Diagnostic technique for abnormal chromatin packaging in male infertility
SUBSTANCE: diagnostic technique for abnormal chromatin packing in male sterility is based on a quantitative estimation of chromatin hydrolysis depth in sperm nuclei immobilised on slides. Hydrolysis by micrococcal nuclease is followed by preparation washing from soluble chromatin. Chromatin DNA without hydrolysis by nuclease and after hydrolysis is stained by fluorochrome DAPI and examined for the distinctions in a degree of fluorescence of the initial and nuclease-processed samples. A check value is a nature of chromatin hydrolyzate of fertile donor sperm cells characterised by the spermogram values. The prepared images are analysed by comparing brightness of the sperm cell images, and the abnormal chromatin packaging is shown by DNA amount extracted by nuclease relatively to total DNA.
EFFECT: use of the declared technique allows simplifying diagnosing of male sterility within the therapies in extracorporeal fertilisation and homologous insemination programs.
6 cl, 1 ex, 2 tbl
The invention relates to the field of medicine and veterinary medicine, namely to the field of assisted reproductive technologies, in particular to increase the pregnancy rate, and can be used in the treatment of infertility in programs of in vitro fertilization and homologous insemination by sperm sorting of samples containing cells with abnormal chromatin packaging, and diagnostics of the need for a specific therapy or restorative treatments with unfavorable prognosis regarding the effectiveness of assisted reproductive technologies.
Now who accepted methods of diagnosing abnormalities of spermatozoa, leading to male infertility, based on the estimate of their quantity, motility and detection of DNA breaks. At the same time it is shown that one common cause of male infertility is abnormal or not fully completed compaction of the DNA genome in semen.
Known (EN, application 2006130740) the method of determining the integrity of chromatin/DNA in the sperm cells of animals, including the stage of processing of a sample containing sperm cells, denaturing the DNA solution, one stage of processing lytic solution for extraction of nuclear proteins, the stage of determining the integrity of chromatin/DNA of sperm, characterizes the Yuexiu fact, that lyse solution does not contain denaturing proteins detergent and does not destroy much of the tails of sperm.
The disadvantage of this method should recognize that assesses the integrity of DNA in the presence or absence of tears, and this figure does not correlate with the pathology associated with incomplete or abnormal chromatin packaging.
Also known (RU, patent No. 2262107) method of determining maturity of sperm by assessing the degree of condensation of the chromatin of spermatozoa in the study of ejaculate. In the process of defining comparing the fluorescence intensities of sperm DNA before and after violent decondense chromatin heparin by means of static processing method Kolmogorov-Smirnov with the definition of the similarity index curves of the fluorescence intensity of n, a value that is less than 48,5 judge immature sperm, and when the value n is greater than 48,5 judge ripeness of sperm.
The disadvantage of this method should recognize that the property of chromatin to deconcentrate under the action of heparin only indirectly reflects anomalies in its packaging.
Also known (RU, patent No. 2118822) a method for the diagnosis of male infertility by flow-cytofluorimetric analysis of sperm chromatin, characterized in that state of chromatin OC is Nivat on the fluorescence intensity of nuclei of cells, painted bromide by ethidium before and after treatment with heparin, and in the presence of semen, in which the intensity of fluorescence increased source more than 30% of the cells, or it increases more than 2 times in more than 50% of the cells after treatment with heparin, diagnose male infertility.
The disadvantage of this method should be recognized that the method is not strictly quantitative, and how and when decondensation heparin, only indirectly reflect anomalies in the packaging of chromatin. In addition, this method requires complex and expensive equipment and cannot be used for widespread screening.
Known (Fernandez J.L. J.Androl. 2003, v.24(1), h/h/59-66) a method for diagnosing anomalies packaging of the chromatin of spermatozoa in male infertility, with the implementation of the method carried out a study of the state of chromatin using a fluorescent microscope and DAPI.
Significant disadvantages of the method should recognize the complexity and long time (about 2 weeks) sample preparation. It is fundamentally important that the method does not allow to quantify accurately the proportion of spermatozoa with damaged DNA compaction and objectively describe the depth of this pathology.
