Abstract:
System and methods treat plasma carrying contaminants and cellular matter that are capable of entraining contaminants. The systems and methods separate cellular matter from the plasma by filtration, thereby removing contaminants entrained within the cellular matter. The system and methods add to the plasma a photoactive material. The systems and methods emit radiation at a selected wavelength into the plasma to activate the photoactive material and thereby eradicate the contaminant that is free of entrainment by cellular matter.

Description:
FIELD OF THE INVENTION 
     The invention generally relates to the eradication of contaminants using photodynamic therapy. The invention also generally relates to the processing of whole blood and its components for storage and transfusion. In a more specific sense, the invention relates to the extracorporeal treatment of collected whole blood and its components with photoactive materials to eradicate viruses and other pathogenic contaminants. 
     BACKGROUND OF THE INVENTION 
     With the coming of blood component therapy, most whole blood collected today is separated into its clinically proven components for storage and administration. The clinically proven components of whole blood include red blood cells, used to treat chronic anemia; platelet-poor plasma, from which Clotting Factor VIII-rich cryoprecipitate can be obtained for the treatment of hemophilia; and concentrations of platelets, used to control thrombocytopenic bleeding. 
     It is well known that blood can carry infectious agents like hepatitis-B virus; the human immunodeficiency (AIDS) virus; the Herpes virus; and the influenza virus. To avoid the transmission of these infectious agents during blood transfusions, donors of blood are routinely screened and also undergo serologic testing to detect the presence of these agents. Still, it is difficult to always assure that these infectious agents are detected. 
     The use of photodynamic therapy has been suggested as a way to eradicate infectious agents from collected blood and its components. Still, there has been a general lack of success in economically adapting the benefits of photodynamic therapy to the demands of the blood banking industry. One reason for this is that not all biological contaminants are carried free within the blood where they can be readily coupled to photoactive agents. Some biological contaminants are entrained on or within white blood cells out of the reach of photoactive agents. 
     For this and other reasons, the promise of photodynamic therapy in treating the nation&#39;s banked blood supply has gone largely unfulfilled. 
     SUMMARY OF THE INVENTION 
     The invention provides improved systems and methods for treating blood constituents to adventitious viral agents. 
     One aspect of the invention provides systems and methods which remove viral agents from plasma. The systems and methods remove from the plasma targeted cellular matter that does or might entrain viral agents. In a preferred embodiment, the targeted cellular matter includes leukocytes. The system and methods add to the plasma a photoactive material, which binds to viral agents that are free of entrainment by the targeted cellular matter. Radiation emitted at a selected wavelength into the plasma activates the photoactive material and thereby eradicates the free viral agents. 
     In a preferred embodiment, a system for treating plasma comprises tubing adapted to be coupled a plasma source, and a filter in the tubing to separate cellular matter from the plasma conveyed from the source. The system includes a transfer container coupled to the tubing to receive cellular matter-reduced plasma from the filter, and a source of photoactive material to be mixed with the plasma. In this embodiment, the tubing includes a path to vent air from the transfer container in a path that bypasses the filter. 
     In a preferred embodiment, systems and methods remove viral agents entrained within the cellular matter by conveying plasma in a first path through a filter. The systems and methods convey the cellular matter-reduced plasma from the filter in a second path, which includes a connected transfer container. The systems and methods mix the cellular matter-reduced plasma with a photoactive material within the transfer container, forming a plasma mixture. 
     In this embodiment, the systems and methods convey a portion of the plasma mixture from the transfer container in a flush path, which includes the second path, to thereby expose residual contaminants in the second path to the photoactive material. The systems and methods then separate the transfer container from the filter by severing the second path. After severance from the filter, a remnant of the second path remains attached to the transfer container. However, due to the prior flushing step, all contaminants in the attached second path remnant have been exposed to the photoactive material. Subsequent radiation of the transfer container thereby eradicates contaminants, which are free of entrainment by cellular matter, both within the transfer container and the attached second path remnant. 
     In a preferred embodiment, the flush path by passes the filter and also provides a path to vent air from the transfer container. 
     Another aspect of the invention provides systems and methods for treating plasma using multi-stage filtration, which targets for removal different species of cellular matter. The systems and methods separate a first species of cellular matter by filtration through a first filter media, thereby removing contaminants entrained within the first species of cellular matter. The systems and methods separating a second species of cellular matter by filtration through a second filter media, thereby removing contaminants entrained within the second species of cellular matter. The systems and methods add to the plasma a photoactive material and emit radiation at a selected wavelength into the plasma to activate the photoactive material, thereby eradicating the contaminant that is free of entrainment by cellular matter. In a preferred embodiment, the first filtration media targets leukocytes for removal, while the second filtration media targets platelets for removal. 
     Another aspect of the invention provides a kit that envelopes photoactive material in an overwrap that includes a light filtering material. The light filtering material absorbs light that activates the photoactive material. The presence of the light filtering material in the overwrap protects the photoactive material from photo-degradation due to absorption of ambient light during handling and storage prior to use. 
     In a preferred embodiment, the photoactive material within the kit includes methylene blue. In this embodiment, the light filtering material includes a blue material having phthalocyanine pigments. 
     In a preferred embodiment, the photoactive material is contained in liquid form within the kit. In this embodiment, the overwrap also includes material that reduces liquid vapor loss from the kit. 
     Other features and advantages of the invention will be pointed out in, or will be apparent from, the drawings, specification and claims that follow. 
    
