METHOD FOR REMOVAL OF VIRUSES FROM BLOOD BY LECTIN AFFINITY HEMODIALYSIS

Abstract

The present invention relates to a method for using lectins that bind to pathogens having high mannose surface glycoproteins or fragments thereof which contain high mannose glycoproteins, to remove them from infected blood or plasma in an extracorporeal setting. Accordingly, the present invention provides a method for reducing viral load in an individual comprising the steps of obtaining blood or plasma from the individual, passing the blood or plasma through a porous hollow fiber membrane wherein lectin molecules are immobilized within the porous exterior portion of the membrane, collecting pass-through blood or plasma and reinfusing the pass-through blood or plasma into the individual.

Claims

1. (canceled)

2. A method of binding viral particles to an affinity matrix comprising: contacting an affinity matrix comprising an immobilized lectin with viral particles, wherein the immobilized lectin is selected from the group consisting of Galanthus nivalis agglutinin (GNA), Narcissus pseudonarcissus agglutinin (NPA), cyanovirin, and Conconavalin A, and mixtures thereof.

3. The method of claim 2, wherein the affinity matrix comprises agarose, glass, polysulfone, polyethylsulfone, polyamide, polyimide, cellulose acetate, polyacrylamide, or aminocelite.

4. The method of claim 2, wherein the affinity matrix comprises an ultrafiltration membrane.

5. The method of claim 2, wherein the affinity matrix further comprises a linker.

6. The method of claim 5, wherein the linker comprises avidin, streptavidin, biotin, protein A, or protein G.

7. The method of claim 2, wherein the affinity matrix is provided in a cartridge.

8. The method of claim 7, wherein the cartridge is configured to receive blood or plasma from an individual, pass the blood or plasma to the affinity matrix, and permit the blood or plasma to exit the cartridge after the blood or plasma has contacted the affinity matrix.

9. The method of claim 7, further comprising reinfusing the blood or plasma to the individual after the blood or plasma has exited the cartridge.

10. The method of claim 2, wherein the viral particles are from Human Immunodeficiency Virus (HIV).

11. The method of claim 2, wherein the viral particles are from Human Hepatitis C Virus (HCV).

12. A method of binding lectin binding fragments of viral particles to an affinity matrix comprising: contacting an affinity matrix comprising an immobilized lectin with lectin binding fragments of viral particles, wherein the immobilized lectin is selected from the group consisting of Galanthus nivalis agglutinin (GNA), Narcissus pseudonarcissus agglutinin (NPA), cyanovirin, and Conconavalin A, and mixtures thereof.

13. The method of claim 12, wherein the affinity matrix comprises agarose, glass, polysulfone, polyethylsulfone, polyamide, polyimide, cellulose acetate, polyacrylamide, or aminocelite.

14. The method of claim 12, wherein the affinity matrix comprises an ultrafiltration membrane.

15. The method of claim 12, wherein the affinity matrix further comprises a linker.

16. The method of claim 15, wherein the linker comprises avidin, streptavidin, biotin, protein A, or protein G.

17. The method of claim 12, wherein the affinity matrix is provided in a cartridge.

18. The method of claim 17, wherein the cartridge is configured to receive blood or plasma from an individual, pass the blood or plasma to the affinity matrix, and permit the blood or plasma to exit the cartridge after the blood or plasma has contacted the affinity matrix.

19. The method of claim 18, further comprising reinfusing the blood or plasma to the individual after the blood or plasma has exited the cartridge.

20. The method of claim 12, wherein the lectin binding fragments of viral particles are from Human Immunodeficiency Virus (HIV).

21. The method of claim 12, wherein the lectin binding fragments of viral particles are from Human Hepatitis C Virus (HCV).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 is a schematic illustration of a longitudinal cross section of an affinity cartridge.

[0022] FIG. 2 is a schematic illustration of a horizontal cross section at plane 2 in FIG. 1.

[0023] FIG. 3 is an illustration of a channel from FIG. 2. A hollow fiber membrane structure 40 is composed of a tubular section comprising a relatively tight ultrafiltration membrane 42 and relatively porous exterior portion 44 in which may be immobilized affinity molecules 46, such as lectins.

[0024] FIG. 4 is a graphical representation of the removal of gp120 from HIV loaded physiological saline. Initial gp120 was 500 ng/ml in PBS (1.6 ml/run). Gp120 was recirculated over a column containing 0.2 ml GNA agarose vs Sepharose 4B control at 0.5-0.58 ml/min and room temperature.

