WO2018221496A1

WO2018221496A1 - Method for separating rare cells in blood sample and method for analyzing genes of said cells

Info

Publication number: WO2018221496A1
Application number: PCT/JP2018/020498
Authority: WO
Inventors: 清太中村; 勝也遠藤
Original assignee: 日立化成株式会社
Priority date: 2017-06-02
Filing date: 2018-05-29
Publication date: 2018-12-06
Also published as: WO2018220835A1

Abstract

This method for separating rare cells in a blood sample comprises: a step for filtering the blood sample by using a filter and capturing cells on the filter; and a step for bringing the cells captured on the filter into contact with a hemolytic agent. The hemolytic agent includes ammonium chloride or a cyclic amine. The rare cells separated according to this invention can be subjected to genetic analysis efficiently.

Description

Method for separating rare cells in blood sample and method for analyzing genes of the cells

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. For example, 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. . As described above, since a lot of information on diseases can be obtained from rare cells, a technique for separating and analyzing these rare cells is required.

As 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). In this method, a blood sample is filtered. By filtration, rare cells are trapped on the filter while blood cells pass through the pores of the filter and are removed along with the filtrate.

International Publication No. 2015/012315

Generally, gene analysis of cells can be performed by extracting RNA from cells, reverse transcription, and then amplifying (reverse transcription PCR). However, in the rare cells separated from the blood sample by the method using the filter described in Patent Document 1, 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. However, there were cases where 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.

As a result of intensive studies in view of the above problems, the present inventor has found that red blood cells that should be removed remain together with rare cells on the filter in the method of Patent Document 1, and the presence of the red blood cells It was found that it prevented the amplification of transcripts in reverse transcription PCR. Since the number of red blood cells in the blood is remarkably larger than the number of rare cells, the method described in Patent Document 1 suggests that red blood cells are likely to remain on the filter. Guessed.

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.

According to the method of the present invention, since 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.

It is a schematic diagram which shows one Embodiment of Forter. It is a perspective view which shows one Embodiment of a cell capture | acquisition cartridge. It is the II-II sectional view taken on the line in FIG. It is a schematic diagram which shows the structure of a CTC acquisition apparatus.

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).

As a blood sample, 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. To the blood sample, 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. On the other hand, it is preferable not to add an additive such as heparin that has an effect of hindering gene analysis to the blood sample.

“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.

In the filtration step, cells in the blood sample are captured on the filter by filtering the blood sample with a filter. Here, “capturing” means that a blood sample is filtered through a filter, and cells in the blood sample remain on the filter. Of the cells in the blood sample, 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. However, since 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. Here, the substrate 107 may be a thin film having a thickness of 3 μm to 50 μm, 5 μm to 40 μm, or 5 μm to 30 μm, 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. For example, when the rare cell is CTC, the pore diameter of the through hole 106 is preferably 5 μm to 15 μm, more preferably 6 μm to 12 μm, and even more preferably 7 μm to 10 μm.

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.

In the subsequent hemolysis step, 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. By filtering the hemolytic agent through the filter, 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. Can be made. Therefore, according to this method, 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 μL / min to 3000 μL / min, more preferably 20 μL / min to 1000 μL / min from the viewpoint of minimizing damage to rare cells and from the viewpoint of efficiently lysing red blood cells. Preferably, 50 μL / min to 600 μL / min is more preferable.

The hemolytic agent contains ammonium chloride or cyclic amine as a component (haemolytic component) having an action of hemolyzing red blood cells. Specific examples of 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.

From the viewpoint of efficiently lysing erythrocytes, 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. In addition to the above hemolytic component, 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.

Note that it is not preferable to use a fixative for the cells trapped on the filter to perform the fixing treatment.

When separating rare cells in a blood sample by the above method, for example, the cartridge shown in FIGS. 2 and 3 can be used. Hereinafter, 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.

First, 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. Next, the filter 105 may be cleaned by passing a cleaning solution through the filter 105.

After the cells are captured on the filter 105, a hemolytic agent is introduced into the cartridge 100, and the hemolytic agent is filtered through the filter 105 (hemolysis step). At this time, 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. Next, the filter 105 may be cleaned by passing a cleaning solution through the filter 105. Through the above steps, rare cells can be separated while minimizing the number of red blood cells remaining on the surface of the filter 105.

Various analyzes can then be performed on the rare cells separated by the above method. Although the type of analysis is not limited, rare cells isolated by the above method are particularly useful when analyzing genes of rare cells. That is, it can be said that 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.

Since 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.

(CTC capture device)
Hereinafter, the CTC acquisition apparatus used in the embodiment will be described with reference to FIGS. 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. Is provided. 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 μm. The through holes have an opening shape having a major axis of 100 μm and a minor axis of 8 μm, 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. Specifically, 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.

(Reference Example 1)
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.

(Comparative Example 2)
Rare cells were separated from a sample prepared by the same method as in Reference Example 2 using a CTC capture device 60 as follows. First, a washing solution was put in the reservoir 10 and the cell capture cartridge 100 in the filter unit 1 was filled with the washing solution. Subsequently, the sample was introduced into the reservoir 10 and fed to the cell capture cartridge 100 at a flow rate of 600 μL / min, thereby filtering the sample. The filter 105 was cleaned by feeding 2 mL of the cleaning liquid from the processing liquid storage container 5. Thereafter, a hemolytic agent having a formaldehyde concentration of 1% was fed to the cell capture cartridge 100, and the filter 105 on which the cells were captured was immersed for 10 minutes. Next, 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. .

The results are shown in Table 1. 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. In Example 1 using a hemolytic agent containing cyclic amine, 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. . On the other hand, 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. Moreover, since Ct of the comparative example 2 using the hemolytic agent containing formaldehyde is significantly larger than Ct of the reference example 1, it turns out that amplification of cDNA of HER2 gene was prevented. If the transcription product is slowly amplified as in Comparative Example 2, it is difficult to efficiently analyze the gene.

DESCRIPTION OF SYMBOLS 1 ... Filter unit, 3, 6 ... Processing liquid flow path, 4 ... Sample flow path, 5 ... Processing liquid storage container, 8 ... Selection valve, 10 ... Reservoir, 12, 13 ... Pinch valve, 14 ... Peristaltic pump, 16 DESCRIPTION OF SYMBOLS ... Waste liquid tank, 48 ... Control part, 49 ... Selection valve driver, 50 ... Valve driver, 51 ... Pump driver, 60 ... CTC capture device, 100 ... Cell capture cartridge, 105 ... Filter, 106 ... Through-hole, 107 ... Substrate, DESCRIPTION OF SYMBOLS 110 ... Upper member, 115 ... Lower member, 120 ... Housing | casing, 125 ... Inflow pipe, 130 ... Inlet, 135 ... Outlet, 140 ... Outlet.

Claims

A method for separating rare cells in a blood sample,
Filtering the blood sample with a filter and capturing cells on the filter;
Contacting a hemolytic agent with cells trapped on the filter, and
Hemolytic agents include ammonium chloride or cyclic amine,
Method.
Analyzing a gene of a rare cell isolated by the method according to claim 1,
A method for analyzing genes of rare cells in blood samples.
The method according to claim 1 or 2, wherein the rare cells are circulating cancer cells in the blood.
The method according to any one of claims 1 to 3, wherein the filter includes a substrate and a plurality of through holes provided on the substrate.