JPWO2017141789A1 - Storage method of blood-derived specimen and determination method of rare cells - Google Patents

Abstract

An object of the present invention is to provide a method capable of storing a blood-derived specimen in a state where an appropriate test result can be obtained even after several days have passed since blood collection. The blood-derived sample storage method of the present invention comprises a separation step of removing red blood cells from a blood-derived sample to produce a cell sample, a cell fixing solution added to the cell sample, and a formaldehyde concentration of 0.001 to 0.4 v Storage step of storing the cell specimen in a state of being in the range of / v%.

Description

  The present invention relates to a method for fixing or storing a cell sample.

  The blood usually contains blood cells such as red blood cells and white blood cells (for example, neutrophils, eosinophils, basophils, lymphocytes, monocytes, etc.), but also circulating tumor cells (CTC). There may be rare cells such as circulating vascular endothelial cells (CEC), circulating vascular endothelial progenitor cells (CEP), or various stem cells. CTC is a cell that exists in peripheral blood according to the pathology of patients such as breast cancer, lung cancer, prostate cancer, pancreatic cancer, etc., and detecting CTC in blood is, for example, whether or not it is affected by cancer. Or the effect of treatment on the patient can be determined and is clinically useful. However, the number of CTCs present in blood is extremely rare (for example, about 1 to 10 per 10 mL of whole blood), and its detection and counting are not easy.

  In order to detect rare cells, for example, in the prior art documents 1 and 2, the target cells are separated from the blood-derived specimen by adding magnetic particles specifically to the target cells. An inspection method in which a fluorescent label is added to an expressed protein for observation is disclosed. As a method for adding a fluorescent label to a protein, an immunostaining method using a fluorescently labeled antibody is generally used. In order to perform immunostaining appropriately, it is important to maintain the morphology and antigenicity of the target cells. As a means for achieving this, many prior literatures have used cell fixatives (4% in Non-Patent Document 1 and non-patents). In Reference 2, it is essential to fix cells by adding 1% formaldehyde) and reacting with cells.

Wen Xu, Lu Cao, Lei Chen, Jing Li, Xiao-Feng Zhang, Hai-Hua Qian, Xiao-Yan Kang, Yu Zhang, Jian Liao, Le-Hua Shi, Ye-Fa Yang, Meng-Chao Wu and Zheng- Feng Yin. Isolation of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma Using a Novel Cell Separation Strategy. Clinical Cancer Research. 2011, vol. 17, p. 3783-3793. Zhian Liu, Alberto Fusi, Eva Klopocki, Alexander Schmittel, Ingeborg Tinhofer, Anika Nonnenmacher and Ulrich Keilholz.Negative enrichment by immunomagnetic nanobeads for unbiased characterization of cyclic tumor cells from peripheral blood of cancer patients. .

  As described above, a test that observes or counts rare cells in blood is clinically useful for evaluating the diagnosis or therapeutic effect of cancer. In general, in the examination, blood collection work and examination work are continuously performed without any time between them. However, if the equipment or technology of a medical institution such as a hospital that collected blood is insufficient, the blood-derived specimen is transported to a laboratory far from the medical institution, and rare cells are observed or counted at the laboratory. Such inspection may be performed. The transportation time varies depending on the distance between facilities, but generally it takes several days. In that case, the morphology and antigenicity of the cells change due to the transport of the blood-derived specimen, and appropriate test results cannot be obtained.

  In addition, in the inspection facility, there are cases in which the inspection cannot be performed immediately due to the convenience of the inspection facility, for example, because there are a large number of samples waiting for the inspection, or because there are many inspection items for one sample, preparation takes time. Even in such a case, a waiting period of several hours to several days occurs from the time of blood collection until the test is performed, and the form and antigenicity of cells in the blood-derived specimen change as in the case of transportation between facilities. Appropriate test results cannot be obtained.

  As described above, in any case, apoptosis or decay may occur in cells in the blood-derived specimen during transportation between facilities or during a waiting period within the facility. Therefore, in order to prevent or delay these occurrences as much as possible, there is a need for a method for storing a blood-derived specimen so that an appropriate test result can be obtained even if a test operation is performed after long-term storage after blood collection.

  In order to solve the above problems, in one aspect, the present invention is a method for storing a blood-derived sample, the separation step of removing red blood cells from the blood-derived sample to generate a cell sample, There is provided a storage method including a storage step of storing a cell sample in a state where a fixative is added and a formaldehyde concentration is within a range of 0.001 to 0.4 v / v%.

