JP2008045955A - Technique for diagnosing peritoneal dialysis compatibility due to peritoneal cell analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To more accurately determine the condition of an examinee subjected to peritoneal dialysis, and to provide data for performing treatment accurately corresponding to the state of the examinee. <P>SOLUTION: A target nuclear cell specifying method includes a step of detecting the nucleus part and non-nucleus part of the nuclear cell and a step of determining the presence of the nuclear cell when both of the nucleus part and the non-nucleus part are detected. For example, the ageing of a mesothelial cell can be accurately determined by this method. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to peritoneal dialysis. More specifically, the present invention relates to a technique for accurately determining the condition of a patient undergoing peritoneal dialysis, diagnosing peritoneal dialysis, and providing useful information about subsequent treatment.

  Peritoneal dialysis involves injecting peritoneal dialysis fluid into the abdominal cavity and allowing it to stay in the abdominal cavity for 4 to 8 hours, dialyzing and draining blood and lymph circulating in the peritoneum using the properties of the peritoneum as a semipermeable membrane, It is a method of injecting a new liquid and repeating this. This method is preferable from the standpoint of maintaining the residual kidney function and pediatric care, as well as being able to return to society without any restrictions on the activities of the patient. It is necessary to constantly monitor whether it can continue. Of particular concern is encapsulating peritoneal sclerosis (EPS) that occurs after prolonged peritoneal dialysis therapy. EPS mortality is extremely high, and the establishment of preventive measures and treatments is urgent. As its etiology, mesothelial cells reach the division limit (aging) due to repeated loss and regeneration of mesothelial cells, and are depleted. In addition to the measurement of dialysate / serum creatinine ratio using a peritoneal equilibrium test (PET) as a test method, analysis of mesothelial cells in peritoneal dialysis effluent is performed by chemical staining and observation with the naked eye. The present invention provides a mesothelial cell aging monitoring technology that is highly objective and quantitative and has advanced automation, and a technology that contributes to peritoneal dialysis therapy and related businesses.

To date, literatures such as Non-Patent Documents 1 to 3 have been reported on morphological measurement of mesothelial cells in peritoneal dialysis drainage and its clinical significance. In these documents, useful knowledge about analyzing the relationship between the development of encapsulating peritoneal sclerosis and the morphology of mesothelial cells has been obtained. Reporting findings. In these documents, the shape of mesothelial cells is measured by centrifugal smear staining, followed by observation with a microscope, computer image processing and area calculation of images, and obtaining measurement average values and standard errors as data. However, it is clear that these operations require highly trained technicians and are labor intensive.
Dialysis Society Vol.30, No.10, P.1225-1231, Tadaji Yamamoto, etc. Renal and dialysis Vol.53 separate volume peritoneal dialysis 2002: 334-337, 2002, Tadaji Yamamoto and others Kidney and dialysis Vol.53 separate volume peritoneal dialysis 2002: 338-341, Tadaji Yamamoto et al.

  An object of the present invention is to contribute to safe peritoneal dialysis by providing a method with a large amount of information that uses a staining method having high specificity for mesothelial cells and is more automated and has increased objectivity.

  Another object of the present invention is to more accurately determine the status of a subject undergoing peritoneal dialysis and provide information for accurately performing treatment according to the status.

  The above object is a method for identifying a target nucleated cell in the present invention, the step of detecting the nucleus and the non-nucleated part of the nucleated cell; and when both the nucleus and the non-nucleated part are detected In addition, the present invention has been solved by a method including the step of determining that the nucleated cell is present, for example, by accurately determining the state of senescence of mesothelial cells.

  For example, by observing the situation of cells that are positively stained with an antibody called HBME-1 that identifies a mesothelial cell marker, the situation becomes an index of suitability in a subject of peritoneal dialysis. The above problem was solved.

Accordingly, the present invention provides the following.
(1) A method for identifying a target nucleated cell,
A) detecting the nucleus and non-nucleus part of the nucleated cell; and B) determining that the nucleated cell is present when both the nucleus and the non-nucleus part are detected;
Including the method.
(2) The method according to item 1, further comprising a step of obtaining a shape parameter of the detected nucleated cell and thereby determining a state of the nucleated cell.
(3) The method according to item 2, wherein the shape parameter is used to determine whether the area of the nucleated cell is measurable or whether the area measurement is significant.
(4) The method according to Item 2, wherein the shape parameter is used to determine a cell in a state inappropriate for measuring the area of the nucleated cell.
(5) The method according to item 4, wherein the state unsuitable for the area measurement is selected from the group consisting of aggregation, adhesion and collapse.
(6) The method according to item 2, wherein the shape parameter is selected from the group consisting of a projected area, a cell surface irregularity, and a ratio of a major axis to a minor axis of a cell.
(7) The method according to item 2, wherein the cell surface irregularities are calculated by a calculation procedure of square of cell circumference of the nucleated cell ÷ cell area of the nucleated cell × 4π.
(8) The method according to item 2, wherein at least one of the cell surface irregularities and the ratio of the major axis to the minor axis of the cell is set to less than 1.5.
(9) The method according to item 2, wherein the cell surface irregularities and the ratio of the major axis to the minor axis of the cell are set to less than 1.5.
(10) The method according to item 1, wherein the nucleated cell is a mesothelial cell.
(11) The method according to item 1, wherein the nucleated cell is a mesothelial cell, and the mesothelial cell is determined by the presence of the nucleus and expression of a mesothelial marker.
(12) An antibody selected from the group consisting of HBME-1, anti-CA-125 antibody, anti-mesothelin antibody and anti-calretinin antibody is used for the detection of the non-nuclear part, and HOECHST 33342 is used for the detection of the nucleus. The method according to item 1, wherein the method is used.
(13) The method according to item 1, wherein the non-nuclear part includes a cell surface.
(14) The method according to item 2, wherein the step of obtaining the shape parameter includes selection of a substantially circular shape.
(15) The method according to item 1, further comprising selecting, as an index, that the negative marker of the nucleated cell is not detected.
(16) The method according to item 15, wherein the negative marker is selected from the group consisting of CD14, CD33, CD45 and CD3.
(17) The method according to item 1, further comprising the step of obtaining the projected area of the nucleated cell.
(18) The method according to item 17, wherein the projected area is used as an index of aging of the nucleated cells.
(19) A method for diagnosing the suitability of peritoneal dialysis in a subject,
A) a step of detecting mesothelial cells in a sample derived from a subject,
A-1) Detecting nuclei and non-nuclear parts of mesothelial cells in the sample, and A-2) detecting the mesothelial cells by determining the cells in which both the nuclei and non-nuclear parts are detected. And B) calculating the projected area of the mesothelial cell, wherein the suitability for peritoneal dialysis is determined by the variation of the mesothelial cell.
(20) The step A) further includes A-3) obtaining a shape parameter of a cell in the sample, and the detection is determined by determining whether the shape parameter is normal. The method according to item 19.
(21) The method according to item 19, wherein an increase in the projected area where the average projected area of the cells reaches 250 square micrometers is an indicator of a decrease in the suitability of peritoneal dialysis.
(22) The method according to item 19, wherein the sample is a peritoneal dialysis drainage sample.
(23) The method according to item 19, further comprising the step of measuring the cell concentration of the mesothelial cells and presenting a sign to the patient to stop peritoneal dialysis when the concentration falls below a certain level.
(24) The method according to item 19, wherein the decrease in the cell concentration of mesothelial cells is an indicator of a decrease in the suitability of peritoneal dialysis.
(25) The method according to item 19, wherein the increase in the average projected area of the mesothelial cells is an indicator of a decrease in the suitability of peritoneal dialysis.
(26) The method according to item 25, wherein the increase in the projected area of the mesothelial cells is determined by an average projected area.
(27) The method according to item 19, wherein in the mesothelial cells, an increase in cells with an increased projected area is an indicator of a decrease in suitability for peritoneal dialysis.
(28) The method according to item 19, wherein the peritoneal dialysis is judged to be inappropriate when the mesothelial cells are less than about 5% of the total number of total cells.
(29) A system for pre-diagnosing or diagnosing whether a subject is compatible with peritoneal dialysis continuation,
A) Nuclear detection means for detecting the nuclei of nucleated cells in a sample from the subject;
B) non-nucleus detection means for detecting a non-nucleated portion of the nucleated cell in the sample from the subject; and C) shape parameter acquisition means for obtaining a shape parameter of the nucleated cell in the sample from the subject. ,system.
(30) The system according to item 29, wherein the nucleus detection means and the non-nucleus detection means are selected from the group consisting of a protein, a nucleic acid molecule, a polypeptide, a lipid, a sugar chain, a small organic molecule, and a complex molecule thereof.
(31) A system according to item 29, wherein the nuclear detection means is a fluorescent dye, and the non-nuclear detection means is an antibody that is labeled or not.
(32) A system according to item 29, wherein the non-nucleus detection means is a cell surface detection means.
(33) A system according to item 29, wherein the nuclear detection unit and the non-nuclear detection unit are labeled or labelable.
(34) The system according to item 29, wherein the shape parameter acquisition means is a photomicrograph apparatus.
(35) The system according to item 29, further comprising means for purifying the sample.
(36) The system according to item 35, wherein the means for purifying is selected from the group consisting of a centrifugal separator, a filtration means, an adhesive means, and an electrical means.
(37) A system according to item 29, wherein the subject includes a mammal.
(38) The system according to item 29, wherein the subject includes a human.
(39) The system according to item 29, further comprising means for measuring the area distribution coefficient of the nucleated cell.
(40) The system according to item 39, wherein the area distribution coefficient is obtained by accumulating the number of cells for each projected area and performing natural logarithmic regression calculation.
(41) The system according to item 40, wherein the area distribution coefficient is 30 as a lower limit in natural logarithmic regression calculation.
(42) A system according to item 40, wherein the calculation means is a computer.
(43) The system according to item 29, wherein the system is a peritoneal dialysis compatibility diagnostic apparatus.
(44) A system according to item 29, wherein the system includes an array scan.
(45) The system according to item 29, wherein the system includes an array scan and a hemocytometer, and the hemocytometer is used for measuring the number of cells during sample preparation.
(46) The system according to item 29, wherein the shape parameter is indicated by a projected area, a cell surface unevenness of the nucleated cell, and a ratio of a major axis to a minor axis of the nucleated cell.
(47) A system according to item 29, wherein the nucleated cell is a mesothelial cell.
(48) In the item 29, the non-nuclear part detection means is an antibody selected from the group consisting of HBME-1, anti-CA-125 antibody, anti-mesothelin and anti-calretinin, and the nuclear detection means is HOECHST 33342 The system described.
(49) The system according to item 29, wherein a substantially circular shape is selected based on the shape parameter.
(50) The system according to item 29, further comprising means for detecting a negative marker for the nucleated cell.
(51) The system according to item 50, wherein the negative marker is selected from the group consisting of CD14, CD33, CD45 and CD3.
(52) The system according to item 46, wherein the projected area is used as an index of aging of the nucleated cells.
(53) A system according to item 29, wherein the sample is a peritoneal dialysis drainage sample.
(54) The system according to item 47, wherein peritoneal dialysis is stopped when the cell concentration of the mesothelial cells reaches a certain level.
(55) The system according to item 47, wherein the decrease in the cell concentration of the mesothelial cell is an indicator of a decrease in the suitability of peritoneal dialysis.
(56) The system according to item 47, wherein the increase in the projected area of the mesothelial cells is an indicator of a decrease in suitability for peritoneal dialysis.
(57) The system according to item 56, wherein the increase in the projected area is determined by an average projected area.
(58) The system according to item 47, wherein an increase in mesothelial cells with an increased projected area is an indicator of a decrease in suitability for peritoneal dialysis.
(59) A system according to item 47, wherein peritoneal dialysis is judged to be inappropriate when the mesothelial cells become less than about 5% of the total number of total cells.
(60) Measure the total cell count with trypan blue stain and total nucleated cells with Hinkelmann's solution using a hemocytometer, determine the preparation conditions of the sample to be measured by array scan, and improve work efficiency 30. The system according to item 29, characterized by:
(61) A) Nuclear detection means for diagnosing whether a subject can continue peritoneal dialysis, a nuclear detection means for detecting the nucleus of nucleated cells in a sample from the subject; B) in a sample from the subject A non-nucleus detection means for detecting a non-nucleated portion of the nucleated cell; and C) use of a shape parameter acquisition means for obtaining a shape parameter of the nucleated cell in a sample from the subject.

