Re: [Histonet] Cryotomy help

[Charles Riley via Histonet]

Thanks for the advice. That's what I feared but was hoping someone may have
found a trick to pull out the methanol to harden the tissue.



On Fri, Jul 12, 2024 at 1:19 PM Colleen Forster  wrote:

> Once the tissues are in any ethanol or methanol you will never be able to
> get a frozen section. The only way I have been able to salvage anything
> like this was to place the OCT block into 10%BNF on a rotator and let them
> thaw/fix and process into a FFPE block.
>
> If they cannot use FFPE because of the experiment I would say they need to
> start over. This time, freeze the sample, NO METHANOL FIRST. Do the
> fixation as  a post fix OR fix in an aqueous fixative such as 4%para or
> 10%BNF, sucrose protect and then freeze.
>
> Believeme, I have tried to get this to work in so many ways and all
> experts I consulted told me the exact same thing. The mehthaol will not
> allow the sample to freeze hard enough to cut. They don't even want to use
> a slurry with methanol to freeze it the OCT block. The tissue will  absorb
> enough of that methanol to prevent good frozen sectioning.
>
> Good Luck
>
> Colleen Forster HT(ASCP)QIHC
>
> On Fri, Jul 12, 2024 at 11:09 AM Davoli, Katherine A via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
>> The initial freeze isn't usually the problem.  It's what happens when it
>> warms back up to Cryostat cutting temperatures of -20 or -25.
>> You'd need to get the methanol out of the tissue completely because it
>> causes the support to melt at the temperatures the Cryostat cuts at.
>>
>> And unfortunately I have not found a method that fully removes ethanol or
>> methanol from a tissue sample that would then allow me to freeze and cut
>> it.  If someone else replies with one I will genuinely be delighted because
>> I have this problem a lot.
>>
>> Katherine Davoli, MDiv, HTL(ASCP)cm(they/them/theirs)
>> Lab Manager, Tissue Culture & Histology Cores, U. Pitt Dept of
>> Ophthalmology
>> 7.373 UPMC Mercy Pavilion    1622 Locust St.,Pittsburgh PA 15219
>> (412) 624-8508   this number cannot receive texts
>>
>> -Original Message-
>> From: Charles Riley via Histonet 
>> Sent: Friday, July 12, 2024 12:04 PM
>> To: histonet@lists.utsouthwestern.edu
>> Subject: [Histonet] Cryotomy help
>>
>> I have a researcher who is trying to mass spectrometry on frozen tissue
>> sections. They submitted the sample in methanol and even after sitting in a
>> -80 freezer overnight the samples are too soft to section.
>>
>> Does anyone have any tips on how to harden the tissue for sectioning that
>> won't damage the results for mass spect (I was thinking liquid nitrogen but
>> want to be sure it won't damage the results)?
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>> ___
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>>
>
>
> --
> Colleen Forster HT(ASCP)QIHC
> BLS Histology and IHC Laboratory
> Jackson Hall, Room 2-155
> 321 Church St. SE
> Minneapolis, MN 55455
> 612-626-1930
>
>
>
>
>
>
>
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[Histonet] Cryotomy help

[Charles Riley via Histonet]

I have a researcher who is trying to mass spectrometry on frozen tissue
sections. They submitted the sample in methanol and even after sitting in a
-80 freezer overnight the samples are too soft to section.

Does anyone have any tips on how to harden the tissue for sectioning that
won't damage the results for mass spect (I was thinking liquid nitrogen but
want to be sure it won't damage the results)?
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Re: [Histonet] Ihc control tissues

[Charles Riley via Histonet]

Awesome thank you Val. I like the multi control option. Can save money in
the end by using less reagents

On Wed, May 8, 2024 at 10:52 AM Val T  wrote:

> Hey Charles-
>
> Our research lab prepares a “multimouse” control. We embed 10 organs on
> the same slide and it serves as a +/- control for just about every IHC
> marker.
> However, if you don’t want to do that- here are some tissues that would
> make nice controls.
>
> > BCL-2- spleen, lymph nodes
> > Caspase-3- spleen, lymph nodes
> > CD117- skin, spleen, brain, lymph nodes
> > CD62E (e-selectin), bone marrow, colon
> > Collagen II, cartilage
> > Desmin, heart, skeletal muscle
> > GFAP, brain, spinal cord
> > HMB45, skin
> > Ki67, spleen, bowel
> > p53, tumors, colon
> > s100, colon, muscle
>
> Brain can serve as a negative control on just about all these. CD117 will
> have some expression in the cerebellum, but will be negative in most other
> areas.
> Heart can be a negative control for GFAP.
> Val
>
> > On May 8, 2024, at 6:59 AM, Charles Riley via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
> >
> > Hello all,
> >
> > I am trying to put together a positive and negative tissue control list
> for
> > the following antibodies at my research core.  I came over from a
> clinical
> > pathology background and we used tonsils for a lot of these antibodies.
> > Since rats and mice don't have tonsils I am looking for something they do
> > have that can be used if possible to keep the controls as similar to the
> > samples as possible.
> >
> > BCL-2
> > Caspase-3
> > CD117
> > CD62E (e-selectin)
> > Collagen II
> > Desmin
> > GFAP
> > HMB45
> > Ki67
> > p53
> > s100
> > ___
> > Histonet mailing list
> > Histonet@lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
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[Histonet] Ihc control tissues

[Charles Riley via Histonet]

Hello all,

I am trying to put together a positive and negative tissue control list for
the following antibodies at my research core.  I came over from a clinical
pathology background and we used tonsils for a lot of these antibodies.
Since rats and mice don't have tonsils I am looking for something they do
have that can be used if possible to keep the controls as similar to the
samples as possible.

BCL-2
Caspase-3
CD117
CD62E (e-selectin)
Collagen II
Desmin
GFAP
HMB45
Ki67
p53
s100
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[Histonet] Plastic embedding and cutting training

[Charles Riley via Histonet]

Hello all,

   I am a research histologist at the University of Delaware. My core is
looking to bring on plastic embedding and microtomy  within the next year.

