RU2524616C1 - Diagnostic technique for early stage of rheumatoid arthritis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: bone marrow and synovium is sampled from a patient to produce preparations for immunohistological and immunocytochemical analyses. That is followed by determining a portion of Mdm2 protein synthesis cells by formula: $$A{I}_{Mdm2}=\frac{N_{Mdm2}\cdot 100}{N_{total}},$$ wherein AI_Mdm2 is a portion of Mdm2 protein synthesis cells, %; N_Mdm2 is a Mdm2 protein synthesis cell count; N_total is the total cell count in the range of vision. An early stage of rheumatoid arthritis is diagnosed if observing AI_Mdm2=81.6-88.8% in the patient's synovium, and AI_Mdm2=85.0-89.2% in the patient's bone marrow.

EFFECT: using the given method enables diagnosing the disease at the early stage and starting adequate basic therapy for treating and delaying the progression of rheumatoid arthritis.

3 ex, 1 tbl, 1 dwg

Description

The invention relates to the field of predictive (predictive) medicine and can be used in the diagnosis of rheumatoid arthritis at an early stage of the disease.

In recent years, rheumatic diseases have attracted increasing attention of scientists due to their high prevalence and social significance. Among rheumatic diseases, rheumatoid arthritis (RA) is of particular theoretical and medical and social significance. The prevalence of RA in the population is 0.5-1.5% [1]. Affecting mainly people of working age, RA leads to early disability, about 50% of patients lose their ability to work during the first 5 years of the disease, the life expectancy of patients is reduced by 8-10 years. According to statistics, in Russia in 2002, 280,000 patients with RA were registered, among whom more than 26,000 were first-time cases. About 300,000 patients are registered annually in the Russian Federation [2].

RA in the advanced stage is characterized by a progressive course with the formation of joint deformities, the development of severe functional disorders and damage to internal organs. According to epidemiological studies, the frequency of cardiovascular events in patients with rheumatoid arthritis is 3.96 times higher than in the population and 30-60% higher than in patients with other joint pathologies. A survey of studies showed that the direct cost of RA by 2000 was about 6,000 euros per year. The deterioration of joint function brings the greatest financial loss to society, since the cost of the disease is proportional to the increase in functional failure (FN). It has been shown that total costs increase from 5,000 euros per patient with minimal FN to 20,000 euros per patient with severe FN. The total cost of RA in Western and Eastern Europe for 2006 is from 42 billion to 3.4 billion euros per year, respectively [1].

Currently, diagnostic criteria for RA have been developed, including: articular syndrome, radiological changes. Laboratory markers of this disease include class M immunoglobulins autoantibodies that react with the Fc fragment of class G immunoglobulin, or rheumatoid factor (RF) - class M immunoglobulin and antibodies to the cyclic citrified peptide (anti-CCP antibodies).

Unfortunately, none of the currently developed RA criteria is strictly specific for this disease. Moreover, using the available criteria, it is very difficult to diagnose RA at a very early and early stage of the disease. For example, in “conditionally” healthy people who subsequently became ill with RA, various immunological abnormalities are found that are characteristic of RA (an increase in the level of the RF, anti-PCP antibodies, C - reactive protein), long before the first clinical symptoms of the disease appear. In 2/3 patients, structural changes (erosion) of the joints occur very quickly, already within the first two years from the onset of the disease.

It was established that the prevention of structural damage in the debut of RA contributes to the preservation of the functional activity of patients in the long term. However, the period of time when active therapy with basic anti-inflammatory drugs can effectively inhibit the progression of joint damage (the so-called “window of opportunity”) is very short and sometimes only takes a few months from the onset of the disease.

Obviously, RA is a disease in which the long-term prognosis directly depends on how early the diagnosis was made and pharmacotherapy started [3]. Thus, the development of new methods for diagnosing RA at an early stage of the disease is a promising area of predictive (predictive) medicine.

A known method for the preclinical diagnosis of juvenile rheumatoid arthritis (see RF patent 2195856, IPC АВВ 3/10, publication date 10.01.2003) by biomicroscopy of the cornea and iris of the eyes using a slit lamp, according to which, potential juvenile rheumatoid arthritis is exposed when keratopathy is detected , Aksenfeld's symptom, pale iris, narrowing or star shape of the pupil girdle, poor differentiation of the Krause line, hypoplasia or dysplasia of the anterior leaflet, gaps in the outer segment s, "embryonic" such as the iris, "scalloped" peripheral parts of the ciliary zone.

The disadvantage of this invention is the low efficiency and narrow scope due to age restrictions of patients.

