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WO2006047252A1 - Solution de conservation de cellules amelioree, et methodes d'utilisation associees - Google Patents

Solution de conservation de cellules amelioree, et methodes d'utilisation associees Download PDF

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Publication number
WO2006047252A1
WO2006047252A1 PCT/US2005/037824 US2005037824W WO2006047252A1 WO 2006047252 A1 WO2006047252 A1 WO 2006047252A1 US 2005037824 W US2005037824 W US 2005037824W WO 2006047252 A1 WO2006047252 A1 WO 2006047252A1
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WIPO (PCT)
Prior art keywords
solution
cells
agent
alcohol
clumping
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PCT/US2005/037824
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English (en)
Inventor
Steven A. Hecht
Wendy E. Swinehart
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Cytyc Corporation
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Publication of WO2006047252A1 publication Critical patent/WO2006047252A1/fr

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    • 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
    • A01N1/122—Preservation or perfusion media
    • A01N1/124—Disinfecting agents, e.g. antimicrobials
    • 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
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients

Definitions

  • a unique cell preservative solution and methods for using that solution are disclosed.
  • the formulation preserves cells in vitro in an ambient liquid suspension; minimizes protein precipitation; reduces cell clumping; selectively eliminates or reduces red blood cell; and retains nucleic acid and protein integrity for further analysis.
  • the present invention relates to a preservative solution for use in the processing of cell and tissue samples and more particularly relates to a novel combination of agents for the preservation of mammalian cell samples for use in preparing specimen slides for microscopic evaluation. It is known in the clinical and research arenas that preservation of cell samples for subsequent analysis is desirable. From a diagnostic standpoint, a specimen is most valuable when it is fresh. The more time that elapses between collection of a specimen and its transfer to a slide or other matrix, the less integrity is retained. Depriving cells of the physiologic conditions of its donor for long periods of time, i.e., minutes, allows autolysis to begin.
  • Cytology is a branch of biology dealing with the study of the formation, structure, morphology, and function of cells. As applied in a laboratory setting, cytologists, cytotechnologists, and other medical professionals make medical diagnoses of a patient's condition based on visual examination of a specimen of the patient's cells.
  • a typical cytological technique is a "pap smear" test, in which cells are scraped from a woman's cervix and analyzed microscopically in order to detect the presence of abnormal cells, a precursor to the onset of cervical cancer. Cytological techniques are also used to detect abnormal cells and disease in other parts of the human body.
  • Cytological techniques are widely employed because collection of cell samples for analysis is generally less invasive than traditional surgical pathological procedures such as biopsies, whereby a tissue specimen is excised from the patient using specialized biopsy needles having spring loaded translatable stylets, fixed cannulae, and the like.
  • Cell samples may be obtained from the patient by a variety of techniques including, for example, by scraping or swabbing an area, or by using a needle to aspirate body fluids from the chest cavity, bladder, spinal canal, or other appropriate area.
  • the tissues are clinically removed from a patient and placed in a container that often contains a preservative and/or fixative and is then transported to the lab for further treatment or conditioning.
  • fixatives were primarily used for the conditioning of the cellular samples.
  • the chemical reagents used as fixatives are those that preserve tissue components for an extended period of time without deterioration.
  • alcohol solutions with or without other additives such as polyethylene glycol and formaldehyde, ranging from 50% to 95% (v/v: methanol, ethanol, isopropanol) are known solutions for use in fixation.
  • v/v methanol, ethanol, isopropanol
  • Protein sedimentation makes the fixed cytologic material difficult to transfer to glass slides for examination, regardless of whether the transfer is done by direct application to the glass slide, by cytofiltration through a small pore filter, or by cytocentrifugation onto glass slides coated with an adhesive such as chrome alum gelatin. Also, alcohol fixatives greater than about 50% (v/v) that are used for collecting and fixing cytologic specimens are not optimum fixatives because the processed cells become distorted in their appearance.
  • cross linking agents such as glutaraldehyde, paraformaldehyde, formalin or formaldehyde
  • Formaldehyde which is a very reactive electrophilic species, fixes tissue by combining with proteins and nucleic acids therein (see e.g., Varshavsky et al., Cell. Jun 17; 53(6):937-47 (1988)).
  • cross linkages inside the preserved tissue prevent the large probe molecules employed in analytical tests, particularly antibodies and oligo- or polynucleotides, from penetrating (see e.g., Ikeda, Journal of Histochemistry and Cytochemistry, Vol.