I am trying to find an online training course to learn the process and or
if anyone along the East Coast near Delaware would be willing to let me
come tour your facility and shadow you sometime that would be great.

So any assistance or suggestions would be greatly appreciated.
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[Histonet] Inflammatory testing options

[Charles Riley via Histonet]

Other than H and Giemsa stains,   what can be used to show inflammation
in paraffin sections?
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[Histonet] IF staining questions

[Charles Riley via Histonet]

Why would one decide to use a primary antibody along with a secondary
antibody rather than a primary antibody conjugated with the secondary?

Example.   I have a researcher who wants to do CD11C staining with
Alexafluor488

Is it better to buy and use a primary antibody CD11C conjugated with
Alexafluor488  or to do the CD11C primary and a Rabbit anti-rat (H_L) IgG
antibody secondary?
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[Histonet] Fixing and processing mouse eyeballs

[Charles Riley via Histonet]

Does anyone have any helpful tips or protocols for fixing and processing
schedules for adult mouse eyes?
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[Histonet] Alizaren Red and von kossa staining

[Charles Riley via Histonet]

Does anyone have any methods to soften bone without decalifier solutions to
perform microtomy for these tests on E 18.5, P7, and 930 mouse skulls
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[Histonet] Von Kossa staining

[Charles Riley via Histonet]

Can anyone out there who performs Von Kossa staining provide me with any
guidelines or suggestions for the light source to use for the Silver
nitrate activation?

Is a standard handheld black light strong enough or does it need to be a UV
sanitizing strength light if using UV versus incandescent bulb exposure?
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[Histonet] Possible causes for tissue tears

[Charles Riley via Histonet]

Hello all,

I tried posting this question earlier but the file size was too big.

I processed a piece of tissue engineered cartilage the other day for a
research project and stained it with a few different stains. Each sample
has various tears through the tissue (that don't appear to be microtomy
artifacts in my opinion). I was hoping to get some feedback as to what
everyone thinks may be the most likely cause.   I have a image I can send
directly to you if you reply for added info.
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[Histonet] Blade holder assitance

[Charles Riley via Histonet]

Hello all,

I am working with a microtome that has an unusual blade holder.  Standard
low profile blades are too short and are covered by the stage so no cutting
occurs. The standard high profile blades stick out above the blade holder
edge significantly and this causes wobble in the blade while sectioning
(creating thick and thin sections and or skipping sections entirely before
cutting a really thick section).

Can anyone suggest what type of blade I should purchase to use with this
holder?  I was thinking maybe sapphire knife blades might fit but didn't
want to waste money buying items I don't need
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[Histonet] IHC staining of tendons and cartilage

[Charles Riley via Histonet]

Hello all,

I am in a new position and it will potentially require doing a lot of IHC
testing on cartilage and tendon samples. I have done some practice runs on
my automated stainer and manually and am running into issues with the
tissue sections falling off completely or folding over on itself during
each process.

If anyone does staining like this routinely and has some pointers/tricks to
try to get the samples to adhere to the slides better it would be greatly
appreciated.

I have tried using charged slides from a variety of vendors and get similar
results across the board.
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[Histonet] Alcian blue van gieson double stain

[Charles Riley via Histonet]

I am working with a researcher and they saw an article where someone
performed an Alcian Blue  and elastic van Gieson double stain.  If anyone
has done this before can you provide your protocol.  I am trying to figure
out the best way to perform this process to get the best staining results.
Here are my questions/thoughts

1. Is it better to run the Alcian blue as usual and then perform the van
gieson stain?
2. Will the differentiating solution in the van gieson stain affect the
alcian blue staining in any way?
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[Histonet] Tissue Processors

[Charles Riley via Histonet]

Has anyone used the Donatello 2 tissue processor from Diapath
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[Histonet] research questions

[Charles Riley via Histonet]

When performing IHC's for research projects is it recommended to use the
same species for control tissues or will any tissues that the antibody has
been validated for by the company be acceptable?
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[Histonet] Antibody control blocks or slides

[Charles Riley via Histonet]

Hello all,

I am working on a research project and I need to obtain control tissues for
the following antibodies for mouse/rat samples.

IL-6
Collagen I
CD68
MMP9
CTGF

If you have a vendor that you recommend for both positive and negative
tissue controls it would be greatly appreciated.
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[Histonet] GFAP IF staining protocols

[Charles Riley via Histonet]

I am trying to set up a new protocol for GFAP IF staining in my research
lab. I have never done this before and would like some suggestions for
antibodies and other necessary secondary reagents that are essential to the
stain.

If you have a protocol you have used in the past that you are also willing
to share that would be amazing.

Thanks to everyone in advance who offers any insights.
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[Histonet] Manual double staining IHC

[Charles Riley via Histonet]

Hello all,

I have never done double staining manually and wanted to know if anyone has
protocols or suggestions for doing this?
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[Histonet] Reconstituting antibodies and dilutions

[Charles Riley via Histonet]

When calculating the dilution of an antibody when do start?

i.e.  If i have 100 ug of lyophilized antibody and its recommended
reconstitution is 500 ug/ml

How do I mix a 1:1000 dilution?

I am thinking it's adding 2mL diH20 to create the 500 ug/mL and
then diluting the sample.
And I would be able to make 2000ml of 1:1000 concentration solution ?  2mL
antibody and 1998mL diluent?


Please let me know if I made a calculation error
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[Histonet] Manual IHC staining help

[Charles Riley via Histonet]

I have worked mostly with pre-built kits for automated platforms. I am in
the process of doing studies on rat tendons using IL-6, MMP3, Collagen I,
and CD68 antibodies.

I am purchasing all Rabbit polyclonal antibodies from
Invitrogen/ThermoScientific.

Can anyone provide me with protocols they use or suggestions as to what
other reagents/antibodies I need to buy to get good quality results (i.e.
secondary antibodies, blocking buffers, antigen retrieval methods, HRP or
streptavidin/biotin conjugates, and or chromagens)?
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[Histonet] Discussion help

[Charles Riley via Histonet]

Hello everyone,

I have been asked to give a presentation on the best practices for
preparing histological study samples for research on animal tendons.

