WO2013038306A1 - System and kit for preparing a cytological sample for examination - Google Patents
System and kit for preparing a cytological sample for examination Download PDFInfo
- Publication number
- WO2013038306A1 WO2013038306A1 PCT/IB2012/054606 IB2012054606W WO2013038306A1 WO 2013038306 A1 WO2013038306 A1 WO 2013038306A1 IB 2012054606 W IB2012054606 W IB 2012054606W WO 2013038306 A1 WO2013038306 A1 WO 2013038306A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sample
- kit
- support means
- stain
- aforementioned
- Prior art date
Links
- 230000002380 cytological effect Effects 0.000 title claims abstract description 33
- 210000004027 cell Anatomy 0.000 claims abstract description 178
- 239000000834 fixative Substances 0.000 claims abstract description 37
- 230000001086 cytosolic effect Effects 0.000 claims abstract description 21
- 239000003607 modifier Substances 0.000 claims abstract description 15
- 230000004048 modification Effects 0.000 claims abstract description 5
- 238000012986 modification Methods 0.000 claims abstract description 5
- 239000000523 sample Substances 0.000 claims description 161
- 238000002360 preparation method Methods 0.000 claims description 65
- 239000000203 mixture Substances 0.000 claims description 61
- 238000000034 method Methods 0.000 claims description 59
- 238000010186 staining Methods 0.000 claims description 46
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 239000003795 chemical substances by application Substances 0.000 claims description 22
- 210000000805 cytoplasm Anatomy 0.000 claims description 20
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 18
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims description 17
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 17
- 239000006185 dispersion Substances 0.000 claims description 12
- 229960000583 acetic acid Drugs 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 206010028980 Neoplasm Diseases 0.000 claims description 9
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims description 9
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 8
- 201000011510 cancer Diseases 0.000 claims description 8
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 8
- 239000002953 phosphate buffered saline Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 7
- 230000007935 neutral effect Effects 0.000 claims description 7
- 210000001519 tissue Anatomy 0.000 claims description 7
- 239000012472 biological sample Substances 0.000 claims description 6
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- 210000003679 cervix uteri Anatomy 0.000 claims description 4
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- 238000000151 deposition Methods 0.000 claims description 4
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 claims description 4
- 238000011179 visual inspection Methods 0.000 claims description 4
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 claims description 3
- VVAVKBBTPWYADW-RVTJCSDESA-L biebrich scarlet Chemical compound [Na+].[Na+].OC1=CC=C2C=CC=CC2=C1\N=N\C(C(=C1)S([O-])(=O)=O)=CC=C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 VVAVKBBTPWYADW-RVTJCSDESA-L 0.000 claims description 3
- AXIKDPDWFVPGOD-UHFFFAOYSA-O [7-(dimethylamino)phenothiazin-3-ylidene]-dimethylazanium;2-(2,4,5,7-tetrabromo-3,6-dihydroxyxanthen-10-ium-9-yl)benzoic acid Chemical compound C1=CC(=[N+](C)C)C=C2SC3=CC(N(C)C)=CC=C3N=C21.OC(=O)C1=CC=CC=C1C1=C(C=C(Br)C(O)=C2Br)C2=[O+]C2=C1C=C(Br)C(O)=C2Br AXIKDPDWFVPGOD-UHFFFAOYSA-O 0.000 claims description 2
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- XJCPMUIIBDVFDM-UHFFFAOYSA-M nile blue A Chemical compound [Cl-].C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4[O+]=C3C=C(N)C2=C1 XJCPMUIIBDVFDM-UHFFFAOYSA-M 0.000 claims description 2
- 238000011275 oncology therapy Methods 0.000 claims description 2
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 claims description 2
- 238000002560 therapeutic procedure Methods 0.000 claims description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims 3
- YYYARFHFWYKNLF-UHFFFAOYSA-N 4-[(2,4-dimethylphenyl)diazenyl]-3-hydroxynaphthalene-2,7-disulfonic acid Chemical compound CC1=CC(C)=CC=C1N=NC1=C(O)C(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=C12 YYYARFHFWYKNLF-UHFFFAOYSA-N 0.000 claims 1
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- 239000001267 polyvinylpyrrolidone Substances 0.000 description 11
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- 150000001875 compounds Chemical class 0.000 description 10
- 206010008342 Cervix carcinoma Diseases 0.000 description 9
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 9
- 201000010881 cervical cancer Diseases 0.000 description 9
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- 230000008569 process Effects 0.000 description 9
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- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 4
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- LOMVENUNSWAXEN-UHFFFAOYSA-N Methyl oxalate Chemical compound COC(=O)C(=O)OC LOMVENUNSWAXEN-UHFFFAOYSA-N 0.000 description 2
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- 229940051132 light green sf yellowish Drugs 0.000 description 2
- 150000003891 oxalate salts Chemical class 0.000 description 2
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 2
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- 238000011120 smear test Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- ZNCPFRVNHGOPAG-UHFFFAOYSA-L sodium oxalate Chemical compound [Na+].[Na+].[O-]C(=O)C([O-])=O ZNCPFRVNHGOPAG-UHFFFAOYSA-L 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- XFTALRAZSCGSKN-UHFFFAOYSA-M sodium;4-ethenylbenzenesulfonate Chemical compound [Na+].[O-]S(=O)(=O)C1=CC=C(C=C)C=C1 XFTALRAZSCGSKN-UHFFFAOYSA-M 0.000 description 2
- ILJSQTXMGCGYMG-UHFFFAOYSA-N triacetic acid Chemical compound CC(=O)CC(=O)CC(O)=O ILJSQTXMGCGYMG-UHFFFAOYSA-N 0.000 description 2
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 description 2
- LBBAKTMYSIFTBS-UHFFFAOYSA-N 3-[(4-aminophenyl)diazenyl]benzene-1,2-diamine Chemical compound C1=CC(N)=CC=C1N=NC1=CC=CC(N)=C1N LBBAKTMYSIFTBS-UHFFFAOYSA-N 0.000 description 1
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- 241000701806 Human papillomavirus Species 0.000 description 1
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- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
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- BDFZFGDTHFGWRQ-UHFFFAOYSA-N basic brown 1 Chemical compound NC1=CC(N)=CC=C1N=NC1=CC=CC(N=NC=2C(=CC(N)=CC=2)N)=C1 BDFZFGDTHFGWRQ-UHFFFAOYSA-N 0.000 description 1
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- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
- 229920001495 poly(sodium acrylate) polymer Polymers 0.000 description 1
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Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
-
- 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
- G01N1/31—Apparatus therefor
- G01N1/312—Apparatus therefor for samples mounted on planar substrates
Definitions
- the invention relates to the field of microscopic examination of cytological samples.
- Cervical cancer is the second most common cancer in women worldwide and the leading cause of cancer deaths in women in developing countries. About 30% of cancers in women are due to cervical cancer with more than 100,000 new cases diagnosed every year, e.g., in India. The estimated compounded annual growth rate (CAGR) for cervical cancer cases is 2.56% and at this growth rate approximately 175,000 new cases of cervical cancer will be detected in the year 2012.
- CAGR compounded annual growth rate
- One of the recommended tools for screening of cervical cancer is to detect cytological precursors of cancer in Papanicolaou tests (also called Pap-smear, Pap-test, cervical smear, or smear test), which is a screening test used in gynaecology to detect premalignant and malignant processes in the cervical canal especially in the transformation zone.
- Papanicolaou tests also called Pap-smear, Pap-test, cervical smear, or smear test
- a speculum is used to gather cells from the outer opening of the cervix of the uterus and the endocervix.
- the cells are examined under a microscope to look for abnormalities.
- the test aims to detect potentially pre-cancerous changes, which are, among others, caused by sexually transmitted human papillomaviruses.
- the test remains an effective, widely used method for early detection of pre-cancer and cervical cancer.
- the test may also detect infections and abnormalities in the endocervix and endometrium.
- a system or kit for preparing a cytological sample for examination comprises a fixative for fixing cells comprised in said sample, a cell surface modifier for modification of the surface of cells comprised in said sample, a first sample support means having at least two sides, and a second sample support means having at least two sides, wherein on at least one side of at least one of the support means a cytoplasmic stain or a nuclear stain is deposited.
- cytological sample is defined as any specimen from an organism, preferably a mammal, in sufficient amount to be characterized and/or analyzed.
- a cytological sample includes cell samples, skin samples, tissue samples, and mucosal samples.
- the term "modification of the surface of cells” means that cells are treated in such way that they are unfolded and/or uncurled, and/or that cell overlapping is reduced. Reduction of cell folding and curling during sample preparation will result in enhanced visualization of cellular and morphological details that are used for the subsequent detection of abnormal cells in the sample, e.g. in cervical cancer screening.
