Re: [Histonet] EDTA decalcification tissue issues
We switched from acid decal to EDTA recently; primarily for molecular testing. We are starting to see some excellent results. At first, we were not decaling long enough (EDTA decalcification is a very slow process). We also use the Leica Bond Max and we have not had any problems with tissue loss. We are only using EDTA for small biopsies. Please not that these specimens must be fixed adequately before the decal process is started. Richard Richard W. Cartun, MS, PhD Director, Histology & The Martin M. Berman, MD Immunopathology & Morphologic Proteomics Laboratory Director, Biospecimen Collection Programs Assistant Director, Anatomic Pathology Hartford Hospital 80 Seymour Street Hartford, CT 06102 (860) 972-1596 (860) 545-2204 Fax -Original Message- From: Pairan, Kelly via Histonet [mailto:histonet@lists.utsouthwestern.edu] Sent: Friday, November 10, 2017 6:39 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] EDTA decalcification tissue issues This email is from outside HHC. BE CAREFUL when opening attachments or links from unknown senders. Good Morning, Recently, our lab has been working on validating an EDTA method of decalcification. When we ran the IHC's on the decalcified bone block, the majority of the tissue lifted off the slide. We use the Leica Bonds for our IHC staining. Does anyone else have a hard time getting EDTA decalcified tissue to stay on positively charged slides during IHC runs? Do you have a trick you use? We really did not have this big of a problem when we were decalcifying with Formical or Nitrical (big bones only). Thanks for your help, ?Kelly Pairan, HT(ASCP), QIHC(ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Reminder: This e-mail and any attachments are subject to the current HHC email retention policies. Please save or store appropriately in accordance with policy. This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure, or distribution is prohibited. If you are not the intended recipient, or an employee or agent responsible for delivering the message to the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message, including any attachments. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] EDTA decalcification solution
Hello, We make our own formic acid EDTA for a slow decal solution. Is this what you are looking for? Thanks Histologistics Hans B Snyder 508.308.7800 h...@histologistics.com > On Dec 1, 2014, at 14:20, Martha Ward-Pathology wrote: > > I am asking this question for our Histology Lab. They are being asked about > using EDTA as a decal solution for bone and wondered if anyone else is using > this?Is this available as a ready to use? Or do you have to make it up? > What vendors are you using and could you provide a procedure about how you > use itlength of time in solution, etc. > > Thanks in advance for your help. > > Martha Ward, MT (ASCP) QIHC > Manager > > Molecular Diagnostics Lab > Medical Center Boulevard \ Winston-Salem, NC 27157 > p 336.716.2109 \ f 336.716.5890 > mw...@wakehealth.edu > > > > > > > > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] EDTA decal
Good idea. I have to soak the edta decaled tissues in an alkaline solution
inorder to restore enzyme histochemical staining for TRAP, might be the same
issue for some IHC.
Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E. Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
prueg...@hotmail.com
rueggihcconsultin...@outlook.com
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-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Teri Johnson
Sent: Friday, August 16, 2013 11:54 AM
To: 'cfors...@umn.edu'
Cc: histonet@lists.utsouthwestern.edu
Subject: [Histonet] EDTA decal
Hi Colleen,
I would say it's unusual, but not completely impossible that EDTA has
interfered with your IHC. We had that problem with demonstrating
B-galactosidase in mouse bones. If we decalcified it in EDTA after whole
mount staining in X-gal, the blue staining was removed. But if we
decalcified it in formic acid, the stain was retained.
I think a few years ago, Biogenex had a room temperature antigen retrieval
solution for acid decalcified bone (not the same as your situation, I know).
But I think it was largely an alkaline solution you let the slides sit for
maybe 30 minutes prior to staining. Might be worthwhile to try a different
retrieval method for these and see what happens.
Otherwise, are you sure you are using a clone that reacts in mouse tissue?
We use Rabbit monoclonal SP6.
