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Due to the increasing popularity of tattoos among the general population, to ensure their safety and quality, there is a need to develop reliable and rapid methods for the analysis of the composition of tattoo inks, both in the ink itself and in already existing tattoos. This paper presents the possibility of using Raman spectroscopy to examine tattoo inks in biological materials. We have developed optical tissue phantoms mimicking the optical scattering coefficient typical for human dermis as a substitute for an in vivo study. The material employed herein allows for mimicking the tattoo-making procedure. We investigated the effect of the scattering coefficient of the matrix in which the ink is located, as well as its chemical compositions on the spectra. Raman surface line scanning has been carried out for each ink in the skin phantom to establish the spatial gradient of ink concentration distribution. This ensures the ability to detect miniature concentrations for a tattoo margin assessment. An analysis and comparison of the spectra of the inks and the tattooed inks in the phantoms are presented. We recommend the utilization of Raman spectroscopy as a screening method to enforce the tattoo ink safety legislations as well as an early medical diagnostic screening tool.
Tattoos have become one of the most popular forms of body modification and people, especially young ones, are no longer perceiving them as a controversial element of their image [1]. The popularity of tattoos is growing, as can be confirmed by surveys conducted in the United States, where, in 2017, as many as 29% of adults said they had at least one tattoo [2]. That is up from 20% in 2015 and 13% in 2007 [3]. Tattoos are made by the injection of a pigment into the body during a series of skin punctures with a needle containing the ink. As a result, the ink reaches the dermis layer, embedding the pigment, and thus, changing the skin color in the desired pattern [4]. Professional tattoos require specialized equipment, like an electric-powered tattoo machine, which allows for making hundreds of punctures per minute [5], multi-colored inks, which are available in hundreds of shades and colors [6], and antiseptics. Tattoo ink producers, to meet the customers’ expectations, provide inks in various embodiments, such as vegan inks and UV-fluorescent inks [7]. The latter might be used in research for the accurate identification of biopsy sites [8]. Besides allergies, tattoos can also cause other health problems. The ink injected into the body can trigger an immune over-response, while small particles of the pigment migrate to the lymph nodes and liver [9]. As a rare consequence, unnecessary surgical treatment can be undertaken due to the misidentification of melanoma metastases in the lymph nodes due to the presence of ink in the organs [10]. There are no regulations for tattoo inks, as they are for cosmetics or medical products. Tattoo inks, especially for cosmetics tattoos, may contain mixtures of unknown pigments and other substances potentially dangerous to human health [11, 12]. The Council of Europe, in a resolution from 2008, recommends two analytical methods for the analysis and detection of hazardous chemical compounds in the composition of tattoo inks: gas and liquid chromatography with mass spectrometry (GC/LC-MS) [13]. However, the applicability of those methods is limited since they require advanced instrumentation, trained personnel, and specific protocols. Sample handling and preparation protocols are available for cosmetics and food but not for tattoo inks. Such methods may be applied to samples of inks, while measurements of tattoos are possible only for biopsy samples, so there must be a premise, such as a severe reaction, warranting the biopsy procedure. Those methods are unavailable for in vivo measurements to ascertain the ink safety, for example, during the screening tests.
The composition of tattoo inks is a mixture of pigments [10], solvents, and supplementary substances that improve the applicative properties of the ink, like viscosity or drying [14, 15]. The most important component in the ink is the pigment, responsible for the ink’s color. Pigments are crystalline particles, often with low water solubility, that remain in a solid state even after injection into living tissue, making them difficult to remove [16]. The pigments are marked with the identifier “C.I.” by the International Color Index, according to their chemical structure [17]. Other utilized components are alcohols, such as glycerin, ethanol, isopropyl alcohol, or benzyl alcohol, which stabilize the dispersion of pigments. In addition, the inks contain numerous preservatives that improve the effectiveness of applying the ink into the skin or accelerate wound healing [14] but may also contain contaminants and hazardous chemicals [18]. The procedure carries a risk of the introduction of hazardous, harmful, allergenic, or cancerogenic compounds into the skin. The growing popularity of tattoos and the lack of regulations on the safety of their composition is an alarming factor in public health. This indicates the need for better tools that would allow for ascertaining compliance with regulations should they inevitably come. Especially in the case of already existing tattoos, non-invasive in vivo measuring methods would allow for pre-emptive screening for hazardous substances in the individuals’ tattoos.
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As GC/LC-MS methods require a sampling of the inks, they cannot be used to measure an existing tattoo in the skin. A non-invasive optical method, like Raman spectroscopy, can be used to measure the skin in vivo. The Raman spectrum is dependent on a specific chemical structure and is often referred to as the "molecular fingerprint" of a substance, allowing qualitative measurements for chemical identification. It is used, for example, in archeology, to determine the details of the carbon-based black ink composition found in mummies’ bodies [19], or in medicine to detect the components of ink causing an allergic reaction [19]. It is also possible to perform quantitative measurements, as the signal intensity depends on the concentration of the substance. Additionally, for the purpose of tattoos, Raman spectroscopy has been used very scarcely, often with the use of very expensive spectrometers with microscope-based systems [20].
However, the assessment of tattoo composition is complicated due to the matrix they reside in, that is, the skin, because of the measurement noise and variability of biological substances [21]. In order to establish Raman spectroscopy as a viable measurement method for this cause, it is necessary to build spectral databases, calibration models, or machine learning methods [22]. These require the acquisition of the spectra of substances in the designated matrix, which in this case, makes it near impossible to obtain since carcinogenic or otherwise harmful ink contaminants would have to be introduced into human skin. This cannot be ethically accomplished in vivo, while ex vivo skin samples are tough to obtain and their parameters change very rapidly over time. This raises the necessity of a stable and reliable material with modifiable properties as a substitute for the skin [23, 24]. Optical tissue phantoms often serve such a purpose in numerous optical methods. In the case of Raman spectroscopy, the phantoms have been used rarely due to changes in their chemical composition and the target material [25]. We propose that this may not necessarily be a major obstacle since the main phantom Raman bands are outside the most important regions for the investigated molecules.
In this paper, we present the possibility of using Raman spectroscopy to detect tattoo inks in biological materials. We determine the characteristic bands of the Raman spectra of tattoo inks. In addition, we developed phantoms for a Raman spectroscopy study of tattoo inks as a substitute for an in vivo study. The material employed herein allows for mimicking the tattoo-making procedure as it normally would be done by the artist’s hand. We used a tattooed porcine skin as a control. We investigated the effect of the scattering coefficient of the matrix in which the ink is located. We performed Raman surface scanning for each ink in the skin phantoms in order to investigate the spatial gradients of the ink concentration distribution in such a medium.
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