Development of blood transfusion product pathogen reduction treatments: a review of methods, current applications and demands.

Transfusion and Apheresis Science 52 (2015) 19–34

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Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i

Review

Development of blood transfusion product pathogen reduction treatments: A review of methods, current applications and demands Vishal Salunkhe a, Pieter F. van der Meer b, Dirk de Korte a,b, Jerard Seghatchian c,*, Laura Gutiérrez a,** a Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre (AMC), University of Amsterdam (UvA), Amsterdam, The Netherlands b Department of Product and Process Development, Sanquin Blood Bank, Amsterdam, The Netherlands c International Consultancy in Blood Components Quality/Safety Improvement and DDR Strategy, London, UK

A R T I C L E

I N F O

Keywords: Blood Plasma Platelets Red blood cells (RBC) Microvesicles Transfusion Pathogen inactivation Pathogen reduction treatment

A B S T R A C T

Transfusion-transmitted infections (TTI) have been greatly reduced in numbers due to the strict donor selection and screening procedures, i.e. the availability of technologies to test donors for endemic infections, and routine vigilance of regulatory authorities in every step of the blood supply chain (collection, processing and storage). However, safety improvement is still a matter of concern because infection zero-risk in transfusion medicine is nonexistent. Alternatives are required to assure the safety of the transfusion product and to provide a substitution to systematic blood screening tests, especially in less-developed countries or at the war-field. Furthermore, the increasing mobility of the population due to traveling poses a new challenge in the endemic screening tests routinely used, because nonendemic pathogens might emerge in a specific population. Pathogen reduction treatments sum a plethora of active approaches to eliminate or reduce potential threatening pathogen load from blood transfusion products. Despite the success of pathogen reduction treatments applied to plasma products, there is still a long way to develop and deploy pathogen reduction treatments to cellular transfusion products (such as platelets, RBCs or even to whole blood) and there is divergence on its acceptance worldwide. While the use of pathogen reduction treatments in platelets is performed routinely in a fair number of European blood banks, most of these treatments are not (or just) licensed in the USA or elsewhere in the world. The development of pathogen reduction treatments for RBC and whole blood is still in its infancy and under clinical trials. In this review, we discuss the available and emerging pathogen reduction treatments and their advantages and disadvantages. Furthermore, we highlight the importance of characterizing standard transfusion products with current and emerging approaches (OMICS) and clinical outcome, and integrating this information on a database, thinking on the benefits it might bring in the future toward personalized transfusion therapies. © 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. International Consultancy in Blood Components Quality/safety Improvement and DDR Strategy, London, UK. E-mail address: [email protected] (J. Seghatchian). ** Corresponding author. Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre (AMC), University of Amsterdam (UvA), Amsterdam, 1066CX, The Netherlands. Tel.: +31205123787; fax: +31205123310. E-mail address: [email protected] (L. Gutiérrez). http://dx.doi.org/10.1016/j.transci.2014.12.016 1473-0502/© 2014 Elsevier Ltd. All rights reserved.

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V. Salunkhe et al./Transfusion and Apheresis Science 52 (2015) 19–34

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Transfusion demands, risk of infection and pathogen reduction treatment criteria .................................................................................... 1.1. Criterion of acceptability for pathogen reduction treatments ................................................................................................................. 1.2. Quality control of transfusion products – storage lesion .......................................................................................................................... Pathogen reduction treatments for plasma products .............................................................................................................................................. 2.1. Pasteurization of plasma products (wet heat treatment) ......................................................................................................................... 2.2. Dry heat treatment ................................................................................................................................................................................................. 2.3. Solvent–detergent method (S-D method) ...................................................................................................................................................... 2.4. Methylene blue, riboflavin and amotosalen .................................................................................................................................................. 2.5. Acid-pH treatment .................................................................................................................................................................................................. 2.6. Nanofiltration ........................................................................................................................................................................................................... Pathogen reduction treatments for platelet products ............................................................................................................................................. 3.1. Amotosalen/UVA (Intercept®; Cerus, Concord, CA) ..................................................................................................................................... 3.2. Riboflavin/UVB based pathogen reduction technology (Mirasol® PRT; Terumo BCT, USA) ......................................................... 3.3. Theraflex UVC-based pathogen reduction treatment (MacoPharma, Mouvaux, France) ............................................................... 3.4. Meta-analyses of clinical trials for pathogen reduction treated platelet concentrates .................................................................. Pathogen reduction treatments for RBC products ..................................................................................................................................................... Pathogen reduction treatments for whole blood ...................................................................................................................................................... Prion elimination .................................................................................................................................................................................................................. Pathogen reduction treatments of immunotherapy products: anti-Ebola serum .......................................................................................... New perspectives to study the characteristics and quality of transfusion products .................................................................................... 8.1. Proteomics ................................................................................................................................................................................................................. 8.2. Microvesicles (MVs) and other biological by-products ............................................................................................................................. Current pathogen reduction treatment scenario in the world ............................................................................................................................. 9.1. USA ............................................................................................................................................................................................................................... 9.2. Europe ......................................................................................................................................................................................................................... 9.3. Developing and poor countries .......................................................................................................................................................................... Conclusion and future perspective ................................................................................................................................................................................. References ...............................................................................................................................................................................................................................

1. Transfusion demands, risk of infection and pathogen reduction treatment criteria The majority of blood transfusions in developed countries are frequently given to patients undergoing transplant and cardiovascular surgery, onco-hematologic patients and trauma patients whereas in less-developed countries, they are mainly given to women due to pregnancy-related complications and blood loss, as well as to children due to anemia [1]. These transfusions are performed mostly in the form of specific products such as red blood cells (RBCs), platelets, granulocytes, fresh frozen plasma (FFP), cryoprecipitate, intravenous immunoglobulins (IVIG) and various coagulation factors [2–4]. Although a meticulous donor selection takes place and blood transfusion products are regularly screened with serological and nucleic acid tests, accompanied by transfusion hemovigilance procedures, the risk of acquiring existing and emerging transfusion transmitted infections (TTIs) cannot be overlooked completely [5]. The estimated risk for a transfusion unit to be contaminated differs from the type of blood product and geographical location. The origin of the contamination can either be the donor’s blood (i.e. endogenous) or an exogenous source; in the latter case the source is primarily the skin of the donor at the phlebotomy site. Contamination during blood processing or product storage can occur but is very rare; alternatively it can take place prior to transfusion (i.e. if thawing of plasma is required and the water bath is not carefully decontaminated) and contamination during transfusion cannot be excluded completely [5,6].

