Method for in vitro determination of skin tuberculin high-grade sensitivity

FIELD: clinical diagnosis, in particular in vitro determination of skin tuberculin high-grade sensitivity.

SUBSTANCE: skin tuberculin high-grade sensitivity is determined based on alteration of fluorescent intensity of common leucocytal CD45 antigen and isoforms thereof CD45RA and CD45RO in test probe (after incubation of peripheral blood with 2TE tuberculin solution) in contrast to control probe with physiological solution by using monoclonal antibodies labeled with fluorescein isothiocyanate (FITC) and laser flow cytofluorometry.

EFFECT: new method for in vitro determination of skin tuberculin high-grade sensitivity.

1 tbl, 4 ex

 

The invention relates to medicine, in particular to a branch of clinical laboratory diagnostics, and can be used as a way of identifying the nature of the tuberculin skin hypersensitivity in vitro at the level of fluorescence intensity (if) labeled fluorostyrene (FITZ) CD45+-, CD45RA+and CD45RO+lymphocytes in the experimental sample (after incubation of peripheral blood with a solution of tuberculin TE) relative to the control sample (after incubation of peripheral blood with saline solution) using directly labeled FITZ monoclonal antibodies (MCAT) to antigens CD45, CD45RA, CD45RO and the flowing laser cytofluorimetry.

A known method for determining allergic hypersensitivity to drugs, antibiotics, anesthetics and muscle relaxants, vitamins in vitro by specific level of fluorescence intensity (WIF) labeled FITZ CD45+lymphocytes, an application for patent which was previously filed (application No. 99123165/14 from 04.11.99) [1, 15]. The method involves the incubation of peripheral blood of the patient with saline solution and medicinal solutions using labeled FITZ, MCAT IR 46 to the antigen CD45, automatic cooking lykousis using ImmunoPrepSystem ("Coulter", USA) and laser flow cytofluorimetry mode of lifegate, where the level change WITH IU the military FITZ CD45+lymphocytes in the experimental sample relative to a control sample to determine the coefficient of sensitization, at exceeding of which the corresponding critical value (for drugs antibiotics and 0.5, for anesthetics and muscle relaxants - 0.3, and for vitamins and 0.3) determine the patient the presence of hypersensitivity to the investigated drug[3, 4, 5].

The advantages of the above method include the use of changes if labeled FITZ CD45+lymphocytes in peripheral blood in the experimental sample (after incubation with drug solutions) relative to a control sample with saline in vitro as an indicator of allergic hypersensitivity on the studied drugs in patients with drug hypersensitivity[4, 5, 7].

The disadvantage of the above method is the lack of instructions for use in the test system in vitro solution of tuberculin TE, there is no technical and methodological recommendations on the use of this method in terms of survey BCG-vaccinated children with tuberculin skin hypersensitivity.

There is a method of determining increased sensitization to tuberculin in vitro the level of total and specific intensities of fluorescence labeled FITZ CD45+lymphocytes, which was chosen as the prototype [2]. The method involves the incubation of peripheral blood of a patient with physio is ogicheskom solution (control sample) and with a solution of tuberculin TE (experimental sample), followed by the use of labeled FITZ, MCAT IR-46 to surface onselaciunea antigen CD45, automatic cooking lykousis using ImmunoPrepSystem ("Coulter", USA) and laser flow cytofluorimetry mode of lifegate, where the change in the level of total and specific intensities of fluorescence labeled FITZ CD45+lymphocytes in the experimental sample relative to a control sample to determine the coefficient of sensitization, above which the standard value of 0.2 in the patient identify allergic hypersensitivity to the components of the solution of tuberculin used in the production of tuberculin tests [2].

The scope of the prototype is laboratory and diagnostic testing of children with doubtful and elevated skin reactions to the introduction of the solution of tuberculin TE (Mantoux test).

The use of the prototype in the laboratory and diagnostic practice allows you to install allergic Genesis clinical manifestations of cutaneous tuberculin hypersensitivity in children.

The advantages of the prototype can be attributed to the use of changes if labeled FITZ CD45+lymphocytes in the experimental sample (after incubation of peripheral blood with a solution of tuberculin TE) relative to a control sample with saline in vitro as an indicator of allergic hypersensitivity to components of the solution of tuberculin in children with clinical manifestations of cutaneous tuberculin the Oh hypersensitivity.

