RE: [Histonet] IHC pos. & neg. control question
This is my understanding about this CAP checklist item - CAP is looking for 2 types of negative controls: 1. Negative reagent control - this is satisfied by running a negative control slide from the same patient block through the whole process without the primary antibody. 2. Negative tissue control - this is satisfied by running a slide of multi tissue control containing tissues that are positive for the primary antibody and tissues that are negative for the primary antibody. The negative tissue on the multi tissue control will serve as the negative tissue control for the specific antibody. If you look at the positive control and the patient tissue only the areas that contain the target antigen will stain. There are other elements in the tissue that are known negative (e.g. smooth muscle in the small blood vessels will not stain with CD3 - then the smooth muscle in either the positive control and the patient tissue would serve as an internal negative tissue control for CD3). This negative result on the tissue elements that are expected not to stain with a specific antibody should be negative - hence - negative tissue control and should be noted in the documentation (preferably in the final report when commenting on the results of IHC stains). At Emory, we just had our CAP inspection (we only had a couple of deficiencies - xylene monitoring in the IHC area, Space and one corrected on site - labeling of chemicals without manufacturer's supplied expiration dates). Prior to inspection, we got clarification from CAP on this checklist item. CAP prefers the use of multi tissue control (containing both positive and negative tissue elements), however using negative tissue elements in the positive control and patient tissue are acceptable as negative tissue control for the specific antibody and it should be noted / documented accordingly. We document the negative control in our final reports when commenting on the results of IHC stains. This is my two cents contribution to the discussion. Godfrey > From: jshea...@roadrunner.com > To: histonet@lists.utsouthwestern.edu > Date: Thu, 19 May 2011 21:11:01 -0400 > Subject: [Histonet] IHC pos. & neg. control question > > Curt is right, according to CAP ... > > ANP.22570 QC - Antibodies Phase II > > Appropriate negative controls are used. > > NOTE: Negative controls must assess the presence of nonspecific staining in > patient tissue as well > > as the specificity of each antibody. Results of controls must be documented, > either in internal > > laboratory records, or in the patient report. A statement in the report such > as, "All controls show > > appropriate reactivity" is sufficient. > > A negative reagent control is used to assess nonspecific or aberrant staining > in patient tissue related > > to the antigen retrieval conditions and/or detection system used. A separate > section of patient tissue > > is processed using the same reagent and epitope retrieval protocol as the > patient test slide, except > > that the primary antibody is omitted, and replaced by any one of the > following: > > ? An unrelated antibody of the same isotype as the primary antibody (for > monoclonal > > primary antibodies) > > ? An unrelated antibody from the same animal species as the primary antibody > (for > > polyclonal primary antibodies) > > ? The negative control reagent included in the staining kit > > ? The diluent/buffer solution in which the primary antibody is diluted > > In general, a separate negative reagent control should be run for each block > of patient tissue being > > immunostained; however, for cases in which there is simultaneous staining of > multiple blocks from > > the same specimen with the same antibody (e.g. cytokeratin staining of > multiple axillary sentinel > > lymph nodes), performing a single negative control on one of the blocks may > be sufficient provided > > that all such blocks are fixed and processed identically. This exception does > not apply to stains on > > different types of tissues or those using different antigen retrieval > protocols or antibody detection > > systems. The laboratory director must determine which cases will have only > one negative reagent > > control, and this must be specified in the department's procedure manual. > > The negative reagent control would ideally control for each reagent protocol > and antibody retrieval > > condition; however, large antibody panels often employ multiple antigen > retrieval procedures. In > > such cases, a reasonable minimum control would be to perform the negative > reagent control using > > the most aggressive retrieval procedure in th
RE: [Histonet] IHC pos. & neg. control question
No problem Amos, I have a one page handout that I use for workshops and presentations on how to do the math on this, it can be tricky, so if anyone is interested just e-mail me. Since we are talking about matched isotype negative controls, essentially three things need to be taken into consideration: 1. The isotype of the primary antibody 2. The protein concentration of the working dilution of the primary antibody 3. The antibody form - (affinity purified, culture supernatant, ascities fluid, etc.) the isotype negative control needs to match the primary antibody - some spec sheets list appropriate isotype negative control reagents some do not There is one caveat that I would like to address for those that are working with animal tissues - you need to check the spec sheet on your negative control reagents to make sure that it does not cross react with the species you are working with. If it does then you may get staining in your negative control slide. Here is a table that was generated from one of the older versions of the dako handbook that addresses the antibody form and suggested negative control reagents. Primary Antibody Type Suggested Negative Control Reagent Monoclonal Mouse - produced in ascites Antibody produced in ascites, same isotype as the primary antibody OR Normal nonimmune mouse serum Monoclonal Mouse - produced in tissue culture Antibody produced in tissue culture, same isotype as the primary antibody Polyclonal Rabbit or Goat - immunoglobulin fraction Normal rabbit or goat immunoglobulin fraction Solid-phase absorbed Polyclonal Rabbit - immunoglobulin fraction Rabbit immunoglobulin fraction - solid phase absorbed Polyclonal Rabbit - whole serum Normal or nonimmune rabbit serum, whole serum Hope this helps, and everyone have a great weekend 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: Amos Brooks [mailto:amosbro...