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preservation of living cells and is a medium for the accumulation, preservation and washing of the sample cell material withdrawn from the patient, for the period until the subsequent cytological and/or immunocytochemical analysis.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.

Manufacturers:

- 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.



 

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