Devices and methods for blood separation

Abstract

A collection device that includes at least one capillary seal provides for the metering and dilution of a fluid sample. A fluid separation module particularly adapted for the separation of plasma from whole blood includes a sample chamber, for receiving the sample and wherein agglomeration is allowed to proceed. The filter separation module is particularly adapted to efficiently transport separated plasma from the sample chamber to a digital microfluidic (DMF) chip.

Description

[0001] DEVICES AND METHODS FOR BLOOD SEPARATION

[0002] This application claims priority to United States provisional patent application no. 63 / 680,369 filed on August 7, 2024.

[0003] TECHNICAL FIELD

[0004] This application relates to blood separation devices and methods.

[0005] BACKGROUND OF THE ART

[0006] For health monitoring, disease diagnosis, and illness management for humans, and animals, biochemistry testing is common. Much of this testing requires the collection of whole blood, separation of the liquid plasma from the cellular matter, aliquoting / dilution, distribution, and then using various biochemistry methods and analytics to measure one or more analytes such as Creatinine, Albumin, Vitamin D etc. in the plasma. In a typical scenario, 3 ml or more of venous blood is collected into vacutainer tubes by a technically trained and certified specialist (phlebotomist or nurse), spun in a centrifuge at 1500 times gravity for about 12 minutes to speed up the natural tendency of blood to sediment, causing the more dense cellular components of blood (erythrocytes: 37-52% typical by volume, leukocytes: 1%, and thrombocytes: <1%) to settle to the bottom, after which the plasma (the remaining liquid) can be carefully collected off the top. Then, again by a technician, the plasma is aliquoted, diluted as needed, and distributed into instrument-specific vials for analysis.

[0007] More recently, to better allow testing near to the subject to be tested, lessen the impact on the subject and speed up reporting, tests using capillary blood (a few drops of blood, approximately 20-200 microliters (pl), taken from a finger prick) have become popular. Concurrent with this is the concept of a single use, disposable ‘lab on a chip’. There are a number of challenges with this concept, but significantly: it is difficult to quickly collect more than a 100 ul of whole blood and it is challenging to efficiently separate plasma from such a small quantity of whole blood.

[0008] BRIEF SUMMARY

[0009] In one aspect there is provided a fluid metering device that includes: a cup having an inlet for receiving the fluid; a flexible diaphragm manipulable to increases pressure in the cup above a first threshold pressure; a diluent chamber; and one or more capillary channels fluidly connecting the cup to the diluent chamber, the one or more capillary channels operable to drain the fluid from the cup into the diluent chamber when pressure in the cup exceeds the first threshold pressure.

[0010] The device suitably further includes a vent for releasing gas from the diluent chamber when it receives the drained fluid.

[0011] In some embodiments, the flexible diaphragm is configured to releasably close the inlet, preferably the diaphragm is a flexible cap for releasably sealing the cup inlet.

[0012] The diluent chamber can contain a known volume of diluent or be configured to receive a known volume of diluent.

[0013] In some embodiments, the device further includes one or more second capillary channels in a wall of the diluent chamber distal the one or more capillary channels and operable to drain fluid from the diluent chamber when pressure in the diluent chamber exceeds a second threshold pressure. The one or more second capillary channels each may include a channel inlet on an interior surface of the wall of the diluent chamber and a channel outlet on an exterior surface of the wall and, in some embodiments, the device further includes an engagement mechanism on the exterior surface of the wall to fluidly connect the one or more second capillary channels with a fluid separation module. The engagement mechanism can be in the form of a wall descending from the exterior surface and defining an engagement recess for receiving a portion of the fluid separation module, with the channel outlet(s) being in the engagement recess. A removable seal may further be provided for sealing a mouth of the engagement recess before use.

[0014] Also provided is a fluid separation device for separating plasma from whole blood that includes: a filtration chamber for receiving a volume of whole blood and a volume of agglutinator, wherein the volume of agglutinator agglutinates red blood cells of the volume of whole blood to red blood cell particles having a minimum average diameter of 10 pm or greater within 10 minutes or less, preferably within 5 minutes or less; an outlet in a wall of the filtration chamber; and a filter having a minimum average pore diameter of between 5 pm and 15 pm secured over the filtration chamber outlet.

[0015] The filtration chamber may include an inner wall surface and an outer wall surface, with the filter secured on the inner wall surface. The filtration chamber outlet includes a hydrophilic surface.

[0016] Also provided is a system that includes a fluid metering device as provided herein and a fluid separation device as provided herein, the diluent chamber being fluidly connected to the filtration chamber by the one or more capillary channels. The filtration chamber may be fluidly connected to a DM F chip via the filtration chamber outlet.

