Re: [Histonet] PAS Stain

[Ray via Histonet]

Indeed, a very curious and interesting way to do this.  And as I said-have done 
it very occasionally in long ago past. 
What I am still curious about is that for those who still do this, how do you 
write it up for CLIA or CAP or GLP when, as the Samuri Pathologist would call 
them, Herrn Inspektors come to visit.  Lot number of spit?  Variation of spit 
reagent with a different lot (person)?  Preparation of spit reagent (before or 
after starchy meal)?  What the step-by-step procedure looks like?  Just 
curious. 
  
Spokane Ray 

- Original Message -

From: "Sherry Martin via Histonet"  
To: histonet@lists.utsouthwestern.edu 
Sent: Thursday, May 5, 2016 5:30:22 PM 
Subject: Re: [Histonet] PAS Stain 

Hello All! I've served in laboratory medicine for well over 35 years and in my 
early years we did indeed spit on the slides. I learned very quickly on that my 
personal spit fails to digest any glycogen. And worse yet, I used to have to 
search for someone else to spit for me. PAS stains were always awkward and 
gross! I'm VERY thankful for the artificial amylase we have now. :-) 
Y'all have a great day! 
Sherry Martin 

    On Thursday, May 5, 2016 7:06 PM, Ingles Claire via Histonet 
 wrote: 
  

 I don't know, I believe Dr. Salk did the same with the polio vaccine. He even 
involved his family! Dedicated doctors... 
Claire 

 
From: Tony Henwood (SCHN) via Histonet [histonet@lists.utsouthwestern.edu] 
Sent: Thursday, May 05, 2016 6:33 PM 
To: Anne Murvosh 
Cc: histonet@lists.utsouthwestern.edu 
Subject: Re: [Histonet] PAS Stain 

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,  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 

Re: [Histonet] PAS Stain

[Sherry Martin via Histonet]

Hello All! I've served in laboratory medicine for well over 35 years and in my 
early years we did indeed spit on the slides. I learned very quickly on that my 
personal spit fails to digest any glycogen. And worse yet, I used to have to 
search for someone else to spit for me. PAS stains were always awkward and 
gross! I'm VERY thankful for the artificial amylase we have now. :-) 
Y'all have a great day! 
Sherry Martin 

On Thursday, May 5, 2016 7:06 PM, Ingles Claire via Histonet 
 wrote:
 

 I don't know, I believe Dr. Salk did the same with the polio vaccine. He even 
involved his family! Dedicated doctors...
Claire


From: Tony Henwood (SCHN) via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Thursday, May 05, 2016 6:33 PM
To: Anne Murvosh
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] PAS Stain

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,  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"
>> 
>> *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 

Re: [Histonet] PAS Stain

[Ingles Claire via Histonet]

I don't know, I believe Dr. Salk did the same with the polio vaccine. He even 
involved his family! Dedicated doctors...
Claire


From: Tony Henwood (SCHN) via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Thursday, May 05, 2016 6:33 PM
To: Anne Murvosh
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] PAS Stain

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,  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"
>> 
>> *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
>> ___
>> 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
>

Re: [Histonet] Slide printers

[Frazier, John via Histonet]

The Thermo Slidemate  and Prima are both good.   The Thermo is small.
It is designed for a microtome work station. If you find another
company, I would be careful to ensure they can interface with your LIS
and or cassette marker. You want continuity between the two makers and
the LIS

Sent from my iPad

> On May 5, 2016, at 6:45 PM, Ginny Achstetter  wrote:
>
>
> I need a recommendation on a slide printer. Tried the Leica and liked it but 
> it only works well with their round edged slides and they aren't charged.
> Sent from my iPhone
>
>

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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,  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" 
>> 
>> *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
>> ___
>> 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
>
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet


--
--
**
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 

[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" 
To: koelli...@comcast.net
Cc: "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] Slide printers

[Ginny Achstetter via Histonet]

I need a recommendation on a slide printer. Tried the Leica and liked it but it 
only works well with their round edged slides and they aren't charged.  
Sent from my iPhone

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Re: [Histonet] picric acid

[Carol G Fields via Histonet]

Hi,
Do not touch it and call the bomb squad to dispose of it.  I have had several 
friends, including myself, find this on a back shelf in Histology and when they 
hauled it off and exploded it they said it would take out a city block. 
 I do not mess with it. it's not worth taking the chance.   Call 
the proper people to dispose of it for you.

