IHC Resource Group Bulletin
December, 1999

Volume 2, Number 3

Antigen Retrieval Question
FDA Regs and IHC
NCCLS Guideline for IHC
IHC Resouce Group Bank

Greetings from the IHC Resource Group.  We hope you all had a wonderful Holiday and wish everyone a Happy and Healthy 2000!!

Antigen Retrieval Question

This query and response recently appeared on Histonet.  We felt it to be worth repeating here.  Many thanks to Catherine Bennett and John Kiernan.

Question:

Do all antigen retrieval procedures do the same thing?  In other words, can an antigen retrieval procedure that is used in one immunohistochemical run be used successively in another IHC run? 

Or is it more a case of  trial and error to see which antigen retrieval will work in any given new IHC procedure?   Let's say for this query that fixation and tissue type are the same, and just the primary antibody is different.

Answer:

Antigen retrieval works by causing either acid- or base-catalysed hydrolysis of methylene cross-links between protein molecules,  which are introduced by fixation in formaldehyde, and possibly also by removing calcium ions (citrate and EDTA are common ingredients of retrieval solutions). Most antigens are retrieved by heating in either acidified (pH 1) or alkaline (pH 8-10) water, some by either or neither of these. Less extreme pH levels (e.g. 6) are often effective and less likely to cause section losses. Something about each antigen seems to determine which retrieval method is most effective. There are some good surveys in the literature, with tables showing the effects of different retrieval methods on many antigens. Here is a very briefly annotated list of 4 such papers:

Taylor,CR; Shi,SR; Cote,RJ (1996a): Antigen retrieval for immunohistochemistry - Status and need for greater standardization. Appl. Immunohistochem. 4(3), 144-166. Review of antigen retrieval procedures for paraffin sections of formaldehyde-fixed tissues.

Taylor,CR; Shi,SR; Chen,C; Young,L; Yang,C; Cote,RJ (1996b): Comparative study of antigen retrieval heating methods: Microwave, microwave and pressure cooker, autoclave, and steamer. Biotech. Histochem. 71(5, Sep), 263-270.

Pileri,SA; Roncador,G; Ceccarelli,C; Piccioli,M; Briskomatis,A; Sabattini,E; Ascani,S; Santini,D; Piccaluga,PP; Leone,O; Damiani,S; Ercolessi,C; Sandri,F; Pieri,F;Leoncini,L; Falini,B (1997): Antigen retrieval techniques in immunohistochemistry: Comparison of different methods. J. Pathol. 183(1, Sep), 116-123.

Shi,SR; Cote,RJ; Chaiwun,B; Young,LL; Shi,Y; Hawes,D; Chen,TY; Taylor,CR (1998): Standardization of immunohistochemistry based on antigen retrieval technique for routine formalin-fixed tissue sections. Appl. Immunohistochem. 6(2,  Jun), 89-96.

Most antigens are retrieved in formalin-fixed paraffin sections treated at pH either 1 or 10. One needed pH 1. The antigen retrieval solutions used in the above publications are all easy to make up, and there does not seem much point in buying one commercially, especially if it turns out to be less than optimal for the antigen you want to detect. Don't use any solution unless you know its composition.


FDA Regs and IHC

The following are the comments of  Peter A. Takes, Ph.D., RAC Director, Clinical & Regulatory Affairs STEREOTAXIS, Inc. in response to a question regarding FDA Regs and Immunohistochemistry, reprinted by permission:

The NCCLS IHC Guideline is in the final stages toward 'Approved Guideline' status.  In the interim, the proposed guideline can be obtained from NCCLS via their web site at http://www.nccls.org

FDA (and hence HCFA) is starting to offer a bit more latitude in the use of IHC by individual labs.  There are basically three types of reagents that you can acquire based on their labeling: "Research Use Only" [RUO], "In Vitro Diagnostic Use" [IVD], and "Analyte Specific Reagents" [ASR].  Most states are getting pretty tight on the use of RUOs for diagnostic purposes, i.e., Medicare won't reimburse tests that employ reagents so labeled.  California is probably among those leading the way. 

ASRs can be used for diagnostic purposes by ANY high complexity lab,  but you have to establish, verify, and validate the tests on your own in order for them to be acceptable to inspectors and reimbursable. This is where the laboratory "disclaimer" on final reports comes in to play (See footnote below).

Manufacturers are restricted, by regulation, from providing a great deal of application help for ASRs.  This is probably how many companies are
labeling their antibodies. IVDs can be used by ANY high complexity lab for diagnostic purposes, and manufacturers can provide application assistance within the context of the product's intended use.

What you need to look for is how the reagent is labeled, its intended use, and what type of other information is provided by the vendor.  True,
because of regulatory costs, most diagnostic reagents (IVDs and ASRs) will come from "big" companies, but that is not universally the case. 

