System and method of plasma separation and measurement in point-of-care diagnostic cartridges

Abstract

A system and method of plasma separation and measurement in point-of-care diagnostic cartridges. The present disclosure relates to a point-of-care blood analysis system, specifically to the disposable, single-use test cartridges used in such a system. The disclosure is a plasma separation STACK and a plasma separation MODULE, and the test cartridges incorporating such STACKS and MODULES. The test cartridges are inexpensive because they are constructed from just 3 or 4 polymer layers. The test cartridges have broad application because they allow analytes in both a whole blood sample and in the plasma separated from that same whole blood sample plasma to be measured in a variety of optical assays including absorbance in standard cuvettes, absorbance in membrane cuvettes, fluorescent sensors and colorimetric sensors.

Description

SYSTEM AND METHOD OF PLASMA SEPARATION AND MEASUREMENT IN POINT-OF-CARE DIAGNOSTIC CARTRIDGESCross Reference to Related Applications

[0001] The application claims priority to and the benefit of US Provisional Patent Application Serial No. 63 / 620,297, entitled “POINT-OF-CARE BLOOD ANALYSIS SYSTEM” filed on Jan 12, 2024 and US Provisional Patent Application Serial No. 63 / 736,659, entitled “PLASMA SEPARATION AND MEASUREMENT IN POINT-OF-CARE DIAGNOSTIC CARTRIDGES” filed on Dec 20, 2024, the disclosures of which are incorporated herein by reference in their entirety.Background

[0002] The present disclosure pertains to the field of fluid analysis systems, in particular diagnostic test cartridges used in point-of-care blood analysis systems.Point-of-Care Blood Analysis

[0003] Point-of-care (POC) blood analysis systems are medical devices that perform in vitro blood analysis in close proximity to the patient (in locations such as a physician’s office, the patient’s home, or a remote ward of a hospital) rather than in a large, dedicated blood-testing laboratory relatively far from the patient. POC blood testing shortens the time to results primarily by eliminating the transportation of the patient and / or the blood sample to the lab. This allows a more rapid clinical response to test results. It also allows a uniformity of care that is independent of the location of blood-testing laboratories. Moreover, a number of analytes can change in concentration with time and transportation, resulting in measurements less reflective of the original sample.

[0004] POC blood analysis systems typically comprise a portable instrument and single-use, disposable test cartridges containing one or more sensors. Each sensor typically measures one analyte of interest in a discrete blood sample applied to the cartridge. The volume of the blood sample is typically 100 microliters or less.Whole Blood versus Plasma

[0005] Whole blood (i.e. , blood in vivo or an unaltered blood sample in vitro) consists primarily offour components: plasma, red blood cells (RBCs), white blood cells and platelets. Plasma is the liquid component and normally has a pale yellow colour.

[0006] Plasma may be obtained by removing the RBCs, white blood cells and platelets from whole blood. For our purposes, we will use the term plasma to indicate whole blood from which at least the RBCs have been removed.Whole Blood versus Plasma in Analyte Measurement

[0007] Some analytes are optimally measured in whole blood. An example is the analyte hematocrit (Het), which is the volume percentage of RBCs in whole blood. Since Het is a measure of RBCs, it follows that Het must be measured in whole blood. Another example is the analyte total hemoglobin (tHb), which is the concentration of hemoglobin in whole blood. Since hemoglobin occurs largely in RBCs, it follows that tHb must be measured in whole blood.

[0008] Some analytes are optimally measured in plasma. An example is the analyte plasma free hemoglobin (pfHb), which is the concentration of hemoglobin in plasma. By definition, pfHb must be measured in plasma.

[0009] Some analytes can be measured in either whole blood or plasma. For example, potassium (K) can be measured in plasma by lab systems such as the Siemens Atellica and is measured in whole blood by POC systems including the Abbott i-STAT and Siemens epoc.Whole Blood versus Plasma in the POC Setting

[0010] Obtaining plasma from a whole blood sample is difficult in the POC setting where the user lacks both the equipment (such as a centrifuge) and the skill required to separate plasma from whole blood. Furthermore, performing plasma separation incorrectly (eg centrifuging for too long) can cause hemolysis (breaking of RBCs).

[0011] If a POC system needs to measure an analyte in plasma, the plasma separation is typically performed not by the user but by the POC system, specifically the test cartridge. For example, the Quidel Triage POC system measures BNP (B-type natriuretic peptide) in plasma using a test cartridge that accepts a whole blood sample in its sample port. The sample port contains a filter that blocks the RBCs so that only plasma enters the interior of the test cartridge. In another example, the NOWDiagnostics COVID-19 test detects antibodies to the SARS-CoV-2 virus in plasma using a lateral flow immunoassay test cartridge that accepts whole blood in its sampleport. Again, the sample port contains a filter that blocks the RBCs so that only plasma enters the interior of the test cartridge.

[0012] In general, POC systems fall into two camps: (1 ) those with test cartridges that accept whole blood as a sample and perform all measurements on that whole blood sample, and (2) those with test cartridges that accept whole blood as a sample, immediately separate out the plasma so that only plasma enters the interior of the test cartridge, and then perform all measurements on the separated plasma.The Need to have Both Whole Blood and Plasma inside the POC Test Cartridge

[0013] Diagnostic scenarios exist that require the measurement of one or more analytes in a whole blood sample and also one or more analytes in the plasma separated from that same whole blood sample. Accordingly, there is a need in the art for a POC test cartridge capable of accepting a whole blood sample, measuring analytes (such as K, Het and tHb) in that whole blood sample, separating plasma from that same whole blood sample, and measuring analytes (such as pfHb) in the separated plasma.

[0014] Performing all these steps in the same test cartridge relieves the user from having to prepare both a whole blood sample and a plasma sample. The simplicity that results from having all steps performed inside a single test cartridge is a key factor to enabling such measurements in the home.Current Plasma Separation Techniques

[0015] POC test cartridges typically separate plasma from whole blood using a plasma separation membrane (PSM). These membranes are porous to permit the flow of plasma but with pore sizes small enough to block the passage of RBCs.

[0016] The typical implementation of plasma separation in POC test cartridges is shown in FIG. 6. FIG. 6 is a schematic of an implementation of a plasma separation in an exemplary POC test cartridge. According to FIG. 6, test cartridge 600 shows a test cartridge component 602 and PSM 604 and a direction of flow direction into the test cartridge 600. Test cartridge component 602 pulls plasma out of the PSM 604 and into the test cartridge 600. Whole blood is applied to its top surface of the PSM 604.Typical Implementation of a Plasma Separation Membrane

[0017] The first required component is the PSM, which is typically located at the test cartridge sample port. The user deposits one or more drops of whole blood directly onto the top surface of the PSM. The small pore size of the PSM blocks RBCs, and plasma collects inside the PSM. Due to the capillarity of the porous PSM, the plasma tends to stay inside the PSM.

[0018] The second required component is a means to pull the plasma out of the PSM and into the test cartridge. The second component is positioned such that it contacts the bottom surface of the PSM. The second component can be any of a number of means (alone or in conjunction), including (1 ) another membrane with greater capillarity than the PSM, (2) one or more small channels, typically in a polymer component, with greater capillarity than the PSM, and (3) a large channel without much capillarity but connected to a vacuum pump. Other means to pull the plasma out of the plasma separation membrane may also be used. The second component typically delivers the plasma to the location inside the test cartridge where analytes in the plasma are to be measured. The total volume of separated plasma needed comprises the following volumes including (1 ) the volume of plasma filling the pores of the PSM, (2) the volume of plasma filling the second component of the test cartridge, and (3) the volume of plasma filling the measurement region of the test cartridge.

[0019] During plasma separation, RBCs accumulate on the surface of the PSM. RBCs are highly deformable, which helps them pass through small blood vessels. This deformability causes the RBCs to pack together tightly on the surface of the PSM. The tight packing can lead to clogging of the PSM. This tendency to clog limits the volume of whole blood that can be successfully processed through a PSM of a given area. Typically, increasing the volume of whole blood that can be successfully processed through a PSM requires increasing the area of that PSM.Hemolysis and its Effect on Analytes such as Potassium

[0020] Hemolysis is breakage of the RBC membranes. Hemolysis can be caused by the act of drawing a blood sample, whether a venous draw using a needle or a fingerstick draw using a lancet. With a fingerstick draw, hemolysis can be exacerbated by “milking” or squeezing the finger in order to elicit blood flow.

[0021] Hemolysis causes the contents of the RBCs to spill out into the plasma, which can change the concentration of analytes in the plasma. One analyte that is particularly affected by hemolysisis K (potassium). The concentration of K inside RBCs is approximately 20X - 30X higher than the concentration of K in plasma. Hemolysed RBCs release this high concentration of K into the plasma which raises the concentration of K in the plasma. Even a small percentage of hemolysed RBCs in a blood sample can lead to spuriously high K measurements. This effect is common enough that it has a name, pseudo-hyperkalemia, meaning false high potassium.

[0022] Pseudo-hyperkalemia can be detected by measuring the degree of hemolysis in the blood sample. Since hemoglobin (Hb) is normally found only inside RBCs, the presence of free Hb in plasma (referred to as plasma free hemoglobin, or pfHb) indicates hemolysis. Plasma can be separated from the whole blood, then the plasma pfHb concentration measured via characteristic absorption by hemoglobin of light incident on the plasma. The pfHb alert levels set by typical central lab analyzers varies between 25-100 mg / dL as discussed in Lippi G, Salvagno GL, Blanckaert N, et al. Multicenter evaluation of the hemolysis index in automated clinical chemistry systems. Clin Chem Lab Med. 2009;47(8):934-939. Excessive pfHb levels may require that the test be re-run on a new blood sample.

[0023] Accordingly, there is a need in the art for POC test cartridges capable of accepting a whole blood sample, measuring analytes (such as K) in that whole blood sample, then separating plasma from that same whole blood sample, and measuring pfHb in the separated plasma. It is critical that the plasma derives from the same volume of whole blood that was used to measure the K, so that any indication of hemolysis pertains directly to the K measurement. Such a test cartridge would enable certain analyte measurements (such as K) to move out of the central laboratory without sacrificing the pfHb measurement that is crucial to clinical trustworthiness and decision-making.Hemoglobin Measurement

[0024] The measurement of Hb (or pfHb) via a characteristic absorption spectrum is complicated by the fact that Hb exists in a variety of forms, each having a different absorbance spectrum. These forms include oxyhemoglobin (where oxygen is bound to the Hb), deoxyhemoglobin (where oxygen is not bound to the Hb) and methemoglobin (which cannot bind to oxygen). This complication can be overcome by the use of one or more reagents to transform all hemoglobin forms to a single form with a single absorbance spectrum. One example method known in the art uses sodium nitrite to transform both oxyhemoglobin and deoxyhemoglobin to methemoglobin, then uses sodium azide to transform all methemoglobin to azide-methemoglobin, a form that iseasily measured optically.Whole Blood Sample Volume

[0025] Finger sticks are the simplest way to draw blood samples at home. A downside of finger sticks is that users may resort to ‘milking’ or squeezing the finger in order to produce larger quantities of blood. Such squeezing can lead to hemolysis. For analytes such as potassium (K), where hemolysis can lead to falsely elevated results, it is important to minimise the ‘milking’ or squeezing. The whole blood sample volume should be <50pl and preferably <25pl.Optical Measurement: Fluorescent Sensors

[0026] Fluorescent sensors contain one or more fluorescent probes chosen so that the excitation spectrum and / or the emission spectrum varies with the concentration of the analyte. The analyte concentration is determined by collecting the appropriate spectra and analyzing them relative to spectra from reference samples.Optical Measurement: Colorimetric Sensors

[0027] Colorimetric sensors contain one or more probes that generate colour or change colour in response to the concentration of the analyte. The analyte concentration is determined by collecting the appropriate spectra and analyzing them relative to spectra from reference samples.Optical Absorbance Measurement: Cuvettes

[0028] Cuvettes are transparent hollow chambers that can be filled with a sample fluid. Cuvettes are used to measure the absorbance of light passing through a sample fluid as follows: a light source is placed on one side of the cuvette and a spectral measurement is made on the opposite side of the cuvette, after the light has passed through the cuvette. A first spectral measurement is made before arrival of the sample fluid and a second spectral measurement is made after arrival of the sample fluid in the cuvette. The difference between the two spectra is the absorbance (or transmission loss) caused by the sample fluid.