  In addition, the present invention provides a method for determining a rare cell, in a further aspect, a pretreatment step including a storage step of storing a cell sample generated from a blood-derived sample, and a target cell contained in the blood-derived sample. A staining step of adding a fluorescent label that specifically binds to the target cell, and determining whether or not the target cell is based on the fluorescence intensity of the fluorescent label specifically added to the target cell. And a determination method for specifying the determination method. In this determination method, the storage method of the present invention described above is used in the storage step.

  According to the present invention, it is possible to store a blood-derived specimen in a state where an appropriate test result can be obtained even after several days have passed since blood collection.

FIG. 1 is a graph showing the transition of immunostaining fluorescence intensity from the storage day 0 to the storage day 10 of the cell specimens stored by the storage method of Example 2 and Comparative Example 2.

<Storage method>
The blood-derived specimen storage method of the present invention includes at least a separation step and a storage step, and may further include a fixing step and other processing steps as necessary. Hereinafter, each step included in the method for storing a blood-derived specimen of the present invention will be described in more detail.

(blood)
“Blood” as described herein is intended to include any component of whole blood, including red blood cells, white blood cells, platelets, endothelial cells, mesothelial cells or epithelial cells. Also, plasma components such as proteins, lipids, nucleic acids, carbohydrates, and any other cells that may be present in the blood due to pregnancy, organ transplantation, infection, trauma or disease (eg, CTC, CEC, CEP, Other rare cells such as progenitor cells).

(White blood cells)
“Leukocytes” as used herein are leukocytes or cells of the hematopoietic lineage that are not reticulocytes or platelets. Leukocytes include lymphocytes such as T cells, B cells and natural killer (NK) cells. Leukocytes also include phagocytic cells and mast cells such as monocytes, macrophages, granulocytes.

(Red blood cells)
As used herein, “erythrocytes” are erythrocytes, particularly non-nucleated red blood cells.
(Blood sample)
In order to evaluate the diagnosis or therapeutic effect of cancer, it is useful to observe rare cells such as CTC in blood, or to examine the count. The “blood-derived sample” described in the present specification is a sample collected from the “blood” described in the present specification in order to observe rare cells such as CTC in blood or to perform a test such as counting thereof. is there.

  A blood-derived specimen is a blood component that is necessary for diagnosis of cancer, and is a rare cell (target cell) such as CTC to be detected, and a blood component that is not necessary for diagnosis of cancer. Cells other than target cells (non-target cells) such as red blood cells and white blood cells that are not included are included.

(Anti-coagulation treatment)
The blood taken out of the body (whole blood), when exposed to air as it is, becomes a gel with the passage of time, and the cells contained therein cannot be collected and observed. Therefore, it is preferable that the collected blood is immediately subjected to anticoagulation treatment.

  Anticoagulant for whole blood, such as ethylenediaminetetraacetic acid (EDTA) and citric acid (including salts such as sodium salt), which inhibits coagulation by binding to calcium ions by chelation and removing it from the reaction system And anticoagulants of the type that block coagulation by forming a complex with antithrombin III in plasma and suppressing thrombin production, represented by heparin. It can be used according to conditions such as general concentration and processing time. Moreover, you may utilize the blood-collecting tube by which the above anticoagulants were previously accommodated. However, the anticoagulant is not limited to these in the present invention.

[Separation process]
The separation step is a step of removing a red blood cell or the like from a blood-derived sample to generate a cell sample described later. The separation step is a process performed to remove blood components (non-target cells such as red blood cells) such as cells other than the target cells contained in the blood-derived specimen, thereby reducing observation obstacles. Detectability of target cells such as CTC can be improved.

(Red blood cell removal)
In order to reduce the obstruction of observation, blood components such as cells (non-target cells) other than the target cells contained in the blood-derived specimen should be removed as much as possible. Originally, it is preferable to specifically extract the target cell from the blood-derived sample, but if the target cell is to be extracted from the blood-derived sample, the target cell may be lost. Therefore, in the present invention, the target cells are not taken out from the blood-derived sample, but are substances that can be an obstacle to observation contained in the blood-derived sample (for example, red blood cells present in large quantities in blood, other diagnostically A method of removing components that are not necessary for the above is adopted.

  Methods for removing red blood cells include hemolyzing red blood cells using ammonium chloride and the like, and density gradient centrifugation that separates red blood cells and other cells by centrifugation and purifies the cell fraction containing at least the target cells. However, the present invention is not limited to these. Even if red blood cells are removed by the method described above, the red blood cells contained in the blood-derived sample are not necessarily completely removed, and a very small amount of red blood cells may remain in the blood-derived sample. There is. The present invention can also be applied under such circumstances.

