Description

The present invention relates to a bi- or multi-layered membrane comprising a track- etched membrane and at least one layer of a porous pre-filter material, a process for preparation of such bi- or multi-layered membrane as well as the use of the membrane for the separation of cells or other larger items from fluid, especially from body fluids and more specifically for the separation of blood into blood cells and serum or plasma.

Background and Prior Art

A huge number of filter materials have been developed for separating larger constituents from aqueous solutions and especially for separating cells from body fluids for analytic or therapeutic purposes. Especially, separation of cells from whole blood to obtain plasma or serum requires a filter, which allows to eliminate blood cells in an efficient manner. Many patent documents describe filters that can be used for such or similar purposes, cf. US 6,045,899, US 5,906,742, US 6,565,782, US 7,125,493, US 6,939,468, EP 0,946,354, EP 0,846,024, US 6,440,306, US 6,110,369, US 5,979,670, US 5,846,422, US 6,277,281 , EP 1 ,118,377, EP 0,696,935 and EP 1 ,089,077. A commercially available example of such plasma separation membrane is the Vivid™ Membrane of Pall Corporation, which is an asymmetric polysulfone membrane coated with a substance like PVP.

However, using such well-known membranes, the cells are caught in the spongelike structure of the membrane and slowly clog the membrane leading to a decreased flow rate. The rough geometry of such membranes and the cells inside the membrane prevent any successful cleaning or regeneration steps. Also track-etched membranes have been considered as filter materials as they possess uniform and smooth surfaces and precise pore sizes. Due to these properties, track-etched membrane filters are an excellent choice for fractionation of particles. The smooth surface of the track-etched membrane can be washed and cells or particles collected on the membranes can be removed by backflushing.

Furthermore, the track-etched membrane is a high precision surface filter that is suitable for separating particles or cells from liquids with a highly specific cut-off size. The pore geometry of track-etched membrane is either cylindrical, hourglassshaped or cigar-shaped. (Apel, Nuclear Instruments and Methods in Physics Research B, 179, 2001 , 55-62; Apel, Radiation Measurements, 34, 2001 , 559-566; Apel, Colloid Journal, 66, 6, 2004, 649-656). In all such cases, the pore is a straight pore without branching. This enables the membrane to catch all objects that have to be separated on the surface of the membrane without loading the membrane with these objects like in case of depth filters (e.g., expanded PTFE membranes, sintered membranes or cast membranes). As explained above, such membranes with sponge-like structures capture the particles within the membrane leading to clogging of the membrane and a continuously decreasing filter performance.

A further advantage of track-etched membranes lies in the fact that the smooth surface is especially suitable for cell separation, because it prevents cells of being destroyed by rough pore surfaces. In order to enable liquids to pass the membrane, the liquid has to be able to wet the membrane. In case of a polyethylene terephthalate (PET) track-etched membrane, the membrane is naturally slightly hydrophilic, whereas in case of a polycarbonate (PC) track-etched membrane, the membrane has to be coated with a hydrophilic agent. In any case, applying a hydrophilic coating onto the membrane supports the wetting and, thus, the fast separation of blood cells from whole blood.

One property that makes separation of whole blood into its constituents very challenging is clotting (blood) or drying (body fluids). If the separation process takes too long, the blood drop clots or the body fluid dries and the clot/residue blocks the pores of porous material, also of track-etched membranes. As a result, no further transport of plasma or serum or other fluids through the membrane will occur.

The object underlying the present invention was to overcome the shortcomings and problems of the prior art and to provide suitable membranes for fast blood cell separation from whole blood, but also for providing improved filtration properties for other filtering applications.

Summary of the Invention

The object has been solved by the invention as defined in the appended claims.

According to a first aspect, the present invention provides a bi- or multi-layered membrane comprising a track-etched membrane and at least one layer of a porous pre-filter material with pores having size which is equal to or larger than the pore size of the track-etched membrane, and wherein the bi- or multi-layered membrane comprises a hydrophilic coating on at least the track-etched membrane.

According to a second aspect of the present invention, a process for the preparation of an inventive bi- or multi-layered membrane is provided, which process comprises a) providing a track-etched membrane and at least one porous pre-filter material with pores having a size which is equal to or larger than the pore size of the track- etched membrane; b) laminating at least one layer of a porous pre-filter material onto the track-etched membrane; c) treating the obtained bi- or multi-layered membrane with a hydrophilic coating solution; and d) optionally, drying the bi- or multi-layered membrane.

