EP3931359A1 - Method for counting cell types or cell markers in a sample, in particular in a blood sample - Google Patents
Method for counting cell types or cell markers in a sample, in particular in a blood sampleInfo
- Publication number
- EP3931359A1 EP3931359A1 EP20707263.8A EP20707263A EP3931359A1 EP 3931359 A1 EP3931359 A1 EP 3931359A1 EP 20707263 A EP20707263 A EP 20707263A EP 3931359 A1 EP3931359 A1 EP 3931359A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sample
- cells
- cell
- lysate
- ribonucleic acids
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- CTCs circulating tumor cells
- Keratin (from the Greek k ⁇ raV keras "horn", genitive keratos) is a collective term for various water-insoluble fiber proteins which are formed by animals and which characterize the horny substance and which are mechanical in cells
- Keratins are the main component of mammalian hair, fingernails and toenails etc.
- a-Keratins or cytokeratins are in the form of loosely organized keratin filaments.
- Nomenclature also simply called keratins are intermediate filaments that form proteins that provide mechanical support and perform a variety of additional functions in cells. They are part of the cytoskeleton and the largest family of intermediate filament proteins. A distinction is made between two types of cytokeratins which form heterodimers, namely acidic type I (cytokeratins 9-23) and basic type II (cytokeratins 1-8).
- Cyto-Keratins are typical markers in diagnostic pathology
- CD45 is an antigen that can be used in particular to recognize leukocytes (white blood cells).
- Antigens are substances to which antibodies and certain lymphocyte receptors can specifically bind (the latter can also cause antibodies to be produced against the antigen). Antigens are mostly proteins, but they can also be carbohydrates, lipids or other substances. They can either be recognized or bound by B-cell receptors, T-cell receptors or antibodies (produced by B cells).
- a cell is of epithelial origin if it comes from epithelial tissue.
- Epithelial tissue is a special type of tissue that is used as a collective name for cover tissue and glandular tissue.
- Epithelial tissue is one or more layers of cells that cover all inner and outer body surfaces of multicellular organisms. Joint capsules and bursa of the musculoskeletal system are an exception.
- epithelial tissue is one of the four basic tissue types in multicellular organisms.
- a nucleus (DAPI proof) is proof that these cells have a nucleus - i.e. they are not nucleus-free cells, such as red cells
- Cells without a nucleus are generally not regarded as CTCs, even if they meet the other criteria mentioned.
- the term “lysing” means that the cells or the cell membranes of the cells that are present in a cell suspension are destroyed so that the individual components of the cells of the cell suspension are freely available in a solution. This enables later analysis.
- CTCs The purpose of counting CTCs is that, in medical studies, a clear connection can be established between overall survival (OS) and the CTC counts measured post-therapeutically. This means that simply counting CTCs can provide important information about OS or the effectiveness of a therapy. In order to obtain a more comprehensive picture of the tumor status, CTC research is currently also focusing on identification genetic traits from CTCs. Systems for automatically sorting, counting and assigning CTCs are therefore extremely relevant.
- a method is therefore desirable in which the absolute number of cells in a sample can be counted.
- Described here is a method for detecting (or even counting) isolated CTCs by identifying genetic traits.
- the process also enables dyeing steps to be dispensed with.
- RNA ribonucleic acids
- the method is particularly preferred when the cell markers in step h) serve to identify CTCs (circulating tumor cells).
- step h comprise at least one of the following markers:
- Staining step is necessary for quantification.
- Quantify here means that the number of CTSs can be determined absolutely.
- the approach also allows the sample to be normalized, that is to say how many cells are in the sample in total, in order to make the sample and the counted CTCs comparable with other samples or CTCs counted in other samples.
- Step a) can include, for example, providing cells to a lab-on-chip system.
- Steps b) and e) are common lysing processes that serve to dissolve membranes of cells and with which the
- the extraction steps c) and f) each serve to prepare the subsequent analysis steps d) or g) and h).
- a reasonable size of a sample for step a) comprises, for example, an amount of blood between 10 ⁇ l and 10 ml. Such a sample contains between 5 ⁇ 10 7 and 5 ⁇ 10 10 cells.
