Re: [Histonet] Cell block processing

[Tony Henwood (SCHN) via Histonet]

Hi Charles,

I have had excellent success with lysing the red blood cells (using  Isotonic 
Ammonium Chloride) prior to cell block preparation with thromboplastin-plasma. 
The lysing solution contains EDTA so you will need to add a few drops of 1% 
calcium chloride. Method as follows:

Lysis solution
Ammonium Chloride4.5g
Potassium carbonate  0.5g
EDTA 0.0186g
Distilled water  500mls

Method:
1.  Centrifuge bloody fluid.
2.  Remove supernatant and add equal volume of lysis solution.
3.  Resuspend and incubate for 5 minutes at 4oC.
4.  Centrifuge, if blood still remains, then repeat from step 2.
5. Rinse in Hanks or RPMI, centrifuge.
6.  Mix pellet in a few drops of plasma.
7. Add thromboplastin and a few drops  of 1% Calcium Chloride, mix gently 
and allow clot to form.
8. Add 10% buffered formalin and fix and process as usual.

Reference:  
Kuenen-Boumiester etal (1996) Acta Cytolog 40:475-479

The lysis solution can also be purchased commercially from several companies 
(eg Biolegend). It is commonly used for sample preparation for flow cytometry. 
Check the SDS to make sure it does not contain formaldehyde.


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: Charles Riley via Histonet 
Sent: Friday, 25 October 2019 23:12
To: Histo List
Subject: [Histonet] Cell block processing

Does anyone have any tips or suggestions on how to better process extremely
bloody FNA  specimens?Is there anyway to clear out some or all of the
blood without destroying the other tissues?

--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] Cell block processing

[Cartun, Richard via Histonet]

I agree with Joe.  We used to use ETOH for cell blocks, but stopped using it 
when we started doing IHC biomarker testing on these specimens.  Alcohol is 
good for some proteomic targets, but can be a disaster for others.  We also fix 
all of our cell block specimens that are collected in saline or RPMI in 
formalin.

Richard

Richard W. Cartun, MS, PhD
Director, Histology & The Martin M. Berman, MD Immunopathology/Morphologic 
Proteomics Laboratory
Director, Biospecimen Collection Programs
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 972-1596 (Office)
(860) 545-2204 (Fax)
richard.car...@hhchealth.org

-Original Message-
From: Joe W. Walker, Jr. via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Friday, October 25, 2019 4:36 PM
To: Charles Riley
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Cell block processing

EXTERNAL email is from outside HHC. DO NOT open attachments or click links from 
unknown senders.

As a cytotech, that wouldn’t be my first choice for collections and FNA 
specimens.  The main reason is that once fixed in 95% ETOH you are limited if 
you need to perform IHC stains on the cell block unless you have validated your 
IHCs on ETOH fixed specimens.  How do you process the FNA rinses that in in 
ETOH: Only Cell blocks or do you have another cytology liquid prep?



Without knowing your prep process, I’d suggest collecting the FNA needle rinses 
in Hank’s Balanced Salt solution.  After making the cell block, you could then 
formalin fix them.  I can send you a procedure that we utilize for this 
process.  The cell blocks cut great, look great, and you can perform IHC an 
molecular testing if needed.



Joe Walker



From: Charles Riley 

Sent: Friday, October 25, 2019 12:57 PM

To: Joe W. Walker, Jr. 

Cc: histonet@lists.utsouthwestern.edu

Subject: Re: [Histonet] Cell block processing



[External Email] This email originated from outside of the organization. Think 
before you click: Don’t click on links, open attachments or respond to requests 
for sensitive information if the email looks suspicious or you don’t recognize 
the sender.



Our tech said they use 95% alcohol to collect the specimen.



On Fri, Oct 25, 2019 at 12:23 PM Joe W. Walker, Jr. 
mailto:jwwal...@rrmc.org>> wrote:

Hi Charles,



What are you collecting the FNA into?  Cytorich? Cytolyt? Other?



Joe W. Walker, Jr. MS, SCT(ASCP)

Anatomical Pathology Manager

joewal...@rrmc.org, 
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.rrmc.org=DwIGaQ=e_HtEeZEQXP5NUOb33qoTj0AVvRFBS9_rhBTQcfkWoA=eCMS5E4UqfGKTlPknIMjMlCkk-KGKHflslRsFz3l5JE=2CdTK8PmO1-CXFcs6R-WWhnxgbCm1AHZ9wJ_1YOqDoA=JIavq_Wfz4S4TppCsMv8kQfnmD9wLQhRVNqSm1eJAO4=
 




-Original Message-

From: Charles Riley via Histonet 
mailto:histonet@lists.utsouthwestern.edu>>

Sent: Friday, October 25, 2019 8:13 AM

To: Histo List 
mailto:histonet@lists.utsouthwestern.edu>>

Subject: [Histonet] Cell block processing



[External Email] This email originated from outside of the organization. Think 
before you click: Don’t click on links, open attachments or respond to requests 
for sensitive information if the email looks suspicious or you don’t recognize 
the sender.





