WO2021052854A1

WO2021052854A1 - Method for determining cell count by means of a reference dna

Info

Publication number: WO2021052854A1
Application number: PCT/EP2020/075310
Authority: WO
Inventors: Wolfgang Wagner
Original assignee: Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen Aör
Priority date: 2019-09-16
Filing date: 2020-09-10
Publication date: 2021-03-25
Also published as: EP4031684A1; DE102019124828A1

Abstract

The invention relates to a method for determining the cell count of eukaryotic cells in a sample, in which at least one specific region in the genome of at least one cell or one cell type is identified and at least one reference DNA is provided, wherein the number of cells is calculated by means of the reference DNA. According to the invention, cell count is determined by using a reference DNA which has a sequence deviating from the corresponding cellular sequence in one or more nucleotides. Prior to DNA quantification, the cells are admixed with said reference DNA in a known concentration. By means of the quantification, it is possible to determine the ratio between cellular DNA and reference DNA. By comparison with a reference curve, the cell count can then be determined therefrom.

Description

Method for determining the number of cells using a reference DNA

The invention relates to a method for determining the cell number of eukaryotic cells in a sample, in which at least one specific region in the genome of at least one cell or cell type is identified and at least one reference DNA is provided, the number of cells being calculated using the reference DNA becomes.

The relative quantification of cell types is of particular importance for many applications in clinical diagnostics, for example when taking blood counts. The determination of the cellular composition of blood samples and in particular the differential blood count, i. H. The determination of the composition of the white blood cells (leukocytes) in the blood is one of the most common diagnostic test procedures and therefore a routine examination in medical laboratory diagnostics. A differential blood count can be made, for example, by microscopic examination of the blood sample and manual counting of the cells. The determination of the number of cells is also necessary for many other diagnostic and cell biological processes. Numerous methods are available for this, which are based in particular on imaging or flow cytometry (impedance or scattered light methods). These automatic cell counters record, for example, the electrical impedance, the optical effects of light scattering or the intensity of fluorescence signals. Alternative measurements are based on counting chambers, automated optical cell counts, or measurements of electrical resistance (CASY).

In addition to methods that enable cellular material to be quantified on the basis of counting individual cells, different flow cytometric methods are also used, which allow cells to be quantified, for example with the aid of reference beads in a certain concentration. However, these methods have the disadvantage that a labor-intensive processing of the sample to be measured has to be carried out beforehand, which for example involves complex steps such as centrifugation, filtration and / or May include sedimentation. Furthermore, many optical flow cytometers use the principle of fluorescence or scattered light measurement. However, both measurement methods require the use of specially prepared and purified blood samples to count individual cells. For example, in order to prepare a blood sample, it is necessary to carry out a hemolysis of the erythrocytes and a specific marking of the cells. The sample preparation is consequently very time-consuming, so that neither fast nor inexpensive cell count determinations can be carried out in a complex sample by means of optical flow cytometry.

The above-mentioned methods of determining the number of cells require relatively fresh material in which the cells are alive and as isolated as possible. Reliable determinations of cell counts in tissues (e.g. from punched cylinders of biopsies with defined volumes, cells in 3D tissue engineering tissue constructs or embryological developments in different organisms) or coagulated blood samples are hardly possible. As a rule, these methods also require a certain minimum volume due to the hose systems. In addition, the samples must be freshly analyzed. However, in the course of sending the sample to a large laboratory or delayed measurements, increased cell deaths can occur, so that the cell count would be estimated accordingly too low. Autolysis begins after a few hours and initially affects the granulocytes in particular. Sending blood samples in a frozen state has not yet been possible because the cells are lysed in the process. In addition, there are measurement inaccuracies in all measurement methods, which may make validation with another method useful. With very small amounts of cells (e.g. liquor diagnostics) or with very small amounts of samples, the analysis is not possible with all methods.

From DE 10 2017 004 108 A1, however, an alternative method is known which is based on the analysis of DNA methylation. Genomic areas are addressed that are basically methylated in the blood cells to be examined. The method comprises the identification of a chromatin region which is reliably methylated in the different blood cells and the provision of an unmethylated reference DNA which can be produced, for example, by PCR. Using this well-known epigenetic process, a determination of the cell number can also be carried out on coagulated blood samples and frozen material.

