Discussion:
Histonet Digest, Vol 140, Issue 5
(too old to reply)
Kevin Bennett
2015-07-07 18:06:29 UTC
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Raw Message
Alkaline Phosphatse bubbles

Hi Tim,
Use Dako ultramount. The bubbles are caused by the naphthyl breaking down
and releasing CO2 under the coverslip. Apply small amount of the ultramount
to cover the muscle tissue and bake in 65 degree oven of 15 to 20 minutes.
If you bake it too long the myofibers will draw up and damage the
morphology of the section.
Kevin
On Jul 7, 2015 1:29 PM, <histonet-***@lists.utsouthwestern.edu> wrote:

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> Today's Topics:
>
> 1. Specimens lost during processing. (STEVEN PINHEIRO)
> 2. Preventing Bubbles in Alkaline Phosphatase (Morken, Timothy)
> 3. Histology on Rat spine. (Clough, Bret)
> 4. 12ml tubes for Dako IHC stainers (Sally Price)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 6 Jul 2015 18:28:21 +0000
> From: STEVEN PINHEIRO <***@lumc.edu>
> To: "***@lists.utsouthwestern.edu"
> <***@lists.utsouthwestern.edu>
> Subject: [Histonet] Specimens lost during processing.
> Message-ID:
> <
> ***@SB01MSTMBX01.sb.trinity-health.org
> >
>
> Content-Type: text/plain; charset="us-ascii"
>
> All,
> Looking for help in analyzing the entire scope of the process. There is
> not much published data (that I can find) and I am hoping this group can
> lend some expertise.
> Our rate is higher than we would like it to be. There is no consistent
> size at risk although GI and Derm biopsies are the biggest involved group.
> We have broken it down into steps.
>
> 1. Can be lost at grossing- either never loaded into the cassette at
> all, or cassette was discarded. Thus we hold on to our waste and can search
> for misdirected cassettes if need be.
>
> 2. Lost in the processor itself. Most are wrapped. If large enough
> not to be wrapped, we would not expect the processor to eat them, so assume
> cassette lid not properly closed. Frankly the highest number of losses
> we're seeing is no tissue found in cassette by embedders.
>
> 3. I am being told that we can't use micromesh cassettes in our
> microwave processors (Milestone Pathos) and want to know if anyone is.
>
> 4. Tissue not seen at embedding. Again no way to tell when the
> tissue disappeared. We know that tiny tissue can spring out during the
> opening at embedding but I don't know how else to examine or limit this
> step.
>
> 5. Tissue can be exhausted during microtomy. Rare but noteworthy.
> I am hoping people can tell me about their procedures for dealing with
> "specimens that don't survive processing", what safeguards they have in
> place, and to some extent what your own lab percentage or experience is.
> Apologies in advance for the length of the message, but could really use
> your help.
>
>
> Steven Pinheiro, MBA, MLS(ASCP)DLMCM
> Manager Anatomic Pathology and Cytology
> Loyola University Medical Center
> 2160 S First Ave, Bldg 110 Rm 2214
> Maywood, IL 60153
> 708-327-2642 (O)
> 708-327-2620 (F)
> ***@lumc.edu<mailto:***@lumc.edu>
>
> "You must do the thing you think you cannot do"
> E. Roosevelt
>
>
> Confidentiality Notice:
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>
> ------------------------------
>
> Message: 2
> Date: Mon, 6 Jul 2015 19:39:35 +0000
> From: "Morken, Timothy" <***@ucsf.edu>
> To: Histonet <***@lists.utsouthwestern.edu>
> Subject: [Histonet] Preventing Bubbles in Alkaline Phosphatase
> Message-ID:
> <***@ex07.net.ucsf.edu>
> Content-Type: text/plain; charset="iso-8859-1"
>
> All knowing Histonet....
>
> We started doing alkaline phosphatase on muscle frozen a little while ago
> and have had an issue with apparent air bubbles forming over the tissue.
> Not trapped air from coverslipping, but forming from apparent reaction in
> the tissue. Does anyone have experience with this and a solution?
>
> Images:
> http://histosearch.com/imageupload/alkaline-phosphatase-bubbles/
>
> We tried longer formalin fixation after the stain (10 min) and an acetic
> acid rinse after the tap water, after the formalin. Still the same problem
>
> The only mention I have found about this is a Histochemistry text by Lojda
> from 1979 that says to fix in formalin for several hours after staining to
> prevent bubble formation. Does anyone have anything shorter? I don't really
> remember seeing this when I did these stains 20 years ago....
>
>
>
> Our procedure (from a method given to our neuropath folks by a group in
> Australia):
>
> Frozen sections of muscle
>
>
> Borate Buffer, pH 8.8:
> 0.992 g Boric Acid
> 2.28 g Sodium Tetraborate
> 200 ml distilled water
> Mix well. Adjust to pH 8.8. Store at 4?C.
>
> 0.1M Magnesium Sulphate
> 0.6 g Magnesium sulphate, anhydrous (M7506-500G)
> 50 ml distilled water
> Store at 4?C.
>
> ALP Incubation Solution:
> 15 ml Borate Buffer
> 2 ml 0.1M Magnesium Sulphate
> 16.5 mg 1-naphthyl phosphate
> 16.5 mg Fast Blue RR
> Mix in well order. Filter.
>
> Glycerin Jelly Mounting Media
>
>
> 1. Place glycerin jelly in 60?C oven to liquify
> 2. Air dry slides 15 minutes.
