Elimination of potential mutagenicity in platelet concentrates that are virally inactivated with psoralens and ultraviolet A light

BACKGROUND: For virus sterilization of platelet concentrates (PCs), treatment with aminomethyltrimethyl psoralen (AMT) and long-wavelength ultraviolet A light (UVA) has shown efficacy. It has been found that treatment with 50 micrograms per mL of AMT and 38 J per cm2 of UVA in the presence of 0.35-mM rutin efficiently kills viruses while maintaining platelet integrity. There is, however, concern about the mutagenic potential of psoralens and UVA (PUVA)-treated PCs. STUDY DESIGN AND METHODS: Adsorption of PUVA-treated PCs with a hydrophobic resin containing C18 as the ligand was used for AMT removal, which was quantitated by the use of radioactive AMT. PUVA-treated PCs, with and without C18 treatment, were examined for solution pH and platelet aggregation response to agonists. In addition, residual AMT activity was determined by AMT's virucidal activity or incorporation into cellular DNA upon a second UVA irradiation and by its mutagenic potential in the Ames test. RESULTS: After PUVA treatment of PCs, residual AMT retained virucidal and adduct-forming ability upon re- exposure to UVA, but activities were less than those observed originally. As has been found previously, AMT had mutagenic potential following incubation in the dark with rat liver S9 microsomal enzymes. The PUVA treatment reduced this potential by 90 percent. C18 adsorption following PUVA treatment had no negative effect on platelet integrity and eliminated 50 percent of the added radioactive AMT. In addition, all detectable virucidal, nucleic acid-modifying, and mutagenic activities of AMT-treated PCs were removed by C18. CONCLUSION: These results suggest that hydrophobic resin adsorption of PUVA-treated PCs will conveniently remove functional psoralens and eliminates their mutagenic potential.     

Virus sterilization in platelet concentrates with psoralen and ultraviolet A light in the presence of quenchers

The virucidal and functional effect of the treatment of platelet concentrates (PCs) with long-wave ultraviolet light (UVA) and the psoralen derivative 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) was studied. Cell-free vesicular stomatitis virus (VSV) was completely inactivated (greater than or equal to 6.5 log10) on treatment of PCs with 25 micrograms per mL (85 microM) of AMT and with 20.7 J per cm2 (30 min) of UVA in the presence of air, or with 82.8 J per cm2 (2 hours) of UVA under conditions of reduced oxygen tension. When treatment was in air, the extent and rate of platelet aggregation in response to collagen measured after overnight storage were reduced to about 70 and 50 percent of control values, respectively; however, aggregation responses were similar to those of controls when PCs were treated under reduced oxygen tension. As a means of eliminating the necessity of oxygen depletion during AMT and UVA treatment, we examined the effects of the addition of quenchers of reactive oxygen species. The presence of 2 mM (2 mmol/L) mannitol during treatment of PCs with 25 micrograms per mL of AMT and 20.7 J per cm2 of UVA in air significantly improved the aggregation response and other in vitro indicators of platelet function and had little or no effect on VSV inactivation. Less benefit was observed with the other quenchers examined. Thus, the nucleic acid specificity of psoralen photoinactivation under reduced oxygen conditions may also be attainable when selected free radical scavengers such as mannitol are present during treatment in air.     

Psoralen-mediated photodecontamination of platelet concentrates: inactivation of cell-free and cell-associated forms of human immunodeficiency virus and assessment of platelet function in vivo

