Pathogen Reduction Technologies: The Best Solution for Safer Blood?

Although it is generally accepted that blood has never been safer than today, transfusion-associated side effects, particularly infective, still occur. Unlike screening strategies, pathogen reduction technologies offer a new approach to increase blood safety by actively/directly targeting possible, also emerging pathogens or donor leukocytes. Advanced technologies for cellular blood products like the psoralen-based INTERCEPT BLOOD SYSTEM or the riboflavin-based Mirasol pathogen reduction technology system have extensively been examined and are on the way to enter the blood bank routine. However, as with any medical treatment, the transfusion of pathogen reduced blood products is not completely risk-free. Due to possible impairment of the treated blood cells the transfusion success is significantly lower as compared to untreated blood products. Long-term side effects concerning the photosensitizers and their photoproducts still remain a matter of debate. This paper outlines current pathogen reduction technologies but also focuses on ethical concerns associated with the employment of these techniques. *Corresponding author: Susanne Maria Picker, University Hospital of Cologne, 65 Lippstadt, Germany, Tel: +49-2941-62842; E-mail: Susanne.picker@uk-koeln.de Received October 01, 2012; Accepted October 22, 2012; Published October 24, 2012 Citation: Picker SM (2012) Pathogen Reduction Technologies: The Best Solution for Safer Blood? J Blood Disorders Transf 3:133. doi:10.4172/2155-9864.1000133 Copyright: © 2012 Picker SM. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


The Rationale for Pathogen Reduction Technologies
Meanwhile, bacterial contamination, especially of platelet (PLT) concentrates (PCs) due to their storage at ambient temperature allowing for bacterial proliferation, is recognized as the most common cause of transfusion-transmitted infections. Up to 0.6% of PCs from routine production might be bacterially contaminated [6], and the estimated mortality risk of severe post-transfusion sepsis ranges from 1:230,000 to 1:625,000 donor exposures [7][8][9]. Thus, bacterial (not viral) contamination might become the driving force leading to broad implementation of pathogen reduction technologies (PRTs) that are able to inactivate small numbers of bacteria (even when anaerobic) below the detection limit of screening methods. The latter require time for the organisms to proliferate prior to detection (generally >36 hours) [10,11] and implicate the risk of false -positive or -negative results [12]. Of note, applied virus inactivation steps like solvent-detergent, β-propiolactone treatment or nanofiltration have markedly increased the safety of plasma (derivates), but are not suitable for cellular blood products, as they irreversibly damage cellular membranes and function.

Pathogen Reduction Technologies for Cellular Blood Products
Since the beginning of the 1990s, considerable progress has been made in the development of PRTs for cellular blood products, and some have already entered routine blood bank users, thus clinical practice. Such PRTs are based on photosensitizers, which are added during processing and, after being activated, generate active oxygen species or utilize electron transfer processes that are oxygen independent to predominantly damage nucleic acids (photodynamic reactions). Another class of agents irreversibly form covalent cross-links in nucleic acids (photochemical reactions) to prevent transcription, translation, and growing of the pathogen. The rationale for targeting nucleic acids is that pathogens and white blood cells require nucleic acid function not required for the therapeutic effects of PLTs, plasma, and red blood cells (RBCs). The reduction capacity should reach at least 4-6 log10 steps. Currently, the most intensive studied dyes with photodynamic properties are the essential vitamin B2 (riboflavin RB) and the phenothiazine derivative methylene blue (MB), while dyes with photochemical properties include psoralens (PS) like S-59 (amotosalen-HCl) and S-303 or the inactine PEN110. The latter compounds also interfere in nucleic acids by alkylation chemistry but become activated by other mechanisms than an external light source, i.e. upon pH shift, which is important for RBCs, whose hemoglobin strongly absorbs ultraviolet (UV) light. 35 ml of RB (at a final concentration of 50 µM) is added to the respective blood product, which then is illuminated with UV light (265-370 nm, 4-6 min, dose 6.2 J/ml). Most of the applied energy is in the UVB range (280-315 nm), a lesser amount in the UVA range (315-400 nm). The peak wavelength (313 nm) preferentially targets RBinduced damage to nucleic acids and does not emit energies, where cytochromes and other essential cofactors for mitochondrial function/ activity absorb. As an essential vitamin, RB and its photoproducts do not require subsequent removal from the treated blood component providing minimal blood cell loss.