As the closest analogue, a test method for the presence of rheumatic disease was taken (see RF patent 2173462, IPC G01N 33/48, G01N 33/68, publication date 09/10/2001), including the study of joint elements, analysis of the results of the study and diagnosis of rheumatic disease according to the results research.

The disadvantage of the closest analogue is the increased complexity of the process and low analysis efficiency due to the subjectivity of the morphological assessment of crystal forms, as well as the lack of a quantitative assessment of diagnostic parameters and their parameters.

The technical task of the claimed invention is the development of an effective diagnostic method for determining rheumatoid arthritis at an early stage of the disease.

The technical result achieved by solving the problem is expressed in expanding the scope due to the possibility of diagnosing the disease at its early stage, as well as increasing the efficiency of diagnosis due to the following factors:

- an additional study of bone marrow samples can improve the information content and reliability of the method;

- conducting immunohistochemical and immunocytochemical studies allows you to determine the number and localization of cells synthesizing the protein Mdm2;

- the detection of anti-apoptotic protein, namely Mdm2, allows you to establish a diagnostic indicator specific for rheumatoid arthritis in the early stages.

The problem is solved in that in a method for diagnosing an early stage of rheumatoid arthritis, including the study of joint elements, analysis of the results of the study and diagnosis of rheumatic disease according to the results of the study, samples of the bone marrow and synovial membrane are taken from the patient, from which preparations are prepared for immunohistochemical and immunocytochemical studies, after which the proportion of cells synthesizing the Mdm2 protein is determined by the formula:

, $$\mathrm{BUT}{\mathrm{AND}}_{Mdm2}=\frac{N_{Mdm2}\cdot \mathrm{one\; hundred}}{N_{\mathrm{about}bu}}$$

,

where AI _Mdm2 is the proportion of cells synthesizing the protein Mdm2,%;

N _Mdm2 - the number of cells synthesizing the protein Mdm2;

N _total - the total number of cells in the field of view;

and in the case when in the samples of the synovial membrane AI _Mdm2 = 81.6-88.8%, and in bone marrow samples AI _Mdm2 = 85.0-89.2%, the patient is diagnosed with an early stage of rheumatoid arthritis.

Figure 1 shows the bone marrow cells of a patient with an early stage of rheumatoid arthritis, synthesizing the protein Mdm2.

A bone marrow smear was stained by the immunohistochemical method with antibodies to the Mdm2 protein (green immunofluorescence) (arrow). The scale bar corresponds to 20 microns.

Morphologically, RA is characterized by hyperplasia of synovial tissue and its invasion into cartilage and bone, followed by their destruction [4, 5]. These changes are the result of an imbalance between the multiplication of the inner layer of the synovial membrane by dividing, in particular, synovial macrophages and fibroblasts on the one hand and programmed cell death (apoptosis) of these cells on the other [5]. The key to blocking the cell cycle and stimulating apoptosis in cells is the synthesis of the P53 protein, which is responsible for the synthesis of another cellular protein, namely P21. Protein P21 inhibits the G1 phase of the cell cycle, and also affects the synthesis of the Mdm 2 protein, which is responsible for blocking apoptosis and the PUMA protein, which is responsible for triggering apoptosis involving mitochondria. [6]. Tumor-like growth of synoviocytes plays a significant role in the progression of RA, and inadequate apoptosis of synovial fibroblasts makes a significant contribution to these processes. Thus, anti- and proapoptotic proteins can act as diagnostic markers of RA at different stages.

In an in-depth study of the role of apoptosis in the pathogenesis of RA and the dependence of the activity of programmed cell death on the stage of RA, a number of researchers obtained the following results. It turned out that the prevalence of apoptosis correlates with the duration and severity of the inflammatory infiltration of the synovial membrane. In the early stages of RA, a low apoptosis rate was recorded. At a late stage, a significant activation of apoptosis was revealed [1]. Accordingly, the determination of an anti-apoptotic protein, namely Mdm2, is a reliable way to diagnose RA at an early stage.

The method is as follows.

At the initial stage, a patient with RA is sampled of biological material. Bone marrow (CM) samples are taken by sternal puncture under local anesthesia; in parallel, at the time of arthroscopy, the samples of the synovial membrane (CO) are taken.

At the second stage, preparations are prepared for further studies.

For this, bone marrow smears are prepared and dried at room temperature for 10 minutes. Then the bone marrow smears are fixed in 4% paraform for 5 minutes, and then dried.