  • Hybridization can also be done on soluble extracts prepared from tissue or cells for a composition assay (e.g., in gels or blots). Fixative modifications can compromise either the extraction efficiency, or the reactivity of the analyte. For example, fixation may affect the extraction efficiency of nucleic acids or the efficiency of subsequent nucleic acid amplification.
  • PreservCyt ® (Cytyc Corporation, Marlborough, Massachusetts), is
  • alcohol-free solutions are commercially available for preserving cell specimens in the interim between sampling and fixation and/or analysis.
  • a few of these solutions includes Hanks' balanced salt solution, a minimal essential tissue culture medium (MEM), and normal saline.
  • alcohol-free solutions that are the most versatile cannot be stored for a long amount of time or transported over long distances due to problems with contamination by microorganisms and may not preserve cellular morphology over extended periods of time. See, Boon, M. E. and Lykles, C, "Imaginative Approach to Fine Needle Aspiration Cytology," Lancet, 1031-1032 (1980).
  • the cells on the slide have a proper spatial distribution, so that individual cells can be examined.
  • a single layer of cells is typically preferred. Accordingly, preparing a specimen from a fluid sample containing many cells typically requires that the cells first be separated from each other by mechanical dispersion, ffuidic shear, or other techniques so that a thin, monolayer of cells can be collected and deposited on the slide. In this manner, the cytotechnologists can more readily discern abnormal cells. The cells are also able to be counted to ensure that an adequate number of cells have been evaluated. Certain methods and apparatus for generating a thin monolayer of cells on a slide advantageous for visual examination are disclosed in U.S. Pat. No. 5,143,627 issued to Lapidus et al. and entitled "Method and Apparatus for Preparing Cells for Examination;" U.S. Pat. No. 5,240,606 issued to Lapidus et al. and entitled
  • the specimen may be manually visually inspected by a cytotechnologists, typically under magnification, and with or without various sources of illumination.
  • automated machine vision systems have been adapted to aid cytological inspection.
  • an automated vision system may perform a preliminary assessment of the entire slide on which the specimen is disposed to alert the cytotechnologists to potentially the most relevant areas of the slide for close inspection, or may be used to rescreen specimens already analyzed by the cytotechnologists.
  • This invention generally relates to a solution and method for the preservation of cells and tissue.
  • the solution is an alcohol buffer solution for in vitro preservation of mammalian cells at ambient temperatures following removal from a mammalian body, and prior to transferring to a slide, staining or other forms of molecular analysis.
  • a method of preserving cells in a solution comprising the steps of collecting cells from a patient and suspending said cells in a cell preservative solution, said solution comprising about twenty to thirty percent alcohol, an anti-clumping agent in an amount sufficient to prevent the cells from clumping in said solution, and a buffering agent which maintains said solution, with the cells, at a pH of about seven, wherein said solution maintains the structural integrity of cells at ambient temperature in vitro while increasing the solubility of hemoglobin.
  • a method of preserving cells in a solution comprising the steps of collecting cells from a patient and suspending said cells in a cell preservative solution, said solution comprising alcohol in a concentration sufficient to preserve but not fix said cells, an anti-clumping agent in an amount sufficient to prevent the cells from clumping in said solution, and a buffering agent which maintains said solution, with the cells, at a pH of about seven, wherein said solution maintains the structural integrity of said cells at ambient temperature in vitro while increasing the solubility of hemoglobin.
  • a method of preserving cervical cells in a solution comprising the steps of collecting cervical cells from a patient and suspending said cervical cells in a cell preservative solution, said solution comprising alcohol in a concentration sufficient to preserve but not fix said cervical cells, an anti- clumping agent in an amount sufficient to prevent the cervical cells from clumping in said solution, and a buffering agent which maintains said solution, with the cervical cells, at apH of about seven, wherein said solution maintains the structural integrity of said cervical cells at ambient temperature in vitro while increasing the solubility of ⁇ hemoglobin.
  • a method of preserving cells in a solution comprising the steps of collecting cells from a patient; and suspending said cells in a cell preservative solution, said solution comprising about 24% methanol or ethanol by volume, about 0.07% ProClin 300 antibacterial agent, about 3 mM EDTA, about 200 parts per million cholic acid, about 0.1% sodium chloride, about 5 mM potassium chloride, about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.0, wherein said solution maintains the structural integrity of cells at ambient temperature in vitro while increasing the solubility of hemoglobin.
