[Histonet] decalcification

[Luz Linares via Histonet]

Does anybody have a good protocol for decalcified teeth?
 I will really appreciated

Luz
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[Histonet] Decalcification for Bone Marrow Biopsies

[Cartun, Richard via Histonet]

Help!  For years we have used Decal-Stat for decalcifying our bone marrow 
biopsies with good results.  For the past month we have been having problems 
with tissue loss and morphological damage with these specimens following 
decalcification.  Unfortunately, this was just brought to my attention.  
Someone told me this morning that the company producing this product was sold 
and the formulation may have changed.  Is that true?

Richard

Richard W. Cartun, MS, PhD
Director, Histology & The Martin M. Berman, MD Immunopathology & Morphologic 
Proteomics Laboratory
Director, Biospecimen Collection Programs
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 972-1596
(860) 545-2204 Fax


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[Histonet] Decalcification with formic acid sodium citrate

[Dorothy Hu via Histonet]

There was a paper 
http://www.genedetect.com/Merchant2/ExampleRefs/Decalcifying_protocols.pdf
Talking about formic acid (Morse solution) can get as good result as EDTA in 
ISH. 
FYI.

Dorothy Hu


> Today's Topics:
> 
>   1. Re.  Decalcification with formic acid sodium citrate
>  (Gayle Callis)
>   2. Re: Re. Decalcification with formic acid 
> 
> Merissa and Tim,  
> 
> 
> 
> This formic acid decalcifying solution is basically the classic Evans and
> Krajian fluid (Sheehan and Hrapchak,   Theory and Practice of
> Histotechnology, 2nd edition, P.92).  Shandon has added other ingredients
> for some reason, and has kept those concentrations proprietary.   You really
> don't need to add a surfactant or PVP emulsifier when making up this
> decalcifying agent.   Simply use the classic recipe for successful
> decalcification.   This is also referred to as buffered formic acid and in
> some publications an "acidic buffer".  It is excellent if IHC is needed and
> less damaging, obviously, than a strong mineral HCL acid decalcifiers.  
> 
> 
> 
> Sodium citrate crystals (a buffering salt) 10 g 
> 
> 90% formic acid stock25 ml  
> 
> Distilled water75 ml   
> 
> 
> 
> One can calculate the concentration of formic acid i.e. approx. 4.5% since
> is it made from 90% formic acid stock.  
> 
> 
> 
> Don't bother with the surfactants or PVP.  
> 
> 
> 
> Enjoy an excellent in house formic acid decalcifying solution.  I also
> suggest you read Sheehan and Hrapchak textbook chapter on bone as a way to
> familiarize yourself with decalcifiying solutions that manufacturers now
> supply with some modifications.  Some manufacturers will refer to these
> methods but probably prefer not to do this since they want you to buy their
> commercial product that is obviously a time saver with elimination of having
> to store stock acid solutions.   The classic methods made in house are
> excellent if you have time to make them up.   Formic acid with sodium
> formate is another popular buffered formic acid.   I suggest you look for
> another source/manufacturer of the your favorite decalcifier in question as
> more than one company will make it.  Decal Corp, recently sold to Stat Lab,
> could also be the source as Shandon isn't the only game in town.   Others
> are Newcomer Supply, Poly Scientific.  Not having to make it up may remain
> your preference. 
> 
> 
> 
> Gayle M. Callis 
> 
> HTL/HT/MT(ASCP) 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> Written by Tim and Merissa:   
> 
> 
> 
> Merissa,
> 
> 
> 
> Water77-80   solvent
> 
>Formic acid  21-23   active ingredient
> 
> Fluorad  >1   surfactant  - a
> wetting agent to make the solution wet the bone more easily
> 
>Sodium citrate   >1   emulsifier , buffer
> 
> Polyvinyl pyrrolidone>1   emulsifier 
> 
> 
> 
> They say less than one percent of the last three, but you really have no
> idea whether that is 1%, .1% or .01%. It could be any of those.
> 
> 
> 
> But all those surfactants and emulsifiers are meant to keep the solution
> viable for long periods on the shelf. When you make it fresh you don't
> really need them.
> 
> 
> 
> You can either buy a different decalcifier, or make your own. Making your
> own with just the water and acid will work just fine. 
> 
> 
> 
> 
> 
> Tim Morken
> 
> Pathology Site Manager, Parnassus 
> 
> Supervisor, Electron Microscopy/Neuromuscular Special Studies
> 
> Department of Pathology
> 
> UC San Francisco Medical Center
> 
> 
> 
> -Original Message-
> 
> From: M.O. via Histonet [mailto:
>  histonet at
> lists.utsouthwestern.edu] 
> 
> Sent: Wednesday, July 22, 2015 1:24 PM
> 
> To:   histonet at
> lists.utsouthwestern.edu
> 
> Subject: [Histonet] understanding reagents in decalcifier; making it
> in-house
> 
> 
> 
> Hello Histonet
> 
> 
> 
> The supplier for our decalcifier, TBD-2 from Shandon, is having issues with
> getting the product out and we will not be receiving it for at least another
> month.  Our samples are piling up and I don't know what I should do, but
> maybe I can make the decalcifier in-house.  I am wondering if I can make my
> own based on the reagents they listed and their percentages and if certain
> reagents are not actually necessary.
> 
> 
> 
> The samples we typically decalcify are mouse knees (decal time = 2 days),
> mouse spines (3 days), human bone slabs about 7mm in thickness (7-12 days).
> Fixation is in zinc buffered formalin, then decalcification, then 70% EtOH.
> Our choice to use TBD-2 is due to the gentle decalcification for IHC and we
> get GREAT results.
> 
> 
> 
> Composition of Shandon TBD-2 Decalcifier:
> 
> ComponentWeight %
> 

