JP5834559B2 - Target cell inspection method - Google Patents

Description

  The present invention relates to a method for recovering target cells from blood, a kit and a system used for the method. More specifically, the present invention relates to a method for lysing red blood cells and collecting leukocytes and rare target cells by filtration (size separation), and a kit and system used in the method.

  One of the diagnosis of malignant tumor (cancer) or a method for discriminating whether the tumor is localized or metastatic is a blood test. Tumor cells such as blood free cancer cells [CTC] are differentiated from morphology by microscopic observation, ie, microscopic examination, but per 10 mL of blood collected from cancer patients regardless of stage (stage). The number of contained tumor cells is very small, about 10.

  Methods for filtering a cell suspension such as blood using a filter are described in many documents. For example, Patent Document 1 discloses parylene having a pore diameter of about 5 μm to about 12 μm as a pretreatment for CTC examination. A method for filtering blood using a membrane filter including a substrate is disclosed. However, for example, when a CTC having a diameter of about 11 μm exists, there is a risk that the CTC may be lost by filtration. In addition, in order to prevent clogging during filtration, it is necessary to dilute blood and perform filtration at a low speed (flow rate). It may take.

  On the other hand, Patent Document 2 describes that red blood cells are lysed using a red blood cell lysing agent as a pretreatment for CTC examination. However, when red blood cells are lysed, cell fragments derived from red blood cells are produced, which may hinder observation. Furthermore, there is a risk of losing the target cells by washing the cells after lysis.

Special table 2008-538509 gazette Special table 2009-525468 gazette

  In order to facilitate observation of rare target cells contained in blood, the present invention reduces the number of red blood cells contained in blood, but recovers the target cells without reducing the number of white blood cells and target cells as much as possible, It is an object to provide kits and systems for use in the method.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have clarified the target cells without clogging or diluting the blood by filtering after dissolving the red blood cells in the blood. The present inventors have found that it is possible to reduce the red blood cell count / white blood cell count ratio to about 1 to 10 at the highest without making a washing operation that may cause loss, and the present invention has been completed.

That is, the method of inspecting of the present invention, the purpose cells,
(a) a step of hemolysis by adding a red blood cell lysing agent to the blood;
(b) Using a filter paper filter having a pore size of 0.4 μm or more and 5 μm or less, among the blood cell components contained in blood or the collapsed blood cell components, target cells containing at least CTC are placed on the filter paper filter. Separating to remain in ; and
(c) confirming the presence or absence of target cells containing at least CTC by observing the filter paper filter;
Only including,
Of the steps (a) to (c), the step (c) is performed last, and this is a method for examining target cells containing at least CTC contained in blood .

Whether the step (b) is performed after the step (a) or the step (a) is performed after the step (b), the step (a) and the step (b) are simultaneously performed. You may implement .

The solubilizer may be ammonium chloride, an amine, or a surfactant, and the surfactant is preferably a nonionic (nonionic) surfactant .

Also, the observation system of the present invention, a separation means using the filter paper-like filter, and a addition unit above Ki溶 solution agent, characterized in that it is used in the method of the present invention.

  According to the present invention, while reducing the number of red blood cells contained in blood, the number of white blood cells and target cells is not reduced as much as possible, and it is possible to easily observe rare target cells contained in blood. Recovery can be performed.

  In particular, when performing the hemolysis step for lysing red blood cells and the size separation step for separating the blood cell components or the collapsed blood cell components by size at the same time or in that order, the red blood cells are lysed and washed. Instead, leukocytes and rare target cells can be separated from erythrocyte fragments and undissolved erythrocytes by filtration (size separation). Even if a filter paper filter is used for filtration (red blood cells can be removed in the filter filtrate and white blood cells can be collected on the filter), a flow path filter is used as an alternative (filter classification is performed to trap or trap white blood cells). It can be recovered as a turbid liquid).

  When filtration is performed after lysis of erythrocytes, it is not necessary to dilute blood (the concentration of target cells does not decrease), and since filtration can be performed quickly, a large amount of blood can be processed in a short time.

