Re: [Histonet] frozen section problem

[Tony Henwood (SCHN) via Histonet]

We have had this issue previously.

We tracked it down to the brain biopsies arriving in "Isotonic" saline (which 
is really not isotonic).
See: Henwood, A., (2007) “Adverse effect of saline on brain intraoperative 
(frozen section) Histology” J Histotechnol 30(3):193.

Ask the surgeons to send the biopsies to the lab on damp shiny (non-absorbent) 
card.

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


From: Manfre, Philip via Histonet 
Sent: Saturday, 17 July 2021 07:54
To: Bonello Dorianne M at Health-MDH;   (histonet@lists.utsouthwestern.edu)
Subject: Re: [Histonet] frozen section problem

Hi Dorianne,
You need to freeze your tissue faster.  Ideally, isopentane placed in a 
metal cup, that is that is then frozen in a dewar of liquid nitrogen, works 
best.  The isopentane, once frozen, is thawed a little with a metal rod to 
produce a small liquid pool and your tissue is placed in this for about one 
minute.  You need some equipment for this procedure, such as the metal cup that 
can sit inside a small, open dewar of liquid nitrogen.  Alternatively, you can 
freeze directly in liquid nitrogen, though you need to beware of the tissue 
fracturing due to the sudden and extreme temperature reduction.  Slower 
freezing of tissue (sitting on dry ice, etc.)  allows ice crystals to form in 
the tissue, creating the vacuoles you describe.
I hope this helps.
Phil.

Philip Manfre, BA, HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP81-406
770 Sumneytown Pike
West Point, PA 19486
215-652-9750
philip_man...@merck.com




-Original Message-
From: Bonello Dorianne M at Health-MDH via Histonet 

Sent: Friday, July 16, 2021 11:25 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] frozen section problem

EXTERNAL EMAIL – Use caution with any links or file attachments.

Dear all,


We are experiencing freezing artifacts on our frozen sections. Basically, we 
are seeing cavity-like structures under the microscope, mostly elongated, 
especially when it's a frozen section on brain tissue. This is most probably 
happening due to ice crystal formation. We're not using cryospray, relying only 
on the cryobar boost function.


Does anyone has a solution to this problem please?


Regards,



Dorianne Bonello
Allied Health Practitioner (MLS)
Histology Laboratory - Pathology
Health-Mater Dei Hospital


[cid:image001.jpg@01D67184.63288530]


T +356 +356 25456434

E dorianne.m.bone...@gov.mt


Mater Dei Hospital, Triq id-Donaturi tad-Demm, l-Imsida, Malta MSD 2090 | Tel 
+356 2545  | 
https://secure-web.cisco.com/108H5-f5Ylbr6vXriY71-fp4JDQxST7FvP8i3GNqBPS05qZlvu_1O6tMXRjgYucUESUfrxOSGo-3VSj277AHeUKp72a2u23a8E1gThp8T_C_qgaCkXVHMnAxY-YyXqqjb0pEmmV-J74L1UJXhwqdO88nHIMcK0Y7_wGmWPWFgvvMfmer4a-NFgWAPDQDYgBdnmJ-UysbSbmKuEVVZzOZFDBgn-f2wXW3JKjvg0h2q9rXdcxCMqWYFuQFY1xiKWJHR9NcNUphJ6xHBSRJSogoK53iKRSk1tEyOBPErMdipZOz4PXDp29G4cAm7LIrI_3TQWhJLOaI2wZTc0wJ7yRUScq8nh2P3hrxSuBaL3ivOfsU9eUtNGDZDo9XeNFEcGmivY-fPo1XKWjQy6pmHUaksuix_Jp9AvaCBh0IWf_zuPVGEv2oeS9aYQXub1b9B0QpJFqSicysNAHY65JYUzhjHXxuuvsaJ-m6zou-55OWaT8dK3Cjh6bvXTnQWzS3C4Uya/https%3A%2F%2Fdeputyprimeminister.gov.mt%2Fen%2FMDH%2FPages%2FHome.aspx
 | https://www.facebook.com/materdeihospital/


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Re: [Histonet] frozen section problem

[Manfre, Philip via Histonet]

