Anaerobic blood storage and pathogen inactivation method

Abstract

To provide a novel oxygen reduced blood product.SOLUTION: An oxygen reduced blood product has a final concentration of approximately 0.2 mM amustaline (S-303), having an oxygen saturation (SO2) of less than 25%, and a carbon dioxide partial pressure (pCO2) of 90 mmHg or less at 37°C.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to methods for improving the quality and safety of blood and blood products for use in transfusion medicine. [Background technology]

[0002] The use of blood and blood components for transfusion is a common practice in current medicine, but it represents a risk to patients due to potential exposure to immunogenic and pathogenic contaminants. The practice of storing collected blood and blood components for up to several weeks exacerbates this risk. Whole blood is typically processed by filtration to remove white blood cells (leukoreduction), then centrifuged and separated into the three major blood components: plasma, platelets, and red blood cells. The leukoreduced packed red blood cells (LRpRBCs) are then typically suspended in an additive solution, such as AS-1 (Adsol®), AS-3 (Nutricel®), AS-5 (Optisol®), and AS-7 (SOLX®) in the US, and SAGGM or PAGGSM in the EU, to extend the shelf life before refrigerated storage for up to 42 days. Plasma is typically frozen within 24 hours of phlebotomy and separation ("fresh frozen plasma" -FFP or FP24). FFP must be thawed prior to use and used within 5 days of thawing. Platelets (PLTs) are collected by apheresis or by pooling the PLT fractions separated from multiple units of whole blood. PLTs collected by apheresis are typically suspended in an additive solution such as PAS-C (Intersol®) in the EU (not yet in the US) or PAS-F (Isoplate®). PLTs are constantly shaken at room temperature to inhibit PLT activation and must also be used within 5-7 days of collection. Due to storage conditions, all blood components are susceptible to donor viral and bacterial contamination, but PLTs are more susceptible to bacterial contamination and proliferation than other blood components.

[0003] Recent advances in the art have provided for the inactivation of bacterial and viral pathogens by utilizing UV light to irradiate blood components prior to storage, both with photosensitizers (see, e.g., the psoralen-based INTERCEPT® system, the riboflavin-based MIRASOL® system) and without photosensitizers (THERAFLEX® UV-Platelet system). These systems crosslink and inactivate DNA in pathogenic species, thereby reducing the risk to the patient. The INTERCEPT® system uses amotosalen HCl (a synthetic psoralen) and 3 J / cm to crosslink pathogen DNA. 2 The Mirasol® system uses UV-A light delivered at a radiant exposure of 100 nm to remove or reduce remaining amotosalen and photoproducts after treatment. The Mirasol® system uses UV light centered around 313 nm to target riboflavin and absorption by riboflavin-nucleotide complexes. Systems without photosensitizers typically use UV-C light at 254 nm. The long-term effects of some of the photosensitizers and photoproducts in these systems are still being established.

[0004] Other advances in the art include the use of S-303 (Cerus Corporation, Concord, CA), a quinacrine mustard-based alkylating agent containing a frangible anchor group, to crosslink nucleic acids and inactivate infectious bacteria and other pathogens (see Henschler et al. “Development of the S-303 pathogen inactivation technology for red blood cell concentrates,” Transfus Med Hemother 38:33-42(2011) (“Henschler 2011”)). Without being limited by theory, it is believed that there are two reactions that form the basis of the S-303 pathogen inactivation process. The first reaction is the reaction with the S-303 molecule to form covalent DNA and RNA adducts. This first reaction is complete within approximately 30 minutes. The second reaction is the breakdown of excess S-303 into a less toxic by-product, S300. This breakdown occurs simultaneously with an additional reaction and is complete within 16-18 hours.

[0005] Without being limited to any particular theory, it is believed that the formation of covalent DNA and RNA adducts with S-303 is based on the intercalation of the molecule with nucleic acid polymers (e.g., DNA or RNA). As is currently understood, when S-303 is added to RBCs, it rapidly (within seconds to minutes) crosses membranes, including those of cells and viral envelopes, due to its amphipathic properties, and burrows into the helical regions of the nucleic acid. It is hypothesized that the presence of fragile anchors on the molecule aids in the intercalation process by attracting the positively charged amine groups on the molecule to the negative charges of the DNA or RNA nucleic acid. The proximity of the S-303 molecules allows for a thermal cycloaddition reaction that rapidly covalently bonds the S-303 molecule to the DNA or RNA. It is believed that the covalent bond inhibits the replication or translation process from occurring, halting further pathogen production. During the process of forming the covalent adduct, the fragile anchors are removed by hydrolysis, resulting in the less toxic compound S300.

[0006] The spontaneous decomposition of S-303 to the less toxic S300 is the second reaction in the pathogen inactivation process. An excess of S-303 (about 0.2 mM) is usually added to the RBCs to provide enough reagent to completely react with all of the DNA and RNA in the sample. However, since S-303 is a toxic compound, the remaining S-303 must be removed in order for the resulting product to be safely transfused. In the Cerus pathogen inactivation process, this is accomplished primarily by decomposing S-303 to the significantly less toxic S-300 compound. The decomposition process occurs by hydrolysis, and when the S-303 reagent is first mixed with the RBCs, a low-to-high pH change induces hydrolysis of S-303. The decomposition kinetics of the remaining S-303 is rapid at concentrations above 10 nm / L with a half-life of about 20 minutes.

[0007] As currently understood, S-303 also has the potential to react with other nucleophiles in the RBC unit, including small molecules such as phosphate, water, and macromolecules such as proteins. Without being limited to any particular theory, 20 mM glutathione (GSH) is simultaneously added to the RBC during the pathogen inactivation process to reduce these non-specific interactions with proteins. (See Henschler 2011). Glutathione (GSH) is a naturally occurring antioxidant present in most cells at an intracellular concentration of about 5 mM. As currently understood, GSH distributes only into the extracellular plasma space, while S-303 diffuses across the membrane and equilibrates inside and outside the cell. This allows GSH to suppress the extracellular response of S-303 without significantly affecting pathogen inactivation (see Olcina et al. Hypoxia and the DNA damage response. Hypoxia and Cancer in Cancer Drug Discovery and Development 2014, Chapter 2:21-30, Melillo G (ed)).

[0008] Recent advances in the art also include the use of packed red blood cells stored anaerobically in additive solutions to reduce the amount of storage damage typically associated with the use of older blood (see Bitensky, et al. U.S. Pat. No. 5,789,152; Bitensky, et al. U.S. Pat. No. 6,162,396; and Bitensky, et al. U.S. Pat. No. 8,071,282). These storage damages are believed to result from metabolic processes and by-products resulting from storing blood in what is not the normal physiological environment of the circulatory system, and it is believed that the removal or reduction of available oxygen in stored blood creates damaging oxidative species within the red blood cells during storage.

[0009] Hemolysis is recognized as an important indicator of blood quality and safety. During storage, the level of hemolysis increases over time, and the presence of free hemoglobin is an indicator that blood has exceeded its shelf life. For this reason, regulations and guidelines have been developed that limit the acceptable storage period for blood product units that can be used for transfusion. Due to the importance of hemolysis for blood safety, a 0.8% upper limit has been set in Europe before blood must be discarded. The FDA recommends that levels of hemolysis not exceed 1.0%. Therefore, methods to reduce hemolysis would extend the safe shelf life of blood, reduce costs, and increase blood utilization.

[0010] Another indicator of blood health and safety are microparticles. See Cognasse et al., “The role of microparticles in inflammation and transfusion: A concise review,” Transfus.Apher.Sci.53(2):159-167 (2015). Microparticles (MPs) are produced by red blood cells, white blood cells, platelets, and endothelial cells. They are thought to be produced as a result of normal physiology, apoptosis, or cell damage. Generally, they are described as particles smaller than 1000 nm. A lower range is sometimes indicated at 50 nm, but there is no clear definition or convention for the lower limit. Flow cytometers in conjunction with fluorescent surface antibodies are usually used for quantification, but there is no universally accepted method for MP measurement, and measurements may depend on the instrument used. See Poncelet et al., “Tips and tricks for flow cytometry-based analysis and counting of microparticles,” Transfus. Apher. Sci. 53(2):110-126 (2015). The composition of MPs reflects the parent cells from which they are derived, but only select molecules are included or exposed on the surface of the resulting MPs. Some MPs are considered to be highly thrombogenic (especially platelet-derived MPs). In general, MPs in the stored RBC component are harmful to the recipient as a source for immunomodulation, hypercoagulation, nitric oxide capture (poor hemoperfusion), or the development of alloimmunity. Thus, methods that result in reduced levels of microparticles would result in improved blood health and safety.

[0011] Here, the inventors have demonstrated that reduced oxygen in whole blood unexpectedly results in reduced amounts of hemolysis and microparticle formation when treating blood products to reduce disease-causing viruses, bacteria, and multicellular parasites, as well as to reduce white blood cells. The methods provided herein provide for an extended usable life of pathogen-reduced blood products by reducing hemolysis.

[0012] Pathogen inactivation of blood and blood products has been developed to improve their safety. Although various bacteria, viruses, and parasites can be inactivated, research studies have demonstrated negative effects on blood components. Currently, plasma and platelet concentrates can be processed using pathogen inactivation systems, but processing of red blood cells is still under development. Pathogen inactivation of whole blood after donation may offer the advantage that, in addition to the destruction of remaining white blood cells, all derived products are pathogen inactivated. However, recent studies have demonstrated that the quality of red blood cells derived from whole blood irradiation using riboflavin / UV light technology (Mirasol, TerumoBCT) is significantly reduced compared to untreated study groups, to the extent that their shelf life under standard storage conditions must be shortened. A feature of these analyses is the promotion of hemolysis, reaching the currently accepted level of 0.8% at approximately 30 days of blood bank storage. The generation of reactive oxygen species (ROS) during UV irradiation contributes to hemolysis.

[0013] Here, we demonstrate that reducing oxygen from whole blood prior to pathogen treatment using the Mirasol system improves blood quality. The Hemanext™ system (New Health Sciences), designed to remove oxygen from whole blood and red blood cell concentrates, combined with pathogen reduction results in improved red blood cell quality compared to pathogen reduction under non-oxygen-reduced conditions. Hemanext™ treatment combined with Mirasol pathogen reduction treatment results in blood with less than 0.8% hemolysis after 42 days under oxygen-reduced conditions. Summary of the Invention [Means for solving the problem]

[0014] The present disclosure provides a method for inactivating blood pathogens and reducing hemolysis during storage by removing oxygen from the blood product, adding riboflavin to a final concentration of 40-60 μM, and irradiating the riboflavin-containing blood product with 265-400 nm UV light.

[0015] The present disclosure provides a method of reducing microparticle formation and inactivating blood pathogens by removing oxygen from the blood product, adding riboflavin to a concentration of 40-60 μM, and irradiating the riboflavin-containing blood product with UV light at 265-400 nm.

[0016] The present disclosure provides deoxygenated whole blood, comprising whole blood collected in a CPD, having 40-60 μM riboflavin, having an oxygen saturation (SO2) of less than 25%, having a pCO2 of 90 mmHg or less at 37° C., and irradiated with UV light of 265-400 nm.

[0017] The present disclosure provides oxygen- and carbon dioxide-reduced whole blood, comprising whole blood collected in a CPD, having 400-60 μM riboflavin, having an oxygen saturation (SO2) of less than 25%, and having a pCO2 of 20 mmHg or less at 37°C, wherein the oxygen-reduced whole blood has been irradiated with UV light of 265-400 nm.

[0018] The present disclosure provides a method for inactivating blood pathogens and reducing hemolysis by removing oxygen from blood products, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-20 mM.

[0019] The present disclosure provides methods for inactivation of blood pathogens and reduction of microparticle formation by removing oxygen and carbon dioxide from blood products, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-20 mM.

[0020] The present disclosure provides oxygen-reduced red blood cells having a final concentration of about 0.2 mM S-303, an oxygen saturation (SO2) of less than 25%, and a pCO2 of 90 mmHg or less at 37°C.