Can anyone provide me with resources to reference and or any practices that
you use for specific animal types or are they pretty much similar across
all animals?
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[Histonet] Automated ihc staining of bone

[Charles Riley via Histonet]

I am working on trying to get IHC stains optimized on bone samples of rat
tibias. The issue I am running into is that the periosteum is
separating from the bone marrow and wrinkling up over top of the rest of
the sample.

If anyone has any techniques to prevent this wrinkling/detachment of tissue
from the main section during automated ihc staining it would be greatly
appreciated.
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[Histonet] Immunoflouresence staining

[Charles Riley via Histonet]

I have been given an antibody that is used for flow cytometry yet the
researcher wants to use it for IF staining.  The protocol they provided is
for the flow cytometry staining. Will this produce the immunoflourescense
results they are looking for or do i need to use a different method?
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[Histonet] staining protocol help

[Charles Riley via Histonet]

I am testing out a new antibody. Alexa-fluor 488 from biolegend.  If anyone
has any tips or protocols they could share on validating the antibody it
would be greatly appreciated.
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[Histonet] Active Caspase-3 IHC-P staining

[Charles Riley via Histonet]

I am currently running a research project and the researcher wants me to
use Caspase-3 for apoptosis staining. I have never worked with this
antibody before and want to know if anyone has any suggestions on what to
use for a positive tissue control and a negative tissue control?


I purchased the antibody from Novus Biologicals and they recommend a Jurkat
cell lysate for positive control but I am not familiar with this as well.

Any guidance would be greatly appreciated
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[Histonet] IHC staining of cartilage

[Charles Riley via Histonet]

I recently tried running an automated ihc test on a few pieces of cartilage
tissue.

All of the sections fell off my slides. I am using the TOMO charged slides
and have never had any issues.

Do cartilage sections require longer drying times in order for the tissue
to adhere better?
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[Histonet] Sakura 4070 coverslipper troubleshooting

[Charles Riley via Histonet]

Can anyone give me suggestions on how to fix an E18 error code?

The machine initiates with no issues and homes itself with no problems.
When I load a rack in to begin coverslipping the base moves to the far back
position and then I get the error code as the basin won't move to the front
position.

However once I hit the stop button to abort it moves to the front on its
own with no issues.

Any guidance or suggestions would be helpful. Trying to get it up and
running by the end of the day if possible
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[Histonet] Cell aspirate composition

[Charles Riley via Histonet]

I am looking to assist a researcher in quantifying bone marrow cell
aspirate compositions.  Does anyone perform this in their histology lab or
is this more of a cytology/hematology lab process?

If you do this, what type of equipment are you using/ what process do you
use?
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[Histonet] Paraffin embedding following storage in 70% alcohol

[Charles Riley via Histonet]

I am working with a grad student on a project dealing with equine articular
cartilage.

The protocol she sent me for embedding the tissue samples goes directly
from 70% alcohol to the embedding step in paraffin.


Correct me if I am wrong but shouldn't the tissue be dehydrated fully and
cleared before embedding the samples?
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[Histonet] Frozen sectioning bones

[Charles Riley via Histonet]

I am starting an all new research center and the researchers are looking to
do frozen sections on bones. I have never done this before and would like
some tips/suggestions.

1. What is/are the best cryostats to use for cutting bones if any?

2. What is the best process to embedding/cutting these specimens?
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[Histonet] FISH testing

[Charles Riley via Histonet]

Does anyone use an automated staining platform to perform FISH testing?  If so 
which one and are all the steps automated or do you have to perform any steps 
manually/ on another platform for any reason?
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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] Detection systems for IHC's

[Charles Riley via Histonet]

DOes anyone know what the comparison detection kit for the Xfinity Matrix from 
Biogenex would be in relation to the Bond Refine detection kits?
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[Histonet] Film coverslipping adjustments

[Charles Riley via Histonet]

We are running into an issue with our automated film coverslipper. The borders 
where the film meets the slide show small distortions (possibly air bubbles or 
incompletely melted adhesive).  Has anyone ran into this issue before and could 
you recommend adjustments or troubleshooting methods to try to clean up the 
slides appearance?
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[Histonet] Tiziano special stainer

[Charles Riley via Histonet]

Does anyone out there currently use the DiaPath Tiziano special stainer?If 
so could you please send me an email about how you think it works?   I am 
trying to see if it would be beneficial to our lab. Our biggest concern is 
creating reliability and consistency in our GMS and Retic staining. If the 
machine can do that it would be great.
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[Histonet] AFB controls

[Charles Riley via Histonet]

Does anyone have any control tissue that they can spare for AFB staining?   I 
am willing to trade for any other controls tissues that you may need in return.
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[Histonet] Toenail staining issues

[Charles Riley via Histonet]

>From time to time, we have issues with our sections of toenail falling off the 
>slide during staining.

Our H sections usually stain without issue but when we do our special stains 
(PASF and GMS) the tissue falls off routinely.

We use charged slides and if a section falls off, we coat the recut slide with 
albumin to help get extra adherence. However sometimes even this does not help 
the tissue stick through the whole process (especially on GMS).


Does anyone have any additional suggestion we could try to get the sections to 
adhere better throughout the process?
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[Histonet] Alcian Blue staining

[Charles Riley via Histonet]

We are having an issue where the goblet cells in our control tissue are not 
staining pink even though the patient tissue is staining beautifully on the 
same slide.

The stain is done manually.  What can be some causes for the issues in staining 
on the control section?
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[Histonet] Microtome Suggestions/Demos

[Charles Riley via Histonet]

My company is seeking thoughts on new microtomes to purchase.  We have demoed a 
Sakura and Leica HistoCore BioCut but would like to test out at least 1/2 more 
options before deciding on where to proceed.

Does anyone have any suggestions on microtomes that are good for both 
experienced tech and new graduates?

We are also specifically searching for microtomes that have the capability to 
reverse the advance wheel directions (i.e spinning away from the body moves the 
block holder backwards but can be changed to do the reverse back on tech 
preference)
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Re: [Histonet] End of shift

[Charles Riley via Histonet]

I have been trying to order the stuff we need for cytoprep as I see we need it 
but I have never ordered anything for them before so its difficult for me to 
find everything and I don't have all the login information for our vendors so 
this is becoming very time consuming and drawing out the process.

