Apparatus and method for preparing biological samples for analytical or diagnostic purposes - Patents.com

Abstract

The present invention relates to an apparatus for the automated processing of body fluid samples, preferably for the analysis of liquid biopsies, configured to obtain one or more informative components of the fluid sample, separate and distinct from one another, a first component comprising cellular elements which are consequently immobilized on a planar support, a second component comprising suspension sample cells and a third component comprising the liquid part of the sample, preferably contained in a test tube. The present invention further relates to a method for the preparation of the components implemented by the apparatus.

Description

[Technical field]

[0001] The present invention relates to the field of devices and methods for preparing biological samples for subsequent analytical or diagnostic purposes.

[0002] In particular, the present invention relates to a device that allows for the automated and standardized processing of body fluid samples for analysis or diagnosis, preferably based on liquid biopsy, configured to provide an informative and stable component of the sample close to the point for collecting the starting sample, particularly suitable for the analysis of biological contents correlated with a number of conditions, among them tumors, or degenerative or infectious lesions, or lesions detectable in the prenatal setting. [Background technology]

[0003] It is known that in the clinical context, the preanalytical stage represents a critical moment within the activity of analyzing biological samples, since it comprises different procedural paths in which the appearance of different variables can affect the quality of the samples themselves, the outcome of the subsequent analysis and their usefulness. Sample suitability and acceptability are fundamental elements of the preanalytical stage, on the basis of which the variability of the results of laboratory experiments should be parameterized.

[0004] There are several preanalytical variables, including biological, physiological events and technical modes of sample collection, preparation, storage, and transportation that often determine uncertainties, errors, and unwanted costs to the laboratory for their management.

[0005] The aforementioned variables can in important ways affect the outcome of laboratory tests by adding additional elements of uncertainty regarding the obtainable results or can give rise to responses that do not correspond to the actual biological situation of the patient, especially in diagnostics based on so-called liquid biopsies.

[0006] Liquid biopsy relates to diagnostics in the field, mainly oncology diagnostics, aimed at the identification and / or characterization of biological components through analysis of a sample of a body fluid, for example peripheral blood, and in particular of components that are correlated with pathology, for example with tumors, released into the blood circulation. The analysis of liquid biopsies can be aimed at prenatal diagnosis or, in the case of other acellular elements (e.g. nucleic acids, proteins, exosomes) extracted from the (mainly acellular) liquid component of the sample after removal of the cellular components, the suspension cellular components can be used for extremely informative genetic analysis to characterize the mutational status of the genomic DNA and to compare it with the mutational characteristics identified in the part of the cell-free DNA present in the liquid component. In the suspension cellular parts it is possible to find genetic mutations that have no pathological significance, the identification of which then essentially does not lead to false positives, this phenomenon, which has a frequency that increases with the age of the individual, is known as CHIP (clonal hematopoiesis of undetermined potential) and represents one of the main causes of misdiagnosis. The suspension cell component can further be used for proteomic and transcriptomic analysis of immune cells. However, the three components have different sample preparation needs and analysis modes that limit the possibility of obtaining clinical information from all of them without having to resort to multiple collections.

[0007] In order to maintain the consistency and composition of the samples, known devices and methods used in sample preparation pre-analytical activities generally face problems due to factors quite different from each other, e.g. storage temperature, possible presence of preservatives, centrifugation, filtration, freezing, transportation and mechanical / physical stress due to the type of test tube / container used.

[0008] From the moment of collection, body fluids start to deteriorate and several degradation phenomena can be observed. Cells decompose by modifying their morphology and releasing the genetic material contained within them, which mixes with the material circulating in the liquid component. Non-cellular material present in the liquid component (exosomes, proteins, vesicles, free nucleic acids), for example in the plasma in the case of blood collection, starts to become damaged with the resulting loss of information, which then requires the use of preservatives to stop or at least slow down the process. These substances can however interfere with the cell biology, for example by altering their morphology or by preventing the expression of proteins. For example, some types of preservatives are contraindicated if one wants to carry out tests that require cell reactivity, such as adhesion to a substrate or reaction to a marker, for example an antibody. Furthermore, freezing, for example, is a known technique to reduce the deterioration phenomena of plasma, but represents a very important mode of preservation that is difficult to control.

[0009] Moreover, in the case of blood samples, the formation of clots is typically noted, and a common method to avoid this formation due to the waiting time and the mode of transport of the sample itself is to add an anticoagulant to the collected blood. The problem is especially felt in cytology, hematology and oncology, especially in liquid biopsy protocols, where excessive clotting is one of the causes of unacceptable samples.

[0010] Another example is the time that elapses between the taking and the analysis of a hematological sample, which is a decisive factor for obtaining reliable results. In fact, it is known that prolonged contact with blood samples causes modifications to the cells, for example in procedures that result in the use of anticoagulants such as ethylenediaminetetraacetic acid (EDTA). In addition to that, if the procedure makes use of preservatives, substances different from EDTA that make it possible to slow down the aforementioned degradation processes as previously explained, the characteristics of the blood and the reactivity of its cellular components will be altered and subsequently downstream tests will be impaired, especially in the case of analyses involving circulating tumor cells (CTCs).

[0011] A need is therefore felt for a solution that overcomes the difficulties of known techniques associated with the time at which analyses should be carried out on samples of body fluids after their separation, and with the aim of not compromising their validity, particularly in liquid biopsy protocols.

[0012] In view of the above, and especially when handling different components of a body fluid sample with the aim of slowing down their degradation process, methods and known devices typically provide for the collection of multiple samples, which are then handled and analyzed at different times and modes depending on the type of analysis that can be retrieved / detected at that moment and that may nevertheless relate to the same diagnostic study. This situation represents an additional drawback, both in terms of the uniformity of the informative content of the samples and in terms of the management costs of the analytical laboratory.

[0013] Additional drawbacks of the known solutions, however, result from the fact that significant operational activities by the operator are provided during the preparation of the sample to be analyzed, thereby inevitably reducing the clinical relevance of the analyses and tests and the applicability of one and the same repeatable procedure for preparing the sample even in different diagnostic situations.

[0014] Another problem of the known techniques is the fact that, to be able to separate the biological material of interest from the other sample components, it is necessary to use techniques that represent a source of stress on the sample itself and / or that cause its partial loss, such as filtration by filters or microfluidic systems, the use of magnetic beads and cytocentrifugation. For example, with regard to the management of liquid samples that contain cells, such as blood, the most widespread technical solution is the use of cytocentrifugation units, in which a centrifuge is able to collect the cells by crushing them directly on a support, typically a slide.

