WO2015034178A1 - Agent d'agrégation de cellules sanguines pour la préparation de bloc de paraffine et procédé de préparation de bloc de paraffine à l'aide de ce dernier - Google Patents

Agent d'agrégation de cellules sanguines pour la préparation de bloc de paraffine et procédé de préparation de bloc de paraffine à l'aide de ce dernier Download PDF

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Publication number
WO2015034178A1
WO2015034178A1 PCT/KR2014/006952 KR2014006952W WO2015034178A1 WO 2015034178 A1 WO2015034178 A1 WO 2015034178A1 KR 2014006952 W KR2014006952 W KR 2014006952W WO 2015034178 A1 WO2015034178 A1 WO 2015034178A1
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blood
blood cell
cells
paraffin block
gelatin
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PCT/KR2014/006952
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English (en)
Korean (ko)
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장희진
김선영
백지연
여현양
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국립암센터
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Priority claimed from KR1020140095898A external-priority patent/KR101643403B1/ko
Application filed by 국립암센터 filed Critical 국립암센터
Priority to US14/917,687 priority Critical patent/US10656058B2/en
Publication of WO2015034178A1 publication Critical patent/WO2015034178A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Definitions

  • the present invention relates to a blood cell coagulant for preparing paraffin block and a method for preparing paraffin block using the same, and more particularly, to preparing a paraffin block including blood cells present in blood from which red blood cells are removed from a small amount of blood.
  • Blood cell coagulant for the production of paraffin block and paraffin block using the same which can implement paraffin block for diagnosing blood cancer cells with high reproducibility and excellent sensitivity and specificity as it can improve the cohesiveness and prevent damage and loss of blood cells at the same time It is to provide a manufacturing method.
  • Circulating tumor cells are rare tumor cells that exist in the blood and circulate in the body, and there is one known one million cells in the blood of cancer patients. In spite of the very small amount present in the blood, the presence of blood cancer cells in the patients with metastatic breast cancer, colorectal cancer and prostate cancer has been found to be an important prognostic factor related to the survival rate. There are reports that monitoring can easily predict treatment response.
  • Diagnosing cancer through blood has conventionally predicted cancer occurrence through increased expression of a specific enzyme
  • Korean Patent Laid-Open No. 2003-0036010 discloses a cancer diagnosis method by measuring enzyme activity of protein kinase in blood. .
  • detection of cancer cells in the blood is mainly limited to quantitative analysis of the amount of tumor cells in blood, that is, the degree of tumor burden. If the detected blood cancer cells can be collected and analyzed pathologically, it can be a diagnostic alternative to biopsy for end-stage cancer patients who have difficulty in biopsy, reducing unnecessary examination costs and appropriate for histological type of cancer. The treatment continues to indirectly reduce the cost of treatment.
  • Representative methods for detecting blood cancer cells known to date through these studies include immunological analysis using monoclonal antibodies and separation by cell size. Most of them are in the early stages of development and are commercially available in some parts. However, the method currently used is a one-time test, which allows only a simple count of blood cancer cells or immunocytochemical staining of blood cancer cells once or twice.
  • the surface marker used is EpCAM, and in the case of hematopoietic cancer cells showing epithelial-mesenchymal transition, hepatocellular carcinoma or malignant melanoma, etc., the marker does not respond to the cancer cells and thus may have false negatives.
  • a conventional method for diagnosing blood cancer cells uses a method of removing red blood cells through chemical treatment and then removing only white blood cells by removing leukocytes by a microfiltration membrane or surface marker, and extracting the final blood cancer cells.
  • a problem of poor reproducibility because it has to go through many steps.
  • paraffin blocks are used for the purpose of diagnosing, treating and researching diseases such as special staining and immunohistochemical staining based on the identification of tissues and cells.
  • the application of the diagnosis using paraffin block is generally through biopsy tissue, and the biopsy tissue is fixed, and the fixed tissue is paraffin block through washing, dehydration, transparent, and paraffin infiltration process for the production of paraffin sections. It is cut to thickness and dried.
  • the sediment fixation method when using the sediment fixation method to precipitate the sample through centrifugation in the blood cells for the blood cells, the amount of the cell population sample is reduced or the tissue or cells are interspersed due to the destruction and loss of the blood cells, the cell population after paraffin infiltration It is difficult to distinguish between the sample and paraffin, and there is a problem in that embedding with tweezers is impossible.
  • the target of the diagnostic method using paraffin block is conventional biopsy tissue or somatic cells, and these biopsy tissue or somatic cells have good intercellular adhesion and contain extracellular matrix components that aggregate cells. It was very easy to manufacture. However, since blood cells have no intercellular junction and do not contain a substance that functions as an extracellular matrix that can aggregate cells, the cells are separated from each other without aggregation. In the blood cell sample in this state, since the blood cells are not aggregated even when the above-described method for preparing the various cell population samples is made, when the paraffin block is manufactured for blood cells, blood cells are frequently lost or lost during the manufacturing process. To make it happen, there is a problem that is very difficult to produce the desired paraffin block.
  • the blood cells are not aggregated properly and processed in formalin fixation or paraffin block production, the blood cells themselves are exposed to the outside and are not protected by the blood during the various intermediate processes of manufacturing the paraffin block. More frequent destruction and loss or loss of cells can occur.
  • blood cancer cells are included in the blood in very small numbers, there is a fatal problem in that the cancer cells cannot be diagnosed through paraffin blocks prepared when the blood cells destroyed, lost or lost are blood cancer cells.
  • the present invention has been made to solve the above problems, the first problem to be solved of the present invention is to facilitate the production of paraffin block, and the destruction, damage, loss, loss of specific blood cells of interest in the manufacturing process
  • the present invention provides a blood cell aggregate that can significantly improve diagnostic reliability of blood cells of interest.
  • the second problem to be solved of the present invention is to provide a kit for diagnosing blood cancer cells with remarkably excellent reproducibility, sensitivity and specificity in the diagnosis of blood cancer cells.
  • the third problem to be solved of the present invention is to provide a method for producing a paraffin block using a blood cell aggregate, which has high reproducibility, and excellent sensitivity and specificity.
  • the present invention provides a paraffin block for agglomerating blood cells against blood cells and performing a function of an extracellular matrix for preventing the damage and loss of blood cells.
  • the blood cell aggregate may include gelatin.
  • the concentration of gelatin may be 5 to 35 mg / ml.
