Differential diagnostic technique of leiomyoma growth patterns
SUBSTANCE: differential pre-operative diagnosis of the fast growth patterns of leiomyoma is ensured by measuring the relative content of CD3+CD56+CD158i lymphocytes in the peripheral venous blood of a woman with the fast-growing leiomyoma. If the derived value is equal to 0.8% or less, the true growth of leiomyoma is diagnosed, while the value of more than 0.8% shows the false growth of myoma.
EFFECT: using the given technique enables the preoperative diagnosis of the fast growth patterns of leiomyoma in the females of reproductive age that enables developing the optimum management of the patient in good time and selecting the conservative and surgical methods of treating.
1 tbl, 3 ex
The invention relates to medicine, namely to gynecology, and can be used as a preoperative differential diagnosis types of rapid growth of leiomyoma of the uterus in women of reproductive age.
The relevance of the proposed method is determined by the fact that fibroids is one of the most common gynecological diseases. Developing in women of reproductive age, uterine fibroids violates their reproductive function, leads to infertility, miscarriage, perinatal loss, which negatively affects the demographic situation in the country (Immune mechanisms of rapid growth of fibroids / A. I. Malyshkina, N. Y. Sotnikova, Y. S. Antsiferov, A. K. Krasilnikov. Ivanovo, 2010. - 272 S.).
There is the concept of rapid growth of fibroids, which refers to the increasing size of the uterus during the last year of observation on the value corresponding to 4-5 weeks of pregnancy. When studying morphological criteria of rapid growth of fibroids distinguish "true" type tumor growth, caused by the proliferation of myocytes, and "false" when the node increases due to secondary changes (edema, hyalinosis, sclerosis, petrification) associated with circulatory disorders (Immune mechanisms of rapid growth of fibroids / A. I. Malyshkina, N. Y. Sotnikova, Y. S. Antsiferov, A. K. Krasilnikov. - vinovo, 2010. - 272 S.). It is known that proliferating uterine fibroids has a less favorable course. Patients with this type of tumor growth is often a malignancy of the tumor and its recurrence. Women with proliferative type of growth of fibroids belong to the risk group for the development of hyperplastic endometrium and mammary glands, as well as on the development of cystic benign and malignant tumors in the ovaries (Fibroids / Ed. by I. S. Sidorova. - M.: MIA, 2003. - 256 S.).
When choosing tactics and treatment of patients is important for defining the types of tumor growth. In this regard, we need to search for new criteria that allow to accurately differentiate between "true" and "false" type growth of leiomyoma of the uterus.
A known method of differential diagnosis of a rapidly growing leiomyoma of the uterus on the content of epidermal growth factor in endometrial. When the value of 6 ng/ml or less, diagnose "false" growth of fibroids, while values greater than 6 ng/ml is the "true" growth of fibroids (patent No. 2258222. Of The Russian Federation. The method of differential diagnosis of rapidly growing leiomyoma of uterus / L. C. Moiseeva, A. I. Malyshkina, A. K. Krasilnikova, etc. // Invention. The utility model. - 2005. No. 22). However, this method has several disadvantages.
1. The method is invasive.
2. Taking endometrial investigated the e is carried out during the hysteroscopy in stationary conditions, which is associated with hospitalisation and use of expensive equipment.
3. The method is extremely time-consuming, because its implementation requires the allocation of endometrial tissue enriched population of mononuclear cells with their subsequent cultivation for 24 hours. For mycobacterial culture required expensive nutrient and CO2-incubator.
A method of differential diagnosis of rapidly growing leiomyoma of the uterus in women of reproductive age, by definition, in the blood content of TNF-α, the absolute content of CD4+ lymphocytes, the relative content of T-cytotoxic lymphocytes CD8+, results of spontaneous NBT-test, and serum total complement activity and calculation of index Y by the formula. When the Y value is less than the forecast of 4.6 nephrolithiasis option growth of uterine fibroids; when Y is greater 6,8 predict proliferating option growth of uterine fibroids; for Y from 4.6 to 6.8 - uncertain prognosis, requiring the use of the method of descriptive statistics, using quality anamnestic and clinical criteria (patent No. 2400757. Of The Russian Federation. The way to diagnose morphological types of uterine leiomyoma / Ie; but, Y. O. Sevastyanova, I. A. Gazieva, etc. // Invention. The utility model. - 2010. No. 27). However, this method it is no several disadvantages.