[0015] However, these very common modes currently used in laboratories are associated not only with cell loss of the sample, but also with morphological loss and adhesion of cells on non-uniform overlapping surfaces, which impair their proper and complete analysis under the microscope by partially masking the cellular content, since the cells generally tend to detach from the support. Attempts have been proposed to mitigate these adverse effects of cell loss and damage to morphology, but these attempts are in practice limited within manual or empirical solutions specific to each laboratory or their effectiveness. These problems, already in relation to cytological analysis, become even more critical in the context of circulating tumor cells, considering their rarity inside fluid samples. Summary of the Invention

[0016] The technical problem recognised and solved by the present invention is therefore to overcome the aforementioned problems and in particular to provide an apparatus as defined in claim 1.

[0017] Further features of the invention are defined in the corresponding dependent claims.

[0018] The present invention relates to a method for detecting a marker comprising the steps of: (a) detecting a marker containing one or more, preferably a plurality of, information-providing components as defined in claim 10 of a sample in combination or alternatively; a first immobilized cellular component, preferably stabilized on a planar support; a second suspension cellular component, preferably stabilized inside the test tube; a third liquid stable component, stabilized inside the test tube; The present invention also relates to a method for the automated preparation of body fluid samples, in particular for chemical analysis of liquid biopsies, which is directed to obtaining a

[0019] The device of the present invention is a device for the automated processing of biological samples, in particular liquid biopsies, for chemical analysis using a body fluid sample as starting material. In a preferred embodiment, the body fluid sample processed by the device is a sample of whole blood.

[0020] The device provides a box-like body with an internal chamber and an opening for accessing the chamber. The internal chamber has first, second and third housing means respectively configured to accommodate a fluid sample, a reagent element and a planar support suitable for immobilizing cells of the sample. The device still comprises means for collecting liquid and solid waste. The device further comprises means for collecting and dispensing the sample and / or reagents and / or for intermediate preparation of the sample in the internal chamber.

[0021] The apparatus further comprises a centrifugation means operatively associated with the collection and distribution means, and a control unit, the control unit configured to control the collection and distribution means and the centrifugation means according to an automated protocol.

[0022] The overall configuration of the device is such that it is capable of obtaining, according to an automated protocol, a first component of the sample and / or a second component of the sample and / or a third component of the sample from a biological fluid sample entering the internal chamber, and a process waste component, each of which is separate and distinct from one another.

[0023] The first component comprises cells of the sample immobilized on a planar support, the second component comprises cells of the sample in liquid suspension preferably made available in a test tube, the third component comprises a liquid portion of the sample, e.g. plasma, preferably made available in a test tube, and the fourth component comprises process liquid waste.

[0024] In a preferred embodiment of the invention, the apparatus is capable of simultaneously acquiring at least two of the three aforementioned information providing components separately from the process liquid waste.

[0025] Advantageously, the device of the invention allows the preparation of analytical biological samples on a planar support, for example blood samples collected in test tubes containing exclusively anticoagulant, preferably EDTA, maintaining their stable state over time, for example up to one year, thanks to their immediate processing and stabilization carried out by the device. It is then possible to interrupt the laboratory workflow, for example allowing the freezing of components of the obtained samples, by significantly improving the mode for managing analytical activities (laboratory feasibility). In other words, from a biological point of view, the device of the invention guarantees the validity of analytical results that are not altered, thanks to intermediate processing after sample collection, while from a sample management point of view, the device of the invention allows the easy implementation of laboratory analytical flows that do not require the operator to work continuously. In particular in the case of blood samples, the device of the invention allows to obtain such advantages, even by carrying out the collection in test tubes containing only anticoagulant, preferably EDTA, thus minimizing sample alterations typically induced by storage.

[0026] Moreover, the device of the invention allows the processing of one single sample from which all three aforementioned informative components can be obtained. In fact, from one single sample entering the device, a first and a second component comprising the cellular elements of the sample, either deposited on a support or in suspension, a third component comprising a liquid component, and an additional component comprising liquid waste, can be obtained. Advantageously, three separate components with extremely informative and clinically useful content can then be obtained simultaneously from one single sample entering the device, by overcoming the limitations imposed by the different needs of the three components to ensure their storage and stability over time.

[0027] A particularly advantageous aspect of the present invention is then represented by the fact that the device is able to obtain from one single incoming sample multiple pieces of information from its different components, without the need to collect samples at different times / moments to perform the required set of analyses and without the need to use during collection multiple containers with specific preservatives or reagents for the different informative components one wishes to obtain from the body fluid.

[0028] Advantageously, the device of the present invention is capable of collecting from one single biological sample: A first cellular component of the incoming sample, which is particularly suitable for identifying circulating tumor cells (CTCs); a second suspension cellular component, mainly consisting of leukocytes, which is particularly suitable for use as a control for the analysis of mutations, for example present in the free nucleic acids present in the liquid portion, or, in the case of specific analyses on the immune cell content, for the analysis of the protein content and the nucleic acids of the leukocytes contained therein; Even in clinical practice, a third component comprising a liquid portion, e.g. plasma, is widely used for the analysis of multiple analytes, such as free nucleic acids (cell-free DNA, cell-free RNA), proteins, vesicles, and exosomes.

[0029] An additional advantageous aspect of the present invention relates to the fact that the device is able to prepare the biological sample to be analyzed in an automated manner, by providing an almost total standardization of the process for preparing the sample and such that subsequent analysis is feasible in a variety of clinical situations and the results have effective clinical utility.

[0030] According to a preferred embodiment, the device is configured to work with a means for supporting the acquired components, in particular a flat supporting means, which are to be functionalized through the surface deposition of nanomaterials as a result. Such a supporting means, in particular in the form of a slide, allows an intermediate and effective attachment of any living cells, advantageously allowing an integrated capture approach in a liquid biopsy protocol.

[0031] An apparatus configured in this manner is capable of dispensing reagents and performing subsequent washes in a generally flat mode, thus avoiding the need for solution providing substrate immersion in high volume reagent filled containers.

[0032] The device is configured so that aspiration and dispensing speed, volume transferred, and arm positioning accuracy can be tailored to the process needs of each of the information providing components.