The technical problem to be solved by the developed method consists in establishing the diagnosis and what omali packaging of the chromatin of spermatozoa in male infertility.
The technical result obtained by the implementation of the developed method consists in the simplification and cheapening of the method while increasing the informativeness of the study.
To achieve the technical result of the proposed use of the developed method for the diagnosis of abnormalities of the packaging of the chromatin of spermatozoa in male infertility. According to the developed method to determine the number of spermatozoa in 1 ml, sperm concentration adjusted to 10-50 million/ml with PBS buffer, the cover glass is covered with a layer of polylysine, conduct immobilization of spermatozoa on the prepared cover glass, fix and paint the kernel part of immobilized spermatozoa DNA-binding fluorochrome DAPI obtaining control samples, the second part of immobilized spermatozoa treated Micrococcus a nuclease, spend fixation and staining of the nuclei of spermatozoa treated Micrococcus a nuclease, DNA-binding fluorochrome DAPI stained preparations of control sperm and sperm treated Micrococcus a nuclease, photographed using a fluorescent microscope and analyze the image by comparing fluorescence intensity images of sperm. The decrease in the fluorescence intensity of the poison is R spermatozoa after treatment Micrococcus the nuclease reflects the release of soluble (available for hydrolysis Micrococcus by nuclease) chromatin of the nuclei. Quantitatively, the proportion of soluble chromatin corresponds to the difference between the fluorescence intensity of nuclei in control and after treatment Micrococcus the nuclease and expressed as a percentage of control. The increase in the share of soluble chromatin shows the presence of anomalies in the packaging of chromatin, which are one of the causes of male infertility.
During the implementation of the developed method, you must perform the following procedures.
1. To determine the number of spermatozoa in 1 ml solution. To do this, use a standard camera Goryaeva, which in Hematology used for counting blood corpuscles. Determine the number of sperm in the sample. The sperm concentration was adjusted to 10-50 million/ml with PBS buffer. This procedure is necessary to calculate the concentration micrococcal nucleases. The resulting suspension cannot be stored in a frozen state!
2. To prepare the glass. To do this, cover a total area of 24×24 mm2cut by the cutter into fragments with an area of about 3×5 mm2. To reduce the size of the glasses should be for use in subsequent procedures standard tubes "Eppendorf" with a volume of 1.7 ml Next glass tear solution polylysine (concentration 25 µg/ml) and left for 30 min at room temperature, covered from dust. The volume of solution should be such that pokr is th most of the glass (10-50 μl, depending on the area). After 30 minutes the pipette away the excess solution polylysine with glass back in the tube for later use. The solution polylysine must be stored at a temperature of 20°C. Further, the glass loosely in a closed Petri dish is placed in a thermostat at 37°C until complete dryness polylysine. Glass is recommended to be prepared immediately prior to diagnosis, as during long-term storage, they lose their adhesive properties.
3. To carry out the immobilization of spermatozoa on the prepared glasses. This covered polylysine glass is placed in a plastic Petri dish with a diameter of 4 cm, in a Cup pour 2 ml of sperm suspension, diluted if necessary, PBS. The sperm is then precipitated in a centrifuge at acceleration 670g, 10 minutes At this stage, the drugs should not be dry.
4. To hold the fixation and staining of the nuclei of the sperm DNA-binding fluorochrome DAPI in control. For this glass with immobilized spermatozoa treated with 0.5% solution of Triton X-100 in PBS for 10 min at room temperature and washed in three changes of PBS for 5 min For staining DNA glass is placed in a solution of DAPI (0.1 mg/ml in PBS) for 10 min, washed off the dye two changes of PBS for 5 min After staining glass with spermatozoa conclude on slides in a drop of Moviol with DABCO (volume drops about µl). Promised so drugs are controlled. Prisoners in Moviol drugs can be stored in the refrigerator for 1-2 months.