From: Alexis M. Hayslett 
Sent: Wednesday, August 12, 2020 8:30 AM
To: Charles Riley ; Coordinators ; 
Pathologists ; histonet@lists.utsouthwestern.edu 
; Debbie Hawkins 
Subject: Re: End of shift

Need EA-36 solution ASAP.Purchase order was approved but not sure who we 
purchase from.  The thermo fischer supplier has a much higher price according 
to their website than what was approved.  I believe the Statlab quoted price 
was also lower than what was approved through precoro.Can someone please 
make a decision and place the order tomorrow and then let me know what the plan 
should be going forward- who is in charge of Cytology inventory/ordering?

From: Charles Riley 
Sent: Tuesday, August 11, 2020 11:04 PM
To: Coordinators ; Pathologists 
; histonet@lists.utsouthwestern.edu 
; Debbie Hawkins 
Subject: End of shift

3 GBS inoculations started at 7:30 pm1 started  at 8:15 pm

42  blocks coming off at 5:51 am

FNA prep kits filtered, refilled, and replaced on stock shelf


Need EA-36 solution ASAP.Purchase order was approved but not sure who we 
purchase from.  The thermo fischer supplier has a much higher price according 
to their website than what was approved.  I believe the Statlab quoted price 
was also lower than what was approved through precoro.Can someone please 
make a decision and place the order tomorrow and then let me know what the plan 
should be going forward.


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[Histonet] End of shift

[Charles Riley via Histonet]

3 GBS inoculations started at 7:30 pm1 started  at 8:15 pm

42  blocks coming off at 5:51 am

FNA prep kits filtered, refilled, and replaced on stock shelf


Need EA-36 solution ASAP.Purchase order was approved but not sure who we 
purchase from.  The thermo fischer supplier has a much higher price according 
to their website than what was approved.  I believe the Statlab quoted price 
was also lower than what was approved through precoro.Can someone please 
make a decision and place the order tomorrow and then let me know what the plan 
should be going forward.


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Re: [Histonet] Levels on biopsies

[Charles Riley via Histonet]

We usually cut 3 lvls/slides for prostate/vaginal/bladder and anus.  6 
lvls/slides for cervical tissues. In the end it is the pathologists comfort 
level with sectioning to make the diagnosis.

From: Ringer, Bradley via Histonet 
Sent: Wednesday, June 3, 2020 10:26 AM
To: histonet@lists.utsouthwestern.edu 
Subject: [Histonet] Levels on biopsies

Hi All,
I'm curious to know how many levels your lab cuts on the following biopsies: 
prostate core, cervical, vaginal, bladder, and anus biopsies.  I ask this 
because we are currently (and have been for years) giving our pathologists 3 
levels/slides for each of these and they recently requested that we only give 
them one slide with two sections. Thank you in advance for any responses.
-Brad

Brad Ringer, HTL (ASCP)
Springfield Clinic Histology Manager
1025 S. 6th Street
Springfield, IL 62703
(217)528-7541 ext. 14078


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[Histonet] ANSI or ASTM certified scales

[Charles Riley via Histonet]

I am prepping our lab for our next CAP inspection. Does anyone know a good 
place to buy analytical scales that meet the CAP COM.30860/70/80/90 checklist 
items?

And does anyone think it is cheaper to buy a new scale each year or to have a 
certified tech come out to your location to re-certify its accuracy each year?

Thanks in advance for any guidance you could provide

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[Histonet] CAP checklist help

[Charles Riley via Histonet]

Does anyone have a policy for assessing professional competency  (checklist 
item ANP. 10010)?

I want to get ideas on how everyone performs this to verify we are meeting the 
best standards
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[Histonet] Ventana question

[Charles Riley via Histonet]

Does anyone know if Ventana offers a FFPE PCR testing platform
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[Histonet] FFPE PRC testing

[Charles Riley via Histonet]

What platform does everyone use to carry out FFPE PCR testing?

Seeking to bring in as many genetic mutation testing options as possible
(BRAF, KRAS, NRAS...etc.)



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[Histonet] CAP proficiency testing

[Charles Riley via Histonet]

Does anyone use another vendor besides CAP to perform their PT
requirements?

If so, who do you use and how do you record it for inspection purposes?

I am looking for a PD-L1 PT specifically but will take any other options as
well as we are trying to help our budget.

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[Histonet] Competency questionnaires

[Charles Riley via Histonet]

Does anyone use a questionnaire for testing a techs competency either by
itself or on top of practical application of the skills needed?   If so
would you mind sharing with me your questions (I am looking for grossing,
microtomy and troubleshooting, embedding, processing, and
immunohistochemistry/special/H staining and troubleshooting.

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[Histonet] BRAF testing

[Charles Riley via Histonet]

Can anyone send me a block that can be used to validate BRAF V600E
 antibody.  I have tried several cases that should work but our
pathologists don't think it is working correctly. I just need a small core
sample that is known to be positive. Any help would greatly be appreciated

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[Histonet] Pap slides

[Charles Riley via Histonet]

Can pap slides be left in 95% alcohol overnight to be stained the next
morning without affecting the quality of the slides? Or should they be
moved to xylene then run back prior to staining?

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Re: [Histonet] Tissue Tek embedding centers

[Charles Riley via Histonet]

Thank you for your help!

On Thu, Mar 12, 2020 at 1:19 PM Greg Dobbin  wrote:

> Hi Charles,
> If memory serves, there is a thermal fuse or switch that will shut the
> unit down if it exceeds a certain temp. Once the unit cools, the switch is
> reset. So either that switch needs to be replaced or there is a problem
> elsewhere that is causing the unit to overheat. Replacing that switch
> should be an easy and cost-effecient thing to try first.
> Good luck,
> Greg
>
> --
> *Greg Dobbin*
> 1205 Pleasant Grove Rd
> RR#2 York,
> PE  C0A 1P0
>
>
> *Everything in moderation...even moderation itself**!*
>


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[Histonet] Tissue Tek embedding centers

[Charles Riley via Histonet]

Has anyone experienced an issue with their embedding center where is had
continuous power outages (machine starts to cycle on then power kicks out,
then immediately restarts)?

