WO2022208506A1

WO2022208506A1 - Bio-ferrographic system and methods of use

Info

Publication number: WO2022208506A1
Application number: PCT/IL2022/050349
Authority: WO
Inventors: Noam Eliaz; Itai Benhar; Ofer Levi; David SVETLIZKY
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2021-04-03
Filing date: 2022-04-03
Publication date: 2022-10-06

Abstract

The technology disclosed herein generally ccoonncceerrnnss a system and method for simultaneously assessing a large number for patient samples for determining disease states.

Description

BIO-FERROGRAPHIC SYSTEM AND METHODS OF USE

TECHNOLOGICAL FIELD

The invention generally contemplates a bio-ferrograph and methods of using same in assessing disease states.

BACKGROUND OF THE INVENTION

Ferrography is a magnetic monitoring technique that allows separation of ferromagnetic and paramagnetic particles from a liquid onto a glass slide under a strong external magnetic field. Based on the number, size, shape, surface morphology and chemical composition of the isolated magnetic particles, the level, origin and the mechanism of wear can be determined. Since its conception in the early 1970s, ferrography has been found reliable and sensitive for monitoring wear evolution in engineering systems such as helicopter gearboxes.

Bio-ferrograph (BF) aims at capturing by means of magnetization target biological matter such as cells and tissue fragments from body fluids. The magnetization of the biological matter may be either non-specific using solvents that contain rare earth salts, most commonly ErCh, or specific using antibodies conjugated to magnetic beads. The BF allows flowing simultaneously up to five fluid samples through five distinguishable bracketed areas on the coverslip slide, without cross-contamination. Other advantages of BF compared to filtration or other immunomagnetic isolation (IMI) techniques include, inter alia, quantitative analysis of the captured biological matter while observing it microscopically, extremely high selectivity and sensitivity, very high recovery rates, small and confined deposition area, preservation of the structure and morphology of the isolated particles, the ability to analyze various isolated cells simultaneously, applicability to any liquid sample (including whole blood), and the ability to capture particles as small as several nanometers.

The Bio-Ferrograph 2100, from Guilfoyle Inc. [1-3], is a bench-top, cytometry- based, high-gradient magnetic field separator. The magnetic field is generated by a ferrite- based (SrFe) permanent magnet assembly and a pair of low-carbon steel pole pieces. An interpolar gap which is made of a magnetic isolator material forms a magnetic barrier to both inherently magnetic and magnetized particles suspended in a liquid. The maximum magnetic flux density formed across the interpolar gap was between 1.67 and 1.8 T. The gradient of the field is maximal at the edges of the interpolar gap, where most of the particle deposition takes place, thus forming two parallel particle strips, primary and secondary, on the ferrogram (the microscope coverslip slide with deposited particles). Consequently, a rectangular deposition band can be observed on the ferrogram, often even by the naked eye.

The Bio-Ferrograph 2100 system utilizes a simple five-syringe pump assembly that allows the simultaneous processing of five samples under identical flow and magnetic field conditions. Such a design is limited in permitting simultaneous testing of control samples together with the actual samples. To-date, bio-ferrography has been used successfully to track bacteria, capture rare magnetic minerals embedded in Vespinae comb, separate between carbon micro- and nanoparticles suspended in ethanol, isolate bone and cartilage tissue fragments from the synovial fluids in human joints for diagnosis of osteoarthritis (OA) and determination of the efficacy of a pain-relief drug treatment and isolation of both polymeric and metallic wear particles from artificial joints for mechanical wear evaluation. To achieve OA evaluation, for example, magnetization of the target tissue fragments was achieved by mixing the synovial fluids with cocktails containing monoclonal anti-collagen I and anti-collagen II antibodies coupled to 50 nm paramagnetic magnetic-activated cell sorting (MACS™) MicroBeads [4, 5].

Bio-ferrography has also been used to isolate and characterize mechanically cancer cells [6-10]. The identification of circulating tumor cells (CTCs) in the blood circulation may play a role in early detection of cancer as well as for follow-up on the progress of disease. Due to the low prevalence of CTCs in the blood circulation, a very high recovery rate of any CTCs isolation technology is needed. In [6], bio-ferrography was used to separate between target (positive) A431 cells, which simulate epidermal growth factor receptor (EGFR)-overexpressing epithelial CTCs, from NIH 3T3 mouse embryo fibroblast nontarget (background, or negative) cells. EGFR-overexpressing tumors include colorectal cancer (CRC), the 3^rd most commonly diagnosed cancer and the 3^rd leading cause of cancer death, which suffers from a lack of diagnostic techniques that are both effective and noninvasive. The target and nontarget cells were mixed at a 1 : 10⁶ ratio, either in phosphate -buffered saline (PBS) or in human whole blood (HWB). IM labeling was based on monoclonal primary antibodies conjugated to magnetic microbeads via a secondary antibody. A proof-of-concept isolation procedure was developed, yielding recovery rates of 78% and 53% and limit-of-detection (LOD) values of 30 and 100 target cells in 1 mL PBS or HWB, respectively.

BACKGROUND PUBLICATIONS

[1] N. Eliaz, “Wear particle analysis,” in: ASM Handbook, Vol. 18: Friction, Lubrication, and Wear Technology, edited by G. E. Totten, ASM International, Materials Park, OH, 2017, pp. 1010-1031.

[2] N. Eliaz and K. Hakshur, “Fundamentals of tribology and the use of ferrography and bio-ferrography for monitoring the degradation of natural and artificial joints,” in Degradation of Implant Materials , edited by N. Eliaz, Springer, NY, 2012, pp. 253-302.

[3] J. B. Desjardins, W. W. Seifert, R. S. Wenstrup, and V. C. Westcott, “Ferrographic apparatus,” US patent 6303030, October 16 (2001).

[4] K. Mendel, N. Eliaz, I. Benhar, D. Hendel, and N. Halperin, “Magnetic isolation of particles suspended in synovial fluid for diagnostics of natural joint chondropathies,” Acta Biomater. 6, 4430-4438 (2010).

[5] K. Hakshur, I. Benhar, Y. Bar-Ziv, N. Halperin, D. Segal, and N. Eliaz, “The effect of hyaluronan injections into human knees on the number of bone and cartilage wear particles captured by bio-ferrography,” Acta Biomater. 7, 848-857 (2011).

[6] O. Levi, A. Shapira, B. Tal, I. Benhar, and N. Eliaz, “Isolating epidermal growth factor receptor overexpressing carcinoma cells from human whole blood by bio- ferrography,” Cytometry B Clin. Cytom. 88, 136-144 (2015).

[7] B. Fang, M. Zborowski, and L. R. Moore, “Detection of rare MCF-7 breast carcinoma cells from mixture of human peripheral leukocytes by magnetic deposition analysis,” Cytometry 36, 294-302 (1999).

[8] P. B. Turpen, “Isolation of cells using bioferrography,” Cytometry 42, 324 (2000).

[9] O. Levi, B. Tal, S. Hileli, A. Shapira, I. Benhar, P. Grabov, and N. Eliaz, “Optimization of EGFR high positive cell isolation procedure by design of experiments methodology,” Cytometry Part B - Clin. Cytom. 88, 338-347 (2015).

[10] D. Svetlizky, O. Levi, I. Benhar, and N. Eliaz, “Mechanical properties of bio- ferrography isolated cancerous cells studied by atomic force microscopy,” J. Mech. Behav. Biomed. Mater. 91, 345-354 (2019). GENERAL DESCRIPTION

The present disclosure provides a novel, high-throughput, sensitive bio- ferrography-based (FB-based) system for separating target cells or tissue fragments, such as circulating tumor cells (CTCs), from human whole blood (HWB) samples or other liquid samples (such as saliva, sweat, urine, stool, cerebrospinal fluid (CSF), synovial fluids, maternal fluids and others. The system of the invention is designed, in its broadest possible configuration, to magnetically separate magnetically labeled cells, tissue fragments or solid wear particles from a sample. The system includes a plurality of flow channels, typically more than 5, and in some configurations several dozen or several hundreds of flow channels, that allow simultaneous analysis of dozens of patient samples simultaneously with suitable control samples. In other words, the system of the invention allows analysis of 20 or more patient samples at a time, a requirement set to make a system of the invention useful in a variety of medical arenas, such as hospitals, general laboratories, as well as research labs.