[0025] FIG. 5 is a graphical representation of the removal of gp120 immune complexes from HIV infected human plasma. Initial gp120 was 500 ng/ml in human HIV+plasma (1.6 ml/run). Assayed with 10 μg/well GNA/NPA plate to capture gp120 immune complexes detected with sheep anti-human IgG. Plasma was recirculated over a Glen Research column containing 0.2 ml GNA agarose vs Sepharose 4B control at 0.5-0.58 ml/min and room temperature. Lines are theoretical exponential best-fit R.sup.2=1.91 for Experimental (◯) and linear for Control (□).

[0026] FIGS. 6A and 6B demonstrate the removal of native HIV on GNA Agarose FIG. 6A is a graphical representation of a plasmapheresis exponential curve where R.sup.2=0.90 (excluding one point at 22 hours). FIG. 6B is a graphical representation of a log plot of initial removal rate, where half time ˜0.9 hours. Conditions Masterflex pump with #14 silicon tubing (1.1 ml/min). Plasma sample 3 ml initial volume (100,000 copies per ml (CPM) BBI ER8-03030-0002 native HIV). Aliquot volume was 250 ul plasma for RNA isolation. Realtime RTPCR with Sybr green tracking dye. Thermocycling 95, 60, 72, 83° C. (15, 30, 60 sec, read 6 sec). Ct calculated from the primary curve at T=20.

[0027] FIG. 7 is a graphical representation of the removal of gp120 from HIV.sup.+ blood. Initial gp120 was 100 ng/ml in human HIV.sup.+ plasma. Assayed with 0.1ug/well GNA-NPA plate with immune complexes disrupted with acid/detergent prior to assay. The blood was recirculated over a Microkros column containing 0.6 ml GNA agarose vs. Sepharose 4B control. Flow rate 0.9 ml/min at 37° C. using a Masterflex pump (1 rpm) and Pharmed 6485-16 tubing. Lines are theoretical exponential best-fit R.sup.2=0.91 for Experimental (□) (t.sub.1/2=22 min) and linear for Control (◯).

[0028] FIG. 8 is a graphical representation of the the removal of Hepatitis C virus infected blood. The blood was recirculated over a Microkros column containing 0.6 ml GNA agarose vs. Sepharose 4B control. Flow rate 0.5 ml/min at room temperature using a Masterflex pump (1 rpm) and Pharmed 6485-16 tubing. The line is a theoretical exponential best-fit R.sup.2=0.85.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The term “viral load” as used herein for the purpose of specification and claims refers to the amount of viral particles or toxic fragments thereof in a biological fluid, such as blood or plasma. Viral load is accordingly related to the number of virus particles in the body. Viral load can therefore be a measure of any of a variety of indicators of the presence of a virus, such as viral copy number per unit of blood or plasma or units of viral proteins or fragments thereof per unit of blood or plasma.

[0030] The term “high mannose glycoprotein” as used herein for the purpose of the specification and claims refers to glycoproteins having mannose-mannose linkages in the form of α-1.fwdarw.3 or α-1.fwdarw.6 mannose-mannose linkages. Some examples of such lectins include GNA, NPA, cyanovirin and Conconavalin A (ConA).

[0031] The present invention relates to a method for using lectins to remove pathogenic organisms and fragments thereof from infected blood or plasma in an extracorporeal setting. Accordingly, the present invention provides a method for reducing viral load in an individual comprising the steps of obtaining blood or plasma from the individual, passing the blood or plasma through a porous hollow fiber membrane wherein lectin molecules which bind to high mannose glycoproteins are immobilized within the porous exterior portion of the membrane, collecting pass-through blood or plasma, and reinfusing the pass-through blood or plasma into the individual.

[0032] In a preferred embodiment, the method of the present invention is carried out by using an affinity cartridge using the device illustrated in FIG. 1. Devices of this general type are disclosed in U.S. Pat. Nos. 4,714,556, 4,787,974 and 6,528,057, the disclosures of which are incorporated herein by reference. In this device, blood is passed through the lumen of a hollow fiber ultrafiltration membrane that is in intimate contact, on the non-blood wetted side of the membrane, with immobilized lectins, which form a means to accept and immobilize viruses and toxic and/or infectious fragments thereof. Thus, the device retains intact virions and viral glycoproteins bound by lectin while allowing other components to pass through the lumen.