- both aims can be achieved by increasing the surface charge on the cells, which leads to an increased repulsion between individual cells and thus provides better dispersion.
- Such system or kit provides the option to prepare a cytological sample in such way that cells in the sample are dispersed in a manner suitable for viewing through a microscope or for analysis by an automated optical cell analysis device.
- one important feature of the invention is that it allows on the spot staining of a given sample, i.e., at the Point of Care. Further, the need to prepare a staining solution at the site is eliminated, as the stains are pre-dispensed in appropriate quantity on the sample support means. Further, the need of going through a complex protocol and a multiple step staining procedure is eliminated, as well as muck problems which are basically due to the dust and impurities during staining. Further, aberrations like over-staining or under-staining are minimized due to the fact that the stains are dispensed in exact measure as required for optimal staining.
- a method for preparing a cytological sample for examination comprising the steps of:
- fixing cells comprised in said sample with a fixative
- first sample support means having at least two sides
- second sample support means having at least two sides, wherein on at least one side of at least one of the support means a cytoplasmic stain or a nuclear stain is deposited
- the invention comprises the use of at least one type of prefabricated sample support means.
- These prefabricated sample support means are coated with a nuclear stain or with a cytoplasmic stain.
- a nuclear stain is deposited on at least one side of the first sample support means and a cytoplasmic stain is deposited on at least one of the second sample support means.
- only one of the two sample support means is coated with a stain (e.g., nuclear stain), and the other type of stain /e.g., the cytoplasmic stain) is added to the cell suspension, e.g. together with the fixative and/or the cell surface modifier.
- the first sample support means and/or the second sample support means are in the form of a slide and/or a cover slip.
- sample means a small transparent plate made from glass or plastic on which samples, like cells can be deposited for examination under an optical magnification device, like a microscope.
- cover slip means a small transparent plate made from glass or plastic which is used to cover samples, like cells, prior to examination under an optical magnification device, like a microscope.
- slides and cover slips differ from one another in size and thickness.
- microscopic slides Under ISO 8255-2, microscopic slides have a size of 26 x 76 mm, and a thickness of 1 mm, while cover slips usually have a size of 18 x 18 mm and a thickness of 100 - 200 ⁇ .
- cover slip and slide can be used interchangeable.
- microscopic slides are used to both support the sample and to cover it, after the smear process has been carried out.
- the first sample support means and the second sample support means are connected to one another by means of a hinge.
- a hinge can, for example, consist of a rebated joint provided between two plastic frames carrying the first and second sample support means.
- Other possibilities to provide such hinge comprise the use of a piece of adhesive tape connecting the first and second sample support means. The skilled person will be able to find other technical embodiments which make use of such hinge without the use of inventive step.
- the fixative and/or the cell surface modifier are provided in liquid form.
- the fixative comprises at least one agent selected from the group consisting of
- the fixative comprises a mixture including ethanol, isopropyl alcohol and Acetic acid, preferably in a volume ratio of 7:2: 1.
- the cell surface modifier comprises at least one agent selected from the group consisting of
- the agent for rendering a positive charge to the cell surface is preferably at least one water-soluble compound selected from the group consisting of Poly-l-Lysin hydrochloride and Polyvinylpyrrolidone (PVP).
- PVP Polyvinylpyrrolidone
- the agent for rendering a negative charge to the cell surface is preferably at least one water-soluble compound selected from the group consisting of Dextran sulfate, Poly sodium 4-styrenesulfonate, Polymethylacrylic acid, Carboxymethyl cellulose and/or a Sodium Polyacrylate.
- the said polymers can be used in different molecular weight and/or different concentrations.
- High molecular weight polymers seem to work well for imparting high charge on the cells.
- high concentration e.g. : 5-10 mg/ml of PVP in alcohol fixative works good
- shows increased charge on cells surface see Fig. 5.
- the chelating agent and/or the anti-coagulating agent is preferably at least one selected from the group consisting of Ethylenediamine tetraacetic acid (EDTA), Hydroxyethylenediamme triacetic acid (HEDTA), Nitriolotriacetic acid (NTA), Sodium citrate, and/or disodium oxalate or dimethyl oxalate, or other citrates or oxalates.
- the demucifying agent is preferably at least one selected from the group consisting of Sodium Hydroxide, N-Acetyl-L-Cystein, and/or Sodium Hypochlorate.
- the cell surface modifier comprises 2 - 5 % w/v NaOH and 0.25g/50ml N-Acetyl- L-Cystein, or 0.5 - 6 % w/v NaOCl.
- the agent for supporting cell dispersion and/or the agent for creating micropores on the cell surface is preferably a detergent, even more preferably at least one selected from the group consisting of Dithiothreitol and/or TritonX-100. Presence of these agents creates controlled pores in the cellular membranes, leading to fast access of stain to the cytoplasm and the nucleus.
- the mentioned compounds support proper fixation, avoid coagulation and enhance a good dispersion of cell samples. They do not affect the staining of cells with Methylene Blue, Eosin Azure (EA) and Orange G (see Fig. 6), and help that cells stay stable for almost a week (see Fig. 7).
- a cell preparation mixture which comprises at least the fixative and the cell surface modifier.
- the steps of fixing the cells comprised in said sample and modifying the surface of the cells comprised in said sample are carried out simultaneously.
- the cell preparation mixture serves to simultaneously fix the cells and modify the surface of the cells in the sample.
- fixative and/or the cell surface modifier can furthermore comprise a stain.
- the cytoplasm stain comprises at least one selected from the group of:
- a smear sample is for example similar or identical to those samples used in the Papanicolaou tests (also called Pap smear, Pap test, cervical smear, or smear test).
- a tissue slice is for example provide by a microtome.
- a liquid sample can preferably consist of a suspension of cells, e.g., obtained by a smear.
- Suitable samples comprise, but are not restricted to, fine needle aspiration cytology (FNAC) samples, abrasive cytology samples and/or exfoliated samples.
- FNAC fine needle aspiration cytology
- a sample is indeed placed on a slide to make it available for investigation, e.g. a tissue slice, or a smear.
- other devices can also be used to carry a sample, e.g. a small cuvette in case the sample is a liquid sample, or a cartridge in case the sample is a brush sample.
- the term slice as used in the flow charts is thus by no means construed to limiting the scope of the present invention.
- Fig. 1 shows schematically, an exemplary embodiment of the system, or kit according to the invention.
- Fig. 2 shows an exemplary method of preparing a cytological slide according to the present invention.
- Fig. 3 shows various problems which occur under protocols for sample preparation according to the state of the art.
- Fig. 4 shows an exemplary workflow, plus the functionality of each of the components of the present invention.
- Fig. 5 shows the increase in zeta potential (i.e., cell membrane potential) for cervical cells with increase in PVP (Polyvinylpyrrolidone) concentration.
- zeta potential i.e., cell membrane potential
- PVP Polyvinylpyrrolidone
- Fig. 6 shows cells in which cell nuclei (Fig. 6A), or cell cytoplasm (Fig. 6B) has been stained with protocols according to the invention.
- Fig. 7 shows the gradual degradation of cells over time in base fixative. This degradation is improved when pH of the fixative is taken closer to neutral without any change in staining pattern.
- Fig. 9 shows an exemplary work flow showing the preparation of stain and, and an exemplary staining process.
- Fig. 10 shows an exemplary embodiment comprising the first and second sample support means.
- Fig. 11 shows results of a pre-dispensed on the spot staining technique according to the invention.
- the invention in an arrangement wherein the cell preparation mixture is supplied in one or more containers, to be mixed on the spot before the sample is dispersed in it. It may be advantageous to pre-dispense the nuclear stain on the slide and the cytoplasmic stain on the cover slip, without deviating from the disclosure. All such variations are considered to be variants of the present disclosure. Further variations and combinations will occur to a practitioner and all such variations are deemed to be within the scope of the disclosed methods.
- FIG. 1 shows schematically, an embodiment of the disclosed system 100.
- Container 101 contains a cell preparation mixture in liquid form.
- the container 101 is shown having a lid 109 that when closed is configured to be airtight and hence protect the liquid 107 contained in the container 101.
- the liquid 107 is constituted to perform a variety of functions simultaneously. It may be deemed to perform two main groups of functions called fixing the cells and cell preparation.
- fixing is used here in the sense that it is to kill, preserve, and harden (tissue, cells, etc.) for subsequent microscopic study.
- Cell preparation is a function by which the cells are readied for being put on a slide. The respective process will be described in detail below.
- the slide 103 is similar to the rectangular piece of glass normally used in cytological slides except that it is coated with a nuclear stain, among other things.
- the slide is meant to be covered with a cover slip which is similar to the cover slip normally used in cytological slides, i.e., a rectangular piece of glass except that it is coated with a cytoplasmic stain.