The disadvantage of the prototype is that on the basis of elevated values obtained coefficient sensitization (>0,2) is able to determine only the Genesis of allergic hypersensitivity to the components of the solution of tuberculin TE used in the production of tuberculin tests. According to our data, this amounts to 20.0% of cases of children with clinical manifestations of cutaneous tuberculin hypersensitivity [2]. While remaining in most cases children using the prototype does not allow you to install the Genesis of cutaneous tuberculin hypersensitivity.

It is known that among other tuberculin skin hypersensitivity (induration within 5-9 mm) in children vaccinated with BCG may arise from nonspecific toxic action of the components of the solution of tuberculin and vaccine-induced hyperresponsiveness of sensitized lymphocytes, and infection of Mycobacterium tuberculosis (MBT) [9].

In addition, we have shown that the diagnostic significance has as a phenomenon of increase and decrease if labeled FITZ CD45+lymphocytes in the test system with drugs in vitro relative to a control sample with saline solution [6]. In this regard, an important disadvantage of the prototype is the lack of guidance on diagnostic the significance of the recorded increase or decrease if labeled FITZ CD45+lymphocytes after incubation of peripheral blood with a solution of tuberculin THE in vitro relative to the level of a control sample [2].

The essence of the proposed method lies in the fact that to identify the nature of the tuberculin skin hypersensitivity use change if labeled FITZ CD45+-, CD45RA+and CD45RO+lymphocytes in the experimental sample (after incubation of peripheral blood with a solution of tuberculin TE) relative to the control sample (after incubation of peripheral blood with saline solution) (table 1).

Table 1.

The nature of the tuberculin skin hypersensitivity to change if labeled FITZ CD45+-, CD45RA+and CD45RO+lymphocytes in the experimental sample relative to a control sample
ConclusionIf in the experimental sample relative to the control
CD45+cellsCD45RA+cellsCD45RO+cells
Option I: Allergic reactivity to components of the solution of tuberculinIncreases >than 25.0%increases (decreases <than 9.0%)increases <than 20,0%
Option II: the Consequence of post-BCG vaccination hyperresponsiveness sensitized lymphocytesIncreases <than 25.0%increases >than 9.0%increases >than 20,0%
III variant a Consequence of the cytotoxic effects of the components of the solution of tuberculin Reduced <than at20,0%reduced<than 8.5%decreases (increases <than 8.8%)
IV variant: Suspected infection MBTReduced >than 20,0%decreases (increases <than 9.0%)decreases (increases <than 8.8%)
Note: allocation of marks leading indicator.

When in the pilot test with tuberculin is logged increase if labeled FITZ CD45+-, CD45RA+and CD45RO+lymphocytes relative to a control sample with the natural solution and this increase mainly affects CD45+lymphocytes (more than 25.0% - variant I in table 1), it is assumed that the patient is allergic reactivity to components of the solution of tuberculin, which can cause skin tuberculin hypersensitivity.

When in the pilot test with tuberculin is logged increase if labeled FITZ CD45+-, CD45RA+and CD45RO+lymphocytes relative to a control sample with the natural solution and this increase mainly affects CD45RO+lymphocytes (more than 20,0%, option II in table 1), it is assumed that the patient is post-BCG vaccination Hyper reactivity of sensitized lymphocytes, which can cause skin tuberculin is howling hypersensitivity.

When in the pilot test with tuberculin is logged reduction if labeled FITZ CD45+and CD45RA+lymphocytes relative to a control sample with the natural solution (less than 25.0% and 8.5%, respectively, option III in table 1), it is assumed that the clinical manifestations of cutaneous tuberculin hypersensitivity patients may be a consequence of the cytotoxic effects of the components of the solution of tuberculin.

When in the pilot test with tuberculin is logged reduction if labeled CD45+lymphocytes relative to a control sample with the natural solution (more than 20,0%, option IV in table 1), the patient does not exclude the state of infection of the office.

