INACTIVATION OF PATHOGENS IN EX VIVO BLOOD PRODUCTS IN STORAGE BAGS USING VISIBLE LIGHT
Disclosed herein are methods and devices for the inactivation of pathogens (e.g., bacteria, viruses, etc.) in ex vivo stored blood products, such as plasma and/or platelets, by means of directing visible light radiation from an illuminating device into blood product storage containers in order to achieve effective pathogen inactivation without the presence of an added photosensitising agent in the blood product. An exemplary apparatus includes a control unit that operates a light source that emits light in the wavelength region of about 380-500 nm which is directed onto blood product storage bags at sufficient intensity to penetrate the bag material and the opaque blood product therein in order to inactivate pathogens in the blood product but at dose levels that cause no significant detrimental effects on the blood product.
This application claims the benefit of U.S. provisional patent application No. 62/236,706 filed Oct. 2, 2015, which is incorporated by reference herein in its entirety.FIELD
This application is related to methods and devices for inactivating pathogens in ex-vivo stored blood and blood products.BACKGROUND
Bacterial and viral contamination of ex vivo stored human blood and blood components is a major complication for transfusion medicine, resulting in discarding of the products, and health risks for recipients of contaminated donor blood. Current pathogen reduction technologies (PRT) rely on the use of chemicals and/or ultraviolet light; however reported adverse events, and concerns about the PRT-treated blood components quality and toxicity, have limited the routine use of PRT worldwide.
For plasma treatment, a number of PRTs have been developed and pathogen-reduced plasma is routinely used, with several of these methods now licensed for use in North America and Europe. The original PRTs developed for plasma treatment included the use of solvent/detergent, and methylene blue in combination with visible light. More recently developed methods have employed ultraviolet (UV) light. Exposure to amotosalen (S-59) plus long-wave ultraviolet (UVA) light, and treatment with riboflavin and UV light, have been developed to treat both plasma and platelets. Many current PRTs have limitations and affect plasma coagulation factors and inhibitors of homeostasis to varying degrees, and concerns about reduced component quality and toxicity have limited or prevented the routine use of PRTs in many countries.
For platelet components, bacterial contamination still poses the highest risk of transfusion-transmitted disease despite the availability of bacterial detection methods. Although platelets usually have very low levels of bacterial contamination at the time of donation, platelets are stored at room temperature and this allows bacterial proliferation to occur throughout the storage period. Over a 5 day storage period even extremely small numbers of contaminating bacteria can multiply to very high and clinically dangerous levels.
A challenge for PRTs with platelet products is to maintain biological function of the treated cells, while achieving adequate pathogen reduction levels. For platelets this means sufficient retention of adhesive, aggregating and procoagulant properties to restore hemostasis in the recipient. Platelet treatment methods that have been developed include amotosalen/UVA treatment and riboflavin/UV-light treatment. While data of clinical and hemovigilance studies indicate that the overall safety profile of pathogen-reduced platelets is comparable to that of conventional platelets, there have been concerns over acute respiratory distress associated with UV light and photo sensitizer-treated platelets.
Because of these uncertainties regarding currently available PRTs for plasma and platelet treatments, and because the full extent of future microbiological challenges cannot be predicted, methods to improve existing pathogen reduction technologies and the introduction of novel technologies remains an active area of investigation in transfusion medicine.SUMMARY
The technology disclosed herein provides a new solution for the decontamination of blood products that overcomes limitations associated with currently available PRTs. This disclosure describes, for example, the application of violet-blue light centered around 405 nm wavelengths for pathogen reduction in blood products such as plasma and platelets. Unlike previous methods that require the decontamination treatment of the blood product prior to deposition in the storage container, methods described herein permit decontamination to take place after the blood products enter the final storage receptacle, thereby minimizing any unnecessary further contamination from environmental sources during subsequent transfer operations.
Another aspect of the technology is that, unlike previous PRTs which utilize light radiation from the ultra-violet (UV) region of the spectrum, disclosed technologies utilize radiation from the visible light spectrum, such as with wavelengths particularly within the range of 380-420 nm and especially at or around 405 nm. This is beneficial as it permits inactivation of pathogens to occur without the photochemical damage to sensitive blood product components caused by high energy UV-light photons associated with UV light. In addition, visible light has longer wavelengths that can more efficiently penetrate through blood storage bags and through the opaque blood products at lower intensities and without damaging the storage bag material. Moreover it does not require the addition of chemical photosensitising agents which can also have a deleterious impact on the quality of blood products.
The disclosed technology makes use of properties of visible light, which have somewhat greater penetrability powers than UV light, thereby permitting the concept of in situ treatment of bagged products by enabling penetration of light through the transparent bag material and into the stored product. The low transmissibility property of blood plasma and platelets is shown in
One aspect of the disclosed technology is to combine the use of a sufficient level of irradiation of visible light into the product which is mixed by agitation to a sufficient extent that the blood product circulates within the container and effective decontamination of most of or substantially the entire product volume is achieved. Agitation also prevents aggregation of platelets and other blood components.
Another aspect is the specification of the levels of irradiation needed to achieve the desired decontamination effect. For an LED light source emitting light at 405 nm with bandwidth of ˜10 nm, for example, the irradiance level at the blood product surface is desirably within a certain range that inactivates pathogens but does not harm the blood products (such as at least 3 mWcm−2 and less than 80 mWcm−2), with exposures ranging from minutes to hours, depending on the level of irradiance used. Use of higher irradiance levels enables faster treatment times, e.g. for rapid treatment (of the order of minutes), and use of lower irradiance levels facilitates longer treatment, e.g. for background decontamination during standard storage conditions (of the order of hours). These values can permit effective microbial decontamination of the blood product without causing any significant deleterious effects on blood product components. Hence in some embodiments, the blood products do not contain chemical photosensitization agent additives.