Teri Johnson
Manager, Histology
GNF - San Diego, CA
858-332-4752
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RE: [Histonet] EDTA
José, I have inserted my ideas below within your email. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 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.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jose Luis Palazon Fernandez Sent: Monday, 10 October 2011 10:34 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] EDTA Dear List members Greetings > I would like to ask you some questions about the use of EDTA as > demineratizing agent. > I have to do histology and histochemistry (including metallothioneins) in > fish larvae preserved in Bouins solution. How long do you fix in Bouins solution? Bouins, being an acidic fixative (picric and ascetic acids) will fix as well as decalcify, especially fish larvae which would have little calcium compared to human trephine specimens. So I am not sure whether you will need to decalcify. > 1) First of all I would like to know what is the best formula (or the one you > recomend) to prepare the EDTA solution, as well as the importance of > maintaining a given pH and if it is necessary to put the > > samples in the > refrigerator during the demineralization process. The following solution was used by Sanderson et al (Biotech & Histochem 70(1):12-18, 1995): 1. To 1 litre of distilled water add 90ml concentrated ammonium hydroxide. 2. Stirring continuously, slowly add 140g EDTA 3. Adjust to pH 7.1 using concentrated ammonia. Adjustment of the pH is essential. If the pH is too low it works only as a too-weak acid. If the pH is too high (above 8) decalcification is accelerated but alkalinity can be damaging to tissues. > 2) Is EDTA recomended only for certain fixatives, or It can be used with any > fixative, including Bouins Again why do you need to decalcify these specimens, since Bouins will also decalcify > 3) When using Bouins solution as fixative, many books recommend not to wash > in water but instaed to remove the excess of picric acid using several washes > in etanol 70%, on the other hand, I´ve read > that EDTA precipitates in the > presence of alcohols. What do you recomend in this case? I also recommend washing in 70% ethanol since protein picrates are believed to be water soluble. I would suspect placing the Bouins fixed tissues in an aqueous EDTA solution would do the same thing (ie allow extraction of the picrates). > 4) Do you think that the demineralization procedure could alter or affect the > detection of metallothioneins in the tissues? It would be possible that the Bouin's fixative might affect the antigens recognised by your metallothionein antibodies but unlikely that EDTA treatment would - only by testing would it become clearer. Are your antigens affected by Bouins? Many thanks in advance Best regards José Luis Dr. José Luis Palazón Fernández Instituto de Investigaciones Científicas Universidad de Oriente Boca del Rio-Isla Margarita-Venezuela Direccion actual: Instituto de Ciencias Marinas de Andalucia-CSIC Campus universitario Rio San Pedro, 11510, Puerto Real, Cádiz, España email: jluis.pala...@icman.csic.es; jose.pala...@ne.udo.edu.ve tlf: +34-956832612; fax: +34-956834701; cell: +34-600487100 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] EDTA 14%
Why don't you prepare it in your lab? By doing so you will cut costs and assure a consistent decal solution. René J. --- On Fri, 7/22/11, pam plumlee wrote: From: pam plumlee Subject: [Histonet] EDTA 14% To: "histonet@lists.utsouthwestern.edu" Date: Friday, July 22, 2011, 9:51 AM Good morning Histonetters: I would like suggestions for a vendor that supplies 14% EDTA for a decal project our lab is starting. Thanks for any help. Pam Plumlee H.T. (ASCP) Biotheranostics ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] EDTA decalcification of bones which have been fixed in 70% ethanol
Since EDTA is essentially an aqueous solution, I would wash out the 70EthOL, fix in NBF for 24 hours and then place the specimen in EDTA until decalcification is completed. René J. --- On Wed, 6/22/11, Orla M Gallagher wrote: From: Orla M Gallagher Subject: [Histonet] EDTA decalcification of bones which have been fixed in 70% ethanol To: histonet@lists.utsouthwestern.edu Date: Wednesday, June 22, 2011, 7:55 AM Dear Histonetters, We would like to decalcify some mouse bones in EDTA pH 7 which have been received by our lab already fixed directly in 70% ethanol rather than in formalin or paraformaldehyde. How would you recommend preparing these bones for decalcification e.g. whether to post-fix in formalin or to wash out the ethanol before transferrring to EDTA? I realise that ethanol is not the best fixative to use, especially as the end user may want to do immunocytochemistry or enzyme histochemistry using TRAP. Thanks, Orla -- ** Ms. Orla Gallagher Bone Analysis Laboratory Mellanby Centre for Bone Research D Floor Medical School University of Sheffield Beech Hill Road Sheffield S10 2RX Website: http://mellanbycentre.dept.shef.ac.uk Tel: 0114-2713337 (office) 0114-2713174 (lab) E-Mail: o.m.gallag...@sheffield.ac.uk Please think about the environment before printing this email ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] EDTA
