A plasma collection device

Abstract

A plasma collection device (1) comprising: a housing (1a) accommodating: a microcapillary tube (2) for collecting a volumetric quantity of blood by capillary action; a membrane separator (4) in fluid communication with the microcapillary tube (2) for separating plasma from said volumetric quantity of blood; a collector (7) for separated plasma; and optionally, a desiccant (8) for drying separated plasma inside the device (1).

Description

A PLASMA COLLECTION DEVICETECHNICAL FIELD

[0001] The present invention relates to a plasma collection device.BACKGROUND ART

[0002] The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

[0003] A blood sample is often collected from a subject to analyse a blood dried component, in particular plasma, for diagnostic reasons. In general, these blood samples are of a small volume and require careful collection and handling.

[0004] The plasma from a sample of whole blood collected from a subject is often required for analysis. Plasma is usually prepared by collecting whole blood into commercially available anticoagulant-treated tubes e.g., EDTA-treated, or citrate-treated tubes, usually with a relatively large volume. Heparinized tubes are indicated for some applications. However, heparin can often be contaminated with endotoxin, which can stimulate white blood cells to release cytokines. Cells are removed from plasma by centrifugation using a refrigerated centrifuge. The resulting supernatant is designated plasma. Following centrifugation, it is considered important to immediately transfer the plasma into a clean polypropylene tube, using a pipette. Plasma samples should be maintained at a temperature of between 2-8°C while handling. If the plasma is not analyzed immediately, the plasma should be stored, and transported at -20°C or lower. It is also important to avoid freeze-thaw cycles. Samples which are hemolyzed, icteric, or lipemic can invalidate certain tests.

[0005] There are presently three commercially available devices for the collection of dried plasma from a subject, and in particular, a human subject that require a smaller sample size. These devices are generally used to collect a small volume of blood from a subject.

[0006] One of these devices is the Telimmune™ card (formally Noviplex™ card), which uses an asymmetric membrane to separate a volumetric plasma sample from a non-volumetric whole blood sample. The card is available in two versions: UNO, for the collection of one 2.5 μL plasma spot, and DUO, for the collection of two 3.8 μL plasma spots (Shimadzu, 2022). The Telimmune™ UNO card requires at least 25 μL of blood for separation while the DUO card requires at least 60 μL. These volumes are higher than what is generally obtained from a single blood drop, indicating that the subject is required to produce multiple drops to properly load either of the UNO or DUO Telimmune™ cards. The plasma collection disc(s) are situated underneath the separation membrane, attached to a base card. After blood application, approximately three minutes is required to separate plasma, after which the top layer is peeled off and discarded. The DPS discs then needs to dry for an additional 15 minutes, after which they can be packaged and transported to the laboratory for analysis. Thus, the amount of blood required is relatively high, and the time required for generation of a plasma disc is also high which may be unsuitable for longitudinal studies where samples need to be collected quickly and easily (e.g., at a professional sporting facility) (Li et al., 2014).

[0007] The second type of blood collection device is the HemaSpot SE (Spot on Sciences), which is a device featuring a spiral-shaped membrane designed for lateral separation of plasma from whole blood (Kaduskar et al., 2021 ). Plasma separation is achieved by adding four or five large blood drops (-150 μL ) to the centre, with the spiral-shaped membrane allowing the separation of the red blood cells through lateral flow along the spiral (Kaduskar et al., 2021 ). Given the large volume required to adequately load the Hemaspot SE, the device may be unusable in some circumstances, such as the use by unsupervised or untrained participants. After drying for two minutes, the sample cartridge is closed and sent to the laboratory for analysis. Depending on the desired sample (e.g. whole blood, or cell free plasma), a sub-punch can be made in different parts of the spiral. To date, there has only been a single, independent peer reviewed publication that has tested the performance of the Hemaspot SE. Here, the authors noted that the device showed considerable variability in results compared to the corresponding serum sample, and that concentration is dependent on where the filter paper is punched (Kaduskar et al., 2021). The authors also noted that there were challenges in determining an appropriate location to punch to collect plasma samples which may indicate haemolysis on the paper, masking the presence of a distinct plasma region (Kaduskar et al., 2021 ).

[0008] The third device is the Roche Cobas card, which is similar in design to theTelimmune card but is specifically designed for HIV plasma viral load testing in conjunction with the Roche cobas® 6800 / 8800 Systems (Velasquez-Orozco et al., 2021 ). The Cobas plasma separation card collects approximately 5-6 drops (-140 μL) onto a porous membrane which allows only plasma to pass and collect on an underlying polyester fleece (Velasquez-Orozco et al., 2021 ). As the COBAS card is intended for HIV viral load testing, the fleece is saturated with an RNA-stabilizing reagent. After drying at room temperature overnight, the top cover of the card is removed and the disc containing a large volume of separated plasma is placed into a test tube for elution of the sample. Similar to the Hemaspot SE, the Roche Cobas card requires a relatively large volume of the blood sample, which likely makes its use by untrained or unsupervised participants in the field or home difficult. The requirement for overnight drying of the sample is also likely to restrict the usefulness of the card as it makes sample collection a lengthy process. Images from a study using the Roche Cobas card show significant haemolysis present of the plasma separation surface (Velasquez- Orozco et al., 2021). Thus, it is possible that the separation membrane is unsuitable for separating red blood cells from the volume of blood that is being applied or may indicate that overloading of the membrane was an issue during this study.

[0009] The plasma collection devices discussed above are considered to be unsuitable for detecting or monitoring important components, including biomarkers in a blood sample. For example, none of the devices are able to monitor the oxidation level of albumin in a plasma sample. Firstly, the available devices require a volume of blood that is larger than the volume capable of being produced from a single blood drop, (approximately 15 μL). This suggests that the commercially available devices require the subject to generate multiple blood drops to sufficiently load the device. Obtaining multiple drops requires “milking” the finger (i.e. applying pressure in a continuous motion from the base to the tip), sometimes forcefully to produce extra blood. This is not always possible by subject without the supervision of a phlebotomist or nurse (Hall et al., 2020). For example, a study has found that 50% of subjects had trouble collecting sufficient blood (-65 μL ) required to load the HemaSpot HF (whole blood version of the HemaSpot SE) (Hall et al., 2020). Excessive milking of the finger is not preferable as it has been shown to cause haemolysis and contamination of the blood with interstitial and intracellular fluid (Fliervoet et al., 2022; Krleza et al., 2015; WHO, 2010). Additionally, if the required volume cannot be produced, multiple fingers may have to be lanced which may cause discomfort for the participant (Serafin et al., 2020).

[0010] Thus, none of plasma devices that are commercially available have the capability to collect a volumetric blood sample prior to separation and thus are susceptible to overloading or as may be more likely owing to their relatively high blood volume requirement, underloading. Non-volumetric blood collection is of concern as underloading may result in little or no plasma generation and overloading would result in haemolysis. Both situations present an opportunity for user error that may result in the collection of samples that are not suitable for analysis.

[0011] Therefore, there is a need to develop a plasma collection device that collects a small, volumetric blood sample for separation and collection of plasma.SUMMARY OF INVENTION

[0012] The device of the present invention is designed to facilitate collection of plasma samples from a subject, either in the field, in a hospital setting, or in the home.

[0013] In one aspect, the present invention provides a plasma collection device comprising: a housing accommodating: a microcapillary tube for collecting a volumetric quantity of blood by capillary action; a membrane separator in fluid communication with the microcapillary tube for separating plasma from said volumetric quantity of blood; a collector for separated plasma; and optionally, a desiccant for drying separated plasma inside the device.

[0014] The plasma collection device conveniently comprises a housing having a body portion and a movable portion, the movable portion being movable relative to the body portion. The movable portion is preferably a clip portion conveniently comprising a body connected at one end to a plurality of prongs and at the other end to a pusher or handle. The prongs may be co-operable with slots provided in the body portion for clipping clip portion and body portion together. The prongs may be provided at each end with hook portions co-operable with the slots of the body portion.

[0015] Conveniently compression of the prongs allows movement of the clip portion from an unclipped position, in which the microcapillary tube does not supply blood to the membrane separator, toward the body portion and a clipped position in which the microcapillary tube is in fluid communication with the membrane separator for extraction of plasma from collected blood.

[0016] In one embodiment, the movable portion holds the microcapillary tube enabling filling with blood when the movable portion is in unclipped position. In the unclipped position, the microcapillary tube does not supply blood to the membrane separator. Movement of the movable portion toward the body portion and into a clipped position brings the microcapillary tube into fluid communication with the membrane separator for extraction of plasma from collected blood.

[0017] The microcapillary tube advantageously has capacity to collect a volumetric blood sample, preferably a single drop of blood with volume in the range 5 to 30 μL, advantageously collecting less than 20 μL of blood, most advantageously collecting between 10 μL and 15 μL of blood. Such a small volume has the advantage of easy sample collection while avoiding or preventing underloading or overloading of the membrane separator.

[0018] The membrane separator comprises at least one separation membrane having an inlet surface and an exit surface, desirably having a highly asymmetric pore structure, suitable for separating plasma from a small blood volume advantageously by drawing blood across the membrane(s) by capillary action. An asymmetric pore structure contains decreasing pore sizes from an inlet surface (conveniently on the top of the membrane) through to the exit surface (conveniently on the bottom of the membrane) and is capable of trapping red blood cells within the membrane whole allowing plasma to pass through. Polysulfone and glass fibre membranes are most suitable.

[0019] The membrane separator may comprise a plurality of membranes wherein a membrane proximate an outlet of the microcapillary tube is defined as an inlet membrane.

[0020] The membrane(s) may be treated prior to inclusion within the device to minimise reactions, such as oxidation which may interfere with later sample analysis, andhemolysis. Membrane treatment may include steps selected from the group consisting of washing, coating, applying saline solution and drying.