5. To handle the sperm Micrococcus the nuclease. For this glass with immobilized sperm transfer in tubes with PBS with 0.5 mM PMSF, leave on for 5 minutes and carefully selected solution. The procedure is repeated 2-3 times. The subsequent stage is carried out in ice. In tubes with glass add PBS with 0.5 mM PMSF and 0.5% Triton X100 and incubated for 10 min in ice. After incubation, the glass is washed 2 times with PBS 0.5 mM PMSF, carefully selecting the liquid. Then hold incubation of the slides in the reaction buffer: 10 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 2 mM MgCl2and add micrococcal nuclease 10 IU/mg, 20 IU/mg, 30 IU/mg, 40 u/mg, 80 IU/mg. Incubation carried out for 10 min at room temperature. To stop reaction for samples add 100 mM EDTA to a final concentration of 10 mM and incubated for 30 min in ice. Glass 2-3 times washed with reaction buffer without EDTA.
6. To hold the fixation and staining of the nuclei of spermatozoa treated Micrococcus a nuclease, DNA-binding fluorochrome DAPI. Drugs placed in fixative (3% paraformaldehyde in PBS) for 15 min at room temperature, washed from release two changes of PBS for 5 min For staining DNA glass is placed in a solution of DAPI (0.1 μg/ml in PBS) for 10 min, washed off the dye is two changes of PBS for 5 minutes After staining glass with spermatozoa conclude on slides in a drop of Moviol with DABCO (volume drops about 5 µl).
7. Registration and image processing. Stained preparations of control sperm and sperm treated Micrococcus a nuclease, photographed on a microscope Zeiss Axiovert 200M equipped with a camera Hamamatsu Orca II-ERG2 and connected to the computer. To register and view images using the Zeiss Axio Vision v4.5. A survey carried out on the lens X20 (you can use X40). The obtained images are exported in .tif and analyzed using the software ImageJ. For image analysis, conduct the following:
7.1. The program ImageJ, you must call the Analyze menu->Set measurements and put the marker opposite the Integrated density, removing all other tokens.
Open the image and enlarge it to a convenient scale (~h).
7.2. Using the menu you can make the dull parts of the sperm is clearly visible;
7.3. To allocate sperm (the free select tool, similar to the Lasso tool in Photoshop), to measure the total brightness. Then, without resetting the selection to move it in the blank area (black background) and re-measure the brightness. The total brightness of the sperm and the background will be automatically added to the table. To select spermatozoa with high accuracy is not required
7.4. To copy the received data into the spreadsheet Excel.
7.5. Subtract pairs of the background brightness from the brightness of the object for each measurement.
7.6. The obtained data are expressed graphically.
In the future, the essence of the developed method will be discussed in detail.
The essence of the proposed method in identifying anomalies packaging of the chromatin of sperm on his availability for hydrolysis Micrococcus the nuclease.
The method consists in quantifying the depth of hydrolysis of DNA chromatin Micrococcus the nuclease. After hydrolysis of chromatin produce staining of the nuclei of sperm fluorochrome DAPI. Control is the nature of the hydrolysis of the chromatin of sperm donors and sperm parameters corresponding norm.
The method is as follows.
Fresh sperm cells taken from healthy donors and infertile patients without treatment from related somatic and not differentiated cells, immobilized on the glass pre-coated with polylysine. For immobilization of spermatozoa, you can use a commercial glass coated with a special adhesive for cell structures. Using centrifugation at 650 g treated with 0.5% solution of Triton X-100 in PBS for 10 min at room temperature and washed in three changes of PBS for 5 minutes in each shift. At this point you can about the tis single washing, and instead of PBS, you can use another buffer with the appropriate pH and ionic strength.
Then the glass is incubated in a solution of 20 mm Tris-Hcl (pH 8), 2 mm CaCl2, 2 mm MgCl2. You can use 10 mm Tris-HCl, however, in this case, the concentration of CaCl2and MgCl2should be 1 mm. To the medium was added micrococcal nuclease in the concentration range from 10 IU/mg to 80 u/mg Sample is incubated for 3 minutes at a temperature of not lower than 10°C and above 40°C. the hydrolysis Reaction is stopped by adding EDTA to 10 mm in ice.