@gmail.com] Sent: Friday, May 20, 2011 3:05 PM To: Liz Chlipala Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IHC pos. & neg. control question Thanks Liz, You are absolutly right there, but have you ever noticed some folks eyes glaze over when you say that they must both be run at the same ug/mL? I would be lying if I said it never happened to me until I forced myself to work it out. Once you do though it isn't too bad. Unfortunately people don't think about this much. They want to put a slide on a machine and walk away without thinking about what they are doing. I guess I was oversimplifying the description. Thanks for pointing that out as it is actually an important aspect to the dilutions. Amos On Fri, May 20, 2011 at 4:25 PM, Liz Chlipala wrote: Amos Isotype negative controls are based upon protein concentration not dilution. They must be the same protein concentration of the primary antibody at the dilution you are using. Using the same dilution will not work unless both stock solutions (primary antibody and isotype control) are at the same protein concentration which is rarely the case. 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 Amos Brooks Sent: Friday, May 20, 2011 2:16 PM To: jm.lapoi...@accellab.com; histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question Hi, This is simply a question of definitions. They are actually both right. If you have the patient tissue as a negative control you are not using the primary antibody on it but are replacing it (ideally) with an Ig with the same isotype AND dilution (universal negative is stupid). So if your primary is an IgG1 from a mouse and you use it at 1:100, your negative control would be a non-immune mouse serum IgG1 diluted to 1:100. Running this on unrelated material will not show you anything in this case. what it will show you is that the detection system you are using is specific to the antibody you put on the tissue and not to A) something else in the tissue or B) something on the Ig that is not the epitope itself. Although, If you run the same primary antibody on tissue that you expect will not react with the primary antibody, this would prove that the antibody reaction is specific to the antigenin question. This does raise the question of what to do about ubiquitous antigens (like ubiquitin) that are actually everywhere. Every cell has a cell cycle so they will all at one poi
Re: [Histonet] IHC pos. & neg. control question
Thanks Liz, You are absolutly right there, but have you ever noticed some folks eyes glaze over when you say that they must both be run at the same ug/mL? I would be lying if I said it never happened to me until I forced myself to work it out. Once you do though it isn't too bad. Unfortunately people don't think about this much. They want to put a slide on a machine and walk away without thinking about what they are doing. I guess I was oversimplifying the description. Thanks for pointing that out as it is actually an important aspect to the dilutions. Amos On Fri, May 20, 2011 at 4:25 PM, Liz Chlipala wrote: > Amos > > Isotype negative controls are based upon protein concentration not > dilution. They must be the same protein concentration of the primary > antibody at the dilution you are using. Using the same dilution will > not work unless both stock solutions (primary antibody and isotype > control) are at the same protein concentration which is rarely the case. > > 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 Amos > Brooks > Sent: Friday, May 20, 2011 2:16 PM > To: jm.lapoi...@accellab.com; histonet@lists.utsouthwestern.edu > Subject: [Histonet] IHC pos. & neg. control question > > Hi, > This is simply a question of definitions. They are actually both > right. > If you have the patient tissue as a negative control you are not using > the > primary antibody on it but are replacing it (ideally) with an Ig with > the > same isotype AND dilution (universal negative is stupid). So if your > primary > is an IgG1 from a mouse and you use it at 1:100, your negative control > would > be a non-immune mouse serum IgG1 diluted to 1:100. Running this on > unrelated > material will not show you anything in this case. what it will show you > is > that the detection system you are using is specific to the antibody you > put > on the tissue and not to A) something else in the tissue or B) something > on > the Ig that is not the epitope itself. > Although, If you run the same primary antibody on tissue that you > expect will not react with the primary antibody, this would prove that > the > antibody reaction is specific to the antigenin question. This does raise > the > question of what to do about ubiquitous antigens (like ubiquitin) that > are > actually everywhere. Every cell has a cell cycle so they will all at one > point or another show proliferation or degeneration for example. Does > this > mean you don't run a negative control? No you need to look at it in the > perspective of expected results. > There are various ways of using negative (and positive) controls. > It > would actually be cost prohibitive to run ALL the possible positive and > negative controls for every test that we as histologists do. We need to > discuss with our pathologists what they want to have done to give them > the > confidence in our testing to support their diagnosis. But remember that > just > because another lab and another pathologist does it differently doesn't > mean > it is wrong. It's just different. > > Happy Friday Folks, > Amos > > > Message: 5 > Date: Thu, 19 May 2011 13:25:49 -0400 > From: "Jean-Martin Lapointe" > Subject: [Histonet] IHC pos. & neg. control question > To: > Message-ID: > < > befd613bd39142499989f836556ddc8301272...