Panel of serums containins antibodies to hcv antigens of various subtypes
SUBSTANCE: positive portion of the panel comprises patient's plasma samples positive to RNA VHS and is presented in the form of 4 boxes accommodating NS5, NS4, NS3 and Core antigen antibodies respectively. A negative portion of the panel comprises donor's plasma samples containing other than HIV-1/2, HBV, HCV, syphilis, HBsAg antibodies, as well as from HCV-noninfected risk groups with additionally included heterophillic serums with rheumatoid factor (RF) and with autoimmune antibodies for strict control of specificity.
EFFECT: method improvement.
5 cl, 5 tbl, 1 dwg
The invention relates to the field of biotechnology, immunology and vaccinology and can be used for fabrication of panels of sera containing antibodies to viruses of the most socially dangerous infections.
Form the collection of plasma samples of infected individuals from different regions of Russia. Plasma samples in Russia necessarily sceneroot for the presence of HBsAg and anti-HIV 1+2, anti-HCV and anti-Tr antibodies. Approximately 2% of all samples sent for re-confirmed in a reference laboratory. However, the effectiveness of the validation of the results depends not only on the quality of the test systems, but also on the availability in the control laboratories standard drugs during infectious markers Recommendations agreed by the Group for the serological diagnosis of hepatitis C.
To prepare anti-HCV genotype reference panel. All panel components must be inactivated and should be prepared from plasma samples (sera) genotypes 1, 2 and 3. In the subsequent accumulation of the material in this panel you can add reactive samples genotypes 4, 5 and the panel should include samples of extremely diluted in recalcification plasma, negative for HBsAg, anti-HCV and anti-HIV 1+2 and negative in Poland assays for HCV RNA, HIV RNA and HBV DNA [meeting of the WHO ECBS 1996, 2000].
When the solution to the problem of assessing the quality of diagnosis of viral infe the Nations in Russia should create a Collection of plasma from patients with diagnosed mainly from regional infectious clinics. And on the basis of this material to form a national panel of sera. This conclusion has and statistical justification, because the diagnosis requires a considerable investment of time and involvement of qualified personnel.
In accordance with the recommendations of the ECBS national panel should include plasma samples with genotypes and subtypes characteristic of Russia.
Use in everyday work products with different HCV subtypes will allow you to extend the range of diagnosed markers and use these materials for quality control analysis of blood for the detection of hepatitis C.
External quality control of laboratory research is an integral part of ensuring the value and reliability of laboratory diagnostics. Serological diagnosis of anti-HCV is the main instrument of control and prevention of hepatitis C. Detection of antibodies to each of the antigens of the hepatitis C virus has a self diagnostic value.
For this purpose, the most widely used screening enzyme-linked immunosorbent assay (ELISA). To perform the ELISA use test systems, representing a multi-component sets of antigens of the virus. On the effectiveness of the ELISA affects not only the quality of these ant the genes but professional training of laboratory staff providing the test . To provide an objective external assessment of the quality of laboratory research is extremely essential specially prepared and certified reference materials.
The famous master panel HBsAg. Described by MP. For the formation of the panel, it is necessary to have a representative sample of plasma or serum prepared from samples of different regions of Russia, and certified by genotype HBV and HBsAg serotype .
Famous national panel of sera (NTC) anti-HCV [3, prototype]. The active components of the set are antibodies to HCV, reacts with portions of the proteins of different HCV subtypes encoded by structural (core) and nonstructural (NS3, NS4, NS5) region of the HCV genome, samples diluted in stable defibrination plasma.
Set includes 20 samples of human serum containing and not containing antibody subtypes 1a, 1b, 2 and 3A of the hepatitis C virus, which can be used for quality control as a screening and confirmatory ELISA test systems. The active components of the set are antibodies to HCV, reacts with portions of the proteins of different HCV subtypes encoded by structural (core) and nonstructural (NS3, NS4, NS5) region of the HCV genome, samples diluted in stable is Noah defibrination plasma.
