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The application of direct immunofluorescence to intraoperative neurosurgical diagnosis
Biomolecular Engineering 17 (2000) 17 – 22 www.elsevier.com/locate/geneanabioeng
The application of direct immunofluorescence to intraoperative neurosurgical diagnosis Satori Iwamoto a,b,*, Robert C. Burrows a, Donald E. Born c, Michael Piepkorn b,d, Mark Bothwell a a
Department of Physiology and Biophysics, Uni6ersity of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA b Department of Medicine/Dermatology, Uni6ersity of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA c Department of Pathology/Neuropathology, Uni6ersity of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA d Department of Pathology/Dermatopathology, Uni6ersity of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA Accepted 7 July 2000
Abstract A diagnostic problem can occur at the time of intraoperative consultation of neurosurgical tumors as to whether the tumor is of neuroectodermal origin or whether it represents an epithelial metastasis from another site. Intraoperative diagnoses based on hematoxylin and eosin stained frozen sections are often later confirmed by immunocytochemical analysis of formalin-fixed, paraffin-embedded tissue sections that are not available at the time of surgery. The objective of the current study was to demonstrate that the application of direct immunofluorescence to the intraoperative diagnosis of neurosurgical tumors would provide unequivocal, and nearly immediate results. This report describes a new application of an existing technique for an optimized, rapid procedure utilizing direct immunocytochemistry with fluorescence-labeled primary antibodies to analyze surgical biopsies intraoperatively. The examination of five neurosurgical biopsies established a neuroectodermal origin of three tumors via immunolabeling for glial fibrillary acidic protein (GFAP) and lack of labeling with keratin markers, whereas several metastatic lung carcinomas were identified by immunostaining for keratin, but not GFAP, markers. The results of the direct immunolabeling method were unequivocal and required only minutes. The same diagnoses were confirmed by standard immunocytochemical labeling of formalin-fixed, paraffin-embedded sections, though it required several days to obtain the results. Direct immunofluorescence using fluorescently conjugated primary antibodies is a practical and rapid method for deciding whether a neurosurgical tumor is a primary glial or an epithelial metastatic tumor in origin. It is the first reported application of the technique for this aspect of rapid neurosurgical diagnosis. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Direct immunofluorescence; Intraoperative diagnosis; Tumor biology
1. Introduction Rapid intraoperative histologic diagnosis of tumor biopsies is often desirable. While some neurosurgical cases involve stereotactic biopsy, where resection and treatment planning occur at a later time, there are cases in which resection is undertaken without a prior biopsy. In these particular cases, the neurosurgeon requires a highly accurate intraoperative histologic diagnosis of the tumor biopsy. The pathologist is usually asked to * Corresponding author. Present address: Box 356524, University of Washington Medical Center, Seattle, WA 98195-6524, USA. Tel.: +1-206-5982112; fax: +1-206-5432489. E-mail address: [email protected] (S. Iwamoto).
decide between a high-grade glial tumor and a metastasis from a carcinoma. These diagnoses are currently obtained by standard histological analyses with hematoxylin and eosin (H&E) staining of smear preparations or frozen sections. The intraoperative diagnosis is confirmed several days later by immunocytochemical evaluation of formalin-fixed paraffin embedded sections. Typically, it is relatively straightforward to distinguish a high grade glioma from an epithelial metastasis on permanent sections, however, this difference may not be as clear on frozen sections if the infiltrative border is not included in the intraoperative sample obtained for diagnosis. More importantly, the accuracy of the diagnosis is highly dependent upon the variable quality of
1389-0344/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 9 - 0 3 4 4 ( 0 0 ) 0 0 0 6 0 - 5
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S. Iwamoto et al. / Biomolecular Engineering 17 (2000) 17–22
the frozen sections. An immunocytochemical evaluation at the time of intraoperative diagnosis would be very helpful in the setting of a difficult frozen section. The recent increase of commercially available directly conjugated fluorescent antibodies prompted us to try these antibodies in optimizing an improved method for the diagnosis of intraoperative biopsies. Direct immunofluorescence differs from routine immunocytochemistry [1] in that a fluorophore tag is directly conjugated to the primary antibody thus eliminating the usual subsequent steps to amplify and visualize the bound antibody (Fig. 1). The direct method eliminates these subsequent steps and is thus faster and simpler to use. In this study, we have examined frozen sections from neurosurgical biopsies obtained at the time of surgery, and analyzed them using fluorescently conjugated primary antibodies against either GFAP or keratin. Our results demonstrate the feasibility of resolving diagnostic uncertainty within minutes using the directly conjugated primary antibodies with results identical to those obtained several days later with standard immunocytochemical preparations on formalin-fixed permanent sections. As far as we know, this is the first report of this technique to be used to expedite this aspect of neurosurgical diagnosis.
