Abstract:
Methods and apparatuses are provided for reduction of pathogens in fluids containing blood products. Preferred methods include the steps of adding an effective, non-toxic amount of a photosensitizer such as riboflavin to the blood product and exposing the fluid to light having a peak wavelength.

Description:
[0001]    This application claims priority to U.S. provisional application Ser. No. 60/822,726, filed Aug. 17, 2006. This application is also a continuation-in-part of Ser. No. 10/904,361, filed Nov. 5, 2004; which is a continuation of Ser. No. 10/357,599 filed Feb. 3, 2003 now U.S. Pat. No. 6,843,961; which is a continuation-in-part of Ser. No. 09/962,029 filed Sep. 25, 2001, now abandoned; which is a continuation-in-part of Ser. No. 09/596,429 filed Jun. 15, 2000 now U.S. Pat. No. 7,094,378. 
     
    
     BACKGROUND 
       [0002]    Whole blood collected from volunteer donors for transfusion into recipients is typically separated into components such as red blood cells, white blood cells, platelets, plasma and plasma proteins, using apheresis or other known methods. Each of these separated blood components may be stored individually for later use and are used to treat a multiplicity of specific conditions and disease states. For example, the red blood cell component is used to treat anemia, the concentrated platelet component is used to control bleeding, and the plasma protein component is used frequently as a source of Clotting Factor VIII for the treatment of hemophilia. 
         [0003]    In cell separation procedures, there are usually some small percentage of other types of cells which are carried over into a separated blood component. When contaminating cells are carried over into a separated component of cells in a high enough percentage to cause some undesired effect, the contaminating cells are considered to be undesirable. For example, white blood cells, are considered undesirable because they may transmit infections such as HIV and CMV may also cause other transfusion-related complications such as transfusion-associated Graft vs. Host Disease (TA-GVHD), alloimmunization and microchimerism. 
         [0004]    Whole blood or blood products may also be contaminated with other undesirable pathogens such as viruses and bacteria. The term pathogen as used in this context includes undesirable contaminants such as donor white blood cells, parasites, bacteria and viruses. 
         [0005]    Blood screening procedures may miss pathogenic contaminants, and sterilization procedures which do not damage cellular blood components but effectively inactivate all donor white blood cells, infectious viruses and other microorganisms have not heretofore been available. 
         [0006]    The use of pathogen reducing agents, which include certain photosensitizers, or compounds which absorb light of defined wavelengths and transfer the absorbed energy to an energy acceptor, have been proposed for reduction of undesirable cells and microorganisms found in blood products or fluids containing blood products. Such photosensitizers may be added to the fluid containing blood or blood components and irradiated. 
         [0007]    A number of systems and methods for irradiating pathogens in a fluid with light either with or without the addition of a photosensitizer are known in the art. For example, U.S. Pat. No. 5,762,867 is directed toward a system for activating a photoactive agent present in a body fluid with light emitting diodes (LEDs). 
         [0008]    U.S. Pat. No. 5,527,704 is directed toward an apparatus containing LEDs used to activate a fluid containing methylene blue. 
         [0009]    U.S. Pat. No. 5,868,695 discloses using LEDs having a red color and emitting light at a wavelength of 690 nm in combination with benzoporphyrin derivative photosensitizers to inactivate red blood cells. As taught in this patent, at a wavelength of 690 nm, red blood cells are essentially transparent to radiation, and as such, the benzoporphyrin derivatives absorb radiation at this wavelength to become activated. Also disclosed in this patent is the use of LEDs having a blue color and emitting light at a peak wavelength of 425 nm to inactivate platelets. 
         [0010]    U.S. Pat. No. 5,658,722 discloses irradiating platelets using UVA1 light having an emission peak near 365 nm. This patent teaches that damage to platelets is caused by short UVA&lt;345 nm, and unlike the present invention, calls for removing UVA wavelengths below 345 nm. 
