Order for individualized patient blood analysis

Abstract

Arrangement for individualized in-vitro patient blood analysis comprising a holography module (1), a Raman spectroscopy module (2) and a biomarker module (3), characterized in that the holography module (1), the Raman spectroscopy module (2) and the biomarker module (3) are microfluidic, wherein - the holography module (1) consists of a holography sample preparation cartridge (11) and a holography sample measurement unit (12) which are microfluidically connected to each other, wherein the holography sample measurement unit (12) is downstream of the holography sample preparation cartridge (11) in the direction of flow, - the Raman spectroscopy module (2) consists of a Raman spectroscopy sample preparation cartridge (21) and a Raman spectroscopy sample measurement unit (22) which are microfluidically connected to each other, wherein the Raman spectroscopy sample measurement unit (22) is downstream of the Raman spectroscopy sample preparation cartridge (21) in the flow direction, - the biomarker module (3) consists of a biomarker sample preparation cartridge (31) and a biomarker sample measurement unit (32) which are microfluidically connected to each other, wherein the biomarker sample measurement unit (32) is downstream of the biomarker sample preparation cartridge (31) in the flow direction, wherein the holography sample preparation cartridge (11), the Raman spectroscopy sample preparation cartridge (21) and the biomarker sample preparation cartridge (31) are fluidically connected via fluid channels through an integrated microfluidic flow control module (4), and the holography sample preparation cartridge (11), the Raman spectroscopy sample preparation cartridge (21), the biomarker sample preparation cartridge (31) and the flow control module (4) data- and information-guiding with a central control and computer unit (5) which has integrated control software by means of which all processes are controlled and regulated,are connected to a database (6) via an information-conducting link, wherein the modules (1, 2 and 3) are fluidically connected via the flow control unit (4) to a common blood sample feeder (73) and further fluid material feeders (7) via fluid guideways, and the fluid guideways can be controlled and the fluid feeders (7) can be selectively switched on via valves (41) controlled by the control and computer unit (5), and the control and computer unit (5) accesses the database (6), so that a data comparison of the recorded and processed data of the modules (1, 2 and 3) with the reference data stored in the database (6) and a complex data evaluation can be generated.

Description

[0001] The invention relates to an arrangement for individualized patient blood analysis, in particular for sepsis examinations.

[0002] According to current technology, large quantities of blood are taken from the patient being examined during clinical diagnostics in order to subsequently subject the blood to a wide variety of analyses, which are often carried out at different laboratory workstations or even in different laboratories.

[0003] In cases of suspected infectious diseases, for example, blood tests are performed to determine the number and shape of the cellular blood components, tests with various specific biomarkers are carried out to qualify and quantify different infectious agents (whereby each biomarker usually has to be measured in a separate test), and microbiological analyses are performed by culturing and subsequently characterizing the pathogens.

[0004] The disadvantage of these common clinical examinations is that several individual tests have to be performed, requiring several milliliters of patient blood, which are also often very time-consuming (microbiological test results are often only available after 2 or more days) and which mostly do not provide all relevant information (e.g., no information on the activation state of the leukocytes is currently recorded).

[0005] A simple system solution based on biomarkers that provides the full range of information desired by the physician has not yet been created.

[0006] The press release from the Institute for Photonic Technologies eV entitled “Less Blood, More Information” dated November 19, 2013 (URL: http: / / www.leibnizipht.de / uploads / media / IPHTPresse2013_Hemospec_Medica.pdf) reveals the goal of the EU project Hemospec: to develop a handy device as a system solution that massively improves the analysis process. The key to improving blood analysis lies in the use of optical technologies, which utilize the unique properties of light to obtain even more information beyond the classic blood count, and in the shortest possible time.

[0007] To achieve this goal, the publication proposes combining various optical techniques on a modular platform.

[0008] A few drops of blood should be enough to be distributed, mixed and filtered via microfluidic channels on the platform.

[0009] Holography, Raman spectroscopy and the reading of fluorescently labeled biomarkers are intended to provide further clues about the patient's condition from their blood.

[0010] The disadvantage of this revelation is that it remains unclear how exactly the individual parameters must be designed when combining holography, Raman spectroscopy and classical blood count, three technologies with completely different requirements for the respective parameters (in particular, no fluid management is specified), in order to obtain even more information from a whole blood sample in the shortest possible time, going beyond a classical blood count.

[0011] WO 2008052221 A2 discloses the use of coherent Raman methods for medical diagnostic and therapeutic purposes, wherein a system and a method are provided to enable non-invasive, in vivo and real-time molecular detection and quantification of molecular species in a sample or animal.

[0012] This enables non-invasive, quantitative detection of molecular species continuously and in real time, for example to track the course of a therapy.

[0013] DE 10 2012 016 318 A1 discloses an arrangement for a lensless, holographic inline reflected-light microscope, preferably used for the detection of extended and / or moving biological samples with high lateral and depth resolution. The proposed arrangement is particularly suitable for three-dimensional reflected-light imaging of a sample from a single measurement (3D snapshot), making it especially suitable for the reconstruction of relatively fast three-dimensional motion sequences of microscopic objects, e.g., in microfluidics. Further applications of the proposed arrangement include underwater microscopy, the microscopy of biological samples, and quantitative phase microscopy. The particularly noteworthy advantage of this arrangement lies in its use for the non-destructive online examination of extended surfaces in materials testing, medicine, etc.