  • a method of preserving cervical cells in a solution comprising the steps of collecting cervical cells from a patient; and suspending said cervical cells in a cell preservative solution, said solution comprising about 24% methanol or ethanol by volume, about 0.07% ProClin 300 antibacterial agent, about 3 mM EDTA, about 200 parts per million cholic acid, about 0.1% sodium chloride, about 5 mM potassium chloride, about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.0, wherein said solution maintains the structural integrity of said cervical cells at ambient temperature in vitro while increasing the solubility of hemoglobin.
  • a method of preserving cells to render them useful for subsequent immunological, genetic, or cytological analysis comprises the steps of collecting cells from a patient suspending said cells in a cell preservative solution, said solution comprising about twenty to thirty percent alcohol an anti-clumping agent in an amount sufficient to prevent the cells from clumping in said solution and a buffering agent which maintains said solution, with the cells, at a pH of about seven, and removing a portion of said preserved cells for immunological, genetic, or cytological analysis.
  • a method of preserving cervical cells to render them useful for subsequent immunological, genetic, or cytological analysis comprises the steps of collecting cervical cells from a patient suspending said cervical cells in a cell preservative solution, said solution comprising about twenty to thirty percent alcohol an anti-clumping agent in an amount sufficient to prevent the cervical cells from clumping in said solution and a buffering agent which maintains said solution, with the cells, at a pH of about seven, and removing a portion of said preserved cervical cells for immunological, genetic, or cytological analysis.
  • a method of preserving cells to render them useful for subsequent immunological, genetic, or cytological analysis comprises the steps of collecting cells from a patient suspending said cells in a cell preservative solution, said solution comprising about 24% methanol or ethanol by volume about 0.07% ProClin 300 antibacterial agent about 3 mM EDTA about 200 parts per million cholic acid about 0.1% sodium chloride about 5 mM potassium chloride about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.O., and removing a portion of said preserved cells for immunological, genetic, or cytological analysis.
  • a method for the preparation of a specimen slide comprising collecting a cell sample from a patient suspending the cell sample in a suitable volume of preservative solution said preservative solution comprising about twenty to thirty percent alcohol an anti-clumping agent in an amount sufficient to prevent the cells from clumping in said solution; and a buffering agent which maintains said solution, with the cells, at a pH of about seven, and applying the preserved, suspended sample to a slide for analysis.
  • a method for the preparation of a specimen slide comprising collecting a cell sample from a patient suspending the cell sample in a suitable volume of preservative solution said preservative solution comprising about 24% methanol or ethanol by volume about 0.07% ProClin 300 antibacterial agent about 3 mM EDTA about 200 parts per million cholic acid about 0.1% sodium chloride about 5 mM potassium chloride about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.0, and applying the preserved, suspended sample to a slide for analysis.
  • a method for the preparation of a specimen slide comprising collecting a cell sample from a patient suspending the cell sample in a suitable volume of preservative solution said preservative solution comprising about twenty to thirty percent alcohol an anti-clumping agent in an amount sufficient to prevent the cells from clumping in said solution; and a buffering agent which maintains said solution, with the cells, at a pH of about seven; and applying the preserved, suspended sample to a slide, wherein said slide is subsequently used for immunological or genetic analysis.
  • a method for the preparation of a specimen slide comprising collecting a cell sample from a patient suspending the cell sample in a suitable volume of preservative solution said preservative solution comprising about 24% methanol or ethanol by volume about 0.07% ProClin 300 antibacterial agent about 3 mM EDTA about 200 parts per million cholic acid about 0.1% sodium chloride about 5 mM potassium chloride about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.0; and applying the preserved, suspended sample to a slide, wherein said slide is subsequently used for immunological or genetic analysis.
  • a cell preservative solution comprising about 24% methanol or ethanol, about 0.07% ProClin 300 antibacterial agent, about 3 mM EDTA, about 200 parts per million cholic acid, about 0.1% sodium chloride, about 5 mM potassium chloride, about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.0.
  • a cell preservative solution comprising about 24% methanol or ethanol, about 0.07%
  • ProClin 300 antibacterial agent about 3 mM EDTA, about 200 parts per million cholic acid, about 0.1% sodium chloride, about 5 mM potassium chloride, about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.0, and said solution maintains the structural integrity of said cells at ambient temperature in vitro while increasing the solubility of hemoglobin.
