Multimodal analysis of body fluid samples containing stabilized cells

Abstract

To provide a method for stabilizing and isolating a plurality of biological targets contained in a cell-containing body fluid.SOLUTION: (A) providing a stabilized cell-containing body fluid sample by contacting a cell-containing body fluid with a stabilizing composition comprising one or more of the following stabilizing factors: (a) at least one primary, secondary or tertiary amide, (b) at least one poly (oxyethylene) polymer, and / or (c) at least one apoptosis inhibitor, (B) maintaining the stabilized cell-containing body fluid sample for a stabilization period, (C) determining the presence or absence of a cell subpopulation, extracellular nucleic acids, extracellular vesicles, and treating the stabilized cell-containing body fluid sample to enrich three or more biological targets selected from the group consisting of intracellular nucleic acids from the stabilized cell-containing body fluid. The method allows for multimodal analysis of different biological targets from a single stabilized cell-containing body fluid sample.SELECTED DRAWING: None

Description

[Technical Field]

[0001] A liquid biopsy-based method and workflow is provided for analyzing different biological targets of interest from a single stabilized cell-containing bodily fluid sample. [Background technology]

[0002] Liquid biopsy (LB), the analysis of biological targets (e.g., cells, proteins, and nucleic acids) in human body fluids (e.g., blood, urine, saliva, and fluids), is a powerful tool for companion diagnostics in clinical practice. Important liquid biopsy specimens, also referred to herein as biological targets, include rare cells, extracellular nucleic acids, extracellular vesicles, intracellular nucleic acids, and specific cell subpopulations. Liquid biopsies in cancer and prenatal testing are attracting the most attention, and some currently available tests have already been implemented in routine patient care.

[0003] Solid tumors and hematological malignancies are known to shed biological materials into the systemic circulation. These biological materials include cells (circulating tumor cells, also known as CTCs) and extracellular vesicles (EVs), such as exosomes and other types of subcellular membrane vesicles. Cell-free circulating nucleic acids are also known to contain information about cancer, e.g., mutations. These biological materials are present in readily accessible bodily fluids, such as peripheral whole blood, peritoneal, or pleural effusions, and carry molecular information, including proteins, nucleic acids, and lipids. The molecular information provided by these circulating biological materials can be correlated with, for example, prognosis, treatment response, recurrence, or treatment resistance mechanisms. There has been significant interest in these biological targets for minimally invasive testing in the prior art. These targets offer the significant advantage of avoiding the challenges of biopsy and can be easily and repeatedly obtained to provide minimally invasive reflection of tumor molecular information. It is understood in the art that extracellular nucleic acids, extracellular vesicles, or circulating tumor cells can provide valuable diagnostic, prognostic, predictive, and monitoring information. This information can be utilized, for example, by analyzing the biomarkers contained therein. Biomarkers are biological molecules that can be measured in the biological sample being analyzed and, either alone or in combination with other biomarkers, can be indicative of some clinically important disease state. Biomarkers can be, for example, diagnostic, surrogate, prognostic, and / or predictive. Biomarkers can be, for example, nucleic acids (e.g., DNA or RNA molecules) or proteins. Blood is the best source of material for liquid biopsy. Cell-based liquid biopsy tests often rely on the analysis of target cell populations, such as CTCs in cancer (further examples include endothelial cells in cancer, diabetes, cardiovascular or acute kidney disease, fetal cells in prenatal testing, and organ-specific cells in transplant histology) (see, for example, Pantel et al., Nat Rev Clin Oncol, Feb 2019; Neumann et al., Comput Struct Biotechnol J, 2018, Vol. 16: 190-195; Lehmann-Werman et al., Proc Natl Acad Sci US A. 2016 Mar 29, Vol. 113(13): E1826-34; Snyder et al., Proc Natl Acad Sci USA, 2011 Apr 12, Vol. 108(15): 6229-34).

[0004] CTCs can be exfoliated from primary or metastatic tumors in cancer patients and found in the blood. These cells represent a rare cell population: 1–10 CTCs per 10 6 ~10 8CTCs can be found in a background of individual blood cells, with a limited half-life in circulation of 2.5 hours. CTCs seed distant metastases. Their presence in the peripheral blood of cancer patients has been validated as a surrogate marker for overall and disease-free survival and can be used as prognostic, predictive, and treatment-guiding biomarkers. In addition to enumeration, examining phenotypic, genotypic, and transcriptional characteristics of CTCs provides information relevant to treatment and outcomes. However, CTC analysis is hampered by 1) the low abundance of CTCs among the high background of white blood cells (WBCs) and 2) the short half-life of CTCs in the circulation. Due to their rarity, CTCs must be enriched before detection / analysis. Existing enrichment methods can be essentially divided into label-dependent and label-independent approaches (Joosse et al., EMBO Mol Med, 2015 Jan, Vol. 7(1):1-11). Label-dependent methods rely on the isolation of target cell populations based on biological characteristics, such as the expression of specific antigens on the cell surface, while label-independent methods exploit the physical properties of tumor cells, such as size, density, deformability, and other characteristics. Detection of CTCs is possible at the cellular level (based on antigen-specific staining of target proteins) and at the molecular level, for example, based on the detection of tumor-associated transcriptomic, genomic, or epigenomic abnormalities.

[0005] Another excellent liquid biopsy specimen is extracellular nucleic acid, such as circulating cell-free DNA (ccfDNA). The primary source of ccfDNA is mononucleosomal DNA fragments derived from apoptotic and necrotic cells. Furthermore, extracellular DNA also exists as vesicle-associated apoptotic bodies, microparticles, microvesicles, exosomes, or histone / DNA complexes, nucleosomes, and virosomes. In addition, extracellular RNA is present inside exosomes and other extracellular vesicles (EVs). In cancer patients, a certain proportion of ccfDNA is circulating tumor DNA (ctDNA) derived from tumor cells. Given tumor-specific abnormalities at the genomic and epigenomic levels, ctDNA can be efficiently detected among a high background of wild-type ccfDNA. Modern technologies (e.g., digital droplet PCR, BEAMing, next-generation sequencing) enable the development and rapid implementation of ccfDNA-based liquid biopsy tests into clinical practice (e.g., cobas EGFR Mutation Test v2, Therascreen KRAS Test). A similar concept is implemented in non-invasive prenatal testing in transplant histology and organ rejection, which rely on the detection of rare fetal DNA fragments in a background of maternal ccfDNA and the detection of organ-specific allogeneic DNA in a background of autologous wild-type ccfDNA, respectively.

[0006] Besides well-established biological targets such as CTCs and ccfDNA, additional target analytes such as extracellular vesicles (EVs), including mRNA and miRNA content, circulating non-coding RNAs (miRNA and others), and thrombocytes (platelets), can be analyzed in the context of liquid biopsies (see Anfossi et al., Nat Rev Clin Oncol, 2018 Sep, Vol. 15(9):541-563; In't Veld, Wurdinger, Blood, 2019 Mar 4, p.i.: blood-2018-12-852830). Furthermore, genomic and epigenomic profiling of cell subpopulations contained in cell-containing body fluid samples, such as peripheral mononuclear blood cells (PMBCs), can be useful biomarkers for early diagnosis and immunosurveillance monitoring in cancer patients (see Shen et al., Nature, 2018 Nov, Vol. 563(7732):579-583; Abu Ali Ibn Sina et al., Nature Communications, 2018, Vol. 9, Article number: 4915 and Nichita et al., Aliment Pharmacol Ther, 2014 Mar, Vol. 39(5):507-17). Despite the well-recognized clinical potential of these biological targets contained in bodily fluid samples such as blood, their utilization remains challenging. Existing methods based on the analysis of molecular biomarkers contained in cell-free circulating nucleic acids, EVs, or CTCs to obtain cancer-related information often suffer from shortcomings in sensitivity and / or robustness. Considering the role of liquid biopsies as companion diagnostics in personalized medicine, a complete and standardized workflow for liquid biopsy analysis is required. Preanalytical conditions can significantly affect the impact of analytical testing. Stabilization is required for all liquid biopsy specimens if testing is performed more than 3–4 hours after blood collection. Stabilization of the biological target of interest must be sufficient and reliable. Currently, blood stabilization tubes (BCTs) are available for either CTC analysis (e.g., CellSave, Transfix) or ctDNA analysis (Streck BCT, PAXgene Blood ccfDNA Tube). Although some of these tubes, such as the Streck BCT, claim compatibility with CTC analysis, such claims are essentially limited to one specific CTC enrichment and detection technique. Furthermore, the use of formaldehyde or formaldehyde-releasing substances (e.g., as utilized in Streck BCT) is problematic because it induces crosslinks between nucleic acid molecules or between proteins and nucleic acids, thereby compromising the effectiveness of extracellular nucleic acid isolation and downstream analysis. Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present invention to overcome at least one drawback of the prior art and to provide an improved liquid biopsy-based analytical method. Specifically, it is an object of the present disclosure to provide a method that allows reliable enrichment and analysis of multiple biological targets from a single-cell-containing bodily fluid sample. [Means for solving the problem]

[0008] The present disclosure provides methods and workflows for, for example, the simultaneous stabilization, enrichment, and detection of cell subpopulations, such as target cells (e.g., CTCs), and extracellular nucleic acids, such as extracellular DNA, from the same cell-containing bodily fluid sample, as well as the simultaneous stabilization, enrichment, and analysis of other biological targets, such as extracellular vesicles (EVs), from such stabilized samples. Furthermore, high-quality intracellular nucleic acids, such as genomic DNA (gDNA), can be isolated from the cellular fraction of stabilized cell-containing bodily fluid samples. In particular, a workflow is provided for the parallel liquid biopsy analysis of extracellular DNA, CTCs, EVs, and gDNA from single-cell-containing bodily fluid samples collected and stabilized by the stabilization techniques according to the present disclosure.

[0009] According to a first aspect, there is provided a method for stabilizing and enriching multiple biological targets in a cell-containing body fluid, the method comprising: (A) The cell-containing body fluid is treated with one or more of the following stabilizing factors: (a) at least one primary, secondary, or tertiary amide; (b) at least one poly(oxyethylene) polymer, and / or (c) at least one apoptosis inhibitor providing a stabilized cell-containing bodily fluid sample by contacting the sample with a stabilizing composition comprising: (B) maintaining the stabilized cell-containing bodily fluid sample for a stabilization period; and (C) processing the stabilized cell-containing body fluid sample to isolate three or more biological targets selected from the group consisting of rare cells, extracellular nucleic acids, extracellular vesicles, and intracellular nucleic acids from the stabilized cell-containing body fluid. The method comprises: (D) analyzing the enriched three or more biological targets. It may further include:

[0010] Other objects, features, advantages, and aspects of the present application will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the present application, are given by way of illustration only. [Brief explanation of the drawings]

[0011] [Figure 1] Immunocytochemical staining of MCF7 breast cancer cell line cells for human pan-cytokeratin (green) and cell nuclei (blue). The top panel shows staining in untreated MCF7 cells. The bottom panel represents staining of MCF7 cells stabilized in the stabilization solution of the present disclosure for 30 minutes. [Figure 2] Blood sample after centrifugation using Ficoll-Paque density gradient medium. Blood sample collected in EDTA-containing BCT and diluted with PBS served as a reference. The layers (from top to bottom) are platelet-rich plasma, PBMC ring, Ficoll, and erythrocyte-enriched fraction. In stabilized samples diluted with PBS, the aforementioned fractions cannot be observed; they only exist after the addition of 5% glucose or 0.9% NaCl + 0.1 M glycerol-containing solutions. This allows for the restoration of correct layering to obtain the various fractions. [Figure 3] Detection of spiked tumor cells by AdnaTest ProstateCancerPanel AR-V7 from blood collected and stored in PAXgene Blood ccfDNA tubes at experimental time points 3, 24, 30, and 48 hours after spiking. Materials: Blood collected in PAXgene Blood ccfDNA tubes and spiked with 20 LNCaP95 cells / 5 mL blood or 20 μL PBS / 5 mL blood and stored at 2–8°C. CTC enrichment and detection: AdnaTest ProstateCancerPanel AR-V7. Figure 3A shows the results for a sample spiked with 20 LNCaP95 cells / 5 mL blood. Figure 3B shows the results for a sample spiked with PBS only (non-spiked control sample). [Figure 4-1] Detection of spiked tumor cells by AdnaTest ProstateCancerPanel AR-V7 in blood collected and stored in PAXgene Blood ccfDNA tubes (Figure 4A; n=11) and Streck Cell-Free DNA BCT (Figure 4B; n=8) at experimental time points 3, 24, 48, and 72 hours after spike. Materials: Blood collected in PAXgene Blood ccfDNA tubes and Cell-Free DNA BCT (Streck), spiked with 20 LNCaP95 cells / 5 mL of blood, and stored at 2–8°C (PAX) and RT (Streck). CTC enrichment and detection: AdnaTest ProstateCancer Panel AR-V7. Figures 4C and 4D show the performance of AdnaTest ProstateCancer Panel AR-V7 on PAXgene Blood ccfDNA-stabilized blood spiked with 20 LNCaP95 cells / 5 mL blood stored at either 2-8°C or room temperature for 3, 24, 48, or 72 hours. Materials: Blood collected in PAXgene Blood ccfDNA tubes and spiked with 20 LNCaP95 cells / 5 mL blood. CTC enrichment and detection: AdnaTest ProstateCancerPanel AR-V7. [Figure 4-2] Figure 4-1 continued. [Figure 5] Figure 1 shows the cell capture efficiency using the Parsortix cell enrichment workflow when processing EDTA-stabilized blood or blood stabilized using the stabilization technique according to the present disclosure. Blood collected in PAXgene Blood ccfDNA tubes is compatible with and can be processed after 3 days of storage at room temperature. [Figure 6] RT-qPCR analysis of RNA obtained from purified EVs. Lower Ct values ​​indicate better results. [Figure 7]Schematic representation of the AdnaTestSelect and -Detect procedure, with the option to collect CTC-depleted blood after CTC enrichment for subsequent ccfDNA and gDNA isolation (see also Figure 11). [Figure 8] Evaluation of absolute differences in expression of 66 and 500 bp fragments of the human 18S rDNA gene (left and right panels, respectively) in samples after CTC enrichment (CTC-depleted blood) and control samples (i.e., samples without CTC enrichment) over storage time. Box plots show the median (horizontal line) and the 25-75% interquartile range (box), as well as the minimum and maximum values ​​of the data range (whiskers) and outliner (points outside the whiskers). P values ​​correspond to unpaired two-tailed t-tests. [Figure 9] Evaluation of gDNA yield from 200 μL of whole blood (i.e., samples without CTC enrichment, n = 3 donors) and post-CTC enrichment samples (n = 8 donors) at 3 hours after spike-in and at all time points (3-72 h, n = 11 donors, 12 samples without CTC enrichment and 32 CTC-depleted samples). All data are shown as box plots, with median and quartiles included within the box and 10 / 90 percentiles as two-sided. Individual data points are overlaid as circles. P values ​​correspond to unpaired two-sided t-tests. [Figure 10] Overview of various options for liquid biopsy-based analysis that are compatible with PAXgene Blood ccfDNA tubes according to the methods of the present invention. [Figure 11] 1 is an exemplary liquid biopsy-based workflow for analyzing multiple targets from a single stabilized blood sample. As disclosed herein, the stabilization techniques according to the present invention allow for long-term storage of the stabilized blood sample at room temperature before processing the stabilized blood sample according to step (D). [Figure 12]Left panel: Blood samples collected in EDTA and PAXgene Blood ccfDNA tubes (left and right, respectively) after centrifugation through Ficoll-Paque. The layers (from top to bottom) are platelet-rich plasma, PBMC ring, and erythrocyte-enriched fraction. In PAX samples diluted with PBS, the above fractions are not clearly separated. Right panel: Relative difference in MNC recovery observed in PAX-stabilized samples compared to EDTA samples (taken as reference, n=8). [Figure 13] AdnaTest ProstateCancerPanel AR-V7 detection of spiked tumor cells from blood collected and stored in PAXgene Blood ccfDNA tubes at experimental time points of 3, 24, 30, 48, 72, 120, and 144 hours after spike. Materials: Blood collected in PAXgene Blood ccfDNA tubes, spiked with 20 LNCaP95 cells / 5 mL blood, and stored at 2-8°C. CTC enrichment and detection: AdnaTest ProstateCancerPanel AR-V7. Figure 13 shows the performance of the AdnaTest ProstateCancer Panel AR-V7 test for detecting tumor cells spiked into blood collected in PAXgene Blood ccfDNA tubes. [Figure 14] Test performance with respect to storage temperature. CTC enrichment and detection by AdnaTest ProstateCancerPanel AR-V7 at experimental time points of 3, 24, and 72 hours after spikes stored at room temperature (Figure 14A) or 3, 24, 30, 48, 72, 120, and 144 hours after spikes stored at 2-8°C (Figure 14B). Materials: Blood collected in PAXgene Blood ccfDNA tubes and spiked with 20 LNCaP95 cells / 5 mL blood. [Figure 15]Test performance with respect to the number of spiked tumor cells to assess the limit of detection (LOD). Detection of spiked tumor cells by AdnaTest ProstateCancerPanel AR-V7 from blood collected and stored in PAXgene Blood ccfDNA tubes at experimental time points 3, 24, 48, 72, 120, and 144 hours after spiking. Materials: Blood collected in PAXgene Blood ccfDNA tubes, spiked with either 5 LNCaP95 cells / 5 mL (Figure 15A) or 20 LNCaP95 cells / 5 mL blood (Figure 15B), and stored at 2-8°C. CTC enrichment and detection: AdnaTest ProstateCancerPanel AR-V7. [Figure 16] Test performance depended on the plasma preparation regimen. Blood samples were used for CTC enrichment, and CTC-depleted blood was used for plasma preparation (Figure 16A). Alternatively, plasma was first prepared (1900g for 15 min), and then the cell fraction was reconstituted to the initial volume with PBS and used for CTC enrichment (Figure 16B). Both plasma preparation methods performed equally well by allowing detection of spiked tumor cells by AdnaTest ProstateCancerPanel AR-V7 from blood collected and stored in PAXgene Blood ccfDNA tubes at experimental time points 3, 24, 48, and 72 hours after spike. [Figure 17] Performance of the EZ1 test depends on the plasma preparation regimen. The same two plasma preparation methods as in Figure 16 were performed by first enriching CTCs and then preparing plasma (Figure 17A), or by first preparing plasma and then enriching CTCs (Figure 17B). Similar satisfactory results were observed when the same experiments as those performed in Figure 16 were performed on the EZ1 instrument (automated solution) using the EZ1-compatible AdnaTest. Tumor cells spiked from blood collected and stored in PAXgene Blood ccfDNA tubes were detected by the AdnaTest ProstateCancerPanel AR-V7 at experimental time points 3, 24, 48, 72, and 144 hours after spike. [Figure 18-1] Detection of spiked tumor cells by AdnaTest ProstateCancerPanel AR-V7 from blood collected and stored in PAXgene Blood ccfDNA tubes (Figures 18A, 18C, 18E, and 18G) compared to AdnaTest Prostate Cancer (also called "ProstateDirect"; Figures 18B, 18D, 18F, and 18H) at experimental time points of 3, 24, 48, and 72 hours after spiking. Materials: Blood collected in PAXgene Blood ccfDNA tubes and spiked with LNCaP95 cells / 5 mL blood. Figures 18A and 18B show the performance of the test by spiking with 20 LNCaP95 cells / 5 mL blood and storing at 2-8°C. Figures 18C and 18D show the performance of the test by spiking with 20 LNCaP95 cells / 5 mL blood and storing at room temperature. Figures 18E and 18F show the performance of the assay by spiking with 5 LNCaP95 cells / 5 mL blood and storing at 2-8° C. Figures 18G and 18H show the performance of the assay using an alternative plasma production technique, where plasma was produced first and the cellular fraction was used for CTC enrichment. [Figure 18-2] Figure 18-1 continued. [Figure 19] Performance of the AdnaTest ColonCancer. Detection of spiked tumor cells by the AdnaTest ColonCancer in blood collected and stored in PAXgene Blood ccfDNA tubes (Figure 19A) and ACD-A BCT (Figure 19B) at experimental time points of 3, 24, 48, and 72 hours after spiking. Materials: Blood collected in PAXgene Blood ccfDNA tubes and ACD-A BCT, spiked with 20 T48 cells / 5 mL of blood, and stored at 2-8°C. CTC enrichment and detection: AdnaTest ColonCancer. Figures 19A and 19B show that the PAXgene Blood ccfDNA tube is compatible with the AdnaTest ColonCancer, allowing detection of tumor cells (100% sensitivity) when samples are stored for less than 72 hours. [Figure 20]Detection rate of spiked tumor cells (50 MCF7) after harvest (Fig. 20A) and in-cassette staining (Fig. 20B). "T" means the number of days of storage at room temperature (0, 1, 2, 3 days). [Figure 21] Presence or absence of tumor cell staining after Parsortix enrichment. Fluorescent green - anti-pankeratinizing antibody staining (specific for tumor cells); fluorescent blue - DAPI (nuclear stain). Storage for 0 (T0) or 2 (T2) days. [Figure 22] Detection of spiked tumor cells after Parsortix-based enrichment stored in PAXgene Blood ccfDNA tubes compared to AdnaTest ProstateCancer (also called "ProstateDirect"; FIG. 22B) using the detection portion of the AdnaTest ProstateCancerPanel AR-V7 (FIG. 22A). FIG. 22 shows that cells spiked into PAX ccfDNA collected blood samples and stored for up to 3 days can be efficiently detected as if spiked into EDTA collected samples. [Figure 23] Multimode workflow for analysis of ccfRNA, ccfDNA, and gDNA used in Example 7. [Figure 24a] CT values ​​of qPCR analysis of miR150, let7a and miR451 microRNAs, ACTB mRNA and 18S rDNA (ccfDNA) in PAXgene and EDTA plasma prepared directly after blood collection (test time point = TTP0) and extracted using the indicated kits. [Figure 24b] Calculated fold changes (relative to TTP0) for qPCR analysis of miR150, let7a, miR451 microRNAs, and ACTB mRNA in PAXgene and EDTA plasma generated after 1, 3, or 6 days of whole blood storage. RNA was extracted using the indicated kits. [Figure 25a]CT values ​​of qPCR analysis of miR150, let7a, and miR451 microRNAs in PAXgene, Streck cfDNA, Streck RNA, and Biomatrica plasma generated directly after blood collection (TTP0) and extracted using the indicated kits. [Figure 25b] Calculated fold changes (relative to TTP0) from qPCR analysis of miR150, let7a, miR451 microRNAs, and 18S rDNA in PAXgene, Streck cfDNA, Streck RNA, and Biomatrica plasma generated after 3 days of storage (T3d). RNA was extracted using the indicated kits. [Figure 26] Concentration and DNA Integrity Index (DIN) assessment of gDNA extracted from whole blood in PAXgene Blood ccfDNA tubes, Streck cfDNA, Streck RNA, and Biomatrica tubes. DNA was extracted from the cellular fraction after the initial plasma centrifugation step using the QIAamp Blood DNA kit and analyzed by Agilent Genomic DNA ScreenTape® on a TapeStation System. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present disclosure provides an advantageous method for stabilizing and enriching multiple biological targets in cell-containing bodily fluids, comprising: (A) The cell-containing body fluid is treated with one or more of the following stabilizing factors: (a) at least one primary, secondary, or tertiary amide; (b) at least one poly(oxyethylene) polymer, and / or (c) at least one apoptosis inhibitor; providing a stabilized cell-containing bodily fluid sample by contacting the sample with a stabilizing composition comprising: (B) maintaining the stabilized cell-containing bodily fluid sample for a stabilization period; and (C) processing the stabilized cell-containing body fluid sample to enrich from the stabilized cell-containing body fluid three or more biological targets selected from the group consisting of at least one cell subpopulation, extracellular nucleic acids, extracellular vesicles, and intracellular nucleic acids. The method comprises: (D) further processing the enriched three or more biological targets for analysis. It may further include: Each individual step of the method, as well as suitable and preferred embodiments of the method, are described in detail below.

[0013] Process (A) In step (A), the cell-containing body fluid is treated with the following stabilizing factors: (a) at least one primary, secondary, or tertiary amide; (b) at least one poly(oxyethylene) polymer, and / or (c) at least one apoptosis inhibitor The stabilized cell-containing biological fluid sample is obtained by contacting the sample with a stabilizing composition comprising one or more, two or more, or all three of the above. The stabilizing composition can be contained in, e.g., pre-filled with, a collection container, e.g., a collection tube. The cell-containing biological sample can be introduced into the collection container. The step of contacting the cell-containing biological fluid with the stabilizing composition is performed ex vivo.