BACKGROUND: Treatment of platelet concentrates (PCs) with psoralens and broad-band ultraviolet A (UVA) radiation is being examined for the elimination of pathogens that might be present in donated blood. Previous studies have demonstrated the inactivation of cell-free viruses and the maintenance of platelet integrity with common in vitro assays. STUDY DESIGN AND METHODS: Human immunodeficiency virus (HIV) in three forms-cell-free, activity replicating, and latently infected cell lines-was added to PCs and treated with 50-microgram per mL of 4'- aminomethyl-4,5',8-trimethylpsoralen (AMT), 0.35 mM rutin, and broad- and narrow-band UVA light (320-400 nm and 360–370 nm [UVA1], respectively). The inactivation of added HIV was assessed in tissue culture; platelet hemostatic activity was assessed in thrombocytopenic rabbits. RESULTS: Each form of HIV was inactivated completely (> or = 10(5) infectious units) on treatment with 30 J per cm2 of UVA1 light. Similar results were obtained on treatment of 2.5 mL of PCs in test tubes or intact PC units (50 mL) in blood bags. Latently infected cell lines were substantially more sensitive than cell-free HIV or HIV that was actively replicating. Human platelets treated with 40 J per cm2 of UVA1 light had a fully corrected bleeding time shortly after treatment or after 5 days' storage, as assessed in thrombocytopenic rabbits. Platelet hemostatic function began to decrease with 81 J per cm2 of UVA1 light and was abolished with 113 J per cm2. At similar fluences, broad-band UVA light was more injurious to platelets than was UVA1 light. CONCLUSION: HIV transmission might be eliminated by PCs after treatment with AMT and UVA1 light and without a reduction in platelet hemostatic function.     

Inactivation of free and cell-associated human immunodeficiency virus in platelet suspensions by aminomethyltrimethylpsoralen and ultraviolet light

BACKGROUND: It has previously been reported that 40 micrograms per mL of aminomethyltrimethylpsoralen (AMT) plus 2.4 to 7.2 J per cm2 of ultraviolet A (UVA) light inactivated 4 to 6 log10 of several model viruses in platelet suspensions. This inactivation was achieved while satisfactory levels of platelet count, pH, morphology, aggregation, and hemostatic effectiveness were maintained. STUDY DESIGN AND METHODS: The efficacy of this procedure for inactivating free and intracellular human immunodeficiency virus (HIV), including integrated proviral sequences, was studied. RESULTS: The kinetics of inactivation for free HIV (4-5 log10 kill with 1.2-4.8 J/cm2) were similar to those obtained for the previously studied viruses. For studies on cell-associated virus, H9 cells productively infected with HIV were added to platelet suspensions and treated with the above regimen of AMT and UVA. The phototreated cells were then cocultivated with uninfected H9 cells for 4 weeks and supernatants were assayed by enzyme-linked immunosorbent assay for HIV p24. No evidence of HIV replication was detectable for cells receiving as little as 2.4 J per cm2 of UVA irradiation in the presence of AMT. Further, it has been demonstrated that stably integrated sequences from the HIV proviral env gene can no longer be amplified by polymerase chain reaction after 1.2 J per cm2 of UVA (with 40 micrograms/mL AMT) exposure. CONCLUSION: These data suggest that AMT and UVA is an effective antiviral treatment for free and cell- associated HIV in platelet suspensions.     

Inactivation of viruses in platelet suspensions that retain their in vitro characteristics: comparison of psoralen-ultraviolet A and merocyanine 540-visible light methods

The ability of two fundamentally different photochemical procedures to inactivate model viruses in platelet suspensions was compared. Merocyanine 540 (MC 540) with visible light was used as an example of an oxygen-dependent chemical-directed at the viral membrane, and aminomethyl trimethyl psoralen (AMT) with ultraviolet A light (UVA) was used as an example of a nucleic acid-directed system. Antiviral conditions in petri dishes were identified and the effects of these procedures on platelet suspensions in plastic storage containers were studied. Concentrations of photochemicals in the 10 to 150 mumol range with 30 to 60 minutes of visible light (MC 540) or 1 to 2 minutes of UVA (AMT) readily inactivated 5 to 6 log10 of vesicular stomatitis virus (VSV) and other model viruses in platelet suspensions, provided the plasma concentration was reduced to about 15 percent by the use of a synthetic platelet storage medium. Extracellular pH, morphology scores, and aggregation response dropped markedly when platelets were treated with MC 540 and visible light. However, treatment with 136 mumol per L of AMT and 1 to 3 minutes of UVA could inactivate 5 log10 of VSV in platelet suspensions with retention of platelet characteristics for 4 days, particularly if oxygen levels were reduced during treatment. These studies demonstrate that AMT-UVA treatment meets the initial requirements for virus inactivation in platelet suspensions.     

