RE: [Histonet] FoxP3 and Geminin Immunohistochemistry
Hello Steven, I have used both of these antibodies for various projects and we performed all of these on the Bondmax from Leica with a 60 minute antibody incubation. We have used two FoxP3 antibodies from eBiosciences; cat# 14-4777 on human tissue and cat# 13-5773 for mouse tissue. (both were 1:100 dilutions and ER2 for 20 mins (EDTA pH9). I have only used Geminin on human tissue. It was from Proteintech Group cat# 10802-1-AP (1:1500 dilution using ER1 for 15 min-ER1=citrate pH6) All of these were on FFPE tissues for brightfield only. We did not use them for IF. If you need any other specifics, let me know. Ashley Troutman BS, MBA, HT(ASCP)QIHC Supervisor-Translational Pathology Shared Resource Vanderbilt University Medical Center S-1310 Medical Center North 1161 21st Avenue South Nashville, TN 37232 Message: 2 Date: Tue, 2 Dec 2014 19:37:51 + From: Swartwood, Steven J steven.swartw...@cshs.orgmailto:steven.swartw...@cshs.org Subject: [Histonet] FoxP3 and Geminin Immunohistochemistry To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: 959202ac61aef942968646ec66e2be3e3bf09...@espwmsgmbx07.csmc.edumailto:959202ac61aef942968646ec66e2be3e3bf09...@espwmsgmbx07.csmc.edu Content-Type: text/plain; charset=us-ascii Hello all, I've done some research on Foxp3 and Geminin and I've seen a few antibodies that look sufficient for IHC in FFPE tissues. My lab is currently looking into purchasing these antibodies for a few different projects. Does anyone on here use these two antibodies for IHC in FFPE tissues? I'm going to use them in bright field, but we may also turn to IF for other studies. I'd be grateful for any information from what clone/company do you buy from and retrieval methods to primary antibody incubations/dilutions. Thank you all for any information, Steven Swartwood HT(ASCP) Cedars Sinai Medical Center steven.swartw...@cshs.orgmailto:steven.swartw...@cshs.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Churukian Silver stain for Fungus
In my experience, periodic acid will not help you demonstrate histoplasma. Chromic acid should be the preferred oxidizer if histoplasmosis is suspected. I have had good results with periodic acid for pneumocystis and aspergillus, but not histoplasma. Ashley Troutman BS, MBA, HT(ASCP)QIHC Supervisor-Translational Pathology Shared Resource Vanderbilt University Medical Center S-1310 Medical Center North 1161 21st Avenue South Nashville, TN 37232 Message: 11 Date: Thu, 5 Jun 2014 09:29:29 -0400 From: Terri Braud tbr...@holyredeemer.commailto:tbr...@holyredeemer.com Subject: [Histonet] RE: Churukian Silver stain for Fungus To: Tony Henwood (SCHN) tony.henw...@health.nsw.gov.aumailto:tony.henw...@health.nsw.gov.au, histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: BFBF7C6B9627B947B74CBEBD45CE368B1359E4A3@hrex-svr.holyredeemer.localmailto:BFBF7C6B9627B947B74CBEBD45CE368B1359E4A3@hrex-svr.holyredeemer.local Content-Type: text/plain;charset=us-ascii I beg to differ. I think you are confusing stains. I am not talking about using a Periodic Acid Schiff's stain (PAS) to stain fungus. I am referring to the Churukian Microwave Ammoniacal Silver stain for fungus. It oxidizes with Periodic Acid and the Silver solution is similar to what is used for most Reticulum stains. It stains the exact same organisms as a Grocott's Methenamine Silver (GMS), just without staining the elastic fibers. Our pneumocystis stains using the Churukian Ammoniacal Silver stain are just beautiful. This method has been taught at NSH workshops and has been widely used in published literature. As soon as I can figure out how to post pictures, I will send some pictures of a a pneumocystis control and an aspergillus control stained with the Churukian method. Terri L. Braud, HT(ASCP) Anatomic Pathology Supervisor Holy Redeemer Hospital Laboratory 1648 Huntingdon Pike Meadowbrook, PA 19046 Ph: 215-938-3676 Fax: 215-938-3874 -Original Message- From: Tony Henwood (SCHN) [mailto:tony.henw...@health.nsw.gov.au] Sent: Wednesday, June 04, 2014 7:20 PM To: Terri Braud; histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: RE: Acid Clean Glassware No, Using Periodic acid instead of chromic acid just gives you a PASM. Pseudo-fungi are PAS Positive but classic GMS (using chromic acid) negative. The literature is quite confusing on pseudo-fungi. Some say that they are GMS positive whereas other claim they are GMS negative. My own experience is that they are GMS (using chromic acid) negative. It is possible that our pathologists aren't aware that the GMS that their lab does might be using Periodic acid instead of Chromic acid. Commercial kits (eg Sigma and Richard-Allan) use periodic acid instead of chromic acid. The literature often does not report the exact GMS used which makes clear understanding of the histochemical results difficult. Pneumocystis will not be easy to see unless chromic acid is used (the mucin stains strongly PAS (and hence PASM) positive obscuring the small microorganisms). Using PAS, Old fungi, Mucor, Actinomyces and Nocordia do not stain well whereas they stain quite well with GMS. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior 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 -Original Message- From: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Terri Braud Sent: Thursday, 5 June 2014 5:58 AM To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: [Histonet] RE: Acid Clean Glassware Chromic acid does a nifty job of removing metal deposits on glassware, but so do many commercial lab detergents. Chromic acid is the oxidizer for the fungus in the GMS stain. Go one better and get rid of Chromic Acid out of your lab. It is probably one of the more toxic / nasty chemicals in your department. Instead, try Churukian's Ammoniacal Silver for Fungus in the microwave. It is a much simpler, faster, prettier stain. It uses Periodic Acid as the oxidizer and does not stain the elastic fibers like a regular GMS. Both you and your pathologists will love it, I promise. Terri L. Braud, HT(ASCP) Anatomic Pathology Supervisor Holy Redeemer Hospital Laboratory 1648 Huntingdon Pike Meadowbrook, PA 19046 Ph: 215-938-3676 Fax: 215-938-3874 Today's Topics: ___ Histonet mailing list Histonet@lists.utsouthwestern.edu
[Histonet] Mouse NK cell marker
Good day, colleagues! Does anyone have a good Natural Killer cell marker for mouse tissue? Any antibody recommendations/protocol information would be a big help as well. This is for IHC, not IF. Thanks in advance! Ashley Troutman BS, MBA, HT(ASCP)QIHC Supervisor-Translational Pathology Shared Resource Vanderbilt University Medical Center S-1310 Medical Center North 1161 21st Avenue South Nashville, TN 37232 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] The Twelve Days of Histo
Hello all, I hope the holidays are treating everyone well. (Especially in light of us having the most hazardous job in the country...) Here's some fun for the season: The Twelve Days of Histo TWELVE Medical Directors ELEVEN logs-a-printing TEN residents reading NINE frozen sections EIGHT administrators SEVEN stainers beeping SIX techs complaining FIVE I...H...Cs :) FOUR calling docs THREE recuts TWO special stains And a block with an H and E... Please feel free to sing along. Merry Christmas in Histoland! Ashley Troutman BS, MBA, HT(ASCP) QIHC Histopathology and Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 ashley.trout...@vanderbilt.edumailto:ashley.trout...@vanderbilt.edu ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Monitoring of IHC staining trends
