RU2726062C2 - Method for differential and early diagnosis of diseases of ent organs of inflammatory and tumor aetiology using raman-fluorescence spectrometry - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, in particular to otorhinolaryngology and oncology, and can be used for differential diagnosis of chronic tonsillitis and squamous cell carcinoma of pharynx. For this purpose, Raman-fluorescence spectroscopy is used to obtain spectral characteristics of palatine tonsillar tissue and patient's pharynx. Spectra are processed by main components method, by projection on latent structures in combination with linear discriminant analysis; Savitzky—Golay method and reference line correction using asymmetric least squares. Most informative Raman scattering strips are identified, the main and additional peaks are used to differentiate the patient's chronic tonsillitis of the compensated, decompensated form or squamous cell carcinoma of the pharynx.EFFECT: invention enables diagnosing chronic tonsillitis and squamous cell carcinoma of the pharynx and evaluating the clinical effectiveness of the dynamics in these diseases.1 cl, 61 dwg

Description

The invention relates to medicine: to otorhinolaryngology and oncology, and can be used in the differential diagnosis of diseases of ENT organs of inflammatory and tumor etiology and other organs of the head and neck. A method for the rapid diagnosis of diseases of a tumor and inflammatory nature using Raman fluorescence spectrometry involves the use of laser radiation of various wavelengths (405 nm, 532 nm) to register individual tissue features at the metabolic, morphometric and functional levels. The Raman effect (Raman scattering of light) allows you to register changes in the tissue at the molecular level and can be used to diagnose pathological changes in the ENT organs and other organs of the head and neck. This effect can be used to increase the efficiency of the diagnosis of inflammatory and tumor diseases of the ENT organs and other organs of the head and neck; for accelerated identification of the nature of the pathological process, including differential and early diagnosis of cancer.

The method is aimed at conducting high-quality, highly sensitive, highly specific, accurate, minimally invasive and rapid diagnosis of diseases of the ENT organs and other organs of the head and neck of inflammatory and tumor etiology, both in vitro and in vivo.

The method according to the invention allows with high accuracy, sensitivity and specificity (close to 100%) to characterize the physiological changes that occur in the tissue during inflammation and to register its structural features, as well as the presence of certain metabolites in it for the purpose of immediate (quick) diagnosis and early identification of pathological process (for carrying out preventive measures, using this method as a screening, as well as monitoring treatment and its correction at various stages of the pathological process).

To date, there are many ways to diagnose inflammatory and tumor diseases.

A known method for the diagnosis of compensated and decompensated forms of chronic tonsillitis based on the method of infrared spectrometry of saliva (Portenko GM et al., 2005, patent No. 2261048). This method is based on the application of the probabilistic-logical training algorithm of the binary tree of CART decisions according to the indicators of the infrared spectrum of saliva. This method relates to rapid methods of identification and differential diagnosis of forms of chronic tonsillitis, in combination with a probabilistic neural network according to the energy-information indicator of a saliva sample. However, this method is indirect in the differential diagnosis of forms of chronic tonsillitis, since a biological fluid, saliva, is being studied, the biochemical changes occurring in the body can thus be judged only indirectly, that is, in situ diagnosis of a biological object, in this case, palatine tonsils and other structures of the pharynx, is not provided in this work, which cannot carry complete information about the studied object.

A known method for the diagnosis of lung cancer in humans, consisting in the study of the composition of exhaled air; if cyclohexyl isothiocyanate is present in the latter, cancer is diagnosed. The study of the composition of exhaled air is based on the method of mass spectrometry with preliminary gas chromatographic separation (Bakhmutov D.N., 2014; G01N 33/497; АВВ 5/08). The method is valuable, however, when registering the concentration of a chemical in exhaled air, the latter is affected by many additional factors, that is, the method is carried out indirectly and is difficult to perform, in particular due to the fact that the equipment occupies a large area (is not portable).

A known method for the diagnosis of cancer and the potential resistance of malignant cells to hypoxia is that a cell fingerprint or suspension of cells is created from a biopsy sample that creates hypoxia conditions and a spectrofluorometric study of these cells is carried out in the dynamics of the NAD (P) H fluorescence photodestruction process . The increase in the fluorescence intensity of NAD (P) H under dark conditions judges the presence of tumor cells and their potential resistance to hypoxia. Spectrofluorometric research is carried out in the range of 440-470 nm at an excitation wavelength of 365-370 nm. This method allows you to diagnose cancer and the potential resistance of malignant cells to hypoxia (Schwartzburd P.M .; G01N 33/52; G01N 33/483; A61B 10/02), however, the main disadvantage of this method is that it is used only in vitro.

Also, as an analogue of the proposed method, one can note a method for the diagnosis of cancer and somatic diseases, which consists in the study of the polarization and spectral characteristics of biological fluids mixed and condensed with other substances either in pure form or in a tissue section. The technical result consists in the effectiveness of the diagnostic method by applying the effect of optical anisotropy by visualizing the anisotropy of the liquid optical media of the body on the monitor with a further indication of anisotropy through devices and sensors, spectroscopy and optical polarization, as well as improving the accuracy of diagnostics and reducing the time of diagnosis (Kutushov M. B., 2011; G01N 21/21). The method is quite informative, however, it is also indirect, that is, it allows registering morpho-functional and metabolic changes occurring in the tissue itself in a given specific period of time only through systemic manifestations.

Also known is a method of endoscopic fluorescence diagnosis of malignant tumors of hollow organs (Sokolov V.V .; RU (11) 2129273 (13) C1; G01N 33/48, G01N 33/52; 2014), which is used using exogenous fluorescence of the photosensitizer and endogenous fluorescence of biological tissues and is characterized in that in the process of fluorescence-diagnostic examination, a spectral parameter is determined, which is the ratio of the integrated fluorescence intensity in the red spectrum (superposition of endogenous and exogenous fluorescence of the biological tissue) and the integral intensity of endogenous fluorescence in the yellow-green region of the spectrum, and with its value 3 rel. units and above diagnose a malignant tumor. The method is quite complicated in execution, including through the use of additional consumables, such as photosensitizers.

Also known is a method for early diagnosis and monitoring of cancer (Laktionov P.P., 2005; G01N 33/53). A method for early diagnosis of cancer by determining the concentration of nucleic acid in the blood of patients, characterized in that the concentration of extracellular nucleic acids associated with the cell surface of blood cells is determined, and cancer is diagnosed at a value of zero. The concentration of nucleic acids in the blood can vary with various diseases, not only with cancer, respectively, the method is valuable, but it is necessary to use the latter in conjunction with other parameters.

A known method for the early diagnosis of malignant tumors of the stomach. The essence of this invention is that during the first endoscopic visual examination, a biopsy is taken from sections of the stomach with endoscopic signs of dysplasia, the coordinates of these sections are determined using a cylindrical coordinate grid and they are entered into the database, and if histological signs of dysplasia or metaplasia are detected, repeated biopsies are performed from areas with previously established coordinates and with an increase in the degree of dysplasia, malignant formation of the stomach is diagnosed. The method improves the reliability of the diagnosis of early forms of cancer (2008791; A61B 5/00, A61B 1/00; A. Balalykin), is informative, but is longer in comparison with the claimed method.

All of the above methods have significant useful properties and advantages, however, the main disadvantage of these methods is the complexity of the execution, the duration, in some cases, the relatively low sensitivity and specificity, as well as the inability to use the in vivo method (inside the body, organ) non-invasively and quickly (with "Speed of light").

