SE2100130A1 - System for integrated automated isolation and analysis of biological vesicles and associated molecules - Google Patents

Abstract

Disclosed herein are methods, instruments and systems for automated integrated isolation of biological microvesicles such as exosomes and analysis of molecular markers carried by these vesicles. The systems comprise reagents for isolation and analysis processing and instrument comprising a core assembly of an integrated isolation and analysis module and a plurality of additional supportive modules. The instrument is totally automated and controlled by embedded instrumental as well as external computer software’s. The isolation and analysis performed apply dedicated analytical methods. Three main groups of methods are implemented in the system presented herein. In one of methods the molecular marker at the surface of the microvesicle is analyzed. The other two types of methods require the microparticles to be lysed to release the intra vesicular molecular markers. In these methods the released intra vesicular molecular protein markers and the intra-vesicular RNA molecular markers are analyzed. The system is applicable for analysis of biological microvesicles such as exosomes in plasma, urine, semen, saliva or breast milk and in other body-fluids, in diagnosis of cancer, brain diseases, chronic inflammation diseases and infection.

Description

1. Field of the lnvention The invention relates to the field of cell biology and to the isolation of cell membrane based micro vesicular structures found in body fluids. The invention also relates to analysis of the membrane-bound protein molecules and the internal free protein as well as nucleic acid molecules enclosed in these vesicles. Microvesicles are produced by and released from living cells and are distributed into most body fluids (see reference AR1, AR2). The invention comprises an integrated totally automated research and routine laboratory analysis system, which includes implemented methods for isolation and analysis of biological membrane based microparticles. The main object for isolation and analysis using the invention is exosomes including all types of intra and extra cellular microvesicles, also including viruses and other biological microvesicles with similar structures. 2. Discussion of the Art Exosomes are a diverse collection of small membrane vesicles with a size distribution of 30 -150 nm. The vesicles are produced in all living cells (AR3, AR4, AR5 and AR6). These vesicles assembled in the cells in a process resulting in an enclosure consisting of a double lipid layer membrane similar to the mother cell membrane (AR1, AR2). The exosome membrane is mainly composed of phospholipids and a number of membrane associated proteins. The exosome surface membrane proteins are related to and have the same Characteristics as the mother cell membrane proteins. This similarity is applicable to use in analysis of isolated exosomes to define the source of the vesicles. ln the internal luminal liquid enclosed by the microvesicle membrane, an extensive number of free soluble molecules of different types are found. Examples of these are proteins and nucleic acids (AR7).

During the last decades cell biology research has shown that the extracellular microvesicles such as exosomes have crucial functions in biology as a key factor in nano-shuttles for transport and delivery of components and information from one location and cell type to near or distant locations and other cell types (AR8 and AR9). lt has also been demonstrated that exosomes are involved in a wide variety of friendly physiological processes such as cardiac, immune system, brain, steam cell functions etc. (AR1O and AR11). Exosomes can on the other hand turn into devastating foes, taking part in pathological processes such as cancer, brain diseases and infections (AR12 and AR13). The ubiquitous presence of circulating microvesicles in body fluids, their association with a broad range of physiological processes, as well as their elevated numbers in human disease, suggests that microvesicles potentially can serve as tools in molecular medicine, to perform disease diagnostics (AR12 and AR14), and to improve therapeutic targeting during treatment (AR15).

A significant technical challenge in current research involving exosomes and other types of microvesicles, is the task to efficiently isolate the microvesicles from various 2 sources. Current methodologies to isolate secreted microvesicles, including but not limited to exosomes, are constrained by technical limitations and other drawbacks. The applied methodologies are labor intensive, time-consuming, costly, and can be unreliable (AR16, AR17 and AR18). ln the present invention the listed drawbacks are resolved, using automated and integrated isolation and analysis in instrumentaily based analytical systems. The systems offer all required functionality for effective microvesicle isolation and analytical processing. The system concept is adaptable to both research investigation and routine diagnostic use. The strategy of the system design is to make available logistics and handling similar to what is common in the present established automated systems used in clinical diagnostics (AR19).

Automated analyzers are well-known for example in the field of clinical chemistry and in the field of immunochemistry. These analyzers are typically configured to perform analysis for a collection of samples according to specific assay methods unique for the particular clinical chemistry and the targeted molecular types (AR20). These analyzers are designed to measure single molecules in a free state, homogenously distributed in a sample (AR21). The systems and instruments defined herein are designed to specifically isolate and analyze complex objects such as microvesicles in samples, where the bulk of objects such as surrounding free soluble molecules are initially discarded (AR22 and AR23).

Clinical chemistry is a medical discipline with the objective to define the pathological state of patients by analyzing body fluids. The analyses are performed using different types of analytical technologies to qualitatively or quantitatively measure the amount and character of various components in mostly blood plasma or urine (AR22). To improve the quality, performance and cost-efficiency of the analyses, these are performed in semi or totally automated instruments (AR22). The instruments are supervised using computer hardware and software, securing the quality and information handling of the analyses. ln modern routine clinical chemistry, analytical processing, handling of the analyses is performed in fine-tuned integrated systems using reagents adapted to the automated instrument processing. The analysis process and the instruments are handled using administrative software in external computers as well as instrument embedded processing software (AR21).

The clinical chemistry discipline comprises an extensive number of disease related marker molecules and to these adapted analytical methods and systems. A significant part of these disease markers are free molecules or molecules associated into larger conglomerate structures (AR18). To identify and quantify these targets a number of different analytical methods and processes are applied. ln the early phase of the evolution of the clinical chemistry discipline, methods using pH, photometry, fluorimetry, luminescence etc. were used to direct qualitative and quantitative measure metabolites, ions, enzymes and non-specific proteins. Later on, the methodology was improved by the development of highly specific immunology-based assays. The immunoassay technology significantly enhanced the analytical sensitivity and a large number of high performance automated immunoassay systems were introduced during the 1980 and 90-ties (AR23).

During the 90-ies, at the onset of the development of analytical methods based on the polymerase chain reaction (PCR) and other molecular biology related technologies, commercial systems for analyzing DNA and RNA sequence-based diagnostics were launched. The general molecular biology analysis processes 3 include extraction and amplification of DNA. ln classical PCR, thermal cycling is required. To the amplification process alternatives have recently been added methods for isothermal amplification, which will simplify the design of automated instruments. ln the following analysis the sequence of the amplified DNA is determined using different methods. To analyze RNA sequences, the RNA molecules are first converted to the relevant DNA sequences, which are thereafter analyzed using DNA-probe methodologies.

During the last decades, it has been introduced a new line of diagnostic methods and system concepts offering multiplexed analysis. These methods are based on principles enabling simultaneous detection of large number different analyte molecules in the same reaction incubate. The principle makes the design of compact and effective multi-parameter instrument possible. This multi-analysis process increases significantly the efficiency of the analysis in comparison to the single analyte measurement performed in earlier system concepts. The methods and systems for multiplexed analyses comprise both specific protein analysis and detection of specific sequences in DNA and RNA (AR21, AR23, AR24 and AR25).

To improve the performance, quality and cost efficiency of clinical chemistry, a wide range of automated systems are commercially available and marketed together with reagent kits and supervising software (AR22 and AR23). The required collection of products has been defined as a clinical diagnostic systems. Such systems are delivered by suppliers such as Abbott Laboratories, Thermo Fisher Scientific, Roche Diagnostics, Siemens AG, Danaher, Beckman-Coulter, Sysmex and Ortho-Clinical Diagnostics (AR24 and AR25). Most of these systems and their instruments were initially designed during the previous millennia and apply established common basic technologies as well as design concepts. ln these instruments a number of general required modules can be recognized. The main function is a reaction chamber, in which chemistry, enzyme, immunological and other type reactions is run. Supporting the reaction module are modules for sample and reagent storage, pipetting, washing, and signal measurement etc. Other required functionalities are hydraulic deliveries and waste handling. The instrument includes electronics hardware and computer processing, which for example comprises operator interface and network communication. To run the assay process the instruments are controlled by process software (AR23, AR24 and AR25).

Exosomes are effectively applied in cellular communication and transformation processes. Exosomes are involved both in health and in sickness. Especially critical and devastating is exosomes in cancer and brain diseases, in which the vesicles interact with cellular structures and processes to effectively spread the disease. Exosomes are able to change the structures and processes in target cells and even in whole organs, both local and distant body environments. This enables diseases to evolve and spread faster. Exosome is specifically loaded with structural protein molecules participating in the organ environment transformation. Also, the exosome contains messenger and micro RNA structures, aimed to be involved in protein synthesis and regulatory processes in target cells. Exosomes are of these reasons, highly interesting in the development of new concepts for early and highly sensitive diagnosis of many diseases. ln the required diagnostic methods, exosomes from plasma and other body fluids are isolated and analyzed. The diagnostic will be based on molecular markers carried by isolated exosomes. These "liquid biopsy" based 4 concepts have been postulated to be the future tool for early diagnosis of diseases such as cancer and brain diseases (AR26).

To make cost-effective exosome diagnostics a reality, it is required highly efficient instrument based automated systems. These systems have to be able to specifically isolate exosomes and analyze exosome molecular markers. Exosomes can be isolated using methods applying basic principles used in immunoassays. ln these methods, the immunological specificity in antibodies for binding to surface proteins located on the outer side of the exosomes are utilized. Potentially this principle offers the possibility to isolate exosomes from specific cell types (AR27). Even if there are limitations in the selectivity of the isolation process, methods to measure the number of exosomes from a specific cell in a bulk of isolated exosomes are a possibility. This application requires assay processes enabling the detection of the different surface antigens at the intact exosome. Methods for exosome isolation use antibodies specific for the protein or glycoprotein protruding out from the surface of the exosome. ln analysis of the cargo internally carried by the exosomes, methods for lysis of the vesicle are applied. The exosomes are lysed, using a reagent containing detergents, buffer ions and other required components. ln the resulting lysate the exosome is decomposed and the liberated cargo proteins and RNA's will be available as free molecules in a buffer solution. ln the following analyses, immunoassay methods are used for the protein measurements. In RNA's analyses, either direct solid phase molecular probe assays of the RNA or analyzed as a DNA transcript after an initial transcription of RNA to DNA. ln this case is the RNA-DNA transcription performed, using the enzyme Reverse Transcriptase. The obtained DNA is there after amplified using PCR methods and the sequences are analyzed applying common molecular biology probe techniques.

Current available methods to isolate and analyse microvesicles such as exosomes are limited by major obstacles like contamination from other particles of the same size, aggregation of soluble proteins (AR28). Typically, isolation of exosomes is based on physical parameters (size, density, charge etc.) or immunocapture. The system defined herein uses immunocapture (IC) applying magnetic beads, which results in process characterized of a high degree of specificity. Also IC techniques make extensive washing possible. This minimizes the impact of contamination of soluble bulk plasma proteins and the isolation step delivers pure exosomes. A number of companies market reagents for exosome investigations employing different technologies (AR29). Thermo Fisher Scientific (AR30, PR1) and Miltenyi (AR31, PR2) offer instruments that are used for exosome isolation in research laboratories. Since the instruments are only designed for isolation, there is no integrated analysis functionality available. Besides these two examples of automated systems, there is at present no other product in the market offering the functionally automation found in the system defined herein. This means, that there at present are no automated systems with integrated isolation and analysis functionalities for microvesicle isolation and analysis in the market. A potential automated procedure alternative is applications, developed by users applying xyz-liquid handling robotics such as platforms from Hamilton, Tecan, Eppendorf and others. These liquid handling platforms can be supplemented with the required modules from other producers of laboratory equipment, to set up an arrangement able to perform both isolation and analyses. This kind of process logistic solutions are not common applied in the design of highly compact integrated isolation and analysis systems, in the context outlined herein. Especially, the concepts are lacking optimized reaction Chambers, which are crucial modules in the analyzers offering high performance required in routine diagnostic system (AR22, AR24 and AR25).

Besides these automated alternatives for exosome isolations there are an extensive numbers of manual methods for microvesicle isolation, which for example have been disclosed in patents (PR 3 - PR19). A number of manual methods including both isolation of exosome and following analysis of exosome surface markers have also been disclosed (PR2O - PR28). There are also an extensive number of methods disclosed for analysis of exosome markers enclosed in the vesicle as well as concepts for exosome based disease diagnosis in filed patent applications and patents (PR29 - PR49). These areas and objects are outside of the field disclosed in the invention defined herein, since the patents disclose no integration of automated isolation and analysis.

