EP3931359A1

EP3931359A1 - Method for counting cell types or cell markers in a sample, in particular in a blood sample

Info

Publication number: EP3931359A1
Application number: EP20707263.8A
Authority: EP
Inventors: Jochen Hoffmann; Tino Frank
Original assignee: Robert Bosch GmbH
Current assignee: Robert Bosch GmbH
Priority date: 2019-03-01
Filing date: 2020-02-27
Publication date: 2022-01-05
Also published as: US20220081717A1; WO2020178134A1; CN113544289A; DE102019202788A1

Abstract

The invention relates to a method for detecting particular characteristic cells in a sample, comprising the following steps: a) providing a sample of cell suspension 1; b) carrying out a first lysis (2) on the sample (1) so that a first lysate (4) of the sample (1) is formed, the lysis proceeding such that cell nuclei of the cells of cell suspension 1 remain intact; c) extracting the ribonucleic acids (RNA) that are freely available in the first lysate (4); d) identifying the extracted ribonucleic acids in order to determine the number of different types of ribonucleic acids in the sample lysate; e) carrying out a second lysis (3) on the remaining sample (1) so that a second lysate (5) of the sample (1) is formed, the lysis proceeding such that cell nuclei of cells in the sample (1) are destroyed; f) extracting the deoxyribonucleic acids (DNA) that are freely available in the second lysate (5); g) calculating a total number of cells in the sample by means of the number of deoxyribonucleic acids; and h) calculating the number of cells with individual cell markers in the sample by means of the number of different types of ribonucleic acids identified in step d) and the total number of cells calculated in step g).

Description

title

Method of counting cell types or cell markers in a sample,

especially in a blood sample

State of the art

The extraction or isolation of circulating tumor cells (CTCs) from blood is an important step in the analysis of blood for the diagnosis of tumor cells. CTCs are defined as cells that are cyto-keratin-positive, CD45-negative, have a nucleus (DAPI detection) and are of epithelial origin. This

Definition is a requirement and largely accepted in the Liquid Biopsy Community. Cells that have these properties should be extracted or isolated.

Keratin (from the Greek kέraV 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

Take over functions.

According to their molecular structure as a-helix or ß-sheet, a distinction is made between a- and ß-keratins. 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. Cytokeratins (CKs or newer

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,

especially for the detection of tumors. Primary tumors and metastases from a given carcinoma share the same pattern of cytokeratins that they do differs from other types of carcinoma, thereby making it possible to differentiate between the various tumors.

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.

Along with muscle tissue, nerve tissue and connective tissue, 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

Blood 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.

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.

For a genetic analysis of CTCs, it is essential to ensure that cells were actually present in the sample. Otherwise, if signals are not present (= genetic trait not present) it is not clear whether there were no CTCs or generally no cells in the sample, for example because the sample was contaminated and the detection reaction therefore did not generate any signals.

A method is therefore desirable in which the absolute number of cells in a sample can be counted.

One possibility is to count cells by performing staining steps and automated processing of samples. However, such methods are difficult to implement in fully automated workflows for clinical use - on the one hand, because image processing is usually necessary in order to optically analyze samples when certain colored features are recognizable - on the other hand, because staining steps often take longer. This also complicates the clinical application of such methods

Disclosure of the invention

Described here is a method for detecting (or even counting) isolated CTCs by identifying genetic traits. The

Under certain circumstances, the process also enables dyeing steps to be dispensed with.

The method is used to detect certain characteristic cells in a sample and has the following steps:

a) Providing a sample of cell suspension

b) performing a first lysing process with the sample, so that a first lysate of the sample is produced, the lysing being carried out in such a way that the nucleus of the cells of the cell suspension remains intact;

c) extracting the ribonucleic acids (RNA) freely available in the first lysate; d) Assigning the extracted ribonucleic acids to the amounts

identify various types of ribonucleic acids in the lysate of the sample;

e) performing a second lysing process with the remaining sample so that a second lysate of the sample is produced, the lysing being carried out in such a way that cell nuclei of cells in the sample are destroyed,

f) Extracting those freely available in the second lysate

Deoxyribonucleic acids (DNA),

g) calculating a total cell number of cells in the sample from the amount of deoxyribonucleic acids

h) Calculating numbers with individual cell markers in the sample on the basis of the amounts of different types of ribonucleic acids determined in step d) and the total number of cells calculated in step g).

The method is particularly preferred when the cell markers in step h) serve to identify CTCs (circulating tumor cells).

The method is also particularly preferred if the cell markers in step h) comprise at least one of the following markers:

epithelial marker;

- EpCAM;

CD45; and

Cyto-keratin.

With this procedure, isolated CTCs are quantified without a

Staining step is necessary for quantification. The term “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.

The procedure lends itself to being based on a lab-on-chip

fully automated workflow for the point-of-care.