By replacing the washing step with a size separation step, the loss of white blood cells can be reduced, and unnecessary residues can be removed by filtration.
In addition, when the hemolysis step is performed after the size separation step, the size at the time of separation can be set slightly larger, thereby reducing the damage to target cells that occurs at the time of separation, and the red blood cells in the subsequent hemolysis step It is further effective in that it can remove an inhibitor of the lysis reaction (eg, unnecessary cells such as disrupted erythrocytes contained in plasma).

FIG. 1 is a schematic diagram of a micro-channel device in an embodiment in which the channel-shaped filter is most simplified, which is FIG. 1 described in Japanese Patent No. 4005022. When blood is introduced into the branch (10), the leukocytes are separated into the second drainage channel (12), and the blood cell component or the collapsed blood cell component is separated into the first drainage channel (11). FIG. 2 shows a schematic diagram of a microchannel device which is an embodiment of the channel filter used in Reference Examples 4 and 5 and Comparative Example 4. The unit of the numbers in the figure is μm. As branching channels, 10 each connected to outlets 2 to 4 and 100 connected to outlet 5 were provided. When blood diluted 10-fold with PBS from inlet1 and PBS from inlet2 are introduced continuously, red blood cells and platelets with an average diameter of less than 7 μm are obtained from outlet 5, 7-10 μm white blood cells from outlet 4, and 10 from outlet 3. The leukocytes of ˜17 μm are designed to flow out of the liquid mainly containing leukocytes of 17 to 30 μm from outlet 2 and leukocytes of 30 μm or more from outlet 1. FIG. 3 is a schematic diagram for explaining the basic principle of the hydraulic filtration method in the microchannel device shown in FIGS. FIG. 4 schematically shows the configuration of each component used when filtering in the state (b) of Example 3 with the filter paper filter sandwiched between the clamp [2] and the support screen [3]. It is a figure.

Next, a method for recovering target cells from blood, a kit and a system used for the method of the present invention will be described in detail.
<Method of collecting target cells from blood>
In the step (a) of the present invention, when a erythrocyte lysing agent is added to the blood to cause hemolysis, fragments of red blood cells after dissolution (redacted red blood cells), small red blood cells (red blood cell debris), and red blood cells that have not been dissolved remain. There is. Conventionally, these unnecessary residues have been removed by washing (after adding a washing liquid and centrifuging, discarding the unnecessary residues as a supernatant and resuspending and collecting leukocytes as a precipitate). However, in the removal by washing, the discarded supernatant contained white blood cells (loss), or the red blood cells were settled and remained (mixed) in the lower layer.

  The method for recovering target cells from blood according to the present invention is characterized in that the above-mentioned step of washing (operation) is unnecessary, and includes the following steps (a) and (b), preferably the following steps: (c) a method of reducing leukocyte loss.

(a) A step of hemolysis by adding a red blood cell lysing agent to blood.
(b) A step of separating a blood cell component contained in blood or a material in which the blood cell component is disrupted by using a separation means depending on its size.

(c) A step of examining a target cell by observing a cell population obtained by performing the steps (a) and (b) (that is, the method for recovering a target cell from blood according to the present invention).
Here, “inspection” includes simply examining the presence or absence of the target cell, identifying the cell type, and the like.

  The present invention is also positioned as a pretreatment for examining target cells. First of all, it is necessary to observe all cells contained in the suspension after pretreatment, so reducing the number of red blood cells and maintaining the number of white blood cells (ie, reducing the ratio of red blood cells / white blood cells) desirable.

  In the present invention, whether the step (b) is performed after the step (a) or the step (a) is performed after the step (b), the step (a) and the step (b) are performed simultaneously. May be. In any case, step (c) is performed last. That is, the method for recovering target cells from blood according to the present invention includes the following first, second and third aspects.

[First aspect]
In the first embodiment, step (b) is performed after step (a), and step (c) is finally performed.