[0033] HIV is the prototypic virus for which this invention is described, but the invention can be adapted to the removal of any blood-borne viruses. The device, described in detail in FIGS. 1-3 includes multiple channels of hollow fiber ultrafiltration membrane that forms a filtration chamber. An inlet port and an effluent port are in communication with the filtration chamber. The ultrafiltration membrane is preferably an anisotropic membrane with the tight or retention side facing the bloodstream. The membrane is conveniently formed of any number of polymers known to the art, for example, polysulfone, polyethersulfone, polyamides, polyimides, cellulose acetate, and polyacrylamide. Preferably, the membrane has pores 200-500 nm in diameter, which will allow passage of intact viruses and viral particles and fragments (e.g., HIV virions of 110 nm diameter), but not most blood cells (red blood cells 2,000 nm diameter, lymphocytes 7,000-12,000 nm diameter, macrophages 10,000-18,000 nm diameter). A diagram of the device is shown in FIG. 1. The device comprises a cartridge 10 comprising a blood-processing chamber 12 formed of interior glass wall 14. Around chamber 12 is an optional exterior chamber 16. A temperature controlling fluid can be circulated into chamber 16 through port 18 and out of port 20. The device includes an inlet port 32 for the blood and an outlet port 34 for the effluent. The device also provides one or more ports 48 and 50, for accessing the extrachannel space in the cartridge. As shown in FIGS. 1 and 2, chamber 12 contains a plurality of ultrafiltration membranes 22. These membranes preferably have a 0.3 mm inside diameter and 0.5 mm outside diameter. FIG. 3 is a cross sectional representation of a channel 22 and shows the anisotropic nature of the membrane. As shown in FIG. 3, a hollow fiber membrane structure 40 is composed of a single polymeric material which is formed into a tubular section comprising a relatively tight ultrafiltration membrane 42 and relatively porous exterior portion 44 in which may be immobilized lectins 46. During the operation of the device, a solution containing the lectins is loaded on to the device through port 48. The lectins are allowed to immobilize to the exterior 22 of the membrane in FIG. 2. Unbound lectins can be collected from port 50 by washing with saline or other solutions.

[0034] For the method of the present invention, blood having viral particles and/or fragments thereof is withdrawn from a patient and contacted with an ultrafiltration membrane. In one preferred embodiment, the blood is separated into its plasma and cellular components. The plasma is then contacted with the lectins to remove the viral particles or fragments thereof by binding between viral high mannose glycoproteins and lectins. The plasma can then be recombined with the cellular components and returned to the patient. Alternatively, the cellular components may be returned to the patient separately. The treatment can be repeated periodically until a desired response has been achieved. For example, the treatment can be carried out for 4 hours once a week.

[0035] The technology to immobilize enzymes, chelators, and antibodies in dialysis-like cartridges has been developed (Ambrus et al. Science 201(4358): 837-839, 1978; Ambrus et al. Ann Intern Med 106(4): 531-537, 1987; Kalghatgi et al. Res Commun Chem Pathol Pharmacol 27(3): 551-561, 1980) and is incorporated herein by reference. These cartridges can be directly perfused with blood from patients through direct venous access, and returned to the patients without further manipulations. Alternatively, blood can be separated into plasma and cellular components by standard techniques. The cellular components may be combined with the plasma before reinfusing or the cellular components can be reinfused separately. Viral load can be assessed in the effluent from the cartridge by standard techniques such as ELISA and nucleic acid amplification and detection techniques. Prototypic cartridges have been used to metabolize excess phenylalanine (Kalghatgi et al., 1980, supra; Ambrus, 1978, supra) or to remove excess aluminum from patients' blood (Anthone et al. Jr Amer Sac Nephrol 6: 1271-1277, 1995). An illustration of preparing proteins for immobilization to the hollow fibers for the method of the present invention is presented in U.S. Pat. Nos. 4,714,556 and 4,787,974, 5,528,057.

[0036] For binding of lectins to the ultrafiltration membrane, the polymers of the ultrafiltration membrane are first activated, i.e., made susceptible for combining chemically with proteins, by using processes known in the art. Any number of different polymers can be used. To obtain a reactive polyacrylic acid polymer, for example, carbodiimides can be used (Valuev et al., 1998, Biomaterials, 19:41-3). Once the polymer has been activated, the lectins can be attached directly or via a linker to form in either case an affinity matrix. Suitable linkers include, but are not limited to, avidin, strepavidin, biotin, protein A, and protein G. The lectins may also be directly bound to the polymer of the ultrafiltration membrane using coupling agents such as bifunctional reagents, or may be indirectly bound. In a preferred embodiment, GNA covalently coupled to agarose can be used to form an affinity matrix.

