Re: [Histonet] cloudy cornea after Hartman's fix

[Tony Henwood via Histonet]

Hartman's (also known as Davidson's) fixative is sometimes used to reveal lymph 
nodes in resections where they appear opaque - white. It is not surprising that 
the tissues of the eye would react the same.
I assume that the alcohol causes the bleaching of the tissue (or is it the 
acetic acid?) - more research needed.


Regards,



Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)

Principal Scientist, the Children’s Hospital at Westmead (Retired)

Adjunct Fellow, School of Medicine, University of Western Sydney.


From: Davoli, Katherine A via Histonet 
Sent: 28 June 2024 04:12
To: histonet@lists.utsouthwestern.edu 
Subject: [Histonet] cloudy cornea after Hartman's fix

Anyone know why the cornea of my pig eye got white/cloudy on dropping it in to 
Hartman's fixative?  I'm used to working with mice where, if this happens I 
didn't notice.

Katherine Davoli, MDiv, HTL(ASCP)cm(they/them/theirs)
Lab Manager, Tissue Culture & Histology Cores, U. Pitt Dept of Ophthalmology
7.373 UPMC Mercy Pavilion1622 Locust St., Pittsburgh PA 15219
(412) 624-8508   this number cannot receive texts
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Re: [Histonet] Inquiry on Tissue Softening for Microtomy

[Tony Henwood via Histonet]

My preference is cold water or Mollifex on faced blocks.

Ammonia is definitely obnoxious (reminds me of wet nappies )


Regards,



Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)

Principal Scientist, the Children’s Hospital at Westmead (Retired)

Adjunct Fellow, School of Medicine, University of Western Sydney.


From: Jay Lundgren via Histonet 
Sent: 13 May 2024 08:37
To: John Kiernan 
Cc: histonet@lists.utsouthwestern.edu ; 
IGNACIO GONZÁLEZ MASSONI 
Subject: Re: [Histonet] Inquiry on Tissue Softening for Microtomy

Pleasant is not a mission parameter.
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Re: [Histonet] Histonet Digest, Vol 245, Issue 20

[Tony Henwood via Histonet]

A few years ago there was an article on the Block - "What is Denatured Alcohol 
and What are the Implications For Histopathology?" that might be useful. The 
URL is

https://www.nsh.org/blogs/tony-henwood/2020/02/11/what-is-denatured-alcohol-and-what-are-the-implica?_gl=1*1mgiiv6*_ga*MTkyMzY5MTQ4Mi4xNzExNDA1ODQ2*_ga_7J0VYE6519*MTcxNDU0NzgyNy4xMC4xLjE3MTQ1NDc4NzcuMC4xLjEzNzYzNTQ4NjQ.&_ga=2.47649445.329273616.1714547828-1923691482.1711405846



Regards,



Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)

Principal Scientist, the Children’s Hospital at Westmead (Retired)

Adjunct Fellow, School of Medicine, University of Western Sydney.


From: John O’Brien via Histonet 
Sent: 01 May 2024 01:49
To: histonet@lists.utsouthwestern.edu 
Subject: Re: [Histonet] Histonet Digest, Vol 245, Issue 20

Ethyl Alcohol,
Reagent 100% alcohol is what is normally used in Pathology processing and 
slide staining
IMEB Inc offer all grades of Reagent alcohol, pure ethyl is a controlled 
alcohol regulated and taxed by government
Regards
John

> On Apr 30, 2024, at 10:18 AM, histonet-requ...@lists.utsouthwestern.edu wrote:
>
> Send Histonet mailing list submissions to
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> Today's Topics:
>
>   1. Alcohol (Naira Margaryan)
>
>
> --
>
> Message: 1
> Date: Mon, 29 Apr 2024 18:49:04 -0500
> From: Naira Margaryan 
> To: Histonet 
> Subject: [Histonet] Alcohol
> Message-ID:
>
> Content-Type: text/plain; charset="UTF-8"
>
> Hello,
>
> What type of ALCOHOL ETHYL you?re using in your histology lab.?
>
> We are thinking to get 20-30-gal drum.
>
> Could you please help me with that?
>
>
>
> Your suggestion is appreciated,
>
> Naira
>
>
> --
>
> Subject: Digest Footer
>
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> --
>
> End of Histonet Digest, Vol 245, Issue 20
> *


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Re: [Histonet] Modified Davidson solution

[Tony Henwood via Histonet]

I agree.

A great book.


Regards,



Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)

Principal Scientist, the Children’s Hospital at Westmead (Retired)

Adjunct Fellow, School of Medicine, University of Western Sydney.


From: John Kiernan via Histonet 
Sent: 06 March 2024 18:32
To: Histonet ; Naira Margaryan 
; Davoli, Katherine A 
Subject: Re: [Histonet] Modified Davidson solution

The Davidson/Hartmann fixative is just another alcoholic formaldehyde mixture 
acidified with acetic acid. Like all others of that ilk it was intended for 
making up in the lab soon before using. Storage causes slow deterioration. The 
ethanol slowly gets esterified by the acetic acid, making ethyl acetate, which 
has no fixative properties. This change is evident from the fruity smell of the 
ester, which becomes evident after a week or two. All this has been in the 
textbooks for more than 50 years.
Why doesn't every lab spend perhaps $200 on a couple of books?  For obvious 
reasons I recommend my 5th edition (ISBN 9781907904325; $82.50,from the 
publisher, which is $30 less than Amazon's price), but there are other 
histotechnology books that are very good.  A book may cost much less than a big 
bottle of a deteriorating mixture of questionable value, and you and your staff 
could learn a lot by reading.
John Kiernan
= = =

From: Davoli, Katherine A via Histonet 
Sent: March 5, 2024 4:40 PM
To: Histonet ; Naira Margaryan 

Subject: Re: [Histonet] Modified Davidson solution

Hi Naira, I get Hartmann's from Electron Microscopy Sciences
Cat# 64133-10 (for 1L, but they have larger sizes I believe)


Katherine Davoli, HTL(ASCP)cm(they/them/theirs)

Lab Manager, Tissue Culture and Histology Core Facilities

U. Pitt Dept of Ophthalmology

7.373 UPMC Mercy Pavilion

1622 Locust St., Pittsburgh PA 15219

(412) 624-8508   this number cannot receive texts


From: Naira Margaryan via Histonet 
Sent: Tuesday, March 5, 2024 1:27 PM
To: Histonet 
Subject: [Histonet] Modified Davidson solution

Hello!

What is the best company to buy a ready to use Modified Davidson solution?

Thanks in advance,
Naira
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From: Davoli, Katherine A via Histonet 
Sent: March 5, 2024 4:40 PM
To: Histonet ; Naira Margaryan 

Subject: Re: [Histonet] Modified Davidson solution

Hi Naira, I get Hartmann's from Electron Microscopy Sciences
Cat# 64133-10 (for 1L, but they have larger sizes I believe)


Katherine Davoli, HTL(ASCP)cm(they/them/theirs)

Lab Manager, Tissue Culture and Histology Core Facilities

U. Pitt Dept of Ophthalmology

7.373 UPMC Mercy Pavilion

1622 Locust St., Pittsburgh PA 15219

(412) 624-8508   this number cannot receive texts


From: Naira Margaryan via Histonet 
Sent: Tuesday, March 5, 2024 1:27 PM
To: Histonet 
Subject: [Histonet] Modified Davidson solution

Hello!

What is the best company to buy a ready to use Modified Davidson solution?

Thanks in advance,
Naira
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Re: [Histonet] Modified Davidson

[Tony Henwood via Histonet]

There are several formulations (some are probably typos), but this seems to be 
one commonly cited.

Modified Davidson's Fixative:

(Latendresse, J. R., Warbrittion, A. R., Jonassen, H., & Creasy, D. M. (2002). 
Fixation of testes and eyes using a modified Davidson's fluid: comparison with 
Bouin's fluid and conventional Davidson's fluid. Toxicologic pathology, 30(4), 
524-533.)

37–40% formaldehyde30ml
Absolute ethanol 15ml
Glacial acetic acid5ml
Distilled Water 50ml

Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.


From: Naira Margaryan via Histonet 
Sent: Friday, February 9, 2024 8:25:47 AM
To: Histonet 
Subject: [Histonet] Modified Davidson

Hello,

Could you please share your best recipe for the Modified Davidson?

Thanks in advance,
Naira
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Re: [Histonet] Processing artifact - delayed start

[Tony Henwood via Histonet]

I would check the level of the formalin after it has been pumped into the 
retort.

I will wait till the pics are posted

Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.


From: Verizon wireless via Histonet 
Sent: Saturday, February 3, 2024 2:36:42 PM
To: histonet@lists.utsouthwestern.edu 
Subject: [Histonet] Processing artifact - delayed start

Dear Histonet Members,

We have terrible processing artifact if tissue sits in the formalin-filled 
retort (at ambient temperature) for too long (more than 10-12 hours) before a 
delayed process starts. The longer the wait, the worse it looks. We have Tissue 
Tek VIP 5 processors, and we process luminal gastrointestinal biopsies 
exclusively. I've attached some photomicrographs of problem cases on the 
Histonet Images website (with the same topic title).
This artifact typically affects a few specimens per day (~2% or less), even 
though everything is done on the same processor; it may affect all tissue 
portions in a cassette or only some of the tissue in that cassette. Some tissue 
portions may only have the artifact on one half with the other half looking 
perfectly fine. The techs sometimes note the tissue feeling "crunchy" at the 
time of embedding and / or at the time of microtomy. These tissues tend to 
suffer greater chatter artifact and have trouble sticking to the slides. The 
sections look just as bad on recuts as the originals. Re-processing does not 
seem to help at all.
If the cassettes sit in formalin in a container outside of the processor for 
days before the processor is loaded (with subsequent immediate start), things 
look perfectly fine. When we have staff around to start the processor 
immediately upon loading cassettes and empty immediately upon completion, the 
tissue looks perfectly fine.

Our current processing program is as follows:

1. 10% Formalin, 30 minutes, ambient temp, p/v on
2. 10% Formalin, 30 minutes, ambient temp, p/v on
3. 65% Alcohol, 10 minutes, ambient temp, p/v on4. 80% Alcohol, 10 minutes, 
ambient temp, p/v on5. 95% Alcohol, 7 minutes, ambient temp, p/v on6. 95% 
Alcohol, 7 minutes, ambient temp, p/v on 7. 100% Alcohol, 10 minutes, ambient 
temp, p/v on
8. 100% Alcohol, 10 minutes, ambient temp, p/v on
9. Xylene, 15 minutes, ambient temp, p/v on
10. Xylene, 15 minutes, ambient temp, p/v on
11. Paraffin, 10 minutes, 60 degrees C, p/v on12. Paraffin, 10 minutes, 60 
degrees C, p/v on13. Paraffin, 15 minutes, 60 degrees C, p/v on14. Paraffin, 15 
minutes, 60 degrees C, p/v on

Obviously there are times we absolutely need to use delayed start. I would 
greatly appreciate guidance, and I'll be happy to provide any other details 
that might be useful.
Sincerely,
Brian Quigley MDLaboratory Director of a GI Pathology Laboratory

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Re: [Histonet] Faded H tissue section

[Tony Henwood via Histonet]

I thought he may have reported this in Histologic.
This trick certainly works. You can notice the nuclear clarity on GIT and to a 
lesser extent skin biopsies stained with PAS.

Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.

From: Akemi Allison<mailto:akemiat3...@gmail.com>
Sent: Friday, 10 November 2023 1:30 PM
To: jayalakshmy p.s<mailto:psjayalaks...@gmail.com>
Cc: Tony Henwood<mailto:afhenw...@outlook.com>; 
histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: Re: [Histonet] Faded H tissue section

Hi Tony:
I actually gave that tip to Lee back in 1979 when he came to OHSU to give a 
seminar to the Oregon Histology Society.  I found out that when I was doing GMA 
and the pathologists didn’t like Gills Hematoxylin. They loved my PAS on GMA so 
I tried using 1% Periodic Acid before using Harris Hematoxylin for H’s on GMA 
and it turned out beautifully. Guess he shared my tip with the rest of our 
society. He and I became closer se friends and we shared several tips, 
including his Movat’s tips which he didn’t publish, but I shared them in 
Frieda’s 4th Ed.

Best,
Akemi Allison-Tacha, BS, HT/HTL (ASCP)

Sent from my iPhone

> On Nov 9, 2023, at 6:11 PM, jayalakshmy p.s via Histonet 
>  wrote:
>
> Thanks, I'll check it out.
>
>> On Fri, Nov 10, 2023, 6:58 AM Tony Henwood  wrote:
>>
>> You could treat the decoverslipped section in 1% periodic acid (same as
>> used in the PAS technique)  for 30 minutes prior to H staining. This
>> might improve the H staining.
>>
>>
>>
>> I believe Lee Luna suggested this but for the life of me, I can’t find the
>> reference!
>>
>>
>>
>> Regards,
>>
>>
>>
>> Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
>>
>> Principal Scientist, the Children’s Hospital at Westmead (Retired)
>>
>> Adjunct Fellow, School of Medicine, University of Western Sydney.
>>
>>
>>
>> *From: *jayalakshmy p.s via Histonet 
>> *Sent: *Friday, 10 November 2023 3:43 AM
>> *To: *histonet@lists.utsouthwestern.edu
>> *Subject: *[Histonet] Faded H tissue section
>>
>>
>>
>> Hello,
>> I would like to know how to effectively restain a faded H tissue section.
>> The color becomes dull when re stained. Somebody please advise.
>> Prof. Jayalakshmy. P S
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>>
>>
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Re: [Histonet] Faded H tissue section

[Tony Henwood via Histonet]

You could treat the decoverslipped section in 1% periodic acid (same as used in 
the PAS technique)  for 30 minutes prior to H staining. This might improve 
the H staining.

I believe Lee Luna suggested this but for the life of me, I can’t find the 
reference!

Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.

From: jayalakshmy p.s via Histonet<mailto:histonet@lists.utsouthwestern.edu>
Sent: Friday, 10 November 2023 3:43 AM
To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: [Histonet] Faded H tissue section

Hello,
I would like to know how to effectively restain a faded H tissue section.
The color becomes dull when re stained. Somebody please advise.
Prof. Jayalakshmy. P S
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Re: [Histonet] Formalin pH

[Tony Henwood via Histonet]

Hew-Shue (1991) has described a useful pH indicator for working neutral 
buffered formalin solutions. Bromocresol purple, when added to formalin 
solutions, serves as an indicator of pH as well as, from a safety aspect, 
labelling the solution as formalin making redundant the dangerous “smell” test. 
At an acidic pH (5.2) the intense indicator colour is yellow whereas at pH 6.8, 
the colour is purple. A saturated solution of the dye is prepared, and 2 to 4 
drops are added to 10 litres of neutral buffered formalin. Other advantages are 
that the fixative is more readily distinguished from other colourless solutions 
such as saline, thus preventing accidental misuse, and formalin spills are more 
easily recognised.

Hew-Shue N (1991) “Bromcresol Purple as a Colored Marker and pH Indicator for 
Ten Percent Neutral Buffered Forrnalin” J Histotechnol 14(4):257-260.


Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.

From: Bacon, Charles via Histonet<mailto:histonet@lists.utsouthwestern.edu>
Sent: Thursday, 14 September 2023 6:42 AM
To: e...@pigs.ag<mailto:e...@pigs.ag>
Cc: HistoNet<mailto:histonet@lists.utsouthwestern.edu>
Subject: Re: [Histonet] Formalin pH

We pull the COA from the vendors website. Some are better than others but the 
are usually listed by lot number. I keep them in a network shared folder so 
they can be pulled by anyone that is asked for the evidence during inspection.

Sent from my iPhone

> On Sep 13, 2023, at 8:22 AM, "e...@pigs.ag"  wrote:
>
> Sooner or later the people have got to rise up
> and tell those brain-dead paper-shuffling regulators
> to just stuff it.  Their mindless interference is
> just wasting time and interfering with people's
> ability to keep their own garden.
>
> -Original Message-
> From: Cooper, Brian via Histonet
> Reply-To: Cooper, Brian
> To: Paula Sicurello, HistoNet
> Subject: Re: [Histonet] Formalin pH
> Date: Today 10:19 AM
>
> Ask your vendor to send a certificate of analysis that includes the pH. 
> That's what we did and now they come with each lot. We do have to bug them 
> for them occasionally.
>
> I'm betting your vendor has at least heard about this from other customers so 
> it should be on their radar at the very least.
>
>
> 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<mailto:bcoo...@chla.usc.edu>
>
> 
> From: Paula Sicurello via Histonet 
> Sent: Tuesday, September 12, 2023 7:12:05 PM
> To: HistoNet 
> Subject: [Histonet] Formalin pH (EXTERNAL EMAIL)
>
> CAUTION: BE CAREFUL WITH THIS MESSAGE*
> This email came from outside CHLA. Do not open attachments, click on links, 
> or respond unless you expected this message and recognize the email address: 
> histonet-boun...@lists.utsouthwestern.edu.
>
> Hello Histoteckies,
> What are y'all doing regarding the CAP requirement to monitor the pH of 
> formalin?
> We buy tons and tons of the 5 gallon cubitainers and we are still debating 
> over how to check the pH.
>
> Looking forward to your replies.
> Toodles!
> Sincerely,
>
> Paula Sicurello
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Re: [Histonet] shelf life of working antibody solutions in IHC

[Tony Henwood via Histonet]

In Australia, we have evolved from Prescriptive accreditation (following the 
standards) to Risk-based accreditation where it is more important to assess the 
risk of a laboratory process rather than only meeting a list of rules, since 
following a list of rules is often not in the patient’s nor in the community’s 
interest.

Years ago, I battled with our accreditation organization over the risk involved 
in fridge temperature recording, presenting evidence that continuous digital 
monitoring (with an alarm system) was less risky than recording max/min 
temperatures daily (eg what happens when the lab is not manned during Christmas 
holidays and weekends?).

The excessive cost involved in automatically disposing of expired antibodies is 
wasteful. Continuous monitoring as well as preventative practices such as 
preservatives (azide, procillin, etc) and pH monitoring with phenol red greatly 
reduces the risk.

As presented in the paper, many working dilutions survive without contamination 
for many years. For example, factor 8 (21 years), factor 13a (19 years) and 
epithelial membrane antigen (17 years). There were no failed verifications and 
to date, the average life after expiration was 6 years; eight antibodies 
exceeded 6 years. All the other antibodies would probably have similar survival 
times but were exhausted before reaching these times.

Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.

From: Gudrun Lang<mailto:gu.l...@gmx.at>
Sent: Sunday, 2 July 2023 7:26 PM
To: 'Tony Henwood'<mailto:afhenw...@outlook.com>
Subject: AW: [Histonet] shelf life of working antibody solutions in IHC

Dear Tony,
thank you for the interesting article. It reflects my personal experience. 
Nevertheless accreditation rules overrule the experience and in an diagnostic 
lab we have to discard the reagenses.

My question regards more the practical approach with working solutions.
What is your experience how long the working solutions are „good enough“ for 
using.

Thank you very much and kind regards
Gudrun

Von: Tony Henwood [mailto:afhenw...@outlook.com]
Gesendet: Samstag, 1. Juli 2023 23:41
An: Gudrun Lang
Betreff: RE: [Histonet] shelf life of working antibody solutions in IHC

Here it is

Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.

From: Gudrun Lang via Histonet<mailto:histonet@lists.utsouthwestern.edu>
Sent: Sunday, 2 July 2023 4:59 AM
To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: [Histonet] shelf life of working antibody solutions in IHC

Hi all!

I have tried to find a general instruction for the shelf life of antibody
working solutions.

With automated IHC you usually fill the container with the working solution
and depending on the frequency of usage they stay on the instrument at
roomtemperature (or higher) or are put in the fridge again.

The working solutions are up to 10 ml and may last for months. The
antibody-diluent is from the same company of the instrument. The titers are
in a range from 1:10 to 1:3000. - so a very heterogen situation.



How do you handle this? Have you a general rule, when the solution has to be
discarded? Is it just a matter of positiv-controls?



Thanks in advance

Gudrun Lang

Biomedical scientist

Austria





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Re: [Histonet] shelf life of working antibody solutions in IHC

[Tony Henwood via Histonet]

Hi Gudrun,

This should be useful ( I will send a copy separately)

Henwood, A. F. (2023). Validation of nominally expired antibodies for 
immunohistochemistry. Biotechnic & Histochemistry, 98(2), 86-93.

Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.

From: Gudrun Lang via Histonet<mailto:histonet@lists.utsouthwestern.edu>
Sent: Sunday, 2 July 2023 4:59 AM
To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: [Histonet] shelf life of working antibody solutions in IHC

Hi all!

I have tried to find a general instruction for the shelf life of antibody
working solutions.

With automated IHC you usually fill the container with the working solution
and depending on the frequency of usage they stay on the instrument at
roomtemperature (or higher) or are put in the fridge again.

The working solutions are up to 10 ml and may last for months. The
antibody-diluent is from the same company of the instrument. The titers are
in a range from 1:10 to 1:3000. - so a very heterogen situation.



How do you handle this? Have you a general rule, when the solution has to be
discarded? Is it just a matter of positiv-controls?



Thanks in advance

Gudrun Lang

Biomedical scientist

Austria





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Re: [Histonet] Get the Short Term or Long Term Lab Staffing Coverage You Need

[Tony Henwood via Histonet]

I agree

Regards,

Tony Henwood
Sydney, Australia

From: John Kiernan via Histonet<mailto:histonet@lists.utsouthwestern.edu>
Sent: Saturday, 3 June 2023 11:09 AM
To: Melissa Owens<mailto:meli...@alliedsearchpartners.com>; Jay 
Lundgren<mailto:jaylundg...@gmail.com>
Cc: Tom Walls<mailto:histonet@lists.utsouthwestern.edu>
Subject: Re: [Histonet] Get the Short Term or Long Term Lab Staffing Coverage 
You Need

Hear, Hear! Let's see less advertising on Histonet.
John Kiernan
(London, Canada).
= = =

From: Jay Lundgren via Histonet 
Sent: June 2, 2023 1:15 PM
To: Melissa Owens 
Cc: Tom Walls 
Subject: Re: [Histonet] Get the Short Term or Long Term Lab Staffing Coverage 
You Need

As far as I know, Histonet doesn't really allow advertising for individuals
or agencies *looking* for jobs.  There are a very few, selective, agencies
that are allowed to post on Histonet  tastefully and infrequently, but they
are buying, not selling.  Or histotechs who let others know about open
positions in their organization.  That's cool.

I'm not a moderator, but this is supposed to be a collegial forum for
practical histopathology advice.  There are plenty of job search sites out
there already.

But if Histonet does start allowing this, then ***%%%$$$HEY SCRIPPS
INSTITUTE HIRE ME$$$***

***%%%$$$LONG OR SHORT CONTRACT!!!CHEAP$$$%%%***

 **%%%$$$BIG TIME IMUNNOHISTOCHEMISTRY$$$%%%***

 ***%%%$$$USAF HTL (ASCP) M.S. 30+ YRS!!!$$$%%%***

 Sincerely,

  Jay A. Lundgren, M.S., HTL (ASCP)

On Fri, Jun 2, 2023 at 10:50 AM 'Melissa Owens' via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> li {display:list-item;text-indent: -1em;}
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Re: [Histonet] Sudanblack B on FFPET

[Tony Henwood via Histonet]

I would also let the saturated solution stand for a few days. Like Oil Red O, 
it probably needs time to “mature”. I would also use a frozen section of skin 
as a control.

Regards,

Tony Henwood
Sydney, Australia

From: Bob Richmond via Histonet<mailto:histonet@lists.utsouthwestern.edu>
Sent: Wednesday, 29 March 2023 4:51 AM
To: Histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: Re: [Histonet] Sudanblack B on FFPET

>
> Gudrun Lang in Austria asks:
>

>>Has anyone experience with Sudanblack B on paraffin slides for staining
[lipofuscin]? A doctor wants the demonstration of the lipoid content of
foamy cells or granulocytes in lung. I've found protocols that have
incubation-times from 10 minutes to over-night. - I've made a saturated
Sudan black B-solution in 70% ethanol and tried it with10 min on liver
without real success.<<

The main thing you need to do is demonstrate that it isn't hemosiderin with
an iron stain (Perls prussian blue reaction), and perhaps also that it
isn't acid-fast. Lipofuscin can be identified an H & E staining, except for
these considerations.

