Title

Flat sheet filter media in the separation of plasma or serum from whole blood Field of invention

The present invention relates to the use of flat sheet filter media for separating blood plasma or blood serum from whole blood.

Background to invention

In medical technology, various kinds of blood and plasma / serum separation and treatment processes are known and state-of-the-art. The most common method to separate blood cells from the liquid part of the blood is centrifugation.

In transfusion medicine, filters are used to remove leucocytes from transfusion blood and to remove blood clots and particles. Furthermore, artery filters are applied during surgeries, e.g. to remove blood clots, particles and gas bubbles. Plasmapheresis filters are used to clean or to substitute plasma from patients, which is poisoned by bacteria, viruses or further components, which are dangerous to life, with artificial blood plasma or plasma from donators. Moreover, microdevices are known for whole blood analysis, which are based either on "test stripes" or on lab-on-a-chip technology. When using these devices, only a few microliters of blood are required for the blood or plasma / serum analysis. With these devices the separation of plasma / serum from whole blood is usually performed by fluid mechanical effects like the wetting behavior of different surfaces or the application of microchannels. Although this method is very attractive concerning the quick obtainment of blood analysis results, the results from these analyses are restricted to a few, test specific components. These applications are unable to replace a plasma / serum based blood analysis with the existing sophisticated systems in labs and hospitals, which comprise the analysis of a plurality of blood components and which are able to give an overall picture of a patient's state of health. Furthermore, also for microdevices, the task of separating blood cells from the liquid part of the blood is still not solved satisfactorily. The blood cell separation based on "test stripes" is highly dependent on the lateral separation efficiency of the separation media and enables preferably a qualitative blood analysis. Often chemical agents are applied to the "test stripes" which lead to a color reaction dependant on the presence and/or concentration of a blood component.

In many countries, it is obligatory to withdraw a sufficient amount of blood from the patients to be able to store the obtained plasma / serum sample for some time to check the analysis result some time later with a so-called retain sample. Until now, the task to obtain enough cell- free plasma / serum can however only be accomplished by centrifugation.

The centrifugation procedures, which are typically used for separating blood plasma / serum from whole blood, are not only cumbersome requiring large amounts of manual and mechanical handling, but are also time consuming, which is particularly disadvantageous in emergency medicine.

Blood plasma / serum analysers, which have a great capacity for plasma / serum samples, cannot operate at full capacity, if a centrifugation process is applied upstream, which works batch-wise and represents the »bottleneck« in the blood sample processing. This bottleneck problem could possibly be overcome with a filtration process instead of a centrifugation process for plasma / serum generation. Such a system would allow a flexible analysis of the samples: Urgent samples from emergency patients could be processed with a higher priority without any need of interrupting a running centrifugation process or of waiting for the centrifugation process to be finished.

It is a further advantage of a simple filtration process for whole blood separation that the whole blood separation into plasma / serum and blood cells can be performed directly after collecting the whole blood sample. This is especially advantageous for the quality of the subsequent blood analysis as the red blood cell stability decreases with increasing sample storage time. This can influence the plasma / serum composition when the plasma / serum separation is not performed immediately after the blood sample withdrawal, but with some time delay. This aspect becomes important in rural areas or developing countries when there is no centrifuge available for the plasma / serum separation and when the blood sample has to be transported for a long period of time and/or distance, sometimes even in a hot and/or humid environment.

A subsequent whole blood separation into plasma / serum can be advantageous for Point-of-Care testing devices, which are used to provide a quick blood analysis at / near the patient to get a quick blood analysis result outside of a clinical laboratory to make immediate decisions about patient care. Typically Point-of-Care testing is performed by non- laboratory personnel. A quick foregoing plasma filtration process facilitates the quick blood analysis and enables new operating conditions for Point- of-Care devices, since most of them work with whole blood or with the aforementioned microdevices which lead to a very small yield of plasma / serum volume. The whole blood separation process can also be integrated within the Point- of-Care device.

Therefore, whole blood separation methods have been developed as an alternative measure for obtaining blood plasma / serum from whole blood. These plasma / serum separation methods known in the art are however problematic in view of e.g. the blood cell concentration, the plasma / serum yield, the molecular adsorbance capacity, the extent of hemolysis, and the leakage of blood cells (erythrocytes, thrombocytes and leukocytes). Hemolysis is one of the important problems because the red blood cells, if ruptured, will alter the concentration of some plasma / serum analytes required for further testing and, in some cases, make an analysis using optical measurements techniques impossible due to the red color of the released hemoglobin. Moreover, the leakage of blood cells is problematic because the cells or even other particles can damage the blood plasma / serum analyzers as the sensitive capillaries and conduits can become plugged. Only (substantially) cell- and hemolysis-free plasma / serum can be used for a reliable blood analysis.

This is also the case in the application of »test stripes« for qualitative blood analysis: As often color reactions are used to indicate the presence and/or concentration of a blood component, no blood cells and no hemolysis may change the color of the plasma / serum and therefore the plasma / serum has to be (substantially) cell- and hemolysis- free. US 5,674,394 discloses a small-volume disposable filtration technology to separate blood plasma from whole blood.

US 5,919,356 relates to a fluid sampling device. US2003/0206828 descibes a whole blood sampling device.

US 5906742 A is directed to micro filtration membranes having high pore density and mixed isotropic and anisotropic structure. WO2012/143894 Al relates to a method and device for the determination of analytes in whole blood.

W093/19831 relates to a blood separation filter assembly and corresponding methods.

US 5,186,843 relates to blood separation media and methods for separating plasma from whole blood.

