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EP2528433A1 - Fixing solution for biological cells - Google Patents

Fixing solution for biological cells

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Publication number
EP2528433A1
EP2528433A1 EP11704659A EP11704659A EP2528433A1 EP 2528433 A1 EP2528433 A1 EP 2528433A1 EP 11704659 A EP11704659 A EP 11704659A EP 11704659 A EP11704659 A EP 11704659A EP 2528433 A1 EP2528433 A1 EP 2528433A1
Authority
EP
European Patent Office
Prior art keywords
fixing solution
cells
alcohol
volume
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11704659A
Other languages
German (de)
French (fr)
Inventor
Eric Peltier
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novacyt SA
Original Assignee
Novacyt SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novacyt SA filed Critical Novacyt SA
Publication of EP2528433A1 publication Critical patent/EP2528433A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/128—Chemically defined matrices for immobilising, holding or storing living parts, e.g. alginate gels; Chemically altering living parts, e.g. by cross-linking
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • the present invention relates to a binding solution for biological cells for preserving in vitro a cytological sample of the type comprising alcohol to fix the cells.
  • the invention applies more particularly to the field of cytology.
  • Fixation solutions are provided to preserve and preserve samples or cytological samples, including cells, for subsequent analysis by a cytologist.
  • These cell samples may for example be obtained by needle aspiration, washing, brushing or compaction of urination in a patient.
  • the cells can come from any organ, for example the uterus, liver, stomach, breast, etc.
  • a sample of cells may for example be a fluid sample such as urine, ascites, pleural or pericardial fluid; a smear such as cervical smear, vaginal ...; an organ puncture, for example a superficial gland such as breast, thyroid or deep, such as pancreas or liver, etc.
  • cytological fixator or "cytological fixation solution” is meant a solution commonly used in cytological analysis to achieve cell fixation.
  • Fixation is an operation intended to preserve the morphology of the cells, as much as possible, in the state they were in before they were taken.
  • the best fixators are those which, while acting quickly, produce as little as possible of secondary modifications or artifices likely to prevent the analysis of the internal morphology of the cells.
  • Acetic formaldehyde alcohol or AFA is known and used for a long time especially in agricultural and veterinary environments.
  • AFA comprises methyl or ethyl alcohol, formalin and glacial acetic acid.
  • Formalin is formic acid, formaldehyde, paraformaldehyde and methanol.
  • An example is Locquin's AFA whose composition is as follows:
  • the glacial acetic acid destroys the red blood cells and lyses their contents involving very troublesome hemoglobin deposits in the analysis after standard staining such as Papanicolaou, or a May-Grunwald Giemsa staining (MGG), or very annoying for the patients.
  • MCG May-Grunwald Giemsa staining
  • alcohol Ethyl is considered at the regulatory level as flammable and formalin as toxic and carcinogenic.
  • the object of the invention is to provide a fixing solution for biological cells allowing good preservation of the integrity of the nucleated cells and red blood cells for their analysis, but nevertheless less dangerous for the user.
  • the subject of the invention is a fixing solution for biological cells of the above-mentioned type, comprising physiological saline in order to avoid osmotic shocks at the level of the cell wall, formalin and polyethylene glycol to preserve the size and integrity of nucleated cells and red cells fixed in said solution.
  • the fixing solution for biological cells has one or more of the following characteristics:
  • the fixing solution does not comprise acetone or compounds of the ketone family, nor acetic acid in order to preserve the integrity of the red blood cells.
  • the alcohol is ethanol or isopropanol
  • the alcohol is a mixture of ethanol and isopropanol
  • the quantity of alcohol is substantially less than 45% by volume of the fixing solution
  • the amount of formalin is substantially between about 0.2 and 1% by volume of the fixing solution.
  • the fixing solution comprises a buffer guaranteeing a pH of the fixing solution substantially between 6.4 and 7.4;
  • the fixing solution comprises between 80% and 95% by volume of:
  • this solution allows a better conservation of cellular proportions, in particular the dimensions of the nucleus relative to the dimensions of the cell, and especially an excellent conservation of red blood cells.