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However, comprehensive epidemiological studies regarding TTIs are currently underrepresented, while they are crucial to shed insight in a country-wise and global manner so as to provide guidance to the current transfusion service guidelines. Current screening procedures applied in North America and Europe include Human Immunodeficiency Virus (HIV), Hepatitis B virus (HBV), Hepatitis C virus (HCV) and syphilis; West Nile virus (WNV) screening is mostly restricted to the USA. However, there are some potential bloodborne infections that might pose a risk, for which diagnostic tests may not be available or that are not routinely performed in each and every hospital or blood supplier, including non-endemic pathogens or new viral strains (virus mutants). Some of these infective agents are Dengue virus, Chikungunya virus, Human Herpes virus 6–8, Human Parvovirus B19, Q fever, malarial parasites, Babesia, Toxoplasma gondii and Leishmania donovani [5,7–9]. Multiple studies have shown that bacterial contaminations of transfusion products (especially platelets, since they are stored at room temperature, which facilitates the expansion of the initial bacterial inoculum) are more frequent than viral infections in Western countries. The infection prevalence ranges between 1:1000 and 1:5000 per platelet concentrate unit, while this risk is 50–250 times lower in RBC units [10–12]. Around 70% of the transfusion-related sepsis (TRS) cases are caused by contamination with Gram-positive bacteria from the skin flora at the phlebotomy site [13,14]. Bacterial screening, mandatory in many countries in Europe and the USA, has decreased the risk, but still bacteriacontaminated units might be transfused. Culture methods


are used for pre-storage screening successfully, however, if the initial bacterial inoculum is low or the bacterium is slowgrowing, microbiological cultures might give false-negative results [15]. Within our global world, and the increase in transcontinental traveling, it is also a matter of concern that potential donors might have been exposed to non-endemic pathogens for which no screening is locally performed. Pathogen reduction treatments are a promising solution to decrease pathogen contamination of any blood transfusion product and can complement routine pathogen screening [7,16]. Although the assumed average cost of a given pathogen reduction treatment is higher than screening when premises are already available ($100 vs. $44 per donation) [17], such treatments appear to be the best complement to or alternative for screening, specially at locations when screening is not fully developed or possible [11,17]. In addition, few screening tests could be probably dismissed if pathogen reduction treatment technologies are introduced, but a formal cost-efficiency analysis needs to be done.

1.1. Criterion of acceptability for pathogen reduction treatments The Food and Drug Administration (FDA)’s approach to measure the characteristics of an ideal pathogen reduction treatment was described already in 2003 by Epstein and Vostal, highlighting three major points: it must target a broad spectrum of pathogens, it must not cause damage to the transfusion product and it must be safe for the recipient [18]. Seghatchian and Putter described in 2013 various criteria of acceptability for pathogen reduction treatments [19], extending remarks on safety, cost-effectiveness and availability: 1. Must be efficacious to eliminate a broad spectrum of pathogens and prevent sepsis. 2. Should cause minimal damage to the blood transfusion product. 3. It must not compromise transfusion safety as assessed by in vivo assays and clinical outcomes. 4. As an uncomplicated and cost-effective technology, it should be non-toxic, maintain functional cell integrity for transfusion purposes, and pass the stringent tests for bio-equivalency and bio-security. 5. It should satisfy the main criteria of availability: being accessible, affordable, and safe, and demonstrate correct usage of the technology. Several pathogen reduction treatments for plasma and platelets have been licensed in European countries and currently, clinical trials are undergoing to assess pathogen reduction treatments on platelet concentrates [16]. However, there is no consensus on one method for pathogen reduction treatment of plasma or platelets, nor a well-established pathogen reduction treatment for whole blood or red cell components. Several pathogen reduction methods have been proven to reduce a broad range of bacteria and enveloped viruses in blood transfusion products, while being less effective

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against non-enveloped viruses, and achievement of complete sterility is still a concern. Furthermore, reports have observed collateral damage to the transfusion products caused by pathogen reduction treatments, i.e. lower recovery or shorter circulation time of these products in blood circulation of recipients [20]. 1.2. Quality control of transfusion products – storage lesion In clinical practice, the acceptability of any blood component for transfusion is dependent on its in vivo functional quality, which is highly dependent on the degree of potential induced damage as well as the concomitant or accompanying storage lesion [21,22]. There are several assays in vitro as well as in vivo, which can measure the degree of storage lesion in platelets and red cells. For platelets, the storage lesion is evaluated by several parameters such as metabolic activity (levels of glucose and ATP, LDH release, blood gases, bicarbonate and MTT assay), platelet activation (P-selectin and phosphatidylserine [PS] exposure) and functional assays (i.e. aggregometry). Other physiological parameters such as platelet morphology (swirl), pH, mean platelet volume (MPV) and thromboelastography (TEG) are used. After transfusion into patients, a platelet count increment up to 30,000/μL is expected within 1 hour. The count increment is usually corrected for the patient’s body surface area and dose resulting in Corrected Count Increment (CCI); CCI after 1 hour post-transfusion (CCI-1h) and 24 hours post-transfusion (CCI-24h) is expected to be more than 7.5–10 × 109/L and 4.5–7×109/L respectively. However, for actively bleeding patients, termination of bleeding is a more important clinical endpoint than the posttransfusion platelet count increment [23,24]. For RBCs, it includes hemolysis, concentrations of 2,3DPG, ATP, blood gases, pH, bicarbonate, glucose, lactate, mean corpuscular volume (MCV) and morphology. In addition to this, there must be more than 75% RBC recovery within 24 hour of transfusion in recipients, usually measured with chromium 51 [21,25]. This review discusses the current scenario of various pathogen reduction treatments, their benefits and disadvantages, technologies to determine modification of biological response of blood components due to the pathogen reduction treatment, and finally the current update of clinical trials evaluating the efficacy and safety of pathogen reduction treatments. 2. Pathogen reduction treatments for plasma products Fresh frozen plasma (FFP) has been in demand by hospitals with tremendous emphasis on viral safety. Bacterial contamination is not a serious threat for two main reasons: it is possible to screen or filter the plasma units (to remove bacteria) and have microbiological results ahead while plasma is frozen (although this is not done in practice), but most importantly, plasma is kept frozen and after thawing, plasma is stored at 4 °C for a limited time; such conditions preclude bacteria of replicating or surviving. Several methods have been developed and/or are in use, which aim at targeting potential viral load in plasma products.

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2.1. Pasteurization of plasma products (wet heat treatment) This process has been in use since as early as 1948 to inactivate viruses from albumin fractions [26]. This method is performed at 60 °C for 10 hours, imparting a 6-log reduction of the viral load to the final product with 60–80% recovery of clotting factors. A drawback of this method is that non-enveloped viruses cannot be destroyed [27]. Mostly clotting factors denature during this process unless additional stabilizers are added. Subsequently, these stabilizers can be removed either by diafiltration or chromatography [26]. 2.2. Dry heat treatment Initially the method contemplated sterilization of a plasma container at 80 °C for 72 hours. With this method, there is a 90% recovery of clotting factors. This process inactivates human immunodeficiency virus (HIV), Hepatitis B virus (HBV) and Hepatitis C virus (HCV); however, nonenveloped viruses are not destroyed completely. To circumvent this, dry heat treatment is nowadays performed at 100 °C for 1 hour, leading to inactivation of lipid enveloped and non-enveloped viruses [27]. 2.3. Solvent–detergent method (S-D method) This method has been in use mainly at industrial scale: plasma is collected from donors (up to 10,000 donors per pool, although this can vary among countries) and treated with organic solvent (1% tri-n-butyl phosphate) in combination with virucidal detergent (1% Triton X-100) for 1.5–4 hours at 30 °C. Later in the process, these chemicals are extracted by oil and removed by chromatographic adsorption respectively. This process disrupts the lipid membrane of viruses and further prevents their replication. Non-lipid enveloped viruses like Parvovirus B19 and Hepatitis A virus (HAV) remain unaffected. However, the risk of infection by Parvovirus B19 or HAV is very low due to the presence of neutralizing antibodies in the product pool that very likely will include previously immunized donors [28–31]. The earlier S-D method produced by Vitex leads to some loss of plasma proteins such as α2-antiplasmin and Protein S. These proteins are crucial in homeostasis and the blood coagulation process. Such disadvantages along with a number of thromboembolic adverse reactions led to removal of S-D Vitex plasma from the USA market. Recently, a new generation of S-D produced by Octapharma, so called Octaplas FFP, introducing shortening of the processing time to avoid alteration in the active principals, has been approved by the FDA [31,32]. Currently, the method is applied to single unit and mini-pools (10–12 units) of plasma and studies show promising results overcoming qualitative disadvantages of the past industrial scale S-D method on the plasma product [26,33]. Large-scale industrial pool of S-D plasma is also widely used in Europe and apparently the loss of α 2 antiplasmin and Protein S is acceptable. In The Netherlands, S-D plasma is not advised and therefore not used in the following situations: Protein S deficiency, IgA deficiency (since these patients are treated with IgA depleted plasma

obtained from a pool of selected donors) or in situations where exchange transfusions have to be performed with reconstituted whole blood (due to lack of clinical safety data).

