[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?

-- 

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] Pap stains

[John Kiernan via Histonet]

As a student in the 1960s I was told by my elders and betters to filter all 
staining solutions before using. It was very good advice. Filtration does not 
prolong the life of a stain, but it does remove crud, a material that can be 
composed of polymerized dyes and bits of previously stained cells or tissues.

Fifty years on, I can identify a few staining solutions that never deteriorate 
into insoluble materials that fall out of solution even after 10+ years. Even 
for these, filtration before use is good laboratory practice. Not filtering any 
stain may therefore be bad laboratory practice.

Happy New Year,  John Kiernan
= = =

From: Haas, Elizabeth via Histonet 
Sent: 31 December 2019 11:46
To: S hay 
Cc: histonet@lists.utsouthwestern.edu 
Subject: Re: [Histonet] Pap stains


I believe filtering stains daily is a CAP requirement


Sent from my iPhone

> On Dec 31, 2019, at 9:23 AM, S hay via Histonet 
>  wrote:
>
> 1. Does everyone filter their pap stains daily?
> 2. Are you chaining all other reagents daily?
>
> Thanks in advance.
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Re: [Histonet] Pap stains

[Haas, Elizabeth via Histonet]

I believe filtering stains daily is a CAP requirement


Sent from my iPhone

> On Dec 31, 2019, at 9:23 AM, S hay via Histonet 
>  wrote:
>
> 1. Does everyone filter their pap stains daily?
> 2. Are you chaining all other reagents daily?
>
> Thanks in advance.
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[Histonet] Pap stains

[S hay via Histonet]

1. Does everyone filter their pap stains daily?
2. Are you chaining all other reagents daily?

Thanks in advance.
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Re: [Histonet] PAP stains

[John Kiernan via Histonet]

Charles,  What do you mean by "dark nuclei"? Are you asking about the normal 
colour for the method, or about something you have not seen before that looks 
wrong?  Also, what is "PAP stain"?


If "PAP stain" means Papanicoloau, the nuclear stain is Mayer's haemalum. This 
is a progressive stain; you may need to shorten the time.


If "PAP stain" means peroxidase-antiperoxidase (unlabelled antibody-enzyme 
complex for amplifying detection of HRP-tagged secondary antibodies in 
immunohistochemistry), nuclei will be dark (usually brown) if they contain the 
antigen sought by the primary antibody. Nuclear staining might also be a 
false-positive artifact; if so, it would be present in the simplest of the 
routine controls (omission of primary antibody).  Dark nuclei might also be due 
to a nuclear counterstain that is too strong. The counterstain should be done 
in a contrasting colour. Usually it is a progressive haemalum (blue) to 
contrast with the oxidation product of DAB (brown).


Is this a Papanicoloau question or an immunohistochemistry question?  Does "PAP 
stain" have a third meaning?


John Kiernan

= = =


From: Charles Riley via Histonet 
Sent: 19 March 2019 14:17
To: Histo List
Subject: [Histonet] PAP stains

What causes dark nuclei in the PAP stain.

--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] PAP stains

[Joe W. Walker, Jr. via Histonet]

There are many reasons why the nuclei may look dark in the Pap stain.
1. What type of hematoxylin are you using?
2. What type of Pap stain are you using, regressive or progressive?

This could be due to a variety of things like: type of preparation utilized, 
too long in hematoxylin, not enough rinsing of excess hematoxylin, not enough 
time in HCL (if used), too long in bluing, etc., personal preference of 
interpreting cytotech or pathologist, just to name a few.

Joe W. Walker, Jr. MS, SCT(ASCP)
Anatomical Pathology Manager
joewal...@rrmc.org, www.rrmc.org

-Original Message-
From: Charles Riley via Histonet 
Sent: Tuesday, March 19, 2019 3:18 PM
To: Histo List 
Subject: [Histonet] PAP stains

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What causes dark nuclei in the PAP stain.

--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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[Histonet] PAP stains

[Charles Riley via Histonet]

What causes dark nuclei in the PAP stain.

-- 

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] pap control

[Terri Braud via Histonet]

Are you referring to a Pap stain control?  If so, we run a self made buccal 
smear every day to check for stain quality.

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: Monday, July 16, 2018 1:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 176, Issue 9

Today's Topics:
   1. Cap PAP Smears Controls (Mary Ann)
Message: 1
Date: Mon, 16 Jul 2018 12:51:53 -0400
From: Mary Ann 
Subject: [Histonet] Cap PAP Smears Controls
Hello,
In preparing for CAP I have a question:
Does anyone run a positive PAP control with their run?