2. Materials and methods
2.1. Surgical materials Intraoperative neurosurgical specimens were obtained from the University of Washington Medical Center, Seattle, WA. Two specimens of astrocytomas, one specimen of a primitive neuroectodermal tumor, and two specimens of primary epithelial metastasis were obtained. A sample from each tissue specimen was placed in embedding medium and frozen in the
cryostat. An adjacent portion was processed for paraffin embedded immunocytochemistry (ICC). Fresh frozen 6-mm cryostat sections were collected and mounted on precoated slides and stored at − 80°C for up to 10 days prior to processing for ICC and for H&E staining. In addition, 6 mm formalin-fixed, paraffin-embedded sections from the same intraoperative biopsies were processed for ICC and H&E.
2.2. Rapid immunofluorescence Cy3 conjugated anti-GFAP (Sigma, St. Louis, MO) was used at 1:250 dilution. Anti-cytokeratin antibodies conjugated to fluorescein isothiocyanate (FITC) used in this study included C-11 and PCK-26 (Sigma) and MNF-116 (DAKO, Carpinteria, CA) used at 1:50 dilution. Incubations with anti-GFAP and anti-cytokeratin antibodies were for 15 min at 37°C, however, incubation times as short as 5 min are adequate to detect the presence of highly abundant antigens including keratin and GFAP. Sections of the fresh frozen surgical specimens mounted on precoated Superfrost slides (Fisher Scientific, Springfield, NJ, USA) were immersed in acetone for five minutes at room temperature and air-dried. Slides were then rehydrated and blocked for nonspecific staining with 5% fetal calf serum in phosphate buffered saline (PBS) for five minutes at 37°C. We have subsequently found that this blocking step is optional. The blocking solution was replaced with fluorophore-conjugated primary antibody (anti-cytokeratin 1:50) or antiGFAP 1:250, or both, diluted in blocking solution and incubated for 15 min at 37°C. Slides were then rinsed in PBS and cover slipped with 80% glycerol containing 5% propyl gallate as an antifade agent. Sections were then examined with the aid of a Nikon Microphot SA microscope with fluorescence and a charged coupled device (CCD) camera interfaced with a Macintosh PPC
Fig. 1. The upper panel depicts the single step direct immunolabeling method, illustrating the single incubation step with a fluorescently conjugated primary antibody. The lower panel shows, for comparison, the standard immunocytochemical labeling method. Several steps, including incubation with the primary antibody, incubation with the secondary antibody, and an enzymatic visualization step, are required.
S. Iwamoto et al. / Biomolecular Engineering 17 (2000) 17–22
9600. Digital images were captured using the IpLab Spectrum software version 3.12 (Scanalytics Inc., Fairfax, VA). After normalizing the pixel scale of collected images, the files were saved in PICT format and assembled into plates using Adobe Photoshop (Adobe Systems, San Jose, CA).
2.3. Standard immunocytochemistry Standard ICC methods using streptavidin-biotin techniques were used for all paraffin embedded neurosurgical specimens. Following formalin fixation and paraffin embedding, 4 mm paraffin sections were collected on slides, deparaffinized and treated with 1% hydrogen peroxide in PBS to quench the endogenous peroxidase activity. Following several PBS rinses, the tissues were incubated for 1 h at room temperature in either anti-cytokeratin antibody 35BH11 (1:500) or rabbit anti-GFAP (1:7000) both from Dako. Following PBS rinses, biotinylated secondary antibodies antimouse (1:100) for the cytokeratin or anti-rabbit (1:250) for the GFAP, diluted in PBS were applied to the sections for 30 min. Following extensive washing with PBS, avidin-peroxidase (Vectastain Elite from Vector Laboratories Inc., Burlingame, CA) and peroxide with diaminobenzedine chromagen, with or without nickel intensification, was used in the visualization steps. A light hematoxylin or methyl green counterstain was then applied to the sections prior to dehydrating and coverslipping.
