Method for the diagnosis of chronic endometritis and nature of the inflammation

 

(57) Abstract:

The invention relates to medicine, namely to gynecology, pathology and immunology, and can be used for the diagnosis of chronic endometritis and determine the nature of the inflammatory changes in the endometrium. The invention consists in immunohistochemical determining local immunity, namely the number of lymphocytes expressing markers of natural killer cells CD56+, CD16+ and the activation markers HLA-DR(II)+. The technical result is the development of criteria for the diagnosis of chronic endometritis, establishing the involvement of the immune system in inflammatory process in the endometrium.

The invention relates to medicine, section of gynecology, pathology and immunology.

The problem of the study of inflammatory diseases of the endometrium in women of reproductive age is relevant, as it causes not only a violation of menstrual function, but also generative. The frequency of occurrence of chronic endometritis and infertility ranges from 20 to 60% (Smetnik B. N., Tumilovich L., non-operative gynecology. - 2nd edition. - SPb., 1995.- KN.2 - S. 46-52), with the usual Nerat on competition of a scientific degree of the doctor of medical Sciences. - SPb., 1993). Clinical manifestations of chronic endometritis are not specific, the disease is often diagnosed incidentally screening women.

For the diagnosis of chronic endometritis using standard histological method that takes into account the totality of the General morphological features of chronic inflammation: presence of focal lymph leukocyte infiltration; focal fibrosis of the stroma; the presence of plasma cells and sclerotic changes of the walls of the spiral arteries (Khmelnitsky O. K. Pathomorphological diagnosis of gynecological diseases. - SPb: Sothis., 1994. S. 136-144; Kuznetsov A. C. Chronic endometritis. Archives of pathology, 2000, No. 3, S. 48-52). The main disadvantage of the existing method is the low diagnostic accuracy. The presence of only one or two of the above criteria inflammation is always doubt as to the morphological diagnosis of chronic endometritis. Other methods of verification of diagnosis to date does not exist (Saveliev, M. and others Hysteroscopy/ Savelyev, M., Prusenko Century BC, Eplosive L. M. - M.: GSTAR Medicine, 1999).

The literature data concerning changes in the parameters of local immunity in chronic vospalitelnoe endometritis. Archives of pathology, 2001, No. 5, S. 8-13; Elliniki C. N., Kalinina N. M. Immunomorphologic endometrium. Infertility treatment: unresolved issues. Saratov, 2001, S. 158-162).

The purpose of the present invention is the development of criteria for the diagnosis of chronic endometritis.

The decision of the specified objective is achieved by identifying inflammation in the tissue of the endometrium based on a comprehensive clinical-morphological examination of women, indicators of local immunity characteristic of inflammation, immunohistochemical method and clarify the nature of the inflammatory process in the comparison of the performance of local and systemic immunity.

According to the proposed method is carried out to determine whether markers of immune cells of the endometrium by immunohistochemical method that allows to reliably detect the presence of chronic inflammation in the endometrium and the degree of severity; this is examined in endometrial cells expressing markers of natural killer cells CD56+ and CD16+ lymphocytes expressing the activation marker HLA-DR+, class II, involved in antigen recognition.

Rationale for the proposed diagnostic method is the fact that inflammation is often resisting and local immunity.

The proposed method for the determination of the expression on the membrane of immune cells specific antigens, i.e., surface phenotype, was chosen due to the fact that it is a method of identifying cells. The advantage of the proposed method is the identification of cells involved in inflammation. Immunohisto-cytochemical method is a modern, highly sensitive method for identification and localization in cells and tissues of different structures that have antigenic properties, based on the reaction of antigen-antibody. The method used vysokoavidnyh and highly specific monoclonal antibodies directed to the same epitope of the antigen. In this regard, the method allows to identify the different immunecompetent cells (T lymphocytes, natural killer cells) involved in inflammation. To visualize the reaction of the antigen-antibody used avidin-Biotin-peroxidase complex, and the manifestation of horseradish peroxidase is diaminobenzidine. As a result, the reaction product is detected in the form of persistent brown staining of the membrane. Immunohistochemical method described in detail in the methodological recommendations (Elliniki C. N., Anikeeva N.In., Maksimova N. A.: Prakticheski is using immunohistochemical method carry out a quantitative assessment of immune cells expressing markers of activation and markers of natural killer cells, which allows to estimate the severity of the inflammatory process, and dynamic changes of the studied parameters to evaluate the effectiveness of therapy.

The proposed diagnostic method allows, by comparison of the local and systemic immunity to clarify the nature of inflammation, to identify patients with autoimmune nature of the inflammatory processes.

The proposed method for the diagnosis of chronic endometritis and nature of the inflammatory disease is as follows.

