RU2766747C2 - Method for comprehensive assessment of the energy supply of immunocompetent blood cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely, to immunology, obstetrics, reproductive medicine, therapy, pediatrics. The method for determining the degree of energy supply of immunocompetent blood cells (ICC) by means of flow cytometry with determination of the percentage of ICC with a reduced membrane potential of mitochondria consists in the fact that a comprehensive assessment of the condition of the immune system is conducted. The indicators of lymphocytes, granulocytes, monocytes are therein used as immunological indicators, determining the percentage of ICC: lymphocytes, granulocytes, monocytes with a reduced membrane potential of mitochondria, and in comparison with similar indicators of blood samples of healthy individuals, deviations from the norm are determined: for the adult population: lymphocytes - more than 35%, granulocytes and monocytes - more than 5%, for pregnant women: lymphocytes - more than 40%, granulocytes and monocytes - more than 10%, for children: lymphocytes - more than 35%, granulocytes - more than 3%, monocytes - more than 5%. Depending on the combinations of ICC with deviations from the norm, the degree of abnormality of energy supply of ICC is determined: the first degree is a monovariant combination of ICC with a low membrane potential of mitochondria: lymphocytes, or granulocytes, or monocytes; the second degree of abnormality of energy supply of ICC is simultaneous bivariation combinations of ICC with a low membrane potential of mitochondria: lymphocytes and granulocytes, or lymphocytes and monocytes, or granulocytes and monocytes; the third degree of abnormality of energy supply of ICC is a simultaneous increase in the amount of cells with a low membrane potential of mitochondria of all types: lymphocytes, granulocytes, monocytes.

EFFECT: invention provides the possibility of early preclinical, technically rapid diagnostics and comprehensive assessment of the degree of energy supply deficiency of immunocompetent blood cells in pregnant women, children, and other groups of the adult population.

1 cl, 2 tbl, 5 ex

Description

The invention relates to medicine, namely to immunology, obstetrics, reproduction, therapy, pediatrics, and can be used to assess the degree of impaired energy supply of immunocompetent blood cells (ICCs) in the adult population, in pregnant women and children.

There are various methods for assessing the functional state of cell mitochondria in various tissues, including immunocompetent cells both in general and in individual populations, for example, in lymphocytes and other blood cells - platelets. The high-risk group is made up of women of reproductive age, especially during the gestation period, children of preschool age, with chronic somatic pathology.

The role of mitochondrial activity in female fertility and assisted reproductive technologies: overview and current insights // Reproductive biomedicine online. - 2018. - №36. - P. 686-697. Проводилось изучение роли митохондриальных функций (например, биоэнергетика, стероидогенез, активность дыхательной цепи, производство активных форм кислорода в опосредованном программировании развития мозга у плода при различных стрессовых ситуациях (физических, социальных, воздействие окружающей среды) у женщин во время гестации по данным плацентарной и пуповинной крови (Anke Hoffmann, Dietmar Spengler. The Mitochondrion as Potential Interface in Early-Life Stress Brain Programming. Frontiers in Behavioral Neuroscience., 2018, Vol. 12, doi: 10.3389/fnbeh.2018.00306. Представлена оценка митохондриальной дисфункции в лимфоцитарных клетках периферической крови методом проточной цитометрии при субклиническом гипотиреозе у женщин в гестационном периоде во взаимосвязи с неблагоприятными исходами беременности (Feldthusen A.-D. et al. Pregnancy-induced alterations in mitochondrial function in euthyroid pregnant women and pregnant women with subclinical hypothyroidism; relation to adverse outcome // Journal of Clinical & Translational Endocrinology. - 2014. - №1. - P. 13-17.)In the works of foreign authors, the state of mitochondrial functions, the ability of mitochondria to generate energy was studied as one of the biomarkers of female fertility, in particular the quality of oocytes and the subsequent viability of the embryo and fetus (Cecchino GN, Alves da Motta EL,

The role of mitochondrial activity in female fertility and assisted reproductive technologies: overview and current insights // Reproductive biomedicine online. - 2018. - No. 36. - P. 686-697. The role of mitochondrial functions (for example, bioenergetics, steroidogenesis, respiratory chain activity, production of reactive oxygen species in the mediated programming of fetal brain development under various stressful situations (physical, social, environmental influences) in women during gestation was studied according to placental and umbilical cord data. Anke Hoffmann, Dietmar Spengler. The Mitochondrion as Potential Interface in Early-Life Stress Brain Programming. Frontiers in Behavioral Neuroscience., 2018, Vol. 12, doi: 10.3389/fnbeh.2018.00306. An assessment of mitochondrial dysfunction in peripheral blood lymphocytic cells is presented. flow cytometry in subclinical hypothyroidism in women in the gestational period in relation to adverse pregnancy outcomes (Feldthusen A.-D. et al. Pregnancy-induced alterations in mitochondrial function in euthyroid pregnant women and pregnant women with subclinical hypothyroidism; relation to adverse outc ome // Journal of Clinical & Translational Endocrinology. - 2014. - No. 1. - P. 13-17.)

