Prospective Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. Nov 15, 2018; 10(11): 439-448
Published online Nov 15, 2018. doi: 10.4251/wjgo.v10.i11.439
Raman spectroscopy for the diagnosis of unlabeled and unstained histopathological tissue specimens
Haruo Ikeda, Hiroaki Ito, Muneaki Hikita, Noriko Yamaguchi, Naoyuki Uragami, Noboru Yokoyama, Yuko Hirota, Miki Kushima, Yoichi Ajioka, Haruhiro Inoue
Haruo Ikeda, Naoyuki Uragami, Haruhiro Inoue, Digestive Disease Center, Showa University Koto Toyosu Hospital, Koto-ku, Tokyo 1358577, Japan
Hiroaki Ito, Noriko Yamaguchi, Noboru Yokoyama, Department of Surgery, Digestive Disease Center, Showa University Koto Toyosu Hospital, Koto-ku, Tokyo 1358577, Japan
Muneaki Hikita, Stem Cell Business Development Department, Nikon Corporation, Sakae-ku, Yokohama, Kanagawa 2448533, Japan
Yuko Hirota, Miki Kushima, Department of Pathology, Showa University Koto Toyosu Hospital, Koto-ku, Tokyo 1358577, Japan
Yoichi Ajioka, Division of Cellular and Molecular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata City, Niigata 9518510, Japan
Author contributions: Ikeda H and Ito H conceived and designed the study, collected clinical data, collected samples, performed the statistical analysis, and interpreted the data; Hikita M performed Raman spectroscopic analysis; Yamaguchi N, Uragami N, and Yokoyama N performed medical examinations and surgical operations; Hirota Y and Kushima M performed pathological examinations; Ajioka Y and Inoue H participated in the study design and coordination; all authors have read and approved the final paper.
Supported by the Japan Society for the Promotion of Science (JSPS), through two JSPS KAKENHI Grants-in-Aid for Scientific Research (C), No. JP26460688 and JP17K09022.
Institutional review board statement: The Institutional Review Board of Showa University approved the study.
Clinical trial registration statement: This prospective study is registered with University Hospital Medical Information Network in Japan, UMIN000017045.
Informed consent statement: We obtained the written consent from the participant before executing this study.
Conflict-of-interest statement: The authors declare that there is no conflict of interest.
Data sharing statement: There is no additional data available.
CONSORT 2010 statement: The guidelines of the CONSORT 2010 Statement have been adopted.
Open-Access: This is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Hiroaki Ito, MD, PhD, Associate Professor, Department of Surgery, Digestive Disease Center, Showa University Koto Toyosu Hospital, 5-1-38 Toyosu, Koto-ku, Tokyo 1358577, Japan. h.ito@med.showa-u.ac.jp
Telephone: +81-3-62046000 Fax: +81-3-62046396
Received: July 20, 2018
Peer-review started: July 20, 2018
First decision: August 31, 2018
Revised: September 6, 2018
Accepted: October 17, 2018
Article in press: October 17, 2018
Published online: November 15, 2018
ARTICLE HIGHLIGHTS
Research background

Histopathological evaluation is the gold-standard for cancer diagnosis. However, the diagnostic accuracy of histopathology staining is low, and the protocols for immunohistochemistry are complicated and time-consuming.

Research motivation

To achieve rapid, accurate and minimally invasive cancer diagnosis, a label-free and non-contact diagnostic technology is useful. Raman scattering spectroscopy has been used to analyze several types of biological tissue specimens; however, the clinical significance and diagnostic accuracy of this approach remain unclear. In addition, there are currently no standardized evaluation methods of gastrointestinal tissue spectroscopy analysis for living organisms.

Research objectives

We used the surgically resected stomach of a patient who underwent.

Research methods

The resected stomach was processed using general histopathological specimen preparation procedures. We produced two consecutive tissue specimens from areas with and without stomach cancer lesions. Each tissue specimen was sliced to a thickness of 3 μm and attached to a low-autofluorescence slide. One of the two tissue specimens was stained with hematoxylin and eosin and used as a reference for laser irradiation positioning by the spectroscopic method. Another tissue specimen was left unstained and used for Raman spectroscopy analysis by a laser light source with a wavelength of 532 nm.

Research results

Raman scattering spectrum intensities of 725 cm-1 and 782 cm-1, are associated with the nucleotides adenine and cytosine, respectively. The Raman scattering spectrum intensity ratios of 782 cm-1/620 cm-1, 782 cm-1/756 cm-1, 782 cm-1/1250 cm-1, and 782 cm-1/1263 cm-1 in the gastric adenocarcinoma tissue were significantly higher than those in the normal stomach tissue. In addition, both adenine and cytosine were presumed to be present at higher concentrations in the non-cancerous lymphocytes infiltration area surrounding cancer compared to the cancer area in the gastric adenocarcinoma tissue specimen.

Research conclusions

This preliminary experiment suggests the feasibility of our spectroscopic method as a diagnostic tool for gastric cancer using unstained pathological specimens. The Molecular biological differences among cells in the resected stomach tissue can be detected by Raman spectroscopy. Adenine and cytosine may be influential substances for histopathological diagnosis by Raman spectroscopy. By focusing on adenine and cytosine, we were able to distinguish qualitative differences in the stomach tissue by Raman spectroscopy. Both adenine and cytosine were presumed to be present at higher concentration in the gastric adenocarcinoma tissue were significantly higher than those in the normal stomach tissue. We measured the Raman scattering spectrum intensities at 620 cm-1 (C-C twisting mode of phenylalanine), 725 cm-1 (adenine), 756 cm-1 (symmetric breathing of tryptophan), 782 cm-1 (cytosine), 1002 cm-1 (phenylalanine), 1250 cm-1 (amide IIIβ-sheet), and 1263 cm-1 (amide IIIα-Helix), corresponding to the Raman scattering wavenumber of the organism constitution organic substance. We then calculated the ratio of the Raman scattering spectrum intensities of 725 cm-1 and 782 cm-1, associated with the nucleotides, to those of the others. We compared the ratio of the Raman scattering spectrum intensities of 725 cm-1 and 782 cm-1, associated with the nucleotides adenine and cytosine to qualitatively evaluate tissue. We found that Raman scattering spectrum intensities associated with the nucleotides adenine and cytosine were higher in adenocarcinoma than in normal tissue specimen of the stomach. In conclusion, we were able to distinguish qualitative differences in the stomach tissue by Raman spectroscopy.

Research perspectives

The Molecular biological differences among cells in the resected stomach tissue can be detected by Raman spectroscopy. In the future, we should raise the accuracy of estimation by Raman spectroscopy and to complete it as a technology that can obtain both high-precision morphological information and qualitative information.