US020040222168A1 describes a device for separating and discharging plasma.

A need remains for filter media for separating blood plasma / serum from whole blood, which allow for an effective separation of blood plasma / serum from whole blood and which are suitable for use in a quick, safe and robust way to get a suitable amount of cell-free plasma / serum, without causing hemolysis. With this kind of separation process a deterioration of the blood quality after the blood withdrawal from the patient or bad analysis results due to a time delay in a centrifugation process or due to transportation will be avoided as the blood cell separation can be performed immediately without a centrifuge in an emergency case or at the point of collection of the blood sample.

One option of a system for the separation of blood plasma/serum from blood cells without using a centrifuge is the separation with a flat sheet filter medium. However such a separation method faces several challenges. Flat sheet filter media only provide a limited capacity for taking up and retaining blood cells inside or on the surface of the filter. Typically, flat sheet filter media are applied in filtration of fluids with a low particle content instead of whole blood, which consists of approximately 40-50% of cells. Particularly, small pores within these filters tend to clog quickly due to the formation of a filter cake on top of the raw side, which leads to a high loss of pressure over the filter. The pressure loss would then require an increase in pressure to continue the filtration process, but high pressure would cause the blood cells to burst and subsequent occurrence of hemolysis in the filtrate. In contrast, if the pores of a flat sheet filter are too big, the clogging of the filter may be reduced, but instead some of the blood cells may then pass through the filter into the filtrate. Further, the amount of blood volume for a flat sheet filter medium is typically highly dependent on the filter surface area, and thus its capacity.

It is therefore an object of the present invention to provide the use of a whole blood filter medium and a process for separating blood plasma / serum from whole blood, which are advantageous over the prior art, in particular regarding the problems of hemolysis and leakage of blood cells (erythrocytes, thrombocytes and leukocytes).

It is another object of the present invention to provide the use of a whole blood filter medium and a process for separating blood plasma / serum from whole blood, wherein the separation of a sufficient amount of cell-free blood plasma / serum is possible with no or substantially no hemolysis. It is yet another object of the present invention to provide the use of a whole blood filter medium and a process for separating blood plasma / serum from whole blood, wherein the separation of blood plasma / serum is possible, preferably in a manual way or in an easy automatic way without using centrifugation means.

It is another object of the present invention to provide the use of a whole blood filter medium and a process for separating blood plasma / serum from whole blood, wherein the separation is less time consuming than the separation with conventional methods such as centrifugation methods.

It should be noted in this regard that there is typically no need that the blood cells are recovered so that the process would require the step of isolating the blood cells from the filter. It is another object of the present invention to provide a blood filter medium, particularly a whole blood filter medium, and a process for separating blood plasma / serum from whole blood from a whole blood sample in an emergency case. Ideally, the cell separation can already take place at the scene of blood withdrawal. Subsequently the obtained plasma / serum sample can be immediately processed and can be directly delivered into the blood plasma / serum analyzer or to a lab-on-a-chip blood analysis product or to a Point-of-Care testing device. The term emergency case comprises not only patient diagnosis from accidents, but also all blood treatment processes as they are provided from medical offices or patient control during surgeries in hospitals. In this regard, it is also an object to overcome the bottleneck problem of centrifugation and/or to avoid a falsification of the blood analysis due to a long treatment or transport of the unseparated whole blood sample.

It is another object of the present invention to provide a whole blood filter medium and a process for separating blood plasma / serum from whole blood, which reduces the risk of a leakage of red blood cells into the filtrate. It is another object of the present invention to provide a whole blood filter medium and a process for separating blood plasma / serum from whole blood, which leads to a cell-free or substantially cell-free plasma / serum as a filtrate, wherein the relative amounts of the molecular components to be analyzed remain substantially unchanged upon filtration. Ideally, the process comprises a filter medium that is inert and hemocompatible, releases no extractables or particles, and neither leads to the adsorption of particular blood plasma / serum components on its solid surface nor to a cross-reaction of particular blood plasma / serum components with its solid surface. It is another object of the present invention to provide a whole blood filter medium, which can be used for separating blood plasma / serum from a whole blood sample, wherein the whole blood filter medium does not induce rupture of blood cells e.g. due to frictional forces or other mechanical stresses. It is another object of the present invention to provide a whole blood filter medium, which can be used for separating blood plasma / serum from a whole blood sample without clogging of the filter medium.

Summary of the invention

In one aspect, the present invention is related to the use of a flat sheet filter medium for separating blood plasma or blood serum from whole blood, wherein the flat sheet filter medium comprises a first material comprising:

a. microglass fibers treated with a binder;

b. microglass fibers treated with a laminate; or

c. combinations thereof.

In yet another aspect, the present invention is directed to a process for separating plasma or serum from whole blood comprising the steps of:

i. providing a flat sheet filter medium comprising microglass fibers treated with a binder and/or a laminate;

ii. optionally positioning the flat sheet filter medium between orifice plates at the raw side and/or the clean side; optionally providing a mesh at the clean side of the flat sheet filter medium;

applying whole blood to the raw side of the flat sheet medium; optionally applying a positive or negative pressure, preferably up to 0.8 bar and more preferably of up to 0.4 bar in respect to ambient pressure; and

collecting plasma or serum filtrate from the clean side of the filter.

In yet another aspect, the present invention is directed to a process for separating plasma or serum from whole blood comprising the steps of:

i. providing a flat sheet filter medium comprising microglass fibers treated with a binder and/or laminate;

ii. applying whole blood to a first zone of the flat sheet medium; iii. lateral propagation of blood plasma or serum from a first zone to a second zone, wherein the second zone at least partially extends over the first zone;

iv. optionally reaction of the blood plasma or serum analytes with a reagent located in the second zone.