The prototype of the proposed method is a method of infrared Raman spectroscopy in low beam for in vivo-in vitro-early diagnosis of cervical cancer (Rebecca Richards-Kortum; 1995; G01N 21/6486), which consists in using the corresponding individual spectral peaks at different wavelengths in combination with the methods of the main components and Fisher's discriminant analysis, so that it becomes possible to differentiate the intact tissue and the tissue involved in the inflammatory process from the pre-tumor (low- and highly differentiated precancer), including based on the determination of the content collagen, nucleic acids, phospholipids and glucose-1-phosphate in tissues. However, in this case, infrared radiation is used, and the equipment used occupies a large area, in addition, the measurements were carried out in only one location, there were no studies of tissue involved in inflammation and tissues of various benign and malignant neoplasms, which limits the application of the method in the corresponding clinical direction . In addition, this method does not provide for the registration of tissue indicators in dynamics with an assessment of the effectiveness of the treatment. From recent studies on the diagnosis of cancer in otorhinolaryngology using spectrometry, mention is made of the dipped infrared spectrometry method used as a diagnostic method for nasopharyngeal cancer (Ming L.C. 2017). The method consists in in vivo-differentiation of intact tissue and nasopharyngeal tissue in cancer, however, infrared radiation is used in this case, however, this paper does not provide an assessment of tissue parameters in dynamics with analysis of not only morphological and metabolic features of the tissue, but also physiological; no indexes are calculated to evaluate the functional characteristics of the tissue.

The claimed method uses a hardware-software complex certified approved for use, however, it is necessary to adapt the Raman-fluorescence technologies in the diagnosis of diseases of the ENT organs and other organs of the head and neck, and therefore a method for calculating special indices for evaluating morphometric metabolic and functional features of tissue in normal and pathological conditions. Work is underway to improve the specialized hardware-software complex (APC) for the rapid diagnosis of diseases of the ENT organs and other organs of the head and neck using special nozzles on the optical fiber, designed specifically for the anatomical structures of the pharynx, larynx, paranasal sinuses, and ear. Also, the agro-industrial complex is being modernized on the basis of Raman fluorescence spectrometry using the combined endoscopy-Raman fluorescence spectrometry-computed tomography technique and other research methods that allow the most accurate diagnosis and rapid diagnosis of diseases of ENT organs and other organs of the head and neck, such as in vivo and in vitro. The inventive method is carried out using hardware and software systems for both in vitro and in vivo applications, which makes it more valuable and variable in the diagnosis of diseases of ENT organs and other organs of the head and neck. Also, this hardware-software complex allows you to record changes in tissue at a given time interval using a special fiber optic fiber, the software allows you to interpret data on a biological object in real time, quickly, with an assessment of the main phases of the pathogenesis of diseases of ENT organs and other organs of the head and neck, which allows the use of the in vivo technique. The proposed new method of Raman fluorescence spectroscopy is a highly sensitive, highly specific and accurate diagnostic method, since it allows you to record minimal changes in physiology, morphometric and metabolic parameters non-invasively and quickly (within a few seconds). In addition, this method is the fastest compared to other methods. Thus, the technique of Raman fluorescence spectroscopy used by us in the diagnosis of inflammatory and tumor diseases of the ENT organs and other organs of the head and neck is a universal, accurate, minimally invasive, highly sensitive, highly specific, accelerated diagnostic method, which is characterized by determining not only the spectral features of the tissue itself , but also with a set of metabolic, structural, as well as functional changes in the studied biological object (in this case, tissues of the ENT organs and other organs of the head and neck). The technique we use for Raman fluorescence spectroscopy based on hardware and software systems EnSpectr M405 and EnSpectr R532 (device and method for Raman fluorescence identification of a substance, I. Kukushkin et al., 2011; measuring instrument RU. P.37.003 .A No. 44570) can be used to record and interpret Raman spectra of ENT tissue in inflammatory and neoplastic (benign and malignant) diseases, as well as registration of types of metabolites in the blood in case of corresponding diseases. In addition, using the in vitro technique, spectral characteristics of microorganisms and / or their associates in the inflammatory process can be recorded, which is an additional factor indicating the possibility of using this method in the diagnosis of inflammatory diseases of ENT organs and other organs of the head and neck (“Inflammatory pharyngeal diseases: a guide for doctors (V.T. Palchun et al., 2013).

A method for the diagnosis (differential and early diagnosis) of inflammatory and tumor diseases of the ENT organs and other organs of the head and neck using Raman fluorescent medical technologies consists in recording spectral data from the surface of the tissue in vivo and in vitro with the corresponding pathological process, as well as spectral characteristics of the blood plasma of patients with these diseases and the characteristics of the microflora of the studied biological object in normal and pathological conditions.

The problem solved by the invention is the difficulty of establishing an accurate diagnosis in a timely manner, in its early stages, including during surgical treatment (to determine the boundaries of the spread of the tumor process), as well as determining indications for surgical or conservative treatment. Using the proposed new method, it seems possible to accelerate the diagnosis of the type of pathological process: inflammatory or tumor (or determination of tissue not involved in the pathological process) for diseases of the ENT organs and other organs of the head and neck, since the method allows for minimal time with high sensitivity and specificity non-invasively, both in vivo and in vitro, using a portable hardware-software complex of Raman-fluorescence spectroscopy to identify the nature of the process, as well as its type in certain diseases.

The technical result is the implementation of a method for differential diagnosis of inflammatory and tumor diseases of the ENT organs and other organs of the head and neck, including their early diagnosis, together with objective examination data, additional research methods (cytological, histological, PCR method, radiation and immunological methods etc.), including recording the spectral characteristics of tissue and biological fluids in case of corresponding diseases, in the accelerated mode (with the “speed of light”), using a highly sensitive, highly specific and minimally invasive express method in dynamics with the assessment of special indices that allow analyzing changes in tissue at this particular time period.

The implementation of the method.

The device for recording fluorescence and Raman light scattering signals includes a green Nd: YAG (2w) solid-state laser with a wavelength of 532 nm, an optical signal focusing and filtering system, a spectrometer with a diffraction grating of 1200 lines / mm with a spectral resolution of 4 cm ^-1 , CCD-matrix, controller and computer with a software product for recording spectra. To check the reproducibility of the spectral features, measurements were made at several points of the test sample. To reduce the measurement error, a 10-fold lens was chosen, which focused the laser beam into a sufficiently wide spot (comparable in size with the microscopic features of the studied tissues) with a diameter of 10 μm. The laser radiation power was 10 mW; the spectra were recorded during the exposure time equal to 1 s. The number of averagings for recording each spectrum was chosen sufficiently large (to effectively suppress readout noise) —it was 20. As an individual reference (object of comparison), a point called intact was chosen — this is the unaffected area of the mucous membrane of the studied anatomical regions. On the removed material, an express indication of the pathological process was carried out by sequential measurement of the spectral characteristics of the tissue at several points in each test sample and a database was accumulated inside each studied class. The recorded data, which are multidimensional (wave shift of scattered radiation, fluorescence intensity and Raman scattering), were analyzed using the regression method of projections onto latent structures (PLS-DA). The essence of the method is that discriminatory rules for classes are defined by linear regression equations. From the recorded data, the intensity of the Raman-fluorescence spectra at certain wave shifts, a matrix is compiled, where for each spectrum from the sample, a value of 0 or 1 is assigned, depending on the class, for example, healthy tissue and a tumor. The regression problem is solved by the projection method on latent structures, which allows us to predict further the ownership of new samples. PLS-DA (projection onto latent structures and linear discriminant analysis) also reveals spectral features for classes that determine specific vibrations of bonds or molecules for each type of tumor. Before applying the PLS-DA method, the recorded data was eliminated by the Savitsky-Golay method [Ch. Ruffin, 1999] and background radiation by the reference line correction method using asymmetric least squares [S.-J. Baek, 2015]. All preprocessing methods and multidimensional data analysis were implemented in the TP ^T cloud ^beta online software.

