Solution for fixing biological cells

G01N1/30 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)

FIELD: chemistry.

SUBSTANCE: invention relates to a solution for fixing biological cells. The fixing solution is intended for in vitro preservation of a cytological sample containing nucleated cells and erythrocytes. The solution contains 80-95 vol. % of a mixture of 590 ml normal saline, 10 ml polyethylene glycol (Carbowax®), 203 ml isopropyl alcohol, 193 ml pure ethanol, 0.01 vol. % sodium azide, and 5-20 vol. % buffered 4% formalin; pH of the fixing solution is in the range of 6.4 to 7.4.

EFFECT: preserving the integrity of nucleated cells.

2 cl, 2 dwg, 1 ex


The present invention relates to a solution for fixing biological cells (fixative solution) intended for in vitro conservation of the cytological specimen, the type containing alcohol solution for fixing cells. More specifically, the invention relates to the field of Cytology.

The fixing solutions or the latches provided for the preservation and conservation of cytological samples, including cells, for subsequent analysis by a pathologist. Cell samples can be, for example, obtained by the puncture needle by washing, scraping or collecting urine from the patient. The cells can be cells of any organ, such as the uterus, liver, stomach, mammary gland, and the like. The sample of cells may be, for example, a sample of fluid, more specifically, urine, ascites, pleural or pericardial fluid; smear, more specifically, cervical smear, smear from the vagina; the puncture body, more specifically the outer body, such as mammary gland, thyroid gland, or an internal organ, such as pancreas or liver and the like.

"Cytological fixative or cytological fixing solution" means a solution commonly used cytological assays for cell fixation.

The commit operation is designed for maximum feasible�th preservation of the morphology of cells in the state in which they were prior to their selection. The best clips are the ones that, providing a fast action, cause the minimum possible number of secondary or artificial changes which can interfere with the analysis of the internal morphology of the cells.

Known and has long been used, particularly in the agricultural and veterinary field, formaldehyde acetic alcohol or AFA. AFA contains methyl or ethyl alcohol, formalin and glacial acetic acid. Formalin is a mixture consisting of formic acid, formaldehyde, paraformaldehyde and methanol. An example is the AFA company Locquin, having the following composition:

Ethyl alcohol strength 80°100 cm3
Laboratory formalin concentration of 38%10 cm3
Glacial acetic acid5 cm3
Sucrose10 g
Distilled water20 cm3

Thus, glacial acetic acid destroys red blood cells and dissolves their contents, resulting in formation of deposits of hemoglobin, very disturbing �ri analysis after standard staining, more specifically on the smear, or staining by May-Grunwald-Giemsa (MGG), or very disturbing when immunocytochemically or other studies. Moreover, from the perspective of normative acts with this concentration, ethanol is considered to be a flammable substance, and the formalin is toxic and carcinogenic substance.

The purpose of the invention is to provide a solution for fixing biological cells, to ensure good preservation of the integrity of nucleated cells and red blood cells for analysis, but less dangerous for the user.

In this regard, an object of the invention is the solution for fixing biological cells of the above type containing the saline solution to prevent osmotic shock on the level of the cell wall, formaldehyde and polyethylene glycol to preserve the size and integrity of nucleated cells and erythrocytes, fixed mentioned solution.

According to other aspects of the invention the solution for fixing biological cells possess one or more of the following properties:

the fixing solution contains no acetone or compounds of the class of ketones or acetic acid to maintain the integrity of red blood cells.

- alcohol is ethanol or isopropanol;

- alcohol is a mixture of ethanol and isopropanol;

- the number of alcohol 45% by about�jemu fixative;

the amount of formaldehyde in substance is from about 0.2 to 1% by volume of fixative.

the fixing solution contains a buffer, ensuring that the pH of the fixing solution is essentially from 6.4 to 7.4; and

the fixing solution contains from 80% to 95% by volume:

- 590 ml of saline,

To 10 ml of polyethylene glycol (Carbowax®),

- 203 ml of isopropyl alcohol,

- 193 ml of pure ethanol,

- 0,01% sodium azide,

and from 20% to 5% by volume of formalin, buffered at the rate of 4%.

So, this solution provides better preservation of cell ratios, more specifically the size of the nucleus relative to the size of the cell, and more specifically the excellent preservation of erythrocytes.

In addition, this fixing solution slavophones, non-toxic and not carcinogenic.

The invention will be better understood when reading the description below is only an example and made with reference to the drawings, in which:

- figure 1 is a photograph of a sample of cells obtained from breast cancer, which was retained in the fixing solution according to the invention,

- figure 2 is a photograph of a sample of cells selected from the thyroid gland, which has been stored in fixative solution according to the invention.

The present invention relates to a fixing solution, PR�naznachennomu for in vitro conservation of the cytological specimen, containing biological cells intended for analysis by a pathologist.

The fixing solution contains an isotonic solution of sodium chloride or saline instead of distilled or deionized water is normally used as the basis of traditionally used fixing solution.