[0014] The essence of this technical solution lies in the fact that, due to the unambiguous separation of reference and illumination wave fields, no additional conditions need to be imposed on the sample. Furthermore, the parallel, sequentially arranged, flat components (illuminator, sensor chip, semi-transparent mirror, etc.) used in the proposed configuration enable an extremely compact overall design, eliminating the need for any type of imaging optics (objective, lens). This design makes it possible to avoid the phase-shifting techniques typically used in interferometry, which otherwise result in the secondary images (twin image) characteristic of inline holography.

[0015] German patent DE 10 2004 034 354 B3 discloses an ultra-compact Raman spectrometer intended for use in the investigation of chemical structures, etc., or for the quasi-simultaneous identification of many bioparticles on substrates. The objective of providing such a spectrometer, which can be used as a true handheld device, is achieved by positioning the sample (31) under investigation directly in the imaging plane of the optical spectral unit (22). The optical excitation of the sample (31) by means of focused radiation from the laser (1) occurs precisely in the plane of the virtual slit position of the imaging spectral unit (22), and a Raman-transmitting filter (21) is arranged exclusively in the beam path between the sample (31) and the spectral unit (22).

[0016] The publication SEO, Sungkytu [et al.]: Lensfree holographic imaging for on-chip cytometry and diagnostics. In: Lab on a Chip, Vol. 9, 2009, No. 6, pp. 777-787. -IDDN 1473-0197 also discloses a lensless, holographic inline system for cytometry and diagnostics.

[0017] US 2014 / 0 327 944 A1 discloses a method for processing holographic intensity data, the method comprising: • Receiving holographic intensity data comprising at least one holographic intensity pattern or image at a discrete location in a propagation space, wherein the propagation space comprises a space over which the illumination associated with generating the holographic intensity pattern propagates at least to facilitate the generation of the holographic intensity data, • Processing the received holographic intensity data to determine one or more data key points of at least one potential object of interest in the received holographic intensity data, and • Comparing the specified one or more data key points with at least one predetermined object descriptor associated with an object to determine a match, where the object descriptor is a propagation space invariant.

[0018] DE 10 2008 047 240 A1 discloses a method for identifying individual viruses in a sample, in which the sample is preferably pretreated for size sorting, bound to a support surface and examined for viruses using an imaging method, wherein the height profile of the support surface with the bound sample is scanned by a probe, that in addition to the height profile scanning of the support surface, at least selected scanning locations determined from the height profile of the support surface are each irradiated with monochromatic excitation light and the spectrum of the Raman scattered light occurring at the scanning location as a result of the light excitation is recorded in or on the probe and wherein the Raman scattered light recorded at the scanning locations is each compared with reference values, from this comparison a conclusion is drawn about the individual virus present at the scanning location.

[0019] The Raman scattered light is simultaneously captured with the height profile scanning of the carrier surface for each scanning location of the carrier surface in or on the probe, whereby the Raman scattered light captured synchronously with the height profile scanning is compared with reference values ​​for each scanning location selected according to the height profile for virus identification at that location.

[0020] The scanning process, using a probe, employs the well-known AFM method. From the height profile obtained through surface scanning, sampling points are selected (either simultaneously with or after the scanning process) that, based on their surface structure (height profile size), suggest the presence of a virus. These sampling points, selected according to the height profile, are each irradiated with monochromatic excitation light and spectrometrically analyzed for the Raman scattering that occurs at the sampling point as a result of the light excitation. By comparing these Raman scattering results with reference values, particularly values ​​from an electronic database, the presence of a single virus at each sampling point is determined.

[0021] This proposed method aims to detect individual viruses in any (solid, liquid or gaseous) sample.

[0022] This method allows for the precise identification of viruses or bacteriophages without time-consuming and material-intensive sample preparation. This makes it possible to obtain reliable information about the type and composition of virus particles in a sample, leading to accurate and unambiguous identification of the particles. This method is universally applicable to all viruses, regardless of which cells the viruses infect or the specific type of virus.

[0023] The publication KRAUSE, Mario [et a.]: Localizing and identifying living bacteria in an abiotic environment by a combination of Raman and fluorescence microscopy. In: Analytical Chemistry, Vol. 80, 2008, No. 22, pp. 8568-8575. -ISSN 0003-2700 discloses the combination of Raman and fluorescence microscopy.

[0024] The disadvantage of the aforementioned technical solutions is that no single arrangement consisting of a lensless, holographic inline reflected-light microscope, an ultra-compact Raman spectrometer, and a biomarker system is provided, nor is any technical solution suggested as to how exactly the individual parameters must be configured when combining holography, Raman spectroscopy, and a classic blood count (three technologies with completely different requirements for the respective parameters) (in particular, no fluid management is specified for combining these three systems into a single arrangement) in order to obtain even more information from a whole blood sample in the shortest possible time, going beyond a classic blood count.

[0025] The object of the present invention is to provide an arrangement for individualized in vitro patient blood analysis, in particular for sepsis examinations, which avoids the aforementioned disadvantages of the prior art and enables a meaningful examination result in less than 1 hour using a blood quantity of only 1 to 2 ml.

[0026] According to the invention, this problem is solved by the characterizing features of claim 1. Further advantageous embodiments of the invention are specified in the dependent claims.

[0027] The essence of the invention lies in the fact that the arrangement for individualized patient blood analysis, in particular for sepsis examinations, simultaneously comprises a holography module, a Raman spectroscopy module and a biomarker module, which are connected in a data and information-conducting manner to a central control and computer unit, which is connected in an information-conducting manner to a database, whereby this arrangement enables a multimodal and very fast blood analysis.

