Apparatus and method using a real-time system for analyzing a biological sample, in particular comprising nucleic acid amplification

Abstract

The invention relates to an apparatus (200) for analyzing a biological sample, in particular comprising nucleic acid amplification, wherein a cartridge (100) having a sample inserted into the cartridge (100) is processed by the apparatus (200) for analytic purposes, wherein the apparatus (200) comprises a first computing system (220) for interacting with a user via a user interface (215) of the apparatus (200), and a second computing system (230) in the form of a real-time system (230) for automatically controlling processing components (241, 242, 243, 244) of the apparatus (200), and wherein the real-time system (230) is designed to receive a protocol for processing the cartridge (100) and to execute the protocol in real time by controlling the processing components. The invention also relates to a method (500) for operating a real-time system (230) of this kind.

Description

[0001] R. 418683

[0002] - 1 -

[0003] Description

[0004] title

[0005] Device and method with real-time system for analyzing a biological sample, in particular comprising nucleic acid amplification

[0006] State of the art

[0007] The detection of pathogens in a biological sample, such as blood or sputum, can be achieved by identifying fragments of the pathogens' nucleic acids within the sample. This is done using nucleic acid amplification, a process in which nucleic acids are amplified section by section, to specifically search for these fragments. Such methods can be implemented at the point of care and as immediate, near-patient diagnostics using microfluidic systems. In this process, a patient sample is introduced into a microfluidic cartridge, which is then processed in an analyzer to perform nucleic acid amplification within the cartridge, as described, for example, in documents DE 102016 222075 A1 and DE 102016 222072 A1.

[0008] The operation of such an analyzer can be carried out via predefined protocols, whereby the protocols define the cartridge processing sequence in the form of successively executed process steps. Each process step comprises commands for controlling components of the analyzer to implement the microfluidic processing within the cartridge through external actuation. These components may include, for example, heaters for setting temperature levels in specific areas of the cartridge, plungers for releasing reagents into the cartridge, or pneumatic interfaces for pneumatically controlling valves and pumps within the cartridge. Furthermore, R. 418683

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[0010] Components also include sensors for detecting a condition in the cartridge, such as temperature sensors, and sensors for detecting the microfluidic process, for example, an optical sensor for observing the progress and result of (quantitative real-time) nucleic acid amplification. R. 418683

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[0012] Disclosure of the invention

[0013] Advantages of the invention

[0014] Against this background, the invention relates to a device for analyzing a biological sample, wherein a cartridge containing a sample is processed by the device for analysis. The device comprises a first computing system for user interaction via a user interface. Furthermore, the device comprises a second computing system configured as a real-time system for the automatic control of processing components of the device. The real-time system is configured to receive a protocol for processing the cartridge and to execute the protocol, in particular the process steps of the protocol, in real time by controlling the processing components.

[0015] The sample contained in the cartridge can, for example, include sputum, urine, blood, or a swab. The analysis of the sample comprises several steps to be performed by the instrument, in particular a usually multi-step sample preparation and a usually multi-step identification of sample components. Sample preparation may require purification to separate the sample components to be analyzed, especially nucleic acid fragments. In the case of nucleic acid fragments, the identification of the sample components preferably includes amplification of these fragments, particularly via a polymerase chain reaction or isothermal amplification. The steps to be performed are included in the protocol applicable to the respective analysis, hereinafter referred to as the assay protocol, as process steps for processing the cartridge.The process steps listed in the assay protocol preferably include instructions, also referred to as commands to the second computing system, specifying which processing components are to be controlled and in what manner, wherein in particular a type or kind of the respective processing component to be used is specified, for example an instruction to heat a heating element not yet specifically selected to a target temperature for heating the sample, preferably with an R. 418683.

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[0017] The instructions provide a functional name for the processing component of the device. However, they preferably do not yet specify the concrete processing component of the device; in the aforementioned example of heating the heating element, for instance, they provide a general specification for using a heating element, but not yet a specific one of several heating elements present in the device.

[0018] The real-time system is preferably configured to receive individual process steps in addition to the protocol, in particular one or more individual process steps that are to be executed before and / or after the steps of the protocol. Alternatively, the process steps contained in the protocol can be viewed as an encapsulated instruction or command, or simply a capsule command, which, along with other individual process steps, is transmitted to the second computing system as other individual instructions or commands. In this respect, the protocol can also be considered a capsule command.