  • a cervical cell preservative solution comprising about 24% methanol or ethanol, about 0.07% ProClin 300 antibacterial agent, about 3 mM EDTA, about 200 parts per million cholic acid, about 0.1% sodium chloride, about 5 mM potassium chloride, about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.0, and said solution maintains the structural integrity of said cervical cells at ambient temperature in vitro while increasing the solubility of hemoglobin.
  • a cell preservative solution comprising about 24% methanol or ethanol, about 0.07% ProClin 300 antibacterial agent, about 3 mM EDTA, about 200 parts per million cholic acid, about 0.1% sodium chloride, about 5 mM potassium chloride, about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.0, and said solution maintains the structural integrity of said cells at ambient temperature in vitro while increasing the lysis of whole red blood cells.
  • a cell preservative solution comprising about 24% methanol or ethanol, about 0.07%
  • ProClin 300 antibacterial agent about 3 mM EDTA, about 200 parts per million cholic acid, about 0.1% sodium chloride, about 5 mM potassium chloride, about 1 mM calcium acetate, and about 6 mM magnesium acetate at a final pH of 7.0, and said solution maintains the structural integrity of said cells at ambient temperature in vitro while solubilizing undesired protein material from a sample specimen such as blood or mucus.
  • a sample of mammalian cells is provided and, within a specified time frame following biopsy, the cells are suspended in a preservation solution of the invention.
  • the sample is placed in the preservation solution to solubilize undesired proteins, such as blood or mucous, from the cell sample.
  • the clean sample may then be transported in the inventive solution for subsequent analysis and/or storage.
  • an aqueous alcohol-buffer solution for maintaining the structural integrity of mammalian cells in vitro, while increasing the solubility of undesirable proteins such as blood or hemoglobin, is provided; said preservative solution comprising about twenty to thirty percent alcohol an anti-clumping agent in an amount sufficient to prevent the cells from clumping in said solution; and a buffering agent which maintains said solution, with the cells, at a pH of about seven.
  • the preserved cells may subsequently be used for immunological, genetic, or cytological analysis or applied to a slide for analysis.
  • the present invention generally relates to an alcohol-buffer solution and methods for the preservation of mammalian cells in suspension at ambient temperature.
  • the solution enhances maintenance of the nuclear and cytoplasmic structure of the cells, in that it maintains cell membranes intact for subsequent cytological staining and maintains nucleic acid or protein integrity for other molecular or immunological analysis.
  • the solution also effectively destroys microbial pathogens, inhibits retroviral activity, and solubilizes undesired protein material from the sample such as blood or mucus.
  • the invention also provides for methods and kits for preparation of a specimen slide by applying the preserved, suspended mammalian cells to a slide for analysis.
  • the preservative of the present invention comprises three components with optional fourth, fifth, and sixth components.
  • a first component is a preservative that maintains cellular DNA and protein integrity and retains the detail of the cell and it's nucleus for subsequent cytological staining, analysis, and molecular diagnosis.
  • the preservative is an alcohol and a preferred alcohol is methanol. Other alcohols that may be used include isopropanol and ethanol among others.
  • the alcohol is present in an amount of approximately 20% to 50%, or 20%- 40%, or 20% to 30% by volume
  • the alcohol is present in an amount of approximately 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%.
  • Solutions containing greater than 50% alcohol tend to exhibit cell clumping, and/or protein coagulation, which interferes with the subsequent ability to affectively stain the sample cells.
  • the concentration of alcohol in this embodiment is at 20% or below, the cells are not sufficiently fixed for relatively long- term preservation, causing the cells to degrade over time, hi a preferred embodiment, the solution contains approximately 24% methanol, by volume.
  • the preservative of the present invention also removes undesired protein material from a sample specimen such as blood or mucus.
  • a sample specimen such as blood or mucus.
  • the term "increasing the solubility of hemoglobin” as used herein is defined as meaning a reduction in the amount of visible blood in a sample.
  • the reduction in the amount of visible blood in a sample can occur through a number of means including, but not limited to the lysing of red blood cells and the increased solubility of red blood cell proteins, including hemoglobin.
  • Solubility is defined as the amount of solute that can be dissolved in a solvent.
  • the solubility of different proteins i.e. solutes
  • Buffer or solvent type, pH, ionic strength, and temperature all affect protein solubility. Changes in these attributes of a solvent will change the solubility of proteins therein.
  • Hemoglobin is one of the major protein components of blood cells.