2.4. Methylene blue, riboflavin and amotosalen Methylene blue (MB–Light; Theraflex®; MacoPharma, Lille, France) is used in many European countries for pathogen inactivation of plasma. Unlike the S-D method, it can be applied to the single unit of plasma. Methylene Blue (MB) has a natural affinity for nucleic acids. When MB-treated plasma is exposed to visible light (red/white), a photodynamic reaction generates reactive oxygen species (mostly singlet oxygen), which specifically target the guanine residues of nucleic acids [34]. The majority of enveloped viruses are inactivated. However, non-enveloped viruses, intracellular viruses, protozoa and bacteria remain unaffected, therefore, plasma is generally leukoreduced by filtration. Furthermore, up to 30% of the activity of fibrinogen and factor VIII is reduced after MB–Light treatment. With MB plasma, coagulation assays are prolonged but the remaining fibrinogen activity is enough to sustain interaction with the fibrinogen platelet receptor (GPIIb/ IIIa) [16]. Millions of MB-treated plasma units have been transfused with no adverse effects in Europe [26], suggesting that the loss of these clotting factors is probably acceptable. MB–Light treatment was unable to inactivate non-enveloped virus and also, apprehensions about possible mutagenic effects of MB and its derivatives led to the incorporation of an additional filtration step to remove the residual dye from the final product. In the UK, MB-Light treated plasma is used for recipients under the age of 16 in clinical settings, and no adverse effects were observed in this group [35]. In addition, it was observed that thrombin generation capacity was reduced and clot formation strength was unaffected, although formation was slower, in MB-Light treated plasma [16,36]. As a consequence of anaphylactic reactions reported in patients transfused with MB-Light treated plasma, there have been concerns regarding its use as a routine practice. These reactions were thought to be due to the formation of autoantibodies against an MB antigen in these patients; however, these anaphylactic reactions are very rare [16,37–39]. Recent hemovigilance data (from 2000 to 2011) on safety and allergic events in Greece found that allergic adverse events were fewer in MB-Light treated FFP (1:24,593) compared to untreated quarantine FFP (1:7489), thereby demonstrating the long-term safety of MB-Light treated plasma. Other, UV activated, photochemicals such as amotosalen and riboflavin are routinely used as pathogen reduction treatment of cellular transfusion products (such as platelets and RBCs; see Fig. 1). These systems are also used for treatment of single plasma units. Riboflavin/UVB-based pathogen inactivation treatment (Mirasol®; Terumo BCT, USA) of plasma resulted in 20–30% reduction in fibrinogen, factor VIII and factor XI activities [40]. Amotosalentreated plasma (Intercept® plasma; Cerus, Concord, CA) has been shown to retain pro-coagulant activity up to 80–90% of fresh plasma. Plasminogen and α2-antiplasmin activities are retained up to 94 and 78% respectively [41].


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Fig. 1. Scheme representing electromagnetic wavelength spectra, zooming into the ultraviolet range and positioning UV-light based pathogen reduction treatments. The UVA, UVB and UVC ranges are indicated, and the absorption curves of DNA and RNA throughout the ultraviolet range. Intercept uses UVA, Mirasol PRT uses UVB and Theraflex uses UVC. Note that Theraflex UVC uses a wavelength that targets DNA/RNA at a much higher extent than protein, when compared to Mirasol or Intercept.

2.5. Acid-pH treatment Human intravenous IgG preparations (IVIG) and equine IgG preparations (antivenoms) are subjected to pathogen inactivation by acid-pH treatment. This method involves incubation at pH 4, with or without pepsin, at temperatures from 30 °C to 37 °C, using protein content close to 50 g/L, and for more than 20–24 hours. At this low pH, most of the enveloped viruses are destroyed yet immunoglobulins remain stable [26,42]. 2.6. Nanofiltration This process involves size exclusion to remove virus particles and is also known as viral filtration. Typically, the size of most viruses range from approximately 20 to 200 nm while a nano-filter has a pore size of 15–40 nm. This filtration process reduces the viral load 5- to 6-log, including non-enveloped viruses and preserves protein activity up to 90–95%. Currently most plasma derived coagulation factors and immunoglobulins are nano-filtered either after heat treatment, after pasteurization or after S-D method [26]. As part of the global filtration approach, copper oxidebased filters were proposed by Borkow and colleagues to reduce pathogens in suspension fluids [43–45]. Using this technology, they have shown that it was possible to reduce (up to 1- to 4-log) the load of several viruses (including

enveloped, non-enveloped, DNA and RNA viruses) spiked into the culture media. Copper has a virucidal and bactericidal property and can be used to inactivate these pathogens by damaging their genetic material, membrane or key proteins. These filters are intended to be used along with or without nano-filters. Although this technology needs further study in terms of clinical applications, it can be costeffective and therefore a promising technology for reducing pathogens, especially in developing and poor countries where blood screening and pathogen reduction treatments are not affordable. In summary, until 2014, four pathogen reduction treatments are at present widely used for plasma products (Fig. 2): (1) Solvent–detergent treatment (Octaplas®), Octapharma AG, Switzerland. Octaplas® is an S-D treated pooled human plasma used internationally for the last 20 years. In the year 2013, it has been approved for use in the US by the FDA [32,46,47]. (2) Methylene Blue (Theraflex®); MacoPharma, Mouvaux, CEDEX, France. Theraflex® MB–Light Plasma is in use for the last 10 years in 18 countries [46,48]. (3) Amotosalen (S-59) + UVA (Intercept® plasma; Cerus, Concord, CA). It has been in routine use for the last 8 years in more than 20 countries. It received CE Class III mark approval in 2006 in Europe [46,49]. As of 16

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Fig. 2. Pathogen reduction treatments: products, demands, solutions and geography. Scheme summarizing the three major transfusion products, their storage conditions, the pathogen threats (summarizing two major pathogen threats either in countries with optimal or suboptimal screening infrastructure), the applied pathogen reduction treatments, and the geographical overview of registered use as of July 2013 (http://www.aabb.org/tm/eid/Documents /prt-systems-in-use-country-listing.pdf) [46] and as provided by Terumo BCT or published by the FDA (www.fda.gov) as of December 2014. Note that: white colored countries, there is no available information; black colored countries, treatment not in use; grey (colored online) countries, treatment in use. The fact that a whole country is grey (or colored online) does not mean that a treatment is performed in its geographical totality and does not give information whether the usage is either continued or discontinued, nor does it give information on the specific pathogen reduction treatment used. FFP, frozen fresh plasma; LP, lyophilized plasma; PLT, platelets; RBC, red blood cells.