  Originally, it is preferable to remove leukocytes which are cells not included in the target cells. When the target cell is a cancer cell such as CTC, erythrocytes that are non-nucleated cells can be removed because their morphology, density, and surface properties are significantly different from those of cancer cells. On the other hand, leukocytes belonging to nucleated cells as well as cancer cells are similar in shape and density to cancer cells, so if you try to remove them by hemolysis or density gradient centrifugation, the target cells will also be removed May be missed and misdiagnosis may occur due to inaccurate counting. In addition, when removal with a leukocyte-specific antibody (for example, an anti-CD45 antibody) is attempted, there is a high possibility that a rare cancer cell may be missed due to the occurrence of a non-specific reaction. Therefore, in such a case, it is preferable not to remove leukocytes from the viewpoint of capturing all the rare cells contained in the blood-derived specimen as much as possible and minimizing the loss.

(Cell sample)
The “cell specimen” described in the present specification refers to a group of cells including target cells that have been pretreated. For example, in the case of cultured cells, it is a group of cells that have been peeled off and washed from the culture vessel. In the case of a blood-derived sample, it is a group of cells mainly composed of white blood cells that have undergone red blood cell removal and washing treatment, and this cell group may contain not only white blood cells but also cancer cells such as CTC. is there. In the examples described in the present specification, washing work was performed as pretreatment of cultured cells, and red blood cell removal and washing work were performed as pretreatment of blood-derived specimens. However, the pretreatment is not limited thereto. Absent.

[Storage process]
The storage step is a step of adding a cell fixing solution composed of a formaldehyde solution of an appropriate concentration to the cell sample and storing the cell sample in the cell fixing solution.

  By performing the storage process, cell morphology and antigenicity changes, apoptosis, and corruption can be prevented even if a test is performed to observe or count rare cells such as CTC in the blood of a cell sample that has passed for a long time since blood collection. Thus, an appropriate inspection result can be obtained.

(Cell fixative)
In the present invention, the “cell fixing solution” refers to a cell fixing agent such as formaldehyde dissolved in a suitable solvent such as phosphate buffered saline (PBS) (or commercially available in the form of an aqueous solution such as formalin). Refers to an aqueous solution diluted.

As the cell fixing solution used in the storage step, a formaldehyde solution is preferable.
When a cell sample is stored for a long time in a conventional high-concentration cell fixing solution (for example, the formaldehyde concentration is 1% or 4%), there is a problem that the immunostaining property of the cell sample is lowered. Therefore, in the present invention, in the storage step, storage is performed using a low-concentration cell fixing solution in which the concentration of formaldehyde in the cell fixing solution is dilute. That is, in the present invention, in the storage step, the amount of formaldehyde is such that the final concentration is 0.001 v / v% to 0.4 v / v% (0.001 v / v% or more and 0.4 v / v% or less). Add. The final concentration of formaldehyde in the cell fixative during the storage process is within the range of 0.001 v / v% to 0.4 v / v%, thereby reducing the immunostaining of the cell specimen in the subsequent staining process You can avoid it.

  In one aspect of the present invention, the final concentration of formaldehyde in the cell fixing solution in the storage step is 0.1 to 0.4 v from the viewpoint that the change in fluorescence intensity with time during immunostaining can be reduced and maintained almost constant. / V% is preferable. If the fluorescence intensity can be maintained almost constant in this way, it becomes easy to standardize cell image processing conditions and compare different data even between cells having different storage days, and one type of target cell in automated measurement. The determination can be performed based on one preset threshold value.

  In another aspect of the present invention, the final concentration of formaldehyde in the cell fixing solution in the storage step is 0.001 v / v% or more and less than 0.1 v / v% from the viewpoint of increasing the fluorescence intensity during immunostaining. It is preferable. When cells are stored in a cell fixative having a final formaldehyde concentration in such a range, the fluorescence intensity during immunostaining tends to be higher when the storage days are longer than when the storage days are shorter. Therefore, when it is necessary to increase the fluorescence intensity during immunostaining, or when it is preferable to increase the fluorescence intensity, the cells may be stored in the cell fixing solution for a relatively long period of time.

Although the contact time (sample storage time) between the cell fixing solution and the cell sample can be appropriately adjusted according to the purpose of examination or the like, for example, it is about 1 to 14 days at room temperature.
In the storage step of the present invention, the cell specimen is stored in a low-concentration cell fixing solution. However, since it has a certain effect in terms of delaying apoptosis and decay of cells, it can also function as a fixing step. Therefore, in the present invention, in order to reduce the number of steps, it is not necessary to perform a fixing step of fixing cells by adding a high concentration fixing solution to the cell specimen before the storage step. However, in order to obtain an effect of delaying cell apoptosis or decay more reliably, a fixing step may be performed before the storage step.