[0021] Advantageously, the plasma collection device further comprises means for cooperating the microcapillary tube with the membrane separator. In one embodiment, a closure of the plasma collection device may be configured to act as a plunger pressing the micro-capillary tube into co-operation with the membrane separator. The closure may include prongs that engage with corresponding apertures in the membrane separator.

[0022] When in co-operation with the membrane separator, the device is configured to maintain an air gap of selected dimension between an outlet of said microcapillary tube and said inlet surface of an inlet membrane included in said membrane separator. The air gap is intended to facilitate unloading of blood from the microcapillary tube to the membrane separator. Dimension of the air gap is selected to optimise the unloading of blood from the microcapillary tube with desired loading of the membrane(s) included within the membrane separator. A desired loading of the membrane(s) with blood depends on factors including efficient utilisation of a membrane surface and avoidance of haemolysis.

[0023] Optionally, the microcapillary tube may be provided with a hydrophobic portion, conveniently a bottom portion of the microcapillary tube proximal the membrane separator. The portion may, for example, be made hydrophobic by coating with a hydrophobic compound. The hydrophobic compound may be selected, without limitation, from the group consisting of silicone resins, acrylic copolymers, polyurethanes, bituminous and asphalt compounds, teflon, polyester resins, fibreglass, polystyrene, calcium carbonate, fluorinated silanes and fluoropolymer coatings.

[0024] The collector for separated plasma preferably includes a wicking means, conveniently a wicking pad, which is desirably impregnated with a conjugating reagent, such as maleimide polyethylene glycol (mal-PEG), to prevent reactions - such as oxidation - that could interfere with later analysis of the plasma sample. A conjugating reagent may also be applied to the membrane(s) of the membrane separator subject to taking account of any issues of sample degradation that may arise.

[0025] The plasma collection device may further comprise a bypass of blood, exceeding a selected volume. Such a feature allows overloading of the plasma separation membrane to be avoided and the selected volume is such as to avoid such overloading. An excess volume of blood may be an issue where the source of blood is not removed from the capillary after filling. For example, where blood is taken from a patient’s finger, failure to remove the finger from the microcapillary tube after filling, there may be potential for overfilling where excess blood collects at the base of the capillary overloading the membrane(s) of the membrane separator.

[0026] Conveniently, the desiccant is silica gel, preferably a silica gel with a high surface area to weight ratio.

[0027] The device advantageously includes a closure that seals the device, particularly in terms of being air-tight and water-tight. This avoids disadvantages such as water or water vapour inactivating the conjugating reagent, leading to rapid protein degradation and subsequently poor quality analysis of the plasma sample.

[0028] The device may include an anti-coagulant, such as EDTA or heparin, provided inside the entirety of the microcapillary. Anticoagulant can enhance the rate of blood flow through the membrane separator and therefore improve plasma separation in the membrane separator.

[0029] The plasma collection device and its components may be fabricated by 3D printing. Sterilisation using chemical or UV processes conveniently follows fabrication.

[0030] In another aspect, the present invention provides a method of collecting blood for biomarker assay comprising: collecting a sample of blood in a plasma collection device comprising: a housing accommodating: a microcapillary tube for collecting a volumetric quantity of blood by capillary action; a membrane separator co-operable with the microcapillary tube for separating plasma from said volumetric quantity of blood; a collection pad for the drying of the plasma component of the blood sample and;optionally, a desiccant for drying separated plasma; and assaying a biomarker present within said separated plasma.

[0031] A range of biomarkers may be assayed using the method, assay potentially allowing user of simpler and faster assay methods. In one embodiment, the biomarker is for oxidative stress, advantageously albumin thiol oxidation. If plasma is collected using the plasma collection device, albumin thiol oxidation may be analysed with capillary electrophoresis which is simpler, faster and more cost effective than using dried blood spots (DBS) techniques, such as DBS with Sodium dodecyl-sulfate polyacrylamide gel electrophoresis.

[0032] The plasma collection device of the present invention can advantageously collect a small volume of blood, typically equivalent to a drop of blood, which increases the likelihood of successful sample collection.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Further features of the plasma collection device and method of use of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:

[0034] Figure 1 (a) is a schematic side view of a plasma collection device according to a first embodiment of the present invention and Figure 1 (b) is a photograph providing a side view of the plasma collection device of Figure 1(a)

[0035] Figure 2A is a schematic side view of the plasma separation membrane and supports for the microcapillary tube of the plasma collection device of Figures 1 (a) and 1 (b).

[0036] Figure 2B is a schematic top view of the microcapillary tube and plasma separation membrane of Figure 2A.

[0037] Figure 3A is a schematic view of VIVID GF, a polysulfone membrane (Pall Life sciences) prepared in a sandwich format as recommended by the manufacturer.

[0038] Figure 3B illustrates the plasma separation performance of the VIVID GF membrane.

[0039] Figure 4A illustrates vertical plasma separation performance of the Ahlstrom CytoSep membrane.

[0040] Figure 4B illustrates lateral plasma separation performance on Ahlstrom CytoSep 1660 membrane (strip 1) and the Whatman Fusion 5 membrane (strip 2).

[0041] Figure 5 is a schematic representation of the plasma separation by the Ahlstron CytoSep membrane using (A) vertical technique and (B) lateral technique.

[0042] Figure 6 is a photographic illustration showing (a) a microcapillary tube used in a first embodiment of the present invention and (b) a microcapillary tube with a hydrophobic portion used in a second embodiment of the present invention.

[0043] Figure 7 is a photographic illustration showing (a) a microcapillary tube as shown in Figure 6(b) filled to capacity with blood and (b) a microcapillary tube as shown in Figure 6(a) with a droplet of blood in excess of capacity of the capillary tube.

[0044] Figure 8 a top view of a plasma collection device according to a second embodiment of the present invention and in the form of a clip in a first unclipped position.

[0045] Figure 9 is a top view of the plasma collection device of Figure 8 in a second clipped position.

[0046] Figure 10 is a top view of a body portion of the plasma collection device of Figures 8 and 9.

[0047] Figure 11 is a front orthogonal view of the portion of the plasma collection device of Figures 8 to 10.

[0048] Figure 12 is a top view of a clip portion of the plasma collection device of Figures 8 to 11 in assembly with the microcapillary tube used in embodiments of the present invention.

[0049] Figure 13 is a detail top view of the clip portion of Figure 12 showing the microcapillary tube pressing on flexible supports of the clip portion.

[0050] Figure 14 is a top view of the plasma collection device shown in Figures 8 to 10 in assembly with the microcapillary tube used in embodiments of the present invention.

[0051] Figure 15 is a top view of the plasma collection device of Figures 8 to 10 in unclipped position and schematically showing the microcapillary tube and blood flow direction.

[0052] Figure 16 is a top view of the plasma collection device of Figures 8 to 10 in clipped position and schematically showing the microcapillary tube and blood flow direction.

[0053] Figure 17 is a graphical representation of the effect of malPEG concentration on the proportion of labelled albumin thiols.

[0054] Figure 18 is a schematic illustration of the thiol disulfide exchange reaction involved in the labeling of reversibly oxidised albumin Cys34 with malPEG.

[0055] Figure 19 is a graphical illustration of the effect of cysteine concentration (graph A) and malPEG concentration (graph B) on the proportion of labeled albumin thiols.

[0056] Figure 20 is a photographic illustration of an SDS-PAGE gel image of plasma obtained through conventional centrifugation and plasma dried onto Whatman Fusion 5 membranes.

[0057] Figure 21 illustrates the effect of malPEG treatment of Fusion 5 membrane on plasma separation.

[0058] Figure 22 illustrates the effect of wicking plasma onto a Whatman 903 wicking pad positioned below a Fusion 5 membrane.

[0059] Figure 23 is a graphical illustration of the effect of the wicking pad diameter on plasma protein stability.

[0060] Figure 24 is a graphical illustration of the washing of Whatman Fusion 5 membranes on the artificial irreversible (A) and reversible (B) oxidation of albumin.

[0061] Figure 25 is a graphical illustration of the optimization of malPEG labelling of albumin on Whatman 903 wicking pads.

[0062] Figure 26 is a photographic illustration of the shape and dimensions of the Fusion 5 membrane for the optimal separation of 10 μL of blood with minimal haemolysis.

[0063] Figure 27 is a photographic illustration of the effect of 150 or 250 mM NaCI and 10% (w / v) sucrose treatments on plasma separation and haemolysis.

[0064] Figure 28 is a graphical illustration of the optimization of protein elution time from plasma dried onto wicking pads.DESCRIPTION OF PREFERRED EMBODIMENTS

[0065] For convenience, the following sections generally outline the various meanings of the terms used herein. Following this discussion, general aspects regarding compositions, use of medicaments and methods of the invention are discussed, followed by specific examples demonstrating the properties of various embodiments of the invention and how they can be employed.Definitions

[0066] The meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0067] Those skilled in the art will appreciate that the plasma collection device of the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[0068] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. None of the cited material or the informationcontained in that material should, however be understood to be common general knowledge.

[0069] Manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0070] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0071] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1 %.

[0072] The invention described herein may include one or more range of values (e.g. size, concentration etc.). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. For example, a person skilled in the field will understand that a 10% variation in upper or lower limits of a range can be totally appropriate and is encompassed by the invention. More particularly, the variation in upper or lower limits of a range will be 5% or as is commonly recognised in the art, whichever is greater.

[0073] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term “portion” can include part of a moiety or the entire moiety.

[0074] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0075] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0076] Features of the invention will now be discussed with reference to the following non-limiting description of preferred embodiments made with reference to the figures.DESCRIPTION OF PREFERRED EMBODIMENTS

[0077] Referring to Figures 1 (a) and 1 (b), a first embodiment of plasma collection device 1 comprises a housing 1 a accommodating a microcapillary tube 2 for collecting a determined volumetric quantity of blood by capillary action. Membrane separator 5 is in fluid communication with the microcapillary tube 2 for separating plasma from said volumetric quantity of blood. Housing 1a also accommodates a collector 7 for separated plasma; and a desiccant 8 for drying separated plasma inside the plasma device 1 .