The main disadvantage of the control panel is a small sample Swatch. 16 positive samples, the NTC does not allow you to display the diversity of genotypic and antigenic representation of HCV in Russia, which reduces the quality control test systems. Similarly, the specificity of the test systems should be tested on a representative sample of negative samples, including donor serum and serum of persons from risk groups.
The technical result of the present invention is:
obtaining a national panel of sera, providing better control of ELISA test systems: for practical situations the analysis of plasma samples (sera) with and without hepatitis C virus, the presence or absence of antibodies to HCV, but with a diagnosis of hepatitis C or a healthy patient.
The technical result is achieved by creating a panel of lyophilized samples of plasma (serum)containing and not containing antibodies to different HCV subtypes and to different antigens of the virus spread in various regions of Russia.
This panel of anti-HCV built on a sample of 100 primary anti-HCV positive plasma samples, genotyped and sublimirovanny for HCV RNA, and 100 negative samples of plasma collected from healthy donors and persons from risk groups with rheumatoid factor and community antibodies.
The positive part of the panel includes plasma samples of patients positive for HCV RNA, and is formed in the form of a set of blocks containing separately antibodies to NS5, NS4, NS3 and Core antigens (samples No. 1-100).
The negative part of the panel includes samples of plasma donors that do not contain antibodies to HIV-1/2, HBV, HCV and syphilis, HBsAg, risk not infected with HCV (samples No. 101-200). For the strict control of specificity in the negative block included heterophile serum rheumatoid factor (RF) and autoimmune antibodies.
Invited panel (P anti-HCV) lyophilized sera containing and not containing anti-HCV, obtained from individuals infected with hepatitis C virus, volunteers and blood donors. The positive part of the panel includes plasma samples of individuals infected with hepatitis C virus (samples No. 1 to No. 100), the negative part of the panel includes samples of plasma donors, persons from risk groups and volunteers not infected with HCV (samples No. 101-200).
The positive part of the panel consists of 4 blocks of plasma samples, mostly positive in PCR for HCV RNA and having high levels of aminotransferases.
Each block positive side of the panel has its own characteristics.
Samples of block 1 (No. 1-25) certified by genotype and subtype of HCV. Block 1 includes serum 1, 2 and 3 genotypes different from tipov (1a, 1b, 2A and 3A), distributed on the territory of Russia. This sample unit 1 contain antibodies to NS5 antigen along with antibodies to other antigens.
2nd block formed from plasma samples with a positive result in PCR, partially sublimirovanny. Note that samples of unit 2 must contain antibodies to the NS4 antigen along with antibodies to other antigens.
3rd block is composed of plasma samples with a positive result in PCR, partially sublimirovanny. This sample block 3, be sure to contain antibodies to NS3 antigen along with antibodies to other antigens.
4th block is composed of plasma samples with a positive result in PCR, partially sublimirovanny. This sample block 4 must contain antibodies to core antigen along with antibodies to other antigens.
Negative block panel composed of samples of plasma donors, volunteers and representatives of the risk groups.
The unit included samples with heterophile antibodies, including human antibodies to mouse serum samples with rheumatoid factor and autoimmune antibodies. This negative part of the unit allows for strict control of specificity of diagnostics.
Each sample panel certified by immuno-biological and biochemical properties. Range of antibodies determined the Yali by asking sample panel sample panel nishtanah sera PHV106 (7) on the same microplate.
The panel is designed to perform the state control of the quality of each series of licensed anti-HCV test systems and for the certification of new diagnostics.
The panel can be used to determine the titer of anti-HCV antibodies of different subtypes.
A panel of sera should be stored at temperature (2-6)°C.
Diluted serum panels are stored at a temperature of (2-6)°With one month at a temperature of minus 24°C for 6 months. Pets 3x freezing and thawing of serum.
Panel of sera containing and not containing antibodies to hepatitis C virus, was obtained as follows.
1. Admission plasma samples.
Plasma samples arrive at the production gemikonagi CPDA-1 with a capacity of 250 ml, hermetically sealed, frozen.