[0033] Thanks to the immediate automated and standardized preparation of the sample, the device then returns components with at least a practically intact repertoire of clinically informative cells from the freshly taken sample (e.g. CTCs), making it possible to obtain maximum sensitivity, even in the context of early stage disease, which is consequently particularly advantageous in liquid biopsy protocols.

[0034] Advantageously, the device of the invention then allows for the subsequent complete analytical evaluation of morphological, proteomic and genetic features on the acellular components (third components obtained from the sample) in a fixed or suspended state or contained in the liquid portion at the single cell level, further allowing both the completion of detectable clinical analytical information and the comparison between different informative features.

[0035] The first component acquired with the device is suitable to be analyzed using conventional techniques of pathoanatomical biology, such as cytology, immunocytochemistry, immunofluorescence, FISH, and molecular analysis.

[0036] The second component consisting of the suspension cell portion is suitable for proteomic analysis by FACS techniques, mass spectrometry, ELISA and for genetic studies by sequencing (NGS, RNAseq, Sanger, digital PCR) both to obtain specific information about the white blood cells contained and to compare the mutations identified in the cell-free DNA present in the third liquid component with possible mutations present in the cellular genomic DNA extracted from the suspension cells present in the second component; such a comparison is essential to validate the results of the mutations identified on the cell-free DNA of the third component and then to exclude the presence of false positives that would invalidate the analytical results.

[0037] In contrast, the third component is suitable for genetic and non-genetic analyses (NGS, Sanger, digital PCR) of free nucleic acids as well as other analyses of other analytes (e.g. blood proteins and exosomes).

[0038] The combination of artificial intelligence guided image analysis of cellular components on a planar support with advanced genetic sequencing on the same cells, and analysis of elements extracted from both the liquid component (acellular content) of the sample and the suspension cellular components prepared with the device of the invention, allows maximum sensitivity and specificity of liquid biopsy testing in any clinical situation. The absence of preservatives during collection, combined with the reduction of stress induced by the most common methods for isolating vital components, can reduce the loss of information obtainable from the sample.

[0039] In essence, the invention makes available a pre-analytical device suitable for implementing the main protocol of liquid biopsy and ensuring standardized and automated preparation of freshly taken body fluid samples, preferably using specific supports, such as slides, functionalized and configured to preserve the morphological characteristics of cells, with the aim of providing a first component of the sample suitable for identifying cellular components. Apart from standard microscope slides, alternative supports can be cover slips, single or multi-well plates.

[0040] The device of the present invention is configured to simultaneously provide, together with the first component, a second component of the sample for the analysis of suspension cells, and a third component of the sample, in particular a component comprising a liquid portion for the analysis of its contents from which the cells have been removed, without the need to use test-specific test tubes for blood collection or the aid of a specialized technician.

[0041] The present invention is consequently particularly advantageous in the context of oncology, in particular in searching for cellular and rare content, such as, for example, CTCs in a first component and circulating nucleic acids or other tumor markers in a third component, and in comparison with the nucleic acids of suspension leukocytes present in a second component. Particularly advantageously, it is possible to compare possible abnormalities in the genomic content of one (or both) of the first and third components with the genomic content of the component containing the suspension cells, in order to distinguish mutations present in the leukocytes that do not have pathological significance. Furthermore, for example, in the context of immunotherapy, it is particularly useful to be able to analyze the specific characteristics of suspension leukocytes using transcriptome analysis.

[0042] Finally, the system is programmable to simultaneously manage a variable number of samples, preferably 1 to 8, resulting from one or more subjects, by then using a variable number of slides, preferably 1 to 80, for each sample, making the system flexible for diagnostic situations that may require different amounts of sample to be analyzed.

[0043] Other advantages, as well as features and modes of use of the invention as a result, will become apparent from the following detailed description of preferred embodiments, given by way of example and not of limitation.

[0044] The figures shown in the enclosed drawings refer to the following: [Brief description of the drawings]

[0045] [Figure 1] 1 is a general view of a preferred embodiment of the device according to the invention; [Diagram 2]FIG. 2 is a schematic top view showing the inside of the device shown in FIG. 1. [Diagram 3] FIG. 2 shows a preferred embodiment of a means for containing a body fluid sample to be processed, implemented in the device of FIG. 1. [Figure 4] FIG. 2 shows a preferred embodiment of the means for containing reagents and waste implemented in the device of FIG. 1. [Diagram 5] FIG. 2 shows a preferred embodiment of a means for accommodating a planar support intended to immobilize and carry cells of a fluid sample to be processed and preferably used in connection with the device of FIG. 1. [Figure 6] FIG. 2 shows a preferred embodiment of the replaceable components of the collection and distribution means incorporated in the device of FIG. 1, in particular the means for accommodating the chips. [Figure 7] FIG. 2 shows a preferred embodiment of the centrifugation means incorporated in the device of FIG. 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The invention is described below with reference to the aforementioned drawings.

[0047] 1, there is shown in a schematic overview an apparatus for the automated processing of samples of biological fluids, in particular blood, for analytical or diagnostic purposes. The apparatus is designated as a whole by the reference numeral 1.

[0048] The device is preferably applied in clinical analysis, in particular in the preparation of blood samples within the procedure of liquid biopsy. However, other applications of liquid biopsy are not excluded by using other types of body fluids such as urine, cerebrospinal fluid, pleural fluid, bronchial lavage, saliva, cytological collection via aspiration needle, samples generally consisting of several informative biological parts (acellular and cellular components). Furthermore, advantageous applications in other fields such as cytology, immunology, medicine for pathogen search and as an alternative to all cytocentrifuge applications are not excluded.

[0049] The apparatus is preferably a bench tool and comprises a box-like body 10 having an internal chamber 20 and an opening 12 for accessing the internal chamber 20. In a preferred embodiment, the components described below are contained within the internal chamber 20 where the preparation of the biological sample is carried out.

[0050] As will be explained below, the device 1 is preferably provided with a system for filtering the air and / or vapour present in the internal chamber 20. Such a system is generally designated by the reference numeral 50.

[0051] The internal chamber 20 is divided into a number of regions or zones intended to receive the fluid sample, its intermediate preparations, reagents, waste and housing means configured to contain information-providing components of the sample acquired by the device 1.

[0052] The housing means is preferably removable and can be inserted / removed through an opening 12 in the box-like body 10 for inserting / retrieving / replacing its contents.