[0017] Also provided is a method for separating plasma from a volume of whole blood, the method comprising: combining the sample of whole blood with a diluent solution comprising an agglutinator, preferably wherein the agglutinator comprises polyvinyl alcohol (PVA); and allowing agglutination of red blood cells to proceed for a time sufficient to form an agglutinated sample comprising agglutinated red blood cell containing particles having a minimum average diameter of 10 pm or greater; and passing the agglutinated sample through a filter having an average pore size between 5 pm and 15 pm. In some embodiments, the time sufficient to form an agglutinated sample is a period of 30 seconds to 10 minutes, preferably 1 to 5 minutes. The agglutinator suitably includes PVA, optionally: the diluent comprises: phosphate buffered saline (PBS); 0.5 % to < 2%, preferably about 1% weight / volume concentration of PVA; and 0.1% - 0.5%, preferably 0.2% weight / volume of a surfactant; preferably an ethoxylated and propoxylated ethylene diamine; optionally wherein: the PVA has a molecular weight between 50,000 and 250,000 Daltons; and / or the PVA is 70% to 100% hydrolyzed, preferably 86% to 90% hydrolyzed, more preferably 88% hydrolyzed. Also provided is a composition for agglutinating components of a biological fluid comprising an agglutinator as described herein .

[0018] The method can further include determining the concentration of an analyte in the diluted plasma.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a front cross-sectional view of a fluid metering and dilution module according to an embodiment.

[0021] Figure 2 is a front cross-sectional view of a fluid metering and dilution module according to an embodiment, including a flexible cap.

[0022] Figure 3 is a top, perspective photograph of a fluid metering and dilution module according to an embodiment, including a flexible cap.

[0023] Figure 4 is a front exploded perspective view of an aligning apertures fluid separation module that may be used in combination with a fluid metering and dilution module according to some embodiments. Figure 5 is a rear exploded perspective view of the separation module shown in Figure 4.

[0024] Figure 6 is a front cross-sectional view of a filtration fluid separation module according to an embodiment.

[0025] Figure 7 is a front cross-sectional view of a filtration fluid separation module integrated with a digital microfluidic (DMF) chip, according to an embodiment.

[0026] DETAILED DESCRIPTION

[0027] Successful point-of-care blood testing using capillary blood i.e. a few drops of blood, requires two steps: dilution (in view of the small quantity of blood available) and plasma separation. Both these steps present challenges.

[0028] In the case of dilution, current methods and devices for blood collection do not allow for easy mixing of blood with a secondary solution (e.g. a diluent solution) in a manner that prevents consumer contact with the mixed fluid. Metering of a fixed volume of blood from a large droplet often requires a consumer to fill a capillary to a marked line that depends on consumer control.

[0029] One approach to separation in micro-volume testing, whether alone or as part of a process that includes other separation steps, is passive separation through use of size-exclusion filter material. Cellular matter in blood has a size whereby filter material with a pore size of about 2-4 urn in diameter will prevent the cellular matter from transiting - getting stuck in the filter material, while plasma can flow through. Filtration typically takes 10 minutes or longer. There are challenges with this approach. For example, there is a potential to rupture the cellular material, which will cause hemolysis and contaminate the plasma, invalidating most tests. Thus, pressure cannot be used to speed up filtering. As another example, there is a potential that some cellular matter will transit the filter, again causing problems with downstream testing, so filter quality must be high. Further, filter material is prone to clogging because of its small pore size and that it is intended to hold back the cellular material. If the surface area of the filter is insufficient, the filter will eventually clog and thereby prevent any further flow of plasma.

[0030] As the surface area is increased to prevent clogging, another issue becomes more pronounced: hold-back volume. All filters are made of a material that has openings to allow fluid to transit. The size of these openings (average pore size) is controlled. For any filter, the total volume of the openings is called void volume and typically after filtration, the void volume contains the filtride and some remaining filtrate. This hold-back affects ‘yield’ i.e. the percentage of filtrate that is recovered after filtration over the amount that was actually available. In small filtrations such as from a few drops of blood, the hold-back volume can be significant and yields using known devices and methods therefore are typically less than 60%. E.g. for a person with a hematocrit 50%, 100 ul of blood (about 2 drops) would yield only about 30 ul of plasma if there is no clogging. In one example of a combination of passive sedimentation with filtration [Membrane-based, sedimentation-assisted plasma separator for point-of-care applications Anal Chem. 2013 Nov 5; 85(21): 10463-10470. Changchun Liu, Michael Mauk, Robert Gross, Frederic D. Bushman, Paul H. Edelstein, Ronald G. Collman, Haim H. Bau], in about 7 minutes the authors recovered about 275 ul from about 2 ml of whole blood, or a 30% yield. With such little amounts of plasma, it becomes very difficult to transfer and work with the volume for testing purposes.