[0078] Housing 1a may be a screw cap test tube of, for example, 5 ml_ capacity. Housing 1a holds a number of components as described below.

[0079] The microcapillary tube 2 is desirably of glass and has a maximum volumetric capacity of 10 μL blood. It is held within the housing 1a by a rotatable support 1 b interlockable or clippable with the membrane separator 5.

[0080] Membrane separator 5 includes a separation membrane 5a, as schematically shown in Figures 2A and 2B having an inlet surface and an exit surface. Membrane 5a has a highly asymmetric pore structure, suitable for separating plasma from a 10 μL blood sample when collected in microcapillary tube 2. An asymmetric pore structure contains decreasing pore sizes from an inlet surface (not shown) on the top of the membrane 5 through to the exit surface (not shown) on the bottom of the membrane 5.

[0081] Polysulfone and glass fibre membranes are most suitable for use as separation membrane 5a. A suitable membrane 5a is available from Whatman under the trade name Fusion 5 and is composed of latex bound glass fibres.

[0082] The separation membrane 5a may have shape selected to maximise plasma yield and a triangular shape 7.2 mm (L) x 7.0 mm (W) membrane.

[0083] Device 1 includes support 1 b, as described above, enabling microcapillary tube 2 to be held - when in co-operation with membrane separator 5 - leaving an air gap between them to assist capillary unloading. The microcapillary tube 2 has an inner diameter of approximately 660 μm while a Whatman Fusion 5 membrane has a thickness of 370 μm , thus covering approximately 55% of the capillary inner diameter leaving 145 μm of open capillary 2a on each side as shown in Figure 2B. This spacing facilitates proper unloading of blood from microcapillary tube 2.

[0084] Channels 6 are disposed between the membrane separator 5 and the inside wall of the housing 1 a of the device. Channels 6 are provided to allow excess blood to bypass the membrane separator 5 avoiding overloading.

[0085] Plasma collection device 1 includes a closure conveniently made by container 50, which allows the device 1 to be maintained water-tight and air-tight. Collector 7 includes a wicking pad 10 impregnated with maleimide polyethylene glycol (malPEG) which, in embodiments, slows down the rate of degradation of protein degradation. As an example, a wicking pad 10 of at least 10mm diameter may be selected. 5 μL of 10 mM malPEG may be impregnated onto the wicking pad 10.

[0086] The wicking pad 10 has a cellulose composition (for example being a filter paper, for example a Whatman 903 wicking pad of 10 mm diameter. Without wishing to be bound by theory, since cellulose is highly hygroscopic and porous, the filter paper may act as a desiccant drawing water away from the plasma sample, helping to preserve the plasma sample. Cellulose fibres have a large surface area that is comparable to that of silica gel crystals (estimated to be 100-200m2 / g). However, cellulose is documented to be able to absorb water up to 24-27 times its own weight, while silica gel can only absorb 0.4 times its own weight which would make cellulose a superior desiccant. Cellulose may also be appropriate in situations where only a small number of silica gel crystals can be included due to their large size and non-uniform shape.

[0087] In a case where albumin is to be detected, the measurable albumin signal may be up to 4 weeks.

[0088] The water-tight and air-tight closure 50 minimises loss of malPEG reactivity. Nevertheless, users of device 1 should be wary of exposing the plasma collection device 1 to atmosphere prior to sample collection.

[0089] A method for using plasma collection device 1 will now be described.

[0090] To collect blood, the screw cap 19 of container 50 is removed and placed aside. A fingertip is lanced by a user of the plasma collection device 1 to produce a hanging drop of blood. The fingertip of the user is gently touched to the top 11 of plasma collection device 1 to collect blood, by capillary action, through inlet 2a of microcapillary tube 2. This step may be assisted by holding the plasma collection device 1 at an angle, for example 45 degrees.

[0091] The user may remove their fingertip from the inlet 2a of the microcapillary tube 2 on visually verifying that the microcapillary tube 2 is full, indicating that a 10pL sample of blood has been collected. Any excess blood may be removed through the channels 6 disposed between the membrane separator 5 and the housing 1a of the plasma collection device 1 to avoid overfilling.

[0092] As described above, screwcap 19 has two prongs 19a. The user inverts screwcap 19 so that the two prongs 19a face down allowing insertion into corresponding holes (not shown) on the top 11 of plasma collection device 1 . The screwcap 19 is then twisted while applying downward pressure. Conveniently, a click or other sound indicates correct use. This action brings the blood filled capillary tube 2 into contact with membrane 5 to allow blood to flow through it by capillary action.

[0093] The separation membrane 5a in Figure 2A retains red blood cells and separated plasma passes through the exit surface of membrane 5a and onto the wicking pad 10. With the triangular shape of membrane 5a, as described above, plasma may be separated in high yield. Dependent on haematocrit level, the indicative plasma separation is as tabulated below:

[0094] Desiccant at the base 12 of the plasma collection device 1 allows for drying of plasma separated onto the wicking pad 10 (figure 1 B).

[0095] Referring to Figures 6 and 7, further advantage in terms of limiting overfilling of capillary tube 2 may be achieved by physically blocking the microcapillary tube 102 with a hydrophobic material. Such a microcapillary tube 102A is provided at one end with hydrophobic end cap 102B as shown on the right hand side (b). A microcapillary tube 102, the same in design as microcapillary tube 2, is shown on the left hand side (a) of Figure 6. An air gap is required to enable microcapillary tube 102A filling and can be achieved by puncturing or otherwise forming a very small hole into the hydrophobic end cap 102B (e.g. by using a fine 30G needle). Hydrophobic compunds that could be used for the hydrophobic end cap 102B include silicone resins, acrylic copolymers, polyurethanes, bituminous and asphalt, teflon, polyester resin, fiberglass, polystyrene, calcium carbonate, fluorinated silanes, and fluoropolymer coatings.

[0096] As shown in Figure 7(a), there is no excess blood, i.e. overfilling of microcapillary tube 102A, when hydrophobic end cap 102B is included at one end. On the other hand, as shown in Figure 7(b), an excess of blood 102D, i.e. overfilling of microcapillary tube 102, is likely when the hydrophobic end cap 102B is absent.

[0097] Another embodiment of plasma collection device 201 is now described with reference to Figures 8 to 16. This embodiment provides the plasma collection device 201 with a body portion 202 and a movable clip portion 203 both of which may be fabricated from a suitable polymer.

[0098] Body portion 202, as shown in Figures 10 and 11 , comprises membrane separator 205 in the form of a separation membrane, circular drying pad 211 and capillary tube 102 supports. The function and nature of the membrane separator 205 and drying pad 211 , as well as the materials used are as above described. The membrane separator 205 has triangular shape and the drying pad 211 has a circular shape being impregnated, in this embodiment, with a malPEG conjugating reagent.

[0099] Body portion 202 is also provided with two slots 204, one on each side of body portion 202. Body portion 202 includes a surface 214 at its base. Surface 214 includes an aperture 221 acting as a user guide for placement of a finger for collection of blood into microcapillary tube 102 and a guide 217 for accommodating and guiding movement of microcapillary tube 102 during a plasma collection procedure.

[0100] The aperture 221 also acts as a hole to allow any fallen blood drops to pass through the plasma collection device 201 instead of being collected on surface 214 and potentially splashing onto the plasma separation membrane 205.

[0101] As shown in Figure 12, movable clip portion 203 comprises a body 203A connected at one end to two flexible prongs 213 and at the other end to a pusher or handle 215. The prongs 213 are provided at each end with hook portions 213A cooperable with the slots 204 of body portion 202 to enable the clipped position of plasma collection device 201 to be achieved by movement of clip portion 203 into co-operation with body portion 202. When hook portions 213A as located in slots 204 are compressed, the clip portion 203 may be slid out of co-operation with body portion 202.

[0102] Body 203A also includes a guide or channel 208 for holding microcapillary tube 102 which is pressed into it. Clip portion 203 is dimensioned such that it can move into co-operation with body portion 202 as above described.

[0103] Clip portion 203 further includes a flexible member 230 bendable in the direction of pusher 215, in particular when a microcapillary tube 102 is pressed into position against it. Flexible member 230 has two portions separated by a gap 209 through which any excess blood may flow into spaces 231. However, with thehydrophobic end 102B cap as described above, overfilling of the microcapillary tube 102 is practically avoided.

[0104] It will be appreciated that plasma collection device 201 does not have an airtight / watertight housing. It should be stored with a dessicant. In this embodiment, the dessicant is not inside plasma collection device 201 .

[0105] Plasma collection device 201 is designed to be used like a side release clip (similar to a buckle found on a backpack) with the clip portion 203 movable between a first unclipped position as shown in Figures 8 and 15; and a second clipped position as shown in Figures 9, 14 and 16. The clip portion 203 is not fully separated from the body portion 202 to ensure that the individual pieces are not lost or damaged when collecting a blood sample.

[0106] The plasma collection device 201 is used as described below.

[0107] First, blood is collected from a patient as described above. Blood is into microcapillary tube 102 as shown in red in Figure 15 with blood flowing in the direction of the blue arrow. Aperture 221 provides a user with a guide of where to place a finger onto microcapillary tube 102. The two red arrows, shown in Figures 13 and 15, indicate a flexible point in flexible member 230 which is compressed by the microcapillary tube 102 to create a pinch point with an air gap 209. This acts to prevent overfilling of the microcapillary tube 102 in addition to the hydrophobic end cap 102B blocking one end, avoiding overfilling. If there is any excess blood, this accumulates in spaces 231 .

[0108] Following collection of the blood sample, clip portion 203 is slid into cooperation with body portion 202. This is done by the user manually compressing the flexible prongs 213 on each side of clip portion 203 by pushing on pusher or handle 215.