- a cytological sample is obtained from a subject in the conventional way.
- the sample maybe extracted from the cervix a female subject for screening for cervical cancer.
- the sample is obtained using a type of wooden spatula or a cotton swab, or brush.
- the sample so obtained is dipped into the cell preparation mixture 107, preferably within the container 109 and stirred or shaken so that the constituents of the sample, especially the cells, are suspended uniformly in the cell preparation mixture.
- the cell preparation mixture essentially contains a fixative whose purpose is to fix the cells for further preparation of the slide.
- the major component of the fixative is a mixture of ethanol, isopropyl alcohol and glacial acetic acid substantially in the ratio of 7: 2 : 1 respectively. Even though other ratios could be used, the said ratio has the appropriate pH to preserve the cells without degradation over some lengths of time. If long term preservation is not envisaged, in the interest of other functions of the fixative, the ratio could be varied, with some experimentation. The other functions could be the speed of staining of the cells - in the steps that follow, for example.
- anti-coagulants are added to the cell preparation mixture.
- One or more of the following are also added to the cell preparation mixture: Ethylenediamine tetraacetic acid (EDTA), Hydroxyethylenediamine triacetic acid (HEDTA), Nitriolotriacetic acid (NTA), Sodium citrate, disodium oxalate ((Na + ) 2 C 2 0 4 2 ⁇ ), and dimethyl oxalate (CH 3 ) 2 C 2 0 4 .
- EDTA 1-1.5 mg/ml of the cell preparation mixture
- Sodium citrate 3.8% v/v of cell preparation mixture
- Oxalate 1% v/v of cell preparation mixture and so on.
- demucifying agents such as 1% NaOH, 2% NaOH + 0.25g N- Acetyl-L-Cystein or 3% NaOCl w/v are added to the cell preparation mixture.
- detergents are added to the cell preparation mixture for better dispersion of cells in the cell preparation mixture and create limited pores in the membrane that help in the staining of the cells.
- a suitable detergent would be Dithiothreitol (DTT).
- DTT Dithiothreitol
- About 1% of the w/v of the cell preparation mixture would be a suitable concentration of the detergent in the cell preparation mixture. With the above concentration, the sample may be stored without much damage to the cells for about a week. If there is no such need, 1-2 % w/v of the detergent may be used.
- the slide 103 is similar to the normal cytological slide but pre-dispensed with a stain.
- a cytoplasmic stain is pre-dispensed on the slide.
- the stain of choice is Methylene Blue (MB) or Cresyl Violet (CV) or mixtures of the two.
- MB Methylene Blue
- CV Cresyl Violet
- Other equivalent nuclear stains which are hydrophilic in nature can be used for this purpose.
- This slide is coated with the stain by spin coating by use of an appropriate protocol whereby a thin uniform layer of the stain is formed over the slide. A coating thickness of 10 nm to 10 ⁇ provides an adequate amount of the stain.
- the cytoplasmic stain pre-dispensed on the slide has a neutral pH and the cell preparation mixture has a pH of 4.5 to 6.5 - i.e. the cell preparation mixture contains acetic acid and the pH of the cell preparation mixture is controlled to be in this range while it is prepared.
- the stain pre-dispensed on the slide has a suitable acid mixed with it before it is pre-dispensed on the slide.
- a suitable acid is Hydrochloric acid (HC1).
- the amount of HC1 mixed with the stain is so controlled that when a predefined quantity of the cell preparation mixture with the sample dispersed in it is disposed on the slide, the pH is changed to the optimum value viz., 4.5 to 6.5.
- the cytoplasmic stains with the cell preparation mixture such that the cytoplasm of the cells is stained when the sample is dispersed in it.
- a small quantity of the prepared sample is deposited on the slide coated with a nuclear stain and a base so that the pH of the prepared sample is changed on the spot and the nucleus of the cells are stained.
- the cover slip has no coating on it and hence is a cover slip normally used in cytological slides.
- Fig. 2 shows a method of preparing a cytological slide.
- the first step in the described in the procedure below is not a step in the disclosed process but a process followed normally in obtaining a cytological sample.
- the act of obtaining a cytological sample is shown as step 211 and in a box with a dotted line and dotted arrow, to indicate that it does not, in fact, form a part of the disclosed method.
- the sample is dispersed in the specially prepared cell preparation mixture described above and dispersed in it in a dispersion step 213.
- the cell preparation mixture prepares the cells in the sample for disposing them to prepare a slide.
- the dispersion may be aided by stirring the sample in the cell preparation mixture using the means with which the sample is obtained, the spatula for example.
- the container containing the cell preparation mixture is shaken gently with the sample obtaining means immersed in it. It is to be understood that the part of the means containing the sample is immersed in the cell preparation mixture.
- This dispersion of the sample in the cell preparation mixture may require a minimum duration. This may depend on the exact composition of the cell preparation mixture.
- a quantity of the sample dispersed in the cell preparation mixture is deposited on the slide pre-dispensed with the cytoplasmic stain and allowed to spread evenly on it.
- the quantity of the prepared mixture deposited on the slide is to be defined since the pH of the prepared mixture is to be changed on the spot either at this step or in a subsequent step and may be from a few drops to several drops at different locations on the slide.
- the nuclear stain dissolves in the cell preparation mixture and starts staining the nucleus of the cells.
- a minimum period of time is to be allowed for the staining of the cells with the nuclear stain before the prepared slides may be dried in an accelerated manner, if need be, by heating for instance.
- the cell preparation mixture may also contain the cytoplasmic stain so that apart from the functions already listed in the first phase, the cytoplasm may also be stained at the same time. It is possible to make suitable changes in the cell preparation mixture such that the nucleus of the cells are stained first and then the cytoplasm. All such variations are deemed to be variations of the disclosed method and hence covered by this disclosure.
- Fig. 6 shows stained cells.
- Fig. 6A cell nuclei have been stained with
- Fig. 9 shows an exemplary work flow showing the preparation of stain, and an exemplary staining process.
- a mixture of Methylene Blue (MB) and Cresyl Violet (CV) is used as nuclear stain.
- Other nuclear stain dyes which are hydrophilic can be used for this purpose, too.
- the stain is coated on a slide by spin coating by an appropriate protocol in order to provide a thin uniform layer of the stain (10 nm - 10 ⁇ ) is formed.
- Orange G (OG) and Eosin Azure (EA) are mixed together to form one solvent, and are then coated on the cover slip.
- the slide and the cover slip i.e., the first and second sample support means
- the slide and the cover slip can be assembled in such way that they are connected to one another by a hinge as shown in Fig. 10.
- a spacer can be provided between the surfaces (2-100 ⁇ thickness) so that when the cells are placed between the two sample support means, the cells are protected from being squeezed.
- Fig. 10 shows an exemplary embodiment comprising a first sample support means (slide 1001) and a second sample support means (cover slip 1002), in which both sample support means are connected by a hinge 1005.
- a spacer 1003 is provided w which may have a thickness of 2-100 ⁇ to avoid squeezing of calls.
- the spacer can have cushioning and/or self adhesive properties.
- the cytoplasmic stain has been pre- dispensed on the slide, and the nuclear stain has been predispensed on the cover slip.
- this arrangement can be flipped (i.e., the cytoplasmic stain can be on the cover slip, and the nuclear stain can be in the slide).
- cover slip and slide can have similar or even same sizes, or differ from one another even more than shown in Fig. 10. As discussed above, either the nuclear stain or the cytoplasm stain can as well be given to the cell suspension earlier.
Abstract
The present invention is related to a system or kit for preparing a cytological sample for examination, which comprises a fixative for fixing cells comprised in said sample, a cell surface modifier for modification of the surface of cells comprised in said sample, a first sample support meanshaving at least two sides, and,a second sample support means having at least two sides, wherein on at least one side of at least one of the support means a cytoplasmic stain or a nuclear stain is deposited (Fig. 1).
Description
TITLE
System and Kit for preparing a cytological sample for examination
FIELD OF THE INVENTION
The invention relates to the field of microscopic examination of cytological samples.
BACKGROUND OF THE INVENTION
Cervical cancer is the second most common cancer in women worldwide and the leading cause of cancer deaths in women in developing countries. About 30% of cancers in women are due to cervical cancer with more than 100,000 new cases diagnosed every year, e.g., in India. The estimated compounded annual growth rate (CAGR) for cervical cancer cases is 2.56% and at this growth rate approximately 175,000 new cases of cervical cancer will be detected in the year 2012.
One of the recommended tools for screening of cervical cancer is to detect cytological precursors of cancer in Papanicolaou tests (also called Pap-smear, Pap-test, cervical smear, or smear test), which is a screening test used in gynaecology to detect premalignant and malignant processes in the cervical canal especially in the transformation zone.