TERMINOLOGY As used herein, the term "sample" means the fluid having one or more substances comprising the chemical, biochemical or biological properties targeted for isolation and analytical processing. Samples typically subjected to isolation and analytical processing include mixtures of substances, which are present in a biological fluid, for such as blood, serum, plasma, urine, cerebrospinal fluid etc. Also relevant samples are for example partially purified lysate, intact cells, subcellular fractions, cell culture or other biological fluids.

As used herein, the expression "aliquot" is a portion of a sample and reagent used for isolation or analysis. An aliquot of a sample is for example added to an isolation or analysis vial to start an assay. A number of aliquots of the same samples are dispensed in different vials, when multiple tests are ordered on a single sample.

As used herein, the term "antigen" or "surface antigen" means a substance, in free liquid such as a lysate or associated to the surface of a microvesicle such as an exosome. An antigen can be bound to one or more antibodies. Examples of antigens are peptides, proteins, nucleic acids (for example DNA and RNA). Antigen can be of different forms (for example, synthetic, recombinant or natural) and be of different sources (for example, isolated, partially purified vesicles, lysate components, intact cells or biological fluids).

As used herein, the expression "immunoassay" means a process for determining the presence and/or concentration of a substance in a sample by means of an immunological technique (that is, a technique employing antibodies to search for antigens). The immunoassays applied herein are a direct sandwich assays (that is, assays that apply an antibody-antigen-antibody complex).

As used herein, the expression "molecular biology probe assay" means a process for determining the presence and/or concentration of a substance in a sample by means of a molecular biology technique (that is, a technique employing probe consisting of a sequence of linked nucleotides to search for complementary RNA or DNA). The analysis are either be performed by applying the probe assay directly on the sample RNA or applying the molecular biology analysis procedure after transcription of RNA to DNA. 6 As used herein, the expression "assay process analyzer" means an automated instrument used for isolation of microvesicles in a sample. The isolation step is followed by analysis of the concentration of the microvesicles and of one or more of the microvesicle associated molecular markers. The analysis is either an immunoassay or a molecular biology probe based assay.

As used herein, the expression "isolation vial" means a container, in which isolation process to capture and wash microvesicles such as exosomes. The vial is also used to perform a lysis of microvesicles to prepare a Iysate.

As used herein, the expression "analysis vial" means a container, in which a biochemical, immunochemical, or molecular biology reaction, or measurement thereof, is carried out.

As used herein, the term "module" means a functionally related and interconnected group of mechanical and electronically components with software, which performs a specific task or class of tasks required to undertake the system assay process. For example, the system instruments described herein can include such as a reaction chamber process module, reagent and sample storage module, pipetting module, wash stations module, signal measuring module, hydraulic module and waste module. A given module in the system instrument is preferably designed to be used with other system modules that are related thereto.

As used herein, the expression "embedded software" means a computer- implemented software program, that is designed to control the operations of various modules and subsystems in the system, including the electrical and mechanical operations required for accomplishing required operations and related functions.

SUMMARY OF THE INVENTION Disclosed herein are instrument system designs for performing automated integrated processing including isolation of microvesicles from liquid sample and direct analysis of microvesicle molecular markers associated to the isolated vesicles. The system instruments include a central process module (a reaction chamber) surrounded by several supporting modules such as reagent and sample storage modules, pipetting module, wash station module, signal measuring module, hydraulic module and waste modules. The process module is equipped with an in discrete steps rotating circular reaction wheel, designed to carry and transport isolation and analysis vials. The vials are placed in circles ranging from the periphery to the center of the wheel. ln each circle there are positions for 50 or more vials. The outer vial circle is used to isolate microvesicles and one or more inner vial circles are dedicated to analyse marker molecules associated to the vesicles.

To the microvesicle isolating vials placed in the outer circle, aliquots of samples containing microvesicles and isolation reagent are pipetted. The total volume of the mixture used in the isolation process is 2 ml of more. To the analysis vials in the inner molecular marker analysis reaction circles, intact or lysed isolated microvesicles and analysis reagent aliquots are pipette. The total volume of the mixture used in the analysis process is in the range of 30 to 200 pl. During the isolation and analysis processes, vials are served by process functions in the pipetting module, wash stations module, signal measuring module, hydraulic module and waste module. 7 Using the resources delivered from the supporting modules, isolation and analytical assay methods are run.

The pipetting module, wash stations module, signal measurlng module and waste module are positioned aligning the reaction chamber process module reaction wheel. At defined working positions along the periphery of the reaction wheel these modules are able to reach vials transported in the circles, to perform a process operation. The arrangement enables the supporting modules to be able to serve isolation and analysis vials with the functionality required to perform the processing actions, defined in isolations and analysis method protocols. The pipetting module is able to aspirate aliquots of samples and reagents in vials placed in the sample and reagent storage module and transfer these to any of the different dispensing positions in the reaction chamber process module. This enable the pipetting module to deliver samples, controls, calibrators and reagents from sample tubes, reagent vials and buffer bottles in the sample and reagent storage module to vials processed in the reaction wheel. ln both the isolation and analysis processes, a solid phase reagent based on paramagnetic beads is used to separate microvesicles and markers from the bulk liquid in samples and reaction mixtures. Besides the solid phase reagents, immunologically and molecular biology related soluble reagents are used, either coupled to the paramagnetic beads or as free conjugated marker reagents. To enable mixing of pipetted samples and reagents, the process module comprises a plurality of positions at which samples and reagents in vials are effectively mixed. After mixing the vials are incubated, while the reaction wheel is stepped to give incubates a proper reaction time. isolation and analysis reaction incubations are terminated when the vial reach the wash station module. At the first position in the wash station the magnetic beads are trapped to the vial wall by a magnet raised up against the outside of the vial. Thereafter an aspiration/dispensing nozzle movable in the z-direction mounted in a wash tower is lowered into the vial. The wash nozzle design includes an inner aspiration tube and an outer dispensing tube. While the wash nozzle is lowered into the vial, the washing module is aspirating the liquid in the vial and transfers it to the liquid waste. Thereafter the dispensing tube of the nozzle is adding wash buffer to the vial, to wash the magnetic beads free from contaminants. Leaving an aliquot of wash buffer in the vial, the aspiration/dispensing nozzle is moved out from the vial. Thereafter the reaction wheel is moved one step and the vial is transferred to a mixing position, at which the magnetic beads are re-suspended in wash buffer. The reaction wheel is again stepped and the magnetic bead trapping, washing and mixing is repeated at the following two positions. To obtain totally pure microvesicles or uncontaminated trapped marker molecules, the vial is treated in a number of wash cycles, while the magnetic bead trapping, waste aspiration, buffer dispensing and mixing is repeated. After leaving the wash station module, the vial reaches a reagent pipetting position, at which reagents for the next reaction step in the assay is added to the vial by the pipetting module.

After finalization of the isolation phase of the assay run in the outer process circle, aliquots of magnetic bead with bound intact microvesicles or aliquots of lysed microvesicles are transferred to one or more of the reaction wheel inner circles. ln these circles one or more analysis processes will thereafter started. ln these 8 immunoassays or molecular biology probe based analyses are performed, using magnetic beads and conjugate reagents to measure specific marker molecules. The analysis processes are ended by a signal measuring step, which is performed either using a luminometer or a fluorometer. Finally after signal measurement, all remaining liquids in the used vial are aspirated to the liquid waste, followed by the removal of the empty vial from the reaction wheel to the solid waste. These actions are performed by the waste handling modules. The reaction chamber process module, pipetting module, wash stations module, signal measuring module and waste module are all supported by the hydraulic module. This module is active during dispensing of buffers or other liquids and is delivering the aspiration vacuum required to remove liquid waste.

The logistic arrangements of modules enable the system instrument to perform assay runs using samples and reagents and to perform three types of isolation and analysis processes, which are disclosed herein. lt is disclosed a process method having the objective to isolate microvesicles and directly analyze one or a plurality of microvesicle surface bound molecular components, such as proteins and glycoproteins using immunoassay techniques. lt is also disclosed is a process method having the objective to isolate microvesicles, directly lyse the vesicle and analyze one or a plurality of the internal cargo components, such as proteins or glycoproteins using immunoassay techniques. A process method is also disclosed having the objective to isolate microvesicles, directly lyse the vesicles and analyze one or a plurality of the internal RNA cargo components, such as messenger RNA or micro RNA. The process is performed using the appropriate molecular biology probes, with or without applying molecular biology amplification such as PCR or comparable isothermal methods. The three analysis method alternatives are implemented in automated instrument based systems using a mechanic assembly, analogue and digital electronics as well as an embedded software applied in the instrument computer hardware, as well as an administrative software implemented in an external computer hardware. The instrument embedded and the external software operator interfaces consist of keyboards, touch screens, mice. Information is stored in solid state disks in the internal and external hardware. The system instrument and the external computer are connected to the laboratory network and Internet.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1: lsometric view of the embodiment of the common reaction chamber process module.

Fig. 2: Side view of the embodiment of the common reaction chamber process module.

Fig. 3: Top view of the embodiment of the common reaction chamber process module.

Fig. 4: Top view of a general layout demonstrating an embodiment of an instrument consisting of the common reaction process chamber module aligned with a design alternative of required supporting modules to perform random-accessed assay runs. 9 Fig. 5: lsometric view of the pipetting module gender mechanics applied for transfer of vials, samples, controls and reagents from positions in the storage modules to the reaction chamber in the random access instrument embodiment.

Fig. 6A and B: Batch analyzer layout demonstrating an embodiment of an instrument consisting of the common reaction chamber process module aligned with a design alternative of required supporting modules to perform a batch analysis of a limited number of samples and diagnostic tests in a closed assay run. The instrument configuration in figure 6A is a top view of the instrument showing the general reaction module to the left and three positions to the right for carrying vials, pipette tips, reagents and samples. The sampling and pipetting module is localized in a position between the two modules. Figure 6B shows an isometric view of the instrument concept Fig. 7A and B: Drawing showing the isolation and analysis vials applied in isolation of microvesicles (7A) and to perform analyses of microvesicle markers (7B).

Fig. 8A to E: Drawings showing the reaction chamber process wheel in side view (8A), top view (8B), isometric view (8C) and the process wheel step drive function in exploded isometric view (8D) and top view (8E). The process wheel illustration is presented including loaded isolation and analysis vials. The process wheel driving function includes the process wheel with the vial mixer, magnets carrying plate and motor drive parts.

Fig. 9A, B and C: Drawing showing the pipette syringe device applied in the pipetting module in the random access instrument configuration. Figure 9A is a side view, figure 9B is a front view and figure 9C is an isometric view of the pipette syringe device.

Fig. 10A, B and C: Drawing showing details of the pipette unit. Figure 10A is a side view, figure 10B is a front view and figure 10C is an isometric view of the pipette unit.

Fig. 11A, B, C and D: Drawing showing the pipette unit applied in the pipetting module in the batch instrument configuration. Figure 11A is a left side view, figure 11B is a front view, figure 11C is a right side view and figure 11D is an isometric view of the pipette.

Fig. 12A and B: Drawing illustrating an alternative pipetting principle applied in the pipette unit applied in pipetting module in the batch instrument configuration. The drawing illustrates the connection of the pipetting nozzle and rotating pipette arm to a remote dilution pump. Figure 12A demonstrates the pipette tip accessing vials and reagent bottles and in 12B is an optional concept of pipette tip rinsing illustrated.

Fig. 13A and B: Drawing showing the vial griper tool for transfer of vials and the vial gripper storage station embodiment. Figure 13A shows the isometric view of the gripper placed in the griper stand. Figure 13B shows an exploded view of the gripper stand with the storage position of the gripper tool and of the gripper tool.

Fig. 14A, B and C: Drawing showing the embodiment of the wash tower. Figure 14A is the front view, figure 14B is the right side view and figure 14C is an isometric view of the wash tower.

Fig. 15A, B and C: Drawing 15A is showing the wash tower nozzle radial movement positions. The radial movement enables the nozzle to reach the rinse cup, isolation vial and the analysis vial positions in the reaction wheel. Drawing 1B is illustrating magnetic bead to the vial wall trapping and figure 15C is showing the wash tower aspiration and dispensing nozzle in position for washing of trapped magnetic beads.

Fig. 16: Drawing showing the interior of the wash station module. The embodiments in the figure demonstrate the assembly of wash tower positions, magnets for bead trapping and mixers for re-suspension of trapped magnetic beads.

Fig. 17A and B: Drawing outlining the embodiment of the luminometer, there as the figure 17A is a side view of the interior of the unit and figure 17B is an outside isometric view.

Fig. 18A, B and C: Drawings showing the general hydraulic module. Figure 18A shows an isometric view of the assemble plate with mounted peristaltic pumps, there as the figure 17B shows the side view of the assembly. Figure 17C is a top view of the assembly.