Also disclosed herein is an exemplary apparatus for inactivating pathogens in ex vivo stored blood products. The apparatus includes a treatment chamber configured to receive at least one sealed container storing a blood product, and at least one light source operable to emit light that is directed to a blood product storage container in the treatment chamber. The emitted light has a wavelength or wavelength spectrum and an intensity sufficient to penetrate through a wall of the container and into the blood product, such that the irradiance of the light reaching the blood product inside the wall of the container is sufficient to inactivate pathogens in the blood product without the presence of an added photosensitizing agent in the blood product and without a detrimental effect on the blood product, such that the blood product is suitable for future medical use. The treatment chamber can include a viewing window that filters out the treatment light wavelengths and allows other visible light wavelengths to pass through so that an operator can see inside the chamber. The chamber can include reflective walls, lenses, diffusers, and/or other optical features for directing light at the blood storage containers. The containers can rest on a tray in the chamber that vibrates or otherwise agitates the blood product in the container to prevent aggregation and/or to circulate the blood product inside the container for increased radiation exposure. The blood product containers can also be suspended in the chamber or positioned in other orientations. Light sources can be positioned on different sides of the container to expose more than one surface of the container at the same time. The apparatus can include any of various other features, such as a fan, air circulation system, temperature monitoring system, sensors, and a control system that controls the light sources and other active features of the apparatus.
The foregoing and other objects, features, and advantages of the disclosed technology will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
The disclosed technology can be used to treat any of various stored blood products. As used herein, the term “blood products” includes whole blood, plasma, platelets, red blood cells, fluids comprising any of these substances, and/or fluids derived from any of these substances. The terms “blood” and “blood products” includes human, animal, and/or synthetic blood and blood products.
As used herein, the terms “pathogen” and “contaminant” and “contaminant microorganism” are used interchangeably and include any undesired bacteria, viruses, fungi, viroids, parasites, and/or other contaminant microorganisms present in a blood product. The term “bacteria” includes Gram positive types of bacteria, such as Staphylococcus, Streptococcus, Bacillus, Clostridium, Enterococcus, etc., species and also includes Gram negative types such as Escherichia, Pseudomonas, Klebsiella, Acinetobacter, etc., species. The term “fungi” includes single celled fungi such as yeasts and filamentous or spore forms such as mold fungi. The term “virus” includes any of the various types of viruses that can be present as contaminants in blood products.
As used herein, the term “inactivation” means that the subject microorganisms/pathogens are killed, damaged, and/or otherwise affected so as to inhibit or prevent their ability to replicate, infect a subject, and/or cause disease. For example, inactivation of a pathogen including viruses and other microorganisms stated in the above paragraph can include damaging the pathogen's genetic material that includes for example DNA, RNA, non-coding small RNAs (ncRNAs including microRNAs), and long non-coding RNAs (lnRNAs), thereby inhibiting its ability to reproduce, replicate and/or multiply. Methods disclosed herein can therefore be considered, in some cases, as bactericidal/fungicidal/viricidal or bacteriostatic/fungistatic/virustatic and this may depend on the species/strain of bacteria or virus, wavelength of light, dose, and/or other factors.
Pathogen levels in blood products can be measured in terms of colony forming units, or “CFU”, which is an estimate of the number of pathogens (e.g., cells, viruses, etc.) in a given volume of blood product that are viable and can multiply, reproduce, or replicate.
As used herein, the term “light” means electromagnetic radiation having specified wavelength properties. If not specifically limited to particular wavelength properties, “light” includes electromagnetic radiation having wavelengths from about 1 nm to about 1 mm, including ultraviolet (UV) light, visible light, and infrared (IR) light. The term “visible light” includes electromagnetic radiation having wavelengths from about 380 nm to about 800 nm. The term “violet-blue light” means electromagnetic radiation having wavelengths from about 380 nm to about 500 nm. The term “wavelength” means the length of one full wave or spatial period of the electromagnetic radiation, and can be defined as the inverse of the radiation's frequency.
As used herein, the terms “irradiance” and “intensity” are used interchangeably to mean the radiant flux received by a given surface per unit area, measured in watts per square meter (W/m2). Radiant flux is the radiant energy emitted/transmitted/received per unit time, and is measured in watts or joules per second. The term “radiation dose” or “light dose” means the product of the irradiance and the duration of exposure, and can be measured in joules per square meter (J/m2).
Light emitted or received can include a range of different wavelengths, and the light at each wavelength can have a different spectral irradiance. “Spectral irradiance” is the irradiance of a surface per unit wavelength. Often, light has a greatest spectral irradiance at a certain wavelength, and reduces in spectral irradiance at longer or shorter wavelengths, such as in a “bell curve” shaped pattern. The wavelength or wavelength range having the greatest spectral irradiance is referred to herein as the center wavelength or center wavelength range of the light, such that the light is referred to herein as being “centered on or around” the center wavelength or center wavelength range.
As used herein, the term “photosensitizing agent” means any chemical additive, not naturally present in blood, that is added to a blood product and that produces a chemical change in another molecule in a photochemical process. Exemplary photosensitizing agents include riboflavin, benzoporphyrin derivative-monoacid ring A (BPD-MA), phenothiazine derivative methylene blue and other dyes with photochemical properties including psoralens, such as S-59 (amotosalen-HCl) and S-303 or the ethylene imine PEN-110.
