Anaerobic blood storage and pathogen inactivation method

Abstract

The present invention relates to anaerobic blood storage and pathogen inactivation methods, in particular to a method for reducing blood dissolution and microparticle formation during pathogen storage. A reduced oxygen blood composition comprises SAGM and riboflavin having a reduced hemolysis effect. A reduced oxygen blood composition comprises SAGM with reduced microparticles and riboflavin. Blood compositions with reduced oxygen and pathogens comprise CPAD and riboflavin with reduced hemolysis. Blood compositions with reduced oxygen and pathogens comprise SAGM with reduced microparticles and riboflavin.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of May 25, 2017, application number 201780031963.X, and invention name “Anaerobic blood storage and pathogen inactivation method”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 342,756, filed May 27, 2016, and U.S. Provisional Application No. 62 / 445,081, filed January 11, 2017. All of the above applications are incorporated herein by reference in their entirety. Technical Field

[0004] The present disclosure relates to methods for improving the quality and safety of blood and blood products used in transfusion medicine. Background Art

[0005] Blood and blood components for transfusion are common practice in current medicine, but pose a risk to patients due to the potential for exposure to immunogenic and pathogenic contaminants. This risk is exacerbated by the practice of storing collected blood and blood components for up to several weeks. Whole blood is typically processed by filtration to remove white blood cells (leukopenia), followed by centrifugation to separate the 3 major blood components of plasma, platelets, and red blood cells. The leukopenia-reduced packed red blood cells (LRpRBCs) are then suspended in an additive solution, such as AS-1 in the United States. AS-3 AS-5 and AS-7 or SAGGM or PAGGSM in the EU to extend storage life during refrigeration for up to 42 days. Plasma is typically frozen within 24 hours of phlebotomy and separation ("fresh frozen plasma" - FFP or FP24). FFP is thawed prior to use and must be used within 5 days of thawing. Platelets (PLTs) are collected by apheresis or by pooling PLT fractions isolated from multiple whole blood units. PLTs collected by apheresis are typically suspended in an additive solution such as PAS-C in the EU (not yet in the US) or PAS-F PLTs are kept agitated at room temperature to prevent PLT activation and must be used within 5 to 7 days of collection. Although all blood components are susceptible to donor viral and bacterial contamination due to storage conditions, PLTs are more susceptible to bacterial contamination and proliferation than other blood components.

[0006] Recent advances in the art have provided the inactivation of bacterial and viral pathogens by irradiating blood components with photosensitizers using UV light prior to storage (see, e.g., psoralen-based System, Riboflavin-based system), nor photosensitizers ( UV-Platelet systems). These systems cross-link and inactivate DNA from pathogenic species, thereby reducing the risk they pose to patients. The system uses amitosalin HCl (a synthetic psoralen) and UV-A light at 3 J / cm 2 The radiation irradiation cross-links to cross-link pathogen DNA and removes or reduces residual amitosamol and photoproducts after treatment. Systems use riboflavin and UV light at around 313 nm to target the absorption of riboflavin-nucleotide complexes. Systems without photosensitizers typically use UV-C light at 254 nm. The long-term effects of some photosensitizers and photoproducts in these systems remain to be determined.

[0007] Other advances in the art include the use of S-303 (Cerus Corporation, Concord, CA), a quinac mustard-based alkylating agent that includes a frangible anchoring group, crosslinks nucleic acids, and inactivates 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 to form covalent DNA and RNA adducts by reaction with the S-303 molecules. This first reaction is complete in about 30 minutes. The second reaction is the degradation of excess S-303 into a less toxic byproduct, S-300. This decomposition occurs simultaneously with the adduct reaction and is complete within 16-18 hours.

[0008] Although not limited to any particular theory, it is believed that the formation of covalent DNA and RNA adducts with S-303 is based on the embedding of molecules with nucleic acid polymers (e.g., DNA or RNA). As currently understood, when S-303 is added to RBCs, due to its amphipathic characteristics, it quickly (within seconds to minutes) passes through membranes, including membranes of cell and viral envelopes, and inserts into the helical region of nucleic acids. It is assumed that the presence of a fragile anchor on the molecule helps the insertion process by attracting negative charges in the nucleic acid chain of DNA or RNA through the positively charged amine groups on the molecule. The S-303 molecules in close proximity allow for rapid thermal cycloaddition reactions to covalently bond the S-303 molecules to DNA or RNA. It is believed that covalent bonding prevents the occurrence of replication or translation processes and further prevents the production of other pathogens. In the process of forming covalent adducts, the fragile anchor is removed by hydrolysis to produce a less toxic compound S-300.

[0009] The spontaneous decomposition of S-303 to the less toxic S300 is the secondary reaction during the pathogen inactivation process. An excess of S-303 (approximately 0.2 mM) is typically added to the RBCs to provide enough reagent to completely react with all the DNA and RNA in the sample. However, S-303 is a toxic compound, so in order to safely deliver the resulting product, residual S-303 must be removed. In the Cerus pathogen inactivation process, this is primarily achieved by degrading S-303 to S-300, a significantly less toxic compound. The degradation process occurs by hydrolysis; the hydrolysis of S-303 is triggered by a change in pH from low to high when the S-303 reagent is initially mixed with the RBCs. The decomposition kinetics of residual S-303 are rapid at concentrations above 10 nM / L, with a half-life of approximately 20 minutes.

[0010] As currently understood, S-303 also has the potential to react with other nucleophiles in the RBC unit, including small molecules such as phosphates, water, and macromolecules such as proteins. While not limited to any particular theory, in order to reduce these nonspecific interactions with proteins, 20 mM glutathione (GSH) was simultaneously added to the RBC during the pathogen inactivation process. (See Henschler 2011). Glutathione (GSH) is a naturally occurring antioxidant that is present in most cells with an intracellular concentration of approximately 5 mM. As currently understood, GSH is distributed only in the extracellular plasma space, while S-303 diffuses across the membrane and equilibrates inside and outside the cell. This allows GSH to quench the extracellular reaction of S-303 without a significant effect on 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)).

[0011] Recent advances in the art also include the use of anaerobically stored packaged red blood cells in additive solutions to reduce the amount of storage damage typically associated with the use of older blood (see Bitensky et al., US 5,789,152; Bitensky et al., US 6,162,396; and Bitensky et al., US 8,071,282). These storage damages are believed to result from the metabolic processes and byproducts produced by storing blood without the normal physiological environment of the circulatory system, and removing or reducing the available oxygen in the stored blood can reduce the production of harmful oxidative species within the red blood cells during storage.

[0012] Hemolysis is considered an important indicator of blood quality and safety. During storage, the level of hemolysis increases over time, and the presence of free hemoglobin indicates that the blood has exceeded its shelf life. As a result, regulations and guidelines have been developed that limit the acceptable storage time of blood product units that can be used for transfusion. The importance of hemolysis to blood safety has led Europe to set an upper limit of 0.8% before blood must be discarded. The FDA recommends that hemolysis levels do not exceed 1.0%. Therefore, methods to reduce hemolysis extend the safe shelf life of blood, reduce costs and increase blood availability.

[0013] Another indicator of the health and safety of stored blood is 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. Microparticles are believed to be produced as a result of normal physiology, apoptosis, or cell damage. Typically, they are described as particles less than 1000 nm. The lower range is sometimes expressed as 50 nm, but there is no clear definition or agreement about the lower limit. Typically, flow cytometry is used for quantification in conjunction with fluorescent surface antibodies, but there is no universally accepted method for measuring MPs, and measurements can 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 parental cells from which they are derived, although only selected 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 stored RBC components are harmful to recipients as a source of immunomodulation, hypercoagulation, nitric oxide scavenging (poor blood perfusion), or the development of alloimmunity. Therefore, methods that result in reduced levels of microparticles provide improved stored blood health and safety.

[0014] Here, we demonstrate that reduced oxygen in whole blood can significantly reduce the amount of hemolysis and particulate generation when processing blood products to reduce pathogenic viruses, bacteria, and multicellular parasites and to reduce leukocytes. The methods provided herein extend the useful life of pathogen-reduced blood products by reducing hemolysis.

[0015] Pathogen inactivation of blood and blood products has been developed to improve their safety. Although a wide range of bacteria, viruses, and parasites can be inactivated, studies have shown that it has negative effects on blood components. Currently, plasma and platelet concentrates are treated with pathogen inactivation systems; however, red blood cell treatment is still under development. Pathogen inactivation of whole blood after donation would offer the following advantages: all derived products are pathogen inactivated as well as destruction of residual leukocytes. However, recent studies have shown that red blood cells derived from whole blood irradiation using riboflavin / UV technology (Mirasol, TerumoBCT) are of significantly reduced quality compared to untreated study groups, if it requires a shortened shelf life under standard storage conditions. A hallmark of these analyses is the accelerated development of hemolysis, reaching currently accepted levels of 0.8% by day 30 of blood bank storage. The generation of reactive oxygen species (ROS) during UV irradiation is one of the causes of hemolysis.

[0016] Here we demonstrate that using the Mirasol system to reduce oxygen in whole blood prior to pathogen treatment can improve blood quality. Hemanext is designed to remove oxygen from whole blood and red blood cell concentrates combined with pathogen reduction TM The system (New Health Sciences) resulted in improved red blood cell quality compared to pathogen reduction under non-oxygen-reduced conditions. TM Treatment in combination with Mirasol pathogen reduction treatment resulted in less than 0.8% hemolysis of blood after 42 days of storage under reduced oxygen conditions. Summary of the invention

[0017] The present invention provides a method for inactivating blood pathogens and reducing hemolysis during storage by the following steps: removing oxygen from a blood product; adding riboflavin to a final concentration of 40 to 60 μM; and irradiating the blood product containing riboflavin using ultraviolet light between 265-400 nm.

[0018] The present invention provides a method for reducing particle formation and inactivating blood pathogens by the following steps: removing oxygen from a blood product; adding riboflavin to a concentration of 40 to 60 μM; and irradiating the blood product containing riboflavin using ultraviolet light between 265-400 nm.

[0019] The present invention provides oxygen-reduced whole blood, including whole blood collected in CPD, having 40 to 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 has been irradiated using UV light between 265 and 400 nm.

[0020] The present invention provides oxygen and carbon dioxide reduced whole blood, including whole blood collected in CPD, having 400 to 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 using UV light between 265 and 400 nm.

[0021] The present invention provides a method for inactivating blood pathogens and reducing hemolysis by the following steps: removing oxygen from a blood product; adding S-303 to a final concentration of 0.2 mM; and adding GSH to a final concentration of 2 to 20 mM.

[0022] The present invention provides a method for inactivating blood pathogens and reducing particulate generation by the following steps: removing oxygen and carbon dioxide from a blood product; adding S-303 to a final concentration of 0.2 mM; and adding GSH to a final concentration of 2 to 20 mM.

[0023] The present invention 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present disclosure is disclosed with reference to the accompanying drawings, in which:

[0025] Figure 1 A diagram illustrating a blood bag collection and storage system according to one aspect of the present specification is shown.

[0026] Figure 2 A diagram illustrating a discontinuous apheresis system according to one aspect of the present specification is presented.

[0027] Figure 3 A diagram illustrating a discontinuous apheresis system according to one aspect of the present specification is shown.

[0028] Figure 4 is a graph showing the results of experiments according to the present invention, comparing the average hemolysis of control whole blood containing sterile saline (1avg), control whole blood containing riboflavin (2avg), oxygen-reduced packaged RBCs containing sterile saline (3avg), oxygen-reduced pRBCs containing riboflavin (4avg), oxygen-reduced whole blood containing sterile saline (5avg), and oxygen-reduced whole blood containing riboflavin (6avg).

[0029] Figure 5is a graph showing the results of an experiment according to the present invention, comparing the average amount of microparticles in control whole blood containing sterile saline (1avg), control whole blood containing riboflavin (2avg), oxygen-reduced packaged RBCs containing sterile saline (3avg), oxygen-reduced pRBCs containing riboflavin (4avg), oxygen-reduced whole blood containing sterile saline (5avg), and oxygen-reduced whole blood containing riboflavin (6avg).

[0030] Figures 6A to 6G is a graph showing experimental results according to the present invention, which compares osmotic fragility (6A), potassium (6B), total hemoglobin (6C), oxygen saturation (6D), glucose (6E), lactate (6F) and pH (6G) of control whole blood containing sterile saline (1avg), control whole blood containing riboflavin (2avg), oxygen-reduced packed red blood cells containing sterile saline (3avg), oxygen-reduced packed pRBCs containing riboflavin (4avg), oxygen-reduced whole blood containing sterile saline (5avg) and oxygen-reduced whole blood containing riboflavin (6avg).

[0031] Corresponding reference numerals indicate corresponding parts throughout the several views.The embodiments set out herein illustrate various aspects of the present specification, but should not be construed as limiting the scope of the present specification in any way. DETAILED DESCRIPTION

[0032] Here, we show that the harmful side effects of irradiating blood samples with UV light can be mitigated by reducing the amount of oxygen present in the sample. Without being limited by 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 inactivation of pathogens in blood samples, whether the sample is whole blood or any blood component, such as plasma, platelets or red blood cells.

