[Histonet] Processing Schedule- ASP-6025

[Tony Henwood (SCHN)]

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
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-----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
RR#2 York,
PE      C0A 1P0


*Everything in moderation...even moderation itself**!*
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