The choice of the method of Raman fluorescence spectroscopy and its software implementation in the EnSpectrR532 instrument for pathology of ENT organs and other organs of the head and neck is justified by the need to increase the sensitivity and specificity of diagnosis with the subsequent interpretation of informative criteria for individual spectral characteristics. To identify and confirm the diagnostic informative value of Raman fluorescence spectroscopy in inflammatory and tumor diseases of ENT organs and other organs of the head and neck, we conducted a comparative study. Also, the removed material was sent for pathological and microbiological examination (to assess the specificity of the method). Software processing of the obtained spectral data was carried out in conjunction with the use of clinical, pathological and microbiological data of the studied tissue samples. The presented medical technology, apparently, can objectively characterize the functional state of the ENT organs and other organs of the head and neck, which was proved by counting special indices (explanation in the text). In addition, using laser radiation with a wavelength of 405 nm, as well as an optical fiber, it seems possible to record tissue in vivo in situ dynamics.

Using the EnSpectr R532 hardware-software complex, it seems possible to analyze in metabolic mode the metabolic, morphometric and functional features of the tissue, as well as the biological fluids of patients (blood) in inflammatory and tumor diseases of the ENT organs and other organs of the head and neck in vitro.

Another modification of the method is to supply laser radiation with a wavelength of 405 nm (the use of this wavelength is based on the Raman effect) to the biological object under study, in this case, tissues of the ENT organs and other organs of the head and neck (palatine and pharyngeal tonsils, paranasal sinuses , ear, larynx, tongue, parotid salivary glands); registration of both the main (probing) signal and the reflected signal during the exposure time - 1-5 seconds, and then in the video recording of the spectrum and calculating the indicators of fluorescence intensity, spectral half-width, normalized fluorescence index (ratio of different wavelengths to each other), aerobic index (the ratio of the wavelength of 665 nm to the wavelength of 670 nm), the characteristic features of peaks recorded at certain wavelengths are informative parameters for identifying the type of tissue: healthy, tissue or tissue affected by the inflammatory process, as well as identifying the type of pathological process using a special index calculation software. The characteristics of the peaks themselves are analyzed: their shape, amplitude, width, wavelength, their number is normal and for various inflammatory and neoplastic diseases of the ENT organs and other organs of the head and neck. Using this method, an accelerated differential diagnosis of diseases of the ENT organs and other organs of the head and neck of inflammatory and tumor etiology is carried out. The method is also carried out using the method of principal components, linear discriminant analysis, the principle of least squares, projection onto latent structures to highlight the most informative Raman lines at various wavelengths in the spectra of intact tissue, as well as tissue involved in the inflammatory and tumor process in ENT diseases - organs and other organs of the head and neck. A combined method of analyzing spectral characteristics was used to describe differences in the norm in inflammation and tumors of ENT organs and other organs of the head and neck. This method includes all of the above parameters, as well as a description of the spectrum as a whole, its fluorescent component, Raman peaks, their height, width, half-width, wavelength ratio, the value of which identifies the same metabolites in inflammation to a value that is strictly individual in these cases, including with various forms of chronic tonsillitis. The sensitivity and specificity of the method were analyzed on the basis of classical diagnostic methods: histological, cytological, microbiological and biochemical. It was found that, depending on various metabolic, morphometric, and functional features of the tissue, it is possible to carry out differential diagnostics of the type of tumor process: malignant, benign, or metaplastic using Raman-fluorescent medical technologies with high accuracy, specificity, sensitivity, non-invasively and in express mode. The existing methods for diagnosing inflammatory and neoplastic diseases of the ENT organs and other organs of the head and neck undoubtedly have many advantages, but they are either long-term, expensive, or provide only an indirect idea of the processes occurring in the body, not allowing diagnosis in vivo in situ, or can be difficult to perform, and are also implemented using large-sized equipment. Also, they are not as sensitive and specific as the Raman fluorescence spectroscopy we use, which is not only highly sensitive, highly specific, but also non-invasive, fast. In addition, it allows you to register numerous parameters in real time, that is, to carry out "diagnostics at the place of examination and treatment." To determine the morphological, functional and metabolic characteristics of the tissue in normal and pathological conditions, including on the basis of recording Raman-autofluorescence characteristics, it is necessary to choose the most optimal diagnostic method, which includes the method of Raman-fluorescence diagnostics. The development and implementation of the presented technology in wide clinical practice is promising and clinically feasible and will make it possible in the future to significantly improve the quality of diagnosis of inflammatory and tumor diseases of the ENT organs and other organs of the head and neck and to prescribe rational treatment in a timely manner. The use of the EnSpectr R532 software and hardware complex also allows for reliable, highly sensitive (10 ⁴ - 10 ⁵ CFU / ml) detection of the etiological factor in various inflammatory diseases of the ENT organs and other organs of the head and neck, as it helps to identify the type, concentration of the microorganism or associates microorganisms.

Differential diagnosis of inflammatory and neoplastic diseases of the ENT organs and other organs of the head and neck is one of the most important tasks facing otorhinolaryngologists, oncologists and other clinicians. Using the method of Raman fluorescence spectroscopy on the studied sample of histological and cytological samples, it was possible to analyze the spectral data. The separation between these classes was carried out with a specificity close to 100%, a sensitivity of about 100%) and an accuracy of approximately 100%. However, it should be understood that the differential diagnosis in this case is possible when analyzing the totality of data obtained in relation to various diseases of the ENT organs and other organs of the head and neck, that is, it is not only the spectral characteristics of the tissue itself, but also the corresponding metabolites in the blood plasma patients, the microbial composition of the pathological focus, the concentration per gram of tissue of microbial bodies in a given period of time at the corresponding stage of the disease, clinical, cytological and histological data, as well as examination data. A systematic approach with the analysis of the above indicators in dynamics at various stages of the disease will also allow us to judge the physiological processes occurring in the tissues, such as blood flow and microcirculation, proliferative activity of cells, the difference in metabolites and other features. It is also valuable in diagnostics to determine characteristic features using the principal component method, linear discriminant analysis, the projection method on latent structures, etc., since they allow you to select the most informative criteria when analyzing spectral data.

Thus, the method of Raman fluorescence spectroscopy can be used in addition to the existing methods for differential diagnosis (including early diagnosis) of various inflammatory and tumor diseases of ENT organs and other organs of the head and neck. Raman fluorescence spectrometry is based on the phenomenon of autofluorescence of biological tissues and the effect of Raman scattering of light, in which, when a tissue is exposed to laser radiation, characteristic molecular vibrations of the substances in its composition are recorded in the spectrum of scattered light. It is known that inflammatory and neoplastic diseases of the ENT organs and other organs of the head and neck are an urgent problem of the modern population and occupy an important place in the structure of diseases of the upper respiratory tract (El-NaggarAK., 2009; 1: O9). It must be borne in mind that the term “diagnosis” used in the present work implies not only a diagnosis of the disease, but also screening of patients to identify and differentiate various pathological conditions, identify the etiological factor, choose a tactic and treatment method, evaluate its effectiveness, choose the preferred treatment the drug, identifying indications and contraindications for its use, monitoring the treatment process, identifying complications and objectively determining the timing of patient rehabilitation.

When analyzing spectral data, separation by the PLS-DA method was carried out between the tumor and inflammation classes, and a comparison was also made with the norm. An intact region not affected by inflammation or tumor process was taken as the norm. The model of data processing and analysis includes smoothing, centering and normalization of data and a method for isolating a signal against a general background (getting rid of fluorescence).

The results of the rapid diagnosis of the type of pathological process (inflammatory or tumor) are presented in the corresponding figures (figures) below.

The invention relates to medicine, namely, otorhinolaryngology and oncology, and can be used to diagnose the inflammatory and tumor process in diseases of the ENT organs and other organs of the head and neck.