  • the adult form of the protein contains 4 polypeptide chains, 2 of one kind ( ⁇ ) and 2 of another ( ⁇ ), held together by non-covalent interactions.
  • Each chain has an iron-containing heme group which is the binding site for oxygen.
  • the solubility of hemoglobin, as with other proteins, is dependent on the solvent being used.
  • Hemoglobin has an isoelectric point of 6.8, which is the pH at which the molecule is electrically neutral. At a pH above 6.8, hemoglobin has a net negative charge, and below 6.8, a net positive charge.
  • one preferred embodiment of the present invention is a cell preservative solution that fixes cellular samples while simultaneously allowing the maintenance of the nuclear and cytoplasmic structure of the cells by maintaining cell membranes intact for subsequent cytologicai staining as well as solubilizing undesired protein material from the sample such as blood or associated blood proteins.
  • a second component of the preservative is an anti-clumping agent in an amount sufficient to prevent cell clumping.
  • anti-clumping agent as used herein is defined as meaning an agent that prevents the reaggregation of cells after they have been dispersed into a solution.
  • the anti-clumping agent is the chelating agent ethylene diamine tetraacetate (EDTA), with the preferred form being the disodium salt.
  • EDTA ethylene diamine tetraacetate
  • Other EDTA salts comprising potassium, cesium, rubidium, and various organic cations may also be effective.
  • Other effective anti-clumping agents include, but are not limited to other derivatives of tetraacetic acid such as l,2-bis(2-aminophenoxy) ethane-N,N,N',N'- tetraacetic acid (BAPTA), ethylene glycol bis(2-aminoethyl ether)-N,N,N'N' -tetraacetic acid (EGTA) and trans- 1,2-diamino cyclohexane-N,N,NTSf '-tetraacetic acid (CDTA). Salts of these compounds, which are soluble in the preservative at the preservative concentration, may also effective.
  • BAPTA l,2-bis(2-aminophenoxy) ethane-N,N,N',N'- tetraacetic acid
  • EGTA ethylene glycol bis(2-aminoethyl ether)-N,N,N'N' -tetraacetic acid
  • CDTA trans
  • a third component is a buffer for adjusting the pH of the solution to help retain characteristic morphology of the cells.
  • the buffer used in the inventive solution has a large buffering range to accommodate for the change in pH resulting from autolytic by ⁇ products from the sample cells suspended in the solution.
  • a solution having a broad buffering range can be used for a wide range of cell types and is optimal for the solution of the invention.
  • Exemplary cells or fluids for which this solution can be used include cervical cells, white blood cells, bronchial cells, urine, ductal lavage, nipple aspirate, and sputum, among other cells and body fluids.
  • the buffer has a pH preferably in the range of about 6 to about 7 but alternatively in the range of about 5 to about 6 or about 7 to about 8.
  • a preferred buffer is an acetate buffer, such as sodium acetate, magnesium acetate, calcium acetate, and combinations thereof.
  • buffers such as N-Q.- Acetamido)-2-aminoethanesulfonic acid (ACES), iV-(2-Acetamido)iminodiacetic acid (ADA), bis(2-Hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-TRIS), 2- Morpholinoethanesulfonic acid (MES), and Piperazine-l,4-bis(2-ethanesulfonic acid (PIPES) may be used, the effective buffering range of these buffers is deemed to be not as broad at the desired PH as that of acetate.
  • An optional fourth component is a substance to maintain the ionic strength within limits that inhibit cell distortion.
  • a specific example is KCl (e.g., at a suggested concentration of about 5mM of the total preservative), and it must be both soluble in the preservative (e.g., methanol) and not cause precipitation of the anti-clumping agent (e.g., sodium EDTA or derivatives).
  • the substance for maintaining the ionic strength may be an additional amount of the buffer previously added or another compatible buffer.
  • An optional fifth component is an anti-microbial that kills pathogens.
  • pathogens For example, in test samples the preservative effectively kills the following organisms: Candida albicans, Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.
  • ProClin® 300 (Rohm-Hass, Philadelphia, Penn.) ranging from about 0.01 to about 0.1%. This anti-microbial component is optional depending on the time lapse between collection, shipment and analysis.
  • An optional sixth component is a mucolytic agent.
  • a mucolytic agent is used to liquefy mucoid cytology specimens to optimize cytological analysis. Excess mucous in a sample can precipitate in alcoholic fixatives and thus may interfere with subsequent slide preparation, staining and analysis.
