Quality Control in Immunohistochemistry: Report of a

Award Articles and Special Reports Special Report: Quality Control in Immunohistochemistry Report of a Workshop Sponsored by the Biological Stain Commission

JULES M. ELIAS, PH.D., ALLEN M. GOWN, M.D., ROBERT M. NAKAMURA, M.D., DAVID C. WILBUR, M.D., GILBERT E. HERMAN, PH.D., M.D., ELAINE S. JAFFE, M.D., HECTOR BATTIFORA, M.D., AND DAVID J. BRIGATI, M.D.

Although immunohistochemical methods are increasingly applied in diagnostic histopathology, there has been little standardization or quality control of immunoreagents; and published reports have not standardized Material and Methods for meaningful comparisons of results among clinicians. The Biological Stain Commission-sponsored workshop was convened to address the following issues: a manufacturers' testing program for probity of commercial antibodies, development of a manual for performance criteria and quality control assurance procedures, standardization of package inserts, standardization of information provided in the Materials and Methods sections of publications, establishment of a reagent and procedure clearing house, study of the effects of different fixation regimes on tissue antigens, and investigation of the environmental conditions needed for antigenantibody interaction. The recommendations of the ad hoc committee and their implications for the future are discussed. (Key words: Antibodies; Diagnostic histopathology; Immunohistochemical methods; Immunoreagents; Standardization) Am J Clin Pathol 1989;92:836-843 THE NUMBER OF REPORTS describing the application of immunohistochemical methods in diagnostic histopathology has grown steadily since the original National Cancer Institute (NCI)-sponsored workshop on immunoperoxidase techniques in diagnostic pathology held in May 1979.3 The major concerns of the NCI group were diagnostic applications for lymphoreticular and endocrine tumors, oncological developmental markers, and method considerations. At the June 1986 annual meeting of the Biological Stain Commission (BSC), there was a discussion of the need for quality control of immunostaining reagents and their technical use that would parallel the successful standardization and certification program of biological stains car-

Received December 30, 1988; received revised manuscript and accepted for publication February 14, 1989. Address reprint requests to: Biological Stain Commission, Box 626, University of Rochester Medical Center, Rochester, NY 14642.

University of New York at Stony Brook School of Medicine, Stony Brook, New York; University of Washington School of Medicine, Seattle, Washington; Scripps Clinic and Research Foundation, La Jolla and City of Hope National Medical Center, Duarte, California; The University of Connecticut Health Center, Farmington, Connecticut; Sinai Hospital of Detroit, Detroit, Michigan; National Institutes of Health, Bethesda, Maryland; and The Milton S. Hershey Medical Center, Hershey, Pennsylvania

ried out by the Commission in the past 50 years.17 The pressing need for such quality control was affirmed although the issues and administrative procedures needed more study. In 1987, the trustees of the BSC accepted a proposal by one of us (J.M.E.) that a panel of experts be identified and solicited as to how testing might be done to obtain quality control in immunohistochemistry and to identify potential problems involved. As a follow-up, in June 1988 the BSC supported and convened a one-day workshop in Washington, D.C., on Standardization of Immunoreagents. This meeting was attended by an ad hoc committee. The BSC workshop was a natural outgrowth of the original NCI group effort and addressed issues not previously considered or those requiring additional review. The following topics were discussed for quality control measures: 1. a voluntary manufacturers' testing program to determine probity of any monoclonal or polyclonal antibody offered for sale; 2. development of a manual to provide guidelines on performance criteria and quality control assurance procedures; 3. standardization of package inserts; 4. standardization of information provided in the Materials and Methods portion of published results;

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AWARD ARTICLES AND SPECIAL REPORTS

