Differential diagnostic technique for cervical dysplasia and cervical cancer
SUBSTANCE: invention refers to medicine and aims at differential diagnostics of cervical dysplasia and cervical cancer. The cervical biopsy material is taken. If a cytological examination shows cervical dysplasia, RNA is recovered from the tissue sample, and iRNA expression of the c-fos gene is analysed in relation to the TATA-related protein (TBP) with using qualitative PCR. If the ratio of iRNA expression of the c-fos gene to TBP iRNA is from 109 to 145 relative units, severe dysplasia is diagnosed; if the related ratio falls within the range of 18 to 35 standard units, pre-invasive cervical cancer (cancer in situ) is diagnosed; the ratio falling within the range of 148 to 208 relative units enables diagnosing squamous microinvasive cervical cancer; the derived value exceeding 208 standard units, squamous invasive cervical cancer is diagnosed.
EFFECT: presented invention enables diagnosing the pre-cancer processes of cervical cancer effectively and differentiating the early stages of squamous cancer.
1 tbl, 4 ex
The invention relates to medicine, namely to the diagnosis of cancer. The invention can be used for differential diagnosis of cervical dysplasia and cervical cancer (cervical cancer).
Currently in the world there is a growing number of patients with malignant neoplasms of the reproductive system, in particular cancer of the cervix (cervical cancer). Cervical cancer takes the first place in the structure of oncological morbidity women 15-39 years in the Russian Federation . For certain reasons it at a young age cervical cancer is more aggressive, has a propensity for early metastasis and rapid occurrence of relapses. The effectiveness of diagnostic and therapeutic measures cervical cancer cannot be considered satisfactory, since the five-year survival rate of patients depending on the stage of the disease ranges from 15% to 80%. This raises the need for extensive measures to identify precancerous lesions and early stages of cervical cancer, in which the existing methods of treatment can lead to complete recovery. It is established that the etiologic agent for the development of cervical cancer is human papilloma virus group "high-risk disease (HPV 16, 18 and related) [2, 3, 4]. At the moment, evidence of mutual influence genomaps and genes of infected cells (for example, P16, Ki67, ProEx C, p53, bcl-2, c-fos, c-jun) on the expression of each other, however, the mechanism and physiological significance of this interaction requires further study. The development of cervical cancer passes through the stages of dysplasia and intraepithelial cancer. The difficulty in diagnosing the disease in the early stages is that the initial forms of neoplasia can be the focal character, leaking clinically asymptomatic, camouflage background diseases.
Modern oncological science has a number of methods of cervical cancer screening diagnostics, allowing to judge the presence or absence of tumor at the time of the survey. In particular, the widespread method of cytological screening for malignant tumors of the cervix , detection of HPV mRNA E6/E7 (APTIMA), PreTect HPV-Proofer (NorChip), designed to detect the full-size mRNA genes E6 and E7 HPV test CINtec p16INK4a and immunocytochemical detection of expression of P16 (INK4a), a marker of cervical dyskaryosis. It is believed that overexpression of p16INK4a is due to inactivation of the retinoblastoma gene oncogenic protein of the virus E7. In addition, often determine HPV DNA (your HC2) to identify women with a high risk of neoplasms of the cervix. However, the use of known methods of screening diagnostics, especially cytological and immunocytochemical, the hour is about leads to false-positive results. The drugs may be sporadic physiological staining unchanged glandular epithelium of the cervix .
A known method for the diagnosis of dysplasia and cervical cancer in cervical mucous (US 7.455.973), based on immunocytochemical determination in a sample of cervical epithelial number of nuclear markers: protein p14ARF protein 1, topoisomerase Toroa, cycline E, proteins belonging to the group of MSM. The expression of these markers increased in severe pathological cervical disorders. Due to the variability of their expression the best specificity and sensitivity of the test in relation to the diagnosis of intraepithelial violations, according to the authors, can be obtained by using combinations of antibodies to three different markers. The disadvantage of this method is the strong variability of the designated markers, which is revealed in the development of abnormal cervical disorders, including benign tumors.
Known way early and preclinical diagnosis of cervical cancer (patent RF №2251699, date of publication, 05.10.2005) , which consists in determining in a biopsy sample of the E7 oncoprotein of human papillomavirus (HPV) using pairs of monoclonal antibodies specific to IgG2a and IgG2b groups, which is judged on the presence of a malignant neoplasm. Nedostatki is this method is the impossibility of a full differential diagnosis of dysplasia of the cervical epithelium and cervical cancer because the method does not clearly reflect the early and later stage of cervical cancer.
There is a method of differential diagnosis of hyperplasia, intraepithelial neoplasms and cancer of the cervix (RF patent No. 2321859, date of publication, 04.10.2008) , based on cytological staining of smears obtained from the cervical canal, a nuclear dye with subsequent research on computer analyzer area of the nuclei of normal and tumor cells. The disadvantage of this method is its complexity and the inability to detect the early stage of malignant growth is diagnosed by the presence of cervical cancer, but not stage oncopathology).
A known method for the diagnosis of cervical cancer (patent RF №2343485, date of publication, 10.01.2009) , carried out by lectin-enzyme immunoassay vaginal discharge, which determine the concentration of lectin-associated CEA/CEA-like antigens (LCA). However, the increase of the titer of the antigen is not specific to cervical cancer, as can occur in inflammatory diseases, benign neoplasms in smokers. The known method of forming the risk of neoplastic disorders in the cervical epithelium (RF patent No. 2437096, publication date 20.12.2011) , which can be used for the cytological diagnosis of cervical dysplasia is petelia and cervical cancer. The method includes immunofluorescent staining smear cervical epithelium using a monoclonal antibody that interacts with a high molecular weight glycoprotein MUC1, followed by counterstaining cells chromogenic nuclear dye. The result of the test allows you to select the cases with potentially high risk of the presence of pathological disorders, but does not allow to fully differentiate severe cervical dysplasia, carcinoma in situ and cervical cancer.