I am trying to determine what the issue is to see if it can be fixed easily
or if I need to look at a service call or if better to just buy new (my
machines are around 11 years old)

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[Histonet] Poll question

[Charles Riley via Histonet]

How often does everyone clean their water-bath?

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[Histonet] IHC troubleshooting

[Charles Riley via Histonet]

Our pathologists are complaining that chunks of the dermis are missing from
IHC slides yet the entire section is present prior to staining.

Does anyone have any ideas what could cause the tissue to not adhere to the
slides throughout the staining process? We use the Leica Bond stainers.

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[Histonet] Warthin starry

[Charles Riley via Histonet]

Is there any way to lighten and over stainer WS stain?

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[Histonet] Grossing manual examples

[Charles Riley via Histonet]

Is anyone willing to share an example of their grossing manual setup?  I am
trying to get ours updated as I feel it doesn't adequately provide
instruction on how to gross specimens.I would like some examples to
show our pathologists what would be helpful so they can modify the current
one we have

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Re: [Histonet] Skin Sample with Tattoo - need to remove for diagnosis

[Charles Riley via Histonet]

The only way I know of is through lasers

On Fri, Jan 17, 2020 at 12:25 PM John Garratt via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> It is a pigment (ie carbon) and not like a soluble ink, so good luck with
> that.
> You could consult your histotech text book on exogenous pigment removal
> and give the recipe a go. I will certainly be interested in other histonet
> replies.
>
> John
>
>
>
> www.cpqa.ca
>
> ‐‐‐ Original Message ‐‐‐
> On Friday, January 17, 2020 8:16 AM, Donna Emge via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
> > Hello fellow histonetters,
> >
> > We received a skin sample that has ink from a tattoo - the sample if from
> > tattooed skin. One of our pathologists would like us to see if we can get
> > rid of the tattoo ink from the sections before H staining. Does anyone
> > out there know how to do this?
> >
> > Thank you,
> > Donna Emge
> > Anatomic Pathology Manager
> > Mercy Hospital and Medical Center, Chicago
> >
> >
> -
> >
> > Histonet mailing list
> > Histonet@lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
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[Histonet] Cell block preparations

[Charles Riley via Histonet]

What is the best way to remove excess blood from FNA sample collections
before spinning them down into cell blocks?

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[Histonet] H protocols

[Charles Riley via Histonet]

Does everyone use the same protocol for routine H's as well as things
like Cell blocks and or lymph nodes?

If you use a different protocol how does it differ from your routine stain?

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Re: [Histonet] guidelines for using a microwave designated for food, but used to heat up histogel

[Charles Riley via Histonet]

There is no direct CAP regulation regarding the use of microwaves
concerning food and chemical use.  However, ANP.29430 says that microwaves
used in the histology lab need to be under a fume hood to capture any
vented chemical fumes.   Also, I am not sure what the actual record is but
I am sure there is an OSHA regulation that is part of the Chemical exposure
regulations that states microwaves used in laboratories can not be used for
both food and chemical processing.

On Thu, Dec 12, 2019 at 8:37 AM Eileen Akemi Allison via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Went on line to get MSDS regarding HistoGel.  Here’s the link.
> https://www.medline.com/media/catalog/Docs/MSDS/MSD_SDSD91968.pdf <
> https://www.medline.com/media/catalog/Docs/MSDS/MSD_SDSD91968.pdf>
>
> Happy eating from a microwave which has been used at MD Anderson for
> HistGel!
>
> Best regards,
> Akemi
>
> > On Dec 12, 2019, at 5:18 AM, Kathleen S Cormier  wrote:
> >
> > Can I share that we had a pathologist  (back in the day) use our lab
> microwave to re-heat his coffee? It had Schiff’s reagent stains all over
> the inside of it. Some people really don’t have any common sense…
> >
> >
> > Kathy Cormier
> > Core Leader
> > Hope Babette Tang Histology Facility
> > Koch Institute for Integrative Cancer Research at MIT
> > 500 Main Street, 76-182
> > Cambridge, MA 02139
> > 617-258-8183
> > corm...@mit.edu 
> > https://ki.mit.edu/sbc/histology 
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >> On Dec 12, 2019, at 7:12 AM, Eileen Akemi Allison via Histonet <
> histonet@lists.utsouthwestern.edu  histonet@lists.utsouthwestern.edu>> wrote:
> >>
> >> Good morning histopeeps:
> >>
> >> We recently brought on an Ophthalmology Pathologist from MD Anderson
> and they use histogel for orienting their thin eye specimens and need a
> microwave to heat it up.  We do not have a microwave in the lab for their
> use but I had ordered one for them.  I just found out they used the
> microwave in our lab lounge to heat up histonet which was opened and being
> stored in our gross area refrigerator.
> >>
> >> Do any of you know the CAP regulation against use of laboratory
> reagents being used in a microwave being used for food consumption?  I
> ended up removing the microwave they used and put it in the histology lab.
> Our Director wants me to write a formal letter to submit to him.
> >>
> >> Thank you in advance for your impute!
> >>
> >> Akemi Allison, BS,  HT/HTL (ASCP)
> >>
> >>
> >> ___
> >> Histonet mailing list
> >> Histonet@lists.utsouthwestern.edu  Histonet@lists.utsouthwestern.edu>
> >> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >
>
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[Histonet] Tissue based PCR FISH and SISH platforms

[Charles Riley via Histonet]

Is anyone aware of a platform that can perform all three of the testing
methods in my subject line besides the Biogenex platforms?

If not, can anyone recommend a good combination of machines that can
perform all three tests ?

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[Histonet] AED machines

[Charles Riley via Histonet]

Doesn anyone know if there are any US regulations (OSHA, CLIA, CAP, DE
state) that require pathology practices to have an AED machine on site?
Especially if we are seeing patients and performing procedures on them in
the lab.




