[Histonet] Microtomes

[HERRINGTON, SHEILA via Histonet]

We are looking to purchase two new microtomes and were wondering on thoughts 
and recommendations from the experience of the people that actually use them.  
Pros and cons would be extremely valuable on ergonomics, reliability and 
overall performance.

Sheila Herrington, RT
Technical Lead, Immunohistochemistry and Histopathology
Kelowna General Hospital
2268 Pandosy Street,
Kelowna, B.C. V1Y 1T2

250-862-4300 x 7510

sheila.herring...@interiorhealth.ca



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[Histonet] RE: Pankeratin- Trouble with Mast Cell Staining

[HERRINGTON, SHEILA]

We found that if using the stronger proteases/enzymes for retrievals, there 
tended to be more mast cell staining seen.  


Sheila Herrington
Technical Lead - Histopathology and Immunohistochemistry
Kelowna General Hospital
2268 Pandosy Street, Kelowna, B.C. V1Y 1T2
250-862-4300 ext 7587 or 7510
sheila.herring...@interiorhealth.ca



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Daniel Gonzalez
Sent: Tuesday, May 20, 2014 1:33 PM
To: 'Miller, Suzie'; histonet@lists.utsouthwestern.edu
Subject: [Histonet] RE: Pankeratin- Trouble with Mast Cell Staining

Posting for my colleague, Jeff Gordon, because his post did not go through 
successfully:

"Suzie, your histonet post was forwarded to me by a colleague, and I thought I 
could help with your mast cell staining dilemma with pan-keratin.  University 
of Pennsylvania published an article a while back about "undesirable" 
reactivity of cytokeratins that focused primarily on sentinel lymph node 
testing.  In their study they found that when cytokeratin markers OTHER than 
strictly AE1/AE3 (with no other components) were used, other non-epithelial 
elements were being labeled that they deemed "undesirable."  This entire 
article can be found here for your's and your pathologists' reference:  
http://www.archivesofpathology.org/doi/pdf/10.1043/0003-9985(2000)124%3C1310%3AUCIONN%3E2.0.CO%3B2
  

Based on these findings, and because another person in the thread also 
indicated that they may be dealing with the same issue, cervical tissue that 
was rich in mast cells was stained on a Ventana Ultra with cytokeratin AE1/AE3 
ONLY as well as with cytokeratin OSCAR (which is another pan-keratin but with a 
lesser sensitivity to squamous epithelium than AE1/AE3) with CC1 mild heat 
retrieval and 16 minute heated primary incubation and Ultraview detection.  To 
confirm the presence of mast cells in the tissue, both CD117 and tryptase were 
also stained on the same tissue with the same protocols.  The results showed 
that though there was an abundance of mast cells present labeled by tryptase 
and CD117, there was no detectable staining of mast cells with either 
cytokeratin AE1/AE3 or with cytokeratin OSCAR, while the epithelial portion of 
the tissue stained with both cytokeratins (though the OSCAR was noticeably 
weaker because of the lower sensitivity to squamous epithelium).  

The stains can be seen here:  
http://www.cellmarque.com/cmc/AO_mastcellreactivity.php  

Hopefully this helps.  If you have any questions, please contact us at your 
convenience."

Thank you

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Miller, Suzie
Sent: Tuesday, May 06, 2014 8:57 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Pankeratin- Trouble with Mast Cell Staining

Hello Everyone,

If you have a Ventana Ultra and use (Ventana# 760-2595) Pankeratin 
AE1/AE3/PCK29, would you consider sharing your staining protocol?

Our Pathologists are displeased with the amount of mast cell staining with our 
current protocol so we are looking to explore other protocols or suggestions.

Thank you for your assistance,
Suzie

Suzie Miller, MLT ASCP
Senior Histotech
Mercy Health System of Maine Laboratory
mill...@emhs.org

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[Histonet] RE: Cornflaking artifact

[HERRINGTON, SHEILA]

We also have recently started to see this artifact more than ever before, and 
nothing in our process has changed.  We have tried everything to correct to no 
avail.  Wonder if it is possible to be a change in some type of supply, either 
xylene or coverslipping film.  Something has changed but am at a loss as to 
what.


Sheila Herrington
Technical Lead Histopathology and Immunohistochemistry
Kelowna General Hospital
2268 Pandosy Street, Kelowna, B.C. V1Y 1T2
250-862-4300 ext 7587 or 7510
sheila.herring...@interiorhealth.ca



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie Colbert
Sent: Thursday, March 13, 2014 6:30 AM
To: Sharon Scalise; histonet@lists.utsouthwestern.edu
Subject: [Histonet] RE: Cornflaking artifact

You will also see the cornflaking if your tissue is lifting off of the slide at 
all.  We used to get this more often on hard, decal specimens than on other 
specimens.  We used the film to coverslip.  If you remove the film from the 
problem slides and recoverslip conventionally with extra mountant and glass 
coverslips, I'm sure you will not see the artifact.

Laurie Colbert

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sharon Scalise
Sent: Wednesday, March 12, 2014 8:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] RE: Cornflaking artifact

I am looking for help with "cornflaking" (tiny, brown dry spots under 
coverslip)artifact.  We have been using fresh xylene on our stainer and 
coverslipper, cleaned and wiped all containers dry before filling, tried 
different lots of coverslipping film and had service on our coverslipper to 
make sure it was functioning properly, including the xylene drip.  We continue 
to have this artifact and it is driving us crazy.  It is sporadic with no 
pattern of tissue type or placement on the slide.  Sometimes it lands on tissue 
other times not.  Most of the time when we remove the coverslip and 
re-coverslip it goes away (I am assuming because the acetone removes any minute 
amounts of water that may be present).  We just cannot figure out where the 
water is coming from.  Has anyone seen this artifact while using the drying 
step on the prisma stainer?  We just recently started using the drying on some 
slides and I am thinking maybe it is causing humidity???  I cannot say for a 
fact that our "cornflaking" started at the same time, but it is suspicious. 
HELP!

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Wait, Trevor 
Jordan
Sent: Wednesday, March 12, 2014 3:57 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Paraffin type and Tetracycline labelling Questions

For those who have done Decalcified bone processing with paraffinwhat is 
the best type of paraffin that you guys are familiar with?

Also, if you are wanting to see a tetracycline label on the bone for bone 
turnover, must undecalcified sections be used? How for a double tetracycline 
label?


Trevor Jordan Wait
University of Texas Health Science Center, San Antonio Class of 2017 MD 
Candidate Abilene Christian University Class of 2013 Graduate B.S.  
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RE: [Histonet] Low Temp question

[HERRINGTON, SHEILA]

I also have been having low temp errors.  I use  Benchmark Ultras and they are 
not a batch stainer, so pads are heated independently.  I noticed the staining 
on the particular pad with the error was not quite to the usual standard so I 
have stopped using that pad.  Have contacted Ventana for a case number, but 
have not got an answer.  Is this a new problem for you as well?  This only 
started a few weeks ago for us.


Sheila Herrington
Technical Lead Histopathology and Immunohistochemistry
Kelowna General Hospital
2268 Pandosy Street, Kelowna, B.C. V1Y 1T2
250-862-4300 ext 7587 or 7510
sheila.herring...@interiorhealth.ca





-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tom McNemar
Sent: Tuesday, October 22, 2013 8:28 AM
To: 'Shawn Leslie '; Histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Low Temp question

We see these continually as well.  We are told that it is a software glitch and 
not really a problem with the heat. I was told that they are working on it.

Tom McNemar, HT(ASCP)
Histology Co-ordinator
Licking Memorial Health Systems
(740) 348-4163
(740) 348-4166
tmcne...@lmhealth.org
www.LMHealth.org


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Shawn Leslie
Sent: Tuesday, October 22, 2013 11:07 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Low Temp question


Goodmorning all,
I have a question concerning the Ventana Benchmark XT. We are constantly 
having low temperature errors during almost every run. The immunos look fine 
but we are still concerned. Has anyone else that uses the Benchmark experienced 
this also ? We have noticed that the silver coating on the heating pads is 
wearing off. Ventana indicated to us that it's nothing to be concerned about. 
Any hel p would be appreciated.