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

Components of the cells become identifiable. 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 μl 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. This makes use of the fact that the features determined in the prior art using staining methods (e.g. epithelial marker, EpCAM, CD45, cytocreatine) can also be detected using corresponding molecules, namely RNA

(Ribonucleic acids), in particular mRNA (Messenger RNA) are detectable in the cells. These cause the cells to react to certain dyes. When using staining methods, the existence of these molecules is used indirectly anyway in order to achieve an assignment of cells. With the method described, it is, so to speak, possible to draw conclusions directly about these molecules. Hence these

Molecules determined in step d) or assigned in order to determine amounts of different types of RNA. In the assignment in step d), genetic evidence is preferably used in order to achieve an assignment of RNA to a specific feature (epithelial marker, EpCAM, CD45, cyto-keratin).

In principle, such genetic detection works in such a way that when detecting RNA, a reverse transcriptase (RT) is first made from RNA (cDNA) so that this cDNA can be replicated using a polymerase chain reaction (PCR), with quantification also taking place at the same time . Everything together is then called RT-PCR. DNA is simply detected by means of PCR (if quantitative, then called qPCR).

Different types of 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. In step d), preference is given to the quantities of RNA using PCR-based methods (especially mRNA amounts) of the above (surface) markers (eg epithelial marker EpCAM, CD45, cytokeratin) are determined. Here, 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. for non-specifically isolated cells; Cytokeratins are proteins that are present in the interior of cells of epithelial origin, i.e. they are again a marker for CTCs. PCR (especially rtPCR = quantitative real-time PCR, or qPCR) is a method of amplification for nucleic acids. This duplication method is based on the principle of the conventional one

Polymerase chain reaction (PCR). In PCR, quantification is carried out with the help of fluorescence measurements, which are recorded in real time during a PCR cycle.

In addition, 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).

The order in which the various steps of the procedure described can be varied. This applies in particular to steps that are not directly based on one another and whose processing sequence can therefore be adjusted. For example, step d) does not have to

necessarily take place before e). You can also use the first lysate

Caching, e.g. after the reverse transcriptase step, and then at the same time as the DNA detection of the second lysis. In particular, 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.

In principle, 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.

Staining processes have the fundamental disadvantage that it is difficult to draw unequivocal conclusions from the images that are evaluated electronically. This requires a lot of intelligence for image evaluation. There are also no uniform standards for determining CTCs. The so-called ACCEPT5 was developed by the CANCER-ID consortium, which is supposed to define a kind of standard for image evaluation. The measurement using the procedure described here (promises) / delivers more meaningful and clearer signals. In particular, a (classic) numerical evaluation can take place in step h).

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

is basically much less available per cell.

These 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. There is a clear normalization of the sample material. 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

Process enables simple automation and thus saves time and money.

In the staining process used in the prior art, with

Antibodies worked, which also always bind unspecifically. With these

With this method, it is possible that undesired staining occurs which falsify the result of a subsequent count of stained cells. Genetic evidence provides a higher specificity here. Antibodies in production can also have a variance and are dependent on

Manufacturing LOT differently sensitive or specific.

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. The term “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.

In general, 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.

In order to make the calculation / evaluation carried out with the aid of the method more precise, 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. In variant embodiments of the method, 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

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

Cell sizes are possible and differences in size of the cells can be standardized and made comparable. 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.

The method is also particularly advantageous if the following step is carried out before step h):

g2) Differentiating between active ribonucleic acids and inactive ribonucleic acids. This results in further improved evaluation options for method steps h) and g).

In particular, the estimation in 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. Such

The data matrix can be structured in the manner of an expert system.

The method is particularly advantageous if the following step is carried out before 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.

It is also advantageous if the lysing in step b) by a

A change in an osmotic equilibrium occurs in the sample, which causes the cell membranes of the cells to burst. In this

Context is particularly beneficial when an osmotic

Equilibrium is adjusted in step b) so that cell nuclei of cells in the sample remain intact. Such a design of the lysing processes can ensure that RNA and DNA do not mix for the analysis steps d), g) and h).

The method is explained below with reference to the figures. The figures show a preferred exemplary embodiment to which the method cannot, however, be reduced. Show it:

1: a flow chart of the method described,

FIG. 2: exemplary parameters for assigning RNA to cell markers, and FIG. 3: another flow chart of the method described

In 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

Cell purification product or a sample sorted on a flow cytometer. As an alternative to a cell suspension, cells adhering to a surface can also be used for the method. In particular, cells can be used which adhere to a biopsy needle functionalized for the method. 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).

With a typical amount of a starting sample of 1 ml [milliliter], 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).

With a typical amount of an initial sample of 2 ml [milliliters] to 4 ml, 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.

In particular, purification of the lysate should not be carried out, since otherwise material to be quantified can be lost. 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. First, 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. As a result, the cytoplasm in which the cell's RNA is located can be extracted. After the extraction of the RNA, 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.

After successful lysis, the 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

Real-time polymerase chain reaction (qPCR) can be detected.

