[Histonet] Fw: Waste paraffin and edge effect

[Jeffrey Silverman]

---







Hello everyone, 
 
Our system's  hazardous chemical waste consultants changed our method of 
disposal- we had been solidifying the paraffin in a hood and then red bagging 
it, ie treating it as regulated medical (biohazard) waste. 
 
But, due to it's contamination with xylene, the very reason for our disposing 
of it, the paraffin is really hazardous chemical waste. Believe it or not, EPA 
regs prohibit most hospitals from treating their chemical waste and the 
consultants worried that solidifying it under the hood was considered 
"treating" the waste.  Yeah right!!   Anyway, the blocks of solidified paraffin 
are now dumped in it's own specially labelled drum and has become a part of our 
hazardous waste stream, manifested like the xylene and dye waste. I'd be 
careful about putting hazardous chemicals into regulated medical waste. 
 
Interestingly, our system recently inquired if formalin in discarded surgicals 
needs to be decanted before disposal- many hospitals are doing just that, but 
most of those, but not all, recycle formalin (yuk!). Our disposal facility 
informed us that as long as the containers are plastic, ie are flammamble, they 
can be incinerated with the formalin and there is no need to decant.  Glory 
be!! 
 
In IHC slides, edge effect, or more intense, sometimes nonspecific, staining at 
the periphery of the tissue, can be caused  by more intense fixation of a block 
at the periphery, drying out of the edges of a block before fiation,  and/or by 
some degree of drying of antibodies and detection chemistry reagents 
during staining- this cause is more common in manually stained slides rather 
than those stained on automated stainers.  
 
Also electrocautery used during the excision can also cause intense staining at 
the edges of specimens. 
 
Jeffrey S. Silverman HT HTL QIHC (ASCP)
Southside Hospital- NSLIJ Health System
Pathologists' Assistant and Laboratory Safety Officer
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RE: [Histonet] RE: Eliminating the edge effect in IHC/IF

[Hermina Borgerink]

I do my immuno staining manually using ProbeOn Plus slides which employ the 
capillary action principle.  I always monitor the taking up/whicking off of my 
solutions for each pair of slides throughout the entire staining process and 
know that my sections never dry out (I incubate using moist chambers with 
plenty of fluid).  I work at a research/diagnostic medical school facility 
where our primary focus is on experimental tissues, using standardized and 
rigid guidelines for fixation: 24 hours at 4 degrees overnight in 4% PF, 
followed by post fixation in 70% ethanol for a few days.  Never see the "edge 
effect" with these sections.  However, I do occasionally see the effect using 
archival diagnostic samples that were fixed in 10% NBF for a minimum of 48 
hours, and probably longer. I have therefore always perceived this effect to be 
caused by extended, rather than incomplete fixation.

Hermina

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Tuesday, January 12, 2010 3:41 PM
To: C.M. van der Loos; histonet@lists.utsouthwestern.edu; MargaretSherwood
Cc: k...@prosci-inc.com
Subject: RE: [Histonet] RE: Eliminating the edge effect in IHC/IF

Again, provided that you are doing IHC manually.
René J.

--- On Tue, 1/12/10, Sherwood, Margaret  wrote:


From: Sherwood, Margaret 
Subject: RE: [Histonet] RE: Eliminating the edge effect in IHC/IF
To: "C.M. van der Loos" , 
histonet@lists.utsouthwestern.edu
Cc: k...@prosci-inc.com
Date: Tuesday, January 12, 2010, 3:28 PM


Actually, when I read about the problem, my thought was that if you don't circle
your tissue with an immunopen or wax pencil, then the reagents (esp. primary
antibody)might not cover the tissue evenly as you say.  So I tend to agree with
you.

Peggy 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der
Loos
Sent: Tuesday, January 12, 2010 2:55 PM
To: histonet@lists.utsouthwestern.edu
Cc: k...@prosci-inc.com
Subject: [Histonet] RE: Eliminating the edge effect in IHC/IF

Hi all,We also have observed this phenomenon many times. But sorry Colleen and
Rene, I don't believe that an fixation issue is the explanation why the edges
are sometimes stronger than the rest. To my opinion this is a bit too easy. One
of my explanations is that the immuno reagents tend to stick to the edges of the
tissue section, especially when no Tween20 is involved. As a result the outer
edges might become a little dry during incubation times and cause darker mostly
non-specific staining. Always try to cover a section completely, including a rim
around the section. However, to be honest, I am sure my explanation is certainly
not always appropriate. Anyone elseCheers,ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
phone:  +31 20 5665631
From: Rene J Buesa 
Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu, Karen Cai 

Usually that is the result of incomplete fixation. Check your fixation protocol.
Ren  J.

