Pathogen-reduction systems for blood components: The current position and future trends

Transfusion and Apheresis Science 35 (2006) 189–196 intl.elsevierhealth.com/journals/tras

Pathogen-reduction systems for blood components: The current position and future trends Jerard Seghatchian a

a,¤

, Gracinda de Sousa

b

Blood Components/Apheresis Technologies and Thrombosis/Haemostasis Consultancy, 50 Primrose Hill Road, London NW3 3AA England, UK b Lisbon Regional Blood Centre, Avenida do Brasil, 53-Pav 17, 1740-005 Lisbon, Portugal

Abstract The current multi-layered interventional approaches to blood safety have dramatically reduced the risk of viral contamination of blood components. Nowadays most of the residual transfusion transmitted infections (TTI) occur as the result of the interval between the time the donor is infected and the moment at which tests are capable of detecting the agent, the so called “window period” which has been considerably reduced by the increased sensitivity of nucleic acid testing (NAT). However, the residual risk of bacterial contamination and the unexpected appearance of some other emerging pathogens, almost every Wve years, are still of major concern to the public, politicians, regulatory agencies and place immense pressures on the organisations responsible for the provision of safe blood and its components. In view of these bleak scenarios, the use of human blood as a raw biological source is inherently unsafe, and screening/testing alone cannot exclude all the potential human pathogens; hence we need to put in place some sort of safer alternatives and/or additional preventative safety measures. Recently, several substitutes (alternatives) to virtual blood components have been developed and tried. Moreover, various mechanical methods such as cell washing and leukoWltration have been implemented as additional preventative safety measures but with limited success in abrogating the risk of transfusion transmitted cell-associated agents. The most promising approaches, so far, are methods that target pathogen nucleic acids (Methylene blue; Psolaren and RiboXavin UV light treatment). These procedures have undergone considerable in vitro studies to ensure their extremely high safety margins in terms of toxicity to the cells or to the recipients. In essence, while the technology of targeting nucleic acid to stop viral proliferation is common to the above three strategies, in practice these procedures diVer in terms of operational, physicochemical and biological characteristics; including the potential impacts of their metabolites and photo-adducts; their eVects on the spectrum of pathogens aVected and the log reductions in culture infective studies. Accordingly, any strategy that involves addition of an extraneous agent or physicochemical manipulation of blood must balance the beneWts of pathogen reduction against the loss or alteration to the cells and plasma functional integrity, short and long term toxicity to the cells and to the recipients, as well as the risk to the personnel involved and the community at large. Moreover, it must be noted that each method will have a diVerent proWle of adverse reactions and may diVer in terms of the risk to particularly vulnerable groups of patients, requiring in depth clinical trials, while taking into consideration the cost beneWt of the Wnal process. Newer diagnostic procedures must be in place to establish the storage stability of products

*

Corresponding author. E-mail address: [email protected] (J. Seghatchian).

1473-0502/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.transci.2006.10.002

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that have undergone pathogen inactivation, in particular tests reXecting the release of platelet-derived cytokines, cellular apoptosis or microvesiculation and their role in immunosupressiveness. This overview aims to provide an update on the continual improvements in blood component safety, in particular using methods that target pathogen nucleic acid. Emphasis is placed on methylene blue light treatment (MBLT) and Intercept or Mirasol PRT systems for platelets and plasma. The status of pathogen reduction of whole blood and red cells is also highlighted, though the progress in this area has been virtually stopped after the Wnding of antibody development in the clinical trial. © 2006 Elsevier Ltd. All rights reserved.