Hello Histonet, I have a question for the group at large. How are labs monitoring drift in IHC staining over time? Here's the scenario: You do lot to lot testing and everything looks fine until one day your pathologists are telling you that the CAM5.2 is too dark. Now, you've been looking at these slides every day for the last year and, sure enough, when you pull out a slide from last year's lot, it is significantly lighter. So what do we do about it? Do we revalidate the stain? Does anyone have a mechanism to monitor this better? What is the threshold for revalidation? Feedback from techs as well as any pathologists would be greatly appreciated. Regards, Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 ashley.trout...@vanderbilt.edumailto:ashley.trout...@vanderbilt.edu ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Validation of Her2
Hi Jim, The way I understand this guideline, you will need to send it to a lab that uses the same Ventana clone and instrument that you use. Regards, Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 ashley.trout...@vanderbilt.edumailto:ashley.trout...@vanderbilt.edu Message: 5 Date: Tue, 19 Mar 2013 17:33:03 -0500 From: Vickroy, Jim vickroy@mhsil.commailto:vickroy@mhsil.com Subject: [Histonet] Validation of Her2 To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: bb0b9f1a8373f14fa2974e8cb24bf9cf2adfb...@mmc-mail.ad.mhsil.commailto:bb0b9f1a8373f14fa2974e8cb24bf9cf2adfb...@mmc-mail.ad.mhsil.com Content-Type: text/plain; charset=us-ascii We are working on validating HER 2 testing in our laboratory. I see that the guidelines state that we should use 25 - 100 cases to complete our validation. In the past we sent our Her2 IHC testing to Clarient and they performed the technical testing and then our pathologists would do the scoring. So for validation we used 25 of the cases we sent to Clarient and then did them in house to compare. Our comparison was nearly 100%. Here is my question: Clarient uses the DAKO method while we are using Ventana's antibody Pathway (4B5). The validation guidelines state parallel by identical method in another lab with the same validated assay is also acceptable. Can I assume that this means comparison with another lab for IHC HER2 testing and does not have to be using the same clone (DAKO vs Ventana)? Jim James Vickroy BS, HT(ASCP) Surgical and Autopsy Pathology Technical Supervisor Memorial Medical Center 217-788-4046 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC validation
Hi Laurie, Ideally, you would like to validate both with tissues processed in your laboratory (controls and patient tissue). Purchased TMAs are okay, but they should not be the only thing used. For the instrument validation, I would definitely use tissue that has been processed in your lab and run the same stain on each instrument (I think we ran serial sections of vimentin) and compare them very closely for any variation (use the same antibody and the same detection kit for each instrument). My recommendation for new antibody validations is to run a set of normal tissues and tumor tissues (we have about 15 normal tissues that we harvested and created our own sausage block) along with about 20 or so different tumors (also created in sausage blocks) to see how they stain under as many circumstances as possible (especially in the conditions your pathologists are most likely to see). In addition to that, run a set of known positives and known negatives from your cases. CAP recommends 10 of each, but I believe the final number is at the Medical Director's discretion. You can also run a representative sample of positives and negatives in your lab and send another set of the same tissues to another lab that has a validated procedure for that antibody and validate it that way. As far as the detection kit goes, we don't actually validate the detection kit per se. We do a lot-to-lot comparison, which involves running tissue from the same block (we created special blocks specifically for detection kits) and run the same antibody (AE1/AE3 or CD3--something we do a LOT of) to compare from lot to lot. I hope I answered your question. Regards, Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 ashley.trout...@vanderbilt.edumailto:ashley.trout...@vanderbilt.edu Message: 21 Date: Fri, 15 Mar 2013 16:51:27 + From: Laurie Colbert lcolb...@pathmdlabs.commailto:lcolb...@pathmdlabs.com Subject: [Histonet] IHC validation To: Histonet Post (histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu) histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: 12ECD7346266D74691EC2BFC75285E451CC048EB@BFL323E10.pathmdlabs.localmailto:12ECD7346266D74691EC2BFC75285E451CC048EB@BFL323E10.pathmdlabs.local Content-Type: text/plain; charset=us-ascii If a lab is just starting up IHC for the first time and has to validate both the IHC stainer and the AB's/detection kit, does the validation have to be done using actual patient cases - or can you use strictly control tissue and purchased microarrays? Laurie Colbert, HT (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: IHC Lead
Hello Dorothy, Here we require HT or HTL certification for IHC positions. I look for individuals that want to learn and have a desire to do IHC. I prefer folks with experience, but since experienced techs are hard to pry away from other employers, I am willing to train the right people. I encourage all of my techs to study and take the QIHC so that each IHC tech is knowledgeable and credible in their profession and be able to speak intelligently to our faculty and residents regarding any aspect of our operation. As for titles, we don't really have a lead here (for IHC specifically), but we have created a Technical Specialist that is part of the career ladder pathway that all of our clinical labs are creating for techs (Med Techs and Histotechs). To qualify for that position you must demonstrate considerable technical skill as well as the ability to train/mentor others. It also requires that you can make sound decisions regarding workflow issues (basically be the go-to person for problems and questions should the supervisor not be available.) Since our histology and IHC labs are not physically separated, IHC techs must also occasionally do routine work (cut HEs, perhaps a special stain now and then). So they must also be technically sound in all areas of histology, not just IHC. I hope that helps. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 ashley.trout...@vanderbilt.edumailto:ashley.trout...@vanderbilt.edu From: Webb, Dorothy L dorothy.l.w...@healthpartners.commailto:dorothy.l.w...@healthpartners.com To: 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Sent: Wednesday, September 12, 2012 2:17 PM Subject: [Histonet] IHC lead What criteria does everyone use to hire for working in your IHC department?? I am looking for feedback as to what qualifications are expected for IHC techs as well as qualifications and expectations of the lead in IHC.? Also, what title do you have for the lead in IHC, technical specialist, coordinator, lead, etc??? Thanks ahead of time for your information!? We are trying to reinvent the position and want to make it more like a community standard (community meaning the world of histology)!! ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] p16