Define endometrial cells expressing markers CD56+, CD16+ and HLA-DR(II)+. The research is being done with the use of antigen-specific monoclonal antibodies of the company Novocastra to the data markers on paraffin sections of endometrial tissue immunohistochemically method comprising the following steps:

Carry out the dewaxing of the sections, followed by demeterova antigen tissue by boiling in citrate buffer (pH 6.0) under pressure in a pressure cooker for 2 minutes. Spend incubation of slices with normal serum for 30 minutes. Spend the incubation of the sections with the first antigen specific monoclonal antibodies CD56+, CD16+ and HLA-DR(II)+the Yat incubation with avidin-Biotin-peroxidase complex for 30 minutes. The achievement of the staining substrate reaction (horseradish peroxidase) for the purpose of visualization is performed with the help of diaminobenzidine 3 minutes. The reaction product appears as a persistent membranous staining in brown color. The results of the study parameters of local immunity (CD56+, CD16+ and HLA-DR(II)+) measure quantitative way, looking at the light microscope stained by immunohistochemical analysis of endometrial tissue (biopsies, swabs, aspirates) at magnification of lens 40, eyepiece 10 3 fields of view.

Count the number of positively stained cells (expressing the studied markers) in each field of view. In the case of a minor variation of quantitative indicators in different fields of view will determine the arithmetic mean.

The number of lymphocytes expressing markers CD56+, CD16+ and HLA-DR(II)+ is determined in the following way.

The number of lymphocytes expressing markers CD56+ is equal to the sum of the number of lymphocytes expressing markers CD56+ in the first, second and third fields of view divided by 3.

The number of lymphocytes expressing markers CD16+ is equal to the sum of the number of lymphocytes expressing markers CD16+ in the first, second and third fields of view is impositon, expressing markers HLA-DR(II)+ in the first, second and third fields of view divided by 3.

Example. The number of lymphocytes, CD56+ in the first field of view 21, in the second field of view 23, in the third field of view is 25.

The calculation is as follows: (21+23+25):3=23.

The increase of quantitative indices of the investigated markers determine the severity of the inflammatory process.

To analyze the obtained results revealed changes of parameters of local immunity natural killer cells (CD56+, CD16+) and lymphocytes expressing the activation marker HLA-DR(II)+ in the endometrial tissue is correlated with the indicators of systemic immunity in women surveyed, which explore the subpopulation composition of peripheral blood lymphocytes (method flow cytometry), production of cytokines and serum immunoglobulin classes a, M, G, E, secretory immunoglobulin A (sIgA) (enzyme-linked immunosorbent assay).

According to the proposed method when the number of cells expressing CD56+, CD16+, HLA-DR(II)+ from 0 to 10 in the field of view, diagnose the absence of endometritis. When the number of cells expressing CD56+ above 10, and the number of cells expressing CD16+ and HLA-DR(II)+ ausich CD56+, CD16+ HLA-DR(II)+ above 10 in the field of view, diagnose exacerbation of autoimmune chronic endometritis. When the number of cells expressing CD16+ and HLA-DR(II)+ above 10, and the number of cells expressing CD56+ from 0 to 10 in the field of view, diagnose chronic endometritis with exacerbation or acute endometritis. When the number of cells expressing CD16+, CD56+ above 10, and the number of cells expressing HLA-DR(II)+ from 0 to 10 in the field of view, diagnose chronic inflammation with an autoimmune component, but without activation process. When the number of cells expressing CD16+ above 10, and the number of cells expressing CD56+ and HLA-DR(II)+ from 0 to 10 in a field of view of diagnosed chronic endometritis.

The method is illustrated by the following examples.

1. no history of the disease 4620.01 outpatient Department of the Institute of obstetrics and gynecology. D. O. Ott RAMS, L. N., 28 years. Clinical diagnosis of primary Infertility. The data of anamnesis, objective examination, gynecological examination revealed no signs of a chronic endometritis. Data transferred urogenital infection no. Patient screened for 19 day of a menstrual cycle. Histological examination N2277.01: endometrium secondary phases proliferatsiya CD56+ - 5 in the field of view, CD16+ 10 in the field of view, HLA-DR(II)+ - 0 in the field of view, indicating the absence of inflammation. After conducting specific hormonal therapy pregnancy ended 05.11.2002 how year of birth full-term baby.

2. no history of the disease 3170.01 outpatient Department of the Institute of obstetrics and gynecology. D. O. Ott, RAMS, C. Y. C., 28 years. Clinical diagnosis of primary Infertility. The data of anamnesis, objective and gynecological examination revealed no signs of a chronic endometritis. Data transferred urogenital infection no. Examined on day 24 of the menstrual cycle. Histological examination N1887.01: endometrium middle phases of secretion - late cell proliferation, developmental delays stroma, patterns "iron gland", isolated mononuclear cells in the stroma, lymphoid infiltration of the stroma. Immunohistochemistry: the number of cells expressing CD56+ - 5 in the field of view, CD16+ 0 -10 in the field of view, HLA-DR(II)+ 0 in the field of view, indicating the absence of inflammation. After conducting specific hormonal therapy pregnancy, 15.01.2003, is progressing 25 weeks.