[0033] According to aspects of this specification, blood processing and storage systems can collect, separate, deoxygenate and irradiate blood components with UV light before storage. The blood processing system can be a gravity-driven bag system, such as a system commonly used for whole blood leukocytopenia. In some aspects, the blood processing system can be a single blood component system of a continuous or discontinuous flow type, as known in the art, in which blood is collected and separated into required components, and then the required components are deoxygenated and irradiated as needed. Use this specification before storage. In some aspects, before storage, blood is collected and deoxygenated before separation and UV irradiation of required blood components. In some aspects, before storage, blood is collected and separated before deoxygenation and UV irradiation of required blood components. In some aspects, before separation and storage of desired blood components, blood is collected, deoxygenated and UV irradiated.

[0034] It will be understood by those of ordinary skill in the art that the following examples and drawings are only for illustration and are not meant to limit the scope of the invention. For the purpose of this specification and description, the following definitions and terms are understood to have their common meanings. The term "UV" refers to ultraviolet light with a wavelength of about 220nm to about 400nm, and generally includes a peak at 405nm from a mercury arc lamp. The term "blood sample" refers to a blood sample from an animal or human, including whole blood and whole blood components, including red blood cells (RBC), platelets (PLT), plasma, leukocytes, proteins found in blood, such as albumin, enzymes, coagulation factors, and also includes a combination of these components commonly found in blood, such as a partially separated portion of whole blood, a previously separated recombinant component, and includes a freshly collected or stored blood sample. The term "apheresis" is intended to have its common meaning in the art, and includes collecting and separating blood by continuous and discontinuous methods, as known in the art. The term "blood component separation system" is intended to have its common meaning in the art, and includes devices and systems for collecting and separating blood by continuous and discontinuous methods, as is known in the art. The term "blood container" refers to any container made of a polymeric material for storing blood, regardless of the duration of storage, and includes the generic term "blood bag" commonly used in the art. The term "PVC" refers to a polymer composed of polyvinyl chloride, and includes PVC with any added materials, such as plasticizers, stabilizers, inhibitors, and other materials known in the art for making PVC.

[0035] Figure 1 One aspect of the present specification is shown, including a blood collection bag 1, a blood processing bag 4, a UV radiation chamber 10, and a blood storage bag 13. The blood collection bag 1 is well known in the art and is typically made of a flexible plastic, such as polyvinyl chloride (PVC), but may also be made of other polymer materials, such as polyurethane, silicone, or other biocompatible materials. The blood collection bag 1 has a blood transmission line 3. .The blood transmission line 3 is also made of a flexible plastic known in the art, and is typically made of PVC, but may also be made of other biocompatible polymer materials. The blood transmission line 3 is equipped with a flow control device 2, such as a clamping clamp, a ratchet clamp, or a frangible seal, to prevent blood from flowing from the collection bag 1 through the transmission line 3 into the processing bag 4 until such flow and blood transfer is required. In some aspects, the flow control device is designed to control the flow rate.

[0036] The processing bag 4 is composed of an outer barrier bag 5 and an inner blood bag 6, wherein the outer barrier bag 5 is substantially impermeable to oxygen. Suitable materials for constructing the barrier bag 5 are well known in the art and include metal foils, such as aluminum foil, polymer films with suitable barrier properties, such as ethyl vinyl alcohol (EVA), polyvinyl alcohol (PVA), polyacrylonitrile, Cyclic polyolefin, polychlorotrifluoroethylene (PCTFE or ), polyvinylidene chloride (PVDC), polymer films with coatings to provide suitable barrier properties, such as polyethylene or nylon films coated with silicon oxide, aluminum oxide or multi-layer coatings, and films containing a combination of polymer films and / or coatings to provide suitable barrier properties. In some aspects, the barrier bag is made of RollPrint Z Film or Renolit Solmed An exemplary method for producing a processed bag is provided in International Patent Application No. PCT / US2016 / 021794, filed on March 10, 2016, which is incorporated herein by reference in its entirety.

[0037] The inner blood bag 6 is made of a flexible polymer material with high oxygen transfer performance known in the art, including PVC, polyurethane, silicone, polyethylene, polypropylene, polyethersulfone, polyvinylidene fluoride (PVDF). In one aspect, the inner blood bag 6 is made of silicone, such as Wacker 30-μm thick silicone film. On the other hand, the inner blood bag 6 is made of MilliporeGVHP29325 PVDF membrane. The oxygen absorbing adsorbent material 7 is disposed between the outer barrier bag 5 and the inner blood bag 6. The oxygen absorbing adsorbent material 7 is well known in the art and is usually composed of an iron-based adsorbent material, such as Mitsubishi A 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 aspects, the outer barrier bag 5 and the inner blood bag 6 can be laminated together with the oxygen absorbing material 7 disposed in a laminate structure, such as Mitsubishi film.

[0038] The oxygen absorbing adsorbent material 7 is typically disposed in a breathable pouch and is adapted to absorb oxygen in the gas head space between the outer barrier bag 5 and the inner blood bag 6. In some aspects, the oxygen absorbing adsorbent material 7 is secured to a plastic mesh structure (not shown) to provide spacing between the inner bag and the outer bag, thereby providing enhanced gas transfer in the gas head space. In some aspects, a plurality of oxygen absorbing pouches are disposed in the gas head space. In some aspects, the oxygen absorbing adsorbent material 7 is formulated to act quickly and absorb high levels of oxygen quickly, having the ability to absorb the entire oxygen content of an entire blood unit. In some aspects, the oxygen absorbing adsorbent material 7 has an oxygen absorption capacity of at least 100cc of oxygen, more preferably at least 200cc. In some aspects, an oxygen indicator connector (not shown) is disposed in the gas head space to indicate the presence or absence of a specific level of oxygen by color, such as a S0rbent Systems Additional details of the processing bag 4 and inner blood bag 6 applicable to the present description are provided in International Patent Application No. PCT / US2016 / 021794 filed on March 10, 2016.

[0039] The fluid path of the inner blood bag 6 is connected to the UV radiation chamber 10 through a blood transfer line 9, which has a flow control device 8, such as a clamp, ratchet clamp, or frangible seal, to prevent blood from flowing from the inner blood bag 6 through the transfer line 9 into the UV radiation chamber 10 until blood flow and blood transfer are required. In some aspects, the flow control device is designed to control the flow rate. The UV radiation chamber 10 is further fluidly connected to a blood storage bag 13 through a blood transfer line 12, which has a flow control device 11, such as a clamp, ratchet clamp, or frangible seal, to prevent blood from flowing from the UV radiation chamber 10 through the transfer line 12 into the blood storage bag 13 until blood flow and blood transfer are required. In some aspects, the flow control device 11 is designed to control the flow rate, and in some aspects, the flow control device is in communication with a UV lamp to provide controlled radiation exposure of the blood sample contained in the UV radiation chamber.

[0040] The UV radiation chamber 10 also includes a UV lamp (not shown) that is operably connected to a power source (not shown) and provides UV radiation of blood contained in or passing through the chamber. In some aspects, the UV radiation chamber is adapted to receive a section of blood transfer tubing 9 and irradiate UV light through the tubing. One of ordinary skill in the art will appreciate that most plastics absorb lower wavelength UV light and are not suitable for treatment with lower wavelength UV light, such as 254nm UV-C light, but for certain photosensitizers that absorb at higher wavelengths, such as UV-B (~290-320nm) or UV-A (~320-400nm), thin sections of plastic may be suitable. Therefore, in some aspects, a portion of the blood transfer tube 9 is adapted to have a very thin wall thickness to accommodate the UV radiation chamber 10 to provide enhanced light penetration through the plastic tube into the blood sample. In some aspects, the wall thickness of the transfer tube section in the UV radiation chamber is about 0.1 to about 1.0 mm, and in some aspects, the transfer tube wall thickness is about 0.2 to about 0.5 mm thick.

[0041] In some aspects, the UV radiation chamber 10 is adapted to have a UV-C transparent portion, such as a portion of quartz (SiO2) or sapphire (Al2O3) material in fluid communication with the blood transfer tube 9 and the blood transfer tube 12. In some aspects, the blood transfer line 9 is adapted to have a UV-C transparent portion, such as a portion of a quartz (SiO2) or sapphire (Al2O3) tube nested between the blood transfer tube 9 and the blood transfer tube 12, so that the UV-C transmissive portion can be easily inserted into the UV radiation chamber 10. In some aspects, the UV radiation chamber 10 is made of a UV-C transparent material, such as quartz (SiO2) or sapphire (Al2O3), and the UV radiation chamber 10 is connectively adapted and fluidly communicated with the blood transfer tube 9 and the blood transfer tube 12. In some aspects, the UV radiation chamber 10 is made of 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, polyurethane, etc., and is connectedly adapted and fluidically connected to the blood transfer tube 9 and the blood transfer tube 12. In some aspects, the UV radiation chamber 10 is made of a polymer material, such as silicone that is sufficiently transparent to UV-C, UV-A, and UV-B wavelengths to effectively transmit UV light into the inner cavity of the radiation chamber.

[0042] The blood storage bag 13 includes an outer barrier bag 14 and an inner blood bag 15, wherein the outer barrier bag 14 is substantially impermeable to oxygen. Suitable blood storage bags 13 are described in International Patent Application No. PCT / US2016 / 029069 filed on April 22, 2016, and the entire contents of which are incorporated herein by reference. In short, materials suitable for constructing the outer barrier bag 14 are provided; the outer barrier bag 14 is 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 also includes a staple port 17, wherein the staple port 1717 is well known in the field of transfusion medicine and is suitable for aseptically connecting an infusion staple to receive blood contained in the inner blood bag 15 when used in a transfusion patient. The blood storage bag 13 also includes an oxygen absorbing material 16 disposed between the outer barrier bag 14 and the inner blood bag 15. In these aspects, the oxygen absorbing material 16 is formulated to function for a long time at refrigerated temperatures and absorb low levels of oxygen.

[0043] Figure 2 A discontinuous three-line apheresis system is shown in which whole blood is withdrawn from a subject through a venous access device 110 that can be inserted into the subject's arm. A blood apheresis line 120 fluidly connects the venous access device 110 to a blood component separation device 150 to separate blood components. A traction pump 122 located on the traction line 120 controls the direction, rate, and duration of flow through the traction line 120.

[0044] When whole blood is drawn from the subject, an anticoagulant can be added to the whole blood to prevent the blood from clotting within the line or within the blood component separation device 150. To this end, the system includes an anticoagulant line 130 that is fluidly connected to an anticoagulant source 134 (e.g., a bag of anticoagulant) at one end, and a venous access device 110 (or aspiration line 120) at the other end. The anticoagulant pump 132 through which the anticoagulant line 130 passes controls the flow through the anticoagulant line 130 and the amount of anticoagulant introduced into the whole blood. The anticoagulant pump 132 operates in proportion to the aspiration pump 122 to ensure that the appropriate amount of anticoagulant is added to the whole blood. The anticoagulant is usually introduced into the whole blood as close to the venous access device 110 as possible. The line / catheter usually includes a clamping valve 164 to prevent flow within the line.

[0045] Once a desired amount of anticoagulated whole blood is removed from a subject and contained within 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.

[0046] Some aspects of the blood processing system 100 include a transfer pump 210 and a dilution / extraction line 160 connected to a plasma bag 158. The transfer pump 210 and the dilution / extraction line 160 can be used for a variety of purposes, including diluting the anticoagulated withdrawn blood into the blood component separation device. For example, if the user desires that the withdrawn blood have a higher plasma content, the system can dilute the withdrawn blood by turning on the transfer pump and introducing plasma from the plasma bag 158 into the withdrawn blood in the extraction line 120. Additionally or alternatively, the transfer pump can be used to introduce plasma from the plasma bag 158 into the blood component separation device 150 during surge elutriation to extract platelets (or other blood components).

[0047] After the blood sample is separated in the blood component separation device 150 and the desired components are removed and stored in the appropriate storage containers 156 or 158, the system returns the unextracted and / or unwanted components to the subject via a dedicated line.

[0048] Figure 3 One aspect of the present disclosure for use with a discontinuous three-line apheresis system is shown, wherein the improved portion is shown within the dashed lines. In particular, the blood separation blood processing system 100 also includes a fluid delivery pump 172, a deoxygenation device 173, a processing reservoir 174, and a UV radiation chamber 177, which are fluidly connected by a delivery line 170 and a recirculation line 175. The flow of fluid in the delivery line 170 and the recirculation line 175 is controlled by controlling flow control valves 171, 176, 178, and 179, which work together to direct the fluid to the desired device.

[0049] After the separation of blood components in the blood separation device 150 is completed, the flow of the desired blood component is directed to the delivery line 170 of the present specification by closing the flow control valve 179 of the present specification and opening the flow control valve 171. Therefore, the desired blood component is directed to the delivery pump 172 of the present specification instead of the delivery line 152 and is ultimately delivered to the plasma bag 158 or the platelet bag 156. The fluid flow in the delivery line 170 can be directed by the fluid flow from the delivery pump 122 or the delivery pump 210, or by the delivery pump 172 of the present specification, or a combination thereof.