[0014] The advantageous stabilizing effects of individual agents, and advantageous stabilizing compositions comprising combinations of these agents, in stabilizing cell-containing body fluid samples are disclosed, for example, in WO 2013 / 045457, WO 2013 / 045458, WO 2014 / 146780, WO 2014 / 146781, WO 2014 / 146782, WO 2014 / 049022, WO 2015 / 140218, and WO 2017 / 085321, which are incorporated herein by reference. Advantageous stabilizing compositions comprising combinations of stabilizing agents (a)-(c) are also described elsewhere herein and are referred to as the present disclosure. As shown in the examples that follow and supported by the documents mentioned above, parallel processing and analysis of different biological targets of interest contained in cell-containing body fluids is possible. The stabilization techniques used in the present methods advantageously stabilize multiple biological targets of interest upon contact with cell-containing body fluids, including rare cells (e.g., circulating tumor cells), extracellular nucleic acids (e.g., extracellular DNA and RNA), extracellular vesicles, and intracellular nucleic acids (e.g., genomic DNA). As shown in the examples that follow, multiple biological targets of interest can be recovered from stabilized cell-containing body fluid samples and subjected to classical analysis and detection methods. This allows for multimodal analysis of various biological targets of great interest from a single stabilized cell-containing body fluid.

[0015] Process (B) In (B), the stabilized cell-containing body fluid sample is maintained for the intended stabilization period. The stabilized cell-containing body fluid sample can be processed, for example, immediately after stabilization, or immediately after stabilization (e.g., within 3 hours), or can be maintained for a longer storage period. It is particularly advantageous to be able to store the stabilized cell-containing body fluid sample for a long storage period. The biological target contained in the stabilized sample is also preserved for a long storage period.

[0016] In some embodiments, (B) includes storing the stabilized cell-containing bodily fluid sample prior to processing step (C). Storing can include, for example, transporting the stabilized cell-containing bodily fluid sample from the location where it was collected and stabilized to a location different from where it was collected and stabilized for further processing. The stabilized cell-containing body fluid sample can be stored for up to 12 hours, or up to 24 hours, prior to processing step (C). As shown in the examples, the stabilized cell-containing body fluid sample can be maintained for up to 30 hours, up to 36 hours, or up to 48 hours prior to processing step (C). In some embodiments, the stabilized cell-containing body fluid sample is maintained for up to 50 hours, or up to 72 hours prior to processing step (C). When maintaining the stabilized cell-containing body fluid sample for the intended stabilization period, it is advantageous if the stabilized sample is not subjected to a freezing step, which can damage the cells contained in the sample. Therefore, avoiding the freezing step is advantageous because it supports the preservation of the cell-containing body fluid sample.

[0017] In some embodiments, the stabilized cell-containing body fluid sample is maintained at room temperature (e.g., 15-25°C) for the intended stabilization period. In other embodiments, the sample is cooled, e.g., maintained at a temperature of 1-14°C, such as 1-12°C, or 2-10°C, or 2-8°C. In some embodiments, the stabilized cell-containing body fluid sample, such as blood, can be maintained at 2-8°C for up to 72 hours. In some embodiments, the stabilized cell-containing body fluid sample is maintained for at least 4 hours, or at least 6 hours, before performing processing step (C). In some embodiments, the stabilized cell-containing body fluid sample is maintained for at least 8 hours, or at least 12 hours, before performing processing step (C). In some embodiments, the stabilized cell-containing body fluid sample is maintained for at least 24 hours, at least 30 hours, or at least 48 to 72 hours (or more) before performing processing step (C).

[0018] Process (C) After the stabilization period, the stabilized cell-containing body fluid sample is processed to enrich the stabilized cell-containing body fluid for three or more biological targets selected from the group consisting of at least one cell subpopulation, extracellular nucleic acids, extracellular vesicles, and intracellular nucleic acids. As disclosed herein, it is highly advantageous that multiple different biological targets of interest can be stabilized within a cell-containing body fluid and subsequently recovered from the same stabilized sample, even after extended stabilization periods, thereby enabling parallel / simultaneous recovery and analysis of multiple different biological targets from a single stabilized cell-containing body fluid in an efficient workflow.

[0019] As disclosed herein, in one embodiment, at least one enriched cell population comprises or consists essentially of target rare cells. In some embodiments, the target rare cells are tumor cells, such as circulating tumor cells (CTCs). As discussed in the Background of the Invention, tumor cells, such as CTCs, represent biological targets of particular interest. As described herein, it is advantageous to separate the stabilized cell-containing bodily fluid sample into at least one cell-depleted fraction and at least one cell-containing fraction. This allows cells contained in the cell-containing bodily fluid sample to be enriched within the provided cell-containing fractions. At least one cell-containing fraction may comprise nucleated cells. In some embodiments, at least one cell-containing fraction consists essentially of nucleated cells. Preferred embodiments for the treatment of the stabilized cell-containing body fluid in step (C), and the biological targets, are described below.

[0020] Embodiment A According to embodiment A, the process in (C) is (aa) separating the stabilized cell-containing body fluid sample into at least one cell-containing fraction and at least one cell-depleted fraction; (bb) further processing the cell-containing fraction, wherein further processing the cell-containing fraction comprises: (i) enriching at least one cell subpopulation from said cell-containing fraction, e.g., comprising a target rare cell; and / or (ii) further processing, including enriching, e.g., purifying, intracellular nucleic acids (e.g., genomic DNA) from the cell-containing fraction; (cc) further processing said cell-depleted fraction, wherein further processing said cell-depleted fraction comprises: (i) enriching, e.g., purifying, extracellular nucleic acids (e.g., extracellular DNA) from the cell-depleted fraction; and / or (ii) further processing, including enriching the cell-depleted fraction for extracellular vesicles.

[0021] In this embodiment, the stabilized cell-containing body fluid sample (e.g., blood) is separated in (aa) into at least one cell-containing fraction (e.g., including nucleated blood cells and CTCs) and a cell-depleted fraction (e.g., plasma). Suitable separation methods are known in the art (e.g., involving centrifugation and / or filtration) and are described elsewhere herein. For example, when processing a (coagulated) stabilized blood sample according to step (aa) using a centrifugation-based separation method, the stabilized blood sample can be separated into a cell-depleted fraction (plasma), a cell-containing fraction (buffy coat, which may include white blood cells, and CTCs, if present, and optionally platelets), and a red blood cell fraction. The buffy coat can be further processed as a cell-containing fraction in step (bb), and the plasma fraction can be further processed as a cell-depleted fraction in step (cc).

[0022] The resulting cell-containing fraction of interest is then further processed in (bb). At least one cell subpopulation, such as target rare cells (e.g., CTCs), can be enriched from the resulting cell-containing fraction (see (i)). Furthermore, intracellular nucleic acids (e.g., genomic DNA) can be enriched and thus purified from the cell-containing fraction (see (ii)). In several embodiments, for example, at least one cell subpopulation comprising rare cells (e.g., CTCs) and intracellular nucleic acids (e.g., genomic DNA) are both enriched from the cell-containing fraction as biological targets of interest. For example, the cell subpopulation of interest, comprising rare cells (e.g., CTCs), can first be isolated from the cell-containing fraction of (i) before purifying intracellular nucleic acids (e.g., genomic DNA) from the remaining cell-containing fraction from which the target cell subpopulation (e.g., rare cells) has been removed / depleted in (ii). Advantageously, this embodiment allows the entire volume of the cell-containing fraction to be used to isolate the target cell subpopulation, which in one embodiment comprises rare cells (e.g., CTCs). In other embodiments, the cell-containing fraction is separated into at least two aliquots, with at least one aliquot being used to enrich for a cell subpopulation of interest (e.g., containing rare cells) and at least one aliquot being used to enrich for intracellular nucleic acids such as genomic DNA. In other embodiments, the cell-containing fraction is subdivided into at least two aliquots, with at least one aliquot being used to enrich for a cell subpopulation of interest (e.g., containing rare cells) and at least one aliquot being used to enrich for intracellular nucleic acids such as genomic DNA. The resulting cell-depleted fraction (e.g., plasma) is further processed in (cc) to isolate extracellular nucleic acids and / or enrich extracellular vesicles from the cell-depleted fraction (e.g., plasma). As disclosed herein, in an advantageous embodiment, extracellular DNA is purified from the cell-depleted fraction (e.g., plasma). Furthermore, as shown in the Examples, extracellular vesicles can be enriched from the cell-depleted fraction. Exemplary suitable and preferred methods for enriching extracellular vesicles are also described below. In several embodiments, both extracellular vesicles and extracellular nucleic acids, preferably extracellular DNA, are enriched from the cell-depleted fraction. For example, extracellular vesicles can be first isolated from the cell-depleted fraction, and then extracellular DNA can be enriched from the remaining cell-depleted fraction from which extracellular vesicles have previously been removed. In a further embodiment, the cell-depleted fraction is divided into at least two aliquots, at least one aliquot being used to enrich for extracellular vesicles and at least one aliquot being used to purify extracellular nucleic acids, such as extracellular DNA, from said aliquot.

[0023] Embodiment B According to embodiment B, the treatment in (C) is (aa) enriching at least one cell subpopulation from the stabilized cell-containing bodily fluid sample, e.g., comprising a target rare cell; (bb) separating the stabilized cell-containing bodily fluid sample enriched for and thus depleted from a cell subpopulation (e.g., including a target rare cell) into a cell-containing fraction and a cell-depleted fraction; (cc) further processing said cell-depleted fraction, wherein further processing said cell-depleted fraction comprises: (i) enriching the cell-depleted fraction for extracellular nucleic acids (which may be extracellular DNA); and / or (ii) enriching extracellular vesicles from the cell-depleted fraction; and further processing including (dd) optionally enriching the cell-containing fraction for intracellular nucleic acids, preferably genomic DNA.

[0024] In step (aa), at least one cell subpopulation, for example, including target rare cells (e.g., CTCs), is enriched from the stabilized cell-containing body fluid sample. By first isolating the target cell subpopulation of interest, for example, including rare cells, from the biological sample before separating the stabilized sample into a cell-containing fraction and a cell-depleted fraction, the overall handling time of the cell subpopulation is reduced. This is particularly advantageous when the cell subpopulation contains or essentially consists of rare cells, in order to prevent damage to these rare and valuable cells. In one embodiment, rare cells (e.g., CTCs) are enriched from the entire stabilized cell-containing body fluid sample. Advantageously, this allows the entire collected sample volume to be used to isolate rare cells (e.g., CTCs). This is advantageous considering that certain cells, such as CTCs, are often rare, and it is desirable to process a larger sample volume to ensure that the rare cells (e.g., CTCs) contained therein are enriched and detectable. In step (bb), the stabilized cell-containing body fluid sample, from which the target rare cells (or other cell subpopulation of interest) have been removed, is separated into a cell-containing fraction and a cell-depleted fraction. Thus, where the complete collected volume of the stabilized cell-containing body fluid sample was used to enrich for rare cells in step (aa), the entire stabilized cell-containing body fluid sample from which the rare cells, or a portion thereof, if desired, have been removed, is processed to provide a cell-containing fraction and a cell-depleted fraction. In step (cc), the cell-depleted fraction is further processed, details of which are described in conjunction with embodiment A above, and reference is also made to the respective disclosures which also apply here. Additionally, intracellular nucleic acids such as genomic DNA can be enriched from the cell-containing fraction in step (dd).

[0025] Embodiment C According to embodiment C, the process in (C) is (aa) dividing the stabilized cell-containing bodily fluid sample into at least two aliquots and enriching at least one cell population of interest, e.g., comprising rare cells, from at least one of the provided aliquots; (bb) providing at least one cell-containing fraction and at least one cell-depleted fraction; (cc) further processing said cell-depleted fraction, wherein further processing said cell-depleted fraction comprises: (i) enriching the cell-depleted fraction for extracellular nucleic acids (e.g., extracellular DNA); and / or (ii) further processing, comprising enriching the cell-depleted fraction for extracellular vesicles; (dd) optionally enriching the cell-containing fraction for intracellular nucleic acids, preferably genomic DNA.

[0026] In step (aa), the stabilized cell-containing body fluid sample is divided into at least two aliquots. At least one aliquot is used to enrich at least one cell population of interest, which can, for example, contain target rare cells (e.g., CTCs) or consist essentially of rare cells. This provides at least one aliquot of the stabilized cell-containing body fluid sample from which rare cells have been removed. The same applies when another cell subpopulation of interest is enriched. At least one additional aliquot corresponds to the original stabilized cell-containing body fluid from which rare cells (or other cell populations of interest) have not been removed. In step (bb), at least one cell-containing fraction and at least one cell-depleted fraction are provided. Step (bb) can include separating the stabilized cell-containing body fluid sample enriched for a target cell population (e.g., containing or consisting essentially of rare cells, such as CTCs) and / or any remaining stabilized cell-containing body fluid sample (aliquot) not used to enrich the target cell population in step (aa) into a cell-containing fraction and a cell-depleted fraction. When the stabilized cell-containing body fluid sample is divided into at least two aliquots, it is possible to process only the aliquot from which the target cells have not been removed to provide the cell-containing fraction and the cell-depleted fraction. Alternatively, at least one aliquot from which the target cells have been removed can be recombined with an additional aliquot of the original stabilized body fluid sample from which the target cells have not been removed. This pooling advantageously increases the volumes of the resulting cell-depleted and cell-containing fractions, which is beneficial for further processing and analysis of these fractions. Step (cc) and optional step (dd) correspond to embodiment B, see above disclosure.

[0027] Exemplary suitable and preferred methods for separating a sample into at least one cell-containing fraction and at least one cell-depleted fraction are also described below, and such methods can be used, in particular, in step (C) in embodiments A to C. In particular, exemplary suitable and preferred methods for enriching rare cells, such as CTCs, and other target cell subpopulations are described below that can be used in step (C) in embodiments A to C. As disclosed herein, the removed target cells, such as rare cells, can be further processed in step (D), for example, to isolate and subsequently detect intracellular nucleic acids (e.g., RNA). In particular, exemplary suitable and preferred methods for enriching extracellular vesicles such as exosomes that can be used in step (C) in embodiments A to C are also described below. As disclosed herein, the collected extracellular vesicles can be further processed in step (D), for example, to isolate and subsequently detect nucleic acids (e.g., RNA).

[0028] Method for separating a cell-containing body fluid sample into a cell-containing fraction and a cell-depleted fraction Methods for separating a cell-containing body fluid into at least one cell-containing fraction and at least one cell-depleted fraction are well known in the art and need not be described in detail. Common methods include, but are not limited to, centrifugation, filtration, and density gradient centrifugation. Different methods can also be combined. Such common methods can be advantageously used in conjunction with stabilization techniques in accordance with the present disclosure, which advantageously allows for the avoidance of the use of cross-linking agents for stabilization, thereby allowing the use of commonly established methods. This method is carried out to maintain the integrity of the cells contained in the sample. This is advantageous because, for example, cells are disrupted during separation, which would contaminate the extracellular nucleic acids contained in the cell-depleted fraction with cellular nucleic acids released from the disrupted cells.

[0029] According to one embodiment, at least one centrifugation step is performed to separate the cell-containing fraction from the cell-depleted fraction. In several embodiments, centrifugation can be performed at a temperature ranging from, for example, 800 to 3000 x g, e.g., 1000 to 2500 x g, or 1500 to 2000 x g. The duration of centrifugation can be, for example, 5 to 20 minutes, e.g., 10 to 15 minutes. Suitable conditions can be selected by those skilled in the art. The cell-depleted fraction can be collected as a supernatant. To ensure that any remaining cells and particulate matter (e.g., cell debris) have been removed from the cell-depleted fraction, the cell-depleted fraction can be removed from the resulting cell fraction(s) and subjected to a second centrifugation step (optionally at a higher speed). This can be advantageous for later purification of extracellular nucleic acids, such as extracellular DNA, from the cell-depleted fraction. It is also within the scope of the present disclosure to perform a filtration step to provide the cell-depleted fraction. Such methods are well known in the art and are used, for example, to obtain plasma from blood samples for subsequent purification of extracellular nucleic acids, such as extracellular DNA (see, e.g., Chiu et al., 2001 Clinical Chemistry 47:9 1607-1613; Sorber et al., Cancers 2019, 11, 458). In cases where it is desirable to recover exosomes and / or platelets from the cell-depleted fraction as the biological target(s) of interest, the separation procedure(s) are selected so that the exosomes and / or platelets remain in the cell-depleted fraction and are available for recovery therefrom. For example, the cell fraction obtained after the first centrifugation step can be used as the cell-containing fraction and further processed as described herein (e.g., to isolate intracellular nucleic acids, such as genomic DNA, and / or enrich for target cells (e.g., CTCs) therefrom).

[0030] Suitable centrifugation and / or filtration based separation methods can include, but are not limited to: - Separate the cell-depleted fraction from the cell fraction(s) by centrifugation at 1900 x g (15 min) and centrifugation of the cell-depleted fraction at 1900 x g (10 min). - Separating the cell-depleted fraction from the cellular fraction(s) by centrifugation at 1600 x g (10 min) and centrifuging the cell-depleted fraction at 16000 x g (10 min). - Separate the cell-depleted fraction from the cellular fraction(s) by centrifugation at 1600 x g (10 min) and then filter the cell-depleted fraction, for example, using a 0.2 µm to 0.8 µm filter. - Centrifugation at 1600 x g (10 min) and 16,000 g (10 min) followed by filtration, for example using a 0.2 μm to 0.8 μm filter. - Centrifuge at 1000 rpm (10 min) and 3000 rpm (10 min). Further combinations and variations are also possible.

[0031] For example, to avoid contamination of the contained biological targets (e.g., extracellular nucleic acids or extracellular vesicles) with cellular components, the provided cell-depleted fraction is in a substantially cell-free embodiment. Such cell-free fractions can be obtained using the centrifugation and / or separation-based methods described above. The resulting cell-depleted / cell-free fraction can be transferred to a new container. The fraction can be directly processed, e.g., to purify extracellular nucleic acids and / or extracellular vesicles therefrom, or can be stored (e.g., cooled or frozen) until use. The resulting cell-containing fraction, which is further processed, can further contain nucleated cells, and target cells (e.g., rare cells) and / or intracellular nucleic acids (e.g., genomic DNA) can be isolated from the fraction.

[0032] According to one core embodiment, the cell-containing body fluid is blood. Blood samples are very important because they are widely used for diagnostic purposes. When the cell-containing body fluid is blood, the stabilizing composition preferably comprises an anticoagulant, e.g., a chelating agent such as EDTA. The stabilized blood sample may be processed to provide a cell-depleted plasma fraction and a cell-containing cell fraction (such as a buffy coat), which are then further processed. Methods for producing plasma are well known in the art and include, but are not limited to, centrifugation and filtration, as well as combinations of such methods.

[0033] Subpopulations of cells and enrichment of such subpopulations, particularly enrichment of rare cells such as circulating tumor cells According to one embodiment, step (C) comprises enriching a cell subpopulation from the stabilized cell-containing body fluid sample. The target cell subpopulation can be enriched directly from the stabilized cell-containing body fluid, or can be enriched from a cell-containing, and therefore cell-depleted, fraction of the stabilized cell-containing body fluid (which can be obtained by separating the stabilized cell-containing body fluid sample into a cell-containing fraction and a cell-depleted fraction). The enriched subpopulation of cells can be processed and further analyzed (e.g., by analyzing the resulting cells and / or isolating intracellular nucleic acids from the population) as described herein. Desired cell subpopulations can be enriched using methods known in the art. A suitable method is disclosed below in combination with the enrichment of rare cells, and similar methods can be used for other cell populations. For example, specific cells can be enriched based on cell surface characteristics using affinity capture-based methods. Furthermore, cells can be separated and therefore enriched based on their density. For example, density gradient centrifugation can enrich PBMCs and other cell types in specific layers. Specific cells, i.e., each cell population, can also be enriched by sorting techniques, such as FACS sorting.

[0034] According to one embodiment, step (C) comprises enriching rare cells. Thus, according to one embodiment, the enriched cell subpopulation comprises the target rare cells. The enriched cell subpopulation can also consist essentially of the target rare cells. This depends on the enrichment method used. Rare cells are cells that are present in small amounts in a large population of background cells. Rare cells are typically present in the 10 5 Rare cells are found at concentrations of less than 1 in cells. Therefore, detecting, quantifying, and enriching rare cells is challenging. Rare cells are of great importance for various applications, such as the diagnosis and prognosis of many cancers, prenatal diagnosis, and the diagnosis of viral infections. Typical rare cells are circulating tumor cells (CTCs), circulating fetal cells (e.g., circulating in maternal blood), stem cells, and cells infected by viruses or parasites. Such rare cells are found, for example, in blood samples and other body fluids and can be enriched therefrom. Further rare cell types that can be enriched are circulating endothelial cells (CECs) and circulating endothelial progenitor cells (EPCs). Circulating mature endothelial cells (CECs), potential biomarkers of endothelial dysfunction in cancer, diabetes, cardiovascular, or acute kidney disease, have been reported in 10 6 ~10 8 A frequency of 10–100 CECs has been observed in 10 leukocytes. In comparison, the estimated frequency of CTCs is much lower, at 10 6 ~10 8 The number ranges from 1 to 10 CTCs per white blood cell.

[0035] Various methods for enriching rare cells, such as CTCs, are known and described in the art, and known methods can be used in combination with the present invention (see, for example, Neumann et al., Comput Struct Biotechnol J, 2018, Vol. 16: 190-195; Haber et al., Cancer Discov. 2014 June; 4(6): 650-661; and Chen, Lab Chip: 2014 February 21; 14(4): 625-645). Enrichment, separation, or quantification of rare cells can be performed by various methods based on physical properties, such as cell size, density, deformability, shape, electric polarizability, and magnetic susceptibility, and / or biological properties, such as surface properties (e.g., marker gene expression on the cell surface). Gradient-based centrifugation (e.g., using a Ficoll gradient) is a commonly used method for enriching specific cell types with a certain density. Filtration allows for enrichment of rare cells based on cell size. Another CTC enrichment principle uses microfluidics. Compared to filtration methods, microfluidic systems allow for the collection of CTC-enriched cell suspensions for downstream analysis, such as immunofluorescence labeling for single-cell isolation. CTCs and other rare cells can also be separated based on their electrical charge differences. Generally, CTC enrichment methods fall broadly into different classes, depending on whether they rely on the physical properties of tumor cells, the expression of specific cell surface markers, or the depletion of abundant cells (e.g., normal white blood cells) to enrich for untagged CTCs. For example, immunomagnetic methods based on antibody-mediated capture of cancer cells can also be used for CTC enrichment.

[0036] According to one embodiment, the target rare cells are tumor cells contained in a cell-containing body fluid sample. Preferably, circulating tumor cells (CTCs) are obtained as target rare cells from a stabilized body fluid sample, such as a stabilized blood sample. As disclosed in the Background of the Invention, circulating tumor cells are well known in the art. Generally, CTCs are cells that are released from primary tumors into the vasculature or lymphatics and transported throughout the body in the circulation. CTCs can be actively or inactively released. They can circulate in the blood and lymphatic systems as single cells or as aggregates (so-called circulating tumor microemboli). Thus, CTCs originate from primary tumors and can constitute viable seeds for the subsequent growth of additional tumors (metastases) in distant vital organs. They are closely associated with cancer metastasis, which is believed to be the primary cause of cancer mortality. CTCs can also originate from metastases. CTCs have been identified in many different cancers, and it is widely accepted that CTCs found in peripheral blood originate from solid tumors and are involved in the hematogenous spread of solid tumors to distant sites. As used herein, the term "CTC" particularly includes circulating cells derived from any type of tumor, particularly solid tumors, especially metastatic solid tumors. As used herein, the term "CTC" particularly includes (i) CTCs identified as cancer cells with intact, living nuclei that express cytokeratin or epithelial marker molecules (such as EpCam) and the absence of CD45; (ii) cytokeratin-negative (CK) CTCs, which are cancer stem cells or cells undergoing epithelial-mesenchymal transition (EMT), which may lack expression of epithelial markers such as cytokeratin or EpCam and CD45. - (iii) apoptotic CTCs, which are traditional CTCs that have undergone apoptosis (cell death); (iv) small CTCs, which are typically cytokeratin-positive and CD45-negative but resemble white blood cells in size and shape; and (v) dormant CTCs, as well as, for example, CTC clusters of two or more individual CTCs of any of the above types of CTCs, or mixtures of the above types of CTCs associated with each other. CTC clusters can be, for example, traditional small and / or CK -It may contain CTCs.