    
     DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a plane view of a blood processing and storage kit for reducing the presence of viral agents in plasma; 
     FIG. 2 is an exploded, perspective view of the laminated walls of the overwrap envelope shown in phantom lines in FIG. 1; 
     FIG. 3 is a side view of the laminated walls of the overwrap envelope shown in FIG. 2; 
     FIG. 4 is a top perspective view of the laminated walls of the overwrap envelope, after having been joined by a peripheral heat seal; 
     FIG. 5 is an exploded side view of the leukocyte reduction filter that forms a part of the kit shown in FIG. 1; 
     FIG. 6 is a top perspective view of the interior of the outlet housing part for the filter shown in FIG. 5; 
     FIG. 7 is a plane view the kit shown in FIG. 1 being used to convey plasma from a source container, through the leukocyte reduction filter, and into the processing and storage container; 
     FIG. 8A is a plane view the kit shown in FIG. 7 being used to vent air and residual plasma from the processing and storage container in a bypass path around the leukocyte reduction filter; 
     FIG. 8B is a plane view of the kit shown in FIG. 8A being used to flush the tubing section next to the container with photoactive material, to assure exposure of residual viruses occupying the tubing section with photoactive material; 
     FIG. 9 is a perspective view of the kit shown in FIGS. 8A and 8B, after separation of the processing and storage container and placement of the processing and storage container in an irradiation chamber; 
     FIG. 10 is a plane view of an alternative embodiment of a blood processing and storage kit for reducing the presence of viral agents in plasma, in which the photoactive material is stored within an auxiliary container whose walls include a light filtering material; 
     FIG. 11 is a plane view of an alternative embodiment of a blood processing and storage kit for reducing the presence of viral agents in plasma, which includes an integrally attached air reservoir; 
     FIG. 12A is a plane view of the kit shown in FIG. 11 being use to vent air and residual plasma from the processing and storage container into the air reservoir; 
     FIG. 12B is a plane view of the kit shown in FIG. 12A being used to flush the tubing section next to the container with photoactive material, to assure exposure of residual viruses occupying the tubing section with photoactive material; and 
     FIG. 13 is a plane view of another alternative embodiment of a blood processing and storage kit for reducing the presence of viral agents in plasma, which reduces the presence of viral agents in plasma by the removal by filtration of least two different cellular blood species which actually do or potentially can entrain viral agents. 
    