Each plasma coming into production for the manufacture of panel must have a passport with the characteristics IN (a unique number) of the donor, the volume of serum, the date of blood sampling, the manufacturer, as well as to meet the following requirements: without signs of hemolysis and hyperlipidaemia, transparent, color - amber or light yellow, extraneous solids, slurry, sludge absent. Each Gemechu assign encoding the room.
Inactivation of sera of healthy donors.
Gemany with plasma placed in thermostate temperature of 56°C. Record the start time of inactivation. Incubation of plasma spend 3 hours At the end of this time gamecon get from a thermostat, record the end time of inactivation.
Measurement of the volume of plasma.
Measure the volume of each plasma using a sterile glass measuring cylinder.
Packing plasma samples.
From each gemikona in a sterile room in the kelp automatic pipette select 10 aliquot (1±0,05) ml plasma into a plastic test tube "Eppendorf" with a capacity of 1.5 ml for the control of biochemical parameters and to study viral markers in PCR and ELISA. The tubes close to each tube permanent marker write the code number of the plasma sample.
The remaining volume of plasma Packed in sterile plastic containers with a capacity of 25 ml In each container permanent marker write the code number.
Storage of plasma samples.
Gemany with donor plasma, and aliquots placed in storage in a freezer at a temperature of from minus 20°C to minus 35°C. Gemany to processing stored under these conditions for up to 10 years. Pets single freezing and thawing of serum.
The control aliquot plasma methods PCR and ELISA.
2 aliquots of each plasma passed in ELISA the laboratory for testing samples for the content of HBs antigen, antibodies to HCV, HIV-1,2 types of the anti-HBs. ELISA analysis carried out on imported or domestic, certified gisk named after. Laurasia, enzyme immunoassay systems (table 3).
Control of biochemical parameters.
One aliquot of each plasma sample in the Eppendorf passed in a biochemical laboratory for monitoring of biochemical parameters (color, aminotransferases, total protein content, pH). The protein content is determined by micromethods Lowry (table 3).
Samples plasmas that do not meet the requirements for biochemical parameters, rejected and disposed of.
Adding to the serum solution preservative.
In each gamecon with plasma add 2% solution of thimerosal and gentamicin at a rate of 0.5 ml each bacteriostatic per 100 ml of serum, and the solution of calcium chloride to precipitate the ballast proteins. Serum (recalcification plasma) are thoroughly mixed. The final concentration of thimerosal in serum is 0.01% and gentamicin of 0.01%.
Preparation of pooled donor sera.
From native serum samples that do not contain markers of viral infections, prepare the pool from at least 10 donors.
Stable pool of sera used as matrix - diluent solution (PP) for the preparation of positive samples MP. Bring the solution pH to 6.8-7.2 at room temperature with n is a power of 0.1 N NaOH or 0.1 N HCl. The solution is poured in sterile containers at 250-500 ml and stored at 2-8°C for no more than 1 month. Frozen at a temperature of minus 35°C, the solutions can be stored for 10 years.
1 Stable pool of sera normalize for total protein and viscosity relative to donor human serum and get the RR. Control the viscosity of the PP is carried out with the use of glass viscometer according to the classical method using a calibrated capillary. Ousterhout viscosity of drugs to the viscosity of the SNP using a 10%solution of BSA or phosphate-saline buffer (FSB).
2. Titration of drugs anti-HCV.
Titration of drugs anti-HCV performed using PP on one tablet three times in the screening and confirmatory tests - Spectrum anti-HCV companies CJSC Vector bis (Novosibirsk, Russia), JSC MBS (Novosibirsk) and NPK DS (N. Novgorod) (figure 1).
Reference drugs anti-HCV. To perform attestation definitions in use pane, anti-HCV, certified in relative units in international tests:
- Nishtana sera panel PHV 106 BBI (Boston, USA).
3. Preparation of positive samples panel.
For the formation of 1, 2, 3, and 4 blocks positive side of the panel study research results positive samples in Poland, in screening and confirmatory ELISA, which determine the levels of aminotransferases.