[0037] CTCs are generally very rare cells in bodily fluids. To provide information about CTCs, enrichment of tumor cells or removal of other nucleated cells in the blood is necessary. Any method can be used in combination with this method, which is suitable for enriching CTCs from stabilized cell-containing bodily fluid samples or their resulting cell-containing fractions. Because CTCs are often rare, most common CTC enrichment procedures involve co-isolation of other cell types along with the desired CTCs to ensure that the enriched CTCs are contained to a certain extent in a background of normal cells. Nevertheless, such methods are useful for enriching CTCs and therefore for analyzing them. Methods for enriching CTCs from various biological samples are well known in the art and have been summarized above. Exemplary suitable methods are briefly described below. CTCs can be enriched using various physical and / or affinity capture-based methods.CTCs can be enriched by methods that include positive selection of CTC cells, for example, by directly targeting CTCs, or by methods that include negative selection, for example, by depleting non-CTC cells (for example, white blood cells in the case of blood).For example, methods that use filtration-based methods, deformability or density, or other physical methods to enrich CTCs by size are also suitable.In addition, a combination of the above methods can be used.

[0038] According to a preferred embodiment, CTCs are enriched by affinity capture. Such affinity-based capture methods specifically bind CTCs to a surface (e.g., beads, membranes, or other surfaces). Specificity for CTCs is achieved by using one or more binding agents (e.g., antibodies) that bind to structures present on CTCs, such as epitopes or antigens. In several embodiments, the one or more binding agents bind to tumor-associated markers present on CTCs. For example, CTCs can be enriched using a solid phase (e.g., magnetic beads) coated with antibodies capable of capturing CTC cells. For CTC capture, a combination of two or more antibodies that bind with high specificity and affinity to epitopes or antigens on desired CTC cells can be used. Binding agents can also be selected to target target epitopes or antigens present on CTCs depending on the type of tumor. For example, depending on the type of primary tumor, various structures, such as epitopes or antigens, that can be targeted by binding agents (e.g., antibodies) may be present on CTCs, taking into account potential EMT or changes in tumor stem cell phenotype. For example, the use of such a binding agent (e.g., antibody)-based capture platform is advantageous because it can also enrich CTCs that undergo phenotypic changes during epithelial-mesenchymal transition (EMT) or exhibit tumor stemness. According to a preferred embodiment, the epitopes targeted by the binding agents are epithelial and / or tumor-associated antigens, such as EpCAM, EGFR, and HER2. A commercially available system for enriching circulating tumor cells is AdnaTest (QIAGEN). Another proposed method for enriching CTCs based on positive selection involves counting epithelial cells isolated from blood using antibody-magnetic nanoparticle conjugates targeting the epithelial cell surface marker EpCAM, followed by subsequent identification of CTCs using fluorescently labeled antibodies against cytokeratins (CK8, 18, 19) and fluorescent nuclear stains. A suitable method is used in a commercially available system from CellSearch (Menarini / Veridex LLC). Other known methods for enriching CTCs, and thus isolating CTCs, include, but are not limited to, the Epic Sciences method, the ISET test, the use of microfluidic cell sorters (μFCS, which uses a modified dam-type physical barrier to separate and capture CTCs based on size differences, e.g., from unprocessed whole blood), ScreenCell (a filtration-based device that enables sensitive and specific isolation of CTCs, e.g., from human whole blood), Clearbridge, Parsortix, and IsoFlux.

[0039] According to one embodiment, the stabilized sample is a blood sample, and step (C) comprises enriching PBMCs from the stabilized sample, optionally using an enrichment method based on density gradient centrifugation. Suitable methods are described below. As disclosed in the Background of the Invention, genomic and / or epigenomic profiling of peripheral mononuclear blood cells (PMBCs) presents biomarkers of interest for early diagnosis and monitoring of immunosurveillance in cancer patients. This can further be used to analyze contained CTCs, for example, by isolating intracellular nucleic acids such as RNA and detecting CTC-specific target nucleic acid molecules. Furthermore, the enriched PBMC fraction can be used to further enrich and purify specific cell types, such as CTCs.

[0040] According to one embodiment, the cell-containing body fluid sample is blood, and step (C) comprises enriching the stabilized sample for target lymphocytes as a cell subpopulation. According to one embodiment, the lymphocytes are selected from T4 and / or T8 lymphocytes. According to one embodiment, the stabilized blood sample is obtained from a patient with an immune deficiency. Analysis of T4 and T8 lymphocytes in such samples is of particular diagnostic value.

[0041] According to one embodiment, the cell-containing body fluid sample is blood, and step (C) comprises enriching platelets as a cell subpopulation from the stabilized sample. Optionally, step (D) is performed, comprising isolating RNA from the enriched platelets. Methods for enriching platelets from a blood sample are known in the art and can be used in conjunction with the present invention. In some embodiments, platelet-rich plasma (PRP) is obtained from the stabilized (anticoagulated) blood sample by centrifugation. Suitable methods for obtaining platelet-rich plasma have been described in the art (see also Sorber et al., 2019) and can be used and / or adapted for the present disclosure. Platelet-rich plasma is depleted of other white blood cells and red blood cells. Platelets can then be isolated from each portion of the obtained platelet-rich plasma using methods known in the art. In some embodiments, the remaining plasma portion not used to isolate platelets can be further processed to isolate extracellular nucleic acids (e.g., ccfDNA) and / or exosomes therefrom. In some embodiments, the remaining plasma portion is centrifuged and / or filtered again to remove any remaining cells or cell debris prior to isolating extracellular nucleic acids and / or exosomes from the resulting supernatant.

[0042] According to one embodiment, the cell-containing body fluid sample is blood, and step (C) comprises enriching blast cells as a target cell subpopulation from the stabilized sample. The blast cells are enriched by affinity capture, optionally using magnetic particles. The blast cells can be enriched, for example, by targeting cell surface markers, which may be CD34 and / or CD117. Analysis of blast cells is useful, for example, when the stabilized blood sample is obtained from a patient with acute myeloid leukemia. As described above, additional rare cell types that can be enriched from stabilized cell-containing bodily fluid samples are circulating endothelial cells (CECs) and circulating endothelial progenitor cells (EPCs). Such target cells can be identified and enriched based on specific markers, including, but not limited to, CD31, CD34, CD105, CD133, and CD146.

[0043] Density gradient centrifugation step According to one embodiment, processing step (C) comprises subjecting the stabilized blood sample or a cellular fraction thereof to a density gradient centrifugation step. This density gradient centrifugation step allows the stabilized cell-containing body fluid sample to be separated into a cell-depleted plasma fraction (or a cell-depleted liquid, if a cellular fraction obtained from the stabilized cell-containing body fluid sample is used as input material) and various cell-containing fractions. In several embodiments, the stabilized cell-containing body fluid sample is first processed in step (C) to obtain cell-containing and cell-depleted fractions. The methods described above (e.g., centrifugation and / or filtration) can be used for this purpose. For example, the stabilized blood sample can be separated into a plasma fraction and a cellular fraction. The obtained plasma fraction can then be used for enrichment of (i) extracellular nucleic acids and / or (ii) extracellular vesicles, as described elsewhere. The obtained cellular fraction can then be subjected to density gradient centrifugation. For this purpose, the cellular fraction can be diluted using a diluent. The diluted cellular fraction is then subjected to density gradient centrifugation. A density gradient centrifugation procedure can then be performed as known and described for cell-containing body fluids, such as blood. An embodiment of density gradient centrifugation is described below in the examples using stabilized blood samples, however, other types of stabilized cell-containing body fluid samples can also be processed appropriately.

[0044] The stabilized blood sample (or its cell fraction) is contacted with a density gradient medium. Suitable density gradient media are commercially available, including, but not limited to, Ficoll®, Ficoll®-Paque, and Lymphopure. Density gradient centrifugation techniques (such as Ficoll® Paque and OncoQuick®) can be used to separate peripheral blood mononuclear cells from other components of whole blood, including red blood cells and polymorphonuclear cells (e.g., granulocytes), based on their different cell densities. The stabilized blood sample (or its cell fraction) is diluted with a diluent before the density gradient centrifugation step, preferably before contacting the stabilized blood sample (or its cell fraction) with the density gradient medium. The dilution may be at least a 1:1 ratio. The diluted stabilized blood sample (or a diluted cellular fraction thereof) can be layered on top of (preferably) or underneath a density gradient medium and centrifuged to separate distinct cell populations from the plasma, typically pelleting red blood cells and granulocytes to the bottom of the tube and leaving mononuclear cells (including rare cells such as CTCs) in an interface layer above the gradient medium layer, accessible for collection and analysis due to their low density. However, as described herein and known in the art, the density of cell populations can be artificially altered to locate within distinct cell-containing layers. For example, the RosetteSep™ CTC Enrichment Cocktail (StemCell technologies) combined with Ficoll® separation utilizes a tetrameric antibody complex that crosslinks CD45-expressing white blood cells to red blood cells, artificially altering the density of labeled white blood cells, which are then pelleted to the bottom, thereby enriching the interface layer for CTCs for CTC enrichment.

[0045] As shown in the examples, the stabilization compositions used in accordance with the present disclosure to stabilize blood samples can result in altered layer patterns after density gradient centrifugation in embodiments using a combination of stabilizing factors (a)-(c). To avoid handling errors, it is advantageous to pre-treat the stabilized blood sample (or its cellular fraction) to ensure that the stabilized blood sample (or its cellular fraction) produces a layer pattern similar to that of a typical EDTA-stabilized blood sample (or its cellular fraction) upon density gradient centrifugation. It has been discovered that this can be achieved by diluting the stabilized blood sample (or its cellular fraction) with a diluent other than the commonly used PBS. The diluent used can be a hypotonic or isotonic solution as described herein. The dilution can be performed at a ratio of at least 1:1.

[0046] In one embodiment, the dilute solution contains a tonicity-adjusting agent. Tonicity-adjusting agents are known in the art and include compounds such as salts (e.g., sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate, calcium carbonate, sodium lactate), and polyols such as sugars (e.g., glucose, dextran, dextrose, lactose, trehalose) and sugar alcohols (e.g., glycerol, mannitol, sorbitol, xylitol). The dilute solution may contain a polyol. As used herein, the term "polyol" refers to a substance having multiple hydroxyl groups, including sugars (reducing and non-reducing sugars) and sugar alcohols. Polyols may contain at least three, at least four, or at least five hydroxyl groups. In certain embodiments, the polyol has a molecular weight of ≦600 Da (e.g., in the range of 120-400 Da). A "reducing sugar" is a sugar that contains a free aldehyde or ketone group and is capable of reducing metal ions or covalently reacting with lysine and other amino groups in proteins. A "non-reducing sugar" is a sugar that lacks a free aldehyde or ketone group and is not oxidized by mild oxidizing agents such as Fehling's solution or Benedict's solution. Examples of reducing and non-reducing sugars are known to those skilled in the art. In some embodiments, the compound (tonicity modifier / polyol) involved is capable of permeating cell membranes.

[0047] In some embodiments, the polyol included as a tonicity adjuster is a sugar or a sugar alcohol. Combinations of sugars and / or sugar alcohols can also be used. The sugar can be a reducing sugar or a non-reducing sugar. In some embodiments, the sugar is a reducing sugar. In some embodiments, the dilute solution includes glucose. In one embodiment, the dilute solution includes a reducing sugar (which may be glucose) at a concentration ranging from 2-10%, 3-7%, or 4-6% (w / v). In further embodiments, the dilute solution includes a sugar alcohol (which may be glycerol). In some embodiments, the dilute solution includes a salt. The salt can act as a tonicity adjuster. The salt can be an alkali metal salt, optionally a chloride salt such as sodium chloride. In some embodiments, the dilute solution includes a sugar alcohol (such as glycerol) and a salt (which may be an alkali metal salt, such as sodium chloride). In one embodiment, the diluent solution comprises up to 0.5 M glycerol and up to 2% sodium chloride; optionally, the diluent solution may comprise 0.7-1.2% sodium chloride and 0.075-0.15 M glycerol. In embodiments, the diluent solution comprises (i) 5% (w / v) glucose, (ii) 0.9% NaCl + 0.1 M glycerol, and (iii) at least one tonicity adjuster, and is selected from a diluent solution having an osmolality that matches the osmolality of the diluent solution defined in (i) or (ii), or an osmolality that is within ±20%, ±15%, or ±10% of the osmolality of the solution defined in (i) or (ii).

[0048] According to one embodiment, the diluent solution comprises DMSO. The diluent solution may comprise DMSO at a concentration of 1% to 10% (v / v), for example 1% to 5% (v / v).

[0049] In some embodiments, the stabilized blood sample is incubated in the dilution solution for 10 minutes or less, 5 minutes or less, or 3 minutes or less, after which the diluted stabilized blood sample (or a cellular fraction thereof) is contacted with the density gradient medium. Preferably, the diluted stabilized blood sample (or a cellular fraction thereof) is processed directly after dilution and contacted with the density gradient medium. As shown in the examples, the use of such a dilution solution advantageously restores the density of the stabilized blood cells, ensuring that essentially the same type of layer as that formed in an EDTA-stabilized blood sample can be formed after density gradient centrifugation. Various layers are formed after density gradient centrifugation, resulting in a distinct PBMC layer. The formed layers may include (from top to bottom) a top layer (e.g., containing plasma in the case of a stabilized blood sample, or primarily containing the dilution solution when processing a cellular fraction of a stabilized blood sample), a PBMC layer (also containing CTCs, if present in the stabilized sample), a density gradient medium layer, and even granulocytes and red blood cells. Additional layers may form below the granulocyte / red blood cell layer. It is important that a distinct PBMC layer is formed, as this layer can be further processed as a cell subpopulation, for example, for CTC analysis. In one embodiment, the method includes collecting the formed PBMC layer to provide a PBMC fraction. The collected PBMC fraction can be washed. Washing can be performed using a buffer solution (which may be PBS buffer or another suitable solution). The collected PBMC layer can be further processed and / or analyzed. As disclosed in the Background of the Invention, genomic and / or epigenomic profiling of peripheral mononuclear blood cells (PMBCs) presents biomarkers of interest for early diagnosis and monitoring of immunosurveillance in cancer patients. Furthermore, the layer may be used to enrich specific cell types, such as CTCs, from the layer. When the stabilized blood sample is subjected to density gradient centrifugation, a plasma fraction that may form above the PBMC layer can also be further processed or discarded. Embodiments for processing plasma are described elsewhere herein.

[0050] In one embodiment, the method comprises using a collected PBMC fraction to enrich for or detect circulating tumor cells. The biological target thus enriched can be further processed and analyzed in step (D). For example, genomic DNA can be purified from the collected PBMC fraction (optionally depleted of circulating tumor cells beforehand). Furthermore, at least a fraction of PBMC cells can be subjected to white blood cell counting or other analysis. Furthermore, specific cell types can be enriched from the collected PBMC fraction.

[0051] Extracellular Nucleic Acids and Enriched Extracellular Nucleic Acids According to one embodiment, step (C) comprises obtaining a cell-depleted fraction from the stabilized cell-containing body fluid sample and enriching, in particular purifying, extracellular nucleic acids from the obtained cell-depleted fraction.

[0052] As used herein, "extracellular nucleic acids" specifically refers to nucleic acids that are not contained within cells but are present in the extracellular fraction of a cell-containing body fluid sample. Each extracellular nucleic acid is also often referred to as cell-free nucleic acid. These terms are used synonymously herein. Cell-free nucleic acids obtained from circulating body fluids (such as blood) are also referred to as circulating cell-free nucleic acids, e.g., ccfDNA or ccfRNA. Extracellular nucleic acids can be enriched from cell-depleted fractions available from cell-containing body fluids (e.g., plasma or serum, preferably plasma). The term "extracellular nucleic acids" refers, for example, to extracellular RNA as well as extracellular DNA. Typical examples of extracellular nucleic acids found in cell-free fractions of bodily fluids include, but are not limited to, mammalian extracellular nucleic acids, such as extracellular tumor-related or tumor-derived DNA and / or RNA, other extracellular disease-related DNA and / or RNA, epigenetically modified DNA, fetal DNA, and / or RNA, small interfering RNAs, such as miRNA and siRNA, and non-mammalian extracellular nucleic acids, such as viral nucleic acids, pathogen nucleic acids released into the extracellular nucleic acid population, e.g., from prokaryotes (e.g., bacteria), viruses, eukaryotic parasites, or fungi. Extracellular nucleic acid populations typically contain a certain amount of intracellular nucleic acids released from damaged or dying cells. For example, extracellular nucleic acid populations present in blood typically contain intracellular globin mRNA released from damaged or dying cells, a natural process occurring in vivo. Such intracellular nucleic acids present in the extracellular nucleic acid population can also serve as controls in subsequent nucleic acid detection methods. The stabilization methods described herein, in particular, reduce the risk that ex vivo handling of the sample will significantly increase the amount of intracellular nucleic acids, such as genomic DNA, contained in the extracellular nucleic acid population after the cell-containing body fluid is collected. Thus, changes in the extracellular nucleic acid population due to ex vivo handling can be significantly reduced or even prevented by the stabilization techniques according to the present disclosure. The enriched, preferably purified, extracellular nucleic acids may preferably comprise or consist essentially of extracellular DNA. Extracellular DNA, such as ccfDNA (circulating cell-free DNA), obtained from circulating body fluids is a valuable tool for diagnostic applications and is therefore widely used in the art for diagnostic and prognostic purposes.

[0053] In one embodiment, the isolated extracellular nucleic acid comprises or consists essentially of extracellular RNA. It is well known and described in the art that cell-depleted fractions obtained from cell-containing body fluid samples (such as plasma in the case of stabilized blood samples) contain extracellular RNA. Suitable methods and kits for purifying extracellular nucleic acids are known in the art and commercially available, such as the QIAamp® Circulating Nucleic Acid Kit (QIAGEN), QIAsymphony DSP Circulating DNA Kit, Chemagic Circulating NA Kit (Chemagen), NucleoSpin Plasma XS Kit (Macherey-Nagel), Plasma / Serum Circulating DNA Purification Kit (Norgen Biotek), Plasma / Serum Circulating RNA Purification Kit (Norgen Biotek), High-Purity Viral Nucleic Acid Large Volume Kit (Roche), and other commercially available kits suitable for extracting and purifying extracellular nucleic acids. See also the methods disclosed in WO 2013 / 045432 and WO 2016 / 198571. The described methods are particularly suitable for purifying extracellular nucleic acids, such as extracellular DNA, from plasma obtained from blood samples stabilized using the stabilization methods described herein.

[0054] In one embodiment, in the case of blood, extracellular nucleic acids are isolated from a cell-depleted fraction such as plasma or serum (preferably plasma) rather than from pre-enriched extracellular vesicles. In one embodiment, a subsequent step (D) is performed, which comprises detecting one or more target molecules in the extracellular nucleic acid purified in step (C).

[0055] Extracellular vesicles and extracellular vesicle enrichment According to one embodiment, step (C) comprises enriching extracellular vesicles from a cell-depleted fraction obtained from the stabilized cell-containing body fluid sample.

[0056] As used herein, the term "extracellular vesicles (EVs)" refers to any type of secreted vesicle, particularly those of cellular origin. EVs can be broadly classified into exosomes, microvesicles (MVs), and apoptotic bodies. EVs, such as exosomes and microvesicles, are small vesicles secreted by cells. EVs have been found to circulate through many different body fluids, including blood and urine, making them readily accessible. Because the composition of EVs is similar to that of their parent cells, circulating EVs are a valuable source of biomarkers. Circulating EVs are likely composed of a mixture of exosomes and MVs. Circulating EVs contain nucleic acids (e.g., mRNA, miRNA, and other small RNAs), DNA, and proteins protected from degradation by a lipid bilayer. The contents are appropriately and specifically packaged, representing a mechanism for local and distant cell communication. EVs can transport RNA between cells. EVs, such as exosomes, are a rich and diverse source of circulating biomarkers. Source cells can be healthy or cancerous. EVs such as exosomes are often actively secreted by cancer cells, especially dividing cancer cells. As part of the tumor microenvironment, EVs such as exosomes appear to play an important role in fibroblast proliferation, desmoplastic responses, and also in the initiation of epithelial-mesenchymal transition (EMT) and SC, as well as the establishment of therapy resistance, as well as the initiation of metastasis and therapy resistance. Exosomes are smaller than CTCs and contain fewer copies per biomarker. Compared to CTCs, EVs are present in much larger quantities in body fluids, e.g., approximately 10 per mL of plasma. 9 ~10 12 It exists in individual vesicles and is therefore easily obtained.

[0057] As described above, in one embodiment, the present method includes enrichment of extracellular vesicles. Any suitable method can be used in combination with the present method for isolating and thus enriching extracellular vesicles from a stabilized cell-containing body fluid sample. As disclosed herein, the stabilized cell-containing body fluid sample can first be processed to provide a cell-depleted fraction, such as plasma in the case of a stabilized blood sample. Different options for providing a cell-depleted fraction are disclosed herein. Extracellular vesicles can then be enriched from the cell-depleted fraction, such as plasma. The term "enrichment" is again used broadly to cover the concentration or purification of extracellular vesicles. Extracellular vesicles can be enriched from virtually any body fluid after cellular components have been collected. Suitable methods for enriching extracellular vesicles, such as exosomes, are known in the art and need not be described in detail herein. Exemplary suitable methods for enriching extracellular vesicles are briefly described herein. Extracellular vesicles containing exosomes can be enriched from cell-depleted fractions of stabilized body fluids, such as plasma or serum. For example, extracellular vesicles can be enriched by ultracentrifugation, ultrafiltration, gradient and affinity capture, or a combination of suitable methods. Numerous procedures and commercial products are available for, for example, extracellular vesicles / exosome isolation and are known to those skilled in the art. Exemplary, non-limiting isolation methods are described below. Extracellular vesicles, and in particular exosomes, can be enriched, for example, by methods involving ultracentrifugation. An exemplary ultracentrifugation isolation method is described by Thery et al. (Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Unit 3.22, Subcellular Fractionation and Isolation of Organelles, in Current Protocols in Cell Biology, John Wiley and Sons Inc., 2006). Thus, according to one embodiment, extracellular vesicles are enriched by ultracentrifugation. To increase the purity of the enriched extracellular vesicles, cells and cell fragments, and optionally, apoptotic bodies, if desired, can be removed before enriching the extracellular vesicles, for example, by centrifugation or filtration. For example, filtration methods that exclude particles of ≧0.8 μm, ≧0.7 μm, or ≧0.6 μm can be used.

[0058] According to one embodiment, extracellular vesicles can be enriched by affinity capture onto a solid phase. According to one embodiment, extracellular vesicles such as exosomes are enriched by immunomagnetic capture using magnetic beads coated with antibodies against proteins exposed on the membrane of extracellular vesicles, e.g., exosomes. According to one embodiment, extracellular vesicles are captured by passing a cell-depleted sample through a vesicle capture agent. The bound extracellular vesicles can be washed and then eluted. Commercial systems based on affinity capture, such as the exoEasy Kit (QIAGEN), are available for extracellular vesicle purification and can be used in conjunction with the present invention. Methods based on the use of volume-excluding polymers, such as PEG, have also been described for the isolation of EVs. In these methods, the polymers bind water molecules and displace less soluble components, such as extracellular vesicles, from the solution, allowing them to be collected by brief, low-speed centrifugation. Commercial products using this principle include ExoQuick (System Biosciences, Mountain View, USA) and Total Exosome Isolation Reagent (Life Technologies, Carlsbad, USA). Thus, according to one embodiment, extracellular vesicles are enriched by precipitation with a volume-excluding polymer. Extracellular vesicles, such as exosomes, can also be enriched based on their density, for example, by layering the sample on a discontinuous sucrose or iodixanol gradient and subjecting it to high-speed centrifugation. Thus, according to one embodiment, extracellular vesicles, such as exosomes, are enriched by density gradient centrifugation.

[0059] According to one embodiment, the extracellular vesicles comprise or consist essentially of exosomes and / or microvesicles. Thus, in one embodiment, the enriched biological targets consist essentially of exosomes. Thus, in some embodiments, the enriched biological targets consist essentially of exosomes.

[0060] As disclosed herein, the collected extracellular vesicles can be further processed in step (D) to isolate nucleic acids, such as RNA, from the extracellular vesicles. Thus, RNA can be purified from enriched extracellular vesicles, particularly enriched exosomes. Therefore, relevant molecular information can be obtained by analyzing the RNA molecules present in extracellular vesicles, such as exosomes. EVs have been shown to contain a variety of small RNA species, including miRNA, piRNA, tRNA (and fragments thereof), vault RNA, Y RNA, and fragments of rRNA, as well as long non-coding RNAs and even mRNAs. Exemplary and preferred methods for isolating RNA are described herein.

