Re: [Histonet] Bone samples

[Simmons, Christopher via Histonet]

Ion Exchange Decal containers will change your experience forever. 

-Original Message-
From: Gudrun Lang via Histonet  
Sent: Friday, January 26, 2024 3:32 AM
To: 'Chakib Boussahmain' 
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Bone samples

Hi,
In my opinion the hardness of the decalcified blocks is often rather due to the 
paraffin-processing than the residual calcium. Especially when the tissue is 
decalcified really long. The hardness comes from the dehydration and "cooking" 
of collagen fibers. So additional decal will not help reducing the calcium, but 
helps to reintroduce water into the collagen-grid. And this is helpful for 
softening and cutting.
For myself, I often scratch the paraffin on the blocksurface away to face the 
bone directly to the water. Then I let them "swim" on my waterbath, until the 
surface is turned rather milky. After cooling again I cut in very very small 
steps to trim the surface. Sometimes it needs repeated swimming and cooling 
(and patience) to get a rather acceptable section. It is advantageous to pick 
them up on adhesive slides and let them dry in an 60°C oven to get rid of any 
residual water under the section.

Hope this helps
Gudrun

-Ursprüngliche Nachricht-
Von: Chakib Boussahmain via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Gesendet: Mittwoch, 24. Jänner 2024 23:34
An: histonet@lists.utsouthwestern.edu
Betreff: [Histonet] Bone samples


Hi guys,

 I hope this messagefinds you well. I am currently working on a study involving 
bone samples thathave been treated with slow decal and embedded in paraffin. I 
am facingchallenges in obtaining nice sections, and I was wondering if you 
could providesome guidance or recommendations.

Thank you in advance for your help!
Chakib
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Re: [Histonet] Bone samples

[Gudrun Lang via Histonet]

Hi,
In my opinion the hardness of the decalcified blocks is often rather due to the 
paraffin-processing than the residual calcium. Especially when the tissue is 
decalcified really long. The hardness comes from the dehydration and "cooking" 
of collagen fibers. So additional decal will not help reducing the calcium, but 
helps to reintroduce water into the collagen-grid. And this is helpful for 
softening and cutting.
For myself, I often scratch the paraffin on the blocksurface away to face the 
bone directly to the water. Then I let them "swim" on my waterbath, until the 
surface is turned rather milky. After cooling again I cut in very very small 
steps to trim the surface. Sometimes it needs repeated swimming and cooling 
(and patience) to get a rather acceptable section. It is advantageous to pick 
them up on adhesive slides and let them dry in an 60°C oven to get rid of any 
residual water under the section.

Hope this helps
Gudrun

-Ursprüngliche Nachricht-
Von: Chakib Boussahmain via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Gesendet: Mittwoch, 24. Jänner 2024 23:34
An: histonet@lists.utsouthwestern.edu
Betreff: [Histonet] Bone samples


Hi guys,

 I hope this messagefinds you well. I am currently working on a study involving 
bone samples thathave been treated with slow decal and embedded in paraffin. I 
am facingchallenges in obtaining nice sections, and I was wondering if you 
could providesome guidance or recommendations.

Thank you in advance for your help!
Chakib
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Re: [Histonet] Bone samples

[Cooper, Brian via Histonet]

Embed the bones diagonally in your molds if you're able (size depending) as 
this will allow for the greatest amount of paraffin support.  Trim them very 
slowly, and if need be, place the blocks into the same "slow decal" solution 
for additional amount of time sufficient to enable better sectioning.  Start 
checking in half hour or 45 minute intervals; rinse the blocks well in running 
water and attempt to section.  If they're not ready, back into decal solution 
they go.  

I feel that very wet ice helps to facilitate sectioning better than ice that is 
drier and fresh out of the freezer.  Just be sure to blot the face of the block 
with gauze before attempting to cut.  

There's my two cents!

Thanks, 

Brian D. Cooper, HT (ASCP)CMQIHCCM| Histology Supervisor 
Department of Pathology and Laboratory Medicine
Children's Hospital Los Angeles 
4650 Sunset Blvd MS#43- Los Angeles, CA 90027 
Ph: 323.361.3357
bcoo...@chla.usc.edu 

-Original Message-
From: Chakib Boussahmain via Histonet  
Sent: Wednesday, January 24, 2024 2:34 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone samples (EXTERNAL EMAIL)

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Hi guys,

 I hope this messagefinds you well. I am currently working on a study involving 
bone samples thathave been treated with slow decal and embedded in paraffin. I 
am facingchallenges in obtaining nice sections, and I was wondering if you 
could providesome guidance or recommendations.

Thank you in advance for your help!
Chakib
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Re: [Histonet] Bone saw

[Terri Braud via Histonet]

We have used the IMEB band saw for years and love the results.  It has a 
similar footprint and will cut through anything, including a steel pin.  It 
also has a diamond blade and optional water cooling.  It does seem a little 
awkward to clean, and I wish the protective housing was a little better.  If 
our IMEB failed, I would certainly take a long hard look at the Exakt 302 
because it seems to address some of the cons of the IMEB.  I don't know about 
cost comparison.  Best of luck.

Terri L. Braud, HT(ASCP)
HNL Laboratories for 
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046

Today's Topics:

   1. Looking for Bone/Pathology saw recommendations (M.O.)
--
Message: 1
Date: Fri, 20 Jan 2023 10:53:59 -0800
From: "M.O." 
Subject: [Histonet] Looking for Bone/Pathology saw recommendations

Hello and Happy Friday!
I work in an arthritis research lab and we are looking into pathology saws like 
the Exakt 302. We study lumbar spine and knees. During our tissue harvesting 
procedure we take slabs of each vertebral disc unit, facet joints, and knee 
osteochondral samples. Does anyone have experience in cutting bone specimens 
with a saw like this? Do you have any recommendations on the brand you prefer?

Sincerely,
Merissa


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Re: [Histonet] Bone marrow clot IHC tissue sections washing

[Regan Fulton via Histonet]

Martha,

We reported our study of 15 brands of adhesive slides for "wash off" and
found little difference among the different slides when well-fixed cell
culture material was examined.
On the other hand, poorly-fixed breast cancer tissues did appear to adhere
more strongly to some slides than others (TOMO being among the best).
Additional factors need to be considered, though, and I note that your
baking procedure is different from what is recommended by many slide
vendors.
In general, baking at 60-65 deg C for 1 hour is said to be optimal,
although we did not examine that parameter specifically.

Please see our poster at https://www.arrayscience.com/publications#Posters

Best regards,



Regan
Regan Fulton, M.D., Ph.D.
CEO and Co-Founder
Array Science, LLC
475 Gate 5 Road, #100
Sausalito, CA 94965
(415) 577-7360
email: ful...@arrayscience.com


www.arrayscience.com



On Thu, Sep 23, 2021 at 1:02 PM Martha Ward-Pathology via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> It was brought to my attention that we had significant washing on  3 of 8
> bone marrow clot sections the other day; this is not the first time so we
> would like to get to the bottom of this.   We use positively charged slides
> and all 8 cases were cut and run the same morning but allowed to air dry
> and then bake at 60C for 20 minutes before being run on our Bond 3
> stainer.   Has anyone out there experienced this type of problem and if so,
> what were your solutions?The repeat of the 3 cases today showed similar
> washing of tissue.
>
> This hasn't just started but has occurred periodically but the
> pathologists have tried to live with it and usually we can finally get
> enough tissue to stay on after 1-2 attempts.   Suggestions include cutting
> and drying the slides overnight and/or going to a gelatinated  slide versus
> a sialylated slide.   We have been using this particular brand of positive
> charged slide with good results for several years and rarely have issues
> with other tissue types unless they are particularly bloody.
>
> Thoughts or suggestions are greatly appreciated.
>
>
> Martha Ward MT(ASCP) QIHC
> Atrium Health Wake Forest Baptist
>
>
>
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Re: [Histonet] Bone decalcification

[hotmail via Histonet]

In my experience soaking the block in water works just as well as surface 
decal. If you need to you can take the bone back, by reverse processing. Run 
the block through the clean cycle back to water, then leave the bone in NBF for 
24-48hrs. The fixative will protect the tissue when you put it back into decal.

Gerard Spoelstra


Sent from my iPad

> On 1 Jul 2021, at 4:37 am, Patricia Latham via Histonet 
>  wrote:
> 
> To Histopeeps,
> Does anyone know if there is a method to decalcify bone once it is FFPE?
> Thank you,
> Pat L
> George Washington University
> Washington, DC
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Re: [Histonet] Bone decalcification

[Bryan Llewellyn via Histonet]

Use surface decalcification:

Surface the block, then remove from the microtome. Do not use the 
microtome for another block, so as to avoid adjusting the position of 
the block face to the knife.


Place the block face down in 4% nitric acid for 2 hours or longer.

Rinse the block and cool down.

Place back into the microtome. Use a fresh blade to ensure sharpness. 
You should be able to get 3 or 4 sections at 5 microns before hitting 
the calcium again.


Bryan Llewellyn


Patricia Latham via Histonet wrote:

To Histopeeps,
Does anyone know if there is a method to decalcify bone once it is FFPE?
Thank you,
Pat L
George Washington University
Washington, DC
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Re: [Histonet] Bone Marrow Assists

[Terri Braud via Histonet]

Our Histology Techs also assist with Bone Marrows in CT, though there is little 
to do.  We collect the aspirate in Heparin Tubes and the core in Bouins.  We 
bring it back to the lab and make our smears from one of the heparin tubes.  
The other tubes go for special studies.  I'd like to think our smears are 
better than many.  We pour the aspirate into a weigh boat and use a pipet to 
pick out the spicules for picture perfect smears. Much better results than the 
old method of making the smears at bedside.

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: Saturday, September 28, 2019 1:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 190, Issue 22
Today's Topics:

   1. Re: Histo techs assisting on BM Bx's (Webster, Thomas S.)
--
Message: 1
Date: Fri, 27 Sep 2019 17:13:29 +
From: "Webster, Thomas S." 
Subject: Re: [Histonet] Histo techs assisting on BM Bx's

Our histotechs assist bone marrows but it used to be Hematology's 
responsibility.  The bone marrows at my institution aren't very time consuming 
and are done in CT.


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Re: [Histonet] Bone PMMA sections

[Patsy Ruegg via Histonet]

Stefano, after cutting the sections, dry them on a heat plate, then they can be 
stored dried stacked next to each other.


Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
prueg...@hotmail.com



From: MANTERO Stefano RIC 
Sent: Thursday, February 21, 2019 7:08 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone PMMA sections

Good morning Histonetters!

I'm starting to cut out of bone samples included in PMMA.
I would like some suggestion how to storage the slides cut.

Many thanks in advance

Stefano

Dott. Stefano Mantero

Human Genome Laboratory  CNR-IRGB
c/o Humanitas Research Hospital

Via Rita Levi Montalcini (Ex Via Dainese)
20090 Pieve Emanuele (MI)

Ph. +39 02 8224 5164 (desk)
Ph. +39 02 8224 5177 (lab)
Fax +39 02 8224 5191



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Re: [Histonet] Bone saw

[Bob Richmond via Histonet]

>
> Terri L. Braud, HT(ASCP). Anatomic Pathology Supervisor, Holy Redeemer
> Hospital, Meadowbrook PA describes:
>
> >>We use an awesome little band saw made by IMEB, Inc.  It has a small
> foot print, 4 blade types and added accessories for a super lab bone
> cutting station, and best of all, very inexpensive.
> It can zip through the densest of bone, or the most delicate.  It can be
> set up as a water cooled station to reduce dust particulate, but we just
> have ours under a hood (It's that tiny!) and we use a standard blade.
> Our pathologists and PA LOVE it, and so do the techs, because we get such
> fabulously decaled thin, consistent sections.<<
>

Is this the item you're describing?
http://www.imebinc.com/necropsy-morgue/imeb-bone-band-saw.html

$1,600 each? No way is a pathologist going to be allowed one of these! With
a new gig, I usually go to a hardware store and buy a hacksaw, and leave it
behind at the job when I'm done.

Bob Richmond
Samurai Pathologist
Maryville TN
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Re: [Histonet] Bone saw

[Morken, Timothy via Histonet]

I agree, we use the IMEB bone saw as well. All human bones though...

-Original Message-
From: Terri Braud via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, October 27, 2016 10:34 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: Re: [Histonet] Bone saw

We use an awesome little band saw made by IMEB, Inc.  It has a small foot 
print, 4 blade types and added accessories for a super lab bone cutting 
station, and best of all, very inexpensive.
It can zip through the densest of bone, or the most delicate.  It can be set up 
as a water cooled station to reduce dust particulate, but we just have ours 
under a hood (It's that tiny!) and we use a standard blade.
Our pathologists and PA LOVE it, and so do the techs, because we get such 
fabulously decaled thin, consistent sections.
Hope this helps, 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

-Original Message-
Sent: Thursday, October 27, 2016 1:00 PM
To: histonet@lists.utsouthwestern.edu

Today's Topics:
   6. bone saw (Lauren Sweeney)
--
Message: 6
Date: Thu, 27 Oct 2016 16:55:06 +
From: Lauren Sweeney <lmari...@uga.edu>
Subject: [Histonet] bone saw

Hello histoworld,
Does anyone out there use a bone saw in their lab? We routinely have research 
cases with hundreds of femur head submissions from avian species. We currently 
use a bone saw made by Buehler from the 70's or 80's and it's a work horse, but 
the blade keeps cracking in the diamond tip from overuse during these surveys 
of hundreds of bones. I was wondering what kind of saws are out there that 
could be used for this purpose and if anyone has any experience with this? I am 
looking for something a little more durable, or if not, at least a little 
cheaper. Each blade costs about $350.
Thanks!




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Re: [Histonet] Bone saw

[Terri Braud via Histonet]

We use an awesome little band saw made by IMEB, Inc.  It has a small foot 
print, 4 blade types and added accessories for a super lab bone cutting 
station, and best of all, very inexpensive.
It can zip through the densest of bone, or the most delicate.  It can be set up 
as a water cooled station to reduce dust particulate, but we just have ours 
under a hood (It's that tiny!) and we use a standard blade.
Our pathologists and PA LOVE it, and so do the techs, because we get such 
fabulously decaled thin, consistent sections.
Hope this helps, 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

-Original Message-
Sent: Thursday, October 27, 2016 1:00 PM
To: histonet@lists.utsouthwestern.edu

Today's Topics:
   6. bone saw (Lauren Sweeney)
--
Message: 6
Date: Thu, 27 Oct 2016 16:55:06 +
From: Lauren Sweeney 
Subject: [Histonet] bone saw

Hello histoworld,
Does anyone out there use a bone saw in their lab? We routinely have research 
cases with hundreds of femur head submissions from avian species. We currently 
use a bone saw made by Buehler from the 70's or 80's and it's a work horse, but 
the blade keeps cracking in the diamond tip from overuse during these surveys 
of hundreds of bones. I was wondering what kind of saws are out there that 
could be used for this purpose and if anyone has any experience with this? I am 
looking for something a little more durable, or if not, at least a little 
cheaper. Each blade costs about $350.
Thanks!




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Re: [Histonet] bone marrow clots

[Boyd, Debbie M via Histonet]

We use the syringe used for the smears and the syringe used for the aspirate.  
We do not put it in an anti-coagulate.  We simply let the syringe specimen clot 
and place it in lens paper in a cassette and place in 10% formalin for 
processing.  If you are doing Flow Cytometry/Cytogenetic studies you can use 
1ml for these studies and any left in this syringe can also be used for the 
aspirate/clot.  One of our pathologist will purposely get 2mls in the aspirate 
syringe.  One ml for Flow and Cyto (green top sodium heparinized tube) and the 
other for the clot.

Debbie M. Boyd HT (ASCP) | Chief Histologist  | Southside Regional Medical 
Center | 200 Medical Park Blvd.  |  Petersburg, Va.  23805 | PH 804-765-5025 | 
FAX 804-765-6058


From: Noelle Linke via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Wednesday, March 23, 2016 7:27 PM
To: histonet@lists.utsouthwestern.edu
Subject: [EXTERNAL] [Histonet] bone marrow clots

Hi all,

Question: Does anyone produce beautiful, well fixed bone marrow clot blocks 
that has a procedure they would be willing to share?  Do you all use the 
specimen from the EDTA tube after prepping the smears?  Any help would be 
greatly appreciated!