In yet another aspect the present invention is directed to a process for separating plasma or serum from whole blood comprising the steps of:

i. providing a flat sheet filter medium comprising microglass fibers treated with a binder and/or laminate;

ii. applying whole blood to a first zone of the flat sheet filter medium;

iii. lateral propagation of blood plasma or serum from a first zone to a second zone, wherein the second zone at least partially extends over the first zone;

iv. collecting the plasma or serum from the second zone.

Detailed description of invention As used herein, the term "whole blood" refers to blood composed of blood plasma, which is typically unclotted, and cellular components. The plasma represents about 50 to about 60% of the volume, and cellular components, i.e. erythrocytes (red blood cells, or RBCs), leucocytes (white blood cells, or WBCs), and thrombocytes (platelets), represent about 40 to about 50% of the volume. As used herein, the term "whole blood" may refer to whole blood of an animal, but preferably to whole blood of a human subject.

Erythrocytes, which contribute with about 90 to about 99% to the total number of all blood cells, have the form of biconcave discs and measure about 7 μιη in diameter with a thickness of about 2 μιη in an undeformed state. During maturation in the bone marrow the erythrocytes lose their nucleus. They contain the plasma membrane protein spectrin and other proteins to provide flexibility to change shape as necessary. Their unique and flexible shape enables them to pass through very narrow capillaries and provides for maximum surface area to transfer oxygen and carbon dioxide. This flexibility makes it particularly difficult to separate the red blood cells from a whole blood sample by filtration as they can elongate themselves and reduce their diameter down to about 1.5 μιη. Normal whole blood has approximately 4.5 to 5.5 million erythrocytes per microliter. The life-span of erythrocytes is approximately 120 days in the circulating bloodstream. One core component of erythrocytes is hemoglobin which binds oxygen for transport to the tissues, then releases oxygen and binds carbon dioxide to be delivered to the lungs as waste product. Hemoglobin is responsible for the red color of the erythrocytes and therefore of the blood in total. Erythrocytes are the major factor contributing to blood viscosity.

Leucocytes make up less than about 1% of the total number of all blood cells and can be differentiated into different white blood cell groups (lymphocytes, granulocytes and monocytes). They can leave capillaries via diapedesis. Furthermore, they can move through tissue spaces by amoeboid motion and positive chemotaxis. They have a diameter of about 6 to about 20 μιη. Leucocytes participate in the body's defense mechanisms e.g. against bacterial or viral invasion. Thrombocytes are the smallest blood cells with a length of about 2 to about 4 μιη and a thickness of about 0.9 to about 1.3 μιη. They are membrane-bound cell fragments that contain enzymes and other substances important to clotting. In particular, they form a temporary platelet plug that helps to seal breaks in blood vessels.

The terms "blood plasma" or "plasma" refer to the liquid part of the blood and lymphatic fluid, which makes up about half of the volume of blood (e.g. about 50 to about 60 vol.-%). Plasma is devoid of cells, and unlike serum, has not clotted. So it contains all coagulation factors, in particular fibrinogen. It is a clear yellowish liquid comprising about 90 to about 95 vol.-% water.

The term "blood serum" or "serum" refers to the clear liquid that separates from blood when it is allowed to clot completely, and is therefore blood plasma from which in particular fibrinogen has been removed during clotting. Like plasma, serum is light yellow in color.

Molecular plasma / serum components can be classified into different groups including electrolytes, lipid metabolism substances, markers, e.g. for infections or tumors, enzymes, substrates, proteins and even pharmaceuticals and vitamins.

As used herein, the term "cell-free" describes a plasma / serum sample with no or substantially no cells (erythrocytes, leucocytes, thrombocytes) in its volume that is prepared by e.g. a centrifuge. A substantially cell-free or cell-free sample is needed for a subsequent plasma / serum analysis to prevent blocking of the analysis system and to prevent color falsification within "test stripes".

For the plasma analysis performed with the plasma, which is obtained by filtration or by pressing out liquid out of the filter medium, the following analytes may be chosen which comprise the relevant molecular groups. The reference concentration ranges of these chosen analytes for whole blood with heparin stabilization depend on the applied measurement technique. The following exemplary reference concentration ranges of these chosen analytes are obtained by the analysis device "Dimension" from Siemens.

Plasma components Reference concentration ranges of analytes for whole blood with heparin stabilization and the chosen measurement device