1. Introduction Despite various proactive interventional approaches to blood safety which embodies: a more stringent donor screening, introduction of new exclusion criteria, registries of previously deferred donors, various speciWc serological and nucleic acid based testing (NAT), post-donation tracking and transfusion haemovigilance etc., transfusion transmitted infections (TTI) still occur. Moreover, plasma fractionation using two or three step pathogen-reduction systems, designed to reduce the risk of pathogens entering into the manufacturing process, have been successful in eVectively eradicating TTI by blood products since 1985. Unfortunately, a similar approach to improve the safety of cellular blood components have not been realised partially because the methods of safeguards used in plasma fractionation are not generally applicable to the spectrum of pathogens aVecting cellular blood components. Some methods target certain speciWc organisms and other methods are broad spectrum but may not suYciently reduce those with high viral loads. Some may have potent activity against lipidenveloped agents present at high titre, but less eVective against non-enveloped viruses and, more importantly, each method will have a speciWc proWle of adverse reactions and may diVer in terms of risk to particularly vulnerable patient groups [1–3]. This overview brieXy discusses the current state of the art pathogen inactivation (reduction) for plasma/cryoprecipitate, highlighting the shortcoming observed for pathogen inactivation of whole blood/red cells and Wnally focusing on procedures for platelet concentrates, namely Intercept and Mirasol PRT. 1.1. Methods for pathogen inactivation of plasma and its derivatives Methods for pathogen inactivation for plasma and derivatives include Wrstly, heat treatment which is not suYciently robust to attenuate non-enveloped viruses, especially human parvovirus B-19, although

dry heat at 100° for 1 h inactivates both enveloped and non-enveloped viruses eVectively, without substantial loss of the active proteins [3–5]. Secondly, the combination of a detergent to disturb lipidenveloped viruses and a non-volatile solvent, so called SD procedure, proven to be eVective in providing added protection against large organisms such as protozoa, but the SD mixture needs to be removed by precipitation of the product or by chromatography at the end of the process. Another drawback of this approach is that non-enveloped viruses such as HAV and HPVB-19 are not inactivated by this technology [5,6]. Moreover some SD methods may lead to loss of some important plasma proteins such as, alpha 2 antiplasmin and protein S, causing clinical concerns in regard to haemostasis and thrombosis [7,8]. Understandably, SD methods are not applicable to the cellular components of blood but have been adapted for plasma with success and have been licensed and are currently in clinical use in Europe and the USA. Another useful approach for pathogen reduction of plasma is nanoWltration, which consists of Wltration through membranes of a very small pore size, typically 15–40 nm, to retain pathogens based on size exclusion [9]. This procedure is simple, robust and reliable and can be applied to all plasma products, attributing against non-enveloped viruses and others emerging infectious agents, by 4–6 logs reduction, with the recovery of 90–95% of the essential components of plasma. This procedure is reported to have some additional promise for removing prion infectivity [10]. Considerable eVorts were also directed to the use of photoactive methylene blue and similar phenothiazine dyes, which have a high aYnity for both nucleic acids and the surface structure of viruses [11,12]. The virucidal activity of MB light treatment (MBLT) has been known for more than half a century and in fact Paul Ehrlish Wrst applied MB clinically in the 19th century. Unfortunately, MBLT method does not eVectively inactivate intracellular