The only IVD p16 available in the US is from MTM. However, Roche just bought MTM so you will have to order it through Ventana. Their Customer support number is 800-227-2155. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 *(Office) 615-875-3311 *(Lab) 615-343-9134 ashley.trout...@vanderbilt.edumailto:ashley.trout...@vanderbilt.edu Message: 20 Date: Fri, 20 Jul 2012 12:57:31 -0400 From: Britton, Josette C jcbrit...@cheshire-med.commailto:jcbrit...@cheshire-med.com Subject: To: Richard Cartun rcar...@harthosp.orgmailto:rcar...@harthosp.org, Gerald Davydov geralddavy...@gmail.commailto:geralddavy...@gmail.com, histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: f644cd64b2313f43bb3d2496b454cdac0a2db...@cmc-ex01.cheshire-med.commailto:f644cd64b2313f43bb3d2496b454cdac0a2db...@cmc-ex01.cheshire-med.com Content-Type: text/plain;charset=us-ascii Where is everyone getting their p16 IVD antibody from? Josie Britton HT Cheshire Medical Center 580 Court Street Keene, NH 03431 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Slide distribution amongst Pathologists
I like Michael's solution. We used to give them out 10 cases at a time as they came off the stainer. Currently, I am in a lab that color codes blocks by service (GI, GYN, ENT, etc.) and these cases are all delivered to one location and whatever pathologist is assigned to that service picks up the cases in the respective box. Hope that helps. Ashley Troutman Message: 3 Date: Thu, 7 Jun 2012 10:29:46 -0400 From: Michael Mihalik m...@pathview.commailto:m...@pathview.com Subject: RE: [Histonet] Slide distribution amongst Pathologists To: 'Rathborne, Toni' trathbo...@somerset-healthcare.commailto:trathbo...@somerset-healthcare.com, 'Podawiltz, Thomas' tpodawi...@lrgh.orgmailto:tpodawi...@lrgh.org, susan.wal...@hcahealthcare.commailto:susan.wal...@hcahealthcare.com, gu.l...@gmx.atmailto:gu.l...@gmx.at, sa...@hotmail.camailto:sa...@hotmail.ca Cc: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: 003501cd44ba$17fb8da0$47f2a8e0$@pathview.commailto:003501cd44ba$17fb8da0$47f2a8e0$@pathview.com Content-Type: text/plain;charset=UTF-8 Good morning, By the way, if anyone wants an LIS perspective, we've been requested to distribute slides according to: 1. slides per case or relative value units per case 2. specimen type 3. pathologist availability for the day 4. requesting facility 5. requesting clinician 6. and of course, 'manual override' I can also tell you when the computer assigns the cases to a pathologist it takes a lot of the political burden off of the accessioning, grossing, and histology personnel. Michael Mihalik PathView Systems | cell: 214.733.7688 | 800.798.3540 | fax: 952.241.7369 -Original Message- From: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu]mailto:[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rathborne, Toni Sent: Tuesday, June 05, 2012 8:41 AM To: 'Podawiltz, Thomas'; susan.wal...@hcahealthcare.commailto:susan.wal...@hcahealthcare.com; gu.l...@gmx.atmailto:gu.l...@gmx.at; sa...@hotmail.camailto:sa...@hotmail.ca Cc: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Slide distribution amongst Pathologists We do the same. -Original Message- From: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu]mailto:[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Podawiltz, Thomas Sent: Tuesday, June 05, 2012 6:06 AM To: susan.wal...@hcahealthcare.commailto:susan.wal...@hcahealthcare.com; gu.l...@gmx.atmailto:gu.l...@gmx.at; sa...@hotmail.camailto:sa...@hotmail.ca Cc: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Slide distribution amongst Pathologists We have one simple way of doing it. He who Grosses is he who reads. -Original Message- From: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu]mailto:[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of susan.wal...@hcahealthcare.commailto:susan.wal...@hcahealthcare.com Sent: Tuesday, June 05, 2012 3:21 AM To: gu.l...@gmx.atmailto:gu.l...@gmx.at; sa...@hotmail.camailto:sa...@hotmail.ca Cc: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Slide distribution amongst Pathologists Boy, they sure like to put us in the middle of what should be their own problem. Thank heavens I only work now for one Dr at a time now but when I was at a larger place they rotated. They still used to tell us to give them particular cases when it was not their turn so that we got the flak when someone did not get what they thought was theirs. You can never win! :) -Original Message- From: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu]mailto:[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Gudrun Lang Sent: Monday, June 04, 2012 2:34 PM To: 'Sheila Adey' Cc: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: AW: [Histonet] Slide distribution amongst Pathologists Here the slides go through the hands of one pathologist, who distributes the cases. Gudrun -Urspr??ngliche Nachricht- Von: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu]mailto:[mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Sheila Adey Gesendet: Montag, 04. Juni 2012 20:19 An: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Betreff: [Histonet] Slide distribution amongst Pathologists Hello Netters: I am looking for
[Histonet] stainer question
Hi Andi, I have had good luck with the Shandon Gemini. Small footprint, too. Ashley Troutman BS, HT(ASCP) QIHC Message: 5 Date: Tue, 8 May 2012 08:25:51 -0700 From: Grantham, Andrea L - (algranth) algra...@email.arizona.edumailto:algra...@email.arizona.edu Subject: [Histonet] stainer question To: HISTONET histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: e19369bf-0454-4275-a716-086ea9e81...@email.arizona.edumailto:e19369bf-0454-4275-a716-086ea9e81...@email.arizona.edu Content-Type: text/plain; charset=us-ascii Good morning Histoland! It is a sad day here because I am loosing my Leica stainer. It was given to me by another lab and now they want it back. So it is back to hand staining for us. I'm considering purchasing a replacement and I was looking at refurbed stainers. Don't have much room (as usual) and the ST5020 actually took up so much space that we had it in the processing/embedding room which was a little inconvenient. Thinking of one of the more linear types of stainers. They also seem cheaper. Does anybody have one to recommend? Andi Grantham U of A ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] bone marrow aspirate
Hi Tim, The best way I have found over the years actually requires the person collecting the specimen to do the most work. What we used to do is after the aspirate is performed, make all of the smears, and then inject the remaining aspirate directly into formalin before it coagulates. This gets rid of all the blood and ensures all that is left is marrow. After sufficient time in formalin, filter the marrow out of the formalin and process. As for a processing protocol, we do a run as follows: Formalin 30 min 70% Alcohol 20 min 90% Alcohol 10 min 100% Alcohol 10 min 100% Alcohol 10 min 100% Alcohol 15 min Xylene1 15 min Xylene2 15 min Xylene3 20 min Paraffin1 15 min Paraffin2 15 min Paraffin3 30 min This protocol was done with pressure/vacuum. We have excellent results with this and the pathologists do not have to spend a lot of time hunting for small areas of marrow on the slide, the whole slide is marrow. Good luck! Ashley Message: 5 Date: Fri, 4 May 2012 16:54:57 + From: Coskran, Timothy M timothy.m.cosk...@pfizer.commailto:timothy.m.cosk...@pfizer.com Subject: [Histonet] bone marrow aspirate To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: 70249e5b79afeb48a47d78568ce216e9027...@ndhamrexde02.amer.pfizer.commailto:70249e5b79afeb48a47d78568ce216e9027...@ndhamrexde02.amer.pfizer.com Content-Type: text/plain; charset=us-ascii Does anyone have a protocol on how to fix and process a bone marrow aspirate to paraffin? Thanks, Tim Coskran Pfizer ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Automated coverslipper with Ventana labels