[0029] Absorbance by the sample fluid is due to one or both of the following factors: (1 ) light is absorbed by materials in the sample fluid (either directly by the analyte itself or by chromogenic reagents added to the sample fluid), and (2) light is scattered by materials in the sample fluid (for example, red blood cells). The degree of absorbance correlates with the concentration of theanalyte.Standard Cuvette

[0030] The cuvettes used to perform absorbance measurements are typically hollow chambers made from optically clear materials, including transparent glass and transparent polymers. For our purposes, we will use the term ‘standard cuvette’ to denote such a hollow chamber.Membrane Cuvette

[0031] It is also possible to perform absorbance measurements through membranes. The membrane may be contained in an optically transparent chamber. For our purposes, we will use the term “membrane cuvette’ to denote a porous membrane that can fill with a sample fluid.

[0032] The membrane itself will cause some absorbance; consequently, the total absorbance is higher with a membrane cuvette than with a standard cuvette. The membrane must have sufficient transparency to allow the absorbance due to the sample fluid to be resolved with the required sensitivity.

[0033] There is a desire to fabricate diagnostic cartridges with both whole blood and plasma inside a simple, inexpensive POC diagnostic cartridges.SummaryPlasma Separation stack (STACK)

[0034] An object of the present invention is to provide a plasma separation stack (STACK) for use in a test cartridge for a POC blood analysis system. The STACK comprises a plasma separation membrane (PSM) stacked on top of a plasma collection membrane (PCM). The STACK accepts whole blood into the PSM, uses the PSM to separate plasma from that whole blood, and collects the separated plasma in the PCM. The PSM contains an opening, or aperture, providing an unobstructed view of the PCM; therefore, light may be passed through the PCM without any interference from the PSM. This allows for optical measurement of the separated plasma in the PCM. The PCM forms a membrane cuvette.

[0035] Both the plasma separation and the optical measurement of the separated plasma occur entirely within the STACK; in other words, the STACK is a self-contained plasma separation and plasma measurement unit. One or more STACKS may be placed inside a whole blood channel ofa test cartridge wherever plasma measurements are desired. The resulting test cartridge can admit a whole blood sample, measure analytes in that whole blood sample, and also measure analytes in plasma separated from that same whole blood sample. The STACK enables test cartridges to be constructed using either a 3-layer or a 4-layer lamination. The layers may be polymer films bonded to one another using pressure-sensitive adhesive (PSA). The polymer film materials could be any known in the art, including acrylic (PMMA), polyester (PET), polystyrene (PS) and cyclic olefin copolymer (COC).

[0036] The STACK comprises a PSM stacked on top of a PCM, as illustrated in Figures 1 A-1 B, 2A-2B and 3A-3B. The PSM may be any shape but preferably an annulus with both an outer diameter (PSM OD) and an inner diameter (PSM ID). The PCM may be any shape but preferably a disc with only an outer diameter (PCM OD). The hole in the PSM annulus is the aperture providing an unobstructed view of the PCM.

[0037] The PSM is a porous membrane with a pore size that varies across its thickness: the top portion has a pore size sufficiently large to admit whole blood (including the RBCs) both through its top surface and its outer perimeter surface, while the bottom portion has a pore size sufficiently small to block RBCs. The PSM can be a single material with pores that vary from large to small across its thickness, as illustrated in Figure 1 F. Examples include any of the asymmetric polyethersulfone (PES) membranes known in the art, including the Cytiva Vivid PES membranes. Alternatively, the PSM can be a multi-material construction where a large pore material is bonded to a small-pore material.

[0038] The PCM is a porous membrane with a capillarity sufficiently high to draw plasma out of the PSM and into the PCM. Examples include PES, chromatography paper such was Whatman CHR1 , and expanded Teflon (ePTFE) with a hydrophilic coating.

[0039] The STACK separates and collects plasma as follows:1 . Whole blood enters the PSM via the large pores in the top portion of the PSM, largely through its outer perimeter but in certain configurations also through its top surface.2. The capillarity of the PSM is high enough to prevent the whole blood in the PSM from leaving the PSM and entering the aperture in the PSM.3. Only plasma fills the small-pore bottom portion of the PSM because the small pores block RBCs.4. The capillarity of the PSM is high enough to prevent the separated plasma in the PSM from leaving the PSM and entering the aperture in the PSM.5. The capillarity of the PCM is high enough to draw the separated plasma out of the PSM and into the PCM.

[0040] The end result is a PCM filled with separated plasma. The PCM constitutes a membrane cuvette. Neither whole blood nor plasma has filled the aperture in the PSM, so that an optical measurement, such as an absorbance measurement, of the plasma-filled PCM may be made through the aperture.

[0041] Test cartridges using one or more STACKS can be constructed using either a 3-layer lamination or a 4-layer lamination, as illustrated in the example cartridges in Figures 1 C-1 D, 2C 2D and 3C-3D. The layers are laminated, or bonded, to one another. The test cartridge contains a sample port where the user may deposit a whole blood sample. One or more of the interior layers contain a cutout forming a whole blood channel that leads from the sample port to the interior of the test cartridge. One or more of the layers may contain hydrophilic surfaces to help draw the whole blood sample from the sample port into the whole blood channel. The test cartridge may contain one or more vent openings to vent air displaced by the whole blood entering the cartridge. The test cartridge may also contain one or more vent openings to vent air displaced by whole blood entering the PSMs and plasma entering the PCMs.

[0042] A test cartridge so constructed accepts a whole blood sample at the sample port and conveys that whole blood sample into a whole blood channel in the interior of the cartridge. All sensor locations in the whole blood channel without a STACK can be used to measure analytes in whole blood. All locations in the whole blood channel with a STACK can be used to measure analytes in plasma.

[0043] The use of STACKS thus enables a test cartridge with broad application, allowing analytes in both a whole blood sample and in the plasma separated from that same whole blood sample to be measured optically via fluorescent sensors, colorimetric sensors and absorbance measurements.

[0044] Such test cartridges have important real-world applications. For example, the test cartridge could accept a whole blood sample, measure potassium (K) in that whole blood sample, and measure pfHb in plasma separated from that whole blood sample. The pfHb measurementindicates the extent of hemolysis in the whole blood sample and can help determine whether, and to what degree, the potassium measurement is falsely elevated. Measuring pfHb in plasma derived from the same whole blood sample used to measure K ensures that the pfHb measurement correlates directly with the K measurement.

[0045] There is also provided an instrument for determining the concentration of analytes in a sample fluid. The instrument comprises a source of excitation light for fluorescent sensors and a means to measure the spectra of the excitation light and / or the emitted fluorescent light. The instrument also comprises a source of light for transmission through standard cuvettes and / or membrane cuvettes in the cartridge, and a means to measure the spectra of the light after it has passed through the standard cuvettes and / or membrane cuvettes. The instrument also comprises a source of light for transmission through colorimetric sensors in the cartridge, and a means to measure the spectra of the light after it has passed through the colorimetric sensors. The instrument includes a computer processing means to convert the measured light intensities to analyte concentrations using proprietary mathematical models.

[0046] There is also provided software on a mobile device that will securely accept information (measurement results) transmissions from the instrument. Furthermore, the software on the mobile device will facilitate the secure transmission of information to the patient’s electronic medical record (EMR) so that test records may be shared with the patient’s physician.Plasma Separation Module (MODULE)

[0047] An object of the present invention is to provide a plasma separation module (MODULE) for use in a test cartridge for a POC blood analysis system. The MODULE is capable of accepting a whole blood sample and then separating plasma from that whole blood sample. The MODULE enables the test cartridge to be constructed using just a 3-layer lamination.

[0048] The MODULE comprises 3 layers and a plasma separation membrane (PSM). The 3 layers are a bottom layer, a middle layer and a top layer. The 3 layers are laminated, or bonded, to one another. The middle layer contains a whole blood channel. The bottom layer forms the bottom of the whole blood channel and the top layer forms the top of the whole blood channel. The PSM is located in the whole blood channel, sandwiched between the bottom layer and the top layer. The bottom surface of the PSM is bonded to the bottom layer, and the top surface of the PSM is bonded to the top layer, leaving only the perimeter of the PSM exposed to whole blood. When the whole blood channel fills with whole blood, the whole blood can enter the PSMonly through its perimeter surface. The MODULE may contain one or more hydrophilic surfaces to help draw whole blood into the whole blood channel

[0049] The PSM prevents RBCs from substantially entering the PSM. RBCs may encroach into the PSM a certain allowable distance. RBCs are prevented from encroaching beyond that allowable distance through any means, including size exclusion by the pore size of the PSM, and / or agglutination reagents that clump RBCs together such that the large clumps no longer fit through the pores, and / or reagents that deform the shape of the RBCs such that they are more likely to be caught / snagged on the interior surfaces of the PSM. Beyond the allowable distance, only plasma enters the PSM. The top and / or bottom layer contain a means, such as one or more holes, to vent the air from the PSM as it fills with plasma. The PSM is a membrane cuvette. The top layer, the PSM and the bottom layer are substantially transparent, so that the concentration of analytes in the separated plasma may be measured via absorbance measurements. The substantial transparency also allows the use of other optical measurement means, including fluorescent sensors and colorimetric sensors.

[0050] There is also provided a test cartridge making use of the MODULE. The test cartridge is capable of accepting a whole blood sample, drawing the whole blood sample into the test cartridge by means of one or more hydrophilic surface(s), and separating plasma from the whole blood sample using the MODULE. The test cartridge can measure analytes in the whole blood (including K, tHb and Het) and in the separated plasma (including pfHb, a measure of hemolysis) in the separated plasma. Measuring all analytes on the same blood sample ensures that, for example, the hemolysis measurement correlates directly with the K measurement.

[0051] There is also provided an instrument for determining the concentration of analytes in a sample fluid. The instrument comprises a source of excitation light for the fluorescent sensors and a means to measure the intensity of the excitation light and / or the emitted fluorescent light across multiple wavelengths. The instrument also comprises a source of light for transmission through the whole blood and / or the separated plasma, and a means to measure the intensity of the transmitted light across multiple wavelengths after it has passed through the whole blood and / or the separated plasma. The various light sources and the means to measure light intensity may also be used to measure colorimetric sensors. The instrument includes a computer processing means to convert the measured light intensities to analyte concentrations using proprietary mathematical models.

[0052] There is also provided software on a mobile device that will securely accept information (measurement results) transmissions from the instrument. Furthermore, the software on the mobile device will facilitate the secure transmission of information to the patient’s electronic medical record (EMR) so that test records may be shared with the patient’s physician.Brief Description of the Drawings

[0053] These and other features of the disclosure will become more apparent in the following detailed description in which reference is made to the appended drawings.

[0054] FIG. 1 A illustrates an exploded view of the STACK.

[0055] FIG. 1 B illustrates a cross-sectional view of the assembled STACK.

[0056] FIG. 1C illustrates an assembled view of the STACK in an example of a 3-layer cartridge.

[0057] FIG. 1 D illustrates an exploded view of the STACK in an example of a 3-layer cartridge.

[0058] FIG. 1 E illustrates a cross-sectional view of the STACK in an example of a 3-layer cartridge.

[0059] FIG. 1 F illustrates an alternate cross-sectional view of the STACK in an example of a 3- layer cartridge, highlighting the asymmetric nature of the pore sizes in the PSM.

[0060] FIG. 2A illustrates an exploded view of the STACK of a second embodiment.

[0061] FIG. 2B illustrates a cross-sectional view of the assembled STACK of a second embodiment.

[0062] FIG. 2C illustrates an assembled view of the STACK in an example of a 3-layer cartridge of a second embodiment.

[0063] FIG. 2D illustrates an exploded view of the STACK in an example of a 3-layer cartridge of a second embodiment.

[0064] FIG. 2E illustrates a cross-sectional view of the STACK in an example of a 3-layer cartridge of a second embodiment.

[0065] FIG. 3A illustrates an exploded view of the STACK of a of a third embodiment.