December 2014, it received approval for use in the US by the FDA (www.fda.gov/BiologicsBloodVaccines /BloodBloodProducts/ApprovedProducts/Premarket ApprovalsPMAs/ucm427204.htm). (4) Riboflavin + UVB (Mirasol®; Terumo BCT, USA). It has been in routine use at many blood centers in many countries. It received CE Class IIb mark approval in 2008 [46,49].

sampling. Therefore, there is a growing demand to eradicate the potential bacterial load in platelet products. Since 2009, FDA approved a rapid test (Pan Genera Detection Test, Verax Biomedical, USA) to detect bacteria from whole blood derived platelets. However, such current bacterial screening methods are not 100% sensitive [50]. The most widely used pathogen reduction methods are summarized below. 3.1. Amotosalen/UVA (Intercept®; Cerus, Concord, CA)

3. Pathogen reduction treatments for platelet products As mentioned before, platelet transfusion products are stored at room temperature (20 °C–24 °C), and this would allow the replication of potential bacterial inoculums present either in the blood of the donor or due to exogenous contamination during collection. Although culture-based systems are proven safeguard methods [6,15], false-negative results have been documented along with loss of product due to the

The synthetic photochemical compound amotosalen hydrochloric acid (also known as S-59) has been extensively used for pathogen reduction in platelets. After illumination with UVA (320–400 nm) light, this compound reacts with the pyrimidine bases of DNA or RNA and forms covalent bonds that result in either intra- or inter-nucleic acid crosslinks (Fig. 1). This crosslinking (one adduct is formed in 1 out of 83 bp) eventually halts replication of a pathogen [51,52]. For treatment with Intercept, platelets are resuspended with plasma


(~30–45%) and platelet additive solution (~55–70%) to which amotosalen (150 μmol/L) is added. The mixture is then exposed to UVA light (3 J/cm2) for 4–6 minutes on continuous agitation. The remaining amotosalen and the photo by-products of this reaction are removed by compound absorption device (CAD) for 4–16 hours at room temperature with agitation. Later, platelets are transferred and stored in standard storage bags. All these transfer steps lead to 10–15% of platelet loss [26,53,54]. A recent study demonstrated that a total of 6 microRNAs (miR-223, miR-17, miR484, miR-146a, let-7g and let7e) and 2 anti-apoptotic mRNAs (Bcl-xl and CLU) were reduced significantly in expression (p < 0.05) after 24 hours in Intercepttreated platelets compared to control platelets [55]. As this effect was evident only in Intercept-treated platelets and not in Mirasol-treated or gamma-irradiated platelets, the authors further speculated that this reduction in expression of microRNAs and mRNAs was related to platelet activation and could result in impairment of platelet function. However, the role of micro-RNAs is not sufficiently established in platelets and needs further confirmatory studies to link the specific loss of expression of micro-RNAs with defects in platelet function. Several clinical trials in Europe and USA were conducted to evaluate the efficacy and safety of Intercepttreated platelets. The data from the euroSPRITE trial suggested that Intercept-treated (buffy coat-derived) platelet components stored for up to 5 days were comparable to conventional platelets used for transfusion support of thrombocytopenic patients [56]. These Intercept-treated platelet concentrates were also tested for safety and carcinogenicity and found to be safe, posing no carcinogenic effect on mice. However, as extrapolated from this study, a tendency to lower – not statistically significant – posttransfusion platelet corrected count increments (CCI) was recorded after 1-hour and 24-hours in patients transfused with Intercept-treated platelets compared to control platelets. The SPRINT trial was performed on a total of 645 patients with thrombocytopenia to determine the hemostatic effectiveness of Intercept-treated platelets compared to control platelets [57]. Although patients transfused with Intercept-treated platelets received more transfusions, had lower platelet CCI post-transfusion and had shorter interval between transfusions, Intercept-treated platelets were hemostatically equivalent to control platelets. The more frequent transfusions needed in the patient group receiving Intercept-treated platelets were explained due to the reduction in effective platelet counts at the time of transfusion in Intercept-treated units compared to control platelet units, despite the treatment being applied to the same amount of platelets/unit (due to 10–15% platelet loss in the process). No unusual toxicity or adverse effects were recorded in the patient group receiving Intercept-treated platelets. Both euroSPRITE and SPRINT trials have used a clinical prototype device of the Intercept processing set. The handling and treatment of platelet units was performed following their own standards and therefore, this led to variability in the platelet number per unit. A clinical trial with 43 thrombocytopenic patients was carried out in Germany with an Intercept commercial prototype device with an integrated processing set and wafer compound adsorption device (CAD)

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[58]. The commercial prototype device introduces standardization of the collection and treatment of the platelet product, assuring less variability in the platelet count per unit between control and treated groups. In this trial, the patient group receiving Intercept-treated platelets did not require more transfusions compared to the control group. Recently, two clinical trials of Intercept-treated platelets have led to different conclusions. A clinical trial in the year 2010 conducted in Dutch hospitals in 278 thrombocytopenic patients revealed that Intercept PCs showed decreased platelet viability and quality (hemostatic capacity) compared to control PCs [59]. In a test group, higher incidences of bleeding were observed compared to the control group (32% Intercept-PC, 19% Control-PC, P = 0·034). This study suggested using this pathogen reduction treatment (Intercept) cautiously in patients in the future as it compromises the hemostatic capacity of platelets. Subsequently, another clinical trial in the year 2011 was conducted in 201 thrombocytopenic patients in several European hospitals [60]. Test and control groups were transfused with PCs treated or not with Intercept and stored up to 7 days. This study suggested that there was not much difference with respect to hemostatic capacity and overall safety profile in PCs of test and control groups. These Intercept clinical trials showed different results and therefore, further phase IV studies are needed to confirm its safety and efficacy. Overall, from these clinical trials that were conducted in different countries, it has been suggested that patients receiving Intercept-treated PCs might require more frequent transfusions due to the somewhat lower platelet quality (function and structure). However, this treatment has not shown any unusual side-effect or toxicity events in patients. 3.2. Riboflavin/UVB based pathogen reduction technology (Mirasol® PRT; Terumo BCT, USA) Riboflavin (vitamin B2) is a naturally occurring photochemical compound and has been used for pathogen inactivation in platelets and plasma. Riboflavin binds to nucleic acids (DNA or RNA) bases and upon UV light illumination (at 265–370 nm wavelengths; see Fig. 1); it oxidizes guanine bases resulting into single strand breaks in nucleic acids. This reaction is a result of direct electron transfer during the oxidation of the guanine bases, which later creates oxygen, peroxide and hydroxyl radicals. These breaks in DNA or RNA are irreversible and cannot be repaired by any endogenous repair mechanism of pathogens. The major advantage of this method is that riboflavin and its byproducts do not need to be removed after UV illumination. This is mainly because riboflavin and its metabolic products are present in natural foods and also in blood. These are “Generally Regarded as Safe” products by the FDA [26,61]. The treatment consists of adding riboflavin to a final concentration of 50 μM in an illumination/storage bag containing the platelet concentrate unit. The riboflavin containing platelet units are then subjected to UV illumination (6.24 J/mL in plasma) in a controlled temperature and agitation for 10 minutes [26,61]. Around 3% of platelets are lost due to transfer from bag to bag [21]. Mirasol PRT is able to reduce the infectivity of many pathogens including HIV, West Nile Virus and several bacteria by 4- to 6-log [62]. Studies have shown