In my experience with the Leica coverslipper, I have to hand coverslip all Ventana slides. It throws more than it coverslips. I have tried running them down quickly and avoiding Xylene on the labels, but that works only marginally better. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Message: 1 Date: Mon, 31 Oct 2011 17:04:43 + From: Theresa (Teri) Johnson tjohn...@gnf.org Subject: [Histonet] Automated coverslipper with Ventana labels To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: 9f3cfee76e51b64991c7485270890b4009ed5...@ex5.lj.gnf.org Content-Type: text/plain; charset=us-ascii Hi all, I wanted to ask if any of you out there are currently using an automated coverslipper and do not have issues with the forks picking up the slides if they have Ventana labels on them. I have used a Leica and had only very occasionally a dropped slide. Is this your experience as well? Are there other units that would work well for this? Feel free to contact me off list if you need to. Thanks! Teri Johnson, HT(ASCP)QIHC GNF Histology Lab Manager Genomics Institute of the Novartis Research Foundation 858-332-4752 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Ventana's OptiView Detection
Hi Matt, I have worked a bit with this detection kit and my impression is that is extremely sensitive and should be able to boost signal considerably. I have also been able to shave off an average of 30 minutes off several stains. (Cyclin D1, which is one of our longer stains at 4 hours is down to 2.5) You can further enhance the stain (or shorten the time) with the amp kit, but I did not find it necessary. I thought it was particularly good on nuclear stains. There is also a good deal of flexibility with the kit and you should be able to get a great stain in less time. Hope that was helpful. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Content-Type: text/plain; charset=us-ascii Hello All, Is anyone using Ventana's OptiView detection kit? If so please let me know what you impression is. Thank you, Matt Brooks, BS, HT (ASCP) Histology Supervisor InCyte Pathology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] MYOD1
Hi Donna, I had a time with this one as well. I use Dako's antibody on the Leica Bondmax. I run it at 1:50 using ER2 for 30 minutes. I would be careful going any stronger on the dilution, I got quite a bit of background with a 1:40, the 1:50 stains clean but can be a little weak if you don't use a fresh cut slide. I ultimately decided to go with the 1:50 because I could get cleaner, consistent staining with it. An equivalent for this protocol would be an EDTA (High pH) retrieval in a pressure cooker for about 20 minutes with a 10 minute cool down. Antibody incubation on the Bond protocol is 15 minutes. I would recommend about 30 minutes on an open system or if you are doing it by hand. If you are running this on a Ventana platform I would start with a CC1 standard and at least 40 minutes on the antibody incubation. You may have to bump the retrieval time up to the extended. Good luck! Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] P63 controls
We use normal prostate and we have also used skin in the past-they both work well. I would avoid pre-cutting a lot of controls, though; it has a tendency to loose antigenicity. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Message: 9 Date: Thu, 8 Sep 2011 09:08:52 -0400 From: Sara Baldwin/mhhcc.org sbald...@mhhcc.org Subject: [Histonet] P63 controls To: histonet@lists.utsouthwestern.edu Message-ID: OF62E3D6D8.5113CF5C-ON85257905.0048390E-85257905.00483911@LocalDomain Content-Type: text/plain; charset=ISO-8859-1 Hi Histonetters We are having trouble with our control for P63 what is everyone using? Thanks Pathology Supervisor S. Kathy Baldwin, SCT (ASCP) Memorial Hospital and Health Care Center sbald...@mhhcc.org Ph 812-996-0210, 0216, Fax 812-996-0232, Pager 812-481-0897, Cell 812-887-3357 Confidential information, Authorized use only. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Immuno. confusion
Hi Valerie, The peroxidase blocking step is to prevent endogenous peroxidase activity that will cause a false positive stemming from your application of DAB (the substrate for your DAB solution is hydrogen peroxide). You can essentially put this blocking step at any point in your stain (following deparaffinization) and before your application of DAB. I have run this step at varying points in my procedures and they all work fine. Some antibodies prefer you do this step before primary application and others prior to DAB, but for most stains, it doesn't matter too much. As for an enhancer, you can make a copper sulfate solution (5mg/mL in buffer). Or you can try DAB Enhancer or DAB Sparkle from Biocare. Good luck, Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 From: Hannen, Valerie valerie.han...@parrishmed.com Subject: [Histonet] Immuno. confusion To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Date: Wednesday, August 3, 2011, 11:57 AM Hi folks... I am going to try to revamp out Immuno. procedure and am looking at a couple of spec. sheets from our primary vendor. I am gettin conflicting information...so I am turning to you all for help. When doing immuno's that require either HIER or enzyme digestion, do you perform the retrieval first or do you do the peroxide step first?? One spec. sheets says retrieve then block endogenous peroxidase...the other says block endogenous peroxidase then do the HIER or enzyme digestion..which is correct?? One other question that I have is...what do you use to enhance your stain?? The enhancing solution that we were using has been discontinued. Thanks so much!! Valerie Hannen,MLT(ASCP), HTL,SU(FL) Histotechnologist Parrish Medical Center 951 N. Washington Avenue Titusville, Florida 32796 (321) 268-6111 ext. 7506 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Vasculature in tumors