A well-known analogue of the claimed invention is a study on the assessment of reduced membrane potential (MPM) of total leukocytes using the Mito Probe™ JC-1 Assay Kit (Life Technologies, USA) in accordance with the instructions for the kit on the BD FACS CANTO II flow cytometer (BD Biosciences , USA) in chronic obstructive pulmonary disease in adults. The revealed increase in the number of leukocytes with reduced MMP indicates a decrease in the energy supply of the cell, oxygen starvation and a predisposition to the development of cellular hypoxia (Denisenko Yu.K., Novgorodtseva T.P., Vitkina T.I., Antonyuk M.V., Zhukova N.V. On the issue of mitochondrial dysfunction in chronic obstructive pulmonary disease // Bulletin of Physiology and Pathology of Respiration - 2016. - Issue 60. - P. 28-33).

The closest analogue of the present invention is a method for diagnosing a violation of the energy metabolism of lymphocytes in community-acquired pneumonia in children, which consists in determining the degree of violations of the energy supply of blood lymphocytes by flow cytometry by the percentage of lymphocytes with reduced mitochondrial membrane potential (RU 2579317 C1 10.04.2016).

The disadvantage of these analogues is:

- assessment of the total leukocyte population or only individual pools of ICC;

- lack of a comprehensive assessment of the energy supply of immunocompetent blood cells;

- the inability to assess the degree of severity of blood ICC energy deficiency, which is especially important for the justification, volume and selection of doses of therapeutic and preventive measures;

- the influence was considered only within the framework of one nosology, although the considered mechanism is universal for any immunodependent pathology.

The problem is to improve diagnostics, with the possibility of preclinical determination and simplification of data interpretation with a high accuracy of a comprehensive assessment of the energy supply of immunocompetent blood cells (lymph-, granulo- and monocytes) in case of risk or specified pathology among the observed patients.

EFFECT: possibility of early preclinical, technically fast diagnostics and comprehensive assessment of the degree of deficiency in the energy supply of immunocompetent blood cells, in particular in adults, pregnant women, children, by taking into account the percentage of peripheral blood lympho-, granular- and monocytes with reduced MMP (Δψ) by flow cytometry .

The method is carried out as follows.

At the initial stage, the number of 3 pools of immunocompetent peripheral blood cells (lymph-, granulo- and monocytes ) with reduced MMM. The upper limits of the norm were determined empirically when examining patients of various age groups (women of reproductive age outside of pregnancy, n=58 studies, during pregnancy, n=170, children, n=30) according to the criteria with minimal risk factors for any pathology or complicated course during gestation. Based on the results obtained, group ranges of reduced MMP values were calculated: for the adult population (lymphocytes - more than 35%, granulocytes and monocytes - more than 5%), for pregnant women (lymphocytes - more than 40%, granulocytes and monocytes - more than 10%), for children (lymphocytes - more than 35%, granulocytes - more than 3%, monocytes - more than 5%).

Depending on the combination of blood ICC with a reduced membrane potential of mitochondria, 3 degrees of energy deficiency are distinguished: the first degree - monovariant compositions of ICC (lymphocytes, or granulocytes, or monocytes); the second degree of energy deficiency - simultaneous bivariate compositions (lymphocytes and granulocytes, or lymphocytes and monocytes, or granulocytes and monocytes); the third degree of deficiency of energy supply of immunocompetent blood cells is a simultaneous increase in the number of cells with a reduced membrane potential of mitochondria of all types (lymphocytes, granulocytes and monocytes).

EFFECT: method makes it possible to comprehensively diagnose ICC energy deficiency at the preclinical stage with high accuracy and reliability, which makes it possible to determine the tactics of therapeutic and preventive measures in the early stages of pathological conditions.

The technical result is also achieved by the fact that, in addition to the simultaneous accounting of the number of lymphocytes, granulocytes and monocytes with a reduced membrane potential of mitochondria, the boundaries of the norm and pathology of these indicators are determined.