A known method for the diagnosis of cervical pathology using optical coherence tomography (OCT) (RF patent 2463958, publication date 20.10.2012) , which registered OCT images in direct polarization and orthogonal polarization. The disadvantages of the method are the identification of the disease only on the clinical stage of development and the use of special equipment, available only in specialized clinics.
Now assume that the most promising for the differential diagnosis of cervical intraepithelial neoplasms and cervical cancer are the methods based on the detection of tumor biomarkers in a biological material from the biopsies (or blood). In particular, there is a method of diagnosing cervical neoplasms, based on immunohistochemical identification of proteins P16,BC1-2 and p53 . The sensitivity of the proposed method achieved an 83.3%, specificity of moderate to 65.4%. This is because the change in the profile of proteins BC1-2 and p53 is not typical only for cervical cancer, this fact occurs when other malignant tumors, inflammatory reactions, other nonneoplastic diseases.
As tumor markers for diagnosis of cervical neoplasms and use of cervical cancer and other biomolecules cells: cyclin D1, Ki67, ProEx C . The most promising combination of markers, the authors consider P16 plus model ProEx C. However, in the study again proposed to use immunohistochemical analysis, which often leads to false-positive results.
There is also known a method for the diagnosis of cervical intraepithelial neoplasms (RF patent No. 2464570, publication date 20.10.2012) , which consists in sampling the studied material from the cervix by scraping followed by analysis of expression of p16INK4a mRNA as a marker of potential progression of dysplastic cervical processes using quantitative PCR analysis of the mRNA concentration of gene gipoksantin-guanine phosphoribosyltransferase (HPRT-1). At this rate the level of relative mRNA expression p16INK4a gene to the expression level of mRNA HPRT-1. The level of expression of mRNA of the gene p16INK4a mRNA HPRT-1 over 93 Rel. unit corresponds to CIN III (carcinoma in situ), the it is more than 201 Rel. unit corresponds to squamous cell cancer. This method can improve the efficiency of diagnosis of precancerous cervical processes.
The disadvantage of this method is that it separates cervical intraepithelial neoplasia and squamous cell carcinoma, not taking into account the initial stage of malignant process, i.e., has low specificity for the differential diagnosis of early-stage cervical cancer, in particular, preinvasive cancer (carcinoma in situ and microinvasive cancer.
The closest in technical essence to the claimed method, which is adopted as a prototype, is a method of determining the mRNA of the gene c-fos in carcinoma of the cervix, which consists in sampling the studied material by biopsy and then determine the mRNA expression of c-fos as one of the prognostic markers . The disadvantage of this method is that it can be differentiated only cervical intraepithelial neoplasia and squamous cell carcinoma, and the initial stages of the malignant process is not possible to diagnose, i.e., the method also has low specificity for the differential diagnosis of early-stage cervical cancer, in particular, preinvasive cancer (carcinoma in situ and microinvasive cancer.
Of the currently known methods of diagnostics the abnormalities of the cervix no, which allows you to clearly differentiate between severe dysplasia, carcinoma in situ and microinvasive cancer is cervical cancer.
The technical result of the proposed method is the detection and differentiation with a high degree of reliability of cervical dysplasia and early-stage cervical cancer, including carcinoma in situ and microinvasive cancer, while maintaining the speed and versatility of the method of diagnosis of these pathologies. In addition, this method of diagnosis will contribute to a more correct choice of tactics of treatment of patients, reducing the length of their stay in hospital, as well as the formation of risk groups for the development of cervical cancer.
The technical result is achieved by the fact that in this way analyze the mRNA expression of the gene c-fos and analysis of mRNA expression of the gene TATA-binding protein (TBP) using quantitative PCR in the diagnostic material of the cervix in the presence of cytologically installed cervical dysplasia, then compare the level of expression of mRNA of the gene c-fos with the level of expression of TBP mRNA and, if the level of expression of mRNA of the gene c-fos mRNA TBP from 109 to 145 Rel. unit - diagnosed with severe dysplasia, if the value of this ratio from 18 to 35 Rel. unit - diagnosed with pre-invasive cervical cancer (carcinoma in situ), if the value of the ratio from 148 to 208 Rel. unit - diagnosed squamous cell mi is reinvasion cervical cancer, if the ratio exceeds 208 Rel. unit - diagnosed with invasive squamous cell cervical cancer.
The method includes sampling the studied material by biopsy of the cervix, the selection of the sample of tissue total RNA, obtaining cDNA amplificatoare mRNA gene c-fos mRNA and gene TBP, the calculation of the relative amount of mRNA of the gene c-fos in the sample in relation to the contents of the reference gene TBP, a comparison with the results of histological analysis and diagnosis.
The method is as follows.
The allocation of total RNA.
Samples of cervical tissue were removed from the solution for stabilization of RNA in the biomaterial EverFresh RNA (Clonogen", St. Petersburg), removed the remains of EverFresh through a sterile filter, homogenized in a lytic buffer "RNA-Extra" ("Synthol", Moscow) using a rotary homogenizer TissueRuptor ("Qiagen, Germany). Further, the allocation of total cellular RNA was performed by standard method of liquid-phase extraction using sets of "RNA-Extra" according to the instructions set ("Synthol", Moscow). The dried precipitate the RNA was dissolved in 50 μl of deionized ddH2O, not containing RNase. The concentration and purity of the RNA preparation was determined by absorption at 260, 280 and 320 nm using a scanning spectrophotometer BioWave II+ ("Biochrom, UK) (2 ál education is CA RNA was diluted 50 times ddH 2O, in the future, the study included only those samples for which a/A>1,8). The native characteristics of bacterial RNA was determined by capillary electrophoresis on microchips RNA StdSens using automatic station Experion (Bio-Rad, USA) according to the manufacturer's instructions. Conclusion about the quality of the RNA preparation was done according to the ratio of the fractions of 18S and 28S rRNA.
Carrying out reverse transcription, obtaining cDNA.
