[Histonet] Processing Schedule- ASP-6025

[John Garratt]

The processing schedule looks fine. I am thinking that if you are having no problems with morphology on the H&E processing is not the issue. The extended period in the warming draw is a good hypothesis.
I do have a question. How long was that particular piece of tonsil kept in formalin before processing? Could over fixation be the issue?
I have found that maintaining tight control of control tissues is important which includes minimum and maximum fixation time.

John

Sent from ProtonMail Mobile

On Tue, May 7, 2019 at 5:52 PM, Greg Dobbin via Histonet <histonet at lists.utsouthwestern.edu> wrote:

> Fascinating Tony!
> We don’t typically leave them in the warming drawer any longer than a
> couple of hours, but maybe this particular tonsil was left linger for some
> reason and no one thought anything of it?! Something to consider for sure!
> Thanks.
> Greg
>
> On Tue, May 7, 2019 at 9:37 PM Tony Henwood (SCHN) <
> tony.henwood at health.nsw.gov.au> wrote:
>
>> Processing seems adequate.
>>
>> After processing, how long do they sit in the embedding centre block
>> holding tank before embedding?
>>
>> We found that quite a few antigens were affected when we stored control
>> tonsil in the embedding centre (dry) at 60oC for a few days before
>> embedding. In summary:
>>
>> Antibody Clone Dried (Normal = 3+)
>> CD4 4B12 0
>> BOB-1 TG14 0
>> CD3 LN10 1+
>> CD79a JCB117 1+
>> Oct-2 Oct-207 1+
>> CD8 4B11 2+
>> CD20 L26 3+
>>
>> So CD20 was unaffected but this process affected most of the antigens with
>> some losing antigen recognition by the antibody (eg CD4 and BOB-1).
>>
>> Another one of those pre-analytical issues we need to consider.
>>
>> And yes I am writing this up for submission!
>>
>>
>> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) |
>> Principal Scientist; Adjunct Fellow, School of Medicine, University of
>> Western Sydney; Visiting Lecturer, School of Life Sciences, Faculty of
>> Science, University of Technology Sydney | Histopathology
>> t: (02) 9845 3306 | f: (02) 9845 3318 | e: tony.henwood at health.nsw.gov.au
>> | w: www.schn.health.nsw.gov.au
>> m:
>>
>>
>> Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
>> Locked Bag 4001, Westmead 2145, NSW Australia
>>
>> ♲ Please consider the environment before printing this email.
>>
>> -----Original Message-----
>> From: Greg Dobbin via Histonet [mailto:histonet at lists.utsouthwestern.edu]
>> Sent: Wednesday, 8 May 2019 5:07 AM
>> To: histonet at lists.utsouthwestern.edu
>> Subject: [Histonet] Processing Schedule- ASP-6025
>>
>> Hello colleagues,
>> I recently stained (IHC) a section of normal tonsil from another facility
>> with p16 and the resulting stain was better than the same stain on a
>> section of my labs own normal tonsil control.
>>
>> This has led us to question our processing schedule. I am not concerned
>> with our fixation because we fix everything for at least 24 hours in 10%
>> formalin (commercially prepared) prior to processing.
>>
>> Does anything jump out at you as being a potential red flag in the
>> following overnight protocol?
>>
>> - Formalin 15 mins; RT
>> - Processing water 1 min; RT
>> - ETOH 70% 30 mins; 35C
>> - ETOH 80% 30 mins; 35C
>> - ETOH 95% 30 mins; 35C
>> - ETOH 100% 30 mins; 35C
>> - ETOH 100% 40 mins; 35C
>> - ETOH 100% 60 mins; 35C
>> - Xylene 60 mins; 35C
>> - Xylene 60 mins; 35C
>> - Xylene 60 mins; 35C
>> - Paraffin 60 mins; 57C; vacuum
>> - Paraffin 60 mins; 57C; vacuum
>> - Paraffin 60 mins; 57C; vacuum
>>
>> Our formalin is changed after every 1100 cassettes and the alcohol,
>> xylenes and paraffins are managed similarly by the instrument. Our specimen
>> mix is a little of everything (skins, GIs, breasts, needle cores, gall
>> bladders, gyne, etc).
>>
>> The one unknown (so far) in this story, is how the tonsil from the other
>> laboratory was handled (ie the fixative used and for how long-I am assuming
>> 10% formalin).
>>
>> Obviously, many of you will have schedules that differ from this one, in
>> any number of ways, but what I am looking for from you is your opinion: *is
>> there anything about this schedule that is particularly concerning?* Thank
>> you, Greg
>>
>>
>> --
>> *Greg Dobbin*
>> 1205 Pleasant Grove Rd
>> <https://maps.google.com/?q=1205+Pleasant+Grove+Rd&entry=gmail&source=g>
>> RR#2 York,
>> PE C0A 1P0
>>
>>
>> *Everything in moderation...even moderation itself**!*
>> _______________________________________________
>> Histonet mailing list
>> Histonet at lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>> This message is intended for the addressee named and may contain
>> confidential information. If you are not the intended recipient, please
>> delete it and notify the sender.
>>
>> Views expressed in this message are those of the individual sender, and
>> are not necessarily the views of NSW Health or any of its entities.
>>
> --
> *Greg Dobbin*
> 1205 Pleasant Grove Rd
> RR#2 York,
> PE C0A 1P0
>
> *Everything in moderation...even moderation itself**!*
> _______________________________________________
> Histonet mailing list
> Histonet at lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet