[Histonet] Dallas Texas Lab Positions

[Pat Patterson via Histonet]

ProPath, a progressive, CAP accredited, high volume, pathology practice, 
located in Dallas, Texas, is seeking candidates for the following positions:



HISTOLOGY TECHNOLOGIST/TECHNICIAN

Responsibilities include embedding tissue specimens, microtomy of 
paraffin-embedded tissue, operation of automated stainer and cover slipper, 
equipment maintenance, and record retention.  HT & HTL (ASCP) registered or 
eligible.  Experience preferred.  Hours are 7:00 a.m. to 3:30 p.m. or 6:00 p.m. 
to 2:30 a.m.


IHC HISTOLOGY TECHNOLOGIST/TECHNICIAN

Responsibilities include slide preparation (paraffin and frozen sections), IHC 
staining using our unique manual system, antibody titer preparation, equipment 
maintenance, supply/reagent inventory maintenance, and QC/QA recording. A 
minimum of 4 years Histology experience with at least 1-2 years 
immunohistochemistry, immunofluorescence or in situ hybridization experience.  
Working knowledge of IHC theory required and hands on IHC performance desired.  
Hours for this position are 5:00 p.m. to 1:30 a.m.


GROSSING HISTOTECHNICIAN


Responsibilities include performing gross dictation and dissect low to moderate 
complexity specimens received in the laboratory for microscopic diagnosis.  You 
will work in Histology and will be responsible for embedding tissue specimens, 
microtomy of paraffin-embedded tissue, special stains, frozen sections, 
operation of automated stainer and coverslipper, equipment maintenance and 
record retention.   Bachelors' degree in a laboratory science or medical 
laboratory technology plus HT/ HTL (ASCP) certification required and 3 years' 
experience in grossing and histology.  Hours for this position are 11:00 p.m. 
to 7:30 a.m.

For more details and to apply, please visit 
www.propath.com

ProPath utilizes leading technology and is a quality oriented pathology 
laboratory.  Benefits include medical, dental, Short and Long Term Disability 
insurance, a matched 401K plan and more!

Don't Follow the Leader!  Join the Leader!

EEO/AA-M/F/disability/protected veteran status


Pat Patterson, HTL(ASCP)
Manager, Immunohistochemistry
ProPath - The Leader in Pathology Services
1355 River Bend Drive
Dallas, TX 75247

214-237-1700 x 2027
214-237-1730 fax

To learn more about ProPath, please visit 
http://www.ProPath.com



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Re: [Histonet] RTU antibodies with 1+ year expiration date

[Morken, Timothy via Histonet]

To the specific question of if lab-made predilutes will last longer than two 
weeks. Yes they can. But be sure you are using reasonably sterile technique so 
they don't become contaminated. Then validate how long they last.  I know of 
one lab in the "old" days of manual staining that kept their antibodies in 
Coplin jars in the fridge and used them for many weeks.  They just pulled out a 
particular antibody jar and added slides when that antibody was ordered. It 
made bulk manual staining much easier.

I agree with all Tony writes below. One observation from experience working for 
a vendor of antibodies... While vendors test for reasonable shelf life, the 
fairly standard 2-year expiration date is implemented more to limit a vendors 
liability than indicate actual shelf life. We tested ours for up to 4 months in 
a 60 C oven and I rarely saw one fail. Indeed, some, as Tony noted, became 
better with age and heat!

While those of us who have worked in IHC for many, many years know that most 
modern antibodies are very stable, and, if frozen, good for decades, the 
various inspection agencies are very focused on expiration dates. Part of that 
may be ease of enforcement (not having to look though hundreds of pages of 
revalidations) and part not trusting labs to do good revalidations. And many 
labs would much rather not revalidate since it takes work to do and if you have 
many antibodies, it could be a lot of work (we have over 250 on our menu). Much 
easier to get new ones. We don't revalidate, but we also make every effort to 
only have on hand that which will not expire before used. We have very few that 
have so few orders that the vial lasts 2 years. And we still dilute a fair 
number that are not available as predilutes from our instrument vendor.



And thanks for all the references!


Tim Morken
Pathology Site Manager, Parnassus 
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center




-Original Message-
From: Tony Henwood (SCHN) via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Monday, December 05, 2016 2:44 PM
To: Allan Wang
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] RTU antibodies with 1+ year expiration date

Hi Alan,

Not every source states that diluted antibodies only last for a few weeks, in 
fact my experience as well as those of the commercial suppliers (see their data 
sheets) indicates otherwise, for example, I just re-validated an antibody 
(CD45RO)  that was prepared 8 years ago (but unfortunately rarely used) and it 
worked very well, in fact we had to re-titre it since it seems our detection 
system has improved its sensitivity (we have a Bond 3). Since it is not 
routinely used (last time was at least 4 years ago) we have retired it.