Fig. 19A to D: Drawings showing the structure of the reaction chamber and the thermostating heat and fan plate. The figure 19A is an exploded isometric view of the reaction chamber lids, plates and cover including devices and parts. Figure 19B is a side view of the thermostating heat and fan plate. The figure 19C is the bottom view, there as the 19 D is an isometric view at the bottom side.

Fig. 20: Figure illustrating the total structure of products and components in the total isolation and analytical system.

Fig. 21: Figure showing the assay processes and methods applied in the isolation and analysis systems.

Fig. 22: Figure summarizing the general process applied in the instrument to sequentially step by step perform isolation and analyses.

Fig. 23: Figure summarizing the chemistry reaction concepts applied by the systems to perform microvesicle isolation and the three marker molecule analysis alternatives. ln row A is the vesicle isolation and the surface protein analysis methodology outlined. ln row B is the cytosolic proteins analysis methodology outlined. ln row C is the RNA sequence analysis methodology illustrated.

Fig. 24 A and B: Fig 24A shows the calculated signal-to-noise ratio (SNR) in an ELISA experiment where the microvesicle prostasomes were isolated and detected using antibodies d-CD9, a-CD63 and or-CD81, and the beads Dynabeads MyOne Streptavidine T1. The results are the measured chemiluminescence signal expressed as SNR values. Fig 24B shows the calculated SNR of control samples in an ELISA experiment where exosomes from cancer dendrite cells were isolated using Dynabeads MyOne Streptavidin T1 immobilized with the antibody d-CD9 and detected using the antibody d-CD81. The results are the measured chemiluminescence signal expressed as SNR values. 11 DETAILED DESCRIPTION ln the following detailed description, references are made to the accompanying figures. ln the figures, similar symbols typically identify similar components, unless context dictates othen/vise. The illustrative embodiments outlined in the detailed description, figures, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. lt will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are contemplated herein.

Disclosed herein are systems, instruments, processes and methods for accomplishing integrated automated isolation of microvesicles such as exosomes or other types of biological microparticles and analysis of molecules associated and carried by the isolated microvesicles. Disclosed are a range of system designs expressed in two process resource levels of system instrument concepts, offering different processing capacity. ln both levels of processing capacity, the instrument system logistics are configured to use a common reaction chamber module to offer conformity in isolation and assay performance for all developed system configurations and levels. The common reaction processing module is shown in Figs. 1, 2 and 3.

Referring to Figs. 4 and 6, the structures and logistics of system instruments representing two levels of processing capacity are outlined. These are designed to either offer continues loading of samples during the processing (Fig. 4) or loading of a defined number of samples at the startup of process runs (Fig. 6). The sample loading area configuration in "the random access layout configuration" shown (Fig. 4 - 100) is designed to offer possibilities for continuous loading of samples. The random access instrument is also designed for extended cold storage of reagents of a number of different test types (Fig 4 - 101). The instrument is also configured to offer common large sized instrument functionalities, required in sampling and test selections to obtain a random-access analyzer capability. The Fig. 6 batch analyzer instrument concept offers a loading area configuration, which enables loading of samples and reagents only at the assay run startups (Fig 6 - 102). In this concept, the reagent handling only allow for temporarily storage of a limited number of different test types during the assay run. This means that the design is defined to offer the common small sized instrument functionalities, which are required in a sample and reagent batch analyzer concept. To gain the required processing performance, the sampling and pipetting module in the large sized random-access instrument is equipped with traverse transfer arms (Fig. 5 - 103 and 104), carrying two pipette devices (Fig. 5 - 105 and 106). The small sized batch instrument is only equipped with a rotation pipette transfer arm carrying one pipette (Fig. 6 - 107) or with one traverse transfer arm carrying one pipette device (F ig. 5), alternatively.

Referring to Fig. 4 embodiment showing a random access instrument configuration, a plurality of modules including the reaction chamber process module (Fig. 1, 2, 3 and Fig. 4 - 122) is outlined. The instrument concept includes a left reagent cold storage module (101) also comprising an area for storing isolation vials and pipette tips (109). The instrument concept includes a right storage module for samples storage (100), storage areas for reagents and buffers (110) and an area for analysis vials and pipette tips storage (111). The instrument concept includes a pipetting module 12 comprising two traverse gender arms (left Fig. 5 -103 and right 104) carrying one pipette device in each arm (left 105 and right 106), wash station module (112) with wash towers, magnetic bead trapping and vial mixing, signal measuring luminometer module (113), hydraulic module (114), buffer and liquid waste storage area and dry waste module (115). All storage modules are accessible by the user during assay run. The instrument electronic module is enclosed in the electronic compartment at the backs side of the instrument (223).

Referring to Fig. 6 embodiment, a general batch analyzer instrument configuration is outlined, comprising a plurality of modules including the reaction chamber process module (122) , isolation vials and pipette tip storage module (117, used for storage vials during assay run preparations and for tips during assay runs), analysis vials and sample storage module (118, used for storing vials during assay run preparations and for samples during assay runs), reagent storage module (119 used for storage reagents during assay runs), pipetting module consisting of a rotating pipette (107), wash station module (120) with wash nozzles, magnetic trapping and vial mixing. The instrument also includes signal measuring, hydraulic, buffer storage, waste and electronic modules, which are not depicted in the figure. These modules are of the same design as the modules applied in the random access instrument shown in Fig. 4. ln an alternative sampling and pipetting module design, a single arm traverse transfer module is used. This is of a similar design as the module applied in figure 5, lacking one arm and pipette device. At the start of the batch instrument assay run, the reaction chamber wheel is first loaded with all vials. These are transferred from the appropriate vial storage modules (117, 118) to the reaction wheel (123). The vials are loaded into the dedicated positions for isolation and analysis vials, e.g. the isolation vials in the outer circle and the analysis vials in the inner circles. After the vial transfer is finalized, the instrument halts and the operator loads the pipette tips, reagents and samples into the storage modules (117, 118 and 119). The instrument is thereafter again started to run the assay, during which the instrument is not accessible to the operator.

By referring to Fig. 1 embodiment, the concepts of the general reaction module design is outlined. ln the reaction module the processing is performed, using a process wheel (123), rotated in discrete steps. The process wheel periphery includes two or more circles of vial positions, used to carry vials. The outer vial circle (124) transport isolation vials (Fig. 7A - 125) during the isolation part of the assay run. ln the inner circles (126), the analysis vials (Fig. 7B - 127) are transported during the analysis part of the assay run. The number of vial positions in each circle ranges between 50 and 100, depending on the size of the reaction chamber and other instrument resources reaction incubation time, step time and throughput of the instrument. ln the Fig.1, 2 and 3 embodiment, the number of vials in each circle is 54. The outer circular process path (124) is used to perform the microvesicle isolation process. One or more inner circular process paths (126) are used to perform analysis processes of markers molecules located on the outside of the microvesicles or contained inside the microvesicles, released after the microvesicles have been lysed. This means the system can run an isolation process to obtain isolated and purified microvesicles in the outer isolation process vial circle, thereafter intact or lysed vesicles are transferred from the outer vial circles to be analyzed in the inner analysis process vial circles. After the intra circle transfer the system can run either one or more immunological or molecular biology related assay methods, using the different inner process paths. The common structure of process paths in the reaction 13 chamber module enables the system to use the process paths for any of the applied analytical methods available in the system.

The processes are essentially based upon six different types of automated steps, 5 started after the operator has loaded vials, pipette tips, reagents, liquids and samples in the instrument storage modules. The six types of automated process steps are: 1. lsolation and washing of microvesicles performed in the outer isolation circle in the reaction wheel using isolation vials and paramagnetic beads coated with antibodies specific for one or more micro vesicle surface proteins. During incubation the microvesicles are specifically bound to the paramagnetic beads by an antibody - antigen binding.

Transfer of intact isolated and washed microvesicles bound to the paramagnetic beads to analysis vials placed in the inner circle, to perform the first step in the analysis of the microvesicle outer surface proteins. The first step of the analysis is performed using an anti-surface protein antibody- enzyme conjugate, specific for surface marker proteins. During the following incubation, the surface protein antibody-enzyme conjugate is specifically bound to the microvesicles on the paramagnetic beads.

Alternatively start the first step in the analysis of the microvesicle internal molecular markers by lysing of the microvesicles. These are lysed in the isolation vial still located in the outer circle. After lysis, the lysate is transferred to an analysis vial placed in the inner circle. To perform the analysis, the vials also contain an anti-microvesicle internal protein antibody or a microvesicle RNA sequence specific molecular biology probe, both coupled to an analysis paramagnetic bead. During the following incubation, the marker molecules are specifically bound to the magnetic bead. ln the following step in the internal marker molecule analysis processes, the washed paramagnetic beads with bound internal marker molecules are incubated with a conjugate either consisting of a second anti-internal marker protein antibody or another marker RNA sequence specific molecular biology probe. Both conjugate carries includes an enzyme used in the final signal generating reaction. During the incubation, the enzyme conjugate is specifically bound the protein or RNA molecule marker bound to the paramagnetic beads.

To measure the amount of bound micro vesicles, internal protein or internal RNA marker molecules, the washed paramagnetic beads carrying the enzyme conjugate coupled to microvesicles or to internal marker molecules is incubated with an enzyme substrate. The reaction generates a luminescence signal and the signal is measured in a luminometer.

The assay run is finalized by aspiration of liquid waste from vials and by the transfer of vials to the dry waste.

All processes in step 1 to 5 are started by activities performed by the pipetting 50 module (e.g. the traverse gender arms Fig. 5 - 103 and 104 using pipette units Fig. 5 - 105 and 106 in the random access instrument or the rotating pipette arm Fig. 6 - 14 107 in the batch instrument). These modules are at assay start up using gripper devices (Fig. 13 - 130) connected to the pipette nozzles (Fig 9A - 166 or Fig. 11A - 132) to transfer isolation and analysis reaction vials from the vial storage sites to the reaction wheel. The pipetting module uses a larger griper tool to transfer the larger separation vials from the vial rack position (Fig. 4 - 109 or Fig. 6B - 117) and place these in the outer circle of the process wheel. The pipetting module uses a smaller griper tool to transfer the smaller analysis reaction vials (Fig. 4 - 111 or Fig. 6B - 118) and place these in the inner circles of the process wheel. There after the pipette devices use standard disposable pipette tips to pipette reagents (200 ul sized tips) and sample (1000 pl sized tips) from the reagent storage modules to the vials in the process wheel.

The magnetic bead-based separation process is performed in larger isolation vials (Fig. 7A - 125), placed in the outer circle of the process wheel. The analysis processes is performed in smaller analysis vials (Fig. 7B - 127) placed in the inner circles of the process wheel. The main part of the processing is done while the samples and reagents are interacted in chemical, immunological or molecular biological reactions taking place while the vials are stepwise moved from position to position by the rotating process wheel. lncubations are ended by either a magnetic bead wash operation (steps listed as 1 to 4 above) or when a signal measuring performed (listed as step 5 above). ln the wash steps, the washing module (Fig 4 - 112 in the random access instrument and Fig. 6A - 120 in the batch instrument) is using wash towers (Fig. 14B -133), magnetic bead trapping positions (Fig. 3 -134), and mixers (Fig. 3 -135) in the wash station, to remove contaminating molecules in the vials. ln the final signal detection step, the analysis vial incubate emitting light, is transferred to a luminometer (Fig. 17B - 113).

The isolation and analysis activities include a number of liquid handling operations and lncubations, performed in the vials carried by the reaction wheel. These activities are done while the reaction wheel is rotated, the number of turns required to undertake the complete isolation and analysis assay run. The process wheel is moved applying a defined exact repeated step time, during which the wheel is stepped to the next position and thereafter is staying still, to enable process operations to take place in the vials. To give the reaction mixture in all vials the same treatment, the reaction wheel transfers the vials from position to position, where as the vials receive the same treatment at each active processing position. Fig. 8 outlines the embodiment of the process wheel indexing functionality, required to secure the process wheel stepping and timing. The index wheel (136) shown in the figure is designed to enable the implementation of 54 positions for isolation and analysis vials in each circle, but other numbers of positions are applicable design alternatives. The index wheel is driven a 200 step/rev stepper motor using a belt transmission (Fig. 8 - 137). The index wheel hub is equipped with a laser cut encoder disc (Fig. 8 - 138), having one slot for every vial position in the wheel and a secondary slot used for homing of the wheel. The encoder disc is read by a set of 2 optical relays (Fig. 8 - 139), screwed onto the motor plate assembly. The encoders enable the system to obtain feedback of the wheel position and to control the wheel movement.