--- On Mon, 1/11/10, Karen Cai  wrote:


From: Karen Cai 
Subject: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu
Date: Monday, January 11, 2010, 2:00 PM


Hi,
I have a question for the generous input. When I do the IHC or IF, it
seems very common that the intensity of the edge area of the tissue is
always stronger than the central tissue part. Is it possible to
eliminate this and make the staining evenly distributed around the whole
tissue section?

Your kind help is greatly appreciated,


Thanks in advance,

Best,
Karen

Karen Cai
Research Scientist
Prosci Incorporated
(858) 513-2638 x 204
(858) 513-2692 Fax
 www.prosci-inc.com
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RE: [Histonet] RE: Eliminating the edge effect in IHC/IF

[Della Speranza, Vinnie]

I think we'll agree that there are different scenarios so that the solution is 
not a one size fits all. For example, darkened staining around the periphery of 
needle core biopsies is not uncommon with even tinctorial stains, often thought 
to be the result of drying of the tissue during the collection of the sample. 
Years ago I came across an article maintaining that the dark staining on the 
periphery of needle cores was in fact due to the "trauma" of the needle cutting 
into the sampled organ. I've since forgotten the author's name and wish I could 
get my hands on that reference.

So I agree with Chris that there doesn't appear to be one simple answer to 
prevent this artifact and while fixation may contribute in some circumstances 
it's unlikely to be the remedy for all.

Vinnie Della Speranza
Manager for Anatomic Pathology Services
165 Ashley Avenue Suite 309
Charleston, SC 29425
tel. 843-792-6353
fax. 843-792-8974
 
-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der 
Loos
Sent: Tuesday, January 12, 2010 2:55 PM
To: histonet@lists.utsouthwestern.edu
Cc: k...@prosci-inc.com
Subject: [Histonet] RE: Eliminating the edge effect in IHC/IF

Hi all,We also have observed this phenomenon many times. But sorry Colleen and 
Rene, I don't believe that an fixation issue is the explanation why the edges 
are sometimes stronger than the rest. To my opinion this is a bit too easy. One 
of my explanations is that the immuno reagents tend to stick to the edges of 
the tissue section, especially when no Tween20 is involved. As a result the 
outer edges might become a little dry during incubation times and cause darker 
mostly non-specific staining. Always try to cover a section completely, 
including a rim around the section. However, to be honest, I am sure my 
explanation is certainly not always appropriate. Anyone 
elseCheers,ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
phone:  +31 20 5665631
 From: Rene J Buesa 
Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu, Karen Cai 

Usually that is the result of incomplete fixation. Check your fixation protocol.
Ren� J.

--- On Mon, 1/11/10, Karen Cai  wrote:


From: Karen Cai 
Subject: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu
Date: Monday, January 11, 2010, 2:00 PM


Hi,
I have a question for the generous input. When I do the IHC or IF, it
seems very common that the intensity of the edge area of the tissue is
always stronger than the central tissue part. Is it possible to
eliminate this and make the staining evenly distributed around the whole
tissue section?

Your kind help is greatly appreciated,


Thanks in advance,

Best,
Karen

Karen Cai
Research Scientist
Prosci Incorporated
(858) 513-2638 x 204
(858) 513-2692 Fax
 www.prosci-inc.com
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[Histonet] RE: Eliminating the edge effect in IHC/IF

[Rene J Buesa]

Hi Dr. van der Loos:
I had also experienced that artifact caused by less than necessary reagents, 
but that happens mostly when IHC is done manually, or when the autostainer 
delivers less amount than required or programmed.
IF we assume that the colleague with the question did the IHC manually, your 
explanation can be accepted, otherwise, poor fixation is a valid cause to the 
problem.
René J.

--- On Tue, 1/12/10, C.M. van der Loos  wrote:


From: C.M. van der Loos 
Subject: RE: Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu
Cc: k...@prosci-inc.com, rjbu...@yahoo.com, cfors...@umn.edu
Date: Tuesday, January 12, 2010, 2:54 PM



Hi all,
We also have observed this phenomenon many times. But sorry Colleen and Rene, I 
don't believe that an fixation issue is the explanation why the edges are 
sometimes stronger than the rest. To my opinion this is a bit too easy. One of 
my explanations is that the immuno reagents tend to stick to the edges of the 
tissue section, especially when no Tween20 is involved. As a result the outer 
edges might become a little dry during incubation times and cause darker mostly 
non-specific staining. Always try to cover a section completely, including a 
rim around the section. However, to be honest, I am sure my explanation is 
certainly not always appropriate. Anyone else
Cheers,
Chris
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
phone:  +31 20 5665631

 
From: Rene J Buesa 
Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu, Karen Cai 

Usually that is the result of incomplete fixation. Check your fixation protocol.
Ren� J.