2.4. Paraffin section microscopy Sections were examined on a Nikon Microphot SA microscope equipped with brightfield and epifluorescent capabilities. Digital images were captured from the microscope via a CCD camera interfaced with a Macintosh 9600 power PC computer and IPLab software. Digital images were normalized to a pixel scale of 0 – 4095, pseudo-colored and saved as PICT files.
3. Results Direct immunofluorescence was applied to intraoperative frozen sections from neurosurgical procedures. The tumor phenotype obtained in minutes using the direct method, was found to be identical to standard immunocytochemical labeling of formalin-fixed paraffin sections, which required several days processing time. Two cases of astrocytoma, one case of GFAP-positive primitive neuroectodermal tumor, and two cases of lung carcinoma metastatic to brain were analyzed for their immunophenotype using our rapid, one-step direct method. For each of these cases, four adjacent sections were processed as follows: one section was stained with
19
H&E, one section was stained with Cy3 conjugated anti-GFAP, one section was stained with FITC conjugated anti-cytokeratin, and one slide was incubated only with buffer. The cases of astrocytomas (Fig. 2, A–F) and the GFAP positive primitive neuroectodermal tumor (Fig. 2, G–I) all showed intense GFAP immunoreactivity (Fig. 2, B,E,H), but no significant keratin immunoreactivity (Fig. 2, A,D,G). In the two cases of metastatic carcinoma (Fig. 2, J,M) there was clear immunolabeling with the anti-cytokeratin antibody, but virtually no labeling with the anti-GFAP (Fig. 2, K,N). All results from the direct immunolabeled sections were confirmatory and appropriate in diagnosis as well as morphology when compared to the routinely processed formalin-fixed, paraffin-embedded sections (data not shown). In addition, none of the control sections incubated with buffer alone showed any degree of autofluorescence (data not shown).
4. Discussion We report here the application of a rapid, single-step direct immunofluorescence procedure for the diagnosis of tumors during neurosurgical procedures. The method requires only a 5–10 min incubation, and the resulting immunophenotype is the same as that using the traditional immunocytochemical methods that require multiple prolonged incubation periods. The main advantage of this method is not only its rapidity, but also its simplicity. It requires only a single short incubation, compared to the standard immunocytochemical methods, as shown in Fig. 1. The simplicity may reduce the chances of error. Intraoperative pathology consultations influence patient care decisions [2]. We have applied the method to neurosurgical specimens in situations where the pathologist is asked to determine intraoperatively whether a tumor is a primary glial tumor or a metastatic epithelial tumor. There are two sources of ambiguity during such an intraoperative diagnosis. First, ambiguity may arise from a specimen that is histopathologically difficult to interpret. Second, and more commonly, ambiguity may arise from the variability in the quality of frozen sections. Immunolabeling with two different antibodies, however, can be definitive because of the unique pattern of intermediate filament expression: glial tumors express GFAP [3,4] while metastatic epithelial tumors express cytokeratin intermediate filaments [5,6]. Furthermore, immunolabeling is interpreted as the presence or absence of labeling, and thus relies less on the quality of frozen section than does H&E interpretation. Currently, when intraoperative diagnoses made by cellular and histologic criteria based on H&E stained frozen sections are ambiguous, the final interpretation is deferred to permanent sections and routine immuno-
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S. Iwamoto et al. / Biomolecular Engineering 17 (2000) 17–22
Fig. 2.
