Systems and methods for bacterial growth detection using dual optical excitation - Patents.com

Abstract

A method for determining the presence of a target analyte in a blood sample is provided in a test device including a sensor in an aqueous medium, the method comprising the steps of transmitting light to the test device at a first excitation wavelength at which optical absorption of the sensor remains substantially constant even as the pH of the aqueous medium changes, measuring an intensity of a first fluorescent signal emitted from the sensor in the test device in response to the first excitation wavelength, transmitting light to the test device at a second excitation wavelength different from the first excitation wavelength, measuring an intensity of a second fluorescent signal emitted from the sensor in the test device in response to the second excitation wavelength, and normalizing the intensity of the second fluorescent signal emitted from the test device in response to the second excitation wavelength to the first fluorescent signal.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 307,907, filed February 8, 2022, which is incorporated by reference in its entirety.

[0002] [Technical field] The present disclosure relates to optimizing fluorescence detection systems, such as optical blood culture measurement systems, and more particularly, to systems and methods for normalizing a fluorescence signal in an optical blood culture measurement system configured to detect the presence of an analyte of interest in a sample. [Background technology]

[0003] In certain technical fields (medical, pharmaceutical, food industries, etc.), a fast and accurate determination of microbial contamination in certain systems (e.g., a patient's blood, a batch of pharmaceutical products, food supplies) is desired. Methods using sensors containing fluorescent substances in combination with indicator substances have been developed to indirectly detect microorganisms in a sample through their biological activity. Measurement systems employing these methods use a fluorescent sensor or detector to detect a fluorescent signal emitted from a container or vial containing the sample and the sensor. Software and / or hardware systems are then utilized to process the data collected by the detector. The signal measured by the detector may be normalized using one or more reference signals, for example, to improve the signal-to-noise ratio in the resulting data measurements. In certain applications, an initial detector reading is taken when the vial is placed in the measurement system, and the initial reading is used as the reference signal. Normalization based on the initial detector reading may be subject to several limitations.

[0004] For example, there is often a delay between when a sample is collected and injected into a test vial and when the vial is placed into a measurement system. In some cases, the inoculated vial is placed into the measurement system after hours or even days, such as over a weekend. This means that the initial detector reading may not occur for 24-72 hours after the vial is inoculated with the sample. The time between when the sample is placed into the vial and when the vial is placed into the measurement instrument is typically referred to as the Delayed Vial Entry (DVE). This DVE time may allow for the growth of bacteria and other microorganisms before the vial is placed into the measurement system. Growth of bacteria and other microorganisms before placement into the measurement system may affect the detector's initial reading reference signal and, therefore, the test data that is normalized using the detector's initial reading reference signal. Current methods for detecting DVE may use a kinematic DVE algorithm, which is an algorithm that measures the rate of change of an output signal reading over time. Such kinematic DVE algorithms also rely on an initial read reference signal from the detector that may be affected by the growth of bacteria and other microorganisms prior to placement in the measurement system.

[0005] There are other drawbacks to using the initial detector reading as a reference signal to normalize the current detector reading output by the measurement system. The initial detector reading reference signal may be affected by temperature fluctuations of the sensor. Sensor temperature fluctuations may be caused by external factors such as improper control of the ambient environment by the end user of the sensor and / or sensor equipment, changes in vial temperature after insertion into the system, air movement through the measurement system, etc. Sensor temperature fluctuations may require compensation in order to provide an accurate reading.

[0006] Current normalization techniques also fail to provide real-time feedback on the signal quality of the measurement system. The system architecture of the measurement system may cause erroneous measurements to be taken. For example, some light source components for exciting the fluorescent material in the sensor may degrade in emission intensity even during their useful life. The performance of the optical detector may also degrade. Changes in either the energy emission from the light source components or the sensitivity of the optical detector may cause the test data reported by the measurement system to be inaccurate. Further causes of inaccurate data measurements may include incorrect use of the measurement system equipment (e.g., slamming the door of the equipment's housing). Mishandling of the measurement system may cause misalignment of the test vial in the optical inspection path during sample testing. Furthermore, inconsistencies in the chemical composition of the sensor in the test vial may cause the test data reported by the measurement system to be inaccurate.

[0007] Embodiments of the disclosed technology address or mitigate these and other shortcomings in optical blood culture measurement systems. Implementations of the disclosed technology may address the aforementioned sources of variability in components of an optical blood culture measurement system. In one implementation, the components reside in a blood culture vial received in the optical blood culture measurement system. For example, implementations of the disclosed technology may address or mitigate the variability of a fluorescent sensor in a blood culture vial received in a test optical blood culture measurement system. Implementations of the disclosed technology may also address or mitigate inconsistencies associated with DVE detection. For example, implementations of the disclosed technology may address or mitigate inconsistencies in DVE detection due to the growth of bacteria or other microorganisms prior to an initial reading of the detector.

[0008] Embodiments of the disclosed technology are described herein with reference to optical blood culture measurement systems, such as, but not limited to, the "BD BACTEC" (registered trademark) blood culture system by Becton Dickinson and Company. However, it will be understood that embodiments of the disclosed technology are not limited to blood culture measurement systems, but may also be applied to other types of optical detection systems that rely on sensors or other substances having invariant or constant characteristics suitable for use in generating a reference signal for normalizing detector readings, such as an isobestic point. For example, embodiments of the disclosed technology may be implemented in immunoassays. Summary of the Invention

[0009] Aspects of the present disclosure include systems and methods for determining the presence of an analyte of interest in a blood sample in a test device that includes a sensor in an aqueous medium.

[0010] In a first embodiment, a method is provided for determining the presence of an analyte of interest in a blood sample in a test device including a sensor in an aqueous medium, the method comprising the steps of transmitting light to the test device at a first excitation wavelength at which optical absorption of the sensor remains substantially constant even as the pH of the aqueous medium changes, measuring an intensity of a first fluorescent signal emitted from the sensor in the test device in response to the first excitation wavelength, transmitting light to the test device at a second excitation wavelength different from the first excitation wavelength, measuring an intensity of a second fluorescent signal emitted from the sensor in the test device in response to the second excitation wavelength, and normalizing the intensity of the second fluorescent signal emitted from the test device in response to the second excitation wavelength to the first fluorescent signal.

[0011] A second embodiment includes the method of the first embodiment, wherein normalizing the intensity of the second fluorescent signal includes generating a ratio comparing the second fluorescent signal to the first fluorescent signal.

[0012] A third embodiment includes the method of the second embodiment, further comprising comparing the ratio to a threshold.

[0013] A fourth embodiment includes the method of the third embodiment, further comprising determining the presence of the analyte of interest when the ratio exceeds the threshold value.

[0014] A fifth embodiment includes the method of any of the first to fourth embodiments, wherein the first excitation wavelength is at an isosbestic point of a component of the sensor.

[0015] A sixth embodiment includes the method of the fifth embodiment, wherein the sensor comprises a pH indicator and a fluorophore, and the isosbestic point is the isosbestic point of the pH indicator.

[0016] A seventh embodiment includes the method of any of the first to sixth embodiments, wherein the first excitation wavelength is in the blue / cyan range.

[0017] An eighth embodiment includes the method of any one of the first to seventh embodiments, wherein the first excitation wavelength is between 485 nm and 495 nm.

[0018] A ninth embodiment includes the method of any of the first to eighth embodiments, wherein the first excitation wavelength is about 490 nm.

[0019] A tenth embodiment includes the method of any of the first to ninth embodiments, wherein the second excitation wavelength is in the green range.

[0020] An eleventh embodiment includes the method of any one of the first to tenth embodiments, wherein the second excitation wavelength is between 550 nm and 560 nm.

[0021] A twelfth embodiment includes the method of any of the first through eleventh embodiments, wherein the second excitation wavelength is about 555 nm.