[0021] The present disclosure is disclosed with reference to the accompanying drawings. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a diagram illustrating a blood bag collection and storage system according to one embodiment of the present disclosure. [Diagram 2] FIG. 2 is a diagram showing a discontinuous apheresis system according to one embodiment of the present disclosure. [Diagram 3] FIG. 3 is a diagram showing a discontinuous apheresis system according to one embodiment of the present disclosure. [Figure 4] FIG. 4 is a graph showing the results of an experiment according to the present disclosure comparing the mean hemolysis of control whole blood with sterile saline (average 1), control whole blood with riboflavin (average 2), oxygen-reduced packed RBCs with sterile saline (average 3), oxygen-reduced pRBCs with riboflavin (average 4), oxygen-reduced whole blood with sterile saline (average 5), and oxygen-reduced whole blood with riboflavin (average 6). [Diagram 5] FIG. 5 is a graph showing the results of an experiment according to the present disclosure comparing the average amount of microparticles in control whole blood with sterile saline (average 1), control whole blood with riboflavin (average 2), oxygen-reduced packed RBCs with sterile saline (average 3), oxygen-reduced pRBCs with riboflavin (average 4), oxygen-reduced whole blood with sterile saline (average 5), and oxygen-reduced whole blood with riboflavin (average 6). [Figure 6A]6A-6G are graphs showing the results of experiments according to the present disclosure comparing the osmotic fragility (6A) of control whole blood with sterile saline (1 average), control whole blood with riboflavin (2 average), oxygen-reduced packed RBCs with sterile saline (3 average), oxygen-reduced pRBCs with riboflavin (4 average), oxygen-reduced whole blood with sterile saline (5 average), and oxygen-reduced whole blood with riboflavin (6 average). [Figure 6B] 6A-6G are graphs showing the results of experiments according to the present disclosure comparing potassium (6B) for control whole blood with sterile saline (1 average), control whole blood with riboflavin (2 average), oxygen-reduced packed RBCs with sterile saline (3 average), oxygen-reduced pRBCs with riboflavin (4 average), oxygen-reduced whole blood with sterile saline (5 average), and oxygen-reduced whole blood with riboflavin (6 average). [Figure 6C] 6A-6G are graphs showing the results of experiments according to the present disclosure comparing total hemoglobin (6C) for control whole blood with sterile saline (1 average), control whole blood with riboflavin (2 average), oxygen-reduced packed RBCs with sterile saline (3 average), oxygen-reduced pRBCs with riboflavin (4 average), oxygen-reduced whole blood with sterile saline (5 average), and oxygen-reduced whole blood with riboflavin (6 average). [Figure 6D] 6A-6G are graphs showing the results of experiments according to the present disclosure comparing oxygen saturation (6D) of control whole blood with sterile saline (1 average), control whole blood with riboflavin (2 average), oxygen-reduced packed RBCs with sterile saline (3 average), oxygen-reduced pRBCs with riboflavin (4 average), oxygen-reduced whole blood with sterile saline (5 average), and oxygen-reduced whole blood with riboflavin (6 average). [Figure 6E] 6A-6G are graphs showing results of experiments according to the present disclosure comparing glucose (6E) for control whole blood with sterile saline (1 average), control whole blood with riboflavin (2 average), oxygen-reduced packed RBCs with sterile saline (3 average), oxygen-reduced pRBCs with riboflavin (4 average), oxygen-reduced whole blood with sterile saline (5 average), and oxygen-reduced whole blood with riboflavin (6 average). [Figure 6F] 6A-6G are graphs showing the results of experiments according to the present disclosure comparing lactate (6F) of control whole blood with sterile saline (1 average), control whole blood with riboflavin (2 average), oxygen-reduced packed RBCs with sterile saline (3 average), oxygen-reduced pRBCs with riboflavin (4 average), oxygen-reduced whole blood with sterile saline (5 average), and oxygen-reduced whole blood with riboflavin (6 average). [Figure 6G] 6A-6G are graphs showing the results of experiments according to the present disclosure comparing the pH (6G) of control whole blood with sterile saline (1 average), control whole blood with riboflavin (2 average), oxygen-reduced packed RBCs with sterile saline (3 average), oxygen-reduced pRBCs with riboflavin (4 average), oxygen-reduced whole blood with sterile saline (5 average), and oxygen-reduced whole blood with riboflavin (6 average). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Corresponding reference characters indicate corresponding parts throughout the several views. The examples described herein illustrate aspects of the present invention but should not be construed as limiting the scope of the present invention.

[0024] Here, we show that the harmful side effects of irradiating a blood sample with UV light can be mitigated by reducing the amount of oxygen present in the sample. Without being limited to theory, it is believed that the reduction in oxygen reduces the generation of reactive oxygen species (ROS). The reduction in ROS is believed to increase the beneficial aspects of UV light irradiation for pathogen inactivation in blood samples, whether the blood sample is whole blood or any blood component, such as plasma, platelets, or red blood cells.

[0025] According to embodiments herein, the blood processing and storage system may be a blood component collection, separation, deoxygenation, and UV light irradiation before storage. The blood processing system may be a gravity-driven bag system, as is commonly used in whole blood leukoreduction. In some embodiments, the blood processing system may be an apheresis system, either continuous or discontinuous flow type, as is commonly known in the art, in which blood is collected, separated into desired components, and then deoxygenated and irradiated according to the present disclosure before storage. In some embodiments, blood is collected and deoxygenated before separation of desired blood components and UV irradiation before storage. In some embodiments, blood is collected and separated before deoxygenation and UV irradiation of desired blood components before storage. In some embodiments, blood is collected, deoxygenated, and UV irradiated before separation of desired blood components and storage.

[0026] Those skilled in the art will appreciate that the following examples and figures are intended to be illustrative only and are not intended to limit the scope of the invention. For purposes of this specification and description, the following definitions and terms are understood to have their general meaning. The term "UV" refers to ultraviolet light having a wavelength of about 220 nm to about 400 nm, generally including a peak from a mercury arc lamp at 405 nm. The term "blood sample" refers to a sample of blood from an animal or human, and includes whole blood and components of whole blood including red blood cells (RBCs), platelets (PLTs), plasma, white blood cells, proteins commonly found in blood, such as albumin, enzymes, clotting factors, and also includes combinations of such components commonly found in blood, such as partially separated fractions of whole blood, pre-separated recombinant components, including freshly collected or stored blood samples. The term "apheresis" is meant to have its general meaning in the art and includes the collection and separation of blood by both continuous and discontinuous methods, as generally known in the art. The term "apheresis system" is meant to have its general meaning in the art and includes devices and systems for the collection and separation of blood by both continuous and discontinuous methods, as generally known in the art. The term "blood container" is meant to refer to any container made of a polymeric material for the purpose of storing blood, regardless of storage period, and includes the general term "blood bag" as generally used in the art. The term "PVC" is meant to refer to a polymer contained in polyvinyl chloride, and includes PVC with any added materials, such as plasticizers, stabilizers, inhibitors, and other added materials known in the art and used in the manufacture of PVC.

[0027] FIG. 1 illustrates one embodiment of the present disclosure, including a blood collection bag 1, a blood processing bag 4, a UV irradiation chamber 10, and a blood storage bag 13. The blood collection bag 1 is generally known in the art and is typically made from a flexible plastic, such as polyvinyl chloride (PVC), but can also be made from other polymeric materials, such as urethane, silicone, or other biocompatible materials. The blood collection bag 1 has a blood transport line 3. The blood transport line 3 is also generally known in the art and is made from a flexible plastic, typically made from PVC, but can also be made from other biocompatible polymeric materials. The blood transport line 3 is fitted with a flow control device 2, such as a pinch clamp, ratchet clamp, or frangible seal, that prevents blood from flowing from the collection bag 1 through the transport line 3 to the processing bag 4 until such flow and transport of blood is desired. In some embodiments, the flow control device is designed to control the flow rate.

[0028] The processing bag 4 is composed of an outer barrier bag 5 and an inner blood bag 6, the outer barrier bag 5 being substantially impermeable to oxygen. Suitable materials for the construction of the barrier bag 5 are generally known in the art and include metal foils, e.g., aluminum foil, polymeric films having suitable barrier properties, e.g., ethyl vinyl alcohol (EVA), polyvinyl alcohol (PVA), polyacrylonitrile (Barex®), cyclic polyolefins, polychlorotrifluoroethylene (PCTFE or Aclar®), polyvinylidene chloride (PVDC), polymeric films having a coating to impart suitable barrier properties, e.g., by coating a polyethylene or nylon film with a coating of silicon oxide, aluminum oxide, or multi-layer films including a combination of polymeric films and / or coatings to impart suitable barrier properties. In some embodiments, the barrier bag is made from RollPrint ClearFoil® Z film or Renolit Solmed Wrapflex® film. Exemplary methods for making processing bags are provided in International Patent Application No. PCT / US2016 / 021794, filed March 10, 2016, which is incorporated by reference in its entirety.

[0029] The internal blood bag 6 is made from flexible polymeric materials with high oxygen transport properties commonly known in the art, including PVC, urethane, silicone, polyethylene, polypropylene, polyethersulfone, polyvinylidene fluoride (PVDF). In one embodiment, the internal blood bag 6 is made from silicone, such as Wacker Silpuran® 30 um thick silicone film. In another embodiment, the internal blood bag 6 is made from Millipore GVHP29325 PVDF membrane. An oxygen absorbing absorbing material 7 is disposed between the external barrier bag 5 and the internal blood bag 6. The oxygen absorbing absorbing material 7 is commonly known in the art, and is typically composed of an iron-based absorbing material, such as the Mitsubishi Ageless® series of oxygen absorbers, or other oxygen absorbing materials or systems, such as ascorbate / metal salt systems, metal catalysts such as platinum, or oxygen absorbing polymers such as nylon MXD6. In some embodiments, the outer barrier bag 5 and inner blood bag 6 can be laminated together with an oxygen absorbing material 7 disposed within the laminate structure, such as Mitsubishi Ageless® OMAC® film.

[0030] The oxygen absorbing sorbent material 7 is typically disposed in a breathable sachet and adapted to absorb oxygen in the gas headspace between the outer barrier bag 5 and the inner blood bag 6. In some embodiments, the oxygen absorbing sorbent material 7 is attached to a plastic mesh structure (not shown) to create a gap between the inner and outer bags, thus providing increased gas transport in the gas headspace. In some embodiments, a plurality of oxygen absorbing sachets are disposed in the gas headspace. In some embodiments, the oxygen absorbing sorbent material 7 is formulated with a capacity to absorb the entire oxygen content of a full unit of blood so that it acts quickly and quickly absorbs high levels of oxygen. In some embodiments, the oxygen absorbing sorbent material 7 has an oxygen absorption capacity of at least 100 cc of oxygen, and more preferably at least 200 cc. In some embodiments, an oxygen indicator tab (not shown) is disposed in the gas headspace to indicate by color the presence or absence of oxygen at a particular level, such as, for example, the adsorption system Tell-Tab®. Further details of the processing bag 4 and internal blood bag 6 suitable herein are provided in International Patent Application No. PCT / US2016 / 021794, filed March 10, 2016.

[0031] The fluid path of the internal blood bag 6 is connected to the UV irradiation chamber 10 by a blood transport line 9 having a flow control device 8, such as a pinch clamp, ratchet clamp, or frangible seal, to prevent blood from flowing from the internal blood bag 6 through the transport line 9 to the UV irradiation chamber 10 until such flow and transport is desired. In some embodiments, the flow control device is designed to control the flow rate. The UV irradiation chamber 10 is further fluidly connected to a blood storage bag 13 by a blood transport line 12 having a flow control device 11, such as a pinch clamp, ratchet clamp, or frangible seal, to prevent blood from flowing from the UV irradiation chamber 10 through the transport line 12 to the blood storage bag 13 until such flow and transport is desired. In some embodiments, the flow control device 11 is designed to control the flow rate, and in some embodiments, the flow control device is in communication with a UV lamp to provide controlled radiation exposure of the blood sample contained within the UV irradiation chamber.

[0032] The UV irradiation chamber 10 further includes a UV lamp (not shown) operably connected to a power source (not shown) to provide UV irradiation of blood contained within the chamber or passing through the chamber interior (not shown). In some embodiments, the UV irradiation chamber is adapted to receive a section of blood-transporting tubing 9 and irradiate UV light through the tubing. Those skilled in the art will appreciate that most plastics absorb UV light at lower wavelengths and would not be suitable for use with lower wavelength treatment UV light, such as UV-C light at 254 nm, but for certain photosensitizers that absorb at higher wavelengths, such as UV-B (about 290-320 nm) or UV-A (about 320-400 nm), thin pieces of plastic may be suitable. Thus, in some embodiments, a portion of the blood-transporting tubing 9 is adapted to have a very thin wall thickness to be fitted to the UV irradiation chamber 10 to enhance light transmission to the blood sample passing through the plastic tubing. In some embodiments, the wall thickness of the transport tube portion within the UV irradiation chamber is about 0.1 to about 1.0 mm, and in multiple embodiments, the wall thickness of the transport tube is about 0.2 to about 0.5 mm.

[0033] In some embodiments, the UV irradiation chamber 10 is adapted to have a UV-C transparent portion, such as a piece of quartz (SiO2) or sapphire (Al2O3) material, in fluid communication with the blood-transporting tube 9 and the blood-transporting tube 12. In some embodiments, the blood-transporting line 9 is adapted to have a UV-C transparent portion, such as a piece of quartz (SiO2) or sapphire (Al2O3) tubing, disposed between the blood-transporting tube 9 and the blood-transporting tube 12 such that the UV-C transparent portion can be easily inserted into the UV irradiation chamber 10. In some embodiments, the UV irradiation chamber 10 is constructed from a UV-C transparent material, such as quartz (SiO2) or sapphire (Al2O3), and is matingly adapted to be in fluid communication with the blood-transporting tube 9 and the blood-transporting tube 12 in and out. In some embodiments, the UV irradiation chamber 10 is constructed from a material that is opaque to UV-C but transparent to UV-A and / or UV-B wavelengths, such as glass or a polymer, such as polycarbonate, acrylic, PVC, urethane, etc., and is in internal and external fluid communication with the blood-carrying tubes 9 and 12. In some embodiments, the UV irradiation chamber 10 is constructed from a polymeric material, such as silicone, that is sufficiently transparent to UV-C, UV-A, and UV-B wavelengths to be effective in delivering UV light to the lumen of the irradiation chamber.

[0034] The blood storage bag 13 is comprised of an outer barrier bag 14 and an inner blood bag 15, the outer barrier bag 14 being substantially impermeable to oxygen. A suitable blood storage bag 13 is provided in International Patent Application No. PCT / US2016 / 029069, filed April 22, 2016, which is incorporated herein by reference in its entirety. Briefly, suitable materials for the construction of the outer barrier bag 14 are provided, the outer barrier bag 14 being substantially equivalent to the outer barrier bag 5. The inner blood bag 15 is substantially equivalent to the blood collection bag 1 described above, except that it further includes a spike port 17, which is generally known in the art of transfusion medicine and is adapted for sterile connection of an infusion spike to receive blood contained within the inner blood bag 15 during patient use. The blood storage bag 13 further includes an oxygen absorbing material 16 disposed between the outer barrier bag 14 and the inner blood bag 15. In embodiments, the oxygen absorbing material 16 is formulated to function at refrigeration temperatures and absorb low levels of oxygen for extended periods of time.

