Process and assembly for preparing a sample

Abstract

Preparation process and assembly (2) for preparing a treated sample (TS); intermediate layers (IL, IL', IL") of initial blood samples (IS, IS', IS") containing leukocytes and target particles are retrieved from the initial blood samples (IS, IS', IS") and moved to a collecting station (5) so as to obtain an intermediate sample (IIS); to the intermediate sample (IIS) arranged inside a selection container (6) selection particles (FF) are added which are selectively bound to the target particles and moved, together with the target particles bound to them, to determined positioning zones inside the selection container (6); part of the intermediate sample (IIS) not including the combined particles (CP) is removed from the selection container.

Description

[0001] “PROCESS AND ASSEMBLY FOR PREPARING A SAMPLE”

[0002] Cross-Reference to Related Applications

[0003] This Patent Application claims priority from Italian Patent Application No. 102024000029487 filed on December 20, 2024, the entire disclosure of which is incorporated herein by reference.

[0004] Technical Field

[0005] The present invention relates to a preparation process, an assembly for preparing a treated sample, an analysis method and an analysis system. Furthermore, the present invention relates to a machine for preparing a treated sample.

[0006] Background of the Invention

[0007] It is known to retrieve a sample of blood or other biological fluid (typically of about 7.5 ml or 4 ml), insert it into a test tube and optionally after removing a portion of plasma to add thereto the ferrofluid (i.e. a colloidal suspension of ferromagnetic particles) appropriately functionalized (i.e. provided with biospecific ligands, such as antibodies, having binding affinity for at least one determining feature present on the target particles, such as for example surface antigens) so that it binds to specific target particles, typically circulating tumour cells (CTC); tumour-derived extracellular vesicles (tdEV); circulating multiple myeloma cells (CMMC); extracellular vesicles in multiple myeloma (mmEV); circulating endothelial cell (CEC) and fragments thereof - (edEV); circulating melanoma cells (CMC) and fragments thereof and / or foetal cells (circulating foetal cells (CFC) such as trophoblasts and / or erythroblasts and / or lymphocytes). At this point, a magnetic field is imposed and the target particles are moved on the wall of the test tube and the portion of the sample not of interest (relatively far from the wall) is more easily taken away and a treated sample is obtained which should contain the target particles at a higher concentration than the original sample. The treated sample can then be used for further analyses, such as genetic analysis and / or by means of staining reagents, and / or for sorting individual cells or assemblies of cells, and / or for enumeration operations and / or for analysis of the expression of biomarkers on target particles (for example cells or vesicles), such as, for example, in the case of tumour cells and / or tdEVs, tumour antigens that are potential targets for therapies. Some examples of targets are listed in the following list:

[0008] - For breast cancer: Her2(+)

[0009] ER(+) or PR (+)

[0010] ER(+) or PR(+) plus Her2(-), Ki-67(+)

[0011] Her2 (-), ER (-) and PR (-)

[0012] - For bowel cancer

[0013] Her2 (+)

[0014] EGFR (+) no RAS-gm, no RAF-gm

[0015] - For lung cancer:

[0016] EGFR (+)

[0017] Her2 (+)

[0018] RO SI -gm

[0019] KRAS G12C-gm

[0020] BRAF V600E-gm

[0021] MET exon 14 skipping-gm

[0022] RET fusion-gm

[0023] - For prostate cancer:

[0024] PARP and low cell repair

[0025] PSMA

[0026] - For myeloma:

[0027] CD38, CD47, CD 138, SLAMF7, GPRC5D,

[0028] FcRH5 and BCMA.

[0029] The above list complies with the American Society of Clinical Oncology (ASCO, August 2023), where (+): cancer has higher than normal target marker expression, (-): normal expression, ER: oestrogen receptor, PR: progesterone receptor, EGFR: epidermal growth factor receptor, PARP: poly-ADP ribose polymerase, genetic mutations are indicated with the suffix -gm after the gene name or aberration description. In multiple myeloma, CD38, CD47, CD 138, SLAMF7, GPRC5D, FcRH5 and BCMA are highly expressed by malignant plasma cells.

[0030] We would like to point out, however, that this type of approach has shown significant limitations.

[0031] Among these we would like to point out the following: the analyses have a low sensitivity, the percentage of patients for whom it is possible to make evaluations through biomarker is low, the percentage of patients for whom it is possible to make evaluations by means of genetic analysis is low, the statistical error linked to a low target particle count is relatively high (J Oncol. 2010; 2010: 617421; Cytometry Pt A, Volume: 71A, Issue: 3, Pages: 154, DOI: 10.1002 / cyto.a.20369).

[0032] These limits make the methods for enriching the target particles in the state of the art ineffective in many cases, for example in the oncological field it is known that carcinomas start from a primary tumour and, over time, can spread to more distant areas of the body. During this evolutionary process, tumour cells acquire or lose genes that favour migration and growth in other organs. Most patients die due to metastases, which can impair vital functions. In this regard, treatment decisions are based on several factors, including the diagnosis of the tumour, the selection of the treatment and the evaluation of the effectiveness thereof. One of the problems is to understand whether the risk of aggravation of the disease justifies treatments with serious side effects, or whether a targeted therapy can be effective. In this context, the information obtained from the CTCs can be useful in this decision-making process. However, CTCs and tdEVs are rare, and their counts may be null even in patients with advanced disease, making enrichment and consequently the evaluation of the presence of biomarkers and / or the genetic characteristics of CTCs and tdEVs ineffective.

[0033] It should be noted that this type of problem has not been observed only for CTCs but in general for all rare cells and / or fragments of rare cells of interest, i.e. present in numbers from 1 to 10,000 in 7.5 ml of blood. For example, circulating multiple myeloma cells (CMMC) and relative fragments, circulating melanoma cells (CMC) and relative fragments (mmEV), circulating endothelial cells (CEC) and relative fragments (edEV), and the foetal cells such as trophoblasts and / or erythroblasts and / or lymphocytes (circulating foetal cells - CFC).

[0034] In this regard, it is recalled that the current methods of prenatal diagnosis require obtaining foetal cells through invasive procedures, such as chorionic villus sampling (CVS) and amniocentesis (amniotic fluid test), which entail associated risks for both the foetus and the mother. Therefore, efforts have been made to develop non-invasive tests through methods for enriching foetal cells from the peripheral blood of the mother, in particular erythroblasts and / or trophoblasts. However, even in this case the foetal cells are extremely rare making it difficult to isolate from 7.5 ml of blood a sufficient number of cells to perform genetic analysis of the foetus, for example in order to identify the possible presence of chromosomal diseases or other genetic diseases.

[0035] US2017211058A1 discloses a method for the automated processing of aggregated samples, in particular blood samples and an apparatus for performing the method and relative uses. The method and, consequently, the apparatus can be used in particular to perform nucleic acid amplification techniques for testing blood donations and blood products.

[0036] EP3371595B1 discloses a method for producing an artificial environment of primary cell populations, in particular an artificial tumour environment of primary tumour cell populations and its use in an ex vivo method for testing the cellular reactivity of primary tumour cell populations to one or more drugs. The method comprises incubating the primary tumour cells with the artificial tumour environment and the drug or the drugs and analysing the response of the primary tumour cell populations.

[0037] US2019338248A1 discloses a method for producing red blood cell-based products, plasma and platelet with uniform dose and volume. The method provides for joining multiple units of blood, the leukodepletion of the blood and inactivation of any pathogen contained in it.

[0038] US2008206757A1 discloses a method and compositions for isolating and detecting rare cells from a biological sample containing other cell types. The method comprises a debulking step using microfabricated filters to filter fluid samples and the enriched rare cells can be used in a downstream process such as identification, characterization or even growth in culture or use in other ways.

[0039] W02022250490A1 discloses a microparticle separation device and a method for separating microparticles using the same and comprises: a rotating platform provided with rotation for transferring a test tube to a working position of each module; a container storage module comprising a first container rack for storing a plurality of test tubes and a first container transfer unit for transferring the test tubes; a first and a second capping module for opening / closing the lid of the test tube transferred by means of the rotating platform; a fluid management module comprising an automatic pipette; a centrifuge module.

[0040] Aim of the present invention is to provide a preparation process, an assembly for preparing a treated sample, an analysis method, an analysis system and a machine for preparing a treated sample, which allow to overcome, at least partially, the drawbacks of the state of the art and are, at the same time, easy and economical to implement.

