Volume XXXV, No. 6, April 2007 Official Publication of the Michigan Society of Histotechnologists The Michigan MIKRO-GRAF Volume XXXV, No. 6 April 2007 IHC PRETREATMENTS Inside this Issue What are they and why do IHC Pretreatments we do them? Laboratory Staff Shortage Rachel Kropf, HTL(ASCP)QIHC Anatech LTD., Battle Creek, MI MSH Officers & Chairpersons Biological Stain Commission HIER, AR, EIER, PIER… what do all of these abbreviations mean? In the world of immunohistochemistry (IHC), these are all abbreviations Around The Region for different ways to pretreat tissue sections before staining begins. Pretreatment is often necessary due to the effects of fixation on tissue sections, and its purpose is to try to expose and unmask the binding MSH Bits From The Board sites where IHC staining will occur. This article will explain some basics of fixation, IHC staining, and some different methods of pretreatment 2007 Annual Meeting Update that are used in today’s labs and why they are used. MSH Website Update … continued page 3 2007-2008 MSH Student Rep LABORATORY STAFF SHORTAGE Dues Increase Update How are we promoting our profession? National Medical Laboratory Week Wendi Edgett, HTL(ASCP) Preposterous Definitions MSH President Winter Seminar Conclusion At some point in your career, you have been or will be asked the question: “What do you do for a living?”… At this point there is usually TechPoints Update a pause where you formulate a response. If you respond “I’m a histotech” or I’m a histotechnologist”, most likely you were asked “What NSH & ASCP Teleconferences exactly is that?” The need for histotechs in the country and in Michigan is growing. Increased retirement and an aging population have Registry Exam Stains attributed to the estimated increase of laboratory professional job openings. The problem is few people know the profession is available. ASCP (BOR) Exam Statistics … continued page 6 Page 1 Michigan MIKRO-GRAF Volume XXXV, No. 6, April 2007 Official Publication of the Michigan Society of Histotechnologists (IHC Pretreatment, cont. from page 1) In any of these procedures that require pretreatment, it is recommended that positively charged slides be Neutral buffered formalin is the most common fixative used. The addition of heat or digestive enzymes can in today’s histology laboratories. It is a universal be rigorous on the tissue and tissue fall-off is often fixative that preserves tissue structure and cell experienced. Also extended slide drying times can be morphology and helpful to retain tissues during pretreatment. “’Pretreatment’ is a generic is easy to term for a method of treating purchase, store, Heat Induced Methods and dispose of. Heat Induced Epitope Retrieval (HIER, at times called the tissue where the fixation Unfortunately, Antigen Retrieval or AR) is the application of heat crosslinks are broken and the formalin works to using different heating sources while immersing the epitope is exposed…” stabilize the slides in a buffered solution to break the crosslinks and tissues by ready the epitope for binding to the antibody. Tissue forming crosslinks between proteins within that tissue. sections are deparaffinized and rehydrated to water. The crosslinks that are formed are strong bonds that Then a buffer is chosen that is preferred to the antigen can cause the configuration of the tissue proteins to that is being detected. These buffers are made with fold and twist upon each other, thus altering the natural different chemicals and are regulated to maintain molecular configuration and masking potential antibody different pH’s. Common buffer solutions at usual binding sites. For routine hematoxylin and eosin (H & manufactured pH’s for HIER are: E) staining and most special stains this abnormal configuration is not an issue so pretreatment is not • Citrate buffer at pH 6.0 necessary, however for some immunohistochemical • EDTA buffer at pH 8.0 reactions these effects of fixation need to be reversed. • Tris buffer at pH 10.0 The basis of IHC staining is an antibody – antigen There are several other buffer solutions that can be interaction. The antigen is present within the tissue used and that are preferred by specific antibodies section. An antibody that has been produced outside of however these are the most common in the clinical IHC the body against that antigen is added to the tissue lab. section and the two will bind. Then a detection system is added, which is a series of reagents that ultimately Common heating elements used for HIER are: bind to each other and yield a colored end product so we can visualize the antibody – antigen interaction. • Vegetable steamer • Pressure cooker The site of an antigen where the antibody will bind is • Microwave called an epitope, and is a unique specific combination • Waterbath of amino acids present within an antigen. When the • Rice cooker formalin ‘fixes’ the tissues and creates the crosslinks that stabilize and preserve the tissues, the epitopes are o o Slides are heated to between 90 C and 120 C, sometimes masked or hidden due to the twisting of the depending on the heating element used. A waterbath molecule on itself. “Pretreatment” is a generic term for will not heat solutions to as high of a temperature as a a method of treating the tissue where the fixation pressure cooker will, due to the restraints of the crosslinks are broken and the epitope is exposed and heating method and equipment. Also different methods becomes available for binding. Pretreatment is will keep the tissues at these elevated temperatures for generally done by the addition of heat or by digestive varying amounts of time. For example, using the enzymes that break these crosslinks. pressure cooker for HIER will keep the slides at a high temperature (and under pressure) for a short amount Primary antibodies prefer specific pretreatment of time (as short as 1 minute). This is a harsh procedures, however at times performing multiple pretreatment, compared to using a vegetable steamer pretreatment procedures will still yield acceptable IHC that keeps slides at a lower temperature for a longer staining. The optimal suggested pretreatment should o amount of time (i.e. 96 C for 25 minutes), which is a be determined by the primary antibody manufacturer gentler pretreatment for the tissue sections. and listed on the data sheet, although at times your lab may find a different pretreatment method to work better. It is often recommended to try a