[0028] The three modules of the arrangement are microfluidic, each consisting of a sample preparation cartridge and a sample measurement unit, the cartridges being fluidically connected to various fluid feeds (including a central blood sample feed) via an integrated microfluidic flow control module, which is also connected to a central control and computer unit in a data and information-conducting manner.

[0029] The integrated microfluidic flow control module allows the sample to be loaded at a single loading position and then automatically divided for the individual sample preparation cartridges and sample measurement units.

[0030] This modular design of the arrangement offers, among other things, the following advantages: - Decoupling of the modules from each other with regard to optical and electrical interference - Optimally short path within the modules - Easy exchange through further developed modules - Service friendliness - Combination of different technologies with integrated evaluation

[0031] Due to the modular, microfluidic design, multimodal analysis of minute amounts of patient blood (only about 1-2 ml) is possible for patient stratification (e.g., of patients with and without infection as well as with and without hyperinflammatory immune response), whereby the final results provided by the modular arrangement are available much faster than the state of the art (less than 1 hour) and simultaneously provide the physician with deeper insights into the disease picture and differentiation possibilities.

[0032] The invention is explained in more detail below with reference to the schematic drawing and the exemplary embodiment. The drawing shows: Fig. 1: A schematic representation of an embodiment of the arrangement for individualized patient blood analysis.

[0033] The one in Fig. The arrangement shown for individualized patient blood analysis comprises a holography module (1), a Raman spectroscopy module (2), a biomarker module (3) and an integrated flow control module (4), which are connected in a data and information-conducting manner to a central control and computer unit (5), which is connected in an information-conducting manner to a database (6).

[0034] The holography module (1), the Raman spectroscopy module (2) and the biomarker module (3) are microfluidically connected via the flow control unit (4), which preferably consists of one or more fluidically and control-technically coupled units, to a common blood sample feed (73) and further fluid material feeds (7).

[0035] These additional fluid material feeds (7) are a dye solution feed (71), a buffer feed (72), a blood sample feed (73), a biomarker 1 feed (74), a biomarker 2 feed (75), a biomarker 3 feed (76), and feeds for service and cleaning fluids (78).

[0036] The holography module (1) consists of a holography sample preparation cartridge (11) and a holography sample measurement unit (12) which are microfluidically connected.

[0037] The Raman spectroscopy module (2) consists of a Raman spectroscopy sample preparation cartridge (21) and a Raman spectroscopy sample measurement unit (22) which are connected microfluidically.

[0038] The biomarker module (3) consists of a biomarker sample preparation cartridge (31) and a biomarker sample measurement unit (32) which are connected microfluidically.

[0039] The flow control module (4) can consist of tubing and / or a microstructured substrate with microfluidic channels in which controllable valves (41) are arranged, such that ordered fluid pathways can be generated in such a way that the fluid pathway from the common blood sample feeder (73) is split into three fluid pathways leading to the cartridges (11, 21 and 31), whereby the fluid feeders (7) can be selectively switched on via the valves (41) controlled by the control and computer unit (5). The valves (41) can be arranged in a valve block and, for example, be designed as pinch valves.

[0040] The fluid control module is implemented as an elastomer chip system (multi-length chip system) with integrated reagent templates, conveying channels, a valve block and a connection block for the fluid connectors to the analysis modules and optionally integrated filter structures for separation tasks.

[0041] Fluid delivery is based on the principle of slip-free linear peristaltic pumps. These convert a feed rate into a defined volume flow. This eliminates the need for integrating additional sensors for flow rate measurement and control.

[0042] Useful monitoring functions, such as checking the absence of bubbles in the pumped fluid and evaluating filtration processes and dyeing procedures in the cartridge, are optionally provided on the flow control module (4) (in the Fig. 1 not shown).

[0043] For the production of the flow control module (4), contactless imaging methods are provided, which, when using optically transparent materials for the base plate, can be integrated as a non-contact sensor and monitoring unit for functional control. For this purpose, an optical monitoring system must be integrated below the chip cartridge in the system, so that sufficient installation space and optical accessibility for image-based monitoring of the system function are realized.

[0044] The elastomer chip system consists of a rigid, optically transparent base plate (e.g., made of glass or plastic) against which a liquid-tight elastomer molded part is arranged. This molded part includes the reagent receptacles, connecting channels, conveying channels, filter elements, valve elements of the valve block, and the connection block for the fluid connectors, as well as optional filter elements and auxiliary structures for efficient fluid mixing. The chip system can be designed to be, for example, the size of a check or credit card.

[0045] The base plate is a plate made of inelastic, optically transparent material, which serves as a carrier for the elastomer molded part and is permanently connected to it by gluing / bonding.

[0046] Base plate materials can be glass, silicon, metals, thermoplastic polymers (polycarbonate, COC, PVC, polystyrene), thermosetting polymers (e.g., electronic circuit boards) or ceramic substrates (LTCC and ceramic plates).

[0047] The elastomer molded part is manufactured, for example, using replication techniques (injection molding, molding of a master made of PDMS, silicone rubber or silicone materials = elastomer materials with Shore hardnesses in the range between 10 and 120).

[0048] The elastomer molded part is attached to the base plate using adhesive or bonding methods. When using a PDMS elastomer molded part and a glass base plate, self-bonding of the molded parts after surface activation with oxygen plasma is recommended.

