TW201311907A - Genomic diagnostics using circulating endothelial cells - Google Patents

Abstract

Circulating Endothelial Cells are isolated from patient blood and gene expression of the cells is analyzed to assess a medical condition or the tissue of origin of the cell. Kits for conducting the method are also provided.

Description

Genetic diagnosis using circulating endothelial cells

The circulating endothelial cell line is isolated from the blood of the patient and the gene expression of the cell is then analyzed to assess the medical condition or the tissue of the cell. A kit for performing this method is also available.

Circulating endothelial cells (CEC) are present in low numbers in healthy individuals, but their number has been observed in a variety of human diseases, including cardiovascular diseases and cancer patients. The characteristics of the CEC will help to understand and monitor these diseases and other conditions. CEC can be isolated from peripheral blood by a variety of techniques, including antibody capture with CD 146 antibodies and magnetic separation, and flow cytometry and other means. Unfortunately, CEC separation and analysis is complicated by the large presence of white blood cells. Helping CEC identification is to associate it with important factors, such as its original organization, to provide a basis for further analysis and to provide relevant medical information based on the analysis.

In one aspect of the invention, the CEC is isolated and then analyzed for CEC gene expression to assess a medical condition. Preferably, the gene expression is performed using microarray analysis or amplification and recognition methods such as reverse transcription PCR (RTPCR).

In another aspect of the invention, a population of 130 specific genes is selected to be low in peripheral blood mononuclear cells (PBMC), but in blood A gene that expresses high in endothelial cells and then assembles the gene into a CEC marker.

In yet another aspect of the invention, the gene-based CEC marker is associated with one or more of the following: cell motility, cell migration, angiogenesis, or cell attachment. Preferably such markers are overexpressed relative to other cells.

In yet a further aspect of the invention, the gene-based marker has different manifestations depending on the type of blood vessel and is capable of identifying the original blood vessel or tissue from which the CEC is drawn. Preferably, the marker is selected from 67 genes that are overexpressed on CEC relative to other cells.

In yet another aspect of the invention, the cell retrieval system is used to obtain a CEC for gene expression analysis. Preferably, the immunomagnetic method is used for the extraction and then the analysis provides a medical assessment, such as a disease or condition diagnosis, prediction or prognosis.

In yet another aspect of the invention, a CEC analysis kit is provided. Preferably, the kit includes reagents for CEC gene expression recognition and analysis. In addition, the kit can contain a draw reagent for separation from CEC. The kit, in turn, can include a mechanical code embodiment, wherein the mechanical code embodiment applies the message and algorithm to the message generated during the CEC separation and/or analysis implementation, thereby generating or facilitating a medical assessment.

The present invention provides compositions, methods and kits for the rapid and efficient isolation and identification of endothelial cells from biological samples. This method describes blood The isolation and identification of CEC in the sample while minimizing the selectivity of non-specifically bound or trapped cells.

Although any effective mechanism for the isolation, concentration, and analysis of CEC in blood can be used to extract and concentrate CEC for analysis, the preferred method of collection is to combine immunomagnetic condensation techniques with immunofluorescence labeling techniques. Made with the appropriate analysis platform. Relevant tests have sensitivity and specificity to detect such rare cells in whole blood samples, and to use these rare cells for clinical process analysis of diseases and conditions, as well as related assessments of many identical aspects including prediction and prognosis.

The extraction and separation techniques used in the preferred embodiments have been widely used to analyze circulating tumor cells (CTCs) by using, for example, a tool to explore the significance of epithelial blast cells in the peripheral circulation of cancer patients. This technique is described in U.S. Patent No. 6,365,362 and U.S. Patent No. 6,645,731. The "CellSearch" system (Veridex, Raritan, NJ) using this technology is an automated system based on fluorescence microscopy of cells isolated from blood. This is also a known use in capturing and separating CEC. The system includes an integrated computer-controlled fluorescent microscope and an automated platform with a yoke kit that will hold a disposable sample cartridge. The yoke is designed such that the candidate rare cells labeled by the magnetic liquid are located in the sample chamber to locally magnetize the observation surface above the sample cartridge for viewing under the microscope. The software detects cells that are labeled by antibodies and contain endothelial cells from the blood. In a preferred embodiment, an example A preservative such as "CellSave" cell preservative is used in 7.5 ml of whole blood to separate cells of interest. After the addition of the cell-specific magnetic particles, it is preferably cultured for about 20 minutes. The CellSave preservative can be placed, for example, in a tube for blood sample collection, or can be part of a kit of the invention. After magnetic separation, the cell line bonded to the immunomagnetic-linked antibody is magnetically attached to the tube wall. The unbound sample is then aspirated and an isotonic solution is added to resuspend the sample. A nucleic acid dye, a monoclonal antibody corresponding to the indicated label, and CD 45 (a broad spectrum white blood cell marker) are incubated with the sample. After magnetic separation, the unbound portion was again aspirated, and the bonded and labeled cells were resuspended in 0.2 ml isotonic solution. The sample is suspended in a cell display chamber and placed in a magnetic field device wherein the magnetic field within the magnetic field device directs the magnetic indicator cell to conform to the fluorescence microscopy in the CellSearch system. The cells are automatically identified using control cells enumerated by the system, and candidate target cells presented to the operator for enumeration to identify the morphology of such a pattern. The captured cells can then be subjected to the gene-based assay of the present invention.

Preferred magnetic particles included in the reagent for performing CEC separation are particles which exhibit a colloid. Such particles are characterized by submicron particle sizes, which are typically less than about 200 nanometers (0.20 micrometers), and maintain long-term stability after gravity separation from solution. In addition to many other advantages, this size range makes it almost impossible to observe in the analytical techniques used in cell analysis. Particles in the range of 90-150 nm with a magnetic mass between 70-90% are also expected to be used this invention. Suitable magnetic particles consist of a crystalline core of one of the superparamagnetic materials surrounded by molecules that are bound, for example physically adsorbed or covalently attached to the core and imparting a stable colloidal nature. Preferably, the coating material is applied in an amount to effectively prevent non-specific interactions between the biomacromolecule and the magnetic core found in the sample. Such biomacromolecules may include, for example, carbohydrates, lectins, glycoproteins, and other membrane components that are residual of sialic acid on the surface of non-target cells. In addition, the material should contain as high a magnetic mass as possible per nanometer particle. The size of the magnetic crystal containing the core is small enough to not contain a complete magnetic domain. The size of the nanoparticle is small enough that its Brownian energy exceeds its magnetic moment. Therefore, the mutual attraction/rejection of the colloidal magnetic particles aligned with the north pole, the south pole, and the subsequent occurrence of the colloidal magnetic particles does not appear to occur, even in a moderately strong magnetic field that affects the stability of the solution. Finally, the magnetic particles should be separable in a high magnetic gradient external field separator. This feature facilitates sample processing and provides a better economic advantage than a more complex internal gradient column filled with ferromagnetic beads or steel wool. The magnetic particles having the above properties can be produced by the substrate material modification described in U.S. Patent Nos. 4,795,698, 5,597,531 and 5,698,271.

Immunomagnetic sample preparation is very important to reduce the amount of sample and the terms obtained after ^104-fold concentration of target cells. The reagents used in the preferred kit of the present invention include: an antibody corresponding to the white blood cell antigen CD 45 to recognize white blood cells (non-target cells); a cell type specific or nucleic acid capable of excluding residual red blood cells, platelets and other non-nuclear articles Dye; used as a biospecific reagent or antibody corresponding to the structure of the target cell, or specific for the target membrane, unlike an antibody used to immunomagnetically select cells.

Morphological analysis can be performed on a variety of analytical platforms, including, for example, the CELLSPOTTER system, which is a magnetic cell immobilization and analysis system using a microscopic detector for artificial cell observation, as in U.S. Patent Nos. 5,876,593, 5,985,153 and 6,136,182, respectively. Said. The above-identified U.S. Patent Application is hereby incorporated by reference herein in its entirety in its entirety in its entirety in the the the the the the the the Other analytical platforms include, but are not limited to, laser scanners, Chromavision, and Biometric Imaging.