@ace.accellab.lan> > Content-Type: text/plain; charset="iso-8859-1" > > Hi Curt, > I agree with your pathologist. The section that you use as a negative > control (without primary) in an IHC run should ideally be tissue that is > a > known positive, and of the same nature as the test sample. Therefore the > best sample is often a section of your positive control tissue. > The purpose of this negative control is to make sure that any positive > staining observed in the test sample is not due to a spurious > cross-reaction, unrelated to the primary. I don't think the issue per se > is > that you are using tissue from the same patient; rather, it is that you > are > using a sample for which the staining characteristics are not known. For > instance, if are using a lymph node section as a negative, for a stain > that > targets an epithelial marker (eg Her2) in the test breast sample, then > your > negative tissu
RE: [Histonet] IHC pos. & neg. control question
Amos Isotype negative controls are based upon protein concentration not dilution. They must be the same protein concentration of the primary antibody at the dilution you are using. Using the same dilution will not work unless both stock solutions (primary antibody and isotype control) are at the same protein concentration which is rarely the case. 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 Amos Brooks Sent: Friday, May 20, 2011 2:16 PM To: jm.lapoi...@accellab.com; histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question Hi, This is simply a question of definitions. They are actually both right. If you have the patient tissue as a negative control you are not using the primary antibody on it but are replacing it (ideally) with an Ig with the same isotype AND dilution (universal negative is stupid). So if your primary is an IgG1 from a mouse and you use it at 1:100, your negative control would be a non-immune mouse serum IgG1 diluted to 1:100. Running this on unrelated material will not show you anything in this case. what it will show you is that the detection system you are using is specific to the antibody you put on the tissue and not to A) something else in the tissue or B) something on the Ig that is not the epitope itself. Although, If you run the same primary antibody on tissue that you expect will not react with the primary antibody, this would prove that the antibody reaction is specific to the antigenin question. This does raise the question of what to do about ubiquitous antigens (like ubiquitin) that are actually everywhere. Every cell has a cell cycle so they will all at one point or another show proliferation or degeneration for example. Does this mean you don't run a negative control? No you need to look at it in the perspective of expected results. There are various ways of using negative (and positive) controls. It would actually be cost prohibitive to run ALL the possible positive and negative controls for every test that we as histologists do. We need to discuss with our pathologists what they want to have done to give them the confidence in our testing to support their diagnosis. But remember that just because another lab and another pathologist does it differently doesn't mean it is wrong. It's just different. Happy Friday Folks, Amos Message: 5 Date: Thu, 19 May 2011 13:25:49 -0400 From: "Jean-Martin Lapointe" Subject: [Histonet] IHC pos. & neg. control question To: Message-ID: < befd613bd39142499989f836556ddc8301272...@ace.accellab.lan> Content-Type: text/plain; charset="iso-8859-1" Hi Curt, I agree with your pathologist. The section that you use as a negative control (without primary) in an IHC run should ideally be tissue that is a known positive, and of the same nature as the test sample. Therefore the best sample is often a section of your positive control tissue. The purpose of this negative control is to make sure that any positive staining observed in the test sample is not due to a spurious cross-reaction, unrelated to the primary. I don't think the issue per se is that you are using tissue from the same patient; rather, it is that you are using a sample for which the staining characteristics are not known. For instance, if are using a lymph node section as a negative, for a stain that targets an epithelial marker (eg Her2) in the test breast sample, then your negative tissue is not appropriate, because since lymph node contains no epithelial tissue, it will not stain no matter what. Therefore if your test sample shows a positive reaction in the epithelial tissue, but for some reason that reaction is a spurious false-positive, then the lymph node negative will not reveal that. I realize that this is all very theoretical and hypothetical, but I understand the pathologist wanting to be confident in the knowledge that all potential technical issues are eliminated before making his diagnosis. Jean-Martin ___ 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] IHC pos. & neg. control question