         [0011]    Use of UV light which is variably pulsed at a wavelength of 308 nm without the addition of a photosensitizer to inactivate virus in a washed platelet product is taught in an article by Prodouz et al., (Use of Laser-UV for Inactivation of Virus in Blood Products; Kristina Prodouz, Joseph Fratantoni, Elizabeth Boone and Robert Bonner;  Blood,  Vol 70, No. 2). This article does not teach or suggest the addition of a photosensitizer in combination with light to kill viruses. 
         [0012]    One of the features associated with the use of UV light to irradiate red cells has been the propensity of this approach to induce methemoglobin (metHb) formation. In general, metHb levels in normal red cells is less than 1-2%. When levels exceed 10%, symptoms begin to appear in individuals suffering from methemoglobinemia. These include nausea, vomiting, difficulty breathing, discoloration of skin, etc. When levels reach 20-30% severe reactions, including death occurs. 
         [0013]    UV light is known to be absorbed by hemoglobin in red cells, primarily through the porphyrin structure of the hemoglobin molecule. Once absorbed, it can result in a change in the oxidation state of the hemoglobin molecule from the Fe2+ (ferrous) to Fe3+ (ferric) state. This occurs via oxidation of the iron chelate. The Fe3+ state is not capable of binding or transporting oxygen. This form of the molecule is known as methemoglobin. Normally, oxidation is reversible through several enzymatic systems in the red cell, which can reduce the Fe3+ state back to Fe2+. Several chemical agents are also known to be able to affect this reduction chemistry. These include methylene blue and riboflavin. 
         [0014]    One study on the ability of riboflavin to cause a reduction in hemoglobin from the metHb form back to the normal hemoglobin form was discussed in Dotsch et al. “Comparison of Methylene Blue, Riboflavin, and N-acetylcysteine for the Reduction of Nitric Oxide-induced Methemoglobinemia” ( Crit. Care Med  2000, Vol. 28, No. 4, pp. 958-961). The reference showed a concentration effect for this chemistry, with optimal levels being observed at 120 μM. 
         [0015]    At the time this study was done, using concentrations of riboflavin greater than 120 μM was not possible due to the solubility limits of riboflavin. 
         [0016]    The present invention is directed toward the reduction of pathogens which may be present in red blood cells using Uv light in combination with an endogenous photosensitizer, without extensive formation of methemoglobin or excessive hemolysis of the red blood cells upon illumination with UV light. 
       SUMMARY 
       [0017]    This invention is directed to a method for reducing pathogens which may be contained in red blood cells and includes the steps of adding an isoalloxazine photosensitizer to the red blood cells to form a mixture, and exposing the mixture of the red blood cells and the photosensitizer to light having a peak wavelength of approximately 302 nm. 
         [0018]    The method of this invention also reduces the formation of methemoglobin and hemolysis of the red blood cells. 
         [0019]    This invention is also directed toward a treatment chamber for reducing pathogens in a fluid containing red blood cells and a photosensitizer which includes at least one radiation emitting source emitting radiation at a peak wavelength of approximately 302 nm, a support platform for holding the fluid containing red blood cells and photosensitizer to be irradiated; and a control unit for controlling the radiation emitting source. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES  
         [0020]      FIG. 1  is a cross-sectional view of a treatment chamber which may be used in the present invention. 
           [0021]      FIG. 2  is a cross-sectional view of a treatment chamber like that of  FIG. 1 , but with an alternative reflective surface that may also be used in the present invention. 
           [0022]      FIG. 3  is a plan view of an array of LEDs that may be used in the present invention. 
           [0023]      FIG. 4  is a graph depicting the light spectrum of one type of bulb which may be used in the present invention. 
           [0024]      FIG. 5  is a graph of log pathogen reduction as a function of energy dose. 
           [0025]      FIG. 6  is a graph of log pathogen reduction at 19 J/mL red blood cells. 
           [0026]      FIG. 7  is a graph comparing the percentage hemolysis of red blood cells as a function of energy dose. 
           [0027]      FIG. 8  is a graph comparing the percentage methemoglobin generated during illumination as a function of energy dose. 
           [0028]      FIG. 9  is a graph comparing the percentage of methemoglobin as a function of storage time. 
           [0029]      FIG. 10  is another embodiment of a treatment chamber which may be used in the present invention. 
