Immunohistochemistry in Bone Marrow Aspirates
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From: | "Hall, Phil" <Phil.Hall@ubht.swest.nhs.uk> |
To: | "'HistoNet Server'" <HistoNet@Pathology.swmed.edu> |
Reply-To: | |
Date: | Sat, 26 Jun 1999 09:32:15 +0100 |
Content-Type: | text/plain; charset="iso-8859-1" |
Dear Jennifer,
We fix in 4% formal saline for 6 - 8 hours (usually overnight) and then
decalcify 6 - 8 hours @37*C in the following EDTA solution:
125g Sodium EDTA
875ml Distilled water
Mix. This gives a cloudy solution.
Add 13.25g sodium hydroxide (pellets) to give a clear solution at pH 7.2
To the best of my knowledge we have had no trouble demonstrating
heamopoietic cell lines or neuroblastoma with immunohistochemistry.
Phil Hall
Paediatric Pathology
St Michael's Hospital
BRISTOL
UK
----------
From: HistoNet Server[SMTP:HistoNet@pathology.swmed.edu]
Sent: 26 June 1999 06:06
To: HistoNet Server
Subject: Daily Digest
----------------------------------------------------------------------
Date: 25 Jun 1999 04:46:15 -0500
From: "Tony Henwood" <henwood@mail.one.net.au>
Subject: Re: AFP Control Tissue
I have found that liver tissue containing a hepatocellular carcinoma
can be a good control if you can get it.
NB Not all HCC's produce AFP, look for one that is associated with
hepatitis B (ie IPX or Mod.Orcein positive in associated benign
liver).
Regards Tony
.
Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital
Sydney, AUSTRALIA
http://www2.one.net.au/~henwood
http://www.pathsearch.com/homepages/TonyHenwood/default.html
----------------------------------------------------------------------
Date: 25 Jun 1999 04:46:43 -0500
From: "Tony Henwood" <henwood@mail.one.net.au>
Subject: RE: Bone IHC
> Date: Sat, 19 Jun 1999 10:34:15 -0600
> From: Patsy.Ruegg@UCHSC.edu
>
>" coat plus slides with a 5% mixture of elmer's white glue, mix
with
dionized water. stand the slides up and allow to air dry."
Elmer's white glue is known in Australia as Aquadere. See:
http://www2.one.net.au/~henwood
Regards, Tony
.
Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital
Sydney, AUSTRALIA
http://www2.one.net.au/~henwood
http://www.pathsearch.com/homepages/TonyHenwood/default.html
----------------------------------------------------------------------
Date: 25 Jun 1999 04:47:09 -0500
From: "Tony Henwood" <henwood@mail.one.net.au>
Subject: Re: water bath method for antigen retrieval. -Reply
Since water boils at less than 100oC, you may have problems with
antigen retrieval (try recording the temperature when the water is
boiling- it can be as low as 96oC!). If you replace the water in the
water bath with oil or PEG (Can't remember the exact solution), you
can obtain a temperature higher than 100oC. This should give you a
chance of consistent antigen retrieval.
This was a trick we used when we used to do the protein binding
radioassay for B12 and folate many years ago (in my biochem days)
Regards, Tony
.
Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital
Sydney, AUSTRALIA
http://www2.one.net.au/~henwood
http://www.pathsearch.com/homepages/TonyHenwood/default.html
----------------------------------------------------------------------
Date: 25 Jun 1999 04:47:39 -0500
From: "Tony Henwood" <henwood@mail.one.net.au>
Subject: Re: stain for crypto
The following procedure may help,
LAZCANOS ETAL COMBINED SILVER-MUCIN STAIN FOR CRYPTOCCUS NEOFORMANS
NOTES:
This combined stain distinctively demonstrates both cell wall and
capsule of
most cryptococci. More organisms are identified than when each stain
was used
individually. The cell wall is stained dark brown to black and the
capsule was
stained blue.
SOLUTIONS:
1. Methenamine Silver Solution (see Grocotts Methenemine Silver).
2. 0.2% Gold Chloride.
3. 5% Sodium Thiosulphate.
4. 3% Acetic Acid.
5. 1% Alcian Blue in 3% Acetic Acid.
6. Kernechtrott.
STAINING PROCEDURE:
1. Dewax and hydrate sections.
2. Rinse slides well in distilled water.
3. Place in preheated Methenamine Silver Solution at 60oC for
15-30 minutes.