[0050] The flow of fluid in the delivery line 170 is controlled by a flow control valve 171 and flows into a delivery pump 172 and continues to flow into a deoxygenation device 173 and a treatment reservoir 174, as indicated by the arrows in the figure. During the deoxygenation process, the flow control valve 178 will be closed and the flow control valve 176 disposed on the recirculation line 175 will be opened, thereby allowing the fluid to be recirculated through the deoxygenation device 173, the treatment reservoir 174 and the recirculation line 175 via the delivery pump 172 until the desired oxygen level is reached in the fluid. In some aspects, the deoxygenation device 173 includes hollow porous fibers known in the art and used for degassing fluids, such as Membrana and Devices, and blood oxygenation for extracorporeal perfusion, such as Medtronic Series Oxygenator and Sorin Series oxygenator. In some aspects, the deoxygenation device 173 is operably connected to a nitrogen supply (not shown) to provide nitrogen to the hollow porous fibers to remove oxygen from the blood. In some aspects, the deoxygenation device 173 is operably connected to a device (not shown) for measuring the oxygen content of the fluid contained therein to provide a means for determining when to deoxygenate the fluid sample to a level of about 25% oxygen or less, and in some aspects to about 10% oxygen or less. In some aspects, the delivery pump is operably connected to a control device for shutting off the pump after a predetermined period of time that has been experimentally proven to be effective for the volume of fluid to be treated.

[0051] After the fluid sample has been fully deoxygenated, the blood sample is pumped through the UV radiation chamber 177 by opening the flow control valve 178 by pump 172. The blood sample is irradiated with UV light from a suitable UV light source (not shown). The UV light source can be contained in the UV radiation chamber 177 and operably connected to a power source (not shown), or the UV light source can be operably connected to the UV radiation chamber, such as by a light pipe or a mirror (not shown). UV light sources are known in the art and are selected based on the desired spectral output and the chemicals involved in the blood component processing, and can be selected from mercury arc lamps, xenon lamps, flash lamps, deuterium lamps, halogen lamps, tungsten lamps, fluorescent lamps, and LEDs that emit ultraviolet light.

[0052] The fluid flow in the UV radiation chamber is preferably controlled, either by timed UV radiation of a static sample contained within the UV radiation chamber 177 or by controlled flow of a dynamic sample through the UV radiation chamber 177 and controlled by a flow valve 178, so that a targeted radiation exposure of UV light is achieved in the fluid. In some aspects, the radiation exposure of UV light is about 1-8 J / cm 2 , and in some aspects about 3 J / cm 2 The deoxygenated and UV irradiated blood components then flow through transfer line 180 into transfer line 152 for collection and storage. The blood components are transferred to a blood storage bag after being processed to reduce pathogens and deoxygenated to a level of less than about 25% oxygen in accordance with the present specification. In some aspects, transfer line 180 includes a component absorber (not shown) to provide reduction of excess photosensitizer and photoproducts, such as used with psoralen photosensitizers and as is known in the art.

[0053] The present disclosure provides and includes a method for pathogen reduction with reduced hemolysis, which includes 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 to 60 μM, and irradiating the blood product containing the riboflavin with UV light between 265 and 400 nm. In certain aspects, the method also includes storing the oxygen-reduced pathogen-reduced blood product under anaerobic conditions. The method for pathogen reduction for reducing hemolysis also included and provided in this specification includes removing oxygen and carbon dioxide from a blood product to prepare an oxygen- and carbon dioxide-reduced blood product, reducing blood pathogens from blood including adding riboflavin to a final concentration of 40 to 60 μM, and irradiating the blood product containing the riboflavin with UV light between 265 and 400 nm. As used herein, "anaerobic conditions" include conditions depleted in carbon dioxide and containing carbon dioxide. In most aspects, anaerobic conditions refer to storage conditions in which oxygen and carbon dioxide are depleted.

[0054] The present disclosure provides and includes methods for pathogen reduction with reduced hemolysis, reducing hemolysis comprising removing oxygen from a blood product to prepare an oxygen-reduced blood product, reducing blood pathogens from a blood product comprising adding S-303 to a final concentration of about 0.1 to 0.5 mM, adding glutathione (GSH) to a final concentration of 2 to 20 mM. In certain aspects, the pathogen reduction method with reduced hemolysis comprises removing oxygen from a blood product before reducing blood pathogens to prepare an oxygen-reduced blood product. In other aspects, the pathogen reduction method with reduced hemolysis comprises removing oxygen from a blood product after reducing blood pathogens to prepare an oxygen-reduced blood product. In other aspects, the pathogen reduction method with reduced hemolysis comprises removing oxygen from a blood product while reducing blood pathogens to prepare an oxygen-reduced blood product. The method for reducing the pathogen reduction of reducing hemolysis also included and provided in this specification includes removing oxygen and carbon dioxide from a blood product to prepare a blood product reduced in oxygen and carbon dioxide, reducing blood pathogens from a blood product includes adding S-303 to a final concentration of about 0.2mM, and adding glutathione (GSH) to a final concentration of 5-20mM. In other aspects, the method for reducing the pathogen reduction of reducing hemolysis includes removing oxygen and carbon dioxide from a blood product to prepare a blood product reduced in oxygen and carbon dioxide, and reducing blood pathogens from a blood product includes adding S-303 to a final concentration of about 0.1 to 0.5mM and adding glutathione (GSH) to a final concentration of 2 to 20mM. On the other hand, the pathogen reduction method with reduced hemolysis includes removing oxygen and carbon dioxide from a blood product to prepare a blood product reduced in oxygen and carbon dioxide, and reducing blood pathogens from a blood product includes adding S-303 to a final concentration of about 0.1 to 0.4mM, and adding glutathione (GSH) to a final concentration of 5 to 20mM. In another aspect, a pathogen reduction method with reduced hemolysis comprises 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 comprises adding S-303 to a final concentration of about 0.1 to 0.3 mM and adding glutathione (GSH) to a final concentration of 5 to 10 mM. In another aspect, a pathogen reduction method with reduced hemolysis comprises 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 comprises 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.

[0055] As used herein, pathogens include viruses, parasites, and bacteria. Also as used herein, low levels of leukocytes that remain after leukopenia are considered pathogens. Thus, the method of pathogen reduction can further provide a reduction in leukocytes that may remain after leukopenia.

[0056] As used herein, the terms "reduced", "lowered" and "reduced" mean a final amount that is less than the initial amount or less relative to a control sample. Reduced pathogen levels mean that the pathogen level is reduced by at least one order of magnitude compared to a similar untreated sample. Typically, for the purposes of transfusion medicine, the level of the pathogen is reduced by at least 1.8 logs. In one aspect of the disclosure, the level of the pathogen is reduced by at least 3 logs. In another aspect, the level of the pathogen is reduced by at least 4 logs. In another aspect, the level of the pathogen is reduced by 3 to 10 logs. In another aspect, the level of the pathogen is reduced by at least 7 logs.

[0057] As used herein, "reduced oxygen" or "reduced oxygen saturation" refers to a reduction in the oxygen saturation of red blood cells to 25% or less. As used herein, "reduced carbon dioxide" refers to a reduction in carbon dioxide to 90 mmHg or less when measured at 37°C. An oxygen-depleted blood product has an oxygen saturation of less than 25%, typically less than 10%, and may be about 5% SO2. A carbon dioxide-depleted blood product is a blood product having a carbon dioxide level of less than 20 mmHg when measured at 37°C.

[0058] As used herein, "blood product" includes whole blood or any component derived from whole blood, including red blood cells, platelets, plasma, and white blood cells.

[0059] As used herein, "whole blood" includes white blood cells (WBC), platelets suspended in plasma, and includes electrolytes, hormones, vitamins, antibodies, etc. In whole blood, white blood cells are usually present at 4.5 and 11.0 × 10 9 The normal RBC range for men is 4.6-6.2×10 12 / L, 4.2-5.4×10 12 / L. Normal hematocrit, or percentage of packed cell volume, is about 40-54% for men and about 38-47% for women. Platelet counts are usually 150-450 × 10 9 / L. Whole blood is collected from a blood donor and is usually combined with an anticoagulant. The collected whole blood is initially at about 37°C and rapidly cooled to about 30°C during and shortly after collection, but slowly cooled to ambient temperature within about 6 hours. At the time of collection, the whole blood can be processed according to the methods of the present disclosure starting at 30-37°C or at room temperature (usually about 25°C).

[0060] As used herein, "red blood cells" (RBCs), stored red blood cells, oxygen-reduced red blood cells, oxygen and carbon dioxide-reduced red blood cells, include red blood cells present in whole blood, leukopenic red blood cells, thrombocytopenic red blood cells, leukopenic and thrombocytopenic red blood cells, and packed red blood cells (pRBCs). Human red blood cells are in a dynamic state in the body. Red blood cells contain hemoglobin, an iron-containing protein that can carry oxygen throughout the body and provide color to red blood. The percentage of blood volume composed of red blood cells is called the hematocrit. As used herein, unless otherwise limited, RBCs also include packed red blood cells (pRBCs). Packaged red blood cells are prepared from whole blood using centrifugation techniques 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) may include oxygen and carbon dioxide-reduced (OCR-) RBCs (OCR-RBCs)

[0061] As used herein, "leukopenic whole blood" (LRWB) includes whole blood with an anticoagulant, which is typically treated by filtration or centrifugation to remove leukocytes and platelets. Leukopenic whole blood has at least a 5 log reduction in leukocyte levels.

[0062] As used herein, "oxygen reduced leukocyte-reduced whole blood" (OR-LRWB) may include oxygen and carbon dioxide reduced leukocyte-reduced whole blood (OCRLRWB).

[0063] As used herein, "leukoreduced packaged red blood cells" (LRpRBC) include packaged red blood cells with oxygen reduced (OR-) whole blood that has been processed to remove leukocytes. As used herein, oxygen reduced leukoreduced packaged red blood cells (OR-LRpRBC) may include oxygen and carbon dioxide reduced leukoreduced packaged red blood cells (OCR-LRpRBC).

[0064] According to aspects of this specification, the viruses reduced by the method include enveloped viruses. In other aspects, the method provides reduction of non-enveloped viruses. In aspects of this specification, the viral pathogens reduced include one or more of: 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, herpes virus (SuHV--1), vesicular stomatitis virus (VSV), Sindbis virus, herpes simplex virus (HSV), Epstein-Barr virus (EBV), porcine pseudorabies virus (PPRV), pseudorabies virus (PRV) or Semliki forest virus (SLFV). In some aspects, the inactivated viruses include non-enveloped viruses, including blue tongue virus, calicivirus, human adenovirus-5, porcine parvovirus (PPV), encephalomyocarditis virus (EMCV), hepatitis A virus (HAV), coxsackievirus, and poliovirus. It should be understood that the method of viral parasite reduction includes all viruses and is not limited to those described above. One of ordinary skill in the art will recognize that the inactivation method targets the genetic material of various pathogens, while oxygen reduction reduces hemolysis of, for example, the red blood cell component of the blood product, and further improves blood quality.

[0065] In accordance with the aspects of the present specification, the method provides the parasite hemolysis and inactivation of reduction. In some aspects of the present specification, the parasite of inactivation includes Plasmodium falciparum (malaria), Trypanosoma cruzi (Chaga disease), Leishmania mexicana, Leishmania major, Leishmania infantum (leishmaniasis), Babesia microti and Babesia divergens (babesiosis). In some aspects, the pathogenic bacteria of inactivation may include Bacillus cereus, Clostridium perfringens, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Propionibacterium acnes, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas fluorescens, Listeria monocytogenes, Streptococcus pyogenes and Acinetobacter baumannii.

[0066] The methods of the present invention provide and include reducing hemolysis of red blood cells after pathogen treatment. The pathogen reduction methods applied to oxygen reduced blood or oxygen and carbon dioxide reduced blood result in a significant reduction in hemolysis levels. The pathogen reduction methods applied to oxygen reduced blood or oxygen and carbon dioxide reduced blood result in a significant reduction in particulate levels. Incorporating an additional oxygen reduction step into the pathogen reduction methods can improve the quality of stored blood, thereby extending shelf life. Importantly, applying the blood oxygen reduction methods to existing pathogen reduction methods results in significant improvements. Therefore, the methods of the present specification can be applied to pathogen reduction methods known in the art.Pathogen removal methods suitable for reduced oxygen pathogen inactivation include, but are not limited to, 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 INTERCEPTBlood System. TM in Transfusion Medicine and Hemotherapy, 38:19-31 (2010), and the methods described in U.S. Pat. No. 5,120,659, issued Jun. 9, 1992 to King et al.

