[Histonet] FW: Formalin collection at grossing stations

[Gagnon, Eric via Histonet]

Jim,

Our PA's collect formalin after each specimen, dumping it into a waste 10-litre 
carboy. Most of the waste containers are in a small lab sink beside each 
grossing area. A funnel positioned in the neck of the carboy not only helps 
channel the formalin, it also keeps the container mostly closed-off re: fumes.

The empty containers are also bagged, and occasionally the bags need to be 
opened and checked for labelling or other issues that come up.

Hope this helps,

Eric Gagnon, MLT
Histology Laboratory
Kingston General Hospital,
Kingston, Ontario, Canada

From: Vickroy, James via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Tuesday, September 12, 2017 4:45 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Formalin collection at grossing stations

We have been told by our chemical waste company that we no longer can discard 
small biopsy containers with formalin in them.  In our workflow process we 
gross small specimens.  We generally use forceps to take the small biopsies 
from the container of formalin and reseal the container with the residual 
formalin.  The container is then discarded in a red bag and held for two days 
incase we have to go back for labeling questions.  Rarely do we have to go back 
to make sure a specimen was not left in a container. (example three fragments 
grossed and the clinician said there were four fragments).  Large amounts of 
formalin from our tissue processors is recycled so the only formalin that has 
been going in the biohazard waste has been what's in the prefilled containers.  
However, I know it adds up.   We have two options that seem to make the most 
sense.  We can collect and recycle the residual formalin from the containers or 
we can collect and neutralize the formalin.   Since we alr!
 eady recycle it seems logical to recycle when we can however at some point we 
will have an abundance and will be forced to neutralize.

Here is my questions:

Do your grossing techs collect the formalin as they gross each specimen or do 
they leave the containers with the residual formalin in them for a period of 
time?  We will still keep the empty containers for two days whether we dump the 
formalin while we are grossing or have to go back to them two days later just 
in case there are labeling questions.

If they collect the formalin as they gross each specimen is there any special 
way they have come up with to keep the formalin fumes down besides removing a 
lid of a waste container each time they discard the formalin.   I realize that 
grossing is done under the hoods however, I'm not in favor of an open container 
of formalin in the grossing station.   It elevates exposure amounts and 
expensive filters become exhausted much faster.  One of my staff members 
immediately went into McGiver mode to come up with a method however I'm not 
sure we aren't overthinking this process.   Your thoughts?

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



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[Histonet] Section adherence issues (IHC)

[Gagnon, Eric via Histonet]

Hi Greg,

Permit me a few thoughts on factors not mentioned in your email:

  *   Do your slides 'look' baked/is the wax melted when they are removed from 
the oven?
  *   Also, is there variance in your oven temperature?
  *   Especially vexing is the fact that control sections are lifting. Are the 
control sections pre-baked or at time of cutting the ER/PR patient sections?
  *   Are the sections completely dry before covertiles are placed on top of 
the slides in the Leica staining rack?
  *   Has Leica checked the instrument to rule out any instrument-related 
issues? Is the poor adherence occurring in one particular location within the 
instrument, or in various slide positions?

Fortunately, special stains and routine staining rarely seem to be affected by 
the various pH/mechanical/detection system variables that exist in IHC.

In our experience with Bond-III, areas of poor adherence tend to be 
longitudinally lifting/up 'the middle' of the slide and are usually resolved by 
leaving slides of only such problem tissues (as you mentioned) for longer times 
in the oven than normal.

Hope this helps,
Eric Gagnon MLT
Kingston General Hospital
Kingston, ON



We have a Bond-III immunostainer and we use the Leica Apex charged slides
for our IHC stains. We have no additives in our water baths. Our sections
drain in a stand and then any trapped water under the section is either
flicked or wicked away as needed prior to placing the slides in a rack in a
60 C oven for 30 mins prior to staining.  We do not do on board baking
(primarily because on board baking is only 10 mins long and with the
section lifting issue we want longer).

Some of the specimen types are more susceptible it would seem. Cervix LEEP
specimens tend to be bad, we use a 4mm punch to obtain some of our control
tissue and so the breast tissue we use for Myosin Heavy Chain seems to be a
bad one and sometimes our ER/PR control sections (but not always). Fine
needle cores where a lot of tumour is present tend to be bad (again not
always).

We have tried baking longer, we turn ourselves inside out trying to get all
of the water out from under the sections, we have tried charged slides from
another manufacturer. I have looked at the HIER protocols and none are
extraordinary in nature. We use very clean covertiles (no scratches or
blemishes). Our specimens are fixed for 24hrs before processing. We use the
same slides for Special Stains and don't have this issue there.

I need some new suggestions to try! All ideas welcome.
Cheers,
Greg

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Re: [Histonet] ER/PR Uneven Staining

[Gagnon, Eric via Histonet]

As Greg has already  mentioned, placing control sections on each patient slide 
will assist in troubleshooting your staining issue. Joanne, I don't believe you 
mentioned it in your initial email, but did you attempt a repeat and if so, did 
the results replicate on the repeat? Occasionally there may be a mixing issue 
or other physical issue with the covertile that inhibits complete reaction 
across the slide.

Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital,
Kingston, Ontario, Canada


From: Joanne Clark via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Tuesday, November 29, 2016 4:09 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] ER/PR Uneven Staining

I had a breast needle core today that when stained with ER and PR the staining 
was uneven throughout the core, even though the cancer cells were present in 
the entire core.  The specimen had 10 hours fixation in 10% NBF.  I could 
understand the uneven staining from inadequate fixation on large grossed in 
breast tissue, but 10 hours with  needle core biopsies has always been more 
than sufficient.  Does anyone have any ideas?  We use Leica's ER and PR 
antibodies on the BOND.



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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[Histonet] White Benchkote product

[Gagnon, Eric via Histonet]

Is anyone in Histoland using white bench-covering products, either in roll form 
or in sheets? (These are white, shiny-coated on one side, and have the 
consistency of filter paper on the other.) In the past, we have taped them to 
benchtops to provide a clean look.



Are these still in widespread use, and if so, have you encountered any 
infection control issues arising from their use i.e. during inspections or with 
your IC folks?



Thanks in advance,



Eric Gagnon MLT

Histology Laboratory

Kingston General Hospital

Kingston, Ontario, Canada


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[Histonet] automated special stains and control tissue

[Gagnon, Eric via Histonet]

Agree with your opinion re: including special stain control tissue and patient 
tissue on same slide, Curt.



Assuming you're referring to FFPE tissue, there is little if any chance of the 
control tissue contaminating the patient tissue on the same slide, thus causing 
a 'false positive' in the patient tissue. (Even if it did, a good pathologist 
could tell it was not native tissue to that section and was sitting 'on top' of 
the patient section :)



Including control and patient on the same slide for automated special staining 
IS a process change and may take some time, commitment and training to 
implement, but for the same reasons that it makes sense in IHC, it makes sense 
in automated special staining. A side benefit to confidence in the correct 
dispensing of reagent on each slide is the saving in slides, reagents, and room 
on your stainer that fewer independent control slides would represent.



Perhaps try some test runs to instill confidence in this process change before 
implementing.



Eric Gagnon, MLT

Histology Laboratory

Kingston General Hospital

Kingston, Ontario, Canada






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[Histonet] bx levels

[Gagnon, Eric via Histonet]

We currently place 3 ribbons of 5-6 sections, at each of three levels, 'down' 
the slide. This may be too many sections for the pathologists' needs. A 
suggestion for us going forward would be as Lacy Normington suggests - one or 
two sections at three or four levels, also, placing these short ribbons 
'across' the slide. Currently with long biopsies (over 5 mm) we are unable to 
fit three ribbons of such longer biopsies on one slide, necessitating the use 
(and cost, and storage) of a second slide.



Eric Gagnon MLT

Histology Laboratory

Kingston General Hospital

Kingston, Ontario, Canada


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[Histonet] Fixation before Decalcification?

[Gagnon, Eric]

What are you placing tissue blocks in before decalcification? We place blocks 
for decal in a jar of Bouin's Fixative from any time on Day 1, for specimens 
received in 10% NBF, until decalcification starts on Day 2. In other words, we 
gross specimens received in 10% NBF, then fix overnight in Bouin's, then begin 
decalcification the next morning. Two reasons for this - a decades-old 
reference, plus we've always done it that way. We manually transfer the 
Bouin's-fixed blocks through graded alcohols to water, then use RDO as our 
decalcifying solution, then place in 10% NBF until blocks loaded on processor.



But, going with the current everything is on the table approach in our 
laboratory, we are looking closely at pre-decalcification fixation of Bone 
Marrow Biopsies in 10% NBF (previously Dacie's then AZF) and we are pulling 
back the focus to treating larger decal blocks i.e. femoral head sections and 
other bone specimens, in the same way.



Interested in your thoughts on pre-decalcification fixation



Eric Gagnon MLT

Histology Laboratory

Kingston General Hospital

Kingston, Ontario, Canada


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[Histonet] Kidney bx transport

[Gagnon, Eric]

We receive kidney biopsies brought directly to our grossing area within a few 
minutes after the cores are obtained. Previously, this was done by 
nephrologists, but since the renal biopsies are performed in radiology, the 
interventional radiologist brings them. We decide adequacy first, then 
apportion specimen to EM, IF and formalin.



Similar to the protocol Tim has mentioned, our cores are received on 
saline-soaked pads in a petri dish. We transfer them to a slide wetted with 
saline to assess under light microscopy.



It seems to come down to a decision of who wants to go where :) The IVR folks 
are willing to bring the biopsies to our laboratory - they get instant 
feedback, and often use the exchange for teaching purposes as we are a tertiary 
care academic centre. Another factor would be the location of microscope used 
for visualizing adequacy of the cores. The distance between the two locations 
is not huge, and there is always an MLT instantly available to assess the cores.