Received the drugs are divided into 2 groups: glass with the sperm cells, not treated with nuclease (control), and glass with spermatozoa treated with nuclease. Drugs placed in fixative (3% paraformaldehyde in PBS) for 15 min at room temperature, washed from release two changes of PBS for 5 minutes Instead of paraformaldehyde you can use any release, stabilizing chromatin in the nuclei of the sperm. For staining DNA glass is placed in a solution of DAPI (0.1 μg/ml in PBS) for 10 min, washed off the dye two changes of PBS for 5 minutes Instead of fluorochrome DAPI you can use any DNA-binding fluorochrome. After staining glass with spermatozoa conclude on slides in a drop of Moviol with DABCO (volume drops about 5 µl). You can use other environment conclusions, not about lagausie own fluorescence. The relative amount of DNA in the control and the treated spermatozoa determined by the fluorescence intensity of fluorochrome. For this purpose, the samples are photographed in fluorescent microscope Zeiss Axiovert 200M equipped with a camera Hamamatsu Orca II-ERG2 and connected to the computer. You can use any fluorescence microscope equipped with a camera and connected to the computer. To register and view images using the Zeiss Axio Vision v4.5. A survey carried out on the lens X20 (you can use X40). The obtained images are exported in .tif and analyzed using the software ImageJ. Method tests the differences between the amount of DNA in the sperm cells, not treated Micrococcus the nuclease and DNA content in spermatozoa treated Micrococcus the nuclease. Differences in the content of insoluble nuclease chromatin are expressed in percentage of the control.
The method was tested on samples of healthy sperm donors and sperm samples infertile patients provided by the family planning Center. The method was implemented as described above. Below are the results.
4.1. Examples of healthy donors:
|No. of donors||Insoluble chromatin (%)|
4.2. Examples infertile patients
|No. of patients||Insoluble chromatin (%)|
Therefore, for semen collected from healthy donors, the amount of DNA extracted by micronucleate, from the total amount of DNA does not exceed 3%, and sperm taken from infertile patients, the content of extracted DNA from total DNA, usually increased 10-20 times and in some cases reaches 60% and above.
Received data corraleros with electronmicroscopical studies of the same donors and patients and represent an accurate assessment of the content in the ejaculate infertile patients of abnormal sperm.
1. The way to diagnose anomalies packaging of the chromatin of spermatozoa in male infertility, including the study of the state of chromatin using a fluorescent microscope and a colorant, characterized in that stoopidest the number of spermatozoa in 1 ml of solution, the sperm concentration was adjusted to 10-50 million/ml with PBS buffer, the cover glass is covered with a layer of polylysine, conduct immobilization of spermatozoa on the prepared cover glass with an area of about 3×5 mm2fix and paint the kernel part of immobilized spermatozoa DNA-binding fluorochrome DAPI obtaining control samples, the second part of immobilized spermatozoa treated Micrococcus a nuclease, spend fixation and staining of the nuclei of spermatozoa treated Micrococcus a nuclease, DNA-binding fluorochrome DAPI stained preparations of control sperm and sperm treated Micrococcus a nuclease, photographed using a microscope and analyze the image by comparing the brightness of the images of sperm, and an anomaly packaging of the chromatin of sperm judged by the amount of DNA extracted by the nuclease, relative to the total amount of DNA in the control.
2. The method according to claim 1, characterized in that the determination of the number of spermatozoa is performed using the standard camera Goryaeva, which in Hematology used for counting blood corpuscles.
3. The method according to claim 1, characterized in that the glass cover with a solution of polylysine with a concentration of 25 μg/ml and left for 30 min at room the first temperature, covering of dust.
4. The method according to claim 1, characterized in that the glass is prepared immediately before diagnosis.
5. The method according to claim 1, characterized in that, during the fixation of the nuclei of the sperm DNA-binding fluorochrome DAPI glass with immobilized spermatozoa treated with 0.5%solution of Triton X-100 in PBS for 10 min at room temperature and washed in three changes of PBS for 5 minutes
6. The method according to claim 1, characterized in that for staining DNA glass is placed in a solution of DAPI concentration of 0.1 μg/ml in PBS for 10 min, washed off the dye two changes of PBS for 5 minutes
SUBSTANCE: invention describes a method for prediction of neurological recovery in acute ischemic stroke by clinical and biochemical studies of total albumin concentration (TAC) in blood serum in g/l where in addition on the 5-7th day of the disease, effective albumin concentration (EAC) is evaluated; an albumin binding reserve (ABR) is determined, and if the related value is less than one, a negative effect of neurological recovery in patients in acute ischemic stroke.