[0067] The method provides a reduction in hemolysis, which extends the permissible storage time after pathogen inactivation by maintaining the hemolysis level below 0.8%, or in some aspects, below 1.0%. In one aspect, after 14 days of storage under anaerobic conditions, the hemolysis is no more than 0.2%. In one aspect, after 21 days of storage under anaerobic conditions, the hemolysis is no more than 0.4%. In another aspect, after 28 days of storage under anaerobic conditions, the hemolysis is no more than 0.5%. In another aspect, after 35 days of storage under anaerobic conditions, the hemolysis is no more than 0.8%. In other aspects, after 42 days of storage under anaerobic conditions, the hemolysis is no more than 0.8%. In other aspects, after 49 days of storage under anaerobic conditions, the hemolysis is no more than 0.8%. In some aspects, after 35 days of storage under anaerobic conditions, the hemolysis is no more than 1.0%. In other aspects, after 42 days of storage under anaerobic conditions, the hemolysis is no more than 1.0%. In other aspects, after 49 days of storage under anaerobic conditions, the hemolysis is no more than 1.0%. In another aspect, after 14 days of storage under anaerobic conditions, the hemolysis is no greater than 0.1%. In another aspect, after 14 days of storage under anaerobic conditions, the hemolysis is 0.01-0.2%. In one aspect, after 21 days of storage under anaerobic conditions, the hemolysis is between 0.2 and 0.4%. In one aspect, after 21 days of storage under anaerobic conditions, the hemolysis is between 0.05 and 0.4%. In another aspect, after 28 days of storage under anaerobic conditions, the hemolysis is no greater than 0.6%. In another aspect, after 28 days of storage under anaerobic conditions, the hemolysis is between 0.1 and 0.4%. In another aspect, after 28 days of storage under anaerobic conditions, the hemolysis is between 0.1 and 0.5%. In another aspect, after 35 days of storage under anaerobic conditions, the hemolysis is no greater than 0.4%. In another aspect, after 35 days of storage under anaerobic conditions, the hemolysis is 0.1-0.8%. In another aspect, after 35 days of storage under anaerobic conditions, the hemolysis is between 0.2 and 1.0%. In another aspect, after 42 days of storage under anaerobic conditions, the hemolysis is between 0.2 and 0.8%. In another aspect, after 42 days of storage under anaerobic conditions, the hemolysis is between 0.2 and 1.0%. In another aspect, after 49 days of storage under anaerobic conditions, the hemolysis is between 0.2 and 0.8%. In another aspect, after 49 days of storage under anaerobic conditions, the hemolysis is between 0.2 and 1.0%. The method of pathogen reduction using oxygen and carbon dioxide reduced blood products provides hemolysis reduction as described above.

[0068] As provided herein, the method provides hemolysis levels in oxygen-reduced erythrocytes that reduce pathogen treatment compared to non-oxygen-reduced erythrocytes. In one aspect, pathogen reduction under oxygen-reduced conditions results in about 30% hemolysis levels observed in pathogen reduction in non-oxygen-reduced preparations. In one aspect, at least about 30% reduction is observed after anaerobic storage for 21 days. In one aspect, hemolysis is observed to reduce by about 30% when anaerobic storage is 35 days. On the other hand, about 30% reduction is observed when anaerobic storage is 42 days.

[0069] As provided herein, the method provides hemolysis levels in oxygen-reduced leukocyte-reduced whole blood (OR-LRWB) that reduces pathogen treatment compared to non-oxygen-reduced leukocyte-reduced whole blood (LRWB). In one aspect, pathogen reduction under oxygen-reduced conditions results in a hemolysis level of about 30% observed in pathogen reduction in a non-oxygen-reduced preparation. In one aspect, at least about 30% reduction is observed after anaerobic storage for 21 days. In one aspect, hemolysis is observed to reduce by about 30% when anaerobic storage is 35 days. On the other hand, about 30% reduction is observed when anaerobic storage is 42 days.

[0070] In some aspects, the reduction in hemolysis is at least 20% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 20% was observed after 21 days of anaerobic storage. In one aspect, a reduction of at least 20% was observed in hemolysis at 35 days of anaerobic storage. On the other hand, a reduction of at least 20% was observed in 42 days of anaerobic storage. In some aspects, the reduction in hemolysis is at least 25% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 25% was observed after 21 days of anaerobic storage. On the other hand, a reduction of at least 25% was observed after 35 days of anaerobic storage. On the other hand, a reduction of at least 25% was observed after 42 days of anaerobic storage. In some aspects, the reduction in hemolysis is at least 35% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 35% was observed after 21 days of anaerobic storage. On the other hand, a reduction of at least 35% was observed after 35 days of anaerobic storage. On the other hand, a reduction of at least 35% was observed after 42 days of anaerobic storage. In some aspects, the reduction in hemolysis is at least 40% compared to conventional pathogen reduction methods. In one aspect, at least a 40% reduction is observed after 21 days of anaerobic storage. In another aspect, at least a 40% reduction is observed after 35 days of anaerobic storage. In another aspect, at least a 40% reduction is observed after 42 days of anaerobic storage.

[0071] In some aspects, the reduction in hemolysis is at least 45% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 45% is observed after 21 days of anaerobic storage. In another aspect, a reduction of at least 45% is observed after 35 days of anaerobic storage. In another aspect, a reduction of at least 45% is observed after 42 days of anaerobic storage. In some aspects, the reduction in hemolysis is at least 50% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 50% is observed after 21 days of anaerobic storage. In another aspect, a reduction of at least 50% is observed after 35 days of anaerobic storage. In another aspect, a reduction of at least 50% is observed after 42 days of anaerobic storage.

[0072] As provided herein, the method provides hemolysis levels in oxygen and carbon dioxide-reduced erythrocytes that reduce pathogen treatment compared to non-oxygen-reduced erythrocytes. In one aspect, pathogen reduction under oxygen and carbon dioxide-reduced conditions results in about 30% hemolysis levels observed in pathogen reduction in non-oxygen and carbon dioxide-reduced preparations. In one aspect, at least about 30% reduction is observed after anaerobic storage for 21 days. In one aspect, hemolysis is observed to reduce by about 30% when anaerobic storage is 35 days. On the other hand, about 30% reduction is observed when anaerobic storage is 42 days.

[0073] As provided herein, the method provides hemolysis levels in oxygen and carbon dioxide-reduced leukocyte-reduced whole blood (OR-LRWB) that reduces pathogen treatment compared to non-oxygen-reduced leukocyte-reduced whole blood (LRWB). In one aspect, pathogen reduction under conditions of oxygen and carbon dioxide reduction results in about 30% hemolysis levels observed in pathogen reduction in non-oxygen-reduced preparations. In one aspect, at least about 30% reduction is observed after anaerobic storage for 21 days. In one aspect, hemolysis is observed to reduce by about 30% when anaerobic storage is 35 days. On the other hand, about 30% reduction is observed when anaerobic storage is 42 days.

[0074] In some aspects, the reduction in hemolysis in oxygen and carbon dioxide depleted blood products is at least 20% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 20% was observed after 21 days of anaerobic storage. In one aspect, a reduction of at least 20% was observed after 35 days of anaerobic storage. In another aspect, a reduction of at least 20% was observed after 42 days of anaerobic storage. In some aspects, the reduction in hemolysis is at least 25% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 25% was observed after 21 days of anaerobic storage. In another aspect, a reduction of at least 25% was observed after 35 days of anaerobic storage. In another aspect, a reduction of at least 25% was observed after 42 days of anaerobic storage. In some aspects, the reduction in hemolysis is at least 35% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 35% was observed after 21 days of anaerobic storage. In another aspect, a reduction of at least 35% was observed after 35 days of anaerobic storage. In another aspect, a reduction of at least 35% was observed after 42 days of anaerobic storage. In some aspects, the reduction in hemolysis is at least 40% compared to conventional pathogen reduction methods. In one aspect, at least a 40% reduction is observed after 21 days of anaerobic storage. In another aspect, at least a 40% reduction is observed after 35 days of anaerobic storage. In another aspect, at least a 40% reduction is observed after 42 days of anaerobic storage.

[0075] In some aspects, the reduction in hemolysis in oxygen and carbon dioxide depleted blood products is at least 45% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 45% is observed after 21 days of anaerobic storage. In another aspect, a reduction of at least 45% is observed after 35 days of anaerobic storage. In another aspect, a reduction of at least 45% is observed after 42 days of anaerobic storage. In some aspects, the reduction in hemolysis is at least 50% compared to conventional pathogen reduction methods. In one aspect, a reduction of at least 50% is observed after 21 days of anaerobic storage. In another aspect, a reduction of at least 50% is observed after 35 days of anaerobic storage. In another aspect, a reduction of at least 50% is observed after 42 days of anaerobic storage.

[0076] By reducing the oxygen level in the blood prior to pathogen inactivation, the safe storage period (e.g., shelf life) of pathogen-reduced blood products can be extended due to improved health of red blood cells as indicated by reduced hemolysis. Without being limited by theory, it is believed that the safety and usefulness of stored blood products are reflected in a number of measurable parameters. One of the parameters is the overall level of hemolysis and the level of microparticles. Therefore, the reduced hemolysis and reduced microparticle formation observed using the methods of the present specification may reflect potential improvements in red blood physiology that have not been characterized or are unknown.

[0077] Because the initial health of the red blood cells provided by reducing oxygen in the blood before pathogen treatment is improved, the present specification provides and includes extending the safe storage period (e.g., shelf life) of the blood products with reduced pathogens. As provided herein, the shelf life of the blood products with reduced pathogens can be increased by one week or longer. In one aspect, the shelf life can be increased by two weeks. In other aspects, the shelf life can be increased by three weeks, wherein the blood remains below a hemolysis level of 0.8%.

[0078] As described above, various pathogen reduction methods provide one or more photosensitizers that are added to the blood product prior to irradiation with light of one or more wavelengths. Table 1 lists a number of suitable photosensitizers. As used herein, photosensitizers include those that produce reaction products as well as photosensitizers that are themselves reactive (e.g., psoralen-related photosensitizers).

[0079] Table 1: Pathogen Reduction Photosensitizers and Methods

[0080]

[0081]

[0082]

[0083] In aspects according to the present specification, the pathogen reduction method may include one or more photosensitizers. In one aspect, the photosensitizer is riboflavin. In one aspect of the present disclosure, the final concentration of riboflavin is 40 to 60 μM. In another aspect, the final concentration of riboflavin is at least 40 μM. In another aspect, the final concentration of riboflavin is at most 60 μM. In another aspect, the final concentration of riboflavin is 40 to 55 μM. In another aspect, the final concentration of riboflavin is 45 to 55 μM. In another aspect, the final concentration of riboflavin is 50 to 60 μM. In one aspect, the final concentration of riboflavin is 50 μM.

[0084] The present disclosure provides and includes irradiating oxygen-reduced blood products with added photosensitizers to reduce pathogen levels.As used herein, the term "irradiation" refers to irradiating blood using visible and ultraviolet wavelengths.

[0085] In one aspect of the invention, the oxygen-reduced blood product with riboflavin is irradiated between 265 and 400 nm. In another aspect, the blood product is irradiated between 300 and 400 nm. In another aspect, the blood product is irradiated between 265 and 350 nm. Suitable wavelengths for irradiation of the oxygen-reduced blood product are determined based on the photosensitizer, such as provided in Table 1. As provided herein, a reduction in oxygen levels results in reduced hemolysis and reduced particle formation, which is typically caused by the pathogen reduction process and reflects an improvement in the health of the red blood cells in the pathogen-inactivated blood product.

[0086] The present invention provides and includes irradiating an oxygen-reduced blood product with riboflavin with a UV radiation exposure f of 3.2 to 7.0 J / cm 2 On the other hand, with 5 to 7.0 J / cm 2 UV radiation exposure irradiates oxygen-reduced whole blood containing riboflavin. On the other hand, exposure to 5 to 6.0 J / cm 2 UV radiation exposure irradiates oxygen-reduced whole blood containing riboflavin. On the other hand, exposure to 4 to 7.0 J / cm 2 In another aspect, the method comprises irradiating the oxygen-reduced whole blood containing riboflavin with at least 3.2 J / cm 2 In another aspect, the UV radiation exposure irradiates the oxygen-reduced whole blood containing riboflavin with at least 5 J / cm 2 UV radiation exposure irradiates oxygen-reduced whole blood containing riboflavin. On the other hand, exposure to up to 7.0 J / cm 2 The UV radiation exposure irradiates the oxygen-reduced whole blood containing riboflavin.

[0087] The present disclosure provides and includes total doses up to 100 J / mL. In one aspect, the dose is 10 to 100 J / mL. In another aspect, the dose is 50 to 100 J / mL. In another aspect, the dose is 50 to 80 J / mL. In other aspects, the dose is less than 150 J / mL. In one aspect, the dose is at least 25 J / mL. Other suitable doses can be determined.

[0088] The present specification provides and includes a method for pathogen inactivation that reduces the level of microparticles in stored blood. In aspects according to the present specification, the method includes obtaining a blood product with reduced oxygen, adding a photosensitizer, and irradiating the photosensitizer containing the blood product with reduced oxygen. In certain aspects, the method also includes storing the irradiated photosensitizer containing the blood product with reduced oxygen for a period of time. In other aspects, the irradiated photosensitizer containing the blood product with reduced oxygen is stored under anaerobic conditions for a period of time.

