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									                                                    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.
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 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.

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NAME: _________________________________________________________________________________________
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CITY: _________________________________________                STATE: __________     ZIP: ____________________
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                                **FEE:            $5.00 for MSH members,        $10.00 for non-MSH members
_____ Yes       _____ No        I require a fee receipt for reimbursement from my employer

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