To identify and confirm diagnostic informativeness of Raman fluorescence spectroscopy, several groups of tissues were compared: intact tissue involved in the inflammatory process in chronic tonsillitis, adenoids, chronic rhinosinusitis, chronic suppurative otitis media, and tissue affected by the tumor process (as benign, and malignant, including transient forms of metaplasia) in squamous cell carcinoma, adenocarcinoma, malignant melanoma of various locations, including the larynx, pharynx, neck, lymph nodes, parotid salivary glands, with pleomorphic adenoma, lipoma and other diseases of ENT organs and other organs head and neck. In total, over 1200 spectra were analyzed. The results obtained are partially presented in the figures, the description of which is given below (see the Appendix “Drawings and Drawings”):

Brief Description of the Drawings

In FIG. Figures 1-60 show the following conventions in English (it is impossible to change them), since the conventions used in the software used are exactly as they are depicted in FIG. 1-60, namely:

Tumor tumor; inflammation inflammation; variables- variables (Fig. 1); correct-correct, false negative- false negative, false positive-false positive, excluded NMC excluded spectra, accuracy-accuracy, Spec.-specificity, Sens.-sensitivity; Y Observed-observed in fact; Y predicted- predicted values (Fig. 2).

In FIG. 3 - Value-value, variables- variables, options- are measured, in this case, in inverse centimeters (wave numbers).

In FIG. 4 OGPKK4, PAC - squamous cell carcinoma of the pharynx, F 1-chronic tonsillitis, compensated form. XT CF - chronic tonsillitis, compensated form.

The legend shown in FIG. 5 were presented in the description of FIGS. 1-4.

The legend in FIG. 7: OGPKK4, PAC-squamous pharyngeal carcinoma, F2; XT DF - chronic tonsillitis, decompensated form.

In FIG. 1-60 (with the exception of Fig. 2, Fig. 5, Fig. 8), the abscissa axis indicates the wave numbers (unit of measurement is inverse centimeters-1 / cm), the ordinate axis represents relative units of fluorescence intensity (OE).

The determination of the most important spectral bands at different wave numbers is an important task for interpreting the morphometric, metabolic and functional characteristics of tissue belonging to different classes: inflammation, benign tumor, malignant process. These Raman lines characterize not only the type of metabolites secreted in various diagnoses, but also structural changes in the tissue. Using the method of Raman spectroscopy in the future it will become possible to minimally invasively and quickly determine the transition zone of a benign process to a malignant one.

We also conducted a comparative study of the spectral data of squamous cell carcinoma of the pharynx and chronic tonsillitis, compensated form (according to the classification of Soldatov IB, 1975) to identify the most significant diagnostic criteria.

In FIG. Figure 4 shows the model (smoothing, isolating the Raman signal against the general background, normalization, centering) and data after pre-processing for differentiation of squamous cell carcinoma of the pharynx (PAC pharynx - indicated by an arrow) and chronic tonsillitis of compensated form (XT CF; indicated by an arrow). The abscissa axis shows the values of the wave numbers, the ordinate axis shows the relative units of signal intensity.

In FIG. 5 we see the result of building this model. Designations: 0 - chronic tonsillitis, compensated form, 1-squamous cell carcinoma of the pharynx, NMC - non missed classified, that is, incorrectly defined spectra. The separation was carried out with a specificity of 100%, a sensitivity of 100%, an accuracy of 100%.

The designations in English in the upper right corner “Correct” are correctly defined, False positives and False Negatives are false positive and false negative, respectively, values that in this case were 0.

Thus, the differentiation of tissue in squamous cell carcinoma of the pharynx (according to the results of histological examination of the tissue) from that in chronic tonsillitis is carried out using the claimed method with a specificity of 100%, sensitivity of 100%, accuracy of 100%, which is extremely important in clinical practice, in particular during a quick intraoperative diagnosis or screening during the examination.

In FIG. 6 the most informative bands are marked (VIP indicators of the PLS-DA model): 573, 1094, 1162, 1520, 2932 cm ^-1

The abscissa axis shows the values of the wave numbers, the ordinate axis shows the relative units of signal intensity. A separation of these classes of tissue with a specificity of 100%, a sensitivity of 100% and an accuracy of 100% was noted, which speaks in favor of the possibility of using this method (Raman fluorescence spectroscopy) in the differential diagnosis of the inflammatory process in the pharynx with chronic tonsillitis, a compensated form and a tumor process with squamous cell carcinoma of the pharynx. When comparing two groups: tissue of the tonsils with chronic tonsillitis, decompensated form and squamous cell carcinoma of the pharynx, reproducibility of the results was noted, but with less specificity, which speaks in favor of the similarity of the inflammatory (during decompensation) and tumor processes at some stages of pathogenesis. The spectra were also analyzed: squamous cell carcinoma of the pharynx and chronic tonsillitis, decompensated form (according to the classification of Soldatov IB, 1975)

In FIG. Figure 7 shows the model (smoothing, highlighting of the Raman signal against the general background, normalization, centering) and data after pre-processing for the differentiation of squamous cell carcinoma of the pharynx (PAC pharynx - indicated by an arrow) and chronic tonsillitis, decompensated form (XT DF; indicated by an arrow). The abscissa axis shows the values of the wave numbers, the ordinate axis shows the relative units of signal intensity.

And the result of building the model is depicted in FIG. 8. Designations (Fig. 8): 0 - chronic tonsillitis, decompensated form, 1 - squamous cell carcinoma of the pharynx, NMC - pop missed classified, that is, incorrectly defined spectra. The separation was carried out with a specificity of 95.8%, a sensitivity of 90%, an accuracy of 94.1%. In this case, in FIG. 9, the most informative bands for differentiation are noted (VIP indicators of the PLS-DA model): 488, 1149, 1358, 1516, 1575, 1627, 2861, 2931 cm ^-1 . The abscissa axis shows the values of the wave numbers, the ordinate axis shows the relative units of signal intensity.

In FIG. 10 shows the spectrum of neoplasm of the pharynx-pleomorphic adenoma (according to the results of histological examination). The abscissa axis shows the values of wave numbers (the unit of measurement is inverse centimeters - 1 / cm, the ordinate axis shows the relative units of signal intensity - OE. Typical Raman lines are noted on the values of the wave numbers 1150, 1500, 2900 / cm, and the last peak is trapezoidal similar changes are not observed in the case of an inflammatory or malignant process in the pharynx.

In FIG. 11 shows the spectrum of intact pharyngeal tissue surrounding the tumor tissue. A characteristic peak at 2950 / cm, the absence of additional peaks makes it possible to distinguish this curve from the spectra in case of inflammation or tumor process.

In FIG. Figure 12 shows the tissue spectrum of the tonsils with chronic tonsillitis, compensated form (classification by I. Soldatov, 1975). There is a smooth curve, a high fluorescence signal, beyond which the corresponding peaks are not visualized, but they can be easily calculated, as shown in the figures. above using the principal component method, linear discriminant analysis, and the least squares principle.

In this figure, FIG. 13- shows the spectrum of tonsils with chronic tonsillitis, decompensated form (hyperplastic and fibrosing process). 1050, 1150, 1510, 2950 / cm, which distinguishes this tissue from intact and tissue affected by the tumor process.

In FIG. Figure 14 shows the tissue spectrum of the tonsils with the p16-positive type of highly differentiated oropharyngeal carcinoma. Compared with the spectrum of tissue in chronic tonsillitis, a decompensated form, there is no intermediate increase in fluorescence, the fluorescence intensity is much lower, some peaks characteristic of inflammation, in particular, a peak at 1050 / cm is not recorded.

In FIG. 15 shows a spectrum of a low differentiated low keratinized squamous p16-positive type of oropharyngeal carcinoma. The serration of the curve with many peaks at different wavelengths is recorded, the peaks merge, are not distinct, there is no intermediate rise in the fluorescence signal, as is observed in chronic inflammation.