Analyzed in this way CD45+lymphocytes in peripheral blood are to antipersonnel the cells of the immune system, which due to morphological and functional characteristics of the molecule CD45 and cooperation with CD45 membrane antigen-receptor complexes lymphocytes (CD3-TCR-CD4; CD3-TCR-CD8) carry out the processes of recognition, initiation, and regulation development response immunological responses to antigens[11, 12, 13, 14]. Analyzed in this way CD45RA+lymphocytes in peripheral blood are to naive, nepilirovanny the cells of the immune system that are responsible for antigen-specific activation [10]. Analyzed in this way CD45RO+-lim is ocity peripheral blood are memory cells [8, 10].

Conducted by the applicant search on scientific, technical and patent information sources and selected from the list of analogues of the prototype [2] allowed us to identify the distinctive features of the claimed technical solution. Therefore, the claimed method meets the criteria of "novelty".

Conducted by the applicant additional search of the known technical solutions [1, 2, 15] to detect in them the characteristics similar to the characteristics distinctive part of the formula of the proposed method, showed that these signs are absent, therefore, the claimed technical solution meets the criterion of "inventive step".

The criteria of the invention "clinical diagnostic applicability is confirmed by the fact that the proposed method of determining the nature of tuberculin hypersensitivity level if labeled FITZ CD45+-, CD45RA+and CD45RO+lymphocytes in the pilot test with tuberculin TE relative to a control sample in vitro can be used in clinical practice as a standardized method specific diagnosis.

The inventive method is carried out as follows: to 480 μl of heparinized peripheral blood from the cubital vein add 120 ál of the working solution of tuberculin TE (experimental sample). Used commercial preparation of allergen tuberculosis purified liquid in a hundred is a standard breeding TE, containing 0.01% of chinosol (phenol) and 0.005% tween-80, manufactured by research Institute of vaccines and sera (, St. Petersburg). A similar method of preparing a control sample, which contains 480 μl of heparinized blood and 120 μl of physiologic saline. Experimental and control samples tightly closed, incubated 1 hour at 37 ° °With constant shaking.

After incubation:

to 100 μl of the control (sample 1) and 100 ál of the experimental samples (sample 2) add labeled FITZ, MCAT to the antigen CD45, at concentrations specified by the manufacturer;

- for the next 100 ál of the control (sample 3) and 100 µl of the test (test 4) add 5 ál of labeled FITZ, MCAT to CD45RA, in concentrations specified by the manufacturer;

- for the next 100 ál of the control (sample 5) and 100 µl of the test (test 6) add 5 ál of labeled FITZ, MCAT to CD45RO, in concentrations specified by the manufacturer.

All 6 samples incubated for 30 minutes at room temperature in the dark. After incubation, all 6 samples processed using equipment and ImmunoPrep reagent System ("Coulter", USA), resulting receive a suspension of leukocyte and lymphocyte labeled complex [FITZ - MCAT], without losing their viability and immunogenicity. Each sample was analyzed by laser flow cytofluorimetry EPICS XL (Coulter, USA) in infogate. The final result of the analysis of samples with p the power of the specified device is the study Protocol, where the data statistical processing of the histogram distribution of labeled FITZ CD45+-, CD45RA+-, CD45RO+lymphocytes at the level of philosophy. To assess changes if labeled FITZ CD45+lymphocytes in the experimental sample relative to a reference use indicator WITH. To assess changes if labeled FITZ CD45RA+and CD45RO+lymphocytes in the experimental sample relative to a reference use the average if (Srif). The level value Sref labeled FITZ CD45RA+and CD45RO+lymphocytes in experimental and control samples obtained from the study Protocol. The level value WITH labeled FITZ CD45+lymphocytes in experimental and control samples is determined by the developed by the author and described in the literature method [3]. Change if labeled FITZ CD45+-, CD45RA+-, CD45RO+lymphocytes in the pilot test with tuberculin TE relative to a control sample with saline solution is expressed as the speakers if (WPPT) and evaluated according to the formula:

where if is the intensity of fluorescence (specific for CD45; the average for CD45RA; middle - CD45RO).

The inventive method of determining the nature of the tuberculin skin hypersensitivity is illustrated by the following examples.

Example 1. Patient K., born in 2001, vaccinated with BCG vaccination scar 5 mm. In history marked fever and allergic rashes after the latter Lanovoy intradermal tuberculin TE. Induration of 5 mm

To 480 μl of heparinized peripheral blood from the cubital vein was added 120 μl of a solution of tuberculin TE. Similarly prepared control sample, which contained 480 μl of heparinized blood of the patient and 120 μl of saline. Experimental and control samples were tightly closed and incubated 1 hour at 37 ° °With constant shaking.