[0061] Intracellular nucleic acids and enrichment of intracellular nucleic acids According to one embodiment, step (C) comprises enriching, e.g., purifying, intracellular nucleic acids as biological targets from the stabilized cell-containing biological fluid sample. The intracellular nucleic acids can be purified from an aliquot of the stabilized cell-containing biological sample, or the stabilized cell-containing biological sample can be separated into a cell-containing fraction and an aliquot / portion of a cell-depleted fraction, respectively, and the intracellular nucleic acids can be purified from the cell-containing fraction. Optionally, a target cell population, e.g., comprising or essentially consisting of rare cells, can be previously removed, and thus intracellular nucleic acids can be enriched from, e.g., the stabilized cell-containing biological fluid depleted of rare target cells and / or its enriched cell-containing fraction. Additionally, a subpopulation of cells can be first enriched from the stabilized cell-containing bodily fluid, and then intracellular nucleic acid can be enriched from that subpopulation. Preferred embodiments are described herein. As disclosed herein, cells can be enriched, and thus concentrated, within a cell-containing fraction. Intracellular nucleic acids can be selected from RNA and genomic DNA. According to one embodiment, genomic DNA is enriched as a biological target. Thus, according to one embodiment, the method comprises obtaining a cell fraction from a stabilized cell-containing body fluid sample and enriching genomic DNA from the cell fraction, and the cell fraction can be stored, or even frozen, prior to isolating the genomic DNA. Suitable methods for purifying intracellular nucleic acids, such as RNA and genomic DNA, are well known in the art and are briefly described herein. According to one embodiment, step (C) comprises enriching at least circulating tumor cells, genomic DNA, and circulating cell-free DNA as biological targets.

[0062] Process (D) Step (D) involves processing the enriched three or more biological targets for analysis. In particular, the analysis can involve detecting one or more biomarker molecules.

[0063] According to one embodiment, step (C) comprises enriching a cell subpopulation, for example comprising or essentially consisting of rare cells (e.g., CTCs), and a subsequent step (D) comprises analyzing the enriched cell subpopulation. Cell analysis can be important for basic cellular research, drug discovery, diagnosis, and prognosis. The analysis can be performed at the molecular level (e.g., DNA, RNA, proteins, secreted molecules, etc.) or at the cellular level (e.g., cell metabolism, cell morphology, cell-cell interactions, etc.). Thus, the subsequent step (D) can comprise analyzing the enriched cell subpopulation (e.g., comprising or essentially consisting of rare cells such as CTCs) at the cellular level and / or enriching intracellular nucleic acids, e.g., RNA, from the enriched cell subpopulation. As disclosed herein, the enriched rare cells are preferably circulating tumor cells. Step (D) may optionally include lysing the enriched cell subpopulation (e.g., comprising or consisting essentially of rare cells) to release intracellular nucleic acids for subsequent purification. Suitable methods for purifying genomic DNA, as well as RNA, are known in the art and need not be described in detail.

[0064] According to one embodiment, step (D) comprises detecting one or more target molecules within the extracellular nucleic acids enriched in step (C). According to one embodiment, step (C) comprises enriching extracellular vesicles from a cell-depleted fraction obtained from the stabilized cell-containing body fluid sample, and a subsequent step (D) comprises enriching RNA from the enriched extracellular vesicles. As disclosed herein, extracellular vesicles can comprise or consist essentially of exosomes. According to one embodiment, step (D) comprises enriching RNA from cells, preferably enriched rare cells and / or enriched extracellular vesicles. The enriched RNA can comprise or consist of mRNA and / or non-coding RNA. In some embodiments, the purified RNA comprises miRNA or consists essentially of small RNAs, including miRNAs, of 350 nt or less in length, 300 nt or less in length, or 250 nt or less in length.

[0065] According to one embodiment, step (C) comprises enriching at least circulating tumor cells and circulating cell-free DNA, and further genomic DNA and / or extracellular vesicles, as biological targets, and step (D) comprises: - analyzing the enriched circulating tumor cells, the analysis comprising enriching RNA from the enriched rare cells and detecting one or more target nucleic acid molecules in the enriched RNA (which allows, for example, to detect and / or characterize the enriched circulating tumor cells); - detecting one or more target nucleic acid molecules within said circulating cell-free DNA. Furthermore, when genomic DNA is further enriched, one or more target nucleic acid molecules can be detected within the genomic DNA. When extracellular vesicles are further enriched, nucleic acids such as RNA can be enriched from the extracellular vesicles, and one or more target nucleic acid molecules can be detected within the enriched nucleic acids. Where platelets are enriched in step (C), nucleic acids such as RNA can be enriched from the platelets in step (D), and one or more target nucleic acid molecules can be detected within the purified nucleic acids.

[0066] Thus, according to a preferred embodiment, step (D) comprises detecting one or more target nucleic acid molecules within the isolated nucleic acid. Step (D) may comprise reverse transcribing the isolated RNA to provide cDNA. Step (D) may further comprise performing at least one amplification step (e.g., polymerase chain reaction, isothermal amplification, whole genome amplification, etc.). According to one embodiment, step (D) comprises performing a qualitative or quantitative polymerase chain reaction. According to one embodiment, step (D) comprises performing a sequencing reaction. According to one embodiment, step (D) comprises analyzing one or more intact cells, which may be circulating tumor cells.

[0067] According to one embodiment, the at least one target nucleic acid molecule detected in step (D) has one or more of the following characteristics: - It is a cancer-related tumor marker; - is a diagnostic, prognostic, and / or predictive biomarker; - is a prognostic or predictive biomarker; - Associated with solid tumors (possibly metastatic cancer); - Associated with breast or prostate cancer, especially metastatic breast and prostate cancer; - is a positive or negative response marker; and / or - It is a therapeutic marker.

[0068] According to one embodiment, the at least one target nucleic acid molecule forms part of a panel of target nucleic acid molecules. Thus, step (D) can comprise detecting the panel of target nucleic acid molecules. The panel can comprise at least 5, at least 10, at least 15, at least 20, at least 25, or at least 50 target nucleic acid molecules. Detecting the panel of target nucleic acid molecules (e.g., using a panel of corresponding primers, and optionally using probes) is advantageous, for example, for characterizing the enriched CTCs. According to one embodiment, step (D) comprises isolating RNA from circulating tumor cells and detecting biomarker RNA molecules within said isolated RNA. In some embodiments, step (D) comprises immunofluorescently staining the enriched cells. The enriched cells can be target cells, e.g., target rare cells. In some embodiments, the CTCs are analyzed by immunofluorescent staining. For example, staining can be performed using a monoclonal or polyclonal antibody against a marker specific to the target cells of interest to be stained. For example, in the case of CTCs, the cells can be stained for cytokeratin, Epcam, EGFR, E-cadherin, HER2, PSA, PSMA, and / or other CTC markers. Additionally, staining can involve staining for exclusion markers to exclude myeloid-derived cells. Such markers can include CD45 and / or CD14.

[0069] RNA enrichment In some embodiments, the method comprises enriching, e.g., purifying, RNA from cells, e.g., rare cells (e.g., CTCs). The method can also comprise isolating RNA from extracellular vesicles. The term "enrichment" is again used broadly and includes, e.g., RNA isolation and purification. Suitable RNA isolation methods are known to those skilled in the art and need not be described in detail herein. Exemplary embodiments are briefly described below. For example, methods based on the use of phenol and / or chaotropic salts can be used to isolate RNA. Examples of suitable methods include, but are not limited to, extraction, solid-phase extraction, polysilicic acid-based purification, magnetic particle-based purification, phenol-chloroform extraction, anion exchange chromatography (using anion exchange surfaces), electrophoresis, precipitation, and combinations thereof. Suitable methods are well known in the art. If DNA is enriched together with RNA, the DNA can be removed, for example, by DNase digestion. Methods for specifically isolating RNA that is substantially free of DNA contamination are also known in the art. As discussed, remaining DNA can be further removed by DNase digestion, and / or primers flanking an intron can be used when the expression of biomarker RNA molecules is detected by amplification. An example of a phenol / chloroform-based organic extraction method for isolating RNA is the Chomczynski method (Chomczynski and Sacchi, 1987: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. (162): 156-159) and its variations. An example of a commercial product based on phenol / chloroform is the miRNeasy Mini Kit (QIAGEN). This kit provides high-quality and high-yield total RNA, including small RNAs, from a variety of different biological samples.

[0070] According to one embodiment, RNA isolation comprises binding RNA to a solid phase and eluting the RNA from the solid phase. The RNA may be washed before elution. Suitable solid phases and compatible chemistries for binding RNA to the solid phase are known to those skilled in the art and include, but are not limited to, silica solid phases and solid phases comprising anion exchange moieties. According to one embodiment, RNA isolation involves binding RNA to a solid phase, particularly a silica solid phase, and at least one chaotropic agent (e.g., a guanidinium salt) and / or at least one alcohol (e.g., isopropanol or ethanol) is used for RNA binding. The chaotropic agent and alcohol concentration are known to those skilled in the art. The bound RNA may optionally be washed, and the RNA is eluted.

[0071] According to one embodiment, RNA isolation involves binding RNA to a solid phase comprising an anion-exchange moiety and eluting the RNA from the solid phase. In particular, isolation methods based on the charge-switching principle may be used. Examples of suitable solid phases comprising anion-exchange moieties include, but are not limited to, particles or columns functionalized with anion-exchange groups. Examples of anion-exchange moieties include monoamines, diamines, polyamines, and nitrogen-containing aromatic or aliphatic heterocyclic groups. The RNA is bound to the solid phase under binding conditions that allow the RNA to bind to the anion-exchange moiety. Suitable pH and / or salt conditions known to those skilled in the art can be used for this purpose. The bound RNA may optionally be washed. Any suitable elution method can be used, and suitable embodiments are known to those skilled in the art. Elution may involve, for example, changing the pH value. Thus, elution can occur, for example, at an elution pH higher than the binding pH. Similarly, ionic strength can be used to assist or cause elution. Elution can also be assisted by heating and / or shaking. Cells (e.g., enriched CTCs) and / or enriched extracellular vesicles can be lysed / disintegrated to liberate RNA from the cells or extracellular vesicles for RNA isolation. Suitable lysis methods are well known in the art. Cells and / or extracellular vesicles can be contacted with one or more lysis agents for disruption, respectively, and these can be included in a disruption reagent, such as a lysis buffer. RNA must be protected from degradation by nucleases during lysis. Generally, the lysis procedure can include, but is not limited to, mechanical, chemical, physical, and / or enzymatic action on the sample. Additionally, a reducing agent, such as β-mercaptoethanol or DTT, can be added for lysis, for example, to aid in denaturing nucleases. According to one embodiment, at least one chaotropic agent, preferably at least one chaotropic salt, is used for lysis and disruption. Suitable chaotropic agents, and in particular suitable chaotropic salts, are known to those skilled in the art.

[0072] According to one embodiment, the RNA fraction enriched in step (D) comprises or consists of mRNA. Step (D) involves purifying RNA containing mRNA (among other RNA species), as well as selectively purifying mRNA. Essentially pure mRNA can be obtained, for example, by using an RNA isolation method that selectively isolates mRNA (but not other RNA species) from a degraded sample. Purified mRNA can also be isolated sequentially, for example, by first enriching total RNA and then selectively enriching mRNA from the isolated total RNA. Suitable methods for selective mRNA isolation are known to those skilled in the art and do not require elaboration. Well-established methods are based on oligo(dT) capture on a solid phase (e.g., a column or magnetic beads), which allows the specific isolation of mRNA via its poly(A) tail. According to one embodiment, mRNA is isolated from the resulting cell lysate, for example, a cell lysate (such as a CTC lysate). According to one embodiment, mRNA is isolated directly from the resulting cell lysate, e.g., CTC lysate, as also shown in the Examples. The mRNA is isolated from the resulting cell lysate by direct addition of an oligo d(T) moiety (e.g., oligo d(T) 25 The extracellular vesicle fragments can be captured from the lysate using a solid phase (e.g., magnetic beads or a column) comprising a soluble portion (e.g., a soluble portion). According to a further embodiment, total RNA is first isolated, e.g., by oligo d(T) capture or other suitable methods, and then mRNA is isolated from the total RNA. According to one embodiment, total RNA is isolated from the resulting extracellular vesicle lysate / digestion. According to one embodiment, mRNA is then isolated from the total vesicle RNA, e.g., by oligo d(T) capture or other suitable methods.

[0073] According to one embodiment, the RNA isolated in step (D) comprises miRNA or consists essentially of small RNAs, including miRNA, having a length of 350 nt or less, 250 nt or less, or 200 nt or less. Thus, step (D) can involve purifying RNA, including miRNA (among other RNA species), as well as specifically purifying small RNA molecules, including miRNA, but depleted of large RNA molecules (e.g., having a length of 400 nt or more). Suitable methods for specifically enriching small RNA molecules in separation from large RNA molecules are well known in the prior art and need not be described here. As disclosed herein, isolated RNA (e.g., mRNA) can be reverse transcribed into cDNA, which can then be amplified. Amplification results in amplicons corresponding to one or more target nucleic acid molecules to be tested. Suitable primers for amplification can be determined by one skilled in the art. According to one embodiment, the expression of two or more target nucleic acid molecules (e.g., biomarker RNAs) is measured in parallel by performing multiplex PCR using the obtained cDNA as a template. Suitable primers for amplification can be determined by one skilled in the art. Furthermore, reverse transcription can be combined with an amplification step, for example, by performing reverse transcription polymerase chain reaction. According to one embodiment, measuring the expression of at least one biomarker RNA molecule among the isolated RNA comprises performing quantitative polymerase chain reaction. In one embodiment, semi-quantitative PCR is performed. In another embodiment, the method is not semi-quantitative. Performing quantitative PCR (qPCR) is advantageous because it allows for determining whether a biomarker RNA molecule is overexpressed, for example, in CTCs and / or EVs. Suitable methods for performing quantitative PCR are well known to those skilled in the art and need not be described in detail herein. The Ct values ​​obtained in quantitative PCR for each analyzed marker RNA molecule or molecules can then be recorded and used to provide an expression profile. According to one embodiment, a pre-amplification step is performed after the reverse transcription step and before the quantitative PCR reaction. Such a pre-amplification step can improve sensitivity, which is advantageous considering that CTCs are often rare. By pre-amplifying cDNA molecules corresponding to the analyzed target nucleic acid molecule(s) (e.g., one or more biomarker RNA molecules), more DNA material is provided for the subsequent amplification step, which is preferably qPCR. This can improve results.

[0074] Cell-containing body fluid samples Advantageously, the cell-containing body fluid sample may be a liquid biopsy sample. In one embodiment, the cell-containing body fluid is a circulating body fluid. The cell-containing body fluid may be selected from blood, urine, saliva, synovial fluid, amniotic fluid, tears, lymphatic fluid, fluid (cerebrospinal fluid), sweat, ascites, breast milk, bronchial lavage, peritoneal and pleural effusions, bone marrow and nipple aspirates, semen / seminal plasma, bodily secretions or bodily excretions. The cell-containing body fluid may be the product of a diagnostic leukapheresis transfusion. In one embodiment, the cell-containing body fluid is selected from blood and urine. In one embodiment, the cell-containing body fluid is blood. In one embodiment, the blood is peripheral blood.

[0075] The methods of the present invention can be performed as in vitro methods using a biological sample obtained from a subject, e.g., a human subject, such as a cancer patient. In one embodiment, where at least one biological target is a rare cell (e.g., a tumor cell such as a CTC), the cell-containing bodily fluid contains or is suspected of containing such rare cells. As shown in the examples, rare cells, or specific subpopulations thereof, such as circulating tumor cells, extracellular nucleic acids (e.g., ccfDNA), extracellular vesicles such as exosomes, and intracellular nucleic acids of cellular fractions, can be enriched from the same stabilized sample (e.g., a blood sample) and analyzed using this method. The described workflow allows for the parallel analysis of multiple different biological targets that can be enriched from the same stabilized cell-containing body fluid.

[0076] Stabilization techniques used in accordance with the present disclosure As disclosed above, step (A) comprises treating the cell-containing body fluid with one of the following stabilizing factors: (a) at least one primary, secondary, or tertiary amide; (b) at least one poly(oxyethylene) polymer, and / or (c) at least one apoptosis inhibitor; with a stabilizing composition comprising one or more, two or more, or preferably all three of: This results in a stabilized cell-containing body fluid sample. Suitable embodiments and concentrations of the stabilizing factors (a) to (c), as well as advantageous embodiments of the stabilizing composition, are disclosed, for example, in WO 2015 / 140218, which is incorporated herein by reference. Suitable embodiments are also briefly described below.

[0077] At least one primary, secondary, or tertiary amide According to one embodiment, the stabilized composition comprises at least one primary, secondary, or tertiary amide. As disclosed herein, the amide may be a carboxylic acid amide, a thioamide, or a selenoamide. Preferably, the amide is a carboxylic acid amide.

[0078] According to one embodiment, the composition optionally comprises one or more compounds according to formula 1: [ka] wherein R1 is hydrogen or an alkyl group, preferably a C1-C5 alkyl group, a C1-C4 alkyl group, or a C1-C3 alkyl group, more preferably a C1-2 alkyl group; R2 and R3 are the same or different and are selected from hydrogen and hydrocarbon groups, preferably alkyl groups having a carbon chain length of 1 to 20 carbon atoms arranged in a linear or branched fashion; and R4 is oxygen, sulfur, or selenium group, preferably oxygen. Combinations of one or more compounds according to Formula 1 can also be used. In embodiments where R1 is an alkyl group, a chain length of 1 or 2 is preferred for R1. R2 and / or R3 of a compound according to Formula 1 are the same or different and are selected from hydrogen and hydrocarbon groups, with alkyl groups being preferred. According to one embodiment, R2 and R3 are both hydrogen. According to one embodiment, one of R2 and R3 is hydrogen and the other is a hydrocarbon group. According to one embodiment, R2 and R3 are the same or different hydrocarbon groups. The hydrocarbon groups R2 and / or R3 can be independently selected from groups including alkyl, including short-chain alkyl and long-chain alkyl, alkenyl, alkoxy, long-chain alkoxy, cycloalkyl, aryl, haloalkyl, alkylsilyl, alkylsilyloxy, alkylene, alkenediyl, arylene, carboxylate, and carbonyl (see, for example, pages 20-21 of WO 2013 / 045457, which is incorporated herein by reference for a description of these groups). The chain length n of R2 and / or R3 can in particular have the values ​​1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. According to one embodiment, R2 and R3 have a carbon chain length of 1 to 10, preferably 1 to 5, more preferably 1 to 2. According to one embodiment, R2 and / or R3 are alkyl groups, preferably C1 to C5 alkyl groups. Preferably, the compounds according to formula 1 are carboxylic acid amides, and therefore R4 is oxygen. The carboxylic acid amides can be primary, secondary, or tertiary carboxylic acid amides.

[0079] According to one embodiment, the compound according to formula 1 is an N,N-dialkylcarboxylic acid amide. Preferred R1, R2, R3, and R4 groups are described above. According to one embodiment, the compound according to formula 1 is selected from the group consisting of N,N-dimethylacetamide, N,N-diethylacetamide, N,N-dimethylformamide, and N,N-diethylformamide. Corresponding thio analogs containing sulfur instead of oxygen as R4 are also suitable. It is preferred to use at least one compound according to formula 1 that is not a toxic agent according to the GHS classification. According to one embodiment, the compound according to formula 1 is an N,N-dialkylpropanamide, such as N,N-dimethylpropanamide.

[0080] The stabilizing composition can include one or more compounds according to Formula 1′: [ka] [wherein R1 is hydrogen or an alkyl group, preferably a C1-C5 alkyl group, more preferably a methyl group; R2 and R3 are the same or different hydrocarbon groups having a chain length of 1 to 20 carbon atoms arranged in a linear or branched fashion; and R4 is an oxygen, sulfur, or selenium group.] Formula 1' is encompassed by Formula 1 above and is compared to Formula 1 with the limitation that R2 and R3 are the same or different hydrocarbon groups (not hydrogen). Alternatively, groups R1-R4 correspond to those described above for Formula 1, and the above disclosure also applies here. Preferably, the composition comprises butanamide and / or N,N-dialkylpropanamide, more preferably N,N-dimethylpropanamide.

[0081] According to one embodiment, the stabilized composition comprises one or more primary, secondary, or tertiary amides at a concentration selected from 0.4% to 38.3%, 0.8% to 23.0%, 2.3% to 11.5%, 3.8% to 9.2%, 5% to 15%, and 7.5% to 12.5%. The above concentrations refer to (w / v) or (v / v) depending on whether the primary, secondary, or tertiary amide is liquid or not. Preferably, at least one primary, secondary, or tertiary carboxylic acid amide is used. According to one embodiment, when a cell-containing body fluid sample is contacted with a stabilizing composition comprising one or more primary, secondary, or tertiary amides (and optionally further additives used for stabilization), the resulting mixture / stabilized cell-containing body fluid sample comprises said amides (or concentrations of amides) in a concentration range ranging from 0.25% to 5%, for example, 0.3% to 4%, 0.4% to 3%, 0.5% to 2%, or 0.75% to 1.5%.

[0082] at least one poly(oxyethylene) polymer According to one embodiment, the stabilizing composition comprises at least one poly(oxyethylene) polymer. As detailed in the referenced WO2015 / 140218, poly(oxyethylene) polymers exhibit advantageous stabilizing properties. Therefore, it is advantageous for the stabilizing composition to comprise a poly(oxyethylene) polymer. The poly(oxyethylene) polymer is preferably polyethylene glycol. Unsubstituted polyethylene glycol may be used. In general, all disclosures made in this application regarding poly(oxyethylene) polymers apply to and specifically reference the preferred embodiment of polyethylene glycol, even if not explicitly stated. Poly(oxyethylene) polymers can be used in a variety of molecular weights. Polyethylene glycol has the formula HO-(CH2CHO) n -H, where n is a whole integer and is dependent on the molecular weight. A correlation has been found between the stabilizing effect of a poly(oxyethylene) polymer and its molecular weight. Higher molecular weight poly(oxyethylene) polymers have been found to be more effective stabilizers than lower molecular weight poly(oxyethylene) polymers. To achieve efficient stabilization with lower molecular weight poly(oxyethylene) polymers, higher concentrations are generally recommended compared to higher molecular weight poly(oxyethylene) polymers. However, for some applications, it is preferable to keep the amount of additive used for stabilization low. Thus, in some embodiments, high molecular weight poly(oxyethylene) polymers are used as stabilizers to allow the use of lower concentrations of poly(oxyethylene) polymers while achieving a strong stabilizing effect on cell-containing body fluids and the biological targets of interest contained therein.

[0083] According to one embodiment, the stabilizing composition comprises a poly(oxyethylene) polymer, which is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500. The high molecular weight poly(oxyethylene) polymer may have a molecular weight in a range selected from 1500-50,000, 1500-40,000, 2000-30,000, 2500-25,000, 3000-20,000, 3500-15,000, and 4000-12,500. Alternatively, or in addition, the stabilizing composition comprises at least one poly(oxyethylene) polymer having a molecular weight of less than 1500, preferably a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less. In one embodiment, the molecular weight of the low molecular weight poly(oxyethylene) polymer is in a range selected from 100-1000, 200-800, 200-600, and 200-500.

[0084] According to one embodiment, the stabilizing composition contacted with the cell-containing body fluid in step (A) comprises a high-molecular-weight poly(oxyethylene) polymer, preferably polyethylene glycol, at a concentration selected from 0.4% to 35% (w / v), e.g., 0.8% to 25% (w / v), 1.5% to 20% (w / v), 2.5% to 17.5% (w / v), 3% to 15% (w / v), 4% to 10% (w / v), or 3% to 5% (w / v). Suitable concentrations can be determined by those skilled in the art and may depend, inter alia, on whether the high-molecular-weight poly(oxyethylene) glycol is used alone or in combination with an additional poly(oxyethylene) polymer, such as a low-molecular-weight poly(oxyethylene) polymer, and the amount, e.g., volume, of the stabilizing composition used to stabilize a certain amount of cell-containing body fluid sample. High-molecular-weight poly(oxyethylene) polymers can be used alone at concentrations of 2.2% to 33.0% (w / v). Suitable concentration ranges can be selected from 4.4% to 22.0% (w / v), 6.6% to 16.5% (w / v), and 8.8% to 13.2% (w / v). When a high molecular weight poly(oxyethylene) polymer is used in combination with a low molecular weight poly(oxyethylene) polymer, the concentration can be in the range of 0.4% to 30.7% (w / v). Suitable concentration ranges can be selected from 0.8% to 15.3% (w / v), 1% to 10% (w / v), 1.5% to 7.7% (w / v), 2.5% to 6% (w / v), 3.1% to 5.4% (w / v), and 3% to 4% (w / v).