       
    
    
     DETAILED DESCRIPTION  
       [0030]    Photosensitizers useful in this invention include endogenous photosensitizers. The term “endogenous” means naturally found in a human or mammalian body, either as a result of synthesis by the body or because of ingestion as an essential foodstuff (e.g. vitamins) or formation of metabolites and/or byproducts in vivo. When endogenous photosensitizers are used, particularly when such photosensitizers are not inherently toxic or do not yield toxic photoproducts after photoradiation, no removal or purification step is required after decontamination, and the decontaminated product can be directly administered to a recipient in need of its therapeutic effect. 
         [0031]    Examples of such endogenous photosensitizers which may be used in this invention are alloxazines such as 7,8-dimethyl-10-ribityl isoalloxazine (riboflavin), 7,8,10-trimethylisoalloxazine (lumiflavin), 7,8-dimethylalloxazine (lumichrome), isoalloxazineadenine dinucleotide (flavin adenine dinucleotide [FAD]) and alloxazine mononucleotide (also known as flavin mononucleotide [FMN] and riboflavine-5-phosphate). The term “alloxazine” includes isoalloxazines. 
         [0032]    One mechanism by which these photosensitizers may inactivate pathogens is by interfering with nucleic acids, so as to prevent replication of the nucleic acid. As used herein, the term “reduction of a pathogen” means totally or partially preventing the pathogen from replicating, either by killing the pathogen or otherwise interfering with its ability to reproduce. Specificity of action of the preferred photosensitizer is conferred by the close proximity of the photosensitizer to the nucleic acid of the pathogen and this may result from binding of the photosensitizer to the nucleic acid. “Nucleic acid” includes ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). 
         [0033]    Upon exposure of the photosensitizer to light, the photosensitizer will absorb the light energy, causing photolysis of the photosensitizer and any nucleic acid bound to the photosensitizer. In this invention, the photosensitizer used in the examples is 7,8-dimethyl-10-ribityl isoalloxazine (riboflavin). 
         [0034]    Besides undesirable donor white blood cells, other pathogens which may be reduced using pathogen reduction agents or photosensitizers include, but are not limited to, viruses (both extracellular and intracellular), bacteria, bacteriophages, fungi, blood-transmitted parasites, and protozoa. Exemplary viruses include acquired immunodeficiency (HIV) virus, hepatitis A, B and C viruses, sinbis virus, cytomegalovirus, vesicular stomatitis virus, herpes simplex viruses, human T-lymphotropic retroviruses, lymphadenopathy virus LAV/IDAV, parvovirus, transfusion-transmitted (TT) virus, Epstein-Barr virus, BVDV, HAV, and others known to the art. Bacteriophages include Φ X174, Φ 6, λ, R17, T 4 , and T 2 . Exemplary bacteria include but are not limited to  P. aeruginosa, S. aureus, S. epidermis, L. monocytogenes, E. coli, K pneumonia, Y. enterocolitica  and  S. marcescens.    
         [0035]    The photosensitizer may be added directly to the fluid to be pathogen reduced, or may be flowed into the photopermeable container separately from the fluid being treated, or may be added to the fluid prior to placing the fluid in the photopermeable treatment container. The photosensitizer may also be added to the photopermeable container either before or after sterilization of the treatment container. 
         [0036]    The fluid containing the photosensitizer may also be flowed into and through a photopermeable container for irradiation, using a flow through type system. Alternatively, the fluid to be treated may be placed in a photopermeable container which is agitated and exposed to photoradiation for a time sufficient to substantially reduce the pathogens, in a batch-wise type system. 
         [0037]    After treatment, the blood or blood product may be stored for later delivery to a patient, concentrated, infused directly into a patient or otherwise processed for its ultimate use. 