1. The method is extremely time-consuming, as it involves a large number of techniques and, consequently, has a higher cost.
2. There is a range of values from 4.6 to 6.8, which is regarded as an uncertain prognosis, requiring the use of additional methods of research that has an impact on the accuracy of the results. According to the mathematical formula of this study, the reliability of the results is 70%. The closest technical solution to the claimed method is a method of differential diagnosis of the type of growth of leiomyoma of the uterus in women of reproductive age. In the peripheral blood of women with growing leiomyomas uterine determine the relative number of lymphocytes with the phenotype CD4+IL-10+ and if the value of this rate of 5.8%, or more, diagnose the "true" growth of uterine leiomyoma, and a value of less than 5.8% - "false" growth (patent No. 2361216. Of The Russian Federation. The method of differential diagnosis of the type of growth of uterine leiomyoma / N. Y. Sotnikova, L. C. Moiseeva, O. M. Boyko, etc. // Invention. Utility models. - 2009. - №19).
However, it was shown that the level of this pool T-helper cells increased in patients with several diseases. It is established that the CD4+IL-10+ increased in patients with connective tissue dysplasia (Regulatory T cells in eripheral blood of patients with mixed connective tissue disease / S. Barath, S. SipKa, M. AleKsza et al. // Scand. J. Rheumatol. - 2006. - Vol.35, No. 4. - P. 300-304), with autoimmune diseases such as systemic lupus erythematosus (Monocytes and T lymphocytes contribute to a predominance of interieukin 6 and interleukin 10 in systemic lupus lupus / S. Mellor-Pita, M. J. Citores, R. Castejon et al. // Cytometry In Clin. Cytom. - 2009. - Vol.76, No. 4. - P. 261-270), and with antiphospholipid syndrome (Immunological features of primary anti-phospholipid syndrome in connection with endothelial dysfunction / P. Soltesz, H. Der, K. Veres et al. // Rheumatology (Oxford). - 2008. - Vol.47, No. 11. - P. 1628-1634). In addition, there are a number of studies A. Bourn, confirming the change of this indicator during infection with herpes simplex virus (Dysregulated expression of IFN-gamma and IL-10 and impaired IFN-gamma-mediated responses at different disease stages in patients with genital herpes simplex virus-2 infection / R. Singh, A. Kumar, W. D. Creery et al. // Clin. Exp.Immunol. - 2003. - Vol.133, No. 1. - P. 97-107). In addition, there is an increase in the pool of T-helper cells in the blood of allergic patients after a course of allergen-specific immunotherapy (Induction of T regulatory cells by standardized house dust mite immunotherapie: an increase in CD4+CD25+ interieukin-10+ T cells expressing peripheral tissue trafficking / L. M. Gardner, F. C. Thien, J. A. Douglass et al. // Clin. Exp. Allergy. - 2004. - Vol.34, No. 8. - P. 1209-1219). Thus, this indicator varies in a wide range of diseases, and therefore the specificity of the method is reduced. The accuracy of the method is 79,1%. These drawbacks are proposed to be eliminated in the present method.
The technical solution of the proposed method is to improve the accuracy of cooperation the th differential diagnosis types growth of leiomyoma of the uterus in women of reproductive age 88,2%, sensitivity to 85.0%, specificity up of 92.9%. The claimed technical result is achieved by the fact that in the peripheral blood of women with growing leiomyomas uterine determine the relative number of lymphocytes with the phenotype CD3+CD56+CD158i and if the value of this indicator more than 0.8% diagnose "false" the growth of uterine leiomyoma, and a value equal to or less than 0.8%, the "true" growth.
