Intraoperative combined spectroscopic diagnostic technique for cerebral and spinal tumours. RU patent 2497558.

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.

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The present invention relates to medicine, namely to the neuro-Oncology, and may be used for intraoperative diagnostic boundaries brain and spinal cord 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 definition of the boundaries of the cerebral and tumors. Urgency of the problem of determining the boundaries of astrocytic tumors caused by features of their growth along nerve fibers and blood vessels, leading to infiltration healthy white matter of neoplastic cells, which affects the high frequency of postoperative recurrence. The complexity of the surgery tumors of the spinal cord is that the tumor is not always a clear line and a high risk of trauma due to the smaller size of the operated plot compared with the brain. Reliable information about the amount tumor can be obtained by intraoperative imaging. The solution to this problem is mainly realized in three directions: use of intraoperative computer tomography, magnetic resonance imaging and ultrasound scanning, and different 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 ways of intraoperative diagnosis do not allow for the study of tissues in the operating wound in real time and/or do not provide simultaneous analysis of structural and metabolic changes.

Meanwhile, clinical studies have demonstrated a significant correlation between the level of accumulation of 5-aminolevulinic acid (5-ALA) - induced protoporphyrin DC and the speed of cell division that allows the use of this substance as a tumor marker. However, due to the fact that in 20-30% of cases, there was only a slight accumulation of drug in the tumor cells, additional criteria for intraoperative diagnosis of the investigated tissues. As is known, the tumor tissue and healthy brain tissue characterized by differences in the structure of tissues and the degree of and hemoglobin oxygenation. Thus, the simultaneous determination of these parameters allows to increase sensitivity of the method, based on the determination of the concentration of tumour marker (5 ALA-induced protoporphyrin IX).

Therefore very important problem is the development of a way to quickly intraoperative analysis of the tissues on the totality of the characteristic features during removal of tumors of the brain and spinal cord. Fluorescence spectroscopy and diffuse reflectance spectroscopy to provide you with accurate, fast and non-invasive methods of metabolic and structural changes in the tissues.

The closest analog to the present invention (prototype) is a method of combined spectroscopy, described in the article [Journal of Biomedical Optics 16 (11), 116007-14 (November 2011]. In this article, we propose a method of combined spectroscopy, combining analysis of spectra diffuse scattering of radiation of a pulsed broadband source in the range from 450 up to 720 nm obtained for the two distances between the lighting and the adoptive fibers, and the fluorescence spectrum of 5-ALA induced protoporphyrin IX and products of its photodecomposition range from 600 to 720 nm caused by radiation with the wavelength of 405 nm. Few hours before the operation the patient's organism is introduced solution hydrochloride 5-ALA. Then during operation resection of tumors produce consistent measure the spectrum of the background radiation of two spectra: diffuse scattering and fluorescence spectrum. The total time required for registration of all spectral dependencies in each point is about three seconds. To obtain the spectral characteristics of the use fiber optic sensor with three lighting fiber and two receptions. Received spectroscopic data treated with diffusion approximation theory of radiation propagation in a turbid medium, the optical characteristics which correspond to the optical characteristics of the tissue studied. The resulting data allow us to quantify the following parameters: the concentration of 5-ALA-induced protoporphyrin IX and products of its photodecomposition, concentration of hemoglobin in the oxygenated and reduced form of the scattering properties of the fabrics. For the implementation of the diagnostic algorithm is used by the support vector machine, which is a method of classification with a «teacher», in the space of five characteristics obtained in the result of mathematical treatment of the spectral dependencies. According to the results of the automatic analysis make a conclusion about radical held resection.

Disadvantages of using this method is consistent record spectra of background light, diffuse scattering and fluorescence, which requires measurement of all parameters required for the classification of tissue, about 3 C, and not allow measurements in real-time and reliably establish that the spectra were obtained from the same point in the same conditions. Another drawback of the method selected by us as a prototype, is the use of fluorescence shortwave radiation (wavelength 405 nm), for which the biological tissue is an environment with low transparency, which leads to reviewing the basics of fluorescence sensing tissue amount not to exceed several microns, and requires pre flush tissue of blood prior to optical measurements. These shortcomings complicate the procedure of tissue analysis by the method of optical biopsy.

Objective of the proposed invention is a method to enhance the reliability of measurements, reduce the time of their implementation, increase the depth of sounding and simplifying the process of registration of spectra in vivo.