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5. establishment of a clearinghouse to facilitate the flow of information; 6. the effects of different fixation regimes on tissue antigens; and 7. investigation of the environmental conditions conducive to antigen-antibody interaction. The recommendations of the workshop panel were presented at the annual BSC meeting for consideration by the Board of Trustees. They have been summarized in the following report. Manufacturers' Testing Program Although this program will be a voluntary one, we expect that the privilege of carrying the commission's "seal of approval" would be a strong positive incentive for voluntary participation. All vendors of antibodies will be asked to submit to the BSC a complete standardized Antibody Specificity Performance Test certification and Product Information Sheet. The information provided in the Product Information Sheet will be incorporated into the manufacturers' package insert (see Standardization of Package Inserts). Antibody Specificity Performance Test Antibody specificity will be documented by vendor's certification that the antibody had successfully passed a "performance test" on a sausage-type block, which will consist of an array of tissues/tumors fixed in common fixatives.1"'25 Results of this test will be indicated in terms of tissue/tumor specificity and optimal fixation requirements. We discussed several ways of implementing this program. In one, the vendor would be required to supply an immunostained sausage slide along with the antibody. As an alternative, vendors could contract with satellite laboratories authorized by our committee and approved by the BSC and/or the College of American Pathologists (CAP). These satellites would test the vendor-supplied antibodies on a sausage-type block. It is possible that, in lieu of the vendor's actually sending out an immunostained slide, only a copy of the satellite laboratory's report need accompany the Product Information Sheet. There was some question as to whether a viable program can be safely left in the hands of the vendors. To assure credibility, some participants preferred that testing be done by independent laboratories. A consensus view on this issue has not been obtained. Manual A manual similar to an NCCI guideline will be developed to address performance criteria, quality assurance

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procedures, technical pitfalls of current methods, and interpretation of results. Standardization of Package Inserts Although some manufacturers already provide some or all of the specific information recommended by the committee on package inserts for primary antibodies, this is not standard practice for all primary antibodies. Guidelines are proposed for a uniform approach to reporting information that will be useful to laboratories using these reagents. The following are recommended for package inserts provided with all primary antibody reagents: 1. clone designation for monoclonal antibodies; 2. animal species and preparation techniques for polyclonal antibodies; 3. lot number and specific antibody concentrations; 4. immunoglobulin class; 5. total protein and specific antibody concentrations; 6. antibody diluent or medium where applicable; 7. description of the antigen (immunogen) against which the antibody was raised; 8. operating characteristics of the antibody, including reactivities in various fixatives, optimal processing techniques, and use of pretreatment regimes (e.g., proteolytic enzyme treatment); 9. brief statement of the reactivity of the antibody, with sensitivity and specificity data for specific tissues or antigen. This section also could identify clinical applications of the reagent, citing references from the science literature; 10. tissue requirements for positive controls; 11. storage requirements for the antibodies; and 12. shelf life of the antibodies based on storage methods. In addition to providing a uniform approach to comparison of the results of one laboratory to another, the inclusion of these data with each antibody sold by commercial manufacturers will greatly assist users to optimize staining reactions in their own laboratories and would facilitate interlaboratory comparison of results. Similar information can be easily transferred to scientific articles concerning the usefulness, sensitivity, and specificity of a particular reagent. Standardization of Material and Methods Sections in Published Reports All standard laboratory licensing procedures are based upon standard methodology (procedure manuals) and standard checks of control material—both external/internal proficiency testing and accurate documentation. High quality communication to allow reproducibility of results is necessary for standardization of diagnosis. Immunohistochemistry, a relatively new technology, lacks a unified national standard. A review of current literature

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from representative pathology and oncology journals reveals significant deficiencies in articles incorporating immunohistochemical techniques. An example of the current state of immunohistochemistry quality control is illustrated in a recent copy of a leading pathology journal. An article in this journal is accepted by many pathologists as established fact; and based upon this communication, diagnosis on patients will be rendered. Of the ten articles in this issue, nine relied on immunohistochemistry to help support the diagnosis and one used only immunohistochemistry to establish a diagnosis not available by other techniques. The quality control process for the immunohistochemistry methodology was undertaken to determine if the results could be easily replicated in other laboratories with only the information given in the articles. Because in eight of nine cases the light microscopic diagnosis was the starting point for case selection, the questions asked were: "How sure were the investigators that the light microscopic diagnosis was correct? What criteria were used to establish the light microscopic diagnosis? What special nonimmunohistochemical tests served as the gold standard to establish the diagnosis?" Neuroendocrine peripheral nerve sheath tumors, myospherulosis, giant cell tumor of tendon sheath, signet-ring carcinoma of prostate, polymorphous adenocarcinoma of minor salivary gland, Kaposi's sarcoma, hepatic adenoma in glycogen storage disease, and adenocarcinoma of rete testis were retrieved by the individual investigators from archival or current case material for point-by-point case comparison. After cases were selected in a retrospective manner, immunohistochemistry was performed, leading to questions about materials and methods, techniques, and protocol. The type of fixative was listed in four papers, with one paper using "fresh tissue." Four articles stated only that "paraffin blocks" were used. The specifics of fixation were not addressed in any article, so time and temperature were unknown. Only two of four papers stated that 10% (v/v) neutral buffered formalin was the fixative used. Tissue processing was not addressed in any paper; thus, in all likelihood, tissue placed in a vacuum processor using heat, vacuum, and pressure was treated the same as an Autotechnicon or hand-processed tissue. The solutions used, be they ethanol, toluene, xylene, synthetic xylene, or chloroform, were not mentioned in any paper. The published immunohistochemical technique was then examined. Antibody panels, ranging from 3 to 24, were used in eight of nine papers. Of these, "private" (i.e., noncommercially available) antibodies were used in four papers, and commercial antibodies used in all papers. The antibodies' lot numbers were not given in any of the papers, whereas the clone numbers were given in three of

