Re: [Histonet] processing artifact - delayed start

[Ruppert, Amysue via Histonet]

I am wondering if you are getting air bubbles/pockets in the cassettes 
themselves.  Are you using mesh cassettes?  Are you loading the trays with 
cassettes into an empty retort and then starting the delayed program?  If so,  
air pockets could be forming in the cassette and around the tissue pieces.  
Since there is no p/v cycle running yet, those air pockets may be just sitting 
there and the exposed tissue pieces may be getting dried out.

I suggest that once the retort is full and in ambient mode, open retort and 
move the cassette trays around a bit and see if air bubbles are rising from the 
cassettes.

When the trays of cassettes sit on the bench in formalin waiting to go on the 
VIP,  there may be enough movement when moving the container, etc,  that any 
air pockets are disrupted and the tissues are  not exposed to air pockets.

good luck,
amysue ruppert
Marshfield Labs Histology

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Today's Topics:

   1. Re: Processing artifact - delayed start (Bacon, Charles)


--

Message: 1
Date: Mon, 5 Feb 2024 13:07:41 +
From: "Bacon, Charles" 
To: Verizon wireless ,
"histonet@lists.utsouthwestern.edu"
    
Subject: Re: [Histonet] Processing artifact - delayed start
Message-ID:



Content-Type: text/plain; charset="utf-8"

We have had 2 reasons why we saw processing issues like this:

1. I recently found out on our VIP 5 they did not turn the level sensors on 
during install. These sensors are known to error so often, Sakura tells 
technicians to set the default to off. All the processor can sense is pressure 
and time. So it may be that the formalin is not filling all the way. You are 
noticing this on delayed runs, but on those runs are you often stacking trays? 
If so, isolate the cases in the upper trays and review.

2. We found a clogged line. This happens with the formalin lines if you don?t 
do the hot water flush often enough. This can be an even bigger issue if you 
recycle and re-buffer your formalin (we do not).

Good luck!

Chuck Bacon, HTL(ASCP)CM
Supervisor Histology
Baystate Medical Center


-Original Message-
From: Verizon wireless 
Sent: Friday, February 2, 2024 10:37 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing artifact - delayed start

Dear Histonet Members,

We have terrible processing artifact if tissue sits in the formalin-filled 
retort (at ambient temperature) for too long (more than 10-12 hours) before a 
delayed process starts. The longer the wait, the worse it looks. We have Tissue 
Tek VIP 5 processors, and we process luminal gastrointestinal biopsies 
exclusively. I've attached some photomicrographs of problem cases on the 
Histonet Images website (with the same topic title).
This artifact typically affects a few specimens per day (~2% or less), even 
though everything is done on the same processor; it may affect all tissue 
portions in a cassette or only some of the tissue in that cassette. Some tissue 
portions may only have the artifact on one half with the other half looking 
perfectly fine. The techs sometimes note the tissue feeling "crunchy" at the 
time of embedding and / or at the time of microtomy. These tissues tend to 
suffer greater chatter artifact and have trouble sticking to the slides. The 
sections look just as bad on recuts as the originals. Re-processing does not 
seem to help at all.
If the cassettes sit in formalin in a container outside of the processor for 
days before the processor is loaded (with subsequent immediate start), things 
look perfectly fine. When we have staff around to start the processor 
immediately upon loading cassettes and empty immediately upon completion, the 
tissue looks perfectly fine.

Our current processing program is as follows:

1. 10% Formalin, 30 minutes, ambient temp,

Re: [Histonet] Processing artifact - delayed start

[Bacon, Charles via Histonet]

We have had 2 reasons why we saw processing issues like this:

1. I recently found out on our VIP 5 they did not turn the level sensors on 
during install. These sensors are known to error so often, Sakura tells 
technicians to set the default to off. All the processor can sense is pressure 
and time. So it may be that the formalin is not filling all the way. You are 
noticing this on delayed runs, but on those runs are you often stacking trays? 
If so, isolate the cases in the upper trays and review. 

2. We found a clogged line. This happens with the formalin lines if you don’t 
do the hot water flush often enough. This can be an even bigger issue if you 
recycle and re-buffer your formalin (we do not).

Good luck!

Chuck Bacon, HTL(ASCP)CM 
Supervisor Histology
Baystate Medical Center


-Original Message-
From: Verizon wireless  
Sent: Friday, February 2, 2024 10:37 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing artifact - delayed start

Dear Histonet Members,

We have terrible processing artifact if tissue sits in the formalin-filled 
retort (at ambient temperature) for too long (more than 10-12 hours) before a 
delayed process starts. The longer the wait, the worse it looks. We have Tissue 
Tek VIP 5 processors, and we process luminal gastrointestinal biopsies 
exclusively. I've attached some photomicrographs of problem cases on the 
Histonet Images website (with the same topic title). 
This artifact typically affects a few specimens per day (~2% or less), even 
though everything is done on the same processor; it may affect all tissue 
portions in a cassette or only some of the tissue in that cassette. Some tissue 
portions may only have the artifact on one half with the other half looking 
perfectly fine. The techs sometimes note the tissue feeling "crunchy" at the 
time of embedding and / or at the time of microtomy. These tissues tend to 
suffer greater chatter artifact and have trouble sticking to the slides. The 
sections look just as bad on recuts as the originals. Re-processing does not 
seem to help at all. 
If the cassettes sit in formalin in a container outside of the processor for 
days before the processor is loaded (with subsequent immediate start), things 
look perfectly fine. When we have staff around to start the processor 
immediately upon loading cassettes and empty immediately upon completion, the 
tissue looks perfectly fine. 

Our current processing program is as follows:

1. 10% Formalin, 30 minutes, ambient temp, p/v on 2. 10% Formalin, 30 minutes, 
ambient temp, p/v on 3. 65% Alcohol, 10 minutes, ambient temp, p/v on4. 80% 
Alcohol, 10 minutes, ambient temp, p/v on5. 95% Alcohol, 7 minutes, ambient 
temp, p/v on6. 95% Alcohol, 7 minutes, ambient temp, p/v on 7. 100% Alcohol, 10 
minutes, ambient temp, p/v on 8. 100% Alcohol, 10 minutes, ambient temp, p/v on 
9. Xylene, 15 minutes, ambient temp, p/v on 10. Xylene, 15 minutes, ambient 
temp, p/v on 11. Paraffin, 10 minutes, 60 degrees C, p/v on12. Paraffin, 10 
minutes, 60 degrees C, p/v on13. Paraffin, 15 minutes, 60 degrees C, p/v on14. 
Paraffin, 15 minutes, 60 degrees C, p/v on

Obviously there are times we absolutely need to use delayed start. I would 
greatly appreciate guidance, and I'll be happy to provide any other details 
that might be useful.
Sincerely,
Brian Quigley MDLaboratory Director of a GI Pathology Laboratory
  

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Re: [Histonet] Processing artifact - delayed start

[Tony Henwood via Histonet]

I would check the level of the formalin after it has been pumped into the 
retort.

I will wait till the pics are posted

Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.


From: Verizon wireless via Histonet 
Sent: Saturday, February 3, 2024 2:36:42 PM
To: histonet@lists.utsouthwestern.edu 
Subject: [Histonet] Processing artifact - delayed start

Dear Histonet Members,

We have terrible processing artifact if tissue sits in the formalin-filled 
retort (at ambient temperature) for too long (more than 10-12 hours) before a 
delayed process starts. The longer the wait, the worse it looks. We have Tissue 
Tek VIP 5 processors, and we process luminal gastrointestinal biopsies 
exclusively. I've attached some photomicrographs of problem cases on the 
Histonet Images website (with the same topic title).
This artifact typically affects a few specimens per day (~2% or less), even 
though everything is done on the same processor; it may affect all tissue 
portions in a cassette or only some of the tissue in that cassette. Some tissue 
portions may only have the artifact on one half with the other half looking 
perfectly fine. The techs sometimes note the tissue feeling "crunchy" at the 
time of embedding and / or at the time of microtomy. These tissues tend to 
suffer greater chatter artifact and have trouble sticking to the slides. The 
sections look just as bad on recuts as the originals. Re-processing does not 
seem to help at all.
If the cassettes sit in formalin in a container outside of the processor for 
days before the processor is loaded (with subsequent immediate start), things 
look perfectly fine. When we have staff around to start the processor 
immediately upon loading cassettes and empty immediately upon completion, the 
tissue looks perfectly fine.

Our current processing program is as follows:

1. 10% Formalin, 30 minutes, ambient temp, p/v on
2. 10% Formalin, 30 minutes, ambient temp, p/v on
3. 65% Alcohol, 10 minutes, ambient temp, p/v on4. 80% Alcohol, 10 minutes, 
ambient temp, p/v on5. 95% Alcohol, 7 minutes, ambient temp, p/v on6. 95% 
Alcohol, 7 minutes, ambient temp, p/v on 7. 100% Alcohol, 10 minutes, ambient 
temp, p/v on
8. 100% Alcohol, 10 minutes, ambient temp, p/v on
9. Xylene, 15 minutes, ambient temp, p/v on
10. Xylene, 15 minutes, ambient temp, p/v on
11. Paraffin, 10 minutes, 60 degrees C, p/v on12. Paraffin, 10 minutes, 60 
degrees C, p/v on13. Paraffin, 15 minutes, 60 degrees C, p/v on14. Paraffin, 15 
minutes, 60 degrees C, p/v on

Obviously there are times we absolutely need to use delayed start. I would 
greatly appreciate guidance, and I'll be happy to provide any other details 
that might be useful.
Sincerely,
Brian Quigley MDLaboratory Director of a GI Pathology Laboratory

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[Histonet] Processing artifact - delayed start

[Verizon wireless via Histonet]

Dear Histonet Members,

We have terrible processing artifact if tissue sits in the formalin-filled 
retort (at ambient temperature) for too long (more than 10-12 hours) before a 
delayed process starts. The longer the wait, the worse it looks. We have Tissue 
Tek VIP 5 processors, and we process luminal gastrointestinal biopsies 
exclusively. I've attached some photomicrographs of problem cases on the 
Histonet Images website (with the same topic title). 
This artifact typically affects a few specimens per day (~2% or less), even 
though everything is done on the same processor; it may affect all tissue 
portions in a cassette or only some of the tissue in that cassette. Some tissue 
portions may only have the artifact on one half with the other half looking 
perfectly fine. The techs sometimes note the tissue feeling "crunchy" at the 
time of embedding and / or at the time of microtomy. These tissues tend to 
suffer greater chatter artifact and have trouble sticking to the slides. The 
sections look just as bad on recuts as the originals. Re-processing does not 
seem to help at all. 
If the cassettes sit in formalin in a container outside of the processor for 
days before the processor is loaded (with subsequent immediate start), things 
look perfectly fine. When we have staff around to start the processor 
immediately upon loading cassettes and empty immediately upon completion, the 
tissue looks perfectly fine. 

Our current processing program is as follows:

1. 10% Formalin, 30 minutes, ambient temp, p/v on
2. 10% Formalin, 30 minutes, ambient temp, p/v on
3. 65% Alcohol, 10 minutes, ambient temp, p/v on4. 80% Alcohol, 10 minutes, 
ambient temp, p/v on5. 95% Alcohol, 7 minutes, ambient temp, p/v on6. 95% 
Alcohol, 7 minutes, ambient temp, p/v on 7. 100% Alcohol, 10 minutes, ambient 
temp, p/v on
8. 100% Alcohol, 10 minutes, ambient temp, p/v on
9. Xylene, 15 minutes, ambient temp, p/v on
10. Xylene, 15 minutes, ambient temp, p/v on
11. Paraffin, 10 minutes, 60 degrees C, p/v on12. Paraffin, 10 minutes, 60 
degrees C, p/v on13. Paraffin, 15 minutes, 60 degrees C, p/v on14. Paraffin, 15 
minutes, 60 degrees C, p/v on

Obviously there are times we absolutely need to use delayed start. I would 
greatly appreciate guidance, and I'll be happy to provide any other details 
that might be useful.
Sincerely,
Brian Quigley MDLaboratory Director of a GI Pathology Laboratory
  
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Re: [Histonet] Processing artefact??

[Greg Dobbin via Histonet]

Since images cannot be attached, if any of you feel you have some
experience that I may find helpful...please email me directly and I will
attach the image to my reply.
Thanks again,
*Greg Dobbin*

Chief Technologist
Anatomic Pathology
Queen Elizabeth Hospital
Charlottetown, PE, Canada
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[Histonet] Processing artefact??

[Greg Dobbin via Histonet]

Hello experts,
Every now and then we will get stained slides that look like the attached
image. We run one processing schedule (about 11 hrs) for everything so yes,
our smaller biopsies are somewhat overprocessed, but 90-95% of the time
they look perfectly fine. We can have GI biopsies in a case that go from A
to P and find only one that looks like this. Moreover, recuts do not
remedy the problem either...the problem seems to be occurring prior to
microtomy.

We use vendor grade Xylenes in our processor (a Leica ASP6025). All
specimens are fixed for 18-24 hours prior to processing. Our GI blocks get
a short soak in phenol prior to microtomy to help with the effects of
overprocessing. All blocks within a case are treated the same. In fact we
have also seen it in skin biopsies occasionally and skins do not get soaked
in phenol.

It is the randomness of the problem that is throwing us!
Any ideas?
Thanks,
*Greg Dobbin*

Chief Technologist
Anatomic Pathology
Queen Elizabeth Hospital
Charlottetown, PE, Canada
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Re: [Histonet] Processing Protocols

[Tony Reilly via Histonet]

Hi Jessica

My advice would be to process your small biopsies during the day allowing 
depending on the times you use at least 2 runs per day.  One in the am and one 
in the pm.  Then run your large blocks over night as per usual.  Any late 
arriving small biopsies can be run on the am run the following day .  This way 
everything is processed within 24 hours of arrival maintaining your TATs.

Regards

Tony
Retired Histotragic

Sent from my iPhone

> On 4 Aug 2021, at 4:46 am, Terri Braud via Histonet 
>  wrote:
> 
> We have encountered a similar problem with one of our two processors is 
> down.  Shortening the longer schedule produced underprocessed fatty tissues 
> and over processed biopsies, so we run the biopsies on the short schedule 
> overnight, run a quick clean cycle, then the larger tissues on the 10hr 
> schedule on the same instrument, pull the them off and leave them in the 
> embedding unit to embed in the afternoon, or next morning.  It does put the 
> larger tissue a day behind, but does not sacrifice the quality of the 
> processing.  It you shorten your large tissue processing run so that you can 
> run small biopsies with the bigger stuff, you will have to revalidate your 
> IHC, so maybe this might be a better alternative.  Good luck, Terri
> 
> Terri L. Braud, HT(ASCP)
> HNL Laboratories for 
> Holy Redeemer Hospital
> 1648 Huntingdon Pike
> Meadowbrook, PA 19046
> Ph: 215-938-3689
> Fax: 215-938-3874
>   Honesty
> AccouNtability
> AgiLity
> CoLlaboration
>   CoMpassion
> 
> Message: 1
> Date: Tue, 3 Aug 2021 10:34:11 +
> From: "Piche, Jessica" 
> Subject: [Histonet] Processing protocols
> 
> Good Morning Everyone,
> Our pathologist wants us to shorten our large tissue processing run so that 
> we can run small biopsies with the bigger stuff when one of our tissue 
> processors are down. We have 2 Excelsiors. Would anyone like to share any 
> protocols they might have for this purpose?
> Thank you!
> Jessica Piche, HT(ASCP}
> Waterbury Hospital Histology Lab
> 
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Re: [Histonet] Processing Protocols

[Terri Braud via Histonet]

We have encountered a similar problem with one of our two processors is down.  
Shortening the longer schedule produced underprocessed fatty tissues and over 
processed biopsies, so we run the biopsies on the short schedule overnight, run 
a quick clean cycle, then the larger tissues on the 10hr schedule on the same 
instrument, pull the them off and leave them in the embedding unit to embed in 
the afternoon, or next morning.  It does put the larger tissue a day behind, 
but does not sacrifice the quality of the processing.  It you shorten your 
large tissue processing run so that you can run small biopsies with the bigger 
stuff, you will have to revalidate your IHC, so maybe this might be a better 
alternative.  Good luck, Terri

Terri L. Braud, HT(ASCP)
HNL Laboratories for 
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
Ph: 215-938-3689
Fax: 215-938-3874
  Honesty
AccouNtability
    AgiLity
    CoLlaboration
  CoMpassion

Message: 1
Date: Tue, 3 Aug 2021 10:34:11 +
From: "Piche, Jessica" 
Subject: [Histonet] Processing protocols

Good Morning Everyone,
Our pathologist wants us to shorten our large tissue processing run so that we 
can run small biopsies with the bigger stuff when one of our tissue processors 
are down. We have 2 Excelsiors. Would anyone like to share any protocols they 
might have for this purpose?
Thank you!
Jessica Piche, HT(ASCP}
Waterbury Hospital Histology Lab

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[Histonet] Processing protocols

[Piche, Jessica via Histonet]

Good Morning Everyone,

Our pathologist wants us to shorten our large tissue processing run so that we 
can run small biopsies with the bigger stuff when one of our tissue processors 
are down. We have 2 Excelsiors. Would anyone like to share any protocols they 
might have for this purpose?