The novelty of the proposed method lies in the fact that for the first time are encouraged to differentiate between "true" and "false" type rapid growth of uterine leiomyoma by definition in the peripheral blood expression of the receptor CD158i on natural killer T-lymphocytes (EC-T), markers which are CD3+CD56+. Previously it was shown that the level of this indicator decreased in endometrial tissue in women with rapidly growing fibroids (Character expression of KIR receptors of natural killer T-lymphocytes in the endometrium uterine fibroid different growth / A. I. Malyshkina, N. Y. Sotnikov, A. N. Voronin and other // problems of reproduction. - 2012. - Special. release. - S. 186). In other works studied receptors (CD1581) on natural killer cells (CD56+). There was an increased CD56+CD158i lymphocytes during HIV infection (Perturbation of the natural killer cell compartment during primary human immunodeficiency virus 1 infection primarily involving the CD56 bright subset / P. Mantegani, G. Tambussi, L. Galli et al. / Immunology. - 2010. - Vol.129, No. 2. - P. 220-233) and chronic hepatitis (High frequency of polyfunctional natural killer cells with high expression of NKG2D is linked to HBV-specific T-helper type 1 immune control / I. Carey, A. Mendes, S. Bansal et al. // Journal of Hepatology. - 2010. - Vol.52. - P. 176). In addition, the marked increase in CD56+CD158i lymphocytes in cancer of blood - leukemia T-cell large granular lymphocyte leukemias have multiple phenotypic abnormalities involving pan-T-cell antigens and receptors for MHC molecules / R. Lundell, L. Hartung, S. Hill et al. // Am J. Clin. Pathol. - 2005. - Vol.124, No. 6. - P. 937-946). For the differential diagnosis of types of rapid growth of uterine leiomyoma content of CD3+CD56+CD158i lymphocytes have not used.
The population of lymphocytes with the phenotype CD3+CD56+ referred to as natural killer T-lymphocytes (EC-T). It is established that the targets for these cells are transformed (infected) and rapidly proliferating cells (Yarylo A. A. Immunology. - M.: GEOTAR-Media, 2010. - 752 C.). Cytotoxic activity of EC-T is regulated by activating and inhibitory KIR receptors that interact with ligands on the target cells (Activating and inhibitory receptors of natural killer cells / J. Pegram Hollie, Daniel M. Andrews, Mark J. Smyth et al. // Immunology and Cell Biology. - 2011. - Vol.89. - P. 216-224). Receptors CD158i are activators of cytotoxic lymphocytes directed against target cells, because of their involvement in tumor immunity (Activating and inhibitory receptors of natural killer cells CD158 Receptor Controls Cytotoxic T-Lymphocyte Susceptibility to Tumor-Mediated Activation-Induced Cell Death by Interfering with Fas Signaling / A. Gati, N. Guerra, C. Gaudin et al. //Cancer Research. - 2003. - Vol.63, No. 1. - P. 7475). Changes in the expression of activating CD158i Rotz is tori, ligands are molecules HLA-Cw on the surface of transformed cells of the myometrium, reflect the state of the anti-tumor surveillance by the immune system. Thus, the EC-T, expressing the activating KIR receptors are involved in the mechanisms of rapid growth of fibroids.
The method is performed by a standard method as follows.
1) To 3 ml of heparinized venous blood obtained from the cubital vein, add 3 ml of medium 199 and produce an enriched population of mononuclear cells standard method of high-speed centrifugation (Bourn A. Isolation of mononuclear cells and granulocytes from human blood and bone marrow // Scand. J. Clin. Invest. - 1968. - Vol.21, No. 97. - P. 77).
2) Suspension of mononuclear cells washed twice with saline, adjusting the cell concentration to 1×106KL/mn. To 0.1 ml of the resulting suspension at the same time add 20 ál of monoclonal antibodies anti-D3 labeled PC 5, anti-CD56, labeled D, anti-SW labeled FICE (JSC "Sorbent", Moscow). Cells incubated for 30 min at room temperature in the dark, washed in 1 ml of saline.
3) Determine the relative (percentage) number of triple-positive CD3+CD56+CD158i lymphocytes in lymphocytic gate on a flow cytometer.
The use of the proposed method with high accuracy 88,2% to differentiate between "true" and "false" is the IPA rapid growth of leiomyoma of the uterus.
Distinctive features of the process are: the establishment of differential diagnostic parameter of the relative content of CD3+CD56+CD158i lymphocytes, when the value of which is 0.8% or less diagnosed "true" growth of uterine leiomyoma, and a value of more than 0.8% is "false" the growth of leiomyoma of the uterus.
The essence of the proposed method is illustrated by the following examples.
Example 1. Patient Z., 39 years old, was admitted for planned surgery. Was observed in the antenatal clinic with uterine myoma of the small sizes (6 weeks). During the next inspection one year after the first treatment discovered the rapid growth of fibroids. The patient experienced pain in the lower abdomen and lower back, heavy menstruation, accompanied by a decrease of hemoglobin. History 1 miscarriage, 3 medical abortion. Method of contraception - barrier. The patient planning to conceive.