In taking a Pap smear, a speculum is used to gather cells from the outer opening of the cervix of the uterus and the endocervix. The cells are examined under a microscope to look for abnormalities. The test aims to detect potentially pre-cancerous changes, which are, among others, caused by sexually transmitted human papillomaviruses. The test remains an effective, widely used method for early detection of pre-cancer and cervical cancer. The test may also detect infections and abnormalities in the endocervix and endometrium.
This procedure has been effective in bringing down the incidence of cervical cancer in the developed countries. However, current pap staining is tedious and time consuming. Moreover, the number of steps required and methodology makes it very difficult to automate the whole process and implement it in a point of care setting, which means that samples cannot be analyzed at the point of care, but have to be delivered to a laboratory.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, a system or kit for preparing a cytological sample for examination is provided. The system or kit comprises a fixative for fixing cells comprised in said sample, a cell surface modifier for modification of the surface of cells comprised in said sample, a first sample support means having at least two sides, and a second sample support means having at least two sides, wherein on at least one side of at least one of the support means a cytoplasmic stain or a nuclear stain is deposited.
As used herein, a "cytological sample" is defined as any specimen from an organism, preferably a mammal, in sufficient amount to be characterized and/or analyzed. A cytological sample, by way of non-limited example, includes cell samples, skin samples, tissue samples, and mucosal samples.
As used herein, the term "fixative" means a composition, which may or may not be in liquid form that fixes the cytological sample such that the cytological sample is adhered to the slide for a period of time sufficient to characterize and/or analyze the cytological sample.
As used herein, the term "modification of the surface of cells" means that cells are treated in such way that they are unfolded and/or uncurled, and/or that cell overlapping is reduced. Reduction of cell folding and curling during sample preparation will result in enhanced visualization of cellular and morphological details that are used for the subsequent detection of abnormal cells in the sample, e.g. in cervical cancer screening.
The reduction of overlapping will result in improved cell dispersion, which is an important issue particularly in cell suspensions, where cells tend to overlay each other, particularly polymorphs that are very small in size and tend to cluster and sit on large cells. In general, both aims can be achieved by increasing the surface charge on the cells, which leads to an increased repulsion between individual cells and thus provides better dispersion.
The nuclear stain serves for staining the nuclei of cells comprised in the sample when the sample is deposited on said first sample support means, or covered by said first sample support means. The cytoplasm stain serves for staining the cytoplasm of cells comprised in the sample when the sample is deposited on said second sample support means, or covered by said second sample support means.
Such system or kit provides the option to prepare a cytological sample in such way that cells in the sample are dispersed in a manner suitable for viewing through a microscope or for analysis by an automated optical cell analysis device. Thus, one important feature of the invention is that it allows on the spot staining of a given sample, i.e., at the Point of Care. Further, the need to prepare a staining solution at the site is eliminated, as the stains are
pre-dispensed in appropriate quantity on the sample support means. Further, the need of going through a complex protocol and a multiple step staining procedure is eliminated, as well as muck problems which are basically due to the dust and impurities during staining. Further, aberrations like over-staining or under-staining are minimized due to the fact that the stains are dispensed in exact measure as required for optimal staining.
Another advantage is that pH conditions can be controlled easily, which is essential for cytoplasm staining (where acidic pH is advantageous under some circumstances) and nuclear staining (where basic pH is advantageous under some circumstances).
The use of sample support means on which the stains are already deposited makes it possible to carry out the staining in an enclosed environment, i.e., in a cartridge, which further eliminates dust and impurities and increases the reproducibility of the staining process.
In one preferred embodiment, only one side of the first and/or second sample support means, is provided with the respective stain. However, it can be advantageous to have both sides of the first and/or second sample support means provided with a stain, in such case, the person handling the system does not have to take care which side of the support is facing the sample. In such embodiment, artifacts due to use of the wrong side of the first and/or second sample support means are avoided.
According to a second aspect of the invention, a method for preparing a cytological sample for examination, the method comprising the steps of:
fixing cells comprised in said sample with a fixative,
modifying the surface of cells comprised in said sample with a cell surface modifier,
providing a first sample support means having at least two sides, and, a second sample support means having at least two sides, wherein on at least one side of at least one of the support means a cytoplasmic stain or a nuclear stain is deposited,
depositing the cells on either the first or the second sample support means, and covering the deposited cells with the second or first sample support means, respectively. It is important to understand that the steps of this method do not have to be performed in the order shown above.
Thus, the invention comprises the use of at least one type of prefabricated sample support means. These prefabricated sample support means are coated with a nuclear stain or with a cytoplasmic stain. In a preferred embodiment of the present invention, a nuclear stain is deposited on at least one side of the first sample support means and a
cytoplasmic stain is deposited on at least one of the second sample support means. Alternatively, only one of the two sample support means is coated with a stain (e.g., nuclear stain), and the other type of stain /e.g., the cytoplasmic stain) is added to the cell suspension, e.g. together with the fixative and/or the cell surface modifier.
In a preferred embodiment of the invention, the first sample support means and/or the second sample support means are in the form of a slide and/or a cover slip.
As used herein, the term "slide" means a small transparent plate made from glass or plastic on which samples, like cells can be deposited for examination under an optical magnification device, like a microscope.
As used herein, the term "cover slip" means a small transparent plate made from glass or plastic which is used to cover samples, like cells, prior to examination under an optical magnification device, like a microscope.
According to common understanding, slides and cover slips differ from one another in size and thickness. Under ISO 8255-2, microscopic slides have a size of 26 x 76 mm, and a thickness of 1 mm, while cover slips usually have a size of 18 x 18 mm and a thickness of 100 - 200 μιη. However, in the context of the present invention, the terms cover slip and slide can be used interchangeable. Particularly in smear preparations, microscopic slides are used to both support the sample and to cover it, after the smear process has been carried out.
According to another preferred embodiment of the invention, the first sample support means and the second sample support means are connected to one another by means of a hinge. This embodiment further facilitates the use of the system or kit according to the present invention. Such hinge can, for example, consist of a rebated joint provided between two plastic frames carrying the first and second sample support means. Other possibilities to provide such hinge comprise the use of a piece of adhesive tape connecting the first and second sample support means. The skilled person will be able to find other technical embodiments which make use of such hinge without the use of inventive step.
According to still another preferred embodiment of the invention, the fixative and/or the cell surface modifier are provided in liquid form. Preferably, the fixative comprises at least one agent selected from the group consisting of
• ethanol
• isopropyl alcohol
• acetic acid (preferably glacial acetic acid)
• formaldehyde, and/or
• glutaraldehyde.
Even more preferred, the fixative comprises a mixture including ethanol, isopropyl alcohol and Acetic acid, preferably in a volume ratio of 7:2: 1.
Alcohol-based fixatives are very good for cytological smears because they act quickly and give good nuclear detail. Preferably, a mixture of ethanol (70% v/v), isopropyl alcohol (70%) v/v) and glacial acetic acid ( 10% v/v) is used. This is a low pH mixture, which can be maintained using either buffer or controlling the concentration of acetic acid.
According to another preferred embodiment, the fixative comprises glutaraldehyde and Phosphate Buffered Saline, with preferably 2% - 20% w/w of glutaraldehyde in a 0.05 M to 1 M Phosphate Buffered Saline (PBS). Preferably, the fixative has a pH of 5 to 6.5.
According to still another preferred embodiment of the invention, the cell surface modifier comprises at least one agent selected from the group consisting of
• an agent for rendering a positive charge to the cell surface,
• an agent for rendering a negative charge to the cell surface,
• a chelating agent,
• an anti-coagulating agent,
• a demucifying agent,
• an agent for supporting cell dispersion, and/or
• an agent for creating micropores on the cell surface.
The agent for rendering a positive charge to the cell surface is preferably at least one water-soluble compound selected from the group consisting of Poly-l-Lysin hydrochloride and Polyvinylpyrrolidone (PVP).
The agent for rendering a negative charge to the cell surface is preferably at least one water-soluble compound selected from the group consisting of Dextran sulfate, Poly sodium 4-styrenesulfonate, Polymethylacrylic acid, Carboxymethyl cellulose and/or a Sodium Polyacrylate.
It is important to understand that any other molecule that produces positive/ negative charge in the solution can also be used in the context of the present invention. However, it is beneficial if said molecule is water-soluble.
The said polymers can be used in different molecular weight and/or different concentrations. High molecular weight polymers seem to work well for imparting high charge on the cells. Similarly, high concentration (e.g. : 5-10 mg/ml of PVP in alcohol fixative works good) shows increased charge on cells surface (see Fig. 5).
The chelating agent and/or the anti-coagulating agent is preferably at least one selected from the group consisting of Ethylenediamine tetraacetic acid (EDTA), Hydroxyethylenediamme triacetic acid (HEDTA), Nitriolotriacetic acid (NTA), Sodium citrate, and/or disodium oxalate or dimethyl oxalate, or other citrates or oxalates.
