Tackling Sepsis after transfusions

UV-LED at 265 nm can potentially disinfect human platelet concentrates

Hayashi et al., 2021


Platelets are essential for stopping bleeding under low or high flow rates through surface molecules, including glycoproteins and released molecules in their granules. One way to stop bleeding in thrombocytopenic patients (with fewer platelets) is through platelet concentrates (PCs) transfusion. PCs can also prevent thrombocytopenia in patients receiving chemotherapy.


Most laboratories store PCs at room temperature (20-24˚C) under continuous agitation. But these storage conditions often allow bacterial contamination. Cold can prolong storage duration. However, cold-stored platelets survive for a shorter time after transfusion. Because of that, the shelf-life of PC products is relatively short, between 3 to 5 days.


Thus, it’s critical to find strategies to prevent PCs contamination and avoid sepsis after transfusion. The most widespread are culture-based detection of bacteria and pathogen inactivation procedures. Culture-based bacteria inactivation is highly sensitive, but it’s not perfect since bacteria grow during storage even with a low count. Pathogen inactivation techniques typically use UVA, UVB, or UVC, which can affect platelets in different ways.

Thus, researchers at the Japanese Red Cross Kinki Block Blood Centre, Japan, investigated the application of UV-LED at 265 nm as a novel disinfection method for platelet transfusion safety.


Study Overview

Platelet concentrates

The researchers obtained the PCs from healthy volunteers’ blood. They kept it at 22˚C with agitation before use.


Bacterial inoculation

The investigators cultured bacteria (E.coli, S.aureus, and B cereus) and suspended them in a saline solution, diluted to a predetermined bacterial concentration. After that, they spiked an aliquot of the suspension to 10 mL of PC.


UV-LED irradiation

Next, the researchers placed approximately 2,750µL of the PC-bacteria mixture in a petri dish to obtain a sample depth of 5mm. Then, they irradiated it with a UV-LED setup containing 8 UV-LED units of 3.5 mm square with a peak emission wavelength of 265 nm, a heat sink, and a cooling fan (Figure 1A and 1B). The incident irradiance at the surface of the mixture was 1.05 mW/cm2.

Fig 1. from Hayashi et al., 2021: UV-LED irradiation setup. (a) Samples were placed in a siliconized quartz glass dish (28-mm inner diameter) with a stir bar. Eight UV-LED units (265 nm) were placed on the board face down for irradiation; the distance between the UV-LED and the sample surface before sampling was 17.7 mm. (b) The emission spectrum of the UV-LED used in this study.

The investigators removed 100 uL samples from the dish every 5 or 10 minutes. These samples allowed them to measure platelet counts, glycoprotein surface expression., agonist-induced platelet aggregation, and platelet adherence and aggregation under flow conditions.


Platelet aggregation and adherence under flow conditions

For this experiment, the researchers loaded 50μL of collagen on a Vena8 Fluoro+ microfluidic biochip and incubated it at 37˚C for one hour. Next, they diluted the platelet samples to a concentration of 5.0 × 10^4/μL. The sample was then pumped through the collagen-coated biochip at a constant flow rate of 3.2 or 32 μL/min. They determined the surface coverage using a fluorescence microscope with imaging software.



Results

Platelet adhesion and aggregation to collagen under flow after UV-LED irradiation

The group analysed platelet function under two flow conditions, 100 s-1, and 1,000 s-1. They measured the percentage of fluorescence coverage, which reflects both platelet adhesion and aggregation at a distance of 8 mm from the input port of the microchannels of the Vena8 Fluoro+ biochips (Fig 6A).


There was no difference between the two treatment groups in platelet adhesion to collagen and aggregation under the two flow conditions (Fig 6B). So, these functions were maintained with or without 30 minutes of irradiation.

Fig 6. from Hayashi et al., 2021: Effect of UV-LED irradiation on adhesion and aggregation to collagen under flow conditions. (a) Representative images from six independent experiments, Non-irradiated (UV-LED (−)) and UV-LED-irradiated (UV-LED (+)) platelets. (b) Six independent experiments of control (non-irradiated, (−)) and UV-LED-irradiated (+) samples are displayed as open and closed circles, respectively. Mean values ± SD are shown as one long and two short horizontal bars.

Other experiments in this study:

The main findings of this study were:

  • UV-LED irradiation produced about 1 log (90%) inactivation of E-coli after 5 minutes. About 10 minutes of irradiation produced the same result in S. aureus while B. cereus needed 20 minutes to reach the limit of detection.

  • Without irradiation, platelet counts remained stable for 30 minutes. With UV-LED irradiation, platelet counts were stable for 10 minutes, gradually decreasing after that (18 ± 7% mean ± SD, n = 7).

  • Platelet aggregates were only visible after 60 minutes of irradiation.

  • The platelet surface markers, CD42b, and CD61 remained stable after UV-LED irradiation.

  • There were no changes in platelet activation with UV-LED irradiation. However, collagen-induced platelet aggregation slightly increased after 30 minutes of irradiation. The ADP-induced aggregation pattern of UV-LED irradiated platelets was comparable with that of platelets treated with a short wavelength UVC.

Conclusion

The authors of the study concluded that the 265 nm UV-LED has high potential as a new disinfection method to ensure the microbial safety of platelet transfusion.


To see more details about the experiments, read the full study.


Would you like to run similar experiments in your lab and don’t know how to start? This is what you will need:

  • Vena8 Fluoro+ biochip – to mimic human blood vessels and model blood clots, see further details below.

  • Mirus Evo pump – to control shear rates (flow rates) in the biochip; this enables you to set the shear rate at a setting which models flow rates for thrombosis in micropillaries or other vessels.

  • Microenvironmental chamber – this is a temperature-controlled frame, the biochip sits in this and it keeps everything at 370C. The microenvironmental chamber sits on the microscope stage.

  • Inverted microscope – we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option.

  • Digital camera – to capture images and video recordings. We supply the Prime BSI Express with both the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies.

  • Image Pro Cell Analysis software – to analyse the images and videos from your experiments.

If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit. Our options suit all budgets. Take a look at them on our eShop.


References

Hayashi, T., Oguma, K., Fujimura, Y., Furuta, R. A., Tanaka, M., Masaki, M., ... & Takahashi, K. (2021). UV light-emitting diode (UV-LED) at 265 nm as a potential light source for disinfecting human platelet concentrates. Plos one, 16(5), e0251650.




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