System design is based on an optimized procedure for bio-ferrographic isolation of cells and other cellular fragments from samples obtained from a subject, i.e., human or non-human. The system incorporates a semi-automated or a fully automated sample separation system that is configured to enable sample preparation, labeling and staining, magnetic isolation, and system recovery. The design process was optimized based on experimental feasibility tests and finite element analysis (FEA).

The system is configured as a decision-making tool for medical staff when monitoring patients in a hospital setting. It opens a new route for early diagnosis, prognosis and treatment of cancers, proliferative diseases, osteoarthritis (OA) and other medical conditions.

Thus, in a first of its aspects the invention provides a high-throughput magnetic isolation (and concentration) system comprising a plurality of flow channels, wherein the plurality of flow channels include more than 5 flow channels, or 10 or more or between 10 and 100 or more flow channels and a magnetic core arranged in a parallel configuration, wherein the plurality of flow channels is configured to (simultaneously) receive one or more samples within at least one of the plurality of flow channels, wherein at least one target component in said one or more samples is configured to be separated magnetically (in other words to be separated by being responsive to an applied magnetic field). The invention further provides a bio-ferrography-based magnetic isolation system, the system comprising: a plurality of flow channels, wherein the plurality of flow channels includes more than 5 or more than 10 flow channels, as defined, and a magnetic unit in a parallel configuration; wherein the plurality of flow channels is configured to (simultaneously) receive one or more samples within the plurality of flow channels, wherein at least one target component in said one or more samples is configured to be separated magnetically.

Further provided is a bio-ferrography-based magnetic isolation system, the system comprising: a magnetic unit; and a plurality of more than 10 flow channels, wherein each of the flow channels is configured to (simultaneously) receive an amount of a sample comprising a magnetically labeled target component or a sample suspected of comprising a magnetically labeled target component and allow flow of said amount of the sample in a direction (e.g., substantially perpendicular to) of a receiving surface; wherein said receiving surface is configured to receive and associate (or configured to capture and immobilize) said magnetically labeled sample component.

The invention further provides a bio-ferrography-based magnetic isolation system, the system comprising a plurality of sampling units, each being configured to receive same or different sample, as defined; a plurality of more than 10 flow channels, wherein each of the flow channels is configured to (simultaneously) receive an amount of a sample from said plurality of sampling units; a magnetic unit; and a receiving surface configured to receive isolated magnetically labeled sample components.

Also provided is a high-throughput magnetic isolation (and concentration) system comprising

-a plurality of flow channels,

-a magnetic core arranged in a parallel configuration, wherein the plurality of flow channels is configured to receive one or more samples within at least one of the plurality of flow channels, wherein at least one target component in said one or more samples is configured to be separated magnetically,

-a sample preparation unit, and

-optionally a system recovery unit.

In some embodiments, each of the systems of the invention comprises between 50 and 150 flow channels, each configured to receive and flow a sample or a control sample.

All systems of the invention may be configured to simultaneously receive and analyze 5 or more, 10 or more, 20 or more, 30 or more samples from different or same human or non-human samples. Thus, high-throughput magnetic isolation systems of the invention, configured for simultaneous analysis of 5 or more different samples, may comprise in some embodiments 100 or more flow channels, and a magnetic unit, wherein each of the flow channels is configured to receive one sample within the flow channel such that the system may receive any number of different samples to determine presence or absence of least one target component in the samples, wherein the target component is configured to be separated magnetically.

In some embodiments, the system comprises:

100 or more flow channels, and a magnetic unit; wherein the flow channels are configured to separately and simultaneously receive 5 or more, or 10 or more samples within the flow channels, and wherein at least one target component in said 5 or more, or 10 or more samples is configured to be separated magnetically.

In some embodiments, the system comprises: a magnetic unit; and

100 or more flow channels, wherein each of the flow channels is configured to receive an amount of a sample comprising a magnetically labeled sample component and allow flow of said amount of the sample in a direction (e.g., substantially perpendicular to) of a receiving surface; wherein said receiving surface is configured to capture and immobilize said magnetically labeled sample component.

In some embodiments, the system is for isolating magnetically labeled sample components from 20 or more samples simultaneously.

In some embodiments, the system is for determining presence or absence of a disease state, severity of a disease state, successful treatment of a disease state, recurrence of a disease state, improvement in a disease state, slowing down of disease progression, slowing down of irreversible damage caused in a progressive chronic stage of a disease, and combination of the aforementioned.

In some embodiments, the system is for determining a disease state, wherein the disease is cancer, a proliferative disease, or osteoarthritis (OA).

Systems of the invention are isolation (and concentration) system that are configured to isolate magnetically labeled sample components (or target components) from non-labeled components and concentrate them so as to analyze, evaluate, diagnose or determine their presence in the sample obtained from a human or animal subject. According to some configurations of a system of the invention, a magnetically labeled sample containing or suspected of containing the target component to be analyzed or which presence is to be determined is flown from a sample unit through one or more flow channels in a direction substantially parallel to a magnet core. Magnetic field applied on the channels causes isolation of the magnetically labeled target components from the unlabeled sample components. The magnetically labeled components are thereafter collected on a receiving surface, which may be subsequently analyzed to determine an isolated sample profile (presence of a disease marker, number of marker components, etc), as defined herein.

As used herein, the expression “at least one target component in said one or more samples is configured to be separated magnetically ” refers to a target component, as defined herein, that is present in the sample and which can be separated, isolated or concentrated by associating thereto, directly or indirectly, at least one magnetically responsive tag or label, as disclosed herein.

In some embodiments, the system comprises a sample preparation unit comprising one or more individually addressable reagent reservoirs for forming the sample and including sample labeling for selective magnetic labeling of a target component present or suspected of being present in the sample.

In some embodiments, the system comprises a mixing unit, e.g., in a form of a sample shaker, a sample mixing element etc.

In some embodiments, the number of sample units is equal to the number of flow channels. In some embodiments, the number of sample units is smaller than the number of flow channels, such that at least one sub-plurality of flow channels is configured to receive and flow an amount of a same sample. In some embodiments, the ratio of samples to flow channels (samples: channels) is between 1:1 and 1:100. In some embodiments, the sample units are arranged as an array of sample chambers or containers. The array of sample units may be arranged in any desirable fashion. The array may be circular, arranged around, e.g., a central sample reservoir, or may be arranged in rows and columns, which enables logical organization of the units with respect to specific samples. For example, where the number of sample units is 100, an array may be arranged as a 1 x 100 array, a 2 x 50 array, 20 x 5 array, etc.

The sample units may be arranged in a rack which may be a reusable rack, while the units are installed manually into the rack and can be replaced when the analysis has been completed. Alternatively, the array of sample units is arranged as an n-well plate, having n number of sample units formed in a monolithic disposable rack.

Systems of the invention may further include one or more fluid distributers configured to distribute predetermined amounts of the one or more samples into the one or more sample units. The fluid distributer may be a manifold unit comprising a main fluid stream input and a plurality of fluid output elements that are arranged to deliver an amount of the sample into the one or more sample units. The fluid distributer, e.g., manifold, may be a consecutive-based manifold, a fractal bifurcation-based manifold, a circular bifurcation manifold or any other manifold known in the art.

The consecutive-based manifold incudes a plurality of ports, each typically having a constant cross-sectional area. The manifold may be configured to receive a main fluid stream through a single input and divide the stream into the different sample units.

In the fractal bifurcation-based manifold, a fluid stream enters the manifold from a single input and divides into two flowlines, wherein each of the two flowlines is further divided into two other flowlines, and so forth. A desired number of output ports may be generated by successive division of the flowlines. For example, to achieve 100 output ports, the fractal bifurcation-based manifold may require a 7-level division from the input port.

The circular bifurcation manifold may be configured to receive a fluid stream through an inlet port and divide the stream into a plurality of lines, each typically having a same or similar cross-sectional area, line length, angle spacing, and circular symmetrical distribution. This manifold design enables equal tube length of each flow line and identical flow parameters for all sample units.

The expression “ an amount of the sample ” or any lingual variation thereof refers to an aliquot or a predetermined amount of a sample that is withdrawn from a sample reservoir. The expression also refers to amounts or aliquots of a reagent which may be withdrawn from a reagent reservoir. The amount is typically predetermined. The amount of the sample that may flow through the flow channels may range from several microliters to several milliliters. The amount of samples prepared in the sample preparation units may range from several microliters to several milliliters.

