Effect of increased agitation speed on pathogen inactivation efficacy and in vitro quality in UVC-treated platelet concentrates.

BACKGROUND AND OBJECTIVES: Pathogen inactivation technologies require continuous development for adjustment to different blood components and products. With Theraflex UV-Platelets, a system using shortwave ultraviolet C (UVC) light (254 nm), efficient mixing of platelet concentrates (PCs) during UVC treatment is essential to ensure homogeneous illumination of the blood components. In this study, we investigated the impact of increasing the agitation speed during UVC treatment on pathogen inactivation capacity and platelet quality. MATERIAL AND METHODS: The pathogen inactivation efficacy of UVC treatment was evaluated at two agitation speeds (110 vs. 180 rpm) using four different transfusion-relevant bacteria strains and three model viruses. Using a pool-and-split design, the in vitro quality of buffy coat-derived PCs stored in SSP+ additive solution for up to 7 days was assessed in UVC-treated PCs agitated at either 110 rpm (standard speed) or 180 rpm (increased speed) and in untreated controls. RESULTS: The higher agitation speed improved bacterial inactivation but did not influence viral inactivation. Metabolic activity (glucose consumption and lactate accumulation) in UVC-treated platelets was slightly higher than in untreated controls. Increases in parameters such as CD62P expression and annexin A5 binding indicated moderate activation of UVC-treated platelets. Quality variables for UVC-treated platelets agitated at standard vs. increased agitation speed were comparable. CONCLUSION: The mixing rate during illumination may be a process parameter for further development of UVC-based pathogen inactivation procedures for PLT concentrates.

Two pathogen reduction technologies--methylene blue plus light and shortwave ultraviolet light--effectively inactivate hepatitis C virus in blood products.

BACKGROUND: Contamination of blood products with hepatitis C virus (HCV) can cause infections resulting in acute and chronic liver diseases. Pathogen reduction methods such as photodynamic treatment with methylene blue (MB) plus visible light as well as irradiation with shortwave ultraviolet (UVC) light were developed to inactivate viruses and other pathogens in plasma and platelet concentrates (PCs), respectively. So far, their inactivation capacities for HCV have only been tested in inactivation studies using model viruses for HCV. Recently, a HCV infection system for the propagation of infectious HCV in cell culture was developed. STUDY DESIGN AND METHODS: Inactivation studies were performed with cell culture-derived HCV and bovine viral diarrhea virus (BVDV), a model for HCV. Plasma units or PCs were spiked with high titers of cell culture-grown viruses. After treatment of the blood units with MB plus light (Theraflex MB-Plasma system, MacoPharma) or UVC (Theraflex UV-Platelets system, MacoPharma), residual viral infectivity was assessed using sensitive cell culture systems. RESULTS: HCV was sensitive to inactivation by both pathogen reduction procedures. HCV in plasma was efficiently inactivated by MB plus light below the detection limit already by 1/12 of the full light dose. HCV in PCs was inactivated by UVC irradiation with a reduction factor of more than 5 log. BVDV was less sensitive to the two pathogen reduction methods. CONCLUSIONS: Functional assays with human HCV offer an efficient tool to directly assess the inactivation capacity of pathogen reduction procedures. Pathogen reduction technologies such as MB plus light treatment and UVC irradiation have the potential to significantly reduce transfusion-transmitted HCV infections.

Testing genotoxicity and cytotoxicity strategies for the evaluation of commercial radiosterilized fetal calf sera.

Effects of 18 commercial lots of fetal calf serum (FCS) after gamma-irradiation and their non-irradiated counterparts were comparatively analyzed on CHO-K1 and MDBK MDL1 cells for genotoxicity [sister chromatid exchange (SCE), micronuclei (MNi), and single cell gel electrophoresis (SCGE)], cytotoxicity [cell-cycle progression (CCP), proliferative replication index (PRI), mitotic index (MI), growth promotion (GP), and plating efficiency (PE)], and microbiological properties (mycoplasma and bovine viral diarrhea virus contamination). SCE and SCGE were the most informative end-points for genotoxicity since significant differences were found in 44.4% (P<0.05-0.001, Student's t-test) and 61.1% (P<0.05-0.001, chi(2) test) samples, respectively. MI was the cytotoxicity assay revealing the greatest variation, showing differences in 66.7% (P<0.05-0.001, chi(2) test) samples. Thus, these three end-points for screening bioproducts such as FCS were found most suitable for detecting potential geno-cytotoxicants in biological samples; their simultaneous use could be strongly recommended.