  The first embodiment will be specifically described. First, a red blood cell lysing agent is added to blood to cause hemolysis. Next, leukocytes that did not disintegrate with the red blood cell lysing agent, blood cell components such as blood free cancer cells [CTC] and red blood cells (all red blood cells may disintegrate and may not exist), and disintegration with the red blood cell lysing agent Depending on the size of the blood cell component (which is the one in which the red blood cells are disintegrated, but may also be the one in which the blood cell components other than the red blood cells are disintegrated) is separated using, for example, a filter filter Alternatively, separation is performed using, for example, a flow channel filter. Finally, the cell population obtained in this way is observed with a microscope or the like, and the target cells such as CTC are examined.

[Second embodiment]
In the second embodiment, step (a) is performed after step (b), and step (c) is finally performed.

  The second aspect will be specifically described. First, blood as it is or diluted with an appropriate buffer is used, for example, using a filter filter or the like, depending on the size of white blood cells, CTCs and red blood cells. Separated or separated using, for example, a flow channel filter. Next, a erythrocyte lysing agent is added to the blood containing white blood cells and CTC on the filter paper filter or white blood cells and CTC separated by the flow path filter to cause hemolysis (mainly only the remaining red blood cells are disrupted). ) Finally, the cell population obtained in this way is observed with a microscope or the like, and the target cells such as CTC are examined.

[Third embodiment]
The third aspect is an aspect in which the step (a) and the step (b) are performed simultaneously, and the step (c) is finally performed.

  The third embodiment will be specifically described. First, blood (or blood diluted with an appropriate buffer) and red blood cell lysing agent are each dropped onto a filter paper filter. Red blood cells that are smaller than the pore size of the filter (for example, platelets) contained in the blood are filtered, and red blood cell debris is filtered at the same time as red blood cells are lysed on the filter. Alternatively, as shown in FIG. 1, blood (which may be blood diluted with an appropriate buffer) and red blood cell lysing agent are continuously introduced from inlets 1 and 2 of the microchannel device, respectively, and lysis and separation are performed. At the same time. Next, the cell population obtained in this way is observed with, for example, a microscope, and a target cell such as CTC is examined.

[filter]
The separation in the step (b) may be performed using a filter paper filter or may be performed using a flow channel filter.

  The pore size of the filter paper filter is preferably 0.4 μm or more and 5 μm or less. As such a filter paper filter, for example, (PC MB 25 mm 3.0 μm) and (PC MB 25 mm 5.0 μm) of “New Clipore Membrane (trademark)” series manufactured by GE Healthcare Japan Co., Ltd. are suitable. is there.

  As shown in FIG. 1, the flow path filter branches an inflow channel (9) for supplying blood to the branch (10), and a blood cell component contained in blood or a fluid having a collapsed blood cell component ( Comprising a flow channel (3) comprising a first discharge channel (11) leading away from 10) and a second discharge channel (12) leading out the fluid having the target cell away from the branch (10), The target cell flows in the second discharge channel (12) much faster than in the first discharge channel (11), and the inner diameter of the first discharge channel (11) is not less than 0.4 μm and not more than 5 μm. Is preferred. As the on-flow path filter used in the present invention, it is more preferable to use a micro flow path device as shown in FIG. 2, for example, “Mucell Sorter (trademark)” (manufactured by Advance Co., Ltd.).

  It is preferable that the pore diameter or the inner diameter of these filters is in the above range because only blood cell components (mainly red blood cell debris) disintegrated by the red blood cell lysing agent are filtered, and blood cell components that have not been disintegrated are not filtered.

For example, Matsuda et al. “Classification of blood cells using hydraulic filtration” (Electronics Theory, Vol. 128, No. 10, 2008) It will be briefly described with reference to FIG. In FIG. 2 (a), a particle suspension is continuously introduced from a lower inlet (inlet) into a channel having a branch point in the middle (the hatched lines in the figure are introduced into the branch channel). Fluid part). At this time, particles having a center on the branch channel side (right side in the figure) with respect to the boundary line A determined by the flow rate to the branch channel and the like, such as the particles a, are introduced into the branch channel. When the center of the particle c is on the left side of the boundary line A, it is not introduced into the branch channel. Fig. 3 (b) shows the flow path produced based on this principle. Here, many outlets and two inlets are provided, and a liquid that does not contain particles (the composition is the same as the continuous phase of the particle suspension) is introduced from one of the inlets (left side of the figure). The particles are allowed to flow in the vicinity of the wall (the right side in the figure) having the branch flow path. Furthermore, when a plurality of branch flow channels adjusted so that particles of a certain size or larger are not introduced into the region d in the figure, the particles of a certain size or larger are gradually attracted to the right channel wall, Eventually it will be aligned to the wall side. Then, in the downstream (region e in the figure), by gradually increasing or decreasing the branch flow path so that small particles are discharged in order, the particles can be separated according to size and can be concentrated simultaneously.