Irrespective of the manifold arrangement or configuration, a system of the invention may comprise two or more fluid distributors, e.g., manifolds, that are each or at least one of which is designed either to receive a single sample and distribute said sample into a plurality of sample units (such a manifold regraded as a l:n manifold, wherein n is the number of output ports or sample units), and/or manifolds that are each or at least one of which is designed to receive a plurality of samples and collect all into a single container (in which case the manifold may be regarded as a n: 1 manifold, wherein n is the number of input ports or sample units). In some embodiments, a system of the invention comprises at least one l:n manifold and at least one n:l manifold.

Systems of the invention are high-throughput multichannel ferrograph systems capable of isolating, concentrating and enabling analysis of target sample components indicative of e.g., a disease state. To achieve high throughput capabilities, systems of the invention may thus include more than 5 (excluding 5) flow channels. The expression “ more than 5 flow channels ” includes 10 or more, 100 or more flow channels, wherein each of the channels may be designed or configured to receive same or different samples. The expression excludes a configuration wherein the number of flow channels is 5 or less.

In some embodiments, the system comprises 10 or more flow channels. In some embodiments, the system comprises between 10 and 100 more flow channels. In some embodiments, the system comprises 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more flow channels. In some embodiments, the system comprises 100 or more flow channels.

In some embodiments, the system comprises between 50 and 150 flow channels.

In some embodiments, the system comprises 100 or more flow channels arranged to receive between 1 and 50 different samples. In some embodiments, the system comprises 100 or more flow channels in an arrangement configured to receive between 10 and 50 different samples. In some embodiments, the system comprises 100 or more flow channels in an arrangement configured between 1 and 100 different samples. In some embodiments, the system comprises 100 or more flow channels that are arranged to receive between 10 and 50 different samples. In some embodiments, the system comprises 100 or more flow channels that are arranged to receive between 20 and 50 different samples.

In some embodiments, the flow channels are configured as flow chambers as disclosed herein.

The magnetic unit of systems of the invention include a magnet assembly which may comprise a magnetic core, which includes a magnetic field source, a magnetic conductor, and one or more magnetic isolators (wherein each includes one or more interpolar gap). The magnet unit forms a concentrated magnetic field generated by one or a plurality of interpolar gaps provided in one or a plurality of triangular-like structures provided on the surface of the (permanent) magnet core. Each of the plurality of interpolar gaps (which may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc) may be made of a magnetic isolator material that forms a magnetic barrier. Due to the distinct geometry of the interpolar gap, the gradient of the magnetic field is maximal at its edges, where effectively most of the particle deposition takes place. The unique assembly creates a closed circuit of magnetic flux. The width-to-depth ratio of each of the interpolar gaps defines the intensity of the magnetic flux at the gap edges. With a proper width-to-depth ratio, a high magnetic flux gradient may be formed, which enables maximal magnetic field at the interpolar gap edges.

The magnet core may be provided in either of two alternative configurations: an extended configuration, and a parallel configuration.

In some embodiments, the magnet core is provided in the extended configuration. This configuration is based on extending the interpolar gap length. In terms of magnetic field value at the interpolar gap, there is no actual difference in analogy to the magnetic core assembly of the bio-ferrograph, due to the plane symmetry of the magnetic core. In the parallel configuration, however, several (or two or more) interpolar gaps (or magnetic isolators) are provided parallel to each other along the magnetic core, such that the magnetic field magnitude at the interpolar gaps (or at the magnetic isolators) is consistent.

As presented herein, the magnetic field is maximal at the edges of the interpolar gap(s) at the upper surface of the magnetic core, and persistently fades as the distance increases from the interpolar gap(s) surface. In some embodiments, the parallel configuration the magnets are configured to simultaneously isolate and concentrate the cells, tissue fragments and solid material debris within the plurality of flow channels.

The “ receiving surface ” onto which magnetically labeled target components are deposited or associated is a glass or a polymer surface defining a plurality of surface regions (spaced apart regions, each with a predetermined location on the surface), wherein each region is designed to receive thereon the isolated target component(s) from a different flow channel. Where the receiving surface is said to be configured or selected to receive and associate a labeled sample component, the association may be by way of capturing and immobilizing the sample component(s). The number of surface regions may be equal to the number of flow channels such that each flow channel is configured to deposit the magnetically labeled target components onto a predesignated region on the receiving surface.

Alternatively, the receiving surface is a plurality of individually and separately addressable receiving surfaces, wherein each is designed to receive thereon magnetically labeled target components from a different flow channel. The number of receiving surfaces may be equal to the number of flow channels such that each flow channel is configured to deposit the magnetically labeled target components onto a predesignated slide.

In some embodiments, each individually and separately addressable receiving surfaces is a curved groove feature formed on a receiving plate, wherein the groove inner surface defining a receiving surface.

In some embodiments, each individually and separately addressable receiving surfaces, e.g., curved groove, is configured as a flow chamber, being provided with an input port and an output port configured to allow flow of the sample through the chamber, allowing settling or deposition of the magnetically labeled sample components on the surface of the coverslip slide. Unlabeled sample components continue to flow away from the receiving surface into a sample disposing unit or drain.

In some embodiments, each receiving surface is provided as a separate glass or polymeric slide having one or more flow chambers; namely having one or more a groove features designated as flow chambers. In some embodiments, each slide includes between 1 and 5 flow chambers. In some embodiments, each slide is configured to assemble into a slide assembly comprising two or more such slides. The slide assembly is designed to contain a plurality of slides, at a predesignated location over the magnetic core. The slide assembly may be installed in a slide frame by inserting the slides into designated openings or slots in the frame.

In some embodiments, the slide frame is provided on the magnetic core surface. In some embodiments, the slide slots are formed over, or engraved on the magnetic core surface.

The flow chambers may be formed on a surface of a slide or a glass or polymeric material surface and may be adapted with a sealing feature such as an O-ring seal that is provided at a back face of the receiving surface (namely the face of the receiving surface that is facing the ferrogram), such that the sample may enter the flow chambers through the input ports and exit through the output ports that are positioned at the front face of the receiving surface (coverslip slide).

Systems of the invention may be provided with a disposable or a reusable cassette which includes the receiving surface and optionally further a flow chamber facilitating the plurality of flow channels. The cassette is configured and designed to assemble into a cassette-magnet subsystem comprising the receiving surface and the magnet unit.

The “ sample ” may be any liquid body sample obtained from a human or an animal subject and which may contain at least one target component which presence and/or amount may provide an indication of a disease state or which presence and/or amount may provide a response to a desired qualitative or quantitative inquiry. The sample may contain cells, tissue fragments and/or solid material debris. The at least one target component may similarly be a cell, a tissue fragment or a solid material debris which presence and/or amount may be indicative of a disease state, as disclosed herein.

The cell(s) may be a cell containing a target component or may itself be a target cell indicative of a disease state, such as a cancer cell. The tissue fragment may be a part of a tissue that contains a target component or a cell or a material that is e.g., indicative of a disease state, e.g., a cancerous tissue or a liquid tissue biopsy sample. The solid material debris may be a solid component which is a decomposition or a degeneration product of e.g., a medical device, an implant, a bone, a cartilage, etc.

Systems of the invention are configured for simultaneous analysis of a plurality of different samples. The differences between the samples may be in at least one of: (a) different subjects; (b) different types of samples (e.g., blood samples, urine samples, etc); (c) samples for the same subject but different types of samples; (d) samples taken from the same subject at different times; (e) true subject samples and control samples; (f) samples of different dilutions; (g) samples containing different magnetically responsive materials; etc.

The sample may alternatively or additionally be a control sample (negative or positive control). The negative control may be a sample that is not expected to contain magnetically labeled components. Such a sample may be a sample that has been cleared out of any components which may undergo magnetic labeling. The negative control sample may also be a reagent used in the sample preparatory stages. The positive control sample may be a sample that is expected or known to contain a predefined amount of magnetically labeled target component.

In systems and methods of the invention both suspected samples (or samples to be evaluated) as well as control samples may be processed under identical conditions to increase confidence that the outcome is caused by presence of the disease state.

The sample may be a blood sample, a saliva sample, a stool sample, a tissue sample (such as a liquid biopsy), a urine sample, sweat sample, a CSF sample, a synovial fluid sample, a maternity sample or any other sample obtained from a human or non human subject. The blood sample is typically whole blood sample or a plasma-free blood sample (a blood sample processed to remove the plasma therefrom).

In some embodiments, the sample comprises a processed or unprocessed sample obtained from the subject, e.g., blood sample, and one or more reagents selected to enable labeling of a predetermined component present or suspected of being present in the sample.