[0037] The following examples are presented to illustrate this invention and are not intended to be restrictive.

EXAMPLE 1

[0038] This Example demonstrates the preparation of an affinity matrix using GNA covalently coupled to Agarose using Cyanogen Bromide. Cyanogen bromide (CNBr) activated agarose was used for direct coupling essentially according to Cuatrecasas, et al (Cuatracasas et al. Proc Natl Acad Sci USA 61(2): 636-643, 1968). In brief, 1 ml of GNA at a concentration of 10 mg/ml in 0.1M NaHCO.sub.3 pH 9.5 is added to 1 ml CNBr activated agarose (Sigma, St. Louis, Mo.) and allowed to react overnight in the cold. When the reaction is complete, unreacted materials are aspirated and the lectin coupled agarose washed extensively with sterile cold PBS. The lectin agarose affinity matrix is then stored cold until ready for use. Alternatively, GNA agarose is available commercially from Vector Labs (Burlingame, Calif.)

EXAMPLE 2

[0039] This Example demonstrates preparation of the lectin affinity matrix using GNA covalently coupled to glass beads via Schiff's base and reduction with cyanoborohydride. The silica lectin affinity matrix was prepared by a modification of the method of Hermanson (Hermanson. Bioconjugate Techniques: 785, 1996). GNA lectin was dissolved to a final protein concentration of 10 mg/ml in 0.1M sodium borate pH 9.5 and added to aldehyde derivatized silica glass beads (BioConnexant, Austin Tex.). The reaction is most efficient at alkaline pH but will go at pH 7-9 and is normally done at a 2-4 fold excess of GNA over coupling sites. To this mixture was added 10 μl 5M NaCNBH.sub.3 in 1N NaOH (Aldrich, St Louis, Mo.) per ml of coupling reaction and the mixture allowed to react for 2 hours at room temperature. At the end of the reaction, remaining unreacted aldehyde on the glass surfaces are capped with 20 μl 3M ethanolamine pH 9.5 per ml of reaction. After 15 minutes at room temperature, the reaction solution was decanted and the unbound proteins and reagents removed by washing extensively in PBS. The matrix was the stored in the refrigerator until ready for use.

EXAMPLE 3

[0040] This Example demonstrates preparation of GNA covalently coupled to aminocelite using glutaraldehyde. Aminocelite was prepared by reaction of celite (silicate containing diatomaceous earth) by overnight reaction in a 5% aqueous solution of aminopropyl triethoxysilane. The aminated celite was washed free of excess reagent with water and ethanol and dried overnight to yield an off white powder. One gram of the powder was then suspended in 5 ml 5% glutaraldehyde (Sigma) for 30 minutes. Excess glutaraldehyde was then removed by filtration and washing with water until no detectable aldehyde remained in the wash using Schiff's reagent. The filter cake was then resuspended in 5 ml of Sigma borohydride coupling buffer containing 2-3 mg/ml GNA and the reaction allowed to proceed overnight at room temperature. At the end of the reaction, unreacted GNA was washed off and the unreacted aldehyde aminated with ethanolamine as described. After final washing in sterile PBS, the material was, stored cold until ready for use.

EXAMPLE 4

[0041] This Example demonstrates the preparation of an exemplary lectin plasmapheresis device. Small volume filter cartridges (Glen Research, Silverton, Va.) were prepared containing 0.2 ml lectin resin, sealed and equilibrated with 5-10 column volumes sterile PBS. The cartridges were used immediately.

EXAMPLE 5

[0042] This Example demonstrates preparation of a GNA lectin affinity hemodialysis device. The viral Hemopurifier was made by pumping a slurry of particulate immobilized GNA on agarose beads or celite in sterile PBS buffer into the outside compartment of a hollow-fiber dialysis column using a syringe. For blood samples up to 15 mls, Microkros polyethersulfone hollow-fiber dialysis cartridge equipped with Luer fittings (200 μID×240 μ OD, pore diameter 200-500 nm, 0.5 ml internal volume) obtained from Spectrum Labs (Rancho Dominguez, Calif.) were used. Cartridges containing the affinity resin were equilibrated with 5-10 column volumes sterile PBS.

EXAMPLE 6

[0043] This Example demonstrates removal of HIV gp120 from physiological saline using an affinity plasmapheresis device. The plasmapheresis device described in Example 4 was equilibrated with 5-10 column volumes sterile PBS. A sample ˜1.5 ml containing gp120 (typically 500 ng/ml) was circulated over the column at a flow rate of 0.5-0.6 ml/min at room temperature. The circulating solution was tested at various time intervals for the presence of gp120 and gp120 immune complexes where appropriate.