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Re: [Histonet] Pap stain without xylene

[E. Wayne Johnson via Histonet]

Xylene is becoming more and more of a nuisance material and a problem 
for us in use and in disposal.  We are still able to use it but with 
increasing concern.


We have been able to eliminate xylene from our staining procedures 
altogether in our small laboratory.


We use a hair dryer to dry the slides in the rack after water or alcohol.

Since we read our slides right away, we have been using cedarwood oil to 
clear and mount the slides.  It's cheap, and makes lovely slides with no 
crystals or "floaties" or other artifacts.  It's easy to clean up 
without any xylene or toluene. It's not permanent but we can remount the 
slides with a permanent mounting medium if we need to keep the slides 
for some reason.


*

We also have found several different methods for eliminating xylene from 
the paraffin infiltration process,


and we have not used xylene for dewaxing for more than 2 years now.


On 05/26/2016 03:07 AM, Mike Toole wrote:

Thank you Beth,

It’s good to know that someone else had tried this and had very good results. 
The method you suggest is very much in line with the recommendations from René. 
He did recommend using a drying oven at 60°C to help ensure complete removal of 
any water or alcohol. And, that absolute dryness was a requirement for 
coverslipping without artifact such as the appearance of sand like grains or 
cornflakes.

Just to reiterate, was the field method performed at ambient temperature 
without the aid of a drying oven? And, just a thought, I suppose if the lack of 
electricity was an issue in a field setting, that perhaps a solar oven made 
with plywood and glass could be used to elevate temperature for drying.

Do you  know if altering the method for final clearing would require validation?

Mike


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[Histonet] Pap stain without xylene

[Mike Toole via Histonet]

Thank you Beth,

It’s good to know that someone else had tried this and had very good results. 
The method you suggest is very much in line with the recommendations from René. 
He did recommend using a drying oven at 60°C to help ensure complete removal of 
any water or alcohol. And, that absolute dryness was a requirement for 
coverslipping without artifact such as the appearance of sand like grains or 
cornflakes.

Just to reiterate, was the field method performed at ambient temperature 
without the aid of a drying oven? And, just a thought, I suppose if the lack of 
electricity was an issue in a field setting, that perhaps a solar oven made 
with plywood and glass could be used to elevate temperature for drying.

Do you  know if altering the method for final clearing would require validation?

Mike

From: Beth Cox [mailto:bethc...@gmail.com]
Sent: Wednesday, May 25, 2016 12:56 PM
To: Mike Toole; Histonet
Subject: [EXTERNAL] Re: Pap stain without xylene


Mike,



As an alternative for your discussion:



I work on medical mission trips where I run Paps in a field setting.  For the 
past 4 years, we have not had access to xylene, and we have done the following:



1. Remove slides from the last alcohol after staining

2. Allow to air dry completely (generally only takes a few minutes)

3. Use standard mounting media to coverslip them dry.



This works beautifully.  Microscopically they look the same as xylene cleared 
slides.  The only caveat is that they must dry completely.



--

BETH COX, HTL/SCT(ASCP)QIHC

AP Consultant | Pathology Solutions, Inc | (810) 240-2190 | 
bethc...@gmail.com<mailto:bethc...@gmail.com>





--



Message: 6

Date: Tue, 24 May 2016 17:08:51 -0500

From: Mike Toole <mto...@dcol.net><mailto:mto...@dcol.net>

To: 
"histonet@lists.utsouthwestern.edu"<mailto:histonet@lists.utsouthwestern.edu>


<histonet@lists.utsouthwestern.edu><mailto:histonet@lists.utsouthwestern.edu>

Subject: [Histonet] Pap stain without xylene

Message-ID: <31530E35E0BAB044B3B56B7FE5CF4EB33E1B2A3240@mail>

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



Ren? and other Histonetters,





After reading the paper:







Buesa RJ, Peshkov, MV. Histology without xylene. Ann Diagn Pathol. 2009 
Aug;13(4):246-56. Epub 2009 Feb 5.







It appears that xylene in the final clearing steps is replaced with isopropanol 
and mineral oil as follows:



?5:1 isopropanol to mineral oil 50?C



?2:1 isopropanol to mineral oil 50?C



?Undiluted mineral oil 50?C



?Drying oven 5 minutes   60?C



?Coverslip







Do you feel these clearing steps be applied to the pap stain in order to 
eliminate xylene?  If so, can it be done at room temperature?



