RU2497558C1 - Intraoperative combined spectroscopic diagnostic technique for cerebral and spinal tumours - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to neurooncology, and may be used for the intraoperative diagnosis of cerebral and spinal tumour borders and determination of the tumour resection quality. What is presented is an intraoperative combined spectroscopic diagnostic technique for cerebral and spinal tumours which involve the preoperative preparation including the oral administration of hydrochloride-5-aminolevulinic acid (several hours before the tumour resection). Actually the intraoperative spectroscopic analysis of the tumour marker content in the analysed tissues (5-aminolevelinic acid-induced protoporphyrin IX), as well as its desintegration products, oxygenated and reduced hemoglobin; the tissue scattering properties are varied using broadband and laser geode light source and a fibre-optic probe. The broadband light source is used within the range of 500÷600 nm, while the exiting fluorescent laser/diode light source - at wave length 620÷640 nm. The diffuse-reflectance spectra of the broadband and laser/diode light, as well as fluorescence are recorded at the same time. After the spectroscopic characteristics have been automatically classified, the operative radicality is assessed.EFFECT: presented method enables higher reliability of the intraoperative measurements in neuronavigation, reducing time needed for implementing them, providing deeper probing and simplifying the spectra recording in vivo.4 cl, 9 dwg, 3 ex

Description

The present invention relates to medicine, namely to neurooncology, and can be used for intraoperative diagnosis of the boundaries of brain and spinal tumors and determine the quality of tumor resection.

Currently, there is a significant increase in the frequency of oncological diseases of the central nervous system. Of particular difficulty is the determination of the boundaries of intracerebral and intramedullary tumors. The urgency of the problem of determining the boundaries of astrocytic tumors is due to the peculiarities of their growth along myelinated nerve fibers and blood vessels, leading to the infiltration of healthy white matter by tumor cells, which affects the high frequency of postoperative relapses. The complexity of surgery for intramedullary tumors of the spinal cord is that the tumor does not always have a clear boundary and there is a high risk of trauma due to the smaller size of the operated area compared to the brain. Reliable information regarding the volume of the resected tumor can be obtained by intraoperative imaging. The solution to this problem is implemented mainly in three directions: the use of intraoperative computed tomography, magnetic resonance imaging and ultrasound scanning, and various combinations of these methods [bitraoperative Imaging in Neurosurgery: MRI, CT, U / S, Springer-Verlag, Vienna, 2003 ; Radiology Research and Practice, Volume 2012 (2012), Article ID 197364, 9 pages; Acta Neurochirurgica (Wien), 2007; 149 (4): 365-78].

Unfortunately, all these methods of intraoperative diagnostics do not allow real-time examination of tissues in the surgical wound and / or do not provide simultaneous analysis of both structural and metabolic changes.

Meanwhile, clinical studies showed a significant correlation between the level of accumulation of 5-aminolevulinic acid (5-ALA) - induced protoporphyrin DC and cell division rate, which allows the use of this substance as a tumor marker. However, due to the fact that in 20-30% of cases there is only a slight accumulation of the drug in the tumor cells, additional criteria are necessary for the intraoperative diagnosis of the studied tissues. As you know, tumor tissue and healthy brain tissue are characterized by differences in both the structure of the tissue and the degree of blood supply and oxygenation of hemoglobin. Thus, the simultaneous determination of these parameters makes it possible to increase the sensitivity of the method based on determining the concentration of the tumor marker (5 ALA-induced protoporphyrin IX).

Therefore, a very urgent problem is the development of a method for rapid intraoperative tissue analysis by the set of characteristic features during the removal of brain and spinal cord tumors. Fluorescence spectroscopy and diffuse reflectance spectroscopy provide accurate, fast and non-invasive methods for studying metabolic and structural changes in tissues.

