It is essential that a pathogen reduction treatment is capable of broad-spectrum antimicrobial efficacy and that these antimicrobial doses do not significantly impact the safety, quality, or effectiveness of the treated blood components. Results demonstrating the compatibility of 405-nm light as a microbicidal treatment for prebagged human plasma are described for the first time in this paper, with demonstration of the ability to reduce contamination in prebagged plasma up to volumes of 300 mL, and a range of proteomics tests assessing the stability and functionality of 405-nm light treated plasma. With microbial contamination being an infectious risk of ex vivo human platelet concentrates (PCs) stored in plasma at room temperature, this pilot study provides strong evidence of the broad-spectrum antibacterial efficacy for advancing to trials utilising large volume platelet concentrates.

Preliminary compatibility studies in an earlier study indicated that low volume (250 µL) human plasma samples treated with an effective microbicidal dose of 360 J cm−2 showed no signs of protein degradation via gel electrophoresis analysis and advanced oxidation protein products (AOPP) assay (Stewart et al. 2020). In the present, significantly scaled-up study, further positive results were obtained, with no signs of oxidative protein damage detected via the AOPP assay in prebagged 300-mL volumes of human plasma treated with light doses up to 403 J cm−2. It was expected that post-treatment fibrinogen levels would be unaffected since AOPP levels are a strong indicator for the stability of fibrinogen (Selmeci et al. 2006). Results from the fibrinogen ELISA confirmed this, with no significant changes in fibrinogen levels detected following exposure to doses up to 403 J cm−2. From these results, it appears that visible 405 nm light has a less adverse effect on the integrity of fibrinogen compared to UV-light, even when over 65× the dose is applied (403 J cm−2 405 nm light versus 6 J cm−2 UV-light), with reports stating that UV-light based PRTs can potentially reduce fibrinogen in human plasma by up to 21% (Hornsey et al. 2009). The level of Protein S, an essential anti-clotting agent, was unaffected following exposure to antimicrobial doses up to 403 J cm−2 at > 94%, is similar to that of plasma treated with commercially available PRTs (Bubinski et al. 2021; Hornsey et al. 2009). The results of this study are also supportive of those in a recent study by Jackson et al (2024) which assessed the activities of a range of other coagulation factors (FV, FVII, FVIII, FIX, FX, FXI) in PCs and platelet poor plasma (PPP) using a dose of 270 J cm−2. The study demonstrated that 405 nm light exposure did not drastically effect activity of coagulation factors in PCs, but in some cases, differences were more notable in PPP, suggesting possible interplay between platelet surface and coagulation factors yields a protective effect on factor functions (Jackson et al 2024).

This study also provides further proof-of-concept results for the broad-spectrum microbicidal efficacy of 405-nm light for treatment of 300 mL prebagged human plasma, with near-complete inactivation (≤ 10 CFU mL−1) of the three representative organisms (Gram+ and − bacteria, and a yeast). Further, a wider panel of organisms tested in 100 mL prebagged volumes (Fig. 2) also demonstrated similar inactivation, supporting results of previous studies using lower volume, 250 µL, plasma samples (Stewart et al. 2020). As the transmissibility of 405-nm light in platelets stored in plasma is within the same region as human plasma, at 0.1–0.3% compared to 0.38–0.70% (based on data from this study and (Maclean et al. 2020)), we envision that similar microbial reductions would be observed in prebagged platelets stored in plasma.

The previous small-scale study by Stewart et al (2020) demonstrated that 405-nm light was capable of inactivating bacteria seeded at a range of densities (101–108 CFU mL−1) in small volume (250 µL) human plasma samples, with a fixed dose of 360 J cm−2 achieving 95.1–100% inactivation across all contamination levels. As naturally occurring levels of bacterial contamination in blood products are typically low, ranging from 10 to 100 cells per unit prior to storage, low level contamination at approximately 103 CFU mL−1 was selected for investigation in the present study (Figs. 2, 3) to reflect a realistic clinical scenario in large prebagged volumes (Hillyer et al. 2003).

Whilst these dose levels are relatively high compared to existing UV-light based technologies (typically delivering doses in the region of 3 J cm−2) treatment by 405 nm light eliminates the need for additive photosensitive agents, which lengthens the processing times required to remove the additive chemicals to reduce the risk of adverse reaction in recipients (Irsch and Lin 2011; Liu and Wang 2021).

To treat or prevent bleeding in patients, it is important to ensure that an antimicrobial treatment of blood transfusion products has little to no effect on the stability and functionality of clotting factors. Prothrombin Time (PTT) and Activated Partial Thromboplastin Time (APTT) tests were used to assess potential changes in coagulation activity in 405-nm light treated human plasma. Analysis of PTT results (Fig. 5a) indicates that 405-nm light has minimal effect on clotting factors involved in the extrinsic (factor VII) and common coagulation (factors I, II, V and X), with no significant differences in time to clot detected between treated and non-treated human plasma following exposure to doses up to 403 J cm−2 (P > 0.05). The time to clot, measured via the APTT assay, was slightly higher in human plasma treated with 405-nm light doses ≥ 345 J cm−2, suggesting that intrinsic clotting factors (factors VIII, IX, XI, and XII) may be more photo-sensitive compared to extrinsic and common coagulation pathway factors. Nevertheless, the overall impact on clotting activity, with a maximum prolongation of 4.3% over the treatment period, is relatively low in comparison to clinically approved, UV-light based PRTs that have shown to prolong clotting times by up to 24% (Hornsey et al. 2009).

In this report, microbial inactivation and compatibility studies were conducted using a fixed 405 nm light treatment using an irradiance of 16 mW cm−2, however previous studies have demonstrated antimicrobial efficacy in human plasma using a range of irradiances between 5 and 100 mW cm−2 (Maclean et al. 2016; Stewart et al 2020). Research has shown that use of lower irradiances is more germicidally energy efficient compared to higher irradiances for pathogen reduction of human plasma and platelets stored in plasma. This is thought to be due to a threshold level of photons that can interact with porphyrins at any one time, referred to as the as the free porphyrin to photon ratio (Maclean et al 2020; Maclean 2016). Whilst an important consideration, the method of dose delivery must be selected in line with the application type, as the irradiance level directly influences the exposure time required to apply an effective antimicrobial dose. It is envisioned that the method of dose delivery may be adjusted to suit the practical application, as per Eq. 1, i.e. utilising higher irradiances (and therefore shorter treatment times) for rapid decontamination pre or post-storage, or lower irradiances to continuously irradiate prebagged plasma or platelets stored in plasma during the inventory period in hospitals. Nevertheless, future work is required to assess how varying the dose delivery regime may impact the compatibility of 405-nm light with human plasma, and platelet concentrates suspended in plasma.

In conclusion, these results indicate that effective antimicrobial light doses up to 403 J cm−2 cause little to no changes to protein stability or in vitro functionality of the factors tested in this study (fibrinogen, Protein S, AOPP, PTT, APTT). This indicates the potential for doses in the region of 270 J cm−2, previously shown to be capable of viral and parasitic inactivation in human plasma (4-log and 9-log reductions respectively), to be applied without comprising plasma quality (Jankowska et al. 2020; Ragupathy et al. 2022). Further, 405-nm light has also shown potential compatibility with human platelets stored in plasma, a more sensitive cellular blood component, using antimicrobial doses up to 288 J cm−2, with the recovery of treated and non-treated platelets shown to be statically similar in a murine model (P > 0.05) (Maclean et al. 2020). This study, together with previous results, provides further evidence supporting the potential compatibility of antimicrobial doses of 405-nm light for treatment of plasma.