In FIG. 16 shows the spectrum of a fragment of a tumor of the base of the tongue (squamous cell carcinoma). In this case, small negative peaks at 1400, 3250 / cm are recorded, there are no peaks characteristic of this histological diagnosis of a different location (for example, palatine tonsil, Fig. 13), a smooth curve is noted, the fluorescence intensity is much higher compared to the PAC of the pharynx and reaches almost 35,000 rel. units

In FIG. 17 shows a tissue spectrum of a lymph node with metastases of a P-16 positive type of oropharyngeal carcinoma. Characteristic peaks at 1050, 1150, 1350, 1500, 1600, 1700, 1800, 1900, 2950 / cm are noted, the last peak has a trapezoidal shape, similar changes with PAC of a different localization are not noted. Accordingly, PACs of different localization are distinguished by different spectra within the studied class of tissue, which can also be taken into account when conducting differential diagnostics.

In FIG. 18, the spectrum of adenoid tissue is illustrated, the curve has a small jagged shape with a corresponding “plateau” from 2000 to 2950 / cm, which is absent in chronic tonsillitis or pharyngeal tumors.

In FIG. 19 shows a tumor spectrum of a shearly differentiated squamous cell carcinoma. In this case, the corresponding peaks at 1050, 1150, 1510, 2950 / cm are also noted, however, they have a higher intensity compared to those in the case of other localizations, and a low fluorescence intensity is also noted — about 1900 rel. units, additional small peaks at 1300, 1650, 1700, 2700 / cm are recorded, the peak at 1510 / cm is two-tooth, and the peak at 2950 / cm has a trapezoidal dentate shape, which is also a characteristic feature.

In FIG. 20 shows the pharyngeal tumor spectrum for intraepithelial neoplasia. Noted a low fluorescence intensity of about 1250 Rel. units And at 3450 / cm a high trapezoidal tooth is recorded, with a fluorescence intensity of about 2100 rel. units, significantly exceeding the main fluorescence signal, which is a characteristic feature in this case and allows you to register early pretumor changes in the spectral curves.

In FIG. Figure 21 shows the spectrum of neck lipoma, which has a characteristic appearance: high-amplitude peaks at 1000, 1200, 1500, 2950 / cm and low-amplitude peaks at 900, 990, 1300, 1600, 2350, 2600 / cm, a low fluorescence intensity of about 7000 rel. units, against the background of a high amplitude of the main peaks, about 15,000 rel. units However, despite the characteristic appearance of the curves, the methods of principal components, linear discriminant analysis, the principle of least squares and the projection method on latent structures should be used to obtain individual Raman bands in the corresponding range characterizing the specific type of fabric and its composition.

In FIG. 22 shows a spectrum of a tumor of the esophagus, a highly differentiated squamous cell carcinoma. In fig. it can be seen that the curve is smoother in comparison with the PAC spectra of another localization, has a fine-toothed surface with low amplitude peaks, the trapezoidal peak recorded with the PAC of other localizations is shifted to the right and has a hump shape in the range from 3100 to 3500 / cm.

In FIG. 23 illustrates the individual spectral features of the tissue of a middle ear glomus tumor. The curve has a characteristic appearance: first, increasing the fluorescence intensity to 9000 rel. units, then a sharp decline. At 1050, 1150, 1500 / cm, low-amplitude peaks are recorded, also the peak at 2950 / cm is smoothed and loses its trapezoidal shape. Based on the data obtained, it should be noted that spectra should be compared only within the same tissue class: that is, ear tumors are benign with malignant and intact, as well as with tissues affected by the inflammatory process. If we study the spectral features of the pharynx, then, accordingly, it is necessary to compare the above types of the pathological process within the tissues of the pharynx, since the tissue also has its own characteristics, which make an additional contribution to the individual characteristics of the spectral curves.

In FIG. 24 shows a spectral curve of the mucous membrane of the middle ear in chronic suppurative otitis media. The curve is characterized by a sharp increase in fluorescence intensity and, in comparison with the spectrum of the intact mucosa, does not have corresponding peaks at different wavelengths (see figure below).

In FIG. 25 shows the spectral curve of scar tissue with adhesive otitis media. In this case, in comparison with chronic inflammation, an increase and a gradual decrease in the fluorescence intensity are noted at first, there are also no peaks characteristic of the normal mucous membrane of the middle ear, a corresponding peak is noted at 2990 / cm. The curve has a jagged shape and negative peaks.

FIG. Figure 26 illustrates the differences in the spectral curve of fresh granulation tissue in the middle ear from the spectra of scar tissue in the pre-adhesion process in the middle ear. A decrease in fluorescence intensity has a steeper decline; no positive peaks are recorded.

In FIG. 27 shows the spectral features of chronic inflammation in the middle ear with the formation of polyps. First, a gradual increase in fluorescence intensity of over 9000 rel. units, then there is a sharp decline and a “plateau” from 2400 to 3500 / cm with a characteristic peak at 3000 / cm, which has a smooth shape.

FIG. Figure 28 illustrates that, in contrast to all the processes in the middle ear noted above, a curve characterized by high fluorescence intensity (over 70,000 rel. Units) is recorded in the middle ear cholesteatoma. The curve is relatively smooth, has no positive peaks.

In FIG. 29 it was noted that in the case of an intact mucous membrane of the middle ear, peaks at 1050, 1150, 1500, 2950 / cm, as well as a second signal of increasing fluorescence intensity from 3000 to 3500 / cm, are noted. The fluorescence intensity is low and equal to approximately 1350 rel. units As can be seen in the figures, normally from the surface of the mucous membrane of the middle ear, curves with characteristic peaks are recorded, with pathology this is not observed. In the case of pharyngeal tumors, the picture is the opposite: normally these peaks are not recorded, with tumors, they are noted. This fact once again confirms the concept that it is necessary to compare the rate of inflammation and the tumor only within the same tissue class, in order to eliminate the “error” in the morphometric characteristics of the tissue itself.

In FIG. 30 we see that in the sclerotic process in the middle ear, the curve resembles the curve in the case of cholesteatoma, but it is clear that the fluorescence intensity is much lower: about 30,000 rel. units, the maximum fluorescence intensity accounts for 1550 / cm, in contrast to that with a cholesteatoma (1700 / cm).

FIG. 31 shows that acoustic neurinoma has a maximum fluorescence peak at 1510 / cm, the fluorescence intensity is lower than in the case of cholesteatoma and sclerotic process (about 20,000 rel. Units). The decrease in fluorescence, starting from 1510 / cm, has a steeper slope and more rapid; it does not have additional small rises, that is, with a decrease in the fluorescence intensity, the curve acquires a more concave shape.

In FIG. 32 shows that in the case of basal cell carcinoma of the external auditory meatus, a linear increase in fluorescence intensity to approximately 13,000 rel. units; at a value of 1750 / cm, it becomes maximum, then its sharp decline by 2100 / cm-minimum follows, then a “plateau” to 3000 / cm and a gradual decrease in fluorescence intensity are noted. Similar changes were observed when recording spectra from the surface of polyps in chronic inflammation in the middle ear, however, in this case, spectra are recorded from the surface of the structures of the external auditory canal of the ear with a malignant process in it.

FIG. 33 illustrates the spectral features of the tissue of the parotid salivary gland in epithelioid malignant melanoma. The main peaks at 1050, 1150, 1500 / cm are noted, the peak at 2950 / cm has a low amplitude, the maximum fluorescence intensity of 3250 rel. units The maximum fluorescence intensity is 2050 / cm. The curve has a finely serrated shape with many small amplitude peaks.