The devices and stations used during processing are designed to handle both isolation and analysis vials, though the vials are different in size. The reaction wheel isolation circle vial positions (Fig. 1C - 124) are designed to carry the dedicated isolation vials (Fig. 7A - 125) and analysis circle vial positions (Fig. 1C - 126) are designed to carry the dedicated analysis vials (Fig. 7B - 127). The shape of vials enables packed storage in storage trays (Fig. 7A - 140) and automated transfer of the vials from storage trays to vial positions in the reaction wheel using gripper tools (Fig. 13 - 130). The design of both the isolation and analysis vials enables mixing of samples, beads and reagent in the vials after reagent dispensing, trapping of magnetic beads to the vial wall, aspiration and dispensing of liquid waste and wash buffer to and from vials during magnetic bead washing and re-dispensing of trapped magnetic bead into reagents and buffers. The design of the analysis vials enables direct detection of luminescence in the vial. The vial is in this case transferred to the luminometer and placed in a sledge (Fig. 17A - 203), moved into a measuring site (Fig. 17A - 142), signals measured by a photomultiplicator (Fig 17A - 205) and transferred back to the wheel. During the last step of the process the liquid in the vials is aspirated to liquid waste (Fig. 4 - 144), using an aspiration functionality delivered by the hydraulic module (Fig. 18 - 114). The empty vials are finally transferred to the dry waste container (Fig. 4 - 115).

The isolation vial (7A - 125) is preferably produced using polypropylene or similar opalescent soft polymer. The total volume of the vial is 4 ml. The vial shape is conical, and the height is 68.1 mm, whereas the width at the upper end is 12.4 mm. The analysis vial (7B - 127) is preferably produced using styrene or similar optically transparent polymer of higher quality material. The total volume of the vial is 0.4 ml. The vial shape is conical, and the height is 21.6 mm, whereas the width at the upper end is 8.4 mm.

The assay processing is using a number of functions, required in the isolation and analysis activities. These functions are all assembled closed to and contained in process chamber environment at different processing positions or in stations placed along the periphery of the reaction wheel (Fig. 1 - 123). The process module wheel and surrounding stations are all enclosed in a thermostated incubation chamber (Fig. 19A - 145). The reaction chamber temperature is regulated and controlled at 37° C by a heater equipped with a fan (Fig. 19D - 146) and a temperature sensor (Fig. 19A - 147) connected to control electronics. The action positions in the reaction wheel, at which supporting modules and devices have access to vials are as follows: 1. Vial transfer (dispensing and aspirating) and main pipetting positions (placed after the last wash tower, Fig. 3 - 149). 2. Main mixer positions placed after bead washing and reagent dispensing (Fig. 3 - 150).

Luminometer transfer position (Fig. 3 - 151).

Mixers (other supporting mixers placed along the wheel periphery - Fig. 3 - 198).

Pipetting positions (other supporting positions along the wheel periphery - Fig. 3 - 153).

Liquid waste handling position with liquid waste aspiration tower (Fig. 3 - 154). Dry waste handling position (Fig. 4 - 115) Wash positions including trapping magnets devices, wash towers and bead re-suspending mixers (Fig. 3 - 112) The process related tasks are performed by supporting modules and devices located around the reaction wheel, having access to positions with processing activities. These modules and devices are: 16 Precision pipette using vial gripper tools (Fig. 13 - 130) and disposal tips. The pipettes are mounted onto the x, y, z-traverses used in the random access instrument (arms Fig. 5 - 103 and 104 carrying the pipettes Fig. 5 - 105 and 106).

Precision pipette (Fig. 6 - 107) using vial gripper tools (Fig. 13 - 130) and disposal tips. The pipette is mounted onto a rotating z-moving arm used in the batch instrument.

Wash towers with z-moving dispensing and aspiration wash nozzle in arrangement with magnetic bead trapping device.

Vial vortex mixer motor bases in arrangement with mixer hats.

Luminometer measuring signal in analysis reaction vial (Fig. 1 and Fig. 17B - 113).

Liquid waste aspiration tower using radial and z-moving arm, in designs based on the wash tower design (Fig. 14D - 133).

Dry waste unloading device using radial and rotating z-moving arm, in designs based on the rotating pipette design (Fig. 11D - 107).

Hydraulic module with buffer storage rack, buffer dispensing pumps and waste aspiration pumps connected to wash towers and liquid waste aspiration nozzles (Fig. 4 and 18A - 114).

Referring to Fig. 4 and Fig. 6B, the instrument design includes a plurality of material storage areas accessible to the operator during preparation of an assay run, to enable loading of consumables, buffers, reagents and samples. ln a random access instrument the areas are also accessible to the operator during assay run. ln the batch analyzer this is not allowed. The structures of the storage areas are optimized for effective and convenient operator manual loading and for the instrument pipetting modules to effectively transfer material from storage areas to the reaction chamber. The following materials are required to be loaded in storage areas, to allow the instruments to be started: 1 lsolation and analysis vials in dedicated racks at positions Fig. 4 - 109 and Fig. 4 - 111 in the random access instrument and positions Fig. 6 - 117 as well as Fig. 6 - 118 in the batch instrument.

Disposal pipette tips placed in position Fig. 4 - 156 and Fig. 4 - 157 in the random access instrument and in position Fig. 6 - 117 in the batch instrument (pipette tip loading is done at the site earlier used for the pre-run loading of vials - Fig. 6 - 117).

Assay specific reagents for isolation and analysis reactions in positions Fig. 4 - 101 in the random access instrument and Fig. 6 - 119 in the batch instrument.

General and buffer system reagents at positions Fig. 4 - 110 in the random access instrument and Fig. 6 - 119 in the batch instrument.

Wash buffers, diluents and water in a hydraulic module buffer shelf to the right side of the instruments in the random access instrument (Fig. 6 - 224) and at the left side of the batch instrument (not shown in figures).

Samples and controls placed at positions Fig. 4 - 100 and Fig. 4 - 158 in the random access instrument and Fig. 6 - 118 in the batch instrument (during pre-run used for vial storage).

Assay curve calibrators or assay curve control placed in positions Fig. 4 - 159 in the random access instrument and Fig. 6 - 118 in the batch instrument (during pre-run used for vial storage). 17 8 Micro titer plate for dilution of samples at position Fig 4 - 160 (not available in batch instrument).

At loading of reagents manual and/or automated control of reagents and sample identities are performed using barcodes or QR-codes. 5 During assay run the instrument pipetting module transfers material from the storage areas to the reaction chamber to be used in the assay processing performed in the reaction chamber. Reagents and samples are transferred to vials placed in the reaction wheel at start of and during the assay run. This done at following dedicated dispensing positions: 1. At the start of an assay run, isolation and analysis reaction vials are dispensed into the reaction wheel at the main dispensing position (F ig. 3 - 149). 2. At start and during assay run, dispensing of samples, magnetic beads and reagents are done at the main dispensing positions (Fig. 3 - 149). 3. During assay run, aspiration of reaction mixtures from vials in an outer reaction wheel circle is followed by a dispensing of the mixture at dedicated dispensing position in an inner circle (Fig. 3 - 161). 4. To start the reactions in a homogenous state, reaction mixture, vials containing samples, magnetic beads and reagents are mixed at a plurality of positions (Fig. 3 - 153).

. At the wash towers in the wash station, magnets are raised to the vial outside wall, magnetic bead is trapped to the inner wall, liquid waste aspirated from the vials and wash buffer dispensed by the wash tower nozzles into the vials (Fig. 1 - 129 at positions Fig. 3 - 134). 6. At the following mixing position in the wash station, trapped magnetic beads are re-dispensed in buffers or reagents (Fig. 3 - 135). 7. To measuring the luminescence signal, the analysis vials are moved from the reaction wheel at a vial transfer position, to be move to the luminometer (Fig. 1 - 151). After measuring of the signal the vial is moved back to the same position in the reaction wheel. 8. At the termination of the assay run, vials are aspirated dry and liquids are transferred to the liquid waste container using a liquid waste aspirating tower (Fig. 1 - 154). 9. At the termination of the assay run vials are moved from the reaction wheel at 35 a vial removal position and discarded in the dry waste (Fig. 1 - 115).

Fig. 9 is a schematic figure illustrating the design of the pipette device used by the pipetting module. The pipette is mounted in and carried by an x, y, z-transfer arm (Fig. 5). ln the random access instrument, the pipetting module includes two x, y, z- 40 transfer arms carrying one pipette each (Fig. 5 - 103 and 104). To enable the instrument to span an extended pipetting volume range, keeping a high volume accuracy and precision, the pipetting module is carrying two different sized pipette syringes (Fig. 9 - 162). One of the pipettes is using a syringe with a total volume of 1000 ul, which is carried by the right arm (Fig. 9 - 105). The other pipette is using a 45 syringe with a total volume of 200 ul, which is carried by the left arm (Fig. 9 - 106). The pipette units are motorized using a stepper motor (Fig. 9 - 163). The accuracy is about 0.05 ul/step for the larger volume pipette and 0.01 ul/step for the smaller volume pipette. The syringe is mounted in a frame (Fig. 10 - 164) equipped with a pipette nozzle block (Fig. 10 - 165), to which the syringe nozzle are fixed. The lower 50 part of the block is shaped as a nozzle (Fig. 9 - 166), to which disposal pipette tips 18 are attached. For the larger pipette 1 ml tips are used and for the smaller volume pipette 200 ul tips are used.

The direct mounting of syringes to nozzle part, will minimize the "non active" air volume and thereby increase system precision. A level sensor tube is placed in between the syringe nozzle and pipette nozzle block (Fig. 10A - 168). ln order to minimize volumes related to the level detection, the level sensor electronics is placed on a small electronic board (Fig. 10A - 169) just above the exit of the level sensor tube. The level sensing is applying two pressure sensing chips (LDESO25BF6S and HMEM250BZ7HS from Sensortechnic Gmbh), mounted at the exit of the levels sensor tube. The two sensors measure the tubing pressure and the flow speed (Fig. 10A - 170). To enable automated transfer of vials from the vials storage racks to the reaction wheel, a griper tool for vial transfer will be mounted to the pipette tip nozzle block, during vial loading at the assay start up (Fig. 13 - 130). ln Fig. 11 an embodiment of an alternative pipetting unit applicable in the batch instrument is outlined (Fig. 6). Pipetting is performed using a rotating arm able to access a pipette tip storage rack, a sample storage rack, reagent storage rack and the dispensing positions in the reaction wheel (Fig. 6 - 225). The pipetting principle used by the pipette is illustrated in Fig. 12, in which Fig. 12A demonstrates the principle for radial positioning to access vials and Fig. 12D demonstrates the z- movement for reaching different levels in vials. Used tips are unloaded at a dry waste position (Fig. 6 - 167). The storage rack holders are linearly movable and the movement allows the pipette to reach all positions in racks placed in the holders (Fig. 6 - 117, 118 and 119). The pipette syringe size and used tips enable pipetting of all required volumes for microvesicle isolation and marker analysis. To the pipette tip, griper tools for vial transfer is mounted before assay start up, to enable automated placement of vials in the reaction wheel (Fig. 13 - 113). To improve the performance in the batch instrument pipetting module, the traverse based pipetting principle outlined in figure 5 is an applicable option.

The embodiment of the griper devices and the griper stand is shown in Fig. 13. The gripper tool (Fig. 13 - 130) is used for vial transfer, performed by the pipetting module. The gripper stand (Fig. 13 - 171) is used to store the gripper tool and enable the attachment of the gripper tool to the pipette nozzle block (Fig. 9 - 166). The gripper tool attached to the pipette is used to transfer isolation and analysis vials from the vial storage racks to the reaction wheel at the assay start up. A dedicated griper tool size is used for each vial type, there as the right transfer arm in the pipetting module is used to transfer isolation vials and the left arm in the gender unit is used to transfer analysis vials. For storage of the gripper tools two gripper stands are applied (Fig. 4 - 171), each located close to the position used for storing the vial racks. ln these stands the grippers are placed, employing an unlocking and locking mechanism. This mechanism enables a safe unloading and reloading of the gripper tool in the stand. During the vial transfer, the gripper tool is temporarily mounted to the pipette nozzle block. The mounted gripper tool transfers vials from the vial rack storage area to the vial dispensing positions in the reaction chamber, to be dispensed in the reaction wheel. The gripper tool is equipped with functions to enable attachment to the pipette nozzles, to perform vial aspiration from vial racks and dispensing of vials into the reaction wheel. The gripper tool storage stand is designed to interact with the functions contained in the gripper tool to support in unloading and reloading of the gripper tool. The storage of the gripper tool at the gripper tool stand, 19 attachment of the gripper tool to the pipette nozzle and the unloading of the gripper tool from the griper tool stand is performed according to the following steps: 1. The gripper tool is during storage fixed at the upper part to the gripper storage stand by latches (Fig. 13 - 175), which are pushed against two pins (Fig. 13 - 178). The gripper tool is in the middle part pushed against a fork like guide (Fig. 13 - 173) in the gripper tool stand frame. The tool is in the lower end is in its lower end pushed into a movable sledge (Fig. 13 - 174) at the gripper tool stand base (Fig. 13 - 177). A spring (Fig. 13 - 176) located in the gripper stand base pushes the gripper tool against the fork like guide in the middle part in the stand frame and lock it to the gripper stand. 2. To release the gripper tool, the pipette nozzle is inserted into the gripper tool top (Fig. 13 - 172) and the gripper tool is pushed down by the pipette z- movement. 3. The spring in the base in the gripper station as well as a spring (Fig. 13 - 180) inside of the gripper is compressed. 4. While the compression takes place, the holes (Fig. 13 - 181) in the gripper spring latches close over the pipette nozzle action pins (Fig. 10 - 166) and the latches and guide are freed from interaction with the gripper station.

