Re: [Histonet] GMS question
Gayle Callis's discourse on GMS staining is must reading for anyone who does the GMS stain. Freida Carson in 1999 showed the importance of using chromic acid - I was distressed that she didn't cite this paper in the 4th edition of her book. The most rigorous test of the GMS stain is probably the dead histoplasma in ancient fibrotic granulomas. I've gotten these things to turn up with GMS more than once - a finding of potential clinical importance. For control material, I don't think there's any substitute for infected human (or mammalian) tissue, preferably though not necessarily with a species identification of the fungus. I think either active histoplasmosis or invasive aspergillosis might provide the best material. The big academic centers that are still doing autopsies could be helpful in getting control material for everyone. Periodic acid should indeed be made up fresh with the dry chemical, but precision weighing isn't required - I used to keep a small plastic measure in the stock bottle so I could simply spoon out what I needed for the day. I'm not sure what the rules are for disposing of chromic acid, but it's a significant hazmat - toxic metal, strong acid, strong oxidant. I think you can neutralize it and precipitate the chromium, but I'd have to look up the method. Bob Richmond Samurai Pathologist Maryville TN ** Gayle Callis wrote: The same site of infection for your control and patient tissues does not mean the fungus species was the same. Now for addtional commentary. The classic GMS uses chromic acid as the oxidizer, stronger than periodic acid. This is something to beware of since you had a false negative result with the kit. Periodic acid should be freshly made up from periodic acid crystals just before use (Culling insisted on fresh periodic acid reagent) then discarded after that day - something that is not going on with a kit where all components are sent to you in ready to use liquid form. I think if you had used the classic GMS with chromic acid to start with, you would have had the results seen with Periodic Acid/Schiffs. Part of the problem is the not all fungus species stain well either PAS or Periodic acid/GMS, and even classic GMS. All these factors can present a problem when trying to diagnose fungal infections.Lee Luna in AFIP manual, pointed out it has been found that the time of exposure to methenamine-silver nitrate solution for complete development may vary according to type and/or strains suspected. He advised if Nocardia asteroides is suspected, then two slides should be run: one for 60 minutes and one for 90 min in the silver solution. Histotechs should be aware these possible problems. Fungi are difficult at best even when doing fungus cultures and treat. Fortunately, there are antibodies for some fungi i.e. Aspergillus, Candida) but you would probably need the specific fungus as a control for the antibody being used. Fungus IHC experts can weigh in on the latter topic. I don't know what fungus species was in your fungus ball control block, but our clinical lab fungus ball control contained Aspergillus sp. from a post mortem human lung. With the researcher, he only had pure Aspergillus sp. infecting the tissues.It would be nice to know what species of fungus is in your control tissue block The publication by Freida Carson along with Jerry Fredenburgh and John Maxwell Inconsistent detection of Histoplasma capsulatum with periodic acid in GMS fungus stain , J Histotechnology, 1999 is a profound statement about what you just experienced. Their tissue control i.e. Aspergillus sp. stained adequately with periodic acid/GMS but the H. capsulatum fungus in tissue did not stain. They studied several different times, temperatures, periodic acid concentrations and fungus species. They had additional controls containing fungi other than Aspergillus sp. for better sampling of PA/GMS on different fungi. Having different fungus species control blocks is a luxury many labs do not enjoy. The reason chromic acid is not in kits is due to shipping, health and safety hazards, must be handled carefully and collected for proper, safe disposal so it is easier to make up GMS kits with periodic acid.If you have to collect your silver solutions for safe chemical disposal, then chromic acid shouldn't be a big problem to do the same. Also, since Periodic acid/Schiffs is popular and commonly used for fungus staining, PAS can also present false negative results or weak staining due to the weaker periodic acid oxidation, even when the periodic acid is made in house, fresh for the day. Chromic acid/Schiffs has been recommended by people on Histonet to improve fungus staining over PAS. PAS stain always seem to work well with Candida sp. and Aspergillus sp. but classic GMS was always better in my hands. I only used classic GMS, prepared in house, and controlled the development with a microscope
[Histonet] Software for Tracking/Archiving Long-term Storage of Various Specimens
What are people using to keep track of tissue specimens that they archive? We have an archive of wet and frozen tissues going back to the early 90's and are looking into a software program that will allow us to keep track of these specimens. We would also like to then generate a variety of reports based on the tissue that we have archived. Ideally, it would be nice if the program interfaced with EPIC Beaker, but that is not essential. Within the program, we would like to be able to delineate different categories that the specimens might fit into. Does such a program exist? If you are currently using a program, what do you like? And what are the limitations? We are currently in the process of implementing ABT with Cerner, but will be moving to Epic in the next couple of years. Thank you in advance Michelle Lamphere Senior Tech, Histology Anatomic Pathology O: 214.456.2318 | Fax: 214.456.0779 E: michelle.lamph...@childrens.com 1935 Medical District Drive | B1.06 | Dallas, Texas 75235 Please consider the environment before printing this e-mail This e-mail, facsimile, or letter and any files or attachments transmitted with it contains information that is confidential and privileged. This information is intended only for the use of the individual(s) and entity(ies) to whom it is addressed. If you are the intended recipient, further disclosures are prohibited without proper authorization. If you are not the intended recipient, any disclosure, copying, printing, or use of this information is strictly prohibited and possibly a violation of federal or state law and regulations. If you have received this information in error, please notify Children's Medical Center Dallas immediately via e-mail at priv...