[Red blood cell lysing agent]
As the erythrocyte lysing agent used in the present invention, various known lysing agents can be used, which may be ammonium chloride, amine, surfactant, or ammonium chloride. Or you may use together an amine and surfactant.

  When the red blood cell lysing agent is ammonium chloride, the generated ammonium ions cause a difference in osmotic pressure on the red blood cell membrane surface, and the water outside the red blood cell flows into the red blood cell through the semipermeable membrane. , Which ruptures (disintegrates) red blood cells. For example, use a commercial product such as “PharmLyse (trademark)” (a 10-fold concentrated buffer based on ammonium chloride) manufactured by Nippon Becton Dickinson Co., Ltd. Can do. According to the manufacturer of “PharmLyse”, “PharmLyse” is diluted 10 times and added to a solution in which red blood cells are suspended (for example, 1 mL of “PharmLyse” diluted 10 times is added to 100 μL of whole blood, etc.) After gentle stirring, it is recommended to react in the dark for 15 minutes at room temperature (20-25 ° C).

When the erythrocyte lysing agent is an amine, the mechanism of action to disrupt erythrocytes is similar to that of ammonium chloride, but it does not generate ammonium ions, so it does not cause pH change, and ammonium chloride is used. Hemolysis occurs more slowly than in the case. Commercially available amines include, for example, “VersaLyse (trademark)” manufactured by Beckman Coulter, Inc. (the main component having activity is a cyclic amine, and when the cyclic amine comes into contact with carbonic anhydrase present in erythrocytes, It is converted into a compound that exhibits high solubility. According to the manufacturer of “VersaLyse”, 1 mL of “VersaLyse” is added to 5 × 10 ⁵ leukocytes / 100 μL (red blood cells 6 × 10 ⁸ cells / 100 μL), and at least 10 minutes at room temperature (18-25 ° C.) It is recommended to react in the dark.

  When the erythrocyte lysing agent is a surfactant, it is preferably a nonionic (nonionic) surfactant. Examples of the nonionic surfactant include general commercially available reagents such as “Triton X-100 (trademark)” and “TWEEN 20 (trademark)” manufactured by GE Healthcare Japan.

  Red blood cells can be lysed in a desired manner by appropriately adjusting the type of red blood cell lysing agent used (the strength of the degree of red blood cell lysis), concentration and reaction time. For example, even if a solubilizing agent having the same degree of dissolution is used, either when the reaction time is increased at a relatively low concentration or when the reaction time is decreased at a relatively high concentration, The ratio of the number of red blood cells / white blood cells after collection can be lowered (specifically, a ratio of about 1 to 10) without depending on the filter used for separation. For example, when time is required for filtration, such as separation with a flow channel filter, the lysis process is performed before separation by allowing the red blood cells to flow down in contact with the red blood cell lysing agent. Can be. Blood added with a red blood cell lysing agent may be introduced into the flow channel, or a red blood cell lysing agent may be introduced through another flow channel and contacted with the red blood cells.

[Target cell]
The target cell is preferably a rare cell contained in blood, particularly preferably a blood free cancer cell [CTC].

The ratio of red blood cells to the number of white blood cells in blood, ie, (number of red blood cells) / (number of white blood cells) is on the order of 10 ³ (about 1000 to 2000). More specific data is shown in the table below.

ＣＴＣの直径は、例えばClin Cancer Res. 2004 Dec 15;10(24):8152-62などＣＴＣに関する多くの報告において記載されており、平均として約20μmである。

The diameter of CTC is described in many reports on CTCs, for example Clin Cancer Res. 2004 Dec 15; 10 (24): 8152-62, and averages about 20 μm.