[0089] As provided herein, the storage period can be up to 9 weeks under aerobic or anaerobic conditions, and reduced particle formation is provided. In one aspect, the storage period under aerobic or anaerobic conditions is 2 weeks. On the other hand, the storage period under aerobic or anaerobic conditions is 3 weeks. On the other hand, the storage period under aerobic or anaerobic conditions is 3 weeks. The present method also provides a storage period under aerobic or anaerobic conditions of 4 weeks. In other aspects, the storage period after pathogen inactivation can be 5 weeks. In other aspects, the aerobic or anaerobic storage period is 6 weeks. On the other hand, the aerobic or anaerobic storage period is 6 weeks. It is worth noting that as the storage period is extended, the improvement in blood cell quality observed increases. Without being limited by theory, it is believed that the reduction in particle formation is the result of the immediately improved red blood cell quality. That is, the red blood cell quality is improved before storage, and the reduced particle formation is evidence of this improvement. It is believed that other uncharacterized changes in red blood cells may be the basis for the observed reduction in particles.

[0090] In aspects according to the present specification, the pathogen reduction method providing reduced particle formation may include one or more photosensitizers. In one aspect, the photosensitizer is riboflavin. In one aspect of the present disclosure, the final concentration of riboflavin is 40 to 60 μM. In another aspect, the final concentration of riboflavin is at least 40 μM. In another aspect, the final concentration of riboflavin is at most 60 μM. In another aspect, the final concentration of riboflavin is 40 to 55 μM. In another aspect, the final concentration of riboflavin is 45 to 55 μM. In another aspect, the final concentration of riboflavin is 50 to 60 μM. In one aspect, the final concentration of riboflavin is 50 μM.

[0091] The present disclosure provides and includes irradiating oxygen-reduced blood products with added photosensitizers to reduce pathogen levels and reduce particle formation. As used herein, the term "irradiation" refers to irradiating blood using visible and ultraviolet wavelengths.

[0092] In one aspect of the invention, the oxygen-reduced blood product with riboflavin is irradiated between 265-400 nm to result in reduced microparticle formation. In another aspect, the blood product is irradiated between 300 and 400 nm. In another aspect, the blood product is irradiated between 265 and 350 nm. Suitable wavelengths for irradiation of the oxygen-reduced blood product are determined based on the photosensitizer, such as provided in Table 1. As provided herein, a reduction in oxygen levels results in reduced microparticle formation, which is typically caused by the pathogen reduction process and reflects an improvement in the health of the red blood cells in the pathogen-inactivated blood product.

[0093] The reduction in particle formation can be measured by methods known in the art. Suitable methods for particle formation include Schubert et al., "Whole particle formation". oo d 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 the absolute value of the number of microparticles can be determined, the reduction of microparticles is usually determined relative to a control sample. In this specification, the control includes a non-oxygen-reduced blood product. Absolute values ​​can be determined by preparing a set of standards.

[0094] The methods of the present specification provide for a reduction in particle formation in stored blood. In some aspects, the stored blood is stored under anaerobic conditions, thereby maintaining an oxygen-reduced state obtained for the pathogen inactivation process. In other aspects, the stored blood is maintained under conventional storage conditions. While conventional storage conditions allow oxygen to enter over time and, for example, reduce improvements to ATP and 2,3-DPG, conventional storage can reduce costs and reduce disruption to existing blood bank facilities. However, in most aspects, it is expected that storage will occur under anaerobic conditions, with or without carbon dioxide.

[0095] As provided herein, prior to completing the pathogen inactivation method, particulate formation is reduced by reducing oxygen to 25% SO2 or less. In one aspect, the particulates in the oxygen-reduced sample are reduced by about two times compared to the equivalently treated aerobic sample after 2 days. The method further provides at least a two-fold reduction in particulates after one week. In one aspect, after two weeks of storage, the level of particulates is reduced by more than five times. In one aspect, the particulates are reduced by 9 times after 3 weeks of storage. In another aspect, after 6 weeks of storage, the number of particulates is reduced by 9 times. The present method provides at least a two-fold reduction in particulate formation after 9 weeks of storage under anaerobic conditions. In other aspects, the method provides at least a two-fold reduction in particulate formation after 9 weeks of storage under aerobic conditions.

[0096] The present method also provides at least a three-fold reduction in particulate formation compared to a pathogen reduction method performed in the presence of oxygen. When measured at 2 to 6 weeks, the level of particulates is maintained at least three-fold compared to similarly treated oxygenated samples. In other aspects, the reduced particulate formation results in at least a five-fold reduction after two weeks of storage.

[0097] In aspects of the present description, the level of microparticle formation is reduced to the level of non-pathogen treated samples. Thus, using the methods of the present description, the increase in microparticle formation caused by pathogen inactivation is reversed.

[0098] The present disclosure provides and includes improved blood compositions with extended shelf life. In one aspect, the present disclosure provides and includes oxygen-reduced whole blood comprising whole blood collected in CPD, having 40 to 60 μM riboflavin, having an oxygen saturation (SO2) of 1 to 25%, and having a pCO2 of 90 mmHg or less at 37°C, wherein the oxygen-reduced whole blood has been irradiated with UV light between 265 and 400 nm.

[0099] In aspects according to the present specification, oxygen-reduced whole blood comprising whole blood having an oxygen saturation (SO2) of less than 25% may have an SO2 of less than 20%. In other aspects, pathogen-reduced blood containing riboflavin may have an SO2 of less than 15%. In one aspect, the SO2 level in riboflavin containing oxygen-reduced blood products may have an SO2 of less than 10%. In certain aspects, pathogen-reduced blood containing riboflavin may have an SO2 of 5%. The present specification also provides oxygen-reduced whole blood having an SO2 of 5% to 20%. In another aspect, the SO2 may be between 5% and 25%. In another aspect, the SO2 may be between 5% and 15%. In another aspect, the SO2 is reduced to 5% to 10%.

[0100] In the aspect according to the present specification, 3.2 to 7.0 J / cm 2On the other hand, the UV dose of 5 to 7.0 J / cm 2 On the other hand, the UV dose of 5 to 6.0 J / cm 2 On the other hand, the UV dose of 4 to 7.0 J / cm 2 On the other hand, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV dose of at least 3.2 J / cm 2 On the other hand, the oxygen-reduced whole blood containing riboflavin is irradiated with a UV dose of at least 5 J / cm 2 On the other hand, the UV dose of up to 7.0 J / cm 2 UV doses irradiate oxygen-reduced whole blood containing riboflavin.

[0101] The present specification provides and includes improved blood compositions with extended shelf life. In one aspect, the present disclosure provides and includes oxygen and carbon dioxide reduced whole blood comprising whole blood collected in a CPD, having 40 to 60 μM riboflavin, having an oxygen saturation (SO2) of 1 to 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 between 265 and 400 nm. On the other hand, oxygen and carbon dioxide reduced whole blood comprising whole blood collected in a CPD has 40 to 60 μM riboflavin, an oxygen saturation (SO2) of 1 to 25%, and a pCO2 of 20 to 40 mmHg at 37°C, wherein the oxygen reduced whole blood has been irradiated with UV light between 265 and 400 nm. In another aspect, the oxygen- and carbon dioxide-reduced whole blood comprising whole blood collected in a CPD has 40 to 60 μM riboflavin, has an oxygen saturation (SO2) of 1 to 25%, and has a pCO2 of between 40 and 70 mmHg at 37° C., wherein the oxygen-reduced whole blood has been irradiated using UV light between 265 and 400 nm. In another aspect, the oxygen- and carbon dioxide-reduced whole blood comprising whole blood collected in a CPD has 40-60 μM riboflavin, has an oxygen saturation (SO2) of 1 to 25%, and has a pCO2 of between 10 and 20 mmHg at 37° C., wherein the oxygen-reduced whole blood has been irradiated using UV light between 265 and 400 nm. In another aspect, oxygen- and carbon dioxide-reduced whole blood comprising whole blood collected in CPD has 40-60 μM riboflavin, has an oxygen saturation (SO2) of 1 to 25%, and has a pCO2 of less than 15 mmHg at 37°C, wherein the oxygen-reduced whole blood has been irradiated using UV light between 265 and 400 nm.

[0102] The method of this specification sheets provides and includes improvement selected from and includes complete blood count (CBC), residual pathogen concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine is exposed to cell membrane surface, %SO2 and S-303 decomposition kinetics, and when compared with the pathogen reduction method carried out in the presence of oxygen, their combination in the blood of storage. In some aspects, the blood of storage is stored under anaerobic conditions, thereby maintaining the oxygen reduction state obtained for the pathogen inactivation process. In other aspects, the blood of storage is maintained under conventional storage conditions. Although conventional storage conditions allow oxygen to enter over time and for example reduce the improvement of ATP and 2,3-DPG, traditional storage can reduce costs and reduce the destruction of existing blood bank facilities. However, in most aspects, it is expected that storage will occur under anaerobic conditions, with or without carbon dioxide.

[0103] The method of the present specification provides a reduction in hemolysis of 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 to 20 mM. On the other hand, the reduction in 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 other aspects, the reduction in 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 to 20 mM. On the other hand, the reduction in 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 to 20 mM, and storing under anaerobic conditions. In another aspect, reducing hemolysis of a blood product includes mixing an additive solution with a 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 to 20 mM, and storing under anaerobic conditions. The method of the present specification also reduces hemolysis in S-303 pathogen inactivation and oxygen-reduced blood products by maintaining the hemolysis level below 1.0%. In one aspect, hemolysis is less than 0.8%. In another aspect, hemolysis is no more than 0.2%. In another aspect, hemolysis is no more than 0.4%. In another aspect, hemolysis is no more than 0.6%. In another aspect, hemolysis is between 0.01 and 0.2%. In certain aspects, hemolysis is between 0.2 and 0.8%. In other aspects, hemolysis is between 0.2 and 0.6%. In another aspect, hemolysis is between 0.5 and 1.0%.

[0104] The method of the present specification provides a blood product with reduced microparticle formation, 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 to 20 mM. On the other hand, the reduction of 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 to 10 mM. In other aspects, the reduction of 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 to 20 mM. On the other hand, the reduction of 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 to 20 mM, and storing under anaerobic conditions. In another aspect, a blood product that reduces particle formation includes mixing an additive solution with a 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 to 20 mM, and storing under anaerobic conditions. In another aspect, a blood product that reduces particle formation includes mixing an additive solution with a 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 to 20 mM, centrifuging the blood product, and storing under anaerobic conditions. The method of the present specification also provides S-303 pathogen inactivation and reduction of particle formation in oxygen-reduced blood products by reducing the level of particles by more than 5 times after storage for at least one week. In one aspect, the level of particles is reduced by more than four times after storage for at least one week. In another aspect, the level of particles is reduced by more than three times after storage for at least one week. In another aspect, the level of particles is reduced by more than two times after storage for at least one week. In one aspect, the level of particles is reduced by more than 10% after storage for at least one week. In another aspect, the level of particles is reduced by more than 25% after storage for at least one week. In another aspect, after at least one week of storage, the level of microparticles is reduced by 10% to 50%. In another aspect, after at least one week of storage, the level of microparticles is reduced by 20% to 60%. In another aspect, after at least one week of storage, the level of microparticles is reduced by more than 60%. In one aspect, after at least one week of storage, the level of microparticles is reduced by 60% to 90%. In another aspect, after at least one week of storage, the level of microparticles is reduced by 90% to 100%. In another aspect, after at least one week of storage, the level of microparticles is reduced by more than 80%. The method of the present specification also provides S-303 pathogen inactivation and reduction of microparticle formation in oxygen-reduced blood products by reducing the level of microparticles by more than five times after storage for at least three weeks. In one aspect, after at least three weeks of storage, the level of microparticles is reduced by more than four times. In another aspect, after at least three weeks of storage, the level of microparticles is reduced by more than three times. In another aspect, after at least three weeks of storage, the level of microparticles is reduced by more than two times.In one aspect, the level of microparticles is reduced by more than 10% after storage for at least three weeks. In another aspect, the level of microparticles is reduced by more than 25% after storage for at least three weeks. In another aspect, the level of microparticles is reduced by 10% to 50% after storage for at least three weeks. In another aspect, the level of microparticles is reduced by 20% to 60% after storage for at least three weeks. In another aspect, the level of microparticles is reduced by more than 60% after storage for at least three weeks. In one aspect, the level of microparticles is reduced by 60% to 90% after storage for at least three weeks. In another aspect, the level of microparticles is reduced by 90% to 100% after storage for at least three weeks. In another aspect, the level of microparticles is reduced by more than 80% after storage for at least three weeks. The method of the present specification also provides S-303 pathogen inactivation and reduction of microparticle formation in oxygen-reduced blood products by reducing the level of microparticles by more than five times after storage for three weeks. In one aspect, the level of microparticles is reduced by more than four times after storage for three weeks. In another aspect, the level of microparticles is reduced by more than three times after storage for three weeks. In another aspect, the level of microparticles is reduced by more than two times after storage for three weeks. In one aspect, after three weeks of storage, the level of microparticles is reduced by more than 10%. In another aspect, after three weeks of storage, the level of microparticles is reduced by more than 25%. In another aspect, after three weeks of storage, the level of microparticles is reduced by 10% to 50%. In another aspect, after three weeks of storage, the level of microparticles is reduced by 20% to 60%. In another aspect, after three weeks of storage, the level of microparticles is reduced by more than 60%. In one aspect, after three weeks of storage, the level of microparticles is reduced by 60% to 90%. In another aspect, after three weeks of storage, the level of microparticles is reduced by 90% to 100%. In another aspect, after three weeks of storage, the level of microparticles is reduced by more than 80%. The method of the present specification also provides S-303 pathogen inactivation and reduction of microparticle formation in oxygen-reduced blood products by reducing the level of microparticles by more than five times after at least six weeks of storage. In one aspect, after at least six weeks of storage, the level of microparticles is reduced by more than four times. In another aspect, after at least six weeks of storage, the level of microparticles is reduced by more than three times. In another aspect, after at least six weeks of storage, the level of microparticles is reduced by more than two times. In one aspect, after at least six weeks of storage, the level of microparticles is reduced by more than 10%. In another aspect, after at least six weeks of storage, the level of microparticles is reduced by more than 25%. In another aspect, after at least three weeks of storage, the level of microparticles is reduced by 10% to 50%. In another aspect, after at least six weeks of storage, the level of microparticles is reduced by 20% to 60%. In another aspect, after at least six weeks of storage, the level of microparticles is reduced by more than 60%. In one aspect, after at least six weeks of storage, the level of microparticles is reduced by 60% to 90%. In another aspect, after at least six weeks of storage, the level of microparticles is reduced by 90% to 100%. In another aspect, after at least six weeks of storage, the level of microparticles is reduced by more than 80%.