We use CD34 for endothelial staining in blood vessels and Podoplanin (D2-40) for lymphatic vessels. These are for humans only. (Mine was worth less than a half-cent...but hopefully helpful.) :) Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Date: Tue, 5 Jul 2011 11:10:02 -0600 From: Elizabeth Chlipala l...@premierlab.com Subject: RE: [Histonet] Vasculature in tumors - long response To: 'Leiker, Merced' lei...@buffalo.edu, 'JR R' rosenfeld...@hotmail.com, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: 14E2C6176416974295479C64A11CB9AE0646F756@SBS2K8.premierlab.local Content-Type: text/plain; charset=us-ascii Hello all I have been following this discussion and I'm going to put in my 2 cents. Tumor vasculature has traditionally been measured with IHC and CD31 or possibly another IHC marker. We use CD31 here for mouse xenograft samples. We have several antibodies we use and it is dependent upon the species. Human tumors (not xenografts) are stained with CD31 but a different antibody (Dako) than the mouse xenografts (Dianova). For other species, canine, porcine, etc we use factor VIII. Factor VIII is not the best marker since it may not pick up fine capillaries but we have been unsuccessful in getting a CD31 to work in FFPE tissue for those species and we have tried several antibodies. The dako antibody will also work on rabbit samples. I can't remember off the top of my head on what we use for rat but it may be Factor VIII. I believe that a large number of publications in which tumor vasculature is measured CD31 has been used. I do not believe that any special stain will provide you with the information that is needed for this type of staining and analysis, especially if you are going to publish the results. The IHC stain for CD31 (for mouse xenograft samples) had been tricky in the past since most antibodies that stain mouse endothelial cells only worked in frozen sections or zinc fixed (not zinc formalin) paraffin embedded samples. Santa Cruz did have a goat polyclonal antibody that did work on FFPE samples for a period of time, early 2000's, but that antibody no longer works in FFPE samples (only frozen, something about the goat dying and the new lots of antisera did not work in FFPE tissues). If you are staining mouse xenograft samples then the Dianova antibody would be the best one to use in my opinion. I need to state that we have not used or even tried VE Cadherin or other IHC markers for vessels, so I really can't comment on those, they may work very well. We use CD31 since we are also running analysis and in order to do that you need a very clean stain and we can achieve that with the Dianova antibody, prior to the Dianova antibody we had used the santa cruz antibody (we had the particular lot number that worked in FFPE tissues, which also worked in multiple species, I do miss that antibody). Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308-1592 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell www.premierlab.com Ship to address: 1567 Skyway Drive, Unit E Longmont, CO 80504 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Leiker, Merced Sent: Tuesday, July 05, 2011 10:39 AM To: 'JR R'; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Vasculature in tumors I agree... I've used CD31 to stain tumor vasculature. Some tumor vasculature isn't even lined with endothelium; there's just blood channels. But most should be lined. Regards, Merced -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of JR R Sent: Friday, July 01, 2011 1:55 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Vasculature in tumors You are probably looking for capillaries maybe arterioles so there wont be any elastic lamina for the pentachrome stain to detect. Instead try IHC with an endothelial marker like VE Cadherin (best) or in a pinch, CD31 or PECAM. Jerry Ricks Research Scientist University of Washington Department of Pathology ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Tumors tumors everywhere
Hi Sarah, Biocare's MACH3 is designed to work with mouse or rabbit antibodies, so it is likely picking up some mouse antigens: http://biocare.net/products/detection/mach-3/ MACH 3(tm) MACH 3 is a two-step, biotin-free detection system which provides excellent specificity, sensitivity and nuclear staining for mouse or rabbit primary antibodies. I would choose a reagent that recognizes ONLY your rabbit antibody. Check out Biocare's Starr Trek components (like the Trekkie Rabbit link. I know the name is goofy... :)). They should be able to help you. Good luck. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Message: 7 Date: Thu, 28 Apr 2011 15:16:16 -0500 From: sgoe...@mirnarx.com Subject: [Histonet] Tumors tumors everywhere To: histonet@lists.utsouthwestern.edu Message-ID: d957f2a7d21959488c492a2680f9920a224...@svrexch.asuragen.us Content-Type: text/plain; charset=us-ascii So I am staining tumors that were implanted as cells subdermally into mice. The cells are human. I am trying to do Caspase staining on these tumors. The primary is an anti-human rabbit polyclonal, and I am using a polymer (Biocare Mach3) in lieu of the secondary antibody. The background is through the roof!! Could the reason be that the tumor was grown in a mouse and is having cross reactivity somehow? What species antibody should I be using instead? All my mouse monoclonal antibodies work perfect on the tissue, it's this stupid rabbit polyclonal!! I am blocking endogenous enzymes (peroxidase etc., DAKO), avidin and biotin (just to see if that would help...it didn't), and protein block (it's literally an hour worth of blocking!!), developing with DAB (Dako) and hematoxylin counterstain. I am so confused as how to get this to work! Also, it isn't just this particular antibody it is any rabbit polyclonal I have tried. Could it be the polymer? It is the one that Biocare suggested? HELP!! Thanks in advance =) Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Input needed
Hi Jason, It is against Federal law to discriminate based on age or gender. And one should not have too much of an issue finding a job, many places are hiring and looking for well-trained techs. Good luck! Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 (Office) 615-875-3311 (Lab) 615-343-9134 Message: 16 Date: Thu, 21 Apr 2011 18:06:19 -0400 From: Mary mhunt...@earthlink.net Subject: [Histonet] Input needed To: histonet@lists.utsouthwestern.edu Message-ID: A17271D060CC4B8D8342DB154F1E0F4D@maryPC Content-Type: text/plain; charset=utf-8 What???s the chance of getting a job in histology as a new grad at age 57. Will gender be an issue? Thanks for the input. Jason ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Positive controls
Hi Cindy, We get ours from American Master Tech: HSV1 Item # CSH0425P (Box of 25) HSV2 Item # CSH0525P (Box of 25) I use Cell Marque's antibodies and they cross-react with each other, so you should be able to get by with one or the other. (I would test this in your lab first, though.) Good luck. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 (Office) 615-875-3311 (Lab) 615-343-9134 Message: 13 Date: Fri, 22 Apr 2011 07:12:48 -0700 (PDT) From: Cindy Bulmer cjbul...@sbcglobal.net Subject: [Histonet] Positive controls To: Histonet histonet@lists.utsouthwestern.edu Message-ID: 988711.32712...@web82302.mail.mud.yahoo.com Content-Type: text/plain; charset=iso-8859-1 Hello Histoland, ? Does anyone have a good source for purchasing HSV I II ( Cocktail) positive controls? ? Thanks, Cindy Cynthia Bulmer HT(ASCP)QIHC IHC Supervisor, CTPL Waco, TX ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC validation
Hematoxylin should not affect the immunoreactivity (since counterstaining occurs following the IHC reaction), so no, changing the hematoxylin would not require a revalidation. Just make sure you are not obscuring the immunostain with an overly-dark counterstain. The point is to provide contrast between the DAB (brown) or fast red chromagen and to offer the reader a sense of context of the surrounding morphology of the tissue being stained. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Message: 10 Date: Tue, 19 Apr 2011 14:04:32 -0500 From: Ring, Mary L mary.l.r...@healthpartners.com Subject: [Histonet] IHC validation To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: 2594d39b69223047b6e2f4adf1e62db9010ffb487...@hpemx3.healthpartners.int Content-Type: text/plain; charset=iso-8859-1 Does a change in the type of hematoxylin counterstain require any re-validation of IHC stains? Thanks for your help! Mary Ring, HT, QIHC Regions Hospital, St Paul, Mn ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] HercepTest