Hi Dorianne,
You need to freeze your tissue faster.  Ideally, isopentane placed in a 
metal cup, that is that is then frozen in a dewar of liquid nitrogen, works 
best.  The isopentane, once frozen, is thawed a little with a metal rod to 
produce a small liquid pool and your tissue is placed in this for about one 
minute.  You need some equipment for this procedure, such as the metal cup that 
can sit inside a small, open dewar of liquid nitrogen.  Alternatively, you can 
freeze directly in liquid nitrogen, though you need to beware of the tissue 
fracturing due to the sudden and extreme temperature reduction.  Slower 
freezing of tissue (sitting on dry ice, etc.)  allows ice crystals to form in 
the tissue, creating the vacuoles you describe.
I hope this helps.
Phil.

Philip Manfre, BA, HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP81-406
770 Sumneytown Pike
West Point, PA 19486
215-652-9750
philip_man...@merck.com




-Original Message-
From: Bonello Dorianne M at Health-MDH via Histonet 
 
Sent: Friday, July 16, 2021 11:25 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] frozen section problem

EXTERNAL EMAIL – Use caution with any links or file attachments.

Dear all,


We are experiencing freezing artifacts on our frozen sections. Basically, we 
are seeing cavity-like structures under the microscope, mostly elongated, 
especially when it's a frozen section on brain tissue. This is most probably 
happening due to ice crystal formation. We're not using cryospray, relying only 
on the cryobar boost function.


Does anyone has a solution to this problem please?


Regards,



Dorianne Bonello
Allied Health Practitioner (MLS)
Histology Laboratory - Pathology
Health-Mater Dei Hospital


[cid:image001.jpg@01D67184.63288530]


T +356 +356 25456434

E dorianne.m.bone...@gov.mt


Mater Dei Hospital, Triq id-Donaturi tad-Demm, l-Imsida, Malta MSD 2090 | Tel 
+356 2545  | 
https://deputyprimeminister.gov.mt/en/MDH/Pages/Home.aspx
 | https://www.facebook.com/materdeihospital/


Think before you print. Kindly consider your environmental responsibility.

This email and any files transmitted with it are confidential, may be legally 
privileged and intended solely

for the use of the individual or entity to whom they are addressed.






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Re: [Histonet] brain freeze artifact

[Terri Braud via Histonet]

First of all, freezing spray should NEVER be used in a cryostat.  It produces 
dangerous aerosolized pathogens that linger in the air, just waiting to infect 
someone. There should be no exceptions.
As to the ice artifact problem,  not surprising to see this in brain since it 
is so fragile and often edematous.  Your best bet to eliminate freeze artifact 
is to "snap freeze" the tissue using the technique used in muscle biopsy 
samples.  A metal sample cup containing 2-Methyl Butane (Isopentane) is  
suspended in Liquid Nitrogen, stirring until it becomes a thickened, white 
slurry.  The mounted tissue sample is immersed in this slurry to freeze, then 
placed in the cryostat to come up to cryostat temps before cutting.  That is 
the short and sweet version, and there are many published versions of the 
technique.  It is cumbersome, but produces no freezing artifact.
Best of luck, Terri

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
HNL Laboratories for 
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
Ph: 215-938-3689
Fax: 215-938-3874
--

Message: 3
Date: Fri, 16 Jul 2021 15:25:08 +
From: Bonello Dorianne M at Health-MDH 
To: "histonet@lists.utsouthwestern.edu"
Subject: [Histonet] frozen section problem

Dear all,
We are experiencing freezing artifacts on our frozen sections. Basically, we 
are seeing cavity-like structures under the microscope, mostly elongated, 
especially when it's a frozen section on brain tissue. This is most probably 
happening due to ice crystal formation. We're not using cryospray, relying only 
on the cryobar boost function.
Does anyone has a solution to this problem please
Regards,
Dorianne Bonello
Allied Health Practitioner (MLS)
Histology Laboratory - Pathology
Health-Mater Dei Hospital



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Re: [Histonet] frozen section problem

[John Kiernan via Histonet]

Yes, definitely ice crystal holes! If the tissues are unfixed you will have to 
freeze much more rapidly (isopentane cooled with liquid nitrogen.) If fixed in 
formaldehyde, cryoprotect by immersing the pieces in 20% sucrose, until they 
sink.