Study on continuous (254 nm) and pulsed UV (266 and 355 nm) lights on BVD virus inactivation and its effects on biological properties of fetal bovine serum.

Both continuous UV lights and pulsed UV lasers have potentials to inactivate known and emerging viruses. Bovine viral diarrhea virus (BVDV), from the Pestivirus genus, is known to be a common viral contamination in (fetal) bovine serum (FBS). Also, BVDV has been used in the blood product industry as a surrogate for Hepatitis C virus (HCV), due to its similarity in structure and genome. Germicidal UV lamp with the wavelength of 254 nm and Nd:YAG laser (pulsed UV laser) in its third and fourth harmonic with the wavelengths of 355 and 266 nm, respectively, were used. BVDV suspended in PBS or FBS were exposed to different intensities and doses and then reduction in BVDV titer were calculated. To complete inactivation of BVDV suspended in PBS and PBS containing 5% FBS, 1.6 (t=30 min) and 3.2 (t=60 min)J/cm(2) were used. The minimum doses for inactivation of BVDV suspended in PBS with the 355 and 266 nm of pulsed UV laser were 352 and 92.25 J/cm(2). Also, the minimum doses for inactivation of BVDV suspended in FBS with 355 and 266 nm wavelengths of pulsed UV laser were 704 and 127 J/cm(2). To evaluate the irradiated FBS quality to support cell culture growth, FBS was treated with the dose of 190.5 J/cm(2) and 266 nm pulsed UV laser and was used to grow Vero cells, in comparison with a control group. The viability of cells in two groups was identical and the statistical evaluation showed no significant difference in 12 passages.

Potential and limitation of UVC irradiation for the inactivation of pathogens in platelet concentrates.

BACKGROUND: Pathogen contamination, causing transfusion-transmitted diseases, is an ongoing concern in transfusion of cellular blood products. In this explorative study, the pathogen-inactivating capacity of UVC irradiation in platelet (PLT) concentrates was investigated. The dose dependencies of inactivation of several viruses and bacteria were compared with the effect on PLT quality. STUDY DESIGN AND METHODS: The potential of UVC irradiation was studied with a range of lipid-enveloped (LE) and non-lipid-enveloped viruses (NLE) and bacteria. LE viruses were bovine viral diarrhea virus (BVDV), human immunodeficiency virus (HIV), pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), and vesicular stomatitis virus (VSV). NLE viruses were canine parvovirus (CPV) and simian virus 40 (SV40). Bacteria were Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Bacillus cereus. After spiking and irradiation, samples were tested for residual infectivity and reduction factors (RFs) were calculated. Furthermore, the effect of UVC irradiation on PLT quality was determined by measuring in vitro quality variables. RESULTS: A UVC dose of 500 J per m(2) resulted in acceptable PLT quality (as measured by pH, lactate production, CD62P expression, and exposure of phosphatidylserine) and high RFs (>4 log) for CPV, TGEV, VSV, S. epidermidis, S. aureus, and E. coli. Intermediate RFs (approx. 3 log) were observed for BVDV, PRV, and B. cereus. Low RFs (approx. 1 log) were found for HIV and SV40. No differences in virus reduction were observed between cell-free and cell-associated virus. CONCLUSION: UVC irradiation is a promising pathogen-reducing technique in PLT concentrates, inactivating bacteria, and a broad range of viruses (with the exception of HIV) under conditions that have limited effects on PLT quality. Further optimization of the UVC procedure, however, is necessary to deal with blood-borne viruses like HIV.

The use of phenothiazine dyes to inactivate bovine viral diarrhea virus in goat colostrum.

The objectives of this study were to determine the optimal concentration of phenothiazine dye required to inactivate bovine viral diarrhea virus (BVDV) in goat colostrum following 60 min of illumination and determine if immunoglobulin concentration is affected by this technique. In addition, the potential of continuous agitation of colostrum during illumination to affect viral kill was investigated. This experiment was designed to more closely approximate on-farm use than a previous pilot study performed by the same investigators. Bovine viral diarrhea virus was used as a model for caprine arthritis-encephalitis virus. Goat colostrum containing BVDV was illuminated for 60 min following the addition of either methylene blue (MB) or methylene violet (MV). Four different concentrations of each dye were evaluated. Illumination was performed in a small, portable chest-type freezer equipped on the inside with white fluorescent lights. Some samples were continuously rocked during illumination, while others remained stationary. Virus levels were determined before and after illumination. Immunoglobulin concentrations were determined for time 0 and 60 min. One microM MB reduced virus to undetectable levels following 60 min of illumination. A concentration of 20 microM MV was required to reduce virus levels to zero. Agitation of colostrum samples had no effect with either MB or MV on whether virus levels were reduced. High concentrations of MB and MV had no important effect on immunoglobulin concentrations.