[0066] FIG. 3B illustrates a cross-sectional view of the assembled STACK of a of a third embodiment.

[0067] FIG. 3C illustrates an assembled view of the STACK in an example of a 4-layer cartridge of a of a third embodiment.

[0068] FIG. 3D illustrates an exploded view of the STACK in an example of a 4-layer cartridge of a of a third embodiment.

[0069] FIG. 3E illustrates a cross-sectional view of the STACK in an example of a 4-layer cartridge of a of a third embodiment.

[0070] FIG. 4A illustrates successful plasma separation using a STACK and test cartridge constructed according to a first embodiment.

[0071] FIG. 4B illustrates successful plasma separation using a STACK and test cartridge constructed according to a second embodiment.

[0072] FIG. 4C illustrates successful plasma separation using a STACK and test cartridge constructed according to a third embodiment.

[0073] FIG. 5A illustrates successful plasma separation using a STACK and test cartridge constructed according to a third embodiment.

[0074] FIG. 5B illustrates the measured absorbance data vs hemolysis.

[0075] Figure 5C illustrates the absorbance as a function of pfHb.

[0076] FIG. 6 is a schematic of an implementation of a plasma separation in an exemplary POC test cartridge.

[0077] FIG. 7A illustrates an embodiment of the plasma separation module with a circular plasma separation membrane and multiple small air vent holes for the plasma separation membrane.

[0078] FIG. 7B illustrates an alternate embodiment of the plasma separation module with a circular plasma separation membrane and a single large air vent hole for the plasma separation membrane.

[0079] FIG. 7C illustrates an alternate embodiment of the plasma separation module with an oblong plasma separation membrane and multiple small air vent holes for the plasma separation membrane.

[0080] FIG. 7D illustrates an alternate embodiment of the plasma separation module with an oblong plasma separation membrane and a single large air vent hole for the plasma separation membrane.

[0081] FIG. 7E illustrates the plasma separation module with the top layer lifted to better show the individual components.

[0082] FIG. 7F illustrates a cross-sectional view of the plasma separation module.

[0083] FIG. 7G illustrates whole blood entry through the plasma separation membrane perimeter surface.

[0084] FIG. 7H illustrates a top view of the plasma separation membrane in the whole blood channel.

[0085] FIG. 8A illustrates the extent of RBC encroachment on membranes treated with ANTI-AB antibodies.

[0086] FIG. 8B illustrates the extent of RBC encroachment on membranes not treated with ANTI- AB antibodies.

[0087] FIG. 9A illustrates the measured absorbance data from membrane cuvettes filled with plasma samples of varying hemolysis levels.

[0088] FIG. 9B illustrates the same data as Fig. 9A but having the hemolysis levels have been converted to pfHb concentrations via theoretical calculations.

[0089] FIG. 9C illustrates measured fluorescence intensity data from the fluorescent K sensor.

[0090] FIG. 9D illustrates measured absorbance data from standard cuvettes filled with whole blood samples of varying Het levels.

[0091] FIG. 9E illustrates the same data as Fig. 9D but with the Het values converted to tHb concentrations via theoretical calculations.

[0092] FIG. 10A illustrates an exemplary assembled test cartridge.

[0093] FIG. 10B illustrates the test cartridge with the top layer lifted to better show the individual components.

[0094] FIG. 10C illustrates an exploded view of the test cartridge to show its construction.Detailed DescriptionPlasma Separation Stack (STACK) and Plasma Separation Module (MODULE)

[0095] The present invention relates to a POC blood analysis system, specifically to a plasma separation stack (STACK) and a plasma separation module (MODULE), and the test cartridges enabled by the STACK and the MODULE. The simple construction of the STACK and the MODULE enables the simple construction of the test cartridge. Despite its simplicity, the test cartridge accepts a whole blood sample and separates plasma from that same whole blood sample. The test cartridge has broad application because it allows analytes in both a whole blood sample and in the plasma separated from that same whole blood sample to be measured optically via fluorescent sensors, colorimetric sensors and absorbance measurements.STACK Design

[0096] The STACK comprises a plasma separation membrane (PSM) stacked on top of a plasma collection membrane (PCM). The STACK accepts whole blood into the PSM, uses the PSM to separate plasma from that whole blood, and collects the separated plasma in the PCM.

[0097] The PCM is a membrane cuvette. The PSM contains an aperture providing an unobstructed view of the PCM so that light may be passed through the PCM without any interference from the PSM. This allows for optical measurement of the separated plasma in the PCM.

[0098] FIGURES 1A-1 F illustrate a first embodiment of the STACK, where the PSM outer diameter (PSM OD) is the same size as the PCM outer diameter (PCM OD) and where the test cartridge may be constructed using 3 layers.

[0099] FIGURES 2A-2E illustrate a second embodiment of the STACK, where the PSM OD is larger than the PCM OD, and where the test cartridge may be constructed using 3 layers.

[0100] FIGURES 3A-3E illustrate a third embodiment of the STACK, where the PSM OD is larger than the PCM OD, and where the test cartridge may be constructed using 4 layers.

[0101] Figures 1A-1 B, 2A-2B and 3A-3B illustrate various possible geometries of a STACK. The PSM may be any shape but preferably an annulus with both an outer diameter (PSM OD) and an inner diameter (PSM ID). The PCM may be any shape but preferably a disc with only an outer diameter (PCM OD).

[0102] According to the disclosure, FIG. 1A, 2A and 3A illustrates an exploded view of embodiments of the STACK. According to FIG. 1A, 2A and 3A, STACK 100, 200 and 300 comprises a top PSM 102, 202, 302 and a bottom PCM 110, 204 and 304. PSM 102 comprises an aperture 104 having PSM Inner Diameter (PSM ID) 106 and PSM Outer Diameter (PSM OD) 108. Plasma collection membrane 1 10 further comprises PCM OD 112.

[0103] According to the disclosure, FIG. 1 B, 2B and 3B illustrates a cross-sectional view of the embodiments of the STACK 120, 210, 310 with a circular PCM 124, 214 and 314 and an annulus PSM 122, 212 and 312.

[0104] The PSM (122, 212 and 312) is a porous membrane with a pore size that varies across its thickness: the top portion has a pore size sufficiently large to admit whole blood (including the RBCs), while the bottom portion has a pore size sufficiently small to block RBCs. The PSM can be a single material with pores that vary from large to small across its thickness, as illustrated in Figure 1 F. Examples include any of the asymmetric polyethersulfone (PES) membranes known in the art, including the Cytiva Vivid PES membranes. Alternatively, the PSM can be a multi-material construction where a large pore material is bonded to a small-pore material.

[0105] The PCM is a porous membrane with a capillarity sufficiently high to draw plasma out of the PSM and into the PCM. Examples include PES, chromatography paper such was Whatman CHR1 , and expanded Teflon (ePTFE) with a hydrophilic coating.

[0106] FIG. 1 C illustrates an assembled view of an example of a 3-layer cartridge. According to FIG. 1 C, cartridge 130 comprises air vent for whole blood channel 132, air vents for STACK 134, STACK for plasma separation and measurement 136, location for whole blood measurement 138 and 140, whole blood channel 142 and sample port 144.

[0107] FIG. 1 D illustrates an exploded view of an example of a 3-layer cartridge. According to FIG. 1 D, cartridge 150 comprises top layer 152 having air vents for STACK 152, middle layer 164, PSM 156, PCM) 158 and bottom layer 160. Bottom layer 160 further comprises air vent for whole blood channel 162.

[0108] FIG. 1 E illustrates a cross-sectional view of an example of a 3-layer cartridge. According to FIG. 1 E, the 3 layers of cartridge 170 are a bottom layer 172, a middle layer 174 and a top layer 176. The 3 layers are laminated, or bonded, to one another. The middle layer 174 contains a whole blood channel, PCM 178 and PSM 180.

[0109] According to FIG. 1 E, the bottom layer forms the bottom of the whole blood channel and the top layer forms the top of the whole blood channel. The bottom surface of PCM 182 may be bonded to the bottom layer 172 The bottom surface of PSM 184 may be bonded to the top surface of PCM 178. The top surface of PSM 186 may be bonded to the top layer 176.

[0110] FIG. 1 F illustrates an alternate cross-sectional view of an example of a 3-layer cartridge, highlighting the asymmetric nature of the pore sizes in the PSM. According to FIG. 1 F, cartridge 170 is constructed so that the bottom layer (small pore) surface of the PSM 180 contacts the top surface of the PCM 178. PSM 180 contains an aperture 186 which is filled with air 188.[0011 1] According to FIG. 1 F, the middle layer 174 and PCM 178 may be bonded to the bottom layer 172. The PSM 180 may be bonded to the top layer 176. Whole blood 184 can only contact and enter the PSM 180 through its perimeter surface. One or both of bottom layer 172 and top layer 176 may contain hydrophilic surfaces to help draw whole blood 184 into the whole blood channel.

[0112] The PSM 180 separates plasma from whole blood 184 and the PCM 178 collects the separated plasma as follows:1 . Whole blood 184 enters the PSM 180 via the large pores in the top portion of the PSM 180. When the STACK is installed in a cartridge, the top surface of the PSM 180 is either completely or substantially covered by the top layer of the cartridge, so that whole blood 184 must enter the PSM 180 either completely or substantially through its outer perimeter. Any processing of the PSM 180 in manufacturing (such as punching or laser cutting, for example) must not close the large pores on that outer perimeter.2. The capillarity of the PSM 180 is high enough to prevent the whole blood in the PSM 180 fromleaving the PSM 180 and entering the aperture 186 in the PSM 180.3. Only plasma fills the small pores in the bottom portion of the PSM 180 because the small pores block red blood cells (RBCs).4. The capillarity of the PSM 180 is high enough to prevent the separated plasma in the PSM 180 from leaving the PSM 180 and entering the aperture 186 in the PSM 180.5. The capillarity of the PCM 178 is high enough to draw the separated plasma out of the PSM 180 and into the PCM 178.

[0113] The PCM 178 constitutes a membrane cuvette. The layers of the test cartridge above and below the STACK are substantially transparent. The plasma filled PCM 178 is also substantially transparent. The aperture 186 in the PSM 180 provides an unobstructed view of the plasma-filled PCM 178. Neither whole blood nor plasma has filled the aperture 186 in the PSM 180, so that an optical measurement, such as an absorbance measurement, of the plasma-filled PCM 178 may be made through the aperture.

[0114] Both the plasma separation and the optical measurement of the separated plasma occur entirely within the STACK; in other words, the STACK is a self-contained plasma separation and measurement unit. One or more STACKS may be placed inside a whole blood channel of a test cartridge wherever plasma measurements are desired. The resulting test cartridge can admit a whole blood sample, measure analytes in that whole blood sample, and also measure analytes in the plasma separated from that same whole blood sample.

[0115] One diagnostic scenario which requires this capability is the measurement of K in a whole blood sample. Even a small degree of hemolysis in the whole blood sample can falsely elevate the K measurement; therefore, the extent of hemolysis must be ascertained using the measurement of pfHb in plasma derived from the same whole blood sample used to measure K. Adding this capability to a point-of-care test cartridge enables the K measurement to move out of the central laboratory without sacrificing the pfHb measurement that is crucial to clinical trustworthiness and decision-making.

[0116] Analytes in the separated plasma may be measured without the addition of reagents or they may be measured with the addition of reagents. For example, the measurement of pfHb may be facilitated by the addition of reagents that change the various forms of Hb to a single form. Examples of such reagents include sodium nitrite and sodium azide. Such reagentscould be incorporated into the PSM and / or the PCM, resulting in a STACK that not only separates plasma but also adds reagents to the whole blood and / or the separated plasma.