[0105] The present specification provides and includes improved blood compositions with extended shelf life. In one aspect, the present disclosure provides and includes oxygen-reduced erythrocytes, which include erythrocytes with a final concentration of about 0.2 mM S-303, which have an oxygen saturation (SO2) of less than 25%, and have a pCO2 of 90 mmHg or less at 37°C. On the other hand, the oxygen-reduced erythrocytes include erythrocytes with a final concentration of about 0.2 mM S-303 and a final concentration of about 5-20 mM GSH. In one aspect, the oxygen-reduced erythrocytes are also carbon dioxide-reduced erythrocytes, which include erythrocytes with a final concentration of about 0.2 mM S-303, whose oxygen saturation (SO2) is less than 25%, and pCO2 is 90 mmHg or less at 37°C. On the other hand, the oxygen- and carbon dioxide-reduced erythrocytes include erythrocytes with a final concentration of about 0.2 mM S-303 and a final concentration of about 2-20 mM GSH. The present specification also provides and includes improved blood compositions having improvements in at least one, at least two, at least three, at least four or at least five parameters selected from CBC, residual pathogen concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed to the cell membrane surface, %SO2 and the decomposition kinetics of S-303. In one aspect, the improved blood composition is improved in two parameters selected from CBC, residual pathogen concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed to the cell membrane surface, %SO2 and the decomposition kinetics of S-303. On the other hand, the improved blood composition is improved in three parameters selected from CBC, residual viral atom concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed to the cell membrane surface, %SO2 and the decomposition kinetics of S-303. In another aspect, the improved blood composition is improved in three parameters selected from the group consisting of CBC, residual pathogen concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed to the cell membrane surface, % SO2, and the decomposition kinetics of S-303. In another aspect, the improved blood composition is improved in four parameters selected from the group consisting of CBC, residual pathogen concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed to the cell membrane surface, % SO2, and the decomposition kinetics of S-303. In another aspect, the improved blood composition is improved in five parameters selected from the group consisting of CBC, residual viral atom concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed to the cell membrane surface, % SO2, and the decomposition kinetics of S-303.On the other hand, the improved blood composition has improvements in 5 to 9 parameters selected from CBC, residual viral atom concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed to the cell membrane surface, %SO2 and decomposition kinetics of S-303.

[0106] This specification provides an improved blood composition of S-303 pathogen inactivation and oxygen reduction, which has an improved residual pathogen concentration by reducing pathogen levels by more than 10%. In some aspects, the residual pathogen concentration is reduced by more than 20%. In other aspects, the residual pathogen concentration is reduced by 30%. On the other hand, the residual pathogen concentration is reduced by more than 40%. On the other hand, the residual pathogen concentration is reduced by more than 60%. On the other hand, the residual pathogen concentration is reduced by more than 80%. In some aspects, the residual pathogen concentration is reduced by 10% to 50%. In other aspects, the residual pathogen concentration is reduced by 50% to 95%. On the other hand, the residual pathogen concentration is reduced by 60% to 100%. In some aspects, this specification provides an improved blood composition of S-303 pathogen inactivation and oxygen reduction, which has an improved residual pathogen concentration, while also having reduced carbon dioxide.

[0107] This specification provides an improved blood composition with S-303 pathogen inactivation and oxygen reduction, with a hemolysis level of less than 1.0%. In one aspect, hemolysis is less than 0.8%. In another aspect, hemolysis is no more than 0.2%. In another aspect, hemolysis is no more than 0.4%. In another aspect, hemolysis is no more than 0.6%. In another aspect, hemolysis is between 0.01 and 0.2%. In certain aspects, hemolysis is between 0.2 and 0.8%. In other aspects, hemolysis is between 0.2 and 0.6%. In another aspect, hemolysis is between 0.5 and 1.0%. In some aspects, this specification provides an improved blood composition with S-303 pathogen inactivation and oxygen reduction by also having reduced carbon dioxide, which has improved hemolysis.

[0108] The present specification provides S-303 pathogen inactivation and oxygen reduction improved blood compositions with improved deformability. In some aspects, the deformability is increased by more than 5%. In other aspects, the deformability is increased by more than 10%. In another aspect, the deformability is increased by 10% to 50%. In other aspects, the deformability is increased by more than 50%. In some aspects, the present specification provides S-303 pathogen inactivation and oxygen reduction improved blood compositions, which have improved deformability by also having reduced carbon dioxide.

[0109] This specification provides S-303 pathogen inactivation and oxygen reduction improved blood compositions by increasing ATP levels, which have improved ATP levels. In some aspects, the ATP level increases by more than 10%. In other aspects, the ATP level increases by 5 to 40%. On the other hand, the ATP level increases after one week of storage. In other aspects, the ATP level increases after two weeks of storage. On the other hand, the ATP level increases after four weeks of storage. On the other hand, the ATP level increases after five weeks of storage. On the other hand, the ATP level increases after 6 weeks of storage. In some aspects, this specification provides S-303 pathogen inactivation and oxygen reduction improved blood compositions, which have ATP levels improved by also having reduced carbon dioxide.

[0110] This specification provides S-303 pathogen inactivation and oxygen reduction of improved blood composition, it has improved 2,3-DPG level by having increased 2,3-DPG level. In some aspects, the level of 2,3-DPG increases by more than 10%. In other aspects, the level of 2,3-DPG increases by more than 20%. In other aspects, the level of 2,3-DPG increases by more than 30%. In other aspects, the level of 2,3-DPG increases by 5% to 40%. On the other hand, the level of 2,3-DPG increases after one week of storage. In other aspects, the level of 2,3-DPG increases after two weeks of storage. On the other hand, the level of 2,3-DPG increases after four weeks of storage. On the other hand, the level of 2,3-DPG increases after five weeks of storage. On the other hand, the level of 2,3-DPG increases after 6 weeks of storage. In some aspects, this specification provides S-303 pathogen inactivation and oxygen reduction of improved blood composition, it has improved 2,3-DPG level by also having reduced carbon dioxide.

[0111] The present specification provides an improved blood composition for S-303 pathogen inactivation and oxygen reduction, which has improved microparticle levels by reducing the microparticle level by five times after storage for at least one week. In one aspect, the level of microparticles is reduced by more than four times after storage for at least one week. In another aspect, the level of microparticles is reduced by more than three times after storage for at least one week. In another aspect, the level of microparticles is reduced by more than two times after storage for at least one week. In one aspect, the level of microparticles is reduced by more than 10% after storage for at least one week. In another aspect, the level of microparticles is reduced by more than 25% after storage for at least one week. In another aspect, the level of microparticles is reduced by 10% to 50% after storage for at least one week. In another aspect, the level of microparticles is reduced by 20% to 60% after storage for at least one week. In another aspect, the level of microparticles is reduced by more than 60% after storage for at least one week. In one aspect, the level of microparticles is reduced by 60% to 90% after storage for at least one week. In another aspect, the level of microparticles is reduced by 90% to 100% after storage for at least one week. In another aspect, the level of microparticles is reduced by more than 80% after storage for at least one week. In one aspect, the level of microparticles is reduced by more than 10% after storage for at least three weeks. In another aspect, the level of microparticles is reduced by more than 25% after at least three weeks of storage. In another aspect, the level of microparticles is reduced by 10% to 50% after at least three weeks of storage. In another aspect, the level of microparticles is reduced by 20% to 60% after at least three weeks of storage. In another aspect, the level of microparticles is reduced by more than 60% after at least three weeks of storage. In one aspect, the level of microparticles is reduced by 60% to 90% after at least three weeks of storage. In another aspect, the level of microparticles is reduced by 90% to 100% after at least three weeks of storage. In another aspect, the level of microparticles is reduced by more than 80% after at least three weeks of storage. In one aspect, the level of microparticles is reduced by more than 10% after at least six weeks of storage. In another aspect, the level of microparticles is reduced by more than 25% after at least six weeks of storage. In another aspect, the level of microparticles is reduced by 10% to 50% after at least six weeks of storage. In another aspect, the level of microparticles is reduced by 20% to 60% after at least six weeks of storage. In another aspect, the level of microparticles is reduced by more than 60% after at least six weeks of storage. In one aspect, the level of microparticles is reduced by 60% to 90% after at least six weeks of storage. In another aspect, the level of microparticles is reduced by 90% to 100% after storage for at least six weeks. In another aspect, the level of microparticles is reduced by greater than 80% after storage for at least six weeks. In some aspects, the specification provides an improved blood composition for S-303 pathogen inactivation and oxygen reduction having improved microparticle levels by also having reduced carbon dioxide.

[0112] This specification also provides S-303 pathogen inactivation and oxygen reduction improved blood composition, which has improved phosphatidylserine exposure on the cell membrane surface by maintaining the asymmetric distribution of phosphatidylserine along the cytoplasmic surface of the cell membrane. In some aspects, phosphatidylserine expression can be measured by marking the cell membrane with fluorescent annexin-V and quantitatively using flow cytometry or microscopy. In some aspects, this specification provides S-303 pathogen inactivation and oxygen reduction improved blood composition, which also has reduced carbon dioxide.

[0113] The present specification provides S-303 pathogen inactivation and oxygen reduction improved blood compositions by making %SO2 less than 30%, which have improved %SO2 levels. In one aspect, %SO2 is less than 25%. In another aspect, %SO2 is less than 20%. In another aspect, %SO2 is less than 20%. In another aspect, %SO2 is less than 10%. In another aspect, %SO2 is less than 5%. In certain aspects, %SO2 is 5 to 20%. In other aspects, %SO2 is 3 to 15%. In some aspects, the present specification provides S-303 pathogen inactivation and oxygen reduction improved blood compositions, which also have reduced carbon dioxide.

[0114] This specification provides an improved blood composition of S-303 pathogen inactivation and oxygen reduction, which has a reduced S-303 level after a 3-hour pathogen inactivation process. In one aspect, after a 6-hour pathogen inactivation process, the level of S-303 is reduced. On the other hand, after a 9-hour pathogen inactivation process, the level of S-303 is reduced. On the other hand, after a 12-hour pathogen inactivation process, the level of S-303 is reduced. On the other hand, after a 24-hour pathogen inactivation process, the level of S-303 is reduced. In some aspects, this specification provides an improved blood composition of S-303 pathogen inactivation and oxygen reduction, which also has reduced carbon dioxide.

[0115] The method of the present specification provides the efficacy of improving the pathogen inactivation S-303 in blood products, including removing oxygen from red blood cells, adding S-303 to a final concentration of 0.2mM, and adding GSH to a final concentration of 2 to 20mM. On the other hand, the improvement of the pathogen inactivation efficacy of S-303 in blood products includes removing oxygen from blood products, adding S-303 to a final concentration of 0.2mM, and adding GSH to a final concentration of 2 to 10mM. In other aspects, the improvement of the pathogen inactivation efficacy of S-303 in blood products includes removing oxygen and carbon dioxide from blood products, adding S-303 to a final concentration of 0.2mM, and adding GSH to a final concentration of 2 to 20mM. On the other hand, the improvement of the pathogen inactivation efficacy of S-303 in blood products includes removing oxygen from blood products, adding S-303 to a final concentration of 0.2mM, adding GSH to a final concentration of 2 to 20mM, and storing under anaerobic conditions. In another aspect, improving the efficacy of S-303 pathogen inactivation in red blood cells includes mixing an additive solution with a 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 to 20 mM, and storing under anaerobic conditions. In some aspects, the improvement in the efficacy of S-303 pathogen inactivation is by reducing the residual pathogen concentration by more than 10%. In some aspects, the residual pathogen concentration is reduced by more than 20%. In other aspects, the residual pathogen concentration is reduced by 30%. In another aspect, the residual pathogen concentration is reduced by more than 40%. In another aspect, the residual pathogen concentration is reduced by more than 60%. In another aspect, the residual pathogen concentration is reduced by more than 80%. In some aspects, the residual pathogen concentration is reduced by 10% to 50%. In other aspects, the residual pathogen concentration is reduced by 50% to 95%. In another aspect, the residual pathogen concentration is reduced by 60% to 100%.

