Re: [Histonet] Ventana Retic problems

[Curt via Histonet]

We have had the same problem our solution was not the second but the wash, 
it for a bad too quickly so we don't make a full carboy and let it sit for a 
week, we make fresh wash almost daily, ESPECIALLY when we have a retic. Our 
stains look great daily with fresh wash solution.
You may try that,  hope it helps.

Curt



Sent from my Verizon, Samsung Galaxy smartphone


 Original message 
From: Jeffrey Robinson via Histonet 
Date: 6/14/17 4:01 PM (GMT-08:00)
To: donna mihalik rossi 
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Ventana Retic problems

Hi Donna-  I have been dealing with these same exact issues for 3 1/2 years now 
so I think I have achieved "expert status" in dealing with this problem.

First, I'll give you my current protocol.  When all things are working 
smoothly, it produces a nice stain.  But it is very frustrating when it starts 
to "fade."

We cut all retics at 5.  Thinner cuts will definitely look lighter.

Protocol:  warmup slide:  47 degrees C.
 Oxidizer:4 minutes
 Decolorizer: 4 minutes
 Sensitizer:  8 minutes
 Optimize Counterstain Intensity:  4 Minutes

I have gone around and around with Ventana on decontamination problems with 
this instrument.  I was performing full decontamination runs every month.  My 
rep finally said to just decon the bulk wash carboy and the wash bottle on the 
instrument when fading begins to show.  Ventana claims there will be a new wash 
out this year that will hopefully take care of the problem once and for all but 
no one at Ventana can give me a release date on that.
In the meantime, this is what I do:

Daily:  run the "Purge Wash" function test 3 times in the morning before 
running any slides.  Be sure to run the "Purge" function and not the "Prime" 
function.
When loading slides, put all of your retic slides on after everything else 
(after the GMS, etc., slides).

When staining starts to fade:  after ruling out "thin cuts" and other obvious 
problems it is time for decon.  The fading will show first on the patient 
tissue (the control may still look OK).
Here is my current decon protocol:  I use the Lysol IC decon solution. I have 
an extra 6 gallon carboy and I leave some made up (diluted) so that it is ready 
to go.  Put some decon solution on a 4X4 guaze and wipe the dispenser tip 
underneath the top (lift lid up for access).  Dump the wash solution in the 
wash bottle on the instrument.  Rinse out with DI water. Fill with decon 
solution.  Swish solution inside bottle.  Replace bottle (on instrument).  Run 
"Purge Wash" function test (3 times).  After Purge #3, let the solution sit in 
the lines for at least 15 minutes (the longer the better).  When you are ready 
to proceed, rinse bulk bottle well several times and replace with DI water.  
Run "Purge Wash" 3 more times.  Time is not a factor here so you can just run 
them one after another.  After the third purge, replace the DI with BM SS wash. 
 Run "Purge Wash" function test 3 more times.  That's it.  I find I need to run 
this procedure about once a month.
Additionally, I decon the 5 gallon bulk wash carboy EVERY TIME I make up a new 
batch.  It doesn't take long (I just let the decon solution sit for 15 minutes) 
and then when I do need to do the modified decon on the instrument I do not 
need to worry about the bulk carboy.

I hope this helps- it takes a little time but it does seems to help with the 
retic stain intensity.

Jeff Robinson, Senior Histotechnologist (HT, HTL), Sierra Pathology Lab, 
Clovis, CA.

-Original Message-
From: donna mihalik rossi via Histonet 
[mailto:histonet@lists.utsouthwestern.edu]
Sent: Wednesday, June 14, 2017 1:45 PM
To: histonet
Subject: [Histonet] Ventana Retic problems

Hi Histonetters!  We are experiencing sporadic staining of retic fibers from 
our Ventana Benchmark machines.  We have 2 machines and are having the same 
problem on both. The machines were both decontaminated 2 weeks ago with all new 
solutions  being made.  The fibers are not crisp and are disconnected.  Our 
control tissue is  at the top of the slide with the patient tissue at the 
bottom. It appears that the control is staining better than the patient tissue 
but still is  not crisp. We have tried different lots with the same result. Any 
comments before we consult Ventana? Your help would be appreciated.  Thanks, 
Donna  Rossi, PSU


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[Histonet] Fixation and IHC

[Cristi Rigazio via Histonet]

Hi histonetters!

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

Thanks,
Cristi
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Re: [Histonet] Ventana Retic problems

[Jeffrey Robinson via Histonet]

Hi Donna-  I have been dealing with these same exact issues for 3 1/2 years now 
so I think I have achieved "expert status" in dealing with this problem.

First, I'll give you my current protocol.  When all things are working 
smoothly, it produces a nice stain.  But it is very frustrating when it starts 
to "fade."

We cut all retics at 5.  Thinner cuts will definitely look lighter.

