Bacterial contamination of injectable stored biological fluids such as blood plasma and platelet concentrates preserved in plasma at room temperature is a major health risk. Current pathogen reduction technologies (PRT) rely on the use of chemicals and/or ultraviolet light, which affects product quality and can be associated with adverse events in recipients. 405 nm violet-blue light is antibacterial without the use of photosensitizers and can be applied at levels safe for human exposure, making it of potential interest for decontamination of biological fluids such as plasma. As a pilot study to test whether 405 nm light is capable of inactivating bacteria in biological fluids, rabbit plasma and human plasma were seeded with bacteria and treated with a 405 nm light emitting diode (LED) exposure system (patent pending). Inactivation was achieved in all tested samples, ranging from low volumes to prebagged plasma. 99.9% reduction of low density bacterial populations (≤103 CFU mL−1), selected to represent typical “natural” contamination levels, was achieved using doses of 144 Jcm−2. The penetrability of 405 nm light, permitting decontamination of prebagged plasma, and the nonrequirement for photosensitizing agents provide a new proof of concept in bacterial reduction in biological fluids, especially injectable fluids relevant to transfusion medicine.
Bacterial contamination of
A number of bacterial reduction methods have been developed for plasma treatment, and pathogen reduced plasma is routinely used [
It is generally accepted that all these methods have limitations [
Here, we report the first proof-of-concept results on the use of a novel visible violet-blue light method that does not require the addition of photosensitive chemicals for inactivation of bacterial pathogens in plasma. This method utilises light with a peak wavelength of 405 nm, which causes photoexcitation of endogenous microbial porphyrin molecules and oxidative damage through reactive oxygen species [
The organisms used in this study were
Lyophilised rabbit plasma (LRP020, E&O Laboratories, UK) was reconstituted using sterile distilled water. Fresh frozen human plasma (approximately 300 mL bag volume) was obtained from the Scottish National Blood Transfusion Service (SNBTS, UK) and defrosted before experimental use. Study involving human subjects protocol was approved by FDA Risk Involved in Human Subjects Committee (RIHSC, Exemption Approval # 11-036B) and by the University of Strathclyde Ethics Committee (letter dated 10 February 2011). Rabbit plasma and human plasma suspensions were seeded with known concentrations of bacterial contaminants by adding bacterial-PBS suspension to the plasma.
The 405 nm light sources used were rectangular arrays of 99 LEDs in an 11 × 9 matrix (Opto Diode Corp., USA). The array had a centre wavelength close to 405 nm, with a bandwidth of approximately 10 nm at full width at half maximum (FWHM). The LED array was powered by a direct current supply, and, for thermal management, the LED array was bonded to a heat sink and fan, thus ensuring that heating had no effect on the test samples exposed to the 405 nm light (device patent pending [
Three arrangements were employed for exposure of three different sample volumes: 3 mL, 30 mL, and approximately 300 mL (whole plasma transfusion bags). For exposure of 3 mL sample volumes, the samples were held in the well of a 12-well microplate (without the lid), and the LED array was mounted in a polyvinyl chloride (PVC) housing which positioned the array approx. 3 cm directly above the sample. Irradiance at the sample surface was measured to be approximately 100 mWcm−2 (measured by using a radiant power meter and photodiode detector; LOT-Oriel Ltd.).
For exposure of 30 mL sample volumes, the human plasma was held in a sterile 90 mm Petri dish with the lid on. The LED array was positioned 8 cm directly above the closed sample dish, providing irradiance of approximately 8 mWcm−2, through the lid, at the centre of the sample dish.
For exposure of plasma bags, a test rig was constructed which held two 405 nm LED arrays at a distance of 12 cm above the horizontally positioned plasma bag. This arrangement provided irradiance of approximately 5 mWcm−2 at the centre position of the plasma bag, taking into account a 20% reduction in irradiance as the light transmits through the bag layer. In order to investigate the influence of higher irradiance on bacterial inactivation, plasma bags were also exposed using irradiance of 16 and 48 mWcm−2. This higher irradiance was achieved by using two high-power 405 nm LED arrays (PhotonStar Technology, UK), with 14 nm FWHM.
All experimental systems were held in a shaking incubator (72 rpm; 25°C) to allow continuous sample agitation and maintain exposure conditions. Samples seeded with bacterial contamination were treated with increasing exposures of 405 nm light. Control samples were held in identical conditions but shielded from the 405 nm light.
The optical profiles of the light distribution across the Petri dishes and transfusion bags (plotted using MATLAB R2012b software) demonstrate the nonuniform irradiance of the plasma (Figures
To ensure that bacterial inactivation was not the result of the plasma becoming toxic upon exposure to 405 nm light,
Following 405 nm light exposure, samples were either plated onto nutrient agar using an automatic spiral plater (Don Whitley Scientific, UK) or manually spread by using sterile L-shaped spreaders, depending on the expected population density of the samples. Sample plates were incubated at 37°C for 24 hours and then enumerated with the surviving bacterial load reported as colony-forming units per millilitre (CFU mL−1).
Results are reported as surviving bacterial load (log10 CFU mL−1) as a function of dose and are presented as mean values from triplicate independent experiments (
The transmission values for rabbit plasma and human plasma, PBS, and the blood bag material were measured by using a BioMate 5 UV-Visible Spectrophotometer (Thermo Spectronic). Analysis was carried out in the wavelength range of 220–700 nm. Fluorescence spectrophotometry (RF-5301 PC spectrofluorophotometre; Shimadzu, US) was used to determine whether plasma or PBS contained photosensitive components which could be excited by 405 nm light. Excitation was carried out at 405 nm and emission spectra were recorded between 500 and 700 nm.