. The gripper/nozzle is then moved along the x-direction and the gripper is free to be lifted in z-direction and start the transfer of vials.

To secure function of the gripper station, it is equipped with a shifter device (Fig. 13 - 182). This can lock the gripper storage stand sledge (Fig. 13 - 174) in two positions, which are one inner gripper tool storage position and one outer lift position to move the gripper tool. There is also a linear ball bearing (Fig. 13 - 184) at the bottom of the sledge to allow a low friction/force movement in x-direction. Furthermore, the inner parking position is equipped with a guide (Fig. 13 - 184) to fix the entrance for the pipette nozzle block at the gripper tool in a repeatable position. The sledge guide is equipped with a slope in order to allow the device to self-align minor misalignments, caused by incorrect gripper release. The rotation of the tool is secured via an interaction between the gripper ejector pin (Fig. 13 - 185) at the bottom of the tool and the gripper station base sledge. When the vials have been distributed from the vial storage racks to the reaction wheel, the gripers are returned to the griper storage stations using the steps above in the order 5 to 1.

As is shown in Fig. 1, a number of wash towers (Fig. 1 - 129) are grouped together in one part in the reaction chamber, with wash nozzles oriented against the periphery of the reaction wheel. This assembly of wash towers (Fig. 14 - 133) comprises the wash module in the random access (Fig. 4 - 112) and in the batch (Fig. 6 - 120) instrument. In the positions after each wash tower, mixing positions are located (Fig. 3 - 135). One exception is the position after the last wash tower in the row towers, at this position is the main reagent dispensing position placed (Fig. 3 - 149). lnstead, the dispensing position is followed by a mixing position (Fig. 3 - 150). During the wash operation, the wash tower is using a wash nozzle (Fig. 14B - 191). The nozzle is movable both in radial (Fig. 14A) and in vertical directions (Fig. 14 and Fig. 15C). The wash tower moves the nozzle in radial positions (Fig. 15A) to enable treatment of vials in different reaction wheel circles as well cleaning of the wash nozzle in a rinsing vial outside of the wheel. The available wash nozzle positions are: 1. Outside the reaction wheel in a rinsing vial (Fig. 15A - 186). 2. ln isolation vials in the outer circle of the reaction wheel (Fig. 15A - 187). 3. ln analysis vials in the inner analysis circles of the reaction wheel (Fig. 15A- 188).

The first step in a vial and magnetic bead wash cycle is trapping of magnetic beads to the vial walls (Fig. 15B). The trapping is performed by placing magnets close to the vial wall (Fig. 2, Fig. 15B and Fig. 16 - 189). The magnetic bead trapping is started by lifting magnets from a lower non-bead-trapping position to a bead trapping position, at which the magnets rests against the vial wall. The embodiment used to transfer of the magnet from the non-trapping to the trapping position is a movable plate (Fig. 2 and Fig. 16 - 190), at which magnets (Fig. 16 - 189) are mounted. This plate is also carrying the vortex mixers (Fig. 16 - 199), required to re-dispense the trapped magnetic beads. When the reaction wheel is stepped, the plate is in the lower non- trapping position (Fig. 16 - 228). When a wash/mix sequence is activated, the plate is raised up into the magnetic bead trapping position (Fig. 16 - 229), at which the magnets are close to vials containing the magnetic beads. The magnets are mounted on the plate to trap the magnetic beads along the side of the vial slightly above the vial bottom (Fig. 15C). This enables the wash nozzles to be lowered to the vial bottom (Fig. 16 - 230), to completely aspirate all liquids in the vial. This will minimize the residual volume of liquid in the vial without any loses of magnetic beads during waste aspiration. The magnets are fixed to steel holders mounted on the plate using an adhesive. To enable magnetic trapping in both isolation and analysis vials, at the movable plate is equipped with magnets in all reaction wheel circles accessed by the wash towers Fig. 15C illustrates the liquid aspiration from vials. This is done while the magnetic beads are kept against the wall by the magnets during the wash. The wash cycle is composed of a liquid aspiration step to remove liquid waste from the vial and a wash buffer dispensing step to add wash buffer. The liquid handling by the wash nozzle is performed using an inner aspiration tube (Fig. 15C - 196) and an outer dispensing tube (Fig. 15C - 195). At the start of the wash cycle, the wash tower moves the wash nozzle (Fig. 14C - 191) between the upper stand-by position to the bottom of the vial. The positions are: 1. Upper position in standby while the reaction wheel is stepped. 2. Lower positions for start of liquid aspiration (dependent of volume in the vial). 3. Vial bottom position for aspiration of last liquid residue. 4. Positions for dispensing of wash buffers during the end of a wash cycle. Position 2 to 4 is positions defined by the analysis method protocols and are set according to the volumes used in the isolation and analysis processes.

The wash tower movements along the radial and vertical axis are achieved using stepper motors. The stepper motors can stack linear stepper actuators with 24 full steps per revolution. The radial motor (Fig. 14 - 192) utilizes an external nut design for compact integration, whilst the vertical motor (F ig. 14 - 193) uses a non-captive actuator in order to allow the tubing and radial actuator to be placed centrally in the tower. The transfer guides are linear glide bushings (Fig. 14 - 194) that slide on stainless steel shafts.

To make wash tower unit as compact as possible (allowing access to the confined space in vials etc.), the wash nozzles consists of two concentrically tubes (Fig. 15C). The outer tube (Fig. 15C - 195) used for the wash buffer dispensing is mounted around the circumference of the inner waste aspiration tube (Fig. 15C - 196). The 21 two tubes are attached to a PEEK block manifold (Fig. 14 - 197), which also comprises a slider bearing and nut support. The layout consisting of an inner tube that aspire liquid and an outer tube that dispense liquid, makes it easier to achieve the required wash buffer volumetric precision during a wash cycle. Also, the design secures that the wash buffer dispensing tube does not come in contact with the liquid waste. The wash cycle is done by running multiple wash sequences in the vial. ln order to avoid accidental trapping of magnetic beads by magnetic interaction with the nozzle, the material in the aspirating tube is using nonmagnetic lnconel. To avoid carry over between samples, the wash nozzle is carefully rinsed in the rinse cup (Fig. 15A - 186), placed on the plate outside the moving reaction wheel. The functionality to move the nozzle in radial direction enables the wash tower to treat vials in both isolation and analysis circles (Fig. 15A). After that the waste liquid has been aspirated from the vial and fresh wash buffer has been dispensed into the vial, the plate carrying the trapping magnets is lowered to the non-trapping position. The reaction wheel is stepped and the vial is moved to the next position in the washing unit, which is a vortex mixing position.

To enable mixing of samples, beads and reagents as well as re-suspension of trapped magnetic beads in new reagents or buffer, the reaction chamber is equipped with a number of positions having a mixing functionality. After each position in the reaction chamber, at which dispensing of samples and reagents into a vial have been done, a mixing device is located. Further mixing positions are located direct after each washing tower. Thus, the mixing positions are either placed outside of the wash station (Fig. 1 and 3 - 198, Fig. 3 - 150) or inside of the wash station (Fig. 1, 2 and 3 - 135). The details of the mixing functions located in the reaction chamber along the reaction wheel are illustrated in Fig. 16. The mixers in the wash station are used to enable the magnetic beads to be re-suspended in new portion of wash buffer after trapping and aspiration of used wash buffer (Fig. 16 - 135). ln the case the reagent dispensing position (Fig. 3 - 149) is located direct after a wash tower position, the required re-suspension of beads in the reagent is done in a mixing position following the dispensing of the reagents (Fig. 3 - 150). Since the magnetic beads after the finalization are still trapped to the vial wall, the beads have to be re-suspended in the dispensed reagents. After re-suspension of the beads in the reagents, the assay processing is continued in the required incubation.

The transfer plate (Fig. 16A - 190), carrying the bead trapping magnets, is also carrying the vortex mixer bases (Fig. 1 and 16 - 190). While the transfer plate is moved up, the mixer seats will be attached to the vials bottom and the vials will be lifted from their positions in the reaction wheel vial holders. The lifted vials are moved into mixer hats (Fig. 2 and 16 - 200). The mixer unit bases on the transfer plate are able to horizontal rotate in an oscillating manner by a 6V DC-motor mounted in the mixer base (Fig. 14 - 201). The mixer hats are flexible, enabling the vial to vortex, while the mixer base is oscillating. ln the inside of the mixer hats O-rings have been placed. The O-ring increases the friction between vial and hat and prevents the vial from spinning around its center axis during mixing. The movable plate carrying the mixers with their motors and the bead trapping magnets is raised using a set of 3 can-stack captive linear stepper actuators (Fig. 2, 16 - 202) supplied by Haydon Kerk. Each motor axis is equipped with an opto relay to enable position control of the plate. 22 Fig. 17 is illustrating the design of the luminometer. The Iuminescence measurement is performed by lifting the analysis vial from the reaction wheel vial position, to be transferred from the reaction wheel and placed in measurement position in the luminometer. At the luminometer the vial is loaded in a sleigh (Fig. 17 - 203), which is moved out from the luminometer house to receive the vial. There after the vial is moved into the luminometer by the sleigh. The sleigh is equipped with a lid (Fig. 17 - 204) closing the luminometer entrance, to keep the measuring chamber free from stray light. At the signal measuring position inside the luminometer, the vial is placed direct below the emitted light measuring unit (Fig. 17 - 205). The signal detection is performed using a photomultiplier with integrated electronics from Hamamatsu (Type H11890-110). The photomultiplier unit is located at the luminometer top (Fig. 17 - 206) and is totally sealed from stray light. During measurement, an aperture is adjusted the light level to fit the signal range of the photomultiplier. The aperture is consisting of a circular plate (Fig. 17 - 207), with holes of different diameter. The plate is rotated using a stepper motor (Fig. 17 - 208) and is positioned by reading fork (Fig. 17 - 209). The temperature in the luminometer is controlled to 37° C and the measurement of the signal is done after the temperature has been adjusted to 37° C.

Fig. 18 is a schematic figure illustrating of the design of the general hydraulics module. The hydraulic module is equipped with an extensive number of peristaltic pumps (Fig. 18 - 210). Each wash tower is connected to two individual peristaltic pumps, one for dispensing of wash buffer and one for aspiration of waste liquid. A pinch valve (Fig. 18 - 211) is positioned before each wash tower dispensing pumps. This allows the system to change between two different wash buffers during the assay run. The hydraulic module is equipped with Watson Marlow pumps using a set of 4 rollers, which means that it's possible to run the pumps in increments of 50 steps in order to maintain precision. A spring feature is present in the pumps to guarantee that a sufficient pressure is present on the tube independent of tolerances. The pumps are also equipped with pinches that prevent the tubes from moving through the pump, as it is running.

Fig. 19 is illustrating the embodiment of the thermostatic function in the process module. An aluminum plate in the bottom of the reaction chamber is heated using a self-adhesive silicone wire wound heater, lHP 180-13859 (Fig. 19 - 212). Air is circulated through the process chamber and forced to pass over the heated aluminum, using a fan of the type SUNON MFC0251V1-000U-A99 (Fig. 19 - 213). The heater box and the process chamber are encapsulated using top and side covers made from thermoformed plastic (Fig. 19 - 214). Pipettes will access the reaction wheel through pipetting holes, machined in the top cover. Holes in the compartment cover are machined for the rinse and waste tubings. Other units such as wash towers and luminometer are enclosed in the reaction chamber compartment. The temperature in the chamber is controlled and regulated at 37° C by a thermostating electronic circuitry, using sensors mounted at the vial level of the reaction wheel.