John Kiernan
Anatomy & Cell Biology, UWO
London, Canada
= = =

From: Bonello Dorianne M at Health-MDH via Histonet 

Sent: July 16, 2021 11:25 AM
To: histonet@lists.utsouthwestern.edu 
Subject: [Histonet] frozen section problem

Dear all,


We are experiencing freezing artifacts on our frozen sections. Basically, we 
are seeing cavity-like structures under the microscope, mostly elongated, 
especially when it's a frozen section on brain tissue. This is most probably 
happening due to ice crystal formation. We're not using cryospray, relying only 
on the cryobar boost function.


Does anyone has a solution to this problem please?


Regards,



Dorianne Bonello
Allied Health Practitioner (MLS)
Histology Laboratory - Pathology
Health-Mater Dei Hospital


[cid:image001.jpg@01D67184.63288530]


T +356 +356 25456434

E dorianne.m.bone...@gov.mt


Mater Dei Hospital, Triq id-Donaturi tad-Demm, l-Imsida, Malta MSD 2090 | Tel 
+356 2545  | 
https://deputyprimeminister.gov.mt/en/MDH/Pages/Home.aspx
 | https://www.facebook.com/materdeihospital/


Think before you print. Kindly consider your environmental responsibility.

This email and any files transmitted with it are confidential, may be legally 
privileged and intended solely

for the use of the individual or entity to whom they are addressed.






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Re: [Histonet] Frozen section problem

[Bob Richmond via Histonet]

Dorianne Bonello, Allied Health Practitioner (MLS), Histology Laboratory -
Pathology Health-Mater Dei Hospital, on the island of Malta asks:


> >>We are experiencing freezing artifacts on our frozen sections.
> Basically, we are seeing cavity-like structures under the microscope,
> mostly elongated, especially when it's a frozen section on brain tissue.
> This is most probably happening due to ice crystal formation. We're not
> using cryospray, relying only on the cryobar boost function.<<
>
> You need to freeze faster. A low-tech solution is a "heat extractor" - a
> flat-bottomed mass of metal with a handle, kept cold in the cryostat, that
> you press on top of the specimen after it's mounted on the chuck with
> frozen section embedding medium. Here's a picture of it:

https://tinyurl.com/4djk85xd

This 82 year old pathologist would expect to trouble-shoot the problem
himself.

Not every day I get e-mail from Malta! If anyone on Histonet doesn't know
where Malta is, it's an island nation in the Mediterranean Sea, south of
Sicily. If you look at the Mater Dei web site, you'll see that it's in
English, but with occasional lines of Maltese, the native language of the
island, a form of North African Arabic, but always written in the Roman
alphabet. Wikipedia has good resources for the island, the language, and
the Mater Dei Hospital.

Bob Richmond
Samurai Pathologist
Maryville, Tennessee, USA
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[Histonet] frozen section problem

[Bonello Dorianne M at Health-MDH via Histonet]

Dear all,


We are experiencing freezing artifacts on our frozen sections. Basically, we 
are seeing cavity-like structures under the microscope, mostly elongated, 
especially when it's a frozen section on brain tissue. This is most probably 
happening due to ice crystal formation. We're not using cryospray, relying only 
on the cryobar boost function.


Does anyone has a solution to this problem please?


Regards,



Dorianne Bonello
Allied Health Practitioner (MLS)
Histology Laboratory - Pathology
Health-Mater Dei Hospital


[cid:image001.jpg@01D67184.63288530]


T +356 +356 25456434

E dorianne.m.bone...@gov.mt


Mater Dei Hospital, Triq id-Donaturi tad-Demm, l-Imsida, Malta MSD 2090 | Tel 
+356 2545  | 
https://deputyprimeminister.gov.mt/en/MDH/Pages/Home.aspx
 | https://www.facebook.com/materdeihospital/


Think before you print. Kindly consider your environmental responsibility.

This email and any files transmitted with it are confidential, may be legally 
privileged and intended solely

for the use of the individual or entity to whom they are addressed.