  Accordingly, it is understood that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description with reference to the accompanying drawings.

  The present invention provides a method for easily determining nucleated cells such as mesothelial cells. Thereby, for example, suitability in peritoneal dialysis can be easily determined.

  The present invention will be described below. Throughout this specification, singular articles (eg, “a”, “an”, “the”, etc. in English, corresponding articles in other languages, adjectives, etc.) It should be understood that it also includes the plural concept. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

(Explanation of terms)
The terms and general techniques used in this specification are explained below.

(General technology)
Techniques relating to peritoneal dialysis used herein are well known and commonly used in the art.

Dialysis is performed by sieving with a molecular weight size through a membrane and removing a predetermined molecule by a concentration gradient. Various solutes accumulated in the body by metabolic activity (urea (U) as a uremic toxin, creatinine ( Cr) etc.), and electrolyte (Ca ²⁺ , Cl ⁻ , Na ⁺ , K ⁺ ), excess water, etc. are eluted from the body fluid into the dialysate, and then discarded as dialysate, It helps the patient's reduced renal function. Blood dialysis (HD; Hemo Dialysis), peritoneal dialysis (PD) depending on the method of purifying blood mechanically by extracorporeal circulation or the method of putting dialysate into the abdominal cavity and purifying blood through the peritoneum There is a distinction;

  Peritoneal dialysis utilizes the patient's own peritoneum as a semipermeable membrane. This peritoneum is the membranous layer of the abdominal cavity of the body. This peritoneum can function as a natural semipermeable membrane.

  In peritoneal dialysis, a sterile aqueous solution is periodically injected into the abdominal cavity. This solution is called peritoneal dialysis solution or dialysate. Diffusion and osmotic exchange occur between the solution and the blood stream across the natural body membrane. These exchanges remove the waste products normally excreted by the kidneys. This waste product typically consists of solutes such as urea and creatinine. The kidneys also maintain levels of other substances such as sodium and water that need to be regulated by dialysis. The diffusion of water and solutes across the peritoneum during dialysis is called ultrafiltration.

  In mobile continuous peritoneal dialysis, the dialysis solution is introduced into the peritoneal cavity using a catheter. The exchange of solutes between dialysate and blood is accomplished by diffusion. Further removal is achieved by providing an appropriate osmotic pressure gradient from the blood to the dialysate to allow water to flow out of the blood. This allows proper acid-base balance, electrolyte balance and fluid balance to be achieved in the body. This dialysis solution is simply drained from the body cavity through the catheter.

  There are various improvements in peritoneal dialysis. One such modification is automatic peritoneal dialysis (“APD”). APD uses an instrument (called a cycler) to automatically infuse, pause, and drain peritoneal dialysis solution into and out of the patient's peritoneal cavity. APD is particularly attractive for peritoneal dialysis patients. This is because it can be performed at night while the patient is sleeping. This frees the patient from the day-to-day requirements of mobile continuous peritoneal dialysis during waking and work hours. The APD procedure typically takes several hours. This often begins with an initial drainage cycle to empty the spent dialysate from the abdominal cavity. The APD procedure then proceeds through a series of sequential filling, resting and draining steps. Each fill / pause / drain procedure is called a cycle. APD can and can be implemented in many different ways.

  In peritoneal dialysis, the patient mainly performs dialysis at home. The patient himself or herself puts the dialysate into the abdominal cavity using a catheter, stores it for several hours, and then repeats the draining step several times a day. The patient records the amount of excess water excreted from the body at each drainage (referred to as the amount of water removed) and presents it to the doctor at the time of a subsequent examination for a prescription. Such a method of peritoneal dialysis is called CAPD (Continuous Ambientatory Peritonal Dialysis). The doctor pays particular attention to the amount of water removed from the patient, and selects an appropriate prescription for the patient depending on the amount of the water.

  As used herein, “shape parameter” refers to an arbitrary parameter indicating the shape of a cell. Examples of the shape parameter include surface area, volume, projected area, cell surface unevenness and ratio of long axis to short axis of cells, sphericity, roundness, cross-sectional area, cross-sectional shape, etc. It is not limited. By using shape parameters, the state of cells such as nucleated cells can be investigated. The shape parameter is also used to determine whether the area of the nucleated cell is measurable (ie, suitable for area measurement) or whether the area measurement is significant. The specific procedure takes into account, for example, the square of the circumference of the projected shape ÷ area × 4π and the ratio of the major axis to the minor axis by utilizing the fact that mesothelial cells that are floating alone are almost spherical. Can be implemented.

  Conditions that are unsuitable for area measurement may be aggregation, adhesion and disintegration.

  The cell surface unevenness is calculated by a calculation procedure of square of cell circumference of the nucleated cell ÷ cell area of the nucleated cell × 4π. This number is 1 for a circle, and 1 or more when the shape is distorted. At least one or both of the cell surface irregularities and the ratio of the major axis to the minor axis of the cell can be set to less than 1.5, for example, less than 1.2. These values are preferably close to the minimum value of 1 which can be present, indicating that the projected shape is a circle, ie, the cell shape is close to a sphere. Therefore, at least one or both of the cell surface irregularities and the ratio of the major axis to the minor axis of the cell can be described in the range of less than 1 to 1.5, preferably less than 1 to 1.2.

  In the step of obtaining the shape parameter, a substantially circular shape may be selected, but is not limited thereto.

  In the present specification, the “state” of a cell refers to whether or not the cell has a “state suitable for area measurement”. This determines the state of the cell based on the shape parameter, and is applied to the measurement of the present invention.

The shape parameter can be calculated by the following calculation procedure.
An exemplary calculation procedure is described below.
1. The major axis, minor axis, area (projection area), and circumference (periphery of the projection shape) of a cell or a projected shape of a cell are obtained by any method commonly known in the art.
2. Calculate the ratio of major axis to minor axis, that is, the ratio of major axis to minor axis.
3. Calculate the square of the circumference / area × 4π as an index of the unevenness of the cells.

  As used herein, as an example, when using Target activation software with an array scan as an example, the shape parameter is by way of example and the ratio of the major axis to the minor axis is less than 1.5, for example, 1.0-1 .2 is set. Similarly, the square of the circumference / area × 4π is also set to 1.0 to 1.2. This provides information useful for semi-automatically selecting mesothelial cells suitable for area measurement, but is not limited thereto.

  The “state inappropriate for area measurement” of a cell refers to a state suspected of having an obstacle in measuring the projected area of the cell, and includes, for example, aggregation, adhesion, and collapse.

  Specifically, in the above software, the determination of “inappropriate state for area measurement” is made when, for example, the ratio of the major axis to the minor axis and the square of the circumference / area × 4π is 1.2 or more, the area measurement Achieved by excluding from the subject.

  A “cell” as used herein is defined in the same way as the broadest meaning used in the art, and is typically a unicellular organism itself or a constituent unit of a tissue of a multicellular organism, An organism that is wrapped in a sequestering membrane structure, has self-renewability inside, and has genetic information and its expression mechanism. The cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells). As used herein, “cell” is a generic term for nucleated cells and non-nucleated cells.

  In the present specification, the “nucleated cell” refers to any cell having a nucleus, and examples thereof include stem cells, differentiated cells, mesothelial cells, and the like. Normal cells have a nucleus.

  In the present specification, the “non-nucleated cell” refers to any cell having no nucleus, and examples thereof include erythrocytes. Such non-nucleated cells can be selected, for example, as an indicator that they are not detected by nuclear staining.

  In the present specification, examples of the negative marker for nucleated cells include various individual CD markers. For example, mesothelial cells include CD14, CD33, CD45, CD3, CD34, CD4, and the like.

  In the present specification, the “nucleus” refers to a cell nucleus, also referred to as a cell nucleus, and is a spherical or elliptical structure that normally exists in one eukaryotic cell and is a structure that is enveloped by the nuclear membrane. The nucleus normally contains genetic material represented by DNA.

  In the present specification, the “non-nuclear part” means a part other than the nucleus in the cell, and includes, for example, a cell surface, a cell membrane, a cytoplasm, and the like. For detection of the non-nuclear portion, an antibody selected from the group consisting of HBME-1, anti-CA-125 antibody, anti-mesothelin antibody and anti-calretinin antibody can be used, and HOECHST 33342 can be used for detection of the nucleus.

  In the present invention, the “cell surface” refers to the cell surface. Various biomolecules (sugars, complex sugars, receptors, transmembrane proteins, etc.) exist on the cell surface, and many are unique to cells. Therefore, in the present invention, they are particularly preferably used for identifying non-nuclear parts. It is done.

  In the present specification, the “projected area of nucleated cells” refers to an area obtained when projected using ordinary optical means. In the present invention, the projected area can be used as an index of aging of nucleated cells. Acquisition of the nucleated cell projection area can be performed using any method commonly known in the art.

  Examples of such indicators of decreased peritoneal dialysis compatibility due to mesothelial cell aging can include the following.

  For example, an increase in projected area where the average projected area of cells reaches 250 square micrometers may be an indication of a decrease in suitability for peritoneal dialysis. In addition, a decrease in the mesothelial cell ratio (4% or less) in the cells in the peritoneal dialysis drainage can be mentioned. Furthermore, it is also possible to evaluate the distribution by logarithmic regression calculation of the cumulative distribution of the mesothelial cells in the projected area order. By this evaluation, it is possible to represent the distribution of the projected area, and it is possible to grasp the increase of mesothelial cells having a larger projected area and more appropriately evaluate the decrease in the suitability of peritoneal dialysis.