[0117] The STACK enables test cartridges to be constructed using either a 3-layer or a 4- layer lamination. The layers may be polymer films bonded to one another using PSA, though other bonding techniques known in the art, such as laser welding, are also possible. The polymer film materials could be any known in the art, including PMMA, PET, PS and COC.First Embodiment of the STACK

[0118] In a first embodiment, the STACK is constructed so that the PSM OD is the same as the PCM OD, as illustrated in Figures 1 A-1 B. This embodiment allows the test cartridge to be constructed using a 3-layer lamination consisting of a bottom layer, a middle layer and a top layer, as illustrated in Figures 1 C-1 D. The middle layer contains a cutout forming the whole blood channel. The STACK is placed inside the whole blood channel and is thus sandwiched between the bottom layer and the top layer. In this embodiment, the following factors need to be considered for successful operation of the STACK, as illustrated in Figures 1 E-1 F:1 . Since the top layer contacts the top surface of the PSM 180, whole blood can only enter the PSM 180 through its outer perimeter, not through its top surface. The upper portion of the PSM 180 must have pores sufficiently large to admit whole blood (including the RBCs). Conversely, the bottom portion of the PSM 180 must have pores sufficiently small to blocks RBCs, so that only plasma can move from the bottom surface of the PSM 180 and into the PCM 178.2. The large-pore side of the PSM 180 contacts the top layer 176. The PSM 180 may be bonded to the top layer 176 but such bonding is not necessary. Bonding may aid manufacturing. Bonding may be accomplished using any means known in the art, including PSA, heat staking and ultrasonic welding.3. The small-pore side of the PSM 180 contacts the PCM 178. Direct contact between the PSM 180 and PCM 178 is necessary to draw the separated plasma from the PSM 180 into the PCM 178. Contact may be achieved by a press fit between the PSM 180 and the PCM 178. A press fit is achieved by compressing the STACK between the bottom layer and top layer of the test cartridge; in other words, by making the distance between the bottom layer and top layer less than the height of the STACK; this is accomplished by making the middle layer thinner than the STACK. A press fit may also aid in preventing RBCs from slipping between the PSM 180 and thePCM 178. Another means to achieve contact is to bond the PSM 180 to the PCM 178 using any means known in the art, including heat staking and ultrasonic welding. One may use only a press fit, or only bonding or a press fit combined with bonding.4. The PCM 178 contacts the bottom layer. The PCM 178 may be bonded to the bottom layer. Bonding may aid manufacturing. Bonding may also help prevent RBCs from slipping between the PCM-to-bottom-layer interface. Bonding may be accomplished using any means known in the art, including PSA, heat staking and ultrasonic welding.5. The perimeter surface of the PCM 178 is in direct contact with the whole blood sample. Since only plasma should be collected in the PCM 178, the PCM should block RBCs from entering the PCM 178. Such blocking may be accomplished by using a membrane with a pore size small enough to block RBCs. Membranes with larger pores may be used if the pores on the perimeter surface are substantially closed by processing techniques known in the art, including laser cutting, punching, heat staking and ultrasonic welding.

[0119] One or more of the 3 layers in the test cartridge may contain a means, such as one or more openings, to vent to atmosphere any air displaced from the STACK as it fills with whole blood and plasma. Alternatively, the STACK may not be vented to atmosphere, so that any air displaced from the STACK enters the whole blood channel.

[0120] FIG. 4A illustrates successful plasma separation in a 3-layer test cartridge 400 constructed as per the first embodiment. According to FIG. 4A, a successful plasma separation using a STACK and test cartridge 400 constructed according to a first embodiment is shown, where the PSM OD is the same size as the PCM OD, and the test cartridge is constructed using a 3-layer lamination. The PSM material is Cytiva Vivid GR polyethersulfone (PES) and the PCM material is expanded Teflon (ePTFE) with a hydrophilic coating. The bottom layer has a hydrophilic pressure-sensitive adhesive (PSA) on its top surface. The middle layer has no PSA. The top layer has a hydrophilic PSA on its bottom surface. The hydrophilic PSA on the bottom layer and the top layer help draw whole blood into the cartridge. This construction is similar to that illustrated in Figures 1A-1 F. This prototype successfully separated plasma from a whole blood sample (42% Het) in 10 seconds.Second Embodiment of the STACK

[0121] In a second embodiment, the STACK is constructed so that the PSM OD is larger than the PCM OD, causing the PSM OD to overhang the PCM OD, as illustrated in Figures 2A- 2B. This geometry embodiment allows the test cartridge to be constructed using a 3-layer lamination consisting of a bottom layer, a middle layer and a top layer, as illustrated in Figures 2C-2D of a second embodiment.

[0122] FIG. 2C illustrates an assembled view of an example of a 3-layer cartridge of the second embodiment. According to FIG. 2C, cartridge 230 comprises air vent for whole blood channel 232, air vents for STACK 234, STACK for plasma separation and measurement 236, location for whole blood measurement 238 and 240, whole blood channel 242 and sample port 244.

[0123] FIG. 2D illustrates an exploded view of an example of a 3-layer cartridge of the second embodiment. According to FIG. 2D, cartridge 250 comprises top layer 252 having air vents for STACK 254, middle layer 264, PSM 256, PCM 258 and bottom layer 260. Bottom layer 260 further comprises air vent for whole blood channel 262.

[0124] The PSM 256 overhang is deformed downward so that both the PSM 256 overhang and the PCM 258 contact the bottom layer 260. In the second embodiment, the PCM 258 is completely covered by the PSM 256 and is never in direct contact with the whole blood. The middle layer 264 contains a cutout forming the whole blood channel. The STACK is placed inside the whole blood channel and is thus sandwiched between the bottom layer and the top layer.

[0125] FIG. 2E illustrates a cross-sectional view of an example of a 3-layer cartridge of the second embodiment. According to FIG. 2E, the 3 layers of cartridge 270 are a bottom layer 272, a middle layer 274 and a top layer 276. The 3 layers are laminated, or bonded, to one another. The middle layer 274 contains a whole blood channel, PCM 278 and PSM 280.

[0126] According to FIG. 2E, the bottom layer forms the bottom of the whole blood channel and the top layer forms the top of the whole blood channel. The bottom surface of PCM 282 may be bonded to the bottom layer 272. The bottom surface of PSM 284 may be bonded to the bottom layer 272. The bottom surface of PSM 288 may be bonded to the top surface of PCM 278. The top surface of PSM 286 may be bonded to the top layer 276.

[0127] According to the second embodiment, the following factors need to be considered for successful operation of the STACK, as illustrated in Figure 2E:1. Since the top layer 276 contacts a portion of the top surface of the PSM, whole blood can enter the PSM 280 through its outer perimeter and through a portion of its top surface. The upper portion of the PSM 280 must have pores sufficiently large to admit whole blood (including the RBCs). Conversely, the bottom portion of the PSM 280 must have pores sufficiently small to blocks RBCs, so that only plasma can move from the bottom surface of the PSM 280 and into the PCM 278.2. The large-pore side of the PSM 280 contacts the top layer 276. The PSM 280 may be bonded to the top layer 276 but such bonding is not necessary. Bonding may aid manufacturing. Bonding may be accomplished using any means known in the art, including PSA, heat staking and ultrasonic welding.3. The small-pore side of the PSM 280 contacts the PCM 278. Direct contact between the PSM 280 and PCM 278 is necessary to draw the separated plasma from the PSM 280 into the PCM 278. Contact may be achieved by a press fit between the PSM 280 and the PCM 278. A press fit is achieved by compressing the STACK between the bottom layer 272 and top layer 276 of the test cartridge; in other words, by making the distance between the bottom layer 272 and top layer 276 less than the height of the STACK; this is accomplished by making the middle layer 274 thinner than the STACK. Another means to achieve contact is to bond the PSM 280 to the PCM 278 using any means known in the art, including heat staking and ultrasonic welding. One may use only a press fit, or only bonding or a press fit combined with bonding.4. The PSM 280 overhang is deformed downward until it contacts the bottom layer 272. The PSM 280 overhang may be bonded to the bottom layer 272 to help prevent RBCs from slipping between the PSM 280 and the bottom layer 272. Bonding may be accomplished using any means known in the art, including PSA, heat staking and ultrasonic welding.5. The PCM 278 contacts the bottom layer 272. The PCM 278 may be bonded to the bottom layer 272. Bonding may aid manufacturing. Bonding may be accomplished using any means known in the art, including PSA, heat staking and ultrasonic welding.6. The perimeter surface of the PCM 278 is not in direct contact with the whole blood sample. This means the PCM 278 does not need to block RBCs and the PCM 278 pore size may be larger than RBCs.

[0128] One or more of the 3 layers in the test cartridge may contain a means, such as one or more openings, to vent to atmosphere any air displaced from the STACK as it fills with whole blood and plasma. Alternatively, the STACK may not be vented to atmosphere, so that any air displaced from the STACK as it fills with whole blood and plasma enters the whole blood channel.

[0129] FIG. 4B illustrates successful plasma separation in a 3-layer test cartridge 410 constructed as per the second embodiment. FIG. 4B illustrates successful plasma separation using a STACK and test cartridge 410 constructed according to a second embodiment, where the PSM OD is larger than the PCM OD, and the test cartridge is constructed using a 3-layer lamination. The PSM material is Cytiva Vivid GR polyethersulfone (PES) and the PCM material is Whatman CHR1 chromatography paper. The bottom layer has a hydrophilic PSA on its top surface. The middle layer has a plain PSA on its top surface. The top layer is a polyester (PET) film with a hydrophilic coating on its bottom surface and has no PSA. The hydrophilic PSA on the bottom layer and the hydrophilic coating on the top layer help draw whole blood into the cartridge. This construction is similar to that illustrated in Figures 2A-2E. This prototype successfully separated plasma from a whole blood sample (42% Het) in 28 seconds.Third Embodiment of the STACK

[0130] In a third embodiment, the STACK is constructed so that the PSM OD is larger than the PCM OD, causing the PSM OD to overhang the PCM OD, as illustrated in Figures 3A- 3B. This embodiment allows the test cartridge to be constructed using a 4-layer lamination consisting of a bottom layer, a plasma layer, a whole blood layer and a top layer, as illustrated in Figures 3C-3D.

[0131] FIG. 3C illustrates an assembled view of an example of a 4-layer cartridge of the third embodiment. According to FIG. 3C, cartridge 330 comprises air vent for whole blood channel 332, air vents for STACK 334, STACK for plasma separation and measurement 336, location for whole blood measurement 338 and 340, whole blood channel 342 and sample port 344.

[0132] FIG. 3D illustrates an exploded view of an example of a 4-layer cartridge of the third embodiment. According to FIG. 3D, cartridge 350 comprises top layer 352 having air vents for STACK 354, whole blood layer 356, PSM 358, plasma layer 360, an air vent for whole blood channel 362 on the plasma layer 360, PCM 364 and bottom layer 366. Bottom layer 366 further comprises air vent for whole blood channel 368.

[0133] FIG. 3E illustrates a cross-sectional view of an example of a 4-layer cartridge of the third embodiment. According to FIG. 3E, the 4 layers of cartridge 370 are a bottom layer 372, a plasma layer 374, a whole blood layer 376 and a top layer 378. The 4 layers are laminated, or bonded, to one another. The whole blood layer 376 contains a whole blood channel and a PSM 382. The plasma layer 374 contains a PCM 380.

[0134] According to FIG. 3E, the plasma layer 374 forms the bottom of the whole blood channel and the top layer 378 forms the top of the whole blood channel. The bottom surface of PCM 384 may be bonded to the bottom layer 372. The bottom surface of PSM 386 may be bonded to plasma layer 374. The top surface of PSM 388 may be bonded to the top layer 378. The bottom surface of the PSM 390 may be bonded to the top surface of PCM 380.

[0135] The plasma layer 374 is approximately the same thickness as the PCM 380 and contains a cutout into which the PCM 380 is placed. The whole blood layer 376 is approximately the same thickness as the PSM 382 and contains a cutout forming the whole blood channel. The PSM 382 is placed in the whole blood channel directly over the PCM 380. The STACK is thus sandwiched between the bottom layer 372 and the top layer 378.