  • the blood cell aggregate is pectin (pectin), chondroitin sulfate (chondroitin sulfate), fibronectin (fibronectin), starch (starch) and laminin (laminin) group It may further include any one or more selected materials.
  • the substance further included in addition to gelatin as a blood cell aggregate may be a weight within 5% of the gelatin weight.
  • the blood cell coagulant further comprises a colorant for developing the blood cell aggregate, the colorant is included in 5 to 10 parts by volume with respect to 100 parts by volume of the blood cell aggregates Can be.
  • the concentration of gelatin may be 10 to 30 mg / ml.
  • the blood cancer cell flocculant for preparing paraffin block may be used for the diagnosis of blood cancer cells.
  • the present invention provides a kit for diagnosing cancer cells in the blood, including a blood cell aggregate for preparing paraffin block according to the present invention, in order to achieve the second object described above.
  • the present invention to achieve the above-mentioned third object, (1) removing the red blood cells from the blood of the subject and extracting blood cells; (2) mixing the blood cells with blood cell aggregates according to the present invention to produce blood cell aggregates; (3) curing the blood cell aggregates; And (4) treating the cured product according to step (3) with a paraffin block forming material.
  • the tube (tube) from which the blood cells used in step (1) are extracted may be coated with gelatin solution.
  • the red blood cell removal in step (1) may use ficoll.
  • the step (2) may mix the blood cells and blood cell aggregates in a 1: 1 to 1.5 volume ratio.
  • the blood cell coagulant of step (2) comprises gelatin, and the gelatin concentration in the blood cell coagulant according to the temperature at which step (2) is performed may be performed under the conditions (a) Any one of (c) may be satisfied.
  • step performance temperature of step (2) When the step performance temperature of step (2) is 5 ° C. or more and less than 15 ° C., the concentration of gelatin in the blood cell coagulant is 5-15 mg / ml.
  • step performance temperature of step (2) When the step performance temperature of step (2) is 15 ° C. or more and less than 25 ° C. The concentration of gelatin in the blood cell coagulant is 15 to 25 mg / ml,
  • step performance temperature of step (2) is 15 ° C. or more and less than 25 ° C.
  • the concentration of gelatin in the blood cell coagulant is 15 to 25 mg / ml
  • the concentration of gelatin in the blood cell coagulant is 25 to 35 mg / ml when the temperature in step (2) is greater than 25 ° C. and less than 35 ° C.
  • the paraffin block forming material of step (4) includes alcohol, xylene and paraffin
  • step (4) is paraffin block in the order of alcohol, xylene and paraffin It can be carried out through the automatic infiltration process by treating the forming material.
  • the paraffin block may be used for histological, immunohistochemical or molecular biological detection methods of circulating tumor cells.
  • step (3) may further comprise the step of fixing the cured body according to the step (3).
  • the fixing of the cured body may be performed by one or more selected from the group consisting of formalin, methanol or ethanol.
  • blood cell refers to a cell contained in blood, preferably peripheral blood, wherein the blood cell includes all of nucleated or nucleated cells, and the blood cells are conventional blood cells (blood In addition to normal cells and / or cancer cells, the term includes all of the blood cells, such as macrophages, lymphocytes (lymphocytes), monocytes (peripheral blood mononuclear cells (PBMC), red blood cells, white blood cells and platelets) It is meant to include all of the cells usually classified as blood cells, such as.
  • PBMC peripheral blood mononuclear cells
  • paraffin block in preparing a paraffin block containing blood cells, it is difficult to immobilize blood cells by a conventional method for preparing a conventional cell population sample, and the blood cell population sample immobilized by a conventional method is a paraffin block.
  • Critical problems that can be damaged, lost and / or lost in the manufacturing process can be solved.
  • paraffin blocks may be used for the purpose of histological, immunohistochemical and molecular biology of certain cells of interest, such as blood cancer cells. It can detect through various methods.
  • the manufacturing process of the paraffin block can be simplified, and the manufacturing time and manufacturing cost can be reduced.
  • paraffin blocks containing blood cells prepared by the present invention can reduce background staining and obtain thin sections, thereby providing excellent resolution.
  • the morphological confirmation of the cancer cells in the blood enables to distinguish from contaminated epithelial cells, thereby reducing errors in false-positive determinations, thereby providing excellent sensitivity and specificity.
  • the count of blood cancer cells per unit volume of blood can be counted by the count of leukocytes and blood cancer cells contained per unit area.
  • specimens are made of paraffin blocks for permanent storage and over 30 different immunohistochemical tests.
  • a paraffin block can be manufactured even with a small amount of blood collection, it is possible to diagnose and diagnose a cancer recurrence and metastatic cancer by measuring and preparing blood sample collected from a sample with a time difference.
  • Figure 1 shows a schematic experimental process for the paraffin block manufacturing method according to an embodiment of the present invention.
  • Figure 2 is a photograph observed at 200-fold magnification using a microscope after performing CK immunohistochemical staining for paraffin sections prepared from colorectal cancer cell line (SW620) -derived blood cancer cells according to an embodiment of the present invention.
  • FIG. 3 shows H & E staining and CK immunity for paraffin fragments prepared after spiking cancer cells: leukocyte derived from colorectal cancer cell line (SW620) in normal blood according to a preferred embodiment of the present invention. After histochemical staining, the image was observed at 200 times magnification using a microscope.
  • SW620 colorectal cancer cell line
  • Figure 4 is carried out CK immunohistochemical staining for paraffin sections prepared from colorectal cancer cell line (SW620) -derived blood cancer cells in accordance with a preferred embodiment of the present invention, 400 times using a microscope to detect false positives due to contamination It is a photograph observed at magnification.
  • SW620 colorectal cancer cell line
  • Figure 5 is a micrograph observing the cytological pattern of blood cancer cells through the paraffin block prepared differently according to the blood standing time after blood collection of blood cancer cells (SKBR3) -derived specimens before red blood cell separation treatment according to an embodiment of the present invention.
  • SKBR3 blood cancer cells
  • FIG. 6 shows H & E staining, c-erbB2 immunohistochemical staining and fluorescence in situ hybridization reaction against Her2 / neu genes for paraffin fragments prepared from breast cancer cell lines (SKBR3) -derived blood cancer cells according to one preferred embodiment of the present invention. ), And then observed at 1000 times magnification using a microscope.
  • SKBR3 breast cancer cell lines
  • FIG. 7 illustrates H & E staining and CK immunohistochemical staining for paraffin fragments prepared from hepatic cancer cell lines (HepG2) -derived blood cancer cells according to an exemplary embodiment of the present invention, followed by cancer specific markers EpCAM and liver cancer specific markers.