The band III ligand dipyridamole protects human RBCs during photodynamic treatment while extracellular virus inactivation is not affected.

BACKGROUND: Recently, the potential usefulness of dipyridamole (DIP) in protecting RBCs against the harmful side effects of photodynamic sterilization was demonstrated. In the present study, the use of DIP for selective protection of RBCs was investigated under conditions more relevant for blood bank practice. STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9-dimethylmethylene blue and red light, and the influence of the inclusion of DIP on photohemolysis was assessed as a function of sensitizer concentration, light dose, and storage time. Furthermore, the possible interference of DIP with inactivation of extracellular virus by use of a panel of different viruses (HIV-1, pseudorabies virus [PRV], bovine viral diarrhea virus [BVDV], VSV, encephalomyocarditis, and canine parvovirus) was investigated. RESULTS: In WBC-reduced RBC suspensions (30% Hct), DIP exerted a clear protective effect against photohemolysis. Part of this protection was achieved with concentrations near the dissociation constant for band III binding. Importantly, efficiency of inactivation of extracellular HIV-1, PRV, BVDV, and VSV was not significantly impaired by the inclusion of DIP. Phototreatment conditions, resulting in a 4 to 5 log inactivation of extracellular HIV-1 and PRV, resulted in a high level of hemolysis after 28 days of storage. This long-term hemolysis could be decreased, but not completely prevented, by the inclusion of DIP. CONCLUSION: Photohemolysis in RBC concentrates can be reduced substantially by the application of DIP, while the efficacy of inactivation of HIV-1 and other viruses remains unchanged.

Strategies for evaluation of enveloped virus inactivation in red cell concentrates using hypericin.

Photodynamically induced virus inactivation appears promising in preventing transmission of enveloped virus infections in transfusible blood products. The potential for utilizing hypericin as a photosensitizer to inactivate key enveloped viruses in packed red cell concentrates (PRC) was evaluated. In addition to inactivating effectively > or = 10(6) TCID50 of human immunodeficiency virus (HIV), inactivation of bovine viral diarrhea virus (BVDV) in PRC was used as a model for hepatitis C virus to overcome the deficiency in reliable experimental systems for hepatitis C virus (HCV) inactivation. BVDV was two orders of magnitude more sensitive to inactivation by hypericin than HIV. As part of the virucidal efficacy analyses, the effects of photosensitization on hemopoietic cell lines carrying quiescent integrated HIV provirus were studied as models for evaluating virus inactivation in latently infected cells. Phorbol ester-induced virus production by these cells was effectively prevented by photosensitization with hypericin. A refinement of the illumination conditions, incorporating a monochromatic sodium light source with an emission spectrum coinciding with the absorption peak of hypericin, was highly virucidal, however, caused unacceptable levels of hemolysis. Red blood cells could be protected from phototoxic cellular damage by complexing hypericin with human serum albumin (albumin-hypericin), but the decrease in hemolysis was at the expense of virucidal efficacy. Thus, excitation of hypericin with a fluorescent source appears to be useful potentially for virus inactivation in PRC.

Efficient inactivation of viruses and mycoplasma in animal sera using UVC irradiation.

Transmission of viruses by animal sera represents a considerable risk for humans and animals particularly when the serum is used for the production of pharmaceutical products such as vaccines. Procedures applicable for inactivating large numbers of different viruses, both enveloped and non-enveloped, are therefore mandatory. For this purpose we have developed and validated UVC irradiation as the virus-inactivation procedure of choice for serum to be used in an industrial setting. Spiking experiments in foetal calf serum (FCS) were performed by independent contract laboratories and revealed constantly high clearance rates for various viruses such as bovine parvovirus, parainfluenza type III virus, bovine diarrhoea virus, foot-and-mouth disease virus and different forms of mycoplasmas. UVC-treated sera maintained their growth-promoting activities for various cell types (MRC-5, Vero, CHO). Conventional growth curves generated in the presence of 10% and 1% UVC-treated FCS differed only slightly from controls, indicating the lack of significant damage during UVC exposure. Experiments using a sensitive photometric-based acid phosphatase assay (APA), which correlates well with the more tedious cell counting procedure, confirmed these findings even in the presence of minimal serum requirements. UVC treatment of animal sera appears advantageous compared to currently recommended inactivation procedures, such as Gamma irradiation, for at least three reasons: (i) it possesses a high inactivation capacity for parvoviruses, a pathogen that cannot be destroyed easily by conventional methods; (ii) it causes no noticeable impairment in cell growth and (iii) it can be performed in a controlled manner at the production site.