Thank you!

Jessica Piche, HT(ASCP}
Waterbury Hospital Histology Lab




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Re: [Histonet] Processing correction

[Thomas Podawiltz via Histonet]

Recovering Tissue That Has Dried Out After Malfunction of Tissue Processor 
  
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|   
|   
|   ||

   |

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|  
|   |  
Recovering Tissue That Has Dried Out After Malfunction of Tissue Processor
 

  |   |

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  |

  


Sent from Yahoo Mail for iPhone


On Monday, March 1, 2021, 2:10 PM, Charles Riley via Histonet 
 wrote:

We had an issue last week where the tech loading the processor forgot to start 
the machine. The tissues we left in an empty chamber for around 8/9 hours after 
having been in formalin from anywhere from 3 hours to 10 hours from the 
surgical sites.

All of the specimens are unreadable. What steps can be taken (if any) to try 
and salvage the samples and get better slides for the pathologists to read?
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[Histonet] Processing correction

[Charles Riley via Histonet]

We had an issue last week where the tech loading the processor forgot to start 
the machine. The tissues we left in an empty chamber for around 8/9 hours after 
having been in formalin from anywhere from 3 hours to 10 hours from the 
surgical sites.

All of the specimens are unreadable. What steps can be taken (if any) to try 
and salvage the samples and get better slides for the pathologists to read?
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[Histonet] Processing descemet's membrane

[Terri Braud via Histonet]

Hello Histopeeps -
I need your help, please.  I need to improve our processing of Descemet's 
membrane, more specifically keeping track of that little wisp of clear 
colorless tissue?  Do you have any tricks to help keep track of it through 
processing?  Inks?  Embedding gel? Anything?
I'm open for any ideas to make this process easier.  Thanks in advance, Terri

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874
Care, Comfort, and Heal

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Re: [Histonet] Processing Schedule- ASP-6025

[Tony Auge via Histonet]

What are you using for your lab's own control. Was that tissue processed
recently? Do you have normal tonsil that you process  in-house regularly
that also stains poorly?



On Wed, May 8, 2019 at 9:16 AM Greg Dobbin via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Great point John!
> We do attempt to have all of the specimen handling for our control tissue,
> match that of our specimens. However,
> tonsillectomies at our hospital are only performed on Thursdays. So we fix
> them for 24hrs (until Friday) then process overnight and remove
> from the processor on Saturday (we don't typically work Saturdays). And I
> have done this a couple of times but can I swear that this particular
> tonsil
> was handled that way? I cannot ...which I know, points to another
> deficiency! :-0  It could very well be that the fixation times of the two
> tonsils I am comparing are sufficiently different that they will require
> different retrieval times to produce stains of similar intensity.
>
> I also appreciate getting your opinion on the processing schedule. You have
> much more experience than I!! Thank you for your insights.
> All the best,
> Greg
>
> On Wed, May 8, 2019 at 10:50 AM John Garratt  wrote:
>
> > The processing schedule looks fine. I am thinking that if you are having
> > no problems with morphology on the H processing is not the issue. The
> > extended period in the warming draw is a good hypothesis.
> > I do have a question. How long was that particular piece of tonsil kept
> in
> > formalin before processing? Could over fixation be the issue?
> > I have found that maintaining tight control of control tissues is
> > important which includes minimum and maximum fixation time.
> >
> > John
> >
> > Sent from ProtonMail Mobile
> >
> >
> > On Tue, May 7, 2019 at 5:52 PM, Greg Dobbin via Histonet <
> > histonet@lists.utsouthwestern.edu> wrote:
> >
> > Fascinating Tony!
> > We don’t typically leave them in the warming drawer any longer than a
> > couple of hours, but maybe this particular tonsil was left linger for
> some
> > reason and no one thought anything of it?! Something to consider for
> sure!
> > Thanks.
> > Greg
> >
> > On Tue, May 7, 2019 at 9:37 PM Tony Henwood (SCHN) <
> > tony.henw...@health.nsw.gov.au> wrote:
> >
> > > Processing seems adequate.
> > >
> > > After processing, how long do they sit in the embedding centre block
> > > holding tank before embedding?
> > >
> > > We found that quite a few antigens were affected when we stored control
> > > tonsil in the embedding centre (dry) at 60oC for a few days before
> > > embedding. In summary:
> > >
> > > Antibody Clone Dried (Normal = 3+)
> > > CD4 4B12 0
> > > BOB-1 TG14 0
> > > CD3 LN10 1+
> > > CD79a JCB117 1+
> > > Oct-2 Oct-207 1+
> > > CD8 4B11 2+
> > > CD20 L26 3+
> > >
> > > So CD20 was unaffected but this process affected most of the antigens
> > with
> > > some losing antigen recognition by the antibody (eg CD4 and BOB-1).
> > >
> > > Another one of those pre-analytical issues we need to consider.
> > >
> > > And yes I am writing this up for submission!
> > >
> > >
> > > Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) |
> > > Principal Scientist; Adjunct Fellow, School of Medicine, University of
> > > Western Sydney; Visiting Lecturer, School of Life Sciences, Faculty of
> > > Science, University of Technology Sydney | Histopathology
> > > t: (02) 9845 3306 | f: (02) 9845 3318 | e:
> > tony.henw...@health.nsw.gov.au
> > > | w: www.schn.health.nsw.gov.au
> > > m:
> > >
> > >
> > > Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
> > > Locked Bag 4001, Westmead 2145, NSW Australia
> > >
> > > ♲ Please consider the environment before printing this email.
> > >
> > > -Original Message-
> > > From: Greg Dobbin via Histonet [mailto:
> histonet@lists.utsouthwestern.edu
> > ]
> > > Sent: Wednesday, 8 May 2019 5:07 AM
> > > To: histonet@lists.utsouthwestern.edu
> > > Subject: [Histonet] Processing Schedule- ASP-6025
> > >
> > > Hello colleagues,
> > > I recently stained (IHC) a section of normal tonsil from another
> facility
> > > with p16 and the resulting stain was better than the same stain on a
> > > section of my labs own normal tonsil control.
> > >
> > > This 

Re: [Histonet] Processing Schedule- ASP-6025

[Greg Dobbin via Histonet]

Great point John!
We do attempt to have all of the specimen handling for our control tissue,
match that of our specimens. However,
tonsillectomies at our hospital are only performed on Thursdays. So we fix
them for 24hrs (until Friday) then process overnight and remove
from the processor on Saturday (we don't typically work Saturdays). And I
have done this a couple of times but can I swear that this particular
tonsil
was handled that way? I cannot ...which I know, points to another
deficiency! :-0  It could very well be that the fixation times of the two
tonsils I am comparing are sufficiently different that they will require
different retrieval times to produce stains of similar intensity.

I also appreciate getting your opinion on the processing schedule. You have
much more experience than I!! Thank you for your insights.
All the best,
Greg

On Wed, May 8, 2019 at 10:50 AM John Garratt  wrote:

> The processing schedule looks fine. I am thinking that if you are having
> no problems with morphology on the H processing is not the issue. The
> extended period in the warming draw is a good hypothesis.
> I do have a question. How long was that particular piece of tonsil kept in
> formalin before processing? Could over fixation be the issue?
> I have found that maintaining tight control of control tissues is
> important which includes minimum and maximum fixation time.
>
> John
>
> Sent from ProtonMail Mobile
>
>
> On Tue, May 7, 2019 at 5:52 PM, Greg Dobbin via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
> Fascinating Tony!
> We don’t typically leave them in the warming drawer any longer than a
> couple of hours, but maybe this particular tonsil was left linger for some
> reason and no one thought anything of it?! Something to consider for sure!
> Thanks.
> Greg
>
> On Tue, May 7, 2019 at 9:37 PM Tony Henwood (SCHN) <
> tony.henw...@health.nsw.gov.au> wrote:
>
> > Processing seems adequate.
> >
> > After processing, how long do they sit in the embedding centre block
> > holding tank before embedding?
> >
> > We found that quite a few antigens were affected when we stored control
> > tonsil in the embedding centre (dry) at 60oC for a few days before
> > embedding. In summary:
> >
> > Antibody Clone Dried (Normal = 3+)
> > CD4 4B12 0
> > BOB-1 TG14 0
> > CD3 LN10 1+
> > CD79a JCB117 1+
> > Oct-2 Oct-207 1+
> > CD8 4B11 2+
> > CD20 L26 3+
> >
> > So CD20 was unaffected but this process affected most of the antigens
> with
> > some losing antigen recognition by the antibody (eg CD4 and BOB-1).
> >
> > Another one of those pre-analytical issues we need to consider.
> >
> > And yes I am writing this up for submission!
> >
> >
> > Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) |
> > Principal Scientist; Adjunct Fellow, School of Medicine, University of
> > Western Sydney; Visiting Lecturer, School of Life Sciences, Faculty of
> > Science, University of Technology Sydney | Histopathology
> > t: (02) 9845 3306 | f: (02) 9845 3318 | e:
> tony.henw...@health.nsw.gov.au
> > | w: www.schn.health.nsw.gov.au
> > m:
> >
> >
> > Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
> > Locked Bag 4001, Westmead 2145, NSW Australia
> >
> > ♲ Please consider the environment before printing this email.
> >
> > -Original Message-
> > From: Greg Dobbin via Histonet [mailto:histonet@lists.utsouthwestern.edu
> ]
> > Sent: Wednesday, 8 May 2019 5:07 AM
> > To: histonet@lists.utsouthwestern.edu
> > Subject: [Histonet] Processing Schedule- ASP-6025
> >
> > Hello colleagues,
> > I recently stained (IHC) a section of normal tonsil from another facility
> > with p16 and the resulting stain was better than the same stain on a
> > section of my labs own normal tonsil control.
> >
> > This has led us to question our processing schedule. I am not concerned
> > with our fixation because we fix everything for at least 24 hours in 10%
> > formalin (commercially prepared) prior to processing.
> >
> > Does anything jump out at you as being a potential red flag in the
> > following overnight protocol?
> >
> > - Formalin 15 mins; RT
> > - Processing water 1 min; RT
> > - ETOH 70% 30 mins; 35C
> > - ETOH 80% 30 mins; 35C
> > - ETOH 95% 30 mins; 35C
> > - ETOH 100% 30 mins; 35C
> > - ETOH 100% 40 mins; 35C
> > - ETOH 100% 60 mins; 35C
> > - Xylene 60 mins; 35C
> > - Xylene 60 mins; 35C
> > - Xylene 60 mins; 35C
> > - Paraffin 60 mins; 57C; vacuum
> > - Paraffin 60 mins; 57C;

Re: [Histonet] Processing Schedule- ASP-6025

[John Garratt via Histonet]

The processing schedule looks fine. I am thinking that if you are having no 
problems with morphology on the H processing is not the issue. The extended 
period in the warming draw is a good hypothesis.
I do have a question. How long was that particular piece of tonsil kept in 
formalin before processing? Could over fixation be the issue?
I have found that maintaining tight control of control tissues is important 
which includes minimum and maximum fixation time.

John

Sent from ProtonMail Mobile

On Tue, May 7, 2019 at 5:52 PM, Greg Dobbin via Histonet 
 wrote:

> Fascinating Tony!
> We don’t typically leave them in the warming drawer any longer than a
> couple of hours, but maybe this particular tonsil was left linger for some
> reason and no one thought anything of it?! Something to consider for sure!
> Thanks.
> Greg
>
> On Tue, May 7, 2019 at 9:37 PM Tony Henwood (SCHN) <
> tony.henw...@health.nsw.gov.au> wrote:
>
>> Processing seems adequate.
>>
>> After processing, how long do they sit in the embedding centre block
>> holding tank before embedding?
>>
>> We found that quite a few antigens were affected when we stored control
>> tonsil in the embedding centre (dry) at 60oC for a few days before
>> embedding. In summary:
>>
>> Antibody Clone Dried (Normal = 3+)
>> CD4 4B12 0
>> BOB-1 TG14 0
>> CD3 LN10 1+
>> CD79a JCB117 1+
>> Oct-2 Oct-207 1+
>> CD8 4B11 2+
>> CD20 L26 3+
>>
>> So CD20 was unaffected but this process affected most of the antigens with
>> some losing antigen recognition by the antibody (eg CD4 and BOB-1).
>>
>> Another one of those pre-analytical issues we need to consider.
>>
>> And yes I am writing this up for submission!
>>
>>
>> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) |
>> Principal Scientist; Adjunct Fellow, School of Medicine, University of
>> Western Sydney; Visiting Lecturer, School of Life Sciences, Faculty of
>> Science, University of Technology Sydney | Histopathology
>> t: (02) 9845 3306 | f: (02) 9845 3318 | e: tony.henw...@health.nsw.gov.au
>> | w: www.schn.health.nsw.gov.au
>> m:
>>
>>
>> Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
>> Locked Bag 4001, Westmead 2145, NSW Australia
>>
>> ♲ Please consider the environment before printing this email.
>>
>> -Original Message-
>> From: Greg Dobbin via Histonet [mailto:histonet@lists.utsouthwestern.edu]
>> Sent: Wednesday, 8 May 2019 5:07 AM
>> To: histonet@lists.utsouthwestern.edu
>> Subject: [Histonet] Processing Schedule- ASP-6025
>>
>> Hello colleagues,
>> I recently stained (IHC) a section of normal tonsil from another facility
>> with p16 and the resulting stain was better than the same stain on a
>> section of my labs own normal tonsil control.
>>
>> This has led us to question our processing schedule. I am not concerned
>> with our fixation because we fix everything for at least 24 hours in 10%
>> formalin (commercially prepared) prior to processing.
>>
>> Does anything jump out at you as being a potential red flag in the
>> following overnight protocol?
>>
>> - Formalin 15 mins; RT
>> - Processing water 1 min; RT
>> - ETOH 70% 30 mins; 35C
>> - ETOH 80% 30 mins; 35C
>> - ETOH 95% 30 mins; 35C
>> - ETOH 100% 30 mins; 35C
>> - ETOH 100% 40 mins; 35C
>> - ETOH 100% 60 mins; 35C
>> - Xylene 60 mins; 35C
>> - Xylene 60 mins; 35C
>> - Xylene 60 mins; 35C
>> - Paraffin 60 mins; 57C; vacuum
>> - Paraffin 60 mins; 57C; vacuum
>> - Paraffin 60 mins; 57C; vacuum
>>
>> Our formalin is changed after every 1100 cassettes and the alcohol,
>> xylenes and paraffins are managed similarly by the instrument. Our specimen
>> mix is a little of everything (skins, GIs, breasts, needle cores, gall
>> bladders, gyne, etc).
>>
>> The one unknown (so far) in this story, is how the tonsil from the other
>> laboratory was handled (ie the fixative used and for how long-I am assuming
>> 10% formalin).
>>
>> Obviously, many of you will have schedules that differ from this one, in
>> any number of ways, but what I am looking for from you is your opinion: *is
>> there anything about this schedule that is particularly concerning?* Thank
>> you, Greg
>>
>>
>> --
>> *Greg Dobbin*
>> 1205 Pleasant Grove Rd
>> <https://maps.google.com/?q=1205+Pleasant+Grove+Rd=gmail=g>
>> RR#2 York,
>> PE C0A 1P0
>>
>>
>> *Everything in moderation...even moderation itself**!*
>> _

Re: [Histonet] Processing Schedule- ASP-6025

[Tony Henwood (SCHN) via Histonet]

Processing seems adequate.