Inactivation of viruses in blood with aluminum phthalocyanine derivatives

The inactivation of viruses added to whole blood and a red cell concentrate with aluminum phthalocyanine and its sulfonated derivatives was studied. A cell-free form of vesicular stomatitis virus (VSV), used as a model, was completely inactivated (greater than 10(4) infectious units; TCID50) on treatment of whole blood with 10 microM (10 mumol/L) aluminum phthalocyanine chloride (AIPs) and visible light dosage of 88 to 176 J per cm2. At 44 J per cm2, complete VSV inactivation was achieved on raising the concentration of AIPc to 25 microM (25 mumol/L). Results at least as good were achieved on similar treatment of a red cell concentrate. Also inactivated were a cell-associated form of VSV and both cell-free and cell-associated forms of human immunodeficiency virus; encephalomyocarditis virus, used as a model for non-lipid-enveloped viruses, was not inactivated by this procedure. This inactivation of cell-free VSV suggests that a similar degree of inactivation could be achieved with a lower concentration of the sulfonated forms of aluminum phthalocyanine. Throughout the above studies, red cell integrity was well maintained, as judged by the absence of hemoglobin release (less than or equal to 2%) during the course of treatment or on subsequent storage. Red cell osmotic fragility was decreased on treatment of whole blood with AIPc. This study indicates that AIPc may be a promising method for the inactivation of viruses in cellular blood products.     

Photochemical inactivation of duck hepatitis B virus in human platelet concentrates: a model of surrogate human hepatitis B virus infectivity

BACKGROUND: Photochemical decontamination of platelet concentrates (PCs) has been demonstrated by the use of 8-methoxypsoralen and ultraviolet A light. Systems for studying the inactivation of blood- borne viruses facilitate the evaluation of photochemical decontamination protocols. STUDY DESIGN AND METHODS: Duck hepatitis B virus (HBV), a model for human HBV, was adapted for the study of hepadnavirus inactivation. A highly specific in vitro infectivity assay used primary duck hepatocyte cultures and was followed by the detection of replicated duck HBV sequences. RESULTS: Duck HBV-infected primary duck hepatocyte cultures produced authentic infectious virus. High- titer (> 10(9) virus genome equivalents/mL) duck HBV-infected sera were completely inactivated in serum or PCs by the use of 100 micrograms per mL of 8-methoxypsoralen and 70 J per cm2 of ultraviolet A light. Intracellular duck HBV (> 4.2 log10) in PCs was also inactivated. Culture results were confirmed by a sensitive duckling infectivity assay that indicated that 6.3 log10 of infectious duck HBV had been inactivated by photochemical decontamination. CONCLUSION: The sensitivity of the culture assay was comparable to that of the duckling assay using polymerase chain reaction gene amplification to detect duck HBV. Duck HBV inactivation in PCs was dependent on the dose of ultraviolet A light and independent of 8-methoxypsoralen concentrations of 100 to 300 micrograms per mL: 100 micrograms per mL 8- methoxypsoralen inactivated 4 to 5 log10 of virus in conjunction with 20 to 40 J per cm2 of ultraviolet A light. The polymerase chain reaction-enhanced duck HBV culture system has utility in optimizing photochemical decontamination protocols.     

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.     

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.     

BLOOD COMPONENTS: A novel approach to pathogen reduction in platelet concentrates using short‐wave ultraviolet light

