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doi:10.1369/jhc.5B6691.2005
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Volume 53 (9): 1167-1170, 2005
Copyright ©The Histochemical Society, Inc.
BRIEF REPORT |
Protein-embedding Technique : A Potential Approach to Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections
Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, California
Correspondence to: Clive R. Taylor, MD, PhD, Department of Pathology, University of Southern California Keck School of Medicine, HMR 204, 2011 Zonal Avenue, Los Angeles, CA 90033. E-mail: taylor{at}pathfinder.hsc.usc.edu
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A serial study was performed to develop a protein-embedding technique for standardization of immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) tissue sections. A protein carrier matrix must have two phases, a liquid phase to allow uniform mixing of a protein and a solid phase forming a ‘block’ that can be fixed and processed in the same manner as human tissue. This standardized protein block would serve as a source of thin sections for control of IHC and therefore must also withstand the boiling conditions of antigen retrieval (AR). After multiple experiments, a method was developed utilizing polymer microsphere (beads) as a support medium for protein. The method showed particular promise for quantitative IHC.
(J Histochem Cytochem 53:1167–1170, 2005)
Key Words: immunohistochemistry • antigen retrieval • standardization • protein-embedding-technique • beads
THE DEMAND for quantitative immunohistochemistry (IHC) continues to escalate due to the widespread utilization of IHC in clinical diagnosis and in translational cancer research. This demand is largely a result of the growing emphasis on prognostic markers and therapeutic indicators, as exemplified by the clinical application of Herceptin (anti-Her 2 antibody) for breast cancer treatment (O'Leary 2001
; Shi et al. 2001). Quantitative IHC that is reproducible among laboratories must be based on standardization as a first step (Taylor 1994,1996). Although great efforts have been made in the past two decades, standardization of IHC is easier said than done. One approach has been to adopt a ‘Total Test’ strategy as advocated by our earlier studies (Taylor 1992), addressing pre-analytical, analytical, and post-analytical phases of the test in concert. From a practical point of view, one of the most difficult issues in the standardization of IHC, as applied to formalin-fixed, paraffin-embedded (FFPE) tissues, relates to extreme variability in sample preparation, particularly the time of fixation in formalin. It is, therefore, not surprising that the preservation of antigenicity in FFPE tissues may vary greatly and may result in variable intensity of immunostaining for formalin-sensitive antigens. This critical issue has recently been reviewed by Leong (2004), who concluded that it is currently impossible to standardize IHC for accurate quantitation in FFPE tissue sections, unless an optimal reference control material is included and treated in a manner identical to the test specimen. This principle for an optimal reference material has previously been adopted for development of the "Quicgel" method that employs an artificial cell control block, which is added to the tissue cassette containing the clinical biopsy specimen and then subjected to routine fixation and embedding in paraffin (Riera et al. 1999). Although the "Quicgel" method provides a satisfactory reference material for quantitative IHC, it has not been widely accepted because of the following drawbacks: (a) it requires embedding an artificial cell line preparation with each specimen as the paraffin tissue block is prepared; thus, the "Quicgel" cannot be used for retrospective studies; (b) it is not a practical design for standardization of IHC in general because of the numerous different analytes (proteins) that the diagnostic pathologist wants to identify by IHC staining; (c) cell lines are not consistent in behavior under cell culture and storage and are difficult to standardize. Establishing a protein standard in a cell or tissue in order that the tissue may serve as a reference material is, therefore, very difficult.
The application of purified protein incorporated within various matrices was used as a model system for cytochemistry more than half a century ago (reviewed by van der Ploeg and Duijndam 1986). The advantages of a protein-embedding technique include consistency in quantity and quality of protein and easier and more accurate measurement of protein. Because standardization and quantitation of IHC are desired, the protein-embedded matrix model must be subjected to identical conditions as the test specimen, including any antigen retrieval (AR) process. To date there is no published report of a satisfactory protein-embedding technique that lends itself to identical treatment as routine FFPE tissues.
Recently, we have conducted an extensive research for an optimal matrix medium in which to embed proteins for establishing a model reference control system. To reach the goal of identical treatment of FFPE tissue sample, this optimal matrix must have several properties: (a) it must be capable of existing in two phases, liquid and solid; (b) the liquid phase must allow even mixing of a protein and should then easily be converted into the solid phase; (c) the solid phase should be amenable to fixation with formalin and embedding in paraffin without excessive hardening or brittleness, i.e., it must be suited to sectioning by a microtome after embedding in paraffin; (d) sections of this embedding material must remain adherent on glass slides after boiling AR treatment; (e) it must be non-reactive and not interfere with subsequent AR or IHC methods. With these requirements in mind, a variety of materials and methods have been evaluated.