It is the object of the present invention to provide a method for determining the cell number of eukaryotic cells in a sample, with which cell numbers in tissues can also be determined and which can be safely used with a wide variety of cell types in humans and other organisms.

According to the invention, the object is achieved by a method for determining the cell number of eukaryotic cells in a sample, which comprises:

Identifying at least one specific region in the genome of at least one cell or cell type, the specific region being a conserved genomic region;

Providing at least one reference DNA which comprises at least one reference nucleotide sequence which is identical to the sequence of the specific region with the exception of at least one nucleotide exchange;

Introducing a predetermined amount of the reference DNA into the sample; Quantifying the DNA of the specific region and the reference DNA; and calculating the number of cells based on the ratio of the genomic DNA of the cells to be tested and the reference DNA.

According to the invention, a reference DNA is used to determine the number of cells, regardless of epigenetic changes, which has a sequence that differs from the corresponding cellular sequence in one or more nucleotides. Before the DNA is quantified, the cells are mixed with this reference DNA in a known concentration. The concentration should preferably correspond approximately to the expected number of cells. The quantification allows the ratio of cellular DNA and reference DNA to be determined relatively precisely, for example by means of digital PCR, pyrosequencing or amplicon sequencing methods. The cell number can then be determined from this in comparison with a reference curve. The method according to the invention can in principle be implemented for determining the number of cells of the most varied of cell types and of the most varied of species. However, a corresponding reference DNA must be generated for each species. in the In contrast to the epigenetic method according to DE 10 2017 004 108 A1, the method according to the invention can be safely used for a wide variety of human cell types. The cells of other organisms can also be quantified with a corresponding reference DNA independently of epigenetic changes. In contrast to conventional methods, the samples can also be stored or shipped frozen, since only the DNA content is determined in relation to the volume. First, a reference DNA is created which basically has the same sequence as a conserved genomic region in the cells to be tested, but which has a sequence difference in one or more bases. The optimal number of exchanges depends on the type of analysis method. For dPCR, for example, 2-10, preferably 2-8 or 2-6, in particular 2-5, 3-5 or 2-4 exchanges in the area of the binding site of the probe have proven to be advantageous, since a single exchange may still be unspecific Allows ties. Theoretically, there is no upper limit for the sequence differences, although a PCR bias may more likely occur if the two sequences were very different. The reference DNA (eg approx. 100 base pairs) can be created synthetically, by means of PCR or as plasmid DNA, for example. It is advantageous if the DNA concentration of the reference DNA can be precisely determined.

The following additional advantages also result from the application of the method according to the invention:

- The samples can be frozen before analysis;

- Cell numbers can be determined in tissues;

- Very small sample quantities are sufficient (theoretically <10 pl);

- Simple analysis;

- Isolated DNA can be sent very easily for analysis purposes;

- No bisulfite treatment is necessary;

- Based on the sequence, a clear assignment (reference DNA versus cellular DNA) is possible;

- In the high throughput process, the process according to the invention can be carried out very inexpensively. In an advantageous embodiment of the invention it is provided that two or more different reference DNAs are introduced into the sample, the reference DNAs corresponding to different specific regions. By using several different reference sequences, a wider range of cell numbers can be reliably covered. In addition, further validation and additional accuracy can be achieved through the parallel determination of different specific genomic regions.

In a further advantageous embodiment of the invention, it is also provided that the reference DNA corresponds in each case to the sequence of a specific species which is differentiated from other species. This would be advantageous in the event that human and other cells are mixed, e.g. cells on murine feeder cells.

In a further advantageous embodiment of the invention it is provided that the reference DNA is introduced into the sample in different concentrations. The reference DNAs can, for example, be mixed beforehand in different concentrations and then, as a “ladder”, an aliquot of the sample can be added.