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viruses, bacteria and protozoa, as in low concentrations it is a membrane stabiliser and only at high concentration leads to cellular damage. Moreover, non-enveloped viruses especially HAV are relatively resistant to MBLT and concerns are expressed about the potential mutagenic eVects of MB and its derivatives. Accordingly, an additional Wltration step is currently used to remove the residual dye in the Wnal product with little changes on various coagulation parameters [13–16]. Another shortcoming of MBLT is that signiWcant changes in the Wbrin polymerisation curve occur, making MBLT plasma behave like aWbrinogenimia plasma [15,17]. Some streamlining is needed to meet FFP speciWcation as plasma derived from some donors, with O groups, fail the current standard for acceptance, based on factor VIII potency [13,14]. Currently MBLT is the only method that is applied to individual units of plasma and has become the common practice in several European countries. In the UK transfusion settings, this procedure is selectively used for recipients under 16 years of age and other vulnerable groups. Up to now no immediate problems have been reported in transfusion of such products to neonates, children and parous women treated by this technology. Other newer technologies, targeting speciWcally the nucleic acid, such as Intercept and Mirasol that are used for pathogen reduction of platelets are also applicable to plasma. The pharmacokinitic of FFP, photo chemically treated with the S-59 system has been studied and it was observed that after freeze/ thawing the functional activity of coagulation factors was well preserved with a range of 73–98% of control, with no signiWcant diVerences in VWF, and its multimers or metalloprotease activity, proteins C and S, ATIII and there was no evidence of activation of thrombin, complements or contact system, making treated FFP and cryoprecipitate suitable for therapeutic use. In a paired cross over S-59 treated and untreated post-transfusion study, the administration of both types of FFP in patients after four days of Warfarin resulted in comparable FVII kinetics, which returned to base line after 8 h [18]. The works on the use of Mirasol for inactivation of plasma is currently in the planning stage. 1.2. Methods for pathogen reduction of whole blood and red cell concentrates An eVective method for pathogen reduction of whole blood and red cell components must fulWl at

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least three essential criteria: Wrstly, to physically remove viruses and the blood cells that contain viruses and secondly, to penetrate selectively into the cells, without damaging the blood cells and Wnally, to eVectively inactivate free viruses, viruses attached to cells and intracellular viruses. Several approaches based on either physical, universal leukoWltration or chemical/photochemical techniques have been validated and are undergoing clinical trials. The earlier methods, which were based on extensive washing and controlled freezing followed by deglycerolization, were found to be eVective in reducing TTI. LeukoWltration that reduces the residual leukocytes by >4 logs to <6 logs per components can also adequately remove some viruses such as CMV and HTLV that are transmitted exclusively by leukocytes from red cell and platelet concentrates [19]. Current Wltration technologies are clearly of value but are not suYciently robust and are far from ideal, particularly during infectivity as shown earlier for HTLV [20,21]. Currently, eVorts are directed toward newer technology, based on chemical/photochemical processes that target nucleic acid to inactivate residual contaminating viruses, bacteria and protozoa in cellular blood components, while sparing the functional integrity of red cells, platelets and plasma. The broad-spectrum pathogen inactivation potential of these technologies makes them the methods of choice for inactivation of both known and unknown DNA/RNA containing pathogens. This is an important virtue as some viruses, such as HPV B19, are a plasma agent that adheres to red cells. Others such as HIV are transmitted both by leukocytes, plasma and other blood cells as they adhere to red cells and can be internalised by platelets [22,23]. A considerable amount of work has been carried out to substantiate that nucleic acid targeted technologies are highly eVective, showing a very high safety margin and merit clinical trials. Moreover, nucleic acid targeted processes have the potential to inactivate residual lymphocytes in these components and prevent transfusion-associated graft versus host disease TA-GVHD [24,25]. Several important criteria must be made for photochemical pathogen reduction of whole blood and red cells. These include: the use of red light, with a wavelength above that of haemoglobin, otherwise it would be either absorbed or scattered; the optimal selection of the appropriate sensitising dye that can easily penetrate cell and viral membrane, bind to the