    
     The invention is not limited to the details of the construction and the arrangements of parts set forth in the following description or shown in the drawings. The invention can be practiced in other embodiments and in various other ways. The terminology and phrases are used for description and should not be regarded as limiting. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     FIG. 1 shows a blood constituent processing and storage set or kit  300 . The kit  300  is intended, during use, to assist in the removal of viral agents from plasma. The viral agents are either carried free within the plasma or are entrained on or within cellular matter (e.g., red blood cells, platelets, and leukocytes) that the plasma carries. The kit  300  shown in FIG. 1 will be described in the context of reducing the presence of viral agents in single donor units of plasma, because it is particularly well suited for this purpose. 
     The kit  300  includes a processing and storage container  302 , which carries an integrally attached length of flexible transfer tubing  304 . In the illustrated embodiment, the transfer tubing  304  is made from medical grade plasticized polyvinyl chloride plastic. However, other flexible medical grade plastic materials can be used. 
     The transfer tubing  304  includes an integrally attached in-line filter  306 . The filter  306  includes a filter media  307  (see FIG. 5) that removes from plasma cellular matter that does actually or potentially entrain viral agents. 
     As FIG. 5 shows, the filter media  307  is encased within a two part housing  348 A and  348 B made, for example, from polycarbonate, although any engineering medical grade plastic with appropriate toxicology characteristics can be used. The housing  348 A/ 348 B is sealed about the filter media  307  by, for example, sonic welding. 
     The pore size of the filter media  307  can be tailored to remove by exclusion all or some species of cellular matter found in plasma, depending upon the extent to which viral agents sought to be eliminated are entrained by the different cellular species. In the illustrated embodiment, the principal cellular species targets of the filter  306  are leukocytes, for it is known that leukocytes entrain many viral agents. With this objective in mind, the filter media  307  comprises a non-fibrous membrane having a pore size smaller than the size of leukocytes, to thereby remove leukocytes by exclusion. In the illustrated embodiment, the media  307  also includes a prefilter material, which removes fibrin clots and other large size aggregates from the plasma. 
     The composition of the membrane for the media  307  can vary. For examples, hydrophilic membranes made from nylon, acrylic copolymers, polysulfone, polyvinylidene fluoride, mixed cellulose esters, and cellulose ester can be used to remove leukocytes by exclusion. Non-hydrophilic membranes can also be treated to serve as a membrane for the filter media  307 . Likewise, the composition of the prefilter for the media  307  can vary. For example, the prefilter can comprise fibers of glass or polyester. Material selection takes into account customer preferences, performance objectives, and manufacturing requirements, including sterilization techniques. 
     In the illustrated and preferred embodiment, (see FIG.  5 ), the filter media  307  includes three filter media layers  342 ,  344 , and  346 . The first filter media layer  342  comprises USP Grade VI glass fiber or the equivalent. The second and third filter media layers  344  and  346  comprise polyethersulfone (PES) membranes, which remove leukocytes by exclusion. The second and third filter media layers  344  and  346  possess pore sizes which are approximately 10 fold smaller than the size of leukocytes and which decrease in the direction of flow. The second filter media layer  344  has a pore size in the range of about 0.9 μm to about 2.0 μm, with an average pore size of about 1.2 μm. The third filter media layer  346  has a smaller pore size in the range of about 0.3 μm to about 1.5 μm, with an average pores size of about 0.8 μm. The second and third filter media layers  344  and  346  also incidently remove red blood cells by exclusion. 
     The filter media  307  should preferably be capable of filtering 310 ml of plasma, suspended at a head height of 3 feet, in 20 minutes. 
     The housing part  348 A includes an inlet  350 , which, in use, conveys plasma and leukocytes into contact with the prefilter layer  342 . The axis  351  of the inlet  350  is generally parallel to the plane of the layer  342  to uniformly perfuse plasma across the entire prefilter layer  342 . 
     The housing part  348 B includes an outlet  352 , which conveys leukocyte-reduced plasma from the second and third PES filter layers  344  and  346 . As FIG. 6 shows, the interior surface of the housing part  348 B is grooved, creating a fluid manifold  354  that uniformly distributes leukocyte-reduced plasma to the outlet  352 . 
     Referring back to FIG. 1, a length of branch tubing  308  is integrally attached to the transfer tubing  304  by conventional Y-connectors  316 . The branch tubing  308  forms a fluid path bypassing the filter  306 . As will be described in greater detail later, the branch tubing  308  serves to vent air. 
     The far end of the transfer tubing  304  carries an air pillow  310 . The air pillow  310  prevents collapse of the tubing  304  and  308  caused by pressure differentials during steam sterilization of the kit  300 . 
     The transfer tubing  304  further includes a conventional in-line frangible cannula  312  between the filter outlet  352  and the processing and storage container  302 . The cannula  312  normally closes fluid the transfer tubing  304  to fluid flow. 
     The cannula  312  can be constructed in various ways. U.S. Pat. Nos. 4,181,140 and 4,294,247 disclose representative constructions for the cannula  312 , which are incorporated herein by reference. 
     Alternatively, an external roller clamp or C-clamp of conventional construction could be used for the same purpose. 
     The branch tubing  308  includes a conventional in-line one-way valve  314 . The valve  314  prevents fluid flow through the branch tubing  308  in the direction of the processing and storage container  302 , while permitting fluid flow in the opposite direction away from the processing and storage container  302 . For redundancy, the branch tubing  308  also includes an external roller clamp or C-clamp  318 . The C-clamp  318  normally closes the tubing  308  between the one-way valve  314  and the processing and storage container  302 . 
