ARTICLE |
Correspondence to: Rainer Malisius, Dept. of Pathology, Medical University of Lübeck, Ratzeburger Allee 160, D 23538 Lübeck, Germany.
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Summary |
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Immunohistochemical methods are widely used for diagnostic purposes in histopathology. However, the use of most monoclonal anti-leukocyte antibodies is limited to frozen tissues. Initially, it was believed that formalin fixation in particular, which is the gold standard for morphological tissue preservation, destroys most of the antigen binding sites. In recent years, protease digestion and the introduction of microwave techniques have significantly enhanced the sensitivity of immunohistochemical techniques, and a variety of hidden antigen sites in formalin-fixed tissue have been retrieved for initially unreactive antibodies. It therefore became clear that many of the leukocyte antigens are not irreversibly destroyed but are most probably masked during the fixation process. We developed a technique combining optimized pretreatment of formalin-fixed tissue with a dramatic enhancement of the immunohistochemical sensitivity and named it the ImmunoMax method. The ImmunoMax method proves that by optimizing the technique at the following three levels it is possible to detect formalin-sensitive leukocyte antigens: (a) standard fixation of the tissue; (b) sufficient antigen unmasking; and (c) increasing the substrate turnover by multiplication of binding sites with subsequent enhancement of the immunohistochemical reaction. Using this optimized ImmunoMax method, we were able to detect CD2, CD3, CD4, and CD5 with conventional monoclonal antibodies in formalin-fixed, paraffin-embedded tissue specimens of various lymphoid tissues. (J Histochem Cytochem 45:1665-1672, 1997)
Key Words: immunohistochemistry, biotin-tyramide, ImmunoMax, paraffin, CD2, CD3, CD4, CD5
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Introduction |
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Since the introduction of monoclonal antibodies, immunohistochemistry has become an important tool in research and in diagnostic pathology (
The first steps were the use of enzyme digestion and the development of immunohistochemical techniques employing multiple secondary antibodies, both of which led to enhanced sensitivity (
Here we describe a combination of antigen retrieval techniques and a highly sensitive immunohistochemical method for detection of the T-cell-associated antigens CD2, CD3, CD4, and CD5 using monoclonal antibodies (MAbs) that were not originally suitable for use in formalin-fixed, paraffin-embedded tissues.
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Materials and Methods |
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Specimens of human tonsils with follicular hyperplasia (n = 10), lymph nodes (n = 10), and spleen (n = 5), and resection specimens from the ileum containing Peyer's patches (n = 5), were investigated. The tissues were routinely fixed in 4% buffered formaldehyde, pH 7.5, and subsequently embedded in paraffin. A portion of each biopsy specimen was snap-frozen and investigated in parallel.
Tissue Preparation
All tissues were fixed within 1 hr of excision. Variable fixation times were used (minimum 2 hr, maximum 72 hr). Subsequently, the specimens were dehydrated in a graded series of alcohols, cleared in xylene, and embedded in paraffin. Paraffin sections were placed on slides coated with 2% 3-(triethoxysilyl)propylamine (Merck; Darmstadt, Germany). The sections were dewaxed in xylene in a series of three steps (10 min each) and rehydrated in a series of 100%, 96%, and 70% alcohol.
Microwave Oven Treatment
A microwave oven, Miele supratronic M 752 (Miele; Stuttgart, Germany), operating at a frequency of 2.45 GHz with five power level settings, was used. Initially, the maximal power setting of 850 W was used. When the boiling point was reached, the power was reduced to 150 W for different periods of time (Table 1, operating time). Coplin jars were filled with 50 mM Tris-buffered saline (TBS), 10 mM citrate buffer, 4 M guanidine chloride (all from Sigma; Munich, Germany), and 0.1 M formic acid in 2% gelatin (Merck) (Table 2). The microwave operations with formic acid have to be carried out under a fume hood. The jars were covered with loosely fitting glass caps. Every 5 min the fluid level was checked and adjusted (TBS and citrate buffer). The treatment was stopped by intensely rinsing the slides several times in TBS. The number of jars, their position in the microwave oven, and the volume were kept constant because these factors may influence the temperature during microwave treatment.
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Enzyme Digestion
Alternatively, the tissue sections were pretreated by protease digestion. Slides were incubated with proteinase K (Boehringer; Mannheim, Germany) 25 µg dissolved in 1 ml TBS at 37C. Protease digestion was stopped after 5 min by rinsing the sections several times in TBS.
Additional immunohistochemical stainings were performed without any procedures for antigen unmasking.
Immunohistochemistry
Before incubation with the primary antibody, slides were pretreated with 0.05% H2O2 to block endogenous peroxidase. The primary antibodies (Table 3) were diluted in 10% normal rabbit serum. As detection system we used the ImmunoMax method recently described by
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After having optimized the pretreatment parameters, we alternatively performed conventional immunohistochemical staining [APAAP and ABC techniques according to
Negative Controls
Negative controls were performed, in which each of the reagents (antibodies, biotinylated tyramine) was omitted. In addition, specimens from kidney and liver as control tissues were used, which were expected to be negative for CD2, CD3, and CD5.