  • this fixing solution is low in flammability, non-toxic and non-carcinogenic.
  • FIG. 1 is a photograph of a sample of cells taken from a breast, said sample being preserved in a fixing solution according to the invention
  • FIG. 2 is a photograph of a sample of cells taken from a thyroid, said sample being preserved in a fixing solution according to the invention.
  • the present invention relates to an attachment solution for preserving in vitro a cytological sample comprising biological cells for analysis by a cytologist.
  • the fixing solution comprises an isotonic sodium chloride solution, or physiological saline, instead of the distilled or deionized water usually used as the base of commonly used fixing solutions.
  • the physiological saline is known for a long time and comprises sodium chloride diluted in distilled water, at a rate of 9 grams of sodium chloride per liter of water.
  • the saline is iso-osmolar which keeps the cells in a state of correct osmolarity to avoid osmotic shock at the cell wall and thus the destruction of cells by bursting cytoplasmic and nuclear membranes.
  • the osmorability of the physiological serum is equal to 308 mosmols per liter.
  • Osmolarity is the concentration of a medium. This calls for the concept of osmosis, which is the diffusion of a solvent through a semipermeable membrane which separates two solutions of different concentrations, for example the fixing solution and the cytoplasmic liquid of the cells.
  • the fixing solution contains alcohol to keep the cells in their state by fixing them in the alcoholic medium.
  • the amount of alcohol is substantially less than 45% by volume of the fixing solution to be low in flammability.
  • this low alcohol content allows easier transport and storage because less dangerous of the fixing solution.
  • the solution according to the invention does not require the use of the safety pictogram signaling the flammability of a product on the packaging of the solution.
  • the alcohol is, for example, ethanol or isopropanol.
  • the alcohol is a mixture of ethanol and isopropanol.
  • alcohol alone is a desiccant reducing agent, thus altering cell size by nuclear and cytoplasmic retraction.
  • the fixing solution further comprises formalin to maintain the size of the cells.
  • the fixation solution may also include Carbowax® or Polyethylene Glycol.
  • formalin may be combined with decalcifying or anti-aggregating agents such as ethylene diamine tetracetic acid (EDTA) and its salts.
  • EDTA ethylene diamine tetracetic acid
  • a mucolytic agent such as dithiothreitol (DTT) or acetylcysteine can be added to the specimens containing mucus.
  • Formalin makes it possible to preserve the red blood cells and the nucleated cells without lysing them, guaranteeing a reference in terms of size making it possible to make a more relevant diagnosis to the cytologist.
  • the amount of formalin is substantially from about 0.2 to 1% by volume of the fixing solution.
  • formalin or formaldehyde is considered as simply irritating according to the toxicological data sheet published by the National Institute for Research and Safety (INRS) in contrast to the generally used concentrations of 1 and 25% in which formalin is corrosive and carcinogenic.
  • the conditioning of the solution according to the invention also does not require the use of the safety pictogram indicating a corrosive product.
  • the fixing solution may comprise an antimicrobial agent.
  • the fixing solution comprises a buffer ensuring a pH of the fixing solution substantially between 6.4 and 7.4. This pH range corresponds to optimal conditions for the storage of cells and blood of the human body.
  • the solution can be formed to 90% by volume of the above mixture and 10% by volume of 4% or 95% by volume buffered formalin of the above mixture and 5% by volume of buffered formalin. at 4%.
  • the fixing solution according to the invention does not contain acetone or compounds of the ketone family, nor acetic acid. These products lyse red blood cells which, when they burst, release the hemoglobin that binds to the cells. Certain types of staining, such as the staining of Papanicolaou, because of the complexity of coloring agents associating several nuclear dyes and hemoglobin, make the cytological analysis, and in particular nuclear, very difficult if not impossible. Similarly, immuno-cytochemical studies are often hampered by hemoglobin deposition.
  • the fixing solution described above thus allows excellent preservation of the integrity of nucleated cells and red blood cells for their analysis.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