The tables show the results of 503 studies of pregnant women during registration at the antenatal clinic in the early stages of gestation and in dynamics (Table 1), as well as 42 children with bronchial asthma (Table 2).

The use of the proposed method for a comprehensive assessment of the energy supply of immunocompetent blood cells, as an additional method to the approved medical standards, performed technically in a short time and with high accuracy, allows for early preclinical diagnosis of deficient conditions in various pathological manifestations, it is possible to reasonably determine the choice, the amount of necessary therapy and assess the effectiveness of treatment.

The advantages of the proposed method:

1. A comprehensive assessment of the energy supply of 3 pools of the leukocyte blood population in different variations makes it possible to judge the severity of mitochondrial insufficiency of ICC;

2. Changes in the MMM of the ICC are ahead of the clinical picture and other data from other diagnostic methods;

3. The use of the high-tech method of laser flow cytometry and the specialized Cell Quest Pro program allow one-time, on a large number of cells, to analyze separately all subpopulations of leukocytes with high measurement accuracy.

4. Determining the degree of energy deficiency of blood ICC is a preclinical diagnosis, which makes it possible to substantiate and select schemes for correcting the identified disorders, as well as to evaluate the effectiveness.

The following are examples of the use of the proposed method among various population groups.

Example 1. Pregnant N., 35 years old. I got registered in the antenatal clinic at 7-8 weeks. This pregnancy is the second, the first pregnancy ended in term, normal delivery, the child was born with a weight of 2900, height 48 cm. Of the extragenital diseases, a spinal hernia was noted, she was not operated on, she observes a regimen of physical activity. Gynecological history is aggravated: endometriosis for 3 years, chronic adnexitis. The social conditions are good. This pregnancy is planned. Harmful factors before and during pregnancy were not noted. The power source is mainly market, cottage, shop, uses filtered water. During the initial examination, complaints of weakness, drowsiness in the evening, shortness of breath, palpitations during exercise, marked pallor and dryness of the skin. A laboratory examination showed no deviations in the general, biochemical blood tests, in terms of ferrodynamics, a decrease in blood iodides to 7.41 µmol/l was noted. A decrease in MMP was determined in all three pools of immunocompetent blood cells: the percentage of lymphocytes with reduced MMP - 55.89%, granulocytes - 17.76%, monocytes - 15.69%. Conclusion: there is an energy deficiency of ICC blood of the 3rd degree (a decrease in all 3 pools: lympho-, granulo-monocytes). At a period of 10-11 weeks, an ultrasound diagnosis was made: a miscarriage and medical measures were taken.

Example 2. Pregnant O., 39 years old. I got registered in the antenatal clinic at 11-12 weeks. This pregnancy is the third (IVF). Of the extragenital diseases, hemorrhoids were noted. The obstetric and gynecological history is burdened: the first pregnancy (16 years ago) was frozen, ended in an early miscarriage for a period of 8-9 weeks, the second (15 years ago) proceeded with anemia, chlamydia, ureaplasmosis was detected, ended in an urgent, normal birth, the child was born with a weight of 3275, a height of 48 cm, with an entanglement of the umbilical cord. In the next 10 years she was observed for infertility, fibroids. The social conditions are good. This pregnancy is planned. Harmful factors before and during pregnancy were not noted. She was sent for an additional examination due to a violation of the utero-placental-fetal blood flow of the 3rd degree for a period of 20-21 weeks. On examination, he complained of recurrent leg cramps. Conducted laboratory examination showed LJD according to the general blood count and ferrodynamics, no deviations in biochemical parameters, a decrease in blood iodides to 8.91 µmol/l. A decrease in MMP was determined in two pools of immunocompetent blood cells: granulocytes - 14.72%, monocytes - 15.69%, in normal MMP lymphocytes - 30.24%. Conclusion: there is an energy deficiency of ICC blood of the 2nd degree (decrease in MMP in 2 pools: granulo-, monocytes). Repeatedly at a period of 28-29 weeks, LJ remained, a decrease in blood iodides to 8.3 μmol / l, according to MMP, the following results were obtained: a decrease in MMP in two pools of blood ICC: in lymphocytes up to 53.07%, monocytes - 15.54%, in granulocytes - the norm is 6.68%. Conclusion: there is an energy deficiency of ICC blood of the 2nd degree (decrease in MMP in 2 pools: lymph and monocytes). At a period of 32-33 weeks at the third screening by ultrasound, FGR of the 1st degree, an asymmetric form, a violation of the uteroplacental-fetal blood flow of the 1a degree were determined. The woman received therapy according to the standard. The pregnancy ended in an urgent delivery, the birth of a child with FGR of the 1st degree, an asymmetric form, with a weight of 2040 g, a height of 49 cm.