The demucifying agent is preferably at least one selected from the group consisting of Sodium Hydroxide, N-Acetyl-L-Cystein, and/or Sodium Hypochlorate. Preferably, the cell surface modifier comprises 2 - 5 % w/v NaOH and 0.25g/50ml N-Acetyl- L-Cystein, or 0.5 - 6 % w/v NaOCl.
The agent for supporting cell dispersion and/or the agent for creating micropores on the cell surface is preferably a detergent, even more preferably at least one selected from the group consisting of Dithiothreitol and/or TritonX-100. Presence of these agents creates controlled pores in the cellular membranes, leading to fast access of stain to the cytoplasm and the nucleus.
The mentioned compounds support proper fixation, avoid coagulation and enhance a good dispersion of cell samples. They do not affect the staining of cells with Methylene Blue, Eosin Azure (EA) and Orange G (see Fig. 6), and help that cells stay stable for almost a week (see Fig. 7).
It is particularly preferred that a cell preparation mixture is provided which comprises at least the fixative and the cell surface modifier. Alternatively, it is preferred that in the method according to the invention the steps of fixing the cells comprised in said sample and modifying the surface of the cells comprised in said sample are carried out simultaneously.
In these embodiments, the cell preparation mixture serves to simultaneously fix the cells and modify the surface of the cells in the sample.
In another preferred embodiment the fixative and/or the cell surface modifier can furthermore comprise a stain.
It is furthermore preferred that the nuclear stain comprises at least one selected from the group of:
• Carmine
• Methylene blue
• Neutral/ Toluylene red
• Haematoxylin
• Safranin
• Nile blue.
Likewise, it is preferred that the cytoplasm stain comprises at least one selected from the group of:
• Eosin
• Alician blue
· Xylidine Ponceau,
• Biebrich scarlet
• Tartazine
• Van Gieson's stain (Picric Acid and Acid Fuchsin)
• Wright stain.
Even more preferred, stain combinations can be used, as for example comprised in the so called Pap staining mixture, and which comprises a combination of haematoxylin, Orange G, Eosin Y, Light Green SF yellowish, and sometimes Bismarck Brown Y.
Another suitable stain combination is used in Masson's trichrome, which comprises Weigert's hematoxylin, acid fuchsin, Xylidine Ponceau, phosphomolybdic acid, and Light Green SF yellowish, or alternatively Fast Green FCF, methyl blue, water blue or aniline blue.
Yet another suitable stain combination is used in Lillie's trichrome, which is similar to Masson's trichrome, but uses Biebrich scarlet instead of acid fuchsin and/or Xylidine Ponceau.
Further, fluorescent stains like DAPI (4',6-diamidino-2-phenylindole) can also be used.
Further, particular stain combinations which are commercially available can also be used, like the HCS cell mask™ staining kits provided by Invitrogen.
The nuclear stain serves for staining the nuclei of cells comprised in the sample when the sample is deposited on said first sample support means, or covered by said first sample support means. The cytoplasm stain serves for staining the cytoplasm of cells comprised in the sample when the sample is deposited on said second sample support means, or covered by said second sample support means.
In another preferred embodiment of the invention, a first and second sample support means are configured for being received by an automated device for staining the sample.
The method according to the invention may, preferably, further comprise at least one step selected from the group consisting of
a) detailed inspection of the cervix by colposcopy;
b) carrying out a HPV DNA test in said biological sample, or in new sample with comparable properties;
c) carrying out a Biomarker test in said biological sample, or in new sample with comparable properties; and/or
d) visual inspection of said biological sample, or of a new sample with comparable properties, by a qualified pathologist.
According to another aspect of the invention, the use of a system, kit or method according for at least one purpose selected from the group of
• cancer screening
• cancer diagnosis
• prediction with respect to a given therapy
• concomitant monitoring of a given cancer therapy.
Preferably, the cyto logical sample is a human sample. Preferably, the cytological sample is a cervical sample. However, because principles of cancer genesis and cell transformation are ubiquitous, the method can also be used with samples from other body tissues which have to be checked for abnormalities, like breast samples, prostate samples, liver samples, lung samples, blood samples, and so forth.
It is preferred that the cytological sample is selected from the group consisting of
• smear sample
• tissue slice
· liquid sample, and/or
• other cytology samples.
A smear sample is for example similar or identical to those samples used in the Papanicolaou tests (also called Pap smear, Pap test, cervical smear, or smear test). A tissue slice is for example provide by a microtome. A liquid sample can preferably consist of a suspension of cells, e.g., obtained by a smear.
Other suitable samples comprise, but are not restricted to, fine needle aspiration cytology (FNAC) samples, abrasive cytology samples and/or exfoliated samples.
In many cases a sample is indeed placed on a slide to make it available for investigation, e.g. a tissue slice, or a smear. However, other devices can also be used to carry a sample, e.g. a small cuvette in case the sample is a liquid sample, or a cartridge in case the sample is a brush sample. The term slice as used in the flow charts is thus by no means construed to limiting the scope of the present invention.
Example: Spin coating of cover slips and slides with nuclear stains and cytoplasm stains Slides and cover slips were cleaned as per standard clean room protocol.
1. Stain preparation
a) EosinY-Azure (EA): EosinY: 0.23% w/v, Fast green F: 0.08%> w/v, Bismark brown:
0.05%) w/v, Phosphotungstic acid; 0.2%> w/v, dissolved in denatured alcohol;
b) Orange G (OG): OG: 0.3% w/v, Phosphotungstic acid: 0.01% w/v, dissolved in denatured alcohol;
c) Methylene Blue (MB) / Cresyl Violet (CV): MB: 80g/l in methanol, CV: 40g/L in methanol;
2. Spin coating (Holmarc Spin Coater, India)
a) MB/CV slide: 1000 rpm for 20 seconds (200 μΐ of dye);
b) EA/OG cover slip: 3000 rpm for 20 seconds (70 μΐ of dye);
3. Coating concentration and spin speed used for coating are as follows: i) Cover slip
Code Speed (rpm) Time (s) Volume (μΐ)
OE1 2000 20 100
OE2 2000 10 100
OE3 1000 20 100
OE4 1000 10 100
OE5 500 10 100
OE6 500 5 100
OE7 500 10 100
OE8 500 5 100
OE9 500 10 100
OE10 500 5 100 ii) Slides
i) Slides
BRIEF DESCRIPTION OF THE DRAWINGS
These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter. In the drawings:
Fig. 1 shows schematically, an exemplary embodiment of the system, or kit according to the invention.
Fig. 2 shows an exemplary method of preparing a cytological slide according to the present invention.
Fig. 3 shows various problems which occur under protocols for sample preparation according to the state of the art.
Fig. 4 shows an exemplary workflow, plus the functionality of each of the components of the present invention.
Fig. 5 shows the increase in zeta potential (i.e., cell membrane potential) for cervical cells with increase in PVP (Polyvinylpyrrolidone) concentration.
Fig. 6 shows cells in which cell nuclei (Fig. 6A), or cell cytoplasm (Fig. 6B) has been stained with protocols according to the invention.
Fig. 7 shows the gradual degradation of cells over time in base fixative. This degradation is improved when pH of the fixative is taken closer to neutral without any change in staining pattern.
Fig. 8 shows an exemplary staining process of cervical cells on slides.
Fig. 9 shows an exemplary work flow showing the preparation of stain and, and an exemplary staining process.
Fig. 10 shows an exemplary embodiment comprising the first and second sample support means.
Fig. 11 shows results of a pre-dispensed on the spot staining technique according to the invention. DETAILED DESCRIPTION OF EMBODIMENTS
While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not
indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.
While the embodiments of the disclosed system and the variants of the disclosed method have been described in detail in the description with reference to the drawings, the description and drawings are to be considered exemplary and not restrictive; the invention is not limited to the disclosed embodiments.
For example, it is possible to practice the invention in an arrangement wherein the cell preparation mixture is supplied in one or more containers, to be mixed on the spot before the sample is dispersed in it. It may be advantageous to pre-dispense the nuclear stain on the slide and the cytoplasmic stain on the cover slip, without deviating from the disclosure. All such variations are considered to be variants of the present disclosure. Further variations and combinations will occur to a practitioner and all such variations are deemed to be within the scope of the disclosed methods.
Fig. 1 shows schematically, an embodiment of the disclosed system 100. Container 101 contains a cell preparation mixture in liquid form. The container 101 is shown having a lid 109 that when closed is configured to be airtight and hence protect the liquid 107 contained in the container 101.