[0109] On completion of the sliding of the clip portion 203 into the body portion 202, the microcapillary tube 102 is also moved into fluid communication with the separation membrane 205 as indicated by a click sound, i.e. audibly, when the plasmacollection device 201 reaches the end point of movement, i.e. the clipped position. Blood is then transferred from the microcapillary tube 102 to the separation membrane 205 for separation of plasma onto drying portion 211 . As the microcapillary tube 102 is not overfilled, as described above, the membrane separator 205 is likewise not overloaded with blood as was problematic with previous plasma collection devices.

[0110] The plasma collection device 1 or 201 is advantageously used as a means to collect a sample for the analysis of a biomarker or blood component. The plasma collection devices 1 , 201 may be used for plasma collection without waiting, overnight drying or additional sample dilution steps.

[0111] Plasma collection device 1 or 201 can be stored for up to one month at room temperature before use for laboratory analysis, for example as described below.

[0112] The current literature suggests that spikes in training load and excessive and cumulative training loads significantly increases the risk of serious muscle injury (e.g. hamstring tear). For example, in studies of rugby league and Australian football players, increased 3-weekly total distance and sprint (18-24 km / h) distance were associated with a greater risk of injury. In some studies, increased training loads have been shown to result in an increase in the concentration of plasma creatine kinase and C-reactive protein which suggest muscle damage and inflammation. These observations highlight muscle damage as a risk factor for muscle injury. Currently, there are no athlete monitoring techniques that are routinely used by professional sports teams capable of detecting muscle damage or inflammation following training. The inventors believe that albumin thiol oxidation could be used to identify periods of increased physiological stress within the muscle during a professional sport’s preseason.

[0113] Furthermore, it is believed by the inventors that changes in plasma albumin thiol oxidation correlates with muscle damage in the mouse model of Duchenne Muscular Dystrophy (Al-Mshhdani et al., 2021 ). Duchenne Muscular Dystrophy is a genetic disease characterized by skeletal muscle damage, chronic inflammation, and immune cell infiltration within the muscle similar to that which occurs in the days after EIMD. In mdx mice, muscle damage can also be exacerbated by physical exercise which has been shown to further increased albumin thiol oxidation. Thus, this workprovides further evidence that albumin thiol oxidation responds to muscle damage, chronic inflammation, and immune cell infiltration within a muscle.

[0114] Therefore, in one embodiment, the present invention provides the use of the device of the present invention in the determination of the level of muscle damage that may have been caused by training or changes in albumin thiol oxidation due to disease, disorders or conditions. The present device and methods thereof provides a simple and cost effective approach, such that it can be used, for example, routinely by sports practitioners to reduce the risk of serious muscle injury in an athlete, or in the analysis of disease, conditions or disorders.EXAMPLES

[0115] The following examples further describe the device of the present invention and its use.

[0116] Preferred embodiments of the plasma collection device, as described above, incorporate a glass microcapillary tube 102 which aims to collect 10±0.4 μL of blood for separation. The device of the present invention aims to enable reproducible and valid sample collection for analysis every by preventing underloading / overloading of the plasma separation membrane 5a. The collection of only about 10 μL of blood is significantly less than known Dried Blood Spot (DBS) devices and based upon the present findings, means that participants can fill the microcapillary with a single drop of blood. Requiring only one drop of blood addresses some of the problems associated with excessive milking of the finger and / or added discomfort from lancing more than one finger.

[0117] Further, the device of the present invention is designed to facilitate the simple collection of plasma samples from participants in the field or the home. Unlike known devices, the inclusion of a desiccant within the device means that there is no need to wait for the sample to dry before storage or transport (such as shipping).

[0118] Additionally, there is no need to manipulate the plasma collection device after the blood is applied, such as removing the top cover as required by devices such as the Telimmune and Roche Cobas cards, the cap is simply re-screwed, and the sample is ready for storage or transport. The present device is both watertight and airtight which allows for the incorporation of stable malPEG without the need for storagein bulky containers with silica gel desiccant, reducing costs associated with transportation and improving the usability of the device in the field.

[0119] The effectiveness of the use of the collected plasma sample in the detection / monitoring of a blood component, such as a biomarker, is described in relation to the oxidation of albumin thiol. However, the person skilled in the art will appreciate that the blood collection device has a wider use, including as a tool to identify and / or quantify biomarkers associated with diseases, disorders or conditions, and may be used to collect a blood sample for use in any detection assay.

[0120] As the present device collects plasma and not hemolyzed whole blood as required by the DBS cards, there is also an advantage for the laboratory analysis of biomarkers, such as albumin thiol oxidation. For example, albumin thiol oxidation can be analysed with capillary electrophoresis which is simpler and faster as it is circumventing the need for often complex and / or time consuming techniques and methods, such as Cibacron blue and SDS-PAGE.

[0121] During the study of albumin thiol oxidation in subjects, the present inventors were investigating the use of Dried Blood Spot (DBS) techniques for blood collection in the field and the use of Cibacron blue purification of albumin following the determination of albumin thiol oxidation. However, throughout the development and field testing of known DBS techniques, the inventors often obtained an invalid sample collection as well as experienced complications with the Cibacron blue isolation methodology. Therefore, the inventors sought to develop a new technique for the collection of dried plasma as opposed to the collection of dried blood.

[0122] As shown in the following examples, it was not possible to separate albumin from DBS (both before and after Cibacron blue isolation) using capillary electrophoresis. The analysis of samples obtained using plasma collection devices with capillary electrophoresis were 4-fold faster than those obtained using the DBS technique with SDS-PAGE while also being more cost-effective. The cost of each method includes labour (assembling of the DBS cards or plasma devices and processing samples in the laboratory) as well as the cost of materials.Example 1 - Development of Plasma Collection Device

[0123] This experiment aimed to develop a plasma collection device capable of being used in any situation outside of a laboratory setting. The following example describes the collection of dried plasma out in the field. The device utilised a microcapillary tube to collect a known volume of blood (<20pL) for separation in a completely passive manner requiring no external equipment or user input and only a filtration membrane. The performance of the device was tested by comparison with plasma separated using conventional centrifugation.

[0124] Double-deionized (DDI) water was used throughout. Polyethylene glycol maleimide (Malpeg), 2000g / mol and 5000 g / mol, was from JenKem Technology (Malpeg, JenKem Technology, USA). Disposable lancets (Accu Check Safe T Pro Plus were purchased from Medline Australia. All other chemicals and reagents were from Sigma-Aldrich (Castle Hill, Australia).

[0125] Microcapillaries were sourced from BLAUBRAND® with a Capacity 10pL of, 29mm length and accuracy of <±0.5% (cat no 7091 09).

[0126] All shapes for paper were outlined using Cricut design space software and cut using a Cricut Joy cutting machine.

[0127] The blood separation membrane to be used in the plasma collection device 1 , 201 was determined by testing the performance of three different membranes; (1 ) Fusion 5 (GE Healthcare), (2) VIVID GF (Pall Corporation), and (3) Cytosep (Ahlstrom Munksjo).

[0128] Whatman Fusion 5 is a proprietary single layer matrix membrane which uses a hydrophilic glass fibre material which acts as a blood separator, with an average particle retention size of 2.3 μm.

[0129] VIVID GF grade was chosen as it is stated to be suitable for small blood volume applications (20pL / cm2), such as those obtained from the fingertip. Additionally, the makers of VIVID state that the membranes have no post-treatment and thus may exhibit higher levels of hemolysis than other grades (GX and GR grades require larger volumes of blood). The VIVID GF membrane is composed of asymmetric polysulfone ofthickness 3.3pm. Length and different shapes of membranes were outlined using Cricut Design Space software and cut using a Cricut Joy cutting machine (Cricut, USA).

[0130] Ahlstrom Cytosep™ 1660 membrane is composed of glass fibers and is intended for use in applications where less than 100 μL of whole blood may be applied to the surface of the material (blood volume (30pL / cm2). The composite material does not contain binders or chemicals that may interfere with analyte measurements in diagnostic assays. Cytosep™ is described retaining the red blood cells on the surface of the single layer of glass fibers while the plasma, along with the platelets and leukocytes, move through the fibrous matrix.

[0131] Whatman 903 cellulose paper was used as a wicking pad to draw plasma through the membrane and to trap the albumin thiol. A 5pL volume of trapping solution comprising 62.5 mM malPEG in 40 mM imidazole, pH 7.4 (trapping solution) was dried onto each wicking pad. A 3.5mm punch device was used to collect a punch from the centre of either the Fusion 5 membrane or Whatman 903 paper circles.

[0132] 5mL polypropylene test tubes with lids were purchased from Technoplas Australia (product number P7512TUU) into which 3D printed components of the microfluidic device were inserted. The tubes provide an airtight seal protecting the inner components from moisture.