Carole 
Los Angeles, CA
 

-Original Message-
From: Mca Werdler via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, May 05, 2016 12:09 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] picric acid

Dear histonetters,

Since a few months, i started working in a histology lab, run only by me ( 
coworkers are not specialized in histology). There has not worked here a person 
at histology for about 2 years.

After many new protocols, i decided to clear out some chemicals.
Now i found around 1 KG of DRY picric acid. I informed my coworkers about this, 
and they said just to dissolve everything in water.

What do you guys think is the best way for handeling with this explosive 
chemical? Thank you all in advance!

Maarten
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Re: [Histonet] PAS/Decal Question

[Marcum, Pamela A via Histonet]

We have no say in the decal procedure and I have been fighting this for 6 
years.  It is determined by our HemePath docs and when they want bone marrows 
completed for clinics the next day.  We have a 24 hour turnaround time, which 
is ridiculous however; we cannot not change it.  

The feedback we get now is the PAS stain on the bone marrows Levels 2 and 4 are 
not staining evenly.  They show a smudginess in some cells on the lower or 
level 4 section/ or they look over processed/cooked.  WE have done manual, 
instrument and now Dako Artisan (Dako has bend over backward to help) and 
nothing changes what they see on the slides.  They will not even consider 
changing the early steps as I have been told decal is fine (no its not) and 
processing is fine.  Funny thing H are always fine and only the PAS is a 
problem.  Just tired of it.  

This has been a losing fight for almost 5 months and I reached out to all of 
you in hopes it would help us make some changes.  Thank you for your 
suggestions unfortunately no one wants to hear about changes if they mean it is 
not a 24 hour turnaround time.  I am almost ready to just say "garbage in 
garbage out".  I feel very bad for the patients at this point.  I have not 
given up just trying to find a way to get change and better results.  

Thank You All for Your Suggestions!! 

Pam 

-Original Message-
From: Gudrun Lang [mailto:gu.l...@gmx.at] 
Sent: Thursday, May 05, 2016 2:19 AM
To: Marcum, Pamela A 
Cc: histonet@lists.utsouthwestern.edu
Subject: AW: [Histonet] PAS/Decal Question

Hi Pam,
my personal opinion is, that 2 hours fixation is too short for sufficient 
tissue-protection before acid decalcification. Formic acid at 50°C must have an 
impact on glycoproteins. Wether it is a kind of solving the "sugars" or 
beginning oxidation of the OH-groups (like periodic acid does in the 
PAS-reaction).

In our lab we do also acid decal with formic acid for at least 6 hours at RT, 
after one day in NBF. Our processing protocol is the routine-protocol over 
night. How thick are your BMT, also 3-4 mm?
In my opinion 4 hours are a challenge. Are the other stainings of the BMT 
optimal or show sometimes similar outcome? "Smudginess" reminds me of 
insufficient infiltration.

I also see that our PAS is not as bright as in the other specimens without 
decal. Sometimes it gives more the impression of a diastase-PAS. 