[0031] The methods and devices provided herein may address one or more of these challenges.

[0032] In one aspect, there is provided a metering and dilution module 100. While reference is made to a “module”, it should be understood that the metering and dilution module 100 may be sold or provided as a standalone device, which may have one or more connection features for connecting the metering and dilution module 100 to further processing and / or testing components.

[0033] In this context, “metering” and variations thereof refers to collecting a known volume of sample. In particular, embodiments provided herein limit the collectible sample volume while enabling a minimum sample volume collection without requiring active measurement by the user - the adequacy of the sample can be verified by simple visual observation.

[0034] Metering and dilution module 100 includes at least one capillary seal. A capillary seal consists of a flexible diaphragm and one or more capillary tubes. When pressure is applied to the seal via the diaphragm, fluid passes into the capillary tube(s). In the metering and dilution module 100 shown in the Figures, a removable cap 102 acts as a flexible diaphragm.

[0035] While in one embodiment, cap 102 acts as the flexible diaphragm, in other embodiments, flexible components may be provided elsewhere on the module, the key requirement being a mechanism that can be used to introduce a pressure difference e.g. a diaphragm in a wall of the metering and dilution module 100 or the module can be pre-compressed with a part (e.g. a pin) that is pulled out to allow decompression. With reference to Figures 1 and 2, dilution and metering module 100 includes a cup 104 for collection of the sample, suitably a whole blood sample, suitably obtained from a pinprick blood collection procedure. Cup 104 has a receiving inlet 106, suitably configured in some embodiments to facilitate blood collection, as described further below. Suitably, prior to use, receiving inlet 106 is covered by a removable or breachable seal 110 (e.g. a seal that can be peeled off or pierced or cut by a user). In some embodiments, suitable cutting or piercing means may be provided on cap 102. In some embodiments, cap 102 is operatively connected to the seal 110 such that removal of the cap 102 breaches or removes seal 110 from cup receiving inlet 106. Seal 110 may be e.g. a polyethylene seal adhered or heat sealed to receiving inlet 106.

[0036] Cup 104 is connected to a diluent chamber 108 via one or more capillary channels 112. As capillary cup 104 fills to the capillary seal 114 i.e. formed at the junction between cup 104 and capillary channel(s) 112, it does not accept more fluid until the seal is breached, which requires the application of pressure. It is thus evident to a user by simple visual inspection when cup 104 is filled or, in some embodiments, filled to a fill line or other visual indication on cup 104 indicating that sufficient sample has been collected. Accordingly, the arrangement provides for metering of a specific volume of fluid (in a preferred embodiment, whole blood).

[0037] Diluent chamber 108 is suitably pre-filled with a diluent solution. In other embodiments, diluent chamber 108 is configured to receive a volume of diluent solution, e.g. diluent chamber 108 may be fluidly connected to a diluent source (e.g. a storage chamber or pouch) operable to dispense the diluent solution into diluent chamber 108; in one example, dilution and metering module 100 may include a breachable storage chamber or pouch, which may be breached by application of cap 102 or through other manipulation of the device. Other arrangements are possible and, in one embodiment, a known volume of diluent solution may be stored in a secondary chamber and moved into diluent chamber 108 by a pumping action on cap 102. In preferred embodiments, diluent chamber 108 is prefilled, preferably with a known volume of diluent solution.

[0038] In some embodiments, the top of cup 104 may be specifically adapted for blood collection e.g. by having a scoop or lancet (not shown), which may be integrally formed with the upper edge of cup 104 to facilitate collection of small amounts of blood. In some embodiments, the composition of the diluent solution is not particularly restricted and the reference to “diluent” simply reflects that the combined volume of the sample (i.e. original sample + diluent solution) will be greater and the concentration of whole blood in the sample will thus be diluted. However, in some embodiments, the diluent is a polyvinyl alcohol (PVA) containing diluent, embodiments of which are described in more detail below.

[0039] As described above, when flexible cap 102 is used to close / seal receiving inlet 106 of cup 104 after blood collection, a pressure is generated that pushes the blood past the capillary seal 114, i.e. through capillary channel(s) 112, and drives the blood into diluent chamber 108 where it interacts with the diluent solution. Repeated application of pressure on cap 102 (e.g. a repeated depression / compression or pumping motion) induces pressure changes within diluent chamber 108 that destabilizes the liquid and promotes mixing.