The number of cells can be calculated, for example, in such a way that different reference DNAs are added to the sample in different concentrations in order to cover a larger area. In other words, in order to broaden the measuring range, further reference DNAs can be added which have different sequence modifications. By using different concentrations of the reference molecules, a reference curve can be created in an advantageous manner, by means of which the number of cells can then be precisely determined. This also enables the concentration of the reference molecules to be determined, which roughly corresponds to the expected number of cells and thus ensures the highest possible accuracy of the measurement. Alternatively, the number of copies of the reference DNA can also be calculated and the number of cells can thus also be determined without a reference curve. With both methods, for example, the reference DNA concentration can be determined which corresponds to the same number of copies of the reference DNA and the genomic (cellular) DNA. Basically, the amount of reference molecules should be around the expected number of copies of the correspond to genomic DNA, as the sensitivity is highest in this measuring range. In addition, a wide range of cell numbers can be reliably covered by using different reference molecules in increasing concentrations. For example, the amount of reference DNA can be set such that the ratio of the number of reference molecules to the approximately expected number of cells is approximately 2: 1. Assuming that the cells to be analyzed each contain two copies of their DNA molecules, this ratio corresponds to the same number of copies of the reference DNA and the genomic (cellular) DNA, so that in this embodiment the highest possible sensitivity of the method according to the invention is guaranteed is.

For example, two or more different reference DNAs could also be introduced into the sample, the reference DNAs corresponding to the same specific region but comprising different nucleotide exchanges. In this way, for example, the ratio of the reference DNAs to one another could be calculated.

In a further advantageous embodiment of the invention it is further provided that the specific region comprises a conserved region without repeats and / or single nucleotide polymorphism, preferably a non-transcribed region. A conserved region without repeats and single nucleotide polymorphism (SNP) should therefore be chosen in an advantageous manner, e.g. a promoter region of a gene.

In an advantageous embodiment of the invention, it is also provided that the DNA is isolated from the sample before the quantification. For the accuracy of the method according to the invention, it is advantageous if the entire DNA of the sample can largely be extracted. However, since large deviations inevitably arise during DNA isolation, the measurement of the DNA concentration cannot be reliably measured. In contrast to this, the reference DNA can be added to the sample at a precisely set concentration, which is then subject to the same fluctuations in the DNA isolation during the DNA isolation. The Mixing of the reference DNA with the sample is therefore preferably carried out before the DNA isolation in order to compensate for fluctuations in the DNA isolation.

After DNA isolation, the ratio of cellular DNA and reference DNA can be determined relatively precisely, e.g. using quantitative PCR methods (especially digital PCR), pyrosequencing or amplicon deep sequencing methods.

According to the invention, the object is also achieved by a nucleic acid molecule which comprises at least one nucleotide sequence which is identical to the sequence of a conserved region in the genome of at least one cell or a cell type with the exception of at least one nucleotide exchange, for use in the above-described method according to the invention. For example, one or more of the reference DNA sequences according to SEQ ID NOs: 1 to 7 according to Table 1 can be used for the method according to the invention.

The invention also comprises a kit which comprises at least one such nucleic acid molecule and optionally at least one oligonucleotide for amplifying and / or quantifying at least one nucleic acid molecule. This kit can be used to determine the number of eukaryotic cells in a sample. For example, such a kit can comprise one or more of the reference DNA sequences according to SEQ ID NOs: 1 to 7 (Table 1) and / or one or more of the primers / probes according to SEQ ID NOs: 8 to 35 (Table 1).

In addition, such a kit can comprise at least one buffer solution and / or a reagent for carrying out one or more of the following methods: DNA amplification, DNA sequencing, for example pyrosequencing, digital droplet PCR, bar-coded bisulfite amplicon sequencing (BBA-seq), bar-coded amplicon sequencing, "Mass Array ^® " and "SNP genotyping".

The reference DNA can be multiplied, for example, by introducing the reference nucleotide sequence into at least one prokaryotic cell, increasing the nucleotide sequence in the prokayotic cell and isolating at least one DNA molecule, which comprises at least one reference nucleotide sequence, can be carried out from the prokaryotic cell. The reference molecule can thus be amplified, for example, in E. coli (for example in the form of a plasmid) and then purified. In an optimized process, the plasmid DNA can be literaryized with a restriction enzyme before it is used as reference DNA.