After processing, how long do they sit in the embedding centre block holding 
tank before embedding?

We found that quite a few antigens were affected when we stored control tonsil 
in the embedding centre (dry) at 60oC for a few days before embedding. In 
summary:

AntibodyClone   Dried (Normal = 3+)
CD4 4B120
BOB-1   TG140
CD3 LN101+
CD79a   JCB117  1+
Oct-2   Oct-207 1+
CD8 4B112+
CD20L26 3+

So CD20 was unaffected but this process affected most of the antigens with some 
losing antigen recognition by the antibody (eg CD4 and BOB-1).

Another one of those pre-analytical issues we need to consider.

And yes I am writing this up for submission!


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) | Principal 
Scientist; Adjunct Fellow, School of Medicine, University of Western Sydney; 
Visiting Lecturer, School of Life Sciences, Faculty of Science, University of 
Technology Sydney | Histopathology 
t: (02) 9845 3306 | f: (02) 9845 3318 | e: tony.henw...@health.nsw.gov.au | w: 
www.schn.health.nsw.gov.au
m: 


Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
Locked Bag 4001, Westmead 2145, NSW Australia

♲  Please consider the environment before printing this email.

-Original Message-
From: Greg Dobbin via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 8 May 2019 5:07 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing Schedule- ASP-6025

Hello  colleagues,
I recently stained (IHC) a section of normal tonsil from another facility with 
p16 and the resulting stain was better than the same stain on a section of my 
labs own normal tonsil control.

This has led us to question our processing schedule. I am not concerned with 
our fixation because we fix everything for at least 24 hours in 10% formalin 
(commercially prepared) prior to processing.

Does anything jump out at you as being a potential red flag in the following 
overnight protocol?

   - Formalin 15 mins; RT
   - Processing water 1 min; RT
   - ETOH 70% 30 mins; 35C
   - ETOH 80% 30 mins; 35C
   - ETOH 95% 30 mins; 35C
   - ETOH 100% 30 mins; 35C
   - ETOH 100% 40 mins; 35C
   - ETOH 100% 60 mins; 35C
   - Xylene  60 mins; 35C
   - Xylene  60 mins; 35C
   - Xylene  60 mins; 35C
   - Paraffin 60 mins; 57C; vacuum
   - Paraffin 60 mins; 57C; vacuum
   - Paraffin 60 mins; 57C; vacuum

Our formalin is changed after every 1100 cassettes and the alcohol, xylenes and 
paraffins are managed similarly by the instrument. Our specimen mix is a little 
of everything (skins, GIs, breasts, needle cores, gall bladders, gyne, etc).

The one unknown (so far) in this story, is how the tonsil from the other 
laboratory was handled (ie the fixative used and for how long-I am assuming 10% 
formalin).

Obviously, many of you will have schedules that differ from this one, in any 
number of ways, but what I am looking for from you is your opinion: *is there 
anything about this schedule that is particularly concerning?* Thank you, Greg


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


*Everything in moderation...even moderation itself**!*
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[Histonet] Processing Schedule- ASP-6025

[Greg Dobbin via Histonet]

Hello  colleagues,
I recently stained (IHC) a section of normal tonsil from another facility
with p16 and the resulting stain was better than the same stain on a
section of my labs own normal tonsil control.

This has led us to question our processing schedule. I am not concerned
with our fixation because we fix everything for at least 24 hours in 10%
formalin (commercially prepared) prior to processing.

Does anything jump out at you as being a potential red flag in the
following overnight protocol?

   - Formalin 15 mins; RT
   - Processing water 1 min; RT
   - ETOH 70% 30 mins; 35C
   - ETOH 80% 30 mins; 35C
   - ETOH 95% 30 mins; 35C
   - ETOH 100% 30 mins; 35C
   - ETOH 100% 40 mins; 35C
   - ETOH 100% 60 mins; 35C
   - Xylene  60 mins; 35C
   - Xylene  60 mins; 35C
   - Xylene  60 mins; 35C
   - Paraffin 60 mins; 57C; vacuum
   - Paraffin 60 mins; 57C; vacuum
   - Paraffin 60 mins; 57C; vacuum

Our formalin is changed after every 1100 cassettes and the alcohol, xylenes
and paraffins are managed similarly by the instrument. Our specimen mix is
a little of everything (skins, GIs, breasts, needle cores, gall bladders,
gyne, etc).

The one unknown (so far) in this story, is how the tonsil from the other
laboratory was handled (ie the fixative used and for how long-I am assuming
10% formalin).

Obviously, many of you will have schedules that differ from this one, in
any number of ways, but what I am looking for from you is your opinion: *is
there anything about this schedule that is particularly concerning?*
Thank you,
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] Processing Veterinary Samples

[e wayne johnson via Histonet]

The vet samples can be processed just any other animal tissue whether they be 
human or rat. People is animals too. Formalin fixed there are no special safety 
issues. E. Wayne Johnson DVM Enable AgTech Beijing ewj Email:e...@pigs.ag 
Signature is customized by Netease Mail Master On 04/16/2019 08:05, Miranda 
Giorgi via Histonet wrote: Hello, Our AP lab was recently approached by a 
Veterinary Pathologist to process, embed, and cut H slides of their 
postmortem animal samples.  If we consider taking on this work are there any 
concerns with variation in processing or safety protocols we might need to be 
aware of with regards to veterinary samples compared to human samples? Any 
information or experience you can share would be appreciated.  Thank you! 
Miranda Giorgi, HTL (ASCP)cm Histology Manager Incyte Diagnostics 509-892-2744 
This e-mail and any attachments may contain CONFIDENTIAL information, including 
PROTECTED HEALTH INFORMATION. If you are not the intended recipient, any use or 
disclosure of this information is STRICTLY PROHIBITED; you are requested to 
delete this e-mail and any attachments, notify the sender immediately, and 
notify the InCyte Privacy Officer at priv...@incdx.com or call (509) 892-2700. 
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[Histonet] Processing Veterinary Samples

[Miranda Giorgi via Histonet]

Hello,

Our AP lab was recently approached by a Veterinary Pathologist to process, 
embed, and cut H slides of their postmortem animal samples.  If we consider 
taking on this work are there any concerns with variation in processing or 
safety protocols we might need to be aware of with regards to veterinary 
samples compared to human samples?

Any information or experience you can share would be appreciated.  Thank you!

Miranda Giorgi, HTL (ASCP)cm
Histology Manager
Incyte Diagnostics
509-892-2744
This e-mail and any attachments may contain CONFIDENTIAL information, including 
PROTECTED HEALTH INFORMATION. If you are not the intended recipient, any use or 
disclosure of this information is STRICTLY PROHIBITED; you are requested to 
delete this e-mail and any attachments, notify the sender immediately, and 
notify the InCyte Privacy Officer at priv...@incdx.com or call (509) 892-2700.
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Re: [Histonet] processing Dosophilla flies

[Hobbs, Carl via Histonet]

Hi
Processing them , how?

To Pwax?
To snapfreeze?
To resin?

Kind regards

Carl


  
 
Carl Hobbs FIBMS 
Histology and Imaging Manager 
Wolfson CARD 
Guys Campus, London Bridge  
Kings College London 
London 
SE1 1UL 
  
020 7848 6813
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[Histonet] processing Dosophilla flies

[Colleen Forster via Histonet]

Can anyone give me a processing schedule for Drosophila flies...very tiny.

Thank you in advance!

Colleen Forster HT(ASCP)QIHC
U of MN
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Re: [Histonet] Processing cystic tissue

[Bob Richmond via Histonet]

Charles Riley HT, HTL(ASCP)CM, a Mohs histopathology coordinator, asks:

>>Can anyone give me an idea how they process cystic tissues? The normal tissue
processes extremely well on my current protocol but the cystic areas just
are too soft to cut. Any tricks anyone can provide to process better or cut
better after processing would be greatly appreciated.<<

Whoever's grossing should remove most of the keratinous contents of an
epidermal inclusion cyst (or the contents of a pilar cyst), leaving a small
amount around the edge so that you don't disrupt the lining much. That's
what I do for myself. There's nothing to see in the keratin, and it
interferes with getting a good section.

The same reasoning applies to other cystic material, such as ovarian cysts.

Bob Richmond
Samurai Pathologist
Maryville TN
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Re: [Histonet] Processing cystic tissue

[Ingles Claire via Histonet]

Charles:
I usually get out as much of the cystic material as I can without damaging the 
cyst wall itself. That is all the docs are looking at anyway. I am assuming you 
are referring to skin cysts, not ovaries, etc.

Claire


From: Charles Riley via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Tuesday, April 04, 2017 9:05 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing cystic tissue

Can anyone give me an idea how they process cystic tissues. THe normal
tissue processes extremely well on my current protocol but the cystic areas
just are too soft to cut. Any tricks anyone can provide to process better
or cut better after processing would be greatly appreciated

--

Charles Riley HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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[Histonet] Processing cystic tissue

[Charles Riley via Histonet]

Can anyone give me an idea how they process cystic tissues. THe normal
tissue processes extremely well on my current protocol but the cystic areas
just are too soft to cut. Any tricks anyone can provide to process better
or cut better after processing would be greatly appreciated

-- 

Charles Riley HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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[Histonet] Processing of frozen material

[Julio Benavides via Histonet]

Hi there,

I have frozen (-20ºC) samples  from skeletal muscle (sheep samples)  
that now requiere processing and cutting for HE and IHC. Any advice on  
how to proceed? better to put them in formalin while still frozen or  
first thaw them? A good way to thaw them preserving the most of the  
histological architecture?



Thanks a lot for your help and have a very nice Chritsmas. All the  
best for 2017!


Julio




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[Histonet] Processing time for small biopsies

[Gareth Davis via Histonet]

Hi,
Recently I saw a post from someone looking for a protocol for processing
small biopsies.  I looked on the histonet search, but found that to be too
time consuming to locate the post.  Anyway, I hoping to get some opinions
on good process times for small biopsies.  I recently changed my paraffin
brand and now I have chatter on my sections.


-- 
Ms. Gareth B. Davis, HT, QIHC (ASCPcm)
Yuma Gastroenterology
Yuma, AZ 85364
928-248-5259
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[Histonet] processing question

[Blanca Lopez via Histonet]

If I have tissues being held in PBS after fixation. It is ok to load them in 
the processor starting with formalin or Do I need to skip this step and started 
in alcohol?

Blanca Lopez
Histotech (ASCP)
UTSW Tissue Resource K1.210
Simmons Comprehensive Cancer Center
UT Southwestern Medical Center
Telephone: 214-648-7598
Email: blanca.lo...@utsouthwestern.edu




UT Southwestern


Medical Center



The future of medicine, today.

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Re: [Histonet] processing cell blocks in formalin

[Cartun, Richard via Histonet]

Yes, I will send you the procedure that we use.

Richard

Richard W. Cartun, MS, PhD
Director, Histology & The Martin M. Berman, MD Immunopathology & Morphologic 
Proteomics Laboratory
Director, Biospecimen Collection Programs
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 972-1596
(860) 545-2204 Fax

-Original Message-
From: Algeo, Lacie A via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, November 01, 2016 3:10 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] processing cell blocks in formalin

Hi All,
Does anyone have a procedure for processing cellblocks in formalin that works 
well?
Thanks,
Lacie

Lacie Algeo, HTL (ASCP) MBCM
Histology Supervisor
Providence Sacred Heart Medical Center Laboratory
101 W 8th Avenue
L-2
Spokane, WA 99204
509-474-4418
FAX 509-474-2052
lacie.al...@providence.org<mailto:lacie.al...@providence.org>


This message is intended for the sole use of the addressee, and may contain 
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information contained in the message.  If you have received this message in 
error, please immediately advise the sender by reply e-mail and delete this 
message.




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message.
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[Histonet] processing cell blocks in formalin

[Algeo, Lacie A via Histonet]

Hi All,
Does anyone have a procedure for processing cellblocks in formalin that works 
well?
Thanks,
Lacie

Lacie Algeo, HTL (ASCP) MBCM
Histology Supervisor
Providence Sacred Heart Medical Center Laboratory
101 W 8th Avenue
L-2
Spokane, WA 99204
509-474-4418
FAX 509-474-2052
lacie.al...@providence.org


This message is intended for the sole use of the addressee, and may contain 
information that is priviledged, confidential and exempt from disclosure under 
applicable law.  If you are not the addressee, you are hereby notified that you 
may not use, copy, disclose or distribute to anyone the message or any 
information contained in the message.  If you have received this message in 
error, please immediately advise the sender by reply e-mail and delete this 
message.




This message is intended for the sole use of the addressee, and may contain 
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applicable law. If you are not the addressee you are hereby notified that you 
may not use, copy, disclose, or distribute to anyone the message or any 
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[Histonet] processing cross-contamination

[Sanders, Jeanine (CDC/OID/NCEZID) via Histonet]

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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[Histonet] Processing Canine Nasal Passages

[Wilson, Carol via Histonet]

Good Afternoon,
I am looking for information on collection (ie. Upper jaw to eye socket?), 
decalcification(approximate timing, tips?), trimming (any tips/suggestions) and 
processing (without mega cassettes if possible) canine FFPE tissues.  If 
someone can please give me some suggestions or ideas I would appreciate it.  Or 
direction to an appropriate article?  Thanks in advance.
Carol

Carol Wilson, HT(ASCP)
Associate Scientist III
Team Leader/Histopathology
Ricerca Biosciences, LLC

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Re: [Histonet] Processing FFPE tissue without alcohol

[Dessasau III, Evan via Histonet]

Will they be able to do IHC stains?

-Original Message-
From: Wanda Shotsberger Gray via Histonet 
[mailto:histonet@lists.utsouthwestern.edu]
Sent: Saturday, March 26, 2016 12:56 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing FFPE tissue without alcohol


While the tissue will still go through alcohol, have you considered preserving 
the fat with osmium tetroxide prior to routine processing? This turns the fat 
black, but it is retained in the tissue.


Hi Histonet, is there a way to process tissue for paraffin embedding without 
using alcohol? One of the labs that send their processing to us will be doing a 
study examining fat. I told them if they want to look at the fat they will have 
to cut frozen sections but I'm being ask again about processing without the 
alcohol. So I said I would ask around.

Please let me know what you think.

Thank you for your help!

E-van



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[Histonet] Processing FFPE tissue without alcohol

[Wanda Shotsberger Gray via Histonet]

While the tissue will still go through alcohol, have you considered 
preserving the fat with osmium tetroxide prior to routine processing? This 
turns the fat black, but it is retained in the tissue.



Hi Histonet, is there a way to process tissue for paraffin embedding 
without using alcohol? One of the labs that send their processing to us 
will be doing a study examining fat. I told them if they want to look at 
the fat they will have to cut frozen sections but I'm being ask again about 
processing without the alcohol. So I said I would ask around.


Please let me know what you think.

Thank you for your help!

E-van



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Re: [Histonet] Processing issues

[Mca Werdler via Histonet]

Hey Charles,

Why are the first 2 steps of formalin that long? If you make sure your
tissue is well fixated, then there is no need for such long fixation in the
machine. You could use step 2 for just washing the formalin out, so for
only like 5 minutes. You could use that time, to add to other chemicals.

Hope it works for you.