  • Gene expressions for Glypican-3 and alpha-fetoprotein (AFP) are photographed at 400-fold magnification using a microscope.
  • FIG. 8 shows CK immunohistochemical staining for paraffin fragments prepared from colorectal cancer cell line (SW620) -derived blood cancer cells according to an embodiment of the present invention, and then CDX2, a cancer-specific marker EpCAM and a colon cancer-specific marker. And gene expression for p53 at 400-fold magnification using a microscope.
  • SW620 colorectal cancer cell line
  • FIG. 9 shows H & E staining and CK immunohistochemical staining of paraffin sections prepared from blood of stage 4 colon cancer patients with liver metastases according to a preferred embodiment of the present invention.
  • FIG. 10 is a photograph taken by H & E staining and CK immunohistochemical staining for blood cancer cells observed in the blood of the patient, and then observed at 400 times magnification using a microscope.
  • paraffin block is used for the purpose of diagnosing, treating and researching diseases such as special staining and immunohistochemical staining based on the identification of tissues and cells.
  • diseases such as special staining and immunohistochemical staining based on the identification of tissues and cells.
  • the paraffin block is prepared by making the cells in the cell population by using conventional methods, when the paraffin block fragments are stained, the background staining is excessive, so that the sensitivity and specificity for the detection of the specific blood cells that are targeted are significantly decreased. There was this.
  • blood cells do not contain substances that perform functions of extracellular matrix, such as intercellular aggregation, on the surface of cell membranes.
  • paraffin block When the paraffin block is prepared for the cells, there is a problem in that it is very difficult to prepare the desired paraffin block due to frequent loss or loss of blood cells. Furthermore, when blood cells undergo formalin fixation or paraffin block production, blood cells are destroyed, lost, and / or lost during various intermediate processes of manufacturing paraffin block as the blood cells themselves are not exposed and protected outside. This occurred frequently. In particular, since blood cancer cells are included in the blood in a very small number, there is a fatal problem in that the cancer cells cannot be diagnosed through paraffin blocks prepared when the blood cells that are destroyed, lost and / or lost are blood cancer cells.
  • the conventional methods for diagnosing blood cancer cells are less reproducible because they have to go through many steps until blood cancer cells are extracted by removing red blood cells through chemical treatment and then extracting only blood cancer cells through a white blood cell removal method using a microfiltration membrane or a surface marker. There was a problem.
  • a blood cell aggregation material which aggregates blood cells with respect to blood cells and performs an extracellular matrix function to prevent damage and loss of blood cells.
  • a blood cell sample as a cell population sample and a method of sampling a conventional biological tissue in a conventional method for preparing a cell population sample for a conventional biological tissue.
  • Blood cell population prepared in the present invention can solve the fatal problem that is damaged, lost and / or lost during the preparation of paraffin block. This enables the inclusion of paraffin blocks in the blood cells without damage, loss and / or loss of the specific cells of interest.
  • paraffin blocks may be used for the purpose of histological, immunohistochemical and molecular biology of certain cells of interest, such as blood cancer cells. It can detect through various methods. In addition, by not undergoing multiple centrifugation processes for blood cells, the manufacturing process of the paraffin block can be simplified, and the manufacturing time and manufacturing cost can be reduced. Furthermore, as in the conventional methods for detecting blood cancer cells, physical or chemical treatments for removing blood cells in multiple stages can be omitted, thereby reducing loss of target blood cancer cells and increasing cell retention.
  • the blood cell aggregation material which aggregates blood cells with respect to blood cells and performs the function of the extracellular matrix which prevents the damage and loss of blood cells is demonstrated.
  • the blood cells refer to cells contained in blood, preferably peripheral blood, and the blood cells may include both nucleated or nucleated cells, and the blood cells may include conventional blood cells and blood cells. It may include both normal cells and / or cancer cells, and the blood cells are typically peripheral blood mononuclear cells (PBMCs) such as macrophages, lymphocytes, monocytes, red blood cells, leukocytes, and platelets. It may include all cells classified as blood cells.
  • PBMCs peripheral blood mononuclear cells
  • the blood cell aggregate can function as an extracellular matrix for blood cells from which red blood cells have been removed.
  • the degree of effect expression may not vary depending on the specific type of blood cells.
  • the blood cell aggregates according to the present invention may be used for cells in which red blood cells are removed from blood cells. Can serve as extra substrate.
  • red blood cells in the blood cells contained in the blood occupy a high proportion in the number of cells, and when prepared with paraffin blocks including red blood cells, the specific cells of the blood cells contained per unit area of the paraffin block fragments are included.
  • the probability can be very low.
  • various enzymes that may affect the cell membrane, DNA, RNA and cytoplasm of a specific type of blood cells may be released. Diagnosis of blood cells can be very disadvantageous or impossible. Accordingly, in order to prepare a paraffin block for diagnosing blood cancer cells in the blood, the blood cell aggregates preferably perform an extracellular matrix function on blood cells except red blood cells.
  • the blood cell aggregate according to the present invention aggregates the blood cells, and performs the function of the extracellular matrix to prevent damage and loss of blood cells, the function of the blood cell aggregates will be described in detail below.
  • an extracellular matrix refers to a collection of biopolymers that fill an intracellular or extracellular space, and is present in all tissues, but especially in connective tissues.
  • the extracellular matrix in addition to the physical functions such as support and binding of tissues, physical boundaries, absorption of force, elasticity, and the like in recent years, cell proliferation, cell migration, intracellular metabolism, cell differentiation, cell morphology, etc. It has been known to play a role in regulating physicochemical roles outside of.
  • Blood cell aggregates included in blood cell aggregates according to the present invention also perform the functions of the extracellular matrix as described above for blood cells, but are commonly known as the function of extracellular matrix, cell proliferation, cell migration, intracellular metabolism, etc. It is not a physiochemical role involved, but it is responsible for physical functions such as agglutination of blood cells, defense of physical elements applied to the outside of blood cells, absorption, and elastic elements, through which blood cell aggregates prevent damage and loss of blood cells. To perform the function.
  • the aggregation of the blood cells means that the blood cells in the state of being separated individually through the medium of blood cell aggregates (or blood cell flocculents including the same) form a physically aggregated form.
  • the substance may be a ligand and may not mean that the blood cells are aggregated due to the aggregation of the blood cells by the binding of the receptors on the cell membrane surface or the chemical change of the blood cell aggregates.