Rarely has this system had hiccups. Occasionally, the adequacy is a 
question-mark, and we err on the side of asking IVR to obtain more tissue. This 
is usually delivered within 5-10 minutes after we request it.



Hope this helps,



Eric Gagnon MLT

Histology Laboratory

Kingston General Hospital

Kingston, Ontario, Canada


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[Histonet] On the lighter side...

[Gagnon, Eric]

AKA Roll-call of the Histo-lifers!



Started in Histo 1985. Spent 3.5 years in Hematology, but had to leave because 
I became bored with their incessant blood testing. Where were the interesting 
Histo bits and pieces I was so used to seeing and glomming?



Our laboratory nearly-completely re-equipped with new tissue processors, Bond 
IHC stainers, Dako Special Stainer, Cerebro tracking system. Now on same level 
of playing field with much newer/younger Histo MLT's. My incessant babbling 
about the Technicons, AO microtomes, freon-cooling of specimens and sharpening 
of real, big ol' 'real' microtome knives is now sounding reminiscent... back 
in the day, I used to... and likely viewed as a quaint anachronistic ramblings 
of a well-fixed, but in need of reprocessing, MLT.



I am reassured, however, that this discipline still remains relevant to patient 
care. Fears of being replaced completely by instrumentation have not been 
realized. With the coming nexus of molecular medicine and pathology, the 
increased use of ISH and IHC and many other developments I have quietly and 
humbly witnessed during my long journey to jadedness and grizzledom, I remain 
optimistic for future Histotechs.



Now, where did I leave my, um...where was I?



Eric Gagnon MLT

Histology Laboratory

Kingston General Hospital,

Kingston, Ontario, Canada


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[Histonet] Peggy Wenk's passing

[Gagnon, Eric]

Peggy's reach in our field went beyond borders. Here and I'm sure in other 
Canadian sites, we were fortunate to benefit from her enthusiasm and expertise 
online in forums such as this, as well as through teleconferences and blogging. 
Such knowledge and years of experience are not easily or quickly replaced. 
Condolences,

Eric Gagnon MLT

Histology Laboratory

Kingston General Hospital

Kingston, Ontario, Canada


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[Histonet] Dako Omnis

[Gagnon, Eric]

London, Ontario.



Eric Gagnon MLT

Histology Laboratory

Kingston General Hospital

Kingston, Ontario, Canada


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[Histonet] Blade Rationing / Blade Conservation

[Gagnon, Eric]

Desperately trying to salvage something positive out of this justifiably 
acrimonious thread...may I suggest the following blade conservation strategy, 
that though perhaps well-known, hasn't come up in this discussion yet.

By using one blade as a trimming blade, the 'edge' on the next blade will be 
conserved for actual sectioning.  Similarly, when cutting levels, one-half of a 
blade can be used for rough trimming, then the same blade pushed across into 
the cutting zone for the actual sectioning.

Also, if during trimming a hard/calcified/stapled section is found, perform 
microtomy on that block last, after trying to minimize the negative effects on 
cutting.

Perhaps by conserving blades in these and other ways, some cost-savings can be 
found for the penny-wise manager!

Hope this helps,
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada

I work in a hospital, there are three of us on this particular shift and we 
cut approx. 200 blocks, give or take a few.  Our histo lab manager is telling 
us we should only be using one pack of blades (50 per pack) a month.  I'm 
wondering what other techs think of this especially lab managers and 
supervisors.
tmoor...@gmail.com

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[Histonet] Leica CV5030

[Gagnon, Eric]

We have this same model which we use for routine staining, as well as the 
smaller Dako product we use for IHC and special stains.  We've recently been 
advised to use microtome oil to lubricate the V-shaped grippers on the CV5030, 
as the previously-suggested grease was only causing more buildup (we also use 
Ventana labels).  We had earlier received a software upgrade which lessened but 
certainly did not eliminate the gripping/dropping problems we had experienced 
that you described, Bruce.

Show me a walkaway, problem-free coverslipper and I'll show you...

I'm sure all models have their problems and challenges.  Certainly the Dako 
CR100 has a simpler in-and-out, less-gravity-challenging mechanism than the 
Leica CV5030.

Eric Gagnon
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada

Bruce wrote:
This coverslipper is over engineered. No self respecting histologist would 
design something that holds the slide a foot in the air by two v shaped 
grippers. Gravity sucks and the slide falls. I have a box of DISTROYED slides 
that I show the repairman. Can you imagine Sorry Mr. Smith we can't read your 
biopsy because it splintered into pieces AFTER we did all the hard work. 
Good luck with that health of yours.
It is NOT compatible with Ventana labels, as the gum sticks to those grippers 
and the slide falls, or jams in the output rack.
To tell you the truth, I loved the old Leica coverslipper. It had a walking 
beam and never broke a slide.
I'm in the mood for a Sakaura.

