RU2735738C1 - Method for early diagnosis of rheumatoid arthritis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to rheumatology, and can be used for early diagnosis of rheumatoid arthritis. Method consists in adding monoclonal antibodies to TNFRI, TNFR2, CD4, CD45R0, CD45RA, CD3 into a blood sample; peripheral blood mononuclear cells are analyzed by flow cytometry. CD3^+--T-lymphocytes, naive T-helper cells (CD4⁺CD45RA⁺), T-helper memory cells (CD4⁺CD45R0⁺) are isolated by gating. Further, the TNFR1 and TNFR2 receptor expression values are determined by counting the number of receptors on the cells of the studied subpopulations, the percentage of cells carrying the TNFRI and TNFR2 receptors is determined in each selected subpopulation, after which based on parametric logistic regression model is calculated probability of presence of rheumatoid arthritis by formula

where e = 2.7182818 - mathematical constant - base of natural logarithm, α₀ = 2.386696717005736 - coefficient, α₁ = -0.00276285111326 - coefficient, α₂ = 0.00189985337763 - coefficient, α₃ = -0.28206543500757 - coefficient, x₁ - number of receptors TNFR2 on CD3⁺ T-lymphocytes, x₂ - amount of TNFR1 on T-helper cells CD4⁺CD45R0⁺, x₃ - percentage TNFR1 positive cells among naive T-helper cells CD4⁺CD45RA⁺. If value P ≥ 0.5 there is a probability of rheumatoid arthritis.

EFFECT: use of this method for mononuclear subpopulations estimation: TNFR1+TNFR2 cells, cells among naive T-helper cells and number of type 1 receptors on T-helper memory cells, as well as type 2 receptors on CD3⁺ T-cells enables distinguishing indicators of healthy donors and rheumatoid arthritis patients at an early stage.

3 cl, 2 tbl, 5 ex, 1 dwg

Description

The invention relates to medicine, biology, molecular immunology and can be used as an examination method for early diagnosis of a patient with suspected rheumatoid arthritis (RA).

Rheumatoid arthritis is one of the most common chronic inflammatory diseases affecting 0.5 to 1% of the world's population. Despite the development of new diagnostic methods, there is a problem of early diagnosis and patients go through a long, many months' journey before a diagnosis is made, which leads to a delay in the start of treatment and the development of irreversible joint damage.

Early detection of rheumatoid arthritis (RA) is an important step in controlling disease progression. As early as the 1970s, evidence emerged to support the importance of early diagnosis and early therapeutic intervention (Arnett FC, Edworthy SM, Bloch DA, et al. The American rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988 Mar; 31 (3): 315e24 PubMed PMID: 3358796 Epub 1988/03/01 Eng). Over the years, data have been collected showing that treating a patient with RA in the first 12-16 weeks from the onset of nonspecific symptoms can indeed lead to clinically significant results. The data obtained show that early diagnosis of rheumatoid arthritis is the best opportunity to achieve remission of the disease (Emery P, Hammoudeh M, FitzGerald O, et al. Sustained remission with etanercept tapering in early rheumatoid arthritis. N Engl J Med 2014 Nov 6; 371 ( 19): 1781e92 PubMed PMID: 25372086).

The serological test currently most commonly used due to its low cost and availability is the determination of rheumatoid factor. Of greatest importance in clinical practice is the definition of immunoglobulin M (hereinafter - IgM) (Rheumatology: Clinical guidelines. Edited by EL Nasonov, Publishing house GEOTAR-Media; 2010: 19-76). The standard methods for the determination of IgM are the agglutination reaction of sensitized IgG latex particles (latex test) or sheep erythrocytes (Waaler-Rose reaction), immunonephelometry and ELISA (A). As a screening test, a semi-quantitative immunochromatographic rapid method for measuring IgM in whole blood using dry test strips can be used. Serum IgM positive results serve as a diagnostic criterion for RA.

The disadvantage of this method is that the marker (IgM) is sensitive, but not specific enough for the diagnosis of RA, as it is found in sera in other rheumatic diseases, chronic infections, malignant neoplasms, etc. Also, the disadvantages of the method include the fact that the absence of rheumatoid factor at the first examination does not exclude the diagnosis of seronegative RA. It should be noted that a negative result in the determination of rheumatoid factor IgM at an early stage of RA is insufficient to exclude this disease and requires re-examination after 12 months.

In addition, in the diagnosis of early RA, it is always necessary to supplement the detection of rheumatoid factor IgM with the detection of anti-citrulline antibodies (ACA). Among anticitrulline antibodies, antibodies to cyclic citrulline peptide (ACCP / anti-CCP) and antibodies to modified citrulline vimentin (AMCV / anti-MCV) have the best clinical and laboratory parameters

The disadvantage of the ACA detection method is false positive results in patients with systemic lupus erythematosus (SLE), with chronic destructive / deforming arthritis and patients with pulmonary tuberculosis, as well as in patients with Down syndrome without articular manifestations. To clarify the diagnosis in patients seronegative for ACA, there is a need to re-determine ACA at an early stage of RA once every 6 months. In ACA-low-positive patients, the study of ACA at an early stage of RA should be carried out once every 3-6 months.

The currently existing methods of serological diagnostics do not fully satisfy the requirements for the timely and reliable diagnosis of rheumatoid arthritis, therefore it is promising to search for additional criteria for determining this disease, which may be immunological markers.

There is a known method of determining whether a patient has or is at risk of rheumatic inflammatory disease or not. The specified method includes the step of measuring in a sample of body fluid obtained from said patient: a) the expression level of furin and / or b) the level of furin activity and / or c) the ratio of the expression levels of the mature form of the substrate furin to the untreated form of the specified substrate furin (WO 2014177719, G01N 33/68, 06.11.2014).