During examination: cervix without mucosal defects, the body of the uterus is increased to 18 weeks of pregnancy due to myoma node, located on the rear wall of the uterus. The body of the uterus is thick, movable, painless. Appendages to palpation without features.
According to CCS: the body of the uterus heterogeneous patterns, sizes together with site - 142×130×122 mm In the rear wall of the uterus is intramurals-subserous site 132×123×107 mm, the uterus is not deformed. The thickness of the end the Tria 13 mm The right ovary 30×18 mm Left ovary 38×30 mm Both ovaries normal patterns.
Blood analysis: hemoglobin - 94 g/l, erythrocytes-4.0 t/l, colour index-0.6, leukocyte - 5,2 g/l, stab neutrophils - 2, segmented neutrophils - 59, monocytes - 8, lymphocytes - 28, ESR - 15 mm/h
Clinical diagnosis: intramurals-subserous leiomyoma of the uterus. Rapid growth. Hypermenorrhea, leading to anemia. Pain syndrome.
According to immunological examination by the present method it is established that the content in peripheral blood CD3+CD56+CD158i lymphocytes was 0.55%, which is lower than the proposed option (0,8%).
Conclusion: diagnosed "true" type rapid growth of leiomyoma of the uterus.
Given the age of the woman and unrealized reproductive function, scheduled organ-preserving volume of surgical intervention. In connection with the results of the growth of uterine fibroids, and since this type of tumor has a less favorable for the patient as a pre assigned agonist RH-RG - buserelin of 3.75 mg of Drug was administered once in 28 days intramuscularly for 2 months. In the second stage, 3 weeks after the last injection, the woman admitted for surgical treatment.
According to CCS after treatment: the body of the uterus heterogeneous patterns, sizes together with node - 125×122×102 mm On Adna of the uterus is intramurals-subserous site 120×108×87 mm The uterus is not deformed. Endometrial thickness of 8 mm Right ovary 30×18 mm Left ovary 38×30 mm Both ovaries normal patterns.
Blood test (after treatment): hemoglobin - 120 g/l, erythrocytes-4.2 t/l, leucocytes - 5,3 g/l, stab neutrophils - 1, segmented neutrophils - 63, monocytes - 8, lymphocytes - 26, ESR -18 mm/h
Transaction - laparotomy, myomectomy.
Histological examination of the surgical material from the proliferating leiomyoma uterus". Thus, the diagnosis of "true" type growth of leiomyoma of the uterus was confirmed.
Example 2. Patient B., 38 years. During the year, was observed in female consultations on uterine myoma of the small sizes (6-7 weeks). For a specified period of menstruation became more abundant, there is a weakness after menstruation. History 1 abortion, 1 timely delivery without complications. Method of contraception - barrier.
At vaginal examination: cervix cylindrical, without defects mucosa. The uterus is increased to 12-13 weeks of pregnancy, due to multiple hosts, much too soft to palpation, mobile, painless. The appendages are not palpable, vaults available.
According to the ultrasonic scanning (CCS): the body of the uterus dimensions 120×80×115 mm, heterogeneous structure. In the front wall of the uterus is intramurals-subserous site diameter is rum 48 mm in the right side wall with a diameter of 38 mm, in the left corner of the node with a diameter of 48 mm, the rear node diameter 57 mm, All nodes of heterogeneous structure. The uterus is not deformed. Endometrial thickness 10 mm Right ovary 38×26 mm, left ovary 37×20 mm Both ovaries normal patterns.
Blood analysis: hemoglobin - 63 g/l, erythrocytes - 2,78 t/l, colour index of 0.7, the leukocyte count was 3.9 g/l, stab neutrophils - 5, segmented neutrophils - 50, monocytes - 9, lymphocytes - 31, ESR - 20 mm/h
Clinical diagnosis: multiple intramural-subserous leiomyoma uterus large sizes. Rapid growth. Hypermenorrhea, leading to anemia.
According to immunological examination by the present method it is established that the content in peripheral blood CD3+CD56+CD158i lymphocytes amounted to 1.35%, which is higher than the proposed option (0,8%).
Conclusion: diagnosed "false" type rapid growth of leiomyoma of the uterus.
Transaction: Subtotal hysterectomy without appendages.
Histological examination of the surgical material was diagnosed: "leiomyoma uterus with severe edema and focal hyalinosis stroma". Thus, the diagnosis of "false" type growth of leiomyoma of the uterus was confirmed.