[0133] Silica gel crystals (also known as flower drying crystals) were purchased from Silica Gel Australia and were chosen for their significantly smaller size compared to regular silica gel beads (0.3 - 0.85mm).3D Printing

[0134] In this study, the plasma collection devices were fabricated using a high- resolution LCD-based SLA3D printer (Anycubic Photon Mono X, Shenzhen, China) featuring 10 μm XY resolution and 192mm(L)x120mm(W)x245mm(H) printing area. The desired microfluidic device is first drafted using a commercial CAD drawing software (TinkerCAD) and then translated into STL format, a suitable file for 3D printer language. The file was then sliced in Z direction using the Chitubox software (Version 4.01 , Miicraft). The slicing in Z direction (slice thickness) can be adjusted from 50 to 200 μm with an increment of 50 μm. The slicing option is related to the complexity of the geometry and was set at 100uM. The sliced file was then sent to the 3D printer with UVwavelength of 358-405 nm. Parts were removed from the build plate and rinsed and washed with water to remove any unpolymerized resin, thoroughly and air-dried by an air nozzle and placed into an oven at 70c to remove any remaining water before curing. Afterward, the 3D printed components were exposed to a LIV light with 405 ± 5 nm wavelength within a curing chamber for post curing process. Finally, components were stored in a desiccated container overnight before assembly to remove any water that may still be present.Testing of the plasma collection device

[0135] In one embodiment of the device, plasma samples were obtained by lancing a subject’s finger and lightly touching the finger to the tip of the device 1 allowing for collection of about 10 μL of blood into the microcapillary tube 2. Once filled, the lid was inserted into the corresponding holes of the device and rotated while applying downward pressure to bring the tube into contact with the Fusion 5 membrane. Plasma was allowed to separate and collected on Whatman 903 paper (containing dried malPEG) and was dried inside the device overnight at ambient temperature.Elution of Proteins

[0136] A 3.5mm punch device was used to collect a disc of paper from the centre of either the Fusion 5 membrane or Whatman 903 paper circles. An additional disc of paper from an area of the 903-paper card with no plasma present was used to remove any residual dried plasma residue from the hole punch after each punch. The resulting dried plasma spot discs were transferred to the wells of a 96 well plate. Wells were filled with 30pL of either 40 mM phosphate buffer pH 8.2, containing 2.5% sodium dodecyl sulfate and 0.10% dimethyl sulfoxide or 30pL of 20 mM phosphate-buffer containing 0.05% Tween 20 pH 7 and allowed to elute at room temperature with gentle agitation. Elution buffer was determined by the separation technique (SDS-PAGE or capillary electrophoresis). The elute was separated into two equal aliquots, referred to as sample A and B from hereafter. Sample A was used to measure total albumin oxidation.Disulphide exchange reaction

[0137] Sample B underwent further reduction and labelling of cys34 to determine the extent of reversible and irreversible oxidation. An equal volume of 20 mM L-cysteine (pH 3) was added to sample B and incubated for 30 minutes at room temperature, and then combined with equal volume of 25 mM malPEG with a further incubation for 15 minutes at room temperature.Determination of Hemolysis in the Plasma Sample

[0138] The extent of hemolysis in plasma samples was determined through absorbance measurement using a hemoglobin standard curve according to Cripps method. The Cripps method determines hemolysis according to the Equation:H - A576.5 - (A560 + A593) / 2 where H is the final absorbance of hemoglobin, A560, A576.5 and A593 are the absorbance values of the sample at 560 nm, 576.5 nm and 593 nm, respectively, in this method, background absorption from other proteins, such as bilirubin, is subtracted from the signal using the fact that the concentration of haemoglobin in a sample is proportional to twice absorbance at 576 nm minus that at 560 and 592 nm. Absorbance was measured using a Powerwave XS Spectrophotometer (KC4, BioTek, USA). An Evaluation of a Spectrophotometric Scanning Technique for Measurement of Plasma Hemoglobin (Numerical Model t Numerical Model to Predict Hemolysis and T edict Hemolysis and Transport in a Membrane-Based Microfluidic Device).Effect of different HOT on plasma separation volume

[0139] Blood samples with defined hematocrit levels (30%, 45% and 60%) were produced by diluting packed red blood cells with the appropriate volume of fresh plasma. A 10pL volume of blood was pipetted onto the top of each membrane and wicked through the membrane by Whatman 903 paper. Volume was determined by the BioRad DC protein assay.BioRad DC Protein assay

[0140] A Bio-Rad detergent compatible (DC) protein assay was used to determine the protein concentration of DBS elute. Standards were prepared from a 10 mg / ml bovine serum albumin (BSA) stock made fresh in SDS / Tris buffer and serially diluted from 2 mg / ml to 0.5 mg / ml. The assay was performed by pipetting 5 μL of each sample and standard in duplicate into a clear flat-bottom 96-well plate. Each well had 50 pL of Reagent A’ and 150 μL of Reagent B (DC Assay kit, Bio-Rad, USA). The platewas gently shaken for 5 minutes and absorbance read at 750 nm using a Powerwave XS Spectrophotometer (KC4, BioTek, USA). Protein concentration was calculated using polynomial regression. mPEG assay using plasma

[0141] The following process was developed based on the method according to Lim et al., (2020). Oxidation of cysteine 34 of plasma albumin as a biomarker of oxidative stress. Free Radio Res, 54(1 ), 91 -103. h tips : / / doi.orq / 10.1080 / 1.0715762.2019.1708347 . Plasma samples containing malPEG were thawed at 37 °C with agitation. Like the DBS extracts, the sample was divided into two samples, A and B. In sample A, SDS / Tris buffer (198 pl) containing 0.5% (w / v) SDS and 0.5 mM Tris (pH 7.4) was added to 2pL of plasma. In sample B 2.5 pl of 20 mM L- cysteine (pH 3) was added to 2.5pL plasma, incubated for 30 minutes at room temperature, and then combined with 5 pl of 25 mM malPEG with a further incubation for 15 minutes at room temperature. A sub-aliquot of this sample (4 pl) was added to 195 pl of SDS / Tris buffer.Gel Electrophoresis

[0142] Gels were hand cast using mini-protean plates (Mini-PROTEAN®, BioRad, Australia) using Laemmli method (Laemmli 1970) modified to generate a 16% resolving gel. For fluorescent imaging, 1 % (v / v) of 2,2,2-trichloroethanol (Ladner et al. 2004) was added to the resolving gel. After polymerisation of the resolving gel, the 4% stacking gel was poured on top of the resolving gel and, after polymerization, the gels were stored in a dark cold room at least 3 hours before use.

[0143] Plasma separation device plasma samples and conventional plasma samples were mixed with equal parts of loading buffer containing 0.5 M TRIS (pH 6.8), 3% (w / v) SDS, 30% (v / v) glycerol and 0.03% (w / v) bromophenol blue in DDI water. Gels were run at 180 V for 2 hours and 30 minutes in a refrigerated and dark room. Following electrophoresis, the gel was washed twice with DDI water before being placed on a UV transilluminator (ChemiDocTM, Biorad, Australia) for 5 minutes and then visualised with Image LabTM software (Biorad, Australia). The gel image was analysed using National Institute of Health (NIH) Imaged software (Version 1.48v, USA; Schneider et al. 2012). The image was inverted, and after background subtraction, and editing for specklingand noise, a signal profile was plotted for each lane from the gel (Image J user guide 1 ,46r, 2012). The area under each peak was calculated using the trapezoid rule to give the intensity for each band (Ladner et al. 2004).Protein Visualisation

[0144] Following electrophoresis, the gel was washed twice with DDI water before being placed on a UV transilluminator (ChemiDoc™, Biorad, Australia) for 5 minutes and then visualised with Image Lab™ software (Biorad, Australia). The gel image was analysed using National Institute of Health (NIH) Imaged software (Version 1.48v, USA; Schneider et al. 2012). The image was inverted, and after background subtraction, and editing for speckling and noise, a signal profile was plotted for each lane from the gel (Image J user guide 1.46r, 2012). The area under each peak was calculated using the trapezoid rule to give the intensity for each band (Ladner et al. 2004).Capillary Electrophoresis

[0145] All capillary electrophoresis separations were performed using Agilent 7100 CE system. Bare-fused silica capillary with a diameter of 50pm was purchased from Polymicro Technologies (Tucson, AZ, USA). New capillary was flushed in sequence with methanol (10minutes), double deionized (DDI) water (2minutes), 0.1 M NaOH (15minutes), DDI (2minutes), and background electrolyte (BGE) (20minutes). Separations were performed in a capillary 32cm long with an effective length of 24cm to the detector. Background electrolyte (BGE) used is 30mM phosphate buffer pH 8.2 with 2.5% SDS. Sample injection was performed hydrodynamically (10mbar for 10 seconds). Running voltage was set at +12kV, generated current is 61 uA. Absorbance at 214nm was recorded. Post conditioning of the capillary was performed every after sample by flushing DDI (1 minute) and BGE (3minutes).Subjects for Device Testing

[0146] To develop and optimise the plasma separation device methodology six healthy adults [age, 26 years ± 4 (range, 22-33 years); height, 171 ± 8 cm (range, 172— 191 cm); weight, 67 ± 9 kg (range, 55-80 kg)], were recruited to provide capillary blood samples. Capillary blood samples were collected as described above. The EthicsCommittee of the University of Western Australia approved this study, and all procedures conformed to the Declaration of Helsinki.RESULTS

[0147] The following discussion of the results details the selection and optimisation of a plasma separation membrane.Selection of a suitable plasma separation membrane

[0148] Plasma separation membranes are composed of polysulfone or glass fibers, as such the performance of each material was investigated. One polysulfone membrane (VIVID GF) and two glass fiber membranes (Ahlstrom Cytosep and Whatman Fusion 5) were evaluated based upon their plasma yield and extent of visible hemolysis.

[0149] VIVID GF, a polysulfone membrane (available from Pall Life Sciences), was prepared in a sandwich format as recommended by the manufacturer (Fig. 3A). The membrane was loaded with 20 μL of 45% hematocrit blood. The device was composed of 1 , 2, or 3 cm squares of VIVID GF plasma separation membrane, 4x1 cm Merck nitrocellulose membrane to wick away separated plasma and a 6x6 cm square of desiccant paper to dry the sample. A plastic sheet was placed on the top and bottom and sealed using a heat sealer to provide tension to the edges of the VIVID membrane. No air gaps / bubbles were observed between the membrane and the receiving nitrocellulose. A sample port was punched out the top sheet directly above the VIVID membrane allowing for blood to be applied.

[0150] Throughout multiple tests, plasma separation performance was inadequate with only a few very small spots of plasma visible on the nitrocellulose membrane and significant hemolysis as shown in Fig. 3B. Larger sizes of VIVID membrane (2 and 3 cm2) were also tested with the same 20 μL 45% hematocrit blood but similarly showed significant visible hemolysis with reduced plasma yield.