The kit of the invention preferably comprises or can be used in conjunction with a kit having reagents for performing molecular analysis of the obtained cells (CEC). These include reagents that aid in the determination of the gene expression pattern of the relevant cells, as well as reagents that aid in the determination of the protein substrate of the gene expression, wherein the protein substrate method includes reverse transcription PCR (RT-PCR), competition. RT-PCR, RT-PCR, differential display RT-PCR, Northern blot analysis and other related tests. While it is possible to perform these techniques using individual PCR reactions, it is preferred to amplify the complementary deoxyribonucleic acid (cDNA) or complementary ribonucleotide (cRNA) produced from the message ribonucleotide (mRNA) and then pass through a microarray analysis. Some different array configurations and methods for which production is applicable are known to those skilled in the art and are described in the following U.S. Patent: 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637; Into this article.

Microarray technology is capable of simultaneously measuring the steady state mRNA levels of thousands of genes, thereby demonstrating a powerful tool to identify effects such as the onset, arrest or regulation of uncontrolled cell proliferation. There are two types of microarray technology that are currently widely used. The first is a cDNA array and the second is an oligonucleotide array. Although there are differences in these wafer structures, substantially all downstream data analysis and output systems are identical. The output of these assays is typically a measure of the intensity of the signal received by the labeled probe from the cDNA sequence of the sample, wherein the sample will be mixed at a known position on the microarray. Typically, the signal intensity is proportional to the amount of cDNA and thus the mRNA expressed in the sample cells. There are a large number of such technologies available and helpful. A preferred method for determining the suitability of a gene is found in U.S. Patent No. 6,271,002 to Linsley et al.; 6, 218,122 to Friend et al; 6, 218,114 to Peck et al; and 6,004,755 to Wang et al. Incorporated herein by reference. The ingredients and reagents to which these procedures are applicable may be included in the kit of the present invention.

In a preferred embodiment of the invention, the patient's blood is collected and then isolated from the CEC using immunomagnetic capture techniques, such as in the CellSearch system. The CEC is then subjected to a separate analysis, such as with RT-PCR or DNA microarrays. The gene expression pattern is determined and processed in accordance with pattern recognition, which is an indicative disease diagnosis, prediction or prognosis to be provided to the clinician and/or patient. The expression of a variety of genes or their expression-based gene-based markers, and for example, the presence or absence of a disease, disease process or development, the likelihood of contracting a disease or condition, and other physical conditions of prediction and prognosis, Get relevance. A pattern recognition soft system can be used to perform these correlations and indicate if a correlation exists. Computer instructions include an execution code used to compare the results of the verification and related performance patterns, and to provide a medical assessment or useful information in the provision of a medical assessment, which may be incorporated into the form of a DVD, a flash drive or any other convenient form. Invented in the kit.

Instance

In the following examples, gene expression profiles from 18 endothelial cell samples from 9 anatomical locations were analyzed. A set of 130 gene-based markers that exhibited high expression in endothelial cells but no in PBMC were identified. These markers were also tested for endothelial cells concentrated from the CellSearch system. Gene-based marker systems are readily detected by QRT-PCR in blood samples spiked by endothelial cells. In addition, for different blood vessel types A panel of 67 markers that differ in expression in endothelial cells is identified, wherein these markers can be used to detect the source of CEC to be analyzed.

Example 1: CECs

Frozen HAEC (human aortic endothelial cells), HUVEC (human umbilical vein endothelial cells), HPAEC (human pulmonary artery endothelial cells), HMVEC-ad (human microvascular endothelial cells, adult dermis) and HASMC (human arterial smooth muscle cells) Obtained from Invitrogen (Invitrogen, Carlsbad, Calif.), frozen HCAEC (human coronary endothelial cells), HUAEC (human umbilical artery endothelial cells), HIAEC (human brachial artery endothelial cells), HMVEC-C ( Human cardiac microvascular endothelial cells were obtained from Lonza (Lonza, Cologne, Germany). Frozen HSaVEC (human saphenous vein endothelial cells) was obtained from PromoCell (PromoCell, Heidelberg, Germany). The endothelial cell line was cultured in EGM®-2 endothelial cell growth medium-2 (Lonza) under a 37 ° C incubator, 95% humidity and 5% carbon dioxide. The smooth muscle cell line was cultured with medium 231, supplemented with smooth muscle growth supplement (SMGS) (Invitrogen). Cell clumps of HAEC, HUVEC, HPAEC, HMVEC-ad, HCAEC, HUAEC, HMVEC-C, HSaVEC were purchased from PromoCell. Human blood peripheral white blood cell (PBMC) total RNA was purchased from Clontech (Clontech, Inc., Mountain View, CA).

Blood from healthy donors was aspirated into a vacuum blood collection tube containing EDTA, and then 4 ml was treated with the CellSearch system (Veridex, Raritan, NJ) to separate the CEC. This is the use of the CEC Spectrum Kit (Veridex), according to the manufacturer's guidelines. In the dowel experiment, 500 or 1000 endothelial cells (HAEC, HPAEC, HUVEC, or HMVEC-ad) were infiltrated into the blood of 4 ml healthy donors and then processed with the CellSearch system.

On cultured endothelial cells or cell aggregates, the approximately 5 × 10 ⁵ cell lines studied taking AllPrep DNA / RNA Micro Kit (Qiagen, Hilden, Germany's) on the isolated RNA. In order to separate RNA from the CEC concentrated in the CellSearch system, 350 μl of RLT plus buffer (Qiagen) was added to the cleaved CEC, followed by 4 μl of a concentration of 5 Ng/μl of poly. (I) (Epicentre, Inc., Madison, Wis.) As a vector RNA, DNA and RNA were isolated using the AllPrep DNA/RNA micro-kit according to the manufacturer's guidelines. Two identical dowel samples were pooled for the next analysis. Quantification and characterization of RNA was performed using a NanoDrop 1000 (NanoDrop, Wilmington, Delaware) and an Agilent Bioanalyzer 2100 (Agilent, Inc., Santa Clara, Calif.).

Endothelial RNA, smooth muscle cell RNA, and PBMC RNA samples were converted to the target antisense RNA (cRNA) using single-strand RNA amplification and the Enzo Biotin labeling system. Targets are recommended according to the supplier (Affymetrix, Santa Clara, Calif.) Follow-up protocol to the Affymetrix human U133 Plus 2.0 array. After hybridization, the array was first cleaned and stained using the standard Affymetrix program, scanned using an Affymetrix GeneChip scanner and data extracted using the Expression console.

For EC nails and donated blood samples processed by the CellSearch Spectrum Suite, the RNA was converted to the target gene cDNA using the Ovation RNA Amplification System V2 (NuGEN Corporation, San Carlos, Calif.). Briefly, 50 ng of total RNA was converted to double-stranded cDNA using isozyme amplification using DNA/RNA chimeric primers suitable for reverse transcription. The cDNA was purified using magnetic beads and quantified using spectroscopy. 3.75 micrograms of purified cDNA was subsequently subjected to a two-step fragmentation and labeling procedure using the Encore Biotin module (NuGEN). First, the purified cDNA line is fragmented to produce a single-stranded cDNA product ranging from 50 to 100 bases. Second, the fragmented product is labeled by attaching the biotin-labeled nucleotide to the 3-hydroxyl end of the fragmented cDNA generated in the first step. For hybridization, the hybrid cocktail was prepared using the hybrid, wash and stain kit (Affymetrix) applied to the array and added to the shredded product, followed by incubation at 45 °C for 18 hours. After hybridization, the array was first cleaned and stained using the standard Affymetrix program, scanned using an Affymetrix GeneChip scanner and data extracted using the Expression console.

Gene expression intensity was extracted using the MAS5 algorithm using the Affymetrix Expression console (version 1.1). data analysis Previously, global scaling was first performed to bring the average signal strength of the wafer to a target value of 600. To minimize the level of noise, remove probes that have fewer than two calls in the queue. Therefore, 31K probe sets were retained for subsequent analysis. A source effect was observed between cells taken from cell culture and samples taken from cell pellets. In order to minimize this effect, probes showing significant differences (p < 0.05) between the two ethnic groups were removed. Therefore, 23K probes were obtained. In order to find the marker for the detection of CEC in the blood, the probes with a strength of more than 200 in either of the 2 PBMC samples or 2 PBMC samples were removed, and 3950 probes were retained to accommodate the next selection. .