In an alternative embodiment of this second aspect of the invention, the process for the preparation of an inventive bi- or multi-layered membrane comprises a’) providing a track-etched membrane and at least one layer of a hydrophilic porous pre-filter material with pores having sizes which are equal to or larger than the pore size of the track-etched membrane; b’) treating the track-etched membrane with a hydrophilic coating solution; c’) optionally, drying the track-etched membrane; and d’) laminating at least one layer of the hydrophilic porous pre-filter material onto the coated track-etched membrane.

In a third aspect of the present invention, the inventive bi- or multi-layered membranes are used for separation of cells or other larger items from fluids, especially from body fluids. Preferably, this aspect includes the separation of blood into blood cells and serum or plasma, especially for diagnostic and point of care or home applications.

A fourth aspect of the present invention relates to a device for vertical blood separation comprising a bi- or multi-layered membrane as herein described.

Detailed Description of the Invention

The present invention is based on the realization that the problems encountered by the prior art can be avoided by providing and using the bi- or multilayered membrane of the present invention.

More specifically, the inventive bi- or multilayered membranes show superior filtering efficiency, especially when filtering samples like body fluids and especially whole blood. The pre-filter material of the bi- or multilayered membrane absorbs the fluid, e.g., the blood sample, and spreads the fluid inside the filter material. The prefilter material also at least partly absorbs larger items and possibly also compounds which lead to clotting of blood. As the fluid passes through the pre-filter material, the wider distribution of the fluid in the pre-filter also leads to a broader application of the fluid to the membrane and an optimized wetting of the track-etched membrane area. Consequently, more of the track-etched membrane’s pores are covered by the fluid compared to the situation in which a blood drop is applied directly to the membrane surface, and the capillary forces of the membrane’s pores can act more efficiently to channel the fluid through the membrane.

The bi- or multi-layered membrane is preferably formed or aligned in that the porous pre-filter material is to receive the sample to be separated, in particular body fluid, and even more preferably blood.

Thus, the inventive membranes have an upper and a lower side, wherein the upper side, i.e. the porous pre-filter material, receives the sample to be separated, such as body fluids and in particular blood. The track-etched membrane on the lower side preferably face an absorber as herein described. The pre-filter material on the upper side serves for pre-separation of larger items and also compounds which might lead to clotting of blood. By using the correspondingly arranged pre-filter material, clearly improved filtration results can be achieved as compared to the prior art. Therefore, first the porous pre-filter material comes into contact with the liquid to be filtrated, and then the track-etched membrane.

Furthermore, the hydrophilic coating applied onto at least the track-etched membrane also enhances the wetting and the spreading of the fluid on the membrane. Thus, especially for filtration of whole blood, the liquid parts of the blood, i.e., serum or plasma can be readily absorbed by the pores before the clotting process sets in.

Larger particles and especially blood cells are retained not only on the membrane surface as with some prior art products, but also within the pre-filter material. Thus, blocking of the pores by retained blood cells is also minimized for the inventive membranes.

Summarizing the above detailed description of the inventive membranes and their properties and effectivity, the invention provides a filter material which exhibits superior properties and is especially suitable for separation of blood cells from whole blood to obtain blood plasma or serum in an efficient manner. Within the context of the invention, the porous pre-filter material can either be made of a porous solid or from suitable fibers in the form of a woven or a non-woven material. The pores or the porous structure of the pre-filter material have to be larger or at least equal to the pore size of the track-etched membrane to generate the prefilter effect. Thus, the pre-filter material absorbs larger constituents of a fluid sample whereas the fluid itself and items smaller than the pore size of the pre-filter material can accumulate on the surface of the track-etched membrane and, depending on the pore size, pass through the track-etched membrane. To enable the pre-filter to absorb for instance whole blood and pass the plasma or serum to the track-etched membrane, which performs the final separation step, the blood preferably also effectively wets the pre-filter. This can either be achieved by using a hydrophilic or other suitable base material for the pre-filter or by providing a hydrophilic coating not only on the track-etched membrane, but also on the pre-filter material.

In a preferred embodiment of the invention, the porous pre-filter material is a natural or synthetic, woven or non-woven polymeric fiber material. In an especially preferred embodiment of the invention, the porous pre-filter material itself has hydrophilic properties, but is usually not water-soluble. Alternatively, if a porous pre-filter material is used, which is a respective natural or synthetic, woven or non-woven polymeric fiber material exhibiting hydrophobic properties, preferably such materials are treated by applying a hydrophilic coating onto such material.

