Chaitanya Kolluru)


--

Message: 1
Date: Tue, 3 Mar 2020 18:36:12 +
From: "Perl, Alison" 
To: 'John Garratt' , Amy Self
, 'John Garratt via Histonet'

Subject: Re: [Histonet] [EXTERNAL] Re: Incomplete cross sections of
all tissue in blocks
Message-ID: <96e787bcf5684af6bb3b701beb772...@mk-exmb02.mkmg.com>
Content-Type: text/plain; charset="utf-8"

Each of our techs is responsible for fully facing their blocks and getting a 
representative section. If the docs see something is amiss (epidermis missing, 
incomplete cross section, etc), they can order a Technical Recut rather than a 
routine Recut. Unfortunately, it sounds like you (or someone else of 
authority), has to give some feedback/coaching that the sections are 
insufficient, particularly if it's a recurring problem. Is it one tech that 
needs a 1-on-1 convo, or a meeting with the whole staff to address widespread 
issues?

Alison Perl, HTL(ASCP)CM
Anatomic Pathology Manager 
CareMount Medical
(914) 302-8424
ap...@caremount.com


-Original Message-
From: John Garratt via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, March 3, 2020 12:02 PM
To: Amy Self; histonet@lists.utsouthwestern.edu
Subject: [EXTERNAL] Re: [Histonet] Incomplete cross sections of all tissue in 
blocks

I suggest that each histotech is responsible for the blocks they cut and they 
cut the deepers on the their own blocks when they are requested. With feedback 
on the reason for the deeper from the pathologists they (the techs) will become 
more confident and learn how deep to cut.

John

On Tue, Mar 3, 2020 at 7:31 AM, Amy Self via Histonet 
 wrote:

> Good Morning HistoNetters,
>
> I am reaching out to the histonet in hopes to get some suggestions from you 
> on how to handle incomplete cross-sections of tissue in blocks. We are a 
> small lab so this has not been an issue in the past but now that we are 
> growing and our staff has increased I am getting feed-back from pathologist 
> that the sections of tissue are not complete. They are asking for too many 
> deepers that possibly could be avoided if it was cut deep enough to begin 
> with. I have been given some managerial type duties ? which I don?t like 
> cause I know nothing about managing people and I need to approach this but I 
> need to approach this issue correctly. Do you have the histotech compare 
> his/her cut slides to the block to make sure that a complete cross-section is 
> obtained and is this documented somehow? Any and all suggestions I need.
>
> Thanks in advance for your help and as always you all rock.. ?
>
> Amy Self
> Histology Lab Senior Tech
> Lab
> Tidelands Georgetown Memorial Hospital
> 606 Black River Road
> Georgetown, SC 29440
> (843) 520-8711
> as...@tidelandshealth.org
> Our mission: We help people live better lives through better health.
>
> NOTE:
> The information contained in this message may be privileged, confidential and 
> protected from disclosure. If the reader of this message is not the intended 
> recipient, or an employee or agent responsible for delivering this message to 
> the intended recipient, you are hereby notified that any 

Re: [Histonet] Histonet Digest, Vol 194, Issue 12btattoo removal

[Steve McClain via Histonet]

I don not know any method for tattoo pigment removal.  Aside from carbon black, 
most of the red, yellow, blue and green pigments are metal salts and polarize 
brightly.  The inflammatory response may be vigorous, especially w repeat or 
Re-do of a tattoo. In some cases a pseudo-carcinoma or KA may result.

I am puzzled to learn why the request!  Can anyone in the group think of a 
reason why or purpose for the pathologist needs to request removal?Perhaps 
Staining of infectious organism?

Steve
Steve A. McClain, MD
631-361-4000  Cell 631-926-3655

Hello fellow histonetters,

We received a skin sample that has ink from a tattoo - the sample if from
tattooed skin. One of our pathologists would like us to see if we can get
rid of the tattoo ink from the sections before H staining. Does anyone
out there know how to do this?

Thank you,
Donna Emge
Anatomic Pathology Manager
Mercy Hospital and Medical Center, Chicago

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Re: [Histonet] Histonet Digest, Vol 189, Issue 26 Tissue Contamination /Floaters

[Steve McClain via Histonet]

I wrote and posted on this subject some years ago.

In our lab, we virtually eliminated floaters 10 years ago, by using a 
combination of behavioral and technical procedures:
1) photographing every specimen since 2004 (in order to achieve report -worthy 
gross images, one must keep the grossing background scrupulously clean for 
every case).
2) wrapping every specimen in lens paper (creates a double barrier, first 
keeping floaters from this specimen inside and second keeps potential floaters 
from other specimens out).
3) folding the lens paper wrapping using forceps (cleaning the forceps as you 
fold).

Thanks,
Steve
Steve A. McClain, MD

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Re: [Histonet] Histonet Digest, Vol 189, Issue 13 PRAME antibody for melanoma

[Steve McClain via Histonet]

Would one of the LIST members kindly share their experience with PRAM antibody 
in paraffin/

Steve A. McClain, MD
*

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Re: [Histonet] Histonet Digest, Vol 188, Glass plate for AO knife sharpener

[Steve McClain via Histonet]

Does anyone know where to find the glass plate for an ancient AO sharpener for 
microtome?

Steve
Steve A. McClain, MD
631-361-4000  Cell 631-926-3655
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Re: [Histonet] Cassette labeler

[Steve McClain via Histonet]

Anyone care to recommend their favorite cassette labeler to replace the 
Microwriter?
Thanks

Steve
Steve A. McClain, MD
631-361-4000  Cell 631-926-3655

On Jun 24, 2019, at 13:19, 
"histonet-requ...@lists.utsouthwestern.edu"
 
mailto:histonet-requ...@lists.utsouthwestern.edu>>
 wrote:

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When replying, please edit your Subject line so it is more specific
than "Re: Contents of Histonet digest..."


Today's Topics:

  1. Equippment for Sale 
(leon...@rrclinicallab.com)


--

Message: 1
Date: Mon, 24 Jun 2019 08:17:29 -0500
From: leon...@rrclinicallab.com
To: cynthia haynes via Histonet 
mailto:histonet@lists.utsouthwestern.edu>>
Subject: [Histonet] Equippment for Sale
Message-ID: 
mailto:ab74b76ac7ef7d9e869cba75b97c3...@rrclinicallab.com>>
Content-Type: text/plain; charset=US-ASCII

Hi Cynthia

My name is Leonard with R Clinical Labs. Sorry to here that the lab is
closing. Can you please email us a list of what you all are selling. We
are definitely interested.

Leonard Ringo

_OPERATION MANAGER_

R Clinical Lab

PH: 773.289.6296

Office:708.783.7441

--

Subject: Digest Footer

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End of Histonet Digest, Vol 187, Issue 18
*
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Re: [Histonet] Histonet Digest, Vol 179, Issue 4 AFB control

[Steve McClain via Histonet]

I was taught it best to use separate controls on a second slide. But in 
practice, the only cases where a floater appeared it was obvious. In my own lab 
14 year experience it has not happened.

However, about 10 years ago we started an experiment and switched to 2 AFB 
slides with pos and neg controls on one slide only but with patients samples on 
both slides. Looking at 2 slides sometimes makes me more confident.

I have not yet competed the study but anecdotally I do not recall or have never 
seen an AFB floater using this practice. We  deliberately keep both our control 
blocks quite small maybe 1-2 mm.

Steve
Steve A. McClain, MD
631-361-4000  Cell 631-926-3655

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Re: [Histonet] Histonet Digest, Vol 170, Issue 13 histology hacks

[Steve McClain via Histonet]

I purchased the book and applaud the effort because there is some decent 
information.  However, the term hack is a poor choice for histology and many of 
the fixes or secrets described are because some hack failed to do her/his job 
at an earlier step in the process. (Definition of hack. transitive verb. 1 a : 
to cut or sever with repeated irregular or unskillful blows. b : to cut or 
shape by or as if by crude or ruthless strokes. As a noun it is used to mean a 
mediocre performer or worker; tiresome drudge.)