  As used herein, “mesothelial cell” refers to the mesothelial (mesoderm epithelial tissue that forms the inner wall of the true body cavity, and in vertebrates, it usually refers to the abdominal epithelium derived from the lateral plate, the pleural cavity, and the pericardial cavity. , Which covers the surface of various internal organs such as the peritoneal cavity and scrotal cavity, and is often a single-layer squamous epithelium). For example, mesothelial cells can be determined by the presence of the nucleus and the expression of mesothelial markers. It can also be used as an indicator of continuation of peritoneal dialysis by measuring the cell concentration of mesothelial cells and presenting a sign to the patient to stop peritoneal dialysis when below a certain level. This is because it is generally known that a decrease in the cell concentration of mesothelial cells is an indicator of a decrease in the suitability of peritoneal dialysis. Also, an increase in the average projected area of mesothelial cells can be an indicator of a decrease in suitability for peritoneal dialysis. The increase in the projected area of mesothelial cells is determined by the average projected area. In mesothelial cells, an increase in cells with an increased projected area is an indicator of a decrease in suitability for peritoneal dialysis.

  Mesothelial cells can be an indicator of whether peritoneal dialysis has been improved. When the mesothelial cells are less than about 4% of the total number of cells in the peritoneal dialysis drainage, it is judged that peritoneal dialysis is inappropriate.

  As used herein, “isolated” or “separated” refers to a material that is at least reduced, preferably substantially free of naturally associated substances in a normal environment. Thus, an isolated cell refers to a cell that is substantially free of other substances (eg, other cells, proteins, nucleic acids, etc.) that accompany it in its natural environment. In this specification, although the cell to be used can be used even if it is not separated, it is preferable that it is separated to some extent.

  The cells used in the present invention may be cells derived from any organism (for example, any kind of multicellular organisms (eg, animals (eg, vertebrates, invertebrates), plants (eg, monocotyledonous plants, dicotyledonous plants, etc.)). ) Etc.)). Preferably, cells derived from vertebrates (eg, eelfish, lamprey, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.) are used, more preferably mammals (eg, single pores, Marsupial, rodent, winged, winged, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed, rodent, scale, sea cattle, cetacean, primate , Rodents, rabbit eyes, etc.). More preferably, cells derived from primates (eg, chimpanzee, Japanese monkey, human) are used. Most preferably, human-derived cells are used. In the present invention, since the identification of mesothelial cells is used in a preferred embodiment, the cells used in the present invention can be cells derived from any organism having mesothelial cells, and such organisms Is understood to be a normal vertebrate.

  As used herein, “sample” refers to any material containing cells used to practice the present invention. An example is a peritoneal dialysis drainage sample.

  As used herein, “marker” refers to a substance or factor that characterizes a cell. Such markers can include genes, gene products, metabolites, enzymes, and the like. A marker for identifying mesothelial cells may be referred to as a “mesothelial (cell) marker”. For detection of the non-nuclear part of mesothelial cells, an antibody selected from the group consisting of HBME-1, anti-mesothelin antibody and anti-calretinin antibody can be used.

  In the present specification, “HBME-1” is a kind of monoclonal antibody that recognizes mesothelial cell markers, and has a high positive rate in mesothelioma. See Arch Gynecol Obstet. 2001 Aug; 265 (3): 151-4.

  In the present invention, the diagnosis of peritoneal dialysis compatibility can be realized using factors or means specific to the mesothelial marker.

  As used herein, an “agent” can be any substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) that can achieve the intended purpose. Good. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, compounds, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.) And the like, but are not limited to these. As a factor specific for a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.

  Therefore, in the present specification, a “factor that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for the biological agent such as the polynucleotide or the polypeptide, The affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides is typically equivalent or higher, preferably significantly (eg, statistically significant) ) Includes the expensive. Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.

  As used herein, a first substance or factor “specifically interacts” with a second substance or factor means that the first substance or factor has a second Interacting with a higher affinity than a substance or factor other than a substance or factor (especially another substance or factor present in a sample containing a second substance or factor). Specific interactions for a substance or factor include, for example, when both nucleic acid and protein are involved, such as hybridization in nucleic acids, antigen-antibody reactions in proteins, ligand-receptor reactions, enzyme-substrate reactions, etc. Examples include, but are not limited to, protein-lipid interactions, nucleic acid-lipid interactions, and the like, such as reactions with transcription factor binding sites. Thus, when both a substance or factor is a nucleic acid, the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor. Further, for example, when both substances or factors are proteins, the fact that the first substance or factor “specifically interacts” with the second substance or factor includes, for example, interaction by antigen-antibody reaction, receptor- Examples include, but are not limited to, interaction by a ligand reaction and enzyme-substrate interaction. When two substances or factors include proteins and nucleic acids, the first substance or factor “specifically interacts” with the second substance or factor means that the transcription factor and the transcription factor Interaction between the binding region of the nucleic acid molecule to be included. Such specific interactions can be applied to diagnosis.

As used herein, “antibody” refers to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotype antibodies, and fragments thereof such as F (ab ′) ₂ and Fab fragments, and other recombinants. Conjugates produced by In addition, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like.

  In this specification, “means” refers to anything that can be an arbitrary tool that achieves a certain purpose. In particular, in this specification, “means for selectively recognizing” refers to an object as another. Means that can be recognized differently. Such means may be labeled or unlabeled. When not labeled, such means may be treated to allow labeling. In the present specification, the means for detecting a certain target (for example, a cell marker) is also referred to as “detection means”.

  As used herein, “nucleus detection means” refers to any means capable of detecting the nuclei of nucleated cells in a sample from a subject. Examples of such means include systems, devices, and kits, and include proteins, nucleic acid molecules, polypeptides, lipids, sugar chains, compounds, small organic molecules, and complex molecules thereof (for example, dyes, Compositions, antibodies, antigens, etc.) may also be suitable as the means.

  As used herein, “non-nuclear detection means” refers to any means for detecting the non-nuclear portion of the nucleated cells in a sample from a subject. When detecting the cell surface, it is called cell surface detection means. Examples of such means include systems, devices, or kits, and include proteins, nucleic acid molecules, polypeptides, lipids, sugar chains, compounds, small molecules, and complex molecules thereof (for example, dyes, Compositions, antibodies, antigens, etc.) may also be suitable as the means.

  Typically, the nuclear detection means is a fluorescent dye, and the non-nuclear detection means can be, but is not limited to, an antibody that is labeled or not.

  As used herein, “shape parameter acquisition means” refers to any means for obtaining the shape parameters of the nucleated cells in a sample from a subject. As such means, for example, a system, an apparatus, a kit, or the like can be cited, and a configuration that can be calculated by connecting a computer or the like may be appropriate as the means. Such a shape parameter acquisition means may be a photomicrograph apparatus.

  As used herein, “means for purifying a sample” may be any means capable of purifying a sample. Such purification means can be, for example, a centrifugal separation means, a filtration means, an adhesion means, and an electrical means.

  In the present specification, “means for measuring the area distribution coefficient of nucleated cells” refers to any means capable of measuring the area distribution coefficient of nucleated cells.

  In this specification, the “area distribution coefficient” of nucleated cells (for example, mesothelial cells) means the number of cells having a projected area smaller than that in the order of projected areas of nucleated cells (for example, mesothelial cells). This is the coefficient of the regression equation obtained by logarithmic regression. In the case of mesothelial cells, the area distribution coefficient is generally 30 as the lower limit of peritoneal dialysis suitability in the natural logarithmic regression calculation. When using as an index of peritoneal dialysis compatibility, the upper limit is not particularly limited. Since the upper limit is not related to the object of the present invention, there is no significance to define it. Although not wishing to be bound by theory, the upper limit is not defined from the purpose of the present invention, that is, “detecting a situation where the size of mesothelial cells increases due to aging and the distribution of size increases”. Because it is a meaning. Therefore, it is considered impossible to specify the upper limit with a reasonable basis.

  As used herein, “antigen” refers to any substrate that can be specifically bound by an antibody molecule. Since cell markers such as CD3 and CD4 are antigens, antibodies against them are detection means.

  It will be understood that antibodies of any specificity may be used as long as false positives are reduced. Since an antibody has a certain specificity, the antibody used in the present invention is not limited to HBME-1. It will be understood that any antibody that recognizes a mesothelial marker or a mesothelial negative marker is useful. Therefore, the antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.

  As used herein, “compound” means any identifiable chemical or molecule, including small molecules, peptides, proteins, sugars, nucleotides, or nucleic acids, including: Without limitation, such compounds can be natural or synthetic.

  In the present specification, the “small organic molecule” means an organic molecule having a relatively small molecular weight. Usually, a low molecular weight organic material has a molecular weight of about 1000 or less, but may have a higher molecular weight. The organic small molecule can be synthesized usually by using a method known in the art or a combination thereof. Such small organic molecules may be produced by living organisms. Examples of small organic molecules include hormones, ligands, signal transmitters, small organic molecules, molecules synthesized by combinatorial chemistry, and small molecules that can be used as pharmaceuticals (for example, small molecule ligands). It is not limited.

  As used herein, “ligand” refers to a substance that specifically binds to a certain protein. Examples of the ligand include lectins, antigens, antibodies, hormones, neurotransmitters, and the like that specifically bind to various receptor protein molecules present on the cell membrane.

  As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also refer to a complex assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is included. When specifically mentioned, the “polypeptide” of the present invention may refer to a marker.

  In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art.

  As used herein, “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a nucleotide or nucleotide derivative or nucleotide analog polymer of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”.

  In the present specification, the “nucleotide” may be natural or non-natural. “Nucleotide derivative” or “nucleotide analog” refers to a substance that is different from a naturally occurring nucleotide but has a function similar to that of the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well known in the art. Examples of such nucleotide derivatives and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, peptide-type nucleic acids (PNA). Not.

  As used herein, “complex molecule” refers to a molecule formed by linking a plurality of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules. Examples of such complex molecules include, but are not limited to, glycolipids and glycopeptides. In the present specification, a nucleic acid molecule encoding a polypeptide having the amino acid of SEQ ID NO: 2, or a variant or fragment thereof, as long as it has biological activity involved in diagnosis, and the like. Can also be used. A complex molecule containing such a nucleic acid molecule can also be used.

  As used herein, a “corresponding” gene (eg, a polypeptide molecule or polynucleotide molecule such as CD3, CD4, etc.) has the same action as a given gene in a species as a reference for comparison in a certain species, Alternatively, it refers to a gene (for example, a polypeptide molecule or a polynucleotide molecule) that is predicted to have, and when there are a plurality of genes having such an action, it refers to one that has the same origin evolutionarily. Thus, a gene corresponding to a gene can be an ortholog of that gene. Therefore, it is understood that when a diagnosis similar to that of a human is performed in an animal other than a human, a gene corresponding to CD3 or the like in that animal is used for the same diagnosis. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in an animal (for example, mouse) is a reference gene of the corresponding gene (for example, CD3) using the sequence of the animal (for example, human or rat) as a query sequence. It can be found by searching a sequence database.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg 11 etc.) are also suitable as lower limits obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, it is understood that such a fragment falls within the scope of the present invention as long as the full-length fragment functions as a marker, as long as the fragment itself also functions as a marker. For example, a fragment of an antibody can also be used in the present invention as long as it has the same detectability as the corresponding antibody.