[0151] Fig. 3B shows the performance of the VIVID membrane at separating blood separation as prepared in accordance with Fig 3A. Images 1 to 3 were taken with the plastic top, base and desiccant paper removed. Image 1 shows two 1 cm2VIVID plasma separation membranes loaded with 20pL of 45% hematocrit blood on top of thenitrocellulose membranes. Image 2 shows the 1cm2VIVID plasma separation membrane removed to reveal separated plasma on the nitrocellulose membrane with significant hemolysis as indicated by the red color of the plasma. Image 3 represents the minimal plasma separation (highlighted in green boxes) resulting from a larger 2cm2VIVID plasma separation membrane.

[0152] An additional complication with VIVID GF was that the membrane could not be compressed or disturbed in anyway, owing to its asymmetric structure which can become disorganized if pressure is applied to the site of blood application. This presented a design challenge regarding how best to apply blood to the VIVID membrane as pressure from an external source such as a fingertip would risk destroying the membrane structure, inhibiting plasma separation.

[0153] The second membrane that was tested was a glass fiber membrane (Ahlstrom CytoSep) which was also prepared in accordance with Fig. 3A. A 1 cm2square of Ahlstrom CytoSep 1660 plasma separation membrane was loaded with 30pL of 45% haematocrit blood (Ahlstrom states that the required volume is 30 μL / cm2). Areas of plasma separation are highlighted in green boxes (see Fig. 4A). Very little plasma was separated onto the nitrocellulose membrane; however, plasma can be seen separating laterally towards the corners of each membrane.

[0154] Plasma separation onto the nitrocellulose was poor. It was observed that plasma tended to separate towards the edges of the membrane rather than through the membrane as observed with the VIVID GF membrane. As illustrated in Fig. 5, Ahlstrom states that plasma can be separated using the CytoSep membrane both vertically (A) and laterally (B). 30pL of blood was applied to a strip of Ahlstrom Cytosep membrane and allowed to separate laterally plasma separation to be optimal. However, the plasma generated by the CytoSep membranes exhibited significant hemolysis following drying figure (as shown in Fig. 4B, strip 1).

[0155] The third of the membranes tested was also a glass fiber membrane (Whatman Fusion 5). Similar to CytoSep membranes, lateral separation of plasma was carried out on Whatman Fusion 5 membrane. The membrane was loaded with 30pL of 45% HCT blood. Referring to Fig. 4B, hemolysis is highlighted by a red rectangle and plasma separation is highlighted by a green. Ahlstrom Cytosep 1660 shows greater hemolysis (see Fig. 4B, strip 1 ) compared to Whatman Fusion 5 (see Fig. 4B, strip 2).Whatman Fusion 5 membrane demonstrated very similar characteristics to Ahlstrom Cytosep as plasma tended to separate laterally as opposed to vertically. However, lateral plasma separation was superior to CytoSep due to a lesser observed hemolysis after drying as shown in Fig. 4B.

[0156] As a result of the above experiments, in preferred embodiments, Fusion 5 was selected as the separation membrane 5.Increases in albumin thiol oxidation following exposure to ambient oxygen

[0157] Albumin can be oxidised after at least 60 minutes of exposure to the atmosphere. To prevent the artifactual thiol oxidation of albumin during plasma separation and drying on the membrane, we tested 5 μL of malPEG at different concentrations dried at the top surface (first point of blood contact) of the Fusion 5 membrane. A concentration of 10 mM of malPEG was sufficient for the full trapping of reduced albumin thiol as shown in Fig. 17. Significantly different from mean maximal value (p <0.05). Data are expressed relative to baseline values as means ± 95% Cl (n=6 participants).

[0158] The quantification of the level of irreversible thiol-oxidised albumin requires a disulfide exchange reaction step (see Fig. 18). The optimal concentration of cysteine in the thiol disulphide exchange reaction and of malPEG that result in the maximal labelling of albumin were evaluated by incubating for 15 minutes the plasma eluate from the Fusion 5 membranes with (A) increasing cysteine concentrations to maximally reduce the reversibly oxidised thiols or (B) increasing malPEG concentrations for maximal labelling of the newly reduced thiols (See Fig. 19). Significantly different from mean maximal value (p < 0.05). Data are expressed relative to baseline values and as means ± 95% Cl (n = 6 participants).

[0159] The incubation of the separated plasma (eluted using 20 mM phosphate- buffer containing 0.05% Tween 20 buffer as discussed in the following section) with cysteine at a concentration of at least 5 mM was sufficient for maximising the disulfide exchange reaction that converts reversibly oxidised albumin back to a reduced sulfide that is susceptible to further malPEG conjugation as shown in Fig. 9 (Column A). The incubation of the resulting reduced albumin with 12 mM of malPEG was sufficient formaximal malPEG-labelling of the newly exposed thiol groups as shown in Fig. 9, column B.Issues with malPEG- membrane blocking and degradation

[0160] Fusion 5 membranes (4 x 1cm) pre-treated with 5 μL of 10 mM malPEG were loaded with 30 μL of whole blood and stored in silica gel desiccant for 1 week (lanes 2, 4, 6), before being compared to malPEG-treated plasma prepared using a conventional blood centrifugation-based protocol, as shown in Fig. 20, lanes 1 , 3, 5. Additional lanes unrelated to this figure have been blocked out. Albumin protein (both malPEG labelled reduced and unlabelled oxidised) is highlighted in a black box, as shonw in Fig. 20.

[0161] Although the application of malPEG to the Fusion 5 membrane prevented the artifactual thiol oxidation of albumin, it resulted in two problems. Firstly, the plasma proteins degraded rapidly if dried while on the Fusion 5 membrane, so it was not possible to measure albumin oxidation level after 1 week of storage in desiccant (Fig. 20).

[0162] Secondly, malPEG decreased the plasma separation yield substantially (Fig. 11 ). Figure 21 illustrates the results of our experiment where a 30 μL sample of blood (45% haematocrit) was loaded on 4 x 1 cm strips made out of Fusion 5 separation membranes that was either (1 ) previously treated with 5 μL of 10 mM malPEG or (2) left untreated. Plasma is highlighted in the green boxes in Fig. 21 .

[0163] In order to address these problems, malPEG was removed from the Fusion 5 separation membrane and instead applied to a “wicking pad” made out of a Whatman 903 filter paper layered immediately below the Fusion 5 membrane to draw the sample across the separation membrane by capillary action (Fig. 22).

[0164] Figure 22 shows the results of a volume of 5 μL of 10 mM malPEG dried onto a 10 mm diameter circle of Whatman 903 paper (wicking pad). A volume of 30 μL of blood (45% haematocrit) was loaded on 4 x 1 cm strips of Fusion 5 membranes which were then placed against the wicking pad. Plasma is highlighted in green boxes. The addition of a wicking pad allowed the plasma to be drawn through the membrane onto the wicking pad which contained malPEG for the trapping of the albumin thiols.

[0165] A volume of 10 μL of 10 mM malPEG was dried onto increasing diameters of Whatman 903 wicking pads followed by the addition of 3 μL of plasma to the pads and their storage in silica gel desiccant. After 1 month, the wicking pads were eluted and subjected to SDS-PAGE. The density of the albumin band on an SDS-PAGE gel was quantified to determine the extent of protein degradation as shown in Figure 13. P represents plasma. An * represents significantly different from plasma (p < 0.05). Data are means ± 95% Cl (n = 6 participants).

[0166] The results of this experiment showed that increasing the size (diameter) of the wicking pad slowed protein degradation up to a size of 10 mm beyond which a further increase in size resulted in no further change to the rate of protein degradation (Fig. 23).Issues with Fusion 5 membrane - increasing in albumin thiol oxidation during separation

[0167] The effect of washing of Whatman Fusion 5 membranes on the artifactual irreversible and reversible oxdiation of alubmin was tested. Fusion 5 membranes were either subjected to 3 x 5-minute washes in DDI or left untreated. Then, 20 μL of whole blood was applied to the Fusion 5 membrane, and plasma was separated and collected onto a wicking pad. The results were compared to those obtained using plasma prepared using a centrifugation-based method as shown in Figure 24. The * represents significantly different from plasma (p < 0.05). Data are means ± 95% Cl (n = 6 participants).

[0168] As a result of both removing the malPEG from the Fusion 5 membrane and adding the malPEG to the Whatman 903 paper wicking pad, there was a significant artifactual increase in the level of thiol-oxidised albumin, as shown in column A of Fig. 24. The increase in oxidation occurred even after preparing the plasma via a conventional centrifugation-based protocol and loading it onto the Fusion 5 membrane, indicating that the presence of cells or cell lysis during plasma separation did not contribute to the increase in oxidation. Only the level of reversibly oxidised albumin increased under this condition, with no change in the level of irreversibly oxidised albumin, as shown in Fig. 24, column B.

[0169] To test whether a component or chemical within the membrane was responsible for the artifactual oxidation of albumin, all Fusion 5 membranes were washed with DDI water and dried at 100°C prior to blood application. The addition ofthis washing step prevented the artifactual increase in the level of thiol-oxidised albumin (see column B of Fig. 24).

[0170] Optimization of malPEG labelling of albumin was performed using Whatman 903 wicking pages. The effect of malPEG concentration on the labelling of protein thiols was examined by drying increasing concentrations of malPEG onto 10 mm Whatman 903 paper circles and measuring its effect on the proportion of oxidised albumin as shown in Fig. 25. The * represents significantly different from mean maximal value (p < 0.05). Data are expressed relative to baseline values and as means ± 95% Cl (n = 6 participants).

[0171] The results showed that a concentration of 10 mM malPEG applied to the wicking pad was sufficient to prevent the artifactual oxidation of albumin that would occur following exposure to the atmosphere (Fig. 25).Optimization of the separation performance of the Fusion 5 membrane

[0172] Optimising the separation performance of the Fusion 5 membrane was determined for (i) the average blood volume from one drop of capillary blood and (ii) the shape of the Fusion 5 membrane and separation volume various hematocrits, as described below.