[Histonet] Decalcification and Neutralization (Huynh,Thomas)

[Steve McClain via Histonet]

I have not found the need for neutralization.
With sufficient washing, common histochemical stains including H&E Gram 
PAS-fungus and trichrome are spectacular.
Please explain your protocol for neutralization.

Steve A. McClain, MD

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[Histonet] Decalcification

[Huynh,Thomas via Histonet]

Hello Histoneters,

I would like to add that washing after decalcifying is still not enough. The 
bone specimens have to be neutralized before processing ( putting them in the 
neutralizer solution), so there are no trace of acid when we process them.

Thomas 


-Original Message-
From: histonet-requ...@lists.utsouthwestern.edu 
[mailto:histonet-requ...@lists.utsouthwestern.edu] 
Sent: Monday, July 20, 2015 12:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 140, Issue 22

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Today's Topics:

   1. Decalcification (Steve McClain)
   2. Cerner PathNet AP tracking (Younes, Pamela S)


--

Message: 1
Date: Sun, 19 Jul 2015 19:00:38 +
From: Steve McClain 
To: ""

Subject: [Histonet] Decalcification
Message-ID: 
Content-Type: text/plain; charset="us-ascii"

One frequently overlooked point is washing after decal. Especially important 
with acid decal, we wash for an hour in running water. 


--

Message: 2
Date: Mon, 20 Jul 2015 13:00:06 +
From: "Younes, Pamela S" 
To: "histonet@lists.utsouthwestern.edu"

Subject: [Histonet] Cerner PathNet AP tracking
Message-ID:

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

Hello everyone,
Has anyone used the Cerner PathNet AP tracking system? I would like to hear 
about any feedback, good or bad.
Many thanks,
Pam Younes

Pamela S. Younes MHS, HTL(ASCP), CPC(AAPC), PA(ASCP) Pathologists' Assistant 
Assistant Professor, Department of Pathology and Laboratory Medicine University 
of Texas Medical School, Houston
6430 Fannin, MSB 2.233
Houston, TX 77030
(713) 704-2053



--

Subject: Digest Footer

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End of Histonet Digest, Vol 140, Issue 22
*

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[Histonet] Decalcification

[Steve McClain via Histonet]

One frequently overlooked point is washing after decal. Especially important 
with acid decal, we wash for an hour in running water. 
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Re: [Histonet] Decalcification of mouse jaw and legs

[iliana Dimitrova via Histonet]

We do mouse legs routinely, either with Cal Ex II or EDTA. We look at the bone 
and the tissue around, so we have to have good fixation, decal., cutting and 
staining for both. 
Cal Ex II (formic acid) is a gentler decal agent than HCL (Plain "Cal EX") - 
and Cal Ex II keeps the H&E staining true - whereas HCL would make the Eosin 
dominate and stain everything!
For future - since Cal Ex II contains Formalin - on theory you can fix and 
decal at the same time, but I always do my fixation first and then decal after. 
Use chemical method to determine the end point.
Other ways to decal bone are with EDTA - I personally prefer it, though it 
takes a really long time, almost 2 months, it is better if you want to do IHC 
for certain markers that the acids might interfere with. We weight the samples 
before we start decal, and after that 2/week. The moment you stop seeing 
decrease in weight and see slight increase, that is the end point.
For cutting, I prefer samples decalc. with Cal Ex II, cuts without a problem.

Iliana Dimitrova, MLT, LHP, B.Tech., M.Sc. 

Histology Supervisor
Medical Education and Laboratory Support Services (MELSS)
Faculty of Medicine
Memorial University of Newfoundland
St. John's, NL Canada A1B 3V6

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-Original Message-
From: Swartwood, Steven J [mailto:steven.swartw...@cshs.org] 
Sent: July-10-15 7:23 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Decalcification of mouse jaw and legs

Hello all,

I hope everyone is having or had a fantastic week,

I'm going to test a few different decal methods on mouse jaws and leg bones. I 
was wondering if anyone does this routinely out there who is willing to share a 
protocol. I've never done this on mouse jaw/leg bones before. I've only done 
this on human tissues. From what I've read it seems that for routine histology 
low % HCl is fine, but if I wanted to perform IHC then anywhere from 5-15% 
formic acid may be the best way to go. Nitric acid is probably too strong of a 
decal agent for these tiny specimens I would assume. Maybe an EDTA based decal 
agent may be best as well. I'm just really inexperienced with this and I'm very 
open to ideas and trying a few different methods.