[0044] Quantitative ELISA assays for HIV-1 gp120 were performed using a modification of the method of Weiler (Weiler et al. J Virol Methods 32(2-3): 287-301, 1991). GNA/NPA plates were prepared on Greiner C bottom plates by adding 100 ul protein (1-100 ug/ml each of GNA and NPA in PBS) to each well and incubating 2 hours at 37° C. The plates were then washed in PBST (PBS containing 0.01% Tween 20) and blocked in Casein blocking buffer for 1 hour at 37° C. Plates not used immediately were stored for up to 2 weeks at 4° C.

[0045] For detection of free gp120, 100 μl samples of test solutions were incubated for 1-2 hours at 37° C. After capture, plates were washed in PBS and 100 μl of the appropriate horse radish peroxidase (HRP) labeled anti-gp120 antibody (1:2500 in blocking buffer) was added. After incubation for 1 hour at 37° C. the antiserum was aspirated and the plates washed 4×300 ul PBSTA and the bound HRP detected with stabilized tetramethylbenzidine (TMB) substrate (BioFx). For the determination of immune complex and immune complex formation, after capture, plates were washed in PBS and 100 μl of affinity purified IIRP labeled sheep anti-human IgG antibody (1:2500 in blocking buffer) was added. After incubation for 1 hour at 37° C. the antiserum was aspirated and the plates washed 4×300 ul PBSTA. Bound HRP was detected with tetramethylbenzidine (TMB) (BioFx).

[0046] FIG. 4 shows that GNA agarose removed gp120 from buffer solution with 99% efficiency in <15 minutes. Because gp120 is a heavily glycosylated protein which can bind non-specifically to a variety of surfaces, it is not surprising that the control column also bound 85% of the input gp120.

EXAMPLE 7

[0047] This Example demonstrates the removal of HIV gp120 from infected plasma using a lectin affinity plasmapheresis device. The plasmapheresis device described in Example 4 was equilibrated with 5-10 column volumes sterile PBS. A plasma sample of about 1.5 ml containing gp120 (typically 500 ng/ml) was circulated over the column at a flow rate of 0.5-0.6 ml/min at room temperature. The circulating solution was tested at various time intervals for the presence of gp120 and gp120 immune complexes where appropriate as in Example 6.

[0048] Since anti-gp120 antibodies are typically abundant in HIV+plasma, removal of gp120 from infected plasma might be expected to be more difficult than removal from simple buffer solutions. In part due to these antibodies, gp120 detection in HIV+plasma and blood typically shows at best low amounts of gp120. In order to measure removal it was therefore necessary to add gp120 to infected patient plasma to provide a sample for measurement. ELISA measurement of the sample confirmed that all of the added gp120 in this sample was complexed with anti-gp120 antibodies (data not shown).

[0049] FIG. 5 shows that the GNA agarose affinity resin effectively removed gp120 in immune complexes from HIV infected plasma samples. Removal was rapid with an apparent half reaction time of 20 minutes. A portion of the gp120 signal was not removed (˜10% of the initial gp120 immune complex) even after 7 hours and appeared to represent background binding of IgG in the assay.

EXAMPLE 8

[0050] This Example demonstrates removal of HIV virions from infected plasma using GNA plasmapheresis. An HIV infected plasma sample (ER8-03030-0002 native HIV, Boston Biomedica, Boston Mass.) containing 100,000 copies per ml (cpm) of the virus was circulated over a 0.2 ml GNA agarose column described in Example 4. At intervals, 250 μl aliquots of the plasma were taken and the viral RNA extracted using TRI-LS reagent according to the manufacturers instructions (MRC Corporation). HIV viral RNA was then quantitated using real time RT PCR and an Access 1 step reagent set from Promega (Madison, Wis.) in 25 μl reaction volumes containing 400 nM SK432 and SK461 gag gene primers, Sybr green (1:10,000), 1× SCA blocking buffer, 3 mM MgCl.sub.2, 400 uM dNTPs and 10 ul of unknown RNA or HIV-1 RNA from armored RNA standards (Ambion Austin Tex.). Amplification and reaction times were: RT (45 minutes at 48° C.) and PCR 40 cycles (94° C./15 sec; 62° C./30 sec; 72° C./60 sec; 83° C./read) in a SmartCycler real time thermocycler (Cepheid, Sunnyvale, Calif.) essentially according to the manufacturers instructions. When necessary for confirmation of amplification, 10 μl aliquots of the amplification mix were subjected to agarose gel electrophoresis 2%(w/v) (Sigma, molecular biology grade) in 0.5× TBE buffer pH 8.3 containing 0.25 ug/ml ethidium bromide for 45 minutes at 120 VDC at room temperature. Gels were photographed on a UV transilluminator with the images subsequently digitized and analyzed using ImageJ.

