RU2826338C1 - Diagnostic technique for autoimmune thyroiditis - Google Patents

Abstract

FIELD: medicine; endocrinology; immunology.

SUBSTANCE: invention can be used for diagnosis of autoimmune thyroiditis. Mononuclear cells are recovered from venous blood on a density gradient of ficoll 1.077, which are incubated with a thyroperoxidase antigen labeled with L-azidohomoalanine. They are fixed in 4% paraformaldehyde solution for 15 minutes. They are washed and incubated in solution of 100 mM of copper (II) sulphate and 1 mcM of the green fluorescent dye, Alexa Fluor 488 in volume of 500 mcl for 15 minutes. Mononuclear cells are washed by centrifugation for 5 minutes, after which 500 mcl of phosphate-buffered saline is added. They are analyzed by laser flow cytometry: the sample is irradiated with a laser beam with wavelength of 488 nm. Result of the irradiation is detected in the FL1-channel of the cytometer with the monitor, wherein the binding of lymphocytes to the thyroperoxidase antigen is established. If observing more than 5% positively fluorescent cells of the sample, autoimmune thyroiditis is diagnosed when exposed to a laser beam. If there are less than 5% positive fluorescent cells of the sample, the absence of autoimmune thyroiditis is diagnosed.

EFFECT: method enables diagnosing autoimmune thyroiditis, which enables to increase the reliability and accuracy of diagnosing autoimmune thyroiditis, simplification (less steps and consumed time) of the diagnostic process by analyzing the binding of lymphocytes to the thyroperoxidase antigen with the L-azidohomoalanine label by laser flow cytometry.

1 cl, 3 dwg, 2 ex

Description

The invention relates to the field of medicine, namely to endocrinology and immunology, more precisely to a method for diagnosing autoimmune thyroiditis. It can be used to diagnose autoimmune thyroiditis based on determining the presence or absence of fluorescence in a blood preparation, which is lymphocytes isolated from the patient's venous blood, incubated with a thyroid peroxidase antigen labeled with L-azidohomoalanine. When said blood preparation is irradiated in a flow cytometer with a laser beam (in the case of fluorescence), it is possible to identify autoimmune thyroiditis, otherwise a diagnosis of the absence of said disease is made.

The technical problem solved by the claimed invention is the insufficient (unsatisfactory) reliability of the results of diagnostics of autoimmune thyroiditis, performed by known methods - physical examinations, laboratory blood tests and studies of biological fluids, ultrasound, as well as the multi-stage nature, high time-consuming process and high cost (high cost of reagents and process) of its implementation; the need for the participation of highly qualified personnel to perform diagnostics.

Terms and definitions used in the text:

An antigen is a substance that can bind to a specific antibody or T-lymphocyte receptor and cause an immune response [https://bigenc.ru/c/antigen-eddc38].

Autoantigens are components of the body's own cells and tissues that are recognized under certain conditions as foreign [https://бмэ.орг/index.php/AUTOANTIGENS].

Autoimmune thyroiditis is a chronic inflammatory autoimmune disease of the thyroid gland, which, as of the date of filing the claimed technical solution, is diagnosed with an error in the range of 10-20%. In this case, the disease is characterized by the destruction of the patient's thyroid tissue by lymphocytes [see https://www.palomahealth.com/learn/hashimotos-thyroiditis-normal-antibodies].

Autoreactive lymphocytes are lymphocytes that react to their own antigens [see Murphy, K. Immunobiology according to Janway / K. Murphy, K. Weaver; trans. from English; edited by G. A. Ignatyeva, O. A. Svitich, I. N. Dyakov. - M .: Logosfera, 2020. - 1184 p.: ill.: 21.3 cm].

Ficoll density gradient is a solution of polysucrose 400 with sodium diatrizoate, optimized to create a solution with a given density gradient, which is used in the separation of cells and organelles by centrifugation. Ficoll is a registered trademark owned by GE Healthcare. It is used in biological laboratories to separate blood into its components (erythrocytes, leukocytes, etc.) [see https://www.dia-m.ru/news/fikoll-polisakharoza-400-v-rastvore/].

Lymphocytes are blood cells from the leukocyte group. They provide humoral immunity (antibody production), cellular immunity (contact interaction with victim cells), and also regulate the activity of other types of cells [see https://meduniver.com/Medical/Physiology/334.html].

Peripheral blood mononuclear cells are peripheral blood cells that have a round nucleus. These cells consist of lymphocytes (T cells, B cells, natural killers) and monocytes, while red blood cells and platelets have no nuclei and granulocytes (neutrophils, basophils, and eosinophils) have multilobed nuclei [see https://www.ncbi.nlm.nih.gov/books/NBK500157/].

Seronegative autoimmune thyroiditis is an autoimmune thyroiditis in which antibodies to thyroid peroxidase and thyroglobulin are not detected [see Rotondi M, de Martinis L, Coperchini F, Pignatti P, Pirali B, Ghilotti S, Fonte R, Magri F, Chiovato L. Serum negative autoimmune thyroiditis displays a milder clinical picture compared with classic Hashimoto's thyroiditis. Eur J Endocrinol. 2014 Jul;171(1):31-6. doi: 10.1530/EJE-14-0147].

Seropositive autoimmune thyroiditis is autoimmune thyroiditis in which antibodies to thyroid peroxidase and/or thyroglobulin are detected.