In some embodiments, the sample is or comprises a human whole blood sample (HWB) or an animal whole blood sample. In some embodiments, the sample is HWB.

In some embodiments, the sample comprises sample components including cells separated from human whole blood (HWB) sample.

In some embodiments, the sample components include circulating tumor cells (CTCs) present in or separated from HWB.

In some embodiments, the sample components include stem cells suspended in a liquid. In some embodiments, the sample components include bone or cartilage tissue fragments present in or separated from synovial fluids or saline.

In some embodiments, the sample is a synovial fluid comprising or suspected of containing bone or cartilage tissue fragments.

In some embodiments, the sample components include bacteria separated from saliva, blood, urine, natural water or a bio-fluid.

Samples may be prepared in advance and stored for future use or prepared via use of a sample preparation unit. The sample preparation unit provided in systems of the invention may comprise one or more reagent reservoirs for delivering metered amounts of the reagents into one or more of the sample units. Thus, each sample unit may be in liquid communication with the reagent reservoirs and may be configured to receive an amount of the sample to be tested, e.g., blood sample, and a reagent formulation configured to provide association between a magnetic label or tag present in the formulation and a component present or suspected of being present in the sample.

The reagent formulation may include one or more of a solvent or a medium (being typically an aqueous medium, a saline), a cell culture medium (such as phosphate buffered saline), a buffer, an oxidizing agent, a reducing agent, a fixative reagent (such as formaldehyde or paraformaldehyde), a staining dye, magnetic particles, an antibody, or any other type of bio-markers.

The reagent formulation may also include a chemical label or a tag that is both magnetically responsive and capable of associating to a target component in the sample. The term “ magnetically responsive ” or any lingual variation thereof refers to a material or target component that includes or which is associated with a label or a tag that responds instantaneously to a magnetic field in a contactless manner, drawing the material component or target component to which it is attached or associated with towards the magnet or the receiving surface to thereby separate it magnetically. The target component may be a cell, a tissue fragment and/or a solid material debris, such as cells, tumor cells or tissue fragments, bone and cartilage tissue fragments and/or a material contained in said cell, tissue fragment and/or solid material debris. The material may be a tumor marker, or a component indicative of the disease state. In some embodiments, the tumor marker is an antibody, a hormone (such as calcitonin), a protein (such as beta-2- microglobulin), and others. Magnetic labeling or magnetic immunolabeling of a target component indicative of a disease state may involve associating or conjugating antibodies indicative of the disease state present in a sample to magnetic micro or nanobeads (the tag or label) via a secondary antibody or another linker moiety. The association of the micro or nanobeads to the sample component, e.g., an antibody, provides for the separation of the cell or tissue in which the component is present from its environment or from other cells or tissues in which the component is not present.

Magnetic labelling may be achieved by a variety of methodologies known in the art. Labelling methods which may be used for associating micro or nanoparticles or beads to a target component, according to methods of the invention, may include any of the methods disclosed in (a) Cytometry B Clin Cytom., 2014 Nov 28. doi: 10.1002/cytob.21212; Levi O., et ah, Isolating EGFR Overexpressing Carcinoma Cells from Human Whole Blood by Bio-Ferrography, (b) Cytometry B Clin Cytom., Sep-Oct 2015; 88(5):338-47. doi: 10.1002/cyto.b.21246; Fevi O., Optimization of EGFR high positive cell isolation procedure by design of experiments methodology, and others.

The magnetic particles used may be particles of a magnetic material such as iron oxide including ferrite and magnetite, chromic oxide, and cobalt. Two or more magnetic particles may be combined. The particle size of the magnetic particles is not particularly limited, and may be, for example, from 5 nm to 100 micrometers. In some embodiments, the magnetic particles are provided encapsulated in liposomes.

In some embodiments, the system is configured to analyze cells, tissue fragments and solid material particles in more than 5 patient samples, and preferably 20 or more patient samples, simultaneously.

In some embodiments, the system is configured to be used in hospitals, research centers and laboratories, testing houses, drug companies, academia, etc.

The magnetically labeled cells or tissues fixed or associated to a receiving surface may be evaluated or assessed using any microscopic, spectroscopic or otherwise biological or chemical means. Qualitative and quantitative evaluation of the receiving surface may be achievable by means of atomic force microscopy (AFM), optical microscopy, scanning electron microscopy (SEM), DNA and RNA analysis, confocal microscopy, chemical composition analysis, and others.

Thus, in some embodiments, a system of the invention may further include an analysis unit comprising microscopic, spectroscopic or biological or chemical means or tools for analyzing the magnetically labeled cells or tissues fixed or associated to a receiving surface.

In some embodiments, the analysis may include microscopic, chemical, biological, physical and mechanical characterization for diagnostics, prognosis, research and development purposes.

In some embodiments, the system may include an artificial intelligence- aided image analysis unit configured and used to characterize the number, shape, morphology, and size of the isolated cells (or particles).

The evaluation of the magnetically labeled components fixed or associated to a region of the receiving surface aims at determining an isolated sample profile. The sample profile includes any one or more of (a) determining presence or absence of a disease marker, (b) number of labeled target components, namely number of micro or nanobeads that are associated or tagged to the target components), (c) mechanical rigidity, (d) sample morphology, (e) DNA or RNA sequence, (f) presence of material agglomerations and others.

Based on the sample profile a determination may be made as to the disease state, or generally to a state of a sample. Determining the disease state includes determination of presence or absence of a disease state, severity of a disease state, successful treatment of a disease state, recurrence of a disease state, improvement in a disease state, slowing down of disease progression, slowing down of irreversible damage caused in a progressive chronic stage of a disease, and others. The sample profile may further assist in determining state of decomposition of a medical device or an implant (by determining presence and/or an amount of debris resulting from decomposition of the device or implant), or state of a solid or a hard tissue such as bone or cartilage.

Systems of the invention are configured as semiautomatic or fully automatic systems comprising reagent addition, mixing, incubation, flowing, magnetically separating and concentrating and readout or detection.

In some embodiments, systems of the invention are configured as a microfluidic device.

In some embodiments, systems of the invention are configured as a bench-top system.

The invention further provides a method comprising: providing a system comprising plurality of flow channels; magnetically isolating at least one target component present in one or more samples (being a cell sample, a tissue fragment sample, or a solid material particle sample), by flowing said one or more samples in a plurality of flow channels (e.g., including more than 5 flow channels); wherein each of the plurality of flow channels is configured to receive the one or more samples within the plurality of flow channels, wherein the at least one target component in said one or more samples is configured to be separated magnetically.

Also provided is a method comprising providing a system comprising plurality of flow channels, wherein e.g., the plurality of flow channels includes more than 5 flow channels, and a parallel magnetic unit; wherein the plurality of flow channels is configured to receive one or more samples within the plurality of flow channels, wherein at least one target component in said one or more samples is configured to be separated magnetically; magnetically separating magnetically labeled target components present in said one or more samples.

In some embodiments, the method involves analyzing the one or more samples from 20 or more patients simultaneously.

In some embodiments, the method involves steps of sample preparation, labeling and staining, magnetic isolation, system recovery, and both automatic and complementary analyses.

In some embodiments, the method is configured to detect different types of cancer and other diseases, components of implants or other medical devices suggesting degradation of the implants or the devices, efficacy of drug treatments, contamination by microorganisms (such as bacteria, fungi, parasites, viruses).

In some embodiments, the method further comprises providing a microscope slide with flow channels, within which the targets are isolated and concentrated, and where targets are configured to be analyzed within the flow channels on the microscope slide.

Also provided is method for simultaneously determining presence of a target component in a plurality of samples obtained from different human or non-human subjects, the method comprising preparing a plurality of sample mixtures, each containing an amount of a different sample from said plurality of samples and at least one medium comprising a magnetically responsive tag or label selected to interact or associate with the target component under conditions permitting selective association of the magnetically responsive tag or label with the target component in each sample; flowing a plurality of aliquots of the plurality of sample mixture in a plurality of flow channels under magnetic field in a direction of a receiving surface configured to receive and capture magnetically labeled or tagged target components; and determining presence or absence of the target component in each of the captured magnetically labeled target components, indicating presence of said target components in one or more of the plurality of samples obtained from different human or non-human subjects.

In some embodiments, the presence or absence of the target component in the labeled components may be achievable by any one or more microscopic, spectroscopic, biological or chemical analyses, or combinations thereof.

In some embodiments, 5 or more, 10 or more, 20 or more, 30 or more samples may be analyzed simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 provides a flowchart depicting a system operation and method according to some embodiments of the invention.