[0041] Summary

[0042] According to the present invention, a preparation process, an assembly for preparing a treated sample, an analysis method, an analysis system and a machine for preparing a treated sample as claimed in the following independent claims and, preferably, in any of the claims directly or indirectly dependent on the independent claims are provided.

[0043] Unless explicitly specified otherwise, in this text the following terms have the meaning indicated below.

[0044] By selection particle is meant a corpuscle (capable of binding substantially selectively to the target particles) having the larger dimension of less than about 1000 nm (advantageously, less than about 200 nm - in particular, greater than 1 nm). According to some non-limiting examples, the selection particles are chosen from: microspheres (magnetic), nanospheres (magnetic, e.g. nanospheres up to 100 nm,), complexes formed by nanospheres; ferrofluid particles (and a combination thereof). Advantageously but not necessarily, the selection particles are magnetic and in particular they show a ferrofluid behaviour in colloidal suspension.

[0045] The expressions “cell fragments” and “fragments thereof’ (referring to cells) are intended to indicate structures enclosed by phospholipid membranes, in particular cellular particles such as, for example, exosomes, vesicles, microvesicles, apoptotic bodies, whose presence and quantity in an organic fluid sample of a patient may indicate the presence of a disease in the patient him- / herself.

[0046] Typically but not necessarily, the target particles have the larger dimension of less than about 50 pm (in particular, less than about 30 pm; in particular, less than about 25 pm). In particular, the target particles have the larger dimension greater than about lOOnm (in particular, greater than 1 pm).

[0047] The dimensions of the target particles can be measured in a standard way with graduated scale microscopes or normal microscopes used with graduated scale slides (on which the particles are deposited) or by using electron microscopes.

[0048] The dimensions of the selection particles are measured by means of electron microscopes or dynamic light scattering (DLS - for example in accordance with the provisions of ISO 22412:2017).

[0049] In this text, by dimensions of a particle is understood to mean the length, width and thickness of the particle.

[0050] The expression “substantially selective” is used to indicate that the selection particles FF bind for the most part only to the target particles (or to fragments of target particles - including vesicles, for example the tumour-derived extracellular vesicle - tdEV) and in a residual or null way to other cells (or corpuscles, or fragments) present in the intermediate sample (or in any case in a blood sample - the intermediate sample is described in more detail below). Advantageously, the substantially selective binding of the selection particles FF provides, considering an equal number of target particles and of other cells (or corpuscles), a ratio between the number of selection particles FF that bind to the target particles and the number of selection particles FF that bind to other cells (or corpuscles) present in the intermediate sample to be at least 5 to 1 (advantageously, greater than 10 to 1).

[0051] To achieve this effect, all known procedures in this regard can be implemented (several examples of how to obtain selective bonds with target particles are provided in the literature) as well as using specific molecules (for example suitably conjugated with ferrofluid particles) capable of binding to the target particles and not to other cells. For example, antibodies and / or antibody portions may be used (such as, for example, anti- EPCAM for CTCs and / or tdEVs, or anti-CD138 for CMMCs and / or mmEVs, or anti- CD146 for CECs and / or edEVs and / or CMCs and / or fragments deriving from CMCs, or anti-CD105 and / or anti-CD90 and / or anti-CD141 and / or anti-EpCAM and / or anti-HLA- G and / or anti-TROP-2 for foetal trophoblasts, or anti-CD71 and / or anti-CD36 and / or anti- GPA for foetal erythroblasts).

[0052] To measure the selectivity of the selection particles FF, a sample can be subjected to the treatment, transfer, combination, moving and removal steps (as described in more detail below) and to an analysis step, during which, in particular, the collected cells are analysed by using specific markers (for example, anti-cytokeratin for CTCs, anti-CD105 for CECs, anti -Mel for CMCs, anti-CD38 for CMMCs, anti-CK for trophoblasts, antiglobin chains e for erythroblasts) and a fluorescence microscope, or alternatively they are sorted and subjected to genetic analysis.

[0053] Brief Description of the Drawings The invention is described below with reference to the attached drawings, which show a non-limiting example of implementation thereof, in which:

[0054] Figure 1 shows schematically and in plan view an analysis system in accordance with the present invention; and

[0055] Figures 2-12 show several successive steps of a process in accordance with the present invention.

[0056] Detailed Description

[0057] In accordance with a first aspect of the present invention, in Figure 1, 1 denotes as a whole an analysis system, which comprises an assembly 2 for preparing a treated sample TS (Figure 12) and an evaluation device 3 configured to (adapted to) implement an examination step, during which combined particles CP (described in more detail below) are subjected to genetic analysis and / or (after marking the cells) to analysis by means of colouring agents and / or to analysis by means of biomarkers and / or to analysis by means of cell imaging and / or cell counting and / or cell sorting and / or cell culture and / or proliferation.

[0058] For example, the evaluation device 3 may be the device and / or the system described in patent application W02010 / 106434 (containing a DEP Array) of the same owner or it may be the device and / or the system described in patent US7777885 or it may be based on FACS (florescence-activated cell sorting) technology.

[0059] In accordance with a second aspect of the present invention, a process for preparing the treated sample TS is provided.

[0060] In particular, the process comprises: a first treatment step, during which a first initial blood sample IS (typically, having a volume ranging from about 4 ml to about 7.5 ml) which comprises a first plasma, first erythrocytes, first leukocytes, and a first quantity of target particles, is treated so as to obtain (Figure 2) a first upper layer HL, which mainly contains the first plasma, a first intermediate layer IL, which contains the first quantity of target particles and (mainly) the first leukocytes, and a first lower layer LL, which mainly comprises the first erythrocytes; and a second treatment step, during which a second initial blood sample IS’ (typically, having a volume ranging from about 4 ml to about 7.5 ml), which comprises a second plasma, second erythrocytes, second leukocytes and a second quantity of target particles, is treated so as to obtain (Figure 4) a second upper layer HL’, which mainly contains the second plasma, a second intermediate layer IL’, which contains the second quantity of target particles and (mainly) the second leukocytes, and a second lower layer LL’, which mainly comprises the second erythrocytes.

[0061] The first initial sample IS has (after the first treatment step): the first upper layer HL, the first intermediate layer IL and the first lower layer LL. In addition or alternatively (after the second treatment step), the second initial sample IS’ has: the second upper layer HL’, the second intermediate layer IL’ and the second lower layer LL’.

[0062] The process comprises a first transfer step (Figure 3), during which the first intermediate layer IL is transferred to a collecting station 5 (in particular from a first test tube T); a second transfer step (Figure 5), which is at least partially subsequent to the first transfer step and during which the second intermediate layer IL’ is added to the first intermediate layer IL (in particular, the second intermediate layer IL’ is transferred to the collecting station 5; in particular, from a second test tube T’) so as to obtain an intermediate sample IIS (which, in this case, is obtained by combining the intermediate layers IL and IL’); and a combination step (Figure 10), during which selection particles FF are substantially selectively bound to the target particles present in the intermediate sample IIS so as to obtain combined particles CP.

[0063] The process further comprises a moving step (Figure 11), which is subsequent to the combination step and during which the combined particles CP are moved to at least one determined positioning zone inside a selection container 6; and a removal step during which at least a part of the intermediate sample IIS not including at least part of the combined particles CP is removed from the selection container 6 (and moved to a container C) so that the treated sample TS comprising the aforementioned at least part of the combined particles CP remains inside the selection container 6.

[0064] Advantageously, the removal step is (at least partially) subsequent to the moving step.

[0065] It has been experimentally observed that, by proceeding as indicated above, several advantages are obtained with respect to the state of the art. Among these we note: a higher percentage of patients for whom it is possible to isolate the target particles; a higher percentage of patients for whom it is possible to analyse biomarkers (such as for example Her2(+), ER(+) or PR (+), ER(+) or PR(+) plus Her2(-), Ki-67(+),RAS-gm, RAF-gm, EGFR (+), RO SI -gm, KRAS G12C-gm, BRAF V600E-gm, MET exon 14 skipping-gm, RET fusion-gm; PARP; PSMA, CD38, CD47, CD 138, SLAMF7, GPRC5D, FcRH5 and BCMA); greater precision in counting the target particles; at the same time, no substantial increase in reagents and costs and limited impact on the processing time.

[0066] It has been hypothesized that this is due to the fact that, in this way, increasing the volume of the sample also increases the number of target particles that can be present in the blood even at very low concentrations.