[0022] A thirteenth embodiment includes the method of any of the first to twelfth embodiments, further comprising measuring a rate of change over time of a fluorescent output configured to change in response to proliferation of the analyte in the test device, and determining the presence of the analyte based on the normalized intensity of the second fluorescent signal and the measured rate of change over time of the fluorescent output configured to change in response to proliferation of the analyte in the test device.

[0023] A fourteenth embodiment includes the method of the thirteenth embodiment, wherein determining the presence of the analyte based on the normalized intensity of the second fluorescent signal and the measured rate of change over time of the fluorescent output configured to change in response to growth of the analyte in the test device includes performing a summation of the normalized intensity of the second fluorescent signal and the measured rate of change over time of the fluorescent output configured to change in response to growth of the analyte in the test device.

[0024] In a fifteenth embodiment, a system for determining the presence of an analyte of interest in a blood sample is provided. The system includes a blood culture test device configured to receive a blood sample including a sensor in an aqueous medium; a first light source; a first excitation filter configured to filter light from the first light source to provide light to the sensor at a first excitation wavelength at which optical absorption of the sensor remains substantially constant even as the pH of the aqueous medium changes; a second light source; a second excitation filter configured to filter light from the second light source to provide light to the sensor at a second excitation wavelength, different from the first excitation wavelength; one or more detectors configured to measure an intensity of a first fluorescent signal emitted from the sensor in the test device in response to the first excitation wavelength and an intensity of a second fluorescent signal emitted from the sensor in the test device in response to the second excitation wavelength; and a processor configured to normalize a measurement of the intensity of the second fluorescent signal emitted from the sensor in the test device in response to the second excitation wavelength using the measurement of the intensity of the first fluorescent signal emitted from the sensor in the test device in response to the first excitation wavelength.

[0025] A sixteenth embodiment includes the system of the fifteenth embodiment, wherein the processor is configured to normalize the measurement of the intensity of the second fluorescent signal by generating a ratio comparing the second fluorescent signal to the first fluorescent signal.

[0026] A seventeenth embodiment includes the system of the sixteenth embodiment, wherein the processor is further configured to compare the ratio to a threshold value.

[0027] An eighteenth embodiment includes the system of the seventeenth embodiment, wherein the processor is further configured to determine the presence of the analyte of interest when the ratio exceeds the threshold value.

[0028] A 19th embodiment includes the system of any of the 15th to 18th embodiments, wherein the first excitation wavelength is at an isosbestic point of a component of the sensor.

[0029] A twentieth embodiment includes the system of the nineteenth embodiment, wherein the sensor includes a pH indicator and a fluorophore, and the isosbestic point is the isosbestic point of the pH indicator.

[0030] A twenty-first embodiment includes the system of any of the fifteenth to twentieth embodiments, wherein the first light source includes a blue or cyan LED light source.

[0031] A twenty-second embodiment includes the system of any one of the fifteenth to twenty-first embodiments, wherein the first excitation wavelength is between 485 nm and 495 nm.

[0032] A twenty-third embodiment includes the system of any of the fifteenth to twenty-second embodiments, wherein the first excitation wavelength is about 490 nm.

[0033] A twenty-fourth embodiment includes the system of any of the fifteenth to twenty-third embodiments, wherein the second light source includes a green LED light source.

[0034] A twenty-fifth embodiment includes the system of any one of the fifteenth to twenty-fourth embodiments, wherein the second excitation wavelength is between 550 nm and 560 nm.

[0035] A twenty-sixth embodiment includes the system of any of the fifteenth to twenty-fifth embodiments, wherein the second excitation wavelength is about 555 nm.

[0036] A 27th embodiment includes the system of any of the 15 to 26 embodiments, wherein the one or more detectors are configured to measure a rate of change over time of a fluorescent output configured to change in response to growth of the analyte in the test device, and the processor is configured to determine the presence of the analyte based on a normalized measure of the intensity of the second fluorescent signal and the measured rate of change over time of the fluorescent output configured to change in response to growth of the analyte in the test device.

[0037] A 28th embodiment includes the system of any of the 15 to 27 embodiments, wherein the processor is configured to determine the presence of the analyte based on a summation of a normalized measure of the intensity of the second fluorescent signal and the measured time rate of change of the fluorescent output configured to change in response to growth of the analyte in the test device. [Brief description of the drawings]

[0038] [Figure 1] FIG. 1 shows a schematic diagram of a sample measurement system according to an exemplary embodiment of the present disclosure.

[0039] [Diagram 2] FIG. 2 is a graph showing the absorption spectra of a pH indicator at various pH conditions, according to an exemplary embodiment of the present disclosure.

[0040] [Figure 3A] FIG. 3A is a graph showing spectra of various optical components of a sample measurement system and the absorption spectrum of a pH indicator at various pH conditions according to an exemplary embodiment of the present disclosure.

[0041] [Figure 3B] FIG. 3B shows a schematic diagram of a sample measurement system according to an exemplary embodiment of the present disclosure.

[0042] [Figure 4]FIG. 4 is a flow chart illustrating a process for optimizing detection of an optical signal indicative of the presence of an analyte of interest in a blood sample, according to an exemplary embodiment of the present disclosure.

[0043] [Diagram 5] FIG. 5 is a graph illustrating the results of an experiment for detecting an optical signal indicative of the presence of an analyte of interest in a blood sample, according to an exemplary embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Any feature or combination of features described herein is included within the scope of the present disclosure, unless the features included in such combination are mutually inconsistent, as is apparent from the context, the description of the present specification, and the knowledge of one skilled in the art. Also, any feature or combination of features may be specifically excluded from any embodiment of the present disclosure. For purposes of summarizing the present disclosure, certain aspects, advantages, and novel features of the present disclosure are described (explained) herein. Of course, it should be understood that not all such aspects, advantages, or features are necessarily present in any particular embodiment of the present disclosure.

[0045] It should be understood that the embodiments presented herein are illustrative and not limiting of the present invention. The following detailed description describes exemplary embodiments, but should be construed to cover all modifications, alternatives, and equivalents of the embodiments that may fall within the spirit and scope of the present disclosure.

[0046] The embodiments described herein relate to systems and methods for optimizing detection of an optical signal indicative of the presence of an analyte of interest in a sample, such as a blood sample. In certain embodiments, the presence of an analyte of interest, such as bacteria or other microorganisms, is determined based on detection of a change in an optical property, such as fluorescence, that correlates with the growth of the analyte in the sample. For example, the sample may be introduced into a test device, such as a vial, cartridge, or other container, that contains a fluorescent material and an indicator material including one or more dyes that undergo an optically measurable change (e.g., fluorescence intensity) in response to the growth of the analyte in the test device. The indicator material may be configured to optically change in response to a change in conditions in the test device or a change in a property of the sample, such as, but not limited to, a pH indicator that undergoes an optically measurable change (e.g., a change in absorbance) in response to a change in pH conditions in the test device. The optical change in the indicator material may change the fluorescent behavior of the fluorescent material, for example, by modulating the excitation of the fluorescent material and / or the emission of a fluorescent signal. In certain embodiments, the fluorescent material and the indicator material may be part of a sensor in the test device.

[0047] One or more detectors may detect the intensity of a fluorescent signal emitted by a fluorescent material in the test device, and data detected by the detectors may be processed to indirectly determine the presence of an analyte of interest above a threshold in the test device by determining a change in the intensity of the fluorescent signal emitted by the fluorescent material in response to different excitation wavelengths.