Maarten Werdler

2016-03-22 9:26 GMT-06:00 Charles Riley via Histonet <
histonet@lists.utsouthwestern.edu>:

> We are having issues with our tissue processing. We use the thermo shandon
> excelsior processor. All steps have agitation and under vacuum. Our routine
> process is  as follows
>
> 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.
>
> Can anyone give any suggestions for altering this process to work better? I
> know the process should probably be longer however medical director does
> not want to delay turn around time. If possible keep the process to under 9
> hours. If this isn't conducive to consistent processing please explain why
> so  I can show my superiors to give myself some leverage to change things
> --
>
> Charles Riley HT(ASCP)CM
>
> Histopathology Coordinator/ Mohs
>
> Doctors Pathology Services, Dover DE
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Re: [Histonet] Processing issues

[WILLIAM DESALVO via Histonet]

Not knowing what issues you have, I suggest you look to your samples for 
processing. Reduce thickness and the shorter times will work. 2-2.5 mm thick. 
Standardize fixation before placing in tissue processor. What time is the 
processor started and what time does the tech remove? You may be able to reduce 
delay and increase alcohols. Specimen thickness will reduce the the time for 
solution to move through the tissue sample and allow exchange of solution. 
There are many options, but be more specific about the problem and you can 
narrow the changes

Sent from my Windows Phone

From: Charles Riley via Histonet<mailto:histonet@lists.utsouthwestern.edu>
Sent: ‎3/‎22/‎2016 8:57 AM
To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: [Histonet] Processing issues

We are having issues with our tissue processing. We use the thermo shandon
excelsior processor. All steps have agitation and under vacuum. Our routine
process is  as follows

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.

Can anyone give any suggestions for altering this process to work better? I
know the process should probably be longer however medical director does
not want to delay turn around time. If possible keep the process to under 9
hours. If this isn't conducive to consistent processing please explain why
so  I can show my superiors to give myself some leverage to change things
--

Charles Riley HT(ASCP)CM

Histopathology Coordinator/ Mohs

Doctors Pathology Services, Dover DE
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[Histonet] Processing issues

[Charles Riley via Histonet]

We are having issues with our tissue processing. We use the thermo shandon
excelsior processor. All steps have agitation and under vacuum. Our routine
process is  as follows

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.

Can anyone give any suggestions for altering this process to work better? I
know the process should probably be longer however medical director does
not want to delay turn around time. If possible keep the process to under 9
hours. If this isn't conducive to consistent processing please explain why
so  I can show my superiors to give myself some leverage to change things
-- 

Charles Riley HT(ASCP)CM

Histopathology Coordinator/ Mohs

Doctors Pathology Services, Dover DE
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[Histonet] Processing human brain xenografts

[Murphy, Valerie via Histonet]

Hello Histonetters,
I was wondering if anyone could offer advice for the  processing of our  human 
brain xenografts harvested from mice. The tumor specimens are approximately 0.5 
x 0.5 x 0.5cm.  We have been putting them on our animal program ( 30 min 
alcohols) but once embedded, the tissue looks brittle and some shrinkage 
occurs. The morphology looks okay. S hould we put them on our routine program ( 
1 hour ethanols and xylenes) or is brain tissue too delicate for that run?
Would appreciate any advice.

Thank you,

Valerie

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Re: [Histonet] Processing cytology and cell block protocols

[Terri Braud via Histonet]

We are a similar sized hospital with an 8,000/yr Surgical load of mixed large 
and small cases.  We process 1000 Non-gyn Cytology cases, assist with FNA 
collection in Interventional Radiology.  We also assist with about 130 bone 
marrow collections, including smears and processing.  There is myself, and 3 
other Histotechs and we do it all! 
 
Our cell block protocol for unfixed fluids is
Centrifuge in 50ml conical tubes at 1200rpm for 10 minutes 
Pour off supernate, resuspend the pellet in buffer wash (for large volumes with 
low cell yields, we keep spinning the fluid until we get a cell pellet in the 
bottom of the tube, or we have exhausted the fluid)
Repeat centrifuge step 
Pour off supernate and fix in 10%NBF/95% Alcohol 50/50 for at least 10 minutes
Gently dislodge the fixed pellet and pour into the corner of a nylon bag into a 
cassette for routine processing
If the fixed pellet is smaller than 0.5cm in greatest dimension, then we pour 
off the fixative and add heated to liquid agar
Let cool, then gently dislodge the fixed agar pellet and place gently into the 
corner of a nylon bag into a cassette for routine processing

Hope this helps

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874


-Original Message-
From: histonet-requ...@lists.utsouthwestern.edu 
[mailto:histonet-requ...@lists.utsouthwestern.edu] 
Sent: Friday, March 04, 2016 1:00 PM

Today's Topics:
   2. Re: Fluids for Cytology 
   6. Cell block protocols 

*


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Re: [Histonet] Processing of breast tissue

[Jennifer MacDonald via Histonet]

Do you using "freezing spray" on these specimens?  Direct spray can cause 
the tissue to appear burnt. 



From:   Charles Riley via Histonet <histonet@lists.utsouthwestern.edu>
To: histonet@lists.utsouthwestern.edu
Date:   01/05/2016 06:00 AM
Subject:    [Histonet] Processing of breast tissue



We have been experiencing some issue lately with cutting our breast
excisions. Recently we had two specimen where after our 8 hr run the 
tissue
was still extremely soft. Our medical director said the fat sectioned
beautifully but the fibrous tissue appeared cooked. Can anyone give a
suggestion as to how we should process our breast excisions from grossing
through to microtomy sectioning.

Currently I have tried to get grossing to limit the specimen to 2mm by 2mm
by 1mm thick. We run an 8hr process using the Thermo Shandon Excelsior
processor. We cut our sections at 4um but have recently had to do them at
6um in order to get the soft samples cut.
-- 

Charles Riley HT(ASCP)CM

Histopathology Coordinator/ Mohs

Doctors Pathology Services, Dover DE
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Re: [Histonet] processing cycle

[Gudrun Lang via Histonet]

Hi Allison,
we doubled the times of the regular processing protocol beginning with
longer absolute ethanol, intermedium and paraffin. Our regular protocol
takes 13 hours and the fatty-protocol takes 17 hours. We start it at about 2
pm with endtime at 8 am. So breast tissue is mostly embedded at the end of
the bulk of cassettes.

This improved cutting really in a great manner.  The problem occurs, that
fixation time is rather short, when grossing of breast is done just shortly
before processing. So we want our pathologists to gross the breast from the
day before rather early in the morning. 

Gudrun

-Ursprüngliche Nachricht-
Von: Eck, Allison via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Gesendet: Dienstag, 17. November 2015 20:15
An: 'histonet@lists.utsouthwestern.edu'
Betreff: [Histonet] processing cycle

Does anyone have a processing cycle that is good for fatty tissues like
breast that they would be willing to share?  This will be used on a VIP5.

Thank you in advance

Allison
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[Histonet] processing cycle

[Eck, Allison via Histonet]

Does anyone have a processing cycle that is good for fatty tissues like breast 
that they would be willing to share?  This will be used on a VIP5.

Thank you in advance

Allison
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[Histonet] Processing and HE Validation

[Manahil via Histonet]

Does anyone have processing and HE auto stainer machines validation procedure 
they would like to share?
If our lab would like to change the bluing and differentiation reagent from in 
house to commercial, do we need to revalidate the stain?
Thanks and appreciate your input.
Manahil
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[Histonet] Processing Validation

[Leslie, Mary]

Does anyone have a processing validation procedure they would like to share?  
Thank you!

Mary Leslie
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Re: [Histonet] Processing Validation

[Michael Ann Jones]

Would love to see also.

Michael Ann




On 6/15/15, 2:18 PM, Leslie, Mary mary.les...@tricore.org wrote:

Does anyone have a processing validation procedure they would like to
share?  Thank you!

Mary Leslie
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Re: [Histonet] processing problem

[Rene J Buesa]

Having morphology problems when re-processing a tissue after an accident as 
you had is the unavoidable consequence. The problem is rooted in the fact 
that usually you remove the paraffin using the tissue processor cleaning cycle 
and that is a very harsh method (albeit the most expeditious). If you  
re-process using more steps than usual in the dehydration you may have very 
slightly better results, but not that much.The mistake you made have the 
consequences you fear.René J.  

 On Monday, February 16, 2015 12:08 PM, Davenport, Martha R 
martha.davenp...@uky.edu wrote:
   

 I am Martha Davenport, supervisor, at the University of KY histology lab. We 
had a processing problem caused  by  accidently placing 70% (instead of 100%) 
into the last dehydration container. Would anyone please give me info on how 
you would remedy this?  We usually reprocess the tissue but have had trouble in 
the past with the tissue morphology being optimal. Any help will be greatly 
appreciated.
Martha Davenport 859-257-1822
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[Histonet] processing problem

[Davenport, Martha R]

I am Martha Davenport, supervisor, at the University of KY histology lab. We 
had a processing problem caused  by  accidently placing 70% (instead of 100%) 
into the last dehydration container. Would anyone please give me info on how 
you would remedy this?  We usually reprocess the tissue but have had trouble in 
the past with the tissue morphology being optimal. Any help will be greatly 
appreciated.
Martha Davenport 859-257-1822
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Re: [Histonet] processing problem

[Caroline Miller]

I am afraid your tissue might be 'toast'. I tried to reprocess from that
same issue and things did not go well. We were processing mouse tissue,
which is dry enough, but then when we put the tissues back through xylene
and into 100% and back again it got even worse.

We had to just get what we could from the blocks and confess to the
researchers what had happened :(

sorry for the bad news, I hope someone has better for you!

mills

On Thu, Feb 12, 2015 at 5:41 AM, Davenport, Martha R 
martha.davenp...@uky.edu wrote:

 I am Martha Davenport, supervisor, at the University of KY histology lab.
 We had a processing problem caused  by  accidently placing 70% (instead of
 100%) into the last dehydration container. Would anyone please give me info
 on how you would remedy this?  We usually reprocess the tissue but have had
 trouble in the past with the tissue morphology being optimal. Any help will
 be greatly appreciated.
 Martha Davenport 859-257-1822
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-- 
Caroline Miller
Director of Histology
3Scan.com
415 2187297
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Re: [Histonet] processing problem

[Barry Rittman]

Hi Martha
I think that Caroline is correct but you have nothing to lose by removing
the  wax and then trying to dehydrate and so on. A more gentle way after
removing the wax and dehydrating is to use chloroform instead of xylene and
hand processing. Chloroform is much more gentle although tissue do usually
float in it an do not become transparent. However unlike xylene, tissue can
be left in chloroform for hours to overnight without any appreciable
hardening.  Then after chloroform you can leave the tissues in a mixture of
chloroform/wax (1:1) at room temperature again for hours to even overnight.
During this process some wax does penetrate into the  tissue and therefore
can leave in wax for less time with less chance of hardening.
Yours is certainly not the first lab that this has happened to.
Good luck to you.
Barry

On Mon, Feb 16, 2015 at 11:30 AM, Caroline Miller mi...@3scan.com wrote:

 I am afraid your tissue might be 'toast'. I tried to reprocess from that
 same issue and things did not go well. We were processing mouse tissue,
 which is dry enough, but then when we put the tissues back through xylene
 and into 100% and back again it got even worse.

 We had to just get what we could from the blocks and confess to the
 researchers what had happened :(

 sorry for the bad news, I hope someone has better for you!

 mills

 On Thu, Feb 12, 2015 at 5:41 AM, Davenport, Martha R 
 martha.davenp...@uky.edu wrote:

  I am Martha Davenport, supervisor, at the University of KY histology lab.
  We had a processing problem caused  by  accidently placing 70% (instead
 of
  100%) into the last dehydration container. Would anyone please give me
 info
  on how you would remedy this?  We usually reprocess the tissue but have
 had
  trouble in the past with the tissue morphology being optimal. Any help
 will
  be greatly appreciated.
  Martha Davenport 859-257-1822
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 --
 Caroline Miller
 Director of Histology
 3Scan.com
 415 2187297
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[Histonet] processing and staining preserved specimen

[Schade, Adelle]

Hello everyone,
I have been following and learning from this listserv for the past two years, I 
have enjoyed all of the advice this forum presents.  This is my first inquiry 
for the group.  I am a high school Anatomy and Physiology teacher and a grad 
student at Thomas Jefferson (Philadelphia, PA).  In our research lab, we 
process, section, embed and image for all research projects.  I was lucky 
enough to acquire some used histology equipment for my high school lab this 
school year.  I am teaching the students about histology and the processing.  
We have an embedding station, microtome, staining dishes and a good microscope 
to image.  We do not have an automatic tissue processor so we are processing 
manually (I prepare all solutions for the students).  We are taking biopsy 
samples from our preserved mink that we are dissecting at the moment.  I have 
processed the tissue using the following method and have pretty good results, 
not great.  (we are doing a basic HE stain to see the cells of the tissue).  
If we do not complete the  manual processing method and need to leave school 
for the day, I move the cassettes into 70% EtOH until the next morning when we 
continue the process.  I am wondering if anyone has manually processed 
preserved specimen tissue in the past and has any advice/ protocol that you 
would suggest?  This is what I am using:

Processing Method

1.  70% EtOH:  1 hour
2.  80%  EtOH:  1 hour
3.  90%  EtOH:  1 hour
4.  95% EtOH:  2 hours
5.  100% EtOH:  2 hours
6.  Histoclear II:  2 hours
7.  Paraffin:  2 hours

Also- I will eventually be looking for grants to try to purchase an automatic 
tissue processor- this would alleviate the time issues we are facing.  Does 
anyone know of grants that would focus on the educational aspect of histology?
Thank you for your time and have a great weekend!
Adelle Schade
Graduate student:  Jefferson School of Biomedical Science


Ms. Adelle L. Schade, B.S., M.Ed.
Anatomy and Physiology
Conrad Weiser High School
44 Big Spring Rd.
Robesonia, PA  19551
a_sch...@conradweiser.org


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[Histonet] processing biopsies on VIP

[Algeo, Lacie A]

Hi there,
Does anyone use P/V on their biopsy runs on the Tissue-Tek VIP 5?  We're having 
really dry biopsies, and I think this could be why
Thanks,
Lacie

Lacie Algeo, HTL (ASCP) MBCM
Histology Supervisor
Providence Sacred Heart Medical Center Laboratory
101 W 8th Avenue
L-2
Spokane, WA 99204
509-474-4418
FAX 509-474-2052
lacie.al...@providence.orgmailto:lacie.al...@providence.org


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may not use, copy, disclose or distribute to anyone the message or any 
information contained in the message.  If you have received this message in 
error, please immediately advise the sender by reply e-mail and delete this 
message.




This message is intended for the sole use of the addressee, and may contain 
information that is privileged, confidential and exempt from disclosure under 
applicable law. If you are not the addressee you are hereby notified that you 
may not use, copy, disclose, or distribute to anyone the message or any 
information contained in the message. If you have received this message in 
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message.
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[Histonet] Processing baskets for Peloris

[Schaundra Walton]

Hey histoland!

Anybody know where I could purchase some gently used divided racks for a Leica 
Peloris machine?  They are very expensive new.  TIA

Schaundra Walton BS, HTL (ASCP)
Histology Supervisor
PathGroup
Nashville, TN
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[Histonet] processing tissue with silicone mesh in it

[pruegg]

Does anyone have any experience with processing tissues with silicone mesh
in them so that the silicone is not dissolved by xylene and we also assume
that it might be dissolved by MMA or GMA polymers since they are strong
solvents in their own right?

 

Thank you,

Patsy

 

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting

40864 E. Arkansas Ave

Bennett, CO 80102

H 303-644-4538

C 720-281-5406

 mailto:prueg...@hotmail.com prueg...@hotmail.com 

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RE: [Histonet] processing tissue with silicone mesh in it

[saby_joseph_a]

Patsy-
We routinely process and section many meshes in paraffin. I would suggest 
processing a sample, embedding and sectioning to see if you can get good 
sections. It is worth a try.
Joe Saby
NAMSA



Sent on the new Sprint Network from my Samsung Galaxy S®4.

 Original message 
From: pru...@ihctech.net
Date:03/27/2014 2:22 PM (GMT-05:00)
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] processing tissue with silicone mesh in it

Does anyone have any experience with processing tissues with silicone mesh
in them so that the silicone is not dissolved by xylene and we also assume
that it might be dissolved by MMA or GMA polymers since they are strong
solvents in their own right?



