Nutrient medium for accumulation of cell sample for following cytological and/or immunocytochemical analysis

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.

3 ex


The invention relates to compositions for preservation of living cells and is a medium for the accumulation, preservation and washing of the sample cell material withdrawn from the patient, for the period until the subsequent cytological and/or immunocytochemical analysis. The inventive composition can also be used for the cultivation of living cells.

A known solution for the preservation of cells, representing water spirit-buffer solution, designed to preserve the sample in vitro morphology of the nuclei of mammalian cells, including miscible with water, alcohol, calcium ions and magnesium and a buffer agent (EP 0772972 A1, 14.05.97). Well-known solution has disadvantages inherent as a buffer, and alcohol environments. Namely:

partial dehydration of the investigated cells due to the presence in the composition of the alcohols. Dehydration of the cytoplasm leads to disruption of intracellular biochemical processes, in particular to the shrinkage of the cytoplasmic membrane and, consequently, to changes in the morphology of many cells, especially malignant, to accelerate their death and, ultimately, to the distortion of the results of the subsequent analysis;

- buffer systems are a good preservative, but is not suitable for the preservation of cellular material for more than ten to twelve hours of sredstv the e absence in the composition of the power source cells: proteins and carbohydrates.

Also known physiological environment for washing, preservation and storage of cells, tissues and organs containing salt components, buffer components, components of the substrate, amino acids, components of the ku-enzymes, vitamin components, the components of proteins (EP 1164841 A1, 02.01.2002). A disadvantage of the known environment that impede the achievement of the following technical results is the presence of large amounts of foreign proteins, particularly protein components of serum, the presence of which in the sample under investigation can lead to a distortion of the thin immune responses for subsequent immunohistochemical analysis.

Closest to the claimed invention is a salt Hanks solution, designed for canning cell sample in the interval between sampling and analysis or fixation (Dawson R., Elliot D., Elliot U., Jones, K., Handbook biochemist, M.: Mir 1991). Adopted for the prototype. A known solution is a buffer system comprising a mixture of water-soluble inorganic salts (g/100 ml), namely: NaCl - 0,80, KCl - 0,04, l2anhydrous - 0,014, MgSO4·6H2O - 0,01, MgCl2·6H2O - 0,01, Na2HPO4·2H2O - 0,006 KN2RHO4- 0,006, Panso3- 0.035 and glucose, and 0.1. However, after the content in solution-FR is the type more than twenty minutes to change the morphology of the investigated cells, which completely distorts objective picture subsequent cytological analysis. This is due to the insufficient energy of the material to maintain the viability of the cells.

The claimed invention is directed to solving the problem of creating an environment for the accumulation, preservation and washing of the sample of cells taken from a patient for subsequent cytological and/or immunocytochemical analysis as close as possible to the natural physiological conditions of their existence.

Use in laboratory practice, the proposed solution can achieve several technical results:

- the ability to store and transport specimen of cellular material for up to 48 hours.

- preservation of morphological, biological and biochemical characteristics of the studied cells, in particular their immune specificity;

- low cost;

- long shelf-nutrient medium for subsequent use;

- can be used for the subsequent implementation of the various techniques of analytical studies, including centrifugation.

These technical results in the implementation of the claimed invention are achieved due to the fact that the nutrient medium accumulation of sample cells for subsequent ecologicheskogo and/or immunocytochemical analysis also known as Hanks solution, contains salts NaCl, KCl, l2anhydrous, MgSO4·6H2O MgCl2·6H2O, Na2HPO4·2H2O KH2PO4, NaHCO3and glucose. The peculiarity of the proposed environment is that it additionally includes a 10% solution of albumin and poliglyukin (dextran 60000) in the following ratio: 10% albumin : Hanks solution : poliglyukin = 1:1:1.

The essence of the invention.

Among the diverse types of analyses of the qualitative and quantitative composition of biological material especially careful attitude to security require tissue samples, which presumably are present in tumor cells. Cells from malignant tumors are characterized by dystrophic changes compared with normal (healthy), which leads to the acceleration of their death in the period from the withdrawal of a sample of cellular material from the patient to the stage of its analysis. This circumstance leads to increased requirements for the composition environment for storage, transportation and preparation of the sample of cellular material for cytological and/or immunocytochemical analysis, especially in the presence of the sample, with a high probability of tumor cells.