[0040] Vents 116a and 116b are fluidly connected to diluent chamber 108, suitably proximate the capillary channel(s) 112 i.e. the top of the diluent chamber 108 as dilution and metering module 100 would be typically oriented during use. Vents 116a and 116b prevent air entrapment that negatively affects movement. While embodiments shown in the figures have two vents, as will be apparent to a person of skill in the art, different configurations are possible e.g. a single vent, multiple vents or an annular vent. Vents 116a, 116b are suitably one-way vents in that they permit entrapped air to escape diluent chamber 108 in response to the pressure on cap 102 and filling of the diluent chamber 108, but do not permit air or contaminants to pass into diluent chamber 108.

[0041] Dilution and metering module 100 suitably includes one or more stabilizing structures that enable the device to be stably positioned in an upright orientation to facilitate sedimentation of the diluted sample. Suitable stabilizing structures may e.g. be legs 122a, 122b.

[0042] In embodiments shown in the figures, a second capillary seal 118 is present at the bottom of diluent chamber 108 (as dilution and metering module 100 would be typically oriented during use). Second capillary seal 118 prevents discharge of mixed fluid (sample + diluent solution) unless contact is made with an external surface. Contact with the external surface can lead to a pressure change enabling passage of the mixed sample through second capillary channel(s) 120 of second capillary seal 118. This can be further facilitated by an external surface with hydrophilic properties, as discussed further below. Suitably, second capillary seal 118 is placed slightly interior to the edge of the outer surface such that discharge is possible when dilution and metering module 100 is made to contact a paired component.

[0043] After mixing of the metered sample and diluent solution, metering and dilution module 100 may be positioned in the stabilized position on the external surface to breach second capillary seal 118. While in some embodiments, the metering and dilution module 100 may be positioned on the external surface immediately, in other embodiments, the metering and dilution module is positioned after a short rest period, suitably of less than 5 minutes, in some embodiments, the metering and dilution module 100 is positioned after 1-2 minutes.

[0044] As mentioned above, dilution and metering module 100 suitably includes one or more components for facilitating engagement with a paired component. In some embodiments, this paired component is a module for separating plasma from whole blood, embodiments of which are described in more detail below.

[0045] With reference to Figure 1 , in some embodiments, dilution and metering module 100 includes an inner engagement surface 124 and outer engagement surface 126.

[0046] In some embodiments, the shape of cup 104 and diluent chamber 108 are not restricted. However, in some embodiments one or both are cylindrical or conical shaped. In a preferred embodiment, both cup 104 and diluent chamber 108 are conical shaped, which has been found to facilitate the unidirectional movement of fluid i.e. from collection through diluent chamber 108.

[0047] In preferred embodiments, a removable airtight seal (not shown), e.g. a removable lid or peelable plastic seal e.g. an adhered or heat sealed polyethylene seal, closes engagement aperture 125. Suitably the airtight seal is applied prior to placing diluent in the diluent chamber 108. The airtight seal creates an airlock and works in cooperation with second capillary seal 118 to ensure that the diluent and sample are not inadvertently discharged from the module, which can be a problem with prior blood collection devices. Before the airtight seal is removed and pressure is applied, the diluted sample is trapped at the second capillary seal 118, and moves down from the capillary seal when both the airlock is removed, and a pressure is applied to make a large, heavy drop that can enable transfer. This reduces the risk of unintended contact with an external surface

[0048] The components of dilution and metering module 100, other than cap 102, are suitably manufactured using a material that is sufficiently rigid to maintain its structure when pressure is applied to cap 102 and that is non-reactive or minimally reactive with blood and does not alter the constituent concentrations of plasma. Suitably, the components are manufactured from a plastic. In some embodiments, one or more of the components of dilution and metering module 100 (including, optionally, the cap) may be fabricated using material that may affect blood or constituent concentrations to a level that may be mitigated, accounted for, and / or tolerated by a testing device or a processor that analyzes an output of same.

[0049] Cap 102 is suitably manufactured from a material that is sufficiently flexible to enable it to be pumped. Like the other components of metering and diluent module 100, cap 102 should be non-reactive or minimally reactive with blood and not alter the constituent concentrations of plasma.

[0050] Suitably, the components are manufactured from a plastic, suitably polypropylene.

[0051] In some embodiments, the diluted and metered blood sample is passed to any suitable plasma separation module.