S. Iwamoto et al. / Biomolecular Engineering 17 (2000) 17–22
cytochemical assays. The confirmation of diagnosis is then delayed by at least one day, and usually longer for immunocytochemical results. In each of the five neurosurgical specimens, we were able to obtain immunophenotype results well within 30 min that matched subsequent standard immunocytochemical studies. Furthermore, as shown in Fig. 2, the results were unequivocal. There are rare cases in which high-grade gliomas may not express GFAP at high levels. It is for that reason that multiple antibodies are used. Expression of keratin suggests an epithelial metastasis. In rare cases, negative GFAP and negative keratin expression could be inconclusive. That is true for permanent sections and for standard immunocytochemistry, but they are still performed. The technique of direct immunolabeling is not new. Direct immunofluorescence methods have been previously employed in the clinical setting mainly in autoimmune diseases, renal diseases and cutaneous bullous disorders, where deposits of immunoglobulins and complement are to be detected [7,8]. In those cases, accumulations of autoreactive immunoglobulins prevent the use of a secondary amplifying antibody. One potential disadvantage of this method is that a fluorescence microscope is required and therefore requires an initial investment. However, many medical and academic institutions may already have ready access to a fluorescent microscope. If so, this technique allows a quick confirmation of a diagnosis in much the same way that a Tzanck preparation or potassium hydroxide preparation in primary care medicine provides much faster results than corresponding culture results. The direct immunofluorescence method was the original method of immunolabeling, associated with the use of darkened rooms and the inability to maintain a permanent record of the results. The availability of new higher intensity fluorochromes such as the cyanine dyes, as well as advances in fluorescence microscopes have obviated the original need for darkened rooms. Digital photography now allows immediate and permanent documentation of the histologic results. Although a one step procedure described in this paper may not provide an advantage in a controlled research setting, our experience is that it is considerably more error-free than the standard two step immunofluorescence when the proce-
21
dure is performed in an actual busy clinical service. We have recently shortened the protocol discussed in this paper and we can obtain results within 10 min, though there can be considerable dependence on tissue section quality. We have also investigated the use of a rapid single-step non-fluorescent method of direct immunolabeling using the EPOS (DAKO, Carpinteria, CA) reagent [9]. Our initial results are that the EPOS reagent is also able to provide rapid results with the slides viewable by a standard light microscope. However, the EPOS method does require two steps, an antibody incubation step and a development step with the chromophore, and the signal is not as intense in our hands, given the same duration of incubation. Furthermore, double labeling with two different antibodies such as shown in Fig. 1p and Fig. 1q is not as simple to perform with the EPOS method, and additional tissue sections may be needed.
5. Conclusion We have demonstrated the application of a rapid, single-step direct immunofluorescence labeling method for the intraoperative diagnosis of neurosurgical tumors. In the present communication, we have preliminary results to suggest a potential use in intraoperatively deciding whether a brain tumor is either a primary glial tumor or a metastatic carcinoma from a different site. The advantages of this method are that it is simple, accurate and rapid.
Acknowledgements We would like to thank Holly Predd and Robert Underwood for technical assistance in photography; and Lorraine Gibbs, Erik Gunther, and Rodney Schmidt MD PhD for helpful discussions with the manuscript. This work was supported by American Cancer Society Grant 91-020-06-IRG, Dermatology Foundation’s American Society of Dermatologic Surgery Grant and Dermatologist-Investigator Award (S.I.); NIH NS01910 (D.E.B.); NIH RO1 DCO2863 and RO1 NS 33200 (M.B.).
Fig. 2. Photomicrographs of neurosurgical specimens. Rapid immunofluorescent analysis of two astrocytomas (A – F), a GFAP-expressing primitive neuroectodermal tumor (G–I), and two metastatic lung small cell carcinomas (J – O) probed with antibodies against cytokeratin (A,D,G,J,M) or GFAP (B,E,H,K,N). Note the absence of keratin immunostaining, (A,D,G), along with the abundance of GFAP immunoreactivity (B,E,H) in the primary neuronal tumors. In contrast, note the high levels of anti-cytokeratin immunoreactivity (J,M) and the paucity of GFAP immunoreactivity (K,N) in the small cell lung metastasis tumors. H&E from frozen sections are shown for each case (C,F,I,L,O). Panels P and Q represent sections of astrocytoma and small cell lung metastasis (cases Nos 1 and 5) respectively. Sections were immunolabeled with both anti-keratin and anti-GFAP antibodies, conjugated to FITC and CY-3 respectively, applied as a cocktail, then counterstained with DAPI. Note the absence of keratin immunoreactivity in the astrocytoma (P) and the abundance of keratin immunoreactivity, along with a few GFAP labeled cells near the tumor border in the small cell lung metastasis (Q).
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References [1] Schetters H. Genet Anal Biomol Eng 1999;16(1–4):73–8. [2] Zarbo RJ, Schmidt WA, Bachner P, Howanitz PJ, Meier FA, Schifman RB, et al. Arch Pathol Lab Med 1996;120(1):19– 25. [3] McComb RD, Burger PC. Neurol Clin 1985;3(4):711–28. [4] Yung WK, Luna M, Borit A. J Neurooncol 1985;3(1):35– 8.