Frozen sections from samples of the synovial membrane are also prepared. Preliminarily, the samples of the synovial membrane are fixed in a 4% paraform solution for 1 hour, and then washed in a solution of phosphate-saline buffer. Next, a fixed sample of the synovial membrane is placed in a 15% sucrose solution in phosphate-buffered saline for 24 hours, and then placed in Neg 50 medium (Thermo Scientific, USA) and frozen on a freezing table. Sections of a thickness of 10-15 μm were prepared from frozen samples of the synovial membrane on a cryostat (Thermo Scientific, USA).

Then, bone marrow smears and frozen sections of the synovial membrane are boiled in citrate buffer (pH 6.0) for 20 minutes. Then the cooled smears of the bone marrow and sections of the synovial membrane are washed three times in distilled water for 5 minutes.

At the third stage, they conduct research.

An immunohistochemical study is performed by staining fixed smears of bone marrow and frozen sections of the synovial membrane, which are previously washed in phosphate-buffered saline with primary antibodies at a dilution of PUMA - 1: 100, P53 - 1: 100, Mdm2 - 1:30, P21 - 1:30 . Then washed three times with smears of bone marrow and frozen sections of the synovial membrane stained with primary antibodies PUMA, P53, Mdm2 and P21 phosphate-buffered saline. Then smears stained with primary antibodies were treated with secondary antibodies (anti-rabbit or anti-mouse depending on the primary) labeled with Alexa (488, 546, 594). Then smears stained with primary antibodies are treated with secondary antibodies (anti-rabbit or anti-mouse, depending on the primary) labeled with Alexa (488, 546, 594).

An immunohistochemical study of bone marrow smears and frozen sections of the synovial membrane was carried out using a biological microscope Axio Imager Z2 (Carl Zeiss, Germany, 2009), which is equipped with a DIC system, a 100 W halogen illuminator, a fluorescent illuminator with an HXR lamp, and highly sensitive digital Axio Cam MRM and Axio Cam HRC cameras. A-Plan lenses: 10x / 0.25, 40x / 0.65, 100x / 1.25 Oil, eyepieces E-PL 10x / 20.

An immunocytochemical study of bone marrow smears and frozen sections of the synovial membrane is also carried out using a Libra 120 transmission electron microscope (Carl Zeiss, Germany, 2005) with the following characteristics: transmission electron microscopy, acceleration voltage of 120 kV, max. magnification 630000, resolution 3.2 A.

At the final stage, an analysis of the results is carried out.

In the process of quantitative analysis, the proportion of cells synthesizing proteins is determined by the formulas:

, $$\mathrm{BUT}{\mathrm{AND}}_{P53}=\frac{N_{P53}\cdot \mathrm{one\; hundred}}{N_{\mathrm{about}bu}}$$

,

, $$\mathrm{BUT}{\mathrm{AND}}_{P21}=\frac{N_{P21}\cdot \mathrm{one\; hundred}}{N_{\mathrm{about}bu}}$$

,

, $$\mathrm{BUT}{\mathrm{AND}}_{PUMA}=\frac{N_{PUMA}\cdot \mathrm{one\; hundred}}{N_{\mathrm{about}bu}}$$

,

, $$\mathrm{BUT}{\mathrm{AND}}_{Mdm2}=\frac{N_{Mdm2}\cdot \mathrm{one\; hundred}}{N_{\mathrm{about}bu}}$$

,

where AI _P53 , AI _P21 , AI _PUMA , AI _Mdm2 — percentage of cells synthesizing P53, P21, PUMA and Mdm2, respectively,%;

_{N P53, N P21, N PUMA} , N Mdm2 - number of cells synthesizing protein, respectively P53, P21, PUMA and Mdm2;

N _total - the total number of cells in the field of view.

At the same time, the number of cells synthesizing the studied proteins (N _P53 , N _P21 , N _PUMA , N _Mdm2 ) is determined by the glow (fluorescence) in green. The data obtained as a result of the research are processed by the method of variation statistics with the determination of the t-criterion of reliability according to Student's test (P <0.05).

In the case when in the samples of the synovial membrane AI _Mdm2 = 81.6-88.8%, and in samples of the bone marrow AI _Mdm2 = 85.0-89.2%, the patient is diagnosed with an early stage of rheumatoid arthritis.

In Vladivostok State Medical University for three years, special research was conducted to develop the proposed method.

For research, 2 groups of patients were formed.

The control group included 20 women with diagnosed osteoarthrosis (OA), the average age of which was 55.75 (38-70 years). In 25% (5 people) of the examined, the stage of OA corresponded to the early stage, in 75% (15 people) of the late stage of OA.