[0052] In some embodiments, a separation module is as taught in International patent application publication no. WO2020142839 entitled “Blood Separation and Analysis Device and Methods” filed January 7, 2020, the contents of which are incorporated herein by reference in their entirety. Briefly, the separation module as taught therein, which is referred to herein as an aligning apertures fluid separation module (200), includes: an inner container 202 forming a sedimentation compartment 204 for receiving a sample of diluted fluid, inner container 202 having a sample receiving inlet port 206 and a first aperture 208; an outer container 210 for receiving the inner container 202 and having a second aperture 212; at least one of inner container 202 and outer container 210 being movable between a first configuration where outer container 210 seals first aperture 208 and a second configuration wherein first aperture 208 and second aperture 212 align to form an outlet for a separated component of the sample fluid. Inner container 202 and outer container 210 are configured and dimensioned to allow transition from a first position to a second position relative to each other. In one embodiment, inner container 202 can be rotated within outer container 210 to align the first aperture and the second aperture to form the outlet. Inner container 202 and outer container 210 can e.g. be conical or cylindrical in shape. In a first position, first aperture 208 (of the inner container) and second aperture 212 (of the outer container) are not aligned and outer container 210 seals first aperture 208 to prevent egress of fluid from sedimentation compartment 204. In a second position, first aperture 208 and second aperture 212 are aligned creating the outlet for egress of fluid from sedimentation compartment 204. Suitably the apertures (208, 212) are sized and / or positioned to limit egress to a component of the fluid, while preventing egress of the remainder of the diluted fluid sample. In one embodiment, the apertures (208, 212) are slots. It one embodiment, the outlet is positioned so that it will be positioned above a sedimented component of the fluid after separation (as aligning apertures fluid separation module 200 would typically be oriented in use). In one embodiment, the sample is whole blood and the cellular components are sedimented beneath the outlet, while plasma passes though the outlet. Optionally, inner container 202 may have a plurality of apertures alignable with a plurality of apertures on outer container 210. The aligning apertures fluid separation module 200 can further include a reaction module in fluid communication with the outlet for receiving the separated component of the fluid sample for subsequent analysis.

[0053] In another aspect of the invention, there is provided a filtration fluid separation module 300. An example of this module is shown according to an embodiment in Figure 6. The filtration fluid separation module 300 includes a sample chamber 302 having a sample chamber inlet 304 and a sample chamber outlet 306 having positioned therein a filter 308. While in some embodiments, the shape of sample chamber 302 is not specifically restricted, in preferred embodiments, sample chamber 302 is cylindrical or frustoconical in shape or includes a cylindrical or frustoconical portion with sample chamber outlet 306 positioned at one end of the cylinder or at the narrower end when the chamber or portion thereof has a frustoconical shape.

[0054] In one embodiment, sample chamber inlet 304 is configured to receive diluted fluid from second capillary channel(s) 120. In these embodiments, sample chamber wall 305 may cooperatively engage with inner engagement surface 124 of metering and dilution module 100.

[0055] Suitably, filter 308 has a pore size of 1pm to 20 pm, more preferably 2 pm to 15 pm, suitably a pore size of ~ 10 pm. In some embodiments, the filter material is not restricted other than that it is minimally or not reactive with the combined solution so as not to interfere with subsequent analysis of analytes contained in the separated portion (which in the preferred embodiment is plasma). Suitable filter materials can include cellulose and glass filber filters. An example of a suitable filter is Whatman™ Fusion 5 Filter Paper. Another example is a Vivid™ plasma separation membrane (Cytiva). The area of filter 308 is suitably selected to reflect the volume of fluid to be separated and in some embodiments each side of the filter has a surface area of between about 1 cm2and 25 cm2, preferably between about 10 cm2and 20 cm2.

[0056] Filter 308 suitably has a thickness between about 50 pm and about 500 pm, more preferably between about 100 pm and about 300 pm, in some embodiments about 200 pm.

[0057] The present inventors have found that agglutinating red blood cells (RBCs) allows the use of a filter having relatively large pores (suitably between about 1pm to 20 pm), which enables efficient separation of the agglutinated RBCs (the retentate) from the filtrate i.e. the plasma containing portion.

[0058] In some embodiments, plasma may be efficiently separated from whole blood using a filtration fluid separation module 300 and, preferably, diluent solutions as taught therein, in some embodiments, in 5 minutes or less, in 4 minutes or less, in 3 minutes or less, in 2 minutes or less or in 1 minute or less.

[0059] Suitably, the filtrate side of filter 308 is in direct contact with a hydrophilic surface 312 to pull the filtrate (in one embodiment plasma) through a capillary 314. Sample chamber wall 305 can suitably be formed of a hydrophilic material and / or the sample facing surface of sample chamber wall 305 may be coated with a hydrophilic coating.