Example 3. Pregnant A., 38 years old. Got registered in the antenatal clinic at 15-16 weeks. This pregnancy is the fifth. Of the extragenital diseases, obesity of 2-3 degrees, esophageal reflux, esophagitis, chronic course, hemorrhoids, vegetative-vascular dystonia of the cardiac type were noted. Obstetric and gynecological history is burdened: 3 medical abortions, chronic adnexitis, chlamydia, candidiasis. The first pregnancy (16 years ago) proceeded with ICI, preeclampsia, ended in urgent, pathological delivery (bleeding), the child was born with a weight of 3250, height 51 cm. Social conditions are good, the second marriage. This pregnancy is planned. Harmful factors before and during pregnancy were not noted, work at the computer for 7-8 hours a day before and during pregnancy. She was referred for additional examination at 21-22 weeks. On examination, complaints of weakness, drowsiness, occasional dizziness, shortness of breath and palpitations during physical exertion, dry skin, brittle nails, rarely cramps in the calf muscles, constipation. A comprehensive laboratory examination revealed: LDH according to the general blood test and ferrodynamics, a violation of the lipid profile and glucose tolerance test. A decrease in MMP was determined in two pools of immunocompetent blood cells: lymphocytes - 49.5%, granulocytes - 14.72%, in monocytes - the norm is 5.1%. Conclusion: there is an energy deficiency of ICC blood of the 2nd degree (decrease in MMP in 2 pools: lymph and granulocytes). Repeatedly for a period of 32-33 weeks, LJ, gestational diabetes mellitus persisted. The woman was under observation, received the necessary therapy in accordance with the standards. According to the MMM data, the following results were obtained: a decrease in MMM in one pool of ICC blood: in lymphocytes up to 57.89%, granulocytes (2.7%) and monocytes (6.25%) corresponded to the norm. Conclusion: there is an energy deficiency of ICC blood of the 1st degree (decrease in MMP of the 1st pool: lymphocytic). At a period of 34-35 weeks, at the third screening, ultrasound determined premature detachment of the placenta, impaired uteroplacental blood flow of 2-3 degrees. Conducted operational benefits for a period of 35 weeks. The child was born prematurely, 7-8 points on the Apgar scale, with a body weight of 2590, a length of 50 cm. The placenta shows signs of chronic placental insufficiency, in the stage of subcompensation, impaired placental circulation of 2-3 degrees.

Example 4. Patient K., 15 years old. Admitted to the hospital with complaints of unproductive cough, shortness of breath at rest and during exercise, noisy breathing. A child from the 2nd pregnancy, which proceeded against the background of toxicosis of the 1st half, gestosis-2nd half of the pregnancy. Childbirth 1st, urgent. Long dry period. Apgar score 3-7-8 points, received mechanical ventilation with a mask - 3 minutes.

From an early age, there were manifestations of an allergic reaction such as urticaria (the cause is not clear), sneezing, difficulty in nasal breathing when in contact with a cat, wormwood, lilac. Hereditary history is not burdened. The dynamics of the development of this disease: in 2014, the child was diagnosed with Bronchial asthma, moderate severity. She received basic therapy: Seretide 125/25 1 t * 2 times until 2015, then flixotide 50 mcg 1 t * 2 times, since 2016 - montelukast with a positive clinical effect, she was regularly observed by the allergist of the Research Institute of OMiD. At the end of 2016, due to the remission of the disease, basic therapy was canceled by the allergist. In September 2017, a child developed a cough against the background of ARVI, attacks of difficulty in breathing, and the peak flow meter was below normal. Due to the exacerbation of the disease, she was hospitalized at the clinic of the Research Institute of OMiD, was discharged with the main diagnosis: Bronchial asthma, mild, persistent, exacerbation. Attack of moderate severity; concomitant diagnosis: allergic rhinitis, moderate course. When examined for allergens by ELISA, sensitization to mold was revealed. After discharge, Seretide 25/50 mcg, 1 push 2 times a day through a spacer, was recommended as basic therapy. On the background of therapy, positive dynamics during the year. Due to the repeated exacerbation of the underlying disease (the attack was removed on an outpatient basis), since April 2018 (according to the examination by an allergist), she received Seretide 25/125 mcg, 1 push 2 times a day. In December 2018, there was an exacerbation against the background of ARVI, the dose of Seretide 25/250 mcg was increased, 1 push 2 times a day. Against the background of increased basic therapy, no signs of bronchial obstruction were observed within 3 months. According to the girl, since March 2019 she received Seretide 25/125 mcg, 1 push 2 times a day with a positive effect. A real aggravation from 06/13/19, associated with flowering and an increase in the number of trees. There was a hacking cough, shortness of breath. She received Seretide in the same dosage, berodual episodically at the time of increased dyspnea during physical exertion. On the background of therapy, cough and difficulty in breathing persisted. We turned to the allergist of the Research Institute of OMiD, sent for hospitalization. On the radiograph of the chest on 06/17/19, there is an increase in the pulmonary pattern, there are no infiltrative changes. Diagnosis: Bronchial asthma, moderate, uncontrolled, exacerbation, attack of severe bronchial obstruction. Persistent allergic rhinitis of moderate severity.