[0085] According to one embodiment, when a cell-containing body fluid sample is contacted with a stabilizing composition comprising a high molecular weight poly(oxyethylene) polymer (and optionally further additives used for stabilization), the resulting mixture / stabilized cell-containing body fluid sample comprises the high molecular weight poly(oxyethylene) polymer at a concentration in the range of 0.05% to 4% (w / v), for example, 0.1% to 3% (w / v), 0.2% to 2.5% (w / v), 0.25% to 2% (w / v), 0.3% to 1.75% (w / v), and 0.35% to 1.5% (w / v). The concentration of the high molecular weight poly(oxyethylene) polymer in the stabilized cell-containing body fluid sample may be in the range of 0.25% to 1.5% (w / v), for example, 0.3% to 1.25% (w / v), 0.35% to 1% (w / v), or 0.4% to 0.75% (w / v). According to one embodiment, the stabilizing composition comprises a low molecular weight poly(oxyethylene) polymer, preferably polyethylene glycol, at a concentration in the range of 0.8% to 92.0%, for example, 3.8% to 76.7%, 11.5% to 53.7%, 19.2% to 38.3%, 20% to 30%, or 20% to 27.5%. According to one embodiment, the concentration is 11.5% to 30%. The above concentrations refer to (w / v) or (v / v) depending on whether the low molecular weight poly(oxyethylene) polymer is liquid or not. According to one embodiment, when a cell-containing body fluid sample is contacted with a stabilizing composition comprising a low molecular weight poly(oxyethylene) polymer (and optionally additional additives used for stabilization), the resulting mixture / stabilized cell-containing body fluid sample contains the low molecular weight poly(oxyethylene) polymer at a concentration ranging from 0.5% to 10%. The concentration of the low molecular weight poly(oxyethylene) polymer in the stabilized cell-containing body fluid sample can range from 1.5% to 9%, for example, 2% to 8%, 2 to 7%, 2.5% to 7%, and 3% to 6%.

[0086] In one embodiment, the stabilizing composition comprises a poly(oxyethylene) polymer that is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500, and a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less. In one embodiment, the stabilizing composition comprises a poly(oxyethylene) polymer that is a high molecular weight poly(oxyethylene) polymer and a poly(oxyethylene) polymer that is a low molecular weight poly(oxyethylene) polymer, wherein the high molecular weight poly(oxyethylene) polymer has a molecular weight in a range selected from 1500 to 50,000, 2,000 to 40,000, 3,000 to 30,000, 3,000 to 25,000, 3,000 to 20,000, and 4,000 to 15,000, and the low molecular weight poly(oxyethylene) polymer has a molecular weight in a range selected from 100 to 1,000, 200 to 800, 200 to 600, and 200 to 500. Suitable concentrations are described above.

[0087] At least one apoptosis inhibitor According to one embodiment, the stabilized composition comprises at least one apoptosis inhibitor. Preferably, the apoptosis inhibitor is a caspase inhibitor. Suitable apoptosis inhibitors and caspase inhibitors are described in WO 2013 / 045457 A1 and WO 2013 / 045458 A1. The caspase inhibitors disclosed therein are incorporated herein by reference. Advantageous stabilized compositions comprising one or more caspase inhibitors that can be used in the methods of the present disclosure are also disclosed in WO 2014 / 146780 A1, WO 2014 / 146782 A1, WO 2014 / 049022 A1, WO 2014 / 146781 A1, WO2015 / 140218, and WO 2017 / 085321. Caspase inhibitors are preferably cell-permeable. Members of the caspase gene family play important roles in apoptosis. The substrate preferences or specificities of individual caspases have been exploited to develop peptides that successfully compete for caspase binding. By conjugating caspase-specific peptides to, for example, aldehyde, nitrile, or ketone compounds, it is possible to create reversible or irreversible inhibitors of caspase activation. For example, peptides derived from fluoromethyl ketone (FMK), such as Z-VAD-FMK, function as effective irreversible inhibitors without additional cytotoxic effects. Synthesized inhibitors with a benzyloxycarbonyl (BOC) group at the N-terminus and an O-methyl side chain exhibit increased cell permeability. Additionally, suitable caspase inhibitors with a phenoxy group at the C-terminus have been synthesized. One example is Q-VD-OPh, a cell-permeable, irreversible, broad-spectrum caspase inhibitor that is much more effective at preventing apoptosis and thus supporting stabilization than the caspase inhibitor Z-VAD-FMK.

[0088] According to one embodiment, the caspase inhibitor is a pan-caspase inhibitor and thus a broad-spectrum caspase inhibitor. The caspase inhibitor comprises or consists of a peptide or protein. According to one embodiment, the caspase inhibitor comprises a modified caspase-specific peptide. The caspase-specific peptide is preferably modified with an aldehyde, nitrile, or ketone compound. According to one embodiment, the caspase-specific peptide is modified, preferably at the carboxyl terminus, with an O-phenoxy (OPh) or fluoromethyl ketone (FMK) group. Suitable caspase inhibitors comprising or consisting of proteins or peptides, and caspase inhibitors comprising modified caspase-specific peptides, are disclosed in Table 1 of WO 2013 / 045457, which is incorporated herein by reference. The table provides examples of caspase inhibitors. In one embodiment, the caspase inhibitor is a peptide caspase inhibitor modified with an O-phenoxy (OPh) group (preferably at the carboxyl terminus) and / or modified with a glutamine (Q) group (preferably at the N terminus). In one embodiment, the caspase inhibitor involved is Q-VD-OPh.

[0089] According to one embodiment, the caspase inhibitor is selected from the group consisting of Q-VD-OPh, Z-VAD(OMe)-FMK, and Boc-D-(OMe)-FMK. According to one embodiment, the caspase inhibitor is selected from the group consisting of Q-VD-OPh and Z-VAD(OMe)-FMK. In a preferred embodiment, Q-VD-OPh, a broad-spectrum inhibitor of caspases, is used for stabilization. Q-VD-OPh is cell-permeable and inhibits apoptotic cell death. Q-VD-OPh is not toxic to cells even at very high concentrations and contains a carboxy-terminal phenoxy group conjugated to the amino acids valine and aspartic acid. It is equally effective in preventing apoptosis mediated by the three major apoptotic pathways: caspase-9 and caspase-3, caspase-8 and caspase-10, and caspase-12 (Caserta et al., 2003). The stabilizing composition used in step (A) can contain one or more caspase inhibitors, particularly caspase inhibitors comprising modified caspase-specific peptides such as Q-VD-OPh, in an amount sufficient to achieve a stabilizing effect on the extracellular nucleic acid population contained in the biological sample. According to one embodiment, the stabilizing composition contains the caspase inhibitor at a concentration that results in a final caspase inhibitor concentration of 0.1 μM to 25 μM, 0.5 μM to 20 μM, 1 μM to 17 μM, 2 μM to 16 μM, or more preferably 3 μM to 15 μM after the stabilizing composition has been contacted and stabilized with a desired volume of cell-containing biological fluid. A final concentration in the range of 5 μM to 15 μM is well suited for stabilizing, for example, blood samples.

[0090] According to one embodiment, the stabilizing composition, and therefore the stabilizing reagent, comprises a caspase inhibitor at a concentration selected from 0.35 μg / mL to 70 μg / mL, 0.7 μg / mL to 63 μg / mL, 1.74 μg / mL to 59 μg / mL, 10.5 μg / mL to 56 μg / mL, or 15 μg / mL to 50 μg / mL, 20 μg / mL to 45 μg / mL, 25 μg / mL to 40 μg / mL, and 30 μg / mL to 38 μg / mL. The concentration can be selected from 0.7 μg / mL to 45 μg / mL and 1.74 μg / mL to 40 μg / mL. According to one embodiment, the stabilizing composition, and therefore the stabilizing reagent, comprises a caspase inhibitor at a concentration selected from 0.68 μM to 136 μM, 1.36 μM to 122.5 μM, 3.38 μM to 114.72 μM, 20.4 μM to 109 μM, or 29.2 μM to 97.2 μM, 38.9 μM to 87.5 μM, 48.6 μM to 77.8 μM, and 58.3 μM to 74 μM. The concentration may be selected from 20.4 μM to 97.2 μM and 29.2 μM to 87.5 μM. The above-mentioned concentrations of caspase inhibitors in the mixture containing the stabilization composition (reagent) and the cell-containing biological fluid to be stabilized apply to the use of a single caspase inhibitor and to the use of a combination of caspase inhibitors. The above-mentioned concentrations are particularly suitable when using pan-caspase inhibitors, particularly modified caspase-specific peptides such as Q-VD-OPh and / or Z-VAD(OMe)-FMK. Another example of a modified caspase-specific peptide is Boc-D-(OMe)-FMK. The above-mentioned concentrations are suitable, for example, for stabilizing blood samples. Suitable concentration ranges for individual caspase inhibitors and / or other cell-containing biological samples can be determined by those skilled in the art, for example, by testing various concentrations of the corresponding caspase inhibitor in the test assays described in the Examples.

[0091] Further components of the stabilizing composition The cell-containing body fluid can also be contacted with additional additives that are preferably included in the stabilizing composition. According to one embodiment, the additional additive is a chelating agent. A chelating agent is an organic compound capable of forming coordinate bonds with a metal at two or more atoms of the organic compound. Examples of chelating agents include, but are not limited to, ethylenedinitrilotetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylene glycol tetraacetic acid (EGTA), and N,N-bis(carboxymethyl)glycine (NTA), as well as salts of carboxylic acids such as citrate or oxalate. According to a preferred embodiment, EDTA is used as the chelating agent. As used herein, the term "EDTA" refers specifically to the EDTA moiety of EDTA compounds, such as K2EDTA, K3EDTA, or Na2EDTA. The use of a chelating agent such as EDTA also has the advantageous effect of inhibiting nucleases such as DNase and RNase, thereby preventing the degradation of extracellular nucleic acids by nucleases. EDTA used / added at a high concentration supports the stabilizing effect. When the cell-containing body fluid sample is blood, an anticoagulant is used as an additional additive. Anticoagulants include, but are not limited to, heparin, chelating agents, and salts of carboxylic acids, such as citrate or oxalate. In an advantageous embodiment, the anticoagulant is a chelating agent such as EDTA. For example, K2EDTA can be used. This embodiment is particularly useful when the body fluid to be stabilized is blood.

[0092] According to one embodiment, the further additive is at least one compound selected from a thioalcohol, which is N-acetyl-cysteine ​​or glutathione, a water-soluble vitamin, and a water-soluble vitamin E derivative. As disclosed in WO 2017 / 085321, this can be advantageous if the stabilized composition further comprises a caspase inhibitor and is provided in sterile form.

[0093] According to one embodiment, the stabilization technique used has one or more of the following characteristics: (i) The stabilization of the cell-containing body fluid sample does not involve the use of additives at concentrations that induce or promote lysis of nucleated cells; (ii) the stabilization does not induce protein-nucleic acid or protein-protein cross-linking; (iii) the stabilization does not involve the use of cross-linking agents that induce protein-nucleic acid and / or protein-protein cross-links, such as formaldehyde, formalin, paraformaldehyde, or formaldehyde-releasing agents; (iv) the stabilization does not involve the use of toxic factors; and / or (v) A stabilizing agent is contained in a stabilizing composition that includes water. In particular, it is preferred that the stabilization composition used to provide a stabilized cell-containing body fluid sample does not contain cross-linking agents that induce protein-DNA and / or protein-protein cross-links. Examples of cross-linking agents that induce protein-DNA and / or protein-protein cross-links include formaldehyde, formalin, paraformaldehyde, and formaldehyde-releasing agents. Cross-linking agents induce intermolecular or intramolecular covalent bonds between nucleic acid molecules or between nucleic acids and proteins. This effect can result in reduced recovery of such stabilized, partially cross-linked nucleic acids after purification or extraction from complex biological samples. For example, the concentration of circulating nucleic acids in whole blood samples is already quite low, and any measures that further reduce the yield of such nucleic acids must be avoided. This can be particularly important when detecting and analyzing extremely rare nucleic acid molecules derived from malignant tumors or from developing fetuses during the first trimester of pregnancy. Therefore, it is preferred that formaldehyde-releasing agents are not included in the sterile stabilization composition or used additionally for stabilization. Thus, according to one embodiment, no cross-linking agents, such as formaldehyde or formaldehyde-releasing agents, are included in the stabilization composition and, respectively, are used for stabilization. Furthermore, as described, the stabilization composition preferably does not contain any additives, such as chaotropic salts, that typically induce nucleated cells or cell lysis. As shown in the examples, this is an important advantage over known state-of-the-art stabilization reagents and methods that involve the use of cross-linking agents, such as formaldehyde or formaldehyde-releasing agents, because it allows for efficient recovery of biological targets of interest (e.g., CTCs, extracellular nucleic acids, cell subpopulations, and intracellular nucleic acids) from stabilized cell-containing body fluid samples. It is advantageous to use a stabilizing composition that does not contain components capable of releasing aldehydes, thereby avoiding obstacles in the subsequent isolation of nucleic acids from the stabilized sample.

[0094] Advantageous combinations of stabilizing factors in the present stabilized compositions According to one embodiment, the stabilizing composition used is (a) at least one primary, secondary, or tertiary amide; (b) at least one poly(oxyethylene) polymer, preferably a high molecular weight polyethylene glycol and a low molecular weight polyethylene glycol; (c) optionally, at least one apoptosis inhibitor, preferably a caspase inhibitor.

[0095] According to one embodiment, the stabilizing composition used is (a) at least one primary, secondary, or tertiary amide; (b) optionally at least one poly(oxyethylene) polymer; (c) at least one apoptosis inhibitor, preferably a caspase inhibitor. According to one embodiment, the stabilizing composition used is (a) optionally at least one primary, secondary, or tertiary amide; (b) at least one poly(oxyethylene) polymer; and (c) at least one apoptosis inhibitor, preferably a caspase inhibitor.

[0096] According to one embodiment, the stabilizing composition used is (a) at least one primary, secondary, or tertiary amide; (b) at least one poly(oxyethylene) polymer; and (c) at least one caspase inhibitor. Suitable and preferred embodiments of the individual stabilizing factors (a) to (c), as well as suitable and preferred concentrations, are described above.

[0097] According to one embodiment, a cell-containing body fluid, preferably blood, a) one or more compounds according to Formula 1 above; b) at least one high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 3000, and optionally a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less; c) at least one caspase inhibitor; and d) optionally a chelating agent, preferably EDTA and bring it into contact with.

[0098] According to one embodiment, the blood is a) one or more compounds according to Formula 1 above; b) at least one high molecular weight poly(oxyethylene) polymer having a molecular weight in the range of 3000 to 40000, such as in the range of 3000 to 30000, or 3500 to 25000, and a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less, such as in the range of 100 to 800, 200 to 800, or 200 to 500; c) at least one caspase inhibitor, preferably a pan-caspase inhibitor, optionally Q-VD-OPh; and d) an anticoagulant, preferably a chelating agent, preferably EDTA and after the blood sample has been contacted with the additive, and optionally further stabilizing additives, the resulting mixture / stabilized blood sample is one or more compounds according to formula 1 in a concentration ranging from 0.3 to 4%, for example from 0.5 to 3%, from 0.5 to 2%, or from 0.75 to 1.5%, - high molecular weight poly(oxyethylene) polymers at a concentration ranging from 0.2% to 1.5% (w / v), for example from 0.25% to 1.25% (w / v), from 0.3% to 1% (w / v), or from 0.4% to 0.75% (w / v), - a low molecular weight poly(oxyethylene) polymer, at a concentration ranging from 1.5% to 10%, for example from 2% to 6%, and - caspase inhibitors at concentrations ranging from 1 μM to 10 μM, e.g., from 3 μM to 7.5 μM Includes.

[0099] The stabilizing composition can be liquid. The concentrations shown are particularly preferred for stabilizing blood samples. For example, 0.5 mL to 2.5 mL, 0.5 mL to 2 mL, preferably 1 mL to 2 mL, or 1 mL to 1.5 mL of liquid stabilizing composition can be used. Such a stabilizing composition containing the stabilizing factors at the concentrations shown below can be used to stabilize, for example, 10 mL of blood.

[0100] Specific Embodiments Further embodiments of the present invention are described again below. The present invention also specifically provides the following:

[0101] Embodiment 1. 1. A method for stabilizing and enriching multiple biological targets in a cell-containing body fluid, comprising: (A) The cell-containing body fluid is treated with one or more of the following stabilizing factors: (a) at least one primary, secondary, or tertiary amide; (b) at least one poly(oxyethylene) polymer, and / or (c) at least one apoptosis inhibitor providing a stabilized cell-containing bodily fluid sample by contacting the sample with a stabilizing composition comprising: (B) maintaining the stabilized cell-containing bodily fluid sample for a stabilization period; and (C) at least one cell subpopulation, - extracellular nucleic acids, -extracellular vesicles, and -Intracellular nucleic acids and processing the stabilized cell-containing bodily fluid sample to enrich from the stabilized cell-containing bodily fluid for three or more biological targets selected from the group consisting of:

[0102] Embodiment 2. 2. The method of embodiment 1, wherein the enriched cell subpopulation comprises target rare cells.

[0103] Embodiment 3. 3. The method of embodiment 1 or 2, wherein the cell subpopulation consists essentially of the target rare cells.

[0104] Embodiment 4. The method according to any one of embodiments 1 to 3, wherein the target rare cells are selected from the group consisting of tumor cells, particularly circulating tumor cells (CTCs), fetal cells, stem cells, cells infected by viruses or parasites, circulating endothelial cells (CECs), and circulating endothelial progenitor cells (EPCs).

[0105] Embodiment 5. 5. The method of any one of embodiments 1 to 4, wherein the target rare cells are circulating tumor cells.

[0106] Embodiment 6. 6. The method according to any one or more of embodiments 1 to 5, wherein the intracellular nucleic acid, which may be genomic DNA, is isolated from a stabilized body fluid sample or a cell-containing fraction thereof.

[0107] Embodiment 7. Step (C) is rare cells, preferably circulating tumor cells, - extracellular nucleic acids, -extracellular vesicles, and -Intracellular nucleic acids 7. The method of any one or more of embodiments 1-6, comprising processing the stabilized cell-containing body fluid sample to enrich from the stabilized cell-containing body fluid for three or more biological targets selected from the group consisting of:

[0108] Embodiment 8. 8. The method of any one or more of embodiments 1 to 7, wherein step (C) comprises obtaining at least one cell-containing fraction and at least one cell-depleted fraction from the stabilized body fluid sample, and optionally, the cell-depleted fraction may be separated from the at least one cellular fraction by a separation method involving centrifugation and / or filtration.

[0109] Embodiment 9. The process in (C) (aa) separating the stabilized cell-containing body fluid sample into at least one cell-containing fraction and at least one cell-depleted fraction; (bb) further processing the cell-containing fraction, wherein further processing the cell-containing fraction comprises: (i) enriching a cell subpopulation, preferably comprising a target rare cell, from said cell-containing fraction; and / or (ii) enriching intracellular nucleic acids (e.g., genomic DNA) from the cell-containing fraction; (cc) further processing said cell-depleted fraction, wherein further processing said cell-depleted fraction comprises: (i) enriching the cell-depleted fraction for extracellular nucleic acids, which may be extracellular DNA; and / or (ii) enriching extracellular vesicles from the cell-depleted fraction; 9. The method of any one of embodiments 1 to 8, comprising:

[0110] Embodiment 10. The process in (C) (aa) enriching a cell subpopulation, preferably comprising a target rare cell, from the stabilized cell-containing body fluid sample; (bb) separating the stabilized cell-containing body fluid sample, from which the target cell subpopulation has been depleted, into a cell-containing fraction and a cell-depleted fraction; (cc) further processing said cell-depleted fraction, wherein further processing said cell-depleted fraction comprises: (i) enriching the cell-depleted fraction for extracellular nucleic acids, which may be extracellular DNA; and / or (ii) enriching extracellular vesicles from the cell-depleted fraction; (dd) optionally enriching intracellular nucleic acids, preferably genomic DNA, from said cell-containing fraction.

[0111] Embodiment 11. The process in (C) (aa) dividing the stabilized cell-containing bodily fluid sample into at least two aliquots and enriching a cell subpopulation, preferably comprising rare cells, from at least one of the provided aliquots; (bb) providing at least one cell-containing fraction and at least one cell-depleted fraction; (cc) further processing said cell-depleted fraction, wherein further processing said cell-depleted fraction comprises: (i) enriching the cell-depleted fraction for extracellular nucleic acids, which may be extracellular DNA; and / or (ii) further processing, comprising enriching the cell-depleted fraction for extracellular vesicles; (dd) optionally enriching intracellular nucleic acids, preferably genomic DNA, from said cell-containing fraction; 9. The method of any one of embodiments 1 to 8, comprising:

[0112] Embodiment 12. (D) The method of any one of embodiments 1 to 11, further comprising processing the enriched three or more biological targets for analysis.

[0113] Embodiment 13. 13. The method of embodiment 12, wherein step (C) comprises enriching target rare cells, and a subsequent step (D) comprises analyzing the enriched rare cells, and optionally, analyzing the enriched rare cells comprises analyzing the enriched rare cells at the cellular level and / or enriching intracellular nucleic acids, preferably RNA, from the enriched rare cells.

[0114] Embodiment 14. 14. The method of embodiment 13, wherein step (D) comprises detecting the enriched intracellular nucleic acids, said detection optionally comprising amplification and / or sequencing.

[0115] Embodiment 15. 15. The method of embodiment 13 or 14, wherein the intracellular nucleic acid comprises mRNA.

[0116] Embodiment 16. 16. The method of any one or more of embodiments 1-15, wherein step (C) comprises obtaining a cell-depleted fraction from the stabilized cell-containing body fluid sample and enriching extracellular nucleic acids from said obtained cell-depleted fraction.

[0117] Embodiment 17. 17. The method of embodiment 16, wherein the extracellular nucleic acid comprises or consists essentially of extracellular DNA.

[0118] Embodiment 18. 18. The method of embodiment 16 or 17, wherein the extracellular nucleic acid comprises or consists essentially of extracellular RNA.

[0119] Embodiment 19. 19. A method according to any one or more of embodiments 12 to 18, wherein step (D) comprises detecting one or more target molecules within the extracellular nucleic acid obtained in step (C).

[0120] Embodiment 20. 20. The method of any one or more of embodiments 6-19, wherein step (C) comprises enriching extracellular vesicles from a cell-depleted fraction obtained from the stabilized cell-containing body fluid sample, and a subsequent step (D) comprises enriching RNA from the isolated extracellular vesicles.

[0121] Embodiment 21. 21. The method according to any one or more of embodiments 1 to 20, wherein the extracellular vesicles comprise or consist essentially of exosomes.

[0122] Embodiment 22. 22. The method of one or more of embodiments 1-21, comprising enriching, preferably purifying, RNA, wherein optionally, said RNA enrichment may comprise binding RNA to a solid phase and eluting the bound RNA from said solid phase.

[0123] Embodiment 23. 23. The method according to any one or more of embodiments 12 to 22, wherein step (D) comprises enriching, preferably purifying, RNA from cells, preferably enriched target rare cells, and / or enriched extracellular vesicles.

[0124] Embodiment 24. 24. The method of embodiment 22 or 23, having one or more of the following features: (i) The enriched RNA comprises or consists essentially of mRNA. (ii) the enriched RNAs include miRNAs or consist essentially of small RNAs, including miRNAs, that are 350 nt or less in length, 300 nt or less in length, or 250 nt or less in length;

[0125] Embodiment 25. 25. The method according to any one or more of embodiments 12 to 24, wherein step (D) comprises detecting one or more target nucleic acid molecules among the enriched nucleic acids, preferably purified nucleic acids.

[0126] Embodiment 26. 26. The method of embodiment 25, wherein at least one target nucleic acid molecule has one or more of the following characteristics: - It is a cancer-related tumor marker; - is a diagnostic, prognostic, and / or predictive biomarker; - is a prognostic or predictive biomarker; - Associated with solid tumors, which may be metastatic; - Associated with breast or prostate cancer, especially metastatic breast and prostate cancer; - A positive or negative response marker; - is a therapeutic marker; and / or - form part of a panel of target nucleic acid molecules, optionally said panel comprising at least 5, at least 10, at least 15, at least 20, at least 25, or at least 50 target nucleic acid molecules.

[0127] Embodiment 27. 27. The method of one or more of embodiments 12-26, wherein step (D) comprises one or more of the following: (i) reverse transcription of purified RNA to provide cDNA; (ii) performing at least one amplification step; (iii) performing a quantitative polymerase chain reaction; and / or (iv) Analyzing intact cells, which may be circulating tumor cells.

[0128] Embodiment 28. 28. The method according to one or more of embodiments 1 to 27, comprising enriching target rare cells and / or extracellular vesicles by affinity capture.