Results from the exposure of PBS, rabbit plasma, and human plasma seeded with bacterial contamination (105 CFU mL−1) to 100 mWcm−2 405 nm light are presented in Figure
Inactivation of bacterial contamination: (a)
Inactivation of
405 nm light exposure of contaminated human plasma transfusion bags. (a) Three-dimensional model demonstrating the irradiance profile across the plasma bag, with irradiance of ~5 mWcm−2 at the centre. (b) Inactivation of
Similar inactivation kinetics was observed for
No significant change in the seeded 103 CFU mL−1 population [
Figure
Inactivation of low density (101–102 CFU mL−1) bacterial contaminants within plasma transfusion bags was achieved using irradiance as low as 5 mWcm−2 (Figure
Comparison of the exposure times (a) and doses (b) required for inactivation of
Spectrophotometric analysis shows that transmission of 405 nm light through plasma is low (1-2%) compared with transparent PBS (99%), and this correlates with the longer exposure times/increased doses required for comparative microbial inactivation in plasma compared to PBS. Figure
Optical analysis. (a) Transmission properties of the Petri dish and blood bag material, highlighting 405 nm and UV-C light wavelengths for reference. (b) Fluorescence emission spectra of PBS and plasma (500–700 nm), detected using an excitation wavelength of 405 nm.
In order to assess the potential of 405 nm light for decontamination of blood plasma, the penetrability and antimicrobial efficacy of 405 nm light in plasma required evaluation, and the aim of this study was to determine the antibacterial effects of 405 nm light at varying irradiance on bacteria seeded in blood plasma ranging from small volume samples up to prebagged plasma.
Initial investigation of the inactivation of bacterial contaminants in low volume (3 mL) plasma samples using 100 mWcm−2 405 nm light demonstrated that successful inactivation could be achieved in both rabbit plasma and human plasma. Significantly greater doses were required for inactivation of bacterial contaminants when being suspended in plasma compared to PBS, and this is accredited to the differing optical properties of these suspending media. The opacity, and consequent low transmissibility of plasma (Figure
Despite the optical transmission properties of rabbit plasma and human plasma being relatively similar, slight differences were recorded between the susceptibilities of the bacterial contaminants when seeded in these media. This is likely due to the batch-to-batch variation in color and opacity of the rabbit plasma and, in particular, the human plasma. Indeed, optical analysis of a number of human plasma bag samples (
The bacterial species used in this study were selected to represent significant contaminants associated with blood components [
The initial exposure tests in this study to establish proof of principle utilised low volumes of plasma seeded with high population densities of bacterial contaminants at a level of 105 CFU mL−1. A more realistic scenario involves larger volumes of plasma contaminated with low microbial densities. Indeed, it has been reported that the levels of naturally occurring bacterial contamination in plasma are likely to be as low as 10–100 bacterial cells per product at the beginning of storage [
In addition to demonstrating efficacy when applied to larger volumes of plasma, these experiments highlighted that the 405 nm light disinfection effect can be achieved through transparent packaging. A similar effect was reported in a recent study which highlighted the ability of 405 nm light to decontaminate biofilms on the underside of transparent materials [
Published studies have identified microbial endogenous porphyrin molecules as the key photosensitive targets which initiate the lethal oxidative damage exerted by 405 nm and other violet light wavelengths [
The 405 nm light doses required in this study for the decontamination of blood plasma have been in the region of 158 Jcm−2 and above. These doses are relatively high compared to those typically required for other light-based methods, and this is due to the higher germicidal efficacy of UV light compared to 405 nm light [
Due to the absence of cells, solvent/detergent treatment, methylene blue and visible light, amotosalen and UV-A light, riboflavin and UV, and UV-C light are generally accepted as being suitable for plasma decontamination. This study has generated significant evidence of the efficacy of 405 nm light for decontamination of blood plasma as a model system to study injectable biological fluids. Since person-to-person variation in the activity of plasma proteins in healthy individuals is known to be significant, any loss in plasma integrity due to 405 nm light treatment is unlikely to have noticeable clinical impact. Further, since violet-blue light (405 nm) is relatively safer compared to already accepted UV light-based methods [
Overall, this study provides the first evidence that 405 nm light has the ability to inactivate bacterial contamination within biological fluids such as blood plasma. Significant inactivation of microbial contaminants was achieved in plasma samples of varying volumes held in different containers including prebagged plasma. The penetrability of 405 nm light and the nonrequirement for photosensitizing agents provide this antimicrobial method with unique benefits that could support its further development as a potential alternative to UV light-based systems. Further work is, however, required not only to extend the microbiological data but also to investigate the compatibility of 405 nm light with plasma components before its potential for plasma decontamination can be fully assessed. Although this study has focused on the antimicrobial effects of 405 nm light for the decontamination of plasma, it will also be of interest to establish whether bacterial reductions can be achieved in platelets stored
The views expressed in this article are an informal communication and represent the authors own best judgment. These comments do not bind or obligate FDA.
The authors declare that there are no competing interests regarding the publication of this paper. The authors have filed a joint US device patent application.
This work was supported by US FDA funding to Chintamani D. Atreya, with experimental work conducted at ROLEST through a collaborative research contract. The authors would like to thank J. Gillespie and HVT technical staff (Department of Electronic & Electrical Engineering (EEE), University of Strathclyde) for technical support with Matlab software and system construction, respectively, and Professor M. H. Grant, K. Henderson, and K. McKenzie for access and technical support with the fluorescence spectrophotometry (Departments of Biomedical Engineering and EEE, University of Strathclyde). The authors would also like to thank the Scottish National Blood Transfusion Service (SNBTS) for provision of blood components.