Thank you,
Noëlle

Noëlle Linke, MS, HTL(ASCP) QIHC
Manager, Anatomic Pathology
Pacific Diagnostic Laboratories
nli...@sbch.org
Phone: (805) 324-9814
Fax: (805) 696-6433






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Re: [Histonet] Bone histology charges

[Shruti Shah via Histonet]

Hi All,

Can any one let me know general price for mouse bone decalcification, 
processing, microtome charges for per block or per slide.

it will great help.

Many thanks,


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RE: [Histonet] Bone Saw

[Jason McGough]

We use a Dremel tool. It works great!!



Jason McGough, HT(ASCP)

Operations Manager

Clinical Laboratory of the Black Hills

605-343-2267

jmcgo...@clinlab.com mailto:jmcgo...@clinlab.com 

www.clinlab.com http://www.clinlab.com 

 
 
-Original message-
 From:Mike Pence mpe...@grhs.net mailto:mpe...@grhs.net 
 Sent: Tuesday, February 17, 2015 1:56 PM
 To: histonet-boun...@lists.utsouthwestern.edu 
 mailto:histonet-boun...@lists.utsouthwestern.edu ; 
 histonet@lists.utsouthwestern.edu mailto:histonet@lists.utsouthwestern.edu 
 Subject: [Histonet] Bone Saw
 
 I am trying to see what everyone is using at your grossing station for bone 
 saw to cut femoral heads and toes for osteo. If you are using a Stryker saw 
 how are you holding the specimens to make good thin sections?
 ___
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RE: [Histonet] Bone Saw

[Mike Pence]

Those double bladed hacksaws work great for femoral heads, but are not good for 
toes! I also have one pathologist that wants to cut their own specimens and 
they do not want anything manual. They want one of those rr thingies 
that cuts bone.

-Original Message-
From: Rathborne, Toni [mailto:toni.rathbo...@rwjuh.edu] 
Sent: Tuesday, February 17, 2015 2:55 PM
To: 'Jason McGough'; Mike Pence; histonet-boun...@lists.utsouthwestern.edu; 
histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone Saw

We use a device from Mopec.

http://media3.mopec.com/media/pdf/AutopsyAccessories(Page76).pdf

It's manual, but works great.


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jason McGough
Sent: Tuesday, February 17, 2015 3:59 PM
To: Mike Pence; histonet-boun...@lists.utsouthwestern.edu; 
histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone Saw

We use a Dremel tool. It works great!!



Jason McGough, HT(ASCP)

Operations Manager

Clinical Laboratory of the Black Hills

605-343-2267

jmcgo...@clinlab.com mailto:jmcgo...@clinlab.com 

www.clinlab.com http://www.clinlab.com 

 
 
-Original message-
 From:Mike Pence mpe...@grhs.net mailto:mpe...@grhs.net 
 Sent: Tuesday, February 17, 2015 1:56 PM
 To: histonet-boun...@lists.utsouthwestern.edu 
 mailto:histonet-boun...@lists.utsouthwestern.edu ; 
 histonet@lists.utsouthwestern.edu mailto:histonet@lists.utsouthwestern.edu 
 Subject: [Histonet] Bone Saw
 
 I am trying to see what everyone is using at your grossing station for bone 
 saw to cut femoral heads and toes for osteo. If you are using a Stryker saw 
 how are you holding the specimens to make good thin sections?
 ___
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 Histonet@lists.utsouthwestern.edu mailto:Histonet@lists.utsouthwestern.edu 
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
 
 


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RE: [Histonet] Bone Saw

[Rathborne, Toni]

We use a device from Mopec.

http://media3.mopec.com/media/pdf/AutopsyAccessories(Page76).pdf

It's manual, but works great.


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jason McGough
Sent: Tuesday, February 17, 2015 3:59 PM
To: Mike Pence; histonet-boun...@lists.utsouthwestern.edu; 
histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone Saw

We use a Dremel tool. It works great!!



Jason McGough, HT(ASCP)

Operations Manager

Clinical Laboratory of the Black Hills

605-343-2267

jmcgo...@clinlab.com mailto:jmcgo...@clinlab.com 

www.clinlab.com http://www.clinlab.com 

 
 
-Original message-
 From:Mike Pence mpe...@grhs.net mailto:mpe...@grhs.net 
 Sent: Tuesday, February 17, 2015 1:56 PM
 To: histonet-boun...@lists.utsouthwestern.edu 
 mailto:histonet-boun...@lists.utsouthwestern.edu ; 
 histonet@lists.utsouthwestern.edu mailto:histonet@lists.utsouthwestern.edu 
 Subject: [Histonet] Bone Saw
 
 I am trying to see what everyone is using at your grossing station for bone 
 saw to cut femoral heads and toes for osteo. If you are using a Stryker saw 
 how are you holding the specimens to make good thin sections?
 ___
 Histonet mailing list
 Histonet@lists.utsouthwestern.edu mailto:Histonet@lists.utsouthwestern.edu 
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
 
 


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Re: [Histonet] Bone Saw

[Richard Yeo]

We use a small band saw from MAR-MED. cuts through a femoral head like a hot 
knife through soft butter. You let the blade do the job, don't force the bone. 
Just very little consistent pressure.

Rich Y

Sent from my iPhone

 On Feb 17, 2015, at 4:01 PM, Mike Pence mpe...@grhs.net wrote:
 
 Those double bladed hacksaws work great for femoral heads, but are not good 
 for toes! I also have one pathologist that wants to cut their own specimens 
 and they do not want anything manual. They want one of those rr 
 thingies that cuts bone.
 
 -Original Message-
 From: Rathborne, Toni [mailto:toni.rathbo...@rwjuh.edu] 
 Sent: Tuesday, February 17, 2015 2:55 PM
 To: 'Jason McGough'; Mike Pence; histonet-boun...@lists.utsouthwestern.edu; 
 histonet@lists.utsouthwestern.edu
 Subject: RE: [Histonet] Bone Saw
 
 We use a device from Mopec.
 
 http://media3.mopec.com/media/pdf/AutopsyAccessories(Page76).pdf
 
 It's manual, but works great.
 
 
 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu 
 [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jason McGough
 Sent: Tuesday, February 17, 2015 3:59 PM
 To: Mike Pence; histonet-boun...@lists.utsouthwestern.edu; 
 histonet@lists.utsouthwestern.edu
 Subject: RE: [Histonet] Bone Saw
 
 We use a Dremel tool. It works great!!
 
 
 
 Jason McGough, HT(ASCP)
 
 Operations Manager
 
 Clinical Laboratory of the Black Hills
 
 605-343-2267
 
 jmcgo...@clinlab.com mailto:jmcgo...@clinlab.com 
 
 www.clinlab.com http://www.clinlab.com 
 
 
 
 -Original message-
 From:Mike Pence mpe...@grhs.net mailto:mpe...@grhs.net 
 Sent: Tuesday, February 17, 2015 1:56 PM
 To: histonet-boun...@lists.utsouthwestern.edu 
 mailto:histonet-boun...@lists.utsouthwestern.edu ; 
 histonet@lists.utsouthwestern.edu mailto:histonet@lists.utsouthwestern.edu 
 Subject: [Histonet] Bone Saw
 
 I am trying to see what everyone is using at your grossing station for bone 
 saw to cut femoral heads and toes for osteo. If you are using a Stryker saw 
 how are you holding the specimens to make good thin sections?
 ___
 Histonet mailing list
 Histonet@lists.utsouthwestern.edu mailto:Histonet@lists.utsouthwestern.edu 
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet
 
 
 ___
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 Histonet@lists.utsouthwestern.edu
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 ___
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 Histonet@lists.utsouthwestern.edu
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet


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RE: [Histonet] Bone Marrow processing using Immunocal

[Patsy Ruegg]

I use a platform shaker and slosh the sample around while it is in decal 
solution at RT, just make sure it is well fixed before you decal, Immunocal is 
5% formic acid decal solution it is not a fixative and it is a bit slower than 
some of the rapid decal reagents out there but is much better for IHC.  When I 
was in HemePath we would fix for about 6 hrs before decaling on the shaker for 
2-4hrs depending on the size (thickness) of the bone bx.

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
prueg...@hotmail.com
pru...@ihctech.net


 Date: Mon, 17 Nov 2014 14:05:28 -0500
 From: carolyn.barn...@va.gov
 To: Histonet@lists.utsouthwestern.edu
 CC: 
 Subject: [Histonet] Bone Marrow processing using Immunocal
 
 I am looking for a Bone Marrow procedure that uses Immunocal as the
 decalcifier. I am particularly interested in how long they fix in
 Immunocal and if heat is used in any way . Our lab  just started using
 this product because we are under the impression that it gave better ISH
 results.
 
 Thank-you all so much for any assistance. I really appreciate everyone's
 help.
 
  
 
 Carolyn K. Barnes
 
 Histology Supervisor
 
 McGuire VA Medical Center
 
 Richmond, VA 23249
 
 Ph: 804-675-5000, x2158
 
  
 
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RE: [Histonet] bone paraffin embedding

[Wineman, Terra]

Yes 1:20 dilution

Terra Wineman, HTL (ASCP)CM
Research Biologist, Nutritional Physiology
636-926-7476 phone
terra.wine...@novusint.com


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Nicole Cosenza
Sent: Monday, November 03, 2014 9:28 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] bone paraffin embedding

Histonetters:

Our lab needs to paraffin embed and cut bone.  Is there a special process for 
fixation of bone, or can it be harvested and dropped right into NBF?

--
Nicole Cosenza
Research Technician
Institute for Plastic Surgery
SIU School of Medicine
Springfield, Il
217.545.3862


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RE: [Histonet] bone paraffin embedding

[Jack Ratliff]

Nicole,
You can very easily fix the bone in 10% NBF and then go into your 
decalcification process. Remember that fixation rate of bone is generally 
around 1mm per 24 hours (in all directions) and that it is good to have a 
minimum of 20:1 ratio of solutions to specimen size for each step. I would also 
recommend either 5% or 10% formic acid for decalcification.
Can you tell us more specifics about the bone and what you wish to accomplish 
histologically? This information would be 
I might also suggest contacting Sarah Mack (copied to this message) from the 
University of Rochester. She is the new Hard Tissue Committee Chairperson for 
the National Society for Histotechnology and an expert in decalcified bone. I 
might also add that Sarah and Kim Simmons, NSH Education Development Manager, 
are working on educational opportunities and resource materials for those 
working with bone, biomaterials and medical device implants. If you are not a 
member of the NSH then you might want to consider becoming a member 
www.nsh.org/content/benefits-membership so that you can be kept up to date with 
what's coming soon from the Hard Tissue Committee.
Best Regards,
Jack

 Date: Mon, 3 Nov 2014 09:28:01 -0600
 From: ncose...@siumed.edu
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] bone paraffin embedding
 
 Histonetters:
 
 Our lab needs to paraffin embed and cut bone.  Is there a special 
 process for fixation of bone, or can it be harvested and dropped right 
 into NBF?
 
 -- 
 Nicole Cosenza
 Research Technician
 Institute for Plastic Surgery
 SIU School of Medicine
 Springfield, Il
 217.545.3862
 
 
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RE: [Histonet] bone paraffin embedding

[Jack Ratliff]

Sorry Nicole I forgot to finish a thought before I hit send. What I was going 
to say was any additional information you could provide regarding the specimen 
and what you wanted to see at the microscope can sometime yield additional 
information regarding specific processing protocols, staining, tips and tricks, 
etc. 
Best,
Jack

From: ratliffj...@hotmail.com
To: ncose...@siumed.edu; histonet@lists.utsouthwestern.edu
CC: sarah_m...@urmc.rochester.edu; k...@nsh.org; ratliffj...@gmail.com
Subject: RE: [Histonet] bone paraffin embedding
Date: Mon, 3 Nov 2014 12:07:06 -0500




Nicole,
You can very easily fix the bone in 10% NBF and then go into your 
decalcification process. Remember that fixation rate of bone is generally 
around 1mm per 24 hours (in all directions) and that it is good to have a 
minimum of 20:1 ratio of solutions to specimen size for each step. I would also 
recommend either 5% or 10% formic acid for decalcification.
Can you tell us more specifics about the bone and what you wish to accomplish 
histologically? This information would be 
I might also suggest contacting Sarah Mack (copied to this message) from the 
University of Rochester. She is the new Hard Tissue Committee Chairperson for 
the National Society for Histotechnology and an expert in decalcified bone. I 
might also add that Sarah and Kim Simmons, NSH Education Development Manager, 
are working on educational opportunities and resource materials for those 
working with bone, biomaterials and medical device implants. If you are not a 
member of the NSH then you might want to consider becoming a member 
www.nsh.org/content/benefits-membership so that you can be kept up to date with 
what's coming soon from the Hard Tissue Committee.
Best Regards,
Jack

 Date: Mon, 3 Nov 2014 09:28:01 -0600
 From: ncose...@siumed.edu
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] bone paraffin embedding
 
 Histonetters:
 
 Our lab needs to paraffin embed and cut bone.  Is there a special 
 process for fixation of bone, or can it be harvested and dropped right 
 into NBF?
 
 -- 
 Nicole Cosenza
 Research Technician
 Institute for Plastic Surgery
 SIU School of Medicine
 Springfield, Il
 217.545.3862
 
 
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RE: [Histonet] bone saw for cutting slabs

[Barbara Tibbs]

Most hospitals that I've worked at that needed to cut bone used a Stryker bone 
saw.  The pathologists never mentioned damaged cartilage.

Barbara S. Tibbs
Histology Supervisor
Accurate Diagnostic Labs
South Plainfield, NJ
barbara.ti...@accuratediagnosticlabs.com
732-839-3374
Cell: 610-809-6508



From: histonet-boun...@lists.utsouthwestern.edu 
histonet-boun...@lists.utsouthwestern.edu on behalf of M.O. 
modz9...@gmail.com
Sent: Tuesday, August 19, 2014 3:45 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] bone saw for cutting slabs

Histoland! Happy Tuesday!

 I just wanted to get your feedback on cutting slabs from human femora
for histopathological analysis.

 At them moment we are just using a hack saw to cut 7mm slabs from
femora.  We notice some marks on the cartilage from sawing, so when we cut
the tissue down after decalcification for histological preparation, we cut
the thickness down to 4mm and remove the damaged tissue.

Would using some sort of bone saw damage the tissue even more or would it
be comparable to using a hack saw?  Is there a saw that you recommend that
is precise and easy to handle that doesn't damage tissue greatly?

Thank you so much for your help!

Sincerely,
Merissa
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Re: [Histonet] bone saw for cutting slabs

[Sean McBride]

Hi Merissa,

Exakt technologies makes a wonderful saw designed specifically for exactly what 
you are trying to do with a hack saw.  It is a bit pricy though.  Contact Linda 
Durbin at 405-848-5800 for a quote.

Alternatively, you can use a wet saw designed for cutting stained glass.  Check 
out the one by Gryphon:  Not too expensive  cuts bone well if you take your 
time.  Other vendors like Mar-Med make alternative blades for this saw as well.


Good luck,


~Sean McBride


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

412-268-8275 (o)
571-989-BONE (m)
412-268-8275 (fax)
smcbr...@andrew.cmu.edu 







On Aug 19, 2014, at 12:45 PM, M.O. wrote:

 Histoland! Happy Tuesday!
 
 I just wanted to get your feedback on cutting slabs from human femora
 for histopathological analysis.
 
 At them moment we are just using a hack saw to cut 7mm slabs from
 femora.  We notice some marks on the cartilage from sawing, so when we cut
 the tissue down after decalcification for histological preparation, we cut
 the thickness down to 4mm and remove the damaged tissue.
 
 Would using some sort of bone saw damage the tissue even more or would it
 be comparable to using a hack saw?  Is there a saw that you recommend that
 is precise and easy to handle that doesn't damage tissue greatly?
 
 Thank you so much for your help!
 
 Sincerely,
 Merissa
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Re: [Histonet] Bone Histology Protocol

[Jack Ratliff]

Trevor,

May I ask first if there is a particular reason why you are wanting to 
decalcify? Have you ever considered resin/plastic embedding of non-decalcified 
bone? Also, what type/species of bone are you wanting to process?

