Discussion:
CAP checklikst for pathology lab
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Пешков Максим
2015-06-30 03:42:17 UTC
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Dear histonetters, can you send me, pleasse, the last pdf version cap checklist for pathology lab? Or reference where I can download it.
Sincerely,
Maxim Peshkov,
Russia,
Taganrog.
Whitaker, Bonnie
2015-06-30 10:22:19 UTC
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The checklists are now customized, and your version must be downloaded from CAP.org.

Good luck!

Bonnie



Sent with Good (www.good.com)


-----Original Message-----
From: Пешков Максим [***@mail.ru<mailto:***@mail.ru>]
Sent: Monday, June 29, 2015 11:43 PM Eastern Standard Time
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] CAP checklikst for pathology lab


Dear histonetters, can you send me, pleasse, the last pdf version cap checklist for pathology lab? Or reference where I can download it.
Sincerely,
Maxim Peshkov,
Russia,
Taganrog.
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Suzanne Martin
2015-06-30 18:37:10 UTC
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Hi all,

We are having trouble troubleshooting our trichrome. It is too blue. We are using Leica's kit with the Weigerts iron with Gills. Most of the small bowel controls have seen improvement but patient tissue is not... strange.

We have tried lessening the time in Gills, adding time for the last acid step, even lessening time and adding time in the Weigerts.


Thoughts?

Thank you.

Suzanne HT
Elizabeth Chlipala
2015-06-30 20:29:56 UTC
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Suzanne

How many times have you used the kit and reagents, I did look up how the kit works but the trichrome stain can be tricky. First of all you need to make sure that the mordant (bouins solution) is at 60C prior to placing your slides in them. We normally heat up our bouins for at least an hour prior to placing the slides in the solution. We leave in bouins for an hour and a half rather than an hour. I see that this is a microwave protocol I cannot comment on that but I don't think that the hematoxylin is the issue, if you leave longer in 1% acetic acid that may pull some of the blue stain out or I would try dehydrating with lower alcohol percentage that can pull some of the blue stain out. I would also try leaving it a bit longer in the bouins after you microwave it - that might help.

Trichrome staining works best with fresh reagents so if you have used these reagents too much that could cause problems. I'm also not a big fan of the one step trichromes, they are quicker but sometimes not as good as the two steps, just my opinion.

FYI - to evaluate your staining look for a smaller vessel, the smooth muscle should be nice a red, if its greyish or blue you have not done the stain properly. Good Luck

Liz

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

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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


-----Original Message-----
From: Suzanne Martin [mailto:***@lcpath.com]
Sent: Tuesday, June 30, 2015 12:37 PM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] Trichrome troubleshooting


Hi all,

We are having trouble troubleshooting our trichrome. It is too blue. We are using Leica's kit with the Weigerts iron with Gills. Most of the small bowel controls have seen improvement but patient tissue is not... strange.

We have tried lessening the time in Gills, adding time for the last acid step, even lessening time and adding time in the Weigerts.


Thoughts?

Thank you.

Suzanne HT
Gayle Callis
2015-06-30 21:05:39 UTC
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Years ago, I was taught by Jerry Fredenburg, a stain guru, never to
microwave the Bouins step but do this for 1 hour at 60C or overnight at RT.
The post-fixation/mordant is very important in order to acidify the
connective tissue fibers properly for a trichrome stain, and the short time
in MW will not do the job. Liz is correct here in letting the sections
stand longer in Bouins after microwaving or simply just do the 1 hour at
60C.

I have removed Gills type hematoxylins from nuclei by over exposure to
acetic acids so remember that ALL Masson's Trichrome reagents are acidified
with acetic acid and will automatically do this, even on Weigert's Iron
hematoxylin. We also examined our sections microscopically during
staining to make sure the check the depth of red staining reagents on smooth
muscle is correct - once again, Liz is correct about this fact.

Gayle Callis

-----Original Message-----
From: Elizabeth Chlipala [mailto:***@premierlab.com]
Sent: Tuesday, June 30, 2015 2:30 PM
To: Suzanne Martin; ***@lists.utsouthwestern.edu
Subject: Re: [Histonet] Trichrome troubleshooting

Suzanne

How many times have you used the kit and reagents, I did look up how the kit
works but the trichrome stain can be tricky. First of all you need to make
sure that the mordant (bouins solution) is at 60C prior to placing your
slides in them. We normally heat up our bouins for at least an hour prior
to placing the slides in the solution. We leave in bouins for an hour and a
half rather than an hour. I see that this is a microwave protocol I cannot
comment on that but I don't think that the hematoxylin is the issue, if you
leave longer in 1% acetic acid that may pull some of the blue stain out or I
would try dehydrating with lower alcohol percentage that can pull some of
the blue stain out. I would also try leaving it a bit longer in the bouins
after you microwave it - that might help.

