Immunohistochemistry in Bone Marrow Aspirates

<< Previous Message | Next Message >>
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!
	



<< Previous Message | Next Message >>