Also relevant is the problem of preservation and transportation of the sample cell material withdrawn from the patient, the venue complex cytological and/or immunocytochemical analysis, requiring the availability of appropriate laboratory equipment and qualified personnel researchers.

At the present time the most efficient way of preparing a tissue sample for conducting a complex cytological and/or immunocytochemical analysis is centrifugation, i.e., the separation into fractions of cellular elements in size with the aim of obtaining a multilayer drugs. The advantage of this method is that in the process of centrifugation is the laundering - remove background elements: mucus, bacteria, decay products. At the same time in the implementation phase centrifugation living cells experience intense mechanical stress, which can lead to distortion of their morphology. In this regard, is of great importance to the composition of the medium, in which is placed a sample of living tissue. On the one hand, the investigated cells must retain their morphology. biological and biochemical characteristics. in particular, their immune specificity, and on the other hand, the environment for the accumulation of cells should not prevent the implementation of the process of centrifugation.

The inventive composition of the nutrient medium, accumulating sample of cells is optimal and meets all specified requirements.

Quantitative and qualitative composition of inorganic ions, Pris is concerned in Hanks solution, is adequately required for normal metabolism of cells, are able to maintain the osmotic pressure within physiological norms and optimal pH level.

Poliglyukin representing plazmozamenjajushchih solution includes a salt component (NaCl) and a balanced complex carbohydrates, thus provides the necessary power and maintaining the viability of living cells taken from the patient tissue sample.

In most cases, nutrient solutions, designed to maintain the viability of tissues in vitro and contains protein components of serum that can distort the thin immune response. At the same time used protein - albumin - does not have this shortcoming and its presence does not distort cytochemical properties of the investigated cells. Albumin contributes to the creation and maintenance of a given cell sample in suspension - colloidal, state, close to the natural.

All used components are produced by the domestic industry, which leads to low cost of the proposed structure, and ensures compliance with the criterion of industrial applicability of the claimed invention.


- Hanks solution (Soluto Hanksi) - RAMS Epps for the production of bacterial and viral drugs IPV is them. M.P. Chumakov;

- polyglucin - JSC “trasforma”, , Krasnoyarsk;

- albumin - Nizhny Novgorod regional station of blood transfusion them. Nassimov.

For preparation of the inventive composition of the above components are mixed in specified proportions.

The process of collecting, washing and preservation of the sample cell material withdrawn from the patient, for the period until the subsequent analysis is as follows. Make the selection of the sample fabrics, such as fine-needle biopsy of the tumor or stroke mucosa special designed for this purpose medical instrument. Next, put the contents of the syringe or other instrument for sampling in a special microprobing with the prepared environment of accumulation, produce at least two washing syringe specified environment to complete cleanup tool from particles of the investigated tissue (volume of sample). This punctures or other procedure sampling can be repeated, if appropriate for qualitative performance analysis. Prepared environment accumulation may be stored in a refrigerator at a temperature of -4° C. Transportation and storage of a sample of living tissue, placed in the inventive culture medium of savings can be made is at ambient temperature for up to two days.

Then, the sample is placed into a centrifuge, where the stage laundering - remove background elements: mucus, bacteria, decay products and the separation into fractions of cellular elements in size with the aim of obtaining monolayer preparations. After centrifugation, the sample is ready for carrying out morphological, cytological and/or immunocytochemical analysis.

The inventive medium can be used to analyze samples of tumor tissue, blood, lymph nodes, mucosa and other

Thus, the claimed nutrient medium for the accumulation of cells has significant advantages compared with the known compounds of the same purpose and meets the criteria of patentability.

For the preparation of a portion of the claimed nutrient medium with a volume of 90 ml solutions Hanks, polyglucin and albumin in an equal volume of 30 ml was placed in a flask with a volume of 100 ml and stirred at room temperature for 1-2 minutes Prepared environment is divided into portions in accordance with the amount needed for a single analytical research.