Some methods, while useful in some settings, have important cons not listed, 
cons which may be counterproductive. For example using Mercurochrome or Eosin 
to mark tissue may preclude further testing with fluorescent endpoints, such as 
FISH.  Plus if you really want to use Eosin to mark the dermis, it is far 
easier to add used Eosin to one alcohol in the tissue processor. That gives a 
visible indicator of carryover, indicating need to change or rotate solutions.

Other methods seem (to me) like workarounds or Band-Aids for Labs w poor 
grossing, poor processing or poor reagents or poor technique or poor method 
choices, eg, 2.14 describes a situation where an incompetent grosser truly 
hacks or crudely cuts into unfixed tissue yielding too thick a slice. The real 
solution is to fix the tissue before slicing. Poor fixation results in poor 
processing and poor sectioning and poor staining reactions.

For another example, Cassette sponges offer few advantages, while folding lens 
paper allows the grossers to see through the paper and know all pieces are 
inside before closing the cassette lid.  The tissue does not stick to it, and 
small flakes can be scraped from the lens paper at embedding. Last during 
folding, the forceps can be cleaned at grossing and during unfolding, forceps 
may be cleaned w the paper after embedding.  Sponges also result in greater 
solution carryover.

Several colloquial naming conventions, eg, chamber saver 2.16 for 
underprocessed tissue may be memorable to some readers, yet seem  are odd to me.

This 2.16 method is an especially useful technique which may also be done to 
extend paraffin time, whenever poor sectioning due to poor processing is 
encountered at the microtome.
Variation 1 Place the block back into the proper sized mold and return to the 
heated side of the embedding center for an hour to extend processing 
(reprocessing). Then remove the old paraffin from the mold w a plastic pipette, 
 then re-embed, replacing the paraffin w new.
Variation 2 for outside blocks we routinely replace an unknown paraffin from 
another lab by melting in a mold, and ‘reprocess’ in our (blue ribbon) paraffin 
for 1 hr in a mold in the embedding center then re-embed.

Good first effort, yet this book could be improved by a good editor, by more 
collaborators, by illustrations, and the addition of variations or other uses 
as described above for 2.16.
The font size and format will cause many readers to suffer because of the small 
font.

Steve
Steve A. McClain, MD
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Re: [Histonet] Histonet Digest, Vol 168, Issue 2 Needing a microwave processor

[Steve McClain via Histonet]

Why do you think you need a microwave processor?
You can certainly use a conventional K3000 or VIP5 or RVG (and probably other) 
processors to accomplish short 2 hours-4 hours processing on small samples by
1. Raising the processing temperatures to 45-50C
2. Removing formalin from the processor (all fixation is done BEFORE loading on 
the processor)
and 3.Maniacally ensuring your reagents are always fresh (frequent solution 
rotations)

We looked to buy a microwave processor 10 years ago but found the equipment we 
had produced better results- identical to 8-12 hour overnight processing.

This method has been posted several times on Histonet.
Steve A. McClain, MD

On Nov 2, 2017, at 10:14, 
"histonet-requ...@lists.utsouthwestern.edu"
 
>
 wrote:

Subject: Re: [Histonet] Needing a microwave processor
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Re: [Histonet] Histonet Digest, Vol 164, Issue Olympus microscope camera DP70 works on Windows 10

[Steve McClain via Histonet]

Yes, to my amazed relief, Olympus microscope camera DP70 works on Windows 10.
These old but reliable cameras can be upgraded from Windows 7 (Vista drivers) 
to Windows 10.
We just upgraded one of my workstations and it works fine.

Steve A. McClain, MD


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Re: [Histonet] Histonet Digest, Vol 164, Issue 10 mycoplasma on agar

[Steve McClain via Histonet]

Too bad, Hoescht stain is rapid and sensitive (UV) for mycoplasma.

They can try staining direct smears w difquik giemsa. 

Or fix agar plate in Carnoy's and slice out and process to paraffin for 
microtomy and staining.  IHC  can be done from sections if needed.
These 1 micron bacteria are at the practical limits of visibility.

Steve A. McClain, MD


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Re: [Histonet] Histonet Digest, Vol 164, Issue 9 slide adhesion

[Steve McClain via Histonet]

Greg, 
It sounds like 2 or 3 problems and LEEP specimens are partially heat fixed, 
giving addition artifacts related to poor adhesion.

1. Short processing cycles w insufficient paraffin times or dirty xylenes can 
do this. Double your maintenance or try the radical by removing formalin from 
processor adding more time in alcohol xylene and paraffins.

2. Any water during annealing on slide warmer. Summer months w high humidity 
may prolong drying times. 

3. 30 min baking sounds short to me-skin usually needs at least an 60-90min 
@90C sometimes 4H, some antibodies overnight. Extra baking does not repair or 
compensate for the damage from #2 in my experience.  
Good luck. 

Steve A. McClain, MD
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Re: [Histonet] Original 1979 sales price for Miles Tissue Tek 3000

[Steve McClain via Histonet]

Does Dr. Richmond or anyone else recall the original sales price for the Miles 
TissueTek VIP 3000?

Some of these vacuum processors are still in operation almost 40 years later!


Steve A. McClain, MD
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Re: [Histonet] Histonet Digest, Vol 163, Issue 22 CPT Code for PGP9.5 free floating tissue stain (Eddie Martin)

[Steve McClain via Histonet]

Depends also on what you are doing w the pgp9.5
The only reason I know for free floating section technique is for 
intraepidermal nerve fiber analysis. Morphometric analysis and nerve fiber 
counting is an additional CPT.

Steve A. McClain, MD

> On Jun 24, 2017, at 13:09, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> CPT Code for PGP9.5 free floating tissue stain (Eddie Martin)

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Re: [Histonet] Histonet Digest, Vol 163, Issue 15 Research histology and animal IHC

[Steve McClain via Histonet]

Underfixation being the greater problem, for animal research I tell everyone 24 
hours fixation. After 24 hours in clean formalin, Stop fixation by pouring off 
and transfer to 50% reagent alcohol. Using standard fixation and slow 
processing, repeatable near-perfect histology and IHC can be made w comparable 
staining in small biopsy and large samples alike. 

There is no right answer but kindly choose one method and teach that in your 
lab, better consistent than correct. IHC is antibody dependent and species 
dependent, and fixation dependent but IHC  is especially section dependent. 
Quite predictably excellent slides and excellent IHC can be made from tissues 
in almost every species given 24 hours fixation and overnight processing, the 
methods we first learned in school 30-40 years ago.

For 2mm samples in human clinical testing using common antibodies, we can 
acceptably shorten this to minimum 8-12 hours fixation and rapid 2 hour 
processing using fresh reagents w a wee bit of heat. But in research what is 
gained by it? Underfixed tissue causes more spurious results than over 
fixation. 

In weird species, having weird collagens weird cells, weird antibodies and 
weird questions all confounding the microscopist who needs spurious results?

In research studies w IHC endpoints  and morphometric work, we found results 
improved by stopping fixation at 24 hours, transferring the whole batch to 50% 
alcohol and then slow 12H to 20H processing wo heat.  For really large slices 
processed in teabags or even toenails, an extended 16-20H processing can be 
useful. Adding more time in paraffin and xylene to the 12 hour.  
Counterintuitive in the era of rapid processing schedules, but slow processing 
produces consistent IHC results between small biopsy and large samples.