  • blood cell aggregates may not include plasma components, more preferably plasma proteins, such as fibrinogen, prothrombin, or thrombin or fibrin in their activated state.
  • the aggregation of blood cells by the blood cell aggregates may not be the aggregation by a conventional coagulation mechanism.
  • blood cells are aggregated by the coagulation mechanism, excessive red blood cells in the aggregates are included so that the amount of blood cells other than the red blood cells contained per unit area is too low, and the diagnostic efficacy is very low.
  • the diagnostic value of blood cell aggregates produced by damaging DNA and RNA of blood cells due to the release of intracellular enzymes is very low, and plasma proteins have problems that cannot achieve the object of the invention.
  • the blood cell aggregate contained in the blood cell aggregate according to the present invention may be very advantageous in preparing the blood cells with paraffin blocks according to the above-described functions.
  • the paraffin block object is prepared as a cell population sample and then treated with paraffin.
  • the conventional paraffin block is manufactured as a biological tissue, and a conventional biological tissue is formed by clustering cells with intercellular junctions. Each of the clustered cells includes an extracellular matrix outside the cell so that the cells can be more aggregated and the cells themselves are physically protected.
  • blood cells unlike biological tissues, each cell independently suspended in the blood, there is no intercellular adhesion, and does not contain substances that perform extracellular matrix outside the cell membrane.
  • the blood cell aggregates included in the blood cell flocculant according to the present invention aggregate the blood cells according to the function of the extracellular matrix to the blood cells so as to solve the above problems. Damage or loss can be prevented.
  • the above-described blood cell aggregate may include gelatin.
  • gelatin is used for various purposes for food and industrial purposes, and it swells in cold water but melts in hot water to become a sol, and when cooled to below room temperature at a concentration of 2 to 3% or more, it becomes a gel. Because it is a heat stable molecule, it may be suitable as the blood cell aggregate of the present invention. In addition, when the aggregated blood cells alone than the material capable of performing other types of extracellular matrix, it is more effective in preventing damage and loss of blood cells to prepare a paraffin block excellent in the physical properties of the present invention. It is more advantageous to do.
  • Gelatin as a blood cell aggregate in accordance with the present invention may mean containing collagen.
  • the gelatin may be included in the form of a gelatin solution, preferably mixed with a solvent.
  • the solvent may include a phosphate buffer solution, distilled water, physiological saline, and the like, and may be distilled water according to a preferred embodiment of the present invention.
  • gelatin included as a blood cell aggregate may be included in the blood cell aggregate for preparing paraffin block according to the present invention preferably at a concentration of 5 to 35 mg / ml, more preferably at a concentration of 5 to 30 mg / ml. More preferably, it may be included at a concentration of 5 to 20 mg / ml. If the concentration of gelatin is less than 5 mg / ml, paraffin block preparation may take time when the paraffin block is prepared for blood cells, resulting in damage to the cells due to gelatin coagulation, or the gelatin may be melted to significantly reduce the sensitivity and specificity of the diagnosis of blood cells. have.
  • the concentration of gelatin exceeds 35 mg / ml, the hardness of paraffin block increases, causing paraffin fragments to bounce off or collapse during the cutting process of paraffin block, causing loss of blood cells and thus sensitivity and specificity of blood cell diagnosis.
  • the degree can be significantly reduced. Specifically, as shown in Table 5 below, which compares the blood cancer cell collection rate according to the gelatin concentration change, when the gelatin concentration is 10 mg / ml, the blood cancer cell collection rate is 93%, whereas the gelatin concentration is 3 mg / ml. It can be seen that the cancer cell collection rate is significantly low as 68%, the gelatin concentration of 50 mg / ml may be confirmed that the cancer cell collection rate is only 6%.
  • the blood cell aggregate may further include any one or more substances selected from the group consisting of pectin, chondroitin sulfate, fibronectin, starch, and laminin. have.
  • the paraffin block is prepared by further including such a substance, the sensitivity and specificity for blood cells can be improved.
  • gelatin when gelatin is included as a blood cell aggregate, gelatin may vary in temperature depending on its concentration. If the concentration of gelatin is increased in order to have a solid or semi-solid in a specific temperature range, the desired properties are realized. There is a problem that the sensitivity and specificity of paraffin blocks for blood cells may be significantly reduced.
  • the gelatin concentration is 20mg / ml If the room temperature is subjected to more than 10 minutes in a room temperature of 28 °C (see Table 6 below) to change the liquid phase Increasing the concentration of gelatin higher than 20 mg / ml to maintain the solid phase or semi-solid phase at 28 ° C. can maintain the solid phase or semi-solid phase as desired. Sensitivity and specificity may not be achieved.
  • the blood cancer cell collection rate of paraffin block is 80%, but when the gelatin concentration is 50 mg / ml, the blood cancer cell collection rate of paraffin block is significantly reduced to 6%, which is higher than a certain concentration. As the concentration of gelatin increases in the blood cancer cell collection rate decreases the sensitivity and specificity of the paraffin block to the cancer cells in the blood can be seen to significantly decrease.
  • pectin, chondroitin sulfate, and fibronectin may be used to preserve the sensitivity and specificity of blood cells, which are reduced with increased gelatin concentrations to maintain the desired properties at specific room temperatures.
  • Any one or more substances selected from the group consisting of starch and laminin may be further included in the blood cell aggregate, and in this case, while maintaining the desired properties at a specific temperature, It may be more advantageous in expressing sensitivity and specificity.
  • the substance further included in addition to gelatin as a blood cell aggregate may be included within 5% of the weight of gelatin. If more than 5% of the weight of gelatin is contained in addition to gelatin, more than 5% of the weight of gelatin increases the hardness of the paraffin block, which causes the loss of blood cells due to the breakdown or fragmentation of the paraffin block. Etc. There may be a problem that the sensitivity and specificity of the blood cell diagnosis is inferior.
  • the blood cell aggregates do not contain gelatin and include only the substances added as blood cell aggregates in addition to the gelatin, it may be difficult to achieve the desired physical properties.
  • the starch is easy to use, but easy to separate, and after adjusting the concentration, there is a disadvantage in that the viscosity or quality does not come out even when the heat is applied again, and also does not melt in cold water, but swells in hot water and luxury.
  • pectin is a polymer of carbohydrate is used as a coagulant, thickener, stabilizer, anti-solidifying agent, emulsifier, etc. in food
  • the thickening power and gelling power of the pectin depends on the balance of acid and ester, acid is not more than 50%
  • acid is not more than 50%
  • non-esterified pectin there is a strong tendency to form gels with calcium ions, which may limit its use alone as a blood cell aggregate.