Thyroid peroxidase is an enzyme contained in the cells of the thyroid gland and responsible for the formation of the active form of iodine for the synthesis of thyroid hormones. It is the main antigen to which antibodies are produced in autoimmune thyroiditis [Jean Ruf, Pierre Carayon. Structural and functional aspects of thyroid peroxidase (English) // Archives of Biochemistry and Biophysics. - 2006-01. - Vol. 445, iss. 2. - P. 269–277. doi:10.1016/j.abb.2005.06.023].

L-azidohomoalanine is an amino acid analogue of methionine, a biochemical reagent required to detect a protein, in this case thyroid peroxidase, which contains a modification, namely an azido group. This compound is incorporated into proteins during protein synthesis in a cell during cultivation. To detect a protein with L-azidohomoalanine, chemoselective ligation or a reaction between the azide group of the protein and the alkyne group of a fluorescent dye is used. Thus, it is necessary to perform the claimed method as an initial reagent for diagnosing autoimmune thyroiditis [https://ru.lumiprobe.com/p/l-azidohomoalanine-aha].

MHC (Major Histocompatibility Complex) – major histocompatibility complex, a complex of genes encoding proteins responsible for the presentation of antigens (to T-lymphocytes) during an immune response [https://www.britannica.com/science/major-histocompatibility-complex].

Physical examination is the inspection, palpation, auscultation, and measurement of the body and its parts. This is the stage that follows the collection of anamnesis and precedes the prescription of laboratory tests and radiological studies [https://meduniver.com/Medical/travmi/fizikalnoe_issledovanie.html?ysclid=lq56je6o39717735985].

The diagnosis of "autoimmune thyroiditis" is made based on the symptoms of the disease, physical examination of the thyroid gland and test results. To exclude diseases with similar manifestations, such as thyroid cancer, diffuse toxic goiter, a number of additional studies are required [https://helix.ru/kb/item/2509/].

Laboratory blood tests for autoimmune thyroiditis:

– antibodies to thyroid peroxidase (AT-TPO);

– antibodies to thyroid-stimulating hormone receptors (AT-rTSH);

– TSH, T3, T4 levels [https://helix.ru/kb/item/2509/].

Ultrasound examination is an instrumental method for diagnosing autoimmune thyroiditis. In relation to the diagnosis of thyroiditis, ultrasound is the only instrumental method. It is performed using an ultrasound machine [http://pmarchive.ru/ultrazvukovoe-issledovanie-autoimmunnogo-tireoidita-v-ambulatornyx-usloviyax/].

The applicant's research into the state of the art has revealed analogs of the proposed invention. The terminology of their (analogs') descriptions has been used in describing the analogs.

An invention is known under patent CN 102735833 "A kit for homogeneous-phase luminescent immunoassay for antibodies to thyroid peroxidase and a detection method". The essence is a method for diagnosing autoimmune thyroiditis by homogeneous-phase luminescent immunoassay for antibodies to thyroid peroxidase. The kit for homogeneous-phase luminescent immunoassay for antibodies to thyroid peroxidase includes a homogeneous-phase luminescent 96-pore plate for detection, a standard product of antibodies to thyroid peroxidase, a solution of donor microspheres coated with streptavidin and biotin-labeled thyroid peroxidase antigen, as well as a solution of receptor microspheres coated with thyroid peroxidase antigen. The thyroid peroxidase antigen in the sample competes with the thyroid peroxidase antigen on the receptor microspheres by combining with the biotin-labeled thyroid peroxidase antigen. The detection method involves measuring the luminescence signals of known standard products with different contents, constructing a standard curve and obtaining a mathematical function, and obtaining the content of an unknown sample using a mathematical function. The detection method combines the advantages of both enzyme immunoassay and immunochemiluminescent assay, accurately determines the level of thyroid peroxidase antibodies in serum, and is used for screening, diagnosis, and evaluation of the therapeutic effect in high-risk groups with autoimmune thyroid diseases.

As can be seen from the description of the invention, the diagnosis is made by the presence of antibodies to thyroid peroxidase. Antibodies to thyroid peroxidase are found in 80-90% of patients, 10-20% of patients are seronegative.

The disadvantage is the unsatisfactory (insufficient) sensitivity for reliable diagnosis.

The invention is known under patent RU 2315313 “Method for determining antibodies and method for diagnosing autoimmune thyroiditis”. The essence is a method for determining autoantibodies by changing the oscillation frequency of a piezoelectric quartz resonator based on volumetric acoustic waves before and after the step-by-step immobilization of L-polylysine, DNA antigen on the solid surface of the piezoelectric quartz resonator electrodes and subsequent incubation of the piezoelectric quartz resonator in blood serum diluted in a buffer, characterized in that the electrodes are silver or gold, L-polylysine is taken in an aqueous solution of 1.75 mg / ml in a volume of 10 μl and after application to the electrode is kept in a chamber saturated with water vapor for 4 hours, then washed and dried, and DNA is taken at a concentration of 0.68 mg / ml, diluted in a buffer of 0.01 M Tris-HCl, 0.01 M EDTA in a volume of 10 μl and after application to the electrode is kept in a chamber saturated with water vapor in for 1 hour, then washed and dried, and the piezoelectric quartz resonator is incubated in blood serum for 1 hour in a chamber saturated with water vapor, washed and dried. A method for diagnosing autoimmune thyroiditis, including clinical and laboratory studies, determining autoantibodies to DNA, characterized in that the determination of autoantibodies to DNA is carried out using the method according to paragraph 1 of the formula, and with an indicator of the frequency shift of the piezoelectric quartz resonator oscillations within Δf = 124.50 ± 13.84 Hz, the presence of the disease in the subject is judged.