Hi Cindy, The protocol is very specific, requiring you use a water bath @ 95 deg for 40 minutes (I think) in the epitope retrieval solution provided. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 (Office) 615-875-3311 (Lab) 615-343-9134 Message: 9 Date: Tue, 22 Mar 2011 09:51:33 -0700 (PDT) From: Cindy Bulmer cjbul...@sbcglobal.net Subject: [Histonet] HercepTest To: Histonet histonet@lists.utsouthwestern.edu Message-ID: 614879.51084...@web82307.mail.mud.yahoo.com Content-Type: text/plain; charset=iso-8859-1 Hello Histoland, ? Anyone using the HercepTest for the Dako autostainer, Code # K5207? If so, what equipment are you using for the Epitope retrieval process? ? Cindy Cynthia Bulmer HT(ASCP)QIHC IHC Supervisor, CTPL Waco, TX ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE:[Histonet] IHC validation
I believe the 25 cases are for ER/PR/Her-2 testing. We have done this recently and we ran about 10 for each antibody. We have over 100 ourselves and it took quite a while. There will be some antibodies that you will be unable to find 10 slides to test, so do as many (or as few) as your Medical Director is comfortable with to validate the stain. Good luck! Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Message: 14 Date: Tue, 8 Feb 2011 18:12:42 -0500 From: Weems, Joyce jwe...@sjha.org Subject: RE: [Histonet] IHC validation To: Liz Chlipala l...@premierlab.com, Joe Nocito jnoc...@satx.rr.com, Histonet histonet@lists.utsouthwestern.edu Message-ID: 92ad9b20a6c38c4587a9febe3a30e164081e09a...@chexcms10.one.ads.che.org Content-Type: text/plain; charset=us-ascii But that is for receptors, correct? Do you do that for everything? Thanks, j -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz Chlipala Sent: Tuesday, February 08, 2011 17:58 To: Joe Nocito; Histonet Subject: RE: [Histonet] IHC validation Joe If you are following the recommendations from the CAP paper on IHC standardization then it would be 25 tissues (10 strong positive, 10 weak to moderate positive and 5 negative). Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC PO Box 18592 Boulder, Colorado 80308 office (303) 682-3949 fax (303) 682-9060 www.premierlab.com Ship to Address: 1567 Skyway Drive, Unit E Longmont, Colorado 80504 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Joe Nocito Sent: Tuesday, February 08, 2011 3:44 PM To: Histonet Subject: [Histonet] IHC validation Greetings Histoland, I need some help. We are about to switch IHC machines from the Richard-Allen Axiom to the Ventana Benchmark Ultra. How many slides, per antibody, do you run for the validation study? We have over 100 primary antibodies. Normally, when we work up a new antibody, we start with a titer. Once that is established, we run 10 cases to check for specificity. Hopefully we can obtain cases that are really positive, some weakly positive and some flat out negative. Once that is completed, we run 10 different tissue types to check for any unexpected cross-reactivity. The ultra holds 30 slides and we are receiving two machines. If we run 10 slides/antibody, that's going to take a while, not to mention the number of detection kits that will be used. Do you think 5 slides/antibody is sufficient? I emailed CAP last week for their take and they never returned my email (I told my medical director to hold their check for the year and see how fast they respond to that). Ah oh, don't go down that road Joe, it's unhealthy. What are your thoughts? Thanks Joe (JTT) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Any problems with Bond Max Heater Assembly (SSA)
Hi, I see this issue from time to time. One thing to note, does the same heater stay out or do they jump around? If they jump around, this is probably due to an issue with the power for the instrument. Make sure all your line conditioners and battery backups are performing properly and this will fix it. If the same heater is going out, the heater is definitely bad and should be replaced. We have had a couple go out and it is a relatively easy fix, even though it can be a bit of a bother until they get replaced. Good luck! Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Parafolicular cells, C cells
Hi Lin, I am not aware of a special stain that is that specific for C-cells, but a calcitonin immunostain is pretty specific. Good luck Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Message: 6 Date: Wed, 27 Oct 2010 15:21:37 -0500 From: Lin Bustamante lbustama...@cvm.tamu.edu Subject: [Histonet] Parafolicular cells, C cells To: histonet@lists.utsouthwestern.edu Message-ID: 4cc8438102b9000e3...@cvm.tamu.edu Content-Type: text/plain; charset=US-ASCII Do you know a good specific special stain (not IHC stain) for this cells of the thyroid gland? Lin S. Bustamante, B.Sc.; HT(ASCP) Research Associate Histology Lab Supervisor Veterinary Integrative Bioscience Texas AM University College Station, TX 77843-4458 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC techs needed
Vanderbilt University is seeking two certified techs for 2nd shift. Primary duties will be IHC staining. Anyone interested please visit www.mc.vanderbilt.eduhttp://www.mc.vanderbilt.edu/ and click on Careers at Vanderbilt. Select Current staff openings and type Histotech in the keyword search. Thank you! Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC Validation on new instrument
Hi Laurie, I have a Benchmark Ultra and a Benchmark XT from Ventana and they follow the basic steps, similar protocols and I needed to revalidate everything. They are sufficiently different (in my experience) that it warranted a complete revalidation. A part of the reasoning for this was some of the bulk reagents were different (a consideration that may lead you to not completely revalidate). For me, there were a few protocols that were quite different from one to the other. I would go to the trouble up front, that way there is no question later. Good luck! Ashley Troutman BS, HT(ASCP) QIHC Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4532 Nashville, TN 37232 615-343-9134 Message: 4 Date: Wed, 19 May 2010 07:59:30 -0700 From: Laurie Colbert laurie.colb...@huntingtonhospital.com Subject: [Histonet] IHC Validation on new instrument To: histonet@lists.utsouthwestern.edu Message-ID: 57be698966d5c54eae8612e8941d768308bca...@exchange3.huntingtonhospital.com Content-Type: text/plain; charset=us-ascii What do others do when validating a new model of a piece of equipment - same manufacturer, same basic staining process, but an updated version of the equipment? I've been told the protocols should be the same and that we only need to run three controls with three different but similar protocols to determine what looks best. Do you all think that is thorough enough, or would you run actual patient cases and compare old and new equipment? I don't see where the CAP checklist refers to new equipment - just new antibodies and new antibody lots. Laurie Colbert ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Special stain to demonstrate Silver