The invention relates to a fixing solution for in vitro preservation of a cell sample including nucleated cells and red blood cells, wherein said solution includes alcohol for fixing the cells. Said fixing solution includes physiological serum so as to avoid osmotic shocks in the cell walls and moreover contains formol and polyethylene glycol for preserving the size and integrity of the nucleated cells and red blood cells that are fixed in said solution. The solution does not contain compounds from either the ketone or acetic acid families.

Description

Solution de fixation pour des cellules biologiques  Fixative solution for biological cells

La présente invention concerne une solution de fixation pour des cellules biologiques destinée à préserver in vitro un échantillon cytologique, du type comprenant de l'alcool pour fixer les cellules. L'invention s'applique plus particulièrement au domaine de la cytologie.  The present invention relates to a binding solution for biological cells for preserving in vitro a cytological sample of the type comprising alcohol to fix the cells. The invention applies more particularly to the field of cytology.

Les solutions de fixation, ou fixateurs, sont prévues pour préserver et conserver des prélèvements ou échantillons cytologiques, comprenant des cellules, en vue de leur analyse ultérieure par un cytologiste. Ces échantillons de cellules peuvent par exemple être obtenus par ponction à l'aiguille, lavage, brossage ou recueil de miction chez un patient. Les cellules peuvent provenir de n'importe quel organe, par exemple l'utérus, le foie, l'estomac, le sein, etc. Un échantillon de cellules peut par exemple être un prélèvement de liquide tel que les urines, l'ascite, le liquide pleural ou péricardique ; d'un frottis tel qu'un frottis cervical, vaginal... ; d'une ponction d'organe, par exemple une glande superficielle telle que le sein, la thyroïde ou profonde, tel que le pancréas ou le foie, etc.  Fixation solutions, or fixatives, are provided to preserve and preserve samples or cytological samples, including cells, for subsequent analysis by a cytologist. These cell samples may for example be obtained by needle aspiration, washing, brushing or compaction of urination in a patient. The cells can come from any organ, for example the uterus, liver, stomach, breast, etc. A sample of cells may for example be a fluid sample such as urine, ascites, pleural or pericardial fluid; a smear such as cervical smear, vaginal ...; an organ puncture, for example a superficial gland such as breast, thyroid or deep, such as pancreas or liver, etc.

Par « fixateur cytologique » ou « solution de fixation cytologique », on entend une solution couramment utilisée en analyse cytologique pour réaliser la fixation de cellules.  By "cytological fixator" or "cytological fixation solution" is meant a solution commonly used in cytological analysis to achieve cell fixation.

La fixation est une opération destinée à conserver la morphologie des cellules, autant que possible, dans l'état où elles se trouvaient avant leur prélèvement. Les meilleurs fixateurs sont ceux qui, tout en agissant rapidement, produisent le moins possible de modifications secondaires ou d'artifices susceptibles d'empêcher l'analyse de la morphologie interne des cellules.  Fixation is an operation intended to preserve the morphology of the cells, as much as possible, in the state they were in before they were taken. The best fixators are those which, while acting quickly, produce as little as possible of secondary modifications or artifices likely to prevent the analysis of the internal morphology of the cells.

L'alcool formolé acétique ou AFA est connu et utilisé depuis longtemps notamment dans les milieux agricoles et vétérinaires. L'AFA comprend de l'alcool méthylique ou éthylique, du formol et de l'acide acétique glacial. Le formol est composé d'acide formique, de formaldéhyde, de paraformaldéhyde et de méthanol. Un exemple est l'AFA de Locquin dont la composition est la suivante :  Acetic formaldehyde alcohol or AFA is known and used for a long time especially in agricultural and veterinary environments. AFA comprises methyl or ethyl alcohol, formalin and glacial acetic acid. Formalin is formic acid, formaldehyde, paraformaldehyde and methanol. An example is Locquin's AFA whose composition is as follows:

- Alcool éthylique à 800 100 ce, - ethyl alcohol at 80 0 100 this,

- Formol de laboratoire à 38% 10 ce,  - Lab formol at 38% 10 this,

- Acide acétique glacial 5 ce,  - glacial acetic acid 5 ce,

- Saccharose 10 g,  - Sucrose 10 g,

- Eau distillée 20 ce.  Distilled water 20 cc.