Thus, in brief, the determination of autoantibodies is carried out by changing the oscillation frequency of a piezoelectric resonator based on volumetric acoustic waves before and after the step-by-step immobilization of L-polylysine, antigen-DNA on the solid surface of the piezoelectric resonator electrodes and subsequent incubation of the piezoelectric resonator in blood serum.

A disadvantage of the known invention is the limitations associated with the use of volumetric acoustic waves as an exciter of oscillations of a piezoelectric quartz resonator - such as the presence of side resonances and temperature instability. In addition, the implementation of the method occurs with large expenditures of time due to the fact that the speed of analysis depends on the duration of the incubation time required for the formation of the antigen-antibody complex and the time spent on complete regeneration of the sensor. Another disadvantage is the low diagnostic specificity of the method. It is known that antibodies to DNA are detected both in autoimmune thyroiditis and in other autoimmune diseases, for example, systemic lupus erythematosus, systemic scleroderma, rheumatoid arthritis.

The existence of similarity (sameness) of observed symptoms and signs of various diseases significantly reduces the reliability of diagnostic results and the results of subsequent treatment based on a questionable (unreliable) diagnosis, which does not allow for a high degree of reliability to conduct an accurate diagnosis, make an accurate diagnosis and prescribe appropriate treatment.

An invention is known according to patent RU 2704232 "Single-domain antibodies directed against tumor necrosis factor alpha and their use". The essence is a polypeptide construct capable of specifically binding TNF-alpha, for the treatment, prevention or alleviation of disorders that are sensitive to modulation by a substance modulating TNF-alpha and are associated with inflammatory processes or autoimmune diseases, containing two to four antagonistic single-domain antibodies directed against TNF-alpha, obtained by a method including a selection step based on determining the antagonistic effect using a cytotoxicity assay and at least IC50 ~ 100 nM, and one single-domain antibody directed against a serum protein, in which said two to four single-domain anti-TNF antibodies do not have the same sequence or all single-domain anti-TNF antibodies have the same sequence.

In brief, the essence is that a polypeptide construct capable of specifically binding tumor necrosis factor alpha (TNF-alpha) is used for diagnostics, containing two to four antagonistic single-domain antibodies directed against TNF-alpha and one single-domain antibody directed against serum protein.

The disadvantage of the invention is the low reliability of diagnostics by determining the tumor necrosis factor alpha in the blood serum. The reason for the low reliability of diagnostics is the observed spontaneous increase in TNF-alpha titers in the blood serum. The increase in titers may be associated not with one (autoimmune thyroiditis), but with several other autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, systemic lupus erythematosus. The existence of similarity of symptoms and signs of various diseases reduces the reliability of the results of thyroiditis diagnostics.

An invention is known according to patent US 10796785 "Method and system for microbiome diagnosis and treatment of endocrine diseases". The essence is a method for diagnosis and treatment of an endocrine disease in a subject, including: obtaining a cumulative set of samples from a population of subjects; for each sample of the cumulative set of samples: determining a microorganism sequence, including: identifying primers for nucleic acid sequences associated with an endocrine disease, and amplifying nucleic acid material using the identified primers; and determining an alignment of the microorganism sequence with a reference sequence associated with an endocrine disease; generating a data set of microbiome characteristics for a population of subjects based on comparisons; generating a characteristic of an endocrine condition based on features extracted from the data set of microbiome characteristics and an additional data set informative about the characteristics associated with the endocrine condition; based on the characteristic, creating a therapy model that determines a therapy for correcting the endocrine condition; and in an output device associated with the subject, providing a therapy to the subject having an endocrine disease based on the characteristic and the therapy model. A method for diagnosing and treating an endocrine disease in a subject, comprising: obtaining a sample from the subject; determining a set of microorganism sequences from the sample, comprising: identifying primers for nucleic acid sequences associated with the endocrine condition and amplifying nucleic acid material of the sample using the identified primers; extracting a data set of microbiome characteristics for the subject based on the set of microorganism sequences; creating a characteristic of the subject after processing the data set of microbiome characteristics with a characteristic model obtained from a population of subjects suffering from the endocrine disease; processing the characteristic with a therapy model that determines a therapy for correcting the endocrine condition; and in an output device associated with the subject, providing a therapy to the subject having an endocrine disease based on the characteristic and the therapy model.

More briefly, the essence of the method consists in diagnosing the state of the endocrine system in a subject, wherein the method includes: obtaining a cumulative set of biological samples from a population of subjects; creating at least one of a set of data on the composition of the microbiome and a set of data on the functional diversity of the microbiome for a population of subjects; creating a characteristic of the state of the endocrine system based on features extracted from at least one of the set of data on the composition of the microbiome and the set of data on the functional diversity of the microbiome; based on the characteristic, generating a therapy model configured to correct the state of the endocrine system; and in an output device associated with the subject, promoting therapy for the subject based on the characteristic and the therapy model.

The disadvantages of the known method are the multi-stage and high time-consuming nature of the process of its implementation, the need to use heterogeneous and expensive equipment. Another disadvantage of the method is the need to attract highly qualified specialists of a narrow profile to implement the known method. Due to its features, the known method is actually feasible mainly in large research centers. It is unlikely to be used in the laboratories of the overwhelming majority of health care organizations that are not equipped with special devices and the appropriate personnel to service this equipment.