[0019] Depending on its specific configuration, the protocol can contain one or more groups comprising one or more process steps. In particular, the protocol can identify several process steps as belonging to a single group. For such groups, a number of iterations for processing can be specified, especially in the protocol, with the second computing system configured to execute the steps of a group multiple times sequentially according to the number of iterations specified for that group. For example, such a group can comprise several process steps that involve controlling, in particular opening or closing, several valves arranged in series within the cartridge, such as the successive opening or closing of the valves, which is performed multiple times sequentially according to the corresponding number of iterations.

[0020] The hardware configuration of the device thus comprises two main components for software execution: the two computing systems. The invention advantageously divides the work required for sample analysis into two tasks and optimizes their execution. Both computing systems can perform their respective tasks, namely the R. 418683

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[0022] Interaction with the user and the processing of the cartridge can be carried out largely independently in real time. In particular, the real-time system can independently translate the block of instructions contained in the received protocol, especially abstract ones, into concrete process steps and preferably execute them.

[0023] The first computing system is preferably connected directly to the device's interaction interfaces, in addition to a connection to an internal network, and serves to interact with a user and external systems. The user interaction interfaces may include, for example, a touch-sensitive display, several LEDs for status feedback, and a speaker. The device may also include, for example, an optical scanner (such as a barcode scanner) for reading cartridge data, as well as a wired or wireless digital communication interface (such as a USB port and a WLAN, Bluetooth, or cellular module) as interaction interfaces for external devices. The first computing system may, in particular, comprise a single-board computer (SBC).According to an advantageous embodiment, the first computing system is configured to transmit the cartridge processing protocol to the real-time system, in particular to a buffer memory of the real-time system. The protocols can, for example, be stored in a data memory of the device and / or received via the communication interface. The protocols are preferably in the form of the assay protocols described above. The first computing system is preferably configured to specify the process steps of the assay protocols for use in the device. In particular, this specification can include defining the specific processing units to be controlled in one or more of the process steps. The specified assay protocol, hereinafter simply referred to as the protocol, is then transmitted from the first computing system to the real-time system.This has the advantage that an assay protocol can be mapped to the specific architecture of the device by the first computing system. R. 418683.

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[0025] The second computing system is connected, preferably via a connection to an internal network, directly or indirectly via other components, to processing components for controlling these components and serves, via the real-time system, to execute the fully automated process steps of the protocols in real time. The processing components can be active components such as, in particular, electromechanical components (e.g., drives), actuators (e.g., plungers), heating and cooling units, pumps, valves, and / or a movable flap for closing an input opening for the cartridges. The processing components can also include passive components, in particular sensors, for example, optical sensors (e.g., a camera or photodiode), pressure or temperature sensors. The real-time system can be configured to receive the respective protocol from the first computing system.Alternatively or additionally, the real-time system can be configured to receive the respective protocol directly from the data storage or via the communication interface. During processing, the received protocol is preferably held in a buffer memory of the real-time system, in particular temporarily stored, and preferably managed by the real-time system along with the process steps contained in the protocol.

[0026] The first and second computing systems can each have their own hardware component. The two computing systems can be configured as two independent components or, alternatively, as two subcomponents, for example, as physically separate processors, as part of a common computing system, such as a shared circuit board. Alternatively or additionally, the first and second computing systems can share at least one hardware component. It is possible for both computing systems to be configured as separate only in software. According to a particular embodiment, the real-time system forms part of the first computing system. In particular, a portion of the first computing system, preferably in the form of a single-board computer (SBC), comprising, for example, one or more processor cores, can be implemented as a real-time system, especially with a guaranteed response time. R. 418683

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[0028] According to a preferred embodiment, the real-time system can be configured to execute process steps of the protocol, particularly the specified assay protocol, taking into account the conditions associated with each step for its execution. These conditions can include, in particular, time requirements, dependencies between process steps, especially regarding the sequence or prerequisites for executing the process steps, and / or requirements for the operating states of components of the device or the cartridge. These conditions can, in particular, already be included, explicitly or implicitly, in the unspecified assay protocol. The protocol thus preferably comprises a protocol extension, which, depending on the specific protocol, can contain such conditions for one, several, or all process steps of the protocol.The protocol received by the real-time system is stored and preferably managed in a memory, preferably a buffer memory, of the real-time system, as described above. The status of the processing of the individual process steps is kept up-to-date, for example, via status codes. Thus, the stored protocol, including the current status, accurately reflects the valid operating state of the device, which advantageously supports the well-defined processing of the protocol.

