[Histonet] -80 freezer question

[Paula Sicurello]

Dear Netters,

What is the rationale for storing frozen patient samples at -80?  Is there
a range that keeps the sample viable but not as cold?

Our -80 (which we just inherited) is struggling and I would like to raise
the temperature a smidge to ease the stress on the compressor.

Thanks!

Paula

-- 
Paula Sicurello, HTL (ASCP)
Supervisor, Clinical Electron Microscopy Laboratory
Duke University Health System
Rm.#251M, Duke South, Green Zone
Durham, North Carolina 27710
P:  919.684.2091

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[Histonet] problem with double IF of brain section

[King,Michael A]

Caroline,
Overnight at 4 degrees may not be long enough for equilibration of the 
antibody binding reaction deep in the section.  Try room temp or longer 
time if cold is important.  The early users of IHC often used 4 days at 
4 deg C, based on antibody binding kinetics data.  I get full-thickness 
labeling assessed by confocal microscopy with overnight incubations 
(both primary and secondary) at room temp in 50 um rodent brain 
sections.  Your secondary incubation may be too short for deep 
labeling--easy to test.  Alternatively, the primary may be depleted (all 
bound to tissue epitopes) before penetrating the section interior.  
Increasing the volume or concentration of primary would help.  Some 
24-well plates (culture-treated) will absorb significant (and variable) 
amounts of antibody, and little critters can gobble it up too. If you're 
not using peroxidase reporters you may want to leave azide or another 
antimicrobial in the incubation solutions.  They don't alter 
fluorescence, though you'll want to convince yourself of this 
empirically.


Message: 12 Date: Tue, 19 Nov 2013 17:43:13 + From: "Bass, 
Caroline"  Subject: [Histonet] problem with double 
IF of brain section To: Histonet  
Message-ID: <3ccee4fb-40b5-49a2-8456-72d71f893...@buffalo.edu> 
Content-Type: text/plain; charset="Windows-1252" Hello everyone, I’m 
trying a double stain for GFP and tyrosine hydroxylase in floating brain 
sections. Rat, perfused with formalin. I’m having a big problem, in 
that it’s clear that the top side of the sections are well stained, as 
are the bottom, but everything in between is not. These sections are 50 
microns. Any suggestions? I assume it’s a penetration problem. But 
I’m not very comfortable with IF in general. Here’s what my tech 
does: Placed sections each in a well of a 24 well dish and rinsed in PBS 
(to remove the Na azide), 1 X 5 min. Rinsed in PBS-0.5% Triton X-100, 3 
X 10 min. Incubated tissue in PBST (0.25% Triton X) + 5% NGS, 1 X 60 to 
120 min. Incubated sections in 1ary antibody in PBS-T(0.25%) overnight 
@40C (covered to minimized evaporation). Both primaries together. 
Tyrosine Hydroxylase 1: 4,000 ImmunoStar #22941 (mouse) a- GFP 1:2,000 
Invitrogen 6455 (rabbit) Next day, rinsed tissue w/ PBS-T, 3 x 10 min. 
Incubated w/ 2ary ab : Alexa 555 donkey anti mouse 1:4,000, Alexa 488 
goat anti rabbit, 1:2,000, 1 x 2hs @RT. 2ary ab diluted in PBS. Rinsed 
w/ PBS, 3 x10 min. Mounted on slides, coverslipped using ProLOng Gold w/ 
DAPI. Cured in dark place @ RT overnight. Thanks, Caroline


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RE: [Histonet] Thank you all.

[Weems, Joyce K.]

Searching for the "like" button!!

We do have an awesome group!

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@emoryhealthcare.org



www.saintjosephsatlanta.org
5665 Peachtree Dunwoody Road
Atlanta, GA 30342

This e-mail, including any attachments is the property of Saint Joseph's 
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regarding the error in a separate email.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Catherine 
Simonson
Sent: Tuesday, November 19, 2013 1:42 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Thank you all.

I wanted to thank everybody, especially Lynn Burton, for their help during my 
time of crisis.  It is nice to be a part of a group that is so supportive and 
willing to lend a hand.

Again, thank you.

Catherine
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[Histonet] Thank you all.

[Catherine Simonson]

I wanted to thank everybody, especially Lynn Burton, for their help during
my time of crisis.  It is nice to be a part of a group that is so
supportive and willing to lend a hand.