Although what you say is true, it is worth noting that there are some antigens/antibodies more amenable to acid decal. I have experienced this myself several times recently and been burned by thinking EDTA will always give better IHC results. At least for murine bone marrow vasculature antigens, this appears to be the case for some antibodies. Unfortunately as with most things in histology, there is always exceptions to rules of thumb! --- On Fri, 2/19/10, Rene J Buesa wrote: From: Rene J Buesa Subject: Re: [Histonet] EDTA To: "'histonet@lists.utsouthwestern.edu'" , "Dorothy LWebb" Date: Friday, February 19, 2010, 9:03 PM EDTA (which stands for Ethylene-diamino-tratacetic-acid) is a chelating agent and the method of choice to decalcify BM biopsies. The thing is that it should be used alone, and not combined with any acid, as you state (not even formic acid). By itself will produce an extremely gentle decalcification that will be completely suitable for IHC studies. René J. --- On Fri, 2/19/10, Webb, Dorothy L wrote: From: Webb, Dorothy L Subject: [Histonet] EDTA To: "'histonet@lists.utsouthwestern.edu'" Date: Friday, February 19, 2010, 1:29 PM I am looking into the various decals on the market and have found one that in addition to formic acid, has EDTA in the mix. I have never worked with EDTA so would appre ciate any help in your comments on the use of EDTA in decalcification methods for bone marrow and routine specimens. Thank you ahead of time for your advice! Dorothy Webb, HT (ASCP) Regions Histology Technical Supervisor 651-254-2962 This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this e-mail in error, please immediately notify the HealthPartners Support Center by telephone at (952) 967-6600. You will be reimbursed for reasonable costs incurred in notifying us. HealthPartners R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] EDTA
EDTA (which stands for Ethylene-diamino-tratacetic-acid) is a chelating agent and the method of choice to decalcify BM biopsies. The thing is that it should be used alone, and not combined with any acid, as you state (not even formic acid). By itself will produce an extremely gentle decalcification that will be completely suitable for IHC studies. René J. --- On Fri, 2/19/10, Webb, Dorothy L wrote: From: Webb, Dorothy L Subject: [Histonet] EDTA To: "'histonet@lists.utsouthwestern.edu'" Date: Friday, February 19, 2010, 1:29 PM I am looking into the various decals on the market and have found one that in addition to formic acid, has EDTA in the mix. I have never worked with EDTA so would appre ciate any help in your comments on the use of EDTA in decalcification methods for bone marrow and routine specimens. Thank you ahead of time for your advice! Dorothy Webb, HT (ASCP) Regions Histology Technical Supervisor 651-254-2962 This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this e-mail in error, please immediately notify the HealthPartners Support Center by telephone at (952) 967-6600. You will be reimbursed for reasonable costs incurred in notifying us. HealthPartners R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] (EDTA)Decal Bone marrows
Hi Patti, Years ago, I used a EDTA Decal Solution which was recommended by Dr. Jaffee, a Hematopathologist at NIH. This formula was particularly great for BM Core BX's. It is much gentler on the tissue, and is less likely to harm the cellular integrity. Dr. Todd Barry also prefers the use of EDTA formula's for BM Bx's. Below is the formula. or you can purchase from Biocare Medical. You should decal the specimen for 30 minutes to 1 hour and check for decalcification. DO NOT leave for any longer than 1 hour. 10% EDTA (Ethyl-media minetetra-acetic-acid Stock EDTA 10 ml DI water 90 ml pH 4.5-5.0 with Sodium Hydroxide If you prefer to purchase the IED Solution, Biocare Medical has it available. They have 2 sizes 140 ml or 500 ml. cat # IED 1204. Ion-Exchange Decalcification Unit (IED Unit, Designed for Bone Marrow Biopsies) An advanced decalcification system that removes calcium from bone quickly while leaving superior cellular detail. The IED Unit incorporates a strong cation ion-exchange resin in a weak acid solution to remove calcium ions from bone, replacing them with hydrogen ions. Because the IED Unit does not require strong concentrated acid solutions, as in traditional decalcification methods, delicate cellular structures antigenicity remain intact. Regards, Akemi Akemi Allison-Tacha BS, HT(ASCP)HTL Histology Manager APMG Laboratories 105A Cooper Court, Los Gatos, CA 95032 Contact: 800.848.2764 V/M: 408.884.2718 Fax: 408.884.2758 Cell: 408.335.9994 E-Mail: aallison-ta...@apmglab.com --- On Fri, 7/10/09, Patti Loykasek wrote: From: Patti Loykasek Subject: [Histonet] Bone marrows To: "histonet" Date: Friday, July 10, 2009, 12:38 PM Hi All. I was wondering what everyone's current favorite bone marrow decal solution/method is. Including commercially available decal solutions. We will be doing IHC after fixation/decal. Thanks for the input (as always). Patti Loykasek BS, HTL, QIHC Clinical Lab Supervisor PhenoPath Laboratories Seattle, WA This e-mail message, including any attachments, is for the sole use of the intended recipients and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by e-mail and destroy all copies of the original message, or you may call PhenoPath Laboratories, Seattle, WA U.S.A. at (206) 374-9000. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