> 3. Incubate in filtered ALP Incubation Solution at 37?C...45 minutes.
> 4. Rinse in tap water.
> 5. Change to 10% formalin...1 min
> 6. Rinse in tap water.
> 7. Air dry.
> 8. Coverslip with Glycerin Jelly.
>
>
> Tim Morken
> Pathology Site Manager, Parnassus
> Supervisor, Electron Microscopy/Neuromuscular Special Studies
> Department of Pathology
> UC San Francisco Medical Center
> 505 Parnassus Ave, Box 1656
> Room S570
> San Francisco, CA 94143
>
> (415) 353-1266 (ph)
> (415) 514-3403 (fax)
> ***@ucsfmedctr.org<mailto:***@ucsfmedctr.org>
>
>
>
> ------------------------------
>
> Message: 3
> Date: Tue, 7 Jul 2015 03:05:14 +0000
> From: "Clough, Bret" <***@medicine.tamhsc.edu>
> To: "Histonet list serv. (***@lists.utsouthwestern.edu)"
> <***@lists.utsouthwestern.edu>
> Subject: [Histonet] Histology on Rat spine.
> Message-ID:
> <***@CSR-Mail1.ad.tamhsc.edu>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hello everyone,
>
>
>
> I have some rat spines that I need to decal, process, and embed for
> sectioning. Is there someone in the histonet community that would be
> willing to either share with me their protocol or at least give me guidance
> with these spines. These are larger than any tissue that I have ever had to
> process. The spines have been fixed in Carson's fixative. Any help would
> greatly be appreciated.
>
>
>
> Sincerely,
>
> Bret Clough
>
> Texas A&M Health Science Center
>
> Temple, Texas
>
>
> ------------------------------
>
> Message: 4
> Date: Tue, 7 Jul 2015 12:29:43 -0400
> From: Sally Price <***@gmail.com>
> To: ***@lists.utsouthwestern.edu
> Subject: [Histonet] 12ml tubes for Dako IHC stainers
> Message-ID:
> <
> ***@mail.gmail.com>
> Content-Type: text/plain; charset=UTF-8
>
> Can anyone recommend a vendor for the 12ml tubes that are used on original
> Dako stainers other than Dako?
>
>
>
> --
> Sally Price
>
>
> ------------------------------
>
> Subject: Digest Footer
>
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> ------------------------------
>
> End of Histonet Digest, Vol 140, Issue 5
> ****************************************
>
Terri Braud
2015-07-07 18:24:42 UTC
Permalink
Raw Message
In the article reference below, they estimate tissue loss at 7%. In my lab, our pathologists would croak if we had tissue loss at this high of a rate. But with that said, we do have a 0.8% "loss of tissue". Without exception, they have been extremely small pieces that could have been mucous, and they have been frequently described as "possibly too small to survive processing".
We've had a cassette discarded during processing twice in 10 years, however we save all trash until the tissue has been embedded and the piece count and block log verified. In both cases, we were able to go back to the gross station trash and retrieve the cassette.
We use blue pads to hold bx tissue in the cassette but the pieces are placed on the pad as if on a grid.
One piece: direct center
Two pieces: [:]
Three pieces: [:.]
Four pieces: [::] and so on.
If they are wrapped in lens paper, we use a special folding technique and write a target on the paper and place the tissue on the bulls eye.
Large sections of skin are held in place with a single blue pad and are always placed in the cassette the same way at gross.
For a bisected punch, [:]
For a serial sectioned ellipse submitted in one cassette, it is tip, x-sect, x-sect, tip [.//.]
For a multicassette skin ellipse, it is both tips in the first cassette, and x-sections in the following serial cassettes.
If it's there, then the grosser should have communicated to the tech, "may not survive processing" or "very tiny" on the gross worksheet notes. Often, the gross person will have another set of eyes look at a questionable piece, if they think it may not survive processing.
We hold techs accountable to match the piece count and it is inexcusable to lose a section of skin at embedding or cut through a specimen.
If a pathologist orders recuts where tissue might be cut away, then the tech must notify the pathologist before proceding and a note is placed in the computer that the tissue will be cut away.
We hold all papers or blue pads of missing specimens until the gross dictation is reviewed and the pathologist notified.
With special care at grossing, we seldom have an incident because the embedders know exactly where to find the tissue and if the tissue is possibly mucous or a spec, that may not survive processing, then it usually is noted at gross and is reported as "tissue not sufficient for processing" with a suggestion to repeat biopsy based on clinical findings.
Reference:
Owens, SR.; Wiehagen, L.; Simmons, C.; Sikorova, A.; Stewart, W.; Kelly, S.; Nestler, R.; Yousem, SA. (Dec 2011). "Numerical fidelity of endoscopic biopsy fragments in the processing sequence of a university surgical pathology laboratory.". Arch Pathol Lab Med 135 (12): 1561-4. doi:10.5858/arpa.2011-0020-OA. PMID 22129184.
Sorry to be so long winded, but I hope this helps.
Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Holy Redeemer Hospital Laboratory
1648 Huntingdon Pike
Meadowbrook, PA 19046
Ph: 215-938-3676
Fax: 215-938-3874