[0049] The conveying channels are designed as preferably semi-circular linear channels, which are actuated using the operating principle of linear peristaltic pumps. For this purpose, a plunger is pressed onto the top of the chip above the conveying channel to be actuated, so that it is completely squeezed at the point of contact. As the plunger moves along the channel direction, the fluid follows the plunger and is thus conveyed.

[0050] The flow rate Q is calculated from the feed rate of the piston u and the cross-sectional area of ​​the conveying channel A as Q = A * u. The conveying channel is connected to a fluid reservoir on one side and opens into the valve block on the opposite side. The channel dimensions are between 0.3 and 6 mm (width) and 0.15 and 3 mm (height).

[0051] The connecting channels are integrated into the elastomer molded part and have smaller channel cross-sections compared to the conveying channels, with channel widths in the range of 0.1 and 1.2 mm and channel heights in the range of 0.05 and 0.8 mm.

[0052] The reagent reservoir is designed as cavities integrated into the elastomer section, optionally equipped with a septum. Volumes range from 5 µl to 500 µl (e.g., for service and cleaning fluids).

[0053] The valve block is implemented using an arrangement of pinch valves. Each valve position contains a valve plunger which can be lowered, thereby pinching off the elastomer channel and sealing it securely.

[0054] The arrangement is designed such that fluids can be directed from any inlet into the valve block to any outlet of the valve block. Inlets can also be operated bidirectionally as outlets from the valve block.

[0055] In this way, fluids from conveying channels can be directed to any outlets - or, in the case of parallel conveying from several conveying channels, combined with each other and either collected in one conveying channel or conveyed as a mixture through one of the chip-side outlets to the analysis stations.

[0056] This simplicity of implementation makes it possible to combine fluid conveying with flexibly configurable sample preparation steps.

[0057] The connection block consists of an array of vertical fluid outlets integrated into the chip cartridge, which are fluidically connected to the valve block. Fluidic contact is achieved by pressing on a connection plate containing the connecting capillaries and a sealing element. The connection plate can be automatically lowered or raised to the connection position, thus enabling a detachable fluidic connection of the flow control module 4 to the system and the analysis modules (1, 2, and 3).

[0058] The filter elements are implemented as column arrays integrated into channels, or by integrating filter membranes into the connecting channels, or by integrating commercially available filter elements into the installation space between two conveying or connecting channels.

[0059] Furthermore, auxiliary structures are provided for the efficient mixing of fluids. This ensures efficient mixing in the conveying channels by pumping the fluid into the fluid reservoirs and pumping it back into the conveying channels.

[0060] Furthermore, by periodically lowering the piston onto the conveying channel, agitation of the fluid can be achieved and used for mixing.

[0061] Furthermore, micromixers based on the multilamination principle, which are known per se, can be integrated into the cartridge.

[0062] For fluid management with high flow rates and Reynolds numbers, well-known micromixers based on chaotic advection principles, such as T-mixers or meander mixers, can be used. These systems act as mixers above a critical Reynolds number (Re > 240 for T-mixers and Re > 80 for zigzag mixers) and can be used when fluids need to be transported at high velocities and Reynolds numbers.

[0063] In fluid management applications with low flow rates and low Reynolds numbers (Re < 10), a special fluid rotation unit is used. This unit consists of two or more microchannel segments arranged in series and connected at their ends. The two connected channel segments are positioned at an angle α to each other, lie in two superimposed planes, have closed ends, and transition to the next channel segment occurs laterally at the ends of the channel segments. The channel segments are alternately placed in the upper and lower chip layers.

[0064] This two-length chip system can also incorporate a hydrodynamic focusing unit before and / or after the arrangement for fluid rotation.

[0065] Because the channel segments are arranged at angles to each other, the flowing medium is subjected to changes in direction, which together cause a rotation of the medium around the axis in the direction of flow, so that this fluid rotation unit is used when fluids are to be transported at low transport velocities and Reynolds numbers.

[0066] Another method for efficient mixing involves the well-known introduction of fluid through a nozzle into a cavity. This arrangement creates circular flow patterns within the cavity, leading to efficient mixing while simultaneously counteracting the sedimentation of particles and cells contained in the fluid.

[0067] The central control and computer unit (5) has integrated control software by means of which all processes are controlled and regulated.

[0068] The holography sample preparation cartridge (11) comprises microstructured fluidic lysis channels or dielectrophoresis channels with microelectrodes that are microfluidically connected to the flow control module (4).

[0069] Example parameters for operating the flow control module (4) are: • Provision of flow rates in the range of 0.1 - 10 nl / s during measurement, • Provision of flow rates up to 10 µl / s for cleaning and service operations • Providing flow rates on the order of 1 µl / s for fluid exchange in the connecting capillaries. • Applying a defined back pressure to the outlets to trap cells at the pore arrays for Raman measurement.

[0070] The operation of the three modules [holography module (1), Raman spectroscopy module (2) and biomarker module (3)] is started simultaneously by means of the central control and computer unit (5) immediately after the sample is loaded into the blood sample feeder (73), so that parallel operation of the three sample measurement units is possible in order to keep the time required for diagnostic testing low.

[0071] The holography sample measurement unit (12) is an arrangement for a lensless inline reflected light microscope with a coherent illumination source and a detector array, as is known, for example, from DE 10 2012 016 318 A1.

[0072] The Raman spectroscopy sample preparation cartridge (21) comprises microstructured fluidic lysis channels or dielectrophoresis channels with microelectrodes that are microfluidically connected to the flow control module (4).

[0073] The integration of the required electrode structures into the respective cartridge and the electronic control of the system is achieved by producing the electrodes on the cartridge substrate using thin-film processes and photolithography / electron beam lithography.