     The processing and storage container  302  can be constructed in various ways. In the illustrated and preferred embodiment, the container  302  includes an interior chamber  320 . The transfer tubing  304  communicates with the chamber  320  for conveying plasma into the chamber  320 . In a preferred implementation, the chamber  320  is capable of holding between 235 to 310 mL of plasma. A normally sealed outlet port  360  also communicates with the chamber  320 . The port  360  is opened when it is time to remove plasma from the chamber  320 . 
     The chamber  320  holds a photoactive material  326 . The photoactive material  326  mixes with the plasma introduced into the chamber  320 . The photoactive material  320  binds to extracellular viruses that plasma introduced into the chamber  326  may carry. When exposed to light energy in a particular spectrum, the photoactive material  326  inactivates the nucleic acids of the bound viruses, rendering them nonviable. 
     In the illustrated and preferred embodiment, the photoactive material  326  comprises 10 mL of liquid solution containing 83 micrograms of methylene blue in water at pH 3.1, without buffers or other additives. Methylene blue, a thiazine dye, possesses the ability to bind to nucleic acids with high affinity, targeting the viruses for destruction upon exposure to a particular spectrum of light energy. Methylene blue absorbs light in the 660 nm region of the visible spectrum, which is the spectrum region where plasma is most transparent. Methylene blue inactivates a broad range of viruses, such as HIV, human hepatitis B (HBV), human hepatitis C (HCV), and Parvo virus B19, with minimal loss of therapeutic plasma proteins. 
     The mixture of plasma and photoactive material  326  is irradiation by light within the chamber  320  as part of a viral inactivation process. The container  302  is therefore made of a material that is substantially transparent to the applied light energy. The material for the container  302  is also adapted to withstand contemplated storage conditions for the plasma. 
     In the illustrated and preferred embodiment, the applied light energy is in the white light spectrum (400 to 700 nm). The container  302  is therefore made of a plastic, poly(ethylene vinyl acetate) material. This material is transparent to white light and is also resistant to the cold temperatures at which frozen plasma is stored. This material is commercially available and is made and sold, for example, by Baxter Healthcare Corporation under the trademark PL-732® Plastic. 
     The container  302  also includes a flap  322 , which extends below the chamber  320 . The flap  322  carries a printed label  324  having identifying indicia. The flap  322  keeps the label  324  away from the chamber  320 , where it could block or impede the irradiating light. 
     The container  302  also serves after the viral inactivation process to store the viral inactivated plasma at temperatures below −30° C., following standard blood banking procedures. 
     Further details of container  302  are found in copending U.S. patent application, Ser. No. 08/121,820, filed Sep. 15, 1993, and entitled “Container for Irradiation of Blood Products.” 
     As FIG. 4 shows, the kit  300  is preferably enclosed for storage and handling before use in an overwrap envelope  328  (FIG. 1 diagrammatically shows the envelope  328  in phantom lines). The overwrap envelope  328  serves multiple functions. 
     To minimize evaporation of the liquid photoactive material  326  from the container  302  prior to use, the envelope  328  includes a material  332  possessing a relatively low water vapor transmission rate (WVTR). In the illustrated and preferred embodiment, the targeted WVTR is about 0.020 gh −1  at 25° C. and 60% relative humidity. 
     The particular composition of the water vapor barrier material  332  can vary. In the illustrated and preferred embodiment, the water vapor barrier material  332  comprises an oriented polypropylene material having a thickness of 25 μm. 
     To prevent degradation of the photoactive material  326  prior to use, the envelope also includes a light filtering material  330  possessing the ability to absorb ambient light energy in the spectrum that activates the photoactive material  326 . It has been discovered that, during storage and handling prior to use, the photoactive material  326  absorbs from ambient visible light (400 nm to 700 nm) the spectrum that initiates photoactivation. The incidental absorption of ambient visible light by photoactive material  326  initiates a photoreduction process, creating byproducts that are either partially or completely ineffective for viral inactivation. 
     For example, exposure of methylene blue to visible ambient light (whose emission spectrum includes the 660 nm region) converts the methylene blue into colorless leucomethylene blue. The leucomethylene blue photoreduction byproduct is not effective in inactivating viruses. 
     The particular composition of the light filtering material  330  will vary according to the light sensitivity spectrum of the particular photoactive material  326  used. In the illustrated and preferred embodiment, the light filtering material  330  comprises a blue die of phthalocyanine pigments. The blue die material  326  transmits not more than 1% of light in the range of 600 nm to 700 nm, which is the spectrum in which methylene blue is activated. 
     As FIGS. 2 and 3 show, in the illustrated and preferred embodiment, the overwrap envelope  328  comprises sheets S 1  and S 2 , each of which comprises a multiple layer laminate L 1  and L 2 . The water vapor barrier material  332  constitutes one of the exterior layers of each laminated sheet S 1  and S 2 . The blue die comprising the light filtering material  330  is printed on the interior face of the water vapor barrier material  332 . 
     Each laminated sheet S 1  and S 2  also preferably includes as another exterior layer a material  334  that flows in response to heat. The presence of the material  334  makes it possible to heat seal the two sheets S 1  and S 2  together, forming the envelope  328 . 
