[Histonet] RE: Histonet Digest, Vol 78, Issue 1

Sun May 2 19:46:32 CDT 2010

I never throw a book away! cbarone <@t> nemours.org 

-----Original Message-----
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Sent: Saturday, May 01, 2010 1:01 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 78, Issue 1

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Today's Topics:

   1. RE: CD68 (Dana Settembre)
   2. Re: CD68 (Fabrice gankam)
   3. Repeat validation when IHC protocol changed?
      (Kevin_Kurtz <@t> ssmhc.com)
   4. CD68??` (Jeffrey Silverman)
   5. disposable blade holder (Liebig, Tyler K.)
   6. How to troubleshoot a cytology slide with dehydration
      problems (Valerie R.)
   7. Re: How to troubleshoot a cytology slide with	dehydration
      problems (Valerie R.)
   8. RE: Re: How to troubleshoot a cytology slide with	dehydration
      problems (IRENA SREBOTNIK KIRBIS)
   9. Does anyone need books? (alineumann <@t> aol.com)

----------------------------------------------------------------------

Message: 1
Date: Fri, 30 Apr 2010 14:15:42 -0400
From: "Dana Settembre" <settembr <@t> umdnj.edu>
Subject: RE: [Histonet] CD68
To: "Histonet" <histonet <@t> pathology.swmed.edu>,	"Drew Sally A"
	<SDrew <@t> uwhealth.org>
Message-ID: <sbdae621.035 <@t> smtpnpc.umdnj.edu>
Content-Type: text/plain; charset=US-ASCII

We are currently using KP-1 and HAM 56

Dana Settembre, HT ASCP
Immunohistochemistry Lab
UMDNJ - University Hospital
Newark, NJ    USA

>>> Drew Sally A <SDrew <@t> uwhealth.org> 04/30/10 12:11 PM >>>
We also use KP1 

Sally

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Cynthia Pyse
Sent: Friday, April 30, 2010 10:49 AM
To: 'Histonet'
Subject: [Histonet] CD68

Happy Friday Everyone

What clone is everyone using for the CD68 antibody for FFPE human tissue?
Thanks for the info in advance. Everyone have a great weekend.

Cindy Pyse, CLT, HT (ASCP)

Histology Supervisor

X-Cell Laboratories

e-mail cpyse <@t> x-celllab.com 

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------------------------------

Message: 2
Date: Fri, 30 Apr 2010 13:32:58 -0500
From: Fabrice gankam <gankam <@t> googlemail.com>
Subject: Re: [Histonet] CD68
To: Cynthia Pyse <cpyse <@t> x-celllab.com>,
	histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<q2g86c37d601004301132i4230308fi790031e00f08ab46 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

I use the ED1 from abdserotec and it works perfectly
can dilute up to 1/500 to 1/1000

2010/4/30 Cynthia Pyse <cpyse <@t> x-celllab.com>

> Happy Friday Everyone
>
> What clone is everyone using for the CD68 antibody for FFPE human tissue?
> Thanks for the info in advance. Everyone have a great weekend.
>
>
>
> Cindy Pyse, CLT, HT (ASCP)
>
> Histology Supervisor
>
> X-Cell Laboratories
>
> e-mail cpyse <@t> x-celllab.com
>
>
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>

------------------------------

Message: 3
Date: Fri, 30 Apr 2010 13:46:16 -0500
From: Kevin_Kurtz <@t> ssmhc.com
Subject: [Histonet] Repeat validation when IHC protocol changed?
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<OF694A8036.F0735D59-ON86257715.00664CDE-86257715.00671D8A <@t> SSMHC>
Content-Type: text/plain;	charset="US-ASCII"

Recently our IHC vendor sent a tech to help us out with some weak staining 
affecting multiple antibodies.  Several staining protocols were changed, 
including adjusting the incubation time and adding amplification.  The 
results were great in the slides that I was shown.

The tech said that revalidation was unnecessary, since the changes to the 
protocol were considered "minor".  However, I personally disagree, since 
we don't know how the change in the staining protocol could potentially 
affect staining of other tissues (especially tissues that would be 
expected to be negative). Before subjecting our lab to the cost and effort 
of revalidation, I'd like to get your opinion.

For new antibodies, I usually validate using 10 cases expected to be 
positive and 10 cases expected to be negative for the antibody in 
question. For revalidation, I was thinking of running 5 positive cases and 
5 negative cases.

Thanks for your input - 

Kevin Kurtz, M.D.
St. Mary's Hospital
Madison, Wisconsin

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------------------------------

Message: 4
Date: Fri, 30 Apr 2010 11:47:17 -0700 (PDT)
From: Jeffrey Silverman <pathmaster <@t> yahoo.com>
Subject: [Histonet] CD68??`
To: cpyse <@t> x-celllab.com
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <821799.98032.qm <@t> web111111.mail.gq1.yahoo.com>
Content-Type: text/plain; charset=us-ascii

Another vote for KP-1. It also stains mast cells. 

