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WO2018221496A1 - Procédé de séparation de cellules rares dans un échantillon de sang, et procédé d'analyse des gènes de ces cellules - Google Patents

Procédé de séparation de cellules rares dans un échantillon de sang, et procédé d'analyse des gènes de ces cellules Download PDF

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Publication number
WO2018221496A1
WO2018221496A1 PCT/JP2018/020498 JP2018020498W WO2018221496A1 WO 2018221496 A1 WO2018221496 A1 WO 2018221496A1 JP 2018020498 W JP2018020498 W JP 2018020498W WO 2018221496 A1 WO2018221496 A1 WO 2018221496A1
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Prior art keywords
cells
filter
blood sample
blood
rare
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English (en)
Japanese (ja)
Inventor
清太 中村
勝也 遠藤
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Resonac Corp
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Hitachi Chemical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler

Definitions

  • the present invention relates to a method for separating rare cells in a blood sample and a method for analyzing genes of the cells.
  • the blood may contain a small amount of cells other than these blood cells, as well as red blood cells, white blood cells, and platelets.
  • cancer cells called circulating tumor cells (CTC) may exist in the blood of cancer patients.
  • the primary cancer cells circulate in the body as a CTC in the flow of blood or lymph and metastasize to other organ tissues. Therefore, cancer metastasis can be detected early by detecting CTC in the blood sample.
  • Circulating endothelial cells (CEC) are present in the peripheral blood.
  • CEC is an endothelial cell (mature cell) that has been detached from the blood vessel wall due to metabolism, and the number of CEC is known to increase due to many diseases such as cardiovascular disease, infectious disease, immune disease, and cancer. .
  • a technique for separating and analyzing these rare cells is required.
  • a method of separating rare cells in blood there is a method of separating rare cells using a filter based on differences in cell size and deformability (for example, Patent Document 1).
  • a blood sample is filtered.
  • rare cells are trapped on the filter while blood cells pass through the pores of the filter and are removed along with the filtrate.
  • RNA analysis of cells can be performed by extracting RNA from cells, reverse transcription, and then amplifying (reverse transcription PCR).
  • many RNAs derived from impurities remaining without separation or transcription products thereof are derived from the gene to be analyzed.
  • the RNA or its transcription product is buried, or the presence of a component that prevents gene amplification in the contaminant does not sufficiently amplify the transcription product, so that gene analysis cannot be performed.
  • efficiency was poor. Accordingly, an object of the present invention is to separate rare cells from a blood sample and to analyze the genes of the separated rare cells so that efficient gene analysis of rare cells can be performed.
  • the method for separating rare cells in a blood sample according to the present invention includes a step of filtering a blood sample with a filter to capture the cells on the filter, a step of bringing a hemolytic agent into contact with the cells captured on the filter, And the hemolytic agent comprises ammonium chloride or cyclic amine.
  • the method for analyzing a gene of a rare cell in a blood sample according to the present invention includes a step of analyzing the gene of a rare cell isolated by the above method.
  • the rare cell may be a circulating cancer cell in the blood.
  • the filter may include a substrate and a plurality of through holes provided on the substrate.
  • the rare cells and erythrocytes are separated using a specific hemolytic agent, the rare cells are separated from the blood sample so that efficient gene analysis of the rare cells is possible. Can do.
  • FIG. 1 It is a schematic diagram which shows one Embodiment of Forter. It is a perspective view which shows one Embodiment of a cell capture
  • the method of separating rare cells in a blood sample according to the present invention includes a step of filtering a blood sample with a filter and capturing the cells on the filter (filtration step), and contacting a hemolyzing agent with the cells captured on the filter A process (hemolysis process).
  • blood collected from a subject may be used as it is, or blood diluted with a buffer solution such as phosphate buffered saline (PBS) or other suitable medium may be used.
  • a buffer solution such as phosphate buffered saline (PBS) or other suitable medium
  • CTAD phosphate buffered saline
  • additives usually added to the blood sample such as CTAD (citrate, theophylline, adenosine, dipyridamole) may be added within a range not impairing the effects of the present invention.
  • CTAD cetylline
  • “Rare cells” are specific types of cells contained in the blood, and usually the number of cells is extremely small relative to the total number of all cells in the blood. Rare cells are, for example, cancer cells such as CTC or cancer stem cells, endothelial cells such as CEC, and stem cells such as hematopoietic stem cells.
  • cells in the blood sample are captured on the filter by filtering the blood sample with a filter.
  • “capturing” means that a blood sample is filtered through a filter, and cells in the blood sample remain on the filter.
  • rare cells are captured on the filter, while many of the blood cells pass through the pores of the filter and are removed along with the filtrate.
  • the number of blood cells in the blood sample is much larger than the number of rare cells, some blood cells also remain on the filter together with the rare cells.
  • the filter is not particularly limited as long as it can capture rare cells present in the blood sample, and a conventionally known filter can be used.
  • FIG. 1 shows an example of the filter.
  • the filter 105 may be, for example, a metal or resin filter, and includes a substrate 107 and a plurality of through-holes 106 provided on the substrate 107 and having a hole diameter corresponding to the diameter of a rare cell.
  • the substrate 107 may be a thin film having a thickness of 3 ⁇ m to 50 ⁇ m, 5 ⁇ m to 40 ⁇ m, or 5 ⁇ m to 30 ⁇ m, for example.
  • the opening shape of the through hole 106 may be, for example, a circle, an ellipse, a square, a rectangle, a rounded rectangle, or a polygon.
  • a rounded rectangle is a shape having a rectangle and two semicircles that have the same radius as the short side of the rectangle and that are adjacent to each other and are adjacent to the two short sides of the rectangle.
  • the arrangement of the through holes 106 may be an aligned arrangement as shown in FIG. 1, a zigzag arrangement, or a random arrangement in which the through holes 106 are arranged at arbitrary positions.
  • the hole diameter of the through hole 106 is the maximum value of the diameter of a sphere that can pass through the through hole 106.
  • the pore diameter of the through hole 106 is preferably 5 ⁇ m to 15 ⁇ m, more preferably 6 ⁇ m to 12 ⁇ m, and even more preferably 7 ⁇ m to 10 ⁇ m.
  • the cells may be washed after the filtration step (washing step).
  • the washing step is performed, for example, by bringing a washing solution containing a known buffer solution such as PBS into contact with cells.
  • the washing solution may contain additives such as bovine serum albumin (BSA) or EDTA. Washing is not limited to after the filtration step, but can be appropriately performed before the filtration step and after the hemolysis step.
  • BSA bovine serum albumin
  • the hemolyzing agent is brought into contact with the cells captured on the filter.
  • This can be done, for example, by filtering the hemolytic agent through a filter in which the cells are captured.
  • the hemolytic agent including the hemolyzed red blood cells is removed through the filter's through-holes, so that the freshly collected hemolytic agent is continuously contacted with the cells captured on the filter.
  • red blood cells can be efficiently hemolyzed with a small amount of hemolytic agent.
  • the flow rate for filtering the hemolytic agent is preferably 10 ⁇ L / min to 3000 ⁇ L / min, more preferably 20 ⁇ L / min to 1000 ⁇ L / min from the viewpoint of minimizing damage to rare cells and from the viewpoint of efficiently lysing red blood cells.
  • the hemolytic agent contains ammonium chloride or cyclic amine as a component (haemolytic component) having an action of hemolyzing red blood cells.
  • cyclic amines include pyrrolidine, heterocyclic N-alkylamine oxide, and lipogramistin A.
  • the hemolysis component may be used alone or in combination of two or more. By using the hemolytic agent containing the specific hemolytic component, it is possible to efficiently analyze the gene of a rare cell separated from a blood sample.
  • the hemolytic agent is preferably a hemolytic agent that does not contain a compound having a fixing action (fixing agent) such as formaldehyde.
  • the concentration of the hemolytic component in the hemolytic agent is, for example, 0.1% by mass to 10% by mass, 0.5% by mass to 5% by mass, or 0 based on the total amount of the hemolytic agent. It may be from 8% to 3% by weight.