According to a most preferred embodiment of the present invention, a hydrophilic coating is provided on both the track-etched membrane and the porous filter material.

According to the present invention, the bi- or multi-layered membrane comprises one layer of a track-etched membrane and one or several layers of a porous prefilter material. The two or more layers of the inventive membrane can either be loosely stacked and held together in an appropriate manner or in a suitable device and appropriate measures can be taken to avoid separation of the layers of such stack. However, in preferred embodiments of the present invention, at least one layer of a pre-filter material is laminated onto the track-etched membrane. In preferred embodiments, at least one layer of a non-woven pre-filter material is laminated onto the track-etched membrane. According to further preferred embodiments of the invention, the membrane contains two or more layers of a prefilter material, and all layers of the membrane are joined by lamination.

Within the context of the present invention, the more than one layers of pre-filter material can include multiple layers of the same pre-filter material or multiple layers of different materials or a combination thereof. In some instances, it is preferred to use layers of different pre-filter materials, e.g., with, towards the track-etched membrane, decreasing pore sizes. Furthermore, it is possible and a further preferred embodiment, to use a pre-filter material which in one layer includes different pore sizes, especially decreasing pore sizes relative to the track-etched membrane. While also in such specific embodiments of the present invention, the pore size of all existing pre-filter material layers is equal to or larger than the pore size of the track-etched membrane, an even higher filtering efficiency can be achieved by using a pre-filter material with decreasing pore sizes. Also, an enhanced spreading of the fluid and wetting of the membrane is obtained due to the fact that clogging of the various layers is minimized due to the continuously decreasing pore size and the fact that the largest items are retained in a different layer than smaller items.

The track-etched membrane layer, which is present in the inventive bi- or multilayered membranes, can be made of any material known to the skilled person as applicable in this context. Track-etched membranes are commercially available or can be obtained by treating a membrane base material under conditions forming pores of the desired size and density by a track-etching treatment. Such tracketching treatment conditions are well known to the skilled person and are disclosed in many prior art documents, e.g., the documents cited in the above Background and Prior Art section. According to preferred embodiments of the present invention, the material of the track-etched membrane is selected from polyethylene terephthalate (PET), polycarbonate (PC), polyimide (PE), polyvinylidine fluoride (PVDF), polyetheretherketon (PEEK), ethylenetetrafluoroethylene (ETFE) or from other polymers having similar properties. In especially preferred embodiments, the material of the track-etched membrane is PET or PC.

As mentioned above, a hydrophilic coating can be applied to the track-etched membrane only, to the track-etched membrane and at least one layer of porous prefilter material, or to all layers of the inventive membrane. In preferred embodiments of the invention, the hydrophilic coating is applied to all layers of the inventive membrane.

The coating can be applied to some of the layers before assembling the bi- or multilayered membrane. In preferred embodiments, however, the coating is applied to all layers of the membrane, and in especially preferred embodiments, the coating is applied to an already assembled bi- or multilayered membrane. Preferably, the layers of the assembled bi- or multilayered membrane are joined by lamination before applying the coating. In such case, the coating will also cover the intersection(s) between the track-etched membrane and the pre-filter material(s).

In case of lamination of an already coated track-etched membrane and a suitable pre-filter material, especially a non-woven pre-filter material, under certain circumstances, the coating of the track-etched membrane could be damaged. Also, in some instances, using different precoated materials of the membrane and the pre-filter could lead to an inhomogeneous coating and, thus, inhomogeneous wetting of the surfaces. A slower passage of the plasma or serum through the membrane could result from such inhomogeneous coating. Accordingly, in especially preferred embodiments of the invention, the bi- or multilayered membrane contains only layers which are first joined by lamination and thereafter treated with the hydrophilic coating.

Depending on the intended use and application of the inventive bi- or multilayered membrane, the thickness of the track-etched membrane can be adapted as desired and as explained above for the pre-filter material. In preferred embodiments of the invention, the track-etched membrane has a thickness of 1 to 100 pm, preferably 2 to 50 pm and more preferably 5 to 25 pm. The pore size of the track-etched membrane is also selected as best suited for the intended use of the membrane. In preferred embodiments of the invention and for well-established uses like separation of whole blood into blood cells and plasma or serum, the pore size of the track-etched membrane is 0.1 to 5 pm and more preferably 0.3 to 1.2 pm. The pore density can also be varied according to the intended use, however, for most purposes a preferred pore density lies between 10,000 to 1 ,000,000,000 pores per cm ² , preferably 2,000,000 to 100,000,000 pores per cm ² .

