Riboflavin and UV light can reduce SARS-CoV-2 virus in plasma and whole blood


Scientists do not yet know if Severe Acute Respiratory Syndrome Coronavirus 2, or SARS-CoV-2—the virus that causes COVID-19 – can be transmitted by blood transfusion.

But given the unknowns around this new pathogen, researchers at Colorado State University used existing technologies to show that exposing the coronavirus to riboflavin and ultraviolet light reduces pathogens in human plasma and whole-blood products.

The study, “Pathogen reduction of SARS-CoV-2 virus in plasma and whole blood using riboflavin and UV light,” was published May 29 in PLOS ONE.

Dr. Izabela Ragan, postdoctoral fellow in the Department of Biomedical Sciences at CSU, said the research team tackled one of the big questions about the novel coronavirus:

If the pathogen can spread through blood or by donating blood, would it be possible to kill the virus?

“The research we conducted answers that question: yes, you can,” said Ragan. “We eliminated a huge amount of virus and we could not detect the virus post-treatment.”

The research team used the Mirasol Pathogen Reduction Technology System to treat nine plasma and three whole-blood products for the study.

The technology – which is owned by medical device company Terumo BCT – was invented by Ray Goodrich, senior author of the study and executive director of the Infectious Disease Research Center at CSU. He is also a professor in the Department of Microbiology, Immunology and Pathology.

Simple process

Dr. Heather Pidcoke, a co-author on the study and chief medical research officer at CSU, said the process used by the research team is quite simple.

The blood product or plasma is placed in a specially designed storage bag, riboflavin solution is added, and the mixture is then exposed to UV light. The Mirasol PRT device gently shakes the bag to circulate the blood cells, so the cells come to the surface where they are exposed to the UV light.

The authors caution that this is not an experiment to try at home. The light does not penetrate the entire bag, so it’s not the same as exposing body parts to UV light.

Goodrich said the research may help to avoid what happened in the 1980s, when HIV was transmitted through blood and blood products while scientists were still trying to isolate and identify what might be causing the spread of the virus.

However, he noted that the Mirasol system is currently only approved for use outside of the United States, mainly in Europe, the Middle East and Africa.

“Our research may help inform people outside the U.S who are using it,” he said. “They may breathe a sigh of relief knowing that while we continue to study this, there is some potential mitigation in place just in case.”

Studying transmission question

CSU researchers are currently studying whether SARS-CoV-2 is transmitted by blood. Ragan said they hope to answer that question very soon.


Due to a combination of factors including rapidly mutating viral strains, increasingly close animal-human contacts, and ever burgeoning rates of travel and urbanization, the rate at which new human pathogens emerge and spread globally appears to be rising over the last 80 years [1].

Climate change is likely to accelerate pandemic emergence because temperate zones encompass a larger area of the globe, expanding vector territories and favoring bacterial pathogens [2,3]. Mass gatherings and higher Basic Reproduction Numbers further contribute to rapid dissemination around the globe [4,5].

The emergence of Coronavirus Disease 2019 (COVID-19), whose causative agent is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is only the latest example of the speed with which a pathogen can travel around the globe causing successive waves of outbreaks [5].

Despite the recent emergence of this pandemic, community spread of COVID-19 is well recognized but transfusion transmission has yet to be reported [4,6]. In the early days of the pandemic experts debated whether asymptomatic transmission was possible, a necessary precondition for transmission through transfusion given rigorous donor screening practices [7].

That question; however, is no longer debated as the degree of community transmission in Italy and now New York have accelerated despite symptomatic screening. COVID-19 is characterized by viral shedding which starts during an initial asymptomatic phase that can last more than 14 days, followed by active disease and post-resolution viral shedding that can persist for up to 37 days [7].

Furthermore, findings of viral RNA in multiple bodily fluids tested including blood raises considerable concern regarding the safety of convalescent plasma [8]. From initial reports in patients from Wuhan, China, viremia was found in 6/41 (15%) patients. The median PCR cycle threshold value was 35.1 (95% CI: 34.7–35.1), suggesting a very low RNA concentration in the range between 102 to 103 copies per mL [8,9].