To remove possible impurities genomic DNA samples RNA was pre-treated with Dnazol I ("Fermentas", Lithuania) according to the Protocol recommended by the manufacturer. The reverse transcription reaction was performed in a two-volume set "FROM-1" firm "Synthol" (Moscow). In reaction took 1 μg of sample RNA in one volume; the number of remaining components of the reaction mixture corresponded to the manufacturer's instructions. As primers for reverse transcription used a mixture of hexanucleotides ("Silex", Moscow). The reaction was carried out in thermostat "Dwarf" (CJSC SPC "DNA-Technology") at a temperature of +37°C for 1 h, followed by inactivation of the reverse transcriptase at +70°C for 10 min 2 µl of the resulting cDNA sample was diluted 50 times ddH2O and spectrophotometric analysis was performed as described above (the ratio of the degree of absorption was varied in the range of 1.7<a/A<1,9). Samples of kankroli at -20°C or, in the case of long term storage at -70°C.
Amplification in "real time" was performed in a volume of 25 µl using the device "iCycler Thermal Cycler" (BioRad, USA) and software iQ5 Optical System Software version 2.0 (BioRad, USA) using reagent kits for PCR in real time in the presence of the intercalating dye SYBR Green ("Synthol", Moscow). To increase the sensitivity and specificity of PCR was applied the principle of "hot start", which was provided by the manufacturer at the expense of inhibiting the activity of Taq polymerase antibodies. The synthesis of oligonucleotides was carried out by the company Synthol (Moscow). When preparing the reaction mixture for PCR adhered amounts of the components recommended by the manufacturer: per test tube was mixed with 2.5 μl of deoxynucleosides (2.5 mm), and 2.5 µl of 10x PCR buffer containing SYBR Green, 2,5 µl MgCl2(25 mm), 1 μl forward and reverse primers (10 mcmol/µl), and 0.25 ál SynTaq-DNA-polymerase (5 u/ál), 2 ál of cDNA sample, deionized water up to 25 µl. Amplification was carried out according to the following Protocol: cycle No. 1 (1 repeat) - 95°C for 3 min; cycle No. 2 (45 repeats) of 15 s at 95°C, 50 s at 62°C.
Detection of the intensity of the fluorescent signal was performed on the stage of the annealing/elongation. To determine with whom ecificity annealing of primers directly after amplification was performed procedure melting PCR fragments: 1 min at 95°C, 1 min at 60°C, 10 s at 60°C (80 cycles, increasing in each cycle the temperature by 0.5°C). Received melting curves were made conclusion about the homogeneity of the PCR products. PCR reactions for each sample were performed in two independent replicates.
On stage optimization of the PCR Protocol, to further confirm the specificity of the primers and the reaction efficiency, carried out the separation of PCR products by electrophoresis in 8% polyacrylamide gel and Tris-borate buffer using low-molecular markers of the lengths of the fragments of pUC19/MspI ("Silex", Moscow); amplicons were visualized by fluorescence of ethidium bromide in UV light, documented through automated analytical system GelDoc-It M-26XV ("UVP, USA). In this work, we used the following primers: gene c-fos (forward 5'-CAGCGAGCAACTGAGAAGCC-3' (exon 1, item 62), reverse 5'-CGCTGTGAAGCAGAGCTGG-3' (exon 1, item 135)); for control TBP gene (forward 5'-GTTCTGGGAAAATGGTGTGC-3', reverse 5'-CCAGGAAATAACTCTGGCTCA-3').
The amplification efficiency (E) was determined by PCR with serial 10-multiple dilutions randomly selected sample and primers for GAPDH (forward 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3', reverse 5'-CATGTGGGCCATGAGGTCCACCAC-3'). The value of "E" was determined according to the schedule according to the threshold cycle of concentration.
Statistical analysis was performed in the program Statistics 6.0. For about Enki the relative amount of mRNA of the gene c-fos in the samples used content analysis of mRNA reference gene TBP in the same sample, characterized by low and stable level of expression of mRNA in the cell. When the ratio of mRNA expression of the gene c-fos mRNA TBP is less than 1, it was assumed that the expression of mRNA of the gene c-fos in the sample is virtually absent, and when the ratio is greater than 1 - about the presence of gene expression of c-fos in the sample.
The calculation of the relative expression was carried out on the basis of a comparative assessment of the values of S(T) X (where X is investigated gene) obtained for each gene by PCR in "real time". Determining the level of expression of mRNA of the gene c-fos relative to TBP mRNA believed method ΔΔ(T)-2(-Delta Delta C(T)) .
where [c-fos]/[TWR] normalized by TBP is the expression of the gene c-fos, measured in relative units, shows how many copies of mRNA of C-fos have 1 copy of TBP;
E - the efficiency of amplification indicates the gain matrix for each cycle of amplification. Theoretically, at each cycle there is a double matrix, so E=2;
ST1 - value threshold cycle model for gene c-fos;
ST2 - value threshold cycle in the sample for TVR.
Analyzed 36 samples of cervical tissue of patients with histologically confirmed invasive squamous cell cervical cancer (6 cases), microinvasive carcinoma (5 cases), cancer in situ (6 cases), severe dysplasia CIN III (7 cases), CIN II (4 Sluch is I), CIN I (5 cases) and reactive changes (3 cases). The material obtained by biopsy from the cervix with a comprehensive gynecological examination. The samples were analyzed for the mRNA expression of the gene c-fos. There were also cytological examination. Criterion validity of cytological and molecular genetic methods have been matching results with histological examination of the biopsy and/or surgical material. Analysis of the investigated material also included quantitative determination of mRNA gene TATA-binding protein (TBP). When comparing the level of expression of mRNA of the gene c-fos with the level of expression of TBP mRNA. The TBP gene expression is carried out at a fairly constant level, which allows to determine the relative level of expression of functionally active genes (in this case, c-fos), based on the values in the expression of TBP. This approach is standard and ensures high reproducibility of the results.
