Re: Daily Digest
On Mon, 12 Aug 2002 00:00:42 -0500 (CDT) HistoNet Server
writes:
>
Hi Barbara Stancel,
I saw your posting and thought I should respond as I may be of some help.
I have been using PLP fixative(periodate-lysine-paraformaldehyde) for
about 20 years. It is a wonderful fixative but has gotten little
recognition. It is great for preserving antigenicity and is even great
for EM. If have had great results fixing eyes as well. You can leave
tissues in it indefinitely and still have good immunostaining and EM
results. Fifteen years is my maximum test. That should be long enough for
most studies. I cannot guarantee that it will work with all monoclonal
antibodies but it works with most. I have always had good luck with
polyclonals. Antigen retrieval is usually necessary but with the DAKO
antigen retrieval solution, results are often as good a cryosections.
Don't even consider trying to get samples out of Russia without
fixation. The samples will undoubtedly get hung up by some bureaucrat
someplace or held at an airport security office or get diverted on the
wrong flight. I have had a lot of experience with such snafu's working
as I do in a foreign animal disease lab. That even happens with samples
from developed countries in the EU.
The only down side of PLP fixative is that it should be made up
relatively close to the time of use. I have stored it for up to two weeks
at 4C and it still was OK. It can also be frozen for a month. It turns
yellow when frozen but as long as it is clear when it is thawed, it is
OK.
The theory of PLP is that lysine binds to sulfhydro groups and
decreases cross-linking by the paraformaldehyde. The periodate apparently
oxidizes S-H groups too. The paraformaldehyde is 2% (half the strength
of 10% formalin-4%).
What antibodies are you going to use? Have they worked in fresh
formalin? If they can work in freshly fixed formalin samples, they will
work as well or better in PLP. My experience is that many antigens can be
stained (with retrieval) in formalin after a brief fixation time but
antigen is lost in weeks to months. Formalin continues to cross link over
time and retrieval may fail.. PLP does not have this problem.
PLP was first published for use in immunoelectron microscopy by
McLean and Nakane. The ref. is:
McLean IW, Nakane PK.Related Articles
Periodate-lysine-paraformaldehyde fixative. A new fixation for
immunoelectron microscopy.
J Histochem Cytochem. 1974 Dec;22(12):1077-83. No abstract available.
PMID: 4374474 [PubMed - indexed for MEDLINE]
The formula is a bit hard to extract from this article. Here is my method
that has worked for years. It makes one liter. Scale up or down for more
or less.
Make it up in three parts:
Part A:
Paraformaldehyde powder 20 grams
10 pellets of Sodium hydroxide to aid dissolving
400 ml DH2O
Dissolve with stirring. Heating is not necessary if paraformaldehyde is
powdered. It can be heated if necessary but must be cooled before mixing
with parts B and C.
Part B: buffers
Na2H2PO4 0.73 grams
Na2HPO4 anhydrous 8.52 grams
L-Lysine 13.7 grams
400 ml DH2O
Part C:
Na periodate 2.13 grams
100 ml H2O
Dissolve all three parts (takes about 10 minutes) with stirring. Mix
together at room temperature in order ( A,B, and then C) with stirring.
QS to one liter. The pH should end up about neutral. I don't test it
anymore.
Use fresh if possible or store at 4C until it turns yellowish-about 1-2
weeks or freeze -20C for a month or two. It will look yellow when frozen
but will clear when thawed. If not, discard it and make fresh.
The fixative will turn yellow after tissue is fixed. That is fine. It may
be a month before this happens.Tissues should be stored in PLP and
embedded as needed from the wet tissue. I have had best results with
freshly embedded paraffin blocks. I have lost some antigens in a month
after embedding in paraffin. I don't know why but re-embedding tissue
from wet fixed tissue (even after years) has worked as well as freshly
fixed tissue.