Gudrun

-Ursprüngliche Nachricht-
Von: Marcum, Pamela A via Histonet
[mailto:histonet@lists.utsouthwestern.edu]
Gesendet: Dienstag, 03. Mai 2016 18:39
An: histonet@lists.utsouthwestern.edu
Betreff: [Histonet] PAS/Decal Question

We are still having issues with our PAS stain on decaled bone marrows.  The 
Pathologists in HemePath are seeing what they refer to as smudginess in cells 
on some areas of the completed PAS slides.  We have looked at everything and 
cannot find where the issue is coming from at this point.  We have done manual 
staining for PAS, automated on the Leica stainer and on the Dako Artisan.  All 
methods show the same result for some slides.  We can go for several days to a 
week or more with no problem and then suddenly it is back and we have changed 
nothing in the way we do the processing, embedding, sectioning, 
deparaffinization and coverslipping.  We do as many as 38 bone marrow cores a 
night or as few as 8 and can find no correlation in the number we have to deal 
with for a given period.  All bone marrows drawn today must be completed by 8AM 
tomorrow morning.

Fixation after pulling the bone marrows is a minimum of 2 hours in AZF with a 
maximum of 7 hours +/-.

Grossed and placed in cassettes for 15 minute rinse in running DI Water

Decal currently in the Milestone Decal Unit for 45 minutes in Immunocal at 50C

Rinsed in running DI water for 15 minutes

Placed in 10% NBF and processed on a 4 hour program with a delay of 4 hours 
minimum to come off at 4:45AM.

If anyone knows of any literature on decal effects on PAS staining in bone 
marrows please contact me.  This has been going on for months and no matter 
what we do manual staining, Leica adaptation for automated or Dako it is not 
helping.  Dako has been great with sending in technical experts repeatedly and 
we cannot get this corrected.

Thanks,
Pam

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Re: [Histonet] picric acid

[Jay Lundgren via Histonet]

https://www.youtube.com/watch?v=IqTZNn0UHdA

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

On Thu, May 5, 2016 at 12:29 PM, STEVEN PINHEIRO via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Well you have the most important part correct- Keep it wet. But that
> doesn't address how to dispose of it. You could choose to make it non
> explosive by using sodium hydroxide and sulfide. But then its toxic and
> needs to be treated and removed accordingly. You can also list it as
> flammable and have it removed by the correctly licensed third party.
> Never pour down the drain as it could react w copper pipes. Many reasons
> to dispose of it properly.
>
> Steve Pinheiro
>
>
>
> -Original Message-
> From: Mca Werdler via Histonet [mailto:histonet@lists.utsouthwestern.edu]
> Sent: Thursday, May 05, 2016 2:09 PM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] picric acid
>
> Dear histonetters,
>
> Since a few months, i started working in a histology lab, run only by me (
> coworkers are not specialized in histology). There has not worked here a
> person at histology for about 2 years.
>
> After many new protocols, i decided to clear out some chemicals.
> Now i found around 1 KG of DRY picric acid. I informed my coworkers about
> this, and they said just to dissolve everything in water.
>
> What do you guys think is the best way for handeling with this explosive
> chemical? Thank you all in advance!
>
> Maarten
> ___
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>
> Confidentiality Notice:
> This e-mail, including any attachments is the property of Trinity Health
> and is intended for the sole use of the intended recipient(s). It may
> contain information that is privileged and confidential.  Any unauthorized
> review, use, disclosure, or distribution is prohibited. If you are not the
> intended recipient, please delete this message, and reply to the sender
> regarding the error in a separate email.
>
>
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Re: [Histonet] PAS Stain

[Anne Murvosh via Histonet]

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,  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" 
>> *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
>> ___
>> Histonet mailing list
>> Histonet@lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
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>
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Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732) 235-4583; fax: -4029
mcaul...@rwjms.rutgers.edu
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Re: [Histonet] PAS Stain

[Ingles Claire via Histonet]

The level of amylase in your saliva also depends on when you ate last. If 
you're spitting on slides after you just ate, the reaction will be weaker as 
the amylase will have been used up on lunch digestion. (also, you need to be 
careful about having your lunch salad contaminate the slide... :)
Claire


From: Ray via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Thursday, May 05, 2016 1:48 PM
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" 
To: koelli...@comcast.net
Cc: "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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Re: [Histonet] PAS Stain

[Geoff via Histonet]

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,  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" 
*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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--
--
**
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Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732) 235-4583; fax: -4029
mcaul...@rwjms.rutgers.edu
**