[0074] The Raman spectroscopy sample measurement unit (22) is, for example, a portable Raman spectroscopy system for mobile use, as is known from DE 10 2004 034 354 B3.

[0075] For plasma separation from whole blood, a lab-in-a-vial platform is used as a sample preparation cartridge. This cartridge contains a sealable, cylindrical vessel with a diameter between 5 and 25 mm and a tapered or rounded lower tip. The vessel walls are equipped with cell-collecting structures.

[0076] For separation, the cylindrical vessel is rotated around its axis. The required angular velocity is determined by the centrifugal accelerations described in the literature for blood cell separation and the diameter of the vessel. During this process, the blood cells are drawn into the trapping structures lined with the vessel walls by the centrifugal forces.

[0077] The cell-free plasma overlays the trapping structures as a fluid film.

[0078] After the rotation has ended, this film sinks downwards under the influence of gravity and collects at the top of the cylindrical vessel, from where it is either manually removed by pipetting or automatically with an autosampler.

[0079] While conventional centrifugation requires pipetting off the plasma as supernatant from the cells collected in the lower part of the vessel, the method described here collects a pure plasma sample at the tip of the vessel, which can be extracted completely and fully automatically, for example, with the needle of the autosampler. This approach thus offers optimal conditions for automated plasma separation.

[0080] Due to the short sedimentation paths (only a few millimeters - compared to several centimeters in conventional Vacutainers), the required process time is reduced to 4 minutes (including loading the vessel and taking the plasma).

[0081] The trapping structures for blood cell separation are designed as three-dimensional surface structures with depressions into which the blood cells are placed during centrifugation and which prevent the blood cells from flowing away under the influence of gravity.

[0082] In the simplest case, pieces of polyester gauze with a mesh size between 30 and 200 µm and a thickness of 200 µm can be placed on the sides of the vessel. During centrifugation, they automatically press themselves against the wall, thereby forming the desired trapping structures.

[0083] Alternatively, depressions with a structure width between 30 and 200 µm and depths of up to 200 µm can be introduced into the side walls.

[0084] Another possibility is the integration of column structures with a spacing between 30 and 300 µm and a height of 100-500 µm.

[0085] For plasma separation, commercially available 2 ml disposable reaction vessels with a rounded bottom made of polypropylene are used. A gauze serves as an example of a collection structure.

[0086] The biomarker sample preparation cartridge (31) comprises microstructured fluidic dielectrophoresis channels with microelectrodes or structured microchannels with mechanical filters or cross-sectional changes that are microfluidically connected to the flow control module (4).

[0087] All cartridges in the modules (1, 2 and 3) can be designed as either disposable or reusable cartridges, with the disposable cartridges being made of plastic (which is cost-effective) and reusable cartridges with optical functions being made of glass, especially quartz glass, and being cleanable, so that these expensive cartridges are regenerated after each sample run by intermediate cleaning steps.

[0088] The biomarker sample measurement unit (32) is a photosensor detector array arrangement with a coherent illumination source, such as is used for the detection of diagnostic and prognostic markers in biomedicine.

[0089] This involves chip-based biomarker detection based on an optically readable surface-bound immunofluorescence assay. Other, already known alternatives for biomarker detection can also be used.

[0090] The central control and computer unit (5) has integrated software control for regulating all processes of the flow control module (4) and all modules (1, 2 and 3) and special software for data processing and evaluation of the measurement data recorded by the three modules (1, 2 and 3), such as for the evaluation of Raman data.

[0091] This evaluation of the Raman data, with the aim of extracting clinically relevant information from the Raman spectra, is carried out using multivariate data analysis techniques, such as principal component analysis, neural networks, linear discriminant analysis, or cluster analysis.

[0092] At the same time, the access of the central control and computer unit (5) to the database (6) enables the comparison of the recorded and processed data from the modules (1, 2 and 3) with the reference data stored in the database (6) and the complex data evaluation, which results in a specified score value. This involves the use of methods that are known per se. The following procedure is used to operate the order:

[0093] Via the blood sample feeder (73), the blood sample is manually injected directly from the cannula with which it was taken from the patient into the microfluidic system of the flow control module (4).

[0094] Alternatively, it is also possible for blood samples to be supplied automatically, e.g. by means of a pipetting robot.

[0095] The blood volume from the smallest commercially available cannula of 2.7 ml is perfectly sufficient, and no further pretreatment is necessary after blood collection from the patient, as the pretreatment of the blood sample takes place directly in the respective microfluidic element of the three different sample preparation cartridges (11, 21 and 31) for the subsequent analysis in the subsequent sample measurement units (12, 22 and 32).

[0096] The blood sample is distributed to the sample preparation cartridges (11, 21 and 31) by means of the flow control module (4) as follows: Holography module (1)

[0097] The holographic detection of blood components is carried out using whole blood on the one hand and enriched leukocytes on the other.

[0098] For holographic detection of blood components, the sample is first diluted (by approximately a factor of 1:500). This is done in the flow control module (4) by adding buffer from the buffer supply (72) and subsequently transferring the diluted whole blood microfluidically into the holography sample preparation cartridge (11).

[0099] A maximum of 4 µl of whole blood is used in total, which is diluted to a maximum of 2 ml and prepared and measured as follows: Raman spectroscopy module (2)

[0100] For Raman spectroscopic analysis of white blood cells (leukocytes), the red blood cells (erythrocytes) are separated from the whole blood in the Raman spectroscopy sample preparation cartridge (21). This is preferably done by lysis of the erythrocytes, e.g. with NH4Cl, but can alternatively also be done by dielectrophoretic deflection.