Hi, This is simply a question of definitions. They are actually both right. If you have the patient tissue as a negative control you are not using the primary antibody on it but are replacing it (ideally) with an Ig with the same isotype AND dilution (universal negative is stupid). So if your primary is an IgG1 from a mouse and you use it at 1:100, your negative control would be a non-immune mouse serum IgG1 diluted to 1:100. Running this on unrelated material will not show you anything in this case. what it will show you is that the detection system you are using is specific to the antibody you put on the tissue and not to A) something else in the tissue or B) something on the Ig that is not the epitope itself. Although, If you run the same primary antibody on tissue that you expect will not react with the primary antibody, this would prove that the antibody reaction is specific to the antigenin question. This does raise the question of what to do about ubiquitous antigens (like ubiquitin) that are actually everywhere. Every cell has a cell cycle so they will all at one point or another show proliferation or degeneration for example. Does this mean you don't run a negative control? No you need to look at it in the perspective of expected results. There are various ways of using negative (and positive) controls. It would actually be cost prohibitive to run ALL the possible positive and negative controls for every test that we as histologists do. We need to discuss with our pathologists what they want to have done to give them the confidence in our testing to support their diagnosis. But remember that just because another lab and another pathologist does it differently doesn't mean it is wrong. It's just different. Happy Friday Folks, Amos Message: 5 Date: Thu, 19 May 2011 13:25:49 -0400 From: "Jean-Martin Lapointe" Subject: [Histonet] IHC pos. & neg. control question To: Message-ID: < befd613bd39142499989f836556ddc8301272...@ace.accellab.lan> Content-Type: text/plain; charset="iso-8859-1" Hi Curt, I agree with your pathologist. The section that you use as a negative control (without primary) in an IHC run should ideally be tissue that is a known positive, and of the same nature as the test sample. Therefore the best sample is often a section of your positive control tissue. The purpose of this negative control is to make sure that any positive staining observed in the test sample is not due to a spurious cross-reaction, unrelated to the primary. I don't think the issue per se is that you are using tissue from the same patient; rather, it is that you are using a sample for which the staining characteristics are not known. For instance, if are using a lymph node section as a negative, for a stain that targets an epithelial marker (eg Her2) in the test breast sample, then your negative tissue is not appropriate, because since lymph node contains no epithelial tissue, it will not stain no matter what. Therefore if your test sample shows a positive reaction in the epithelial tissue, but for some reason that reaction is a spurious false-positive, then the lymph node negative will not reveal that. I realize that this is all very theoretical and hypothetical, but I understand the pathologist wanting to be confident in the knowledge that all potential technical issues are eliminated before making his diagnosis. Jean-Martin ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. & neg. control question
"If an inspector saw that, you would be on immediate suspension." This was the last statement from the original post, I chose not to include it initially but it's just too juicy to keep from everyone else. Having forwarded several of the responses I think he has come to understand that it's not necessarily a requirement that will have us on immediate suspension but rather a preference that, being a private lab, I will gladly provide to keep the business. Thanks again everyone and have a good weekend. Curt From: Jay Lundgren [mailto:jaylundg...@gmail.com] Sent: Friday, May 20, 2011 11:57 AM To: Curt Tague Cc: Ingles Claire; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IHC pos. & neg. control question Curt, Thank you. I'm here all week, try the veal. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC pos. & neg. control question
Curt, Thank you. I'm here all week, try the veal. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. & neg. control question
All very insightful input. I took the liberty of forwarding some of the comments to the client, I think I errantly sent some of the critical comments about him too. I'll probably hear about it later, the best was the clown residency, I was in tears laughing. i'll let you all know what he says about CAPs protocol, don't exactly know how he can argue against that. Thanks all, Curt -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Ingles Claire Sent: Friday, May 20, 2011 6:30 AM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question And the block in question has already been proven positive using THAT procedure and antibody during validation. Claire From: histonet-boun...@lists.utsouthwestern.edu on behalf of Thomas Jasper Sent: Thu 5/19/2011 2:39 PM To: pete.peder...@healthonecares.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Pete, When you run a positive control. The tissue is already a known positive (or it should be) for whichever antibody you are running regardless of prior handling. It would be impossible for this not to be so. However, with a negative, the concern is seeing how the patient tissue turns out when subjected to all the same conditions, minus the antibody. tj -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pete.peder...@healthonecares.com Sent: Thursday, May 19, 2011 12:31 PM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt _
RE: [Histonet] IHC pos. & neg. control question
And the block in question has already been proven positive using THAT procedure and antibody during validation. Claire From: histonet-boun...@lists.utsouthwestern.edu on behalf of Thomas Jasper Sent: Thu 5/19/2011 2:39 PM To: pete.peder...@healthonecares.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Pete, When you run a positive control. The tissue is already a known positive (or it should be) for whichever antibody you are running regardless of prior handling. It would be impossible for this not to be so. However, with a negative, the concern is seeing how the patient tissue turns out when subjected to all the same conditions, minus the antibody. tj -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pete.peder...@healthonecares.com Sent: Thursday, May 19, 2011 12:31 PM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet m
RE: [Histonet] IHC pos. & neg. control question
