Method of automated morphometric myelofibrosis diagnostics

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to a method of automated morphometric myelofibrosis diagnostics. The method essence consists in the fact that overview images of zones with different optical properties with determinable fibrous and heamopoietic properties of a biological tissue are performed. Ratios of areas of the said zones of at least three paraffin cuts of trepanobiopsy samples are calculated. The coefficient (Cop) is calculated as the ratio of the fibrous tissue area to the area of the heamopoietic tissue by formula. If the value Cop ≥14.5%, myelofibrosis is diagnosed.

EFFECT: application of the claimed method makes it possible to increase the accuracy and improve the efficiency of myelofibrosis diagnostics.

7 cl, 1 tbl, 4 dwg, 1 ex

 

The invention relates to medicine, in particular to Hematology, namely to clinical research methods using automated morphometric methods (including the use of computers), and can be used to diagnose lesions of fibrous tissue, in particular bone marrow tissue (myelofibrosis).

In recent years, thanks to the development and use in clinical practice of new drugs, transplantation of hematopoietic stem cells and many other modern methods of treatment, significantly increased overall survival of patients with neoplastic diseases, in particular hematopoietic tissue. It is obvious that with the increase in life expectancy of cancer patients is significantly increased role of reliable qualitative diagnosis of malignant tumors.

Known conventional methods for diagnosis of myelofibrosis with the help of bone marrow biopsy bone marrow. The scale adopted in the European consensus in 2005 (Thiele J., Kvasnicka Η.Μ, F. Facchetti et al. European consensus for grading of bone marrow fibrosis and assessment of cellularity // Haematilogica. - 2005. - 90:1128 - 1132), allows to carry out qualitative and semi-quantitative assessment of myelofibrosis. Qualitative assessment of myelofibrosis lies in the differentiation between reticulin and collagen, semiquantitative assessment involved determining tightly�ti retikulinovye fiber.

Also known method for automated quantitative assessment of myelofibrosis in myeloproliferative disorders, which consists in determining the average percentage of black pixels in the 3 hematopoietic squares of 0.5 mm2(Teman J. arolin, 2010).

However, the known methods of diagnosis of myelofibrosis do not provide sufficient accuracy and rapidity in obtaining the relevant results.

Also known a method of diagnosing an inflammatory pathology of the maxillary sinuses (RF patent for the invention №2234859 publication of 27.04.2004), which in particular includes the performance review of the radiographs of the paranasal sinuses in neopatriomonal projection, characterized in that the x-ray emit a "region of interest" corresponding to the x-ray contours of the maxillary sinus and the namesake of the orbit, build a histogram and calculate the density ratio (KP) as the ratio of the density of the maxillary sinus, which is expressed in units of gray scale, and density of same orbit, expressed in units of grayscale colors moreover, "one-humped" curve area of interest is the x-ray contour of the entire maxillary sinus, and in the presence of "mnogogolos curve areas of interest are the areas of the sinuses with different densities.

The known method, due and�use, in particular, x-ray technicians, also does not have a high sufficient accuracy and efficiency of diagnosis.

Closest to the claimed invention identified the solution known from the publication (Domnikov N. P. . Structural analysis of trypanosomiases in aggressive and indolent Nahodkinskij lymphomas // Fundamental research, No. 9, 2001, pp. 27-61 (page 58 column 1 and 2), which, in particular, is characterized in that it is determined by the relative area of tumor tissue in different types lympany infiltration of bone marrow by aggressive and indolent non-Hodgkin's lymphoma, which, in any case, facilitates the diagnosis of myelofibrosis.

The main disadvantage of the known method, taken as a prototype, is the lack of opportunities automated morphometric diagnosis of myelofibrosis that hinder improving the accuracy of setting the appropriate diagnosis.

The purpose of the claimed invention is to eliminate the above disadvantages and achieve these technical results, increase accuracy and improve efficiency of diagnosis of myelofibrosis, primary myelofibrosis (chronic idiopathic myelofibrosis), and secondary myelofibrosis that develops when: 1) hematologic diseases (polycythemia Vera, �ssentially thrombocythemia, chronic myelogenous leukemia, hairy cell leukemia, rarely acute leukemia, myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma); 2) solid tumors with metastases in the bone marrow (cancer of breast, lung, prostate); 3) diffuse diseases of connective tissue (systemic lupus erythematosus, systemic sclerosis).