         [0038]      FIG. 1  shows, in a cross-sectional view, the inside of a radiation or treatment chamber of one type of apparatus that may be used in the present invention. The treatment chamber shown in  FIG. 1  may be used in batch-wise systems; however, it should be noted that similar elements may also be used in flow-through systems. It should be noted that throughout the description of the invention, like elements have been given like numerals. The apparatus  55 , used for inactivating a fluid which may contain pathogens, consists of an internal chamber  33  having at least one source of radiation  26 . In one preferred embodiment, the internal chamber may contain a second source of radiation  36 . Each radiation source  26  and  36  respectively, is depicted as including a plurality of discrete radiation-emitting elements. The internal chamber  33  further consists of a support platform, shelf or platen  25  for supporting the fluid container  10  containing the fluid to be irradiated, and a control unit  11 . 
         [0039]    As introduced above, two sources of radiation are shown within internal chamber  33 . Radiation source  26  may be located along the top portion of the internal chamber  33  above the container  10  which holds or contains the fluid to be irradiated, while radiation source  36  may be located along the bottom portion of the internal chamber  33  below the container  10 . Although not shown, radiation sources may also be located along some or all of the sides of the internal chamber  33  perpendicular to the container  10 . The radiation or treatment chamber  55  may alternatively contain a single radiation source at any location within the internal chamber  33  and still comply with the spirit and scope of the present invention. 
         [0040]    The radiation source including a plurality of radiation-emitting elements collectively designated as source  26  includes an upper support substrate  15  containing a plurality of discrete radiation emitting elements or discrete light sources (see discrete source  20  as one example) mounted thereon. The support substrate  15  may be in an arcuate shape as shown, in a flat shape, or in other configurations which are not shown but are known in the art. Thus, the upper support substrate  15  could also be in a shape other than arcuate without departing from the spirit and scope of the invention. 
         [0041]    As further depicted in  FIG. 1 , the radiation source collectively designated as discrete source  36  includes a lower support substrate  35  which also contains a plurality of discrete radiation emitting elements or discrete light sources (see discrete source  30  as another example). Lower support substrate  35  preferably runs parallel to support platform  25 . The lower support substrate  35  may be substantially flat as shown, or may be in an arcuate shape similar to element  15  above, or may be in a shape other than arcuate, without departing from the spirit and scope of the invention. 
         [0042]    As shown in  FIG. 1 , the support substrates  15  and  35  may include at least one reflective surface, and as shown, may include two or more reflective surfaces  17  and  37  thereon. Reflective surface  17  is shown as running contiguous with upper support substrate  15 . Reflective surface  37  is shown as running contiguous with lower support substrate  35 . The reflective surfaces  17  and  37  may also run contiguously with only a portion of support substrates  15  and  35 . As shown in  FIG. 1 , discrete light source devices  20  and  30  extend outwardly away from the surface of the support substrates  15  and  35 . Alternatively, a discrete light source could be recessed into the surface such that the surface surrounds each discrete light source in a parabolic shape (not shown). The support substrate may or may not have reflective surfaces. In a further alternative configuration, the reflective surface may not contain any light sources. Such a reflective surface containing no light sources (not shown) may be located within the treatment chamber on a side opposite from the radiation source. As shown in  FIG. 2 , the support platform  25  may have a reflective surface  39 . This reflective surface  39  on support platform  25  may be in place of, or may be in addition to, another reflective surface (see element  17  as one example) within the treatment chamber. There may also be no reflective surfaces at all within the treatment chamber. 
         [0043]    In any of these reflective surface embodiments, the reflective surface may be coated with a highly reflective material which serves to reflect the radiation emitted from the lights back and forth throughout the treatment chamber until the radiation is preferably completely absorbed by the fluid being irradiated. The highly reflective nature of the reflective surface reflects the emitted light back at the fluid-filled bag or container  10  with minimum reduction in the light intensity. 
         [0044]    In  FIG. 1 , support platform  25  is positioned within the internal treatment chamber  33 . The support platform  25  may be located substantially in the center of the radiation or treatment chamber (as shown in  FIG. 1 ), or may be located closer to either the top portion or the bottom portion of the treatment chamber without departing from the spirit and scope of the present invention. The support platform  25  supports the container  10  containing the fluid to be irradiated. The support platform  25  may also be defined as a platen or a shelf. Additionally or alternatively, the platform  25  may be made of a photopermeable material to enable radiation emitted by the lights to be transmitted through the platform and penetrate the fluid contained within the container  10 . The platform may also be a wire or other similar mesh-like material to allow maximum light transmissivity therethrough. 