The liquid 107 is constituted to perform a variety of functions simultaneously. It may be deemed to perform two main groups of functions called fixing the cells and cell preparation. The word fixing is used here in the sense that it is to kill, preserve, and harden (tissue, cells, etc.) for subsequent microscopic study. Cell preparation is a function by which the cells are readied for being put on a slide. The respective process will be described in detail below.
The slide 103 is similar to the rectangular piece of glass normally used in cytological slides except that it is coated with a nuclear stain, among other things. The slide is meant to be covered with a cover slip which is similar to the cover slip normally used in cytological slides, i.e., a rectangular piece of glass except that it is coated with a cytoplasmic stain. Having introduced the essential organs of the disclosed system, the details regarding them and the intended method of their use are described below.
A cytological sample is obtained from a subject in the conventional way. As an example, the sample maybe extracted from the cervix a female subject for screening for cervical cancer. The sample is obtained using a type of wooden spatula or a cotton swab, or brush. The sample so obtained is dipped into the cell preparation mixture 107, preferably
within the container 109 and stirred or shaken so that the constituents of the sample, especially the cells, are suspended uniformly in the cell preparation mixture.
In one embodiment the cell preparation mixture essentially contains a fixative whose purpose is to fix the cells for further preparation of the slide. The major component of the fixative is a mixture of ethanol, isopropyl alcohol and glacial acetic acid substantially in the ratio of 7: 2 : 1 respectively. Even though other ratios could be used, the said ratio has the appropriate pH to preserve the cells without degradation over some lengths of time. If long term preservation is not envisaged, in the interest of other functions of the fixative, the ratio could be varied, with some experimentation. The other functions could be the speed of staining of the cells - in the steps that follow, for example.
In an alternative embodiment, the major component of the fixative is a mixture containing 2-4% by volume of glutaraldehyde in 0.1 M Phosphate Buffer Saline (PBS). The PBS can either be of Sodium salts or Potassium salts.
However, for the preparation of good slides i.e., slides that have a substantially mono layer of cells without overlap and uniformly distributed over the slide surface, other compounds are added to the fixative and these may be collectively called the cell preparation. It is found that the cells do not clump together if they have a surface charge on them. To make the cells acquire surface charge other components are added to the fixative. Poly-l-Lysin hydrochloride (PLL) or Polyvinylpyrrolidone (PVP) when mixed in appropriate quantities provide positive charge to the cells and hence they repel each other facilitating a mono layer of cells. Alternatively compounds that provide negative charge could also be used. Some of the compounds that could be used are Dextran sulfate or Poly sodium 4-styrenesulfonate or Poly methyl acrylic acid or Carboxymethyl cellulose or a sodium salt or Polyacrylic acid. This list is by no means exhaustive. Knowing the principle involved a variety of other compounds may be used.
Additionally, the clotting of blood and other cells in the sample is to be avoided. Thus anti-coagulants are added to the cell preparation mixture. One or more of the following are also added to the cell preparation mixture: Ethylenediamine tetraacetic acid (EDTA), Hydroxyethylenediamine triacetic acid (HEDTA), Nitriolotriacetic acid (NTA), Sodium citrate, disodium oxalate ((Na+)2C204 2~), and dimethyl oxalate (CH3)2C204. The various concentrations of these compounds that may be used are EDTA: 1-1.5 mg/ml of the cell preparation mixture; Sodium citrate: 3.8% v/v of cell preparation mixture; Oxalate: 1% v/v of cell preparation mixture and so on. The appropriate quantities for the other mentioned compounds may also be found with some experimentation.
Additionally demucifying agents such as 1% NaOH, 2% NaOH + 0.25g N- Acetyl-L-Cystein or 3% NaOCl w/v are added to the cell preparation mixture.
Further, detergents are added to the cell preparation mixture for better dispersion of cells in the cell preparation mixture and create limited pores in the membrane that help in the staining of the cells. A suitable detergent would be Dithiothreitol (DTT). About 1% of the w/v of the cell preparation mixture would be a suitable concentration of the detergent in the cell preparation mixture. With the above concentration, the sample may be stored without much damage to the cells for about a week. If there is no such need, 1-2 % w/v of the detergent may be used.
Thus this one component, viz., the cell preparation mixture, alone prepares the cells for on the spot preparation of slides for cytological examination.
The slide 103 is similar to the normal cytological slide but pre-dispensed with a stain. In one embodiment a cytoplasmic stain is pre-dispensed on the slide. The stain of choice is Methylene Blue (MB) or Cresyl Violet (CV) or mixtures of the two. Other equivalent nuclear stains which are hydrophilic in nature can be used for this purpose. This slide is coated with the stain by spin coating by use of an appropriate protocol whereby a thin uniform layer of the stain is formed over the slide. A coating thickness of 10 nm to 10 μιη provides an adequate amount of the stain.
However, good cytoplasmic staining is achieved when it takes place in a medium whose pH is in the range of 4.5 to 6.5. To achieve this, in one embodiment the cytoplasmic stain pre-dispensed on the slide has a neutral pH and the cell preparation mixture has a pH of 4.5 to 6.5 - i.e. the cell preparation mixture contains acetic acid and the pH of the cell preparation mixture is controlled to be in this range while it is prepared. In another embodiment wherein the cell preparation mixture contains glutaraldehyde and PBS, the stain pre-dispensed on the slide has a suitable acid mixed with it before it is pre-dispensed on the slide. A suitable acid is Hydrochloric acid (HC1). The amount of HC1 mixed with the stain is so controlled that when a predefined quantity of the cell preparation mixture with the sample dispersed in it is disposed on the slide, the pH is changed to the optimum value viz., 4.5 to 6.5.
The cover slip 105 is pre-dispensed with nuclear stains. Orange G (OG) and
Eosin Azure (EA) are mixed and pre-dispensed on the cover slip 105. The cover slip is also pre-dispensed with the nuclear stain combination by spin coating, with a thickness of 10 nm to 10 μιη.
Further, nuclear staining is most effective when the pH of the medium is basic with a pH of 7 - 10. Nuclear staining can occur at a pH of 7, or even in the range of 6 to 7 but the staining is weak or slow. Thus, the nuclear stain pre-dispensed on the cover slip along with a base such as Sodium Hydroxide (NaOH) or Ammonium Hydroxide (NH4OH). Thus, when the cell preparation mixture itself is acidic and is deposited on the slide which is neutral or when the cell preparation mixture is neutral (7 pH) and its pH is changed on the slide to 4.5 to 6.5 for cytoplasmic staining, its pH is changed again to 7 - 10 by the base mixed with the nuclear stain pre-dispensed on the cover slip.
Though a few embodiments of the disclosed system are described above, many other embodiments may be thought of, following the principles behind the disclosed system. For example, it is possible to mix the cytoplasmic stains with the cell preparation mixture such that the cytoplasm of the cells is stained when the sample is dispersed in it. Once a predefined time has elapsed after the sample is introduced into the cell preparation mixture, a small quantity of the prepared sample is deposited on the slide coated with a nuclear stain and a base so that the pH of the prepared sample is changed on the spot and the nucleus of the cells are stained. In such an embodiment, the cover slip has no coating on it and hence is a cover slip normally used in cytological slides. Another variation that may be thought of is to stain the nucleus of the cells first by having a cell preparation mixture with a pH above 7 and containing a nuclear stain and then changing the pH on the spot on the slide to 5 - 6.5 with the slide being pre-dispensed with a cytoplasmic stain and an acid. All such other embodiments are deemed to be covered by this disclosure.
Fig. 2 shows a method of preparing a cytological slide. The first step in the described in the procedure below is not a step in the disclosed process but a process followed normally in obtaining a cytological sample. However the act of obtaining a cytological sample is shown as step 211 and in a box with a dotted line and dotted arrow, to indicate that it does not, in fact, form a part of the disclosed method.
However, once the sample is obtained in a known way using a spatula or a swab or a brush or by any other suitable means, the sample is dispersed in the specially prepared cell preparation mixture described above and dispersed in it in a dispersion step 213. The cell preparation mixture prepares the cells in the sample for disposing them to prepare a slide. The dispersion may be aided by stirring the sample in the cell preparation mixture using the means with which the sample is obtained, the spatula for example. Alternatively the container containing the cell preparation mixture is shaken gently with the sample obtaining means immersed in it. It is to be understood that the part of the means
containing the sample is immersed in the cell preparation mixture. This dispersion of the sample in the cell preparation mixture may require a minimum duration. This may depend on the exact composition of the cell preparation mixture.
In a deposition step 215, a quantity of the sample dispersed in the cell preparation mixture, called the prepared sample, is deposited on the slide pre-dispensed with the cytoplasmic stain and allowed to spread evenly on it. The quantity of the prepared mixture deposited on the slide is to be defined since the pH of the prepared mixture is to be changed on the spot either at this step or in a subsequent step and may be from a few drops to several drops at different locations on the slide. Once the prepared sample comes into contact with the pre-dispensed stain and the stain dissolves in the cell preparation mixture, it starts staining the cytoplasm of the cells in the prepared sample.