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Results |
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The results obtained using different microwave pretreatment parameters are depicted in Table 1, Table 2, Table 5, and Table 6. Table 5 also shows a comparison of staining results after microwave pretreatment, enzyme digestion, and without any pretreatment of the tissue. Without any pretreatment of the tissue, only for CD5 (Becton-Dickinson, Mountain View, CA; Novocastra, Newcastle, UK) was a weak signal obtained in a primary antibody dilution of 1:50 by use of the ImmunoMax method (Table 5). As Table 2 indicates, the optimal microwave operating time varies from reagent to reagent. Formic acid proved to be superior to citrate buffer and guanidine chloride. Using it for an operating time of 1 min resulted in high sensitivity combined with well-preserved morphology. Moreover, microwave treatment was also superior to enzyme digestion (in the form of pretreatment with 25 µg/ml proteinase K in TBS for 5 min at 37C). Different fixation times, ranging from 2 to 72 hr (2-24 hr for tissue sections with a maximal thickness of 0.5 cm), in PBS-buffered 4% formaldehyde, pH 7.5, did not have any influence on staining intensity (data not shown). Optimized fixation and microwave treatment with formic acid enabled CD5 to be detected with conventional APAAP and ABC techniques, using the primary antibody in a dilution of up to 1:50. These results were obtained equally for the Becton-Dickinson MAb (claimed not to be suitable for use in paraffin sections) and the Novocastra MAb (claimed to be suitable for use in paraffin sections). The combination of microwave pretreatment and the ImmunoMax method resulted in additional enhancement of sensitivity, which allows the primary antibody to be diluted up to 1:10,000 and more (1000-fold and more compared with conventional APAAP or ABC; Table 4). However, only with the ImmunoMax method was a constantly reliable and highly sensitive detection of CD5 possible in formalin-fixed, paraffin-embedded tissue. This was not possible with either the APAAP or the ABC technique. Paradoxically, only a low signal was recognized, or none at all, when the ImmunoMax method was used in combination with high antigen concentrations (e.g., when CD5 was used at a concentration higher than 1:100). This may be due to an effect like that demonstrated by
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All snap-frozen and paraffin-embedded tissues were tested in parallel. They showed identical reaction patterns with regard to the quantity and topography of cells stained by defined antigens. This was true for lymph nodes, tonsils, and Peyer's patches of the ileum. In formalin-fixed, paraffin-embedded tissues the cytology was well preserved, enabling a detailed study of the T-cells in the different compartments. In the T-zone and in interfollicular areas, the cells that stained positive for CD2, CD3, CD4, and CD5 consisted mostly of small lymphocytes (Figure 1). In exceptional cases a blast cell was positive. In the mantle zone and in the border between mantle zone and germinal center, the CD5 MAb stained two different populations of cells (Figure 2). Some cells were strongly stained and had the morphology of small lymphocytes or pleomorphic T-cells. Other cells with almost the same nuclear irregularity showed less intense staining and were interpreted as precursor cells of the mantle zone lymphocytes. Pleomorphic T-cells were detected particularly in germinal centers by CD2 and CD3 staining (Figure 3 and Figure 4). Macrophages in the germinal centers showed weak staining for CD4 (Figure 5).
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In the spleen, the staining pattern was also identical in both frozen and formalin-fixed tissues. Kidney and liver specimens as control tissues showed the expected negative reaction for all antibodies tested. No staining and enhancement was obtained in lymphatic tissue when one of the reagents (antibodies, ABC complex, or biotin-tyramide) was omitted.
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Discussion |
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Since the introduction of MAbs that can detect leukocyte differentiation antigens, we have gained a better understanding of the biology of lymphoid tissues (
In the past, various approaches have been tested for detecting T-cell antigens in formalin-fixed, paraffin-embedded tissues. One strategy was to synthesize new MAbs by using synthetic peptide sequences, as has been described for CD8 (
The fact that T-cell antigens are, in principle, available in formalin-fixed tissues stimulated us to develop procedures for unmasking such antigens, combined with highly sensitive staining techniques. Our staining technique, termed the ImmunoMax method, increases the sensitivity up to about 100-fold compared to conventional ABC and APAAP methods (
Monoclonal and polyclonal antibodies have been described that were prepared by immunization with synthetic peptide sequences and are available for detection of CD3 and CD8 in paraffin-embedded tissue (
Another strategy is to improve the immunohistochemical technique by starting with optimized fixation of the tissue, in the hope of being able to detect formalin-sensitive antigens in paraffin-embedded material. Many different fixatives, such as Bouin's solution, glutaraldehyde, lysine-phosphate-buffered formalin, acetic acid, or zinc-based solutions, have been tested (
The T-cell associated-antigens CD2, CD3, CD4, and CD5 were detected in paraffin-embedded specimens of tonsils, lymph nodes, spleen, and ileum with Peyer's patches. The staining patterns were identical to those seen in cryostat sections tested in parallel and to staining patterns described previously (
In the future, the use of CD4 and CD8 may help us to better describe T-cell lymphomas when immunohistochemistry can be combined with well-preserved morphology. Our approach will enable other leukocyte differentiation antigens to be detected in formalin-fixed material. This study demonstrates that it is possible to detect a number of T-cell antigens with highly sensitive but conventional immunohistochemical techniques, and it can be assumed that other B-cell or myelomonocytic antigens can also be detected by the use of equivalent techniques. They can then also be studied in combination with the morphology.
It must be stressed that formalin fixation is still a critical issue in immunohistochemistry. Our experiments also show that it is necessary to standardize formalin fixation by using PBS-buffered formaldehyde with defined fixation times. Such a standardization would also increase the availability of intact DNA and, to some extent, RNA for molecular genetic studies.
In summary, we have described a three-step system of standardized fixation, optimal antigen unmasking, and highly sensitive immunohistochemistry, which is a reliable and feasible technique that can open new perspectives in diagnostic and scientific pathology.
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Acknowledgments |
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Supported by Deutsche Forschungsgemeinschaft, grants Me 1037/4-1 and Me 1037/5-1.
Received for publication April 7, 1997; accepted July 3, 1997.
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