[0136] According to the third embodiment, the following factors need to be considered for successful operation of the STACK, as illustrated in Figure 3E:1 . Since the top layer 378 contacts the top surface of the PSM 382, whole blood can only enter the PSM 382 through its outer perimeter, not through its top surface. The upper portion of the PSM 382 must have pores sufficiently large to admit whole blood (including the RBCs). Conversely, the bottom portion of the PSM 382 must have pores sufficiently small to blocks RBCs, so that only plasma can move from the bottom surface of the PSM 382 and into the PCM 380.2. The large-pore side of the PSM 382 contacts the top layer 378. The PSM 382 may be bonded to the top layer 378 but such bonding is not necessary. Bonding may aid manufacturing. Bonding may be accomplished using any means known in the art, including PSA, heat staking and ultrasonic welding.3. The small-pore side of the PSM 382 contacts the PCM 380. Direct contact between the PSM 382 and PCM 380 is necessary to draw the separated plasma from the PSM 382 into the PCM 380. Contact may be achieved by a press fit between the PSM 382 and the PCM 380. A press fit is achieved by compressing the STACK between the bottom layer and top layer of the testcartridge; in other words, by making the distance between the bottom layer and top layer less than the height of the STACK; this is accomplished by making the combined thickness of the plasma layer and the whole blood layer thinner than the STACK. A press fit may also aid in preventing RBCs from slipping between the PSM 382 and the PCM 380. Another means to achieve contact is to bond the PSM 382 to the PCM 380 using any means known in the art, including heat staking and ultrasonic welding. One may use only a press fit, or only bonding or a press fit combined with bonding.4. The PSM 382 overhang contacts the plasma layer 374. The PSM 382 overhang may be bonded to the plasma layer 374 to help prevent RBCs from slipping between the PSM 382 and the plasma layer 374. Bonding may be accomplished using any means known in the art, including PSA, heat staking and ultrasonic welding.5. The PCM 380 contacts the bottom layer 372. The PCM 380 may be bonded to the bottom layer 372. Bonding may aid manufacturing. Bonding may be accomplished using any means known in the art, including PSA, heat staking and ultrasonic welding.6. The perimeter surface of the PCM 380 is not in direct contact with the whole blood sample. This means the PCM 380 does not need to block RBCs and the PCM 380 pore size may be larger than RBCs.

[0137] One or more of the 4 layers in the test cartridge may contain a means, such as one or more openings, to vent to atmosphere any air displaced from the STACK as it fills with whole blood and plasma. Alternatively, the STACK may not be vented to atmosphere, so that any air displaced from the STACK as it fills with whole blood and plasma enters the whole blood channel.

[0138] Figure 4C illustrates successful plasma separation in a 4-layer test cartridge 420 constructed as per the third embodiment where the PSM OD is larger than the PCM OD, and the test cartridge 420 is constructed using a 4-layer lamination. The PSM material is Cytiva Vivid GR PES and the PCM material is Whatman CHR1 chromatography paper. The bottom layer has a plain PSA on its top surface. The plasma layer has a hydrophilic PSA on its top surface. The whole blood layer has a plain PSA on its top surface. The top layer is a polyester (PET) film with a hydrophilic coating on its bottom surface and has no PSA. The hydrophilic PSA on the plasma layer and the hydrophilic coating on the top layer help draw whole blood into the cartridge. This construction is similar to that illustrated in Figures 3A-3E. This prototype successfully separated plasma from a whole blood sample (42% Het) in 17 seconds.STACK PCM is a Membrane Cuvette

[0139] The PCM in the STACK is a membrane cuvette. The layers of the test cartridge above and below the STACK are substantially transparent. The plasma-filled PCM is also substantially transparent. The aperture in the PSM provides an unobstructed view of the plasma- filled PCM. Neither whole blood nor plasma has filled the aperture in the PSM, so that an optical measurement, such as an absorbance measurement, of the plasma-filled PCM may be made through the aperture.

[0140] Figures 5A-5C illustrate experimental results of plasma separation and subsequent pfHb measurement using the plasma-filled PCM as a membrane cuvette. Blood samples with various levels of hemolysis were created by adding lysed RBCs to 42% Het whole blood. The light source was a broadband LED (white light) located above the membrane cuvette. The intensity of the transmitted spectra was measured below the membrane cuvette before and after arrival of the separated plasma in the PCM. The difference between the spectra gives the absorbance.

[0141] Figure 5A illustrates successful plasma separation in a 4-layer test cartridge constructed as per the third embodiment. FIG. 5A illustrates successful plasma separation using a STACK and test cartridge 500 constructed according to a third embodiment, where the PSM OD is larger than the PCM OD, and the test cartridge is constructed using a 4-layer lamination. The PSM material is Cytiva Vivid GR PES and the PCM material is Cytiva Vivid GF PES. The bottom layer has a plain PSA on its top surface. The plasma layer has a hydrophilic PSA on its top surface. The whole blood layer has no PSA. The top layer has a hydrophilic PSA on its bottom surface. The hydrophilic PSA on the plasma layer and the top layer help draw whole blood into the cartridge. This construction is similar to that illustrated in Figures 3A-3E.

[0142] FIG. 5B illustrates the measured absorbance data vs hemolysis. According to FIG. 5B, chart 510 is shown illustrating the measured absorbance data vs hemolysis (% of red blood cells). Whole blood samples with varying levels of hemolysis were prepared by adding lysed RBCs to whole blood.

[0143] Figure 5C illustrates the absorbance as a function of pfHb. According to FIG. 5C, chart 520 illustrates the same data as Fig. 5B but the hemolysis levels have been converted to pfHb concentrations via theoretical calculations. The pfHb results illustrate the ability to discriminate between the 25-100 mg / dL alert levels set by typical central lab analyzers.Alternate Uses of the STACK

[0144] Use of a STACK for purposes other than a membrane cuvette are possible:1 . The STACK may be used to provide plasma to a fluorescent sensor. Fluorescent sensor material is printed and dried to a thin film on the bottom layer, at the location of the STACK, directly under the PCM and in line with the aperture in the PSM. The PSA on the top surface of the bottom layer may be locally removed at the location of the fluorescent sensor to form a shallow well into which the fluorescent sensor material is printed and dried. When the PCM is bonded to the bottom layer PSA, the bottom of the PCM may contact the fluorescent sensor, so that plasma collected in the PCM may be delivered to the fluorescent sensor. It follows that such a construction also works for other optical sensors that can be printed on the bottom layer, such as colorimetric sensors.2. The STACK may be used to carry a fluorescent sensor. Fluorescent sensor material may be printed and dried into the PCM membrane itself. Plasma collected in the PCM contacts the fluorescent sensor material. It follows that such a construction also works for other optical sensors that can be printed and dried onto the PSM, such as colorimetric sensors.Test Cartridge using the STACK

[0145] In accordance with another aspect of the invention, there is provided a single-use test cartridge, or ‘consumable’, for optical measurement of analytes in a sample fluid.

[0146] Figures 1 C-1 D illustrate one possible design of a 3-layer test cartridge constructed as per the first embodiment. Figures 2C-2D illustrate one possible design of a 3-layer test cartridge constructed as per the second embodiment. Figures 3C-3D illustrate one possible design of a 4-layer test cartridge constructed as per the third embodiment.

[0147] The illustrated test cartridges all contain 1 STACK for plasma separation and measurement and 2 whole blood sensor locations for whole blood measurement. Other test cartridge designs are possible having a different number of STACKS and a different number of whole blood sensor locations.

[0148] The whole blood sensor locations may be used to measure whole blood optically using fluorescent sensors or colorimetric sensors. Such sensors may be printed and dried directly in the whole blood sensor locations, though other sensor fabrication techniques are possible.T1Examples of analytes that may be measured in whole blood using such sensors include K and pH.

[0149] The whole blood sensor locations may also be used to measure whole blood optically via an absorbance measurement. In this case, the cartridge layers above and below the whole blood sensor locations are substantially transparent and the whole blood sensor location constitutes a standard cuvette. Examples of analytes that may be measured in whole blood using absorbance measurements include Hb and Het.

[0150] The user applies whole blood from a fingerstick to the sample port. The volume of whole blood required is approximately 25pl (though other volumes may be used).

[0151] One or more of the layers may contain a hydrophilic surface to draw the whole blood into the cartridge. The hydrophilic surface may be a hydrophilic PSA but other means, including a polymer film with a hydrophilic coating, are possible.

[0152] The test cartridge contains a sample channel air vent used to vent to atmosphere the air displaced by the whole blood entering the test cartridge. The test cartridge may contain a vent element, such as a hydrophobic porous plug, that allows air to pass but contains fluid inside the test cartridge.

[0153] The test cartridge may contain one or more air vents to vent to atmosphere the air displaced from each STACK as it fills with whole blood and plasma. Alternatively, the STACKS may not be vented to atmosphere, so that any air displaced from the STACK enters the whole blood channel.

[0154] The particular cartridge design illustrated herein can measures 3 analytes: 1 analyte in plasma and 2 analytes in whole blood. Examples of analytes that may be measured are K and Het in whole blood and pfHb in plasma. Another example of analytes that may be measured are K and pH in whole blood and pfHb in plasma. Test cartridges that measure other analytes are also possible. Test cartridges that measure fewer or more analytes are also possible.MODULE Design

[0155] According to further embodiments of the disclosure, an alternate plasma separation module (MODULE) for the separation of plasma from a whole blood sample isdisclosed. FIGURES 7A-7H illustrate the alternate MODULE. Figures 7A-7D illustrate four variations of the design of the MODULE; other variations are possible.

[0156] According to the disclosure, FIG. 7A illustrates an embodiment of the MODULE 700 with a circular plasma separation membrane (PSM) 702 and multiple small air vent holes 704 for the PSM 702.

[0157] FIG. 7B illustrates an alternate embodiment of the MODULE 710 with a circular PSM 712 and a single large air vent hole 714 for the PSM 712.

[0158] FIG. 7C illustrates an alternate embodiment of the MODULE 720 with an oblong PSM 722 and multiple small air vent holes 724 for the PSM 722.

[0159] FIG. 7D illustrates an alternate embodiment of the MODULE 730 with an oblong PSM 732 and a single large air vent hole 734 for the PSM 732.

[0160] The MODULE comprises 3 layers and a PSM. FIG. 7E illustrates an exploded view of an embodiment of the MODULE. According to FIG. 7E, MODULE 740 comprises a top layer 742, a middle 746 layer and a bottom layer 748. Top layer 742 contains air vent holes 744 for the PSM 750.

[0161] According to FIG. 7E, bottom layer 748 further comprises PSM 750 and a whole blood channel 752.

[0162] The PSM 750 is located in the whole blood channel and is sandwiched between the bottom layer and the top layer. FIG. 7F illustrates a cross-sectional view of an embodiment of the MODULE. According to FIG. 7F, MODULE 760 is shown having a top layer 762 and bottom layer 764. The bottom layer 764 and the top layer 762 are lined with pressure sensitive adhesive (PSA) 766.

[0163] According to FIG. 7F, sandwiched between top layer 762 and bottom layer 764 is a middle layer comprising a PSM 768 and whole blood channel 770. The PSM perimeter surface 772 is open to whole blood entry. The bottom surface of the PSM 774 is bonded to the bottom layer PSA 766. The top layer 762 further comprises air vents 778 for the PSM 768.

[0164] According to FIG. 1 F, the top surface of the PSM 776 is bonded to the top layer PSA 766. With both the bottom surface and top surface of the PSM 768 sealed by PSA 766,whole blood can only contact and enter the PSM 768 through its perimeter surface 772. One or both of the PSAs may be hydrophilic to help draw whole blood into the whole blood channel 770.

[0165] When whole blood fills the whole blood channel 770, the whole blood contacts the perimeter surface 772 of the PSM 768. The whole blood is then drawn inside the PSM 768 due to the capillarity of the PSM 768. The PSM 768 prevents red blood cells (RBCs) from substantially entering the PSM 768. RBCs may encroach into the PSM 768 a certain allowable distance. Beyond the allowable distance, only plasma enters the PSM 768.

[0166] Plasma separation membranes are designed to receive whole blood on a relatively broad top surface having a sufficiently large surface area to enable the separation of relatively large volumes of plasma before that top surface clogs with RBCs. The PSM 768 receives whole blood on its perimeter surface 772, which has a relatively small surface area. As just one example, a PSM 768 with a diameter of 5mm and a thickness of 0.25 mm has a top surface area of 19.6 mm2and a perimeter surface area of 3.9 mm2which makes the perimeter surface area approximately 5X smaller than the top surface area. Nevertheless, by separating relatively small volumes of plasma, PSM 768 can completely fill with plasma before the perimeter surface 772 clogs with RBCs.