Serum samples containing anti-NS5 antibodies and positive in PCR (with a certain genotype and subtype HCV), form block 1; serum samples containing anti-NS4 antibodies positive in PCR, form block 2; serum samples containing anti-NSS antibodies positive in PCR or high levels of aminotransferases, form a 3-th block; serum samples containing anti-Core antibody positive in PCR or high levels of aminotransferases, form 4-th block of the positive side of the panel.
Using PP serum blocks from the 1 St to 4-th normalized in the range of 1.5-5 OPCretefor the desired antibodies (figure 1).
OPCrete- On the scale of the optical spectrometer entire range of values of optical density (OD) is from 0 to 4 PU serum Samples human blood split this range into 2 areas: area of OP healthy donors and area infected patients. The boundary between the area values OP donors and infected patients determines the OD value at which the sample test of blood (serum) with equal probability can be evaluated as positive (infectious)and negative (healthy donor). This boundary value OP called in ELISA diagnosis of critical OP and denote abbreviated OPCreteor international designation is Cut off.
In accordance with this ODA is by dividing all samples serum panels must be in ELISA OD value> OPCrete(positive samples) or less OPCrete(negative samples).
In the manufacture of candidates in positive samples nizkotatranski standard toolbar developer selects the coefficients of the cultivation of native sera such that, ELISA OD diluted sera exceeded OPCrete1.5-2.0 times.
Likewise, when the selection of candidates for negative samples panel a developer selects sera with OD<OPCrete.
Calculate OPCreteaccording to the formula given in the Instructions for use of the used test system.
Just so prepare 120 samples for the positive side of the panel.
4. Preparation of negative samples panel.
For the manufacture of negative samples panels use the native serum of healthy donors, vaccinated and persons from risk groups, stabilized and filtered through 0.45 µm. Part MP included 100 negative sera (No. 201-301).
Purification of the samples panel filter.
Fine polishing of the samples is carried out in a laminar box by filtering using disposable syringes 10 ml disposable syringe nozzles Minisart with the pore size of the membrane of 0.8 and 0.45 μm. Serum of each number after the filter is placed in a sterile glass vial with a capacity of 150 cm3and sealed with a cork.
Filling about ascov panel vials for freeze drying.
Of the boxes in the cassette stack 350 bottles with a capacity of 2-3 cm3. The cassette is placed in the kelp-Boxing, which make the filling dispenser (Diluter model 203, Beckman, USA) serum vials. Boot capacity with serum No. 1 is placed in an ice bath and stirred serum during the filling process. On the scale of the dispenser determine the volume of a dose of 0.8 ml of the serum is dispensed into vials, placed in a cassette labeled "serum No. 1". After emptying the boot capacity dispenser and a boot capacity of washed residues from serum No. 1 and sterilized. In flushed with sterile boot capacity load serum # 2 and start filling in vials, placed tape labeled "serum No. 2". Thus poured all 200 sera panels intended for simultaneous freeze drying at the facility. The vials poured serum cover tightly with rubber stoppers to limit the access of air into the bottles.
In the cassette with the bottles placed the tag, in which you specify:
the name of the drug, the volume of solution in the vial, the number of serum, total number of vials, date, name and signature of the operator, making the filling. Tapes bottles pass through lyophilization.
Freeze drying the samples panel.
Freeze drying of samples is performed on set is VCE TG-50 (Germany). Include a compressor unit for cooling the condenser and shelves of the drying chamber. Set in the temperature setter shelf temperature of -35°C. after 2.0 to 2.5 h, when the temperature of the capacitor minus (75-80°C., and the temperature of the shelves minus (35)°C, in the installation of load tapes vials with solutions sera. The loading cassettes with bottles in the drying chamber should be minimized and should not exceed 10-15 minutes.
Each shelf is placed on the bottle with serum from deep in sensor temperature measurement. Door camera installation, close and tighten the locking screws. Freezing should be performed within 8 hours the Temperature in the freezing process is automatically maintained and controlled by the operator through every hour with the introduction of temperature data in the log. After the time of the freezing process is conducted stage of freeze drying.