Steven Swartwood HT(ASCP)
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[Histonet] Decalcification of mouse jaw and legs

[Swartwood, Steven J]

Hello all,

I hope everyone is having or had a fantastic week,

I'm going to test a few different decal methods on mouse jaws and leg bones. I 
was wondering if anyone does this routinely out there who is willing to share a 
protocol. I've never done this on mouse jaw/leg bones before. I've only done 
this on human tissues. From what I've read it seems that for routine histology 
low % HCl is fine, but if I wanted to perform IHC then anywhere from 5-15% 
formic acid may be the best way to go. Nitric acid is probably too strong of a 
decal agent for these tiny specimens I would assume. Maybe an EDTA based decal 
agent may be best as well. I'm just really inexperienced with this and I'm very 
open to ideas and trying a few different methods.

Steven Swartwood HT(ASCP)
IMPORTANT WARNING: This message is intended for the use of the person or entity 
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reader of this message is not the intended recipient, or the employee or agent 
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Re: [Histonet] Decalcification of bone marrows

[Bob Richmond]

Adrienne (where?) asks: "I have a really quick question: about how long
does it take to decal a bone marrow biopsy?" to which Jessica (where? -
apparently in the US though) replies "It all depends on what you use for
decal. We use 5% Nitric acid for 1 hour or so. Sometimes it needs a bit
more time."

To decalcify a Jamshidi needle bone marrow biopsy specimen in one of the
ordinary commercial decalcifiers (usually hydrochloric acid, all deep dark
trade secrets) takes about two hours.

Nitric acid is NOT an acceptable decalcifier today, since it destroys
immunoreactivity. I used to use it for most decalcification in the days
before immunohistochemistry, but remember I've been practicing pathology
for more than fifty years.

Successful processing of bone marrow biopsy specimens requires cooperation
among hematologist-oncologists, pathologists, and histotechnologists.
(Dream on!) Practically speaking, you have to set a deadline - if your
specimen arrives after 2 PM (for example) it doesn't get processed until
tomorrow.

Once again - Decal is the registered trademark of Decal Chemical
Corporation's proprietary decalcifier. It is not a generic word for
decalcifiers. (No, I don't work for them.)

Bob Richmond
Samurai Pathologist
Maryville, TN
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[Histonet] Decalcification using EDTA

[Dorothy Hu]

It is optional to decal in 4oC, but it is much better for preserve
antigenicity and get good result from enzymatic staining.
Similar way as you do fixation, you can choose RT or 4oC.
Dorothy Hu


Hello Histonetters

Is it imperative OR optional to maintain samples at below 4 degrees celcius
when using EDTA to decalcify bone samples

Thank You
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[Histonet] Decalcification using EDTA

[Rooki Parak]

Hello Histonetters

Is it imperative OR optional to maintain samples at below 4 degrees celcius
when using EDTA to decalcify bone samples

Thank You
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[Histonet] decalcification after dehydration

[Rui TAHARA]

I have an adult bird skull that fixed with formalin and then has been stored in 
70% ethanol.  
I have seen the post that the sample stored in 70% ethanol can be walking back 
through to series of ethanol to water and can be decalcified if it needs to be. 

I am wondering if anybody has done this and there is any side effects from 
decalcification after going through dehydration and rehydration of a sample 
compared to a general straight forward protocol from decalcification to 
dehydration? 

Thank you,


rui  
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RE: [Histonet] decalcification of premolar teeth (dog)

[Rittman, Barry R]

Alice
I would recommend using sodium formate/formic acid mixture for demineralization 
as this is more gentle than most agents.
I would not use hydrochloric acid unless you are shipwrecked on a desert island 
and that is the only chemical available to you.
I am assuming that EDTA demineralization is not an option for you?
Barry

From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Alice Fraser 
[toxpat...@gmail.com]
Sent: Tuesday, August 07, 2012 8:56 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] decalcification of premolar teeth (dog)

Dear Members
I would be really interested to hear your advice on the currently preferred
procedure for decalcification of premolar teeth from dogs. Do laboratories
find the HCl/water solution or the formic acid solution or another solution
to be optimal for decal without obliterating the tissues to be evaluated? A
procedure/method would be hugely appreciated if poss.
Many thanks.
Alice
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[Histonet] decalcification of premolar teeth (dog)

[Alice Fraser]

Dear Members
I would be really interested to hear your advice on the currently preferred
procedure for decalcification of premolar teeth from dogs. Do laboratories
find the HCl/water solution or the formic acid solution or another solution
to be optimal for decal without obliterating the tissues to be evaluated? A
procedure/method would be hugely appreciated if poss.
Many thanks.
Alice
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Re: [Histonet] Decalcification of bone

[Rene J Buesa]

As a matter of fact any decalciying technique presented in any histotechnique 
book works. The differences reside in the time it will take and the 
decalcifying method you use.
I will be very complicated to indicate you one, but a commercial productuct 
called "RDO" is one I used for hard bones and EDTA solution for bone marrow 
biopsies.
I this you will better of in consulting a techniques book and follow the 
instructions, especially those related to the "end point" of the 
decalcification.
René J.