My name is Jack Ratliff and I Chair the Hard Tissue Committee for the National 
Society for Histotechnology and as a professional histology organization and 
committee focusing on bone, biomaterials and medical device implants, we offer 
educational solutions to help those like yourself that are in search of 
information. This is accomplished through the presentation of workshops at the 
state, regional and national levels or even by providing free reference 
materials like processing manuals, books for purchase at non-member or member 
discounts, and free access to the archives of The Journal of Histotechnology to 
society members. If any of this interests you please check out the NSH website 
(www.nsh.org) where you can navigate around to view all of what the society has 
to offer, become a member and even connect with the Hard Tissue Committee.

One last thing I can tell you now is that there will be several bone workshops 
available at the NSH Symposium/Convention this August in Austin, TX. I will 
also be one of the speakers at this meeting giving a workshop on resin 
embedding techniques in support of bone, biomaterials and medical device 
implants.

Best Regards,

Jack



 On Feb 28, 2014, at 10:40 PM, Wait, Trevor Jordan 
 wa...@livemail.uthscsa.edu wrote:
 
 Hello colleagues! I'm currently trying to construct a complete Bone 
 Processing Protocol that includes fixation (10%NBF), decalcification (EDTA), 
 Dehydration, Clearing of the decalcificant (Xylene), infiltration, and 
 Embedding in Paraffin. I would like to look at some procedures just to get a 
 good backbone of what a complete procedure is displayed like and I was hoping 
 somebody could give me a website or a source where I could see some. I'm kind 
 of new to bone histological processing so any procedures that are reliable 
 will help!
 
 
 Trevor Jordan Wait
 University of Texas Health Science Center, San Antonio
 Class of 2017 MD Candidate
 Abilene Christian University Class of 2013 Graduate
 B.S.  Biochemistry
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RE: [Histonet] Bone Histology Protocol

[Wait, Trevor Jordan]

Thanks so much for the reference and I will visit that site and use that site 
most definitely as a resource.  However, the reason I am wanting to decalcify 
is that my researcher (my employer) has instructed me that that is the way we 
will be doing it lol.  I have read about resin/plastic embedding and that 
method does seem pretty efficient to use so I'm not sure why he opted to not go 
the plastic resin route.  I do know that we have a limited budget to work with 
so maybe this is the cheapest route we can afford, along with a microtome that 
might not be sufficient to cut through hard bone and plastic.  As far as the 
species of bone that we are processing, that too is something I'm not exactly 
sure about, but is something I should probably find out because from my 
readings, this is an important aspect. 

Trevor Jordan Wait
University of Texas Health Science Center, San Antonio
Class of 2017 MD Candidate
Abilene Christian University Class of 2013 Graduate
B.S.  Biochemistry


From: Jack Ratliff ratliffj...@hotmail.com
Sent: Saturday, March 1, 2014 3:16 AM
To: Wait, Trevor Jordan
Cc: histonet@lists.utsouthwestern.edu; Jack Ratliff
Subject: Re: [Histonet] Bone Histology Protocol

Trevor,

May I ask first if there is a particular reason why you are wanting to 
decalcify? Have you ever considered resin/plastic embedding of non-decalcified 
bone? Also, what type/species of bone are you wanting to process?

My name is Jack Ratliff and I Chair the Hard Tissue Committee for the National 
Society for Histotechnology and as a professional histology organization and 
committee focusing on bone, biomaterials and medical device implants, we offer 
educational solutions to help those like yourself that are in search of 
information. This is accomplished through the presentation of workshops at the 
state, regional and national levels or even by providing free reference 
materials like processing manuals, books for purchase at non-member or member 
discounts, and free access to the archives of The Journal of Histotechnology to 
society members. If any of this interests you please check out the NSH website 
(www.nsh.org) where you can navigate around to view all of what the society has 
to offer, become a member and even connect with the Hard Tissue Committee.

One last thing I can tell you now is that there will be several bone workshops 
available at the NSH Symposium/Convention this August in Austin, TX. I will 
also be one of the speakers at this meeting giving a workshop on resin 
embedding techniques in support of bone, biomaterials and medical device 
implants.

Best Regards,

Jack



 On Feb 28, 2014, at 10:40 PM, Wait, Trevor Jordan 
 wa...@livemail.uthscsa.edu wrote:

 Hello colleagues! I'm currently trying to construct a complete Bone 
 Processing Protocol that includes fixation (10%NBF), decalcification (EDTA), 
 Dehydration, Clearing of the decalcificant (Xylene), infiltration, and 
 Embedding in Paraffin. I would like to look at some procedures just to get a 
 good backbone of what a complete procedure is displayed like and I was hoping 
 somebody could give me a website or a source where I could see some. I'm kind 
 of new to bone histological processing so any procedures that are reliable 
 will help!


 Trevor Jordan Wait
 University of Texas Health Science Center, San Antonio
 Class of 2017 MD Candidate
 Abilene Christian University Class of 2013 Graduate
 B.S.  Biochemistry
 ___
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RE: [Histonet] Bone Histology Protocol

[Wait, Trevor Jordan]

I'm terribly sorry, when you asked about species...my inclination was that you 
were curious which type of bone we were using (femur, humerus, tibia, etc. and 
cortical/cancellous) but we will be using human bone for this research project 
that has been donated from various orthopedic surgeries. 

Trevor Jordan Wait
University of Texas Health Science Center, San Antonio
Class of 2017 MD Candidate
Abilene Christian University Class of 2013 Graduate
B.S.  Biochemistry


From: Jack Ratliff ratliffj...@hotmail.com
Sent: Saturday, March 1, 2014 3:16 AM
To: Wait, Trevor Jordan
Cc: histonet@lists.utsouthwestern.edu; Jack Ratliff
Subject: Re: [Histonet] Bone Histology Protocol

Trevor,

May I ask first if there is a particular reason why you are wanting to 
decalcify? Have you ever considered resin/plastic embedding of non-decalcified 
bone? Also, what type/species of bone are you wanting to process?

My name is Jack Ratliff and I Chair the Hard Tissue Committee for the National 
Society for Histotechnology and as a professional histology organization and 
committee focusing on bone, biomaterials and medical device implants, we offer 
educational solutions to help those like yourself that are in search of 
information. This is accomplished through the presentation of workshops at the 
state, regional and national levels or even by providing free reference 
materials like processing manuals, books for purchase at non-member or member 
discounts, and free access to the archives of The Journal of Histotechnology to 
society members. If any of this interests you please check out the NSH website 
(www.nsh.org) where you can navigate around to view all of what the society has 
to offer, become a member and even connect with the Hard Tissue Committee.

One last thing I can tell you now is that there will be several bone workshops 
available at the NSH Symposium/Convention this August in Austin, TX. I will 
also be one of the speakers at this meeting giving a workshop on resin 
embedding techniques in support of bone, biomaterials and medical device 
implants.

Best Regards,

Jack



 On Feb 28, 2014, at 10:40 PM, Wait, Trevor Jordan 
 wa...@livemail.uthscsa.edu wrote:

 Hello colleagues! I'm currently trying to construct a complete Bone 
 Processing Protocol that includes fixation (10%NBF), decalcification (EDTA), 
 Dehydration, Clearing of the decalcificant (Xylene), infiltration, and 
 Embedding in Paraffin. I would like to look at some procedures just to get a 
 good backbone of what a complete procedure is displayed like and I was hoping 
 somebody could give me a website or a source where I could see some. I'm kind 
 of new to bone histological processing so any procedures that are reliable 
 will help!


 Trevor Jordan Wait
 University of Texas Health Science Center, San Antonio
 Class of 2017 MD Candidate
 Abilene Christian University Class of 2013 Graduate
 B.S.  Biochemistry
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Re: [Histonet] Bone samples long-term storage in 10% formalin or 4% paraformaldehyde

[Sean McBride]

Dear Orla,

Post fixation, we have stored our bone specimens in 1x PBS while having them 
sent out for MicroCT analysis.  We have also stored them in 1x PBS at 4°C post 
fixation when necessary until further processing without having adverse affects 
on our staining.  However, we chose conventional staining over IHC for our 
results.


Best regards,


~Sean McBride


Scientific Specialist
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)
412-268-8275 (fax)
smcbr...@andrew.cmu.edu 






On Dec 9, 2013, at 8:44 AM, Orla M Gallagher wrote:

 Thanks to everyone for your comments.
 
 I may not have been clear in my question - our researchers don't wish to
 decalcify these formalin-fixed bones yet, but rather to store them for more
 than a couple of weeks, in case they need to carry out MicroCT followed by
 histology later. I'm aware that the formalin or paraformaldehyde will
 degrade over time, but I just wondered if anyone has a protocol for storage
 without decalcification? I guess transfer to 70% ethanol is an option but
 this is also not ideal for longterm storage, and would need to be removed
 before decal in EDTA.
 
 All the best,
 Orla
 
 
 On 6 December 2013 16:12, Wineman, Terra terra.wine...@novusint.com wrote:
 
 I would suggest a different protocol if the tissue will not be processed
 for a while.  I would say a week in 10%NBF and then transfer the bones to
 an EDTA decal solution.  The bones will decal slowly without the affects of
 the formic acid.  I am in research and this is what we do with our bones.
 
 Terra Wineman, HTL (ASCP)CM
 Research Biologist
 636-926-7476 phone
 terra.wine...@novusint.com
 
 
 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu [mailto:
 histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pru...@ihctech.net
 Sent: Thursday, December 05, 2013 2:50 PM
 To: gu.l...@gmx.at; 'Orla M Gallagher'
 Cc: histonet@lists.utsouthwestern.edu
 Subject: RE: AW: [Histonet] Bone samples long-term storage in 10% formalin
 or 4% paraformaldehyde
 
 i would think u are correct in advising formic acid decal and then
 processing into paraffin for the best protection of the trap enzyme,
 immunoreactivity, etc.  A couple of weeks in formalin should be fine.
 Paraformaldehyde show be the same as formalin.  I do know a way to restore
 the enzyme activity for TRAP that may have been lost so if u need that let
 me know.
 
 - Original Message - Subject: AW: [Histonet] Bone samples
 long-term storage in 10% formalin or 4% paraformaldehyde
 From: Gudrun Lang gu.l...@gmx.at
 Date: 12/5/13 11:42 am
 To: 'Orla M Gallagher' o.m.gallag...@sheffield.ac.uk
 Cc: histonet@lists.utsouthwestern.edu
 
 Paraformaldehyd is formaldehyd in solid form. Formalin is the aequous
 solution of formaldehyd.
 So the main characteristics are the same.
 
 Gudrun Lang
 
 -Urspruuml;ngliche Nachricht-
 Von: histonet-boun...@lists.utsouthwestern.edu
 [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Orla M
 Gallagher
 Gesendet: Donnerstag, 05. Dezember 2013 19:31
 An: histonet@lists.utsouthwestern.edu
 Betreff: [Histonet] Bone samples long-term storage in 10% formalin or 4%
 paraformaldehyde
 
 Dear Histonetters,
 
 What is your opinion on storing bone samples long-term (more than a
 couple  of weeks) in 10% formalin? As I was taught, best practice has
 always been to  fix only as long as necessary, depending on the size of the
 sample, then  decalcify and process to wax, and I always stress this to
 everyone I advise.
 
 However, research colleagues sometimes wish to do histology on bone
 samples  that have been stored for months ..or even years! As the formalin
 pH becomes  more acidic, there is formalin pigment and the immunoreactivity
 and TRAP  enzyme activity is diminished or destroyed during long fixation,
 is there  any way of minimising this e.g. has anyone tried regularly
 replacing the old  formalin with fresh buffered formalin, or storing
 formalin-fixed bones in  any other medium? I'm also interested in how best
 to fix in 4%  paraformaldehyde and whether the problems are the same with
 long-term  storage.
 
 Thanks for your comments.
 
 All the best,
 Orla
 
 --
 **
 Ms. Orla Gallagher
 Bone Analysis Laboratory
 Mellanby Centre for Bone Research
 Department of Human Metabolism
 D Floor Medical School
 University of Sheffield
 Beech Hill Road
 Sheffield
 S10 2RX
 UK
 
 Website: http://mellanbycentre.dept.shef.ac.uk
 
 Tel: 0044114-2713337 (office)
 0044114-2713174 (lab)
 E-Mail: o.m.gallag...@sheffield.ac.uk
 
 
 *STOP*: Do you really need to print this e-mail?
 
 *BE GREEN:* Keep it on the screen.
 
 
 *Times Higher Education University of the Year*
 
 
 
 Data protection and confidentiality:
 The information contained in this message or any appended documents may
 be  privileged and confidential and is intended for 

RE: [Histonet] Bone/cartilage/epithelial tissue stain

[Hobbs, Carl]

A HE will allow you to identify those three tissue types, in  normal tissues, 
of most organisms ( certainly 
in avian embryos, imho), IF you  know your Histology.

There is no stain/demonstration technique that will substitute for this 
knowledge, imho.
Sure, a special stain will help to confirm the tissue type to a high degree.
I usually start with staining the section with Alcian blue pH3, then place in 
Hx ( I use Gill's 2sure, they state you must use Iron Hx...not necessary, 
for me) for 10 mins.
Then place in Van Gieson's solution until what I know is collagen type I is red.

I hope this adds to previous excellent posts.
Best regards,

Carl

Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson CARD
Guys Campus, London Bridge 
Kings College London
London
SE1 1UL
 
020 7848 6813

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Re: [Histonet] Bone/cartilage/epithelial tissue stain

[E. Wayne Johnson 朱稳森]

At one time I did a lot of work on cartilage and growth plate
and used Toluidine Blue and Fast Green.  T-blue (buffered appropriately) 
stains the
proteoglycan a lovely metachromatic blue and the bone and most 
everything else
is green.  The nuclei of cells are also green since we used no nuclear 
stain, but

that was not a problem for our work.

The cartilage is in stark contrast to the bone because of the high 
carbohydrate

(glycosaminoglycan/proteoglycan) content of cartilage.

On 3:59, Rui TAHARA wrote:

Hi,
Would anyone suggest me what staining is
best to color differentiate between cartilage and bone and epithelial tissues
in avian embryos?

I have been trying Mallory Trichrome for
embryos but recently I was suggested that Mallory Trichrome stains cartilage 
differently
in embryos compared to adult samples since Aniline blue stains fiber that may
not develop in early embryos. There is some protocol that modified the Mallory 
Trichrome
that could be applied to embryos. However, the resulting colors of all tissues
look all purple-ish and difficult to tell the cartilage from the weak blue
stain from aniline blue.

Currently I am thinking to try out Alcian
blue/Hematoxylin and Eosin stain (Ehrlich’s hematoxylin). The purpose of the
staining is to look at interaction between ossification and epithelial 
development
so I think alcian blue for staining cartilage works but I am wondering if there
is any other staining combination with alcian blue exist for visualizing bone
and epithelial tissue (e,g. alcian blue/alizarine red with other staining?).
   



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RE: [Histonet] Bone decalcifying/processing problem

[Tony Henwood (SCHN)]

Orla,

You seem to have introduced many poor histologic processing practices into your 
technique.

Firstly ???4% paraformaldehyde. How did you make this up? The nomenclature is 
incorrect (see Histonet archives and Manoonkitiwongsa PS, Schultz RL. (2002) 
Proper nomenclature of formaldehyde and paraformaldehyde fixatives for 
Histochemistry Histochem J 34: 365-367).

Secondly, why fix and decal at 4oC. Low temperatures slow down the rate of 
diffusion and therefore fixation and can cause the formation of ice crystal 
artefact, which seems like what you have here.

Also if you are going to decalcify tissues then I would recommend doubling your 
fixation time. Remember one of the big advantages of aldehyde fixation is that 
it cross-links and thereby protects cell and tissue constituents from 
processing.

Try doing it all at room temperature, I bet that the artefacts will disappear.