[0067] In this regard, it was observed that: breast cancer patients with more than 10 CTCs in 7.5 ml of blood are less than 39%; bowel cancer patients with more than 10 CTCs in 7.5 ml of blood are less than 12%; and prostate cancer patients with more than 10 CTCs in 7.5 ml of blood are less than 45% (J Oncol. 2010; 2010: 617421).

[0068] A significant reduction in processing times and reagent costs was observed compared to processing the same volume of sample processed in accordance with the state of the art in 7.5 ml or 4 ml aliquots. For example, processing 15 ml of blood according to the traditional methodology requires about twice the time and costs compared to the process of the present invention; processing 22.5 ml of blood according to the traditional methodology requires about three times the time and costs compared to the process of the present invention; processing 30 ml of blood according to the traditional methodology requires about four times the time and costs compared to the process of the present invention.

[0069] Regarding the processing time, it should be noted that it is decidedly higher for processing two blood samples in an absolutely independent manner. Similarly, if it is proceeded in this way, one needs twice as many reagents.

[0070] Moreover, by proceeding as described above, it is possible, from time to time, to handle an easily manageable quantity of sample.

[0071] In this regard, advantageously but not necessarily, the first initial sample IS and the second initial sample IS’ (and, optionally, the third initial sample IS” and the fourth initial sample- described in greater detail below) each have a volume ranging from about 3 mL (in particular, from about 4 mL; more in particular, from about 6 mL) to about 10 mL (in particular, to about 8 mL; more in particular, to about 7.5 mL).

[0072] In particular, at least at the beginning of the removal step the combined particles CP are in the area of the positioning zone. More in particular, during the removal step the combined particles are (are held) in the area of the positioning zone. Note that the first initial sample IS does not necessarily comprise only the first quantity of target particles. It is possible that the first initial sample IS also contains other target particles which, for example, are not contained in the first intermediate layer IL. In some advantageous but non-limiting cases, the first quantity of target cells includes most (advantageously but not necessarily all) of the target particles of the first initial sample IS.

[0073] Similarly, the second initial sample IS’ does not necessarily comprise only the second quantity of target particles. It is possible that the second initial sample IS’ also contains other target particles which, for example, are not contained in the second intermediate layer IL’. In some advantageous but non-limiting cases, the second quantity of target cells includes most (advantageously but not necessarily all) of the target particles of the second initial sample IS’.

[0074] Advantageously but not necessarily, during the removal step the part of the intermediate sample IIS not including at least part of the combined particles CP is removed from the selection container 6 (and moved to a container C) so that at least 10% (in particular at least 20%; more in particular at least 30%; in particular up to 90%), by weight with respect to the total weight of the combined particles CP, of the combined particles CP remains inside the selection container 6. In other words, the aforementioned at least part of the combined particles CP remaining in the selection container 6 after the removal step is at least 10% in particular, at least 20%; more in particular, at least 30%; in particular, up to 90%, by weight with respect to the total weight of the combined particles CP (obtained during the combination step).

[0075] In particular, the process comprises at least a third treatment step, during which a third initial blood sample IS” (typically, having a volume ranging from about 4 ml to about 7.5 ml), which comprises a third plasma, third erythrocytes, third leukocytes and a third quantity of target particles, is treated so as to obtain (Figure 6) a third upper layer HL”, which mainly contains the third plasma, a third intermediate layer IL”, which contains the third quantity of target particles and (mainly) the third leukocytes, and a third lower layer LL”, which mainly comprises the third erythrocytes.

[0076] The third initial sample IS” has (after the third treatment step): the third upper layer HL”, the third intermediate layer IL” and the third lower layer LL”.

[0077] Advantageously but not necessarily, the process comprises at least a third transfer step (Figure 7), which is at least partially subsequent to the second transfer step and during which the third intermediate layer IL” is added to the first intermediate layer IL and to the second intermediate layer IL’ (in particular, the third intermediate layer IL” is transferred to the collecting station 5; in particular, from the selection container 6) so as to obtain the intermediate sample IIS. In other words, in this case, the intermediate sample IIS is the combination of the intermediate layers IL, IL’ and IL”.

[0078] It has been experimentally observed that, in this way, the various advantages described above (a higher percentage of patients for whom it is possible to isolate the target particles; a higher percentage of patients for whom it is possible to analyse biomarkers (such as for example Her2(+), ER(+) or PR (+), ER(+) or PR(+) plus Her2(- ), Ki-67(+),RAS-gm, RAF -gm, EGFR (+), ROS 1 -gm, KRAS G12C-gm, BRAF V600E- gm, MET exon 14 skipping-gm, RET fusion-gm; PARP; PSMA, CD38, CD47, CD138, SLAMF7, GPRC5D, FcRH5 and BCMA); a greater precision in counting the target particles; at the same time, no substantial increase in reagents and costs and limited impact on processing time are obtained in an even more relevant manner.

[0079] According to some embodiments not shown, the process comprises at least a fourth treatment step, during which an initial fourth blood sample (typically, having a volume ranging from about 4 ml to about 7.5 ml), which comprises a fourth plasma, fourth erythrocytes, fourth leukocytes and a fourth quantity of target particles, is treated so as to obtain a fourth upper layer, which mainly contains the fourth plasma, an intermediate fourth layer, which contains the fourth quantity of target particles and (mainly) the fourth leukocytes, and a fourth lower layer, which mainly comprises the fourth erythrocytes.

[0080] The fourth initial sample has (after the fourth treatment step): the fourth upper layer, the fourth intermediate layer and the fourth lower layer.

[0081] Note that the third initial sample IS” (and / or the fourth initial sample) does not necessarily comprise only the third quantity of target particles (and / or the fourth quantity of target particles). It is possible that the third initial sample IS” (and / or the fourth initial sample) also contains other target particles which, for example, are not contained in the third intermediate layer IL’ (and / or in the fourth intermediate layer). In some advantageous but non-limiting cases, the third (and / or the fourth) quantity of target cells includes most (advantageously but not necessarily all) of the target particles of the third initial sample IS” (and / or of the fourth initial sample). Advantageously but not necessarily, the process comprises at least a fourth transfer step, during which the fourth intermediate layer is transferred to the collecting station 5 (in particular from the selection container 6) so as to obtain the intermediate sample IIS. In other words, in this case, the intermediate sample IIS is the combination of the first intermediate layer IL, the second intermediate layer IL’, the third intermediate layer IL” and the fourth intermediate layer.

[0082] According to some non-limiting embodiments, the target particles are chosen from the group consisting of: stem cells (and fragments thereof), erythroblasts, trophoblasts, lymphocytes, neuronal cells (and fragments thereof), epithelial cells (and fragments thereof), tumour cells (and fragments thereof), stromal cells (and fragments thereof), spermatozoa, circulating tumour cells (CTC - and fragments thereof), tumour- derived extracellular vesicles (tdEV), foetal cells, circulating multiple myeloma cells (CMMC), extracellular vesicles in multiple myeloma (mmEV), melanoma cells (CMC) (and fragments thereof), and a combination thereof.

[0083] If possible, when referring to a cell, it is also understood to include a fragment thereof.

[0084] In particular, the target particles are tumour cells (and fragments thereof), in particular chosen from the group consisting of: circulating tumour cells (CTC), tumour- derived extracellular vesicles (tdEV), circulating multiple myeloma cells (CMMC), extracellular vesicles in multiple myeloma (mmEV), melanoma cells (CMC) (and fragments thereof) and a combination thereof.

[0085] According to some non-limiting embodiments, the target particles are chosen from the group consisting of: foetal cells, tumour cells and a combination thereof.

[0086] More precisely but not necessarily, the target particles are chosen from the group consisting of: circulating tumour cells (CTC), tumour-derived extracellular vesicles (tdEV), foetal cells (erythroblasts, trophoblasts and / or lymphocytes), circulating multiple myeloma cells (CMMC), extracellular vesicles in multiple myeloma (mmEV), melanoma cells (CMC) (and fragments thereof) and a combination thereof.

[0087] In some non-limiting cases, the target particles comprise (are) foetal cells (erythroblasts, trophoblasts, and / or lymphocytes).

[0088] According to non-limiting embodiments not shown, the selection container 6 is arranged in the collecting station 5 during the moving step and during the removal step (and, in particular, also during the combination step). In other words, in these cases, the collecting container 8 acts as a selection container 6. In particular, in these cases, the process does not comprise a transfer step for transferring the intermediate sample IIS (this step is described in more detail below).

[0089] In some alternative cases, during the moving step and during the removal step (Figure 11) (and, in particular, also during the combination step), the selection container

[0090] 6 is located in the area of a treatment station 7.