  • the hemolytic agent is a component that is usually added to the hemolytic agent, such as hydrochloric acid, sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, as long as the effects of the present invention are not impaired May be added.
  • the cartridge shown in FIGS. 2 and 3 can be used.
  • a method for separating rare cells in a blood sample using a cartridge according to an embodiment of the present invention will be described.
  • a cell capture cartridge (cartridge) 100 shown in FIGS. 2 and 3 has a housing having an inlet 130 to which an inflow pipe 125 into which a liquid flows is connected and an outlet 140 to which an outflow pipe 135 from which a liquid flows out is connected.
  • the body 120 and the filter 105 shown in FIG. 1 are provided.
  • the filter 105 is fixed by a casing 120 including an upper member 110 and a lower member 115.
  • a blood sample, a hemolyzing agent, a washing solution, and other reaction solutions are introduced into the housing 120 through the inflow pipe 125, and are discharged to the outside through the filter 105 through the outflow pipe 135.
  • Such a liquid flow can be created, for example, by connecting a pump upstream of the inflow pipe 125 or downstream of the outflow pipe 135. Further, a cock may be provided upstream of the inflow pipe 125 and / or downstream of the outflow pipe 135 to control the flow of the liquid.
  • a blood sample is introduced into the cartridge 100 from the inflow tube 125, and the blood sample is filtered by the filter 105 (filtration process). Rare cells in the blood sample cannot pass through the through hole 106 of the filter 105 and remain on the surface of the filter 105. Most components in the blood sample including red blood cells pass through the through-hole 106 and are discharged out of the cartridge 100, but some red blood cells remain on the surface of the filter 105 together with rare cells.
  • the filter 105 may be cleaned by passing a cleaning solution through the filter 105.
  • a hemolytic agent is introduced into the cartridge 100, and the hemolytic agent is filtered through the filter 105 (hemolysis step).
  • the flow rate of the hemolytic agent is adjusted so that the cells on the filter 105 are in contact with the hemolytic agent for a predetermined time.
  • Red blood cells hemolyzed by the hemolytic agent pass through the through-hole 106 together with the hemolytic agent, and are discharged out of the cartridge 100.
  • the filter 105 may be cleaned by passing a cleaning solution through the filter 105.
  • the present invention provides, as one aspect thereof, a method for preparing a rare cell sample for gene analysis from a blood sample.
  • the gene of rare cells can be analyzed by a known method, for example, by the following method. First, RNA is extracted from the cells separated by the above method, isolated and purified, and then the cDNA of the desired gene is amplified by reverse transcription PCR.
  • the gene to be analyzed is not limited and may be the entire genome or a specific gene. For example, when the rare cell is CTC, the HER2 gene known to be overexpressed in cancer cells may be analyzed.
  • the expression of the target gene in rare cells can be analyzed using the results of reverse transcription PCR, and further analysis of the target gene can be performed using the amplification product.
  • the rare cell sample isolated by the method of the present invention or the rare cell sample prepared by the method of the present invention can sufficiently amplify the cDNA of the gene of the rare cell, the gene analysis as described above is efficient. Can be done automatically.
  • a CTC capturing device 60 shown in FIG. 4 is a device that captures CTC contained in a sample by filtering a sample such as a blood sample with a filter.
  • the CTC capturing device 60 includes a filter unit 1 having a cell capturing cartridge 100 shown in FIG. 3 inside, a processing liquid channel 3 for supplying a processing liquid to the filter unit 1, and a sample channel 4 for supplying a sample to the filter unit 1.
  • the filter 105 in the cell trapping cartridge 100 includes a plurality of through holes on a thin metal substrate having a thickness of 18 ⁇ m.
  • the through holes have an opening shape having a major axis of 100 ⁇ m and a minor axis of 8 ⁇ m, and are arranged in alignment.
  • a plurality of processing liquid storage containers 5 containing different processing liquids are provided on the upstream side of the processing liquid flow path 3.
  • Examples of the processing liquid that is put into the processing liquid storage container 5 include a cleaning liquid for cleaning the hemolytic agent and the rare cells captured by the filter 105.