[0053] Figure 2 shows a schematic top view of the interior of the device 1. In a preferred embodiment, the internal chamber 20 comprises guide means 21 configured to couple with the housing means to facilitate extraction and insertion into and from the device and to ensure their correct positioning. In Figure 2 the guide means is exemplarily shown as a track carried by the support surface 20a of the internal chamber 20, configured to receive and slide the housing means as a platform.

[0054] The device 1 is further provided with a transfer system (of samples, of reagents and their intermediate preparation) comprising collection and distribution means adapted to work with exchangeable tips, for example of 1 mm or 5 mm.

[0055] The actions for collection and distribution of liquids are preferably carried out via a movable arm, for example along three mutually orthogonal axes, which advantageously comprises two mutually independent collection / distribution channels.

[0056] The arm allows movement of the tip in space, particularly in the housing means, for positioning within points of interest within the internal chamber 20. The arm can move liquids for multiple sample preparations at intermediate positions within the internal chamber 20.

[0057] For example, the arm movement allows positioning of the chip on the xy plane, while the relative movement of the channel with respect to the movable arm allows positioning of the chip along the z direction perpendicular to the xy plane. The channel can be slidably coupled to the movable arm by a guide integrated with the movable arm to implement the movement along the z direction. The distance between the two channels (axis-to-axis) is preferably fixed.

[0058] The transfer system preferably has a resolution and positioning accuracy of the order of tenths of a millimeter. The transfer system, in particular the collection and dispensing means, only transfers liquids and not containers, bottles, test tubes or slides, which may for example be received within the housing means.

[0059] The collecting and dispensing means are configured to carry out procedures at different speeds depending on the type of procedure to be carried out. In a preferred embodiment, the collecting and dispensing means are provided with a stepping motor, ideally with a precision of less than μl (microliter), preferably 0.3 μl, in order to be able to control the shifts of small fluid volumes and to obtain a precision in the definition of a speed of 3 μl / s (microliters per second), preferably 1 μl / s. This advantageously makes it possible to obtain a constant and uniform aspiration and dispensing at low speeds, a feature that is not guaranteed in the manual processing of samples and that affects their stability.

[0060] In a preferred embodiment of the device 1, even the tip is rechargeable within the housing means, as shown by way of example in Figure 6 and designated by reference number 34. Advantageously, it is possible to exchange the tips used by the collection and distribution channels, in order to avoid contamination of the reagents and samples.

[0061] The device further comprises a centrifugation means 40 operatively associated with the collection and distribution means, and a control unit, the latter configured to control the centrifugation means 40 and the collection and distribution means according to an automated sample preparation protocol, as described in more detail below. The control unit is preferably located outside the inner chamber 20.

[0062] The overall configuration of the device enables an automated protocol to obtain from the sample entering the inner chamber 20 a final preparation comprising one or more, preferably multiple, informative components of the sample separate and distinct from one another, being the first component of the sample and / or the second component of the sample and / or the third component of the sample, apart from process waste components.

[0063] In particular, the first component comprises or consists of cells to be fixed on a planar support and results for the analysis of a liquid biopsy intended to be received by the housing means. The second component comprises cells suspended in a fluid and is contained in a dedicated collection means. The third component comprises or consists of the sample liquid portion, e.g. plasma, also contained in a dedicated housing means. The waste component collects waste of the sample processing, preferably liquid waste, and is contained in a dedicated collection means.

[0064] With further reference to Figures 3-6, example embodiments of housing means contained within interior chamber 20 are described below.

[0065] For example, Fig. 3 shows a preferred embodiment of a first housing means 31, adapted to receive a biological fluid sample entering the device 1. As can be seen, such housing means comprises a number of sheets 31a, shaped to receive test tubes containing the samples, for example to form a rack. For example, each sample can be contained in a 10 ml test tube. In particular, blood samples can be contained in 10 ml test tubes, such as Vacutainer®. In a preferred embodiment, the device can accommodate a variable number of samples, preferably from 1 to 8 samples, preferably coming from one or more different subjects, preferably with two identical vials for each sample. The sheets 31a can have a side opening 31b to enable a reading means to carry out the reading of the identification and / or traceability code present on the test tube.

[0066] 4 shows a preferred embodiment of the second housing means 33 configured to receive reagents and to contain waste component collection means. In particular, the second housing means 33 comprises a plurality of seats 33a, each shaped to receive a respective bottle 33b containing a reagent or waste. Examples of reagents used in connection with the procedures implemented by the device 1 of the present invention are PBS (phosphate buffered saline), RBL (red blood cell lysing buffer), and a fixer solution, preferably containing formaldehyde, preferably between 2% and 4%. A fully loaded device 1 can contain, for example, 400 ml of PBS, 300 ml of RBL, 45 ml of fixer solution, and 500 ml of waste.

[0067] FIG. 5 illustrates a preferred embodiment of a third housing means configured to receive a means for supporting a first component of a sample obtained using the apparatus of FIG. 1, the sample comprising a cell.

[0068] Similar to what has been described above with respect to the first housing means, the third housing means preferably comprises a plurality of seats 32a, each preferably shaped to receive a planar support, in particular a slide, e.g. to form a rack. In a preferred embodiment, the apparatus 1 can accommodate eight racks, each rack capable of receiving up to ten slides.

[0069] The transfer system executes a process for distributing the cells on the planar support and the output of the automated protocol preferably comprises a planar support comprising a homogenous layer of cells, e.g. white blood cells. Preferably, the third housing means 32, each sheet 32a and / or each slide contained thereby, comprises an identification code suitable to enable the device 1 to generate a traceability report.

[0070] The second component of the sample obtained with one of the protocol formats implemented is collected in a container, preferably a test tube, carried by a fourth housing means or by a suitable seat (designated with reference number 42 in FIG. 7) of the centrifugation means 40, which will be described shortly.

[0071] A third component of the sample obtained with the protocol implemented by the device, e.g. plasma, is collected in a container, preferably a test tube, carried by a fourth housing means, not shown in the figures, but having a shape similar to what has been described above for the first housing means 31. The fourth housing means is provided within the internal chamber 20 to receive even intermediate preparations of the sample. In an alternative embodiment of the invention, a part 31p of the rack of the first housing means 31 is loaded into the internal chamber 20 with empty test tubes and extracted with test tubes containing the second and / or third informative components obtained by processing the sample.

