Pathogen inactivation technologies represent a shift in blood safety from a reactive approach to a proactive protective strategy. Commercially available technologies demonstrate effective killing of most viruses, bacteria, and parasites and are capable of inactivating passenger leukocytes in blood products. The use of pathogen inactivation causes a decrease in the parameters of products that can be readily measured in laboratory assays but that do not seem to cause any alteration in hemostatic effect of plasma or platelet transfusions. Effort needs to be made to further develop these technologies so that the negative quality impact is ameliorated without reducing the pathogen inactivation effectiveness.
Key points
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Pathogen inactivation technologies (PITs) permit a shift in the blood safety paradigm from reactive to proactive approaches.
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PITs developed for fresh blood components achieve a high degree of pathogen killing as well as inactivation of passenger leukocytes in blood products.
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Although there is some loss of quality using in vitro tests, the clinical performance of treated products is sufficient to warrant acceptance of these first-generation technologies.
Safety of blood products
A major focus in blood transfusion over the past 3 decades has been the improvement of the safety of transfusions with respect to the risk of transfusion-transmitted diseases. Huge amounts of effort and resources have gone into bringing a high level of safety to modern blood products. The approach to increasing the level of blood safety, however, has been reactive. A pathogen is identified and then a test is developed to screen the donor or the blood product for that pathogen. The limitations of a society’s financial resources that can be devoted to blood safety in turn limit the success of this strategy. Although large reductions in the risk of transmission of those pathogens for which blood is screened (eg, HIV, hepatitis B, hepatitis C, and West Nile virus) have been generated, there are other agents that are transmissible in blood for which there currently are not tests readily available or for which implementing testing has not been chosen (eg, Babesia and dengue virus). Because the development of blood screening tests is driven by market forces, it is unlikely that new blood screening tests will become available without the demonstration of a high degree of disease burden placed on the recipients of infected blood products. This reactive thinking means that in the months to years between the recognition of the risk of a blood-borne pathogen and the availability of a blood screening test for that agent, recipients are harmed.
This reactive blood safety paradigm was shifted in the plasma proteins industry some time ago. In response to the rise of HIV and the demonstration of its ready transmissibility in fractionated products, especially factor VIII, a multilayered approach to blood safety was developed that not only relied on the screening of donors for known pathogens but also added a series of PITs to the purification and preparation processes for human plasma–derived protein products. These have been highly successful with no transmissions of HIV, hepatitis B, or hepatitis C by fractionated products for approximately 30 years.
For unfractionated blood products, both plasma and cellular, concerns about pathogen transmission remain. Cases of transfusion-transmitted infections and transfusion-associated sepsis still occur, even with the improvement of test sensitivity and concomitant reduction in the window period brought by nucleic acid testing (NAT). Detection limits due to low concentration of pathogens at the time of testing, as well as the specter of unknown species not tested for, remain risk factors.
Although the testing of blood for transmissible viruses prior to its release for clinical use has significantly reduced pathogen transmission, not all testing strategies are equally efficacious. An examination of the risk of bacterial transmission in platelet products illustrates the role that the microbial ecology plays in efforts to minimize risk. The contamination of blood products by bacteria most commonly arises from a small number of bacteria that enter the collection bag at the time of donation. With storage at room temperature in a nutrient-rich suspension, the bacteria may multiply many-fold in the days prior to transfusion yet not be at a sufficient concentration to be detected by the gold standard culture methods shortly after preparation. Thus, concerns over potential bacterial contamination of platelet concentrates have limited the shelf life of this product to 5 to 7 days. Other approaches besides testing have also been implemented to increase blood safety. These have included extensive donor screening with improved exclusion criteria, registries of previously deferred donors, prequalification of donors with extended wait periods prior to donation, quarantine of plasma donations until a subsequent donation, donor arm disinfection, and diversion of the first few milliliters of the donation.
In an effort to further improve blood safety, lessons from the plasma protein industry began to be applied to component therapy. The advent of PITs brings a profound shift to the overall approach to blood safety. Finally, transfusion medicine can bring a proactive strategy to blood safety rather than remain locked in reactive paradigms. The focus of this review is the current state of PITs for fresh blood components.