  • a soft tube is inserted into each processing liquid storage container 5 to form individual processing liquid flow paths 6. These flow paths are connected to the selection valve 8, and the processing liquid connected to the processing liquid flow path 3 is selected by rotating the selection valve 8.
  • a reservoir 10 is connected to the sample flow path 4, and the sample is supplied to the reservoir 10.
  • the filter unit 1 is configured to supply either the processing liquid or the sample, and control of which liquid of the processing liquid and the sample is supplied is attached to each of the flow paths 3 and 4.
  • the pinch valves 12 and 13 are used for switching.
  • the treatment liquid and the sample are supplied by being sucked by a peristaltic pump 14 provided downstream of the filter unit 1.
  • the sample or the processing liquid flows through the processing liquid channel 3 or the sample channel 4 and is supplied to the filter unit 1, and then flows into the waste liquid tank 16.
  • Rare cells in the sample are captured by the filter 105 provided in the cell capturing cartridge 100 in the filter unit 1.
  • the above units are controlled by the control unit 48.
  • the selection valve 8 is controlled by the selection valve driver 49 based on an instruction from the control unit 48.
  • the pinch valves 12 and 13 are controlled by two valve drivers 50 connected to each other.
  • the driving of the peristaltic pump 14 is controlled by a pump driver 51.
  • SKBR3 HER2 positive
  • a cell line derived from human breast cancer contained in a culture flask was cultured at 37 ° C. in a carbon dioxide incubator. Trypsin-EDTA with a concentration of 0.25% was added to the culture flask, and the cultured cells attached to the flask were detached from the flask. After the detached cells were counted using a hemocytometer and a phase contrast microscope, a sample containing 100 cells was prepared. Cells were lysed by adding the sample to 0.7 mL of RNA extraction reagent and incubating for 5 minutes.
  • RNA was isolated from the cell lysate, and cDNAs of the HER2 gene and GAPDH gene were obtained from the purified RNA by reverse transcription. These cDNAs were amplified by pre-amplification, and real-time PCR was performed on the by-product to analyze the expression levels of the HER2 gene and the GAPDH gene.
  • the GAPDH gene is a housekeeping gene and was used as a reference gene for real-time PCR.
  • Reference Example 2 Samples were prepared by adding 100 cultured cells of Reference Example 1 to 1 mL of healthy human blood collected in a “BD Vacutainer (registered trademark) blood collection tube” containing CTAD solvent from Becton Dickinson and Company (BD) did. The expression levels of the HER2 gene and GAPDH gene were analyzed by the same method as in Reference Example 1.
  • Comparative Example 1 To a sample prepared by the same method as in Reference Example 2, 10 mL of a hemolytic agent having a formaldehyde concentration of 1% was added and incubated for 10 minutes. Thereafter, this was centrifuged at 400 g for 3 minutes, the supernatant was removed, and the pellet was suspended in a PBS solution containing 0.5% BSA and 2 mM EDTA (hereinafter referred to as “washing solution”). In the same manner as in Reference Example 1, RNA was isolated from the suspension, and the expression levels of the HER2 gene and GAPDH gene were analyzed.
  • the washing liquid was sent to the cell capture cartridge 100 to wash the filter 105.
  • the cell capture cartridge 100 was removed from the filter unit 1, and 0.7 mL of RNA extraction reagent was introduced into the cell capture cartridge 100 and incubated for 5 minutes to lyse the cells.
  • the cell lysate was collected, RNA was isolated by the same method as in Reference Example 1, and the expression levels of the HER2 gene and GAPDH gene were analyzed.
  • Example 1 Samples were prepared and analyzed for the expression levels of the HER2 gene and the GAPDH gene by the same method as in Comparative Example 2 except that a hemolytic agent having a cyclic amine pyrrolidine concentration of 2% by mass was used. .
  • Ct (Threshold Cycle) in Table 1 indicates the number of cycles of real-time PCR required until a signal having a constant intensity is detected.
  • Ct equivalent to Ct of the positive control (Reference Example 1) was obtained, and thus it was found that the cDNA of the HER2 gene was normally amplified.
  • Ct of Comparative Example 1 in which the sample was not filtered with a filter is equivalent to Ct of the negative control (Reference Example 2), so that it can be seen that the HER2 gene cDNA was not significantly amplified.