Hi Fanny, I don't know of one, but perhaps you could modify a GMS. I don't think you would need to oxidize it at the beginning with the periodic acid or chromic acid. Make the working Methenamine silver solution without the silver. (Don't forget to add the Borate) and try and visualize it that way. I probably wouldn't do the hypo and gold chloride until I looked at it under the scope to see if it was actually staining, you wouldn't want to remove the silver you are trying to visualize. I would be careful in interpreting that because I don't know what else it might be picking up--it might pick up other metals. That's just a suggestion off the cuff. If you try that, let us know what happens! Good luck! Thanks, Ashley Troutman BS, HT(ASCP) QIHC Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4532 Nashville, TN 37232 615-343-9134 Message: 15 Date: Fri, 7 May 2010 07:00:25 -0500 From: DeGuzman, Maria mdeguz...@lifecell.com Subject: [Histonet] Special stain to demonstrate Silver To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: 5476245379016b4d8212e8bcd11ecffbb77a5...@amwpvex01.kci.com Content-Type: text/plain; charset=us-ascii Hello Histoneters, Is there a special stain to detect silver in tissue? If there is please share the procedure. Thanks, Fanny ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Histonet Digest, Vol 77, Issue 36
Hello, We do a two stage validation procedure here. First, I will work up an antibody to get a working protocol. I give the best slides along with a form recording the processes used for each slide (dilution, antigen retrieval solution, A-R time, chromogen, antibody incubation time, etc.) and I make the pathologist sign off on the one they like the best. Next, we pull a number of cases (we do 10-15) that include known positives and negatives. (If you are switching platforms, just pull the old slides and compare them to the new method.) Record this data (block numbers, etc.) along with the pathologist's signature. If it is a new antibody, I have to tell the pathologist to find a minimum of 10 cases that the stain will be used on and I can validate it that way. If you are validating ER, PR and/or Her-2, you are in for a lot more work. ASCO/CAP recommends 20 to 100 cases that must be compared to a known method. ie someone else's lab or your old method. We did 50 and it took awhile. Sorry this is a bit choppy. Feel free to email me with any other specific questions that you might have or if you want the forms I use. We are in the same boat! CAP is due any day, now! Ashley Troutman BS, HT(ASCP) QIHC Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4532 Nashville, TN 37232 615-343-9134 Message: 7 Date: Wed, 28 Apr 2010 12:30:44 -0400 From: thisis...@aol.com Subject: [Histonet] IHC Validation Procedure To: histonet@lists.utsouthwestern.edu Message-ID: 8ccb50748d97178-1c98-9...@webmail-d051.sysops.aol.com Content-Type: text/plain; charset=us-ascii Can anyone share their IHC Validation procedure. I am preparing for a CAP inspection and want to make sure that the procedure I (presently do/may change to) is acceptable. Thank you very much! ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Phospho antibodies and fixation
Good day colleagues, Does anyone have any information on phospho antibodies and fixation? Is there any reason to NOT fix specimens in formalin vs say, 70% EtOH? (and then process them through formalin later?) I can't seem to find any information on whether or not fixation in formalin does something strange to a phosphorylated protien and makes it a less accessible antigen. Also, on that same note, does retreival do anything to it? I am assuming that these antibodies go through the same testing for cross reactivity, etc (depending on the vendor) and are reliable when used properly (like any other antibody). I don't, however, know if there is enough of a difference in the epitope (the fact that it is phosphorylated) that would make it more susceptible to some strange cross linking with formalin (especiallly with phosphate buffered formalin). Any help with this topic would be greatly appreciated as I am uneducated in this area. Thanks, Ashley Troutman BS, HT(ASCP) QIHC Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4532 Nashville, TN 37232 615-343-9134 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Benchmark Ultra
Hello, We currently use a Benchmark Ultra and it is worth several XTs. There are some features about it that you should know about before you start a run. What we currently do in our lab is load all of the antibodies we are likely to run every day. This can be troublesome if you run the same 30 antibodies every day (unless you have more than one Ultra, then it is much easier). We currently only run 30 total antibodies on our Ventana platform and of those 30 we run 10 or 12 every day. When we come in, we pull a list of stains for the AM run off the computer and we have a good idea if we need to load something less common along with the list of 12 more common antibodies and a detection kit. For the next run, if we have already started a run and we need to add an antibody, we will be forced to use the Landing Zone feature. Using this, we are able to load the missing antibodies/reagents and remove what we don't need. With regard to the important or unimportant cases, you can flag them with the blue LED, (which looks really cool, by the way...) so that you know visually which cases you are looking for without having to hover over a position on the computer. As far as loading important cases after unimportant cases, I don't think I can offer any advice there. The instrument is essentially 30 individual stainers and you can choose whether or not to load the slides. We don't really encounter this issue because of the way we have structured our workflow, but then again, we have enhanced our workflow based upon the capabilities of the instrumentation that we use. I hope this has helped. If there are any other questions, feel free to email me. Ashley Troutman BS, HT(ASCP) QIHC Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4532 Nashville, TN 37232 Date: Wed, 24 Mar 2010 19:37:25 +0100 From: Gudrun Lang gu.l...@gmx.at Subject: [Histonet] benchmark ultra and continuous workflow To: histonet@lists.utsouthwestern.edu Message-ID: b646f3f46af8408cafc5b4887d948...@dielangs.at Content-Type: text/plain; charset=us-ascii Hi Benchmark Ultra users! I would like to hear of your experiences with the continuous workflow of this instrument. Does it really make life easier or even more complicated? I think of handling one slide every few minutes after the staining is completed and of the problems, that occur when the stainer is started with unimportant cases and the later coming important cases have not enough place to complete them in time. Gudrun Lang ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: immunoperoxidase background staining
A couple of suggestions. First, what kind of fixative are you using? If you are using a heavy metal fixative, sometimes that can be the reason. We have had this issue with B-plus fixative (B5 replacement) and I have seen it with some protocols in zinc formalin and non-buffered fixatives. Second, try backing off of the CC1 or 2. I use a Biocare's Background Terminator in an option container to help with my most troublesome antibodies. You can also use the green diluent they provide you with as a blocking reagent. I found it works on most things. If you are having issues with tons of background on ALL your slides, I would send a slide to a place that has established protocols to see if it is the instrument or the tissue. Hope this helps. Ashley Troutman BS, HT(ASCP) QIHC Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4532 Nashville, TN 37232 615-343-9134 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] immunohistochemistry