[0035] 2 shows a discontinuous three-line apheresis system in which whole blood is drawn from a subject through a venous access device 110, which may be inserted into the subject's arm. A blood draw line 120 fluidly connects the venous access device 110 to a blood component separation device 150 for separation of blood components. A draw pump 122 located on the draw line 120 controls the direction, rate, and duration of flow through the draw line 120.

[0036] As the whole blood is being drawn from the subject, an anticoagulant can be added to the whole blood to prevent the blood from clotting in the line or in the blood component separation device 150. To that end, the system includes an anticoagulant line 130 fluidly connected to an anticoagulant source 134 (e.g., a bag of anticoagulant) at one end and a venous access device 110 (or draw line 120) at the other end. An anticoagulant pump 132 through which the anticoagulant line 130 passes controls the flow rate through the anticoagulant line 130 and the amount of anticoagulant introduced into the whole blood. The anticoagulant pump 132 operates in proportion to the draw pump 122 to ensure that an appropriate amount of anticoagulant is added to the whole blood. The anticoagulant is typically introduced into the whole blood as close as possible to the venous access device 110. The line / conduit typically includes a clamp valve 164 to stop flow in the line.

[0037] Once a desired amount of anticoagulated whole blood has been withdrawn from a subject and contained within the blood component separation device 150, the blood component separation device separates the whole blood into several blood components, typically plasma, platelets, red blood cells, and optionally white blood cells.

[0038] Some embodiments of the blood processing system 100 include a transport pump 210 and a dilution / extraction line 160 connected to the plasma bag 158. The transport pump 210 and the dilution / extraction line 160 can be used for various purposes, including diluting the anticoagulated draw blood introduced into the blood component separation device. For example, if a user desires draw blood with a higher plasma content, the system can dilute the draw blood by switching on the transport pump and introducing plasma from the plasma bag 158 into the removed blood in the draw line 120. Additionally or alternatively, the transport pump may be used during surge elutriation to introduce plasma from the plasma bag 158 into the blood component separation device 150 to extract platelets (or other blood components).

[0039] After the blood sample has been separated in the blood component separation device 150 and the desired components have been removed and stored in appropriate storage containers 156 or 158, the system returns the unextracted and / or unwanted components to the target again via dedicated lines.

[0040] 3 illustrates one embodiment of the present disclosure for use with a discontinuous three-line apheresis system, with improvements shown within the dashed lines. Specifically, the apheresis blood processing system 100 further includes a fluid transport pump 172, a deoxygenation device 173, a processing reservoir 174, and a UV irradiation chamber 177, which are fluidly connected by a transport line 170 and a recirculation line 175. Control of fluid flow rates in the transport line 170 and the recirculation line 175 is maintained by control of flow control valves 171, 176, 178, and 179, which act in conjunction to direct the fluid to the desired devices.

[0041] Upon completion of separation of the blood components in blood separation device 150, the flow of the desired blood component is directed to transport line 170 herein by closing flow control valve 179 herein and opening flow control valve 171 herein. Thus, the desired blood component is directed to transport pump 172 herein, rather than to transport line 152 and eventual plasma bag 158 or platelet bag 156. The fluid flow in transport line 170 can be directed by the fluid flow from transport pump 122 or transport pump 210, or by transport pump 172 herein, or by a combination thereof.

[0042] Fluid flow in transport line 170 is controlled by flow control valve 171 and flows into transport pump 172 and subsequently into deoxygenation device 173 and processing reservoir 174, as indicated by the arrows in the figure. During the deoxygenation process, flow control valve 178 may be closed and fluid control valve 176 located on recirculation line 175 may be opened, thereby allowing the fluid to be recirculated by transport pump 172 through deoxygenation device 173, processing reservoir 174, and recirculation line 175 until a desired level of oxygen is achieved in the fluid. In some embodiments, deoxygenation device 173 is constructed of hollow porous fibers commonly known in the art for use in fluid degassing, such as Membrana Liqui-Cel® and MiniModule® devices, and for blood oxygenation in cardiopulmonary bypass perfusion, such as the Medtronic Affinity® series oxygenators and the Sorin Inspire® series oxygenators. In some embodiments, the deoxygenation device 173 is operably connected to a nitrogen gas source (not shown) to provide nitrogen gas to the hollow porous fibers to remove oxygen from the blood. In some embodiments, the deoxygenation device 173 is operably connected to a means for measuring the oxygen content of the fluid contained therein (not shown) to provide a means for measuring whether the fluid sample has been deoxygenated to about 25% oxygen or less, in embodiments, about 10% oxygen or less. In some embodiments, the transport pump is operably connected to a control means for turning the pump off after a predetermined time period that has been experimentally shown to be effective for the volume of fluid being processed.

[0043] After the fluid sample is sufficiently deoxygenated, the blood sample is pumped by pump 172 through UV illumination chamber 177 by opening flow control valve 178. The blood sample is illuminated with UV light from a suitable UV light source (not shown). The UV light source can be housed within UV illumination chamber 177 and operably connected to a power source (not shown), or the UV light source can be operably connected to the UV illumination chamber by a light pipe or mirror (not shown), or the like. UV light sources are known in the art and are selected based on the desired spectral output and the chemistry involved in the processing of the blood components, and can be selected from a group including mercury arc lamps, xenon lamps, flash lamps, deuterium lamps, halogen lamps, tungsten lamps, fluorescent lamps, and UV emitting LEDs.

[0044] Preferably, the fluid flow rate within the UV irradiation chamber is controlled such that a target radiant exposure of UV light is achieved in the fluid, either by time-dependent UV irradiation of a static sample contained within the UV irradiation chamber 177, or by controlling the flow rate of a dynamic sample passing through the interior of the UV irradiation chamber 177 and controlled by flow valve 178. In some embodiments, the radiant exposure of UV light is about 1-8 J / cm. 2 and in some embodiments, about 3 J / cm 2 The deoxygenated and UV irradiated blood components then flow through transport line 180 to transport line 152 for collection and storage. Once treated to reduce pathogens in accordance with the present disclosure and deoxygenated to an oxygen level of less than about 25%, the blood components are transported to a blood storage bag. In some embodiments, transport line 180 includes a component absorption device (not shown) to provide reduction of excess photosensitizer and photoproducts, as used, for example, with psoralen photosensitizers and commonly known in the art.

[0045] The present disclosure provides and includes a method for pathogen reduction with reduced hemolysis, comprising removing oxygen from a blood product to prepare an oxygen-reduced blood product, reducing blood pathogens from the blood product, including adding riboflavin to a final concentration of 40-60 μM, and irradiating the riboflavin-containing blood product with UV light at 265-400 nm. In certain embodiments, the method further includes storing the oxygen-reduced, pathogen-reduced blood product under anaerobic conditions. Also included and provided herein is a method for pathogen reduction with reduced hemolysis, comprising removing oxygen and carbon dioxide from a blood product to prepare an oxygen-reduced, carbon dioxide-reduced blood product, reducing blood pathogens from the blood product, including adding riboflavin to a final concentration of 40-60 μM, and irradiating the riboflavin-containing blood product with UV light at 265-400 nm. As used herein, "anaerobic conditions" includes both carbon dioxide depleted and carbon dioxide containing conditions. In most embodiments, anaerobic conditions refer to storage conditions that are depleted of both oxygen and carbon dioxide.

[0046] The present disclosure provides and includes methods for pathogen reduction with reduced hemolysis, including removing oxygen from a blood product to prepare an oxygen-reduced blood product, reducing blood pathogens from the blood product, and including adding S-303 to a final concentration of about 0.1-0.5 mM, and adding glutathione (GSH) to a final concentration of 2-20 mM. In certain embodiments, the methods for pathogen reduction with reduced hemolysis include removing oxygen from the blood product to prepare an oxygen-reduced blood product prior to reducing blood pathogens. In other embodiments, the methods for pathogen reduction with reduced hemolysis include removing oxygen from the blood product to prepare an oxygen-reduced blood product after reducing blood pathogens. In other embodiments, the methods for pathogen reduction with reduced hemolysis include removing oxygen from the blood product to prepare an oxygen-reduced blood product simultaneously with reducing blood pathogens. Also included and provided herein are methods for pathogen reduction with reduced hemolysis, including removing oxygen and carbon dioxide from a blood product to prepare an oxygen-reduced, carbon dioxide-reduced blood product, and reducing blood pathogens from the blood product, including adding S-303 to a final concentration of about 0.2 mM, and adding glutathione (GSH) to a final concentration of 5-20 mM. In other aspects, methods for pathogen reduction with reduced hemolysis include removing oxygen and carbon dioxide from a blood product to prepare an oxygen-reduced, carbon dioxide-reduced blood product, and reducing blood pathogens from the blood product, including adding S-303 to a final concentration of about 0.1-0.5 mM, and adding glutathione (GSH) to a final concentration of 2-20 mM. In another aspect, a method for pathogen reduction with reduced hemolysis includes removing oxygen and carbon dioxide from a blood product to prepare an oxygen-reduced, carbon dioxide-reduced blood product, reducing blood pathogens from the blood product, including adding S-303 to a final concentration of about 0.1-0.4 mM, and adding glutathione (GSH) to a final concentration of 5-20 mM.In yet another embodiment, a method for pathogen reduction with reduced hemolysis includes removing oxygen and carbon dioxide from a blood product to prepare an oxygen and carbon dioxide reduced blood product, and reducing blood pathogens from the blood product, including adding S-303 to a final concentration of about 0.1-0.3 mM, and adding glutathione (GSH) to a final concentration of 5-10 mM. In a further embodiment, a method for pathogen reduction with reduced hemolysis includes removing oxygen and carbon dioxide from a blood product to prepare an oxygen and carbon dioxide reduced blood product, and reducing blood pathogens from the blood product, including adding S-303 to a final concentration of about 0.1-0.3 mM, and adding glutathione (GSH) to a final concentration of 2-10 mM.

[0047] As used herein, pathogens include viruses, parasites, and bacteria. Also, as used herein, low levels of white blood cells remaining after leukoreduction are also considered pathogens. Thus, the method of pathogen reduction may further provide for reduction of white blood cells that may remain after leukoreduction.

[0048] As used herein, the terms "reducing", "reduced" or "reduced" refer to a final amount that is less than the initial amount or less relative to a control sample. A reduced level of a pathogen means that the pathogen level is reduced by at least one order of magnitude compared to a similar untreated sample. Generally, for transfusion medicine purposes, the pathogen level is reduced by at least 1.8 logs. In one embodiment of the present disclosure, the pathogen level is reduced by at least 3 logs. In another embodiment, the pathogen level is reduced by at least 4 logs. In another embodiment, the pathogen level is reduced by 3-10 logs. In another embodiment, the pathogen level is reduced by at least 7 logs.

[0049] As used herein, "reducing oxygen" or "reducing oxygen saturation" means reducing the oxygen saturation of red blood cells to 25% or less. As used herein, "reducing carbon dioxide" means reducing carbon dioxide to 90 mmHg or less when measured at 37°C. Oxygen-depleted blood products have oxygen saturation of less than 25%, typically less than 10%, and may have SO2 of about 5%. Carbon dioxide-depleted blood products are blood products that have carbon dioxide levels below 20 mmHg when measured at 37°C.

[0050] As used herein, "blood product" includes whole blood or any components derived from whole blood including red blood cells, platelets, plasma, and white blood cells.

[0051] As used herein, "whole blood" includes white blood cells (WBCs), platelets suspended in plasma, and includes electrolytes, hormones, vitamins, antibodies, etc. In whole blood, the white blood cells typically range from 4.5 to 11.0 x 10 9 cells / L, and the normal RBC range at sea level is 4.6–6.2 × 10 for men. 12 / L, and 4.2–5.4 × 10 for women. 12 / L. A normal hematocrit, or percent packed cell volume, is about 40-54% for men and 38-47% for women. The platelet count is usually between 150 and 450 × 10 for both men and women. 9 / L. Whole blood is collected from a blood donor and is usually combined with an anticoagulant. When collected, the whole blood is initially at about 37° C., rapidly cooled to about 30° C. during and immediately after collection, but gradually cooled to ambient temperature over a period of about 6 hours. Whole blood may be processed according to the methods of the present disclosure either at 30-37° C. upon collection or at room temperature (typically about 25° C.).

[0052] As used herein, "red blood cells" (RBCs), stored red blood cells, oxygen-reduced red blood cells, and oxygen- and carbon dioxide-reduced red blood cells include RBCs present in whole blood, leukocyte-reduced RBCs, platelet-reduced RBCs, leukocyte- and platelet-reduced RBCs, and packed red blood cells (pRBCs). Human red blood cells in vivo are in a dynamic state. Red blood cells contain hemoglobin, an iron-containing protein that carries oxygen throughout the body and gives red blood cells their color. The percentage of the corpuscular volume that is composed of red blood cells is called the hematocrit. As used herein, unless otherwise limited, RBCs also include packed red blood cells (pRBCs). Packed red blood cells are prepared from whole blood using centrifugation techniques commonly known in the art. As used herein, unless otherwise specified, the hematocrit of pRBCs is about 70%. As used herein, oxygen-reduced RBCs (OR-RBCs) can include oxygen and carbon dioxide-reduced (OCR-) RBCs (OCR-RBCs).