[0129] Embodiment 29. 29. The method according to one or more of embodiments 1 to 28, wherein the cell-containing body fluid has one or more of the following characteristics: - Circulating body fluids; - selected from blood, urine, saliva, synovial fluid, amniotic fluid, tears, lymph, secretions, cerebrospinal fluid, sweat, ascites, breast milk, bronchial washings, peritoneal and pleural effusions, bone marrow and nipple aspirates, semen / seminal plasma, body secretions or excretions; - selected from blood and urine; and / or - It's blood.

[0130] Embodiment 30. 22. The method of any one or more of embodiments 1-21, wherein the stabilizing composition comprises at least one primary, secondary, or tertiary amide.

[0131] Embodiment 31. 31. The method of embodiment 30, wherein the stabilizing composition comprises at least one primary, secondary, or tertiary amide according to Formula 1: [ka] wherein R1 is hydrogen or an alkyl group, preferably a C1-C5 alkyl group, a C1-C4 alkyl group, or a C1-C3 alkyl group, more preferably a C1-C2 alkyl group; R2 and R3 are the same or different and are selected from hydrogen and hydrocarbon groups, preferably alkyl groups having a carbon chain length of 1 to 20 carbon atoms arranged in a linear or branched fashion; and R4 is oxygen, sulfur, or selenium group, preferably oxygen.

[0132] Embodiment 32. 32. The method of embodiment 31, wherein at least one compound according to Formula 1 is a primary, secondary, or tertiary carboxylic acid amide.

[0133] Embodiment 33. 32. The method of embodiment 30 or 31, wherein the stabilizing composition comprises an N,N-dialkylpropanamide, preferably N,N-dimethylpropanamide, and / or butanamide.

[0134] Embodiment 34. 34. The method of any one or more of embodiments 1-33, wherein the stabilizing composition comprises at least one poly(oxyethylene) polymer.

[0135] Embodiment 35. 35. The method of embodiment 34, wherein the poly(oxyethylene) polymer is polyethylene glycol.

[0136] Embodiment 36. 36. The method of embodiment 34 or 35, wherein the stabilized composition has one or more of the following characteristics: a) the poly(oxyethylene) polymer involved is unsubstituted polyethylene glycol; b) the composition comprises a poly(oxyethylene) polymer, which is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500; c) the composition comprises at least one poly(oxyethylene) polymer having a molecular weight of less than 1500, preferably a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less, optionally the molecular weight may be in a range selected from 100 to 1000, 200 to 800, 200 to 600, and 200 to 500; d) the composition comprises a poly(oxyethylene) polymer that is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500 and a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less; and / or e) The composition comprises a poly(oxyethylene) polymer that is a high molecular weight poly(oxyethylene) polymer and a poly(oxyethylene) polymer that is a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less, wherein the high molecular weight poly(oxyethylene) polymer has a molecular weight in a range selected from 1500 to 50,000, 2000 to 40,000, 3000 to 30,000, 3000 to 25,000, 3000 to 20,000, and 4000 to 15,000, and / or the low molecular weight poly(oxyethylene) polymer has a molecular weight in a range selected from 100 to 1000, 200 to 800, 200 to 600, and 200 to 500.

[0137] Embodiment 37. 37. The method according to any one or more of embodiments 1-36, wherein the stabilized composition comprises at least one inhibitor of apoptosis, preferably a caspase inhibitor.

[0138] Embodiment 38. 38. The method of embodiment 37, wherein the apoptosis inhibitor, caspase inhibitor has one or more of the following characteristics: a) the caspase inhibitor is a pan-caspase inhibitor; b) the caspase inhibitor comprises a caspase-specific peptide; c) the caspase inhibitor comprises a modified caspase-specific peptide, preferably modified at the carboxyl terminus with an O-phenoxy (OPh) group; d) the caspase inhibitor comprises a modified caspase-specific peptide modified (preferably at the N-terminus) with a glutamine (Q) group; e) the caspase inhibitor is selected from the group consisting of Q-VD-OPh, Boc-D-(OMe)-FMK, and Z-Val-Ala-Asp(OMe)-FMK; f) the caspase inhibitor is selected from the group consisting of Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK; and / or g) The caspase inhibitor is Q-VD-OPh.

[0139] Embodiment 39. The stabilizing composition comprises: According to aspect A, (a) at least one primary, secondary, or tertiary amide, preferably as defined in any one of embodiments 31 to 33, and (b) at least one poly(oxyethylene) polymer, preferably as defined in embodiment 35 or 36, and (c) optionally at least one apoptosis inhibitor, preferably a caspase inhibitor as defined in embodiment 38; According to aspect B, (a) at least one primary, secondary, or tertiary amide, preferably as defined in any one of embodiments 31 to 33; (b) optionally, preferably, at least one poly(oxyethylene) polymer as defined in embodiment 35 or 36, and (c) at least one apoptosis inhibitor, preferably a caspase inhibitor as defined in embodiment 38; According to aspect C, (a) optionally, preferably, at least one primary, secondary, or tertiary amide, as defined in any one of embodiments 31 to 33; (b) at least one poly(oxyethylene) polymer, preferably as defined in embodiment 35 or 36, and (c) at least one apoptosis inhibitor, preferably a caspase inhibitor as defined in embodiment 38; 39. The method of any one or more of embodiments 1-38, comprising:

[0140] Embodiment 40. The stabilizing composition comprises: (a) at least one primary, secondary, or tertiary amide, preferably as defined in any one of embodiments 31 to 33; (b) at least one poly(oxyethylene) polymer, preferably as defined in embodiment 35 or 36, and (c) at least one apoptosis inhibitor, preferably a caspase inhibitor as defined in embodiment 38; 40. The method of embodiment 39, comprising:

[0141] Embodiment 41. 41. The method according to one or more of embodiments 1 to 40, having one or more of the following features: (i) The stabilization of the cell-containing body fluid sample does not involve the use of additives at concentrations that induce or promote lysis of nucleated cells; (ii) the stabilization does not induce protein-nucleic acid or protein-protein cross-linking; (iii) the stabilization does not involve the use of cross-linking agents that induce protein-nucleic acid and / or protein-protein cross-links, such as formaldehyde, formalin, paraformaldehyde, or formaldehyde-releasing agents; (iv) the stabilization does not involve the use of toxic factors; and / or (v) A stabilizing agent is contained in a stabilizing composition that includes water.

[0142] Embodiment 42. 42. The method of any one or more of embodiments 1-41, wherein the stabilizing composition comprises a chelating agent, which may be EDTA.

[0143] Embodiment 43. 43. The method according to any one or more of embodiments 1-42, wherein the cell-containing body fluid is blood and the stabilised composition comprises an anticoagulant, preferably a chelating agent.

[0144] Embodiment 44. A cell-containing body fluid, preferably blood, a) one or more compounds according to Formula 1; b) at least one high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 3000, and optionally a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less; c) at least one caspase inhibitor; and d) optionally a chelating agent, preferably EDTA; 44. The method of any one or more of embodiments 1-43, wherein the contacting is with

[0145] Embodiment 45. The cell-containing body fluid is blood, a) one or more compounds according to Formula 1; b) at least one high molecular weight poly(oxyethylene) polymer having a molecular weight in the range of 3000 to 40000, such as in the range of 3000 to 30000, or 3500 to 25000, and a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less, such as in the range of 100 to 800, 200 to 800, or 200 to 500; c) at least one caspase inhibitor, preferably a pan-caspase inhibitor, optionally Q-VD-OPh; and d) an anticoagulant, which is preferably a chelating agent, preferably EDTA and after the blood sample has been contacted with the additive and, optionally, with further additives for stabilization, the resulting mixture / stabilized blood sample is one or more compounds according to formula 1 in a concentration ranging from 0.3 to 4%, for example from 0.5 to 3%, from 0.5 to 2%, or from 0.75 to 1.5%, - said high molecular weight poly(oxyethylene) polymer at a concentration ranging from 0.2% to 1.5% (w / v), for example from 0.25% to 1.25% (w / v), from 0.3% to 1% (w / v), or from 0.4% to 0.75% (w / v); - a low molecular weight poly(oxyethylene) polymer, at a concentration ranging from 1.5% to 10%, for example from 2% to 6%, and caspase inhibitors, at a concentration ranging from 1 μM to 10 μM, for example from 3 μM to 7.5 μM, 45. The method of any one or more of embodiments 1-44, comprising:

[0146] Embodiment 46. 46. ​​The method of any one of embodiments 1 to 45, wherein the processing step (C) comprises subjecting the stabilized cell-containing body fluid sample or a cell-containing fraction obtained from the stabilized cell-containing body fluid sample to a density gradient centrifugation step, and optionally, the cell-containing body fluid sample may be blood.

[0147] Embodiment 47. 47. The method of embodiment 46, wherein the stabilized blood sample, or a cell-containing fraction obtained from the stabilized blood sample, is contacted with a density gradient medium.

[0148] Embodiment 48. 48. The method of embodiment 46 or 47, wherein the stabilized blood sample or a cell-containing fraction obtained from the stabilized blood sample is diluted with a dilution solution before performing the density gradient centrifugation step, preferably before contacting the diluted sample with the density gradient medium.

[0149] Embodiment 49. 49. The method of embodiment 48, wherein the stabilized blood sample or a cell-containing fraction obtained from the stabilized blood sample is diluted using a diluent solution having one or more of the following characteristics: (a) It is a hypotonic or isotonic solution; (b) contains a tonicity modifier; (c) a polyol, optionally including a sugar or sugar alcohol; (d) sugar, optionally glucose; (e) a sugar alcohol, optionally including glycerol; and / or (f) salts, optionally alkali metal salts, optionally chloride salts;

[0150] Embodiment 50. 50. The method of embodiment 48 or 49, wherein the diluted solution comprises a reducing sugar, which may be glucose, at a concentration in the range of 2 to 10%, 3 to 7%, or 4 to 6% (w / v).

[0151] Embodiment 51. 51. The method of any one of embodiments 48 to 50, wherein the dilute solution comprises a sugar alcohol, which may be glycerol, and a salt, optionally an alkali metal salt.

[0152] Embodiment 52. 52. The method of embodiment 51, wherein the diluted solution comprises up to 0.5 M glycerol and up to 2% sodium chloride, and optionally the diluted solution may comprise 0.7 to 1.2% sodium chloride and 0.075 to 0.15 M glycerol.

[0153] Embodiment 53. A method according to any one of embodiments 48 to 52, wherein the diluted solution achieves, after density gradient centrifugation, recovery of at least 60% or at least 70% of leukocytes from the stabilized sample compared to an EDTA-stabilized blood sample.

[0154] Embodiment 54. The diluted solution is (i) 5% (w / v) glucose; (ii) 0.9% NaCl + 0.1 M glycerol, and (iii) at least one tonicity modifier and having an osmolality that matches the osmolality of the diluted solution defined in (i) or (ii), or an osmolality that is within ±20%, ±15%, or ±10% of the osmolality of the solution defined in (i) or (ii); 54. The method according to any one of embodiments 48 to 53, wherein the method is selected from the group consisting of:

[0155] Embodiment 55. 55. The method of any one or more of embodiments 48 to 54, wherein the stabilized blood sample, or a cell-containing fraction obtained from the stabilized blood sample, is incubated in the dilution solution for no more than 10 minutes, no more than 5 minutes, or no more than 3 minutes before contacting the diluted sample with the density gradient medium, preferably wherein the diluted sample is processed directly after dilution by contacting the diluted sample with the density gradient medium.

[0156] Embodiment 56. 56. A method according to any one or more of embodiments 46 to 55, wherein different layers are formed after density gradient centrifugation, said formed layer comprising a PBMC layer.

[0157] Embodiment 57. 57. The method of embodiment 56, comprising providing a PBMC fraction by collecting the formed PBMC layer.

[0158] Embodiment 58. 58. The method of embodiment 56 or 57, comprising isolating circulating tumor cells from the collected PBMC fraction.

[0159] Embodiment 59. 59. The method of any one of embodiments 46 to 58, comprising isolating genomic DNA from a collected PBMC fraction (which may have previously been depleted of circulating tumor cells).

[0160] Embodiment 60. 60. The method of any one of embodiments 46 to 59, comprising washing the collected PBMC fraction using a buffer, which may be a PBS buffer.

[0161] Embodiment 61. 60. The method of any one of embodiments 46 to 59, wherein at least a portion of the PBMC cells are subjected to leukocyte counting.

[0162] Embodiment 62. 62. The method of any one of embodiments 1 to 61, comprising obtaining a cellular fraction from the stabilized cell-containing body fluid sample and isolating genomic DNA from said cellular fraction, wherein said cellular fraction may be stored and optionally frozen prior to genomic DNA isolation.

[0163] Embodiment 63. 2. The method of any one of the preceding embodiments, comprising enriching the cell population or individual cells using cell sorting.

[0164] Embodiment 64. 3. The method of any one of the preceding embodiments, wherein the cell-containing body fluid sample is blood, and step (C) comprises enriching target lymphocytes as a cell subpopulation from the stabilized sample.

[0165] Embodiment 65. 65. The method of embodiment 64, wherein the lymphocytes are selected from T4 and / or T8 lymphocytes.

[0166] Embodiment 66. 66. The method of embodiment 64 or 65, wherein the stabilized blood sample is obtained from a patient with an immune deficiency.

[0167] Embodiment 67. The method of any one of the preceding embodiments, wherein the cell-containing body fluid sample is blood, and wherein step (C) comprises enriching platelets as a cell subpopulation from the stabilized sample, and optionally, step (D) is performed to comprise isolating RNA from the enriched platelets.

[0168] Embodiment 68. 3. The method of any one of the preceding embodiments, wherein the cell-containing body fluid sample is blood, and step (C) comprises enriching blast cells as a cell subpopulation from the stabilized sample.

[0169] Embodiment 69. 69. The method of embodiment 68, wherein the blast cells are enriched by affinity capture, optionally using magnetic particles.

[0170] Embodiment 70. The method of embodiment 68 or 69, wherein the blast cells are enriched by targeting cell surface markers, which may be CD34 and / or CD117.

[0171] Embodiment 71. The method of any one of embodiments 68 to 70, wherein the stabilized blood sample is obtained from a patient with acute myeloid leukemia.

[0172] Embodiment 72. 72. The method of any one of embodiments 1 to 71, wherein step (B) comprises transporting and / or storing the stabilized cell-containing body fluid sample prior to (C).

[0173] Embodiment 73. 73. The method of embodiment 72, wherein storing comprises transporting the stabilized cell-containing bodily fluid sample from the location of collection and stabilization to a different location for processing.

[0174] Embodiment 74. A method according to any one of embodiments 1 to 73, wherein the stabilized cell-containing body fluid sample is maintained for up to 12 hours, or up to 24 hours, prior to the processing step (C).

[0175] Embodiment 75. A method according to any one of embodiments 1 to 74, wherein the stabilized cell-containing body fluid sample is maintained for up to 36 hours, or up to 48 hours, prior to the processing step (C).

[0176] Embodiment 76. A method according to any one of embodiments 1 to 75, wherein the stabilized cell-containing body fluid sample is maintained for up to 60 hours, or up to 72 hours, prior to the processing step (C).

[0177] Embodiment 77. 77. The method of any one of embodiments 1 to 76, comprising maintaining the stabilized cell-containing body fluid sample for at least 6 hours, at least 8 hours, or at least 12 hours prior to the processing step (C).

[0178] Embodiment 78. 78. The method of any one of embodiments 1 to 77, comprising maintaining the stabilized cell-containing body fluid sample for at least 16 hours, at least 24 hours, or at least 48 hours prior to the processing step (C).

[0179] Embodiment 79. 80. The method according to any one or more of embodiments 1 to 79, wherein step (C) comprises isolating at least circulating tumor cells, genomic DNA, and circulating cell-free DNA as biological targets.

[0180] Embodiment 80. 80. The method of embodiment 79, wherein step (D) is performed, comprising isolating RNA from circulating tumor cells and detecting biomarker RNA molecules within said isolated RNA.

[0181] Embodiment 81. 82. The method of embodiment 81, wherein the isolated RNA is mRNA.

[0182] Embodiment 82. Use of a dilution solution as defined in any one of embodiments 49 to 54 for processing a stabilized blood sample or a cell-containing fraction thereof, wherein the blood sample is stabilized with a stabilizing composition comprising (a) at least one primary, secondary, or tertiary amide, (b) at least one poly(oxyethylene) polymer, and / or at least one apoptosis inhibitor, optionally a stabilizing composition as defined in any one of embodiments 30 to 44.

[0183] Embodiment 83. 84. Use according to embodiment 83 for restoring the density of contained mononuclear cells, preferably for gradient density centrifugation.

[0184] Embodiment 84. 84. An embodiment according to embodiment 82 or 83, wherein the diluted solution is contacted with the stabilized blood sample or a cell-containing fraction thereof before being contacted with the gradient density medium.

[0185] The present invention is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention. Numerical ranges are intended to be inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be read by reference to the specification as a whole. As used in the subject specification and claims, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. The terms "include," "having," "comprise," and variations thereof are used interchangeably and should be construed as open-ended. Throughout the specification, where a composition is described as comprising components or materials, it is construed that the composition in multiple embodiments also consists essentially of or consists of any combination of the recited components or materials, unless otherwise stated. References to "the disclosure," "the present invention," and the like include single or multiple aspects, etc., taught herein. The aspects taught herein are encompassed by the term "invention." It is preferable to select and combine the preferred embodiments described herein, and the specific subject matter resulting from each combination of preferred embodiments also belongs to this disclosure. The terms "enriching," "enrichment," and similar terms are used broadly herein to encompass any form of enrichment, such as, inter alia, the isolation and purification of a target (e.g., nucleic acids such as DNA and / or RNA, rare cells such as circulating tumor cells, extracellular vesicles from a sample). [Example]

[0186] The following examples demonstrate that methods according to the present disclosure have important advantages, allowing for multimodal analysis to be performed based on bodily fluid samples containing single cells collected and stabilized using stabilization techniques according to the present disclosure. 1) The antigenic makeup of cells contained in samples stabilized using the stabilization techniques of the present disclosure is preserved. 2) The stabilization techniques of the present disclosure can be used in combination with different rare cell enrichment techniques (e.g., density gradient centrifugation, Parsortix device, AdnaTest technology, CellSearch). 3) The stabilization techniques of the present disclosure can be used for analysis of the cellular transcriptome (e.g., the RNA content of cells in a sample, such as rare and / or abundant cells). 4) The stabilization techniques of the present disclosure can be used for analysis of the circulating transcriptome (e.g., RNA from extracellular vesicles). 5) The stabilization technology of the present disclosure allows for multi-modal testing (e.g., analysis of CTCs, ccfDNA, and leukocytes derived from genomic DNA (gDNA) of a single stabilized blood sample).

[0187] The following examples demonstrate that the stabilization technique used in this method advantageously achieves stabilization of spiked tumor cells, preserving their core surface structure, transcriptome, and genome. Immunocytochemical staining of MCF7 tumor cell line cells stabilized with PAXgene Blood ccfDNA solution (a stabilization composition according to the present invention) showed results comparable to those of unstabilized MCF7, indicating that cellular antigen composition and morphology are preserved. Furthermore, cell density can be restored by adding specific solutions to stabilized samples, such as blood samples, enabling cell separation using gradient density centrifugation. This approach enables classical density-based blood fraction separation, for example, to enrich and thus concentrate PBMCs and CTCs in a single layer. The suitability of collected blood stabilized using the disclosed stabilization technique as a front-end solution for various CTC analysis workflows is demonstrated based on label-independent enrichment and cellular readout (Parsortix, ANGLE plc) and label-dependent enrichment with molecular readout (AdnaTest ProstateCancerPanel AR-V7, QIAGEN GmbH). The results demonstrate that both approaches are compatible with the disclosed stabilization technique, with high levels of CTC stabilization and recovery. Furthermore, enriched CTCs may be advantageously used for RNA-based analysis. These data provide evidence of sufficient transcriptome stabilization of cells collected in collection tubes containing the disclosed stabilization composition. The following examples further demonstrate that not only cellular RNA but also circulating RNA (packaged in extracellular vesicles, EVs) is available for analysis. Cell-containing body fluid samples stabilized using the disclosed stabilization technique are suitable for multimodal testing of various biological targets contained in cell-containing body fluid samples, such as blood. Based on the workflow established for the AdnaTest ProstateCancerPanel AR-V7 test, a single stabilized blood sample can be used for analysis of CTCs, ccfDNA, and genomic DNA from leukocytes, as exemplarily shown. The examples carried out are described below:

[0188] 1. Example 1: Evaluation of antigenic organization maintenance in cells stabilized using the stabilization technique according to the present disclosure. Immunocytochemical staining of untreated and stabilized MCF7 cancer cell line cells. Preparation of MCF7 cytospins Human breast cancer cell line (MCF7) cells were used as a CTC model to evaluate the effect of PAXgene Blood ccfDNA stabilization solution (PAXccfDNA) on antigen preservation and accessibility. PAXgene Blood ccfDNA stabilization solution is a commercially available stabilization composition according to the present invention, containing stabilization factors (a) to (c) and an anticoagulant. The solution is contained in a commercially available PAXgene Blood ccfDNA tube (PreAnalytiX) (1.5 mL). Cultured MCF7 cells were trypsinized, washed in PBS, and incubated with either PBS or PAXccfDNA solution for 30 minutes at room temperature. Cytospins were then prepared, dried overnight at room temperature, and stored at +4°C until staining.

[0189] Immunocytochemical staining Cells on cytospins were fixed, permeabilized, and treated to prevent nonspecific antibody binding (blocking step), then stained with a fluorescently labeled antibody against human pan-cytokeratin and DAPI for nuclear staining at room temperature for 1 hour. Cytospins were then washed, covered, and analyzed by fluorescence microscopy within 1 week.

[0190] result The presence of specific signals from fluorescently labeled anti-human pan-cytokeratin antibodies on unstabilized and stabilized (in stabilization solution) cancer cells demonstrates the feasibility of assessing antigen composition in cells stabilized in PAXgene Blood ccfDNA tubes (Figure 1). Pan-cytokeratins were well detected on the cell surface in stabilized samples, indicating preservation of cell surface antigens. Nuclear staining confirmed cytoplasmic staining of cytokeratins and preserved nuclei (i.e., morphology) of stained cells.

[0191] 2. Example 2: Use of the present stabilization technique for CTC enrichment and analysis 2.1. Combining this stabilization technique with Ficoll density centrifugation for CTC enrichment Ficoll density centrifugation is a commonly used method for separating blood fractions, and therefore cell populations, into fractions based on their density. Nucleated blood cells have a density of approximately 1.062 g / mL and can be efficiently separated from red blood cells (1.092 g / mL) and platelets (1.030 g / mL) when centrifuged through a Ficoll layer (1.077 g / mL) or similar density gradient medium. The resulting interface layer contains the PBMC fraction, which includes CTCs and other rare nucleated cells. It was observed that blood stabilized with PAXgene Blood ccfDNA stabilization solution, when diluted with common PBS buffer, did not form a plasma / PBMC / erythrocyte layer as typically observed with EDTA-preserved blood (taken as a reference) (see Figure 2). Based on these observations, various dilutions, particularly those slightly hypotonic and isotonic, were tested to restore the density of PAXccfDNA-stabilized blood cells. Isotonic 0.9% NaCl was used as a reference. Next, isotonic solutions containing substances capable of permeating cell membranes (e.g., glycerol) were included in the dilutions tested. The aim was to obtain a typical layering suitable for obtaining various density-based fractions of interest, especially for multimodal analysis. Efficacy was measured as the number of recovered white blood cells (WBCs) after Ficoll density centrifugation.

[0192] Blood sample processing Whole blood collected in EDTA tubes (BD) or PAXgene Blood ccfDNA tubes (PreAnalytiX) was used. BD Vacutainers were used to obtain EDTA-stabilized blood (the EDTA concentration in stabilized blood is approximately 1.8 mg / mL). Four milliliters of whole blood was collected, diluted with 4 mL of each dilution solution for the indicated time periods (see below), and layered onto 4 mL of Ficoll-Paque PLUS (GE Healthcare, density 1.077 g / mL). The samples were immediately centrifuged at 400 × g for 40 minutes without acceleration or deceleration. After centrifugation, the upper plasma fraction was discarded, and the PBMC ring was transferred to a new 15 mL tube, filled with PBS, and centrifuged at 300 × g for 10 minutes (maximum acceleration and deceleration). After removing the supernatant, the pellet was resuspended in 200 μL of PBS and used for WBC counting (Beckman Coulter). The amount of WBC per mL of whole blood was calculated taking into account a dilution factor of 1.15 relative to the PAX ccfDNA tube.