Or l'acide acétique glacial détruit les hématies et lyse leur contenu impliquant des dépôts d'hémoglobine très gênant dans l'analyse après coloration standard telle que le Papanicolaou, ou une coloration de May-Grunwald Giemsa (MGG), ou très gênant pour les études immuno-cyto-chimique, ou autre. En outre, à cette concentration, l'alcool éthylique est considéré au plan réglementaire comme inflammable et le formol comme toxique et cancérigène. However, the glacial acetic acid destroys the red blood cells and lyses their contents involving very troublesome hemoglobin deposits in the analysis after standard staining such as Papanicolaou, or a May-Grunwald Giemsa staining (MGG), or very annoying for the patients. immuno-cytochemical, or other studies. In addition, at this concentration, alcohol Ethyl is considered at the regulatory level as flammable and formalin as toxic and carcinogenic.

Le but de l'invention est de fournir une solution de fixation pour des cellules biologiques permettant une bonne conservation de l'intégrité des cellules nucléées et des hématies en vue de leur analyse, mais néanmoins moins dangereuse pour l'utilisateur.  The object of the invention is to provide a fixing solution for biological cells allowing good preservation of the integrity of the nucleated cells and red blood cells for their analysis, but nevertheless less dangerous for the user.

A cet effet, l'invention a pour objet une solution de fixation pour des cellules biologiques du type précité, comprenant du sérum physiologique afin d'éviter les chocs osmotiques au niveau de la paroi des cellules, du formol et du polyéthylène glycol pour conserver la taille et l'intégrité des cellules nucléées et des hématies fixées dans ladite solution.  For this purpose, the subject of the invention is a fixing solution for biological cells of the above-mentioned type, comprising physiological saline in order to avoid osmotic shocks at the level of the cell wall, formalin and polyethylene glycol to preserve the size and integrity of nucleated cells and red cells fixed in said solution.

Selon d'autres aspects de l'invention, la solution de fixation pour des cellules biologiques comporte l'une ou plusieurs des caractéristiques suivantes :  According to other aspects of the invention, the fixing solution for biological cells has one or more of the following characteristics:

- la solution de fixation ne comprend pas d'acétone ou de composés de la famille des cétones, ni d'acide acétique afin de préserver l'intégrité des hématies.  the fixing solution does not comprise acetone or compounds of the ketone family, nor acetic acid in order to preserve the integrity of the red blood cells.

- l'alcool est de l'éthanol ou de l'isopropanol ;  the alcohol is ethanol or isopropanol;

- l'alcool est un mélange d'éthanol et d'isopropanol ;  the alcohol is a mixture of ethanol and isopropanol;

- la quantité d'alcool est sensiblement inférieure à 45% en volume de la solution de fixation ;  the quantity of alcohol is substantially less than 45% by volume of the fixing solution;

- la quantité de formol est sensiblement comprise entre environ 0,2 et 1 % en volume de la solution de fixation.  the amount of formalin is substantially between about 0.2 and 1% by volume of the fixing solution.

- la solution de fixation comprend un tampon garantissant un pH de la solution de fixation sensiblement compris entre 6,4 et 7,4 ; et  the fixing solution comprises a buffer guaranteeing a pH of the fixing solution substantially between 6.4 and 7.4; and

- la solution de fixation comprend entre 80% et 95% en volume de :  the fixing solution comprises between 80% and 95% by volume of:

- 590 ml de sérum physiologique,  - 590 ml of physiological saline,

- 10 ml de PEG (Carbowax®),  - 10 ml of PEG (Carbowax®),

- 203 ml d'alcool iso-propylique,  203 ml of isopropyl alcohol,

- 193 ml d'éthanol pur,  193 ml of pure ethanol,

- 0.01 % en volume d'azide de sodium,  0.01% by volume of sodium azide,

et entre 20% et 5% en volume de formol tamponné à 4%.  and between 20% and 5% by volume of 4% buffered formalin.