.
[5] Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. Cell 1982;31(1):11 – 24. [6] Lane EB, Alexander CM. Semin Cancer Biol 1990;1(3):165– 79. [7] Valenzuela R, Deodhar SD. Pathobiol Ann 1980;10:183–221. [8] Maize JC, Provost TT. Am J Dermatopathol 1983;5(1):67– 72. [9] Chilosi M, Lestani M, Pedron S, Montagna L, Benedetti A, Pizzolo G, et al. Biotech Histochem 1994;69(4):235– 9.
The application of direct immunofluorescence to intraoperative neurosurgical diagnosis Satori Iwamoto a,b,*, Robert C. Burrows a, Donald E. Born c, Michael Piepkorn b,d, Mark Bothwell a a
Department of Physiology and Biophysics, Uni6ersity of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA b Department of Medicine/Dermatology, Uni6ersity of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA c Department of Pathology/Neuropathology, Uni6ersity of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA d Department of Pathology/Dermatopathology, Uni6ersity of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA Accepted 7 July 2000
Abstract A diagnostic problem can occur at the time of intraoperative consultation of neurosurgical tumors as to whether the tumor is of neuroectodermal origin or whether it represents an epithelial metastasis from another site. Intraoperative diagnoses based on hematoxylin and eosin stained frozen sections are often later confirmed by immunocytochemical analysis of formalin-fixed, paraffin-embedded tissue sections that are not available at the time of surgery. The objective of the current study was to demonstrate that the application of direct immunofluorescence to the intraoperative diagnosis of neurosurgical tumors would provide unequivocal, and nearly immediate results. This report describes a new application of an existing technique for an optimized, rapid procedure utilizing direct immunocytochemistry with fluorescence-labeled primary antibodies to analyze surgical biopsies intraoperatively. The examination of five neurosurgical biopsies established a neuroectodermal origin of three tumors via immunolabeling for glial fibrillary acidic protein (GFAP) and lack of labeling with keratin markers, whereas several metastatic lung carcinomas were identified by immunostaining for keratin, but not GFAP, markers. The results of the direct immunolabeling method were unequivocal and required only minutes. The same diagnoses were confirmed by standard immunocytochemical labeling of formalin-fixed, paraffin-embedded sections, though it required several days to obtain the results. Direct immunofluorescence using fluorescently conjugated primary antibodies is a practical and rapid method for deciding whether a neurosurgical tumor is a primary glial or an epithelial metastatic tumor in origin. It is the first reported application of the technique for this aspect of rapid neurosurgical diagnosis. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Direct immunofluorescence; Intraoperative diagnosis; Tumor biology
1. Introduction Rapid intraoperative histologic diagnosis of tumor biopsies is often desirable. While some neurosurgical cases involve stereotactic biopsy, where resection and treatment planning occur at a later time, there are cases in which resection is undertaken without a prior biopsy. In these particular cases, the neurosurgeon requires a highly accurate intraoperative histologic diagnosis of the tumor biopsy. The pathologist is usually asked to * Corresponding author. Present address: Box 356524, University of Washington Medical Center, Seattle, WA 98195-6524, USA. Tel.: +1-206-5982112; fax: +1-206-5432489. E-mail address: [email protected] (S. Iwamoto).
decide between a high-grade glial tumor and a metastasis from a carcinoma. These diagnoses are currently obtained by standard histological analyses with hematoxylin and eosin (H&E) staining of smear preparations or frozen sections. The intraoperative diagnosis is confirmed several days later by immunocytochemical evaluation of formalin-fixed paraffin embedded sections. Typically, it is relatively straightforward to distinguish a high grade glioma from an epithelial metastasis on permanent sections, however, this difference may not be as clear on frozen sections if the infiltrative border is not included in the intraoperative sample obtained for diagnosis. More importantly, the accuracy of the diagnosis is highly dependent upon the variable quality of
1389-0344/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 9 - 0 3 4 4 ( 0 0 ) 0 0 0 6 0 - 5
18
S. Iwamoto et al. / Biomolecular Engineering 17 (2000) 17–22
the frozen sections. An immunocytochemical evaluation at the time of intraoperative diagnosis would be very helpful in the setting of a difficult frozen section. The recent increase of commercially available directly conjugated fluorescent antibodies prompted us to try these antibodies in optimizing an improved method for the diagnosis of intraoperative biopsies. Direct immunofluorescence differs from routine immunocytochemistry [1] in that a fluorophore tag is directly conjugated to the primary antibody thus eliminating the usual subsequent steps to amplify and visualize the bound antibody (Fig. 1). The direct method eliminates these subsequent steps and is thus faster and simpler to use. In this study, we have examined frozen sections from neurosurgical biopsies obtained at the time of surgery, and analyzed them using fluorescently conjugated primary antibodies against either GFAP or keratin. Our results demonstrate the feasibility of resolving diagnostic uncertainty within minutes using the directly conjugated primary antibodies with results identical to those obtained several days later with standard immunocytochemical preparations on formalin-fixed permanent sections. As far as we know, this is the first report of this technique to be used to expedite this aspect of neurosurgical diagnosis.