[0029] According to a preferred embodiment, the first computer system is configured to receive feedback on the progress of the protocol processing (status information) and, depending on the content of the feedback, to output information via a user interface of the device and / or to give the real-time system an instruction, in particular an instruction to continue or terminate the processing. The first computer system thus advantageously monitors the processing of the protocol by the real-time system, can intervene if necessary, and in particular can terminate the processing prematurely. Preferably, the real-time system transmits such feedback (status message) to the first computer system whenever the status of a process step changes. The feedback can, for example, simply contain an identification of the process step and the current status. R. 418683

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[0031] Preferably, the first computing system is configured to evaluate measured values ​​acquired by the real-time system after or during the processing of the protocol as part of the analysis. A result of the evaluation can then preferably be output via an interface of the device, in particular via the display to the user, or via a communication interface to an external storage device or an external device, for example in the cloud, for further processing.

[0032] According to an advantageous embodiment of the invention, the real-time system is configured to at least partially evaluate the measured values ​​acquired via one or more processing components as part of the analysis and preferably transmit the result of the evaluation to the first computing system. Thus, the real-time system at least partially takes over the task of analyzing the acquired data from the first computing system. This advantageously leads to even greater independence for the real-time system and relieves the first computing system of this task. For example, the evaluation can include pre-filtering the acquired data, such as filtering out data of insufficient quality, and deriving relevant parameters for the analysis, such as determining or estimating relevant parameter values ​​of a (particularly quantitative real-time) PCR, for example, a C t - or C q-value, a form of PCR curve, or even an estimate of an initial concentration of a sought-after nucleic acid segment in the sample.

[0033] The invention also relates to a method for operating a real-time system of the device, wherein the real-time system receives a protocol for processing a cartridge with the device and wherein the real-time system controls processing components of the device for processing the protocol in real time.

[0034] Brief description of the drawings

[0035] Exemplary embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description. For the ones in R. 418683

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[0037] The same reference symbols are used for elements depicted in different figures and which appear similar, without repeating the description of the elements.

[0038] They show

[0039] Figure 1 shows an oblique view of an embodiment of the device according to the invention with a cartridge.

[0040] Figure 2 shows a side view of the embodiment shown in Figure 1.

[0041] Figure 3 shows a flowchart of an embodiment of the method according to the invention, as well as

[0042] Figure 4 illustrates an exemplary interaction between the first computing system and the real-time system, as well as between the real-time system and processing components of the device.

[0043] Embodiments of the invention

[0044] Figure 1 schematically shows an embodiment of the device 200 according to the invention for analyzing a biological sample (analyzer) in a particularly microfluidic cartridge 100, for example based on a processing device and cartridge as described in documents DE 102016222 075 A1 and DE 102016222072 A1. The device 200 and cartridge 100 are in particular part of a microfluidic system 1000, wherein the cartridge 100 is received into the device 200 via an opening 210 for carrying out the analysis. The analysis may in particular include testing for the presence of one or more pathogens in the sample by detecting nucleic acid fragments of the pathogens, wherein the nucleic acid fragments are amplified by nucleic acid amplification, in particular a polymerase chain reaction (PCR) or isothermal amplification, carried out in the cartridge 100 during the analysis.R. 418683.

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[0046] The device 200 comprises a first computing system 220 and a second computing system 230, which can communicate with each other. The first computing system 220 preferably comprises a single-board computer (SBC), for example, a Raspberry Pi. The first computing system 220 is configured to communicate with a user of the device and external devices via interfaces of the device 200, in particular via a preferably touch-sensitive display 215 or an interface 205 for wired or wireless digital communication, for example, a WLAN, Bluetooth, or cellular module.

[0047] The second computing system 230 is configured as a real-time system and comprises at least one processor (processing unit) or microcontroller, for example, an STM32F103. The first computing system 220 is configured to transmit protocols for processing the cartridge 100 to the real-time system 230, the second computing system 230 preferably having a buffer memory 232 for storing the protocols. The buffer memory 232 can, in particular, be part of a processor or microcontroller of the second computing system 230. The device 200 can have a data memory 225, for example, as part of the first computing system 220, wherein one or more protocols are stored in the data memory. Alternatively or additionally, the device can be configured to receive the protocols via the preferably wireless interface 205.The device 200 is preferably configured to perform various analyses, in particular the detection of different pathogens (tests). Since these tests may differ in their procedure, different protocols are assigned to the tests. The first computing system 220, as described above, is preferably configured to specify the protocols, which are, for example, in XML encoding, in particular to define the processing components of the device 200 to be controlled for one or more process steps. For example, it is specified which specific heater is to be controlled to heat the sample to a target temperature or which specific pneumatic port is to be controlled to open a predefined valve in the cartridge. R. 418683.