Jeff Silverman

------------------------------

Message: 5
Date: Fri, 30 Apr 2010 12:40:45 -0700
From: "Liebig, Tyler K." <tyler.liebig <@t> thermofisher.com>
Subject: [Histonet] disposable blade holder
To: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<6746115DFA761840A605BB27624424AAEF0F10D5 <@t> USPHO-MXVS01.amer.thermo.com>

Content-Type: text/plain; charset="us-ascii"

Hello Lucy,

In regards to your trouble finding an HM310 blade holder we do have one in our refurbished pool that I could have sent to you.
It is used but in working order so we can let you have it at no charge.  If you let me know where to ship it I will have it sent.

Hope that helps, It's hard to cut without a blade holder.

Regards,

Tyler Liebig
Product Manager
Histology Instrumentation and IHC
Anatomical Pathology
Thermo Fisher Scientific
46360 Fremont Blvd
Fremont, CA 94538
USA
Office: (510) 979-5000
Mobile: (510) 299-1751
Fax: (510) 979-5239
tyler.liebig <@t> thermofisher.com<mailto:tyler.liebig <@t> thermofisher.com>
www.thermo.com<http://www.thermo.com>

------------------------------

Message: 6
Date: Fri, 30 Apr 2010 17:34:39 -0700
From: "Valerie R." <vrodriguez10 <@t> gmail.com>
Subject: [Histonet] How to troubleshoot a cytology slide with
	dehydration	problems
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<h2mb9208e7b1004301734v980366abn287b0b0132dbc706 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

I am an HTL but I have to stain pap smears. In the Papanicalou procedure for
fine needle aspirations I have to stain the slides in sodium chloride first
for 25 seconds in total (15 seconds in one container and 10 in another
container). This for me is the trickiest part when staining cytology because
if slides are not well dehydrated in saline then the cells are not going to
absorb the stains well, and the result will be a pinkish slide, when it is
suppose to look blue-greenish.

I had this problem today where I stained an entire rack of slides, and all
the slides turned blue (which means they stained correctly) except 4 slides
which turned out pinkish (which is a sign that they did not dehydrated
properly in sodium chloride (h20 saline).

Is there a solution to this issue. Can these slides be re-stained?

I know this newsgroup is for histology only but I would like to know if
histotech and cytotechs have encountered this issue in their labs and how
they ended up solving the problem.

Thanks in advance

------------------------------

Message: 7
Date: Fri, 30 Apr 2010 17:48:58 -0700
From: "Valerie R." <vrodriguez10 <@t> gmail.com>
Subject: [Histonet] Re: How to troubleshoot a cytology slide with
	dehydration problems
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<m2sb9208e7b1004301748g2cf5d358p510b87eb857f2415 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

I forgot to include the complete protocol that my lab uses so you can help
me better:

1.After dehydrating in sodium chloride for 25 seconds you have to dip the
slides in alcohol 95.
2. 10 dips in water then 1.30 minutes in hematoxylin, it used to be 1 min,
then rinse in water.
3. 1 min in rinse solution
4.10 dips in water.
5. 1 min in bluing solution
6. 10 dips in water.
7. 10 dips in alcohol.
8. 10 dips in alcohol.
9. 1 min in orange solution
10. 15 dips in 95 alcohol.
11. 15 dips in 95 alcohol.
12.  3.15 mins in EA solution
13. The slides are then dipped in 3 containers of 95 alcohol (20 dips each)
14. Slides are dipped in 3 containers of 99 alcohol (15 dips in each)
15. Then they are dipped in Isoxylene which is a combination of xylene and
99 alcohol (15 dips)
16. And lastly Xylene 10 dips

When the slides do not absorb the h20 saline seems that the slides absorb
more EA than hematoxylin.  I don't know why.

On Fri, Apr 30, 2010 at 5:34 PM, Valerie R. <vrodriguez10 <@t> gmail.com> wrote:

> I am an HTL but I have to stain pap smears. In the Papanicalou procedure
> for fine needle aspirations I have to stain the slides in sodium chloride
> first for 25 seconds in total (15 seconds in one container and 10 in another
> container). This for me is the trickiest part when staining cytology because
> if slides are not well dehydrated in saline then the cells are not going to
> absorb the stains well, and the result will be a pinkish slide, when it is
> suppose to look blue-greenish.
>
> I had this problem today where I stained an entire rack of slides, and all
> the slides turned blue (which means they stained correctly) except 4 slides
> which turned out pinkish (which is a sign that they did not dehydrated
> properly in sodium chloride (h20 saline).
>
> Is there a solution to this issue. Can these slides be re-stained?
>
> I know this newsgroup is for histology only but I would like to know if
> histotech and cytotechs have encountered this issue in their labs and how
> they ended up solving the problem.
>
> Thanks in advance
>
>
>

------------------------------

Message: 8
Date: Sat, 1 May 2010 11:16:27 +0200
From: IRENA SREBOTNIK KIRBIS <irena.kirbis <@t> hotmail.com>
Subject: RE: [Histonet] Re: How to troubleshoot a cytology slide with
	dehydration problems
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <COL121-W30DF9C2B98E327D8EF69F098F00 <@t> phx.gbl>
Content-Type: text/plain; charset="iso-8859-2"