     The particular composition of the heat flowing material  334  can vary. In the illustrated and preferred embodiment, the material  334  comprises a cast polypropylene material having a thickness of about 25 μm. The heat flowing material  334  can be attached to the layer  332 , for example, by a polyurethane-polyester resin-epoxy. 
     Laminated sheets S 1  and S 2  as described, with the layers  330 ,  332 , and  334  and suited for use as the overwrap envelope  328 , can be purchased from Hosokawa Yoko Co., LTD. (Japan). The sheet material from this company has a weight of 50 g/m 2  and density 1.0 g/cm 3 . 
     The envelope  328  is created by laying the sheets S 1  and S 2  of the overwrap laminate together (as FIG. 3 shows) and applying pressure and heat H along the sheet edges in a heat sealing die. The pressure and heat H form a peripheral heat seal  336 , which joins the sheets S 1  and S 2  together, forming the envelope  328  (as FIG. 4 shows). 
     Despite the presence of the light filtering material  330 , the overwrap envelope  328  as above described nevertheless retains sufficient transparency to other visible light spectrums to allow visual inspection of the contents of the overwrap envelope  328 , for quality control or customer inspection purposes. 
     The overwrap envelope  328 , including an appropriate light filtering material  330  as just described, can be used in association with other containers or in other systems which hold liquids or other materials sensitive to ambient light degradation. For example, photoactive materials  326  activated in different spectrum regions will require accordingly different light filtering material  330 . For example, 4′-(4-Amino-2-oxa)butyl-4,5′8-trimethylpsoralen (S-59) is a photoactive material usable in conjunction with platelet-containing blood suspensions. S-59 is activated by ultraviolet-A light and can undergo intramolecular reactions when exposed to ambient UV-A and short wavelength regions of visible light. To protect against such degradation of S-59 material, the light filtering material  330  can comprise a UV-A absorbent red die. 
     For another example, as FIG. 10 shows, instead of using a light filtering overwrap envelope  328 , the kit  300  (or another system) can include an auxiliary container  362  to store the light activated material  326  before use. The walls of the container  362  include an appropriate light filtering material  330  to protect the light activated material  326  from ambient light degradation before use. In this arrangement, the photoactivated material  326  is transferred from the auxiliary container  362  to plasma before the light activation process, either before or during filtration, or after filtration when the plasma occupies the processing and storage container  302 . Of course, a container (like the container  302 ), which is intended to ultimately serve as a light transparent chamber, must remain free or essentially free of a light filtering material. In this arrangement, it is still desirable to provide an overwrap envelope  364  (shown diagrammatically in FIG. 10) to decrease water vapor loss of the liquid photoactive material  326  during storage and handling prior to use. 
     The overwrap envelope  328  (or  364  in the FIG. 10 embodiment) is torn away when it is time to use the kit  300 . As FIG. 7 shows, a container  338  holding the plasma P is connected in a sterile fashion to the transfer tubing  304  near the air pillow  310 . The source container  338  can, for example, hold fresh plasma or plasma that has been frozen and thawed. The plasma is harvested by conventional blood banking procedures. These procedures, which are accomplished through centrifugation of whole blood, yield plasma that is essentially free of red blood cells. 
     Known sterile connection mechanisms (not shown) like that shown in Spencer U.S. Pat. No. 4,412,835 can be used for connecting the container  338  to the transfer tubing  304 . These mechanisms form a molten seal between tubing ends, which, once cooled, forms a sterile weld  360 . The air pillow  310  is discarded after sterile connection between the source container  338  and the transfer tubing  304  is made. 
     As FIG. 7 shows, once the sterile connection is made, the source container  338  is suspended above the processing and storage container  302 . The operator checks to assure that the clamp  318  is closed on the bypass branch tubing  308 . The operator breaks the cannula  312 , and the plasma P flows by gravity head pressure through the filter  306 . The leukocyte-reduced plasma exits the filter  306  and drains into the chamber  320  of the container  302 . 
     It has been observed that the triple layer membrane filter  306  described above provides plasma having a leukocyte level that is below the limit of flow cytometer detection (i.e., less than about one leukocyte per μL). The actual residual level of leukocytes in the plasma after filtration by the filter  306  is estimated not to exceed an average theoretical level of 0.004 leukocyte per μL. Based upon an initial leukocyte level of 0.79×10 8  per L, the leukocyte reduction percentage of the filter  306  is estimated to be about 99.99% (log reduction≧4.0). 
     The methylene blue photoactive material  326  is mixed with the leukocyte-reduced plasma within the container  302  by manual inversion. 
     As FIG. 8A shows, after mixing plasma P and photoactive material  326  within the container chamber  320 , the clamp  318  is opened and the container  302  squeezed. Air A is vented from the container  302 , through the bypass branch tubing  308  back into the source container  338 . As FIG. 8A also shows, the venting of air A also displaces residual plasma P, out of the transfer tubing  304  between the filter  306  and the container  302  and into the bypass branch tubing  308 . Viruses in the residual plasma P, having never entered the container chamber  320  have not been exposed to the photoactive material  326  and therefore should be removed before undertaking the desired photoactivation process. 