--- On Thu, 8/11/11, Rachael Glebocki  wrote:


From: Rachael Glebocki 
Subject: [Histonet] Decalcification of bone
To: "histonet@lists.utsouthwestern.edu" 
Date: Thursday, August 11, 2011, 4:10 AM


Dear Histonet users,

I was wondering if anyone has a operation procedure for bone decalcification 
that works. I am having no joy in decalcifying the bone and making a good slide 
from it.

Thank you for your time.

Rachael Glebocki
Teaching Technician
School of Veterinary Medicine & Science
University of Nottingham

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Re: [Histonet] Decalcification of bone

[Adam .]

Hi Rachael,

I work with mouse bones on a regular basis, and I assure you that they are
incredibly difficult to work with, but with practice you can get decent
sections. It would be useful if you gave us a bit more information. What
kind of bones are you decalcifying? How are they fixed? How are you
currently decalcifying? Are you paraffin embedding or cutting frozen
sections? What is exactly happening when you're cutting the bones that gives
you poor quality? What is your end goal (IHC, IF, H&E)?

Adam

On Thu, Aug 11, 2011 at 3:10 AM, Rachael Glebocki <
rachael.glebo...@nottingham.ac.uk> wrote:

> Dear Histonet users,
>
> I was wondering if anyone has a operation procedure for bone
> decalcification that works. I am having no joy in decalcifying the bone and
> making a good slide from it.
>
> Thank you for your time.
>
> Rachael Glebocki
> Teaching Technician
> School of Veterinary Medicine & Science
> University of Nottingham
>
> This message and any attachment are intended solely for the addressee and
> may contain confidential information. If you have received this message in
> error, please send it back to me, and immediately delete it.   Please do not
> use, copy or disclose the information contained in this message or in any
> attachment.  Any views or opinions expressed by the author of this email do
> not necessarily reflect the views of the University of Nottingham.
>
> This message has been checked for viruses but the contents of an attachment
> may still contain software viruses which could damage your computer system:
> you are advised to perform your own checks. Email communications with the
> University of Nottingham may be monitored as permitted by UK
> legislation.___
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[Histonet] Decalcification of bone

[Rachael Glebocki]

Dear Histonet users,

I was wondering if anyone has a operation procedure for bone decalcification 
that works. I am having no joy in decalcifying the bone and making a good slide 
from it.

Thank you for your time.

Rachael Glebocki
Teaching Technician
School of Veterinary Medicine & Science
University of Nottingham

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RE: [Histonet] decalcification of articular cartilage

[sgoebel]

You could always get formical which fixes and decals at the same time.  This 
would eliminate the waiting for fixation.  Although I do usually let it fix in 
straight formalin overnight first.

Sarah Goebel, BA, HT(ASCP)
Histotechnologist
Mirna Therapeutics
2150 Woodward Street
Suite 100
Austin, Texas  78744
(512)901-0900 ext. 6912


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz Chlipala
Sent: Thursday, April 21, 2011 12:59 PM
To: Nejat Yilmaz; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] decalcification of articular cartilage

We do a lot of IHC staining on bone and cartilage samples and we use 10% formic 
acid for decalcification for immunohistochemistry.  I'm afraid you may not be 
able to turnaround this in a week.  The tissue needs to be adequately fixed 
prior to decalcification, and properly decaled.  I would trim the tissue to 
about 4 mm thick if you can on a saw you might be able to turn this around in  
your time frame, but I have learned from experience if you rush decalcification 
you have a greater chance of running into problems.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Nejat Yilmaz
Sent: Wednesday, April 20, 2011 8:12 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] decalcification of articular cartilage

Dear Members,

We're planning to study sheep knee articular cartilage with histochemistry and 
immunohistchemistry. Proximal parts of the tibia bones fixing in formaldehyde 
at the moment. We need an effective, quick and cartilage tissue protective 
decalcification method. We'll perform only light microscopic examination in 
this study and we have to complete the study within one week. In these 
circumstances what are your advices?
Thanks in advance.