  Therefore, CTC has the same diameter as many of the white blood cells contained in the blood. Therefore, by recovering many of the white blood cells without reducing the number of them as much as possible, the CTC as the target cell is lost. This can be prevented.

<White blood cell observation kit>
The leukocyte observation kit of the present invention comprises the filter paper filter or the flow channel filter and the erythrocyte lysing agent, and is used for the method of recovering target cells from blood according to the present invention. To do.

  For example, CTC can be detected with high accuracy by using the leukocyte observation kit of the present invention and blood collected from a subject. From this result, the presence of a preclinical noninvasive cancer (carcinoma in situ) that cannot be detected by palpation or the like can be predicted with high accuracy.

  Specifically, such a kit includes, in addition to a filter and a lysing agent, an appropriate buffer for diluting blood, and various equipment, materials, and devices required for carrying out the method of the present invention. You can also.

<System for pretreatment of observation>
The observation pretreatment system of the present invention includes a separation means using the filter paper filter or the flow channel filter and a means for adding the erythrocyte lysing agent, and is used in the method of the present invention. It is characterized by being able to.

Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these.
[Example 1]
First, as a step (a), “PharmLyse (trademark)” diluted 10-fold with distilled water as a solubilizing agent in a tube containing 100 μL of commercially available blood (“normal human whole blood / type B” manufactured by Kojin Bio Inc.) 1 mL of Nippon Becton Dickinson Co., Ltd. was added. And after stirring gently, it left still at room temperature for 15 minutes in the dark place.

  Next, as a step (b), the whole amount was filtered using a filter paper filter having a pore diameter of 0.4 μm (“New Clipore Membrane (PC MB 25 mm 0.4 μm) (trademark)” manufactured by GE Healthcare Japan).

  The filter paper-like filter was observed with a microscope, the white blood cell count and red blood cell count were counted, and the recovery rate of white blood cells and red blood cells was calculated using the ratio of the number of cells after treatment to the number of cells before treatment as the recovery rate. It is desirable that the recovery rate of target cells (CTC, leukocytes in Examples and Comparative Examples) is high and the recovery rate of unnecessary cells (mainly red blood cells) is low. For unnecessary cells, the low recovery rate indicates the high percentage of removed cells. The results are shown in Table 2.

It should be noted that the number of red blood cells / white blood cells is considered to be slightly smaller than the numbers shown in Table 1, since commercially available blood has been collected for about 2 to 4 weeks.
[Example 2]
In Example 1, the pore size of the filter paper filter used in step (b) was changed to 2 μm (that is, “New Clipore Membrane (PC MB 25 mm 2 μm) manufactured by GE Healthcare Japan Co., Ltd.) was used as the filter paper filter. The filter paper filter was observed with a microscope in the same manner as in Example 1, except that the number of white blood cells and red blood cells was measured, and the recovery rate of white blood cells and red blood cells was calculated. The results are shown in Table 2.

[Example 3]
First, as a step (b), 100 μL of commercially available blood (“normal human whole blood / type B” manufactured by Kojin Bio Co., Ltd.) is used as a filter paper filter having a pore diameter of 2 μm, “Nuclear pore membrane (PC MB 25 mm 2 μm)” As shown in FIG. 4, the sample was placed on a “support screen 19193004” ([3]) manufactured by Advantech Toyo Co., Ltd., and filtered while sandwiched and fixed by “clamp 19111003” ([2]).

  Next, as step (a), 1 mL of “PharmLyse” diluted 10-fold with distilled water is dripped onto the filter paper filter, and the filter is gently squeezed so that the PharmLyse spreads over the entire filter paper filter surface. The mixture was shaken using Max 1040 5 ° (trademark) ”(manufactured by Heidolph). Then, it left still at room temperature for 15 minutes in the dark place.

The filter paper filter was observed with a microscope, the white blood cell count and red blood cell count were counted, and the recovery rate of white blood cells and red blood cells was calculated. The results are shown in Table 2.
[ Reference Example 4]
First, as a step (a), 200 μL of “PharmLyse ™” (manufactured by Nippon Becton Dickinson Co., Ltd.) diluted 10-fold with distilled water was added to a tube containing 20 μL of commercially available blood. And after stirring gently, it left still at room temperature for 15 minutes in the dark place.