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[Histonet] Waterbath cleaning between specimens

[Charles Riley via Histonet]

Random question,

Does anybody use anything other the KimWipes (or similar light-duty tissue
wipe) to clean the surface of their waterbath between cases?
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[Histonet] Trouble shooting issues

[Charles Riley via Histonet]

Can anyone let me know what possible causes for the poor staining quality
might be?

I believe it to be a processing issue as all other adjustments I have made
so far have not improved the quality,   however pathologists feel it is a
microtomy issue. Trying to get outside answers to improve the quality of
specimen if possible

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[Histonet] PLAG1

[Charles Riley via Histonet]

My pathologists want to bring this test in house. Can anyone suggest a
company I can purchase this antibody for testing via IHC on the Leica Bond
platform?

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[Histonet] BRAF KRAS NRAS by PCR

[Charles Riley via Histonet]

Does anyone do these tests using PCR on FFPE blocks?

If so what platform do you use/recommend?

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Re: [Histonet] Cell block processing

[Charles Riley via Histonet]

Our tech said they use 95% alcohol to collect the specimen.

On Fri, Oct 25, 2019 at 12:23 PM Joe W. Walker, Jr. 
wrote:

> Hi Charles,
>
> What are you collecting the FNA into?  Cytorich? Cytolyt? Other?
>
> Joe W. Walker, Jr. MS, SCT(ASCP)
> Anatomical Pathology Manager
> joewal...@rrmc.org, www.rrmc.org
>
> -Original Message-----
> From: Charles Riley via Histonet 
> Sent: Friday, October 25, 2019 8:13 AM
> To: Histo List 
> Subject: [Histonet] Cell block processing
>
> [External Email] This email originated from outside of the organization.
> Think before you click: Don’t click on links, open attachments or respond
> to requests for sensitive information if the email looks suspicious or you
> don’t recognize the sender.
>
>
> Does anyone have any tips or suggestions on how to better process extremely
> bloody FNA  specimens?Is there anyway to clear out some or all of the
> blood without destroying the other tissues?
>
> --
>
> Charles Riley BS  HT, HTL(ASCP)CM
>
> Histopathology Coordinator/ Mohs
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> https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.com%2Fv3%2F__http%3A%2F%2Flists.utsouthwestern.edu%2Fmailman%2Flistinfo%2Fhistonet__%3B!5JlSGkcjda6Eqs5J!rntZr6E7tVvCc2tzoJy_L8lFMF6EvHUTKJOsc3NTMLFSI35SgrcseW2ly3GyamY%24data=02%7C01%7Cjwwalker%40rrmc.org%7C6e4cdd79edfa4e1ef23408d75944c458%7C0e55647d438e4a448437e959c3cf2240%7C0%7C0%7C637076024216531651sdata=xAxqyyCfRepB3DlAl%2Fw651nk3B5ViHQLjqdToa2iAhw%3Dreserved=0
> [
> https://www.rrmc.org/app/files/public/2633/2019_hyht_sig-_jan2019_final.jpg
> ]
>


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[Histonet] Instrument feed water

[Charles Riley via Histonet]

Where does everyone purchase their IFW from?  or how do you produce it in
house?

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[Histonet] Cell block processing

[Charles Riley via Histonet]

Does anyone have any tips or suggestions on how to better process extremely
bloody FNA  specimens?Is there anyway to clear out some or all of the
blood without destroying the other tissues?

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Re: [Histonet] Freezing spray in cryostats

[Charles Riley via Histonet]

I have to agree with Terri. I also believe that CAP is going to be putting
in regulations forbidding the use of freeze sprays in cryostats soon (at
least I think I heard a rumor about this).

Anyway another useful item is liquid nitrogen if you really need a quick
freeze. Dip a brush into the liquid and lightly apply to the specimen. Not
always recommended as could cause artifacts.

On Wed, Sep 25, 2019 at 1:37 PM Terri Braud via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> My question is why would you ever want to use an aerosol in the cryostat,
> taking a chance on aerosolizing some nasty bug?
> Is the 30 seconds you save in using freezing spray worth the exposure to
> TB or goodness-knows-what?
> We use a simple metal heat extractor (comes standard in our Leicas) and
> place it on top of the freezing block to accelerate the process.  We never
> have issues with turn around times.
> For me, no freezing spray.  Ever.
>
> 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
>
> -Original Message-
> From: histonet-requ...@lists.utsouthwestern.edu [mailto:
> histonet-requ...@lists.utsouthwestern.edu]
> Sent: Wednesday, September 25, 2019 1:00 PM
> To: histonet@lists.utsouthwestern.edu
> Subject: Histonet Digest, Vol 190, Issue 18
>
> CAUTION: This is an EXTERNAL EMAIL. Stop and think before clicking links
> or opening attachments.
>
> Send Histonet mailing list submissions to
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>
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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. Flammable Sprays in Cryostats (Knutson, Deanne)
>2. Re: Flammable Sprays in Cryostats (Akemi)
>3. Re: Flammable Sprays in Cryostats (Hannen, Valerie)
>4. Re: Flammable Sprays in Cryostats (Ingles Claire)
>
>
> --
>
> Message: 1
> Date: Wed, 25 Sep 2019 07:50:44 -0500
> From: "Knutson, Deanne" 
> To: "'histonet@lists.utsouthwestern.edu'"
> 
> Subject: [Histonet] Flammable Sprays in Cryostats
> Message-ID:
> <
> 1e0e2b14c709174b8ac2be0ae7f768330159b5756...@exchange2k7.staprimecare.org>
>
> Content-Type: text/plain; charset="us-ascii"
>
> I just received a letter from Leica Biosystems where they are prohibiting
> the usage of flammable
> freezing sprays in their cryostats.  What are others using in their
> cryostats to instantly freeze specimens?
>
> Thank you.
>
>
> Deanne Knutson
> Supervisor
> Anatomic Pathology
> dknut...@primecare.org
>
>
>
>
>
>
>   
> This email may include confidential and privileged information. If this is
> not intended for your use, please destroy immediately and contact the
> sender of the message.
>
> This email and attachments contain information that may be confidential or
> privileged. If you are not the intended recipient, notify the sender at
> once and delete this message completely from your information system.
> Further use, disclosure, or copying of information contained in this email
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> of privilege or other confidentiality protections.
>
>
> --
>
> Message: 2
> Date: Wed, 25 Sep 2019 09:05:20 -0500
> From: Akemi 
> To: "Knutson, Deanne" , Histonet
> 
> Subject: Re: [Histonet] Flammable Sprays in Cryostats
> Message-ID: <19db25eb-9944-406b-917c-0878d6f15...@gmail.com>
> Content-Type: text/plain;   charset=us-ascii
>
> We are purchasing two of their newer UV cryostats and they were adamant
> that we use a non flammable freezing spray because it can damage the UV
> unit and have potential for exploding.
>
> Akemi Allison
>
> Sent from my iPhone
>
> > On Sep 25, 2019, at 7:50 AM, Knutson, Deanne via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
> >
> > I just received a letter from Leica Biosystems where they are
> prohibiting the usage of flammable
> > freezing sprays in their cryostats.  What are others using in their
> cryostats to instantly freeze specimens?
> >
> > Thank you.
> >
> >
> > Deanne Knutson
> > Supervisor
> > Anatomic Pathology
> > dknut...@primecare.org
> >
> >
> >
> >
> >
> >
> >  
> > This email may include confidential and privileged information. If this
> is not intended for your use, please destroy immediately and contact the
> sender of the 