Thank you,

Patsy



Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting

40864 E. Arkansas Ave

Bennett, CO 80102

H 303-644-4538

C 720-281-5406

mailto:prueg...@hotmail.com prueg...@hotmail.com 

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[Histonet] Processing frozen brain samples

[Murphy, Valerie]

Hi,

I am new to Histonet. Here is my question.

We have brain samples that were fixed in 4% paraformaldehyde then placed in 30% 
sucrose (cryoprotectant) prior to freezing on dry ice. These samples have been 
stored at -80 for a couple of years. We want to thaw these samples then process 
them to make paraffin embedded blocks. What is the best way to remove the 
sucrose prior to processing? Should we just  rinse with PBS?



Thank you,



Valerie



Valerie Ratliff  B.Sc HTL(ASCP)

Research Assistant

Department of Oncology

Karmanos Cancer Institute

4100 John R

Detroit, MI 48201



Telephone: (313) 576 8282

Fax: (313) 576 8306

E-mail: murp...@karmanos.orgmailto:murp...@karmanos.org



Better treatments. Better outcomes.

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[Histonet] Processing:

[Jb]

I have one tech telling me that when the entire processor is changed the tissue 
is too dry. We run a lot of fatty tissues, breast, etc on this processor. (Our 
biopsies are run on a separate processor). Is this correct, or should we only 
rotate reagents?  No other techs complain. I have a hard time believing this, 
my experience is the opposite. Any input is appreciated.



Sent from my iPhone
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Re: [Histonet] Processing:

[Will Chappell]

This depends on so many different factors, however, I prefer a frequent 
rotation over a complete change.

Do what is best for your tissue!

Sent from my iPhone

 On Jan 13, 2014, at 9:46 AM, Jb craiga...@gmail.com wrote:
 
 I have one tech telling me that when the entire processor is changed the 
 tissue is too dry. We run a lot of fatty tissues, breast, etc on this 
 processor. (Our biopsies are run on a separate processor). Is this correct, 
 or should we only rotate reagents?  No other techs complain. I have a hard 
 time believing this, my experience is the opposite. Any input is appreciated.
 
 
 
 Sent from my iPhone
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RE: [Histonet] Processing:

[Curt]

This gets me back to another recent topic, soaking the blocks.

I've seen this a little in the past, just soak them on an ice block,tray for a 
couple minutes and you'll be fine. To me, another indicator would be that if 
you're getting dry tissue when changed but not later could there be some kind 
of variation in results??? How often do you change the processors, all the 
tissue A complete change every other day would probably get you consistent 
results, at least, even if they are a little dry. 

Just my experience.

Curt


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jb
Sent: Monday, January 13, 2014 8:46 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing:

I have one tech telling me that when the entire processor is changed the tissue 
is too dry. We run a lot of fatty tissues, breast, etc on this processor. (Our 
biopsies are run on a separate processor). Is this correct, or should we only 
rotate reagents?  No other techs complain. I have a hard time believing this, 
my experience is the opposite. Any input is appreciated.



Sent from my iPhone
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[Histonet] Processing thin brain slices in. Histogel

[kgrobert]

We are trying to process thin slices of mouse cerebellum embedded in
Histogel into paraffin, and we have encountered two problems: 1) the
Histogel button with the slices inside it has been curling up in the
processor ( before anyone asks, we're using our own cassettes, not the
histoscreen ones that come in that starter kit), and pressing it flat with
weights while embedding puts stress on the tissuehas anyone else seen
this?

2) The Histogel, once embedded in paraffin, has occasionally chipped out
of the block on sectioning.  This, to me, implies an infiltration problem,
but this stuff is supposed to be compatible with paraffin processing.  Why
would it not infiltrate?  We ran the buttons on the biopsy program that
came with our VIP 5.  If you want details, I'll send them once the
processor is done.

The Histogel is still within its expiration date, and the curly batches
were done with a fresh tube of Histogel from the fridge.  Any and all
ideas/suggestions/solutions are most welcome.

Happy Friday,
Kathleen


Principal Lab Technician
Neurotoxicology Labs
Molecular Pathology Facility Core
Dept of Pharmacology  Toxicology
Rutgers, the State University of NJ
41 B Gordon Road
Piscataway, NJ 08854
(848) 445-1443
FAX (732) 445-6905

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Re: [Histonet] Processing Problem

[Rene J Buesa]

A sample 2mmx2mmx1mm is quite small and there should not be any problems with 
either fixation of processing but there is where the problem my reside.
1- fixing in 10% formalin: is it neutral buffered? For such a small piece 24 
hours will be enough but to be absolutely sure try leaving the pieces for 48 
hours.
2- sectioning should not be a problem but I advise that you check the times in 
the reagents. Such small pieces should be less time in the alcohols. Since it 
is pig skin try using more time in the ante-medium. What are you using, 
xylene? And give extra time in the infiltration steps (melted paraffin wax).
With these precautions you should have no problems sectioning the samples.
René J.



From: Sandra Cheasty cheas...@svm.vetmed.wisc.edu
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu 
Cc: Sandra Cheasty cheas...@svm.vetmed.wisc.edu 
Sent: Tuesday, September 17, 2013 11:26 AM
Subject: [Histonet] Processing Problem


Hi All,

                I need help with two issues. I have a researcher who is sending 
us porcine skin, about 2mm x 2mm x 1mm tall.



Issue 1: There is a lesion in the center, and although he wants the skin 
sectioned through the lesion eventually, he says if he bisects the chunk of 
skin before fixation, the lesion becomes distorted. So, he is fixing them in 
the 2x2x1 chunks, and the 10% formalin (and subsequent processing reagents) are 
not penetrating. Does anyone know of either a more penetrating fixative or a 
less distorting one so we can bisect the skin before fixation?



Issue 2: Even on smaller sections that fixed and processed well, we are having 
issues with the porcine skin sections staying on the slides. We use Superfrost 
Plus slides, drain them, air dry them overnight, and then they go on the Extra 
Oven program on the stainer. (25 minutes in the oven.) Any suggestions on 
other slides, drying techniques to try?



My background is that of a certified Histologist for 30 years, with experience 
in many labs in various parts of the country. This research project has me 
stymied!



Thanks for your help!

Sandy

UW Madison

School of Veterinary Medicine



























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[Histonet] processing eyes

[pruegg]

Can someone direct me to a protocol for processing eyes which have been
injected with Davidson's fixative and are now in 10% NBF.  We had an eye
expert at the U for years I was going to send my techs to but apparently she
has finally retired because we cannot get a hold of her.  Where are you Mary
Jo?


We need insight on how to process non-human primate eyes? They have been
injected with Davidson's fixative and are now in 10%NBF. We need a specific
processing schedule, embedding and sectioning advice.

 

Thank you,

 

Patsy


 


 

 

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting

40864 E. Arkansas Ave

Bennett, CO 80102

H 303-644-4538

C 720-281-5406

 mailto:prueg...@hotmail.com prueg...@hotmail.com 

 mailto:rueggihcconsultin...@outlook.com rueggihcconsultin...@outlook.com

 

 

 

 


This email is confidential and intended solely for the use of the Person(s)
('the intended recipient') to whom it was addressed. Any views or opinions
presented are solely those of the author. It may contain information that is
privileged  confidential within the meaning of applicable law. Accordingly
any dissemination, distribution, copying, or other use of this message, or
any of its contents, by any person other than the intended recipient may
constitute a breach of civil or criminal law and is strictly prohibited. If
you are NOT the intended recipient please contact the sender and dispose of
this e-mail as soon as possible.

 

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RE: [Histonet] processing eyes

[Bea DeBrosse-Serra]

Patsy, 

18-24 hours in Davidson's followed by 70% ethanol prior to processing. Some 
people also store the eyes in 10%NBF. I will attach a schedule for you off 
line. 

Bea

Beatrice DeBrosse-Serra HT(ASCP)QIHC
Isis Pharmaceuticals
Antisense Drug Discovery
2855 Gazelle Ct.
Carlsbad, CA 92010
760-603-2371




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
pru...@ihctech.net
Sent: Thursday, August 08, 2013 1:06 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] processing eyes


Can someone direct me to a protocol for processing eyes which have been 
injected with Davidson's fixative and are now in 10% NBF.  We had an eye expert 
at the U for years I was going to send my techs to but apparently she has 
finally retired because we cannot get a hold of her.  Where are you Mary Jo?


We need insight on how to process non-human primate eyes? They have been 
injected with Davidson's fixative and are now in 10%NBF. We need a specific 
processing schedule, embedding and sectioning advice.

 

Thank you,

 

Patsy


 


 

 

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting

40864 E. Arkansas Ave

Bennett, CO 80102

H 303-644-4538

C 720-281-5406

 mailto:prueg...@hotmail.com prueg...@hotmail.com 

 mailto:rueggihcconsultin...@outlook.com rueggihcconsultin...@outlook.com

 

 

 

 


This email is confidential and intended solely for the use of the Person(s) 
('the intended recipient') to whom it was addressed. Any views or opinions 
presented are solely those of the author. It may contain information that is 
privileged  confidential within the meaning of applicable law. Accordingly any 
dissemination, distribution, copying, or other use of this message, or any of 
its contents, by any person other than the intended recipient may constitute a 
breach of civil or criminal law and is strictly prohibited. If you are NOT the 
intended recipient please contact the sender and dispose of this e-mail as soon 
as possible.

 

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Re: [Histonet] processing eyes

[b427297]

We transfer eyes out of Mod Davidsons after 24 hours and process with the other 
soft tissues in formalin- why transfer to alcohol? We have beautiful  eye 
sections.
Jackie O'

Sent from my iPhone

On Aug 8, 2013, at 3:12 PM, Bea DeBrosse-Serra bdebrosse-se...@isisph.com 
wrote:

 Patsy, 
 
 18-24 hours in Davidson's followed by 70% ethanol prior to processing. Some 
 people also store the eyes in 10%NBF. I will attach a schedule for you off 
 line. 
 
 Bea
 
 Beatrice DeBrosse-Serra HT(ASCP)QIHC
 Isis Pharmaceuticals
 Antisense Drug Discovery
 2855 Gazelle Ct.
 Carlsbad, CA 92010
 760-603-2371
 
 
 
 
 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu 
 [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
 pru...@ihctech.net
 Sent: Thursday, August 08, 2013 1:06 PM
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] processing eyes
 
 
 Can someone direct me to a protocol for processing eyes which have been 
 injected with Davidson's fixative and are now in 10% NBF.  We had an eye 
 expert at the U for years I was going to send my techs to but apparently she 
 has finally retired because we cannot get a hold of her.  Where are you Mary 
 Jo?
 
 
 We need insight on how to process non-human primate eyes? They have been 
 injected with Davidson's fixative and are now in 10%NBF. We need a specific 
 processing schedule, embedding and sectioning advice.
 
 
 
 Thank you,
 
 
 
 Patsy
 
 
 
 
 
 
 
 
 
 Patsy Ruegg, HT(ASCP)QIHC
 Ruegg IHC Consulting
 
 40864 E. Arkansas Ave
 
 Bennett, CO 80102
 
 H 303-644-4538
 
 C 720-281-5406
 
 mailto:prueg...@hotmail.com prueg...@hotmail.com 
 
 mailto:rueggihcconsultin...@outlook.com rueggihcconsultin...@outlook.com
 
 
 
 
 
 
 
 
 
 
 This email is confidential and intended solely for the use of the Person(s) 
 ('the intended recipient') to whom it was addressed. Any views or opinions 
 presented are solely those of the author. It may contain information that is 
 privileged  confidential within the meaning of applicable law. Accordingly 
 any dissemination, distribution, copying, or other use of this message, or 
 any of its contents, by any person other than the intended recipient may 
 constitute a breach of civil or criminal law and is strictly prohibited. If 
 you are NOT the intended recipient please contact the sender and dispose of 
 this e-mail as soon as possible.
 
 
 
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 Histonet@lists.utsouthwestern.edu
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[Histonet] Processing Fumes

[MaryK Mendell]

I run a very small Derm lab and starting having a new problem processing and 
cleaning cycle.  Nothing has changed processing wise but it seems we have more 
fumes than before.  I have the fume hood running plus a  Aller Air 5000 Voc D 
portable air purifer, all filters have been changed.  There seems to be more 
fumes while the  processor is running and durining the clean cycle.  I'm at a 
loss here as what it could be.  The processor is a Tissue Tek VIP 2000 and I 
can't see any leaks any where and no error codes present.  I have called our 
HVAC guy to have him look at venting to see if an issue, but with the hood 
check showing no blockage not sure it that is the issue.  Any suggestions?




Kate Mendell
Histopathology/Lab Manager

Anyone who stops learning is old, whether at twenty or eighty.  ~Henry Ford

HOWARD S. GOLDBERG, M.D., INC
990 Paradise Road
Swampscott, MA  01907
TEL:  781.595.0151
FAX:  781.592.6780
kmend...@goldbergmd.netmailto:kmend...@goldbergmd.net
www.cosmesticdermcenter.comhttp://www.cosmesticdermcenter.com/
PRIVACY NOTICE: This e-mail message may contain confidential patient or other 
information belonging to the sender that is legally privileged.  This 
information is intended only for the use of the individual or authorized entity 
named above. The authorized recipient of this patient or other confidential 
information is prohibited from disclosing the information to any other party.  
If you have received this message in error, please notify the sender 
immediately and delete.  Please keep any information you may have viewed 
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Re: [Histonet] Processing Fumes

[Rene J Buesa]

Stop using xylene!
René J.



From: MaryK Mendell kmend...@goldbergmd.net
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu 
Sent: Friday, August 2, 2013 7:58 AM
Subject: [Histonet] Processing Fumes


I run a very small Derm lab and starting having a new problem processing and 
cleaning cycle.  Nothing has changed processing wise but it seems we have more 
fumes than before.  I have the fume hood running plus a  Aller Air 5000 Voc D 
portable air purifer, all filters have been changed.  There seems to be more 
fumes while the  processor is running and durining the clean cycle.  I'm at a 
loss here as what it could be.  The processor is a Tissue Tek VIP 2000 and I 
can't see any leaks any where and no error codes present.  I have called our 
HVAC guy to have him look at venting to see if an issue, but with the hood 
check showing no blockage not sure it that is the issue.  Any suggestions?




Kate Mendell
Histopathology/Lab Manager

Anyone who stops learning is old, whether at twenty or eighty.  ~Henry Ford

HOWARD S. GOLDBERG, M.D., INC
990 Paradise Road
Swampscott, MA  01907
TEL:  781.595.0151
FAX:  781.592.6780
kmend...@goldbergmd.netmailto:kmend...@goldbergmd.net
www.cosmesticdermcenter.comhttp://www.cosmesticdermcenter.com/
PRIVACY NOTICE: This e-mail message may contain confidential patient or other 
information belonging to the sender that is legally privileged.  This 
information is intended only for the use of the individual or authorized entity 
named above. The authorized recipient of this patient or other confidential 
information is prohibited from disclosing the information to any other party.  
If you have received this message in error, please notify the sender 
immediately and delete.  Please keep any information you may have viewed 
confidential.
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Re: [Histonet] Processing Fumes

[Tony Auge]

I have the same processor. If you remove the back panel there is a drip jar
for waste just inside. It might be getting full.

Tony Auge HTL (ASCP) QIHC
Cell: (651) 373-4768
Email: tony.a...@gmail.com

On Fri, Aug 2, 2013 at 4:58 AM, MaryK Mendell kmend...@goldbergmd.netwrote:

 I run a very small Derm lab and starting having a new problem processing
 and cleaning cycle.  Nothing has changed processing wise but it seems we
 have more fumes than before.  I have the fume hood running plus a  Aller
 Air 5000 Voc D portable air purifer, all filters have been changed.  There
 seems to be more fumes while the  processor is running and durining the
 clean cycle.  I'm at a loss here as what it could be.  The processor is a
 Tissue Tek VIP 2000 and I can't see any leaks any where and no error codes
 present.  I have called our HVAC guy to have him look at venting to see if
 an issue, but with the hood check showing no blockage not sure it that is
 the issue.  Any suggestions?