Bob Richmond
Maryville, Tennessee
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Re: [Histonet] Alcianblue for fast frozen sections

[Tony Henwood (SCHN) via Histonet]

Hello Gudrun,
Alcian blue for fast frozen sections

I was not aware that alcian blue would be routinely used on frozen sections of 
transplant liver biopsies.
There are other staining techniques that contain a blue dye, that have been 
used in liver biopsies including Masson's Trichrome (aniline blue) and Millers 
Stain (Victoria blue). Sulphated Alcian Blue has been used for the 
demonstration of amyloid in liver biopsies, though not usually on transplant 
liver biopsies.
Alcian blue has been used on frozen sections to demonstrate carboxylated mucins 
(J Clin Pathol 42(1): 101-105, 1989).

Alcian blue staining has been used on FFPE sections of transplant liver 
sections to show loss of heparan sulphate from endothelial cells of liver graft 
vessels. Alcian blue at pH 5.8 with 0.3 M MgCl2 on 3-micrometre 
paraffin-embedded liver section. At this MgCl2 concentration only sulfate 
glycosaminoglycans such as heparan sulfate stain. Loss of heparin sulphate 
reduces the ability of the graft to maintain an anti-coagulant environment and 
can result in diffuse microvascular thrombosis and vascular congestion (see 
Remuzzi et al (2005). Hemolytic uremic syndrome: a fatal outcome after kidney 
and liver transplantation performed to correct factor H gene mutation. American 
Journal of Transplantation, 5(5), 1146-1150).
If this is the alcian blue technique needed, then the question arises whether 
heparan sulphate is insoluble, and therefore stainable, following cryotomy and 
fixation. Heparan sulphate has a solubility of 100mg/mL in water but is 
insoluble in ethanol. I would suggest validating the technique on frozen 
sections of kidney and liver, comparing the staining results to corresponding 
FFPE sections.

Please tell us how you go.

Tony

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

From: Gudrun Lang via Histonet 
Sent: 28 January 2023 04:38
To: histonet@lists.utsouthwestern.edu 
Subject: [Histonet] Alcianblue for fast frozen sections

Dear histonetters!

Today I've heard about alcianblue staining on fast frozen sections, but I've
got no details.

I would like to know, if the staining result is the same as for staining AB
on paraffinslides.



They use the stain on transplantation liver.

Is this a usual procedure?



I would be glad about any information.

Thanks in advance

Gudrun

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Re: [Histonet] IHC background

[Tony Henwood (SCHN) via Histonet]

Paula,

What does the artefact look like?

Another cause could be partial lifting of parts of the section, allowing 
reagents to become trapped under the section.
You commonly see it in thyroids where the colloid traps antibody and/or 
detection reagents under the colloid giving a particulate brown dust-like 
staining (sometimes looking like cocci).
You can also sometimes see it in fibrous tissue in sections.

Suggestions to solve this: check your sticky adhesive slides, or allow the 
slides to dry longer at room temperature before placing on the Bond (allows the 
adhesive time to work).

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

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Re: [Histonet] Clarification about Immunohistochemistry

[Tony Henwood (SCHN) via Histonet]

"Bond wash" is the propriety buffer used in the Bond Immunostainer.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

From: jayalakshmy p.s 
Sent: 12 April 2022 22:01
To: Tony Henwood (SCHN) 
Cc: histonet@lists.utsouthwestern.edu 
Subject: Re: [Histonet] Clarification about Immunohistochemistry

Thank you so much Susan and Tony. Let me prepare the reagents 
To Susan- can you please elaborate on the steps of the giemsa method.
To Tony Henwood - please tell what is meant by "bond washing"
Thanks
Dr. Jayalakshmy

On Tue, Apr 12, 2022, 9:58 AM Tony Henwood (SCHN) 
mailto:tony.henw...@health.nsw.gov.au>> wrote:
Hi there,

I have had good results with Giemsa-like counterstaining (Stefanović  et al 
2013, Ravishankar et al 2016)  :

Azure blue, when substituted for hematoxylin as a counterstain in immunostain 
preparation, has been used to help differentiate melanocytes from melanophages. 
Azure blue preferentially stains cytoplasmic melanin granules blue-green, 
whereas melanocytes are highlighted by brown DAB chromogen. Melanophages, which 
contain melanin and lack melanocytic determinants, appear clear with blue-green 
granules in the cytoplasm (Hillesheim et al 2011).

After the slides were bond washed for 4min and rinsed in distilled water, they 
were stained with a mixture of the following solution: 100 mg of Azure blue 
(Sigma) in 4 ml of distilled water. Solution 2 was prepared as follows: 0.6ml 
of 0.1M sodium acetate and 3.4 ml 0.1M acetic acid were added to 27ml distilled 
water. Both solutions 1 and 2 were combined and 5ml of acetone was added. The 
slides were incubated for 60 min at room temperature, differentiated in 95% 
ethanol and dehydrated in several changes of absolute ethanol, followed by 
clearing in xylene with subsequent mounting (Kamino & Tarn 1991, Hillesheim et 
al 2011).

An alternate method is to counterstain the immunohistochemical reaction with a 
methylene blue solution (method courtesy of Dr Vince Munro, St Vincents 
Hospital in Sydney):

Staining Solution
2.38gm Sodium acetate
4.7ml Acetic acid
5gm Methylene Blue
Make up to 1 litre with distilled water

Procedure
1.  After immunostaining, wash slides in tap water
2.  Stain in Methylene Blue solution for 2 minutes
3.  Wash well in water
4.  Counterstain in Haematoxylin, wash well & blue as usual.
5.  Dehydrate, clear and mount

Result: Melanin should stain green-blue.

Hillesheim, P. B., Slone, S., Kelley, D., Malone, J., & Bahrami, S. (2011). An 
immunohistochemical comparison between MiTF and MART‐1 with Azure blue 
counterstaining in the setting of solar lentigo and melanoma in situ. Journal 
of cutaneous pathology, 38(7), 565-569

Kamino H, Tarn ST (1991) Immunoperoxidase technique modified by counterstain 
with azure B as a diagnostic aid in evaluating heavily pigmented melanocytic 
neoplasms. Journal of cutaneous pathology, 18(6):436-439.

Ravishankar, S., Nagarajan, P., Curry, J. L., Tetzlaff, M. T., Ivan, D., 
Torres-Cabala, C. A., ... & Prieto, V. G. (2016). Giemsa is the optimal 
counterstain for immunohistochemical detection of BRAF V600E mutation status in 
pigmented melanomas. Journal of cutaneous pathology, 43(8), 722-724.

Stefanović, D., Stefanović, M., & Nikin, Z. (2013). Romanowsky-Giemsa as a 
counterstain for immunohistochemistry: optimizing a traditional reagent. 
Biotechnic & Histochemistry, 88(6), 329-335.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA




-Original Message-
From: jayalakshmy p.s via Histonet 
[mailto:histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>]
Sent: Tuesday, 12 April 2022 1:59 PM
To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: [Histonet] Clarification about Immunohistochemistry

Hai all Histonetters
 Please anybody clarify my this doubt if possible.
When doing Immunohistochemistry for confirmation of Melanoma with DAB 
chromogen(brown color)the interpretation is not possible because of obscuring 
by the dense melanin pigment. We dont have any other color chromogen. I tried 
ihc after bleaching but the section gets detached even from charged slides. Is 
there any other effective

Re: [Histonet] Clarification about Immunohistochemistry

[Tony Henwood (SCHN) via Histonet]

Hi there,

I have had good results with Giemsa-like counterstaining (Stefanović  et al 
2013, Ravishankar et al 2016)  :

Azure blue, when substituted for hematoxylin as a counterstain in immunostain 
preparation, has been used to help differentiate melanocytes from melanophages. 
Azure blue preferentially stains cytoplasmic melanin granules blue-green, 
whereas melanocytes are highlighted by brown DAB chromogen. Melanophages, which 
contain melanin and lack melanocytic determinants, appear clear with blue-green 
granules in the cytoplasm (Hillesheim et al 2011).

After the slides were bond washed for 4min and rinsed in distilled water, they 
were stained with a mixture of the following solution: 100 mg of Azure blue 
(Sigma) in 4 ml of distilled water. Solution 2 was prepared as follows: 0.6ml 
of 0.1M sodium acetate and 3.4 ml 0.1M acetic acid were added to 27ml distilled 
water. Both solutions 1 and 2 were combined and 5ml of acetone was added. The 
slides were incubated for 60 min at room temperature, differentiated in 95% 
ethanol and dehydrated in several changes of absolute ethanol, followed by 
clearing in xylene with subsequent mounting (Kamino & Tarn 1991, Hillesheim et 
al 2011).

An alternate method is to counterstain the immunohistochemical reaction with a 
methylene blue solution (method courtesy of Dr Vince Munro, St Vincents 
Hospital in Sydney):

Staining Solution
2.38gm Sodium acetate
4.7ml Acetic acid
5gm Methylene Blue
Make up to 1 litre with distilled water

Procedure
1.  After immunostaining, wash slides in tap water
2.  Stain in Methylene Blue solution for 2 minutes
3.  Wash well in water
4.  Counterstain in Haematoxylin, wash well & blue as usual.
5.  Dehydrate, clear and mount

Result: Melanin should stain green-blue.

Hillesheim, P. B., Slone, S., Kelley, D., Malone, J., & Bahrami, S. (2011). An 
immunohistochemical comparison between MiTF and MART‐1 with Azure blue 
counterstaining in the setting of solar lentigo and melanoma in situ. Journal 
of cutaneous pathology, 38(7), 565-569

Kamino H, Tarn ST (1991) Immunoperoxidase technique modified by counterstain 
with azure B as a diagnostic aid in evaluating heavily pigmented melanocytic 
neoplasms. Journal of cutaneous pathology, 18(6):436-439.

Ravishankar, S., Nagarajan, P., Curry, J. L., Tetzlaff, M. T., Ivan, D., 
Torres-Cabala, C. A., ... & Prieto, V. G. (2016). Giemsa is the optimal 
counterstain for immunohistochemical detection of BRAF V600E mutation status in 
pigmented melanomas. Journal of cutaneous pathology, 43(8), 722-724.

Stefanović, D., Stefanović, M., & Nikin, Z. (2013). Romanowsky-Giemsa as a 
counterstain for immunohistochemistry: optimizing a traditional reagent. 
Biotechnic & Histochemistry, 88(6), 329-335.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA




-Original Message-
From: jayalakshmy p.s via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, 12 April 2022 1:59 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Clarification about Immunohistochemistry

Hai all Histonetters
 Please anybody clarify my this doubt if possible.
When doing Immunohistochemistry for confirmation of Melanoma with DAB 
chromogen(brown color)the interpretation is not possible because of obscuring 
by the dense melanin pigment. We dont have any other color chromogen. I tried 
ihc after bleaching but the section gets detached even from charged slides. Is 
there any other effective way to do this?
Thanks in advance
Regards
Dr. P S Jayalakshmy
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Re: [Histonet] Histonet Digest, Vol 220, Issue 8

[Tony Henwood (SCHN) via Histonet]

Hello Jayalakshmy,

iPassport is our digital quality management system. It is a commercial product 
that allows us to collect data, record audits, document Corrective Actions, 
manage controlled documents (eg manuals), expiry data and verification of 
antibodies, staff competencies and training and equipment maintenance reports 
etc in order to aid us in meeting the ISO 15189 standard for laboratory 
accreditation.

The “History” screen is available on the Bond automated immunostainer.

This is our policy for quality assurance of out-dated antibodies, so the 
mechanics are our department’s procedure. I did not try to make it generic (too 
little time to edit ☺ ).


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) | Principal 
Scientist; Adjunct Fellow, School of Medicine, University of Western Sydney; 
Visiting Lecturer, School of Life Sciences, Faculty of Science, University of 
Technology Sydney | Histopathology
t: (02) 9845 3306 | e: 
tony.henw...@health.nsw.gov.au<mailto:tony.henw...@health.nsw.gov.au> | w: 
www.schn.health.nsw.gov.au<http://www.schn.health.nsw.gov.au>
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Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
Locked Bag 4001, Westmead 2145, NSW Australia
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♲  Please consider the environment before printing this email.
From: jayalakshmy p.s [mailto:psjayalaks...@gmail.com]
Sent: Friday, 18 March 2022 12:45 AM
To: Tony Henwood (SCHN) 
Subject: Re: [Histonet] Histonet Digest, Vol 220, Issue 8

Hello Henwood,
Thank you for the reply to my post with elaborate explanation and appropriate 
references. We doubted about the validity of using after expiry date. We keep 
onslide controls for every case. So with your suggestion we are confident of 
using it looking at the control. We, in developing country has not much finance 
to replenish the reagents past expiry date frequently when it is sparingly used.
Sorry to say that I didnt understand the words ipassport & history screen. Can 
you elaborate a little more about these terms?
Thanks & Regards
Jayalakshmy


On Thu, Mar 17, 2022, 6:48 AM Tony Henwood (SCHN) 
mailto:tony.henw...@health.nsw.gov.au>> wrote:
Hi Jayalakshmy,

This our policy at the Kid's hospital in Sydney:

Validation of Expired Antibodies
Usually when a new concentrated antibody is received it will have an expiry 
date of around 2 years from receipt but usually we can continue to use 
antibodies well past this expiry date.

If the antibody continues to stain control sections appropriately, with no loss 
of sensitivity and no increase in non-specific staining then its use should be 
continued. If positive control samples are deemed unsatisfactory, even if the 
antibody is within the manufacturer’s printed expiration date, evaluation of 
the clinical specimen is aborted and the test deemed invalid. The quality of 
the primary antibody is therefore not based on an expiration date, but rather 
on its performance on a case-by-case basis with appropriate positive and 
negative control samples (1).

Several authors have investigated whether the shelf-life of diagnostic 
antibodies was longer than the expiry date on the label. They found them to 
work perfectly on routine histology sections (1-4). Monoclonal antibodies 
originally supplied as culture supernatants or as ascites (neat or diluted), of 
all isotypes, as well as all of the polyclonal antibodies, produced 
satisfactory staining irrespective of their age. Notable exceptions were 
ammonium-precipitated, IgM or conjugated antibodies.

The policy at CHW is, when an antibody has reached past its expiry date, its 
control is tested to ensure that there has been no loss of sensitivity in the 
test. This is now controlled through iPassport, where a task is attached to the 
antibody requesting validation of control when the antibody is expired. This 
can be easily done by using the History Screen and looking for use of this 
antibody within the last two weeks. If results of control are acceptable, 
another task is instigated for 6 months hence.

For antibody concentrates that are received without an expiry date, a 
verification is scheduled 12 months after receipt of the antibody.

If an antibody fails to perform to expectations then a Corrective Action 
Request is instigated in iPassport and appropriate investigation is instituted.

References:
1. Savage, E. C., & DeYoung, B. R. (2010). Antibody Expiration in the Context 
of Resource Limitation What Is the Evidence Basis?. American journal of 
clinical pathology, 134(1), 60-64.

Re: [Histonet] Histonet Digest, Vol 220, Issue 8

[Tony Henwood (SCHN) via Histonet]

Hi Jayalakshmy,

This our policy at the Kid's hospital in Sydney:

Validation of Expired Antibodies
Usually when a new concentrated antibody is received it will have an expiry 
date of around 2 years from receipt but usually we can continue to use 
antibodies well past this expiry date.

If the antibody continues to stain control sections appropriately, with no loss 
of sensitivity and no increase in non-specific staining then its use should be 
continued. If positive control samples are deemed unsatisfactory, even if the 
antibody is within the manufacturer’s printed expiration date, evaluation of 
the clinical specimen is aborted and the test deemed invalid. The quality of 
the primary antibody is therefore not based on an expiration date, but rather 
on its performance on a case-by-case basis with appropriate positive and 
negative control samples (1).

Several authors have investigated whether the shelf-life of diagnostic 
antibodies was longer than the expiry date on the label. They found them to 
work perfectly on routine histology sections (1-4). Monoclonal antibodies 
originally supplied as culture supernatants or as ascites (neat or diluted), of 
all isotypes, as well as all of the polyclonal antibodies, produced 
satisfactory staining irrespective of their age. Notable exceptions were 
ammonium-precipitated, IgM or conjugated antibodies.

The policy at CHW is, when an antibody has reached past its expiry date, its 
control is tested to ensure that there has been no loss of sensitivity in the 
test. This is now controlled through iPassport, where a task is attached to the 
antibody requesting validation of control when the antibody is expired. This 
can be easily done by using the History Screen and looking for use of this 
antibody within the last two weeks. If results of control are acceptable, 
another task is instigated for 6 months hence.

For antibody concentrates that are received without an expiry date, a 
verification is scheduled 12 months after receipt of the antibody.

If an antibody fails to perform to expectations than a Corrective Action 
Request is instigated in iPassport and appropriate investigation is instituted.

References:
1. Savage, E. C., & DeYoung, B. R. (2010). Antibody Expiration in the Context 
of Resource Limitation What Is the Evidence Basis?. American journal of 
clinical pathology, 134(1), 60-64.
2. Balaton, A. J., Drachenberg, C. B., Rucker, C., Vaury, P., & Papadimitriou, 
J. C. (1999). Satisfactory performance of primary antibodies beyond 
manufacturers' recommended expiration dates. Applied Immunohistochemistry & 
Molecular Morphology, 7(3), 221.
3. Argentieri, M. C., Pilla, D., Vanzati, A., Lonardi, S., Facchetti, F., 
Doglioni, C., & Cattoretti, G. (2013). Antibodies are forever: a study using 
12-26‐year‐old expired antibodies. Histopathology, 63(6), 869-876.
4. Drachenberg, C. B., Papadimitriou, J. C., Balaton, A. J., & Vaury, P. 
(2001). The total test approach to standardization of immunohistochemistry. 
Archives of pathology & laboratory medicine, 125(4), 471-471.


Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA



-Original Message-
From: jayalakshmy p.s via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Thursday, 10 March 2022 1:27 PM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Histonet Digest, Vol 220, Issue 8

Hello all, I would like to know whether Immunohistochemistry markers can be 
used after its expiry date(atleast in resource poor countries). If yes, for how 
much period of time and what is the criteria to look for?
Thanks and regards
Jayalakshmy

On Wed, Mar 9, 2022, 11:30 PM 
wrote:

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> Today's Topics:
>
>1. Re: Post message to Histonet (Chenoa Hardwick)
>2. Re: formalin in OR (Greg Dobbin)
>3. posting (Chenoa Hardwick)
>4. Re: formalin in OR (John Garratt)
>
>
>
> -- Forwarded message --
> From: Chenoa Hardwick 
> To: histonet@lists.utsouthwestern.edu
> Cc:
> Bcc:
> Date: Tue, 8

Re: [Histonet] Dr Freida Carson

[Tony Henwood (SCHN) via Histonet]

I agree,

She will be missed


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


-Original Message-
From: Shirley A. Powell via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Thursday, 13 January 2022 7:59 AM
To: Willis, Donna G 
Cc: Histonet (histonet@lists.utsouthwestern.edu) 

Subject: Re: [Histonet] Dr Freida Carson

Hi Donna,
So sorry to hear of this. I am proud to have known her and been a recipient of 
her wealth of knowledge.
She contributed so  much to the Histology Community.

Shirley

Shirley Powell, HTL(ASCP)
Technical Director Histology Curricular Support Laboratory Pathology Department 
Mercer University School of Medicine
1550 College St, Macon, GA  31207
O: 478-301-2374/F:478-301-5489
medicine.mercer.edu




-Original Message-
From: Willis, Donna G via Histonet 
Sent: Wednesday, January 12, 2022 3:19 PM
To: Histonet (histonet@lists.utsouthwestern.edu) 

Subject: [Histonet] Dr Freida Carson

For those of you that are not on Facebook you may not already know that 
yesterday Freida Carson, PhD became one of our Histology Angels.  I had the 
privilege of having her as my Histology Educator long before she wrote her 
first edition of Histotechnology.  But more precious to me than having her as 
my educator was that she was a friend.  She will be missed.  Thank you Freida 
for all that you have given to the Histology Profession and to me.  Rest In 
Peace singing in the Angels Choir.

Donna Willis
Anatomic Pathology Manager
Baylor Scott Health
Baylor University Medical Center
3500 Gaston Ave|Dallas, Texas 75246
214-820-2465 office|214-725-6184 mobile



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Re: [Histonet] tissue disposal

[Tony Henwood (SCHN) via Histonet]

Hi Pam,

We have used the Milestone Tissue Safe Plus unit with Integrated Data Logger 
card reader to seal formalin-fixed placentas (and other specimens if needed) 
since 2019.
Placentas are drained of excess formalin in a chemical hood, placed into a 
labelled bag and sealed.

So possibly for long term storage, the Tissue Safe Plus might be worth looking 
at.

To save the environment, we try and re-cycle the larger plastic containers. As 
clean as possible but we try to not spend too much time on them.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Richardson, Pam K via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 14 October 2021 7:37 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] tissue disposal

I am interested to learn how others are handling tissue disposal. How long to 
you keep wet tissue? Are you reusing containers? We currently dispose of the 
tissue and wash the containers and I am questioning if there is a better 
system? 

Best Wishes

Pam ~
National Histology Professionals Day 3/10/21 Pathologists' Assistant Day 
4/14/2021 Medical Laboratory Professionals Week April 18-24, 2021 National 
Cytotechnology Day 5/13/2021

+++
Pam Richardson
Clinical Manager
Gundersen Health System Laboratory Services
Email: pkric...@gundersenhealth.org
Phone: 608 775-4133
Fax: 608 775-6136
Interdepartmental Mail Stop: H04-007
E-visit us at: 
http://secure-web.cisco.com/1NGyXQUNr0j3MPQIjvmHqDwbUHBOGTSAJGY8v49mWsvp1_J2JSv0VL0YU2v5fBMwm9P9gkTaqYKmqnmP-QuNOClh6woo0CZiF0odkeVPTcoU5KS5ryxDxp16U_vnAsVLijWu1CwBGDPkJLePBE4OKDeXzL3cX-bzf0r-dKm0B9hw9e_yFECTUr-x3L_AVKV3OEhuj1kxg-ohIeIRtgqr4e0OlWI1MflK2moiDwp3XHYOx1kk8j4WPwEsuQqG1sLi6FKEDmyeUND1yNAGvWc5mhpyyyEHi9-3_9hd-8JUIk379SYu2qAHtKWCIdXHaovxUfgrnfqQp4X8G2t6pER6kt9RMNNAg7H_Kp9rl_V_SspLOu3QVAddkglNqSBHC3oFxLtxU5-HyiKOdHf5WqYOXdldkvybWkSXOf78Vl0gzJyIE4_lFYSwx26nqcd78P3vMVctVFNiNZry0Yo0oU6m-TjQlve8-a6kR98wk7nR04dQ/http%3A%2F%2Fwww.gundersenhealth.org

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Re: [Histonet] Vacuum packing of formalin fixed tissues for archiving

[Tony Henwood (SCHN) via Histonet]

Hi Michelle,

We have used the Milestone Tissue Safe Plus unit with Integrated Data Logger 
card reader to seal formalin-fixed placentas since 2019.
Placentas are drained of excess formalin in a chemical hood, placed into a 
labelled bag and sealed.

We instituted the process to reduce the formalin fume leakage that often 
accompanies the storage of these specimens and to decrease the storage space 
required.