As used herein, the term “detrimental effect” means a substantial change in the normal physiological condition of a stored blood product (e.g., a substantial reduction in the ability of platelets to rapidly aggregate in response to TRAP introduction). Further, the phrase “without detrimental effect on the blood product” means without causing a substantial change in the normal physiological condition of the blood product.Exemplary Methods, Systems, and Devices
According to one aspect of the disclosed technology, there is provided methods and devices that utilize visible light at a wavelength and irradiation sufficient to inactivate pathogens (e.g., bacteria, viruses, fungi, viroids, prions, parasites) that are present in stored human and animal blood products, such as whole blood, plasma and platelets.
According to another particularly beneficial aspect, the disclosed technology utilizes visible light wavelengths (e.g., violet-blue light) without the use of an added photosensitising agent. Hence, in some embodiments a photosensitizing agent is not added to the blood products. Also since disclosed methods utilize visible light, this avoids the chemical and physical damage caused by the conventional methods of exposure to the higher energy photons of UV light.
According to another aspect of the disclosed technology, there is provided a method for inactivating bacteria, fungi, viruses, and/or other pathogens that can be present as contaminants in blood products, comprising exposure of the blood products to visible light having wavelengths in the range 380-500 nm. The light may have wavelengths in the range 400-450 nm. The light may have wavelengths in the range 380-420 nm. The light may have a wavelength spectrum centered on or around 405 nm. In some embodiments, at least 50% or at least 90% of the light has a wavelength within any of the disclosed ranges. In some embodiments, at least 50% or at least 90% of the light has a wavelength within 10 nm of 405 nm.
According to another aspect of the disclosed technology there is provided a method and apparatus for the inactivation of pathogens in blood products while these are contained within sealed blood storage containers, such as blood transfusion bags. Microbicidal wavelengths of light are used that penetrate through the storage bag material and to sufficient depth within the blood product as to achieve effective pathogen inactivation over an exposure period.
An exemplary treatment device 2 is illustrated in
The device 2 can utilize a lighting system 8 which can comprise one or more LED sources 10 and/or other types of light sources that emits light in a selected wavelength range and intensity sufficient to inactivate pathogens in stored blood products. The lighting system 8 can be configured to deliver light in any direction above, below and/or to the sides of the stored blood products in order to achieve focused or uniform illumination of the blood products as required. For example, light sources can be mounted on any surface of the chamber and/or on shelves, racks, trays, and/or other structures within the chamber. Blood storage bags can thereby be irradiated from two or more different directions, such as from the top and bottom, or two other opposing sides, at the same time.
The treatment chamber 4 can have various dimensions, even up to the size of an entire room, in which the lighting system 8 is installed at one or more locations around the treatment chamber, and the internal surfaces 12 of which can be lined with or made from highly reflective material. Exemplary reflective materials include various mirrored surfaces, polished stainless steel, polished aluminum, silver coated surfaces, various retroreflective material surfaces and surfaces that are optimal for reflecting light of wavelengths between 380-500 nm and/or 400-420 nm and/or 405 nm.
According to another aspect of the disclosed technology, the treatment device can include a mechanically operated reciprocating tray or platform 14 on which stored blood products may be continuously agitated during exposure to the inactivating light. Agitation can help circulate the blood product inside the container for a more even irradiation and/or to reduce aggregation of platelets or other blood components. The reciprocating tray or platform 14 can be operated at different reciprocation rates (e.g., 10-100 rpm or 30-100 rpm) and for differing periods of time throughout the duration of the light treatment in order to provide sufficient mixing so that the opaque blood product receives sufficient light treatment within the illuminated zone under the semi-transparent bag material. As shown, the device 2 includes one tray 14, though in other embodiments the device can include two or more independent trays, such as trays that are stacked on top of each other and/or side-by-side. In some embodiments, the tray 14 or trays can be at least partially transparent or transmissive of the treatment light wavelengths such that blood product bags can be irradiated from below while resting on a tray. In embodiments with multiple trays, the light sources can be arranged and positioned around the chamber roof, walls, floor, and/or on the tray bottoms to provide the desired light exposure to the blood products on each tray. In various embodiments, the treatment chamber 4 can simultaneously contain two or more blood product containers in any relative orientation, such as lying side-by-side flat on one or more trays, one above the other on different trays, suspended from above or hanging, etc.
According to another aspect, the device 2 can comprise a temperature control system, ventilation system, and/or air circulation system 16 in order to accurately control the environment inside the chamber 4 throughout the treatment process.
According to another aspect of the disclosed technology, the lighting system 8 can include lenses or diffusers positioned adjacent to the light sources 10 in order to achieve directed or diffuse light outputs depending on the process requirements. For example,
According to yet another aspect of the disclosed technology, the lighting system 8 can deliver light at different intensities during the treatment period so that, for example, a high radiation dose may be delivered at the beginning of the process to achieve effective initial inactivation followed by a lower dose regime to maintain the decontamination effect. Predetermined intensity patterns and cycles can be used during a treatment. Other variations in dose delivery may also be used, such as depending upon the blood product type and nature of the pathogens present. For example, the intensity of the light emitted from the light sources can be sufficient such that irradiance levels at the surface of the blood product within the storage container can be from about 3 mWcm−2 to about 80 mWcm−2 in some embodiments. In some embodiments, the light reaching the surface of the blood product has an irradiance of 3 mWcm−2 or less. In some embodiments the light reaching the surface of the blood product has an irradiance of 80 mWcm−2 or greater. In some embodiments, the light emitted from the light sources can have any of the irradiance or intensity values provided herein, or greater values due to the reduced area of the light sources relative to the blood product. In some embodiments, the light reaching the external surface of the blood product storage container can have any of the irradiance or intensity values provided herein. In various embodiments, the light reaching at least some portion of the blood product within the storage container can have any of the irradiance or intensity values provided herein. The irradiance of the light can decrease as it passes through the storage container material, and thus the irradiance at the surface of the blood product can depend on the material and thickness of the storage container walls, as well as various other factors.