Thanks,

Mike



Mike Toole, BS, CT(ASCP)CM

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Re: [Histonet] Pap stain without xylene

[Rene J Buesa via Histonet]

Hi Mike:The steps you desrcibe are wrong.After you finish staining your PAP 
smear, just wash them in the last ethanol → oven dry at 60ºC for 5 minutes or 
as required if the smear is too thick and when completely dried → 
coverslip.This final drying has to take place at temperatures above room temp. 
because you have to be absolutely sure the smear (or stained tissue section)  
is absolutely dried before applying the mounting medium → coverslip.The steps 
you write about 2-propanol and mineral oil are those the tissues have to go 
through before paraffin infiltration. René 

On Tuesday, May 24, 2016 6:36 PM, Mike Toole via Histonet 
 wrote:
 

 René and other Histonetters,


After reading the paper:



Buesa RJ, Peshkov, MV. Histology without xylene. Ann Diagn Pathol. 2009 
Aug;13(4):246-56. Epub 2009 Feb 5.



It appears that xylene in the final clearing steps is replaced with isopropanol 
and mineral oil as follows:

·        5:1        isopropanol to mineral oil            50°C

·        2:1        isopropanol to mineral oil            50°C

·        Undiluted mineral oil                                    50°C

·        Drying oven 5 minutes                                  60°C

·        Coverslip



Do you feel these clearing steps be applied to the pap stain in order to 
eliminate xylene?  If so, can it be done at room temperature?

Thanks,
Mike

Mike Toole, BS, CT(ASCP)CM

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[Histonet] Pap stain without xylene

[Mike Toole via Histonet]

René and other Histonetters,


After reading the paper:



Buesa RJ, Peshkov, MV. Histology without xylene. Ann Diagn Pathol. 2009 
Aug;13(4):246-56. Epub 2009 Feb 5.



It appears that xylene in the final clearing steps is replaced with isopropanol 
and mineral oil as follows:

·5:1 isopropanol to mineral oil 50°C

·2:1 isopropanol to mineral oil 50°C

·Undiluted mineral oil 50°C

·Drying oven 5 minutes   60°C

·Coverslip



Do you feel these clearing steps be applied to the pap stain in order to 
eliminate xylene?  If so, can it be done at room temperature?

Thanks,
Mike

Mike Toole, BS, CT(ASCP)CM

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Re: [Histonet] Pap stain troubleshooting

[Bob Richmond via Histonet]

Thanks for these excellent comments on troubleshooting the EA and OG-6
components I've copied them for my files. The important thing is to have
frequent feedback from the cytotechnologist to whoever is doing the
staining.

In one job I had the staining was done by a clerical person who took great
pride in the fact that she'd never looked down a microscope in her life.
(She never took a day's vacation - always an eosinophlic clupeid, as any
accountant will tell you - got caught with her hand in the till - I hope
her jump suit's dyed with orange G.) The cytotechnologists were
moonlighters in a distant place - we saw them once a year at the Christmas
party. I got burned doing the 10% QC - we had no way to feed back if we
found an error - we all missed a cervical adenocarcinoma - only time I've
been sued in >50 years in practice.

In another crazy practice the cytotechnologist only worked at night, and
was forbidden - for reasons I never understood - to ever be in the hospital
in the daytime - she'd never even met the histotechnologist who did her
staining, and who never looked at a slide. Fortunately we didn't get sued,
but we certainly could have.

Bob Richmond
Samurai Pathologist
Maryville TN
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Re: [Histonet] PAP stain troubleshooting

[Terri Braud via Histonet]

Hi Charles - Not to worry.  Many of us Histo folks don't have a Cytologist to 
help.  Beth is spot on in her advice.  I just wanted to add  Orange G should 
appear yellow to orange; 15 sec to 1 minute is the usual range of staining 
times. EA is often problematic because of fundamental limitations in its 
chemical composition. Ideally, one should see clearcut hues of green and red in 
separate cells. Staining times less than about 3 minutes usually favor the 
uptake of eosin, with eosin and light green often occupying different areas of 
the same cells. Most EA formulations perform optimally in the 6-8 minute range. 
Note particularly that the OG and EA staining times are interdependent: 
relatively too much time in OG will overload cells with orange G and block the 
subsequent uptake of eosin.  Make sure you record your lots of stain as they 
are changed out, and try using a self-made buccal smear to check new lots of of 
the stain components before they are put into use.  Then, if you see a te
 st problem, you can repeat the buccal smear and compare to the original.  It 
may help you to pinpoint the problem.
Best of luck - Terri
Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874