Tj, Amen brother! GD -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Thomas Jasper Sent: Thursday, May 19, 2011 2:39 PM To: pete.peder...@healthonecares.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Pete, When you run a positive control. The tissue is already a known positive (or it should be) for whichever antibody you are running regardless of prior handling. It would be impossible for this not to be so. However, with a negative, the concern is seeing how the patient tissue turns out when subjected to all the same conditions, minus the antibody. tj -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pete.peder...@healthonecares.com Sent: Thursday, May 19, 2011 12:31 PM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://
RE: [Histonet] IHC pos. & neg. control question
Pete, OK, time for an example: A pathologist orders 4 IHC's on a block. I run 5 slides total: 4 IHC slides with a section of patient tissue and a known positive. 1 slide with patient tissue only for the negative control. The one negative control is put through the retrieval/protocol that is most likely to cause nonspecific staining. I don't run the known positive control tissue used on the 4 actual IHC slides as negative controls. Perhaps I didn't point out that my original post was addressing the negative control? I'm a bit surprised by the confusion. As for the question of "how I know the staining seen in a positive control is truly positive?": All known positive controls are tested previously so I know that they work for the antibody that I'm using them for and I would assume the pathologist uses morphology and localization of staining to determine that the positive control is working. That's what I do. I've gone through 7 CAP inspections utilizing the practices above with no problems thus far. Perhaps you could enlighten me on IHC requirements that I haven't come across. Glen D. -Original Message- From: pete.peder...@healthonecares.com [mailto:pete.peder...@healthonecares.com] Sent: Thursday, May 19, 2011 2:31 PM To: Dawson, Glen; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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] IHC pos. & neg. control question
"either would fulfill the purpose of detecting non-specific positive staining" - NO Not in the patients sample unless it is included (which for diagnostic uses is the most important). 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 Jean-Martin Lapointe Sent: Friday, 20 May 2011 7:50 AM To: Angela Bitting; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question I can certainly agree with that. Whether it’s the patient’s sample or the known positive control that are stained without primary, either would fulfill the purpose of detecting non-specific positive staining. Jean-Martin From: Angela Bitting [mailto:akbitt...@geisinger.edu] Sent: Thursday, May 19, 2011 4:50 PM To: Jean-Martin Lapointe; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IHC pos. & neg. control question I agree with the majority. A patient slide stained without primary antibody, and a patient section with primary antibody and a known positive control tissue, on the same slide when possible, is sufficient. I do agree, however, that the use of a negative tissue control is important in your initial antibody optimization and validation. Angela Bitting, HT(ASCP), QIHC Technical Specialist, Histology Geisinger Medical Center 100 N Academy Ave. MC 23-00 Danville, PA 17822 phone 570-214-9634 fax 570-271-5916 email. Thank you. * 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] IHC pos. & neg. control question
It still comes down to why test the positive control for false positive reactions after it has already been determined that they are not present. But what you say about positive controls matching patient's tissue with regards to pre-fixing, fixing and processing is correct but in the real world we cannot guarantee that this would happen. We can only work on averages and hope that conditions are as similar as possible. In a perfect world we would not need negative, nor dare I say positive, controls. 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 pete.peder...@healthonecares.com Sent: Friday, 20 May 2011 5:31 AM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.e
[Histonet] IHC pos. & neg. control question
Curt is right, according to CAP ... ANP.22570 QC - Antibodies Phase II Appropriate negative controls are used. NOTE: Negative controls must assess the presence of nonspecific staining in patient tissue as well as the specificity of each antibody. Results of controls must be documented, either in internal laboratory records, or in the patient report. A statement in the report such as, "All controls show appropriate reactivity" is sufficient. A negative reagent control is used to assess nonspecific or aberrant staining in patient tissue related to the antigen retrieval conditions and/or detection system used. A separate section of patient tissue is processed using the same reagent and epitope retrieval protocol as the patient test slide, except that the primary antibody is omitted, and replaced by any one of the following: ? An unrelated antibody of the same isotype as the primary antibody (for monoclonal primary antibodies) ? An unrelated antibody from the same animal species as the primary antibody (for polyclonal primary antibodies) ? The negative control reagent included in the staining kit ? The diluent/buffer solution in which the primary antibody is diluted In general, a separate negative reagent control should be run for each block of patient tissue being immunostained; however, for cases in which there is simultaneous staining of multiple blocks from the same specimen with the same antibody (e.g. cytokeratin staining of multiple axillary sentinel lymph nodes), performing a single negative control on one of the blocks may be sufficient provided that all such blocks are fixed and processed identically. This exception does not apply to stains on