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that Mirasol PRT leads to increased platelet activation [63], shape change from discoid to sphere, induces partial platelet aggregation and also increased platelet glycolysis. The latter was shown to be related to the UV induced stress [64]. Recent studies have found that Mirasol-treated platelets undergo spontaneous aggregation throughout storage, i.e. they are hyperactivated, while ristocetin- and convulxininduced aggregation in Mirasol-treated platelets was reduced compared to control platelets. In addition, Mirasol platelets showed significantly lesser spreading on collagencoated slides compared to control platelets [65]. The functional defects were claimed to be due to uncontrolled basal degranulation, which would result in hyperactivation of platelets and concomitant reduction of the maximum response capacity to aggregation upon specific agonists [65]. The randomized controlled trial (RCT) conducted in France in different hospitals (56 Mirasol-treated PC transfused patients vs. 54 control PC transfused patients) showed that CCI-1h and CCI-24h were lower in the Mirasol-treated compared to the control transfusion group. However, these values were well above the required threshold (CCI-1h and CCI-24h, less than 7.5–10 × 109/L and 4.5–7 × 109/L are indicative of platelet refractoriness), suggestive of successful transfusions. Also, Mirasol-treated platelets did not induce any adverse reactions in patients, indicating that Mirasol PRT is safe for transfusion. However, this trial was not powered to demonstrate a difference in the risk of bleeding after Mirasol-treated platelet transfusions in patients [66]. In a study performed in rats, transfusion of Mirasol-treated platelets prevented alloimmunization and inhibited subsequent heart transplant rejection. Furthermore, the primary and secondary immunoglobulin (IgG and IgM) responses were completely absent in these rats [67]. In a separate study performed with mice [68], transfusion with Mirasol-treated platelets prevented transfusion-associated graft-versushost disease (TA-GVHD) in 14 out of 14 mice whereas 12 mice of 14 transfused with control platelets developed TA-GVHD. 3.3. Theraflex UVC-based pathogen reduction treatment (MacoPharma, Mouvaux, France) The Theraflex UVC treatment uses exposure to short wavelength monochromatic UVC light (254 nm) that has a unique ability to interfere with DNA/RNA replication while preserving protein integrity, exploiting the maximum absorption capacity of DNAs or RNAs which occurs at 260 nm of wavelength (Fig. 1). Short wavelength UVC light leads to the formation of intra-strand or inter-strand cyclobutanepyrimidine and/or pyrimidine-pyrimidone dimers on DNA or RNA, subsequently halting replication [69,70]. The Theraflex UVC treatment of PCs consists of an illumination device (Macotronic UV illuminator) and processing kit (that contains an illumination bag, a storage bag and a sampling bag). The platelet units (in illumination bag) are placed in an UV illuminator under continuous high-speed agitation and exposed to UVC light (0.2 J/cm2 energy with double sided exposure) for one minute. Platelets are then transferred to the storage bag. This whole treatment takes approximately 8 minutes. With this treatment, it is possible to reduce the bacterial load by >4 log; in addition, many viruses are

inactivated, except HIV (~1 log reduction) [69,70]. A recent study has shown that Theraflex UVC treatment also targets T-cell proliferation by reducing it 5-log (in limiting dilution assays) [71]. The in vitro quality of platelets was analyzed with and without Theraflex UVC treatment and it was found that UVC-treated platelets showed slightly higher metabolic activity (glucose consumption and lactate accumulation) and moderate platelet activation (higher P-selectin expression) compared to untreated platelets [69]. In addition, the tolerability and immunogenicity was studied in a dog transfusion model [72]. When dogs were transfused with UVC-irradiated PCs, they showed no indicators for local or systemic intolerance and also no antibodies were detected against UVCexposed plasma or platelet proteins. 3.4. Meta-analyses of clinical trials for pathogen reduction treated platelet concentrates More specific to Intercept and Mirasol, meta-analyses were performed based on previous clinical trials [73,74]. The first meta-analysis study conducted on clinical data derived from 5 RCTs in the year 2011 found that the bleeding complications were mild to moderate in pathogen reduction treated PCs compared to control PCs transfusions [56–59,66,73]. However, in this meta-analysis, 4 out of 5 RCTs were performed with Intercept-treated platelets while one RCT was performed with Mirasol PRT. The second metaanalysis study conducted in the year 2012 included available data of 5 RCTs that only comprised Intercept-treated platelets, showing that Intercept treatment may increase the risk of all (World Health Organization – WHO – Grades 1–4) and clinically significant (WHO Grades 2–4) bleeding complications compared to control platelets, but not the risk of severe bleeding (WHO Grade 4) [56–60,74]. In contrast to the findings of the previous meta-analyses, there was another one performed in 2012 that was based on the same 5 RCTs with a different approach [56–60,75]. The difference between this and previous meta-analysis was the integration of different parameters such as CCI-1h, CCI24h, the intervals between transfusion and bleeding outcomes, but not mild or severe bleeding or clinically significant bleeding as reported earlier [74,75]. This analysis concluded that there were no differences in bleeding risk between Intercept-treated PC and control PC transfusion groups, despite the reduced post-transfusion CCI-24h in the Intercept-treated PCs transfusion group [75]. As both these meta-analyses were performed with different approaches and also all RCTs were not homogenous with respect to the outcome measurements and design, it is a necessity to count on additional clinical studies, where not only CCI, but also bleeding, is taken into consideration. In order to provide further insight, Sanquin Blood Bank (The Netherlands) in collaboration with Terumo BCT has started a randomized controlled Phase III clinical trial in 2010 – Pathogen Reduction Evaluation & Predictive Analytical Rating Score (PREPAReS) – to study the effect of Mirasol PRT in hemato-oncological patients (~618 patients) which require prophylactic transfusions due to severe thrombocytopenia. The primary endpoint for this clinical trial is bleeding (WHO grade ≥ 2 bleeding complications). The results of this clinical trial will be available soon in the public domain [76].