The process has lived up to its aims (formalin fume containment, no liquid 
leakage and reduced storage requirements) and we are currently sampling the 
early specimens (3 years in storage) to analyse any adverse morphology and 
Immunohistochemical staining that might occur.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-Original Message-
From: Michelle Perrins via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 6 October 2021 11:25 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Vacuum packing of formalin fixed tissues for archiving

Hello
I would like to know if any histology laboratory stores their fixed specimens 
this way and what instrument do you use?
Regards
Michelle

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[Histonet] Fw: Fixative in diff-quick

[Tony Henwood (SCHN) via Histonet]

Hi Clay,

The first solution is methanol.
So lab grade methanol will do.
The blue colour is from an innocuous dye added to differentiate from water (and 
ethanol used in cytology fixation). This helps us poor cytologists when we are 
doing ROSE at FNAs.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA



-Original Message-
From: Corbin, Clay via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, 5 October 2021 7:58 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Fixative in diff-quick

Hey folks,
I am shopping for a diff-quick kit.  However, all I really need is the 
fixative.  Generally, there is a blue stain (triarylmethane) added to the 
methanol in the fixative solution.  I have a giant jug of lab grade methanol.  
What would I lose by using methanol alone compared to the fixative solution 
included in a diff-quick kit?
Thanks!
Clay

Clay Corbin, PhD
Professor of Biology
Bloomsburg University
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Re: [Histonet] Jones' Methenamine Silver Stain for Basement Membranes of Kidney - Issues and Questions

[Tony Henwood (SCHN) via Histonet]

I agree with Bryan,

The introduction of thiosemicarbazide before the silver step improves the 
staining immensely.

I would also look at the periodic acid. Is it too dilute, though 0.5% should 
work? I usually cover this by using a 1% solution for 20 minutes.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Bryan Llewellyn via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 24 September 2021 7:47 AM
To: Jordan ; Histonet 

Subject: Re: [Histonet] Jones' Methenamine Silver Stain for Basement Membranes 
of Kidney - Issues and Questions

Hi,
Try the method given in StainsFile at:
http://stainsfile.info/stain/metallic/jones.htm

Bryan Llewellyn


Hood, Jordan via Histonet wrote:
> Hello,
> 
> I'm new to histology (and new to histonet), and I work in a small histology 
> lab specializing in animal tissues that receives requests/submissions from 
> researchers. I tried (and failed) to perform a Jones' Methenamine Silver 
> stain on a client's submission of pig kidneys (formalin-fixed, 
> paraffin-embedded, cut at 2.5 microns), and I need some help troubleshooting 
> this stain since my co-workers are stumped, too.  I used the following 
> procedure from Rowley Biochemical:
> 
> 
> ~
> "Fixation: 10% Buffered Neutral Formalin (F-113) or Bouin's Solution 
> (F-40) or Zenker's (F-155)
> 
> Sections: Paraffin, 2 microns
> 
> Procedure: Acid washed glassware must be used
> 1. Deparaffinize and hydrate to distilled water.
> 2. Oxidize in Periodic Acid 0.5% (F-396-1) 11 minutes. Wash in chloride-free 
> water.
> 3. Prepare Methenamine Silver solution by mixing: 42.5 ml Methenamine 3% 
> (F-396-2), 2.5 ml Silver Nitrate, 5% (F-396-3) and 12.0 ml Borate Buffer, pH 
> 8.2 (F-396-4).
> 4. Place slides in the solution and the entire jar in a water bath at 70°C 
> for approx. 60-75 minutes. Check under microscope when slides appear medium 
> brown microscopically. Every 10 minutes, once the medium brown color has been 
> established, rinse a slide in 70°C, chloride free water and check under a 
> microscope. Rinse again in hot water and return to the hot staining solution. 
> As the staining time approaches the end point, check the slides, as above, 
> every 1-2 minutes. The entire procedure must be performed quickly to prevent 
> an uneven staining of the tissues. The slides should exhibit a brownish- 
> yellow background, intense black reticulum fibers, and black basement 
> membranes. If the slides become oversaturated, i.e. too black, destain in a 
> dilute Potassium Ferricyanide Solution (F-396-11) for one or two dips.
> 5. Rinse well in distilled water. Tone in Gold Chloride 0.2% (F-396-5), 1 
> minute. If sections are overtoned place in Sodium Metabisulfite, 3% 
> (F-396-12) for 1-3 minutes. Rinse well in distilled water.
> 6. Sodium Thiosulfate 3% (F-396-9), 1-2 miutes. Wash in running tap water, 10 
> minutes. Rinse well in distilled water.
> 7. Stain in Harris' Hematoxylin (F-396-6) containing 2-4ml of Glacial Acetic 
> Acid per 100 ml for 5-15 minutes. Wash in water.
> 8. Differentiate in Acid Alcohol 1% (F-396-13) until the sections turn red.
> 9. Blue section in Ammonia Water, 0.3% (F-396-14). Wash thoroughly.
> 10. Counterstain in Eosin Y, 1%, Alcoholic Solution (F-396-7).
> 11. Dehydrate in 95% alcohol, absolute alcohol and clear in xylene 3 changes 
> each. Mount.
> 
> Stain Results:
> Basement membranes, reticulum fibers: Black
> Nuclei: Blue
> Cytoplasm, collagen, connective tissue: Pink-orange
> 
> References: Jones, D.B., Amer.J.Path. 27:99 (1951). AFIP Manual of 
> Histolocical Staining Methods, 3rd ed., Ed. L. Luna: NY: McGraw-Hill 
> Publications, c. 1968, p. 97."
> ~
> 
> 
> It became apparent that something went wrong during Step 4 when the slides 
> were in the glass container (not a coplin jar - we have ten slides that we 
> need to stain so we're using a rectangular glass container that holds ten 
> slides on their sides - it does require a metal handle to move, but the 
> handle is flexible and easy to remove after the glass slide rack has been 
> transferred between containers) of silver solution in the water bath because 
> there was lots of precipitate on the slides and floating on the surface of 
> the silver solution.
> 
> In my first test, I used five test slides (extra slides that we cut from the 
> same blocks that were submitted to us). I deparaffinized them in

Re: [Histonet] Retirement in sight!

[Tony Henwood (SCHN) via Histonet]

I am reminded of the Beatles classic:

"Will you still need me, will you still feed me, When I'm sixty-four"

40 years! - a sh.. load of experience, a sh.. load of knowledge.

Histotechnology worldwide still needs your wisdom so we hope you can keep an 
eye on us through Histonet and the Block.

Enjoy retirement and hope to see you soon

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 



-Original Message-
From: Morken, Timothy via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 10 September 2021 2:26 AM
To: Histonet 
Subject: [Histonet] Retirement in sight!


After 40 years in the lab I've decided to retire this year - in a week actually!

It has been an interesting 4 decades...

I started out in an EM lab after getting a degree in Physiology and then  
competing a 2 year EM course at Delta College in Stockton, CA - the only 
dedicated EM program at that time. I started out running a scanning EM lab for 
an electronics company looking at microchips but after a couple years moved to 
a hosptial lab in Fresno, CA running their EM lab. I was the only one, so from 
day one was the "Manager" of the lab! I did about 150 EM cases a year and in 
those days it was a mix of kidney and tumor cases - there was no IHC yet so 
some tumor diagnostics depended on EM. I did not have quite enough work to keep 
me busy so I started hanging out in the histology lab. As with many people in 
this field the day I started working there was the first I had heard of 
"histology."  At first it was helping set up grossing, coverslipping slides and 
doing immunofluorescence for the kidney cases (and taking "kodachromes" of the 
results! Does anyone under 30 know what a Kodachrome is?!). But then our 
director wanted to bring in IHC and so had a tech from a lab at Cedars Sinai in 
LA come to teach us how to do it. We did all of 10 stains at first. Of course 
it was all manual and so had to know what was going on with every step. I 
didn't use an automated stainer for the first 12 years that I did IHC, and at 
times was doing 150 slides a day manually.

Gradually I ended up doing half time in histology and learned cutting, special 
stains, muscle histochemistry, immunofluorescence for kidney cases. I decided 
to work on the HT exam since I was doing all that work anyway. We had a lab of 
four men - pretty rare, Imagine - and we started a study group to all take the 
test. We met after work a couple times a week for 6 months pretty  much 
memorizing the Sheehan book. We all took the HT and all but one passed. Later I 
passed the HTL as well.

After 11 years of that I moved on to a job in Saudi Arabia - and my wife and 
daughter went along. I managed the IHC and muscle lab at King Faisal Specialist 
Hospital in Riyadh. My wife was lucky enough to get a teaching position at the 
American School where our daughter was in 9th grade. That made all the 
difference in our life there because if she had not gotten a job I don't think 
we would have stayed there  5 years. She would have been stuck doing pretty 
much nothing. I moved on to managing the histology lab as  whole. Living in 
another country is a great experience, even if it is a totally different 
culture. It certainly changed our outlook on the world and I would not trade 
that experience for anything. We also did a lot of travelling during those 
years - being on "that" side of  world makes traveling there much easier!

Once we decided to leave Saudi I looked for a job back in the States and was 
lucky enough to land one at the Centers for Disease Control in Atlanta in their 
Infectious Disease Pathology division. I worked with 5 infectious disease 
pathology specialists and a dozen technologists from histotechs to EM techs, to 
microbiologists to molecular biologists. We worked on routine cases to 
world-wide outbreak cases. During the 5 years I was there we identified at 
least one novel human virus every year that caused outbreaks. And that was in 
addition to numerous cases of outbreaks of known diseases for which we received 
samples from all over the world. Probably the most notorious case was the 
anthrax attack after 9/11. Four of us histotechs manned the lab 24 hours a day, 
7 days a week for 6 weeks running IHC tests on endless samples while trying to 
get on top of that case. In the middle of it all the power went out to the 
facility and we had to work on generator power with temporary lighting set up 
in the lab and battery packs to keep the equipment running. After 9/11 and then 
anthrax everyone was thinking it was a bioterror attack by the same 

Re: [Histonet] New cryostat validation procedure

[Tony Henwood (SCHN) via Histonet]

Hi Greg,

I would just grab some kidneys from the butcher, freeze and section as usual. 
They should give equivalent results to the older unit.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Greg Dobbin via Histonet 
Sent: Monday, 23 August 2021 22:16
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] New cryostat validation procedure

Hello experts!
I would like to find out what others have done to validate a new cryostat.
I believe it would basically be the same as validating a new microtome
except specimens (ie blocks) are plentiful for microtomy whereas for us,
fresh tissue for cryosectioning is not readily available.

Background:
We are an acute care hospital and we only get about 50 intraoperative
consult requests per year. Our older unit is still functional so I can do
side-by-side comparisons with the new unit. Our frozens are only stained
for H and a very rare (one every couple of years) Oil Red O.

So I have two questions:

   1. Can I use formalin-fixed tissue soaked overnight in a saturated
   sucrose solution for the validation? This seems reasonable since I can do
   side-by-side with the older unit. {autopsy tissue could be an option if the
   timing coincided with our validation but Murphy's Law will probably prevent
   this option! lol}

   2. How many tissues would be satisfactory? Is one specimen of each of
   our typical frozen specimen types enough? We typically get: ovary, thyroid,
   skin and lymph node. Any other tissue type would be rare.

I look forward to hearing from you!
Thank you,
Greg

--
*Greg Dobbin*
1205 Pleasant Grove Rd
RR#2 York,
PE  C0A 1P0


*Everything in moderation...even moderation itself**!*
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Re: [Histonet] cytology non-gyn screening

[Tony Henwood (SCHN) via Histonet]

do your cytotechs screen the non-gyn cases? Yes
Do they regularly assist with FNA's? Yes
How many paps are they expected to screen in a day? Not as important as the 
Turn-around-time (TAT) - if the TAT is short, regularly, then all is good.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Nancy Schmitt via Histonet 
Sent: Saturday, 7 August 2021 02:55
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] cytology non-gyn screening

Hello-
I know a lot of places have overlap with histology/cytology - do your cytotechs 
screen the non-gyn cases?
Do they regularly assist with FNA's?
How many paps are they expected to screen in a day?
This seems like a lot..
Input appreciated,
Nancy



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Re: [Histonet] frozen section problem

[Tony Henwood (SCHN) via Histonet]

We have had this issue previously.

We tracked it down to the brain biopsies arriving in "Isotonic" saline (which 
is really not isotonic).
See: Henwood, A., (2007) “Adverse effect of saline on brain intraoperative 
(frozen section) Histology” J Histotechnol 30(3):193.

Ask the surgeons to send the biopsies to the lab on damp shiny (non-absorbent) 
card.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Manfre, Philip via Histonet 
Sent: Saturday, 17 July 2021 07:54
To: Bonello Dorianne M at Health-MDH;   (histonet@lists.utsouthwestern.edu)
Subject: Re: [Histonet] frozen section problem

Hi Dorianne,
You need to freeze your tissue faster.  Ideally, isopentane placed in a 
metal cup, that is that is then frozen in a dewar of liquid nitrogen, works 
best.  The isopentane, once frozen, is thawed a little with a metal rod to 
produce a small liquid pool and your tissue is placed in this for about one 
minute.  You need some equipment for this procedure, such as the metal cup that 
can sit inside a small, open dewar of liquid nitrogen.  Alternatively, you can 
freeze directly in liquid nitrogen, though you need to beware of the tissue 
fracturing due to the sudden and extreme temperature reduction.  Slower 
freezing of tissue (sitting on dry ice, etc.)  allows ice crystals to form in 
the tissue, creating the vacuoles you describe.
I hope this helps.
Phil.

Philip Manfre, BA, HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP81-406
770 Sumneytown Pike
West Point, PA 19486
215-652-9750
philip_man...@merck.com




-Original Message-
From: Bonello Dorianne M at Health-MDH via Histonet 

Sent: Friday, July 16, 2021 11:25 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] frozen section problem

EXTERNAL EMAIL – Use caution with any links or file attachments.

Dear all,


We are experiencing freezing artifacts on our frozen sections. Basically, we 
are seeing cavity-like structures under the microscope, mostly elongated, 
especially when it's a frozen section on brain tissue. This is most probably 
happening due to ice crystal formation. We're not using cryospray, relying only 
on the cryobar boost function.


Does anyone has a solution to this problem please?


Regards,



Dorianne Bonello
Allied Health Practitioner (MLS)
Histology Laboratory - Pathology
Health-Mater Dei Hospital


[cid:image001.jpg@01D67184.63288530]


T +356 +356 25456434

E dorianne.m.bone...@gov.mt


Mater Dei Hospital, Triq id-Donaturi tad-Demm, l-Imsida, Malta MSD 2090 | Tel 
+356 2545  | 
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Re: [Histonet] "cooked" biopsy

[Tony Henwood (SCHN) via Histonet]

Hi John,


There is anecdotal evidence that recycled xylene is of a better quality than 
the original purchased product. We have found it to be so. No processing nor 
staining problems unless there has been a recycling issue (as shown by the CBG 
xylene purity test).


I suppose we have to access each reagent that is recycled and determine the 
risk of using it. For example, unless care is taken, recycling alcohol can be 
an issue (eg xylene contamination). We do not recycle alcohol for this reason.


"When we are continuously challenged by pre-analytic variables in 
histotechnology why do labs continue to use recycled reagents? "


>From your survey, has re-cycling xylene been found to be a major, or minor 
>issue?


We definitely need evidence that re-cycling is a risk.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

From: John Garratt 
Sent: Saturday, 10 July 2021 05:31
To: Paula; Tony Henwood (SCHN); 'Erick Rodriguez'
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] "cooked" biopsy

Interesting discussion.
At CPQA we recently started a H EQA program that includes fixation and 
processing in the feedback to participants and in a recent mini-survey I found 
that a third of labs use recycled product somewhere in the pre-analytic phase.
When we are continuously challenged by pre-analytic variables in 
histotechnology why do labs continue to use recycled reagents?

John



On Thu, Jul 8, 2021 at 5:59 AM, Paula via Histonet 
mailto:histonet@lists.utsouthwestern.edu>> 
wrote:
Hi Tony,
Thank you for the procedure. I do send out the recycled xylene to have its 
analysis done by an outside company every month, and I'll put into place this 
testing procedure that you outlined below in place ase well.
Paula

-Original Message-
From: Tony Henwood (SCHN) [mailto:tony.henw...@health.nsw.gov.au]
Sent: Wednesday, July 07, 2021 1:25 PM
To: 'Erick Rodriguez'; Paula
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] "cooked" biopsy

Hi Paula,

We can check the purity of the xylene quite easily:

Xylene Purity Test Procedure

Note: The recommended and most accurate method of determining the purity of the 
recycled xylene is by doing a Gas Chromatography analysis. The following method 
can be used to obtain an acceptable confidence level in the purity of the 
recycled xylene (CBG Biotech).

Testing Procedure

1. To a clean, dry 100 ml mixing cylinder graduate, add sufficient recovered 
xylene so that the bottom of the meniscus is aligned with the top edge of the 
85 ml mark on the graduate.

2. Add water to the graduate until the bottom of the meniscus aligns with the 
top edge of the 100 ml mark on the graduate. At this point, 15 ml of water will 
have been added to 85 ml of recovered xylene.

3. Stopper the graduate and invert the mixture. Allow the mixture to settle, 
making sure that all of the water settles to the bottom of the graduate. No 
water should remain clinging to the sides of the graduate above the 
xylene/water separation point. This separation point should be near the 15 ml 
level of the graduate. (Note: xylene floats on top of the water).

4. Carefully inspect and record the point of separation between the water and 
xylene using the bottom of the meniscus as the separation point.

5. Subtract 15 ml from the quantity of water indicated in step 5. The remainder 
plus an additional 0.1 correction factor equals the percentage of recovered 
xylene impurities.

EXAMPLE:

Xylene/Water separation point is indicated to be 15.5 ml.
(15.5 - 15) + 0.1 = 0.6% impurities.
Therefore, the recovered xylene is 99.4% pure.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Paula via Histonet 
Sent: Thursday, 8 July 2021 04:53
To: 'Erick Rodriguez'
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] "cooked" biopsy

Thank you, everyone...
I looked at my reagents and saw the color pink in the xylene, which tells me 
that there is water or too much water in it so I changed it.
We recycle xylene, so I need to get the recycler looked at now.
Thanks again,
Paula


-Original Message-
From: Erick Rodriguez [mailto:rodriguez.er...@icloud.com]
Sent: Wednesday, July 07, 202

Re: [Histonet] "cooked" biopsy

[Tony Henwood (SCHN) via Histonet]

Hi Paula,

We can check the purity of the xylene quite easily:

Xylene Purity Test Procedure 

Note: The recommended and most accurate method of determining the purity of the 
recycled xylene is by doing a Gas Chromatography analysis.  The following 
method can be used to obtain an acceptable confidence level in the purity of 
the recycled xylene (CBG Biotech).

Testing Procedure

1.  To a clean, dry 100 ml mixing cylinder graduate, add sufficient 
recovered xylene so that the bottom of the meniscus is aligned with the top 
edge of the 85 ml mark on the graduate. 

2.  Add water to the graduate until the bottom of the meniscus aligns with 
the top edge of the 100 ml mark on the graduate. At this point, 15 ml of water 
will have been added to 85 ml of recovered xylene.

3.  Stopper the graduate and invert the mixture.  Allow the mixture to 
settle, making sure that all of the water settles to the bottom of the 
graduate.  No water should remain clinging to the sides of the graduate above 
the xylene/water separation point. This separation point should be near the 15 
ml level of the graduate. (Note: xylene floats on top of the water).

4.  Carefully inspect and record the point of separation between the water 
and xylene using the bottom of the meniscus as the separation point.

5.  Subtract 15 ml from the quantity of water indicated in step 5.  The 
remainder plus an additional 0.1 correction factor equals the percentage of 
recovered xylene impurities.

EXAMPLE:

Xylene/Water separation point is indicated to be 15.5 ml.
(15.5 - 15) + 0.1 = 0.6% impurities.  
Therefore, the recovered xylene is 99.4% pure.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Paula via Histonet 
Sent: Thursday, 8 July 2021 04:53
To: 'Erick Rodriguez'
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] "cooked" biopsy

Thank you, everyone...
I looked at my reagents and saw the color pink in the xylene, which tells me 
that there is water or too much water in it so I changed it.
We recycle xylene, so I need to get the recycler looked at now.
Thanks again,
Paula


-Original Message-
From: Erick Rodriguez [mailto:rodriguez.er...@icloud.com]
Sent: Wednesday, July 07, 2021 11:24 AM
To: Paula
Subject: Re: [Histonet] "cooked" biopsy

Did you change the processor reagents before running your tissues? Cooked 
tissue usually means the tissue wasn’t dehydrated properly and the leftover 
water boiled and fried your tissue. I would double check the alcohols.

> On Jul 7, 2021, at 11:00 AM, Paula via Histonet 
>  wrote:
>
> Hello, good day,
>
>
>
> Our pathologist is complaining about the tissues today that they are
> "cooked, burnt, crushed, shrunken" those are the adjectives she is using.
>
>
>
> Can you tell me the cause?  Usually, the work comes out beautiful but today
> they are not. Nothing has changed on our processing times.
>
>
>
> What should I investigate?
>
>
>
> Thank you in advance,
>
> Paula
>
> Bio-Path Medica Group
>
>
>
> ___
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Re: [Histonet] Unstained slides precut for IHC

[Tony Henwood (SCHN) via Histonet]

ation of heated detergent dewaxing and 
rehydration to immunohistochemistry. Biotechnic & Histochemistry, 87(1), 46-50.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Carrie Disbrow via Histonet 
Sent: Monday, 14 June 2021 12:04
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Unstained slides precut for IHC

Hi! Current lab is cutting extra slides for IHC and putting in 60 degree oven 
for 45 mins to dry.  If IHC is ordered a precut control and the already baked 
slides are again put in the 60 degree oven for 45 mins. Sometimes the control 
is cut and added to the dry slide or a separate slide.  So, the questions  have 
been should the   slides be air dried first using a fan before baking and 
slides not baked until the pathologist places an order? Should the tissue and 
the control all have the same amount of time in the oven to ensure consistency? 
Also, is it better to rack slides standing or on edge for IHC? Additionally, 
when sending slides for IHC to other labs is it preferred to send dry slides or 
baked slides? Thanks for your input!
Carrie Disbrow, HTL

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Re: [Histonet] Prussian Blue Reaction

[Tony Henwood (SCHN) via Histonet]

The following might be useful:

Iron Histochemistry - A Review

It is convenient to divide iron-containing complexes in human tissues into two 
categories: those in which the iron is loosely bound to proteins and easily 
released by mild acid treatment (eg hemosiderin) and those in which the iron is 
more strongly bound (masked iron) and cannot be released by mild acid 
hydrolysis (eg haemoglobin) (1).

Iron in the body is stored in the forms of hemosiderin (ferric hydroxide 
polymer) or ferritin (a ferrous iron-protein complex) (1). Iron in tissues 
occurs mainly in the ferric state (2,3).

The reactions that have been used for the detection of iron in tissues include 
(2-5):

1.  The Quincke reaction using ammonium sulphide
2.  The Perls reaction, using ferrocyanide, for ferric and the Turnbull 
Blue reaction, using ferricyanide for ferrous iron.
3.  Coloured lakes, eg haematoxylin (Mallory's Method)
4.  Coloured precipitates with organic chemicals not classified as dyes (eg 
bathophenanthroline). 

Ferric iron may be converted into ferrous iron by ammonium sulphide (Quinke's 
reaction) and the ferrous sulphide thus formed can then be demonstrated using 
the Turnbull blue reaction (3,5).

Some iron-containing compounds (hemoglobin, malaria pigment, formalin pigment) 
do not react with the Perl's method because the iron is present in bound form. 
These compounds can be unmasked using hydrogen peroxide and then demonstrated 
using the Perl's reaction (1).

Interestingly, it is possible to remove excess iron pigment from tissue 
sections. Iron can be removed by (5):

.   15 min in 1% sodium dithionite in 0.1M acetate-HCl  buffer (pH 4.5)
.   3 hours in 2.4N HCl
.   30 min in 3.7N H2SO4
.   15 min in 5% Oxalic acid

Heavily pigmented tissues may need to have these times extended (5).