[0072] As mentioned above, Figure 6 shows a preferred embodiment of a housing means 34 configured to receive the aforementioned tips. In particular, the replaceable tips are preferably carried within the internal chamber 20 near the dispensing and / or collecting means.

[0073] Apart from means for collecting components consisting of waste in liquid form, the device can even comprise means for collecting waste in solid form obtained during the sample preparation.

[0074] As mentioned above, the device 1 of the invention advantageously comprises a reading means, preferably integrated in the movable arm. In particular, the reading means advantageously comprises a first and a second reading device, for example of optical type, capable of reading the unique identification codes of the samples, of the reagent elements and of the supports (test tubes or slides) used for the first, second or third components obtained from the preparation of the sample. In this way, their full traceability is guaranteed. The codes read by the reading means are transmitted, for example, to a control unit capable of generating a traceability report. Preferably, the first reading device is configured for reading codes associated with the test tubes, vials of the samples and bottles containing the reagents. Preferably, the light beam of the first reading device is included in a plane substantially parallel to the support surface 20a of the internal chamber 20. The light beam of the second reading device is preferably included in a plane substantially perpendicular to the support surface 20a of the internal chamber 20 and is preferably configured for reading codes that can be associated with the planar support means and / or the respective sheet 32a.

[0075] With further reference to FIG. 7, a preferred embodiment of the centrifugation means is shown (the casing is not shown in the figure) and is generally designated by the reference number 40. The centrifugation means preferably comprises a rotatable drum or basket 41 provided with a plurality of sheets 42 arranged radially around the circumference with respect to the axis of rotation α of the drum 41. The motor driving the centrifugation means is preferably a stepper motor. Each of the plurality of sheets 42 develops along a direction substantially parallel to the axis of rotation α and is shaped to receive a test tube containing the sample, an intermediate preparation thereof or the aforementioned second information providing component of the sample. Several sheets can be provided, for example, to contain up to eight test tubes of 30 ml each. In an alternative embodiment of the invention, each sheet 42 is shaped to receive several test tubes having different sizes containing a sample of intermediate preparation.

[0076] Preferably, the drum 41 and / or the seat 42 are tiltable, the rest or rest position of the seat 42 determining the right angle between its own direction of deployment and the support surface 20a of the inner chamber 20. The drum 41 is capable of reaching a rotational speed equal to 400 g. During the centrifugation process, depending on the speed reached by the centrifuge itself, the seat 42 is tiltable until it reaches a position parallel to the support surface 20a of the inner chamber 20.

[0077] Advantageously, the centrifugation means 40 is configured so that the drum 41 and / or the sheet 42 can be stopped by the control unit of the device 1 in a predefined position, so that the movable arm can access the test tubes carried thereby in an automated manner.

[0078] Additional structural components implementing the movement of the centrifugal separation means 40 are within the understanding of those skilled in the art and will not be described in more detail.

[0079] As mentioned above, the device 1 is preferably provided with a system 50 for filtering the air and / or vapors present in the internal chamber 20. The automated protocol for preparing the samples preferably provides for the use of a fixative solution. In the case of blood samples, such a solution comprises, for example, 4% formaldehyde, and advantageously, the filtering system 50 allows the device 1 to be used safely without the need for additional external containment means such as a chemical hood. Additional embodiments of the invention can provide for the use of other organic compounds, such as alcohol-based fixatives, which can be used without the use of additional containment means, thanks to the presence of the filtering system 50.

[0080] Returning to Fig. 2, the filtering system 50 is an active system, preferably assembled on top of the box-like body 10. The filtering system 50 implements a recess capable of trapping the reagent vapors inside the device by filtering them with a suitable active filter. The filtering system 50 comprises a fan and filters, e.g. one to three filters (not visible), to create a forced air flow to contain the generated vapors inside the internal chamber 20.

[0081] The speed of the fan is adjustable depending on the step of the automated protocol. For example, the airflow generated by the fan is adjusted during the first step of the protocol, when the fixer is still in the bottle, to be minimal enough to prevent the escape of vapors from the device 1. During the dispensing of the fixer, i.e. when the evaporation rate is at a maximum, the fan will ensure a larger flow in order to reduce the concentration of the fixer vapors. The filtering system 50 can be sized to ensure a workload equivalent to at least 6 months of operation. The control unit of the device is preferably configured to signal the user of the need for a maintenance intervention to replace the filter. In a preferred embodiment, the fan can be made to operate at its maximum speed at the end of the automated protocol in order to speed up the drying time of the planar support from remaining reagent residues.

[0082] In a preferred embodiment, the apparatus 1 is operatively associated with a device configured to count the number of cells present in a biological sample. In a preferred embodiment of the invention, such a device is configured to count the number of white blood cells.

[0083] Such a device configured to count the number of cells can be integrated into the apparatus 1 or can be an external device operatively coupled to the apparatus 1 and can actually determine the appropriate dilution for sample preparation. The cell counting is preferably performed in a step carried out during sample loading in the internal chamber 20 and can be performed by grading the incoming sample during the loading step.

[0084] For example, a linear correlation between the count of white blood cells present in whole blood and the height of the sediment (white blood cells that have settled at the bottom of the test tube) obtained in the lysis process has been experimentally verified. Using this correlation, the concentration of white blood cells is known for each sample and the height of the sediment relative to the supernatant is obtained. It is then advantageously possible to remove the supernatant portion in a predetermined manner in the lysis protocol. Furthermore, the sediment volume and even the total number of white blood cells present in a single vial can be easily calculated. By grading the buffer volume in the test tube according to the two above parameters, it is possible to obtain a cell resuspension with an equivalent density for each sample. This process allows distributing an equivalent number of cells on each planar support for each sample.

[0085] The cell count process then advantageously provides several operating parameters of the automated protocol implemented by the device 1. As will be explained in detail below, the parameters include the height of the cellular elements (or sediment) that have settled in the test tube during sample processing, and the resuspension volume to obtain an equivalent density of cells to be distributed on the planar support.

[0086] As mentioned above, the control unit of the device implements an automated protocol for preparing a sample capable of providing as output a first component comprising cells fixed on a planar support and / or a second component comprising suspension cells and / or a third component comprising a liquid portion preferably received in a test tube, and separately a component preferably receiving liquid waste, each of these components being separate and distinct from each other.

[0087] A preferred embodiment of the automated protocol implemented by the device 1 for a sample of biological fluid consisting of whole blood is described below, by way of example and without limiting the scope of the invention.