- the core of this invention are methods for the quantitative detection of isolated CTCs via a multi-parameter measurement of genetic characteristics. This takes place in step h) where the numbers of individual cell types are calculated.
- staining methods e.g. epithelial marker, EpCAM, CD45, cytocreatine
- RNA RNA RNA cleavage protein
- RNA reverse transcriptase
- PCR polymerase chain reaction
- ribonucleic acids which are assigned in step d) in order to determine their amounts in the sample are in particular ribonucleic acids which are markers for certain cancer cells.
- EpCAM is considered a marker for cells of epithelial origin, and thus for cells that come from a tissue composite, i.e. CTCs; CD45 is a marker for white blood cells, i.e.
- Cytokeratins are proteins that are present in the interior of cells of epithelial origin, i.e. they are again a marker for CTCs.
- PCR Polymerase chain reaction
- the total amount of isolated cells is determined via DNA. This is done using steps e), f) and g). Finally, the amounts determined at the mRNA level are related to the total amount of cells (step h).
- step d does not have to
- steps c) and f) can take place in parallel.
- the method can also be referred to as a genetic counting method, the term “counting method” here particularly relating to step d).
- the method has compared to known methods of detection
- characteristic cells in a sample have a number of advantages.
- the main advantage of the method is that based on the measurement of genetic characteristics, the number or quantity ranges of tumor cells in an isolated sample are measured. So it doesn't have to be in any a quantification of the sample amount takes place in an additional step. A quantification of the sample is necessary in order to be able to relate the measurement on the sample to other data and to give the sample a meaningfulness.
- the method is also suitable for detecting cancer-specific mutations, deletions or insertions - often using PCR-based methods - on the basis of CTCs.
- the method offers the advantage that such evidence and the method presented here by the
- Process steps are basically the same, and are therefore particularly suitable for co-integration on a lab-on-chip, because here too the quantity of the sample quantity is an important basis for the design.
- the method according to the invention promises signals that are readily detectable even when cells are present in only small amounts in a sample, since in the majority of cases a large number of mRNA molecules are present in a cell in the case of transcribed, active genes.
- This method therefore relies on a marker which is advantageous in terms of the quantities present, this marker being particularly advantageous compared to markers based on cell DNA, of which
- mRNA markers are combined with information at the DNA level as a reliable source for measuring the total amount of cells in a sample.
- This has the advantage that the relative ratios determined on the mRNA level can be converted / converted into absolute values by referring to DNA measurements.
- the method described can also reduce the number of manual steps involved in sample analysis. Depending on the insulation system, staining requires a large number of process steps. These process steps can be dispensed with thanks to the method described. The one suggested here
- Antibodies worked, which also always bind unspecifically.
- the method presented here allows an individual combination of targets for which a sample is to be examined. This can be done by adapting the assignment in step d) and the calculation / evaluation in step h) accordingly. This universal method thus allows application-specific use.
- target here refers to certain cell markers whose existence the cell is to be examined for or for which it is to be determined how often these occur in the cells of the sample.
- the method is not limited to applications in oncology.
- this method offers a simplified process approach for the analysis of expression patterns of cells for diagnostics.
- the method can be used particularly advantageously in fully automated point-of-care applications.
- a variance of the cell sizes of the cells in the sample is preferably taken into account in at least one of the two steps g) and h).
- This variance in cell sizes can be determined, for example, by optical analysis (image analysis) of the sample or obtained from statistical data.
- the variance is permanently stored as a parameter for the calculation evaluation in steps g) and h).
- the term “calculate” in steps g) and h) is not to be understood as meaning that the result in step g) is necessarily an exact one
- step h Total cell count comes out, which definitely or actually corresponds to the exact number of cells in the sample. Nor is it meant here that in step h) exactly calculate which numbers of cells with individual cell markers are definitely or actually contained in the sample. Because of uncertainties or stochastic inaccuracies in the input variables described for the calculations (amount of deoxyribonucleic acids for step g) and amounts of different types of ribonucleic acids for step h)) this may not even be possible. Rather, the step term “calculate” here means that the determination of the total number of cells or the determination of the number of cells with individual cell marks is determined on the basis of the available input variables according to a mathematical rule and preferably no further estimated values or Assumptions are used.