[0116] In another aspect, a blood product that reduces microparticle formation comprises mixing an additive solution with a 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 to 20 mM, centrifuging the blood product, and storing under anaerobic conditions. In certain aspects, the reduction in microparticle formation in the blood product comprises reducing the level of microparticles by five times after storage for at least one week. In one aspect, the level of microparticles is reduced by more than four times after storage for at least one week. In another aspect, the level of microparticles is reduced by more than three times after storage for at least one week. In another aspect, the level of microparticles is reduced by more than two times after storage for at least one week. In one aspect, the level of microparticles is reduced by more than 10% after storage for at least one week. In another aspect, the level of microparticles is reduced by more than 25% after storage for at least one week. In another aspect, the level of microparticles is reduced by 10% to 50% after storage for at least one week. In another aspect, the level of microparticles is reduced by 20% to 60% after storage for at least one week.

[0117] The present specification provides S-303 pathogen inactivated and oxygen-reduced blood compositions having improved parameters selected from CBC, residual viral atom concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed on the cell membrane surface, %SO2, and decomposition kinetics of S-303 compared to pooled blood samples. In certain aspects, CBC, residual viral atom concentration, hemolysis percentage, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed on the cell membrane surface, %SO2, and the improvement of decomposition kinetics of S-303 in stored blood are as described above.

[0118] The present specification provides units of S-303 pathogen inactivation and oxygen-reduced blood having improved parameters when compared to blood units having a pathogen reduction method performed in the presence of oxygen, the parameters being selected from CBC, residual viral atom concentration, percent hemolysis, ATP, 2,3-DPG, deformability, microparticle formation, phosphatidylserine exposed to the cell membrane surface, % SO2 in stored blood, and decomposition kinetics of S-303.

[0119] The terms "including", "comprising", "having" and their conjugations mean "including but not limited to".

[0120] The term "consisting of" means "including but limited to".

[0121] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only when the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0122] 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 a plurality of compounds, including mixtures thereof.

[0123] Although the present disclosure has been described with reference to specific aspects, it will be appreciated by those skilled in the art that various changes may be made and elements thereof may be replaced with equivalents without departing from the scope of the present disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present disclosure without departing from the scope of the present disclosure.

[0124] Therefore, it is intended that the disclosure not be limited to the particular aspects disclosed as the best mode contemplated for carrying out this disclosure, but that the disclosure will include all aspects falling within the scope and spirit of the appended claims.

[0125] Example

[0126] Example 1: Blood collection and sample preparation

[0127] Six ABO-matched whole blood units were collected, pooled, and divided into six samples in three pairs as shown in Table 2. Six units of whole blood (450 mL + 10%) were collected in CPD and kept on a cooling tray until pathogen inactivation treatment. On the day of donation (D0), these six whole blood units were pooled and separated. Whole blood deoxygenation was performed as described below. Saline or riboflavin was provided to the treated units as shown in Table 2. Samples 3 and 4 were oxygen reduced after pathogen treatment and component separation and packed red blood cell preparation. Samples 5 and 6 were oxygen reduced at the whole blood stage and pathogen reduced prior to component separation and preparation of packed red blood cells. The units containing riboflavin were transferred to All units were placed on a cooling tray in whole blood illumination bags. Within 24 hours of donation, whole blood units were processed by the buffy coat method and stored as red blood cell concentrates after addition of SAGM additive solution. Oxygen-reduced red blood cell concentrates were prepared as described in Example 2.

[0128] Table 2: Samples with riboflavin photosensitizer for pathogen reduction

[0129] treat <![CDATA[Sample 1]]> <![CDATA[Sample 2]]> <![CDATA[Sample 3]]> <![CDATA[Sample 4]]> <![CDATA[Sample 5]]> <![CDATA[Sample 6]]> Reduced oxygen no no RBC RBC Whole blood Whole blood Riboflavin (final 50 μM) no 35ml no 35ml no 35ml Sterile saline 35ml no 35ml no 35ml no <![CDATA[Remove O2 before pathogen reduction]]> n / a no n / a no n / a yes

[0130] Example 2: Whole blood collection, leukopenia and gas consumption

[0131] After Mirasol treatment and component separation, the pooled and split red blood cell units ("blood units") in CPD were leukoreduced according to the manufacturer's instructions.

[0132] Each whole blood unit (samples 5 and 6) was processed by transferring whole blood into a collection bag connected to a Sorin D100 membrane oxygenator to deplete oxygen and using a mixture of 95% N2 and 5% CO2 gas at a flow rate of 700 ml / min to achieve a pre-storage %SO2 less than 3%, pCO2 less than 70 mmHg (37°C). For sample 6, Disposables (excluding riboflavin solution) were O2 purged of oxygen prior to processing. Immediately following preparation of each sample, ABL90 blood gas levels were determined according to the manufacturer's instructions to establish baseline SO2 and pCO2 levels (e.g., T0). For Sample 6, the oxygen-reduced blood was then transferred to a Mirasol treatment bag. After the addition of riboflavin, it was placed in a Mirasol photosensitive device and exposed to UV light according to the manufacturer's instructions. After Mirasol treatment (Sample 6), the contents were transferred back to the original blood collection bag and the components were then processed according to the standard upper and lower buffy coat coating method. The isolated RBCs were leukoreduced with an attached leukoreduction filter and stored in an anaerobic tank. For Sample 5, the Mirasol treatment step was omitted from above.

[0133] For samples 3 and 4, the separated and leukocyte-reduced RBC bags were connected to a Sorin D100 membrane oxygenator and connected to a mixture of 95% N2 and 5% CO2 gas at a flow rate of 700 ml / min to achieve a pre-storage % SO2 of less than 3% and a pCO2 of 70 mmHg (37°C). Immediately after preparation of each sample, ABL90 blood gas levels were determined according to the manufacturer's instructions to establish baseline SO2 and pCO2 levels (e.g., T0). The O2-reduced RBCs were transferred back to the original RBC storage bags and stored in anaerobic jars.

[0134] Embodiment 3: Pathogen reduction

[0135] Samples were processed using a Mirasol Illuminator according to the manufacturer's instructions. The experiment was repeated five times for a total of n=5 samples at each data point.

[0136] Example 4: Storage of Anaerobic Test Products

[0137] Blood in oxygen-reduced, oxygen- and carbon dioxide-reduced transfer bags was wrapped in mesh, secured with elastic and placed in an anaerobic canister with 4 sorbent pouches (Mitsubishi, SS-300). The canister was sealed and the canister was purged of air with nitrogen. Anaerobic and aerobic blood was placed in a blood bank refrigerator at 1 to 6°C. The canister pressure gauge was monitored daily to ensure that it read 5 ± 1 psi. Canisters that fell below 2 psi were adjusted.

[0138] Example 5: Sample Analysis

[0139] The following measurements were taken from each of the six samples prepared according to the above example at day 2, day 7, day 14, day 21, day 28, and day 42. The experiment was repeated five times for a total of n=5 samples at each data point.

[0140] a. Sample preparation

[0141] These methods are known to those skilled in the art. Red blood cell supernatant was prepared for microvesicle 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)). Red blood cell count, mean microvolume (MCV), and total hemoglobin in each sample were determined in a hematology analyzer (ADVIA 120, Siemens). Hematocrit was determined using a HAEMATOKRIT 210 device from Hettrich Zentrifugen according to the manufacturer's instructions. Metabolites (glucose and lactate) and potassium (K) were quantified using a GemPremier 3000 blood gas analyzer (Instrumentation Laboratories). + ). pH was measured with an Orion Ross Ultra Semi-Micro pH probe (Thermo Scientific). After measurement on day 1, hemoglobin and blood gas status (% SO2, pCO2, pH, K+, glucose, %Hb-O2, %Hb-CO, %met-Hb, %Hb) were determined using a Gem Premier 3000 blood gas analyzer (Instrumentation Laboratories). The degree of hemolysis was determined by the Harboe method of Han et al. (2010) VoxSang: 98: 116-23). ​​After perchloric acid extraction of erythrocytes, ATP levels in erythrocytes were quantified by HPLC. Bacterial testing was performed on day 42 using the BacT / ALERT system (bioMérieux).

[0142] The results of the hemolysis analysis are shown in Figure 4. Figure 4 As shown, oxygen reduction prior to pathogen reduction significantly reduced hemolysis at all time points. Improvements in storability of erythrocytes became evident starting at day 14, although improvements were seen at earlier times.

[0143] Particle analysis results showing relative reduction of particles such as Figure 5 As shown. Figure 5 As shown, oxygen reduction prior to pathogen reduction significantly reduced microparticle formation at all time points. Improvements in storability of erythrocytes became evident starting at day 14, although improvements were seen at earlier times.

[0144] Results for osmotic fragility (6A), potassium (6B), total hemoglobin (6C), oxygen saturation (6D), glucose (6E), lactate (6F), and pH (6G) of control whole blood with sterile saline (1avg), control whole blood with riboflavin (2avg), oxygen-reduced RBCs with sterile saline (3avg), oxygen-reduced pRBCs with riboflavin (4avg), oxygen-reduced whole blood with sterile saline (5avg), and oxygen-reduced whole blood with riboflavin (6avg).

[0145] Example 6: Oxygen-reduced blood pathogen inactivation method

[0146] Blood samples to be processed for pathogen inactivation were first processed for oxygen depletion by transfer to a whole blood collection bag connected to a Sorin D100 membrane oxygenator (Sorin Group, Arvada, CO) with a mixture of 95% N2 and 5% CO2 gas pumped at a flow rate of 700 mL / min to achieve a pre-treatment %SO2 less than 25% and pCO2 of approximately 70 mmHg at approximately 23° C. For the required blood component of whole blood with anticoagulant (WB), a volume of 500+ / -50 mL was used: For the required blood component of leukoreduced packed red blood cells with additive solution (LRpRBC), a volume of 500+ / -50 mL was used: For the required blood component of suspended platelets (PLT), samples were combined for a total volume of approximately 400+ / -50 mL: For the required blood component of plasma, two units of approximately 200 mL were combined for a volume of 400+ / -50 mL. The blood sample is transferred to a polyvinyl chloride illumination bag (TerumoBCT, Lakewood, CO) and filled into the bag and mixed with the calculated dose of cross-linking agent. According to the table, the illumination bag is then placed on a tray and irradiated with the appropriate illumination source based on the cross-linking agent, and the irradiation duration is calculated based on the dose required for a given volume and dose required for a given cross-linking agent. The tray is gently agitated during irradiation to expose the contents to the illumination source evenly during the exposure. After the illumination cycle is completed, the blood sample is transferred from the illumination bag to an anaerobic storage bag for refrigeration. The whole blood sample can be further processed and separated into individual blood components by centrifugation. Pathogen inactivation methods suitable for the methods of this specification include the methods shown in Table 1.

[0147] Example 7: Apheresis Collection and Ultraviolet Treatment of Blood Components

[0148] The donor is approached by venipuncture using a 17-gauge hypodermic needle and connected to a plasma separation system. About 450 mL of whole blood (WB) is drawn from the donor into the apheresis system and mixed with an anticoagulant before centrifugation and separation of blood components. The separated red blood cells (RBC) are then mixed with an additive solution and riboflavin, then passed through a UV radiation chamber and irradiated with UV light to inactivate pathogens and also inactivate any residual white blood cells. After UV irradiation, the RBCs are collected in a separate storage bag. The separated PLTs are then mixed with a PLT additive solution and riboflavin, then passed through a UV radiation chamber and irradiated with UV light to inactivate pathogens. After UV irradiation, the PLTs are collected in a separate storage bag. The separated plasma is then mixed with riboflavin, then passed through a UV radiation chamber and irradiated with UV radiation to inactivate pathogens. After UV irradiation, the plasma is collected in a separate storage bag. Before removing the phlebotomy needle, the replacement fluid volume of 0.9% saline and crystalloids is returned to the donor.

[0149] Example 8: Whole Blood Apheresis Collection and Ultraviolet Treatment

[0150] The donor is approached by venipuncture using a 17-gauge hypodermic needle and connected to the plasma separation system. Approximately 450 mL of whole blood (WB) is drawn from the donor into the apheresis system and mixed with an anticoagulant and riboflavin before passing through an irradiation chamber and irradiated with UV light to inactivate pathogens, then centrifuged and the blood components are separated. After centrifugation and separation of the 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 collected in a separate storage bag. The separated plasma is collected in a separate storage bag. Before removing the phlebotomy needle, the replacement fluid volume of 0.9% saline and crystalloids is returned to the donor.