3. no history of the disease 3440.02 outpatient Department And the. the before survey was received combined therapy for urogenital infection: from the cervical canal were isolated Mycoplasma and Ureaplasma in the blood was determined by the diagnostic titer of immunoglobulin G to chlamydia 1/64. Examined on day 12 of the menstrual cycle: a Histological study N: 1515.02: endometrium middle stage phase proliferation of the stroma with a tendency to vibrazioni expressed diffuse focal mononuclear infiltration. Immunohistochemistry: the number of cells expressing CD56+ 50 in the field of view, CD16+ 60 field of view, HLA-DR(II)+ - 5 in the field of view, indicating that chronic inflammation in the endometrium without signs of activation, but with an autoimmune component. After conducting a comprehensive therapy pregnancy, 15.01.2003, - progress - 29 weeks.

4. no history of the disease 4408.02 - patient Department of the Institute of obstetrics and gynecology. D. O. Ott, RAMS, P. M., 32 year history of primary infertility for 7 years. Clinical diagnosis of Primary infertility. Bacteriological examination of data for infection is not received. Histological examination N2033.02: 7 day cycle: the endometrium middle phases of proliferation with merenneito cells expressing CD56+ - 3 in the field of view, CD16+ 17 in the field of view, HLA-DR(II)+ - 30 field of view, indicating that chronic endometritis without autoimmune component, but with signs of acute (active inflammation).

After conducting a comprehensive treatment of pregnancy, at the moment is progressing to 26 weeks.

5. no history of the disease 58,01 - patient Department of the Institute of obstetrics and gynecology. D. O. Ott, RAMS, T. E. C., 24 years. The clinical diagnosis of secondary Infertility. History of miscarriage early period. Bacteriological examination of the data for the presence of genital infection is not obtained. Histological examination N1316.01: 22 day cycle: an early phase of secretion, the coils of the spiral arteries is well expressed, weak diffuse mononuclear infiltration of the stroma, the tendency to vibrazioni under the surface epithelium. Immunohistochemistry: the number of cells expressing CD56+ - 45 in sight, CD16+ 0 - 45 in sight, HLA-DR(II)+ - 45 field of view, indicative of autoimmune chronic endometritis in the acute phase. After conducting a comprehensive therapy pregnancy ended with the birth of the unwanted child 05.02.2002,On the basis of the proposed method for the diagnosis of chronic endometritis, a survey of 156 women of reproductive age in clinics, research Institute of obstetrics and gynecology. D. O. Ott RAMS (including 5 healthy female control group).

Before performing invasive examinations (laparoscopy, hysteroscopy, hysterosalpingography, endometrial biopsies) all women were clinically examined. The survey was conducted to exclude syphilis, HIV, hepatitis b and C. Genital infection was excluded by bacteriological study of the secrets of the vagina and cervical canal, including conducted cultural diagnosis of Mycoplasma, chlamydia, method of immunofluorescence was detected herpes type I and II, were performed serological testing blood for the presence of antichlamydial antibodies of class G and A, antibodies of class G to pathogens herpes infection, and when their availability was determined by the avidity index. All infected was 44.5% of women identify 2 or more pathogens was observed in 66% of those infected. To conduct immunological examination of all infected IATA - the number of natural killer cells CD56+and CD 16+, lymphocytes expressing the activation marker HLA-DR(II)+) was studied in the endometrium of all 156 women.

When using conventional survey (histological) diagnosis of chronic endometritis was installed only 18 of 151 women (11.9 percent). The use of immunohistochemical studies helped to establish the diagnosis of chronic endometritis 81 of 151 women (53,6%), which increased the accuracy of clinical diagnosis and facilitated the correct tactics of treatment of women.

After conducting a comprehensive therapy at the moment, different terms of ongoing pregnancy are 28 women, the pregnancy ended urgent delivery in 27 women.

Thus, the proposed method allowed us to develop criteria for evaluating indicators of local immunity of the endometrium by immunohistochemical method on paraffin sections. The proposed method for the diagnosis of chronic endometritis and nature of the inflammatory changes in the endometrium in women immunohistochemically method differs in that it allows you to accurately establish the involvement of the immune system in inflammatory process in the investigated tissue (endometrium). Apostal the II)+) and systemic immunity has allowed to confirm the nature of the autoimmune inflammatory process in the endometrium.