         [0045]    The support platform  25  is preferably capable of movement in multiple directions within the treatment chamber. One type of agitator, such as a Helmer flatbed agitation system available from Helmer Corp. (Noblesville, Ind., USA) may be used. This type of agitator provides to and fro motion. Other types of agitators may also be used to provide a range of motion to the fluid contained within the container  10 , without departing from the spirit and scope of the invention. For example, the support platform might be oriented in a vertical direction and the light sources may be rotated about a horizontal axis. The support platform  25  may alternatively rotate in multiple possible directions within the radiation chamber in varying degrees from between 0° to 360°. Support platform  25  may also oscillate back and forth, or side to side along the same plane. As a further alternative, one or more of the light sources may also move in a coordinated manner with the movement of the support platform. Such oscillation or rotation would enable the majority of the photosensitizer and fluid contained within the container  10  to be exposed to the light emitted from each of the discrete radiation sources (e.g. discrete sources  20  and  30 ), by continually replacing the exposed fluid at the light-fluid interface with fluid from other parts of the bag not yet exposed to the light. Such mixing continually brings to the surface new fluid to be exposed to light. 
         [0046]    The movement of both the support platform  25  and/or the radiation sources  26  and  36  may be controlled by control unit  11 . The control unit  11  may also control the rate of light emission. 
         [0047]    In a preferred embodiment each discrete light source  20  and  30  emits a peak wavelength of light to irradiate the fluid contained in bag  10 . The peak wavelength of light emitted by each discrete light source is selected to provide irradiation of a sufficient intensity to activate both the photosensitizer in a pathogen inactivation process as well as to provide sufficient penetration of light into the particular fluid being irradiated, without causing significant damage to the blood or blood components being irradiated. The preferred photosensitizer is riboflavin. To irradiate a fluid containing red blood cells and riboflavin, it is desirable that each discrete light source  20  and  30  be selected to emit light at a peak wavelength of between 302-306 nm. 
         [0048]      FIG. 4  is a graph showing the spectral output of lamps which may be used in this invention. As can be seen, the bulbs emit light energy in a range of approximately 270-400 nm, with a phosphor peak intensity occurring at a wavelength of between 302-306 nm. 
         [0049]    The term peak wavelength as defined herein means that the light is emitted in a narrow range centered around a wavelength having a particular peak intensity. In an embodiment, the light may be centered around a narrow range of UV light at an approximate wavelength of 302 nm. The term “approximate” means the light may be ±5 nm around 302 nm. The term light source or radiation source as defined herein means an emitter of radiant energy, and includes energy in the ultraviolet range, as further described below. 
         [0050]    As shown in  FIG. 3 , each radiation source  26  may consist of a bank or array of a plurality of discrete LEDs devices. LED devices  20 ,  21  and  22  are self-contained emitters of radiation. Each LED emits a single color of light when an electrical current is applied. Each of the LED devices in the array  26  may also emit light in the same peak wavelength, which for red blood cells is preferably selected to be around approximately 302 nm. 
         [0051]    The discrete radiation sources or lights may be arranged in banks or arrays containing multiple rows of individual lights, or may be arranged in a single row (not shown). As shown in  FIG. 5 , if LED devices are used, a plurality of discrete LED devices may be arranged in multiple rows. The lights may also be staggered or offset from each other (not shown). If a bank or an array of LED lights is located in both the top and the bottom of the irradiation chamber  55  (see  FIG. 1 ), or in a vertical orientation as described above, each bag or container  10  containing fluid to be irradiated will be exposed to light on both the top and the bottom surfaces (or on both sides of the bag if in a vertical orientation). A reflective surface  17  (like that shown in  FIG. 1 ) may also be part of the array. 
         [0052]    One or more light sources may be used in the irradiation apparatus, depending on the output required to substantially reduce pathogens which may be present in the blood product, and without substantially damaging the blood component being irradiated. 