Ainsi, cette solution permet une meilleure conservation des proportions cellulaires, en particulier les dimensions du noyau par rapport aux dimensions de la cellule, et surtout une excellente conservation des hématies.  Thus, this solution allows a better conservation of cellular proportions, in particular the dimensions of the nucleus relative to the dimensions of the cell, and especially an excellent conservation of red blood cells.

En outre, cette solution de fixation est peu inflammable, non toxique et non cancérigène.  In addition, this fixing solution is low in flammability, non-toxic and non-carcinogenic.

L'invention sera mieux comprise à la lecture de la description qui va suivre, donnée uniquement à titre d'exemple, et faite en se référant aux dessins, sur lesquels : - la figure 1 est une photographie d'un échantillon de cellules prélevées d'un sein, ledit échantillon étant conservé dans une solution de fixation selon l'invention, The invention will be better understood on reading the description which follows, given solely by way of example, and with reference to the drawings, in which: FIG. 1 is a photograph of a sample of cells taken from a breast, said sample being preserved in a fixing solution according to the invention,

- la figure 2 est une photographie d'un échantillon de cellules prélevées d'une thyroïde, ledit échantillon étant conservé dans une solution de fixation selon l'invention.  FIG. 2 is a photograph of a sample of cells taken from a thyroid, said sample being preserved in a fixing solution according to the invention.

La présente invention concerne une solution de fixation destinée à préserver in vitro un échantillon cytologique comprenant des cellules biologiques destinées à être analysées par un cytologiste. The present invention relates to an attachment solution for preserving in vitro a cytological sample comprising biological cells for analysis by a cytologist.

La solution de fixation comprend un soluté isotonique de chlorure de sodium, ou sérum physiologique, au lieu de l'eau distillée ou désionisée habituellement utilisée comme base des solutions de fixation couramment utilisées.  The fixing solution comprises an isotonic sodium chloride solution, or physiological saline, instead of the distilled or deionized water usually used as the base of commonly used fixing solutions.

Le sérum physiologique est connu de longue date et comprend du chlorure de sodium dilué dans de l'eau distillée, à raison de 9 grammes de chlorure de sodium pour 1 litre d'eau. Le sérum physiologique est iso-osmolaire ce qui maintient les cellules dans un état d'osmolarité correcte pour leur éviter tout choc osmotique au niveau de la paroi des cellules et donc la destruction des cellules par éclatement des membranes cytoplasmiques et nucléaires. L'osmoralité du sérum physiologique est égale à 308 mosmols par litre.  The physiological saline is known for a long time and comprises sodium chloride diluted in distilled water, at a rate of 9 grams of sodium chloride per liter of water. The saline is iso-osmolar which keeps the cells in a state of correct osmolarity to avoid osmotic shock at the cell wall and thus the destruction of cells by bursting cytoplasmic and nuclear membranes. The osmorability of the physiological serum is equal to 308 mosmols per liter.

L'osmolarité est la concentration d'un milieu. Ceci fait appel à la notion d'osmose, qui est la diffusion d'un solvant à travers une membrane semi-perméable qui sépare deux solutions de concentrations différentes, par exemple la solution fixante et le liquide cytoplasmique des cellules.  Osmolarity is the concentration of a medium. This calls for the concept of osmosis, which is the diffusion of a solvent through a semipermeable membrane which separates two solutions of different concentrations, for example the fixing solution and the cytoplasmic liquid of the cells.

En outre, la solution de fixation comporte de l'alcool pour conserver les cellules en leur état en les fixant dans le milieu alcoolique.  In addition, the fixing solution contains alcohol to keep the cells in their state by fixing them in the alcoholic medium.

De préférence, la quantité d'alcool est sensiblement inférieure à 45% en volume de la solution de fixation afin d'être peu inflammable. Ainsi, cette faible teneur en alcool permet un transport et un stockage plus facile car moins dangereux de la solution de fixation. Par peu inflammable, on entend en particulier que la solution selon l'invention ne nécessite pas l'emploi du pictogramme de sécurité signalant l'inflammabilité d'un produit sur le conditionnement de la solution.  Preferably, the amount of alcohol is substantially less than 45% by volume of the fixing solution to be low in flammability. Thus, this low alcohol content allows easier transport and storage because less dangerous of the fixing solution. By little flammable means in particular that the solution according to the invention does not require the use of the safety pictogram signaling the flammability of a product on the packaging of the solution.