Again, thank you.

Catherine
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RE: [Histonet] omnimap/chromomap kit for ventana

[Burton, Lynn]

I have two chromo map kits for the Discovery I can send you.

Lynn M Burton
Histology
Animal Disease Lab
Galesburg, Il
309-344-2451



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Catherine 
Simonson
Sent: Tuesday, November 19, 2013 11:09 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] omnimap/chromomap kit for ventana

Hello Fellow Histonetters,

I find myself in a bit of a bind.  I have a couple of urgent studies/cases that 
need to be done by the end of this week, but our kit has run out and is on 
back-order from Ventana.  I need to do Caspase  and Major Basic
Protein   (both rabbit antibodies).  Would it be the acme of foolishness to
ask if anyone would have a spare kit? Or any other suggestions?  is it possible 
to do antigen retrieval on the machine and use the dead volume in the 
containers to do the rest of the stain by hand?

Thank you all in advance,

Catherine Simonson, HT
The Jackson Laboratory
Bar Harbor, ME
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[Histonet] problem with double IF of brain section

[Bass, Caroline]

Hello everyone,

I’m trying a double stain for GFP and tyrosine hydroxylase in floating brain 
sections. Rat, perfused with formalin. I’m having a big problem, in that it’s 
clear that the top side of the sections are well stained, as are the bottom, 
but everything in between is not. These sections are 50 microns. Any 
suggestions? I assume it’s a penetration problem. But I’m not very comfortable 
with IF in general.

Here’s what my tech does:

Placed sections each in a well of a 24 well dish  and rinsed in PBS (to remove 
the Na azide), 1 X 5 min. Rinsed in PBS-0.5% Triton X-100, 3 X 10 min. 
Incubated tissue in PBST (0.25% Triton X) + 5% NGS, 1 X 60 to 120 min. 
Incubated sections in 1ary antibody in PBS-T(0.25%) overnight @40C (covered to 
minimized evaporation). Both primaries together.
Tyrosine Hydroxylase  1: 4,000 ImmunoStar #22941 (mouse)
a- GFP  1:2,000  Invitrogen 6455 (rabbit)
Next day, rinsed tissue w/ PBS-T, 3 x 10 min. Incubated w/ 2ary ab : Alexa 555 
donkey anti mouse 1:4,000, Alexa  488 goat anti rabbit, 1:2,000, 1 x 2hs @RT. 
2ary ab diluted in PBS. Rinsed w/ PBS, 3 x10 min. Mounted on slides, 
coverslipped using ProLOng Gold w/ DAPI. Cured in dark place @ RT overnight.

Thanks,
Caroline
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[Histonet] microtome

[Rui TAHARA]

Hello, 
 
I have undecalcified biological sample embedded in plastic media (MMA). 
I am looking for faclities that offers self-service microtome (for plastic 
embedding sectioining) or short time rental microtome with some training around 
Quebec, Ontraio, NY. I have been trying to find ones in Montreal, however, its 
been difficult to find self-service microtome. 
I appreciate if you would provide some information for this. 
 
Thank you in advance, 
 
Rui 
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RE: [Histonet] New CPT code for IHC

[Murray Anderson]

I think that everyone is waiting to see if CMS makes a "policy Change"
to accommodate 88342 +88343 code changes.  The first indication may be
"via the 2014 Medicare Physician Fee Schedule final rule due out for
release on or before November 27, or through  the 2014 version of the
NCCI Policy Manual, which could be available as early as December 1"
(care of Dennis Padget).

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Leann M.
Murphy
Sent: Tuesday, November 19, 2013 8:32 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] New CPT code for IHC

How will everyone be handling the new CPT code 88343 for IHC going into
effect 1/1/14?  The CPT code 88342 has a new description stating it is
to be used for the "first" antibody and 88343 is to be used for each
additional separately identifiable antibody.  The concern is that each
antibody at any point and time could be the primary or additional.

LeAnn Murphy
Aultman Hospital
Technical Specialist
lmurp...@aultman.com

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[Histonet] omnimap/chromomap kit for ventana

[Catherine Simonson]

Hello Fellow Histonetters,

I find myself in a bit of a bind.  I have a couple of urgent studies/cases
that need to be done by the end of this week, but our kit has run out and
is on back-order from Ventana.  I need to do Caspase  and Major Basic
Protein   (both rabbit antibodies).  Would it be the acme of foolishness to
ask if anyone would have a spare kit? Or any other suggestions?  is it
possible to do antigen retrieval on the machine and use the dead volume in
the containers to do the rest of the stain by hand?