Bruce Gapinsk HT (ASCP)
Chief Histologist
Marin Medical Laboratories
PathGroup SF


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[Histonet] Ergonomics

[Gagnon, Eric]

Hi Karen,

Here's a response to your question, as I haven't seen any others (?)  While I 
haven't used the Newcomer handgrip device, I developed have DeQuervain's 
Tenosynovitis , a repetitive strain injury from turnin' the ol' microtome wheel 
for 28 or so years now.  I would definitely recommend the foot pedal, since you 
have a microtome equipped with one.  Yes, there is some loss of 'feeling' and 
it's a change from two hands on, but there is still the option of taking the 
microtome 'off line' and turning the wheel manually for difficult blocks.

I can only speak for myself, but the more I used the foot pedal, the more 
confident and at ease I became.  Start off slowly, working up towards more 
challenging blocks as soon as you can.  Not that one can ever let one's guard 
down with anything with a motor...car, microtome, what have you.

But in terms of ergonomics and saving your joints, I would try anything and 
everything you have at your disposal.  It's never too late to change for the 
better.

Hope this helps,

Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital,
Kingston, Ontario, Canada
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[Histonet] Block Alignment Tool

[Gagnon, Eric]

Interesting thread about block alignment.  Bernice makes a good point about 
Newcomer's level, and the possible effects of benchtop or floor not being 
level; perhaps the stage-mounted tools are preferable.  We have a stage-mounted 
tool, which brings me to another point.

Before this type of tool was invented, we used a block of wax, no tissue, for 
alignment.  This required some eye-balling and often subsequent adjustment of 
the head.  This was a skill learned by experience and practice, as most skills 
are.  The results were usually good.

Now that we have Leica RM2255 motorized microtomes, I find their method of 
aligning the block to be excellent, with red cylinders showing aligned or not 
on both axes.

We receive many cases from referring hospitals for subsequent IHC in our 
laboratory.  Many of these blocks contain very thin biopsies which have already 
been cut.  At this point, when microtomy for IHC begins, the alignment almost 
inevitably has to be adjusted.  This takes us back to the eye-balling mentioned 
earlier, and very careful initial turns of the wheel to gauge proper alignment 
and not waste tissue.

But in terms of daily microtomy of just-embedded blocks, either the Leica 
microtome method or the block alignment tool will go a long way to ensuring 
that all microtomes are as closely aligned as possible.  They all rely on 
proper training and experience to ensure this.

Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital,
Kingston, Ontario, Canada
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[Histonet] Friday funny...block alignment

[Gagnon, Eric]

Speaking of block alignment, when this topic comes up, someone invariably pipes 
up... Hey, is your head on straight?

Eric
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[Histonet] HIS-Copath-Paperless Order Entry

[Gagnon, Eric]

Have any histonetters had experience with paperless order entry using a 
HIS-Copath interface?  All our clinical laboratories have just gone to 
paperless order entry on patient floors and clinics, and would be interested in 
any other sites' experiences that use these same systems.

We have already found that pathology order entry is quite different from other 
clinical laboratories that now receive pre-barcoded tubes of blood that are 
instrument-ready.  Pathology has not been quite that straightforward.

Thanks for any experiences or advice you wish to share,

Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
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[Histonet] Ventana XT Closed System misnomer

[Gagnon, Eric]

Responding to Del who asked about pros/cons of Ventana BenchMark XT:

 

Ventana is often equated with 'closed system'.  This is a misnomer.  The
only thing I regard as closed (besides some colleagues' minds on this
issue) is the detection system kit and bulk reagents that are used on
the instrument.  While these are proprietary, they are also of extremely
high quality, resiliency, consistency, and in my opinion, dependability.
Preparing one's own reagents can lead to a lack of consistency of
staining, and we have confidence in Ventana reagents.

 

As others have correctly noted...other non-Ventana antibodies can be
used in Ventana dispensers.  We use an equal mix of ready-to-use
antibodies with our own diluted antibodies from other suppliers.  How is
this a closed system? 

 

Pre-treatments can also be used in Ventana dispensers. There is a range
of non-ab reagents such as hematoxylins and proteases.  There are a
range of detection system kits as well - at least four that I can think
of.

 

Perhaps this sounds like my personal crusade, as a Ventana user;  to try
to demolish the 'closed system' myth.  In my professional interactions
with non-Ventana users, this is often heard as a derogatory, negative
reaction to Ventana, which I certainly speak truth to at every
opportunity.

 

Del, we have found the BenchMark XT's very reliable, with good support
and service, plus excellent quality reagents including antibodies.  

 

Eric Gagnon MLT

Histology Laboratory

Kingston General Hospital,

Kingson, Ontario, Canada

 

 

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[Histonet] Saffron

[Gagnon, Eric]

Beth, as Bob Richmond has noted regarding saffron,
 
The most common use is as the hematoxylin-phloxin-saffron (HPS)
trichrome stain. It was in use as a general oversight stain in a few
pathology services when I was a resident in the 1960's...
 
and is still in use here in Ontario by about 10% of the province's histology 
laboratories as a routine oversight stain. 
 