(Diagnosis method)
As used herein, “diagnosis” refers to identifying various parameters related to a disease, disorder, condition, etc. in a subject and determining the current state or future of such a disease, disorder, condition. By using the methods, devices, and systems of the present invention, conditions within the body can be examined, and such information can be used to formulate a disease, disorder, condition, treatment to be administered or prevention in a subject. Alternatively, various parameters such as methods can be selected. In the present specification, in a narrow sense, “diagnosis” refers to diagnosing the current state, but diagnosing before onset is also referred to as “preliminary diagnosis”.

  In principle, the diagnostic method of the present invention can be used from the body. This is industrially useful because it can be performed away from the hands of medical personnel such as doctors. In this specification, in order to make it clear that it can be performed away from the hands of medical personnel such as doctors, it is sometimes referred to as “assisting diagnosis”.

  As used herein, “treatment” refers to preventing a disease or disorder from deteriorating, preferably maintaining the status quo, more preferably reducing, when a certain disease or disorder becomes such a condition. Preferably, it means elimination.

  Herein, for example, the number of mesothelial cells can be measured and a sign can be presented to the patient to stop peritoneal dialysis when below a certain level. Thus, a decrease in mesothelial cells can be an indication of a decrease in suitability for peritoneal dialysis. Also, an increase in the average projected area of mesothelial cells can be an indicator of a decrease in suitability for peritoneal dialysis. The increase in the projected area of mesothelial cells can also be determined by the average projected area.

  An increase in mesothelial cells may be an indicator of a decrease in suitability for peritoneal dialysis. In an exemplary embodiment, peritoneal dialysis may be determined to be inadequate when mesothelial cells are less than about 5% of the total total number of cells. This number can vary from sample to sample.

  As used herein, the “subject” refers to an organism to which the treatment of the present invention is applied, and is also referred to as a “patient”, and can typically be a mammal. The patient or subject may preferably be a human.

(system)
In this specification, “system” refers to an arbitrary system for diagnosis, and generally includes one or a plurality of components, and when there are a plurality of components, these components act and relate to each other. It means a system that satisfies the three conditions of exhibiting harmonious behavior and function as a whole. The system can be in any form, such as a device, composition, diagnostic agent. Accordingly, the system can be used, for example, from a large-scale system including a measuring device to a system including chromatography, a kit using an immune reaction, a composition including an antibody (that is, a diagnostic agent that is an in-vitro drug including a monoclonal antibody of a marker) ) And the like. In the present invention, typically the system is a peritoneal dialysis compatible diagnostic device. The system may include an array scan or the like. In the system of the present invention, the shape parameter may include a projected area, a cell surface irregularity of a nucleated cell, and a ratio of a major axis to a minor axis of a nucleated cell. Peritoneal dialysis compatibility diagnosis is realized by commanding to stop peritoneal dialysis when the cell concentration of mesothelial cells reaches a certain level.

  In such a system, the projected area can be used as an indicator of nucleated cell senescence, and a decrease in mesothelial cell concentration can be used as an indicator of decreased suitability of peritoneal dialysis or mesothelial cell projection. The increase in area can be used as an indicator of a decrease in suitability for peritoneal dialysis. The increase in the projected area is determined by the average projected area, but is not limited to this. An increase in mesothelial cells with increased projected area can be used as an indicator of decreased suitability for peritoneal dialysis. For example, when mesothelial cells become less than about 5% of the total number of total cells, it can be determined that peritoneal dialysis is inadequate, but is not limited thereto.

  The system of the present invention may include a hemocytometer, and this hemocytometer is used for measuring the number of cells during sample preparation. It is possible to improve the working efficiency by measuring the total number of cells with trypan blue staining solution and the total nucleated cells with Hinkelmann's solution using a hemocytometer and determining the preparation conditions of the sample to be measured by array scan.

(kit)
As used herein, the term “kit” refers to a unit provided with a portion (eg, antibody, label, etc.) to be provided, usually divided into two or more compartments. This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use. Such a kit preferably comprises a provided part (eg, instructions or instructions describing how the reagent should be processed. In this specification the kit is a reagent kit. When used as a kit, the kit usually contains instructions describing how to use the antibody, etc. The instructions are so-called package inserts, which are usually provided on paper media, However, the present invention is not limited to this, and may be provided in the form of, for example, an electronic medium (for example, a home page or electronic mail provided on the Internet).

(Preferred embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(Significance of mesothelial cytology and data analysis)
Tadaji Yamamoto et al. About the significance of mesothelial cytodiagnosis 1997 Dialysis Society Vol. 30, No. 10, P.1225-1231 49 patients, CAPD period of 3 to 161 months (average 63.9 months) Mesothelium in peritoneal dialysis drainage We report the result of analyzing cells by conventional technology. As a conclusion, the area of mesothelial cells in peritoneal drainage is useful as an index of peritoneal degeneration in peritoneal dialysis patients, and the current name indicates that it is useful in avoiding the development of encapsulated peritoneal sclerosis. Yes.
→ (Correlation between peritoneal dialysis period and mesothelial cell area in peritoneal dialysis drainage)
Yamamoto et al. Regarding the significance of mesothelial cytodiagnosis, the Journal of Dialysis Society, Vol. 30, No. 10, P. 1225-1231 showed a significant positive correlation between the mesothelial area and the CAPD period. It is useful as an index of denaturation.

  For mesothelial cytology, the following references can be referred to (these references are incorporated by reference in their relevant parts, which may be in their entirety): (1) Yamamoto T, et al. Morphological studies of mesothelial cells in CAPD effect and therological clinical science. Am J Kidney Dis 32: 946-952, 1998; (2) Tadaji Yamamoto, et al .: Morphology and clinical significance of CAPD drained mesothelial cells. Dialysis Journal 30: 1225-1231, 1997; (3) Izumotani T, et al. : Correlation between peripheral mesothelial cell cytology and peritonal histopathology with respect to prognosis in patients on continu ous ambitions. Nephron 89: 43-49, 2001; (4) Tadaji Yamamoto, et al .: Usefulness of HD combination therapy from the viewpoint of drainage mesothelial cells. Peritoneal dialysis 2003: 101-103, 2003.

(About data analysis)
According to the above paper, the correlation between the PD period and the area of drainage mesothelial cytology is shown with a certain width. The degree of mesothelial cell injury is indicated by a certain area cut-off value.

(About cancellation criteria for PD)
It can be determined with reference to literature on mesothelial cytology (Yamamoto T, et al .; Tadaji Yamamoto, et al., 1997; Izumotani T, et al .; Tadaji Yamamoto, et al. 2003).

For example, as a typical example, when the area in PD is 400 μm ² or more and the PD period is 120 months or more, the prediction rate of developing EPS (or ileus) after discontinuation is 29% at 400 μm ² or more, 120 months or more 21%. If an area of 400 μm ² or more continues for 3 months or more, it is desirable to stop PD. When the area in PD is 350 μm ² or more and the PD period is 72 months or more, the prediction rate of developing EPS after discontinuation is 25% at 350 μm ² or more, and 17% at 72 months or more. When an area of 350 μm ² or more continues for more than 3 months, it is desirable to consider PD suspension along with other findings.

(About CAPD cancellation criteria)
It can be determined with reference to literature on mesothelial cytology (Yamamoto T, et al .; Tadaji Yamamoto, et al., 1997; Izumotani T, et al .; Tadaji Yamamoto, et al. 2003).

For example, as a representative example, among 469 cases in which Yamamoto et al. Performed drainage mesothelial cytology, 63 cases in which CAPD was discontinued (average age: 50.7 years, average CAPD period: 81.6 months) About 46 cases without abdominal symptoms after discontinuation, 14 cases with some ascites including ileus and EPS (ascites group), 11 cases with ileus symptoms including EPS (ileus group), EPS group It was divided into 3 cases and examined by EBM. When the area cut-off is 400 μm ^2, the EPS group has a sensitivity of 100% and specificity of 81.7%, and when the cut-off is 350 μm ² the sensitivity of the ileus group is 90.9%, the specificity is 36.5%, and the cut-off is 300 μm ² . In the ascites group, the sensitivity was 64.7% and the specificity was 60.9%. Then, the positive predictive rate and negative predictive rate when dividing the area cut off every 50 [mu] m ^2, the most established was high 300 [mu] m ² in ascites group was 400 [mu] m ² at 350 .mu.m ^2, SEP groups with ileus group .

  The CAPD cancellation standard for EPS prevention is based on the area around 400 μm 2, and if the ascites retention is considered to be in the pre-EPS state, the area around 350 μm 2 is considered to be a temporary standard. In cases with an area of 350 μm 2 or more, careful observation is necessary even after CAPD is discontinued (see Tadaji Yamamoto, et al .: CAPD discontinuation criteria by drainage mesothelial cytology. See Peritoneal Dialysis 2002: 334-337, 2002).

(About cancellation criteria for intraperitoneal washing)
The criterion for discontinuation of intraperitoneal lavage for EPS prevention is considered to be an approximate standard with an area of 300 μm ² if ascites retention is considered to be the previous EPS state. There is a debate about whether it is appropriate or not to place a catheter and perform intraperitoneal lavage after CAPD is discontinued, but if placement is possible, confirm the area decrease by mesothelial cytodiagnosis with lavage fluid, and then remove it I think that is one way. Intra-abdominal lavage after discontinuation of CAPD for EPS prevention should be continued until the area is 300 μm ² or less (Tadaji Yamamoto, et al .: Criteria for discontinuation of intra-abdominal lavage by drained mesothelial cytology. Peritoneum Dialysis 2002: 338-341, 2002).

  As a sample, mesothelial cytodiagnosis is possible even with ascites. In principle, it is possible to test both drainage and ascites. However, it may not be possible to measure with high fibrin deposition or blood drainage.

  In the present invention, since there is a step of counting nucleated cells in a sample, the range of cell concentrations in the sample that can be handled is wider than in the conventional method. However, considering obtaining data such as the total number of mesothelial cells, drainage during PET is considered to be most preferable.

  Even if it is pointed out by the conventional method that the number of cells is insufficient for the above reasons, it can be measured.

  If the decreased mesothelial cell area has increased after discontinuation of PD, literature on mesothelial cytology (Yamamoto T, et al .; Tadaji Yamamoto, et al., 1997; Izumotani T, et al .; Tadaji Yamamoto) , Et al. 2003).

  For example, as a typical example, it can be interpreted as follows. The peritoneal mesothelium is a cell with a very strong regenerative capacity, and it is regenerated immediately even if it is damaged by CAPD. After CAPD is discontinued, the area is usually in the direction of regeneration, and as a result, the area decreases. However, in cases where the peritoneum is significantly damaged (peritoneal sclerosis, etc.), the mesothelium does not recover. It is expected that the area will not recover or will take considerable time to recover.