Thank you all in advance,

Catherine Simonson, HT
The Jackson Laboratory
Bar Harbor, ME
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Re: [Histonet] New CPT code for IHC

[Will Chappell]

88343 refers to subsequent antibodies placed on the same slide such as 
multiplex staining or the PIN-4. 

Hope that helps. 

Will Chappell, HTL(ASCP), QIHC

Sent from my iPhone

> On Nov 19, 2013, at 8:31 AM, "Leann M. Murphy"  wrote:
> 
> How will everyone be handling the new CPT code 88343 for IHC going into 
> effect 1/1/14?  The CPT code 88342 has a new description stating it is to be 
> used for the "first" antibody and 88343 is to be used for each additional 
> separately identifiable antibody.  The concern is that each antibody at any 
> point and time could be the primary or additional.
> 
> LeAnn Murphy
> Aultman Hospital
> Technical Specialist
> lmurp...@aultman.com
> 
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[Histonet] New CPT code for IHC

[Leann M. Murphy]

How will everyone be handling the new CPT code 88343 for IHC going into effect 
1/1/14?  The CPT code 88342 has a new description stating it is to be used for 
the "first" antibody and 88343 is to be used for each additional separately 
identifiable antibody.  The concern is that each antibody at any point and time 
could be the primary or additional.

LeAnn Murphy
Aultman Hospital
Technical Specialist
lmurp...@aultman.com

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[Histonet] Leica RM2255 block clamp

[Finlay Finlay]

Hello

 

Hoping someone might be able to help with this microtome issue. I have a 
problem with the block clamp on one of my Leica RM2255's. It has recently 
loosened up and it is now possible to move the clamped cassette from 
side-to-side when the clamp is shut. 

When I turn the bolt on the underside of the clamp it neither tightens up nor 
loosens off so there does not seem to be a way of taking the clamp apart any 
further.

 

Leica have suggested that I buy a new block clamp (£292) but it just feels like 
something that could be easily fixed, perhaps with a new spring? Has anyone had 
experience with this problem? 

 

Thank you

 

Finlay Finlay

 

Histology Technician

Forensic Medicine and Science 

School of Medicine 

College of Medical, Veterinary and Life Sciences

University of Glasgow 

Joseph Black building

 

Direct Line: +44 (0) 141 3303443

E-mail: finlay.fin...@glasgow.ac.uk 
  

 

The University of Glasgow Charity Number SC004401

 

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[Histonet] Methyl Carnoy's processing

[Hans B Snyder]

Hello,

I have a researcher who has fixed their specimens in methyl Carnoy's
but would like to skip the methyl benzoate step before processing (we
do not have methyl benzoate).  Does anyone have another method that
works well without the methyl benzoate?

Thank you

Hans B Snyder
Histologistics
60 Prescott Street
Worcester, MA 01605
508-308-7800
h...@histologistics.com

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[Histonet] Vendor suggestions for IgG subclass antibodies

[Martha Ward-Pathology]

I am looking for a vendor for FITC labeled IgG1, IgG2 IgG3 and IgG4 antibodies 
for use in our immunofluorescence panels when staining renal biopsies. We 
get most of our FITC antibodies for our panel from Dako but I didn't see these 
others.   Any suggestions are appreciated.   Thanks in advance for your help!


 
Martha Ward, MT (ASCP) QIHC
Manager

Molecular Diagnostics Lab
Medical Center Boulevard  \  Winston-Salem, NC 27157
p 336.716.2109  \  f 336.716.5890  
mw...@wakehealth.edu  
 
 






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RE: [Histonet] Specimen collection/transportation

[joelle weaver]

The usual procedure where I have worked is that the placenta is delivered fresh 
to pathology in a large container, or refrigerated until next specimen pick up 
and formalin is added by the lab staff ( who have PPE, formalin training, spill 
kit) etc.  