   9. PAP staining quality (Beth Cox)
Message: 9
Date: Thu, 4 Feb 2016 18:06:53 -0500
From: Beth Cox <bethc...@gmail.com>
Subject: [Histonet] PAP staining quality
Hi Charles,
A couple things to check on:
1.  The first concern I would have is your EA stain.  Poor EA staining 
will give too much orange staining and pale other counterstains (making 
them look aged).What brand are you using? Have you changed brands? 
Is your EA close to the expiration date? Is the bulk stored with light 
exposure?  I think fixing your EA will fix all the other problems.
2.  The other question I have regards your alcohol.  Have you changed 
types/brands?  Pap staining is very delicate and the different alcohols 
used can make a big difference.
Beth Cox, HTL/SCT(ASCP)QIHC
--
Message: 3 Date: Thu, 4 Feb 2016 09:50:18 -0500
From: Charles Riley <cri...@dpspa.com>
Subject: [Histonet] PAP stain quality
Not sure if anyone out the would know the answer to this. We are having 
an issue with our PAP stained slides appearing too orange and look aged. 
If you have any idea for causes I appreciate any help

-- Charles Riley HT(ASCP)CM
Histopathology Coordinator/ Mohs
  Doctors Pathology Services, Dover DE
--



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[Histonet] PAP staining quality

[Beth Cox via Histonet]

Hi Charles,

A couple things to check on:

1.  The first concern I would have is your EA stain.  Poor EA staining 
will give too much orange staining and pale other counterstains (making 
them look aged).What brand are you using? Have you changed brands? 
Is your EA close to the expiration date? Is the bulk stored with light 
exposure?  I think fixing your EA will fix all the other problems.


2.  The other question I have regards your alcohol.  Have you changed 
types/brands?  Pap staining is very delicate and the different alcohols 
used can make a big difference.


Beth Cox, HTL/SCT(ASCP)QIHC

--
Message: 3 Date: Thu, 4 Feb 2016 09:50:18 -0500
From: Charles Riley <cri...@dpspa.com>
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] PAP stain quality
Message-ID: 

[Histonet] PAP stain quality

[Charles Riley via Histonet]

Not sure if anyone out the would know the answer to this. We are having an
issue with our PAP stained slides appearing too orange and look aged. If
you have any idea for causes I appreciate any help

-- 

Charles Riley HT(ASCP)CM

Histopathology Coordinator/ Mohs

Doctors Pathology Services, Dover DE
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Re: [Histonet] PAP stain quality

[Jamal Rowaihi via Histonet]

You need to review the slides fixation and what chemicals you are using


Regards
Jamal RowaihiAnatomic Pathology SupervisorAl Borg Medical Laboratories Sent 
from my cell phone Original message From: Charles Riley via 
Histonet <histonet@lists.utsouthwestern.edu> Date: 2/4/2016  5:50 PM  
(GMT+03:00) To: histonet@lists.utsouthwestern.edu Subject: [Histonet] PAP stain 
quality 
Not sure if anyone out the would know the answer to this. We are having an
issue with our PAP stained slides appearing too orange and look aged. If
you have any idea for causes I appreciate any help

-- 

Charles Riley HT(ASCP)CM

Histopathology Coordinator/ Mohs

Doctors Pathology Services, Dover DE
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Re: [Histonet] Pap stain quality

[Bob Richmond via Histonet]

Charles Riley HT(ASCP)CM, Histopathology Coordinator/ Mohs, Doctors
Pathology Services, Dover DE asks:

>>Not sure if anyone out there would know the answer to this. We are having
an issue with our PAP stained slides appearing too orange and looking aged.
If you have any idea for causes I appreciate any help.<<

The Pap stain (short for Papanicolaou, not an acronym, not capitalized)
requires frequent adjustments - it's somewhat finicky at best. A
cytotechnologist should know how to trouble-shoot the Pap stain - if you're
using the stain, I suppose you have a cytotechnologist available.

Bob Richmond
Samurai Pathologist
Maryville TN
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[Histonet] PAP stains done by hand

[Nancy Schmitt via Histonet]

Happy Friday-

Could you please share how you are handling the potential for cross 
contamination in non-gyn pap specimens?  Are you filtering/changing out 
solutions between each case?