The disadvantage of this method is that the role of furin in the pathogenesis of RA is very poorly studied, as well as the fact that this protein has an intracellular location, which makes it difficult to determine.

There is a known method for diagnosing the inflammatory process in early RA (RU 2417263, C12Q 1/68, 04.24.2011), which consists in the fact that mononuclear cells are isolated from peripheral blood, from which RNA is isolated, based on the quantitative determination of mRNA expression of cytokine genes and terleukin-2 ( IL-2), interleukin-4 (IL-4) and interleukin-10 (IL-10) by the method of reverse transcription and polymerase chain reaction with registration of the accumulation of reaction products by fluorescence directly during the reaction, the balance of cytokines is assessed by the formula:

[IL '] / [IL' '] = E IL' '^ Ct' '/ E IL' ^ Ct ',

where [IL '] - the concentration of mRNA of the cytokine gene, characterized by increased expression;

[IL ''] - the concentration of mRNA of the cytokine gene, characterized by reduced expression;

E - amplification efficiency - a value characterizing the growth of the template at each amplification cycle;

Ct 'and Ct' 'is the value of threshold cycles for the corresponding cytokine, and the presence of an inflammatory process is noted when the following balances are obtained:

IL-10 / IL-2> 1.5;

IL-10 / IL-4> 1.0.

The disadvantage of this method is that the level of cytokines can increase in various inflammatory diseases, which cannot be used for accurate diagnosis of RA.

A known method for diagnosing RA in a subject, and said method includes the step of detecting in a biological sample obtained from the subject, one or more autoantibodies that recognize one or more protein biomarkers selected from the group of proteins consisting of WIBG protein, TPM2 protein, C17orf85- protein, TCP10L protein, ZNF706 protein, MGC17403 protein, CCDC72 protein, ELOF1 protein and GABARAPL2 protein (US 2015133322, G01N 33/564, 05/14/2015).

The disadvantage of this method is the complexity of the identification of these proteins.

There is a known diagnostic method, RA, which refers to pairs of oligonucleotides (primers) that provide amplification of CDR3-V-β-Jbeta TCR regions, in RA, a method for diagnosing and / or monitoring a specified disease using said oligonucleotides, a diagnostic kit for diagnostics and / or monitoring rheumatoid arthritis containing said oligonucleotides, nucleotide sequences amplified from said oligonucleotides as markers for PA, amino acid sequences encoded by said nucleotide sequences having amino acid sequences such as markers of rheumatoid arthritis and the peptides themselves, together with one or more acceptable immunostimulatory adjuvants and pharmaceutical adjuvants with acceptable adjuvants, auxiliary substances such as polypeptide vaccines against rheumatoid arthritis (WO 2009019657, C12Q 1/6883, 12.02.2009).

The disadvantage of this method is that the T-cell response can be manifested in other diseases and / or conditions.

A known method for diagnosing RA using differentially methylated regions identified in peripheral blood mononuclear cells, T cells, B cells and monocytes. A method for diagnosing RA in a mammalian subject, comprising the following stages: isolating DNA from a patient; determining the methylation status of a panel of at least 3 DNA loci from said patient; as well as collectively analyzing the methylation state of the loci on the panel using a computer to establish the likelihood that the patient has PA (WO 2014036314, C12Q 1/68, 03/06/2014).

The disadvantage of this method is that DNA is available in limited quantities, is highly fragmented, and it is often necessary to determine the methylation status of individual molecules that make up a minor part of the pool of all DNA, which reduces the accuracy of the data obtained.

A known method for the diagnosis of RA based on multivariate analysis of 131 biomarkers. It was found that only the detection of anti-citrulline antibodies (ACA), alone or in combination with the determination of interleukin-6 levels, is most important for the diagnosis of RA with a sensitivity of 75.8% and a specificity of 94.0% (Diagnosis of Rheumatoid Arthritis: Multivariate Analysis of Biomarkers N Wild et al. Biomarkers 13 (1), 88-105. Feb 2008.)

The disadvantage of this method is that not all biomarkers that play an important role in the pathogenesis of RA have been studied, which reduces the diagnostic value of this method.

A known method for the diagnosis of RA using the ACR / EULAR criteria, including joint damage, autoimmune serology, acute phase reactants and the duration of symptoms. The method uses a scoring system. Patients with a score <6/10 are not classified as RA patients. The criteria are intended for the classification (diagnosis) of primary patients. Also, patients with erosive disease typical of rheumatoid arthritis (RA) with a history that meets this year's criteria should be classified as having rheumatoid arthritis. Patients with long-term illness, including those in whom the disease is inactive (without or with treatment) but who have met these criteria in the past, should be classified as rheumatoid arthritis (Daniel Aletaha et al. 2010 Rheumatoid Arthritis Classification Criteria An American College of Rheumatology / European League Against Rheumatism Collaborative Initiative arthritis & rheumatism Vol. 62, No. 9, September 2010, pp 2569-2581).

The disadvantage of this method is that the method can be false-negative in patients with an atypical clinical picture of RA, or false-positive in diseases such as systemic lupus erythematosus, psoriatic arthritis and gout.

In general, currently available laboratory tests for the early diagnosis of RA are inaccurate and imperfect, including those approved by the APP (Association of Rheumatologists of Russia) in 2013 "Federal clinical guidelines for" rheumatology "with additions from 2016.

Immunological examination is important for the diagnosis of RA in the early stages of the disease, since this disease is often characterized by an atypical clinical picture precisely at the onset of the disease.

The pathogenesis of RA is closely related to various immune cells, and each cell type contributes to the development of this disease. There are several types of immunomodulatory molecules, mainly cytokines (eg TNF-α) secreted by immune cells that are involved in the pathogenesis of RA.