[0088] First, the test tubes with the blood samples to be processed are loaded into the device 1 of the invention apart from possible reagents. In particular, appropriately labeled test tubes are introduced into the internal chamber 20 and accommodated in the respective racks 31. Inserting the racks 31 into the corresponding areas of the support area 20a activates a procedure for reading the traceability code associated with the samples. Preferably, the insertion of each type of housing means into the corresponding areas of the support surface 20a activates a procedure for reading the relative position and traceability code present both on the housing means itself and on the support inserted therein. If not already present in the internal chamber 20, the housing means are also loaded, comprising planar supports or slides of reagents and of flasks for the collection of waste liquids, exchangeable (disposable) tips of test tubes for the collection of plasma and of suspended cellular components, and test tubes in the seat 42 of the basket 41 of the centrifugation means 40. A first aliquot of blood is transferred from the collection and distribution means by a number of pipettes into the test tubes present in the basket 41. The aspiration and dispensing speed of blood is preferably comprised between 460 and 650 μl / s, and the volume transferred for each pipette is preferably comprised between 3 and 5 ml. Once the blood sample transfer process is completed, the separation of the plasma from the cellular part of the blood is started by the centrifuge. Once the centrifugation means 40 reaches the resume speed, it is preferably maintained there for 10 minutes and then decelerates in a gentle and controlled manner to avoid resuspension of the contents in the test tube. In a preferred embodiment of the invention, the stepper motor of the centrifugation means can control the position, the rotation speed, the acceleration and the deceleration. Two initial components of the blood sample are so obtained: a component containing cells and a component containing plasma.

[0089] The basket 41 stops at a predefined position and the movable arm collects and transfers the sample components, including the plasma, in a dedicated storage element. The plasma aspiration speed is preferably comprised between 20 and 150 μl / s, preferably between 90 and 110 μl / s, advantageously reducing damage to the cellular contents, similar to that caused by natural deterioration of the sample over time. The dispensing speed is comprised between 300 and 400 μl / s. The volume transferred for each pipette is preferably comprised between 2 and 3 ml.

[0090] In a first embodiment of the invention, such a storage element is preferably carried by a compartment of the housing means 31 of the inner chamber 20, in particular in a new test tube intended for subsequent storage and disposal. In a second embodiment of the invention, the storage element is carried in a previously unused seat 42 of the centrifugation means 40, in particular in a new test tube for carrying out a second centrifugation.

[0091] The transfer procedure is preferably carried out by a series of pipettes. Preferably, for each blood test tube, a plasma volume approximately equal to 3 ml is transferred. In a preferred embodiment of the invention, the movable arm is provided with a stepper motor, allowing a positioning accuracy of approximately one tenth of a millimeter on the z-axis. Such accuracy allows the adjustment of the need to aspirate as much plasma volume as possible without aspirating the cellular contents from the underlying layer.

[0092] Subsequently, the reagents are collected and distributed in the tubes of the basket 41 containing the cellular part of the blood sample (i.e. the plasma has been removed) for subsequent lysis of the red blood cells, preferably in a fixed volume, for example with a dilution ratio of 1:4 or a maximum final volume of 30 ml. Preferably, even a resuspension procedure is carried out in order to homogenize the contents of the tubes containing only the cellular part, after which an intermediate preparation is obtained.

[0093] The speeds for aspirating, dispensing and resuspending the reagents are preferably comprised between 500 and 2000 μl / s, more preferably between 1400 and 1600 μl / s. The volume transferred for each pipette is preferably comprised between 4 and 5 ml. In an alternative embodiment of the invention, resuspension can be performed by rotating at high speed a small magnet which joins the rotation in the centrifuge base, using a rotating magnetic field generated by a support with a permanent magnet, placed at the bottom of each test tube in the centrifuge.

[0094] The automated protocol then preferably provides a cyclical series of procedures on the intermediate preparation, including, in sequence, the steps of dilution, resuspension, centrifugation, and removal of the supernatant (as described above), until components of the blood sample containing cells, in particular white blood cells, are obtained. If a second centrifugation is provided, such centrifugation is performed simultaneously with the first procedure cycle on the intermediate preparation.

[0095] In particular, in the dilution step, the kinetic means collects the reagent and distributes (resuspends) it within the test tube containing the cell fraction in order to homogenize the contents of the test tube. The final procedure is preferably repeated five times without interruption.

[0096] This is followed by a waiting period, preferably 5 minutes.

[0097] In the subsequent centrifugation step, the control unit activates the centrifugation means up to a rotation speed preferably equivalent to 400 g, followed by a deceleration step after a centrifugation time preferably equal to 5 minutes. The deceleration can be performed via mechanical braking. In an alternative embodiment of the invention, the deceleration can be controlled by a stepper motor. When the basket 41 stops, the movable arm is positioned in a place for collecting, typically around 29 ml of supernatant, to be dispensed in a bottle for disposal purposes. The supernatant aspiration speed is comprised between 450 and 550 μl / s. The volume transferred for each pipette is preferably comprised between 2 and 5 ml. In particular, the residual volume of the cell portion contained in the intermediate preparation is determined by an automated protocol depending on the number of white blood cells determined by a counting device.

[0098] If a second centrifugation is provided, at the end of the first cycle the movable arm aspirates plasma from the test tubes in the seat 42 and distributes it into previously unused test tubes placed in the housing means 31. The plasma aspiration speed is preferably comprised between 20 and 150 μl / s, more preferably between 90 and 110 μl / s, while the dispensing speed is comprised between 300 and 400 μl / s. The volume transferred for each pipette is preferably comprised between 2 and 3 ml.

[0099] The cyclic sequence is preferably repeated three times until the test tube contained in the centrifugation means 40 contains preferably approximately 1 ml of sediment. The step for diluting the intermediate product with a reagent is carried out, for example by using as reagent RBL, preferably in a ratio of 1:4. The final suspension step is preferably carried out by using PBS, preferably in a ratio of 1:10.

[0100] Once a sample component carrying an optimal concentration of cells, and in particular of leukocytes, has been obtained, the automated protocol preferably provides for five resuspension cycles.

[0101] Subsequently, a portion of the cellular components is collected and dispensed onto a planar support housed within the second housing means 32 of the inner chamber 20. The aspiration speed is comprised between 450 and 500 μl / s, while the dispensing speed is preferably comprised between 100 and 500 μl / s, more preferably between 300 and 380 μl / s. The volume transferred for each pipette is preferably comprised between 700 and 900 μl.