Douglas Gregg DVM PhD
Veterinary pathologist
Plum Island Animal Disease Center
Greenport, NY 11944
d.gregg@juno.com
> ----------------------------------------------------------------------
>
> Date: 11 Aug 2002 04:31:10 -0500
> From: Barbara Stancel
> Subject: IHC, tissues & shipping
>
> Dear Histonet friends,
>
> We are considering a special project involving IHC to be done on
> tissues
> from a remote area of the world! Specifically how to get them here
> quickly
> and while preserving the antigencity (sp). We have considered
> shipping in
> formalin and formalin-fixed-70%ETOH-stored. But shipping could take
>
> weeks...or longer. Hand carried tissues may get hung up in customs.
> The
> pathologist would like to somehow process the tissue. The tech who
> usually
> handles these questions is unavailable. I do not perform IHC myself.
> (I am
> feeling a little like a fish flopping on the dock!)
>
> I have begun looking and calling about international shippers and
> carriers
> and their regulations, but I believe getting advice from people who
> daily
> handle these types of shipping circumstances is the most practical
> approach.
>
> Does anyone have any experience with samples for IHC being shipped
> from the
> Russian area of the world? What is the best method for shipment and
> by what
> carrier?
>
> Also, I have been asked to look for a laboratory in Kazakhstan or
> Moscow or
> the Moscow area that processes human, veterinary, or plant tissues.
>
> Any advice on the tissue preservation, carriers, or locating a lab
> would be
> most appreciated.
>
> Histologically yours,
>
> Barbara H. Stancel, HTL(ASCP)HT
> Pathology, RRC
> Athens, Georgia 30604
> phone: (706) 546-3556
> fax: (706) 546-3589
>
>
>
>
> _________________________________________________________________
> Chat with friends online, try MSN Messenger:
> http://messenger.msn.com
>
>
>
> ----------------------------------------------------------------------
>
> Date: 11 Aug 2002 19:00:59 -0500
> From: "Diane G. Miller"
> Subject: Michelle Silverman
>
> Hi,
>
> I'm trying to contact Michelle Silverman, she use to work for Biotek
> before
> Ventana bought them out. Please forward my information to her if
> you're in
> contact with her.
>
> Thanks
> Diane
>
>
>
> ----------------------------------------------------------------------
>
> Date: 11 Aug 2002 19:15:55 -0500
> From: Bernard Ian R SSgt 59 CRES/MSROP
>
> Subject: Need help finding portable vacuum dessicator
>
> I need a purchase source for a good vacuum dessicator that has a
> control
> stop cock to allow pressure in and shuts it off to maintain
> pressure. This
> dessicator will then be place in a 4 deg C fridge for a number of
> days. I
> hope to maintain the same vacuum pressure all through out while in
> the
> refrigerator. Any ideas? (a source will be nice).
>
> Boring a hole in the fridge to maintain pressure is not an option.
> I will
> be using a plastic/monomer organic solvent(Methyl Methacrylate) in
> the
> dessicator. It needs to be under vacuum pressure.
>
> Thanks histonetters(especially in plastic)
>
>
> ----------------------------------------------------------------------
>
> Date: 11 Aug 2002 19:30:35 -0500
> From: Melissa Jensen
> Subject: Re: Histotech's grossing
>
>
> By Clia standards..Histotechs can only gross in if they have a B.A.
> ----- Original Message -----
> From: Barbara.Davies@memhospcs.org
> To: Jason Fowler
> Cc: histonet@pathology.swmed.edu
> Sent: Wednesday, August 07, 2002 2:22 PM
> Subject: Re: Histotech's grossing
>
>
>
> The histotechs gross in tissue, frankly we do 70% of the gross
> dictation,,,,we have it very clear what type/diagnosis that histo
> techs do and
> what type our PA does. Histo does all biopsies, currettings,
> shavings,
> skins which are not oriented, gallbladders, appendix, some
> placentas, some
> uteri. The PA does the rest. We vary from 70 to 100 cases per
> day.
>
> Barb Davies
> Memorial Hospital
> Colorado Springs, CO
>
>
>
> The mission of Memorial Hospital is to optimize the health and
> comfort of
> our patients, their families and the community we serve by providing
> the best
> and most sensitive professional care and service.