According to yet another aspect of the disclosed technology, the device 2 can have a lighting control system 18 that allows for the setting or programming of different irradiation intensities and durations throughout the blood product treatment period. The control system 18 can control the chamber air temperature and/or humidity, air circulation, tray agitation/vibration, light source selection, light source intensity, treatment duration, cycles, patterns, and/or automatic light source turn-off (e.g., when door is open or temperature is too high). The control system 18 can also include or be coupled to one or more sensors that measure desired properties and provide feedback to the control system 18. Exemplary sensors or sensor systems can include temperature sensors, light intensity or irradiance sensors, light wavelength or frequency sensors, tray weight sensors, and/or other sensors.Exemplary Investigations, Results, and Analysis
To investigate the effects of visible light, and particularly 405 nm light, on pathogens in stored blood products, the optical properties of blood, blood plasma, platelets, and other blood products were initially determined. To do this, spectrophotometric analysis was carried out over the wavelength range 220-700 nm using a Biomate 5 UV-Visible Spectrophotometer. The results shown in
The effectiveness of the visible light for inactivating pathogents in blood products was investigated by seeding bacteria into test liquids and then exposing them to visible light irradiation. Bacteria used in these tests were Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli and these were selected to represent significant contaminants associated with blood components.
The use of the bacteria described above was for illustrative purposes only. In addition, a wide range of other pathogens are susceptible to visible light, including 405 nm light. For example, Gram-positive and Gram-negative bacteria, yeasts, fungi, bacterial endospores and viruses can be inactivated by 405 nm light and/or other visible light, with up to 9-log reduction in populations demonstrated. Such pathogens can be successfully inactivated when suspended in blood products such as plasma and platelets when exposed to inactivating doses of 405 nm light and/or other visible light having bandwidths near 405 nm.
Disclosed devices and methods can be used for decontamination (e.g., inactivation of pathogens) of blood products, either within test containers or within blood transfusion storage bags. To test the effect of visible light on the inactivation of bacteria in selected blood products, defrosted fresh frozen human plasma and human platelets were seeded with known concentrations of bacterial contaminants. The light source used for exposure was an array of 405 nm LEDs powered by a direct current supply. For thermal management, the LED arrays were bonded to a heat sink and fan, thus ensuring that heating had no effect on the test samples exposed to the 405 nm light.
To demonstrate the principle, three different sample volumes (3 ml, 30 ml and approximately 300 ml (whole transfusion bags)) were used for exposure. For exposure of 3 ml samples, samples were held in 12-well microplates (without the lid), with the LED array positioned 3 cm above, providing an irradiance at the sample surfaces of ˜100 mWcm−2. 30 ml samples were exposed in 90 mm dishes (lid on), with the LED array positioned above to provide an irradiance of ˜8 mWcm−2. For exposure of platelet and plasma bags, LED arrays were held above the horizontally-positioned bags to give an irradiance of 3-5 mWcm−2 at the center position of the bag. Experimental systems were held in a shaking incubator (72 rpm; 25° C.) for continuous sample agitation to maintain exposure conditions. After light exposure, samples were plated onto growth medium, incubated, and enumerated to determine the number of surviving colony-forming units (CFU)/ml. The light dose (J cm−2) used to expose the blood samples was calculated as the product of the irradiance (W cm−2) multiplied by the exposure time (sec).
Tests carried out using 3 ml volumes of platelets seeded with different levels of bacterial contamination also demonstrate the ability of light of this wavelength to inactivate microbial contamination in platelets.
Pathogens suspended in clear media, such as phosphate buffered saline (PBS), can be inactivated using lower doses of visible light. The need for higher doses in the case of inactivation in blood plasma and platelets can be accredited to the differing optical properties of these suspending media. The opacity, and consequent relatively low transmissibility of plasma and platelets (
In addition to using high (e.g., 100 mWcm−2) irradiance levels at the blood product surface, experiments were carried out to establish that low density pathogen contamination (101 CFU/ml) can be inactivated when using a lower irradiance level, such as 14 mW/cm2. Results, shown in
To demonstrate that decontamination of larger volumes of plasma and platelets can be achieved (i) using lower irradiance visible light, and (ii) in sealed containers without the presence of an added photosensitizing agent, tests were conducted wherein 30 ml volumes of human plasma and platelets seeded with different levels of S. aureus were exposed, in a closed Petri dish, to an irradiance of ˜8 mWcm−2. An example of the results for plasma with a seeding density of 103 CFUml−1 is shown in
In order for the applied light to sufficiently affect the blood product, a sufficient amount of the applied light has to first pass through the storage container wall. For this reason, the transmissibility of blood product bag materials was measured and tested.
To further demonstrate the application of the disclosed technology, tests were carried out to establish that bacteria can be inactivated in human blood plasma when the applied light was transmitted through a layer of blood bag material. Tests were carried out by placing samples of the contaminated plasma into the wells of a 12-well microplate (without the lid), but with the wells covered with a layer of PVC blood-bag material, and the sample was exposed to 14 mWcm−2 405 nm light at the sample surface. Samples were treated in the rotary incubator (37° C.; 72 rpm). Exposure of S. aureus suspended in human plasma to 405 nm light transmitted through the blood-bag material resulted in successful inactivation with complete inactivation achieved after a 4-hour exposure period as shown in
To demonstrate the effectiveness of the disclosed technology for decontamination of plasma and platelets when these are contained within sealed blood bags, experiments were conducted to expose whole PVC transfusion bags, seeded with contamination, to 405 nm light. Inactivation of low density (101-102 CFU ml−1) bacterial contaminants within PVC plasma transfusion bags was achieved using irradiances as low as 3 mWcm−2 at the plasma surface. An example of inactivation using 5 mWcm−2 at the plasma surface is shown in
The preceding investigations and results demonstrate that the disclosed technology effectively inactivates pathogens contained within blood products, such as blood plasma and platelets. Furthermore the disclosed technology establishes that visible light and particularly 405 nm light can be used for inactivation of pathogens in plasma and platelets while these blood products are contained within transfusion bags. The fact that light of 405 nm wavelength can penetrate the transfusion bag material enables the blood products to be decontaminated (i.e., inactivation of pathogens) while sealed in the bags, thereby significantly reducing the risk of any post treatment contamination that can occur with other procedures.