[0102] The step for distributing the cellular components on the slide advantageously provides for the deposition of a single, substantially uniform layer of leukocytes, preferably about 2 million leukocytes, on each planar support, resulting in substantially no overlap.

[0103] The dispensing step is advantageously carried out with one single continuous movement of the dispensing tip, in particular the tip being positioned on the functionalized surface of the slide and carrying out continuous dispensing during a shift of the tip itself along the larger size, in particular larger axis, of the functionalized surface of the slide, this dispensing having the advantage of being rapid and of guaranteeing substantial uniformity of adhesion.

[0104] Other distribution methods are possible. For example, two fixed tips positioned on the functionalized surface of a slide can provide a modern distribution of cellular components, even if such modes require longer time periods. The distribution step is preferably completed within 20 minutes of the last performed resuspension cycle.

[0105] At the end of the dispensing step, a portion of the cell suspension remains available inside the test tube in the seat 42 of the basket 41 of the centrifugation means 40. This portion constitutes a second informative component obtainable with the device of the invention. This portion can be aspirated by the movable arm and dispensed in a previously unused test tube placed in the housing means 31. The aspiration speed is comprised between 450 and 500 μl / s, while the dispensing speed is comprised between 300 and 380 μl / s.

[0106] The protocol then provides a step for adhering the cellular components onto the planar support, advantageously treated to preserve the morphological characteristics of the cells. In the automated protocol described herein, the planar support used in connection with the device of the invention has specific surface characteristics and is preferably functionalized with a surface deposition of nanomaterials. The adhesion of the cellular components is carried out at room temperature and lasts approximately 20 minutes.

[0107] A step of aspirating the supernatant from the planar support follows, preferably with two tips in two separate points so as to leave only a liquid portion (e.g., 100 μl) on the planar support. The aspiration speed is comprised between 80 and 120 μl / s, while the dispensing speed is comprised between 1400 and 1600 μl / s. The volume transferred for each pipette is preferably comprised between 600 and 700 μl / s.

[0108] The steps then alternate between dispensing and aspirating (supernatant) first of PBS, then of the fixing solution and finally of the washing solution onto / from the planar support. The aspiration speed is comprised between 1400 and 1600 μl / s, while the dispensing speed is comprised between 300 and 380 μl / s. The volume transferred for each pipette is preferably comprised between 500 and 1000 μl. Such a procedure is always carried out on predefined points, for example four points or two points of the planar support, all of which are at the same distance from the support itself calculated along the z-direction.

[0109] In particular, reagent dispensing is modernly performed via two fixed tips positioned on the functionalized surface of the slide. When the tips are loaded with reagents, they are positioned above a predefined point at a given distance from the slide surface and release the desired amount of reagent. The optimal dispensing and aspiration distance from the slide is defined based on a range of experimentally obtained values. Beyond the upper limit of this range (with a tolerance lower than a millimeter), the residue of the reagent remaining on the slide has a volume comparable to the subsequent reagent, which is responsible for the dilution of the subsequently dispensed reagent. Below the lower limit of this range, the adhesion of the cellular components is hindered by the presence of the tips, which determines the slide areas that are free or actually devoid of cells at the position assumed by the tips. As for all liquids dispensed on the slide (containing the cellular components of the sample), the presence of the external hydrophobic edges of the slide and of the internal surface functionalized with a hydrophilic material, preferably a hydrophilic nanomaterial, allows the liquid to expand and cover the entire area inside the confinement.

[0110] Preferably, the dispensing of the fixer is followed by a fixing period comprised between 30 seconds and 20 minutes depending on the type of fixative used. In a preferred embodiment of the invention, for a fixative comprising 4% formaldehyde, the fixing time is approximately equal to 20 minutes. In an alternative embodiment of the invention, for a fixative comprising an alcohol solution, the fixing time is approximately equal to 30 seconds.

[0111] It will be appreciated that advantageously the collection and dispensing means of the device is further configured to then allow a "temporary" stabilisation of the components of the blood sample by collecting and dispensing a fixative liquid.

[0112] In particular, in the case of immobilized cellular components (first information providing components), the stabilization step is implemented by administering a fixative solution.

[0113] Advantageously, the suspension cellular components (second informative components) are available for subsequent analysis without the need for a stabilization step via a fixative. In an alternative embodiment of the invention, reagents for similar preparation for subsequent analysis are placed in the test tube in which such components are collected.

[0114] In the case of a liquid component (third information providing component), the stabilization step can be implemented via management of the fixative and / or freezing. Advantageously, if such a liquid component is subjected to a second centrifugation, a stabilization step is not necessary.

[0115] Such a preferred configuration makes it possible to further improve the quality of the informative content of the components of the blood sample and to make it possible to use a single automated device that is able to separate in an optimal manner the pre-analytical steps for preparing the incoming blood sample, both in terms of time (stabilization) and space (fixation), from the subsequent analytical steps of its components, which are obtained as output.

[0116] For example, a fixative solution containing formaldehyde at a concentration of up to 4%, i.e., an alcohol-based solution containing a compound that inhibits cellular apoptosis to stabilize blood components including plasma when a stabilization step is required, and a fixative solution can be used to stabilize blood components including cells attached to a support.

[0117] In relation to what the known solutions offer and then the associated advantages that the device itself guarantees, it is convenient to explain in a distinguishing manner the automated protocol described in the examples and implemented by the device of the present invention when using a fixing solution for components of a blood sample.

[0118] As mentioned above, currently, preservatives are typically used for whole blood samples, which are useful for CTC analysis to avoid cell deterioration and death.However, due to the presence of liquid parts in blood samples, such preservation modes are only successful in practice in limiting this process.For plasma analysis, preservatives are instead useful to avoid the death of existing cells and the release of healthy DNA, which would contaminate the sample.In practice, then, two complementary needs are addressed: on the one hand, to preserve the cellular components of the sample to be analyzed, and on the other hand, to prevent contamination of the plasma to be analyzed.

[0119] In a preferred embodiment of the invention, the device and the protocol it implements, unlike known solutions, does not use any preservatives during the step of collecting the blood sample, since the sample can be processed immediately via insertion into the device 1, but it is possible to use only EDTA, which ensures that the sample is affected as little as possible.

