[Histonet] Processing artifact - delayed start

[Tony Henwood]

I would check the level of the formalin after it has been pumped into the retort.

I will wait till the pics are posted

Regards,

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) (Retired)
Principal Scientist, the Children’s Hospital at Westmead (Retired)
Adjunct Fellow, School of Medicine, University of Western Sydney.

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Sent: Saturday, February 3, 2024 2:36:42 PM
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Processing artifact - delayed start

Dear Histonet Members,

We have terrible processing artifact if tissue sits in the formalin-filled retort (at ambient temperature) for too long (more than 10-12 hours) before a delayed process starts. The longer the wait, the worse it looks. We have Tissue Tek VIP 5 processors, and we process luminal gastrointestinal biopsies exclusively. I've attached some photomicrographs of problem cases on the Histonet Images website (with the same topic title).
This artifact typically affects a few specimens per day (~2% or less), even though everything is done on the same processor; it may affect all tissue portions in a cassette or only some of the tissue in that cassette. Some tissue portions may only have the artifact on one half with the other half looking perfectly fine. The techs sometimes note the tissue feeling "crunchy" at the time of embedding and / or at the time of microtomy. These tissues tend to suffer greater chatter artifact and have trouble sticking to the slides. The sections look just as bad on recuts as the originals. Re-processing does not seem to help at all.
If the cassettes sit in formalin in a container outside of the processor for days before the processor is loaded (with subsequent immediate start), things look perfectly fine. When we have staff around to start the processor immediately upon loading cassettes and empty immediately upon completion, the tissue looks perfectly fine.

Our current processing program is as follows:

1. 10% Formalin, 30 minutes, ambient temp, p/v on
2. 10% Formalin, 30 minutes, ambient temp, p/v on
3. 65% Alcohol, 10 minutes, ambient temp, p/v on4. 80% Alcohol, 10 minutes, ambient temp, p/v on5. 95% Alcohol, 7 minutes, ambient temp, p/v on6. 95% Alcohol, 7 minutes, ambient temp, p/v on 7. 100% Alcohol, 10 minutes, ambient temp, p/v on
8. 100% Alcohol, 10 minutes, ambient temp, p/v on
9. Xylene, 15 minutes, ambient temp, p/v on
10. Xylene, 15 minutes, ambient temp, p/v on
11. Paraffin, 10 minutes, 60 degrees C, p/v on12. Paraffin, 10 minutes, 60 degrees C, p/v on13. Paraffin, 15 minutes, 60 degrees C, p/v on14. Paraffin, 15 minutes, 60 degrees C, p/v on

Obviously there are times we absolutely need to use delayed start. I would greatly appreciate guidance, and I'll be happy to provide any other details that might be useful.
Sincerely,
Brian Quigley MDLaboratory Director of a GI Pathology Laboratory

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