Shawn Leslie HT (ASCP)
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RE: [Histonet] IHC mystery

[HERRINGTON, SHEILA]

We have had issues like this in the past on occasion and it usually traces back 
to a slide hydrophobicity problem, and completely random.  It also seem to be 
affected by certain tissues more than others when it occurred, namely breasts.

Sheila

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Truscott, Tom
Sent: Friday, August 09, 2013 10:13 AM
To: Cindy Pyse; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] IHC mystery

Hi Cindy, I don't use the Dako system or any of the antibodies you mention, but 
this kind of sounds like an antibody diluent problem, where either what you 
dissolve the antibody in doesn't spread out evenly on the slide or the antibody 
doesn't dissolve well in the diluents you use. In an automated system, it just 
could be that the rinse cycle in between reagents is incomplete. I have seen 
something like this happen where oily or waxy blotches contaminated the slide 
before the run . Good luck, Tom T 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Cindy Pyse
Sent: Friday, August 09, 2013 9:51 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC mystery

Happy Friday Histonetters

 

I have a mystery. First a little background. We stain our immuno’s slides on a 
Dako 48 with the PT modules for pretreatment. We have not changed any protocols 
or antibodies since we optimized and validated the instruments. The majority of 
our antibodies are Dako but we do use other vendors, our detection is the Dako 
Flex. The pretreatment used is either high or low Dako pretreatment solution 
with the Her-2 and p16 using there FDA approved pretreatment. The her-2 and p16 
slides are deparaffinized with xylene and rehydrated to water as the FDA 
protocol requires. All other antibodies are placed directly into the preheated 
pretreatment solution. All controls are precut and placed on Dako slides and 
prebaked for 30 minutes in a 58⁰ oven for 30 minutes. We cut our patient tissue 
and place them on the bottom of the slides and bake them in the same oven for 
45 minutes. 

 

Recently we have noticed that some of the ER and PR (breast tissue) is not 
staining completely, with an occasional Her-2 giving us the same staining 
problem. Sometime it is part of the section not staining at all, almost like a 
hydrophobic effect with part of the patient tissue not staining past an almost 
perfect line. Sometimes it seems like the parts of the patient tissue within 
the section is lifted off, but when you look at the slide the tissue is there 
it just does not stain. All of our controls stain perfectly. All other 
antibodies are not effected.

 

Some of the things we have tried. Leaving the slides longer in the oven. Since 
it is breast tissue and the pretreatment has a surfactant in it. We have 
checked the level of the slides on the instruments. Since I do have two 
instruments, we have plotted the slides to see if it is a certain rack or 
instrument. We have checked if the pretreatment modules are running correctly 
since we have three. I have three tech running the slides. I am having an 
engineer coming in to look at both instruments.

 

There is no common denominator I can find. I have talked to my TRS from Dako 
and their Tech Service. No one can come up with the cause of the problem. I 
have been doing histology for over 30 years and immuno’s for over 20 years and 
I have never had a problem that I couldn’t figure out the cause. This time I 
need expert help. Does anyone have ideas? I am willing to trying anything to 
solve this problem. 

 

Please help!

Cindy

 

Cindy Pyse CLT, HT(ASCP)

Laboratory Manager

X-Cell Laboratories of WNY

20 Northpointe Parkway Ste 100

Amherst, NY 14228

716-250-9235 Ext. 232

cp...@x-celllab.com

 

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[Histonet] RE: WT1 overstaining on Benchmark Ultra

[HERRINGTON, SHEILA]

I have been experiencing the same random over staining issues with WT1 as well. 
I use the Benchmark Ultras with Ultraview.  It started around the same time as 
a new lot number changed a few months back.  I have tried everything, but being 
so random it is hard to find a solution.