  In cases where the mesothelial area has stabilized at a low value after discontinuation, the area does not increase again, but in the case of a rare increase in the case, there is a literature on mesothelial cytology ( Yamamoto T, et al .; Tadaji Yamamoto, et al., 1997; Izumotani T, et al .; Tadaji Yamamoto, et al. 2003).

For example, as a typical example, it is presumed that in the above case, the peritoneum is damaged for some reason and the area is increased. There is no much of a problem is some of the upper and lower area of less than 350μm ^2, If you've been raised to 350μm ² or more is considered to be careful. EPS may suddenly develop after 2 or 3 years from discontinuation, and careful observation is necessary for cases of increased area.

  It is said that the mesothelial area increases with the CAPD period, but in the case of decreasing cases, the literature on mesothelial cytology (Yamamoto T, et al .; Tadaji Yamamoto, et al., 1997; Izumotani T, et al .; Tadaji Yamamoto, et al. 2003).

  For example, as a representative example, this case is interpreted as follows. In previous studies, it is speculated that PD does not worsen unilaterally, but there is a period of recovery. The reason for this is currently unknown, but considering that the area decreases due to the change to neutral dialysate and HD combination therapy once a week, the change in dialysate GDPs concentration ( It is thought that the area slightly changes depending on various conditions such as the influence of certain drugs and the recovery function of the peritoneum itself.

When PET is at a level from L to LA but the mesothelial cell area exceeds 400 μm ^2, and it is pointed out that caution is required, literature on mesothelial cytology (Yamamoto T, et al .; Tadaji Yamamoto, et al., 1997; Izumotani T, et al .; Tadaji Yamamoto, et al. 2003).

  For example, as a representative example, in this case, the following determination is made. Yamamoto et al. Interprets drainage mesothelial cytology as reflecting the degree of peritoneal mesothelial damage and regeneration, and does not correlate with peritoneal function as expressed by PET, particularly peritoneal permeability. Is done. That is, even if the peritoneal mesothelium is damaged, if the substance permeability of the submucosal connective tissue and vascular endothelium is normal, PET will be maintained well, and conversely, even if the peritoneal mesothelial is normal, If the mid-subcutaneous connective tissue and vascular endothelium are damaged, PET is considered to show a high value. For this reason, a contradiction occurs between peritoneal permeability and mesothelial cytodiagnosis, and if EPS is caused by peritoneal mesothelial damage, disappearance, or adhesion, PET is low. However, if the area is high, it is a case that needs attention. Actually, even in cases where PET is low average, disappearance of the mesothelium and sclerosis of the subcutaneous tissue are experienced by histopathological examination. Whether or not PD can be continued is considered to require comprehensive judgment including cytology and PET, including pathological tissue.

When PET is H and there is also water removal failure, it is pointed out that there is no problem with the mesothelial cell area of 250 μm ² or less. Also, literature on mesothelial cytology (Yamamoto T, et al .; Tadaji Yamamoto, Et al., 1997; Izumotani T, et al .; Tadaji Yamamoto, et al. 2003).

  For example, as a representative example, in this case, the following determination is made. Yamamoto et al. Interprets that drainage mesothelial cytology reflects the degree of peritoneal mesothelial injury and regeneration and does not correlate with peritoneal function as expressed by PET, particularly peritoneal permeability. is doing. That is, even if the peritoneal mesothelium is damaged, if the mesothelial connective tissue and vascular endothelium are normal, the peritoneal permeability will be maintained. If the vascular endothelium is damaged, PET is considered to show a high value. For this reason, a contradiction occurs between the peritoneal permeability and the results of mesothelial cytodiagnosis, and if EPS is caused by injury, disappearance, or adhesion of the peritoneal mesothelioma, Even if is high, if the area is low, the peritoneal mesothelium is well maintained. Whether or not PD can be continued is considered to require comprehensive judgment including cytology and PET, including pathological tissue.

  EPS can be prevented using the mesothelial cytology of the present invention. The origin of EPS is still unknown, but EPS is thought to be caused by inflammation and adhesion due to disappearance of the peritoneal mesothelium. Therefore, it seems that the current best preventive treatment is to detect and deal with the pre-EPS condition at an early stage. Senescence of mesothelial cells, which is a precursor to mesothelial cell loss, can be better monitored in the present invention.

(Automatic detection technology of the present invention)
In one aspect, the present invention provides a method for identifying a nucleated cell of interest. The method comprises: A) detecting the nucleus and non-nucleated part of the nucleated cell; B) obtaining the shape parameter of the nucleated cell as required; and C) detecting both the nucleus and the non-nucleated part. A step of determining that the nucleated cell is present when it is determined that the shape parameter of the cell is the shape parameter of the nucleated cell, if necessary. Here, the shape parameter is used to determine that the detected mesothelial cell is capable of measuring the area or is meaningful to measure the area. That is, it is used for picking out mesothelial cells that have not aggregated, adhered, or collapsed. Judgment of mesothelial cells is based on the presence of nuclei and the expression of mesothelial markers. Regarding the detection of mesothelial cells and the like, conventional techniques cannot be used as they are for the purpose of measuring the area of mesothelial cells contained in dialysate or the like. This is because most of the mesothelial cells that have detached from the peritoneum and are in a free state have aggregated, adhered, or collapsed, and are not suitable for measuring the area. Therefore, the measuring technician picks up clean mesothelial cells with the eyes of a chemical dyeing + trained engineer, acquires a digital image manually, measures the area with image processing software, and totals it. This measurement is impossible unless a human trained engineer. However, in the present invention, as a method for causing the machine to perform, shape recognition by fluorescence staining and image analysis is the first practical example. The shape parameter and the cell surface unevenness are calculated by a calculation procedure of square of the cell circumference of the nucleated cell ÷ cell area of the nucleated cell × 4π. This number is 1 for a circle, and 1 or more when the shape is distorted. At least one or both of the cell surface irregularities and the ratio of the major axis to the minor axis of the cell can be set to less than 1.5, but can also be varied depending on the size of the cell.

  In one preferred embodiment, the method of the present invention comprises obtaining a projected area of nucleated cells. For example, if light having the same fluorescence wavelength as that of the fluorescent dye used for the label of HBME-1 is detected, the attached scan scan software (Targetactivation) calculates and records the area. Therefore, the shape recognition function can be used as a means for detecting mesothelial cells that is meaningful for area measurement.

In one embodiment, the specific recognition function procedure is as follows.
1. The major axis, minor axis, area (projection area), and circumference (periphery of the projection shape) of a cell or a projected shape of a cell are obtained by any method commonly known in the art.
2. Calculate the ratio of major axis to minor axis, that is, the ratio of major axis to minor axis.
3. Calculate the square of the circumference / area × 4π as an index of the unevenness of the cells.

As used herein, as an example, when using Target activation software with an array scan as an example, the shape parameter is by way of example and the ratio of the major axis to the minor axis is less than 1.5, for example, 1.0-1 .2 is set. Similarly, the square of the circumference / area × 4π is also set to 1.0 to 1.2. Thereby, information useful for selecting mesothelial cells suitable for area measurement semi-automatically can be obtained. The square of the cell circumference as an index of the cell surface irregularity / the cell area of the nucleated cell × 4π is 1 in a circle, and is 1 or more when the shape is irregular. These values are preferably close to the minimum value of 1 which can be present, indicating that the projected shape is a circle, ie, the cell shape is close to a sphere.

  In one embodiment of the automatic detection technique of the present invention, the nucleated cell can be a mesothelial cell. For detection of the non-nuclear portion, an antibody selected from the group consisting of HBME-1, anti-CA-125, anti-mesoterin and anti-calretinin is used, and HOECHST 33342 is used for detection of the nucleus.

In one embodiment, obtaining the shape parameter includes selecting a projection shape that is approximately circular. This is because mesothelial cells are usually spherical when they are floating alone.
“Almost circular” is determined as follows.
1. The major axis, minor axis, area (projection area), and circumference (periphery of the projection shape) of a cell or a projected shape of a cell are obtained by any method commonly known in the art.
2. Calculate the ratio of major axis to minor axis, that is, the ratio of major axis to minor axis.
3. Calculate the square of the circumference / area × 4π as an index of the unevenness of the cells.

  As used herein, as an example, when using Target activation software with an array scan as an example, the shape parameter is by way of example and the ratio of the major axis to the minor axis is less than 1.5, for example, 1.0-1 .2 is set. Similarly, the square of the circumference / area × 4π is also set to 1.0 to 1.2. Thereby, information useful for selecting mesothelial cells suitable for area measurement semi-automatically can be obtained. The square of the cell circumference as an index of the cell surface irregularity / the cell area of the nucleated cell × 4π is 1 in a circle, and is 1 or more when the shape is irregular. These values are preferably close to the minimum value of 1 which can be present, indicating that the projected shape is a circle, ie, the cell shape is close to a sphere.

  In another embodiment, the method of the present invention further comprises selecting as an indicator that it is not detected by the nucleated cell negative marker. Examples of negative markers include CD14, CD33, CD45 and CD3 in the case of mesothelial cells. By using a negative marker, mesothelial cells can be selected more reliably.

  In one embodiment, the present invention can use the projected area as an indicator of nucleated cell aging.

  In another aspect, the present invention provides a method for diagnosing the suitability of peritoneal dialysis in a subject. This method is a step of A) detecting mesothelial cells in a sample derived from a subject, and A-1) detecting nuclei and non-nuclear portions of mesothelial cells in the sample and A-2) nuclei and non-nuclear portions. Detection is achieved by determining the cells detected together as mesothelial cells, and B) calculating the projected area of the mesothelial cells, wherein peritoneal dialysis due to mesothelial cell fluctuations Including suitability is determined.

  In one embodiment, step A) further comprises A-3) obtaining a shape parameter of a cell in the sample, the detection depending on whether the shape parameter is in a state suitable for area measurement. Can be determined.

  In one embodiment, an increase in projected area where the average projected area of cells reaches 250 square micrometers is an indication of a decrease in peritoneal dialysis suitability. This numerical value is a presently preferable numerical value, and may change due to changes in the population to be measured in the future. Such variations are within the scope of those skilled in the art, and such variations can be determined using techniques well known in the art.

  In one embodiment, the sample used in the present invention is a peritoneal dialysis drainage sample. This is because the peritoneal dialysis drainage sample often contains mesothelial cells, and the analysis result thereof can be used as an index of peritoneal dialysis compatibility.

  In another embodiment, peritoneal dialysis can be discontinued when the number of mesothelial cells is significantly reduced. The reduction is believed to mean a loss of suitability, but a measure can be determined using techniques well known in the art. Therefore, it is preferable to measure the cell concentration of mesothelial cells, and when it falls below a certain level, it is preferable to present a sign to the patient to stop peritoneal dialysis, but is not limited thereto. In addition, such measurement is possible if the same patient is followed for a long time. Further, by analyzing the size distribution of mesothelial cells according to the present invention, it becomes possible to know a decrease in compatibility. There is no need to count mesothelial cells that have been forced to disintegrate or aggregate. It can be a significant effect that it is not necessary to count the number of mesothelial cells. In particular, it should be noted that assessment within a patient can be performed. As compatibility decreases, mesothelial cells decrease. By using the present invention, a decrease in mesothelial cells can be captured by measurement over time. As a method for realizing this, for example, in addition to fluorescent staining and counting manually, automatic counting may be performed. The array scan can also be used as an imaging device. Thus, in one embodiment, a decrease in the mesothelial cells, particularly cell concentration, may be an indication of a decrease in peritoneal dialysis suitability.