This object is achieved in that the method automated morphometric diagnosis of myelofibrosis, including the execution of overviews of biological tissue, the allocation of such image areas with different optical properties, defined fibrous and hematopoietic properties of biological tissue, and calculating the relationship of these zones, characterized by including the fact that the review image is accomplished by the formation of at least three paraffin sections of trypanosomatid obtained sections were treated as follows: first impregnert silver by the method Gomory, the second stained by the method of van Gizon and third were stained with hematoxylin and eosin, thus obtained observation image is subjected to fixation and analysis so that as a result of this analysis get the value of the areas, colored in black and pink color, corresponding to fibrous tissue, and the magnitude of p�Asadi areas painted in blue and pale pink color corresponding to hematopoietic tissue; then calculate the ratio of the relative areas as a ratio of the area of fibrous tissue to the area of hematopoietic tissue by the formula:

where:

Kop- the ratio of the relative area ( % );

Sblack- an area of fabric, painted black;

Sroses- an area of tissue, colored in pink;

SBL-rose- an area of fabric, painted in pale pink color;

Ssin- an area of tissue, colored blue,

moreover, if set Toop≥14,5% are diagnosed with myelofibrosis.

Method automated morphometric diagnosis of myelofibrosis can be characterized by the fact that paraffin sections of trypanosomatid formed in a thickness of 4 microns.

Method automated morphometric diagnosis of myelofibrosis can be characterized by fixation of overviews of paraffin sections by using a color fotovideostore.

Method automated morphometric diagnosis of myelofibrosis can be characterized by the fact that the area sizes of the areas, colored in black and pink color, corresponding to fibrous tissue, and the area sizes of the areas, colored in blue and pale pink, respectively�adequate hematopoietic tissue, get with the light source and the corresponding RGB sensor colour definition, so that the size of the colored areas correspond to the electrical voltage output from the color sensors.

Method automated morphometric diagnosis of myelofibrosis can be characterized by the size of the painted areas get installed in the circuit after the RGB sensors of the digital signal processor.

Method automated morphometric diagnosis of myelofibrosis can be characterized by the analysis to obtain the values of the squares is colored areas produced using a computerized microscope.

Method automated morphometric diagnosis of myelofibrosis can be characterized by the fact that as a light source using RGB led light source.

In some cases the task is, for example, is solved by the fact that the identification of these indicators of myelofibrosis is carried out using image analysis AxioVision 4.6, using, for example, color packageexists photo/video camera AxioCam, Zeiss microscope and a standard PC. In paraffin sections of trypanosomatid, for example, the Ilium, the thickness of 4 microns, impregnated with silver by the method Gomory and stained by the method of van Gisone, increasing 200 absolute measure�Yu square fibrous (collagen and retikulinovye) tissue and hematopoietic tissue. Studied all the medullary cavity of one of the slice that corresponds to 5-7 fields of view of the microscope and 20-30 fields of view of the camera (depending on the size of the slice). The area of one field of view of the camera is known and 364000 μm2(0.7 mm2). Folded area of fibrous tissue in all fields of view of the camera, get the total absolute area of fibrous tissue within the same slice. Similarly get the total absolute area of hematopoietic tissue. Then calculate the percent area of fibrous tissue from hematopoietic tissue (Kop). Kopreflects the severity of myelofibrosis.

Often the task of diagnostics is empowerment evaluation of myelofibrosis for the purpose of determining such a very meaningful measure, as inhibition of erythroid sprout. The state of erythroid Rostock is evaluated in sections, stained with hematoxylin and eosin.

The proposed method is investigated, for example, trainability Ilium 30 patients with diffuse large b-cell lymphoma (DLBL): 20 men, 10 women, 21 to 59 years, mean age 50,36±14,31 years, 30 patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphomas of small lymphocytes with neoplastic bone marrow lesion (NHL of small lymphocytes): 18 men, 12 women, from 44 to 78 years, average age - to 59.13±9.5 years, 10 patients with multiple myeloma (MM): 5 males, 5 females aged 50 to 68 years, mean age 59,67±6,58 years who were treated in GBUZ NSO "Novosibirsk State regional clinical hospital from 2010 to 2012.