The Sterways process: a new approach to inactivating viruses using gamma radiation.

The Sterways process (Process) provides a means of terminally sterilizing and virally inactivating blood products. The Process uses gamma radiation in a controlled manner to achieve maximum viral kill with minimum product loss. This goal is achieved by controlling the dose rate for the particular product and formulation in question. Because the Process uses gamma radiation it not only inactivates known viruses but also emerging or mutilating viruses.

Preservation of red cell properties after virucidal phototreatment with dimethylmethylene blue.

BACKGROUND: All published reports have described methods for virus photoinactivation which significantly alter red cell (RBC) properties during storage. In order to improve virucidal activity and reduce damage to RBCs, a series of phenothiazine derivatives were either synthesized or purified and screened for bacteriophage inactivation and red cell potassium efflux. One compound, 1,9-dimethylmethylene blue (dimethyl-methylene blue), had superior screening results and was chosen for further characterization. STUDY DESIGN AND METHODS: White cell reduced RBC suspensions (30% hematocrit) were deliberately inoculated with extracellular virus or virus-infected VERO cells, incubated with 4 microM dimethyl-methylene blue and illuminated with cool-white fluorescent light. Control and treated samples were titered for virus inactivation. In parallel studies, RBC suspensions were exposed to dimethylmethylene blue and light under identical conditions and assayed for in vitro RBC storage properties. RESULTS: Phototreatment of RBC suspensions inactivated > 4.4 log10 of extracellular vesicular stomatitis virus (VSV), > 3.0 log10 of intracellular VSV, > 5.0 log10 of extracellular pseudorabies virus (PRV), > 4.8 log10 of intracellular PRV, > 4.7 log10 of extra-cellular bovine virus diarrhea virus, 5.8 log10 of bacterio-phage phi 6 and > 7 log10 of bacteriophage R17. Encephalo-myocarditis virus, a nonenveloped picornavirus, was resistant to photoinactivation. Virucidal conditions resulted in no detectable IgG binding in 11 of 13 samples, unchanged RBC morphology, normal banding patterns of RBC membrane proteins on SDS PAGE, and unaltered characteristics of 12 of 13 RBC antigens during storage as measured by antibody titrations. In addition, minimal changes were observed in RBC osmotic fragility, lysis, potassium efflux, ATP and 2,3-DPG levels, and the strength of one RBC antigen during storage of phototreated samples compared with controls. CONCLUSION: Dimethylmethylene blue photo-treatment can inactivate several intracellular and extracellular model viruses under conditions which minimally alter RBC properties during 42 days storage at 1-6 degrees C.

Comparison of two different methods for inactivation of viruses in serum.

In order to compare protocols for inactivation of viruses potentially present in biological specimens, three different model viruses were treated in bovine serum by two different inactivation methods: samples were subjected either to chemical inactivation with ethylenimine (El) at concentrations of 5 and 10 mM at 37 degrees C for periods up to 72 h or to electron-beam irradiation in frozen and liquid form with doses varying between 11 and 46 kGy. The chemical inactivation resulted in nonlinear tailing curves in a semilogarithmic plot of virus titer versus inactivation time showing non-first-order kinetics with respect to virus titer. The time for inactivation of 7 log10 units of porcine parvovirus (PPV) was about 24 h for both El concentrations, whereas 5 log10 units of bovine viral diarrhea virus (BVDV) was inactivated in 2 h for both El concentrations and 6 log10 units of porcine enterovirus (PEV) was inactivated within 3 h. The inactivation with electron-beam irradiation resulted in almost linear curves in a semilogarithmic plot of virus titer versus irradiation dose, reflecting a first-order inactivation. The rate of inactivation was almost twice as fast in the liquid samples compared to the rate in frozen ones, giving values of the doses needed to reduce virus infectivity 1 log10 unit for inactivation of PPV of 11.8 and 7.7 kGy for frozen and liquid samples, respectively, whereas the corresponding values for BVDV were 4.9 and 2.5 kGy, respectively, and those for PEV were 6.4 and 4.4 kGy, respectively. The nonlinear inactivation with El makes it impossible to extrapolate the curves beyond the virus detection limit and thereby predict the necessary time for complete inactivation, i.e., to a level beyond the detection limit, of virus in a given sample. The first-order inactivation obtained with electron-beam irradiation makes such a prediction possible and justifiable. The two methods are discussed with respect to their different kinetics and applicability under different circumstances and criteria for inactivation, and considerations for choice of method are discussed.