 Kate Mendell
 Histopathology/Lab Manager

 Anyone who stops learning is old, whether at twenty or eighty.  ~Henry Ford

 HOWARD S. GOLDBERG, M.D., INC
 990 Paradise Road
 Swampscott, MA  01907
 TEL:  781.595.0151
 FAX:  781.592.6780
 kmend...@goldbergmd.netmailto:kmend...@goldbergmd.net
 www.cosmesticdermcenter.comhttp://www.cosmesticdermcenter.com/
 PRIVACY NOTICE: This e-mail message may contain confidential patient or
 other information belonging to the sender that is legally privileged.  This
 information is intended only for the use of the individual or authorized
 entity named above. The authorized recipient of this patient or other
 confidential information is prohibited from disclosing the information to
 any other party.  If you have received this message in error, please notify
 the sender immediately and delete.  Please keep any information you may
 have viewed confidential.
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-- 

Tony Auge HTL (ASCP) QIHC
Cell: (651) 373-4768
Email: tony.a...@gmail.com
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[Histonet] Processing Drosophila brains into paraffin

[kgrobert]

I was given a protocol for processing Drosophila brains into paraffin
manually, and it involves methyl benzoate.  It is as follows:

Fixation: 3.5-4 hours in Carnoy's fixative, in hood (room temperature)

Processing: 2x30 min 99% ethanol (Would 95% be okay?)

60 min in absolute ethanol

Overnight in methyl benzoat (that's how it's written in the protocol), can
be up to 3 days (*Here is my question-I looked on Sigma's website for
this, and found a bunch of similar chemicals, but no straight methyl
benzoat.  What do I order?)

1 hour methylbenzoat-paraffin 1:1 mix at 60 degrees C

6x20 min 60 degrees C paraffin (we use Paraplast X-tra)

Thanks so much!

Kathleen




Principal Lab Technician
Neurotoxicology Labs
Molecular Pathology Facility Core
Dept of Pharmacology  Toxicology
Rutgers, the State University of NJ
41 B Gordon Road
Piscataway, NJ 08854
(848) 445-1443
FAX (732) 445-6905

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Re: [Histonet] Processing Guinea Pig

[Joseph Saby]

Heather-
 
I do not know why, but to properly process guinea pig tissues you need a much 
more rigorous program than what would work for mice. It is very easy to over 
process mouse tissue.  Even rat tissue needs more processing.  Guinea pig 
tissue need a program designed for processing larger animals/tissues, such as 
one would use to process dogs or even swine.
 
Let me know what programs you have, and I will get back with you with what 
would work. 
 
Joe Saby BA HT
NAMSA, Inc.
 


 From: Heather Marlatt hmarlat...@gmail.com
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu 
Sent: Monday, June 17, 2013 10:33 AM
Subject: [Histonet] Processing Guinea Pig
  

Hello Histonet! I'm a long time reader first time poster. Does anyone have
experience processing guinea pig tissues? I have been processing kidney and
heart but it is consistently coming out mushy in the middle. The mouse
tissue comes out fine even when processed on the same run. I had the tissue
grossed in thinner (2.5mm) thinking that perhaps it was too thick but it
didn't seem to help. Also, it has been fixed in 10%NBF for several days.

I was just wondering if anyone else had similar problems with guinea pig?

I appreciate in advance any advice or tips.

Here is the protocol:

Formalin 1hr
70% etOH 1hr
95% 1hr
100% 30min
100% 1hr
100% 1hr
100%1hr
Clearify 1hr
Clearify  1hr
Clearify 1hr
Paraffin 1hr
paraffin 1hr


All under pressure and heat only on the paraffin.

Thanks
Heather
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[Histonet] Processing Guinea Pig

[Heather Marlatt]

Hello Histonet! I'm a long time reader first time poster. Does anyone have
experience processing guinea pig tissues? I have been processing kidney and
heart but it is consistently coming out mushy in the middle. The mouse
tissue comes out fine even when processed on the same run. I had the tissue
grossed in thinner (2.5mm) thinking that perhaps it was too thick but it
didn't seem to help. Also, it has been fixed in 10%NBF for several days.

I was just wondering if anyone else had similar problems with guinea pig?

I appreciate in advance any advice or tips.

Here is the protocol:

Formalin 1hr
70% etOH 1hr
95% 1hr
100% 30min
100% 1hr
100% 1hr
100%1hr
Clearify 1hr
Clearify  1hr
Clearify 1hr
Paraffin 1hr
paraffin 1hr


All under pressure and heat only on the paraffin.

Thanks
Heather
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Re: [Histonet] Processing Guinea Pig

[Grantham, Andrea L - (algranth)]

Heather,
Looking at your protocol for processing the kidney and heart tissue I can't 
figure out why it is mushy especially if you are putting it in the cassettes so 
thin and it has had a chance to fix well before processing. In fact, looking at 
your protocol I might think that your tissue might be dried out and need to sit 
in cold icy water before sectioning.

At any rate, I would rinse well in running water after fixing and skip the 
formalin on the processor.

I'd start in 70% alcohol, maybe two changes and move on up to 80%, 2- 95%'s and 
3-100%'s.
I'm not familiar with the clearing agent that you use. I use Clear Rite 3 and 
never have a problem - 2 changes of Clear Rite 3 and 4 paraffins.

I don't know how many cassettes you are processing but make sure there is 
adequate room for the reagents on the processor to move around the cassettes 
and that you have a good ratio of the reagents to the tissue and that the 
reagents are fresh. If you can increase the agitation in some of the 
dehydration steps it might help.


Andrea Grantham, HT (ASCP)
Senior Research Specialist
University of Arizona
Cellular and Molecular Medicine
Histology Service Laboratory
P.O.Box 245044
Tucson, AZ 85724

algra...@email.arizona.edumailto:algra...@email.arizona.edu
Tel: 520.626.4415 Fax: 520.626.2097





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RE: [Histonet] Processing thin slices of mouse cerebellum

[Hobbs, Carl]

http://www.cellpath.us.com/magento/cellpath-on-line-shop/specimen-processing-and-embedding/small-biospy-processing/cellsafe/cellsafe-biopsy-capsule-blue.html

I  find them very good for brain slices.
There is a deeper and a shallower space, depending on which way they are folded.

Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson Centre for Age-Related Diseases
School of Biomedical Sciences
King's College London
Guys Campus
London SE1 1UL
 
Tel.020 7848 6810
fax 020 7848 6816
carl.ho...@kcl.ac.uk



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AW: [Histonet] Processing thin slices of mouse cerebellum

[Gudrun Lang]

Is the tissue fixed well enough? At least two days?
Processing through graded ethanols with little difference should minimize
shrinkage. Instead of 70-96-100, take 50-60-70-80-96-100. Times shouldn't be
too short. Fatty brain needs more time for reagens-exchange than other small
biopsies.
...smooth protocol.
For embedding one can try to press slightly the tissue with a warm small
metalblock on the ground of the mold 

Gudrun


-Ursprüngliche Nachricht-
Von: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von
kgrob...@rci.rutgers.edu
Gesendet: Mittwoch, 29. Mai 2013 20:14
An: histonet
Betreff: [Histonet] Processing thin slices of mouse cerebellum

To all,

We have these very thin slices of mouse cerebellum that need to be processed
into paraffin.  The problem is that they need to be kept flat. 
We have tried sponges, but some of the slices are so small that they shrink
(processed with our biopsy program, of course; if you want details, let me
know) and get lost in the holes of the sponges.  We have biopsy cassettes,
but the slices will still tumble and not stay flat. We have processed other
things in lens paper, but I had to scrape the tissue with the paraffin into
the mold.  I have not tried Histogel, though-maybe embed the slices flat in
that first and then process it?  Is there anything else?


Thanks so much!

Kathleen



Principal Lab Technician
Neurotoxicology Labs
Molecular Pathology Facility Core
Dept of Pharmacology  Toxicology
Rutgers, the State University of NJ
41 B Gordon Road
Piscataway, NJ 08854
(848) 445-1443
FAX (732) 445-6905

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RE: [Histonet] Processing thin slices of mouse cerebellum

[Truscott, Tom]

Hi Kathleen, Several years and a couple employers ago we attached small 
biopsies (for proper orientation) to small squares of cucumber (which had been 
dehydrated in several changes of alcohol) with a egg-albumin/glycerol mixture. 
The bx/cucumber unit was embedded together after processing and cut and stained 
as a unit with no adverse effects. Tom Truscott

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
kgrob...@rci.rutgers.edu
Sent: Wednesday, May 29, 2013 11:14 AM
To: histonet
Subject: [Histonet] Processing thin slices of mouse cerebellum

To all,

We have these very thin slices of mouse cerebellum that need to be processed 
into paraffin.  The problem is that they need to be kept flat. 
We have tried sponges, but some of the slices are so small that they shrink 
(processed with our biopsy program, of course; if you want details, let me 
know) and get lost in the holes of the sponges.  We have biopsy cassettes, but 
the slices will still tumble and not stay flat. We have processed other things 
in lens paper, but I had to scrape the tissue with the paraffin into the mold.  
I have not tried Histogel, though-maybe embed the slices flat in that first and 
then process it?  Is there anything else?


Thanks so much!

Kathleen



Principal Lab Technician
Neurotoxicology Labs
Molecular Pathology Facility Core
Dept of Pharmacology  Toxicology
Rutgers, the State University of NJ
41 B Gordon Road
Piscataway, NJ 08854
(848) 445-1443
FAX (732) 445-6905

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[Histonet] Processing thin slices of mouse cerebellum

[kgrobert]

To all,

We have these very thin slices of mouse cerebellum that need to be
processed into paraffin.  The problem is that they need to be kept flat. 
We have tried sponges, but some of the slices are so small that they
shrink (processed with our biopsy program, of course; if you want details,
let me know) and get lost in the holes of the sponges.  We have biopsy
cassettes, but the slices will still tumble and not stay flat. We have
processed other things in lens paper, but I had to scrape the tissue with
the paraffin into the mold.  I have not tried Histogel, though-maybe embed
the slices flat in that first and then process it?  Is there anything
else?


Thanks so much!

Kathleen



Principal Lab Technician
Neurotoxicology Labs
Molecular Pathology Facility Core
Dept of Pharmacology  Toxicology
Rutgers, the State University of NJ
41 B Gordon Road
Piscataway, NJ 08854
(848) 445-1443
FAX (732) 445-6905

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[Histonet] Processing question

[Connolly, Brett M]

Histonetters,

Has anyone tried to process an intact mouse brain to paraffin?


Brett M. Connolly, Ph.D.
Principal Scientist, Imaging Dept.
Merck  Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
brett_conno...@merck.com
T- 215-652-2501
F- 215-993-6803








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[Histonet] Processing Protocol Using Mineral Oil

[Lyn Stadler]

We have been using this protocol on mostly mouse tissues with great results!  
We run this on a VIP 3000.

70% Ethanol, room temp, 1 hour
100% Isopropyl Alcohol, room temp, 1 hour
100% Isopropyl Alcohol, room temp, 1 hour
100% Isopropyl Alcohol, room temp, 1 hour
5:1 Iso:Mineral Oil, 50 degrees, 1 hour
2:1 Iso:Mineral Oil, 50 degrees, 1 hour
100% Mineral Oil, 50 degrees, 1 hour
100% Mineral Oil, 50 degrees, 1 hour
100% Mineral Oil, 50 degrees, 6 hours
Paraffin, 60 degrees, 1 hour
Paraffin, 60 degrees, 1 hour
Paraffin, 60 degrees, 1 hour

Followed by a cleaning cycle after cassettes are removed and put into molten 
paraffin at the embedding center

Lyn M. Stadler, BS, HTL(ASCP)CM
Research Histotechnologist
Department of Histopathology
Cleveland Biolabs, Inc.
73 High Street
Buffalo, NY 14203
716-849-6817, ext 417


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
histonet-requ...@lists.utsouthwestern.edu
Sent: Wednesday, December 05, 2012 1:05 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 109, Issue 6

Send Histonet mailing list submissions to
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To subscribe or unsubscribe via the World Wide Web, visit
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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. RE: automated microtomes (Lynette Pavelich)
   2. HistoTALK (David Kemler)
   3. RE: Tissue Processors (Marcum, Pamela A)
   4. RE: Tissue Processors (Boyd, Debbie M)
   5. Re: Immunohistochemical detection of cytokines in older
  frozen sections (Rob Day)
   6. RE: Histonet Digest, Vol 109, Issue 5 (Riesen, Rebecca)
   7. Re: Fixation time (Geoff)
   8. Histology Positions in Little Rock AR  (Marcum, Pamela A)
   9. Re: Fixation time (Teri Johnson)
  10. Re: Tissue Processors (Teri Johnson)
  11. job opening in PA (Hutton, Allison)
  12. Re: Re: Tissue Processors (Rene J Buesa)
  13. RE: Re: Tissue Processors (Ellenburg, Deborah)


--

Message: 1
Date: Wed, 5 Dec 2012 12:10:06 +
From: Lynette Pavelich lpave...@hurleymc.com
Subject: RE: [Histonet] automated microtomes
To: Rene J Buesa rjbu...@yahoo.com, Rathborne, Toni
trathbo...@somerset-healthcare.com,
histonet@lists.utsouthwestern.edu
histonet@lists.utsouthwestern.edu
Message-ID:
89f4666a496dc949a819ecc40e11c867bf56c...@exchangemb1.hmc.hurleymc.com

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

I am purchasing my second Leica RM2255 automated microtome. Rene' is correct in 
saying that the carpal tunnel syndrome will eventually affect every advanced 
tech by using any microtome manually. On the RM2255, the flywheel additionally 
is automated. You can choose to use the flywheel manually or automated by the 
simultaneous pushing of two buttons. Nice feature for us oldies to ease us 
into automation or if you have a tiny specimen that you need to take extra care 
with and want to use manually.
Companies are very happy to send in a demo to try for a couple weeks.

Happy shopping!! ;)
Lynette


From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] on behalf of Rene J Buesa 
[rjbu...@yahoo.com]
Sent: Tuesday, December 04, 2012 1:19 PM
To: Rathborne, Toni; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] automated microtomes

The advantage of the so called automated microtomes (the only thing automated 
about them is the block advance) is that they alleviate wrist effort and in 
some ways prevent carpal tunnel syndrome that affects some histotechs (mostly 
of the senior persuasion).
I would go with the Leica.
Ren? J.

From: Rathborne, Toni trathbo...@somerset-healthcare.com
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
Sent: Tuesday, December 4, 2012 11:48 AM
Subject: [Histonet] automated microtomes

I'm looking for some opinions about the automated microtomes currently 
available. Which ones do most techs prefer? Which are more reliable? Is there 
an advantage to having a semi-automated microtome?
Thanks in advance for your replies.

Toni


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--

Message: 2
Date: Wed, 5 Dec 2012 04:28:54 -0800 (PST)
From: David Kemler histot...@yahoo.com

Re: [Histonet] processing protocol using isopropyl alcohol, mineral oil paraffin

[Maria Mejia]

I'd like to be included in the protocol processing exchange too!

Best
Maria Mejia
On Dec 5, 2012, at 10:55 AM, Lynette Pavelich wrote:

 Yes, please share! Thanks!
 
 Lynette
 
 
 From: histonet-boun...@lists.utsouthwestern.edu 
 [histonet-boun...@lists.utsouthwestern.edu] on behalf of Davis, Cassie 
 [cda...@che-east.org]
 Sent: Wednesday, December 05, 2012 1:41 PM
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] RE: processing protocol using isopropyl alcohol, mineral 
 oil  paraffin
 
 I'd really like to see a copy of the protocol if any one would be willing to 
 share. We are still using reagent alcohol  xylene, the waste disposal prices 
 are a significant portion of our budget.
 
 Cassandra Davis
 cda...@che-east.org
 302-575-8095
 Confidentiality Notice:
 This e-mail, including any attachments is the
 property of Catholic Health East and is intended
 for the sole use of the intended recipient(s).
 It may contain information that is privileged and
 confidential.  Any unauthorized review, use,
 disclosure, or distribution is prohibited. If you are
 not the intended recipient, please delete this message, and
 reply to the sender regarding the error in a separate email.
 