Using PCs from PREPAReS study, Bakkour and colleagues have developed a mitochondrial DNA (mtDNA) amplification real-time PCR assay to see the effectiveness (DNA damage) of Mirasol PRT [77]. They have found that Mirasol PRT inhibits the amplification of long amplicon mtDNA by 1 log compared to the control group and that their assay might be used as a quality control checkpoint for effective pathogen reduction treatment. Intercept, Mirasol and Theraflex pathogen reduction treatments (Fig. 1) received CE certification in 2002, 2007 and 2008 respectively. Until 2014, Intercept and Mirasol are routinely used in some CE countries (i.e. Belgium), while Theraflex is undergoing Phase III clinical trial [78]. As of 19 December 2014, Intercept applied to PCs has been approved for use in the US by the FDA (www.fda.gov /BiologicsBloodVaccines/BloodBloodProducts/Approved Products/PremarketApprovalsPMAs/ucm427204.htm) (Fig. 2). 4. Pathogen reduction treatments for RBC products The demand for pathogen reduction treatments in red blood cell products is not as high as for platelet transfusion products, because the storage temperature (2 °C– 6 °C) does not promote bacterial replication in RBC bags. A pathogen reduction treatment developed by Cerus Corporation, Concord, USA uses S-303 to inactivate pathogens in RBCs. S-303 is a positively charged alkylating compound with 2 groups. The first group is an intercalating agent that locates to the helical region of nucleic acids and the second group is the effector molecule that allows covalent modification of nucleic acid and degrades S-303 to S-300. Glutathione is added to prevent protein damage caused by the procedure. The pathogen reduction process of S-303 compound is intracellular; however it also may interact with other proteins present extracellularly in plasma. A quenching agent (glutathione) is also added along with S-303 to minimize the interactions of S-303 with other proteins in the extracellular plasma. During a Phase III trial, 2 patients developed antibodies against neoantigens (acridine moiety of S-303 on the surface of RBCs). Therefore, this Phase III trial study in chronic anemia patients and other parallel study in cardiovascular patients were halted [79,80]. Recently, Cerus has developed a similar second generation pathogen reduction treatment to be applied in RBC products, with more glutathione (approximately 10 times more) and adjusted pH for the additive solution to minimize acridine moieties on the RBC surface. A Phase I study has shown promising results with no antibody formation in 27 healthy volunteers in the USA, along with demonstration of satisfactory RBC recovery after autologous transfusion [80]. The primary end-point of this study was 24-hour post transfusion recovery and it was similar in the S-303 RBC transfused group and control RBC group (FDA accepted criteria). Other parameters such as hemolysis (reflection of RBC quality) and ATP concentration (reflection of RBC glycolytic metabolism) were measured and found to be similar in both groups; however, the mean life span of S-303 RBCs was reduced (75 days) compared to the control RBCs (88 days). This study also showed complete absence of antibodies against acridine moieties on the RBC surface [80].

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Phase III studies have recently begun in Europe and results will shed more insight into the use of S-303 pathogen reduction treatment in RBCs [21,49]. Of particular interest, as of 2014, a trial has been initiated by Cerus Corporation (S-303 Intercept) in Europe in a cohort of thalassemia major patients. The results of this clinical trial will be in the public domain in 2015 [81] (Fig. 2). 5. Pathogen reduction treatments for whole blood Among all pathogen reduction treatments, Mirasol (Riboflavin/UVB) PRT is currently the single platform used to reduce pathogens in whole blood, either prior to whole blood transfusions, or prior to separation of whole blood into different blood components (plasma, platelets and RBCs). In a recent study, the recovery and viability of RBCs separated from Mirasol treated whole blood using three different UV light doses (22, 33, or 44 J/mL) were analyzed (IMPROVE study Phase I, USA) and it was found that on average, MirasolPRT RBCs (radiolabeled) did not show more than 75% count recovery in the circulation at 24-hour post-transfusion (which is the FDA criteria for acceptability of RBC transfusion) [82]. However, only 3 of the total 11 subjects showed less than 70% recovery while 5 of them showed more than 75% recovery. The RBC life span was 24 ± 9 days in all three UV-light groups, which indicates that survival was also affected in Mirasol-treated RBCs (FDA criteria for acceptability of RBC storage up to 42 days). Hemolysis was 1–1.5% in Mirasol-PRT RBCs stored for 42 days (requirement for hemolysis is less than 1% in USA, and in Europe, less than 0.8%), indicative of storage lesion of RBCs. In another study, Mirasol-treated and control whole blood units stored for 7 days were analyzed for platelet and RBC quality [83,84]. It was found that RBC hemolysis was similar between Mirasol-treated and control RBCs. Also, there was no significant difference between platelet aggregation and adhesion functions in both groups. Investigation in baboons (Mirasoltreated RBCs and during subsequent storage injected on days 0, 21, 42 and 49) found that there was no immunoreactivity or adverse events with Mirasol-treated RBCs and did not induce any antibodies on the surface of RBCs [85]. As of 2014, the prototype for pathogen reduction in fresh whole blood (FWB) was developed by Terumo BCT, USA and it has the same equipment and disposable kit as the CEmarked Mirasol PRT System for platelets and plasma [84]. Phase II and Phase III studies will shed insight into the safety and efficacy of Mirasol PRT on whole blood and/or subsequently produced components. 6. Prion elimination Most pathogen reduction treatments have not been studied sufficiently in order to examine the elimination of prions from whole blood or its components. Several process steps used during plasma fractionation produced a significant reduction (>2 log) in prion load [47,86]. Quinacrine and related heterocyclic compounds have been described to exert anti-prion activity [87,88], however, continuous treatment with quinacrine might lead to the development of drug-resistant or drug-induced mutated prions [89,90]. Therefore, further studies evaluating and comparing other anti-prion agents are ongoing [91]. To circumvent the lack

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of specific, efficient and safe targeting agents, prion reduction filters for blood components have been developed [92,93]. Octaplas has recently included a filter step (OctaplasLG), which as shown by Heger et al. has achieved reproducible and high prion infectivity binding capacity of ≥5·64 log(10) ID(50)/mL gel [94]. This product, prepared exclusively from Dutch donor plasma, has been introduced in The Netherlands under the name OmniplasmaTM in 2013 (Sanquin Blood Bank, Amsterdam, The Netherlands) [95]. 7. Pathogen reduction treatments of immunotherapy products: anti-Ebola serum The recent outbreak of Ebola virus (EBOV) in Western African countries (Guinea, Liberia and Sierra Leone) has produced several concerns about blood transfusion safety. Recently, the WHO has drafted guidelines toward “Potential Ebola therapies and Vaccines” to assist in developing a framework at a global level. WHO suggested the use of “convalescent serum or plasma” as a first therapeutic option to treat patients with EBOV, as currently antiviral treatment or a vaccine is not available. Convalescent blood products are obtained by collecting blood (or plasma) from a donor, who preferably lives in the same geographically infected area to achieve passive immunization in a recipient’s blood [96–98]. Because in most of these areas the frequency of infection with blood borne pathogens is high, measures should be taken to avoid TTIs by treatment with convalescent blood products. Therefore, licensed pathogen reduction treatments such as MB–Light, Intercept and Mirasol-PRT can be used to inactivate possible pathogens in the convalescent plasma. Moreover, although there are no studies available showing inactivation of residual EBOV in the convalescent blood products using these technologies, these treatments should be able to reduce the load of an enveloped virus such as EBOV [99]. 8. New perspectives to study the characteristics and quality of transfusion products As briefly outlined in the first section, there are a number of in vitro tests and clinical parameters that have been well established as to evaluate the functional capacity of a specific transfusion product. The fact that a transfusion product might display functional differences due to a number of parameters such as storage time or, as evidenced in this review, a pathogen reduction treatment would be very valuable to fully characterize the different standardized transfusion products, with the appropriate technologies and/or physiological approaches. Some of these parameters or approaches to consider are mentioned below. 8.1. Proteomics In the last decade, proteomics approaches have been applied in the field of transfusion medicine, which could provide unbiased deep insight into the protein expression changes, protein–protein interactions and post-translational protein modifications in blood components due to the storage or due to a treatment procedure. Various blood components (plasma, platelets, RBCs) have been studied under