Tumor necrosis factor (TNF-α) has a wide range of biological effects on cells and is involved in many physiological and pathological processes. TNF-α exerts its effects through two types of receptors - TNFR1 and TNFR2. In this case, both types of receptors carry out signal transmission into the cell, and, despite the functional differences, their effects overlap to some extent, and the final effect on the cell may depend, inter alia, on the ratio of the number of molecules of both types of receptors on the cell membrane. Each of the types of receptors can be in both membrane-bound and soluble forms. The biological effects of TNF are directly realized through membrane-bound receptors. (Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ. 2003 Jan; 10 (1): 45-65) It is known that the regulation of receptor expression can be carried out at the gene level (allelic polymorphism of receptor genes), on transcriptional level, and also regulated by various factors, in particular, cytokines, including TNF-α.

Binding of the TNF-α mediator to type I receptors (TNFR1) can lead to the formation of two different complexes, which determine different signaling pathways: 1) Complex I (pro-inflammatory pathway), which determines cell survival; 2) Complex II (proapoptotic pathway), causes cell death (Micheau O, Tschopp J. Induction of TNF receptor l-mediated apoptosis via two sequential signaling complexes. Cell. 2003 Jul 25; 114 (2): 181-90.)

At the moment, it is generally accepted that the balance of signaling pathways of TNFRI receptors, which determines the further fate of the cell, is regulated by transcriptional proteins, and possibly the number of receptors bound to the cytokine (Wajant H, Scheurich P. TNFR1-induced activation of the classical NF-κB pathway. FEBS. J. 2011 Apr; 278 (6): 862-76. Doi: 10.1111 / j.1742-4658.2011.08015.x) by the ratio of different types of receptors in subpopulations (Fotin-Mleczek M, Henkler F, Samel D, Reichwein M , Hausser A, Parmryd I, Scheurich P, Schmid JA, Wajant H. Apoptotic crosstalk of TNF receptors: TNF-R2-induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1-dependent activation of caspase-8. J Cell Sci. 2002 Jul 1; 115 (Pt 13): 2757-70.0). Since a change in the number of receptors for cytokines on cells is a powerful regulator of the biological effects of cytokines and the variability of their functions (Conti L, Cardone M, Varano B, Puddu P, Belardelli F, Gessani S. Role of the cytokine environment and cytokine receptor expression on the generation of functionally distinct dendritic cells from human monocytes. Eur J Immunol. 2008 Mar; 38 (3): 750-62), studying the relationship between the number of TNF-α receptors on target cells and the effects of TNF-α on these cells provides additional knowledge about the functioning of TNF-α in the body.

Expression indices of different types of receptors for TNF-α on individual subpopulations of cells actively involved in pathological processes can significantly change in pathologies. It has been shown to significantly increase the level of TNF-α and soluble receptors sTNF-RI and sTNF-RII in the serum of patients with acute RA. The data obtained indicate differences in the expression of TNFα receptors on various subpopulations of immunocompetent cells in health and disease, and the involvement of not only TNFα itself, but also its receptors in the pathological process in rheumatoid arthritis. The results of the study suggest that the different ability of individual subpopulations of immunocompetent cells to respond to TNFα is associated with both a different percentage of cells in this subpopulation carrying TNFα receptors and with a different number of receptors expressed on cells. At the same time, monocytic cells of patients with RA had a significantly higher level of expression of membrane-bound receptors compared to healthy donors, which confirms the active involvement of immunocompetent cells of patients with rheumatoid arthritis in TNF-mediated processes (Alshevskaya A.A., Vasiliev F.F., Lopatnikova Yu. A., Sennikov S.V. Membrane-bound and soluble receptors for tumor necrosis factor-α in norm and in rheumatoid arthritis.Acta Biomedica Scientifica. 2012; (3 (2)): 23-28.), Which created the preconditions for a more in-depth studying the level of expression of membrane-bound and soluble receptors on various subpopulations of immune cells in health and disease. To calculate the number of receptors and estimate the percentage of cells carrying TNF-α receptors, a calibration protocol was developed that allows obtaining data for various subpopulations of immune cells in order to further process the results obtained (Lopatnikova JA, Vasilyev FF, Alshevskaya AA, Sennikov SV. Quantitative flow cytometric analysis of expression of tumor necrosis factor receptor types I and II on mononuclear cells. J Recept Signal Transduct Res. 2013; 33 (1): 49-55).

Next, we studied the number of TNFR1 and TNFR2 receptors and the percentage of cells carrying receptors on monocytes, total pools of T-lymphocytes and B-lymphocytes of patients with RA and atopic dermatitis, in which mononuclear cells isolated from the whole peripheral blood of RA patients were cultured for 24 h in the presence and absence of lipopolysaccharide. Analysis of phenotypic characteristics was carried out by flow cytometry. BD Quantibrite ™ PE Beads were used to determine the absolute number of TNFR1 and TNFR2 receptors. It was found that the density of expression of receptors on the surface of monocytes in RA patients is higher than in healthy donors and is an indicator reflecting the response of cells upon activation, depending on the state of health and the stage of the disease. (Sennikov SV, Alshevskaya AA, Shkaruba NS, Chumasova OA, Sizikov AE, Lopatnikova JA. Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients. Cytokine. 2015 Jun; 73 (2): 288-94.). The data obtained indicated the role of the number of TNFR1 and TNFR2 receptors and the percentage of cells carrying receptors in the pathogenesis of rheumatoid arthritis.