Research processing for science
RT 12 hours fresh reagents NO heat. NO formalin. We run ALL tissue processors 
wo formalin (station 1 is 50% alcohol). Long processing wo heat or prolonged 
50% alcohol storage for days or weeks at RT does not appreciably harden tissue. 
 

Removing formalin completely from the tissue processors was done gradually, 
first on the animal research machines and rapid processing machines. Eventually 
the last VIP5 w the overnight run is where I met w resistance from my 
histotechs. Even though we had shown no issues w research samples or when short 
processing human samples, they wanted formalin on the processor- in case. 

Quite logically once fixed tissues are in 50% alcohol additional processing 
time in formalin serves no purpose.  My histotechs belatedly gave in after we 
ran the processors for 6 months with only 1 minute in the formalin station 1. 
Our medical director covertly reprogrammed formalin station 1 to one minute and 
added back time to the alcohol in station 2 wo their knowledge (Choose your 
battles.)

Steve A. McClain, MD
McClain Laboratories, LLC

On Jun 16, 2017, at 13:20, "histonet-requ...@lists.utsouthwestern.edu" 
 wrote:

>> Can anyone tell me what, if any, guidelines there are for fixation time for
>> animal tissue with potential subsequent IHC stains.  I am very familiar
>> with CAP guidelines for receptor testing with breast cases, but I can't
>> seem to find much on IHC for animal tissue.
>> 
>> Thanks,
>> Cristi

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[Histonet] (no subject)

[Steve McClain via Histonet]

If it is any help, A dollar bill weighs 1 gram and a quarter weighs 5.67 grams. 
It is simpler to write up some calibration procedure based on knowns. 

Steve A. McClain, MD

> On Jun 8, 2017, at 13:20, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> Balance calibration
> Message-ID:

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Re: [Histonet] Histonet Digest, Vol 162, Issue 26 butterflies

[Steve McClain via Histonet]

I have no experiences w butterflies, but the chitin in ticks, carpenter ants 
and spiders does not cut well.  
For insects and arthropods we post-fix in Carnoy's for 60 minutes
Then soften with 10% KOH RT for 10-20 minutes 
then wash for 30-60minutes in water. Long processing cycle. 

Steve A. McClain, MD

On May 30, 2017, at 13:26, "histonet-requ...@lists.utsouthwestern.edu" 
 wrote:

>> Does anyone have any experience processing and paraffin embedding
>> butterflies?
>> 
>> Rhonda Gregoire, MLT
>> Supervisor, Clinical Pathology
>> Veterinary Diagnostic Services
>> Manitoba Agriculture
>> 545 University Crescent, Winnipeg, MB, R3T 5S6
>> rhonda.grego...@gov.mb.ca
>> T : 204-945-7641  F : 204-945-7646

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[Histonet] Blades

[Steve McClain via Histonet]

http://www.emsdiasum.com/microscopy/products/preparation/blades.aspx

Shared via the Google app

Try electron microscopy sciences. They have a variety of blades.

Steve A. McClain, MD
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[Histonet] PCR for tick bite vasculitis

[Steve McClain via Histonet]

Does anyone have experience or suggestions for where to send FFPE biopsy 
sections from a patient with erythema migrans and known Amblyomma tickbite.
Dieterle negative, anti-Borrelia burgdorferii negative
Gram negative
PAS negative.
Thanks for your help.
Steve A McClain, MD
631-361-4000
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Re: [Histonet] Histonet Digest, Vol 162, Issue 6

[Steve McClain via Histonet]

Olympus microscopes win in my experience.

You can't go wrong with any of the 3 major manufacturers. Modern scopes all 
have have better optics than what we trained on.  I place a high priority on 
durability and in my experience with Nikon and Olympus, Olympus has 
consistently shown better overall quality and durability and wins hands down.


Why?  In my personal experience, in scopes getting used 8-10 hours a day, and 
multiple users, Olympus survives better in terms of rubber parts such as ocular 
cups and the ring around the objective turret which failed on both Nikons. In 
other words, where the rubber meets the road ( or the pathologist touching the 
rubber), both Nikons suffered.


During my first 10 years, I used Nikon scopes exclusively, and I still own one 
Nikon scope (albeit stored in a cabinet). After 2-4 years both stages wore out 
and those rubber parts needed replacing.  I purchased  3 different digital 
cameras in my first 5 years of slide imaging (Polaroid and Spot) and 2 
different Nikon scopes 400 and e600.


But in my own private lab experience Olympus is clearly more durable. I wore 
out 1 stage and wore out 1 motorized microscope head in 14 years of rigorous 
daily use since changing to Olympus in 2004. That's it. 3 pathologists 3 
scopes, 100,000 slides a year. 2 repairs* We rely on our microscopes to work to 
a greater degree than most labs because we

IMAGE EVERY SPECIMEN.  Having a scope down or having suboptimal  performance 
inevitably shows up in the images.


Olympus scopes BX 61 BX41 with digital cameras DP70 DP71 have held up really 
well.  3 student grade scopes in the dirty lab environ at each microtome 
cutting station, used for immediate wet slide inspection and KOH exams, have 
held up well. Perhaps most surprisingly, both Olympus digital camera models 
(surprisingly because they are now 12-14 years old) still produce great slide 
images and remain in daily use.  The scopes likewise.


If you are buying a scope to be replaced every 3 years, you may see little 
difference.  But if you plan to use for 3-10 years with little maintenance 
aside from cleaning, odds favor Olympus.


Steve A. McClain, MD

*PS truth be told I also left the UV lamp on for a week 240 hours and burned up 
the UV lamp housing and had to replace that. Adding of course a $12 darkroom 
timer to prevent that accident from repeating.


On May 8, 2017, at 13:09, 
"histonet-requ...@lists.utsouthwestern.edu"
 
>
 wrote:


Subject: [Histonet] Microscope selection

Message-ID:

   

Re: [Histonet] Histonet Digest, Vol 161, Issue 24 ICD10

[Steve McClain via Histonet]

1 Google it or look it up 
Or
2 use an allpurpose icd10 as a placeholder, e.g. For skin one can use r23.8 
other skin changes, unless you can find an icd10 for "no icd10".
3 feeling bold-
use V91.07XA (burn due to water skis) or V96.00XS, which outlines an 
unspecified balloon accident injuring occupant.

Steve A. McClain, MD

> On Apr 25, 2017, at 13:21, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> What do you do when you receive a pathology consult and it is missing an 
> ICD-10 code?  I am being told that we cannot accession it until we get a code 
> from the submitting doctor's office, and that we cannot add one.  Thank you.
> 
> Richard
> 
> Richard W. Cartun, MS, PhD

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Re: [Histonet] Histonet Digest, Vol 161, Issue 5Subject: Re: Processing cystic tissue

[Steve McClain via Histonet]

Skin cysts are difficult for everyone.  Cysts and toenails lead the top of our 
list of cases with catastrophic tissue loss - which we define as tissue loss w 
"holes" >1mm2 square.

The cyst center generally does not fix readily, composed of unfixed cornified 
cells and waxy lipid. On the scalp, wens or isthmus-catagen cysts are also  
calcified. The outer aspect may include dermis and scar and inflammation and 
also the squamous cyst lining. All of these factors limit diffusion and 
penetration of fixatives and processing alcohols/clearants/paraffins.

We open all cysts by slicing into them and allow them to fix another 6-8 hours 
in fresh 10%NBF, then process on a long cycle of 12-16 hours, beginning in 50% 
alcohol in station 1. 12 hours is our standard overnight cycle and 16 hours is 
used for toenails.

Our cyst slides are still not perfect, but are sufficient to make a 
photomicrograph-our usual method-we photograph every specimen.