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-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Orla M Gallagher
Sent: Saturday, 13 July 2013 12:52 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone decalcifying/processing problem

Dear Histonetters,

Has anyone ever seen large holes/vacuoles in the bone marrow of mouse long 
bones after decalcification and processing to wax? We've had this problem with 
a study which was fixed in 4% paraformaldehyde and decalcified at 4 degrees C 
over a 14 day period according to a routinely-used protocol involving changes 
of 15% EDTA/0.5% PFA in PBS pH 8.0, rinsed in PBS to remove EDTA before 
processing to paraffin wax. The marrow is filled with large holes, almost like 
balloons. I'm happy to send images to anyone who might be able to help!

Best wishes,
Orla

--
**
Ms. Orla Gallagher
Bone Analysis Laboratory
Mellanby Centre for Bone Research
Department of Human Metabolism
D Floor Medical School
University of Sheffield
Beech Hill Road
Sheffield
S10 2RX
UK

Website: http://mellanbycentre.dept.shef.ac.uk

Tel: 0044114-2713337 (office)
  0044114-2713174 (lab)
E-Mail:o.m.gallag...@sheffield.ac.uk


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RE: [Histonet] Bone decalcifying/processing problem

[Elizabeth Chlipala]

Orla

I know this might sound silly but normal mouse bone marrow contains both fat 
and hemoatopoietic marrow,  the areas that are fat will appear like round 
voids.  Is that what you are seeing?

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Orla M Gallagher 
[o.m.gallag...@sheffield.ac.uk]
Sent: Friday, July 12, 2013 8:52 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone decalcifying/processing problem

Dear Histonetters,

Has anyone ever seen large holes/vacuoles in the bone marrow of mouse
long bones after decalcification and processing to wax? We've had this
problem with a study which was fixed in 4% paraformaldehyde and
decalcified at 4 degrees C over a 14 day period according to a
routinely-used protocol involving changes of 15% EDTA/0.5% PFA in PBS
pH 8.0, rinsed in PBS to remove EDTA before processing to paraffin
wax. The marrow is filled with large holes, almost like balloons. I'm
happy to send images to anyone who might be able to help!

Best wishes,
Orla

--
**
Ms. Orla Gallagher
Bone Analysis Laboratory
Mellanby Centre for Bone Research
Department of Human Metabolism
D Floor Medical School
University of Sheffield
Beech Hill Road
Sheffield
S10 2RX
UK

Website: http://mellanbycentre.dept.shef.ac.uk

Tel: 0044114-2713337 (office)
  0044114-2713174 (lab)
E-Mail:o.m.gallag...@sheffield.ac.uk


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The information contained in this message or any appended documents
may be privileged and confidential and is intended for the exclusive
use of the addressee(s). If you are not the addressee, any disclosure,
reproduction, distributions, other dissemination or use of this
message is strictly prohibited and may be unlawful. If you receive
this correspondence in error please contact the sender immediately and
permanently delete/destroy what you have received.

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RE: [Histonet] bone marrow aspirations

[Cathy]

We are using a similar method at our site.  The aspirate is placed into EDTA
and then into formalin to fix.  We pour the aspirate through a biopsy bag,
open the bag and scrape the spicules together, transfer them to another
biopsy bag and process as usual.  I have received several compliments on the
quality of the slides.

Cathy
Kelowna, B.C.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Shea's
Sent: Saturday, June 1, 2013 4:26 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] bone marrow aspirations

I am also interested in how others are processing bone marrow aspirates.
Currently, we let the aspirate clot on a watch glass for about 20 minutes,
gentle slide it onto filter paper, roll it around to get rid of the excess
fluid, place the clot between sponges, and process as usual.
However, I am interested in knowing if any one out there is using a
technique that we use to use at another hospital 33 years ago.
 If I remember correctly, the pathologist or oncologist would collect the
aspirate using a heparin syringe. Some how, the aspirate smears for
Hematology were made on 22x22 cover slips instead of slides. Then the EDTA
tube was then given to Histology. I can't remember if we added formalin or
B5 fixative to it at that time, but it was filtered into an obex tea bag
type filter paper (The blood never clotted and ran through the filter paper,
leaving only bone marrow spicules in the paper).  We would fold the paper,
place it in the cassette, and process as usual. The pathologist preferred
this method because he wouldn't have to look at levels of clot to find
spicules.  Is there anyone out there using this method? 
Jan
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RE: [Histonet] Bone Marrow Issues

[Shirley A. Powell]

I had this happen when I was doing IHC and changed to regular slides and used 
StayOn from Leica. Had more success with keeping the tissue on the slides.  I 
now do autopsy slides and do the same.

Shirley

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jordan Phillips
Sent: Tuesday, May 28, 2013 7:56 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone Marrow Issues

HI everyone.  We are currently having problems with all of our bone marrows, 
mainly clots, washing off the slides.  We fix our bone marrows in Zenkers.  We 
use positive charged slides, put the slides in the oven for a minimum of 2 
hours and hand depar with no agitation.  When we get to the water, the sections 
just float off the slides, leaving just a rim of the tissue.   Any tips or 
suggestions would be greatly appreciated! 
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RE: [Histonet] Bone Marrow Issues

[C.D.G.]

Shiley: positive charged slides do not mantain certain tissues on them well. 
Blood is one of them,
and it is advisable to mount your bloody bone marrows on slides treated with 
vinyl glue, like Elmer's or
the one you prefer.
My regards,
Carlos.

  *** REPLY SEPARATOR  ***

On 29/05/2013 at 11:31 a.m. Shirley A. Powell wrote:

I had this happen when I was doing IHC and changed to regular slides and
used StayOn from Leica. Had more success with keeping the tissue on the
slides.  I now do autopsy slides and do the same.

Shirley

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jordan
Phillips
Sent: Tuesday, May 28, 2013 7:56 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone Marrow Issues

HI everyone.  We are currently having problems with all of our bone
marrows, mainly clots, washing off the slides.  We fix our bone marrows in
Zenkers.  We use positive charged slides, put the slides in the oven for a
minimum of 2 hours and hand depar with no agitation.  When we get to the
water, the sections just float off the slides, leaving just a rim of the
tissue.   Any tips or suggestions would be greatly appreciated!
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Re: [Histonet] Bone processing help

[Jack Ratliff]

Dr. De la Vega,

My name is Jack Ratliff and I Chair the Hard Tissue Committee for the
National Society for Histotechnology. I am not sure if anyone has provided
you with a response to your message, but maybe I can be of assistance. Just
so you know, the NSH prides itself on being an educational resource for
those working in the field of Histology. The Hard Tissue Committee
represents the educational arm of the society for those working with hard
tissues like bone, biomaterials and medical device implants and especially
those type of histological specimens embedded into resins or plastics!


I would like to first direct your attention to the NSH website at
www.nsh.org. If you go to the following link:
http://www.nsh.org/content/benefits-membership, you will find information
concerning membership benefits and how to join so that you can take
advantage of what the society can do for you and your laboratory! Please
visit this site first and then feel free to get back to me with any
questions.


As for your current issues, I can definitely help you with all that you are
experiencing. At the moment I am currently away from the US and in Germany
until the 26th of March working with a group (Rowiak GmbH - www.rowiak.de)
that has developed a non-contact laser microtome (Tissue
Surgeonhttp://www.rowiak.de/index.php?id=19)
that can do what you are doing now and produce stained slides @ 10-15
microns in 30 minutes from a resin/plastic embedded block! If you can give
me a few more hours, I will personally respond to your message privately.
In the meantime, please visit the NSH website (www.nsh.org), consider its
membership and also look to join the Hard Tissue
Committeehttp://www.nsh.org/committee-detail/1044 so
that I can provide you with meeting updates.


With that said and seeing that you are in Massachusetts, on May the 4th,
2013, Polysciences, Inc. is hosting a full day educational event focusing
on the *Histological Applications  Techniques for Bone, Biomaterials and
Medical Device Implants*. I will also be a speaker at this event. Those
that attend can expect to further their knowledge, understanding and
training of specialized histology techniques associated with bone,
biomaterials and medical device implant specimen types and attendees will
learn of the applicational relevance of the techniques used in the
evaluation safety and efficacy of therapeutic treatments. Registration is
currently open with an *Early Bird registration set to end this Friday,
March 22nd*. The National Society for Histotechnology (NSH) will be
providing 6 CEUs and Polysciences, Inc. will be providing a discount to any
current NSH member as outlined below:


*WORKSHOP FEES*
*
*
*Before March 22, 2013:  $149.00 for NSH Members / $199.00 for Non-Members*
*
*
*After March 22, 2013:  $179.00 for NSH Members / $229.00 for Non-Members*


For the complete details of this full day *Histological Applications 
Techniques for Bone, Biomaterials and Medical Device Implants *event and to
register online, please visit the following link: *
http://www.polysciences.com/Interactive-Histology-Forum-About/185/* and
sign up today to participate in the discussion of these specialized
histology specimen applications and techniques that are rarely shared or
even discussed on Histonet! You will not want to miss out on the
information presented by 4 expert speakers in the field, all course
materials, meals and a complete program book also containing technique
specific protocols that you can repeat back in your lab!


Best Regards,

Jack


Jack L. Ratliff
Owner/Histologist, Ratliff Histology Consultants, LLC
Chairman, Hard Tissue Committee - National Society for Histotechnology

389 Nichol Mill Lane
Franklin, TN 37067
(317) 281-1975 (c)
(615) 236-4901 (o)
(615) 236-4962 (f)
jratl...@ratliffhistology.com




On Sun, Mar 17, 2013 at 8:09 PM, Jack Ratliff ratliffj...@hotmail.comwrote:




 Begin forwarded message:

 *From:* De La Vega Amador, Rodolfo Enrique rdelavegaama...@partners.org
 
 *Date:* March 15, 2013, 9:41:23 PM GMT+01:00
 *To:* histonet@lists.utsouthwestern.edu 
 histonet@lists.utsouthwestern.edu
 *Subject:* *[Histonet] Bone processing help*

 Hi everybody,

 I am fairly new to all Histotechnology processes. We mainly work in our
 lab with mineralized bone with implants, so we fix them, dehydrate them,
 embed them in MMA, cut them, glued them to slides, ground them manually and
 then stain them. I need help in some parts of the process that need
 improvement:


  1.  We use Loctite 4471 on the samples, put them under vacuum, apply them
 to the slide and more vacuum. There's good results but there are some
 bubbles that still appear. Suggestions?
  2.  I can't seem to get the yellow/orange on bone with the Van Gieson
 stain. I've been doinga preheat at 55 °C, etching, rinse in DI water,
 Sanderson´s Rapid Bone Stain, running tap water, Van Gieson (commercial
 from DHM) for 30 seconds to 5 minutes, paper dry and quickly dehydrate with
 100% EtOH. What 

Re: [Histonet] bone marrow specimens

[Rene J Buesa]

I fix in NBF at pH7 exactly and decalcify with EDTA
René J.

From: Clare Thornton cthorn...@dahlchase.com
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu 
Sent: Thursday, February 14, 2013 9:22 AM
Subject: [Histonet] bone marrow specimens

What fixative and decal solution is everyone using for bone marrow specimens 
which will have subsequent IHC and Kappa/Lambda ISH staining?  We are currently 
using B+ fixative and Decal A (formic acid and formaldehyde).  Our pathologists 
demand a quick turn around time, and are willing to sacrifice some quality for 
this, but we are having issues with our Kappa/Lambda ISH stains not 
highlighting all the plasma cells.  The staining tends to be stronger on the 
outer edge of the tissue, and weakens or disappears entirely towards the 
middle, so I'm thinking the fixation and/or decal is the problem.  We run our 
K/L ISH using Ventana instrumentation and reagents.

Thank you in advance!

Clare J. Thornton, HTL(ASCP)QIHC
Assistant Histology Supervisor
Dahl-Chase Diagnostic Services
417 State Street, Suite 540
Bangor, ME 04401
cthorn...@dahlchase.com


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Re: [Histonet] Bone Marrows

[DKBoyd]

Yes we do.  We assist with all bone marrows regardles of where they are 
(not hematology).   We dress out in scrubs, etc.

Debbie M. Boyd, HT(ASCP) l Chief Histologist l Southside Regional Medical 
Center I 
200 Medical Park Boulevard l Petersburg, Va.  23805 l T: 804-765-5050 l F: 
804-765-5582 l dkb...@chs.net





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Re: [Histonet] Bone Marrow HE Protocols

[Dawud AlYasa]

What's up Tena! 
Long time. The bone marrows will be fixed in B-5.
Dawud
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RE: [Histonet] bone marrow biopsy decals

[Patsy Ruegg]

Diana,

I have a lot of experience with decal reagents and Formic acid is the best
in my experience for IHC.  It is not as fast as the rapid decals but is the
kindest at not adversely affecting the antigens and more gentle on iron
leaching, although all decals will leach iron, we usually do iron on the
smear or clot section.  Not only does the formic acid leach the iron so does
the alcohols and solvents in tissue processing.

Regards,

Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Diana
Martinez-Longoria
Sent: Thursday, March 24, 2011 11:01 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] bone marrow biopsy decals

Our pathologist wants to know why Formical-4 is the best decal for bone
marrow biopsies, since we heard about in Histonet? I have experimented with
four different decals: Rapid-Immuno, Formical-4, Nitric Acid, and HCL\Formic
Acid and we are still not sure which will be the best decal. Also we a decal
that will  minimize the leeching out of iron. If any out there has any
suggest, please let me know. Thanks in advance! 

Diana Martinez-Longoria
Histotechnician  HT(ASCP)CM
El Centro Regional Medical Center
1415 Ross Ave
El Centro CA 92243
(760) 339-7267
(760)482-5365 fax

dmlongo...@ecrmc.org






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Re: [Histonet] bone marrow biopsy decals

[Akemi Allison]

Hi Diana,

I recently made a histonet posting Great B5 substitute which also  
addresses BM decal.  Since there are a number of companies who sell  
decal solutions with similar names, I am not sure you tried the  
products from BBC Chemical.  It is best to use both products  
together.  My post goes into my findings.


Regards,
Akemi

Akemi Allison BS, HT (ASCP) HTL
Director
Phoenix Lab Consulting
Tele: 408.335.9994
E-Mail: akemiat3...@yahoo.com

On Mar 24, 2011, at 10:00 AM, Diana Martinez-Longoria wrote:

Our pathologist wants to know why Formical-4 is the best decal for  
bone marrow biopsies, since we heard about in Histonet? I have  
experimented with four different decals: Rapid-Immuno, Formical-4,  
Nitric Acid, and HCL\Formic Acid and we are still not sure which  
will be the best decal. Also we a decal that will  minimize the  
leeching out of iron. If any out there has any suggest, please let  
me know. Thanks in advance!


Diana Martinez-Longoria
Histotechnician  HT(ASCP)CM
El Centro Regional Medical Center
1415 Ross Ave
El Centro CA 92243
(760) 339-7267
(760)482-5365 fax

dmlongo...@ecrmc.org






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distribution is prohibited. If you are not the intended recipient,  
please contact the sender at the phone number above and promptly  
destroy this e-mail and its attachments.



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Re: [Histonet] bone marrow aspirations

[Rene J Buesa]

Either air dry → methanol (most labs) or air dry → PAP fixative (I always 
preferred methanol).
René J.

--- On Wed, 3/2/11, Debra Siena dsi...@statlab.com wrote:


From: Debra Siena dsi...@statlab.com
Subject: [Histonet] bone marrow aspirations
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
Date: Wednesday, March 2, 2011, 10:43 AM


Hi All,

I would appreciate some insight as to how most labs are treating bone marrow 
smears, in regards to fixation.  I am not embarrassed to say that it has been 
awhile since I worked with them.  With the smears are most labs air drying, 
methanol (alcohol fixation) or spray fixation (pap fixative) or something 
else?  I do thank you for your help.

Debbie Siena HT(ASCP)QIHC
Technical Manager | StatLab Medical Products
407 Interchange St. | McKinney, TX 75071
Direct: 972-436-1010  x229 | Fax: 972-436-1369
dsi...@statlab.commailto:maub...@statlab.com | 
www.statlab.comhttp://www.statlab.com/


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Re: [Histonet] bone marrow aspirations

[Mark Turner]

I've generally used the methanol fixation.

Mark Turner


 Rene J Buesa rjbu...@yahoo.com wrote: 
 Either air dry → methanol (most labs) or air dry → PAP fixative (I always 
 preferred methanol).
 René J.
 