Fig. 2 provides a block diagram of an example system according to some embodiments of the invention. PBS is used in all stages of IM labeling, fixation, priming and recovery.

Fig. 3 provides a flow system piping and instrumentation flow diagram of a system according to the invention.

Figs. 4A-B provide (A) Flowline connection in exploded view. (B) Cross-section assembly view of the tubes array sealing.

Fig. 5 provides schematics of a magnetic isolation system based on a single interpolar gap and the resultant magnetic flux flow.

Figs. 6A-B provide (A) Parallel and extended configurations of the magnetic core. Magnetic flux direction (arrows), isolation material components (I), and conduction material components (C) are labeled. (B) A parallel configuration of the magnetic core with four-capture-band assembly, 3D isometric with a cross-section view (green plane).

Figs. 7A-D provides (A) The magnetic field vector flow, demonstrating a closed circuit of the magnetic flux. (B) The magnetic field magnitude of the magnetic core assembly. (C) Magnification of the magnetic field magnitude at the labeled area in (B). (D) Magnification of the magnetic field magnitude at the labeled area in (C), demonstrating the high magnetic field gradient very close to the interpolar gap.

Fig. 8 illustrates a change in the simulated magnetic field of the four interpolar gaps along the lateral axis on the magnetic core surface.

Figs. 9A-C provides (A) Top view of a coverslip slide rack allowing the installation of 20 slides. (B) Top view of the designed cassette with general dimensions. (C) Cross-section view (A-A) from figure (B) of five O-ring grooves. (D) Cross-section view (B-B) from figure (B) of a single O-ring groove. All dimensions are in mm.

Figs. 10A-C provides (A) The tightening subsystem assembly. (B and C) 3D mechanical FEA simulation results (isometric view) of a simplified tightening subsystem: (B) equivalent (von-Mises) stresses, and (C) total displacement.

DETAILED DESCRIPTION OF EMBODIMENTS

The technology disclosed herein provides a novel design of a high-throughput bio- ferrography-based isolation system that may be used as a medical system for simultaneous analysis of a great number of patient samples. The system design is based on an optimized procedure for bio-ferrographic isolation of target components from various samples obtained from human or non-human subjects, e.g., HWB samples, and takes into account the needs of different practitioners, e.g., system operator, medical doctor, and hospital. The designed system allows an effective isolation of target components in dozens or hundreds of flow channels, suitable for many patients simultaneously. For each patient, only a small amount of the sample (e.g., about 1 mL blood) per channel may be required. The flow system of systems of the invention allows efficient delivery of reagents in a controlled and accurate manner for labeling of target components in the samples. The flow system also promotes automatic priming, isolation, and system recovery procedures. The gradient magnetic core with a plurality of interpolar gaps in a parallel configuration allows running a plurality of slides simultaneously with the most compact dimensional configuration being presented. The novel design of the magnetic core was validated using FEA magnetostatic simulation and calibrated by physical measurements on the Bio-Ferrograph 2100. The results confirmed that the maximal absolute magnetic field magnitude is at the interpolar edges, where the magnitude of the magnetic field decrease sharply as the distance increases from the surface of the interpolar gaps. Interestingly, the maximal magnetic field magnitude exhibited a "bath" type distribution between the four parallel interpolar gaps, where the magnetic field at the interior gaps was slightly lower (by ca. 0.08 T) than at external gaps. The magnetic field magnitude at all interpolar gaps was analyzed according to the used coverslip slide's tolerance thickness integrated into the system. The results show that target components that pass through the magnetic core are subjected to a magnetic field in the range of 1.43 to 1.71 T. The presented magnetic core design was confirmed to produce a suitable magnetic field for an efficient magnetization of the target components. Following the isolation process, the isolated cells can be further counted and identified using advanced artificial intelligence- aided image analysis, and characterized by complementary chemical, biological, and mechanical analyses. Overall, the presented high-throughput bio-ferrography-based isolation system may serve as an important decision-making tool for medical doctors when monitoring cancer patients in a hospital setting. It opens a new route for early diagnosis, prognosis and treatment of cancers, as well as other diseases, for example osteoarthritis.

A general configuration of a system 100 and method of the invention is depicted by way of a flowchart in Fig. 1. As depicted, a sample containing a variety of reagents and a body sample, as defined, are premixed (10) and subsequently labeled (20). In some configurations, sample labeling by immunomagnetic labeling or generally by magnetic labeling may be achievable while preparing the sample. Thus, sample labeling may be carried out in a sample preparation unit or step (10). The magnetic labeling is allowed to form and the system may be primed (30), as disclosed herein. Flowing of samples (including between 1 and 100 or more samples) in the plurality of flow channels, 10 or more or 100 or more flow channels, carries the labeled components present in the sample(s) in a direction of a magnet unit to cause magnetic isolation (40), as explained herein. Following isolation of the magnetically labeled components, they are analyzed to determine presence or absence of the target component which information regarding its presence or absence was desired. The system may thereafter be recovered and reused (60) with another sample(s) from the sample preparation unit (10), which may be processed as before under identical, similar, or different conditions (reagents, temperatures, etc).

A non-limiting configuration of a system design according to the invention is depicted in the block diagram presented in Fig. 2. As depicted, the system is composed of two main subsystems: (1) a controlled flow subsystem (for reagents delivery, priming, and system recovery), and (2) an isolation subsystem.

In the exemplified system, the system is sustained with the following external inputs: (1) tubes filled with samples, such as blood samples, (2) biological and recovery reagents, (3) a slide rack, which in the particular examples comprises 20 slide rack but may contain any number of slides. Thus, in this example, the system output includes 20 slides, which are associated with 20 patients, where each slide has a plurality of channels of isolated CTCs, e.g., 5 channels. The bio-ferrography-based CTCs isolation procedure utilizes several operations with different pieces of equipment: centrifuge, orbital shaker, cooling devices, optical and fluorescence microscope. Specific changes in the isolation protocol can be excluded if they do not result in an apparent change in recovery rate. Hence, procedures that involve centrifuge were excluded from the system requirements and were replaced by primary washing of the patient blood sample. The antibodies and microbeads excess washing step, which follows the cocktail-blood incubation, was excluded too. In contrast to the single-use BF flow system (disposable cassette, priming cups, and syringes), the designed system is reusable. It utilizes high-precision metering pumps, reusable tubes, and manifolds (tube rack, needles, and coverslip slides are disposable). Therefore, a system recovery procedure was developed.

Each of the components (e.g., reagents, number of tubes and slides) shown are exemplary and may be varied or replaced by others.

The flow system is responsible for the delivery of reagents in both accurate and controlled manner. Each stage in the system operation requires its own reagents, as listed in Table 1.

Table 1. Exemplary, non-limiting reagents used in stages of a method or system operation according to the invention.

To meet both the biological procedure and technical requirements, a conceptual design of the flow system was developed, as illustrated by the flow diagram in Fig. 3. This design includes the definition of the flow direction in any pipeline, the intersections, and the locations of the pumps, reservoirs, valves, manifold, and tubes. This is a non limiting design.

The particular design of the flow system was divided into two main phases: (1) accurate portion delivery of the ingredients from the required reservoir tubes into a control volume manifold; and (2) transportation of the ingredients from the control volume manifold into the target tubes. According to this system's flow pattern, the ingredients are prepared and installed by the operator in the designated location in the refrigeration system. Each ingredient (specific ingredients are labeled; these may vary based on the sample desired) is pumped by its corresponding pump (A-D) through a designated disposable needle and tubing to the entry of the control volume manifold (priming). This step facilitates the ability to deliver a precise amount of fluid at each stage of the delivery of the ingredients using a metering pump with a precisely controlled volume (low-flow miniature original equipment manufacturer pump with a dispense rate which may vary, for example being 0-25 mE per revenue). The control volume manifold is for example equipped with one-way check valves in order to ensure that the inlet lines remain primed and that the pump dispense accuracy will not decrease due to pressure changes in the control volume manifold. Following the priming stage, the delivery flow system is ready for use. According to each process, the appropriate pump may deliver a precise required volume of fluid into the control volume manifold, then delivered by a nitrogen gas pressure flow, which 'pushes' the fluid through tubes in the circular manifold into the corresponding test tubes. The gas flow rate may be controlled via a mass flow controller which enables a stable flow rate with responsive control of the process flow rates and pressures with real-time readings. This delivery concept enables the delivery of the reagents with minimal residues in the tubes and minimal waste. In order to reduce the inlet pressure of the mass flow-up controller to a workable level (e.g., maximum 1 bar), a two-stage gas pressure regulator may be integrated into the flow system. It may provide a constant delivery pressure, with no need for periodic readjustment.