Steve A. McClain, MD

> On Apr 5, 2017, at 13:23, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> Subject: Re: [Histonet] Processing cystic tissue

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Re: [Histonet] Histonet Digest, Vol 160, Issue 30 Subject: disposable grossing pad for specimen imaging

[Steve McClain via Histonet]

We use squares of absorbent paper towels to first blot excess fixative and then 
place on a 2mm thickness piece of art foam.
Art foam is used as a background for 
photography and slicing.
By sliding or moving the foam we position the specimen under the camera during 
specimen photography. We have photographed every specimen since 2004. 
Send me an email and I will reply w images to illustrate our method.
Steve A. McClain, MD

> On Mar 31, 2017, at 13:25, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> Subject: Re: [Histonet] disposable grossing pad

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Re: [Histonet] Histonet Digest, Vol 159, Issue 20Subject: simple tests for xylene contamination and alcohol conc. in Recycled alcohol

[Steve McClain via Histonet]

Eventually your recycler retort will become contaminated. When you suspect your 
recycled alcohol product is contaminated by xylene, here is one fairly 
sensitive yet simple test. 

Use a 100 ml glass graduated cylinder
Add 50ml recycled 95%
Add 50ml water. Shake well.
When xylene is present it will form a thin layer on top. Cover w parafilm and 
let sit over night. Each ml of xylene=1%

A second test one can do is measure specific gravity by hydrometer in another 
50ml sample and graduated cylinder.
Specific Gravity should be about 0.82 to 0.85. 

Steve A. McClain, MD

> On Feb 23, 2017, at 13:22, Steve McClain  wrote:
> 
> We only use recycled reagents (except 100%) on processor.
> We only use Fresh reagents on stainer and coverslipper. 
> Steve A. McClain, MD

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Re: [Histonet] Histonet Digest, Vol 159, Issue 20Subject: Recycled reagents in VIP processor

[Steve McClain via Histonet]

We only use recycled reagents (except 100%) on processor.
We only use Fresh reagents on stainer and coverslipper. 
Steve A. McClain, MD

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Re: [Histonet] Histonet Digest, Vol 159, Issue 16 Colloidal iron leaching

[Steve McClain via Histonet]

After checking reagents and glassware, look for other sources of iron 
contamination, e.g. Metal forceps, steel staining racks, water bath. 

Inadequate rinsing after the colloidal iron step - increase wash times, using DI

Check concentration of working reagent. Decrease the time in colloidal iron 
working reagent to 5 min. Hales method specified 5-20 min.

> Steve A. McClain, MD
> 
>>> Hello netters,
>>> We have had a problem with our colloidal iron leaching for about a year 
>>> now. We have changed all reagents and tried different kits. Has anyone else 
>>> had this problem? I cannot figure out what it is it's making me crazy! We 
>>> go directly into clean 100% alcohol then clearing. Any advice would be 
>>> greatly appreciated, thanks in advance!
>>> 
>>> Sent from my iPhone

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Re: [Histonet] Histonet Digest, Vol 158, Issue 22 Breast Specimen

[Steve McClain via Histonet]

In my experience, rushing to process fatty or inadequately fixed specimens is a 
fool's game.
In my opinion, this problem cannot be solved by the histotechs- it begins with 
the grossers and is one for the pathologists to solve at the grossing bench.  

Suggestion #1 Do nothing and let the medical director pathologist/ sign it 
out/deal w this individual case.

Suggestion #2  Sometimes a decent section can be obtained after changing 
paraffin. 
[place the blocks in molds and melt the blocks and change to new paraffin- let 
them sit in the embedding center in the new paraffin for 60 minutes. Re-embed 
in new paraffin (2 changes) and then re-embed.]

Suggestion #3 Reprocess these blocks if permitted, recognizing that breast 
markers (if cancerous) may be erroneous.

Suggestion #4 Seek to prevent future occurrences by adjusting behavior at the 
grossing bench.
a) first ensuring adequate fixation and b) second ensuring adequate length/time 
of processing.  


Steve A. McClain, MD
631 361 4000

What is the best way to handle Breast specimens that were grossed too thick and 
did not process well?  Our medical director does not want us to reprocess the 
tissue but it is almost impossible to get even a remotely decent section. If 
anyone has any other tips please let me know as soon as possible

-- 

Charles Riley HT(ASCP)CM

Histopathology Coordinator/ Mohs**

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Re: [Histonet] Histonet Digest, Vol 157, Issue 21 the center

[Steve McClain via Histonet]

What paraffin are you using?
I heard a rumor one manufacturer changed formulations recently. Same brand name 
different formula. 

Center problems often  manifest where  insufficient fixation is a primary event 
or where rapid processing meets the limits of solution maintainance and tissue 
size. Rapid processing w inadequate alcohols is a silent offender. Sometimes 
you just replace every solution, just so you know where you a new starting 
point. 

If you suspect a processing problem as a cause, one can often achieve a better 
result by melting and re-embedding in new paraffin. By this I mean Allow to sit 
in the embedding center melted in a new mold, in fresh new paraffin for 
60minutes. Then Change paraffin again and embed and cool. Not sure why it 
works, but cooking for a few more minutes does.improve sectioning in marginal 
tissues.

Pathologists can also be the cause- Tissues which are crushed have aberrant 
staining patterns. 
XS handling prior to fixation can mess up staining. Raw tissues rushed through 
and on the processor. I am guilty of all of them in the past.

Steve A. McClain, MD


> On Dec 28, 2016, at 13:30, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> Our pathologists are complaining that the center of tissues are not
> staining properly in both our H's and IHC's. Can anyone provide some
> thoughts as to where this problem could be occurring?
> 
> 
> As a separate situation we are experiencing the tissues folding or falling
> off during the staining process. Both this issue and the staining issue are
> recent occurrences and we have not changed our process in any way from
> previous years.

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[Histonet] Rapid processing With Conventional Processors and conventional (old school) reagents

[Steve McClain via Histonet]

I posted this previously.
We have done this more than 2400 times with a number of processors.

It can be done if you pay close attention to processing only FIXED tissues, 
use fresh reagents
and lay out the cassettes not more than two high.
Steve
631 361 4000

Station 1 50% 10 min
Station 2 70% 10 min 
Station 3 95% 5 min
Station 4 95% 10 min
Station 5 100% 5 min
Station 6 100% 10 min
Station 7 100% 20 min
Station 8 Xylene 5 min
Station 9 Xylene 10 min
Station 10 Xylene 20 Min
Station 11 Paraffin 5 min
Station 12 Paraffin 10 min
Station 13 Paraffin 20 Min (VIP5 Station 14 Paraffin 20min)
Total Time to complete ~3hours



I posted our 7 year experience on this topic previously on histonet.
Remember, this machine (K3000, VIP5) was not designed specifically for this 
purpose, but it can be made to work admirably.  
IOW Short processing on VIP is unreliable UNLESS you are willing to accept or 
control certain conditions, 
1) only process well-fixed tissue 
2) take formalin off this processor, making 14 changes beginning in 50%. If you 
want to keep station 1 formalin and standardize fixation with time X in 
formalin at temp Y, that may work. I prefer to change the tissues to 50% 
alcohol at the grossing bench so that all tissues are not only fixed, but 
rinsed in alcohol and started processing at the growing bench. This simplifies 
processing to dehydration, clearing and paraffin infiltration.  Simple is 
reliable and short processing cycles on this machine leave little room for 
error.
2) Program heat to 50C in all alcohols and xylene. Mixing pressure and vacuum 
yes yes yes 
4) Keep all solutions fresh, rotate after 200- 400 blocks.
5) Lay blocks out not more than 2 cassettes back to back, max 100 for VIP5.
4 hour is reliable for tissues up to 3mm thickness such as punch biopsy. 
2 hour is reliable for up to 1.5 mm tissue thickness such as core biopsies and 
shave biopsies of skin.