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[Histonet] Job Opportunity in Tampa FL

[Newman-Bodden, Cheryl L. via Histonet]

External JOA Link: https://www.usajobs.gov/GetJob/ViewDetails/607696600 

VA in Tampa has several openings for HTL's with a four year degree.
See listing above for details

Cheryl  Bodden, BS, HTL(ASCP)
Histology Supervisor
Supervisor Health Science Specialist
Pathology and Laboratory Medicine
James A. Haley Veterans Hospital
13000 Bruce B. Downs Blvd.
Tampa, FL 33612
(813) 972-2000 ext. 7844

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Re: [Histonet] Histonet Digest, Vol 212, Issue 7

[richard.wild--- via Histonet]

Message: 1
Date: Wed, 14 Jul 2021 22:03:24 + (UTC)
From: Cheryl 
To: "histonet@lists.utsouthwestern.edu"

Subject: [Histonet] PathCentre (ThermoShandon) protocols - help?
Message-ID: <7393119.276682.1626300204...@mail.yahoo.com>
Content-Type: text/plain; charset=UTF-8


Help?? Starting a refurbished PathCentre and was hoping someone could share a 
biopsy protocol for xylene processing?? Routine human biopsies with specials 
and some IHC and molecular down the road.
Open to all -?
Much appreciated!!
Cheryl Kerry, HT(ASCP)


Hi Cherryl

it's a good machine (we have 3 of them)

Here under some "official" protocole

If unreadable, i'll send you pdf - just ask for it

Richard Wild

-

SHANDON PATHCENTRE PROCESSING SCHEDULE
RAPID BIOPSY PROGRAMME TOTAL DURATION 1hr 14mins
STEP REAGENT CONC
%
TEMP
°C
VAC/PRES TIME
Hrs:Mins
DRAIN AGIT
1 Formalin 10 45 V 00:01 15 S
2 Formalin 70 45 V 00:01 15 S
3 Alcohol 95 45 V 00:01 15 S
4 Alcohol 100 45 V 00:01 15 S
5 Alcohol 100 45 V 00:01 15 S
6 Alcohol 100 45 V 00:01 15 S
7 Alcohol 100 45 V 00:01 15 S
8 Xylene 45 V 00:01 15 S
9 Xylene 45 V 00:01 15 S
10 Xylene 45 V 00:01 15 S
11 Wax 60 V 00:01 60 S
12 Wax 60 V 00:01 60 S
13 Wax 60 V 00:01 60 S
14 Wax 60 V 00:01 60 S

SHANDON PATHCENTRE PROCESSING SCHEDULE
RAPID PROCESSING TOTAL DURATION 0hr 56mins
STEP REAGENT CONC
%
TEMP
°C
VAC/PRES TIME
Hrs:Mins
DRAIN AGIT
1 Formalin 10 45 V 00:02 15 S
2 Formalin 70 45 V 00:02 15 S
3 Alcohol 95 45 V 00:02 15 S
4 Alcohol 00:00 S
5 Alcohol 00:00 S
6 Alcohol 100 45 V 00:02 15 S
7 Alcohol 100 45 V 00:02 15 S
8 Xylene 00:00 S
9 Xylene 45 V 00:02 15 S
10 Xylene 45 V 00:02 15 S
11 Wax 60 00:00 S
12 Wax 60 00:00 S
13 Wax 60 V 00:02 60 S
14 Wax 60 V 00:02 60 S

SHANDON PATHCENTRE PROCESSING SCHEDULE
LARGE SURGICALS TOTAL DURATION 22hrs 34mins
STEP REAGENT CONC
%
TEMP
°C
VAC/PRES TIME
Hrs:Mins
DRAIN AGIT
1 Buffered Formalin 10 A N 02:00 15 S
2 Alcohol Formalin 10 A N 02:00 15 S
3 90% IMS * 60 A V 01:30 15 S
4 IMS 80 A V 01:30 15 S
5 IMS A V 01:30 15 S
6 IMS A V 01:30 15 S
7 Xylene/IMS A V 01:30 15 S
8 Xylene A V 01:30 30 S
9 Xylene A V 01:30 60 S
10 Xylene A V 01:30 60 S
11 Wax 60 V 01:00 120 S
12 Wax 60 V 01:00 120 S
13 Wax 60 V 01:00 120 S
14 Wax 60 V 01:30 120 S





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