I appreciate your input-

Nancy
Histology Coordinator
Dubuque, IA  52001
Check us out at www.uclaccess.com





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[Histonet] PAP Pen With Aqueous Mounting Media

[Langsdorf, Aliete]

Hello All,
I would like to use a PAP pen to separate sections on my slide, but have been 
using an aqueous (non-xylenes based) mounting media. How do you remove the PAP 
pen without using xylenes? Is this done?
Thank you,
~Ally



Research Technologist
Comander Lab - Ocular Genomics Institute
Massachusetts Eye  Ear Infirmary
Lab: (617)-573-6485

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Re: [Histonet] PAP Pen With Aqueous Mounting Media

[Emily Sours]

We just leave the pap pen on.  Since it's not covering the tissue, it won't
matter.

Emily

By bitching and bitching and bitching, they could exhaust the drama of
their own horror stories. Grow bored. Only then could they accept a new
story for their lives. Move forward.

-Chuck Palahniuk, Haunted


On Thu, Apr 11, 2013 at 3:29 PM, Langsdorf, Aliete 
aliete_langsd...@meei.harvard.edu wrote:

 Hello All,
 I would like to use a PAP pen to separate sections on my slide, but have
 been using an aqueous (non-xylenes based) mounting media. How do you remove
 the PAP pen without using xylenes? Is this done?
 Thank you,
 ~Ally



 Research Technologist
 Comander Lab - Ocular Genomics Institute
 Massachusetts Eye  Ear Infirmary
 Lab: (617)-573-6485

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[Histonet] pap supply processing

[Gale Limron]

Happy Holidays!
Can anyone tell me what their current per-test pricing is for ThinPrep pap 
supplies? I'm putting together a study to present numbers to possibly bring our 
Gyn Cytology back in-house.
Thank you,
Gale

Gale Limron CT,HT (ASCP)
Histology Supervisor
Union Hospital
659 Boulevard
Dover, Ohio 44622
330-343-3311 ext 2562



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RE: [Histonet] PAP

[Tony Henwood]

This is our method:

1.  Tap water   dip gently 5-10 times
2.  Harris haematoxylin 5 minutes
3.  Wash in running water
4.  Differentiate in acid/alcohol   one dip
5.  Wash in running water
6.  Scott's Blueing Solutionone minute
7.  Wash in running water.
8.  95% Alcohol dip gently 5-10 times
9.  Modified  PAP Stain (see below) three minutes
10. Rinse excess dye off in 95% Alcohol  
11. Absolute Alcoholdip gently 5-10 times
12. Absolute Alcoholdip gently 5-10 times
13. Xylol   dip gently 5-10 times
14. Xylol   dip gently 5-10 times
15. Xylol   dip gently 5-10 times
16. Mount in D.P.X. Mounting Medium.

This modification uses a single solution, omitting the OG6 solution and
replacing the light green with the more fade resistant Fast Green FCF.

Modified PAP stain

Stock solutions: 
Prepare 10% solutions of each of the stains as follows:
2.5g Eosin Y (CI 45380) in 25ml distilled water 
1g Fast Green FCF (CI 42053) in 10ml distilled water 
  
  Working Solution:
Mix (for 400ml stain)
12ml Eosin Y stock 
2.5ml Fast Green FCF Stock

Make mixture up to 400ml with 95% alcohol.
Add:
0.8g Phosphotungstic acid
4 drops saturated lithium carbonate.

Mix well. 
Store solution in dark brown, tightly capped bottle.


Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager  Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Aazath
Raj
Sent: Thursday, 12 August 2010 10:40 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] PAP



Dear friends,

   does anyone have rapid PAP(cytostain) preparation,for
rapid PAP staining.

 

Aazathraj.P

Technical Officer

Department of Histopathology and Cytology

Apollo Hospitals chennai

India
 
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[Histonet] PAP

[Aazath Raj]

Dear friends,

   does anyone have rapid PAP(cytostain) preparation,for rapid PAP 
staining.

 

Aazathraj.P

Technical Officer

Department of Histopathology and Cytology

Apollo Hospitals chennai

India
  
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[Histonet] Pap sign out

[Martin, Gary]

We have always had our Pathologist sign out all Pap results, and are now
considering having out Cytotech sign out Pap's. Is there an agency we
have to report o before we start this process. Also, how are others
choosing the 10% for review by the Pathologist? 

Thanks 

Gary

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[Histonet] pap pen with vecter antifade

[Perry, Samuel]

Hi All,

I am wondering how people do remove pap pen when they need to cover slip with an
aqueous mounting media.  Thanks Sam  


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