Electrolytes Potassium 3.5 - 5.1 mmol/1

Sodium 136 - 145 mmol/1

Calcium 2.12 - 2.52 mmol/1

Magnesium 0.74 - 0.99 mmol/1

Chloride 98 - 107 mmol/1

Phosphate 0.80 - 1.60 mmol/1

Lipids Triglycerides 75 - 175 mg/dl

Cholesterol 110 - 200 mg/dl

HDL-cholesterol 35 - 60 mg/dl

LDL-cholesterol < 150 mg/dl

Infection markers CRP 0 - 5.00 mg/1

Enzymes AST/GOT 0 - 35 Unit/1

Lipase 114 - 286 Unit/1 Substrates Albumin 3.4 - 5.0 g/dl

Bilirubin 0 - l .O mg/dl

Glucose 74 - 106 mg/dl

Creatinine 0.60 - 1.30 mg/dl

Proteins IgG 6.81 - 16.48 g/1

Ferritine 3.0 - 244 ng/1

Hormones TSH basal 0.36 - 16.00 mUnit/1

The analysis device "Dimension" from Siemens may not only be used for the analysis of blood plasma, but also for the analysis of blood serum. As used herein, the expression "ensuring permeability", for example "to blood plasma or serum" or "to whole blood", preferably means that none of the above mentioned plasma or serum components to be analyzed is retained completely upon separation. Preferably, the concentrations of the plasma or serum components to be analyzed are not significantly changed compared to the whole blood sample before separation. More preferably, the concentrations of the plasma or serum components to be analyzed are changed by not more than about 50%, preferably by not more than about 35%, more preferably by not more than about 10%, most preferably by not more than about 8%. As used herein, the term "hemolysis" refers to the rupture of erythrocytes, e.g. due to chemical, thermal or mechanical influences, causing the release of the hemoglobin and other internal components into the surrounding fluid. Hemolysis can be visually detected by showing a pink to red tinge in the plasma / serum. Hemolysis is a common occurrence seen in serum and plasma samples and may compromise the laboratory's test parameters for blood analysis. Hemolysis can occur from two sources. In vivo hemolysis may be due to pathological conditions such as autoimmune hemolytic anemia or transfusion reaction. In vitro hemolysis may be due to improper specimen sample collection, specimen sample processing or specimen sample transport. In particular, hemolysis may be caused by a high pressure drop and high shear or elongation rate, which may e.g. occur during filtration processes, when the sample is passed through a porous filter medium. Other important factors for hemolysis are bacterial contamination, pressure, temperature, osmotic environment, pH value, contact with surfaces, factional forces, blood age and storage time of the unseparated whole blood sample.

The degree of hemolysis can be detected visually in comparison to a plasma reference solution having a certain concentration of hemoglobin (Hb, Hgb). Blood plasma samples having the same color as a reference solution comprising no hemoglobin show no hemolysis. Blood plasma samples being equally or less red than a solution comprising about 50 mg/dl hemoglobin show substantially no hemolysis. In this respect, "substantially no hemolysis" means that the blood plasma samples show such a degree of hemolysis that is still sufficiently low to ensure that the samples can be analyzed with satisfactory results, e.g. by the plasma analysis device "Dimension" from Siemens. Blood plasma samples being equally or less red than a solution comprising about 100 mg/dl hemoglobin show a medium degree of hemolysis. Blood plasma samples with a color corresponding to a solution with a higher hemoglobin content than 100 mg/dl show a high degree of hemolysis.

Any medium or material which shows no interaction with whole blood is generally described as "hemocompatible". No interaction means especially that the medium or material does not cause blood clotting, e.g. by interacting with the blood coagulation system or the blood platelets. Accordingly, a hemocompatible material has no thrombotic effect. It is preferred that the bulk filter media according to the present invention are hemocompatible. Furthermore, it is preferred that the filter media do not modify any blood component concentrations by adsorption or reaction and that the contact with whole blood does not cause hemolysis. The term "diagnostic marker" as used herein refers to a molecular parameter, wherein its presence can be measured in whole blood, or preferably in blood plasma or serum or a dilution thereof. A diagnostic marker can preferably also be quantified, and it reflects the severity or presence of a physiological state or other disease state. Further, a diagnostic marker may even indicate a risk or progression of a disease, or the susceptibility of the disease to a given treatment. Diagnostic markers can be categorized in different groups, e.g.

(a) according to their molecular structure, diagnostic markers may belong to the group comprising atomic ions, lipids, lipoproteins, steroids, sugars, nucleic acids, proteins, peptides, amino acids, alcohols and porphyrins;

(b) according to their function, diagnostic marker may belong to the group comprising electrolytes, enzymes, substrates , antibodies, hormones, toxins, neurotransmitters, drugs, metabolites, lipid metabolites, transport proteins, vitamins, or

(c) according to their molecular weight; diagnostic markers may belong to the group comprising small molecule analytes of a molecular weight between 10 and 2000 Da or large molecules, which comprise proteins and protein complexes with a molecular weight higher than 2000 Da; or

(d) according to their application in the detection of a specific disease; diagnostic markers may belong to the group comprising cancer markers, cardiac markers, autoimmune markers, metabolic markers.

Examples of diagnostic markers comprise potassium cation, sodium cation, calcium cation, magnesium cation, chloride, phosphate, triglycerides, cholesterol, high density lipoprotein (HDL)-cholesterol, low density lipoprotein (LDL)-cholesterol, C- reactive protein (CRP), aspartate transaminase/ glutamic-oxaloacetic transaminase (AST/GOT), lipase, albumin, bilirubin, glucose, creatinine, IgG, ferritine, TSH, insulin, rheumatoid factors, prostate-specific antigen (PSA), S100B, cytochrome C, creatine kinase or troponin.

As used here, the term "microglass fibers" refers to glass fibers used in filter media that have diameters in the range of micrometers. The term "mesh" as used herein refers to a solid medium, preferably a filter medium which is preferably flat and preferably produced of polymeric or metal fibers which are combined geometrically as e.g. square mesh, reverse plain Dutch weave, single plain Dutch weave or Dutch twilled weave by textile weaving technologies. Preferably, the mesh does not contribute to the separation of plasma from whole blood and preferably provides a stabilizing effect and thereby counteracts the deformation of the flat sheet filter to avoid filter rupture. Preferably, the mesh provides a mesh opening between 50 μιη and 1000 μιη, preferably between 150 μιη and 400 μιη, and even more preferably between 200 and 350 μιη. Preferably, the mesh is made of hydrophobic fibers or with fibers that are coated with a hydrophobic coating.