[0053] As used herein, "leukoreduced whole blood" (LRWB) includes whole blood with an anticoagulant that has been treated to remove white blood cells and platelets, usually by filtration or centrifugation. Leukoreduced whole blood has white blood cell levels reduced by at least 5 logs.

[0054] As used herein, "oxygen-reduced leukoreduced whole blood" (OR-LRWB) can include oxygen and carbon dioxide-reduced leukoreduced whole blood (OCR-LRWB).

[0055] As used herein, "leukoreduced packed red blood cells" (LRpRBCs) include packed red blood cells with oxygen-reduced (OR-) whole blood that has been treated to remove leukocytes. As used herein, oxygen-reduced leukoreduced packed red blood cells (OR-LRpRBCs) can include oxygen and carbon dioxide reduced leukoreduced packed red blood cells (OCR-LRpRBCs).

[0056] According to aspects herein, the viruses include enveloped viruses that have been reduced by the recited methods. In other aspects, the methods provide for the reduction of non-enveloped viruses. In embodiments herein, the reduced viral pathogen comprises one or more of the following: HIV-1, HIV-2, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human T-lymphotropic virus I and II (HTLV-1 and -II), Cell-associated cytomegalovirus (CMV), Bovine viral diarrhea virus (BVDV), Duck hepatitis B virus (DHBV), Pseudorabies virus (PRV), West Nile virus, Human coronavirus, Chikungunya virus, Influenza virus, Sug herpes virus (SuHV-1), Vesicular stomatitis virus (VSV), Sindbis virus, Herpes simplex virus (HSV), Epstein-Barr virus (EBV), Porcine pseudorabies virus (PPRV), Pseudorabies (PRV), or Semliki Forest virus (SLFV). In some embodiments, the inactivated viruses include non-enveloped viruses, including bluetongue virus, calicivirus, human adenovirus-5, porcine parvovirus (PPV), encephalomyocarditis virus (EMCV), hepatitis A virus (HAV), coxsackievirus, and poliovirus. It will be understood that the methods of viral parasite reduction include all viruses and are not limited to those listed above. Those skilled in the art will recognize that the inactivation methods target the genetic material of various pathogens, while oxygen reduction reduces hemolysis of, for example, red blood cell components of blood products, or otherwise improves blood quality.

[0057] In embodiments according to the present disclosure, the methods provide for reduced hemolysis and inactivation of parasites, including, in some embodiments herein, Plasmodium falciparum (malaria), Trypanosoma cruzi (Chagas' disease), Leishmania mexicana, Leishmania major, Leishmania infantum (leishmaniasis), Babesia microti, and Babesia divergens (babesiosis). In some embodiments, the inactivated pathogenic bacteria is selected from the group consisting of Bacillus cereus, Clostridium perfringens, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Propionibacterium acnes, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas fluorescens, and the like. These may include Bacterial and Mortality Rates, Bacterial and Mortality Rates, Bacterial and Mortality Rates of Bacterial Infection ...

[0058] The method of the present disclosure provides and encompasses reduced hemolysis of red blood cells after pathogen treatment. Pathogen reduction methods applied to oxygen-reduced blood or oxygen and carbon dioxide-reduced blood result in significantly reduced levels of hemolysis. Pathogen reduction methods applied to oxygen-reduced blood or oxygen and carbon dioxide-reduced blood result in significantly reduced levels of microparticles. Incorporating an additional oxygen reduction step into the pathogen reduction method results in improvements in the quality of stored blood, leading to improved shelf life. Importantly, applying blood oxygen reduction methods to existing methods of pathogen reduction results in significant improvements. Thus, the methods herein can be applied to pathogen reduction methods known in the art. Suitable methods for pathogen removal compatible with reduced oxygen pathogen inactivation include Wagner et al., “Developing pathogen reduction technologies for RBC suspensions” in Vox Sanguinis,100:112-121(2011); Pidcoke et al., “Primary hemostatic capacity of whole blood: a comprehensive analysis of pathogen reduction and refrigeration effects over time” in Transfusion,53:137-149(2013); Henschler et al., “Development of the S-303 pathogen inactivation technology for red blood cell concentrates” in Transfusion Medicine and Hemotherapy,38:33-42(2011); Guignard et al., “The clinical and biological impact of new pathogen inactivation technologies on platelet concentrates” in Blood Reviews,28: 235-241(2014); Picker et al., “Current methods for the reduction of blood-borne pathogens: a comprehensive literature review” in Blood Transfusion, 11:343-348 (2014); Irsch et al., “Pathogen inactivation of platelet and plasma blood components for transfusion using the INTERCEPT Blood System™” in Transfusion Medicine and Hemotherapy, 38:19-31 (2010); and U.S. Pat. No. 5,120,659, issued June 9, 1992 to King, et al.

[0059] The method provides reduced hemolysis resulting in extended acceptable storage time after pathogen inactivation by maintaining hemolysis levels below 0.8%, or in certain embodiments, below 1.0%. In one embodiment, hemolysis is 0.2% or less after 14 days of storage under anaerobic conditions. In one embodiment, hemolysis is 0.4% or less after 21 days of storage under anaerobic conditions. In another embodiment, hemolysis is 0.5% or less after 28 days of storage under anaerobic conditions. In a further embodiment, hemolysis is 0.8% or less after 35 days of storage under anaerobic conditions. In other embodiments, hemolysis is 0.8% or less after 42 days of storage under anaerobic conditions. In other embodiments, hemolysis is 0.8% or less after 49 days of storage under anaerobic conditions. In some embodiments, hemolysis is 1.0% or less after 35 days of storage under anaerobic conditions. In another embodiment, the hemolysis is 1.0% or less after storage under anaerobic conditions for 42 days. In another embodiment, the hemolysis is 1.0% or less after storage under anaerobic conditions for 49 days. In another embodiment, the hemolysis is 0.1% or less after storage under anaerobic conditions for 14 days. In another embodiment, the hemolysis is 0.01-0.2% after storage under anaerobic conditions for 14 days. In one embodiment, the hemolysis is 0.2-0.4% after storage under anaerobic conditions for 21 days. In one embodiment, the hemolysis is 0.05-0.4% after storage under anaerobic conditions for 21 days. In another embodiment, the hemolysis is 0.6% or less after storage under anaerobic conditions for 28 days. In another embodiment, the hemolysis is 0.1-0.4% after storage under anaerobic conditions for 28 days. In another embodiment, the hemolysis is 0.1-0.5% after 28 days of storage under anaerobic conditions. In another embodiment, the hemolysis is 0.4% or less after 35 days of storage under anaerobic conditions. In another embodiment, the hemolysis is 0.1-0.8% after 35 days of storage under anaerobic conditions. In another embodiment, the hemolysis is 0.2-1.0% after 35 days of storage under anaerobic conditions. In another embodiment, the hemolysis is 0.2-0.8% after 42 days of storage under anaerobic conditions. In another embodiment, the hemolysis is 0.2-1.0% after 42 days of storage under anaerobic conditions. In another embodiment, the hemolysis is 0.2-0.8% after 49 days of storage under anaerobic conditions. In another embodiment, the hemolysis is 0.2-1.0% after 49 days of storage under anaerobic conditions.Pathogen reduction methods using oxygen and carbon dioxide reduced blood products provide reduced hemolysis as provided above.

[0060] As provided herein, the method provides a reduction in hemolysis levels in pathogen-treated oxygen-reduced red blood cells when compared to non-oxygen-reduced red blood cells. In one embodiment, pathogen reduction under oxygen-reduced conditions results in about 30% of the hemolysis levels observed in pathogen-reduced in non-oxygen-reduced preparations. In one embodiment, at least about 30% reduction is observed after 21 days of anaerobic storage. In one embodiment, about 30% reduction in hemolysis is observed on the 35th day of anaerobic storage. In a further embodiment, about 30% reduction in hemolysis is observed on the 42nd day of anaerobic storage.

[0061] As provided herein, the method provides a reduction in hemolysis levels in pathogen-treated oxygen-reduced leukocyte-reduced whole blood (OR-LRWB) when compared to non-oxygen-reduced leukocyte-reduced whole blood (LRWB). In one embodiment, pathogen reduction under oxygen-reduced conditions results in about 30% of the hemolysis levels observed in pathogen-reduced in non-oxygen-reduced preparations. In one embodiment, at least about 30% reduction is observed after 21 days of anaerobic storage. In one embodiment, about 30% reduction in hemolysis is observed at 35 days of anaerobic storage. In a further embodiment, about 30% reduction is observed at 42 days of anaerobic storage.

[0062] In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 20%. In one embodiment, at least about 20% reduction is observed after 21 days of anaerobic storage. In one embodiment, at least 20% reduction in hemolysis is observed at 35 days of anaerobic storage. In a further embodiment, at least 20% reduction is observed at 42 days of anaerobic storage. In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 25%. In one embodiment, at least 25% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 25% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 25% reduction is observed after 42 days of anaerobic storage. In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 35%. In one embodiment, at least 35% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 35% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 35% reduction is observed after 42 days of anaerobic storage. In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 40%. In one embodiment, at least 40% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 40% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 40% reduction is observed after 42 days of anaerobic storage.

[0063] In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 45%. In one embodiment, at least 45% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 45% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 45% reduction is observed after 42 days of anaerobic storage. In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 50%. In one embodiment, at least 50% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 50% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 50% reduction is observed after 42 days of anaerobic storage.

[0064] As provided herein, the method provides a reduction in hemolysis levels in pathogen-treated oxygen and carbon dioxide reduced red blood cells when compared to non-oxygen reduced red blood cells. In one embodiment, pathogen reduction under oxygen and carbon dioxide reduced conditions results in about 30% of the hemolysis levels observed in pathogen reduction in non-oxygen and carbon dioxide reduced preparations. In one embodiment, at least about 30% reduction is observed after 21 days of anaerobic storage. In one embodiment, about 30% reduction in hemolysis is observed on the 35th day of anaerobic storage. In a further embodiment, about 30% reduction is observed on the 42nd day of anaerobic storage.

[0065] As provided herein, the method provides a reduction in hemolysis levels in pathogen-treated oxygen and carbon dioxide reduced leukocyte reduced whole blood (OR-LRWB) when compared to non-oxygen reduced leukocyte reduced whole blood (LRWB). In one embodiment, pathogen reduction under oxygen and carbon dioxide reduced conditions results in about 30% of the hemolysis levels observed in pathogen reduction in non-oxygen reduced preparations. In one embodiment, at least about 30% reduction is observed after 21 days of anaerobic storage. In one embodiment, about 30% reduction in hemolysis is observed at 35 days of anaerobic storage. In a further embodiment, about 30% reduction is observed at 42 days of anaerobic storage.

[0066] In some embodiments, the reduction in hemolysis in oxygen and carbon dioxide depleted blood products compared to conventional pathogen reduction methods is at least 20%. In one embodiment, at least 20% reduction is observed after 21 days of anaerobic storage. In one embodiment, at least 20% reduction in hemolysis is observed on the 35th day of anaerobic storage. In a further embodiment, at least 20% reduction is observed on the 42nd day of anaerobic storage. In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 25%. In one embodiment, at least 25% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 25% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 25% reduction is observed after 42 days of anaerobic storage. In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 35%. In one embodiment, at least 35% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 35% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 35% reduction is observed after 42 days of anaerobic storage. In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 40%. In one embodiment, at least 40% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 40% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 40% reduction is observed after 42 days of anaerobic storage.

[0067] In some embodiments, the reduction in hemolysis in oxygen and carbon dioxide depleted blood products compared to conventional pathogen reduction methods is at least 45%. In one embodiment, at least 45% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 45% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 45% reduction is observed after 42 days of anaerobic storage. In some embodiments, the reduction in hemolysis compared to conventional pathogen reduction methods is at least 50%. In one embodiment, at least 50% reduction is observed after 21 days of anaerobic storage. In another embodiment, at least 50% reduction is observed after 35 days of anaerobic storage. In a further embodiment, at least 50% reduction is observed after 42 days of anaerobic storage.

[0068] Due to the improved health of red blood cells indicated by reduced hemolysis, the safe storage period (e.g., shelf life) of pathogen-reduced blood products can be extended by reducing the oxygen level in the blood prior to pathogen inactivation. Without being limited by theory, it is believed that the safety and usefulness of stored blood products is reflected in a number of measurable parameters, among which are the overall hemolysis level and the microparticle level. Thus, the reduced hemolysis and reduced microparticle formation observed using the methods herein may reflect fundamental improvements to uncharacterized or unknown blood physiology.

[0069] Due to the improved initial health of red blood cells provided by reducing oxygen in the blood prior to pathogen treatment, the present disclosure provides and encompasses an extended safe storage period (e.g., shelf life) of pathogen-reduced blood products. As provided herein, the shelf life of pathogen-reduced blood products can be increased to one week or more. In one embodiment, the shelf life can be increased to two weeks. In other embodiments, the shelf life can be increased to three weeks while the blood maintains a hemolysis level of less than 0.8%.

[0070] As provided above, various pathogen reduction methods provide one of many photosensitizers to be added to the blood product prior to irradiation with one or more wavelengths of light. Table 1 shows a number of suitable photosensitizers. As used herein, photosensitizers include reactive products as well as photosensitizers that are themselves reactive (e.g., psoralen-related photosensitizers).