In FIG. 34 it is illustrated that with squamous cell carcinoma of the parotid salivary gland, high-amplitude peaks at 1010, 1150, 1500 / cm, a low-amplitude peak at 2650 / cm, a trapezoid peak at 2950 / cm are observed. The fluorescence intensity of about 3000 / cm, the peak fluorescence intensity of about 4000 rel. units, the maximum increase in fluorescence intensity at a value of 2050 / cm

According to FIG. 35, unlike the spectrum in PAC, with pleomorphic adenoma of the parotid salivary gland, the peaks become low-amplitude, recorded at 1000, 1100, 1300, 1490, 1510 / cm, followed by a sharp increase in fluorescence intensity to 5100 rel. units, then a decrease in fluorescence intensity, the maximum value of the latter is recorded at 2100 / cm, at a value of 2950 / cm there is a trapezoidal peak, which has a higher amplitude compared to that of a squamous cell carcinoma.

In accordance with FIG. 36, with a parotid salivary gland adenocarcinoma, the following features of the spectral curve are observed: first, a gradual increase in fluorescence intensity is observed, the maximum value of which falls at 2010 / cm, then a sharp decrease in fluorescence intensity to 2850 / cm follows; spiky high-amplitude peaks are not recorded, as with squamous cell carcinoma of the parotid salivary gland, however, a trapezoid peak at 2950 / cm remains.

In FIG. 37 it was noted that with chronic inflammation in the submandibular salivary gland, a gradual increase in fluorescence intensity to 30,000 rel. units, followed by its gradual decrease to 2850 / cm, a characteristic trapezoidal peak is noted, which has a lower amplitude compared to a similar peak with parotid salivary gland adenocarcinoma. The fluorescence intensity in this case is also higher, in addition, negative peaks are recorded.

FIG. 38 illustrates that with chronic hyperplastic rhinosinusitis, a curve is observed without characteristic peaks, the fluorescence intensity of about 4500 rel. units, its maximum value falls on 1990 / cm, the curve has a finely jagged shape.

According to FIG. 39, with polypous rhinosinusitis, the main peaks at 1200, 1510, 2950 / cm are noted, the maximum fluorescence intensity at 4100 rel. units, a second rise in fluorescence intensity is also observed after recording a peak at 2950 / cm. During the formation of polyps, the appearance of corresponding peaks is observed, which are not observed in chronic inflammation without the formation of polyps.

In accordance with FIG. 40, with dysplasia in case of chronic rhinosinusitis, a decrease in fluorescence intensity is noted, peaks shift to the left and are recorded at 1050, 1150, 1500 / cm, the peak after a decrease in fluorescence intensity shifts slightly to the right and is recorded at 3000 / cm. Instead of a gradual decrease in the fluorescence intensity, a “plateau” is observed from 1900 to 2800 / cm, which gradually passes into a peak at 3000 / cm. The second increase in fluorescence intensity has a higher amplitude compared to the last peak, which is not observed in the polypous process and in chronic inflammation without the formation of polyps.

FIG. 41 shows that during the papillomatous process in the maxillary sinus there is a characteristic “bumpy” appearance of the spectral curve, the fluorescence intensity reaches 11,000 rel. units, then declining, the second increase of 10500 rel. units Positive peaks are not recorded.

In FIG. 42, it is seen that in the transition from a chronic inflammatory process to squamous cell carcinoma of the pharynx with metastases to the lymph nodes, the spectral curve shown in Fig. High amplitude peaks with a peak intensity of 11800 rel. units recorded at 1150, 1500 / cm, also recorded a peak at 1000 / cm and a trapezoidal peak at 2950 / cm.

In FIG. 43 shows the spectrum from the surface of metastases to the regional lymph node with primary tumor localization in the parotid salivary gland (adenocarcinoma). At 1100, 1500, 2950 / cm the corresponding peaks are recorded, 1650 rel. units - maximum fluorescence intensity, maximum peak fluorescence intensity reaches 1900 / cm, there is also a second increase in fluorescence intensity in the range of 3050-3400 / cm. The amplitude of the peaks is low compared to peaks in squamous cell carcinoma (with metastasis to the lymph nodes).

In accordance with FIG. 44, during the transition from a chronic inflammatory process to squamous cell carcinoma of the pharynx with metastases to the lymph nodes, the spectral curve shown in Fig. High amplitude peaks with a peak intensity of 11800 rel. units recorded at 1150, 1500 / cm, also recorded a peak at 1000 / cm and a trapezoidal peak at 2950 / cm.

FIG. 45 illustrates that with malignant melanoma (when metastasizing to regional lymph nodes, high-amplitude peaked peaks at 1200, 1500 / cm, as well as low-amplitude peaks at 850, 990, 1000, 1300, 1450, 1650, 1750, 2300, 2700 / cm, are also observed a trapezoidal peak is recorded at 2950 / cm. The fluorescence intensity is low and approaches 6000 rel.units. The peak intensity reaches 14000 rel.

In FIG. Figure 46 shows that with squamous cell carcinoma of the tongue, a smooth spectral curve with a maximum fluorescence intensity of about 24,000 rel. units at 2100 / cm.

FIG. 47 illustrates that, unlike the spectral curve, with squamous cell carcinoma of the tongue, in the absence of a malignant process, characteristic peaks at 1200, 1500, 2950 / cm are observed. The fluorescence intensity in this case is lower and equal to approximately 14000 rel. units The maximum fluorescence intensity falls on the value of the wave number of 1400 / cm.

In accordance with FIG. 48, malignant melanoma of the nasal mucosa is characterized by the spectral curve shown in Fig. and differs in the presence of peaks at 1200, 1500, 2950 / cm. The fluorescence intensity of about 4000 rel. units, the peak fluorescence intensity is slightly higher, the curve has a finely jagged shape.

In FIG. 49 it is seen that a smooth spectral curve is observed with synonasal carcinoma, however, in contrast to the spectra with chronic hyperplastic rhinosinusitis, a fluorescence intensity of about 14,000 rel. units, its maximum value falls on 1990 / cm, the curve has a finely jagged shape. With a decrease in fluorescence intensity, the curve has a more convex shape, a positive peak at 2990 / cm and a negative peak at 3250 / cm.

According to FIG. 50, the spectral curve of the intact laryngeal mucosa is smooth, has no positive peaks, and has a maximum fluorescence intensity of over 20,000 rel. units at 1650 / cm.

In FIG. 51 it was noted that in case of cancer of the vocal folds, a spectral curve is shown, shown in the figure and characterized by a maximum fluorescence intensity of more than 5000 rel. units and with the appearance of a high-amplitude wide trapezoidal tooth with a secondary increase in fluorescence intensity from 3000 to 3500 / cm, other positive peaks are not recorded.

In FIG. 52 it is noted that with dysplasia in the area of the vocal folds, a peak at 2950 / cm is observed, as well as a smoothing of the trapezoidal peak in the range from 3000 to 3500 / cm, clearly visualized with cancer of the vocal folds, that is, the more malignant the process in the area of the vocal folds in this case, the trapezoidal peak takes on a more distinct form.

FIG. 53 illustrates that with squamous cell carcinoma of the larynx (low-grade, non-keratinizing), a characteristic bumpy spectral curve is observed without additional peaks. At 1800 / cm, a secondary increase in fluorescence intensity is noted, however, it is less pronounced compared to the first.

In FIG. 54 it is illustrated that the medium-differentiated non-keratinizing squamous cell carcinoma with metastasis to the regional lymph nodes is characterized by a smooth curve, without additional peaks with a maximum fluorescence intensity of 45,000 rel. units by 1550 / cm.

FIG. 55 demonstrates that with squamous cell carcinoma of the larynx (low-grade, non-keratinizing), a characteristic two-humped spectral curve is observed without additional peaks. At 1800 / cm, a secondary increase in fluorescence intensity is noted, however, it is less pronounced compared to the first. In contrast to the spectral curve obtained from the region of the lymph node with metastases, the bumpy nature of the curve is noted.