References

1.  Barka, T., Anderson, P.J., (1963) "Histochemistry: Theory, practice and 
bibliography" Harper & Row Publishers Inc, New York, p172-174.
2.  Davenport, H.A., (1961) "Histological and Histochemical Technics" W.B. 
Saunders Co., Philadelphia, 280-284.
3.  Gabe, M., (1976) "Histological Techniques" Masson, Paris, p311-317.
4.  Lynch, M.J., Raphael, S., etal "Medical Laboratory Technology and 
Clinical Pathology" 2nd Ed, W.B Saunder Co, Philadelphia, p1135-1136.
5.  Morton, D., (1978) "A comparison of iron histochemical methods for use 
on glycol methacrylate embedded tissues" Stain Tech 53(4):217-223.



Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-Original Message-
From: Mac Donald, Jennifer via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, 8 June 2021 6:41 AM
To: Jay Lundgren 
Cc: histonet@lists.utsouthwestern.edu; John Kiernan 
Subject: Re: [Histonet] Prussian Blue Reaction

Thank you.

From: Jay Lundgren 
Sent: Monday, June 7, 2021 1:35 PM
To: Mac Donald, Jennifer 
Cc: John Kiernan ; Gudrun Lang ; 
histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Prussian Blue Reaction

  EXTERNAL SENDER- Exercise caution with requests, links, and attachments.
Supposed to be insoluble.  Try potassium permanganate followed by oxalaic acid. 
 But book says insoluble.

On Monday, June 7, 2021, Mac Donald, Jennifer via Histonet 
mailto:histonet@lists.utsouthwestern.edu>> 
wrote:
The instrument malfunction and it was overstained.

From: John Kiernan mailto:jkier...@uwo.ca>>
Sent: Monday, June 7, 2021 9:50 AM
To: Gudrun Lang mailto:gu.l...@gmx.at>>; Mac Donald, Jennifer 
mailto:jmacdon...@mtsac.edu>>
Cc: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: Re: [Histonet] Prussian Blue Reaction

  EXTERNAL SENDER- Exercise caution with requests, links, and attachments.
Overstained? Doesn't that mean the tissue contains a lot of iron and you are 
seeing where it is - which was the reason for doing Prussian blue 
histochemistry. Gudrun Lang correctly says that mineral acids won't remove it. 
Oxalic acid is said to dissolve Prussian blue (? by chelation); I've never 
tried this. If it works, you will no longer see where the iron is. To see 
features other than the distribution of iron, why not just stain another 
section from the block with a general-purpose stain like Giemsa or H?
John Kiernan
London, Canada
= = =

From: Mac Donald, Jennifer via Histonet 
mailto:histonet@lists.utsouthwestern.edu><mailto:histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>>>
Sent: June 7, 2021 12:47 AM
To: Gudrun Lang 
mailto

Re: [Histonet] Teaching color blind Histo student

[Tony Henwood (SCHN) via Histonet]

With Red-Green colour-blindness, I have found that a multi-stain approach works 
for example:

Often the ZN counterstain used is light green. A second ZN stained with 
Loefler's Methlene Blue for comparison seems to help.
The same can be done with the Masson's Stain (compare the Fast Green FCF or the 
Light Green collagen stain with the Masson's Variant using methyl blue, aniline 
blue or direct sky blue)
If the Twort's variant of the Gram stain  is used, compare with a Brown-Hopp’s 
Gram Stain.

Those with Red-Green colour blindness seem to compensate excellently and I have 
found no issues with staff (including pathologists) in differentiating the two 
colours.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: B kB via Histonet 
Sent: Saturday, 27 March 2021 20:50
To: Ruppert, Amysue
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Teaching color blind Histo student

Hi,

Colourblindness ain’t a problem at all.
I know, because I already work for 35 years in the histopathogy.
Just a few things that causes problems for me:
Grossing: I describe tissue in the way I see, mostly I call something with
2 colours, p.e. “ the serose is brownish grey” (while collegaes mostly
mention 1 colour. But I can see the difference between normal and abnormal
tissue, that’s more important.
Embedding: only skin biopsies is it difficult to oriëntate when the colour
of the dermis is the same as the subcutis (in my eyes  ;-). So with that, I
ask my collegaes to embed these biopsies for me.
Staining: When I have to do the stainings, I just have to following the
protocol. In the SOP, There is always a example of the control slide. I
just have to compare the determination with the example of the SOP .

These are just a few example.
A colourblind technologist could be a great technician, with just a very
small restriction.

Regards,
Bert klein Brink
Chief histotechologist



Op za 27 mrt. 2021 om 02:41 schreef Ruppert, Amysue via Histonet <
histonet@lists.utsouthwestern.edu>

> Hello, we currently have a color blind Histology student.  Does anyone
> have any helpful hints to share about learning in the Histology setting
> with colorblindness?  Note,  we had previous student with colorblindness,
> many years ago, and he did fine.  But most of the teachers that helped him
> are now retired.
>
>
> Thank you
>
> amysue ruppert
>
> __
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Re: [Histonet] IHC with zamboni's fixed tissues

[Tony Henwood (SCHN) via Histonet]

Hi Colleen,

I was involved in an Immunohistochemical study of the nerves in the human 
ureter that used Zamboni's fixative and cryostat sections. This study was 
before the invention of HIER.

If you are using Zamboni-fixed, paraffin embedded tissue then you will probably 
need to do HIER before many of the localisation antibody incubations since the 
fixative contains 2% formaldehyde. The same might be the case with some 
antibodies when applied to cryostat sections.

Edyvane, K. A., Trussell, D. C., Jonavicius, J., Henwood, A., & Marshall, V. R. 
(1992). Presence and regional variation in peptide-containing nerves in the 
human ureter. Journal of the autonomic nervous system, 39(2), 127-137.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Principal 
Scientist, the Children's Hospital at Westmead Adjunct Fellow, School of 
Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead 
NSW 2145, AUSTRALIA 




-Original Message-
From: Colleen Forster via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Friday, 26 March 2021 9:42 AM
To: histonet-request 
Subject: [Histonet] IHC with zamboni's fixed tissues

Hello Histonet,

Can you give me feedback on trying to do IHC on samples fixed in Zamboni's 
fixative? I have an idea but need confirmation .

Thank you in advance

Colleen Forster HT(ASCP)QIHC
BLS Histology and IHC Laboratory
Jackson Hall, Room 2-155
321 Church St. SE
Minneapolis, MN 55455
612-626-1930
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[Histonet] Source of Bombesin antibody for IHC

[Tony Henwood (SCHN) via Histonet]

Hi all,
Our usual supplier of Bombesin antibody (ABCAM AB86037) (used for 
neuroendocrine cell hyperplasia of infancy) no longer carries this antibody.

Does anyone know of an alternative that works well in FFPE sections?

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

 



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Re: [Histonet] Dipp Kwik Stain

[Tony Henwood (SCHN) via Histonet]

There is a generally accepted scheme of staining which is expected of 
Romanowsky stained preparations, namely purple chromatin, blue leucocyte 
cytoplasm, purple-black basophil granules, red-pink eosinophil granules, purple 
neutrophil granules, purple platelet granules, and pink red-cells (1).

According to recommendations of the French Society of Clinical Cytology and of 
the French Association for Quality Assurance in Anatomic and Cytologic 
Pathology (2), red blood cells and polymorphonuclear leukocytes are the target 
cells for the quality evaluation of Romanowsky stains, and not the tumour 
cells. The tinctorial quality of Romanowsky stains is better and reliably 
judged on these target cells. Even if it is known that a ‘good MGG’ allows 
pink–orange (acidophilic) or buff-coloured RBCs to be obtained, many 
laboratories produce MGG slides showing blue or green erythrocytes, mainly 
because of a high (alkaline) pH of dilution/rinse solutions (3).

Horobin (3) has provided a useful table to trouble-shoot Romanowsky staining.

1.Marshall PN, Bentley SA, Lewis SM. An evaluation of some commercial 
Romanowsky stains. Journal of clinical pathology. 1975 Aug 1;28(8):680-5.
2.Piaton E, Fabre M, Goubin‐Versini I, Bretz‐Grenier MF, Courtade‐Saïdi M, 
Vincent S, Belleannée G, Thivolet F, Boutonnat J, Debaque H, Fleury‐Feith J. 
Guidelines for May‐Grünwald–Giemsa staining in haematology and 
non‐gynaecological cytopathology: recommendations of the French Society of 
Clinical Cytology (SFCC) and of the French Association for Quality Assurance in 
Anatomic and Cytologic Pathology (AFAQAP). Cytopathology. 2016 Oct;27(5):359-68.
3.  Horobin RW. How Romanowsky stains work and why they remain 
valuable—including a proposed universal Romanowsky staining mechanism and a 
rational troubleshooting scheme. Biotechnic & Histochemistry. 2011 Feb 
1;86(1):36-51.




Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Estela Martinez via Histonet 
Sent: Friday, 29 January 2021 02:53
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Dipp Kwik Stain

Hello All,

Do any of your pathologist use Dipp Kwik and if so, what kind of control do you 
use and do you document the control?  TIA!

Estela Martinez
Histology Supervisor
Medical Center Hospital
Odessa, TX 79761
432-640-2348
emartin...@echd.org
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Re: [Histonet] hexamethyleneimine

[Tony Henwood (SCHN) via Histonet]

I agree



Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: John Garratt via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Monday, 12 October 2020 9:58 AM
To: 1...@comcast.net; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] hexamethyleneimine

It is better known as Hexamine in histo circles. Gomori Hexamine Silver, for 
example, can be used to nicely demonstrate Glomeruli.

John

On Sun, Oct 11, 2020 at 3:45 PM, LEROY H BROWN via Histonet 
 wrote:

> What do you use hexamethyleneimine for in your lab. I found an old 
> bottle of this reagent and cannot recall why I have it.
>
> It must be used in making up a stain but I am not remembering what stain.
>
> Anyone know?
>
> Thanks
>
> LeRoy Brown HT(ASCP)HTL
>
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Re: [Histonet] JB4 embedded specimens

[Tony Henwood (SCHN) via Histonet]

I have found that that this gives a pleasing tinctorial result:

Haematoxylin and Eosin

Principle

Traditional HE procedures generally worked poorly on GM sections, with 
background staining of the plastic embedment and the loosening of sections from 
glass slides when alcoholic eosin is used. Aqueous solutions tend to stain 
poorly.

Sections are firstly stained with Celestine blue, followed by a regressive 
Harris's haematoxylin (excess stain is removed from the plastic with acid 
alcohol). The sections are then counterstained with buffered eosin Y/Phloxine B.

Solutions

1.   Celestine Blue - Iron Alum
2.   Harris's Haematoxylin
3.   Buffer for Eosin Y/Phloxine B.
  Solution A2.875ml Glacial Acetic acid in 500ml distilled water.
  Solution B4.1g Sodium Acetate in 500ml distilled water.

  150 ml Solution A + 350 ml Solution B. pH to 4.85.

4.   Eosin Y/Phloxine B.
  Eosin Y (CI 45380)5g.
  Phloxine B (CI 45410) 0.5g.
  Buffer500ml.
  Add 2 crystals Thymol.


Technique

1.  Hydrate sections.
2.  Stain in Celestine Blue 15 minutes.
3.  Wash in running water.
4.  Stain in Harris's Haematoxylin 15 minutes.
5.  Wash, differentiate and blue till surrounding plastic is clear.
6.  Stain in buffered Eosin for 6 minutes.
7.  Wash lightly in running water.
8.  Differentiate in alcohol till surrounding plastic is clear.
9.  Clear in Xylene and mount.


REFERENCES:

Castro,M.D., (1985) "A Haematoxylin - Eosin Phloxine Stain for tissues embedded 
in Glycol Methacrylate" J. Histotechn 8(1); 23-24.

Green,G.H.,Kurnein,F., (1981)  "Glycol Methacrylate embedding in General 
Histopathology " ACP Broadsheet 97.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 



-Original Message-
From: Keena Leon Guerrero via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 25 September 2020 8:47 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] JB4 embedded specimens

Hello All,

I was wondering if anyone has a good Hematoxylin and Eosin (H) staining 
protocol for JB4 embedded specimens?

Thanks!

Keena Leon Guerrero
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Re: [Histonet] Fwd[2]: Re: AFIP Manual

[Tony Henwood (SCHN) via Histonet]

Hi Maxim,

Excellent
At least this amazing piece of history can live on.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Пешков Максим via Histonet 
Sent: Saturday, 11 July 2020 03:23
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Fwd[2]: Re:  AFIP Manual

Dear Bob!
Free download AFIP manuals:
4th ed:  
https://books.google.ru/books/about/Laboratory_methods_in_histotechnology.html?id=R1xrMAAJ_esc=y
3rd ed:  
https://archive.org/details/ManualHistologicalStaining/mode/2up?q=staining+methods+afip+manual
2nd ed:  https://archive.org/details/manualofhistolog00arme/page/n1/mode/2up  
или  
https://books.google.ru/books?id=CFRBYAAJ=frontcover=ru=gbs_ge_summary_r=0#v=onepage=false
1st ed:  
https://books.google.ru/books?id=vx1rMAAJ=frontcover=ru=gbs_ge_summary_r=0#v=onepage=false
Enjoy, please.
Maxim Peshkov,
Russia,
Taganrog.


--
Пешков Максим
--

--
Пешков Максим

--

--
Пешков Максим

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Re: [Histonet] Formalin fixation for COVID-19 positive tissues .....

[Tony Henwood (SCHN) via Histonet]

Hi Richard,

It will depend on the size of the tissue and the source.
Lung tissue is the major concern. Other tissues not affected as much (based on 
the burgeoning literature on Covid-19).
Routine fixation time are applicable, remembering that the alcohols and heated 
wax will also inactivate the virus (triple whammy).
I suppose that I better add a reference:

https://www.tandfonline.com/doi/full/10.1080/01478885.2020.1734718


-Original Message-
From: Cartun, Richard via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, 21 April 2020 6:51 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Formalin fixation for COVID-19 positive tissues .

How long are you fixing surgical tissue specimens from COVID-19 positive 
patient's before tissue processing?  I know that the CDC is recommending "72" 
hours for autopsy tissues, but, to me, that seems excessive for surgical 
pathology specimens.  Any information that you can share on this subject would 
be appreciated.  Thank you and stay safe.

Richard

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


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Re: [Histonet] Recommended thickness of Amyloid sections

[Tony Henwood (SCHN) via Histonet]

I agree,

Slightly thicker makes the polarisation easier to see (personal experience).

I would love to see a study comparing section thickness Vs polarisation 
characteristics. 
Anyone interested? (being a kids hospital we rarely see amyloidosis)

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Paula Keene Pierce via Histonet 
Sent: Friday, 17 April 2020 06:24
To: histonet@lists.utsouthwestern.edu; Ken M
Subject: Re: [Histonet] Recommended thickness of Amyloid sections

As this is my 41 year of being a registered HT, I was taught that slides for 
amyloid are to be cut at 8-10µm.
Paula Keene Pierce, BS, HTL(ASCP)HTPresidentExcalibur Pathology, Inc.5830 N 
Blue Lake DriveNorman, OK 73069PH 405-759-3953http://www.excaliburpathology.com

A sharp knife is nothing without a sharp eye. - Klingon Proverb

On Thursday, April 16, 2020, 03:18:44 PM CDT, Ken M via Histonet 
 wrote:

 Hello All.  We have always cut all of our histology control slides at 5m.  We 
were told today that it is common practice to cut Amyloid at 8m?  Is this your 
experience?

Ken
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Re: [Histonet] On-line references

[Tony Henwood (SCHN) via Histonet]

And each edition gets better (and better).


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: jkiernan--- via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, 7 April 2020 10:13 AM
To: jon.st.o...@agilent.com; Tom Wells 
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] On-line references

Just for the record, there's a 5th edition (2015), ISBN 9781907904325.  (The 
4th edition was in 2008.) John Kiernan.
= = =

From: jon.st.o...@agilent.com 
Sent: 06 April 2020 17:40
To: John Kiernan ; Tom Wells 
Subject: RE: On-line references


There is also a wonderful book called:

Histological and Histochemical Methods;  Theory and Practice, 4th Edition, by 
one J.A. Kiernan that I would highly recommend (and it's at a very good price).



I'd be happy to give you more information if needed.



Happy Monday,

Jon







-Original Message-
From: John Kiernan via Histonet 
Sent: Wednesday, March 25, 2020 11:12 PM
To: histonet@lists.utsouthwestern.edu; Tom Wells 
Subject: Re: [Histonet] On-line references



Hello, Tom.



Some old classics are there for free, most notably JR Baker's "Principles of 
Biological Microtechnique" (1958), but almost anything more recent has to be 
bought.



There are plenty of cheap older editions of histotechnology books on sites like 
AbeBooks. Check it out for the last edition of  Pearse's Histochemistry!  I was 
amazed.



Even the latest editions of books in our field cost only about $100 from the 
publisher and most are good for several years.  Compare this with the price of 
a few drops of an antibody or (more realistically) a staining machine in which 
you must only use the liquids sold by its vendor.



John Kiernan

= = =



From: Tom Wells via Histonet 
mailto:histonet@lists.utsouthwestern.edu>>

Sent: 25 March 2020 14:34

To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu> 
mailto:histonet@lists.utsouthwestern.edu>>

Subject: [Histonet] On-line references



Given that our Institute's library is closed due to the pandemic, is anyone 
aware of on-line versions of Histotechnology/ Histochemistry textbooks? Thanks. 
Tom



Tom Wells BSc, MEd, MLT, ART

Faculty

Medical Laboratory Science

School of Health Sciences

SW03-3088

(604) 412-7594

BCIT



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[Histonet] Flippin on the Bond

[Tony Henwood (SCHN) via Histonet]

Hi all,

I was reading an article entitled “Evaluation of Tissue Adhesion and Staining 
Performance of BOND Adhesive Microscope Slides: A Comparative Study on the 
BOND-III and BOND-MAX Platforms” 
(https://pdfs.semanticscholar.org/f5f1/ad0741bc5445d0e1079a0d07a32c8a3a26c1.pdf)
 and they make mention of the “Flippin Protocol”, an amended protocol widely 
used by BOND customers.
It is used for those antibodies that are adversely affected by the endogenous 
peroxidase block that is used in the Bond kits (and possibly with other 
non-Bond kits as well).
In this protocol, the peroxidase block is applied after the localisation 
antibody is applied. Examples given by Leica are PAX5 and AMACR.

The paper refers to several other antibodies as well (CD3, CD34, SMA and TTF1) 
that uses the “Flippin protocol”. Now I have not had issues with the first 
three antibodies (they work quite well with the standard protocol, ie 
peroxidase block before the localisation antibody) and have no experience with 
TTF1 (we are a pediatric laboratory).

Has anyone experienced this issue with antibodies and use the Flippin protocol?

What antibodies are an issue?

Thanks for your input

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) | Principal 
Scientist; Adjunct Fellow, School of Medicine, University of Western Sydney; 
Visiting Lecturer, School of Life Sciences, Faculty of Science, University of 
Technology Sydney | Histopathology
t: (02) 9845 3306 | e: 
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Re: [Histonet] Frozen section slides fading

[Tony Henwood (SCHN) via Histonet]

Hi Dane,

Fading and especially leaching of the eosin, will occur if slides are 
incompletely dehydrated. Flip out the subsidiary condenser lens (or drop the 
condenser) and look for refractile water droplets.
Certain mountants can also cause fading of H
I would also be concerned about the xylene substitute.

Alternatively, after staining and ethanol dehydration, air dry the slides 
(making sure they are completely dry) and directly coverslip with your mountant 
(without the substitute added). This will reduce the risk of xylene exposure.

Hopefully this is useful


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Hill, Dane via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, 17 March 2020 1:01 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Frozen section slides fading


Hello all,


Thank you, in advance, for your help and for the information you've already 
posted online. It is helpful. I am hoping you can help me troubleshoot an issue 
we are having. I am the Mohs fellow at Texas Tech dermatology. For the last 9 
months or so we have been noticing that the longer our frozen section slides 
are away from the day of surgery, the more they develop these pink, amorphous 
spots in certain areas of the tissue. The further away from the surgery, the 
more faded they get. The actual tissue remains intact on the slide, with 
complete epidermis evaluable, but it's almost as if all the hematoxylin fades 
or something because it just turns into that bright pink color, but does not 
hold any of the purple. We have tried changing our xylene substitutes and even 
changed the hematoxylin and eosin stains. The thinner (5 micron cuts) are 
affected long before the 20 micron cuts. Photos were too big to be attached, 
but I can PM someone with them, if needed. They are from slides prepared just a 
few weeks ago and are just starting to fade. In about 6 months they will turn 
all pink.

Interestingly, this is not happening on any of our permanent sections. They 
remain perfectly stained for years. And it will happen, even if we don't "bake" 
any of the slides or heat them in storage. The only thing the techs say they do 
differently for the frozen sections is use xylene substitute in place of 
xylene. They also mix a little xylene substitute in the glue to cover slip the 
slides. I wasn't sure if that could be contributing. If there are any testing 
protocols of specific reagents we use or adjustments in staining that have 
previously remedied the issue, I'd be very grateful as we have been 
unsuccessful at pinning it down. Thank you, again!

Dane Hill


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[Histonet] Fw: [EXTERNAL] Pap slides

[Tony Henwood (SCHN) via Histonet]

We regularly leave smeared slides in 95% overnight and often over the weekend 
before staining.
There have not been any morphological issues noted.

Remember to seal the lid since alcohol will evaporate quite quickly. You will 
notice an increased eosinophilia in the dried upper area of the slide.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Perl, Alison via Histonet 
Sent: Saturday, 14 March 2020 04:19
To: 'Charles Riley via Histonet'
Subject: Re: [Histonet] [EXTERNAL]  Pap slides

We routinely leave our ThinPrep Paps in 95 overnight and stain the next day. If 
I remember our Hologic training correctly, they said the slides can be left up 
to 5 days - best to check with an FAS before trying it, though

Alison Perl, HTL(ASCP)CM
Anatomic Pathology Manager
CareMount Medical
(914) 302-8424
ap...@caremount.com


-Original Message-
From: Charles Riley via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Friday, March 13, 2020 12:52 PM
To: Histo List
Subject: [EXTERNAL] [Histonet] Pap slides

Can pap slides be left in 95% alcohol overnight to be stained the next
morning without affecting the quality of the slides? Or should they be
moved to xylene then run back prior to staining?

--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] Need a procedure

[Tony Henwood (SCHN) via Histonet]

Good points but:
1. Good QC of the technique is required. The plasma has been tested for known 
infectious agents and (in Australia) has been treated to remove/kill these 
organisms. When we received the expired plasma, usually in a bag, we 
immediately aliquot to smaller volumes and store frozen. They are defrosted 
when needed.

2. The issue with molecular testing has not been proven to be an issue (I have 
read the paper: Balassanian, R., Ng, D. L., & van Zante, A. (2019). Stop using 
expired plasma for cell blocks. Cancer cytopathology, 127(12), 737-738). If 
contaminating human DNA is a problem, then I prefer to scrape the material from 
an alcohol fixed, PAP stained slide rather than a cellblock. In fact the DNA 
and RNA retrieved is of better quality than from a formalin-fixed cell block 
anyway (also refer to: Knoepp, S. M., & Roh, M. H. (2013). Ancillary techniques 
on direct‐smear aspirate slides: a significant evolution for cytopathology 
techniques. Cancer cytopathology, 121(3), 120-128). Also, has anyone assessed 
the deleterious effect of heat on molecular testing?

3. It needs to be remembered that an accurate diagnosis is the aim of the 
biopsy. Using the plasma clot method followed by formalin fixation is the same 
as routine core biopsy fixation, so the expected IPX results should be the 
same. Molecular testing will only be requested after the correct diagnosis is 
made.