A.J.C.P. • December 1989

five monoclonal studies. The antibody titer was given in only four of nine papers. In two of nine papers, the initial handling of the immunohistochemistry slides described a blocking step for endogenous peroxidase, and only one of nine papers described a serum blocking step for nonspecific protein staining. The actual immunohistochemical staining technique was described in all papers: ABC (four), PAP (six), indirect (one). Most papers did not state if the techniques were performed exactly as published by the original investigators or if modifications had been used. Enzyme pretreatment was done in three of nine papers, although many articles were studies of paraffin blocks with antibodies known to require predigestion for maximal activity. When enzyme was used, only two of three papers indicated the conditions of pretreatment, such as enzyme concentration, buffer, temperature, and digestion time. The chromogen was listed in three of nine papers, with most articles referring the reader to books on immunohistochemistry for "standard methodology" without mention of specific page numbers for development of the color reaction. Regarding quality control procedures, only four of nine papers indicated a serum control; three of nine mentioned a ± tissue control. Internal tissue controls were described in the published Results sections in two of nine papers. Of interest, normal liver adjacent to an adenoma was reported as negative for alpha-1-antitrypsin, the liver representing the primary organ in which this protein is manufactured. None of the papers used adsorption or neutralization antibodies controls; one used radioimmunoassay for determining the antibodies' specificity. Grading of immunohistochemical reactions was discussed in three of nine papers, with two semiquantitatively and one quantitatively. This critical journal review graphically illustrates some of the problems we face in immunohistochemistry. A recommendation, therefore, will be made to journal editorial boards that minimum standards be introduced for reporting immunohistochemical data to permit objective comparisons of potentially conflicting data from different laboratories. The Committee will recommend that Materials and Methods sections of papers include specifics of: fixation and tissue processing; antibodies' lot numbers, clone numbers and titrations; enzyme pretreatments and blocking steps; whether the specific technique is as published by the original investigators or modified (if modified, how?); chromogens; positive and negative controls; determination of antibody specificity; and grading systems. It would facilitate matters if the recommendations of the BSC were accepted by the Committee of Medical


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Journal Editors so that uniform technical requirements peculiar to immunohistochemistry would be automatically implemented by participating journals. 8 Facilitating the Flow of Information The results of test performances will be disseminated to the science community through a variety of formats. Package inserts will include performance standards for an antibody, and companies also will identify their products as certified by the BSC through labeling of package containers and commercial advertising. The Commission or other agency will maintain a list of tested and approved antibodies, available upon request. This listing will state performance standards for each antibody evaluated: titrations, diluents, staining protocol used, tissues tested, and recommended fixatives. All tested and approved lot numbers also will be listed. A number of other possibilities exist. A newsletter published by the BSC has been proposed, perhaps in collaboration with a cooperating organization such as CAP, or inclusion of the same information in the CAP newsletter. The newsletter also could serve an educational purpose by publishing brief invited articles on the use of antibodies in differential diagnosis and by disseminating information on specific antibodies tested or used in experimental protocols, new techniques, fixation procedures, technique pitfalls, and other tips of relevance to the clinical pathologist or investigator. The BSC might maintain a bibliography of publications relevant to tested antibodies, which could then be distributed to investigators upon request or periodically printed in appropriate BSC publications such as Stain Technology. The BSC could distribute, at a reasonable cost, a paraffin sausage-type block containing normal tissues with appropriate fixation.'"-25 A slide stained in a BSC laboratory would be distributed with the paraffin block. This slide, stained with a commonly used reagent like antikeratin or antivimentin, would provide a representative example of well-stained tissue section to serve as a comparison with normal controls prepared for pathologists in their own laboratories. Testing laboratories sponsored by the BSC also could serve as technical advice centers for advice about fixation, staining procedures, or other methodology questions. They also could be used as sites for BSC-sponsored continuing medical education programs or workshops for hands-on instruction in immunohistochemical techniques. Effects of Tissue Fixation on Immunohistochemistry The availability of monoclonal antibodies and reliable kits for immunohistochemistry could theoretically lead