[Histonet] PRN pay rates

[Charles Riley via Histonet]

What does everyone think is a good PRN rate for an experienced (4+ years)
Histotechnician/technologist? Obviously location would play a roll but
what do everyone think is a good average rate?

And for anyone with knowledge what would a good PRN rate be for a Pathology
assistant?

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[Histonet] Lab temperature

[Charles Riley via Histonet]

Hello everyone,

Where does everyone set their lab temperature ideally?  I know in some
places its difficult to keep the temp stable but what would everyone
recommend as the best setting?

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[Histonet] Salivary Amylase by IHC

[Charles Riley via Histonet]

Does anyone run salivary amylase testing via IHC.

I use the bond platforms and am looking for an antibody that is not
Research only based

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[Histonet] Sectioning over processed GI biopsy

[Charles Riley via Histonet]

What is the best way to get a section from an over-processed GI biopsy?
Paraffin keeps pulling away from the tissue and nothing sections. Tissue
very hard even after lenghty time on ice water bath

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Re: [Histonet] BOND question

[Charles Riley via Histonet]

I have not seen any lately.   A few quick things to check that may help

1. Do you see any bubbles in the syringe on the side of the machine when it
is running?
2. Have you had any liquid level sensing error messages?


On Thu, May 30, 2019 at 11:28 AM Nancy Schmitt via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hello-
> Checking in to see if anyone else has seen any staining irregularities
> with a new BOND Max (OR III) or DS9800 detection kits.
> I appreciate your thoughts,
>
> Nancy Schmitt MLT, HT(ASCP)
> Pathology Support Services Manager
>
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[Histonet] Retic staining

[Charles Riley via Histonet]

I am having trouble getting my retic stain to come out strong. It is
working but Pathologists are saying they shouldn't have to go to high power
just to see it (and I agree).

I am using a brand new American MasterTech Chandler's Precision kit. I have
followed all the steps correctly (one difficulty is getting the Ammonium
hydroxide to go drop by drop so this may be the issue).

I have tried staining longer in ammoniacal silver. more time in 20%
formalin,  more and less time in gold chloride and sodium thiosulfate.

If anyone has any suggestions or uses this kit with time modifications
please let me know. Willing to try anything to get it working.


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[Histonet] Lab Week

[Charles Riley via Histonet]

Anyone have any ideas on things I can do in our lab to celebrate lab week?

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[Histonet] Cell Block trouble shooting

[Charles Riley via Histonet]

Is there any way to save cell blocks that have been rehydrated to much?
Tech left them on their ice bath too long and they have become really soft.

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Re: [Histonet] Fridge temp

[Charles Riley via Histonet]

Some facilities run their immunostainers on a continuous basis all week
long and antibodies may never leave the machine for multiple days. The
antibody is still valid and still works. Like a previous  responder said
you can test each one and compare to previously stained slides to validate
it is still working.I don't remember where I heard it but someone
published that for each hour an antibody is not keep within it's storage
temperature range you lose 1 day of effective usage. (so if the antibodies
were out of temp for 24 hours you lose 24 days from the printed expiration
date)

On Wed, Mar 27, 2019 at 1:24 PM MONICA D. LOCKHART via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> I agree !! The integrity of the antibody is suspect after being exposed to
> temps like that.  Even if the fridge door was kept closed during the
> outtage.
>
> Monica D. Lockhart
>
>
> -Original Message-
> From: WILLIAM DESALVO [mailto:wdesalvo@outlook.com]
> Sent: Tuesday, March 26, 2019 7:20 PM
> To: MARY ANN; histonet@lists.utsouthwestern.edu
> Subject: Re: [Histonet] Fridge temp
>
> Simple, revalidate or pitch
>
> William DeSalvo
> 
> From: MARY ANN via Histonet 
> Sent: Tuesday, March 26, 2019 4:09:01 PM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Fridge temp
>
> Let's say, hypotheticaly, if you discover your fridge with all you
> antibodies and detection kits were discovered to have been at 19c. for an
> undisclosed amout of time. 12 24 48 hours due tona power surge..south
> Florida weather.
>
> Let's also propose your lab CFO/Owner dosent think its a big deal.
>
> First how would you handle the issue given the frisge has been restored ?
>
>
>
>
> Sent from Xfinity Connect Application
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[Histonet] PAP stains

[Charles Riley via Histonet]

What causes dark nuclei in the PAP stain.

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Re: [Histonet] P16

[Charles Riley via Histonet]

We by predilute from BioGenix

On Wed, Mar 13, 2019 at 12:27 PM Bernice Frederick via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Who is the best and current supplier for p16? We were using biocare,but
> evidently it is no longer available from them. And no Santa Cruz per my IHC
> tech.
> Thanks,
> Bernice
>
> Bernice Frederick
> Pathology Core Facility
> Robert H. Lurie Cancer Center
> 710 North Fairbanks Court
> Olson 8-421
> 312-503-3723
> b-freder...@northwestern.edu
>
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[Histonet] Job opening

[Charles Riley via Histonet]

Hello all,


We are offering a full time histotechnician position in Dover Delaware.
Morning shift (start time semi negotiable). Chance to be cross trained in
other departments as well as advancement to leadership roles. Seeking
someone with 5+ years experience (less acceptable but not preferred) and HT
or HTL certified or certification eligible.