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nucleic acid, absorb light in the presence of red cells, inactivate a wide spectrum of pathogens without damaging red cells, inactivating both free and cellassociated viruses and retaining not only the storage stability of red cells but also not grossly aVecting red cell survival or immunogenecity. Up to now methods for pathogen reduction of whole blood and red cell concentrates did not optimally fulWl the above criteria despite extensive studies as described below: A positively charged, alkylating agent derived from quinacrine mustard, with a planar structure, so called S-303 (Helink), found to rapidly intercalate into helix regions and inactivates nucleated pathogen by cross-linking DNA/RNA. Activation and cross-linking occurs when the dye rapidly and fully undergoes hydrolysis following changes in its low pH environment to the neutral pH of blood. In practice, the majority of the covalently bound degradation product remains associated with red cells, without inXuencing its in vivo survival [26]. Unfortunately, in the initial study two subjects enrolled in the clinical trials developed antibodies and despite the earlier enthusiasm, the trials are temporarily suspended. Moreover a more hydrophobic dye than methylene blue, so called dimethylene blue, which can easily penetrate cells, is currently under investigation for pathogen inactivation of red cells. Despite a promising broad-spectrum virucidal activity, with only slight loss of ATP and 2,3 DPG levels of treated cells, the use of this procedure is a relatively poor choice for commercialisation as the haemolysis of treated cells exceeds 1%. Another broad-spectrum pathogen-reduction technology for red cell is the use ethyleneamine oligmers, so called Inactine or PEN110 [27]. Inactine is a highly water soluble cation that diVuses rapidly across the cell membrane, and selectively binds to nucleic acid guanidine bases and undergoes activation by bringing a negatively charged phosphate group and disrupts transcription and replication. Unfortunately, like S-303, PEN 110 treatment is not harmful to red cells but causes red cell antibody formation making the future of this substance uncertain, despite the fact that latent CMV and WNV are highly susceptible to this process [27–30]. Recently a riboXavin-based pathogen inactivation process has been proposed for red cell concentrates, where red cells are washed and combined with a riboXavin solution with a Wnal concentration of 50 mol/l in normal saline. The mixture is then exposed to visible light of 450 nm, a wavelength with minimum absorbance by haemoglobin. The red cells

are then concentrated and stored for up to 42 days, with an excellent metabolic function and storage stability [31]. Another candidate photosensitizer, using positively charged porphyrins exhibits excellent activity against model viruses and acceptable red cell quality, storage stability and survival has been recently evaluated [32]. To sum up, despite several years of preclinical studies and substantial commitment for inactivation of pathogens in red cells, no method yet has been licensed. 1.3. Methods for pathogen reduction of platelets Two approaches are currently employed for pathogen reduction of platelet concentrates. The Intercept technology uses small planar molecules (psoralens) that pass through cell membrane and capsid viruses, intercalating reversibly into helical regions of nucleic acids. Upon illumination with the long wavelength UV-A light (300–400 nm) psoralens cross-link covalently to pyrimidines in RNA and DNA of both free and HIV incorporated in the genome and block the replication and transcription of nucleic acids [26]. Like any technology used for inactivation of cellular components, several criteria must be met. The platelet product must be suspended in a plasma-reduced medium as plasma inhibits the inactivation of viruses. Since high UV dosage damages platelets, the appropriate psoralen must be selected and the optimal UV dose should be employed. The use of amniomethyl-trimethyl psoralen (AMT, S-59) is preferred, as it requires lower doses of UV, with shorter periods, without loss of virucidal activity for single stranded DNA or RNA. Some psoralen derivatives are less damaging than others and have the advantages of not being mutagenic in the absence of UVA [33]. The addition of agents that neutralize oxygen free radicals are required to protect the platelet membrane. After UV treatment, approximately 20% of the original psoralen remains while 80% has photo degraded, two thirds to dimmers of S-59 and one third to photoproducts that bind covalently to large molecular weight lipids. The potential of mutagenicity of psolaren is reduced, theoretically, by absorbing the residual psolaren on a compound absorbing disk, in a ligand Wxed on silicon as used for SDplasma [33]. Extensive preclinical toxicological and pharmacological studies indicate that there is no evidence of genotoxicity, phototoxicity or excess carcinogenicity. The intercept technology is currently