In FIG. 56 shows the spectral characteristics of the blood of a patient not suffering from any disease (healthy volunteer). A smooth curve is noted, without additional peaks with a maximum fluorescence intensity of 65,000 rel. units at a value of 2100 / cm.

In accordance with FIG. 57, with chronic tonsillitis, the following features of the spectral curve obtained from a blood sample are noted: the curve acquires additional peaks at the following wave numbers: 500, 1000, 1200, 1250, 1400, 1500, 1600, 1650, 1900 / cm, that is, it has fine-toothed form; the maximum fluorescence intensity is much lower and is about 12000 rel. units, the maximum falls on the value of 1150 / cm.

In FIG. 58 it can be seen that in chronic tonsillitis, a compensated form (classification of I. Soldatov, 1975), when registering blood spectra, a gradual increase in the fluorescence intensity is first observed, as shown in FIG. 58, then a “plateau” from 2000 to 2950 / cm is noted, then a gradual decrease in the fluorescence intensity, additional peaks are not visualized due to the strong fluorescence signal.

FIG. 59 demonstrates that with chronic tonsillitis, a decompensated form, as can be seen in the figure, there is a sharper decrease in fluorescence intensity without the formation of a “plateau”, however, changes between these forms of chronic tonsillitis are visible only when the main components of the methods are used, linear discriminant analysis, the principle least squares method of projection onto latent structures, since the fluorescence signal for chronic tonsillitis is quite high, additional peaks are not visible against the background of a powerful fluorescence signal.

FIG. 60 illustrates that with sensorineural hearing loss, many peaks are recorded throughout, the spectral curve, as can be seen in the figure, has a “frequency-like shape”, the maximum fluorescence intensity falls at 2000 / cm and is about 1750 rel. units, the maximum peak fluorescence intensity is 2000 rel. units, after a decrease in the fluorescence intensity, 2 additional peaks are recorded, in contrast to the normal spectra for chronic tonsillitis.

In FIG. 61 a shows spectral curves obtained from the surface of the palatine tonsils of a patient with chronic tonsillitis, compensated form, before treatment-washing of the gaps of the palatine tonsils with a solution of antiseptic-chlorhexidine 0.02%.

что говорит как о регистрации патогенной флоры, так и о повышении концентрации условно патогенной флоры, что может приводить к заболеванию.In FIG. 61 a shows that with inflammatory changes in the tonsils with XT, additional peaks appear on the graph at 6370, 7050

which indicates both the registration of pathogenic flora and the increase in the concentration of conditionally pathogenic flora, which can lead to disease.

Indicators of spectral characteristics of the tissue of the tonsils with XT are graphically depicted in FIG. 61 a; digitally presented below:

IA 3-1.05; IF 3-0.381; IA 2-0.893; IF 2-1,29; IA 1-1,46; IF 1-0.188; IA intact .- 1.84; IF intact. - 5.63 E + 06, where IF = NPF-normalized fluorescence index, And A - aerobicity index at the studied point (points 1-4, including intact).

которого в норме никогда не наблюдается. Данный пик свидетельствует о наличии процесса хронической гипоксии в ткани и выделении протопорфирина IX; в норме подобных изменений не регистрируется (рис. 61 г- графики спектральных характеристик ткани небных миндалин здоровых добровольцев. Интенсивность флюоресценции интактных тканей миндалин, измеренная в разных точках практически одинакова и крайне низка по интенсивности, по сравнению с амплитудно- спектральными показателями области сравнения (кожные покровы)).FIG. 61a illustrates a graph of the spectral characteristics of the tissue of the tonsils in chronic tonsillitis before treatment. A characteristic peak at 6370

which normally is never observed. This peak indicates the presence of a process of chronic hypoxia in the tissue and the release of protoporphyrin IX; normal, such changes are not recorded (Fig. 61d graphs of the spectral characteristics of the tissue of the tonsils of healthy volunteers. The fluorescence intensity of the intact tissues of the tonsils, measured at different points, is almost the same and extremely low in intensity, compared with the amplitude and spectral parameters of the comparison area (skin integument)).

The values of the aerobic indices and normalized fluorescence indices at the studied points are calculated as follows:

The spectrum will be specified as a function of intensity versus wavelength: F = F (x)

These two indices are integral fluorescence indices (over the entire spectral range) for the Intact and Fundus functions.

Intact is an intact point, in this case, the skin of the inner surface of the forearm, Fundus measurements at point 1 are at the upper pole of the palatine tonsil, Middle measurements at point 2 are on the mucous membrane of the palatine tonsil near its lower pole, Internal measurements are at point 3 - in the lacuna of the palatine tonsil. The corresponding formulas indicate the designations described. All functions are embedded in the software of the hardware-software systems used in the claimed method.

These three indices are normalized (by the integrated fluorescence index at the intact point) fluorescence indices for the functions Fundus, Middle, Internal.

The described indices are indicated in the description as “normalized fluorescence index” (hereinafter referred to as “NPF” or “IF”).

These are four aerobic indexes for the functions Intact, Fundus, Middle, Internal. These indices are indicated in figures 61 a-d as the “aerobic index” (hereinafter referred to as “IA”). You can also calculate the following indices for each point in the dynamics before and after the treatment:

This is the normalized index of reflected laser radiation (center of the laser line 637 nm) - it is the ratio of the integrated intensity of the reflected laser light (in the range of 630 - 642 nm) for the intact point (in the numerator) and the studied point (in the denominator). It shows how much more intact tissue reflects compared to point 1. Using this index, you can evaluate the state of the tissue of the palatine tonsil at point 1.

These are 2 microbial contamination indexes for intact tissue and palatine tonsil tissue. They show the ratio of the integrated fluorescence intensity of protoporphyrin IX (which is an indicator of bacterial contamination) to the integrated fluorescence intensity of tissues in the spectral range [665-675 nm], in which this line is not observed.

These are two oxygenation indices. The higher they are, the more oxygenation should be (that is, the more oxidized forms of hemoglobin should be contained in the tissues).

Such integration ranges are selected from the following considerations: at the point of 660 nm, the greatest difference is observed in the absorption spectra of the oxidized and ordinary forms of hemoglobin, and at the point of 820 nm their absorption spectra intersect. Thus, the more oxidized hemoglobin is in the tissue (it absorbs weaker near the point of 660 nm), the greater the oxygenation index.

All of the above indices allow us to judge the physiological, morphometric, metabolic changes in the tissue normal and with inflammation or tumor (benign and malignant process). Assessing these indices using special software, it becomes possible to immediately diagnose the pathological process in the tissues of the ENT organs with tumors and the inflammatory process, monitor treatment, evaluate its effectiveness, and differentiate tissues in the early stages of the disease.

FIG. 61 b illustrates a significant change in all of the above indices, as well as the nature of the curve after short-term exposure (about several seconds) of antiseptic-chlorhexidine 0.02% when washing the lacunae of the tonsils, that is, the fluorescence intensity increases, which indicates the death of microorganisms and the release of protoporphyrins in mass quantity. In FIG. 61 b shows the spectral characteristics of the tissue of the tonsils in graphic form, in digital form they are presented below:

IA 3-1.88; IF3-0.966; IA2-2.35; IF 2 - 0.549; IA 1 - 1.9; IF1-1.23; IA intact.-1.89; IF intact.-7.6 E + 06, where the IA is the aerobic index at the studied point, IF is the fluorescence intensity normalized to the intact point, or the normalized fluorescence index (hereinafter NPF).