Table 1 presents the data obtained by quantitative analysis of mRNA gene c-fos for 36 samples of cervical tissue, as well as data cytological and histological analyses.
The comparison of the results of cytological examination and the level of expression of mRNA of the gene c-fos showed that reactive changes in the cervical epithelium level EC is pressey was insignificant and amounted to 5.0, 7.0 and 11.0 Rel. units Increased expression of mRNA of the gene c-fos was observed in CIN I - 46,8±12.5 Rel. units (M±σ, where M is the mean, σ - standard deviation), CIN II 78,5±19.5 Rel. unit and severe dysplasia 123,6±11.9 Rel. units In the stage of pre-invasive cancer (cancer in situ) expression of mRNA of the gene c-fos was sharply decreased and amounted to 25.3±6.9 Rel. units In stage microinvasive cancer, this figure was again increased to 180,2±21.6 Rel. unit Maximum value of the relative level of mRNA expression of the gene c-fos characteristic of invasive squamous cell cancer is more 208,0 Rel. units Obtained results showed that the proposed method has high specificity and allows one to obtain reliable and selective results, as illustrated in the following examples.
Patient, 27 years of age (sample 14) Cytology diagnosed with CIN II. In a sample of tissue taken by biopsy, the correlation between the level of expression of mRNA of the gene c-fos and the expression of TBP mRNA was 109 Rel. unit - diagnosed with severe dysplasia, which is confirmed by histological examination.
Patient N. 34 years (sample 20) Cytology diagnosed with CIN II. In biopsy material from the ratio between the expression level of mRNA of the gene c-fos and the expression of TBP mRNA was 18 Rel. unit - diagnosed cancer in situ confirmation of the config histological examination.
The patient M. the age of 38 (sample 28) Cytology diagnosed with CIN III (carcinoma in situ). In the sample tissue after biopsy correlation between the expression level of mRNA of the gene c-fos and the expression of TBP mRNA was 148 Rel. unit - diagnosed with microinvasive squamous cell cancer (IA1), confirmed by histological examination.
The patient Century. 46 years (sample 30) Cytology diagnosed with squamous cell carcinoma. In biopsy material from the ratio between the expression level of mRNA of the gene c-fos and the expression of TBP mRNA was 208 Rel. unit - diagnosed with invasive squamous cell cancer, confirmed by histological examination.
The above experimental data indicate that the inventive method can be reliably used for the detection and differential diagnosis of cervical dysplasia and early-stage cervical cancer, including carcinoma in situ and microinvasive cancer. In addition, this method of diagnosis will contribute to a more correct choice of tactics of treatment of patients, reducing the length of their stay in hospital, as well as the formation of risk groups for the development of cervical cancer.
|That the person 1.|
|The amount of mRNA of the gene c-fos in samples of cervical tissue|
|Sample||Cytological analysis||The ratio of mRNA of the gene c-fos TBP mRNA (Rel. unit)||Histological analysis|
|1||Reactive epithelial changes, inflammation||7||Reactive epithelial changes|
|2||CIN I||11||Reactive epithelial changes|
|3||Reactive epithelial changes, inflammation||5||Reactive epithelial changes|
|4||CIN I||47||CIN I|
|5||Pronounced inflammation||31||CIN I|
|6||CIN I||35||CIN I|
|7||CIN I||58||CIN I|
|8||CIN I||63||CIN I|
|9||CIN I||55||CIN II|
|10||CIN I||69||CIN II|
|11||CIN II-III||108||CIN II|
|12||CIN II||82||CIN II|
|13||CIN III (carcinoma in situ)||145||Severe dysplasia|
|14||CIN II||109||Severe dysplasia|
|15||CIN III (carcinoma in situ)||128||Severe dysplasia|
|16||CIN III (carcinoma in situ)||134||Severe dysplasia|
|17||CIN III (carcinoma in sit)||120||Severe dysplasia|
|18||CIN II||117||Severe dysplasia|
|19||CIN II||112||Severe dysplasia|
|20||CIN II||18||Carcinoma in situ|
|21||CIN III (carcinoma in situ)||30||Carcinoma in situ|
|22||CIN II||15||CIN III (carcinoma in situ)|
|23||CIN III (carcinoma in situ)||25||Carcinoma in situ|
|24||CIN III (carcinoma in situ)||29||Carcinoma in situ|
|25||Squamous cell carcinoma||35||Carcinoma in situ|
|26||CIN III (carcinoma in situ)||Microinvasive squamous cell cancer|
|27||Squamous cell carcinoma||183||Microinvasive squamous cell cancer|
|28||CIN III (carcinoma in situ)||148||Microinvasive squamous cell cancer|
|29||Squamous cell carcinoma||197||Microinvasive squamous cell cancer|
|30||Squamous cell carcinoma||208||Microinvasive squamous cell cancer|
|31||Squamous cell carcinoma||261||Squamous cell invasive cancer|
|32||Squamous cell carcinoma||202||Squamous cell invasive cancer|
|33||Squamous cell carcinoma||280||Squamous cell invasive cancer|
|34||Squamous cell carcinoma||293||Squamous cell invasive cancer|
|35||Squamous cell carcinoma||215||Squamous cell invasive cancer|
|36||Squamous cell carcinoma||230||Squamous cell invasive cancer|
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5. Guzick D. S. Efficacy of screening for cervical cancer // A review. Am. J. Public Heath, 1978. Vol.68. P. 125-134.
6. Samarawardana P., Dehn, D. L., Singh, M. et al. p16(INK4a) is superior to high-risk human papillomavirus testing in cervical cytology for the prediction of underlying high-grade dysplasia // Cancer Cytopathol., 2010. Vol.25, 118 (3). P. 146-156.
7. Pat. 2251699 OF THE RUSSIAN FEDERATION, IPC G01N 33/574, G01N 33/577. Way early and preclinical diagnosis of cervical cancer / Kiselev Century. And., Sveshnikov, P. No. 2003128660/15; Statements. 25.09.2003; Publ. 05.10.2005, bull. 13.-11 S.: ill.