 --- On Wed, 3/2/11, Debra Siena dsi...@statlab.com wrote:
 
 
 From: Debra Siena dsi...@statlab.com
 Subject: [Histonet] bone marrow aspirations
 To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
 Date: Wednesday, March 2, 2011, 10:43 AM
 
 
 Hi All,
 
 I would appreciate some insight as to how most labs are treating bone marrow 
 smears, in regards to fixation.  I am not embarrassed to say that it has been 
 awhile since I worked with them.  With the smears are most labs air drying, 
 methanol (alcohol fixation) or spray fixation (pap fixative) or something 
 else?  I do thank you for your help.
 
 Debbie Siena HT(ASCP)QIHC
 Technical Manager | StatLab Medical Products
 407 Interchange St. | McKinney, TX 75071
 Direct: 972-436-1010  x229 | Fax: 972-436-1369
 dsi...@statlab.commailto:maub...@statlab.com | 
 www.statlab.comhttp://www.statlab.com/
 
 
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Re: [Histonet] bone marrow aspirations

[Jennifer MacDonald]

We always air dried them and fixed them in methanol, otherwise they washed 
off.




Debra Siena dsi...@statlab.com 
Sent by: histonet-boun...@lists.utsouthwestern.edu
03/02/2011 07:49 AM

To
histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
cc

Subject
[Histonet] bone marrow aspirations






Hi All,

I would appreciate some insight as to how most labs are treating bone 
marrow smears, in regards to fixation.  I am not embarrassed to say that 
it has been awhile since I worked with them.  With the smears are most 
labs air drying, methanol (alcohol fixation) or spray fixation (pap 
fixative) or something else?  I do thank you for your help.

Debbie Siena HT(ASCP)QIHC
Technical Manager | StatLab Medical Products
407 Interchange St. | McKinney, TX 75071
Direct: 972-436-1010  x229 | Fax: 972-436-1369
dsi...@statlab.commailto:maub...@statlab.com | www.statlab.com
http://www.statlab.com/


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Re: [Histonet] Bone marrow charges

[histot...@imagesbyhopper.com]

It is my understanding that you may now charge for every stain, even if it's 
two blocks from one bottle.  The CMS requirements changed in Oct of 2009, if 
memory serves correctly.  Perhaps some one else can site the reference or go 
back into the archives for it.  I'm not at my computer, or I would do it myself!

Sent from my iPhone

On Feb 23, 2011, at 10:06 AM, Rathborne, Toni 
trathbo...@somerset-healthcare.com wrote:

 Are your blocks (1 bx and 1 clot) separate specimens, or just one specimen 
 with 2 blocks?
 
 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu
 [mailto:histonet-boun...@lists.utsouthwestern.edu]On Behalf Of Goodwin,
 Diana
 Sent: Monday, February 21, 2011 11:40 AM
 To: 'histonet@lists.utsouthwestern.edu'
 Subject: [Histonet] Bone marrow charges
 
 
 Greetings, Histonetters.
 
 How many times can I charge 88313 for a bone marrow case that has an Iron 
 stain on 2 separate blocks (one for the bx and one for the aspirate clot) and 
 also on 1 smear made from the aspirate clot, a PAS on the 2 blocks, a 
 Trichrome and a Retic on the bx. block, and a Wright/ Giemsa on 4 smears?
 
 Thank you!!!
 Diana Goodwin
 Supervisor, Histology Laboratory
 RWJUHHNJ
 
 
 Diana Goodwin
 Supervisor, Histology Laboratory
 xt. 6996
 
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RE: [Histonet] Bone marrow charges

[Rathborne, Toni]

Are your blocks (1 bx and 1 clot) separate specimens, or just one specimen with 
2 blocks?

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu]On Behalf Of Goodwin,
Diana
Sent: Monday, February 21, 2011 11:40 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Bone marrow charges


Greetings, Histonetters.

How many times can I charge 88313 for a bone marrow case that has an Iron stain 
on 2 separate blocks (one for the bx and one for the aspirate clot) and also on 
1 smear made from the aspirate clot, a PAS on the 2 blocks, a Trichrome and a 
Retic on the bx. block, and a Wright/ Giemsa on 4 smears?

Thank you!!!
Diana Goodwin
Supervisor, Histology Laboratory
RWJUHHNJ


Diana Goodwin
Supervisor, Histology Laboratory
xt. 6996

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by e-mail or you may call Somerset Medical Center's computer Help Desk
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Be sure to visit Somerset Medical Center's Web site - 
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RE: [Histonet] Bone IHC

[Kuhnla, Melissa]

Are you placing this bone section on a pre-cut control slides?  The
section will adhere better on its own slide.

Are you drying your slides at room temp prior to baking?  It always
helps to remove all trapped water from under the section prior to
baking.

Post fixing with formalin fumes is said to help.  Remove slides from
oven. Place in a rack in a staining cup w formalin just covering the
bottom of the cup (not covering the section). Cover and allow the fumes
to 'post fix' for 15 minutes.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Vanessa
J. Phelan
Sent: Thursday, October 07, 2010 3:14 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone IHC

Hi everyone,

I was wondering if anyone has any tricks on how to get bone sections to
stop lifting off the slide through the IHC process? I leave them in the
oven for quite a while to make sure they are baked on, however after
antigen retrieval  (pressure cooker for 20mins) most of the boney part
of the tissue comes off and the marrow and muscle stays put! The
sections are cut onto superfrost plus slides.

Any help would be much appreciated, thanks.

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RE: [Histonet] Bone IHC

[Liz Chlipala]

We lower the temp of retrieval to 70C for 2 hours and have good success
with that.

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 Vanessa
J. Phelan
Sent: Thursday, October 07, 2010 1:14 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone IHC

Hi everyone,

I was wondering if anyone has any tricks on how to get bone sections to
stop lifting off the slide through the IHC process? I leave them in the
oven for quite a while to make sure they are baked on, however after
antigen retrieval  (pressure cooker for 20mins) most of the boney part
of the tissue comes off and the marrow and muscle stays put! The
sections are cut onto superfrost plus slides.

Any help would be much appreciated, thanks.

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Re: [Histonet] Bone IHC

[Rene J Buesa]

If you have a bone that will require IHC, you will have to make sure that the 
Gross section is no more than 1.5 mm thick and decalcify it in EDTA. The you 
have to make sure that the infiltration is complete. These are the 2 initial 
factors.
Later you will have to section it as thin as you can, place the sections in (+) 
slides let them drain completely before going into the oven. Extra time in the 
oven is not really required if the aforementioned steps are done.
Then add enough dishwasher soap to the HIER solution to get a solution of 2% 
and you will dewax and retrieve the antigen simultaneously. The sections will 
survive to complete the IHC procedure.René J.

--- On Thu, 10/7/10, Vanessa J. Phelan vjp2...@columbia.edu wrote:



From: Vanessa J. Phelan vjp2...@columbia.edu
Subject: [Histonet] Bone IHC
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
Date: Thursday, October 7, 2010, 3:13 PM


Hi everyone,

I was wondering if anyone has any tricks on how to get bone sections to stop 
lifting off the slide through the IHC process? I leave them in the oven for 
quite a while to make sure they are baked on, however after antigen retrieval  
(pressure cooker for 20mins) most of the boney part of the tissue comes off and 
the marrow and muscle stays put! The sections are cut onto superfrost plus 
slides.

Any help would be much appreciated, thanks.

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Re: [Histonet] Bone IHC

[Adam .]

I've been experimenting with different ways to solve this problem myself. I
fix my tissues in 10% zinc buffered formalin and decalcify in formic acid
for 72 hours, followed by embedding and cutting 5 uM sections.

From trial and error, I've determined that incubating the slides flat on a
slide warmer at 37C (or in my case, the bottom of an unused bacterial
incubator) can prevent nearly all the detachment you observe but it's time
dependent. I think the problem is that the sections get small amounts of
water underneath them when you scoop them up off the water surface during
sectioning and during HIER, that water boils and shears off the slide.

If you leave the slides overnight, the slides were get destroyed during
HIER. However, if you leave them for a week, they tend to be nearly
untouched even at 95C for 10 mins. I'm currently in the process of
determining if a few days is enough time.

Adam

On Thu, Oct 7, 2010 at 2:20 PM, Liz Chlipala l...@premierlab.com wrote:

 We lower the temp of retrieval to 70C for 2 hours and have good success
 with that.

 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 Vanessa
 J. Phelan
 Sent: Thursday, October 07, 2010 1:14 PM
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Bone IHC

 Hi everyone,

 I was wondering if anyone has any tricks on how to get bone sections to
 stop lifting off the slide through the IHC process? I leave them in the
 oven for quite a while to make sure they are baked on, however after
 antigen retrieval  (pressure cooker for 20mins) most of the boney part
 of the tissue comes off and the marrow and muscle stays put! The
 sections are cut onto superfrost plus slides.

 Any help would be much appreciated, thanks.

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RE: [Histonet] Bone Marrow Clots - falling off slides

[Lynette Pavelich]

When I start losing tissue on the slide, I have found that it is usually
a processing issue.  Is the clot too thick?  Try cutting them in half
before processing. 

Hope this helped,
Lynette

Lynette Pavelich, HT(ASCP)
Histology Supervisor
MSH Competency Coordinator
Hurley Medical Center
One Hurley Plaza
Flint, MI  48503
email: lpave...@hurleymc.com
ph:  810-257-9948
Lab:  810-257-9138
fax:  810-762-7082


 MARY T HODGES hodges...@msn.com 4/27/2010 12:18 AM 

recycled anything is not back to it original state always use a hydo
meter and add new to back product of a better quality you will find this
also with alcohols
 
 From: histot...@imagesbyhopper.com 
 To: histonet@lists.utsouthwestern.edu 
 Date: Mon, 26 Apr 2010 23:26:43 -0400
 Subject: [Histonet] Bone Marrow Clots - falling off slides
 
 Hi Histonetters!
 
 We have started to have an issue with our bone marrow clots falling
off the
 slides. We are using plus slides, making sure they drain well (just
like
 our other slides), but when we stain them routinely, we are getting a
fair
 amount of tissue coming off the slides.
 
 It has been suggested that it's related to our using recycled xylene.
Does
 anyone have any experience with recycled xylene and this type of
tissue
 fall-off?
 
 Another suggestion was that the tissues sat in xylene too long. I
don't see
 how that could happen, under routine conditions, but is that a
possibility
 for causing this?
 
 All thoughts will be appreciated!
 
 Michelle
 
 
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RE: [Histonet] Bone Marrow Clots - falling off slides

[histotech]

Mary,
Thanks for the suggestion.  We do check the alcohols with a hydrometer and
if not 100%, we will use that alcohol as the base for dilution alcohols.  We
check quality of the xylene by putting 10ml of water in a graduated cylinder
and then add 10ml of the recycled xylene to it and shake it vigorosly.
Good, clean xylene should have a perfect separation and no  mixing with
the water.

Lynette,
I am leaning towards the processing too, but which part??  The clot seems to
be more dry than moist, so it's not under dehydrated.  Fixation? It spends 3
hours in formalin on the tissue processor and whatever time it can be in
formalin prior to the processor.  Paraffin impregnation?  About the only
thing I could think of here was if the wax wasn't changed fast enough and/or
we got too much carry over from the xylene.  Could that cause this?

Thanks for the help!

Michelle



-Original Message-
From: Lynette Pavelich [mailto:lpave...@hurleymc.com] 
Sent: Tuesday, April 27, 2010 6:56 AM
To: histot...@imagesbyhopper.com; histonet@lists.utsouthwestern.edu; MARY
HODGES
Subject: RE: [Histonet] Bone Marrow Clots - falling off slides


When I start losing tissue on the slide, I have found that it is usually a
processing issue.  Is the clot too thick?  Try cutting them in half before
processing. 

Hope this helped,
Lynette

Lynette Pavelich, HT(ASCP)
Histology Supervisor
MSH Competency Coordinator
Hurley Medical Center
One Hurley Plaza
Flint, MI  48503
email: lpave...@hurleymc.com
ph:  810-257-9948
Lab:  810-257-9138
fax:  810-762-7082


 MARY T HODGES hodges...@msn.com 4/27/2010 12:18 AM 

recycled anything is not back to it original state always use a hydo meter
and add new to back product of a better quality you will find this also with
alcohols
 
 From: histot...@imagesbyhopper.com
 To: histonet@lists.utsouthwestern.edu 
 Date: Mon, 26 Apr 2010 23:26:43 -0400
 Subject: [Histonet] Bone Marrow Clots - falling off slides
 
 Hi Histonetters!
 
 We have started to have an issue with our bone marrow clots falling
off the
 slides. We are using plus slides, making sure they drain well (just
like
 our other slides), but when we stain them routinely, we are getting a
fair
 amount of tissue coming off the slides.
 
 It has been suggested that it's related to our using recycled xylene.
Does
 anyone have any experience with recycled xylene and this type of
tissue
 fall-off?
 
 Another suggestion was that the tissues sat in xylene too long. I
don't see
 how that could happen, under routine conditions, but is that a
possibility
 for causing this?
 
 All thoughts will be appreciated!
 
 Michelle
 
 
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RE: [Histonet] Bone Marrow Clots - falling off slides

[Lynette Pavelich]

In my experience, it can be all of the above that can cause it.  By the
tissue not getting enough penetration by any of the solutions will alter
the outcome.  That is why I asked about the thickness (remember...only
as thick as a nickel).  Blood is also so dense, so it would really apply
here.  Try that.  Do 1 change at a time.  I've been in your shoes, it is
very frustrating.  Then if the thickness is good, work on something
else.starting with fixation times.


 histot...@imagesbyhopper.com 4/27/2010 7:32 AM 
Mary,
Thanks for the suggestion.  We do check the alcohols with a hydrometer
and
if not 100%, we will use that alcohol as the base for dilution
alcohols.  We
check quality of the xylene by putting 10ml of water in a graduated
cylinder
and then add 10ml of the recycled xylene to it and shake it vigorosly.
Good, clean xylene should have a perfect separation and no  mixing
with
the water.

Lynette,
I am leaning towards the processing too, but which part??  The clot
seems to
be more dry than moist, so it's not under dehydrated.  Fixation? It
spends 3
hours in formalin on the tissue processor and whatever time it can be
in
formalin prior to the processor.  Paraffin impregnation?  About the
only
thing I could think of here was if the wax wasn't changed fast enough
and/or
we got too much carry over from the xylene.  Could that cause this?

Thanks for the help!

Michelle



-Original Message-
From: Lynette Pavelich [mailto:lpave...@hurleymc.com] 
Sent: Tuesday, April 27, 2010 6:56 AM
To: histot...@imagesbyhopper.com; histonet@lists.utsouthwestern.edu;
MARY
HODGES
Subject: RE: [Histonet] Bone Marrow Clots - falling off slides


When I start losing tissue on the slide, I have found that it is
usually a
processing issue.  Is the clot too thick?  Try cutting them in half
before
processing. 

Hope this helped,
Lynette

Lynette Pavelich, HT(ASCP)
Histology Supervisor
MSH Competency Coordinator
Hurley Medical Center
One Hurley Plaza
Flint, MI  48503
email: lpave...@hurleymc.com 
ph:  810-257-9948
Lab:  810-257-9138
fax:  810-762-7082


 MARY T HODGES hodges...@msn.com 4/27/2010 12:18 AM 

recycled anything is not back to it original state always use a hydo
meter
and add new to back product of a better quality you will find this also
with
alcohols
 
 From: histot...@imagesbyhopper.com 
 To: histonet@lists.utsouthwestern.edu 
 Date: Mon, 26 Apr 2010 23:26:43 -0400
 Subject: [Histonet] Bone Marrow Clots - falling off slides
 
 Hi Histonetters!
 
 We have started to have an issue with our bone marrow clots falling
off the
 slides. We are using plus slides, making sure they drain well (just
like
 our other slides), but when we stain them routinely, we are getting
a
fair
 amount of tissue coming off the slides.
 
 It has been suggested that it's related to our using recycled
xylene.
Does
 anyone have any experience with recycled xylene and this type of
tissue
 fall-off?
 
 Another suggestion was that the tissues sat in xylene too long. I
don't see
 how that could happen, under routine conditions, but is that a
possibility
 for causing this?
 
 All thoughts will be appreciated!
 