[0091] Advantageously but not necessarily, during the first transfer step, the first intermediate layer IL is transferred from the treatment station 7 to the collecting station 5 (in particular to the collecting container 8, located in the area of the collecting station 5); during the second transfer step, the second intermediate layer IL’ is transferred from the treatment station 7 to the collecting station 5 (in particular to the collecting container 8, located in the area of the collecting station 5). In particular, also during the third transfer step, the third intermediate layer IL” is transferred from the treatment station 7 to the collecting station 5 (in particular to a collecting container 8, located in the area of the collecting station 5). According to some non-limiting embodiments, even during the fourth transfer step, the fourth intermediate layer is transferred from the treatment station

[0092] 7 to the collecting station 5 (in particular to the collecting container 8, located in the area of the collecting station 5).

[0093] In this way it is possible to reduce the presence of unwanted components within the intermediate sample IIS and simultaneously process several initial samples.

[0094] In particular, the intermediate sample IIS is obtained in the collecting container 8 located in the area of the collecting station 5.

[0095] According to some non-limiting embodiments, during the combination step (Figure 10), during the moving step and during the removal step (Figure 11), the intermediate sample IIS lies inside the selection container 6.

[0096] In these cases, the process further comprises a transfer step for transferring the intermediate sample IIS (Figure 9), during which the intermediate sample IIS is moved from the collecting station 5 (in particular, from the collecting container 8, arranged in the collecting station 5) to the selection container 6.

[0097] In particular, it should be noted that, in consideration of the fact that, during the first transfer step, it is the first intermediate layer IL (as such) that is transferred to a collecting station 5, during the first transfer step, the first intermediate layer IL is (at least partially) separated from the first lower layer LL (Figures 3 and 4). Similarly (on the basis of similar considerations), during the second transfer step, the second intermediate layer IL’ is (at least partially) separated from the second lower layer LL’ (Figures 5 and 6). Similarly (on the basis of similar considerations), when the third treatment step is envisaged, during the third transfer step, the third intermediate layer IL” is (at least partially) separated from the third lower layer LL” (Figures 5 and 6). Similarly (on the basis of similar considerations), when the process also comprises the fourth treatment step, during the fourth transfer step, the fourth intermediate layer is (at least partially) separated from the fourth lower layer (Figures 7 and 8).

[0098] Advantageously but not necessarily (in these cases), the intermediate sample IIS consists (mainly; in particular at least 90%, more in particular at least 95%, by weight, with respect to the total weight of the intermediate sample IIS) of a mixture obtained (substantially) exclusively from the combination of the first intermediate layer IL and the second intermediate layer IL’ (and optionally the third intermediate layer IL” and optionally the fourth intermediate layer).

[0099] More precisely but not necessarily, in cases where, during the first transfer step, the first intermediate layer IL is transferred from the treatment station 7 to the collecting station 5, during the first transfer step, the first lower layer LL remains (at least mainly) in the treatment station 7. Similarly (more precisely but not necessarily), in cases where, during the second transfer step, the second intermediate layer IL’ is transferred from the treatment station 7 to the collecting station 5, during the second transfer step, the second lower layer LL’ remains (at least mainly) in the treatment station 7. Similarly (more precisely but not necessarily), when the third treatment step is envisaged, in cases where, during the third transfer step, the third intermediate layer IL” is transferred from the treatment station 7 to the collecting station 5, during the third transfer step, the third lower layer LL” remains (at least mainly) in the treatment station 7. Similarly (more precisely but not necessarily), when the fourth treatment step is envisaged, in cases where, during the fourth transfer step, the fourth intermediate layer is transferred from the treatment station 7 to the collecting station 5, during the fourth transfer step, the fourth lower layer remains (mainly) in the treatment station 7.

[0100] Advantageously but not necessarily, the process comprises an emptying step (Figure 8), which is subsequent (to the first transfer step and) to the second transfer step and during which the second lower layer LL’ is removed from the selection container 6. In particular, in this way the container 6 is substantially emptied.

[0101] In these cases, during the second treatment step, the second initial sample IS’ lies inside the selection container 6 (or, in other words, the selection container 6 also acts as a test tube T’).

[0102] This reduces the number of containers to be used and therefore also mitigates the environmental impact of the process.

[0103] Advantageously but not necessarily, the second treatment step is substantially simultaneous with the first treatment step. This makes the process faster and more efficient.

[0104] In particular, according to some advantageous but non-limiting embodiments, the third treatment step is also substantially simultaneous with the first treatment step (and the second treatment step). Similarly, in some cases, the fourth treatment step is also substantially simultaneous with the first treatment step (and the second treatment step and the third treatment step).

[0105] In particular, during the second transfer step, the second intermediate layer IL’ is transferred from the selection container 6 located in the area of the treatment station 7 to the collecting station 5 (in particular, collecting container 8, located in the area of the collecting station 5).

[0106] Alternatively, advantageously but not necessarily, when the third transfer step (and optionally also the fourth transfer step) is envisaged and the intermediate sample IIS is obtained in the collecting container 8 located in the area of a collecting station 5, the process comprises an emptying step, which is subsequent (to the first transfer step, to the second transfer step and) to the third transfer step (and optionally also to the fourth transfer step) and during which the third lower layer LL” (or the fourth lower layer) is removed from the selection container 6. In particular, in other words, in this case, the selection container 6 is substantially emptied.

[0107] This also reduces the number of containers to be used and therefore also mitigates the environmental impact of the process.

[0108] In particular, during the third treatment step, the third initial sample IS” lies inside the selection container 6. In particular, during the third transfer step, the third intermediate layer IL” is transferred from the selection container 6 located in the area of the treatment station 7 to the collecting container 8 (located in the area of the collecting station 5).

[0109] In particular, during the fourth treatment step, the fourth initial sample lies inside the selection container 6. In particular, during the fourth transfer step, the fourth intermediate layer is transferred from the selection container 6 located in the area of the treatment station 7 to the collecting container 8 (located in the area of the collecting station 5).

[0110] According to some non-limiting embodiments, during the moving step (fig. 11), the combined particles CP are moved to (an inner surface of) at least one outer wall of the selection container 6. In other words, the positioning zone is in the area of the at least one outer wall of the selection container 6.

[0111] In this way, the quantity of combined particles CP that is (mistakenly) removed during the removal step is reduced.

[0112] Note that, in particular, the outer wall externally delimits (an inner chamber of) the selection container 6.

[0113] More precisely, but not necessarily, during the moving step, the combined particles CP are (at least partially) brought in contact with the outer wall of the selection container 6. In other words, the positioning zone is (at least partially) in contact with the outer wall of the selection container 6.

[0114] Advantageously but not necessarily, during the removal step, the at least a part of the intermediate sample IIS not including the aforementioned at least part of the combined particles CP is removed from the selection container 6 by means of at least one suction mouth arranged inside the selection container 6 in a suction zone different from the at least one positioning zone (i.e. where the combined particles CP were arranged following the moving step).

[0115] More precisely but not necessarily, the suction zone is spaced apart from the outer wall of the selection container 6. Even more in particular, the suction zone is arranged approximately in the centre of the selection container 6 with respect to the lateral perimeter of the selection container 6 itself.

[0116] According to some non-limiting embodiments, the process also comprises a first retrieving step (Figure 2), which is subsequent to the first treatment step and prior to the first transfer step and during which at least part of (in particular, at least most of; more in particular, substantially all of) the first upper layer HL is retrieved from the first initial sample IS; and a second retrieving step (Figure 4), which is subsequent to the second treatment step and prior to the second transfer step and during which at least part of (in particular, at least most of; more in particular, substantially all of) the second upper layer is retrieved from the second initial sample IS’.

[0117] In particular, the process also comprises at least a third retrieving step (Figure 6), which is subsequent to the third treatment step and prior to the third transfer step (Figure 7) and during which at least part of (in particular, at least most of; more in particular, substantially all of) the third upper layer HL” is retrieved from the third initial sample IS”.

[0118] According to some non-limiting embodiments not shown, the process also comprises at least a fourth retrieving step, which is subsequent to the fourth treatment step and prior to the fourth transfer step and during which at least part of (in particular, at least most of; more in particular, substantially all of) the fourth upper layer is retrieved from the initial fourth sample.

[0119] In this way, the subsequent first transfer step, second transfer step and third transfer step are facilitated and made more precise.