In the case of hierarchical grouping, the signal intensity of each probe set is regularized to medium; the hierarchical grouping method is performed using Partek Genomics Suites (Partek, St. Louis, Miss., version 6.5). The hierarchical grouping method uses an average ligation method and is performed on both the probe and the sample. Euclidean distance is used to calculate dissimilarity. For the unsupervised hierarchical fractionation method, 23K probes after removal of genes with significant differences between cell culture and cell mass were used. The supervised clustering method was performed using 130 CEC markers or 67 skin, arterial and venous endothelial cell specific markers.

Functional annotations were analyzed by using the Gene Ontology (GO) classification system of DAVID Software (NCIF).

In order to evaluate the genes selected by the microarray analysis, a set of 10 donations processed by CellSearch using the CEC spectrum suite was used. RNA was extracted from the sample and 8 cell strain nail samples (2 HAEC nails, 2 HPAEC nails, 2 HMVEC nails, and 2 HMVEC-ad nail samples). In a 20 microliter reaction, 2 microliters of RNA was used for cDNA synthesis using a high capacity cDNA reverse transcription kit (Applied Biosystems, Inc., Foster, CA). In order to be able to perform multiple gene analyses, 14 microliters of cDNA was used for 14-cycle preamplification using a TaqMan® PreAmp Master (Applied Biosystems) mixing kit in a 20 microliter reaction. The preamplification product was diluted 1:20, and then 5 microliters of the diluted product was used as an input for quantitative PCR. The real-time PCR system was performed on an ABI PRISM 7900HT Sequence Detector (Applied Biosystems) using the Applied Biosystems gene expression Taqman assay.

Example 2: Gene Expression Analysis

RNAs obtained from 18 endothelial cell samples, 2 PBMC samples, and 2 smooth muscle cell samples were analyzed by microarray using an Affymetrix human U133 Plus 2.0 array, which contained more than 54,000 probe sets covering 38,500 grants Good characteristics of 47,000 transcriptions and variants of human genes. The endothelial cell sample group represents 9 different anatomical sites, including 5 different blood vessels (aorta, coronary artery, pulmonary artery, radial artery, umbilical artery), 2 different veins (umbilical vein and saphenous vein), and 2 different tissues ( Skin, heart). In addition to the brachial artery and skin, including one from the cultured cells Two samples taken from frozen cell pellets were obtained from various sources. For the brachial artery and skin, 2 samples taken from the same cultured cells were used as a technical copy. In order to obtain a profile of gene expression patterns, an unsupervised hierarchical analysis of gene array data was performed using 31K probe sets showing the presence of calls in at least 2 samples. Preliminary analysis of endothelial cell populations showed that samples taken from cell culture and samples taken from cell pellets may form different ethnic groups due to differences in sample types. To eliminate this difference, a T test was performed between the two sample groups. The probe set with a P value <0.05 was eliminated and the result was 23K probe sets. Unsupervised analysis on these 23K probe sets yielded three major populations, the PBMC sample population, the smooth muscle cell sample population, and the endothelial cell sample population, reflecting endothelial cells compared to PBMC and smooth muscle cells, overall. similar. Within the endothelial cell population, the sample taken from the cell mass and taken from the cell culture was not isolated, indicating that the source effect was eliminated. However, samples taken from the same anatomical location are not always clustered together, probably due to relatively small sample sizes and different conditions for cell culture.

Example 3: The CellSearch system was applied to CEC concentration.

The CellSearch system is used to separate CEC from the above donated samples. An anti-system corresponding to the most prominent endothelial cell membrane antigen CD146 was used for the immunological extraction reagent. However, after the concentration process, there are still about 1000 to 5000 white blood cells remaining in the concentrated CEC population. In order to identify potential CEC-specific markers, the extent of expression of a specific marker in endothelial cells must be substantially higher than that in PBMC. Therefore, one of the two PBMC samples was removed, and one of the probes or one of the two PBMC samples with a strength of more than 200 was present. 3950 probes were retained for further analysis. Probes with a minimum intensity below 1000 in 18 samples were not considered for subsequent analysis. Therefore, 130 probe sets (106 unique genes) were selected as candidates for CEC markers. Functional annotation and path analysis were performed on these 130 probe sets using the DAVID functional annotation software. The GO (Gene Onlogy) terminology and the KEGG (Kyoto Gene and Genomic Encyclopedia) path with significant over-representation are shown in Table 1. Top biological processes that are overrepresented in the 130 probe sets, including genes involved in cell motility regulation (n=11), cell migration regulation (n=10), and vascular development (n=11). The over-represented population of cellular components includes the plasma membrane (n=44) and the concentrated attachment point (n=6). This overrepresented molecular functional population includes transmembrane receptor protein tyrosine kinase activity (n=7) and protein tyrosine kinase activity (n=8). The major pathways associated with CEC-specific genes are concentrated attachment points (n=12) and ECM receptor interactions (n=5). One of the functions of endothelial cells is angiogenesis, during which significant changes occur in the attachment interaction between cells and the extracellular matrix (ECM), causing endothelial cells to migrate.

Example 3: Gene-based markers

The specific gene line of CEC was identified according to Example 2. These include: integration of related genes, integration of α2 ( I TGA2 ), AXL receptor tyrosine kinase ( AXL ), EPH receptor A2 ( EPHA2 ), TEK tyrosine kinase, endothelial ( TEK ), proto-oncogene ( MET ) , neuropilin 1 ( NRP1 ), VEGFR2 ( KDR ) and TIE1 ; angiogenesis-related genes, activin A receptor type II 1 ( ACVRL1 ), connective tissue growth factor ( CTGF ), endothelial cell-specific chemotactic regulator ( ECSCR ), endothelin 1 ( EDN1 ) and convoluted homolog 4 ( ROBO4 ); genes CAV1, CAV2, COL4A1, COL5A2, CCND1, FLNB, ITGA2, KDR, LAMA4, which maintain vascular integrity through cell-cell interaction LAMB1, PARVA, MET; and independent MCAM (CD146), KDR (VEGFR-2), TEK (Tie-2). However, some genes associated with endothelial cells are not currently labeled in the context because of their high expression in PBMC, such as PECAM (CD31), CXCR4 , or because they are under-performing in one or more endothelial cell lines (eg Diagnostic assistance is not seen in KIT (SCF R/c-group) and SELE (E-selectin). The CEC markers identified in these examples are overrepresenting biological processes or pathways associated with endothelial cell function.

Example 4: Source organization.

3950 genes with no expression or intensity less than 200 were shown in 2 PBMC samples, which were used to identify genes based on genes used to identify CEC-derived tissues. These median signal intensities in endothelial cells obtained from arteries exceed 500, and those greater than the largest manifestations in endothelial cells obtained from other sources are recognized as arterial endothelial cell-specific genes. Similarly, in order to identify venous endothelial cell-specific genes, the median signal intensity in endothelial cells obtained from veins was identified to be more than 500, and the genes larger than the maximum expression in endothelial cells obtained from other sources. A total of 38 arterial endothelial cell-specific genes and 14 venous endothelial cell-specific genes met these criteria (see Table 3). A representative arterial-specific gene is the previously reported arterial endothelial cell-specific gene, HEY2 , which is a member of the Hairy-associated transcription factor family that has been implicated in the embryonic development of mouse embryos. Other arterial-specific CEC genes include CXADR ; it is a tightly joined component and has been reported to be asymmetrical in the heart, with manifestations occurring in the subendothelial layer of the vessel wall but not on the surface of the lumen endothelial cells. SOX17 , an HMG-box transcription factor, has been shown to play an important role in both endoderm formation and cardiovascular development, and its promoter activity has been shown in arterial vascular endothelial cells in the cardiovascular system of mouse models. But does not appear in the veins. In order to initiate detection of tissue-specific markers that are particularly suitable for use with immunomagnetic platforms, such as the CellSearch system, the degree of performance of selected markers should be significantly higher than in PBMC. For skin endothelial cell-specific gene recognition, the lowest performance in endothelial cells obtained from the skin was selected, which is five times higher than the highest performance of all other endothelial cell samples. There are 15 such genes.