different types of tissues or those using different antigen retrieval protocols or antibody detection systems. The laboratory director must determine which cases will have only one negative reagent control, and this must be specified in the department's procedure manual. The negative reagent control would ideally control for each reagent protocol and antibody retrieval condition; however, large antibody panels often employ multiple antigen retrieval procedures. In such cases, a reasonable minimum control would be to perform the negative reagent control using the most aggressive retrieval procedure in the particular antibody panel. Aggressiveness of antigen retrieval (in decreasing order) is as follows: pressure cooker; enzyme digestion, boiling; microwave; steamer; water bath. High pH retrieval should be considered more aggressive than comparable retrieval in citrate buffer at pH 6.0. It is also important to assess the specificity of each antibody by a negative tissue control, which must show no staining of tissues known to lack the antigen.The negative tissue control is processed using the same fixation, epitope retrieval and immunostaining protocols as the patient tissue. Unexpected positive staining of such tissues indicates that the test has lost specificity, perhaps because of improper antibody concentration or excessive antigen retrieval. Intrinsic properties of the test tissue may also be the cause of "non-specific" staining. For example, tissues with high endogenous biotin activity such as liver or renal tubules may simulate positive staining when using a detection method based on biotin labeling. A negative tissue control must be processed for each antibody in a given run. Any of the following can serve as a negative tissue control: 1. Multitissue blocks.These can provide simultaneous positive and negative tissue controls, and are considered "best practice" (see below). 2. The positive control slide or patient test slides, if these slides contain tissue elements that should not react with the antibody. 3. A separate negative tissue control slide. The type of negative tissue control used (i.e. separate sections, internal controls or multitissue blocks) should be specified in the laboratory manual (refer to ANP.22250). Multitissue blocks may be considered best practice and can have a major role in maintaining quality. When used as a combined positive and negative tissue control as mentioned above, they can serve as a permanent record documenting the sensitivity and specificity of every stain, particularly when mounted on the same slide as the patient tissue. When the components are chosen appropriately, multitissue blocks may be used for many different primary antibodies, decreasing the number of different control blocks needed by the laboratory. Multitissue blocks are also ideal for determining optimal titers of primary antibodies since they allow simultaneous evaluation of many different pieces of tissue. Finally, they are a useful and efficient means to screen new antibodies for sensitivity and specificity or new lots of antibody for consistency, which should be done before putting any antibody into diagnostic use. Evidence of Compliance: ? Written pro
RE: [Histonet] IHC pos. & neg. control question
Pete, Can't argue with that. I think for the sake of expediency most clinical services run a "known" positive and a patient slide for negative. In the case of H. Pylori, for instance, we may cut a box of control slides and it's possible to go through the area where the organisms were. This also happens with controls that demonstrate positivity by other means epithelium, tumor, etc. We may have to re-run tests in these situations. I believe we are similar to many clinical labs in our reliance on known positives. tj -Original Message- From: pete.peder...@healthonecares.com [mailto:pete.peder...@healthonecares.com] Sent: Thursday, May 19, 2011 2:54 PM To: Thomas Jasper; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Thomas, Agreed, however, how can you say with certainty that the control is still good, or the antibody is still performing optimally? Hypothetically speaking, if you had a known positive control and ran it like a patient specimen (positive and negative) and had staining in the negatively stained control that you had been only running as a positively stained control prior, how would you proceed? What good is a positive control without if it is not treated identically as patient tissue. If you had none specific staining in a patient negative but is was also there in your known positive control which you stained negatively as well, then you could mark up to nonspecific staining to reagent or IHC user error. If the negatively stained positive control stains truly negative and the patient negative has nonspecific staining then you would know patient tissue is compromised or has been mistreated somewhere along the way because your positively stained and negatively stained positive controls demonstrate the staining was done correctly, correct? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: Thomas Jasper [mailto:tjas...@copc.net] Sent: Thursday, May 19, 2011 1:39 PM To: Pedersen Pete; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Pete, When you run a positive control. The tissue is already a known positive (or it should be) for whichever antibody you are running regardless of prior handling. It would be impossible for this not to be so. However, with a negative, the concern is seeing how the patient tissue turns out when subjected to all the same conditions, minus the antibody. tj -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pete.peder...@healthonecares.com Sent: Thursday, May 19, 2011 12:31 PM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject
RE: [Histonet] IHC pos. & neg. control question