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Re: [Histonet] PAS Stain

[Anne Murvosh via Histonet]

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,  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" 
> *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
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
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Re: [Histonet] picric acid

[STEVEN PINHEIRO via Histonet]

Well you have the most important part correct- Keep it wet. But that doesn't 
address how to dispose of it. You could choose to make it non explosive by 
using sodium hydroxide and sulfide. But then its toxic and needs to be treated 
and removed accordingly. You can also list it as flammable and have it removed 
by the correctly licensed third party.
Never pour down the drain as it could react w copper pipes. Many reasons to 
dispose of it properly.

Steve Pinheiro



-Original Message-
From: Mca Werdler via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, May 05, 2016 2:09 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] picric acid

Dear histonetters,

Since a few months, i started working in a histology lab, run only by me ( 
coworkers are not specialized in histology). There has not worked here a person 
at histology for about 2 years.

After many new protocols, i decided to clear out some chemicals.
Now i found around 1 KG of DRY picric acid. I informed my coworkers about this, 
and they said just to dissolve everything in water.

What do you guys think is the best way for handeling with this explosive 
chemical? Thank you all in advance!

Maarten
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information that is privileged and confidential.  Any unauthorized review, use, 
disclosure, or distribution is prohibited. If you are not the intended 
recipient, please delete this message, and reply to the sender regarding the 
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[Histonet] picric acid

[Mca Werdler via Histonet]

Dear histonetters,

Since a few months, i started working in a histology lab, run only by me (
coworkers are not specialized in histology). There has not worked here a
person at histology for about 2 years.

After many new protocols, i decided to clear out some chemicals.
Now i found around 1 KG of DRY picric acid. I informed my coworkers about
this, and they said just to dissolve everything in water.

What do you guys think is the best way for handeling with this explosive
chemical? Thank you all in advance!

Maarten
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[Histonet] Grossing of oriented skin biopsies and small lymph nodes

[Vickroy, James via Histonet]

Currently our non-pathologists do not gross oriented skin excisions or small 
lymph nodes.   I do understand that CAP requires a list of tissues that can be 
handled by non-pathologists but am wondering how others are handling these 
specimens.

I know there are labs, for example  some large dermatopathology labs that are 
having the oriented skin biopsies grossed by techs.   Of course a accepted 
protocol has to be listed for the dissection.

Let me know how your institution are handling these specimens.

Jim

Jim Vickroy
Histology Manager
Springfield Clinic, Main Campus, East Building
1025 South 6th Street
Springfield, Illinois  62703
Office:  217-528-7541, Ext. 15121
Email:  jvick...@springfieldclinic.com



This electronic message contains information from Springfield Clinic, LLP that 
may be confidential, privileged, and/or sensitive. This information is intended 
for the use of the individual(s) or entity(ies) named above. If you are not the 
intended recipient, be aware that disclosure, copying, distribution, or action 
taken on the contents of this information is strictly prohibited. If you have 
received this electronic message in error, please notify the sender 
immediately, by electronic mail, so that arrangements may be made for the 
retrieval of this electronic message. Thank you.
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Re: [Histonet] PAS Stain

[Ray via Histonet]

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"  
To: koelli...@comcast.net 
Cc: "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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Re: [Histonet] PAS Stain

[Ray via Histonet]

Hello 
  
Agree with comments about temperature of 60 degrees killing an enzyme.  If you 
plot enzyme activity on "y"  axis and temperature on "x" axis it is not a 
straight line.  Every enzyme has an optimal temperature and can function slowly 
at non-optimal or optimally at correct temperature.  Diastase/amylase from your 
body work at 37 degrees, less so or dead at RT or 60 degrees.  Nuking a mixture 
to 37 degrees might nuke protein.  Nuke buffer to 37, verify temp and add the 
enzyme. 
  
pH-if you plot enzyme activity on "y" axis and pH on x-axis, it is not a 
straight line.  There is an optimal pH for each enzyme.  The enzyme might work 
fine at different pH; just not nearly as well. 
  