As mentioned above, the porous pre-filter material can be made of any material that is suitable for achieving the intended filtration effect. In preferred embodiments, the pre-filter material is a woven or a non-woven material, non-woven materials being especially preferred. Such materials can be produced from any polymeric fiber, with polyester; polyolefin, polyimide, cellulose, and nylon being fiber materials, which are considered as preferred within the context of the present invention.

According to the invention, the pore size of the porous pre-filter material is equal to or larger than the pore size of the track-etched membrane. Preferred pore sizes of the pre-filter material are 5 to 10,000 pm, more preferably between 10 and 500 pm, and especially preferably between 20 and 100 pm.

Pore sizes mentioned in the context for the present invention are defined as the pore diameter, especially the mean pore diameter.

The thickness of the layer or layers of porous pre-filter material can be adapted to the intended use and should especially take into account the volumes to be treated per unit of filter area. Also, the nature of the fluid applied onto the filter and the proportion or mass of substances or cells to be removed from the fluid by filtration need to be considered for determining the appropriate size and thickness of the prefilter material in the inventive membranes. In preferred embodiments, the thickness of the layers of porous pre-filter material is between 10 and 2,000 pm, more preferably between 20 and 1 ,000 pm and most preferably between 50 and 250 pm. In further preferred embodiments, the overall thickness of the porous pre-filter material on the track-etched membrane is 75 to 300 pm.

Hydrophilic coating solutions, which can be applied to the bi- or multilayered membrane of the present invention, can be selected from commercially available coating solutions or custom made for the intended use by selecting appropriate hydrophilic materials and preparing a respective solution in a suitable solvent. In preferred embodiments of the invention, the hydrophilic coating solution is a polyvinylpyrrolidone (PVP) solution or a sulphonated coating, a coating containing alcoholic or carboxylic groups or any other coating that supports the wetting process of the membrane. In some embodiments, a preferred grade of hydrophilic property is reached if a contact angle of <69° is reached.

The bi- or multi-layered membranes according to the present invention may contain or be coated with agents that serve for stabilizing components in the liquid to be filtered, in particular blood. Examples for such agents include alkyl polyethoxylates, i.e. mild tensides, which are suitable for isolating functional membrane complexes, such as Brij® 35 (polyoxyethylene(23)lauryl ether) or tocopherol. The use of tocopherol and Brij® 35 is in particular preferred.

In addition to the above detailed description regarding the inventive bi- or multilayered membrane itself, also processes for producing such membranes are subjects of the present invention. All features mentioned above which include or refer to process steps for obtaining the respective materials, are also considered disclosed within the manufacturing process. Basic and additional information regarding the process is provided in the following:

In a second aspect of the present invention, a process for the preparation of an inventive bi- or multilayered membrane is disclosed. Such process comprises provision of the various layers of the bi- or multilayered membrane as defined above and joining the layers in an appropriate manner. According to a preferred embodiment of the present invention, such process comprises a) providing a track-etched membrane and at least one porous pre-filter material with pores having a pore size, which is equal to or larger than the pore size of the track-etched membrane, b) laminating at least one layer of a porous pre-filter material onto the track- etched membrane, and c) treating the obtained bi- or multilayered membrane with a hydrophilic coating solution and, d) optionally, drying the bi- or multilayered membrane.

In an alternative preferred embodiment of the inventive process, the following steps are taken: a’) providing a track-etched membrane and at least one layer of a hydrophilic porous pre-filter material with pores having a pore size, which is equal to or larger than the pore size of the track-etched membrane, b’) treating the track-etched membrane with a hydrophilic coating solution, c’) optionally, drying the track-etched membrane, and d’) laminating at least one layer of the hydrophilic porous pre-filter material onto the track-etched membrane.