The term "capillary effects" refers to the flow of a liquid in narrow spaces without the assistance of an external force like gravity or pressure. It is based on intermolecular forces between the liquid and solid surrounding surfaces, wherein the combination of surface tension and adhesive forces between the liquid and surrounding material act to move the liquid. As used herein the "raw side" or "upstream" side of a filter is the side or surface through which the fluid enters the filter medium. It is considered as the entering side or surface. The "clean side" or "downstream" side of a filter is the side or surface through which the fluid exits the filter medium. It is also considered as the exiting side or surface.

The term "hydrophilic" refers to a surface, which leads to a water or blood droplet contact angle smaller than 90° and therefore to a spreading of the droplet over the surface, "hydrophobic" surfaces lead to a water or blood droplet contact angle bigger than 90° and therefore to a water repelling effect. The contact angle may for example be determined by contact angle measurement systems commonly known in the art, e.g. contact angle measurement systems by Kruss. As used herein, terms such as "hydrophilic binder" or "hydrophilic laminate" refer to a binder or laminate that renders a surface, for example of a microglass fiber or of a flat sheet filter medium, hydrophilic. Likewise, terms such as "hydrophobic binder" or "hydrophobic laminate" refers to a binder or laminate that renders a surface, for example of a microglass fiber or of a flat sheet filter medium, hydrophobic.

The term "flat sheet filter" as used herein relates to filters wherein the filter media is relatively "flat" and thus preferably provides a thickness of less than 5 mm, more preferably less than 2 mm and even more preferably less than 1 mm. A flat sheet filter may comprise one or more layers of filter media.

The term "orifice plate" refers to a thin plate with at least one hole, preferably in the middle of the plate. Preferably the plate is impenetrable to whole blood or to any blood components. Therefore, the orifice plate per se does not constitute a filter medium. In this context, the term "multi-bore orifice plates" refers to orifice plates that have more than one hole.

The term "lateral filtration" refers to a so-called "wicking" separation process, wherein a specific volume of blood is applied to a surface area of the filter medium and then the blood cells of the whole blood are separated from the plasma or serum by different transport mechanisms and/or different transport kinetics on the same side or in the depth of the filter medium. Lateral filtration is, for example, commonly used in test stripes for blood analysis, wherein a drop of blood is applied to a loading zone and the plasma or serum is then transported through the stripe into a detection zone in which specified analytes of the plasma or serum can undergo a chemical reaction with a detection agent. The resulting signal can be measured as qualitative and quantitative signals, e.g. in a colorimetric or fluorescent assay.

The term "grammage" as used herein refers to the density of a flat filter medium as a measure of mass of the filter medium per unit of filter medium area. The unit of grammage is g/m ² . Grammage is also known as paper-density. The term "MFP" refers to the mean flow pore size which is the pore diameter at a pressure drop at which the flow through a wetted medium is 50% of the flow through the dry medium. To determine the mean flow pore size experimentally the method of capillary flow porometry is applied. This method uses the simple principle of gas pressure to force a wetting liquid out of through-pores in a sample. Through-pores are simply those that connect from one side of the sample to the other. The pressure at which pores empty is inversely proportional to the pore size, larger pores require a lower pressure than do smaller pores. The resulting volumetric flow of gas through emptied pores is also measured. The corresponding pore size d opened and throughflown by the gas at a certain differential pressure Δρ is calculated using the Washburn equation

4 v cos ϋ

d =— Δ,—ρ

With the interfacial tension γ between the wetting liquid and ambient gas phase and the contact angle 0 between the wetting liquid and the solid surface.

The largest pore to be emptied (at the lowest pressure at which flow is sensed) defines the so-called bubble point. After all pores have been emptied (up to the highest pressure achievable) during a wet run, a second dry run is performed on the same sample. As a result complete data sets comprising the volume flux (for the wet and dry run) versus the imposed pressure difference is obtained. From this data set the point at which the flow through a wetted medium is 50% of the flow through the dry medium is determined as the mean flow pore size. For example, the mean flow pore size may be determined according to ASTM F 316-03.

The term "binder" as used herein, according to the„Dictionary of Filtration and Separation" (Steve Tarleton and Richard Wakeman), is an organic or inorganic solid or liquid that is used to help fuse/bond fibers or particles, such as those of a filter medium. For instance, resins may be used to bring about resin bonding where the web is dipped in, or sprayed with, resin and subsequently dried and/or set to produce the final product. As used herein, the term "laminated media" refers to, according to the„Dictionary of Filtration and Separation" (Steve Tarleton and Richard Wakeman), two or more of layers of media fixed together. As used herein, the term "laminate" refers to an organic or inorganic solid or liquid that is used to fix two or more layers of media together in a "laminated media" as described above.

In a first embodiment, a flat sheet filter medium is used for separating blood plasma or blood serum from whole blood, wherein the flat sheet filter medium comprises a first material comprising:

a. microglass fibers treated with a binder;

b. microglass fibers treated with a laminate; or

c. combinations thereof.

In another embodiment, the flat sheet filter medium comprises a blend of glass fibers treated with a binder and/or a laminate.

In another preferred embodiment, the binder is selected from the group consisting of epoxy, latexacryl or acrylic resins or combinations thereof. In one preferred embodiment, the flat sheet filter medium comprises microglass fibers treated with a binder and/or a laminate, wherein the binder is a hydrophilic binder, a hydrophobic binder or combinations thereof.