Does anyone have any tips or suggestions on how to better process extremely

bloody FNA  specimens?Is there anyway to clear out some or all of the

blood without destroying the other tissues?



--



Charles Riley BS  HT, HTL(ASCP)CM



Histopathology Coordinator/ Mohs

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Re: [Histonet] waterbath/paraffin

[Tony Henwood (SCHN) via Histonet]

Are the tissues taken directly from the molten wax and placed in the 
wax-containing mold or are they allowed to cool before embedding?

One cause of tissue separation is the difference in temperature between tissue 
and embedding wax. 
Try taking directly from molten wax and embedding directly in the wax-filled 
mold.

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: Betsy Molinari via Histonet 
Sent: Saturday, 26 October 2019 00:49
To: 'Histonet@lists.utsouthwestern.edu'
Subject: [Histonet] waterbath/paraffin

Hi,
When I place my sections on the waterbath the paraffin pulls away from it. 
Leaving just the edge of the tissue. It is very weird. I have tried at 
different temps . The tissue itself is fine so I do not believe it is the 
processing. I use the same paraffin for processing as well as embedding. I use 
Fisher Histoplast IM.  I was considering melting a block or two down and 
embedding them in another paraffin from another lab.
Suggestions?

Betsy Molinari HT, ASCP
Texas Heart Institute
6770 Bertner Ave.
O-511
Houston, TX 77030
832-355-6524 (lab)
832-355-6812 (fax)

Betsy Molinari
Sr. Histology Research Technician
CV Pathology Research

Texas Heart Institute
6770 Bertner Avenue, MC 1-283
Houston, TX 77030

Office: 832-355-6524 | Fax: 832-355-6812
Email: bmolin...@texasheart.org
texasheart.org | 
facebook | 
twitter

This email and attachments contain information that may be confidential or 
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the sender.

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Re: [Histonet] Cell block processing

[Joe W. Walker, Jr. via Histonet]

As a cytotech, that wouldn’t be my first choice for collections and FNA 
specimens.  The main reason is that once fixed in 95% ETOH you are limited if 
you need to perform IHC stains on the cell block unless you have validated your 
IHCs on ETOH fixed specimens.  How do you process the FNA rinses that in in 
ETOH: Only Cell blocks or do you have another cytology liquid prep?

Without knowing your prep process, I’d suggest collecting the FNA needle rinses 
in Hank’s Balanced Salt solution.  After making the cell block, you could then 
formalin fix them.  I can send you a procedure that we utilize for this 
process.  The cell blocks cut great, look great, and you can perform IHC an 
molecular testing if needed.

Joe Walker

From: Charles Riley 
Sent: Friday, October 25, 2019 12:57 PM
To: Joe W. Walker, Jr. 
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Cell block processing

[External Email] This email originated from outside of the organization. Think 
before you click: Don’t click on links, open attachments or respond to requests 
for sensitive information if the email looks suspicious or you don’t recognize 
the sender.

Our tech said they use 95% alcohol to collect the specimen.

On Fri, Oct 25, 2019 at 12:23 PM Joe W. Walker, Jr. 
mailto:jwwal...@rrmc.org>> wrote:
Hi Charles,

What are you collecting the FNA into?  Cytorich? Cytolyt? Other?

Joe W. Walker, Jr. MS, SCT(ASCP)
Anatomical Pathology Manager
joewal...@rrmc.org, 
www.rrmc.org

-Original Message-
From: Charles Riley via Histonet 
mailto:histonet@lists.utsouthwestern.edu>>
Sent: Friday, October 25, 2019 8:13 AM
To: Histo List 
mailto:histonet@lists.utsouthwestern.edu>>
Subject: [Histonet] Cell block processing

[External Email] This email originated from outside of the organization. Think 
before you click: Don’t click on links, open attachments or respond to requests 
for sensitive information if the email looks suspicious or you don’t recognize 
the sender.


Does anyone have any tips or suggestions on how to better process extremely
bloody FNA  specimens?Is there anyway to clear out some or all of the
blood without destroying the other tissues?

--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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[https://www.rrmc.org/app/files/public/2633/2019_hyht_sig-_jan2019_final.jpg]


--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs


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Re: [Histonet] Questions on how to operate a ThermoFisher Lab Vision autostainer 3602D

[Jeanine Ronkowski via Histonet]

Katherine:Our lab did the same thing a few years ago and I received operating 
instructions for this system from a sales rep with Biocare that I know.  I'll 
send this document to you offline.  I can also put you in touch with the guy 
from Biocare.  As Colleen said, Biocare doesn't sell or support this instrumemt 
any more, but you can still get reagents from them.  I know of only three 
companies that can provide service and PMs.  Let me know if you need that info 
too.Good Luck,Jeanine
(jeanine.ronkow...@yahoo.com)
* * * * * * * * * * * * * * *
Hello Histonetters,

My company has acquired a refurbished ThermoFisher Lab Vision autostainer 3602D 
and I have questions on how to operate.  It is no longer supported by 
ThermoFisher so I am unable to get tech support through them.  Is there anyone 
who is familiar and available/willing to answer a few questions?