INSTRUMENT, FUNCTIONALITIES AND SYSTEM PROCESSES Fig. 20 is a schematic illustration the structure of the total isolation and analysis system. Besides the instrument, the system consists of a number of other products. These are chemistry based special reagents, buffers, liquids and calibrators arranged in test kits. The kits are applied in different types of vesicle isolation and marker 23 molecule analysis tests. The system is controlled and managed by the instrument electronics and embedded processing software, which is an integrated module in the instrument. The system is managed by the operator using an administrative system software. The software enables the operator to handle samples, reagents, test kits, instruments and to organize and prepare assay runs. During the assay run the software is used to control the processing and handle result reporting. The administrative software is run in an external desktop computer. Besides the reagents, consumables such as isolation and analysis vials, pipette tips and instrument spare parts are also included in the system.

Fig. 21 outlines the integrated isolation and analysis processes handled by the system. Using reagents, the instrument isolates biological microvesicles such as exosomes from a sample. The isolated microvesicles are washed and analyzed according to three main routes presented as A, B and C in the figure: A. The intact microvesicle surface proteins are analyzed in a process analog to a solid phase enzyme immunoassay, in which the micro vesicle isolation magnetic beads as solid phase is used.

The microvesicles are lysed, and the internal cargo components are released. ln the lysate, the protein components are analyzed in a process analog to a solid phase enzyme immunoassay, using as solid phase magnetic beads coated with antibodies specific for the component protein.

. Alternatively, micro-RNA or messenger-RNA in the obtained lysate is analyzed using molecular biology probe based methods, using reagents comprising a nucleotide probe coated magnetic bead and a nucleotide probe-enzyme conjugate.

The analytical processes are directly run after the microparticle isolation step and performed using the isolated and purified microvesicles. ln the Fig. 22 is schematically illustrated the detailed process steps to isolate the microvesicles and to after lysis analyze the vesicle contained protein molecules (type B analysis above).

The figure shows the pipetting of samples and reagents as well as the incubations, washings and signal measurement. The analysis processes applicable in the system are further illustrated in Figs. 23A, B and C. ln the figures are graphically shown the assay principles to isolate, lyse and analyse the microvesicle marker molecules. ln Fig. 23 row A is the process alternative A in Fig. 21 further outlined. ln the method is an outer surface protein molecule in membrane of the isolated microvesicle bound to the isolation magnetic bead analysed. To start the analysis, an aliquot of the microvesicle - magnetic bead suspension is transferred to an analysis vial. To this vial is a reagent consisting of a conjugate composed of an antibody against the surface protein and the enzyme alkaline phosphatase added. The mixture of magnetic beads and conjugate is incubated, and the antibody-enzyme conjugate is attached to the bead bound microvesicle surface protein. After incubation, the magnetic beads are carefully washed, and a luminescence substrate to the alkaline phosphatase enzyme is added to the washed beads (Adamantyl dioxethan phosphate, lnvitrogen lnc.). The mixture is incubated and the vial is transferred to the luminometer. While the conjugate enzyme splits the substrate, light is generated. The emitted light is measured by the luminometer. The signal is converted to a concentration of beads or surface antigens, using a calibration curve. ln Fig. 23 row B is the process alternative row B in Fig. 21 further outlined. ln the method is the isolated microvesicle lysed, the microvesicle internal cargo released 24 and cargo proteins analyzed using a sandwich immunoassay. The process applies a magnetic bead for isolation of microvesicles coated with an antibody specific for cargo proteins from the inside of the microvesicles. After Iysis of the microvesicles using a detergent containing buffer, an aliquot of the lysate is added to an analysis vial. The vial contains a magnetic bead coated with an antibody against a specific epitope of the protein component to be measure. ln the first of two immunoreaction incubation steps, the proteins in the lysate are bound to the magnetic beads. The beads are thereafter washed, and a conjugate reagent composed of an antibody against another epitope the protein molecule and the enzyme alkaline phosphatase is added to the washed magnetic beads. The mixture of magnetic beads and conjugate is incubated and the antibody-enzyme conjugate is bound to the protein molecules on the magnetic beads. After the incubation, the magnetic beads are carefully washed, and a luminescence substrate is added to the washed beads (Adamantyl dioxethan phosphate, lnvitrogen lnc.). The mixture is incubated and the vial transferred to the luminometer. While the conjugate enzyme splits the substrate, the emitted light is measured by the luminometer. The signal is converted to a concentration using a calibration curve. ln Fig. 23 row C is the process alternative row C in Fig. 21 further outlined. ln accordance with the method in row B, the isolated microvesicle is Iysed. The objective with the assay is to detect specific sequences in micro or messenger RNA. The analysis is performed using two types of nucleotide probe specific sequences in different parts of the RNA. The analysis is done in two steps, there as the RNA to be detected in a first step is bound onto magnetic beads coated with one of the nucleotide probe specific for the analyzed RNA sequence. ln a second step the RNA bound to the magnetic beads is detected, using a conjugate consisting of the other nucleotide probe coupled to the enzyme alkaline phosphatase. Thus, the RNA analysis method comprises of a first incubation to isolate the RNA in the lysate, using probe coated magnetic beads. The beads are there after carefully washed. The washed magnetic beads are in a second incubation incubated with a probe - enzyme conjugate. This conjugate is bound to the RNA trapped on the beads. After incubation, the magnetic beads are carefully washed and a luminescence substrate is added to the beads. The mixture is incubated and the vial transferred to the luminometer. While the conjugate enzyme is splitting the substrate and light is generated, emitted light is measured by the luminometer. The signal is converted to a concentration using a calibration curve.

INSTRUMENT OPERATION AND ASSAY PROCESSING To start an assay run, the operator enters all required analysis run data into the system administrative computer. The required data to be entered includes in the case of a clinical routine diagnostic run, sample identity and the assay methods to be tested on each sample. After that all analysis requests have been entered in the system software, the information is converted into "an assay run file". This file will be sent to the instrument computer and the instrument process software. The assay run file includes besides sample identities and type of tests to be run, also information defining the type of calibration applied for each method to be run. The file also includes information on the identity and number of method controls to be used. After the assay run information has been compiled by the instrument software, the system responds by printing a load list describing the vials, pipette tips, liquids, buffers, reagents, calibrators, controls and samples to be loaded in the instrument. ln instruments equipped with a module for internal cold storage of reagents, the software only requests loading of the reagents not available in the instrument. Othen/vise all required reagents as well as vials, pipette tips and samples have to be manually loaded by the operator.

Referring to Fig. 6 outlining the small sized batch analyzer, the operator first loads racks for the isolation and analysis vials in the appropriate storage modules (Fig. 6 - 117 and 118). There after the operator initiates the transfer of the vials to the reaction chamber process wheel (Fig. 6 - 123), applying the automated vial dispensing routine. This routine uses the gripper tools (Fig. 13 - 130), stored in the gripper tool stand stands (Fig. 6 and 13 - 171). The pipette nozzle (Fig. 11 - 132) in the rotating pipetting module used in the batch analyzer is able to handle two types of gripper tools. One tool is used to transfer the isolation vials and the other transfers the analysis vials. After that nozzle has been attached to a gripper tool (Fig. 13 - 130), the tool is released from the stand. The pipetting module is now using the vial gripper tools to transfer vials from the vial storage racks to the reaction wheel. The tools unload the vials at the vial loading position in the reaction wheel (Fig. 3 - 149). After all required vials have been loaded in the reaction wheel, the vial gripper tool is returned to the vial gripper tool stands. ln the case of the random access instrument no special vial loading run is required, since the vial transfer is included in the total assay processing. ln this instrument, the operator is loading two racks each of isolation vials and reaction vials at the appropriate positions (Fig. 4 - 109 and 111) to be used during the continuously performed assay run (manual reloading of vial racks are possible during the processing). After loading of vials into the reaction wheel in the batch instrument, the vial racks are removed and a pipette tip rack is loaded in the dedicated rack tray (Fig. 6 - 117). ln the random access instrument two pipette tip racks of each size is loaded at positions Fig. 4 - 156 and 157.

Referring to both Figs. 4 and 6, the operator thereafter loads reagent strips in the reagent storage modules (Fig. 4 - 101 and 120) in the random access instrument or the reagent strip storage rack (Fig. 6 - 119) in the batch instrument. The operator loads calibrator or calibration control strips (Fig. 4 - 159), assay methods controls (Fig. 4 - 158), system reagent bottles (Fig. 4 - 101), wash buffers and liquids bottles (Fig. 4 - 215 in their respective loading positions in the random access instruments. ln the batch instrument the operator loads calibrators, calibration controls and system reagents in the reagent storage rack (Fig. 6 - 119). Finally the operator loads the sample tubes in sample tube racks in the sample loading positions in the random access instrument (Fig. 4 - 100) and sample tube loading positions in the sample transfer tray in the batch instrument (F ig. 6 - 118). ln the case of the random access instruments, sample racks can be reloaded during the assay run.

The reagent storage module in the random access instrument is equipped with a QR- code reader automatically verifying the identity of reagents. With the QR-reader the required calibration parameters are entered into the assay run software. Furthermore the identities of sample tubes are in the random access instrument read by an inbuilt barcode reader. Consumables, system reagents and liquids are in the random access instrument checked using a manual QR-code reader. During loading of the batch instrument all identities of reagents, samples and consumables QR-codes are identified using a manual QR-code reader. ln the random access instrument, the QR- and barcodes are automatically verified during loading and continuously during the assay run. 26 After finishing all preparation steps, the operator starts the automated assay run. ln the random access instrument, isolation and analysis reaction vials are transfer from the vial storage positions to the reaction wheel as a part of the integrated automated process. Using the gripper tools, the pipette unit picks up the vials from the vial racks and dispenses these in the isolation and analysis vial dispensing positions in the reaction wheel (Fig. 3 - 149). The number of vials to be transferred depends on the number of assayed samples, controls and calibrator as well as the number of different methods to be run on each sample. Referring to Fig. 4 - 171, the two pipettes in the pipetting module are moved to the vial gripper tool stands, storing the vial gripper tools (Fig. 13 - 171). Each of the two pipette nozzles (Fig. 5 - 106 and 107) are attached to a gripper tool (Fig. 13 - 130) and the tool is released from the gripper tool stand. One of the pipette nozzles carries a gripper tool for handling the isolation vials and the other pipette nozzle carries the tool for handling the analysis vials (Fig. 4 - 231 and 232). The sample pipetting module is now using the vial gripper tools to transfer vials from the vial storage racks (Fig. 4 - 109 and 110) to the reaction wheel. The tools unload the vials at the vial loading position in the reaction wheel (Fig. 3 - 149). After all vials have been loaded in the reaction wheel, the vial gripper tools are returned to the vial gripper tool stands.

After the vials have been loaded in the reaction wheel, the microparticle isolation process is started by pipetting of isolation magnetic beads, other required isolation reagents and samples to the isolation vials. During pipetting, the pipettes (Fig. 5 - 105 and 106 and Fig. 9) are using disposal pipette tips stored in and picked from the pipette tip storage racks (Fig. 4 - 156 and 157). Magnetic beads for microparticle isolation, reagents and samples are aspirated by the pipetting module and dispensed in the vials in the reaction wheel at the main dispensing position (Fig. 3 - 149). The magnetic beads and reagents are transferred from the isolation reagent storage strip in the reagent storage module. The isolation reagent storage strips comprises vials carrying magnetic beads and other required reagents (Fig. 4 - 101). Samples and assay method controls are transferred from the sample and assay method control storage racks (Fig. 4 - 100). The pipetting in the batch analyzer is performed by the rotating pipette (Fig. 6 - 107) using disposal tips (Fig. 6 - 117), there as reagents (Fig. 6 - 119) and samples (Fig. 6 - 118) is pipette from the storages trays. The used pipette tips are discarded in the dry waste handling module (Fig. 4 and Fig 6 - 115). ln the random access instrument the pipetting of isolation magnetic beads, reagents and samples is performed during one operation cycle. The operation cycle comprises the time the reaction wheel between each step is stopped. The operation cycle time is defined according to the time needed to perform all required operations while the wheel is stopped. The operation step cycle step time is in most assay runs 60 seconds. To limit the number of pipette tips used in the batch analyzer, the assay run is started by an initial pipetting of only reagents. The reagent pipetting at the start up phase uses an accelerated step time. This enables the instrument to prepare the isolation vial in shorter time, before the sample pipetting is performed. The sample pipetting and the following processing are there after done, using the defined assay operation cycle step.

After dispensing of the samples and isolation reagents into the isolation vial, the reaction wheel is moved one step from the dispensing position (Fig. 3 - 149) to the mixing position (Fig. 3 - 150). At this position the sample and reagents are carefully mixed. During the following isolation incubation step, the reaction wheel is 27 transferring the vials from position to position in discrete steps, applying a defined operation cycle step time. When the reaction wheel is stopped, active processing such as mixing, addition of further reagents or washing is possible. This is done at positions there the supporting modules have access to the vials. lf no active processing module is able to operate at a position, the mixture in the vial is continued to be incubated.