  Next, as step (b), the cell suspension obtained in step (a) was further diluted 5-fold with PBS and separated using “Mucell Sorter” (manufactured by Advance Co., Ltd.). This “Mucell sorter (abbreviated as“ μ cell sorter ”in the table)” has the same basic structure as the microchannel device shown in FIG. The time from the introduction of the sample into the inlet until the cells were collected at the outlet (that is, the reaction time) was 45 minutes.

The liquids collected from Outlets 1 to 4 (shown in FIG. 1) were observed with a microscope, the white blood cell count and red blood cell count were measured, and the white blood cell and red blood cell recovery rates were calculated. The results are shown in Table 2.
[ Reference Example 5]
In step (b), 20 μL of commercially available blood was diluted 50 times with PBS (total volume: 1 mL) and introduced into the inlet of “Mucell Sorter”. At the same time, “PharmLyse” diluted 10 times with distilled water as step (a) 1 mL was flowed from the other inlet to the flow path and separated with a “Mucell sorter”. The time from the introduction of the sample into the inlet until the cells were collected at the outlet (that is, the reaction time) was 50 minutes.

  The liquids collected from Outlets 1 to 4 (shown in FIG. 1) were observed with a microscope, the white blood cell count and red blood cell count were measured, and the white blood cell recovery rate and red blood cell removal rate were calculated. The results are shown in Table 2.

[Example 6]
In Example 1, the dissolving agent used in step (a) was changed to “VersaLyse ™” (manufactured by Beckman Coulter, Inc.), and the reaction time in the dark was reduced from 15 minutes to 10 minutes. Except that the pore size of the filter paper filter used in (b) was changed to 2 μm, the filter paper filter was observed with a microscope in the same manner as in Example 1, and the white blood cell count and red blood cell count were measured. Was calculated. The results are shown in Table 2.

[Example 7]
In Example 1, 0.05% “Triton X-100 (trademark)” (manufactured by GE Healthcare Japan Co., Ltd.) and 0.0001% “TWEEN 20 (trademark) were used in the step (a). ”” (Manufactured by GE Healthcare Japan Co., Ltd.), except that the reaction time was reduced from 15 minutes to 30 seconds, and the pore size of the filter paper filter used in step (b) was changed to 2 μm. The filter paper filter was observed with a microscope in the same manner as in 1, and the white blood cell count and red blood cell count were measured, and the recovery rate of white blood cells and red blood cells was calculated. The results are shown in Table 2.

  In Examples 1 to 7 above, none of the dilution or washing is performed, but the present invention can be performed by further dilution and washing in addition to the steps (a) and (b) according to the present invention. Of course, this effect can be obtained. For example, PBS is added as a washing solution to leukocyte precipitates or suspensions, centrifuged sufficiently so that leukocytes are not contained in the supernatant after centrifugation, and the supernatant after centrifugation is discarded. A process can be performed. In addition, for example, after step (a), the supernatant after centrifugation by washing is not discarded, and each of the supernatant and leukocyte precipitate or leukocyte precipitate resuspended in the step ( It is also possible to carry out the separation of b) separately and recover them.

[Comparative Example 1]
In Example 1, a washing step was performed instead of step (b). That is,
First, as step (a), 1 mL of “PharmLyse” diluted 10-fold with distilled water was added to a tube containing 100 μL of commercially available blood. And after stirring gently, it left still at room temperature for 15 minutes in the dark place.

Next, as a washing step, the tube was centrifuged at room temperature for 5 minutes (200 × g), the supernatant was discarded, and 2 mL of PBS was added. Then, it was centrifuged (200 × g) at room temperature for 5 minutes and the supernatant was discarded.
Finally, 1 mL of PBS was added and suspended. It was diluted to an appropriate concentration and observed with a hemocytometer, the number of white blood cells and red blood cells was counted, and the recovery rate of white blood cells and red blood cells was calculated. The results are shown in Table 2.