The diagnosis of lymphoma was established in accordance with the REAL classification (1994) and who (2008), the diagnosis of chronic lymphocytic leukemia was made on the basis of the classification Binet (Binet J. L. et. al., 1981). To verify the diagnosis of multiple myeloma criteria used Y. Huang et al. (Y. Huang W. et al.,1 999).

The first group consisted of sections 14 trypanosomatid noted, most likely, only retikulinovye myelofibrosis, in the second group sections 13 trypanosomatid, where there was probably only collagen myelofibrosis, in the third - slices 15 trypanosomatid in which with the greatest certainty was identified as collagen and retikulinovye myelofibrosis. In the remaining 28 trypanosomatid - fibrous tissue is not detected. Assessment of severity of myelofibrosis was carried out in each group.

The Table presents example indicators of myelofibrosis associated with erythroid hypoplasia of Rostock, in particular the data of the results of automated morphometric evaluation of myelofibrosis with lymphoproliferative diseases identified indicators associated with inhibition of eritr�LiDE Rostock.

These tables allow to conclude that these indicators include: in the first group - Kop≥36.7 per cent, in the second group - Kop≥22.5 percent, in the third group To theop- ≥14.5 percent. Thus, the implementation of the method, in the part of these values, such asop>14.5%, and can be used in the diagnosis of myelofibrosis, without a clear separation into groups.

Fig. 1 schematically shows an example of an installation for analysis of paraffin sections trainability claimed method. Fig. 2, Fig. 3 and Fig. 4 shows an enlarged 200 times photographic prints paraffin sections trainability Ilium, subject to such analysis for the purpose of diagnosing the lesion tissue myelofibrosis. In the presented figures the numbers indicate the following:

1. The source of light.

2. Photographic print drug cutoff trainability.

3. Frame photographic print.

4. The direction of light transmitted through the photo of the drug.

5. The sensor matrix colour definition.

6. The connectors.

7. A digital signal processor.

8. Indicator.

9. Zone, black in color.

10. Zone pink color.

11. Area bright pink color.

12. Area pale pink color.

13. Area blue color.

In the presented example, the value of the squares of the desired zones in paraffin sections trainability defined�s as follows. Obtained in the standard way, three paraffin slice trainability, for example, the Ilium, the thickness of 4 μm, sequentially processed as follows: first impregnert silver by the method Gomory, the second stained by the method of van Gizon and third were stained with hematoxylin and eosin. Thus obtained observation image photographed, thereby fixing the obtained color-coded zones.

With the help of light source 1, for example in the form of a matrix of LEDs, a beam of white light is directed to a photographic picture of the drug cut trainability 2 mounted in a frame 3. Further, passing through a photographic picture of the drug cut trainability 2, the light is colored in colors corresponding to the colors selected for the analysis of photographic print of the drug cutoff trainability 2 in direction of 4, and gets on RGB matrix electronic sensors color definition 5 (e.g., digital). The output matrix of electronic sensors color definition 5 is formed voltage (Ur, Ug, Ub), is proportional to the flow of light of the respective colors. Using connectors 6 outputs matrix electronic sensors determine the color 5 pieces, in particular a digital signal processor, which identifies the area of the painted areas analyzed photographic print of the drug cut the tre�adobeupdate, on the basis of proportionality from the output voltages of the respective colors (URUGUB), the results of the determination of the required areas is read from the indicator 8, for example, in the form of monitor.

All three of the analyzed slice is separated from the test sample of the Ilium in a row one after the other, and considering that the slice thickness does not exceed a few microns, they can be found almost identical drugs. The first drug in paraffin slice trainability (Fig. 2) impregnert silver, with black painted retikulinovye fabric (black color 9). A second drug paraffin slice trainability (Fig. 3) stained by the method of van Gisone, collagen tissue becomes pink zone pink color 10). The third drug paraffin slice trainability (Fig. 4) was treated with hematoxylin and eosin, wherein the hematopoietic tissue is colored in pale pink (cytoplasm) (zone pale pink color 12) and in blue (cell nucleus) (zone blue color 13). Bone preparations, stained by van gieson and hematoxylin-eosin, has a bright pink shade (zone of bright pink fabric 11), the area of fibrous tissue is determined by the sum of the areas of the zones of the black color zones 9 and pink color 10. The area of hematopoietic tissue is determined by summo� areas blue zones 12 and pale pink 13 colors.