- the variance of the cell sizes is particularly preferably determined via a household gene of the cells. With the help of a household budget, estimates are the
- Household genes are typically genes that encode the structural molecules and enzymes that are related to the basic metabolism of cells, such as glucose metabolism. Based on such genes, it is possible to infer a cell size.
- step g) takes place with a comparison of the quantities determined in step d) with a data matrix in which data relating to ribonucleic acids present are stored for each cell marker.
- the data matrix can be structured in the manner of an expert system.
- step c) b2) extracting cytoplasm.
- This additional step simplifies the later process steps. This step also makes it possible to avoid errors in steps d), g) and h) which could be caused by the cytoplasm.
- the method is also advantageous if the ribonucleic acids are converted into deoxyribonucleic acids in order to determine the amounts in step d). This conversion offers advantageous options for mapping.
- step b) it is also advantageous if the lysing in step b) by a
- Context is particularly beneficial when an osmotic
- FIG. 2 exemplary parameters for assigning RNA to cell markers
- FIG. 3 another flow chart of the method described
- FIG. 1 the applied process steps of the method are shown very schematically.
- a (mostly) preprocessed cell suspension 1 is lysed.
- the cell suspension can be whole blood, serum, a
- the first lysing process 2 (step b)) is shown here by an arrow.
- the first lysing process 2 generates a first lysate 4 which can be analyzed with steps c) and d).
- step b) preferably produces an amount of 2 ml to 4 ml of the first lysate.
- the second lysing process 3 (step e)) is also shown here by an arrow.
- the second lysing process 3 generates a second lysate 5 which can be analyzed with steps f), g) and h).
- step e) preferably produces an amount of 4 ml to 8 ml of the second lysate.
- the lysis mode used in step b) (first lysis process) can be selected as desired, but must be compatible with the subsequent RT-qPCR.
- a suitable lysing process can be carried out by immobilizing the cell material in a cannula.
- a lysis buffer can then be drawn into the cannula in order to extract nucleic acids such as DNA or RNA from the cell material.
- a hypotonic solution (the lysis buffer) is drawn in, the salt content of the buffer being higher than that of the cells.
- the cell membrane bursts, but not the cell nucleus.
- the cytoplasm in which the cell's RNA is located can be extracted.
- the DNA can be extracted in a second step in which a hypertonic lysis buffer is drawn up.
- the cell nucleus bursts and the DNA can be extracted. It is also possible to use just a hypertonic buffer and extract the DNA and RNA in one step.
- Lysis buffers can be composed differently. A distinction must be made between hypotonic (lysis) buffer and hypertonic (lysis) buffer.
- An exemplary composition of hypotonic lysis buffer contains 20 mM [millimoles] Tris final concentration. That is, in a mixture of sample and Tris, a final concentration of 20 mM NaCl is established.
- An exemplary composition of hypertonic buffer contains 3% sodium chloride (NaCl). This means that in a mixture of sample and NaCl, a final concentration of 3% NaCl is established.
- RNA is converted into DNA by means of reverse transcription. This creates a solution that consists only of DNA. This can now be used for a detection reaction using quantitative
- qPCR Real-time polymerase chain reaction
- the table describes the design strategy of the PCR system for the successful application of the described method.
- the basis here is that the composition of a cell suspension of one cell type or cell population a and another cell type or cell population ß of a eukaryotic individual is detected.
- a specific example is the detection of circulating tumor cells (type a) in a background of blood cells (type ⁇ ).
- the basic strategy is to use the transcriptome (mRNA information) to determine the cell types and the relative cell pattern between the cell types. By measuring the genome, the absolute number of all cells in the sample can then be determined. If this number is known, together with the ratios, the absolute numbers of cells with individual cell markers or the numbers of the individual subpopulations (different types of cells within the sample) can be estimated. The amount of all mRNA in a cell is called the “transcriptome”. It is a cell type-specific parameter. This transcriptome is the total amount of RNA that is available for the assignment in step d).