Method of processor validation for short cycle in machine previously validated 
in your lab.

To validate create your evaluation form and find a set of cases with sufficient 
tissue small tissue to divide into 2 equal blocks. This can (and will) be done 
on clinical cases if you are careful. 
Print duplicate sets of cassettes. 
Mark one half of case 1 yellow and one half blue (tissues will be processed 
separately, 1 regular and one short cycle and then recombined into 1 final 
block at embedding).
We held the short processed tissue cassettes in formalin and timed the short 
cycle to end when the regular program ends.
Then the two blocks are recombined or embedded together so that direct 
comparisons/measures can be made on one H and one on your most common IHC for 
that tissue. If the H and IHC both work, chances are everything else will 
also.
Modify your regular validation form or Create a form for scoring shrinkage, 
measured thickness and length, staining reactions, shrinkage, holes and flaws, 
have your path score.   photo graph yellow and blue tissues under H and IHC. 
IF you do observe shrinkage or other undesirable artifact, be certain that your 
tissues are completely fixed at grossing and repeat.

If they are comparable in the oculars and to the camera, then show your work 
and voila, there is your validation. 

Steve A. McClain, MD

>

Message: 7
Date: Fri, 9 Dec 2016 17:30:35 + (UTC)
From: Cheryl 
To: Histonet 
Subject: [Histonet] short biopsy process for a Tissue Tek VIP -
Message-ID: <1005303688.1255120.1481304635...@mail.yahoo.com>
Content-Type: text/plain; charset=UTF-8

Help??
Anyone have a good 3-hour process on a VIP they can share? ?We're using StatLab 
reagents and all small biopsy work-- VIP-5 smaller version.?
Would love to start from a proven process that's been tweaked to perfection 
?than start from scratch...
thank you!!?Cheryl Kerry, HT(ASCP) ?

tkngfl...@yahoo.com


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Re: [Histonet] Histonet Digest, Vol 157, Issue 1Glypican-3

[Steve McClain via Histonet]

Victoria Verno
You might try normal Placenta 
or fetal liver from POC 
as non-neoplastic stain controls.

That strategy may allow you enough repetitions to develop the stain until you 
find a case of HCC to test.


Steve A. McClain, MD

*

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Re: [Histonet] Histonet Digest, Vol 156, Issue 20a hematoxylin-eosin Y-Azure-II stain

[Steve McClain via Histonet]

hematoxylin-eosin Y-Azure-II stain
Agree w Ren.
But try it anyway. 

To bring out the azure, 
you may wish to post fix.

Steve A. McClain, MD

> On Nov 22, 2016, at 13:29, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> a hematoxylin-eosin Y-Azure-II stain

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Re: [Histonet] Histonet Digest, Vol 154, Issue 13 tissue loss

[Steve McClain via Histonet]

Wendy
Develop your own data.
Check your early steps first. Tissue loss can be simple -a function of poor 
fixation, dirty processing or rush processing or slightly large tissue w 
slightly dirty reagents.

Airdry wet slides before annealing on hot plate. 
Check your baking/annealing. Some tissues require a bit more. 

We do not use that stainer.

Check your heat antigen retrieval against a manual retrieval.
Skin can handle 90 degrees for twice as long as 98. 98 degrees may cause 
excessive collagen loss. 

Steve A. McClain, MD

> On Sep 16, 2016, at 13:25, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> Wendy

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Re: [Histonet] Histonet Digest, Vol 154, Issue 7ubject: fixing fatty skin specimens

[Steve McClain via Histonet]

Not sure I understand your question.  
But assume you are looking for margins on a re-excision of cancer or melanoma.
Are you using a sufficient -20 times volume of clean 10% nbf? 
And changing it often?
Switch to 20% nbf
Or switch to 100% formaldehyde! True dat- Stanford's Dr. McNeal fixed prostates 
~6 hours in 37% raw unbuffered in 1986. (We also used Mercury B5 in 1986)

The question is whether what are you studying (IHC, PCR ) will stand up to 
additional fixation or post fixation methods treatment.

My plan A approach is to use plenty of  clean 10-20% formalin and change it 
every 6-24hours. Once it firms up, gross and bread loaf slice and get into 2x3 
INCH teabags.
lay those out for another 3-12-24 hours in 70% alcohol, then process teabags 
for 12-16-18 hours ROOM TEMPs  program 3.

heat fixation- I've seen processor demos where microwave cooked tissues 
processed well and according to the rep 'cut like butter' but 12 years ago I 
could not photograph the results.

We've had decent results w 1 hour RT Carnoy's postfixation in some samples. 
Love this stuff.  It firms up fat, fixes mucin. It can over harden tissue (not 
in 1hr) and causes shrinkage. But DO NOT HEAT Carnoy's !!! I read it generates 
phosgene gas. 



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Re: [Histonet] Histonet Digest, Vol 154, Issue 2 movats

[Steve McClain via Histonet]

Liz, please send SOP ASAP!
The vvg is a hematoxylin-that's why it works so well. 
The problem w Movat is the photography. It is complex w 5 color signals, hard 
to show all 5 in one image. 
Liz, What control did you use?
Steve A. McClain, MD

> On Sep 2, 2016, at 13:13, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> weigerts hemastoxylin, stain with the elastic portion of the VVG, 
> differentiate like you would for elastic fibers and then continue on with the 
> rest of the trichrome, it turns out great.  I have an SOP if anyone wants it.
> 
> Liz
> 
> Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
> Premier Laboratory, LLC

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Re: [Histonet] Histonet Digest, Vol 153, Issue 24 davidsons fixative fish

[Steve McClain via Histonet]

Something sounds fishy
Dr. Richmond can solve it.
http://www.ihcworld.com/_protocols/histology/davidson_fixative.htm

Steve A. McClain, MD

On Aug 30, 2016, at 13:22, 
"histonet-requ...@lists.utsouthwestern.edu"
 
>
 wrote:

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than "Re: Contents of Histonet digest..."


Today's Topics:

  1. pediatric facilities only (Houston, Ronald)
  2. Eosin (Cameron, Elizabeth)


--

Message: 1
Date: Tue, 30 Aug 2016 11:42:10 +
From: "Houston, Ronald" 
>
To: 
"histonet@lists.utsouthwestern.edu"
   >
Subject: [Histonet] pediatric facilities only
Message-ID:
   
>

Content-Type: text/plain; charset="us-ascii"

If you do not work in a pediatric facility please disregard

I need some help getting statistics for benchmarking for staffing evaluation

What is your annual volume of cases/blocks?
Continual flow processing?
# of IHC/Special stains
EM?
Shifts?
Days only?
Weekend coverage?
How many HTs do you have?
How many PAs?
Lab Assistants?
How many pathologists?
Do you perform any research?
What do you do regarding transcription? # of transcriptionists, outsource, 
voice recognition?

Thanks
Ronnie

Ronnie Houston, MS HT(ASCP)QIHC FIBMS
Anatomic Pathology Manager
Laboratory Services
700 Children's Drive
Columbus, OH 43205
(P) 614-722-5450
(F) 614-722-2899
ronald.hous...@nationwidechildrens.org
www.NationwideChildrens.org

"Without continual growth and progress, such words as improvement, achievement, 
and success have no meaning."
~ Ben Franklin



--

Message: 2
Date: Tue, 30 Aug 2016 14:25:24 +
From: "Cameron, Elizabeth" 
>
To: 
"histonet@lists.utsouthwestern.edu"
   >
Subject: [Histonet] Eosin
Message-ID:
   
>

Content-Type: text/plain; charset="us-ascii"

Hi,
I have been staining fish tissues fixed in Davidsons with H, and the 
researcher would like the eosin to be more intense.  Our standard protocol 
works well for our own tissue, but the fish look much more washed out.  I am 
using alcoholic eosin Y, have tried both water and alcohol before and I have 
varied the alcohol differentiation steps after the Eosin.  I also extended the 
time in Eosin and increased the wash after bluing to make sure the sections are 
not basic.  Any suggestions would be appreciated.
Thank you.