Dr. Necat Yilmaz
University of Mersin
School of Medicine


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RE: [Histonet] decalcification of articular cartilage

[Liz Chlipala]

We do a lot of IHC staining on bone and cartilage samples and we use 10% formic 
acid for decalcification for immunohistochemistry.  I'm afraid you may not be 
able to turnaround this in a week.  The tissue needs to be adequately fixed 
prior to decalcification, and properly decaled.  I would trim the tissue to 
about 4 mm thick if you can on a saw you might be able to turn this around in  
your time frame, but I have learned from experience if you rush decalcification 
you have a greater chance of running into problems.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Nejat Yilmaz
Sent: Wednesday, April 20, 2011 8:12 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] decalcification of articular cartilage

Dear Members,

We're planning to study sheep knee articular cartilage with histochemistry and 
immunohistchemistry. Proximal parts of the tibia bones fixing in formaldehyde 
at the moment. We need an effective, quick and cartilage tissue protective 
decalcification method. We'll perform only light microscopic examination in 
this study and we have to complete the study within one week. In these 
circumstances what are your advices?
Thanks in advance.

Dr. Necat Yilmaz
University of Mersin
School of Medicine


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[Histonet] decalcification of articular cartilage

[Nejat Yılmaz]

Dear Members,

We're planning to study sheep knee articular cartilage with histochemistry and 
immunohistchemistry. Proximal parts of the tibia bones fixing in formaldehyde 
at the moment. We need an effective, quick and cartilage tissue protective 
decalcification method. We'll perform only light microscopic examination in 
this study and we have to complete the study within one week. In these 
circumstances what are your advices?
Thanks in advance.

Dr. Necat Yilmaz
University of Mersin
School of Medicine


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Re: [Histonet] Decalcification

[Langenberg, Stacey]

American master tech makes a great surface decal called Easy cut.

Good luck

Stacey
Sent via BlackBerry from T-Mobile

-Original Message-
From: "Sherwood, Margaret " 
Sender: "histonet-boun...@lists.utsouthwestern.edu"

Date: Wed, 8 Dec 2010 11:40:36 
To: histonet@lists.utsouthwestern.edu
Subject: Re:  [Histonet] Decalcification

We have small arteries that were FFPE and sectioned that should have been
decacified before processing.  (The investigator did not indicate as such when
submitting them).  Cutting was extremely difficult and the sections, as you can
imagine, had terrible knife marks and chatter.  Of course, they are important
(for a paper)!  

Is there any way tissue, that has been embedded, can be decalcified at this
point?  Or some other way to treat the block so that we can get better sections?

Thanks for all your help.

Peggy

Peggy Sherwood
Lab Associate, Photopathology
Wellman Center for Photomedicine (EDR 214)
Massachusetts General Hospital
55 Fruit Street
Boston, MA 02114-2696
617-724-4839 (voice mail)
617-726-6983 (lab)
617-726-1206 (fax)
msherw...@partners.org 




The information in this e-mail is intended only for the person to whom it is
addressed. If you believe this e-mail was sent to you in error and the e-mail
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RE: [Histonet] Decalcification

[Sherwood, Margaret]

Could you explain in more detail the process, Liz?

Thanks!
Peggy  

 

-Original Message-
From: Liz Chlipala [mailto:l...@premierlab.com] 
Sent: Wednesday, December 08, 2010 1:47 PM
To: Sherwood, Margaret ; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Decalcification

Try surface decaling the samples in the block.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Sherwood, Margaret 
Sent: Wednesday, December 08, 2010 11:41 AM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Decalcification

We have small arteries that were FFPE and sectioned that should have
been
decacified before processing.  (The investigator did not indicate as
such when
submitting them).  Cutting was extremely difficult and the sections, as
you can
imagine, had terrible knife marks and chatter.  Of course, they are
important
(for a paper)!  

Is there any way tissue, that has been embedded, can be decalcified at
this
point?  Or some other way to treat the block so that we can get better
sections?

Thanks for all your help.

Peggy

Peggy Sherwood
Lab Associate, Photopathology
Wellman Center for Photomedicine (EDR 214)
Massachusetts General Hospital
55 Fruit Street
Boston, MA 02114-2696
617-724-4839 (voice mail)
617-726-6983 (lab)
617-726-1206 (fax)
msherw...@partners.org 




The information in this e-mail is intended only for the person to whom
it is
addressed. If you believe this e-mail was sent to you in error and the
e-mail
contains patient information, please contact the Partners Compliance
HelpLine at
http://www.partners.org/complianceline . If the e-mail was sent to you
in error
but does not contain patient information, please contact the sender and
properly
dispose of the e-mail.


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RE: [Histonet] Decalcification

[Liz Chlipala]

Try surface decaling the samples in the block.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Sherwood, Margaret 
Sent: Wednesday, December 08, 2010 11:41 AM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Decalcification

We have small arteries that were FFPE and sectioned that should have
been
decacified before processing.  (The investigator did not indicate as
such when
submitting them).  Cutting was extremely difficult and the sections, as
you can
imagine, had terrible knife marks and chatter.  Of course, they are
important
(for a paper)!  