The closest to the proposed invention, a prototype in terms of a set of coinciding features, is the invention according to patent US 7902121 "MHC antigen arrays for detecting and characterizing immune responses". The essence is a method for profiling T cells, including: bringing into contact a cell population containing T cells with a matrix, wherein said matrix contains a plurality of discrete regions of MHC-antigen complexes stably associated with the surface of a solid carrier. The method according to claim 1, characterized in that said array contains a plurality of different MHC-antigen complexes. The method according to claim 1, characterized in that said solid carrier is coated with polyacrylamide. The method according to claim 1, characterized in that said spot contains at least about 0.01 ng of said MHC-antigen complex. The method according to claim 1, characterized in that each of said regions contains a plurality of different antigen complexes. The method of claim 1, wherein one or more of said MHC-antigen complexes comprises an antigen selected from the group consisting of tumor antigens; viral antigens, bacterial antigens; parasitic antigens; environmental antigens; allergens; and autoimmune antigens. The method of claim 1, wherein said population of cells has multiple T-cell antigen specificities. The method of claim 1, wherein said population of cells comprises multiple cell types. The method of claim 1, wherein said sample is selected from the group consisting of blood, lymph, cerebrospinal fluid, and synovial fluid. The method of claim 9, wherein one or more of said cell types is differentially labeled with a detectable marker prior to said contacting step, and wherein said site-specific assay detects the presence of said marker. The method of claim 1, wherein said MHC-antigen complexes comprise a library of antigenic peptides that form a complex with MHC. The method of claim 1, wherein said array additionally comprises one or more regions containing signal probes. The method of claim 12, wherein said signal probes comprise possible pharmacologically active drugs. The method of claim 12, wherein said signal probes comprise a peptide library. The method of claim 12, wherein said signal probes are printed together with said MHC-antigen complexes. The method of claim 12, wherein said signal probes are adjacent to said MHC-antigen complexes. The method of claim 12, wherein said signal probes comprise candidate agents. The method of claim 12, wherein said signaling probes cause a change in the ability of said T cells to bind to said array, and wherein said site-specific assay comprises determining the ability of the cells to bind to said MHC antigen complex in the presence or absence of said signaling probe. The method of claim 12, wherein said signaling probe is selected from the group consisting of cytokines, chemokines and interleukins. The method of claim 1, further comprising determining a change in the phenotype of said T cells upon contact with said array. The method of claim 20, wherein said change in phenotype comprises secretion of a protein in response to antigen stimulation. The method of claim 1, wherein said array further comprises one or more spots containing wells. The method of claim 22, wherein said cells are antigen-presenting cells. The method of claim 1, further comprising the step of contacting said array with a second population of cells and determining binding of said cells to said T cells. The method of claim 1, further comprising the step of contacting said array with a second population of cells and determining a change in the phenotype of said T cells or said second population of cells. The method of claim 1, further comprising the step of contacting said matrix with an exogenous agent and determining a change in the phenotype of said T cells. The method of claim 26, wherein said exogenous agent is selected from the group consisting of growth factors and immunomodulatory agents. The method of claim 1, further comprising the step of determining expression of cell surface antigens by said T cells after said contacting step. The method of claim 1, further comprising the step of isolating said cells from said array. The method of claim 1, further comprising the step of reproducing said T cells after said contacting step. The method of claim 30, wherein said expansion step comprises bringing said T cells into contact with one or more growth factors, cytokines, cell adhesion molecules and extracellular matrix material. The method of claim 1, wherein said matrix comprises a plurality of spots having different concentrations of MHC-antigen complexes. The method of claim 32, further comprising the step of constructing a scaling curve for binding of T cells to said MHC-antigen complexes. The method of claim 1, further comprising the step of determining binding of said cells to said MHC-antigen complexes using a site-specific assay. An array comprising a plurality of discrete regions of MHC-antigen complexes stably bound to the surface of a solid support, wherein the density of spots is at least 1/cm2 and no more than 40,000/cm2, and a population of cells bound to said MHC-antigen complexes. The matrix of claim 35, wherein said solid support is coated with polyacrylamide. The array of claim 35, wherein said spot comprises at least about 0.01 ng of said MHC-antigen complex. The array of claim 35, wherein said population of cells is differentially labeled. The array of claim 35, wherein each of said regions comprises a plurality of different antigen complexes. The array of claim 35, wherein one or more of said MHC-antigen complexes comprises an antigen selected from the group consisting of tumor antigens; viral antigens, bacterial antigens; parasitic antigens; environmental antigens; allergens; and autoimmune antigens. The array of claim 35, wherein said population of cells has multiple T cell antigen specificities. The array according to claim 35, wherein said MHC-antigen complexes comprise a library of antigenic peptides that form a complex with MHC. The array according to claim 35, wherein said array additionally comprises one or more spots containing signal probes. The array according to claim 43, wherein said signal probes contain candidate pharmacologically active drugs. The array according to claim 43, wherein said signal probes contain a peptide library. The matrix according to claim 43, wherein said signal probes are printed together with said MHC-antigen complexes. The array according to claim 43, wherein said signal probes are located next to said MHC-antigen complexes. The array according to claim 43, in which said signal probes contain candidate agents. The array according to claim 43, characterized in that said signal probe is selected from the group consisting of cytokines, chemokines and interleukins. The matrix according to claim 35, characterized in that said matrix contains a plurality of spots having different concentrations of MHC-antigen complexes.