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to better reproducibility of immunoresults among various laboratories. It is evident, however, that such is not yet the case; the literature reveals many examples of discrepancies, even when the same antibodies are used. Variation in tissue processing is one of the most important factors of this problem. Unfortunately, there is no perfect fixative for immunohistochemistry. Some antigens are well preserved with one but not with another fixative; some are destroyed by any type of fixation and are available for immunohistochemistry only in frozen tissue. Thus, the safest—although not the most practical—approach would be to use a variety of fixatives for every biopsy specimen. The ideal treatment, covering all bases, would be to snap-freeze an aliquot of each biopsy sample, fix portions in absolute ethanol or an alcohol-based fixative such as methacarn, and fix in buffered formalin. 111 ' 25 Because this is somewhat impractical, and often impossible, in the daily practice of pathology, it is imperative that a search for a universal fixative be undertaken. An ideal fixative should penetrate the tissues rapidly, preserve most of the antigens, be nontoxic, and result in conventional histologic preparations that are not much different from those obtained with formalin fixed tissues. Formalin Formalin, the most popular fixative, is unfortunately not the best suited for immunohistochemical techniques. It has been hypothesized that formalin and other aldehydes cross-link proteins around antigenic sites, resulting in a masking effect that interferes with binding of the antibody. This masking can at times be removed by proteolytic enzymes, providing that the antigen itself (or the epitope, in the case of monoclonal antibodies) is resistant to the enzyme. To complicate matters further, this crosslinking and the resulting masking effect are gradual and progressive. Tissues fixed for brief periods (12-24 hours) may show relatively well-preserved antigenicity. The same tissues immersed in fixative for longer periods may no longer be immunoreactive. Because pathologists either do not pay much attention to the duration of fixation in formalin or have no control over it, this is one of the most serious drawbacks of formalin fixation and accounts for much of the lack of reproducibility of immunohistochemistry among laboratories and within the same laboratory. If formalin fixation is too short (3-6 hours), the tissues are fixed partially in formalin and partially in alcohol, as fixation is completed in the automatic processor by the alcohol baths used in dehydrating the samples. Thus, the periphery of the specimen may be relatively well fixed (and cross-linked) in formalin, whereas the core is fixed


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by coagulation by the alcohol. Such tissues are prone to show a peripheral effect, a phenomenon in which the periphery of the section does not stain or, conversely, is the only part of the specimen that does stain, depending on which antigen is sought and by what method. Although much useful information can be obtained from formalin-fixed tissues by immunohistochemical methods, false negative results are commonplace when it is used. As a minimum recommendation, pathologists should be urged to fix tissues in buffered formalin and not to exceed 12, or at most 24, hours of fixation. B5 and Bouin Fixatives B5 and Bouin's fixatives often provide better preservation of antigenic molecules than buffered formalin. This is not always the case, however, because some antigens can be better demonstrated with formalin fixation. Moreover, they have the disadvantage that prolonged fixation must be avoided because it often renders the tissue difficult or impossible to cut. It is conceivable that their apparent superiority over formalin may be attributable, in great part, to the fact that their fixation times usually are kept short. Alcohol-Based Fixatives Alcohol-based fixatives include ethanol (95% [v/v] or absolute), Carnoy's, or methacarn. There is ample evidence from the literature and our own experience that these coagulating fixatives are superior to formalin and most other popular fixatives in preserving antigenicity of tissues. Their distinct advantage over formalin is that a protease digestion step usually is not needed when immunohistochemistry is performed in tissues fixed in these noncross-linking reagents. Ethanol. Tissues may be fixed directly in absolute alcohol, preferably reagent grade. If the sample is kept thin (3 mm or less), penetration and fixation are relatively fast. Tissues are then processed directly to clearing agents (xylenes) and embedded in paraffin. Excellent antigen preservation is possible regardless of duration of fixation, and the tissues are not difficult to section. Its major drawback is that it introduces shrinkage artifact in excess of that usually seen with other fixatives. Carnoy's and Methacarn. These fixatives give similar results to each other and are comparable to plain ethanol in antigen survival. They have the advantage over ethanol in affording faster penetration, so thicker sections can be used. Also, they do not cause tissues to become brittle as plain ethanol does. There are indications that alcohol based proprietary formulas preserve antigens as well as or better than ab-


