Ethics of the practice have a long history but formal documents containing information about the ethical education and practices in research involving human subjects were developed in the middle of the 20th century. Fundamental ethical standards for research involving humans were first codified into the Nuremberg Code in 1947 E$. The World Health Organization (WHO) recognized a need for guidelines or ethical standards that were broader in scope than the Nuremberg Code, and The Declaration of Helsinki: Recommendations Guiding Medical Doctors in Biomedical Research Involving Human Subjects was adopted by the World Medical Society in 1964. The concept of ethical committee was first appeared in the Declaration of Helsinki. The Declaration of Helsinki guidelines are key for ethics in medicine and these have been revised periodically
The term "Research" is defined as any systematic investigation designed to develop or contribute to generalizable knowledge. Globally, multi-disciplinary research is conducted for better outcomes in the welfare of humankind
Raman spectroscopy has wide range of utility not only in the field of science and technology but also growing research towards forensic, food, archeological and biomedicine
The present study uses human surgical samples of cancer patients who give consent to perform the study. The biological samples were obtained from the Modified Radical Mastectomy (MRM) surgical procedure. The breast was removed during this surgery. The normal tissue sections were obtained from 2 to 3 cm away from gross tumour volume with negative margin of the tissue removed during the surgery. The study was conducted under a protocol approved by the institutional bioethical committee at the Medical College (3095/MC/EC/12/04/2017). Five clinically unprocessed, fresh human surgical tissue samples were obtained from a patient. All excised samples in our study were normal tissue of the breast. The adoption of this methodology reduced selection bias in the standardization procedure.
The Raman spectra were recorded with a Confocal micro Raman spectrometer system with Photoluminescence (PL) STR 500 system (AIRIX Corp., Tokyo, Japan). In the present experimental work, confocal Spontaneous Raman Spectroscopy (SRS) in reflection mode was performed using incident excitation laser monochromatic beam of 532 nm with different combination of average laser excitation power and integration time 1mW, 5mW and 5sx3, 10sx3 respectively. It has Diode-Pumped Solid State (DPSS) Nd-YAG Green laser (DL 532, AIRIX Corp., Tokyo, Japan). The incident laser beam of the Nd:YAG laser (532 nm) was focused on the sample through a 20x dry objective, and a 4.0–2.9 mm working distance. The notch filter (Kaiser Optics, Ann Arbor, MI, USA) was used to remove Rayleigh scattered light. Further, all data processing and chemometrics analysis have been performed using the data analysis and graphing software OriginPro version 8.5 (OriginLab Corporation, Northampton, MA, United States).
Prior to the analysis, each raw spectral data was processed to auto-polynomial background correction and intensity normalization, cosmic ray removal, and spectral smoothening using OriginPro software. The removal of sharp spikes attributed to cosmic rays for spectra measurement was removed by using frequency and spatial filtering. Raman output data was processed with OriginPro software to quickly analyze and present meaningful information for visualizing, processing and managing spectroscopy data.
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In this section, we described our experimental setup for the acquisition of biological samples and handling protocol for Raman spectroscopy experiments and presented our results. The goal for this work was not only to create the best practices based on bioethical considerations for Raman spectroscopy, but also to provide comprehensive information for spectroscopy of biological samples involving human subjects. Finally, we report the results and experiment details for our experiments.
The biological samples were immediately mounted on acetate paper. The absorption of excessive liquid around the tissue shall reduce the fluorescence background in the experiment. The biological samples were placed in an airtight sterile vial. The vial should withstand at low temperature for transportation to the Raman spectroscopy acquisition facility. The microscope slide was used as a platform for microscopic specimen observation. They are nearly transparent to visible light and chemically inert in nature. Following spectral data acquisition, formaldehyde was used to preserve tissue samples for histopathological analysis. Hematoxylin and Eosin (H&E) was used as staining agents. Samples were prepared and stained using standard H&E-stained protocol. This allowed direct comparisons of tissue to group in normal or cancerous for spectral analysis in case multiple samples are involved.
The effect of formaldehyde on Raman spectroscopy is illustrated in
The working distance between the sample and spectroscope eyepiece should be kept a minimum to reduce the fluorescence background. In the present study, the spectra of human surgical tissue samples were obtained and monitored at 22°C temperature. It is advised that the room temperature should be kept identical throughout the experiment. The spectra obtained at a high spectral resolution of 0.5 cm-1 and within the identical spectral range of 100 – 3000 cm-1 in the present study due to instrument limitation. However, the biological sample covers a spectral range of 500 – 4000 cm-1 for Raman spectroscopy depending on the sample type. After conformal focus, Raman spectroscopy was performed three consecutive times for each sample and average spectra were obtained to reduce uncertainty in the measurements.