[0120] In accordance with a third aspect of the present invention, there is provided an analysis method, which comprises the process for preparing a treated sample TS as described above (in accordance with the second aspect of the present invention) and an examination step, which is subsequent to the removal step and during which the combined particles CP (remained in the selection container 6, after the removal step) are subjected to genetic analysis and / or (after marking of the cells) to analysis by means of colouring agents and / or to analysis by means of biomarkers and / or to analysis by means of cell imaging and / or cell counting and / or cell sorting and / or cell culture and / or proliferation.

[0121] According to some non-limiting embodiments, the analysis method comprises a step of marking (for example, by means of antibodies with fluorophores) the aforementioned at least part of the combined particles CP (and optionally also the other cells with different exclusion markers to improve specificity) prior to the examination step (in particular, prior to analysis by means of colouring agents and / or to analysis by means of biomarkers and / or to analysis by means of cell imaging and / or cell counting and / or cell sorting). More precisely but not necessarily, during the marking step, the aforementioned at least part of the combined particles CP (remained in the selection container 6, after the removal step) is placed in contact with fluorophore antibodies so that the fluorophore antibodies bind to the target particles, in particular to (the aforementioned part of) the combined particles CP (and colour them).

[0122] In accordance with a fourth aspect of the present invention, there is provided a (evaluation) method for evaluating a state and / or development of a state of an individual. The (evaluation) method comprises: a process for preparing a treated sample TS, which process is as described above in accordance with the second aspect of the present invention and wherein the first initial sample IS and the second initial sample IS’ each have a volume ranging from about 4 ml to about 7.5 ml; and an analysis step, which is subsequent to the removal step (in particular, subsequent to the preparation process) and during which one or more of the following sub-steps is implemented: marking sub-step, during which (for example, by means of antibodies with fluorophores) target particles (in particular, of said at least part of the combined particles CP) of the treated sample are marked; counting sub-step, during which the target particles (in particular, of said at least part of the combined particles CP) of the treated sample TS are counted and / or the aggregates of the target particles (in particular, of said at least part of the combined particles CP) of the treated sample TS are counted; a sub-step of analysis with biomarkers, during which the expression of at least one biomarker (for example that is a target for disease therapies, such as for example HER2, PSMA, MUC1, EGFR, ER, PR, KI67, PDL1, RAS, RAF, ROS1, KRAS, BRAF, MET, RET, PARP) on the target particles (in particular, of said at least part of the combined particles CP) of the treated sample TS is evaluated; a sorting sub-step, during which the target particles (in particular, of said at least part of the combined particles CP) of the treated sample TS are selected (for example by means of DEP Array and / or FACS); a molecular analysis sub-step, during which the target particles (in particular, of said at least part of the combined particles CP) of the treated sample TS are analysed by means of molecular analyses (for example: analysis of the genetic profile and identification of DNA mutations, analysis of gene expression, detection of chromosomal aberrations and gene amplifications, analysis of microsatellite instability, analysis of the loss of heterozygosity, methylation analysis, analysis of the variation of the number of copies, proteomic analysis, sequencing of the whole genome or of the exome, etc.).

[0123] According to some non-limiting embodiments, the examination step is as described above in relation to the process referred to in the second aspect of the present invention.

[0124] Advantageously but not necessarily, the (evaluation) method comprises a correlation step, which is subsequent to the examination step and during which the results obtained from the examination step (for example the number of cells of interest and / or a number of cell fragments of interest and / or number of cell aggregates of interest and / or measurement of at least one biomarker or a combination thereof and / or result of the molecular analysis) is correlated with the presence or progression of a disease or with the response to a treatment in the individual, and / or is used to aim a therapy.

[0125] According to some non-limiting forms of implementation, a report is prepared based on the result of these operations (examination step and / or correlation step). In particular, the report provides information on counts and / or analysis of biomarkers and / or sorting and / or molecular analyses.

[0126] Advantageously but not necessarily: the target particles are circulating tumour cells (CTC) and / or tumour-derived extracellular vesicles (tdEV) and the disease is a carcinoma (in particular, chosen from breast cancer, prostate cancer, bowel cancer); alternatively or in addition, the target particles are circulating endothelial cells (CEC) and / or endothelium-derived extracellular vesicles (edEV) and the disease is a cardiovascular disease; alternatively or in addition the target particles are circulating multiple myeloma cells (CMMC) and / or extracellular vesicles in multiple myeloma (mmEV) and the disease is a myeloma; alternatively or in addition the target particles are melanoma cells (CMC) and / or extracellular vesicles of melanoma and the disease is a melanoma; alternatively or in addition the target particles are foetal cells (erythroblasts and / or trophoblasts and / or lymphocytes) and the disease affects the foetus (for example genetic anomalies, chromosomal anomalies and structural anomalies). In particular, in the latter cases, and the report includes the probability that the foetus has genetic anomalies.

[0127] In particular, the (evaluation) method is a method (in vitro) for diagnosing and / or evaluating the progression of a disease in the individual and / or for evaluating the response to a treatment in the individual. With particular reference to Figure 1, in accordance with a fifth aspect of the present invention, there is provided the assembly 2 for preparing the treated sample TS and for implementing the process described above with reference to the second aspect of the present invention.

[0128] The assembly 2 (which can also be used separately from the other components of the analysis system 1) comprises: at least one centrifuge 4 configured to (adapted to) implement the first treatment step and the second treatment step (and optionally the third treatment step; and optionally the fourth treatment step); at least one first housing 9 for the collecting container 8 located in the area of the collecting station 5; and at least one second housing 10 (for the test tube T and optionally for the test tube T’ and / or for the selection container 6) located in the area of a treatment station 7.

[0129] Advantageously but not necessarily, the assembly 2 comprises the collecting container 8 and optionally the selection container 6 (and / or the test tube T and optionally the test tube T’).

[0130] The assembly 2 further comprises: at least one transfer device 11, which in turn comprises a movable head (known per se and not shown), provided with a suction mouth, an operating system configured to move the head with at least two degrees of freedom (in particular, at least three degrees of freedom) and a suction unit for sucking through said suction mouth; and at least one control unit 12 configured to (adapted to) control the transfer device 11 so as to move the first intermediate layer IL and the second intermediate layer IL’ (and, optionally, the third intermediate layer IL”; and, optionally, the fourth intermediate layer) to the collecting container 8 and so as to remove at least a part of the intermediate sample IIS not including at least part of the combined particles CP from the selection container 6.

[0131] Advantageously but not necessarily the transfer device 11 comprises (is) a robot (in particular, Cartesian one - with at least two axes, in particular with at least three axes). According to alternatives embodiments not shown, the transfer device is an anthropomorphic robot.

[0132] The assembly 2 also comprises at least one attraction device 13 (Figures 2-11) which (is located in the area of the treatment station 7) and is configured to (adapted to) move the combined particles CP to at least one outer wall of the selection container 6.

[0133] Advantageously but not necessarily, the control unit 12 is configured to activate the attraction device 13 when the intermediate sample IIS lies inside the selection container 6 (in the area of the treatment station 7).

[0134] In particular, the attraction device 13 is provided with at least one magnet, for example electromagnet or with a magnet whose position can be controlled, in particular by approaching it (the selection container 6) during the moving step.

[0135] More precisely but not necessarily, the attraction device 13 comprises a movement unit for moving the at least one magnet between a first position far from the outer wall of the selection container 6 and a second position close to (in particular, in contact with) said outer wall (in particular, in other words, the first position is farther from the outer wall than the second position). In particular, in the second position the at least one magnet is able to exert its force on the inside of the selection container 6 (thus moving the combined particles CP to the positioning zone near - in particular in contact with) the inner surface of said outer wall.

[0136] Advantageously but not necessarily, when the attraction device 13 comprises the movement unit, during the removal step and during the moving step, the magnet is in the second position.

[0137] Alternatively, when the magnet is an electromagnet, during the removal step and during the moving step, the electromagnet is activated.

[0138] Advantageously but not necessarily, the assembly 2 comprises an image detector 23 (in particular, a camera) for acquiring one or more images of the container 6, and of the test tubes T and T’ (and therefore of the content thereof). In these cases, the control unit 12 is connected to the image detector 23 and is configured to control the transfer device 11 (and optionally the attraction device 13) as a function of the detection of the image detector 23. For example, the control unit 12 is configured to move the suction mouth to the correct heights inside the test tubes T and T’ (and optionally the third and fourth test tubes) to retrieve the upper layers HL and / or HL’ and / or HL” and / or the fourth upper layer without sucking the intermediate layers IL and / or IL’ and / or IL” and / or the fourth intermediate layer as a function of the detection by the image detector 23.