  • a mucolytic agent dissolves or breaks up clumps of mucous which may contain cells of interest. Examples of mucolytic agents are methyl cysteine, N-acetyl-L-cysteine, dithiothreitol, dihydroxy dithiolbutane, or other agents which are able to break or reduce disulfide bonds.
  • a detergent may be used in the preservative.
  • the detergent may be non-ionic, cationic, anionic or zwitterionic. Mixtures of detergents may also be used.
  • Exemplary classes of detergents include alcohol ether sulfates, alcohol sulfates, alkanolamides, alkyl sulfonates, amine oxides, amphoteric detergents, anionic detergents, betaine derivatives, cationic detergents, disulfonates, dodecylbenzene sulfonic acid, ethoxylated alcohols, ethoxylated alkyl phenols, ethoxylated fatty acids, glycerol esters hydrotropes, lauryl sulfates, mono and diglycerides, non-ionic detergents, phosphate esters, quaternary detergents, and sorbitan derivatives.
  • the present invention also provides for methods of preserving cells in a solution.
  • the method comprising the steps of collecting cells from a patient and suspending the cells in a cell preservative solution.
  • the preservative solution may be comprised of about twenty to thirty percent alcohol, an anti-clumping agent in an amount sufficient to prevent the cells from clumping in the solution, and a buffering agent which maintains said solution, with the cells, at apH of about seven, wherein said solution maintains the structural integrity of cells at ambient temperature in vitro while increasing the solubility of hemoglobin.
  • Cells of the present invention can be from any source of biological material that can be obtained from a living organism directly or indirectly, including cells, tissue or fluid.
  • the sample include blood, urine, semen, milk, sputum, mucus, plueral fluid, pelvic fluid, sinovial fluid, ascites fluid, body cavity washes, eye brushing, skin scrapings, a buccal swab, a vaginal swab, a pap smear, a rectal swab, an aspirate, a needle biopsy, a section of tissue obtained for example by surgery or autopsy, plasma, serum, spinal fluid, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, tumors, organs, a microbial culture, a virus, and samples of in vitro cell culture constituents.
  • the preservative of the present invention can be used with cells collected from the cervix, the breast (including ductal lavage), urinary tract malignancies (both biopsy tissue samples and urine cytology smears), colon, lung, bladder, skin, larynx, esophagus, bronchus, lymph nodes, and haematological malignancies.
  • the preservative may additionally be employed in assessment of pre-malignant abnormalities of cervical squamous epithelial cells (squamous intra-epithelial lesion, SIL) or pre-malignant abnormalities in other tissues.
  • the preservative of the present invention maybe particularly appropriate for employment in cytological or biochemical assessment of other clinical specimens where detection of neoplastic cells, or their distinction from cells showing reactive changes, can be very difficult.
  • specimens include sputum, bronchio-alveolar lavage specimens, urine and brushings from the alimentary tract (including oesophagus, stomach and pancreas, both bile duct and pancreatic duct).
  • the present invention may be applied in histological or biological assessment of tissue where assessment of proliferation may enable more accurate prediction of clinical outcome, and/or more rational selection of therapy.
  • Cell samples may be obtained from the patient by a variety of techniques including, for example, by scraping or swabbing an area, or by using a needle or catheter to aspirate body fluids from the chest cavity, bladder, breast duct, spinal canal, or other appropriate area.
  • the cell samples are placed in solution and subsequently collected and transferred to a glass slide for viewing under magnification.
  • Fixative and staining solutions may be applied to the cells on the glass slide for preserving the specimen for archival purposes and for facilitating examination.
  • preparing a specimen from a fluid sample containing many cells typically requires that the cells first be separated from each other by mechanical dispersion, fluidic shear, or other techniques so that a thin, monolayer of cells can be collected and deposited on the slide. In this manner, the cytotechnologists can more readily discern abnormal cells. The cells are also able to be counted to ensure that an adequate number of cells have been evaluated.
  • Samples may be removed from the body using any convenient means and technique.
  • a spatula or swab may be used to remove endothelium cells, e.g. from the cervix or buccal cavity.
  • Blood and other fluid samples may be removed using a syringe or needle.
  • Other tissue samples may be removed by biopsy or tissue section.
  • An automated processor such as the ThinPrep ® 2000 Processor (Cytyc
  • the patient's cells in a preservative fluid in a sample container are dispersed using a spinning sample collector disposed therein.