In one embodiment, the binder and/or laminate is a hydrophilic binder, wherein preferably the hydrophilic binder is selected from the group consisting of epoxy, latexacryl or combinations thereof.

In another embodiment, the binder and/or laminate is a hydrophobic binder, wherein preferably the hydrophobic binder comprises acrylic resin. In another embodiment, the laminate is a hydrophilic laminate, wherein preferably the hydrophilic laminate is selected from the group consisting of epoxy or latexacryl or combinations thereof. In another embodiment, the laminate is a hydrophobic laminate, wherein preferably the hydrophobic laminate comprises acrylic resin.

In another preferred embodiment the binder is a hydrophilic binder for lateral filtration, and preferably is epoxy or latexacryl or combinations thereof.

In another preferred embodiment, the lamination by a hydrophobic laminate or the treatment with a hydrophobic binder preferably leads to a hydrophobic surface for the separation of blood plasma from the raw side of the flat sheet filter medium to the clean side.

In yet another preferred embodiment, the glass fibers are selected from borosilicate fibers, alkali-free borosilicate fibers, quartz fibers or soda lime glass or combinations thereof, and are preferably borosilicate fibers. In another embodiment, the first material of the flat sheet filter medium has a thickness between about 0.2 mm and about 1 mm, preferably between about 0.2 mm and about 0.9 mm, more preferably between about 0.3 mm and about 0.8 mm and even more preferably between about 0.4 mm and about 0.6 mm. In yet another embodiment, the first material of the flat sheet filter medium provides a mean flow pore size between about 2 μιη and about 8 μιη, preferably between about 2 μιη and about 5.5 μιη, even more preferably between about 2 μιη and about 4 μιη and most preferably between about 2.5 μιη and about 3 μιη. In a preferred embodiment, the microglass fibers of the first material are treated with a hydrophobic binder. In another embodiment, the grammage of the first material of the flat sheet filter medium is between about 70 g/m ² and about 100 g/m ² and preferably between about 72 g/m ² and about 85 g/m ² . In a particularly preferred embodiment, the flat sheet filter medium comprises borosilicate fibers with a laminate and/or hydrophobic binder to obtain a hydrophobic surface, wherein the laminate and/or the binder is an acrylic resin and the flat sheet filter medium provides a thickness between about 0.4 mm and about 0.6 mm, a mean flow pore size between about 2.5 μιη and about 3 μιη and a grammage between about 72 g/m ² and about 85 g/m ² .

In yet another embodiment, the flat sheet filter medium comprising a first material comprising microglass fibers treated with a hydrophilic binder and/ or laminate provides a mean flow pore size between about 2 μιη and about 10 μιη, preferably between about 2.5 μιη and about 9 μιη, even more preferably between about 3 μιη and about 8.5 μιη and most preferably between about 3.5 μιη and about 8.0 μιη.

In a particularly preferred embodiment, the first material of the flat sheet filter medium comprises fibers treated with a hydrophilic binder and/or laminate to obtain a hydrophilic surface, wherein the hydrophilic binder and/or laminate preferably is selected from the group consisting of epoxy, latex acryl binder or combinations thereof and the flat sheet filter medium provides a thickness lower than 0.3 mm, a mean flow pore size between about 3.5 μιη and about 8 μιη and a grammage lower than 80 g/m ² .

In another preferred embodiment, the flat sheet filter comprises a mesh downstream of the first material. Preferably, the mesh is a polymeric woven mesh. A mesh with a hydrophobic surface with a pore size ensuring permeability to whole blood, blood plasma or serum is preferred. Even more preferably, the mesh provides a mesh opening of between about 250 μιη and about 350 μιη. In a preferred embodiment, the mesh is a woven mesh, preferably a polymeric woven mesh, preferably of polyester fibers with a mesh opening of about 250 to about 300 μιη and an open area of 44% that provides a mesh count of about 23/cm, a wire diameter of about 145 μιη, a weight of about 110 g/m ² and a thickness of about 255 μιη, wherein the mesh is coated with a hydrophobic coating.

In another preferred embodiment, the mesh is a woven uncoated mesh of hydrophobic fibers. In another embodiment, a second material is located upstream of the first material of the flat sheet filter medium, wherein preferably the second material is a hydrophobic material. The hydrophobic material can be selected from the group consisting of polyester, polypropylene, polyethylene terephthalate or a combination thereof. The hydrophobic material can be a nonwoven material, preferably a meltblown nonwoven material.

In another embodiment, the hydrophobic material comprises one or more layers of polypropylene nonwoven. In yet another preferred embodiment, the flat sheet filter medium is positioned between orifice plates or multi-bore orifice plates.

These orifice plates are made of a material which is not permeable to blood, plasma or serum.

In one embodiment the orifice plates are made of a polymeric material, preferably a hydrophobic material, e.g. polypropylene.

In another embodiment the plates are made of a flat material with a thickness between about 0.3 mm and about 3 mm. In yet another embodiment the orifice plates show one central hole with a circular cross section which allows the blood, plasma or serum through this hole.

In yet another preferred embodiment the orifice plates show some annular holes with material fillets between which allow the blood, plasma or serum through these holes. In yet another preferred embodiment the orifice plates show some holes with different geometries which are combined within one orifice plate which allow the blood, plasma or serum through these holes. In yet another preferred embodiment different orifice plates with different hole shapes are combined within a filter. Preferably the orifice plate with the central hole is positioned downstream of the flat sheet filter medium, and the orifice plate with annular holes is positioned upstream of the flat sheet filter medium. In one embodiment, the flat sheet filter medium is used for the filtration from the raw side towards the clean side.