Rinse slides in distilled water and observe if understained and
check every 5
minutes.
4. Rinse slides in distilled water.
5. Gold Chloride 1 minute.
6. Rinse in distilled water.
7. Sodium Thiosulphate 1 minute.
8. Rinse in running water.
9. Rinse in 3% Acetic acid.
10. Stain in Alcian Blue 20 minutes.
11. Rinse in acetic acid.
12. Wash in water.
13. Counterstain in Kernechtrott 3 minutes.
14 Wash in water, dehydrate, clear and mount.
REFERENCE:
Lazcano, O., etal (1991) Arch. Pathol. Lab. Med 115:1145-1149.
NOTE:
Since Alcian Blue also stains connective tissue mucins, Mucicarmine
can be
used in such instances where ground substance may obscure the
results.
Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital
Sydney, AUSTRALIA
http://www2.one.net.au/~henwood
http://www.pathsearch.com/homepages/TonyHenwood/default.html
----------------------------------------------------------------------
Date: 25 Jun 1999 07:31:12 -0500
From: Susan Walzer <slw_histo@yahoo.com>
Subject: Peutz-Jagert ?
Does anyone know what this is? Boy, do I hate to come in to work in
the
morning and find a request for something I have never heard of!
Thanks, Susan
_________________________________________________________
Do You Yahoo!?
Get your free @yahoo.com address at http://mail.yahoo.com
----------------------------------------------------------------------
Date: 25 Jun 1999 07:47:35 -0500
From: Tim Morken <timcdc@hotmail.com>
Subject: Tony, thanks for the web page!
tony,
thanks for posting your web page address. It looks very interesting.
- ----Original Message Follows----
From: "Tony Henwood" <henwood@mail.one.net.au>
To: jim.manavis@imvs.sa.gov.au, Patsy.Ruegg@UCHSC.edu
CC: histonet@Pathology.swmed.edu
Subject: RE: Bone IHC
Date: Fri, 25 Jun 1999 19:41:22 +0000
> Date: Sat, 19 Jun 1999 10:34:15 -0600
> From: Patsy.Ruegg@UCHSC.edu
>
>" coat plus slides with a 5% mixture of elmer's white glue, mix
with
dionized water. stand the slides up and allow to air dry."
Elmer's white glue is known in Australia as Aquadere. See:
http://www2.one.net.au/~henwood
Regards, Tony
..
Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital
Sydney, AUSTRALIA
http://www2.one.net.au/~henwood
http://www.pathsearch.com/homepages/TonyHenwood/default.html
_______________________________________________________________
Get Free Email and Do More On The Web. Visit http://www.msn.com
----------------------------------------------------------------------
Date: 25 Jun 1999 09:45:41 -0500
From: "Instrumedics, Inc." <info@instrumedics.com>
Subject: freezing core biopsy
Hi Timothy,
We have had very good results freezing core biopsies in the
Gentle-Jane
Snap Freezer. We dispense a small amount of CryoGel(or OCT) on the
specimen
holder and freeze to make a "safety" layer. This will assure that
the
specimen will not freeze into the surface of the specimen holder.
Then we dispense another small amount of Gel on the frozen "safety"
layer
and orient the core(s) on the top of the gel. The heat extractor
that has
been stored in LN2 and it equilibrated to -196C is placed on the
Gentle-Jane
device. It falls at a controlled rate and contacts the cores
first(best
freezing) and in 8-10 sec. the cores and the embedding medium are
snap
frozen . The block is flat with the cores uppermost in the block
which
minimizes trimming to reveal a full face. This snap freezing method
depends
not only on the low temperature of LN2. but also on good thermal
exchange
that results from the highly polished chrome-plated 3/4 lb heat
extractor.
The morphology of the cores has been excellent with this method.
Bernice
schiller@instrumedics.com
----------------------------------------------------------------------
Date: 25 Jun 1999 10:31:10 -0500
From: vandeplas@aurion.nl (Peter van de Plas)
Subject: Re:cell pellets for IHC/ISH
Brett Connoly wrote:
I looking for advice on proper fixing/freezing of cell pellets for
cryostat
sectioning and subsequent fluorescent IHC and ISH. These will be
separate
populations of monocytes, PMNs, eosinophils, and lymphocytes from
peripheral
blood and we'll be staining for monos (CD68), PMNs (CD18,) T & B
cell
markers, other various cytokines and chemokine receptors. Your
suggestions/secrets and protocols will be greatly appreciated.