[0048] In some implementations, embodiments of the systems and methods described herein may account for or mitigate inconsistencies due to bacterial or other microbial growth prior to an initial detector reading. For example, implementations of the disclosed technology may use a reference signal based on a constant or fixed characteristic of a component present in the measurement system (e.g., a constant or fixed characteristic of a sensor in the test device) to detect the presence of an analyte of interest above a threshold in a test device, such as a vial, cartridge, or other container. For example, the reference signal may be independent of the pH in a test device inoculated with a test sample. In one non-limiting example described below, the reference signal is at the isosbestic point of a component of the sensor present in the test device. The component may be a fluorescent substance or an indicator substance of the sensor. In some embodiments, the reference signal is a first fluorescent signal measured after exciting the sensor with a first excitation wavelength at the isosbestic point of a component of the sensor, such as an indicator substance. In some embodiments, a second fluorescent signal measured after exciting the sensor with a second wavelength different from the first wavelength may be compared to the first fluorescent signal to detect the presence of an analyte of interest in the test device above a threshold. In some examples, the first excitation wavelength is in the blue / cyan range. Some implementations of the disclosed technology may improve detection of DVEs in optical blood culture measurement systems by detecting the presence of an analyte of interest in a test device above a threshold using a reference signal based on a constant or fixed characteristic of a component present in the measurement system.

[0049] Additionally, embodiments of the systems and methods described herein may account for or mitigate variability in components of an optical blood culture measurement system. Implementations of the disclosed technology may address the aforementioned sources of variability in components of an optical blood culture measurement system. In one implementation, the components reside in a test device, such as a blood culture vial present in the optical blood culture measurement system. For example, embodiments of the disclosed technology may address or mitigate variability in a fluorescent sensor in a blood culture test device that is received in an optical blood culture measurement system for testing. Embodiments of the systems and methods described with reference to this implementation normalize the detector readings using a reference signal that does not vary as a function of the performance characteristics of the measurement system itself. Advantageously, a reference signal according to the present disclosure may be based on a constant or fixed characteristic of a component present in the measurement system, such as a constant or fixed characteristic of a sensor in the test device. In some cases, a reference signal according to the present disclosure is independent of the pH of the test device inoculated with the test sample. In one non-limiting example described below, the reference signal is at the isosbestic point of a component of the sensor present in the test device, which may be a fluorescent or indicator material of the sensor. In some implementations, the reference wavelength corresponds to a wavelength in the blue / cyan range and serves as a reference channel for a second channel in the green range.

[0050] Embodiments of the present disclosure are described with respect to normalizing a fluorescent signal emitted by a testing device, such as a test vial, to address inconsistencies caused by the presence of an analyte of interest prior to the initial reading of a detector. Embodiments of the present disclosure may also address variations in the components of an optical blood culture measurement system. It will be understood that the methods of adjusting, dividing and comparing fluorescent signals using reference signals and reference ratios described herein may also be described as calibrating a fluorescent signal.

[0051] Implementations of the present disclosure that use a reference signal to normalize a fluorescent signal emitted by a testing device, such as a test vial, to address inconsistencies caused by the presence of an analyte of interest prior to the initial reading of a detector are described with reference to FIGS. 1-5. The reference signal does not vary as a function of the amount of analyte present in the testing device and can be used to normalize a fluorescent signal emitted from a test vial inoculated with a sample under test. Although the embodiments described with reference to FIGS. 1-5 describe a test vial, it will be understood that embodiments of the present disclosure are not limited to systems and methods that use a test vial as a testing device. In other non-limiting embodiments of the present disclosure, other testing devices, such as a test cartridge or other container, can be used.

[0052] 1 shows a schematic diagram of a measurement system 100 according to an exemplary embodiment of the present disclosure. The measurement system 100 includes a test device in the form of a test vial 102, a light source 108, and a detector 110.

[0053] The test vial 102 is configured to receive a sample 104, such as a blood sample. The measurement system 100 is configured to determine the presence or absence of an analyte of interest in the sample 104 received in the test vial 102. The analyte of interest may be, for example, a microorganism or a bacterium. The test vial 102 may further contain a sensor 106 that includes a fluorescent material and an indicator material. The test vial 102 may also contain a liquid medium that may support the growth of the microorganism in the test vial 102. The test vial 102 may be, for example, a blood culture bottle.

[0054] The light source 108 may be activated to emit light at one or more wavelengths or ranges of wavelengths to excite the fluorescent material of the sensor 106. In certain embodiments, the light source 108 may include one or more light emitting diodes (LEDs).

[0055] The detector 110 may be configured to detect the fluorescence emitted by the fluorescent material of the sensor 106 after excitation thereof. The detector 110 may be a silicon photodiode, a PIN silicon diode, a GaAsP photodiode, or any other suitable photodetector. In some embodiments, the detector 110 may include a photovoltaic device, a photoresistive device, a photoconductive device, or any other suitable device for detecting a signal emitted from the sensor 106. In certain embodiments, multiple detectors 110 may be employed to measure the fluorescent signal emitted by the sensor 106.

[0056] The system 100 may include one or more excitation filters 114 configured to filter light from the light source 108 to provide only a particular wavelength or range of wavelengths to the fluorescent material. For example, in certain embodiments, the one or more excitation filters 114 may filter light to provide a particular wavelength or range of wavelengths to the fluorescent material that corresponds to the absorption spectrum of the fluorescent material.

[0057] In certain embodiments, the system 100 may be configured to provide light at multiple excitation wavelengths or excitation wavelength ranges. For example, the light source 108 may emit light at multiple emission spectra. For example, the light source 108 may include multiple LEDs that emit light at different emission spectra. The system 100 may include multiple filters 114 configured to filter the light from the multiple LEDs to provide only a particular wavelength or wavelength range of light from each LED to the fluorescent material. Alternatively, the light source 108 may include a single LED that may be filtered by multiple excitation filters 114 to provide multiple excitation wavelengths or excitation wavelength ranges to the sensor.

[0058] System 100 may include one or more emission filters 116 configured to filter light to provide a wavelength or range of wavelengths to detector 110. For example, in certain embodiments, one or more emission filters 116 may filter light to provide a wavelength or range of wavelengths to detector 110 that corresponds to the emission spectrum of a fluorescent material.

[0059] The fluorescent material used in the system 100 may be selected based on the emission spectrum of the light source 108 and / or the specifications of the detector 110. In certain embodiments, the fluorescent material may include one or more fluorophores. Examples of fluorophores that may be suitable for use in the embodiments described herein include, but are not limited to, thionine, naphthofluorescein, carboxynaphthofluorescein, 3,3'-dimethyloxadicarbocyanine, sulforhodamine B, pyronine B, rhodamine B, Nile red phenoxazone 9, Evans blue, rhodamine 6G perchlorate, sulforhodamine G, 7-aminoactinomycin D, eosin, rhodamine 110, and rhodamine 123.

[0060] As described herein, the indicator material in the sensor 106 may undergo an optical change in response to a change in the analyte of interest in the sample, such as a change in the concentration of the analyte in the sample. In certain embodiments, an indicator material is selected that changes an optical property based on a change in the concentration of one or more of CO2, O2, H2S, NH3, or any other suitable compound known in the art, present in the test vial. In certain embodiments, an indicator material is selected that changes an optical property based on a change in pH in the test vial, the change in pH being due to a change in the concentration of the analyte of interest in the sample.

[0061] In certain embodiments, the indicator may include a pH indicator. Examples of pH indicators that may be suitable for use in the embodiments described herein include, but are not limited to, propyl red, p-nitrophenol, azolimine, chlorophenol red, 3,6-dihydroxyxanthone, alizarin, bromoxylenol blue, m-dinitrobenzoylurea, bromthymol blue, aurin (aosolic acid), neutral red, cresol red, bromocresol red, bromocresol purple, resolic acid, nile blue, phenol red, nitramine, cresol purple, and methyl yellow.

[0062] The optical change in the indicator material may act as an optical filter and change the amount of light exciting the fluorescent material or emitted from the fluorescent material of the sensor 106. Thus, a change in the concentration of the analyte of interest in the sample may cause a change in the fluorescent signal detected by the detector 110 by changing the optical properties of the indicator material of the sensor 106. As a result, a change in the intensity of the fluorescent signal detected by the detector 110 may be indicative of a change in the concentration of the analyte of interest in the sample.