[0173] Average Blood volume from one drop of capillary blood: For ease of use of the separation device we were developing, no more than one drop of blood from the fingertip should be required. To determine the volume of blood that a typical untrained individual can produce from a fingertip, a group of participants were asked to lance their finger and provide one drop of blood into an Eppendorf tube, and the volume of blood was then measured by weight. This process was repeated 3 times for 6 participants. The average volume of a drop of blood was 15.1 ± 3.6 μL (standard error). Therefore, a target collection volume of 10 μL would ensure that the participants only need to provide one drop of blood for the separation of plasma and subsequent analysis.

[0174] Shape of the Fusion 5 membrane and separation volume at various hematocrits: For the optimal separation of 10 μL of blood, a variety of shapes of the Fusion 5 membrane were tested. The highest plasma yield was obtained from a triangular shape, 7.2 mm (L) x 7 mm (W) (Fig. 26). This size and shape were specifically designed to separate 10 μL of capillary blood from the fingertip with a 30, 45and 60% haematocrit. The volume of blood separated was determined by a protein quantification assay. A hematocrit of 30, 45 and 60% led to the separation of 3.9 ± 0.6, 3.5 ± 0.6 and 2.0 ± 0.4 μL (standard error) of plasma, respectively.Optimization of hemolysis reduction in separated plasma

[0175] The extent to which blood was haemolysed on membranes was evaluated by measuring the haemoglobin concentration in the extracted plasma, with haemoglobin being a good indicator of red blood cell rupture. Using the blood from 6 participants (-45% haematocrit), there was no significant difference in the concentration of haemoglobin between the plasma obtained from conventional centrifugation (22.1 ± 1.54 mg / dL) and the plasma obtained from membranes treated as described above (25.3 ± 6.4 mg / dL). Complete hemolysis resulted in a hemoglobin concentration of 14500 ± 840 mg / dL, indicating that only trace level of hemolysis took place on the Fusion 5 membrane.Optimization of the elution time, punch size, and volume for the extraction of proteins from dried wicking pads

[0176] The optimal protein elution time from plasma dried onto wick pads was measured. The results are shown in Figure 28. The effectiveness of a 20 mM phosphate-buffer containing 0.05% Tween 20 buffer (see Fig. 28, graph A) or a 2.5% (w / v) sodium dodecyl sulfate plus 0.10% (w / v) dimethyl sulfoxide buffer (see Fig. 28, graph B) at eluting proteins from dried plasma spots previously placed in a 96 well plate kept on a vortex mixer was evaluated over time at room temperature.

[0177] As shown in Fig. 27, there was complete protein elution of the plasma dried on wicking pad within 30 minutes of agitation at room temperature in the presence of either 2.5% (w / v) sodium dodecyl sulfate, 0.10% (w / v) dimethyl sulfoxide BGE buffer used for capillary zone electrophoresis, or 20 mM phosphate-buffer containing 0.05% (v / v) Tween 20 for SDS-PAGE analysis. Proper elution of the protein may not occur within 30 minutes if the microplate is not agitated during the elution process.

[0178] Based on the results described above, the following design criteria were developed for optimizing the separation of 10 μL of capillary blood into approximately 3 pL of plasma:1. The Fusion 5 membrane produced the highest plasma yield and the lowest sample hemolysis.2. The collection of 10 μL of blood (one drop of capillary blood) provided enough plasma to analyze the thiol oxidation state of albumin.3. Protein degradation was delayed when in contact with a 10 mm diameter wicking pad pre-treated with 5 μL of 10 mM malPEG.4. A triangular shape 7.2 x 7 mm Fusion 5 membrane effectively separated 10 μL of blood with a haematocrit ranging between 30-60%.5. The pre-washing of the Fusion 5 membrane with DDI water prevented the artifactual thiol oxidation of albumin.6. Complete protein elution from the dried plasma on a wicking pad occurred after 30 minutes of incubation at room temperature.DISCUSSION

[0179] The study described above reports the successful development of a plasma collection device. One reason that the device is effective is because it requires a small volume of whole blood providing an analytically useful volume of plasma, with minimal hemolysis. The device is unique, as it is capable of collecting a volume of 10 pL of whole blood in order to separate plasma from the whole blood within the range of 30% - 65% haematocrit.

[0180] The plasma collection device 1 can be stored for one month or longer at room temperature before use and is capable of being used by participants with minimal training and without supervision. Furthermore, the inventors have demonstrated the effectiveness of the device by measuring a biomarker, (i.e. albumin thiol oxidation).

[0181] The use of a membrane to separate plasma from blood is known, and there are many studies which demonstrate successful plasma separation using known membranes, such as Cytosep (glass fiber) and VIVID (polysulfone) membranes. However, the present applicant has identified several limitations of these devices featuring the membranes described in the literature. One limitation is the size of thatdevice, which dictates the volume of blood required for separation, being significantly larger than the volume of sample required by the device 1 , 201 described herein.

[0182] For example, the three devices as detailed in the literature require about 40 μL of whole blood applied to, for example, the Cytosep membrane, and 75 μL or 500 μL of whole blood to be applied to the VIVID membrane. The usability of these devices is therefore questionable, as the average capillary blood drop was found to be only about 15 μL. To achieve the volumes required by the aforementioned devices, a user must be capable of producing multiple large drops of blood or lancing of several fingers, both of which is unlikely without the assistance of a trained practitioner (e g., nurse).

[0183] A further limitation of the known plasma collection devices is that the performance of membranes is tested with blood samples which contain anticoagulants (EDTA or heparin). It is not practical to add anticoagulant in the field as it involves the use of additional equipment (i.e., pipettes), so it is uncertain whether previous studies are relevant because anticoagulants can affect blood flow characteristics. For example, as we have described in the above examples, adding EDTA to whole blood increased the wicking distance and capability to separate blood plasma on cotton membranes. Additionally, some studies have evaluated membrane performance by using blood from cows or pigs. It has not been shown that blood from animals behaves similarly to humans.

[0184] Another limitation of the known devices is that in some previous studies, whole blood has not been applied to the membranes directly from the finger; rather blood is pipetted onto the membrane. Pipettes control the speed at which blood is applied onto the membrane which can alter the pressure as blood flows through the membrane. A reduced flow rate from controlled and gradual pipetting results in lower pressure which in turn may reduce the extent of hemolysis. In contrast, hemolysis and plasma separation was tested with the blood sample collected directly from the finger.

[0185] To increase the analytically useful volume of plasma, drying salt onto the membrane prior to blood application may be effective. The addition of a saline solution immediately prior to blood sample application has previously been shown to increase plasma yields on a cellulose filter paper. It was demonstrated that the addition of salt changes the blood solution from isotonic to hypertonic condition which generates differences in osmotic pressure across the cell membrane. Red blood cells lose waterdue to the osmotic pressure and show crenation, followed by an increase in red blood cell viscosity. The red blood cells formed closely packed lumps, which were unable to travel with the plasma phase by capillary wicking and are left behind the wicking front of the plasma phase.

[0186] Without wishing to be bound by theory, it is possible that the 20 μm pore size of the Whatman No 4 paper used by Nilghaz et al would allow for greater cell packing achieved by using higher salt concentrations then what was possible with the 2.5 μm pore size of Whatman Fusion 5 membrane.

[0187] A significant loss of malPEG reactivity was seen after 10 minutes of exposure to air at 60% humidity. Although it has previously been documented that maleimide can lose reactivity due to hydrolysis in aqueous solutions, this phenomenon is yet to be documented following the drying of maleimide onto paper cards. The rapid rate at which reactivity was lost is an important finding as this means participants must be wary of exposing collection devices containing dried malPEG to the atmosphere prior to sample collection.

[0188] The hydrolysis reaction of malPEG after drying may be due to the hygroscopic nature of the PEG molecule. Polyethylene glycol sorbs atmospheric moisture by the mechanism of deliquescence, furthermore that the critical relative humidity at which deliquescence commences varies with PEG weight, with the hygroscopicity of PEG has been shown to increase with lower molecular weight. PEG'S ability to sorb large amounts of moisture is due to the presence of ether oxygen atoms (-O-) in the oxyethylene polymer backbone as well as hydroxyl (-OH) end groups, both of which can form numerous hydrogen bonds with water. Joao et al has documented that complete maleimide hydrolysis occurred after 20 hours in PBS (pH 7.4, 10 % DMSO, 37 °C). We observed a 4% loss in reactivity after 60 minutes of exposure to 60% humidity following drying. If the rate of degradation should remain the same, complete degradation would occur within approximately 24 hours which is consistent find the findings of PEG in aqueous solution. When conjugated to proteins PEG conveys its properties, including its high affinity for water which is proposed to lead to an increased rate of reaction for protein hydrolysis when blood / plasma samples are not stored in desiccant.

[0189] Extensive protein degradation may be observed depending upon where the plasma was dried, even in the presence of a silica gel desiccant. When plasma was collected and dried onto Whatman Fusion 5 membrane, complete albumin degradation occurred within 1 week. In contrast, a wicking pad of at least 10 mm diameter in combination with silica gel desiccant was effective at slowing the rate degradation and resulting in measurable albumin signal for 4 weeks. The cellulose composition of the wicking pad may explain the effectiveness at slowing protein degradation. Since cellulose is highly hygroscopic and porous, the paper may act as a desiccant to draw water away from the plasma sample, helping to preserve the sample if there is sufficient cellulose paper surrounding the plasma spot that does not contain malPEG. In this regard, cellulose fibers have a large surface area which is comparable to that of silica gel crystals (estimated to be 100-200 m2 / g) (Lavoine et al. 2012). However, cellulose is documented to be able to absorb water up to 24 - 27 times its own weight, while silica gel can only absorb 0.4 times of its own weight which may make cellulose a superior desiccant. Cellulose may also be more appropriate in situations where only a small number of silica gel crystals can be included due to their large size and non-uniform shape (such as the inside of the plasma separation device).