Should 4%
paraformaldehyde be avoided?
- -------------------------------------------------------
Dear Brett,
In ISH you can use, in general, aldehyde fixation. Your probe will
still
recognize target DNA or RNA. A problem that might arise is
accessibility of
the specimen when using a cross-linking fixative like
glutaraldehyde. In
ISH your primary antibody will determine the fixation protocol that
can be
used. In my opinion the supplier should include information on
fixation
compatibillity in the package insert.
The most convenient way to handle single cells/cell pellets is by
embedding
them in gelatin. I always use a concentration of 10 to 20% in PBS.
If this
concentration has a negative effect on signal intensity (masking)
you have
to go down with the concentration (2-5%).
Embedding must be homogeneous and cells evenly distributed to achive
an
optimum effect. After fixation (if this is compatible with your
primary)
the cell pellet is brought on top of a 37 oC gelatin/PBS solution
and spun
down. Remove as much gelatin as possible. Resuspend to obtain a
loosly
packed cell pellet. The gelatin is soldified on ice. Best handling
and
sectioning properties are achieved when a second fixation with
(glutar)aldehyde is possible (again depending on primary antibody).
Remove
the gelatin/cell pellet from the tube. Optionally the pellet can be
cryoprotected in sucrose, otherwise directly submerged in e.g. LN2.
Hope this is of help, Peter
========================================
Peter van de Plas
AURION
Costerweg 5
6702 AA Wageningen
The Netherlands
phone: (31)-317-497676
fax: (31)-317-415955
http://www.aurion.nl
----------------------------------------------------------------------
Date: 25 Jun 1999 14:15:36 -0500
From: jennifer.hoover@pharma.Novartis.com
Subject: Immunohistochemistry in Bone Marrow Aspirates??
Hi Histonetters! Any suggestions for a good protocol to fix
and
decalcify
bone marrow aspirates prior to processing and embedding in paraffin
for
subsequent immunohistochemistry. I have not worked with bone/bone
marrow in
the
past; although I did consult Sheehan's Histotechnology book and
realize that
most conventional decalcifying methods may destroy endogenous enzyme
activity.
Any thoughts or ideas on the subject??? Thank you very much as
always!!
Jennifer Hoover
Histologist
Transplantation Biology
Novartis Pharmaceuticals
Summit, NJ
----------------------------------------------------------------------
Date: 25 Jun 1999 14:36:51 -0500
From: maliniakrm@worldnet.att.net
Subject: Uneven Keratin Staining
We are currently experiencing uneven...areas of Positive and areas
of
Negative immunostaining on skin....with our DAKO keratin AE1/AE3.
Protocol: primary antibody at 1:100 for 30 min at room temp, pepsin
pretreat(4mg pepsin/ml buffer, incubate 37C for 30 minutes),LSAB+
detector system(30 minutes at room temp)on DAKO autoimmunostainer.
Our skins are formalin fixed for an assorted amount of times. Any
suggestions?
Thanks in Advance.
----------------------------------------------------------------------
Date: 25 Jun 1999 15:17:15 -0500
From: "Jennings-Siena, Debbie"
<ds.jennings-siena@baylordallas.edu>
Subject: RE: CPT codes, is their a website or anything?
Hi Victoria,
I am the NSH Legislative Chair person and I have been checking into
a lot of
CPT codes. If you have a histology related question I can try to
help out
with the answer. I also have a committee of very qualified people
who will
also address your questions. As far as CPT code references the
American
Medical Association is the organization that is over CPT codes and
if you go
to their web site or call them they have books of the most recent
CPT codes
that can be purchased. The site is
http://www.ama-assn.org/ <http://www.ama-assn.org/> and the phone
number is
312-464-5930. If we can be of assistance, please do not hesitate
to let
me know.
Debbie Jennings-Siena
214-820-2465
-----Original Message-----
From: Victoria Baker [SMTP:vbaker60@yahoo.com]
Sent: Thursday, June 24, 1999 1:17 AM
To: HistoNet Server; Paul Kwan
Subject: CPT codes, is their a website or anything?
Hi Everyone!