[0063] As an example, in certain embodiments, the system 100 is configured to detect the presence of bacteria or microorganisms in a sample placed in the vial 102. In embodiments where bacteria is the analyte of interest, the indicator may be a pH indicator configured to change absorbance when the pH changes. When bacteria grow, carbon dioxide (CO2) is respired (exhaled). The carbon dioxide may mix with the aqueous medium in the vial 102 and produce carbonic acid. The increasing amount of carbonic acid results in a decrease in pH. At a particular excitation wavelength, when the pH in the vial 102 decreases, the absorbance of the pH indicator decreases. This may allow more excitation energy to reach the fluorescent material in the sensor 106, resulting in an increase in the intensity of the fluorescent emission from the fluorescent material. As described herein, the fluorescent material may include one or more fluorophores. The detector 110 may detect the increased fluorescent emission intensity, which may serve as an indirect measure of an increase in carbon dioxide (CO2) concentration. As previously mentioned, the concentration of carbon dioxide directly correlates with bacterial growth, and therefore detection of increased fluorescence intensity by detector 110 may be indicative of the presence of bacteria within the sample.

[0064] In certain embodiments, the measurement system 100 may further include a processor 112 configured to perform signal processing to determine the presence of an analyte of interest based on changes in fluorescence intensity measured by the detector 110. In certain embodiments, the processor may be part of a computing system. Such a computing system may also include one or more of a memory, an input, and a display. The memory may include a read-only memory (ROM) or both a ROM and a random access memory (RAM) and may be configured to provide instructions and data to the processor 112. For example, the memory may store one or more modules that may store data values ​​that define instructions for configuring the processor 112 to perform signal processing functions.

[0065] In certain embodiments, the fluorescent signals detected by one or more detectors may be normalized by the processor 112 using a reference signal.

[0066] In a particular embodiment, the system 100 may be a "BD BACTEC" (registered trademark) blood culture system by Becton Dickinson and Company. The BACTEC® sepsis test relies on a silicon matrix-based sensor that includes an indicator compound with pH-dependent UV absorption characteristics and a fluorescent dye system. If bacteria are present in the blood test specimen, the growth of the bacteria results in the production of carbon dioxide, which is converted to carbonic acid when dissolved in the BACTEC® media solution. In use, incident light from an LED light source positioned below the BACTEC® sensor bottle is intensity-modulated by the pH-dependent indicator compound in the silicon matrix-based sensor. This intensity modulation of the incident light modulates the amount of incident light that reaches the fluorescent dye system, resulting in a change in the intensity of the fluorescent emission from the fluorescent dye system.

[0067] In current use of the BACTEC® test measurement system, the output of the measurement system at a particular time is generated based on a ratio of the current detector reading to the initial detector reading obtained when the test bottle is first placed in the system ("time zero"). In this system, the current detector reading is normalized by dividing the current detector reading by the initial detector reading at time zero. Variations in the reported measurement system readings can reduce the sensitivity of the measurement system. As a result, detector variability can affect the threshold measurement required to determine the presence of the analyte of interest in the test sample. Additional performance issues within the measurement system, such as delayed vial entry (DVE), instrument temperature variations during sample measurement, and degradation of the LED light source and fluorescence detector, are not currently considered. The embodiments of the present disclosure solve these and other problems.

[0068] In certain embodiments, the measurement system normalizes the detector readings using a reference signal that does not change as a function of the performance characteristics of the measurement system itself. For example, a reference signal can be selected that is based on a constant or fixed characteristic of a component present in the measurement system, such as a constant or fixed characteristic of a sensor in a test vial. In one non-limiting example of the present disclosure, a reference signal is selected that is independent of the pH in the test vial inoculated with the test sample. In a non-limiting example described below with reference to Figures 2 to 4, the reference signal is at the isosbestic point of a component of the sensor present in the test vial.

[0069] An isosbestic point in this application is defined as a wavelength, wavenumber, or frequency at which the overall absorbance of a sample remains constant or substantially constant during a chemical or physical change of a component present in a test vial inoculated with a sample. The chemical or physical change may include, for example, a change in pH of the medium in the test vial. The isosbestic point of a component present in a measurement system may be used to generate a reference signal for normalizing a detector reading. The component may be a pH-dependent indicator compound present in a test vial inoculated with a sample. In one non-limiting example, described in detail below with reference to FIG. 2, the isosbestic point of an indicator of the sensor 106 may be determined and used to generate a reference signal. If the indicator is a pH indicator, the isosbestic point may be a particular wavelength where the absorption spectrum of the pH indicator at various pH conditions intersect. For example, as shown in FIG. 2, a pH-dependent indicator compound may exist in two states in the sensor 106. The first state exists at a relatively low pH level and the second state exists at a relatively high pH level. Each state has its own characteristic maximum absorption wavelength. These two states are in pH-dependent equilibrium with each other. This pH-dependent equilibrium results in one invariant or constant crossover absorption wavelength, referred to herein as the isosbestic point. At the isosbestic point, the optical absorption of the sensor remains substantially constant as the pH of the aqueous medium changes. At the isosbestic point, the optical absorption of the sensor does not substantially change as the pH of the aqueous medium changes.

[0070] The isosbestic point of a component (e.g., a pH indicator) is an inherent property of a substance. As a result, the use of the fluorescence output from an excitation wavelength at the isosbestic point as a reference signal in accordance with the present disclosure is not dependent on or altered by the performance characteristics of the measurement system. Advantageously, the isosbestic point of a component present in a measurement system may allow for real-time continuous normalization of blood culture readings detected by the measurement system.

[0071] The isosbestic point of the sensor's indicator can be used to generate a reference signal for normalizing the detector readings. If the indicator is a pH indicator, the isosbestic point can be a point (i.e., a specific wavelength) in the absorption spectrum where the absorption curves of the pH indicator at various pH conditions intersect.

[0072] An example of such an isosbestic point is shown in FIG. 2 at isosbestic point 205. FIG. 2 is a graph showing the absorption spectrum of a pH indicator at various pH states, including an absorption spectrum at pH 2.12 (low pH state) and an absorption spectrum at pH 5.9 (high pH state). As shown in FIG. 2, the absorption spectrum of the pH indicator at each of the illustrated pH states overlaps at an isosbestic point 205. In this non-limiting example, the isosbestic point 205 is about 525 nm. It will be understood that the isosbestic point of a pH indicator depends on the concentration of the pH indicator in the sensor. The particular isosbestic point of a pH indicator provided at a particular concentration in the sensor of the test vial may be determined empirically or using any other suitable method.

[0073] [Dual excitation light measurement system using the isosbestic point of a pH indicator excited with blue / cyan light as a reference signal] In one non-limiting embodiment described with reference to Figures 3A-4, the ratio of the absorbance of a pH indicator at two specific wavelengths (one of which corresponds to the isosbestic point) can be used to detect the presence of an analyte of interest (such as bacteria or other microorganisms) in a test vial.

[0074] In certain embodiments, light at a first excitation wavelength or range of wavelengths at or near the isosbestic point of the pH indicator may be provided to the sample, and light at a second excitation wavelength or range of wavelengths different from the first excitation wavelength or range of wavelengths may be provided to the sample. The ratio of the absorbance of the pH indicator at the two specified wavelengths or ranges of wavelengths may be used to determine the presence of an analyte of interest, such as bacteria or other microorganisms, in the test vial, for example by comparison to an empirically determined threshold value.

[0075] FIG. 3A is a graph showing the absorption spectrum of an indicator, in this case showing the positive (corresponding to a relatively high pH, ​​indicating the absence of the analyte of interest) and negative (corresponding to a relatively low pH, indicating the presence of the analyte of interest) states of the pH indicator. In this example, the pH indicator is bromocresol purple. As shown in FIG. 3A, when the pH indicator is excited with light having a wavelength of about 490 nm, the absorption spectrum of the positive state pH indicator and the absorption spectrum of the negative state pH indicator overlap at the isosbestic point 305. Thus, in this non-limiting example, the isosbestic point 305 is about 490 nm. The absorbance of the pH indicator excited at the constant crossover absorption wavelength of 490 nm remains constant or substantially constant. In other words, the absorbance of the pH indicator does not change or does not change substantially. Thus, the isosbestic point of this example pH indicator occurs when the pH indicator is excited using blue or cyan light at about 490 nm, and is a constant that can be used to generate a reference signal (or reference channel), as described in more detail below.

