RU2706220C1 - Method of producing cytogenetic epithelial cell preparations for carrying out a fluorescent in situ hybridisation reaction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology, and can be used for producing cytogenetic preparations of epithelial cells for carrying out a fluorescent in situ hybridisation (FISH) reaction. That is ensured by epithelial cells slipped off by the cytobrush. They are centrifuged with phosphate-salt buffer for 10 minutes. Then supernatant is removed. A fixing solution consisting of a mixture of methanol and acetic acid in ratio 3:1 is added to the precipitate. Cell suspension is stored at -20 °C. Cytogenetic preparations are prepared by applying cells on zoned slides while monitoring their density. To increase brightness of fluorescent signals, cells are treated with 2 % Carbowax solution for 2–20 hours at 4 °C.

EFFECT: invention prolongs storage of epithelial cell samples and increases brightness of the fluorescent signal during FISH.

1 cl, 2 dwg, 3 ex

Description

The invention relates to medicine, namely to Oncology, and is used for interphase fluorescence in situ hybridization.

Currently, the method of fluorescent in situ hybridization (FISH) is actively used in clinical practice for the diagnosis, treatment tactics and assessment of the prognosis of the disease, as well as in scientific research to gain knowledge in the biology of malignant tumors. Since this method has a high cost and complexity, the stages of fixing the biological material and obtaining preparations become key points for the successful conduct of FISH studies using commercial DNA probes. When used in clinical practice to achieve the reliability of a FISH study, the hybridization result can be considered successful if the fluorescence signals from the hybridized DNA regions are bright, i.e. contrastively visualized with respect to background fluorescence and their number can be calculated reliably. In addition, it is required that the cells lie separately from each other on the preparation, their boundaries are clearly defined, and their number in the hybridized region is sufficient to obtain statistically reliable results. For malignant tumors of epithelial origin, such as cancer of the oral mucosa, cervical cancer, FISH studies are performed on smears, followed by their fixation, or on preparations obtained by applying a suspension of pre-fixed cells (Liquid cytology in the diagnosis of cervical diseases : teaching aid. - M: RMAPO. - 2012.23 p.). Constantly increasing demand for FISH studies necessitates low-cost methods of fixing biological material and its long-term storage.

A known method in which the FISH reaction is carried out on a traditional smear from the cervix (Pap smear) presented in the article by Heselmeyer-Haddad K. et al. (Heselmeyer-Haddad K., Sommerfeld K., White NM et al. Genomic amplification of the Human Telomerase Gene (TERC) in Pap smears predicts the evelopment of cervical cancer / American Journal of Pathology, 2005, Vol. 166, No. 4 , p. 1229-1238, doi: 10.1016 / S0002-9440 (10) 62341-3). In the method, a cytological examination is first performed, for which all smears are fixed, stained according to standard procedures and filled with special glue under a coverslip. When the preparations are prepared for FISH, the coverslips are removed by incubating the preparations in xylene for 2-4 days, then the preparations are washed twice in xylene, dehydrated and decolored in a mixture of 0.5% HCl and 70% ethanol for 1-2 hours .

The disadvantages of the method are the uneven distribution of cells on a glass slide, multilayer, loss of cells during fixation, the presence of elements leading to high background fluorescence.

The known method is “Standardized analytical technology“ liquid cytology method ”(Shabalova I.P., Dzhangirova T.V. Nikitina L.V. et al. Problems of standardization in healthcare, 2012, No. 7-8, p. 48-58). In the method, the biological material is placed and stored in a special transport medium, the cytological preparation is prepared by depositing cells on a glass slide by centrifuging the cellular material in a cytocentrifuge, and the smears are fixed either as a preliminary step before staining or simultaneously with staining.

The disadvantage of this method is the low availability and high cost due to the use of only unique standardized reagents, consumables and the need for expensive imported equipment, the inability to apply cell suspensions of several people to one glass.

The well-known "Improved method for the preparation of fixed cell preparations for fluorescence in situ hybridization" (patent RU 2490635), in which a suspension of fixed cells in a volume of 5 μl is applied to the bottom of the holes formed by the surface of a glass slide and 8 round holes with a diameter of 9 mm in a hermetically glued to the glass a parafilm strip measuring 26 × 56 mm ² . Attach a strip of parafilm to a slide, heating the glass with parafilm at a temperature of 60 ° C and simultaneously rolling with a roller for 3-5 seconds. Subsequently, the specified strip together with a slide is heated at 95 ° C for 2 minutes and then peeled off from the glass.