Alternatively, the reference DNA can be amplified or synthesized in vitro, preferably by means of the polymerase chain reaction (PCR). In this advantageous way, the reference DNA can be produced quickly and reliably. The PCR product could, for example, be used as an independent DNA double strand or as cloned plasmid DNA as a standard.

The invention further relates to at least one artificial nucleic acid molecule which comprises at least one nucleotide sequence which is selected from the group consisting of: a) at least one nucleotide sequence which contains at least one sequence of the nucleotide sequences SEQ ID NO: 1 to SEQ ID NO: 35 (see table 1) comprises, b) at least one nucleotide sequence which is at least 90%, preferably at least 95%, identical to the nucleotide sequence according to a) and c) at least one nucleotide sequence which corresponds to the strand complementary to one of the nucleotide sequences according to a) or b) corresponds to.

The artificial nucleic acid molecules according to the invention can also advantageously be used to produce the kit for carrying out the method according to the invention.

The invention also relates to the use of the nucleic acid molecule according to the invention and / or the artificial nucleic acid molecule according to the invention and / or the kit according to the invention for determining the cell count of eukaryotic cells in a sample, ie for the absolute quantification of cells or cell types. Both the method according to the invention and the nucleic acid molecules and kits according to the invention can in principle be used for determining the number of cells of the most varied of cell types in various areas of application. In particular, these are also suitable, for example, for determining the number of nucleated cells in the blood or other body fluids (liquor, urine, etc.). With the aid of the method according to the invention, for example, the number of nucleated blood cells can be reliably determined. Cell counts are routinely determined in blood samples using flow cytometric measuring methods. However, the determination is not possible in all blood samples (for example due to coagulation or insufficient blood volume) and can no longer be reliably carried out on older blood samples. The samples must be freshly sent for analysis and measured promptly. In contrast to this, the determination of the cell number according to the invention can also be carried out on blood samples in which the cells are no longer isolated. The method according to the invention is also useful in combination with other methods, since quantitative information is obtained without great additional effort, while otherwise only relative ratios of the hematopoietic cell types can be determined. In the high throughput process, the process according to the invention can also be carried out very inexpensively. The measuring accuracy is comparable with the accuracy of conventional methods.

A “conserved genomic region” within the meaning of the invention denotes a sequence segment in the genome of a cell (DNA) which comprises a nucleotide sequence which has been largely unchanged in the course of evolution, i.e. has undergone no or only minimal changes in the sequence. The term also includes, in particular, non-coding DNA sequences, such as introns or promoter sequences.

The invention is further illustrated by way of example with the aid of the following figures and advantageous embodiments.

FIG. 1 shows an exemplary image of a result of a digital droplet PCR (ddPCR). X-axis: reference, Y-axis: blood FIG. 2 shows a diagram for evaluating the results of the positive droplets for genomic DNA or reference DNA in order to determine the ratio of these DNA strands in the starting material. For this purpose, 150 μl of blood were mixed with various concentrations of the reference DNAs (R2 and R3 according to Table 1). X-axis: dilution or reference DNA Y-axis: ratio of blood to reference DNA

Figure 3 shows a diagram for determining the ratio of blood DNA to reference DNA or the number of copies. For this purpose, different amounts of the reference DNAs (R2-R6, R8 and R9 according to Table 1) were mixed with 150 μl of blood. X-axis: number of copies (ratio of blood to reference)

Y-axis: amount of reference DNA used

FIG. 4 shows diagrams of an example of the quantification of human mesenchymal stem cells (MSC) by means of digital droplet PCR (ddPCR).

(A) X-axis: log cell number, Y-axis: log MSC, cell number ddPCR

(B) X-axis: log Neubauer cell number, Y-axis: log MSC (human mesenchymal stem cells), cell number ddPCR

FIG. 5 shows diagrams of an example of the determination of the number of cells in human blood samples by means of digital droplet PCR (ddPCR).