solute ethanol. 1 '"' 25 Although some tissue shrinkage occurs, it is less than that seen in ethanol-fixed tissues. Morphologic detail approaches that of formalin-fixed tissues. Its drawback is that tissue enzymes do not appear to be destroyed by fixation. Prolonged incubation, therefore, could result in focal tissue autolysis. However, this is not a problem with the usual shorter incubation periods used in immunohistochemistry. In summary, it will evidently be necessary to pay more attention to tissue handling if reproducibility of immunohistochemical results is to be improved. We must search for a fixative that can be used instead of formalin for both routine histologic work and immunohistochemistry. In view of formalin's known toxicity, such a fixative would provide additional health benefits. Microenvironment of the Antigen-Antibody Interaction The moment of creation in any immunohistochemical assay is the precise instant that a primary antibody contacts, recognizes, and subsequently unites with the epitopes of its tissue antigen to form the initial immune complex. All detection systems are simply visualization tools for this event, the magnitude of which depends entirely on the physicochemical microenvironment in which the antibody is placed and the state of the antigen at the tissue surface. In the properly performed immunohistochemical assay, it is the microenvironment of the primary immune complex that governs the ultimate signal-to-noise ratio of the immunoassay. Ideally, when the correct chemical microenvironment is achieved, only specific unions of the primary antibody and its tissue antigen rapidly form without the occurrence of lower affinity, nonspecific interactions. If the antigen is correctly oriented, the strength of the reaction should depend on the number of available epitopes and the corresponding quantity, type, and variability of immune molecules in the medium. However, the ingredients in any medium can affect its performance. Available evidence indicates that immunoreactivity is not only affected by the physical effects of heat but also by the detergent properties, protein concentration, buffer capacity, trace metals, and animal sera sources of the medium in which a primary immune reaction takes place. Many immunohistochemistry laboratories use only one buffer to dilute all their primary antibodies. This buffer may contain a single carrier protein, but some of these proteins can block the effect of the primary antibody instead of optimizing its effect. The primary antibody may contain preservatives to retard bacterial growth that can also weaken immunoreactivity in storage. One of the challenges of the future will be to develop a set of universal standard media that contain the best


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combination of reagents needed by the broadest spectrum of antibodies for optimal reactivity. The production of standardized media for primary antibody reactions will require a systematic study both in the physicochemical state of the antibody and its antigen to determine if the immunoreactivity of monoclonal antibodies can be improved by optimizing the availability of their more restricted epitope sites through changes in the type, strength, and range of the medium used and the mixture of monoclones selected to form an immune cocktail. We will have to know which media improve the presentation of the antigen to the microenvironment of the primary antibody, which systems of enzyme predigestion truly reverse formalin fixation, and which cause the loss of more target than they are worth. The data should result in a set of consensus media that generally optimize immunostaining in an identifiable subset of antigen targets. A comprehensive study to determine the effects of the most widely used fixatives on tissue antigens could be supported by revenues generated from charges for evaluating commercial immunoreagents. Researchers will need a vehicle through which to share data on the media they have found most effective in improving titer, strength, and specificity of a particular antibody. The formulas for these media can then be universally tested and distributed to industrial production sites and clinical laboratories to help ensure the uniformity of an immune reaction. We foresee that the standardization of immunohistochemistry will eventually require international cooperation. Conclusions Although immunohistochemistry is a relatively young science, it already has proved a useful adjunct to conventional morphologic diagnosis.23 However, even its strongest advocates would not hesitate to express concern about the very real risks associated with interpretation of immunostaining if poorly characterized primary antibody is used. The proliferation of commercial kits that provide prediluted antibodies for antigen localization in formaldehyde-fixed, paraffin-embedded human tissue specimens has fostered a false sense ofsecurity within the community of users unfamiliar with ramifications of immunologic reagents that lack defined specificity. Even experienced immunopathologists can have difficulty with interpretations when they encounter unexpected or anomalous staining with these reagents. Anecdotal reports about specificity problems of polyclonal and monoclonal antibodies continue to appear in the literature.4'6-71012-15'18'21'24 Two of the major contributing factors to this problem are contaminating antibodies in polyclonal sera and intrinsic cross-reactivity of mono-