[0071] [Table 1]

[0072] [Table 2]

[0073] In embodiments according to the present disclosure, the pathogen reduction method may include one or more photosensitizers. In one embodiment, the photosensitizer is riboflavin. In one embodiment of the present disclosure, the final concentration of riboflavin is 40-60 μM. In another embodiment, the final concentration of riboflavin is at least 40 μM. In another embodiment, the final concentration of riboflavin is at most 60 μM. In another embodiment, the final concentration of riboflavin is 40-55 μM. In another embodiment, the final concentration of riboflavin is 45-55 μM. In a further embodiment, the final concentration of riboflavin is 50-60 μM. In one embodiment, the final concentration of riboflavin is 50 μM.

[0074] The present disclosure provides and encompasses irradiation of oxygen-reduced blood products having photosensitizers added to reduce pathogen levels. As used herein, the term "irradiation" refers to illuminating the blood using both visible and ultraviolet wavelengths.

[0075] In one embodiment of the present disclosure, the oxygen-reduced blood product with riboflavin is irradiated at 265-400 nm. In another embodiment, the blood product is irradiated at 300-400 nm. In another embodiment, the blood product is irradiated at 265-350 nm. The wavelength suitable for irradiation of the oxygen-reduced blood product is determined based on the photosensitizer, for example, as provided in Table 1. As provided herein, reduced oxygen levels result in reduced hemolysis and reduced microparticle formation, which are normally attributed to the pathogen reduction process and reflect improved health of red blood cells in the pathogen-inactivated blood product.

[0076] This specification defines the range as 3.2 to 7.0 J / cm 2 In another embodiment, the method provides for irradiating an oxygen-reduced blood product having riboflavin with a UV radiation exposure of 5 to 7.0 J / cm. 2 In another embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation exposure dose of 5 to 6.0 J / cm. 2 In another embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation exposure of 4 to 7.0 J / cm. 2 In another embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation exposure of at least 3.2 J / cm. 2 In another embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation exposure of at least 5 J / cm. 2 In a further embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation exposure of at most 7.0 J / cm 2 is irradiated with a UV radiation exposure of. The present disclosure provides and encompasses a total radiation dose of up to 100 J / mL. In one embodiment, the radiation dose is 10-100 J / mL. In another embodiment, the radiation dose is 50-100 J / mL. In another embodiment, the radiation dose is 50-80 J / mL. In another embodiment, the radiation dose is less than 150 J / mL. In one embodiment, the radiation dose is at least 25 J / mL. Other suitable radiation doses can be determined.

[0077] The present disclosure provides and encompasses a pathogen inactivation method that reduces microparticle levels in stored blood. In an embodiment according to the present disclosure, the method includes obtaining an oxygen-reduced blood product, adding a photosensitizer, and irradiating the photosensitizer-containing oxygen-reduced blood product. In certain embodiments, the method further includes storing the irradiated photosensitizer-containing oxygen-reduced blood product for a storage period. In other embodiments, the irradiated photosensitizer-containing oxygen-reduced blood product is stored under anaerobic conditions for a storage period.

[0078] As provided herein, the storage period may be up to 9 weeks under either aerobic or anaerobic conditions, resulting in reduced microparticle formation. In one embodiment, the storage period under either aerobic or anaerobic conditions is 2 weeks. In another embodiment, the storage period under either aerobic or anaerobic conditions is 3 weeks. In a further embodiment, the storage period under either aerobic or anaerobic conditions is 3 weeks. The method further provides a storage period of 4 weeks under either aerobic or anaerobic conditions. In other embodiments, the storage period after pathogen inactivation may be 5 weeks. In yet another embodiment, the aerobic or anaerobic storage period is 6 weeks. In a further embodiment, the aerobic or anaerobic storage period is 6 weeks. In particular, the observed improvement in blood cell quality increases as the storage period is extended. Without being limited by theory, it is believed that the reduced microparticle formation is a result of the immediate improvement in red blood cell quality. That is, the quality of red blood cells is improved prior to storage and the reduction in microparticle formation is evidence of that improvement. It is believed that other uncharacterized changes in red blood cells may underlie the observed microparticle reduction.

[0079] In an embodiment according to the present disclosure, a pathogen reduction method that provides reduced microparticle formation may include one or more photosensitizers. In one embodiment, the photosensitizer is riboflavin. In one embodiment of the present disclosure, the final concentration of riboflavin is 40-60 μM. In another embodiment, the final concentration of riboflavin is at least 40 μM. In another embodiment, the final concentration of riboflavin is at most 60 μM. In another embodiment, the final concentration of riboflavin is 40-55 μM. In another embodiment, the final concentration of riboflavin is 45-55 μM. In a further embodiment, the final concentration of riboflavin is 50-60 μM. In one embodiment, the final concentration of riboflavin is 50 μM.

[0080] The present disclosure provides and encompasses irradiation of oxygen-reduced blood products with photosensitizers added to reduce pathogen levels and reduce microparticle formation. As used herein, the term "irradiation" refers to illuminating the blood using both visible and ultraviolet wavelengths.

[0081] In one embodiment of the present disclosure, an oxygen-reduced blood product having riboflavin is irradiated at 265-400 nm, resulting in reduced microparticle formation. In another embodiment, the blood product is irradiated at 300-400 nm. In another embodiment, the blood product is irradiated at 265-350 nm. The wavelengths suitable for irradiation of the oxygen-reduced blood product are determined based on the photosensitizer, for example, as provided in Table 1. As provided herein, reduced oxygen levels result in reduced microparticle formation, which is normally attributed to the pathogen reduction process and reflects improved health of red blood cells in the pathogen-inactivated blood product.

[0082] The reduction in microparticle formation can be measured by methods known in the art. Suitable methods for microparticle formation include Schubert et al. (Schubert, et al., "Whole blood treated with riboflavin and ultraviolet light: Quality assessment of all blood components produced by the buffy coat method," Transfusion 55(4):815-823(2015)). Although absolute values ​​of the number of microparticles can be determined, generally, the reduction in microparticles is determined against a control sample. In this specification, the control includes a non-oxygen-reduced blood product. The absolute value may be determined by preparing a set of standards.

[0083] The method herein provides for the reduction of microparticle formation in stored blood. In some embodiments, stored blood is stored under anaerobic conditions, thus maintaining the oxygen-reduced state obtained for the pathogen inactivation process. In other embodiments, stored blood is maintained under conventional storage conditions. Conventional storage conditions allow oxygen ingress over time, reducing the improvement to ATP and 2,3-DPG, but conventional storage, for example, may reduce costs and reduce disruption to existing blood bank facilities. However, in most embodiments, it is desired that storage can be performed under anaerobic conditions, either with or without carbon dioxide.

[0084] As provided herein, microparticle formation is reduced by reducing oxygen to 25% SO2 or less prior to completing the pathogen inactivation method. In one embodiment, microparticles are reduced by about 2-fold in oxygen-reduced samples compared to comparably treated aerobic samples after 2 days. The method further provides at least a 2-fold reduction in microparticles after 1 week. In one embodiment, the level of microparticles is reduced by more than 5-fold after 2 weeks of storage. In one embodiment, microparticles are reduced by 9-fold after 3 weeks. In a further embodiment, the number of microparticles is reduced by 9-fold after 6 weeks of storage. The method provides at least a 2-fold reduction in microparticle formation after 9 weeks of storage under anaerobic conditions. In other embodiments, the method provides at least a 2-fold reduction in microparticle formation after 9 weeks of storage under aerobic conditions.

[0085] The method also provides at least a three-fold reduction in microparticle formation compared to pathogen reduction methods performed in the presence of oxygen. When measured at 2-6 weeks, the levels of microparticles remain at least three-fold lower compared to similarly treated oxygen-containing samples. In other embodiments, reduced microparticle formation results in at least a five-fold reduction after two weeks of storage.

[0086] In aspects herein, the levels of microparticle formation are reduced to the levels of non-pathogen treated samples. Thus, using the methods herein, the increase in microparticle formation due to pathogen inactivation is reversed.

[0087] The present disclosure provides and encompasses improved blood compositions having extended shelf life. In one aspect, the disclosure provides and encompasses deoxygenated whole blood, comprising whole blood collected in a CPD, having 40-60 μM riboflavin, having an oxygen saturation (SO2) of less than 1-25%, having a pCO2 of 90 mmHg or less at 37° C., and irradiated with UV light of 265-400 nm.

[0088] In some embodiments according to the present specification, the oxygen-reduced whole blood, including whole blood having an oxygen saturation (SO2) of less than 25%, may have an SO2 of less than 20%. In other embodiments, the riboflavin-containing pathogen-reduced blood may have an SO2 of less than 15%. In one embodiment, the SO2 level in the riboflavin-containing oxygen-reduced blood product may have an SO2 of less than 10%. In a particular embodiment, the riboflavin-containing pathogen-reduced blood may have an SO2 of 5%. The present specification further provides an oxygen-reduced whole blood having an SO2 of 5-20%. In another embodiment, the SO2 may be 5-25%. In another embodiment, the SO2 may be 5-15%. In a further embodiment, the SO2 is reduced to 5-10%.

[0089] In some embodiments according to the present specification, the oxygen-reduced whole blood containing riboflavin has a blood oxygen concentration of 3.2 to 7.0 J / cm 2 In another embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation dose of 5 to 7.0 J / cm. 2 In another embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation dose of 5 to 6.0 J / cm. 2 In another embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation dose of 4 to 7.0 J / cm. 2 In another embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation dose of at least 3.2 J / cm. 2 In another embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation dose of at least 5 J / cm. 2 In a further embodiment, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV radiation dose of at most 7.0 J / cm. 2 is exposed to a UV radiation dose of

[0090] The present disclosure provides and encompasses improved blood compositions having extended shelf life. In one aspect, the disclosure provides and encompasses oxygenated and carbon dioxide reduced whole blood comprising whole blood collected in a CPD, having riboflavin of 40-60 μM, having an oxygen saturation (SO2) of 1-25%, and having a pCO2 of 20 mmHg or less at 37° C., where the oxygenated whole blood has been irradiated with UV light of 265-400 nm. In another aspect, oxygenated and carbon dioxide reduced whole blood comprising whole blood collected in a CPD, having riboflavin of 40-60 μM, having an oxygen saturation (SO2) of 1-25%, and having a pCO2 of 20-40 mmHg at 37° C., where the oxygenated whole blood has been irradiated with UV light of 265-400 nm. In another embodiment, oxygenated and carbon dioxide reduced whole blood comprises whole blood collected in a CPD, has 40-60 μM riboflavin, has an oxygen saturation (SO2) of 1-25%, and has a pCO2 of 40-70 mmHg at 37° C., wherein the oxygenated whole blood is irradiated with UV light at 265-400 nm. In one embodiment, oxygenated and carbon dioxide reduced whole blood comprises whole blood collected in a CPD, has 40-60 μM riboflavin, has an oxygen saturation (SO2) of less than 1-25%, and has a pCO2 of 10-20 mmHg at 37° C., wherein the oxygenated whole blood is irradiated with UV light at 265-400 nm. In another aspect, the oxygen and carbon dioxide reduced whole blood comprises whole blood collected in a CPD, has 40-60 μM riboflavin, has an oxygen saturation (SO2) of 1-25%, and has a pCO2 of less than 15 mmHg at 37° C., wherein the oxygen reduced whole blood has been irradiated with UV light of 265-400 nm.

[0091] The methods herein provide and encompass improved parameters selected from the group including complete blood count (CBC), residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on cell membrane surface, %SO2, and decomposition kinetics of S-303, and combinations thereof, in stored blood, as compared to pathogen reduction methods performed in the presence of oxygen. In some embodiments, stored blood is stored under anaerobic conditions, thus maintaining the oxygen-reduced state obtained for the pathogen inactivation process. In other embodiments, stored blood is maintained under conventional storage conditions. Conventional storage conditions allow oxygen ingress over time, reducing improvements to ATP and 2,3-DPG, but conventional storage, for example, may reduce costs and reduce disruptions to existing blood bank facilities. However, in most embodiments, it is desired that storage can be performed under anaerobic conditions, either with or without carbon dioxide.

[0092] The methods herein provide a method for reducing hemolysis in a blood product comprising removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-20 mM. In another embodiment, reducing hemolysis comprises removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 5-10 mM. In another embodiment, reducing hemolysis comprises removing oxygen and carbon dioxide from the blood product, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-20 mM. In a further embodiment, reducing hemolysis comprises removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, adding GSH to a final concentration of 2-20 mM, and storing under anaerobic conditions. In yet another embodiment, reducing hemolysis in a blood product includes mixing an additive solution with the blood product, removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, adding GSH to a final concentration of 2-20 mM, and storing under anaerobic conditions. The methods herein also provide for reducing hemolysis in S-303 pathogen inactivated and oxygen reduced blood products by maintaining hemolysis levels below 1.0%. In one embodiment, the hemolysis is less than 0.8%. In another embodiment, the hemolysis is 0.2% or less. In a further embodiment, the hemolysis is 0.4% or less. In yet another embodiment, the hemolysis is 0.6% or less. In another embodiment, the hemolysis is 0.01-0.2%. In a particular embodiment, the hemolysis is 0.2-0.8%. In another embodiment, the hemolysis is 0.2-0.6%. In another embodiment, the hemolysis is between 0.5 and 1.0%.