BACKGROUND: Transfusion of platelet concentrates (PCs) is the basic treatment for severe platelet disorders. PCs carry the risk of pathogen transmission, especially bacteria. Pathogen reduction (PR) by addition of photochemical reagents and irradiation with visible or ultraviolet (UV) light can significantly reduce this risk. We present a novel approach for PR in PCs employing UVC light alone. STUDY DESIGN AND METHODS: UVC PR was evaluated by bacteria and virus infectivity assays. PC quality was investigated by measuring pH, lactate, glucose, hypotonic shock response, platelet aggregation, CD62P expression, and annexin V binding as in vitro parameters. The impact of UVC PR on the platelet proteome was assessed by differential in‐gel electrophoresis and compared with changes caused by UVB and gamma‐irradiation, respectively. RESULTS: Vigorous agitation of loosely placed PCs generated thin fluid layers that allow penetration of UVC light for inactivation of the six bacteria and six of the seven virus species tested. HIV‐1 was only moderately inactivated. UVC light at the dose used (0.4 J/cm²) had a minor impact on in vitro parameters and on storage stability of treated PCs. Proteome analysis revealed a common set of 92 (out of 793) protein spots being affected by all three types of irradiation. Specific alterations were most pronounced for gamma‐irradiation (45 spots), followed by UVB (11 spots) and UVC (2 spots). CONCLUSION: UVC irradiation is a potential new method for pathogen reduction in PCs. The data obtained until now justify further development of this process.     

Virucidal levels of ozone induce hemolysis and hemoglobin degradation

The animal virus, vesicular stomatitis virus (VSV), and the bacterial virus, phi 6, were inactivated by greater than 4 log10 in response to incubation with 13 to 14 mL of 1.4 mmol per L (65 micrograms/mL) to 1.6 mmol per L (75 micrograms/mL) of overlaid ozone in virus-spiked, dilute, red cell suspensions. Virus inactivation was greatly inhibited when ozone was overlaid in the presence of high-hematocrit red cells or, to a lesser degree, high levels of plasma. At hematocrits at which 5 to 6 log10 of VSV were inactivated, ozone caused 30-percent hemolysis, as measured by the loss of total cellular hemoglobin. Unexpectedly, this level of hemolysis could not be observed in supernatants because of the ozone-induced destruction (bleaching) of extracellular hemoglobin. These results suggest that ozone may have little biological specificity for damaging viruses over red cells.     

Psoralen photochemical inactivation of Orientia tsutsugamushi in platelet concentrates

BACKGROUND: The risk of transfusion transmission of disease has been reduced by the combination of predonation questions and improved transfusion-transmitted disease assays, but the risk is still present. This study was conducted to determine if psoralen photochemistry could inactivate an obligate intracellular bacterium, with documented potential for transfusion, in PCs to further improve safety. STUDY DESIGN AND METHODS: PCs were inoculated with MNCs infected with Orientia tsutsugamushi. The concentrates were treated with amounts ranging from 0.86 to 138 micromol per L of 4'-(aminomethyl)-4,5',8-trimethylpsoralen hydrochloride (AMT) combined with a constant long-wave UVA light (320-400 nm) exposure of 5 J per cm(2). The effects of photochemical treatment were analyzed by using a mouse infectivity assay along with in vitro testing by PCR, indirect fluorescence antibody, direct fluorescence antibody, and Giemsa staining. RESULTS: AMT, at 0.86 micromol per L or more, combined with UVA light of 5 J per cm(2), inactivated O. tsutsugamushi that contaminated PCs. The PCs that did not receive the combined treatment caused infection. CONCLUSIONS: The psoralen AMT, in conjunction with UVA light exposure, effectively abolished the infectivity of PCs deliberately contaminated with the scrub typhus organism O. tsutsugamushi, as tested in a mouse infectivity assay.     

Trypanosoma cruzi inactivation in human platelet concentrates and plasma by a psoralen (amotosalen HCl) and long-wavelength UV