We have gone the same route as other respondents have noted over the years, 
utilizing a variety of suppliers, including a Mediterranean health food store 
in Ottawa for saffron.  Now we are using the Sun Brand saffron produced in 
Spain, that is available at the check-out counter at our local bulk foods 
store.  One might think that there would be a total shift to HE as a routine 
stain, especially with automated stainers becoming prevalent, but we have 
successfully automated the stain on successive automated stainers.  Since our 
newest pathologists were trained as residents here, they are quite used to HPS, 
and there appears to be little impetus to change.
 
I still think the wafting of the boiling saffron is quite a pleasant aroma.
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada


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[Histonet] Training and Competency Assessment for HE Slide Review

[Gagnon, Eric]

Yes Victoria, Canadian laboratories can participate in HistoQIP as well.
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital,
Kingston, Ontario, Canada


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[Histonet] CBG Recycler Giveaway

[Gagnon, Eric]

CBG Biotech Benchtop Solvent Recycler Model MSLV-03U, has only been used 
occasionally, free to a good laboratory home.  If you would like this 
instrument, only requirement is that you pay shipping, please send email 
off-list to:
 
gagn...@kgh.kari.net
 
Recycler product information here:
http://www.cbgtechnologies.com/solvent-bts-spec.aspx
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 


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[Histonet] How many tissues...

[Gagnon, Eric]

To add to the recent discussion about how many blocks can be cut per hour, the 
College of Medical Laboratory Technologists of Ontario published Practice 
Guidelines for Medical Laboratory Technologists Practising in Histology fairly 
recently, in 2008, which may be of use in this context.  The practice 
guidelines are intended to support, not replace, the exercise of professional 
judgment by medical laboratory technologists practising in histology...and are 
maintained...in consultation with CMLTO members and stakeholders. 
 
The usual workplace variables are taken into account in the guideline, but at 
least there are some daily ranges presented which may help Joanne in her quest 
for a reasonable goal.  
 
Link follows:
http://tinyurl.com/histology-guidelines 
http://tinyurl.com/histology-guidelines 
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario

 
 


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[Histonet] C4d stain for IF

[Gagnon, Eric]

Patrick, or anyone else using the alpco product...can you give some 
details/feedback on how this anti human C4d (FITC-Conjugated) is working for 
you, and whether you were able to replace two steps with one for C4d IF 
staining?  I checked that website, and the pdf on this product reads:
 
The  antibody detects human complement split product C4d and has been tested 
for use on paraffin sections and frozen sections of human Tissue. 
 
We've switched our goat anti-mouse currently used for C4d immunofluorescence, 
and would prefer to switch to a conjugated product.   It sounds like something 
we'd like to try.
 
Thanks in advance,
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada

Try the C4d directly labeled antibody from alpco
www.alpco.com

anti human C4d  (FITC-Conjugated)
Cat #:   04-BI-RC4D-FITC

Good luck.

On Wed, Apr 27, 2011 at 2:54 PM, Butler, Roszetta rbut...@ameripath.com wrote:
 I'm having trouble with my C4d IF stain.  I'm using AbDirect/Serotec, mouse 
 monoclonal diluted 1:500 for 1 hr, and the Vector is AbDirect/Serotec  
 diluted 1:25 for ½ hour.  This is not working and I've tried different 
 dilutions with no success.  Can anyone share their protocol?

 Rosie Butler,
 AmeriPath/3000 United Founders Blvd./Suite 234/Oklahoma City, OK 
 73112/405.842.7575 - Office
 405.650.8921 - Cell/405.841.2002 - Fax
--


Patrick Laurie HT(ASCP)QIHC
CellNetix Pathology  Laboratories
1124 Columbia Street, Suite 200
Seattle, WA 98104
plau...@cellnetix.com





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[Histonet] Immunohistochemistry slides drying and baking protocol

[Gagnon, Eric]

Hi Milton,
 
We've begun baking ALL IHC slides for 2 hours at 60 degrees C.  This also 
applies to unstained extra slides that may potentially used for IHC, including 
extra sections on breast cores, lymph node cores, cytology cell blocks etc.  No 
specific room temperature drying time before the oven though.  This has not 
resulted in any increase in washoffs.  We specify time for removal of 
individual baskets in the oven if oven is filling up, which it often does these 
days.  I should add we are using Ventana BenchMark XT, for which the 
recommendation initially was 30 mins at 37 degrees C before placing slides on 
the instrument.  
 
We've also started discarding unused unstained slides 3 months after they're 
cut.  Now if we can just reduce the number of unstained slides we cut that we 
don't need, that will be really great.
 
Hope this helps,
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada


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[Histonet] Whole mount histology

[Gagnon, Eric]

Hi Dorothy,
 
I concur with what Rene has mentioned.  Main cost would be microtome-related, 
if you don't have microtomes that can handle mega cassettes.  Other 
process-related costs i.e. fixation, tech time, supplies wouldn't be much 
different once process was optimized.  In fact, tech time is likely reduced, as 
it may be quicker (depending on the tech) to cut 4-6 mega blocks than 18-24 
regular blocks.  
 