After incubation:

to 100 μl of the control (sample 1) and 100 µl of the test (sample 2) sample was added to 5 μl of labeled FITZ, MCAT to CD45 ("Caltag, USA);

- for the next 100 ál of the control (sample 3) and 100 µl of the test (test 4) was added to 5 μl of labeled FITZ, MCAT to CD45RA ("Caltag, USA);

- for the next 100 ál of the control (sample 5) and 100 µl of the test (sample 6) was added to 5 μl of labeled FITZ, MCAT to CD45RO ("Caltag, USA).

All 6 samples were incubated for 30 minutes at room temperature in the dark. After incubation, all 6 samples were processed using equipment and ImmunoPrep reagent System ("Coulter, USA). All obtained samples of analizirali using laser flow cytofluorimetry EPICS XL (Coulter, USA) in infogate.

Level WITH labeled FITZ CD45+lymphocytes in this case, in the control sample 1 was 0.2 in the test sample 2 (tuberculin) is 0.27. Using the formula for the calculation of the WPPT, get: ((0,2-0,27):0,27)×100%=-35,0%. In this case, ( - ) indicates that the experimental sample showing the tel WITH more than in the control. From this we conclude that the experimental sample WITH labeled FITZ CD45+lymphocytes increased to 35.0%.

Level Sref labeled FITZ CD45RA+lymphocytes in this case, in the control sample 3 was 6,85 in the test sample 4 (tuberculin) - 6,72. Using the formula for the calculation of the WPPT, get: ((6,85-6,72):6,85)×100%=1,89%. In this case, it is obtained that in the experimental sample rate Srif less than in the control. From this we conclude that the experimental sample Srif labeled FITZ CD45RA+lymphocytes decreased by 1.9%.

Level Sref labeled FITZ CD45RO+lymphocytes in this case, in the control sample 5 was 1,89 in the test sample 6 (tuberculin) is 2.0. Using the formula for the calculation of the WPPT, get: ((1,89-2,0):1,89)×100%=-5,8%. In this case, ( - ) indicates that the experimental sample rate Srif more than in the control. From this we conclude that the experimental sample Srif labeled FITZ CD45RO increased by 5.8%.

The results of the survey suggest that the clinical manifestations of cutaneous tuberculin hypersensitivity are a consequence of allergic reactivity to components of the solution of tuberculin (option 1 in table 1).

Example 2. Patient T., born in 2002, vaccinated with BCG vaccination scar 5 mm. the results of the annual planned tuberculidiagnostic: 2003 increased the size of the PA is uly after setting Mantoux test - 8-10 mm. Diagnosis: post-vaccination Allergy.

To 480 μl of heparinized peripheral blood from the cubital vein was added 120 μl of a solution of tuberculin TE. Similarly prepared control sample, which contained 480 μl of heparinized blood of the patient and 120 μl of physiologic saline. Experimental and control samples were tightly closed and incubated 1 hour at 37 ° °With constant shaking.

After incubation:

to 100 μl of the control (sample 1) and 100 µl of the test (sample 2) sample add 5 ál of labeled FITZ, MCAT to CD45 ("Caltag, USA);

- for the next 100 ál of the control (sample 3) and 100 µl of the test (test 4) was added to 5 μl of labeled FITZ, MCAT to CD45RA ("Caltag, USA);

- for the next 100 ál of the control (sample 5) and 100 µl of the test (sample 6) was added to 5 μl of labeled FITZ, MCAT to CD45RO ("Caltag, USA).

All 6 samples were incubated for 30 minutes at room temperature in the dark. After incubation, all 6 samples were processed using equipment and ImmunoPrep reagent System ("Coulter, USA). All the samples were analyzed using a laser flow cytofluorimetry EPICS XL (Coulter, USA) in infogate.

Level WITH labeled FITZ CD45+lymphocytes in this case, in the control sample 1 was 0.3 in the test sample 2 (tuberculin) is 0.35. WITH labeled FITZ CD45+lymphocytes increased by 16,66%.