  In another embodiment, an increase in the (preferably average) projected area of mesothelial cells may be an indication of a decrease in suitability for peritoneal dialysis. In addition, since there are reports that a small number of large mesothelial cells are clinically meaningless in peritoneal dialysis, such cells may be excluded and measured. Of particular concern is the size distribution pattern of the entire mesothelial cell. The standard deviation in size also seems to increase with years of peritoneal dialysis experience. Evaluating the cumulative distribution graph by regression calculation may be more appropriate than the average for expressing the size distribution pattern. Also, without being bound by theory, this distribution pattern is like a Poisson distribution.

  In another embodiment, an increase in mesothelial cells with increased projected area may be an indication of a decrease in peritoneal dialysis suitability.

  In other embodiments, peritoneal dialysis is determined to be inadequate when mesothelial cells fall below, for example, less than about 10%, preferably less than about 5% of the total number of cells. It can be determined by multiplying individual differences in the number of mesothelial cells by transient errors due to individual differences in infiltrating cells and patient physical condition. Therefore, it is possible to create a database by measuring many examples.

  In another aspect, the present invention provides a system for pre-diagnosing or diagnosing whether a subject is compatible with peritoneal dialysis continuation. The system comprises: A) a nuclear detection means for detecting nuclei of nucleated cells in a sample from the subject; B) a non-nuclear detection means for detecting a non-nuclear portion of the nucleated cells in the sample from the subject; And C) a shape parameter obtaining means for obtaining a shape parameter of the nucleated cell in a sample from the subject. If peritoneal dialysis has not begun, there is no peritoneal dialysis drainage, but prior diagnosis is possible by measuring the equivalent. It is possible that mesothelial cells have aged before onset, and in rare cases it may be less compatible (such as a history of peritonitis).

  In a preferred embodiment, the nuclear detection means and the non-nuclear detection means are independently selected from the group consisting of proteins, nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof. It will be understood that any molecule can be used as long as it is a factor that specifically binds to the target nucleus and non-nuclear moiety, and preferably is biocompatible.

  In one specific embodiment, the nuclear detection means is a fluorescent dye and the non-nuclear detection means is an antibody that is labeled or not. Here, a measuring instrument using fluorescence is used as the detection means. Therefore, it is preferable that the nuclear detection means and the non-nuclear detection means are labeled or can be labeled.

  In one embodiment, the shape parameter acquisition means is selected from the group consisting of a photomicrograph apparatus, an equivalent, and combinations thereof. The main point is input to image analysis software. These settings can generally be made.

  In one embodiment, the present invention further comprises means for purifying the sample. Usually, it is assumed that the drainage is used as a sample as it is. Preferably, the cells are collected by centrifugation. Any method can be used as the centrifugation method. Therefore, the same technique as that of a normal clinical test (such as a urine sediment test) can be used. In most cases, centrifugation may be essential. This is because peritoneal dialysis drainage has a low cell concentration and cannot be measured unless the cells are substantially concentrated. For methods other than centrifugation, filtration, adhesion, electrical means, and the like can be considered. In particular, filtration and adhesion may be the next preferred embodiment after centrifugation.

  In one embodiment, the subject targeted by the method of the present invention comprises a mammal, more preferably a human.

  In one embodiment, the system of the present invention may further comprise means for measuring the area distribution coefficient of nucleated cells. The area distribution coefficient is obtained by accumulating the number of cells for each projected area and performing natural logarithmic regression calculation.

  In another embodiment, the system of the present invention further comprises means for performing a count of nucleated and non-nucleated portions of nucleated cells. The quantitative unit may further include a determination unit that determines whether the measurement result is within an allowable range. The allowable range may be anything as long as it is set appropriately. For example, the allowable lower limit is a coefficient of about 40 in natural logarithmic regression calculation. The area distribution coefficient may be 30 as a lower limit in natural logarithmic regression calculation. Since the upper limit is not related to the object of the present invention, there is no significance to define it. The purpose of this technology is to determine whether the change induced in the mesothelial cells due to continuation of peritoneal dialysis is within the allowable range, so the specific values of the allowable range may vary depending on the population. Those skilled in the art can easily determine this. In the presently preferred embodiment, it appears that the average area is 350 and the natural logarithmic regression calculation has a factor of about 40. The means for measuring the area distribution coefficient preferably includes a computer.

  In one embodiment, the determination means is a computer. It will be understood that the present invention can be used with any computer configuration as long as image analysis and subsequent processing are possible.

  In a preferred embodiment, the system of the present invention is provided as a peritoneal dialysis compatible diagnostic device. In addition, since this technique does not have equipment dependence, it understands that what kind of composition may be sufficient. As an example, if there is a fluorescence microscope capable of photographing the same part with a plurality of wavelengths and appropriate image analysis software, it can be carried out without an array scan.

  In one embodiment, the array scan is used alone on the hardware side. There is no particular configuration. In this configuration, the number of cells is measured with a hemocytometer during sample preparation. A hemocytometer is also a powerful tool for samples with many contaminants, such as peritoneal dialysis drainage, similar to an array scan. Specifically, the total cell count is measured with trypan blue stain, the total nucleated cells are measured with a Hinkkelman solution using a hemocytometer, the preparation conditions of the sample to be measured by array scan are determined, and work efficiency is improved. . As an experiment, it can be understood that it can be carried out by ordinary operations.

  In one embodiment, the shape parameter may be indicated by a projected area, a cell surface roughness of the nucleated cell, a ratio of the major axis to the minor axis of the nucleated cell, and the like.

  In another embodiment, the nucleated cell is a mesothelial cell.

  In a further embodiment, the non-nuclear portion detection means is an antibody selected from the group consisting of HBME-1, anti-CA-125, anti-mesothelin and anti-calretinin, and said nuclear detection means is HOECHST 33342. Here, a substantially circular shape is selected as the shape parameter.

  In one embodiment, it further comprises means for detecting the negative marker of the nucleated cell. As a negative marker, CD14, CD33, CD45, CD3 and the like can be used.

  In the system of the present invention, the projected area can be used as an index of aging of the nucleated cells.

  In the system of the present invention, a peritoneal dialysis drainage sample can be used as a sample.

  It will be understood that any index utilized in the method of the invention described above can be used in the system of the invention.

  In another aspect, the present invention provides A) a nuclear detection means for detecting the nucleus of nucleated cells in a sample from the subject for diagnosing whether the subject can continue peritoneal dialysis; B) the test Providing a non-nuclear detection means for detecting a non-nuclear portion of the nucleated cell in a sample from the body; and C) a shape parameter obtaining means for obtaining a shape parameter of the nucleated cell in the sample from the subject.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(Example 1)
In this example, a semiautomatic detection method of peeled mesothelial cells in peritoneal dialysis drainage by Image Cytometry was examined.

(Materials and methods)
(Sample)
Peritoneal dialysis drainage source: *** hospital (dialysis department)
Patient name: Temporary name A for this example (peritoneal dialysis, about 14 years)
Dedicated temporary specimen identification code MaT-A for this example
(reagent)
Name: HBME-1 (primary antibody): Lot number: 0012C (source: Dako Cytomation)
Name: AlexaFluor 546 Goat-anti mouse IgM μchain (secondary antibody) 2 mg / mL: Lot number: 93E1-1 (obtained from: Molecular Probes)
Name: Bizbenzimide H 33342: Lot number: 122K4021 (Source: SIGMA)
(Total cell count)
A sample of 2510 mL was centrifuged at 300 g for 5 minutes at 4 ° C., and the supernatant was discarded to 5 mL.
When the cells were mixed with an equal volume of Hinkelmann's solution and the number of cells in 0.8 μL was counted with a Neubauer hemocytometer, the total number was 58. From this result, the number of cells in 2510 mL of the sample was calculated to be 7.3 × 10 ⁵ .

(Cell staining)
685 μL (10 ⁵ samples) concentrated at the time of cell counting were centrifuged at 300 × g for 5 minutes at 4 ° C., the supernatant was discarded, and the cell concentration was adjusted to 10 ⁶ / mL with PBS containing 0.1% BSA. . After stirring, 2 μL of HBME-1 was added and allowed to react on ice for 30 minutes. This was centrifuged and washed twice with PBS containing 0.1% BSA, and then stained with a secondary antibody for fluorescent antibody. This was centrifuged and washed twice with PBS containing 0.1% BSA, and then 2 μL / 200 μL of Hoechst33342 (2 mg / mL) was added for nuclear staining. After washing by centrifugation twice with PBS containing 0.1% BSA, cells were fixed by adding 300 μL of CellFix.

(Measurement of mesothelial cells)
Measurements were made using an array scan. The suspension was 300 μL / well (cell count 10 ⁵ ) of cells stained with a transparent bottom black 96 well plate (Coster). The measurement items were as follows.
The number of nuclear staining positive cells, the number and area of HBME-1 positive cells and images automatically recognized for HBME-1 positive cells were visually checked, and multinucleated cells were counted.
Cell counts and multinucleated cells were counted for aggregated HBME-1-positive cells.

(Measurement result)
The total amount of 2510 mL of the sample was concentrated to 5 mL, diluted twice with Hinkelmann's solution, and counted in 8 fields using a hemocytometer.
58 cells
From (58 x 2 dilution x 5000 μL) / (0.8 μL), total number of cells: 7.3 x 10 ⁵ Number of cells detected by image analysis: 6323
Same mesothelial (HBME-1 positive) cell number: 405 mesothelial cell ratio 6.4%
Total number of mesothelial cells 4.6 × 10 ⁴ Projection area measurement Of 84 mesothelial cells, 19 multinucleated cells (23%)
Projected product average 308 ± 19μm ² (Mean ± SEM) (SD = 175μm ² )
Area distribution coefficient 46 (y = 46.465Ln (x) -207.42, R ² = 0.9889)
1-3 show further results.

  FIG. 1 shows calculation of area distribution counts using a cumulative distribution graph of MaT-A.

  FIG. 2 shows the distribution of the projected area in the specimen MaT-A.

  FIG. 3 shows a pseudo color composite photograph of the fluorescence photographed images of HBME-1 (red) and Hoechst33342 (blue) of specimen MaT-A.