[0167] Figures 7G-7H illustrate RBC encroachment into the PSM. FIG. 7G illustrates whole blood entry through the PSM perimeter surface. According to FIG. 7G, PSM 780 is shown with perimeter surface 786. With the top and bottom surfaces of the PSM 780 sealed, whole blood can only enter through the perimeter surface 786. Element 784 shows the extent of the RBC encroachment into the PSM 780. The portion 782 of the PSM 780 is shown filled with plasma.

[0168] FIG. 7H illustrates a top view of the PSM in the whole blood channel. According to FIG. 7H, PSM 790 is shown having a whole blood channel 792 and a portion 794 of the PSM 790 filled with plasma. Furthermore, element 796 also shows the extent of the red blood cell encroachment into the PSM 790.

[0169] The RBCs are prevented from encroaching beyond the allowable distance through any means, including one or more of the following mechanisms:• Size Exclusion - the PSM pore size is small enough to prevent the passage of individual RBCs.• Agglutination - the PSM has been coated throughout with one or more reagents causingRBCs to agglutinate in clumps that are larger than individual RBCs, and where the membrane pore size is small enough to prevent the passage of clumped RBCs. Agglutination can be accomplished by any means known in the art, including blood typing antibodies and lectins derived from plant seeds.• Deformation - the PSM has been coated throughout with one or more reagents causing RBCs to deform in such a way that they are preferentially caught by the interior features of the PSM.

[0170] According to the disclosure, the top and / or bottom layer contains a means, such as one or more holes, to vent the air from the PSM as it fills with plasma. Figures 7A-7D illustrate four possible designs for the shape of the PSM and the configuration of the air vent holes. There may be one air vent hole, or there may be multiple air vent holes. In some embodiments, the PSM may be circular in shape, and the vent holes may be centred on the circular membrane. In other embodiments, the PSM may be oblong in shape, and the vent holes may be off-centre. Noncircular membrane shapes that leave a substantial portion of the PSM free from vent holes may be advantageous for those cases where vent holes may interfere with optical measurements.

[0171] Figures 8A and 8B illustrate experimental results from prototype MODULES. FIG. 8A illustrates the extent of RBC encroachment in membranes treated with ANTI-AB antibodies as shown in image 800. FIG. 8B illustrates the extent of RBC encroachment on membranes not treated with ANTI-AB antibodies as shown in image 810. According to Figures 8A and 8B, the prototype MODULES labeled “HV+” were constructed using 0.35mm thick glass fiber membranes (Ahlstrom Cytosep 1668 HV+). The manufacturer does not provide the pore size but the membrane is intended to be used for plasma separation. According to the manufacturer, this membrane is pre-treated with chemicals that help deform the shape of RBCs and so limit their diffusion into the membrane. The membranes are vented by not fully covering the membrane with the top layer, leaving a small portion exposed to atmosphere.

[0172] According to the Figures 8A and 8B, the prototype MODULES labeled “5000” were constructed using 3.5-7.0 mils (0.089-0.178 mm) thick polyethersulfone (PES) membranes with a 5pm pore size (Cytiva Supor5000). According to the manufacturer, this membrane is not pretreated. The membranes are vented by not fully covering the membrane with the top layer, leaving a small portion exposed to atmosphere.

[0173] According to the disclosure, FIG. 8A illustrates the experimental results formembranes that were coated (fully soaked, then left to air-dry overnight) with anti-A and anti-B antibodies (Bio-Rad part number 801370 Seraclone ANTI-AB). This mixture of antibodies is used in blood typing and agglutinates blood types A, B and AB. Our blood donor was known to be blood type A; consequently, these membranes agglutinated the whole blood sample. The agglutination reduces the distance the RBCs encroach into the membrane.

[0174] According to the disclosure, FIG. 8B illustrates the experimental results for membranes that were not treated with anti-A and anti-B antibodies. These membranes did not agglutinate the whole blood sample. The absence of agglutination increases the distance the RBCs encroach into the membrane.MODULE PSM is a Membrane Cuvette

[0175] The PSM in the MODULE is a membrane cuvette. The top and bottom layers are substantially transparent. These layers may be made from clear, transparent polymers including PMMA, COC, and PET. It is also important to keep any RBCs out of the light path. Bonding the bottom surface of the PSM to the bottom layer via the bottom layer PSA prevents RBCs from slipping between the bottom layer and the PSM. Similarly, bonding the top surface of the PSM to the top layer via the top layer PSA prevents RBCs from slipping between the top layer and the PSM.. In this manner, analytes in the separated plasma may be measured via absorbance measurements.

[0176] According to the disclosure, FIGURES 9A-9E illustrate experimental results from prototype test cartridges. Figures 9A and 9B illustrate experimental results from prototype membrane cuvettes with a construction similar that shown in Figures 8A and 8B. The bottom layer is a glass microscope slide covered in hydrophilic PSA (Adhesives Research part number AR93450) to draw the whole blood sample into the whole blood channel. The middle layer is made from plastic spacers approximately the same thickness as the membrane (cut from plastic shim stock, McMaster-Carr part number 9513K43). The top layer is a hydrophilic PSA with one of its liners left on to provide some stiffness (Adhesives Research part number AR93450). The membrane is 0.33mm thick polyethersulfone (Cytiva part number Vivid GF). All membranes are oblong, measuring approximately 5mm wide by 8mm long. The membranes are vented by not fully covering the membrane with the top layer, leaving a small portion exposed to atmosphere. Note that this particular membrane has a pore size that is too large to be used as a PSM in our application; however, the polyethersulfone material and the 0.33mm thickness are bothcharacteristics that appear to work well in our application. In this particular experiment, there was no need for plasma separation because the sample fluid was plasma to which varying amounts of hemolysed RBCs had been added. The amounts of hemolysed RBCs were chosen to reflect an escalating degree of hemolysis in a typical patient with 40% Het blood. The light source was a broadband LED (white light) located above the membrane cuvette. The intensity of the transmitted spectra was measured below the membrane cuvette before and after arrival of the plasma in the membrane. The difference between the spectra gives the absorbance.

[0177] According to the disclosure, FIG. 9A and Fig 9B illustrates measured absorbance data from membrane cuvettes filled with plasma samples of varying hemolysis levels. FIG. 9A illustrates the absorbance as a function of hemolysis level. FIG. 9B illustrates the same data as Fig. 9A but here the hemolysis levels have been converted to pfHb concentrations via theoretical calculations. The pfHb results illustrate the ability to discriminate between the 25-100 mg / dL alert levels set by typical central lab analyzers.Alternate Uses of the MODULE

[0178] Use of the MODULE for purposes other than a membrane cuvette are also possible:• The MODULE may also be used to provide plasma to a fluorescent sensor. Fluorescent sensor material is printed and dried to a thin film on the bottom layer, at the location of the PSM. When the PSM is bonded to the bottom layer, the bottom of the PSM contacts the top of the fluorescent sensor. During use, plasma collected in the PSM first contacts and then diffuses into the hydrophilic fluorescent sensor. It follows that such a construction also works for other optical sensors that can be printed on the bottom layer, such as a colorimetric sensor.• The PSM may also be used to carry a fluorescent sensor. Fluorescent sensor material is printed onto the PSM. During use, plasma collected in the PSM diffuses into the fluorescent sensor material. It follows that such a construction also works for other optical sensors that can be printed onto the PSM, such as a colorimetric sensor.

[0179] With some changes, the plasma separation module can become a whole blood module for optical sensors:The whole blood module requires a membrane with a pore size large enough to freelyadmit RBCs and must not be coated with any reagents that prevent RBCs from entering the membrane. The whole blood module may be used to carry a fluorescent sensor. Fluorescent sensor material is printed onto the whole blood membrane. During use, the whole blood collected in the whole blood membrane diffuses into the fluorescent sensor material. It follows that such a construction also works for other optical sensors that can be printed onto the whole blood membrane, such as a colorimetric sensor.

[0180] With some changes, the whole blood module can become a co-oximetry module. Co-oximetry analytes, such as tHb, are typically measured using absorbance measurements on hemolysed whole blood:• The co-oximetry module requires a membrane with a pore size large enough to freely admit RBCs and must not be coated with any reagents that prevent RBCs from entering the membrane. The membrane does need to be coated in part or throughout with a reagent, such as sodium deoxycholate, that chemically hemolyses RBCs. During use, whole blood enters the membrane and is hemolysed by the reagent. Absorbance measurements are used to calculate the concentration of co-oximetry analytes such as tHb.

[0181] As shown above, simple changes to the membrane pore size and removal / changes to the reagent(s) coating the membrane can turn a plasma separation module into a whole blood module or into a co-oximetry module. The 3 polymer layers do not need to change. This versatility means 3-layer test cartridges can be constructed containing one or more plasma separation modules, and / or one or more whole blood modules, and / or one or more co- oximetry modules. In addition, the whole blood channel can also contain standard cuvettes. In addition, the whole blood channel can also contain optical sensors, including fluorescent sensors and colorimetric sensors.Test Cartridge using the MODULE

[0182] In accordance with another aspect of the invention, there is provided a single-use test cartridge, or ‘consumable’, for optical measurement of analytes in a sample fluid. The simple 3-layer construction of the MODULE extends to the test cartridge, which can also be constructed using the same 3-layer construction.

[0183] Figures 10A-10C illustrate a test cartridge using the MODULE. FIG. 10A illustratesan exemplary assembled test cartridge 1000.

[0184] FIG. 10B illustrates the test cartridge 1010 with the top layer lifted to better show the individual components for greater clarity. According to FIG. 10B, the top layer 1012 of test cartridge 1010 is shown with air vents holes 1014 for the PSM 1024. The bottom layer 1016 of test cartridge 1010 further comprises a sample port 1018, a standard cuvette (i.e., hollow space) 1020, a printed fluorescent sensor 1022, a membrane cuvette filled with the PSM 1024 and a sample channel air vent 1026.

[0185] FIG. 10C illustrates an exploded view of the test cartridge 1030 to show its construction. According to FIG. 10C, test cartridge 1030 comprises a top layer 1032, a channel layer 1036 and a bottom layer 1038. Top layer 1032 further comprises PSA on the bottom surface and air vent holes 1034 for the PSM 1040.

[0186] PSM 1040 is sandwiched between bottom layer 1038 and top layer 1032. Bottom layer 1038 further comprises a sample channel air vent 1044 and cutout 1046. The cutout 1046 is in the bottom layer PSA to make a well for the printed fluorescent sensor 1042.

[0187] The user applies whole blood from a fingerstick to the sample port. The volume of whole blood required is approximately 25pl (though other volumes may be used). One or both of the bottom layer and the top layer may contain a hydrophilic surface to draw the whole blood into the cartridge. The hydrophilic surface may be a hydrophilic PSA but other means, including a hydrophilic polymer film, are possible.

[0188] The test cartridge contains a sample channel air vent used to vent the air displaced by the sample fluid entering the test cartridge. The test cartridge may contain a vent element, such as a hydrophobic porous plug, that allows air to pass but contains fluid inside the test cartridge.

[0189] This particular design measures 3 analytes: Het, K and pfHb. Test cartridges that measure fewer or more analytes are also possible.

[0190] The first sensor is a hollow space forming a standard cuvette. This standard cuvette will fill with whole blood. Absorbance measurements will be used to calculate the Het of the whole blood. If the bottom layer and top layer are lined with PSA, the PSA may be locally removed at the standard cuvette location, avoiding absorbance due to the PSA.

[0191] Figures 9D and 9E illustrate the absorbance data from standard cuvettes filled with whole blood samples of varying Het levels. FIG. 9D illustrates measured absorbance data from standard cuvettes filled with whole blood samples of varying Het levels. FIG. 9E illustrates the same data as FIG. 9D but here the Het values have been converted to tHb concentrations via theoretical calculations.

[0192] The second sensor is a fluorescent K sensor. The fluorescent sensor is printed and dried onto the bottom layer. As illustrated in FIG. 10C, the bottom layer 1038 may be lined with a PSA. The PSA may be locally removed at the fluorescent sensor location. This would create a well with a depth equal to the thickness of the PSA (typically 1 -2mils or 25-50pm). The well could help contain the printed sensor material prior to drying, while it is still in liquid state. The well would also allow printing and drying of the fluorescent sensor directly onto the bottom layer material (likely a clear, transparent polymer such as COC, PMMA or PETG) which may be preferable to printing and drying onto the PSA. Emitted fluorescence measurements will be used to calculate the concentration of K in the whole blood.

