RU2504783C1 - Diagnostic technique for autoimmune versions of diabetes mellitus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: diagnostic technique for the autoimmune versions of diabetes mellitus, with the autoimmune versions of diabetes mellitus being diagnosed by comparing the production of tumour necrosis factor alpha (TNFα) (pcg/ml) in supernatant after lymphocyte culture taken from patient's blood sample in the autoantigen insulin stimulation environment. The test is considered to be positive if the TNFα in the insulin post-stimulation sample increases its value in the no-stimulation environment.

EFFECT: invention enables diagnosing the autoimmune inflammatory process in the pancreatic tissue that enables assessing a degree of autoimmune aggression on the pancreatic tissue.

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Description

A method for diagnosing autoimmune variants of diabetes mellitus is characterized in that the autoimmune variant of diabetes mellitus is diagnosed by comparing the production of tumor necrosis factor alpha (TNFα) (PCG / ml) in the supernatant after culturing lymphocytes obtained from a patient’s blood sample under conditions of stimulation with autoantigen by insulin. The test is considered positive if the value of TNFα in the sample after stimulation with insulin exceeds its value in the absence of stimulation.

The invention relates to medicine, namely to therapy, endocrinology, diabetology, laboratory diagnostics.

In the structure of diabetes mellitus (DM) a special place is occupied by its autoimmune variant. Latent autoimmune diabetes of adults (LADA) refers to autoimmune variants of diabetes and occupies an intermediate position between diabetes and diabetes but does not stand out in a separate nomenclature unit according to the latest WHO classification (1999). T1DM is a serious disease associated with both acute and chronic complications leading to early disability of patients. LADA is characterized by a milder course compared to T1DM, while the onset of the disease is practically no different from the clinical picture of T2DM, which complicates its diagnosis and the timely initiation of insulin therapy.

The fundamental difference between autoimmune diabetes and its non-immune forms is that with autoimmune inflammation of the pancreatic tissue, the mass of β-cells gradually decreases, which ultimately leads to a decrease in insulin secretion and its absolute deficiency.

To assess the level of insulin secretion is judged by the concentration of C-peptide, measured after prolonged fasting (more than 10 hours) and on the background of stimulation (GST, OGTT, MMTT). Insulin and C-peptide are secreted by the pancreas in equimolar amounts, but 50% or more of insulin is broken down when passing through the liver. Currently, in international studies, as a "gold standard" to assess the secretory function of β-cells, it is customary to use MMTT with a quantitative determination of the concentration of C-peptide in the blood [Greenbaum C., 2008].

The method requires additional preparation (prolonged fasting), significant material costs, it does not allow to diagnose the autoimmune process in its initial stages.

Known analogues

As the closest analogue, a method for the early diagnosis of autoimmune variants of diabetes is adopted, which consists in the simultaneous determination of autoantibodies circulating in the blood, characteristic of diabetes:

1) to glutamate decarboxylase (GADA), by indirect enzyme immunoassay (sensitivity of the method - 87.1%, specificity - 85%);

2) to cytoplasmic structures of beta cells (ICA), by enzyme-linked immunosorbent assay (sensitivity - 87%, specificity - 85%);

3) to insulin (IAA), using indirect enzyme-linked immunosorbent assay (method sensitivity - 89%, specificity - 78%);

4) to tyrosine phosphatase of lymphocytes (IA-2) by indirect enzyme-linked immunosorbent assay (ELISA) (sensitivity of the method - 93%, specificity - 75%).

The disadvantages of the method include false positive and false negative results (according to various estimates from 10 to 15%), which complicates the diagnosis by this method. In addition, autoantibody titer levels do not reflect the activity of inflammatory changes in pancreatic tissue in autoimmune diabetes.

The objective of the invention is to create a highly sensitive and specific method for the early diagnosis of autoimmune variants of diabetes.

A method for diagnosing autoimmune variants of diabetes mellitus, characterized in that the autoimmune variant of diabetes mellitus is diagnosed by comparing the production of tumor necrosis factor alpha (TNFα) (PCG / ml) in the supernatant after culturing lymphocytes obtained from a patient’s blood sample under conditions of stimulation with autoantigen by insulin . The test is considered positive if the value of TNFα in the sample after stimulation with insulin exceeds its value in the absence of stimulation.

The method is as follows. Before determining the interleukins - IL-2, TNFα, IFNγ characteristic of TH1, as well as IL-4 produced by TH2, the lymphocytes of patients and healthy donors are isolated from peripheral blood by gradient centrifugation using a mixture of ficoll-verographin, with a density of 1.077. Cells were washed twice with phosphate-buffered saline with a pH of 7.2 and resuspended in serum-free medium RPMI 1640 (Paneko, Russia) with the addition of 20 μg / ml gentamicin, 2 mM L-glutamine and 2 mM HEPES. A suspension of lymphocytes at a concentration of 10 ⁶ cells per 1 ml of medium is cultured in plastic 24-well plates (Nunc, Denmark) for 18-20 hours in a CO2 incubator.