The supervised clustering method used 67 source-specific genes and performed on cell line microarray data. The 67 genes are correctly assembled from different There is only one exception to the source of endothelial cells (an arterial endothelial cell is clustered with venous endothelial cells). Cross-validation of genes was also performed.

Example 5: Labeling validation using endothelial cell dowels.

The endothelial cell system was nailed to healthy donor blood, and then the CEC system was condensed using the CellSearch system with a CEC spectrum kit. Each 4 ml of healthy donated blood line was nailed together with 500 or 1000 cultured endothelial cells obtained from a single source. Four selected cultured endothelial cell samples were examined, including two obtained from arteries (HAEC and HPAEC), one obtained from vein (HUVEC), and one obtained from skin (HMVEC-ad). Ten healthy donated samples of concentrated CEC and sputum-free nails obtained from dowels were subjected to RNA detachment and microarray analysis. The CEC values obtained from 10 donors were determined to be 2 to 107 in 4 ml of blood. Among the 130 CEC-specific markers obtained from the microarray data of the cell line gene expression profiles, the average performance in the endothelial cell nail samples was higher than that in the donor samples, and in the donor samples in the endothelial cell nail samples. The average performance ratio is between 2 and 655, and the proportion of 93 genes exceeds 20. Using QRT-PCR, 21 markers with a ratio between donated samples ranging from 8 to 654 were validated in the nail samples. The PCR results showed that all the selected markers were good between the nail samples and the non-nail nail samples. seperate effect. To verify arterial, venous, and skin-specific markers, principal component analysis was performed on the nail microarray data using 67 source-specific markers. Donated samples and arterial, venous, and skin endothelial cell nail samples The lines are clearly separated in the PCA. This marker is therefore suitable for distinguishing CECs obtained from different sources.

Example 6: Correlation between disease and CEC analysis (predicted)

Whole blood obtained from the patient was placed directly into a test tube containing CellSave preservative. This step was performed according to the same collected blood protocol suitable for circulating tumor cell (CTC) analysis using the CellSave system. The CEC is condensed with the CEC Cell Capture Kit via the CellSearch system. In this system, immunomagnetic concentration is first performed with the AUTOPREP separation system to produce a concentrated fraction. The kit used contained CD146 magnetic fluid and reagent for cell staining after concentration with the nucleic acid dye DAPI, endoglin (CD105)-PE and pan-white blood cell marker CD45APC. The sample was reduced to approximately 300 microliters and placed in an analysis chamber fixed to a magnetic "bunker" corresponding to the inner side of the magnetic monolayer. The CEC count and morphological analysis are then performed.

The CEC is then extracted from the magnetic masking device. Total RNA was extracted using the RNeasy micro-kit (Qiagen, Hilden, Germany). The RNA was converted to cDNA as follows: First, the total amount of extracted RNA was pre-incubated with a 300 ng random hexamer for 5 minutes at 65 °C. Then 200 U M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant, M-MLV Reverse Transcriptase 1x Reaction Buffer, 10 U RNasin® Plus RNase Inhibitor (all purchased from Promega, Madison, Wis.), 50 nm molar mixture of dATP, dTTP, dCTP and dGTP (Amersham Biosciences, Freiburg, Germany) and water added to 20 Microliters within the final reaction volume. The pre-incubation step was carried out at 20 ° C for 10 minutes and then at 55 ° C for 50 minutes. Finally, after heating to 94 ° C for 5 minutes, the reaction was stopped.

Quantitative reverse transcription PCR (qRTPCR) was then performed as follows. Gene performance analysis was repeated using a separate TaqMan® pre-developed assay reagent designated by the arterial marker in Table 3. In each case, two unlabeled PCR primers and one FAMTM dye used in conjunction with the above system were labeled with a TaqMan® MGB probe. The total reaction volume was 14 |xl, containing 7 μl of 2-fold TaqMan® Universal PCR Master (general PCR core reagent), 0.7 μl of TaqMan® pre-developed assay reagent, and 4×xl 5-fold diluted cDNA template. PCR amplification was performed using the AB 7900HT Fast Real-Time PCR System, which was initially incubated at 50 °C for 2 minutes, then at 95 °C for 10 minutes, followed by 50 cycles at 95 °C for 15 seconds, then at 60 °C. 1 minute and other steps. Data analysis was performed using the AB7900 sequence detection software version 2.2.2 with automatic baseline correction and cycle threshold settings. The generated cycle threshold (Ct) data is remitted for further analysis. Consumables, equipment, and soft systems were purchased from Applied Biosystems, Inc., Foster City, California.

Some arterial CEC-derived tissue markers show significant overexpression. The data is downloaded to a file used by the program, which compares the performance pattern to various performance patterns performed by matching CEC marker performance to known clinical outcomes. The program is included on the storage device, and the storage device also contains executable code that is more suitable for use with statistical algorithms. This pattern suggests that the CEC is essentially an artery, more specifically the aorta. Together with other diagnostic messages, it can be concluded that if left untreated, the patient is likely to have a thoracic aortic aneurysm.

>gi|86787911|ref|NM_004105.3|human fibulin-like extracellular matrix protein 1 (EFEMP1), transcription Variant 1, message ribonucleic acid

>gi|13375818|ref|NM_024609.1|Human Nestin (NES), Message RNA

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>ENA|U67280|U67280.1 Human calcium cavity protein message ribonucleic acid, complete coding sequence.

>ENA|AB032261|AB032261.1 Human sterol supplement? A desaturated? Scd message ribonucleic acid, complete coding sequence.

>refseq|NM_001554|NM_001554 Human-rich cysteine, angiogenesis-inducing factor, 61 (CYR61), message Ribonucleic acid.

>refseq|NM_001423|NM_001423 Human epithelial membrane protein 1 (EMP1), message ribonucleic acid.

>refseq|NM_001387|NM_001387 Human dihydropyrimidine? Sample 3 (DPYSL3), transcript variant 2, message ribonucleic acid.

>refseq|NM_001839|NM_001839 Human Calcine Protein 3, Acidic (CNN3), Message RNA.

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>refseq|NM_015577|NM_015577 Human retinoic acid induced 14 (RAI14), transcript variant 1, message ribonucleic acid.

>refseq|NM_015472|NM_015472 Human WW-containing transcriptional regulator 1 (WWTR1), transcript variant 1, message ribose Nucleic acid.

>refseq|NM_006169|NM_006169 Human Nicotinamide Nitrogen Methyl Transfer? (NNMT), message ribonucleic acid.

>refseq|NM_006169|NM_006169 Human Nicotinamide Nitrogen Methyl Transfer? (NNMT), message ribonucleic acid.

>ENA|AI754404|AI754404.1 cr24g06.x1 Human bone marrow stromal cells human complementary DNA clone HBMSC_cr24g06 3', message ribonucleic acid sequence.

>refseq|NM_000935|NM_000935 Human procollagen lysine, 2-oxoglutarate 5-diox? 2 (PLOD2), transcriptional change Body 2, message ribonucleic acid.

>refseq|NM_000602|NM_000602 Human serpin wins? ? Inhibitory factor, branch E (nexin, first type of fibrin egg White soluble? Pro-activator inhibitor), member 1 (SERPINE1), transcript variant 1, message ribonucleic acid.

>refseq|NM_021913|NM_021913 Human AXL receptor tyrosine kinase (AXL), transcript variant 1, message ribonucleic acid.

>refseq|NM_004199|NM_004199 Human amidoxime-4-hydroxylation? , α more? II (P4HA2), transcript variant 1, message Ribonucleic acid.

>refseq|NM_000138|NM_000138 Human Fibrillin 1 (FBN1), message ribonucleic acid.

>refseq|NM_014899|NM_014899 Human Rho-related BTB area containing 3 (RHOBTB3), message ribose Nucleic acid.

>refseq|NM_002318|NM_002318 Human lysyl oxidase-like 2 (LOXL2), message ribonucleic acid.

>refseq|NM_016201|NM_016201 Human angiomotin like 2 (AMOTL2), message ribonucleic acid.

>ENA|BF197655|BF197655.1 7m95b04.x1 NCI_CGAP_Brn23 Human Complementary Deoxyribonucleic Acid Like: 3562710 3', message ribonucleic acid sequence.