>
> www.memorialhospital.com
>
>
>
> The information contained in or attached to this electronic
> message is
> privileged and confidential, intended only for the use of the
> individual(s)
> named above. If the reader of this message is not the intended
> recipient, or
> the employee or agent responsible to deliver it to the intended
> recipient, you
> are hereby notified that any dissemination, distribution, or copying
> of this
> communication is strictly prohibited. If you have received this
> communication
> in error, please inform the sender immediately and remove any record
> of this
> message.
>
>
>
>
> Jason Fowler
>
>
>
>
>
>
>
>
> Jason Fowler
>
> 08/07/2002 11:24 AM
>
>
> To: histonet@pathology.swmed.edu
> cc:
> Subject: Histotech's grossing
>
>
>
> I have a couple of questions for everyone in histology land. How
> many
> of you are required to do specimen grossing, and if you do, what
> types
> of specimens do you gross? If you have a PA who does some
> grossing, I'm
> curious to know that too.
> Thanks,
> Jason
>
> Jason C. Fowler, PA
> Clinical Instructor
> Department of Pathology
> West Virginia University School of Medicine
> Morgantown, WV 26506
> 304-598-4130 phone
> 304-293-6249 fax
> jfowler@hsc.wvu.edu
> >>> Merkey Marjorie 08/07/02
> 13:20
> PM >>>
> Good Afternoon:
> We are aware that some procedures offer quick mordanting/time
> savings by
> heating Bouin's in the microwave and are seeking more information
> from
> labs
> who have been doing this routinely. We are concerned about the
> picric
> acid
> explosions hazard. If you heat Bouin's in the microwave, we would
> like
> to
> know the safe temperature range, possible time/power setting, and
> the
> amount
> of time sections should sit in heated Bouin's for mordanting
> before
> continuing with the stain.
> Thanks
>
> Marjorie Merkey
> Clinical Support Manager
> HCA Richmond Hospital Laboratories
> office: 804-327-4075 pager: 759-6942
> marjorie.merkey@hcahealthcare.com
>
>
>
>
>
>
>
>
>
>
> ******************* NOTE *******************
> There may be important message content
> contained in the following MIME Information.
> ********************************************
>
>
> - ------------------ MIME Information follows ------------------
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> - --Boundary_(ID_BuQUeLBOTLskH1jIe1UWpQ)
> Content-type: text/plain; charset=iso-8859-1
> Content-transfer-encoding: 7BIT
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> <<<<<< See above "Message Body" >>>>>>
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>
>
>
>
>
>
> By Clia standards..Histotechs can only gross in if
> they have
> a
> B.A.
> style="PADDING-RIGHT: 0px; PADDING-LEFT: 5px; MARGIN-LEFT: 5px;
> BORDER-LEFT:
> #000000 2px solid; MARGIN-RIGHT: 0px">
> ----- Original Message -----
>
>
>
> Sent: Wednesday, August 07,
> 2002 2:22
> PM
> Subject: Re: Histotech's
> grossing
>
The histotechs
> gross in
> tissue, frankly we do 70% of the gross dictation,,,,we have it
> very clear
> what
> type/diagnosis that histo techs do and what type our PA does.
> Histo
> does all biopsies, currettings, shavings, skins which are
> not
> oriented,
> gallbladders, appendix, some placentas, some uteri. The PA
> does the
> rest. We vary from 70 to 100 cases per day.
>
face=sans-serif size=2>Barb Davies
face=sans-serif
> size=2>Memorial Hospital
size=2>Colorado
> Springs, CO
>
>
>
color=#808080
> size=1>Jason Fowler <jfowler@hsc.wvu.edu>
>
>
>
> | size=2>
>
> |
> |
> |
> |
> |
> |
> |
> |
> |
> |
> |
> |
> |
> | |
>
>
>
> |
> | Jason Fowler
>
> <jfowler@hsc.wvu.edu>
> 08/07/2002 11:24 AM
>
> |
>
>
>
> To:
>
> histonet@pathology.swmed.edu
>
face=sans-serif size=1> cc:
>
>
>
> Subject:
> Histotech's
> grossing |
size=2>I
> have a
> couple of questions for everyone in histology land. How
> many
of you
>
> are required to do specimen grossing, and if you do, what
> types
of
> specimens do you gross? If you have a PA who does some
> grossing,
> I'm
curious to know that
> too.