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[0049] The real-time system 230 is configured to process the received protocol in real time by controlling processing components of the device 200. Such processing components can be, in particular, as described above, heaters 241 for setting temperature levels in areas of the cartridge, plungers 242 for releasing reagents in the cartridge, or pneumatic interfaces 243 for pneumatically controlling valves and pumps in the cartridge 100, as schematically illustrated in Figure 2. The processing components can be, in particular, sensors, for example, temperature sensors and optical sensors, which are also operated by the real-time system 230 and which transmit their measurement signals to the real-time system 230. Figure 2 shows, as an example, an optical sensor 244, for example, a camera, for detecting radiation emitted from the cartridge.If the analysis involves nucleic acid amplification, such as quantitative real-time PCR, fluorescent probes can be used in the amplification to measure the progress or success of the amplification via emitted fluorescence radiation.

[0050] Figure 3 shows a flowchart for an embodiment of the method 500 according to the invention, wherein the method 500 can be carried out, for example, with the embodiment of the device 200 according to the invention described above.

[0051] Figure 4 illustrates, by way of example, the interaction between the first computing system 220 and the real-time system 230, as well as between the real-time system 230 and processing components 241, 242, 243 of the device 200, with the progressive temporal sequence being indicated by the arrows extending vertically downwards from top to bottom. The arrows extending between systems 220, 230 and components 241, 242, 243 indicate that information, in particular a control command or a status message, is transmitted in the direction of the respective arrow.

[0052] As shown in Figures 3 and 4, the real-time system 230 (represented by the four parallel arrows from 220 to 230 in Figure 4) receives a protocol to be processed in a first process step 501, in particular a R. 418683.

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[0054] The assay protocol, specified by the first computing system 220, is preferably stored in the buffer memory 232 of the real-time system 230. In a second process step 502, the process steps contained in the protocol are interpreted as instructions and transmitted by the real-time system 230 to the relevant processing components. This allows the process steps to be executed in real time, and thus, in particular, with very short time intervals between the individual process steps. For example, several valves of the cartridge can be switched in rapid succession, for example, at intervals of 100 milliseconds or less. The real-time system 230 can also receive individual instructions or directions from the first computing system 220 in addition to the protocol and thus process further process steps, particularly before or after the protocol has been executed, alongside the process steps of the protocol.

[0055] The process steps can be interpreted as commands by the real-time system 230 and transmitted to the relevant processing components, particularly depending on the fulfillment of any conditions associated with the process steps. These conditions can include, in particular, time requirements or requirements regarding the operating states of the device 200, especially the operating states of one or more of the processing components. For example, a process step involves the release of a reagent located upstream in the cartridge, such as a lysis buffer (release of nucleic acids from cells of the sample), for mixing with the sample contained in the cartridge 100. For this purpose, a plunger 242 is first actuated into the cartridge 100 to rupture a reagent container.The pneumatic interface is then controlled such that, via increased pressure, a diaphragm pump in cartridge 100 is deflected to convey the reagent to the sample. The command to control the pneumatic interface, and thus indirectly the diaphragm pump, is therefore contingent upon the successful execution of the command to move the plunger. These conditions, linked to the process steps, are part of the protocol received by the real-time system 230 as a protocol extension. The individual process steps of the received protocol, which is preferably stored in the buffer memory 232 of R. 418683.

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[0057] Each step of the real-time system 230 is assigned a status code indicating the current status of the respective process step. Table 1 shows several statuses with their corresponding status codes for illustrative purposes only.

[0058] Tabelle 1: Codierung von Status der Prozessschritte

[0059] Enumerator Systemzustand / Status

[0060] 0 Unprocessed

[0061] 1 Condition check

[0062] 2 Timeout condition(s) not fulfilled, skip step, start step wait

[0063] 3 Timeout condition(s) not fulfilled, skip step, no step wait

[0064] 4 Timeout condition(s) not fulfilled, abort 5 Start of execution

[0065] 6 Timeout execution

[0066] 7 Finished execution, no step wait

[0067] 8 Finished execution, start waiting for step duration

[0068]

[0069] 9 Finished waiting for step duration

[0070] As illustrated by the arrows in Figure 4, which point from the real-time system 230 to the first computer system 220, the real-time system 230 can be configured to send feedback to the first computer system 220 during or after the execution of a process step, for example, a status message regarding a change in the status of the respective process step. In particular, feedback can be sent to the first computer system 220 if the execution of a process step is unsuccessful. The first computer system 220 can then decide and instruct the real-time system 230 whether this process step should be repeated or skipped, or whether the entire protocol execution should be modified, for example, paused until a defined event occurs (e.g., after user interaction) or aborted.