You'll also have to stain and coverslip the slides creatively, as they may not 
fit on your stainer.  Our pathologists did many cases, but eventually went with 
regular cassettes and processing.  It's a nice procedure to have available.  
 
Email me if you need more information i.e. specifics on supplies/process.
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada


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Re:[Histonet] Manual Coverslipping Safety Issues

[Gagnon, Eric]

A word of thanks for the plethora of responses regarding justification of 
additional automated coverslipping capacity for our laboratory.  We will be 
considering all your suggestions, combining safety benefits with productivity 
gains to improve our workflow.  You are all truly a valued and trustworthy 
resource.
 
Thanks again,
Eric Gagnon
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada


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[Histonet] Manual Coverslipping Safety Issues

[Gagnon, Eric]

Has anyone successfully lobbied their institution for an automated coverslipper 
for safety reasons?
 
Still coverslipping manually-stained IHC, neuro autopsy and special stains, 
sometimes hundreds per day. There has to be a better way.  Under budget 
constraints. That's why I'm wondering if anyone has used concerns about 
histology staff safety, specifically techs under direct exposure to 
toluene/xylene, to enable purchase of an automated/robot coverslipper.
 
I'd be interested in anyone's experience with this approach, successfully or 
unsuccessfully.
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada


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Re: [Histonet] Manual Coverslipping Safety Issues

[Gagnon, Eric]

I should add to my earlier post this morning:
-we do use an automated coverslipper for our routine HPS staining, which is 
several hundred slides daily as well
-we do have a fumehood over the main manual coverslipping area
-we have had airborne solvent levels measured, but my concern relates to direct 
manual exposure i.e. hands in toluene
-just because we can manually coverslip quickly doesn't mean the prolonged 
exposure is safe
 
Thanks for your answers, keep 'em coming
Eric
 
Has anyone successfully lobbied their institution for an automated 
coverslipper for safety reasons?

Still coverslipping manually-stained IHC, neuro autopsy and special stains, 
sometimes hundreds per day. There has to be a better way.  Under budget 
constraints. That's why I'm wondering if anyone has used concerns about 
histology staff safety, specifically techs under direct exposure to 
toluene/xylene, to enable purchase of an automated/robot coverslipper.

I'd be interested in anyone's experience with this approach, successfully or 
unsuccessfully.

Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada




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[Histonet] Rusting in Pathology Department

[Gagnon, Eric]

In our laboratory, we have also experienced this, especially with microtomes.  
As Jennifer has said, it seemed decal solution containing RDO was the culprit, 
when used in proximity to microtomes.  Techs who used RDO for surface 
decalcification had more rusting on exposed metal parts of the microtome, 
probably due to incomplete rinsing of the block after immersion in the RDO.  
Parts of the microtomes not exposed, where the acid seems to have been 
penetrated, included locking mechanisms and under the blade clamp.  I would 
suggest anyone using RDO surface decal do so by immersing the block in a 
shallow dish of RDO (square Coplin jar lid works well).  We now store RDO away 
from the microtomes.
 
We have no data on how the techs may have been rusting as well.  Effects may 
rival fixation of techs by close proximity to formalin x years. It's Friday :)
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 
 


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[Histonet] Direct immunofluorescence question

[Gagnon, Eric]

Barb,
 
Have you tried a diamond pencil?  Available from a variety of sources, these 
pencils can be used to etch a circle or other shape around the tissue to be 
viewed.  I believe they use lesser-quality industrial diamonds to make the tips 
on the pencils (i.e. not the diamond ring quality). Coming across the etched 
line under the fluorescent microscope produces brightness that helps the 
pathologist find the tissue.
 
We circle our DIF's on the back of the slide - it won't wash off and won't 
interfere with reagents this way, but is still visible in a darkened room.
 
Hope this helps,

Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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[Histonet] Lab Refrigerator

[Gagnon, Eric]

Ours is a Sanyo Medical MPR-1013 R double glass-door model with both pullout 
and stationary shelves.  We looked at buying a second-hand unit, but were able 
to buy brand-new.  We call it our dream fridge having made do with some 
older, much less satisfactory ones before the Sanyo arrived.  It holds all our 
immuno supplies, and the pullout drawers make finding antibodies much easier.
 
Hope this helps,
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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[Histonet] IHC Slides, Hotplate vs Oven

[Gagnon, Eric]

Tony's answer (62 degrees C for 25 mins) is the minimum to ensure section 
adheres to slide.  We all have to stain slides that have been baked longer than 
30 mins.  However, especially for Her2, longer baking times can result in false 
negatives or at least diminished staining.  The justification for baking longer 
is an attempt to prevent the section washing off or lifting.  We are seeing 
evidence that section adhesion is not a good tradeoff for diminished staining.  
Ideally both can be achieved with no tradeoff.
 