The disadvantage of this method is the complexity, as well as the fact that within one well, the researcher does not have the opportunity to choose the best area for hybridization.

The prototype of the invention is a method for the preparation, fixation and preparation of a preparation of epithelial cells desquamated from the cervical canal described in the article by Li T. et al. (Li T, Tang L, Bian D, Jia Y, Huang X, Zhang X. Detection of hTERC and C-MYC genes in cervical epithelial exfoliated cells for cervical cancer screening. Int J Mol Med. 2014 May; 33 (5): 1289-97. Doi: 10.3892 / ijmm. 2014.1699.). The method is implemented in the following sequence:

1. Epithelial cells are shed by a cervical brush (Pap Brush; Beijing TST Medical Technology Company, Ltd., Beijing, China).

2. Place the desquamated cells in phosphate-buffered saline (PBS) and store at 4 ° C until a FISH study is performed.

3. Approximately 5-10 ml of PBS containing desquamated cells is placed in a centrifuge tube, 3 ml of collagenase are added and centrifuged at 1300 rpm for 10 minutes and the supernatant is removed.

4. Resuspend the pellet, add water and incubate at 37 ° C for 20-30 minutes.

5. The resulting cell suspension is centrifuged at 1300 rpm for 10 minutes and the supernatant is removed.

6. Resuspend the pellet, add 5 ml of water and incubate at 37 ° C for 20 minutes.

7. Add 2 ml of fixing solution included in the washing kit (the composition is a commercial secret), centrifuged at 1300 rpm for 10 minutes and remove the supernatant.

8. Then add 5 ml of fixing solution, centrifuged at 1300 rpm for 10 minutes and remove the supernatant.

9. Once again add 5 ml of fixing solution, centrifuged at 1300 rpm for 10 minutes and remove the supernatant.

10. The precipitate is resuspended and digged onto glass slides, which are left to dry in air overnight at room temperature.

The disadvantages of the method are the short shelf life of biological material, time-consuming fixation procedure (90-100 min), a small number of cells in the hybridization region, unknown composition of the fixing solution.

The technical result of the invention consists in increasing the storage time of samples of biological material, providing the ability to control cell density in the field of DNA probe hybridization, increasing the brightness of the fluorescent signal, reducing the consumption of reagents and increasing labor productivity.

The essence of the proposed solution includes desquamation of epithelial cells by a cytobrush, placing them in a test tube with phosphate-buffered saline, centrifuging the resulting mixture for 10 min, followed by removal of the supernatant and adding to the sediment a fixing solution consisting of a mixture of methanol and acetic acid in a ratio of 3: 1, double purification of the resulting suspension by centrifugation for 15 min at 1000 rpm, removal of the supernatant with the addition of a fixing solution and storage of the cell suspension in condition ditions -20 ° C, when they are used repeatedly purified, resuspended and applied to the slides with zoned control cell density, to increase the brightness of the fluorescent signals was treated with 2% Carbowax solution for 2-20 hours at 4 ° C.

Enumeration of figures.

FIG. 1. Examples of zoning of a glass slide: 1 - circles, 2 - rectangles, 3 - squares.

FIG. 2a. Photos of cells of desquamated epithelium after treatment with Carbowax solution: bright (contrasting to background fluorescence) fluorescent signals 4 - green, 5 - red.

FIG. 2b. Photos of cells of desquamated epithelium without treatment with Carbowax solution: dim (non-contrast to background fluorescence) fluorescent signals 4 - green, 5 - red.

The order of implementation of the method.

1. Epithelial cells are desquamated using a disposable cervical brush (Urogenital probe, disposable sterile Type D Model 2, Jiangsu SUYUN Medical Materials Co., Ltd, China).

2. Immediately after taking the material, the brush is placed in a centrifuge tube with 10 ml of phosphate-saline buffer (PBS) (pH = 7.0-7.2) (if necessary, this material can be stored at 4 ° C, but not more than 3 days).

3. Cells from the brush are washed off on a laboratory vortex, holding it with tweezers for 15 seconds, then the brush is removed from the tube.

4. The peeled cells are centrifuged for 15 min at 1000 rpm. After centrifugation, the supernatant is removed.

5. Gently, constantly stirring, add 1 ml of freshly prepared fixing solution (fixative) (methanol and acetic acid in a ratio of 3: 1) to the resuspended precipitate, then add the fixative to 10 ml, centrifuge for 15 min at 1000 rpm and remove the supernatant.