4. I have followed those researchers that have promoted formal-substitute 
fixatives as being the bee's knees and undoubtedly morphology, 
immunohistochemistry and molecular testing are good (sometimes better) than 
formalin-fixed biopsies BUT I have yet to see a follow-up study on these same 
tissues that were fixed 5, 10 or more years ago. Is the morphology just as good 
or, I fear, without the protection of formalin, just mushy! Is the IPX and 
nucleic acids of the same quality. The lack of these follow-up studies make me 
worried that maybe this may have been a mistake!

5. I am not sure what the ethical issues are. Surely we are just utilising 
anonymous, de-identified material that would have been disposed of?

Hey, my rant for the year!!



-Original Message-
From: John Garratt [mailto:john.garr...@ciqc.ca] 
Sent: Friday, 24 January 2020 11:36 AM
To: Garrey Faller 
Cc: Tony Henwood (SCHN) ; Muhammad Azam 
; Terri Braud ; 
histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Need a procedure

Good point regarding the molecular and another good reason to move away from 
plasma. Plus taking plasma, even outdated plasma, is fraught with ethical and 
regularity problems which are best to avoid now that ever milliliter of blood 
product needs to be accounted for.


www.cpqa.ca

‐‐‐ Original Message ‐‐‐
On Thursday, January 23, 2020 3:41 PM, Garrey Faller  wrote:

> Great helpful information.
> We use plasma /thrombin.
> It’s easy to use, and we have plenty of access to it from our blood bank.
>
> However, we are looking to switch to Gel.
>
> Why?
>
> 1.  If you don’t keep an eye on your plasma, at some point you could see 
> fungi in your specimen with a chance you might think it’s real and not a 
> contaminant. That’s a problem. Don’t keep it for too long in the 
> refrigerator. It’s a great culture medium for fungi.
> You could freeze small aliquots I guess.
>
> 2.  Molecular testing on cell blocks:
> With the increasing use of molecular testing on cell blocks, plasma 
> thrombin method poses a problem. The plasma introduces other peoples DNA into 
> the patients sample.Think about that.
>
> Didn’t know about the heat issue with Gel. Important to know. Thanks !
>
>     Garrey
>
> Sent from my iPhone
>
>
> > On Jan 23, 2020, at 6:12 PM, Tony Henwood (SCHN) via Histonet 
> > histonet@lists.utsouthwestern.edu wrote:
> > Hi all,
> > Be careful of using cell block matrix that requires heat to solubilise the 
> > matrix (eg agar or other commercial matrixes like Histogel).
> > Adding a heated matrix to unfixed, or even formalin fixed, material can 
> > denature some antigens (eg CEA) resulting in a false negative IPX.
> > Unfortunately the importance of heat as a pre-analytical factor in 
> > immunohistochemistry is often not appreciated.
> > Regards
> > Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
> > Principal Scientist, the Children’s Hospital at Westmead Adjunct 
> > Fellow, School of Medicine, University of Western Sydney
> > Tel: 612 9845 3306
> > Fax: 612 9845 3318
> > Pathology Department
> > the children's hospital at westmead
> > Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, 
> > Westmead NSW 2145, AUSTRALIA -Original Message-
> > From: John Garratt via Histonet 
> > [mailto:histonet@lists.utsouthwestern.edu]
&g

Re: [Histonet] Need a procedure

[Tony Henwood (SCHN) via Histonet]

Hi all,

Be careful of using cell block matrix that requires heat to solubilise the 
matrix (eg agar or other commercial matrixes like Histogel).
Adding a heated matrix to unfixed, or even formalin fixed, material  can 
denature some antigens (eg CEA) resulting in a false negative IPX.
Unfortunately the importance of heat as a pre-analytical factor in 
immunohistochemistry is often not appreciated.



Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: John Garratt via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 24 January 2020 8:21 AM
To: Muhammad Azam 
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Need a procedure

http://www.avantec.fr/content/dam/tfs/SDG/APD/APD%20Documents/Product%20Manuals%20&%20Specifications/Histology%20Equipment%20and%20Supplies/Embedding%20Cassettes%20and%20Molds/92957066-Richard-Allan-Scientific-HistoGel-Instructions-for-Use.pdf


The above link will help.



www.cpqa.ca

‐‐‐ Original Message ‐‐‐
On Thursday, January 23, 2020 12:13 PM, Muhammad Azam  wrote:

> Anybody has validated procedure for histogel
>
> Sent from my iPhone
>
> > On Jan 23, 2020, at 1:07 PM, John Garratt via Histonet 
> > histonet@lists.utsouthwestern.edu wrote:
> > Hi Terri, I suggest you use Histogel for block preparation. It works 
> > exceptionally well, it is good for IHC and does not have the pitfalls of 
> > plasma/thrombin.
> > Plasma/thrombin does work well for cell blocks but you will have to 
> > consider an ethical and safe source for your plasma.
> > The instructions for using Histogel are in the package insert though I have 
> > one comment. Be careful how you warm the Histogel and use a heat block. Do 
> > NOT use a microwave since there is a tendency to overheat the gel and you 
> > will end up with poor quality IHC.
> > John
> > www.cpqa.ca
> > ‐‐‐ Original Message ‐‐‐
> >
> > > On Thursday, January 23, 2020 6:10 AM, Terri Braud via Histonet 
> > > histonet@lists.utsouthwestern.edu wrote:
> > > Hi fellow Histonetters - I'm in need of some help, please 
> > > Background - We currently use agar to capture our scant cell 
> > > blocks for processing. I am unfamiliar with the Plasma/Thrombin method of 
> > > cell block preparation and am interested in comparing it to our current 
> > > method Request - Could you please send me your procedures for this 
> > > method, specifically where you purchase your plasma and thrombin and what 
> > > species are used?
> > > Thanks in advance. Histotechs rock!
> > > 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
> > > Histonet mailing list
> > > Histonet@lists.utsouthwestern.edu
> > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >
> > Histonet mailing list
> > Histonet@lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet



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Re: [Histonet] Cell block preparations

[Tony Henwood (SCHN) via Histonet]

Hi Jennifer,

I have had excellent success with lysing the red blood cells (using  Isotonic 
Ammonium Chloride) prior to cell block preparation with thromboplastin-plasma. 
The lysing solution contains EDTA so you will need to add a few drops of 1% 
calcium chloride. Method as follows:

Lysis solution
Ammonium Chloride4.5g
Potassium carbonate  0.5g
EDTA 0.0186g
Distilled water  500mls

Method:
1.  Centrifuge bloody fluid.
2.  Remove supernatant and add equal volume of lysis solution.
3.  Resuspend and incubate for 5 minutes at 4oC.
4.  Centrifuge, if blood still remains, then repeat from step 2.
5. Rinse in Hanks or RPMI, centrifuge.
6.  Mix pellet in a few drops of plasma.
7. Add thromboplastin and a few drops  of 1% Calcium Chloride, mix gently 
and allow clot to form.
8. Add 10% buffered formalin and fix and process as usual.

Reference:  
Kuenen-Boumiester etal (1996) Acta Cytolog 40:475-479

If you donot use the plasma clot method for cell block preparation, then use 
your preferred method after step 5.
The lysis solution can also be purchased commercially from several companies 
(eg Biolegend). It is commonly used for sample preparation for flow cytometry. 
Check the SDS to make sure it does not contain formaldehyde.


-Original Message-
From: Mac Donald, Jennifer via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 17 January 2020 4:53 AM
To: Charles Riley ; Histo List 

Subject: Re: [Histonet] Cell block preparations

Acetic acid would work.

Get Outlook for iOS 
From: Charles Riley via Histonet 
Sent: Thursday, January 16, 2020 8:55:21 AM
To: Histo List 
Subject: [Histonet] Cell block preparations

  EXTERNAL SENDER- Exercise caution with requests, links, and attachments.

What is the best way to remove excess blood from FNA sample collections before 
spinning them down into cell blocks?

--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] H protocols

[Tony Henwood (SCHN) via Histonet]

Hi Charles,

Yes we use the same protocol for all, though sometimes we need to modify if for 
example tissue has been overly decalcified (eg mandible).

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Charles Riley via Histonet 
Sent: Saturday, 4 January 2020 04:42
To: Histo List
Subject: [Histonet] H protocols

Does everyone use the same protocol for routine H's as well as things
like Cell blocks and or lymph nodes?

If you use a different protocol how does it differ from your routine stain?

--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] Delay in embedding?

[Tony Henwood (SCHN) via Histonet]

Hi Paula,

We routinely do this, especially for our fetal autopsy blocks.
We are then able to process and let them set at room temp until we are able to 
embed and cut.
Some cases are more urgent than others so these can be expedited a lot easier 
since they will only need embedding, sectioning and staining. We also get best 
usage of our limited processing capabilities.
It is more efficient for us.

Morphology, staining and immunohistochemistry is not affected.

It is better to do this rather than leave them at 64oC plus for extended times 
prior to embedding since many antigens will be adversely affected.

Take home point - do not overcook your tissues.



Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Theresa Dalton via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 18 December 2019 7:34 AM
To: P Sicurello 
Cc: HistoNet 
Subject: Re: [Histonet] Delay in embedding?

We have done this - only on an emergency basis. 

Sent from my iPhone

> On Dec 17, 2019, at 2:48 PM, P Sicurello via Histonet 
>  wrote:
> 
> Good Morning Listers,
> 
> 
> 
> How many out there will process tissue and then leave the cassettes at 
> room temperature and embed it at a later time (hours or the next day)?
> 
> 
> 
> Please send me your opinions about doing this.  I think it’s a bad 
> idea, others I speak with disagree.
> 
> Sincerely,
> 
> Paula Sicurello, HTL (ASCP)CM
> 
> Histotechnology Specialist
> 
> UC San Diego Health
> 
> 9300 Campus Point Drive
> 
> La Jolla, CA 92037
> (P): 858-249-5610
> 
> 
> 
> *Confidentiality Notice*: The information transmitted in this e-mail 
> is intended only for the person or entity to which it is addressed and 
> may contain confidential and/or privileged material.  Any review, 
> retransmission, dissemination or other use of or taking of any action 
> in reliance upon this information by persons or entities other than 
> the intended recipient is prohibited.  If you received this e-mail in 
> error, please contact the sender and delete the material from any computer.
> ___
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Re: [Histonet] Cell block processing

[Tony Henwood (SCHN) via Histonet]

Hi Charles,

I have had excellent success with lysing the red blood cells (using  Isotonic 
Ammonium Chloride) prior to cell block preparation with thromboplastin-plasma. 
The lysing solution contains EDTA so you will need to add a few drops of 1% 
calcium chloride. Method as follows:

Lysis solution
Ammonium Chloride4.5g
Potassium carbonate  0.5g
EDTA 0.0186g
Distilled water  500mls

Method:
1.  Centrifuge bloody fluid.
2.  Remove supernatant and add equal volume of lysis solution.
3.  Resuspend and incubate for 5 minutes at 4oC.
4.  Centrifuge, if blood still remains, then repeat from step 2.
5. Rinse in Hanks or RPMI, centrifuge.
6.  Mix pellet in a few drops of plasma.
7. Add thromboplastin and a few drops  of 1% Calcium Chloride, mix gently 
and allow clot to form.
8. Add 10% buffered formalin and fix and process as usual.

Reference:  
Kuenen-Boumiester etal (1996) Acta Cytolog 40:475-479

The lysis solution can also be purchased commercially from several companies 
(eg Biolegend). It is commonly used for sample preparation for flow cytometry. 
Check the SDS to make sure it does not contain formaldehyde.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Charles Riley via Histonet 
Sent: Friday, 25 October 2019 23:12
To: Histo List
Subject: [Histonet] Cell block processing

Does anyone have any tips or suggestions on how to better process extremely
bloody FNA  specimens?Is there anyway to clear out some or all of the
blood without destroying the other tissues?

--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] waterbath/paraffin

[Tony Henwood (SCHN) via Histonet]

Are the tissues taken directly from the molten wax and placed in the 
wax-containing mold or are they allowed to cool before embedding?

One cause of tissue separation is the difference in temperature between tissue 
and embedding wax. 
Try taking directly from molten wax and embedding directly in the wax-filled 
mold.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Betsy Molinari via Histonet 
Sent: Saturday, 26 October 2019 00:49
To: 'Histonet@lists.utsouthwestern.edu'
Subject: [Histonet] waterbath/paraffin

Hi,
When I place my sections on the waterbath the paraffin pulls away from it. 
Leaving just the edge of the tissue. It is very weird. I have tried at 
different temps . The tissue itself is fine so I do not believe it is the 
processing. I use the same paraffin for processing as well as embedding. I use 
Fisher Histoplast IM.  I was considering melting a block or two down and 
embedding them in another paraffin from another lab.
Suggestions?

Betsy Molinari HT, ASCP
Texas Heart Institute
6770 Bertner Ave.
O-511
Houston, TX 77030
832-355-6524 (lab)
832-355-6812 (fax)

Betsy Molinari
Sr. Histology Research Technician
CV Pathology Research

Texas Heart Institute
6770 Bertner Avenue, MC 1-283
Houston, TX 77030

Office: 832-355-6524 | Fax: 832-355-6812
Email: bmolin...@texasheart.org
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Re: [Histonet] Metals

[Tony Henwood (SCHN) via Histonet]

☺

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) | Principal 
Scientist; Adjunct Fellow, School of Medicine, University of Western Sydney; 
Visiting Lecturer, School of Life Sciences, Faculty of Science, University of 
Technology Sydney | Histopathology
t: (02) 9845 3306 | e: 
tony.henw...@health.nsw.gov.au<mailto:tony.henw...@health.nsw.gov.au> | w: 
www.schn.health.nsw.gov.au<http://www.schn.health.nsw.gov.au>
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<http://res.schn.health.nsw.gov.au/signatures/chw_twitter.php>
[http://res.schn.health.nsw.gov.au/signatures/chw.gif]
Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
Locked Bag 4001, Westmead 2145, NSW Australia
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♲  Please consider the environment before printing this email.
From: John Kiernan [mailto:jkier...@uwo.ca]
Sent: Wednesday, 2 October 2019 3:01 PM
To: Sanders, Jeanine (CDC/DDID/NCEZID/DHCPP) ; Tony Henwood 
(SCHN) 
Cc: Histonet 
Subject: Re: Metals

Timm's sulphide-silver method is very sensitive, and modifications (mostly by 
Danscher) even more so. Sulphide-silver methods detect only those metals that 
have insoluble sulphides (copper and zinc but not aluminium, in Jeanine's 
list). It is necessary to fix in a special solution containing hydrogen 
sulphide - stink and also serious safety precautions!

Tony's mention of Mallory & Parker's fresh hematoxylin stain prompted me to 
look it up. The 1939 paper is a free PDF download (Google Scholar: Mallory 
Parker Hematoxylin Stain Metals). Mallory FB, Parker F (1939) Fixing and 
staining methods for lead and copper in tissues. Am. J. Pathol. 15: 517-522 and 
Plates 83-85.
The authors noted the importance of fixation (neutral formalin was OK for 
copper but no good for lead, which needed 95% or 100% alcohol). Like F.B. 
Mallory's other papers about staining methods, it's rather vague on technical 
details and has no references.

The late Ralph D. Lillie reported more thorough investigations of staining for 
metals with haematoxylin in his classic book Histopathologic Technic and 
Practical Histochemistry (4th and last edn 1976, ISBN 0070378622), giving 
colours of the complexes with 30 metals introduced into tissues. I have tried 
Lillie's method on paraffin sections of rat tissues containing a few of these, 
and it works.  ISBN 9781907904325 (p.333-334)  may be more accessible than 
Lillie's book, which has become an expensive classic.

For the more specific stains mentioned in Tony's message you need to do some 
critical reading. The best place to start may be Frieda Carson's 
Histotechnology textbook. ISBN  978-0891896401.

Enough about metals for now!

John Kiernan
London, Canada
= = =
________
From: Tony Henwood (SCHN) via Histonet 
mailto:histonet@lists.utsouthwestern.edu>>
Sent: 30 September 2019 07:22
To: Sanders, Jeanine (CDC/DDID/NCEZID/DHCPP) mailto:j...@cdc.gov>>
Cc: Histonet 
mailto:histonet@lists.utsouthwestern.edu>>
Subject: Re: [Histonet] Metals

Two good screening stains are Mallory and Parker’s Fresh Hematoxylin Stain for 
Metals and  Timm’s Silver Sulphide Method for Metals. Malloy's results:
Aluminium   Blue-black
Copper  Greenish-blue
IronBlue-black
LeadBlue
ZincBlue

For more specific staining:
Aluminon Stain for Aluminium Hydroxide
Walton’s Stain for Aluminium  (Phloxine binds the aluminium)
Bedrick et al (1986) method for Zinc
Rubeanic Acid Technique for Copper
Rhodanine Technique for Copper

These methods are quite sensitive but  there are some specificity issues. I can 
provide further details and references if required. Here are some:

Ohtsuki, Y., Yamaguchi, T., Sonobe, H., Takahashi, K., Hayashi, K., Takenaka, 
A., ... & Terao, N. (1989). Stain Technology: A Simplified Aluminum Stain in 
Paraffin Sections of Bone from Hemodialysis Patients. Stain technology, 64(2), 
55-59.

Walton, J. R., Diamond, T. H., Kumar, S., & Murrell, G. A. C. (2007). A 
sensitive stain for aluminum in undecalcified cancellous bone. Journal of 
inorganic biochemistry, 101(9), 1285-1290.

Bedrick, A. E., Ramaswamy, G., & Tchertkoff, V. (1986). Histochemical 
determination of copper, zinc, and iron in some benign and malignant tissues. 
American journal of clinical pathology, 86(5), 637-640.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

_

Re: [Histonet] Metals

[Tony Henwood (SCHN) via Histonet]

Two good screening stains are Mallory and Parker’s Fresh Hematoxylin Stain for 
Metals and  Timm’s Silver Sulphide Method for Metals. Malloy's results:
Aluminium   Blue-black
Copper  Greenish-blue
IronBlue-black
LeadBlue
ZincBlue

For more specific staining:
Aluminon Stain for Aluminium Hydroxide
Walton’s Stain for Aluminium  (Phloxine binds the aluminium)
Bedrick et al (1986) method for Zinc 
Rubeanic Acid Technique for Copper
Rhodanine Technique for Copper 

These methods are quite sensitive but  there are some specificity issues. I can 
provide further details and references if required. Here are some:

Ohtsuki, Y., Yamaguchi, T., Sonobe, H., Takahashi, K., Hayashi, K., Takenaka, 
A., ... & Terao, N. (1989). Stain Technology: A Simplified Aluminum Stain in 
Paraffin Sections of Bone from Hemodialysis Patients. Stain technology, 64(2), 
55-59.

Walton, J. R., Diamond, T. H., Kumar, S., & Murrell, G. A. C. (2007). A 
sensitive stain for aluminum in undecalcified cancellous bone. Journal of 
inorganic biochemistry, 101(9), 1285-1290.

Bedrick, A. E., Ramaswamy, G., & Tchertkoff, V. (1986). Histochemical 
determination of copper, zinc, and iron in some benign and malignant tissues. 
American journal of clinical pathology, 86(5), 637-640.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Sanders, Jeanine (CDC/DDID/NCEZID/DHCPP) via Histonet 

Sent: Monday, 30 September 2019 20:49
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Metals

Morning all!

I need some advice re: protocols to demonstrate metals in FFPE tissues. Metals 
such as copper, aluminum and zinc.

Thanks much!

Jeanine Sanders, BS, HT(ASCP), QIHC(ASCP)
Centers for Diseases Control and Prevention
1600 Clifton Road NE
MS H18-SB
Bldg. 18, Rm SB-114
Atlanta, GA 30329
404-639-3590

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Re: [Histonet] Victoria blue for lung tissue

[Tony Henwood (SCHN) via Histonet]

Whoops,

The Ventana does not use Victoria blue, my mistake. They have a resorcin 
fuchsin solution (known as Hart's method) not Miller Victoria blue.



Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Tony Henwood (SCHN) 
Sent: Saturday, 27 July 2019 10:44 AM
To: Bob Richmond 
Cc: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Victoria blue for lung tissue

Victoria Blue is the dye used in Miller's Stain for Elastic Tissue.
It is also used in the Roche Ventana Benchmark Stainer to stain elastic Tissue:

Miller PJ (1971) An elastin stain. Med Lab Technol 28, 148-149

Karen Percival & Zaher Radi (2017) Comparison of five elastin histochemical 
stains to identify pulmonary small vasculature, Journal of Histotechnology, 
40:3, 73-78

Yufeng Yu & Clifford M. Chapman (2000) "Elastic Tissue Staining in Human Skin" 
Histologic 32(1): 12

Roten SV, Bhat S, Bhawan J. Elastic fibers in scar tissue. J Cutan Pathol 1996: 
23: 37-42.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Principal 
Scientist, the Children's Hospital at Westmead Adjunct Fellow, School of 
Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead 
NSW 2145, AUSTRALIA


From: Bob Richmond via Histonet 
Sent: Saturday, July 27, 2019 4:10 AM
To: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Victoria blue for lung tissue

Amy (where?) asks: >>I was asked to do Victoria Blue stain on rodent FFPE lung 
tissue to exam thickness of artery. Could anybody recommend a good vendor of 
this reagent kit?<<

You can get Victoria Blue R (Colour Index 44040) from Sigma-Aldrich and several 
others. I couldn't find anyone who offers a kit, only the dry dye.
There are other dyes called Victoria Blue, reported to give the same results.

You'd have to find a method for preparing it as an elastic stain, and I 
couldn't find such a method either with Google or in my old books. The 
requester needs to supply you with a method. I suspect the requester is reading 
an old article. There are stains for elastic tissue (which is I suppose what 
you want to "exam thickness of artery") that are a lot easier to get.

Bob Richmond
Samurai Pathologist
Maryville TN
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Re: [Histonet] Victoria blue for lung tissue

[Tony Henwood (SCHN) via Histonet]

Methods found here:

https://www.ihcworld.com/_protocols/special_stains/miller's_elastic_ellis.htm

http://www.histosearch.com/histonet/Dec98/Millersstainforelasticfib.html

https://www.sakuraus.com/getattachment/774d89be-4b32-4d69-bb02-8dea75c52c0c/858

?



Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

From: Bob Richmond 
Sent: Saturday, July 27, 2019 11:27:54 AM
To: Tony Henwood (SCHN)
Subject: Re: [Histonet] Victoria blue for lung tissue

Is this method published anywhere that "Amy" would likely have access to? This 
looked to me like one of those cases of the blind leading the blind that are 
all to common in research involving histochemistry.

On Fri, Jul 26, 2019 at 8:44 PM Tony Henwood (SCHN) 
mailto:tony.henw...@health.nsw.gov.au>> wrote:
Victoria Blue is the dye used in Miller's Stain for Elastic Tissue.
It is also used in the Roche Ventana Benchmark Stainer to stain elastic Tissue:

Miller PJ (1971) An elastin stain. Med Lab Technol 28, 148-149

Karen Percival & Zaher Radi (2017) Comparison of five elastin histochemical 
stains to identify pulmonary small vasculature, Journal of Histotechnology, 
40:3, 73-78

Yufeng Yu & Clifford M. Chapman (2000) "Elastic Tissue Staining in Human Skin" 
Histologic 32(1): 12

Roten SV, Bhat S, Bhawan J. Elastic fibers in scar tissue. J Cutan Pathol 1996: 
23: 37-42.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Bob Richmond via Histonet 
mailto:histonet@lists.utsouthwestern.edu>>
Sent: Saturday, July 27, 2019 4:10 AM
To: Histonet@lists.utsouthwestern.edu<mailto:Histonet@lists.utsouthwestern.edu>
Subject: Re: [Histonet] Victoria blue for lung tissue

Amy (where?) asks: >>I was asked to do Victoria Blue stain on rodent FFPE
lung tissue to exam thickness of artery. Could anybody recommend a good
vendor of this reagent kit?<<

You can get Victoria Blue R (Colour Index 44040) from Sigma-Aldrich and
several others. I couldn't find anyone who offers a kit, only the dry dye.
There are other dyes called Victoria Blue, reported to give the same
results.