EFFECT: invention allows extending the range of methods and providing higher accuracy of prediction of neurological recovery in patients in acute ischemic stroke on the 5-7th day from the onset of the disease that matters for timely selection of an adequate therapy.
SUBSTANCE: on the first day after operation in liquor activity of proteinase of broad substrate specificity - plasmin is determined. If index increases to 0.200 CE/ml development of tumour recurrence within 4-5 months after operation is predicted and if plasmin activity is over 0.200 CE/ml is recurrence-free course of postoperative period during two years is predicted.
EFFECT: method makes it possible to determine at early stages after operation terms of continuous development of malignant brain tumour.
SUBSTANCE: invention represents method of estimating efficiency of neoadjuvant chemical therapy of urinary bladder cancer by patient examination during which performed is registration of maximal intensity of autofluorescence of tumour tissues in green spectrum area at the stage of primary diagnostics and 1 month after carrying out pre-operation chemical therapy, and in case of increase in patient of values of maximal intensity of autofluorescence of tumour tissue by 15% from initial ones and more, treatment efficiency is estimated as partial regression of tumour process, if there are no changes in indices of tumour tissue autofluorescence intensity in comparison with initial ones, stability of process is determined, if indices of tumour tissue autofluorescence intensity reduce by 15% and more in comparison with initial ones, tumour process progressing is determined.
EFFECT: method makes it possible to estimate treatment results with high reliability.
3 ex, 1 tbl
SUBSTANCE: oil-in-water microemulsion for a fluorimetry method of quantitative analysis of flumequine contains in an aqueous phase an analysed solution, an acetate-ammoni buffer solution pH 7.2-8.0, terbium salt Tb(NO3)3·5H2O; a surfactant - sodium tetradecyl sulphate, a cosolvent representing n-pentanol, with 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline, in an oil phase - n-octane. The ingredients of the microemulsion are taken in the following proportions: oil phase - 15-30 wt %; sodium tetradecyl sulphate - 7-8 wt %; cosolvent with 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline - 14-15 wt %; water phase - 46-63 wt %.
EFFECT: invention allows analysing flumequine in muscular tissues, blood serum and foods.
SUBSTANCE: technique is implemented by determining abnormal chromatin packaging by its availability for hydrolysis by micrococcus nuclease. The technique is based on quantitative estimation of an intensity of chromatin hydrolysis by micrococcus nuclease. Chromatin hydrolysis is followed by dividing a hydrolysate material on 2 fractions: soluble chromatin and insoluble chromatin. A check value is a nature of chromatin hydrolysate of donor sperm cells characterised by the regular spermogram values.
EFFECT: improved clinical effectiveness in infertility within extracorporeal fertilisation and homologous insemination programs.
10 cl, 2 ex
SUBSTANCE: optical density of produced dinitrophenylhydrazones at four wave lengths: 356 nm, 370 nm, 430 nm, 530 nm and if protein peroxidation products are detected in saliva, a risk of further allergic diseases is predicted.
EFFECT: higher prediction accuracy.
2 cl, 3 ex
SUBSTANCE: substance of the laboratory diagnostic technique for sepsis consists in a quantitative analysis of blood serum for nonprotein microorganism metabolism products, particularly n-hydroxyphenyllactic (HPLA), phenyllactic (PLA), n-hydroxyphenylacetic (HPAA) and homovanillic (HVA) acids. If the contents of said four acids listed above synchronously excess the a healthy level by 5 times or more, sepsis is diagnosed.
EFFECT: use of said technique allows higher reliability of diagnosing sepsis in the patients with hyperthermia of an uncertain aetiology, in impaired state of the patients in hopital, in the intensive care patients, and in surgical postoperative complications.