However, for a more complete understanding of the mechanism of influence of the expression level of TNFR1 and TNFR2 on the functional response of the cell, it was necessary to study more subpopulations of the general pools of immunocompetent cells. In order to determine the role of the number of TNFR1 and TNFR2 receptors and the percentage of cells carrying receptors, a study of these parameters was carried out on various subpopulations of immune cells in healthy donors. Determination of TNFRI and TNFR2 expression on subpopulations was carried out by adding monoclonal antibodies to TNFRI, TNFR2, CD4, CD8, CD45R0, CD45RA, CD3, CD14, CD19, CD25, CD45 and CD127 to blood samples. Analysis of peripheral blood mononuclear cells was performed by flow cytometry for monocytes, B-lymphocytes, T-lymphocytes, as well as among cytotoxic T cells (CD8 ⁺ ), T-helper cells (CD4 ⁺ ), activated CD8 ⁺ cells, activated CD4 ⁺ cells , subpopulations of T-cells of memory (CD4 ⁺ CD45R0 ⁺ ) and naive T-cells (CD4 ⁺ CD45RA ⁺ ) among cytotoxic and T-helper cells, T-regulatory cells (CD4 ⁺ CD25highCD127low). Counting the number of receptors on individual cells of the studied subpopulations was performed using calibration particles BD Quantibrite ™ PE Beads. The comparison was carried out for all populations. It was determined what percentage of cells of the subpopulation carry only TNFRI receptors, only TNFR2 and simultaneously TNFRI and TNFR2 double positive cells). The highest percentage of double positive cells was found in activated cytotoxic T cells (25.8%), with a slightly lower percentage of double positive cells observed in monocytes (15.2%), cytotoxic T helper memory cells (14.3 %), and the total pool of B-cells and activated T-helpers (11.5% each). For other subpopulations, the percentage of double positive cells was below 10%, which is considered as a potential marker for the diagnosis of diseases pathogenetically associated with these subpopulations of cells and the density of expression of TNF-α receptors. This study formed the basis for determining the normative expression of the TNFR1 and TNFR2 receptors and created the prerequisites for studying these parameters in RA in order to develop a diagnostic model for determining the likelihood of RA in patients without specific clinical and laboratory manifestations (Co-expression of membrane-bound TNF-alpha type 1 and 2 receptors differ in the subsets of immunocompetent cells Sergey Sennikov, Immunology Letters Volume 207, March 2019, Pages 1-5). Further studies were aimed at creating a diagnostic model to determine the likelihood of patients having rheumatoid arthritis.

This study formed the basis for determining the normative parameters of expression of TNFR1 and TNFR2 receptors and created the prerequisites for studying these parameters in RA in order to develop a diagnostic model for determining the likelihood of RA in patients without specific clinical and laboratory manifestations.

The objective of the invention is to provide a method for early diagnosis of RA by assessing subpopulations of mononuclear cells in patients with suspected RA.

The problem is solved in the method of early diagnosis of rheumatoid arthritis, which consists in the fact that monoclonal antibodies to TNFRI, TNFR2, CD4, CD45R0, CD45RA, CD3 are added to blood samples, mononuclear cells of peripheral blood are analyzed by flow cytometry, and CD3 ^{+ -} - are isolated by gating T-lymphocytes, naive T-helper cells (CD4 ⁺ CD45RA ⁺ ), T-helper cells of memory (CD4 ⁺ CD45R0 ⁺ ); the expression indices of TNFR1 and TNFR2 receptors are determined by counting the number of receptors on the cells of the studied subpopulations, the percentage of cells carrying TNFRI and TNFR2 receptors in each selected subpopulation is determined, after which the probability of RA is calculated on the basis of a parametric logistic regression model by the formula:

Where

e = 2.7182818 - mathematical constant - the base of the natural logarithm,

α ₀ = 2.38669717005736 - coefficient,

α ₁ = -0.00276285111326 - coefficient,

α ₂ = 0.00189985337763 - coefficient,

α ₃ = -0.28206543500757 - coefficient,

x ₁ - the number of TNFR2 receptors on CD3 ⁺ T-lymphocytes,

x ₂ - the number of TNFR1 on T-helper cells of the memory CD4 ⁺ CD45R0 ⁺ ,

x ₃ - percentage of TNFR1 positive cells among naive T-helper cells CD4 ⁺ CD45RA ⁺ ,

moreover, if P≥0.5, then there is a possibility of rheumatoid arthritis.

The method formula contains specific numerical values of the coefficients:

The method contains the final formula for calculating the probability:

The invention, in our opinion, is new and meets the inventive step criterion.

The proposed method is based on the following. In the studies described above, monoclonal antibodies to TNFRI, TNFR2, CD4, CD8, CD45R0, CD45RA, CD3, CD14, CD19, CD25, CD45, and CD127 were first added to blood samples from healthy donors. Further, the level of expression and co-expression of TNFRI and TNFR2 on subpopulations of immune cells from healthy donors was investigated. The main subpopulations of T cells vary in the combined expression of TNFR1 and TNFR2 receptors, which can lead to a different level and type of cell response to the cytokine TNFα. Thus, all the studied parameters were established in healthy donors.

When creating this method, blood samples were taken from patients with known RA patients, and significant differences between known patients with RA and healthy donors were revealed in terms of:

- the amount of TNFR2 on CD3 ⁺ T-lymphocytes;

- the amount of TNFR1 on CD4 ⁺ CD45R0 ⁺ (T-helper memory cells);

- percentage of TNFR1 positive for CD4 ⁺ CD45RA ⁺ (naive T-helper cells).

Comparison of TNFR1 and TNFR2 expression in RA patients and healthy donors is shown in Figures 1-3. Data are presented as medians with an interquartile range.

In fig. 1 shows the amount of TNFR2 on cells of the total pool of CD3 ⁺ T-lymphocytes; in fig. 2 shows the amount of TNFR1 on T-helper memory cells (CD4 ⁺ CD45R0 ⁺ ); in fig. 3 shows the percentage of TNFR1 positive cells (CD4 ⁺ CD45RA ⁺ ) among naive T helper cells.