Significantly, no difference was observed between patients with severe disease and patients with mild symptoms. Several of these reports have indicated viremia in asymptomatic patients. This may pose particular risk in blood donation due to the potential to escape health screening during donation. In one study, viral RNA was detected in the blood of 96.8% of affected patients [10].

While SARS-CoV, the causative agent of the Severe Acute Respiratory Syndrome (SARS) outbreak of 2002 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) have not been documented to cause transfusion-transmitted disease, those pathogens resulted in higher mortality, but lower infectivity due to a lower binding efficiency with the angiotensin-converting enzyme 2 (ACE2) and dipeptidyl peptidase 4 (DPP4) receptors, respectively.

By contrast, SARS-CoV-2 binding strength to the ACE2 receptor is higher, suggesting a cause for the greater propensity for human to human transmission [11,12]. Observations regarding the absence of documented transfusion transmission of SARS and MERS may not be a good indication of whether COVID-19 poses a threat to the blood supply.

Pathogen reduction with riboflavin and ultraviolet light (R+UV PRT) has demonstrated excellent activity against MERS CoV, suggesting that R+UV PRT could be protective against the possibility of transfusion transmission of SARS-CoV-2 [13]. In this study, we used infectivity assays to test the efficacy of R+UV PRT to reduce the level of infectious SARS-CoV-2 inoculates in plasma and whole blood.

Materials and methods

All human blood products used in this study were obtained via informed consent at an accredited blood banking institution. The protocol was approved by an Institutional Review Board (Advarra, 6940 Columbia Gateway Drive, Suite 110, Columbia, Maryland 21046) at Vitalant (717 Yosemite St., Denver, Colorado, The United States), the blood collection location and the Colorado State University Institutional Review Board which is part of the Research Integrity and Compliance Review Office (RICRO) at Colorado State University, Office of the Vice President of Research. Informed consent was obtained in writing from all donors prior to donation and was witnessed by blood collection staff at Vitalant.

Plasma products

Nine plasma products were collected at an accredited blood bank and shipped to Colorado State University on dry ice. The products were classified as PF24, prepared from whole blood products collected in Citrate Phosphate Double Dextrose (CP2D) and frozen within 24 hours after phlebotomy to ≤ -20°C.

Products were thawed in a water bath at 36°C and pooled in sets of 3 by ABO type to create 3 unique pools. We utilized a pooling method in order to reduce donor variability that might affect the overall outcomes observed in the assays. This was performed for two distinct volumes of plasma with average of 170 mLs and average of 250 mLs each.

Whole blood products

Three non-leukoreduced whole blood (WB) products were collected in Citrate Phosphate Dextrose (CPD) anticoagulant at an accredited blood bank and shipped to Colorado State University at room temperature.

SARS-CoV-2 propagation

Vero cells (CCL-81) were obtained originally from the International Reagent Resource and frozen stocks prepared. Those stocks screened negative for mycoplasma contamination and were used between total passage level 35 and 41.

Virus (isolate USA-WA1/2020) was acquired through BEI Resources (product NR-52281) and amplified in Vero C1008 (Vero E6) cell culture. Vero E6 cells (ATCC CRL-1568) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with glucose, L-glutamine, sodium pyruvate, 5% fetal bovine serum (FBS) and antibiotics. Inoculation of Vero E6 cells with SARS-CoV-2 was carried out directly in DMEM containing (1%) FBS. The identity of the virus as SARS-CoV-2 was established by shotgun sequencing and confirmation with a database on sequence data for the virus.

Medium harvested from infected cells 3–4 days after inoculation was clarified by centrifugation, supplemented with FBS to 10% and frozen to -80°C in aliquots. All virus propagation occurred in a BSL-3 laboratory setting. We measured all values for virus concentration using a standard plaque assay. Results are presented in number of infectious virus (plaque forming units per mL).