(Materials and methods)
(Sample) Temporary Sample Identification Symbol MaT-B for this Example
Peritoneal dialysis drainage source: *** Hospital (dialysis department)
Patient name: Tentative name B for this example (elapsed years of peritoneal dialysis approximately 5 years)
(reagent)
Name: HBME-1 (primary antibody): Lot number: 0012C (source: Dako Cytomation)
Name: AlexaFluor 546 Goat-anti mouse IgM μchain (secondary antibody) 2 mg / mL: Lot number: 93E1-1 (obtained from: Molecular Probes)
Name: Bizbenzimide H 33342: Lot number: 122K4021 (Source: SIGMA)
(Total cell count)
600 mL of the total sample volume of 2475 mL was centrifuged at 4 g at 300 g for 5 minutes, and the supernatant was discarded to 0.5 mL. When the number of cells in 0.8 μL was counted with a Neubauer hemocytometer after mixing with 3 times the volume of Hinkelmann's solution, the total number was 138. From this result, the cell concentration in the concentrated specimen was calculated to be 690 cells / μL. (138 pcs x 4 ÷ 0.8 μL = 690 pcs / μL)
The total number of cells of MaT-B was calculated to be 1.4 × 10 ⁶ from (2475 mL ÷ 600 mL) × 0.5 mL × 690 cells / μL.

(Cell staining)
A sample of 600 mL was centrifuged at 4 g at 300 g for 5 minutes, and the supernatant was discarded to 0.5 mL. Collect 145 μL (10 ⁵ pieces), add 5 mL of PBS containing 0.1% BSA, centrifuge at 300 xg for 5 minutes at 4 ° C, discard the supernatant, and wash with 0.5% PBS containing 0.1% BSA. mL / tube. After stirring, 2 μL of HBME-1 was added and allowed to react on ice for 30 minutes. This was centrifuged and washed twice with 5 mL of PBS containing 0.1% BSA, adjusted to 0.5 mL / tube with PBS containing 0.1% BSA, added with 2 μL of the secondary antibody, reacted on ice for 30 minutes, and stained with fluorescent antibody. This was centrifuged and washed twice with PBS containing 0.1% BSA, and 2 μL of Hoechst33342 (2 mg / mL) was added to stain the nucleus. After 10 minutes of reaction on ice, the cells were fixed by adding 300 μL of CellFix after centrifugation and washing once with 5 mL of PBS containing 0.1% BSA.

(Measurement of mesothelial cells)
Measurement was performed using Image Cytometry. A suspension of cells stained and fixed with a transparent bottom black 96 well plate (Coster) was 150 μL / well. The measurement items were as follows.

The number of nuclear staining positive cells, the number of HBME-1 positive cells and the projected area The images automatically recognized for both positive cells stained with nuclear staining and HBME-1 fluorescent antibody were visually checked, and multinucleated cells were counted. In addition, mesothelial cells whose projected area could not be measured accurately due to damage, aggregation, etc. were excluded from the tabulated projection area as a target for measuring the number of cells and the number of multinucleated cells.

(Measurement result)
Sample total volume 2475mL
Measurement with hemocytometer: total cell count 1.4 × 10 ⁶

Measurement by Image Cytometry Number of HBME-1-positive cells: 141 out of 2282 (6.2%)
54 HBME-1-positive cells capable of measuring projected area Of 56 mesothelial cells measuring projected area, 13 multinucleated cells (23%)
Projected product mean ^{352 ± 35μm 2 (Mean ± SEM} ) (SD = 259μm 2)
Area distribution coefficient 41 (y = 40.779Ln (x) -179.24, R ² = 0.9765)
Further results are shown in FIGS.

  FIG. 4 shows the area distribution count calculation by the cumulative distribution graph of MaT-B.

  FIG. 5 shows the distribution of the projected area in the specimen MaT-B.

  FIG. 6 shows a pseudo color composite photograph of the fluorescence photographed images of HBME-1 (red) and Hoechst33342 (blue) of specimen MaT-B.

(Materials and methods)
(Sample) Temporary Sample Identification Symbol MaT-C
Peritoneal dialysis drainage source: *** Hospital (dialysis department)
Patient name: Temporary name C for this example (peritoneal dialysis, less than 1 year)
(reagent)
Name: HBME-1 (primary antibody): Lot number: 0012C (source: Dako Cytomation)
Name: AlexaFluor 546 Goat-anti mouse IgM μchain (secondary antibody) 2 mg / mL: Lot number: 93E1-1 (obtained from: Molecular Probes)
Name: Bizbenzimide H 33342: Lot number: 122K4021 (Source: SIGMA)
(Total cell count)
600 mL of the total sample volume of 1720 mL was centrifuged at 4 g at 300 g for 5 minutes, and the supernatant was discarded to 0.5 mL. When the number of cells in 0.8 μL was counted with a Neubauer hemocytometer after mixing with 3 volumes of Hinkelmann's solution, the total number was 189. From this result, the cell concentration in the concentrated specimen was calculated to be 945 cells / μL. (189 × 4 ÷ 0.8μL = 945 / μL)
The total number of cells of MaT-C was calculated to be 1.4 × 10 ⁶ from (1720 mL ÷ 600 mL) × 0.5 mL × 945 cells / μL.

(Cell staining)
A sample of 600 mL was centrifuged at 4 g at 300 g for 5 minutes, and the supernatant was discarded to 0.5 mL. 106 μL (10 ⁵ ) of this was collected, 5 mL of PBS containing 0.1% BSA was added, centrifuged at 4 ° C. at 300 × g for 5 minutes, the supernatant was discarded, and 0.5% PBS containing 0.1% BSA was added. mL / tube. After stirring, 2 μL of HBME-1 was added and allowed to react on ice for 30 minutes. This was centrifuged and washed twice with 5 mL of PBS containing 0.1% BSA, adjusted to 0.5 mL / tube with PBS containing 0.1% BSA, added with 2 μL of the secondary antibody, reacted on ice for 30 minutes, and stained with fluorescent antibody. This was washed by centrifugation twice with PBS containing 0.1% BSA, and 2 μL / 200 μL of Hoechst33342 (2 mg / mL) was added for nuclear staining. After 10 minutes of reaction on ice, the cells were fixed by adding 300 μL of CellFix after centrifugation and washing once with 5 mL of PBS containing 0.1% BSA.

(Measurement of mesothelial cells)
Measurement was performed using Image Cytometry. A suspension of cells stained and fixed with a transparent bottom black 96 well plate (Coster) was 300 μL / well. The measurement items were as follows.

The number of nuclear staining positive cells, the number of HBME-1 positive cells and the projected area, and images automatically recognized for both positive cells stained with nuclear staining and HBME-1 fluorescent antibody were visually checked, and multinucleated cells were counted. In addition, mesothelial cells whose projected area could not be measured accurately due to damage, aggregation, etc. were excluded from the tabulated projection area as a target for measuring the number of cells and the number of multinucleated cells.

(Measurement result)
Sample total volume 1720mL
Measurement with hemocytometer: total cell count 1.4 × 10 ⁶

Measurement by Image Cytometry Number of HBME-1-positive cells: 375 out of 5881 (6.4%)
Projected area measurement Of 160 mesothelial cells, 4 multinucleated cells (3%)
Projected product average 179 ± 7.1μm ² (Mean ± SEM) (SD = 90μm ² )
Area distribution count 61 (y = 61.225Ln (x) -260.47, R ² = 0.9791)
Further results are shown in FIGS.

  FIG. 7 shows the area distribution count calculation by the cumulative distribution graph of MaT-C.

  FIG. 8 shows the distribution of the projected area in the specimen MaT-C.

  FIG. 9 shows a pseudo-color composite photograph of the fluorescence photographed images of HBME-1 (red) and Hoechst33342 (blue) of specimen MaT-C.

(Summary and discussion)
The total peritoneal dialysis drainage received was 1530 mL. The total number of cells of Ma-**** was calculated to be 8.2 × 10 ⁵ from the result of concentrating the specimen and counting the cells using Hinkelmann's solution and a modified Neubauer hemocytometer.

In the measurement using fluorescent antibody staining and Image Cytometry, 1126 out of 5443 (21%) were HBME-1 positive cells and were determined to be mesothelial cells. The total number of HBME-1 positive cells was calculated to be 1.7 × 10 ⁵ . As a result of analyzing the images of the HBME-1-positive cells, it was possible to measure 110 projected areas, and the average area was 170 ± 8.6 μm ² (Mean ± SEM, SD = 90). Moreover, the number of HBME-1-positive cells having two or more nuclei in one cell was 12 out of 110 (11%).

  In addition, a large number of floating substances considered to be macroscopic white cotton-fibrous cell aggregates were observed in the specimen. For this reason, it was estimated that errors such as the total number of cells were large.

The detection of exfoliated mesothelial cells in the peritoneal dialysis effluent by Image Cytometry was examined. The total amount of the specimen was 1530 mL, and the total number of cells was 8.2 × 10 ⁵ . Of the detected cells, 21% were positive for nuclear staining and HBME-1 fluorescent antibody staining and were judged to be mesothelial cells. The total number of HBME-1 positive cells was calculated to be 1.7 × 10 ⁵ . 110 of 1126 mesothelial cells detected by Image Cytometry can be image-analyzed, the average area is 170 ± 8.6 μm ² (Mean ± SEM), and there are two or more nuclei in one cell There were 12 cells (11%). In addition, this specimen was in a poor state and macroscopically floating of what appeared to be a cotton-fibrous cell aggregate was observed. Large and dense cell aggregates that were difficult to measure were also observed in the image cytometry.

(Summary of Examples 1 to 3)
This technique revealed not only the average size of mesothelial cells, but also the total cell number, total nucleated cell number, total mesothelial cell number, and area distribution coefficient. Moreover, it became easy to grasp the state of peritoneal mesothelial cells by graphing the size distribution.

(Example 4: Example about negative marker)
Next, a confirmation experiment for a negative marker was performed.

(Materials and methods)
(Sample) Temporary Sample Identification Symbol MaT-D for this Example
Peritoneal dialysis drainage source: *** Hospital (dialysis department)
Patient name: Tentative name D
(reagent)
Name: HBME-1 (primary antibody)
Obtain from: Dako Cytomation
(reagent)
Name: Alexa Fluor546 Goat-anti mouse IgM μchain (secondary antibody)
Obtain from: Molecular Probes
(reagent)
Name: FITC-labeled anti-CD14 monoclonal antibody Source: BD
(reagent)
Name: Bizbenzimide Hoechst 33342
Obtain from: SIGMA.

(Cell staining)
4 × 10 ⁴ nucleated cells were collected from the specimen, and PBS containing 0.1% BSA was added to make 0.1 mL / tube. 2 μL of HBME-1 was added thereto and reacted at 4 ° C. for 30 minutes. This was centrifuged and washed twice with 2 mL of PBS containing 0.1% BSA, and then 0.1 mL / tube with PBS containing 0.1% BSA, and 2 μL each of the secondary antibody (Alexa Fluor546 Goat-anti mouse IgM) and FITC-labeled anti-CD14. Was added, and light-shielded at 4 ° C. for 20 minutes, followed by fluorescent antibody staining. To this was added 2 μL of Bizbenzimide Hoechst33342 (2 mg / mL) and 0.1 mL / tube of PBS containing 0.1% BSA, and the mixture was reacted at 4 ° C. for 20 minutes while light-shielded for nuclear staining. After the reaction, the mixture was washed once by centrifugation with 2 mL of PBS containing 0.1% BSA, and then fixed with 150 μL of Cell Fix.