[0193] FIG. 9C illustrates fluorescence intensity data from our fluorescent K sensor.STACK and MODULE Enable Simple Test Cartridge Design

[0194] According to the disclosure, the test cartridge design enabled by the simplicity of the STACK and the MODULE design has the following advantages:1 . Simplicity of construction - Performing all aspects of plasma separation directly in the whole blood channel means that no additional channels are required above or below the whole blood channel:• The whole blood is not applied to the top surface of the PSM, which eliminates the need for a whole blood supply channel above the PSM.• The separated plasma is not removed from the PCM (in the STACK) or the PSM (in the MODULE), which eliminates the need for a plasma removal channel below the PCM (in the STACK) or the PSM (in the MODULE).2. Reel-to-reel manufacturing - The test cartridge layers can all be made from polymer films, enabling high-speed, reel-to-reel manufacturing. The combination of inexpensive polymer films with high-speed manufacturing enables low-cost test cartridges.3. Enable measurements in both whole blood and plasma - The ability to separate plasma downstream of the sample port means that the interior of the test cartridge can contain both whole blood and plasma; therefore, the same test cartridge can measure analytes in both whole blood and in plasma.4. Minimise sample volume - Separating only the plasma needed by the plasma sensor(s) minimises the volume of plasma separated, which helps minimise the volume of the whole blood sample needed. There is no wasting of plasma by, for example, separating plasma at the sample port and then using a substantial portion of that separated plasma just to transport the plasma from the sample port to the plasma sensor(s) inside the test cartridge. The test cartridges described herein are anticipated to require a whole blood volume of approximately 25pl, though other volumes are possible.5. Speed of plasma separation - Minimising the volume of plasma separated also minimises the time required to complete the plasma separation. This enables a rapid test response. Test results indicate that plasma separation can be completed in less than 30 seconds, though other separation times are possible.

[0195] In some embodiments, the test cartridge, or its packaging, contains a means to store information. The information stored may include regulatory information such as test cartridge serial number, lot number, expiry date and unique device identifier (UDI) code. The information stored may also include factory test data such as measured calibration curves for the fluorescent sensors and measured optical path lengths for the cuvettes. The means of information storage are known in the art and include printed 1 D barcodes, printed 2D barcodes, RFID tags and so forth.

[0196] In some embodiments, the test cartridge contains a label on one or both faces. The labels may be used to close certain fluidic channels and may also contain printed regulatory information such as test cartridge serial number, lot number, expiry date and UDI code.Instrument

[0197] In accordance with another aspect of the invention, there is provided an instrument for determining the concentration of analytes in a sample fluid.

[0198] According to the disclosure, the instrument is configured to accept the test cartridges of the disclosure.

[0199] There is also provided an instrument for determining the concentration of analytes in a sample fluid. The instrument comprises a source of excitation light for fluorescent sensors and a means to measure the spectra of the excitation light and / or the emitted fluorescent light. The instrument also comprises a source of light for transmission through standard cuvettes and / or membrane cuvettes in the cartridge, and a means to measure the spectra of the light after it has passed through the standard cuvettes and / or membrane cuvettes. The instrument also comprises a source of light for transmission through colorimetric sensors in the cartridge, and a means to measure the spectra of the light after it has passed through the colorimetric sensors.

[0200] In some embodiments the instrument comprises a means to read information stored on the test cartridge or its packaging. The means of information reading are known in the art and include an optical barcode reader and a RFID tag reader.

[0201] In some embodiments the instrument comprises a computer processing means to convert the measured light intensities to analyte concentrations using proprietary mathematical models. These proprietary mathematical models may make use of any factory test data that may be stored on the test cartridge or its packaging such as measured factory calibration curves for the fluorescent sensors and measured optical path lengths for the cuvettes.

[0202] In some embodiments the instrument comprises a screen to display information to the user; examples of the information that may be displayed include instrument status, instructions for use, measured analyte concentrations from the current test, and summaries of measured analyte concentrations from past tests. In some embodiments the screen may be a touch screen allowing the user to enter information via an on-screen keyboard; examples of the information that may be entered include patient details and physician details.

[0203] In some embodiments the instrument comprises a means to transmit test records to software on a mobile device such as a mobile phone or a tablet computer. The means for transmission from the instrument to the device include methods known in the art such as Bluetooth and wi-fi. The screen of the mobile device may be used instead of and / or in addition to, the instrument screen to display and enter information.Software

[0204] In some embodiments the software on the mobile device will securely accept test record transmissions from the instrument. Furthermore, the software on the mobile device willfacilitate the secure transmission of those test records to the patient’s electronic medical record (EMR) so that test records may be shared with the patient’s physician.

[0205] In some embodiments the software on the mobile device will include an interface allowing the physician to contact and communicate with the patient; such communication may be accomplished with a number of means known in the art, including texting, voice calling and video calling.

[0206] According to the disclosure, a plasma separation stack apparatus for use in a test cartridge for a point-of-care (POC) blood analysis system is disclosed. The plasma separation stack apparatus comprises a top layer, a middle layer, a bottom layer, a plasma separation membrane (PSM) configured to separate plasma from whole blood, and a plasma collection membrane (PCM) configured to collect the separated plasma. The PSM is placed directly on top of, and in contact with the PCM.

[0207] According to the disclosure, the plasma separation stack apparatus is constructed from a PSM with an opening in its centre functioning as an optical aperture, and a PCM without an opening in its centre. The plasma separation stack apparatus is constructed with a PSM having a pore size that varies across its thickness, with the top portion having pores large enough to admit whole blood and red blood cells (RBCs) both through its top surface and its outer perimeter, and the bottom portion having pores small enough to block RBCs.

[0208] According to the disclosure, the plasma separation stack apparatus is constructed with a PSM that freely admits whole blood and red blood cells through its outer perimeter, even when the entire top surface of the PSM is obstructed by a cartridge layer. The plasma separation stack apparatus is constructed with a PSM having sufficient capillarity to prevent the whole blood and the separated plasma from leaving the PSM and entering the aperture. The plasma separation stack apparatus is constructed with a PCM having sufficient capillarity to draw the separated plasma out of the PSM and into the PCM.

[0209] According to the disclosure, the PSM of the plasma separation stack apparatus has the same outer diameter as the PCM, wherein the STACK is located in the middle layer of a 3-layer test cartridge, wherein the top of the PSM contacts and is be bonded to the top layer, wherein the bottom of the PCM contacts and is bonded to the bottom layer, and wherein the bottom of the PSM is in direct contact with and is bonded to the top of the PCM.

[0210] According to the disclosure, the PSM plasma separation stack apparatus has a larger outer diameter than the PCM, wherein the apparatus is located in the middle layer of a 3- layer test cartridge, wherein the top of a portion of the PSM contacts and is bonded to the top layer, wherein the bottom of a portion of the PSM contacts and is bonded to the bottom layer, wherein the bottom of the PCM contacts and is bonded to the bottom layer, and wherein the bottom of the PSM is in direct contact with and is bonded to the top of the PCM.[0021 1] According to the disclosure, the PSM plasma separation stack apparatus has a larger outer diameter than the PCM, wherein the apparatus is located in the middle 2 layers of a 4-layer test cartridge, the 4-layer test cartridge further comprising a bottom layer, a plasma layer, a whole blood layer and a top layer, wherein the top of the PSM contacts and is bonded to the top layer, wherein the bottom of the PSM contacts and is bonded to the plasma layer, wherein the bottom of the PCM contacts and is bonded to the bottom layer, and wherein the bottom of the PSM is in direct contact with and is bonded to the top of the PCM.

[0212] According to the disclosure, the PSM plasma separation stack apparatus functions as a membrane cuvette for measuring the absorbance of light in the separated plasma. The PSM or the PCM of the plasma separation stack apparatus incorporate reagents that facilitate the absorbance measurement of analytes in the separated plasma.

[0213] The apparatus of Claim 1 , wherein the PCM of the plasma separation stack apparatus is used to deliver the separated plasma to a fluorescent sensor or colorimetric sensor located under the PCM and in line with the aperture in the PSM, wherein the sensor is optically measured through the aperture in the PSM. The fluorescent or colorimetric sensor is incorporated into the PCM, wherein the separated plasma entering the PCM also contacts the sensor, resulting in the apparatus that functions as the fluorescent or colorimetric sensor.

[0214] According to the disclosure, the plasma separation stack apparatus further comprises a chemical hemolysis reagent printed directly into the PSM to hemolyse the whole blood in the PSM, wherein the PCM collects the hemolysed whole blood, to function as a membrane cuvette for measuring the absorbance of light in hemolysed whole blood. The PSM or the PCM of the plasma separation stack apparatus incorporate reagents that facilitate the absorbance measurement of analytes in the hemolysed whole blood.

[0215] According to the disclosure, a test cartridge apparatus for a point-of-care (POC) blood analysis system comprising a plasma separation stack is disclosed. The plasma separation stack further comprises a top layer, a middle layer, a bottom layer, a plasma separation membrane (PSM) configured to separate plasma from whole blood, and a plasma collection membrane (PCM) configured to collect the separated plasma wherein the PSM is placed directly on top of, and in contact with the PCM.

[0216] According to the disclosure, the test cartridge apparatus further contains one or more locations where analytes in whole blood may be measured using fluorescent sensors, colorimetric sensors or absorption measurements. The test cartridge apparatus further contains a sample port that accepts a whole blood sample, a whole blood channel, one or more air vents for the whole blood channel, and one or more air vents for the plasma separation stack.

[0217] According to the disclosure, the test cartridge apparatus further contains one or more hydrophilic surfaces that help draw the whole blood sample into the test cartridge apparatus. The test cartridge apparatus, further contains a standard cuvette for the measurement of Het in whole blood, a fluorescent or colorimetric sensor for the measurement of K in whole blood, and the plasma separation stack configured as a membrane cuvette for the measurement of pfHb in separated plasma.

[0218] According to the disclosure, the test cartridge apparatus further contains a fluorescent or colorimetric sensor for the measurement of pH in whole blood, a fluorescent or colorimetric sensor for the measurement of K in whole blood, and the plasma separation stack configured as a membrane cuvette for the measurement of pfHb in separated plasma.

[0219] According to the disclosure, a plasma separation module for a point-of-care (POC) blood analysis system is disclosed. The plasma separation module comprises a top layer, a middle layer, a bottom layer, and a plasma separation membrane (PSM) configured to separate plasma from whole blood, wherein the bottom layer, middle layer, and top layer are bonded to one another.

[0220] According to the disclosure, the middle layer of the plasma separation module contains a whole blood channel. The PSM is located in the whole blood channel of the middle layer, and has its top surface bonded to the top layer, and has its bottom surface bonded to thebottom layer. The plasma separation module of further contains one or more hydrophilic surfaces to help draw a fluid sample into the module.

[0221] According to the disclosure, whole blood enters the plasma separation membrane through its perimeter surface, and wherein the plasma separation membrane hinders the encroachment of RBCs into the plasma separation membrane by RBC size exclusion, or added RBC agglutination agents, or added RBC deformation agents. The plasma separation membrane functions as a membrane cuvette for measuring absorbance in the separated plasma.

[0222] According to the disclosure, the plasma separation module is configured with a plasma separation membrane to separate plasma from whole blood, and to function as a means to deliver plasma to a printed fluorescent sensor or colorimetric sensor located directly under the plasma separation membrane. The plasma separation module is configured with a plasma separation membrane to separate plasma from whole blood, and having fluorescent sensor or colorimetric sensor material printed directly onto the plasma separation membrane, and to function as a plasma fluorescent sensor or colorimetric sensor.

[0223] According to the disclosure, the plasma separation module is configured with a membrane that admits RBCs, and having fluorescent sensor or colorimetric sensor material printed onto the membrane, and to function as a whole blood fluorescent sensor or colorimetric sensor. The plasma separation module is configured with a membrane that admits RBCs, and having a chemical hemolysis reagent printed directly onto the membrane, and to function as a membrane cuvette for measuring absorbance in hemolysed whole blood.