(Immunostaining)
As a method for detecting target cells, immunostaining is generally used in which a dye-modified antibody (hereinafter labeled antibody) is bound to a target antigen of a cancer cell such as CTC and stained. Diagnosis of abnormal expression of genes and proteins is performed based on the result of the immunostaining. In the immunostaining, a fixing process, a staining process, and a determination process are usually performed as described below.

[Fixing process]
The fixing step is a step in which cell fixation using a cell fixing solution prepared using a cell fixing agent described later is performed before cell staining in order to delay cell apoptosis and decay. In addition, from the viewpoint of delaying apoptosis and decay of cells, in the storage method of the present invention, it is preferable to perform a fixing step before performing the storage step. In particular, from the viewpoint of maintaining the same form and antigenicity as a living cell, it is preferable to perform cell fixation at the time of collecting the cell.

  As cell fixing agents, aldehydes such as formaldehyde, glutaraldehyde and glyoxal, ketones such as acetone and methyl ethyl ketone, and alcohols such as ethanol and methanol are generally used. Among these, aldehydes such as formaldehyde and ketones such as acetone are fixatives that act as a crosslinking agent by reacting an aldehyde group and a ketone group with a specific amino acid residue, and have a protein structure. It is a preferred immobilizing agent in the present invention because it can stabilize and gel the cell protoplasm to suppress enzyme activity. However, in the present invention, the cell fixing agent is not limited to these.

  In addition, although it does not act directly as a fixing agent, a donor that releases the fixing agent upon hydrolysis or the like (for example, formaldehyde donor) can be used as one form of the fixing agent.

  Cell fixation can be performed by contacting a cell sample with an appropriate concentration of an immobilizing agent for an appropriate time. Although the density | concentration of the fixing agent in a cell fixing solution can be adjusted suitably, it exists in the range of 0.1-10 v / v%, for example. The contact time between the cell fixing solution and the cell specimen (cell fixing time) can also be adjusted as appropriate. For example, the contact time is preferably 6 hours or less at room temperature.

[Dyeing process]
In the staining process, an antibody modified with a dye (labeled antibody) is specifically bound to a target cell or a non-target cell to a cell sample generated by removing red blood cells from a blood-derived sample, and these cells are bound to each other. This is a step of specifically staining.

  As the labeled antibody, an antibody labeled with a known fluorescent dye or the like can be used. When fluorescent dyes are used, different excitation wavelengths and fluorescent wavelengths are used so that the fluorescent dyes of labeled antibodies that stain target cells and the fluorescent dyes of labeled antibodies that stain non-target cells can be measured separately. It is preferable to use a fluorescent dye.

  As a method for specifically staining the target cell, a fluorescent label is specifically added to the target cell by contacting a cell sample with a solution containing a labeled antibody that specifically binds to the target antigen of the target cell. Can do. An anti-cytokeratin antibody, an anti-EpCAM antibody, an anti-vimentin antibody, or the like can be used as an antibody that shows a specific reaction with the antigen of the target cell, but is not particularly limited in the present invention.

  The method of specifically staining non-target cells can be performed by contacting a cell sample with a solution containing a labeled antibody that specifically binds to a target antigen possessed by the non-target cells. Examples of antibodies showing a specific reaction with the antigen of non-target cells include anti-CD45 antibody, anti-CD2 antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD16 antibody, anti-CD19 antibody, anti-CD203c antibody and the like. Although it can be used, it is not particularly limited in the present invention.

  In addition, as a method for staining all cells (both target cells and non-target cells), staining can be performed by staining cell nuclei. Examples of dyes that stain the nuclei of whole cells include DAPI (4 ', 6-diamidino-2-phenylindole), Hoechst dyes (Hoechst 33342, Hoechst 33258, etc.), Acridine Orange, DAMO, Ethidium Bromide (Ethidium bromide) , Ethidium Homodimer, Propidium Iodide and the like.

[Judgment process]
In the determination step, based on the fluorescence intensity of the fluorescent label that is added in the staining step and specifically binds to the target cell contained in the blood-derived sample, whether or not the cell contained in the blood-derived sample is the target cell. And determining (detecting) the target cell.

(Cell detection)
As a method for detecting a target cell, there is a method of performing cell photography of the stained cell and then detecting the target cell by image analysis.