[0139] According to some non-limiting embodiments, the image detector 23 is configured to (also) acquire one or more images of the containers 8, 15, 17 and C.

[0140] Advantageously but not necessarily, the image detector 23 is movable in front of the container 6 (optionally of the containers 8, 15, 17 and C) and test tubes T and T’ so as to be able to frame them (individually) according to different needs.

[0141] According to some non-limiting embodiments, the assembly 2 also comprises a third housing 14 for a plasma collecting container 15. The control unit 12 is configured to (adapted to) control the transfer device 11 so as to move the first upper layer HL and the second upper layer HL’ (and, optionally, the third upper layer HL” - in particular, also the fourth upper layer) to the plasma collecting container 15.

[0142] According to some non-limiting embodiments, the assembly 2 comprises a fourth housing 16 for an erythrocyte collecting container 17. The control unit 12 is configured to (adapted to) control the transfer device 11 so as to move the first lower layer LL and the second lower layer LL’ (and, optionally, the third lower layer LL” - in particular, also the fourth lower layer) to the erythrocyte collecting container 17.

[0143] Advantageously but not necessarily, the transfer device 11 comprises a tip, defining the aforementioned suction mouth, mounted in a replaceable manner. In these cases, the assembly 2 comprises a store 18 for further tips. The control unit 12 is configured to (adapted to) control the transfer device 11 so as to replace the tip with a further tip (automatically).

[0144] This reduces the risk of contaminating the different samples.

[0145] In accordance with alternative embodiments not shown, the mentioned tip is not replaceable and the assembly 2 comprises a washing device for washing the tip whenever necessary to avoid contaminations between the different samples.

[0146] According to some non-limiting embodiments, the assembly 2 also comprises at least one tank 19 containing the selection particles FF. The control unit 12 is configured to (adapted to) control the transfer device 11 so as to move the selection particles FF from said tank 19 to the selection container 6 located in the area of the treatment station 7.

[0147] In some non-limiting cases, the assembly 2 also comprises other tanks for further reagents that can be retrieved in an automated manner from the transfer device 11.

[0148] According to some non-limiting embodiments, the assembly 2 further comprises a waste area 20 where the transfer device 11 stores the used-up tips and liquid waste.

[0149] According to some advantageous non-limiting embodiments, the assembly 2 also comprises a fifth housing 21 for the container C (which is configured to contain part of the intermediate sample IIS substantially not including at least part of the combined particles CP - in particular, which has been removed from the selection container 6). The control unit 12 is configured to (adapted to) control the transfer device 11 so as to move part of the intermediate sample IIS (substantially not including the aforementioned at least part of the combined particles CP) to the container C (from the selection container 6).

[0150] In particular, the control unit 12 is configured to control the transfer device 11 so as to move the first intermediate layer IL to the collecting container 8 at least partially separating it from the first lower layer LL; the control unit 12 is configured to control the transfer device 11 so as to move the second intermediate layer IL’ to the collecting container 8 at least partially separating it from the second lower layer LL’. More in particular, the control unit 12 is configured to control the transfer device 11 so as to move the third intermediate layer IL” to the collecting container 8 at least partially separating it from the third lower layer LL”. More in particular, the control unit 12 is configured to control the transfer device 11 so as to move the fourth intermediate layer to the collecting container 8 at least partially separating it from the fourth lower layer.

[0151] More in particular, the control unit 12 is configured to control the transfer device 11 so as to move the first intermediate layer IL from the treatment station 7 to the collecting container 8 leaving at least a main part of the first lower layer LL in the treatment station 7. More in particular, the control unit 12 is configured to control the transfer device 11 so as to move the second intermediate layer IL’ from the treatment station 7 to the collecting container 8 leaving at least a main part of the second lower layer LL’ in the treatment station 7. More in particular, the control unit 12 is configured to control the transfer device 11 so as to move the third intermediate layer IL” from the treatment station 7 to the collecting container 8 leaving at least a main part of the third lower layer LL” in the treatment station 7. More in particular, the control unit 12 is configured to control the transfer device 11 so as to move the fourth intermediate layer from the treatment station 7 to the collecting container 8 leaving at least a main part of the fourth lower layer in the treatment station 7.

[0152] Advantageously but not necessarily, the process referred to in the second aspect of the present invention is implemented by the assembly 2 described above.

[0153] The analysis system 1 is configured to implement an analysis method as described above (in accordance with the third aspect of the present invention). The system 1 comprises the assembly 2 and the evaluation device 3 configured to implement the examination step, during which the aforementioned at least part of the combined particles CP is subjected to genetic analysis and / or to analysis by means of colouring agents and / or to analysis by means of biomarkers and / or to analysis by means of cell imaging and / or cell counting and / or cell sorting and / or cell culture and / or proliferation.

[0154] In accordance with a sixth aspect of the present invention, a machine 22 for preparing the treated sample TS is also provided. The machine 22 comprises: at least the first housing 9 for the collecting container 8 located in the area of the collecting station 5; at least the second housing 10 (for the test tube T and optionally for the test tube T’ and / or for the selection container 6) located in the area of the treatment station 7; and at least the transfer device 11, which comprises the movable head, provided with the suction mouth, the operating system configured to move the head with at least two degrees of freedom (in particular, at least three degrees of freedom) and the suction unit for sucking through said suction mouth.

[0155] The machine 22 further comprises at least the control unit 12 configured to (adapted to) control the transfer device 11 so as to move the first intermediate layer IL of the first initial sample IS and the second intermediate layer IL’ of the second initial sample IS’ (and, optionally, the third intermediate layer IL” of the third initial sample - and, optionally, the fourth intermediate layer IL” of a fourth initial sample) to the collecting container 8 and so as to remove at least a part of the intermediate sample IIS not including the at least part of the combined particles CP from the selection container 6; and at least the attraction device 13 which is (located in the area of the treatment station 7 y) configured to (adapted to) move the combined particles CP to the area of at least one outer wall of a selection container 6 configured to contain the intermediate sample IIS.

[0156] In particular, the control unit 12 is as described above in relation to the assembly 2.

[0157] In particular, the assembly 2 comprises the machine 22.

[0158] Further characteristics of the present invention will become apparent from the following description of a merely illustrative and non-limiting example.

[0159] Example

[0160] This example describes tests performed using blood samples retrieved from some donors. In particular, four blood aliquots of 7.5 ml were retrieved from each donor, to each of which 200 PC3-9 cells (cell line characteristic of prostate cancer) were added.

[0161] A first blood aliquot for each donor was treated by means of the traditional methodology. The other aliquots were treated together according to the process in accordance with the present invention.

[0162] With the process in accordance with the present invention, approximately three times the number of PC3-9 cells isolated with the traditional methodology were isolated, however, using (in both cases) substantially the same quantity of reagents. The process in accordance with the present invention has, moreover, taken a time of only 20% longer than the time taken by the traditional methodology.

[0163] Furthermore, by comparing the processing time of three aliquots (each equal to 7.5 ml) according to the method described with respect to the traditional way, there is an increase in the throughput equal to 3 / 1.2 = 2.5x.