 Michelle
 
 
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Re: [Histonet] Bone Marrow Clots - falling off slides

[histot...@imagesbyhopper.com]

Thanks for all the input.  I will check the collection method.  B-plus  
is the fixative we normally use but I am not convinced that this clot  
ended up in it!  Other tissues are not being dramatically affected and  
not even all the bone marrows!  We do soak the blocks too.


This is very frustrating.  :o(

Michelle



On Apr 27, 2010, at 7:59 AM, Lynette Pavelich  
lpave...@hurleymc.com wrote:


In my experience, it can be all of the above that can cause it.  By  
the
tissue not getting enough penetration by any of the solutions will  
alter

the outcome.  That is why I asked about the thickness (remember...only
as thick as a nickel).  Blood is also so dense, so it would really  
apply
here.  Try that.  Do 1 change at a time.  I've been in your shoes,  
it is

very frustrating.  Then if the thickness is good, work on something
else.starting with fixation times.



histot...@imagesbyhopper.com 4/27/2010 7:32 AM 

Mary,
Thanks for the suggestion.  We do check the alcohols with a hydrometer
and
if not 100%, we will use that alcohol as the base for dilution
alcohols.  We
check quality of the xylene by putting 10ml of water in a graduated
cylinder
and then add 10ml of the recycled xylene to it and shake it vigorosly.
Good, clean xylene should have a perfect separation and no  mixing
with
the water.

Lynette,
I am leaning towards the processing too, but which part??  The clot
seems to
be more dry than moist, so it's not under dehydrated.  Fixation? It
spends 3
hours in formalin on the tissue processor and whatever time it can be
in
formalin prior to the processor.  Paraffin impregnation?  About the
only
thing I could think of here was if the wax wasn't changed fast enough
and/or
we got too much carry over from the xylene.  Could that cause this?

Thanks for the help!

Michelle



-Original Message-
From: Lynette Pavelich [mailto:lpave...@hurleymc.com]
Sent: Tuesday, April 27, 2010 6:56 AM
To: histot...@imagesbyhopper.com; histonet@lists.utsouthwestern.edu;
MARY
HODGES
Subject: RE: [Histonet] Bone Marrow Clots - falling off slides


When I start losing tissue on the slide, I have found that it is
usually a
processing issue.  Is the clot too thick?  Try cutting them in half
before
processing.

Hope this helped,
Lynette

Lynette Pavelich, HT(ASCP)
Histology Supervisor
MSH Competency Coordinator
Hurley Medical Center
One Hurley Plaza
Flint, MI  48503
email: lpave...@hurleymc.com
ph:  810-257-9948
Lab:  810-257-9138
fax:  810-762-7082



MARY T HODGES hodges...@msn.com 4/27/2010 12:18 AM 


recycled anything is not back to it original state always use a hydo
meter
and add new to back product of a better quality you will find this  
also

with
alcohols


From: histot...@imagesbyhopper.com
To: histonet@lists.utsouthwestern.edu
Date: Mon, 26 Apr 2010 23:26:43 -0400
Subject: [Histonet] Bone Marrow Clots - falling off slides

Hi Histonetters!

We have started to have an issue with our bone marrow clots falling

off the

slides. We are using plus slides, making sure they drain well (just

like

our other slides), but when we stain them routinely, we are getting

a
fair

amount of tissue coming off the slides.

It has been suggested that it's related to our using recycled

xylene.
Does

anyone have any experience with recycled xylene and this type of

tissue

fall-off?

Another suggestion was that the tissues sat in xylene too long. I

don't see

how that could happen, under routine conditions, but is that a

possibility

for causing this?

All thoughts will be appreciated!

Michelle


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RE: [Histonet] Bone Marrow Clots - falling off slides

[Shirley A. Powell]

I cut the autopsies for the ME/FBI here and have a lot of bloody tissues I 
lovingly call blood bricks.  I was using charged slides but these tissue 
would wash really badly.  I started using regular slides and StayOn from 
Surgipath and have had great success with keeping these bloody tissues on the 
slides.  I drain the water well, heat the slides up just to the point of 
melting the paraffin and dry them overnight in the slide dryer to make sure, 
but have also dried them for 45 minutes and they have stayed on.  
Shirley

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
histot...@imagesbyhopper.com
Sent: Tuesday, April 27, 2010 7:33 AM
To: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone Marrow Clots - falling off slides

Mary,
Thanks for the suggestion.  We do check the alcohols with a hydrometer and
if not 100%, we will use that alcohol as the base for dilution alcohols.  We
check quality of the xylene by putting 10ml of water in a graduated cylinder
and then add 10ml of the recycled xylene to it and shake it vigorosly.
Good, clean xylene should have a perfect separation and no  mixing with
the water.

Lynette,
I am leaning towards the processing too, but which part??  The clot seems to
be more dry than moist, so it's not under dehydrated.  Fixation? It spends 3
hours in formalin on the tissue processor and whatever time it can be in
formalin prior to the processor.  Paraffin impregnation?  About the only
thing I could think of here was if the wax wasn't changed fast enough and/or
we got too much carry over from the xylene.  Could that cause this?

Thanks for the help!

Michelle



-Original Message-
From: Lynette Pavelich [mailto:lpave...@hurleymc.com] 
Sent: Tuesday, April 27, 2010 6:56 AM
To: histot...@imagesbyhopper.com; histonet@lists.utsouthwestern.edu; MARY
HODGES
Subject: RE: [Histonet] Bone Marrow Clots - falling off slides


When I start losing tissue on the slide, I have found that it is usually a
processing issue.  Is the clot too thick?  Try cutting them in half before
processing. 

Hope this helped,
Lynette

Lynette Pavelich, HT(ASCP)
Histology Supervisor
MSH Competency Coordinator
Hurley Medical Center
One Hurley Plaza
Flint, MI  48503
email: lpave...@hurleymc.com
ph:  810-257-9948
Lab:  810-257-9138
fax:  810-762-7082


 MARY T HODGES hodges...@msn.com 4/27/2010 12:18 AM 

recycled anything is not back to it original state always use a hydo meter
and add new to back product of a better quality you will find this also with
alcohols
 
 From: histot...@imagesbyhopper.com
 To: histonet@lists.utsouthwestern.edu 
 Date: Mon, 26 Apr 2010 23:26:43 -0400
 Subject: [Histonet] Bone Marrow Clots - falling off slides
 
 Hi Histonetters!
 
 We have started to have an issue with our bone marrow clots falling
off the
 slides. We are using plus slides, making sure they drain well (just
like
 our other slides), but when we stain them routinely, we are getting a
fair
 amount of tissue coming off the slides.
 
 It has been suggested that it's related to our using recycled xylene.
Does
 anyone have any experience with recycled xylene and this type of
tissue
 fall-off?
 
 Another suggestion was that the tissues sat in xylene too long. I
don't see
 how that could happen, under routine conditions, but is that a
possibility
 for causing this?
 
 All thoughts will be appreciated!
 
 Michelle
 
 
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RE: [Histonet] Bone Marrow Clots - falling off slides

[MARY T HODGES]

recycled anything is not back to it original state always use a hydo meter and 
add new to back product of a better quality you will find this also with 
alcohols
 
 From: histot...@imagesbyhopper.com
 To: histonet@lists.utsouthwestern.edu
 Date: Mon, 26 Apr 2010 23:26:43 -0400
 Subject: [Histonet] Bone Marrow Clots - falling off slides
 
 Hi Histonetters!
 
 We have started to have an issue with our bone marrow clots falling off the
 slides. We are using plus slides, making sure they drain well (just like
 our other slides), but when we stain them routinely, we are getting a fair
 amount of tissue coming off the slides.
 
 It has been suggested that it's related to our using recycled xylene. Does
 anyone have any experience with recycled xylene and this type of tissue
 fall-off?
 
 Another suggestion was that the tissues sat in xylene too long. I don't see
 how that could happen, under routine conditions, but is that a possibility
 for causing this?
 
 All thoughts will be appreciated!
 
 Michelle
 
 
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RE: [Histonet] Bone marrow trephine decalcification

[McMahon, Loralee A]

They have commercial rapid decals that are formic acid based that do not affect 
the immunohistochemistry.  But it does affect some of the enzyme 
histochemistry.  


Loralee McMahon, HTL (ASCP)
Immunohistochemistry Supervisor
Strong Memorial Hospital
Department of Surgical Pathology
(585) 275-7210

From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Marinez 
[mbba...@uol.com.br]
Sent: Wednesday, April 21, 2010 4:14 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone marrow trephine decalcification

I'm in a bit of trouble. I work in the south of Brazil (Porto Alegre) for some 
years and now in my hospital a lot of bone marrow biopsies are being performed 
(leukemia and lymphomas). With decalcification with strong acids (nitric) I'm 
getting very poor results with the immunohistochemistry. I'm trying to use EDTA 
but we really are not getting speed or good results (we  controled de pH but 
still precipitates). Could some one be kind enough to send me some formula 
(simple one please) that will work in reasonable time (24 or 48 hours) and 
perform well with immunohistochemistry? I would deeply grateful.



M. Barra
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Re: [Histonet] bone marrow fixative

[Amy Farnan]

Acetic Zinc Formalin



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Re: [Histonet] bone marrow fixative

[Rene J Buesa]

Buffered 10% NBF, but with the pH exactly at 7.0, checked for each case. IHC is 
perfect.
René J.

--- On Tue, 3/2/10, Amy Farnan farn...@nehealth.com wrote:


From: Amy Farnan farn...@nehealth.com
Subject: [Histonet] bone marrow fixative
To: histonet@lists.utsouthwestern.edu
Date: Tuesday, March 2, 2010, 2:12 PM


Good afternoon everyone,

In past years our lab used B-5 fixative on bone marrow biopsies for better 
immunostaining.
With the chemical hazards of B-5 we switched to AZF fixative and have been 
using this for the past few years. I am just curious what everyone else is 
using for fixative on their bone marrows and how is your immunoreactivity?  
Maybe a better question is what is the preferred fixative for bone marrows?

Have a nice day,
Amy Farnan
Histology Supervisor
Northeast Health

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RE: [Histonet] bone marrow fixative

[Rathborne, Toni]

We use zinc formalin for the bone marrow specimens, and the pathologists are 
satisfied with the results.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu]on Behalf Of Amy
Farnan
Sent: Tuesday, March 02, 2010 2:12 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] bone marrow fixative


Good afternoon everyone,
 
In past years our lab used B-5 fixative on bone marrow biopsies for better 
immunostaining.
With the chemical hazards of B-5 we switched to AZF fixative and have been 
using this for the past few years. I am just curious what everyone else is 
using for fixative on their bone marrows and how is your immunoreactivity?  
Maybe a better question is what is the preferred fixative for bone marrows?
 
Have a nice day,
Amy Farnan
Histology Supervisor
Northeast Health

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Fwd: Re: [Histonet] Bone tissue

[John Kiernan]

 Just for the record, Mollifex isn't alkaline. It is probably either Baker's 
1941 mixture (see below) or the following (parts by volume):
95% ethanol 952
Glycerol 32
Acetone 80 
Liquid phenol 8
 - - -
Liquid phenol is probably liquefied phenol, USP, which contains about 10% 
water and at least 89% phenol (w/w), or something similar.
 For more information and discussion, see Maria Wynnchuk (1992) Minimizing 
artifacts in tissue processing: Part 1. Importance of softening agents. J. 
Histotechnol. 15(4): 321-323. This paper also discusses effects of softening 
faced blocks on HE staining.
  
John R Baker (1941) used 9 parts 60% ethanol and 1 part glycerol (J. Roy. 
Microsc. Soc. 61: 75), whereas R. C. Pearlman and Buell C. Cole (1951) sang the 
praises of 1% dishwashing detergent and similar solutions (Stain Technol. 
26(2): 115-118). Manfred Gabe (Histological Techniques, Paris, 1976, a great 
book) favoured soaking faced paraffin blocks in cold water. For what it's 
worth, I have found water helpful when sectioning decalcified rats' heads. With 
all these well documented and inexpensive softening liquids it should never be 
necessary to resort to a proprietary product whose composition is kept secret 
from the buyer and user.
  
 John Kiernan
 Anatomy, UWO
 London, Canada
  = = =
 - Original Message -
 From: Rene J Buesa rjbu...@yahoo.com
 Date: Monday, February 22, 2010 16:43
 Subject: Re: [Histonet] Bone tissue
 To: histonet@lists.utsouthwestern.edu, Reuel Cornelia 
 reuel.corne...@tsrh.org
 
  First of all, Mollifex or any other alkaline substance will do 
  nothing useful to the bone.
  I tend to think that you processed the bone before it was 
  completely decalcified and that is the cause for an incomplete 
  infiltration and a subsequent difficult sectioning.
  René J.
  
  --- On Mon, 2/22/10, Reuel Cornelia reuel.corne...@tsrh.org 
  wrote:
  
  From: Reuel Cornelia reuel.corne...@tsrh.org
  Subject: [Histonet] Bone tissue
  To: histonet@lists.utsouthwestern.edu
  Date: Monday, February 22, 2010, 4:36 PM
  
  
  We have a difficulty cutting metatarsal bone . It seems that our 
  sections are so dried up. I was thinking that our dehydration 
  have something to do with this which we have placed it in a 
  wrong processing procedure for our large bone. The tissue is 4 
  mm thick and 1-2 cm in length and width and  was dehydrated in 
  70% - 4 hrs, 80%-4 hrs,95% -4 hrs and 2 changes of 100% 3 hrs 
  each, paraffin is 4 hrs each 2 changes. The tissue was 
  decalcified in 14% EDTA. When we start cutting them it is so 
  brittle and we could not even create a section. I have surfaced 
  decal it and also place in a softener Mollifex some of it work 
  but some does not work. Please help us remedy this tissue. Thank you.
  
  
  
  
  Reuel Cornelia, BS MT, AMT
  Cellular Pathology
  Texas Scottish Rite Hospital for Children
   Welborn Street
  Dallas, TX 75219
  Tel: 214-559-7766
  fax: 214-559-7768
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Re: [Histonet] Bone tissue

[Rene J Buesa]

First of all, Mollifex or any other alkaline substance will do nothing useful 
to the bone.
I tend to think that you processed the bone before it was completely 
decalcified and that is the cause for an incomplete infiltration and a 
subsequent difficult sectioning.
René J.

--- On Mon, 2/22/10, Reuel Cornelia reuel.corne...@tsrh.org wrote:


From: Reuel Cornelia reuel.corne...@tsrh.org
Subject: [Histonet] Bone tissue
To: histonet@lists.utsouthwestern.edu
Date: Monday, February 22, 2010, 4:36 PM


We have a difficulty cutting metatarsal bone . It seems that our sections are 
so dried up. I was thinking that our dehydration have something to do with this 
which we have placed it in a wrong processing procedure for our large bone. The 
tissue is 4 mm thick and 1-2 cm in length and width and  was dehydrated in 70% 
- 4 hrs, 80%-4 hrs,95% -4 hrs and 2 changes of 100% 3 hrs each, paraffin is 4 
hrs each 2 changes. The tissue was decalcified in 14% EDTA. When we start 
cutting them it is so brittle and we could not even create a section. I have 
surfaced decal it and also place in a softener Mollifex some of it work but 
some does not work. Please help us remedy this tissue. Thank you.




Reuel Cornelia, BS MT, AMT
Cellular Pathology
Texas Scottish Rite Hospital for Children
 Welborn Street
Dallas, TX 75219
Tel: 214-559-7766
fax: 214-559-7768



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Re: [Histonet] Bone Marrow clot

[Percival Karen]

Gary, absolutely the heparin is the problem.  We (histologist) would 
participate in the bone marrow procedure to make slides immediately with the 
unheparinized sample.  If someone is making those slides for you, they need to 
use an unheparinized specimen.  Of course, that makes the task of slide 
preparation more difficult, as the time is very limited.  It helps to have an 
experienced person performing that procedure.
 
Karen

 Martin, Gary gmar...@marshallmedical.org 2/4/2010 1:19 PM 

We have recently been receiving bone marrow specimens where the syringe
has been heparinized before the aspiration is retrieved.  We are having
problem getting the clot sections to stay on the slide.  Could the
heparinization be causing this problem, and if so, is there a solution? 

