EA042351B1 - NUTRIENT MEDIUM FOR CELL PRESERVATION AND TRANSPORT FOR FURTHER CYTOLOGICAL AND IMMUNOCYTOCHEMICAL STUDIES - Google Patents

Description

The invention relates to compositions for the conservation of living cells and is a nutrient medium for the accumulation, preservation, washing and transportation of a sample of cellular material taken from a patient for a period before the subsequent cytological and/or immunocytochemical analysis.

In modern conditions of the development of cytological diagnostics and the introduction of additional clarifying diagnostic methods (immunocytochemistry, PCR), there is a need for storage and transportation of cellular material. In the media currently used, the main preservative solution is derivatives of alcohols (ethyl, isopropyl) or aldehydes (formaldehyde), which by their nature cause protein denaturation, leading to morphological changes (wrinkling) and disruption of the antigenic composition of cells. This fact prompted the creation of a nutrient medium, where the preservative is sodium azide, the effect of which is to disrupt cellular respiration (inhibition of phosphorylating oxidation, suppressing the electron transfer associated with phosphorylation in intact mitochondria), without violating the antigenic composition of cells, while suppressing the growth and reproduction of microorganisms .

Known nutrient medium protected by RF patent No. 2435842, C12N 1/20, C12R 1/32, publ. 12/10/2011, containing yeast water, beef meat hydrolyzate, sodium chloride, glucose, glycerin, sodium citrate, sodium metabisulphite and distilled water. EFFECT: invention makes it possible to provide optimal conditions for the growth and reproduction of the brucellosis microbe when transporting the nutrient medium over any distance.

The known solution has disadvantages: it is not possible to analyze samples of tumor tissue, blood, lymph nodes, mucous membranes, etc.; beef hydrolyzate is not effective in improving cytology.

Also known environment for establishing the presence and (or) type of microorganism; use of selective media for testing antibiotics or bactericides; compositions containing a chemical indicator for these purposes, described in the patent of the Russian Federation No. 2529711 C12Q 1/04, C12N 1/20, publ. 09/27/2014

According to a known method, the sensitivity of bacteria that cause intestinal infections to complex antibacterial drugs is determined. Antibacterial drugs are diluted at a concentration of 10 μg/ml in a nutrient medium with a pH of 7.2-7.6. The prepared suspension of bacteria is added to the wells containing the prepared dilutions. An indicator is introduced - a 0.5% solution of bromcresol purple in an amount of 10 μl. Incubate for 3-4 hours. Conduct a visual assessment of bacterial growth and assessment of the color of the medium in the well when the pH reaches 5.2-6.8 of the medium. A conclusion is made about the sensitivity of bacteria to complex antibacterial drugs by the presence or absence of bacterial growth and a change in the color of the medium from red to yellow. Bacteria are considered sensitive if they do not grow and change the color of the nutrient medium with antibacterial drugs. Bacteria that grow and change the color of the medium with antibacterial drugs are considered resistant.

The disadvantages of the known environment are the inability to analyze samples of tumor tissue, blood, lymph nodes, mucosa, etc., and the fact that the antibacterial components that make up the environment do not have a positive effect on the performance of cytological studies.

Closest to the claimed invention, selected as a prototype, is a nutrient medium for the accumulation of a cell sample for subsequent cytological and/or immunocytochemical analysis, protected by RF patent No. 2246110, G01N 33/48, G01N 33/53, A01N 1/02, publ. 02/10/2005 The medium is a Hanks solution containing salts NaCl, HCl, CaCl ₂ anhydrous, MgSO ₄ -6H ₂ O, MgCl ₂ -6H ₂ O, Na ₂ HPO ₄ -2H ₂ O, KH ₂ PO ₄ , NaHCO ₃ and glucose and additionally includes 10% albumin solution and polyglucin (dextran 60000) in the following ratio: 10% albumin solution, Hank's solution, polyglucin = 1:1:1.