Briefly, the essence is T-lymphocyte profiling, in which cells are profiled for their expression of antigen receptors and the ability to respond to external stimuli in the microenvironment. External stimuli include cell-cell interactions, response to factors, etc. The cells are arrayed on a flat or three-dimensional support by binding to immobilized or partially diffusible cells and MHC-antigen complexes. Additional probes can also be arrayed in conjunction with MHC-antigen complexes, including signaling cues that regulate cellular responses, adhesion molecules, differentiation factors, etc. Once the cells are arrayed, they can be characterized for antigen receptor expression and other phenotypic features, such as expression of other cell surface markers; or maintained in culture for a period of time sufficient to determine the response to the stimulus of interest. Each of the MHC-peptide complex "spot" arrays forms a multivalent plane of antigen, self-antigen, presented in the context of a specific MHC molecule. This array is used to select T cells in a complex population that are capable of binding this antigen.

The disadvantage of the prototype is the low specificity of detection of autoreactive T-lymphocytes, which results in low sensitivity of the method and, accordingly, low reliability of its results. The reason is the known fact that autoimmune T-lymphocytes carry T-cell receptors with low affinity to the related antigen, as a result of which MHC molecules do not bind to the T-cell receptor, which leads to a false negative reaction, namely, detection of less than 0.05% of the population of autoreactive T-lymphocytes. Another disadvantage of the prototype is the multi-stage nature, time-consuming and high cost of both the analysis itself and the process of synthesis of MHC molecules. MHC molecules are unstable when they are not part of a complex with an antigen. For this reason, the technology is limited by the long-term production of antigen-MHC molecules, since each antigen-MHC peptide requires an individual assembly and purification procedure. In addition, the development of high-throughput strategies for T-cell identification is limited by the stage that includes the creation of large antigen-MHC libraries, the need to synthesize more than 3 different sequences of antigen-MHC complexes. The disadvantage of the method is also the need to involve highly qualified specialists and specialists of a narrow profile. Due to its features, the method is realistically feasible mainly in large research centers. It is unlikely to be used in the laboratories of the overwhelming majority of health care organizations that are not equipped with special devices and the appropriate personnel to service this equipment.

Thus, in more detail the disadvantages of the prototype are:

1 – insufficient (unsatisfactory) reliability of diagnostic results performed by laboratory testing and detection of autoreactive lymphocytes;

2 – multi-stage, time-consuming process and high cost (high cost of reagents and process) of its implementation;

3 – the need for the participation of highly qualified personnel to implement the prototype.

The technical result of the claimed technical solution is the development of a method for diagnosing autoimmune thyroiditis, the use of which achieves:

1 – increasing the reliability and accuracy of diagnostics of autoimmune thyroiditis by performing diagnostics using the thyroid peroxidase antigen with L-azidohomoalanine label;

2 – simplification (lower steps and time consumption) of the diagnostic process;

3 – no need to attract highly qualified personnel.

The essence of the claimed technical solution is a method for diagnosing autoimmune thyroiditis, which consists in isolating mononuclear cells from venous blood on a Ficoll 1.077 density gradient, which are incubated with a thyroid peroxidase antigen labeled with L-azidohomoalanine; then fixing them in a 4% paraformaldehyde solution for 15 minutes; then washing them and incubating them in a solution of 100 mM copper (II) sulfate and 1 μM green fluorescent dye Alexa Fluor 488 in a volume of 500 μl for 15 minutes; then the mononuclear cells are washed by centrifugation for 5 min, after which 500 μl of phosphate-buffered saline solution are added and they are analyzed using flow laser cytofluorometry: the sample is irradiated with a laser beam with a wavelength of 488 nm, the irradiation result is detected in the FL1 channel of the cytometer with a monitor, while the binding of lymphocytes to the thyroid peroxidase antigen is established; if there are more than 5% of positively fluorescent cells in the sample when irradiated with a laser beam, autoimmune thyroiditis is diagnosed, if there are less than 5% of positively fluorescent cells in the sample, the absence of autoimmune thyroiditis is diagnosed.

The claimed technical solution is illustrated in Fig. 1–3.

Fig. 1 shows the detection by cytofluorometry of the binding of lymphocytes from the peripheral blood of a healthy donor to the thyroid peroxidase antigen, which contains L-azidohomoalanine.

Fig. 2 shows the detection by cytofluorometry of the binding of peripheral blood lymphocytes of a patient with seropositive AIT to the thyroid peroxidase antigen, which contains L-azidohomoalanine.

Fig. 3 shows the detection by cytofluorometry of the binding of peripheral blood lymphocytes of a patient with seronegative AIT to the thyroid peroxidase antigen, which contains L-azidohomoalanine.

The applicant then provides a description of the claimed technical solution.

The claimed technical result is achieved by implementing the claimed method for diagnosing autoimmune thyroiditis.

The claimed method is generally carried out according to the following sequence of actions:

1 – mononuclear cells are isolated from venous blood on a Ficoll 1.077 density gradient, for example, by centrifugation;

2 – then the isolated mononuclear cells are incubated with thyroid peroxidase antigen labeled with L-azidohomoalanine for, for example, 1 min;

3 – then fix the incubated mononuclear cells in a 4% paraformaldehyde solution for 15 minutes;

4 – then the cells are washed and incubated in a solution of 100 mM copper (II) sulfate and 1 μM green fluorescent dye Alexa Fluor 488 in a volume of 500 μl for 15 min; then the incubated sample is centrifuged for 5 min, after which 500 μl of phosphate-buffered saline solution is added, a sample is obtained for analysis by flow cytofluorometry;

5 – then the mononuclear cells are washed and analyzed using flow laser cytofluorometry in the FL1 channel of a cytometer with a monitor, detecting the presence or absence of fluorescence, and establishing the presence or absence of lymphocyte binding to the thyroid peroxidase antigen;

6 – if there is fluorescence of the sample (in the area designated in Fig. 2 under the number 2), the presence of autoimmune thyroiditis is diagnosed; if there is no fluorescence of the sample (in the area designated in Fig. 1 under the number 1), the absence of autoimmune thyroiditis is diagnosed.