Tony or others who may wish to answer:
 
1. How do you monitor the shortened baking time, especially when multiple racks 
of slides are cut throughout the day?
2. Assuming charged slides and cell conditioning/antigen retrieval are 
employed, are you seeing major problems with section adhesion?
3. What if slides are received from another institution for staining, or slides 
get baked longer than 25 mins?
 
Thanks in advance,
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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[Histonet] Biocare Universal Negative Control Serum may be positive

[Gagnon, Eric]

Having just validated and started using Biocare Universal Negative Control 
Serum, we were surprised when a pathologist brought back two cases in which 
tumour and/or epithelium came up positive.  This staining was in the same 
locations as positive staining with some markers.  We use Ventana BenchMark 
XT's, and the staining was in both iView and UltraView protocols.  Mechanical 
problems, contamination and dilution of the Negative Control Serum did not 
appear to be factors.
 
Biocare knows about this and is meeting with Ventana to iron out the problem.  
Would have been nice to know about this if it was a known issue before we 
bought and started using the product, but better late than never.  Posting this 
in case anyone else on Histonet is experiencing similar staining challenges 
using this product.  As Biocare states in the product insert sheet, when used 
in conjunction with non-Biocare kits, results may vary.  We'll keep using it 
and are currently waiting for advice from Biocare.
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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[Histonet] HPS Stain

[Gagnon, Eric]

Hi Sheila,
 
HPS (Hematoxylin-Phloxine-Saffron) is the routine stain we use.  There are a 
few institutions in Canada that also still use this stain; around Ottawa and a 
few other places in Ontario and Quebec.  Perhaps one of these institutions sent 
slides to your pathologist for referral/consult?
 
It is quite a nice trichrome stain, but somewhat more complex and expensive, 
uses more staining dishes, and it's sometimes tricky to get a proper 
phloxine/saffron balance.  Due to the use of HE being more widespread, our 
pathology residents usually ask for HE slides for their files when preparing 
for exams.
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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[Histonet] Negative controls

[Gagnon, Eric]

Hi Anita,
 
We are currently examining the negative control issue in our laboratory.  We 
also have Ventanas. We currently use a normal goat serum as our non-immune 
serum.  We are considering changing our SOP's to use the Ventana Neg Control 
Rabbit and Neg Control Mouse in dispensers.  
 
Linda's suggestion of Biocare's Universal Negative Control Serum is intriguing. 
 It would seem regressive to go back to the days of delineating mouse/rabbit 
antisera, using separate dispensers for each, and a universal negative seems to 
be the way to go, especially when using universal DAB kits.  Using such a 
reagent would mean it could be applied to each case's negative control slide.
 
Linda or others, would such a negative control serum protocol include your most 
aggressive pretreatment? 
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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[Histonet] IHC in Ontario Canada

[Gagnon, Eric]

Hi Sheila,
 
What are the Ontario Laboratory Accreditation requirements questions that you 
have regarding IHC?  We were assessed in spring/09.  We run well over a hundred 
markers using three Ventana BenchMark XT's.
 
Eric Gagnon MLT
Histology Laboratory 
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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[Histonet] Prostate Whole Mounts

[Gagnon, Eric]

Hi Allison,
We cut our prostate whole mounts on the Leica RM2255 automated microtome.  We 
obtained their Super Mega Cassette Clamp, (part no 140502 38967).  This clamp 
mounts easily onto the microtome, and holds SurgiPath SuperCassettes ( Cat No 
VSP59067B-BX grey in colour).  The beauty of this microtome is that the stroke 
is 10 mm longer than our previous Reichert-Jung 2035 microtomes we used to cut 
these on, I believe it is now 70 mm vertically.  In terms of slides, we use 
75x38 up to 75x80 mm slides, with coverslips ranging from 35x50 to 48x65 mm, 
depending on specimen size.  The slides go on our Leica automated stainer, held 
diagonally in the basket by other slides to keep them vertical, and 
coverslipped by hand.

In terms of processing, we use our normal overnight processing cycle as we 
would for our routine surgical blocks.  These sections are well-fixed before 
processing, cut at 4-5 mm during crossing.  We have had some issues when the 
pathologist assistants couldn't get the sections this thin, but it hasn't 
happened often enough to use a longer/different processing cycle.  We have been 
getting 4-6 slides blocks per case, and we've done 35 cases in the last year. 
 
Hope this helps,
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 

 
 


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[Histonet] Control slides for Oil Red O

[Gagnon, Eric]

I was always of the opinion that since a delipidized frozen section, treated 
with toluene/methanol, run in parallel with the patient slide constituted 
adequate control.  Recently though, I've mellowed and realized that only shows 
a difference in staining between the two slides, not the effectiveness of your 
stain.  Using a piece of fat for a control slide confirms microscopically what 
we can confirm macroscopically...yep, it's a piece of fat.  
 
The adrenal and skin ideas sound better to me than using a piece of 
breast/fatty tissue. 
 
 The era of performing staining methods without controls seems to be at an end, 
which isn't necessarily a bad thing.
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario,Canada


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RE: [Histonet] IF Protocol on skin

[Gagnon, Eric]

Hi Angela,
 
We used Ventana FITC Primary Antibodies.
 