Trichrome staining works best with fresh reagents so if you have used these
reagents too much that could cause problems. I'm also not a big fan of the
one step trichromes, they are quicker but sometimes not as good as the two
steps, just my opinion.

FYI - to evaluate your staining look for a smaller vessel, the smooth muscle
should be nice a red, if its greyish or blue you have not done the stain
properly. Good Luck

Liz

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

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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


-----Original Message-----
From: Suzanne Martin [mailto:***@lcpath.com]
Sent: Tuesday, June 30, 2015 12:37 PM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] Trichrome troubleshooting


Hi all,

We are having trouble troubleshooting our trichrome. It is too blue. We are
using Leica's kit with the Weigerts iron with Gills. Most of the small bowel
controls have seen improvement but patient tissue is not... strange.

We have tried lessening the time in Gills, adding time for the last acid
step, even lessening time and adding time in the Weigerts.


Thoughts?

Thank you.

Suzanne HT




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Bernice Frederick
2015-07-01 12:28:06 UTC
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Actually, I have microwaved the Bouins (and still do) for small numbers of slides and the results are the same. I ran a liver bx both ways as well as larger tissue to compare. I use the 10 slide plastic coplin jar and generally have 5 or less slides when I do this. One microwaves the Bouins for 30 seconds on power level 7 in a lab grade microwave. A higher level will cause the Bouin's to spill. Slides are then added and left for 5 minutes. Excess rinsed out and then proceed as per your SOP. As for the blue- I rinse out excess and do 1 dip in 1% GAA. Rinse and dehydrate (10 dips each solution)
Bernice

Bernice Frederick HTL (ASCP)
Senior Research Tech
Pathology Core Facility
Robert. H. Lurie Cancer Center
Northwestern University
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
312-503-3723
b-***@northwestern.edu

-----Original Message-----
From: Elizabeth Chlipala [mailto:***@premierlab.com]
Sent: Tuesday, June 30, 2015 3:30 PM
To: Suzanne Martin; ***@lists.utsouthwestern.edu
Subject: Re: [Histonet] Trichrome troubleshooting

Suzanne

How many times have you used the kit and reagents, I did look up how the kit works but the trichrome stain can be tricky. First of all you need to make sure that the mordant (bouins solution) is at 60C prior to placing your slides in them. We normally heat up our bouins for at least an hour prior to placing the slides in the solution. We leave in bouins for an hour and a half rather than an hour. I see that this is a microwave protocol I cannot comment on that but I don't think that the hematoxylin is the issue, if you leave longer in 1% acetic acid that may pull some of the blue stain out or I would try dehydrating with lower alcohol percentage that can pull some of the blue stain out. I would also try leaving it a bit longer in the bouins after you microwave it - that might help.

Trichrome staining works best with fresh reagents so if you have used these reagents too much that could cause problems. I'm also not a big fan of the one step trichromes, they are quicker but sometimes not as good as the two steps, just my opinion.

FYI - to evaluate your staining look for a smaller vessel, the smooth muscle should be nice a red, if its greyish or blue you have not done the stain properly. Good Luck

Liz

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

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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


-----Original Message-----
From: Suzanne Martin [mailto:***@lcpath.com]
Sent: Tuesday, June 30, 2015 12:37 PM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] Trichrome troubleshooting


Hi all,

We are having trouble troubleshooting our trichrome. It is too blue. We are using Leica's kit with the Weigerts iron with Gills. Most of the small bowel controls have seen improvement but patient tissue is not... strange.

We have tried lessening the time in Gills, adding time for the last acid step, even lessening time and adding time in the Weigerts.


Thoughts?

Thank you.

Suzanne HT




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http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Gayle Callis
2015-06-30 20:33:09 UTC
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Suzanne,

I don't understand what you wrote here " We are using Leica's kit with the
Weigerts iron with Gills". In a long histo career, I never used Gills half
oxidized hematoxylin for a trichrome stain nor heard of the combined
staining you mentioned. Could you explain what you are doing here? Is
this part of the Leica staining protocol? This is a new one for me to
combine Gills hematoxylin with Weigerts iron hematoxylin and why it would
even be necessary??? Both of Leica's trichromes kits use Weigerts Iron
hematoxylin and do NOT have Gills in the method. Their kits look more like
classic Mass Tri methods. Personally I would only use Weigerts Iron
hematoxylin in a classic Trichrome method. It is important to know
that iron hematoxylin is not stable over a long period of time since the
ferric chloride continues to oxidize the hematoxylin so it becomes very weak
or doesn't stain. We never used iron hematoxylin for more than one day
although some people may have success over a week. We never took a chance
in having weak staining with that continued oxidation by ferric chloride,
and only used freshly mixed iron hematoxylin for a day.