[0224] According to the disclosure, a test cartridge apparatus configured for plasma separation from whole blood by incorporating a plasma separation module is disclosed. The test cartridge apparatus contains a sample port that accepts a whole blood sample, a whole blood channel, and an air vent for the whole blood channel.

[0225] According to the disclosure, the test cartridge apparatus contains one or more hydrophilic surfaces that help draw the whole blood sample into the test cartridge and over all sensors contained within the test cartridge. The test cartridge apparatus contains a standard cuvette for the measurement of Het in whole blood, a fluorescent sensor for the measurement of K, and a plasma separation membrane configured as a membrane cuvette for the measurement of pfHb in the separated plasma.

[0226] According to the disclosure, the test cartridge apparatus wherein the same blood sample is used to measure Het in whole blood, K in whole blood, and pfHb in the separated plasma so that all measurements come from the same blood sample and are directly correlated with one another. The test cartridge apparatus contains a multitude of sensors including standard cuvettes, fluorescent sensors, colorimetric sensors.

[0227] Although the disclosure has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the disclosure. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

[0228] Implementations disclosed herein provide systems, methods, and apparatus for generating or augmenting training data sets for machine learning training. The functions described herein may be stored as one or more instructions on a processor-readable 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 a medium may comprise RAM, ROM, EEPROM, flash memory, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store desired program code in the form of instructions or data structures and that can be accessed by a computer. It should be noted that a computer-readable medium may be tangible and non- transitory. As used herein, the term “code” may refer to software, instructions, code, or data that is / are executable by a computing device or processor. A “module” can be considered as a processor executing computer-readable code.

[0229] A processor as described herein can be a general-purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general-purpose processor can be a microprocessor, but in the alternative, the processor can be a controller, or microcontroller, combinations of the same, or the like. A processor can also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration. Although described herein primarily with respect to digital technology, a processor may also include primarily analog components. Forexample, any of the signal processing algorithms described herein may be implemented in analog circuitry. In some embodiments, a processor can be a graphics processing unit (GPU). The parallel processing capabilities of GPUs can reduce the amount of time for training and using neural networks (and other machine learning models) compared to central processing units (CPUs). In some embodiments, a processor can be an ASIC including dedicated machine learning circuitry custom-build for one or both of model training and model inference.

[0230] The disclosed or illustrated tasks can be distributed across multiple processors or computing devices of a computer system, including computing devices that are geographically distributed. The methods disclosed herein comprise one or more steps or actions for achieving the described method. The method steps and / or actions may be interchanged with one another without departing from the scope of the claims. In other words, unless a specific order of steps or actions is required for proper operation of the method that is being described, the order and / or use of specific steps and / or actions may be modified without departing from the scope of the claims.

[0231] As used herein, the term “plurality” denotes two or more. For example, a plurality of components indicates two or more components. The term “determining” encompasses a wide variety of actions and, therefore, “determining” can include calculating, computing, processing, deriving, investigating, looking up (e.g., looking up in a table, a database, or another data structure), ascertaining and the like. Also, “determining” can include receiving (e.g., receiving information), accessing (e.g., accessing data in a memory) and the like. Also, “determining” can include resolving, selecting, choosing, establishing and the like.

[0232] The phrase “based on” does not mean “based only on,” unless expressly specified otherwise. In other words, the phrase “based on” describes both “based only on” and “based at least on.” While the foregoing written description of the system enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The system should therefore not be limited by the above-described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the system. Thus, the present disclosure is not intended to be limited to the implementations shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

ClaimsWhat is claimed is:1 . A plasma separation stack apparatus for use in a test cartridge for a point-of-care (POC) blood analysis system comprising: a top layer; a middle layer; a bottom layer; a plasma separation membrane (PSM) configured to separate plasma from whole blood; and a plasma collection membrane (PCM) configured to collect the separated plasma; wherein the PSM is placed directly on top of, and in contact with the PCM.

2. The apparatus of Claim 1 , wherein the plasma separation stack apparatus is constructed from a PSM with an opening in its centre functioning as an optical aperture, and a PCM without an opening in its centre.

3. The apparatus of Claim 1 , wherein the plasma separation stack apparatus is constructed with a PSM having a pore size that varies across its thickness, with the top portion having pores large enough to admit whole blood and red blood cells (RBCs) both through its top surface and its outer perimeter, and the bottom portion having pores small enough to block RBCs.

4. The apparatus Claim 1 , wherein the plasma separation stack apparatus is constructed with a PSM that freely admits whole blood and red blood cells through its outer perimeter, even when the entire top surface of the PSM is obstructed by a cartridge layer.

5. The apparatus Claim 1 , wherein the plasma separation stack apparatus is constructed with a PSM having sufficient capillarity to prevent the whole blood and the separated plasma from leaving the PSM and entering the aperture.

6. The apparatus of Claim 1 , wherein the plasma separation stack apparatus is constructed with a PCM having sufficient capillarity to draw the separated plasma out of the PSM and into the PCM.

7. The apparatus of Claim 1 , wherein the PSM has the same outer diameter as the PCM, wherein the STACK is located in the middle layer of a 3-layer test cartridge; wherein the top of the PSM contacts and is be bonded to the top layer; wherein the bottom of the PCM contacts and is bonded to the bottom layer; and wherein the bottom of the PSM is in direct contact with and is bonded to the top of the PCM.

8. The apparatus of Claim 1 , wherein the PSM of the plasma separation stack apparatus has a larger outer diameter than the PCM, wherein the apparatus is located in the middle layer of a 3-layer test cartridge; wherein the top of a portion of the PSM contacts and is bonded to the top layer; wherein the bottom of a portion of the PSM contacts and is bonded to the bottom layer; wherein the bottom of the PCM contacts and is bonded to the bottom layer; and wherein the bottom of the PSM is in direct contact with and is bonded to the top of the PCM.

9. The apparatus of Claim 1 , wherein the PSM of the plasma separation stack apparatus has a larger outer diameter than the PCM, wherein the apparatus is located in the middle 2 layers of a 4-layer test cartridge, the 4- layer test cartridge further comprising a bottom layer, a plasma layer, a whole blood layer and a top layer; wherein the top of the PSM contacts and is bonded to the top layer; wherein the bottom of the PSM contacts and is bonded to the plasma layer; wherein the bottom of the PCM contacts and is bonded to the bottom layer; and wherein the bottom of the PSM is in direct contact with and is bonded to the top of the PCM.

10. The apparatus of Claim 1 , wherein the PCM of the plasma separation stack apparatus functions as a membrane cuvette for measuring the absorbance of light in the separated plasma.11 . The apparatus of Claim 1 , wherein the PSM or the PCM of the plasma separation stack apparatus incorporate reagents that facilitate the absorbance measurement of analytes in the separated plasma.

12. The apparatus of Claim 1 , wherein the PCM of the plasma separation stack apparatus is used to deliver the separated plasma to a fluorescent sensor or colorimetric sensor located under the PCM and in line with the aperture in the PSM, wherein the sensor is optically measured through the aperture in the PSM.

13. The apparatus of Claim 1 , wherein the fluorescent or colorimetric sensor is incorporated into the PCM, wherein the separated plasma entering the PCM also contacts the sensor, resulting in the apparatus that functions as the fluorescent or colorimetric sensor.

14. The apparatus of Claim 1 , further comprising a chemical hemolysis reagent printed directly into the PSM to hemolyse the whole blood in the PSM, wherein the PCM collects the hemolysed whole blood, to function as a membrane cuvette for measuring the absorbance of light in hemolysed whole blood.

15. The apparatus of Claim 14, wherein the PSM or the PCM of the plasma separation stack apparatus incorporate reagents that facilitate the absorbance measurement of analytes in the hemolysed whole blood.

16. A test cartridge apparatus for a point-of-care (POC) blood analysis system comprising a plasma separation stack, the plasma separation stack further comprising: a top layer; a middle layer; a bottom layer; a plasma separation membrane (PSM) configured to separate plasma from whole blood; and a plasma collection membrane (PCM) configured to collect the separated plasma; wherein the PSM is placed directly on top of, and in contact with the PCM.

17. The test cartridge apparatus of Claim 16, further containing one or more locations where analytes in whole blood may be measured using fluorescent sensors, colorimetric sensors or absorption measurements.

18. The test cartridge apparatus of Claim 16, further containing a sample port that accepts a whole blood sample, a whole blood channel, one or more air vents for the whole blood channel, and one or more air vents for the plasma separation stack.

19. The test cartridge apparatus of Claim 16, further containing one or more hydrophilic surfaces that help draw the whole blood sample into the test cartridge apparatus.

20. The test cartridge apparatus of Claim 16, further containing a standard cuvette for the measurement of Het in whole blood, a fluorescent or colorimetric sensor for the measurement of K in whole blood, and the plasma separation stack configured as a membrane cuvette for the measurement of pfHb in separated plasma.

21. The test cartridge apparatus of Claim 16, containing a fluorescent or colorimetric sensor for the measurement of pH in whole blood, a fluorescent or colorimetric sensor for the measurement of K in whole blood, and the plasma separation stack configured as a membrane cuvette for the measurement of pfHb in separated plasma.

22. A plasma separation module for a point-of-care (POC) blood analysis system comprising: a top layer; a middle layer; a bottom layer; and a plasma separation membrane (PSM) configured to separate plasma from whole blood; wherein the bottom layer, middle layer, and top layer are bonded to one another.

23. The plasma separation module of Claim 22, wherein the middle layer contains a whole blood channel.

24. The plasma separation module of Claim 22, wherein the PSM is located in the whole blood channel of the middle layer, and has its top surface bonded to the top layer, and has its bottom surface bonded to the bottom layer.

25. The plasma separation module of Claim 22, further containing one or more hydrophilic surfaces to help draw a fluid sample into the module.

26. The plasma separation module of Claim 22, wherein whole blood enters the plasma separation membrane through its perimeter surface, and wherein the plasma separation membrane hinders the encroachment of RBCs into the plasma separation membrane by RBC size exclusion, or added RBC agglutination agents, or added RBC deformation agents.

27. The plasma separation membrane of Claim 22, functioning as a membrane cuvette for measuring absorbance in the separated plasma.

28. The plasma separation module of Claim 22, configured with a plasma separation membrane to separate plasma from whole blood, and to function as a means to deliver plasma to a printed fluorescent sensor or colorimetric sensor located directly under the plasma separation membrane.

29. The plasma separation module of Claim 22, configured with a plasma separation membrane to separate plasma from whole blood, and having fluorescent sensor or colorimetric sensor material printed directly onto the plasma separation membrane, and to function as a plasma fluorescent sensor or colorimetric sensor.

30. The plasma separation module of Claim 22, configured with a membrane that admits RBCs, and having fluorescent sensor or colorimetric sensor material printed onto the membrane, and to function as a whole blood fluorescent sensor or colorimetric sensor.31 . The plasma separation module of Claim 22, configured with a membrane that admits RBCs, and having a chemical hemolysis reagent printed directly onto the membrane, and to function as a membrane cuvette for measuring absorbance in hemolysed whole blood.

32. A test cartridge apparatus configured for plasma separation from whole blood by incorporating the plasma separation module of Claim 22.

33. The test cartridge apparatus of Claim 32, containing a sample port that accepts a whole blood sample, a whole blood channel, and an air vent for the whole blood channel.

34. The test cartridge apparatus of Claim 32, containing one or more hydrophilic surfaces that help draw the whole blood sample into the test cartridge and over all sensors contained within the test cartridge.

35. The test cartridge apparatus of Claim 32, containing a standard cuvette for the measurement of Het in whole blood, a fluorescent sensor for the measurement of K, and a plasma separation membrane configured as a membrane cuvette for the measurement of pfHb in the separated plasma.

36. The test cartridge apparatus of Claim 35, wherein the same blood sample is used to measure Het in whole blood, K in whole blood, and pfHb in the separated plasma so that all measurements come from the same blood sample and are directly correlated with one another.

37. The test cartridge apparatus of Claim 32, containing a multitude of sensors including standard cuvettes, fluorescent sensors, colorimetric sensors.

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