8. Pat. 2321859 OF THE RUSSIAN FEDERATION, IPC G01N 3/483. The method of differential diagnosis of hyperplasia, intraepithelial neoplasms and cancer of the cervix / Avtandil gg, Glukhova Y. K. No. 2005110965/15; Statements. 15.04.2005; Publ. 04.10.2008, bull. 10-6 C.
9. Pat. 2343485 OF THE RUSSIAN FEDERATION, IPC G01N 33/53. The way to diagnose cervical cancer / Bulgakov, A. A., Rodionov, O. M., Apanasevich C. I., Petrova I. Y., Liseykina M, No. 2007123039/15; Statements. 14.06.2007; Publ. 10.01.2009, bull. 1.-8 C.
10. Pat. 2437096 OF THE RUSSIAN FEDERATION, IPC G01N 33/483. The method of forming the risk of neoplastic disorders in the cervical epithelium / Jakubowski, R. I., Karmakova T. A., Volchenko N. N., Novikov E. G., O. Trushin, I. No. 2010121228/15; Statements. 27.05.2010; Publ. 20.12.2011, bull. No. 35.-14 S.: ill.
11. Pat. 2463958 OF THE RUSSIAN FEDERATION, IPC AV 6/00. Method for diagnosis of cervical pathology / Kuznetsov, I. A., Shakhova N. M., Kachalina Ie, Gladkova N.D., Kiseleva E. B., Karabut M M No. 2011119422/14; Statements. 13.05.2011; Publ. 20.10.12, bull. 29.-20 S.: ill.
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13. Conesa-Zamora P., Trujillo-Santos j, Orantes-Casado F. J. et al. Analysis of performance characteristics of five cell cycle-related immunohistochemical markers and human papillomavirus genotyping in the diagnosis of cervical squamous cell carcinoma precursor lesions // Anal. Quant Cytol. Histol., 2012. Vol.34 (1). P. 49-55.
14. Pat. 2464570 OF THE RUSSIAN FEDERATION, IPC G01N 33/53. Method of diagnosis of cervical intraepithelial neoplasms / Bozhenko C. K., Kudinov E. A., Bliznakov O. P., Melnikov N. In., Murmanskaya O. C. No. 2010147074/15; Statements. 19.11.2010; Publ. 20.10.2012, bull. 29.-9 C.
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The method of differential diagnosis of cervical dysplasia and cervical cancer based on the determination of the relative content of mRNA of the gene c-fos using quantitative PCR, which includes the intake of the studied material by biopsy of the cervix, the selection of the sample of tissue total RNA, obtaining cDNA and amplificatoare mRNA gene c-fos, characterized in that in the presence of cervical dysplasia in cytological study analyzed the mRNA expression of the gene TATA-binding protein (TBP), and then comparing the level of expression of mRNA of the gene c-fos with the level of expression of TBP mRNA and, if the level of expression of mRNA of the gene c-fos mRNA TBP from 109 to 145 Rel. unit, diagnosed with severe dysplasia, if the value of this ratio from 18 to 35 Rel. unit, diagnosed with pre-invasive cervical cancer (carcinoma in situ), if the value of the ratio from 148 to 208 Rel. unit, diagnosed with microinvasive squamous cell cervical cancer, if the ratio exceeds 208 Rel. unit, diagnosed with invasive squamous cell cervical cancer.
SUBSTANCE: incubation medium, oxidation substrate and tetraphenylphpsphonium chloride as indicator are placed into respective measuring cell of installation for measuring mitochondria potential, provided with tetraphenylphosphonium-selective electrode, change of tetraphenylphosphonium concentration is registered and when constant tetraphenylphosphonium concentration is achieved, mitochondria, isolated from animal organism, are added. Change of membrane potential of mitochondria is registered by change of electrode signal, when constant potential is achieved, respective water suspensions of analysed samples of nanodiamonds with pH 7.2-7.4 are added, and value of rate of change of mitochondria membrane potential is measured. Presence of statistically reliable difference of rates of mitochondria membrane potential change testifies to biological inequivalence of compared samples of nanodiamonds.
EFFECT: expressive and available method of determining biological inequivalence of nanodiamonds.
2 cl, 1 tbl, 1 dwg, 1 ex
SUBSTANCE: hose of an artificial air feed apparatus is inserted into a lobar bronchus of the estimated injured lobe of the lung. The hose is fixed by ligaturing the bronchus. The air is pumped into the examined lobe of the collapsed lung until it extends completely, and a microinjury is visualised.
EFFECT: technique can provides the more reliable diagnosis of the lung microinjury improve in dead bodies, which is achieved by filling the collapsed lung with air providing opening the microinjury.
SUBSTANCE: invention relates to biology, forensic and analytical chemistry and specifically to methods of determining procaine in blood plasma. The method comprises adding sodium fluoride to blood plasma containing procaine to achieve concentration of 10 mg/ml; treating the obtained mixture with acetone; separating the extract from the precipitate by filtering; evaporating acetone from the filtrate in an air current at room temperature; diluting the aqueous residue by adding water; saturating the obtained solution with ammonium sulphate; alkalising with an ammonium buffer solution to pH 9.0-9.5; extracting twice with portions of an organic extraction agent in the form of 30% camphor solution in methyl acetate, with ratio of the aqueous to the organic phase of 1:1 by volume; separating the organic extracts; combining; evaporating the solvent from the combined extract in an air current at room temperature; chromatographing the residue in a thin layer of silica gel STKH-1A on Sorbfil PTSKH-AF-A-UF plates, using a dichloromethane-ethanol mobile phase in ratio of 6:4 by volume; developing the chromatogram in UV light; eluting the analysed substance from the sorbent with a mixture of acetonitrile-methanol-0.025 M potassium dihydrogen phosphate solution with pH 3.0 in ratio of 10:10:90 by volume; chromatographing by HPLC method using a reversed-phase sorbent Nucleosil C18, a polar mobile phase acetonitrile-methanol-0.025 M potassium dihydrogen phosphate solution with pH 3.0 in ratio of 10:10:90 by volume and a UV detector; measuring optical density at wavelength 298 nm and calculating the amount of the analysed compound from the area of the chromatographic peak.