  For example, when staining is performed using an antibody labeled with a fluorescent dye, first, the fluorescent dye of the labeled antibody that stains the target cell and the non-target cell are stained with respect to the cell sample to which the fluorescent label is added. Excitation light that excites each of the fluorescent dye of the labeled antibody to be stained and the fluorescent dye that stains all cells is sequentially irradiated, and a cell image based on the fluorescence emitted from each fluorescent dye is taken. After that, image analysis such as cell images specifically stained for target cells obtained by photographing, cell images specifically stained for non-target cells, cell images stained for all cells, etc. are performed, and preset fluorescence is set. Target cells are detected based on the intensity threshold.

<Judgment method>
The rare cell determination method of the present invention includes a pretreatment step including the storage step, the staining step, and the determination step, and may further include the fixing step and other processing steps as necessary.

  The pretreatment process includes at least the storage process. When using cultured cells as specimens, washing treatment is included. When using blood-derived specimens, erythrocyte removal and washing treatment are included in the pretreatment process, but other treatments may be performed as necessary. A process may be included.

Hereinafter, the present invention will be described in more detail with reference to examples.
[Example 1]
The storage method of Example 1 is such that a cell sample generated by removing red blood cells from a blood-derived sample is subjected to cell fixation using a high-concentration fixing solution, and then the cell sample subjected to cell fixation is treated with a low-concentration fixing solution. It is characterized by being stored for several days.

(cell)
In Example 1, two types of cells derived from human breast cancer cell line (MDA-MB-231) and cells derived from blood of healthy volunteers collected with a commercially available blood collection tube (Terumo) pre-filled with an anticoagulant. Cells were used. MDA-MB-231 used cultured cells that were subjected to trypsin treatment and washing treatment described later twice with phosphate buffered saline (PBS). As the blood-derived cells, a cell specimen mainly composed of white blood cells subjected to an operation for removing red blood cells was used. In order to remove red blood cells from the blood-derived specimen, red blood cell removal treatment was performed as described below.

[Separation process]
(Red blood cell removal)
In immunostaining, cell fixation is performed using a cell fixing solution, which will be described later. When a large amount of cells (for example, erythrocytes) other than the target cells are present in the blood-derived specimen, the number of CTCs is extremely small compared to that. Therefore, the effect of cell fixation may not be sufficiently exhibited. Furthermore, even when cells have been fixed, blood-derived specimens to which cells have been fixed change the properties, morphology, and specific gravity of the cell membrane, so that components such as red blood cells cannot be separated by density gradient centrifugation. It is difficult and it is desirable to remove red blood cells in advance. Therefore, in this embodiment, cell fixation is performed after removing red blood cells.

  In order to remove erythrocytes, first, 2 mL of blood collected by a blood collection tube and 3 mL of a density gradient centrifugal separation solution (Ficoll, specific gravity 1.077) pre-filled with a centrifuge tube (Becton Dickinson) The mixture was layered from above and centrifuged at 400 × g for 40 minutes at room temperature. After centrifugation, all the layers other than the layer containing a large amount of red blood cells are collected, and centrifuged at 300 × g for 1 minute at room temperature using PBS containing 0.1% bovine serum albumin (BSA) to obtain a cell pellet. The supernatant was removed, and washing was performed twice by adding 1 mL of PBS and washing. After washing, a cell specimen suspension was prepared with PBS.

(Cell fixative)
As a high concentration fixative, 20% formalin solution (containing 8% formaldehyde) (Wako Pure Chemical Industries) is diluted with phosphate buffered saline (PBS) so that the formaldehyde concentration is 4v / v%. Was used. As described later, the low concentration fixing solution was prepared so that the formaldehyde concentration in the centrifuge tube (Proteo Save) was about 0.008 v / v% with the same material.

[Fixing process]
(Cell fixation method)
1 mL of each cell sample suspension was placed in Proteo Save (Sumitomo Bakelite) and centrifuged at 300 × g for 1 minute at room temperature. After centrifugation, the supernatant of the cell pellet was removed, 1 mL of a high concentration fixative was added, and the mixture was reacted at room temperature for 20 minutes while rotating with a rotator. Ideally, the high-concentration fixing solution should be finely adjusted to a predetermined concentration again after being added to the proteo save. However, since the high-concentration fixative has a high formaldehyde concentration, the amount of PBS contained in the cell pellet from which the supernatant was removed has no effect on the formaldehyde concentration without fine-tuning the concentration after addition to Proteo Save. Is slight.