- Transcriptome is preferably checked step by step for targets (i.e. for cell markers or cell types that are to be recognized with the method described).
- the individual targets are preferably processed in a specific order.
- the targets to be examined are labeled with indices A, B, C and so on.
- the sample or the first lysate is tested for target A, then for target B, then target C and so on. This is done in step d). If a certain gene is active in a cell, this corresponding DNA sequence is converted into RNA
- a cell type can be identified using active and inactive genes. Each cell type has a defined group of genes which are particularly active or must be switched off. This gene activity can be demonstrated by detecting the corresponding mRNA or proteins. RNA detection by qPCR is particularly sensitive here.
- step d) it is also possible to detect sequences typical for cell types by means of qPCR.
- the mRNA is then transcribed into DNA as already described in order to subsequently carry out a gene-specific qPCR.
- the transcription measurements allow conclusions to be drawn about the relationships between the transcription patterns of the cell (s).
- at least one target A is selected which is expressed in the same way in all cell types, ie is active in both types. This is a so-called household gene, which is generally constantly active across all cell types. Typical examples are GAPDH, actin, SDHA, HSP, COX8 or MYH9.
- the second target B used is a target which is upregulated for cell type a, while in cell type ⁇ the target is downregulated accordingly.
- the third target C has a chiasmatic transcription profile to target B, ie downregulated in cell type a and upregulated in cell type ⁇ .
- the normalization via target A is necessary because cell populations are very heterogeneous in their measurement parameters.
- the cell size in particular usually shows a high variance. Small cells usually have fewer absolute numbers of mRNA, but their ratios of up and down are conserved. Therefore, a household gene is used to control the
- target B could be an epithelial marker such as EpCAM, CDK, catenin.
- Hematopoietic markers such as CD45 can be used as target C for blood cells (mainly leukocytes such as T cells, neutrophils, dendrites, macrophages).
- a target D is used, which should be the same for all cell types of the individual to be measured.
- genes with high SNP proportions or high mutation rates are not suitable because such genes are present in different amounts in different cells of the sample or the amount of target cells (cell markers) based on such genes with the described method is to be determined.
- hLINE1 or APEX1 highly conserved, multiple abundant genes such as an hLINE1 or APEX1 are used here. These are present several times and are easier to quantify than a classic gene such as EGFR, which occurs exactly twice in the genome. This is particularly advantageous if few cells are available in a sample, as in the case of conventional CTC examinations.
- a target E can be added to the transcriptome, which is used for finer selection of cell types, for statistical determination of quality or for
- EpCAM e.g. CDK19 or vimentin
- vimentin can also be measured. So an additional one
- the additional target E can also be used to introduce a third cell type g.
- the additional target E can be used as a marker for mesynchimal tumor cells, such as twist, N-Notch, Vimentin or Snaill.
- the population of CTCs can be further divided into two subtypes.
- the method can also be used in the context of
- EMT epithelial to mesenchymal transition
- mesynchemical cells are present.
- the respective threshold cycle CT value is determined from the qPCR curves of the individual components A, B, C, D.
- Expression ratios of the individual cell types can be determined.
- the absolute number of cells in the reaction mixture is determined from the genetic material.
- the transcriptome can be separated from the DNA in a first step.
- Selective lysis methods are used, in which first only the cytosol in which the mRNA is located is lysed using hypertonic buffer (lysis buffer), followed by lysis of the DNA-containing cell nucleus via hypotonic lysis buffer.
- lysis buffer hypertonic buffer
- FIG. 3 shows that steps b), c) and d) relating to the isolation and evaluation of the RNA are in principle separate from steps e), f) and g) relating to the isolation and
- Process step g) each provide information that is the basis for performing process step h) and is therefore made available to process step h).
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US20060286558A1 (en) * | 2005-06-15 | 2006-12-21 | Natalia Novoradovskaya | Normalization of samples for amplification reactions |
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