>refseq|NM_001233|NM_001233 Human caveolin 2 (CAV2), transcript variant 1, message ribonucleic acid.

>refseq|NM_004431|NM_004431 Human EPH receptor A2 (EPHA2), message ribonucleic acid.

>ENA|BG170541|BG170541.1 602322942F1 NIH_MGC_89 Human Complementary Deoxyribonucleic Acid Clone Image: 4425947 5', message ribonucleic acid sequence.

>refseq|NM_007034|NM_007034 Human DnaJ (Hsp40) homolog, subfamily B, member 4 (DNAJB4), message core Sugar nucleic acid.

>refseq|NM_002253|NM_002253 Humans? Insertion region receptor (a third type of receptor tyrosine stimulator?) (KDR), Message RNA.

>refseq|NM_014890|NM_014890 Human silk protein A binding protein 1 (FILIP1L), transcript variant 2, message ribose Nucleic acid.

>refseq|NM_002067|NM_002067 Human guanine nucleic acid binding protein (G protein), α11 (Gq class) (GNA11), message ribonucleic acid.

>refseq|NM_003213|NM_003213 Human TEA region family member 4 (TEAD4), transcript variant 1, message ribonucleo acid.

>ENA|AL514445|AL514445.3 Human neuroblastoma CL0BB010ZF08 cloned human full-length complementary deoxyribose Nucleic acid 3' end (human)

>refseq|NM_005613|NM_005613 Human G-protein signal delivery 4 regulatory factor (RGS4), transcript variant 2, message core Sugar nucleic acid.

>ENA|BC000737|BC000737.1 Human G-protein signal delivery 4 regulatory factor, message ribonucleic acid (complementary deoxyribose Nucleic acid clone MGC: 2124 image: 3510260), complete coding sequence.

>refseq|NM_005424|NM_005424 Human tyrosine with immunoglobulin-like region and EGF-like region 1 (TIE1) Excited? , message ribonucleic acid.

>ENA|BE962749|BE962749.2 601656143R1 NIH_MGC_66 Human Complementary Deoxyribonucleic Acid Clone Image: 3855754 3', message ribonucleic acid sequence.

>refseq|NM_012242|NM_012242 Human Dickkopf homolog 1 (Xenopus laevis) (DKK1), message core Sugar nucleic acid.

>refseq|NM_001424|NM_001424 Human epithelial membrane protein 2 (EMP2), message ribonucleic acid.

>ENA|U29586|U29586.1 Human beta-myosin dystrophin-associated glycoprotein message ribonucleic acid, complete Coding sequence.

>refseq|NM_015976|NM_015976 Human Sorting Protein 7 (SNX7), Transcriptional Variant 1, Message RNA.

>refseq|NM_000950|NM_000950 Human rich in proline Gla (G-carboxy glutamic acid) 1 (PRRG1), transcript variant 1 , message ribonucleic acid.

>refseq|NM_005795|NM_005795 Human calcitonin receptor-like (CALCRL), message ribonucleic acid.

>refseq|NM_000459|NM_000459 Human TEK tyrosine acid stimulation? , endothelium (TEK), message ribonucleic acid.

>refseq|NM_003662|NM_003662 Human pirin (iron ion binding nucleoprotein) (PIR), transcript variant 1, message nucleus Sugar nucleic acid.

>refseq|NM_001235|NM_001235 Human serine protein? Inhibitor wins? ? Inhibitory factor, branch H (heat shock protein) 47), member 1, (collagen binding protein 1) (SERPINH1), transcript variant 2, message ribonucleic acid.

>refseq|NM_003483|NM_003483 human high mobility group AT-hook 2 (HMGA2), transcript variant 1, message core Sugar nucleic acid.

>ENA|AV712733|AV712733.1 Human Complementary Deoxyribonucleic Acid Clone: DCAACE01, 5' End, Expressed in Human Tree In the cell.

>ENA|M73554|M73554.1 Human bcl-1 message ribonucleic acid, complete coding sequence.

>ENA|BC004295|BC004295.1 Human, clone image: 3622356, message ribonucleic acid, partial coding sequence.

>ENA|AF312393|AF312393.1 Human leucine zipper protein FKSG13 (FKSG13) message ribonucleic acid, complete coding sequence.

>ENA|AL078459|AL078459.8 Human DNA sequence from RP4-621F18 clone on chromosome 1p11.4-21.3, package Contains a part of the novel gene, and the hydrolysis of dimethyl arginine dimethylamine? 1 DDAH1 gene 3' end.

>ENA|M92934|M92934.1 Human connective tissue growth factor message ribonucleic acid, complete coding sequence.

>ENA|U84895|U84895.1 Human transmembrane 4 superfamily A15 homolog message ribonucleic acid, complete coding sequence.

>ENA|AL037401|AL037401.1 Human Message RNA; EST DKFZp564K1671_s1 (from clone DKFZp564K1671)

>ENA|M90657|M90657.1 Human tumor antigen (L6) message ribonucleic acid, complete coding sequence.

>ENA|D84109|D84109.1 Human RBP-MS/Group III message ribonucleic acid, complete coding sequence.

>ENA|D84109|D84109.1 Human RBP-MS/Group III message ribonucleic acid, complete coding sequence.

>ENA|J03225|J03225.1 Human Lipoprotein Associated Condensation Inhibitor Information RNA, Complete Coding Sequence

>ENA|BC001258|BC001258.1 Human Glucose Phosphorylation? 3, message ribonucleic acid (complementary DNA gram Long MGC: 5002 image: 3451677), complete coding sequence.

>ENA|BC004241|BC004241.1 Human laminin, α4, message ribonucleic acid (complementary DNA clone) Image: 3623597), complete coding sequence.

>ENA|AF026219|AF026219.1 Human HP protein (HP) message ribonucleic acid, complete coding sequence.

>ENA|AF003114|AF003114.1 Human CYR61 message ribonucleic acid, complete coding sequence.

>ENA|U17473|U17473.1 Human calcitonin-like receptor message ribonucleic acid, complete coding sequence.

>ENA|BC004908|BC004908.1 Human Complementary Deoxyribonucleic Acid Clone Image: 3530459, **** Warning: Chimeric clone****.

>ENA|M28882|M28882.1 Human MUC18 glycoprotein message ribonucleic acid, complete coding sequence.

>ENA|BC003096|BC003096.1 Human PDZ and LIM Region 4, Message Ribonucleic Acid (Complementary DNA Cloning MGC: 1645 image: 3501794), complete coding sequence.

>ENA|M20206|M20206.1 Human laminin B1 message ribonucleic acid, complete coding sequence.

>ENA|AI922605|AI922605.1 wm90c05.x1 NCI_CGAP_Ut2 Human Complementary Deoxyribonucleic Acid Clone Image: 2443208 3', message ribonucleic acid sequence.

>ENA|AI695017|AI695017.1 we45d07.x1 NCI_CGAP_Lu24 Human Complementary Deoxyribonucleic Acid Like: 2340445 3', message ribonucleic acid sequence.

>ENA|BE552421|BE552421.1 hw26b02.x1 NCI_CGAP_Kid11 Human Complementary Deoxyribonucleic Acid Like: 3184011 3' Similar to TR: 094872 094872 KIAA0774 protein, message ribonucleic acid sequence.

>ENA|AU147399|AU147399.1 Human complementary DNA clone: MAMMA1000563, 3' end, expressed in the mammary gland.

>ENA|N95026|N95026.1 zb45d12.s1 Soares_fetal_lung_NbHL19W Human Complementary Deoxyribonucleic Acid Clone image: 306551 3' similar to the element MSR1 repeat element, message ribonucleic acid sequence.

>ENA|BE620457|BE620457.1 601483690F1 NIH_MGC_69 Human Complementary Deoxyribonucleic Acid Clone Image: 3886055 5', message ribonucleic acid sequence.

>ENA|BF115739|BF115739.1 7n64b08.x1 NCI_CGAP_Lu24 Human Complementary Deoxyribonucleic Acid Like: 3569246 3', message ribonucleic acid sequence.