The reusable tubes that may be integrated into the reagent delivery subsystem design are medical grade PVC (DEHP-free), with an inner diameter (ID) of, for example, 1 mm and an outer diameter (OD) of for example 2.1 mm. This tubing material may be selected due to its high chemical resistance against the chemicals in use. An essential subsystem requirement is that the flow system facilitates full system recovery for subsequent system use. However, the cleaning of the outer tube surface area that is in contact with blood is highly challenging, time demanding, and expansive. Hence, the use of disposable needles and tubes was chosen. In this subsystem, contamination of the outer surface of both the tubes that pump the reagents (Fig. 3) and the tube array outlet (Fig. 4) might occur. The needles are designed to be easily mounted and replaced in the tube mounting plate.

The sample tube rack may be assembled of e.g., 100 tubes; therefore, in such embodiments, manual disassembly of 100 needles is not practical. The solution concept involves any design which in some cases may be of 10 x 10 disposable tubes array, which may be efficient. This disposable-tubes array may be immersed in the blood tubes on one side (Fig. 4A), and on the other side is connected to a negative array of tubes connector, which is connected to the outlet tube lines of a 1:100 manifold (Fig. 4A). This connection fitting between the arrays facilitates proper flow in two directions: the first is the delivery of reagents into the blood tubes, while the second is the delivery of the blood samples from the tube through the isolation system in the CTCs isolation process (Fig. 3). In total, each of the connection fitting arrays is assembled of 200 tube fittings - 100 tubes for the reagents delivery into the blood samples, and another 100 tubes that deliver the blood through the isolation system (Fig. 4). At the end of the outlets, a flat, gasket (e.g., 50 Shore A hardness) may be glued. Following the connection of the two, the gasket may be squeezed between the mating surfaces as they are clamped together (Fig. 4B). This facilitates proper flow for both the reagents to the blood test tubes and from the blood tubes towards the isolation system, with no dead volume for blood clotting. The loading force that is required to achieve proper sealing of the gasket and to prevent leakage of the tube fitting was experimentally determined via a compression test. The tested gasket was made of EPDM, 50 Shore A hardness, width = 1 mm, ID = 1 mm, OD = 3.9 mm. Compression test results present that a load of 6.2 N is needed for 20% deflection of the gasket seal. In total, the tubes array fitting is assembled with 200 gasket seals; thus, a compressive force of 1,240 N in total is required to facilitate a proper seal between the tubes array fitting and the tubes array. This is established by an operator manual tightening of designated four socket head cap screws, which are located at each corner of the tubes array fitting.

Following a pre-isolation stage, which involves IM labeling and incubation, a priming stage may be included. The isolation flow lines are filled with PBS (or other buffer compatible with biological samples) to prevent air bubbles formed in the flow lines and flow channels. The formation of air bubbles might significantly reduce the recovery rate due to the change in the flow regime and possible blood encapsulation in the formed air bubbles. Since the designed ingredients flow subsystem is integrated with buffer reservoir and an assigned metering pump, the concentration system flow lines priming can be easily performed without adding an additional buffer reservoir. Using a three-way controlled valve system (valve 1, path I-II; valve 3, path I-II), made of Series 1 three-way diaphragm polytetrafluoroethylene (PTFE) valve, through which the buffer is pumped, and a 1:100 manifold, the buffer shall flood the cassette flow channels and fill the blood sample tubes to a final volume of 2 mL. Following the priming stage and using the assigned buffer pump, pipetting and mixing of the buffer-blood samples are executed to improve the sample homogeneity.

Following the pre-isolation stage, which may include IM labeling, incubation and priming, the system is ready for the CTCs isolation stage. Since one of the flow system requirements is to facilitate continuous flow through the flow lines, a diaphragm pump operated in the vacuum mode may be utilized. It allows continuous flow in the system’s flow lines when integrated with a liquid trap design, which acts as a pulse flow dumper, at the specified flow rate to meet the specified requirement. In addition, the liquid trap may allow meeting the requirement for non -contamination of the flow matter since the flow matter does not flow through the pump (Fig. 3).

The flow pattern in the isolation stage is as follows. The diaphragm pump (pump E) draws existing air in the liquid trap to a specific, constant and negative pressure value. The pump cannot lock pressure; therefore, a gas dual pressure controller (DPC, PCD series, -15 to 0 psi) was integrated to maintain a constant and stable pressure during the whole isolation stage. The defined pressure value in the liquid trap is the main parameter that determines the accepted flow rate through the isolation flow system’s lines. The liquid trap outlet may be connected to a liquid flow controller (LFC), which controls the concentration flow rate at a precise and constant value. When the three-way controlled valve (valve 3, path I-III, Fig. 3) is opened, the 100 blood samples are drawn from the tube rack through the tube array fitting and the magnetic core flow channels at a constant flow rate of 0.021 mL/min. As the samples pass through the magnetic core, the samples flow through the controlled valve and are gathered to a single flow line via a 100:1 manifold, and are poured into the liquid trap. The LFC stops the isolation process after pumping 1.9 mL of sample (which takes ca. 90 min), to avoid the entrance of air into the flow lines.

The fixation stage includes delivery of 4% paraformaldehyde (PFA) into the flow channels, 20 min incubation, and washing of the whole flow system with buffer. This is done using the reagents delivery flow subsystem and the isolation flow subsystem, as described above. The LFC stops the isolation process once the PFA fills the flow channels (the sample tubes are filled with PFA up to the tube array fitting tubes inlet) in order to avoid entrance of air into the flow lines.

Once the isolation process is completed, the system is ready for the recovery stage. The slides are disassembled and replaced with a new recovery set slide case assembled with 20 new slides. In order to prepare the flow system for the recovery stage, the manifold and the connected flow lines are first washed with ethanol, using pump A and the nitrogen flow system through valve 2 (path I-II). Next, reservoirs B-D are disassembled, followed by drainage of pumps B-D of remaining residues in the connecting flow lines into the control volume manifold. Using the nitrogen delivery system and valve 2 (path I-III), the remaining residues are delivered to the liquid trap. At this stage, the disassembled reservoir needles are replaced with new ones by installing three reservoirs (B-D) filled with bleach (chemical calcium hypochlorite solution or other suitable disinfectant). The sample tube rack and the disposable tube array fitting are replaced with new ones to prevent flow line contamination during the recovery stage.

The recovery stage itself comprises two wash steps: bleach and ethanol. It starts by washing the flow lines with bleach, by drawing bleach to the tube rack (pumps B-D, valve 2, path TII), and by nitrogen flow, followed by drawing of the bleach through the magnetic core using pump E and valve 3 (path I-III) to the liquid trap. The system priming flow lines are washed as well, using pump B, through valve 1 (path I- II) and valve 3 (path II- III) into the liquid trap. Before proceeding to the second stage, reservoirs B-D are replaced with three reservoirs filled with ethanol. Pumps A-D draw ethanol to the tube rack and the magnetic core, as described in stage 1. In addition, using pump B and valve 3 (path II- III), ethanol is drawn through the priming flow lines to the liquid trap.

When both the isolation and recovery stages are completed, the waste is evacuated from the liquid trap through valve 4 (two-way diaphragm PTFE valve) and a drainage pump F to an external drainage collection vessel. In order to ensure that the blood samples waste is not drawn into the diaphragm pump, a magnetic liquid level switch was integrated into the system design.

The magnetic isolation system of the Bio-Ferrograph 2100 forms a high gradient magnetic field which is generated by a single interpolar gap of a permanent magnet core. The unique assembly of the SrFe permanent magnets, the magnetic isolator prism (pole shim) made of Aluminum 1060 alloy, and the magnetic pole piece conductors made of AISI 1010 low-carbon steel create a close circuit of the magnetic flux (Fig. 5). The width of the interpolar gap defines the intensity of the magnetic flux at the gap edges. With a proper width a high magnetic flux gradient is formed, which enables maximal magnetic field at both edges (in opposite directions) of the interpolar gap. The magnetic field flux absolute values are maximal at the edges of the interpolar gap at the upper surface of the assembly and decreases sharply as the distance from the interpolar gap surface increases. As part of the system design requirements, the CTCs isolation system is able to analyze 100 or more flow channels simultaneously.