Safety of blood products
A major focus in blood transfusion over the past 3 decades has been the improvement of the safety of transfusions with respect to the risk of transfusion-transmitted diseases. Huge amounts of effort and resources have gone into bringing a high level of safety to modern blood products. The approach to increasing the level of blood safety, however, has been reactive. A pathogen is identified and then a test is developed to screen the donor or the blood product for that pathogen. The limitations of a society’s financial resources that can be devoted to blood safety in turn limit the success of this strategy. Although large reductions in the risk of transmission of those pathogens for which blood is screened (eg, HIV, hepatitis B, hepatitis C, and West Nile virus) have been generated, there are other agents that are transmissible in blood for which there currently are not tests readily available or for which implementing testing has not been chosen (eg, Babesia and dengue virus). Because the development of blood screening tests is driven by market forces, it is unlikely that new blood screening tests will become available without the demonstration of a high degree of disease burden placed on the recipients of infected blood products. This reactive thinking means that in the months to years between the recognition of the risk of a blood-borne pathogen and the availability of a blood screening test for that agent, recipients are harmed.
This reactive blood safety paradigm was shifted in the plasma proteins industry some time ago. In response to the rise of HIV and the demonstration of its ready transmissibility in fractionated products, especially factor VIII, a multilayered approach to blood safety was developed that not only relied on the screening of donors for known pathogens but also added a series of PITs to the purification and preparation processes for human plasma–derived protein products. These have been highly successful with no transmissions of HIV, hepatitis B, or hepatitis C by fractionated products for approximately 30 years.
For unfractionated blood products, both plasma and cellular, concerns about pathogen transmission remain. Cases of transfusion-transmitted infections and transfusion-associated sepsis still occur, even with the improvement of test sensitivity and concomitant reduction in the window period brought by nucleic acid testing (NAT). Detection limits due to low concentration of pathogens at the time of testing, as well as the specter of unknown species not tested for, remain risk factors.
Although the testing of blood for transmissible viruses prior to its release for clinical use has significantly reduced pathogen transmission, not all testing strategies are equally efficacious. An examination of the risk of bacterial transmission in platelet products illustrates the role that the microbial ecology plays in efforts to minimize risk. The contamination of blood products by bacteria most commonly arises from a small number of bacteria that enter the collection bag at the time of donation. With storage at room temperature in a nutrient-rich suspension, the bacteria may multiply many-fold in the days prior to transfusion yet not be at a sufficient concentration to be detected by the gold standard culture methods shortly after preparation. Thus, concerns over potential bacterial contamination of platelet concentrates have limited the shelf life of this product to 5 to 7 days. Other approaches besides testing have also been implemented to increase blood safety. These have included extensive donor screening with improved exclusion criteria, registries of previously deferred donors, prequalification of donors with extended wait periods prior to donation, quarantine of plasma donations until a subsequent donation, donor arm disinfection, and diversion of the first few milliliters of the donation.
In an effort to further improve blood safety, lessons from the plasma protein industry began to be applied to component therapy. The advent of PITs brings a profound shift to the overall approach to blood safety. Finally, transfusion medicine can bring a proactive strategy to blood safety rather than remain locked in reactive paradigms. The focus of this review is the current state of PITs for fresh blood components.
Current status of pathogen inactivation technologies
The general approach of PITs is to mediate inactivation of pathogens by termination of growth or proliferation rather than an actual reduction (in concentration) of pathogens. Because an organism must be able to reproduce in order to be infectious, this is an effective strategy for preventing transmission of pathogens by blood products. Several pathogen reduction technologies (PRTs) and PITs have been designed that either target the lipid structure of membranes or the RNA/DNA of pathogens that can be used to treat either plasma or cellular products (platelets and red cells). These procedures rely on the illumination of the blood product with UV light in the presence or absence of a photosensitizer. Numerous systems are on the market ( Table 1 ) with different approval status in various countries. The available PITs for various blood products are summarized.
| System | Manufacturer | UV (nm) | Photosensitizer | Mechanism of Action |
|---|---|---|---|---|
| Intercept | Cerus | 320–400 | Psoralens | Irreversible cross-linking of nucleic acids. |
| Mirasol | Terumo BCT | 280–360 | Riboflavin |
|
| Theraflex | MacoPharma | 254 | None | Nucleic acid damage presumably occurs due to cyclobutyl ring formation |
Pathogen Inactivation Technologies for Plasma
Solvent/detergent method
Several different approaches have been undertaken to reduce or prevent the transmission of blood-borne pathogens in plasma. The most widespread pathogen inactivation method currently available for plasma involves the combination of a detergent (D) to disturb lipid-enveloped viruses and a nonvolatile solvent (S), in a so-called SD procedure. This approach has proved effective in providing added protection against a wide spectrum of pathogens. Due to their direct negative effects on membranes, SD methods are not applicable to the cellular components of blood but have been adapted for plasma with success and have been licensed and marketed worldwide. The SD method has primarily been applied to pooled plasma, but recently technologies have been developed that allow the treatment of individual plasma units.