If the portion of the composition is intended for long-term storage, it is placed in the freezer and stored at a temperature of 5-10° C. In the case, if the portion of the composition before oznacena for current analyses in the time period up to 10 days, it is placed in a refrigerator and stored at a temperature of +5-10° C. a Nutrient medium stored in the freezer, defrost before conducting the required tests at room temperature.

Examples of the nutrient medium.

1. Patient M 61 years, turned to the clinic of Moscow them. Apericena 19.05.02 with complaints about the seal in the right breast. After the ultrasound examination and cytological study punctate tumor, was the preliminary diagnosis: breast cancer with metastases in the axillary lymph nodes. It was decided to hold chemohormonal preoperative treatment. To determine the hormonal status of the tumour was appointed immunocytochemical study, and therefore the patient M 26.05.02 at 11.00 was made to puncture a tumor of the right breast and lymph nodes under ultrasound control. The resulting material is punctate was immediately placed in a nutrient medium accumulation, prepared in advance (04.04.02) in the Department of ecolology of Moscow them. PageRank and taken to the hospital, where she was kept at a temperature of -10° C. the Medium was thawed before puncture at room temperature. The resulting material (nutrient medium with tumor cells) for immunocytochemical analysis was delivered to the Department of ecolology Tits And them. PageRank 27.05.02 at 14.00 (after 27 hours after removal of material-punctate). All this time he was kept at room temperature (+18 to 20°). After centrifugation apparatus Cytospin-3 (data processing system of cells and preparation then get monolayer of tumor cells on glass was carried out immunocytochemical study 27.05.02 in C (28 hours). The following results were obtained: the expression of estrogen receptor and progesterone in tumor cells positive. Morphological and immunological properties of tumor cells of breast cancer after finding in nutrient accumulation within 28 hours have not changed. Nutrient medium accumulation does not invalidate the hormonal status of tumor cells of the breast.

2. Patient S., 53 years old, came to the clinic of Moscow them. Apericena 26.08.02 with complaints about not heal bleeding upon contact with clothing education in the field of the left forearm. After clinical examination and cytological study of scraping wounds was the preliminary diagnosis: undifferentiated carcinoma of the skin? Melanoma?

For an accurate diagnosis was assigned to conduct immunohistochemical study, therefore, 27.08.02 at 10.00 hours in the clinic of Moscow them. Apericena narrow sterile spatula was taken by scraping with isyas is certain the wound and immediately placed in a nutrient medium accumulation, thawed in advance at room temperature. Nutrient medium was prepared in the Department of ecolology Institute 15.06.02 and sent to the clinic for subsequent use. The environment was kept in the freezer of the refrigerator at -10° C. the Material was delivered to the Institute 28.08.02 at 15.00 (over 29 hours). Immunocytochemical study conducted 29.08.02 at 10.00 hours (48 hours after placing the material in the nutrient medium). All this time the environment with tumor cells patients were kept in the refrigerator at +8° C. Immunocytochemical study in accordance with the procedure described in example 1 confirmed the diagnosis of malignant melanoma of the skin. It was thus established that the storage of the investigational product containing tumor cells in a nutrient medium accumulation within 48 hours, does not distort the immune and morphological properties of the cells and does not affect the results of immunocytochemical analysis.

3. Patient K., 42 years old, came of Moscow in them. Apericena 10.10.02 for examination. When conducting an ultrasound examination of the thyroid gland was found a small education in the right lobe. After puncture of education and cytological studies punctate was the preliminary diagnosis: medullary carcinoma? Papillary thyroid cancer? U is Oceania diagnosis was assigned to conduct immunocytochemical analysis, what a sick 11.10.02 at 10.00 was produced by puncture of the right lobe of the thyroid gland under ultrasound control.