The problem is solved by way of intraoperative holding spectroscopic diagnostics of tumors of the brain and spinal cord, including preoperative introduction to the patient hydrochloride solution of 5-ALA and intraoperative spectroscopic analysis of the content of the investigated tissues tumour marker (5-ALA induced protoporphyrin IX), products photodecomposition, and reduced hemoglobin, and changes in the scattering properties with the use of sources of broadband and laser and fiber-optical probe, with subsequent automatic classification spectroscopic characteristics of serving to assess efficacy of conduct resection. The proposed method is carried out using the source of broadband radiation in the wavelength range from 500 to 600 nm nm and the source of the laser/led radiation with a wavelength in the 620-640 nm, registration of spectra of the diffuse reflection and fluorescence produce simultaneously, then the accumulation level tumour marker of 5-ALA induced protoporphyrin DC determined by the degree of affiliation of the measured sample of tissue to the previously defined classes, the results of which assess the radical resection of a tumor.

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

The task is solved by the fact that according to the results of automatic classification form a sound signal, modulated by the loudness and frequency depending on the degree of malignancy of the tissue studied.

The task can be solved by the fact that according to the results of automatic classification form the maps of distribution of biomarkers.

The proposed method is using cross system of lighting and imaging filters, which implements the splitting of the spectral range on the area of registration the spectrum of the diffuse reflection of broadband radiation source (in the range from 500 to 600 nm nm), the intensity of scattering of laser radiation in the range 620-640 nm, which corresponds to the area of long-wave peak Q-absorption bands of protoporphyrin DC, in which biological tissue is optically transparent) and fluorescence spectrum (in the range 640-850 nm). Device for intraoperative combination of spectroscopic diagnostics of tumors of the brain and spinal cord matches the variant of the device described in the patent of Russian Federation №2169590 (IPC A61N 5/06, 2000).

The technical result, at the expense of the totality of features is to provide fast and reliable obtaining spectroscopic characteristics of one and the same volume of tissue in the area of operations for all of the above characteristics and simplification of process due to eliminating the need to pre flush tissue of blood prior to diagnosis.

In accordance with the invention, in the framework of preoperative preparation is performed oral administration to the patient hydrochloride solution of 5-aminolevulinic acid for a few hours before the beginning of the removal of the tumor.

Classification of recorded thus spectroscopic characteristics may be performed via the automatic mapping of spectroscopic and/or physiological characteristics of a database of relevant spectroscopic and/or physiological characteristics of fabrics, the condition of which was verified by histological analysis, methods of automatic classification, the results of which formed the conclusion about the degree of relationship of the tissue studied to one group or another in the framework of the adopted classification of tissues.

To inform the surgeon about the state of fabric on the results of the automatic classification of spectroscopic data may be used as visual and sound indication. For emphasis, the surgeon on the change of the spectral characteristics in the process of operation based on the results of their automated processing possible to use sound indication modulated by the loudness and frequency depending on the degree of malignancy tissues. Also, the formation on the screen two-dimensional maps of distribution of spectroscopic and/or physiological characteristics of fabrics to visualize the presentation of joint distribution of the studied biomarkers.

The invention is illustrated by the following figures.

Figure 1 provides for A patient under paragraph (a - preoperative MRI; b - photography cortex over the tumor in the white light; in - picture distribution of fluorescence cortex over the tumor mode Blue 400 intraoperative microscope Opmi Pentera.

Figure 2 shows for patient A view of the surgical bed in the mode of Blue 400 (time 18.16.07) before the spectroscopic data, (b) at the time of the combined spectroscopy (time 18.16.19-18.16.21), as well as in the mode of Blue 400 (in) after a short aspiration in the position of holding combined spectroscopic analysis. Presents the spectra 208-209 (g)registered in the moment shown in fig.2, as well as the corresponding values of oxygenation, the intensity of light scattering and index fluorescence, rated maximum values obtained in this study (e). Intraoperative were obtained the following values of these parameters are: range 208 - oxygenation 53%, the amplitude of backscattered laser radiation 800, index fluorescence 8.7; range 209 - oxygenation 26%, the amplitude of backscattered laser radiation 210 index fluorescence 7.5.