  • a controlled vacuum is applied to the sample collector to draw the fluid through a screen filter thereof until a desired quantity and spatial distribution of cells is collected against the filter.
  • the sample collector is removed from the sample container and the filter portion impressed against a glass slide to transfer the collected cells to the slide in substantially the same spatial distribution as collected.
  • the specimen may be manually visually inspected by a cytotechnologists, typically under magnification, and with or without various sources of illumination.
  • automated machine vision systems have been adapted to aid cytological inspection.
  • the preservative of the present invention may also be used in the preparation of a specimen for selective staining of a macromolecular species (protein, nucleic acid) or a smaller molecule (protein adduct, drug, metabolite, signal transduction species, lipid, etc.). Analysis of preserved tissue is often performed using an antibody that binds specifically and with high affinity to the analyte in the tissue.
  • a detectable complementary oligo- or poly-nucleotide sequence can be used for hybridization. Hybridization can be done on intact cell structures (in situ) for cytometric assay (e.g., by microscopy or flow cytometry).
  • a variety of analytical tests can be performed with better sensitivity and quality control when practiced either directly upon biological samples prepared using the present invention, or upon extracts prepared therefrom. These tests comprise the categories of immunoassays (e.g., IHC, flow immunocytochemistry, ELISA, immunoprecipitation, immunoblotting), assays for nucleic acid quantitation and sequence without amplification (e.g.
  • amplification methods e.g., PCR, in situ PCR, solution PCR, RT-PCR, ligase chain reaction, strand displacement amplification, NASBA
  • chromatographic methods e.g., gas or liquid phase analyte transport
  • electrophoretic methods capillary, slab gel
  • photometric methods e.g., UV or visible or infra-red spectrophotometry, fluorimetry
  • other methods for analysis of molecular compositions e.g., mass spectroscopy, NMR.
  • room temperature (approximately 15-30° C) is approximately three weeks. This duration
  • the remaining cell-preserving viability of the solution may be limited.
  • a cell sample or body fluid is obtained from a patient or other cell source.
  • a preservation solution of the type described above is placed either in a vial, on a welled slide, or on an appropriate membrane.
  • the collected cells are then placed in the solution, preferably within one minute following collection. The sooner the collected cells are placed in the preservative solution, the longer the cells can be preserved at ambient temperature suspended in the solution, since the trauma to the cells is minimized.
  • a device can be used to remove suspended cells, along with the suspension preservation medium, and place them on a slide or other appropriate surface for further processing.
  • the function of the calcium and magnesium ions is the preservation of nuclear morphology of cytologically significant cells.
  • the acetate is present as a buffer that will both stabilize the pH of the solution, and not form precipitates of calcium and magnesium. Such precipitation would happen with a phosphate buffer.
  • the sodium and potassium salts are present to help stabilize the cells and prevent precipitation and coagulation of hemoglobin and other serum proteins.
  • the methanol is present to aid in the lysing of red blood cells, to act as a preservative against bacterial growth, and to help preserve cytologically significant cells.

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Abstract

L'invention concerne une solution tampon aqueuse à base d'alcool, permettant la conservation in vitro, à température ambiante, de cellules mammifères pendant une période déterminée. La solution selon l'invention contient: de 20 à 30 % d'alcool; un agent anti-agglutination; et un agent tampon destiné à maintenir la solution, ainsi que les cellules, à un pH d'environ 7.