Cultivation of lymphocytes is carried out in 3 different versions: without additives - a control sample (spontaneous test), with the addition of PHA (10 μg / ml) (positive control of the culture system) and a sample with the addition of insulin at a concentration of 0.01 IU / ml. The supernatants of each sample are collected in microtubes and centrifuged for 5 minutes at 3000 rpm, in a centrifuge with cooling.

Interleukin production is evaluated by flow fluorimetry on a two-beam laser automatic analyzer (BioPlex Protein Assay System, Bio-Rad, USA).

In the control group, there is no T-cell response to insulin. In autoimmune variants of diabetes, the value of the level of TNFα production compared with other studied interleukins in response to insulin increases compared to the spontaneous level of production of TNFα, as well as after stimulation of PHA. To register T-cell sensitization to insulin and evaluate its intensity, we first introduced a coefficient of autoimmune inflammation (KAIV). It is calculated as the ratio of the rate of production of TNFα in response to insulin to the spontaneous value of production of TNFα (PCG / ml). With a positive response to insulin, this coefficient is greater than 1. In our sample, the coefficient values varied from 3.09 to 19.23.

The technical result of the claimed invention is a method for the diagnosis of autoimmune inflammatory process in pancreatic tissue, which also allows to evaluate the degree of autoimmune aggression on pancreatic tissue.

The technical result is achieved by determining the sensitization of T-lymphocytes to insulin, the production of TNFα in the in vitro system.

The advantages of the method are:

- ease of implementation,

- the study requires a small amount of material (whole blood),

- profitability - does not require large material costs,

- deadlines - the result is ready within 24 hours,

- the method is minimally invasive,

- the sensitivity of the method is 99%, specificity is 100%,

- the method does not require preliminary preparation of patients (performed in the morning on an empty stomach),

- it can be done on an outpatient basis.

The method was tested at the Federal State Budgetary Institution Endocrinological Research Center of the Ministry of Health of the Russian Federation, at the Institute of Diabetes. Studied 34 people.

Example 1. Patient K., 58 years old, was admitted to the Federal State Budgetary Institution Endocrinology Scientific Center of the Ministry of Health of the Russian Federation with complaints of weight loss, the appearance of acetone in the urine, high glycemia, a rise in blood pressure to 170/110 mm Hg, palpitations, swelling of the legs in afternoon. From the anamnesis it is known that at the age of 46 years (in 1995), thirst, polyuria, dry mouth appeared on the background of stress, lost 8 kg in 2 months. Glycemia of 7 mmol / L was detected. The patient was diagnosed with type 2 diabetes mellitus on insulin therapy, decompensation stage. In the study of spontaneous production of TNFα and after stimulation with insulin, it was found that the level of TNFα after stimulation with insulin [66, 78] is higher than the spontaneous level [14], which indicates the presence of an autoimmune response to insulin (KAIV = 4.77). The obtained data served as the basis for changing the diagnosis with the replacement of the diagnosis of T2DM with T1DM.

Example 2. Patient T., 46 years old, was observed on an outpatient basis at the Federal State Budgetary Institution Endocrinological Research Center of the Ministry of Health of the Russian Federation with a diagnosis of diffuse toxic goiter, II st. (WHO), moderate thyrotoxicosis in the stage of subcompensation. The patient received treatment with thyreostatic drugs without a clear effect, in connection with which she underwent surgery - thyroidectomy. Upon further examination, the patient revealed positive titers for GADA, specific for immune variants of diabetes. In the study of spontaneous production of TNFα and after stimulation with insulin, it was found that the level of TNFα after stimulation with insulin was higher than the spontaneous level: 5.28 and 18.99, respectively, which indicates the presence of an autoimmune response to insulin (KAIV = 3.6). The data obtained served as the basis for the preclinical diagnosis of T1DM.

Claims (1)

A method for diagnosing autoimmune variants of diabetes mellitus, including a patient’s blood test, characterized in that they determine the value of spontaneous production of TNFα (pcg / ml) in the supernatant after culturing the patient’s lymphocytes and after stimulation with a polyclonal T-cell activator - phytohemagglutinin (PHA) and specific T- cellular activator - insulin; compare these indicators with each other and when obtaining large values after stimulation with insulin compared with spontaneous production of TNFα, an autoimmune response to insulin is recorded and an autoimmune variant of diabetes is diagnosed.