In another embodiment, the flat sheet filter medium is used for lateral filtration. In a further embodiment, the whole blood is diluted with isotonic sodium chloride solution. Preferably, the whole blood sample is diluted with isotonic sodium chloride solution, in a ratio of from about 0.5: 1 to about 1 :5, preferably in a ratio of from about 1 :1 to about 1 :4. In a preferred embodiment, the isotonic sodium chloride solution is a 0.9% sodium chloride solution (w:v).

In a preferred embodiment, the whole blood of the sample is stabilized with an anticoagulation agent selected from the group consisting of EDTA, citrate, heparin and combinations thereof. In another preferred embodiment, the whole blood of the sample is pre-treated with a cell agglomeration agent, such as lectin. In another embodiment, the separation of plasma or serum from whole blood comprises the steps of:

i. providing a flat sheet filter medium comprising microglass fibers treated with a binder and/or a laminate ;

ii. optionally positioning the flat sheet filter medium between orifice plates at the raw side and/or the clean side;

iii. optionally providing a mesh at the clean side of the flat sheet filter medium;

iv. application of whole blood to the raw side of the flat sheet filter medium;

v. optionally applying a positive or negative pressure, preferably up to 0.8 bar and more preferably of up to 0.4 bar in respect to ambient pressure; and

vi. collecting plasma or serum filtrate from the clean side of the filter medium.

The pressure in step iv can be positive pressure applied to the raw side or negative pressure applied to the clean side. In one embodiment, the positive or negative pressure is induced with a syringe. Preferably the pressure is less than 0.8 bar, even more preferably less than 0.4 bar and even more preferably less than 0.3 bar.

In a preferred embodiment, the flat sheet filter medium of step i comprises microglass fibers treated with a binder and/or laminate, wherein the binder and/or laminate is a hydrophobic binder and/or laminate, wherein preferably the hydrophobic binder and/or laminate comprises acrylic resin.

In yet another aspect, the present invention is directed to a process for separating plasma or serum from whole blood comprising the steps of:

i. providing a flat sheet filter medium comprising microglass fibers treated with a binder and/or laminate;

ii. applying whole blood to a first zone of the flat sheet filter medium; iii. lateral propagation of blood plasma or serum from a first zone to a second zone, wherein the second zone at least partially extends over the first zone;

iv. optionally reaction of the blood plasma or serum analytes with a reagent located in the second zone.

In a preferred embodiment, the flat sheet filter medium of step i comprises microglass fibers treated with a binder and/or laminate, wherein the binder and/or laminate is a hydrophilic binder and/or laminate and more preferably, the binder and/or laminate is selected from the group consisting of epoxy, latexacryl and combinations thereof.

In yet another aspect the present invention is directed to a process for separating plasma or serum from whole blood comprising the steps of:

i. providing a flat sheet filter medium comprising microglass fibers treated with a binder and/or laminate;

ii. applying whole blood to a first zone of the flat sheet filter medium;

iii. lateral propagation of blood plasma or serum from a first zone to a second zone, wherein the second zone at least partially extends over the first zone;

iv. collecting the plasma or serum from the second zone.

In a preferred embodiment, the flat sheet filter medium of step i comprises microglass fibers treated with a binder and/or laminate, wherein the binder and/or laminate is a hydrophilic binder and/or laminate and more preferably, the binder and/or laminate is selected from the group consisting of epoxy, latexacryl and combinations thereof.

In another embodiment of the process, the whole blood is pre-treated with a) isotonic sodium chloride solution, preferably with a 0.9% sodium chloride solution (w:v), in a ratio of from 0.5 : 1 to 1 :5, preferably in a ratio of from 1 : 1 to 1 :4;

b) an anti-coagulation agent selected from the group consisting of EDTA, citrate, heparin and combinations thereof; or

c) a cell agglomeration agent, preferably with lectin.

In another embodiment, the flat sheet filter media does not contain cellulose fibers. Example 1 Drop test

Every filter medium reacts differently upon direct contact with a drop of fluid or blood. Consequently, in a first assay, one drop of blood was applied to the flat sheet filter media. The test was carried out on both sides of the filter, the raw side and the clean side. The observations of a drop test allow a first evaluation of the filter material, e.g. regarding hydrophobicity, hydrophilic capillary effects or instantaneous hemolysis upon contact. The blood samples were stabilized with heparine.

Glass fiber media without binder, Ahlstrom Grades 111 and 151, were wetted with a drop of whole blood. It was instantaneously observed that the surrounding ring inside the filter medium was red instead of yellow/orange, indicating instant hemolysis. A reason for the generation of hemolysis may be the sharp edges of the untreated microglass fibers.

Glass fiber media from Lydall with binder, LyPore XL Grade 9102-F and Grade 9104-D, were wetted with a drop of whole blood after delamination and rejection of the coarse pre-filter layer. Droplets on the treated glass fiber filter medium were quickly soaked up, building a central red dot out of blood cells, and a yellow colored wreath made of hemo lysis-free plasma around the red dot. Glass fiber media with binder and laminate, Ahlstrom Grade MFPS0201, shows a hydrophobic behavior: The contact angle between a whole blood droplet and the filter medium in air is higher than 90° and the droplet remains on the surface without being soaked up.