[0190] Thus, the devices 1 , 201 of the present disclosure are capable of volumetric blood collection prior to plasma separation. As discussed, there are three plasma separation devices that are commercially available (Telimune card, Hemaspot SE and Roche Cobas card) and none incorporate a technique to control the volume of blood applied for separation. As such, all currently available devices are susceptible to overloading or underloading of blood. This is of concern as underloading may result in no plasma generation and overloading may result in increased hemolysis present in the plasma collection area. Both situations present an opportunity for user error and lead to the collection of compromised plasma samples. Reviews of the available plasma separation devices are highly limited (likely due to their recent availability) and as such the issue of non-volumetric sample collection has not yet been addressed for these devices in the literature. However, the issues of non-volumetric collection were addressed in relation to a dried blood collection device (Mitra device manufactured by Neoteryx). A study by Veenhof et al, (2020) Volumetric absorptive microsampling and dried blood spot microsampling vs Conventional venous sampling for tacrolimus trough concentration monitoring. Clin. Chem. Lab. Med., 58, 1687-1695 observed differences of up to 40% in the quantification of tacrolimus blood concentrations between samplereplicates using the Mitra device. The underfilling of some devices was demonstrated to be the cause of the differences.

[0191] The embodiments of plasma collection device 1 , 201 described herein aim to address some of the issues with the devices of the prior art in that the applicant’s device has a low blood volume requirement of only about 10 μL for separation of approximately 3.5 μL of plasma. The requirement for a low blood volume increases the likelihood of successful sample collection. As demonstrated by the applicant, the average participant produces a drop of blood of approximately 15 μL, and thus the device of the present invention only requires a low volume of blood. Two other devices have been described in the literature that can separate plasma from 10 μL of blood, but both have limitations.

[0192] For example, Nakahima et al developed a blood plasma separation and extraction from 5 μL of blood using dielectrophoretic and capillary force. A dielectrophoretic technique allows blood separation and blood cell handling using an electrostatic force that is caused by an inhomogeneous electric field between two electrodes. As such, this device requires an electric power supply unit and control circuits outside the microfluidic device. Additionally, the device has low yields of plasma, separating only -300 nL of plasma which may restrict which assays can be performed on the sample. Finally, the device does not dry the plasma and as such the liquid plasma that is extracted from the whole blood must be collected using pipettes and stored at -20 °C or-80 °C prior to analysis. Cold storage and transport make this device impractical for use in the home or in the field.

[0193] Nilghaz et al. also developed a paper-based separator capable of separation between 3 and 10 μL of whole blood. The device was made by creating microfluidic channels using hydrophobic wax on Whatman No 4 paper. Plasma separation was achieved by pipetting blood onto the paper, which is not practical in the field or home. Unfortunately, the authors did not quantify the volume of plasma that was separated at each blood volume, instead choosing to measure the distance in mm that plasma separated. After loading with 10 μL of whole blood, plasma separation. The design, as disclosed by Nilghaz, does not include any desiccant to dry the plasma sample which may lead to increased rate of protein degradation.

[0194] Capillary electrophoresis is a rapid technique to accurately determine the extent of, for example, albumin thiol oxidation from plasma separation devices. Quantification is achieved with acceptable Intra and Inter-assay coefficients of variability which indicate the technique produces reliable, precise, and accurate measures of albumin thiol oxidation. The automated nature of capillary electrophoresis significantly reduces the labor required to analyze a sample and results in a more cost-effective method compared to SDS-PAGE. With access to the appropriate number of CE analyzers it is estimated that for throughput is increased 4-fold (-200 samples) over SDS-PAGE Cost is an important consideration when the collection of numerous samples is concerned (e.g. tracking muscle recovery) as a technique that is too expensive can limit the scope of a study. Limiting the number of samples collected may lead to underestimation of the oxidative stress response.

[0195] In summary, the plasma collection device of the present disclosure enables plasma sample collection in the field that can be used in analytical studies.

[0196] The applicant has been able to accurately detect oxidized albumin Cys34. In addition, the device cannot be over or underloaded, and requires only about 10 μL of whole blood to separate plasma across a range of hematocrits and separates plasma that is hemolysis free.

[0197] Preferably, Whatman Fusion 5 membrane should be washed if the sample is to be used, for example, when measuring the concentration of a biomarker that may be oxidized. For example, to prevent artifactual thiol oxidation.

[0198] Finally, the device for collecting plasma is usable by participants with minimal training on the field or in the home.

[0199] Modifications and variations to the plasma collection device and its method of use may be apparent to skilled readers of this disclosure. Such modifications and variations are deemed within the scope of the present invention.

Claims

CLAIMS1 . A plasma collection device comprising: a housing accommodating: a microcapillary tube for collecting a volumetric quantity of blood by capillary action; a membrane separator in fluid communication with the microcapillary tube for separating plasma from said volumetric quantity of blood; a collector for separated plasma; and optionally, a desiccant for drying separated plasma inside the device.

2. The plasma collection device of claim 1, comprising a housing having a body portion and a movable portion, the movable portion being movable relative to the body portion.

3. The plasma collection device of claim 2, wherein the movable portion is a clip portion comprising a body connected at one end to a plurality of prongs and at the other end to a pusher or handle, the prongs being co-operable with slots of the body portion for clipping clip portion and body portion together.

4. The plasma collection device of claim 3, wherein each of said plurality of prongs is provided at each end with hook portions co-operable with slots of the body portion.

5. The plasma collection device of claim 3 or 4, wherein compression of the prongs allows movement of the clip portion from an unclipped position, in which the microcapillary tube does not supply blood to the membrane separator, toward the body portion and a clipped position in which the microcapillary tube is in fluid communication with the membrane separator for extraction of plasma from collected blood.

6. The plasma collection device of any one of claims 2 to 5, wherein the movable portion holds the microcapillary tube enabling filling with blood when the movable portion is in unclipped position.

7. The plasma collection device of any one of the preceding claims, wherein the microcapillary tube has capacity to collect a volumetric blood sample.

8. The plasma collection device of claim 7, wherein the microcapillary tube has a volumetric capacity of 5 to 30 μL , preferably less than 20 μL of blood, more preferably 10 μL and 15 μL of blood.

9. The plasma collection device of any one of the preceding claims, wherein the membrane separator comprises at least one separation membrane having an inlet surface and an exit surface for separating plasma from blood volume by drawing blood across the membrane by capillary action.

10. The plasma collection device of claim 9, wherein the at least one separation membrane has a highly asymmetric pore structure containing decreasing pore sizes from the inlet surface through to the exit surface for trapping red blood cells within the membrane while allowing plasma to pass through.

11. The plasma collection device of claim 10, wherein the at least one separation membrane is selected from the group consisting of polysulfone and glass fibre membranes.

12. The plasma collection device of any one of claims 9 to 11 , wherein the membrane separator comprises a plurality of membranes.

13. The plasma collection device of any one of claims 9 to 12, wherein the membrane(s) are treated prior to inclusion within the device to minimise reactions, optionally selected from the group consisting of oxidation and hemolysis.

14. The plasma collection device of claim 13, wherein the membranes are treated by steps selected from the group consisting of washing, coating, applying saline solution and drying.

15. The plasma collection device of any one of claims 9 to 14, wherein said at least one separation membrane has a triangular shape.

16. The plasma collection device of any one of the preceding claims, further comprising an air-tight and water-tight closure which is configured to act as a plunger for pressing the micro-capillary tube into co-operation with the membrane separator.

17. The plasma collection device of claim 16, wherein the closure includes prongs that engage with corresponding apertures in the membrane separator.

18. The plasma collection device of any one of claims 9 to 15, wherein when the microcapillary tube is in co-operation with the membrane separator, an air gap of selected dimension is maintained between an outlet of said microcapillary tube and an inlet surface of an inlet membrane included in the membrane separator.

19. The plasma collection device of any one of the preceding claims, wherein a bottom of the microcapillary tube is provided with a hydrophobic portion disposed proximal the membrane separator.

20. The plasma collection device of claim 19, wherein said hydrophobic portion is coated with a hydrophobic compound.

21. The plasma collection device of claim 20, wherein said hydrophobic compound is selected from the group consisting of silicone resins, acrylic copolymers, polyurethanes, bituminous and asphalt compounds, teflon, polyester resins, fibreglass, polystyrene, calcium carbonate, fluorinated silanes and fluoropolymer coatings.

22. The plasma collection device of any one of the preceding claims, wherein the collector for separated plasma includes a wicking means, optionally a wicking pad.

23. The plasma collection device of claim 22, wherein the wicking means has a cellulose composition.

24. The plasma collection device of claim 22 or 23, wherein at least one of the wicking means and at least one membrane is impregnated with a conjugating reagent, optionally maleimide polyethylene glycol (mal-PEG).

25. The plasma collection device of any one of the preceding claims, comprising a bypass of blood, exceeding a selected volume, past the membrane separator to prevent overloading of the plasma separation membrane, to be avoided and the selected volume is such as to avoid such overloading.

26. The plasma collection device of any one of the preceding claims is a silica gel with a high surface area to weight ratio.

27. The plasma collection device of any one of the preceding claims, wherein an anticoagulant, optionally EDTA or heparin, is provided inside the entirety of the microcapillary.

28. A method of collecting blood for a biomarker assay comprising: collecting a sample of blood in a device comprising: a housing accommodating: a microcapillary tube for collecting a volumetric quantity of blood by capillary action; a membrane separator co-operable with the microcapillary tube for separating plasma from said volumetric quantity of blood; and a desiccant for drying separated plasma; and assaying a biomarker present within said separated plasma.

29. The method of claim 28, wherein the biomarker is assayed to determine changes in albumin thiol oxidation.

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