I would recommend the following: Citrate for 35 min at 95 deg. Cool down for 10 min. (This is our protocol.) Be sure you are using charged slides and I would let them air dry for at least 1 hour before heating and deparaffinizing. That might help, too. Unfortunately, I don't think there is an effective method of retrieival that will completely eliminate the possibilty of floating tissue. Good luck. Ashley Troutman BS, HT(ASCP)QIHC Histopathology Laboratory Department of Pathology Vanderbilt University Medical Center Nashville, TN http://www.vanderbilthealth.com/main/ http://www.vanderbilt.edu/ http://www.mc.vanderbilt.edu/ Date: Thu, 4 Jun 2009 20:01:50 -0700 (PDT) From: Hatem Salim dr.hatemsa...@yahoo.com Subject: [Histonet] immunohistochemistry To: histonet@lists.utsouthwestern.edu Message-ID: 782588.78944...@web46103.mail.sp1.yahoo.com Content-Type: text/plain; charset=iso-8859-1 HI I am Research assistant at Physiology department of Michigan state university. I am using the (ß-Catenin Antibody (Carboxy-terminal Antigen) #9587 for IHC on mice femur sections. I have used this antigen unmasking method: For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes. The problem is that bone detachment always occurs so I wonder if there is a method of retrieval that prevents bone detachment. Waiting to hear from you soon. Thank you for your consideration Best wishes Hatem Salim http://www.vanderbilthealth.com/main/ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Double-stain for immune cell markers
Hi Naira, Try Biocare. They make fantastic reagents for research as well as some great double-stain kits. As for the antibodies, it looks like you might have to create these either as cocktails (a lot of front-end work) or just make your stain longer (a lot of overall work) by staining seprately. Abcam has some esoteric reagents that you might be able to use for some non-mainstream species like horse or rat or guinea pig. Also I did not see which markers you are planning to stain for (there are quite a few immune cell markers). I might be able to help you find good reagents if I had that information. Good luck. Ashley Troutman BS, HT(ASCP)QIHC Histopathology Laboratory Department of Pathology Vanderbilt University Medical Center Nashville, TN http://www.vanderbilthealth.com/main/ http://www.vanderbilt.edu/ http://www.mc.vanderbilt.edu/ http://www.vanderbilthealth.com/main/ Message: 3 Date: Wed, 18 Mar 2009 14:20:34 -0500 From: Margaryan, Naira nmargar...@childrensmemorial.org Subject: [Histonet] Double-stain for immune cell markers To: histonet@lists.utsouthwestern.edu Message-ID: 6a2230bac92e3b4084dae06869b89fb601e6c...@cmhexc01evs.childrensmemorial.org Content-Type: text/plain; charset=us-ascii Hi Histofriends, I was requested to make the double-staining for immune cell markers together with another genes (IgG goat) that our lab researching on human tumors xenografted in mouse. Which companies kits you will suggest to use for this experiment (the double-staining) and, what Abs (not mouse and not goat) are the best to represent the immune cell markers. Respectfully, Naira ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Human tissue for research purposes
Human tissue for research purposes can be rather complicated. Keep in mind, your institution has requirements, the FDA has requirements and the patient has requirements. If you get the tissue from another institution...you guessed it, more requirements. I have worked with Institutional Review Boards (IRBs) with this and with some things you need the patient's permission. Some hospitals (usually teaching hospitals and large research institutions) have this covered when a patient is admitted. When the patient signs the stack of paperwork to have their surgery, there will be a small sentence that releases that tissue to be used for research purposes. The first thing I would check, though, is what the requirements for the research study are. Sometimes that can decide where your tissue comes from. Second, check with your facility administrator and the chief investigator (I don't know if you are in a hospital or a private lab--it will be different for each) about the legal issues that may surround you obtaining tissue from a source other than your own lab. The paperwork for such an endeavor can be mind-numbing. Another option would be to talk to the investigator to see if they have any colleagues that would be willing to donate tissue. (Sometimes you can get it already processed!) Getting it that way tends to be easier, especially if good documentation on the tissue already exists. If none of this works, call a major university near you and see if they have a research tissue bank or other such department. These guys and gals do nothing but collect various tissues and tumors for the myriad researchers at thier own institutions and may be willing to part with it. (We have something like that here at Vanderbilt, but I don't know if they sell it to outside institutions.) Sorry this is long, but I hope it helps some. Ashley Troutman BS, HT(ASCP)QIHC Histopathology Laboratory Department of Pathology Vanderbilt University Medical Center Nashville, TN http://www.vanderbilthealth.com/main/ http://www.vanderbilt.edu/ http://www.mc.vanderbilt.edu/ Message: 17 Date: Thu, 20 Nov 2008 15:18:49 -0500 From: Elizabeth M Heimrich [EMAIL PROTECTED] Subject: [Histonet] Human tissue for research purposes To: histonet@lists.utsouthwestern.edu Message-ID: [EMAIL PROTECTED] Content-Type: text/plain; charset=iso-8859-1 Hello Histonetters, I have an investigator that is in need of human tissue samples. We would need IBD/UC colon and RA joint/synovium as of right now. Does anyone know where I could locate these tissues? I found three suppliers online, but I was wondering if there are more out there. Do hospitals donate tissue for research purposes? I know there are a lot of legal/ethical issues surrounding human tissue... I know this topic has been discussed before, but the histonet archives are not available to me right now (technical issue). Thanks, Beth http://www.vanderbilthealth.com/main/ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] help
The chicken came first--but she did not have a belly button... Ashley Troutman BS, HT(ASCP)QIHC Histopathology Laboratory Department of Pathology Vanderbilt University Medical Center Nashville, TN http://www.vanderbilthealth.com/main/ http://www.vanderbilt.edu/ http://www.mc.vanderbilt.edu/ -Original Message- From: Barone, Carol Sent: Thursday, November 20, 2008 3:17 PM To: '[EMAIL PROTECTED]' Subject: help Importance: High Histo friends...I am working on a five year strategic plan for a Research Histotechnology Core Lab with very diverse services. So I thought I might ask... What trend for histology services do you all see for the future of histo? We now do routine, specials, HC, IF and IHC, In situ, TUNEL, BrdU, LMD, morphotmetrics, paraffin, plastics, and frozensyou name it, we do it. So, other than TMAwhat do we see as technical trends in histology for the next five years? Another question, what came first the chicken or the egg? http://www.vanderbilthealth.com/main/ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