Dear Valerie,
I can only agree that Papanicolaou stain is very tricky, having 20 yrs experiencies with it! looks very simple but you need to know some basic rules. 
first of all the slides should be hydrated at the beginning of staining procedure since the hematoxylin is water based stain!
it seems to me thatyour slides are not enough hydrated, I never heard about sodium chloride solution as a hydrating but I guess cannot harm, however then you put slides in alcohol it's a bit confusing! and 25 seconds is a very short time for any incubation!
beginning of a staining procedure depends on a fixation of the slides! in a case of a wet fixed slides you can proceed directly to staining (95% alcohol) if the slides are wet fixed and then dried you need to rehydrate them before staining and if you have spray fixed slides you need to remove the wax from slides before staining!

my suggestions:
1. 
- leave slides in a sodium chloride longer, perhaps 2-3 minutes
- alcohol 95%
- alcohol 70%
- water at least 3 min better 5 minutes, you'll probably need to shorten staining time in hematoxylin to 1 minute because the slides will be better hydrated and ready to accept stain.
- staining time in EA solution is too short to allow proper staining and this is probably the reason to get reddish staining, you should leave the slides in EA 5 minutes since only then the light green will start to stain.
hope this help! do you stain by hand or in a stainer? how often do you change stains and other solutions? you should also be very carefull to have the proper level of all solutions in a dishes! the basic rules is to keep the level of stains bellow the level of rinsing solutions.
let me know about your staining results or even better to send me a picture! sometimes you can get the all redish staining also becouse of improper fixation! you can prepare your proprely fixed slide and stain it to see the results!
best regards!
Irena 

> From: vrodriguez10 <@t> gmail.com
> Date: Fri, 30 Apr 2010 17:48:58 -0700
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Re: How to troubleshoot a cytology slide with dehydration problems
> 
> I forgot to include the complete protocol that my lab uses so you can help
> me better:
> 
> 1.After dehydrating in sodium chloride for 25 seconds you have to dip the
> slides in alcohol 95.
> 2. 10 dips in water then 1.30 minutes in hematoxylin, it used to be 1 min,
> then rinse in water.
> 3. 1 min in rinse solution
> 4.10 dips in water.
> 5. 1 min in bluing solution
> 6. 10 dips in water.
> 7. 10 dips in alcohol.
> 8. 10 dips in alcohol.
> 9. 1 min in orange solution
> 10. 15 dips in 95 alcohol.
> 11. 15 dips in 95 alcohol.
> 12. 3.15 mins in EA solution
> 13. The slides are then dipped in 3 containers of 95 alcohol (20 dips each)
> 14. Slides are dipped in 3 containers of 99 alcohol (15 dips in each)
> 15. Then they are dipped in Isoxylene which is a combination of xylene and
> 99 alcohol (15 dips)
> 16. And lastly Xylene 10 dips
> 
> 
> When the slides do not absorb the h20 saline seems that the slides absorb
> more EA than hematoxylin. I don't know why.
> 
> On Fri, Apr 30, 2010 at 5:34 PM, Valerie R. <vrodriguez10 <@t> gmail.com> wrote:
> 
> > I am an HTL but I have to stain pap smears. In the Papanicalou procedure
> > for fine needle aspirations I have to stain the slides in sodium chloride
> > first for 25 seconds in total (15 seconds in one container and 10 in another
> > container). This for me is the trickiest part when staining cytology because
> > if slides are not well dehydrated in saline then the cells are not going to
> > absorb the stains well, and the result will be a pinkish slide, when it is
> > suppose to look blue-greenish.
> >
> > I had this problem today where I stained an entire rack of slides, and all
> > the slides turned blue (which means they stained correctly) except 4 slides
> > which turned out pinkish (which is a sign that they did not dehydrated
> > properly in sodium chloride (h20 saline).
> >
> > Is there a solution to this issue. Can these slides be re-stained?
> >
> > I know this newsgroup is for histology only but I would like to know if
> > histotech and cytotechs have encountered this issue in their labs and how
> > they ended up solving the problem.
> >
> > Thanks in advance
> >
> >
> >
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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------------------------------

Message: 9
Date: Sat, 01 May 2010 12:41:03 -0400
From: alineumann <@t> aol.com
Subject: [Histonet] Does anyone need books?
To: histonet <@t> pathology.swmed.edu, members <@t> lists.cytopathology.org,
	soamail <@t> comcast.net
Message-ID: <8CCB76438FB2942-1C08-14475 <@t> webmail-m084.sysops.aol.com>
Content-Type: text/plain; charset="us-ascii"

Hello all, I have some older microbiology, cytology, electron microscopy and immunohistochemical books, as well as a 1985 Internal Medicine text, would anyone like them for an underserved area?  Thanks, 

Alice Neumann MD
Western Wyoming Pathology
2001 Corner Creek Lane #67 
Jackson, WY 83001 

Cell: 307-413-4092 
Home: 307-734-4410 

------------------------------

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End of Histonet Digest, Vol 78, Issue 1
***************************************