You'd have to find a method for preparing it as an elastic stain, and I
couldn't find such a method either with Google or in my old books. The
requester needs to supply you with a method. I suspect the requester is
reading an old article. There are stains for elastic tissue (which is I
suppose what you want to "exam thickness of artery") that are a lot easier
to get.

Bob Richmond
Samurai Pathologist
Maryville TN
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the sender.

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Re: [Histonet] Victoria blue for lung tissue

[Tony Henwood (SCHN) via Histonet]

Victoria Blue is the dye used in Miller's Stain for Elastic Tissue.
It is also used in the Roche Ventana Benchmark Stainer to stain elastic Tissue:

Miller PJ (1971) An elastin stain. Med Lab Technol 28, 148–149

Karen Percival & Zaher Radi (2017) Comparison of five elastin histochemical 
stains to identify pulmonary small vasculature, Journal of Histotechnology, 
40:3, 73-78

Yufeng Yu & Clifford M. Chapman (2000) "Elastic Tissue Staining in Human Skin" 
Histologic 32(1): 12

Roten SV, Bhat S, Bhawan J. Elastic fibers in scar tissue. J Cutan Pathol 1996: 
23: 37-42.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Bob Richmond via Histonet 
Sent: Saturday, July 27, 2019 4:10 AM
To: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Victoria blue for lung tissue

Amy (where?) asks: >>I was asked to do Victoria Blue stain on rodent FFPE
lung tissue to exam thickness of artery. Could anybody recommend a good
vendor of this reagent kit?<<

You can get Victoria Blue R (Colour Index 44040) from Sigma-Aldrich and
several others. I couldn't find anyone who offers a kit, only the dry dye.
There are other dyes called Victoria Blue, reported to give the same
results.

You'd have to find a method for preparing it as an elastic stain, and I
couldn't find such a method either with Google or in my old books. The
requester needs to supply you with a method. I suspect the requester is
reading an old article. There are stains for elastic tissue (which is I
suppose what you want to "exam thickness of artery") that are a lot easier
to get.

Bob Richmond
Samurai Pathologist
Maryville TN
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Re: [Histonet] Processing Schedule- ASP-6025

[Tony Henwood (SCHN) via Histonet]

Processing seems adequate.

After processing, how long do they sit in the embedding centre block holding 
tank before embedding?

We found that quite a few antigens were affected when we stored control tonsil 
in the embedding centre (dry) at 60oC for a few days before embedding. In 
summary:

AntibodyClone   Dried (Normal = 3+)
CD4 4B120
BOB-1   TG140
CD3 LN101+
CD79a   JCB117  1+
Oct-2   Oct-207 1+
CD8 4B112+
CD20L26 3+

So CD20 was unaffected but this process affected most of the antigens with some 
losing antigen recognition by the antibody (eg CD4 and BOB-1).

Another one of those pre-analytical issues we need to consider.

And yes I am writing this up for submission!


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) | Principal 
Scientist; Adjunct Fellow, School of Medicine, University of Western Sydney; 
Visiting Lecturer, School of Life Sciences, Faculty of Science, University of 
Technology Sydney | Histopathology 
t: (02) 9845 3306 | f: (02) 9845 3318 | e: tony.henw...@health.nsw.gov.au | w: 
www.schn.health.nsw.gov.au
m: 


Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
Locked Bag 4001, Westmead 2145, NSW Australia

♲  Please consider the environment before printing this email.

-Original Message-
From: Greg Dobbin via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 8 May 2019 5:07 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Processing Schedule- ASP-6025

Hello  colleagues,
I recently stained (IHC) a section of normal tonsil from another facility with 
p16 and the resulting stain was better than the same stain on a section of my 
labs own normal tonsil control.

This has led us to question our processing schedule. I am not concerned with 
our fixation because we fix everything for at least 24 hours in 10% formalin 
(commercially prepared) prior to processing.

Does anything jump out at you as being a potential red flag in the following 
overnight protocol?

   - Formalin 15 mins; RT
   - Processing water 1 min; RT
   - ETOH 70% 30 mins; 35C
   - ETOH 80% 30 mins; 35C
   - ETOH 95% 30 mins; 35C
   - ETOH 100% 30 mins; 35C
   - ETOH 100% 40 mins; 35C
   - ETOH 100% 60 mins; 35C
   - Xylene  60 mins; 35C
   - Xylene  60 mins; 35C
   - Xylene  60 mins; 35C
   - Paraffin 60 mins; 57C; vacuum
   - Paraffin 60 mins; 57C; vacuum
   - Paraffin 60 mins; 57C; vacuum

Our formalin is changed after every 1100 cassettes and the alcohol, xylenes and 
paraffins are managed similarly by the instrument. Our specimen mix is a little 
of everything (skins, GIs, breasts, needle cores, gall bladders, gyne, etc).

The one unknown (so far) in this story, is how the tonsil from the other 
laboratory was handled (ie the fixative used and for how long-I am assuming 10% 
formalin).

Obviously, many of you will have schedules that differ from this one, in any 
number of ways, but what I am looking for from you is your opinion: *is there 
anything about this schedule that is particularly concerning?* Thank you, Greg


--
*Greg Dobbin*
1205 Pleasant Grove Rd
RR#2 York,
PE  C0A 1P0


*Everything in moderation...even moderation itself**!*
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Re: [Histonet] Derm IHC question

[Tony Henwood (SCHN) via Histonet]

Possibly, the edges have been allowed to dry prior to immersion in fixative.

Also is there evidence of cautery artefact?


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Debra Siena via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Saturday, 9 February 2019 3:51 AM
To: 'histonet'
Subject: [Histonet] Derm IHC question

Hello fellow Histonetters
I would like to ask you a question about IHC staining and derm cases.  I am 
seeing a peculiar issue going on, where the melanocytes in the middle of the 
tissues are staining pretty well but when you get to the ends of the tissues 
either shaves or ellipses, they are not staining. This is sporadic, not every 
case and there is no consensus as to a common thread between the cases.I 
feel that this may be a fixation issue but was just wondering if anyone had 
ever seen the same phenomena and would be willing to share the theory or even 
better what was the remedy behind this issue.  The fixative is 10% Neutral 
Buffered Formaliln  and the cells in question that are "dropping out" which is 
what the pathologist is describing are melanocytes, especially with Sox-10 and 
Mart 1 antibodies.
Thanks for the assistance, I definitely appreciate it very much.

Best wishes,

[image001]  Debbie Siena, HT(ASCP)QIHC
Empowering Anatomic Pathology
Technical Support Manager, StatLab
2090 Commerce| McKinney, TX 75069
t: 800.442.3573 ext 229 | m: 469-400-6897 | f: 972-436-1369 
dsi...@statlab.com<mailto:dsi...@statlab.com>|www.statlab.com<http://www.statlab.com/>

StatLab is an ISO 13485 Certified Company

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[Histonet] FW: ER/PR question

[Tony Henwood (SCHN) via Histonet]

This paper might be useful:

Ehinger, A., Bendahl, P. O., Rydén, L., Fernö, M., & Alkner, S. (2018). 
Stability of oestrogen and progesterone receptor antigenicity in formalin‐fixed 
paraffin‐embedded breast cancer tissue over time. Apmis, 126(9), 746-754.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 



-Original Message-
From: Heckford, Karen - SMMC-SF via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 8 February 2019 12:19 AM
To: 
histonet@lists.utsouthwestern.edu
Subject: [Histonet] ER/PR question

Good Morning,
One of my Pathologists wants me to do some ER/PR's on 9 year old tissue blocks. 
  I know ER and PR can be sensitive are the IHC's going to work or will there 
be too much antigen decay?

Thanks,

Karen Heckford HT ASCP CE
Lead Histology Technician
St. Mary's Medical Center
450 Stanyan St.
San Francisco, Ca. 94117
415-668-1000 ext. 6167
karen.heckf...@dignityhealth.org

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Re: [Histonet] Opinion on dye on biopsies

[Tony Henwood (SCHN) via Histonet]

One article that might be useful is:

Surprenant D, Garib G, Hutchens K, Dreifke M, Speiser J, Winterfield L, 
Peterson A, Krol C, Adams W, Tung R. Novel Use of Preoperative Epidermal 
Coloring of Very Small Dermatological Specimens-Protocol for Reduction of Lost 
Specimens. The American Journal of Dermatopathology. 2016 Jul 1;38(7):510-2.

I would expect that this would colour the squamous cell layer of the 
oesophageal mucosa.

Regards 

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 



-Original Message-
From: Gareth Davis via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 10 January 2019 9:15 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Opinion on dye on biopsies

So, I work in a small GI lab, and I put Eosin in my first formalin on my 
processor.  My biopsies are very small and this helps, somewhat, to see the 
specimens for embedding and cutting.  But, unfortunately, the esophagus tissues 
do not absorb the eosin much.  Anyway, the hospital lab I work, part-time, in 
has started using hematoxylin to help see their biopsies.  I happen to embed 
there and I think it just makes a big mess and the tissue does not absorb much 
of the stain.
What are other labs doing to aid in making their small biopsies easier to see?  
What are pros and cons to doing this, in your opinion?
Thanks!

--
*Ms. Gareth B. Davis*, B.S., HT, QIHC  (ASCP)cm Yuma Gastroenterology Yuma, AZ 
85364
928-248-5259
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Re: [Histonet] IHC-H HELP...

[Tony Henwood (SCHN) via Histonet]

Yep,

Definitely an issue.

You can easily stain the IPX slides with H, though the discernibility of the 
cells and tissue structure with the H will depend on the degree of DAB 
product laid down (eg I would expect it to be difficult with a Vimentin IPX 
compared to a CD15).

(Grosset, A. A., Loayza-Vega, K., Adam-Granger, É., Birlea, M., Gilks, B., 
Nguyen, B., ... & Trudel, D. (2017). Hematoxylin and Eosin Counterstaining 
Protocol for Immunohistochemistry Interpretation and Diagnosis. Applied 
immunohistochemistry & molecular morphology: AIMM.)

As for doing another IPX on the existing IPX stained section (with DAB as the 
chromogen), you will have to use a different label (eg alkaline phosphatase). 
The result will depend on the cell compartment the two antigens exist. If the 
DAB is nuclear, then a cytoplasm or cytoplasmic membrane localisation with the 
Alk Phosphatase will work. If both antigens are cytoplasmic, then you will not 
see co-localisation in the same cell since the DAB will prevent any antibody 
binding in the same compartment.

Assuming the above is good, then since the antigen retrieval tends to reverse 
over time, I would include a short retrieval before the second antibody.

Now after all that, I hope the section stays on the slide!


Regards 

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Curt via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 4 January 2019 4:05 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC-H HELP...

Need help before I turn a mistake into an irreparable mistake...

We have some unstained slides that were supposed to get stained with H but my 
guy stained them with IHC. It's complicated, we received slides and a block, 
the block was for IHC, the unstained slides were for H, he inverted the 
process) The point is, now the unstained slides are stained with IHC... I know 
we cannot destain the IHC but we can simply run and H over them... the real 
question I have is subsequent to the H this pathologist generally likes to 
see the H then order IHC on them based on what he sees (we only have these 
few unstained slides, don't have blocks to recut)...

So the question is... if we've already run IHC, then followed that with and 
H, can we return to run IHC on the slides again? would you want to skip any 
pre-treatment, antigen retrieval

I don't see this working too well myself, if they're already stained with DAB, 
that would be present on the second stain...

Thoughts?

Thanks for your help.

Curt

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Re: [Histonet] IHC Negative reagent controls

[Tony Henwood (SCHN) via Histonet]

Hi Cayman,

Unfortunately, applying HIER to a negative control for an antibody that 
requires enzyme retrieval (or no retrieval at all) is not appropriate.
The pre-treatment processes are different and could unmask different epitopes.
If you are using a negative control then the whole procedure needs to be same 
with the exception or replacing the localisation antibody with an Isotypic 
antibody solution. (Isotype controls are primary antibodies that lack 
specificity to the target, but match the class and type of the primary antibody 
used in the application.)

For example, applying citrate or EDTA HIER to sections prior to using the CD99 
antibody (clone 12E7) can reveal perinuclear (golgi-like) staining of some 
tumours (eg some colonic carcinomas) but this is not seen if enzyme retrieval 
is used.

Hope this is useful

Regards 

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-Original Message-
From: Cayman Fleck via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 3 January 2019 1:48 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC Negative reagent controls

A question that came up regarding negative reagent controls for IHC...currently 
using Ventana i-View.  Our regular negative control goes through the standard 
antigen retrieval steps, like 99% of our antibodies.  However there are a small 
number of antibodies that require enzyme as well (Protease 1).  I've seen a 
number of suggestions regarding this for the negative reagent control...some 
say use an additional negative control protocol that includes the protease, 
some say to use a single negative control protocol and just include the 
harshest cell conditioning that any of your protocols use (so basically use the 
cell conditioning + protease negative control for all antibodies)...i-View is 
not polymer-based so we need to continue using negative controls.  Any thoughts 
or advice?

Frank

Sent from Outlook<http://aka.ms/weboutlook> 
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Re: [Histonet] Alizarin Red Stain

[Tony Henwood (SCHN) via Histonet]

Hi Richard,

We sure do


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Cartun, Richard via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 26 October 2018 9:02 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Alizarin Red Stain

Does anyone do the Alizarin Red histochemical stain for calcium in clinical 
samples?  Thank you.

Richard

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


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Re: [Histonet] H for H. pylori?

[Tony Henwood (SCHN) via Histonet]

The following might be useful:

Tazawa, K., & Tsutsumi, Y. (1998). Effect of prolonged staining with 
hematoxylin on detecting Helicobacter pyiori in hematoxylin‐eosin‐stained 
gastric mucosa. Pathology international, 48(6), 448-452.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 



-Original Message-
From: Rene J Buesa via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 10 October 2018 6:46 AM
To: P Sicurello; HistoNet
Subject: Re: [Histonet] H for H. pylori?

Paula:"Optimizing" H to detect H. piloris is a rout you should not attempt 
because H cannot be "optimized" for that.Giemsa is one way or, even better, 
modified Steiner stain. The only disadvantage modified Steiner has is using 
radioactive uranium nitrate, but I developed a procedure where 0.02% aq. 
phosphotungstic acid solution can be used instead. IHC will be way more 
expensive.
René 

On Tuesday, October 9, 2018 2:03 PM, P Sicurello via Histonet 
 wrote:
 

 Good Morning Listers,

Has anyone out there optimized an H for H. pylori?

Sure we can run a giemsa or and IHC but what fun is that?

Send me your ideas and make the day of our GI pathologists.

Thanks oodles.

Sincerely,

Paula Sicurello, HTL (ASCP)CM

Histotechnology Specialist

UC San Diego Health

9300 Campus Point Drive

La Jolla, CA 92037
(P): 858-249-5610



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Re: [Histonet] AFB STAIN

[Tony Henwood (SCHN) via Histonet]

Cross-contamination has not been proven in FFPE tissues.

I have not seen it in nearly 40 years of practice

Do you have any evidence of cross-contamination in FFPE?

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Laurie Colbert via Histonet 
Sent: Thursday, October 4, 2018 10:36 PM
To: adesupo2...@hotmail.com; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] AFB STAIN

You should put the AFB control on a separate slide to prevent 
cross-contamination.

Laurie Redmond


-Original Message-
From: ADESUPO ADESUYI via Histonet 
To: histonet 
Sent: Wed, Oct 3, 2018 7:58 pm
Subject: [Histonet] AFB STAIN

Hello,
I have a question please. For the AFB Stain, do we put both the control tissue 
and the patient on the same slide?

Thanks,
Ade
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Re: [Histonet] AFB STAIN

[Tony Henwood (SCHN) via Histonet]

We do,

It works well


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: ADESUPO ADESUYI via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 4 October 2018 12:54 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] AFB STAIN

Hello,
 I have a question please. For the AFB Stain, do we put both the 
control tissue and the patient on the same slide?

Thanks,
Ade
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Re: [Histonet] Unstained slides - how long are they good for?

[Tony Henwood (SCHN) via Histonet]

Will definitely depend on the antibody you are using. Some references:

Jacobs, T. W., Prioleau, J. E., Stillman, I. E., & Schnitt, S. J. (1996). Loss 
of tumor marker-immunostaining intensity on stored paraffin slides of breast 
cancer. JNCI: Journal of the National Cancer Institute, 88(15), 1054-1059.

Manne, U., MYERS, R. B., SRIVASTAVA, S., & GRIZZLE, W. E. (1997). Re: loss of 
tumor marker-immunostaining intensity on stored paraffin slides of breast 
cancer. Journal of the National Cancer Institute, 89(8), 585-586.

Bertheau, P., Cazals-Hatem, D., Meignin, V., de Roquancourt, A., Vérola, O., 
Lesourd, A., ... & Janin, A. (1998). Variability of immunohistochemical 
reactivity on stored paraffin slides. Journal of clinical pathology, 51(5), 
370-374.

Olapade-Olaopa, E. O., Mackay, E. H., & Habib, F. K. (1998). Variability of 
immunohistochemical reactivity on stored paraffin slides. Journal of clinical 
pathology, 51(12), 943.

Wester, K., Wahlund, E., Sundström, C., Ranefall, P., Bengtsson, E., Russell, 
P. J., ... & Busch, C. (2000). Paraffin section storage and 
immunohistochemistry: effects of time, temperature, fixation, and retrieval 
protocol with emphasis on p53 protein and MIB1 antigen. Applied 
Immunohistochemistry & Molecular Morphology, 8(1), 61-70.

van den Broek, L. J., & van de Vijver, M. J. (2000). Assessment of problems in 
diagnostic and research immunohistochemistry associated with epitope 
instability in stored paraffin sections. Applied Immunohistochemistry & 
Molecular Morphology, 8(4), 316-321. 

Olapade-Olaopa, E. O., Ogunbiyi, J. O., MacKay, E. H., Muronda, C. A., Alonge, 
T. O., Danso, A. P., ... & Wong, A. J. (2001). Further characterization of 
storage-related alterations in immunoreactivity of archival tissue sections and 
its implications for collaborative multicenter immunohistochemical studies. 
Applied Immunohistochemistry & Molecular Morphology, 9(3), 261-266.

Mirlacher, M., Kasper, M., Storz, M., Knecht, Y., Dürmüller, U., Simon, R., ... 
& Sauter, G. (2004). Influence of slide aging on results of translational 
research studies using immunohistochemistry. Modern pathology, 17(11), 1414.

DiVito, K. A., Charette, L. A., Rimm, D. L., & Camp, R. L. (2004). Long-term 
preservation of antigenicity on tissue microarrays. Laboratory investigation, 
84(8), 1071.

Fergenbaum, J. H., Garcia-Closas, M., Hewitt, S. M., Lissowska, J., Sakoda, L. 
C., & Sherman, M. E. (2004). Loss of antigenicity in stored sections of breast 
cancer tissue microarrays. Cancer Epidemiology and Prevention Biomarkers, 
13(4), 667-672.

Hameed, O., & Humphrey, P. A. (2009). Immunohistochemical evaluation of 
prostate needle biopsies using saved interval sections vs new recut sections 
from the block: a prospective comparison. American journal of clinical 
pathology, 131(5), 683-688.

Xie, R., Chung, J. Y., Ylaya, K., Williams, R. L., Guerrero, N., Nakatsuka, N., 
... & Hewitt, S. M. (2011). Factors influencing the degradation of archival 
formalin-fixed paraffin-embedded tissue sections. Journal of Histochemistry & 
Cytochemistry, 59(4), 356-365.

Seidu, M. A., Adams, A. R., Gyasi, R. K., Tettey, Y., Nkansah, D. O., & Wiredu, 
E. K. (2013). Immunoreactivity of some epitopes in longtime inappropriately 
stored paraffin-embedded tissues. Journal of Histotechnology, 36(2), 59-64.

Nuovo, A. J., Garofalo, M., Mikhail, A., Nicol, A. F., Vianna-Andrade, C., & 
Nuovo, G. J. (2013). The effect of aging of formalin-fixed paraffin-embedded 
tissues on the in situ hybridization and immunohistochemistry signals in 
cervical lesions. Diagnostic Molecular Pathology, 22(3), 164-173.

Grillo, F., Bruzzone, M., Pigozzi, S., Prosapio, S., Migliora, P., Fiocca, R., 
& Mastracci, L. (2017). Immunohistochemistry on old archival paraffin blocks: 
is there an expiry date?. Journal of Clinical Pathology, jclinpath-2017.

Giunchi, F., Degiovanni, A., Daddi, N., Trisolini, R., Dell'Amore, A., 
Agostinelli, C., ... & Fiorentino, M. (2018). Fading With Time of PD-L1 
Immunoreactivity in Non-Small Cells Lung Cancer Tissues: A Methodological 
Study. Applied Immunohistochemistry & Molecular Morphology, 26(7), 489-494.


-Original Message-
From: P Sicurello via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 17 August 2018 9:49 AM
To: HistoNet
Subject: [Histonet] Unstained slides - how long are they good for?

Hello My Fellow Histologists,

Happy Friday Eve.

The question has come up..  How long are *unstained* slides good for?
Not for H but tests like IHC and molecular testing.  These slides have been 
cut, stored at room temperature, not sealed in anyway, and kept in a cardboard 
box.

Please let me know what your opinions are and what your retention policy is 
concerning *unstained* slides.

Thanks oodles.

Sincerely,

Paula Sicurello, HTL (ASCP)CM

Histotechnology Specialist

UC San Diego Health

200 Arbor Drive

San Diego, CA 92103

(P): 619-543-2872



*Confidentiality Notice*: The information 

Re: [Histonet] Iron Stain

[Tony Henwood (SCHN) via Histonet]

Try your eosin stain used in your H or 1% Neutral red in 2% acetic acid

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Campbell, Tasha M. via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 8 August 2018 9:34 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Iron Stain

Is nuclear fast red the only counter stain for Prussian blue stain?  I have a 
Masson's trichrome kit and was wondering if the Bierbech Scarlet could be a 
counterstain.  I won't be doing the iron stain very often at all so I am trying 
to keep dry reagents on hand to make up as needed so they do not expire so 
quickly.  Thanks!




Tasha Campbell, B.S.,HTL(ASCP)
Frederick Gastroenterology Associates
310 W. 9th St.
Frederick, MD 21701
301-695-6800 ext. 144

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Re: [Histonet] pH of formalin

[Tony Henwood (SCHN) via Histonet]

We routinely use the following

Hew-Shue (1991) has described a useful pH indicator for working neutral 
buffered formalin solutions. Bromocresol purple (CAS: 115-40-2) , when added to 
formalin solutions, serves as an indicator of pH as well as, from a safety 
aspect, labelling the solution as formalin making redundant the dangerous 
"smell" test. At an acidic pH (5.2) the indicator colour is yellow whereas at 
pH 6.8, the colour is purple.

A saturated solution of the dye (about 0.2g/100mls) is prepared and 2 to 4 
drops are added to 10 litres of neutral buffered formalin.

Hew-Shue N (1991) "Bromcresol Purple as a Colored Marker and pH Indicator for 
Ten Percent Neutral Buffered Forrnalin" J Histotechnol 14(4):257-260.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 



-Original Message-
From: Baldwin, Kathy via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 7 June 2018 4:09 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] pH of formalin

Hi Histonetters
Is anyone out there monitoring their Ph of the formalin they use?  If do how 
often?