L'alcool est par exemple de l'éthanol ou de l'isopropanol.  The alcohol is, for example, ethanol or isopropanol.

Selon une variante, l'alcool est un mélange d'éthanol et d'isopropanol.  According to one variant, the alcohol is a mixture of ethanol and isopropanol.

Cependant l'alcool seul est un réducteur déshydratant altérant ainsi la taille des cellules par une rétraction nucléaire et cytoplasmique.  However, alcohol alone is a desiccant reducing agent, thus altering cell size by nuclear and cytoplasmic retraction.

Pour pallier cet inconvénient, la solution de fixation comprend en outre du formol pour conserver la taille des cellules. De façon connue et déjà publiée par Saccomanno et collaborateurs, la solution de fixation peut également comprendre du Carbowax® ou Polyéthylène Glycol. De façon connue et déjà publiée, le formol peut être associé à des agents décalcifiants ou antiagrégants tels que l'acide éthylène diamine tetracétique (EDTA) et ses sels. De façon également connue et déjà publiée, un agent mucolytique tel que le dithiothreitol (DTT) ou de l'acetylcystéine peut être ajouté aux prélèvements renfermant du mucus. To overcome this disadvantage, the fixing solution further comprises formalin to maintain the size of the cells. In a known manner and already published by Saccomanno and co-workers, the fixation solution may also include Carbowax® or Polyethylene Glycol. In a known manner and already published, formalin may be combined with decalcifying or anti-aggregating agents such as ethylene diamine tetracetic acid (EDTA) and its salts. Also known and already published, a mucolytic agent such as dithiothreitol (DTT) or acetylcysteine can be added to the specimens containing mucus.

Le formol permet de conserver les hématies et les cellules nucléées sans les lyser garantissant une référence en termes de taille permettant de faire un diagnostic plus pertinent au cytologiste.  Formalin makes it possible to preserve the red blood cells and the nucleated cells without lysing them, guaranteeing a reference in terms of size making it possible to make a more relevant diagnosis to the cytologist.

En particulier, comme on peut le constater sur les figures 1 et 2 de prélèvements cellulaires conservés dans la solution de fixation selon l'invention, les hématies, référencées 1 sur les figures, sont parfaitement conservées et intactes, ce qui permet de réaliser une analyse pertinente de ces prélèvements.  In particular, as can be seen in FIGS. 1 and 2 of cell samples preserved in the fixing solution according to the invention, the red blood cells, referenced 1 in the figures, are perfectly preserved and intact, which makes it possible to carry out an analysis relevant of these samples.

De préférence, la quantité de formol est sensiblement comprise entre environ 0,2 et 1 % en volume de la solution de fixation. A cette concentration, le formol ou formaldhéhyde est considéré comme simplement irritant selon la fiche toxicologique éditée par l'Institut National de Recherche et de Sécurité (INRS) contrairement aux concentrations généralement utilisées de 1 et 25% auxquelles le formol est corrosif et cancérogène. Ainsi, le conditionnement de la solution selon l'invention ne nécessite pas non plus l'emploi du pictogramme de sécurité signalant un produit corrosif.  Preferably, the amount of formalin is substantially from about 0.2 to 1% by volume of the fixing solution. At this concentration, formalin or formaldehyde is considered as simply irritating according to the toxicological data sheet published by the National Institute for Research and Safety (INRS) in contrast to the generally used concentrations of 1 and 25% in which formalin is corrosive and carcinogenic. Thus, the conditioning of the solution according to the invention also does not require the use of the safety pictogram indicating a corrosive product.

Par conséquent, à une concentration inférieure à 1 %, la solution peut être manipulée sans danger, le niveau de précaution étant moins élevé pour le manipuler.  Therefore, at a concentration of less than 1%, the solution can be handled safely, the level of caution being lower to handle it.