     As FIG. 8B shows, as air venting proceeds, an amount of the mixture M of photoactive material  326  and plasma P will enter the section  305  of the transfer tubing  304  between the filter  306  and the container  302 . The mixture M is allowed to drain back into the container  302 . The mixture M flushes this section of the transfer tubing  304  with the photoactive material  326  and plasma mixture. The flushing process assures that viruses still occupying this section of the tubing  304  after air venting will become mixed with the photoactive material  326 . This assures that all viruses present in the container  302  and adjacent section  305  of tubing  304  are exposed to the material  326 , to thereby assure the desired virucidal effect during subsequent exposure to light irradiation. 
     After air venting and flushing, as just described, the tubing  305  next to the container  302  is sealed closed using, for example, a dielectric tube sealer. As FIG. 9 shows, the remaining portion of the kit  300  containing the filter  306  is removed and discarded. A remnant of the tubing  305  remains connected to the container  302 . 
     The container  302  holding the methylene blue and leukocyte-reduced plasma, and carrying a remnant of the tubing section  305 , is placed into a white light chamber  356  (see FIG.  9 ). The chamber  356  comprises twelve fluorescent lamps  358 , which supply output in the visible range (400 to 700 nm) to both sides of the container  302 . The chamber  356  monitors the light intensity and adjusts exposure time to control total light dosage delivered to the container  306 . The light activates the methylene blue to release singlet oxygen, which inactivates viruses in the plasma. The approximate time of illumination to deliver a targeted dose of 33 J per cm 3  is 30 minutes. Further details of a light chamber can be found in Wolf et al. U.S. Pat. No. 5,290,221 and Bischof et al. U.S. Pat. No. 5,300,019. 
     After the illumination step, the leukocyte-reduced plasma is frozen within the container  302  at less than −30° C. for storage using conventional blood bank practices. The plasma within the container  302  is thawed when fractionation or transfusion is required. 
     In the illustrated embodiment (see FIG.  1 ), the kit  300  includes written instructions  374  for using the kit for its intended purpose. The instructions  374  direct the technician to handle the kit in a prescribed way to best accomplish the desired therapeutic objectives, as set forth in the preceding description and shown in FIGS. 7 to  9 . 
     The instructions  374  may take various forms. Representative instructions  374  direct the technician, upon removal of the overwrap  328 , to convey plasma through the tubing  304  from the source  338  through the filter  306  to separate leukocytes from the plasma. The representative instructions  374  also direct the technician to convey leukocyte-reduced plasma through the tubing  304  from the filter  306  to the transfer container  302 . The representative instructions  374  also instruct the technician to mix the photoactivated material  326  with the plasma and to expose leukocyte-reduced plasma mixed with the photoactive material  326  to light that activates the photoactive material  326 . The representative instructions  374  also direct the technician to store the plasma in the container  302  after the photoactivation process. 
     The instructions  374  can, of course, include further details based upon the particular configuration of the kit  300 . For example, in the context of the kit  300  shown in FIG. 1, the instructions  374  can direct the technician to mix the photoactivated material with leukocyte-reduced plasma within in the container chamber  320 . In this context, the instructions  374  can also direct the technician to expose the container chamber  320  to light that activates the photoactive material  326  mixed within the chamber  320  with the leukocyte-reduced plasma. The instructions  374  can also direct the technician to vent air from the container chamber  320  in a path that bypasses the filter  306 , which in FIG. 1 comprises the branch tubing  308 . The instructions  374  can also instruct the technician to flush the tubing  304  downstream of the filter  306  with plasma and photoactive material  326  from the chamber  320 . 
     EXAMPLE 
     A study was conducted to demonstrate the ability of the kit  300  when used in accordance with the instructions  374  to inactivate viruses under intended use conditions. In the study, a maximum plasma volume of 310 mL was employed to provide the lowest concentration of methylene blue and the greatest fluid thickness to be illuminated. In addition, the nominal targeted light dose of 33 J/cm 2  was reduced to 24 or 30 J/cm 2  to further stress the study conditions. 
     Plasma was collected from CPD anticoagulated whole blood units following routine blood bank procedures, yielding plasma that is essentially free of red blood cells. The plasma was not frozen prior to treatment during the study. 
     A panel of viruses was selected to represent the most significant agents that can contaminate fresh frozen plasma and to represent a broad spectrum of physical/chemical forms of viruses (i.e., lipid enveloped and non-lipid enveloped RNA and DNA viruses, as well as intra-cellular viruses). The panel included the following viruses: BVDV (strain Singer); HIV Type 1 (HIV-1, strain III B ); human herpes simplex virus Type 1 (HSV-1, strain MacIntyre); pseudorabies virus (PRV, strain Aujeszky); simian virus Type 40 (SV-40, strain Pa-57); duck hepatitis B DHBV; and cell associated HIV (H-9/HIV, HIV III B  chronically infected H-9 cells). 
     These viruses were added to units of plasma before treatment in physiologically representative concentrations. A process control comprising an aliquot of virus-spiked plasma, was collected from each unit prior to processing in the kit  300 . The process control served as the baseline value for the calculation of the virus load reduction, called the log reduction value (LRV). LRV represents either (i) the difference in log virus titers between the process control and the processed sample, or (ii) the difference in log virus titers between the process control and the validated sensitivity limit of the assay, if there was no recoverable virus (indicated by the use of the symbol “&gt;” in the Table 1 below). 
     The virus panel and the log reduction values (LRV&#39;s) obtained by processing the plasma in the kit  300  in accordance with the instructions  374  are summarized in the following Table 1: 
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Results of Study on Viral 
               