>ENA|AI935123|AI935123.1 wp13h09.x1 NCI_CGAP_Kid12 Human Complementary Deoxyribonucleic Acid Like: 2464769 3', message ribonucleic acid sequence.

>ENA|AI088622|AI088622.1 qb14f06.x1 Soares_pregnant_uterus_NbHPU Human Complementary Deoxygen Glyconucleotide clone image: 1696259 3' similar gb: X06409 RAF proto-oncogene serine/threonine-protein activation? (people Class); contains PTR5.t3 TAR1 repetitive element, message ribonucleic acid sequence.

>ENA|AA917899|AA917899.1 o176e05.s1 NCI_CGAP_Kid3 Human Complementary Deoxyribonucleic Acid Like: 1535552 3', message ribonucleic acid sequence.

>ENA|AW149379|AW149379.1 xf36d11.x1 NCI_CGAP_Brn50 Human Complementary Deoxyribonucleic Acid Like: 2620149 3' Similar to TR: 043251 043251 Unknown function 39.5 KD protein, message ribonucleic acid sequence.

>ENA|AW129021|AW129021.1 xe93h03.x1 NCI_CGAP_Brn35 Human Complementary Deoxyribonucleic Acid Like: 2615477 3' Similar to TR: 095034 095034 WUGSC: H_RG041D11.1 protein, message ribonucleic acid sequence.

>refseq|NM_018212|NM_018212 Human-enhanced homolog (Drosophila) (ENAH), transcript variant 2, message ribonucleo acid.

>refseq|NM_018222|NM_018222 Human parvin, alpha (PARVA), message ribonucleic acid.

>refseq|NM_012193|NM_012193 Human Curl Family Receptor 4 (FZD4), message ribonucleic acid.

>refseq|NM_017734|NM_017734 Human palmdelphin (PALMD), message ribonucleic acid.

>refseq|NM_001955|NM_001955 Human Endothelin 1 (EDN1), Transcriptional Variant 1, Message RNA.

>refseq|NM_014344|NM_014344 Human four junction box 1 (Drosophila) (FJX1), message ribonucleic acid.

>refseq|NM_017805|NM_017805 Human Ras Binding Protein 1 (RASIP1), message ribonucleic acid.

>refseq|NM_000393|NM_000393 Human collagen, fifth class, α2 (COL5A2), message ribonucleic acid.

>ENA|AK025108|AK025108.1 Human Complementary Deoxyribonucleic Acid: FLJ21455 fis, clone COL04696.

>ENA|BG107577|BG107577.1 602277726F1 NIH_MGC_86 Human Complementary Deoxyribonucleic Acid Clone Image: 4365271 5', message ribonucleic acid sequence.

>ENA|AF322916|AF322916.1 Human uveal autoantigen message ribonucleic acid, complete coding sequence.

>ENA|AF278532|AF278532.1 Human beta-conductin message ribonucleic acid, complete coding sequence.

>ENA|AY009951|AY009951.1 Human thorn? Factor interacting protein message ribonucleic acid, complete coding sequence.

>ENA|AA524250|AA524250.1 ng34a11.s1 NCI_CGAP_Co3 Human Complementary Deoxyribonucleic Acid Clone Image: 936668 3', message ribonucleic acid sequence.

>ENA|BF247906|BF247906.1 601858274F1 NIH_MGC_58 Human Complementary Deoxyribonucleic Acid Clone Image: 4068810 5', message ribonucleic acid sequence.

>ENA|BG285417|BG285417.1 602409786F1 NIH_MGC_91 Human Complementary Deoxyribonucleic Acid Clone Image: 4539428 5', message ribonucleic acid sequence.

>ENA|BF000162|BF000162.1 7h18f09.x1 NCI_CGAP_Co16 Human Complementary Deoxyribonucleic Acid Like: 3316361 3', message ribonucleic acid sequence.

>ENA|N30138|N30138.1 yx81c07.s1 Soares melanocyte 2NbHM human complementary DNA clone Like: 268140 3', message ribonucleic acid sequence.

>ENA|AL040051|AL040051.2 Human Message RNA; EST DKFZp434P1112_s1 (from clone DKFZp434P1112)

>ENA|AA156022|AA156022.1 zo48c05.s1 Stratagene endothelial cells 937223 human complementary deoxyribose nucleus Acid clone image: 590120 3', message ribonucleic acid sequence.

>ENA|AA554833|AA554833.1 ni34g07.s1 NCI_CGAP_Lu1 Human Complementary Deoxyribonucleic Acid Clone Image: 978780 3', message ribonucleic acid sequence.

>ENA|BG290908|BG290908.1 602387068F1 NIH_MGC_93 Human Complementary Deoxyribonucleic Acid Clone Image: 4515905 5', message ribonucleic acid sequence.

>ENA|AW193693|AW193693.1 xm29d12.x1 NCI_CGAP_GC6 Human Complementary Deoxyribonucleic Acid Clone Image: 2685623 3', message ribonucleic acid sequence.

>ENA|T63524|T63524.1 yc07b04.s1 Stratagene Lung (#937210) Human Complementary Deoxyribonucleic Acid Clone Image: 79951 3', message ribonucleic acid sequence.

>ENA|N95414|N95414.1 zb66d10.s1 Soares_fetal_lung_NbHL19W Human Complementary Deoxyribonucleic Acid Clone image: 308563 3', message ribonucleic acid sequence.

>ENA|BF511276|BF511276.1 UI-H-BI4-aoj-d-05-0-UI.s1 NCI_CGAP_Sub8 Human Complementary Deoxygen Glyconucleotide clone image: 3085089 3', message ribonucleic acid sequence.

>ENA|AL571557|AL571557.3 Human placenta COT 25 normalized CS0DI024YP08 clone human full complement complement 3' end of ribonucleic acid (human)

>ENA|AA173223|AA173223.1 zp31b04.s1 Stratagene Neuroepithelial (#937231) Human Complementary Deoxygen Glyconucleotide clone image: 611023 3 similarly contains Alu repetitive elements;, message ribonucleic acid sequence.

>ENA|AI623211|AI623211.1 ts78e11.x1 NCI_CGAP_GC6 Human Complementary Deoxyribonucleic Acid Clone Image 2237420 3' Similar to gb: M33552 lymphocyte-specific protein LSP1 (human), message ribonucleic acid sequence.

>ENA|AI807004|AI807004.1 wf37a06.x1 Soares_NFL_T_GBC_S1 Human Complementary Deoxyribonucleic Acid Image: 2357746 3', message ribonucleic acid sequence.

>ENA|AA181256|AA181256.1 zp58c07.s1 Stratagene endothelial cells 937223 human complementary deoxyribose nucleus Acid clone image: 624396 3', message ribonucleic acid sequence.

>ENA|AI653117|AI653117.1 wb43b10.x1 NCI_CGAP_GC6 Human Complementary Deoxyribonucleic Acid Clone Image: 2308411 3', message ribonucleic acid sequence.

>ENA|BE566894|BE566894.1 601341011F1 NIH_MGC_53 Human Complementary Deoxyribonucleic Acid Clone Image: 3683306 5', message ribonucleic acid sequence.

>ENA|AI670852|AI670852.1 wa06a05.x1 NCI_CGAP_Kid11 Human Complementary Deoxyribonucleic Acid Like: 2297264 3', message ribonucleic acid sequence.

>ENA|AL035086|AL035086.12 Human DNA sequence from RP1-44A20 clone on chromosome 6q23.1-24.3, Contains a novel protein similar to the C1-tetrahydrofolate complex (comprising DKFZP586G1517 protein and FLJ21145, containing tetrahydrofolate Dehydrogenation? / cyclization hydrolysis? Regional) partial genes, some novel genes (including FLJ31738 and KIAA1209) and a novel pseudo-base Because it is similar to a photoprotein-like protein. Contains CpG islands.

>ENA|AI657069|AI657069.1 tt55g01.x1 NCI_CGAP_GC6 Human Complementary Deoxyribonucleic Acid Clone Image: 2244720 3' Similar to SW: KLP1_CHLRE P46870 kinetin-like protein KLP1, message ribonucleic acid sequence.