2. Materials and methods
2.1. Surgical materials Intraoperative neurosurgical specimens were obtained from the University of Washington Medical Center, Seattle, WA. Two specimens of astrocytomas, one specimen of a primitive neuroectodermal tumor, and two specimens of primary epithelial metastasis were obtained. A sample from each tissue specimen was placed in embedding medium and frozen in the
cryostat. An adjacent portion was processed for paraffin embedded immunocytochemistry (ICC). Fresh frozen 6-mm cryostat sections were collected and mounted on precoated slides and stored at − 80°C for up to 10 days prior to processing for ICC and for H&E staining. In addition, 6 mm formalin-fixed, paraffin-embedded sections from the same intraoperative biopsies were processed for ICC and H&E.
2.2. Rapid immunofluorescence Cy3 conjugated anti-GFAP (Sigma, St. Louis, MO) was used at 1:250 dilution. Anti-cytokeratin antibodies conjugated to fluorescein isothiocyanate (FITC) used in this study included C-11 and PCK-26 (Sigma) and MNF-116 (DAKO, Carpinteria, CA) used at 1:50 dilution. Incubations with anti-GFAP and anti-cytokeratin antibodies were for 15 min at 37°C, however, incubation times as short as 5 min are adequate to detect the presence of highly abundant antigens including keratin and GFAP. Sections of the fresh frozen surgical specimens mounted on precoated Superfrost slides (Fisher Scientific, Springfield, NJ, USA) were immersed in acetone for five minutes at room temperature and air-dried. Slides were then rehydrated and blocked for nonspecific staining with 5% fetal calf serum in phosphate buffered saline (PBS) for five minutes at 37°C. We have subsequently found that this blocking step is optional. The blocking solution was replaced with fluorophore-conjugated primary antibody (anti-cytokeratin 1:50) or antiGFAP 1:250, or both, diluted in blocking solution and incubated for 15 min at 37°C. Slides were then rinsed in PBS and cover slipped with 80% glycerol containing 5% propyl gallate as an antifade agent. Sections were then examined with the aid of a Nikon Microphot SA microscope with fluorescence and a charged coupled device (CCD) camera interfaced with a Macintosh PPC
Fig. 1. The upper panel depicts the single step direct immunolabeling method, illustrating the single incubation step with a fluorescently conjugated primary antibody. The lower panel shows, for comparison, the standard immunocytochemical labeling method. Several steps, including incubation with the primary antibody, incubation with the secondary antibody, and an enzymatic visualization step, are required.
S. Iwamoto et al. / Biomolecular Engineering 17 (2000) 17–22
9600. Digital images were captured using the IpLab Spectrum software version 3.12 (Scanalytics Inc., Fairfax, VA). After normalizing the pixel scale of collected images, the files were saved in PICT format and assembled into plates using Adobe Photoshop (Adobe Systems, San Jose, CA).
2.3. Standard immunocytochemistry Standard ICC methods using streptavidin-biotin techniques were used for all paraffin embedded neurosurgical specimens. Following formalin fixation and paraffin embedding, 4 mm paraffin sections were collected on slides, deparaffinized and treated with 1% hydrogen peroxide in PBS to quench the endogenous peroxidase activity. Following several PBS rinses, the tissues were incubated for 1 h at room temperature in either anti-cytokeratin antibody 35BH11 (1:500) or rabbit anti-GFAP (1:7000) both from Dako. Following PBS rinses, biotinylated secondary antibodies antimouse (1:100) for the cytokeratin or anti-rabbit (1:250) for the GFAP, diluted in PBS were applied to the sections for 30 min. Following extensive washing with PBS, avidin-peroxidase (Vectastain Elite from Vector Laboratories Inc., Burlingame, CA) and peroxide with diaminobenzedine chromagen, with or without nickel intensification, was used in the visualization steps. A light hematoxylin or methyl green counterstain was then applied to the sections prior to dehydrating and coverslipping.