The actual core of the described method lies in the construct of the PCR system as indicated in Fig. 1 (steps g) and h)) and shown in the following table:

The evaluation of the qPCR curves enables quantitative determination and conclusions to be drawn about the composition of the sample at the cell type level.

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 β). 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).

The sample or the first lysate of the sample with the total amount of RNA

(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. For example, the targets to be examined are labeled with indices A, B, C and so on. First, 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

transcribed, which then migrates from the cell nucleus into the cytosome and is translated into a protein there.

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.

Instead of examining the transcriptome completely and assigning all the RNA in the transcriptome, in 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). For this purpose, 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 β 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 β. 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

To normalize size differences of the cells and make them comparable. In the example of CTC in the blood, 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).

The information on how much sample material (i.e. what number of cells) was used is now measured via the genome. A target D is used, which should be the same for all cell types of the individual to be measured. In particular, genes with high SNP proportions or high mutation rates (eg oncogenes) 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.

Ideally, 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.

The concept of the method described here can be scaled as required. 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

Introduction of additional cell types.

In the context of a liquid biopsy as part of a

Tumor therapy monitoring, the method described can be carried out as follows. EpCAM, e.g. CDK19 or vimentin can also be measured. So an additional one

Tumor cell markers. If the two expression patterns are too different, it can be concluded that there are reaction problems. The check whether the two expression patterns are the same can be used as a control element for the validity and Quality of the sample material are used. The additional target E can also be used to introduce a third cell type g. In the context of CTC monitoring, the additional target E can be used as a marker for mesynchimal tumor cells, such as twist, N-Notch, Vimentin or Snaill. Thus, the population of CTCs can be further divided into two subtypes. Thus, the method can also be used in the context of

Investigations in the area of the epithelial to mesenchymal transition are used (EMT). This is particularly interesting because it avoids a bias of EpCAM, since there is also a considerable part of

mesynchemical cells are present.

2 shows the analysis chain of the method described in the table above. The respective threshold cycle CT value is determined from the qPCR curves of the individual components A, B, C, D. For the

Transcriptional elements, the relative expression patterns are then determined, via the standard DDOT procedure. The

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.

3 shows an alternative schematic representation of the process management of the method described. Instead of measuring the targets in one

Reaction mix, 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. The illustration in 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

Evaluating the DNA can be considered. The process step d) and the

Process step g) each provide information that is the basis for performing process step h) and is therefore made available to process step h).

Claims

Expectations

1. A method for the detection of certain characteristic cells in a sample comprising the following steps:

a) Providing a sample of cell suspension 1

b) performing a first lysing process (2) with the sample (1) so that a first lysate (4) of the sample (1) is produced, the lysing being carried out in such a way that cell nuclei of the cells of the cell suspension 1 remain intact;

c) Extracting those freely available in the first lysate (4)

Ribonucleic acids (RNA);

d) Assigning the extracted ribonucleic acids to the amounts

identify various types of ribonucleic acids in the lysate of the sample;

e) Carrying out a second lysing process (3) with the remaining sample (1), so that a second lysate (5) of the sample (1) is produced, the lysing being carried out in such a way that the nuclei of cells in the sample (1) are destroyed ,

f) Extracting those freely available in the second lysate (5)

Deoxyribonucleic acids (DNA),

g) Calculating a total number of cells in the sample based on the amount of deoxyribonucleic acids

h) Calculating numbers of cells with individual cell markers in the sample on the basis of the amounts of different types of ribonucleic acids determined in step d) and the total number of cells calculated in step g).

2. The method according to claim 1, wherein the cell markers in step h) serve to CTCs (circulating tumor cells) using cell markers

identify.

3. The method according to any one of the preceding claims, wherein the

Cell markers in step h) comprise at least one of the following markers:

- epithelial marker;

- EpCAM; - CD45; and

- cyto-keratin.

4. The method according to any one of the preceding claims, wherein a blood count is created in step h).

5. The method according to any one of the preceding claims, wherein a variance of the cell sizes of the cells in the sample is taken into account in at least one of the two steps g) and h).

6. The method according to any one of the preceding claims, wherein the following step is carried out before step h):

g2) Differentiating between active ribonucleic acids and inactive ones

Ribonucleic acids.

7. The method according to claim 3, wherein the estimation in step g) is carried out with a comparison of the quantities determined in step d) with a data matrix in which for each cell marker data relating to the existing

Ribonucleic acids are deposited.

8. The method according to any one of the preceding claims, wherein the following step is carried out before step c):

b2) extracting cytoplasm.

9. The method according to any one of the preceding claims, wherein for

Determination of the amounts in step d) a conversion of the ribonucleic acids into deoxyribonucleic acids takes place.

10. The method according to any one of the preceding claims, wherein the lysing in step b) takes place by changing an osmotic equilibrium in the sample, which causes the cell membranes of the cells to burst.

11. The method according to claim 10, wherein an osmotic equilibrium is set in step b) so that cell nuclei of cells in the sample remain intact.