Figure 3 presents the maps of joint distribution of the studied spectroscopic values for normal, tumor tissue, as well as in the operational couch after removal of the tumor, the patient A. The following notation is used points in three-dimensional space, belonging to different classes: ▲ - the edge of the tumor ( area),, I - tumor, x - box after removal of the tumor, ✖ - intact neural tissue. All values are normalized to the maximum values obtained in result of the study. a - x-axis intensity of backscattered laser radiation, on the axis of ordinates index fluorescence; b - the x-axis is the degree of saturation of hemoglobin oxygen, on the axis of ordinates index fluorescence; the abscissa axis postponed the value of degree of saturation of hemoglobin oxygen, op axis shows the intensity of backscattered laser radiation.

Figure 4 shows the results of morphological study tissue samples taken during the surgery the patient And, allowed to make the final conclusion - : a - tumor, b - tumor.

Figure 5 present intraoperative image capture operation remove the tumor patient B in the white light of (a) and in the mode of Blue 400 Carl Zeiss Opmi Pentero (b)showing the presence of blood in the operational bed and weak heterogeneous fluorescence (the time 11.04.40-45), not allowing to make an unambiguous conclusion about the degree of accumulation of tumour marker in the tissue.

Figure 6 shows the results of the combined spectroscopic analysis of tissue B in the area of a box, shown in figure 4: range №36 (a), as well as the corresponding index values fluorescence (b) and oxygenation (). Index fluorescence 26, the amplitude of backscattered laser radiation 350, oxygenation 56%.

Figure 7 shows the results of morphological study of a patient's tumor B., with the final conclusion - anaplastic : a - zone infiltration of the tumor; b - zone infiltration of the tumor; the tumor; Mr. swelling and infiltration area.

On Fig.8 shows the intraoperative spectra obtained by the method of combined spectroscopic analysis of patient Century Range, marked with green, corresponds to a normal brain, the range indicated by the , obtained from tumor tissue, range, marked in orange, registered from the edge of the tumor.

Figure 9 shows the results of morphological study of the tumor, the patient - drugs detected ependymoma.

The following examples illustrate the proposed invention.

Example 1.

The patient, a 23-year (final diagnosis - the left frontal lobe (Grade II classification of the world health organization) operated 17.04.2012 (first time). 2 hours before surgery, the patient orally introduced a solution hydrochloride 5-aminolevulinic acid () in the amount of 20 mg/kg of patient's weight. After conducting trephination and dissection of Dura rendered bark of the left frontal region on the surface of the tumor site, bright fluorescent highlighting mode BL 400 on the operating microscope Carl Zeiss Opmi Pentero with fluorescent module (see intraoperative pictures figure 1). In figure 1. presents preoperative MRI (a) in patients with intracerebral tumor is visualized big tumor left frontal lobe (Grade II); intraoperative photo in white light (b) and in the mode of Blue 400 Carl Zeiss Opmi Pentero (), demonstrate a bright visible fluorescence of the first phase of operation. Started removal of the tumor. In the process of operation periodically assessed visual fluorescence of the wound and control markers of the state examined tissues by using the proposed method of a combined spectroscopy.

To use this source of broadband radiation in the range of 500 to 600 nm for analysis the concentration of hemoglobin in the oxygenated and reduced form and source of laser radiation with a wavelength in the 632.8 nm for the analysis of parameters of the light scattering and fluorescence excitation of 5-ALA induced protoporphyrin IX and products of its photodecomposition. When casting a fiber-optic probe in soft contact with the cloth produced simultaneous registration of spectra of the diffuse reflectance and fluorescence.

The places of intensive accumulation of 5-ALA removed completely, was a gradual decline in the intensity visible fluorescence in the course of the operation as the deepening of the wound. During the operation, the tumor visually almost no different from the medulla. At this stage the most sensitive tool for assessing the condition of the tissue in the operating wound was the method of combined spectroscopy. A series of intra-frames and spectral dependences submitted in figure 2, shows spectroscopic detection of fluorescent focus (2, b)not submitting visualization using mode Opmi Pentera Blue 400 (figure 2, a). In the study of combined spectroscopy revealed a range of 208 index fluorescence 8.7, the amplitude of backscattered laser radiation 800, oxygenation, 53%; range 209 index fluorescence 7.5, the amplitude of backscattered laser radiation 210, oxygenation, 26% (figure 2 g, d).

When conducting combined spectroscopic analysis in real time produced a classification of the tissue studied based on the automatic analysis of spectroscopic characteristics of the method To the nearest neighbours. As a method of display used two-dimensional maps of the distribution of values of the studied physiological markers (figure 3).