The following is an extract from the handout of a workshop we gave on an 
Immunohistochemistry Validation workshop we gave this year:

Expired Antibodies
Usually when a new concentrated antibody is received it will have an expiry 
date of around 2 years from receipt, but most of us will find that we can 
continue to use antibodies well past this expiry date. So where are we at?
Recent discussions on Histonet reveal:
?   At least five labs admit to routinely discarding expired antibodies.
?   Why don’t companies extend expiry dates?
?   Why not freeze aliquots of antibody to extend the shelf life?
?   If controls continue to stain appropriately, then why can’t expiry be 
extended?
An expiration date labelled on a vial merely reflects the time span tested by 
the manufacturer during which performance of a reagent has remained stable. It 
does not imply per se, that the reagent will not function properly beyond the 
indicated date. In fact, because it is unrealistic for a manufacturer to test 
an antibody for aging during decades before putting it on the market, most 
expiration spans for primary antibodies are set arbitrarily within 6 to 24 
months (Balaton et al 1999).

Dr Hadi Yaziji explains: Vendors do stability testing on their antibodies, 
where they leave them at room temperature for an extended time (one month, six 
months, etc.), or they do what's referred to as 'accelerated' testing, where 
they put them in a microwave which is supposed to accelerate the degradation of 
the protein. That's usually how they decide to assign an expiration date.

Dr Hadi Yaziji also suggests that if you aliquot the antibody before its 
expiration date and store it in the -20oC freezer, the clock is in effect 
"frozen" too. For example, if there are 6 months left before the antibody 
expires and you freeze the aliquots for 5 years, then if you thaw one of the 
frozen aliquots, the thawed aliquot will still be good for 6 months. He also 
recommends that you make sure you don't thaw and re-freeze the aliquot. 
Unfortunately, some antibody data sheets state “do not freeze”. One 
Histotechnologist reported that their CAP inspector told them it 

Re: [Histonet] Weak Hematoxylin Staining?

[Tony Henwood (SCHN) via Histonet]

Vivian,

After cutting the frozen sections, how are you fixing them?

If they are air-dried then they will tend to give a washed out nuclear stain.
I would suggest immediate fixation of the sections in either methanol or to 
make even a brighter H: 1% acetic acid in ethanol (1ml glacial acetic acid + 
99ml absolute ethanol), about 1 minute should suffice for both fixations.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-Original Message-
From: Vivian Hou via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, 6 December 2016 7:58 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Weak Hematoxylin Staining?

Dear all,

We are doing some immunofluorescent stains that we want to pair with H 
slides. We are using fresh frozen sections for both (unfixed tissue, human 
diseased coronary arteries) but I am seeing very weak hematoxylin staining to 
the point where I have to dial back the eosin (3 min hematoxylin, 1 dip eosin).


I am using Gills 2 (newly purchased), I know its not necessary to filter it but 
we have been just in case using Whatman no. 5 filters. No differentiation due 
to the weak staining right now.


I would appreciate any thoughts on why the staining might be so weak, we do a 
great deal of processing and imaging on the arteries (about 12 hrs @ 25C) prior 
to histo and I wonder if this has cause the tissue to decay?


Thank you for your time,
V

--
-
V Hou
Bioengineering | Human Photonics Laboratory
e: ho...@uw.edu | c: 206-999-3708
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Re: [Histonet] RTU antibodies with 1+ year expiration date

[Tony Henwood (SCHN) via Histonet]

Hi Alan,

Not every source states that diluted antibodies only last for a few weeks, in 
fact my experience as well as those of the commercial suppliers (see their data 
sheets) indicates otherwise, for example, I just re-validated an antibody 
(CD45RO)  that was prepared 8 years ago (but unfortunately rarely used) and it 
worked very well, in fact we had to re-titre it since it seems our detection 
system has improved its sensitivity (we have a Bond 3). Since it is not 
routinely used (last time was at least 4 years ago) we have retired it.

The following is an extract from the handout of a workshop we gave on an 
Immunohistochemistry Validation workshop we gave this year:

Expired Antibodies
Usually when a new concentrated antibody is received it will have an expiry 
date of around 2 years from receipt, but most of us will find that we can 
continue to use antibodies well past this expiry date. So where are we at?
Recent discussions on Histonet reveal:
?   At least five labs admit to routinely discarding expired antibodies.
?   Why don’t companies extend expiry dates?
?   Why not freeze aliquots of antibody to extend the shelf life?
?   If controls continue to stain appropriately, then why can’t expiry be 
extended?
An expiration date labelled on a vial merely reflects the time span tested by 
the manufacturer during which performance of a reagent has remained stable. It 
does not imply per se, that the reagent will not function properly beyond the 
indicated date. In fact, because it is unrealistic for a manufacturer to test 
an antibody for aging during decades before putting it on the market, most 
expiration spans for primary antibodies are set arbitrarily within 6 to 24 
months (Balaton et al 1999).

Dr Hadi Yaziji explains: Vendors do stability testing on their antibodies, 
where they leave them at room temperature for an extended time (one month, six 
months, etc.), or they do what's referred to as 'accelerated' testing, where 
they put them in a microwave which is supposed to accelerate the degradation of 
the protein. That's usually how they decide to assign an expiration date.