Thermal management sublimation is performed manually, by setting the desired temperature of the shelves using a temperature controller.
The desired temperature throughout the drying is maintained automatically with an accuracy of 3-5°C. the temperature of the shelf and the material temperature and the temperature of the condenser is recorded continuously by a data logger and periodically recorded by the operator in the log drying 1 times the hour. The vacuum in the chamber controls the gauge.
After 8 h after the onset of freezing include vacuum-pump installation. The material temperature at the time of activation of the vacuum pump must not exceed minus 30°C. four hours after the pressure of the residual air in the chamber reaches the value of 20 PA, turn up the heat shelves and set in the temperature setter shelves temperature of 0°C.
Eight hours after this install the setpoint temperature of the shelves plus 10°C. two hours after reaching the shelves temperature 10°C set the temperature setter shelves temperature of 25°C. the Material is maintained at this temperature for 6-8 hours
The control end of the drying process is carried out based on the sensor readings residual pressure. Upon reaching the camera a residual pressure of less than 20 PA, the drying process is complete. The total duration of the drying 32 PM
Capping of vials under vacuum.
Capping of bottles produced inside the chamber lyophilic installation using pressure plates. At the end of the drying process pressure plate is lowered by means of a hydraulic device manually. The process of capping shall be checked visually through the viewing window door drying chamber. After a beep, the process of lowering the pressure plates stop and raise the pressure plate to its original position. Ur univelt pressure in the chamber with atmospheric and open the door to the drying chamber installation. Vials of the serum is passed to the operation of crimp vials caps.
Certification sample panel containing and not containing HBsAg.
Upon completion of receipt of lyophilised serum (200 samples), intended to form the panel, the manufacturer sends 2 panel set in gisk named after Lautareasca for state testing. Together with sets of the manufacturer shares the results of specific tests and physico-chemical properties of the sera panel, as well as data study the stability of the sera at different temperatures.
Description of figures
Figure 1. Titration curves for samples panel in razvodami solution.
This is followed by examples of specific applications of the master panel.
Example 1. Receiving panel.
1. Blank plasma samples of infected individuals diagnosed with hepatitis b and healthy donors was performed in 7 remote regions of Russia. The samples are encoded according to the following cities:
Krasnodar - 1
Nizhny Novgorod - 2
Gorno-Altaisk - 3
St.Petersburg - 4
Novosibirsk - 5
Khabarovsk - 6
Barnaul - 7.
Preparation of plasma is carried out by the method of plasmaphoresis with the written consent of the patient.
2. Plasma samples encode, certificate 10 indicators (table 1), poured into vials of 25 ml and transferred to a specialized store. The plasma stored until use PR is -35°C.
3. On the literary writings  the plasma is transferred to the serum, and inactivate certificate in PCR and ELISA for the preparation of anti-HCV panel.
4. According to the results of the 3rd evaluation samples positive sera used for forming the positive side of the panel.
In unit 1 include serum, which genotypic and subtyping and confidently identify in ELISA for the presence of 2 bands in the immunoblot or "Spectrum" , one of which is necessarily anti-C. Just trying to enter the 26 samples (table 1).
In unit 2 include serum, positive or negative in PCR, but with elevated levels of aminotransferases. In unit 2 include serum, which confidently identify in ELISA for the presence of 2 bands in the immunoblot or "Spectrum", one of which is necessarily anti-m. Trying to enter 25 samples (table 2).
In block 3 include serum, positive or negative in PCR, but with elevated levels of aminotransferases. In block 3 include serum, which confidently identify in ELISA for the presence of 2 bands in the immunoblot or "Spectrum", one of which is necessarily anti-m. Trying to enter 25 samples (table 4).
In unit 4 include serum, positive or negative in PCR, but with elevated levels of aminotransferases. In unit 4 include serum, which confidently identify in ELISA for the presence of 2 bands in the immunoblot or"Spectrum", one of which is necessarily anti-Core. Trying to enter 25 samples.