               
                 Inactivation Using the Kit 300 
               
             
          
           
               
                   
                 Virus 
                 Model for 
                 Size (nm) 
                 LRV 
               
               
                   
                   
               
               
                   
                 HIV 
                 Self 
                 110 
                 &gt;6.6 
               
               
                   
                   
                   
                   
                 at 24 J/cm 2   
               
               
                   
                 BVDV 
                 HCV 
                 60-70 
                 &gt;5.93 ± 0.07 
               
               
                   
                   
                   
                   
                 at 24 J/cm 2   
               
               
                   
                 DHBV 
                 HBV 
                 40 
                 3.5 
               
               
                   
                   
                   
                   
                 at 30 J/cm 2   
               
               
                   
                 PRV 
                 enveloped DNA 
                 150-180 
                 5.52 ± 0.38 
               
               
                   
                   
                 virus 
                   
                 at 30 J/cm 2   
               
               
                   
                 HSV 
                 enveloped DNA 
                 150-180 
                 &gt;6.16 ± 0.06 
               
               
                   
                   
                 virus 
                   
                 at 24 J/cm 2   
               
               
                   
                 SV-40 
                 non-enveloped 
                 55 
                 4.27 ± 0.30 
               
               
                   
                   
                 DNA virus 
                   
                 at 24 J/cm 2   
               
               
                   
                 HIV/H9 
                 virus- 
                   
                 No 
               
               
                   
                   
                 infected 
                   
                 Recoverable 
               
               
                   
                   
                 leukocytes 
                   
                 Viruses after 
               
               
                   
                   
                   
                   
                 challenge 
               
               
                   
                   
                   
                   
                 with 1 × 10 8   
               
               
                   
                   
                   
                   
                 HIV/H9 cells 
               
               
                   