Thank you 

Gary

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RE: [Histonet] Bone Marrow clot

[Mike Pence]

Nice tip!

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Lynette
Pavelich
Sent: Thursday, February 04, 2010 2:19 PM
To: histonet@lists.utsouthwestern.edu; Gary Martin
Subject: Re: [Histonet] Bone Marrow clot


One of our physicians also uses this method of heparin when obtaining
their BM's.  
To help it clot, we make up a solution of formaldehyde and methanol. 
Using the same syringe, pull some of the formal methanol into the
syringe and let it sit.say 10 minutes to give it time to clot. Then,
take the plunger out of the syringe and spin it down in the centrifuge
to compact the specimen.  It usually turns out to be a larger specimen
than the usual clot, but the method works well.  The pathologists' don't
have any problems with this method either. (yea!)  Below is the solution
of formal methanol that we use.

1 part 37% formaldehyde
9 parts 100% methanol  

hope this helps!
Lynette

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Re: [Histonet] Bone marrow processing on Peloris

[Rene J Buesa]

A good tissue processor can handle those specimens without the need of any 
protocol modification.
René J.

--- On Tue, 1/26/10, debra.or...@uchospitals.edu debra.or...@uchospitals.edu 
wrote:


From: debra.or...@uchospitals.edu debra.or...@uchospitals.edu
Subject: [Histonet] Bone marrow processing on Peloris
To: histonet@lists.utsouthwestern.edu
Date: Tuesday, January 26, 2010, 10:36 AM


Good morning,

I am currently looking at the possibility of running bone marrow cores
and clots on our Peloris. Has anyone had success with this, and if so
would you be willing to share your protocol? We use xylene on our runs.



Debra Ann Ortiz

Chief Medical Technologist

The University of Chicago Medical Center

Room E-602-A

5841 S. Maryland Avenue

Chicago, Il 60637

phone: 773.702.5237




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Re: [Histonet] Bone labelling for Microdamage

[Jack Ratliff]

Karie,

If you are looking for microdamage or are using basic fuchsin, I have  
been taught to bulk stain with 1% basic fuchsin in your processing  
solutions (i.e. 1% BF in 70% EtOH, 1% BF in 80% EtOH, 1% BF in 95%  
EtOH, and 1% BF in 100% EtOH) and then finish as you would with the  
remaining steps for your MMA protocol (i.e. xylenes, MMA +DBP, etc.)  
to achieve a fully polymerized bulk stained specimen. You then cut,  
grind, and polish as required for use with the EXAKT system. I was  
told the reason you process with the BF is because the cutting and  
grinding causes additional microdamage and if you stain first the  
additional damage will not be stained.


If you are talking about labeling bone for calculations of BFR or MAR  
and the animal did not receive timed injections of calcein, alizarin,  
tetracycline, or some other fluorescent bone label before necropsy,  
then you could try doing a 0.6% FA etch for 30 seconds followed with  
Sanderson's Rapid Bone Stain for 3 minutes, brief tap water rinse, and  
blot dry.


What is the exact specimen you are working with and what do you  
specifically wish to demonstrate


Jack



On Dec 1, 2009, at 10:04 AM, Karie Reaser krea...@vet.upenn.edu wrote:

Is it ok to process cortical bone, embed in MMA, section and grind  
on the Exakt system. Last do the bone labelling stain? Or do I have  
to do the bone labelling stain prior to embedding, sectioning and  
mounting on slides? Any suggestions would be greatly appreciated.


--
Karie L Reaser A.S.
New Bolton Center
University of Pennsylvania
School of Veterinary Medicine
Comparative Orthopedic Research Laboratory
382 W Street Road
Kennett Square, PA. 19348
Phone:610-925-6278
Fax:610-925-8120
Email:krea...@vet.upenn.edu


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Re: [Histonet] bone fixation and decal question

[Rene J Buesa]

You answered most of your own questions.
The bone should be fixed before decalcifying it.
After that it could be cut in the cryostat, but why to do that?
It will be quite difficult. The best solution is to fix → decalcify → process 
routine by embedding in paraffin → cut conventional sections.
The whole idea behind the FS is the speed of the intraoperative diagnosis..
Once that is lost (by fixing → decalcifying) I don't think it matter much 
waiting a little bit more.
Hydrochloric acid does not fix (answering to one of your questions).
René J.

--- On Sun, 11/29/09, nap...@siscom.net nap...@siscom.net wrote:


From: nap...@siscom.net nap...@siscom.net
Subject: [Histonet] bone fixation and decal question
To: histonet@lists.utsouthwestern.edu
Date: Sunday, November 29, 2009, 1:53 PM


Question regarding fresh tissue, in this case bone.
Scenario:

Mohs surgeon removes layers of skin on a patient's scalp,
eventually discovering the basal cell carcinoma extends to
the periosteum and so a bit of bone is taken and tested to
ensure the margins are free of cancer. The only problem is
that since it is bone, in order to cut it is needs to be
decalcified. 

In order to decalcify this tissue in order to cut it either
in cryostat or on regular microtome, it of course must be
fixed, correct? Reason being is I have heard of Mohs
dermatologic surgeons taking bones frags, letting their
histotech put the fresh bone in hydrochloric acid solution
for decal overnight and then cutting it in the morning on
the cryostat. This seems flawed to me as the acid would seem
to destroy architecture and critical proteins? Isnt this
practice flawed as it doesnt allow fixation? Does the
hydrochloric solution have fixative properties? Does decal
halt autolysis? Also, is there any real reason why formalin
fixed tissues cannot be mounted in OCT and cut frozen? Would
it hurt the frozen section for the specimen to be placed
into one of the decals i have seen that are both fixative
and decal? (Like Surgipath Decal I)It is said to work
reasonably quickly and the bone would be thin. Seems like
this would be the thourough way of getting quick
intraoperative results with a bit of bone on cryo.

Any comments? thanks in advance

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RE: [Histonet] bone fixation and decal question

[gayle callis]

The only way you can do a frozen section on undecalcified bone is using a
tungsten carbide knife($12200 to $1500 per knife and need reconditioning)
but you need a universal knife holder in your cryostat. Even then, doing an
frozen on undecalcified fresh bone is difficult, it tends to crumble if not
held together is some way.  Instrumedics Cryojane (sold by Leica
Microsystems) can solve this problem as you would get the margins of an
intact section (soft tissue and attached bone) you need at the time of Mohs
surgery. There would be no waiting for fixation, decalcification or
cryoprotection into the next day.  Cryojane sectioning does not require
fixation or decalcification. There is a wonderful demonstration of Cryojane
on http://www.alphelys.com/site/us/pHGP_CoupesCongelees.htm

There is another frozen section tape method available now, and you stain the
section while it is on the tape, then cover slip the tape (section side
down) onto a slide.  Go to this website  http://section-lab.jp and read
about the - Kawamoto Cryofilm method.  It is less expensive than Cryojane,
but does NOT transfer the section onto a slide surface - the section stays
on the tape.  However, transferring to the slide surface is something you
may not need to do anyway since diagnosis can be made very quickly on day of
Mohs surgery.   This part of the Kawamoto website has references with all
pdf's downloadable,  http://section-lab.jp/English/Referece.htm and his
original publication is there along with many others on how this Cryofilm
technology is used. I am not sure if permanent mounting medias can be used
with Cryofilm.  

Your assessment of the problem was right on.  If the Surgipath Decal I is
the same kind of fixative/decalcifier as is Calrite (a combination
formalin/formic acid mixture) then this is a better option that protects
tissue integrity with fixation at the same time as decalcifying the bone
fragment. HCl or any acid used without fixing the tissue first should NEVER
be done - this will macerate the tissue and bone, destroying cellular and
tissue morphology. The staining will be horrible.  HCl does not have
fixative qualities, and any enzymes causing autolysis are certainly
destroyed by the acid along with everything else in tissue - without
possibility of rescuing the tissue.  The practice of HCl alone is not only
flawed but potentially a legal issue if the tissue/cells are destroyed by
acid without using fixation first - it could be considered mishandling of a
tissue sample so diagnosis isn't possible.  These labs need to do some
reading on working with bone fixation, decalcification, etc. and to order a
good histotechnology textbook!

You should cryoprotect fixed/decalcified sample with 20 to 30% sucrose
before freezing or large ice crystal freezing artifact damages tissue
morphology, often making tissue unrecognizable. Cryoprotection can be
speeded up by vacuuming during cryoprotection plus exchanging the
fixative/decalcifier should rinse the residual acid from tissue.  Residual
acid can damage staining and also damage metal parts in cryostat. 

The other option is being able to raise the fibrous periosteum without
taking bone fragments - there are surgical instruments designed for doing
this. Question is:  is basal cell cancer also found in the bone? and that
means taking the bone fragment too.   

Good luck

Gayle Callis
HTL/HT/MT(ASCP)  



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
nap...@siscom.net
Sent: Sunday, November 29, 2009 11:54 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] bone fixation and decal question

Question regarding fresh tissue, in this case bone.
Scenario:

 Mohs surgeon removes layers of skin on a patient's scalp,
eventually discovering the basal cell carcinoma extends to
the periosteum and so a bit of bone is taken and tested to
ensure the margins are free of cancer. The only problem is
that since it is bone, in order to cut it is needs to be
decalcified. 

In order to decalcify this tissue in order to cut it either
in cryostat or on regular microtome, it of course must be
fixed, correct? Reason being is I have heard of Mohs
dermatologic surgeons taking bones frags, letting their
histotech put the fresh bone in hydrochloric acid solution
for decal overnight and then cutting it in the morning on
the cryostat. This seems flawed to me as the acid would seem
to destroy architecture and critical proteins? Isnt this
practice flawed as it doesnt allow fixation? Does the
hydrochloric solution have fixative properties? Does decal
halt autolysis? Also, is there any real reason why formalin
fixed tissues cannot be mounted in OCT and cut frozen? Would
it hurt the frozen section for the specimen to be placed
into one of the decals i have seen that are both fixative
and decal? (Like Surgipath Decal I)It is said to work
reasonably quickly and the bone would be thin. Seems like
this would be 

Correction on tungsten knife blade prices RE: [Histonet] bone fixation and decal question

[gayle callis]

Sorry Folks, 

I wrote: tungsten carbide knife ($12200 to $1500 per knife and need
reconditioning) a typo, fault of the computer obviously.

Tungsten carbide knives do not cost $12200 although at times seem like they
do. pricey little things.  $1200 sounds better, and there are
several vendors, Dorn and Hart, DDK, and probably microtome manufacturers
too.  

Gayle Callis 


 

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RE: [Histonet] bone marrow assisting?

[Mike Pence]

Same here!

Mike

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Lecorchick, William
Sent: Monday, September 21, 2009 12:50 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] bone marrow assisting?


The current protocol that we follow here is as follows:  Two green tops
are obtained first thing for Flow and Cyto followed by eight smears,
from that same syringe then draw air in and place on its side allow a
clot to form. From the bone bx make a touch prep rolling the bone
between two slides prior to dropping the bone in 10%NBF all slides are
air dried. You can pick out the best slide for iron and the rest get a
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RE: [Histonet] bone sections

[Garfield, Jacqueline]

Peggy,

Have you used this method of adhesion for special stains and IHC staining as 
well?  If so, have you had any interference or background staining?

Thank you,
Jackie

Jacqueline D. Garfield | Manager, Histology

Main 908.947.1100 Fax   908.947.1085
Direct   908.947.1182 Emailjgarfield@ lifecell.com
www.lifcell.com
 
LifeCell Corporation | One Millennium Way | Branchburg, NJ | 08876
 

 
 
-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of DiCarlo, 
Margaret
Sent: Thursday, August 20, 2009 8:03 AM
To: Williams, Sean; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] bone sections

Marie,

I make a 5% Titebond solution from Titebond II which can be purchased at
any hardware store such as Home Depot, etc. and I rarely lose sections
when HE staining on paraffin embedded bones.  

Right before sectioning, I take a pipette using the 5% titebond and dab
all the corners and center of the slide, take a kimwipe and spread it
zigzag across one way all the way down the slide and then turn the slide
1/4 and do it again.  Let it air dry a few seconds and then pick up your
section.  

I should mention that I first clean the slides in acid alcohol for 5
minutes, followed by two 95% rinses for 5 minutes each and then dry in a
60 degree oven for 1 hour.  Remove from oven, cool, wrap individually
with either a kimwipe or paper towel to keep them protected from dust
and store in an empty coverslip box.

Hope this helps.

Peggy DiCarlo HT (ASCP)
Orthopedic Bone Lab
Buffalo General Hospital
Buffalo, NY  14203
716-859-1293

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Williams, Sean
Sent: Tuesday, August 18, 2009 07:51
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] bone sections

Hi Everyone

Could any one tell me how they keep bone sections adhered to slides when
staining. I'Ve tried polysine slides, pva glue slides, different ways of
drying the sections and for different lenghts of time but nothing seems
to work for every section.
I'm only staining them with HE.

Thanks
Marie O'Brien (liverpool vet path - histology)
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Re: [Histonet] bone sections

[Jack Ratliff]

Are you working with resin or paraffin sections?

Jack

Sent from my iPhone

On Aug 18, 2009, at 6:50 AM, Williams, Sean s.a.willi...@liverpool.ac.uk 
 wrote:



Hi Everyone

Could any one tell me how they keep bone sections adhered to slides  
when staining. I'Ve tried polysine slides, pva glue slides,  
different ways of drying the sections and for different lenghts of  
time but nothing seems to work for every section.

I'm only staining them with HE.

Thanks
Marie O'Brien (liverpool vet path - histology)
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Re: RE: [Histonet] Bone marrows

[John Kiernan]

Patsy Ruegg pru...@ihctech.net  wrote:
 I prefer formic acid decal for IHC work.  You could make 
 your own 5% but I
 always buy it from Decal Chemicals it is called Immunocal.

Why? Does it cost you less to buy pre-diluted formic acid? 
 
John Kiernan
Department of Anatomy  Cell Biology
The University of Western Ontario
London, Canada
= = =
 
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RE: [Histonet] Bone marrows

[Patsy Ruegg]

Patti,

I prefer formic acid decal for IHC work.  You could make your own 5% but I
always buy it from Decal Chemicals it is called Immunocal.  I have found the
rapid decal solutions are usually nitric acid and not good for IHC.

Best regards,
Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Patti
Loykasek
Sent: Friday, July 10, 2009 1:38 PM
To: histonet
Subject: [Histonet] Bone marrows

Hi All. I was wondering what everyone's current favorite bone marrow decal
solution/method is. Including commercially available decal solutions. We
will be doing IHC after fixation/decal. Thanks for the input (as always).


Patti Loykasek BS, HTL, QIHC
Clinical Lab Supervisor
PhenoPath Laboratories
Seattle, WA




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RE: [Histonet] Bone!

[Kemlo Rogerson]

As I say I don't have experience of preservatives but question if the
effects of ethanol (70%) over a long time may also 'overfix' the bone?
Anyway 70% ethanol has perfectly preserved me!!


Kemlo Rogerson  
e-mail kemloroger...@nhs.net if not at work.
DD   01934 647057 or extension 3311 Mob 07749 754194; 
Embrace uncertainty. Hard problems rarely have easy solutions. --Jonah
Lehrer
This e-mail is confidential and privileged. If you are not the intended
recipient please accept my apologies; please do not disclose, copy or
distribute information in this e-mail or take any action in reliance on
its contents: to do so is strictly prohibited and may be unlawful.
Please inform me that this message has gone astray before deleting it.
Thank you for your co-operation

 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rittman,
Barry R
Sent: 22 May 2009 13:21
To: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone!

Lets face it there is no really ideal medium for long term storage.
However this depends on what techniques you want to carry out later and
when.
I agree that 10% NBF is a good fixative for bone, I would definitely not
advise leaving it in this fixative for extended periods of time.
The reason is that formalin continues cross linking.
This means that many stains and immunohistochemistry may no longer be
possible depending on the amount of time left in fixative.
Also Kemlo is correct in that temporary formalin bonds can be broken by
extended washing in watre. This only applies to the weak temporary bonds
and not to the more stable bonds. The longer the fixing the greater the
number of stable bonds.
I would recommend having a mixture of 70% ethanol plus glycerin up to
10-20%.  The glycerin will prevent any drying out of the solution over
extended periods of tim.
Barry



From: histonet-boun...@lists.utsouthwestern.edu
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kemlo Rogerson
[kemlo.roger...@waht.swest.nhs.uk]
Sent: Friday, May 22, 2009 2:24 AM
To: Robert Edward Pogue
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone!