A disadvantage of the known environment is the insufficient period of storage and transportation of a sample of cellular material (up to 48 hours), as well as the need to store it under freezing conditions and the availability of appropriate low-temperature equipment.

The technical problem solved by the present invention is the creation of a nutrient medium that contributes to the preservation of the morphological properties of the cellular composition, with simplified storage conditions and long periods of storage and transportation.

The technical result from the use of the invention is to increase the duration of storage and transportation of a sample of cellular material and to simplify the storage conditions of the medium.

This technical result is achieved by the fact that the nutrient medium for transporting cells for further cytological and immunocytochemical studies, containing a sterile 10% solution of human albumin, additionally includes a solution of the medium molecular fraction of partially hydrolyzed dextran in isotonic sodium chloride solution and sodium azide solution in the following ratio, wt .%:

sterile 10% human albumin solution - 33.0-33.3;

solution of the medium molecular fraction of partially hydrolyzed dextran - 33.0-33.3;

sodium chloride - 33.32-33.94;

- 1 042351 solution of sodium azide - 0.06-0.08.

The claimed composition of the nutrient medium for the accumulation of a cell sample is optimal in terms of obtaining the best results of cytological and immunocytochemical (ICC) studies.

Albumin contributes to the creation and maintenance of the studied cell sample in a state of suspension - a colloidal state close to natural. A smaller amount of albumin leads to a decrease in the size and distortion of the shape of the cells placed there, and a larger amount leads to swelling of the cells, up to their lysis.

A solution of the medium molecular fraction of partially hydrolyzed dextran in an isotonic sodium chloride solution provides the necessary nutrition and maintenance of the viability of living cells in a tissue sample taken from a patient. With a smaller amount of it, swelling and lysis of cells occur, with a larger amount - distortion of cells in the form of wrinkling.

Sodium azide (NaN ₃ ) is a colorless salt of hydronitrous acid. In a solution of sodium azide, almost all biological objects retain their original color, shape and size. The range of working concentrations of sodium azide varies from 0.5 to 0.05%. At high concentrations, there is evidence of effective preservation of biological tissue samples. However, at a concentration of 0.5%, degradation of biological material at the molecular level is observed. Studies were conducted on the effect of different concentrations of sodium azide on the reaction with antigen (RA), the presence of concomitant microflora (MF), the state of the membrane and cell organelles (https://monographies.ru/en/book/section?id=4951).

The data is summarized in a table.

Concentration Cross-sectional study After 1 week In 2 weeks 0.5% RA in the positive control is negative. MF: not available. MO: destruction. RA in the positive control is negative. MF: not available. MO: destruction. RA in the positive control is negative. MF: not available. MO: destruction. 0.4% RA in the positive control is negative. MF: not available. MO: destruction. RA in the positive control is negative. MF: not available. MO: destruction. RA in the positive control is negative. MF: not available. MO: destruction. 0.3% RA in the positive control is negative. MF: not available. MO: destruction. RA in the positive control is negative. MF: not available. MO: destruction. RA in the positive control is negative. MF: not available. MO: destruction. 0.2% RA in the positive control is negative. MF: not available. MO: weak destruction. organelles are visible. RA in the positive control is negative. MF: not available. MO: weak destruction. organelles are visible. RA in the positive control is negative. MF: not available. MO: weak destruction. organelles are visible. 0.1% RA in the positive control is negative. MF: not available. MO: weak destruction. organelles are visible. RA in the positive control is negative. MF: not available. MO: weak destruction. organelles are visible. RA in the positive control is negative. MF: not available. MO: weak destruction. organelles are visible. 0.08% RA in the positive control is weakly positive. MF: not available. MO: weak destruction. organelles are visible. RA in the positive control is weakly positive. MF: not available. MO: weak destruction. organelles are visible. RA in the positive control is weakly positive. MF: not available. MO: weak destruction. organelles are visible. 0.06% RA in the positive control is positive. MF: not available. MO: destruction absent. organelles are visible. RA in the positive control is positive. MF: not available. MO: destruction absent. organelles are visible. RA in the positive control is positive. MF: not available. MO: destruction absent. organelles are visible.