[0151] Example 9: Checking the quality of pathogen active (S-303) treated RBCs

[0152] Measure a complete blood count (CBC) and percent oxygen saturation (%SO2) of five units of leukoreduced packed red blood cells with additive solution (e.g., AS3) (LRpRBC). Pool these five units together into a 2-3 liter bag of blood to form a homogenous pool. Determine the CBC and %SO2 of the pooled LRpRBC. Place aliquots of 300 mL of LRpRBC in 5 storage containers, labeled A through E, process as shown in Table 3, and store at 4°C in a standard blood bank refrigerator for 42 days.

[0153] Using the method described in Example 5, aliquots of samples A to E were collected at days 0 (before storage), 7, 14, 21, and 42, and the following parameters were tested: CBC, percent hemolysis, ATP, 2,3-DPG, deformability using MVA, microparticles, phosphatidylserine (PS) exposed to the red blood cell membrane, % SO2, RBC morphology, and RBC aggregation. The concentrations of S-303 and S-300 were determined from aliquots of samples A to E collected at 0, 3, 6, 9, 12, and 24 hours. The experiment was repeated five times, with a total of n = 5 samples at each data point.

[0154] Use the mean and median to measure the central tendency of the data, and the standard deviation to determine the spread of the data.

[0155] Table 3: Treatment conditions of LRpRBC

[0156]

[0157]

[0158] Example 10: Examination of the effect of S-303 pathogen inactivation under anaerobic conditions

[0159] Measure the complete blood cell count (CBC) and percent oxygen saturation (%SO2) of 5 units of leukocyte-reduced packaged red blood cells (LRpRBC) with AS3 additive solution. Pool these five units together to form a 2-3 liter blood bag to form a homogenous pool. Determine the CBC and %SO2 of the pooled LRpRBC. The pooled blood is spiked with the selected pathogen or model virus according to the manufacturer's instructions. Aliquots of 300 mL LRpRBC aliquots are placed in 5 storage containers, labeled A to E, and processed as shown in Table 3. Sample B is treated with about 0.2 mM S-303 and about 2-20 mM glutathione (GSH) under aerobic conditions. Samples C and D are treated with about 0.2 mM S-303 and about 2-20 mM GSH before and after oxygen reduction, respectively. Sample E is treated with about 0.2 mM S-303 and about 2-20 mM GSH in ORB. Following the outlined treatments, all samples were stored in a standard blood bank refrigerator at 4°C for 42 days. Alternatively, residual amounts of S-300 in samples were removed by centrifugation and replacement with fresh AS3.

[0160] Using the method described in Example 5, aliquots of samples A to E were collected on days 0 (before storage), 7, 14, 21, and 42 and tested for the following parameters: CBC, residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability using MVA, microparticles, phosphatidylserine (PS) exposed to erythrocyte membranes, and % SO2. The concentrations of S-303 and S-300 were determined from aliquots of samples A to E collected at 0, 3, 6, 9, 12, and 24 hours. The experiment was repeated five times for a total of n=5 samples at each data point.

[0161] The central tendency of the data was measured using the mean and median, and the spread of the data was determined with the standard deviation. Repeated measures analysis of variance with the Neuman-Keuls multiple comparison test was used to analyze the differences between the various sample conditions, and a probability level of less than 0.05 was considered significant.

[0162] The following content corresponds to the original claims in the parent application and is now incorporated herein as part of the specification:

[0163] 1. A method for reducing blood pathogens with reduced hemolysis, comprising:

[0164] removing oxygen from blood products to prepare oxygen-reduced blood products;

[0165] Reduction of blood-borne pathogens from the blood products includes:

[0166] Add riboflavin to a final concentration of 40 to 60 μM; and

[0167] The riboflavin-containing blood product is irradiated using ultraviolet light between 265-400 nm.

[0168] 2. The method of claim 1 , further comprising storing the oxygen-reduced pathogen-reduced blood product under anaerobic conditions.

[0169] 3. The method of claim 1, further comprising reducing carbon dioxide from the blood product.

[0170] 4. 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.

[0171] 5. The method of claim 1 , wherein the blood product is whole blood or leukopenic whole blood.

[0172] 6. The method of claim 1, wherein the blood product containing the riboflavin is an oxygen-reduced blood product.

[0173] 7. The method of claim 1, wherein the blood product is collected in an anticoagulant solution of citrate phosphate dextrose (CPD), citrate phosphate diglucose (CP2D), or citrate phosphate dextrose adenine (CPDA1).

[0174] 8. The method of claim 1 , wherein the oxygen-reduced pathogen-reduced blood product is whole blood, leukopenic whole blood, or packed red blood cells.

[0175] 9. A method of reducing blood pathogens with reduced particle formation, comprising:

[0176] removing oxygen from blood products to prepare oxygen-reduced blood products;

[0177] Reduction of blood-borne pathogens from the blood products includes:

[0178] adding riboflavin to a concentration of 40 to 60 μM; and

[0179] The blood product containing the riboflavin is irradiated using ultraviolet light at 265-400 nm.

[0180] 10. The method of claim 9, further comprising storing the oxygen-reduced pathogen-reduced blood product under anaerobic conditions.

[0181] 11. The method of claim 9, further comprising reducing carbon dioxide from the blood product.

[0182] 12. The method of claim 11, further comprising storing the oxygen-reduced pathogen-reduced blood product under anaerobic and carbon dioxide-reduced conditions.

[0183] 13. The method of claim 9, wherein the number of particles is reduced by at least two-fold at day 14, by at least two-fold at day 21, or by at least two-fold at day 42 relative to a sample treated in the presence of oxygen.

[0184] 14. The method of claim 9, wherein the number of particles is reduced by at least 5-fold at 14 days, by at least 5-fold at 21 days, or by at least 5-fold at 42 days relative to a sample treated in the presence of oxygen.

[0185] 15. The method of claim 9, wherein the riboflavin concentration is about 50 μM.

[0186] 16. The method of claim 9, wherein the blood product is collected in citrate phosphate dextrose (CPD), citrate phosphate diglucose (CP2D), or citrate phosphate dextrose adenine (CPDA1).

[0187] 17. The method of claim 9, wherein the blood product is whole blood or leukopenic whole blood.

[0188] 18. The method of claim 9, wherein the oxygen-reduced pathogen-reduced blood product is whole blood, leukopenic whole blood, or packed red blood cells.

[0189] 19. Oxygen-reduced whole blood, including whole blood collected in CPD, having 400 to 60 μM riboflavin, an oxygen saturation (SO2) of less than 25%, and a pCO2 of 90 mmHg or less at 37°C, wherein the oxygen-reduced whole blood has been irradiated with UV light between 265 and 400 nm.

[0190] 20. The oxygen-reduced whole blood of claim 19, wherein the pCO2 is 5 to 90 mmHg at 37°C.

[0191] 21. The oxygen-reduced whole blood of claim 19, wherein the pCO2 is 70 mmHg at 37°C.

[0192] 22. Oxygen- and carbon dioxide-reduced whole blood, including whole blood collected in CPD, having 400 to 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 between 265 and 400 nm.

[0193] 23. The oxygen and carbon dioxide reduced whole blood of claim 19, wherein the pCO2 is 5 to 20 mmHg at 37°C.

[0194] 24. The oxygen-reduced whole blood of claim 19, wherein the pCO2 is 5 mmHg at 37°C.

[0195] 25. The oxygen-reduced whole blood of claim 19, wherein the radiation comprises 3.2 to 7.0 J / cm 2 of UV dose.

[0196] 26. A blood pathogen reduction method for reducing hemolysis, comprising:

[0197] removing oxygen from blood products to prepare oxygen-reduced blood products;

[0198] Reduction of blood-borne pathogens from oxygen-reduced blood products includes:

[0199] Add S-303 to a final concentration of 0.2 mM; and

[0200] GSH was added to a final concentration of 2 to 20 mM.

[0201] 27. The method of claim 26, further comprising storing the oxygen-reduced pathogen-reduced blood product under anaerobic conditions.

[0202] 28. The method of claim 26, further comprising reducing carbon dioxide from the blood product.

[0203] 29. The method of claim 26, wherein the blood product is whole blood, leukopenic whole blood, or packed red blood cells.

[0204] 30. The method described in claim 26 further includes centrifuging the blood product after adding S-303.

[0205] 31. The method of claim 30, further comprising mixing in an additive solution having an acidic pH after the centrifugation.

[0206] 32. A method of reducing blood pathogens with reduced particle formation, comprising:

[0207] removing oxygen from blood products to prepare oxygen-reduced blood products;

[0208] Reduction of blood-borne pathogens from the blood products includes:

[0209] Add S-303 to a final concentration of 0.2 mM; and

[0210] GSH was added to a final concentration of 2 to 20 mM.

[0211] 33. The method of claim 32 further comprising storing the oxygen-reduced pathogen-reduced blood product under anaerobic conditions.

[0212] 34. The method of claim 32 further comprising reducing carbon dioxide from the blood product.

[0213] 35. The method of claim 32, wherein the blood product is whole blood, leukopenic whole blood, or packed red blood cells.

[0214] 36. The method described in claim 32 further includes centrifuging the blood product after adding S-303.

[0215] 37. The method of claim 36, further comprising mixing in an additive solution having an acidic pH after the centrifugation.

[0216] 38. Oxygen-reduced erythrocytes 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.

[0217] 39. The oxygen-reduced red blood cells of claim 38, further comprising GSH at a final concentration of about 2 to 20 mM.

[0218] 40. The oxygen-reduced red blood cells of claim 38, further comprising improvements in at least two parameters selected from the group consisting of complete blood count (CBC), residual pathogen concentration, percent hemolysis, ATP, 2,3-DPG, deformability, particulate formation, %SO2, and decomposition kinetics of S-303.

[0219] 41. The method of claim 40, wherein the percentage of hemolysis is less than 0.8%.

[0220] 42. The method of claim 40, wherein the deformability is increased by greater than 10% compared to pathogen-inactivated oxygenated blood.

[0221] 43. The method of claim 40, wherein the ATP is increased by greater than 10% compared to pathogen-inactivated oxygenated blood.

[0222] 44. The method of claim 40, wherein 2,3-DPG is increased by greater than 10% compared to virion-inactivated oxygenated blood after storage for at least one week.

[0223] 45. The method of claim 40, wherein the particle formation is reduced by greater than 4-fold compared to virion-inactivated oxygenated blood after storage for at least one week.

[0224] 46. ​​A method for improving the pathogen inactivation efficacy of S-303 in red blood cells, comprising:

[0225] removing oxygen from red blood cells to prepare oxygen-reduced blood products;

[0226] Add S-303 to a final concentration of 0.2 mM; and

[0227] GSH was added to a final concentration of 2 to 20 mM.

[0228] 47. The method of claim 46, further comprising reducing carbon dioxide from the blood product.

Claims

1. A method for blood pathogen reduction with reduced hemolysis comprising: removing oxygen from a blood product containing red blood cells to produce an oxygen-reduced blood product having an oxygen saturation (SO2) of less than 25%; Reducing blood pathogens in the oxygen-reduced blood product, comprising: Add riboflavin to a final concentration of 40 to 60 μM, and UV light at 265 to 400 nm at 3.2 to 7.0 J / cm 2 irradiating an oxygen-reduced blood product containing riboflavin with a UV dose of 500 to produce a pathogen-reduced and oxygen-reduced blood product containing red blood cells; wherein hemolysis is reduced in the pathogen-reduced and oxygen-reduced red blood cell-containing blood product as compared to a pathogen-reduced blood product, the pathogen-reduced blood product being a non-oxygen-reduced blood product.

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

3. The method of claim 1, further comprising reducing carbon dioxide from the oxygen-reduced red blood cell-containing blood product.

4. The method of claim 1, wherein red blood cell hemolysis is less than 0.2% at 14 days.

5. The method of claim 1, wherein the pathogen-reduced and oxygen-reduced red blood cell-containing blood product is whole blood, leukopenic whole blood, or packed red blood cells.

6. The method of claim 1, wherein the red blood cell-containing blood product further comprises reduced microparticle formation compared to oxygenated blood.

7. The method of claim 9, wherein the number of microparticles is reduced by at least two-fold at 14 days relative to a pathogen-reduced blood product processed in the presence of oxygen.

8. The method of claim 1, wherein red blood cell hemolysis is less than 0.4% at 21 days.

9. The method of claim 1, wherein red blood cell hemolysis is less than 0.5% at 28 days.

10. The method of claim 9, wherein the number of particulates is reduced by at least 2-fold at 21 days relative to a sample treated in the presence of oxygen.

11. The method of claim 9, wherein the number of particulates is reduced by at least 2-fold at 42 days relative to samples treated in the presence of oxygen.

12. The method of claim 1, wherein red blood cell hemolysis is less than 0.8% at 35 days.

13. The method of claim 1, wherein red blood cell hemolysis is less than 1.2% at 42 days.

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