The INTERCEPT BLOOD SYSTEM for PCs and plasma
The PS-based system is available and utilized in several European countries including Germany. A total of over 700,000 PS-UVA treated blood products have been transfused, and neither observational studies [26,29] nor the hemovigilance program [30,31] showed any unexpected safety concern. Like Mirasol PRT, the INTERCEPT BLOOD SYSTEM has proven sufficient reduction capacity against many pathogens (up to 4-7 log10 steps) including bacteria. The efficiency, with which nonenveloped viruses can be inactivated, varies strongly. Of note, HAV is not susceptible to inactivation [32].
17.5 ml of amotosalen-HCl (at a final concentration of 150 µM) is added to plasma or PCs resuspended in InterSol (Fenwal, Deerfied, USA) or SSP+ (MacoPharma). Thereafter, the mixture is illuminated with UVA light (320-400 nm, 4-6 min, dose 3 J/cm2). Shorter wavelengths were shown to have detrimental effects on proteins through the generation of active oxygen species. After the photoreduction process, amotosalen and free photoproducts are adsorbed in a compound adsorptions device (CAD) for 10-20 min (plasma) or 4-16 hours (PCs). The treated blood component is then transferred into the final storage bag. Due to CAD treatment and several transfer steps, a volume and blood cell loss of about 12% may be observed [33].

UVC irradiation of PCs and plasma
The THERAFLEX UV-Platelets system is currently under evaluation for its efficacy and safety. The process is based on the application of UVC light (200-280 nm) combined with intensive agitation of the blood component. Since no photosensitizer needs to be added, toxicity-related adverse events associated with such agents can be excluded. The irradiation process results predominantly in the formation of cyclobutane pyrimidine and pyrimidine-pyrimidine dimers blocking the elongation of nucleic acid transcripts. Due to the different absorption characteristics of nucleic acids and proteins, the irradiation process mainly affects leukocytes and pathogens (by at least 4-6 log10 steps), while coagulation proteins and PLT function are largely preserved [34]. UVC irradiation failed to effectively inactivate spores (having a low impact in blood products), West Nile virus and especially HIV (for which screening is performed) [35,36]. Disruption irradiation to prevent transfusion-associated graft versus host disease due to the capacity to inactivate donor leukocytes [13][14][15].

Overview Over the Most Advanced Pathogen Reduction Technologies
Long-term effects of photochemicals used in current PRT methods ( Figure 1) followed by irradiation with visible or UV light cannot completely be excluded even when phase-III trials have been performed. Additionally, photosensitizers bear the potential to induce immune responses in the recipient, who can form antibodies that can bind to the altered blood cells and cause them to be cleared from the circulation. Until now, such an immune response has not been reported for PS-UVA, RB-UV, MB-light, nor UVC irradiation but has been seen for S-303 and PEN110 treated RBCs [16,17]. Studies on PRT treated plasma or PCs are more advanced than those with RBCs or even whole blood (WB). This may be because bacterial contamination is highest in PCs. Moreover, RBCs represent a more difficult environment due to the absorption spectrum of hemoglobin, the higher viscosity, and the prolonged storage time increasing e.g. hemolysis and potassium leakage.

