Raman Spectroscopic Evaluation of Efficacy of Current Paraffin Wax Section Dewaxing Agents
School of Physics (EOF), Radiation and Environmental Science Centre (FML), School of Biological Sciences (HAL), Facility for Optical Characterisation and Spectroscopy (HJB), Dublin Institute of Technology, Dublin, Ireland and Department of Histology (MBH,JMB), Department of Pathology and Laboratory Medicine (PK), National Maternity Hospital, Dublin, Ireland
Correspondence to: Eoghan Ó Faoláin, School of Physics, Dublin Institute of Technology, Dublin, Ireland. E-mail: eoghanofaolain{at}eircom.net
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Summary |
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(J Histochem Cytochem 53:121129, 2005)
Key Words: dewaxing paraffin sections xylene Histoclear hexane HMAR Trilogy immunohistochemistry Raman cancer
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Introduction |
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Formalin-fixed paraffin-processed (FFPP) tissues are used extensively for immunohistochemical detection of normal and tumor cell markers. A major step forward was made in the 1990s with the discovery that some antigens previously unreactive in FFPP tissue, even after protease treatment, could be "retrieved" by heating sections in a microwave oven. Originally, this was carried out in a solution of rather toxic heavy metal salts (Shi et al. 1991). Subsequently, these salts were replaced with buffers such as citrate buffer at (pH 6.0) (Cattoretti et al. 1993
). It was shown that heat, rather than microwaves per se, is important in the retrieval process. Boiling the section in a pressure cooker (Norton et al. 1994
) or autoclaving (Bankfalvi et al. 1994
) in the buffer solution achieved the same effect (Polak and Van Noorden 1997
). A more recent dewaxing protocol involves the application of a reagent for simultaneous dewaxing and antigen unmasking, namely, Declere and Trilogy (www.cellmarque.com). These protocols are used extensively for immunohistochemistry and in situ hybridization techniques.
Raman microspectroscopy is an analytical, non-destructive technique that provides information about the molecular structure of the investigated sample (Long 2002). The Raman effect arises when the incident light excites molecules in the sample, which subsequently scatter the light. While most of this scattered light is at the same wavelength as the incident light (
1), some is scattered at a different wavelength (
2). This inelastically scattered light (
2) is called Raman scatter and results from the interaction of the incident light with the molecular motions or vibrations (Long 2002
). The positions, intensities, and linewidths of the Raman lines, corresponding to vibrational energy levels, yield information on the composition, secondary structure, and interaction of molecules, including the chemical microenvironment of molecular subgroups. Many molecules are Raman active with fingerprint spectra, providing molecular-specific information that can be used as a marker of cellular damage (Conroy et al. 2003
; Ó Faoláin et al. 2003
). The molecular structure of nucleic acids, proteins, and lipids differ between normal and tumor tissues and, therefore, Raman spectroscopy has been considered promising for the molecular characterization of cancer cells (Liu et al. 1992
).
Raman spectroscopy has been employed in the examination of a variety of common cancers, including cervical precancers (Mahadevan-Jansen et al. 1998), gastrointestinal lesions (Dacosta et al. 2002
), benign and malignant skin lesions (Gniadecka et al. 1997
), breast cancer (Frank et al. 1995
; (Shafer-Peltier et al. 2002
), colon polyps (Molckovsky et al. 2003
), prostate cancer, and bladder cancer (Crow et al. 2003a
,b
).
To examine the biochemical structure of the FFPP tissue using Raman spectroscopy, the sample must be as close to its original/in vivo state as possible. This requires the removal of the paraffin wax and rehydration to the aqueous phase. In this present study multiple methods of dewaxing were examined and the efficacy of each assessed using Raman spectroscopy.
This study compared the effectiveness of the most commonly used dewaxing agents, namely, xylene, and Histoclear, as well as heat-mediated antigen retrieval (HMAR) using xylene followed by a citrate buffer and HMAR using Trilogy alone. The effectiveness of hexane as a dewaxing agent was also examined, due to its industrial use as a cleaning and degreasing solvent.
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Materials and Methods |
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Sample Preparation
All cervical FFPP sections were obtained from the National Maternity Hospital, Holles St., Dublin. All specimens were automatically processed to wax as follows: (a) vacuum fixed in 10% buffered formal saline histo-grade, pH 6.87.2 (J.T. Baker; Deventer, The Netherlands) heated to 35C, (b) vacuum dehydration in industrial methylated spirit IMS T100 (Lennox; Dublin, Ireland) heated to 35C, (c) vacuum clearing in xylene (Serosep; Limerick, Ireland) heated to 35C, and (d) vacuum impregnation with Tissue Tek III Embedding Wax with polymer added (Sakura, Zoeterwoude, The Netherlands) and heated to 59C.
Three parallel FFPP cervical sections were dewaxed using each of the five protocols outlined below.