PCT/US2005/037824 2004-10-26 2005-10-21 Solution de conservation de cellules amelioree, et methodes d'utilisation associees WO2006047252A1 (fr)

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US60,622,100 2004-10-26

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013011000A1 (fr) * 2011-07-19 2013-01-24 Ovizio Imaging Systems NV/SA Procédé et dispositif holographique pour diagnostics cytologiques
US9684281B2 (en) 2011-07-19 2017-06-20 Ovizio Imaging Systems NV/SA Method and system for detecting and/or classifying cancerous cells in a cell sample
CN107250346A (zh) * 2015-06-30 2017-10-13 希森美康株式会社 细胞保存液及其利用、以及细胞保存液的制造方法
US9846151B2 (en) 2011-11-21 2017-12-19 Ovizio Imaging Systems NV/SA Sample vial for digital holographic analysis of a liquid cell sample
US9904248B2 (en) 2012-09-20 2018-02-27 Ovizio Imaging Systems NV/SA Digital holographic microscope with fluid systems
US10578541B2 (en) 2012-02-13 2020-03-03 Ovizio Imaging Systems NV/SA Flow cytometer with digital holographic microscope
CN111238890A (zh) * 2020-01-16 2020-06-05 江西业力医疗器械有限公司 基于离心法的液基制片技术用于液基真菌的检测方法
US11067379B2 (en) 2016-01-19 2021-07-20 Ovizio Imaging Systems NV/SA Digital holographic microscope with electro fluidic system, said electro-fluidic system and methods of use
CN113647377A (zh) * 2021-09-07 2021-11-16 华东医院 一种组织标本管
CN113884671A (zh) * 2021-09-27 2022-01-04 苏州东岭生物技术有限公司 一种流式染色试剂盒及其配置方法和应用方法
CN116195575A (zh) * 2022-12-29 2023-06-02 广州普希盛科技有限公司 一种液基细胞保存液及其制备方法和应用
EP4413857A4 (fr) * 2021-10-08 2025-04-09 Daan Gene Co Ltd Solution de conservation de cellules à base liquide et son procédé de préparation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0511430A2 (fr) * 1991-05-01 1992-11-04 Cytyc Corporation Solution pour la préservation de cellules
EP1085098A2 (fr) * 1999-09-10 2001-03-21 Becton Dickinson and Company Un composé pour fournir la stabilité à longue échéance aux cellules pour les essais diagnostiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0511430A2 (fr) * 1991-05-01 1992-11-04 Cytyc Corporation Solution pour la préservation de cellules
EP0772972A1 (fr) * 1991-05-01 1997-05-14 Cytyc Corporation Solution pour la préservation des cellules
EP1085098A2 (fr) * 1999-09-10 2001-03-21 Becton Dickinson and Company Un composé pour fournir la stabilité à longue échéance aux cellules pour les essais diagnostiques

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9684281B2 (en) 2011-07-19 2017-06-20 Ovizio Imaging Systems NV/SA Method and system for detecting and/or classifying cancerous cells in a cell sample
WO2013011000A1 (fr) * 2011-07-19 2013-01-24 Ovizio Imaging Systems NV/SA Procédé et dispositif holographique pour diagnostics cytologiques
US10025271B2 (en) 2011-07-19 2018-07-17 Ovizio Imaging Systems NV/SA Method and system for detecting and/or classifying cancerous cells in a cell sample
US10060905B2 (en) 2011-11-21 2018-08-28 Ovizio Imaging Systems NV/SA Liquid medium and sample vial for use in a method for detecting cancerous cells in a cell sample
US9846151B2 (en) 2011-11-21 2017-12-19 Ovizio Imaging Systems NV/SA Sample vial for digital holographic analysis of a liquid cell sample
US10578541B2 (en) 2012-02-13 2020-03-03 Ovizio Imaging Systems NV/SA Flow cytometer with digital holographic microscope
US9904248B2 (en) 2012-09-20 2018-02-27 Ovizio Imaging Systems NV/SA Digital holographic microscope with fluid systems
CN107250346B (zh) * 2015-06-30 2019-11-15 希森美康株式会社 细胞保存液及其利用、以及细胞保存液的制造方法
CN107250346A (zh) * 2015-06-30 2017-10-13 希森美康株式会社 细胞保存液及其利用、以及细胞保存液的制造方法
US11067379B2 (en) 2016-01-19 2021-07-20 Ovizio Imaging Systems NV/SA Digital holographic microscope with electro fluidic system, said electro-fluidic system and methods of use
CN111238890A (zh) * 2020-01-16 2020-06-05 江西业力医疗器械有限公司 基于离心法的液基制片技术用于液基真菌的检测方法
CN111238890B (zh) * 2020-01-16 2024-03-19 南昌准好生物科技有限公司 基于离心法的液基制片技术用于液基真菌的检测方法
CN113647377A (zh) * 2021-09-07 2021-11-16 华东医院 一种组织标本管
CN113884671A (zh) * 2021-09-27 2022-01-04 苏州东岭生物技术有限公司 一种流式染色试剂盒及其配置方法和应用方法
EP4413857A4 (fr) * 2021-10-08 2025-04-09 Daan Gene Co Ltd Solution de conservation de cellules à base liquide et son procédé de préparation
CN116195575A (zh) * 2022-12-29 2023-06-02 广州普希盛科技有限公司 一种液基细胞保存液及其制备方法和应用

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