[0060] In some embodiments, a rim 310 is positioned about and proximate or immediately adjacent the sample chamber outlet 306. Rim 310 may surround filter 308 or be positioned beneath filter 308. Suitably, rim 310 has a height of about half the thickness of filter 308. Rim 310 improves the performance of filter 308 by facilitating passage of sample onto filter 308.

[0061] Filtration fluid separation module 300 includes a locking mechanism to hold filter 308 in place, prevent leakage from the sides of filter 308 and provide hydrophilic contact with filter 308 to provide a conduit for the filtered plasma to exit the separation filter 308 using gravity. Suitable locking mechanisms include, but are not limited to: ultrasonic welding, deforming sample chamber wall 305 to hold the filter in place, and providing a mechanical structure to hold the filter in place e.g. a raised ring having a sharp edge

[0062] Exit of filtrate (preferably plasma) may be facilitate by a hydrophilic surface position adjacent filter 308 on the filtrate side. The filtered liquid, in a preferred embodiment plasma, contacts a transfer medium 322, suitably a transfer paper. Transfer medium 322 can be replaced with a structure that facilitates fluid transfer, e.g., in one embodiment, a structure designed to promote fluid transfer through capillary action. A suitable transfer paper is P8 filter paper from Fisherbrand. In another embodiment, the filter may be replaced with a deposited film that facilitates transfer (e.g. a sugar film). Transfer medium 322 suitably transfers the separated plasma to a reaction module, in one embodiment a digital microfluidic (DMF) chip 318.

[0063] In some embodiments, filtration fluid separation module has a diluted plasma yield (i..e. after hold-back volume) from whole blood of > 30%, > 40%, >50%, >60%, >70%, >80% or >90% (relative to the original diluted whole blood sample).

[0064] With reference to Figure 7, in one embodiment, filtration fluid separation module 300 is configured to operatively engage with DMF chip 318. Again with reference to Figure 7, DMF chip 318 includes a conductive top plate 320, transfer medium 322, a top hydrophobic layer 324, a bottom hydrophobic layer 326, a dielectric layer 328, a DMF reservoir electrode 330 and a DMF printed circuit board 332.

[0065] The filtration fluid separation module 300 is suitably received in an aperture in conductive top plate 320. In some embodiments, filtration fluid separation module 300 is integrally formed with or integrally connected to or adhered to top plate 320. In some embodiments, filtration fluid separation module 300 may be releasably connected to DMF chip 318, e.g. filtration fluid separation module 300 may be received in an aperture in a releasable push fit arrangement, which may be facilitated by biasing means (e.g. a spring) in or on wall 305. Transfer medium 322 (or an alternative structure designed to promote fluid transfer through capillary action) brings the separated plasma in contact with the top and bottom hydrophobic layers 324, 326 of the DMF and establishes electrical contact with the DMF system. Following electrical contact, electrowetting is used to draw the plasma out from transfer medium 322 and onto DMF chip 318. In some embodiments, the electrowetting components and methods may be those described in provisional patent application 63 / 552,990, filed February 13, 2024, and international patent application PCT / CA2025 / 050191, filed February 13, 2025, both having the title DEVICES AND METHODS FOR BLOOD ANALYSIS USING ELECTROWETTING-ON- Dl ELECTRIC and having the same applicant as the present application. In the absence of the transfer medium 322, the exit of the filtration fluid separation module 300 acts as a capillary lock and prevents flow of liquid into the DMF 318.

[0066] Also provided is a novel method of separating plasma from whole blood. Specifically, there is provided a method for sedimentation of blood using PVA. This can be used to facilitate and / or enable measurements or testing of one or more analytes in blood plasma, for example. While in some embodiments, this method of sedimentation may be used in association with metering and dilution module 100 and optionally a fluid separation module 200 or 300, preferably 300, as described herein, in other embodiments, this novel method may be applied independent of any specific device as described herein.

[0067] This method for separating components in blood via sedimentation is particularly suited for microfluidic plasma separation.

[0068] The present inventors have surprisingly found that PVA of a specific molecular weight range may be used in small quantities for the efficient sedimentation of plasma from whole blood and, in particular, for the efficient separation of microfluidic quantities of plasma from whole blood. Further, the diluent may be formulated to facilitate subsequent processing on a DMF system and, in particular, to facilitate movement of a sample using electrowetting techniques, while enabling efficient agglutination of RBCs.

[0069] Accordingly, in one embodiment, there is provided a method of treating whole blood for the purpose of plasma separation that separates plasma from whole blood based on size exclusion, preferably, filtration. The sample or portion thereof is sedimented using PVA, a non-toxic, highly hydrophilic polymer. In some embodiments, the diluent includes a buffer such as one or more of a phosphate buffered saline (PBS) at physiological pH (i.e., ~ 7.4) and a tris(hydroxymethyl)aminomethane (TRIS) buffered saline at physiological pH (i.e., ~ 7.4). In an example, a buffer of the diluent can have a pH at or between 7.5 and 9. Preferably, the diluent further comprises a surfactant.