Further, to obtain the desired final diagnosis of lesions of the studied sample myelofibrosis compute the ratio of the relative area ( % ) are presented in the claimed invention formula. Suppose that in the proposed example, Kopcalculated for slices trainability, which noted only retikulinovye myelofibrosis, equal to 37%, then according to the above regulations, this indicator is important for the inhibition of erythroid sprout. In sections of trypanosomatid only with collagen myelofibrosis important for inhibition of erythroid city is home To theopfor example , 23%. In slices with retikulinovye and collagen myelofibrosis important to the oppression of Central erythron component is, for example, Kop15%. Thus, when getting To theop≥14,5% in any case clearly diagnosed myelofibrosis, even if no unambiguous dividing it into groups.

The high accuracy of the proposed method is achieved by using the automated estimation of small quantities of the variation in fibrous areas and hematopoietic tissues, and the efficiency is improved due to the use of automated (computerized) method of determining the ratio of said areas.

Thus, the inventive method is based on automatic�teachers morphometric study allows for non-Hodgkin's lymphoma, chronic lymphocytic leukemia, multiple myeloma and other diseases to maintain accurate and rapid diagnosis of myelofibrosis.

The inventive method is industrially applicable, as it does not require any specific complex equipment, in addition to the standard set of medical and computer equipment that makes it possible to widely implement it in clinics, research and educational institutions.

1. Method automated morphometric diagnosis of myelofibrosis, including the execution of overviews of biological tissue, the allocation of such image areas with different optical properties, defined fibrous and hematopoietic properties of biological tissue, and calculating the relationship of these zones, characterized in that the observation image is performed by forming at least three paraffin sections of trypanosomatid obtained sections were treated as follows: first impregnert silver by the method Gomory, the second stained by the method of van Gizon and third were stained with hematoxylin and eosin, thus obtained observation image is subjected to fixation and analysis, the result of this analysis get the value of p�Asadi areas painted in black and pink colors, corresponding to fibrous tissue, and the area sizes of the areas, colored in blue and pale pink color corresponding to hematopoietic tissue; then calculate the ratio of the relative areas as a ratio of the area of fibrous tissue to the area of hematopoietic tissue by the formula:

where:
Kop- the ratio of the relative area ( % );
Sblack- an area of fabric, painted black;
Sroses- an area of tissue, colored in pink;
SBL-rose- an area of fabric, painted in pale pink color;
Ssin- an area of tissue, colored blue,
moreover, if set Toop≥14,5% are diagnosed with myelofibrosis.

2. Method automated morphometric diagnosis of myelofibrosis according to claim 1, characterized in that the paraffin sections of trypanosomatid formed in a thickness of 4 microns.

3. Method automated morphometric diagnosis of myelofibrosis according to claim 1, characterized in that the fixation overviews of paraffin sections by using a color fotovideostore.

4. Method automated morphometric diagnosis of myelofibrosis according to claim 1, characterized in that the area sizes of the areas, colored in black and pink color, corresponding to fibrous tissue, and in�mask areas, painted in blue and pale pink color corresponding to hematopoietic tissue, are produced by the light source and the corresponding RGB sensor colour definition, so that the size of the colored areas correspond to the electrical voltage output from the color sensors.

5. Method automated morphometric diagnosis of myelofibrosis according to claim 4, characterized in that the area of the painted areas get installed in the circuit after the RGB sensors of the digital signal processor.

6. Method automated morphometric diagnosis of myelofibrosis according to claim 4, characterized in that the analysis to obtain the values of the squares is colored areas produced using a computerized microscope.

7. Method automated morphometric diagnosis of myelofibrosis according to claim 4, characterized in that the light source using RGB led light source.



 

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