During the following incubation the microvesicles are bound to the magnetic beads. After an incubation time of 40 minutes, the isolation vial enters into the wash station (Figs. 3 - 129). The incubation is now ended and the magnetic bead washing is started. This is initiated by raising magnet to the vial wall, using the magnet and mixer plate (Fig 16A - 190). The magnetic beads are trapped to the wall (Fig. 15B). After the magnetic beads are fixed to the vial wall, the wash nozzle is lowered into the vial. While the nozzle is lowered into the vial, an aspiration force is applied to the nozzle (Fig. 15 C and Fig. 14). After aspiration of the liquid containing non-bound components, portions of wash buffer are dispensed into and simultaneously aspirated from the vial. At the end of the wash cycle, a portion of wash buffer is left in the vial. There after the reaction wheel moves the vial to the next position in the wash module. At this position a vial mixing device applies a mixing force to the vial (Fig. 16 - 135). During the mixing, the magnetic beads are re-suspended into the wash buffer. The wash buffer acts on the magnetic beads to release unspecific components associated to the beads. Again, the reaction wheel is stepped and the vial reaches a second wash tower. The washing operation comprising magnetic bead trapping and buffer aspiration/dispensing are repeated. After this step wheel is again moved and the vial reaches a second mixing position, there mixing and magnetic bead re-suspension is undertaken. After a number of wash and mixing cycles, the unspecific associated components have been removed. The microvesicles carrying magnetic beads are considered to be washed.

After leaving the wash station module, the isolation vial contains magnetic bead carrying bound purified microvesicles. These beads are still trapped to the vial wall and have to be re-suspended in a buffer or a reagent. The vial has been transported one turn by the reaction wheel and has again reached the main dispensing position (Fig. 3 - 149). ln this position an aliquot of a buffer or other type of reagent is added to the vial using the pipetting module. For analysis of non-lysed intact microvesicles, a non-detergent containing buffer is added. To analyse the microvesicles internal components, a detergent supplemented buffer is added. ln the general both intact vesicles and luminal components is analyzed in the same assay run. To enable this process, the buffer added in the main dispensing position contains no detergent. Again, the wheel is stepped, and the vial reaches the main mixing position (Fig. 3 - 150), at which the magnetic beads are re-suspended in the non-detergent buffer. Thus the microvesicle membrane structure is kept intact and only the outer surface component is available in the following analysis processing.

Again the reaction wheel is stepped and the vial reaches the first between vial circles aliquot transfer position (Fig. 3 - 161). This position is used for transfer of incubates from the isolation circle to the analysis circles. The aliquot from the isolation circle is dispensed in the main analysis dispensing position (Fig. 3 - 220). The reaction wheel carries the analysis vials in the analysis circles. These vials have during the isolation processing been prepared for the analysis by addition of the reagents, required for the first analysis reaction. ln the case both intact and lysed microvesicles are 28 analyzed in the assay run, detergent containing lysis reagents are added to the isolation vials, still standing in the first aliquot transfer position by the pipetting module. After dispensing of the lysis reagent, the reaction wheel is stepped. The isolation vial now reaches the second mixing position (Fig. 3 - 218), at which the vial is mixed and the microvesicle is lysed. Again the reaction wheel is stepped and the isolation vial reaches the second transfer position (Fig. 3 - 219). ln this position, the isolation magnetic beads are trapped to the vial wall and aliquots of the lysate are transferred to an analysis vial in the inner analysis circles.

During the ongoing assay processing, the pipetting module has supplemented the analysis vials in the analysis circles with either antibody-enzyme conjugates (for analysis of surface proteins on intact microvesicles still bound onto the isolation magnetic bead), antibody coated magnetic beads (for analysis of protein components in the lysate released from the inside of the microvesicles) or molecular biology nucleotide probes coated magnetic beads (for analysis of RNA components in the lysate released from the inside of the microvesicles). To enable a parallel processing of these three types of methods, the random access analyzer is designed with three inner circles for these processing. After the pipetting of the non-lysed microvesicles bound to magnetic beads or the lysed microvesicles, the reaction wheel is stepped and the analysis reaction vial reaches the first analysis mixer in position (Fig. 3 - 221). After mixing, the wheel is stepped and the first analysis incubation is started.

During the first analysis incubation comprising intact microvesicles on the isolation magnetic beads, the antibody enzyme conjugate is bound to a surface protein on the microvesicle (the specificity of the antibody is directed to another surface antigen than the one used for binding of the microvesicle to the magnetic bead). The reaction wheel will stepwise rotate and approximately after 40 minutes incubation, the vial is reaching the wash module. The magnetic beads with the bound microvesicles are now carefully washed. After leaving the wash module, the vial enters the main analysis dispensing position (Fig. 3 - 220). At this position an enzyme substrate reagent is added to the vial. Thereafter, the reaction wheel is stepped and the vial reaches a mixing position (Fig. 3 - 221). ln this position the magnetic beads are re- dispensed in the substrate solution.

During the first analysis incubation of lysed microvesicles with antibody coated magnetic beads or nucleotide probe coated beads, marker molecules in the lysate from the microvesicles will be specifically bound to the beads. ln the case of antibody coated magnetic beads, the marker protein antigen is bound by the antibody coupled to the magnetic bead. ln the case of molecular biology probe coated magnetic beads, the marker micro- or messenger-RNA is bound by the nucleotide probe coupled to the magnetic bead. The reaction wheel will stepwise rotate and approximately after 40 minutes incubation, the vial is reaching the wash module. The magnetic beads with bound analyte protein or RNA are carefully washed. After leaving the wash module, the vial enters the main reagent dispensing position (Fig. 3 - 220). ln this position an aliquot of an antibody enzyme conjugate or a nucleotide probe enzyme conjugate is pipetted to the vial. The antibody enzyme conjugate is specific for binding to the marker protein to be measured, while the specificity is targeting another epitope in the marker than the one used to bind the protein to the magnetic bead. The nucleotide probe enzyme conjugate is specific for the RNA to be detected, while the nucleotide probe is specific for a part of the RNA not used in the binding of the RNA to the magnetic bead. The enzyme is alkaline phosphatase. The reaction 29 wheel is stepped and the vial is reaching a mixing position (Fig. 3 - 221). The vial is mixed and the second analysis incubation is started and the incubation the conjugates are during the incubation bound to the magnetic bead associated target marker molecules. Again, after 40 minutes incubation the vial is reaching the wash module and the magnetic beads with the bound molecules are carefully washed.

After leaving the wash module, the vial enters the main analysis dispensing position (Fig. 3 - 220). At this position an enzyme substrate reagent is added to the vials. Thereafter, the reaction wheel is stepped and the vial reaches a mixing position (Fig. 3 - 221). ln this position the magnetic beads are re-dispensed in the substrate solution. The substrate added to the magnetic beads is adamantyI-dioxethane- phosphate, which in the following conjugate enzyme reaction is split by the enzyme. During this reaction light is emitted from the reaction mixture. The emitted light is measured in the luminometer. During the enzyme reaction the reaction wheel with the analysis vials is stepped a number of steps, while the enzyme reaction and the light emission is reaching a steady state level. The vials are stepwise moved to the luminometer transfer position (Fig. 3 - 151), at which the pipette in the pipetting module using the gripper tool, transfers the vials to the luminometer. The vial is placed in the luminometer sledge (Fig. 17 - 203) and moved inside the luminometer. The luminescence signal is measured by the luminometer photomultiplier (Fig. 17 - 205) and a signal response value is registered by the process computer. The response is used to establish a calibration curve, control an established calibration curve or calculate concentration of unknown sample. After the signal is measured, the vial is returned to the reaction wheel.

To enable calculations of concentrations, a calibration curve is used. ln the assay run either a whole calibration curve is established by running a set of calibrators or an earlier obtained curve stored in the computer memory is used. ln the case a stored curve is applied, calibration controls have been included in the assay run, to check the validity of the stored curve. Using the calibration curve, the concentration of analyzed sample components is calculated. lf intact microvesicles are analyzed, the concentration of specific the surface antigens or total amount of microvesicles is calculated. lf lysed microvesicles are analyzed, the concentration of specific proteins or RNA from the interior of the microvesicle is calculated.

At the finalization of the assay run the liquid waste in the vial is aspirated at a liquid waste aspiration position (Fig. 3 - 154). There after the reaction wheel moves the vial to the dry waste removal position (Fig. 3 - 167), at which the vial is discarded to the dry waste container. This is done using a special vial transfer tower. ln the end of the assay run shift, the instrument is automatically rinsed and returned to stand by position.

The methods and processes applied by the instrument are developed to follow a general standard protocol, enabling all types of methods to use modules and devices included in the instrument in a similar way. The general process uses the same isolation and analysis vials, process and pipetting module. Also all modules supporting the process module as well as electronics, software control architecture and operator administrative software are adaptable to different instrument configurations. The resulting integrated instruments offer flexibility not previously available in automated analytical instruments used in microvesicle isolation and analysis. Furthermore due to the reduction of parts and common use of assay processing resources, the resulting systems are compact, cost effective and reliable.

While the embodiments have been described in connection with the preferred embodiments of systems, instruments, methods and their use to conduct isolation of biological entities and both immunoassay and nucleotide probe based methods are described herein in text and various figures, it is to be understood that similar embodiments may be used and that modifications and additions may be made to the described embodiments for performing the same function without deviating there from. Therefore the disclosed embodiment should not be limiting to any single embodiment, but rather should be constructed in breath and scope in accordance with the appended claims.

EXOSOME ANALYSIS PATENT EXAMPLES.

The following non-limiting example illustrates the operations of the two types of analytical systems described herein. The following examples employ modules and sub-systems of the type shown in Figs. 4 and 6 using processes outlined in Fig. 21A and Fig 23A, comprising isolation of microvesicles and analysis of micro vesicles surface proteins.

Example 1; Evaluation of antibodies for isolation of prostasomes and for detection of surface antigens on intact vesicles: For the isolation of the prostasomes magnetic beads with coupled antibodies were prepared using DynabeadsW' MyOneTM Streptavidine T1, 10 mg/ml (ThermoFisher, lnvitrogen, 65601) in PBS, pH 7.4, 0.1 % BSA and 0.02 % Sodium azide. The antibodies to be coupled were Biotinylated Anti- human CD9, Mouse lgG1Monoclonal (Biolegend 312112), Biotinylated Anti-human CD63, Mouse lgG1Monoclonal (Biolegend 353018) and Biotinylated Anti-human CD81, Mouse lgG1 Monoclonal (Biolegend 349514). 7.5 mg of the bead were washed 4 times in 1000 ul bead wash buffer, pH 7.4 (1.3 M NaCI, 0.1 M Phosphate). The washed beads were suspended in 750 ul wash buffer and divided in three vials with 2.5 mg beads in each vial. To the vials Biotinylated Anti-human CD9 lgG1 antibodies, Biotinylated Anti-human CD63 lgG1 antibodies or Biotinylated Anti- human CD81 antibodies (Mouse lgG1) in a volume of 100 ul was added (0.5 mg/ml in PBS, Phosphate Saline buffer, pH 7.2 and 0.09 % sodium azide). The mixture was incubated during 30 minutes at room temperature using a SARM|X®). Thereafter the beads were washed 5 times with 1000 ul PBS. The washed antibody coated beads were suspended in 1000 ul of the pH 7.4 Phosphate wash buffer.