-----Original Message-----

1. Specimens lost during processing. (STEVEN PINHEIRO)
Message: 1
Date: Mon, 6 Jul 2015 18:28:21 +0000
From: STEVEN PINHEIRO <***@lumc.edu>
All,
Looking for help in analyzing the entire scope of the process. There is not much published data (that I can find) and I am hoping this group can lend some expertise.
Our rate is higher than we would like it to be. There is no consistent size at risk although GI and Derm biopsies are the biggest involved group. We have broken it down into steps.

1. Can be lost at grossing- either never loaded into the cassette at all, or cassette was discarded. Thus we hold on to our waste and can search for misdirected cassettes if need be.

2. Lost in the processor itself. Most are wrapped. If large enough not to be wrapped, we would not expect the processor to eat them, so assume cassette lid not properly closed. Frankly the highest number of losses we're seeing is no tissue found in cassette by embedders.

3. I am being told that we can't use micromesh cassettes in our microwave processors (Milestone Pathos) and want to know if anyone is.

4. Tissue not seen at embedding. Again no way to tell when the tissue disappeared. We know that tiny tissue can spring out during the opening at embedding but I don't know how else to examine or limit this step.

5. Tissue can be exhausted during microtomy. Rare but noteworthy.
I am hoping people can tell me about their procedures for dealing with "specimens that don't survive processing", what safeguards they have in place, and to some extent what your own lab percentage or experience is.
Apologies in advance for the length of the message, but could really use your help.


Steven Pinheiro, MBA, MLS(ASCP)DLMCM
Manager Anatomic Pathology and Cytology
Loyola University Medical Center
2160 S First Ave, Bldg 110 Rm 2214
Maywood, IL 60153
708-327-2642 (O)
708-327-2620 (F)
***@lumc.edu<mailto:***@lumc.edu>

"You must do the thing you think you cannot do"
E. Roosevelt
Simmons, Christopher
2015-07-07 19:13:21 UTC
Permalink
Raw Message
Thanks for citing the article I helped participate in!


Sent from my iPhone

> On Jul 7, 2015, at 2:25 PM, Terri Braud <***@holyredeemer.com> wrote:
>
>
> In the article reference below, they estimate tissue loss at 7%. In my lab, our pathologists would croak if we had tissue loss at this high of a rate. But with that said, we do have a 0.8% "loss of tissue". Without exception, they have been extremely small pieces that could have been mucous, and they have been frequently described as "possibly too small to survive processing".
> We've had a cassette discarded during processing twice in 10 years, however we save all trash until the tissue has been embedded and the piece count and block log verified. In both cases, we were able to go back to the gross station trash and retrieve the cassette.
> We use blue pads to hold bx tissue in the cassette but the pieces are placed on the pad as if on a grid.
> One piece: direct center
> Two pieces: [:]
> Three pieces: [:.]
> Four pieces: [::] and so on.
> If they are wrapped in lens paper, we use a special folding technique and write a target on the paper and place the tissue on the bulls eye.
> Large sections of skin are held in place with a single blue pad and are always placed in the cassette the same way at gross.
> For a bisected punch, [:]
> For a serial sectioned ellipse submitted in one cassette, it is tip, x-sect, x-sect, tip [.//.]
> For a multicassette skin ellipse, it is both tips in the first cassette, and x-sections in the following serial cassettes.
> If it's there, then the grosser should have communicated to the tech, "may not survive processing" or "very tiny" on the gross worksheet notes. Often, the gross person will have another set of eyes look at a questionable piece, if they think it may not survive processing.