Bibliography

1. Demidov E. M. Habitual miscarriage (Pathogenesis, obstetric tactics): author's abstract on competition of a scientific degree of the doctor of medical Sciences. SPb - 1993. - page 42

2. Kuznetsov A. C. Chronic endometritis. Archives of pathology, 2000, N3, pp. 48-52.

3. Kuznetsov A. C., Spiders, S. C., Voloshchuk, I. N., Demidov E. M., Kazarian S. M. Morphological features of chronic endometritis. Archives of pathology, 2001, N5, pp. 8-13.

4. Saveliev, M. and others Hysteroscopy/ Savelyev, M., Breusenko Century BC, Eplosive L. M.-M.: GSTAR Medicine, 1999. - 176 S.

5. Smetnik B. N., Tumilovich L., non-operative gynecology. - 2nd edition. - SPb., 1995. - KN.2. - S. 46-52.

6. Freidlin, I. S., totolan A. A. cells of the immune system. - SPb.: Science, 2001. - 390 S. - (So 3; T. 4; T. 5).

7. Khmelnitsky O. K. Pathomorphological diagnosis of gynecological diseases. - SPb: Sothis., 1994. - S. 136-144.

8. Ellinida C. N., Anikeeva N.In., Maksimova N. A. Practical immunohistochemistry (methodical recommendations), St. Petersburg, 2002, pp. 1-36.

9. Ellinida C. N., Kalinina N. M. Immunomorphologic endometrium. Infertility treatment: unresolved issues. Saratov, 2001. - page 158-162.

Method for the diagnosis of chronic endometritis, including clinical-morphologies the performance of local immunity: lymphocytes, expressing markers of natural killer cells CD56+, CD16+ and the activation markers HLA-DR(II)+, and the calculation is carried out in a light microscope at magnification lens X40, X10 eyepiece in three fields of view, and when the number of cells expressing CD56+, CD16+, HLA-DR(II)+, from 0 to 10 in the field of view is diagnosed in the absence of endometritis; when the number of cells expressing CD56+, higher than 10 and the number of cells expressing CD16+ and HLA-DR(II)+, from 0 to 10 in sight diagnose autoimmune chronic endometritis; when the number of cells expressing CD56+, CD16+, HLA-DR(II)+ above 10 in sight diagnosed with exacerbation of autoimmune chronic endometritis; when the number of cells expressing CD16+ and HLA-DR(II)+, higher than 10 and the number of cells expressing CD56+, from 0 to 10 in a field of view of diagnosed chronic endometritis with exacerbation or acute endometritis; when the number of cells expressing CD16+, CD56+, higher than 10 and the number of cells expressing HLA-DR(II)+, from 0 to 10 in a field of view of diagnosed chronic inflammation with an autoimmune component, but without activation process; when the number of cells expressing CD16+, higher than 10 and the number of cells expressing CD56+ and HLA-DR(II)+, from 0 to 10 in sight diagnosed the

 

Same patents:
The invention relates to medicine, namely, neurology
The invention relates to medicine, in particular, to gastroenterology, Hepatology, surgery and gynecology

The invention relates to medicine
The invention relates to medicine, namely to traumatology and orthopedics

The invention relates to medicine and can be used to predict pregnancy and determine the degree of risk of impaired development of the embryo/fetus in women of childbearing age before or during pregnancy

The invention relates to the field of immunology, and can be used in the diagnosis of diseases associated with N. pylori using reaction coagglutination (RCA)

The invention relates to the field of health, but rather to Microbiology and vaccination, and can be used to assess the immunogenic properties of the pertussis vaccine, manufactured in serial production

The invention relates to medical Virology and Microbiology, and in particular to identification of a new strain of measles virus NovO/96 to obtain the antigen component of the diagnostic test system and the test system for the diagnosis of antibodies to measles virus

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, medicinal microbiology.

SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.

EFFECT: improved assay method.

3 tbl, 3 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

3 ex

FIELD: medicine, cardiology.

SUBSTANCE: in peripheral blood one should detect the level of CD95(+) and CD16(+) neutrophilic granulocytes and at combination of increased level of CD95(+) neutrophilic granulocytes by 4 times and more and CD16(+) neutrophilic granulocytes by 0.6 times against the norm with ECG signs of myocardial infarction one should predict lethal result of large-focal myocardial infarction.

EFFECT: higher accuracy of prediction.

FIELD: medicine, parasitology.

SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.

EFFECT: higher efficiency and accuracy of diagnostics.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, immunology.

SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.

EFFECT: higher efficiency of detection.

2 ex

FIELD: medicine.

SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.

EFFECT: enhanced accuracy of prediction.

FIELD: medicine, medicinal immunology.

SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.

EFFECT: improved method for assay.

5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.

EFFECT: high accuracy of diagnosis.

Up!