[Histonet] RE: Decalcifying bone

Wed Jul 25 12:44:52 CDT 2012

  Ms. Clark
We are currently use 10% formic Acid in a microwave oven to decal ours for about 2 and 1/2 hour after they were fix in 10% neutral buffer Formalin. We were told that they are Ok for immuno-stains.

Thomas Huynh, HT( ASCP)
Clinical Histology Technician
Pathology/ Histology/ Bone Lab
thuynh <@t> mdanderson.org
T 713-745-4759
F 713-792-2046

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Today's Topics:

   1. Decalcifying bone (Jeanne Clark)
   2. Re: RE: Secondary antibody causing nuclear staining (Eva Permaul)
   3. Thank you for coverslipper responses (Brendal Finlay)
   4. CAP Sample Exchange Registry (jgoldsmi <@t> bidmc.harvard.edu)
   5. Re: Markers for Rat Samples (Hobbs, Carl)
   6. Thanks for all the good wishes (Louise Renton)

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

Message: 1
Date: Wed, 25 Jul 2012 07:14:06 -0700 (PDT)
From: Jeanne Clark <paintedsplashes <@t> yahoo.com>
Subject: [Histonet] Decalcifying bone
To: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<1343225646.39594.YahooMailNeo <@t> web161703.mail.bf1.yahoo.com>
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In looking through old procedures, I have found several different 'solutions' that have been used for fixation and decalcification of bone (particularly bone marrow cores). ?I would very much appreciate hearing what people are using today for optimal fixation and decalcification of bone for routine pathology and IHC testing.

Thank you,

Jeanne Clark
Histology/IHC
Stanford Hospital and Clinics

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

Message: 2
Date: Wed, 25 Jul 2012 10:59:58 -0400
From: Eva Permaul <eca9 <@t> georgetown.edu>
Subject: Re: [Histonet] RE: Secondary antibody causing nuclear
	staining
To: "Reynolds, Donna M" <dreynold <@t> mdanderson.org>,	histonet
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<CAEBNytUc2fe1o4Z2xv5AT_JFM46+c3Ya-g=xTgMneumntKHWNQ <@t> mail.gmail.com>
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I do see positive nuclei in the NC. That is what I am asking about. I know
I could switch methods but my question is also why if it is happening is it
not as strong all the time? Why are the cells very light one day and dark
the next? What is causing them to stain? Just curious is all.
Eva

On Wed, Jul 25, 2012 at 10:04 AM, Reynolds,Donna M
<dreynold <@t> mdanderson.org>wrote:

>
> If you are running a negative control (no primary)with your ABC staining
> wouldn't you see the same nuclear labeling in the NC ? Thus alerting you to
> false staining and indicating that you should try a HRP conjugated
> secondary or use a polymer system.
> Good discussion thank Tony.
> Donna Reynolds HT(ASCP) Chief Histologist IHC Lab
>
> -----Original Message-----
> > I understand the point about the biotin and I should have said that
> > when using the ABC method we have taken to always using an
> > avidin/biotin blocking kit. We are using biotinylated secondary
> > antibodies from Vector. I have seen the same problem occur in our
> > anti-mouse, anti-rabbit and anti-goat. In my last run I had stomach
> > fundus as well as skin melanoma, both had pos.nuclei in the negative
> > (no primary). In another run I had colon ca and breast ca, the breast
> > ca had fewer pos. nuclei than the colon ca but they were still there.
> > Some days the positive nuclei are stronger in a sample that was just
> > weakly positive before. Just want to understand what it is and what
> effects it.
> > Thank you all for your ideas.
> > Eva Permaul
> > Georgetown University
> >
> > On Mon, Jul 23, 2012 at 7:16 PM, Tony Henwood (SCHN) <
> > tony.henwood <@t> health.nsw.gov.au> wrote:
> >
> >> I should have added that this was from the workshop notes on a
> >> Hypotheticals Workshop I ran last year at our Australian National
> Meeting.
> >>
> >> Regards
> >> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
> >> 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-bounces <@t> lists.utsouthwestern.edu [mailto:
> >> histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tony Henwood
> >> (SCHN)
> >> Sent: Tuesday, 24 July 2012 9:00 AM
> >> To: 'Eva Permaul'; histonet <@t> lists.utsouthwestern.edu
> >> Subject: RE: [Histonet] Secondary antibody causing nuclear staining?
> >>
> >> It is possible that this is due to "Biotin nuclei" where excess
> >> biotin is found in the nuclei of some cells, see below:
> >>
> >> Optically clear nuclei have been reported in endometrial epithelium
> >> associated with first and second trimester abortions (Sickel & di
> >> Sant'Agnese 1994). Optically clear nuclei have also been found in
> >> different types of tissues of diverse organs such as ovary, thyroid
> >> and lung (Nakatani et al 1994, Mount & Cooper 2001). The optically
> >> clear nuclei contain excess biotin.
> >>
> >> Endogenous biotin immunoreactivity is generally not visualized in
> >> formalin fixed, paraffin-embedded tissues unless a heat-induced
> >> antigen retrieval step has been introduced (Mount & Cooper 2001).
> >>
> >> In this placental section, optically clear nuclei (containing biotin)
> >> bind to the streptavidin of the ABC technique giving a reaction
> >> similar to that seen with CMV containing cells. If a polymer method
> >> (or even the original Sternberger's PAP method) is used then this
> >> anomalous staining will disappear, thus allowing confident
> demonstration of CMV infected nuclei.
> >>
> >> The false-positive staining pattern caused by endogenous biotin can
> >> be cytoplasmic or nuclear. A report of positive immunoreactivity of
> >> hepatocellular carcinomas for inhibin was later determined to be a
> >> false-positive finding due to cytoplasmic endogenous biotin. Steroid
> >> cell tumours of the ovary were found to demonstrate endogenous biotin
> >> cytoplasmic staining in 36% of cases. Immunoreactivity for
> >> anti-Herpes virus immunohistochemical staining in a series of
> >> endometria was also later determined to be a false-positive result
> >> due to biotin. The prominent intranuclear inclusions, resembling
> >> herpes virus cytopathic effect, were caused by intranuclear biotin
> >> and not viral particles. Similar false positive staining for CMV in
> >> products of conception has also been reported (Mount & Cooper 2001).
> >>
> >> False-positive staining can be cytoplasmic or nuclear. When
> >> cytoplasmic, the appearance of the false signal is that of a dull
> >> brown granular or fluffy staining pattern. If this quality of
> >> staining is observed with several different antibodies, endogenous
> >> staining by biotin should be considered. When nuclear, a
> >> false-positive reaction may be associated with optically clear nuclei
> >> identified on H&E stained sections. False-positive staining due to
> >> endogenous biotin, however, does not occur in a cell membrane pattern
> (Mount & Cooper 2001).
> >>
> >> Mount SL & Cooper K (2001) "Beware of biotin: a source of
> >> false-positive immunohistochemistry" Current Diagnostic Pathology
>  7:161-167.
> >> Nakatani et al (1994) Am J Surg Pathol 18(6):637-642.
> >> Sickel & di Sant'Agnese (1994) Arch Pathol Lab Med 118:831-833
> >>
> >>
> >> Regards
> >> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
> >> 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
> >>
>
> _______________________________________________
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> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>

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

Message: 3
Date: Wed, 25 Jul 2012 10:12:34 -0500
From: "Brendal Finlay" <brendal.finlay <@t> medicalcenterclinic.com>
Subject: [Histonet] Thank you for coverslipper responses
To: histonet <@t> lists.utsouthwestern.edu
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <929594e776a7d135c058e3f19d1aa73f <@t> medicalcenterclinic.com>
Content-Type: text/plain; charset="utf-8"

Thank you everyone for your input on the different types of
coverslippers.?? I'll be sharing the information with the others that
I work with so we can make a decision.?? I'll be happy to share my
thoughts after using whichever we choose for a while. 