  • fibronectin fibronectin
  • laminin laminin
  • the gelatin is included as the blood cell aggregate of the present invention, since the gelatin is colorless and transparent, it is difficult to confirm whether or not it is jelled in a container, and thus, the extracellular matrix is developed in the blood cell aggregate of the present invention.
  • Coloring agent for further may be included.
  • the colorant may be bromophenol blue, carbon black, N, N-dimethyl laminoethanol, copper phthaloc yanine, pennsperse orange ),
  • a coloring agent for coloring blood cell aggregates is preferably a loading buffer containing bromophenol blue (Takara Bio Inc.).
  • the colorant may be included in the blood cell aggregate for paraffin block production in 5 to 10 parts by volume with respect to 100 parts by volume of blood cell aggregate. If more than 10 parts by volume of the coloring agent is added to 100 parts by volume of blood cell aggregates, there is a problem that the sample cohesion is weakened or the sample aggregation is delayed, and when the coloring agent is added below 5 parts by volume, the formalin is fixed. There is a problem in that the effect of the disappearance of color is difficult to embed the specimen.
  • kits for diagnosing blood cancer cells including a blood cell aggregate for preparing paraffin block according to the present invention described above.
  • CTCs Circulating tumor cells
  • Blood cancer cell diagnostic kit of the present invention may further comprise a coloring agent for coloring the blood cell aggregates in 5 to 10 parts by volume with respect to 100 parts by volume of the blood cell aggregates contained in the blood cell aggregates for preparing paraffin block.
  • a coloring agent for coloring the blood cell aggregates in 5 to 10 parts by volume with respect to 100 parts by volume of the blood cell aggregates contained in the blood cell aggregates for preparing paraffin block.
  • the blood cells are 40 ⁇ l, 40 ⁇ l of gelatin solution and 2 ⁇ l of colorant are added to the blood cells to confirm that the blood cells and the gelatin solution are aggregated in the form of jelly.
  • Critical significance for the color developer content is the same as described above and will not be described.
  • the method for producing a paraffin block according to the present invention through the above-described paraffin block for blood cell aggregation agent (1) removing red blood cells from the blood of the subject and extracting blood cells; (2) mixing the blood cells with a blood cell aggregate for preparing paraffin block according to the present invention to produce blood cell aggregates; (3) curing the blood cell aggregates; And (4) treating the cured product according to step (3) with a paraffin block forming material.
  • cell pellets obtained from a small amount of blood can be prepared with paraffin block without damaging or losing the cells.
  • the prepared paraffin block can be obtained by only one blood sample of about 5 to 6 ml from the subject.
  • Assays can be performed on specific types of blood cells up to 50 to 60 times, which can be used for pathological as well as clinically very useful and useful assays. Specifically, by securing new new technologies for screening, retrieval and biological analysis of blood cancer cells, it is possible to simplify, reduce the cost and achieve high reliability through the development of comprehensive cancer diagnostic tools and methods.
  • the step (1) includes the step of removing red blood cells from the blood of the subject and extracting blood cells.
  • the red blood cells occupy the largest number of blood cells, and when manufactured with paraffin blocks including red blood cells, the probability of containing a specific type of blood cells among the blood cells contained per unit area of the paraffin block fragment may be very low. Can be.
  • various enzymes that may affect the cell membrane, DNA, RNA and cytoplasm of a specific type of blood cells may be released. Diagnosis of blood cells can be very disadvantageous or impossible. Accordingly, in order to prepare paraffin blocks for the diagnosis of blood cancer cells, only blood cells from which red blood cells are removed from blood collected from a subject should be extracted.
  • the conventional method for diagnosing blood cancer cells in blood removes white blood cells and extracts the final blood cancer cells through various chemical and physical processes as well as red blood cells in the blood of a subject.
  • the centrifugation there is a problem that can cause the loss of cancer cells in the blood, and can lower the collection rate of cancer cells in the blood.
  • the present invention samples blood cells from which blood is removed from blood, only red blood cells are sampled, which greatly simplifies the manufacturing process, shortens the manufacturing time, and reduces cell loss due to no physical or chemical treatment of the cancer cells. It is a very efficient method with high cell retention.
  • the red blood cell removal method that can be used in step (1) according to the present invention may include, but is not limited to, a conventional centrifugal method, inverted centrifugal method, filter method, destran method, and cell lysis method, but is not limited thereto. May be performed by Piccol treatment. Specifically, as shown in Experimental Example 3 below, after spiking 4000 colorectal cancer cell lines SW620 in normal blood (spicoll) and treated with ficoll (cell) and cell lysis (lysis buffer), respectively, red blood cells were removed. In the paraffin block prepared for blood cells, the blood cancer cell collection rate (the total number of blood cancer cells contained in the paraffin block was counted after cytokeratin staining and counted), and red blood cells were removed by picol treatment. The production of paraffin block is 1.7 times higher on average than the cell lysis method.
  • the tube (tube) from which the blood cells used in the step (1) are extracted may be coated with blood cell aggregates.
  • the coating means applying a blood cell aggregate of the same composition as the blood cell aggregate to be mixed with the blood cells in the following step (2) to the bottom surface of the container from which the blood cells are to be extracted.
  • the coating thickness is important to apply a constant so as not to be too thin or thick so that the collection of blood cells, the preferred coating thickness may be 1 ⁇ 2mm.
  • the reason for coating the tube for extracting blood cells with blood cell agglomerate in advance is that when the cell pellet jelly obtained through step (2) is separated from the cell, In order to prevent damage, loss and / or loss of the blood cell aggregates in advance in the container to be used in step (1) can be facilitated collection of blood cell aggregates.
  • step (2) the step of preparing a blood cell aggregate by mixing the blood cells extracted in step (1) with a blood cell aggregate for preparing paraffin block according to the present invention.
  • Paraffin block preparation for the production of paraffin blocks according to the present invention performs an extracellular matrix function on blood cells from which red blood cells have been removed from the blood, thereby maintaining loss of blood cells and damage to cytoplasm. Can maintain the antigenicity of cancer cells.
  • the blood cell flocculant for preparing paraffin block is preferably mixed in an amount of 80 to 150 parts by volume with respect to 100 parts by volume of blood pellets (cell pellet), and through this, the physical properties of the desired paraffin block. It may be more advantageous to the implementation.