The coefficients α ₀ , α ₁ , α ₂ , α ₃ were calculated from the ratio of the expression rates of TNFR1 and TNFR2 receptors in RA patients and healthy donors.

The studied indicators were used to construct a diagnostic formula using univariate and multivariate logistic regression analysis with a gradual reduction of insignificant indicators.

The method is carried out as follows.

1. Preparation of peripheral blood samples

1.1. Carrying out fasting blood sampling from the cubital vein under sterile conditions, 9 ml in vacuum tubes with an anticoagulant. Performing a general blood test, counting the total number of leukocytes.

1.2. Aliquot the required volume of blood containing 1 million leukocytes into cytometric tubes.

2. Sample preparation:

2.1. Introduction of monoclonal antibodies: anti-human CD3 APC / Cy7 (Allophycocyanin-cyanine7), anti-human CD45R0 FITC (isothiocyanate fluorescein), anti-human CD45RA PacificBlue (Pacific blue), anti-human CD4 Fe / Cy7 (phycoerythrin-cyanine 7). , anti-human TNFRI-PE (phycoerythrin), anti-human TNFRII-PE (phycoerythrin), anti-human TNFRI-APC (Allophycocyanin), anti-human TNFRII-APC (Allophycocyanin) (R&D Systems, Minneapolis, MN), the volume specified by the manufacturer for painting 1 million cells,

2.2. Adding tenfold diluted in PBS (sodium phosphate buffer) lysis buffer BDFACSLysingSolution (cat. Number 349202; BD, USA) according to the manufacturer's instructions in tenfold volume relative to the sample volume. Incubation for 15 minutes at room temperature.

2.3. PBS addition in a volume of 1 ml per sample.

2.4. Centrifugation at 1500 rpm, 10 minutes.

2.5. Removal of the supernatant fluid, adding 100 μl. PBS.

3. Analysis on a flow cytometer

3.1. Adjustment of front and side light scattering on sample 1.

3.2. Adjustment of the laser voltage and the sensitivity of the PMT (photomultiplier device) of all channels using control samples No. 2, 6, 10, 14.

3.3 Gating

CD3 + T lymphocytes were gated as CD3 ⁺ cells from the lymphocyte pool according to FSC on SSC (forward and lateral light scattering) scores.

Memory T cells (CD4 ⁺ CD45R0 ⁺ ) were gated as CD4 ⁺ and CD45R0 ⁺ from the total lymphocyte pool as measured by FSC on SSC.

Naive T-helper cells (CD4 ⁺ CD45RA ⁺ ) were gated as CD4 ⁺ and CD45RA ⁺ from the total lymphocyte pool as measured by FS on SSC.

4. Determination of the percentage of cells carrying TNFR1 and TNFR2 receptors,

Determination of the percentage of cells carrying TNFR1 and TNFR2 receptors is performed using BD FACSDiva ™ Software | BDBiosciences-US As a result, we obtain data on the percentage of cells carrying TNFR1 receptors

5. Determination of expression parameters of TNFR1 and TNFR2 receptors

Determination of expression parameters of TNFR1 and TNFR2 receptors is carried out by analysis of BD QuantiBRITE PE (phycoerythrin) particles (BD, ref. 340495) to convert the fluorescence intensity values for the PE channel into the number of PE molecules on subpopulations of CD3 + T-lymphocytes and T-helper memory cells (CD4 ⁺ CD45R0 ⁺ ). As a result, we obtain data on the amount of TNFR2 on CD3 ⁺ T-lymphocytes and TNFR1 on T-helper memory cells (CD4 ⁺ CD45R0 ⁺ )

6. Calculation based on the parametric logistic regression model of the probability of the presence of RA by the formula

Where,

x ₁ = number of TNFR2 receptors on CD3 ⁺ T lymphocytes

x ₂ = amount of TNFR1 on T-helper memory cells (CD4 ⁺ CD45R0 ⁺ )

x ₃ = percentage of TNFR1 positive cells among naive T helper cells (CD4 ⁺ CD45RA ⁺ ).

Next, we substitute the obtained data into the formula and calculate the value of P.

With the obtained value p≥0.5, there is a probability of the presence of RA.

The proposed method is demonstrated by specific examples.

Example 1. Patient A., a woman of 46 years old, came to the clinic with complaints of pain in the wrist joints, arising periodically throughout the year. Objectively: the condition is satisfactory, the consciousness is clear, the constitutional type is normosthenic, the skin is of normal color. Peripheral lymph nodes are not enlarged, there is no edema. Examination of the osteoarticular apparatus did not reveal any deformities, pain was noted on palpation of the wrist joints, more on the right. The volume of active and passive movements is not changed. On the part of the respiratory and digestive organs, as well as the cardiovascular system, no pathology was revealed. NPV 16 / min, heart rate 66 / min, BP 120/70 mm Hg.

In laboratory and instrumental research: hemoglobin 125 g / l, leukocytes 8.8 × 10 ³ , ESR 20 mm / h. According to the ACR / EULAR 2010 classification, 3 points (tests for rheumatoid factor and ACCP are negative), no radiological changes characteristic of RA were revealed on the radiograph. When analyzing mononuclear cells of peripheral blood by flow cytometry, the following indicators were identified:

Number of TNFR2 on CD3 ⁺ T-lymphocytes-728 (X1)

Number of TNFR1 on CD4 ⁺ CD45R0 ⁺ -452 (X2)

Percentage of TNFR1 positive cells among CD4 ⁺ CD45RA ⁺ -0.6 (X3)

We use the formula

where X ¹ = 728

X ² = 452

X ³ = 0.6

We substitute the data into the formula:

P = 0.7436

As a result, P = 0.7436, which is more than 0.5, therefore there is no likelihood of rheumatoid arthritis.