2.4. Paraffin section microscopy Sections were examined on a Nikon Microphot SA microscope equipped with brightfield and epifluorescent capabilities. Digital images were captured from the microscope via a CCD camera interfaced with a Macintosh 9600 power PC computer and IPLab software. Digital images were normalized to a pixel scale of 0 – 4095, pseudo-colored and saved as PICT files.
3. Results Direct immunofluorescence was applied to intraoperative frozen sections from neurosurgical procedures. The tumor phenotype obtained in minutes using the direct method, was found to be identical to standard immunocytochemical labeling of formalin-fixed paraffin sections, which required several days processing time. Two cases of astrocytoma, one case of GFAP-positive primitive neuroectodermal tumor, and two cases of lung carcinoma metastatic to brain were analyzed for their immunophenotype using our rapid, one-step direct method. For each of these cases, four adjacent sections were processed as follows: one section was stained with
19
H&E, one section was stained with Cy3 conjugated anti-GFAP, one section was stained with FITC conjugated anti-cytokeratin, and one slide was incubated only with buffer. The cases of astrocytomas (Fig. 2, A–F) and the GFAP positive primitive neuroectodermal tumor (Fig. 2, G–I) all showed intense GFAP immunoreactivity (Fig. 2, B,E,H), but no significant keratin immunoreactivity (Fig. 2, A,D,G). In the two cases of metastatic carcinoma (Fig. 2, J,M) there was clear immunolabeling with the anti-cytokeratin antibody, but virtually no labeling with the anti-GFAP (Fig. 2, K,N). All results from the direct immunolabeled sections were confirmatory and appropriate in diagnosis as well as morphology when compared to the routinely processed formalin-fixed, paraffin-embedded sections (data not shown). In addition, none of the control sections incubated with buffer alone showed any degree of autofluorescence (data not shown).
4. Discussion We report here the application of a rapid, single-step direct immunofluorescence procedure for the diagnosis of tumors during neurosurgical procedures. The method requires only a 5–10 min incubation, and the resulting immunophenotype is the same as that using the traditional immunocytochemical methods that require multiple prolonged incubation periods. The main advantage of this method is not only its rapidity, but also its simplicity. It requires only a single short incubation, compared to the standard immunocytochemical methods, as shown in Fig. 1. The simplicity may reduce the chances of error. Intraoperative pathology consultations influence patient care decisions [2]. We have applied the method to neurosurgical specimens in situations where the pathologist is asked to determine intraoperatively whether a tumor is a primary glial tumor or a metastatic epithelial tumor. There are two sources of ambiguity during such an intraoperative diagnosis. First, ambiguity may arise from a specimen that is histopathologically difficult to interpret. Second, and more commonly, ambiguity may arise from the variability in the quality of frozen sections. Immunolabeling with two different antibodies, however, can be definitive because of the unique pattern of intermediate filament expression: glial tumors express GFAP [3,4] while metastatic epithelial tumors express cytokeratin intermediate filaments [5,6]. Furthermore, immunolabeling is interpreted as the presence or absence of labeling, and thus relies less on the quality of frozen section than does H&E interpretation. Currently, when intraoperative diagnoses made by cellular and histologic criteria based on H&E stained frozen sections are ambiguous, the final interpretation is deferred to permanent sections and routine immuno-
20
S. Iwamoto et al. / Biomolecular Engineering 17 (2000) 17–22
Fig. 2.