[Storage process]
(Specimen storage method)
After completion of the cell fixation reaction, centrifugation was performed at room temperature at 300 × g for 1 minute. After centrifugation, the cell pellet supernatant was removed so that about 20 μL of the high concentration fixative remained, and PBS was finally added so that the formaldehyde concentration of the low concentration fixative was about 0.008 v / v%. On the other hand, unlike the high-concentration fixing solution, the low-concentration fixing solution has a low formaldehyde concentration, and thus the formaldehyde concentration may be greatly influenced by the amount of PBS contained in the cell pellet from which the supernatant is removed. Therefore, in the present embodiment, PBS is added while performing fine adjustment. As a guide, about 1000 μL of PBS was added to the cell pellet in which 20 μL of the high concentration fixative was left.

  After fine adjustment of the formaldehyde concentration, the cell specimen was stored for 0, 3, or 7 days in the low concentration fixative, with the fixed date as day 0. When cell specimens are stored for a long time in the conventional high-concentration fixing solution (formaldehyde concentration of 1% or 4%) as described above, the immunostaining properties (fluorescence luminance) of the cells are subsequently lowered. Therefore, in the present embodiment, the specimen is stored by using a low concentration fixative with extremely dilute formaldehyde concentration. As a result, the immunostaining property of the cell sample does not deteriorate even after the sample is stored in the fixing solution for a long period of time.

  In the above-described fixing step and storage step, in order to minimize the target cells from being missed, the cell sample is not moved from the centrifuge tube, and a high concentration fixing solution or a low concentration fixing solution is added to the centrifuge tube. Although fixation and specimen storage were performed, the method of cell fixation and specimen storage is not limited to these. For example, the cell sample may be moved to a container in which the fixing solution is accommodated, and it is sufficient if the reaction can be advanced by bringing the fixing solution and the cell sample into contact with each other.

(Immunostaining)
In order to evaluate the immunostaining properties of the cells after the storage step, immunostaining was performed as described below.

  First, centrifuge at 300 × g for 1 minute at room temperature, remove the cell pellet supernatant after centrifugation, add PBS, and centrifuge again at room temperature at 300 × g for 1 minute. The supernatant was removed. Subsequently, the primary antibody reaction solution was added to perform the primary antibody reaction. As a primary antibody reaction solution, an anti-cytokeratin (CK) antibody (Becton Dickinson) is used for MDA-MB-231 cell samples, and an anti-CD45 antibody (BioLegend) is used for cell samples generated from blood samples of healthy individuals. Was added and allowed to react at room temperature for 30 minutes.

After the primary antibody reaction, centrifugation was performed at 300 × g for 1 minute at room temperature, the cell pellet supernatant was removed, and washing was performed 3 times by adding 1 mL of PBS and washing.
After washing, a secondary antibody reaction was performed. To perform the secondary antibody reaction, the MDA-MB-231 cell specimen was analyzed using AlexaFluor 647-labeled anti-mouse IgG2a antibody (Invitrogen) that specifically binds to the anti-CK antibody used in the primary antibody reaction. AlexaFluor488-labeled anti-mouse IgG1 antibody (Invitrogen) that specifically binds to the anti-CD45 antibody used in the primary antibody reaction was added to the generated cell specimen. In order to stain the cell nucleus, Hoechst33342 (Invitrogen) that binds to and stains DNA in the cell nucleus is added to the cell sample generated from the MDA-MB-231 cell sample and the blood-derived sample, and then for 30 minutes at room temperature. Reacted.

After the secondary antibody reaction, the mixture is centrifuged at 300 × g for 1 minute at room temperature, the supernatant of the cell pellet is removed, 1 mL of PBS is added and washing is performed twice, and finally PBS is added to add 10 A cell suspension of ⁵ to 10 ⁶ cells / mL was prepared.

(Immunostaining evaluation)
After the immunostaining, the immunostaining property was evaluated with a fluorescence microscope as described below.
First, 20 μL from the cell specimen suspension after immunostaining was filled in a hemocytometer, and cell images were taken with a fluorescence microscope.

  Next, image analysis of the cell images obtained by imaging was performed. For each cell, the fluorescence intensity of AlexaFluor 647 for MDA-MB-231 cells, and the fluorescence intensity of AlexaFluor 488 for blood-derived specimen cells Calculated according to Equation 1. In each cell sample, the average value of the fluorescence intensity of 30 cells was calculated as an index of immunostaining of the cell sample. Table 1 shows the values normalized with the value of the storage day 0 as 1.

(Formula 1)
[(Maximum fluorescence intensity per cell) − (average background fluorescence intensity)] / (standard deviation of background fluorescence intensity)
[Comparative Example 1]
In Example 1, the immunostaining property of each cell sample was evaluated in the same manner as in Example 1 except that the sample storage method was not performed. That is, in Example 1, after cell fixation, centrifugation was not performed, and cell samples were stored for 0, 3, or 7 days in a high concentration fixative solution, and then immunostaining and calculation of fluorescence intensity were performed. The results are shown in Table 1.

