Re: [Histonet] Processing Schedule- ASP-6025
What are you using for your lab's own control. Was that tissue processed recently? Do you have normal tonsil that you process in-house regularly that also stains poorly? On Wed, May 8, 2019 at 9:16 AM Greg Dobbin via Histonet < histonet@lists.utsouthwestern.edu> wrote: > Great point John! > We do attempt to have all of the specimen handling for our control tissue, > match that of our specimens. However, > tonsillectomies at our hospital are only performed on Thursdays. So we fix > them for 24hrs (until Friday) then process overnight and remove > from the processor on Saturday (we don't typically work Saturdays). And I > have done this a couple of times but can I swear that this particular > tonsil > was handled that way? I cannot ...which I know, points to another > deficiency! :-0 It could very well be that the fixation times of the two > tonsils I am comparing are sufficiently different that they will require > different retrieval times to produce stains of similar intensity. > > I also appreciate getting your opinion on the processing schedule. You have > much more experience than I!! Thank you for your insights. > All the best, > Greg > > On Wed, May 8, 2019 at 10:50 AM John Garratt wrote: > > > The processing schedule looks fine. I am thinking that if you are having > > no problems with morphology on the H 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@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.henw...@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.henw...@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@lists.utsouthwestern.edu > > ] > > > Sent: Wednesday, 8 May 2019 5:07 AM > > > To: histonet@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
Re: [Histonet] Processing Schedule- ASP-6025
Great point John! We do attempt to have all of the specimen handling for our control tissue, match that of our specimens. However, tonsillectomies at our hospital are only performed on Thursdays. So we fix them for 24hrs (until Friday) then process overnight and remove from the processor on Saturday (we don't typically work Saturdays). And I have done this a couple of times but can I swear that this particular tonsil was handled that way? I cannot ...which I know, points to another deficiency! :-0 It could very well be that the fixation times of the two tonsils I am comparing are sufficiently different that they will require different retrieval times to produce stains of similar intensity. I also appreciate getting your opinion on the processing schedule. You have much more experience than I!! Thank you for your insights. All the best, Greg On Wed, May 8, 2019 at 10:50 AM John Garratt wrote: > The processing schedule looks fine. I am thinking that if you are having > no problems with morphology on the H 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@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.henw...@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.henw...@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@lists.utsouthwestern.edu > ] > > Sent: Wednesday, 8 May 2019 5:07 AM > > To: histonet@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;
Re: [Histonet] Processing Schedule- ASP-6025
The processing schedule looks fine. I am thinking that if you are having no problems with morphology on the H 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 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.henw...@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.henw...@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@lists.utsouthwestern.edu] >> Sent: Wednesday, 8 May 2019 5:07 AM >> To: histonet@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=gmail=g> >> RR#2 York, >> PE C0A 1P0 >> >> >> *Everything in moderation...even moderation itself**!* >> _
Re: [Histonet] Processing Schedule- ASP-6025
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: AntibodyClone Dried (Normal = 3+) CD4 4B120 BOB-1 TG140 CD3 LN101+ CD79a JCB117 1+ Oct-2 Oct-207 1+ CD8 4B112+ CD20L26 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.henw...@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@lists.utsouthwestern.edu] Sent: Wednesday, 8 May 2019 5:07 AM To: histonet@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**!* ___ Histonet mailing list Histonet@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. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[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**!* ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