S. Iwamoto et al. / Biomolecular Engineering 17 (2000) 17–22
cytochemical assays. The confirmation of diagnosis is then delayed by at least one day, and usually longer for immunocytochemical results. In each of the five neurosurgical specimens, we were able to obtain immunophenotype results well within 30 min that matched subsequent standard immunocytochemical studies. Furthermore, as shown in Fig. 2, the results were unequivocal. There are rare cases in which high-grade gliomas may not express GFAP at high levels. It is for that reason that multiple antibodies are used. Expression of keratin suggests an epithelial metastasis. In rare cases, negative GFAP and negative keratin expression could be inconclusive. That is true for permanent sections and for standard immunocytochemistry, but they are still performed. The technique of direct immunolabeling is not new. Direct immunofluorescence methods have been previously employed in the clinical setting mainly in autoimmune diseases, renal diseases and cutaneous bullous disorders, where deposits of immunoglobulins and complement are to be detected [7,8]. In those cases, accumulations of autoreactive immunoglobulins prevent the use of a secondary amplifying antibody. One potential disadvantage of this method is that a fluorescence microscope is required and therefore requires an initial investment. However, many medical and academic institutions may already have ready access to a fluorescent microscope. If so, this technique allows a quick confirmation of a diagnosis in much the same way that a Tzanck preparation or potassium hydroxide preparation in primary care medicine provides much faster results than corresponding culture results. The direct immunofluorescence method was the original method of immunolabeling, associated with the use of darkened rooms and the inability to maintain a permanent record of the results. The availability of new higher intensity fluorochromes such as the cyanine dyes, as well as advances in fluorescence microscopes have obviated the original need for darkened rooms. Digital photography now allows immediate and permanent documentation of the histologic results. Although a one step procedure described in this paper may not provide an advantage in a controlled research setting, our experience is that it is considerably more error-free than the standard two step immunofluorescence when the proce-
21
dure is performed in an actual busy clinical service. We have recently shortened the protocol discussed in this paper and we can obtain results within 10 min, though there can be considerable dependence on tissue section quality. We have also investigated the use of a rapid single-step non-fluorescent method of direct immunolabeling using the EPOS (DAKO, Carpinteria, CA) reagent [9]. Our initial results are that the EPOS reagent is also able to provide rapid results with the slides viewable by a standard light microscope. However, the EPOS method does require two steps, an antibody incubation step and a development step with the chromophore, and the signal is not as intense in our hands, given the same duration of incubation. Furthermore, double labeling with two different antibodies such as shown in Fig. 1p and Fig. 1q is not as simple to perform with the EPOS method, and additional tissue sections may be needed.
5. Conclusion We have demonstrated the application of a rapid, single-step direct immunofluorescence labeling method for the intraoperative diagnosis of neurosurgical tumors. In the present communication, we have preliminary results to suggest a potential use in intraoperatively deciding whether a brain tumor is either a primary glial tumor or a metastatic carcinoma from a different site. The advantages of this method are that it is simple, accurate and rapid.
Acknowledgements We would like to thank Holly Predd and Robert Underwood for technical assistance in photography; and Lorraine Gibbs, Erik Gunther, and Rodney Schmidt MD PhD for helpful discussions with the manuscript. This work was supported by American Cancer Society Grant 91-020-06-IRG, Dermatology Foundation’s American Society of Dermatologic Surgery Grant and Dermatologist-Investigator Award (S.I.); NIH NS01910 (D.E.B.); NIH RO1 DCO2863 and RO1 NS 33200 (M.B.).
Fig. 2. Photomicrographs of neurosurgical specimens. Rapid immunofluorescent analysis of two astrocytomas (A – F), a GFAP-expressing primitive neuroectodermal tumor (G–I), and two metastatic lung small cell carcinomas (J – O) probed with antibodies against cytokeratin (A,D,G,J,M) or GFAP (B,E,H,K,N). Note the absence of keratin immunostaining, (A,D,G), along with the abundance of GFAP immunoreactivity (B,E,H) in the primary neuronal tumors. In contrast, note the high levels of anti-cytokeratin immunoreactivity (J,M) and the paucity of GFAP immunoreactivity (K,N) in the small cell lung metastasis tumors. H&E from frozen sections are shown for each case (C,F,I,L,O). Panels P and Q represent sections of astrocytoma and small cell lung metastasis (cases Nos 1 and 5) respectively. Sections were immunolabeled with both anti-keratin and anti-GFAP antibodies, conjugated to FITC and CY-3 respectively, applied as a cocktail, then counterstained with DAPI. Note the absence of keratin immunoreactivity in the astrocytoma (P) and the abundance of keratin immunoreactivity, along with a few GFAP labeled cells near the tumor border in the small cell lung metastasis (Q).
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