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licensed in Europe as a medical device and in some countries becoming integrated into routine blood components manufacturing [34]. In practice, platelets produced either from pooled buVy coats or apheresis technology, is suspended in approximately 35% of the plasma and 65% storage media, consisting of sodium chloride, acetate, citrate and phosphate. The platelet pack is then sterile docked to a single use integrated photo-activation system consisting of a series of closed systems of disposable plastic containers. The content of the platelet pack (350 ml) is passed through the Wrst container containing S-59 and collected in an illumination container (STEP 1). After 5 min incubation the integrated set is then placed in the UV illumination device that provides a Wxed dose of UVA light, for 3 min to the mixture (STEP 2). After illumination, the mixture is transferred to a second container for treatment with a compound adsorbing device, to lower the levels of residual S-59 and its free photoproducts (STEP 3) and platelets are agitated for 16 hours at room temperature before transferring to a Wnal platelet storage container (STEP 4). This is a relatively complex operation where platelets come in contact with many surfaces, and may undergo shape changes and release of their granule contents. Of particular interest is the release of TGB beta and RANTES, which are considered to be good markers of platelet storage lesion and contribute to the generation of apoptotic cells and immunosupressiveness [35]. In a recent study during validation of Intercept technology in Lisbon regional blood centre, we have observed that the expression of CD 62 on platelets increased from 27.6% in the untreated product in day one to 31.6% after treatment at day two and increased even further to 58.7% at the end of storage. Moreover, PS exposure on the platelet membrane as the hallmark of apoptosis/necrosis rises almost two fold at day two and three fold at the end of storage. The clinical signiWcance of these Wndings, in terms of haemostasis and transfusion reactions, remains to be elucidated. As far as pathogen-reduction eYciency is concerned, it is established that the intercept system inactivates by 5–6 logs both cell free and cell-associated HIV, bovine viral diarrhoea, as surrogate model virus of HCV, duck hepatitis B virus as HBV surrogate and CMV and a wide range of bacteria and protozoa [35,36]. The Intercept treated platelets have been subject to three clinical trials. Two of 166 thrombocytopenic patients in Europe, showing comparable platelet

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count increments and adverse reactions to these in the conventional control [37]. However in a large study (n D 645), aiming to assess the haemostatic function and the safety of apheresis platelets, it was found that 1 h post-transfusion increment, average number of days to next platelet transfusions and the number of platelet transfusion of Intercept treated platelets diVered signiWcantly, though the trial demonstrated equivalence in prevention and treatment of grade 2 and higher grade bleeding, according to WHO criteria [38]. A newer technology using RiboXavin, an essential nutrient (vitamin B2) that absorbs both UV and visible light, has been recently validated for both platelets and plasma components. The ability of RiboXavin to interact with nucleic acid after photoactivation has been extensively studied and it appears that the three-ringed planar structure of this molecule intercalates between DNA and upon light treatment, oxidises guanosine through electron transfer reactions, resulting in single strand breaks of nucleic acids and formation of covalent adducts [39,40]. The damage to DNA nucleic acid can proceed in the absence of oxygen, as the combination of RiboXavin and the UV light, with a wavelength of 280–360 nm damages nucleic acid by direct electron transfer, production of singlet oxygen and production of hydroxyl peroxide with the formation of hydroxyl radicals. Interestingly, RiboXavin is classiWed as a generally-regarded-as-safe (GRAS) compound by the US FDA, which makes it a promising candidate for human use in a pathogen-reduction technology [41]. Moreover, RiboXavin breakdown products, including lumichrome are present and consumed in a wide range of food and natural products. Activated RiboXavin is the essential principle of the standard course of photo-therapy treatment of neonatal jaundice, with no toxicological outcome [42,43]. The early prototype of the Mirasol PRT bag system, is quite similar to S-59 set, except for the CAD removal process. A recent study revealed that this process successfully reduces the window period of transmission of HIV by reducing the levels well below the required levels, eliminating the viremic period for WNV and possibly the chronic phase of HPV B-19 and highly eVective on several pathogens associated with fatal transfusion [43]. While infectious agents by tradition are blood borne, the residual immunocompotent lymphocytes in blood components can engraft in recipients, proliferate and lead to TA-GVHD, fever, rash, nausea, vomiting,