[0193] Test Setup The following dilutions containing various tonicity modifiers and concentrations were tested (Table 1): [Table 1]

[0194] result EDTA blood diluted with PBS without incubation was collected as a reference for WBC counting. Among the most effective dilution solutions for whole blood to obtain a classic gradient density centrifugation layer pattern essentially identical to that of EDTA-stabilized blood are 5% glucose (glucose is taken up by blood cells) and 0.9% NaCl + 0.1 M glycerol. Dilution solutions containing 0.9% NaCl + 0.1 M glycerol also appear to have a normalizing effect on atrophic cells due to the permeation of cell membranes by glycerol. In final experiments, 5% glucose and 0.9% NaCl + 0.1 M glycerol, added without prolonged incubation, gave very good and comparable results in terms of WBC recovery (79% and 80% of the reference, respectively) (see Table 2, Figure 2). Various dilution solutions containing at least one tonicity modifier and having osmolality similar to that of the lead dilution solution demonstrated in this experiment (e.g., ±20%, ±15%, or ±10%) can also be used, and their positive effect on achieving the desired layer pattern and WBC recovery of at least 50%, at least 60%, and preferably at least 65% can be determined by routine experimentation. [Table 2]

[0195] 2.2 Combination of the present stabilization technology with AdnaTest ProstateCancerPanel AR-V7 for CTC detection. AdnaTest CTC enrichment relies on immunomagnetic separation of cells that are captured based on the expression of target proteins on their cell surface. Detection of enriched cells relies on the detection of tumor cell-specific transcripts. Freshly collected EDTA blood (within 4 hours of collection) or blood collected in ACD-A tubes and stored at +4°C for a maximum of 30 minutes can be used, according to the manufacturer's recommendations.

[0196] (a) Materials and Methods cell culture LNCaP95 cells were cultured as monolayers in phenol red-free RPMI 1640 containing 10% charcoal stripped serum and 10% penicillin / streptomycin at 37°C and 5% CO2. Blood collection and sample preparation After giving signed informed consent, whole blood from 21 healthy volunteers was collected by venipuncture into the cubital vein into PAXgene Blood ccfDNA tubes (PreAnalytiX, Switzerland), and the tubes were inverted eight times immediately after blood collection according to the manufacturer's instructions. For the comparative study (see (c) below), blood was drawn from healthy donors and collected in PAXgene Blood ccfDNA tubes and Provider Streck BCT according to the manufacturer's instructions. Blood samples were pooled per donor and blood collection tube (BCT), aliquoted into 15 mL conical tubes within 30 minutes of collection, and spiked immediately. After manual spiking with 20 LNCaP95 cells or 20 μL of PBS per sample, blood samples were stored at 2–8°C or room temperature until processed according to the study design.

[0197] Tumor Cell Enrichment and Detection Using AdnaTest ProstateCancerPanel AR-V7 The AdnaTest ProstateCancerPanel AR-V7 utilizes the CTC enrichment step covered by the AdnaTest ProstateCancerSelect procedure. For CTC detection, cDNA is generated from the CTC-enriched fraction. The AdnaTest ProstateCancerPanel AR-V7 relies on a real-time PCR-based readout to detect prostate-specific PSA, PSMA, AR, and AR-V7 transcripts, GAPDH as a housekeeper, and CD45 as a leukocyte marker. A test was considered positive if at least one of the cancer-specific transcripts was detected. The AR-V7 assay includes a nonspecific cDNA preamplification step to increase the sensitivity of the assay. After the preamplification step (18 cycles), amplification is no longer linear, and quantification of target gene expression is not feasible. AR-V7 testing was performed according to the manufacturer's recommendations.

[0198] Data evaluation LNCaP95 cells are known to be positive for PSMA, AR, and AR-V7 and to have unstable expression of PSA, therefore, all studies were evaluated based on the detection of PSMA, AR, and AR-V7, but PSA was excluded from the analysis. Statistical evaluation of ccfDNA yield and gDNA yield was performed using unpaired two-tailed T-tests (R-statistics version 3.5.1 using the ggplot2 and ggpubr packages).

[0199] (b) Compatibility of Stabilized Compositions According to the Present Disclosure with CTC Detection In the first set of experiments, we evaluated the compatibility of blood collected and stabilized using the AdnaTest ProstateCancerPanel AR-V7 and the stabilization technology described herein for detecting spiked tumor cells. Whole blood samples from 10 donors collected in tubes containing the stabilization composition described herein were pooled for each donor and aliquoted into 15 mL conical tubes as 5 mL samples. Blood samples were manually spiked with either 20 LNCaP95 cells or 20 μL of PBS as a non-spiked control. This setup allowed us to assess whether CTCs could be detected in the collected stabilized blood and whether the stabilization reagent itself had any effect on test performance (spiked samples and non-spiked controls, respectively). All samples were stored at 2–8°C until processing at 3, 24, 30, and 48 hours after spiking. The data show that tumor cell-spiked samples tested positive at all experimental time points (3, 24, 30, and 48 hours after spiking) (see Figure 3A), while all non-spiked control tests were negative (see Figure 3B). Thus, this established workflow demonstrates the compatibility of PAXgene Blood ccfDNA tubes containing a stabilizing composition according to the present disclosure with the AdnaTest ProstateCancerPanel AR-V7 for CTC isolation and detection. The stabilizing solution according to the present disclosure itself does not cause any nonspecific false-positive results. Currently, the commercially available AdnaTest is recommended for use with either EDTA- or ACD-A-collected blood within 4 and 30 hours of collection, respectively, when the blood is stored at 2-8°C. (14) The sensitivity of the assay has been reported to be 90%. Data presented herein demonstrate 100% sensitivity within 30 hours for blood collected in tubes containing a stabilized composition according to the present disclosure, and 90% sensitivity after 48 hours of storage at 2-8°C.

[0200] (c) Comparison of the disclosed stabilization technique with other commercially available stabilization techniques for CTC preservation and detection. We then evaluated the efficiency of CTC detection from samples stored for up to 72 hours and collected in tubes containing a stabilized composition according to the present disclosure (PAXgene Blood ccfDNA tubes) and from samples collected in Cell-Free DNA BCTs from Supplier Streck (also intended for CTC preservation). As in previous experiments, 20 LNCaP95 cells per 5 mL of blood were used as a CTC model. PAXgene Blood ccfDNA-stabilized samples (n=11) were stored at 2–8°C, and Cell-Free DNA BCT (n=8) samples collected at room temperature (per manufacturer's recommendations) were subsequently processed with AdnaTest ProstateCancerPanel AR-V7 at 3, 24, 48, and 72 hours after spiking, as described above. After 72 hours of storage, spiked tumor cells could be efficiently detected in PAXgene Blood ccfDNA-stabilized samples in 91% of cases (see Figure 4A). In contrast, in blood collected in Supplier Streck's BCT, spiked tumor cell detection was positive only in blood stored for less than 3 hours (see Figure 4B). In contrast to the non-crosslinking blood stabilization chemistry of PAXgene Blood ccfDNA tubes, Supplier Streck's Cell-Free DNA BCT relies on crosslinker-based cell preservation, thus hindering RNA detection. The obtained data are consistent with observations made with these BCTs by others (see CTC-mRNA (AR-V7) Analysis from Blood Samples—Impact of Blood Collection Tube and Storage Time. Luk et al., Int J Mol Sci. 2017 May 12;18(5)). Further experiments further demonstrated that CTCs could also be enriched after storage at room temperature (see Figures 4C and 4D).

[0201] 2.3 Compatibility of PAXgene Blood ccfDNA tubes with the Parsortix device for CTC enrichment in the context of the all-from-one solution. research design This experiment tested the general compatibility of PAXgene ccfDNA-stabilized blood with Parsortix (Angle plc, Guildford, UK) enrichment equipment and the capture efficiency of spiked cells from (un)stabilized blood. Parsortix technology enriches for highly deformable and less deformable cells (e.g., CTCs) from the cellular blood component by capturing cells in microscopic disposable cassettes. Cells can be stained and counted within the cassette and recovered using a reverse flow system. In this experiment, we used a model system approach for CTC enrichment. Blood was collected from a single healthy donor into EDTA tubes and PAXgene Blood ccfDNA tubes. Blood was first aliquoted into 5 mL (EDTA) or 6 mL (PAXgene) samples, and additional liquid (stabilizing solution included in the tubes) was examined in the PAXgene ccfDNA tubes. All samples were then spiked with 2,000 cells stably expressing green fluorescent protein (purchased as MCF7-GFP cells). The advantage of this cell line is that captured cells can be detected and counted under a fluorescent microscope within the enrichment cassette without further staining or processing.

[0202] EDTA and PAXgene-stabilized blood samples were processed on the Parsortix instrument on the day of collection (TTP0) and the number of GFP cells captured in the cassettes was counted. EDTA blood served as the reference because capturing CTCs from unpreserved EDTA blood is the workflow recommended by the instrument supplier and remains the primary sample quality used in clinical studies. After 3 days of blood storage at room temperature, cells were enriched from PAXgene-stabilized whole blood (PAXgene) or whole blood was centrifuged (15 min, 1900 × g) at a time, plasma was discarded, and blood was reconstituted with 3 mL of PBS to restore viscosity (PAXgene reconstituted) before Parsortix processing. The number of GFP cells trapped in the cassette was again counted using a fluorescent microscope.

[0203] result On the day of collection, the number of cells captured and counted was similar in blood collected in PAXgene ccfDNA tubes compared to the EDTA control (103% for PAXgene). Regardless of the centrifugation step prior to blood processing, a comparable but slightly higher number of cells could be captured and counted in the cassettes after 3 days of storage (see Figure 5). conclusion Blood collected in PAXgene Blood ccfDNA tubes is compatible with the Parsortix cell enrichment workflow and can be processed even after 3 days of storage at room temperature and after plasma separation. Thus, the All From One solution is advantageously suitable for obtaining both ccfDNA as well as CTCs from blood samples collected and stabilized using the stabilization techniques according to the present disclosure.

[0204] 3. Example 3: PAXgene Blood ccfDNA Tubes can be used to analyze the cellular transcriptome (RNA content of a cell) A proof-of-principle experiment for CTC enrichment and RNA analysis is presented in Section 2.2. AdnaTest relies on RNA-based CTC detection using RT-PCR. The successful detection of spiked tumor cells described above in Section 2.2 demonstrates that the RNA content of individual cells is preserved for at least 72 hours when blood is collected in PAXgene Blood ccfDNA tubes.

[0205] 4. Example 4: PAXgene Blood ccfDNA Tubes Can Be Used for Analysis of the Circulating Transcriptome (RNA from Extracellular Vesicles) research design The compatibility of blood stabilized with the disclosed stabilization technique with subsequent EV analysis was demonstrated in the following study: PAXgene Blood ccfDNA tubes were again used for blood stabilization. Whole blood from four healthy donors was collected into three different blood collection tubes: 10 mL K2-spray-dried EDTA tubes (BD Vacutainer), 10 mL Streck cfDNA BCT, and 10 mL PAXgene Blood ccfDNA tubes. After collection, 5 mL of blood was processed from each tube. Plasma was prepared by double centrifugation and filtered through a 0.8 μm filter. RNA was isolated using the exoRNeasy Serum / Plasma Maxi kit (QIAGEN) and eluted in 20 μL of water. The purified RNA was analyzed by RT-qPCR β-actin assay for amplification of a 294 bp fragment using Quantitect Primer / Probe RT PCR Master Mix and 2 μL of eluate.

[0206] Quantitative real-time PCR assay to measure relative differences in β-actin copies To measure the amount of ccfDNA, a RGQ (QIAGEN) real-time PCR assay was performed using 2 μL of the eluate on a Rotor-Gene Q instrument (Table 3). QuantiTect Multiplex PCR Kit reagents (QIAGEN GmbH) were used to amplify a 294 bp fragment of the human β-actin gene in a 20 μL assay volume. [Table 3]

[0207] result Extracellular vesicles (EVs) can be enriched from plasma generated from whole blood collected in blood collection tubes containing a stabilized composition according to the present disclosure. RNA obtained from purified EVs could be analyzed by RT-qPCR without inhibition (see Figure 6). In contrast, analysis of RNA isolated from EVs derived from whole blood collected in the Streck cfDNA BCT led to increased Ct values ​​and therefore unfavorable results, most likely due to inhibition of RT-qPCR due to cross-linking on RNA molecules induced by the formaldehyde-releasing agent-based stabilization technique.

[0208] 5. Example 5: Samples stabilized in PAXgene Blood ccfDNA tubes can be used for multi-mode testing This example demonstrates that multimodal testing of different biological targets contained in stabilized bodily fluids is feasible, as further illustrated below by an example using a three-pronged (3 from 1) workflow for the analysis of (1) CTCs, (2) ccfDNA, and (3) leukocyte-derived genomic DNA (gDNA) obtained from a single blood sample collected and stabilized with the stabilization technique according to the present disclosure. The AdnaTest Select procedure allows for the collection of whole blood residues after the recovery of bead-bound CTCs (CTC-depleted blood) (see Figure 7). Therefore, to demonstrate the feasibility of multimodal testing on collected blood, CTC-depleted cells from all of the experiments described above were collected in PAXgene Blood ccfDNA tubes. PAXgene Blood ccfDNA tubes allow for the simultaneous analysis of ccfDNA and leukocyte gDNA. It was subsequently demonstrated that CTC-depleted cells from the experiments listed in Section 2.2 could be used for ccfDNA isolation, and yields were advantageously unaffected by CTC depletion. Control samples collected in parallel from each donor, aliquoted into 5 mL samples, spiked with 20 LNCaP95 cells, and stored at 2–8°C for the same time but not used for CTC enrichment, served as references for ccfDNA and gDNA yields.

[0209] The CTC-depleted cell samples, along with their respective control samples, were centrifuged at 1900 x g for 15 minutes, and the resulting blood fractions (plasma and cellular fractions) were used for ccfDNA extraction (after a second centrifugation at 1900 x g for 10 minutes) and gDNA isolation, respectively. The yields of ccfDNA from CTC-depleted blood samples and blood used only for plasma production are shown in Table 4. Statistical analysis did not reveal any significant differences in ccfDNA yield between arms or between the first and last test time points within an arm of the same experiment (see Figure 8). Thus, CTC depletion did not have a significant effect on ccfDNA yield in terms of yield and in situ stability. [Table 4]

[0210] Similarly, the yield of gDNA extracted from the cellular fractions obtained after centrifugation of CTC-depleted cell samples (n = 8) was within the range of values ​​reported for blood stabilized in PAXgene ccfDNA tubes. From a 200 μL cellular fraction, we were able to isolate an average of 10.3 μg of gDNA from CTC-depleted samples (range 5.31–21.97 μg) compared to 9.43 μg of DNA from samples without CTC depletion (range 7.66–11.23 μg). There was no statistically significant difference in the yield of gDNA extracted from CTC-depleted versus blood used for plasma production alone, either 3 h after spiking and processing or overall (all time points, 3–72 h) (see Figure 9). The purity of extracted gDNA was 1.86 ± 0.05 and 1.85 ± 0.06 for CTC-depleted and control samples (i.e., prepared from whole blood), respectively (average across all time points), which is within the expected range of values ​​(1.7-1.9).

[0211] Materials and Methods Preparation of plasma and cell fractions Plasma from PAXgene Blood ccfDNA tubes was prepared according to the manufacturer's instructions. Briefly, cells were centrifuged at 1900 x g for 15 minutes. The cellular and plasma fractions were separated. The plasma-containing fraction was further centrifuged at 1900 x g for 10 minutes, and the plasma was collected without disturbing the respective pellets and stored at -20°C. The cellular fraction obtained after the first spin was immediately frozen at -20°C until processed for gDNA extraction.

[0212] ccfDNA workflow Automated ccfDNA purification on the QIAsymphony From 1.6–2.0 mL of PAXgene plasma, ccfDNA was isolated by a magnetic bead-based extraction procedure using the QIAsymphony PAXgene Blood ccfDNA kit (both PreAnalytiX) on a QIAsymphony instrument (QIAGEN). Quantitative real-time PCR assay for measuring absolute differences in 18S ribosomal DNA copies Absolute quantification of the 66- and 500-bp fragments of the human 18S rDNA gene was performed using standard curves for ccfDNA samples derived from CTC-depleted and non-spiked blood samples (see Figure 8 and the workflow shown in Figure 11). Real-time PCR assays were performed with 8 μL of eluate in a 20 μL assay volume using QuantiTect Multiplex PCR Kit reagents (QIAGEN) on an ABI 7900HT Fast Real-Time PCR-System (ThermoFisher). The calculated amounts of the 66- and 500-bp fragments were normalized to the volume of plasma used.

[0213] gDNA workflow Automated gDNA purification on the QIAsymphony Genomic DNA from 200 μL of the separated cell fraction obtained after plasma separation was isolated by a magnetic bead-based extraction procedure using the QIASymphony DSP DNA Mini Kit on a QIAsymphony instrument (QIAGEN). The elution volume was 200 μL per sample. Quantification of gDNA and assessment of gDNA purity The absorbance of gDNA was measured using a NanoDrop 8000 (Thermo Scientific). Absorbance was measured at 260 nm, 280 nm, and 320 nm. The concentration of gDNA (µg / mL) was calculated as 50 × (A260 - A320), and the total volume was calculated as the concentration multiplied by the sample volume. The purity of extracted gDNA was calculated as the ratio of the corrected absorbance at 260 nm to the corrected absorbance at 280 nm, i.e., (A260 - A320) / (A280 - A320). Pure DNA is characterized by an A260 / A280 ratio of 1.7 to 1.9.

[0214] Overall conclusions - Examples 1 to 5 Cells, including CTCs and other rare cells, rapidly degrade in unstabilized blood. The stabilization technique used in the method of the present disclosure (shown here based on the PAXgene Blood ccfDNA tube) allows for effective stabilization and analysis of ccfDNA levels, CTCs, and extracellular vesicles, thereby enabling parallel analysis of multiple different biological targets that can be enriched from stabilized samples. As demonstrated herein, the stabilization technique according to the present disclosure allows for stabilization of cellular antigen composition, genome, and transcriptome levels, as well as stabilization of the blood circulating transcriptome. Thus, a workflow according to the present invention is suitable for the analysis of individual liquid biopsy specimens (CTCs and other rare cells, ccfDNA, ctDNA, EVs, gDNA from leukocytes, cell subpopulations, etc.) collected in a single collection tube containing a stabilized composition according to the present invention, as well as combinations of such specimens from the same blood sample (see FIG. 10). An exemplary workflow is also shown in FIG. 11.

[0215] According to one embodiment, a blood sample-based workflow according to the present disclosure comprises: - Collection of blood into a collection tube (e.g., PAXgene Blood ccfDNA tube, e.g., blood volume of at least 5 mL, e.g., 10 mL; volume containing stabilizing solution, e.g., 11.5 mL) containing a stabilizing composition according to the present disclosure, transport to the laboratory. A portion of the stabilized blood is used for CTC enrichment (e.g., 5 mL). The unprocessed blood (e.g., 6.5 mL) and the remaining blood after CTC enrichment (approximately 4.5 mL) may be used for plasma production (cell-depleted fraction). Plasma production can be performed using a two-step centrifugation procedure. For example, the cell fraction obtained after the first centrifugation is used for total gDNA extraction from PBMCs or for FACS sorting for DNA extraction from target PBMC subpopulations. The resulting plasma is centrifuged in a second centrifugation step and can be further aliquoted for ccfDNA and / or EV isolation. The enriched CTCs can be further processed. For example, the enriched CTCs can be lysed to isolate intracellular nucleic acids (e.g., RNA, particularly mRNA) for analysis (e.g., CTC transcript detection). Furthermore, the intracellular nucleic acids obtained from the enriched CTCs can be sequenced.

[0216] According to one embodiment, a blood sample-based workflow according to the present disclosure comprises: - collection of blood into a collection tube (e.g., PAXgene Blood ccfDNA tube, e.g., blood collection volume of at least 5 mL, e.g., 10 mL; volume containing stabilizing solution, e.g., 11.5 mL) containing a stabilizing composition according to the present disclosure, transport to the laboratory, - Separating the stabilized blood sample into plasma and cellular fractions by centrifugation (e.g., using a two-step centrifugation procedure). An aliquot of the resulting plasma is used for the direct purification of ccfDNA. A further aliquot of plasma is used for the enrichment of EVs and subsequent isolation of RNA from EVs. - One aliquot of the cell fraction can be used for gDNA isolation. Alternatively, or in addition, an aliquot of the cell fraction (preferably a majority thereof) is used for subsequent gDNA isolation from the remaining PBMCs that have been captured and depleted of CTCs. Again, the enriched CTCs can be further processed as described above.

[0217] 6. Example 6: Further Use of Stabilization Techniques for CTC Enrichment and Analysis in Accordance with the Invention 6.1. Further Experiments on Combining Stabilization Techniques with Ficoll Density Centrifugation for CTC Enrichment Ficoll density centrifugation is described above in conjunction with Example 2, to which reference is made for brevity. Using the same methodology described above, further experiments were conducted aimed at optimizing mononuclear cell (MNC) enrichment from blood collected and stored in PAXgene Blood ccfDNA tubes. The resulting interface layer contains the PBMC fraction, including CTCs and other rare nucleated cells. In Example 2, it was observed that PAX ccfDNA-stabilized blood did not form a plasma / PBMC / erythrocyte layer as typically observed for EDTA-preserved blood (taken as a reference). Thus, in a relative comparison of MNC recovery to EDTA samples, PAX-preserved blood samples often exhibited only 75% of the MNC recovery achievable for EDTA samples (Figure 12). To improve MNC recovery when processing samples stabilized by the techniques of the present invention, additional and different solvents aimed at restoring cell density were evaluated.

[0218] result In addition to Example 2, additional concentrations were tested as well as other supplementary solutions. Comparisons were made with PAX samples diluted with PBS only. The results are shown in Table 5 below. [Table 5]

[0219] Based on these observations, various hypotonic and isotonic solutions were tested to restore the density of PAXccfDNA-stabilized blood cells. Sufficient and excellent recovery rates were observed for the solutions tested, indicating the success of the approach. Different dilutions having similar osmolality (e.g., ±20%, ±15%, or ±10%) to the lead dilutions specified in Table 5 can also be used and their positive effect on achieving the desired layer pattern and achieving a WBC recovery of at least 50%, at least 60%, and preferably at least 65% can be determined by routine experimentation.

[0220] 6.2. Further Experiments on the Combination of PAXgene Blood ccfDNA Tubes and AdnaTest Prostate Cancer Panel for CTC Detection AdnaTest CTC enrichment and associated materials and methods are described above in conjunction with Example 2, to which reference is made here for brevity. Detection of enriched cells relies on the detection of tumor cell-specific transcripts. Freshly collected EDTA blood (within 4 hours of collection) and blood collected in ACD-A tubes and stored at +4°C for up to 30 hours can be used, according to the manufacturer's recommendations. In multiple experiments, we evaluated whether blood collected and stored in PAXgene Blood ccfDNA tubes was compatible with three different AdnaTests, and to what extent it was compatible depending on the duration of blood storage, storage conditions (room temperature, RT vs. 2-8°C), and LOD (20 tumor cells / 5 mL blood vs. 5 cells / 5 mL blood).

[0221] A. Combination of PACgene Blood ccfDNA and AdnaTest Prostate Cancer Panel AR-V7 This set of experiments further evaluated and confirmed the previously discovered compatibility of blood collected and stabilized using the stabilization technology according to the present disclosure with the AdnaTest ProstateCancerPanel AR-V7 for detecting spiked tumor cells. Thus, multiple experiments using the AdnaTest ProstateCancerPanel AR-V7 demonstrated that tumor cell detection in mock samples (20 LNCaP95 cells / 5 mL blood) was 100% within 30 hours of storage at 2-8°C, decreasing to 93% after 72 hours (see Figure 13). Even after 120 hours, 67% were still detected. Again, this confirmed that the stabilization solution according to the present disclosure itself does not cause any nonspecific false-positive results and can therefore be successfully incorporated into the workflow described herein. When assay performance was evaluated with respect to storage conditions (room temperature vs. 2-8°C), a slight decrease in assay performance was observed (75% test positive in samples stored at room temperature vs. 84% in samples stored at 2-8°C) (see Figures 14A and 14B). However, total CTC enrichment was still possible after storage at room temperature.