[0071] The optical sensor 244 allows the real-time system 230 to observe the progress of the analysis through, for example, transparent walls and chambers of the cartridge 100 and, in a third process step 503, report this information back to the first computing system 220. In particular, when optical probes are used in the reaction, for example, fluorescent probes, to verify the success of nucleic acid amplification, the first computing system 220R. 418683

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[0073] The system must be configured to evaluate the measured values ​​transmitted by the real-time system for fluorescence radiation detected by the optical sensor 244 in a fourth process step 504 and output a result of the analysis to the user, for example, via the display 215. A result may include, for example, information on whether and which pathogens were detected in the sample, preferably at what concentration, as well as, in particular, relevant parameters of the detection, such as a C q -value or C t-value or PCR curves. Alternatively or additionally, the real-time system 230 can be set up, for example with powerful processors or microcontrollers and appropriate programming, to perform the fourth process step 504, i.e., to evaluate the recorded measured values ​​or at least prepare them for evaluation. In this way, a result or a partial result can already be transmitted from the real-time system 230 to the first computing system 220.

Claims

R. 418683 - 15 - Claims 1. Device (200) for analyzing a biological sample, in particular comprising nucleic acid amplification, wherein for the analysis a cartridge (100) with sample entered into the cartridge (100) is processed by the device (200), wherein the device (200) comprises a first computing system (220) for interaction with a user via a user interface (215) of the device (200) and a second computing system (230) configured as a real-time system (230) for automatic control of processing components (241, 242, 243, 244) of the device (200), wherein the real-time system (230) is configured to receive a protocol for processing the cartridge (100) and to execute the protocol in real time by controlling the processing components.

2. Device (200) according to claim 1, wherein the received protocol is held in a buffer memory (232) of the real-time system (230) during processing and is in particular managed by the real-time system (230).

3. Device (200) according to one of the preceding claims, wherein the first computing system (220) is configured to receive feedback on the progress of the processing of the protocol and, depending on the content of the feedback, to output information via a user interface (205, 215) and / or to give the real-time system (230) an instruction, in particular an instruction to continue or terminate the processing.

4. Device (200) according to one of the preceding claims, wherein the real-time system (230) is configured to transmit feedback on the status of the processing of the process step to the first computing system (220) when processing each of several, preferably all, process steps of the protocol.

5. Device (200) according to one of the preceding claims, wherein the real-time system (230) is configured to preferably update the status upon each change of a status. 418683 - 16 - To transmit feedback about the change to the first computer system (220) after processing a process step of the protocol.

6. Device (200) according to one of the preceding claims, wherein the real-time system (230) is configured to execute process steps of the protocol taking into account conditions associated with the process steps for the execution of each step, wherein the conditions include in particular time requirements or requirements for operating states of process components (241, 242, 243, 244) of the device (200) or the cartridge (100).

7. Device (200) according to one of the preceding claims, wherein the first computing system (220) is configured to evaluate measured values ​​acquired after or during the processing of the protocol by the real-time system as part of the analysis and preferably to output a result of the evaluation via an interface (205, 215) of the device (200).

8. Device (200) according to one of the preceding claims, wherein the real-time system (230) is configured to at least partially evaluate measured values ​​acquired via one or more processing components (244) as part of the analysis and preferably to transmit a result of the evaluation to the first computing system (220).

9. Device (200) according to one of the preceding claims, wherein the first computing system (220) and the second computing system (230) each have their own hardware component, for example as two independent components or as two subcomponents as part of a common computing system.

10. Device (200) according to one of the preceding claims, wherein the first computing system (220) and the second computing system (220) share at least one hardware component, in particular wherein the first computing system (220) and the real-time system (230) are only separated by software or wherein a part of the first computing system (220) is configured as the R. 418683 - 17 - Real-time system (230) is implemented 11. Method (500) for operating a computing system (230) configured as a real-time system (230) in a device (200) according to one of the preceding claims, wherein the real-time system (230) receives a protocol for processing a cartridge (100) with the device (200) and wherein the real-time system (230) controls processing components of the device (200) for processing the protocol in real time.

12. Computer program comprising instructions which, when the program is executed by a device (200) according to claim 11, cause the device to execute a method (500) according to any one of claims 1 to 10.

13. Computer-readable medium on which the computer program according to claim 12 is stored.

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