Level Sref labeled FITZ CD45RA+-l is Mazitov in this case, in the control sample 3 were 5.0, in the experimental sample 4 (tuberculin) is 5.5. Using the formula for the calculation of the WPPT, get: ((5,0-5,5):5,0)×100%=-10,0%. In this case, ( - ) indicates that the experimental sample rate Srif more than in the control. From this we conclude that the experimental sample Srif labeled FITZ CD45RA+lymphocytes increased by 10%.

Level Sref labeled FITZ CD45RO+lymphocytes in this case, in the control sample 5 was 1.5 in the test sample 6 (tuberculin) is 1.9. Using the formula for the calculation of the WPPT, get: ((1,5-1,9):1,5)×100%=-26,66%. In this case, ( - ) indicates that the experimental sample rate Srif more than in the control. From this we conclude that the experimental sample Srif labeled FITZ CD45RO+lymphocytes increased by 26.6%.

The results of the survey suggest that the clinical manifestations of cutaneous tuberculin hypersensitivity in a patient can be a consequence of post-BCG vaccination hyperresponsiveness cells of the immune system (option II in table 1).

Example 3. Patient P., born in 1995 vaccinated with BCG vaccination scar 5 mm. Referring diagnosis - "turn tuberculin tests since 1999". Tuberculin sensitivity since 1999 is normalizekey, monotonous (the size of the papules after the annual planned production tuberculin Mantoux test was in the range of 8-12 mm).

To 480 μl of perifericheskoi heparinised blood from the cubital vein was added 120 μl of a solution of tuberculin TE. Similarly prepared control sample, which contained 480 μl of heparinized blood of the patient and 120 μl of physiologic saline. Experimental and control samples were tightly closed and incubated 1 hour at 37 ° °With constant shaking.

After incubation:

to 100 μl of the control (sample 1) and 100 µl of the test (sample 2) sample add 5 ál of labeled FITZ, MCAT to CD45 ("Caltag, USA);

- for the next 100 ál of the control (sample 3) and 100 µl of the test (test 4) was added to 5 μl of labeled FITZ, MCAT to CD45RA ("Caltag, USA);

- for the next 100 ál of the control (sample 5) and 100 µl of the test (sample 6) was added to 5 μl of labeled FITZ, MCAT to CD45RO ("Caltag, USA).

All 6 samples were incubated for 30 minutes at room temperature in the dark. After incubation, all 6 samples were processed using equipment and ImmunoPrep reagent System ("Coulter, USA). All the samples were analyzed using a laser flow cytofluorimetry EPICS XL (Coulter, USA) in infogate.

Level WITH labeled FITZ CD45+lymphocytes in this case, in the control sample 1 was 0,675 in the test sample 2 (tuberculin) is 0.6. Using the formula for the calculation of the WPPT, get: ((0,675-0,6):0,675)×100%=11.1 per cent. In this case, it is obtained that in the experimental sample rate WITH less than in the control. From this we conclude that the experimental sample WITH labeled FITZ CD45+lymphocytes decreased the camping 11.1%.

Level Sref labeled FITZ CD45RA+lymphocytes in this case, in the control sample 3 was 5.9 in the test sample 4 (tuberculin) to 5.7. Using the formula for the calculation of the WPPT, in a similar way we obtain that in the experimental sample Srif labeled FITZ CD45RA+lymphocytes decreased by 3.4%.

Level Sref labeled FITZ CD45RO+lymphocytes in this case, in the control sample 5 was 1.77 in the test sample 6 (tuberculin) was 1.69. Using the formula for the calculation of the WPPT, in a similar way we obtain that in the experimental sample Srif labeled FITZ CD45RO+lymphocytes decreased by 4.5%.

The results of the survey suggest that the clinical manifestations of cutaneous tuberculin hypersensitivity are a consequence of the cytotoxic action of components of the solution of tuberculin TE used for the production of Mantoux test (option III in table 1).

Example 4. Patient N., 1997 born in vaccinated with BCG vaccination scar 5 mm Atopiceski burdened history. The results of the annual planned tuberculidiagnostic from 1998 to 2003 - negative. Since 2003 marked induration after setting Mantoux test - 8 mm Diagnosis - turn tuberculin tests since 2003.