Example 2. Patient T., a 33-year-old man, came to the clinic with complaints of pain in the shoulder joints, arising periodically for 5 months. Objectively: satisfactory condition, clear consciousness, constitutional type - hypersthenic, normal color of the skin. Peripheral lymph nodes are not enlarged, there is no edema. Examination of the osteoarticular apparatus did not reveal any deformities, there was pain on palpation of the shoulder joints, more on the right. The volume of active and passive movements is not changed. On the part of the respiratory and digestive organs, as well as the cardiovascular system, no pathology was revealed. NPV 18 / min, heart rate 76 / min, blood pressure 120/80 mm Hg.

In laboratory and instrumental research: hemoglobin 155 g / l, leukocytes 8.8 × 10 ³ , ESR 15 mm / h. According to the ACR / EULAR 2010 classification, 2 points (tests for rheumatoid factor and ACCP are negative), no radiological changes characteristic of RA were detected on the radiograph. When analyzing mononuclear cells of peripheral blood by flow cytometry, the following indicators were identified:

Number of TNFR2 on CD3 ⁺ T-lymphocytes-457

Number of TNFR1 on CD4 ⁺ CD45R0 ⁺ -3522

Percentage of TNFR1 positive cells among CD4 ⁺ CD45RA ⁺ -0.1

The data were put into the formula, as a result P = 0.999597, which is more than 0.5, therefore there is no likelihood of rheumatoid arthritis.

Example 3. Patient X., a 58-year-old woman came to the clinic with complaints of pain in the proximal interphalangeal joints, periodically for 2 years. Objectively: the condition is satisfactory, the consciousness is clear, the constitutional type is normosthenic, the skin is of normal color. Peripheral lymph nodes are not enlarged, there is no edema. Examination of the osteoarticular apparatus did not reveal any deformities, there was pain on palpation of the shoulder joints, more on the right. The volume of active and passive movements is not changed. On the part of the respiratory and digestive organs, as well as the cardiovascular system, no pathology was revealed. NPV 17 per min, heart rate 76 per min, blood pressure 110/70 mm Hg.

In laboratory and instrumental research: hemoglobin 137 g / l, leukocytes 7.2 × 10 ³ , ESR 22 mm / h. According to the ACR / EULAR 2010 classification, 4 points (tests for rheumatoid factor and ACCP are negative), no radiological changes characteristic of RA were found on the radiograph. When analyzing mononuclear cells of peripheral blood by flow cytometry, the following indicators were identified:

Number of TNFR2 per CD3 ⁺ T-lymphocytes-889

Number of TNFR1 on CD4 ⁺ CD45R0 ⁺ -2416

Percentage of TNFR1 positive cells among CD4 ⁺ CD45RA ⁺ -2.8

The data were put into the formula, as a result P = 0.999597, which is more than 0.5, therefore there is no likelihood of rheumatoid arthritis.

Example 4. Patient G., a 45-year-old woman came to the clinic with complaints of pain in the wrist joints, arising periodically for 2 years. Objectively: the condition is satisfactory, the consciousness is clear, the constitutional type is normosthenic, the skin is of normal color. Peripheral lymph nodes are not enlarged, there is no edema. Examination of the osteoarticular apparatus did not reveal any deformities, pain was noted on palpation of the wrist joints, more on the right. The volume of active and passive movements is not changed. On the part of the respiratory and digestive organs, as well as the cardiovascular system, no pathology was revealed. NPV 17 per min, heart rate 68 per min, blood pressure 120/80 mm Hg.

In laboratory and instrumental research: hemoglobin 104 g / l, leukocytes 4.4 × 10 ³ , ESR 21 mm / h. According to the ACR / EULAR 2010 classification, 3 points (tests for rheumatoid factor and ACCP are negative), no radiological changes characteristic of RA were revealed on the radiograph. When analyzing mononuclear cells of peripheral blood by flow cytometry, the following indicators were identified:

Number of TNFR2 on CD3 ⁺ T-lymphocytes - 834

Number of TNFR1 on CD4 ⁺ CD45R0 ⁺ - 832

Percentage of TNFR1 positive cells among CD4 ⁺ CD45RA ⁺ -9.3

The data were put into the formula, as a result P = 0.2768, which is less than 0.5, therefore there is a possibility of rheumatoid arthritis

Example 5. Patient S., a 59-year-old man, came to the clinic with complaints of pain in the proximal interphalangeal joints periodically for 3 years. Objectively: the condition is satisfactory, the consciousness is clear, the constitutional type is normosthenic, the skin is of normal color. Peripheral lymph nodes are not enlarged, there is no edema. Examination of the osteoarticular apparatus did not reveal any deformities, there was pain on palpation of the shoulder joints, more on the right. The volume of active and passive movements is not changed. On the part of the respiratory and digestive organs, as well as the cardiovascular system, no pathology was revealed. NPV 16 per minute, heart rate 78 per minute, blood pressure 130 \ 90 mm Hg.

In laboratory and instrumental research: hemoglobin 133 g / l, leukocytes 6.6 × 10 ³ , ESR 12 mm / h. According to the ACR / EULAR 2010 classification, 3 points (tests for rheumatoid factor and ACCP are negative), no radiological changes characteristic of RA were revealed on the radiograph. When analyzing mononuclear cells of peripheral blood by flow cytometry, the following indicators were identified:

Number of TNFR2 per CD3 ⁺ T-lymphocytes-2517

Number of TNFR1 on CD4 ⁺ CD45R0 ⁺ -736

Percentage of TNFR1 positive cells among CD4 ⁺ CD45RA ⁺ -4.35

The data were put into the formula, as a result P = 0.01217, which is less than 0.5, therefore there is a possibility of rheumatoid arthritis.