The authors of the proposed method examined 30 women whose average age was 60.52 years (35-76 years) with a reliable diagnosis of RA (according to diagnostic criteria ACR / EULAR 2010). In 35% of the examined patients, the stage of RA corresponded to the very early and early stages, in 22% to the advanced stage and in 43% to the late stage of RA.

In all examined patients, regardless of the stage of RA and the degree of activity of the disease, the presence of synovial membrane stavas and smears of red bone marrow proteins P53, P21, PUMA and Mdm2 was revealed in the cells of the studied tissue samples (Table 1).

Table 1 Expression of small molecules (P53, P21, PUMA and Mdm2) in patients with RA according to the results of immunohistochemical studies Apoptotic proteins The studied biomaterial (bone marrow - KM, synovial membrane - СО) KM With KM CO Early stage of RA (n = 30) Late stage of RA (n = 30) P53 11.5% ± 1.40 12.1% ± 2.80 90.9% ± 1.6 * 90.4% ± 3.4 * P21 9.6% ± 1.8 5.4% ± 1.9 15.8% ± 1.6 16.1% ± 2.7 PUMA 2.1% ± 1.10 5.4% ± 2.30 98.6% ± 1.5 * 99.1% ± 3.1 * Mdm2 87.1% ± 2.1 * 85.2% ± 3.6 * 15.4% ± 2.40 13.6% ± 2.80 Note: * - significance of differences with the control group (p <0.05); ◇ - significance of differences between groups of early and late RA (p <0.05)

In the early stage of RA, the maximum amount of Mdm2 protein was determined (a significant difference with the control group), which decreased in the late stage of RA (significant difference with the group of early RA). At the same time, the number of cells containing P53 and PUMA protein molecules was low at the early stage of RA (did not differ from the control) and increased sharply towards the late stage of RA, significantly differing from the control group and the group of early RA. The presence of the studied proteins in cells in patients from the control group did not exceed 10%, which corresponds to published data [7].

Currently, the phenomenon of apoptosis is considered as one of the leading links in the pathogenesis of autoimmune diseases, in particular, RA. After analyzing the results of this study, it can be argued that the number of cells expressing the Mdm2 molecule in the cells of the bone marrow and synovial membrane (synoviocytes) exceeds normal values, namely more than 10% of the total number of cells in the preparation at an early stage of RA.

Example 1. Patient N., 45 years old. I turned to a rheumatologist about pains and swelling in the joints of the hands, morning stiffness for up to two hours. The above complaints worry the patient about 6 months. An objective examination revealed: soreness of 6 joints, swelling of 4 joints. On examination:

- clinical blood test: red blood cells 4.5 · 1012, hemoglobin 130 g / l, white blood cells 8.7 · 109, platelets 380 · 109, ESR 40 mm / hour;

- rheumatoid factor immunoglobulin M = 204.9 (RU / ml);

- antibodies to CCP = 50.9 (RU / ml);

- Radiography of the hands and feet: signs of periarticular osteoporosis.

According to the results of clinical and laboratory data, the patient was given a preliminary diagnosis: rheumatoid arthritis, early stage, high degree of activity, non-erosive version, R I st., Seropositive for RF and Ig M and antibodies to PCP, FC-3 st.

A bone marrow was taken from the patient for the purpose of conducting immunohistochemical and immunocytochemical studies and subsequent determination of the number of cells synthesizing the Mdm2 protein.

As a result of the analysis of bone marrow smears, 87.4% of the cells synthesizing the Mdm2 protein were found from the total number of cells in the studied smears. The result allows us to make a final diagnosis of rheumatoid arthritis at an early stage of the disease and, accordingly, to prescribe adequate basic therapy to treat and slow the progression of rheumatoid arthritis.

Example 2. Patient P., 36 years old. I turned to a rheumatologist about pain and swelling in the knee joints, morning stiffness up to one hour. The above complaints worry the patient about 4 months. An objective examination revealed: soreness and swelling of both knee joints. On examination:

- clinical blood test: red blood cells 3.6 · 1012, hemoglobin 110 g / l, white blood cells 10.4 · 109, platelets 280.3 · 109, ESR 45 mm / hour;

- rheumatoid factor immunoglobulin M = 520.4 (RU / ml);

- antibodies to CCP = 100.6 (RU / ml);

- Radiography of the hands and feet: signs of periarticular osteoporosis;

- X-ray of the knee: signs of synovitis of the knee.

According to the results of clinical and laboratory data, the patient was given a preliminary diagnosis: rheumatoid arthritis, early stage, high degree of activity, non-erosive version, R I st., Seropositive for RF and Ig M and antibodies to PCP, FC-3 st.