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clonal antibodies. Contaminating antibodies in polyclonal antiserum may result from impurities in the antigen or may be present prior to immunization (e.g., autoantibodies or natural antibodies). The disappearance of immunohistochemical staining after preadsorption of the antibody with the antigen does not necessarily validate specificity because the contaminating antibodies may be adsorbed as well as the specific antibody.22 Only antigens with proven purity should be used for immunization and adsorption, but this is not always practical. Preimmune sera should be tested for the presence of autoantibodies or natural antibodies.7 The intrinsic cross-reactivity of monoclonal antibodies due to either shared or common epitopes in either normal or tumor tissue can not always be anticipated.2 An excellent example is the unexpected cross-reactivity of monoclonal antibodies due to shared antigenic determinants between hematopoietic cells and components of the nervous system.5 Although this shared antigenicity is not generally a disadvantage, it is often not determined until the antibodies are in use, causing interpretive difficulties. The use of radioimmunoassay (RIA) to characterize the specificity of a monoclonal antibody will not reveal the intrinsic cross-reactivity. Some investigators actually maintain that RIA specificity testing may not hold any predictive value for specificity in immunohistochemistry.20'22 The immunohistochemical technique used to examine the distribution of antigens in normal and pathologic human tissues will, in most cases, reveal any peculiar reactivity of primary antibody regardless of the cause.19 The level of quality control achieved is directly related to the size and diversity of the sample of normal and pathologic tissues tested and the experience of the investigator. Users generally have derived specificity information of various markers from studies of nonneoplastic tissues and of classical and unambiguous tumors, not anaplastic or ambiguous tumors; and the latter were the basis for many of the published reports that conflict with the original specificity data. Adequate testing is essential for learning more about the antigenic phenotype of various tumors. 14 Because there are no industry-wide standards for assessing antibody specificity, each supplier decides what constitutes an adequate quality control program for guaranteeing antibody specificity. The kind of extensive testing required to detect cross-reactivity is beyond the scope of most manufacturers.9'26 The user is forced either to perform internal quality control or to seek expert assistance. It is essential that commercial antibodies be subjected to a rigorous trial under controlled conditions to determine if they are as reliable as their noncommercial equivalents. Although the BSC stamp of approval will do much to alleviate the anxieties of both the consumer and the ven-


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dor, there still will be some unexpected but specific staining in tissues that did not previously express a particular antigen.1319 A central clearinghouse to oversee the performance characteristics of a primary antibody will provide all users with access to the information required to make intelligent interpretations. The formation of an independent agency, an "Antibody Staining Committee" of the BSC, supported by funds from the professional organizations representing pathologists (e.g., CAP, American Society of Clinical Pathologists, International Academy of Pathologists) is needed to guarantee that quality control standards are adequate and uniform so that the purchase of a reliable commercial antibody is not a problem.16 A trial period is needed to determine the feasibility and adequacy of the tactics and logistics for a limited number of selected target antigens; recall that the testing protocols for additions of new dyes to the BSC certification list has taken years. The trend toward commercial kits that provide the disclosing reagents and not the primary antibody is very desirable because the user then has a wider choice of immunologic reagents. However, with this change, the user assumes more responsibility for the quality (ergo, the specificity) by choosing one antibody over so many others with purported similar specificity. Because there are more than 200 vendors of immunologic reagents, this can be a formidable, if not impossible, task. Although there are some suppliers who derive their product from the same source, that information is presently not available. Clearly, the BSC committee will not only service the user but afford the provider with the opportunity for quality control measures not routinely obtainable in the marketplace. In particular, the assortment of human tumors and normal tissue needed for adequate testing coupled with the different fixation regimes needed to determine the optimal fixative is not achievable by most vendors. Immunologic reagents whose high quality is.assured by an independent agency will place the vendor in partnership with the BSC and afford the buyer the opportunity to select only those reagents that have been properly evaluated. In summary, although we tend to assume that tissue antigen detection with ready-to-use reagents in commercial kits poses no serious problems, the increasing number of reports describing aberrant or unexpected staining with these reagents would argue the contrary. It is somewhat paradoxical that, although manufacturers make bold disclaimers about the use of their reagents for diagnosis because they lack FDA approval, the most prestigious pathology journals publish reports by equally prestigious investigators attesting to the value of immunohistochemistry in diagnosing human tumors. The development of an Antibody Staining Committee with all of its ramifications,