When the disclosed technology is used as a platelet decontamination technology, it can be important that the visible light treatment has no adverse effects on the platelet cells, and that they are suitable for transfusion post treatment. Testing and establishing such features of the disclosed technology included the establishment of information about the physiological effects of visible light on platelet cells. To this end, the standard tests that are performed on platelets in blood bank storage conditions, such as platelet count, pH of the platelet solution, and platelet aggregation in response to platelet agonists [e.g. Thrombin Receptor Agonist Peptide (TRAP)], were evaluated following a selected time (in hours) of 405 nm light exposure of platelets in the bags (see Tables 1a, 1b and 1c below). Rapid aggregation associated with TRAP induction is a positive indicator that platelets are in normal physiological conditions during storage. Thus, the test to control ratio of 1 (i.e., 100%) or close to 1 (i.e., close to 100%) is a good indicator of the quality of platelets in any given context.
Analysis was performed using platelets processed from a donor split into two equal halves and each half was stored in a storage bag. At least two donor samples were used for each experiment (n=2), where feasible. For each 405 nm light exposure treatment, one platelet bag was used as control and the other as test sample. Platelet bags were exposed to three different treatment conditions:  At 10 mW/cm2 405 nm light for up to 6 hrs;  At 40 mW/cm2 405 nm light for up to 6 hrs; and  At 80 mW/cm2 405 nm light for up to 6 hrs. The non-exposed control samples (in a light-safe box) and the test samples were held under rotary conditions during exposure (˜25° C. and 72 rpm) in the same incubation chamber. Samples were collected and analyzed at 2 h intervals from both light-exposed and control samples.
Tables 1a, 1b, and 1c below show platelet counts, pH measurements and TRAP-induced platelet aggregation of non-exposed platelets and platelet samples exposed to 405 nm light at 10 mW/cm2 (Table 1a) 40 mW/cm2 (Table 1b) and 80 mW/cm2 (Table 1c) for up to 6 hrs while in transfusion bags. Note that “Control” and “Test” are two individual bags originated from the same donor and the same donation (split sample).
Overall, the results of the analytical studies carried out (platelet count, pH and TRAP-induced aggregation) on platelets exposed to 405 nm light show that there are no major physiological changes in the platelet cells when exposed in containers or transfusion bags to 405 nm irradiances at 10 mW/cm2 for up to 6 hours (Table 1a), or 40 mW/cm2 for 4 hours (Table 1b), or 80 mW/cm2 for up to 2 hours (Table 1c). However, change in pH and TRAP-induced aggregation was prominent when 405 nm irradiance at 80 mW/cm2 was extended to 6 hours. These irradiance levels and treatment times have been shown to successfully inactivate the low levels of microbial contamination that can occur in whole blood platelet and plasma transfusion bags due to the light transmission through the sealed bag material and into the stored product. These exposure conditions can be compatible with the typical storage conditions of the platelets, so that one application for the technology is to utilize a combined storage and treatment regime involving exposing the platelets to light having wavelength spectrum centered on or around 405 nm while agitating the platelets during storage until they are used for transfusion.
Some embodiments of the disclosed technology utilize high irradiance treatment (e.g., up to 80 mW/cm2) for exposure of whole platelet/plasma bags in order to achieve shorter exposure periods (e.g. minutes) for the effective inactivation of pathogens in the blood products without having detrimental effects on the blood products. Results from the analysis of high irradiance light-exposed platelets showed that there were no significant detrimental changes apparent in the platelet suspensions. Accordingly, some embodiments of the disclosed technology utilize high-irradiance exposure of whole bags to enable platelet/plasma bags to be treated quickly (e.g., 30-60 mins) immediately after collection from the donor before being stored awaiting transfusion.
The disclosed technology can be used to treat blood products stored in many different types of storage containers comprising various materials, so long as the container provides at least some transmissivity to the applied light. Exemplary container/bag materials suitable for use with the disclosed technology include PVC (polyvinyl chloride), EVA (ethylene-vinyl acetate), PE (polyethylene), PP (polypropylene), PS (polystyrene), combinations of such materials, variations of such materials, and/or other at least partially light-transmissive materials.Viral Inactivation
The study described here was designed to illustrate the interaction of narrowband 405 nm light with FCV as a model to study the antiviral effects of this light on NoVs. FCV was selected as a NoV surrogate, as there is currently no standardized cell-culture system for NoV. Our data demonstrates the influence of the suspending media, including biologically-relevant fluids, on viral susceptibility. As such, this study provides evidence of the antiviral efficacy and discusses the potential mechanism of 405 nm light viral inactivation.
Feline embryonic cells, strain FEA (Jarrett, Laird & Hay 1973) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate and 240 U mL−1 penicillin streptomycin (Gibco, Life Technologies, UK), to form 10% FBS-DMEM. Cells were maintained at 37° C. in 5% CO2.
To prepare a virus pool of the FCV vaccine strain F9, virus inoculum (School of Veterinary Medicine, University of Glasgow) was added to FEA monolayers in 850 cm2 cell culture roller flasks (Corning, USA). After 90-min incubation of the inoculated cells on a rotating roller stand at 37° C. in 5% CO2, fresh culture medium was added and flasks incubated for 24 hours. This resulted in virus-induced destruction of nearly 90% of the cell monolayer.