[0076] A wavelength of 490 nm generally corresponds to cyan light, which has a range of about 490 nm to about 520 nm, or blue light, which has a range of 490 nm to 450 nm. Implementations of the present disclosure are described herein with reference to blue / cyan light, but it will be understood that these color descriptions are not limiting. It will be understood that in this example, the color of light at the isosbestic point is described as blue / cyan for ease of reference, because light of particular wavelengths may be described as having different colors when the wavelengths fall within a range of overlapping colors.

[0077] 3A also shows an example spectrum of optical components that may be used in a dual LED measurement system 100 according to the present disclosure. The system 100 includes a first LED light source (emitting excitation light centered at an isosbestic point of about 490 nm), a first excitation filter, a second LED light source (emitting excitation light centered at 555 nm), a second excitation filter, and an emission filter. An example of an implementation of the measurement system 100 with the first LED light source 108a, the second LED light source 108b, the first excitation filter 114a, and the second excitation filter 114b is shown in FIG. 3B.

[0078] FIG. 3A shows the emission spectrum 315 of the first LED light source 108a (shown in FIG. 3B). A first filter window 310 filters the light from the first LED light source 108a. The first filter window 310 is located at or near the isosbestic point 305 of the absorbance spectrum of the pH indicator. Thus, the absorbance of the pH indicator at the first excitation wavelength does not change as a function of pH. As mentioned above, in the non-limiting example shown in FIG. 3A and FIG. 3B, the first LED light source 108a can be a blue or cyan LED light source. In the non-limiting example shown in FIG. 3A and FIG. 3B, the pH indicator can be bromocresol purple. It will be understood that the embodiments of the present disclosure are not limited to systems and methods using bromocresol purple as a pH indicator. In another non-limiting embodiment of the present disclosure, the pH indicator can be, for example, cresol red.

[0079] FIG. 3A shows the emission spectrum 325 of the second LED light source 108b (shown in FIG. 3B). A second filter window 320 filters the light from the second LED light source 108b. As shown in FIG. 3A, the absorbance of the pH indicator in the second filter window 320 is significantly higher when the pH-dependent indicator is in a positive state compared to when it is in a negative state. This is in contrast to the substantially unchanged or constant absorbance of the pH indicator in the negative and positive states at the isosbestic point 305 in the first filter window 310. In a non-limiting example shown in FIG. 3A and FIG. 3B, the second LED light source 108b can be a green LED light source emitting a wavelength centered around 555 nm.

[0080] Due to the invariant absorbance of the pH indicator at the first excitation wavelength or wavelength range, the luminescence reading from the test vial after excitation using the first excitation wavelength can serve as a invariant reference signal for comparison with the luminescence reading from the test vial after excitation using the second excitation wavelength or wavelength range. It has been found that the ratio of the luminescence reading from the test vial after excitation using the first excitation wavelength or wavelength range to the luminescence reading from the test vial after excitation using the second excitation wavelength or wavelength range is proportional to the pH in the vial. This ratio is proportional to the relative slope of the absorbance spectrum between the first and second excitation wavelengths.

[0081] Emission reading at the first excitation wavelength (1 st λ emission), emission reading at the second excitation wavelength (2 nd The relationship between the λ emission and the pH within the vial can be expressed by Equation 1: (Formula 1) TIFF2025506120000002.tif26153

[0082] The relative slope of the absorbance spectrum between the first and second excitation wavelengths can be represented by Equation 2, where Δ nd λ emission is the change in emission reading between a first emission reading at a second excitation wavelength and a second emission reading at a second excitation wavelength. (Formula 2) TIFF2025506120000003.tif26153

[0083] The relative slope of the absorbance spectrum may also be represented by Equation 3 below, where "pH1" is the first pH value at the first emission reading at the second excitation wavelength, "pH1 λ emission" is the first emission reading at pH1 at the second excitation wavelength, "pHn" is the pH value at a time point after the first emission reading, "pHn λ emission" is the emission reading at pHn at the second excitation wavelength, "pH1 slope" is the slope of the absorbance spectrum at pH1, and "pHn slope" is the slope of the absorbance spectrum at pHn. (Formula 3) TIFF2025506120000004.tif26153

[0084] FIG. 4 is a flow chart showing a process 400 for determining the presence of a target analyte, such as bacteria or other microorganisms, using a measurement system similar to the measurement system 100 described with respect to FIG. 1 and FIG. 3B. The measurement system in this implementation is a dual light source measurement system according to the present disclosure. The measurement system includes an excitation filter and an emission filter with certain selection criteria. According to the implementation of the present disclosure, one of the two light sources excites the sensor with light of a first wavelength that coincides with the isosbestic point of the sensor, and the other of the two light sources excites the pH indicator with light of a second wavelength different from the first light source. The measurement system measures the emission after excitation by the first light source and uses these measured emissions as a reference signal or channel to evaluate the emission after excitation by the second light source.

[0085] 4, process 400 includes exciting a sensor (such as sensor 106) that includes a pH indicator and a fluorophore at two separate wavelengths (or ranges of wavelengths), where one of the wavelengths correlates with an isosbestic point in the absorbance spectrum of the pH indicator.

[0086] The process 400 begins at step 405, where a first light source, such as the first LED light source 108a, is activated to emit light at a first wavelength or across a first wavelength range to excite the phosphor. For example, the first light source may be configured to emit light across the emission spectrum 315 shown in FIG. 3A.

[0087] In step 410, a first excitation filter, such as excitation filter 114a, filters the light emitted by the first light source. The excitation filter may be selected such that a filter window 310 defined by the first excitation filter encompasses or is located near an isosbestic point of the absorbance spectrum of the pH indicator. In this non-limiting example, the isosbestic point of the pH indicator is about 490 nm. It will be understood that embodiments of the present disclosure are not limited to pH indicators having an isosbestic point of about 490 nm. The isosbestic point of a pH indicator is an inherent property of the indicator and may be determined in any suitable manner, including empirical methods. After the first excitation filter filters the light emitted by the first light source, the pH indicator absorbs at least a portion of the light emitted by the first light source in step 415. As previously mentioned, the amount of light absorbed by the pH indicator is the same or substantially the same regardless of whether the pH indicator is in a high pH state (indicating the absence of the analyte of interest) or a low pH state (indicating the presence of the analyte of interest).

[0088] After the pH indicator absorbs at least a portion of the light emitted by the first light source, a fluorophore in the sensor absorbs at least a portion of the light emitted by the first light source and, in response, emits (radiates) a first fluorescent signal in step 420. The first fluorescent signal emitted by the fluorophore is then filtered in step 425 by an emission filter, such as emission filter 116.

[0089] After the first fluorescent signal emitted from the fluorophore is filtered by an emission filter, the resulting signal is detected by a photodiode, such as photodiode 110, in step 430. The photodiode may then generate an electronic signal proportional to the intensity of the detected light in step 435. The electronic signal "signal 1" obtained in step 435 due to excitation of the pH indicator at a wavelength at or near the isosbestic point of the absorbance spectrum of the pH indicator may be used as a reference signal that does not change as a result of changes in the pH of the pH indicator.

[0090] In step 440, a second light source, such as the second LED light source 108b, is activated to emit light over a second wavelength or wavelength range to excite the phosphor. For example, the second light source can be configured to emit light over the emission spectrum 325 shown in FIG. 3A. The second wavelength or wavelength range is different from the first wavelength or wavelength range. In one example, the second light source emits light at a second wavelength that is not included in the first wavelength range emitted by the first light source. In another example, the second light source emits light over a second wavelength range that does not overlap with the first wavelength emitted by the first light source. In yet another example, the second light source emits light over a second wavelength range that does not overlap with the first wavelength range emitted by the first light source.