The closest analogue to the proposed invention (prototype) is the combined spectroscopy method described in the article [Journal of Biomedical Optics 16 (11), 116007-14 (November 2011]. This article proposes a combined spectroscopy method that combines the analysis of diffuse radiation scattering spectra pulsed broadband source in the range from 450 to 720 nm, obtained for two distances between the illuminating and receiving fibers, and the fluorescence spectrum of 5-ALA induced protoporphyrin IX and its photodecay products in the range from 6 00 to 720 nm, caused by radiation with a wavelength of 405 nm. A solution of 5-ALA hydrochloride is injected into the patient’s body several hours before the operation. Then, during the tumor resection operation, sequential measurements of the background radiation spectrum, two spectra: diffuse scattering and fluorescence spectrum are performed The total time required to record all spectral dependences at each point is about three seconds. To obtain spectral characteristics, an optical fiber probe is used that contains three illuminating fibers and two receiving fibers. The obtained spectroscopic data are processed using the diffusion approximation of the theory of radiation propagation in a turbid medium, the optical characteristics of which correspond to the optical characteristics of the tissue under study. The data thus obtained make it possible to quantitatively evaluate the following parameters: the concentration of 5-ALA-induced protoporphyrin IX and its photodecay products, the concentration of hemoglobin in oxygenated and reduced form, and the scattering properties of the tissue. To implement the diagnostic algorithm, the support vector method is used, which is a “with a teacher” classification method in the space of five characteristics obtained as a result of mathematical processing of spectral dependencies. According to the results of automatic analysis, a conclusion is made about the radical nature of the resection.

The disadvantages of this method are the sequential recording of the spectra of background illumination, diffuse scattering and fluorescence, which requires about 3 seconds to measure all the parameters necessary for tissue classification, and does not allow real-time measurements and reliably state that the spectra were obtained from one points in the same conditions. Another disadvantage of the method we have chosen as a prototype is the use of short-wavelength radiation (wavelength 405 nm) to excite fluorescence, for which biological tissue is a medium with low transparency, which leads to fluorescence sensing of a tissue volume not exceeding a few micrometers and requires prior rinsing tissue from blood before performing an optical measurement. These shortcomings complicate the procedure of tissue analysis by optical biopsy.

The objective of the invention is to develop a method that improves the reliability of measurements, reduces the time of their implementation, increases the depth of sounding and simplifies the process of recording spectra in vivo.

The problem is solved by the method of intraoperative spectroscopic diagnosis of tumors of the brain and spinal cord, including preoperative administration of a 5-ALA hydrochloride solution to the patient and intraoperative spectroscopic analysis of the content of the tumor marker (5-ALA induced protoporphyrin IX), its photodecay products, oxygenated and reduced hemoglobin as well as changes in scattering properties using sources of broadband and laser radiation Fiber-optic probe, with subsequent automatic classification spectroscopic characteristics serving to conduct evaluation radical resection. The proposed method is carried out using a broadband radiation source in the wavelength range from 500 nm to 600 nm and a laser / LED radiation source with a wavelength in the range of 620-640 nm, the diffuse reflection and fluorescence spectra are recorded simultaneously, after which the tumor marker accumulation level 5-ALA-induced protoporphyrin DC determines the degree of belonging of the measured tissue sample to the previously defined classes, the results of which evaluate the radicality of tumor resection.

The problem is also solved by the fact that for the automatic processing of spectroscopic characteristics using data classification method. To the nearest neighbors in the space of the above characteristics.

The problem is solved in that, according to the results of automatic classification, an audio signal is generated that is modulated in volume and frequency depending on the degree of malignancy of the tissue under study.

The problem can be solved by the fact that according to the results of automatic classification, maps of the distribution of biomarkers are formed.

The proposed method is carried out using a cross-system of lighting and film filters, which implements the separation of the spectral range in the area of registration of the spectrum of diffuse reflection of radiation of a broadband source (in the range from 500 nm to 600 nm), the intensity of laser radiation scattering (in the range of 620-640 nm, which corresponds to the region of the long-wavelength peak of the Q-absorption band of protoporphyrin DC, in which the biological tissue is optically transparent) and the fluorescence spectrum (in the range of 640-850 nm). A device for conducting intraoperative combined spectroscopic diagnosis of brain and spinal cord tumors corresponds to a variant of the device described in RF patent No. 2169590 (IPC A61N 5/06, 2000).

The technical result provided by the given set of features is the ability to quickly and reliably obtain spectroscopic characteristics from the same tissue volume in the operating area for all these characteristics and to simplify the process by eliminating the need for preliminary washing of the tissue from the blood before diagnosis.

In accordance with the invention, as part of the preoperative preparation, a solution of 5-aminolevulinic acid hydrochloride is orally administered to the patient's body several hours before the removal of the tumor.

The proposed method consists in conducting intraoperative spectroscopic analysis by bringing the distal end of the optical fiber probe and the tissue site under investigation into soft contact. As a result of the measurement, the spectrometer input receives broadband radiation diffusely reflected by the tissue in the wavelength range from 500 to 600 nm, diffusely reflected by the fabric and attenuated by three orders of magnitude with a notch filter located at the spectrometer input, laser radiation, as well as laser-induced fluorescence radiation. The recorded spectral dependences are mathematically processed in special software for calculating physiological characteristics (biomarkers) that have an impact on spectral dependences, such as the concentration of protoporphyrin IX, hemoglobin in oxygenated and reduced form, and changes in the scattering properties of the studied tissues.