Example 3. Patient B., 41. Was observed in the antenatal clinic for 4 years with uterine myoma of the small sizes (5-6 weeks is). In the past, there were profuse menstruation, accompanied by a decrease of hemoglobin. History 1 timely delivery without complications, 3 medical abortion. Method of contraception - barrier.
At vaginal examination: cervix without defects mucosa, uterus increased to 15 weeks of pregnancy, due to a host that is located in the anterior wall of the uterus. The uterus is a dense, mobile, painless. The appendages are not palpable, vaults available.
According to CCS: the body of the uterus dimensions 120×75×100 mm, heterogeneous structure. The front wall is visualized intramurals-subserous site 95×65×80 mm with the phenomenon of swelling. The uterus is not deformed. Endometrial thickness 12 mm Right ovary size 38×25 mm, left ovary size 37×20 mm, regular patterns.
Blood analysis: hemoglobin - 121 g/l, erythrocytes - 4,55 m/l, color index is 0.8, leukocyte - 4.0 g/l, segmented neutrophils - 62, monocytes - 6, lymphocytes - 26, ESR - 19 mm/h
Clinical diagnosis: intramurals-subserous leiomyoma of the uterus. Rapid growth. Hypermenorrhea. Pain syndrome.
According to immunological examination by the present method it is established that the content in peripheral blood CD3+CD56+CD158i lymphocytes was 0.8%, which corresponds to the edge value of the requested parameter.
Conclusion: diagnosed "true" type b is strictly growth of leiomyoma of the uterus.
Transaction: Subtotal hysterectomy without appendages.
Histological examination of the surgical material obtained result is: "proliferating leiomyoma with a moderate number of vessels and stromal edema". Thus, the diagnosis of "true" type growth was confirmed.
In this way were examined 34 women of reproductive age with growing leiomyomas of the uterus. These studies are listed in table 1.
|Index||The number of the examined|
|a true-negative result||13|
|a false-negative result||3|
|Total: the accuracy of the proposed method - 88,2%, the sensitivity of the proposed method - 85,0%, specificity of the proposed method - of 92.9%|
The advantages of the proposed method.
1. The way n which increases accuracy, sensitivity, specificity differential diagnosis types growth of leiomyoma of the uterus.
2. The method expands the Arsenal of tools for the differential diagnosis of types of growth of leiomyoma of the uterus.
The method of differential diagnosis types rapid growth of uterine leiomyoma by immunological studies of peripheral venous blood in women of reproductive age, characterized in that to determine the relative number of CD3+CD56+CD158i lymphocytes, and if the value of this indicator, equal to 0.8% or less, diagnose the "true" growth of uterine leiomyoma, and a value of more than 0.8% is "false" the growth of uterine leiomyoma.
SUBSTANCE: invention refers to immunological methods of analysis and can be used for mass screening of congenital diseases in newborn children. That is ensured by immobilising in a microplate well first immune-specific components to thyrotropin, immunoreactive trypsin, thyroxin and 17α-OH-progesterone in the form of discrete microregions, placing in the well a paper disk of a dry blood sample, extracting the detectable markers from dry blood stains by adding into the well a buffer containing danazol and 8-anilinonaphthalene-1-sulfonic acid and 17α-OH-progesteron antibodies, which are bound to extracted 17α-OH-progesterone to form an immune complex with anti-species antibodies in the respective microregion simultaneously, then adding into the microplate well a reaction solution of second immune-specific components, containing a mixture of biotin-conjugated antibodies to thyrotropin, immunoreactive trypsin and thyroxin, and a 17α-OH-progesteron-protein-biotin conjugate for preparing the immune biotin-labelled complexes in the discrete microregions, removing the paper disk of the dry blood sample, adding a streptavidin and Pt-coproporphyrin conjugate to prepare the phosphorescent biotin-streptavidin complexes in the microregions on the bottom of the microplate well and detecting the label phosphoresce emission, by scanning the discrete microregions by a focused laser beam sequentially.
EFFECT: using the given method enables detecting biomarkers specifically and qualitatively in the newborn's capillary blood stain sample that enables diagnosing three newborn's congenital conditions.