Dr Hadi Yaziji also suggests that if you aliquot the antibody before its 
expiration date and store it in the -20oC freezer, the clock is in effect 
"frozen" too. For example, if there are 6 months left before the antibody 
expires and you freeze the aliquots for 5 years, then if you thaw one of the 
frozen aliquots, the thawed aliquot will still be good for 6 months. He also 
recommends that you make sure you don't thaw and re-freeze the aliquot. 
Unfortunately, some antibody data sheets state “do not freeze”. One 
Histotechnologist reported that their CAP inspector told them it was not 
acceptable, so had to allow him to watch them throw all the frozen aliquots 
away.

Both Dr Terry Marshall (UK) and Dr Richard Cartun suggest that if the antibody 
continues to stain control sections appropriately, with no loss of sensitivity 
and no increase in non-specific staining then its use should be continued. If 
positive control samples are deemed unsatisfactory, even if the antibody is 
within the manufacturer’s printed expiration date, evaluation of the clinical 
specimen is aborted and the test deemed invalid. The quality of the primary 
antibody is therefore not based on an expiration date, but rather on its 
performance on a case-by-case basis with appropriate positive and negative 
control samples (Savage & DeYoung 2010).

Savage & DeYoung (2010) had previously examined the staining patterns of 26 
recently acquired primary antibodies and their expired counterparts. Two 
reviewers examined sequential sections of formalin-fixed, paraffin-embedded 
tissue samples for staining intensity and percentage of positivity. Appropriate 
positive and negative control studies were performed. Of the 26 antibodies, 20 
exhibited no difference in percentage of positivity or staining intensity. Of 
the remaining 6, 3 showed better performance with the expired cohort and 3 with 
non-expired antibodies. However, no antibody staining characteristics varied by 
more than 1 step and in no case was positive staining lost after antibody 
expiration. Negligible differences exist in immunostaining between outdated and 
current antibodies. They concluded that exemption for primary antibodies from 
existing regulations would conserve resources without adversely impacting 
patient care. 

Balaton et al (1999) compared 55 antibodies that had expired 6 to 134 months 
previously and found there was no significant difference observed in the 
specificity, sensitivity, and pattern of staining between old versus new 
antibodies.
Recently Maria Argentieri et al (2013) investigated whether the shelf-life of 
diagnostic antibodies was longer than the expiry date on the label. They had 
four independent laboratories test a small number of diagnostic antibodies kept 
at +4°C for 12?26 years, and found them to work perfectly on routine histology 

[Histonet] What are science faculty members doing when evaluating anti-science papers in science classes?

[Jorge A. Santiago-Blay via Histonet]

What are science faculty members doing when evaluating anti-science papers
in science classes?

Dear Colleagues:

To encourage civilized face to face or digital discussions, in my science
classes, I like to ask questions about topics some people find
controversial (e.g. age of Earth, age of human lineage, global climate
change, etc.).

What are science faculty members doing when evaluating anti-science papers
in science classes? I would be interested in knowing whether there are
differences between what people do in different countries.

If you have any constructive response, please feel free to send it directly
to me at

blayjo...@gmail.com

Apologies for potential duplicate emails.

Gratefully,

Jorge
blaypublishers.com
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[Histonet] Weak Hematoxylin Staining?

[Vivian Hou via Histonet]

Dear all,

We are doing some immunofluorescent stains that we want to pair with H
slides. We are using fresh frozen sections for both (unfixed tissue, human
diseased coronary arteries) but I am seeing very weak hematoxylin staining
to the point where I have to dial back the eosin (3 min hematoxylin, 1 dip
eosin).


I am using Gills 2 (newly purchased), I know its not necessary to filter it
but we have been just in case using Whatman no. 5 filters. No
differentiation due to the weak staining right now.


I would appreciate any thoughts on why the staining might be so weak, we do
a great deal of processing and imaging on the arteries (about 12 hrs @ 25C)
prior to histo and I wonder if this has cause the tissue to decay?


Thank you for your time,
V

-- 
-
V Hou
Bioengineering | Human Photonics Laboratory
e: ho...@uw.edu | c: 206-999-3708
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[Histonet] RTU antibodies with 1+ year expiration date

[Allan Wang via Histonet]

Hello list,

Thanks for your responses to my previous dehydration question. I pretty
much got conflicting responses about going straight to 100% alcohol, but I
think I will just start them out in 80% alcohol to be safe.

Something that's been bugging me: Every source states that diluted
antibodies shouldn't be kept for more than a few weeks. Yet Ventana
dispensers of pre-diluted antibodies have expiration dates of 1 - 1.5
years, plus they sit out at room temperature for 5 hours at a time whenever
they're used. Ventana provides user-fillable dispensers, but if we dilute
antibodies ourselves and can't use them for more than a few weeks, it would
be kind of pointless for low-volume needs.

Their specs say: "The antibody is diluted in 0.08 M PBS with 3% carrier
protein and 0.05% ProClin 300, a preservative."
Isn't this pretty much the same stuff that antibody diluent is composed of,
except with sodium azide instead of ProClin 300 (I think they're
equivalent).

I'm already planning on doing some long-term testing of my own over a year
with HER-2 and Ki67, but hopefully someone can provide some insight.

Thanks,
Allan Wang
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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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