Pathogen reduction process


After pooling, each pool was divided in 3 equal volumes of 175 mL dispensed into an illumination bag (Mirasol Illumination Bag, Terumo BCT, Lakewood, CO). Riboflavin solution (35 mL, 500 μmol/L: P/N 777702–140, Lot 19BCT5T01) was added to each product, followed by inoculation with 5 mL SARS-CoV-2 virus, and the bags were placed into the Illuminator (Mirasol PRT System, Terumo BCT, Lakewood, CO) for treatment with UV light.

The products in each set of 3 were treated to either 30%, 60%, or 100% of the total recommended light dose, calculated based on the volume of each product (a full treatment consists of exposure to 6.24 J/mL UV light).

Average time of treatment for these products was 4 minutes. Samples were removed from each product pre- and post-illumination for viral titer determination via plaque assay. All processing occurred in a BSL-3 laboratory setting.

Whole blood.

Each WB product was transferred to an illumination bag per the manufacturer’s instructions. All solution and disposables were obtained from the manufacturer (Terumo BCT, Lakewood, Colorado, The United States of America).

Riboflavin solution (35 mL, 500 μmol/L) was added to each product, followed by inoculation with 20 mL SARS-CoV-2 virus, and the bags were placed into the illuminator for treatment with UV light -calculated using the measured hematocrit and volume of each product—to a dose of 80 J/mL RBC.

Average time of treatment for these samples was 52 minutes. Samples were removed from each product pre- and post-illumination for viral titer determination via plaque assay. All processing occurred in a BSL-3 laboratory setting.

Viral plaque assay

All pre- and post- illumination samples were serially diluted in sterile PBS. Plaque assays were performed using Vero E6 cells at confluency in 6-well cell culture plates. Briefly, plates were washed with sterile PBS. All samples were then plated in duplicates at 100 μL per well. Plates were incubated at 37°C for 45 minutes with occasional rocking.

Then 2 mL of 0.5% agarose in minimal essential media (MEM) containing 2% FBS and antibiotics was added per well. Plates were incubated at 37°C for 72 hours. The cells were fixed with 10% buffered formalin, followed by the removal of the overlay, and then stained with 0.2% crystal violet to visualize plaque forming units (PFU). All assays were performed in BSL-3 laboratory setting. Fig 1.

Fig 1. SARS-CoV-2 cultures and plaque assay for titer determinations.
A) Left- Vero cells at confluency, uninfected; Right- SARS-CoV-2 infection in Vero cells. 3 days after inoculation. CPE present. Cells at 40x magnification. Scale bar = 1mm. B) Plaque assay results from SARS-CoV-2 in media with R + UV treatment. Top left and right- virus titration, pre-pathogen reduction; Bottom left- pathogen reduction at 50J; Bottom right pathogen reduction at 100J.

Calculation of limit of detection and log reduction

When the posttreatment samples were negative for the presence of virus, the limit of detection had been reached. All values at the limit of detection were considered less than or equal to the calculated limit of detection. The theoretical limit of detection and overall log reduction was calculated using the following equations:


where N is the number of replicates per sample at the lowest dilution tested; V is the volume used for viral enumeration (volume inoculated/ well in mL). No cytotoxicity was observed at the zero dilution, hence all replicates for determination of LOD and final titer were done at zero dilution with 10 replicates using 0.1 mL per replicate well.


Pathogen reduction with riboflavin and UV light results in high levels of reduction of SAR-CoV-2 infectivity in inoculated plasma and whole blood. While the infectivity of blood from COVID-19 patients and its transmissibility via transfusion is not proven, the presence of viral RNA in blood is of concern and rapidly expanding community spread combined with a long asymptomatic latent period is limiting the ability of blood centers to identify who should be deferred. The rapid rise of cases suggests the US is still in the exponential phase of pandemic. Pathogen reduction could be a viable strategy to protect the blood supply during the COVID-19 pandemic.


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Journal information:PLoS ONE


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