>ENA|AL135787|AL135787.13 Human DNA sequence from RP11-16L21 clone on chromosome 9, including KIAA1962 The protein is similar to the 3' end of the zinc finger protein HIT-10 gene change, and the leukotriene B412-hydroxy dehydrogenation? (MGC34943) LTB4DH base Gene, KIAA0563 related gene (LOC286331) gene, guanine nucleic acid binding protein (G protein) GNG10 gene, γ 10. Novel gene, chromosome 3 open reading frame 84 of the 3' end of the C90rf84 gene and six CpG islands.

>ENA|BG540685|BG540685.1 602570561F1 NIH_MGC_77 Human Complementary Deoxyribonucleic Acid Clone Image: 4694894 5', message ribonucleic acid sequence.

>ENA|AW960748|AW960748.1 EST372819 MAGE Rearrangement Sequence, MAGF Human Complementary Deoxyribonucleic Acid, The ribonucleic acid sequence.

>ENA|AI583530|AI583530.1 ts12c08.x1 NCI_CGAP_Pan1 Human Complementary Deoxyribonucleic Acid Like: 2228366 3', message ribonucleic acid sequence.

>ENA|BF792126|BF792126.1 602252573F1 NIH_MGC_84 Human Complementary Deoxyribonucleic Acid Clone Image: 4344858 5', message ribonucleic acid sequence.

>ENA|AW014647|AW014647.1 UI-H-BI0p-abd-b-12-0-UI.s1 NCI_CGAP_Sub2 Human Complementary Deoxygenation Ribonucleic acid cloning image: 2711375 3', message ribonucleic acid sequence.

>ENA|AW444502|AW444502.1 UI-H-BI3-akb-g-03-0-UI.s1 NCI_CGAP_Sub5 Human Complementary Deoxygen Glyconucleotide clone image: 2733869 3', message ribonucleic acid sequence.

>ENA|BE218803|BE218803.1 hv44h08.x1 NCI_CGAP_Lu24 Human Complementary Deoxyribonucleic Acid Like: 3176319 3' Similar SW: RTC0_ human P17081 GTP binding protein TC10, message ribonucleic acid sequence.

>ENA|BE218803|BE218803.1 hv44h08.x1 NCI_CGAP_Lu24 Human Complementary Deoxyribonucleic Acid Like: 3176319 3' Similar SW: RTC0_ human P17081 GTP binding protein TC10, message ribonucleic acid sequence.

>ENA|AI628689|AI628689.1 ty43a06.x1 NCI_CGAP_Ut2 Human Complementary Deoxyribonucleic Acid Clone Image: 2281810 3' similarly contains Alu repetitive elements; contains element MER22 repetitive elements, message ribonucleic acid sequence.

>ENA|W72516|W72516.1 zd64g05.s1 Soares_fetal_heart_NbHH19W Human Complementary Deoxyribonucleic Acid Clone image: 345464 3', message ribonucleic acid sequence.

>refseq|NM_001387|NM_001387 Human dihydropyrimidine? Sample 3 (DPYSL3), transcript variant 2, message ribonucleic acid.

>refseq|NM_000943|NM_000943 Human amidinoisomer? C (cyclophilin C) (PPIC), message ribonucleoside acid.

>refseq|NM_002841|NM_002841 Human protein tyrosine dephosphorylation? , receptor type, G (PTPRG), message ribose Nucleic acid.

>refseq|NM_013409|NM_013409 Human follicle inhibitor (FST), transcript variant FST344, message ribonucleic acid.

>refseq|NM_006207|NM_006207 Human platelet-derived growth factor receptor-like (PDGFRL), message ribonucleic acid.

>refseq|NM_004791|NM_004791 human insert, beta-like 1 (with EGF-like repeat region) (ITGBL1), message core Sugar nucleic acid.

>refseq|NM_004186|NM_004186 human sema region, immunoglobulin region (Ig), short alkaline region, secretory Type, (semaphorin) 3F (SEMA3F), message ribonucleic acid.

>ENA|U38276|U38276.1 Human brain signaling protein III family homologous message ribonucleic acid, complete coding sequence.

>ENA|AF055585|AF055585.1 Human neuronal extracellular fissure protein Slit2 message ribonucleic acid, complete coding sequence.

>ENA|AF056085|AF056085.1 Human GABA-B receptor message ribonucleic acid, complete coding sequence.

>ENA|AF095784|AF095784.1 Human GABA-B receptor R2 (GABBR2) message ribonucleic acid, complete coding sequence.

>ENA|AL359052|AL359052.1 Human message ribonucleic acid full-length insertion complementary DNA clone EUROIMAGE 1968422.

>ENA|AF095723|AF095723.nu11 The public inspection was stopped on February 15, 2002.

N

>refseq|NM_012193|NM_012193 Human Curl Family Receptor 4 (FZD4), message ribonucleic acid.

>refseq|NM_021939|NM_021939 Human FK506 binding protein 10,65 kDa (FKBP10), message ribonucleic acid.

>refseq|NM_012259|NM_012259 is associated with YRPW motif 2 (HEY2) human hair/split enhancer, message ribonucleo acid.

>refseq|NM_022454|NM_022454 Human SRY (Gender Sensing Zone Y) - Box 17 (SOX17), Message RNA.

>ENA|AK023795|AK023795.1 Human Complementary Deoxyribonucleic Acid FLJ13733 fis, clone PLACE3000147, Highly similar to the first type of coagulation? Sensitive protein ADAMTS1 motif (ADAMTS1) human metalloprotein? Message RNA.

>ENA|AF060152|AF060152.1 Human METH1 protein (METH1) message ribonucleic acid, complete coding sequence.

>ENA|AF232238|AF232238.1 Human HES-associated repressor protein 1 HERP1 message ribonucleic acid, complete coding sequence.

>ENA|AI049973|AI049973.1 an38g03.x1 Gessler Wilms tumor human complementary DNA clone Image: 1700980 3', message ribonucleic acid sequence.

>ENA|BG260087|BG260087.1 602371673F1 NIH_MGC_93 Human Complementary Deoxyribonucleic Acid Clone Image: 4479642 5', message ribonucleic acid sequence.

>ENA|BF438173|BF438173.1 7q68a03.x1 NCI_CGAP_Lu24 Human Complementary Deoxyribonucleic Acid Like: 3703205 3', message ribonucleic acid sequence.

>ENA|H16409|H16409.1 ym22h04.s1 Soares baby brain 1NIB human complementary DNA clone Like: 49055 3', message ribonucleic acid sequence.

>ENA|BF338870|BF338870.1 602036126F1 NCI_CGAP_Brn64 Human Complementary Deoxyribonucleic Acid Like: 4183999 5', message ribonucleic acid sequence.

>ENA|AI742043|AI742043.1 wg38c09.x1 Soares_NSF_F8_9W_OT_PA_P_S1 Human Complementary Deoxygen Glyconucleotide clone image: 2367376 3', message ribonucleic acid sequence.

>ENA|BE644809|BE644809.1 7e57e09.x1 Soares_NSF_F8_9W_OT_PA_P_S1 Human Complementary Deoxygen Glyconucleotide clone image: 3286600 3' similar to the element MER37 repeat element, message ribonucleic acid sequence.

>ENA|AI963304|AI963304.1 wt61d01.x1 NCI_CGAP_Pan1 Human Complementary Deoxyribonucleic Acid Like: 2511937 3' Similar to gb: J03464 PROCOLLAGEN A2 (I) chain precursor (human), message ribonucleic acid sequence.

>ENA|AW006182|AW006182.1 wz93c05.x1 NCI_CGAP_Brn25 Human Complementary Deoxyribonucleic Acid Like: 2566376 3', message ribonucleic acid sequence.

>ENA|AB037820|AB037820.1 Human Message RNA for KIAA1399 Protein, Partially Encoded Sequence.

>ENA|AI912122|AI912122.1 wd63a12.x1 NCI_CGAP_Lu24 Human Complementary Deoxyribonucleic Acid Like: 2336254 3', message ribonucleic acid sequence.

>ENA|AI376433|AI376433.1 tc36a08.x1 Soares_total_fetus_Nb2HF8_9w Human Complementary Deoxygen Glyconucleotide clone image: 2066678 3', message ribonucleic acid sequence.

>ENA|AW960748|AW960748.1 EST372819 MAGE Rearrangement Sequence, MAGF Human Complementary Deoxyribonucleic Acid, The ribonucleic acid sequence.