Elizabeth M. Cameron, HT(ASCP), QIHCCM
Lead Histologist
Mid Coast Hospital
123 Medical Center Drive
Brunswick, ME 04011
(207) 373-6573



--

Subject: Digest Footer

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End of Histonet Digest, Vol 153, Issue 24
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Re: [Histonet] Histonet Digest, Vol 153, Issue 10

[Steve McClain via Histonet]

For once I almost refuse to answer because there is No good answer. This is 
absolutely ludicrous. 
Heck no is my first response but that probably doesn't pass muster.
My state inspectors are probably wanting to have me fill out more firms   forms 
for monthly bleaching. Or adding a bleach step to corrode the delicate inner 
bits.

Perhaps the most cost effective method of disposal i am presently consider it 
may be least expensive to require all lab medical directors to put it in their 
will 
my wish is to be entombed with all 4 of my Kseries VIPs. 
They've been working longer than I have and refuse to die. I promise to retire 
those processors with me buried upright.  Let me stand guard the rest of 
eternity to prevent the currently rampant spread of all diseases spread by 
contaminated.

Shucks. I may have to be buried in Jamaica. I shipped a couple of those 
hazardous contaminated VIPs to Kingston. 

Heck I really do not know why we are pondering a non significant disease. Let 
someone identify a problem first before making a regulation or procedure.

In my search for more data on  the rampant waterski is on fire v71.0xA ICD10 
problem, the processor contamination problem just snuck up on me!!  

I guess we need a decontamination room just to contain the outpouring of 
biohazardous waste every time we rotate solution or annual maintenance. 

Yep, let them cite me if my burial protocol fails the stink test.
Steve A. McClain, MD


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Re: [Histonet] Histonet Digest, Vol 153, Issue 3

[Steve McClain via Histonet]

Never had one of fancy new ones! But I have  one V5 and 4 K Sakuras. Those back 
to the future Delorean stainless steel  models run circles around the already 
rusting needs to be painted 8 year old model down more in its short life than 
all 4 flux capacitor Ks put together.  

Start by analyzing your history.
What tissue tissues are you processing?
How long is your cycle?
What clearant-xylene?
How long is your cleaning cycle?
What tissue wrapping are you using?

Steve A. McClain, MD

> On Aug 3, 2016, at 13:15, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> Send Histonet mailing list submissions to
>histonet@lists.utsouthwestern.edu
> 
> To subscribe or unsubscribe via the World Wide Web, visit
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> or, via email, send a message with subject or body 'help' to
>histonet-requ...@lists.utsouthwestern.edu
> 
> You can reach the person managing the list at
>histonet-ow...@lists.utsouthwestern.edu
> 
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Histonet digest..."
> 
> 
> Today's Topics:
> 
>   1. Wax clogs in VIP 6 processor (cynthia haynes)
>   2. Re: Histonet Digest, Vol 153, Issue 2 microtomes (Steve McClain)
>   3. slides on the Benchmark Ultra (Amy Johnson)
> 
> 
> --
> 
> Message: 1
> Date: Tue, 2 Aug 2016 18:49:17 + (UTC)
> From: cynthia haynes 
> To: Histonet 
> Subject: [Histonet] Wax clogs in VIP 6 processor
> Message-ID:
><1833276652.9872747.1470163757288.javamail.ya...@mail.yahoo.com>
> Content-Type: text/plain; charset=UTF-8
> 
> Hello Histo NetI am having a problem with wax clogs on my VIP 6 processor. 
> This unit vents to the outside and I was just wondering if that may?have 
> something to do with my problem. It started 2 weeks ago. I've had the unit 
> serviced?2 x's. It has been very humid in our city. I am?changing sol'n 
> regularly as per company supplied maintenance program. HELP!. I am at my wits 
> end. This relatively a new unit. 3 year old. Has anyone experience this 
> problem before.Thanks in advance.Cynthia Haynes-James H.T.
> 
> --
> 
> Message: 2
> Date: Tue, 2 Aug 2016 18:56:51 +
> From: Steve McClain 
> To: "histonet@lists.utsouthwestern.edu"
>
> Subject: Re: [Histonet] Histonet Digest, Vol 153, Issue 2 microtomes
> Message-ID: 
> Content-Type: text/plain; charset="us-ascii"
> 
> Depends on how long you plan to be in business and whether you are using 
> other people's money.  But buy all the same model and make if you can.
> 
> I've owned the same Leica 2125 and 2135 microtomes for 12 years.  
> We usually cut mainly small biopsies but have pushed those to occasionally 
> cut large  research blocks up to 45mm by 60mm.
> I cannot speak to other models. Since I've owned Leica, I haven't had any 
> other experience. 
> 
> Like all mechanical equipment, maintenance is critical or none will cut 
> properly.
> Steve A. McClain, MD
> 
>> On Aug 2, 2016, at 13:19, "histonet-requ...@lists.utsouthwestern.edu" 
>>  wrote:
>> 
>> Thanks to everyone who helped me out by providing their opinions on
>> embedding centers.  This time, I need everyone's thoughts on microtomes.
>> My lab is debating between the Thermo/Microm HM355S and the Leica RM2255.
>> Your thoughts and advice are very much appreciated!  If there are any more
>> I should try, let me know!
>> 
>> Thanks again,
>> Mary Faith
> 
> 
> 
> --
> 
> Message: 3
> Date: Wed, 3 Aug 2016 14:06:09 +
> From: Amy Johnson 
> To: "histonet@lists.utsouthwestern.edu"
>
> Subject: [Histonet] slides on the Benchmark Ultra
> Message-ID:
>
> Content-Type: text/plain; charset="us-ascii"
> 
> Hello Histonetters,
> 
> WE currently run the Benchmark Ultra for our IHC staining.  We have been 
> using SuperFrost Plus slides on the recommendation by Ventana.  What are 
> others using if you aren't using SuperFrost Plus slides.
> Our vendor that we get SuperFrost Plus from no longer carries them and has 
> replaced them with another type..Just wondering what others think
> 
> 
> Thanks
> Amylin Johnson, B.S. HTL(ASCP)
> Associates in Pathology
> Wausau Wi 54401
> 715-847-2130
> 
> 
> 
> --
> 
> Subject: Digest Footer
> 
> ___
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> --
> 
> End of Histonet Digest, Vol 153, Issue 

Re: [Histonet] Histonet Digest, Vol 153, Issue 2 microtomes

[Steve McClain via Histonet]

Depends on how long you plan to be in business and whether you are using other 
people's money.  But buy all the same model and make if you can.

I've owned the same Leica 2125 and 2135 microtomes for 12 years.  
We usually cut mainly small biopsies but have pushed those to occasionally cut 
large  research blocks up to 45mm by 60mm.
I cannot speak to other models. Since I've owned Leica, I haven't had any other 
experience. 