--- On Mon, 1/11/10, Karen Cai  wrote:


From: Karen Cai 
Subject: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu
Date: Monday, January 11, 2010, 2:00 PM


Hi,
I have a question for the generous input. When I do the IHC or IF, it
seems very common that the intensity of the edge area of the tissue is
always stronger than the central tissue part. Is it possible to
eliminate this and make the staining evenly distributed around the whole
tissue section?

Your kind help is greatly appreciated,


Thanks in advance,

Best,
Karen

Karen Cai
Research Scientist
Prosci Incorporated
(858) 513-2638 x 204
(858) 513-2692 Fax
 www.prosci-inc.com




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RE: [Histonet] RE: Eliminating the edge effect in IHC/IF

[Rene J Buesa]

Again, provided that you are doing IHC manually.
René J.

--- On Tue, 1/12/10, Sherwood, Margaret  wrote:


From: Sherwood, Margaret 
Subject: RE: [Histonet] RE: Eliminating the edge effect in IHC/IF
To: "C.M. van der Loos" , 
histonet@lists.utsouthwestern.edu
Cc: k...@prosci-inc.com
Date: Tuesday, January 12, 2010, 3:28 PM


Actually, when I read about the problem, my thought was that if you don't circle
your tissue with an immunopen or wax pencil, then the reagents (esp. primary
antibody)might not cover the tissue evenly as you say.  So I tend to agree with
you.

Peggy 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der
Loos
Sent: Tuesday, January 12, 2010 2:55 PM
To: histonet@lists.utsouthwestern.edu
Cc: k...@prosci-inc.com
Subject: [Histonet] RE: Eliminating the edge effect in IHC/IF

Hi all,We also have observed this phenomenon many times. But sorry Colleen and
Rene, I don't believe that an fixation issue is the explanation why the edges
are sometimes stronger than the rest. To my opinion this is a bit too easy. One
of my explanations is that the immuno reagents tend to stick to the edges of the
tissue section, especially when no Tween20 is involved. As a result the outer
edges might become a little dry during incubation times and cause darker mostly
non-specific staining. Always try to cover a section completely, including a rim
around the section. However, to be honest, I am sure my explanation is certainly
not always appropriate. Anyone elseCheers,ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
phone:  +31 20 5665631
From: Rene J Buesa 
Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu, Karen Cai 

Usually that is the result of incomplete fixation. Check your fixation protocol.
Ren  J.

--- On Mon, 1/11/10, Karen Cai  wrote:


From: Karen Cai 
Subject: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu
Date: Monday, January 11, 2010, 2:00 PM


Hi,
I have a question for the generous input. When I do the IHC or IF, it
seems very common that the intensity of the edge area of the tissue is
always stronger than the central tissue part. Is it possible to
eliminate this and make the staining evenly distributed around the whole
tissue section?

Your kind help is greatly appreciated,


Thanks in advance,

Best,
Karen

Karen Cai
Research Scientist
Prosci Incorporated
(858) 513-2638 x 204
(858) 513-2692 Fax
 www.prosci-inc.com
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contains patient information, please contact the Partners Compliance HelpLine at
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RE: [Histonet] RE: Eliminating the edge effect in IHC/IF

[Sherwood, Margaret]

Actually, when I read about the problem, my thought was that if you don't circle
your tissue with an immunopen or wax pencil, then the reagents (esp. primary
antibody)might not cover the tissue evenly as you say.  So I tend to agree with
you.

Peggy 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der
Loos
Sent: Tuesday, January 12, 2010 2:55 PM
To: histonet@lists.utsouthwestern.edu
Cc: k...@prosci-inc.com
Subject: [Histonet] RE: Eliminating the edge effect in IHC/IF

Hi all,We also have observed this phenomenon many times. But sorry Colleen and
Rene, I don't believe that an fixation issue is the explanation why the edges
are sometimes stronger than the rest. To my opinion this is a bit too easy. One
of my explanations is that the immuno reagents tend to stick to the edges of the
tissue section, especially when no Tween20 is involved. As a result the outer
edges might become a little dry during incubation times and cause darker mostly
non-specific staining. Always try to cover a section completely, including a rim
around the section. However, to be honest, I am sure my explanation is certainly
not always appropriate. Anyone elseCheers,ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
phone:  +31 20 5665631
 From: Rene J Buesa 
Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu, Karen Cai 

Usually that is the result of incomplete fixation. Check your fixation protocol.
Ren  J.