While in principle the layers can be stacked and held together by appropriate means, in preferred embodiments of the inventive process, the laminating steps b) or d’) are performed. These steps include stacking the membrane layers and applying a pressure of 1 to 5 bars, preferably 2 to 3 bars. The temperature applied in preferred embodiments of this lamination step is 60 to 180°C, preferably 120 to 180°C, more preferably 130 to 160°C and most preferably 135 to 155°C. In further preferred embodiments, the treating steps c) or b’), respectively, are performed by applying an aqueous solution of polyvinylpyrrolidone or a sulphonated coating, a coating containing alcoholic and/or carboxylic groups or another suitable coating, such as, for example, agents that serve for stabilizing components in the liquid to be filtered, in particular blood. Examples for such agents include alkyl polyethoxylates, i.e. mild tensides, which are suitable for isolating functional membrane complexes, such as Brij® 35 (polyoxyethylene(23)lauryl ether) and/or tocopherol. Most preferably, a polyvinylpyrrolidone solution is applied at a concentration of 0.1 to 50 g/L. The use of tocopherol and Brij® 35 is in particular preferred.

Further conditions and process steps can be adapted as appropriate for the production of bi- or multilayered membranes, and especially further characteristics of the materials to be employed can be chosen based on the definitions provided above. More specifically, the material of the porous pre-filter material, its thickness and its pore size, the nature of the track-etched membrane material, its thickness, its pore size and pore density are as defined above. Furthermore, instead of laminating the track-etched membrane to at least one pre-filter material layer, which is preferred, in alternative embodiments also a stacking of the layers and an attachment or mounting of the layers can also be achieved by securing or pinning or clamping the layers together in an appropriate frame or device.

A third aspect of the present invention is the use of an inventive bi- or multilayered membrane or of a membrane prepared according to the inventive process for separation of cells or other items from fluids. The particularly effective wetting of the track-etched membrane, which can be achieved by the inventive combination of the various layers and the resulting superior wetting of the membrane leads to a highly efficient separation of material and is especially useful in the application of separating cells from body fluids. Considering the problems encountered in the prior art with clotting of blood during such separation processes, the inventive bi- or multilayered membrane provides a novel and superior concept for such applications. The inventive membranes can be applied in larger separation units for diagnostic purposes; however, it can also be effectively used in smaller devices for point of care and home applications. One example of such point of care or home applications is a lateral flow analysis test system, in which the inventive membrane can be used as a first filter, which separates the cellular components form the fluid, especially from blood, while the fluid moves through the test system for determination of the presence or absence of certain substances and molecules.

Thus, a fourth aspect of the present invention relates to a device for vertical blood separation comprising a bi- or multi-layered membrane as herein described.

Such device preferably comprises an absorber and/or a means for even distribution of the sample to be separated, in particular blood. In a preferred embodiment, the bi- or multi-layered membrane, the absorber layer and the means for distribution are aligned in vertical direction as follows from bottom to top: a) absorber layer, b) bi- or multi-layered membrane, and c) means for even distribution.

In other words, according to the invention, the porous pre-filter material of the bi- or multi-layered membrane receives the sample to be separated, in particular blood, the track-etched membrane faces the absorber.

Devices according to the present invention are, for example, illustrated in FIG. 1.

Besides the bi- or multi-layered membrane, absorber and distribution means the device can further comprise a base plate, which preferably uptakes the absorber, and a cover plate as well as screws to tighten the assembly.

Suitable absorber materials are known to the person skilled in the art and, for example, comprise cellulose-based materials, materials comprising microfluidic channels, and/or suitable foams, in particular foams made of polymeric bonded fibers and/or polyurethane such as Porex HRM (high-release media) fiber media. The means for distribution can be, for example, a grid and/or the cover plate of the device itself. Such cover plate preferably comprises a sample receiving hole.

Furthermore, the bi- or multilayered membranes of the present invention can also be used in other automated analysis systems.

Figures:

Figure 1 : Figure 1A and Figure 1 B show inventive devices for vertical blood separation.

Figure 2: Calibration curves for plasma yield (Figure 2A) and hemolysis (Figure 2B).

Examples

The following Examples further illustrate the invention. Examples 1 to 5 demonstrate the importance of a hydrophilic or other suitable coating and show that standard track-etched membranes themselves, even with a hydrophilic coating (PVP), are only of limited use and success. Without the combination with a pre-filter, the blood clots and blocks the pores before the plasma or serum can pass through the membrane. If a pre-filter, in the present Examples a non-woven material connected to the membrane, is used, the blood is absorbed and spread inside the pre-filter. This leads to a pre-filtering of the clotting substances and a wide distribution of the blood over the membrane surface, instead of the blood drops staying at a certain spot. In the inventive Examples, the plasma or serum is dragged inside the pores of the track-etched membrane by the capillary force and all objects larger than the pore diameter stay on the side with the pre-filter. Typically, the plasma or serum are then either absorbed by a suitable material or transported away by microfluidic structures for further diagnostic tests.