[0101] This leukocyte-enriched blood (approx. 4 µl) is then transferred to the Raman spectroscopy sample measurement unit (22). Biomarker module (3)

[0102] For the analysis of biomarkers in the separated plasma, the cellular components of the blood are separated in the biomarker sample preparation cartridge (31). For this purpose, filtration techniques or, alternatively, deflection techniques using electric fields (dielectrophoresis) or microcentrifugation are used.

[0103] In this step, approximately 1 ml of the total blood sample is used, whereby approximately 500 µl of plasma is transferred to the subsequent biomarker sample measurement unit (32).

[0104] In the holographic sample measurement unit (12), the cellular components, with a particular focus on erythrocytes and leukocytes, are recorded in terms of number and shape. This is done using a lensless setup in a single-channel microfluidic system. The image acquisition frequency must be adapted to the flow velocity (or vice versa).

[0105] Image acquisition is followed by image analysis, which also makes it possible to differentiate various leukocyte subtypes based on nuclear morphology. To achieve optimal results, the whole blood sample can undergo a further pretreatment step before analyzing the leukocyte subtype count. This involves first removing the numerically far superior erythrocytes (approximately 1000 erythrocytes per leukocyte) from the sample by lysis or deflection techniques using electric fields (dielectrophoresis) or microcentrifugation. Staining the cell nucleus (e.g., with Kimura stain) is also possible to achieve better contrast between the subtypes.

[0106] In the Raman spectroscopy sample measurement unit (22), the leukocytes are first arranged on a regular measuring grid for effective spectroscopic characterization.

[0107] This is achieved, for example, through an integrated microfluidic hole chip structure, in which the fluid flows through holes approximately 4 µm in size due to slight negative pressure within the chip structure. Under this slight negative pressure, large leukocytes adhere precisely to these holes, thus ensuring they are available at well-defined locations for further spectroscopic characterization. Platelets are not retained by the holes and therefore do not remain on the integrated microfluidic hole chip structure.

[0108] The excitation of the Raman spectra is performed using commercially available lasers, e.g., 785 nm. If borofloat glass is used for the fabrication of the microhole chip microfluidics, a laser in the green range (e.g., 532 nm) must be used for the excitation of the Raman spectra, otherwise the background of the glass will mask the Raman spectra of the cells.

[0109] The spectra are acquired directly over the holes with a diameter of approximately 1 to 5 µm to avoid any spectral contribution from the chip membrane. This can be achieved by acquiring a single spectrum with a broadened laser beam or by acquiring multiple individual spectra in the relevant area. Great emphasis is placed on the rapid and parallel characterization of multiple cells on the chip, as this reduces the analysis time. A minimum of 500 cells are analyzed.

[0110] In addition to the cells, a spectrum of the perforated membrane is also always recorded. This serves as an internal control for the automated analysis. Acquiring a single spectrum takes approximately 1 second. The spectra are measured in the fingerprint range (approximately 600–1860 cm⁻¹). -1 ) evaluated. If necessary, the CH range (2750 - 3100 cm) can also be evaluated. -1 ) may also be consulted.

[0111] The automated evaluation includes spectrum pretreatment (with background correction and normalization) as well as an assignment of the spectrum to the leukocyte subtype and “activation state”.

[0112] Raman analysis focuses particularly on common leukocytes, such as neutrophils and lymphocytes. Possible "activation states" that can be detected with this module are: a) dormant, meaning the patient has no infection or inflammation; b) activated by sterile inflammation (such as that which can occur after surgery or a heart attack); c) activated by infection. For the last state, further differentiation is useful, providing insights into the pathogen (fungus, bacterium, virus) and the severity of the immune response (appropriate vs. excessive, leading to organ failure).

[0113] A classification model is used to determine leukocyte subtype and leukocyte "activation state," utilizing specific spectral characteristics and changes to better characterize the immune response. To identify the leukocyte subtype, specific fluorescence staining with surface markers on the integrated microfluidic hole chip structure is also possible after Raman measurement. For data readout, the Raman module must then be equipped with a suitable excitation lamp and camera.

[0114] In the biomarker sample measurement unit (32), fluorescence measurement is performed to determine various biomarker concentrations in plasma by simultaneously analyzing at least three fluorescent biomarkers in a microfluidic system. The selection of biomarkers includes both diagnostic markers (e.g., CRP, IL-6, PCT) and prognostic markers (e.g., suPar).

[0115] The central control and computer software (5) with access to a database (6) is also important for operating the arrangement.

[0116] Firstly, it processes the data flow (81) for controlling the valves (41), the data flow (82) for transferring the measured values ​​from the modules (1, 2, and 3) to the control system, and the data flow (83) for controlling the measurement processes in the modules (1, 2, and 3). Secondly, it serves for internal data comparison with the database (6) and the output (100) of the results.

[0117] The database (6) serves for external evaluation and feedback of the results (101), e.g. control by the doctor, and for the input (102) of data from external sources (specific patient data).

[0118] The control and computer software (5), which both connects the individual components of the arrangement to enable easy control from a user platform, but also performs integrated multivariate data analysis so that the individual results of each analysis contribute optimally to the overall result, provides a user interface that can be easily operated by a doctor or nurse.