For inclusion in the panel of serum of each unit is calibrated by titration results plasma samples for each antigen: Core, NS3, NS4 and NS5. Vysokotirazhnyh samples diluted in the range from 5 to 1.5 ODCrete. As diluent solution using a pool of negative sera, stable introduction of EDTA, gentamicin and thimerosal as bacteriostatic (PP).
Example 2. Panel application for controlling the quality of immunoassay test kits for detection of anti-HCV antibodies.
For certification test-system for detection of anti-HCV using a specific set of swatches panel in accordance with the purpose of the test:
Testing the activity of the antigens of the test system. Use a set of samples of 4 blocks and a few negative samples.
Sensitivity testing of the test system. Use the set of samples with the lowest level of anti-HCV and several negative samples.
Testing the specificity of the test system. Use the set of samples negative side of the panel, not less than 90 units.
In each vial with lyophilized serum panel make 300 µl of distilled water. The contents of the vial thoroughly stirred until complete dissolution of the serum. Serum incubated at room temperature in accordance with their 15 minutes. Next, ELISA set in accordance with the Instructions for use on certified test system.
Analysis on samples of sera panel should be undertaken only if the value of the OD of the control conjugate, positive (+) and negative ( -) controls set are within the regulated pharmacopoeial article tested on the test system.
1. Detection of hepatitis C infection at an earlier stage accelerates the cure of the patient and reduces the invalid period of the patient.
2. Using panel significantly increases the level of assessment of the quality of the licensed test kits.
3. National panel allows to unify the procedure of licensing of the various test systems coming to the Russian market, and is the legal basis for state control.
4. Of practical importance is the establishment of a national panel of sera for assessing the quality of immunoassay test kits for the basic parameters: sensitivity to different HCV genotypes and to various antigens - the percentage of detected™ positive sera panel; specificity is the percentage of detected™ negative sera, and the shelf life - the length of time maintain the specific characteristics of the samples panel, held at t is mperature (2-8°C; to develop a methodology for application of the national panel of sera to perform proficiency testing, quality test systems for screening and confirming to detect antibodies to HCV.
Sources of information
1. WHO Working Group on Reference Preparations for testing Diagnostic Kits used for detection of HBsAg, Anti-HCV and Anti-HIV antibodies in blood screening // Report of the third meeting: Geneva, Switzerland, 3-6 October 2003.
2. The principles for production and approval of international and other standards and reference biological preparations // WHO Expert Committee on Biological Standardization. Rep.37, WHO, Geneva, 1990.
3. WHO Working Group on Reference Preparations for testing Diagnostic Kits used for detection of HBsAg, Anti-HCV and Anti-HIV antibodies in blood screening // Report of the third meeting: Geneva, Switzerland, 17-19 January 2000.
4. Kano A.N., Shalunova NV, Netesov S.V. and other Development reference panels HCV with a normalized level of IgG antibodies, Virology 2000, 45(4), 42-47.
5. RF patent №2314540 Master panel of sera containing and not containing HBsAg, and how to get it.
6. Newsletter company Boston Biomedica, Inc., Anti-HCV Low Titer Performance Panel. PHV 106. BBI Diagnostics. A Sera Care Company. 02.2006.
7. Potapov A.A. Bogush p. g, Radchenko E.B., Dronov VM optimizations intralaboratory quality control for mass screening survey for antibodies to hepatitis C virus // Klin. Laborde. Diagnosis 2008, 3, 35-38.
8. Alter MJ, Kuhnert WL, Finelli L. Guidelines for laboratory testing and result reporting antibody to hepatitis With virus. Centers for Disease Control and Prevention. MMWR Recomm Rep 2003, 52: 1013, 15.