Like all mechanical equipment, maintenance is critical or none will cut 
properly.
Steve A. McClain, MD

> On Aug 2, 2016, at 13:19, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> Thanks to everyone who helped me out by providing their opinions on
> embedding centers.  This time, I need everyone's thoughts on microtomes.
> My lab is debating between the Thermo/Microm HM355S and the Leica RM2255.
> Your thoughts and advice are very much appreciated!  If there are any more
> I should try, let me know!
> 
> Thanks again,
> Mary Faith

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Re: [Histonet] Histonet Digest, Vol 153, Issue 1

[Steve McClain via Histonet]

I agree w Dr. Richmond. 
> 
> Message: 2
> Date: Sun, 31 Jul 2016 17:43:20 -0400
> From: Bob Richmond 
> To: "Histonet@lists.utsouthwestern.edu"
>
> Subject: Re: [Histonet] B-5 Fixative
> Message-ID:
>
> Content-Type: text/plain; charset=UTF-8
> 
> Jillian A. Russell, HT (ASCP)CM, QIHCCM, Supervisor, CDx Histology
> Operations, R at Dako in Carpinteria CA asks:
> 
>>> I am wondering how many labs are using B-5 fixatives and how many are
> using B-5 alternatives due to the mercury issue? - For those who have
> switched to an alternative, have you noticed many differences from the
> traditional B-5 fixative?<<
> 
> This is a very controversial topic. B-5 fixative contains a large quantity
> of mercuric chloride, along with formaldehyde, and you really can't use it
> any more. I gave up B-5 and Zenker's fixatives very reluctantly.
> 
> My personal opinion is that B-5 substitutes are no improvement on neutral
> buffered formalin, and may compromise immunostains (which often specify NBF
> fixation).
> 
> Bob Richmond
> 

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Re: [Histonet] Histonet Digest, Vol 152, Issue 18

[Steve McClain via Histonet]

PAS crystals. The problem may not be the stain at all. occurring  due to 
conditions  to leaching of biochemical substances, in the tissue. Dirty 
processors, or use of KOH or acids applied to the block just before sectioning. 
Those may not be washed out.  
These crystals are especially common in thin 1-2mm fungal toe nails. I can tell 
you how to get at it, but it's a long story. And a bit of science to move you 
toward a solution.  Kindly send me a private email. 

The fungus stuff is too weird to share in mixed company.  Your pathologist will 
resist or not believe it. 
Heck if I didn't have calibrated photos, I wouldn't believe it either.  

The toenails are so weird, I am working to improve w 3D gross images.
Steve A. McClain, MD


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[Histonet] 360 video of specimens

[Steve McClain via Histonet]

The next 3 weeks we are turning our lab cameras and our specimens toward making 
a 360 photographic orbits around specimens to create a 360 stitched image or 
video. 
If you know any pathologists who have tried 360 imaging around a specimen, 
kindly send me their name. I have not seen any prior art.

Steve A. McClain, MD

> On Jul 17, 2016, at 13:23, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> Send Histonet mailing list submissions to
>histonet@lists.utsouthwestern.edu
> 
> To subscribe or unsubscribe via the World Wide Web, visit
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> or, via email, send a message with subject or body 'help' to
>histonet-requ...@lists.utsouthwestern.edu
> 
> You can reach the person managing the list at
>histonet-ow...@lists.utsouthwestern.edu
> 
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Histonet digest..."
> 
> 
> Today's Topics:
> 
>   1. plastic sectioning advice requested (daniel blackburn)
> 
> 
> --
> 
> Message: 1
> Date: Sat, 16 Jul 2016 19:51:23 + (UTC)
> From: daniel blackburn 
> To: 
> Subject: [Histonet] plastic sectioning advice requested
> Message-ID:
><682357368.319688.1468698683058.javamail.ya...@mail.yahoo.com>
> Content-Type: text/plain; charset=UTF-8
> 
> My lab hopes to get into plastic sectioning.  We need to be able to process 
> tissue pieces as large and thick as possible, but see that the largest 
> embedding molds for JB4 are only 13x19mm by 5mm deep.   We have two 
> questions:  (1) Do any of the available media (plastic or resins) allow one 
> to embed and section large  pieces (for example eggs with dimensions of 2 cm 
> or larger)?   (2) Is a special microtome (such as a retracting microtome) 
> needed?   Our reason for considering plastic is that we must section yolk, 
> which splits out of standard paraffin during sectioning. Any advice is 
> appreciated. -- Daniel Blackburn, Trinity College 
> 
> 
> 
> --
> 
> Subject: Digest Footer
> 
> ___
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> 
> --
> 
> End of Histonet Digest, Vol 152, Issue 15
> *

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Re: [Histonet] Histonet Digest, Vol 150, Issue 31 5. Re: those slippery lab floors (Normington Lacy)

[Steve McClain via Histonet]

I did see many slip and fall injuries in histolabs over the years.

When we built this lab we accounted for that 
and deliberately left the bare concrete rough, 
and professionally painted polyurethane 
of the type used in firehouse floors.

Has held up reasonably well.
No slips or falls in 12 years.
Some stains; 
Would not use in grossing room again however.
Drop a specimen fragment on that floor and you may be searching for an hour. - 

Steve A. McClain, MD
631 361 4000

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Re: [Histonet] Histonet Digest, Vol 150, Issue 21 floaters

[Steve McClain via Histonet]

Never seen that happen in 34 years of training and practice. 
Sounds like an inadvertent transfer by the grosser.  

But Floaters are so frequent that it behooves labs processing small biopsies to 
wrap each specimen to minimize or eliminate transfers.  We have the embeddets 
clean the forceps w the wrappers to further minimize transfer.  I have posted 
long messages previously, detailing methods. 

However tiny floaters can persist for months in the tissue processor pumps and 
bottles and lines.  20 years ago I inherited an old Fishermatic 160, previously 
used to process placentas.

I saw placenta fragments in skin samples for at least 6 months.   
Steve 

Morning everyone,

I would like to know how many labs experience issues where tissue from a 
typical, sealed cassette is lost in the processor and ends up inside another 
cassette.

Really.

Thanks,
Jeanine Sanders
CDC Atlanta

Steve A. McClain, MD

> On May 19, 2016, at 13:31, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> Morning everyone,
> 
> I would like to know how many labs experience issues where tissue from a 
> typical, sealed cassette is lost in the processor and ends up inside another 
> cassette.
> 
> Really.
> 
> Thanks,
> Jeanine Sanders
> CDC Atlanta

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Re: [Histonet] Histonet Digest, Vol 149, Issue 7 : Re: specimen submission pads ordinary lens paper

[Steve McClain via Histonet]

There is much commendable about ordinary lens paper cut from 9x12 sheets to 
~2x2 inch and folded 4 times like an origami to enclose small tissue fragments. 
Keeps floaters out. Translucent so you can count the fragments before and after 
opening. Unfolds reliably at embedding wo flinging pieces. Cleans your forceps 
after embedding.  Doesn't seem to clog processors.  2 hour processing in 
regular cassettes. 
We wrap nearly every specimen. 
I can send a brief video demonstration if you email me directly.  

Steve A. McClain, MD

> On Apr 7, 2016, at 13:26, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> bject: Re: [Histonet] specimen submission pads

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Re: [Histonet] Histonet Digest, Vol 148, Issue 20 processing

[Steve McClain via Histonet]

I don't really like wasting valuable rapid-processing time on fixation, when I 
cab icontrol that at the bench 
Your schedule is made more difficult because of the length of time in formalin. 
See if your director can ensure only fixed tissue on processor. If not increase 
temp to 45C station #1 and change frequently allowing only 1 hour in warm 
formalin. This will  gain an 45 min for 100% and paraffin (two stages where you 
are disproportionately short).
Then remove the formalin station #2  replace w 50% alc 15 minutes rinses buffer 
salts off the seals lengthening life of processor seals.

3 75% alc 30
4 95% alc decrease to 15 min
5 95% alc 45 min
6 100% alc increase to 15 min
7 100% alc decrease to 30 min
8 100 % alc increase to 45
9 xylene 1 decrease to 15
10 xylene 2 same 30
11 zylene 3 increase to 45
12 paraffin 1 decrease to 15
13 paraffin 2 same 30
14 paraffin 3 increase to 90 min
 Should give you just under 9 hours.
Since you are constrained on time and depend on consistent, rapid diffusion, 
rotate solutions frequently. 