--- On Mon, 1/11/10, Karen Cai  wrote:


From: Karen Cai 
Subject: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu
Date: Monday, January 11, 2010, 2:00 PM


Hi,
I have a question for the generous input. When I do the IHC or IF, it
seems very common that the intensity of the edge area of the tissue is
always stronger than the central tissue part. Is it possible to
eliminate this and make the staining evenly distributed around the whole
tissue section?

Your kind help is greatly appreciated,


Thanks in advance,

Best,
Karen

Karen Cai
Research Scientist
Prosci Incorporated
(858) 513-2638 x 204
(858) 513-2692 Fax
 www.prosci-inc.com
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The information in this e-mail is intended only for the person to whom it is
addressed. If you believe this e-mail was sent to you in error and the e-mail
contains patient information, please contact the Partners Compliance HelpLine at
http://www.partners.org/complianceline . If the e-mail was sent to you in error
but does not contain patient information, please contact the sender and properly
dispose of the e-mail.


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[Histonet] RE: Eliminating the edge effect in IHC/IF

[C.M. van der Loos]

Hi all,We also have observed this phenomenon many times. But sorry Colleen and 
Rene, I don't believe that an fixation issue is the explanation why the edges 
are sometimes stronger than the rest. To my opinion this is a bit too easy. One 
of my explanations is that the immuno reagents tend to stick to the edges of 
the tissue section, especially when no Tween20 is involved. As a result the 
outer edges might become a little dry during incubation times and cause darker 
mostly non-specific staining. Always try to cover a section completely, 
including a rim around the section. However, to be honest, I am sure my 
explanation is certainly not always appropriate. Anyone 
elseCheers,ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
phone:  +31 20 5665631
 From: Rene J Buesa 
Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu, Karen Cai 

Usually that is the result of incomplete fixation. Check your fixation protocol.
Ren� J.

--- On Mon, 1/11/10, Karen Cai  wrote:


From: Karen Cai 
Subject: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet@lists.utsouthwestern.edu
Date: Monday, January 11, 2010, 2:00 PM


Hi,
I have a question for the generous input. When I do the IHC or IF, it
seems very common that the intensity of the edge area of the tissue is
always stronger than the central tissue part. Is it possible to
eliminate this and make the staining evenly distributed around the whole
tissue section?

Your kind help is greatly appreciated,


Thanks in advance,

Best,
Karen

Karen Cai
Research Scientist
Prosci Incorporated
(858) 513-2638 x 204
(858) 513-2692 Fax
 www.prosci-inc.com
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[Histonet] IHC

[Marian Powers]

Hi:  We are looking to start N-ras and B-raf for IHC, is anyone running
these antibodies on paraffin?  If so, what clone and vendor are you using?
Thanks in advance!



-- 
Marian L. Powers, HT(ASCP)
Manager, Technical Operations

Doctors Pathology Services
1253 College Park Drive
Dover, DE 19904
302-677-
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[Histonet] Histotech, Mohs opportunity New Hampshire

[Melissa Ribeiro]

I am recruiting around a Histotech - Mohs position in Southern New Hampshire. 
Right on the border with Massachusetts. 

 

BRIEF SUMMARY:  

- Under the general supervision of the Microbiology/Histology supervisor will 
specifically be working performing Mohs cryotonomy.  

- This includes but is not limited to specimen receipt, mapping the piece of 
tissue, cutting and slide preparation of frozen tissue, and staining of 
prepared slides.  

- Instrument maintenance and quality control documentation is also required. 

-  Previous Mohs experience desired.  

 

CONTACT: Melissa Ribeiro @ (781) 272-3400 ext. 228 or via e-mail at 
mribe...@brinegroup.com 





Melissa Ribeiro Passos
Healthcare Division 
Brine Group Staffing Solutions 
20 Mall Road, Suite 225
Burlington, MA 01803
mribe...@brinegroup.com
Ph.  (781) 272-3400 ext. 228
Fax (781) 494-3401

Save a Tree! Consider the environment before you print this e-mail. 

Visit our newly redesigned web site at www.brinegroup.com, the intelligent 
choice for staffing solutions.
 

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[Histonet] Problems with MSB stain

[Stephen.Eyres]

Hi,
 
I have just encountered a problem I have never seen before and would
welcome opinions as to the cause. I work in pharma R&D and had a request
to perform MSBs on some liver slides. The results varied within the run,
with a distinct difference in the overall colour of the  slides; some
appearing blue and others red. The person performing the staining is
experienced, and our neqas scores are good. When we investigated the
problem the only difference we could identify was that the bluer slides
were stained after  40 minutes drying after being cut, whereas the
redder slides were cut the day before. This suggests that maybe slides
were incompletely dried and that this affected the staining. Comments
please.
 
Cheers
 
Steve 
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