[0091] In step 445, a second excitation filter, such as excitation filter 114b having a filter window, such as filter window 320, filters the light emitted by the second light source. After the second excitation filter filters the light emitted by the second light source, the pH indicator absorbs at least a portion of the light emitted by the second light source in step 450. As previously described, a pH-dependent indicator compound has a single isosbestic point (a single, unchanging crossover absorption wavelength) where absorption (extinction) by the indicator is substantially constant or does not change regardless of changing pH conditions. The first light source excites the pH indicator with a wavelength of light at or near the isosbestic point of the pH indicator, and the second light source excites the pH indicator with a different wavelength of light. Thus, when excited by the second light source, the pH indicator will absorb a different amount of light when in a high pH state (indicating the absence of the analyte of interest) than when in a low pH state (indicating the presence of the analyte of interest).

[0092] After the pH indicator absorbs at least a portion of the light emitted by the second light source, the fluorophore in the sensor absorbs at least a portion of the light emitted by the second light source and, in response, emits (emits) a second fluorescent signal in step 455. The second fluorescent signal emitted by the fluorophore is then filtered by an emission filter in step 460.

[0093] After the second fluorescent signal emitted from the fluorophore is filtered by the emission filter, the resulting signal is detected by a photodiode in step 465. The photodiode may then generate an electronic signal, "signal 2," in step 470 that is proportional to the intensity of the detected light.

[0094] In step 475, a ratio is generated by comparing signal 1, which is proportional to the light intensity detected at the photodiode due to excitation by the first excitation wavelength, generated in step 435, with signal 2, which is proportional to the light intensity detected at the photodiode due to excitation by the second excitation wavelength, generated in step 470. This ratio has been found to be proportional to the pH of the medium in the test vial. This ratio also indicates the average slope of the absorbance spectrum of the pH indicator between the first and second excitation wavelengths (between about 490 nm and 580 nm).

[0095] As described herein, the pH in the test vial may be correlated to the presence of an analyte, such as bacteria or other organisms, in the test vial. For example, as previously described, when bacteria grow, carbon dioxide (CO2) is breathed (exhaled). The carbon dioxide may mix with the aqueous medium in the vial 102 to produce carbon dioxide. An increasing amount of carbon dioxide results in a decrease (reduction) in the pH. Thus, the pH of the medium may serve as an indirect measure of the presence of bacteria in the medium. In step 480, the ratio determined in step 475 may be compared to a predetermined threshold value to determine the presence of the analyte of interest in the test vial. In certain embodiments, a threshold value may be determined once (e.g., empirically) for a particular pH indicator in a particular measurement system, such as measurement system 100. The threshold value may then be used for subsequent measurements of test vials using the particular indicator in the particular measurement system. For example, the threshold value may be determined for a particular pH indicator when the measurement system is initially set up or when a new type of test vial bottle is implemented in the measurement system.

[0096] In certain implementations, process 400 may be used to detect a DVE. Process 400 may be performed when a vial is placed in a measurement system for the first time to determine the presence of an analyte of interest in the vial at the time the vial is placed in the measurement system. Advantageously, determining the presence of an analyte of interest above a threshold value at the time the vial is placed in the measurement system in accordance with the present disclosure may indicate bacterial or other microbial growth prior to placing the vial in the measurement system. Using this principle, the ratio determined in step 475 may be compared to a threshold value to indirectly detect the presence of an analyte of interest in the vial above the threshold value, thereby detecting a DVE.

[0097] Furthermore, because the reference signal is based on the isosbestic point, which is a substantially invariant characteristic of the sensor, the ratio determined in step 475 is not affected by variations in the optical blood culture measurement system components, temperature, or vial position.

[0098] Determining the ratio in step 475 is a non-limiting example of normalizing the second fluorescent signal emitted in response to a second excitation wavelength compared to the first fluorescent signal emitted in response to a first excitation wavelength at the isosbestic point of the pH indicator. In certain embodiments, normalizing the second fluorescent signal may include calculating the ratio of the first fluorescent signal to the second fluorescent signal, calculating the ratio of the second fluorescent signal to the first fluorescent signal, subtracting the first fluorescent signal from the second fluorescent signal, or subtracting the second fluorescent signal from the first fluorescent signal. In certain embodiments, normalizing the second fluorescent signal may include applying any function that uses inputs based on the first fluorescent signal and inputs based on the second fluorescent signal such that the resulting variation of the function is less than the combined variation of both fluorescent signal inputs. Variations in both fluorescent signal inputs, such as excitation intensity and fluorophore concentration, may vary in tandem and therefore may be offset or otherwise compensated for.

[0099] FIG. 5 shows the results of an experiment performed using process 400. In the experiment, 96 vials were provided with several types of media. Bagged blood was introduced into the vials and the vials were inoculated with microorganisms including several common bacterial strains. In the experiment, the test vials were inoculated with Bacteroides fragilis, Escherichia coli, Enterococcus faecalis, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. After inoculation, the test vials were allowed to incubate for 24 hours before being placed into a measurement system such as measurement system 100. After 24 hours, 96 test vials (bottles) were loaded into the measurement system at a rate of 48 per day and data was collected for the next 24 hours. In the measurement system, the test vials were excited with cyan / blue light and the intensity of the fluorescent signal emitted from the test vials was measured and recorded. After approximately 1 minute of excitation with the cyan / blue light, the test vial was excited with green light and the intensity of the fluorescent signal emitted from the test vial was measured and recorded. During the data collection period, independent excitation and measurement using each of the cyan / blue and green lights was repeated approximately every 10 minutes.

[0100] After the initial 24-hour incubation period, each vial was deemed to have sufficient bacterial growth to mimic a DVE scenario. The threshold for DVE detection was empirically calculated based on previous experimental data using fresh blood. As shown in Figure 5, the threshold in this non-limiting example was 1.18.

[0101] In the graph shown in FIG. 5, the Y-axis represents the ratio determined in step 475 of process 400. Each circle on the graph represents one of the 96 vials. As shown in FIG. 5, 84 vials had emission that resulted in a ratio above the threshold of 1.18 (shown in FIG. 5 as 84 circles above the dashed 1.18 threshold). This indicates that process 400 detected DVE for these vials, which corresponds to a sensitivity of 87.5%. Twelve vials had a ratio below the threshold (shown in FIG. 5 as 12 circles below the dashed 1.18 threshold), indicating that 12.5% ​​of the test vials were determined to be false negatives.

[0102] The graph shown in FIG. 5 also illustrates the detection of DVEs using a kinematic (kinetic) DVE algorithm. Each dotted circle represents a vial in which a DVE was detected by the kinematic DVE algorithm, and each filled circle represents a vial in which a DVE was not detected by the kinematic DVE algorithm. As shown, the kinematic DVE algorithm detected DVEs in 69 vials, which corresponds to a sensitivity of 71.9%. These results indicate a higher sensitivity (higher incidence of DVE detection) to DVE events using the process 400 according to an implementation of the present disclosure compared to detecting DVE events using only the kinematic DVE algorithm.

[0103] Advantageously, the process 400 according to the implementation of the present disclosure can be used in combination with a kinematic DVE algorithm to detect DVEs in vials, increasing the overall rate at which DVE events are correctly detected. For example, as shown in FIG. 5, one combination of the process 400 and the DVE algorithm produced only three false negatives (shown in FIG. 5 as three filled circles below the dashed threshold of 1.18), corresponding to a sensitivity of 96.9%. Thus, detecting DVE events using a combination of the current kinematic DVE algorithm and the system and method according to the present disclosure can result in a substantial increase in sensitivity. In a non-limiting experiment corresponding to FIG. 5, the sensitivity increased from 71.9% (27 of 96 vials were not recognized as DVE events) to 96.9% (3 of 96 vials were not recognized as DVE events).