Thanks
S Kathy Baldwin ASCP, SCT
Pathology and Cytology Manager
Ph 812-996-0210
Fax 812-996-0232


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Re: [Histonet] Acid Phosphatase Protocol

[Tony Henwood (SCHN) via Histonet]

Hi April,
Try this

Control tissues: liver, kidney and prostate
Fixation and Sectioning: Air dried unfixed 8µm frozen sections

Solutions:

1.  Substrate solution
Naphtol AS-B1 phosphate 0.005g (Fluka 70494  1g)
Dimethylfomamide0.5ml
Warning: Suspected Carcinogen – see MSDS

2.  Acetate buffer pH 4.8
Sodium acetate  4.8g 
Distilled water 100ml
0.6% Acetic Acid14.75ml
pH to 4.8
Make up to 1000 ml with distilled water

3.  Sodium nitrite: 
Warning: Toxic – see MSDS
Sodium nitrite  1.0g
Distilled water 25.0ml

4.  Pararosaniline-HCl stock solution. 
Warning: Suspected Carcinogen – see MSDS
Pararosaniline hydrochloride 
(Basic Fuchsin CI 42510)0.8g
Distilled water 16ml
Concentrated Hydrochloric acid  4ml
Heat gently, cool to room temperature and filter (store 4C)

5.  Incubation solution:
Take 0.4ml solution 3 + 0.4ml solution 4 and mix. Stand for 2 minutes and then 
add to:
Substrate Solution (Solution 1) 0.5ml
Acetate buffer pH 4.8   25ml
Adjust with 0.1M NaOH to pH 4.5-5.0.

Procedure:

1.  Take air-dried sections and incubate in the incubation solution at 37oC 
for 1 hour
2.  Wash with distilled water and counterstain with Harris's Haematoxylin 
for 1 minute.
3.  Wash in running tap water
4.  Dehydrate rapidly through fresh alcohols to xylene and mount.

Results:

Acid phosphatase activity   red
Nuclei  blue

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


From: Oler, April via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Friday, January 26, 2018 6:26 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Acid Phosphatase Protocol

Good afternoon,

I am writing to you all because I am in search of Acid Phosphatase muscle 
protocols that do not utilize sodium barbital. I'm hoping to eliminating sodium 
barbital from my lab in the near future. If anyone is willing to share their 
protocol, or advice, I would very much appreciate any assistance.

Thank you!

April Oler, HT(ASCP)
Senior Clinical Technologist
IPOX/DIF/Muscle Laboratories
Michigan Medicine
Ann Arbor, MI




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Re: [Histonet] Elastic stain

[Tony Henwood (SCHN) via Histonet]

I would recommend the Orcein stain:

Henwood, A., (2003) "Current applications of orcein in histochemistry. A brief 
review with some new observations concerning influence of dye batch variation 
and aging of dye solutions on staining" Biotech Histochem. 78(6):303-8.

And you can pair the orcein stain with several counterstains (Picro light 
green, Periodic acid Blue Schiff's, Perls, H etc.):

Henwood, A.F., (2002) "The Orcein Stain: A versatile stain for Histopathology" 
J. Histotechn. 25(1):29-32.


Regards 

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 



-Original Message-
From: Nirmala Srishan via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, 7 November 2017 3:13 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Elastic stain

Histonetters,

Is there someone who can recommend a Elastic stain other than Verhoeff?

Thanks
Mala









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Re: [Histonet] validation

[Tony Henwood (SCHN) via Histonet]

Immunohistochemistry validation is much more than simply 20 positives and 20 
negative cases, though this will allow you to meet most accreditation 
requirements.

Validation is a multi step process:
1.  Optimisation: following a thorough literature review (what should and 
should not stain, clones used (any difference), recommended assay conditions 
(eg antigen retrieval type), choose an appropriate control to confirm staining 
conditions and perform an optimum titre.
2.  Validation: based on the literature review, choose appropriate known 
(or expected) positives and negative cases that the pathologists would 
entertain in the differential diagnosis that should be negative.

Take Prostate Specific Antigen as an example, include known prostatic 
carcinomas with a mix of well, moderately and poorly differentiated prostatic 
carcinomas and include negative tumours that should not express PSA (eg 
melanomas, lymphomas, colonic and lung carcinomas).

This is a scientific approach that inspires confidence in the usefulness of the 
test.

3.  Another task that we find extremely useful is on-going Verification, 
where we compare staining results with final diagnosis of cases immunostained 
for the marker.

Food for thought?

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Nancy Schmitt via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 25 October 2017 4:16 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] validation

Good Day!
When performing validation on IHC, we use 20 positives and 20 negative cases.

When testing for negative, can the normal tissue surrounding the tumor be 
counted as negative?

Does the negative tissue need to be tumor that is negative for the AB or just 
normal tissue?

Thank you for your consideration,

Nancy
Dubuque, IA
Ph. 563-589-9810



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Re: [Histonet] Adenovirus antibody?

[Tony Henwood (SCHN) via Histonet]

Hi Paula,

We use Adenovirus antibody from BioSB (Diagnostic Technology)
Cat Number  BSB 5040
Clone   20/11 and 2/6
Isotype IgG1/K
Dilution1:200 with citrate retrieval on the Bond 3

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: P Sicurello via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 4 August 2017 8:50 AM
To: HistoNet
Subject: [Histonet] Adenovirus antibody?

Hello Listers,

I am listing to starboard on this one.  We are using the Ventana Ultra staining 
platform and have had a bit of trouble with our Adenovirus stains.  We have 
used Abcam and Millipore to no avail.

Do you have suggestions about another vendor to try?  We are a clinical lab so 
we only stain human tissue.  IVD is preferred but ASR and RUO are OK too.

Any suggestions are gratefully accepted.

Thank you in advance.

Sincerely,

Paula Sicurello, HTL (ASCP)CM

Histotechnology Specialist

UC San Diego Health

200 Arbor Drive

San Diego, CA 92103

(P): 619-543-2872 <#>



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Re: [Histonet] IHC questions

[Tony Henwood (SCHN) via Histonet]

Hi Vicki,



We recently did a study that we published in our local journal (Histograph June 
2017) that migh be of use:



Controlled Section Baking for Immunohistochemistry
Tony Henwood, Principal Scientist, Histopathology, The Children's Hospital at 
Westmead

One source of poor immunostaining is overheating of tissue and sections. 
Several authors have reported that heated slide drying adversely affects 
sensitivity in immunohistochemistry (1). Therefore, some have advocated the use 
of lower temperature drying using adhesive-coated slides to improve the 
sensitivity of the test (2-5).

In one study (1), half the antigens were adversely affected by section drying 
at 80oC including 5D3, CMV, S100, HMB45 and CEA. Oates (4) used antisera to 
epithelial membrane antigen from three different companies and found that for 
slides dried at 58"C, staining was often paler than slides dried at room 
temperature or at 37°C.

Low heat attachment of sections to slides can cause several issues including 
inadequate attachment of the tissue sections so that tissue sections may be 
lost during antigen recovery and/or immunostaining, the inability of some 
paraffins to melt well at 58oC, and the requirement of more than 1 hr before an 
immunohistochemical procedure may be started. It has been recommended that the 
most efficient protocol for mounting tissue sections to microscopic slides 
would be to attach the tissues overnight before applying the 
immunohistochemical procedure at a temperature at which all tissue mounting 
paraffins should melt (e.g., 65oC) (6). It should be remembered that a 
significant dewaxing of sections occurs when slides are heated a few degrees 
above the melting point of the wax.
Laboratory ovens seem to be variable in their ability to maintain a constant 
temperature with the implication that it is possible to either over-cook 
sections thus adversely affecting antigens or under-heat them, possibly 
compromising subsequent de-waxing. There is also the human element. How often 
are slides removed from the oven at the required time?

The modern automatic immunostainers have excellent on-board slide heating in 
order to achieve reproducible, accurate antigen retrieval. This feature also 
allows controlled "baking" of sections and being able to programme a set time, 
removes the possibility of human error. At the Children's Hospital, the Bond 3 
is used for automated immunohistochemistry. A study was designed to assess the 
usefulness of on-board baking in routine immunohistochemistry.

Control sections were immunostained for several antigens (see table) using the 
Bond 3 on-board baking and dewax facility. Freshly cut sections were dried at 
37oC for 5 minutes to remove excess water. Slides were then loaded onto the 
Bond and the baking procedure used was 35 minutes at 63oC. Stained controls 
were compared with control slides stained prior to the instigation of the 
on-board bake procedure. The historic procedure involved heating sections at 
63-65oC for 35 minutes in a large fan-forced dry-air oven (7).

BCL-2

Mum-1

CD31

BCL-6

Calretinin

SATB2

BOB-1

S100

CyclinD1

CD20

ALK-1

MPO

CD21

Ki67

INI-1

CD3

Synaptophsin

BRG-1

HMB-45

Chromogranin

Inhibin

Melan A

Myogenin

Desmin


The results showed that there was no difference between controls stained with 
the historic compared to the on-board baking procedure except for BCL-6 which 
the new procedure gave stronger staining. (see figure).

In conclusion, we expect that on-board baking of sections should allow 
laboratories to have better control over the pre-analytical variables that can 
adversely affect the immunohistochemistry staining.

References

1.   Henwood, A. F. (2005). Effect of slide drying at 80oC on 
immunohistochemistry. Journal of Histotechnology, 28(1), 45-46.

2.   Wakins, J., Kellock, D., Gillet, C., Egan, M., Pontin, J. E., Millis, 
R. R., & Levinson, D. A. (1990). Enhancement of immunostaining. Histopathology, 
17(2), 185-185.

3.   Dodson, A., Davies, E., & Waring, J. (1991). APTES, a section adhesive 
for immunocytochemistry; and experiences of slide drying at room temperature. 
Histopathology, 19(5), 484-485.

4.   Oates J. (1993) The effect of temperature on immunostaining. Br J 
Biomed Sci 50: 157-158,

5.   Williams, J. H., Mepham, B. L., & Wright, D. H. (1997). Tissue 
preparation for immunocytochemistry. Journal of clinical pathology, 50(5), 
422-428.

6.   Jones, W. T., Stockard, C. R., & Grizzle, W. E. (2001). Effects of 
time and temperature during attachment of sections to microscope slides on 
immunohistochemical detection of antigens. Biotechnic & Histochemistry, 76(2), 
55-58.

7.   Henwood, A. F. (2012). The application of heated detergent dewaxing 
and rehydration to immunohistochemistry. Biotechnic & Histochemistry, 87(1), 
46-50.





Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)

Princ

Re: [Histonet] PAX-5

[Tony Henwood (SCHN) via Histonet]

Hi Beth,
We have found the same thing.

You get a moderate staining (half of what you see with most B cell lymphomas) 
with clone 1EW (as supplied with the Leica Bond rtu).
You will get similar results with clone 24 (38%:  Adams, et al (2009). 
Clinical, phenotypic and genetic similarities and disparities between 
post-transplant and classical Hodgkin lymphomas with respect to therapeutic 
targets. Expert opinion on therapeutic targets, 13(10), 1137-1145.)

We have found that using the Rabbit monoclonal antibody to Pax-5 (Clone SP34), 
it seems that, though staining a similar proportion of B cell lymphomas, 
staining is absent from the Reed-Sternberg cells of Hodgin lymphoma.

Our assessment is in its early stages so with further cases, it might be 
clearer.

It seems that not all Pax-5 antibodies react the same

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Linda Margraf via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Saturday, 22 July 2017 7:00 AM
To: histonet@lists.utsouthwestern.edu
Cc: 'one...@wvumedicine.org'
Subject: [Histonet] FW: PAX-5

Here is a message I am posting for Beth….


From: "O'neil, Beth" <one...@wvumedicine.org<mailto:one...@wvumedicine.org>>
Date: July 21, 2017 at 2:31:24 PM CDT
To: 
"histonet-ow...@lists.utsouthwestern.edu<mailto:histonet-ow...@lists.utsouthwestern.edu>"
 
<histonet-ow...@lists.utsouthwestern.edu<mailto:histonet-ow...@lists.utsouthwestern.edu>>
Subject: PAX-5
My pathologists are not happy with PAX-5 on the Ventana Ultra; it will 
occasionally show cytoplasmic staining of the Reed-Sternberg cells (supposed to 
be a nuclear stain).  We use Ventana’s clone SP34. I have been trying to 
optimize with the Optiview kit but we are getting so much background staining 
and only sporadic staining of the R-S cells.  I tried a different clone, Cell 
Marque EP156 which was really clean but would not stain the R-S cells at all, 
no matter what we did.   My Ventana technical specialist is stumped as to why 
we’re having so much trouble.  Does anyone else have the same problem or have 
any suggestions?

Beth Ann O’Neil, MT(ASCP)SC, HTL, QIHC
Histology Supervisor, Technical Specialist
Lab:  304 – 293 – 6014
Office:  304 – 293 – 7629
 one...@wvumedicine.org<mailto:one...@wvumedicine.org>





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[Histonet] Method for Patzelt stain

[Tony Henwood (SCHN) via Histonet]

Hi all,

I am having difficulty locating the method for the Patzelt stain.

I came across it while reading: Kimura, S., Hirai, A., & Shimizu, H. (1981). 
Epidermal vacuolation: an artifact due to injection of local anesthetics. 
Archives of dermatological research, 270(4), 413-419.

This paper references a German publication entitled: Patzelt V (1926) Zum Bau 
der menschlichen Epidermis. Z Mikroskop Anat Forsch 5:371- 462

Would anyone out there have an English translation of this method?

One of my Grad students is interested.

Thanks muchly

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


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Re: [Histonet] Inhibin

[Tony Henwood (SCHN) via Histonet]

We use Leica rtu PA0110 on the Bond 3

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Pairan, Kelly via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 26 May 2017 2:30 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Inhibin

Hi Everyone,
I went to order more Inhibin yesterday from our current supplier and that 
antibody has been discontinued.  Are you running this antibody in your lab and 
if so, which supplier do you use?  Also, does anyone use Dianova antibodies?  
We have one from this company but I cannot seem to locate a distributer in this 
country.

Thanks,
Kelly
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Re: [Histonet] IHC testing for Parvovirus (Human)

[Tony Henwood (SCHN) via Histonet]

Hi Richard,
We use NCL-PARVO from Leica

Details as follows:

ANTIGEN NAME:Parvovirus B19
OTHER NAMES: 

Clone:  R92F6
Isotype:IgG1  


DESCRIPTION:  Mouse monoclonal to native parvovirus B19 purified from human 
plasma. Human parvovirus B19 (VP1 and VP2 capsid proteins).

STORAGE CONDITIONS:4-8oC undiluted

SUPPLIER:Leica  NCL-PARVO
PROCEDURE:

Methodology:HRP-POLYMER Working Dilution:   Bond: 1/100
Special Conditions:  Citrate HIER required (ER1) 
CONTROL TISSUE: Placenta containing Parvovirus   

Inbuilt Controls 
CLINICAL SIGNIFICANCE:
Diagnosis of parvovirus B19 infection in formalin-fixed, paraffin embedded 
tissue can be made through the identification of the characteristic brick-red 
intranuclear inclusions in normoblasts within the fetal circulation in 
histological sections stained with HE. It has been found that 
immunohistochemistry was the best detection method. It is highly specific and 
sensitive, preserves the morphology and reveals a larger number of positive 
cells than does HE with the advantage of showing cytoplasmic and nuclear 
positivity, making it more reliable.
REFERENCES:
Li, J. J., Henwood, T., Van Hal, S., & Charlton, A. (2015). Parvovirus 
infection: an immunohistochemical study using fetal and placental tissue. 
Pediatric and Developmental Pathology, 18(1), 30-39.
Quemelo PRV, Lima DM, Fonseca BAL, Peres LC (2007) "Detection of parvovirus B19 
infection in formalin-fixed and paraffin-embedded placenta and fetal tissues" 
Rev. Inst. Med. trop. S. Paulo, 49(2): 103-107,

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Cartun, Richard via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 24 May 2017 4:33 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC testing for Parvovirus (Human)

If you do this test, where do you get your antibody?  Thank you.

Richard

Richard W. Cartun, MS, PhD
Director, Histology & The Martin M. Berman, MD Immunopathology & Morphologic 
Proteomics Laboratory Director, Biospecimen Collection Programs Assistant 
Director, Anatomic Pathology Hartford Hospital
80 Seymour Street
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(860) 972-1596
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Re: [Histonet] A Question About Paraffin Times

[Tony Henwood (SCHN) via Histonet]

It is best to remove the blocks and let them set at room temperature.

Leaving them at 60 or more degrees can adversely affect antigens.

We routinely do this with our autopsies and have not noted any issues.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: P Sicurello via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 17 May 2017 1:34 AM
To: HistoNet
Subject: [Histonet] A Question About Paraffin Times

Good Morning Listers,

I am asking the collective wisdom of the Histonet this question:


Is it better to remove baskets from the processor on Saturday morning and:

A.  Let the cassettes freeze, then melt them down and embed Monday morning?
OR
B.  Leave the cassettes in molten paraffin and embed Monday morning?


I am of the opinion that leaving the samples (not fatty, like breast cores) in 
molten paraffin (62 degrees C)  is bad practice, and causes them to get 
"crunchy", among other things.

What do you think?

Thank in advance.

Sincerely,

Paula Sicurello, HTL (ASCP)CM

Histotechnology Specialist

UC San Diego Health

200 Arbor Drive

San Diego, CA 92103

(P): 619-543-2872 <#>



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Re: [Histonet] Cytology pap staining

[Tony Henwood (SCHN) via Histonet]

I would also check the fixation.
Do the smears look air-dried. The larger nuclei, following air-drying do not 
concentrate the Hx as much as prompt alcohol fixation resulting in paler 
stained nuclei. 

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Charles Riley via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 20 April 2017 2:31 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Cytology pap staining

I have been put in charge of figuring out why our pap stains are light on the 
hematoxylin. Everything was filtered and used fresh as per usual using the same 
protocol we have used for the past two years.

If anyone has any suggestions as to how to fix this problem I would greatly 
appreciate it. If you need more information please let me know and I will get 
it for you to help assist you in assisting me

-- 

Charles Riley HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] processor died overnight

[Tony Henwood (SCHN) via Histonet]

Yep start at last alcohol in other processor, xylene & wax as usual.
If the tissues have remained covered in alcohol, you should not have a problem

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Lauren Sweeney via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 19 April 2017 10:44 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] processor died overnight

Hello Histoworld,

I came in this morning to find that the processor died halfway through process 
last night. The tissues are in 100% ETOH exactly half point. We do have a back- 
up processor. In your professional experiences, would these tissues be 
salvageable? Could I create a new program on the backup processor that finishes 
the process from that point and transfer the tissues over?

Thanks.


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Re: [Histonet] help!!

[Tony Henwood (SCHN) via Histonet]

You can use DAB but the issue of endogenous peroxidase will rear its ugly head.

And I suppose IF being around since 1955, when Mellors first applied the 
technique to renal tissue, it has a long history of diagnostic application that 
is hard to replace.

There are other enzymes that could be used that, not being present in human 
tissue, would not require endogenous enzyme blocking for example glucose 
oxidase. The advantage of this enzyme is that there is no endogenous glucose 
oxidase activity in mammalian tissues.

Weening, J. J., & Jennette, J. C. (2012). Historical milestones in renal 
pathology. Virchows Archiv, 461(1), 3-11.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

From: Allan Wang [mailto:alla...@gmail.com]
Sent: Tuesday, 18 April 2017 5:51 PM
To: Tony Henwood (SCHN)
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] help!!

Tim and Tony,

Why couldn't DAB be used on frozen sections in your example?

Allan

On Mon, Apr 17, 2017 at 9:03 PM, Tony Henwood (SCHN) via Histonet 
<histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>> 
wrote:
Hi Bianca,
Well for most Pathology departments, Immunofluorescence (IF) is used for Renal 
and Skin biopsies; looking for human Immunoglobulin (Igs) deposition on 
basement membranes. The advantage here is not so much the fluorescence, but 
that we use unfixed frozen sections. The buffer rinse before antibody 
application, removes un-bound serum immunoglobulins, leaving any pathological 
bound Igs for the IF antibody to bind to. This gives a clean result.

If one would do IF on formalin-fixed paraffin sections of renal or skin 
biopsies, you would find heavy background due to the fixative cross-linking 
serum Igs to tissue and cells (which would usually be removed by the buffer 
rinse if unfixed frozen sections were used - see above).

IF, apart from being a historic method, also does not suffer from endogenous 
peroxidase that would need to be blocked if peroxidase was used in place of 
fluorescence.



Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

-Original Message-
From: Blanca Lopez via Histonet 
[mailto:histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>]
Sent: Thursday, 13 April 2017 11:10 PM
To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: [Histonet] help!!
Hello!
I just need a help with a simple question...Is anyone can explain me what is 
the purpose between performing immunohistochemistry and Immunofluorescence?
Thanks  :)

Blanca Lopez
Histotech (ASCP)
UTSW Tissue Resource K1.210
Simmons Comprehensive Cancer Center
UT Southwestern Medical Center
Telephone: 214-648-7598
Email: blanca.lo...@utsouthwestern.edu<mailto:blanca.lo...@utsouthwestern.edu>




UT Southwestern


Medical Center



The future of medicine, today.

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Re: [Histonet] help!!

[Tony Henwood (SCHN) via Histonet]

Hi Bianca,
Well for most Pathology departments, Immunofluorescence (IF) is used for Renal 
and Skin biopsies; looking for human Immunoglobulin (Igs) deposition on 
basement membranes. The advantage here is not so much the fluorescence, but 
that we use unfixed frozen sections. The buffer rinse before antibody 
application, removes un-bound serum immunoglobulins, leaving any pathological 
bound Igs for the IF antibody to bind to. This gives a clean result.

If one would do IF on formalin-fixed paraffin sections of renal or skin 
biopsies, you would find heavy background due to the fixative cross-linking 
serum Igs to tissue and cells (which would usually be removed by the buffer 
rinse if unfixed frozen sections were used - see above).

IF, apart from being a historic method, also does not suffer from endogenous 
peroxidase that would need to be blocked if peroxidase was used in place of 
fluorescence.



Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Blanca Lopez via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 13 April 2017 11:10 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] help!!

Hello!
I just need a help with a simple question...Is anyone can explain me what is 
the purpose between performing immunohistochemistry and Immunofluorescence?
Thanks  :)

Blanca Lopez
Histotech (ASCP)
UTSW Tissue Resource K1.210
Simmons Comprehensive Cancer Center
UT Southwestern Medical Center
Telephone: 214-648-7598
Email: blanca.lo...@utsouthwestern.edu




UT Southwestern


Medical Center



The future of medicine, today.

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Re: [Histonet] Tissue Fixation

[Tony Henwood (SCHN) via Histonet]

If the specimen is placed on absorbent paper prior to fixation, the paper will 
gradually absorb the water from the specimen causing it to dry out.
Microscopically the tissue will look  a little like what you see when a laser 
scalpel is used to excise a skin specimen. The heat affected eosin stained 
collagen will look quite brilliant (even fluorescent like) and if you do a 
Masson stain for connective tissue will give a red colouration, rather than a 
green or blue you would normally see with well-fixed collagen.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: T H via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 31 March 2017 11:51 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Tissue Fixation

Good Morning,


I have a pathologist that is not happy with the fixation on some of our LEEP 
specimens.  She swears its histology doing something to the specimen to cause 
the tissue to look unfixed on only "part" of the LEEP specimens (all the same 
client specimens).  She claims we must be diluting our formalin to cause this 
issue or "something".  We mentioned maybe it was on the clients end not placing 
them in 10% formalin right away, she wouldn't hear of it.


Let me give you some back ground on how our process works.  Our clients send us 
all our specimens to us via Overnight FedEx or UPS in 10% formalin they will 
then they sit in 10% formalin in-house until the processors are started around 
3pm and sits an additional 5 hours in 10% formalin on the processor before the 
processor actually starts.  That being said the fixation process has had a 
pretty good start before we ever even touch it.


My question is, and I thought I had read this in the past is, when a specimen 
is left out prior to fixation and lying on a absorbent surface such as a paper 
towel, won't the area of the tissue touching the absorbent surface begin the 
disintegration processes faster in that exact area then the rest of the 
specimen?  Or if you have any other suggestion on what might be happening to 
only "certain" specimens would be great as well.


Thanks for your help!