I know some still use 0.5% or 1.0% in di water.  I'd advise not to.  Use a 
buffer at a given pH.  "pHíng" di or distilled water with ordinary lab 
electrodes is an act of futility.  From a standard lab electrode point of view, 
there is NO pH to read.  Not enough ions present so meter might say anything.  
If anything, it will pick up CO2 from air which becomes carbonic acid and meter 
will flail wildly in acidic region.  But that is not a stable buffer.  di water 
might work.  Just that sometimes, it might not.  Why chance it?  di water has 
unstable (no reliably readable) pH. 
  
Ray Koelling, golfing in Spokane WA and enjoying the low humidity 

- Original Message -

From: "Joanne Clark via Histonet"  
To: histonet@lists.utsouthwestern.edu 
Sent: Wednesday, May 4, 2016 1:02:48 PM 
Subject: [Histonet] PAS Stain 

Hi Histonetters, I'm hoping someone can help me troubleshoot our PAS diastase 
method.  We have been digesting the tissue in 0.5% diastase of malt in a 60 
degree oven for 30 minutes, but do not see the glycogen being digested out.  I 
have tried alpha amylase and beta amylase also without any luck.  Does anyone 
have any suggestions to get the digestion to work 

Joanne Clark, BAAS, HT(ASCP)CM 
Director of Histology 

P.   (575) 622-5600 
C.   (575) 317-6403 
F.   (575) 622-3720 
TF. (800) 753-7284 

pcnm.com 




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Re: [Histonet] PAS Stain

[Jennifer MacDonald via Histonet]

We switched form malt diastase to alpha amylase and have seen a 
significant improvement in digestion, but as others have said enzyme 
activity will be destroyed at 60 degrees C. We put ours into a 37 degree C 
water bath.  Enzymes work optimally at 37C, body temperature.  As the 
temperature rises above 37C you will see a decrease in enzyme activity and 
then none at all.
Jennifer



From:   Joanne Clark via Histonet 
To: "histonet@lists.utsouthwestern.edu" 

Date:   05/04/2016 01:04 PM
Subject:[Histonet] PAS Stain



Hi Histonetters, I'm hoping someone can help me troubleshoot our PAS 
diastase method.  We have been digesting the tissue in 0.5% diastase of 
malt in a 60 degree oven for 30 minutes, but do not see the glycogen being 
digested out.  I have tried alpha amylase and beta amylase also without 
any luck.  Does anyone have any suggestions to get the digestion to work

Joanne Clark, BAAS, HT(ASCP)CM
Director of Histology

P.   (575) 622-5600
C.   (575) 317-6403
F.   (575) 622-3720
TF. (800) 753-7284

pcnm.com




Disclaimer: This electronic message may contain information that is 
proprietary,
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the use
of the individual(s) and entity named in the message. If you are not an 
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recipient of this message, please notify the sender immediately and delete 
the
material from your computer. Do not deliver, distribute or copy this 
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do not disclose its contents or take any action in reliance on the 
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Re: [Histonet] PAS Stain

[Bob Richmond via Histonet]

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,  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" 
> *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
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
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Re: [Histonet] PAS Stain

[Ray via Histonet]

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 

- Original Message -

From: "Bob Richmond via Histonet"  
To: "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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Re: [Histonet] PAS Stain

[Bob Richmond via Histonet]

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] Stat Lab IHC's

[Charles Riley via Histonet]

Does anyone have experience using any of STATLAb's new antibodies or their
new IHC platform?   Can you please tell me what you think the advantages
and disadvantages are to their products?

-- 

Charles Riley HT(ASCP)CM

Histopathology Coordinator/ Mohs
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[Histonet] Animals Need Histologist Too!