through its acceptance by suppliers as well as users, can only hasten the process of FDA approval so desperately needed in this burgeoning discipline. Acknowledgments. Other contributors, not in attendance, were: Henry A. Azar, M.D., Veterans Administration Hospital, Tampa, Florida; Richard Cartun, Ph.D., Hartford Hospital, Hartford, Connecticut; Robert A. Erlandson, Ph.D., Sloan Kettering Cancer Center, New York, New York; Frederick H. Kasten, Ph.D., Louisiana State University Medical Center, New Orleans, Louisiana; Francis P. Kuhajda, M.D., The Johns Hopkins Hospital, Baltimore, Maryland; Paul D. Millikin, M.D., Proctor Community Hospital, Peoria, Illinois; Azordes R. Morales, M.D., University of Miami School of Medicine, Miami, Florida; Juan Rosai, M.D. and Lawrence D. True, M.D., Yale University, New Haven, Connecticut; Roger A. Warnke, M.D., Stanford University Medical Center, Stanford, California; and Theresa L. Whiteside, Ph.D., Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.

References 1. Battifora H. The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest 1986;55: 244-248. 2. Dardenne M, Savino W, Bach J-F. Thymomatous epithelial cells and skeletal muscle share a common epitope denned by a monoclonal antibody. Am J Pathol 1987;126:194-198. 3. De Lellis RA, Sternberger LA, Mann RB, et al. Immunoperoxidase technics in diagnostic pathology. Report of a workshop sponsored by the National Cancer Institute. Am J Clin Pathol 1979;71:483488. 4. Fishleder A, Tubbs RR, Levin HS. An immunoperoxidase technique to aid in the differential diagnosis of prostatic carcinoma. Cleveland Clinic Quarterly 1981;48:331-335. 5. Garson JA, Beverley PCL, Coakham HB, et al. Monoclonal antibodies against T lymphocytes label Purkinge neurons of many species. Nature 1982;298:375-377. 6. Goodman ZD, Langloss JM, Bratthauer GL, et al. Immunohistochemical localization of hepatitis B surface antigen and hepatitis B core antigen in tissue sections. A source of false positive staining. Am J Clin Pathol 1988;89:533-537. 7. Harrison F. Contamination of a commercial anti-fibronectin antiserum by antibodies directed against glycosaminoglycan determinants. Histochem J 1985;17:1053-1057. 8. International Committee of Medical Journal Editors. Uniform requirements for manuscripts submitted to biomedical journals. Med Lab Sci 1988;45:209-219. 9. Kelly PMA, Bliss E, Morton JA, et al. Monoclonal antibody EBM/ 11: high cellular specificity for human macrophages. J Clin Pathol 1988;41:510-515. 10. Kobayashi M, Yanagihana E, Hayashi T. Specificity problem of polyclonal rabbit antibody. J Clin Pathol 1988;41:705. 11. Kraaz W, Risberg B, Hussein A. Multibock: an aid in diagnostic immunohistochemistry (letter). J Clin Pathol 1988;41:1337. 12. Leader M, Collins M, Patel J, et al. Desmin: its value as a marker of muscle derived tumors using a commercial antibody. Virchow's Arch [A] 1987;411:345-349. 13. Leader M, Patel J, Collins M, et al. Anti-alpha- 1-antichymotrypsin in staining of 194 sarcomas, 38 carcinomas, and 17 malignant melanomas. Am J Surg Pathol 1987; 11:133-139. 14. Leong AS-Y, Wright J. The contribution of immunohistochemical staining in tumor diagnosis. Histopathology 1987; 11:1295-1305. 15. Millard PR, Chaplin AJ, Heryet AR, et al. Factor VIII related antigen


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positive macrophages and acquired immune deficiency syndrome (AIDS): a problem of antigen specificity. J Clin Pathol 1987;40: 262-266. 16. Milliken PD. Antibody Staining Commission. An idea whose time has come? Pathology 1985;39:43. Mowry RW. The Biological Stain Commission: its goals, its past and its present status. Stain Technology 1980;55:1-4. Nap M, ten Hook KA, Fleuren G-J: Cross-reactivity with normal antigens in commercial anti-CEA sera used for immunohistology. The need for tissue controls and absorptions. Am J Clin Pathol 1983;79:25-31. 19. Nowells K, Kent E, Rinsho K, et al. Prostate specific antigen and acid phosphatase-reactive cells in cystitis cystica and glandularis. Arch Pathol Lab Med 1988,112:734-737. 20. Petrusz P, Sar M, Ordonneau P, et al. Can specificity ever be proved in immunocytochemical staining? J Histochem Cytochem (letter) 1977;25:388-389.