Thomas, Agreed, however, how can you say with certainty that the control is still good, or the antibody is still performing optimally? Hypothetically speaking, if you had a known positive control and ran it like a patient specimen (positive and negative) and had staining in the negatively stained control that you had been only running as a positively stained control prior, how would you proceed? What good is a positive control without if it is not treated identically as patient tissue. If you had none specific staining in a patient negative but is was also there in your known positive control which you stained negatively as well, then you could mark up to nonspecific staining to reagent or IHC user error. If the negatively stained positive control stains truly negative and the patient negative has nonspecific staining then you would know patient tissue is compromised or has been mistreated somewhere along the way because your positively stained and negatively stained positive controls demonstrate the staining was done correctly, correct? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: Thomas Jasper [mailto:tjas...@copc.net] Sent: Thursday, May 19, 2011 1:39 PM To: Pedersen Pete; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Pete, When you run a positive control. The tissue is already a known positive (or it should be) for whichever antibody you are running regardless of prior handling. It would be impossible for this not to be so. However, with a negative, the concern is seeing how the patient tissue turns out when subjected to all the same conditions, minus the antibody. tj -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pete.peder...@healthonecares.com Sent: Thursday, May 19, 2011 12:31 PM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive c
RE: [Histonet] IHC pos. & neg. control question
I can certainly agree with that. Whether it’s the patient’s sample or the known positive control that are stained without primary, either would fulfill the purpose of detecting non-specific positive staining. Jean-Martin From: Angela Bitting [mailto:akbitt...@geisinger.edu] Sent: Thursday, May 19, 2011 4:50 PM To: Jean-Martin Lapointe; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IHC pos. & neg. control question I agree with the majority. A patient slide stained without primary antibody, and a patient section with primary antibody and a known positive control tissue, on the same slide when possible, is sufficient. I do agree, however, that the use of a negative tissue control is important in your initial antibody optimization and validation. Angela Bitting, HT(ASCP), QIHC Technical Specialist, Histology Geisinger Medical Center 100 N Academy Ave. MC 23-00 Danville, PA 17822 phone 570-214-9634 fax 570-271-5916 email. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC pos. & neg. control question
I think that this is indeed what is happening here, that there is confusion between a negative control stain (positive tissue, stained without the primary ab) and a negative control tissue (tissue known to not express the marker, stained normally). I had assumed we were talking about the former, because this is what the pathologist cited by Curt wrote in his email. While a known negative control tissue is useful in an IHC run, I am somewhat alarmed to gather from the responses sent that a section without primary is NOT being included in standard IHC runs at your respective labs. To me, this is an absolute necessity in order to properly evaluate IHC results. But that might be because I work in research rather than in a clinical setting, where i'm assuming that priorities are different. Also, I do not agree with Glen's post, for the reasons outlined by Pete. Jean-Martin -- Message: 11 Date: Thu, 19 May 2011 13:37:38 -0500 From: "Sebree Linda A" Subject: RE: [Histonet] IHC pos. & neg. control question To: "Dawson, Glen" , Message-ID: Content-Type: text/plain; charset="us-ascii" I basically agree with you Glen. I think some people are mixing up Negative Reagent Controls (substituting negative serum, Ab diluent, etc. for antibody) and Negative Tissue Controls (substituting a tissue known to be negative for the antibody being run). It CAN be confusing. Linda A. Sebree University of Wisconsin Hospital & Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 1:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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 -- Message: 12 Date: Thu, 19 May 2011 14:53:57 -0400 From: Mary Helie Subject: [Histonet] neg control To: histonet@lists.utsouthwestern.edu Message-ID: <4dd56745.3030...@yale.edu> Content-Type: text/plain; charset=ISO-8859-1; format=flowed What??? News to me as well. -- Message: 13 Date: Thu, 19 May 2011 14:01:45 -0500 From: Subject: [Histonet] Slides for
RE: [Histonet] IHC pos. & neg. control question
Pete, When you run a positive control. The tissue is already a known positive (or it should be) for whichever antibody you are running regardless of prior handling. It would be impossible for this not to be so. However, with a negative, the concern is seeing how the patient tissue turns out when subjected to all the same conditions, minus the antibody. tj -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pete.peder...@healthonecares.com Sent: Thursday, May 19, 2011 12:31 PM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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 ___ 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] IHC pos. & neg. control question
Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC pos. & neg. control question
I agree 100% with Glen. Jan Shivers UMN VDL - Original Message - From: "Dawson, Glen" To: Sent: Thursday, May 19, 2011 1:32 PM Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. & neg. control question
I basically agree with you Glen. I think some people are mixing up Negative Reagent Controls (substituting negative serum, Ab diluent, etc. for antibody) and Negative Tissue Controls (substituting a tissue known to be negative for the antibody being run). It CAN be confusing. Linda A. Sebree University of Wisconsin Hospital & Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 1:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. & neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. & neg. control question
IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) & QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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] IHC pos. & neg. control question
The ideal situation is as follows: a known (+) control with the patient's tissue to make sure that the reaction worked, and a (-) using a section from the patient's tissue to rule out any false (+). René J. From: Curt Tague To: histonet@lists.utsouthwestern.edu Sent: Thursday, May 19, 2011 12:04 PM Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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
AW: [Histonet] IHC pos. & neg. control question
I think this approach mixes up antibody-validation and analysis. The pathologist should be confident, that the used antibody is validated with several types of tissue and that the special antibody stains positive epitopes positive and negative epitopes negative. But the point is, that the patient tissue is unknown and shows perhaps characteristics, that lead to unspecific staining. And this has to be compared to the specific staining in the sample. Gudrun Lang -Ursprüngliche Nachricht- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Jean-Martin Lapointe Gesendet: Donnerstag, 19. Mai 2011 19:26 An: histonet@lists.utsouthwestern.edu Betreff: [Histonet] IHC pos. & neg. control question Hi Curt, I agree with your pathologist. The section that you use as a negative control (without primary) in an IHC run should ideally be tissue that is a known positive, and of the same nature as the test sample. Therefore the best sample is often a section of your positive control tissue. The purpose of this negative control is to make sure that any positive staining observed in the test sample is not due to a spurious cross-reaction, unrelated to the primary. I don't think the issue per se is that you are using tissue from the same patient; rather, it is that you are using a sample for which the staining characteristics are not known. For instance, if are using a lymph node section as a negative, for a stain that targets an epithelial marker (eg Her2) in the test breast sample, then your negative tissue is not appropriate, because since lymph node contains no epithelial tissue, it will not stain no matter what. Therefore if your test sample shows a positive reaction in the epithelial tissue, but for some reason that reaction is a spurious false-positive, then the lymph node negative will not reveal that. I realize that this is all very theoretical and hypothetical, but I understand the pathologist wanting to be confident in the knowledge that all potential technical issues are eliminated before making his diagnosis. Jean-Martin __ Jean-Martin Lapointe, DMV, MS, dACVP Vice-President, Pathologie AccelLAB Inc 1635 Lionel-Bertrand, Boisbriand Québec, Canada J7H 1N8 tel: 450-435-9482 ext.247 fax: 450-435-4795 jm.lapoi...@accellab.com -- Message: 24 Date: Thu, 19 May 2011 09:04:24 -0700 From: "Curt Tague" Subject: [Histonet] IHC pos. & neg. control question To: Message-ID: <019801cc163e$6c1487d0$443d9770$@ta...@pathologyarts.com> Content-Type: text/plain; charset="us-ascii" I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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] IHC pos. & neg. control question
Hi Curt, I agree with your pathologist. The section that you use as a negative control (without primary) in an IHC run should ideally be tissue that is a known positive, and of the same nature as the test sample. Therefore the best sample is often a section of your positive control tissue. The purpose of this negative control is to make sure that any positive staining observed in the test sample is not due to a spurious cross-reaction, unrelated to the primary. I don't think the issue per se is that you are using tissue from the same patient; rather, it is that you are using a sample for which the staining characteristics are not known. For instance, if are using a lymph node section as a negative, for a stain that targets an epithelial marker (eg Her2) in the test breast sample, then your negative tissue is not appropriate, because since lymph node contains no epithelial tissue, it will not stain no matter what. Therefore if your test sample shows a positive reaction in the epithelial tissue, but for some reason that reaction is a spurious false-positive, then the lymph node negative will not reveal that. I realize that this is all very theoretical and hypothetical, but I understand the pathologist wanting to be confident in the knowledge that all potential technical issues are eliminated before making his diagnosis. Jean-Martin __ Jean-Martin Lapointe, DMV, MS, dACVP Vice-President, Pathologie AccelLAB Inc 1635 Lionel-Bertrand, Boisbriand Québec, Canada J7H 1N8 tel: 450-435-9482 ext.247 fax: 450-435-4795 jm.lapoi...@accellab.com -- Message: 24 Date: Thu, 19 May 2011 09:04:24 -0700 From: "Curt Tague" Subject: [Histonet] IHC pos. & neg. control question To: Message-ID: <019801cc163e$6c1487d0$443d9770$@ta...@pathologyarts.com> Content-Type: text/plain; charset="us-ascii" I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC pos. & neg. control question
Curt, Nonsense. The negative control is used to evaluate endogenous staining in the *patient* tissue. Your Pathologist needs to do another residency at clown college. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. & neg. control question
That's a new one on me! Linda A. Sebree University of Wisconsin Hospital & Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. & neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ 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] IHC pos. & neg. control question
I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: "I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately." Curt ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
