Pathogen inactivation

Pathogen reduction technology (PRT) has been available for pooled plasma derivatives for many decades. The pasteurization process used to inactivate pathogens in human serum albumin solutions has effectively prevented viral transmissions in these products since it was first introduced. Unfortunately, the development of pathogen inactivation (PI) processes for other more labile components in human blood have proven more difficult, and it is only in the last decade or so that PRT has been available for plasma and more recently, for platelets. The implementation of comprehensive measures has enabled the reduction of risks of established transfusion transmitted infections (TTI) such as human immunodeficiency virus (HIV), hepatitis B (HBV), hepatitis C (HCV) and syphilis, in most countries worldwide. The addition of nucleic acid amplification technique (NAT) testing for HIV, HBV and HCV in developed countries has further reduced these risks to very low levels. In the Asia–Pacific region, the introduction of HBV-NAT in particular has resulted in the interdiction of significant number of serological window period donations as well as occult HBV infections (OBI) in countries where HBV infection is endemic. Nonetheless, a minimum residual risk still remains from NAT window period donations and OBI where levels are below current NAT detection limits, as well as from viral variants that are not detected by current tests [1]. There is also continued concern in the Asia–Pacific region from infections such as malaria which are not addressed adequately through existing measures, infections such as cytomegalovirus (CMV) and human T-lymphotropic virus which are not universally tested for, and infections such as dengue (DEN) and chikungunya (CHIK) where tests are not yet established for the blood supply. Transfusion-associated sepsis continues to occur despite the introduction of bacterial testing [2]. Epidemiological evidence indicates that many emerging and re-emerging infections are expanding, and have potential to affect blood safety [3]. Furthermore, the possibility remains that a new pathogen may yet emerge that will be efficiently transmitted through blood transfusion. There is therefore a very obvious advantage to being able to inactivate pathogens in donated blood. Established PRT such as solvent detergent treatment (SD-plasma; OctaplasLG, Octapharma, Lachen, Switzerland), and methylene blue/UV light treatment (MB-plasma; Theraflex, Macopharma, Lille, France) have been applied to plasma for many years with millions of units of SD-plasma and MB-plasma clinically transfused worldwide. However, the development of an effective PI process for cellular products had eluded research efforts until only recently. Currently, two PI systems are licensed for clinical use in Europe and one is in clinical trials. The Intercept Blood System (IBS; INTERCEPT Blood System; Cerus Corp., Concord, CA, USA) received CE Mark approval for apheresis and buffy coat derived platelets in 2002, and for plasma in 2006. The Mirasol Pathogen Reduction Technology System (MPRT; Mirasol Pathogen Reduction Technology System; CaridianBCT Biotechnologies, Lakewood, CO, USA) received CE Mark approval for apheresis and buffy coat derived platelets in 2007, and for plasma in 2008. The IBS uses a synthetic psoralen (amotosalen HCl) that intercalates into DNA and RNA. Ultraviolet A light (320–400 nm) is delivered at a dose of 3 J/cm in approximately 5 min, upon which the psoralen undergoes a reaction with the pyrimidine bases to form irreversible bonds creating adducts and crosslinks. Unreacted amotosalen and photoproducts then adsorbed during incubation in a separate container containing the compound adsorption device (CAD) for at least 4 h and up to 16 h before the platelets are released for transfusion. The IBS has been shown to inactivate a broad spectrum of enveloped viruses, bacteria, and protozoa, with more variable effect on non-enveloped viruses. It also results in inactivation of mitochondrial DNA, with in vitro studies demonstrating spontaneous platelet activation and accelerated metabolic changes. The MPRT system uses riboflavin and UV light (265–370 nm) delivered at 6·2 J/cm in 10 min to induce oxidation of guanine residues causing irreversible nucleic acid damage. Unlike the IBS, the product is ready for transfusion after illumination without need for further processing. Riboflavin treatment has also proven effective against enveloped viruses, bacteria and protozoa, and some non-enveloped viruses. It also leads to platelet activation and increased platelet metabolic activity, although mitochondrial function seems not to be affected. Recently, a new short-wave ultraviolet light technology (THERAFLEX UV-Platelets; Macopharma, Mouvaux, France) has been developed based on UVC-irradiation of platelets without any chemical compound. Platelet concentrates are subjected to short-wave UVC light (254 nm) in combination with strong agitation, which leads to the formation of cyclobutane pyrimidine and pyrimidine–pyrimidine dimers that block nucleic acid replication. In vitro studies have so far demonstrated effective inactivation of bacteria and leucocytes, and a range of viruses, with comparable preservation of platelet function and viability [4,5]. The ability to close current window periods for known pathogens, address the continued risk of transfusion-associated sepsis and prevent most emerging TTI would undoubtedly ease the minds of many a blood service manager. Both the IBS and MPRT system have demonstrated ability to inactivate established TTI such as HIV, HBV, HCV, CMV and syphilis by at least 4–6 logs, which should be sufficient to close the NAT window periods for these infections and eliminate OBI. The effects against non-enveloped viruses are unfortunately more variable, with 4–5 log reduction in parvovirus B19 and little or no effect with hepatitis A virus [6]. Both systems demonstrate varying log