Methylene blue
Considerable efforts were directed to the use of photoreactive methylene blue and similar phenothiazine dyes, which have a high affinity for both nucleic acids and the surface structure of viruses. The virucidal activity of methylene blue light treatment has been known for more than a half-century. Some concerns have been expressed about the potential mutagenic effects of methylene blue and its derivatives. Accordingly, an additional filtration step was added to remove the residual dye in the final product with little change in various coagulation parameters.
Amotosalen/UV and riboflavin/UV
Newer technologies that crosslink nucleic acids or generate reactive oxygen species are applicable to plasma. The Intercept system (Cerus, Concord, California) uses the natural compound amotosalen, which intercalates between the nucleotide base pairs of RNA and DNA. The antipathogen activity requires photoactivation by long-wave UV-A light at 320 nm to 400 nm, turning the compound into a reactive intermediate and structural element in the cross-linking of hydrogen-bonded complementary nucleic acid residues. Using a compound absorption device containing activated charcoal, unincorporated amotosalen and residual photoproducts are removed from the product prior to use. Potential carryover of remaining amotosalen and its photoproducts in the final blood product required toxicologic evaluation studies that revealed no activity in mutagenicity assays. The Mirasol technology (Terumo BCT, Lakewood, Colorado) works by photoactivation of riboflavin (vitamin B 2 ) to inactivate pathogens. This compound shares a similarity of structure and mechanism with methylene blue as a photoreactive heterocyclic compound targeting primarily nucleic acids and producing strand cleavage either through an electron transfer process, with oxidation of guanine nucleotide residues and helix fragmentation, or through the production of reactive oxygen intermediates.
From an operational perspective, the Intercept and Mirasol technologies are similar processes. One other difference is the concern with the Intercept process over the unknown long-term safety profile of amotosalen; however, this concern has not been an impediment to regulatory approval. Such concerns are minimal for riboflavin because the compound has an extensive pharmacologic history and has been used in high doses in neonates in the treatment of bilirubinemia.
The treatment of plasma with UV-C alone to inactivate pathogens (Theraflex, Macopharma, Tourcoing, France) is a new development.
In summary, methods currently in the marketplace all kill pathogens in plasma albeit with some differences among them as to the degree of kill of specific pathogens. All methods also have the trade-off of increased safety profile for some reduction in the function of the proteins, particularly in the coagulation pathway.
Pathogen Inactivation Technologies for Red Blood Cells
Numerous photosensitizers and alkylating agents have been evaluated for pathogen inactivation of red blood cells (RBCs), but many of these caused unacceptable hemolysis or neoantigen formation, precluding commercialization. Candidates currently in late-stage development include the application of a Cerus-developed PIT that is based on an approach similar to Intercept.
Pathogen Inactivation Technologies for Platelets
Two PITs, Mirasol and Intercept, discussed previously, are also used for pathogen inactivation of platelet concentrates. There is an additional filtration step in the Intercept procedure that causes a small loss of platelets. For platelets, these PITs are designed to optimize pathogen killing and minimize platelet damage. Using Intercept’s amniomethyl trimethylpsoralen (amotosalen [S-59]) requires lower doses of UV, with shorter exposure periods, to achieve virucidal activity for single-stranded DNA or RNA containing viruses. Extensive preclinical toxicologic and pharmacologic studies indicate that there is no evidence of genotoxicity, phototoxicity, or excess carcinogenicity.
For the Mirasol technology, the inactivation principles are the same as for its use in plasma. Like Intercept, the application of the PRT also causes some damage to the platelets, which is expressed as reduced survival and recovery in healthy volunteers with normal platelet counts or as shortened interval to the next transfusion in patients receiving repeated platelet support.
Another approach to pathogen inactivation for platelets is under development by Macopharma, which uses UV-C light alone. This method uses aggressive agitation of the units during UV exposure to ensure penetration of the UV light throughout the product. Because there are no additives, there is no post-treatment processing required other than placement in a platelet storage container. The inactivation principle is based mainly on UV-C light absorbance by nucleic acids, resulting in the formation of cyclobutane pyrimidine and pyrimidine pyrimidone dimers, which block nucleic acid replication and hence infectivity.
Pathogen Inactivation Technologies for Whole Blood
Ideally, treatment of whole blood prior to component production or without component production is a preferable approach. The development of PITs for whole-blood treatment also is being pursued, although few clinical data are yet available. Treatment of whole blood prior to component production offers some significant advantages to blood operators, even though it is likely that the allowable storage time for the RBCs would be shortened.
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