Probably of all the fixatives 10% neutral buffered formalin is least
likely to overfix and I've left tissues in it for years. Formalin, as
you know, is one of those fixatives that you can wash out. There are
preservatives, rather than fixatives, you could try but I don't have
experience of those; my best advise is formalin but make sure that it is
neutral and buffered. I think the effects of pH would be more damaging
than the relatively 'soft' formalin fixation.



Kemlo Rogerson
e-mail kemloroger...@nhs.net if not at work.
DD   01934 647057 or extension 3311 Mob 07749 754194;
Embrace uncertainty. Hard problems rarely have easy solutions. --Jonah
Lehrer This e-mail is confidential and privileged. If you are not the
intended recipient please accept my apologies; please do not disclose,
copy or distribute information in this e-mail or take any action in
reliance on its contents: to do so is strictly prohibited and may be
unlawful.
Please inform me that this message has gone astray before deleting it.
Thank you for your co-operation



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robert
Edward Pogue
Sent: 21 May 2009 16:33
To: Kemlo Rogerson
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone!

Thanks Kemlo...

I thought of that, but was worried about over-fixation,- what do you
think?

Redward.


--

In 10% neutral buffered formalin?



Kemlo Rogerson
e-mail kemloroger...@nhs.net if not at work.
DD   01934 647057 or extension 3311 Mob 07749 754194;
Embrace uncertainty. Hard problems rarely have easy solutions. --Jonah
Lehrer This e-mail is confidential and privileged. If you are not the
intended recipient please accept my apologies; please do not disclose,
copy or distribute information in this e-mail or take any action in
reliance on its contents: to do so is strictly prohibited and may be
unlawful.
Please inform me that this message has gone astray before deleting it.
Thank you for your co-operation



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robert
Edward Pogue
Sent: 21 May 2009 14:47
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone!

Friends,

Can someone recommend to me how to store bones (juvenile rat femur) that
I have fixed and decalcified, but am not yet ready to cut (they're for
vibratome, so not embedded).

Thanks!

Redward.

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RE: [Histonet] Bone!

[Kemlo Rogerson]

Probably of all the fixatives 10% neutral buffered formalin is least
likely to overfix and I've left tissues in it for years. Formalin, as
you know, is one of those fixatives that you can wash out. There are
preservatives, rather than fixatives, you could try but I don't have
experience of those; my best advise is formalin but make sure that it is
neutral and buffered. I think the effects of pH would be more damaging
than the relatively 'soft' formalin fixation.



Kemlo Rogerson  
e-mail kemloroger...@nhs.net if not at work.
DD   01934 647057 or extension 3311 Mob 07749 754194; 
Embrace uncertainty. Hard problems rarely have easy solutions. --Jonah
Lehrer
This e-mail is confidential and privileged. If you are not the intended
recipient please accept my apologies; please do not disclose, copy or
distribute information in this e-mail or take any action in reliance on
its contents: to do so is strictly prohibited and may be unlawful.
Please inform me that this message has gone astray before deleting it.
Thank you for your co-operation

 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robert
Edward Pogue
Sent: 21 May 2009 16:33
To: Kemlo Rogerson
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone!

Thanks Kemlo...

I thought of that, but was worried about over-fixation,- what do you
think?

Redward.


--
 
In 10% neutral buffered formalin?



Kemlo Rogerson  
e-mail kemloroger...@nhs.net if not at work.
DD   01934 647057 or extension 3311 Mob 07749 754194; 
Embrace uncertainty. Hard problems rarely have easy solutions. --Jonah
Lehrer This e-mail is confidential and privileged. If you are not the
intended recipient please accept my apologies; please do not disclose,
copy or distribute information in this e-mail or take any action in
reliance on its contents: to do so is strictly prohibited and may be
unlawful.
Please inform me that this message has gone astray before deleting it.
Thank you for your co-operation

 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robert
Edward Pogue
Sent: 21 May 2009 14:47
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone!

Friends,

Can someone recommend to me how to store bones (juvenile rat femur) that
I have fixed and decalcified, but am not yet ready to cut (they're for
vibratome, so not embedded).

Thanks!

Redward.

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RE: [Histonet] Bone!

[Rittman, Barry R]

Lets face it there is no really ideal medium for long term storage.
However this depends on what techniques you want to carry out later and when.
I agree that 10% NBF is a good fixative for bone, I would definitely not advise 
leaving it in this fixative for extended periods of time.
The reason is that formalin continues cross linking.
This means that many stains and immunohistochemistry may no longer be possible 
depending on the amount of time left in fixative.
Also Kemlo is correct in that temporary formalin bonds can be broken by 
extended washing in watre. This only applies to the weak temporary bonds and 
not to the more stable bonds. The longer the fixing the greater the number of 
stable bonds.
I would recommend having a mixture of 70% ethanol plus glycerin up to 10-20%.  
The glycerin will prevent any drying out of the solution over extended periods 
of tim.
Barry



From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kemlo Rogerson 
[kemlo.roger...@waht.swest.nhs.uk]
Sent: Friday, May 22, 2009 2:24 AM
To: Robert Edward Pogue
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone!

Probably of all the fixatives 10% neutral buffered formalin is least
likely to overfix and I've left tissues in it for years. Formalin, as
you know, is one of those fixatives that you can wash out. There are
preservatives, rather than fixatives, you could try but I don't have
experience of those; my best advise is formalin but make sure that it is
neutral and buffered. I think the effects of pH would be more damaging
than the relatively 'soft' formalin fixation.



Kemlo Rogerson
e-mail kemloroger...@nhs.net if not at work.
DD   01934 647057 or extension 3311 Mob 07749 754194;
Embrace uncertainty. Hard problems rarely have easy solutions. --Jonah
Lehrer
This e-mail is confidential and privileged. If you are not the intended
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-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robert
Edward Pogue
Sent: 21 May 2009 16:33
To: Kemlo Rogerson
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Bone!

Thanks Kemlo...

I thought of that, but was worried about over-fixation,- what do you
think?

Redward.


--

In 10% neutral buffered formalin?



Kemlo Rogerson
e-mail kemloroger...@nhs.net if not at work.
DD   01934 647057 or extension 3311 Mob 07749 754194;
Embrace uncertainty. Hard problems rarely have easy solutions. --Jonah
Lehrer This e-mail is confidential and privileged. If you are not the
intended recipient please accept my apologies; please do not disclose,
copy or distribute information in this e-mail or take any action in
reliance on its contents: to do so is strictly prohibited and may be
unlawful.
Please inform me that this message has gone astray before deleting it.
Thank you for your co-operation



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robert
Edward Pogue
Sent: 21 May 2009 14:47
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone!

Friends,

Can someone recommend to me how to store bones (juvenile rat femur) that
I have fixed and decalcified, but am not yet ready to cut (they're for
vibratome, so not embedded).

Thanks!

Redward.

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RE: [Histonet] Bone!

[Robert Edward Pogue]

Thanks Kemlo...

I thought of that, but was worried about over-fixation,- what do you think?

Redward.


--
 
In 10% neutral buffered formalin?



Kemlo Rogerson  
e-mail kemloroger...@nhs.net if not at work.
DD   01934 647057 or extension 3311 Mob 07749 754194; 
Embrace uncertainty. Hard problems rarely have easy solutions. --Jonah
Lehrer
This e-mail is confidential and privileged. If you are not the intended
recipient please accept my apologies; please do not disclose, copy or
distribute information in this e-mail or take any action in reliance on
its contents: to do so is strictly prohibited and may be unlawful.
Please inform me that this message has gone astray before deleting it.
Thank you for your co-operation

 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robert
Edward Pogue
Sent: 21 May 2009 14:47
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone!

Friends,

Can someone recommend to me how to store bones (juvenile rat femur) that
I have fixed and decalcified, but am not yet ready to cut (they're for
vibratome, so not embedded).

Thanks!

Redward.

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RE: [Histonet] Bone saw

[Jack Ratliff]

IMEB also has a Bone Band Saw.

 

http://www.imebinc.com/Item/BBS-82203.htm

 

Jack


 
 Date: Thu, 21 May 2009 09:05:50 -0700
 From: cb...@memorialcare.org
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Bone saw
 
 Good Morning,
 I was wondering if anyone can help me find a really good saw
 for bones (femoral/humeral heads mainly). We currently have a MarMed
 bone saw that works great for knees and such but it's just not strong
 enough for the femurs. 
 Thank you,
 
 Christine Bark HT(ASCP) CM
 Lead Histotech, Pathology
 Saddleback Memorial Medical Center
 949-452-3548
 cb...@memorialcare.org
 
 
 
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 Confidentiality Notice: This e-mail message, including any attachments, is 
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 privileged information. Any unauthorized review, use, disclosure or 
 distribution 
 is prohibited. If you are not the intended recipient, please contact the 
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RE: [Histonet] bone tissue

[Jack Ratliff]

Haupts Gelatin works really well at a 1:1 ratio (Concentrate:50% EtOH). The 
only limitation is background staining from say a hematoxylin counterstain 
after the IHC. You can purchase the concentrate from Fisher (Haupts Fixative 
#785-71).

 

Jack


 
 Date: Thu, 21 May 2009 10:12:23 -0500
 From: reuel.corne...@tsrh.org
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] bone tissue
 
 Can somebody help me with my bone tissue to remain intact during antigen 
 retrieval for IHC staining. I have used subbed slides but It did not help me. 
 Is there more better adhesive. Does Haupts Gelatin works better? I know this 
 is a hundred year old question and hopefully this problem have been resolved 
 since my undated knowledge could remember. 
 I would like to thank everybody for responding to my last e-mail on gross 
 photography. It gives me more idea what to purchase. Thanks again 
 histonetters and I really appreciate your help.
 
 
 
 Reuel Cornelia, BS MT, AMT
 Cellular Pathology
 Texas Scottish Rite Hospital for Children
  Welborn Street
 Dallas, TX 75219
 Tel: 214-559-7766
 fax: 214-559-7768
 
 
 
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RE: [Histonet] Bone!

[Jack Ratliff]

I do not routinely perform decalcified bone preparations as I predominantly 
utilize undemineralized resin (MMA) applications, so maybe someone else can 
better respond to this question. However, I would say that you could store your 
bone in 70% EtOH until you are ready to process, but the length of time in 
solution storage may cause you problems down the road since you have already 
decalcified. I think that a prolonged storage time might make your bone hard 
againif that makes any senseand cause you a fit later when you go to 
the microtome. Typically, I store undemineralized bones at either 10% NBF or 
70% EtOH depending upon what I care to see at the microscope.

 

Jack
 
 Date: Thu, 21 May 2009 10:46:59 -0300
 From: redw...@ucb.br
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Bone!
 
 Friends,
 
 Can someone recommend to me how to store bones (juvenile rat femur) that I 
 have fixed and decalcified, but am not yet ready to cut (they're for 
 vibratome, so not embedded).
 
 Thanks!
 
 Redward.
 
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Re: [Histonet] Bone marrow aspirates from mouse

[Rene J Buesa]

Use the same thing you would use for a blood smear: methanol 5 minutes and air 
dry it.
René J.

--- On Mon, 3/9/09, Vanessa J. Phelan vjp2...@columbia.edu wrote:

From: Vanessa J. Phelan vjp2...@columbia.edu
Subject: [Histonet] Bone marrow aspirates from mouse
To: histonet@lists.utsouthwestern.edu
Date: Monday, March 9, 2009, 11:39 AM

Hi everyone,

I was wondering if anyone had any experience with taking bone marrow
aspirates from mice for analysis, staining with Giemsa and for IHC?
My query is, once the aspirate has been taken and then cytospinned down and
spread on the slide, what fixative would you use at this point before
staining? Maybe formal alcohol? PFA? Or maybe even cytofix spray? also where
to store the slides? -80 freezer?

Any help on this would be much appreciated, as this procedure has not been
carried out here before and I have no experience in this either.

Thanks a mill, 

Vanessa


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Re: [Histonet] Bone Marrow Smears

[Rene J Buesa]

In bone marrow smears it is caused by improper fixation. In the same way that 
Cabot rings in red blood cells are caused by improper fixation with methanol.
René J.

--- On Thu, 2/12/09, Sheila Haas micropathl...@yahoo.com wrote:

From: Sheila Haas micropathl...@yahoo.com
Subject: [Histonet] Bone Marrow Smears
To: histonet@lists.utsouthwestern.edu
Date: Thursday, February 12, 2009, 10:12 AM

Hi all. I have a pathologist complaining about naked nuclei
in his bone marrow smears. It's fairly random, some are great and
some are naked. Anyone heard of this and have any idea what
may be causing this nakedness?
Any input would be helpful.Thank you.
 
Sheila Haas
Laboratory Supervisor
Micro Path Laboratories



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RE: [Histonet] Bone Marrow smears fixation

[Joyce Cline]

We give our Hematology department Superfrost+ slides to use for smears,
because we had the same problem. We also went back to the old fashioned way
of fixing the smears. We use a coplin jar with a small piece of papertowel
in the bottom and place a few drops of formalin in the coplin jar. The
smears are fixed in formalin fumes. We had a lot of smears that were not
staining for iron before using the methanol method.

Joyce Cline, H.T. (ASCP)
Technical Specialist
Hagerstown Medical Lab.
301-665-4980
fax 301-665-4941


-Original Message-
From: [EMAIL PROTECTED]
[mailto:[EMAIL PROTECTED] On Behalf Of Georgine
Whitman
Sent: Friday, November 14, 2008 9:51 AM
To: [EMAIL PROTECTED]
Subject: [Histonet] Bone Marrow smears fixation

we are trying to do an iron  stain on  our bone marrow smears on the Ventana
Nexus. We are having trouble with the specimen staying on the slide. I
personally have not done iron on smears in years.  Our hematology department
has been doing them by hand and they are fixed in methanol alcohol.  
  Please Help Thank
You Georgine
 



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RE: [Histonet] bone marrow biopsies

[Della Speranza, Vinnie]

You are being placed in a difficult position that will only translate into 
physician unhappiness, so even though they are in a hurry (and usually for 
valid clinical reasons) the fact is that compromises will affect something else.

I'm assuming you are a one shift operation. My lab is 24 hours, which allows us 
sufficient time for fixation and decalcification while enabling slides to be 
out the next morning.

While Dr. Richmond is correct that there are faster decalcifiers, I'm guessing 
they want immunohistochemistry in at least some cases and the mineral acids 
will destroy your hopes of good immuno staining. We use 10% formic acid and 
still have to decalcify for about six hours in order to have blocks that 
section well.

If you had an evening shift I'd suggest shortening your processing time. 
Shortening fixation time is counter productive to good morphology and good IHC.

Lastly, you don't indicate if your bone marrows arrive at all times of the day. 
If you are trying to complete fixation and decalcification on specimens 
arriving in the afternoon, all to be on the processor at the end of your shift, 
 you end up in the position you find yourself currently, with blocks poorly 
decalcified that section poorly.

The only way to make this work to everyone's satisfaction is to have a longer 
work day, if it is possible to stagger work shifts. This also presumes that you 
have multiple tissue processors and can place your bone marrows on a shorter 
cycle, perhaps along with other biopsies.

Others here may be able to advise you regarding using microwave technology for 
fixation and decalcification which may ultimately save the day for you.



Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, South Carolina 29425
Tel: (843) 792-6353
Fax: (843) 792-8974
 

-Original Message-
From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Cynthia Robinson
Sent: Thursday, October 30, 2008 11:44 AM
To: histonet
Subject: [Histonet] bone marrow biopsies

We are currently using 10% formalin fixation on our bone marrow cores.  We fix 
for 2 hours minimum prior to decal.  We are using Immunocal from Decal Corp. 
for 2-4 hrs followed with processing overnight in VIP.  Cores are still crunchy 
upon sectioning and we are doing surface decal for up to 30 min. Our paths want 
cores turned out within 24 hrs following procedure. Any suggestions? 
 
Thanks.
Cindi 
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