Each corrected spectrum was processed and smoothened to increase the Signal-to-Noise Ratio (SNR) via spectral smoothing (Savitzky - Golay algorithm) procedure across the full wavenumber region recorded (100 – 3000 cm-1). This reduces the influence of intensity changes caused by differences in cellular density and thickness of the tissue. The baseline subtraction was performed manually (using Origin) and the Raman spectral intensities were normalized. Baseline subtraction eliminates of the Rayleigh scattering. After baseline removal, the dominant remaining source of the distinction between spectra is the intensity of the Raman features, arising from the variable amount of biological material within the sample. Average Raman spectra were calculated as an arithmetic average of the recorded Raman spectra. For comparisons, Raman spectra were vector normalized in the Origin software (Raman intensity divided by the norm). The normalization allows comparing the Raman spectra from different measurements and samples. Apart from spectrometer calibration, the different combination of parameters in a spectroscope shall be tested for biological samples and analyzed the data to get maximum sensitivity and meaningful output, as shown in
The investigator/researcher should specify the inclusion and exclusion criteria for biological samples to conduct the scientifically valid research study. The inclusion and exclusion criteria are devised after careful consideration as per the demand of the research design. This is performed while formulating the research question and defining the population/ group involved in conducting the research study. In general, the cross-sectional research study (such as surveys) involving healthy human participants, healthy volunteers/ controls poses no risk to the investigator/researcher. It is advised and preferable in case the research study can be performed with voluntary participation excluding the patients with pre-existing diseases. However, any prospective, experimental research and clinical trial involving humans or biological samples shall only be conducted after obtaining the ethical approval from the concerned ethical committee/ authority mandatorily.
The research study involving human participants with confirmed/ suspected diagnosis of non-communicable diseases such as diabetes, cancer, hypertension etc., pose almost no risk/ minimal risk to the researcher depending on the study or acquired sample. However, it should be noted that an infected researcher/ surveyor may transmit the infection to a cancer patient while conducting research. Since the cancer patient is immune-compressed. Hence, the research study shall be conducted only if the researcher in good health condition. Research studies involving biological samples shall develop a research protocol for the handling of biological samples. Appropriate measures shall be taken such as wearing gloves, masks/ respirators, sterile tongs, airtight, sterile chemical-free container and kept in a cool dust-free environment if they are fresh clinically unprocessed biological samples. These measures are enough to handle, transport and short duration storage for data acquisition. For long-term storage of the biological sample, samples shall be immersed into formalin solution or in the form of paraffin-embedded tissue blocks, also known as Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimen. It is advisable to keep samples with tissue repositories and biobanks for long term storage of biological specimen. Several research studies conducted involving FFPE tissue blocks from tissue repositories for retrospective analysis of samples and understanding the process of carcinogenesis using spectroscopy
After proper handling of biological samples, gloves and masks have to be disposed of as biomedical waste of the institute and washing hands with soap and water is recommended. WHO recommended 7 step washing of hand with water and soap for hand hygiene to lowering the chances of any suspected infectious disease transmission. The use of masks to cover the mouth and nose is standard practice. The purpose is to prevent contamination and prevent exposure of the mouth and nose of the investigator/researcher from blood or other fluids from patients during a procedure involving biological samples. The use of masks significantly reduces contamination. It is well known that sterilization is the process by which an item is purged of all microorganisms and spores. Appropriate methods for sterilization of materials/ instruments are important in case they are reused during the research study involving biological samples.
Formalin is commonly used for long term storage of biological samples. Formalin contains aqueous solution of formaldehyde. Formaldehyde presents evidence for increased cancer risk in exposed individuals. WHO categorized formaldehyde in carcinogenic risks to humans. Formaldehyde presents strong evidence for increased cancer risk of Nasopharynx and Leukaemia/ lymphoma in exposed individuals. Appropriate preventive measures for inhalation and ingestion of formaldehyde shall be taken care of while handling with use of facemask and hand gloves.
The research study involving human participants with confirmed/ suspected diagnosis of communicable disease such as HIV, Hepatitis positive patients, Covid-19 and other biohazard patients, a great precaution has to be taken as they pose high risk of disease transmission. In case the research is not concerned with positive net benefit for these patients, one shall exclude these patients from the research study. Further, the researcher may seek the help from expert healthcare professional to develop research protocol to conduct study involving these patients/ biological samples. It is important to mention that the output discharge of waste generated by these patients shall strictly be disposed of as per the recent national guidelines of the Bio-Medical Waste Management Rules.
Before conducting the study for the individual, well documented informed consent, preferably in writing, must be obtained from study participants in a language that the participant comprehends. Prospective individuals (patient/participant) are independent in decision making for participation in the study and the participation is voluntary and they are free to withdraw from the study at any point. Potentially vulnerable subjects may include elderly people, prisoners, children, fetuses, cognitively impaired individuals, pregnant women, ill patients, tribals, or economically or educationally disadvantaged people. All vulnerable populations should receive specifically considered protection. Medical research with a vulnerable population is only justified in case the research is responsive to health needs or priorities of the vulnerable group and the research study is not feasible to conduct in a non-vulnerable group. Also, there is a reasonable likelihood that vulnerable population should be benefited from outcomes of the research. For vulnerable patients who are often unable to give consent, investigator/researcher shall obtain written informed consent from the approving authority, who is in close relation with the vulnerable patient and understand the condition of the patient. Moreover, it is the ethical responsibility of investigator/researcher that inclusion of vulnerable group is completely justified and these patients should not be exploited to generate clinical data in the research study.
The present study describes basic bioethical education and sample handling procedures for researchers to plan and conduct research studies involving human biological specimens obtained to develop new technologies and advance science to improve preventive, diagnostic and therapeutic potential. Fresh unprocessed tissue samples yield better spectroscopic information. Informed consent is good way and mandatory for the protection of patient rights. The researchers shall present and get approval from the concerned ethical committee/ authority prior to conduct research studies involving human subjects. The scope of this article is not limited to Raman spectroscopy and further can be used for other spectroscopic techniques involving biological samples. The standardization of procedure under bioethical considerations will provide the best outcomes in an optimized way for conducting the research studies involving human subjects.