different conditions using proteomics approaches in the last decade [100]. It is important to consider that the proteome of RBCs and platelets is on the magnitude of 1000, which allows the analysis of whole-cell proteome differences semiquantitatively. An example of proteomics approach pipeline would consist of protein extraction, separation and protein identification by mass spectrometry (MS). One dimensional or two dimensional sodium dodecyl sulfate– polyacrylamide gel electrophoresis (1D or 2D SDS PAGE) is used commonly to separate proteins (gel-based approach) [101]. In addition, gel-free approaches are also used by some laboratories to analyze protein expression changes, i.e. platelet or RBC lysates, PC supernatants or plasma are processed in solution prior to MS. There are several reports and reviews that consider the use of proteomics related to transfusion medicine [100,102]. A comparative proteomics study found that the majority of proteins that change quantitatively in Mirasol-treated platelets were related to cytoskeleton rearrangements, especially actin, tubulin, filamin, cofilin, VASP, IQGAP2 and ARP 2/3 complex [103]. These evidence supports our previous findings that Mirasol-treated platelets are impaired to spread over collagen coated slides in static conditions [65]. The effect of Mirasol PRT on apheresisderived platelets was studied at the proteome level in the supernatant of PCs using 2-DIGE (2-dimensional gel electrophoresis) technology and it was found that there was not much difference in the supernatant of control and Mirasoltreated PCs at day 5 of storage, except for proteins involved in oxidative stress (accumulation of oxidative glutathione GSSG – a marker for oxidative stress) and metabolism (presence of lactate) in supernatants of Mirasol-treated platelets [104]. Proteomic studies on Intercept-treated platelets identified proteins that showed consistent changes early and late during storage, such as platelet endothelial aggregation receptor 1 precursor, chloride intracellular channel protein 4, and protein-tyrosine sulfotransferase 2, using a gel-free proteomics approach. In this study a total of 721 proteins were identified that showed significant changes comparing Intercept-treated with control platelets [105]. However, in a similar study performed using two-dimensional gelelectrophoresis (2-DIGE), it was found that there were not much protein alterations between the two groups (Intercept platelets and control platelets) in a total of 1882 protein spots [106]. A quantitative proteomics study analyzed the impact on the platelet proteome before and after UVB, UVC and gamma irradiation using 2D-DIGE. In 2D-DIGE, the platelet proteins were labeled with fluorescent dyes and then separated by two-dimensional gel-electrophoresis (2DPAGE) [107]. Very low impact (2 protein spots only) of UVC light was observed on platelet proteome compared to UVB (11 protein spots) and gamma irradiation (45 protein spots) of platelets. None of these proteomics studies clearly explained or linked proteome differences to clinical parameters after transfusion (such as the reduced post-transfusion CCI of platelets or survival after amotosalen treatment). In a recent study, the oxidative damage on peptides was analyzed by LC–MS/MS analysis after Intercept and riboflavin/ UVB (Mirasol) PRT on platelet concentrates and it was found that Mirasol PRT induced more oxidation of peptides compared to Intercept [108]. The identification of oxidative


species containing either disulfide bridge forming dimers or adducts of oxygen atoms (cC, cH, cW, YY and M) was considered for this analysis and these oxidized species were increasingly induced in Mirasol PRT. 8.2. Microvesicles (MVs) and other biological by-products Blood and its components contain MVs that are very small in size (50 nm–5 μm in diameter) [109]. MVs carry different biological materials (proteins, lipids, surface markers, RNAs and microRNAs) from their mother cell and therefore, are able to modulate biological responses of surrounding cells. The content of MVs depends on the mother cell origin and stimulus. Also, the heterogeneous contents present in vesicles bring different downstream functional effects. The isolation and detection of MVs is extremely challenging due to their heterogeneity, small size (less than 100 nm) and sensitivity to handling and collection conditions. Although flow cytometry is a method of choice (see Table 1), vesicles are still sometimes too small to detect as a “single event”. There are a variety of techniques available to study MVs and their effect on blood components, such as Flow cytometry (FACS), electron microscopy (EM), Dynamic light scattering (DLS), Enzyme linked immunosorbent assay (ELISA), Nanoparticle tracking analysis (NTA) or Atomic force microscopy (AFM) [109]; however, there is no gold standard developed yet for the detection of MVs and the study of their impact on other cell components. There are also specialized techniques such as a Raman microspectroscopy, micro Nuclear magnetic resonance (μNMR), Small-angle x-ray scattering (SAXS) and Anomalous Small-angle x-ray scattering (ASAXS) to determine the structure, chemical composition, size distribution, shape, cellular origin and content of MVs. However, these instruments are highly expensive, skill intensive and with limited availability [110,113,114]. Several studies investigated the isolation, characterization and side effect of MVs related to transfusion products. Aatonen and co-workers isolated platelet micro-particles by differential centrifugation [115]. The size distribution of platelet vesicles was studied by NTA and electron

Table 1 Proposed markers for identification of MVs. Exosomes Alix, CD9, CD63, CD81, heat shock proteins, Tumor Susceptibility Gene 101 Microvesicles Phosphatidylserine (detected using annexin V or lactadherin) Cell type marker Leukocyte: CD45, CD11a, CD11b, ICAM-3 Granulocyte: CD66b, ICAM-3 Monocyte: CD14, ICAM-3 Lymphocyte: CD4+ and CD8+ T cells, CD20 (B cells) Platelet: CD41a, CD42a, CD42b, CD31+/CD42+, CD61, CD62b, CD62p, Clec2 Megakaryocyte: CD41, Clec2, GPVI Erythrocyte: CD235a Endothelial cell: CD31+/CD41–, CD62e, CD51, CD105, CD144, CD146 Apoptotic bodies Phosphatidylserine and/or DNA (detected using annexin V or propidium iodide), histones, DNA. Adapted and modified from refs. [110–112].

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microscopy and they found that thrombin-stimulated platelets induced more platelet microparticles than collagen-, LPSand Ca2+ ionophore-stimulated platelets. They have also used a proteomics approach and found that platelet MVs were very heterogeneous within the same control subpopulation or between treatment groups (agonist-stimulated platelets). Recently it has been shown that platelet MVs cannot be removed completely by any of the filters used for leukoreduction, although they are reduced in whole blood, RBC and PCs [111,116]; however, Plasmaflex PLAS 4 filter has been shown to be effective on MV removal. The proteome of platelet MVs was studied by Garcia and co-workers [117]. They found that platelet vesicles express P-selectin, GPIIb, and GPIIIa and also contain chemokines such as CXCL4, CXCL7 and CCL5. Such proteins are able to modify cellular responses and therefore, platelet MVs might have a role not only in hemostasis but also in inflammation, angiogenesis, and cancer metastasis and progression [118,119]. The proteome of RBC MVs was analyzed and it was found that cytoskeleton proteins were absent in vesicles; however, they contain band 3 and actin [120]. Another study found that RBC MVs contain hemoglobin and other proteins less than 70 kDa in molecular weight [121]. Using flow cytometry analysis, a significant increase in the number of RBC MVs per mL in stored RBCs was observed [122]. The presence of several proteins such as factor XI in vesicles initiates and modifies thrombin generation [123]. Future studies are needed to understand better the biological post-transfusion impact of MVs present in transfusion products. Considering this, we think that, especially in pathogen reduction treatment conditions, the study of the impact on MV formation and function should be implemented in in vitro studies and in clinical trials. Since all components in the storage bag are transfused – including MVs and/or released cargo – it should be acknowledged that not only the transfusion product, but its accompanying by-products might fine-tune its response in the recipient. Alternatively, it might be considered in the future to use filters that can remove MVs prior transfusion – such as leukodepletion validated filters also used for prion removal [19,111] – in cases where MVs might cause a threat. Along these lines, it has been recently described that stored platelets release mitochondria [124], either naked or encapsulated within MVs, and it was demonstrated that the amount of mitochondria in transfusion units was higher in those units that caused acute reactions after transfusion. Measuring mitochondria load in transfusion products might be a relevant parameter to include, especially in patients with higher risk of developing acute reactions. Considering the fact that Bakkour et al. measured a reduction in mtDNA in Mirasol-treated platelets [77], it might be that these treated products are, once more, safer for the recipient, in the way that they might result innocuous to the recipient’s immune balance. These are just a few examples of parameters that could be considered as to fully characterize a transfusion product, and correlate it with in vitro and clinical functional outcomes. Overall, we foresee that a transfusion product database generated with relevant datasets derived from global collaborative efforts might be very informative and