The results obtained indicate that at a sodium azide concentration of 0.06-0.08%, no changes in the structure of the membrane and cell organelles were observed, no signs of the presence of

- 2 042351 microflora, and the negative effect of sodium azide on the ability of antibodies to interact specifically with the antigen under study was not manifested.

All used components of the claimed environment are produced by the domestic industry:

sterile 10% solution of human albumin - GUS Nizhny Novgorod Regional Blood Transfusion Station. N.Ya. Klimov;

solution of the medium molecular fraction of partially hydrolyzed dextran - Biosintez LLC;

sodium chloride - Biosynthesis LLC;

sodium azide solution - Bioline Ltd.

To prepare the claimed composition, the above components are mixed in the specified proportion. Store the culture medium in refrigerators at 4-8°C.

Use the nutrient medium as follows. A sample of the studied tissues is taken, for example, thyroid punctate, the cap of the tube is pierced after taking the material, the needle is washed with a nutrient medium by sucking the nutrient medium into the syringe, and all the material will be in the test tube.

Transportation and storage of a living tissue sample placed in the inventive nutrient medium can be carried out at ambient temperature for up to 10 days.

The claimed medium can be used to analyze samples of tumor tissue, blood, lymph nodes, mucosa, etc.

An example of using a nutrient medium for immunocytochemical analysis: a cell pellet is introduced into a test tube with a nutrient medium by piercing the lid and placing the material (cells taken from the patient) inside the test tube. Then the tube is centrifuged until a precipitate is formed, opened no later than 10 days later (depending on the needs of the specialist).

The prepared cell sediment is placed on a glass slide and dried at room temperature.

50 μl of a 3% hydrogen peroxide solution are applied to the cell sediment on glass (hereinafter referred to as the sample) for 10 minutes. Wash the preparations in two portions of the buffer solution, 2 min each.

IMPORTANT! From this stage, the preparation must not be allowed to dry until the reaction is complete. If necessary, staying in the buffer may be longer than the specified time.

50 µl of the protein block is applied to the cell sample for 10 min.

Wash the preparations in two portions of the buffer solution, 2 min each.

30 μl of primary antibodies are applied to the sample. Incubation time 30 min at room temperature.

Wash the preparations in two portions of the buffer solution, 2 min each.

30 μl of secondary labeled antibodies are applied to the sample. Incubation time 20 min at room temperature.

Wash the preparations in two portions of the buffer solution, 2 min each.

Prepare a working solution of diaminobenzidine (DAB) by dissolving it with a working solvent solution in a ratio of 1:1.

30 μl of DAB solution is applied to the sample for 3-5 minutes.

The preparations are washed in three portions of water, 2 minutes in two and 10 minutes in the third.

30 µl of Mayer's hematoxylin solution is applied to the sample for 1 minute.

Wash the preparations in water, dry and microscopically.

The invention is illustrated by clinical examples and illustrations, which show: in Fig. 1 - cytological preparation immediately after sampling (for example 1);

in fig. 2 - cytological preparation after 10 days in the claimed nutrient medium (for example 1);

in fig. 3 - immunocytochemical study of the material after storage in the claimed nutrient medium (antibody NCAM (CD 56)) (for example 1);

in fig. 4 - cytological preparation immediately after sampling (for example 2);

in fig. 5 - cytological preparation after 10 days in the claimed nutrient medium (for example 2);

in fig. 6 - immunocytochemical study of the material after storage in the claimed nutrient medium (antibody NCAM (CD 56)) (for example 2).