Steve A. McClain, MD

> On Mar 22, 2016, at 13:53, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> 1. Formalin   1hr
> 2. Formalin   1hr
> 3. 75% alcohol 30 mins
> 4.85% alcohol 30 mins
> 5. 95% alcohol 30 mins
> 6.95% alcohol 30 mins
> 7. 100% alcohol 30 mins
> 8. 100% alcohol 30 mins
> 9 xylene 30 mins
> 10. xylene 30 mins
> 11 xylene 30 mins
> 12. Wax for 30 mins
> 13. Wax 30 mins
> 14. Wax 30 minutes.

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Re: [Histonet] Histonet Digest, Vol 146, Issue 10 tissue processors

[Steve McClain via Histonet]

Sakura VIP you can run for decades if maintained. Extremely reliable. The parts 
are available. 
 I have 3 K series  and a VIP5. The newest is 17 years old, the oldest maybe 35 
years. All acquired used and refurbished.

Our newest processor, 7 years, a demo acquired almost new, failed miserably 
twice this year after 2000 processing runs. Began leaking from multiple sources 
and reagents, spent hours. Troubleshooting, 2 service calls, etc. 
 I won't name the brand, but every single reagent bottle failed. New isn't 
always more reliable than old VIP.

Steve A. McClain, MD
PS extremely 
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Re: [Histonet] Histonet Digest, Vol 146, Issue 4 validation suggestions for short cycle processing with conventional equipment

[Steve McClain via Histonet]

I posted our 7 year experience on this topic previously on histonet.
Remember, this machine (K3000, VIP5) was not designed specifically for this 
purpose, but it can be made to work admirably.  
IOW Short processing on VIP is unreliable UNLESS you are willing to accept or 
control certain conditions, 
1) only process well-fixed tissue 
2) take formalin off this processor, making 14 changes beginning in 50%. If you 
want to keep station 1 formalin and standardize fixation with time X in 
formalin at temp Y, that may work. I prefer to change the tissues to 50% 
alcohol at the grossing bench so that all tissues are not only fixed, but 
rinsed in alcohol and started processing at the growing bench. This simplifies 
processing to dehydration, clearing and paraffin infiltration.  Simple is 
reliable and short processing cycles on this machine leave little room for 
error.
2) Program heat to 50C in all alcohols and xylene. Mixing pressure and vacuum 
yes yes yes 
4) Keep all solutions fresh, rotate after 200- 400 blocks.
5) Lay blocks out not more than 2 cassettes back to back, max 100 for VIP5.
4 hour is reliable for tissues up to 3mm thickness such as punch biopsy. 
2 hour is reliable for up to 1.5 mm tissue thickness such as core biopsies and 
shave biopsies of skin.

Method of processor validation for short cycle in machine previously validated 
in your lab.

To validate create your evaluation form and find a set of cases with sufficient 
tissue small tissue to divide into 2 equal blocks. This can (and will) be done 
on clinical cases if you are careful. 
Print duplicate sets of cassettes. 
Mark one half of case 1 yellow and one half blue (tissues will be processed 
separately, 1 regular and one short cycle and then recombined into 1 final 
block at embedding).
We held the short processed tissue cassettes in formalin and timed the short 
cycle to end when the regular program ends.
Then the two blocks are recombined or embedded together so that direct 
comparisons/measures can be made on one H and one on your most common IHC for 
that tissue. If the H and IHC both work, chances are everything else will 
also.
Modify your regular validation form or Create a form for scoring shrinkage, 
measured thickness and length, staining reactions, shrinkage, holes and flaws, 
have your path score.   photo graph yellow and blue tissues under H and IHC. 
IF you do observe shrinkage or other undesirable artifact, be certain that your 
tissues are completely fixed at grossing and repeat.

If they are comparable in the oculars and to the camera, then show your work 
and voila, there is your validation. 

Steve A. McClain, MD

> 

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Re: [Histonet] Histonet Digest, Vol 145, Issue 15. 6. Solvent recycler (Rachel)

[Steve McClain via Histonet]

BR for 11 years. We recycle alcohol and xylene for reuse on processors.
Extremely reliable.

Steve A. McClain, MD

> On Dec 16, 2015, at 13:11, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> 6. Solvent recycler (Rachel)

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[Histonet] Miniature arrays for control

[Steve McClain via Histonet]

We have used miniature arrays for 9 years. We place both positive and negative 
controls on every IHC. Minimal design is one positive and one negative control.
To start make  a simple one with positive and negative using a  2mm skin punch 
biopsy with plunger to cut out known tissue and mount together.

If you can't punch out tissue, or a valuable control is thin, then one can 
still make a miniature array. 
Make an accordion or fan by cutting a 50 micron section and scrunch together.
Repeat with negative. Repeat with another positive. Wrap the whole thing in 
parafilm and Mount your layers on edge in paraffin. For example HHV8, KS 
marker, we have 5 controls 2 cases of KS, PG, hemangioma and normal skin.

Steve A. McClain, MD

> On Dec 10, 2015, at 13:20, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> We are looking at the possible use of microarrays or tissue rolls for immuno 
> controls on a routine basis. I would like to talk to people who are currently 
> doing their immunos this way.
> We run about 120 slides a day with about 170 different primaries. Anyone with 
> previous experience in this area (good or bad) who has any suggestions, I 
> would be glad to hear about them.
> Thank you,
> Tim Vickroy
> Hershey Medical Center
> Penn State University

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Re: [Histonet] Histonet Digest, Vol 145, Issue 6 Oct separating from epidermis

[Steve McClain via Histonet]

Skin is often difficult to cut. Not sure I have seen that artifact, but here is 
what I use when having difficulties.
 Gently blot the tissue/epidermis dry before immersing in liquid OCT/Neg50. 
Then freeze in cryostat. Avoid refrigerant spray.
Sometimes we allow tissue to sit in oCT for 5 min and then freeze.
If tissue was in 20% sucrose, we blot, then rinse in OCT for 5 min then 
replace/mount in new OCT and freeze.

Steve A. McClain, MD

> On Dec 6, 2015, at 13:10, "histonet-requ...@lists.utsouthwestern.edu" 
>  wrote:
> 
> [Histonet] Oct separating from epidermis

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Re: [Histonet] Histonet Digest, Vol 144, Issue 21: Rapid tissue Programs

[Steve McClain via Histonet]

Since processing is not the issue, first standardize fixation, eg 4-6 hours 
before processing or in the first reagent on processor. Change that first 
formalin every 200 blocks or so.  When well-fixed, small tissues will tolerate 
rapid processing. Finally, if your pathologists can read the slides and your 
immunostains work, then only make small changes. And ignore the CAP.

Steve A. McClain, MD


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[Histonet] Tissue processing validation (Ann Specian)

[Steve McClain via Histonet]

This seems like overkill. Just moving a processor previously validated should 
not require re-validation, just verify the programming and make certain the 
paraffins are melted completely. 
This may not apply to all processors, but for all Sakura machines manufactured 
since 1980,I would not revalidate.

Steve A. McClain, MD
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[Histonet] Decalcification and Neutralization (Huynh,Thomas)

[Steve McClain via Histonet]

I have not found the need for neutralization.
With sufficient washing, common histochemical stains including HE Gram 
PAS-fungus and trichrome are spectacular.
Please explain your protocol for neutralization.

Steve A. McClain, MD

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[Histonet] Decalcification

[Steve McClain via Histonet]

One frequently overlooked point is washing after decal. Especially important 
with acid decal, we wash for an hour in running water. 
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