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diarrhoea, hepatitis, fatal lymphadenopathy and pancytopenia [44]. In fact, all blood components including plasma have been associated with TAGVHD [45]. Therefore the current pathogenreduction technologies that interfere with the replication of nucleic acid can, in theory, replace gamma irradiation as a preventative measure against TAGVHD. In vitro assessment of platelets by the Mirasol technology revealed signiWcant diVerence between control and treated cells after Wve days of storage, in particular, increase in glycolysis [46]. The increased glycolytic Xux could result either from damage in platelet mitochondria and/or increased ATP consumption. Further study to address this issue revealed that subsequent to the Mirasol treatment, platelet mitochondria structure and functional activity is well maintained, both immediately after treatment and subsequent to storage. Therefore the increased demand for ATP in treated platelets may be driven by the observed oxidative metabolism [47]. Based on currently available data, it is safe to say that the Mirasol PRT system may promise high eYciency, low cellular damage, little toxicity and ease of operation in the transfusion setting [43]. Clinical trails using Mirasol technology are already begun. 2. Future trends Human blood, by nature, will always carry certain residual infectious risks, despite the multi-faceted proactive interventional programme of stringent screening and the use of new generation nucleic acids testing. Moreover, there are always some emerging viruses for which no test will be available. Furthermore, while the production of purer components and the implementation of universal prestorage leukoreduction had some impacts in reducing the viral risk of blood components, the addition of a more proactive approach of pathogen inactivation that targets nucleic acids and can inactivate free, surface membrane-bound and intracellular viruses will undoubtedly improve the safety margin of blood component therapy. A robust pathogen-reduction technology, apart from good clinical eYcacy, must fulWl a high safety margin; to become adopted for those patients with severely compromised renal, hepatic and immune system function. The newer PRT must also be capable of preventing some new DNA/RNA infective agents as well as being eVective in prevention of

TA-GVHD, eliminating the need for irradiation of blood components for selective susceptible patients. The unresolved problem is to optimally inactivate pathogens without altering to a large extent the acceptable biological functions of the active components. Thus one needs always to balance between the optimal viral reduction and the minimal loss of useful biological substances. In considering future trends, it is noteworthy to highlight that pathogen-reduction technology, in use for blood products, have fully eliminated the recognised infectious risks of commercial pooled plasma protein fractions. However the same degree of success has not been achieved for blood components, though pooled SD-plasma, single unit methylene blue light treated FFP and cryoprecipitate are used in selective patients in some countries, despite major shortcoming in their haemostatic functions. Moreover, while considerable progress has been made in the actions of Intercept and Mirasol, for pathogen reduction of both plasma and platelets, the clinical application of these procedures are still in their infancy and whether the current acceptance criteria for therapeutic plasma, following the newer developments, in terms of transfusion complications are still adequate remains to be elucidated [48]. The future tests for storage stability of pathogen-inactivated cellular blood components should also include cellular apoptosis, microvesiculation, mitochondria fragmentation and development of platelet-derived cytokines, having impact on immunosupressiveness. Finally, in regard to considerable progress in pathogen-reduction strategies for whole blood and red cell, further investigation in this area is hampered by the Wndings of antibody developments, requiring additional in-depth review. Should newer DNA targeted pathogen-reduction technologies pass the more and more stringent regulatory requirements, there will still be the question of whether or not the current health care system will pay the premium for the relatively small improvement in levels of added safety. References [1] Klein HG. Pathogen inactivation technology: cleaning of blood supply. J Internal Med 2005;257:224–37. [2] Allain JP, Seghatchian J. Current strategies on pathogen removal/inactivation: an overview. Transfus Apheres Sci 2001;25:195–7. [3] Seghatchian J, Allain JP. Current strategies for the provision of safer plasma and its derivatives: an update. Transfus Apheres Sci 2001;25:185–7.

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