The classification of the spectroscopic characteristics recorded in this way can be carried out by automatically comparing the spectroscopic and / or physiological characteristics with a database of the corresponding spectroscopic and / or physiological characteristics of the tissues, the state of which was verified by histological analysis, by automatic classification methods, based on which a conclusion is drawn on the degree of relationship the studied tissue to a particular group within the framework adopted classification of tissues.

To inform the surgeon about the state of the tissues according to the results of automatic classification of spectroscopic data, it is possible to use both visual and sound indications. To emphasize the surgeon's attention to changing spectral characteristics during the operation according to the results of their automatic processing, it is possible to use sound indications with modulation in volume and frequency depending on the degree of tissue malignancy. It is also possible to form on the monitor screen two-dimensional maps of the distribution of spectroscopic and / or physiological characteristics of tissues for clarity of presentation of the joint distribution of the studied biomarkers.

The invention is illustrated by the following figures.

Figure 1 shows for patient A under point a - preoperative MRI; b - a photograph of the cerebral cortex above a tumor in white light; c - photograph of the distribution of fluorescence of the cerebral cortex over a tumor in the Blue 400 mode of an Opmi Pentera intraoperative microscope.

Figure 2 shows for patient A (a) the image of the operating bed in Blue 400 mode (time 18.16.07) before spectroscopic examination, (b) at the time of the combined spectroscopy (time 18.16.19-18.16.21), and in Blue 400 mode (c) after a short aspiration in the position of conducting combined spectroscopic analysis. Spectra 208-209 (g) recorded at the time shown in FIG. 2b are shown, as well as the corresponding oxygenation degree, light scattering intensity, and fluorescence index, a posteriori normalized to the maximum values obtained during this study (e). The following values of these parameters were obtained intraoperatively: spectrum 208 — oxygenation 53%, amplitude of backscattered laser radiation 800, fluorescence index 8.7; spectrum 209 — oxygenation 26%, amplitude of backscattered laser radiation 210, fluorescence index 7.5.

Figure 3 presents maps of the joint distribution of the studied spectroscopic values for normal, tumor tissue, as well as in the surgical bed after removal of the tumor in patient A. The following notation of points in three-dimensional space belonging to different classes is used: ▲ - edge of the tumor (perifocal zone), ■ - tumor, ✕ - bed after tumor removal, ✖ - intact nerve tissue. All values of the characteristics are normalized to the maximum values obtained as a result of the study. a - the abscissa axis shows the intensity values of the backscattered laser radiation, the ordinate axis represents the fluorescence index; b - the abscissa axis shows the values of the degree of saturation of hemoglobin with oxygen, the ordinate axis represents the fluorescence index; c - the abscissa axis shows the values of the degree of saturation of hemoglobin with oxygen; the op axis — the intensity of the backscattered laser radiation.

Figure 4 shows the results of a morphological study of tissue samples taken during the operation of patient A, which made it possible to draw a final conclusion - oligoastrocytoma: a - astrocomponent of the tumor, b - oligocomponent of the tumor.

5 there are intraoperative still images of the operation to remove the tumor of patient B in white light (a) and in the Blue 400 Carl Zeiss Opmi Pentero mode (b), showing the presence of blood in the surgical bed and weak inhomogeneous fluorescence (time 11.04.40- 45), which does not allow an unambiguous conclusion about the degree of accumulation of the tumor marker in the tissue.

Figure 6 shows the results of a combined spectroscopic analysis of the tissues of patient B in the bed area shown in Figure 4: intraoperative spectrum No. 36 (a), as well as the corresponding values of the fluorescence index (b) and oxygenation (c). Fluorescence index 26, the amplitude of the backscattered laser radiation 350, oxygenation 56%.

Figure 7 shows the results of a morphological study of a tumor of patient B., with the final conclusion - anaplastic astropitoma: a - zone of tumor infiltration; b - tumor infiltration zone; in - a tumor; g - tumor and infiltration zone.

Figure 8 shows the intraoperative spectra obtained by the combined spectroscopic analysis of patient B. The spectrum marked in green corresponds to the normal brain, the spectrum indicated in violet is obtained from tumor tissue, the spectrum marked in orange is recorded from the edge of the tumor.

Figure 9 shows the results of a morphological study of the tumor in patient B - on the preparations revealed intramedullary ependymoma.

The following examples illustrate the invention.