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Abstract

Le procédé de séparation de cellules rares dans un échantillon de sang de l'invention comporte : une étape au cours de laquelle l'échantillon de sang est filtré au moyen d'un filtre, et des cellules sont retenues sur le filtre ; et une étape au cours de laquelle un agent hémolytique est mis en contact avec les cellules retenues sur le filtre. L'agent hémolytique contient un chlorure d'ammonium ou une amine cyclique. Selon l'invention, dans le de cas cellules rares séparées, il est possible d'effectuer une analyse génétique de manière efficace.
PCT/JP2018/020498 2017-06-02 2018-05-29 Procédé de séparation de cellules rares dans un échantillon de sang, et procédé d'analyse des gènes de ces cellules Ceased WO2018221496A1 (fr)

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PCT/JP2017/020661 WO2018220835A1 (fr) 2017-06-02 2017-06-02 Procédé de séparation de cellules rares dans un échantillon de sang, et procédé d'analyse des gènes de ces cellules
JPPCT/JP2017/020661 2017-06-02

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PCT/JP2018/020498 Ceased WO2018221496A1 (fr) 2017-06-02 2018-05-29 Procédé de séparation de cellules rares dans un échantillon de sang, et procédé d'analyse des gènes de ces cellules

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11352125A (ja) * 1998-05-07 1999-12-24 Immunotech Sa 赤血球の溶解のための新規の試薬及び方法
JP2002148255A (ja) * 2000-09-14 2002-05-22 Immunotech Sa カルバメートを用いる赤血球のための多機能試薬及びその適用
WO2004056978A1 (fr) * 2002-12-19 2004-07-08 Ivonex Gmbh Procede de separation de fractions cellulaires
WO2010135603A2 (fr) * 2009-05-20 2010-11-25 California Institute Of Technology Méthode de dépistage, de diagnostic et de pronostic du cancer
WO2012173097A1 (fr) * 2011-06-13 2012-12-20 日立化成工業株式会社 Agent pour l'amélioration de l'adhésivité de cellules cancéreuses
WO2014162810A1 (fr) * 2013-04-04 2014-10-09 日立化成株式会社 Filtre pour capturer une substance biologique
WO2016047444A1 (fr) * 2014-09-24 2016-03-31 株式会社カネカ Matériau de séparation de cellules et procédé de séparation de cellules

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11352125A (ja) * 1998-05-07 1999-12-24 Immunotech Sa 赤血球の溶解のための新規の試薬及び方法
JP2002148255A (ja) * 2000-09-14 2002-05-22 Immunotech Sa カルバメートを用いる赤血球のための多機能試薬及びその適用
WO2004056978A1 (fr) * 2002-12-19 2004-07-08 Ivonex Gmbh Procede de separation de fractions cellulaires
WO2010135603A2 (fr) * 2009-05-20 2010-11-25 California Institute Of Technology Méthode de dépistage, de diagnostic et de pronostic du cancer
WO2012173097A1 (fr) * 2011-06-13 2012-12-20 日立化成工業株式会社 Agent pour l'amélioration de l'adhésivité de cellules cancéreuses
WO2014162810A1 (fr) * 2013-04-04 2014-10-09 日立化成株式会社 Filtre pour capturer une substance biologique
WO2016047444A1 (fr) * 2014-09-24 2016-03-31 株式会社カネカ Matériau de séparation de cellules et procédé de séparation de cellules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAKACSOVA, G., SUBIK, J.: "The hemolytic activity of heterocyclic N-Alkyl Amine Oxides", EXPERIENTIA, vol. 33, no. 11, 1977, pages 1415 - 1416, XP055634507, DOI: 10.1007/BF01918776 *

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