The study revealed a decrease in MMP in all three pools of immunocompetent blood cells: the percentage of lymphocytes with reduced MMP - 95.6%, monocytes - 87.43%, granulocytes - 90.21%. Conclusion: the child has an energy deficiency of blood ICC of the 3rd degree (a sharp decrease in all 3 pools: lympho-, granulo-monocytes), which corresponds to the severity of the condition at the moment - the attack period - and the uncontrolled course of the underlying disease.

Example 5. Patient A., aged 15. Complaints at admission does not actively show. The dynamics of the development of this disease: in 2007, the diagnosis was made: Bronchial asthma, mild persistent, received basic therapy with flixotide. Was observed at the Research Institute of OMiD, hospitalization in the hospital in 2007, 2009. about the exacerbation of the disease. Sensitization to house dust mite. He received basic therapy for a short time. He was observed by an allergist-immunologist, in the dynamics there was a significant improvement. There were no episodes of obstruction for 10 years. He is actively involved in sports (hockey), tolerates physical activity well. He was sent for a scheduled inpatient examination. Taking into account the anamnesis, the duration of the disease, the lack of examination for a long time, he was hospitalized in the pediatric department in order to assess the state of the bronchopulmonary system. Objectively, there were no signs of obstruction, respiratory function was within the normal range, the test with broncholithin was negative, and sensitization to house dust mites was confirmed. The diagnosis was established: Bronchial asthma, intermittent, controlled, long-term clinical remission. Does not require basic therapy.

The study revealed a slight decrease in MMP in lymphocytes to 44.9%, in the remaining pools, the percentage of MMP reduction within the reference age indicators: monocytes - 1.4%, granulocytes - 0.46%. Conclusion: the child has a slight energy deficiency of blood ICC, 1 degree (monovariant decrease in MMP in lymphocytes).

Thus, the above examples have shown the diagnostic significance of determining the degree of energy supply of immunocompetent blood cells, the need for early diagnosis, and the development of prognostic and treatment-and-prophylactic recommendations.

Claims (1)

A method for determining the degree of energy supply of immunocompetent blood cells (ICC) using flow cytometry with determining the percentage of ICC with a reduced membrane potential of mitochondria, characterized in that a comprehensive assessment of the state of the immune system is simultaneously carried out, moreover, indicators of lymphocytes, granulocytes, monocytes are used as immunological indicators, determining the percentage of ICC: lymphocytes, granulocytes, monocytes with a reduced membrane potential of mitochondria, and in comparison with similar indicators of blood samples from healthy individuals, deviations from the norm are determined: for the adult population: lymphocytes - more than 35%, granulocytes and monocytes - more than 5%, for pregnant women are lymphocytes - more than 40%, granulocytes and monocytes - more than 10%, for children these are lymphocytes - more than 35%, granulocytes more than 3%, monocytes - more than 5%; at the same time, depending on combinations of ICC with deviations from the norm, the degree of violation of the energy supply of ICC is determined: the first degree is a monovariant combination of ICC with a reduced membrane potential of mitochondria: lymphocytes, or granulocytes, or monocytes; the second degree of violations of the energy supply of ICC - simultaneous bivariate combinations of ICC with a reduced membrane potential of mitochondria: lymphocytes and granulocytes, or lymphocytes and monocytes, or granulocytes and monocytes; the third degree of disturbances in the energy supply of ICC is a simultaneous increase in the number of cells with a reduced membrane potential of mitochondria of all types: lymphocytes, granulocytes, monocytes.