Thanks,
Jason
size=2>Jason C. Fowler, PA
Clinical
> Instructor
Department of
> Pathology
West Virginia University School of
> Medicine
Morgantown, WV
> 26506
304-598-4130 phone
304-293-6249
> fax
jfowler@hsc.wvu.edu
>>> Merkey Marjorie
> <Marjorie.Merkey@HCAHealthcare.com> 08/07/02 13:20
PM
> >>>
Good Afternoon:
We are aware that some
> procedures offer
> quick mordanting/time savings by
heating Bouin's in the
> microwave and are
>
> seeking more information from
labs
who have been doing this
> routinely.
>
> We are concerned about the picric
acid
explosions hazard. If
> you heat
> Bouin's in the microwave, we would like
to
know the safe
> temperature
> range, possible time/power setting, and the
amount
of time
> sections
> should sit in heated Bouin's for mordanting before
continuing
> with the
> stain.
Thanks
Marjorie
> Merkey
Clinical Support Manager
HCA Richmond Hospital
> Laboratories
office: 804-327-4075 pager:
>
>
759-6942
marjorie.merkey@hcahealthcare.com
>
> - --Boundary_(ID_BuQUeLBOTLskH1jIe1UWpQ)--
>
>
> ----------------------------------------------------------------------
>
> Date: 11 Aug 2002 19:31:03 -0500
> From: Bernard Ian R SSgt 59 CRES/MSROP
>
> Subject: FW: Need help finding portable vacuum dessicator PART 11
>
> It does not have to be a dessicator. A vaccum chamber that will hold
> vaccum
> for days with a stopcock that is portable and can go in the fridge
> as well.
>
> > -----Original Message-----
> > From: Bernard Ian R SSgt 59 CRES/MSROP
> > Sent: Sunday, August 11, 2002 6:49 PM
> > To: 'histonet@pathology.swmed.edu'
> > Subject: Need help finding portable vacuum dessicator
> >
> > I need a purchase source for a good vacuum dessicator that has a
> control
> > stop cock to allow pressure in and shuts it off to maintain
> pressure.
> > This dessicator will then be place in a 4 deg C fridge for a
> number of
> > days. I hope to maintain the same vacuum pressure all through out
> while
> > in the refrigerator. Any ideas? (a source will be nice).
> >
> > Boring a hole in the fridge to maintain pressure is not an option.
> I will
> > be using a plastic/monomer organic solvent(Methyl Methacrylate) in
> the
> > dessicator. It needs to be under vacuum pressure.
> >
> > Thanks histonetters(especially in plastic)
>
>
> ----------------------------------------------------------------------
>
> Date: 11 Aug 2002 19:40:17 -0500
> From: Melissa Jensen
> Subject: Fw: Social Security
>
> The certification ..certificate I received for passing my
> H.T....Didn't have
> my SS number on it...Only a number they gave me...I would think if
> they can
> assign a random number to certification,they could issue a random
> number for
> applicants...Just like we do when receive specimens.Everyone has a
> number
> unique to there case..No SS...SS is only used for billing
>
> - ----- Original Message -----
> From: "Manju Kaushal"
> To:
> Sent: Thursday, August 08, 2002 8:49 AM
> Subject: Social Security
>
>
> > Yes it is annoying to put your SSN & yes we can fight it.
> > But NSH is not the place to fight an issue of why are we required
> to
> > put SSN in NSH or all those other places.
> > Let us fight the battle worth fighting for on the right grounds.
> > Let the system of SSN change outside the NSH when it does I am
> sure it
> > will change in NSH too but until than on this platform we should
> raise
> > more scientific & histotechs related issues than SSN.Are we
> running out
> > of those issues?
> >
> >
> > Manju Kaushal
> > Senior Manager
> > Immunopathology Service
> > Johns Hopkins Medical System
> > Weinberg Building - Rm 2323
> > 401 North Broadway
> > Baltimore MD 21287
> > PH 410 614 6650
> > Fax 410 502 1732
> >
> >
> >
>
>
>
> Here are the messages received yesterday!
>
>
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