battery of … continued page 4 pretreatment methods for each primary antibody to find your optimal procedure in your lab. Page 3 Michigan MIKRO-GRAF Volume XXXV, No. 6, April 2007 Official Publication of the Michigan Society of Histotechnologists (IHC Pretreatment, cont. from page 3) the method that you are used to at the most standardized temperature and time. For example if you o In addition to using different buffers for different generally use a pressure cooker for 2 minutes at 100 C antibodies, different heating methods are sometimes for most of your antibody pretreatments, use this with also preferred by specific antibodies. The primary the buffers listed above. antibody data sheet that is provided by each manufacturer, with the antibody, should give the Present the 6 slides to your pathologist(s) and leave it recommended beginning guideline for which to their discretion as to what pretreatment method is pretreatment method to use. the best. Do these tests on a piece of control tissue that is known to be positive (usually on the weakly- Digestive Enzyme Methods positive side, to ensure that you can find those weaker- Some antibodies prefer a digestive enzyme expressing cells), and that has been routinely fixed and pretreatment instead of the high heat methods (EIER – processed. Enzyme Induced Epitope Retrieval). After deparaffinization and rehydration, a digestive enzyme An example of specific antibodies preferring different is added to the tissue for generally 5 to 20 minutes, at pretreatments is seen with CD3 (clone PS1). If using o times with the addition of gentle heat (37 C). the battery recommended above, the results one might Examples of digestive enzymes are: see include: • Pepsin 1. No pretreatment – no staining • Trypsin 2. Citrate buffer – light staining • Protease 3. EDTA buffer – dark specific staining • Pronase 4. Tris buffer – overly dark staining, tissue fall-off 5. Pepsin- no staining These enzymes are also called proteolytic enzymes 6. Protease – faint staining (PIER – Proteolytic Induced Epitope Retrieval), as they break down proteins into smaller pieces thus breaking These slides could be evaluated and through your the crosslinks and exposing the epitopes. Each experiments, it could be determined that CD3 (PS1) enzyme will break the fixation crosslinks at different would be best with an EDTA buffer pretreatment in sites. your lab. Pretreatment Battery Pretreatment is an integral step in IHC staining and its Since specific antibodies prefer different pretreatments, importance should not be overlooked or simplified. If and fixation effects can vary due to times or reagents you are having trouble with a particular antibody not used, a recommendation is to have a set battery of working, the pretreatment method being used could be pretreatments to use in your lab to determine the a major part of your troubleshooting procedure. optimal pretreatment conditions for each antibody Remember that different antibodies prefer different when they are used in your hands. pretreatments, and in your hands it could be different than what a manufacturer recommends. There are A recommended battery of pretreatment methods to try many different ways to pretreat tissues prior to IHC, so might include: be sure to realize this when working up an antibody. 1. No pretreatment References 2. Citrate buffer, pH 6.0 in vegetable steamer, 25 Elias, JM. Immunohistopathology: A Practical nd o minutes at 96 C Approach to Diagnosis. 2 edition, ASCP Press, 3. EDTA buffer, pH 8.0 in vegetable steamer, 25 2003. o minutes at 96 C Fredenburgh, JL, Myers, RB. Effects of Fixation and 4. High pH buffer, ie. Tris buffer, pH 10.0 in vegetable Antigen Retrieval. 2002. o steamer, 25 minutes at 96 C o 5. Pepsin digestive enzyme, 37 C for 15 minutes 6. Protease digestive enzyme, room temperature for 10 minutes If a different method of heating is used routinely in your lab (like a pressure cooker) then use the buffers above (citrate, EDTA, and Tris at the recommended pH’s) in Page 4 Michigan MIKRO-GRAF Earn 0.5 contact hours of continuing education by reading articles in the Michigan Society of Histotechnologists newsletter MIKRO-GRAF. MSH contact hours can be used for CMP required by ASCP BOR to maintain certification. It is the responsibility of the participant to retain their MSH CE certificates as proof of continuing education. ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ DATE OF ARTICLE: April 2007 TITLE: IHC Pretreatments AUTHOR: Rachel Kropf, HTL(ASCP)QIHC DIRECTIONS: 1. Answer the following questions by circling the one (1) BEST answer for each question. 2. Complete the information required at the bottom of the page. 3. Submit questions & check (MSH) to: Peggy Wenk, HTL(ASCP)SLS, 3840 Elmhurst Rd., Waterford, MI 48328 To earn Continuing Education credit from MSH, completed form must be submitted within twelve (12) months of original date of the article. 1. Which of the following causes the most amount of alteration in protein molecular configuration that can mask potential antibody binding sites? A. Fixation B. Dehydration C. Clearing D. Infiltration 2. All of the following are common buffer solutions used for HIER except: A. Citrate, pH 6.0 B. Phosphate, pH 7.0 C. EDTA, pH 8.0 D. Tris, pH 10.0 3. All of the following are enzymes used in digestive EIER except: A. Pepsin B. Peroxidase C. Pronase D. Protease 4. TRUE or FALSE (circle one): When establishing an IHC protocol for a new antibody, it is recommended to try out a variety of HIER and EIER pretreatments. PLEASE PRINT NEATLY DATE and YEAR Completed/Submitted Test: __________________________ NAME: _________________________________________________________________________________________ STREET: ________________________________________________________________ APT. _____________ CITY: _________________________________________ STATE: __________ ZIP: ____________________ PHONE: ____________________________________ Email: _____________________________________________ _____ Yes _____ No I am a Michigan Society of Histotechnologists (MSH) member _____ Yes _____ No I have included a check made out to “MSH” on US funds **FEE: $5.00 for MSH members, $10.00 for non-MSH members _____ Yes _____ No I require a fee receipt for reimbursement from my employer Certificate documenting 0.5 hours MSH continuing education (CE) will be mailed to the participant within 4 weeks.
Pages to are hidden for
"MIKRO-GRAF"Please download to view full document