Protocol:  warmup slide:  47 degrees C.
 Oxidizer:4 minutes
 Decolorizer: 4 minutes
 Sensitizer:  8 minutes
 Optimize Counterstain Intensity:  4 Minutes

I have gone around and around with Ventana on decontamination problems with 
this instrument.  I was performing full decontamination runs every month.  My 
rep finally said to just decon the bulk wash carboy and the wash bottle on the 
instrument when fading begins to show.  Ventana claims there will be a new wash 
out this year that will hopefully take care of the problem once and for all but 
no one at Ventana can give me a release date on that.
In the meantime, this is what I do:

Daily:  run the "Purge Wash" function test 3 times in the morning before 
running any slides.  Be sure to run the "Purge" function and not the "Prime" 
function.
When loading slides, put all of your retic slides on after everything else 
(after the GMS, etc., slides).

When staining starts to fade:  after ruling out "thin cuts" and other obvious 
problems it is time for decon.  The fading will show first on the patient 
tissue (the control may still look OK).
Here is my current decon protocol:  I use the Lysol IC decon solution. I have 
an extra 6 gallon carboy and I leave some made up (diluted) so that it is ready 
to go.  Put some decon solution on a 4X4 guaze and wipe the dispenser tip 
underneath the top (lift lid up for access).  Dump the wash solution in the 
wash bottle on the instrument.  Rinse out with DI water. Fill with decon 
solution.  Swish solution inside bottle.  Replace bottle (on instrument).  Run 
"Purge Wash" function test (3 times).  After Purge #3, let the solution sit in 
the lines for at least 15 minutes (the longer the better).  When you are ready 
to proceed, rinse bulk bottle well several times and replace with DI water.  
Run "Purge Wash" 3 more times.  Time is not a factor here so you can just run 
them one after another.  After the third purge, replace the DI with BM SS wash. 
 Run "Purge Wash" function test 3 more times.  That's it.  I find I need to run 
this procedure about once a month.
Additionally, I decon the 5 gallon bulk wash carboy EVERY TIME I make up a new 
batch.  It doesn't take long (I just let the decon solution sit for 15 minutes) 
and then when I do need to do the modified decon on the instrument I do not 
need to worry about the bulk carboy.

I hope this helps- it takes a little time but it does seems to help with the 
retic stain intensity.

Jeff Robinson, Senior Histotechnologist (HT, HTL), Sierra Pathology Lab, 
Clovis, CA.

-Original Message-
From: donna mihalik rossi via Histonet 
[mailto:histonet@lists.utsouthwestern.edu]
Sent: Wednesday, June 14, 2017 1:45 PM
To: histonet
Subject: [Histonet] Ventana Retic problems

Hi Histonetters!  We are experiencing sporadic staining of retic fibers from 
our Ventana Benchmark machines.  We have 2 machines and are having the same 
problem on both. The machines were both decontaminated 2 weeks ago with all new 
solutions  being made.  The fibers are not crisp and are disconnected.  Our 
control tissue is  at the top of the slide with the patient tissue at the 
bottom. It appears that the control is staining better than the patient tissue 
but still is  not crisp. We have tried different lots with the same result. Any 
comments before we consult Ventana? Your help would be appreciated.  Thanks, 
Donna  Rossi, PSU


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[Histonet] Ventana Retic problems

[donna mihalik rossi via Histonet]

Hi Histonetters!  We are experiencing sporadic staining of retic fibers from 
our Ventana Benchmark machines.  We have 2 machines and are having the same 
problem on both. The machines were both decontaminated 2 weeks ago with all new 
solutions  being made.  The fibers are not crisp and are disconnected.  Our 
control tissue is  at the top of the slide with the patient tissue at the 
bottom. It appears that the control is staining better than the patient tissue 
but still is  not crisp. We have tried different lots with the same result. Any 
comments before we consult Ventana? Your help would be appreciated.  Thanks, 
Donna  Rossi, PSU


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Re: [Histonet] Histologist In Oregon

[Isabel Soto via Histonet]

HI Rueben. There is 2 HT positions in Salem if you are interested. Calleven for 
details 407-307-8604 

Sent from Yahoo Mail on Android 
 
  On Wed, Jun 14, 2017 at 5:17 AM, RC via 
Histonet wrote:   Hey fellow histologists!


Top of the morning. I’m looking for histology positions in the state of Oregon. 
16 years of experience covering all facets of histology (R, Drug Discovery, 
Clinical Diagnostic, Reference) If any of your laboratories are in need, please 
contact me. Grazie!

Rueben Carter
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[Histonet] Installing a new auto stainer

[Katie via Histonet]

Hello,

I am a small lab that has just relocated to a new building.  As a part of the 
new lab construction we are installing our first auto-stainer.  The plumber is 
concerned about any required permitting or precautions regarding back flow of 
reagents.  I haven’t been able to find any information either way, because most 
labs have building operations management teams handle that end.  Has anyone 
installed a new auto-stainer that knows if any special permitting, etc. is 
required?

Thanks so much and feel free to contact me,

Katie Riley
Technical Supervisor of Dermatopathology
Puyallup Dermatology Clinic
ka...@puyallupderm.com
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[Histonet] 3D plastic

[Mary Lou Norman via Histonet]

Hello Histonet,

I've been asked to section 3D plastic with tissue attached. I will be finding 
out what type of plastic it is. Has anyone done this?  Will I still be able to 
use paraffin?
Thanks for any information.