Small pieces of different matrix media were immersed in a solution containing a known amount of proteins for defined periods of time at 4C as documented previously (Brandtzaeg and Rognum 1984). In one example, normal rabbit serum (DAKO; Carpinteria, CA) was polymerized with glutaraldehyde to form a gel. The gel was stored at 4C in several changes of phosphate-buffered saline (PBS, pH 7.6) for at least 3 days and then sliced into small pieces (
1.5 x 1.5 x 5 mm). These fragments were soaked in serial dilutions of protein and then transferred to 10% neutral-buffered formalin (NBF) for fixation and subsequent paraffin embedding along with a gel fragment piece that had not been exposed to antigen (negative control). Thin sections were cut for IHC staining. It was expected that a positive color would be detected in the protein-impregnated gel matrix. However, there were no significant observed differences of staining among positive and negative protein sections, due in large part to cross reactions and apparent nonspecific staining of the rabbit serum gel. Another drawback of this method was an uneven distribution of the protein in this medium (the peripheral area showed much stronger intensity than the center). Finally, the exact amount of protein absorbed into the media was difficult to calculate. In addition to this classic polymerized rabbit serum method, other support gels were evaluated including egg white, duck salted egg white (purchased from a Chinese supermarket), and plastic sponges (for example, regular sponge used for packaging). None were satisfactory due to similar issues as described for the rabbit serum gel. The sponge failed to adequately retain protein due to the holes that were in fact very large in ‘microscopic’ terms and due to lack of covalent coupling to protein.
Having learned the drawbacks of the protein absorption methods described above, a direct protein mixing method was tested by adding known concentrations of protein solution into the liquid phase of the matrix medium (polymerized rabbit serum, agarose gel, alginate beads, gelatin, etc.) and then attempting to induce a solid phase by fixation. Some materials such as agarose and alginate were unable to withstand the boiling condition of AR, i.e., sections made of agarose or alginate were totally lost after the AR heating procedure. Materials such as polyacrylamide (material used for gel electrophoresis of Western blotting) were excessively hardened by formalin fixation and/or other treatments such as dehydration or paraffin embedding. Other materials, such as gelatin and polymerized rabbit serum, did not allow an even distribution of protein in the medium and showed nonspecific background staining as a further complication.
Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago (van der Ploeg and Duijndam 1986), beads have not been used as a protein-embedding matrix for routine IHC on FFPE tissue. We recently tested primary-coated beads (Dynabeads; Dynal, New York, NY) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (50 µl/34.5 µg; DAKO) was bound to the beads by incubating with the beads (150 µl with a concentration of 109 beads/1 µl) at 4C overnight in a cold room with an automatic shaker. Incubation was followed by three PBS washes, then the addition of biotinylated horse anti-mouse antibody (20 µl of concentrated reagent; Vector Labs, Burlingame, CA), and further incubation under the same conditions for 3 hr. The beads, now coated with biotin-conjugated protein, were then fixed in 10% NBF for 20 min, mixed into 1% agarose gel in a small tube, and fixed in 10% NBF overnight. The blocks of agarose gel containing biotinylated protein-coated beads were subjected to the routine tissue-embedding procedure. The AR technique was routinely performed using a microwave oven as previously documented (Shi et al. 2000a). Both AR-treated and untreated slides were then incubated with avidin–biotin–peroxidase (ABC; Vector Laboratories) reagent. For the second experiment, a purified S-100 protein was bound to the beads by the following two steps: (a) a monoclonal (mouse) antibody to S-100 (30 µl of concentrated reagent; Sigma, St. Louis, MO) was incubated with beads for 1 hr at the same conditions described above; (b) after three PBS washes, a purified S-100 protein (5 µl of a concentration of 10 mg/ml; Sigma) was then incubated at the same conditions for 1 hr, followed by three PBS washes and all the subsequent procedures of fixation, paraffin embedding, and AR treatment described above. Slides were then incubated with the primary monoclonal antibody to S-100, followed by biotinylated anti-mouse antibody and the ABC incubation. Sections of a human melanoma were used as positive control for S-100 (Figure 1A). Biotinylated anti-mouse antibody, precipitated onto a slide, was employed as a baseline positive control (data not shown). One section of beads–gel that was treated by AR heating was used for negative control by omitting the ABC complex or primary antibody incubation for first and second experiments, respectively. Amino-ethyl carbazole was employed as the chromogen, yielding a red color in a positive staining result.