reductions of between 2 and 5 logs against emerging arboviruses such as West Nile Virus (WNV), DEN, CHIK and protozoa such as Plasmodium and Trypanosoma Cruzi [6]. We have demonstrated effective inactivation of both the IBS and MPRT systems on the DEN virus and CHIK virus in comparative studies using apheresis platelets spiked with 107 infectious units of viruses. The IBS showed a 4–6 log reduction in both DEN and CHIK viruses and the MPRT system showed a 1–4 log reduction in DEN virus and 2 log reduction in CHIK virus; this is likely to be sufficient for the low levels of viraemia noted in asymptomatic donors. Studies of the effect of IBS in bacteria show log reductions of 4–6 logs in both Gram positive and negative bacteria, while the MPRT system shows slightly lower log reductions of between 1 and 4 logs [6]. It is postulated that PRT is likely to be sufficient in eliminating the residual risk of transfusion-associated sepsis which commonly occur as a result of low levels of bacterial contamination [7]. There is added evidence that the IBS and MPRT treatment processes are able to replace gamma irradiation for prevention of transfusion-associated graft-vs.-host disease (TA-GvHD). Experiments have shown that both the IBS and MPRT system are able to reduce T-cell proliferation at levels comparable to that achieved by gamma irradiation [8]. The MPRT treatment is also able to reduce both cytokine secretion and the ability to induce proliferative change in allogeneic cells [9]. The CE approval for Mirasol includes an indication for use in the inactivation of WBCs in the products, and specific indication for IBS to replace gamma irradiation for TA-GvHD prevention received CE Mark approval in 2008. Blood services now have the option of incorporating PRT into their blood processing operations, enabling the provision of PI-platelets (PI-PLT) and plasma for clinical use. Yet, other than in a handful of European countries, few have adopted this technology into routine practice. Concerns continue to revolve around the effect of PI processes on the yield and function of the final product and possible side effects that may occur in the patient. A meta-analysis of RCTs involving PI-PLT concluded that PI-PLT were associated with a significant reduction in 1- and 24-h post-transfusion CCI as well as significant increase in all and in clinically significant bleeding complications. The frequency of severe bleeding complications did not differ. Based on the total evidence, it was assessed that the transfusion of PI-PLT is associated with a 58% increase in all bleeding complications and a 54% increase in clinically significant bleeding complications [10]. The loss of platelets during the PI process is postulated to be the major cause of the decrease in post-transfusion platelet recovery and survival, as well as the increase in the number of transfused platelet concentrates and shortened interval between transfusions that was observed in RCT. It is reasoned that this could therefore be overcome by increasing the number of transfused concentrates. However, it is still unclear whether PRT is associated with loss of viability in a proportion of the platelets alone or whether it results in functional impairment of all treated platelets as well. There is some evidence that part of the damage may be due to the use of platelet additive solutions (PASs) for storage of the PI-PLT. Studies have shown that the use of PASs during platelet storage affect platelet function, although the newer PASs can maintain platelet integrity and moderate metabolism similar to plasma for at least 5 days [11]. So far, observational studies after the introduction of PRT have not recorded any increase in bleeding in recipients of PI-PLT or increase in number of transfused concentrates [12]. However, it is possible that an increase in mild or moderate bleeding complications may not have been noticed outside the framework of a RCT. A retrospective review of the impact of the phased introduction of PASs and PRT into routine use in the EFS Alsace over a 5-year period showed that the platelet content per unit was significantly less in platelet concentrates stored in PAS compared with plasma, and also significantly less in platelet concentrates prepared with PRT and PAS compared to untreated platelet concentrates. When adjustments were made for differences in platelet content of individual components, there was no significant difference in the total platelet dose required per patient or in the utilization of red cells. The incidence of adverse events imputed to transfusions of platelet concentrates decreased on both a per-transfusion and a per-patient basis [13]. The impact of introducing PRT into routine operations as well as the additional resources required must be considered carefully by the blood service. For example, the introduction of PRT to the processing workflow in EFS Alsace shows the earliest release of apheresis platelets in the evening of the first day after collection, and on the beginning of the second day after collection for whole blood derived platelets [13]. Much of this is largely due to the time required for incubation in the IBS CAD which requires 6–16 h before the platelets could be released for transfusion. We have estimated the space and manpower requirements required to introduce PRT for platelets in our institution, and the impact on process turn-around-time and platelet release for transfusion (Tables 1 and 2). The introduction of PRT must be accompanied by the implementation of stringent quality systems to ensure the safety and quality of the PI-PLT. Our studies of PI treatment on apheresis platelets showed platelet losses of nearly 20% in platelet yields measured at Day 5 of storage, with no significant difference between the IBS and MPRT system. Significant decrease in platelet concentrate pH levels were also demonstrated at Day 5 of storage compared to post-treatment levels, although the mean pH values were