Abstract
Background/Aim: Endoscopic ultrasonography (EUS)-guided fine-needle aspiration biopsy (EUS-FNB) enhances the diagnostic capabilities of EUS by providing additional pathological samples. However, detecting the target specimens within the collected samples can be challenging. The aim of this study was to determine the optimal wavelength of light for detection of target specimens within EUS-FNB samples in an animal experiment. Materials and Methods: EUS-FNB pancreatic tissue samples were collected from a male beagle (weight, 10 kg), and the samples were illuminated with monochromatic light ranging from 430 to 700 nm in 5-nm intervals. The intensities of the target specimen and blood samples were analyzed using the densitometry of the images obtained through irradiation. Results: We found that transmitted monochromatic light of 605 nm most vividly enhanced the contrast between the target specimens and blood in the samples in the impression of appearance. Conclusion: Thus, microscopical observations under transmitted light of 605 nm are optimal for target tissue identification within EUS-FNB samples.
Recently, the usefulness of endoscopic ultrasonography (EUS)-guided fine-needle aspiration biopsy (EUS-FNB) has been reported for the histological diagnosis of pancreatic masses and mediastinal and abdominal lymph nodes (1). The results of EUS-FNB are used to guide treatment decisions. However, identifying a histologic core within a sample can be challenging because samples collected using fine needles are microscopic and contain blood. EUS-FNB must be conducted promptly to avoid potential complications; thus, the rapid on-site evaluation (ROSE) of samples during EUS-FNB is beneficial. If no ROSE has been introduced, the procedure is generally completed with 3-4 punctures. If ROSE is introduced and it is confirmed that pathological evidence has been acquired in the puncture specimen, puncture can be terminated at that point, leading to a reduction in the number of punctures and complications. However, many healthcare facilities do not have enough cytopathologists to perform ROSE (2-7).
In order to solve this problem, target sample check illuminator (TSCI) was developed and its usefulness has been reported (8, 9). By using this device on a puncture-collected sample, it is possible to determine the presence or absence of histologic core in the sample. However, it has not been tested in basic experiments. Therefore, we conducted this animal experiment with the aim to identify the optimal wavelengths for the detection of target specimens within samples collected using EUS-FNB.
Materials and Methods
This study was approved by the institutional review board of Tottori University (approval number h33-T015) and performed in accordance with the ethical standards of the 1964 Declaration of Helsinki.
EUS-FNB pancreatic tissue samples were collected from a male beagle (weight, 10 kg) by using a 19G biopsy needle (Boston Scientific, Marlborough, MA, USA). A beagle dog underwent laparotomy under anesthesia and its pancreas was manually punctured directly. The puncture site was determined following identification of the target lesion. The central stylet was removed, and the needle was moved in the pancreas. A single puncture was performed for sample collection, and all collected samples were treated as specimens for histological diagnosis.
Light with a half-width of 6 nm, emitted by a 150-W xenon lamp (model no. P-250, Nikon Corporation, Tokyo, Japan), was diffracted using a sweeping-type spectroscope to obtain monochromatic light of variable wavelengths (430-700 nm).
The samples were irradiated with monochromatic light at 5-nm increments to identify the most appropriate wavelength for target specimen detection within the samples. The collected samples were placed on a glass plate. After monochromatic light observation, the formalin-fixed sample was observed with a microscope (DP12, Olympus Optical, Tokyo, Japan). Specimens with an approximate width of 0.9 mm in blood drops were deemed target specimens. We searched for the wavelength that gave the most contrast between the blood and the target specimen in terms of visual impression. Then, we compared the visibility of the target specimen with this wavelength and the reflected light of white light.
Densitometry was used to evaluate the contrast by dividing the transmitted light intensity of the target specimen by the transmitted light intensity of the blood sample, and the wavelength that yielded the largest contrast value was determined. When densitometry and visual evaluation differed, priority was given to visual evaluation in consideration of practical clinical usefulness.
Results
Monochromatic light of 430-585 nm did not penetrate the blood drops; therefore, the target specimens were not identifiable within the aspirated samples in this range. For wavelengths >590 nm, light penetration was enhanced at the margins of the accumulated blood owing to the similarity in thickness between the sample and the light. The target specimens in the samples were detectable at wavelengths of 595-635 nm. However, at wavelengths ≥640 nm, the contrast between the target specimen and blood drop decreased. Transmitted monochromatic light of 605 nm maximized the contrast between the target specimens and the blood in the samples in the impression of appearance (Figure 1A). Compared with observation using reflected white light, observation with transmitted light of 605 nm was more effective in distinguishing target specimens within the EUS-FNB samples (Figure 1A). The detected target specimen was confirmed microscopically (Figure 1B). The intensities of both the target specimen and blood in the sample were quantified and analyzed via densitometry to obtain an image for irradiation at each wavelength. The contrast between the target specimens and blood peaked at 600-605 nm (Figure 1C).