> We hold techs accountable to match the piece count and it is inexcusable to lose a section of skin at embedding or cut through a specimen.
> If a pathologist orders recuts where tissue might be cut away, then the tech must notify the pathologist before proceding and a note is placed in the computer that the tissue will be cut away.
> We hold all papers or blue pads of missing specimens until the gross dictation is reviewed and the pathologist notified.
> With special care at grossing, we seldom have an incident because the embedders know exactly where to find the tissue and if the tissue is possibly mucous or a spec, that may not survive processing, then it usually is noted at gross and is reported as "tissue not sufficient for processing" with a suggestion to repeat biopsy based on clinical findings.
> Reference:
> Owens, SR.; Wiehagen, L.; Simmons, C.; Sikorova, A.; Stewart, W.; Kelly, S.; Nestler, R.; Yousem, SA. (Dec 2011). "Numerical fidelity of endoscopic biopsy fragments in the processing sequence of a university surgical pathology laboratory.". Arch Pathol Lab Med 135 (12): 1561-4. doi:10.5858/arpa.2011-0020-OA. PMID 22129184.
> Sorry to be so long winded, but I hope this helps.
> Terri L. Braud, HT(ASCP)
> Anatomic Pathology Supervisor
> Holy Redeemer Hospital Laboratory
> 1648 Huntingdon Pike
> Meadowbrook, PA 19046
> Ph: 215-938-3676
> Fax: 215-938-3874
>
> -----Original Message-----
>
> 1. Specimens lost during processing. (STEVEN PINHEIRO)
> Message: 1
> Date: Mon, 6 Jul 2015 18:28:21 +0000
> From: STEVEN PINHEIRO <***@lumc.edu>
> All,
> Looking for help in analyzing the entire scope of the process. There is not much published data (that I can find) and I am hoping this group can lend some expertise.
> Our rate is higher than we would like it to be. There is no consistent size at risk although GI and Derm biopsies are the biggest involved group. We have broken it down into steps.
>
> 1. Can be lost at grossing- either never loaded into the cassette at all, or cassette was discarded. Thus we hold on to our waste and can search for misdirected cassettes if need be.
>
> 2. Lost in the processor itself. Most are wrapped. If large enough not to be wrapped, we would not expect the processor to eat them, so assume cassette lid not properly closed. Frankly the highest number of losses we're seeing is no tissue found in cassette by embedders.
>
> 3. I am being told that we can't use micromesh cassettes in our microwave processors (Milestone Pathos) and want to know if anyone is.
>
> 4. Tissue not seen at embedding. Again no way to tell when the tissue disappeared. We know that tiny tissue can spring out during the opening at embedding but I don't know how else to examine or limit this step.
>
> 5. Tissue can be exhausted during microtomy. Rare but noteworthy.
> I am hoping people can tell me about their procedures for dealing with "specimens that don't survive processing", what safeguards they have in place, and to some extent what your own lab percentage or experience is.
> Apologies in advance for the length of the message, but could really use your help.
>
>
> Steven Pinheiro, MBA, MLS(ASCP)DLMCM
> Manager Anatomic Pathology and Cytology
> Loyola University Medical Center
> 2160 S First Ave, Bldg 110 Rm 2214
> Maywood, IL 60153
> 708-327-2642 (O)
> 708-327-2620 (F)
> ***@lumc.edu<mailto:***@lumc.edu>
>
> "You must do the thing you think you cannot do"
> E. Roosevelt
>
>
>
>
> _______________________________________________
> Histonet mailing list
> ***@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Hugh Luk
2015-07-09 00:28:34 UTC
Permalink
Raw Message
Hi Sally,