Two configurations are proposed, Fig. 6A - extended configuration and parallel configuration. The extended configuration is based on extension of the interpolar gap length. In this approach, there is no actual difference in the magnetic field value at the interpolar gap in comparison to the magnetic core assembly of the Bio-Ferrograph 2100 due to the plane symmetry of the magnetic core. The main drawback of this configuration is the resulted large physical dimensions of the magnetic core assembly. The parallel configuration approach is based on a parallel assembly of several interpolar gaps parallel to each other as shown in Fig. 6B.

FEA magneto- static analysis (Maxwell 16.0, ANSYS, Inc., Canonsburg, PA, USA) was utilized to validate that the formed magnetic field magnitude established at the four-interpolar-gap magnetic core is consistent and satisfying. The FEA results showed good correlation with accordance to the measured magnetic field. The magneto-static simulation results show that the four-capture-band parallel configuration magnetic core creates a close circuit of the magnetic flux, enabling the appearance of a maximal magnetic field gradient at the interpolar gap (Fig. 7A). As shown in Fig. 7A-C, the magnetic field is maximal at the edges of the interpolar gap at the magnetic core's upper surface, and decreases sharply as the distance increases from the interpolar gap surface.

Fig. 7 demonstrates the magnetic field gradient around the interaction interface of the pole piece, the insulator material, and the air region. Though the maximal value of the magnetic field magnitude at the edges of the interpolar gap is allegedly 5.464 T, this is not the apparent magnetic field that the CTCs passing through the flow channel sense. One should consider that the maximal magnetic field magnitude is located at the sharp corner of the gaps, where a singularity of the magnetic field solution occurs. Hence, the actual magnetic field magnitude the CTCs sense while passing through the flow channel should be considered across the actual thickness of the glass coverslip. Fig. 8 shows the change of the magnetic field along the lateral axis at 0.15 mm from the magnet’s top plane.

The influence of coverslip thickness on the simulated magnetic field with a significant distance of the singularity points was analyzed (Table 2). The manufacture tolerance of coverslip glass slide # 0 (0.08-0.15 mm) is considered. As expected, due to the high magnetic field gradient at the interpolar gap edge, a significant decrease in the magnetic field occurs with the increase of the slide thickness. An interesting phenomenon arises, resulting from the parallel magnetic core configuration - a "bath" type distribution of the maximal magnetic field magnitude at the interpolar gap edges (corresponds to the results presented in Table 2). This type of behavior was expected due to the plane symmetry of the problem relative to the y-axis and due to the change in distance of the interior gaps (i.e. gaps 2 & 3) and the external gaps (i.e. gaps 1 & 4) relative to the magnetic cores that are placed on both sides of the magnetic core assembly. In spite of 9he described phenomenon the difference in the simulated magnetic field between the interior interpolar gap edges 2 & 3 and the exterior gap edges 1 & 4 is relatively negligible (-0.07 T). Thus, the suggested design is expected to be suitable for the magnetization of CTCs.

Table 2. The influence of slide thickness on the maximal magnetic field magnitude at the interpolar gap edges (L and R correspond to the left and right edges of the interpolar gap, respectively).

The obtained FEA magneto- static analysis results of the parallel configuration magnetic core were also compared with the single interpolar gap magnetic core configuration. A calculated magnetic field of 1.67 T was measured at the interpolar edges at average coverslip #0 thickness of 0.11 mm. The field value was extrapolated from the field measurements taken as a function of distance from the interpolar gap. This report is consistent with the FEA of the four-band parallel magnetic core configuration (Table 2). The above discussion shows that the suggested magnetic core with a parallel configuration is applicable for the isolation of CTCs.

The novel system of the invention is designed to have a reusable cassette system that facilitates many, e.g., 100 or more, flow channels. The cassette-magnet subsystem design was divided into two parts: (1) Cassette and slide assembly, and (2) cassette- magnet tightening.

The conventional Bio-Ferrograph 2100 uses a disposable cassette, which is attached to a designated silicone gasket and a single coverslip slide. This type of assembly forms five individual flow channels after installation onto the magnetic core. The cassette installation is facilitated using two latches, which generate a force of approximately 250 N on each side of the cassette in order to prevent leakage. In order to facilitate 100 reusable flow channels, a new concept of cassette and slide assembly was designed. The cassette- slide assembly design may allow the operator to install 20 slides (Fig. 9A) while establishing 100 flow channels quickly and conveniently in a precise location over the magnetic core. The installation of the slides is done by the operator by inserting the slides into a designated slot on the magnet surface. The flow channels are formed by tightening permanent O-ring seals, which are installed in curved grooves at the backside of the cassette, towards the coverslip slide (Fig. 9B). Tubes are permanently connected to the cassette's backside, so the samples are able to enter the flow channels and exit through the outlet Tygon tubes to the drain (Fig. 9C-D). The permanent O-ring seal is a key component in the tightening system and the establishment of the flow channel. As shown, the magnetic field decreases sharply as the distance increases from the interpolar gap surface; therefore, the flow channel's minimal thickness is essential for optimal magnetization of the IM labeled CTCs. The dimensions of the individual flow channel were experimentally determined (see Supplementary Material).

The flow channel is formed by tightening permanent O-ring seals placed in a curved groove at the cassette's front side towards the coverslip slide and the magnetic core. EPDM O-ring seals with 50 Shore A hardness were selected due to their low hardness and good chemical resistance. The load required to facilitate a leakage-free flow channel of a single O-ring was experimentally found using a compression test. The compression test revealed that for a single O-ring deformation of 0.35 mm, a load of 16.5 N is required (at 20% deformation of the initial O-ring width). Although the load necessary for tightening of a single O-ring is easy to achieve, the need for 100 flow channels requires the generation of 1,650 N to promote a leakage-free flow through the flow channels. The cassette-magnetic core tightening system was thus designed to meet the following requirements: (1) Facilitate continuous axial load (for several hours); (2) Fully automated and controllable; (3) Foad-based control; (4) High accuracy and repeatability; (5) Facilitate dispersed load over the cassette; (6) Minimal maintenance; (7) Minimal size and weight; (8) Horizontal installation option; (9) Equal dispersion of the load over all 100 flow chambers. An electrical -based linear actuator meets the abovementioned requirements, thus it was selected as most suitable for tightening. The isolation of the loads applied by the tightening system on the outer system enclosure is essential to prevent its mechanical failure. An isolation frame was designed to absorb the subjected load and eliminate the applied stress on the enclosure unit (Fig. 10A). 3D FEA structural static analysis was performed to validate that the designed frame absorbs the applied stresses with minimal displacements of its structural elements (Fig. lOB-C). ANSYS Structural software was used to solve the linear static structural analysis. The 3D problem was simplified and analyzed. Structural elements of the tightening assembly which do not affect the solution were eliminated from the FEA model, Fig. 10A-C. Material definition was done according to the material selection design. For analysis settings, boundary conditions of fixed support were defined: top and bottom surfaces of the back plate (magnetic core side) and displacement support ( u_x = u_z = 0, u = free ). To be on the safe side, a safety factor was added to the estimated applied load (4,000 N dispersed on the corresponding surface area perpendicular to the simplified cassette). This simulates the subjected compression load on the frame.

The FEA simulation confirmed that all inspected frame structure elements show considerably small displacements. Furthermore, the resulting stresses were, without exception, substantially lower than the yield strength specification with respect to the examined structure element. This implies a linear elastic material behavior (i.e. without nonlinear material plasticity behavior). Hence, after tightening, the load discharges and the frame elements will recover to their original form. This ensures a proper, long service life operation of an independent cassette-magnetic core tightening subsystem, with minimal effect on the system's enclosure unit.

Claims

CLAIMS:

1. A high-throughput magnetic isolation system comprising a plurality of flow channels, wherein the plurality of flow channels includes 10 or more flow channels and a magnetic core arranged in a parallel configuration, wherein the plurality of flow channels is configured to simultaneously receive one or more samples within at least one of the plurality of flow channels, wherein at least one target component in said one or more samples is configured to be separated magnetically.

2. A bio-ferrography-based magnetic isolation system, the system comprising: a plurality of flow channels, wherein the plurality of flow channels includes more than 10 flow channels and a magnetic unit in a parallel configuration; wherein the plurality of flow channels is configured to simultaneously receive one or more samples within the plurality of flow channels, and wherein at least one target component in said one or more samples is configured to be separated magnetically.