 
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Re: [Histonet] Processing issue

[Rene J Buesa]

That will depend on the tissue and the size/thickness of the specimens. 
Appreciate how they turned out and you will have the answer to your question. 
At least for me it is very difficult to answer your question without knowing 
the tissue characteristics.
René J.



From: Danny Zapata danny...@yahoo.com
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu 
Sent: Wednesday, June 13, 2012 12:57 AM
Subject: [Histonet] Processing issue

Would It be a grave problem if I run the tissue in a short run although it 
needed it to be placed in a routine regular run? 

Danny Zapata
Dr. Norman Dermatology Office
Tampa, Fl 




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[Histonet] Processing issue

[Danny Zapata]

Would It be a grave problem if I run the tissue in a short run although it 
needed it to be placed in a routine regular run? 
 
Danny Zapata
Dr. Norman Dermatology Office
Tampa, Fl 




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[Histonet] Processing Autopsies

[Meryl Roberts]

Our lab processes a high number of autopsies; however we always seem to have 
tissue that needs to be reprocessed; particularly brains. Does anyone out there 
have any suggestions as to what an optimal processing cycle would be? We are 
finding it hard to find a happy medium as there always seems to be something 
that is underprocessed, or sometimes even overprocessed. Thanks.
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Re: [Histonet] Processing Autopsies

[Kim Donadio]

Brains in particular need to be fixed real well If it's a whole brain what I've 
done is hang the brain by a mesh or strings into a large brain bucket so it's 
not touching the sides or bottom. Fix for few days then get you sections. I'd 
go textbook on the  3 mm thick sections for processing and don't over process 
that will cause them to be friable. Hate that. Try a few blocks a couple 
different ways and what kind of alcohol are you using? Reagent grade is fine. 
For processing well fixed brain I've had good success with a straight 30 min 
for every thing. Hope this helps 
Kim D 

I'm out :)

Sent from my iPhone

On Apr 10, 2012, at 7:14 PM, Meryl Roberts mery...@hotmail.com wrote:

 
 
 
 Our lab processes a high number of autopsies; however we always seem to have 
 tissue that needs to be reprocessed; particularly brains. Does anyone out 
 there have any suggestions as to what an optimal processing cycle would be? 
 We are finding it hard to find a happy medium as there always seems to be 
 something that is underprocessed, or sometimes even overprocessed. Thanks.
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RE: [Histonet] Processing adipose tissue

[Rene J Buesa]

I agree that 21 hours is too much, but using isopropyl alcohol does not a 
clearing step because isopropyl alcohol will dissolve paraffin wax at 50ºC 
and above.
This is the method I have published else were with the intermediate step of 
mineral oil (which is paraffin of low molecular weight) to reduce the gradient 
and protect the tissue structure.
The Peloris processor routinely uses this sequence (isopropyl → paraffin wax) 
and is widely used in Australia.
The problem resides in the dehydration time. I will also suggest an 
intermediate step of isopropyl alcohol+paraffin wax 1:1 between the last 
alcohol and the first paraffin wax bath.
René J.

--- On Mon, 1/30/12, Jerry Ricks rosenfeld...@hotmail.com wrote:


From: Jerry Ricks rosenfeld...@hotmail.com
Subject: RE: [Histonet] Processing adipose tissue
To: histonet@lists.utsouthwestern.edu
Date: Monday, January 30, 2012, 7:15 PM



Hi David,

21 hours in isopropyl seems likw ea lot, and I don't see any clearing step to 
remove the IPA.

I've been using Slide Brite instead of Xylene, but I see that Rene Buesa 
published a study indicating that mineral oil is the cat's meow for xylene 
substitutes.

http://www.google.com/url?sa=trct=jq=esrc=ssource=webcd=10ved=0CGMQFjAJurl=http%3A%2F%2Fwww.ebsciences.com%2Fpapers%2FMineral%2520oil%2520as%2520xylene%2520substitute.pdfei=ny4nT73bM8axiQLk3dmQAQusg=AFQjCNFw894if_bFAYgv6Uurw2MkO3Th5A



Jerry



 Date: Mon, 30 Jan 2012 12:20:24 -0600
 From: david.b...@pbrc.edu
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Processing adipose tissue
 
 Esteemed experts,
 
  
 
 We have many clients who want to process mouse and human adipose tissue
 and are having some quality issues in the resultant slides.  
 
 We have tried processing small chunks of tissue (1 cm x 0.5 cm) on our
 automated processor (Excelsior) as follows:
 
 Tissue fixed for ~24 hrs in 10% NBF
 
 70% Isopropyl alcohol (IPA) for 3 hrs
 
 90% IPA, 3hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
  
 
 Embed and section at 5 um prior to HE.  An example of what the sections
 look like can be found here http://imgur.com/7RTGR .  
 
 We also ran a sample on a traditional overnight EtOH/Xylene processor
 (not at our facility) to compare results.  That image is here:
 http://imgur.com/GjJPg .  
 
 What is obvious is that the membranes in the IPA processed tissue seem
 to flap over and don't look as crisp as the Xylene processed tissue.  
 
 We did notice structural defects in both samples (not shown) typically
 toward the middle of the specimens.
 
  
 
 Does anyone know what is causing our IPA processed fat to have these
 wide membrane artifacts?  
 
 We are going to repeat the process with an additional 30 minutes per
 step and raise the temperature of the steps to ~ 35 C.  
 
 We are also going to cut the blocks at 2-3 um to see if it can reduce
 the appearance of the membranes.  
 
  
 
 Thanks very much for any advice you may have for us.  We are pretty
 locked in to using xylene-free processing methodology if at all possible
 but will entertain any suggestions you may have.  
 
 If I can provide any further details about what we are doing on our end,
 please let me know and I'll be happy to provide them.
 
  
 
 Best,
 
 David Burk 
 
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RE: [Histonet] Processing adipose tissue

[David Burk]

René,
Thanks very much for your input on this matter.  I also want to go ahead and 
thank the other two (Jerry and Karen) who have chimed in on my question.  René, 
could you please elaborate on the problem resides in the dehydration time?  
Am I over doing it with 21 hours of IPA?  As for the suggestion about 
introducing an intermediate step, I am going to find out if that is doable on 
our Excelsior and, if so, replace the last (9th) IPA step with either an 
IPA/Paraffin mix or perhaps a mineral oil mix.

If anyone else has further suggestions as to help us get nice crisp membrane 
structure from our adipose sections I will be very happy to hear them.  Also, 
if you need additional example images from our sectioned material, I can 
provide them easily.

Best,
David B.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Tuesday, January 31, 2012 8:34 AM
To: histonet@lists.utsouthwestern.edu; Jerry Ricks
Subject: RE: [Histonet] Processing adipose tissue

I agree that 21 hours is too much, but using isopropyl alcohol does not a 
clearing step because isopropyl alcohol will dissolve paraffin wax at 50ºC 
and above.
This is the method I have published else were with the intermediate step of 
mineral oil (which is paraffin of low molecular weight) to reduce the gradient 
and protect the tissue structure.
The Peloris processor routinely uses this sequence (isopropyl → paraffin wax) 
and is widely used in Australia.
The problem resides in the dehydration time. I will also suggest an 
intermediate step of isopropyl alcohol+paraffin wax 1:1 between the last 
alcohol and the first paraffin wax bath.
René J.

--- On Mon, 1/30/12, Jerry Ricks rosenfeld...@hotmail.com wrote:


From: Jerry Ricks rosenfeld...@hotmail.com
Subject: RE: [Histonet] Processing adipose tissue
To: histonet@lists.utsouthwestern.edu
Date: Monday, January 30, 2012, 7:15 PM



Hi David,

21 hours in isopropyl seems likw ea lot, and I don't see any clearing step to 
remove the IPA.

I've been using Slide Brite instead of Xylene, but I see that Rene Buesa 
published a study indicating that mineral oil is the cat's meow for xylene 
substitutes.

http://www.google.com/url?sa=trct=jq=esrc=ssource=webcd=10ved=0CGMQFjAJurl=http%3A%2F%2Fwww.ebsciences.com%2Fpapers%2FMineral%2520oil%2520as%2520xylene%2520substitute.pdfei=ny4nT73bM8axiQLk3dmQAQusg=AFQjCNFw894if_bFAYgv6Uurw2MkO3Th5A



Jerry



 Date: Mon, 30 Jan 2012 12:20:24 -0600
 From: david.b...@pbrc.edu
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Processing adipose tissue
 
 Esteemed experts,
 
  
 
 We have many clients who want to process mouse and human adipose 
 tissue and are having some quality issues in the resultant slides.
 
 We have tried processing small chunks of tissue (1 cm x 0.5 cm) on 
 our automated processor (Excelsior) as follows:
 
 Tissue fixed for ~24 hrs in 10% NBF
 
 70% Isopropyl alcohol (IPA) for 3 hrs
 
 90% IPA, 3hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
  
 
 Embed and section at 5 um prior to HE.  An example of what the 
 sections look like can be found here http://imgur.com/7RTGR .
 
 We also ran a sample on a traditional overnight EtOH/Xylene 
 processor (not at our facility) to compare results.  That image is here:
 http://imgur.com/GjJPg .
 
 What is obvious is that the membranes in the IPA processed tissue seem 
 to flap over and don't look as crisp as the Xylene processed tissue.
 
 We did notice structural defects in both samples (not shown) typically 
 toward the middle of the specimens.
 
  
 
 Does anyone know what is causing our IPA processed fat to have these 
 wide membrane artifacts?
 
 We are going to repeat the process with an additional 30 minutes per 
 step and raise the temperature of the steps to ~ 35 C.
 
 We are also going to cut the blocks at 2-3 um to see if it can reduce 
 the appearance of the membranes.
 
  
 
 Thanks very much for any advice you may have for us.  We are pretty 
 locked in to using xylene-free processing methodology if at all 
 possible but will entertain any suggestions you may have.
 
 If I can provide any further details about what we are doing on our 
 end, please let me know and I'll be happy to provide them.
 
  
 
 Best,
 
 David Burk
 
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RE: [Histonet] Processing adipose tissue

[Rene J Buesa]

David:
21 hours dehydration is too much, but the main problem resides in the 
abrupt transition from alcohol to paraffin wax, especially with fat tissue.
Under separate cover I am sending you the article I referred to.
René J.

--- On Tue, 1/31/12, David Burk david.b...@pbrc.edu wrote:


From: David Burk david.b...@pbrc.edu
Subject: RE: [Histonet] Processing adipose tissue
To: histonet@lists.utsouthwestern.edu
Date: Tuesday, January 31, 2012, 9:51 AM


René,
Thanks very much for your input on this matter.  I also want to go ahead and 
thank the other two (Jerry and Karen) who have chimed in on my question.  René, 
could you please elaborate on the problem resides in the dehydration time?  
Am I over doing it with 21 hours of IPA?  As for the suggestion about 
introducing an intermediate step, I am going to find out if that is doable on 
our Excelsior and, if so, replace the last (9th) IPA step with either an 
IPA/Paraffin mix or perhaps a mineral oil mix.

If anyone else has further suggestions as to help us get nice crisp membrane 
structure from our adipose sections I will be very happy to hear them.  Also, 
if you need additional example images from our sectioned material, I can 
provide them easily.

Best,
David B.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Tuesday, January 31, 2012 8:34 AM
To: histonet@lists.utsouthwestern.edu; Jerry Ricks
Subject: RE: [Histonet] Processing adipose tissue

I agree that 21 hours is too much, but using isopropyl alcohol does not a 
clearing step because isopropyl alcohol will dissolve paraffin wax at 50ºC 
and above.
This is the method I have published else were with the intermediate step of 
mineral oil (which is paraffin of low molecular weight) to reduce the gradient 
and protect the tissue structure.
The Peloris processor routinely uses this sequence (isopropyl → paraffin wax) 
and is widely used in Australia.
The problem resides in the dehydration time. I will also suggest an 
intermediate step of isopropyl alcohol+paraffin wax 1:1 between the last 
alcohol and the first paraffin wax bath.
René J.

--- On Mon, 1/30/12, Jerry Ricks rosenfeld...@hotmail.com wrote:


From: Jerry Ricks rosenfeld...@hotmail.com
Subject: RE: [Histonet] Processing adipose tissue
To: histonet@lists.utsouthwestern.edu
Date: Monday, January 30, 2012, 7:15 PM



Hi David,

21 hours in isopropyl seems likw ea lot, and I don't see any clearing step to 
remove the IPA.

I've been using Slide Brite instead of Xylene, but I see that Rene Buesa 
published a study indicating that mineral oil is the cat's meow for xylene 
substitutes.

http://www.google.com/url?sa=trct=jq=esrc=ssource=webcd=10ved=0CGMQFjAJurl=http%3A%2F%2Fwww.ebsciences.com%2Fpapers%2FMineral%2520oil%2520as%2520xylene%2520substitute.pdfei=ny4nT73bM8axiQLk3dmQAQusg=AFQjCNFw894if_bFAYgv6Uurw2MkO3Th5A
 



Jerry



 Date: Mon, 30 Jan 2012 12:20:24 -0600
 From: david.b...@pbrc.edu
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Processing adipose tissue
 
 Esteemed experts,
 
  
 
 We have many clients who want to process mouse and human adipose 
 tissue and are having some quality issues in the resultant slides.
 
 We have tried processing small chunks of tissue (1 cm x 0.5 cm) on 
 our automated processor (Excelsior) as follows:
 
 Tissue fixed for ~24 hrs in 10% NBF
 
 70% Isopropyl alcohol (IPA) for 3 hrs
 
 90% IPA, 3hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
  
 
 Embed and section at 5 um prior to HE.  An example of what the 
 sections look like can be found here http://imgur.com/7RTGR .
 
 We also ran a sample on a traditional overnight EtOH/Xylene 
 processor (not at our facility) to compare results.  That image is here:
 http://imgur.com/GjJPg .
 
 What is obvious is that the membranes in the IPA processed tissue seem 
 to flap over and don't look as crisp as the Xylene processed tissue.
 
 We did notice structural defects in both samples (not shown) typically 
 toward the middle of the specimens.
 
  
 
 Does anyone know what is causing our IPA processed fat to have these 
 wide membrane artifacts?
 
 We are going to repeat the process with an additional 30 minutes per 
 step and raise the temperature of the steps to ~ 35 C.
 
 We are also going to cut the blocks at 2-3 um to see if it can reduce 
 the appearance of the membranes.
 
  
 
 Thanks very much for any advice you may have for us.  We are pretty 
 locked in to using xylene-free processing methodology if at all 
 possible but will entertain any suggestions you may have.
 
 If I can provide any further details about what we are doing on our 
 end, please let me know and I'll be happy to provide them.
 
  
 
 Best,
 
 David Burk
 
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RE: [Histonet] Processing adipose tissue

[Pheneger, Tracy]

Jerry-  Ispropanol is completely miscible in Paraffin and doesn't need
to be cleared.  

David - I am also a proponent of Isopropanol processing of tissues, but
I don't usually have really fatty tissues (mostly I deal with animal
tissues).  Unfortunately, I don't think that the Isopropanol methodology
is infiltrative enough for really fatty tissues.  I worry that if you
increase your times and temps that you may damage the tissues.  You may
have to use an ETOH/Xylene - based system for these tissues.  

Please keep us informed on what you find out - there may be a paper or
poster for you!

Tracy

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jerry
Ricks
Sent: Monday, January 30, 2012 5:15 PM
To: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Processing adipose tissue


Hi David,

21 hours in isopropyl seems likw ea lot, and I don't see any clearing
step to remove the IPA.

I've been using Slide Brite instead of Xylene, but I see that Rene Buesa
published a study indicating that mineral oil is the cat's meow for
xylene substitutes.

http://www.google.com/url?sa=trct=jq=esrc=ssource=webcd=10ved=0CGM
QFjAJurl=http%3A%2F%2Fwww.ebsciences.com%2Fpapers%2FMineral%2520oil%252
0as%2520xylene%2520substitute.pdfei=ny4nT73bM8axiQLk3dmQAQusg=AFQjCNFw
894if_bFAYgv6Uurw2MkO3Th5A



Jerry



 Date: Mon, 30 Jan 2012 12:20:24 -0600
 From: david.b...@pbrc.edu
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Processing adipose tissue
 
 Esteemed experts,
 
  
 
 We have many clients who want to process mouse and human adipose
tissue
 and are having some quality issues in the resultant slides.  
 