The proposed method for early diagnosis of RA by assessing the subpopulations of mononuclear cells in patients with suspected RA makes it possible to distinguish between healthy donors and RA patients at an early stage. The formula includes the percentage of TNFR1 + TNFR2 - cells among naive T-helper cells, the number of type 1 receptors on T-helper cells of memory and the number of type 2 receptors on CD3 ⁺ T cells and allows differentiation of patients with RA with a sensitivity of 93% and specificity 90%.

Additional materials

EXPLANATION ON THE MATHEMATICAL FORMULA

To assess the level of expression and co-expression of TNFRI and TNFR2 receptors, we used mononuclear cells (MNC) isolated from the peripheral blood of conventionally healthy donors (whole blood samples were obtained at the blood collection point No. 1 of the Novosibirsk Blood Center) and patients with rheumatoid arthritis undergoing inpatient treatment in the rheumatology department of the NIIFKI immunopathology clinic.

The study included 46 apparently healthy donors aged 18-77 years (median (ICR) 36.5 (30:54) years), including 16 men (34.8%) and 30 (65.2%) women, 64 patients with rheumatoid arthritis in age 22-83 years (median (RBI) 55 (45:65) years), among whom the majority of women are 54 (85.4). Demographic and clinical characteristics of the included patients and healthy donors are presented in Table 1.

Preparation of peripheral blood samples

1.1. Carrying out fasting blood sampling from the cubital vein under sterile conditions, 9 ml in vacuum tubes with an anticoagulant K3-EDTA (3-substituted potassium salt of ethylenediaminetetraacetic acid, Vacuette K3-EDTA, GreinerBio-One GmbH, Austria).

1.2. Performing a general blood test, counting the total number of leukocytes

1.3. Aliquot the required blood volume containing 1 million leukocytes into cytometric tubes.

2. Sample preparation:

2.1. Introduction of monoclonal antibodies with fluorochrome (dye): anti-human CD3 APC / Cy7 (Allophycocyanin-cyanine7), anti-human CD19 PE / Cy7 (phycoerythrin-cyanin 7), anti-human CD8 APC / Cy7, anti-human CD14 PerCP ( peridinin-chlorophyll protein), anti-human CD25 PITC (isothiocyanate fluorescein), anti-human CD127 (IL-7Rα) APC / Cy7 (Allophycocyanin-cyanin7), anti-human CD45R0 FITC (isothiocyanate fluorescein), antiB-human Pacific blue CD45RA ), anti-human CD45 PerCP (peridinine-chlorophyll protein), anti-human CD4 Pe / Cy7 (phycoerythrin-cyanin 7)., anti-human TNFRI-PE (phycoerythrin), anti-human TNFRII-PE (phycoerythrin), anti -human TNFRI-APC (Allophycocyanin), anti-human TNFRII-APC (Allophycocyanin) (R&D Systems, Minneapolis, MN), in the amount indicated by the manufacturer for coloring 1 million cells,

2.2. Addition of BDFACS Lysing Solution (cat. No. 349202; BD, USA) diluted tenfold in PBS (sodium phosphate buffer) according to the manufacturer's instructions in tenfold volume relative to the sample volume. Incubation for 15 minutes at room temperature

2.3. PBS addition in a volume of 1 ml per sample.

2.4. Centrifugation at 1500 rpm, 10 minutes.

2.5. Removal of the supernatant fluid, adding 100 μl. PBS.

3. Analysis on a flow cytometer

3.1. Adjustment of the front and side light scattering on sample No. 1

3.2. Adjustment of the laser voltage and the sensitivity of the PMT (photomultiplier tube) of all channels using control samples No. 2, 6, 10, 14.

3.3 Gating

1. CD3 ⁺ T-lymphocytes were gated as CD3 ⁺ cells from the lymphocyte pool by FSC on SSC (forward and lateral light scattering).

2. Monocytes were gated as CD14 ⁺ cells from the total monocyte pool according to FSC on SSC

3. B cells were gated as CD19 ⁺ cells from the total pool of lymphocytic cells according to FSC values on SSC

4. Cytotoxic T cells were gated as CD8 ⁺ cells from the total pool of lymphocytic cells in terms of FSC on SSC

5. T-helper cells were gated as CD4 ⁺ cells from the total pool of lymphocytic cells in terms of FSC on SSC

6. Activated cytotoxic T cells are gated as CD8 ⁺ CD25 ⁺ cells from the general pool of lymphocytic cells according to FSC on SSC

7. Activated T-helper cells are gated as CD4 ⁺ , CD25 ⁺ cells from the total pool of lymphocytic cells according to FSC on SSC

8. T-helper memory cells are gated as CD4 ⁺ , CD45R0 ⁺ cells from the general pool of lymphocytic cells according to FSC on SSC

9. Naive T-helper cells are gated as CD4 ⁺ , CD45RA ⁺ cells from the general pool of lymphocytic cells according to FSC on SSC

10. Cytotoxic T-cells of memory are gated as CD8 ⁺ , CD45R0 ⁺ cells from the general pool of lymphocytic cells according to FSC on SSC

11. Naive cytotoxic T cells are gated as CD8 ⁺ , CD45RA ⁺ cells from the general pool of lymphocytic cells according to FSC on SSC

12. T-regulatory cells are gated as CD4 ⁺ , CD25high, CD127low cells from the total pool of lymphocytic cells according to FSC on SSC

4. Determination of the percentage of cells carrying TNFR1 and TNFR2 receptors,

Determination of the percentage of cells carrying TNFR1 and TNFR2 receptors was carried out using the BDFACSDiva ™ Software | BDBiosciences-US for each of the 12 studied subpopulations was determined by 2 parameters of the expression of TNFR1 and TNFR2 receptors (the percentage of TNFR1 positive cells among the cells of the subpopulation, the percentage of TNFR2 positive cells among the cells of the subpopulation.)