[Comparative Example 2]
In Comparative Example 1, instead of “PharmLyse”, using “VersaLyse” manufactured by Beckman Coulter, Inc., except that the reaction time in the dark was reduced from 15 minutes to 10 minutes, the same as Comparative Example 1, Cells diluted to an appropriate concentration were observed with a hemocytometer, the number of white blood cells and red blood cells were counted, and the recovery rate of white blood cells and red blood cells was calculated. The results are shown in Table 2.

[Comparative Example 3]
In Example 3, step (a) was not performed. However, commercially available blood was diluted with PBS to an appropriate concentration, and the pore size of the filter was changed to 8 μm. That is,
As step (b), after diluting commercially available blood to an appropriate concentration, it is filtered through a filter paper filter with a pore size of 8 μm (“New Clipore Membrane (PC MB 25 mm 8.0 μm)” manufactured by GE Healthcare Japan). did.

The filter paper filter was observed with a microscope, the white blood cell count and red blood cell count were counted, and the white blood cell recovery rate and red blood cell removal rate were calculated. The results are shown in Table 2.
[Comparative Example 4]
In Reference Example 4, step (a) was not performed. That is,
As step (b), 20 μL of commercially available blood was diluted 50-fold with PBS and introduced into the “Mucell Sorter” inlet for separation. The time from the introduction of the sample into the inlet until the cells were collected at the outlet (that is, the reaction time) was 60 minutes.

  The liquids collected from Outlets 1 to 4 (shown in FIG. 1) were observed with a microscope, the white blood cell count and red blood cell count were measured, and the white blood cell and red blood cell recovery rates were calculated. The results are shown in Table 2.

  When cells collected from blood (10 mL) collected from a subject using the method of collecting target cells from blood according to the present invention are observed, the ratio of the number of red blood cells / white blood cells can be reduced to about 1 to 10, for example Cells with a small number such as CTC can be efficiently inspected.

DESCRIPTION OF SYMBOLS 1 ... Microchannel device 3 ... Flow channel 8 ... Inlet 9 ... Inflow channel 10 ... Branch 11 ... First discharge channel 12 ... Second discharge channel 14 ... Outlet 15 ... Outlet 16 ... Perforation a ... Small particle centered on the branch flow path side (right side in the figure) from the boundary line A b ... Centered on the left side from the boundary line A, Large particles c: Small particles centered on the left side of the boundary line A d: Regions provided with a plurality of branch channels adjusted so that particles of a certain size or more are not introduced e ... Regions with multiple branch channels that are gradually thickened or shortened so that small particles are discharged in order from upstream to downstream.
[1] ・ ・ ・ Funnel
[2] ・ ・ ・ Clamp
[3] ・ ・ ・ Support screen
[4] ・ ・ ・ PTFE gasket
[5] ・ ・ ・ Ring plate
[6] ・ ・ ・ Base
[7] ・ ・ ・ Rubber stopper

Claims (9)

(a) a step of hemolysis by adding a red blood cell lysing agent to the blood ;
(b) Using a filter paper filter having a pore size of 0.4 μm or more and 5 μm or less, among the blood cell components contained in blood or the collapsed blood cell components, target cells containing at least CTC are placed on the filter paper filter. Separating to remain in ; and
(c) confirming the presence or absence of target cells containing at least CTC by observing the filter paper filter;
Only including,
Of the steps (a) to (c), the step (c) is carried out last, and a method for examining target cells containing at least CTC contained in blood. The method of claim 1, implementing the step (b) after the step (a). The method of claim 1, implementing the step (a) after the step (b). The method of claim 1, implementing the steps (a) and said step (b) at the same time. The dissolution agent A method according to any one of claims 1 to 4 which is ammonium chloride. The dissolution agent A method according to any one of claims 1 to 4 which is an amine. The dissolution agent A method according to any one of claims 1 to 4, which is a surfactant. The method according to claim 7 , wherein the surfactant is a nonionic (nonionic) surfactant. Includes a separating means for using the filter paper-like filter, and the addition means of the dissolving agent,
Characterized in that it is used in the method according to any one of claims 1-8, saw sash stem.

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