A bone marrow and synovial membrane was taken from the patient during arthroscopy in order to carry out immunohistochemical and immunocytochemical studies and then determine the number of cells synthesizing the Mdm2 protein.

As a result of the analysis, 87.4% of bone marrow cells and 82.9% of the synovial membrane cells synthesizing the Mdm2 protein were found from the total number of cells in the studied smears. The result allows us to make a final diagnosis of rheumatoid arthritis at an early stage of the disease and, accordingly, to prescribe adequate basic therapy to treat and slow the progression of rheumatoid arthritis.

Example 3. Patient B., 54 years old. I turned to a rheumatologist about pains and swelling in the joints of the hands, morning stiffness up to four hours, weakness and an increase in body temperature to 37.8 ° C. The above complaints worry the patient about 7 months. An objective examination revealed: soreness of 10 joints, swelling of 6 joints. On examination:

- clinical blood test: red blood cells 3.8 · 1012, hemoglobin 98 g / l, white blood cells 10.6 · 109, platelets 306 · 109, ESR 35 mm / hour;

- rheumatoid factor immunoglobulin M = 102.5 (RU / ml);

- antibodies to CCP = 3 0.4 (RU / ml);

- Radiography of the hands and feet: signs of erosive arthritis.

According to the results of clinical and laboratory data, the patient was given a preliminary diagnosis: rheumatoid arthritis, early stage, high degree of activity, erosive version, R I st., Seropositive for RF and Ig M and antibodies to PCP, FC-3 st.

A bone marrow was taken from the patient for the purpose of conducting immunohistochemical and immunocytochemical studies and subsequent determination of the number of cells synthesizing the Mdm2 protein.

As a result of the analysis of bone marrow smears, 88.9% of the cells synthesizing the Mdm2 protein were found from the total number of cells in the studied smears. The result allows us to make a final diagnosis of rheumatoid arthritis at an early stage of the disease and, accordingly, to prescribe adequate basic therapy to treat and slow the progression of rheumatoid arthritis.

Thus, the claimed invention is characterized by a wide scope and increased diagnostic efficiency.

Bibliography

1. Dubikov A.I. Rheumatoid arthritis, apoptosis, nitric oxide: new aspects of pathogenesis: Monograph. - Vladivostok: - Publishing house Dalnevost. University, 2004.

2. Chichasova N.V., Vladimirov S.A., Igolkina E.V., Imametdinova G.R. The burden of rheumatoid arthritis: medical and social problems. Scientific and Practical Rheumatology 2009; 1: 105-108.

3. Rheumatology: national leadership / ed. E.L. Nasonova, V.A. Nasonova. - M .: GOETAR-Media, 2008 .-- 720 p.

4. You X., Boyle D.L., Hammaker D., Firestein G.S. PUMA-mediated apoptosis in fibroblast-like synoviocytes does not require p53. Arthritis Research & Therapy 2006: Vol. 8 No. 6: 1-7.

5. Cha H.S., Bae E.K., Ahn J.K., Lee J., Ahn K.S., Koh E.M. Slug suppression induces apoptosis via Puma transactivation in rheumatoid arthritis fibroblast-like synoviocytes treated with hydrogen peroxide. Experimental and molecular medicine 2010; Vol. 42, No. 6: 428-436.

6. Castedo M., Perfettini J. Z., Roumier T., Andreau K., Medema R., Kroemer G. Cell death by mitotic catastrophe: a molecular definition. Oncogene 2004: 23: 2825-2837.

7. Tu Y., Xue H., Xia Z., Cai M., Liu X., Ma T., Zhang C. Effect of different concentrations of dexamethasone on apoptosis and expression of Fas / FasL in human osteoarthritis chondrocytes. Zhongguo Xiu Fu Chong Jian Wai Ke ZaZhi. 2012 May; 26 (5): 536-41.

Claims (1)

A method for diagnosing an early stage of rheumatoid arthritis, including examining joint elements, analyzing the results of the study, and diagnosing rheumatic disease according to the research results, characterized in that samples of the bone marrow and synovial membrane are collected from the patient, from which preparations for immunohistochemical and immunocytochemical studies are prepared, which determines the proportion of cells that synthesize the Mdm2 protein, according to the formula:

,
where AI _Mdm2 is the proportion of cells synthesizing the protein Mdm2,%;
N _Mdm2 - the number of cells synthesizing the protein Mdm2;
N _total - the total number of cells in the field of view;
and in the case when in the samples of the synovial membrane AI _Mdm2 = 81.6-88.8%, and in bone marrow samples AI _Mdm2 = 85.0-89.2%, the patient is diagnosed with an early stage of rheumatoid arthritis.

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