Example 6 describes the performance of the inventive membrane compared to competitor products. The plasma or serum can be used for different purposes, diagnostic test are not a restriction of the present invention. The membrane presented in the inventive Examples is not only restricted to applications related to blood, but it can also be used for other body fluids like saliva and urine that contain objects that have to be separated from the liquid.

Example 1

A polyester track-etched membrane (pore density of 100,000,000 pores/cm ² , thickness 12 pm and pore size 0.4 pm) was laminated with a polyester sheath-core non-woven by applying a pressure of 3.0 bar and a temperature of 144°C. The laminated membrane was afterwards dip-coated with an aqueous solution of PVP (molecular weight of 40,000 g/mol and a concentration of 1.5 g/L) and dried. To verify the functionality, a drop of blood was placed on the non-woven side of the membrane and the track-etched membrane side pressed on a filter paper. The drop of blood was fully absorbed within 20 seconds and the plasma or serum detected on the filter paper after passing the track-etched membrane.

Example 2

A polyester track-etched membrane (pore density of 22,000,000 pores/cm ² , thickness 22 pm and pore size 1 .0 pm) was laminated with a polyester sheath-core non-woven by applying a pressure of 3.0 bar and a temperature of 144°C. The laminated membrane was afterwards dip-coated with an aqueous solution of PVP (molecular weight of 40’000 g/mol and a concentration of 1.5 g/L) and dried. To verify the functionality, a drop of blood was placed on the non-woven side of the membrane and the track-etched membrane side pressed on a filter paper. The drop of blood was fully absorbed within 20 seconds and the plasma or serum detected on the filter paper after passing the track-etched membrane.

Example 3

A polyester track-etched membrane (pore density of 22,000,000 pores/cm ² , thickness 22 pm and pore size 1 .0 pm) was laminated with a polyester sheath-core non-woven by applying a pressure of 3.0 bar and a temperature of 144°C. To verify the functionality, a drop of blood was placed on the non-woven side of the membrane and the track-etched membrane side pressed on a filter paper. The drop of blood was only partially absorbed by the non-woven side after 10 minutes. Without applying the hydrophilic coating, the blood clotted and blocked the pores before it could be fully absorbed by the pre-filter.

Example 4

A polyester track-etched membrane (pore density of 22,000,000 pores/cm ² , thickness 22 pm and pore size 1 .0 pm) was used without lamination and non-woven material as pure membrane. The membrane was dip-coated with an aqueous solution of PVP (molecular weight of 40,000 g/mol and a concentration of 1 .5 g/L) and dried. To verify the functionality, a drop of blood was placed on the membrane and the track-etched membrane pressed on a filter paper. The drop of blood clotted after roughly 20% of the plasma or serum passed the track-etched membrane and no further transport of plasma or serum through the membrane could be observed.

Example 5

A polyester track-etched membrane (pore density of 22,000,000 pores/cm ² , thickness 22 pm and pore size 1 .0 pm) was used without lamination and non-woven material as pure membrane. To verify the functionality, a drop of blood was placed on the membrane and the track-etched membrane pressed on a filter paper. The drop of blood clotted, and no visual passing of plasma or serum could be observed.

Example 6

The performance of the inventive membrane was compared to competitor products.

6.1.1. Membrane Material

Invention:

- laminated PET Track-Etched Membrane, pre-filter material nonwoven PET with a basis weight of 60 g/m ² , with additional coating, composed of Brij35 and Tocopherol

Competitor material: Ahlstrom CytoSep® Next Generation plasma separation pad, Grade 1668 (HV+)

Pall Vivid Plasma Separation - GR, Part No: T9EXPPA0200S00R

Pall Vivid Plasma Separation - GX, Part No: T9EXPPA0200S00X

6.1.2. Blood Samples

Whole blood samples, EDTA stabilized, have been obtained from the Zurich blood donation center (Swiss Transfusion SRC). All samples have been tested and are negative for critical infectious markers, in accordance with Swiss Transfusion SRC requirements for clinical use of donated blood samples. Blood samples were anonymized, but information on blood group, sex and year of birth are given.

6.1.3. Absorber

To collect separated plasma within the test procedure, various absorbers have been used:

Porex HRM S016107 1 mm

Toilet paper

Tempo tissue (similar to Kleenex tissue I paper handkerchief)

Paper towel

6.1.4. Main evaluation values

Time [seconds], till blood is absorbed by membrane

Plasma yield

Level of hemolysis

6.1.5. Devices and test setup

Devices and test setup are shown in Figures 1 A and 1 B.