Consideration for a novel monochromatic light to detect target specimens within endoscopic ultrasonography-guided fine-needle aspiration biopsy samples. (A) Visual inspection and discrimination of the target specimen in the samples. The samples are readily detectable at 595-635 nm. (B) The target specimen, detected by transmitted light of 605 nm, is confirmed as pancreatic tissue in microscopic analysis. (C) The contrast peaks at 600-605 nm because the absorbance of the tissue component increases, as shown in the comparative contrast analysis between the target specimen and blood, by using densitometry. Contrast analysis is performed on the target specimen and the blood in the sample. The contrast between the target specimens and blood peaks at 600-605 nm.
Discussion
If a white part is present in the sample collected with the biopsy needle when observed with the naked eye, transmitted light observation like this is unnecessary. However, since it is extremely rare to be able to collect under the above conditions, we conducted this study.
The absorption spectrum of visible light for deoxidized hemoglobin peaks at approximately 550 nm, while that of oxidized hemoglobin peaks at 540 and 585 nm (10). Transmitted light is the most effective modality for distinguishing target specimens in EUS-FNB samples; this study was conducted to identify the optimal irradiation wavelength of transmitted light in an animal experiment. Target specimens covered with a blood drop were difficult to identify using reflected light because the blood absorbed the light. Conversely, transmitted light allowed specimen identification owing to an enhanced contrast. Observation with transmitted light using white light has been reported so far (11). However, even in this study, the target specimen could not be identified with the transmitted light of white light, but it was found with this method.
White specimens in the EUS-FNB samples provided histological data, while the red fraction was the blood component. After collecting samples with a 19G needle, a histological core was observed in 78.9% and 9.3% of the white and red specimens, respectively (12). Target specimens are challenging to identify using reflected light because tissue fragments are entrapped within blood droplets. Such specimens can be observed via transmitted light.
In our observational experiment, among various wavelengths, monochromatic light of 605 nm was optimal for identification of target specimens within EUS-FNB samples. Conversely, previous studies have shown that an optimal discrimination ability was obtained at 540 and 585 nm, which are the absorption maxima of hemoglobin (10). In this study, the light of wavelengths of 540 and 585 nm were unable to penetrate the blood drop. The possible cause of this divergent result is the difference in methodologies among studies. Specifically, in this study, dog pancreatic tissue was not specially treated before monochromatic light observation, but in past reports, hemoglobin obtained from human blood was used, and the blood was anticoagulated with sodium heparin and plasma was removed. Our results also indicated that light with longer wavelengths could equally penetrate blood and tissues in the EUS-FNB samples. In general, the longer the wavelength of light, the lower the transmittance. It is thought that the oxygenated hemoglobin in the sample collected by EUS-FNB absorbed light with a longer wavelength than in previous reports, resulting in a clearer contrast with the target specimen. Therefore, it is considered that the light can be transmitted with a longer wavelength than that in previous report, and the contrast is maximized in visual observation. However, since there is no document or scientific basis for this phenomenon, it is not definitive and may be new knowledge.
Study limitations. First, even if a white specimen was identified in the sample, the target sample check illuminator could not distinguish between tumor components and non-tumorous tissue in the sample. Second, visual inspection revealed maximal contrast at 605 nm, whereas densitometric evaluation yielded maximal contrast at wavelengths <600 nm. Although the reason for this difference is unclear, we deemed 605 nm the best wavelength in this study, as we believe that visual inspection will play a crucial role in future devices for target specimen evaluation. Third, the cost-effectiveness of this method should be addressed in addition to its accuracy and safety. In the future, subjective impressions could also be objectively assessed by performing a multicenter prospective study on the detection of target specimens in endoscopic ultrasound-guided fine-needle biopsy samples.
Conclusion
In conclusion, transmitted light of 605 nm effectively maximized the contrast for identification of target tissues within EUS-FNB samples in the impression of appearance. Owing to the feasibility of this method, we hope that the frequency of use of tissue sample check illuminators in clinical practice will increase. If the usefulness of the target sample check illuminator is proven in clinical trials, it will contribute to reducing the frequency of EUS-FNB procedures, which will in turn lower the risk of adverse events and the burden on practitioners.
Acknowledgements
The Authors would like to thank Editage (www.editage.jp) for English language editing.
Footnotes
Authors’ Contributions
Conceptualization, K.M., M.U., K.I., K.U., S.S., and H.I.; data collection and sample management, K.M., M.U., T.T., Y.O., Y.T., T.O., S.K., H.Ka., H.Ku., and T.Y.; data analysis and interpretation; K.M., M.U., K.I., K.U., S.S., and H.I; writing—review, K.M., M.U., K.I., T.T., K.U., Y.T., T.O., S.K., H.Ka., H.Ku., T.Y., Y.O., S.S., and H.I. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received July 30, 2023.
- Revision received August 20, 2023.
- Accepted August 31, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.