Claims

C L A I M S1.- A process for preparing a treated sample (TS); the process comprises: a first treatment step, during which a first initial blood sample (IS), which comprises a first plasma, first erythrocytes, first leukocytes, and a first quantity of target particles, is treated so as to obtain a first upper layer (HL), which mainly contains the first plasma, a first intermediate layer (IL), which contains the first quantity of target particles and the first leukocytes, and a first lower layer (LL), which mainly comprises the first erythrocytes; and a second treatment step, during which a second initial blood sample (IS’), which comprises a second plasma, second erythrocytes, second leukocytes and a second quantity of target particles, is treated so as to obtain a second upper layer (HL’), which mainly contains the second plasma, a second intermediate layer (IL’), which contains the second quantity of target particles and the second leukocytes, and a second lower layer (LL’), which mainly comprises the second erythrocytes; the process being characterized by further comprising: a first transfer step, during which the first intermediate layer (IL) is transferred to a collecting station (5); a second transfer step, which is subsequent to the first transfer step and during which the second intermediate layer (IL’) is added to the first intermediate layer (IL) so as to obtain an intermediate sample (IIS); a combination step, during which selection particles (FF) are substantially selectively bound to the target particles present in the intermediate sample (IIS) so as to obtain combined particles (CP); a moving step, which is subsequent to the combination step and during which the combined particles (CP) are moved to at least one positioning zone within a selection container (6); and a removal step, which is at least partially subsequent to the moving step and during which at least a part of the intermediate sample not including at least part of the combined particles (CP) is removed from the selection container (6) so that the treated sample (TS) comprising said at least part of the combined particles (CP) remains inside the selection container (6).2.- The process according to claim 1, wherein, during the second transfer step, thesecond intermediate layer (IL’) is transferred to said collecting station (5); in particular, during the first transfer step, the first intermediate layer (IL) is transferred to a collecting container (8) (in particular, from a treatment station (7)); in particular, during the second transfer step, the second intermediate layer (IL’) is transferred to the collecting container (8) (in particular, from a treatment station (7)).3.- The process according to claim 1 or 2, wherein, during the moving step, the combined particles (CP) are moved to an area of at least one outer wall of the selection container (6); during the removal step, said at least one part of the intermediate sample (IIS) not including said at least part of the combined particles (CP) is removed from the selection container (6) by means of at least one suction mouth placed inside the selection container (6) in a suction zone different from the at least one positioning zone; in particular, the suction zone is spaced apart from said at least one outer wall.4.- The process according to any one of the preceding claims, wherein, during the removal step, the combined particles (CP) are held at the positioning zone; in particular, during the moving step, the combined particles (CP) are at least partially brought in contact with the outer wall of the selection container (6).5.- The process according to any one of the preceding claims, and comprising: a first retrieving step, which is subsequent to the first treatment step and prior to the first transfer step and during which at least part (in particular, at least most) of the first upper layer (HL) is retrieved from the first initial sample (IS); and a second retrieving step, which is subsequent to the second treatment step and prior to the second transfer step and during which at least part (in particular, at least most) of the second upper layer (HL’) is retrieved from the second initial sample (IS’).6.- The process according to any one of the preceding claims, wherein the selection particles (FF) comprise ferrofluid particles combined with at least one molecule capable of substantially selectively binding to the target particles.7.- The process according to claim 6, wherein said molecule is an antibody or an antibody portion (for example, it comprises anti-EPCAM and / or anti-CD146 and / or anti- CD105 and / or anti-CD90 and / or anti-CD141 and / or anti-EpCAM and / or anti-HLA-G and / or anti-TROP-2).8.- The process according to any one of the preceding claims, wherein the targetparticles are chosen from the group consisting of: stem cells and fragments thereof, erythroblasts, trophoblasts, lymphocytes, neuronal cells and fragments thereof, epithelial cells and fragments thereof, tumour cells and fragments thereof, stromal cells and fragments thereof, spermatozoa, circulating tumour cells (CTC), tumour-derived extracellular vesicles (tdEV), foetal cells, circulating multiple myeloma cells (CMMC), extracellular vesicles in multiple myeloma (mmEV), melanoma cells (CMC) (in particular, and fragments thereof) and a combination thereof.9.- The process according to any one of the preceding claims, wherein the intermediate sample (IIS) is obtained in a collecting container (8) located in the area of the collecting station (5); during the combination step, during the moving step and during the removal step, the intermediate sample (IIS) lies inside the selection container (6); the process further comprising a transfer step for transferring the intermediate sample (IIS), during which the intermediate sample (IIS) is moved from the collecting station (5) (in particular, from the collecting container (8)), to the selection container (6).10.- The process according to claim 9, wherein, during the moving step and during the removal step, the selection container (6) is located in the area of a treatment station (7); during the first transfer step, the first intermediate layer (IL) is transferred from the treatment station (7) to the collecting container (8); during the second transfer step, the second intermediate layer (IL’) is transferred from the treatment station (7) to the collecting container (8).11.- The process according to claim 10, wherein the intermediate sample (IIS) is obtained in the collecting container (8), located in the area of the collecting station (5); the process comprising an emptying step, which is subsequent to the second transfer step and during which the second lower layer (LL’) is removed from the selection container (6); during the second treatment step, the second initial sample (IS’) lies inside the selection container (6); during the second transfer step, the second intermediate layer is transferred from the selection container (6) located in the area of the treatment station (7) to the collecting container (8), located in the area of the collecting station (5).12.- The process according to any one of the preceding claims, and comprising: a third treatment step, during which an initial third blood sample (IS”), which comprises a third plasma, third erythrocytes, third leukocytes and a third quantity of target particles, is treated so that a third upper layer (HL”), which mainly contains the third plasma, a third intermediate layer (IL”), which contains the third quantity of target particles and mainly the third leukocytes, and a third lower layer (LL”), which mainly comprises the third erythrocytes, are created; and a third transfer step, which is subsequent to the second transfer step and during which the third intermediate layer (IL”) is added to the first intermediate layer (IL) and to the second intermediate layer (IL’) so as to obtain the intermediate sample (IIS); in particular, during the third transfer step, the intermediate layer (IL”) is transferred to said collecting station (5); in particular, during the first transfer step, the first intermediate layer (IL) is transferred to a collecting container (8) (in particular, from a treatment station (7)); in particular, during the second transfer step, the second intermediate layer (IL’) is transferred to the collecting container (8) (in particular, from a treatment station (7)); in particular, during the third transfer step, the third intermediate layer (IL”) is transferred to the collecting container (8) (in particular, from a treatment station (7)).13.- The process according to claim 12, and also comprising a third retrieving step, which is subsequent to the third treatment step and prior to the third transfer step and during which the third upper layer (HL”) is retrieved from the third initial sample.14.- The process according to claim 12 or 13, wherein the intermediate sample (IIS) is obtained in the collecting container (8) located in the area of a collecting station(5); the process comprising an emptying step, which is subsequent to the third transfer step and during which the third lower layer (LL”) is removed from the selection container(6); during the third treatment step, the third initial sample (IS”) lies inside the selection container (6); during the third transfer step, the third intermediate layer (IL”) is transferred from the selection container (6) located in the area of the treatment station (7) to the collecting container (8) located in the area of the collecting station (5).15.- The process according to any one of the preceding claims, wherein, duringthe first transfer step, the first intermediate layer (IL) is at least partially separated from the first lower layer (LL); during the second transfer step, the second intermediate layer (IL’) is at least partially separated from the second lower layer (LL’); in particular, during the third transfer step, the third intermediate layer (IL”) is at least partially separated from the third lower layer (LL”).16.- The process according to any one of the preceding claims, wherein during the first transfer step, the first intermediate layer (IL) is transferred from a treatment station (7) to the collecting station (5); during the first transfer step, the first lower layer (LL) remains at least mainly in the treatment station (7); during the second transfer step, the second intermediate layer (IL’) is transferred from the treatment station (7) to the collecting station (5), during the second transfer step, the second lower layer (LL’) remains at least mainly in the treatment station (7); during the third transfer step, the third intermediate layer (IL”) is transferred from the treatment station (7) to the collecting station (5), during the third transfer step, the third lower layer (LL”) remains (at least mainly) in the treatment station (7).17.- An analysis method, comprising the process according to any one of the preceding claims and an examination step, which is subsequent to the removal step and during which said at least part of the combined particles (CP) is subjected to genetic analysis and / or to analysis by means of colouring agents and / or to analysis by means of biomarkers and / or to analysis by means of cell imaging and / or cell counting and / or cell sorting and / or cell culture and / or proliferation.18.