I'm currently setting up a lab in NYC, we are also setting
up a very
new computer system. This system does a lot, but it runs on
the
"input" the "user" has. Given this I was handed the
computer
companys'
CPT coding list. As I read through it,, I came across some
items
that
I'm sort of unsure of. I'd like to find out where I can
either get
an
un-biased copy of CPT codes, or if there is a website that
has this
information that I can accurately check what I'm reading
from the
computer company.
As always I appreciate any assistance I can get. Thanks
much.
Vikki Baker
_________________________________________________________
Do You Yahoo!?
Get your free @yahoo.com address at http://mail.yahoo.com
----------------------------------------------------------------------
Date: 25 Jun 1999 15:38:40 -0500
From: "John C. Dennis" <dennijc@vetmed.auburn.edu>
Subject: Re: Uneven Keratin Staining
Richard
I, too, have problems from time to time with uneven cytokeratin
signal. I
use DAKO's Z0622, which is a polyclonal wide spectrum antibody.
I would say that the problem is remedied with fresh antibody (until
recently I've not been keeping alloquoits in the -80 but only in the
4
deg.) but I've recently seen the phenomenon with other antibodies.
I've experimented with various detergents, no detergent,
paraformaldehyde
fixed tissue, Bouin's fixed tissue, standing on my head and etc.
I don't know yet. I'm starting an assay tomorrow with fresh
antibody so I
can let you know sunday what fresh antibody does, at the least.
More later
John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL 36849
On Fri, 25 Jun 1999, Richard Maliniak wrote:
> We are currently experiencing uneven...areas of Positive and areas
of
> Negative immunostaining on skin....with our DAKO keratin AE1/AE3.
> Protocol: primary antibody at 1:100 for 30 min at room temp,
pepsin
> pretreat(4mg pepsin/ml buffer, incubate 37C for 30 minutes),LSAB+
> detector system(30 minutes at room temp)on DAKO autoimmunostainer.
> Our skins are formalin fixed for an assorted amount of times. Any
> suggestions?
>
> Thanks in Advance.
>
----------------------------------------------------------------------
Date: 25 Jun 1999 17:01:21 -0500
From: angelrj@email.uc.edu
Subject: Lexington Job Posting
So sorry to trouble you, but could you please post the job opening
that
was available in Lexington, Ky on the histonet again? I thought I
had
transferred the message into my folder, but I couldn't find it.
Also if you could send me more detailed info about the job to my
personal email address I would be grateful.
Thank you,
Rita Angel
----------------------------------------------------------------------
Date: 25 Jun 1999 18:15:45 -0500
From: "Michelle M. Sutorik" <istsmmmc@umich.edu>
Subject: re:no messages today!
Hey everybody,
Has anyone received any messages today from Histonet. I certainly
have
not. Is the server down?
Michelle M. Sutorik
Univ. of Michigan
----------------------------------------------------------------------
Date: 25 Jun 1999 19:01:17 -0500
From: vdella@path.som.sunysb.edu
Subject: AP MANAGER POSITION AVAILABLE (repeat post)
this is a repeat post
I would like to inform my friends and colleagues in the histology
community that I am leaving my position here at the University
Hospital & Medical Center at Stony Brook University in New York to
join the staff at the Medical University of South Carolina in
Charleston in late July.
Anyone wishing to reach me there may do so at that time by email at
dellav@musc.edu
or by telephone (843) 792-6553
My goal prior to leaving a terrific technical staff behind is to
help
to find an experienced and accomplished manager who will be a
resource to them. The posting for my position follows. Anyone having
questions about this position may feel free to contact me back
channel.
Immediate Opening !!!!!
Assoc. Technical Director for Anatomic Pathology (AP Manager)
The Stony Brook University Hospital & Medical Center, Long Island's
premiere tertiary care Hospital and School of Medicine located on
Suffolk County's beautiful north shore, has an immediate opening for
an experienced manager and technical supervisor for its Anatomic
Pathology Laboratories. Situated just 75 minutes east of New York
City, the campus' geographical location offers access to the east
end's vacation playgrounds as well as New York City's finest
cultural
events.
The successful candidate for this position will be a self-starter
experienced in managing a diversified group of technical
specialists
in Cytology, Surgical Pathology, Immunohistochemistry, Electron
Microscopy as well as the Autopsy Service.