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Figure 1 shows the results of IHC staining using Dynabeads coated with biotinylated anti-mouse IgG and protein S-100 for the first and second experiments, respectively. Strong positive staining results were obtained after AR, appearing as red circles surrounding beads (Figures 1B and 1C). Slides without AR treatment also showed positive results but staining was much less intense (data not shown). Negative control slides showed clean background and no evidence of positive staining (Figure 1D).
Identification of a suitable matrix to carry protein is a key issue in attempting to identify a protein-embedding reference material for standardization of IHC. It appears that the Dynabeads tested in these experiments have potential to serve as the matrix based on the following results: (a) beads that are able to bind a variety of mouse monoclonal antibodies and their corresponding protein antigens are commercially available; (b) these beads are suitable for formalin fixation and all subsequent processes of dehydration, clearing, and embedding in paraffin; (c) various proteins (antigens) can be applied consistently to coat polymer beads uniformly; (d) cut sections of embedded beads can be boiled in water for the AR treatment; (e) IHC staining demonstrates specificity and sensitivity comparable with human tissue sections; (f) in one example, the quantitative IHC of certain surface markers such as Her-2/neu appear particularly suited to this method in that the surface-positive label on the beads mimics Her-2/neu cell-surface marking.
Further studies are necessary to develop more sophisticated materials for a protein–matrix in such a way that protein can be mixed into the matrix evenly while meeting all requirements described above. To accomplish this goal, it will be necessary to develop a cooperative study among experts in chemical engineering, biochemistry, and IHC. Methods to assure a firm covalent bond coupling protein on the surface of the matrix will be necessary, similar to the protected isocyanate microscope slide-coating technology proposed by Sompuram et al. (2003).
It is important to recognize that establishing a model of reference material, such as the protein-embedding model described in this work, is just the first step for standardization of IHC. Further studies will be required to develop conversion factors and to explore the potential utility and limitations of this approach (Taylor 1994; Shi et al. 1998,2000b).
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Acknowledgments |
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This study is supported by NIH Grant 1 R33 CA-103455-01.
We greatly appreciate Henry Lin, PhD student, University of Southern California School of Medicine, Department of Pathology, for his kind help in handling the Dynabeads.
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Footnotes |
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Received for publication March 17, 2005; accepted March 23, 2005
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Brandtzaeg P, Rognum TO (1984) Evaluation of nine different fixatives. 2. Preservation of IgG, IgA and secretory component in an artificial immunohistochemical test substrate. Histochemistry 81:213–219[Medline]
Leong AS-Y (2004) Quantitation in immunohistology: fact or fiction? A discussion of variables that influence results. Appl Immunohistochem Mol Morphol 12:1–7[Medline]
O'Leary TJ (2001) Standardization in immunohistochemistry. Appl Immunohistochem Mol Morphol 9:3–8[Medline]
Riera J, Simpson JF, Tamayo R, Battifora H (1999) Use of cultured cells as a control for quantitative immunocytochemical analysis of estrogen receptor in breast cancer. The Quicgel method. Am J Clin Pathol 111:329–335[Medline]
Shi S-R, Cote RJ, Chaiwun B, Young LL, Shi Y, Hawes D, Chen T, Taylor CR (1998) Standardization of immunohistochemistry based on antigen retrieval technique for routine formalin-fixed tissue sections. Appl Immunohistochem 6:89–96
Shi S-R, Cote RJ, Shi Y, Taylor CR (2000a) Antigen retrieval technique. In Shi S-R, Gu J, Taylor CR, eds. Antigen Retrieval Techniques: Immunohistochemistry and Molecular Morphology. Natick, MA, Eaton Publishing, 255–272
Shi S-R, Gu J, Cote RJ, Taylor CR (2000b) Standardization of routine immunohistochemistry: where to begin? In Shi S-R, Gu J, Taylor CR, eds. Antigen Retrieval Technique: Immunohistochemistry and Molecular Morphology. Natick, MA, Eaton Publishing, 255–272
Shi Y, Brands FH, Chatterjee S, Feng AC, Groshen S, Schewe J, Lieskovsky G, et al. (2001) Her-2/neu expression in prostate cancer: high level of expression associated with exposure to hormone therapy and androgen independent disease. J Urol 166:1514–1519[CrossRef][Medline]
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