 We have tried processing small chunks of tissue (1 cm x 0.5 cm) on
our
 automated processor (Excelsior) as follows:
 
 Tissue fixed for ~24 hrs in 10% NBF
 
 70% Isopropyl alcohol (IPA) for 3 hrs
 
 90% IPA, 3hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
  
 
 Embed and section at 5 um prior to HE.  An example of what the
sections
 look like can be found here http://imgur.com/7RTGR .  
 
 We also ran a sample on a traditional overnight EtOH/Xylene
processor
 (not at our facility) to compare results.  That image is here:
 http://imgur.com/GjJPg .  
 
 What is obvious is that the membranes in the IPA processed tissue seem
 to flap over and don't look as crisp as the Xylene processed tissue.

 
 We did notice structural defects in both samples (not shown) typically
 toward the middle of the specimens.
 
  
 
 Does anyone know what is causing our IPA processed fat to have these
 wide membrane artifacts?  
 
 We are going to repeat the process with an additional 30 minutes per
 step and raise the temperature of the steps to ~ 35 C.  
 
 We are also going to cut the blocks at 2-3 um to see if it can reduce
 the appearance of the membranes.  
 
  
 
 Thanks very much for any advice you may have for us.  We are pretty
 locked in to using xylene-free processing methodology if at all
possible
 but will entertain any suggestions you may have.  
 
 If I can provide any further details about what we are doing on our
end,
 please let me know and I'll be happy to provide them.
 
  
 
 Best,
 
 David Burk 
 
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[Histonet] Processing adipose tissue

[David Burk]

Esteemed experts,

 

We have many clients who want to process mouse and human adipose tissue
and are having some quality issues in the resultant slides.  

We have tried processing small chunks of tissue (1 cm x 0.5 cm) on our
automated processor (Excelsior) as follows:

Tissue fixed for ~24 hrs in 10% NBF

70% Isopropyl alcohol (IPA) for 3 hrs

90% IPA, 3hrs

100% IPA, 3 hrs

100% IPA, 3 hrs

100% IPA, 3 hrs

100% IPA, 3 hrs

100% IPA, 3 hrs

100% IPA, 3 hrs

100% IPA, 3hrs

Paraffin, 3 hrs

Paraffin, 3 hrs

Paraffin, 3 hrs

 

Embed and section at 5 um prior to HE.  An example of what the sections
look like can be found here http://imgur.com/7RTGR .  

We also ran a sample on a traditional overnight EtOH/Xylene processor
(not at our facility) to compare results.  That image is here:
http://imgur.com/GjJPg .  

What is obvious is that the membranes in the IPA processed tissue seem
to flap over and don't look as crisp as the Xylene processed tissue.  

We did notice structural defects in both samples (not shown) typically
toward the middle of the specimens.

 

Does anyone know what is causing our IPA processed fat to have these
wide membrane artifacts?  

We are going to repeat the process with an additional 30 minutes per
step and raise the temperature of the steps to ~ 35 C.  

We are also going to cut the blocks at 2-3 um to see if it can reduce
the appearance of the membranes.  

 

Thanks very much for any advice you may have for us.  We are pretty
locked in to using xylene-free processing methodology if at all possible
but will entertain any suggestions you may have.  

If I can provide any further details about what we are doing on our end,
please let me know and I'll be happy to provide them.

 

Best,

David Burk 

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RE: [Histonet] Processing adipose tissue

[Jerry Ricks]

Hi David,

21 hours in isopropyl seems likw ea lot, and I don't see any clearing step to 
remove the IPA.

I've been using Slide Brite instead of Xylene, but I see that Rene Buesa 
published a study indicating that mineral oil is the cat's meow for xylene 
substitutes.

http://www.google.com/url?sa=trct=jq=esrc=ssource=webcd=10ved=0CGMQFjAJurl=http%3A%2F%2Fwww.ebsciences.com%2Fpapers%2FMineral%2520oil%2520as%2520xylene%2520substitute.pdfei=ny4nT73bM8axiQLk3dmQAQusg=AFQjCNFw894if_bFAYgv6Uurw2MkO3Th5A



Jerry



 Date: Mon, 30 Jan 2012 12:20:24 -0600
 From: david.b...@pbrc.edu
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Processing adipose tissue
 
 Esteemed experts,
 
  
 
 We have many clients who want to process mouse and human adipose tissue
 and are having some quality issues in the resultant slides.  
 
 We have tried processing small chunks of tissue (1 cm x 0.5 cm) on our
 automated processor (Excelsior) as follows:
 
 Tissue fixed for ~24 hrs in 10% NBF
 
 70% Isopropyl alcohol (IPA) for 3 hrs
 
 90% IPA, 3hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3 hrs
 
 100% IPA, 3hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
 Paraffin, 3 hrs
 
  
 
 Embed and section at 5 um prior to HE.  An example of what the sections
 look like can be found here http://imgur.com/7RTGR .  
 
 We also ran a sample on a traditional overnight EtOH/Xylene processor
 (not at our facility) to compare results.  That image is here:
 http://imgur.com/GjJPg .  
 
 What is obvious is that the membranes in the IPA processed tissue seem
 to flap over and don't look as crisp as the Xylene processed tissue.  
 
 We did notice structural defects in both samples (not shown) typically
 toward the middle of the specimens.
 
  
 
 Does anyone know what is causing our IPA processed fat to have these
 wide membrane artifacts?  
 
 We are going to repeat the process with an additional 30 minutes per
 step and raise the temperature of the steps to ~ 35 C.  
 
 We are also going to cut the blocks at 2-3 um to see if it can reduce
 the appearance of the membranes.  
 
  
 
 Thanks very much for any advice you may have for us.  We are pretty
 locked in to using xylene-free processing methodology if at all possible
 but will entertain any suggestions you may have.  
 
 If I can provide any further details about what we are doing on our end,
 please let me know and I'll be happy to provide them.
 
  
 
 Best,
 
 David Burk 
 
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[Histonet] processing program for biopsies

[Carol Bryant]

Would you experienced histotechs mind to look at this program and see if it 
would cause skin biopsies to be poorly processed? I think too much time in 
alcohols.
  The pathologists say they look bad.
Thanks in advance for your help!

   Station  SolutionTime   Temp   P/VMix
   ---  --  -  -  -  --
   1Formalin 0:20   OFF   P/VSlow
   2Formalin 0:20   OFF   P/VSlow
   370% Alcohol  0:15   OFF   P/VSlow
   480% Alcohol  0:15   OFF   P/VSlow
   595% Alcohol  0:15   OFF   P/VSlow
   695% Alcohol  0:25   OFF   P/VSlow
   7100% Alcohol 0:25   OFF   P/VSlow
   8100% Alcohol 0:25   OFF   P/VSlow
   9Xylene   0:35   OFF   P/VSlow
  10Xylene   0:35   OFF   P/VSlow
  11Paraffin 0:15   63C.  P/VSlow
  12Paraffin 0:15   63C.  P/VSlow
  13Paraffin 0:15   63C.  P/VSlow
  14Paraffin 0:15   63C.  P/VSlow


Carol Bryant, CT (ASCP)
Cytology/Histology Manager
Lexington Clinic
Phone (859) 258-4082
Fax (859) 258-4081
cb...@lexclin.com



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[Histonet] processing program for biopsiesIn-Reply-To=

[Ruppert, Amysue]

I agree that the time may be too short. I would suggest removing the 80% 
alcohol station and adding another 100%, with the time of 25 minutes. If you 
have a VIP tissue processor, you may also want to change the mix to fast, 
with such short times in the stations, the processor does not have any time to 
do a pump in and pump out when set on slow. If set on fast,  you should get 
at least one mix per station. This is helpful because it breaks any surface 
tension created around the tissue pieces and will allow for better penetration 
of the solutions. I would also suggest to put the heat on the paraffins to 58 
C. Too much exposure to heat can cause poor, hazy staining. If you have had 
this program set for awhile, and had good luck before, but now are having 
issues, than you need to look into the quality of the solutions on the 
processor. And make sure the processor is set up correctly..meaning the correct 
solution is in the correct station. It can happen that someone set up the solut
ions incorrectly, since we are all only human and mistakes happen.

good luck

AmySue Ruppert,HT(ASCP) MB(ASCP)
Marshfield Labs, Histology Lab





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[Histonet] processing schedule for GI'S

[anuradha shrivastava]

Hello Histo gurus,
Could u please advice me on gi’s  processing schedule for Leica asp 300.
thanks in advance.
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[Histonet] Processing and embedding mice lungs.

[Clough, Bret]

Hello everyone!
I have a student that wants to paraffin embed mice lungs, and I’ve only worked 
with bone tissue. I was wondering if someone in histo-land would be willing to 
share with me their protocol or their thoughts on how to proceed. The tissue 
will arrive inflated in a formalin fixative.
Thank you,
Bret Clough

Texas AM Health Science Center
College of Medicine
Temple, TX.
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Re: [Histonet] Processing of derm specimens

[Nicole Tatum]

How many 100%  do you have.  I have 3. This is where the dehydration comes
in. The 100 takes all the mositure out of the specimen. So this step is
critical. Water and xylene are not soluable so if the specimens are not
getting dehydrated properly, the xylene will not penertate the specimens
either. Next is make sure your specimen are not thicker then 3mm.
Espceially lipomas and cyst. Try to cut them as thin as possible. Note,
the caseous inside of a cyst will likely not process and usually expoldes
on a water bath. Next, make sure the specimen cassettes are propely placed
inside the tissue rack. If flow between cassettes is restricted, a portion
of a specimen could be raw.

Hope this helps.

Nicole Tatum HT ASCP

I have recently had a problem with my skin specimens being
 underprocessed. I use a Leica 300ASP. The schedule is as follows:
 10% NBF x 2 for 1 hour ea.
 80% Reagent Alcohol for 1 hour
 95% Reagent Alcohol x 2 for 1 hour each
 100% Reagent Alcohol for 1 hour each
 Xylene x 3 for 1 hour each
 Paraffin x 3 for 1 hour each

 The specimens are mushy and swell on the ice
 Any input is welcome.

 send response to :
 _scrochiere@nedlc.com_ (mailto:scrochi...@nedlc.com)

 thanks
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[Histonet] Processing of derm specimens

[CrochiereSteve]

I have recently had a problem with my skin specimens being  
underprocessed. I use a Leica 300ASP. The schedule is as follows:
10% NBF x 2 for 1 hour ea.
80% Reagent Alcohol for 1 hour
95% Reagent Alcohol x 2 for 1 hour each
100% Reagent Alcohol for 1 hour each
Xylene x 3 for 1 hour each
Paraffin x 3 for 1 hour each
 
The specimens are mushy and swell on the ice
Any input is welcome.
 
send response to :
_scrochiere@nedlc.com_ (mailto:scrochi...@nedlc.com) 
 
thanks
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RE: [Histonet] Processing of derm specimens

[Elizabeth Chlipala]

That processing schedule should be fine for skin samples, we add an additional 
100% alcohol step so we have three absolute steps at 1 hour each (I would 
remove one 95%).  Thickness of your samples is also important they should be 
around 3mm in thickness if they are thicker than that they may not process 
properly.  The other thing is that your solutions need to be fresh for samples 
to process properly.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308-1592
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
www.premierlab.com

Ship to address:

1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
crochierest...@aol.com
Sent: Tuesday, August 02, 2011 4:46 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing of derm specimens

I have recently had a problem with my skin specimens being
underprocessed. I use a Leica 300ASP. The schedule is as follows:
10% NBF x 2 for 1 hour ea.
80% Reagent Alcohol for 1 hour
95% Reagent Alcohol x 2 for 1 hour each
100% Reagent Alcohol for 1 hour each
Xylene x 3 for 1 hour each
Paraffin x 3 for 1 hour each

The specimens are mushy and swell on the ice
Any input is welcome.

send response to :
_scrochiere@nedlc.com_ (mailto:scrochi...@nedlc.com)

thanks
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[Histonet] Processing

[Nicole Schuster]

For large tissue specimens (prostate, placenta, etc.) what temperature do you 
use for the solutions on your processor? We currently have them all set at 40 
degrees Celsius. For paraffin it is set at 60 degrees Celsius. I read the 
dehydrating and clearing solutions should be at ambient temperatures... Thought 
I'd check into it. Thanks! :)

Sent from my iPhone
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Re: [Histonet] Processing

[Rene J Buesa]

Set your formalin at 45ºC, dehydration at room temp with vacuum/pressure and 
the paraffin as usual.
René J.

--- On Wed, 7/6/11, Nicole Schuster njbate...@yahoo.com wrote:


From: Nicole Schuster njbate...@yahoo.com
Subject: [Histonet] Processing
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
Date: Wednesday, July 6, 2011, 3:57 PM


For large tissue specimens (prostate, placenta, etc.) what temperature do you 
use for the solutions on your processor? We currently have them all set at 40 
degrees Celsius. For paraffin it is set at 60 degrees Celsius. I read the 
dehydrating and clearing solutions should be at ambient temperatures... Thought 
I'd check into it. Thanks! :)

Sent from my iPhone
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RE: [Histonet] Processing

[Laurie Colbert]

We heat from 38-40 degrees for all reagents except the paraffin, which
is heated to 60 degrees.  I don't use any heat at all on my small
biopsies.

Laurie Colbert

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Nicole
Schuster
Sent: Wednesday, July 06, 2011 12:58 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing

For large tissue specimens (prostate, placenta, etc.) what temperature
do you use for the solutions on your processor? We currently have them
all set at 40 degrees Celsius. For paraffin it is set at 60 degrees
Celsius. I read the dehydrating and clearing solutions should be at
ambient temperatures... Thought I'd check into it. Thanks! :)

Sent from my iPhone
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[Histonet] Processing- Grossing Personnel requirement

[Sara Baldwin/mhhcc.org]

Hi  Histonetters:
 We are Joint Commission inspected and I was wondering if anyone can shoot me 
ane-mail with the requirements from the CAP checklist for techs grossing.  
Any help would be greatly appreciated!

Thanks
Pathology Supervisor
S. Kathy Baldwin, SCT (ASCP)
Memorial Hospital and Health Care Center
sbald...@mhhcc.org
Ph 812-482-0210, 0216,  Fax 812-482-0232, 
Pager 812-481-0897, Cell 812-887-3357
Confidential information, Authorized use only.
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[Histonet] Processing schedule for fatty tissues

[Sheila Adey]

Hello Everyone:
 
I am looking for a processing schedule for fatty specimens. 
 
Could anyone share theirs?
 
Thanks in Advance. 
:) 
Sheila
Bluewater Health  
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RE: [Histonet] Processing schedule for fatty tissues

[Carol Bryant]

Please respond to everyone.  I would like to have the information also. 
Thank you,
Carol 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sheila Adey
Sent: Friday, June 10, 2011 4:23 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing schedule for fatty tissues


Hello Everyone:
 
I am looking for a processing schedule for fatty specimens. 
 
Could anyone share theirs?
 
Thanks in Advance. 
:) 
Sheila
Bluewater Health  
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protected by the State of Kentucky and/or Federal regulations.  If you are not 
the intended recipient, do not read, copy, retain or disseminate this message 
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sender immediately at (859)258-4000 and delete all copies of this message and 
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Re: [Histonet] Processing schedule for fatty tissues

[Rene J Buesa]

The best approach is to increase the fixation time to make sure that the 
tissues are really fixed.
Then cut the dehydration times of the lower alcohols by 20% and add that 20% of 
saved time as increase in the clearing agent (supposedly xylene or alkane 
substitute) times.
René J.

From: Sheila Adey sa...@hotmail.ca
To: histonet@lists.utsouthwestern.edu
Sent: Friday, June 10, 2011 4:22 PM
Subject: [Histonet] Processing schedule for fatty tissues


Hello Everyone:

I am looking for a processing schedule for fatty specimens. 

Could anyone share theirs?

Thanks in Advance. 
:) 
Sheila
Bluewater Health                          
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