Mary Lou Norman
NYS College of Veterinary Medicine
Cornell University
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Re: [Histonet] Microwave processors

[Lester Raff MD via Histonet]

We are quite happy with Milestone Histos3 for processing our prostate and 
bladder biopsies quickly.

Lester J. Raff, MD MBA
UroPartners
Medical Director Of Laboratory
2225 Enterprise Dr. Suite 2511
Westchester, Il 60154
Tel: 708-486-0076
Fax: 708-492-0203


-Original Message-
From: Charles Riley via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, June 14, 2017 8:39 AM
To: Histo List
Subject: [Histonet] Microwave processors

Does anyone have any suggestions for microwave processing units? My lab 
processes about 110 blocks max a day from GI biopsies to breast tissue on size

Sent from my iPhone
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Re: [Histonet] Microwave processors

[Walter Benton via Histonet]

Charles,

Milestone Medical http://www.milestonemed.com/

The processors are great. Easy to operate and use minimal chemicals based on 
the protocols you select.  H and IHC are very good.

Check out the various product offerings to see which unit will meet your needs 
best.


Walter Benton HT(ASCP)QIHC
Lab Operations Manager
Chesapeake Urology Associates
806 Landmark Drive, Suite 127
Glen Burnie, MD 21061
410-768-5961 (Lab)
410-768-5965 (Fax)
Chesapeakeurology.com

Voted a Best Place to Work by
Baltimore and Modern Healthcare
Magazines.



-Original Message-
From: Charles Riley via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Wednesday, June 14, 2017 9:39 AM
To: Histo List 
Subject: [Histonet] Microwave processors

Does anyone have any suggestions for microwave processing units? My lab 
processes about 110 blocks max a day from GI biopsies to breast tissue on size

Sent from my iPhone
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[Histonet] Microwave processors

[Charles Riley via Histonet]

Does anyone have any suggestions for microwave processing units? My lab 
processes about 110 blocks max a day from GI biopsies to breast tissue on size

Sent from my iPhone
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[Histonet] Paraffin histology with Muscle

[Lucas Olson via Histonet]

Dear all,

Does anyone have optimization tips for using paraffin histology with
muscle? We do have access to a cryostat to do frozen histology, but it's
not our lab's thus we have to pay to use it. We do have all the materials
necessary for paraffin-based histology though. I've noticed that muscle can
be more difficult to use with paraffin. Do you guys have any processing or
cutting tips?

Thanks!
Lucas Olson
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[Histonet] H + E Staining protocols

[Lucas Olson via Histonet]

Dear All,

There are many variants to the standard H + E protocol. I'm seeking to
optimize the current protocol our laboratory uses (pasted below). Do you
guys have any suggestions of optimization (e.g. cutting down on the long
water rinse after hematoxylin and increasing bluing reagent time)? Or if
you have protocols that you'd think would be better please let me know. Our
lab deals primarily with bone/cartilage/muscle.

Our current protocol:
Staining Preparation
1. After sectioning samples, dry in oven.
Time Temp C
Overnight 37
2-3 hours 45
30 minutes 55

Deparaffinization
1. Remove paraffin with xylene
a. 2 minutes xylene
b. 2 minutes in fresh xylene
c. 2 minutes in fresh xylene
d. 2 minutes in fresh xylene
2. Remove excess xylene
3. Rehydrate samples
a. 1 minute absolute EtOH
b. 1 minute different bucket of absolute EtOH
c. 30 seconds in 95% absolute EtOH
d. 45 seconds in 70% EtOH
4. Rinse with water for 1 minute
Hematoxylin and Eosin Staining
1. Stain in hematoxylin (VWR 95057-858) for 5-10 minutes
a. Optimized time for calvarium bone: 10 minutes
b. Solution may be filtered to remove oxidized particles
2. Rinse in running tap water for 20 minutes
3. Decolorize in Clarifier 1 (1 second)
a. Longer time (up to 3 seconds) will yield a lighter color. Discard after
each use.
4. Immerse in VWR Bluing Reagent (30 sec- 1 minute)
a. Dilute 25mL bluing reagent in 475 mL ultrapure H2O to working stock
b. Optimized time for calvarium bone: 90 seconds
5. Rinse well in tap water (5 minutes)
6. Rinse in 95% ETOH (30 sec- 1 minute)
7. Counterstain in Eosin (30 sec- 90 sec)
a. Optimized time for calvarium bone: 30 seconds
8. Dehydrate
a. ETOH 95% (3 minutes)
b. ETOH 95% (3 minutes)
c. ETOH 100% (3 minutes)
d. ETOH 100% (3 minutes)
9. Clear in Xylene (5 minutes)
10. Clear in new Xylene (5 minutes)
11. Mount with Cytoseal in fume hood and coverslip




Thanks!
Lucas Olson
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[Histonet] Histologist In Oregon

[RC via Histonet]

Hey fellow histologists!


Top of the morning. I’m looking for histology positions in the state of Oregon. 
16 years of experience covering all facets of histology (R, Drug Discovery, 
Clinical Diagnostic, Reference) If any of your laboratories are in need, please 
contact me. Grazie!

Rueben Carter
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