  As can be seen from Table 1, the specimen fixed and stored by the method of Example 1 has almost no decrease in fluorescence intensity even after 7 days of storage compared to Comparative Example 1, and particularly against the antigen present in the cytoplasm. It was remarkable with the antibody (anti-CK antibody used for the MDA-MB-231 cell specimen). From this result, it is possible to store a cell sample without reducing immunostaining by the configuration of the present invention such as storing in a state in which red blood cells are removed from a blood-derived sample and storing in a low concentration fixative. I understand.

[Example 2]
In the storage method of Example 2, cell samples generated by removing red blood cells from a blood-derived sample are not subjected to cell fixation using a high concentration fixative, and the cell sample is stored in a low concentration fixative. The cell specimen is also left as it is for the purpose of cell fixation, and is stored until the storage days.

(cell)
A human breast cancer cell line (MDA-MB-231) in the same state as in Example 1 was used.
(Cell fixative)
In Example 2, 20% formalin solution (containing 8% formaldehyde) (Wako Pure Chemical Industries) was phosphate buffered saline (PBS), and the formaldehyde concentration was (A) 0.4 v / v%, (B) Four types of low-concentration fixing solutions (A) to (D) prepared by diluting to 0.04 v / v%, (C) 0.01 v / v%, and (D) 0.001 v / v%. D) was used.

[Storage process]
(Specimen storage method also used for cell fixation)
Cell samples were put in 1.5 mL each in Proteo Save (Sumitomo Bakelite), and centrifuged at 300 × g for 1 minute at room temperature. After centrifugation, the supernatant of the cell pellet was removed, 1 mL each of the low concentration fixatives (A) to (D) was added, and the mixture was reacted at room temperature for 20 minutes while rotating with a rotator. As described above, since the low concentration fixative has a low formaldehyde concentration, the formaldehyde concentration may be greatly affected by the amount of PBS contained in the cell pellet from which the supernatant has been removed. Therefore, also in Example 2, fine adjustment of the formaldehyde concentration after addition to Proteo Save is performed.

  In sample storage that also serves as cell fixation, after adding low concentration fixatives (A) and (B), the cell sample is left as it is, and 0, 1, 3, or 7 days, starting on day 0 when the fixative is added Stored. In addition, after adding the low-concentration fixing solutions (C) and (D), the cell specimen was left as it was, and stored for 0 or 10 days on the 0th day when the fixing solution was added.

(Immunostaining)
After elapse of storage days, immunostaining was performed as described below in order to evaluate the immunostaining property of the cell specimen.

  First, as a primary antibody reaction, an anti-cytokeratin antibody (CK) or a phycoerythrin (PE) labeled anti-vimentin (Vmt) antibody (CST Japan Co., Ltd.) was added and reacted at room temperature for 30 minutes. In addition, the cell sample fixed and stored using the low concentration fixing solutions (C) and (D) was only labeled with an anti-CK antibody.

After the primary antibody reaction, centrifugation was performed at 300 × g for 1 minute at room temperature, the cell pellet supernatant was removed, and washing was performed 3 times by adding 1 mL of PBS and washing.
After washing, as a secondary antibody reaction, the anti-CK antibody-conjugated MDA-MB-231 cell specimen binds to AlexaFluor647-labeled anti-mouse IgG2a antibody (Invitrogen) that specifically binds to the anti-CK antibody, and to DNA in the cell nucleus Hoechst 33342 (Invitrogen) was added and allowed to react at room temperature for 30 minutes. Only Hoechst 33342 (Invitrogen) was added to the anti-Vmt antibody-bound MDA-MB-231 cell specimen and allowed to react at room temperature for 30 minutes.

After the secondary antibody reaction, the mixture is centrifuged at 300 × g for 1 minute at room temperature, the supernatant of the cell pellet is removed, 1 mL of PBS is added and washing is performed twice, and finally PBS is added to add 10 A cell suspension of ⁵ to 10 ⁶ cells / mL was prepared.

(Immunostaining evaluation)
After the immunostaining, the immunostaining property was evaluated with a fluorescence microscope as described below.
First, 20 μL of the cell specimen suspension after immunostaining was filled in a hemocytometer and photographed with a fluorescence microscope.

  Next, as described above, the fluorescence intensity of AlexaFluor 647 or PE for each cell was calculated using Equation 1, and the average value of the fluorescence intensity of 30 cells was calculated as an index of immunostaining of the corresponding cell specimen. . Table 2 shows the values normalized with the value on the 0th storage day set to 1.