5. Determination of expression parameters of TNFR1 and TNFR2 receptors

Determination of expression values for TNFR1 and TNFR2 receptors is performed by analyzing BD QuantiBRITEPE (phycoerythrin) particles (BD, ref. 340495) to convert the fluorescence intensity values for the PE channel to the number of PE molecules. The average number of TNFR1 receptors on the cells of the subpopulation and the average number of TNFR2 receptors on the cells of the subpopulation is carried out with the same parameters of the PMT voltage according to the PE detector as in the analysis of calibration beads, this allows us to convert the fluorescence intensity values into the number of PE molecules per cell. Further, the number of PE molecules per cell can be converted to the number of antibody molecules per cell using the known ratio of PE molecules per antibody, equal to 1: 1, according to the manufacturer's instructions.

.Thus, 48 parameters were obtained for each person. These 48 parameters were included in univariate logistic regression analysis to determine their association with rheumatoid arthritis. Based on the results of univariate analysis, 7 parameters were selected that were statistically significantly associated with RA, and they were included in the multivariate analysis. As a result of gradual reduction of statistically insignificant parameters, a set of models was built, from which the optimal model was selected according to the quality criterion AIC [Sakamoto, Y., Ishiguro, M., and Kitagawa G. (1986). AkaikeInformationCriterionStatistics. D. Reidel Publishing Company], and the final multivariate model included 3 indicators: the number of TNFR2 receptors on CD3 + T-lymphocytes, the number of TNFR1 on T-helper memory cells (CD4 ⁺ CD45R0 ⁺ ) and the percentage of TNFR1 positive cells among naive T-helper cells (CD4 ⁺ CD45RA ⁺ ) (Table 2). The resulting model had R ² = 0.68 (coefficient of determination). For the resulting logistic regression model, below we give the Odds Ratios for each of the independent variables, defined as

...

A parametric logistic regression model was constructed based on differences in expression rates between healthy donors and patients with RA, and model parameters were determined (Kuhn, M., & Johnson, K. (2013). AppliedPredictiveModeling.doi: 10.1007 / 978-1-4614-6849link https://link.springer.com/book/10.1007/978-1-4614-6849-3) using a free software toolkit (R-Studio software package version 1.0.136 (FreeSoftwareFoundation Inc., USA) with R packages version 3.3.1 (The R Foundation for Statistical Computing, Austria)) with algorithms for constructing regression models and formulas for calculating the probability of a predicted event (McCullagh P. and Nelder, J.A. (1989) GeneralizedLinearModels. London: Chapman and Hall .; Dobson, AJ ( 1983) An Introduction to Statistical Modeling. London: Chapman and Hall .; Cox, DR and Snell, EJ (1981). Applied Statistics; Principles and Examples. London: Chapman and Hall .; Hastie, TJ and Pregibon, D. (1992) ) Generalized linear mo dels. Chapter 6 of Statistical Models in S eds J.M. Chambers and T.J. Hastie, Wadsworth & Brooks / Col).

The constructed logistic regression model included the following coefficients:

α ₀ = 2.386696717005736

α ₁ = -0.00276285111326

α ₂ = 0.00189985337763

α ₃ = -0.28206543500757

These coefficients were calculated from the ratio of the expression rates of TNFR1 and TNFR2 receptors in RA patients and healthy donors.

The formula for calculating the probability of RA:

Where,

the calculated coefficients α are used as the coefficients as independent parameters -

x ₁ - the number of TNFR2 receptors on CD3 ⁺ T-lymphocytes

x ₂ - the number of TNFR1 on T-helper memory cells (CD4 ⁺ CD45R0 ⁺ )

x ₃ - percentage of TNFR1 positive cells among naive T-helper cells CD4 ⁺ CD45RA ⁺ ,

e = 2.7182818 - mathematical constant (base of natural logarithm)

Substituting the obtained values of the coefficients into the formulas, we obtain:

The final formula for calculating the probability:

With the obtained value of p≥0.5, there is a possibility of rheumatoid arthritis.

Claims (16)

1. A method for early diagnosis of rheumatoid arthritis, which consists in adding monoclonal antibodies to TNFRI, TNFR2, CD4, CD45R0, CD45RA, CD3 to blood samples, analyzing mononuclear cells of peripheral blood by flow cytometry, isolating CD ^{3 + -} -T by gating - lymphocytes, naive T-helper cells (CD4 ⁺ CD45RA ⁺ ), T-helper cells of memory (CD4 ⁺ CD45R0 ⁺ ), determine the expression indices of TNFR1 and TNFR2 receptors by counting the number of receptors on the cells of the studied subpopulations, determine the percentage of cells carrying TNFRI receptors and TNFR2 in each selected subpopulation, after which the probability of the presence of RA is calculated on the basis of a parametric logistic regression model by the formula:

Where e = 2.7182818 - mathematical constant - the base of the natural logarithm, α ₀ = 2.386696717005736 - coefficient, α ₁ = -0.00276285111326 - coefficient, α ₂ = 0.00189985337763 - coefficient, α ₃ = -0.28206543500757 - coefficient, x ₁ - the number of TNFR2 receptors on CD3 ⁺ T-lymphocytes, x ₂ - the number of TNFR1 on T-helper cells of the memory CD4 ⁺ CD45R0 ⁺ , x ₃ - percentage of TNFR1 positive cells among naive T-helper cells CD4 ⁺ CD45RA ⁺ , moreover, if P≥0.5, then there is a possibility of rheumatoid arthritis. 2. The method according to claim 1, the formula of which contains specific numerical values of the coefficients:

3. The method according to claim 1, containing the final formula for calculating the probability:

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