[0119] The patient ID of the blood sample is read from external sources via input (102), as is the patient's clinical data from the hospital system, which is necessary for statistical analysis. The reading and identification of individual patient blood samples can be performed using barcode scanners. The user interface documents the successful completion of the analyses in each module (1, 2, and 3) and displays any errors that may occur.

[0120] As a result, the results of the individual modules (1, 2 and 3), i.e. biomarker concentrations, cell counts and a Raman score, as well as the correlation of these individual results are displayed, in which they are internally evaluated by the control and computer software (5) together with the read-in clinical data and displayed to the physician as a “sepsis score”.

[0121] This “sepsis score” value provides information about the probability that the patient has an infection and the probability that the patient will experience a critical development (e.g. organ failure, septic shock), i.e., require special medical attention.

[0122] The underlying classification model, which enables these statements from the multitude of parameters, is trained and evaluated through the external evaluation and feedback (101) of the results into the database of well-defined patients whose disease course is known.

[0123] One advantage of the parallel operation of the three modules coupled to the flow control module (4) [holography module (1), Raman spectroscopy module (2) and biomarker module (3)] according to the present technical solution is that, compared to previously known individual solutions which only provide the biomarker concentrations, cell counts or a Raman score as a result, all individual results of the three modules are available simultaneously in a much shorter period of time, with the correlation of these individual results being displayed by the control and computer software (5) internally evaluating them together with the read-in clinical data and evaluating and displaying them for the physician as a “sepsis score”.

[0124] Another advantage is that the parallel operation of the three modules (1, 2 and 3), made possible by the special functionality of the flow control module (4) and the central control and computer unit (5), creates a synergy effect, so that the entire analysis [i.e., from injecting a small amount of blood, only 1 to 2 ml, to displaying the so-called "sepsis score" as a meaningful final result on the user interface of the control and computer software (5)] takes a maximum of one hour.

[0125] An advantage of this is that [in the event that no error message is generated by the control and computer software (5)], no external access to the arrangement is necessary during this short measurement period of a maximum of one hour.

[0126] It is also advantageous that, in the event that the biomarker concentrations, cell counts and Raman score supplied by the modules (1, 2 and 3) do not provide clear or contradictory intermediate results on the way to the final result in the form of the "sepsis score", the control and computer software (5) provides feedback to the flow control module (4) in such a way that a renewed automated pass of the sample in question through the modules (1, 2 and 3) is effected.

[0127] The miniaturization of the holography module (1), Raman spectroscopy module (2) and biomarker module (3), the compact biomarker sample measurement unit (32) and the portable Raman spectroscopy sample measurement unit (22) also enable analysis directly in close proximity to the patient (usually an intensive care patient for the primary application or emergency room situation), which represents a further advantage of the present technical solution.

[0128] All features described in the description, the exemplary embodiments and the following claims can be essential to the invention, either individually or in any combination. Reference symbol list 1 Holography module 11 Holography Sample Preparation Cartridge 12 Holography Sample Measurement Unit 2 Raman Spectroscopy Module 21 Raman spectroscopy sample preparation cartridge 22 Raman spectroscopy sample measurement unit 3 Biomarker module 31 Biomarker Sample Preparation Cartridge 32 Biomarker Sample Measurement Unit 4 River control module 41 controllable valve 5 central control and computer unit with integrated software control 6 Database 7 Liquid material feeds 71 Color solution feed 72 Buffer feed 73 Blood sample delivery 74 Biomarker 1 administration 75 Biomarker 2 administration 76 Biomarker 3-infusion 81 Data flow for controlling the valves 82 Data flow for transferring measured values ​​from the modules to the controller 83 Data flow for controlling the measurement processes in the modules 100th edition of the results 101 External evaluation and return of the results to the database 102 Inputting data from external sources