Based on the above, it seems possible to draw logically sound conclusions that in the claimed technical solution, in contrast to the prototype (where detection of autoreactive lymphocytes is performed using a fluorescent antigen-MHC complex), for the diagnosis of autoimmune thyroiditis, an analysis of the number of lymphocytes in % (percentage) associated with the L-azidohomoalanine-labeled thyroid peroxidase antigen is used, with the receipt of an instant and 100% reliable result.

Below is a detailed sequence of actions for the claimed method:

1 – mononuclear cells are isolated from venous blood on a Ficoll 1.077 density gradient, for example, by centrifugation, for which:

a blood sample is taken from the cubital vein in a known manner, for example, 3 ml, into a test tube containing sodium heparin;

then dilute with phosphate buffered saline solution in equal volume proportions;

then take a test tube containing a density gradient solution (ρ) Ficoll ρ = 1.077;

Next, take a blood sample diluted with phosphate-buffered saline solution and carefully apply it to the Ficoll along the wall of the test tube;

then the diluted blood sample is centrifuged, for example, for 40 minutes at 1500 rpm, to obtain a fraction of venous blood mononuclear cells;

then an equal volume of phosphate buffered saline solution is added to the suspension obtained after centrifugation (venous blood mononuclear cells) and centrifuged for 5 minutes at 1500 rpm to obtain a cell sediment;

2 – then 500 μl of phosphate-buffered saline solution is added to the cell sediment and the contents of the test tube are mixed to obtain a homogeneous sample, to which 2 μl of L-azidohomoalanine-labeled thyroid peroxidase antigen are added, a cell suspension is obtained, which is incubated for 1 min and centrifuged for 5 min at 1500 rpm,

3 – after which the cell sediment is fixed in a 4% paraformaldehyde solution for 15 minutes;

4 – then the fixed cells are washed by centrifugation, for example, 5 min, add, for example, 500 μl of a solution containing 100 mM copper (II) sulfate and 1 μM green fluorescent dye Alexa Fluor 488 and incubate for 15 min;

5 - then the mononuclear cells are washed by centrifugation for 5 minutes, after which 500 μl of phosphate-buffered saline solution is added and the analysis is performed using flow laser cytofluorometry in the FL1 channel of a cytometer with a monitor, while the presence or absence of fluorescence is detected, thereby establishing the presence or absence of binding of lymphocytes to the thyroid peroxidase antigen;

6 – if there is fluorescence of the sample (in the area designated in Fig. 2 under the number 2), the presence of autoimmune thyroiditis is diagnosed; if there is no fluorescence of the sample (in the area designated in Fig. 1 under the number 1), the absence of autoimmune thyroiditis is diagnosed.

To explain the claimed technical solution, flow cytofluorimetry graphs are provided (see Figs. 1–3), where the x-axis shows the logarithmic scale of fluorescence intensity from 10 ⁰ to 10 ⁴ , the y-axis shows the number of cells, number 1 shows the percentage of lymphocytes not bound to the L-azidohomoalanine-labeled thyroid peroxidase antigen, and number 2 shows the percentage of lymphocytes bound to the L-azidohomoalanine-labeled thyroid peroxidase antigen.

Fig. 1 shows the percentage of lymphocytes not bound to L-azidohomoalanine-labeled thyroid peroxidase in the peripheral blood of healthy donors.

The characteristic indicators of lymphocytes in the blood of a healthy donor are as follows: no binding to the thyroid peroxidase antigen.

Fig. 2 shows the percentage of lymphocytes bound to L-azidohomoalanine-labeled thyroid peroxidase in the peripheral blood of a patient with seropositive autoimmune thyroiditis.

The indices of blood lymphocytes of a patient with seropositive autoimmune thyroiditis are as follows: the presence of a positive signal of binding to the thyroid peroxidase antigen. Detection in 5 - 99% of the lymphocyte population.

Fig. 3 shows the percentage of lymphocytes bound to L-azidohomoalanine-labeled thyroid peroxidase in the peripheral blood of a patient with seronegative autoimmune thyroiditis.

The indices of blood lymphocytes of a patient with seronegative autoimmune thyroiditis are as follows: the presence of a positive fluorescent signal of binding to the thyroid peroxidase antigen. Detection in 5-99% of the lymphocyte population.

Based on the analysis of the percentage of lymphocytes bound to the antigen of thyroid peroxidase, the presence or absence of autoimmune thyroiditis in the diagnosed patient is diagnosed. If the sample has fluorescence, the presence of autoimmune thyroiditis is diagnosed; if the sample has no fluorescence, the absence of autoimmune thyroiditis is diagnosed. The developed technology allows detecting the presence of autoreactive lymphocytes in patients with both seropositive autoimmune thyroiditis and seronegative autoimmune thyroiditis.

The implementation of the claimed method is shown by the given examples.