@childrens.com. Children's Medical Center Dallas and its affiliates hereby claim all applicable privileges related to this information. Please consider the environment before printing this e-mail This e-mail, facsimile, or letter and any files or attachments transmitted with it contains information that is confidential and privileged. This information is intended only for the use of the individual(s) and entity(ies) to whom it is addressed. If you are the intended recipient, further disclosures are prohibited without proper authorization. If you are not the intended recipient, any disclosure, copying, printing, or use of this information is strictly prohibited and possibly a violation of federal or state law and regulations. If you have received this information in error, please notify Children's Medical Center Dallas immediately via e-mail at priv...@childrens.com. Children's Medical Center Dallas and its affiliates hereby claim all applicable privileges related to this information. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Passing onto PAS discussion RE: GMS question
Bob, Culling said exactly the same thing about not needing precision weighing for periodic acid. I discovered, by a huge error in PAS staining results, that pre-weighing 1 gm of PA into a clear sample vial and letting the pre-weighed PA stand at RT should NOT be done. The PA degraded to the point of very poor staining. I thought I was doing myself a favor to have faster solution preparation, and the pre-weighing efficiency idea was abandoned. We sometimes learn the hard way. Daily freshness is the key and we liked 1% periodic acid (or 0.5% per the original McManus and Mowry method). Our lab used 1% PA for a standard PAS stain for 10 minutes (never used for fungus) most of the time. It is interesting to note how many variations of the PAS stain exist in this world.It's important to check what PAS method variation works best for the component you are looking for as one variation may not be the best for the tissue component you want to see. If over oxidation is suspected, reduce oxidation time to 5 min (original McManus/Mowry method) and/or reduce concentration of PA to 0.5% periodic acid instead of 1% PA OR do both. Good positive controls are mandatory for any particular tissue component in mind. The only time we used a periodic acid/methenamine silver staining was for 2 micrometer renal biopsies aka Jones Basement membrane stain, and our methenamine silver was kept fresh with a 6 month expiration date on in house preparation. Fresh methenamine silver stock solution is important too. Chromic acid cannot be drain dumped and hopefully people will have hazmat collection available. If you have to collect DAB, silver solutions, then chromic acid (chromium trioxide is the stock chemical) can be collected. It isn't that difficult. Thanks for the supportive comment on GMS. Your comments on Histoplasma sp. staining were interesting as not many of us have the opportunity to work with ancient tissues. Gayle Callis -Original Message- From: Bob Richmond [mailto:rsrichm...@gmail.com] Sent: Saturday, May 02, 2015 7:30 AM To: Histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] GMS question Gayle Callis's discourse on GMS staining is must reading for anyone who does the GMS stain. Freida Carson in 1999 showed the importance of using chromic acid - I was distressed that she didn't cite this paper in the 4th edition of her book. The most rigorous test of the GMS stain is probably the dead histoplasma in ancient fibrotic granulomas. I've gotten these things to turn up with GMS more than once - a finding of potential clinical importance. For control material, I don't think there's any substitute for infected human (or mammalian) tissue, preferably though not necessarily with a species identification of the fungus. I think either active histoplasmosis or invasive aspergillosis might provide the best material. The big academic centers that are still doing autopsies could be helpful in getting control material for everyone. Periodic acid should indeed be made up fresh with the dry chemical, but precision weighing isn't required - I used to keep a small plastic measure in the stock bottle so I could simply spoon out what I needed for the day. I'm not sure what the rules are for disposing of chromic acid, but it's a significant hazmat - toxic metal, strong acid, strong oxidant. I think you can neutralize it and precipitate the chromium, but I'd have to look up the method. Bob Richmond Samurai Pathologist Maryville TN ** Gayle Callis wrote: The same site of infection for your control and patient tissues does not mean the fungus species was the same. Now for addtional commentary. The classic GMS uses chromic acid as the oxidizer, stronger than periodic acid. This is something to beware of since you had a false negative result with the kit. Periodic acid should be freshly made up from periodic acid crystals just before use (Culling insisted on fresh periodic acid reagent) then discarded after that day - something that is not going on with a kit where all components are sent to you in ready to use liquid form. I think if you had used the classic GMS with chromic acid to start with, you would have had the results seen with Periodic Acid/Schiffs. Part of the problem is the not all fungus species stain well either PAS or Periodic acid/GMS, and even classic GMS. All these factors can present a problem when trying to diagnose fungal infections.Lee Luna in AFIP manual, pointed out it has been found that the time of exposure to methenamine-silver nitrate solution for complete development may vary according to type and/or strains suspected. He advised if Nocardia asteroides is suspected, then two slides should be run: one for 60 minutes and one for 90 min in the silver solution. Histotechs should be aware these possible problems. Fungi are difficult at best even when doing fungus cultures and treat.