The mirasol PRT system for PCs, plasma and WB
This RB-based system is currently investigated in ongoing clinical trials (IPTAS, PRESS, PREPARES) and available for routine use in several locations in Europe and the Middle East. The system has been shown to be effective against a variety of clinically relevant pathogens (reduction up to 4-6 log10 steps). It has demonstrated 98% efficacy against bacterial strains responsible for most of severe infections following transfusion [27]. To date, the Mirasol PRT system is the only PRT technology that has demonstrated inactivation of the nonenveloped viruses such as the hepatitis A virus (HAV) that is highly resistant to chemical and heat-mediated interventions [28]. The methodology is currently under development for the treatment of WB. Preliminary results suggest good retention of blood cell functionality so that PRT of all blood products using the same system may become achievable in the near future.  of disulfide bonds of the fibrinogen receptor (glycoprotein IIb-IIIa) described for UVC irradiation as "PLT sunburn" [37] appears to only be slightly increased using the THERAFLEX UV procedure as seen from the low increase of free thiol groups on the PLT surface [34].
Resuspended PLTs are transferred into a UVC permeable 19×38 cm illumination bag. Then UVC irradiation (254 nm, 20-30 s, dose 0.2 J/cm2 (1 J/cm2 for plasma)) is performed using a special UVC irradiation device (Mactronic, MacoPharma) where the bags are loosely placed on a quartz plate. Since the pathogen reduction capacity was shown to have its maximum at ≥ 100 rotations per minute (rpm), the plate is agitating at 110 rpm. After illumination, the PLTs are transferred into the final storage container and ready for transfusion without further processing.

The S-303 PRT system for RBCs
The photochemical-based PRT system for erythrocytes has the capacity to effectively reduce pathogens by 4-6 log10 steps but is currently not yet available in routine use [38]. S-303 is composed of an effector (an acridine moiety), a linker (alkyl chain) and an anchor (mustard hydrochloride moiety). It is designed to target nucleic acids, cross-link them via a bis-alkylating group and release a negatively charged nonreactive byproduct (S-300). S-300 is then captured by glutathione (GSH) also added to the blood component to minimize the non-specific reactions with proteins. A second generation system was developed after the observation of an unexpected immune response in 2 of 16 patients suffering from chronic anemia, who required more than a single transfusion of RBCs for therapeutic support [11]. The technology has also shown promise for application to WB [39]. 30 ml of GSH and S-303 in saline are mixed with the blood product (to a final concentration 200 mM GSH and 0.2 mM S-303). The whole mixture is then transferred into a 2nd container to allow both, the pathogen reduction process (30 min) and the decomposition of S-303 to S-300 (6-18 hours). After centrifugation, the supernatant is removed, and the treated RBCs are transferred into the final storage container containing additive solution for storage for up to 35 days at 4 ± 2°C.

The THERAFLEX MB (TMB) Plasma system for plasma
In contrast to PRT treated PCs and RBCs, PRT treated plasma has been in clinical use for several years and proven effective in a variety of therapeutic settings [40,41]. More than 4 million MB treated plasma units including about 2 million TMB treated plasma units have been generated to date [42]. For most enveloped viruses the pathogen kill power reaches at least 6 log10 steps [43], but is considerably less for non-enveloped viruses. TMB treated plasma induces a reduction of clotting factor activities/life spans of 10-35% including ADAMTS-13 [44]. Observational studies in Spain [45] and hemovigilance data from France [46], however, raise concern that MB treated plasma is probably less effective than quarantine plasma in the treatment of thrombotic thrombocytopenic purpura and may induce more severe, partly fatal allergic reactions.
At the start of the current TMB treated plasma system a 0.65 µm membrane filter (Plasmaflex PLAS4, MacoPharma) removes residual leukocytes, RBCs, and PLTs as well as microvesicles and microparticles. Thereafter, the filtered plasma flows pass a dry "pill" containing 85 mg MB ensuring a final concentration of 1 µM for a plasma volume ranging between 235 to 315 ml The following illumination with visible light (590 nm, 20 min, dose 180 J/cm 2 ) is achieved by sodium lowpressure lamps or light emitting diodes in a special device (Mactronic, MacoPharma). After treatment, residual MB and its photoproducts are removed by a special filter (Blueflex, MacoPharma) to an average level of 2 µg/l (0.5 µg /plasma unit).