(A) X-axis: blood cells, automatic counter, Y-axis: blood cells ddPCR

(B) Logarithmic representation of the data according to A.

X-axis: log blood cells, automatic counter Y-axis: log blood cells ddPCR

As an example, nine genomic regions were selected for the design of the reference DNAs. These sequences do not have any homologous sections in the genome and are suitable for the design of the ddPCR primers. They should preferably not be located in translated gene sequences, since an influence by RNA cannot be ruled out with certainty. Of the nine initial sequences, seven have proven successful, which are listed in Table 1. These sequences were provided, cloned into plasmid pBR322 using restriction enzymes, and amplified in E. coli. The plasmid DNA was isolated and quantified. These Reference DNAs are either used individually or combined in different concentrations as reference ladder. A certain amount of this reference DNA is added to the cell sample (eg 150 μl blood). A digital droplet PCR (ddPCR) is then carried out (FIG. 1). The results of the positive droplets for genomic DNA or reference DNA are evaluated using Poisson statistics in order to determine the ratio of these DNA strands in the starting material (FIG. 2). The ratio of blood DNA to reference DNA was then determined in the ddPCR measurements. This allows conclusions to be drawn about the number of cells. The data show very consistent results both for the dilutions and with both reference plasmids. If necessary in the case of large fluctuations in the number of cells, or as an additional internal control, the measurement of a further reference DNA can also be included, since this additional sequence does not interfere with the method (FIG. 3). Different amounts of the reference DNAs were mixed with a certain volume of blood. The ratio of blood DNA to reference DNA was then determined. The measurements show a clear correlation without the different plasmids affecting the measurement.

10-fold dilution series from three MSC donors (MSC1 - MSC3) were each mixed with the same amount of the reference DNA R5 (0.0000897 ng) and measured by means of ddPCR in order to estimate the cell concentration (cells / mI). The cell concentrations calculated by means of ddPCR were correlated with a cell concentration which was determined by means of Neubauer counting for the initial dilution (FIG. 4 A). The cell concentrations calculated by means of ddPCR were correlated with cell concentrations that were determined by means of Neubauer counting for each dilution (the conventional counting method could not be used with very low cell numbers (cell dilutions less than 10 cells / ml) (FIG. 4 B).

40 blood samples (150mI) were each mixed with the same amount of the reference DNA R6 (0.01282 ng) and the same amount of the reference DNA R5 (0.0038943 ng) and then measured by means of ddPCR in order to estimate the cell concentration (cells / ml ). The cell concentration calculated by means of ddPCR was correlated with a cell concentration which was determined by means of an automatic cell counter (FIG. 5).

Claims

1. A method for determining the cell number of eukaryotic cells in a sample, which comprises:

2. The method according to claim 1, characterized in that two or more different reference DNAs are introduced into the sample, the reference DNAs corresponding to different specific regions.

3. The method according to claim 1 or 2, characterized in that the reference DNA corresponds in each case to the sequence of a certain species, which is differentiated from other species.

4. The method according to claim 1, 2 or 3, characterized in that the reference DNA is introduced into the sample in different concentrations.

5. The method according to one or more of claims 1 to 4, characterized in that the specific region comprises a conserved region without repeats and / or single nucleotide polymorphism, preferably a non-transcribed region.

6. The method according to one or more of claims 1 to 5, characterized in that the DNA is isolated from the sample before quantification.

7. The method according to one or more of claims 1 to 6, characterized in that the quantification of the DNA is carried out by means of quantitative PCR methods (in particular digital PCR), pyrosequencing or amplicon deep sequencing methods.

8. Nucleic acid molecule which comprises at least one nucleotide sequence which is identical to the sequence of a conserved region in the genome of at least one cell or a cell type with the exception of at least one nucleotide exchange, for use in the method according to one or more of claims 1 to 7.

9. Kit, which comprises at least one nucleic acid molecule according to claim 8 and optionally at least one primer oligonucleotide for amplifying the nucleic acid molecule.

10. Use of the kit according to claim 9 for determining the cell number of eukaryotic cells in a sample.