10 cl, 7 dwg, 10 tbl
SUBSTANCE: invention refers to medicine, namely to child neurology. A method includes detecting the clinical signs of a disease, measuring erythrocytes with micronuclei in a child's peripheral blood. The novel in the method for determining a degree of severity of the disease in the children with ICP is that the clinical signs of the disease are assessed by the Rivermead Mobility scale, and then an enzyme immunoassay is used to determine the qualitative content of the tumour necrosis factor (TNF-α) in the ICP children's saliva; the measured and determined values are compared to the clinical signs, and if the measured erythrocytes with micronuclei make 0.55±0.14%, saliva TNF-α increases to 13.23±5.2 pg/ml, a moderate severity is stated, while erythrocytes with micronuclei making 1.27±0.87% and more, and saliva TNF-α being 25.41±5.42 pg/ml and more show the severe disease.
EFFECT: invention enables providing the simple, non-invasive, accessible instant method for determining a degree of severity of the disease and prescribing the timely treatment.
5 dwg, 8 tbl
SUBSTANCE: invention represents an instant diagnostic technique for acute intestinal infections (AIIs), involving detecting indication markers of the AII aetiology with the use of laboratory immunology tests, differing by the fact that the AII aetiology is stated in children of an early age category, preferentially in the newborn children; that is accompanied by measuring the concentration of cytokine, interleukin IL-10 in coprofiltrate and diagnosing chronic placental insufficiency (CPI); a probability (P) of the bacterial AII aetiology is calculated; the value P of more than 50% testifies to the bacterial AII aetiology, while the value P being less than 50% shows the absence of the bacterial AII aetiology, and enables considering the diagnostics second stage to be necessary, which implies measuring the concentration of cytokine, interleukin IL-4 in coprofiltrate; the time of latching the newborn child to the breast is established with considering the type of feeding; that is combined with calculating a probability (P) of the viral or viral-bacterial AII aetiology, with the value P of more than 50% testifying to the viral AII aetiology, while the value being less than 50% makes it possible to state the viral-bacterial AII aetiology.
EFFECT: more accurate diagnosing of the aetiology of acute intestinal infection and simplifying the diagnostic procedure.
SUBSTANCE: venous blood and oral fluid are sampled from a patient in any sequence. An oral fluid biomarker is presented by carcino-embryonal antigen (CEA). A blood plasma biomarker is presented by neuron-specific enolase. If the oral fluid CEA is found in an amount of 196.8-318.4 ng/ml, while an amount of plasma neuron-specific enolase is 26.3-34.2 ng/ml, sarcoma of jaw bones is diagnosed. If the oral fluid CEA is found in an amount of 1228.2-1762 ng/ml with an amount of plasma neuron-specific enolase being 38.67-57.67 ng/ml, squamous cell carcinoma of jaw bones is diagnosed. The findings are used to predict a therapeutic approach to the patient.
EFFECT: invention provides high-sensitive and accurate differential diagnostic technique of maxillary and mandibular malignant new growths on the day of visit to a doctor.
2 cl, 6 ex
SUBSTANCE: group of inventions relates to medicine and deals with a method of determining in an individual's sample of a protein of neutrophilic origin, gelatinase-associated lipocalin (NGAL), for determination of acute kidney injury in the individual, where the predominating amount of monomer and/or heterodimer forms of NGAL protein, in comparison with the dimer form of NGAL protein, shows that NGAL protein originates from the individual's kidneys and that the individual has acute kidney injury, whereas equal or predominating amount of the dimer form of NGAL protein, in comparison with the monomer or heterodimer form of NGAL protein shows that NGAL protein originates from the individual's neutrophils and that the said individual does not have acute kidney injury; a set for determination of relative amounts of monomer, dimer or heterodimer forms of NGAL; application of the device in the said method.
EFFECT: group of inventions ensures more accurate diagnosis and therefore contributes to better directed treatment.