Sheila Herrington, IHC Kelowna General
Grade 3, Immunohistochemistry, Histolopathogy
Kelowna General Hospital



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Clare Thornton
Sent: Tuesday, March 26, 2013 8:57 AM
To: 'Dessoye, Michael J'; Histonet
Subject: [Histonet] RE: WT1 overstaining on Benchmark Ultra

I've seen the exact same thing happen...WT-1 antibody from Cell Marque, on 
Ventana instrumentation.  Except we use Ultraview detection.  I had 3 WT-1 
slides running at the same time.  Two were overstained like you describe, one 
was fine.  The repeats were fine.  The overstained slides almost look like they 
had been "cooked".  Interesting!


Clare J. Thornton, HTL(ASCP)QIHC 
Assistant Histology Supervisor
Dahl-Chase Diagnostic Services
417 State Street, Suite 540
Bangor, ME 04401
cthorn...@dahlchase.com




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dessoye, 
Michael J
Sent: Tuesday, March 26, 2013 11:45 AM
To: Histonet
Subject: [Histonet] WT1 overstaining on Benchmark Ultra

Hello Histonet,
 
I have two strange occurrences that I'm trying to get to the bottom of.
Running WT1 from Cell Marque on a Benchmark Ultra using iView detection and the 
recommended protocol.  Two specimens (both of which happen to be pleural fluid) 
severely overstained, even leaving the slide with a brown background stain.  
This has only happened twice, and the slides were on different positions on 
different staining runs.  They used the same antibody and detection kit, 
however other pleural fluid slides with WT1 are fine.  Other slides that run on 
the same positions also stain ok.  I am repeating the stains on the same 
tissues to try to repeat this but in the meantime has anyone experienced this 
condition or have any advice?
 
The only time I've seen this condition before was due to a clogged vortex 
mixer, but it affected all the slides and is obviously not the case here.
 
Thanks!
 
Michael J. Dessoye, M.S. | Histology Supervisor | Wilkes-Barre General Hospital 
| An Affiliate of Commonwealth Health | mjdess...@commonwealthhealth.net | 575 
N. River Street | Wilkes Barre, PA 18764 | Tel: 570-552-1432 | Fax: 
570-552-1526 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 
_ _ _ _ _

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RE: [Histonet] H&E Destain protocol

[HERRINGTON, SHEILA]

Not if it is just from processing. Eosin from processing will not affect any 
IHC or Special stains. If it was H&E stained you just need to take back through 
Xylene, alcohol to water.  The alcohol will remove some of the eosin, and the 
IHC process will not be affected by the stains. It will simply replace them. 
Usually I do the same process as well with a control just to quality control 
it.  


Sheila Herrington, IHC Kelowna General
Grade 3, Immunohistochemistry, Histolopathogy
Kelowna General Hospital



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Maribel Santiago
Sent: Thursday, February 14, 2013 11:52 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] H&E Destain protocol


Hello Histonet,
I have samples that were stained during fixation with Eosin. First question, 
since I  will do IHC on them, do I need to get rid of the eosin like you do 
with an already H&E stained slide? If so, that brings me to my second question, 
does anyone have a protocol that are willing to share with me along with 
helpful hints?? 
Thanks,Minnie
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 



  
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[Histonet] RE: Water in molds

[HERRINGTON, SHEILA]

Are you using mold release?  Sometimes that can cause what you are describing.  

Sheila Herrington, RT (CSMLS)

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Bonnie 
Gambichler
Sent: Tuesday, January 29, 2013 3:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Water in molds

Hello everyone,

My question concerns the possible presence of water in the embedding molds.
Lately I've been noticing that the sections made from blocks embedded in our 
lab are melting very quickly around the edges.  The sections are also coming 
apart from each other when placed on the waterbath.  The tissue remains intact, 
so I don't think it's a processing problem.  Could this be due to residual 
water left in the mold mixing with the paraffin?

Bonnie Gambichler, HT (ASCP)
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