In the course of the operation was conducted fence material for morphological analysis of tissue samples in comparison with data of spectral analysis (figure 4). Differences in the level of scattering shown on fig.2 corresponding equally effective accumulation of protoporphyrin IX (fig.2) can be attributed to morphological difference plots tumour containing as Astro-and (fig.4, b). After obtaining parameters of combined spectroscopy from the bed walls of the removed tumor, identical to the normal brain, the main stage of the operation ended.

Example 2.

In the combination spectroscopy maximum index fluorescence was 26 Rel. units, the amplitude of backscattered laser radiation at the same point amounted to one third of the value of this parameter in normal tissue oxygenation at the same point was 56% (6). Of the points of realization of combined spectroscopic analysis were taken examined tissue biopsies (fig.6, b, C, d). The results of intraoperative mapping of spectral dependencies and morphological data reports served to enrich the database of spectroscopic/physiological to increase the accuracy of determining the membership of the investigated tissues to certain classes.

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

Example 3.

The patient, a 31-year (final diagnosis - tumor (ependymoma) at the level of Th2-Th3 vertebrae) was operated for the first time 03.04.2012, 2 hours before surgery, the patient orally introduced a solution hydrochloride 5-aminolevulinic acid () in the amount of 20 mg/kg of patient's weight. After approaching a tumor assessed visible fluorescence with the help of an endoscope with a fluorescent module - there were lots of bright homogeneous-type visible fluorescence. Resection of residual tumor, because the sensitivity of endoscopic module is insufficient, was combined spectroscopic analysis in the operational bed. For combined modality spectroscopy were used two sources of radiation - halogen lamp with a filter that removes the radiation in the range of 500 to 600 nm and helium-neon laser (632.8 nm). When casting a fiber-optic probe in soft contact with the cloth was made simultaneous registration of spectra of the diffuse reflectance and fluorescence. Real-time occurred classification of the tissue studied based on the automatic analysis of spectroscopic characteristics. According to the results of classification formed a sound signal, modulated by the loudness and frequency depending on the degree of malignancy examined tissue, on screen display the recorded spectra. In Fig.8 (b) shows the three spectra obtained intraoperative during the removal of the tumor, the patient Century Range, marked with orange, registered from the tumor margins, as evidenced intermediate between normal and tumor index value of fluorescence intensity of backscattered laser radiation. Easy normal tissue may be due to traction spinal cord tissue during surgery. The results of the spectroscopic analysis were confirmed morphological study tissue samples taken from the same points that hosted the spectral analysis (fig.9, b). The main stage of the operation was completed after the receipt of the spectral characteristics of the tumor bed, corresponding to normal tissue.

The proposed method is based on the actual material received by us at the analysis of 99 patients with gliomas of the brain Grade I-IV (WHO), 16 patients with metastatic tumors to the brain and 21 patients with meningiomas of the various levels of brain malignancy.

Thus, the proposed method solves the problem of increasing the reliability of intraoperative measurements at , reduce the time of their implementation, increase the depth of sounding and simplify the process of registration of spectra in vivo.

1. Method of combined intraoperative spectroscopic diagnostics of tumors of the brain and spinal cord, including preoperative introduction to the patient orally hydrochloride solution of 5-aminolevulinic acid (5-ALA) and the intraoperative spectroscopic analysis of the content in the studied tumor tissues marker (5-ALA induced protoporphyrin IX), products of its photodecomposition, and reduced hemoglobin, and changes in the scattering properties of the tissues with the use of sources of broadband and laser and fiber-optical probe, with subsequent automatic classification of spectroscopic characteristics used for evaluation of the efficacy of conduct resection, wherein the a source of broadband radiation in the wavelength range from 500 to 600 nm nm and a source of excitation fluorescence of laser radiation with a wavelength in the 620-640 nm, the registration of spectra of the diffuse reflection of broadband and laser radiation, as well as fluorescence are performed simultaneously.

2. The method according to claim 1, wherein the automatic processing of spectroscopic characteristics of the use automatic classification algorithm method To nearest neighbors with the definition of a weight facilities this point to each of the previously defined classes of tissue.

3. The method according to claim 1, characterized in that the results of automatic classification form a sound signal, modulated by the loudness and frequency depending on the degree of malignancy of the tissue studied.