         [0053]    As described above, the lights used in this invention may be LED devices or other narrow bandwidth sources such as fluorescent light sources. The Ushio GT25T8E UV-B lamp is one such fluorescent source which may be used which has 10th percentile bandwidth points at approximately 278 nm and 353 nm. The peak phosphor power is at 306 nm. The UV-B region of the spectrum is 290 nm to 320 nm. 
         [0054]    Emitting light in a narrow spectrum may be beneficial to the blood product being irradiated because all non-useful wavelengths of light which might damage the blood or blood component being irradiated are reduced or eliminated. 
       EXAMPLE 1 
       [0055]    To determine the effect UV light and riboflavin have on the pathogen reduction of red blood cells, whole blood units were leukoreduced with a leukoreduction filter and centrifuged to initially separate the plasma component from the cellular component. The plasma component was removed and saline was added to wash the cellular component. The washed red blood cell component was diluted with 500 μM riboflavin in 0.9% saline to a hematocrit of 30%. The red blood cells were illuminated with UV light in a range of energies, from 5.6 J/cm 2  (19 J/mL RBCs) to 16.6 J/cm 2  (40 J/mL RBCs), with bulbs having a peak wavelength of approximately 302 nm. After treatment, the cells were centrifuged to express the riboflavin solution and the commercially available storage solution AS3 was added. The pathogen reduced cells were stored at 4° C. These cells are referred to as treated red blood cells. 
         [0056]    As shown in  FIG. 5 , at 40 J/mL RBCs, bovine viral diarrhea virus (BVDV, an enveloped virus and model for human Hepatitis C) was reduced in red blood cells by 1.1 logs.  Yersinia enterocolitica  (a bacterium) was reduced by 1.6 logs. 
         [0057]    As shown in  FIG. 6 , at half the energy (19 J/mL RBCs), the HIV virus is reduced by 2.25 logs, while BVDV and hepatitis A virus (HAV, a non-enveloped virus that infects humans) were reduced by 0.5 logs. This is an expected result, since half the energy was used as compared to  FIG. 5  above. 
         [0058]      FIGS. 5 and 6  show that approximately 302 nm of light with riboflavin is capable of reducing pathogens in red blood cells. 
       EXAMPLE 2 
       [0059]    In addition to preventing replication of pathogens which may be present in red blood cells, the addition of riboflavin appears to prevent oxidation of hemoglobin to the methemoglobin (metHb) form as well as to decrease red cell hemolysis due to the exposure to UV light. 
         [0060]    Studies which compare both metHb formation and hemolysis in samples with varying doses of riboflavin (0, 50, 500 Micromolar) were conducted to determine if there is a concentration dependence and if this effect can be correlated with the presence of riboflavin. Since riboflavin&#39;s absorbance of light in this region is minimal (unless bound to DNA or RNA), it is not expected that these results are due to simple screening or shielding of the hemoglobin from UV light. 
         [0061]      FIG. 7  shows the % hemolysis of treated red blood cells at varying energy levels (from 0-40 J/mL red blood cells and 80 J/mL red blood cells having a peak wavelength of approximately 302 nm) for each unit illuminated in saline, 50 μM (shown in legend as 50 uM RF) and 500 μM (500 uM RF) riboflavin. One replicate was illuminated for each riboflavin concentration tested. Hemolysis values were consistently lower for the red blood cells containing 500 μM riboflavin, with the next highest values observed for the red blood cells containing saline and then 50 μM riboflavin. The lower hemolysis values observed in the 500 μM riboflavin samples may indicate that higher concentrations of riboflavin protect the red cells from damage by UV light. There may be a threshold in riboflavin concentration at which UV light can no longer easily affect the RBCs. Any exposure below this threshold would potentially damage the RBCs. This may explain why the 50 μM samples and the saline samples are similar, with the 50 μM samples higher than the saline samples for most of the energies assessed. 
         [0062]      FIG. 8  shows the % metHb generation during illumination of treated red blood cells at varying energy levels (from 0-40 J/mL red blood cells and 80 J/mL red blood cells) for each unit illuminated in saline, 50 μM (shown in legend as 50 uM RF) and 500 μM (500 uM RF) riboflavin. One replicate was illuminated for each riboflavin concentration tested.  FIG. 8  shows that methemoglobin measurements were essentially the same for each riboflavin concentration tested at each energy level. 