[0104] Current methods of detecting DVE using kinetic DVE algorithms rely on detecting a change in a signal over time. For example, measurements may be taken periodically (e.g., every 10 minutes) and a rate of change may be determined. This rate of change may be proportional to the rate of change of pH in the test vial. In a test vial that is not experiencing a DVE, the rate of change may be initially low. In a test vial that is experiencing a DVE, the initial measurements may be high. In such cases, all subsequent measurements may also be high, and the kinetic DVE algorithm may detect a relatively low rate of change. The growth of bacteria and other microorganisms in a test vial is limited by the amount of nutrients in the vial. After the nutrients are depleted, the growth of bacteria and other microorganisms stops, which stops the pH in the vial from changing further. If no rate of change is detected (or if the rate of change is low), the current kinetic DVE algorithm will not determine a DVE and will result in a false negative. In current practice, an operator may have to monitor DVE measurements of a patient's test vial for 12-18 hours before determining that the test result is indeterminate. Using the systems and methods described herein, without relying on the pH in the test vial during the initial detector reading, an operator can immediately determine that a DVE has occurred and proceed to obtain another blood sample from the patient for a new test without having to wait 12-18 hours.

[0105] The use of a reference signal that is independent of the measured intensity of the fluorescent signal emitted from the test vial, such as the reference signals described with respect to Figures 2 to 4, may have many advantages over a reference signal that is dependent on the intensity of the fluorescent signal emitted from the test vial. The use of a reference signal according to the embodiments described herein may improve the sensitivity of the detector by eliminating the variation of the initial detector reading that may cause the variation of the reported measurement system reading. The variation of the initial detector reading proportionally affects the uncertainty of the measurement system output. The latter corresponds to the resolution of the measurement system itself. In the embodiments described herein, the variation of the initial sensor reading for normalizing the detector reading is eliminated, improving the sensitivity of the detector. This may advantageously result in a measurement system with higher resolution.

[0106] Furthermore, the use of a reference signal according to embodiments described herein may improve or eliminate errors due to DVEs because the output signal of the measurement system is not normalized using the initial reading of the detector. The use of a reference signal according to embodiments described herein also eliminates the need to compensate for temperature variations of the sensor.

[0107] As previously discussed, in certain embodiments, detecting DVE events using a combination of a kinetic DVE algorithm and a system and method according to the present disclosure may result in a substantial increase in sensitivity. For example, in certain embodiments, a combination of a measured rate of change over time of a fluorescent output configured to change in response to growth of an analyte in the test device and a normalized measure of intensity (e.g., a ratio generated in step 475 of process 400) normalized using a reference signal generated using an isosbestic point (e.g., "signal 1" in step 435 of process 400, which does not change as a result of a change in pH of a pH indicator) may result in a substantial increase in sensitivity. In certain embodiments, the combination may be the addition (sum) of a measured rate of change over time of a fluorescent output configured to change in response to growth of an analyte in the test device and a normalized measure of intensity normalized using a reference signal generated using an isosbestic point.

[0108] In certain embodiments, the systems and methods described herein may be used in conjunction with other algorithms to reduce noise associated with measurements from a single (e.g., green) channel. In certain embodiments, the systems and methods described herein may be used in conjunction with other algorithms to detect degradation of measurement system equipment, such as LEDs or photodiodes.

[0109] There are also additional advantages associated with the embodiments of the present disclosure. A reference signal that includes or is related to the isosbestic point of a component in a measurement system does not change its specific wavelength position for a given concentration of that component. The lack of fluctuation in the reference signal makes it ideally suited to normalize the readings of the detector of the measurement system. At the same time, a reference signal that does not change based on the measured intensity of the fluorescent signal emitted from the test vial as described herein can serve as a real-time quality indicator of a measurement system such as the measurement system 100.

[0110] For example, because embodiments of a reference signal according to the present disclosure should be substantially constant or substantially unchanged during testing of a vial sample placed in the measurement system, the reference signal may be measured during assay vial testing to determine errors in the measurement system. In certain embodiments, the measurement system may be configured to ignore data if the reference signal changes during the assay vial testing period. Alternatively, the measurement system may be programmed to modify or adjust the sample output signal based on detected variations in the reference signal described herein.

[0111] Additionally, the absolute level of the isobestic signal reading from the measurement system can help determine the health of the measurement system itself. The isobestic signal can be expected to vary only based on the amount and / or concentration of fluorophores in the sensor and the optical properties of the bottle (vial), and the expected range can be determined by empirical testing or other suitable methods. An isobestic signal below an established threshold can indicate a faulty bottle / sensor or a measurement system degradation or failure. Identification of an isobestic signal reading below an established threshold can alert an operator of the measurement system to check and remedy these conditions.

[0112] Additionally, the use of the reference signal described herein may allow for identification of a particular test vial or other test device. By design, the reference signal according to the present disclosure does not vary between test devices. As a result, the reference signal may serve as an identification marker that may be correlated with a test instrument and measured to confirm its identity. This identification marker may be used as verification that a test device containing a sensor is supplied by a particular manufacturer. The identification marker may also serve as an indication that a competitor has replicated the sensor chemistry of a test device. For example, an isosbestic reference signal may be determined for a competitor's test device and compared to the reference signal of a known test device to determine that the competitor has replicated the sensor chemistry of a particular known test device.

[0113] Additional examples of invariant reference signals and their use in optical blood culture measurement systems are described in U.S. Patent Application Publication No. 2021 / 0262936, which is incorporated by reference in its entirety and for all purposes.

[0114] Implementations disclosed herein provide systems, methods, and devices for optimizing the detection of an optical signal indicative of the presence of an analyte of interest in a sample. Those skilled in the art will recognize that these embodiments may be implemented in hardware, software, firmware, or any combination thereof.

[0115] In addition to the aforementioned advantages, embodiments of the systems and methods described herein may be advantageously implemented without modifying consumable components of current blood culture measurement systems. For example, implementations of the techniques disclosed herein may be implemented without modifying the assay bottles containing the contents of media, nutrient solutions, and sensors. Furthermore, embodiments of the systems and methods described herein may be implemented at a one-time cost that may be limited to the total cost of adding optical interrogation at a specified wavelength and associated algorithmic code for processing the optical measurements to determine a reference signal, as described herein. In certain embodiments, a light source (e.g., a blue / cyan LED in the non-limiting examples described herein) that excites a pH indicator with a wavelength of light at or near the isosbestic point of the pH indicator may be selectively omitted from the detection process. This selective omission of the light source may allow a user to use a blood measurement system implementing the techniques disclosed herein in the same manner as a current blood culture measurement system.

[0116] It will be further understood that embodiments of the technology disclosed herein are not limited to blood culture measurement systems, but may also be applied to other types of optical detection systems that rely on sensors or other substances that have invariant properties, such as isosbestic points, suitable for use in generating a reference signal for normalizing detector readings. For example, in certain embodiments, the technology disclosed herein may be applied to immunoassays, which may include immunoassays that monitor output as a function of time, and immunoassays that are point-in-time tests (i.e., episode tests). When an immunoassay is a point-in-time test (episode test), the ratio of the test output to a reference signal generated using the isosbestic point may be used as an indicator of assay quality or as a monitoring method to indicate that the immunoassay has been replicated or counterfeited.

[0117] The functions described herein may be stored as one or more instructions on a processor-readable medium or computer-readable medium. The term "computer-readable medium" refers to any available medium that can be accessed by a computer or processor. By way of example and not limitation, such media may include RAM, ROM, EEPROM, flash memory, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage, or any other medium that can be utilized to store desired program code in the form of instructions or data structures and that can be accessed by a computer. As used herein, disk and disc include compact discs (CDs), laser discs, optical disks, digital versatile discs (DVDs), floppy disks, and Blu-ray discs. Disks usually reproduce data magnetically, while discs reproduce data optically with lasers. It should be noted that computer-readable media may be tangible and non-transitory. The term "computer program product" refers to a computing device or processor in combination with code or instructions (e.g., a "program") that can be executed, processed, or computed by that computing device or processor. As used herein, the term "code" may refer to software, instructions, code, or data that is executable by a computing device or processor.