4 tbl, 3 ex
SUBSTANCE: initial (3 to 12 day) assessment of clinical symptoms of disease (fever and exanthema) is combined with measuring blood serum C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and specific antibodies (AT) and if observing: - fever response for less than 2 days, exanthema duration for less than 1.5 days, the CRP value less than 7.5 mg/l, ESR less than 15 mm/hour; the IFN-γ level less than 75 pg/ml, circulating immune complex 0.115 optical density units and lower, and specific antibody production more than 8.32 log2, a chronic clinical course of yersiniosis is predicted; - fever response for more than 12 days, exanthema duration for more than 3 days, the CRP value more than 15 mg/l, ESR more than 30 mm/hour; the IFN-γ level more than 150 pg/ml, circulating immune complex 0.130 optical density units and higher, specific antibody production less than 7.32 log2, a severe yersiniosis with associating organ lesions is predicted; - fever response for more than 12 days, exanthema duration for more than 9 days, the CRP value more than 5 mg/l, ESR more than 50 mm/hour; the IFN-γ level less than 150 pg/ml, circulating immune complex 0.250 optical density units and higher, and specific antibody production more than 9.32 log2, clinical outcome of yersiniosis in a systemic disease is predicted.
EFFECT: prevention of a chronic and prolonged clinical course of the disease.
3 tbl, 3 ex
SUBSTANCE: invention relates to medicine, namely to oncology and can be used for predicting efficiency of local chemical therapy of brain in patients with malignant tumours of central nervous system. For this purpose during surgical intervention for ablation of brain tumour from perifocal zone of tumour taken is brain tissue, which is divided into samples, 100 mg each. Number of samples is limited by number of studied chemical preparations. Chemical preparationbs are added to samples in dose 0.2 mg. Incubation at temperature 37°C for 30 minutes is performed. Activity of alpha-2-macroglobulin is determined before and after incubation. If index increases by 40% and more, in comparison with the value before incubation, efficiency of chemical preparations is predicted.
EFFECT: method ensures high specificity, possibility of objective evaluation of efficiency of chemical preparation impact in performing analysis on operation day, which makes it possible to start adequate therapeutic measures, including cancelling or replacement of chemical preparation in each specific case.
SUBSTANCE: diagnosing of early congenital latent syphilis is enabled by determining the values of acute cell-mediated, humoral components of the immune system and phagocytosis. If the CD19 +-lymphocyte percentage values exceed 12.1 as related to the reference group, the absolute and relative HLA-DR-antigen number values: more than 80.6×103/mcl and 1.5 % respectively, than in the reference group, and also a phagocytic neutrophil index is more than 48.2 %, latent ECS is diagnosed.
EFFECT: use of the method faster and more accurate diagnosing of latent ECS.
2 tbl, 2 ex
SUBSTANCE: invention relates to medical equipment, namely to devices for analysis of live organism gases. Spectrometer contains mass-analyser, interface of transcutant gas with input surface, input diaphragm in it and system of electrodes, contact transmission line for switching electrodes to generators of electric signals, canal of gas mixture transportation to mass-analyser. To region between input diaphragm and transcutant gas interface electrodes via gate-regulator of inhaled gas switched is canal of gas mixture transportation, connected with device for collection of exhaled gas. In input diaphragm of transcurant gas interface installed is gas gate-regulator of transcutant gas with mechanism for regulation of incoming gas flow.
EFFECT: application of invention makes it possible to realise fast multifunctional diagnostics of live organism simultaneously in exhaled and transcutant gases in real time mode.