Claims

[1] Arrangement for individualized in vitro patient blood analysis comprising a holography module (1), a Raman spectroscopy module (2) and a biomarker module (3), characterized by , that the holography module (1), the Raman spectroscopy module (2) and the biomarker module (3) are implemented microfluidically, wherein - the holography module (1) consists of a holography sample preparation cartridge (11) and a holography sample measurement unit (12) which are microfluidically connected to each other, wherein the holography sample measurement unit (12) is downstream of the holography sample preparation cartridge (11) in the direction of flow, - the Raman spectroscopy module (2) consists of a Raman spectroscopy sample preparation cartridge (21) and a Raman spectroscopy sample measurement unit (22) which are microfluidically connected to each other, wherein the Raman spectroscopy sample measurement unit (22) is downstream of the Raman spectroscopy sample preparation cartridge (21) in the flow direction, - the biomarker module (3) consists of a biomarker sample preparation cartridge (31) and a biomarker sample measurement unit (32) which are microfluidically connected to each other, wherein the biomarker sample measurement unit (32) is downstream of the biomarker sample preparation cartridge (31) in the flow direction, wherein the holography sample preparation cartridge (11), the Raman spectroscopy sample preparation cartridge (21) and the biomarker sample preparation cartridge (31) are fluidically connected via fluid channels through an integrated microfluidic flow control module (4), and the holography sample preparation cartridge (11), the Raman spectroscopy sample preparation cartridge (21), the biomarker sample preparation cartridge (31) and the flow control module (4) data- and information-guiding with a central control and computer unit (5) which has integrated control software by means of which all processes are controlled and regulated,are connected to a database (6) via an information-conducting link, wherein the modules (1, 2 and 3) are fluidically connected via the flow control unit (4) to a common blood sample feeder (73) and further fluid material feeders (7) via fluid guideways, and the fluid guideways can be controlled and the fluid feeders (7) can be selectively switched on via valves (41) controlled by the control and computer unit (5), and the control and computer unit (5) accesses the database (6), so that a data comparison of the recorded and processed data of the modules (1, 2 and 3) with the reference data stored in the database (6) and a complex data evaluation can be generated. [2] Arrangement for individualized in-vitro patient blood analysis according to claim 1, characterized by, that the flow control component (4) consists of tubes and / or a microstructured substrate with micro-fluidic channels in which the controllable valves (41) are arranged, so that the fluid pathways can be generated in such a way that the fluid pathway from the common blood sample supply (73) is split into three fluid pathways leading into the cartridges (11, 21 and 31). [3] Arrangement for individualized in-vitro patient blood analysis according to claim 1, characterized by , that the holography sample preparation cartridge (11) consists of microstructured microfluidic lysis channels or dielectrophoresis channels with microelectrodes which are microfluidically connected to the flow control unit (4) and the holography sample measurement unit (12) is an arrangement for a lensless inline reflected light microscope with a coherent illumination source and a detector array. [4] Arrangement for individualized in-vitro patient blood analysis according to claim 1, characterized by , that the Raman spectroscopy sample preparation cartridge (21) consists of microstructured microfluidic lysis channels or dielectrophoresis channels with microelectrodes which are microfluidically connected to the flow control unit (4) and the Raman spectroscopy sample measurement unit (22) is a portable Raman spectroscopy system for mobile use. [5] Arrangement for individualized in-vitro patient blood analysis according to claim 1, characterized by , that the biomarker sample preparation cartridge (31) consists of microstructured microfluidic dielectrophoresis channels with microelectrodes or microchannels with mechanical filters or cross-sectional changes that are microfluidically connected to the flow control unit (4) and the biomarker sample measurement unit (32) is a photosensor detector array arrangement with a coherent illumination source. [6] Method using an arrangement according to any one of claims 1 to 5, wherein - the sample supply via the blood sample supply (73) is carried out by manually or automatically injecting the blood sample directly from the cannula with which it was taken from the patient into the microfluidic system of the flow control unit (4) by means of a pipetting robot, whereby no further pretreatment after blood collection from the patient is necessary. - the blood sample is then distributed to the sample preparation cartridges (11, 21 and 31) by means of the flow control module (4), - in the holography module (1) the holographic detection of the blood components is carried out using whole blood or enriched leukocytes, in which the sample is diluted in the flow control module (4) by adding buffer from the buffer supply (72) and subsequent microfluidic transfer of the diluted whole blood into the holography sample preparation cartridge (11), - in the Raman spectroscopy module (2) the analysis of the white blood cells is carried out by separating the red blood cells (erythrocytes) from the whole blood in the Raman spectroscopy sample preparation cartridge (21) by lysis of the erythrocytes or by dielectrophoretic deflection, so that the leukocyte-enriched blood is transferred to the Raman spectroscopy sample measurement unit (22) and is examined there by Raman spectroscopy and - in the biomarker module (3) the analysis of the biomarkers in the separated plasma is carried out by separating the cellular components of the blood in the biomarker sample preparation cartridge (31) by filtration or dielectrophoresis or microcentrifugation. [7] Method according to claim 6, characterized by , that - in the holography sample measurement unit (12) the cellular components are detected by image acquisition using a lensless setup in a single-channel microfluidics system, whereby the image acquisition frequency is adapted to the flow rate and subsequently an image evaluation is performed to distinguish the different leukocyte subtypes based on the nuclear morphology, - in the Raman spectroscopy sample measurement unit (22) the leukocytes are arranged on a regular measuring grid in which an integrated microfluidic hole chip structure allows the liquid to pass through holes of approximately the size of the chip structure by means of a slight negative pressure.4 µm is removed, so that the large leukocytes adhere precisely to these holes under only slight negative pressure, in order to be available at defined locations for further spectroscopic characterization; the arranged leukocytes are then excited with laser light and the spectra are recorded precisely above the holes to avoid a spectral contribution from the hole chip membrane; a spectrum is recorded with a widened laser beam or several individual spectra are recorded in the corresponding area; in addition to the leukocytes, a spectrum from the hole membrane is also recorded as an internal control for the automated evaluation; and. - in the biomarker sample measurement unit (32) a fluorescence measurement is performed to determine different biomarker concentrations in the plasma by the simultaneous analysis of at least 3 fluorescent biomarkers in a microfluidic. [8] Method according to claim 6 or 7, characterized by, that the central control and computer software (5) with its access to the database (6) processes, on the one hand, the data flow (81) for controlling the valves (41), the data flow (82) for transferring the measured values ​​from the modules (1, 2, and 3) to the controller, and the data flow (83) for controlling the measurement processes in the modules (1, 2, and 3), and, on the other hand, serves for internal data comparison with the database (6) and the output (100) of the results for external evaluation and the feedback of the results (101) and the input (102) of data from external sources, wherein the control and computer software (5) connects the individual components of the arrangement to enable simple control from a user interface and also to perform integrated multivariate data analysis, and wherein a patient ID of the blood sample is obtained via the input (102) of data from external sources, which contains the patient's clinical data from the hospital system,The data is read in and the user interface documents the successful completion of the analyses performed in each module (1, 2 and 3) and displays any errors that may occur. [9] Use of an arrangement according to one or more of claims 1 to 5 for determining a sepsis score from a whole blood sample within one hour. [10] Use of a method according to one or more of claims 6 to 8 for determining a sepsis score from a whole blood sample within one hour.

Images