[0118] Software or instructions may also be transmitted over a transmission medium, such as when the software is transmitted from a website, server, or other remote source using coaxial cable, fiber optic cable, twisted pair cable, Digital Subscriber Line (DSL), or wireless technologies such as infrared, radio waves, microwaves, etc., all of which are included in the definition of transmission media.

[0119] The methods described herein include one or more steps or actions for achieving the disclosed method. The steps and / or actions of the methods may be interchangeable with one another without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for the proper operation of the described method, the order and / or use (or lack thereof) of specific steps and / or actions may be modified without departing from the scope of the present disclosure.

[0120] The term "determining" encompasses various actions, and thus, the term "determining" may include calculating, operating, processing, deriving, investigating, looking up (e.g., looking up in a table, database, or another data structure), ascertaining, and the like. The term "determining" may also include receiving (e.g., receiving information), accessing (e.g., accessing data in a memory), and the like. The term "determining" may also include resolving, selecting, choosing, establishing, and the like.

[0121] The phrase "based on" does not mean "based only on," unless expressly specified otherwise. In other words, the phrase "based on" refers to both "based only on" and "based at least on."

[0122] In the above description, specific details are given to provide a thorough understanding of the embodiments. However, it will be understood by those skilled in the art that the embodiments may be practiced without these specific details. For example, electrical components / devices may be shown in block diagrams so as not to obscure the embodiments with unnecessary detail. In other instances, such components, other structures, and techniques may be shown in detail to further explain the examples.

[0123] Headings are included herein for reference and to aid in locating the various sections. These headings are not intended to limit the scope of the concepts described therein. Such concepts may have applicability throughout the entire specification.

[0124] It is also noted that the embodiments may be described as a process. The process may be depicted as a flowchart, flow diagram, finite state diagram, structure diagram, or block diagram. Although a flowchart may describe operations as a sequential (continuous) process, many of the operations may be performed in parallel or simultaneously, and the process may be repeated. In addition, the order of operations may be rearranged. A process is terminated when an operation is completed. A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, etc. When a process corresponds to a software function, its termination corresponds to a return of the function to the requested function or the main function.

[0125] The previous description of the disclosed implementations is provided to enable any person skilled in the art to make or use the embodiments of the present disclosure. Various modifications to these implementations (embodiments) will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other implementations (embodiments) without departing from the spirit or scope of the present disclosure. Thus, the present disclosure is not intended to be limited to the implementations (embodiments) shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. 1. A method for determining the presence of an analyte of interest in a blood sample in a test device including a sensor in an aqueous medium, comprising: transmitting light to the test device at a first excitation wavelength at which the optical absorption of the sensor remains substantially constant even as the pH of the aqueous medium changes; measuring an intensity of a first fluorescent signal emitted from the sensor in the test device in response to the first excitation wavelength; transmitting light to the test device at a second excitation wavelength different from the first excitation wavelength; measuring an intensity of a second fluorescent signal emitted from the sensor in the test device in response to the second excitation wavelength; normalizing the intensity of the second fluorescent signal emitted from the test device in response to the second excitation wavelength with the first fluorescent signal; A method comprising:

2. Normalizing the intensity of the second fluorescent signal includes generating a ratio comparing the second fluorescent signal to the first fluorescent signal.

2. The method of claim 1 .

3. comparing said ratio to a threshold value.

3. The method of claim 2 further comprising:

4. determining the presence of said analyte of interest when said ratio exceeds said threshold value.

4. The method of claim 3 further comprising:

5. The first excitation wavelength is at an isosbestic point of a component of the sensor.

2. The method of claim 1 .

6. The sensor includes a pH indicator and a phosphor; The isosbestic point is the isosbestic point of the pH indicator.

6. The method of claim 5 .

7. The first excitation wavelength is in the blue / cyan range.

2. The method of claim 1 .

8. The first excitation wavelength is between 485 nm and 495 nm.

2. The method of claim 1 .

9. The first excitation wavelength is about 490 nm.

2. The method of claim 1 .

10. The second excitation wavelength is in the green range.

2. The method of claim 1 .

11. The second excitation wavelength is between 550 nm and 560 nm.

2. The method of claim 1 .

12. The second excitation wavelength is about 555 nm.

2. The method of claim 1 .

13. measuring a rate of change over time of a fluorescent output configured to change in response to growth of the analyte in the test device; determining the presence of the analyte based on the normalized intensity of the second fluorescent signal and the measured rate of change over time of the fluorescent output configured to change in response to growth of the analyte in the test device; 2. The method of claim 1 further comprising:

14. Determining the presence of the analyte based on the normalized intensity of the second fluorescent signal and the measured rate of change over time of the fluorescent output configured to change in response to the growth of the analyte in the test device includes performing a summation of the normalized intensity of the second fluorescent signal and the measured rate of change over time of the fluorescent output configured to change in response to the growth of the analyte in the test device.

14. The method of claim 13.

15. 1. A system for determining the presence of an analyte of interest in a blood sample, comprising: a blood culture test device including a sensor in an aqueous medium and configured to receive a blood sample; A first light source; a first excitation filter configured to filter light from the first light source to provide light to the sensor at a first excitation wavelength at which optical absorption of the sensor remains substantially constant even as the pH of the aqueous medium changes; A second light source; a second excitation filter configured to filter light from the second light source to provide light to the sensor at a second excitation wavelength different from the first excitation wavelength; one or more detectors configured to measure an intensity of a first fluorescent signal emitted from the sensor in the test device in response to the first excitation wavelength and an intensity of a second fluorescent signal emitted from the sensor in the test device in response to the second excitation wavelength; a processor configured to normalize a measurement of the intensity of the second fluorescent signal emitted from the sensor in the test device in response to the second excitation wavelength using the measurement of the intensity of the first fluorescent signal emitted from the sensor in the test device in response to the first excitation wavelength; A system comprising:

16. The processor is configured to normalize the measurement of the intensity of the second fluorescent signal by generating a ratio comparing the second fluorescent signal to the first fluorescent signal.

16. The system of claim 15.

17. The processor is further configured to compare the ratio to a threshold value.

17. The system of claim 16.

18. The processor is further configured to determine the presence of the analyte of interest when the ratio exceeds the threshold.

20. The system of claim 17 .

19. The first excitation wavelength is at an isosbestic point of a component of the sensor.

16. The system of claim 15.

20. The sensor includes a pH indicator and a phosphor; The isosbestic point is the isosbestic point of the pH indicator.

20. The system of claim 19.

21. The first light source includes a blue or cyan LED light source.

16. The system of claim 15.

22. The first excitation wavelength is between 485 nm and 495 nm.

16. The system of claim 15.

23. The first excitation wavelength is about 490 nm.

16. The system of claim 15.

24. The second light source includes a green LED light source.

16. The system of claim 15.

25. The second excitation wavelength is between 550 nm and 560 nm.

16. The system of claim 15.

26. The second excitation wavelength is about 555 nm.

16. The system of claim 15.

27. the one or more detectors are configured to measure a rate of change over time of a fluorescent output configured to change in response to growth of the analyte in the test device; The processor is configured to determine the presence of the analyte based on a normalized measure of the intensity of the second fluorescent signal and the measured rate of change over time of the fluorescent output configured to change in response to growth of the analyte in the test device.

16. The system of claim 15.

28. The processor is configured to determine the presence of the analyte based on a summation of a normalized measure of the intensity of the second fluorescent signal and the measured time rate of change of the fluorescent output configured to change in response to growth of the analyte in the test device.

28. The system of claim 27.

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