The resulting material is punctate was immediately placed in a nutrient medium accumulation. Wednesday was cooked the day before (10.10.02), in the Department of ecolology and stored in the refrigerator at +5 - 8° C. a Suspension of cells in a nutrient medium was subjected to centrifugation on a system of Cytospin-3 to obtain monolayer preparations. Conducted 11.10.02 at 12.00 Immunocytochemical study confirmed the diagnosis of medullary thyroid cancer. Being within 2 hours in a nutrient medium accumulation, cells preserved their immune and morphological properties.

Examples can illustrate the above description of the invention the advantages of the inventive composition, nutrient accumulation, namely:

- the ability to store and transport specimen of cellular material for up to 48 hours.

- preservation of morphological, biological and biochemical characteristics of the studied cells, in particular their immune specificity;

- long shelf-nutrient medium for subsequent use;

- can be used for the subsequent implementation of the various analytical studies: cytological analysis of cells, immunocytokine the definition of research study the morphometry of tumor cells in monolayer smears;

- can be used to analyze samples of tumor tissue of various organs, blood, lymph and lymph nodes, mucous and other

Nutrient medium accumulation of sample cells for subsequent cytological and/or immunocytochemical analysis, representing the Hanks solution containing salts NaCl, KCl, CaCl2anhydrous, MgSO4·6H2O MgCl2·6H2O, Na2HPO4·2H2O KH2PO4, NaHCO3and glucose, characterized in that it additionally includes a 10%solution of albumin and poliglyukin (dextran 60000), with a ratio of 10%solution of albumin: Hanks solution: poliglyukin = 1:1:1.


Same patents:

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, 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.

2 ex

The invention relates to immunodiagnostics and can be used for the detection of recurrence and metastases to their clinical manifestations in patients with lung cancer and determine the timing and type of anticancer therapy after surgical removal of the lesion

The invention relates to medicine, in particular to obstetrics

The invention relates to the field of veterinary medicine and can be used for diagnosis of the causative agent of brucellosis in an L-shape

The invention relates to medicine, namely to laboratory diagnosis

The invention relates to medicine, namely to immunology, Allergology, pulmonology, dermatology, infectious Microbiology, and veterinary

The invention relates to an immunochemical methods of analysis of the sera of humans and animals and can be used for simultaneous detection in the samples of specific antibodies (ATA) to a number of markers of infectious, parasitic, allergic and somatic diseases in medicine, veterinary medicine, biotechnology, environmental studies and scientific experiments

The invention relates to the field of biology and medicine and can be used for the diagnosis of prostate cancer, monitoring the effectiveness of the treatment of prostate tumors, in screening programs to identify risk, forecast of development of tumor and detection of recurrence

The invention relates to medicine, namely to Hematology and Oncology

FIELD: medicine, urology.

SUBSTANCE: the present innovation deals with differential diagnostics of prostatic cancer and other prostatic diseases at the stage of primary inspection. The method includes the detection of PCA and calculation of probability coefficient for prostatic cancer (PCC) by the following formula: where e - the foundation of natural logarithm (e=2.718…), PCA - the level of total blood PCA in ng/ml, V - patient's age in years. At PCC value being above 0.2 one should diagnose prostatic cancer and to establish final diagnosis one should perform polyfocal prostatic biopsy. The method enables to increase accuracy of diagnostics at decreased number of unjustified prostatic biopsies.

EFFECT: higher efficiency of diagnostics.

2 ex

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: 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.

SUBSTANCE: method involves carrying out microscopic examination of blood serum samples taken from femoral vein and cubital vein. Femoral vein sample is taken on injured side. The examination is carried out before and after treatment. The blood serum samples are placed on fat-free glass slide in the amount of 0.01-0.02 ml as drops, dried at 18-30°C for 18-24 h. The set of pathological symptoms becoming larger or not changed after the treatment in comparison to sample taken before treatment, and morphological picture of samples under comparison taken from the cubital vein showing no changes or being changed to worse, the treatment is considered to be effective.

EFFECT: enabled medicamentous treatment evaluation in course of treatment to allow the treatment mode to be changed in due time; avoided surgical intervention (amputation); retained active life-style of aged patients.

4 dwg

FIELD: medicine, biotechnology, pharmacy.