To 480 μl of heparinized peripheral blood from the cubital vein was added 120 μl of a solution of tuberculin TE. Similarly prepared control samples is, which contained 480 μl of heparinized blood of the patient and 120 μl of physiologic saline. Experimental and control samples were tightly closed and incubated 1 hour at 37 ° °With constant shaking.

After incubation:

to 100 μl of the control (sample 1) and 100 µl of the test (sample 2) sample add 5 ál of labeled FITZ, MCAT to CD45 ("Caltag, USA);

- for the next 100 ál of the control (sample 3) and 100 µl of the test (test 4) was added to 5 μl of labeled FITZ, MCAT to CD45RA ("Caltag, USA);

- for the next 100 ál of the control (sample 5) and 100 µl of the test (sample 6) was added to 5 μl of labeled FITZ, MCAT to CD45RO ("Caltag, USA).

All 6 samples were incubated for 30 minutes at room temperature in the dark. After incubation, all 6 samples were processed using equipment and ImmunoPrep reagent System ("Coulter, USA). All the samples were analyzed using a laser flow cytofluorimetry EPICS XL (Coulter, USA) in infogate.

Level WITH labeled FITZ CD45+lymphocytes in this case, in the control sample 1 amounted to 0.62 in the test sample 2 (tuberculin) - 0,34. WITH labeled FITZ CD45+lymphocytes in the experimental sample decreased by 45.2%.

Level Sref labeled FITZ CD45RA+lymphocytes in this case, in the control sample 3 was 5,78 in the test sample 4 (tuberculin) is 6.0. Using the formula for the calculation of the WPPT, get: ((5,78-6,0):5,78)×100%=-3,8%. In this case, ( - ) indicates the and what in the experimental sample rate Srif more than in the control. From this we conclude that the experimental sample Srif labeled FITZ CD45RA+lymphocytes increased by 3.8%.

Level Sref labeled FITZ CD45RO+lymphocytes in this case, in the control sample 5 was 2,16 in the test sample 6 (tuberculin) - 2,16. Srif labeled FITZ CD45RO+lymphocytes in the test system with tuberculin in vitro has not changed.

The results of the survey do not allow the exclusion condition of infection of the office (option IV in table 1).

Application of the proposed method is a laboratory diagnostic testing BCG-vaccinated children in the process of conducting TB immunoprophylaxis events. In particular, the appointment of the proposed method is shown as optional in the examination of children with clinical manifestations of cutaneous tuberculin hypersensitivity to establish their origin, and as an alternative - in the management of children with atopic and/or burdened vaccination history, for which the production of tuberculin tests in vivo is associated with the possibility of developing dangerous to life and health side effects.

It should be noted that the use of the proposed method in the management of BCG-vaccinated children with clinical manifestations of cutaneous tuberc is Lanovoy hypersensitivity provides the following benefits:

1. Allows you to confirm the Genesis of allergic hypersensitivity to tuberculin used in the formulation of skin tuberculin tests in vivo.

2. Allows to establish the fact that the toxic effects of the components of the solution of tuberculin.

3. Allows to establish the fact that post-BCG vaccination hyperresponsiveness sensitized lymphocytes.

4. Allows you to identify the children at risk who have not excluded the state of infection of the office, with a view to their further further evaluation.

For BCG-vaccinated children with atopic and/or burdened vaccination history using the proposed method as an alternative provides the following benefits:

1. Allows to assess specific functional activity of sensitized lymphocytes in the test system with antigens office in vitro.

2. Allows you to identify the children at risk who have not excluded the state of infection of the office, with a view to their further further evaluation.

SOURCES of INFORMATION

1. Vasneva GP, Toropova N.E. // Method flow cytofluorimetry (PPB) for determination of drug sensitization // Modern problems of Allergology, clinical immunology and pharmacology: proc. Dokl. 1st national conference of allergologists and clinical immunologists (28-31 January 1997). - Moscow. - 1997. - S.

2. Vasneva IP, T is rapava N.E. // Examination of patients with elevated or questionable results response to intra - or subcutaneous injection of tuberculin // Modern diagnostic technology in the health service: Mater. nauch.-the practical. Conf., dedicated to the 10th anniversary of Omsk diagnostic center / Ed. by A.V. Kononov, P. Malkov// Omsk diagnostic center. - Omsk, 1998. - S - 123.