Xylene
After the wax impregnation, tissue was embedded and sliced into 5-µm sections using a microtome, mounted on glass slides, and dried. The unstained samples were immersed in a series of baths consisting of two baths of xylene (BDH; Dorset, UK) for 5 min and 4 min, respectively, two baths of Ethanol Absolut (Merck; Dorset, UK) for 3 min and 2 min, respectively, and a final bath of Industrial Methylated Spirits 95% (Lennox) for 1 min.
Histoclear
The same procedure was used for dewaxing using General Purpose Grade Histoclear (Fisher Scientific; Loughborough, UK), where xylene was substituted with Histoclear in protocol A.
Hexane
Again, the same procedure was used for dewaxing using Hexane (BDH), whereas xylene was substituted with hexane in protocol A.
Each of the sections examined was put through the dewaxing procedure outlined four successive times. Finally, each of the specimens was left sitting in a bath of xylene, Histoclear, and hexane for 18 hr.
Spectra were recorded between each of the successive cycles and after immersion in reagents for 18 hr. Raman spectra were taken from 10 random points from each of the three sections. All spectra were recorded from normal ectocervical squamous epithelial cells.
Xylene Dewaxing and HMAR in Citrate Buffer
To examine the effect of the HMAR technique using xylene and a citrate buffer on the wax content, sections were processed according to the following HMAR protocol. Sections (mounted on positively charged slides) were passed through two changes of xylene for 6 min each and three changes of spirit for 3 min each. Slides were then submerged in 600 ml of 0.1 M citrate buffer (pH 6) and placed in a pressure cooker for 20 min.
Simultaneous Dewaxing and HMAR in Trilogy
Two staining dishes were filled with 200 ml of Trilogy (Cell Marque Corporation; Hot Springs, AR), and the sections (mounted on positively charged slides) were submerged in the first staining dish. Both dishes were placed in a pressure cooker for 8 min on the high-pressure setting. After 8 min the pressure was released, and the slides were transferred to the second dish (hot rinse) to soak in Trilogy for an additional 10 min. The slides were agitated and rinsed in deionized water.
Raman spectra were recorded from 10 random points of normal ectocervical squamous epithelial cells from the HMAR-treated sections.
Raman spectra were also recorded from paraffin wax sections, tissue sections prior to deparaffinization, and frozen sections not embedded in paraffin wax. All sections were air dried and examined spectroscopically.
Immunohistochemistry
Sections were dewaxed for 18 hr at room temperature in hexane or xylene. Sections were rehydrated to water through Ethanol Absolut (Merck) and Industrial Methylated Spirits 95% (Lennox). The sections were placed in 0.1 mM citrate buffer (pH 6) and microwaved for 12 min at 750 W. They were kept in the hot solution for an additional 20 min, washed in water, treated with 0.3% hydrogen peroxide in methanol for 10 min, and transferred to phosphate buffered saline (pH 7.2). The Vector Elite Avidin-Biotin immunoperoxidase kit (Vector Laboratories; Peterborough, UK) was employed for the immunoperoxidase method. Slides were treated with normal horse serum 1:50 for 10 min, and then the monoclonal antibody to cytokeratin MNF 116 (Dakocytomation; Glostrup, Denmark) was applied at a dilution of 1:100 in PBS for 60 min at room temperature. Sections were washed in PBS for 10 min, and biotinylated secondary antibody (1:200) was applied for 15 min. Following a wash in PBS for 10 min, the avidin-biotin complex solution (1:50) was applied for 15 min, sections were washed in PBS for a final 10 min, and incubated in the chromogenic DAB substrate for 5 min (0.003% hydrogen peroxide and 0.06 g/ml diaminobenzidene in PBS). A nuclear counterstain with hematoxylin was applied, the slides were dehydrated through alcohols, placed in xylene, and cover slipped with DPX resin medium.
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Results |
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The effectiveness of xylene (Figure 4) and Histoclear (Figure 5) on the wax content was examined and found to be ineffective at complete removal of wax. The effect of a single dewaxing cycle (Figure 4A) and subsequent dewaxing cycles (Figures 4B4D) left the signature wax contributions at 1062 cm1, 1296 cm1, and 1441 cm1. These contributions were not eliminated even after immersing slides for 18 hr in xylene (Figure 4E). The same residual wax contributions were present after a single dewaxing cycle (Figure 5A), subsequent dewaxing cycles (Figures 5B5D), and immersion in Histoclear for 18 hr (Figure 5E).
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Following dewaxing with xylene and HMAR using citrate buffer, residual wax contributions were observed at 1062 cm1, 1296 cm1, and 1441 cm1 (Figure 7A). Following simultaneous dewaxing and HMAR with Trilogy, the same residual wax contributions were also observed (Figure 7B). Due to the high pressure and temperature involved in the pressure cooker technique, the tissue begins to degrade after multiple cycles. This tissue degradation is evident in the Raman spectra in Figure 7, where an overall deterioration in signal intensity compared with the signals measured in Figures 36 is easily seen. However, multiple-cycle investigations were carried out and residual wax remained (results not shown).