[0093] The methods herein provide for reducing microparticle formation in blood products, comprising removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-20 mM. In another embodiment, reducing microparticle formation comprises removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-10 mM. In another embodiment, reducing microparticle formation comprises removing oxygen and carbon dioxide from the blood product, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-20 mM. In a further embodiment, reducing microparticle formation comprises removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, adding GSH to a final concentration of 2-20 mM, and storing under anaerobic conditions. In yet another embodiment, reducing microparticle formation in a blood product includes mixing an additive solution with the blood product, removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, adding GSH to a final concentration of 2-20 mM, and storing under anaerobic conditions. In yet another embodiment, reducing microparticle formation in a blood product includes mixing an additive solution with the blood product, removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, adding GSH to a final concentration of 2-20 mM, centrifuging the blood product, and storing under anaerobic conditions. The methods herein also provide for reducing microparticle formation in S-303 pathogen inactivated and oxygen reduced blood products by reducing the level of microparticles by more than 5-fold after at least one week of storage. In one embodiment, the level of microparticles is reduced by more than 4-fold after at least one week of storage. In another embodiment, the level of microparticles is reduced by more than 3-fold after at least 1 week of storage. In yet another embodiment, the level of microparticles is reduced by more than 2-fold after at least 1 week of storage. In one embodiment, the level of microparticles is reduced by more than 10% after at least 1 week of storage. In another embodiment, the level of microparticles is reduced by more than 25% after at least 1 week of storage.In a further embodiment, the level of microparticles is reduced by 10-50% after at least one week of storage. In another embodiment, the level of microparticles is reduced by 20-60% after at least one week of storage. In a further embodiment, the level of microparticles is reduced by more than 60% after at least one week of storage. In one embodiment, the level of microparticles is reduced by 60-90% after at least one week of storage. In another embodiment, the level of microparticles is reduced by 90-100% after at least one week of storage. In another embodiment, the level of microparticles is reduced by more than 80% after at least one week of storage. The method herein also provides for reducing microparticle formation in S-303 pathogen inactivated and oxygen reduced blood products by reducing the level of microparticles by more than 5-fold after at least three weeks of storage. In one embodiment, the level of microparticles is reduced by more than four-fold after at least three weeks of storage. In another embodiment, the level of microparticles is reduced by more than three-fold after at least three weeks of storage. In yet another embodiment, the level of microparticles is reduced by more than two-fold after at least three weeks of storage. In one embodiment, the level of microparticles is reduced by more than 10% after at least three weeks of storage. In another embodiment, the level of microparticles is reduced by more than 25% after at least three weeks of storage. In a further embodiment, the level of microparticles is reduced by 10-50% after at least three weeks of storage. In another embodiment, the level of microparticles is reduced by 20-60% after at least three weeks of storage. In a further embodiment, the level of microparticles is reduced by more than 60% after at least three weeks of storage. In one embodiment, the level of microparticles is reduced by 60-90% after at least three weeks of storage. In another embodiment, the level of microparticles is reduced by 90-100% after at least three weeks of storage. In another embodiment, the level of microparticles is reduced by more than 80% after at least 3 weeks of storage. The method herein also provides for reducing microparticle formation in S-303 pathogen inactivated and oxygen-reduced blood products by reducing the level of microparticles by more than 5-fold after 3 weeks of storage. In one embodiment, the level of microparticles is reduced by more than 4-fold after 3 weeks of storage.In another embodiment, the level of microparticles is reduced by more than 3-fold after 3 weeks of storage. In yet another embodiment, the level of microparticles is reduced by more than 2-fold after 3 weeks of storage. In one embodiment, the level of microparticles is reduced by more than 10% after 3 weeks of storage. In another embodiment, the level of microparticles is reduced by more than 25% after at least 3 weeks of storage. In a further embodiment, the level of microparticles is reduced by 10-50% after 3 weeks of storage. In another embodiment, the level of microparticles is reduced by 20-60% after 3 weeks of storage. In a further embodiment, the level of microparticles is reduced by more than 60% after 3 weeks of storage. In one embodiment, the level of microparticles is reduced by 60-90% after 3 weeks of storage. In another embodiment, the level of microparticles is reduced by 90-100% after 3 weeks of storage. In another embodiment, the level of microparticles is reduced by more than 80% after at least 3 weeks of storage. The methods herein also provide for reducing microparticle formation in S-303 pathogen inactivated and oxygen reduced blood products by reducing the level of microparticles by more than 5-fold after at least 6 weeks of storage. In one embodiment, the level of microparticles is reduced by more than 4-fold after at least 6 weeks of storage. In another embodiment, the level of microparticles is reduced by more than 3-fold after at least 6 weeks of storage. In yet another embodiment, the level of microparticles is reduced by more than 2-fold after at least 6 weeks of storage. In one embodiment, the level of microparticles is reduced by more than 10% after at least 6 weeks of storage. In another embodiment, the level of microparticles is reduced by more than 25% after at least 6 weeks of storage. In a further embodiment, the level of microparticles is reduced by 10-50% after at least 3 weeks of storage. In another embodiment, the level of microparticles is reduced by 20-60% after at least 6 weeks of storage. In a further embodiment, the level of microparticles is reduced by more than 60% after at least 6 weeks of storage. In one embodiment, the level of microparticles is reduced by 60-90% after at least 6 weeks of storage. In another embodiment, the level of microparticles is reduced by 90-100% after at least 6 weeks of storage.In another embodiment, the level of small particles is reduced by greater than 80% after at least 6 weeks of storage.

[0094] The present disclosure provides and encompasses improved blood compositions having an extended shelf life. In one aspect, the disclosure provides and encompasses oxygen-reduced red blood cells comprising red blood cells having a final concentration of S-303 of about 0.2 mM, an oxygen saturation (SO2) of less than 25%, and a pCO2 of 90 mmHg or less at 37°C. In another aspect, the oxygen-reduced red blood cells comprise red blood cells having a final concentration of S-303 of about 0.2 mM and a final concentration of GSH of about 5-20 mM. In one aspect, the oxygen-reduced red blood cells are also carbon dioxide-reduced red blood cells comprising red blood cells having a final concentration of S-303 of about 0.2 mM, an oxygen saturation (SO2) of less than 25%, and a pCO2 of 90 mmHg or less at 37°C. In another aspect, the oxygen and carbon dioxide-reduced red blood cells comprise red blood cells having a final concentration of S-303 of about 0.2 mM and a final concentration of GSH of about 2-20 mM. The present specification also provides and encompasses an improved blood composition having an improvement in at least one, at least two, at least three, at least four, or at least five parameters selected from the group consisting of CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on cell membrane surface, %SO2, and degradation reaction rate of S-303. In one embodiment, the improved blood composition has an improvement in two parameters selected from the group consisting of CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on cell membrane surface, %SO2, and degradation reaction rate of S-303. In another embodiment, the improved blood composition has an improvement in three parameters selected from the group consisting of CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on cell membrane surface, %SO2, and degradation kinetics of S-303. In a further embodiment, the improved blood composition has an improvement in three parameters selected from the group consisting of CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on cell membrane surface, %SO2, and degradation kinetics of S-303.In yet another embodiment, the improved blood composition has an improvement in four parameters selected from the group consisting of CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on the cell membrane surface, %SO2, and degradation reaction rate of S-303. In another embodiment, the improved blood composition has an improvement in five parameters selected from the group consisting of CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on the cell membrane surface, %SO2, and degradation reaction rate of S-303. In another embodiment, the improved blood composition has an improvement in five to nine parameters selected from the group consisting of CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on the cell membrane surface, %SO2, and degradation reaction rate of S-303.

[0095] Provided herein are improved S-303 pathogen inactivated and oxygen reduced blood compositions having improved concentrations of residual pathogens by reducing the pathogen levels by greater than 10%. In certain embodiments, the concentration of residual pathogens is reduced by greater than 20%. In other embodiments, the concentration of residual pathogens is reduced by greater than 30%. In another embodiment, the concentration of residual pathogens is reduced by greater than 40%. In yet another embodiment, the concentration of residual pathogens is reduced by greater than 60%. In a further embodiment, the concentration of residual pathogens is reduced by greater than 80%. In certain embodiments, the concentration of residual pathogens is reduced by 10-50%. In other embodiments, the concentration of residual pathogens is reduced by 50-95%. In another embodiment, the concentration of residual pathogens is reduced by 60-100%. In some embodiments, the present disclosure provides improved S-303 pathogen inactivated and oxygen reduced blood compositions having improved levels of remaining pathogens and also having reduced carbon dioxide.

[0096] Provided herein is an improved S-303 pathogen inactivated and oxygen reduced blood composition having a hemolysis level of less than 1.0%. In one embodiment, the hemolysis is less than 0.8%. In another embodiment, the hemolysis is 0.2% or less. In a further embodiment, the hemolysis is 0.4% or less. In yet another embodiment, the hemolysis is 0.6% or less. In another embodiment, the hemolysis is 0.01-0.2%. In a particular embodiment, the hemolysis is 0.2-0.8%. In another embodiment, the hemolysis is 0.2-0.6%. In another embodiment, the hemolysis is 0.5-1.0%. In some embodiments, provided herein is an improved S-303 pathogen inactivated and oxygen reduced blood composition having improved hemolysis and also having reduced carbon dioxide.

[0097] Provided herein are improved S-303 pathogen inactivated and oxygen reduced blood compositions with improved deformability. In certain embodiments, the deformability is increased by more than 5%. In other embodiments, the deformability is increased by more than 10%. In other embodiments, the deformability is increased by 10-50%. In other embodiments, the deformability is increased by more than 50%. In some embodiments, provided herein are improved S-303 pathogen inactivated and oxygen reduced blood compositions with improved deformability and also with reduced carbon dioxide.

[0098] Provided herein are improved S-303 pathogen inactivated and oxygen reduced blood compositions having improved levels of ATP by having increased levels of ATP. In certain embodiments, ATP levels are increased by more than 10%. In other embodiments, ATP levels are increased by 5-40%. In other embodiments, ATP levels are increased after 1 week of storage. In other embodiments, ATP levels are increased after 2 weeks of storage. In other embodiments, ATP levels are increased after 4 weeks of storage. In yet another embodiment, ATP levels are increased after 5 weeks of storage. In a further embodiment, ATP levels are increased after 6 weeks of storage. In some embodiments, provided herein are improved S-303 pathogen inactivated and oxygen reduced blood compositions having improved ATP levels and also having reduced carbon dioxide.

[0099] Provided herein is an improved S-303 pathogen inactivated and oxygen reduced blood composition having an improved level of 2,3-DPG by having an increased level of 2,3-DPG. In certain embodiments, the level of 2,3-DPG is increased by more than 10%. In other embodiments, the level of 2,3-DPG is increased by more than 20%. In a further embodiment, the level of 2,3-DPG is increased by more than 30%. In other embodiments, the level of 2,3-DPG is increased by 5-40%. In another embodiment, the level of 2,3-DPG is increased after 1 week of storage. In another embodiment, the level of 2,3-DPG is increased after 2 weeks of storage. In another embodiment, the level of 2,3-DPG is increased after 4 weeks of storage. In yet another embodiment, the level of 2,3-DPG is increased after 5 weeks of storage. In a further embodiment, the level of 2,3-DPG is increased after 6 weeks of storage. In some embodiments, the present disclosure provides improved S-303 pathogen inactivated and oxygen reduced blood compositions having improved 2,3-DPG levels and also having reduced carbon dioxide.

[0100] Provided herein is an improved S-303 pathogen inactivated and oxygen reduced blood composition having improved levels of microparticles by reducing the level of microparticles by 5-fold after at least one week of storage. In one embodiment, the level of microparticles is reduced by more than 4-fold after at least one week of storage. In another embodiment, the level of microparticles is reduced by more than 3-fold after at least one week of storage. In yet another embodiment, the level of microparticles is reduced by more than 2-fold after at least one week of storage. In one embodiment, the level of microparticles is reduced by more than 10% after at least one week of storage. In another embodiment, the level of microparticles is reduced by more than 25% after at least one week of storage. In a further embodiment, the level of microparticles is reduced by 10-50% after at least one week of storage. In another embodiment, the level of microparticles is reduced by 20-60% after at least one week of storage. In a further embodiment, the level of microparticles is reduced by more than 60% after at least one week of storage. In one embodiment, the level of microparticles is reduced by 60-90% after at least one week of storage. In another embodiment, the level of microparticles is reduced by 90-100% after at least one week of storage. In another embodiment, the level of microparticles is reduced by more than 80% after at least one week of storage. In one embodiment, the level of microparticles is reduced by more than 10% after at least three weeks of storage. In another embodiment, the level of microparticles is reduced by more than 25% after at least three weeks of storage. In a further embodiment, the level of microparticles is reduced by 10-50% after at least three weeks of storage. In another embodiment, the level of microparticles is reduced by 20-60% after at least three weeks of storage. In a further embodiment, the level of microparticles is reduced by more than 60% after at least three weeks of storage. In one embodiment, the level of microparticles is reduced by 60-90% after at least 3 weeks of storage. In another embodiment, the level of microparticles is reduced by 90-100% after at least 3 weeks of storage. In another embodiment, the level of microparticles is reduced by more than 80% after at least 3 weeks of storage. In one embodiment, the level of microparticles is reduced by more than 10% after at least 6 weeks of storage.In another embodiment, the level of microparticles is reduced by more than 25% after at least 6 weeks of storage. In a further embodiment, the level of microparticles is reduced by 10-50% after at least 6 weeks of storage. In another embodiment, the level of microparticles is reduced by 20-60% after at least 6 weeks of storage. In a further embodiment, the level of microparticles is reduced by more than 60% after at least 6 weeks of storage. In one embodiment, the level of microparticles is reduced by 60-90% after at least 6 weeks of storage. In another embodiment, the level of microparticles is reduced by 90-100% after at least 6 weeks of storage. In another embodiment, the level of microparticles is reduced by more than 80% after at least 6 weeks of storage. In some embodiments, the present disclosure provides improved S-303 pathogen inactivated and oxygen reduced blood compositions having improved microparticle levels and also having reduced carbon dioxide.