SUBSTANCE: holder includes housing with surface on which there arranged is investigated object and pressure sponge containing a set of parallel located needles. Housing is made so that surface on which the investigated object is located is made in the form of flat base with holes, along the perimetre of which a guide is fixed on the side opposite to investigated object. Inside the guide there arranged is pressure sponge protruding beyond its limits and copying the inner shape of the guide with possibility of coaxial movement of pressure sponge inside guide. At that, pressure sponge is made in the form of upper part connected as one piece to lower part. In upper part there rigidly fixed are needles protruding beyond the limits of upper part towards the housing so that needles can be arranged in holes of the housing. At that, needles are located along the perimetre of upper part of pressure sponge at equal distance between each other, and holes of housing coincide with axis of needles the protruding pointed tips of which have chamfer opposite direction of microtome blade stroke. In lower part of pressure sponge on the side of microtome blade stroke there is longitudinal slot and transverse cavity interconnected with it. Spring-loaded limit stop made in the form of plate having corrugation in upper part of the plate and equal to the length of the guide is installed in longitudinal slot. At that, corrugation is made on the plate side facing inner side of the housing guide with possibility of interaction of corrugation with corrugation made on surface of the guide adjacent to plate of spring-loaded limit stop. Plate has bent end arranged in transverse cavity. Besides, length of upper and lower parts of pressure sponge, which are connected as one piece, is equal to sum of length of the guide at the position of upper part when it is combined with flat base of housing, and protruding beyond the limits of the guide of length of pressure sponge, which is enough to ensure possible arrangement of handles on side surfaces of pressure sponge. Push-button having the possibility of sliding in groove of microtome rod is fixed on the level of arrangement of handles on outer surface of spring-loaded limit stop plate.
EFFECT: lower deformation degree of soft biological tissue during sampling.
SUBSTANCE: automated system comprises a station of seeds loading to separate seeds from multiple seeds and a system of seeds orientation to receive separated seeds from the station of seeds loading and orientation of the specified seeds. The system also comprises a station of sampling for removal of a seed material sample from the specified seeds. The automated system may also be made as comprising a treatment station to remove at least a part of seed shell material and a station of sampling to extract a sample of seed material with stripped shells. At the same time the system comprises a subsystem for collection and transportation of samples to grip an extracted sample into a collecting tube installed on a device for positioning of a collecting tube in the subsystem of collection and transportation of samples, and a subsystem to deposit samples for transportation of a sample from the subsystem of collection and transportation of samples to a selected nest in a tray for samples. The automated method includes a stage of separate seeds separation from the multiple seeds and to align separate seeds in the system of orientation. Afterwards the sample is removed from at least one of the oriented seeds in the sampling station, and separate seeds are transported into nests of the tray for seeds, and then the sample is transported into the nest of the tray for samples.
EFFECT: selected sample is not contaminated, improved validity of measurement results.
33 cl, 19 dwg
SUBSTANCE: sections are prepared by freezing a biopsy material plate in n-hexane cooled to temperature -95°C, and making sections in thickness no more than 7 mcm in a cryostat. The section is placed on a slide, drowned in an alcohol-aldehyde solution at room temperature (20-25°C) for 3 minutes, then washed in flowing water from a fixative for 30 seconds, stained with histological stains and enclosed by a cover glass consists under integumentary glass in the usual way. The preparations produced as described above preserve completely the structure of tissues and separate cells and allow describing lymphatic tumours according to the existing classifications. The method can be used in urgent histological examinations (intraoperative biopsies) as allows drawing up a conclusion decision for 20 minutes.
EFFECT: use of the method allows reducing working hours for the histological examinations required for diagnostics, in comparison with a conventional method, does not deteriorate the performed operations, and accelerates considerably drawing up the conclusion decisions.
3 dwg, 3 ex
SUBSTANCE: ventricular cerebral system is examined in fatal foetuses and fatal newborns of 22-27 weeks of gestation, namely immunohistochemistry of a germinal matrix within a postcornu is carried out by estimating an expression level of S-100 protein shown by stained neuroblast count. If observing 30 % or less of neuroblasts, ventriculomegaly is diagnosed, while stained 50% or more neuroblasts show hydrocephaly.
EFFECT: use of the declared technique enables cellular differentiation of ventriculomegaly and hydrocephaly in fatal foetuses and fatal newborns of 22-27 weeks of gestation.
SUBSTANCE: method of detecting pollution of surface air using artificial dew is carried out by detecting heavy metals in said dew, where the artificial dew is collected by condensing water vapour on a cooled sorption surface, which is extracted by creating a temperature gradient in the air boundary surface, and concentrating the water vapour, and the sorption substrate used is moulded thin-fibre plates with a microporous structure made from hydrophilic material. Cooling with a coolant and subsequent exposure of the moulded plates takes place in flat shallow cuvettes made from chemically inert material (plastic, carbon fibre-reinforced plastic, composite materials), the number and installation of which is determined by observation conditions within the territory.