>ENA|AW058459|AW058459.1 wx21b12.x1 NCI_CGAP_Kid11 Human Complementary Deoxyribonucleic Acid Like: 2544287 3', message ribonucleic acid sequence.

>ENA|AA904430|AA904430.1 ok07f12.s1 Soares_NFL_T_GBC_S1 Human Complementary Deoxyribonucleic Acid Image: 1507151 3' Similar to TR: 000808 Q00808 B transduction protein-like protein, message ribonucleic acid sequence.

>ENA|AF493879|AF493879.1 Human guanine nucleic acid binding protein γ12 (GNG12) message ribonucleic acid, complete editing Code sequence.

>ENA|AI753143|AI753143.1 cr05h01.x1 Human bone marrow stromal cells human complementary DNA clone HBMSC_cr05h01 3', message ribonucleic acid sequence.

>refseq|NM_015577|NM_015577 Human retinoic acid induced 14 (RAI14), transcript variant 1, message ribonucleic acid.

>refseq|NM_005045|NM_005045 Human Shake White (RELN), Transcriptional Variant 1, Message RNA.

>ENA|AL037401|AL037401.1 Human Message RNA; EST DKFZp564K1671_s1 (from DKFZp564K1671 clone)

>ENA|BC004241|BC004241.1 Human laminin, α4, message ribonucleic acid (complementary DNA clone) Image: 3623597), complete coding sequence.

>ENA|U77706|U77706.1 Human laminin α4 chain (LAMA4*-1) message ribonucleic acid, complete coding sequence.

>ENA|U56725|U56725.1 Human heat shock protein message ribonucleic acid, complete coding sequence.

>refseq|NM_018214|NM_018214 Human is rich in leucine repeat 1 (LRRC1), message ribonucleic acid.

>refseq|NM_005092|NM_005092 Human Tumor Necrosis Factor (Ligangology), Family Member 18 (TNFSF18), Message Nucleus Sugar nucleic acid.

>ENA|AI279819|AI279819.1 qm26h04.x1 NCI_CGAP_Lu5 Human Complementary Deoxyribonucleic Acid Clone Image: 1882999 3', message ribonucleic acid sequence.

>ENA|BE857360|BE857360.1 7g29b12.x1 NCI_CGAP_Brn23 Human Complementary Deoxyribonucleic Acid Like: 3307871 3', message ribonucleic acid sequence.

>ENA|AI801973|AI801973.1 tx29d05.x1 NCI_CGAP_Lu24 Human Complementary Deoxyribonucleic Acid Like: 2270985 3', message ribonucleic acid sequence.

>ENA|AW451115|AW451115.1 UI-H-BI3-alg-g-01-0-UI.s1 NCI_CGAP_Sub5 Human Complementary Deoxygen Glyconucleotide clone image: 2736936 3', message ribonucleic acid sequence.

>ENA|AI472310|AI472310.1 tj87b01.x1 Soares_NSF_F8_9W_OT_PA_P_S1 Human Complementary Deoxygen Glyconucleotide clone image: 2148457 3', message ribonucleic acid sequence.

>gi|18089116|gb|BC020718.1|Human complement factor I, message ribonucleic acid (complementary DNA clone) MGC: 22501 image: 4716122), complete coding sequence

>ENA|BF967657|BF967657.1 602287358T1 NIH_MGC_96 Human Complementary Deoxyribonucleic Acid Clone Image: 4374495 3', message ribonucleic acid sequence.

>refseq|NM_006474|NM_006474 Human glomerular podocyte cell mucin (PDPN), transcript variant 1, message ribonucleo acid.

>refseq|NM_003619|NM_003619Human protein? , serine, 12 (neurotrypin?, motor protein) (PRSS12), message ribonucleic acid.

>refseq|NM_003149|NM_003149 Human SH3 and cysteine-rich region (STAC), message ribonucleic acid.

>ENA|AW007532|AW007532.1 ws52h07.x1 NCI_CGAP_Brn25 Human Complementary Deoxyribonucleic Acid Like: 2500861 3', message ribonucleic acid sequence.

>ENA|AI810767|AI810767.1 tu04d02.x1 NCI_CGAP_Pr28 Human Complementary Deoxyribonucleic Acid Like: 2250051 3', message ribonucleic acid sequence.

>ENA|AF154054|AF154054.1 Human DRM (DRM) message ribonucleic acid, complete coding sequence.

>refseq|NM_013372|NM_013372 Human gremlin 1 (GREM1), transcript variant 1, message ribonucleic acid.

>ENA|AU154455|AU154455.1 Human Complementary Deoxyribonucleic Acid Clone: NT2RP4001145, 3' End, Introducing Retinal Acid (RA) was expressed in NT2 neural precursor cells two weeks later.

>ENA|AW590196|AW590196.1 hg41a02.x1 NCI_CGAP_GC6 Human Complementary Deoxyribonucleic Acid Clone Image: 2948138 3', message ribonucleic acid sequence.

>ENA|BG494007|BG494007.1 602542289F1 NIH_MGC_59 Human Complementary Deoxyribonucleic Acid Clone Image: 4673182 5', message ribonucleic acid sequence.

>ENA|AI540210|AI540210.1 te55e07.x1 Soares_NFL_T_GBC_S1 Human Complementary Deoxyribonucleic Acid Image: 2090628 3' Similar to TR: Q63571 Q63571 Rat growth and transformation depends on the message ribonucleic acid sequence.

>ENA|BF589515|BF589515.1 naa05c06.x1 NCI_CGAP_Pr28 Human Complementary Deoxyribonucleic Acid Like: 3254002 3', message ribonucleic acid sequence.

>gi|11444320|gb|BF432206.1|BF432206 nab81e11.x1 Soares_NSF_F8_9W_OT_PA_P_S1 human Complementary DNA cloning image: 3274101 3', message ribonucleic acid sequence

>gi|283806665|ref|NM_153448.3|Human ESX homeobox 1 (ESX1), message ribonucleic acid

Claims (20)

A method of assessing a medical condition comprising identifying different manifestations of a gene-based marker in a CEC (relative to the performance of the same gene in a normal population). The method of claim 1, wherein the label does not differ in performance in the PBMC. The method of claim 1, wherein the performance in the regulatory gene is at least 2-fold different. The method of claim 1, wherein the p-value of the different regulation is less than 0.05. The method of claim 1, wherein the label is selected from Table 2. A method of identifying a CEC-derived tissue comprising identifying different expressions of a marker based on a tissue-specific gene in CEC (relative to the performance of the same gene in a normal population). The method of claim 6, wherein the label does not differ in performance in the PBMC. The method of claim 1, wherein the performance in the regulatory gene is at least 2-fold different. For example, the method of claim 1 of the patent scope, wherein the p-value of different regulation is less than 0.05. The method of claim 1, wherein the label is selected from Table 3. The method of claim 4, wherein the expression in the regulatory gene is at least 2-fold different. The method of claim 4, wherein the p-value of the different regulation is less than 0.05. A diagnostic kit comprising reagents suitable for single CEC separation and reagents suitable for detecting the performance of the markers of Table 2. A diagnostic kit according to claim 13 wherein the reagent suitable for detecting the label is as listed in Table 3. The kit of claim 13 further comprises reagents suitable for performing a microarray analysis. The kit of claim 13 further comprising a reagent suitable for amplifying the label. The kit of claim 13 further includes a component containing an executable guide, wherein the guide utilizes a pattern to correlate with performance detection. A kit according to claim 13 wherein the reagent suitable for isolated CEC comprises an immunomagnetic reagent. A method of assessing a medical condition, comprising arranging a single CEC, amplifying a gene expression marker obtained from the CEC, detecting the performance of the marker, and correlating the performance with the marker using a prediction or a prognosis; wherein the singularization and amplification system It is executed using the kit as in claim 13 of the patent application. A method of assessing a medical condition, comprising arranging a single CEC, amplifying a gene expression marker obtained from the CEC, detecting the performance of the marker, and correlating the performance with the marker using a prediction or a prognosis; wherein the singularization and amplification system This diagnostic kit is used as described in claim 14 of the scope of the patent application.