6.1.6. Reagents and equipment

PBS buffer: 10x PBS Buffer, pH7.4, Invitrogen/ThermoFisher Scientific Ref# AM9624, use at 1 :10 dilution with deionized water

Lysis buffer: Red Blood Cell Lysis Buffer, Roche Ref# 11814389001 , add 50ul EDTA-whole blood to 1 ml of lysis buffer and mix well Centrifuge: Eppendorf centrifuge 5425, for plasma preparation (10min at 2800rcf) and hematocrit

Nanodrop: NanoDrop OneC Spectrophotometer, ThermoFisher Scientific; used to measure human serum albumin levels to evaluate plasma yield, and to measure oxy-hemoglobin to evaluate level of hemolysis

6.2. Procedure

6.2.1. Blood separation tests, no chase buffer

Cut absorber material, use same number of layers for all samples in a run (including blank), e.g. 20x20mm, 4 layers

Cut membrane material, size needs to be larger than absorber stack, e.g. 25x25mm

Use grey plastic grid, size needs to be smaller than membrane, e.g. 20x20mm

Prepare blank for Nanodrop measurement: absorber (e.g. 4 layers of tissue 20x20mm) into Eppendorf tube (1.5ml), add 1 ml PBS, mix well by shaking Assembly of test device

Orientation of membranes: o Inventive membrane: membrane side faces absorber, nonwoven fabric side receives blood o Ahlstrom membrane: fine side faces absorber, rough side receives blood o Pall membranes: fine side faces absorber, rough side receives blood

Mix blood sample well by inverting sample tube several times to have an homogeneous solution

Immediately after mixing, add blood sample via pipetting at sample hole of device onto the membrane, volumes e.g. 20-45ul of EDTA-whole blood Note time until blood sample is fully absorbed through membrane/absorber and membrane surface seems to be dry

Disassemble device and separate membrane from absorber: o Inventive membrane: exactly after 60 seconds o Ahlstrom membrane: exactly after 60 seconds o Pall membranes: exactly after 120 seconds (recommendation by manufacturer)

Add absorber stack (exactly same number of layers for all samples and compared membranes) into an Eppendorf tube (tube size 1.5ml)

Add 1 ml of 1x PBS buffer and mix well by shaking, to extract plasma from absorber into PBS solution.

Repeat for next blood sample (5-10 samples for each membrane and setting) After one run, squeeze absorber within PBS buffer, e.g. by using a 1000ul pipet tip onto the inner wall of the Eppendorf tube, to extract remaining plasma proteins from absorber into solution. Use new tip for each sample.

Proceed with Nanodrop measurement: Human serum albumin to evaluate plasma yield, and oxy-hemoglobin to evaluate level of hemolysis. Nanodrop methods, calibration curves and calculations are described in more detail in6.2.4.

6.2.2. Blood separation tests, with chase buffer

Test procedure was done essentially as described above (6.2.1 .)

Mix blood sample well and add sample onto membrane (volumes: 20ul, 45ul) To add chase buffer, add same volume or volume of PBS buffer (volumes 20/1 Oul, 45/22.5ul) onto membrane: o Inventive membrane: add chase buffer exactly after 15 seconds o Ahlstrom membrane: add chase buffer exactly after 15 seconds o Pall membranes: add chase buffer exactly after 60 seconds Disassemble device after same amount of time as described above (6.2.1 .) Add absorber into Eppendorf tube (size 2ml) Add 1 ,5ml PBS buffer and mix well by shaking Repeat with next blood sample

Squeeze absorber with pipet tip to potentially get more protein into solution and proceed with Nanodrop measurements 6.2.3. Blood separation tests with Porex absorber

Porex HRM 1 mm absorber was laminated onto the membrane side of inventive laminated membrane by heat and moderate pressure.