SUBSTANCE: invention relates to agents used for treatment of pathological states associated with disorder of synthesis of neuromediating substances. Method involves the development of pharmaceutical composition and a method for it preparing. Pharmaceutical composition represents subcellular synaptosomal fractions: synaptic membranes, "light" synaptosomes and "heavy" synaptosomes prepared from gray matter of cerebral hemispheres from experimental animals based on the goal-seeking modification of humoral mediators of nerve endings transformed to synaptosomes in development and regression of malignant processes. The composition provides inhibiting the growth of tumor cells, to elevate span-life of patients with ascite Ehrlich's sarcoma, breast adenocarcinoma Ca-755, Wolker's carcinosarcoma-256.

EFFECT: valuable medicinal and anti-tumor properties of composition.

12 cl, 3 tbl, 3 ex

FIELD: medicine, phthisiology, microbiology.

SUBSTANCE: diagnostic material is poured preliminary with chlorohexidine bigluconium solution, homogenized, kept at room temperature for 10-12 h and centrifuged. Precipitate is poured with Shkolnikova's liquid medium, incubated at 37oC for 3 days, supernatant part of Shkolnokova's medium is removed, fresh Shkolnikova's medium is added, and precipitate is stirred and inoculated on the dense cellular egg media. Sensitivity of the strain is determined in 3 weeks by the presence of growth in the control tube only. Invention provides enhancing precision and reducing time for assay. Invention can be used in assay for medicinal sensitivity of tuberculosis mycobacterium.

EFFECT: improved assay method.

3 ex

FIELD: molecular biology.

SUBSTANCE: the suggested innovation deals with the fact that nucleic acids should be isolated directly out of the sample without pipetting stage but with the help of interconnected reservoirs being prepared beforehand. The above-mentioned vessels should be applied either separately or being interconnected according to standard microtitrating format. The sample should be mixed with a lyzing buffer and nucleic acids are bound with matrix in closed system including, at least, two interconnected reservoirs. Forced movement of sample's mixture and buffer back and forth from one reservoir into another one for several times through narrow passage provides their thorough intermixing. The method provides quick and safe isolation of nucleic acids.

EFFECT: higher efficiency.

44 cl, 4 dwg, 1 ex

FIELD: medicine, urology.

SUBSTANCE: one should conduct subcutaneous prevocational tuberculin test and, additionally, both before the test and 48 h later it is necessary to perform the mapping of prostatic vessels and at decreased values of hemodynamics one should diagnose tuberculosis. The information obtained should be documented due to printing dopplerograms.

EFFECT: more reliable and objective information.

1 ex, 1 tbl

FIELD: medicine, psychiatry.

SUBSTANCE: one should isolate DNA out of lymphocytes of peripheral venous blood, then due to the method of polymerase chain reaction of DNA synthesis one should amplify the fragments of hSERT locus of serotonin carrier gene and at detecting genotype 12/10 one should predict the risk for the development of hallucino-delirious forms of psychoses of cerebro-atherosclerotic genesis.

EFFECT: more objective prediction of disease development.

3 ex

The invention relates to medicine, namely to laboratory methods of skin diseases and can be used to diagnose common progressive psoriasis

FIELD: medicine.

SUBSTANCE: method involves using product containing spermatozoa treated by drying with freezing to humidity level of 1% and having injured membrane or spermatozoon head so that spermatozoon nucleus retains its genetic validity enough for fertilization. The spermatozoon heads fertilize an isolated oocyte after rehydration and microinjection being done. The retained genetic integrity is enough for fertilizing an oocyte and producing living descendants. Method involves collecting living mature spermatozoa, making spermatozoa suspension in special purpose physiological medium, freezing the spermatozoa suspension for producing frozen spermatozoa, drying the frozen spermatozoa or spermatozoa heads in vacuum to humidity level of 1%, making rehydration of spermatozoa or spermatozoa heads with injured membrane and selecting those retaining nucleus of genetic integrity. The selected spermatozoa or spermatozoa heads are used for fertilizing isolated oocytes with living descendants being produced.

EFFECT: enhanced effectiveness in producing living descendants.

39 cl, 5 dwg, 1 tbl