Tim

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Re: [Histonet] Reference for this Recipe and Use

[Tony Henwood (SCHN) via Histonet]

This is sometimes known as Newel's solution for revealing lymph nodes in fatty 
specimens:

Newell KJ, Sawka BW, Rudrick BF, Driman DK. (2001) "GEWF solution" Arch Pathol 
Lab Med 125:642 645.

Absolute ethanol1000ml
Water   340ml
Concentrated formalin (37%) 160ml
Glacial acetic acid 100ml



Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: ian bernard via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Saturday, 25 March 2017 3:04 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Reference for this Recipe and Use

500 ML OF 100% ALCOHOL

170 ML OF DISTILLED WATER

80 ML OF 40% FORMALIN

50 ML OF GLACIAL ACETIC ACID

 

V/R

IB

 

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Re: [Histonet] Bob Richmond

[Tony Henwood (SCHN) via Histonet]

All the Best Bob,

Have a great retirement. 
Maybe we will see you in Australia on one of those big boats that make their 
way here regularly.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


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Re: [Histonet] Dirty H after break

[Tony Henwood (SCHN) via Histonet]

Hi Allan,

With the quality of the eosin dyes today, filtering should not be required. The 
same for xylene and ethanols.

You will need to monitor the usage and solution levels (evaporation being the 
issue here).

There are no hard and fast rules but a suggestion is one slide per ml of 
solution. Therefore a bath of 400ml of haematoxylin should stain 400 slides 
consistently before a noticeable deterioration in staining is noted. The same 
with the other solutions.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

From: Allan Wang [mailto:alla...@gmail.com]
Sent: Saturday, 7 January 2017 4:01 PM
To: Tony Henwood (SCHN)
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Dirty H after break

Yep, filtering the hematoxlyn fixed it. Thanks for the tip.

Is it worthwhile to filter the eosin, alcohols, and xylene substitutes as well?

Allan


On Jan 4, 2017 1:04 AM, "Tony Henwood (SCHN)" 
<tony.henw...@health.nsw.gov.au<mailto:tony.henw...@health.nsw.gov.au>> wrote:
Hi Allan,

The flakes are alum precipitates.

I would suggest filtering the haematoxylin before use.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

-Original Message-
From: Allan Wang via Histonet 
[mailto:histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>]
Sent: Wednesday, 4 January 2017 4:45 PM
To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: [Histonet] Dirty H after break

Hello all,

Does someone know what would cause dirty flakes of light purple that showed 
across the whole slide?

http://i.imgur.com/1nIXwAy.jpg

Reagents are relatively fresh but the H stainer hadn't been used for a week 
over the holiday break.
Is it surface film on the hematoxylin? I didn't notice anything in the 
container. We are using Mayer's, followed by differentiation with HCl acid 
alcohol, with 2-3 minute long wash steps in between.

Thanks,
Allan
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Re: [Histonet] Dirty H after break

[Tony Henwood (SCHN) via Histonet]

Hi Allan,

The flakes are alum precipitates.

I would suggest filtering the haematoxylin before use.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Allan Wang via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 4 January 2017 4:45 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Dirty H after break

Hello all,

Does someone know what would cause dirty flakes of light purple that showed 
across the whole slide?

http://i.imgur.com/1nIXwAy.jpg

Reagents are relatively fresh but the H stainer hadn't been used for a week 
over the holiday break.
Is it surface film on the hematoxylin? I didn't notice anything in the 
container. We are using Mayer's, followed by differentiation with HCl acid 
alcohol, with 2-3 minute long wash steps in between.

Thanks,
Allan
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Re: [Histonet] Staining issues

[Tony Henwood (SCHN) via Histonet]

Hi Charles,

Many issues could be in play.
Since the centre of the tissue is causing problems, then I would look at 
fixation; are the spaces around cells in the centre of the block wider than at 
the edges (conversely do the cells look like they are smaller than their 
counterparts at the edge)?

If part of this tissue is lightly fixed, they seem to be more prone to heating 
effects. If the haematoxylin is paler (washed-out?) in the centre then it is 
possible that the slides have been heated above 70oC before H staining. This 
will also affect the IPX's. Maybe the oven is over-heating.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Charles Riley via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 29 December 2016 12:19 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Staining issues

Our pathologists are complaining that the center of tissues are not staining 
properly in both our H's and IHC's. Can anyone provide some thoughts as to 
where this problem could be occurring?


As a separate situation we are experiencing the tissues folding or falling off 
during the staining process. Both this issue and the staining issue are recent 
occurrences and we have not changed our process in any way from previous years.


Does anyone think these problems are related? Why or why not?   I have
racked my brain with all the trouble shooting techniques I can think of so any 
help will greatly be appreciated

-- 

Charles Riley HT(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] Weak Hematoxylin Staining?

[Tony Henwood (SCHN) via Histonet]

Vivian,

After cutting the frozen sections, how are you fixing them?

If they are air-dried then they will tend to give a washed out nuclear stain.
I would suggest immediate fixation of the sections in either methanol or to 
make even a brighter H: 1% acetic acid in ethanol (1ml glacial acetic acid + 
99ml absolute ethanol), about 1 minute should suffice for both fixations.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Vivian Hou via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, 6 December 2016 7:58 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Weak Hematoxylin Staining?

Dear all,

We are doing some immunofluorescent stains that we want to pair with H 
slides. We are using fresh frozen sections for both (unfixed tissue, human 
diseased coronary arteries) but I am seeing very weak hematoxylin staining to 
the point where I have to dial back the eosin (3 min hematoxylin, 1 dip eosin).


I am using Gills 2 (newly purchased), I know its not necessary to filter it but 
we have been just in case using Whatman no. 5 filters. No differentiation due 
to the weak staining right now.


I would appreciate any thoughts on why the staining might be so weak, we do a 
great deal of processing and imaging on the arteries (about 12 hrs @ 25C) prior 
to histo and I wonder if this has cause the tissue to decay?


Thank you for your time,
V

--
-
V Hou
Bioengineering | Human Photonics Laboratory
e: ho...@uw.edu | c: 206-999-3708
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Re: [Histonet] RTU antibodies with 1+ year expiration date

[Tony Henwood (SCHN) via Histonet]

 routine histology 
sections. Monoclonal antibodies originally supplied as culture supernatants or 
as ascites (neat or diluted), of all isotypes, as well as all of the polyclonal 
antibodies, produced satisfactory staining irrespective of their age. Notable 
exceptions were ammonium-precipitated, IgM or conjugated antibodies.

Drachenberg et al (2001) suggest that instead of a mechanical adherence to the 
manufacturers’ recommendations, a rational quality assurance system would be 
preferable.

References:
Argentieri, M. C., Pilla, D., Vanzati, A., Lonardi, S., Facchetti, F., 
Doglioni, C., ... & Cattoretti, G. (2013). Antibodies are forever: a study 
using 12?26‐year‐old expired antibodies. Histopathology, 63(6), 869-876.
Balaton, A. J., Drachenberg, C. B., Rucker, C., Vaury, P., & Papadimitriou, J. 
C. (1999). Satisfactory performance of primary antibodies beyond manufacturers' 
recommended expiration dates. Applied Immunohistochemistry & Molecular 
Morphology, 7(3), 221.
Drachenberg, C. B., Papadimitriou, J. C., Balaton, A. J., & Vaury, P. (2001). 
The total test approach to standardization of immunohistochemistry. Archives of 
pathology & laboratory medicine, 125(4), 471-471.
Savage, E. C., & DeYoung, B. R. (2010). Antibody Expiration in the Context of 
Resource Limitation What Is the Evidence Basis?. American journal of clinical 
pathology, 134(1), 60-64.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Allan Wang via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, 6 December 2016 7:42 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] RTU antibodies with 1+ year expiration date

Hello list,

Thanks for your responses to my previous dehydration question. I pretty much 
got conflicting responses about going straight to 100% alcohol, but I think I 
will just start them out in 80% alcohol to be safe.

Something that's been bugging me: Every source states that diluted antibodies 
shouldn't be kept for more than a few weeks. Yet Ventana dispensers of 
pre-diluted antibodies have expiration dates of 1 - 1.5 years, plus they sit 
out at room temperature for 5 hours at a time whenever they're used. Ventana 
provides user-fillable dispensers, but if we dilute antibodies ourselves and 
can't use them for more than a few weeks, it would be kind of pointless for 
low-volume needs.

Their specs say: "The antibody is diluted in 0.08 M PBS with 3% carrier protein 
and 0.05% ProClin 300, a preservative."
Isn't this pretty much the same stuff that antibody diluent is composed of, 
except with sodium azide instead of ProClin 300 (I think they're equivalent).

I'm already planning on doing some long-term testing of my own over a year with 
HER-2 and Ki67, but hopefully someone can provide some insight.

Thanks,
Allan Wang
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Re: [Histonet] Competency for Supervisors

[Tony Henwood (SCHN) via Histonet]

Hi Mighnon,

I find the so-called competency for all staff, including laboratory managers 
one of the stupidest items ever to be put in a standard (ie ISO 15189).

All staff who perform a test need to be recorded as being competent in it, no 
problem with this but managers as well?

How ridiculous! Why you ask? Well please forgive me in advance for the 
following rant:

The Manager/Supervisor is responsible for instituting the test, training 
his/her staff in the test and importantly responsible for the quality of the 
test and yet His/her competency is called into question?

So how does a Manager obtain a competency record for the test?

1.  Ask a manager from another laboratory (?rival) to sign their 
competency. Could you imagine Donald Trump asking Ed McMahon to sign his 
competency? Since many of us sign confidentiality and privacy agreements, this 
will cause problems.

2.  The manager could ask his staff to sign off his competency. A brilliant 
example of a BAD personal management decision. Why put your staff into the 
stressful position of deciding whether their supervisor is competent or not? If 
they say their supervisor is competent even though they believe they are not, 
then they are forced to lie possibly in order to keep their job. OR in the 
worst case scenario, they could hold this over the supervisor's head (and 
visa-versa) in order to keep their job, obtain favours or monetary gain.

I often wonder whether the authors of this item in the standard were complete 
idiots. They probably never had to face the stresses of managing a laboratory 
and especially valuable scientific staff. 

Absolutely stupid!!

We stepped up our competency assessment by evolving it into a Continuing 
Education exercise, including questions and scenarios for my staff to consider 
and hopefully learn from.

I was then asked why I had not done the same tests. Well I wrote the tests so 
one would assume that I would know the answers - a little self-serving?

Unfortunately I could not test myself when I was attempting my driving licence, 
I would have passed the first time!!

Enough of a rant - please advise whether I am driving up the wrong tree!

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Mighnon Lashus via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 2 December 2016 7:38 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: Re: [Histonet] Competency for Supervisors

Did anyone have a form for the Histology Supervisor Competency?

Thanks,

Mighnon Lashus, HT (ASCP)
Laboratory Manager

PathGroup
4071 S. Access Rd, Suite 107
Chattanooga, TN 37406
423-493-0207
423-493-0208 fax
mlas...@pathgroup.com

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Re: [Histonet] Transport training for formalin fixed specimens

[Tony Henwood (SCHN) via Histonet]

Hi Gareth,
You will probably have to design your own Continuing Education Session.
I would recommend the following topics:
1.  Nature of formaldehyde - safety aspects, explanation of the MSDS.
2.  What formaldehyde is used for
3.  Appropriate packaging of pots containing specimens in formalin
4.  Spill containment, neutralisation, clean-up procedures and Notification 
requirements.

Probably only take 30 minutes or so.
Issue each attendee with a certificate and record attendees for CAP.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Gareth Davis via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 30 June 2016 9:18 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Transport training for formalin fixed specimens

Okay, is there a training course for just transportion formalin fixed 
specimens.  CAP told me last year that anyone picking up specimens for us has 
to go through a training course.  The inspector seemed to be more concerned 
with the employees knowledge of formalin and possible spills.
So, does anyone know what course I am suppose to be using?
 I have looked on the CDC website, but all the courses I find refer to specific 
infectious disease.
Thanks in advance.
--
Gareth B. Davis, HT (ASCPcm), QIHC
Yuma Gastroenterology
Yuma, AZ 85364
928-248-5259
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Re: [Histonet] Melanin Bleach

[Tony Henwood (SCHN) via Histonet]

The KMnO4/oxalic acid sequence will weaken the tissue adherence.
If you do not have to use Peroxidase/DAB, then I would recommend alkaline 
phosphatase labelling (+red chromagen).

Remember KMnO4/oxalic acid can be deleterious to some antigen-antibody 
reactions. 
We found that LCA, CAM5.2, L26, alpha-fetoprotein, and NSE were labile, whilst 
alpha-1-antitrypsin, HBsAg, CMV, CEA, Thyroglobin and chromogranin showed 
decreased staining. EMA, desmin, HSVII, S100, GFAP, PSA, PAcP, Calcitonin, ACTH 
and Prolactin were found to be resistant to permanganate treatment.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Heckford, Karen - SMMC-SF via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 11 May 2016 1:45 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Melanin Bleach

Good Morning,
I need some help.  Yesterday I bleached some heavily pigmented tissue.  I have 
to run some IHC's on them.   I bought the bleaching kit from American Master 
Tech.  I had to put the Permanganate for about 6 hours to get the melanin and 
then a couple of minutes in Oxalic Acid.  I had to let them set overnight 
because I could not get another IHC run in that day.   It looks like the tissue 
fell off during decloaking.I used Apex slides.   I rarely ever have to 
bleach anything here.  So I am not sure if I did this correctly.   I am 
thinking I need to bleach and do the run in the same day and not let them set 
over night in DiH20.

Thanks,


Karen Heckford HT ASCP CE
Lead Histology Technician
St. Mary's Medical Center
450 Stanyan St.
San Francisco, Ca. 94117
415-668-1000 ext. 6167
karen.heckf...@dignityhealth.org

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Re: [Histonet] Cytology/Histology Staining Question

[Tony Henwood (SCHN) via Histonet]

Yep,
We do.
We have a separate dehydration sequence for PAP and other special stains 
(separate from the eosin dehydration sequence).

We have a Leica (used to be Vision Biosystems) Autostainer

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Mullen, Mary via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, 11 May 2016 12:55 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Cytology/Histology Staining Question

Hello all,



I work in a small, low volume community hospital and was recently asked by a 
coworker why we do not just run both our cytology and histology slides on the 
same automated stainer (with their respective protocols).



What I am wanting to know is if there is anyone currently running both staining 
protocols on a single automated stainer using common alcohols/xylenes/water? 
What are the pros/cons? Has there been any cross-contamination issues?



We only run non-gyn cytology, all gyn cytology is sent out.







Thanks,



Mary K. Mullen, HTL(ASCP)CM
Histotechnologist
UPMC Northwest
Seneca, PA

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Re: [Histonet] PAS Stain

[Tony Henwood (SCHN) via Histonet]

Only a crazy Aussie would do this!!

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Anne Murvosh via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, 6 May 2016 6:03 AM
To: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] PAS Stain

You clearly don't know your histo history. The reason we know that H pylori 
exists is because a Scientist, Dr. Barry Marshall wanted to prove bacteria 
caused ulcers and not stress.  No one believed him.  So he took the organisms 
from a patient, mixed it in a broth and drank it. He then biopsied himself and 
treated it. There's a non-uniform method that saved a lot of suffering.  Bravo 
we crazy scientists.  Anne


-Original Message-
From: Geoff via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Thursday, May 05, 2016 12:31 PM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] PAS Stain

I cannot believe any scientist would advocate such a non-uniform method as 
spitting on a slide.
Buy a bottle of what ever enzyme and use a reproducible buffer and temperature.

Geoff

On 5/5/2016 3:19 PM, Anne Murvosh via Histonet wrote:
> Yes, spitting is the tried and true way to do it.  Not to mention no 
> measuring and cheaper.  The reason we switched to a powder is because I just 
> don't spit well I used to have someone do it for me cause I would end up 
> drooling. YUCK! The best way to find out is do the amylase method and the 
> spit method at the same time and have the doctor pick the best.  A fun 
> experiment  Anne  
>
> -Original Message-
> From: Bob Richmond via Histonet 
> [mailto:histonet@lists.utsouthwestern.edu]
> Sent: Thursday, May 05, 2016 11:36 AM
> To: koelli...@comcast.net
> Cc: Histonet@lists.utsouthwestern.edu
> Subject: Re: [Histonet] PAS Stain
>
> Spokane Ray points out something I've wondered about for years - can 
> just anybody spit on the slide and remove the glycogen? I've never 
> heard of any variation, but the number of people I've asked is very 
> limited. This
> reference:
> http://www.ncbi.nlm.nih.gov/gene/276
> certainly suggests that different people have different salivary alpha 
> amylase activity.
>
> Bob Richmond
>
> On Thu, May 5, 2016 at 2:27 PM, <koelli...@comcast.net> wrote:
>
>> I love having the Samuri Pathologist on this forum for wisdom and 
>> real-laboratory life knowledge.  And yes, I have in the past spit on 
>> slide ON OCCASSION when faced with a dire necessity.  Although I know 
>> there are those who would wretch about this; it remains a fact of 
>> viable laboratory life for some.
>>
>> My problem now is that in this era of (MUCH TOO MUCH) regulation, how 
>> do you "test lots" or control from "lot-to-lot variation" in this 
>> SOP?  When Jane or Joe do this routinely and then goes on vacation, 
>> what about Sally or Jim spit?  There is a variation in copy number of 
>> the AMY1 gene
>> (amylase) and resulting difference in amylase protein concentration 
>> amongst individuals.
>>
>> Why not just standardize it from the start, reagent, pH, temperature 
>> and it really cannot fail.
>>
>> Spokane Ray
>>
>> --
>> *From: *"Bob Richmond via Histonet" 
>> <histonet@lists.utsouthwestern.edu>
>> *To: *"Histonet@lists.utsouthwestern.edu" < 
>> histonet@lists.utsouthwestern.edu>
>> *Sent: *Thursday, May 5, 2016 11:10:40 AM
>> *Subject: *Re: [Histonet] PAS Stain
>>
>>
>> Amylase (diastase) for the PAS stain queries:
>>
>> Whatever happened to spitting on the slide (30 min at room temperature)?
>> John Kiernan advises "thinking of lemons and drooling into a small beaker"
>> though I'd advise chewing on a rubber band for a few seconds.
>>
>> He notes that alpha amylase is preferred. I'd go with the cheapest 
>> one in the Sigma-Aldrich catalog. Room temperature is usual, but I 
>> note that Sigma offers a heat-stable alpha amylase.
>>
>> Bob Richmond
>> Samurai Pathologist
>> Maryville TN
>> ___
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>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
> ___
> Histonet mailing list

[Histonet] PAS Stain

[Tony Henwood (SCHN) via Histonet]

Yep,

I have had a few techs in my time who could not digest glycogen if their life 
depended on it.
Their salivary amylase just did not work.
It was not a major (not even a minor) health issue for them. They looked 
healthy enough (actually healthier than me).

This is one of the reasons we developed a 10 minute room temp amylase method:

 Mangan, V-M, Farago, V., Kelly, M., Henwood, A.F., (2002) "An Amylase Reagent 
with a Long Shelf Life for the removal of Glycogen from Tissue Sections" J. 
Histotechnol 25:153.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Principal 
Scientist, the Children’s Hospital at Westmead Adjunct Fellow, School of 
Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead 
NSW 2145, AUSTRALIA 


-Original Message-
From: Ray via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Friday, 6 May 2016 4:48 AM
To: Richmond, Bob
Cc: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] PAS Stain

An excellent point.  For anyone wanting to investigate-simply do a PubMed 
search on variation of AMY1 gene.  Sorry; I guess I should say this is, 
strictly speaking, non-histology related topic and I don't want to get into 
trouble as some before me.  Tons of research about this linking back (in 
theory) to positive selection in hunter-gatherers versus agricultural 
ancestors, race, obesity, phenotypic and dietary differences as to why maybe 
there can be big differences. 
Spokane Ray 

- Original Message -

From: "Bob Richmond" <rsrichm...@gmail.com>
To: koelli...@comcast.net
Cc: "Histonet@lists.utsouthwestern.edu" <histonet@lists.utsouthwestern.edu>
Sent: Thursday, May 5, 2016 11:35:42 AM
Subject: Re: [Histonet] PAS Stain 

Spokane Ray points out something I've wondered about for years - can just 
anybody spit on the slide and remove the glycogen? I've never heard of any 
variation, but the number of people I've asked is very limited. This reference: 
http://www.ncbi.nlm.nih.gov/gene/276
certainly suggests that different people have different salivary alpha amylase 
activity. 

Bob Richmond 

On Thu, May 5, 2016 at 2:27 PM, < koelli...@comcast.net > wrote: 



I love having the Samuri Pathologist on this forum for wisdom and 
real-laboratory life knowledge.  And yes, I have in the past spit on slide ON 
OCCASSION when faced with a dire necessity.  Although I know there are those 
who would wretch about this; it remains a fact of viable laboratory life for 
some. 
  
My problem now is that in this era of (MUCH TOO MUCH) regulation, how do you 
"test lots" or control from "lot-to-lot variation" in this SOP?  When Jane or 
Joe do this routinely and then goes on vacation, what about Sally or Jim spit?  
There is a variation in copy number of the AMY1 gene (amylase) and resulting 
difference in amylase protein concentration amongst individuals. 
  
Why not just standardize it from the start, reagent, pH, temperature and it 
really cannot fail. 
  
Spokane Ray 


From: "Bob Richmond via Histonet" < histonet@lists.utsouthwestern.edu >
To: " Histonet@lists.utsouthwestern.edu " < histonet@lists.utsouthwestern.edu >
Sent: Thursday, May 5, 2016 11:10:40 AM
Subject: Re: [Histonet] PAS Stain 


Amylase (diastase) for the PAS stain queries: 

Whatever happened to spitting on the slide (30 min at room temperature)? 
John Kiernan advises "thinking of lemons and drooling into a small beaker" 
though I'd advise chewing on a rubber band for a few seconds. 

He notes that alpha amylase is preferred. I'd go with the cheapest one in the 
Sigma-Aldrich catalog. Room temperature is usual, but I note that Sigma offers 
a heat-stable alpha amylase. 

Bob Richmond
Samurai Pathologist
Maryville TN
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[Histonet] Reference for erythrosin-eosin counterstain for H

[Tony Henwood (SCHN) via Histonet]

Hi all,

I am doing an H workshop next month and one of the Hx counterstains I will be 
including is Erythrosin-eosin (see below).
This counterstain is the preferred by some of our major Histopathology 
departments in Australia.

The issue I have is that I cannot find a reference for this variant of the H

Anyone have any ideas?


Eosin Y (CI 45380) 5g
Erythrosin B (CI 45430)   5g
Sodium Hydrogen Carbonate 1.25g
Magnesium Sulphate 10g
Distilled water   500ml
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principle Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


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Re: [Histonet] Porcessing FFPE tissue without alcohol??

[Tony Henwood (SCHN) via Histonet]

You might find these articles to be useful

Tracy, R. E., & Walia, P. (2002). A method to fix lipids for staining fat 
embolism in paraffin sections. Histopathology, 41(1), 75-79.

Turello, R., Snyder, D., & Hartman, H. A. (1984). A modification the osmium 
tetroxide post-fixation technique for the demonstration of extracellular lipid 
in paraffin-embedded tissue sections. Journal of Histotechnology, 7(2), 75-77.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Hobbs, Carl via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Saturday, 26 March 2016 5:30 AM
To: histonet
Subject: Re: [Histonet] Porcessing FFPE tissue without alcohol??



Fix the tissue in Formalin, wash well in dw, then place very small pieces into 
Osmium tetroxide solution ( std soln for TEM post-fixation) Processing to Pwax 
as usual.
Basically, you will see lipids as black ( oxidised osmium) That's the only way 
to demonstrate solvent- soluble lipids, using Pwax processing.
Sure, there are caveats but, in the main...it will be Ok, imho.
I invite comments as I may be doing exactly this very soon, to count myelinated 
nerve fibres in a sciatic nerve.



  
 
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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