[0101] The present disclosure also provides improved S-303 pathogen inactivated and oxygen-reduced blood compositions with improved phosphatidylserine exposure on the cell membrane surface by maintaining asymmetric distribution of phosphatidylserine along the cytosolic surface of the cell membrane. In certain embodiments, phosphatidylserine expression can be measured by labeling the cell membrane with fluorescent Annexin-V and quantitating by flow cytometry or microscopy. In some embodiments, the present disclosure provides improved S-303 pathogen inactivated and oxygen-reduced blood compositions that also have reduced carbon dioxide.

[0102] Provided herein are improved S-303 pathogen inactivated and oxygen reduced blood compositions having improved levels of %SO2 by having a %SO2 of less than 30%. In one embodiment, the %SO2 is less than 25%. In another embodiment, the %SO2 is less than 20%. In yet another embodiment, the %SO2 is less than 20%. In a further embodiment, the %SO2 is less than 10%. In another embodiment, the %SO2 is less than 5%. In a particular embodiment, the %SO2 is between 5-20%. In other embodiments, the %SO2 is between 3-15%. In some embodiments, provided herein are improved S-303 pathogen inactivated and oxygen reduced blood compositions that also have reduced carbon dioxide.

[0103] Provided herein are improved S-303 pathogen inactivated and oxygen reduced blood compositions having reduced levels of S-303 after a 3 hour pathogen inactivation process. In one embodiment, the levels of S-303 are reduced after a 6 hour pathogen inactivation process. In another embodiment, the levels of S-303 are reduced after a 9 hour pathogen inactivation process. In yet another embodiment, the levels of S-303 are reduced after a 12 hour pathogen inactivation process. In a further embodiment, the levels of S-303 are reduced after a 24 hour pathogen inactivation process. In some embodiments, provided herein are improved S-303 pathogen inactivated and oxygen reduced blood compositions that also have reduced carbon dioxide.

[0104] The methods herein provide an improvement to the pathogen inactivation efficacy of S-303 in blood products comprising removing oxygen from red blood cells, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-20 mM. In another embodiment, the improvement to the pathogen inactivation efficacy of S-303 comprises removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-10 mM. In another embodiment, the improvement to the pathogen inactivation efficacy of S-303 in blood products comprises removing oxygen and carbon dioxide from the blood product, adding S-303 to a final concentration of 0.2 mM, and adding GSH to a final concentration of 2-20 mM. In a further embodiment, the improvement to the pathogen inactivation efficacy of S-303 in a blood product comprises removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, adding GSH to a final concentration of 2-20 mM, and storing under anaerobic conditions. In yet another embodiment, the improvement to the pathogen inactivation efficacy of S-303 in a blood product comprises mixing an additive solution with the blood product, removing oxygen from the blood product, adding S-303 to a final concentration of 0.2 mM, adding GSH to a final concentration of 2-20 mM, and storing under anaerobic conditions. In certain embodiments, the improvement to the pathogen inactivation efficacy of S-303 is by reducing the concentration of residual pathogens by more than 10%. In certain embodiments, the concentration of residual pathogens is reduced by more than 20%. In other embodiments, the concentration of residual pathogens is reduced by up to 30%. In another embodiment, the concentration of remaining pathogens is reduced by more than 40%. In yet another embodiment, the concentration of remaining pathogens is reduced by more than 60%. In a further embodiment, the concentration of remaining pathogens is reduced by more than 80%. In a particular embodiment, the concentration of remaining pathogens is reduced by 10-50%. In another embodiment, the concentration of remaining pathogens is reduced by 50-95%. In another embodiment, the concentration of remaining pathogens is reduced by 60-100%.

[0105] In yet another embodiment, reducing microparticle formation in a blood product includes mixing an additive solution with the blood product, deoxygenating the blood product, adding S-303 to a final concentration of 0.2 mM, adding GSH to a final concentration of 2-20 mM, centrifuging the blood product, and storing under anaerobic conditions. In a particular embodiment, reducing microparticle formation in a blood product includes reducing the level of microparticles by up to 5-fold after at least one week of storage. In one embodiment, the level of microparticles is reduced by more than 4-fold after at least one week of storage. In another embodiment, the level of microparticles is reduced by more than 3-fold after at least one week of storage. In yet another embodiment, the level of microparticles is reduced by more than 2-fold after at least one week of storage. In one embodiment, the level of microparticles is reduced by more than 10% after at least one week of storage. In another embodiment, the level of microparticles is reduced by more than 25% after at least one week of storage. In a further embodiment, the level of microparticles is reduced by 10-50% after at least one week of storage, hi another embodiment, the level of microparticles is reduced by 20-60% after at least one week of storage.

[0106] The present disclosure provides S-303 pathogen inactivated and oxygen reduced blood compositions having improved parameters selected from the group consisting of CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on cell membrane surfaces, %SO2, and degradation kinetics of S-303 in stored blood when compared to pooled blood samples. In certain embodiments, the CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on cell membrane surfaces, %SO2, and degradation kinetics of S-303 in stored blood are as described above.

[0107] The present disclosure provides S-303 pathogen inactivated and oxygen reduced blood having improved parameters selected from the group including CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposure on cell membrane surfaces, %SO2, and degradation kinetics of S-303 in stored blood when compared to blood units having pathogen reduction methods performed in the presence of oxygen.

[0108] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0109] The term "consisting of" means "including and limited to."

[0110] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or parts only if the additional ingredients, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0111] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include mixtures of a plurality, including mixtures thereof.

[0112] Although the present disclosure has been described with reference to specific embodiments, it will be understood by those skilled in the art that various modifications may be made and equivalents may be substituted for the components without departing from the scope of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from its scope.

[0113] Therefore, it is intended that the disclosure not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this disclosure, but that the disclosure will include all embodiments falling within the scope and spirit of the appended claims. EXAMPLES

[0114] Example 1: Blood collection and sample preparation

[0115] Six ABO compatible whole blood units are collected, pooled and split into six samples in triplicate pairs as shown in Table 2. Six units of whole blood (450mL+10%) are collected in the CPD and kept on a cooling tray until pathogen inactivation processing. On the day of donation (D0), these six whole blood units are pooled and split. Deoxygenation of the whole blood is performed as described below. Units are donated with saline or riboflavin as shown in Table 2. Samples 3 and 4 are oxygen-reduced after pathogen treatment and component separation and preparation of packed red blood cells. Samples 5 and 6 are oxygen-reduced while in whole blood form and pathogen-reduced prior to component separation and preparation of packed red blood cells. The riboflavin-containing units are transferred to MIRASOL® whole blood irradiation bags and all units are placed on a cooling tray. Within 24 hours of donation, whole blood units are processed by the buffy coat method and red blood cell concentrates are stored after addition of SAGM additive solution. The oxygen-reduced red blood cell concentrates are prepared as described in Example 2.

[0116] [Table 3]

[0117] Example 2: Whole Blood Collection, Leukoreduction, and Gas Depletion

[0118] Pooled and divided red blood cell units ("blood units") in CPD are leukoreduced according to the manufacturer's instructions after Mirasol treatment and component separation.

[0119] Each whole blood unit (samples 5 and 6) is processed for oxygen depletion by transporting the whole blood in a collection bag connected to a Sorin D100 membrane oxygenator with a mixture of 95% N2 and 5% CO2 gas at a flow rate of 700 ml / min to achieve a pre-stored %SO2 of less than 3% and a pCO2 of 70 mmHg (37°C). For sample 6, O2 from a Mirasol® disposable (without riboflavin solution) is deoxygenated prior to processing. Immediately after preparation of each sample, ABL90 blood gas levels are measured according to the manufacturer's instructions to establish baseline SO2 and pCO2 levels (e.g., T0). For sample 6, the deoxygenated blood is then transported to a Mirasol processing bag. After adding riboflavin, it is placed in a Mirasol photosensitizer device and exposed to UV according to the manufacturer's instructions. After Mirasol processing (sample 6), the contents are returned to the original blood collection bag and the components are then processed according to the standard top-bottom buffy coat method. The separated RBCs are leukoreduced with a leukoreduction filter attached and stored in an anaerobic canister. For sample 5, the Mirasol treatment step is omitted from the above.

[0120] For samples 3 and 4, the separated, leukoreduced RBC bag is connected to a Sorin D100 membrane oxygenator with a mixture of 95% N2 and 5% CO2 gas at a flow rate of 700 ml / min to achieve a pre-storage of %SO2 less than 3% and pCO2 of 70 mmHg (37°C). Immediately after preparation of each sample, ABL90 blood gas levels are measured according to the manufacturer's instructions to establish baseline SO2 and pCO2 levels (e.g., T0). The O2-reduced RBCs are transferred back into the original RBC storage bag and stored in an anaerobic canister. Example 3: MIRASOL® Pathogen Reduction

[0121] Samples are processed using a Mirasol illuminator according to the manufacturer's instructions. Experiments are repeated 5 times for a total of n=5 samples at each data point. Example 4: Storage of Anaerobic Test Products

[0122] The oxygen-reduced and oxygen- and carbon dioxide-reduced blood in the transport bag is wrapped in mesh, secured with rubber, and placed into an anaerobic canister (Mitsubishi, SS-300) with four sorbent sachets. The canister is sealed and the air is removed using nitrogen gas. The anaerobic and aerobic blood is placed in a blood bank refrigerator at 1-6°C. The canister gauge is monitored daily to ensure a reading of 5±1 psi. Canisters that drop below 2 psi are adjusted. Example 5: Sample Analysis

[0123] The following measurements are made from each of the six samples prepared according to the above example on days 2, 7, 14, 21, 28, and 42. The experiment is repeated five times for a total of n=5 samples at each data point. Sample preparation

[0124] These methods are known to those skilled in the art. Erythrocyte supernatant is prepared for vesicular MV counting using the method of Schubert et al. (Schubert, et al., “Whole blood treated with riboflavin and ultraviolet light: Quality assessment of all blood components produced by the buffy coat method”, Transfusion 55(4):815-823(2015)). The number of erythrocytes, mean corpuscular volume (MCV), and total hemoglobin in each sample are measured on a hematology analyzer (ADVIA120, Siemens). Hematocrit is measured using a HAEMATOKRIT 210 device from Hettrich Zentrifugen according to the manufacturer's instructions. Metabolites (glucose and lactate) and potassium (K + ) is quantified using a Gem Premier 3000 blood gas analyzer (Instrumentation Laboratories). pH is measured using an Orion Ross Ultra Semi-Micro pH probe (Thermo Scientific). After the first day of measurement, hemoglobin and blood gas status are measured (%SO2, pCO2, pH, K) using a Gem Premier 3000 blood gas analyzer (Instrumentation Laboratories). + , glucose, %Hb-O2, %Hb-CO, %met-Hb, %Hb). Hemolysis is measured by the Harboe method of Han et. al, (2010) Vox Sang;98:116-23). ​​ATP levels in erythrocytes are quantified by HPLC after perchloric acid extraction of erythrocytes. Bacteriological testing is performed on day 42 using the BacT / ALERT system (bioMerieux).

[0125] The results of the hemolysis analysis are presented in 4. As shown in Figure 4, oxygen reduction prior to pathogen reduction significantly reduces hemolysis at all time points. Although improvements to red blood cell storage are seen at earlier time points, the improvement begins to become evident at day 14.

[0126] The results of the microparticle analysis showing the relative reduction in microparticles are presented in Figure 5. As shown in Figure 5, oxygen reduction before pathogen reduction significantly reduces the formation of microparticles at all time points. Although improvements to red blood cell storage are seen at earlier time points, the improvement begins to become evident at 14 days.