The tissue culture supernatant, and medium from a single wash step, was collected from each roller bottle and subjected to two freeze-thaw cycles before clarification by centrifugation at 3300×g for 10 minutes. The virus-containing supernatant was then stored at −80° C. until required. The infectious titre of FCV was approximately 2×107 plaque forming units per millilitre (PFU mL−1), determined by standard plaque assay techniques (Ormerod & Jarret 1978).
The light source used was a 405 nm light emitting diode (LED) array (ENFIS PhotonStar Innovate UNO 24; PhotonStar Technologies, UK) powered by a 40 V Phillips Xitanium LED Driver (Phillips, Netherlands). The array had a peak wavelength around 405 nm and a bandwidth of approximately 19 nm (
FCV stock virus was defrosted at room temperature and diluted to 2×105 PFU mL−1 in Dulbecco's phosphate buffered saline, supplemented with calcium and magnesium (DPBS; Hyclone, Thermo Fischer Scientific, UK). This was used as a ‘minimal medium’ (MM). 1.5 mL volumes of viral suspension were transferred into the central 4 wells of a 24-well plate (Techno Plastic Products, Switzerland) and the plate positioned on a raised stand, with the sample wells 4 cm directly below the light source and the plate lid kept on to prevent evaporation. Test samples were exposed to increasing doses of 405 nm light at room temperature, with the dose calculated as the product of irradiance (mW cm−2)×exposure time (s). Control samples were set up under identical environmental conditions but without 405 nm light illumination. Post-exposure, FCV samples were immediately removed from the well and serially diluted in MM for enumeration by plaque assay.
Exposures were repeated with FCV suspended in ‘organically-rich media’ (ORM): DMEM, 10% FBS-DMEM, artificial saliva, artificial faeces and blood plasma. The artificial saliva was a modified version of that used by Margomenou et al. (2000) [5.2 g NaHCO3, 0.88 g NaCl, 1.36 g K2HPO4, 0.48 g KCl, 2000 units α-amylase and 2 g pig gastric mucin (Sigma Aldrich, UK) in 1 L sterile water], and was adjusted to pH 7-7.5 to emulate the variability of pH in human saliva, and also to ensure no FCV inactivation occurred (Duizer et al. 2004b; Edgar et al. 2004). The artificial faeces was a modified version of that by Colon et al. (2015) [30 g inactivated yeast (Marigold, UK), 7 g physillum (Buy Whole Foods Online, UK), 11 g miso paste (Yutaka, UK), 8 g cellulose, 1.6 g NaCl, 0.8 g CaCl, 1.6 g KCl (Sigma Aldrich, UK) in 920 mL sterile water], and was also adjusted to pH 7. The modifications to the artificial saliva and faeces formulations were to ensure compatibility with the FEA cells. Fresh frozen human blood plasma was obtained from the Scottish National Blood Transfusion Service (SNBTS, UK), and defrosted before use. FCV was also exposed whilst suspended in MM supplemented with riboflavin, with and without tyrosine, tryptophan, pyridoxine and folic acid (used at the same concentrations as found in DMEM: 0.4, 104, 16, 4 and 4 mg L−1 respectively).
Prior to experiments, 6-well cell culture plates (Thermo Fischer Scientific) were seeded with 7.5×105 FEA cells per well. 3 mL of the cell suspension in growth medium was pipetted into each well, and incubated at 37° C. in 5% CO2 for 20-h, resulting in confluent monolayers.
Post-exposure of FCV, the growth medium was aspirated from the FEA cells and replaced with 1 mL FCV sample. Plates were co-incubated at 37° C. in a humidified 5% CO2 incubator for 90-min, with the plates gently rocked every 15-min to ensure even distribution of the inoculum over each monolayer.
After the viral incubation period, the inoculum was aspirated and the well washed with medium (10% FBS-DMEM or DPBS) before adding 4 mL overlay mixture consisting of 2× supplemented DMEM 1:1 with 2× agarose. 2× supplemented DMEM was prepared using 20 mL from a filter sterilized stock of 10×DMEM, adding the same supplements as detailed earlier, plus 9.86 mL sodium bicarbonate solution (Gibco), and was made up to 100 mL with sterile water. 2× agarose was prepared by dissolving 2 g agarose (Sigma Aldrich) in 100 mL deionized distilled water and sterilized by autoclaving. The overlay was left to set before the plates were incubated for 44-48 hours at 37° C. in 5% CO2.
Post-incubation, the monolayers were fixed and stained overnight with 0.5% crystal violet in 10% buffered neutral formalin. The agarose plugs and stain were then removed, the plates left to dry, plaques counted, and the virus infectivity titre expressed as PFU mL−1.
The presence of porphyrins, or other components with the ability to absorb 405 nm light and emit fluorescence, within the suspending media was determined by fluorescence spectrophotometry. Media were freshly prepared and fluorescence measurements were carried out using a RF-5301 PC spectrofluorophotometer (Shimadzu, USA). Excitation was carried out at 405 nm and emission spectra recorded between 425-700 nm.
Data points represent mean results ±standard deviation (SD), taken from triplicate independent experiments (n=3). The antiviral activity of 405 nm light was determined by calculating the reduction in the level of infectivity from the difference between Log10 values for exposed and control samples. Significant differences were calculated using one-way ANOVA (Minitab 16 Statistical Software) with results found to be significant when P<0.05.