EFFECT: invention enables more accurate and quality assessment of air pollution on a new technological level.
2 tbl, 1 ex
SUBSTANCE: invention refers to medicine, namely to laboratory investigations. Renal functional reserve is evaluated by sampling and analysing meat-loaded urine. Liquid 2.5-3 l is prescribed with underlying protein-free diet, on the following day, 4-6 hours later, meat-loaded urine is sampled. The sample is applied on a test card of a Lytos-system diagnostic kit, and the structures are evaluated in polarising microscopy ×60 - ×100. If observing oily many-coloured irregular formations, reduced renal functional reserve is stated, and the observed regular salt crystals show physiological renal functional reserve.
EFFECT: method simplifies a procedure of evaluating renal reserve in both patients with kidney diseases, and those suffering latent renal pathology during prophylactic medical examination.
3 dwg, 3 ex
FIELD: machine building.
SUBSTANCE: device consists of reservoir, tank, drain cock, vertical pipes, pump and control unit of system of cocks duplication. The control unit is connected to a driving rocker arm via an axle and to driven rocker arms through rods of cocks duplication system which in turn is connected to duplication cocks set on pipes of product samples intake.
EFFECT: raised operational reliability of sampling system; meeting requirements of specifications.
FIELD: machine building.
SUBSTANCE: plate for prevention of twisting consists of overlapping section which overlaps replaceable blade. Also, the plate consists of the section preventing twisting having a protective section connected with the overlapping section and positioned further in front of the cutting edge of the component of the replaceable blade. Between the overlapping section and preventing twisting section of the plate there is formed a slit for opening the cutting edge of the component of the replaceable blade overlapped with the overlapping section of the blade. Also, the slit passes along lengthwise direction of the overlapping section of the plate and crosses the direction of cutting. The plate for prevention of twisting has such configuration, that a thin cut from a sample is transferred at the preventing twisting section of the plate at transfer of the replaceable blade and a sample relative to each other in the direction of cutting. The component of the replaceable blade is attached to the overlapping section of the plate.
EFFECT: prevention of thin cut twisting, protection of operator from injure during cutting, facilitation of visual inspection of condition of sample cuts in process of cutting.
9 cl, 19 dwg
SUBSTANCE: evaluating a degree of mucosal atrophy of an antral portion is enabled by morphological study of biopsy materials of the antral portion of stomach. A depth of a gland body of the antral portion is measured. A degree of mucosal atrophy of the antral portion is calculated by formula: Y=5.03-0.02X1, where X1 is the depth of the gland body of the antral portion. If observing Y ≤-1.6, a zero degree of mucosal atrophy of the antral portion is stated that is the absence of atrophy. If the Y value falls within -1.5-0.61, degree I is observed that is a slight degree of mucosal atrophy of the antral portion. The Y value being within 0.62 to 1.2 shows degree II that is a moderate degree of mucosal atrophy in the antral portion. If the Y value exceeds 1.2, degree III is detected that is a high degree of mucosal atrophy in the antral portion.
EFFECT: method provides more objective evaluation of a degree of mucosal atrophy of the antral portion ensured by developing estimated values in a numerical equivalent.
5 dwg, 2 ex
SUBSTANCE: method involves application of a diagnostic multiplex panel (DMP) designed for simultaneous identification of a set of possible microorganisms which can be found in a biological sample, with applying primer extension reaction to high conserved nucleotide sequences in sampled microorganisms. The biological sample is immobilised either on, or in a solid substratum in a first position, then transferred to the other position and extracted on the solid substratum so that to extract DNA of any microorganism which is found in the sample. It is followed by amplification of nucleic acid on the extracted DNA of microorganisms, then target sequences are mixed with primers with using the DMP. It results in genotyping of any microorganisms found and identifying of the required microorganisms. For implementation of the method, the diagnostic multiplex panel (DMP) applicable for genotyping of pathogenic microorganisms, and a testing kit for microorganisms is used.
EFFECT: use of the invention allows reliable selection of the biological samples and their testing, providing simultaneous testing of a set of microorganisms.
22 cl, 2 dwg, 3 tbl, 1 ex