Thank you in advance.

Kate

Katherine Bummer
Nektar Therapeutics
455 Mission Bay Boulevard South
San Francisco, CA  94158
k.bum...@nektar.com
415.482.5716
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Re: [Histonet] Cell block processing

[Charles Riley via Histonet]

Our tech said they use 95% alcohol to collect the specimen.

On Fri, Oct 25, 2019 at 12:23 PM Joe W. Walker, Jr. 
wrote:

> Hi Charles,
>
> What are you collecting the FNA into?  Cytorich? Cytolyt? Other?
>
> Joe W. Walker, Jr. MS, SCT(ASCP)
> Anatomical Pathology Manager
> joewal...@rrmc.org, www.rrmc.org
>
> -Original Message-
> From: Charles Riley via Histonet 
> Sent: Friday, October 25, 2019 8:13 AM
> To: Histo List 
> Subject: [Histonet] Cell block processing
>
> [External Email] This email originated from outside of the organization.
> Think before you click: Don’t click on links, open attachments or respond
> to requests for sensitive information if the email looks suspicious or you
> don’t recognize the sender.
>
>
> Does anyone have any tips or suggestions on how to better process extremely
> bloody FNA  specimens?Is there anyway to clear out some or all of the
> blood without destroying the other tissues?
>
> --
>
> Charles Riley BS  HT, HTL(ASCP)CM
>
> Histopathology Coordinator/ Mohs
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
>
> https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.com%2Fv3%2F__http%3A%2F%2Flists.utsouthwestern.edu%2Fmailman%2Flistinfo%2Fhistonet__%3B!5JlSGkcjda6Eqs5J!rntZr6E7tVvCc2tzoJy_L8lFMF6EvHUTKJOsc3NTMLFSI35SgrcseW2ly3GyamY%24data=02%7C01%7Cjwwalker%40rrmc.org%7C6e4cdd79edfa4e1ef23408d75944c458%7C0e55647d438e4a448437e959c3cf2240%7C0%7C0%7C637076024216531651sdata=xAxqyyCfRepB3DlAl%2Fw651nk3B5ViHQLjqdToa2iAhw%3Dreserved=0
> [
> https://www.rrmc.org/app/files/public/2633/2019_hyht_sig-_jan2019_final.jpg
> ]
>


-- 

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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Re: [Histonet] Cell block processing

[Joe W. Walker, Jr. via Histonet]

Hi Charles,

What are you collecting the FNA into?  Cytorich? Cytolyt? Other?

Joe W. Walker, Jr. MS, SCT(ASCP)
Anatomical Pathology Manager
joewal...@rrmc.org, www.rrmc.org

-Original Message-
From: Charles Riley via Histonet 
Sent: Friday, October 25, 2019 8:13 AM
To: Histo List 
Subject: [Histonet] Cell block processing

[External Email] This email originated from outside of the organization. Think 
before you click: Don’t click on links, open attachments or respond to requests 
for sensitive information if the email looks suspicious or you don’t recognize 
the sender.


Does anyone have any tips or suggestions on how to better process extremely
bloody FNA  specimens?Is there anyway to clear out some or all of the
blood without destroying the other tissues?

--

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
___
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[Histonet] waterbath/paraffin

[Betsy Molinari via Histonet]

Hi,
When I place my sections on the waterbath the paraffin pulls away from it. 
Leaving just the edge of the tissue. It is very weird. I have tried at 
different temps . The tissue itself is fine so I do not believe it is the 
processing. I use the same paraffin for processing as well as embedding. I use 
Fisher Histoplast IM.  I was considering melting a block or two down and 
embedding them in another paraffin from another lab.
Suggestions?

Betsy Molinari HT, ASCP
Texas Heart Institute
6770 Bertner Ave.
O-511
Houston, TX 77030
832-355-6524 (lab)
832-355-6812 (fax)

Betsy Molinari
Sr. Histology Research Technician
CV Pathology Research

Texas Heart Institute
6770 Bertner Avenue, MC 1-283
Houston, TX 77030

Office: 832-355-6524 | Fax: 832-355-6812
Email: bmolin...@texasheart.org
texasheart.org | 
facebook | 
twitter

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[Histonet] Instrument feed water

[Charles Riley via Histonet]

Where does everyone purchase their IFW from?  or how do you produce it in
house?

-- 

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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[Histonet] Cell block processing

[Charles Riley via Histonet]

Does anyone have any tips or suggestions on how to better process extremely
bloody FNA  specimens?Is there anyway to clear out some or all of the
blood without destroying the other tissues?

-- 

Charles Riley BS  HT, HTL(ASCP)CM

Histopathology Coordinator/ Mohs
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