Sarstedt (Germany) used to make these. Cat #62.732.512, but my web inquiry leads to a null field(??). Per Histosearch, distributors used to include Labvision (purchased by Thermo) and Fisher (also purchased by Thermo). My search on Thermo-Fisher shows they still have (excerpt below), but my institutional price is $371 for 100-15ml tubes. Hope your price is less ...traumatic.

If your price is also high, Sarstedt's Nevada or North Carolina office might be helpful? [Sarstedt.com, (West Coast Sales) NV-USA, 800-257-5101 (only in the USA), E-Mail: ***@sarstedt.us]



Anyone else found a 750 mm x 200 mm (hopefully) sterile vial or tube that can be used as a replacement? I am having no luck.

Hugh
Hawaii

ps. not affiliated with any groups above. Just old.

> ------------------------------
>
> Date: Tue, 7 Jul 2015 12:29:43 -0400
> From: Sally Price <***@gmail.com>
> To: ***@lists.utsouthwestern.edu
> Subject: [Histonet] 12ml tubes for Dako IHC stainers
> Message-ID:
> <***@mail.gmail.com>
> Content-Type: text/plain; charset=UTF-8
>
> Can anyone recommend a vendor for the 12ml tubes that are used on original
> Dako stainers other than Dako?
> --
> Sally Price






via histosearch Tue Jul 24 09:18:40 CDT 2007


From Joanne Mauger
mauger <@t> email.chop.edu




Hazel,

We order reagent vials from Labvision through Fisher#S20002. You get
100-15 ml. vials for $40. They are exactly the same as the ones from
Dako.

Jo
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