De façon connue, la solution de fixation peut comporter un agent antimicrobien. La solution de fixation comporte un tampon garantissant un pH de la solution de fixation sensiblement compris entre 6,4 et 7,4. Cette gamme de pH correspond à des conditions optimales de conservation des cellules et du sang du corps humain.  In known manner, the fixing solution may comprise an antimicrobial agent. The fixing solution comprises a buffer ensuring a pH of the fixing solution substantially between 6.4 and 7.4. This pH range corresponds to optimal conditions for the storage of cells and blood of the human body.

Les exemples suivants illustrent l'invention sans en limiter la portée.  The following examples illustrate the invention without limiting its scope.

Exemple 1 :  Example 1

solution formée à 80% en volume de :  solution formed at 80% by volume of:

- 590 ml de sérum physiologique,  - 590 ml of physiological saline,

- 10 ml de PEG (Carbowax®),  - 10 ml of PEG (Carbowax®),

- 203 ml d'alcool iso-propylique,  203 ml of isopropyl alcohol,

- 193 ml d'éthanol pur,  193 ml of pure ethanol,

- 0.01 % en volume d'azide de sodium,  0.01% by volume of sodium azide,

et à 20 % en volume de formol tamponné à 4%. En variante, la solution peut être formée à 90 % en volume du mélange ci-dessus et à 10 % en volume de formol tamponné à 4% ou à 95 % en volume du mélange ci- dessus et à 5 % en volume de formol tamponné à 4%. and 20% by volume of 4% buffered formalin. Alternatively, the solution can be formed to 90% by volume of the above mixture and 10% by volume of 4% or 95% by volume buffered formalin of the above mixture and 5% by volume of buffered formalin. at 4%.

On notera en outre que la solution de fixation selon l'invention ne contient pas d'acétone ou de composés de la famille des cétones, ni d'acide acétique. Or ces produits lysent les hématies qui, en éclatant libère l'hémoglobine qui se fixe sur les cellules. Certains types de coloration, telle que la coloration de Papanicolaou, du fait de la complexité des agents colorants associant plusieurs colorants nucléaires et de l'hémoglobine, rendent alors l'analyse cytologique, et notamment nucléaire très difficile voire impossible. De même, les études immuno-cyto-chimique sont souvent gênées par les dépôts d'hémoglobine.  It should further be noted that the fixing solution according to the invention does not contain acetone or compounds of the ketone family, nor acetic acid. These products lyse red blood cells which, when they burst, release the hemoglobin that binds to the cells. Certain types of staining, such as the staining of Papanicolaou, because of the complexity of coloring agents associating several nuclear dyes and hemoglobin, make the cytological analysis, and in particular nuclear, very difficult if not impossible. Similarly, immuno-cytochemical studies are often hampered by hemoglobin deposition.

Selon l'invention, les analyses ultérieures de l'échantillon par coloration de Papanicolaou ou les études immuno-cyto-chimique du prélèvement fixé dans la solution de fixation, décrite ici et ne contenant pas d'acétone ou de composés de la famille des cétones, ni d'acide acétique, sont améliorées.  According to the invention, subsequent analyzes of the sample by staining Papanicolaou or immuno-cyto-chemical studies of the sample fixed in the fixing solution, described herein and not containing acetone or compounds of the ketone family or acetic acid are improved.

La solution de fixation décrite ci-dessus permet donc une excellente conservation de l'intégrité des cellules nucléées et des hématies en vue de leur analyse.  The fixing solution described above thus allows excellent preservation of the integrity of nucleated cells and red blood cells for their analysis.