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Fig. 3. Characterization and quality control of transfusion products. Scheme summarizing the current in vitro and clinical functional readouts to evaluate the quality and characteristics of blood transfusion products, including additional perspectives and approaches on their characterization. Generation of a comprehensive database compiling this information will be a fine tool, especially thinking ahead on the possibility and need of developing personalized transfusion therapies.

useful, especially thinking toward personalized transfusion therapies (Fig. 3).

9. Current pathogen reduction treatment scenario in the world 9.1. USA Most pathogen reduction treatments are not available due to several concerns from the FDA (except for Octaplas S-D plasma, and Intercept for plasma and platelet products, recently approved). It is assumed that pathogen reduction treatments could modify, degrade a transfusion product or create toxic products that could be detrimental for the health of recipients of different ages. In order to get approval by FDA, treatments must provide direct evidence of elimination of pathogens from transfusion products. More Phase 3 clinical trials are advised for their implementation [20].

9.2. Europe Most European countries approved to use pathogen reduction treatments in their blood banks with CE mark certification. Four of them (S-D, Intercept, Mirasol PRT and Theraflex MB) have been approved for plasma while three of them (Intercept, Mirasol PRT, Theraflex UVC) have received CE mark for platelets. These treatments are currently in use in several European countries while Phase III clinical trial for Theraflex UVC-treated platelets is planned by MacoPharma [78].

9.3. Developing and poor countries Prevention, detection and inactivation of pathogens are three pillars of any successful and efficient blood safety program. As far as prevention in developing and poor countries is concerned, donor education, donor selection, skin


sterilization before phlebotomy and training of staff personnel are very essential steps [125]. Management of the blood supply chain is still very expensive in developing and poor countries. All viral markers are not screened routinely in blood banks and hospitals, mainly due to costly technologies and reagents. Half of blood donations in developing and poor countries are sourced from family members or paid donors. Voluntary blood donation is not as common as in more developed nations. Such paid blood donors potentially hide important disease information or donate within a time-window in which a potential infectious disease is not yet detected. This practice puts blood supply chain at higher risk of carrying viral infections. This unsafe blood supply is detrimental to public health, results into 8–16 million HBV infections, 2.3–4.7 million HCV infections and 80,000–160,000 HIV cases every year in these countries [26]. Pathogen reduction treatments can be a solution in these countries to reduce pathogens in the blood banks and also to improve current blood safety; however, it will require an investment for the blood banks with an additional increase in the cost per transfusion unit. Use of pathogen reduction treatments at great extent compels the use of specialized instrumentation, along with trained staff. Therefore, use of blood screening with or without pathogen reduction treatments is still not a practically and economically viable option for many of these countries. New viable, economical, inexpensive alternative pathogen reduction methods should be studied and addressed as early as possible [125] (Fig. 2 summarizes worldwide usage of pathogen reduction treatments). Along these lines, a number of alternative pathogen reduction treatment approaches, such as CryoFacets (CryoFacets, Raleigh, NC, USA) technology, are being documented [126]. In particular, this treatment is conceived to be performed prior to transfusion (which would minimize costs). Nevertheless, the problem with post-storage pathogen reduction treatment is the potential presence of lipopolysaccharide (LPS) if the transfusion product was contaminated with bacteria, and thereby might cause a problem to the patient even if bacteria have been inactivated prior to transfusion. More studies (such as in vitro, in vivo and clinical trials) are needed in order to bring these technologies into the market. 10. Conclusion and future perspective For the last two decades, pathogen reduction treatments have been grasping considerable importance in the blood transfusion community. So far, there have been numerous in vitro, in vivo and clinical studies, which ratified their effectiveness in reducing pathogens at several blood banks. Pathogen reduction treatments are easy to perform and take few minutes to reduce pathogens from a given transfusion product, however, it comes with an added cost per transfusion unit because they require specific instrumentation. To date, there is no universal pathogen reduction treatment for all different blood products. Among all treatments, Mirasol PRT and Theraflex-UVC have a good chance to position themselves as Universal PRT (with several confirmatory studies needed) in the future. Nevertheless, pathogen reduction treatments cannot replace completely

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viral screenings at blood banks to avoid TTIs; they can possibly complement them to strengthen blood safety and transfusion service. For plasma, it is an almost universally accepted standard to have complete viral protection. In developed countries there is a sense that the current blood supply is safe and that pathogen reduction treatments might result into products with poorer clinical effectiveness. Nevertheless, safety is an issue, and at the end of 2014, in various blood centers and/or countries in Europe one or more of the available pathogen reduction treatments for either plasma, platelets or both are implemented. The fear of possible side effects and adverse events in recipients or patients has halted progress of pathogen reduction treatment entry into the US market for a long time, and although FDA approval has been recently given to some of the treatments, wide usage at blood centers in the USA on a day-to-day basis will require considerable time. However for developing and poor countries, the cost and infrastructure of pathogen reduction treatments can be detrimental factors for their initial introduction in the blood transfusion chain, suggesting that a parallel strategy tackling the costs of existing pathogen reduction treatments (or alternatives) should be developed. It is in these countries, however, when an investment on such infrastructure (pathogen reduction treatment) might be advantageous to substitute viral screenings and still guarantee a safer product, which will be more cost-efficient in the long term. Similarly, whole blood pathogen reduction treatments prior blood component separation need to be considered in such countries to improve blood safety overall. Efforts should focus on improvement of storage of the pathogen reduction treated transfusion products, for example by using alternative storage solutions or storage bags; on examining their effect on cellular by-products (such as MVs); or developing alternatives, such as using other (photo) sensitizers that have less effect on the cells and/or fewer clinical side effects to recipients. In summary, many groups worldwide are focusing their research from divergent points of view into transfusion products, treatments and their function. We foresee that a transfusion product database generated with relevant datasets derived from global collaborative efforts might be very informative and useful, especially thinking toward personalized transfusion therapies, when a patient has a certain deficiency, complication or incompatibility (Fig. 3). Having knowledge at even the molecular level of the different transfusion products might even condition to use a specific treated-transfusion product or to complement it with a missing factor. Acknowledgements V.S. was supported by the Center for Translational Molecular Medicine (CTMM, www.ctmm.nl), project Innovative Coagulation Diagnostics (INCOAG, grant 01C-201), and the Dutch Heart Foundation. References [1] WHO. Blood safety and availability fact sheet. p. Fact Sheet Nr. 279; 2014. [2] American Cancer Society. Blood transfusion and donation; 2013.

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