Brendal C. Finlay, HT (ASCP)
West Florida Medical Center Clinic
brendal.finlay <@t> medicalcenterclinic.com
phone - 850.474.8758
fax - 850.474.8584

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Message: 4
Date: Wed, 25 Jul 2012 11:33:00 -0400
From: <jgoldsmi <@t> bidmc.harvard.edu>
Subject: [Histonet] CAP Sample Exchange Registry
To: <histonet <@t> lists.utsouthwestern.edu>
Cc: pvasalo <@t> cap.org
Message-ID:
	<D26DC9CC50EA7F4F8F915888A464288108963BCDE8 <@t> EVS5CCR.its.caregroup.org>
Content-Type: text/plain; charset="us-ascii"

All,

I am the current chair of the CAP's Immunohistochemistry Committee.  One of the things we've been grappling with is how to help IHC laboratories evaluate difficult-to-validate antibodies (e.g. HSV, spirochetes, etc.).  Some years ago, our molecular pathology colleagues at the CAP instituted a 'Sample Exchange Registry' that is designed to facilitate interlaboratory validation.   The College has now expanded this service to all laboratory disciplines, including IHC laboratories.   This service is free of charge.
Additional information can be found below, and on the referenced website.
Please direct any questions to me or Ms. P. Vasalos (email:  pvasalo <@t> cap.org).

Jeff

The College of American Pathologists (CAP) has announced the expansion of its current Sample Exchange Registry for Alternative Assessment. This service now includes all clinical laboratory disciplines.

The Sample Exchange Registry is an Internet-based service designed to connect laboratories performing testing where no formal proficiency testing (PT) is available. Laboratories can participate in the registry service at any time. When at least three laboratories are identified as testing for the same analyte, the CAP will facilitate the sample exchange.
Website:
http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=laboratory_resources%2Fexc.html&_state=maximized&_pageLabel=cntvwr

Jeffrey Goldsmith, MD
Director, Surgical Pathology Laboratory and Gastrointestinal Pathology Fellowship
Department of Pathology and Laboratory Medicine
Beth Israel Deaconess Medical Center
Assistant Professor of Pathology, Harvard Medical School
330 Brookline Avenue
Boston, MA  02215

Consultant in Gastrointestinal Pathology
Children's Hospital Boston
300 Longwood Avenue
Boston, MA  02215

BIDMC Office:  617 667 2586
BIDMC Fax:  617 975 5620

CHB Pathology Department (for CHB adminstrative matters only):  617 355 7431

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

Message: 5
Date: Wed, 25 Jul 2012 17:32:22 +0100
From: "Hobbs, Carl" <carl.hobbs <@t> kcl.ac.uk>
Subject: [Histonet] Re: Markers for Rat Samples
To: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<11D9615B89C10747B1C985966A63D7CA386298F0B6 <@t> KCL-MAIL04.kclad.ds.kcl.ac.uk>

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You could have a look here, in the image gallery- top left link under Immunhistochemistry . Then scroll down to individual protein/ab hyperlinks.

Maybe you will find some suitable Abs.

NB: Sure you can use rat primaries on rat tissue, it just depends on whether you are looking for NK cells within B-cell/plasma cell population.
Include a no -primary, just to see..
Sure, ...happy to be proved

Good luck.

Best wishes.

Carl

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

Message: 6
Date: Wed, 25 Jul 2012 18:53:45 +0200
From: Louise Renton <louise.renton <@t> gmail.com>
Subject: [Histonet] Thanks for all the good wishes
To: Histonet <Histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<CABL-0cCSvUSaJR=NKwDcnXf2yJFDZbVrEbrwPw_4C3bx1Oi47g <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

Also....I'll try to get the "UNSUSCIBE" "UNSCRIBE" "UNSUSCRIBE"  thingy
right when the time comes!

-- 
Louise Renton
Bone Research Unit
University of the Witwatersrand
Johannesburg
South Africa
+27 11 717 2298 (tel & fax)
073 5574456 (emergencies only)
A good head and a good heart are always a formidable combination.
Nelson Mandela<http://www.brainyquote.com/quotes/quotes/n/nelsonmand101682.html>

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