Example 1. A method for diagnosing autoimmune thyroiditis, i.e. identifying autoreactive lymphocytes, performed in accordance with the formula of the declared technical solution

Patient B.A.V., 23, suffers from autoimmune thyroiditis. Clinical euthyroidism, hypertrophic form. First diagnosed at the age of 18. Heredity - autoimmune thyroiditis in the mother, father, maternal grandmother and paternal grandmother. There are no concomitant autoimmune diseases. Clinical examination: thyroid peroxidase antibody titer 2235 IU/ml, free thyroxine (T4) 14.33 pmol/l, thyroid stimulating hormone (TSH) 2.56 μIU/ml. Ultrasound examination: diffuse enlargement of the thyroid gland, the presence of multiple hypoechoic areas, single enlarged submandibular lymph nodes on both sides. Thyroid gland volume is 27.4 cm ³ .

A repeat diagnosis of autoimmune thyroiditis was carried out using the stated method:

– a blood sample of 3 ml was taken from the cubital vein in a known manner into a test tube containing sodium heparin (manufactured by EUROTUBO, Spain);

– then the collected blood sample was diluted with phosphate-buffered saline solution in equal proportions sample: buffer solution = 1: 1;

– then we took a 50 ml test tube (Nunc) containing a density gradient solution ρ Ficoll (ρ = 1.077);

– then they took a blood sample diluted with a buffer solution and carefully applied it to the Ficoll along the wall of the test tube;

– then the diluted blood sample was centrifuged for 40 minutes in a bucket rotor at 1500 rpm in an Eppendorf-5810R centrifuge (Germany, Hamburg) to obtain a fraction of mononuclear cells;

– then an equal volume of phosphate-buffered saline solution was added to the fraction obtained after centrifugation (peripheral blood mononuclear cells) and centrifuged for 5 minutes at 1500 rpm to obtain a cell pellet;

– then 500 μl of phosphate-buffered saline solution is added to the resulting cell sediment and the contents of the test tube are mixed to obtain a homogeneous sample; 2 μl of L-azidohomoalanine-labeled thyroid peroxidase antigen are added to the homogeneous sample to obtain a cell suspension;

– then the cell suspension is incubated for 1 min and centrifuged for 5 min at 1500 rpm, after which the cell sediment is fixed in a 4% paraformaldehyde solution for 15 min;

– then the fixed cells are centrifuged for 5 min, 500 µl of a solution containing 100 mM copper (II) sulfate and 1 µM green fluorescent dye Alexa Fluor 488 are added and incubated for 15 min;

– then the incubated sample is centrifuged for 5 minutes, after which 500 µl of phosphate-buffered saline solution is added to the sample, and a sample is obtained for analysis by flow cytometry;

– then the sample is irradiated with a laser beam with a wavelength of λ = 488 nm, the result of the irradiation is detected in the FL-1 channel of the flow cytometer and the presence or absence of fluorescence of the sample is observed on the monitor screen in the form of a diagram, for example, Fig. 2;

– if there are more than 5% positively fluorescent cells in the sample, autoimmune thyroiditis is diagnosed; if there are less than 5% positively fluorescent cells in the sample, the absence of autoimmune thyroiditis is diagnosed.

According to the flow cytometry data obtained using the claimed method, 99% of lymphocytes bound to the thyroid peroxidase antigen containing the L-azidohomoalanine label.

The presence of lymphocytes associated with the antigen of thyroid peroxidase containing the L-azidohomoalanine label allows diagnosing the presence of autoimmune thyroiditis. The diagnosis, instrumentally determined by the declared method, reliably coincides with the previously established diagnosis established by traditional methods - with the results of a physical examination of the thyroid gland, the results of laboratory blood tests and ultrasound studies.

Example 2. Method for diagnosing autoimmune thyroiditis by determining autoreactive lymphocytes

Patient A.L.I., 21 years old. Heredity is burdened - her grandmother has autoimmune thyroiditis. Clinical examination: titer of antibodies to thioreoperoxidase 3.51 IU/ml, free thyroxine (T4) 13.18 pmol/l, thyroid-stimulating hormone (TSH) 2.2816 μIU/ml. As a result of treatment based on a questionable diagnosis, no noticeable improvement in the patient's condition was observed. Additional ultrasound examination: hypoplasia of the thyroid gland. Thyroid gland volume is 9.1 cm ³ .

Autoimmune thyroiditis was diagnosed using the stated method:

– a blood sample of 3 ml was taken from the cubital vein in a known manner into a test tube containing sodium heparin (manufactured by EUROTUBO, Spain);

– then the collected blood sample was diluted with phosphate-buffered saline solution in equal volume ratios sample: buffer solution = 1: 1;

– then we took a 50 ml test tube (Nunc) containing a density gradient solution ρ Ficoll (ρ = 1.077);

– then they took a blood sample diluted with a buffer solution and carefully applied it to the Ficoll along the wall of the test tube;

– then the diluted blood sample was centrifuged for 40 minutes in a bucket rotor at 1500 rpm in an Eppendorf-5810R centrifuge (Germany, Hamburg) to obtain a fraction of mononuclear cells;

– then an equal volume of phosphate-buffered saline solution is added to the fraction obtained after centrifugation (peripheral blood mononuclear cells) and centrifuged for 5 minutes at 1500 rpm to obtain a cell sediment;

– then 500 μl of phosphate-buffered saline solution is added to the cell sediment and the contents of the test tube are mixed to obtain a homogeneous sample; 2 μl of L-azidohomoalanine-labeled thyroid peroxidase antigen is added to the homogeneous sample to obtain a cell suspension;