Saline has long been known, it contains sodium chloride dissolved in distilled water at the rate of 9 grams of sodium chloride in 1 liter of water. Saline is ISO-osmolar that supports cells able to correct osmolarity, prevent any osmotic shock on the level of the cell wall and, consequently, the destruction of cells by rupture of the cytoplasmic and nuclear membranes. The osmolarity of normal saline is 308 mosmol ' per liter.

Osmolarity is a concentration of the medium. This brings us to the concept of osmosis which is the diffusion of solvent through a semipermeable membrane separating two solutions with different concentrations, for example, the fixing solution and the cytoplasmic fluid of the cell.

In addition, the fixing solution contains alcohol for maintaining the cells in their condition, locking them in alcohol medium.

Preferably, the amount of alcohol 45% by volume of fixative so that it was slavophones. Thus�Ohm, it is a small amount of alcohol makes it easier to transport and store the fixative solution, because it is less dangerous. More specifically, under the weak Flammability imply that the solution according to the invention does not require application to the packaging solution for a security icon, warning about the Flammability of the product.

More specifically, the alcohol is ethanol or isopropanol.

According to one of the embodiments of the invention, the alcohol is a mixture of ethanol and isopropanol.

However, alcohol is dehydrating, reducing substance, thus changing the size of cells due to reduction of nucleus and cytoplasm.

To compensate this disadvantage of fixing solution also contains formalin to preserve the size of the cells. The fact that the fixing solution may also contain Carbowax® or other polyethylene glycol, known and published Saccomanno (Saccomanno) and colleagues. Known and publicized the fact that formalin can be combined with substances that remove calcium salts or antiplatelet agents such as ethylenediaminetetraacetic acid (EDTA) and its salts. Also known and publicized the fact that the samples containing mucus, you can add a mucolytic substance, in particular dithiotreitol (DTT) or acetylcysteine.

Formalin allows you to maintain the red blood cells and nuclear cells, not on them and ensuring that the ratio in size that allows the cytologist to have more accurate diagnosis.

More specifically, as can be seen in figures 1 and 2, samples of cells that were stored in the fixative solution according to the invention, the red blood cells, marked by the numeral 1 in the figures, perfectly preserved and not compromised, which allows a precise analysis of these samples.

Preferably, the amount of formaldehyde in substance is from about 0.2 to 1% by volume of fixative. With this concentration, formalin or formaldehyde is considered as a simple irritant tool on Toxicological registry issued by the National Institute of research and safety (INRS), in contrast to the commonly used concentrations from 1 to 25%, in which the formaldehyde is corrosive and carcinogenic. Thus, the packaging solution of the invention also does not require the application of security badge, alerting you that the product is corrosive.

Therefore, when the concentration is below 1% with a solution you can safely make the manipulation, as the level of precautions for performing manipulation with it is below.

It is known that the fixing solution may contain an antimicrobial additive.

The fixing solution contains a buffer, ensuring the pH of the fixing solution essentially from 6.4 to 7.4. This pH range corresponds to about�optimal conditions of storage cells and blood of the human body.

The following examples illustrate the invention without limiting its scope.

Example 1

A solution formed of 80% by volume of:

- 590 ml of saline,

To 10 ml of polyethylene glycol (Carbowax®),

- 203 ml of isopropyl alcohol,

- 193 ml of pure ethanol,

- 0,01% sodium azide,

and 20% by volume buffered 4% formalin.

In one embodiment, the solution can be formed from 90% by volume of the above mixture and from 10% by volume buffered 4% formalin or 95% by volume of the above mixture and of 5% by volume buffered 4% formalin.

It should also be noted that the fixing solution according to the invention contains no acetone or compounds of the class of ketones or acetic acid. These products lisarow red blood cells, with destruction which releases hemoglobin, which is deposited on the cells. Some types of staining, such as staining of PAP smears, given the complexity of dyes that combine multiple nuclear dyes and hemoglobin do in this case, cytological analysis, and more specifically the study of the nucleus is very difficult, if not impossible. Immunocytochemical studies also often obstructed by deposits of hemoglobin.

The use of a fixative solution according to the invention improves the subsequent analyses of the sample with staining on Papanek�Lau or Immunocytochemical examination fixed in fixation solution described in this document and does not contain any acetone or compounds of the class of ketones or acetic acid.

Thus, as described above, the fixing solution provides excellent preservation of the integrity of nucleated cells and red blood cells for analysis.

1. The fixing solution used for in vitro conservation of cytological sample containing nuclear cells and red blood cells, characterized by the fact that it contains from 80% to 95% by volume:
- 590 ml of saline,
To 10 ml of polyethylene glycol (Carbowax®),
- 203 ml of isopropyl alcohol,
- 193 ml of pure ethanol,
- 0,01% sodium azide,
and from 20% to 5% by volume buffered 4% formalin.

2. The fixing solution according to claim 1, characterized in that it comprises a buffer providing a pH of the fixing solution from 6.4 to 7.4.


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1 tbl, 3 ex

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19 cl, 2 tbl, 30 ex

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2 ex

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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.

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