[0070] In a preferred embodiment, a PVA based diluent is used in combination with Whatman™ Fusion 5 Filter Paper.

[0071] In some embodiments, the diluent includes PVA, phosphate buffered saline (PBS) and a surfactant. A particularly preferred surfactant is Tetronic™, an ethoxylated and propoxylated ethylene diamine. In one embodiment, the diluent comprises 1x PBS, 0.1% - 0.5% Tetronic™ T904 / T904R (BASF), more preferably about 0.2% surfactant, and ,5%-2% PVA, preferably 1% PVA.

[0072] Suitably, the diluent is added to whole blood with volumes of about 50 pl-70 pl : 30 pl-50pl diluent : blood, preferably about 60 pl : 40 pl diluent : blood. For example, the sample is diluted by one part diluent to one part blood.

[0073] In preferred embodiments, the PVA has a molecular weight of 50,000-250,000 Daltons, in one embodiment, 50,000 to 205,000 Daltons, in one embodiment 205,000 Daltons and the PVA 70- 100% hydrolyzed, in one embodiment 88% hydrolyzed (e.g., Mowiol 40-88 from Sigma). This method is suitable for sedimenting small volumes of whole blood and, in particular, volumes less than 5 ml, including volumes less than 1 ml, and less than 0.5 ml. The use of a PVA-based diluent as described above enables complete or substantially complete sedimentation of cellular material from whole blood in a matter of minutes, and in the case of sample volumes described here, less than 5 minutes.

[0074] Agglutination may be facilitated by physical displacement, for example, by inversion or movement of a sample container. This can be further facilitated by a mixing structure (e.g., mixing ball bearing) contained within the container. The mixing structure is suitably denser than the components to be mixed without taking up much volume.

[0075] The sedimented sample or portion thereof is separated to remove the plasma component by a size exclusion means, suitably filtration with a filter pore size of 2 pm to 15 pm, suitably a pore size of ~ 10 pm, which excludes red blood cells as agglutinated using the diluents as described from the remaining blood components. By enabling the use of a filter having a relatively large pore size i.e. that would not otherwise be suitable for plasma separation, challenges associated with known filtration methods may be avoided. The separated plasma may optionally be further filtered.

[0076] The filtered components (e.g., filtrate) can be used in analyses, for example, that detect a quantity of one or more components. These analyses may involve chemi- or bioluminescence reactions or electrochemistry techniques for example. Other examples can include spectroscopy, spectrometry, photometric, fluorescence, or other analytical quantification techniques. In this way, one or more components in a sample of blood can be detected and / or measured. This description is made with reference to the separation of plasma from whole blood, however, it is to be understood that devices provided herein may be used to separate any suitable filtrate.

[0077] In addition to separation of plasma from whole blood, methods of separation using PVA and separation devices according to the present invention can be used in various diagnostic and other medical applications.

[0078] It will be apparent to those skilled in the art that various modifications and variations may be made in the materials, devices and methods disclosed herein. It will be understood that elements of embodiments are not necessarily mutually exclusive, and many embodiments can suitably combined with other embodiments.

[0079] The examples described above and illustrated are intended to be exemplary only. The description shall be understood to encompass all equivalents.

[0080] Parts List: metering and dilution module 100 cap 102 cup 104 receiving inlet 106 diluent chamber 108 seal 110 first capillary channel 112 first capillary seal 114 vents 116a, 116b second capillary seal 118 second capillary channel 120 stabilizing legs 122a, 122b inner engagement surface 124 engagement aperture 125 outer engagement surface 126 aligning-apertures fluid separation module 200 inner container 202 sedimentation compartment 204 sample receiving inlet port 206 first aperture (inner container) 208 outer container 210 second aperture (outer container) 212 filtration fluid separation module 300 sample chamber 302 sample chamber inlet 304 sample chamber wall 305 sample chamber outlet 306 filter 308 rim 310 hydrophilic surface 312 capillary 314 digital microfluidic (DMF) chip 318 conductive top plate 320 transfer medium 322 top hydrophobic layer 324 bottom hydrophobic layer 326 dielectric layer 328

[0081] DMF reservoir electrode 330

[0082] DMF printed circuit board 332

Claims

WHAT IS CLAIMED IS:

1. A fluid metering device comprising: a cup having an inlet for receiving the fluid; a flexible diaphragm manipulable to increase pressure in the cup above a first threshold pressure; a diluent chamber; and one or more capillary channels fluidly connecting the cup to the diluent chamber, the one or more capillary channels operable to drain the fluid from the cup into the diluent chamber when pressure in the cup exceeds the first threshold pressure.