I would be happy to personally send you a modified Weigerts Iron hematoxylin
that does NOT differentiate out so the iron hematoxylin staining is always
excellent. It was found in a J of Histotechnology publication many years
ago, and is made up in house - even using other trichrome reagents from the
above vendors I mentioned. The modified Weigerts iron hematoxylin is more
concentrated compared to the original iron hematoxylin recipe.

Gayle M. Callis
HTL/HT/MT(ASCP)



-----Original Message-----
From: Suzanne Martin [mailto:***@lcpath.com]
Sent: Tuesday, June 30, 2015 12:37 PM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] Trichrome troubleshooting


Hi all,

We are having trouble troubleshooting our trichrome. It is too blue. We are
using Leica's kit with the Weigerts iron with Gills. Most of the small bowel
controls have seen improvement but patient tissue is not... strange.

We have tried lessening the time in Gills, adding time for the last acid
step, even lessening time and adding time in the Weigerts.


Thoughts?

Thank you.

Suzanne HT
Michael Ann Jones
2015-06-30 20:51:43 UTC
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Raw Message
Don¹t know if this matters, but we have found that the trichrome stain
fades on tissues that are not cut fresh.
We use uterus as a control but they must be cut fresh, no batching this
one.
The trichrome is not as brilliant on pre-cut tissues.
Just my 2 cents :)

Michael Ann
Michael Ann Jones, HT (ASCP)
Histology Manager
Metropath
7444 W. Alaska Dr. #250
Lakewood, CO 80226
303.634.2511
Post by Suzanne Martin
Suzanne
How many times have you used the kit and reagents, I did look up how the
kit works but the trichrome stain can be tricky. First of all you need
to make sure that the mordant (bouins solution) is at 60C prior to
placing your slides in them. We normally heat up our bouins for at least
an hour prior to placing the slides in the solution. We leave in bouins
for an hour and a half rather than an hour. I see that this is a
microwave protocol I cannot comment on that but I don't think that the
hematoxylin is the issue, if you leave longer in 1% acetic acid that may
pull some of the blue stain out or I would try dehydrating with lower
alcohol percentage that can pull some of the blue stain out. I would
also try leaving it a bit longer in the bouins after you microwave it -
that might help.
Trichrome staining works best with fresh reagents so if you have used
these reagents too much that could cause problems. I'm also not a big
fan of the one step trichromes, they are quicker but sometimes not as
good as the two steps, just my opinion.
FYI - to evaluate your staining look for a smaller vessel, the smooth
muscle should be nice a red, if its greyish or blue you have not done the
stain properly. Good Luck
Liz
Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
www.premierlab.com
March 10, 2014 is Histotechnology Professionals Day
Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504
-----Original Message-----
Sent: Tuesday, June 30, 2015 12:37 PM
Subject: [Histonet] Trichrome troubleshooting
Hi all,
We are having trouble troubleshooting our trichrome. It is too blue. We
are using Leica's kit with the Weigerts iron with Gills. Most of the
small bowel controls have seen improvement but patient tissue is not...
strange.
We have tried lessening the time in Gills, adding time for the last acid
step, even lessening time and adding time in the Weigerts.
Thoughts?
Thank you.
Suzanne HT
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Histonet mailing list
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
_______________________________________________
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Terri Braud
2015-07-01 17:20:29 UTC
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Please clarify if you are referring to the Aniline blue, or the Hematoxylin blue. I, also, have never heard of using Gills in association with any Trichrome stain, but as a long time user and educator of using a one-step (Gomori's) trichrome stain (which is the Leica blue collagen stain), if you rinse with any water between the stain step and the acetic acid step, you will end up with a stain that is overpoweringly blue.
Here are the steps copied from the Leica blue collagen kit instructions. Notice NO WATER RINSE between the stain and Acetic Acid Solution. It is a common mistake.
10. Gently mix the Gomori's Trichrome Blue Solution by swirling and allow to stand for 6 minutes.
11. Place sections in 1% Acetic Acid solution for 1 minute.
I hope this helps. Sincerely, Terri

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Holy Redeemer Hospital Laboratory
1648 Huntingdon Pike
Meadowbrook, PA 19046
Ph: 215-938-3676
Fax: 215-938-3874

2. Trichrome troubleshooting (Suzanne Martin)
From: "Suzanne Martin" <***@lcpath.com>
Hi all,

We are having trouble troubleshooting our trichrome. It is too blue. We are using Leica's kit with the Weigerts iron with Gills. Most of the small bowel controls have seen improvement but patient tissue is not... strange.

We have tried lessening the time in Gills, adding time for the last acid step, even lessening time and adding time in the Weigerts.
Thoughts?
Thank you.
Suzanne HT

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