Example 1

Patient A, 23 years old, (the final diagnosis is oligoastropitoma of the left frontal lobe (Grade II according to the classification of the World Health Organization) was operated on 04/17/2012 (for the first time). 2 hours before surgery, the patient was orally administered a solution of 5-aminolevulinic acid hydrochloride (Alasens) in an amount of 20 mg / kg of patient weight. After trephination and dissection of the dura mater, the cortex of the left frontal region was visualized, on the surface of which a tumor node emerged, brightly fluorescent when illuminated in BL 400 mode using a Carl Zeiss Opmi Pentero operating microscope with a fluorescent module (see intraoperative photographs in Fig. 1). In figure 1. preoperative MRI (a) is presented in a patient with an intracerebral tumor - a large tumor of the left frontal lobe is visualized (Grade II); intraoperative photography in white light (b) and in the Blue 400 Carl Zeiss Opmi Pentero mode (c) demonstrate bright visible fluorescence at the first stage of the operation. Tumor removal started. During the operation, the visual fluorescence of the surgical wound was periodically evaluated and the condition markers of the studied tissues were monitored using the proposed method of combined spectroscopy.

For this, a broadband radiation source in the range of 500--600 nm was used to analyze the hemoglobin concentration in oxygenated and reduced form and a laser radiation source with a wavelength in the region of 632.8 nm was used to analyze the light scattering and fluorescence excitation of 5-ALA induced protoporphyrin IX and its photodecay products. When bringing the optical fiber probe into soft contact with the tissue, the diffuse reflection and fluorescence spectra were simultaneously recorded.

The areas of intensive accumulation of 5-ALA were completely removed; a gradual decrease in the intensity of visible fluorescence during the operation was noted as the surgical wound deepened. In the course of further surgery, the tumor did not visually differ from the brain substance. At this stage, the most sensitive means of assessing the state of tissue in the surgical wound was the combined spectroscopy method. The series of intraoperative frames and spectral dependences shown in Fig. 2 shows spectroscopic detection of a fluorescent focus (Fig. 2, b) that cannot be visualized using the Opmi Pentera Blue 400 video fluorescence mode (Fig. 2, a). In the study of combined spectroscopy, spectrum 208 is revealed — a fluorescence index of 8.7, an amplitude of backscattered laser radiation of 800, oxygenation of 53%; spectrum 209 - fluorescence index 7.5, the amplitude of the backscattered laser radiation 210, oxygenation 26% (Fig.2 g, d).

When conducting combined spectroscopic analysis in real time, the studied tissue was classified based on the automatic analysis of its spectroscopic characteristics by the K method of nearest neighbors. As an indication method, two-dimensional maps of the distribution of the magnitude of the studied physiological markers were used (Fig. 3).

During the operation, material was taken for morphological analysis of tissue samples in comparison with the data of spectral analysis (figure 4). The differences in the scattering level shown in Fig. 2c, corresponding to the equally effective accumulation of protoporphyrin IX (Fig. 2d) can be attributed to the morphological differences in the areas of the tumor containing both astro- and oligocomponents (Figs. 4a, b). After obtaining indicators of combined spectroscopy from the walls of the bed of the removed tumor, identical to the normal brain, the main stage of the operation ended.

Example 2

Patient B, 23 years old, (the final diagnosis was anaplastic astrocytoma of the right temporal lobe (Grade III according to the classification of the World Health Organization)) was operated on for the first time on June 7, 2011. 2 hours before surgery, the patient was orally administered a solution of 5-aminolevulinic acid hydrochloride (Alasens) in the amount of 20 mg / kg of patient weight. During the operation, in solid light and in the Opmi Pentera Blue 400 mode, a solid part of the tumor was visualized in the form of areas of weak fluorescence, periodic filling of the surgical bed with blood made video navigation difficult (Fig. 5a, b). When conducting a tumor resection, a combined spectral analysis was constantly performed in the surgical bed (Figs. 5, 6a, b, c) with the control of the base signal level from the temporal lobe cortex. A broadband radiation source in the range of 500-600 nm was used to record diffuse reflectance spectra and determine on its basis the hemoglobin concentration in oxygenated and reduced form and a laser radiation source with a wavelength of 633 ± 5 nm to analyze the parameters of light scattering and 5- fluorescence excitation ALA-induced protoporphyrin IX and its decay products. When bringing the optical fiber probe into soft contact with the tissue, the spectra of diffuse reflection and fluorescence were simultaneously recorded. In real time there was a classification of the studied tissue based on an automatic analysis of its spectroscopic characteristics. Based on the classification results, an audio signal was generated, modulated in volume and frequency depending on the degree of malignancy of the test tissue, the recorded spectra and distribution maps of measurements in the coordinates of the recorded characteristics were displayed.