Claims

REVENDICATIONS 1 . - Solution de fixation destinée à préserver in vitro un échantillon cytologique comprenant des cellules nucléées et des hématies, comprenant de l'alcool pour fixer les cellules, du sérum physiologique afin d'éviter les chocs osmotiques au niveau de la paroi des cellules, du formol et du polyéthylène glycol pour conserver la taille et l'intégrité des cellules nucléées et des hématies fixées dans ladite solution, la solution de fixation étant caractérisée en ce qu'elle ne comprend pas d'acétone ou de composés de la famille des cétones, ni d'acide acétique afin de préserver l'intégrité des hématies.  1. - Fixation solution intended to preserve in vitro a cytological sample comprising nucleated cells and red blood cells, comprising alcohol to fix the cells, physiological saline to avoid osmotic shocks on the cell wall, formalin and polyethylene glycol to maintain the size and integrity of nucleated cells and red cells fixed in said solution, wherein the fixing solution is characterized by not including acetone or ketone family compounds, or of acetic acid to preserve the integrity of red blood cells. 2. - Solution de fixation selon la revendication 1 , caractérisée en ce que l'alcool est de l'éthanol ou de l'isopropanol.  2. - Fixing solution according to claim 1, characterized in that the alcohol is ethanol or isopropanol. 3. - Solution de fixation selon la revendication 1 , caractérisée en ce que l'alcool est un mélange d'éthanol et d'isopropanol.  3. - fixing solution according to claim 1, characterized in that the alcohol is a mixture of ethanol and isopropanol. 4. - Solution de fixation selon l'une quelconque des revendications 1 à 3, caractérisée en ce que la quantité d'alcool est sensiblement inférieure à 45% en volume de la solution de fixation.  4. - fixing solution according to any one of claims 1 to 3, characterized in that the amount of alcohol is substantially less than 45% by volume of the fixing solution. 5. - Solution de fixation selon l'une quelconque des revendications 1 à 4, caractérisée en ce que la quantité de formol est sensiblement comprise entre environ 0,2 et 1 % en volume de la solution de fixation.  5. - Fixing solution according to any one of claims 1 to 4, characterized in that the amount of formaldehyde is substantially between about 0.2 and 1% by volume of the fixing solution. 6. - Solution de fixation selon l'une quelconque des revendications 1 à 5, caractérisée en ce qu'elle comprend un tampon garantissant un pH de la solution de fixation sensiblement compris entre 6,4 et 7,4.  6. - fixing solution according to any one of claims 1 to 5, characterized in that it comprises a buffer ensuring a pH of the fixing solution substantially between 6.4 and 7.4. 7. - Solution de fixation selon l'une quelconque des revendications 3 à 6, caractérisée en ce qu'elle comprend entre 80% et 95% en volume de :  7. - fixing solution according to any one of claims 3 to 6, characterized in that it comprises between 80% and 95% by volume of: - 590 ml de sérum physiologique,  - 590 ml of physiological saline, - 10 ml de PEG (Carbowax®),  - 10 ml of PEG (Carbowax®), - 203 ml d'alcool iso-propylique,  203 ml of isopropyl alcohol, - 193 ml d'éthanol pur,  193 ml of pure ethanol, - 0,01 % en volume d'azide de sodium,  0.01% by volume of sodium azide, et entre 20% et 5% en volume de formol tamponné à 4%.  and between 20% and 5% by volume of 4% buffered formalin.
EP11704659A 2010-01-27 2011-01-20 Fixing solution for biological cells Withdrawn EP2528433A1 (en)

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FR1050536A FR2955458B1 (en) 2010-01-27 2010-01-27 FIXING SOLUTION FOR BIOLOGICAL CELLS
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CN111972396A (en) * 2019-05-21 2020-11-24 威海威高医用材料有限公司 Cervical exfoliated cell preservation solution, preparation method and cell preservation method
CN112504793B (en) * 2020-10-14 2021-10-01 核工业总医院 Reagent for permeating and fixing blood cells and analysis method
CN113068683B (en) * 2021-03-03 2022-04-01 北京诚智光辉科技有限公司 Cell preservation solution for liquid-based cell examination and preparation method thereof
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KR102870918B1 (en) * 2024-01-23 2025-10-14 주식회사 이에이치엘바이오 Urine neutralizing liquid composition for preserving cells in urine and urine neutralizing treatment method using the same

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CN102791124A (en) 2012-11-21
RU2012136482A (en) 2014-03-10
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WO2011092414A1 (en) 2011-08-04
FR2955458B1 (en) 2014-09-05

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