EFFECT: method improves sensitivity of determination.
3 tbl, 2 ex
SUBSTANCE: invention refers to medicine, namely to hepatology, and describes a method for examining the liver detoxification function state. The method involves measuring fasting blood AWM (average weigh molecule), ALT (alanine aminotransferase) and AST (aspartate aminotransferase), measuring the same values the night before at bedtime, as well as measuring a circulating blood volume and a packed cell volume in the morning and in the evening followed by determining a coefficient of the liver detoxification function state; and if observing the derived coefficient K≤0.6, the liver detoxification function state is considered to be satisfactory, while the coefficient K>0.6 shows the depressed liver detoxification function.
EFFECT: method provides the more reliable assessment of the liver detoxification function state.
SUBSTANCE: method involves the preliminary sorption of a specific ligand in polystyrene tray wells; the above ligand is specified in: haemoglobin, myoglobin, collagen, fibrinogen, fibronectin, immunoglobulin A; that is followed by adding a microorganism cell suspension into the tray wells with the sorbed ligand, incubating the cell suspension in the tray wells for 15 minutes, sampling a suspension aliquot from the well and adding it to wells of another or the same tray containing 0.5% sodium chloride or 0.2 M sodium phosphate; the bacterial cell adhesion is assessed by measuring a decrease of the optical density of the prepared diluted suspensions at wave length 600 nm as compared to the well references free from ligands.
EFFECT: invention is characterised by a high test rate of the microorganism adhesion to ligands of various nature, high reproducibility, using a minimum amount of the microorganism biomass, with no need for hazardous chemicals to be used.
7 tbl, 7 ex
SUBSTANCE: invention relates to biology and toxicological chemistry and can be used in practice of sanitary and epidemiological stations, chemical-toxicological, forensic and veterinary laboratories. A biological material, containing substituted 2-methoxyhydroxybenzene, is two times (each time for 30 minutes) infused with ethylacetate with mixing, separate extracts are separated from solid particles of the biological material, combined, ethylacetate is evaporated in an air flow at 18-22°C, residue is repeatedly processed with acetone, acetone extracts are separated, combined, dehydrated, evaporated in an air flow at 18-22°C, and then in a nitrogen flow until a solvent is completely removed, residue is dissolved in hexane, extracted with a buffer solution with pH 12-13, a water-alkaline extraction is separated, acidified to pH 2-3, saturated with sodium sulphate, extracted with diethyl ether, the ether extract is separated, dehydrated, evaporated in an air flow at 18-22°C, and then - in a nitrogen flow until the solvent is completely removed, residue is dissolved in a mixture of solvents hexane-dioxane-propanol-2, taken in a ratio of 20:5:1 by volume, chromatographed in macrocolumn with silicagel KSS No 3 80/120 mcm with the application of a mobile phase hexane-dioxane-propanol-2 in a ratio of 20:5:1 by volume, eluate fractions, containing the analysed substance, are combined, the eluent is evaporated first in an air flow at a temperature of 18-22°C, and then in a nitrogen flow until the solvent is completely removed, residue is dissolved in dichloromethane, processed for 20 minutes with N-tert-butyl-dimethylsilyl-N-methyltrifluoroacetamide under conditions of heating at a temperature of 60°C with carrying out determination by a chromatography-mass spectrometry method with the application of a capillary column 25 m long with an internal diameter of 0.2 mm with an immobile phase (5%-phenyl)-methylpolysiloxane, with the application of a mass-selective detector, working in an electron impact mode, an initial temperature of the column thermostat constitutes 70°C, the said temperature is kept for 3 minutes, further the temperature is programmed from 70°C to 290°C at a rate of 20°C per minute, the final column temperature is kept for 10 minutes, the injector temperature constitutes 250°C, the quadrupole temperature is 150°C, the temperature of a ion source is 230°C, the temperature of the detector interface is 300°C, intensity of a signal, conditioned by charged particles, formed in the bombardment of the analysed substance, leaving the capillary column and getting into the ion source, with a ionising beam of electrons with the energy of 70 eV, is registered, the mass-spectrum by the complete ion flow is registered and an amount of substituted 2-methoxyhydroxybenzene is calculated by the area of a chromatographic peak of its trimethylsilyl derivative.
EFFECT: achievement of an increased analysis sensitivity.
4 tbl, 3 ex
SUBSTANCE: invention relates to biology and toxicological chemistry and can be used in chemical-toxicology, expert forensic and clinical laboratories. The method includes: crushing a biological object containing N-(4-nitro-2-phenoxyphenyl)-methanesulphonamide, settling twice for 45 minutes with portions of an organic isolating agent which is methyl acetate, combining the obtained extracts, evaporating the solvent from the resultant extract, treating the residue with acetone, separating the acetone extract, evaporating the solvent from resultant extract, dissolving the residue in diethyl ether, extracting the ether solution with a buffer solution at pH 9-10, acidifying the aqueous alkaline extract with 24% hydrochloric acid to pH 2-3, saturating the obtained solution with sodium bromide, extracting with ethyl acetate, evaporating the obtained extract in an air current at 20-22°C until a dry residue is obtained, dissolving the residue in a mixture of hexane and acetone taken in volume ratio of 8:2, performing chromatography on a macrocolumn with silica gel L 40/100 mcm using a hexane-acetone mobile phase in volume ratio of 8:2, combining eluate fractions containing the analysed substance, evaporating the eluent in an air current at 20-22°C until complete removal of the solvent, dissolving the residue in methanol and performing determination via a combined physical-chemical method in the form of chromatography-mass spectrometry, using a DB-5 MS EVIDEX capillary column with a mobile phase which is 5% phenyl-95% methylpolysiloxane, using a mass-selective detector operating in electron impact mode, the initial thermostat temperature of the column is 70°C, maintaining said temperature for 3 minutes, further raising the temperature from 70°C to 290°C at a rate of 20°C per minute, maintaining the final temperature of the column for 16 minutes, the temperature of the injector is 250°C, the temperature of the quadrupole is 150°C, the temperature of the detector interface is 300°C, detecting strength of the signal resulting from charged particles formed when bombarding the analysed substance coming from the capillary column and falling into an ion source with an ionising electron beam with energy of 70 eV, recording the mass spectrum on the full ion current, while calculating the amount of N-(4-nitro-2-phenoxyphenyl)-methanesulphonamide from the area of the chromatographic peak.