  • the properties of the blood cell aggregate at a specific temperature may vary depending on the concentration of gelatin contained in the blood cell aggregate.
  • the concentration of gelatin may be changed in consideration of the performing temperature of step (2), and preferably any one of the following conditions (a) to (c) may be satisfied.
  • the concentration of gelatin in the nucleated cell aggregates in the blood is 5-15 mg / ml when the temperature in step (2) is 5 ° C. or more and less than 15 ° C.
  • Gelatin concentration in the nucleated cell coagulant in the blood is 15 ⁇ 25 mg / ml when the (2) step temperature is more than 15 °C less than 25 °C
  • the concentration of gelatin in the nucleated cell coagulant in the blood is 25-35 mg / ml when the performing temperature of step (2) is greater than 25 ° C but less than 35 ° C
  • gelatin is included as a blood cell aggregate, if the above conditions (a) to (c) are not satisfied, the gelatin melts in the blood cell aggregate prepared in step (2) and the blood cell aggregate cannot be separated from the container. It is difficult to produce the desired blood cell aggregates, and the following (3) step may be difficult to proceed the following process.
  • the concentration at which coagulated gelatin begins to melt differs depending on the temperature at which step (2) is performed, it is preferable to change the concentration of the solution in consideration of the temperature at which step (2) is performed.
  • the step (2) may further include a coloring agent for coloring the blood cell aggregates, and may further include a colorant in 5 to 10 parts by volume with respect to 100 parts by volume of the blood cell aggregates contained in the blood cell aggregates.
  • a coloring agent for coloring the blood cell aggregates may further include a colorant in 5 to 10 parts by volume with respect to 100 parts by volume of the blood cell aggregates contained in the blood cell aggregates.
  • red blood cells are separated from the blood of the subject, and blood cells are aggregated by administering the blood cell coagulant for preparing paraffin block such as gelatin (gelatin) in step (2).
  • the blood cell coagulant for preparing paraffin block such as gelatin (gelatin)
  • the process of manufacturing the blood is preferably finished within 2 hours after collecting the blood of the subject. This is because the blood cancer cells contained in the collected peripheral blood are more likely to undergo cell death than white blood cells.
  • the results of the cell spiking test sprinkled on normal blood using breast cancer cell lines were measured for blood which was left in time-dependent manner immediately after blood collection, 1 hour after blood collection, and 2 hours after blood collection.
  • the results of the cell spiking test sprinkled on normal blood using breast cancer cell lines were measured for blood which was left in time-dependent manner immediately after blood collection, 1 hour after blood collection, and 2 hours after blood collection.
  • step (3) the step of curing the cell aggregates prepared in step (2) is carried out.
  • the blood cell aggregate prepared in step (2) undergoes a curing step, whereby the cells can be separated from the blood cell aggregates in the container of, and the processing time of the general biological tissue that is the target of the paraffin block before the conventional paraffin block preparation ( Typically 12 hours or more) can be shortened to within 6 hours, it becomes easier to process the paraffin block forming material through step (4) described later, it may be more advantageous to implement as a paraffin block expressing the desired physical properties have.
  • the curing of step (3) may leave the blood cell aggregate prepared in step (2) at 2 ° C. to 5 ° C. for 15 to 30 minutes to cure the blood cell aggregate into a jelly form. Cured blood cell aggregates may be immobilized in formalin, methanol and the like for preservation of cells.
  • step (4) the step of treating the paraffin block forming material to the cured product prepared in step (3).
  • the paraffin block forming material may include alcohol, xylene, and paraffin, and the paraffin block forming material is treated in the order of alcohol, xylene and paraffin in the order of the cured product prepared in step (3). Blocks can be produced.
  • the automatic osmosis process may be performed by wrapping a cured product in paper and then treating alcohol, xylene and paraffin in this order.
  • the reason for the automatic infiltration process is as follows. First, in order to thinly thin a sample sample to be observed under the microscope, water may be removed from the sample sample fixed through the fixing solution to enable paraffin penetration (alcohol treatment step). Next, even though the water in the sample is removed by the treated alcohol, alcohol and paraffin are not mixed with each other, so that the alcohol may be removed using xylene (the xylene treatment step). Finally, in order to perform microscopic observation of the specimen, a thin specimen is required to be cut, and thus, a paraffin penetration process may be performed on the specimen in order to perform the cutting procedure (paraffin treatment step).
  • the alcohol treatment step may be performed by dividing into several steps, for example, by first treating a low concentration of alcohol (multiple times) and then processing a high concentration of alcohol (multiple times). May be performed. Such multiple treatments may be equally applied to the xylene treatment step and the paraffin treatment step.
  • the present invention provides a method for observing a specimen sample through a microscope using a paraffin block produced by the above-described method, which is cut into pieces of the paraffin block produced by the above-described method to a suitable size, Staining and observing the chopped paraffin block.
  • the cutting step may be carried out using a conventional cutting device such as a microtome (microtome), and the dyeing process may be performed using a dyeing reagent commonly used in the art.
  • a conventional cutting device such as a microtome (microtome)
  • the dyeing process may be performed using a dyeing reagent commonly used in the art.
  • Paraffin blocks according to the present invention prepared by performing the auto-infiltration process can be sliced by cutting to 3 to 10 ⁇ m thickness, and can be observed through the immunohistochemical staining of the cut block.
  • the paraffin block is thinned to a thickness of 4 to 5 ⁇ m, but the paraffin block of the present invention has a thickness of 3 to 10 ⁇ m, preferably 5 to 8 ⁇ m, more preferably in consideration of the size of blood cancer cells. Can be cut to a thickness of 7 to 8 ⁇ m to count the number of cancer cells in the blood.
  • the paraffin block according to the present invention When the paraffin block according to the present invention is cut to a thickness of 7 to 8 ⁇ m, 30 tests are possible at the time of blood collection of 6 ml blood, and when the thickness is cut to a thickness of 3 to 4 ⁇ m, 60 tests are possible. Dozens of tests are possible with just one small sample of blood, allowing different markers to be applied each time, and multiple tests at different time intervals. This can be
  • the immunohistochemical staining may be performed using staining reagents commonly used in the art, but the dyeing process is not limited thereto, but according to a preferred embodiment of the present invention, H & E staining or cytokeratin (CK) immunity Histochemical staining.
  • staining reagents commonly used in the art, but the dyeing process is not limited thereto, but according to a preferred embodiment of the present invention, H & E staining or cytokeratin (CK) immunity Histochemical staining.