6. Resuspend the precipitate, add the fixative to 10 ml, centrifuge for 15 min at 1000 rpm and remove the supernatant.

7. Resuspend the pellet, top up the fixative to 10 ml and place in the freezer (-20 ° C) for at least 8 hours to complete cell fixation or storage. In the fixative, the suspension of fixed cells can be stored for a long time (at least 5 years) at -20 ° C.

8. Place a clean glass slide (Menzel-Glaser, Thermo Fisher Scientific, article AA00000112E) on a thermostat (30 ° C) and zoning it by applying strips of rubber glue (Fixogam Rubber Cement, Kreactech (cat. Number LK-071A) or any other rubber glue from natural rubber), limiting the area required for applying the cell suspension and waiting for the glue to dry completely.

9. The tube with the cell suspension is centrifuged for 15 min at 1000 rpm, the supernatant is removed, leaving 0.2-1.0 ml and the sediment is resuspended.

10. On a glass slide lying on a thermostat (30 ° C), in a region limited by strips of glue, apply a cell suspension of 5-30 μl.

11. Estimate the cell density under a light microscope and, if it is insufficient, then add another cell suspension until the desired cell density is reached in the proposed hybridization region.

12. After the cytogenetic preparation has completely dried, glue strips are removed from it with tweezers.

13. Then the drug is placed in a glass with a 2% solution of Carbowax (2 ml of polyethylene glycol (Sigma-Aldrich, cat. Number P 5402) and 98 ml of a mixture of 95% ethanol and distilled water in a ratio of 1: 1) for 2-20 hours at 4 ° C.

14. Take the drug out of Carbowax solution, gently shake off excess liquid and place it in a thermostat (37 ° C) for 16-20 hours for dehydration.

Examples of the method.

Example 1

Using the presented method in 32 patients with cancer of the oral mucosa, admitted for treatment at the MRRC them. A.F. Tsyba in 2014-2019, with the tumor, epithelial cells were taken and fixed. The shelf life of the samples before performing the FISH reaction ranged from 0 to 5 years (median 3 years). In accordance with the proposed method, cytogenetic preparations were prepared. Cell suspensions were applied to 8 slides, each of which was zoned into eight square parts (Fig. 1), i.e. on each slide a suspension of cells of 4 people was dug. Under a light microscope, cell densities were monitored in putative hybridization regions. With insufficient cell density, a cell suspension was added. Cytogenetic preparations were treated with a 2% Carbowax solution and dehydrated in a thermostat at 37 ° C for 16 hours. Pre- and post-hybridization washes of cytogenetic preparations were performed in accordance with the protocol of the manufacturer of DNA probes (Kreatech Biotechnology, Holland). Each of the pre- and post-hybridization washing procedures was performed simultaneously for 4 cytogenetic preparations (i.e., for 16 people or 32 areas of hybridization). Thus, when performing pre- and post-hybridization washes for each patient, the consumption of washing reagents and the time taken to complete them decreased by 4 times. Two sets of commercial DNA probes stained with the same fluorochromes were studied for each patient: the CCND1 gene with the centromere of chromosome 11 (fluorochromes red and green, respectively) and the EGFR gene with the centromere of chromosome 7 (fluorochromes red and green, respectively). DNA probes with a volume of 2.2 μl for each studied gene were applied to a region preliminarily selected within the limits of zoning and closed with a 10 mm diameter round coverslip. Hybridization was successful in all cases, i.e. the fluorescence signals of hybridized DNA regions were contrasted with respect to background fluorescence (Fig. 2a). Therefore, the proposed solution makes it possible to see the quasiparticles of living matter, analyze their structure, and also study the physicochemical and functional features.

For each patient, at least 100-200 cells were analyzed (depending on the proportion of cells with abnormalities) for each gene studied, which indicates a high density of epithelial cells in the area of hybridization. Moreover, due to a decrease in the area of hybridization, the cost of expensive DNA probes decreased by 4 times (from 10 μl to 2.2 μl recommended by the manufacturer).