EFFECT: high sensitivity of analysis.
2 ex, 3 tbl
SUBSTANCE: invention relates to medicine, namely to clinical, laboratory diagnostics, microbiological methods of research, and is aimed at standardisation of saliva analysis by method of wedge dehydration/crystallography. Claimed is method of obtaining standard, quality sample of mixed saliva facia for crystallography with application of portable laboratory device with inbuilt levels on axes X and Y, legs, regulated by height and isolating cover. From 20 to 40 microscope slides are simultaneously placed on the surface of portable laboratory device after obtaining smooth horizontal without inclination angle surface. After that, one sample of mixed saliva in amount 0.02 ml, preliminarily centrifuged for 20 min at 3000 rev/min, is applied on each microscope slide by means of micropipette. As a result a round 1.0 mm high drop of mixed saliva with diameter from 4.0 to 5.0 mm, corresponding to standard parameters, is obtained. Then, standard in dimensions drop on microscope slide placed on the surface of portable laboratory device, adjusted by levels, is dried at room temperature +18…+25°C under isolating cover for 5 hours, with further performance of microscopy of standard quality sample of saliva facia.
EFFECT: obtained sample makes it possible to interpret indices of crystallography without distortions, as standard in volume, dimensions and shape drop of mixed saliva is obtained, and there is no displacement of crystallisation centre and impairment of figures of saliva facia (crystallography pattern) in drop, dried on ideal horizontal surface of the device, ratio of central and peripheral zones of facia exactly correspond to organism's condition, which reduces percentage of false results of crystallography.
3 ex, 1 tbl, 3 dwg
SUBSTANCE: invention aims at asserting the maximum allowable blood concentrations (MAC) of heavy metals in the children living in the dirty environment as shown by health risk criteria after the chronic integrated exposure. An environmentally neglected zone is selected; a representative sampling of the children for the examination is drawn that is a basic group with using biological, social and hygienic criteria; the same criteria are used to draw a representative sampling of the children to a reference group living in the environmentally friendly zone. In the territory of the above zones, the chronic exposure of the analysed heavy metal is qualitatively assessed by establishing its average daily concentration in the ambient environment; the derived value is used to calculate a total average daily doses of a heavy metal supplied from various sources into a child's body averaged over the annual exposure for the children of both groups. Blood is sampled from the children every three months for one year to determine the content of the analysed heavy metal and also to measure the biochemical values of blood plasma and serum characterizing body responses presented by actual or potential health problems that are response markers. That is followed by calculating the average blood concentration of the analysed heavy metal and comparing it to the reference for the same heavy metal with using a Student two-sample test, thereby stating whether the children were sampled from the main and reference groups adequately. A mathematical modelling procedure is used to establish a relation between the exposure that is the total average daily doses of the analysed metal, and the exposure marker that is the average blood metal concentration. A sliding window technology is used to assert the response markers selected. The maximum allowable concentration of the exposure marker and respective marker is determined by a technique based on ratio analysis.
EFFECT: enabled measurement of the blood MAC of the heavy metals in the children after the integrated exposure with using sparing techniques making it possible to avoid a health risk.
4 tbl, 2 dwg, 1 ex
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.
FIELD: medicine, hepatology.
SUBSTANCE: one should detect the level of hepato-specific enzymes (HSE) in blood plasma, such as: urokinase (UK), histidase (HIS), fructose-1-phosphataldolase (F-1-P), serine dehydratase (L-SD), threonine dehydratase (L-TD) and products of lipid peroxidation (LP), such as: dienic conjugates (DC), malonic dialdehyde (MDA). Moreover, one should detect the state of inspecific immunity parameters, such as: immunoregulatory index (IRI) as the ratio of T-helpers and T-suppressors, circulating immune complexes (CIC). Additionally, one should evaluate the state of regional circulation by applying rheohepatography (RHG), the system of microhemocirculation with the help of conjunctival biomicroscopy (CB) to detect intravascular index (II). In case of increased UK, HIS levels up to 0.5 mcM/ml/h, F-1-P, L-SD, L-Td, LP products, CIC by 1.5 times, higher IRI up to 2 at the norm being 1.0-1.5, altered values of regional circulation, increased II up to 2 points at the norm being 1 point, not more one should diagnose light degree of process flow. At increased level of UK, HIS up to 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 1.5-2 times, increased IRI up to 2.5, altered values of regional circulation, increased II up to 3-4 points one should diagnose average degree of process flow. At increased level of UK, HIS being above 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 2 and more times, increased IRI being above 2.5, altered values of regional circulation, increased II up to 5 points and more one should diagnose severe degree of process flow.
EFFECT: higher accuracy of diagnostics.
FIELD: medicine, infectology, hepatology.
SUBSTANCE: in hepatic bioptate one should detect products of lipid peroxidation (LP), such as: dienic conjugates (DC), activity of antioxidant enzymes, such as: catalase (CAT)and superoxide dismutase (SOD). One should calculate by the following formula: C = DC/(SOD x CAT)x100, where DC - the content of dienic conjugates, SOD - activity of superoxide dismutase, CAT - activity of catalase. At coefficient (C) values being above 65 one should predict high possibility for appearance of cirrhosis, at 46-645 - moderate possibility and at 14-45 -low possibility for appearance of cirrhosis.