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Qualitatively, it is clear from Figure 8 that there is a stronger positivity in Figure 8B (dewaxed using hexane). To quantify this increase in positivity, both images were converted to greyscale, and the number of gray pixels and hence the overall intensity of the stained region was calculated using the ImageJ analysis program (National Institutes of Health, Bethesda, MD). The overall intensity after dewaxing using xylene was 124.99 ± 5.03 a.u., whereas the value measured from the slide dewaxed using hexane was 97.46 ± 1.52 a.u. This reduction in intensity quantifies the increase in positivity, which has been improved by 28% when dewaxed using hexane as opposed to xylene.
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Discussion |
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Xylene is the most commonly used solvent, due to its rapid and supposedly efficient dewaxing for common histological, histochemical, and immunohistochemical procedures. It is a highly toxic, flammable substance and must be handled with care. Histoclear, on the other hand, is non-toxic, non-flammable, and biodegradable but is a less-efficient clearing agent and potentially causes the hematoxylin stain to fade (Culling et al. 1985). The HMAR technique uses xylene as a clearing agent and reverses formaldehyde fixation effects using citrate buffer in a pressure cooker (MacIntyre 2001
). Trilogy, on the other hand, is a novel product that combines deparaffinization, rehydration, and unmasking of antigens during pressure cooking. However, Trilogy is under evaluation for application in diagnostic immunohistochemistry, and this is the first report comparing the dewaxing efficacy of Trilogy to the more standard reagents using Raman spectroscopy. Hexane is a colorless liquid with a slight odor and, like many solvents, is highly flammable. It should be handled with care and used only in a fume hood. The US Department of Health and Human Services, International Agency for Research on Cancer, and the US Environmental Protection Agency have not classified hexane for carcinogenicity. This paper is also the first to examine the dewaxing properties of hexane and to directly compare it to all commonly used dewaxing agents.
During investigations into the biochemical changes in tissue with the onset of carcinogenesis (Ó Faoláin et al. 2003), it was noticed that the standard dewaxing procedure used on FFPP tissue did not completely remove the wax as previously supposed. A quantity of residual wax remained. From a spectroscopic perspective, with a view to detection of biochemical changes in the composition of FFPP sections, residual wax is problematic. Its spectral contributions add a substantial degree of variation to the overall spectrum. Contributions at 1062 cm1, 1296 cm1, and 1441 cm1, corresponding to CC skeletal stretch, CH2 deformation, and CH2 bending, respectively, mask contributions from these molecules emanating from the tissue. Thus, potential biochemical changes in these regions are rendered useless. Also, the application of an automated analysis technique is not possible while there are random variables in the datasets.
Ineffective removal of wax from sections can cause birefringence of cell nuclei (Nedzel 1951). In addition, failure to completely remove the wax from the sections will result in impairment of staining. This can result in the Pink Disease artifact (Vlachos 1968
). The artifact, which is most noticeable in lymphoid and epithelial tissue, is an extremely patchy distribution of stains and results in the loss of distinction of nuclear margins (Drury and Wallington 1980
). There are also implications for antigen demonstration by immunohistochemistry. It is only in recent times that immunohistochemistry has been carried out on paraffin-fixed sections. It is clear from the results of both HMAR and Trilogy investigations that all the wax is not being removed.
This research is the first study to report that a low level of wax is residual in solvent-treated paraffin wax sections. The majority of histochemical and immunohistochemical methods employed in histopathology laboratories utilize these solvents as their dewaxing agents. Many dyes stain more intensely on cryostat sections, and some do not work well on paraffin sections, particularly metachromatic stains for carbohydrates (H. Lambkin, unpublished data); thus, the residual wax may be a factor contributing to these differences. Immunostaining has been optimized for paraffin sections with hundreds of antigens now detectable in these preparations. Heat-based antigen unmasking has been introduced since the 1990s and has contributed to a lowering of the antigen detection threshold in paraffin sections (Shi et al. 1991); however, the heat effects are considered to be related to reversing of the effects of formaldehyde cross-linking of proteins, rather than removal of wax.
In this study it has been demonstrated that current dewaxing procedures are not completely effective. Hexane has been identified as a superior dewaxing solvent to xylene as well as to Histoclear and Trilogy. It has also been demonstrated that increased wax removal using hexane results in better antigen/antibody binding and, hence, a stronger positivity. In addition, care should be taken when using FFPP sections for spectroscopic investigation of diseased tissue. This study recommends dewaxing using hexane to minimize wax contributions.
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Acknowledgments |
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The authors would like to express thanks to all the staff in the Histology Department in the NMH Holles St. for their generous cooperation. We also thank Stephen M. Hewitt for his advice and encouragement.
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Footnotes |
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