9. Development of Control Serum for Microbial Anibody Tests // Biologicals (2001) 29, 39-43.
Setting panel 1 dried
1. Vector anti-HCV from 02.06.09
2. The best anti-HCV - Range s from 05.06.09
3. HCV - DSM screen, pp.31 from 17.09.09
1. A panel of sera containing and not containing antibodies to HCV antigens of different subtypes, including a positive part and a negative part, characterized in that the positive part of the panel contains samples of plasma or serum of patients and consists of a first block including serum of different HCV subtypes 1a, 1b, 2A and 3A, containing antibodies to NS5 antigen; a second block that includes a positively certified sera containing anti-NS4, diluted to medium and low concentrations in the range from 1.5 to 5.0 VPCretethe third block, including sera with OD values close to or below OPCretepositive PCR or having high levels of aminotransferases, containing anti-NS3 antibodies, and the fourth block containing anti-Core antibody negative portion of the panel includes samples of plasma donors and persons from risk groups.
2. Sera panel according to claim 1, characterized in that as the basis for the preparation of arr is scov master panel use another serum, derived from the stable pool of sera from pH 6.8 to 7.2,
3. Sera panel according to claim 2, characterized in that a stable pool of sera obtained from native serum samples from at least 10 donors that do not contain markers of viral infections.
4. Sera panel according to claims 1 to 3, characterized in that for obtaining positive samples panel take serum samples with a defined genotype and subtype and positive in the ELISA, which form the blocks 1-4 in the form of antibodies.
5. Sera panel according to claim 1, characterized in that the serum of the second block to throw in basis to a concentration of from 1.5 to 5.0 VPCrete.
SUBSTANCE: sequential staining by Romanowsky-Giemsa and conducting an immunocytochemistry reaction are used to analyse destructive processes in epitheliocytes nuclei, specifically changed lymphoid and epithelial cells, high colonisation activity of opportunistic and pathogenic microorganisms, and phagocytic activity. If observing brown-black inclusions in cells under optical microscopy ensured by specific staining of the viral antigens in the cell structures, mono- or mixed herpes viral infection Herpex simplex 1, 2, EBV, CMV are diagnosed.
EFFECT: using the technique enables higher accuracy of etiological diagnosis, expressivity and reduced injures of material sampling for research.
SUBSTANCE: recipient of a cadaver kidney graft is examined to compare recipient's and donor's HLA alloepitopes. The alloepitopes found in the donor, but not found in the recipient are recovered. It is followed by comparing the recipient's HLA-DR antigens and the recovered alloepitopes, the derived combinations are compared with the following combinations: A9C-DR7; A10C- DR3; A10C-DR6; A28C-DR1; Bw4-DR6; B8C-DR3; B12C-DR2; B27C-DR5. Detecting said combinations enables stating an adequacy of the preventive application of monoclonal antibodies blocking an alpha-chain of an interleukin-2 receptor molecule. The preventive application of the monoclonal antibodies blocking an alpha-chain of the interleukin-2 receptor molecule shall be indicated to the patients with said combinations.
EFFECT: improved outcome of the cadaver kidney grafting that is combined with reducing the price of the method.
1 tbl, 2 ex
SUBSTANCE: method is ensured by the double evaluation of total endotoxin (LPS) of gram-negative bacteria: prior to the beginning of the inhalation antibacterial therapy and one hour after the first antibiotic inhalation. It involves controlling the total endotoxin of the gram-negative bacteria in patient's blood serum by activated particle method. Double and higher endotoxin increase as compared with the reference testifies to a bactericidal action of the antibiotic and proves the effectiveness of the conducted antibacterial therapy.
EFFECT: using the method enables the early estimation of the effectiveness of the antibacterial therapy.
SUBSTANCE: invention describes a method for prediction of reduced transport function of band-3 protein of red-cell membranes of newborns delivered by mothers suffered aggravated herpes viral infection in the third trimester of pregnancy wherein newborn's peripheral blood is examined for the TNFa content; band-3 protein is detected in the newborn's red-cell membranes by disk electrophoresis in polyacryl amide gel (10%) with sodium dodecyl sulphate; a discriminant function is calculated by formula Df=(5.167×band-3)+(2.196×TNFα), wherein Df is the discriminant function, band-3 is red-cell membrane stromal protein, TNFα is newborn's blood interleukine, and if Df is more than 218.67, reduced transport anion function by protein band-3 through newborn's red-cell membranes is predicted.