After a suitable amount of time allowing for the staining of cytoplasm of the cells in the prepared sample, the cover slip, pre-dispensed at least with the nuclear stain is used to cover the deposited prepared sample in a covering step 217 such that the surface of the cover slip pre-dispensed with the nuclear stain comes into contact with the prepared sample deposited thereon. The duration of time required to elapse before the placing of the cover slip may depend on the exact composition of the different embodiments of the cell preparation mixture described before. This may have to be experimented with and specified as the minimum time required elapsing before the cover slip can be placed over the sample.
When the cover slip pre-dispensed with nuclear stain comes into contact with the prepared sample deposited on the slide, the nuclear stain dissolves in the cell preparation mixture and starts staining the nucleus of the cells. As in the case of the time required for staining after deposition of the prepared sample on to the slide, a minimum period of time is to be allowed for the staining of the cells with the nuclear stain before the prepared slides may be dried in an accelerated manner, if need be, by heating for instance.
With this, a slide ready for examining through a microscope or being read by an automated optical cell analysis device will be ready. As can be seen by the description above, the preparation of the cytological slide is oriented towards on the spot preparation of slides with a smaller number of steps than used in the methods known hitherto. It is to be understood that even though a preparation of single slide is referred to in the description, the same system may have to be used for the preparation of more than one slide, with the same prepared sample, at a time, as may be required by normal laboratory standards or guidelines. The volume of the cell preparation mixture and the amount of sample obtained from the
subject may be standardized such that the required number of slides may be prepared at a time.
It may be visualized that a practical kit based on the system and method disclosed may have a predefined amount of the cell preparation mixture for preparing the number of slides required by the related laboratory practice and also standardize the amount of sample obtained, the preferred means of obtaining it, the method of dispersal of the sample to obtain the prepared sample and the minimum time required at the various steps of the method so that the required number of good slides may be prepared on the spot for further study.
In essence, the method may be described as having three stages. The first being the dispersing a cytological sample in a cell preparation mixture wherein multiple functions are performed simultaneously, such as, dispersion of the cells in the cell preparation mixture, cell surface modification, providing the surface of the cells with electrical charge so that they are distributed evenly and do not clump together and preparing the cell surface for staining. Additionally the cell preparation mixture may also have a pH that is optimum for staining. The second stage is staining the cytoplasm of the cells. The third stage being the changing of the pH of the prepared sample, on the spot, and staining the nucleus of the cells in the prepared sample. Even though only this variant of the disclosed method is described above, many variants of this method may be thought of based on it. For instance, the cell preparation mixture may also contain the cytoplasmic stain so that apart from the functions already listed in the first phase, the cytoplasm may also be stained at the same time. It is possible to make suitable changes in the cell preparation mixture such that the nucleus of the cells are stained first and then the cytoplasm. All such variations are deemed to be variations of the disclosed method and hence covered by this disclosure.
Fig. 3 shows various problems which occur under protocols for sample preparation according to the state of the art. In Fig. 3A, a polymorph is superimposed to a cell, which will hamper later visual inspection of said cell or create artifacts in automatic image analysis. Fig. 3B shows cells which are folded, which leads to similar problems as discussed on Fig. 3A. Fig. 3C shows cells which are partially overlapping, again, later visual inspection will be hampered artifacts will be created in automatic image analysis. Fig. 3D shows cells as they should be to allow proper image analysis, either visually or with automatic image analysis, i.e., cells are unfolded and cells are properly dispersed with no overlaps.
Fig. 4 shows an exemplary workflow, plus the functionality of each of the components of the present invention. Note that this example is by no means restricting. Further note that components I and II (i.e., fixative and cell surface modifier) can be combined.
Fig. 5 shows the increase in zeta potential (i.e., cell membrane potential) for cervical cells with increase in PVP (Polyvinylpyrrolidone) concentration. PVP affects the charge of cellular membranes positively. In sample 1 , only an alcohol based fixative is used. In samples 2 - 4, the same alcohol fixative is used which contains either 2, 4, or 5 mg/ml PVP.
Fig. 6 shows stained cells. In Fig. 6A, cell nuclei have been stained with
Methylene Blue, and in Fig. 6B cell cytoplasm has been stained with Eosin Azure (EA) and Orange G (OG). It is important to mention that the staining has not been affected by prior steps carried out according to the invention, i.e., the use of chelating and anti-coagulant agents (to avoid cell clotting), like Ethylenediamine tetraacetic acid (EDTA), Hydroxyethylenediamme triacetic acid (HEDTA), Nitriolotriacetic acid (NTA), Sodium citrate, Oxalates, the use of demucifying agents, like 1% NaOH, 2% NaOH + 0.25g/50ml N- Acetyl-L-Cystein, 3% NaOCl, the use of detergents (for better dispersion of cells and created limited pores in the membrane), like Dithiothreitol (DTT), TritonX-100, etc.
Fig. 7 shows the gradual degradation of cells over time in an alcohol-based fixative. The degradation is retarded when the pH of the fixative is made closer to neutral (e.g., 4.5 - 6.5) by addition of a base or a respective buffer.
Fig. 8 shows, exemplarily, a workflow for the preparation of stains in combination with a detergent and a base (for on-the-spot pH change). A detergent (such as Triton X-100 or DTT is mixed directly in fixative (either alcohol- or glutaraldehy de-based) in which the samples are then collected. Optionally, the said solution may also contain a stain, e.g. cytoplasmic stain (e.g., Eosin Azure (EA) and/or Orange G (OG)). The pH of the solution is preferably maintained in the range of 4.5 to 6.5. Another stain (nuclear stain such as hematoxylin, methylene blue, etc) is coated on the slide along with any base compound (such as NaOH, NaCl, NH4OH, etc.). The concentration of the base should be selected in such way that the pH of the desired volume of fixative (acidic in nature) can be changed quickly to basic (7.5 to 9.5. Note that also the nuclear stain can be added to the cell suspension, and the cytoplasmic stain can be coated on the slide.
Fig. 9 shows an exemplary work flow showing the preparation of stain, and an exemplary staining process. A mixture of Methylene Blue (MB) and Cresyl Violet (CV) is
used as nuclear stain. Other nuclear stain dyes which are hydrophilic can be used for this purpose, too. The stain is coated on a slide by spin coating by an appropriate protocol in order to provide a thin uniform layer of the stain (10 nm - 10 μηι) is formed. For staining of the cytoplasm, Orange G (OG) and Eosin Azure (EA) are mixed together to form one solvent, and are then coated on the cover slip. This ensures that both the nuclear staining and cytoplasmic staining are pre-dispensed and can be assembled into a single device for staining. The slide and the cover slip (i.e., the first and second sample support means) can be assembled in such way that they are connected to one another by a hinge as shown in Fig. 10. A spacer can be provided between the surfaces (2-100 μηι thickness) so that when the cells are placed between the two sample support means, the cells are protected from being squeezed.
Fig. 10 shows an exemplary embodiment comprising a first sample support means (slide 1001) and a second sample support means (cover slip 1002), in which both sample support means are connected by a hinge 1005. Further, a spacer 1003 is provided w which may have a thickness of 2-100 μηι to avoid squeezing of calls. The spacer can have cushioning and/or self adhesive properties. In Fig. 10, the cytoplasmic stain has been pre- dispensed on the slide, and the nuclear stain has been predispensed on the cover slip. However, this arrangement can be flipped (i.e., the cytoplasmic stain can be on the cover slip, and the nuclear stain can be in the slide). Further, cover slip and slide can have similar or even same sizes, or differ from one another even more than shown in Fig. 10. As discussed above, either the nuclear stain or the cytoplasm stain can as well be given to the cell suspension earlier.
Fig. 11 shows the results of a pre-dispensed on-the-spot staining technique according to the invention. The slides and cover slips have been coated with stains by spin coating as described in example 1. The pre-dispensed slides were then used for staining experiments on cervical cells. For this purpose the cervical sample collected in a media were placed on the bottom substrate and the thereafter the cover slip were placed on it. The slides were examined on the microscope after 3 min staining, and images were obtained. Fig. 11 A shows staining with Orange G. Fig. 11 B shows staining with Eosin Azure, and Fig. 11 C shows staining with Methylene Blue Methylene Blue (MB) and Cresyl Violet (CV). Fig. 11 D shows the staining of cytoplasm in cervical cells with a combination of Orange G (OG) and Eosin Azure (EA) after 3 min.