Data from Preclinical Investigations
PLTs treated with the INTERCEPT BLOOD SYSTEM or the Mirasol PRT system were associated with increased acidity and cell activation (increased p-selectin (CD62P) surface expression), enhanced metabolism (glucose consumption, lactate production), and reduced functional properties (aggregation, extent of shape change, hypotonic shock response HSR, etc.). Probably due to the fact that the applied wavelength energy differs from the absorbance energy of mitochondrial enzymes (370-450 nm), treatment with the Mirasol PRT system allowed the maintenance of the oxidative phosphorylation pathway, which was in contrast to amotosalen-UVA PRT treatment [47,48]. Mitochondrial respiration plays a critical role on PLT behavior during clot formation at sites of vascular injury [49]. In the absence of this functionality, reduced viability [50] and haemostatic effectiveness [51]

Data from Clinical Investigations
Patients transfused with PRT treated PLTs demonstrated reduced post-transfusional corrected count increments (CCIs) leading to an average of 35% more transfusions [24,25,55]. This was mainly considered as the result of the lower PLT dose after PRT treatment due to multiple bag transfers and CAD treatment, but an intrinsic storage lesion development might also have contributed to this finding. From the frequency of bleeding events PRT treated PLTs were hemostatically as effective as their untreated counterparts reinforced by data of a hemovigilance program [30,31] and similar values for invitro aggregability under flow conditions [56] relative to untreated PLTs. However, a recent clinical investigation on PS-UVA treated PCs was stopped prematurely due to significantly more hemorrhagic events [57]. Indeed, decreased shear induced adhesion properties of PS-UVA treated PLTs (although not significant) were observed in our investigation [51]. Hemovigilance programs are likely too underpowered to really detect such discrepancies that might be overcome by increased transfusion doses. Due to equal frequencies of adverse events, PRT treated PLTs were considered as safe as conventional PCs. Although reduced, recovery and survival rates of radiolabeled, PRT treated PLTs were considered as being still acceptable for transfusion [58], even after UVC irradiation [59]. Nevertheless, study sizes appear far too small to draw any firm conclusion in this respect.
Nearly all phase-III studies on S-303 treated RBCs were suspended when 2 of 16 chronically transfused patients developed positive crossmatch reactions to S-303 treated RBCs. The underlying low-titer antibodies (that also could naturally occur [60]) were directed against the surface-bound acridine moiety of S-303 [61]. A 2 nd generation pathogen inactivation process was developed minimizing the amount of RBC-bound acridine. Preliminary results using this container (n=27) [38] indicated that the treated RBCs maintained sufficient viability (24 hour recovery rates of about 88%) and did not induce positive crossmatches [62].

Conclusions
Open questions of current PRTs still remain concerning the extent to which all (un)known pathogens including non-enveloped viruses or prions can be reduced. Before broad implementation, it has to be shown, that the photosensitizers and their photoproducts are extremely safe, robust in daily routine, cost-effective and controllable in their efficacy. Toxicological studies are difficult to perform and may not reveal rare events like carcinogenicity that only can be detected during long-term observation. Any benefit gained from the use of PRT treated blood products may be offset by any incidence of an unanticipated adverse event [63]. Finally, PRTs must preserve sufficient therapeutic effectiveness of the treated blood cells. Compared to untreated PLTs, however, PRT treated PLTs seem to be functionally inferior and led to increased transfusion requirements due to lower CCIs. Whether CCIs have sufficient clinical sensitivity and specificity is still a subject of debate [64]. Evaluation of PLT function before and after transfusion via new approaches (i.e. thromboelastography) or the careful evaluation of bleeding events appears more appropriate highlighting the role of further clinical trials.