When conducting combined spectroscopy, the maximum fluorescence index was 26 Rel. units, the amplitude of the backscattered laser radiation at the same point was one third of the value of this parameter in normal tissue, oxygenation at the same point was 56% (Fig.6). From the points of the combined spectroscopic analysis were taken biopsy samples of the investigated tissue (figa, b, c, d). A comparison of the intraoperative spectral dependences and the morphological conclusion data served to replenish the database of spectroscopic / physiological characteristics to increase the accuracy of determining the belonging of the studied tissues to certain classes.

After obtaining indicators of combined spectroscopy, from the tissue of the bed of the removed tumor, identical to the normal brain, the main stage of the operation ended.

Example 3

Patient B, 31 years old, (the final diagnosis was an intramedullary tumor (ependymoma) at the level of Th2-Th3 vertebrae) was operated on for the first time on April 3, 2012. 2 hours before surgery, the patient was orally administered a solution of 5-aminolevulinic acid hydrochloride (Alasens) in an amount of 20 mg / kg patient weight. After the approach to the tumor was carried out, the apparent fluorescence was evaluated using an endoscope with a fluorescence module - areas of a bright homogeneous type of visible fluorescence were noted. During resection of the tumor residues, due to the fact that the sensitivity of the endoscopic video fluorescence module was insufficient, a combined spectroscopic analysis was performed in the surgical bed. To conduct combined spectroscopy, two radiation sources were used - a halogen lamp with a filter that transmits radiation in the range of 500-600 nm and a helium-neon laser (632.8 nm). When bringing the optical fiber probe into soft contact with the tissue, the spectra of diffuse reflection and fluorescence were simultaneously recorded. In real time there was a classification of the studied tissue based on an automatic analysis of its spectroscopic characteristics. Based on the classification results, an audio signal was formed, modulated in volume and frequency depending on the degree of malignancy of the test tissue, and recorded spectra were displayed on the screen. Fig. 8 (a, b, c) shows three spectra obtained intraoperatively during removal of a tumor in patient B. The spectrum marked in orange was recorded from the edge of the tumor, as evidenced by the intermediate between the normal and the tumor values of the fluorescence index and the intensity of the backscattered laser radiation. Mild deoxygenation of normal tissue may be due to traction of the tissue of the spinal cord during surgery. The results of spectroscopic analysis were confirmed by morphological examination of tissue samples taken from the same points at which the spectral analysis was carried out (figa, b). The main stage of the operation was completed after obtaining the spectral characteristics of the tumor bed corresponding to normal tissue.

The inventive method is based on the actual material obtained by us in the analysis of 99 patients with brain gliomas Grade I-IV (WHO), 16 patients with metastases of various tumors in the brain, as well as 21 patients with brain meningiomas of various degrees of malignancy.

Thus, the proposed method solves the problem of increasing the reliability of intraoperative measurements during neuronavigation, reducing the time of their implementation, increasing the depth of sounding and simplifying the process of recording spectra in vivo.

Claims (4)

1. A method for conducting combined intraoperative spectroscopic diagnosis of brain and spinal tumors, including preoperative administration of 5-aminolevulinic acid hydrochloride (5-ALA) oral solution to a patient and intraoperative spectroscopic analysis of the content of a tumor marker (5-ALA-induced protoporphyrin IX) in the studied tissues, products of its photodecay, oxygenated and reduced hemoglobin, as well as changes in the scattering properties of tissues using a source of of broadband and laser radiation and a fiber optic probe, followed by automatic classification of spectroscopic characteristics, which serves to assess the radical nature of the resection, characterized in that they use a broadband radiation source in the wavelength range from 500 nm to 600 nm and a source of laser fluorescence excitation with wavelength in the range of 620-640 nm, and the registration of diffuse reflectance spectra of broadband and laser radiation, as well as fluorescence, is performed simultaneously permanently. 2. The method according to claim 1, characterized in that for the automatic processing of spectroscopic characteristics, an algorithm for automatically classifying the nearest neighbors by the K method with determining the weight of a given point to each of the previously defined fabric classes is used. 3. The method according to claim 1, characterized in that according to the results of the automatic classification, an audio signal is generated that is modulated in volume and frequency depending on the degree of malignancy of the test tissue. 4. The method according to claim 1, characterized in that according to the results of automatic classification, distribution maps of spectral and / or physiological characteristics corresponding to them are formed.

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