Example 1

Patient X, 48 years old, with a diagnosis of volumetric formation of the upper lobe of the left lung, was treated in the diagnostic department of the Regional Tuberculosis Dispensary. During an urgent intraoperative cytological examination (09/26/18, 11:30 a.m.), a conclusion was issued: a cytological picture of a malignant neoplasm. Poorly differentiated adenocarcinoma with elements of small cell differentiation. To clarify the histological type of the tumor, a scraping with a sterile scalpel was taken from the incision of the removed tumor (September 26, 2018, 12:00) using a sterile scalpel and placed in a nutrient medium containing a sterile 10% solution of human albumin 33.3 wt.%, a solution of the medium molecular fraction partially hydrolyzed dextran 33.3 wt.% in isotonic sodium chloride solution 33.32 wt.% and sodium azide solution 0.08 wt.%. Mate

- 3 042351 rial was sent for immunocytochemical study and delivered to the PJSC Regional Clinical Hospital on September 27, 2019 at 10:00 (22 hours after collection) with intermediate storage at a temperature of +4°C. The immunocytochemical study was carried out on September 28, 2018 at 13:00 (49 hours after the material was taken). The study was carried out according to the methodology described above. The result of the study confirmed the diagnosis: Small cell carcinoma of the upper lobe of the left lung. Thus, it has been established that storage of the test preparation containing tumor cells in the claimed nutrient medium for 49 hours does not distort the immune and morphological qualities of the cells and does not affect the results of immunocytochemical analysis.

Example 2

The patient is 58 years old. With a diagnosis of Ascites, she was taken to the emergency department of a surgical hospital for the purpose of laparocentesis. During the manipulation, 1.5 l of liquid was removed, the material was sent for cytological examination on May 13, 2018, 15:00. The study issued a cytological conclusion: Cytological picture of a specific exudate. Adenocarcinoma. To determine the primary localization of the tumor, the remaining cell sediment was placed in a nutrient medium containing a sterile 10% solution of human albumin 33.0 wt.% solution of the average molecular fraction of partially hydrolyzed dextran 33.0 wt.% in isotonic sodium chloride solution 33.94 wt.% and sodium azide solution 0.06 wt.% on May 13, 2018 at 16:30 and left at a temperature of +4°C for the purpose of immunocytochemical study. On May 24, 2018, at 8:00 (232 hours after sampling), the material was sent to the PJSC of the regional hospital for further research. Immunocytochemical study was carried out on May 24, 208 at 17:30 (233.5 hours after the sampling). The study was carried out according to the methodology described above. The result of the study confirmed the diagnosis: Serous adenocarcinoma of the ovary. Thus, it has been established that the storage and transportation of the test preparation containing tumor cells in the claimed nutrient medium for 233.5 hours does not distort the immune and morphological qualities of the cells and does not affect the results of immunocytochemical analysis.

Thus, the proposed nutrient medium in comparison with the prototype has the following advantages:

longer shelf life with the material (up to 233.5 hours), which creates conditions for transporting the biomaterial to the laboratory from the sampling site (if necessary, transporting from remote areas to central laboratories);

an easier way to store (no need for freezing storage and the availability of appropriate low-temperature equipment).

In addition, the use of sodium azide as a preservative does not change the organoleptic properties of the nutrient medium (no smell) and the nutrient medium contributes to the preservation of the morphological properties of the cellular composition similar to those at the time of sampling.

Claims (1)

Nutrient medium for transporting cells for further cytological and immunocytochemical studies, containing a sterile 10% solution of human albumin, characterized in that it additionally includes a solution of the medium molecular fraction of partially hydrolyzed dextran in an isotonic sodium chloride solution and a solution of sodium azide in the following ratio of components, wt.%: sterile 10% human albumin solution - 33.0-33.3; solution of the medium molecular fraction of partially hydrolyzed dextran - 33.0-33.3; sodium chloride - 33.32-33.94; sodium azide solution - 0.06-0.08.