17 cl, 2 ex, 10 dwg, 1 tbl
SUBSTANCE: invention is a method of determining the nonspecific resistance of pathogenic microorganisms to antibiotics and the fact of the presence of bacterial biofilms on the basis of measurement of catalytic activity of phosphodiesterases cleaving the cyclic diguanosine monophosphate, with a threshold sensitivity of 50 pg/ml, comprising: 1) isolating the target-phosphodiesterase from lysed bacterial cells; 2) binding of phosphodiesterase with biotin-conjugated antibodies specific for non-catalytic domains of phosphodiesterase; 3) affinity purification of complexes formed by target-phosphodiesterase and biotin-conjugated antibody using paramagnetic particles containing neutravidin or its analogs that bind biotin; 4) interacting of the complexes of phosphodiesterase/biotin-conjugated antibody, immobilised on paramagnetic particles with complexes containing a-di-GMP in the form of G-quadruplex systems with intercalate dye, which is accompanied by decrease in the intensity while destruction of complexes of intercalate dye with c-di-GMP; 5) measurement of decrease of fluorescence upon hydrolysis with c-di-GMP and destruction of complex of c-di-GMP with intercalate dye, followed by quantitative estimation of the phosphodiesterase activity based on calibration curves made using known amounts of the recombinant enzyme of phosphodiesterase identical to the test target; 6) identification of increased level of phosphodiesterase activity detected by the test antibiotic-resistant bacterial strains capable of biofilm formation, as compared with the level of phosphodiesterase activity that can be detected for the control strains of bacteria of the same species not having the antibiotic resistance and the ability to form biofilms.
EFFECT: method enables to determine the nonspecific resistance of pathogenic microorganisms to antibiotics and to establish the fact of the presence of bacterial biofilms.
4 dwg, 5 ex
SUBSTANCE: invention relates to the field of immunology, namely to enzyme-immunoassay, in particular to a method of detecting forms of vascular endothelial growth factor (VEGF) with a size more than 110 amino acids in a biological sample. The method includes the following stages: contact and incubation of the biological sample with an uptake reagent, immobilised on a solid substrate, where the uptake reagent contains a monoclonal antibody, which recognises and specifically binds with residues, in quantity more than 110, from human VEGF; separation of the biological sample from the immobilised uptake reagents; contact of the immobilised molecular complex of the reagent of the uptake-target with detected antibody, which binds with VEGF domains, responsible for binding with KDR and/or FLT1 receptor, or which binds with an epitope in VEGF1-110; measurement of the level of VEGF110+, bound with reagents of the uptake, with application of means of detection for the detected antibody. Set of immune assay reagents for detection of VEGF110+ forms in the biological sample. An antibody 5C3, obtained from hybridoma 5C3.1.1 with a depositary number PTA-7737, with the said antibody 5C3 binding VEGF110+ forms, including VEGF121+. Hybridoma 5C3.1.1, deposited in ATCC with the depositary number PTA-7737, to obtain the monoclonal antibody 5C3.
EFFECT: application of the claimed invention makes it possible to increase accuracy of detecting VEGF isoforms, which must not include isoform VEGF110 and must obligatory include isoform VEGF121.
25 cl, 3 dwg, 2 tbl, 1 ex
SUBSTANCE: invention represents an immunoassay reagent which contains an agent binding to an analyte in a diluent, and glycosaminoglycan in an amount sufficient to decrease non-specific binding in an analyte sample. In the presented immunoassay reagent, the analyte is troponin I binding to the analyte; the agent is a biotin-modified anti-troponin I antibody, and glycosaminoglycan is chondroitin sulphate. Also, the invention provides a composition containing the troponin I binding agent, and chondroitin sulphate in an amount sufficient to decrease non-specific binding in the troponin I sample. What is also provided is a method of detecting the analyte in the sample wherein non-specific binding is decreased by the use of glycosaminoglycan.
EFFECT: method improvement.
5 ex, 5 dwg
SUBSTANCE: in the method for assessing action of biologically active substances on the antigen-antibody interaction based on sampling whole blood, stabilising it by an anticoagulant, adding a biologically active substance to the whole blood samples of the groups O(I)-AB(IV), incubating for 3-5 minutes, introducing standard monoclonal anti-A and anti-B antibodies, and 3-5 minutes later, rating the agglutination intensity relevant to the blood group.
EFFECT: improved assessment accuracy.
SUBSTANCE: flow cytofluorimetry is used to examine peripheral blood for the content of CB34+CB45+hemopoietic precursors on 1-3 pot-injury day, but not earlier than in 8 hours. If the content is lower than 3×106/l, an unfavourable clinical outcome is predicted, while the content 3×106/l and more shows a favourable clinical outcome of a moderate and severe craniocerebral injury.
EFFECT: using the method enables higher accuracy of the early prediction of clinical outcomes in the patients with the craniocerebral injury and the prescription of the adequate differentiated therapy.
SUBSTANCE: method involves evaluating proliferating processes by calculating index of positive cell nuclei (Ki-67). The Ki-67 value being from 6 to 16%, erosive ulcerating stomach lesions accompanied by stomach hemorrhage and hemorrhagic shock is to be predicted. The value being from 17 to 30%, erosive ulcerating stomach lesions without hemorrhage is to be predicted.