Is there any way tissue, that has been embedded, can be decalcified at
this
point?  Or some other way to treat the block so that we can get better
sections?

Thanks for all your help.

Peggy

Peggy Sherwood
Lab Associate, Photopathology
Wellman Center for Photomedicine (EDR 214)
Massachusetts General Hospital
55 Fruit Street
Boston, MA 02114-2696
617-724-4839 (voice mail)
617-726-6983 (lab)
617-726-1206 (fax)
msherw...@partners.org 




The information in this e-mail is intended only for the person to whom
it is
addressed. If you believe this e-mail was sent to you in error and the
e-mail
contains patient information, please contact the Partners Compliance
HelpLine at
http://www.partners.org/complianceline . If the e-mail was sent to you
in error
but does not contain patient information, please contact the sender and
properly
dispose of the e-mail.


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Re: [Histonet] Decalcification

[Sherwood, Margaret]

We have small arteries that were FFPE and sectioned that should have been
decacified before processing.  (The investigator did not indicate as such when
submitting them).  Cutting was extremely difficult and the sections, as you can
imagine, had terrible knife marks and chatter.  Of course, they are important
(for a paper)!  

Is there any way tissue, that has been embedded, can be decalcified at this
point?  Or some other way to treat the block so that we can get better sections?

Thanks for all your help.

Peggy

Peggy Sherwood
Lab Associate, Photopathology
Wellman Center for Photomedicine (EDR 214)
Massachusetts General Hospital
55 Fruit Street
Boston, MA 02114-2696
617-724-4839 (voice mail)
617-726-6983 (lab)
617-726-1206 (fax)
msherw...@partners.org 




The information in this e-mail is intended only for the person to whom it is
addressed. If you believe this e-mail was sent to you in error and the e-mail
contains patient information, please contact the Partners Compliance HelpLine at
http://www.partners.org/complianceline . If the e-mail was sent to you in error
but does not contain patient information, please contact the sender and properly
dispose of the e-mail.


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Re: [Histonet] decalcification

[Joseph Saby]

Dorothy-

If your bones are indeed large, 24 hours in 10% NBF will not be sufficient for 
fixation.  If the fixation isn't sufficient, none of the processes following 
that will be as effective as they might be.  Even after trimming, I would 
recommend fixing an additional 24-48 hours for every mm of thickness of your 
bone section, at a minimum.  If you are in a hospital setting and need reults 
quickly, you can use a processor (with heat, pressure and vacuum) to speed up 
the fixation.

I use buffered formic acid to decal.  The final concentration of formic acid is 
about 20-25%, so it is rather aggressive, but it also allows you to read the 
bone marrow after decal.  If you need results in a short time (hospital setting 
again) you can use an aggressive reagent like RDO for decal, but keep in mind 
that nuclear detail will be lacking, but bone structure should still be 
readable.  If you do not have the reagents to determine decal endpoints, you 
can 
(with training/practice) use a needle to pass through the sections to help 
determine if decal is complete.  The needle should move through the specimen 
smoothly and not get hung up and stiff in the middle of the section.  
Flexibility of the bone section can tell you when you are appoaching the time 
when the needle test will give you useable results.

Please do not read this as an endorsement for RDO or any other super aggressive 
decal solution.  Personally, I always insist on being able to read all tissue 
elements, and these solutions, although fast, give less than optimal staining 
results.

If you have further questions, please ask me off line.

Joe Saby, BA HT






From: "Webb, Dorothy L" 
To: "histonet@lists.utsouthwestern.edu" 
Sent: Mon, August 2, 2010 10:14:35 AM
Subject: [Histonet] decalcification

I would appreciate any feedback on what all are using in your decalcification 
process.  We get a lot of large bones in and the past 2-3 months have noticed a 
huge problem in our microtomy process with these samples.  We have been 
grossing 
the bones in and leaving the sample in the cassette in 10% formalin for 24 
hours 
befoere placing in decal for up to 8 hours and still having the inner portion 
of 
the sample look underprocessed and crunchy!  Any suggestions would be 
appreciated!

Dorothy Webb, HT
Regions Hospital Technical Supervisor
651-254-2962



  
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Re: [Histonet] decalcification

[Sean McBride]

> Hi Dorothy,

I'm not sure what size bone specimens with which you are working, but I 
generally work with femurs & calvaria from rabbits, canine & other small 
rodents.  I fix my specimens longer in 10% NBF using an automated vacuum 
infiltration processor.  Following fixation, if decal is required, I will decal 
in buffered formic acid solution and use a calcium reagent set to titrate to 
the decalcification endpoint. If you would like any specifics, contact me 
offline.  