Claims
1. A system or kit (100) for preparing a cyto logical sample for examination, the system or kit comprising:
a fixative for fixing cells comprised in said sample
a cell surface modifier for modification of the surface of cells comprised in said sample,
a first sample support means (103) having at least two sides, and, a second sample support means (105) having at least two sides, wherein on at least one side of at least one of the support means a cytoplasmic stain or a nuclear stain is deposited.
2. A method for preparing a cytological sample for examination, the method comprising the steps of:
fixing cells comprised in said sample with a fixative,
modifying the surface of cells comprised in said sample with a cell surface modifier,
providing a first sample support means (103) having at least two sides, and, a second sample support means (105) having at least two sides, wherein on at least one side of at least one of the support means a cytoplasmic stain or a nuclear stain is deposited,
depositing the cells on either the first or the second sample support means, and covering the deposited cells with the second or first sample support means, respectively.
3. The system or kit according to claim 1 or the method of claim 2, wherein a nuclear stain is deposited on at least one side of the first sample support means and a cytoplasmic stain is deposited on at least one of the second sample support means.
4. The system, kit or method according to any of the aforementioned claims, wherein the first sample support means and/or the second sample support means are in the form of a slide and/or a cover slip.
5. The system, kit or method according to any of the aforementioned claims, wherein the first sample support means and the second sample support means are connected to one another by means of a hinge.
6. The system, kit or method according to any of the aforementioned claims, wherein the fixative and/or the cell surface modifier are provided in liquid form.
7. The system, kit or method according to any of the aforementioned claims, wherein the fixative comprises at least one agent selected from the group consisting of
• ethanol
• isopropyl alcohol
• acetic acid (preferably glacial acetic acid)
• formaldehyde
· glutaraldehyde.
8. The system, kit or method according to any of the aforementioned claims, wherein the fixative comprises a mixture including ethanol, isopropyl alcohol and glacial acetic acid, preferably in a volume ratio of 7:2: 1.
9. The system, kit or method according to any of the aforementioned claims, wherein the fixative comprises glutaraldehyde and Phosphate Buffered Saline, with preferably 2% - 20% w/w of glutaraldehyde in a 0.1 M to 1 M Phosphate Buffered Saline.
10. The system, kit or method according to any of the aforementioned claims, wherein the fixative has a pH of 5 to 6.5.
11. The system, kit or method according to any of the aforementioned claims, wherein the cell surface modifier comprises at least one agent selected from the group consisting of
• an agent for rendering a positive charge to the cell surface,
• an agent for rendering a negative charge to the cell surface,
• a chelating agent, • an anti-coagulating agent,
• a demucifying agent,
• an agent for supporting cell dispersion, and/or
• an agent for creating micropores on the cell surface.
12. The system, kit or method according to any of the aforementioned claims, wherein a cell preparation mixture is provided which comprises at least the fixative and the cell surface modifier.
13 The method according to any of the aforementioned claims, in which method the steps of fixing the cells comprised in said sample and modifying the surface of the cells comprised in said sample are carried out simultaneously.
14. The system, kit or method according to any of the aforementioned claims, wherein the nuclear stain comprises at least one selected from the group of:
• Carmine
• Methylene blue
• Neutral/ Toluylene red
• Haematoxylin
• Safranin
• Nile blue.
15. The system, kit or method according to any of the aforementioned claims, wherein the cytoplasm stain comprises at least one selected from the group of:
• Eosin
• Alician blue
• Xylidine Ponceau,
• Biebrich scarlet
• Tartazine
• Van Gieson's stain
• Wright stain.
16. The system, kit or method according to any of the aforementioned claims, wherein a first and second sample support means are configured for being received by an automated device for staining the sample.
17. The method according to any of the aforementioned claims, which method further comprises at least one step selected from the group consisting of a) detailed inspection of the cervix by colposcopy;
b) carrying out a HPV DNA test in said biological sample, or in new sample with comparable properties;
c) carrying out a Biomarker test in said biological sample, or in new sample with comparable properties; and/or
d) visual inspection of said biological sample, or of a new sample with comparable properties, by a qualified pathologist.
18. Use of a system, kit or method according for at least one purpose selected from the group of
• cancer screening
• cancer diagnosis
· prediction with respect to a given therapy
• concomitant monitoring of a given cancer therapy.
19. The method, system or kit according to any of the aforementioned claims, wherein the cytological sample is a cervical sample.
20. The method, system or kit according any of the aforementioned claims, wherein the cytological sample is selected from the group consisting of
• smear sample
• tissue slice
· liquid sample, and/or
• other cytology samples.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201280044631.2A CN103827654B (en) | 2011-09-13 | 2012-09-06 | System and kit for preparing the cytological sample for being checked |
| MX2014002844A MX2014002844A (en) | 2011-09-13 | 2012-09-06 | System and kit for preparing a cytological sample for examination. |
| RU2014114524A RU2619784C2 (en) | 2011-09-13 | 2012-09-06 | System and kit for cytological specimens acquisition for research |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161534045P | 2011-09-13 | 2011-09-13 | |
| US61/534,045 | 2011-09-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2013038306A1 true WO2013038306A1 (en) | 2013-03-21 |
Family
ID=47018337
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IB2012/054606 WO2013038306A1 (en) | 2011-09-13 | 2012-09-06 | System and kit for preparing a cytological sample for examination |
Country Status (4)
| Country | Link |
|---|---|
| CN (1) | CN103827654B (en) |
| MX (1) | MX2014002844A (en) |
| RU (1) | RU2619784C2 (en) |
| WO (1) | WO2013038306A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016041890A3 (en) * | 2014-09-17 | 2016-06-09 | Ventana Medical Systems, Inc. | Compositions, methods, and systems for tissue fixation |
| US10126216B2 (en) | 2011-02-17 | 2018-11-13 | Ventana Medical Systems, Inc. | Method for tissue sample fixation |
| JP2021192039A (en) * | 2015-09-14 | 2021-12-16 | エッセンリックス コーポレーション | Device and system for analyzing sample, particularly blood, and methods of using the same |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104390832A (en) * | 2014-11-07 | 2015-03-04 | 刘志军 | Formula of rapid Wright's stain suitable for blood cells, and preparation method of rapid Wright's stain |
| RU178938U1 (en) * | 2017-06-20 | 2018-04-23 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Амурская государственная медицинская академия" Министерства здравоохранения Российской Федерации | A device for preparing a cytological smear of biological fluids for rapid analysis of the cellular composition |
| CN108303416A (en) * | 2018-01-29 | 2018-07-20 | 青岛浩铂生物科技有限公司 | A kind of epithelial tissue dyeing liquid kit and preparation method thereof |
| CN108287240A (en) * | 2018-01-29 | 2018-07-17 | 青岛浩铂生物科技有限公司 | The detection cervical carcinoma reagent and preparation method dyed based on cervical exfoliated cell in urine |
| CN112574938B (en) * | 2019-09-29 | 2023-02-03 | 体必康生物科技(广东)股份有限公司 | Sputum treatment fluid for membrane filtration enriched bacteria and application thereof |
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| CN1144034C (en) * | 1998-06-30 | 2004-03-31 | 莱密纳股份有限公司 | Cytological and histological fixative composition and methods of use |
| RU2184966C1 (en) * | 2000-11-30 | 2002-07-10 | Боев Сергей Федотович | Method to test blood cellular composition according to a smear |
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- 2012-09-06 WO PCT/IB2012/054606 patent/WO2013038306A1/en active Application Filing
- 2012-09-06 CN CN201280044631.2A patent/CN103827654B/en not_active Expired - Fee Related
- 2012-09-06 MX MX2014002844A patent/MX2014002844A/en unknown
- 2012-09-06 RU RU2014114524A patent/RU2619784C2/en not_active IP Right Cessation
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| US3796594A (en) * | 1970-10-30 | 1974-03-12 | Gen Electric | Stain coated slides for differentially staining blood |
| US3834874A (en) * | 1972-10-18 | 1974-09-10 | Gen Electric | Detection of malarial parasites in blood |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US10126216B2 (en) | 2011-02-17 | 2018-11-13 | Ventana Medical Systems, Inc. | Method for tissue sample fixation |
| US11624684B2 (en) | 2011-02-17 | 2023-04-11 | Ventana Medical Systems, Inc. | Method for tissue sample fixation |
| WO2016041890A3 (en) * | 2014-09-17 | 2016-06-09 | Ventana Medical Systems, Inc. | Compositions, methods, and systems for tissue fixation |
| JP2021192039A (en) * | 2015-09-14 | 2021-12-16 | エッセンリックス コーポレーション | Device and system for analyzing sample, particularly blood, and methods of using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| MX2014002844A (en) | 2014-07-09 |
| CN103827654B (en) | 2017-06-13 |
| RU2014114524A (en) | 2015-10-20 |
| CN103827654A (en) | 2014-05-28 |
| RU2619784C2 (en) | 2017-05-18 |
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