3. A bio-ferrography-based magnetic isolation system, the system comprising: a magnetic unit; and a plurality of more than 10 flow channels, wherein each of the flow channels is configured to receive an amount of a sample comprising a magnetically labeled target component and allow flow of said amount of the sample in a direction of a receiving surface; wherein said receiving surface is configured to receive and associate or configured to capture and immobilize said magnetically labeled sample component.

4. A bio-ferrography-based magnetic isolation system, the system comprising a plurality of sampling units, each being configured to receive same or different sample; a plurality of more than 10 flow channels, wherein each of the flow channels is configured to simultaneously receive an amount of a sample from said plurality of sampling units; a magnetic unit; and a receiving surface configured to receive isolated magnetically labeled sample components.

5. The system according to any one of the preceding claims for analyzing, evaluating, diagnosing or determining presence of at least one target component in a sample obtained from a human or animal subject.

6. The system according to any one of the preceding claims, comprising a sample preparation unit comprising individually addressable one or more reagent reservoirs for the sample and including sample labeling for selective magnetic labeling of a target component present or suspected of being present in the sample.

7. The system according to any one of the preceding claims, comprising a mixing unit.

8. The system according to any one of the preceding claims, comprising one or more sample units.

9. The system according to claim 8, wherein the number of sample units is equal to the number of flow channels.

10. The system according to claim 8, wherein the number of sample units is smaller than the number of flow channels, such that at least one sub-plurality of flow channels is configured to receive and flow an amount of a same sample.

11. The system according to any one of the preceding claims, wherein a ratio of samples to flow channels (samples: channels) is between 1:1 and 1:100.

12. The system according to any one of claims 8 to 11, wherein the one or more sample units are arranged as an array of sample chambers or containers.

13. The system according to any one of the preceding claims, comprising one or more fluid distributers configured to distribute predetermined amounts of the one or more samples into the one or more sample units.

14. The system according to any one of the preceding claims, wherein the system comprises between 10 and 100 flow channels.

15. The system according to any one of the preceding claims, wherein the system comprises 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more flow channels.

16. The system according to any one of the preceding claims, wherein the system comprises between 50 and 150 flow channels or 100 or more flow channels.

17. The system according to any one of the preceding claims, wherein the system comprises 100 or more flow channels arranged to receive between 1 and 50 different samples.

18. The system according to any one of the preceding claims, wherein the system comprises 100 or more flow channels in an arrangement configured to receive between 10 and 50 different samples, or between 1 and 100 different samples, or between 20 and 50 different samples.

19. The system according to any one of the preceding claims, wherein the magnetic unit includes a magnet assembly comprising a magnetic core.

20. The system according to claim 19, wherein the magnetic unit includes a magnetic field source, a magnetic conductor, and one or more magnetic isolators.

21. The system according to claim 19, wherein the magnet unit is configured to form a concentrated magnetic field generated by one or a plurality of interpolar gaps provided in one or a plurality of structures provided on the surface of the magnet core.

22. The system according to claim 21, wherein each of the interpolar gaps is formed of a magnetic isolator material.

23. The system according to claim 20, wherein each of the one or more magnetic isolators defines an interpolar gap.

24. The system according to any one of claims 19 to 23, wherein a width-to-depth ratio of each of the interpolar gaps defines a magnetic flux intensity at the gap edges.

25. The system according to any one of the preceding claims, wherein the receiving surface is a glass or a polymer surface defining a plurality of surface regions, wherein each region is designed to receive thereon isolated target component(s) from a different flow channel.

26. The system according to claim 25, wherein the number of surface regions is equal to the number of flow channels such that each flow channel is configured to deposit the magnetically labeled target components onto a predesignated region on the receiving surface.

27. The system according to any one of claims 1 to 24, wherein the receiving surface is a plurality of individually and separately addressable receiving surfaces, wherein each is designed to receive thereon magnetically labeled target component(s) from a different flow channel.

28. The system according to any one of the preceding claims, wherein each of the receiving surfaces is an individually and separately addressable curved groove feature formed on a receiving plate, wherein the groove inner surface defining a receiving surface.

29. The system according to claim 28, wherein each of the individually and separately addressable curved groove features is configured as a flow chamber, provided with an input port and an output port configured to flow the sample through the chamber, permitting settling or deposition of the magnetically labeled sample components on the receiving surface.

30. The system according to any one of the preceding claims, wherein the sample is a liquid body sample obtained from a human or an animal subject, containing or suspected of containing at least one target component which presence and/or amount provides an indication of a disease state or which presence and/or amount provides a response to a desired qualitative or quantitative inquiry.

31. The system according to claim 30, wherein the sample contains cells, tissue fragments and/or solid material debris.

32. The system according to any one of the preceding claims, wherein the sample is a blood sample, a saliva sample, a stool sample, a tissue sample, a urine sample, sweat sample, a CSF sample, a synovial fluid sample, or a maternity sample.

33. The system according to any one of the preceding claims, wherein the sample is whole blood sample.

34. The system according to any one of the preceding claims, wherein the magnetically separable components or target components are labeled or tagged with a magnetic responsive material.

35. The system according to claim 34, wherein the magnetically responsive material is magnetic particles.

36. The system according to any one of the preceding claims, wherein the system is configured to analyze cells, tissue fragments and solid material particles in more than 5 patient samples, simultaneously.

37. The system according to any one of the preceding claims, configured as a microfluidic device.

38. A method comprising: providing a system comprising plurality of more than 10 flow channels; magnetically isolating at least one target component present in one or more samples by flowing said one or more samples in the plurality of flow channels; wherein each of the plurality of flow channels is configured to receive the one or more samples, wherein at least one target component in said one or more samples is configured to be separated magnetically.

39. The method according to claim 38, comprising providing a system comprising plurality of flow channels, wherein the plurality of flow channels includes more than 10 flow channels and a parallel magnetic unit; wherein the plurality of flow channels is configured to receive one or more samples within the plurality of flow channels, wherein at least one target component in said one or more samples is configured to be separated magnetically; magnetically separating magnetically labeled target components present in said one or more samples.

40. The method according to claim 38 or 39, comprising analyzing the one or more samples from 20 or more patients simultaneously.

41. The method according to any one of claims 38 to 40, comprising sample preparation, labeling and staining, magnetic isolation, and system recovery.

42. The method according to any one of claims 38 to 41, for detecting different types of diseases, components of implants or other medical devices suggesting degradation of the implants or the devices, efficacy of drug treatments, contamination by microorganisms.

43. A method for simultaneously determining presence of a target component in a plurality of samples obtained from different human or non-human subjects, the method comprising preparing a plurality of sample mixtures, each containing an amount of a different sample from said plurality of samples and at least one medium comprising a magnetically responsive tag or label selected to interact or associate with the target component under conditions permitting selective association of the magnetically responsive tag or label with the target component in each sample; flowing a plurality of aliquots of the plurality of sample mixture in a plurality of flow channels under magnetic field in a direction of a receiving surface configured to receive and capture magnetically labeled or tagged target components; and determining presence or absence of the target component in each of the magnetically labeled or tagged target components.

44. The method according to claim 43, wherein the presence or absence of the target component is achievable by any one or more microscopic, spectroscopic, biological or chemical analyses.

45. The isolation system according to any one of claims 1 to 4, the system comprising 100 or more flow channels, and a magnetic unit, wherein each of the flow channels is configured to receive a sample, wherein at least one target component in said sample is configured to be separated magnetically.

46. The isolation system according to claim 45, the system comprising:

100 or more flow channels, and a magnetic unit; wherein the flow channels are configured to separately and simultaneously receive 10 or more samples within the flow channels, and wherein at least one target component in said 10 or more samples is configured to be separated magnetically.

47. The isolation system according to claim 45, the system comprising: a magnetic unit; and

100 or more flow channels, wherein each of the flow channels is configured to receive an amount of a sample comprising a magnetically labeled sample component and allow flow of said amount of the sample in a direction of a receiving surface; wherein said receiving surface is configured to capture and immobilize said magnetically labeled sample component.

48. The isolation system according to any one of claims 45 to 47, for isolating magnetically labeled sample components from 20 or more samples simultaneously.

49. The isolation system according to any one of claims 45 to 48, for determining presence or absence of a disease state, severity of a disease state, successful treatment of a disease state, recurrence of a disease state, improvement in a disease state, slowing down of disease progression, slowing down of irreversible damage caused in a progressive chronic stage of a disease, and combination of the aforementioned.

50. The isolation system according to any one of claims 45 to 49, for determining a disease state, wherein the disease is cancer, a proliferative disease, or osteoarthritis (OA).