Orientation of inventive membranes: membrane side faces absorber, nonwoven fabric side will receive blood

Lamination at 110°C for 30-40min between two aluminum plates, bottom plate had a depression of 1 mm

1 . Base plate with 1 mm depression

2. Orientation bottom to top: base plate, aluminum foil, Porex absorber, inventive membrane (membrane side faces absorber)

3. Aluminum foil, cover plate

Mix blood sample well by inverting sample tube several times to have an homogeneous solution

Immediately after mixing, add blood sample via pipetting directly onto the nonwoven fabric side, volumes e.g. 20-45ul of EDTA-whole blood

1 . add blood

2. Peel off membrane

3. Cut absorber into small strips

4. Add absorber strips into PBS

Note time till absorption of blood into membrane/absorber

Peel off membrane from absorber

Cut absorber area, were plasma is visible, into small strips; add strips to 1 ml of PBS in an Eppendorf tube and mix well by shaking

Repeat with next blood sample

Squeeze absorber with pipet tip to potentially get more protein into solution and proceed with Nanodrop measurements

6.2.4. Measurements

6.2.4.1 Calibration curve preparation

To evaluate plasma yield and level hemolysis of the blood separation membrane tests, calibration curves have been prepared by dilution series. Plasma yield

For each new blood sample, plasma has been prepared by centrifugation for 10min at 2800rcf. Hematocrit values have been determined after centrifugation to assess percentage of cells versus percentage of plasma and evaluate maximal possible plasma yield. Then, plasma by centrifugation was used in a dilution series to calculate plasma yield for blood separation membrane tests. Increasing volumes of plasma (2.5ul - 40ul or more) were added to 1 ml of PBS for first set of experiments, or 1 ,5ml of PBS for chase buffer experiments. Human serum albumin concentrations were measured by Nanodrop, volumes were plotted against protein concentration. Y was taken to convert Nanodrop protein concentration into plasma volume per 1 ml PBS (plasma volume = Nanodrop protein concentration I y), or 1.5ml PBS respectively.

Hemolysis

EDTA stabilized whole blood was mixed well by inverting sample tube. Blood was lysed with a red blood cell lysis buffer, by adding 50ul of blood to 1 ml of lysis buffer and mixed well. Then, increasing volumes of lysed blood (2ul - 300ul) was added to 1 ml of PBS. Oxy-hemoglobin concentrations were measured by Nanodrop, volumes were plotted against protein concentration. Y was taken to convert Nanodrop protein concentration into volume of lysed red blood cells per 1 ml PBS (volume lysed blood = Nanodrop protein concentration x y).

6.2.4.2. Nanodrop measurement

A custom method to measure human serum albumin (HSA) was programmed as a protein measurement method (A280) with the following settings:

Absorption: 280nm

Extinction coefficients s/1000: 35.7 moles/L

Molecular weight: 66.40 (kDa)

Baseline correction: 340nm

Result of measurement: Protein concentration [mg/mL] A custom method to measure oxy-hemoglobin was downloaded from

ThermoFisher Scientific technical support onto the Nanodrop device.

Measurement range: UV-Vis range 350-850nm

Analysis wavelength : 414nm

Baseline correction 750nm

Extinction coefficients: 542480 moles/L

Molecular weight: 64500 (kDa)

Correction for analysis: sloping baseline 360 - 500nm

Result of measurement: Protein concentration [pmole/uL]

6.2.4.3. Calculations

Plasma yield

Nanodrop protein concentration was converted into plasma volume per 1 ml PBS (plasma volume = Nanodrop protein concentration I y).

Hematocrit value and according percentage of plasma within blood sample was used to calculate maximal possible plasma yield (maximal possible plasma yield [uL] = blood volume added onto membrane [uL] x percentage of plasma within blood sample).

Plasma yield [%] was calculated (plasma yield [%] = converted plasma volume [uL] / maximal possible plasma yield [uL])

Hemolysis

Nanodrop protein concentration was converted into volume of lysed red blood cells per 1 ml PBS (lysed blood volume [uL] = Nanodrop protein concentration [pmole/uL] x y).

Lysed blood per plasma was calculated by percentage of plasma yield in respective sample (lysed blood per plasma [uL] = lysed blood volume [uL] I plasma yield [uL])

Lysed blood total was calculated (Lysed blood total [uL] = lysed blood per plasma x volume of blood added onto membrane x percentage of plasma within blood sample)

Percentage of hemolysis was calculated (Percentage of hemolysis [%] = Lysed blood total [uL] / volume of blood added onto membrane [uL]) .3. Results

Experiments (NO chase buffer), Device A

Experiments (NO chase buffer), Device B (3D printed)

Experiments with chase buffer, Device A

Results on hemolysis

The significantly changed hemolysis data for the paper towel and the Tempo tissue can be explained by surfactants diffusing from the tissues through the membrane into the blood drop.