[0222] Next, the limit of detection (LOD) of the test was evaluated. Samples collected in PAX ccfDNA tubes were spiked with either 5 or 20 cells / 5 mL of blood. The results show that samples spiked with 20 cells / 5 mL of blood (see Figure 15B) were better detected, while 5 cells / 5 mL of blood (see Figure 15A) was sufficient only for a shorter storage time. It is preferable to use a larger number of cells, such as 20 cells / 5 mL, to achieve high sensitivity (>90%) throughout the entire workflow (see Figures 15A and 15B). Finally, various regimens for plasma production were tested. In the workflow used throughout the present examples, blood samples were first used for CTC enrichment, and the CTC-depleted blood was used for plasma production for further multimodal testing (see Figure 16A). In an alternative plasma production method, plasma was produced as the first step (1900 g for 15 minutes), and then the cellular fraction was reconstituted to the initial volume with PBS and used for CTC enrichment (see Figure 16B). The results of tumor cell detection are shown in Figure 16. Specifically, in both plasma production methods, 100% of spiked tumor cells were detected up to 72 hours of storage. This indicates that samples stabilized by the method according to the present invention can be used for both types of plasma production methods without negatively affecting CTC enrichment and detection. Consistent with this, similar results were observed when the same experiment comparing plasma production methods was performed with the prototype AdnaTest versus EZ1 on the EZ1 instrument (automated solution) (see Figures 17A and 17B). The results of this example demonstrate that either method of plasma production (i.e., multi-mode use) is applicable.

[0223] B. Combination of PAXgene Blood ccfDNA Tube and AdnaTest Prostate Cancer In this example, the AdnaTest ProstateCancer (also called "ProstateDirect") is compared to the AdnaTest ProstateCancerPanel AR-V7. The AdnaTest ProstateCancer is less sensitive than the AdnaTest ProstateCancerPanel AR-V7 and relies on end-point PCR assessment (whereas the AR-V7 test is an RT-PCR test). In this comparison, the samples used in the experiments described above were also used in the AdnaTest Prostate Cancer evaluation, therefore, for the sake of brevity, reference is made to the corresponding section above.

[0224] The comparative results are shown in Figure 18 and confirm the findings made by AdnaTest Prostate Cancer Panel AR-V7. Specifically, the following results were obtained: - The tumor cell detection rate in mock samples (20 LNCaP95 cells / 5 mL blood) was 100% within 30 hours of storage at 2-8°C, decreasing to 93% after 72 hours (see Figure 18A for AdnaTest ProstateCancerPanel AR-V7 and Figure 18B for AdnaTest ProstateCancer). When test performance was evaluated with respect to storage conditions (room temperature vs. 2-8°C), a slight decrease in test performance was observed (AdnaTest ProstateCancerPanel AR-V7: 75% test positive for samples stored at room temperature vs. 84% for samples stored at 2-8°C; AdnaTest ProstateCancer: 50% test positive for samples stored at room temperature vs. 80% for samples stored at 2-8°C; see Figures 18C and 18D, respectively). Again, total CTC enrichment was achieved even after storage at room temperature. - As described above, the limit of detection (LOD) was assessed by spiking either 5 cells / 5 mL of blood and testing with the AdnaTest ProstateCancerPanel (see Figure 18E) or AdnaTest ProstateCancer (see Figure 18F). The results confirmed that samples spiked with 20 cells / 5 mL of blood (see above) provided better detection, indicating that 5 cells / 5 mL of blood is sufficient only for very short storage times. Preferably, a higher cell number, e.g., 20 cells / 5 mL, would be detected by both tests. Finally, different plasma production methods were tested. Specifically, an alternative plasma production method was used in which plasma was produced as a first step and a cell fraction was used for CTC enrichment. The enriched CTC fraction was used for AdnaTest ProstateCancerPanel AR-V7 (see Figure 18G) or AdnaTest ProstateCancer (see Figure 18H). The alternative plasma production method allowed 100% detection of spiked tumor cells for up to 48 hours of storage, indicating that samples stabilized by the method according to the present invention can be used for both types of plasma production methods without negatively affecting CTC enrichment and detection. This can therefore provide an advantageous workflow.

[0225] 6.3. PAXgene Blood ccfDNA Tube and AdnaTest ColonCancer The performance of the AdnaTest ColonCancer was tested in conjunction with the AdnaTest ProstateCancer and AdnaTest ProstateCancerPanel AR-V7 in a similar spike-in system as described above. Specifically, 20 T84 cells were spiked into 5 mL of healthy donor blood. Samples were stored at 2-8°C using PAXgene Blood ccfDNA tubes and compared to test performance using similarly spiked samples collected in an ACD-A BCT. Performance was tested at 3, 24, 48, and 72 hours after spiking. The results show that the PAXgene Blood ccfDNA tube, which is preferred for use in the workflow described herein, is compatible with the AdnaTest ColonCancer and allows for tumor cell detection (100% sensitivity) within 72 hours of sample storage (see Figure 19A). Furthermore, comparable results were obtained with the ACD-A BCT at 3 and 24 hours (see Figure 19B).

[0226] 7. Example 7: Compatibility of PAXgene Blood ccfDNA tubes with the Parsortix device for CTC enrichment in the context of the all-from-one solution. In Parsortix-based CTC (spiked tumor cells as a spike-in model) detection, already tested in Example 2, the following options were further evaluated: A. Detection of tumor cells based on immunofluorescence detection of tumor cells - staining of epithelial tumor-specific antigens. B. Detection of spiked tumor cells based on transcriptome signature (RT-PCR with AdnaTest AR-V7 panel). For further information regarding the Parsonix device and related materials and methods, reference is made to Example 2 for the sake of brevity.

[0227] A. Detection of tumor cells based on immunofluorescence (IF) detection of tumor cells - staining of epithelial tumor-specific antigens The Parsortix instrument (Angle PLC) offers two modes for quantitative (IF-based) detection of tumor cells. After completion of the CTC enrichment program, the CTC-enriched fraction is collected and delivered as a concentrate of approximately 100 μL. This concentrate is placed on a microscope slide for further IF staining and microscopic examination. Alternatively, antibody staining can be performed directly in the separation cassette. The latter approach is more efficient because it reduces potential loss of CTCs due to the collection, centrifugation, and staining steps. Spiked tumor cells (50 MCF7 cells) were detected by immunofluorescence staining for pan-cytokeratin either after collection of the CTC-enriched fraction or in the in-cassette staining (see Figures 20A and 20B, respectively). Storage of the spiked blood (either in the in-cassette staining or in the collected cells) does not affect the stainability of the cells. The spiked tumor cells appear to be stainable without any limitations (see Figure 21). Thus, the cells can be easily enriched and stained, and are therefore useful in the multi-modal workflow described herein.

[0228] B. Detection of spiked tumor cells based on transcriptome signature (RT-PCR with AdnaTest AR-V7 panel). As an alternative to IF staining, enriched tumor cells can be detected based on their transcriptome signature. Therefore, enriched CTCs were collected after Parsortix runs and detected using the aforementioned AdnaTest ProstateCancerPanel AR-V7 (detection part only), referenced herein. As shown in Figure 22, cells spiked into PAX ccfDNA-collected blood samples and stored for up to 3 days (TTP indicates days) are as efficiently detectable as if spiked into EDTA-collected samples. These data highlight the compatibility of the PAXgene Blood ccfDNA tube with the Parsortix instrument for CTC enrichment by either IF staining or RT-PCR-based assays.

[0229] 8. Example 8: Multimodal analysis of circulating cell-free RNA (ccfRNA), circulating cell-free DNA (ccfDNA), and genomic DNA (gDNA) from blood samples collected in PAXgene Blood ccfDNA tubes In addition to circulating cell-free DNA (ccfDNA) from blood, circulating cell-free RNA (ccfRNA) is also gaining importance for biomarker research. Combining insights from both analytes promises to enhance understanding of the underlying molecular processes. Example 8 demonstrates multimodal extraction and analysis of ccfRNA, ccfDNA, and gDNA from a single blood sample collected using PAXgene® blood ccfDNA tubes, resulting in an advantageous stabilized composition according to the present invention. Whole blood samples from healthy consenting donors were collected into PAXgene blood ccfDNA tubes (PreAnalytiX), BD Vacutainer® K2EDTA tubes (BD), cell-free DNA BCT® (Streck®), RNA Complete BCT™ (Streck), and LBgard® blood tubes (Biomatrica). Plasma was produced by double centrifugation immediately after collection or after up to 3 days of storage. Cell-free nucleic acids were extracted as shown in Figure 23.

[0230] result The ccfRNA yields from plasma after blood storage in EDTA and PAXgene blood ccfDNA tubes are shown in Figure 24A (comparison at TTP0) and Figure 24B (relative fold change during whole blood storage). Quantitative PCR analysis revealed comparable yields of miRNA, mRNA, and ccfDNA targets from plasma of blood collected in PAXgene blood ccfDNA tubes and EDTA tubes. After up to 3 days of blood storage in PAXgene blood ccfDNA tubes, RNA targets (both intervesicular and extravesicular, extracted with exoRNeasy and miRNeasy, respectively) remained detectable, demonstrating improved stabilization over ETDA. The miRNA yield in plasma after blood storage in stabilized tubes is shown in Figure 25A (comparison at TTP0) and Figure 25B (relative fold change during whole blood storage). High C at TTP0 (day 0) after 3 days of storage. T RNA extraction and detection sensitivity was affected by blood collection tubes containing formaldehyde-releasing formulations (Streck and Biomatrica), as indicated by the low RNA stabilization efficiency and low RT values. Genomic DNA yield and integrity are shown in Figure 26. Furthermore, PAXgene Blood ccfDNA tubes enabled efficient extraction of gDNA from post-plasma separation blood cells after 3 days of whole blood storage, and the DNA was intact, as indicated by stable DNA integrity index. In contrast, gDNA yield and integrity were reduced upon collection and storage in Streck RNA and Biomatrica tubes.

[0231] The results provided by the multimodal analysis in Example 8 further demonstrate that the non-crosslinking technology of the stabilized compositions of the present invention is highly advantageous, enabling the isolation and analysis of cell-free miRNA, mRNA, ccfDNA, and even genomic cellular gDNA from a single sample. In addition, and as shown by other examples, additional rare cell populations, such as CTCs, can be enriched and detected. The overall data demonstrate that the present invention provides an advantageous multimodal workflow that is highly useful in liquid biopsy research.

[0232] Other stabilization techniques have shown impaired analytical efficiency following whole blood storage of test targets of interest, as demonstrated by several examples contained herein.

Claims

1. 1. A method for stabilizing and enriching multiple biological targets in a cell-containing body fluid, comprising: (A) providing a stabilized cell-containing body fluid sample by contacting the cell-containing body fluid with a stabilizing composition comprising one or more of the following stabilizing factors: (a) at least one primary, secondary, or tertiary amide; (b) at least one poly(oxyethylene) polymer, and / or (c) at least one apoptosis inhibitor; (B) maintaining the stabilized cell-containing bodily fluid sample for a stabilization period; (C) - at least one cell subpopulation, - extracellular nucleic acids, - extracellular vesicles, and -Intracellular nucleic acids processing the stabilized cell-containing bodily fluid sample to enrich from the stabilized cell-containing bodily fluid for three or more biological targets selected from the group consisting of: The method comprising:

2. 2. The method of claim 1, wherein the enriched cell subpopulation comprises target rare cells, and optionally the target rare cells are selected from the group consisting of tumor cells, in particular circulating tumor cells (CTCs), fetal cells, stem cells, cells infected by viruses or parasites, circulating endothelial cells (CECs), and circulating endothelial progenitor cells (EPCs).

3. Step (C) comprises obtaining at least one cell-containing fraction and at least one cell-depleted fraction from the stabilized body fluid, and the processing in (C) comprises: According to aspect A, (aa) separating the stabilized cell-containing body fluid sample into at least one cell-containing fraction and at least one cell-depleted fraction; (bb) further processing the cell-containing fraction, wherein further processing the cell-containing fraction comprises: (i) enriching a cell subpopulation, preferably comprising a target rare cell, from said cell-containing fraction; and / or (ii) enriching the cell-containing fraction for intracellular nucleic acids, which may be genomic DNA; Including, (cc) further processing said cell-depleted fraction, wherein further processing said cell-depleted fraction comprises: (i) enriching the cell-depleted fraction for extracellular nucleic acids, which may be extracellular DNA; and / or (ii) enriching extracellular vesicles from the cell-depleted fraction; Contains, or According to aspect B, (aa) enriching a cell subpopulation, preferably comprising a target rare cell, from the stabilized cell-containing body fluid sample; (bb) separating the stabilized cell-containing bodily fluid sample, from which the target cell subpopulation has been removed, into a cell-containing fraction and a cell-depleted fraction; (cc) further processing said cell-depleted fraction, wherein further processing said cell-depleted fraction comprises: (i) enriching the cell-depleted fraction for extracellular nucleic acids, which may be extracellular DNA; and / or (ii) enriching extracellular vesicles from the cell-depleted fraction; and (dd) optionally enriching intracellular nucleic acids, preferably genomic DNA, from said cell-containing fraction; Contains, or According to aspect C, (aa) dividing the stabilized cell-containing bodily fluid sample into at least two aliquots and enriching a cell subpopulation, preferably comprising rare cells, from at least one of the provided aliquots; (bb) providing at least one cell-containing fraction and at least one cell-depleted fraction; (cc) further processing said cell-depleted fraction, wherein further processing said cell-depleted fraction comprises: (i) enriching the cell-depleted fraction for extracellular nucleic acids, which may be extracellular DNA; and / or (ii) enriching extracellular vesicles from the cell-depleted fraction; and (dd) optionally enriching intracellular nucleic acids, preferably genomic DNA, from said cell-containing fraction; 3. The method of claim 1 or 2, comprising:

4. The method of any one of claims 1 to 3, further comprising (D) processing the enriched three or more biological targets for analysis.

5. 5. The method of claim 4, having one or more of the following features: (i) step (C) comprises enriching target rare cells, followed by step (D) comprising analysing the enriched target rare cells at the cellular level and / or analysing the target enriched rare cells by isolating intracellular nucleic acids from the enriched target rare cells and detecting one or more target molecules in the isolated intracellular nucleic acids, optionally wherein the intracellular nucleic acids comprise mRNA; (ii) step (C) comprises obtaining a cell-depleted fraction from said stabilized cell-containing body fluid sample and isolating extracellular nucleic acid from said obtained cell-depleted fraction, optionally said extracellular nucleic acid comprising or consisting essentially of extracellular DNA, and a subsequent step (D) comprises detecting one or more target molecules within said isolated extracellular nucleic acid; (iii) step (C) comprises enriching extracellular vesicles from a cell-depleted fraction obtained from the stabilized cell-containing body fluid sample, and a subsequent step (D) comprises isolating RNA from the enriched extracellular vesicles and detecting one or more target molecules within the isolated RNA; and / or (iv) step (C) comprises isolating at least (i) circulating tumor cells, (ii) genomic DNA, and (iii) circulating cell-free DNA as biological targets, and step (D) comprises (i) isolating RNA from the circulating tumor cells and detecting biomarker RNA molecules in the isolated RNA; (ii) detecting, e.g., amplifying and / or sequencing, genomic DNA, and (iii) detecting biomarker molecules in the isolated circulating cell-free DNA.

6. 6. The method of one or more of claims 1 to 5, comprising enriching target rare cells and / or extracellular vesicles by affinity capture.

7. 7. The method according to one or more of claims 1 to 6, wherein the cell-containing body fluid has one or more of the following characteristics: - It is a circulating fluid; selected from blood, urine, saliva, synovial fluid, amniotic fluid, tears, lymph, fluid (cerebrospinal fluid), sweat, ascites, breast milk, bronchial lavage, peritoneal and pleural effusions, bone marrow and nipple aspirates, semen / seminal plasma, body secretions or excretions; - selected from blood and urine; and / or - It's blood.

8. 8. The method according to claim 1, wherein the stabilized composition comprises at least one primary, secondary, or tertiary amide, said stabilized composition comprising at least one primary, secondary, or tertiary amide according to formula 1, optionally wherein at least one compound according to formula 1 is a primary, secondary, or tertiary carboxylic acid amide, optionally an N,N-dialkylpropanamide such as N,N-dimethylpropanamide and / or butanamide: 【Chemistry 1】 wherein R1 is hydrogen or an alkyl group, preferably a C1-C5 alkyl group, a C1-C4 alkyl group, or a C1-C3 alkyl group, more preferably a C1-C2 alkyl group; R2 and R3 are the same or different and are selected from hydrogen and hydrocarbon groups, preferably alkyl groups having a chain length of 1 to 20 carbon atoms arranged in a linear or branched fashion; and R4 is oxygen, sulfur, or selenium group, preferably R4 is oxygen.

9. The method of any one or more of claims 1 to 8, wherein the stabilizing composition comprises at least one poly(oxyethylene) polymer, which may be polyethylene glycol.

10. 10. The method of claim 9, wherein the stabilized composition has one or more of the following characteristics: a) the poly(oxyethylene) polymer involved is unsubstituted polyethylene glycol; b) the composition comprises a poly(oxyethylene) polymer, which is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500; c) the composition comprises at least one poly(oxyethylene) polymer having a molecular weight of less than 1500, preferably a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less, optionally the molecular weight of the polymer is in a range selected from 100-1000, 200-800, 200-600, and 200-500; d) the composition comprises a poly(oxyethylene) polymer that is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500 and a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less; and / or e) the composition comprises a poly(oxyethylene) polymer that is a high molecular weight poly(oxyethylene) polymer and a poly(oxyethylene) polymer that is a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less, wherein the high molecular weight poly(oxyethylene) polymer has a molecular weight in a range selected from 1500 to 50,000, 2,000 to 40,000, 3,000 to 30,000, 3,000 to 25,000, 3,000 to 20,000, and 4,000 to 15,000; and / or the low molecular weight poly(oxyethylene) polymer has a molecular weight in a range selected from 100 to 1,000, 200 to 800, 200 to 600, and 200 to 500.

11. 11. The method according to one or more of claims 1 to 10, wherein the stabilized composition comprises at least one caspase inhibitor as an apoptosis inhibitor, optionally said caspase inhibitor having one or more of the following characteristics: a) the caspase inhibitor is a pan-caspase inhibitor; b) the caspase inhibitor comprises a caspase-specific peptide; c) the caspase inhibitor comprises a modified caspase-specific peptide, preferably modified at the carboxyl terminus with an O-phenoxy (OPh) group; d) the caspase inhibitor comprises a modified caspase-specific peptide, preferably N-terminally modified with a glutamine (Q) group; e) the caspase inhibitor is selected from the group consisting of Q-VD-OPh, Boc-D-(OMe)-FMK, and Z-Val-Ala-Asp(OMe)-FMK; f) the caspase inhibitor is selected from the group consisting of Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK; and / or g) The caspase inhibitor is Q-VD-OPh.

12. The stabilizing composition comprises: (a) at least one primary, secondary, or tertiary amide (preferably as defined in claim 8); (b) at least one poly(oxyethylene) polymer (preferably as defined in claim 9 or 10), and (c) at least one caspase inhibitor (preferably as defined in claim 11); and 12. The method of claim 1, optionally comprising (d) a chelating agent such as EDTA.

13. The cell-containing body fluid is blood, a) one or more compounds according to Formula 1; b) at least one high molecular weight poly(oxyethylene) polymer having a molecular weight in the range of 3,000 to 40,000, such as in the range of 3,000 to 30,000, or 3,500 to 25,000, and a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1,000 or less, such as in the range of 100 to 800, 200 to 800, or 200 to 500; c) at least one caspase inhibitor, preferably a pan-caspase inhibitor, optionally Q-VD-OPh; and d) an anticoagulant, which may optionally be a chelating agent such as EDTA; and After the blood sample has been contacted with the additive, and optionally further stabilizing additives, the resulting mixture / stabilized blood sample is one or more compounds according to formula 1 at a concentration ranging from 0.3 to 4%, for example from 0.5 to 3%, from 0.5 to 2%, or from 0.75 to 1.5%, - said high molecular weight poly(oxyethylene) polymer at a concentration ranging from 0.2% to 1.5% (w / v), for example from 0.25% to 1.25% (w / v), from 0.3% to 1% (w / v), or from 0.4% to 0.75% (w / v); - said low molecular weight poly(oxyethylene) polymer at a concentration ranging from 1.5% to 10%, for example from 2% to 6%, and - The method according to one or more of claims 1 to 12, comprising said caspase inhibitor at a concentration ranging from 1 μM to 10 μM, for example from 3 μM to 7.5 μM.

14. 14. The method according to claim 1, having one or more of the following characteristics: (i) said stabilization of the cell-containing body fluid sample does not involve the use of additives at concentrations that induce or promote lysis of nucleated cells; (ii) the stabilization does not induce protein-nucleic acid or protein-protein cross-linking; (iii) the stabilization does not involve the use of cross-linking agents that induce protein-nucleic acid and / or protein-protein cross-links, such as formaldehyde, formalin, paraformaldehyde, or formaldehyde-releasing agents; (iv) the stabilization does not involve the use of toxic factors; and / or (v) A stabilizing agent is contained in a stabilizing composition that includes water.

15. 15. The method of claim 1, wherein the stabilization used in step (A) does not induce protein-nucleic acid or protein-protein crosslinking in the stabilized sample, and optionally, step (C) comprises enriching extracellular vesicles from a cell-depleted fraction obtained from the stabilized cell-containing body fluid sample, and a subsequent step (D) comprises isolating RNA from the enriched extracellular vesicles and detecting one or more target molecules in the isolated RNA.

16. A cell-containing body fluid, preferably blood, a) one or more compounds according to Formula 1; b) at least one high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 3000, and optionally a low molecular weight poly(oxyethylene) polymer having a molecular weight of 1000 or less; c) at least one caspase inhibitor; and d) optionally a chelating agent, preferably EDTA; The method of claim 14 or 15, wherein the contacting is performed with

17. Step (C) - rare cells, preferably circulating tumor cells, - extracellular nucleic acids, - extracellular vesicles, and -Intracellular nucleic acids 17. The method of any one of claims 14 to 16, comprising processing the stabilized cell-containing body fluid sample to enrich from the stabilized cell-containing body fluid for three or more biological targets selected from the group consisting of:

18. 18. The method of claim 14, wherein step (C) comprises obtaining at least one cell-containing fraction and at least one cell-depleted fraction from the stabilized body fluid sample, and step (C) further comprises enriching extracellular vesicles from the cell-depleted fraction obtained from the stabilized cell-containing body fluid sample, and a subsequent step (D) comprises isolating RNA from the enriched extracellular vesicles.

19. 20. The method of claim 18, wherein step (D) comprises detecting one or more target molecules within the isolated RNA.

20. 20. The method of claim 18 or 19, comprising isolating genomic DNA from the cell-containing fraction.

21. 21. The method according to any one of claims 1 to 20, wherein the processing step (C) comprises subjecting the stabilized cell-containing body fluid sample or a cell-containing fraction obtained from the stabilized cell-containing body fluid sample to a density gradient centrifugation step, and optionally, the cell-containing body fluid sample is blood.

22. 22. The method of claim 21, wherein the stabilized blood sample, or a cell-containing fraction obtained from the stabilized blood sample, is diluted with a dilution solution before the density gradient centrifugation step is performed.

23. 23. The method of claim 22, wherein the diluted solution has one or more of the following characteristics, and after density gradient centrifugation, distinct layers are formed, and the formed layers comprise a PBMC layer: (a) a hypotonic or isotonic solution; (b) containing a tonicity modifier; (c) a polyol, optionally a sugar or sugar alcohol; (d) sugar, optionally glucose; (e) a sugar alcohol, optionally glycerol; and / or (f) salts, optionally alkali metal salts, optionally chloride salts.

24. (a) the dilute solution comprises a reducing sugar, which may be glucose, at a concentration in the range of 2-10%, 3-7%, or 4-6% (w / v); (b) the dilute solution comprises a sugar alcohol and a salt, and optionally the dilute solution comprises up to 0.5 M glycerol and up to 2% sodium chloride; (c) the dilution solution comprises 0.7-1.2% sodium chloride and 0.075-0.15M glycerol; and / or (d) the diluted solution is (i) 5% (w / v) glucose; (ii) 0.9% NaCl + 0.1 M glycerol, and (iii) a diluted solution comprising at least one tonicity adjusting agent and having an osmolality that matches the osmolality of the diluted solution defined in (i) or (ii), or an osmolality that is within ±20%, ±15%, or ±10% of the osmolality of said solution defined in (i) or (ii); 24. The method of claim 23.

25. 23. The method of claim 22, wherein the diluent solution comprises DMSO.

26. 26. Use of the dilution solution of any one of claims 22 to 25 for processing a stabilized blood sample or a cell-containing fraction thereof, wherein the blood sample is stabilized with a stabilizing composition comprising (a) at least one primary, secondary, or tertiary amide, (b) at least one poly(oxyethylene) polymer, and / or at least one apoptosis inhibitor, optionally with the stabilizing composition of any one of claims 8 to 13, or 14.

27. 27. The use according to claim 26, for restoring the density of contained mononuclear cells, preferably for gradient density centrifugation, wherein the diluted solution is contacted with the stabilized blood sample or its cell-containing fraction before contacting the diluted solution with the gradient density medium.

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