                   
               
             
          
         
       
     
     Table 1 demonstrates that use of the kit  300  is effective against small and large lipid enveloped viruses with either RNA or DNA genomes. Table 1 also demonstrates the capability of the kit  300  to inactivate certain non-enveloped viruses, which are not resistant to the virucidal action of methylene blue (for example, non-enveloped encephalomyocarditis virus (EMC) has demonstrated a resistance to the virucidal action of methylene blue). 
     The kit  300  provides more reliability and ease of use than the removal of leukocytes from plasma by lysing using conventional freeze-thaw processes. The kit  300  also provides greater removal of adventitious agents (i.e., viruses) than mere light inactivation (which does not remove intracellular agents) and/or bed-side filtering of plasma (which only removes fibrin clots, and not leukocytes). 
     FIG. 11 shows, as an alternative embodiment, a kit  300 ′ sharing many of the component parts of the kit  300  shown in FIG.  1 . The common elements (which are assigned the same reference numbers as in FIG. 1) include the processing and storage container  302 , the transfer tubing  304 , the filter  306 , the photoactive material  326 , and the frangible cannula  312 . 
     However, the kit  300 ′ shown in FIG. 11 does not include the branch tubing  308  and the air pillow  310 . 
     Instead, the far end of the tubing  304  in the kit  300 ′ is closed by a plug  372 . The kit  300 ′ also includes an air reservoir  370  integrally connected to the tubing  304  by the Y-connector  316  between the filter  306  and the container  302 . 
     The air reservoir  370  takes the place of the air pillow  310 . Like the pillow  310 , the reservoir  370  contains a residual amount of air to prevent collapse of the tubing  304  during steam sterilization. The reservoir  370  also serves as a chamber to receive vented air and residual plasma from the container  302  at the end of the filtration process. 
     More particularly, using the kit  300 ′, plasma from the source container  338  is passed for leukocyte reduction through the filter  306  and mixed with the photoactive material  326  in the container  320  in the same manner previously described and shown in FIG.  7 . 
     As FIG. 12A shows, after filtration and mixing, air A is vented from the container  302  into the reservoir  370 . Residual plasma P is also displaced out of the tubing section  305  and into the reservoir  370 . As FIG. 12B shows, as air venting proceeds, an amount of the mixture M of photoactive material  326  and plasma P will enter the section  305  of the transfer tubing  304  between the filter  306  and the container  302 . The mixture M flushes this section of the transfer tubing  304  with the photoactive material  326  and plasma mixture. 
     In all other respects the process for handling the kit  300 ′ is the same as previously described with respect to the kit  300 . 
     FIG. 13 shows, as another alternative embodiment, a kit  300 ″ sharing many of the component parts of the kit  300  shown in FIG.  1 . The common elements (which are assigned the same reference numbers as in FIG. 1) include the processing and storage container  302 , the transfer tubing  304 , the branch tubing  308 , the filter  306 , the photoactive material  326 , the air pillow  310 , and the frangible cannula  312 . The kit  300 ″ shown in FIG. 13 includes an additional in-line filter  376  in the transfer tubing  304  downstream of the filter  306 . The filter  376  includes a filter media  378  that removes from plasma a second cellular species different than the species removed by the filter media  307 , which second cellular species does actually or potentially entrain viral agents. In the illustrated and preferred embodiment, where the principal cellular species targeted by the filter media  307  are leukocytes, the second cellular species targeted by the second filter media  378  are platelets. 
     As described above in connection with the filter media  307 , the pore size of the filter media  378  can be tailored to remove platelets from plasma by exclusion. It is believed that candidate materials for the media  307  formed with a pore size range of between 0.3 μm and 0.45 μm (which is smaller than the pore size range of the media  307 ) will serve to remove platelets from plasma by exclusion. 
     The presence of the second, downstream media  378 , having a smaller pore size than the first, upstream media  307 , also provides added assurance that the cellular species targeted for removal by the first media  307  (i.e., leukocytes) will, in fact, be depleted or essentially depleted from the plasma. In this respect, the smaller pore size media  378  serves both a redundant function of removing leukocytes and an added second step function of removing the smaller platelet species. 
     It should be appreciated that the second filter media  378  can, instead of being separately housed as the filter  378 , be integrated as another layer with the already multi-layer filter media  307 . 
     In all other respects the process for handling the kit  300 ″ is the same as previously described with respect to the kit  300 . 
     Features and advantages of the invention are set forth in the following claims.