[Sandra Cheasty via Histonet]

Hello All!
We are looking for a dedicated Histologist, with at least 1 year of experience 
in a full service histology lab, to join our Veterinary Pathology team. Our 
Histology lab serves the Veterinary Medical Teaching Hospital in Madison, 
Wisconsin, as well as many research needs at the School of Veterinary Medicine. 
We are a full service lab providing processing, embedding, microtomy, 
histochemical and immunohistochemical staining.
Full-time, Days, Monday through Friday
You can reply to me directly, or follow the link below to apply.
http://www.ohr.wisc.edu/Weblisting/External/PDSummaryApply.aspx?vacid=97933=35062
Thank you!
Sandy
Sandra J. Cheasty, HT (ASCP)
Histology & Necropsy Supervisor
UW-Madison, School of Veterinary Medicine

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[Histonet] Open invite

[Travis Herndon via Histonet]

Hello Histonet users,

I would like to welcome anyone who is in the Minneapolis MN area to visit our 
facilities in Ramsey MN.
Even though I have seen some back and forth tension on here. Overall it seems 
like a great group of people that are passionate about their work.
This would not be a  pressure sales thing (we are not going to trap you in a 
room and throw why we are great at you).
Just a chance to see who GMI is as a company and what we do. If you're in the 
area let me know.

Kind regards,
Travis Herndon
Technical Sales Consultant
GMI

thern...@gmi-inc.com | www.gmi-inc.com
Advancing Science with Affordable Solutions
ISO 9001:2008 Certified

T: 763 712-8717 ext. 6826 | F: 763 712-8724
Cell:612-803-1820
www.linkedin.com/in/travisherndon

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[Histonet] pas diastase

[Fortune, James A. via Histonet]

We use a .5% to 1% solution of diastase for our fungal pas (so the docs can see 
the fungus better) and leave it in for 25-30 min at room temp and have had 
great results.  We use American mastertech brand of diastase.

Andy Fortune
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Re: [Histonet] replacing a stainer and a coverslipper question

[Rene J Buesa via Histonet]

Productivity and quality sake, Sakura film coverslipper has no match. If you 
use Sakura tape and the xylene dispenser is properly calibrated, storage is not 
an issue.Sakura stainer was also what I used at my lab and I highly recommend 
both.René 

On Wednesday, May 4, 2016 3:19 PM, Jenn via Histonet 
 wrote:
 

 Dear Histonetters,
We are going to replace our automated stainer (LeicaXL) and coverslipper within 
the next few months.  I would really appreciate any suggestions that people may 
have about which stainer/coverslipper units they use/used and are happy with.

I am also curious about tape vs glass coverslippers.  We have always had glass 
here, but I know that there are people who love their tape units.  I would be 
willing to look at a tape unit, but I am concerned about long term storage 
issues.  I have seen a few postings lately about the tape coming off of the 
slides and that scares me!  I would appreciate any advice on what brands work 
well and which ones to avoid, if you are willing to share...

If you have any insight that you would like to share, you can post it or e-mail 
me directly, especially if it is about which units/tapes to avoid.  I don't 
want to vendor bash, so please feel free to email me directly  at 
jennifer.john...@genzyme.com
Thanks!
Jenn

Jennifer Johnson, B.S., HTL (ASCP)

Scientist
Department of Pathology
TEL.: 508-271-3610  -  Fax.: 508-872-9080
5 The Mountain Road, Framingham, MA 01701-9322
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Re: [Histonet] PAS Stain

[Manfre, Philip via Histonet]

We switched to alpha amylase since the diastase we were ordering was not 
working.  We use a 2% solution of alpha amylase for 40 minutes at room 
temperature and it has been working fine.

Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com



-Original Message-
From: Joanne Clark via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, May 04, 2016 4:03 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] PAS Stain

Hi Histonetters, I'm hoping someone can help me troubleshoot our PAS diastase 
method.  We have been digesting the tissue in 0.5% diastase of malt in a 60 
degree oven for 30 minutes, but do not see the glycogen being digested out.  I 
have tried alpha amylase and beta amylase also without any luck.  Does anyone 
have any suggestions to get the digestion to work

Joanne Clark, BAAS, HT(ASCP)CM
Director of Histology

P.   (575) 622-5600
C.   (575) 317-6403
F.   (575) 622-3720
TF. (800) 753-7284

pcnm.com




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Re: [Histonet] PAS/Decal Question

[Gudrun Lang via Histonet]

Hi Pam,
my personal opinion is, that 2 hours fixation is too short for sufficient
tissue-protection before acid decalcification. Formic acid at 50°C must have
an impact on glycoproteins. Wether it is a kind of solving the "sugars" or
beginning oxidation of the OH-groups (like periodic acid does in the
PAS-reaction).

In our lab we do also acid decal with formic acid for at least 6 hours at
RT, after one day in NBF. Our processing protocol is the routine-protocol
over night. How thick are your BMT, also 3-4 mm?
In my opinion 4 hours are a challenge. Are the other stainings of the BMT
optimal or show sometimes similar outcome? "Smudginess" reminds me of
insufficient infiltration.

I also see that our PAS is not as bright as in the other specimens without
decal. Sometimes it gives more the impression of a diastase-PAS. 

Gudrun

-Ursprüngliche Nachricht-
Von: Marcum, Pamela A via Histonet
[mailto:histonet@lists.utsouthwestern.edu] 
Gesendet: Dienstag, 03. Mai 2016 18:39
An: histonet@lists.utsouthwestern.edu
Betreff: [Histonet] PAS/Decal Question

We are still having issues with our PAS stain on decaled bone marrows.  The
Pathologists in HemePath are seeing what they refer to as smudginess in
cells on some areas of the completed PAS slides.  We have looked at
everything and cannot find where the issue is coming from at this point.  We
have done manual staining for PAS, automated on the Leica stainer and on the
Dako Artisan.  All methods show the same result for some slides.  We can go
for several days to a week or more with no problem and then suddenly it is
back and we have changed nothing in the way we do the processing, embedding,
sectioning, deparaffinization and coverslipping.  We do as many as 38 bone
marrow cores a night or as few as 8 and can find no correlation in the
number we have to deal with for a given period.  All bone marrows drawn
today must be completed by 8AM tomorrow morning.

Fixation after pulling the bone marrows is a minimum of 2 hours in AZF with
a maximum of 7 hours +/-.

Grossed and placed in cassettes for 15 minute rinse in running DI Water

Decal currently in the Milestone Decal Unit for 45 minutes in Immunocal at
50C

Rinsed in running DI water for 15 minutes

Placed in 10% NBF and processed on a 4 hour program with a delay of 4 hours
minimum to come off at 4:45AM.

If anyone knows of any literature on decal effects on PAS staining in bone
marrows please contact me.  This has been going on for months and no matter
what we do manual staining, Leica adaptation for automated or Dako it is not
helping.  Dako has been great with sending in technical experts repeatedly
and we cannot get this corrected.

Thanks,
Pam

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Re: [Histonet] PAS Stain

[Gudrun Lang via Histonet]

As far as I remember the incubation temperature is at roomtemperature. 60°C
would rather denature the native enzyme than increase activity.

Look at the optimal working temp of the reagens you have bought.

regards
Gudrun

-Ursprüngliche Nachricht-
Von: Joanne Clark via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Gesendet: Mittwoch, 04. Mai 2016 22:03
An: histonet@lists.utsouthwestern.edu
Betreff: [Histonet] PAS Stain

Hi Histonetters, I'm hoping someone can help me troubleshoot our PAS
diastase method.  We have been digesting the tissue in 0.5% diastase of malt
in a 60 degree oven for 30 minutes, but do not see the glycogen being
digested out.  I have tried alpha amylase and beta amylase also without any
luck.  Does anyone have any suggestions to get the digestion to work

Joanne Clark, BAAS, HT(ASCP)CM
Director of Histology

P.   (575) 622-5600
C.   (575) 317-6403
F.   (575) 622-3720
TF. (800) 753-7284

pcnm.com




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