[0120] Indeed, it is advantageously possible to fix the first components, including the cells obtained as output, in a well-defined manner that makes them stable and analyzable, in a situation already prepared for analysis, and then to immobilize them on a support, exactly as occurs when fixing tissue sections on slides, where the fixative freezes the state of the cells that can be analyzed, if necessary, in a subsequent step.

[0121] For suspension cellular components, in both analytical tests for identifying proteins, metabolites and for molecular analysis, it is possible to have a defined amount of cells available that are not fixed in suspension, which is available after adding specific reagents (such as lysates with extraction of various cellular components).

[0122] With regard to the informative components, including plasma, since it has already been separated from the cells, it will at most have a minimal cellular content with a greatly reduced risk of contamination. Advantageously, the fact of performing a second centrifugation further reduces this risk. Furthermore, it will be appreciated that the plasma obtained with the device to which the present invention relates does not need to be frozen or have preservatives added for most applications.

[0123] In effect, then, it is possible to drastically reduce (or even completely eliminate) the use of preservatives as far as plasma is concerned, since the cells can advantageously be fixed on a unique support (a condition that cannot otherwise be obtained with whole blood), interrupting the cell degradation process and resulting in the immediate elimination of most of the potentially contaminating cellular components.

[0124] The operator can then extract the housing means 31, 32 from the internal chamber 20 and retrieve the first and / or second component and / or the third component with which the blood sample entering the device 1 was prepared.

[0125] The present invention has been described above with reference to preferred embodiments. Each of the technical solutions implemented in the preferred embodiments is described herein as an example and can advantageously be combined in different ways to create other embodiments that belong to the same inventive core, but all within the scope of protection of the claims set forth below.

Claims

1. An apparatus (1) for the automated processing of a body fluid sample, preferably whole blood, especially for liquid biopsy, for clinical analysis, comprising: A box-shaped body (10) having an internal chamber (20) and an opening (12) for accessing the internal chamber (20); First housing means (31) configured to receive the sample within the internal chamber (20); Second housing means (33) configured to receive a reagent within the internal chamber (20); Third housing means (32) configured to receive a planar support suitable for receiving cells of the sample; Fourth housing means (31p) configured to receive a container that tends to contain the liquid portion of the sample; Means for collecting and dispensing the sample and / or the reagent and / or for intermediate preparation of the sample within the internal chamber (20); Centrifugation means (40) operably associated with the collection and dispensing means; A control unit configured to control the collection and dispensing means and the centrifugation means (40) according to an automated protocol, wherein the overall configuration of the apparatus is such that the automated protocol enables the acquisition of one or more information-providing components and waste components of the fluid sample from the fluid sample before it enters the internal chamber (20), The one or more information-providing components include, separately and distinct from each other, a first component, a second component, and / or a third component of the fluid sample, The first component includes the cells of the sample immobilized on the planar support, The second component includes cells of the suspension sample, The third component includes the liquid portion of the sample contained in the container, especially a test tube, apparatus (1).

2. The apparatus (1) according to claim 1, wherein the collection and dispensing means are further configured to stabilize the first component by collecting and dispensing a fixing liquid.

3. The apparatus (1) according to claim 1 or 2, wherein the collection and dispensing means are further configured to stabilize the third component by collecting and dispensing a fixing liquid.

4. The apparatus (1) according to claim 1 or 2, operably associated with a device configured to count the number of cells, especially white blood cells, present in the fluid sample.

5. ​ The device (1) according to claim 1 or 2, wherein the dispensing means is configured to dispense a single uniform layer of cells in a substantially non-overlapping manner on the planar support.

6. The device (1) according to claim 1 or 2, wherein the collection and dispensing means is configured to identify the body fluid sample, the reagent, the first component, the second component, and / or the third information-providing component.

7. The device (1) according to claim 1 or 2, wherein the collection and dispensing means is configured to work with a replaceable chip that can be charged within a fifth housing means (34).

8. The device (1) according to claim 1 or 2, further comprising a filtering system (50) configured to confine and filter the vapor generated by the reagent within the internal chamber (20).

9. A system comprising the device (1) according to claim 1 or 2 for the automated processing of a body fluid sample, preferably whole blood, especially for liquid biopsy, wherein at least the planar support is intended to receive the first component and has a surface for immobilizing cell elements, the surface being treated with a nanomaterial, the system.

10. A method for the automated conditioning of cells immobilized on a planar support, suspension cells, and the liquid portion starting from a body fluid sample via the device according to claim 1 or 2, the method comprising: a) providing and introducing the fluid sample into a first housing means present within the internal chamber (20) of the device (1); b) collecting the sample from the first housing means (31) and dispensing it into a centrifugation means (40); c) centrifuging the sample to obtain a component containing cells and a component containing the liquid portion; d) collecting the component containing the liquid portion from the centrifugation means (40) and dispensing it into a container, especially a test tube, accommodated by an additional housing means (31p) present within the internal chamber (20). e) collecting the component containing the cells from the centrifugation means (40), and as an alternative or in combination, distributing this into an additional container, in particular a test tube, accommodated by the additional housing means (31p) on a planar support accommodated by additional housing means (32) present in the internal chamber (20); f) immobilizing the cells on the planar support; A method comprising the steps of.

11. providing a reagent element, in particular a fixing solution, in a third housing means (33) of the internal chamber (20); g) administering the fixing solution to the first component and / or the third component in order to obtain stabilization; The method according to claim 10, further comprising the steps of.

12. The method according to claim 10 or 11, further comprising the step of counting the cells present in the fluid sample to obtain a reference value and distributing some of the cells on the planar support according to the reference value.

13. The body fluid sample is a sample of whole blood, The method according to claim 10 or 11, wherein the third component comprises plasma.

14. The centrifugation step c) enables the acquisition of two initial components in the form of an intermediate preparation containing cells and a component containing plasma, Downstream of the step d), the method further comprises d') collecting a reagent element and distributing it into the intermediate preparation; and d'') centrifuging the intermediate preparation to obtain an intermediate component substantially containing white blood cells; The method according to claim 10 or 11, wherein the step e) comprises collecting the white blood cells from the centrifugation means (40) and distributing a single homogeneous layer of substantially non-overlapping white blood cells on the planar support.

15. The method according to claim 11, wherein the reagent element comprises a solution containing phosphate buffered saline, erythrocyte lysis buffer, formaldehyde.

Images