3. Vasneva GP / Diagnostic value of the test on the level of expression of CD45 lymphocytes in drug Allergy: author. dis. ... candles. Biol. Sciences. - Chelyabinsk, 1999.

4. Vasneva GP, Balmasova I.P. / Development of methodical approaches to the assessment of the effects of drugs group antihistamines and analgesics on the performance of the immune system in peripheral blood of patients with drug Allergy in vitro // Simulation in medical and biological research: Collection of scientific articles / edited by Professor S.M. Babkina. - Samara, Samara state medical University, 1999. - Pp.33-42.

5. Vasneva GP, Balmasova I.P. / Implementation of new technologies in the process of the diagnostic examination of patients with drug allergies // proceedings of the VI International Congress "Ecology and health", 12-15 October 1999, Samara. - P.45-48.

6. Vasneva GP, Belyaeva L.V. // features CD45, CD45RA, CD50 in patients with drug hypersensitivity to anesthetic agents is am. Abstracts of the IX International Congress of Physiology and pathology of the immune system"/ International Jornal on Immunorehabilitation, 2003. - V.5. No. 1. 49 - 50.

7. Vasneva GP, Balmasova I.P. // Features of the system of General and specific immunity in patients with drug intolerance / Allergy and immunology, 2003, No. 1. - P.41-47.

8. Medunitsyn NV Vaccinology. - M.: Triada - X, 2004. - 448 S.

9. Chervonsky G.P. Vaccinations: myths and reality. Fundamentals of vaccinology. - M., 2002. - 416 S.

10. The Yarylo A.A. fundamentals of immunology: a Textbook. - M.: Medicine, 1999. - 608 S.

11. Koretzky GA // Role of the CD45 tyrosine phosphatase in signal transduction in the immune system / FASEB J. - 1993. No. 7. - P.420-426.

12. Novak TJ, et al. // Isoforms of the transmembrane tyrosine phosphatase CD45 differentially affect T cell recognition / Immunity. - 1994. - V.1 - P.109.

13. I.S. Trowbridge, Ostergaard H.L., Johnson P. // CD45: a Le - specific member of the protein tyrosine phosphatase family / Biochim. Biophys. Acta. - 1991. - V.1095. - P.46.

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The method of determining the nature of the tuberculin skin hypersensitivity in vitro at the level of fluorescence intensity (if) labeled FITZ CD45+ lymphocytes, using labeled FITZ monoclonal antibodies (MCAT) to onselaciunea antigen CD45, characterized in that the peripheral is such blood is incubated with a solution of tuberculin TO as the experimental samples and saline solution as a control sample, add labeled FITZ, MCAT to CD45 and isoforms CD45RA and CD45RO, incubated to measure the level of philosophy, determine the change if labeled lymphocytes in the experimental sample relative to a reference by the formula

and with the increase of the specific Interfax CD45+ lymphocytes more than 25.0% define allergic skin tuberculin hypersensitivity, when the increase in the average if CD45RA+ lymphocytes more than 9.0% and CD45RO+ lymphocytes more than 20,0%, the nature of the tuberculin skin hypersensitivity is defined as post-BCG vaccination Hyper reactivity of sensitized lymphocytes, while reducing specific Interfax CD45+ lymphocytes less than 20.0%of the average CD45RA+ lymphocytes less than 8.5% character tuberculin skin hypersensitivity is defined as the cytotoxic effects of the components of the solution of tuberculin, while reducing specific Interfax CD45+ lymphocytes more than 20,0% character tuberculin skin hypersensitivity is defined as a possible infection with Mycobacterium tuberculosis.



 

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FIELD: medicine, analytical immunology.

SUBSTANCE: invention relates to a set of reagents used in quantitative determination of secretory immunoglobulin A (sIgA) that provides assaying status of topical immunity and immunodeficient states in carrying out analysis of serum and secrets of human body. Proposed set comprises a plate with immobilized monoclonal antibodies, five calibrating samples containing 0; 1.0; 5.0; 10.0 or 20.0 mcg of sIgA/ml, conjugate of murine monoclonal antibodies with horse radish peroxidase and a reagent for carrying out the enzymatic reaction. Murine monoclonal antibodies produced by hybrid strain of animal Mus musculus L., № PKKK (P) 676D cultured cells, are immobilized on a carrier. Conjugate comprises murine monoclonal antibodies produced by another strain - Mus musculus L., № PKKK (P) 677D. The advantage of invention involves enhancing sensitivity in assay of sIgA.