EFFECT: high accuracy of prognosis.
FIELD: medicine, virology.
SUBSTANCE: invention relates to diagnosis of infection caused by the Epstein-Barr virus. Method involves carrying out the indirect immune peroxidase reaction followed by histochemical staining and detection of the Epstein-Barr virus antigen. Additionally, method provides diagnosis of severity for the infectious mononucleosis course based on the expression of Epstein-Barr virus antigen. Invention provides enhancing precision in diagnosis of infection caused by the Epstein-Barr virus and assay for severity in course of the infectious mononucleosis.
EFFECT: improved diagnosis method.
1 tbl, 2 ex
FIELD: medicinal biochemistry.
SUBSTANCE: the present innovation deals with detecting oncoprotein E7 of human papilloma virus (HPV) in biopsy sample with the help of the pairs of monoclonal antibodies referring to IgG2a and IgG2b groups chosen out of the following groups: 716-321, 716-325, 716-332, 716-343, 716-281, 716-288 one of which is indicated for primary protein binding and another, being the antibody conjugate with enzymatic label - to detect the complexes developed.
EFFECT: higher sensitivity of the method.
5 cl, 4 dwg, 4 ex, 2 tbl
FIELD: medicine, therapy, obstetrics.
SUBSTANCE: at gestation terms of 20-28 wk in peripheral blood one should detect the index for the ratio of relative content of CD4+ to CD8+ lymphocytes. At values of CD4+/CD8+ being equal to or below 2.4 it is possible to predict positive effect of common therapy, and at values being above 2.4 one should predict thorough and prolonged therapy. The method enables to match another therapeutic tactics in due time.
EFFECT: higher accuracy of prediction.
3 ex, 1 tbl
FIELD: medicine, obstetrics.
SUBSTANCE: one should detect during pregnancy-free period relative content of CD38+ lymphocytes in peripheral blood, at its value being either equal to or above 12% one should predict efficient restoration of reproductive function. The method is very simple in application.
EFFECT: higher accuracy and sensitivity of detection.
3 ex, 1 tbl
FIELD: medicine, pediatrics.
SUBSTANCE: in 5-10-d-aged neonatals one should detect relative content of CD8+HLA-DR+ lymphocytes in peripheral blood and at its value being below 2.4% it is possible to predict the healing at different types of neonatal infections.
EFFECT: higher accuracy of prediction.
3 ex, 1 tbl
FIELD: medicine, gynecology.
SUBSTANCE: invention relates to a method for diagnosis of internal endometriosis in peripheral venous blood of women wherein the relative content of lymphocytes CD25+ is determined. Internal endometriosis is diagnosed at values of this index 6% or above. Proposed method provides carrying out diagnosis of internal endometriosis in women with high precision, sensitivity and specificity that allows carrying out the correct and well-timed necessary complex of curative-prophylactic treatment.
EFFECT: improved method for diagnosis.
1 tbl, 3 ex
FIELD: medicine, immunological laboratory diagnostics.
SUBSTANCE: at terms from 6 to 12 wk of gestation one should study relative content of CD3+CD16+ lymphocytes in peripheral venous blood and at its values being either equal or above 5.4% one should predict the development of light-degree gestosis to carry out the complex of curative-prophylactic means.
EFFECT: higher efficiency of prediction.
3 ex, 1 tbl
FIELD: medicine, laboratory diagnostics.
SUBSTANCE: during the 1st trimester of pregnancy (6-13 wk) in peripheral venous blood in women at risk of failed pregnancy one should detect relative content of CD16+CD56- lymphocytes and at its value being either equal or below 11% it is possible to predict the development of infectious diseases in full-term neonatals during the first 7-10 d of their lives. The innovation enables to predict the development of local form of infectious-inflammatory diseases in full-term neonatals.
EFFECT: higher accuracy of prediction.
3 ex, 1 tbl
FIELD: medicine, clinical laboratory diagnostics.
SUBSTANCE: in the sample of peripheral venous blood one should determine relative content of CD45RO+ lymphocytes and at its value being equal to 31% or lower it is possible to diagnose external genital endometriosis. The method is atraumatic and enables to diagnose external genital endometriosis at high accuracy.
EFFECT: higher efficiency of diagnostics.
3 ex, 1 tbl