Many "small" manufacturers also have IVD and ASR reagents.  For more current information, contact the  Joint Council of Immunochemical
Manufacturers [JCIM; formerly Joint Council of Immunohistochemical
Manufacturers] via their web site at http://www.jcim.org .

JCIM keeps up-to-date on end-user requirements and can better provide the current perspective.  They represent international companies and contract research organizations as well as many of the smaller companies in the industry, and are a primary source of regulatory and usage information for end-user labs as well.

Please note the views expressed are solely observations and interpretations from my position as a regulatory professional, and are not to be interpreted as and are not necessarily the views or positions of my employer, Stereotaxis, Inc.

For further, more detailed, information, refer to: Microscopy Today 99-7:8. September, 1999. Clinical Laboratory News 24(4):10. 1998. and 21(7):43-44. 1995.

Reference:
Peter A. Takes, Ph.D.

Footnote:

The disclaimer on diagnostic reports should resemble the following:

"This test was developed and its performance characteristics determined by (your laboratory).  It has not been cleared or approved by the U.S. Food and Drug Administration.  The FDA has determined that such clearance or approval is not necessary.  This test is used for clinical purposed.  It should not be regarded as investigational or for research.  This laboratory is regulated under the Clinical Laboratory Improvement Amendments of 1988 (CLIA) as qualified to perform high complexity clinical testing.

References:     1.   Debbie Jennings-Sienna, Chair, NSH Legislative Committee       2.   College of American Pathologists




NCCLS Guideline for IHC: More Info

New Guideline Coming Soon From NCCLS (www.nccls.org)

NCCLS is a globally recognized, voluntary consensus standards-developing organization that enhances the value of medical testing within the healthcare community through the development and dissemination of standards, guidelines, and best practices.

The use of immunocytochemical methods, though well-established,
nevertheless continues to challenge the pathologist.  Despite the clear
benefits of having another method by which to gain insight into the nature of disease processes, issues of sensitivity, specificity, and
reproducibility mandate the application of more stringent practices in many laboratories.  To ensure the success of immunocytochemical diagnostic methods, pre-analytic, analytic and post-analytic factors influencing test performance may all warrant attention. 

The upcoming Approved-Level NCCLS guideline, MM4-A, Quality Assurance for Immunocytochemistry, addresses the following topics as they relate to the use of immunocytochemical methods in diagnostic pathology and laboratory medicine:

• tissue collection and sample handling
• fixation
• postfixation processing
• "antigen retrieval" methods
• immunocytochemical staining protocols
• automation
• internal and external controls
• troubleshooting
• validation of new immunocytochemical assays
• reporting of assay results.
  

The importance of laboratory participation in external quality assurance
activities, such as proficiency testing programs, is a given.  In this
document the subcommittee assumes that the laboratory will take part in
such programs, when available.  However, it is beyond the scope of this
document to suggest the elements required of such external quality
assurance activities. The document should be useful to pathologists,
histotechnologists, manufacturers of immunocytochemical devices and
reagents, and regulatory personnel.

MM4-A has recently been submitted to the NCCLS Board of Directors for review and approval.  Upon approval by the Board, the document may be published as an approved guideline by early January 2000.

Reference:
Timothy J. O'Leary, M.D., Ph.D. Marc R. Schlank, M.T. (ASCP), M.S.



FROM THE IHC RESOURCE GROUP BANK

The IHC Resource Group is once again gearing up to help support the efforts of applicants who have applied for the spring IHC Qualification exam.  I have just received a copy of the May 2000 exam, and there are a few changes from the last period.  The chart below describes what is being required.

Board Of Registry Exam For Immunohistochemistry Qualification, May 2000

     

The bank is very low on some tissues, on probe and probe detection systems.  I am earnestly asking for your help.  We are out of probe and probe kits, and as you can see, the applicants will now have to perform HPV on infected cervix.  We have no tissue or reagents presently to support this part of the exam.  If anyone can donate anything, tissue being in the greatest demand,  please let me know.  Ask your vendors for samples for donation to this effort.  This might help us
further.

Additionally we are low on prostate, ER pos breast CA, melanoma, and kidney tissue that has both medulla and cortex, as well as frozen lymph node or frozen tonsil.

The antibody stores are pretty good.  If anyone can contribute a paraffin
block for any of the  above tissue, please email me at emacrea@ventanamed.com, call me at 800-227-2155 ext 2739,  or simply mail the blocks to me at the address below.  Thank you for your cooperation!


Ethel R Macrea
4740 N Brookeview Dr
Tucson, AZ 85704
Or
C/o Ventana Medical Systems Inc.
3865 N Business Center Dr
Tucson, AZ 85705