EFFECT: higher accuracy of prediction.
FIELD: medicine, clinical toxicology.
SUBSTANCE: at patient's hospitalization one should gather the data of clinical and laboratory values: on the type of chemical substance, patient's age, data of clinical survey and laboratory values: body temperature, the presence or absence of dysphonia, oliguria being below 30 ml/h, hemoglobinuria, erythrocytic hemolysis, exotoxic shock, glucose level in blood, fibrinogen and creatinine concentration in blood serum, general bilirubin, prothrombin index (PTI), Ph-plasma, the state of blood clotting system. The state of every sign should be evaluated in points to be then summed up and at exceeding the sum of points being above "+20" one should predict unfavorable result. At the sum of "-13" prediction should be stated upon as favorable and at "-13" up to "+20" - prediction is considered to be doubtful.
EFFECT: higher accuracy of prediction.
2 ex, 3 tbl
FIELD: medicine, juvenile clinical nephrology.
SUBSTANCE: disease duration in case of obstructive pyelonephritis should be detected by two ways: either by detecting the value of NADPH-diaphorase activity, as the marker of nitroxide synthase activity in different renal department and comparing it to established norm, or by detecting clinico-laboratory values, such as: hemoglobin, leukocytes, eosinophils, urea, beta-lipoproteides, lymphocytes, neutrophils, the level of glomerular filtration, that of canalicular reabsorption, urinary specific weight, daily excretion of oxalates, arterial pressure, and estimating their deviation against average statistical values by taking into account a child's age.
EFFECT: higher efficiency of detection.
7 dwg, 1 ex, 6 tbl
FIELD: clinical medicine, pulmonology.
SUBSTANCE: one should carry out complex estimation of interleukin-1β) concentration in blood, saliva, bronchoalveolar liquid. Moreover, one should detect distribution coefficient (DC) for IL-1β as the ratio of IL-1β blood content to IL-1β salivary content. At increased IL-1β blood content by 10 times and more, by 2 times in saliva, unchanged level of bronchoalveolar IL-1β, at DC for IL-1β being above 1.0 one should predict bronchial obstruction. The method enables to conduct diagnostics of the above-mentioned disease at its earlier stages.
EFFECT: higher efficiency of prediction.
FIELD: medicine, diagnostics.
SUBSTANCE: the present innovation deals with genetic trials, with diagnostic field of oncological diseases due to analyzing DNA by altered status of gene methylation that take part in intracellular regulation of division, differentiating, apoptosis and detoxication processes. One should measure the status of methylation in three genes: p16, E-cadherine and GSTP1 in any human biological samples taken out of blood plasma, urine, lymph nodes, tumor tissue, inter-tissue liquid, ascitic liquid, blood cells and buccal epithelium and other; one should analyze DNA in which modified genes of tumor origin or their components are present that contain defective genes, moreover, analysis should be performed due to extracting and purifying DNA out of biological samples followed by bisulfite treatment of this DNA for modifying unprotected cytosine foundations at keeping 5-methyl cytosine being a protected cytosine foundation followed by PCR assay of bisulfite-treated and bisulfite-untreated genes under investigation and at detecting alterations obtained according to electrophoretic result of PCR amplificates, due to detecting the difference in the number and electrophoretic mobility of corresponding fractions at comparing with control methylated and unmethylated samples containing normal and hypermethylated forms of genes one should diagnose oncological diseases. The method provides higher reliability in detecting tumors, detection of remained tumor cells after operation.
EFFECT: higher efficiency of therapy.
1 cl, 3 dwg, 4 ex
FIELD: medicine, gastroenterology.
SUBSTANCE: one should carry out diagnostic studying, moreover, on the 5th -6th d against the onset of exacerbation in case of gastric and duodenal ulcerous disease one should detect the content serotonin, histamine and acetylcholine in blood, then during 2-3 wk one should conduct medicinal therapy to detect serotonin, histamine and acetylcholine level in blood again and at serotonin content being by 2-3 times above the norm, histamine - by 1.15-1.4 times above the norm and acetylcholine - by 20-45% being below the norm one should predict the flow of gastric and duodenal ulcerous disease as a non-scarring ulcer.
EFFECT: higher accuracy of prediction.
SUBSTANCE: method involves taking blood from ulnar vein (systemic blood circulation) and from large vein of the injured extremity proximal with respect to lesion focus (regional blood circulation). Spontaneous NST-test value is determined and difference is calculated in systemic and regional blood circulation as regional-to-systemic difference. The difference value is used for predicting clinical course of pyo-inflammatory disease in extremities.
EFFECT: high accuracy of diagnosis.
4 cl, 2 tbl
FIELD: medicine, gastroenterology.
SUBSTANCE: one should introduce biologically active substance, moreover, in patient's blood serum one should detect the content of acetyl choline and choline esterase activity followed by 2-h-long intragastric pH-metry at loading with biologically active substance as warm 40-45%-honey water solution at 35-40 C, and at increased content of acetyl choline being above 1.0 mM/l, choline esterase being above 0.5 mM/l/30 min and pH level being 6.0-6.9 it is possible to consider apitherapy to be useful for treating ulcerous duodenal disease.
EFFECT: higher efficiency and accuracy of detection.
FIELD: medicine, gastroenterology.
SUBSTANCE: it has been suggested a new method to detect pharmacological sensitivity to preparations as acidosuppressors. After the intake of the preparation a patient should undergo fibrogastroduodenoscopy 3 h later, then, through endoscopic catheter one should introduce 0.3%-Congo red solution intragastrically and the test is considered to be positive at keeping red color that indicates good sensitivity to the given preparation, and in case of dark-blue or black color the test is considered to be negative that indicates resistance to this preparation. The suggested innovation widens the number of diagnostic techniques of mentioned indication.
EFFECT: higher efficiency of diagnostics.