Thanks,
Eric



From: Angela Bitting [mailto:akbitt...@geisinger.edu]
Sent: Wed 2/11/2009 5:11 PM
To: Gagnon, Eric
Subject: Re: [Histonet] IF Protocol on skin



Hi eric,
You said you experimented with using the FITCs on your XT. Was that using Dako 
antibodies or Ventana's?

Angela Bitting, HT(ASCP)
Technical Specialist, Histology
Geisinger Medical Center
100 N Academy Ave. MC 23-00
Danville, PA 17822
phone  570-214-9634
fax  570-271-5916
 Gagnon, Eric gagn...@kgh.kari.net 02/11/09 4:02 PM 
Hi Gudrun,

Our IF protocol is very similar to yours...we use 1/30 dilutions as well, 
although we rinse before the FITC-antisera (Dako) are applied, with frozen 
sections cut at 5 microns. 

We did experiment with this procedure on our BenchMark XT's, with some success. 
 The pathologist who reads most of renal biopsies IF's decided that our manual 
method was robust enough (i.e. foolproof enough) that we could continue using 
it.  Manual staining saves us trying to fit in a new or current run when the XT 
is already running.

Regarding our dilutions, we make 3 ml at a time, and have not had any problems 
with the staining diminishing before the antisera are used up.

Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada


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RE: [Histonet] IF Protocol on skin

[Gagnon, Eric]

Hi Gudrun, 
 
We've rarely done Ig's on FFPE renal sections on the Ventana -  our renal 
pathologist prefer these on IF frozen sections only.  We do have the IgG, IgA, 
IgM, IgE antisera for lymphoma cases.  We use Dako Antibody Diluting Buffer, 
Ref. S0809 to dilute our FITC antisera.
 
As I said, we didn't persist with validating these antibodies for use with 
renal or skin IF's on the XT, mainly due to our pathologist's satisfaction with 
the manual IF staining method.  This was back in January 2006, with the 
antibodies incubating for 4-8 minutes.
 
Hope this helps,
Eric



From: Gudrun Lang [mailto:gu.l...@gmx.at]
Sent: Thu 2/12/2009 10:41 AM
To: Gagnon, Eric
Subject: AW: [Histonet] IF Protocol on skin



Hi Eric,
thanks for your response. I have several further questions.
Did you stain frozen renal sections in the benchmark? Or did you also try
NBF-fixed tissue?
What diluent do you use for the fitc-antisera? We have also the
ventana-machine. And I consider to use their antibody-diluent for this.

This week I have stained NBF-fixed skin (Pemphigoid) with IgG, IgM, IgA in
the Benchmark XT. The IF of the frozen part showed IgG and C3C linear
staining. The IHC of the FFPET showed very strong staining with all
Immunoglobulins. Perhaps we can find a diagnostic value.

Bye
Gudrun


-Ursprüngliche Nachricht-
Von: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Gagnon,
Eric
Gesendet: Mittwoch, 11. Februar 2009 22:03
An: histonet@lists.utsouthwestern.edu
Betreff: [Histonet] IF Protocol on skin

Hi Gudrun,

Our IF protocol is very similar to yours...we use 1/30 dilutions as well,
although we rinse before the FITC-antisera (Dako) are applied, with frozen
sections cut at 5 microns. 

We did experiment with this procedure on our BenchMark XT's, with some
success.  The pathologist who reads most of renal biopsies IF's decided that
our manual method was robust enough (i.e. foolproof enough) that we could
continue using it.  Manual staining saves us trying to fit in a new or
current run when the XT is already running.

Regarding our dilutions, we make 3 ml at a time, and have not had any
problems with the staining diminishing before the antisera are used up.

Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada


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[Histonet] Histology Workload Guidelines

[Gagnon, Eric]

To add to the recent discussion about how many blocks can be embedded per hour, 
the College of Medical Laboratory Technologists of Ontario recently published 
MLT practice guidelines which may be of use.  The practice guidelines are 
intended to support, not replace, the exercise of professional judgment by 
medical laboratory technologists practising in histology...and are 
maintained...in consultation with CMLTO members and stakeholders. Check out 
quality assurance at:

http://cmlto.com

or more specifically:

http://cmlto.com/quality_assurance/MLT_practice_guidelines/learning/10_hsto_gdlne.pdf

Eric Gagnon MLT

Histology Laboratory

Kingston General Hospital, 

Kingston, Ontario, Canada

 
 


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[Histonet] GI biopsies

[Gagnon, Eric]

Andria, another factor may be trimming the GI biopsy blocks.  If trimming 
increments are too large, it may rip some of the deeper tissue out of the 
block, causing holes or possibly the chatter you are seeing.  Check the block 
after trimming to see if there is any unevenness to the surface.  Also, is it 
limited to one person?  If not, it may not be microtomy, look at processing as 
others have already mentioned.
 
Hope this helps,
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada


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