2. The device of claim 1, further comprising a vent for releasing gas from the diluent chamber when it receives the drained fluid.

3. The device of claim 1 or 2, wherein the flexible diaphragm is configured to releasably close the inlet, preferably wherein the diaphragm is a flexible cap for releasably sealing the cup inlet.

4. The device of any one of claims 1 to 3, wherein the diluent chamber contains a known volume of diluent or is configured to receive a known volume of diluent, preferably wherein the diluent comprises polyvinyl alcohol (PVA).

5. The device of any one of claims 1 to 4, further comprising one or more second capillary channels in a wall of the diluent chamber distal the one or more capillary channels and operable to drain fluid from the diluent chamber when pressure in the diluent chamber exceeds a second threshold pressure.

6. The device of claim 5, wherein each of the one or more second capillary channels comprises a channel inlet on an interior surface of the wall of the diluent chamber and a channel outlet on an exterior surface of the wall and wherein the device further comprises an engagement mechanism on the exterior surface of the wall to fluidly connect the one or more second capillary channels with a fluid separation module.

7. The device of claim 6, wherein the engagement mechanism comprises a wall descending from the exterior surface and defining an engagement recess for receiving a portion of the fluid separation module and wherein the channel outlet(s) are in the engagement recess.

8. The device of claim 7, further comprising a removable seal for sealing a mouth of the engagement recess before use.

9. The device of any one of claims 1 to 8, further comprising a fluid separation module.

10. A fluid separation device for separating plasma from whole blood comprising: a filtration chamber for receiving a volume of whole blood and a volume of agglutinator, wherein the volume of agglutinator agglutinates red blood cells of the volume of whole blood to red blood cell containing particles having a minimum average diameter of 10 pm or greater within 10 minutes or less, preferably within 5 minutes or less; an outlet in a wall of the filtration chamber; a filter having a minimum average pore diameter of between 5 pm and 15 pm secured over the filtration chamber outlet.

11. The device of claim 10, wherein the filtration chamber comprises an inner wall surface and an outer wall surface, wherein the filter is secured on the inner wall surface.

12. The device of claim 10 or 11 , wherein the filtration chamber outlet comprises a hydrophilic surface.

13. A system comprising the fluid metering device of any one of claims 1 to 8 and the fluid separation device of any one of claims 10 to 12, wherein the diluent chamber is fluidly connected to the filtration chamber by the one or more second capillary channels.

14. The system of claim 13, wherein the filtration chamber is fluidly connected to a DMF chip via the filtration chamber outlet.

15. A method for separating plasma from a volume of whole blood comprising: combining the sample of whole blood with a diluent solution comprising an agglutinator, preferably wherein the agglutinator comprises polyvinyl alcohol (PVA); and allowing agglutination of red blood cells to proceed for a time sufficient to form an agglutinated sample comprising agglutinated red blood cell particles having a minimum average diameter of 10 pm or greater; and passing the agglutinated sample through a filter having an average pore size between 5 pm and 15 pm.

16. The method of claim 15, wherein the time sufficient to form an agglutinated sample is a period of 30 seconds to 10 minutes, preferably 1 to 5 minutes.

17. The method of claim 15 or 16, wherein the agglutinator comprises PVA, optionally wherein: the diluent comprises: phosphate buffered saline (PBS);0.5 % to < 2%, preferably about 1% weight / volume concentration of PVA;0.1% - 0.5%, preferably 0.2% weight / volume of a surfactant; preferably an ethoxylated and propoxylated ethylene diamine; optionally wherein: the PVA has a molecular weight between 50,000 and 250,000 Daltons; and / or the PVA is 70% to 100% hydrolyzed, preferably 86% to 90% hydrolyzed, more preferably 88% hydrolyzed.

18. The method of claim 17, wherein the concentration of an analyte is determined in the diluted plasma.

19. A composition for agglutinating components of a biological fluid comprising: phosphate buffered saline (PBS);0.5 % to < 2%, preferably about 1% weight / volume concentration of PVA;0.1% - 0.5%, preferably 0.2% weight / volume an ethoxylated and propoxylated ethylene diamine; optionally wherein: the PVA has a molecular weight between 50,000 and 250,000 Daltons; and / or the PVA is 70% to 100% hydrolyzed, preferably 86% to 90% hydrolyzed, more preferably 88% hydrolyzed.

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