If interested please reply here and I can give you more information about
the position and give you instructions on how to apply if you are interested

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[Histonet] H Staining question

[Charles Riley via Histonet]

Has anyone tried using Hologic's Thin Prep solutions (Nuclear stain, rinse,
and bluing) to do their H stains?

I am trying to validate a new stainer and in the process the pathologists
want to tweak our protocol to get a better stain and thought maybe we could
try this to see about saving money in the process

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[Histonet] Breast lumpectomy fixation

[Charles Riley via Histonet]

 How does everyone gross and process your breast lumpectomy / mastectomy
specimens?



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[Histonet] Stainer Quotes

[Charles Riley via Histonet]

Are there any vendors out there selling any of the following refurbished
item?

1. Leica ST5010  or ST5020 stainer
2. Sakura DRS 2000
3. DiaPath Giotto
4. Shur/Stain 3030


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[Histonet] Cost per test averages

[Charles Riley via Histonet]

What is a good cost per test average for a standard H slide?

I am trying to see if I should be fighting for better prices or if  my
current costs are below the average and am getting a good deal already.
(Obviously the lower i can go the better

Also what is your average cost per test for IHC's ?
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[Histonet] Cost calculations

[Charles Riley via Histonet]

How does everyone calculate their cost to create things in your lab?

I was tasked to figure out how much it costs us to make the following
1. A paraffin block
2. A single H slide
3. Average cost of an IHC slide
4. Average cost of a Special stain slide

What I need to figure out is beside the cost on the inventory to create
these items what other factors do I need/should use to calculate final
costs (i.e. tech time, energy costs, machine maintenance)?

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[Histonet] Competency signoff CAP

[Charles Riley via Histonet]

I just recently got put in charge of managing the personnel records for
training and am in the process of trying to update our personnel records
for our next CAP inspection. I feel our previous files were over detailed
and excessive in what they covered and wanted to try and consolidate some
of the information under broader categories.

Is anyone willing to send me what they use in their labs for Grossing and
Histology Competency and proficiency testing sign off for their staff so I
can get an idea of how much information I need to provide.

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[Histonet] Cellblock H

[Charles Riley via Histonet]

My pathologists are complaining that our cell blocks appear light on
hematoxylin.

Does anyone use a different protocol for their routine H vs. Cellblocks?
 If so what is the difference in protocols?

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[Histonet] braf (v6001e)

[Charles Riley via Histonet]

Does anyone use this antibody on the Leica Bond systems?


I have used several different vendors to validate this antibody but the
Docs say all of them are over staining or not working correctly (I've gone
down to 1:5000 on the dilution on all of them).

If anyone has any suggestions on protocols or vendors that have worked for
you (other than Ventana) please let me know. Thanks

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[Histonet] Histology slide stainer questions

[Charles Riley via Histonet]

 Does anyone out there use the General Data Healthcare™ SHUR/Stain™ 3030
H Slide Stainer?If so can this machine be used to run PAP stains for
gyn and non-gyn specimens?  I am looking to replace my Gemini stainer and
routine H stainer with a single unit if possible to free up some lab
space.  If anyone has any other suggestions it would be greatly appreciated





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[Histonet] Expired SurePath vials

[Charles Riley via Histonet]

Does anyone know how to dispose of expired SurePath vials in Delaware?

Or is anyone able to use expired SurePath vials for research testing or any
other purpose?


Thanks in advance for any replies!!

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[Histonet] Saukara Prisma Plus

[Charles Riley via Histonet]

Does anyone out there use this for their routine H stains as well as GYN
and Non GYN staining?


I am trying to figure out staining protocols. If anyone would be willing to
share their procedures as well as who you purchase your supplies from it
would be greatly appreciated.

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Re: [Histonet] BRAF

[Charles Riley via Histonet]

I am interested in the same question. If responding please remember to use
the reply all option so everyone can hear the answers.

On Tue, Jul 24, 2018 at 9:13 AM, Alminawi, Samira via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hi,
> Anybody using BRAF antibody for melanoma, what is the recommended clone
> and protocol?
> samira
>
>
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[Histonet] Hazy/cooked cells in H

[Charles Riley via Histonet]

Our pathologists have said that we have had a few cases the past few days
that are unreadable. They say the cells appear "cooked".

Nothing in our process has changed, we have been using the same lot of
reagents since the middle of June, and no errors were reported in our
processing runs.

Do anyone have any ideas to what could be causing this issue?


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[Histonet] Trouble shooting question

[Charles Riley via Histonet]

My pathologists says that 1 out of 100 (his estimate) of our FNA's
cellblocks  apears to be "cooked" when looking at the slides.  The process
has never been changed and all reagents are the same all the time.

 What is/are the possible cause(s) and what can be done to prevent this 1
out of 100 situation?




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[Histonet] RET PTC Antibody

[Charles Riley via Histonet]

Does anyone use RET antibody to test for Papillary thyroid carcinoma?   If
so who do you purchase the antibody from.


We are looking to add it to our panel along with hbme, gal-3, and BRAF.
The one antibody I purchased is not highlighting the target cells the way
our pathologists hoped and they want me to look into another vendor



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[Histonet] Microscope objectives

[Charles Riley via Histonet]

Are all microscope objectives universal between brands or do you have to
buy the objectives from the company that manufactured it?

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[Histonet] Anti-RET

[Charles Riley via Histonet]

Does anyone out there run the Anti-RET IHC from ABCAM (ab134100) on the
Leica bond platform. I am about to work it up on out system and want a good
starting point rather than testing every option and wasting time and money.

1. Do you use any enzyme treatment
2. Do you use ER1 or ER2 and for what times?
3. What do you find to be a good dilution ratio?

Any assistance will be greatly appreciated

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