FCV was suspended in MM and ORM and exposed to increasing doses of 405 nm light at an irradiance of 155.8 mW cm2. Results (
Antiviral efficacy was found to differ significantly when suspended in ORM. When exposed in 10% FBS-DMEM, a significantly lower dose was required for viral inactivation (
Furthermore, components of DMEM have been shown to be photosensitive to light (Grzelak et al. 2001), therefore exposures were repeated with riboflavin added to MM with and without tyrosine, tryptophan, pyridoxine and folic acid in the same concentrations found in DMEM (see Table 2 below). Results demonstrated that exposure of FCV suspended in MM with riboflavin only resulted a 1.3 Log10 reduction after 421 J cm−1, however when all components were present enhanced inactivation occurred and a 5.1 Log10 inactivation was achieved.
Artificial saliva, artificial faeces, and blood plasma were selected as ORM which are biologically-relevant in terms of media in which viral particles may be found in the environment, with Norovirus regularly identified in faeces. Exposure of FCV when suspended in artificial saliva yielded results similar to those in DMEM, with a 5.1 Log10 reduction of infectivity achieved after a dose of 421 J cm−2 (
The fluorescence emission spectra (
Although there has been a recent move towards using murine norovirus and tulane virus as NoV surrogates (Cromeans et al. 2014; Kniel 2014), FCV was chosen as it has physiochemical and genomic similarities to NoV, and is a well-established surrogate with a standardized cell-culture protocol (Doultree et al. 1999; Bidawid et al. 2003; Duizer et al. 2004a, b; Chander et al. 2012). Similarly, studies investigating the virucidal effects of UV-light, ozone, hydrogen peroxide vapour and cold atmospheric gas plasma technologies have also used FCV as a NoV surrogate (Nuswalen et al. 2002; Hudson et al. 2007; Bentley et al. 2012; Aboubaktar et al. 2015; Holmdahl et al. 2016).
The virucidal efficacy of 405 nm light was determined using FCV suspended in both minimal medium (MM) and organically-rich media (ORM). Exposure in MM would provide a better indication of the interaction of 405 nm light and the virus alone, whilst suspension in ORM, which is likely to contain photosensitive components, would assess how viral susceptibility can potentially be influenced by the surrounding media.
Successful FCV inactivation was achieved when suspended in MM, although the dose required was great, with 2.8 kJ cm−2 achieving a 3.9 Log10 reduction (
Inactivation in MM may be associated with the LED emission spectrum extending slightly into the UVA region (
To investigate whether exposure in ORM had any effect on viral susceptibility, FCV was first suspended in DMEM with and without 10% FBS (thought to aid protection against ROS (Grzelak et al. 2001)). Results (
The photosensitization of components of DMEM has also been demonstrated upon exposure to light, with riboflavin shown to produce ROS which is further enhanced by tryptophan, tyrosine, pyridoxine and folic acid (Grzelak et al. 2001). Furthermore, blue light wavelengths are thought to be the most efficient for the photo-decomposition of riboflavin and generation of ROS (Cheng et al. 2015). To investigate this, riboflavin was added to MM with and without tyrosine, tryptophan, pyridoxine and folic acid in the same concentrations found in DMEM (Table 2 above). Results support this with only 1.3 Log10 reduction when only riboflavin was present, however when all components were present enhanced inactivation of FCV was achieved with complete inactivation of a 105 PFU mL−1 population.
It is important to consider how light-induced inactivation would be influenced when viral particles were suspended in more biologically-relevant, naturally occurring matrices such as body fluids or secretions. As artificial saliva and artificial faeces can be prepared, these were used alongside human plasma, as model human secretions in which many viruses can be transmitted (Aitken & Jeffries, 2001).
The disclosed technology can also provide for beneficial application of 405 nm light for the decontamination of air, surfaces and equipment in healthcare settings, as well as in other indoor locations, where transmission of viral pathogens is a significant occurrence.
For purposes of this description, certain aspects, advantages, and novel features of the embodiments of this disclosure are described herein. The disclosed methods, apparatuses, and systems should not be construed as limiting in any way. Instead, the present disclosure is directed toward all novel and nonobvious features and aspects of the various disclosed embodiments, alone and in various combinations and sub-combinations with one another. The methods, apparatuses, and systems are not limited to any specific aspect or feature or combination thereof, nor do the disclosed embodiments require that any one or more specific advantages be present or problems be solved.
Integers, characteristics, materials, and other features described in conjunction with a particular aspect, embodiment, or example of the disclosed technology are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
Although the operations of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed methods can be used in conjunction with other methods.
As used herein, the terms “a”, “an”, and “at least one” encompass one or more of the specified element. That is, if two of a particular element are present, one of these elements is also present and thus “an” element is present. The terms “a plurality of” and “plural” mean two or more of the specified element. As used herein, the term “and/or” used between the last two of a list of elements means any one or more of the listed elements. For example, the phrase “A, B, and/or C” means “A”, “B,”, “C”, “A and B”, “A and C”, “B and C”, or “A, B, and C.” As used herein, the term “coupled” generally means physically, magnetically, electrically, wirelessly, or otherwise coupled or linked together and does not exclude the presence of intermediate elements between the coupled items absent specific contrary language.
In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is at least as broad as the following claims. We therefore claim at least all that comes within the scope of these claims.
1. An apparatus for inactivating pathogens in ex vivo stored blood products, comprising:
- a treatment chamber configured to receive at least one sealed container storing a blood product; and
- a light source operable to emit light that is directed to a blood product storage container in the treatment chamber, the emitted light having a wavelength and intensity sufficient to penetrate through a wall of the container and into the blood product, such that the irradiance of the light reaching the blood product inside the wall of the container is sufficient to inactivate pathogens in the blood product without the presence of an added photosensitizing agent in the blood product and without a detrimental effect on the blood product such that the blood product is suitable for future medical use.
2. The apparatus of claim 1, wherein at least 50% of the light emitted by the light source has a wavelength in a range from 380 nm to 500 nm.