above the AABB standard of 6·2. This highlights the need for vigilant monitoring of the platelet yield and pH quality indicators in PI-PLT. The residual level of amotosalen HCl in IBS-treated platelets must also be measured. Our studies of IBS-treated apheresis platelets have shown levels of 0·2–0·4 μm per unit, which is well below the acceptable level of 50 μm per unit recommended by Cerus. Nonetheless, routine quality control monitoring of amotosalen HCl levels should also be instituted. Cost remains a major hurdle for most blood services contemplating PRT. Custer et al. estimated the cost effectiveness of PRT in Canada using a multiple risk reduction model. Whole blood PRT was estimated to have a cost effectiveness of $1 276 000/quality adjusted life-year (QALY; 95% CI approximation 600 000–3 313 000) compared to current screens and interventions, while PLTs and plasma PRT was estimated to have a cost effectiveness of $1 423 000/QALY (95% CI approximation 834 000–2 818 000) [14]. This was modelled as an addition to rather than a replacement for current interventions, and it is possible that cost effectiveness may be higher if interventions such as bacterial culture for PLTs and gamma irradiation could be eliminated, and testing for WNV, HTLV and T. cruzi modified or avoided. The study did not include the risks of an unknown pathogen. Cost effectiveness would be lower if increased component use and potential adverse events for recipients are included. Nonetheless, the authors observe that the cost effectiveness estimates are consistent with established thresholds for value in blood safety. A separate study modelled the risks of an emerging pathogen entering the Canadian blood supply and estimated approximately 700 infections (2 SD range 0–2103) and 3500 infections (2 SD range 0–11 370) could occur from an emerging infection that establishes an acute and chronic infection, respectively. Assuming a best-case scenario of 100% effectiveness, it was calculated that PI for platelets or plasma alone would be expected to reduce the number of transmitted infections by 20% for either an acute or chronic agent, and by 40% if both platelet and plasma processes were available, with correspondingly lowered medical costs associated with the recipient outcomes [15]. The cost effectiveness estimates for PRT do not include consideration for the cost of introducing new tests to the blood supply. As an example, an assessment of the incremental direct cost of introducing PRT to the blood supply in Singapore suggests that this will still be lower than the incremental direct cost of adding new NAT-based tests for DEN, CHIK and WNV, although it is recognized that the effectiveness of this intervention will not be fully effective against these pathogens until PRT for red cells or whole blood is also available. The costs of introducing PRT will also be reduced with the introduction of larger capacity PRT processing sets for both the IBS and MPRT system. These will be able to accommodate double dose platelet units, which will reduce the cost of processing and increase process efficiency. There is no doubt that the full impact of PRT on blood safety (and on cost effectiveness) can only be realized when systems are available for red cells. Cerus has developed a PRT system for red blood cells based on the use of S-303, a frangible anchor linker effector (FRALE) compound. S303 has been shown to inactivate viruses, bacteria, protozoa and leucocytes efficiently and toxicology studies have also demonstrated safety. Following successful phase I and II trials, a phase III trial in cardiac surgery and sickle cell/thalassaemia patients was put on hold because of the development of antibodies reactive with the S-303-treated red cells in some of the patients [16]. A second generation process has since been developed, and is undergoing early clinical trials. The MPRT system is being developed for whole blood, which will enable a single PI step. The PI product could then be used as whole blood or separated into components. The PI profile is similar to that for PRT of platelets and plasma. A clinical evaluation in 12 patients has enabled the establishment of parameters predictive of 24-h red cell survival and which will be used to develop the final system [17]. Ultimately, the decision to introduce PRT will depend on factors specific to the country, which include the efficacy of existing blood safety measures, the disease epidemiology, healthcare system, and financial and resource considerations. It is crucial that PRT systems are carefully evaluated by the blood service in the context of its own routine operations and expectations. While current PRT systems have demonstrated effective inactivation of most pathogens, the extent of reduction achieved is variable depending on the pathogen. Some pathogens may not be effectively inactivated. The blood service needs to be aware of the inactivation profile to ensure that the limits of PI are clearly communicated and taken into consideration when determining risk reduction outcomes. Questions also remain unanswered with regard to the effect of PRT on platelet function. Further studies are needed to elucidate the nature of PR-induced damage, and an increase in mild and moderate bleeding complications may have to be anticipated and tolerated if PRT is implemented at this point in time. Because toxicity may not be revealed until larger scale clinical use, it is also important that phase IV postmarketing studies and haemovigilance programmes are in place. Notwithstanding these considerations, public and patient expectations and perceptions will no doubt have a major influence on any decisions regarding PRT implementation. It is therefore imperative that blood services are able to communicate the risks and benefits properly to all stakeholders involved, so that educated decisions are made. No potential conflict of interests to declare.

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