To start an assay testing the efficiency of the prepared magnetic beads and avidity of the antibodies, 6 aliquots of 10 ul of each bead type were pipetted into isolation vials (three different antibody beads and 6 vials of each type e.g. 18 vials were used in the assay run). To each vial 600 ul wash buffer were added. The beads were washed three times in 700 ul using the wash buffer. The beads were after washing suspended in 700 ul. Thereafter 3 ul of exosome samples (Prostasomes collected from healthy men semen, suspended in PBS after preparation using ultracentrifugation) were added to 9 of the vials (three vials of each bead type). To the remaining 9 vials no samples were added (zero-samples). To all 18 vials 3 ul FC- blocker (Trustain FCX, Biolegend 422302) were added. The vials were incubated at 37° during 40 minutes. The beads were thereafter washed three times in 700 ul wash buffer. After the last washing, the trapped beads were suspended in primary antibody solutions according to the set up listed in TABLE I. 31 Analysis of the isolated prostasomes were performed using three types of primary antibodies Anti-CD9 antibody (Monoclonal, rabbit, 0.602 mg/ml, Abcam 92726), Anti- CD63 antibody (Monoclonal, rabbit, 0.602 mg/ml, Abcam 134045) and Anti-CD81 antibody (Monoclonal, rabbit, 0.602 mg/ml, Abcam 219209). ln background controls an iso-type antibody was used (Rabbit Purified lgG, AS10 916, Agrisera, 11.1 mg/ml). The Primary antibodies were diluted (1.5 ul antibody solution + 1500 ul wash buffer, dilution 1:1000). The iso-type antibody was diluted to match the primary antibody solutions (2 ul antibody solution + 738 ul wash buffer (dilution1:370), followed by 40 ul of the 1:370 dilution + 1960 wash buffer (dilution 1:18 500), which is comparable to the primary antibody concentrations in dilutions of 1: 1000. These 4 antibody dilutions were used as primary antibodies as is shown in TABLE I, listing the set up of the second immune reaction step. ln this is the efficiency of primary antibody binding to the surface antigens at the magnetic bead trapped prostasomes measured Capture Vial Prostasome Primary antibody nr Sample antibody volume (ul) CD9 1 0 CD81 2 0 CD63 3 0 lsotype 4 3 CD81 5 3 CD63 6 3 lsotype CD63 7 0 CD9 8 0 CD81 9 0 lsotype 10 3 CD9 11 3 CD81 12 3 lsotyp CD81 13 0 CD9 14 0 CD63 15 0 lsotype 16 3 CD9 17 3 CD63 18 3 lsotype TABLE I Patent example "lsolation of prostasomes and detection of surface antigens on intact vesicle" experiment set up The bead containing vials, primary antibodies and iso-type diluted antibodies reagent solutions were pipetted according to the TABLE l. The beads were re-suspended in the antibody reagents and the vials were incubated during 40 minutes at 37° C. The incubation was stopped by washing of the beads 5 times using 700 ul wash buffer. To each vial 200 ul of a secondary antibody-enzyme conjugate was added (Goat Anti- Rabbit lgG Fc -Alkaline Phosphatase pre-adsorbed 1 x 500 UG, Polyclonal, 0.5 mg/ml, Abcam 98461, diluted 115000 in wash buffer). The beads were re-suspended in the antibody-enzyme conjugate reagent and the vials were incubated during 40 minutes at 37° C. The incubation was stopped by washing of the beads 5 times using 32 700 pl wash buffer. . The beads were re-suspended in 50 pl analyse buffer (1.5 M NaCl, 0.5 M Tris-base, 10 mM MgClz, 0.25 % NaN3, pH 9.7). The detection enzyme reaction was started by adding 50 pl substrate (CDP-Star Ready-to-Use substrate solution with Emerald ll, Tropix, lnvitrogen, used according to the packet insert). The beads were incubated during 8 minutes at 37° C. The vial was transferred to analysis vials and thereafter entered into the luminometer. The luminescence signal was measured. The signal-to-noise for each positive sample was calculated relative the zero-sample. The data is presented in Fig. 24A. The data shows that exosomes are isolated and detected by the primary antibodies. lt is also shown that anti-CD9 is most effective for trapping and anti-CD 81 is a suitable primary antibody.

Example 2; Investigation of background effects in assay for isolation of cancer dendrite cell exosomes and for detection of surface antigens on intact vesicles: For the isolation of the exosomes magnetic beads with coupled antibodies were prepared using DynabeadsW' MyOneW' Streptavidine T1, 10 mg/ml (ThermoFisher, lnvitrogen, 65601) in PBS (pH 7.4, 0.1 % BSA and 0.02 % Sodium azide). The antibodies to be coupled were Biotinylated Anti-human CD9. 2.5 mg beads were washed 4 times in 1000 pl bead wash buffer pH 7.4 (1.3 M NaCl, 0.1 M Phosphate). The washed beads were suspended in 750 pl. To the vial, antibody reagent consisting of Biotinylated Anti-human CD9 lgG1 in a volume of 100 pl was added (0.5 mg/ml in PBS, Phosphate Saline buffer, pH 7.2 and 0.09 % sodium azide). The mixture was incubated during 30 minutes at room temperature using a SARMIX®). Thereafter the beads were washed 5 times with PBS. The washed antibody coated beads were suspended in 1000 pl of the pH 7.4 Phosphate buffer.

To start an assay to investigate background effects such the unspecific background of assay concept, 8 times 10 pl of anti-CD9 coated bead suspension was pipetted into isolation vials. To each vial 700 pl wash buffer was added. The beads were washed three times using 700 pl the wash buffer. After washing were the beads suspended in 700 pl wash buffer. To the beads were 20 pl exosomes added to 3 of the vials (isolated from dendrite melanoma cancer cells grown in a cell culture using ultracentrifugation). To the remaining 3 vials were no samples added (zero-samples). To all 6 vials were 3 pl FC-blocker (Trusting FCX, Biolegend 422302) added. The vials were incubated at 37° during 40 minutes. The beads were washed three times in 700 pl wash buffer. After the last washing, were the magnetic beds suspended in wash buffer.

To prepare the following immune incubation were reagents added to each vial according to Table Il. The relevant detection primary e.g. antibody anti-CD81 antibody (Monoclonal, rabbit, 0.602 mg/ml, Abcam 219209) was added to vials 1 and 4, of which 4 is zero-samples since no exosomes were added to the beads. ln other background, controls an iso-type antibody was used as primary antibody (Rabbit Purified lgG, AS10 916, Agrisera, 11.1 mg/ml). This antibody was added to vials 2 and 5. The primary antibody anti-CD81 was diluted (1 .5 pl antibody solution + 1500 pl wash buffer (dilution 121000). The iso-type primary antibody was diluted to match the primary anti-CD81 antibody solutions (2 pl antibody solution + 738 pl wash buffer (dilution1:370), followed by 40 pl of the 11370 dilution + 1960 wash buffer (dilution 1:18 500), which is comparable to the primary antibody concentrations in dilutions of 1: 1000. These 2 antibody dilutions were used as primary antibodies in the Table ll below. ln vials 3 and 6 were only aliquots of buffer pipette. The objective of the following reaction is to couple the primary antibody to the surface protein CD81 at the 33 magnetic bead trapped exosomes. The beads were re-suspended in the antibody reagents and the vials were incubated during 40 minutes at 37° C.

Vial Beads 10 Sample Primary lso-type- Seondary no pl (20 pl) antibody antibody antibody (25 pg) 200 pl 200 pl enzyme Capture- (0,602 pglml) (0,602 pglml) conjugate 200 antibody pl 1:10 000 1 T1 - oi-CD9 DC Exo oi-CD81 - d-Rabbit lgG 2 T1 - a-CD9 DC Exo - Rabbit lgG d-Rabbit lgG 3 T1 - d-CD9 DC Exo - - d-Rabbit lgG 4 T1 - oi-CD9 - d-CD81 - a-Rabbit lgG 5 T1 - oi-CD9 - - Rabbit lgG d-Rabbit lgG 6 T1 - d-CD9 - - - oi-Rabbit lgG TABLE ll Patent example "Isolation and analysis of dendrite cell exosomes investigation of background effects" experimental set up The incubation was stopped by washing of the beads 5 times using 700 pl wash buffer. To all vials, 200 pl of a secondary antibody-enzyme conjugate was added (Goat Anti-Rabbit lgG Fc - Alkaline Phosphatase pre-adsorbed 1 x 500 UG, Polyclonal, 0.5 mg/ml, Abcam 98461, diluted 115000 in wash buffer). The beads were re-suspended in the antibody-enzyme conjugate reagent and the vials were incubated during 40 minutes at 37° C. The incubation was stopped by washing of the beads 5 times using 700 pl wash buffer. The beads were re-suspended in 50 pl analyse buffer (1.5 M NaC|, 0.5 M Tris-base, 10 mM MgClg, 0.25 % NaN3, pH 9.7). The detection enzyme reaction was started by adding 50 pl substrate (CDP-Star Ready-to-Use substrate solution with Emerald ll, Tropix, lnvitrogen, used according to the packet insert). The beads were incubated during 8 minutes at 37° C. The vial was transferred toanalysis vials and thereafter entered into the luminometer. The luminescence signal was measured. The signal-to-noise for each positive sample was calculated relative the zero-sample. The data is presented in Fig. 24B. The data shows that all zero-samples and background controls have low signal levels and the positive sample show a high signal level.

Claims (2)

1. A system with instruments for performing automated integrated isolation biological microvesicles such as exosomes from biological samples and analysis of molecular components contained on in these microvesicles there as, said system is comprising: (a) a common isolation and analysis processing sub system reaction module; (b) a pipetting module; (c) detection module such as a luminometer, fluorimeter or multiplex detection fluorimeter; (d) a plurality of additional required modules, Wherein the common for the process required subsystem module is configured to position one or more isolation and analysis vials containing aliquots of the microvesicle containing sample from which microvesicles such as exosomes are isolated and directly analyzed by the instrument using a variety of different biochemical, immunological and molecular biology methods.

2. The system with instruments of claim 1, wherein the isolated and purified microvesicles such as exosomes are analyzed in a plurality of procedure with the objective to detect and measure: (a) the molecules at and protruding out from the outer surface of the microvesicles such as surface proteins using immunoassay-based methods and other relevant surface analyzing technologies; (b) the free internal cargo proteins contained in the microvesicle, released by lysing the microvesicle using detergents and other analogous methods analyzed using immunoassay-based methods and other relevant analogous technologies; (c) the free internal cargo RNA such as micro- and messenger-RNA contained in the microvesicle, released by lysing the microvesicle using detergents and other analogous methods, analyzed using molecular biology methodology involving probes, reverse transcription, and amplification technologies. (d) the free internal cargo enzymes from the microvesicle, released by lysing the microvesicle using detergents and other analogous methods, analyzed using enzymatic methodology and other relevant technologies. 4. The system with instruments of claim 3, wherein the applied immunoassay technologies is configured for fluorescent and luminescence detection and measurement of assay response driven with or without enzymes. 5. The system with instruments of claim 4, wherein the applied immunoassay technologies configured for fluorescent detection and measurement of assay response includes an optional methodology applying multiplex fluorescence analysis on a plurality of different molecules contained in the microvesicle. 6. The system with instruments of claim 1 is comprising a number of configurations including alternatives consisting batch analyzing or random access analyzing including continued loading of samples, reagents and consumables. 7. The system with instruments of claim 1 is comprising a functionality for export and unloading of the isolated and purified microvesicles into discrete vials or into vials integrated in a micro titer plate. 8. The system with instruments of claim 7, wherein the common process subsystem module comprises: 38 (a) an assembly comprising a carousel having a plurality of isolation and analysis vials positions placed in concentric circles, enabling isolation and analysis to be performed both separately and in close contact; and (b) a plurality of isolation and analysis vials designed to optimally be applied in the isolation and the analysis processing, wherein the plurality of isolation and analysis vials is positioned within the plurality of vial positions to enable aliquots to be transfer between the vials to integrated perform isolation and analysis. 9. The system with instruments of claim 8, wherein the plurality of additional modules comprise a pipetting module, one or more sample, controls, calibrator and reagent modules, a mixing modules, a washing station modules, a reaction vial loader and response signal measuring modules. 10. The system with instruments of claim 9, wherein the plurality of modules is designed to service both the isolation and the analysis vials in the different isolation and analysis reaction circles of the reaction module. 11. The system with instruments of claim 10, wherein the washing station modules is designed to service both the isolation and the analysis processing vials in the different isolation and analysis circles of the reaction module wheel. 12. The system with instruments of claim 11, wherein the vial transfer tool devices is designed to move both the isolation and the analysis vials from storage racks in storage modules to, between and from the isolation and analysis vial position in all circles in the reaction in the reaction module wheel. 14. The system with instruments of claim 12, wherein the solid phase immunoassay analysis processes is performed using para-magnetic beads or discrete molecule coated vials as solid phase. 15. The system with instruments of claim 12, wherein the analysis reaction response detection and signal measurement is performed either by transfer the analysis reaction vial to a separated dedicated detection module separated from the reaction vials holders in the reaction wheel or the reaction solution is aspirated into the measuring cell of an analysis measurement module equipped with an aspiration nozzle moved into the vial. 16. The system with instruments of claim 11, wherein the plurality of isolation vials processing samples in the range of 10 - 2000 ul. 16. The system with instruments of claim 11, wherein the plurality of analysis process vials processing samples in the range of 10 - 300 ul having an optical clarity consisting of plastic. 17. The system with instruments of claim 11, wherein the pipetting module includes pipettes covering a volume range of 1 - 1000 ul. 18. The system with instruments of claim 11, wherein the one or more samples and reagent storage modules each comprises a plurality of reagent vials and bottle storage compartments.