[0051] FIGS. 6A and 6B show that GNA agarose effectively removes HIV virions from infected plasma. FIG. 6A is a linear plot of the data curve fit to a exponential decay (R.sup.2=0.9). The curve predicts essentially quantitative removal of HIV in about 10 hours. FIG. 6B is a log plot of the HIV removal rate which gives an estimate of 0.9 hours as the half time of HIV removal. Virus removal appears first order as expected for GNA in excess over virus. CPM indicates HIV copies/ml.

EXAMPLE 9

[0052] This Example demonstrates removal of gp120 from HIV infected blood using a GNA lectin affinity hemodialysis device. Since most HIV+plasma samples have low or undetectable amounts of gp120, simulated HIV infected blood samples were prepared by mixing 5 ml type O+ fresh packed red cells with 5 ml HIV infected plasma (typically 10.sup.5 cpm) to which was added sufficient gp120 MB to make the sample 100 ng/ml

[0053] The affinity hemodialysis devices described in Example 5 were equilibrated with 5-10 column volumes sterile PBS. A control column containing only Sepharose 4B was prepared as a control. The infected blood sample ˜10 ml containing gp120 was recirculated over the column at a flow rate of 0.9 ml/min at 37° C. using a Masterflex roller pump (1 rpm) and Pharmed 6485-16 silicon tubing. The circulating solution was tested at various time intervals for the presence of free gp120 after acid denaturation and neutralization to disrupt immune complexes.

[0054] FIG. 7 shows that as the blood samples were recirculated over the cartridge, the initial gp120 of 100 ng/ml was reduced to background levels in 4 to 6 hours (apparent t.sub.1/2=22 min). The control cartridge removed gp120 very slowly.

EXAMPLE 10

[0055] This example demonstrate removal of HCV from infected blood using GNA lectin affinity hemodialysis. In order to show the broad specificity of GNA lectin removal of viruses, we performed lectin affinity hemodialysis on HCV infected blood. The lectin affinity hemodialysis devices described in Example 4 were equilibrated with 5-10 column volumes sterile PBS. HCV infected blood samples were prepared by mixing 1 ml type O+ fresh packed red cells with 1 ml HIV infected plasma (typically 10.sup.5 cpm). The infected blood sample was recirculated over the column at a flow rate of 0.5 ml/min at room temperature using a Masterflex roller pump (1 rpm) and Pharmed 6485-16 tubing. The circulating solution was tested at various time intervals for the presence HCV viral RNA.

[0056] Viral RNA was isolated using TRI-LS (MRC Corporation) from 100 μl of plasma according to the manufacturers instructions. HCV viral RNA was then measured by quantitative RT PCR performed using an ImpromII reagent set from Promega (Madison, Wis.) in 25 ul reaction volumes containing 400 nM EY80 and EY78 HCV specific primers, Sybr green (1:10,000), 1× SCA blocking buffer, 3 mM MgCl.sub.2, 400 uM dNTPs, 0.2 units/ul each of Tfl polymerase and AMV reverse transcriptase. Typically 50 ul of the mix was used to dissolve RNA isolated from 100 μl plasma and the mix split into two identical duplicate samples. Amplification and reaction times were: RT (45 minutes at 48° C.) and PCR 40 cycles (94° C./15 sec; 62° C./30 sec; 72° C./60 sec; 87° C. readout) in a SmartCycler real time thermocycler (Cepheid, Calif.) essentially according to the manufacturers instructions. The amount of viral RNA was estimated by comparison to the signal strength of the viral RNA standards in the initial phase of the amplification reaction (C.sub.t=20).

[0057] FIG. 8 shows that as the blood was recirculated over the cartridge, the initial HCV was reduced about 50% in 3 hours (apparent t.sub.1/2=3 hours). The curve fit reasonably well to an exponential decay.

[0058] From the foregoing, it will be obvious to those skilled in the art the various modifications in the above-described methods, and compositions can be made without departing from the spirit and scope of the invention. Accordingly, the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Present examples and embodiments, therefore, are to be considered in all respects as illustrative and not restrictive, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.