Best regards,


~Sean McBride


Senior Researcher
Bone Tissue Engineering Center
Carnegie Mellon Research Institute
Suite 4311
700 Technology Drive
Pittsburgh, PA 15219-3124

412-268-8275 (o)
412-915-1683 (m)
412-268-9807 (fax)
smcbr...@andrew.cmu.edu 

> 


 
> --- On Mon, 8/2/10, Webb, Dorothy L  wrote:
> 
> 
> From: Webb, Dorothy L 
> Subject: [Histonet] decalcification
> To: "'histonet@lists.utsouthwestern.edu'" 
> Date: Monday, August 2, 2010, 10:14 AM
> 
> 
> I would appreciate any feedback on what all are using in your decalcification 
> process.  We get a lot of large bones in and the past 2-3 months have noticed 
> a huge problem in our microtomy process with these samples.  We have been 
> grossing the bones in and leaving the sample in the cassette in 10% formalin 
> for 24 hours befoere placing in decal for up to 8 hours and still having the 
> inner portion of the sample look underprocessed and crunchy!  Any suggestions 
> would be appreciated!
> 
> Dorothy Webb, HT
> Regions Hospital Technical Supervisor
> 651-254-2962
> 
> 
> 
>   
> This e-mail and any files transmitted with it are confidential and are 
> intended solely for the use of the individual or entity to whom they are 
> addressed. If you are not the intended recipient or the individual 
> responsible for delivering the e-mail to the intended recipient, please be 
> advised that you have received this e-mail in error and that any use, 
> dissemination, forwarding, printing, or copying of this e-mail is strictly 
> prohibited.
> 
> If you have received this e-mail in error, please immediately notify the 
> HealthPartners Support Center by telephone at (952) 967-6600. You will be 
> reimbursed for reasonable costs incurred in notifying us. HealthPartners 
> R001.0
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Re: [Histonet] decalcification

[Rene J Buesa]

You cannot set a fixed time for decalcification. You have to leave the bone in 
the decalcifying solution until iy is soft and ready for processing.
René J.

--- On Mon, 8/2/10, Webb, Dorothy L  wrote:


From: Webb, Dorothy L 
Subject: [Histonet] decalcification
To: "'histonet@lists.utsouthwestern.edu'" 
Date: Monday, August 2, 2010, 10:14 AM


I would appreciate any feedback on what all are using in your decalcification 
process.  We get a lot of large bones in and the past 2-3 months have noticed a 
huge problem in our microtomy process with these samples.  We have been 
grossing the bones in and leaving the sample in the cassette in 10% formalin 
for 24 hours befoere placing in decal for up to 8 hours and still having the 
inner portion of the sample look underprocessed and crunchy!  Any suggestions 
would be appreciated!

Dorothy Webb, HT
Regions Hospital Technical Supervisor
651-254-2962



  
This e-mail and any files transmitted with it are confidential and are intended 
solely for the use of the individual or entity to whom they are addressed. If 
you are not the intended recipient or the individual responsible for delivering 
the e-mail to the intended recipient, please be advised that you have received 
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RE: [Histonet] decalcification

[sgoebel]

   I  have  used formical for years and it works great.  You don'= t have
   to  pre-fix your samples because there is formalin in with the decal.  
It  fixes  and  decals at the same time.  I don't know how many sp   ecimens  
you  have,  but a sure fire way to check is to weigh the bone
   after  g=  rossing.   Then  everyday  weigh  it  and replace the decal
   solution. =  It  should  start  losing  weight  because the calcium is
   coming  out.   As  = soon as the sample starts gaining weight, take it
   out.   Now  it is just= absorbing fluid.  In the past when I have done
   huge bones, I use this= technique (learned at a NSH convention) and it
   works like a charm!  H= ope that helps =)

   PS-The formical is made by Dec= al company.

   Sarah Goebel, B.A., HT (ASCP)

   Histotechnician
   <= /span>

   XBiotech USA Inc.

   8201 East Riverside Dr. Bldg 4 = Suite 100

   Austin, Texas  78744

   
   (512)386-5107
    Original Message ----
   Subject: [Histonet] decalcification
   From: "Webb, Dorothy L" <[1]dorothy.l.w...@healthpartners.com>
   Date: Mon, August 02, 2010 7:14 am
   To: "'[2]histo...@lists.u= tsouthwestern.edu'"
   <[3]histo...@lists.uts= outhwestern.edu>
   I  would  appreciate  any  feedback  on  what  all  are  using in your
   decalcificati= on process. We get a lot of large bones in and the past
   2-3 months have no= ticed a huge problem in our microtomy process with
   these  samples.  We  have = been grossing the bones in and leaving the
   sample  in the cassette in 10% fo= rmalin for 24 hours befoere placing
   in decal for up to 8 hours and still ha= ving the inner portion of the
   sample  look  underprocessed  and  crunchy! Any = suggestions would be
   appreciated!
   Dorothy Webb, HT
   Regions Hospital Technical Supervisor
   651-254-2962
   