[Comparative Example 2]
In Example 2, the immunostaining and the immunostaining ratio of each cell specimen were evaluated in the same manner as in Example 2 except that a high concentration fixing solution (formaldehyde concentration was 4 v / v%) was used instead of the low concentration fixing solution. did. That is, in Comparative Example 2, each cell sample was fixed with a high concentration fixative and left as it was for 7 days. Thereafter, immunostaining was performed, and the immunostaining property was evaluated. The results are shown in Table 2. The data of Example 2 and Comparative Example 2 are collectively shown in FIG.

  As can be seen from Table 2, the cell samples subjected to the sample storage process that also fixed by the method of Example 2 were compared with Comparative Example 2 for any of the low concentration fixing solutions (A) to (D). Thus, the fluorescence intensity was maintained or improved even after 7 days of storage. In particular, the cell specimen fixed and stored with the low-concentration fixing solution (A) showed little change in fluorescence intensity from the 0th storage day, and the value was maintained almost constant. In addition, the fluorescence intensity of the cell specimen fixed and stored with the low concentration fixing solutions (C) and (D) was remarkably improved, with the storage days being 10 days and the storage days being about 12 times.

  From this result, it is possible to store cells in a low concentration fixative in a state where erythrocytes have been removed from the blood-derived sample without performing cell fixation using a conventional high concentration fixative in advance. It is clear that the fixation proceeds effectively and the specimen can be stored without a decrease in immunostaining. In particular, since the fluorescence intensity of the cell specimen fixed and stored in the low concentration fixing solution (A) is maintained at a substantially constant value, it is possible to standardize the cell image processing conditions and compare different data.

  For example, in immunostaining, antibodies that specifically bind to target cells are used, and ideally, non-target cells are preferably not stained, but non-target cells are stained by non-specific adsorption or the like. There is also. In such a situation, it is necessary to determine whether the cell is a target cell based on the fluorescence intensity of the stained cell to identify the target cell. When a threshold value of fluorescence intensity is set in advance and the above-described target cell is automated based on the threshold value of fluorescence intensity, whether the target cell is based on the threshold value when the fluorescence intensity changes for each examination. Cannot be properly determined. That is, when determining whether or not a target cell is based on one preset threshold value, it is difficult to determine cells having different fluorescence intensities as the same cell. On the other hand, if the target cell is to be automatically detected in response to the fluorescence intensity that changes depending on the number of storage days, etc., it is necessary to perform calibration for each examination and set different threshold values.

  However, according to the present invention, since the change in fluorescence intensity from the 0th storage day is small, it is possible to determine one type of target cell based on one preset threshold value in automated measurement.

  Further, by storing in a low concentration fixing solution, cell fixation proceeds even during transportation, so that the effect of reducing the number of steps can be obtained.

Claims (7)

A method for storing a blood-derived specimen,
A separation step of removing red blood cells from the blood-derived sample to produce a cell sample;
A storage step of adding a cell fixing solution to the cell sample and storing the cell sample in a state where the formaldehyde concentration is within a range of 0.001 to 0.4 v / v%;
Storage method. A cell fixing solution is further added to the cell sample, and the cell sample is further fixed in a state where the formaldehyde concentration is in the range of 0.1 to 10 v / v%,
The storage method according to claim 1, wherein the fixing step is performed before the storage step.   The storage step is a storage step of adding a cell fixing solution to the cell sample and storing the cell sample in a state where the formaldehyde concentration is within a range of 0.1 to 0.4 v / v%. The storage method according to claim 1.   The storage method according to claim 2, wherein in the fixing step, the cell specimen is fixed for 6 hours or less.   The storage step is a storage step in which a cell fixing solution is added to the cell sample and stored in a state where the formaldehyde concentration is within a range of 0.001 v / v% or more and less than 0.1 v / v%. The storage method according to claim 1, wherein the storage method is characterized by the following. A pretreatment step including a storage step for storing a cell sample generated from a blood-derived sample;
A staining step of adding a fluorescent label that specifically binds to a target cell contained in the blood-derived specimen;
A determination step of determining whether or not the target cell is based on the fluorescence intensity of the fluorescent label specifically added to the target cell and identifying the target cell;
A method for determining a rare cell having
The determination method according to claim 1, wherein the storage method uses the storage method according to claim 1.   The said determination step compares the said fluorescence intensity with the specific threshold value preset according to the kind of said object cell, and determines whether it is the said object cell, It is characterized by the above-mentioned. The determination method described.

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