[0127] Osmotic fragility (6A), potassium (6B), total hemoglobin (6C), oxygen saturation (6D), glucose (6E), lactate (6F) and pH (6G) results for control whole blood with sterile saline (1 mean), control whole blood with riboflavin (2 mean), oxygen-reduced packed RBC with sterile saline (3 mean), oxygen-reduced pRBC with riboflavin (4 mean), oxygen-reduced whole blood with sterile saline (5 mean), and oxygen-reduced whole blood with riboflavin (6 mean). Example 6: Pathogen inactivation method for oxygen-depleted blood

[0128] Samples of blood to be processed for pathogen inactivation are first processed for oxygen depletion by transporting to a whole blood collection bag connected to a Sorin D100 membrane oxygenator (Sorin Group, Arvada, CO) and pumped at a flow rate of 700 ml / min with a mixture of 95% N2 and 5% CO2 gas to achieve a preconditioning of less than 25% %SO2 and a pCO2 of approximately 70 mmHg at approximately 23°C. For the desired blood component of whole blood (WB) with anticoagulant, a volume of 500 + / - 50 mL is used, for the desired blood component of leukoreduced packed red blood cells (LRpRBC) with additive solution, a volume of 500 + / - 50 mL is used, for the desired blood component of suspended platelets (PLT), samples are combined for a total volume of 400 + / - 50 mL, and for the desired blood component of plasma, two units of approximately 200 mL each are combined for a total volume of 400 + / - 50 mL. The blood samples are transported to polyvinyl chloride irradiation bags (TerumoBCT, Lakewood, CO), the bags are filled and mixed with a calculated dose of crosslinker. The irradiation bags are then placed on a tray and irradiated with the appropriate irradiation source according to the crosslinker, and the irradiation time is calculated according to the radiation dose required for a given volume and the dose required for a given crosslinker according to a table. The tray is gently shaken during irradiation, resulting in uniform exposure of the contents to the irradiation source for the duration of the exposure. Upon completion of the irradiation cycle, the blood sample is transported from the irradiation bag to an anaerobic storage bag for refrigerated storage. The whole blood sample may be further processed by centrifugation and separation into separate blood components. Pathogen inactivation methods suitable for the methods herein include those presented in Table 1. Example 7: Apheresis Collection and UV Treatment of Blood Components

[0129] Blood donors are accessed by puncturing a vein using a 17-gauge hypodermic needle and connected to the apheresis system. Approximately 450 mL of whole blood (WB) is drawn from the donor into the apheresis system and mixed with anticoagulant prior to centrifugation and separation of blood components. Separated red blood cells (RBCs) are then mixed with additive solution and riboflavin, and subsequently passed through a UV irradiation chamber to be irradiated with UV light to inactivate pathogens and also inactivate any remaining white blood cells. After UV irradiation, the RBCs are collected in a separate storage bag. Separated PLTs are then mixed with PLT additive solution and riboflavin, and subsequently passed through a UV irradiation chamber to be irradiated with UV light to inactivate pathogens. After UV irradiation, the PLTs are collected in a separate storage bag. Separated plasma is then mixed with riboflavin, and subsequently passed through a UV irradiation chamber to be irradiated with UV light to inactivate pathogens. After UV irradiation, the plasma is collected in a separate storage bag. Separated plasma is then mixed with riboflavin, and subsequently passed through a UV irradiation chamber to be irradiated with UV light to inactivate pathogens. After UV irradiation, the plasma is collected in a separate storage bag. The replacement fluid volume of 0.9% saline and crystalloid is returned to the donor before removing the phlebotomy needle. Example 8: Apheresis collection and UV treatment of whole blood

[0130] Blood donors are accessed by puncturing a vein using a 17-gauge hypodermic needle and connected to the apheresis system. Approximately 450 mL of whole blood (WB) is drawn from the donor into the apheresis system, mixed with anticoagulant and riboflavin, and then passed through an irradiation chamber to irradiate with UV light to inactivate pathogens, followed by centrifugation and separation of blood components. After centrifugation and separation of blood components, the RBCs are mixed with an additive solution and collected in a separate storage bag. The separated PLTs are then mixed with a PLT additive solution and collected in a separate storage bag. The separated plasma is collected in a separate storage bag. A replacement volume of 0.9% saline and crystalloids is returned to the donor before removing the phlebotomy needle. Example 9: Examination of the quality of pathogen inactivated (S-303) treated RBCs

[0131] Five units of leukoreduced packed red blood cells (LRpRBCs) with additive solution (e.g., AS3) are measured for complete blood count (CBC) and oxygen saturation (%SO2). The five units are pooled together in a 2-3 liter blood bag to create a homogenous pool. CBC and %SO2 are measured on the pooled LRpRBCs. Equal aliquots of 300 mL of LRpRBCs are placed into five storage containers, labeled A-E, processed as outlined in Table 3, and stored at 4 °C in a standard blood bank refrigerator for 42 days.

[0132] Using the method described in Example 5, aliquots of samples A-E are collected on days 0 (before storage), 7, 14, 21, and 42 and tested for the following parameters: CBC, percent hemolysis, ATP, 2,3-DPG, deformability using MVA, microparticles, phosphatidylserine (PS) exposure on cell membrane, %SO2, RBC morphology, and RBC aggregation. Concentrations of S-303 and S-300 are measured from aliquots of samples A-E collected at 0, 3, 6, 9, 12, and 24 hours. The experiment is repeated 5 times for a total of n=5 samples at each data point.

[0133] The central tendency of the data is determined using the mean and median, and the dispersion of the data is determined using the standard deviation.

[0134] [Table 4] Example 10: Examination of S-303 pathogen inactivation efficacy under anaerobic conditions

[0135] Five units of leukocyte-reduced packed red blood cells (LRpRBCs) with AS3 additive solution are measured for complete blood count (CBC) and oxygen saturation (%SO2). The five units are pooled together in a 2-3 liter blood bag to create a homogenous pool. CBC and %SO2 are measured on the pooled LRpRBCs. The pooled blood is spiked with a selected pathogen or model virus according to the manufacturer's instructions. Equal aliquots of 300 mL of LRpRBCs are placed into five storage containers, labeled A-E, and processed as outlined in Table 3. Sample B is treated with approximately 0.2 mM S-303 and approximately 2-20 mM glutathione (GSH) under aerobic conditions. Samples C and D are treated with approximately 0.2 mM S-303 and approximately 2-20 mM GSH, respectively, before and after oxygen reduction. Sample E is treated with approximately 0.2 mM S-303 and approximately 2-20 mM glutathione (GSH) overnight in ORB. After the treatment outlined, all samples are stored at 4°C in a standard blood bank refrigerator for 42 days. Alternatively, residual amounts of S-300 in the sample are removed by centrifugation and replacement with fresh AS3.

[0136] Using the method described in Example 5, aliquots of samples A-E are collected on days 0 (before storage), 7, 14, 21, and 42 and tested for the following parameters: CBC, residual pathogen concentration, percent hemolysis, hemolysis, ATP, 2,3-DPG, deformability using MVA, phosphatidylserine (PS) exposure on red blood cell membranes, and %SO2. Concentrations of S-303 and S-300 are measured from aliquots of samples A-E collected at 0, 3, 6, 9, 12, and 24 hours. The experiment is repeated 5 times for a total of n=5 samples at each data point.

[0137] The central tendency of the data is determined using the mean and median, and the variance of the data is determined using the standard deviation. Differences between the various sample conditions are analyzed using repeated measures analysis of variance with the Neuman-Keuls multiple comparison test, with a probability level of less than 0.05 considered significant.

Claims

1. 1. A method for reducing blood pathogens with reduced hemolysis, comprising: removing oxygen from the blood product to prepare an oxygen-reduced blood product; Reducing blood pathogens from the blood product; and wherein the reduction of blood pathogens comprises: adding riboflavin to a final concentration of 40-60 μM; and irradiating the riboflavin-containing blood product with UV light of 265-400 nm.

2. 10. The method of claim 1, further comprising storing the oxygen-reduced, pathogen-reduced blood product under anaerobic conditions.

3. 10. The method of claim 1, further comprising reducing carbon dioxide from the blood product.

4. 10. The method of claim 1, wherein hemolysis is less than 0.2% at 14 days, less than 0.4% at 21 days, less than 0.5% at 28 days, less than 0.8% at 35 days, or less than 1.2% at 42 days.

5. 2. The method of claim 1, wherein the blood product is whole blood or leukoreduced whole blood.

6. 2. The method of claim 1, wherein the riboflavin-containing blood product is an oxygen-depleted blood product.

7. 10. The method of claim 1, wherein the blood product is collected in an anticoagulant solution of citrate phosphate dextrose (CPD), citrate phosphate double dextrose (CP2D), or citrate phosphate dextrose adenine (CPDA1).

8. 10. The method of claim 1, wherein the oxygen-reduced, pathogen-reduced blood product is whole blood, leukoreduced whole blood, or packed red blood cells.

9. 1. A method for blood pathogen reduction with reduced microparticle formation, comprising: removing oxygen from the blood product to prepare an oxygen-reduced blood product; Reducing blood pathogens from the blood product; and and wherein the reduction of blood pathogens comprises: adding riboflavin to a concentration of 40-60 μM; and irradiating the riboflavin-containing blood product with UV light of 265-400 nm.

10. 10. The method of claim 9, further comprising storing the oxygen-reduced, pathogen-reduced blood product under anaerobic conditions.

11. 10. The method of claim 9, further comprising reducing carbon dioxide from the blood product.

12. 12. The method of claim 11, further comprising storing the oxygen-reduced, pathogen-reduced blood product under anaerobic and carbon dioxide-reduced conditions.

13. 10. The method of claim 9, wherein the number of microparticles is reduced by at least 2-fold in 14 days, reduced by at least 2-fold in 21 days, or reduced by at least 2-fold in 42 days relative to samples treated in the presence of oxygen.

14. 10. The method of claim 9, wherein the number of microparticles is reduced by at least 5-fold at 14 days, at least 5-fold at 21 days, or at least 5-fold at 42 days relative to samples treated in the presence of oxygen.

15. 10. The method of claim 9, wherein the riboflavin concentration is about 50 μM.

16. 10. The method of claim 9, wherein the blood products are collected in citrate phosphate dextrose (CPD), citrate phosphate double dextrose (CP2D), and citrate phosphate dextrose adenine (CPDA1).

17. 10. The method of claim 9, wherein the blood product is whole blood or leukoreduced whole blood.

18. 10. The method of claim 9, wherein the oxygen-reduced, pathogen-reduced blood product is whole blood, leukoreduced whole blood, or packed red blood cells.

19. 1. The oxygen-reduced whole blood, comprising whole blood collected during CPD, having 400-60 μM riboflavin, having an oxygen saturation (SO2) of less than 25%, and having a pCO2 of 90 mmHg or less at 37° C., wherein the oxygen-reduced whole blood is irradiated with UV light of 265-400 nm.

20. 20. The oxygen-reduced whole blood of claim 19, wherein the pCO2 is 5 to 90 mmHg at 37°C.

21. 20. The oxygen-reduced whole blood of claim 19, wherein the pCO2 is 70 mmHg at 37°C.

22. 1. Oxygen- and carbon dioxide-reduced whole blood comprising whole blood collected during CPD, having 400-60 μM riboflavin, having an oxygen saturation (SO2) of less than 25%, and having a pCO2 of 20 mmHg or less at 37° C., wherein the oxygen-reduced whole blood is irradiated with UV light of 265-400 nm.

23. 20. The oxygen and carbon dioxide reduced whole blood of claim 19, wherein the pCO2 is 5 to 20 mmHg at 37°C.

24. 20. The oxygen-reduced whole blood of claim 19, wherein the pCO2 is 5 mmHg at 37°C.

25. The irradiation is 3.2 to 7.0 J / cm 2 20. The oxygen-depleted whole blood of claim 19, comprising a UV dose of

26. 1. A method for reducing blood pathogens with reduced hemolysis, comprising: removing oxygen from the blood product to prepare an oxygen-reduced blood product; Reducing blood pathogens from the oxygen-reduced blood product; and wherein the reduction of blood pathogens comprises: adding S-303 to a final concentration of 0.2 mM; adding GSH to a final concentration of 2-20 mM.

27. 27. The method of claim 26, further comprising storing the oxygen-reduced, pathogen-reduced blood product under anaerobic conditions.

28. 27. The method of claim 26, further comprising reducing carbon dioxide from the blood product.

29. 27. The method of claim 26, wherein the blood product is whole blood, leukoreduced whole blood, or packed red blood cells.

30. 27. The method of claim 26, further comprising centrifuging the blood product after adding the S-303.

31. 31. The method of claim 30, further comprising mixing in an additive solution having an acidic pH after said centrifugation.

32. 1. A method for blood pathogen reduction with reduced microparticle formation, comprising: removing oxygen from the blood product to prepare an oxygen-reduced blood product; Reducing blood pathogens from the blood product; and and wherein the reduction of blood pathogens comprises: adding S-303 to a final concentration of 0.2 mM; adding GSH to a final concentration of 2-20 mM.

33. 33. The method of claim 32, further comprising storing the oxygen-reduced, pathogen-reduced blood product under anaerobic conditions.

34. 33. The method of claim 32, further comprising reducing carbon dioxide from the blood product.

35. 33. The method of claim 32, wherein the blood product is whole blood, leukoreduced whole blood, or packed red blood cells.

36. 33. The method of claim 32, further comprising centrifuging the blood product after adding the S-303.

37. 37. The method of claim 36, further comprising mixing in an additive solution having an acidic pH after said centrifugation.

38. Deoxygenated red blood cells having a final concentration of about 0.2 mM S-303, an oxygen saturation (SO2) of less than 25%, and a pCO2 of 90 mmHg or less at 37°C.

39. 39. The oxygen-depleted red blood cells of claim 38, further comprising GSH at a final concentration of about 2-20 mM.

40. 39. The oxygen-reduced red blood cells of claim 38, further comprising an improvement in at least two parameters selected from the group including complete blood count (CBC), residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, SO2%, and decomposition kinetics of S-303.

41. 41. The method of claim 40, wherein the percent hemolysis is less than 0.8%.

42. 41. The method of claim 40, wherein the deformability is increased by more than 10% compared to pathogen-inactivated oxygenated blood.

43. 41. The method of claim 40, wherein the ATP is increased by more than 10% compared to pathogen-inactivated oxygenated blood.

44. 41. The method of claim 40, wherein 2,3-DPG is increased by more than 10% after at least 1 week of storage compared to pathogen-inactivated oxygenated blood.

45. 41. The method of claim 40, wherein the formation of microparticles is reduced by more than four-fold after at least one week of storage compared to pathogen-inactivated oxygenated blood.

46. 1. A method for improving the pathogen inactivation efficacy of S-303 in red blood cells, comprising: removing oxygen from red blood cells to prepare an oxygen-reduced blood product; adding S-303 to a final concentration of 0.2 mM; adding GSH to a final concentration of 2-20 mM.

47. 47. The method of claim 46, further comprising reducing carbon dioxide from the blood product.

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