  • Table 1 ⁇ Tissue specific diagnostic marker by primary site currently used in pathology diagnosis> Primary part Diagnostic marker Breast GCDFP-15, ER Colon CK 20, CDX2 Lung TTF1 Ovary ER, mesothelin Prostate PSA Stomach or pancreas CK7, CK20, MUC5AC
  • FISH fluorescence in situ hybridization
  • EDTA tube Three 15 ml tubes were prepared for each 6 ml of collected peripheral blood, and 5 ml of Ficoll was dispensed into each 15 ml container. Next, all the blood collected in the EDTA tube (tube) was put in a 50 ml container, and then phosphate buffer (PBS) was added to a volume of 2 times the blood and mixed.
  • PBS phosphate buffer
  • the cells were centrifuged at 2000 rpm for 20 minutes, and the serum, picol and three layers separated in the centrifuged container. The white buffy coat layer between serum and picol was identified, and only the white cell layer concentrated in the buffy coat layer was carefully collected. Thereafter, the collected cells were transferred to a new 50 ml container, phosphate buffer solution was added to adjust the final volume to 50 ml, and centrifuged at 1500 rpm for 10 minutes.
  • the gelatin solution was dissolved in a concentration of 10 mg / ml in hot water at 90 to 100 ° C. to form a gelatin solution, and then cooled to room temperature (22 ° C.).
  • the cured product was separated from the container. The cured product was put in a ml container, and 10 ml of neutral formalin was administered, and then stored at room temperature for 18 hours.
  • a paraffin block was prepared using a cured product of blood cell aggregates prepared in Examples 1-3.
  • the manufacturing method of the paraffin block of the present invention can reduce the manufacturing time by shortening the tissue processing process to 6 hours, it can be simplified.
  • alcohol was used as ethanol (Ethanol, Fisher Scientic), xylene as Xylene (DUKSAN), and paraffin as Histosec Pastillen (MERCK).
  • Example 2 Prepared in the same manner as in Example 1, in order to separate red blood cells in 1-2 of Example 1 to prepare a paraffin block using a cell lysate instead of picol.
  • Example 2 Prepared in the same manner as in Example 1, except that the concentration of gelatin solution in Examples 1 to 3 was 3 mg / ml, 20 mg / ml, 30 mg / ml, and 40 mg / ml instead of 10 mg / ml. Paraffin block was prepared at 50 mg / ml.
  • Example 1 Prepared in the same manner as in Example 1, except that the cancer cells spiked in 1-1 of Example 1 was used instead of colon cancer cell line SW620 breast cancer cell line SKBR3, after performing Example 1-1 Example 1- Instead of immediately performing 2, blood was left for 1 hour after performing Example 1-1, followed by Example 1-2 to prepare a paraffin block.
  • Example 1 Prepared in the same manner as in Example 10, but instead of leaving blood for 1 hour after performing Example 1-1, the paraffin block was prepared in the same manner as in Example 1 after 2 hours.
  • Paraffin block was prepared by changing blood aggregates in 1-3 as shown in Table 7 below.
  • the paraffin block prepared in Example 1 was subjected to cytokeratin (CK) staining for sections sliced to a thickness of 8 ⁇ m, and the photographs observed through the microscope are shown in FIG. 2.
  • CK cytokeratin
  • the CK immunohistochemical staining of the paraffin block thin sections shows that the brown cells are blood cancer cells.
  • paraffin blocks were made into continuous sections of 8 ⁇ m thick blocks, and cytokeratin staining was then performed on each section to count the number of blood cancer cells contained in the entire block.
  • Example 1 and Comparative Example The average value of the results for the paraffin block prepared by performing three times each 1 is shown in Table 3 below.
  • Example 1 TABLE 3 Number of sections Number of cancer cells per section Total cancer cell harvest rate Comparative Example 1 56 22 ⁇ 1.3 1017 (25%) Example 1 30 122 ⁇ 4.8 3732 (93%)
  • the collection rate of blood cancer cells through paraffin blocks prepared by Example 1 was 93% on average 3.7 than the 25% collection rate of blood cancer cells through paraffin blocks prepared by Comparative Example 1 It can be seen that the ship is high.
  • the number of sections cut from the paraffin block prepared according to Example 1 was 30, which is less than that of Comparative Example 1, but the number of blood cancer cells contained in each section was significantly higher than that of Comparative Example. have.
  • the paraffin block prepared in Example 1 can be inspected 30 times when cut to 8 ⁇ m thickness, 60 times can be carried out when cut to 4 ⁇ m thickness, it is possible to apply a different marker every time. can confirm.
  • Cancer cell collection rate (calculated after cytokeratin immunocytochemical staining the total number of cancer cells contained in the paraffin block in the paraffin block prepared in Example 1 and Example 2) Indicated.
  • Ficol treatment can confirm that the cancer cell collection rate is 1.7 times higher than that of the cell lysis method, thereby removing red blood cells from the blood in order to prepare paraffin blocks of better physical properties. It can be seen that processing picol is better.
  • Cancer cell harvesting rate (the total number of cancer cells included in the paraffin block was counted after cytokeratin immunocytochemical staining at room temperature 20 ° C. for paraffin blocks prepared in Examples 1 and 3 to 7) The calculation is shown in Table 5 below.
  • Example 5 As a result of comparing the cancer cell collection rate according to the gelatin solution concentration change, as shown in Table 5, in the case of Examples 1, 4, and 5 at room temperature, the cancer cell collection rate is high, whereas Examples 3, 6, and 7 are the cancer cell collection rates It can be seen that the remarkably decreased. In particular, in Example 5, when the concentration of gelatin solution was 30 mg / ml, the blood cancer cell collection rate was 80%, whereas in Example 6, when the concentration of gelatin solution was 40 mg / ml, the blood cancer cell collection rate was 23%. It can be seen that the decrease is 57% (reduction rate is 71.2%).
  • the concentration of the gelatin solution should be different according to the room temperature or the season, and in the winter season when the room temperature is 15 ° C. or less, even if the concentration of the gelatin is 10 mg / ml, it is possible to prepare a gel-type blood cell aggregate. If the gelatin concentration is 10mg / ml in the summer when the temperature is 25 °C or more, it can be confirmed that the gelatin concentration should be changed to a concentration exceeding 20mg / ml in order to prepare a gel-type blood cell aggregate according to the gelatin recording. have.