[Histonet] Bleach as Cleaning Agent or Decontamination
Our facility is moving towards standardization of decontaminants or disinfectants. They prefer all areas use a Sani wipes that kills most pathogens. However, we contend for Anatomic pathology we need our liquid bleach not only as disinfectant or decontaminant but as a cleaning agent for stained lab-ware. You thoughts? Also, what concentration of Bleach (5.25 or 10%) is acceptable for use as both a disinfectant and cleaning agent or should we keep them separate? We used to buy the hospital grade premade bleach at a 5.25% concentration but now they want us instead to buy the 99.9% commercial Bleach and dilute from there. Any suggestions on opaque containers for us to purchase since bleach break down after a time period, at least for disinfecting? V/r Ian ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC and oven temperature
John your points do seem to make it seem somewhat counter-intuitive in regards to the temperatures suggested in the literature for high points for each step. Perhaps someone will be able to provide a complete theoretical basis for the differences. It seems though that there wouldn't be much of a directed point or purpose in heating slides dry at such high temperature for very long at that stage in the process. But during AR , we have moist and the eletrolytic conditions, so use of the higher temperature is applied for a more directed and specific effect that benefits us in identifying the particular epitope of interest..any thoughts? Seems like high temp acheives a goal in the second instance, but not much purpose is gained at the higher temperature in the first instance, and potentially damage to some aspects. I'll await further information and discussion from the group. Thanks Joelle Weaver MAOM, HTL (ASCP) QIHC From: jkier...@uwo.ca To: Histonet@lists.utsouthwestern.edu; tony.henw...@health.nsw.gov.au Date: Thu, 30 Apr 2015 18:24:10 -0500 Subject: Re: FW: [Histonet] IHC and oven temperature CC: The statement quoted by Tony from the Dako manual cannot be true because many antigens have to be exposed to water at 100C in order to be immunostained - antigen retrieval. Denaturation of a macromolecule by heat increases the number of exposed epitopes, which typically are short amino acid sequences that bind specifically to the Fab segments of antibody molecules. On the other hand, it is easy to believe that 60C would denature antibody molecules enough to damage their binding sites and impair or prevent immunostaining. According to AWP Vermeer and W Norde (2000), the Fab segments of IgG were denatured when the temperature of a solution slightly exceeded 60C. (The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain Protein Biophysical Journal 78: 394–404.) They found that further heating denatured the Fc segment, but the changed molecules became entangled and aggregated before denaturation was complete. Microwave heating is sometimes used to accelerate immunostaining, but control of the temperature is critical. For example: ME Boon E Marani (1991) The major importance of temperature data in publications concerning microwave techniques European Journal of Morphology 29: 181–183. John Kiernan London, Canada = = = On 30/04/15, Tony Henwood (SCHN) tony.henw...@health.nsw.gov.au wrote: Yes, I read the Dako IPX educational guides (5th ed) and on page 32: No processes should raise tissue temperature to higher than 60oC as this will cause severe loss of antigenicity that may not be recoverable Unfortunately there is no evidence given or cited that validates this statement. Even though this could be right (and there are several papers that have looked at this), this statement is scientifically weak and we should not cite this as truth. Now I do recommend the Dako reference series to my students, and I have contributed to one of these texts myself (Microscopic control of routine HE - know your histology) but I request my students to continue to question what they read and confirm the scientific validity of the information. Regards, Tony From: Joelle Weaver [joellewea...@hotmail.com] Sent: Saturday, 25 April 2015 5:51 AM To: Tony Henwood (SCHN); WILLIAM DESALVO; Preiszner, Johanna Cc: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC and oven temperature I remember reading that the preferred temperature was about 60 degrees Celsius. I think that this was in the Dako education guides if I'm not mistaken. If that is the case, the citation for the source is probably in that resource available as pdf from their website. Joelle Weaver MAOM, HTL (ASCP) QIHC From: tony.henw...@health.nsw.gov.au To: wdesalvo@outlook.com; preis...@mail.etsu.edu Date: Fri, 24 Apr 2015 09:43:59 + Subject: RE: [Histonet] IHC and oven temperature CC: histonet@lists.utsouthwestern.edu Hi temp drying shown to be a bad idea: Henwood, A., (2005) “Effect of Slide Drying at 80°C on Immunohistochemistry” J Histotechnol 28(1):45-46. Abstract Prolonged high temperature dry heating has been found to be deleterious to the immunohistochemical demonstration of several antigens in formalin-fixed, paraffin- embedded sections. Paraffin sections were dried at 80°C for 7 h and their immunoreactivity was compared with mirror sections dried for 1 h at 60°C. NCL-5D3, CMV, S100, HMB45, and CEA were quite labile to dry overheating whereas AElAE3, HBsAg, HBcAg, HSVII, EMA, chromogranin, and NSE were found to be quite resistant. It is recommended that coated slides (poly-L-lysine or aminopropyltriethoxysilane) and low-temperature