3. The apparatus of claim 1, wherein at least 50% of the light emitted by the light source has a wavelength in a range from 400 nm to 420 nm.
4. The apparatus of claim 1, wherein the light emitted by the light source has a wavelength spectrum centered on or around about 405 nm.
5. The apparatus of claim 1, wherein the light reaching the blood product has a wavelength and intensity sufficient to inactivate bacteria, viruses, and fungi present in an ex vivo blood product stored in a sealed container.
6. The apparatus of claim 1, further comprising an agitation mechanism positioned within or coupled to the treatment chamber and configured to agitate a blood product stored in a container within the treatment chamber while the blood product is irradiated with light.
7. The apparatus of claim 6, wherein the agitation mechanism comprises a oscillating or vibrating tray on which a blood product storage container rests during treatment.
8. The apparatus of claim 6, wherein the apparatus comprises at least two independent agitation mechanisms.
9. The apparatus of claim 6, wherein the agitation mechanism is operable to oscillate a blood product storage container at from 30 to 100 reciprocations per minute during treatment.
10. The apparatus of claim 1, wherein the light source comprises at least a first light source and a second light source positioned on opposing walls of the treatment chamber, the first light source being positioned to direct light at a first surface of a blood product storage container, and the second light source being configured to direct light at a second surface of the blood product storage container that is opposite from the first surface.
11. The apparatus of claim 1, wherein the apparatus comprises a temperature control system operable to maintain the air temperature within the treatment chamber within a predetermined range during treatment.
12. The apparatus of claim 1, wherein walls of the treatment chamber comprise reflective surfaces configured to reflect light having wavelengths between 380 nm and 500 nm.
13. The apparatus of claim 1, further comprising a lens configured to focus or direct light from the light source to a particular region of the treatment chamber.
14. The apparatus of claim 1, further comprising a diffuser configured to diffuse light from the light source or blend light from more than one light source.
15. The apparatus of claim 1, wherein the light source is operable to emit light with an intensity low enough that light reaching a blood product has an irradiance of 3 mW/cm2 or less.
16. The apparatus of claim 1, wherein the light source is operable to emit light with an intensity high enough that light reaching a blood product has an irradiance of 80 mW/cm2 or greater.
17. The apparatus of claim 1, wherein the light source is operable to emit light with an intensity such that light reaching a blood has an irradiance in a range from 3 mW/cm2 to 80 mW/cm2.
18. The apparatus of claim 1, further comprising a control system operable to vary the intensity of light emitted from the light source during treatment.
19. The apparatus of claim 1, wherein the treatment chamber comprises a hanging mechanism for holding blood product storage containers in a suspended or hanging orientation during treatment.
20. The apparatus of claim 1, further comprising a heat sink or a cooling fan arranged to dissipate heat produced by the light source during treatment.
21. A method for inactivating pathogens in ex vivo stored blood products, comprising:
- providing a sealed storage container containing an ex vivo blood product without an added photosensitizing agent present in the blood product, the storage container comprising at least one wall that is at least partially transmissive to light having wavelengths from 380 nm to 500 nm; and
- irradiating at least one wall of the sealed storage container with light of sufficient intensity and wavelength to penetrate through the at least one wall of the sealed storage container and enter into the blood product, and doing so for sufficient duration to inactivate pathogens in the blood product without causing a substantial detrimental effect on the blood product such that the blood product is suitable for future medical use.
22. The method of claim 21, wherein storage container is a blood product transfusion bag.
23. The method of claim 21, wherein the storage container comprises PVC.
24. The method of claim 21, wherein at least 50% of the light emitted by the light source has a wavelength in a range from 380 nm to 500 nm.
25. The method of claim 21, wherein at least 50% of the light emitted by the light source has a wavelength in a range from 380 nm to 420 nm.
26. The method of claim 21, wherein the light emitted by the light source has a wavelength spectrum centered on or around about 405 nm.
27. The method of claim 21, wherein the light reaching the blood product has an irradiance of from 3 mW/cm2 to 80 mW/cm2.
28. The method of claim 21, wherein the light reaching the blood product has an irradiance of 3 mW/cm2 or less.
29. The method of claim 21, wherein the light reaching the blood product has an irradiance of 80 mW/cm2 or greater.
30. The method of claim 21, further comprising agitating the storage container during the irradiation such that the contained blood product moves within the storage container.
31. The method of claim 30, wherein agitating comprising causing the storage container to reciprocate at from 30 to 100 reciprocations per minute.
32. The method of claim 21, wherein the irradiating comprises adjusting the intensity of the light emitted by the light source during the treatment.
33. The method of claim 21, wherein the irradiating comprises irradiating the storage container from at least two different directions simultaneously.
34. The method of claim 21, further comprising circulating air around the storage container during the irradiating to maintain a desired temperature range for the storage container and the blood product.
35. The method of claim 21, wherein the irradiating has a duration of at least one hour.
36. The method of claim 21, wherein the irradiating has a duration of at least two hours.
37. A method comprising using light having a wavelength of between 380 nm and 500 nm for deactivating pathogens in stored blood products.
38. The method of claim 37, wherein deactivating pathogens in stored blood produces comprises deactivating virus contamination in stored blood products.
Filed: Sep 29, 2016
Publication Date: Mar 7, 2019
Applicants: The US of America, as represented by the Secretary Dept. of Health and Human Servics (Bethesda, MD), The University of Strathclyde (Glasgow, Scotland)
Inventors: Chintamani Atreya (Bethesda, MD), Michelle Maclean (Renfrewshire, Scotland), John G. Anderson (Glasgow, Scotland), Scott J. MacGregor (Glasgow, Scotland)
Application Number: 15/765,424