EFFECT: invention provides detecting the effect of herpes viral infection on the content of protein band-3 in newborn's red-cell membranes.
SUBSTANCE: peripheral venous blood of the patients with autoimmune thyroiditis is examined for the absolute transferring receptor (CD71+) T-lymphocyte count and the interleukin-2 (IL-2) concentration. If the CD71+ T-lymphocyte count exceeds 0.3×109 cell/l, and the IL-2 concentration is less than 25 pg/ml, a favourable clinical course of the disease is predicted, while the CD71+ T-lymphocyte count less than 0.3×109 cell/l and the IL-2 concentration exceeding 25 pg/ml shows an unfavourable clinical course of autoimmune thyroiditis.
EFFECT: method provides more accurate and objective prediction with using the material, reagents and research methods widely applied in common clinical-laboratory practise.
SUBSTANCE: patient's peripheral blood is examined for the content of cytotoxic lymphocytes, %: (CD8/CD16), (CP8/GranzymeB), (CP16/GranzymeB), (CDS), (CD16/CD56) and (CD8/HLA DR). It is conmbined with determining cytotoxic activity (CTA), %. It is followed by calculating a RA activity index (AI) by formula: If observing the condition 11.8%<AI≤18.2%, a moderate degree of rheumatoid arthritis activity is diagnosed. If observing the condition AI>18.2%, a high degree of rheumatoid arthritis activity is diagnosed.
EFFECT: using the technique enables high-reliability differential diagnosis of the degree of rheumatoid arthritis activity.
SUBSTANCE: there are involved recording clinical and laboratory manifestations of a CNS injury during the first days of the disease that is followed by the calculation of total diagnostic coefficients related to the detected graduation levels of pathognomonic signs of the disease. Total (+)7 points and more is related to predicting the developing mixed tick-borne encephalitis and borreliosis infection. Total (-)8 points and less shows the developing potential monoinfection of tick-borne encephalitis. If deriving the intermediate values of total diagnostic coefficients when none of said limits is reached, the prognosis is uncertain.
EFFECT: using the method for stating basic tendencies of the developing pathological process at the early stages of the developing disease.
1 tbl, 2 ex
SUBSTANCE: with using indirect immunofluorescence and monoclonal antibodies, the patients suffering gastric ulcer (GU) are examined for an expression level of CD8, CDDR markers by peripheral blood lymphocytes with the CD8 level exceeding 24.5% and the CDDR level exceeding 15% enabling diagnosing a severe clinical course of GU.
EFFECT: use of the offered diagnostic technique provides high information value in diagnosing the severe clinical course of gastric ulcer, well-timed prediction of the severe clinical course of GU, instant diagnosing, provides well-timed and adequate complex of therapeutic measures.
1 tbl, 1 ex
SUBSTANCE: method involves the peripheral blood analysis for T-lymphocyte count with receptor expression in relation to CD3+ and CD25+ cells by cell count in 1 mcl of blood before the beginning of treatment and 10 days after. If observing the count increased 7% and more in comparison with the reference, a therapeutic effect in non-psychotic versions of psycho-organic syndrome is predicted to be positive.
EFFECT: use of the method enables more precise prediction of the clinical effectiveness in the given disease that promotes selecting an adequate therapy and reducing length of treatment.
SUBSTANCE: invention describes a method of detecting the decreased phosphatidyl ethanolamine content in red-cell membranes in a pregnant woman suffered aggravated herpes-virus infection in their third trimester of pregnancy with underlying increasing peripheral blood TNFα, characterised by the fact that peripheral blood of the pregnant woman is analysed for the TNFα content to detect phosphatidyl ethanolamine in red-cell membranes by two-dimensional thin-layer chromatography, and a discriminant equation is presented. Df=+1.743×TNFα+(-1.079×phosphatidyl ethanolamine), and if Df is more than a boundary value 78.86, the decreased phosphatidyl ethanolamine content in red-cell membranes is detected.
EFFECT: higher red-cell membrane stability in peripheral blood of the pregnant woman.
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.
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.
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
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.
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
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.