   This e-mail and any files transmitted with it are confidential and are
   inte= nded solely for the use of the individual or entity to whom they
   are  addres=  sed.  If  you  are  not  the  intended  recipient or the
   individual  responsible  fo=  r  delivering the e-mail to the intended
   recipient,  please  be advised that y= ou have received this e-mail in
   error  and  that  any  use,  dissemination, forw= arding, printing, or
   copying of this e-mail is strictly prohibited.
   If  you  have received this e-mail in error, please immediately notify
   the  He=  althPartners  Support Center by telephone at (952) 967-6600.
   You  will  be  rei= mbursed for reasonable costs incurred in notifying
   us. HealthPartners R001.= 0
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References

   1. 3D"mailto:dorothy.l.w...@healthpartners   2. 
3D"mailto:histonet@lists.utsouthwestern.edu";
   3. 3D"mailto:histonet@lists.utsouthwestern.edu";
   4. 3D"mailto:Histonet@lists.utsouthwestern.edu";
   5. 3D"http://lists.utsouthwestern.edu/mailman/listinfo/histonet";
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[Histonet] decalcification

[Webb, Dorothy L]

I would appreciate any feedback on what all are using in your decalcification 
process.  We get a lot of large bones in and the past 2-3 months have noticed a 
huge problem in our microtomy process with these samples.  We have been 
grossing the bones in and leaving the sample in the cassette in 10% formalin 
for 24 hours befoere placing in decal for up to 8 hours and still having the 
inner portion of the sample look underprocessed and crunchy!  Any suggestions 
would be appreciated!

Dorothy Webb, HT
Regions Hospital Technical Supervisor
651-254-2962



  
This e-mail and any files transmitted with it are confidential and are intended 
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you are not the intended recipient or the individual responsible for delivering 
the e-mail to the intended recipient, please be advised that you have received 
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[Histonet] Decalcification and processing of large bone

[Victor Wong]

Dear all,
 
I am going to decal and process PIG femur and lumbar vertebrates.  I only have 
experience on soft tissues.  Anyone can suggest any protocols in 
decalcification and processing?  Many thanks in advance.
 
Cheers,
Victor



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RE: [Histonet] Decalcification prior to Alizarine red or von Kossa possible?

[Jack Ratliff]

Alexandra,
 
It sounds like to me that you might be working with some sort of calcium 
phosphate material in the presence of demineralized bone and/or soft tissue in 
say a muscle pocket and looking for the presence/absence of bone formation. If 
you decal the tissue you will lose the ability to detect any clear mineralized 
bone as the alizarin red and Von Kossa reaction both identify mineralization 
(calcium). Also, if there are heavily mineralized nodules as you indicate, then 
I would say you do not have the option to cut as a paraffin block.
 
If I was doing this project, I would embed the tissue in methyl methacrylate 
(MMA), cut thin sections (4-6 microns) on a motorized rotary microtome with a 
tungsten-carbide knife, deplastify the tissue sections (similar to 
deparaffinization), and stain with the Von Kossa reaction and MacNeal's 
tetrachrome stain. This combination would then clearly yield the presence or 
absence of mineralization where the increasing degree of mineralization (dense 
older bone) would be seen as black and any newly formed mineralization (vital 
bone - osteoid) would be grayish-green. Furthermore, this staining combination 
will also allow for the identification of bone forming (osteoblasts/blue) and 
bone resorbing (osteoclasts/bluish-green) cells. In fact, this stain will 
clearly identify any active mineralization (osteoid) that has formed (osteoid) 
and show the active bone forming osteoblasts lining the surface as nice plump 
(single nuclei) cuboidal cells.
 
Additionally, if all you care about is mineralization or newly formed bone, you 
could also perform a Goldner's trichrome stain where any mineralization or 
newly formed bone (osteoid) will be highlighted as red. Hope this helps and let 
me know if you need any further assistance or advice.
 
Best Regards,
 
Jack



> Date: Thu, 9 Oct 2008 15:17:07 +0200> From: [EMAIL PROTECTED]> To: 
> histonet@lists.utsouthwestern.edu> Subject: [Histonet] Decalcification prior 
> to Alizarine red or von Kossa possible?> > Hello all,> > I have to stain some 
> specimens with bone formation in soft tissue. They want> me to do Alizarine 
> red or Kossa stain.> But there are some heavily mineralized nodules in the 
> tissue, I think they> can't be cut nicely without some kind of 
> decalcification.> How should I treat these samples? Is there a way to 
> decalcify them without> negative effects on the calcium stain?> > Thanks in 
> advance!> > Alexandra> ___> 
> Histonet mailing list> Histonet@lists.utsouthwestern.edu> 
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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[Histonet] Decalcification prior to Alizarine red or von Kossa possible?

[Alexandra Meinl]

Hello all,

I have to stain some specimens with bone formation in soft tissue. They want
me to do Alizarine red or Kossa stain.
But there are some heavily mineralized nodules in the tissue, I think they
can't be cut nicely without some kind of decalcification.
How should I treat these samples? Is there a way to decalcify them without
negative effects on the calcium stain?

Thanks in advance!

Alexandra
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