– then the cell suspension is incubated for 1 min and centrifuged for 5 min at 1500 rpm, after which the cell sediment is fixed in a 4% paraformaldehyde solution for 15 min;

– then the fixed cells are centrifuged for 5 min, 500 µl of a solution containing 100 mM copper (II) sulfate and 1 µM green fluorescent dye Alexa Fluor 488 are added and incubated for 15 min;

– then the incubated sample is centrifuged for 5 minutes, after which 500 µl of phosphate-buffered saline solution is added to the sample, and a sample is obtained for analysis using the known flow cytometry method;

– then the sample is irradiated with a laser beam with a wavelength of λ = 488 nm, the result of the irradiation is detected in the FL-1 channel of the flow cytometer and the presence or absence of fluorescence of the sample is observed on the monitor screen in the form of a diagram, for example, Fig. 2;

– if there are more than 5% positively fluorescent cells in the sample, autoimmune thyroiditis is diagnosed; if there are less than 5% positively fluorescent cells in the sample, the absence of autoimmune thyroiditis is diagnosed.

According to the flow cytometry data obtained by the claimed method, 91.5% of lymphocytes bound to the thyroid peroxidase antigen containing the L-azidohomoalanine label, which ensures highly reliable diagnosis of the presence of autoimmune thyroiditis. The diagnosis determined instrumentally by the ultrasound method reliably coincides with the diagnosis by the claimed method.

The diagnosis determined instrumentally by the stated method does not coincide with the previously established (without the use of ultrasound) diagnosis established on the basis of a study of the symptoms of the disease, a physical examination of the thyroid gland and the results of laboratory tests to detect antibodies to thyroid peroxidase in the blood serum - according to the prototype (the disadvantage of the prototype is the low specificity of detection of autoreactive T-lymphocytes).

The claimed method, based on the detection of antigen-associated thyroid peroxidase and fluorescent autoreactive lymphocytes, ensures unambiguous diagnosis of the presence of autoimmune thyroiditis.

Based on the diagnosis established using the claimed method, the patient's treatment process was adjusted. After 6 months of treatment, according to the adjusted course of the treatment process, physical examinations showed positive changes in the course of the patient's disease - the thyroid gland pain during palpation disappeared, the patient's well-being improved.

Improvement in the patient's health indicates the correctness of the diagnosis and the adjusted course of treatment established using the claimed method.

The applicant explains that Examples 1 and 2 are given as an illustration and do not limit the possibilities of implementing the claimed method for diagnosing autoimmune thyroiditis.

From the described Examples 1 and 2, it can be concluded that the applicant has achieved the claimed technical result, namely: a method for diagnosing autoimmune thyroiditis has been developed, expanding the arsenal of methods for diagnosing autoimmune thyroiditis, thereby achieving:

– increasing the reliability and accuracy of diagnostics by using the thyroid peroxidase antigen with an L-azidohomoalanine label and identifying from 5% to 99% of autoreactive lymphocytes (Example 1, Example 2, Fig. 1, Fig. 2, Fig. 3), detecting autoreactive lymphocytes in both seropositive and seronegative autoimmune thyroiditis (Examples 1, Example 2, Fig. 2, Fig. 3);

– simplification (lower steps and time consumption) of the diagnostic process;

– no need to attract highly qualified personnel.

The claimed method is applicable for diagnosing diseases in endocrinology and immunology for reliable diagnosis of autoimmune thyroiditis in the presence of doubts about the reliability of the diagnosis, for example, caused by unsatisfactory results of the treatment process and/or the reliability of the diagnosis previously established on the basis of the use of known methods - physical examinations, laboratory blood tests, ultrasound of the person being diagnosed.

The claimed technical solution satisfies the patentability condition of “novelty” imposed on inventions, since when determining the level of technology, no means was found that have features identical (that is, coinciding in the function they perform and the form of execution of these features) to all the features listed in the invention formula, including the characteristic of the purpose.

The claimed technical solution invention satisfies the patentability condition of “inventive step” imposed on inventions, since no technical solutions have been identified that have features that coincide with the distinctive features of this invention, and the knowledge of the influence of the distinctive features on the specified technical result has not been established.

The claimed invention satisfies the patentability condition “industrial applicability” imposed on inventions, since it (the technical solution) can be implemented in the industrial production of diagnostic equipment, in the activities of healthcare organizations, through the use of known standard technical devices and equipment.

Claims (1)

A method for diagnosing autoimmune thyroiditis, which consists of isolating mononuclear cells from venous blood on a Ficoll 1.077 density gradient, incubating them with a thyroid peroxidase antigen labeled with L-azidohomoalanine; then fixing them in a 4% paraformaldehyde solution for 15 min; then washing them and incubating them in a solution of 100 mM copper (II) sulfate and 1 μM Alexa Fluor 488 green fluorescent dye in a volume of 500 μl for 15 min; then the mononuclear cells are washed by centrifugation for 5 min, after which 500 μl of phosphate-buffered saline solution are added and they are analyzed using flow laser cytofluorometry: the sample is irradiated with a laser beam with a wavelength of 488 nm, the irradiation result is detected in the FL1 channel of the cytometer with a monitor, while the binding of lymphocytes to the thyroid peroxidase antigen is established; if there are more than 5% of positively fluorescent cells in the sample when irradiated with a laser beam, autoimmune thyroiditis is diagnosed, if there are less than 5% of positively fluorescent cells in the sample, the absence of autoimmune thyroiditis is diagnosed.