         [0063]    Crossmatch scores were measured for all illumination samples using autologous plasma and Ortho-Clinical gel cards (not shown). All IgG scores were zero—indicating no binding of protein to the red blood cells and therefore no detectable damage. 
         [0064]    As shown in  FIG. 9 , the percentage of methemoglobin decreases over time during storage. Methemoglobin values decrease to acceptable levels (&lt;3%) by Day 4 (96 hours) for all samples at the various riboflavin concentrations tested. 
         [0065]    It is believed that the reason for the successful treatment of red cells using UV light in the wavelength range of approximately 270-400 nm and having an approximate peak wavelength of 302 nm is due to a reduction in the absorbance of light by the hemoglobin molecule in this region. Another potential reason for this unique and unexpected observation and outcome is that the presence of riboflavin at a concentration of 500 μM is able to protect the hemoglobin in the cells from oxidation due to UV light chemistry via the chemical reducing capacity of the riboflavin. 
         [0066]    Any type of bulbs, either fluorescent or LEDs which emit light in a wavelength range between 270-400 nm may be used. Filters which filter out undesired wavelengths of light except light in the range of approximately 302 nm may also be used to obtain an approximate peak wavelength. 
         [0067]    If desired, the light sources  20  and  30  may be pulsed. Pulsing the light may be advantageous because the intensity of light produced by the light sources may be increased dramatically if the lights are allowed to be turned off and rested between light pulses. Pulsing the light at a high intensity also allows for greater depth of light penetration into the fluid being irradiated, thus allowing a thicker layer of fluid to be irradiated with each light pulse. 
         [0068]      FIG. 10  shows an alternative embodiment of an irradiation or treatment chamber to be used with the present invention. A bank of light sources  50  which emit peak wavelengths of light and which may or may not be capable of being pulsed, may be located within the top of the irradiation chamber extending from lid  40 . Although not shown in  FIG. 10 , a bank of lights may also be located in the bottom of the irradiation chamber as well. A reflective surface  57  is shown as part of the inner surface of lid  40 ; however, reflective surface  57  or another one or more surfaces (not shown) may be located anywhere within the radiation chamber as introduced above. 
         [0069]    The lid  40  is capable of being opened and closed. During exposure of the bag  10  containing the fluid to be irradiated to the light sources, the lid  40  is in a closed position (not shown). To add or remove the bag  10  containing the fluid to be irradiated from the irradiation chamber, a drawer  45  located on the front of the irradiation chamber may be disposed in an open position (as shown). During the irradiation procedure, the drawer  45  is placed in a closed position (not shown). 
         [0070]    The light sources  50 , as shown in  FIG. 10 , may be fluorescent or incandescent tubes, which stretch the length of the irradiation chamber, or may be a single light source which extends the length and width of the entire chamber (not shown). The LEDs shown in  FIG. 3  may also be used in this embodiment. 
         [0071]    As shown in  FIG. 10 , the support platform or platen  67  may be located within and/or forming part of drawer  45 . The support platform  67  may contain gaps  60  or holes or spaces within the platform  67  to allow radiation to penetrate through the gaps directly into the container  10  containing fluid to be irradiated. 
         [0072]    A cooling system may also optionally be included. As shown in  FIG. 10 , air cooling using at least one fan  65  may be preferred but it is understood that other well-known systems can also be used. Although not shown in  FIG. 10 , the method may also include the use of temperature sensors and other cooling mechanisms where necessary to keep the temperature below temperatures at which desired proteins and blood components in the fluid being irradiated are damaged. Preferably, the temperature is kept between about 0° C. and about 45° C., more preferably between about 4° C. and about 37° C., and most preferably about 28° C. 
         [0073]    Although described primarily with reference to a stand alone irradiation device used to irradiate individual bags (batch process), peak wavelengths of light may be used to irradiate blood or blood components in a flow-through irradiation system as well, without departing from the scope of the present invention.