Joelle Weaver MAOM, HTL (ASCP) QIHC
 
> Date: Mon, 18 Nov 2013 14:50:15 -0600
> From: ll...@aipathology.com
> To: foreig...@gmail.com
> Subject: RE: [Histonet] Specimen collection/transportation
> CC: histonet@lists.utsouthwestern.edu
> 
> That was my first initial reaction too to be honest and that they are
> trying to put it on us.
> 
>  
> 
> From: Patrick Laurie [mailto:foreig...@gmail.com] 
> Sent: Monday, November 18, 2013 2:37 PM
> To: LeAnn Lang
> Cc: Rathborne, Toni; Will Chappell; histonet@lists.utsouthwestern.edu
> Subject: Re: [Histonet] Specimen collection/transportation
> 
>  
> 
> Ah, I think that the key thing is that the OBT's are not following
> procedure and their procedures are in violation of the OSHA regs.  If
> they were to use the proper PPE and instructions, there should not be an
> issue.  Also, make sure that there is some kind of spill procedure/kit
> available.  In my state, only certain people who are trained can clean
> up any spill that is over 1 gallon.  
> 
>  
> 
> Patrick Laurie
> 
>  
> 
> On Mon, Nov 18, 2013 at 3:13 PM, LeAnn Lang 
> wrote:
> 
> This was the message I received this morning: (I asked for the specific
> documentation on the violation).  I also asked why the OBTs are NOT
> using precautions when working with the placenta and formalin.
> 
> 
> Just discovered this am that we are using formalin filled
> buckets for placentas going to pathology
> This is a huge safety issue for the staff and an OSHA violation.
> The standard practice for placentas going to pathology is to
> store them in a refrigerator and then pathology picks them up.
> The key component is the elimination of the formalin.
> Handling of formalin requires safety goggles, chemical resistant
> gloves and protective clothing, Venting under a hood is also
> recommended.
> The OBTs that place the placentas in a bucket of formalin have
> not been doing any of this or using any precautions.
> The upshot is if this stuff spills  it can cause severe health
> problems (at the last hospital I was at it was spilled and EVS worker
> tried to clean it upand was in the ICU for two weeks).
> 
> 
> -Original Message-
> From: Rathborne, Toni [mailto:trathbo...@somerset-healthcare.com]
> Sent: Monday, November 18, 2013 2:08 PM
> To: 'Will Chappell'; LeAnn Lang
> Cc: 
> 
> Subject: RE: [Histonet] Specimen collection/transportation
> 
> Did they state which OSHA standard you were in violation of?
> 
> -Original Message-
> From: histonet-boun...@lists.utsouthwestern.edu
> [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Will
> Chappell
> Sent: Monday, November 18, 2013 3:06 PM
> To: LeAnn Lang
> Cc: 
> Subject: Re: [Histonet] Specimen collection/transportation
> 
> This to me seems very odd. Almost exclusively specimens are sent to my
> lab in formalin. Placentas are usually sent fresh simply because of
> their size.
> 
> If anything, the birthing unit may not be in compliance, but it has
> nothing to do with the lab. The formalin containers must be properly
> labelled, and appropriate SOPs in use on the floor, usually to include a
> spill kit. I wrote the procedures for the floor units, but it is their
> responsibility to be in compliance.
> 
> Will Chappell, HTL(ASCP)
> 
> Sent from my iPhone
> 
> > On Nov 18, 2013, at 12:02 PM, "LeAnn Lang" 
> wrote:
> >
> > We were recently contacted by our hospital indicating that we are in
> > violation of OSHA by using the process we currently are using.
> > Currently, we provided prefilled 10% neutral buffered formalin
> > containers to the surgical suites, birthing units, etc.   They fill
> the
> > containers with the specimens and return them to the pathology lab.
> We
> > have done this process for many many years and have never been
> > questioned for this by either CAP or Joint Commission.  What is your
> > process for specimen collection/transport?  Are the specimens put in
> > formalin in the surgery suites/birthing unit/etc or in the pathology
> > laboratory?  How about placentas, are they sent in formalin from the
> > floor or are they put in formalin in the histology lab?
> >
> >
> >
> > Thank you!
> >
> > LeAnn
> >
> >
> >
> >
> >
> > <>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>
> >
> > LeAnn Lang
> >
> > Associates in Pathology
> >
> > Practice Administrator
> >
> > Phone:  715-847-0075 (ext 50259)
> >
> > ll...@aipathology.com 
> >
> >
> >
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