Example 2 filtration using glass microfibers treated with hydrophobic binder The separation efficiency of flat sheet filter media was typically investigated in at least two experiments. For filter media that were asymmetric, i.e. wherein the two surface sides of the medium were different, each side was used as the raw side. An effective filtration process to obtain liquid blood plasma out of whole blood was observed with the microglass filter media MFPS from Ahlstrom which contains an acrylic resin as binder and which possess hydrophobic surface properties. For the following test description the filter media were applied with the fine structured side downstream as the clean side. Other experiments have shown that the orientation of the filter medium does not influence the test results. For filter media that were asymmetric, i.e. wherein the two surface sides of the medium were different, each side was as the raw side. Between 0.5 and 1 ml whole blood stabilized with heparine were applied to the raw side of the filter medium in each filtration experiment. If necessary, pressure was applied using a syringe to transport the fluid through the filter medium. The coloration of the filter media on the clean side and the obtained droplets of filtrate on the clean side were analyzed to assess the separation efficiency.

In all experiments, a woven mesh of polyester fibers called Hyphobe 285/44 from Saatitech, which provides a hydrophobic coated surface with a mesh opening of 285 μιη, as a mesh was located downstream of the flat sheet filter media as a stabilizing mesh.

The flat sheet filter media were used as a round flat sheet filter in small housings. Typically, a polypropylene housing with a filter diameter of about 27.7 mm and a filter area of about 5.81 cm ² was used. Downstream of the filter medium, the housing was open to observe the filtration result. In one experiment, the filter medium was MFPS0201 from Ahlstrom with a mean flow pore size of 2.8 μιη. Further, MFPS0301 was used with a mean flow pore size of 3.5 μιη as a filter medium. The results of the filtration experiments with different filter media are summarized in Table 1

Table 1

Example 3 analysis of diagnostic markers after blood plasma filtration process

In order to obtain adequate diagnostic marker results, the whole blood plasma filtration was performed in a housing of medically applicable polypropylene. The flat sheet filter comprised Ahlstrom MFPS0201 and a Hyphobe mesh as described above. To focus on possible deviations of plasma diagnostic markers due to interactions between the plasma and the flat sheet filter medium, pre-centrifuged plasma samples were used in the filtration process.

A syringe was filled with the pre-separated and pre-centrifuged plasma sample and tapped on the upper inlet of the filter, pushing the plasma through the flat sheet filter medium, eluting the filtrate into an Eppendorf tube followed by a plasma analysis using the "Dimension" analysis device from Siemens for the analysis of a representative set of diagnostic markers. The experiment was independently repeated twice with two different plasma samples. The analytes of diagnostic markers and the deviations of the filtered plasma sample compared to the reference plasma sample, which had no contact with the filter, are shown in the following table 2 with quite low deviations. Only CRP, Bilirubin and IgG showed bigger concentration changes after filter passage, but this effect could not be reproduced. Especially sample 1 showed a very low Bilirubin content in the reference sample, so that a slight change in the small value lead to high percentage of deviation.

Table 2: Deviations of analytes after passage through the flat sheet filter MFPS0201

Example 4 use of glass fiber with binder in lateral whole blood filtration

The drop test was performed using LyPore XL 9102-F. The filter medium was an asymmetric filter material that could be delaminated into a first layer and a second layer. The drop test was performed with both layers. The drop test showed that a lateral filtration, i.e. the formation of a yellow ring around the area of blood drop application, occurred using only one of the layers, whereas the other layer did not separate plasma from blood cells and no yellow plasma front was obtained around the whole blood application area.

Example 5 use of glass fiber with hydrophilic binder in lateral whole blood filtration

The wicking test was performed using LyPore XL 9102-F and LyPore XL 9104-D from Lydall. Both filter media represent an asymmetric filter material that can be easily and manually delaminated into a first fine structured layer and a second fluffy and coarse layer.

A first drop test with LyPore XL 9102-F and 9104-D showed that a lateral filtration, i.e. the formation of a yellow ring around the area of blood drop application, occurred using only the first layer, whereas the other fluffy layer did not separate plasma from blood cells and no yellow plasma front was obtained around the whole blood application area (see also Example 4). Only the first layer was used in the subsequent experiments. Thus, only the first layer was applied for the wicking test.

To compare the results with common wicking filter media for blood separation, the filter grades CytoSep 1660 and 1662 from Ahlstrom were applied (see above mentioned patent application US 5,186,843), which are used for lateral separation of whole blood.

For the wicking test, the filter medium was cut into strips of 5 cm length and 8 mm width. Each of these strips was vertically applied into a glass tube which was filled with 0.3 -0.35 ml whole blood, so that one end of the strip was in direct contact with the whole blood sample. It could be observed that a wetting of the filter media took place and that the filter strip pores became filled with liquid as the liquid frontier moved upward to the second end of the strip. All strips showed a separation effect within the filter medium: Plasma could be separated from blood cells, meaning that there were two frontiers visible. A frontier of liquid plasma (with bigger height of capillary rise) and a frontier of blood cells (with smaller height of capillary rise).

In all filter media, the part of the strip which was filled with plasma showed a yellow coloration with translucent appearance. This is an indicator that none of the filter media generated hemolysis. The different heights of capillary rise could be measured. The difference of height was calculated by reducing the height of plasma rise by the height of blood rise. The quotient of height was calculated by difference of height divided by the height of plasma rise.

In one run, the filter media strips were removed from the glass tubes after 2 minutes contact time with blood. The results are shown in table 3.

Table 3 : Height of capillary rise [mm] for different filter media after contact time of

2 minutes

The test shows that the filter media from Lydall comprising microglass fibers and hydrophilic binder showed a better separation efficiency and a more effective lateral wicking than the Ahlstrom filter medium.