In FIG. It is seen in Fig. 61c that all spectral characteristics after a full course of conservative treatment, washing the lacunae of the tonsils with a chlorhexidine solution of 0.02%, return to normal values, which indicates the effectiveness of conservative therapy: namely, the structure of the tissue, oxygenation, microcirculation are normalized, the aerobicity index increased, spectral data in favor of pathogenic flora are not visualized, the maximum fluorescence peak lies in the lower third of the curve recorded from the surface of the intact point, the fluorescence intensity becomes lower, the structure of the spectra is restored, additional peaks are not recorded, the fluorescence intensity measured at all points of the palatine tonsils, located in the lower third of the area under the curve characterizing the spectral features of the intact point - the skin of the inner surface of the forearm. Aerobic indices also increase, normalized fluorescence indicators decrease, which indicates an increase in tissue oxygenation and a decrease in microbial contamination and the degree of the inflammatory process during therapy, and also indicates a clinical improvement and morphological restoration of tonsil tissue.

In FIG. 61c shows the spectral characteristics of the tissue of the tonsils in graphic form, in digital form they are presented below:

IA 3-1.74; IF 3-0,282; IA 2-1.74; IF 2-0,278; IA 1-1.8; IF 1-0.187; IA intact .- 1.85; IF intact. - 9.05 E + 06

On the graph (Fig. 61 c) we see that the structure of the tissue, oxygenation, microcirculation are normal, the aerobicity index has increased, spectral data in favor of the pathogenic flora are not visualized, the maximum fluorescence peak lies in the lower third of the curve recorded from the surface of the intact point, which indicates clinical improvement and morphological restoration of the tissue of the tonsils.

FIG. 61 g illustrates the spectral characteristics of the tissue of the tonsils of healthy volunteers, which differ significantly from those for chronic tonsillitis: the fluorescence intensity is low and is in the lower third of the area under the curve describing the fluorescence intensity at the intact point on the skin of the inner surface of the forearm, no additional peaks are recorded, structured spectra are normal: at each point, the spectra coincide with each other, which indicates the absence of a pathological process in the tissue.

In FIG. 61 g shows the spectral characteristics of the tissue of the tonsils in graphic form, in digital form they are presented below:

IA 3-1,12; IF 3-0.137; IA 2-1.26; IF 2-0,159; IA 1-1.61; IF 1-0.0942; IA intact .- 1.97; IF intact .- 4.85 E + 06

площади под кривой интактной точки, структурированность спектров практически восстановилась, что говорит об эффективности проведенного лечения. Т.е. все исследуемые показатели качественно и количественно приближаются к показателям интактной ткани небных миндалин, не вовлеченной в воспалительный процесс.After determining the sensitivity and specificity of the method, individual spectral characteristics were obtained in digital and graphical form of patients with chronic tonsillitis before and after a course of standard conservative treatment (washing of tonsil lacunae with chlorhexidine solution 0.02% No. 8, daily treatment of tonsils with Lugol solution, physiotherapy) . The aerobic indices, normalized fluorescence indices for this group of patients before and after treatment were significantly different - the fluorescence intensity became 2 times less and almost began to approach the indices of the intact tissue of the tonsils and, compared with the initial values,

the area under the curve of the intact point, the structure of the spectra has practically recovered, which indicates the effectiveness of the treatment. Those. all the studied parameters qualitatively and quantitatively approach the indicators of the intact tissue of the tonsils, not involved in the inflammatory process.

Below are the aerobic indices (IA) and normalized fluorescence (NPF) at the studied points 2, 3, 4, as well as at the intact point of a patient with chronic tonsillitis before and after the standard course of conservative treatment. 1 - the same indicators in the intact region.

Indicators before treatment:

IA 3-1.05; NPF3-0.38; IA2-0.9; NPF2-1.29; IA1-1.46; NPF1-0.19; IA intact.-1.84; IF intact. -5.63 E + 06

Indicators after treatment:

IA 3-1.5; NPF3-0.21; IA2-1.55; NPF2-0.14; IA1-1.79; NPF1-0.08; IA intact. - 2.24; IF intact .- 6.91 E + 06.

After the course of treatment, there is an increase in the increase in the values of aerobic indices at the studied points, which indicates that microcirculation has recovered in the tissue and the level of oxygenation has increased. You can also observe a decrease in NPF (IF) at the studied points, which speaks in favor of reducing the inflammatory process (microbial contamination per gram of tissue) and restoration of the previous tissue structure.

Thus, the use of Raman fluorescence spectroscopy in combination with the main component method, linear discriminant analysis and the principle of least squares when conducting minimally invasive accelerated differential diagnosis of inflammatory and tumor processes at various stages of pathogenesis and for various diagnoses is a promising area of clinical otorhinolaryngology and can serve further not only as an express intraoperative diagnosis, but also as a screening determination of the stage of the pathological process in the body and monitoring and rehabilitation of the patient. The method allows one to determine with high accuracy, sensitivity and specificity that a tissue belongs to an intact, inflamed, or tumor tissue if there is an appropriate spectral database on the qualitative and quantitative characteristics of the metabolites of the samples, as well as their morphometric parameters. Thus, the concept was confirmed that Raman fluorescence spectroscopy objectively reflects the individual characteristics of the tissue in normal and pathological conditions, allows you to register changes in it that occur at the molecular level in dynamics with high sensitivity and specificity. Thus, it can also be concluded that using the method of Raman fluorescence spectroscopy, we can identify the type of tissue both in vitro and in vivo and accelerate the diagnostic process to prescribe appropriate treatment in the early stages. In addition, after the development of a special algorithm, this method can be used as a screening method for recording early tissue changes and prophylactic measures. From the presented results it follows that the choice of the method and its software implementation in the EnSpectr R532 and EnSpectr L405 hardware and software systems for pathology of ENT organs and other organs of the head and neck, based on the data presented, is objectively justified by its high sensitivity and specificity. The analysis of spectral and digital tissue data in normal and pathological conditions is an important aspect in diagnosis and allows monitoring of the pathological process both at the local and systemic levels. Optical spectral methods are promising for in vivo diagnosis of cancer of various localization. Using the method of Raman-fluorescence spectroscopy in the future it will become possible non-invasive way to determine the border of the tumor, the type of tissue, the area of the resection during the oncological process. Currently, the database of the Raman scattering of light and fluorescence of tissues of the ENT organs and other organs of the head and neck during various inflammatory and oncological processes, including the dynamics during treatment, is ongoing. After refinement of the technique on representative clinical material using EnSpectr hardware and software systems with probing radiation in a wide spectral range (from 405 nm to 1064 nm) and the creation of a database of typical spectra of various ENT diseases - organs and other organs of the head and neck, including inflammatory and tumor diseases, this diagnostic method can be used in wide clinical practice in the interests of the patient and the doctor.

Claims (1)

A method for differential diagnosis of chronic tonsillitis and squamous cell carcinoma of the pharynx using Raman fluorescence spectroscopy, which consists in obtaining spectral characteristics of the tissue of the tonsils and pharynx of the patient, and if the patient has the main peaks on the spectra obtained from the surface of the tissue of the tonsils, wave numbers: 1050, 1150, 1510, 2950 cm ^-1 conclude that the patient has chronic tonsillitis; and if the spectra obtained from the surface of the pharynx of the patient have additional low-intensity peaks at the wave numbers of 1300, 1650, 1700, 2700 cm ^-1 , a two-tooth peak at 1510 cm ^-1 and a trapezoid peak at 2950 cm ^-1, they conclude that a patient with squamous cell carcinoma, after processing the spectra using the main component methods, projection onto latent structures in conjunction with a linear discriminant analysis, Savitsky-Golei and correction of the reference line using asymmetric least squares to differentiate the compensated chronic tonsillitis and pharyngeal squamous cell carcinoma with the most informative stripes scatterings are 573, 1094, 1162, 1520, 2932 cm ^-1 , and with differentiation of chronic tonsillitis of the decompensated form and squamous cell carcinoma of the pharynx, the most informative bands of Raman scattering are 488, 1149, 1358, 1516, 1575, 1627, 2861, 2931 cm ^-1 .

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