- An assembly for preparing a treated sample (TS); the assembly (2) is configured to implement the process according to one of claims from 1 to 16, and comprises: at least one centrifuge (4) configured to implement the first treatment step and the second treatment step; at least one first housing (9) for a collecting container (8) located in the area of a collecting station (5); at least one second housing (10) (in particular, for the selection container (6)) located in the area of a treatment station (7); at least one transfer device (11), which, in turn, comprises a movable head, provided with a suction mouth, an operating system configured to move the head with atleast two degrees of freedom and a suction unit for sucking through said suction mouth; at least one attraction device (13) which is configured to move the combined particles (CP) to the area of at least one outer wall of a selection container (6); and at least one control unit (12) configured to control the transfer device (11) so as to move the first intermediate layer (IL) and the second intermediate layer (IL’) (and, optionally, the third intermediate layer (IL”)) to the collecting container (8) and so as to remove at least a part of the intermediate sample (IIS) not including at least part of the combined particles (CP) from the selection container (6); in particular, the attraction device (13) is located at the treatment station (7)19.- The assembly according to claim 18, wherein the control unit (12) is configured to activate the attraction device (13) when the intermediate sample (IIS) lies inside the selection container (6) (in particular, in the area of the collecting station (7)); the attraction device (13) being provided with at least one magnet.20.- The assembly according to claim 18 or 19, and comprising a third housing (14) for a plasma collecting container (15); the control unit (12) being configured to control the transfer device (11) so as to move the first upper layer (HL) and the second upper layer (HL’) (and, optionally, the third upper layer (HL”)) to the plasma collecting container (15).21.- The assembly according to any one of claims from 18 to 20, and comprising a fourth housing (16) for an erythrocyte collecting container (17); the control unit (12) being configured to control the transfer device (11) so as to move the first lower layer (LL) and the second lower layer (LL’) (and, optionally, the third lower layer (LL”)) to the erythrocyte collecting container (17).22.- The assembly according to any one of claims from 18 to 21, wherein the transfer device (11) comprises at least one tip, defining said suction mouth, mounted in a replaceable manner; the assembly (2) comprising a store (18) for further tips; the control unit (12) being configured to control the transfer device (11) so as to replace the tip with a further tip.23.- The assembly according to any one of claims from 18 to 22, and comprising at least one tank (19) containing said selection particles (FF); the control unit (12) being configured to control the transfer device (11) so as to move the selection particles (FF) from said tank (19) to said selection container (6)located at the treatment station (7).24.- The assembly according to any one of claims from 18 to 23, and comprising an image detector (23); the control unit (12) is configured to control the transfer device (11) as a function of the detection of the image detector (23); in particular, the control unit (12) is configured to control the attraction device (13) as a function of what is detected by the image detector (23).25.- The assembly according to any one of claims from 18 to 24, wherein the control unit (12) is configured to control the transfer device (11) so as to move the first intermediate layer (IL) to the collecting container (8) at least partially separating it from the first lower layer (LL); the control unit (12) is configured to control the transfer device (11) so as to move the second intermediate layer (IL’) to the collecting container (8) at least partially separating it from the second lower layer (LL’); in particular, the control unit (12) is configured to control the transfer device (11) so as to move the third intermediate layer (IL”) to the collecting container (8) at least partially separating it from the third lower layer (LL”); in particular, the control unit (12) is configured to control the transfer device (11) so as to move the first intermediate layer (IL) from the treatment station (7) to the collecting container (8) leaving at least a main part of the first lower layer (LL) in the treatment station (7); in particular, the control unit (12) is configured to control the transfer device (11) so as to move the second intermediate layer (IL’) from the treatment station (7) to the collecting container (8) leaving at least a main part of the second lower layer (LL’) in the treatment station (7); in particular, the control unit (12) is configured to control the transfer device (11) so as to move the third intermediate layer (IL”) from the treatment station (7) to the collecting container (8) leaving at least a main part of the third lower layer (LL”) in the treatment station (7).26.- An analysis system for implementing the analysis method according to claim 17, the analysis system (1) comprises the assembly (2) for preparing a treated sample (TS) according to any one of claims from 18 to 25 and an evaluation device (3) configured to implement an examination step, during which said at least part of the combined particles (CP) is subjected to genetic analysis and / or to analysis by means of colouring agents and / or to analysis by means of biomarkers and / or to analysis by means of cell imaging and / or cell counting and / or cell sorting.27.- The analysis system according to claim 26, wherein the evaluation device (3)is configured to mark (in particular, to colour) said at least part of the combined particles (CP) before subjecting it to analysis by means of colouring agents and / or to analysis by means of biomarkers and / or to analysis by means of cell imaging and / or cell counting and / or cell sorting.28.- A machine for preparing a treated sample (TS); the machine comprises: at least one first housing (9) for a collecting container (8) located in the area of a collecting station (5); and at least one second housing (10) for a selection container (6) located in the area of a treatment station (7); the machine being characterized in that it also comprises: at least one transfer device (11), which comprises a movable head, provided with a suction mouth, an operating system configured to move the head with at least two degrees of freedom and a suction unit for sucking through said suction mouth; at least one control unit (12) configured to control the transfer device (11) so as to move a first intermediate layer (IL) of a first initial sample (IS) and a second intermediate layer (IL’) of a second initial sample (IS’) (and, optionally, a third intermediate layer (IL”) of a third initial sample (IS”); and optionally a fourth intermediate layer of a fourth initial sample) to the collecting container (8) and so as to remove at least a part of an intermediate sample (IIS) not including at least part of the combined particles (CP) from the selection container (6); and at least one attraction device (13) which is located in the area of the treatment station (7) and is configured to move the combined particles (CP) to the area of at least one outer wall of a selection container (6) configured to contain the intermediate sample (IIS).29.- The machine according to claim 28, wherein the control unit (12) is configured to control the transfer device (11) so as to move the first intermediate layer (IL) to the collecting container (8) at least partially separating it from the first lower layer (LL); the control unit (12) is configured to control the transfer device (11) so as to move the second intermediate layer (IL’) to the collecting container (8) at least partially separating it from the second lower layer (LL’); in particular, the control unit (12) is configured to control the transfer device (11) so as to move the third intermediate layer (IL”) to the collecting container (8) at least partially separating it from the third lowerlayer (LL”); in particular, the control unit (12) is configured to control the transfer device (11) so as to move the first intermediate layer (IL) from the treatment station (7) to the collecting container (8) leaving at least a main part of the first lower layer (LL) in the treatment station (7); in particular, the control unit (12) is configured to control the transfer device (11) so as to move the second intermediate layer (IL’) from the treatment station (7) to the collecting container (8) leaving at least a main part of the second lower layer (LL’) in the treatment station (7); in particular, the control unit (12) is configured to control the transfer device (11) so as to move the third intermediate layer (IL”) from the treatment station (7) to the collecting container (8) leaving at least a main part of the third lower layer (LL”) in the treatment station (7).30.- A method for evaluating a state and / or a development of a state of an individual; the method comprises: a process for preparing a treated sample (TS) according to any one of claims from 1 to 18, wherein the first initial sample (IS) and the second initial sample (IS’) each have a volume ranging from about 4 ml to about 7.5 ml; an examination step, which is subsequent to the preparation process and during which one or more of the following sub-steps is implemented: marking sub-step, during which for example, by means of antibodies with fluorophores) target particles (in particular of said at least part of the combined particles CP) of the treated sample are marked; counting sub-step, during which target particles (in particular of said at least part of the combined particles CP) of the treated sample are counted and / or the aggregates of the target particles (in particular, of said at least part of the combined particles CP) of the treated sample are counted; a sub-step of analysis with biomarkers, during which the expression of at least one biomarker (for example that is a target for disease therapies, such as for example HER2, PSMA, MUC1, EGFR, ER, PR, KI67, PDL1, RAS, RAF, ROS1, KRAS, BRAF, MET, RET, PARP) on the target particles (in particular, of said at least part of the combined particles CP) of the treated sample is evaluated; a sorting substep, during which the target particles (in particular, of said at least part of the combined particles CP) of the treated sample are selected (for example by means of DEP Array and / or FACS); a molecular analysis sub-step, during which the target particles (in particular, of said at least part of the combined particles CP) of the treated sample are analysed by means of molecular analyses (for example: analysis of the genetic profile andidentification of DNA mutations, analysis of gene expression, detection of chromosomal aberrations and gene amplifications, analysis of microsatellite instability, analysis of the loss of heterozygosity, methylation analysis, analysis of the variation of the number of copies, proteomic analysis, sequencing of the whole genome or of the exome). 31.- The method according to claim 30, and comprising a correlation step, which is subsequent to the examination step and during which the results obtained from the examination step is correlated with the presence or progression of a disease or with the response to a treatment in the individual, and / or is used to aim a therapy.32.- The method according to claim 30 or 31, wherein: the target particles are circulating tumour cells (CTC) and / or tumour-derived extracellular vesicles (tdEV) and the disease is a carcinoma (in particular, chosen from breast cancer, prostate cancer, bowel cancer); or the target particles are circulating endothelial cells (CEC) and / or endothelium-derived extracellular vesicles (edEV) and the disease is a cardiovascular disease; or the target particles are circulating multiple myeloma cells (CMMC) and / or multiple myeloma extracellular vesicles (mmEV) and the disease is a myeloma; or the target particles are melanoma cells (CMC) and / or extracellular vesicles of melanoma and the disease is a melanoma; or the target particles are foetal cells (in particular, erythroblasts and / or trophoblasts and / or lymphocytes) and the disease affects the foetus (for example such as trisomy, sex chromosome anomaly and structural anomaly).

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