(Measurement of mesothelial cells)
Measurement was performed using Image Cytometry. A suspension of cells stained and fixed with a transparent bottom black 96 wellplate (Coster) was 150 μL / well. The measurement results were output as a pseudo-color composite image by assigning red to AlexaFluor 546, yellow to FITC, and blue to Hoechst33342. FIG. 10 shows this image.

  FIG. 10 shows an example of mesothelial cell identification using a negative marker. A pseudo-color composite image in which red is assigned to mesothelial cell marker staining with HBME-1, yellow is assigned to monocyte marker staining with anti-CD14, and blue is assigned to nuclear staining with Hoechst33342. It can be easily identified and analyzed by appropriate image analysis software.

(Results and summary)
Cells other than the mesothelial cells displayed in red are almost yellow. The yellow color suggests that it is CD14 positive and peritoneal macrophage. Therefore, it is presumed that mesothelial cells can also be detected by selecting CD14-negative cells.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The present invention has utility in the pharmaceutical industry and the like. In particular, it has industrial applicability in the production of peritoneal dialysate and therapeutic, preventive and diagnostic agents for peritoneal or mesothelial cell related diseases.

About the result of Example 1, area distribution count calculation by the cumulative distribution graph of MaT-A is shown. About the result of Example 1, distribution of the projection area in specimen MaT-A is shown. About the result of Example 1, the pseudo color synthetic photograph of the fluorescence photography image of HBME-1 (red) and Hoechst33342 (blue) of sample MaT-A is shown. About the result of Example 2, area distribution count calculation by the cumulative distribution graph of MaT-B is shown. About the result of Example 2, distribution of the projection area in specimen MaT-B is shown. About the result of Example 2, the pseudo color composite photograph of the fluorescence imaging | photography image of HBME-1 (red) and Hoechst33342 (blue) of specimen MaT-B is shown. About the result of Example 3, area distribution count calculation by the cumulative distribution graph of MaT-C is shown. About the result of Example 3, distribution of the projection area in specimen MaT-C is shown. About the result of Example 3, the pseudo | simulation color synthetic | combination photograph of the fluorescence imaging | photography image of HBME-1 (red) and Hoechst33342 (blue) of specimen MaT-C is shown. FIG. 10 shows an example of mesothelial cell identification using a negative marker. A pseudo-color composite image in which red is assigned to mesothelial cell marker staining with HBME-1, yellow is assigned to monocyte marker staining with anti-CD14, and blue is assigned to nuclear staining with Hoechst33342. It can be easily identified and analyzed by appropriate image analysis software.

Claims (61)

A method for identifying a target nucleated cell, comprising:
A) detecting the nucleus and non-nucleus part of the nucleated cell; and B) determining that the nucleated cell is present when both the nucleus and the non-nucleus part are detected;
Including the method. The method of claim 1, further comprising obtaining a shape parameter of the detected nucleated cell, thereby determining the state of the nucleated cell. The method of claim 2, wherein the shape parameter is used to determine whether the area of the nucleated cell is measurable or whether the area measurement is significant. The method according to claim 2, wherein the shape parameter is used to determine a cell in a state unsuitable for measuring the area of the nucleated cell. The method of claim 4, wherein the state unsuitable for area measurement is selected from the group consisting of agglomeration, adhesion and disintegration. The method of claim 2, wherein the shape parameter is selected from the group consisting of a projected area, a cell surface irregularity, and a ratio of a major axis to a minor axis of a cell. The method according to claim 2, wherein the cell surface unevenness is calculated by a calculation procedure of square of cell circumference of the nucleated cell ÷ cell area of the nucleated cell × 4π. The method according to claim 2, wherein at least one of the cell surface irregularities and the ratio of the major axis to the minor axis of the cell is set to be less than 1.5. The method according to claim 2, wherein the cell surface irregularities and the ratio of the major axis to the minor axis of the cell are set to less than 1.5. The method according to claim 1, wherein the nucleated cell is a mesothelial cell. The method according to claim 1, wherein the nucleated cell is a mesothelial cell, and the mesothelial cell is determined by the presence of the nucleus and expression of a mesothelial marker. An antibody selected from the group consisting of HBME-1, anti-CA-125 antibody, anti-mesothelin antibody and anti-calretinin antibody is used for the detection of the non-nuclear part, and HOECHST 33342 is used for the detection of the nucleus. The method of claim 1. The method of claim 1, wherein the non-nuclear portion comprises a cell surface. 3. The method of claim 2, comprising obtaining the shape parameter and selecting a substantially circular one. Furthermore, the method of Claim 1 including selecting as a parameter | index that the negative marker of the said nucleated cell is not detected. 16. The method of claim 15, wherein the negative marker is selected from the group consisting of CD14, CD33, CD45 and CD3. The method according to claim 1, further comprising obtaining a projected area of the nucleated cell. The method according to claim 17, wherein the projected area is used as an index of aging of the nucleated cells. A method for diagnosing the suitability of a subject for peritoneal dialysis, comprising:
A) a step of detecting mesothelial cells in a sample derived from a subject,
A-1) detecting the nuclei and non-nuclear parts of the mesothelial cells in the sample; and A-2) detecting the cells in which both the nuclei and the non-nuclear parts are detected as mesothelial cells. Achieved, and B) calculating the projected area of the mesothelial cells, wherein the variability of the mesothelial cells determines suitability for peritoneal dialysis.   The step A) further includes A-3) obtaining a shape parameter of a cell in the sample, and the detection is determined by determining whether the shape parameter is in an unsuitable state for area measurement. 20. The method of claim 19, wherein: 20. The method of claim 19, wherein an increase in projected area where the average projected area of the cells reaches 250 square micrometers is an indication of a decrease in peritoneal dialysis suitability. 20. The method of claim 19, wherein the sample is a peritoneal dialysis drainage sample. 20. The method of claim 19, further comprising the step of measuring the cell concentration of the mesothelial cell and presenting a sign to the patient to stop peritoneal dialysis when below a certain level. 20. The method of claim 19, wherein the decrease in cell concentration of mesothelial cells is an indicator of decreased suitability for peritoneal dialysis. 20. A method according to claim 19, characterized in that an increase in the average projected area of the mesothelial cells is an indicator of a decrease in suitability for peritoneal dialysis. 26. The method of claim 25, wherein the increase in projected area of mesothelial cells is determined by an average projected area. 20. A method according to claim 19, characterized in that in the mesothelial cells, an increase in cells with an increased projected area is an indicator of a decrease in peritoneal dialysis suitability. 20. The method of claim 19, wherein peritoneal dialysis is determined to be inappropriate when the mesothelial cells are less than about 5% of the total total cell count. A system for pre-diagnosing or diagnosing whether a subject is suitable for continued peritoneal dialysis,
A) Nuclear detection means for detecting the nuclei of nucleated cells in a sample from the subject;
B) a non-nuclear detection means for detecting a non-nuclear portion of the nucleated cell in the sample from the subject; and C) a shape parameter acquisition means for obtaining a shape parameter of the nucleated cell in the sample from the subject. ,system. 30. The system of claim 29, wherein the nuclear detection means and the non-nuclear detection means are selected from the group consisting of proteins, nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof. 30. The system of claim 29, wherein the nuclear detection means is a fluorescent dye and the non-nuclear detection means is an antibody that is labeled or not. 30. The system of claim 29, wherein the non-nucleus detection means is a cell surface detection means. 30. The system of claim 29, wherein the nuclear detection means and the non-nuclear detection means are labeled or labelable. 30. The system according to claim 29, wherein the shape parameter acquisition means is a photomicrograph apparatus. 30. The system of claim 29, further comprising means for purifying the sample. 36. The system of claim 35, wherein the means for purifying is selected from the group consisting of centrifuge means, filter means, adhesive means, and electrical means. 30. The system of claim 29, wherein the subject comprises a mammal. 30. The system of claim 29, wherein the subject comprises a human. 30. The system of claim 29, further comprising means for calculating an area distribution coefficient of the nucleated cell. 40. The system according to claim 39, wherein the area distribution coefficient is obtained by accumulating the number of cells for each projected area and performing natural logarithmic regression calculation. 41. The system of claim 40, wherein the area distribution coefficient is 30 as a lower limit. 41. The system of claim 40, wherein the means for measuring the area distribution coefficient includes a computer. 30. The system of claim 29, wherein the system is a peritoneal dialysis compatibility diagnostic device. 30. The system of claim 29, wherein the system includes an array scan. 30. The system of claim 29, wherein the system comprises an array scan and a hemocytometer, the hemocytometer being used for measuring cell numbers during sample preparation. 30. The system of claim 29, wherein the shape parameter is indicated by a projected area, a cell surface irregularity of the nucleated cell, and a ratio of a major axis to a minor axis of the nucleated cell. 30. The system of claim 29, wherein the nucleated cell is a mesothelial cell. 30. The system of claim 29, wherein the non-nuclear portion detection means is an antibody selected from the group consisting of HBME-1, anti-CA-125, anti-mesothelin and anti-calretinin, and the nuclear detection means is HOECHST 33342. . 30. The system of claim 29, wherein the shape parameter is selected to be approximately circular. The system according to claim 29, further comprising means for detecting a negative marker of the nucleated cell. 51. The system of claim 50, wherein the negative marker is selected from the group consisting of CD14, CD33, CD45 and CD3. The system according to claim 46, wherein the projected area is used as an index of aging of the nucleated cells. 30. The system of claim 29, wherein the sample is a peritoneal dialysis drainage specimen. 48. The system according to claim 47, wherein peritoneal dialysis is stopped when the cell concentration of the mesothelial cells reaches a certain level. 48. The system of claim 47, wherein the decrease in cell concentration of the mesothelial cell is an indicator of decreased peritoneal dialysis suitability. 48. The system of claim 47, wherein an increase in the projected area of the mesothelial cell is an indicator of a decrease in peritoneal dialysis suitability. 57. The system of claim 56, wherein the projected area increase is determined by an average projected area or area distribution coefficient. 48. The system of claim 47, wherein the increase in mesothelial cells with increased projected area is an indicator of decreased suitability for peritoneal dialysis. 48. The system of claim 47, wherein peritoneal dialysis is determined to be inadequate when the mesothelial cells are less than about 5% of the total total cell count. Measure the total cell count with trypan blue staining solution and total nucleated cells with Hinkelmann's solution using a hemocytometer, determine the preparation conditions of the sample to be measured by array scan, and improve working efficiency 30. The system of claim 29. A) a nuclear detection means for diagnosing whether the subject can continue peritoneal dialysis, detecting the nucleus of nucleated cells in the sample from the subject; B) the nucleated in the sample from the subject A non-nuclear detection means for detecting a non-nuclear portion of the cell; and C) use of a shape parameter acquisition means for obtaining a shape parameter of the nucleated cell in a sample from the subject.