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21. Sobin LH, Hjerstad BM, Sesterhenn IA, et al. Prostatic acid phosphatase activity in carcinoid tumors. Cancer 1986;58:136-138. 22. Swaab DF. Comments on the validity of immunocytochemical methods. In: Chan-Palay V, Palay S, eds. Cytochemical methods in neuroanatomy. New York: Alan R. Liss, 1982,423-440. 23. Taylor CR. Immunomicroscopy: A diagnostic tool for the surgical pathologist. Philadelphia: WB Saunders Co, 1986. 24. Thomas A, Lindsay J, Wilkinson M, et al. HLA-D region alphachain monoclonal antibodies: cross-reaction between an anti-DP alpha-chain antibody and smooth muscle. J Pathol 1988; 154: 353-363. 25 Wan W-H, Fortuna MB, Furmanski P. A rapid and efficient method for testing immunohistochemical reactivity of monoclonal antibodies against multiple tissue samples simultaneously. J Immunol Methods 1987;103:121-129. 26. Woodley, DT, Ernst T, Reese MJ, et al. Neonatal foreskin substrate has limitations for the immunofluorescent screening of monoclonal antibodies. J Invest Dermatol 1987;88:167-171.

NEWS AND NOTICES American Society of Clinical Pathologists/ College ofAmerican Pathologists Joint Annual Spring Meeting (San Francisco, CA) October 20-26, 1990 Joint Annual Fall Meeting (Dallas, TX) For further information contact: Membership Services, American Society of Clinical Pathologists, 2100 Harrison Street, Chicago, Illinois 60612, or call (312) 738-1336.

For further information contact: Henry A. Wilkinson, M.D., at (704) 355-2251.

March 24-29, 1990

9th Annual Pathology Snowmass Ski Retreat (Snowmass Conference Center, Snowmass, CO) For further information contact: John R. Craig, M.D., St. Jude Hospital, 100 East Valencia Mesa Drive, Fullerton, California 92623, or call American Registry of Pathology/Armed Forces Institute of Pathology (714)992-3907.

January 22-26, 1990 February 14-15, 1990 February 16-21, 1990 February 24-25, 1990 February 26-March 2, 1990 March 17-18, 1990

Orthopedic Pathology (Bethesda, MD) Uroradiology (Bethesda, MD) Genitourinary Pathology (Bethesda, MD) Neuroradiology Review (Bethesda, MD) Neuropathology Review (Bethesda, MD)

Hyperbaric Chamber Awareness (Silver Springs, MD) March 19-23, 1990 Forensic Dentistry (Bethesda, MD) Gastrointestinal Pathology Review (Silver March 18-30, 1990 Springs, MD) March 31-April 1, 1990 Gastrointestinal Radiology Review (Bethesda, MD) For further information contact: American Registry of Pathology, Room 1062, Armed Forces Institute of Pathology, Washington, DC 20306-6000, or call (202) 576-2980.

February 12-16, 1990

University of Texas Southwestern Medical Center at Dallas February 9, 1990

Research Advances in Alzheimer's Disease (National Teleconference) For satellite down-linking and technical information contact: Julia Laudenberg, UT Southwestern Medical Center at Dallas, Department of Gerontology and Geriatric Services, P.O. Box 45567, Dallas, Texas 75245, or call (214) 688-7125.

Hans Popper Hepatopathology Society March 4, 1990

North Carolina/South Carolina Societies of Pathologists February 3, 1990

16th Annual William M. Shelley Memorial Lectureship (Charlotte Memorial Hospital, Charlotte, NC)

First Scientific Session of the Hans Popper Hepatopathology Society: Liver Development, Growth and Transformation (Boston Sheraton, Boston, MA) For further information contact: Michael A. Gerber, M.D., Department of Pathology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112.