EFFECT: improved assay method.

3 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: method involves determining availability of antibodies to basic myelin protein at the first and the third week after burn, by applying immunoenzyme analysis approach. The third week values belonging to the range of 0.1-0.19 ODU, vegetovascular dystonia syndrome is predicted to develop. The values being within the 0.2-0.274 ODU limits, disseminated cerebral microsymptoms are to be predicted. The values being within the 0.275-0.3 ODU limits, focal symptom manifestations syndrome is to be predicted.

EFFECT: high accuracy in predicting cerebral disorders severity degree.

FIELD: medicine.

SUBSTANCE: method involves determining relative content of CD16+ monocytes in monocytic gate in peripheral venous blood of barren woman. The value being equal to or less than 45%, external genital endometriosis diagnosis is to be set.

EFFECT: high accuracy, specificity and sensitivity of diagnosis method.

1 tbl

FIELD: medicine; microbiology.

SUBSTANCE: method an be used for specific identification and evaluation of potential ability of non-cultivated forms of vibrio cholerae O1 and O139 serous groups to reversion, which vibrios take place in water solutions. Method is based upon preliminary preparation of bacterial suspension of non-cultivated forms of vibrio cholerae O1 and O139 serous groups and bacterial suspension of reference culture of Vibrio Cholerae O1 and O139 serous groups. The working dilution of bacterial suspensions and monoclonal antibodies conjugate is determined and specific monoclonal antibodies are selected for conservative and labile lactose-peptone medium epitopes followed by dot-immune-enzyme analysis or solid phase immune-enzyme analysis reaction. Then appearance of color point or optical density, being higher than 0,2-0,65, is registered and conclusion is made on presence of non-cultivated forms of Vibrio Cholerae O1 and O139 serous groups in sample and their potential reversion ability. Preliminary preparation of non-cultivated forms is performed due to increase in their concentration up to 106-107 mxkl/ml followed by inactivation. For the purpose 20 mkl of 4% sterile gentamicine solution is added to 1 ml of microbe suspension followed by subsequent cooling of samples down to 3-4 deg C during 2 hours. Bacterial suspension of control culture Vibrio Cholerae O1 or Vibrio Cholerae O31 is made from 18-hours culture, grown onto Martin's agar. After that the suspension is used for preparation of bacterial suspension in physiological solution, pH 7,6 till final concentration of 107 mxkl/ml. Method uses set of specific monoclonal antibodies, got to different epitopes of O-antigen of lactose-peptone medium cultures of Vibrio Cholerae O1 and O139 serous groups: 3A9, D11, 3E4, 4H5, D8D6, C3B4, F8G12, F11E5.

EFFECT: ability of getting express-answer at low concentration of tested cells.

5 cl, 2 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: it is necessary to study saliva due to carrying out immunoenzymatic assay by applying mycobacterial antigen specifically binding with human secretory immunoglobulin A only, and specific monoclonal antibodies directed against human secretory immunoglobulin A. The present method is considered to be of high efficiency, noninvasive, simple in application, of high information value, specificity and sensitivity to tuberculosis.

EFFECT: higher accuracy of diagnostics.

3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: method involves determining concentrations of carcinogens like aniline, toluyline, diethyl alanine, diphenyl and 1-naphtholamine in urine and urodynamic disorders and proliferative urothelium activity from Ki-67 expression degree data. High carcinogen concentrations and urodynamic disorders being detected apart from the fact of tumor removal and unchanged mucosa biopsy, transurethral prostate resection or urinary bladder neck resection is carried out. Next, liquid consumption increase in postoperative period and carcinogen contact reduction are advised. Three months later, control study is carried out. Carcinogen concentration level remaining high and no proliferative urothelium activity reduction being observed on the background of urodynamic disorders, cystectomy is to be repeatedly carried out.

EFFECT: enhanced effectiveness of treatment; high accuracy of diagnosis.

FIELD: medicine.

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.

1 tbl

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

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