The preferred candidate will possess technical expertise in one or
more of these disciplines, as well as a proven track record in day
to
day operations management, maintaining compliance with all
laboratory
accrediting agencies and billing compliance issues,
familiarity with QA and CQI activities in a busy academic AP
department, preparation and oversight of operational budgets
totaling
approximately $ 1.5 million annually, experience with a laboratory
information system as it applies to AP laboratory operations needs,
all human resource functions supervising a staff of about 30 and
sufficient expertise to permit the evaluation and implementation of
new methodologies, programs and instrumentation to ensure that the
highest standards of excellence are maintained for the delivery of
patient care at the University Hospital.
A B.S. degree in Medical Technology, or one of the biological
sciences
with a minimum of six years experience in a clinical laboratory
setting is required, four years of which must be in the specialty
area
of Histology or Cytology, at least two of which have been in a
supervisory capacity. Relevant registry certification ( ASCP) or
registry eligible and experience with a Laboratory Information
System
is also required. A masters degree and experience in a teaching
hospital laboratory are strongly preferred.
Salary for this challenging position is commensurate with experience
and prior accomplishments.
Interested candidates should submit a detailed curriculum vita
outlining relevant experience and accomplishments to:
Nancy Moran, Laboratory Administrator
University Hospital & Medical Center
Department of Laboratories
Stony Brook, New York 11794-7300
******************************************************************
Vinnie Della Speranza
Technical Director
Anatomic Pathology Laboratories
University Hospital & Medical Center
State University of New York at Stony Brook 11794-7025
(516) 444-8249
fax: (516) 444-3419
vdella@path.som.sunysb.edu
----------------------------------------------------------------------
Date: 25 Jun 1999 19:01:48 -0500
From: "Cindy Higgerson" <chiggerson@memhosp.com>
Subject: AP Computer Systems
Hi Histonetters!
We are starting to look at AP computer systems (we are currently
doing
everything manually) and would appreciate any input. The systems we
are
considering are PowerPath 2000 (Tamtron), CoPath (Dynamic), Window
Path
(Psyche), Soft Path (SCC), Cortex (Cortex Medical Management), and
the
new LabFusion module (Keane). We would like to hear both positives
and
negatives on the above systems and any others that may be worth
looking
into.
Thanks!!
Cindy Higgerson
Department of Surgical Pathology
Memorial Hospital
Belleville, Illinois
(618) 257-5227
fax (618) 257-6868
chiggerson@memhosp.com
----------------------------------------------------------------------
Date: 25 Jun 1999 22:01:04 -0500
From: burch007@mc.duke.edu
Subject: Re[2]: Uneven Keratin Staining
Try the MNF116 cytokeratin from Dako for skin's. Our dermpath
attendings like
a cocktail of AE1/AE3, MNF116 and CAM5.2.
The MNF116 works well after digesting with 0.25% pepsin, pH 2.0.
JB
_______________________ Reply Separator _______________________
Subject: Re: Uneven Keratin Staining
Author: dennijc@vetmed.auburn.edu at internet
Date: 6/25/99 4:23 PM
Richard
I, too, have problems from time to time with uneven cytokeratin
signal. I
use DAKO's Z0622, which is a polyclonal wide spectrum antibody.
I would say that the problem is remedied with fresh antibody (until
recently I've not been keeping alloquoits in the -80 but only in the
4
deg.) but I've recently seen the phenomenon with other antibodies.
I've experimented with various detergents, no detergent,
paraformaldehyde
fixed tissue, Bouin's fixed tissue, standing on my head and etc.
I don't know yet. I'm starting an assay tomorrow with fresh
antibody so I
can let you know sunday what fresh antibody does, at the least.
More later
John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL 36849
On Fri, 25 Jun 1999, Richard Maliniak wrote:
> We are currently experiencing uneven...areas of Positive and areas
of
> Negative immunostaining on skin....with our DAKO keratin AE1/AE3.
> Protocol: primary antibody at 1:100 for 30 min at room temp,
pepsin
> pretreat(4mg pepsin/ml buffer, incubate 37C for 30 minutes),LSAB+
> detector system(30 minutes at room temp)on DAKO autoimmunostainer.
> Our skins are formalin fixed for an assorted amount of times. Any
> suggestions?
>
> Thanks in Advance.
>
Here are the messages received yesterday!
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