Example 2

Using the presented method in 8 patients with cancer of the oral mucosa, admitted for treatment at the MRRC them. A.F. In 2014, a swab of epithelial cells was carried out from the tumor, their fixation, storage (1 year) and application to 4 zoned glass slides. Each slide was zoned with rubber glue into 4 rectangular areas (Fig. 1), into which cell suspensions were applied in the form of drops, i.e. A suspension of 2 people was placed on each slide in 2 areas for each. One area was intended for hybridization with DNA probes of the CCND1 gene and centromere of chromosome 11, the second of the EGFR gene with centromere of chromosome 7. Pre- and post-hybridization washes of cytogenetic preparations were performed in accordance with the protocol of the manufacturer of DNA probes (Kreatech Biotechnology, Holland). Treatment with 2% Carbowax was not performed. Each of the pre- and post-hybridization washes was performed simultaneously for 4 cytogenetic preparations. In all cases, the hybridization could not be considered successful, since the fluorescent signals (especially green) were not contrasted with the background fluorescence (Fig. 26) and this did not allow to reliably determine the number of fluorescent signals in the studied cells.

Example 3

Using the presented method in 12 patients with cervical cancer admitted for treatment at the MRRC them. A.F. Tsyba in 2013-2015, epithelial cells were taken, their fixation and conservation. The shelf life of the samples before performing the FISH reaction ranged from 3 to 6 years (median 4 years). In accordance with the presented method, cell suspensions were applied onto 4 zoned onto 3 circular regions (Fig. 1) of a glass slide, i.e. on each slide a suspension of 3 people was dug up. Cytogenetic preparations were treated with a 2% Carbowax solution and dehydrated in a thermostat at 37 ° C for 16 hours. Pre- and post-hybridization washes of cytogenetic preparations were performed in accordance with the protocol of the manufacturer of DNA probes (Abbott Molecular, USA). Each of the procedures for pre- and hybridization washes was performed simultaneously for 4 slides (i.e., for 12 people or 12 areas of hybridization), thus, for each patient, the consumption of washing reagents and the time taken to complete them decreased by 3 times. For each patient, 2 commercial DNA probes were tested: TERC gene (red fluorochrome), CEP3 (green fluorochrome). Hybridization was successful in all cases, i.e. the fluorescence signals of hybridized DNA regions were contrasted with respect to background fluorescence (Fig. 2a). For each patient, at least 100-200 cells were analyzed (depending on the proportion of cells with abnormalities) for each gene studied, which indicates a high density of epithelial cells in the area of hybridization. Moreover, due to a decrease in the area of hybridization, the cost of expensive DNA probes decreased by 4 times (from 10 μl to 2.2 μl recommended by the manufacturer).

Confirmation of the achievement of a technical result.

On cytogenetic preparations obtained using the presented method, a FISH study was performed for 32 patients with cancer of the oral mucosa and 12 patients with cervical cancer. In all cases, hybridization was successful, i.e. the fluorescent signals of hybridized DNA regions were contrast-visualized with respect to background fluorescence, which allowed us to reliably determine the number of fluorescent signals, while hybridization could not be considered successful in preparations that were obtained without treatment with Carbowax solution. On the preparations obtained in accordance with the proposed method, for all patients, the number of cells that was necessary to obtain reliable results was analyzed. The present invention, when used, provides the following technical effect: due to the fixation of the epithelial cells for a long time (5-6 years) they retain DNA suitable for hybridization with DNA probes when stored under -20 ° C. Carboax solution pretreatment of the preparations creates good conditions for the passage of hybridization, which is observed in the form of an increase in the brightness of the fluorescent signal compared to the brightness of fluorescent signals without such processing: zoning allows applying several cell suspensions to one glass slide, which leads to significant (3 -4 times) reducing the consumption of pre- and post-hybridization washing solutions, and also increases labor productivity. The ability to control cell density reduces the hybridization area, which in turn leads to a several-fold reduction in the cost of expensive DNA probes.

Claims (1)

A method for producing cytogenetic preparations of epithelial cells for carrying out a fluorescence in situ hybridization reaction, including desquamation of epithelial cells by a cytobrush, placing them in a tube with phosphate-buffered saline, centrifuging the resulting mixture for 10 min, followed by removal of the supernatant, characterized in that it is added to the sediment fixing solution, consisting of a mixture of methanol and acetic acid in a ratio of 3: 1, store cell suspensions at a temperature of -20 ° C, cytogenetic preparations are prepared by applying to tapes on zoned glass slides while controlling their density, to increase the brightness of fluorescent signals, cells are treated with a 2% Carbowax solution for 2-20 hours at 4 ° C.

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