  • the cancer cell collection rate of the paraffin block is significantly changed according to the concentration of gelatin, and the cancer cell collection rate is better when the concentration of gelatin solution is 10 mg / ml than when the concentration of gelatin solution is 30 mg / ml
  • the room temperature is 5 ⁇ 15 °C to prepare the best blood cell aggregates in the concentration of gelatin solution to prepare a paraffin block 5 ⁇ 15mg / ml and at the same time to prepare a paraffin block having significantly superior physical properties It can be seen that.
  • the concentration of gelatin solution to 20 ⁇ 30mg / ml can be prepared the best blood cell aggregates and at the same time can be produced a paraffin block having significantly superior physical properties Able to know.
  • the concentration of gelatin solution was found to be the best conditions for making the aggregate when the concentration of 30mg / ml, the coagulated gelatin solution is As time goes by, the problem easily melts, and as it can be seen in Table 5 to solve this problem, when using too high concentration of gelatin solution, another problem occurs that the collection rate of blood cancer cells decreases.
  • the results of Table 6 show that at room temperature 25-35 ° C., paraffin blocks with remarkably excellent physical properties can be produced at the same time when the concentration of gelatin solution is 25-35 mg / ml. It can be seen that it can be produced.
  • Examples 12 to calculate the cancer cell collection rate (calculated after the cytokeratin (cytokeratin) immunohistochemical staining at 20 °C the total number of cancer cells contained in the paraffin block for the paraffin block prepared in Table 7) Shown in
  • Example 12 and Example 17 containing the gelatine solution alone as a blood coagulant can be confirmed that the blood cancer cell collection rate is generally excellent compared to the case of further containing other types of blood aggregates. .
  • the cancer cell collection rate may be further improved, which can be confirmed through Examples 17, 19 and 21.
  • Cancer cells and normal cells can be distinguished cytopathologically regardless of staining.
  • contaminated normal epithelial cells could be read as false-positive because the presence or absence of blood cancer cells is determined according to the surface markers positive or negative.
  • CK-positive squamous epithelial cells were removed from the skin during treatment of skin keratinocytes or erythrocytes and paraffin block during blood collection. It was confirmed that there could be contamination.
  • FIG. 6 the result of performing normal H & E staining, c-erbB2 immunohistochemistry and fluorescence in situ hybridization reaction test (FISH) on the breast cancer cell line (SKBR3) -derived blood cancer cells, Expression of the gene was observed.
  • EpCAM hepatic cancer cell lines
  • the naked eye and tissue findings of the primary colorectal cancer of the primary colorectal cancer and liver cancer recurrence of the patient can be confirmed, and the paraffin according to the manufacturing process of the present invention with blood collected in July 2012. Blood cancer cells could be identified from the section of the block.
  • tissue-specific markers already used for pathological diagnosis of metastatic carcinoma whose primary site is unclear can detect blood cancer cells more sensitively.
  • the method for preparing paraffin block according to the present invention is a non-invasive and simple method of detecting blood cancer cells present in blood through the collection of blood samples, which is very useful for the diagnosis and prognosis of cancer recurrence and metastatic cancer. Analysis of cancer onset, metastasis, and relapse mechanisms can provide a breakthrough in cancer treatment.

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Abstract

La présente invention concerne un matériau pour l'agrégation de cellules sanguines, utilisé dans la préparation d'un bloc de paraffine pour le diagnostic de cellules tumorales circulantes (CTC), et un procédé de préparation d'un bloc de paraffine à l'aide de ce dernier, et plus particulièrement, un procédé de détection des CTC présentes dans le sang par le recueil d'un échantillon de sang dans la préparation d'un bloc de paraffine à partir des CTC, qui sont présentes en très petite quantité dans le sang. La présente invention est non invasive et simple, et est donc très utile pour le diagnostic de récurrence cancéreuse et de cancer métastatique et pour la prédiction du pronostic, et peut constituer un progrès remarquable dans le traitement du cancer par l'analyse des mécanismes d'apparition, de métastase et de récurrence du cancer.
PCT/KR2014/006952 2013-09-09 2014-07-29 Agent d'agrégation de cellules sanguines pour la préparation de bloc de paraffine et procédé de préparation de bloc de paraffine à l'aide de ce dernier WO2015034178A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105699142A (zh) * 2016-02-18 2016-06-22 石河子大学 用于质地紧密坚硬植物材料的石蜡切片制作方法
CN112082836A (zh) * 2020-09-15 2020-12-15 海南医学院第一附属医院 一种临床血性胸水细胞蜡块及其制备方法
CN113074996A (zh) * 2021-03-15 2021-07-06 陈建华 一种血浆凝固细胞块法及其临床应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3970427A (en) * 1973-03-22 1976-07-20 American Hospital Supply Corporation Process for reducing sticking of blood cells to glass in blood testing procedures
US6913921B2 (en) * 2002-10-31 2005-07-05 University Of Massachusetts Rapid cell block embedding method and apparatus
JP2005192521A (ja) * 2004-01-09 2005-07-21 Hiroko Sato 部分分解重合性タンパク質からなる造血幹細胞を含む未熟な血液細胞（ｈｓｃ）の増殖促進用支持体およびその支持体を使用したｈｓｃの製造方法。

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3970427A (en) * 1973-03-22 1976-07-20 American Hospital Supply Corporation Process for reducing sticking of blood cells to glass in blood testing procedures
US6913921B2 (en) * 2002-10-31 2005-07-05 University Of Massachusetts Rapid cell block embedding method and apparatus
JP2005192521A (ja) * 2004-01-09 2005-07-21 Hiroko Sato 部分分解重合性タンパク質からなる造血幹細胞を含む未熟な血液細胞（ｈｓｃ）の増殖促進用支持体およびその支持体を使用したｈｓｃの製造方法。

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BROWN DA ET AL.: "Gelatin-embedded cell -polymer constructs for histological cryosectioning", J BIOMED MATER RES PART B: APPL BIOMATER, vol. 72B, 2005, pages 79 - 85 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105699142A (zh) * 2016-02-18 2016-06-22 石河子大学 用于质地紧密坚硬植物材料的石蜡切片制作方法
CN105699142B (zh) * 2016-02-18 2018-04-03 石河子大学 用于质地紧密坚硬植物材料的石蜡切片制作方法
CN112082836A (zh) * 2020-09-15 2020-12-15 海南医学院第一附属医院 一种临床血性胸水细胞蜡块及其制备方法
CN113074996A (zh) * 2021-03-15 2021-07-06 陈建华 一种血浆凝固细胞块法及其临床应用

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