Trypanosoma cruzi, the protozoan pathogen that causes Chagas' disease, can be found in the blood of infected individuals for their entire life span. This presents a serious challenge in safeguarding blood products. Transmission of T. cruzi from blood products is a frequent occurrence in Latin America, where Chagas' disease is endemic. This study was designed to determine whether T. cruzi could be inactivated in human platelet concentrates and plasma by a photochemical treatment process with long-wavelength UV A light (UVA, 320 to 400 nm) plus the psoralen amotosalen HCl (Cerus Corporation). Units of platelet concentrates (300 ml) and plasma (300 ml) were intentionally contaminated with approximately 10(6) T. cruzi trypomastigotes, the T. cruzi form found in the bloodstream, per ml. The viability of T. cruzi after photochemical inactivation was determined by their ability to replicate in 3T3 fibroblasts. Controls, including treatment with 150 micro M amotosalen or 3 J/cm(2) UVA alone, did not lead to reduction of the viability of T. cruzi in plasma or platelet concentrates. However, treatment with 150 micro M amotosalen plus 3 J/cm(2) UVA inactivated T. cruzi to undetectable levels in plasma and platelet concentrates. This represented a >5.4-log reduction of T. cruzi in platelet concentrates and >5.0-log reduction of T. cruzi in plasma. We conclude that the amotosalen plus UVA photochemical inactivation technology is effective in inactivating high levels of protozoan pathogens, such as T. cruzi, in platelet concentrates and plasma, as has been previously shown for numerous viruses and bacteria.     

Effects of intercept pathogen inactivation on platelet function as analysed by free oscillation rheometry.

INTRODUCTION: The Intercept Blood System, using InterSol as additive solution, is used for inactivation of contaminating pathogens in PCs, thus reducing the risk for transfusion transmitted infection and making it possible to prolong the storage period. This study aimed at investigating the ability of Intercept treated platelets to induce clot formation, as measured by coagulation time using free oscillation rheometry (FOR), and to compare with that of platelets in concentrates with the additive solution T-Sol or plasma. METHODS: Seventy-four single-donor platelet units were diluted in InterSol (n=27) or T-Sol (n=47) to a mean plasma concentration of 38%. The Intercept treatment was performed by addition of amotosalen HCl to the InterSol PCs followed by UVA irradiation and treatment with a compound adsorption device (CAD). Forty-six units were collected and stored in 100% plasma for comparison. Clotting time was measured by FOR in fresh PCs (within 26h after collection) after stimulation by a platelet activator. Soluble P-selectin was analysed as a marker of platelet activation in the Intercept and T-Sol PCs. RESULTS: The clotting time was shorter for Intercept treated platelets compared to platelets in T-Sol and plasma (p<0.05). There was no difference in clotting time between T-Sol and plasma PCs. Soluble P-selectin was higher for Intercept platelets than platelets in T-Sol (p<0.05). CONCLUSIONS: The platelets treated with the Intercept procedure had good clot promoting capacity.     

Inactivation of viruses in labile blood derivatives. II. Physical methods

The thermal inactivation of viruses in labile blood derivatives was evaluated by addition of marker viruses (VSV, Sindbis, Sendai, EMC) to anti-hemophilic factor (AHF) concentrates. The rate of virus inactivation at 60 degrees C was decreased by at least 100- to 700-fold by inclusion of 2.75 M glycine and 50 percent sucrose, or 3.0 M potassium citrate, additives which contribute to retention of protein biologic activity. Nonetheless, at least 10(4) infectious units of each virus was inactivated within 10 hours. Increasing the temperature from 60 to 70 or 80 degrees C caused a 90 percent or greater loss in AHF activity. An even greater decline in the rate of virus inactivation was observed on heating AHF in the lyophilized state, although no loss in AHF activity was observed after 72 hours of heating at 60 degrees C. Several of the proteins present in lyophilized AHF concentrates displayed an altered electrophoretic mobility as a result of exposure to 60 degrees C for 24 hours. Exposure to lyophilized AHF to irradiation from a cobalt 60 source resulted in an acceptable yield of AHF at 1.0, but not at 2.0, megarads. At 1 megarad, greater than or equal to 6.0 logs of VSV and 3.3 logs of Sindbis virus were inactivated.     

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.     

Inactivation of viruses in platelet concentrates by photochemical treatment with amotosalen and long-wavelength ultraviolet light

BACKGROUND: Viral contamination of platelet (PLT) concentrates can result in transfusion-transmitted diseases. A photochemical treatment (PCT) process with amotosalen-HCl and long-wavelength ultraviolet light (UVA), which cross-links nucleic acids, was developed to inactivate viruses and other pathogens in PLT concentrates. STUDY DESIGN AND METHODS: High titers of pathogenic or blood-borne viruses, representing 10 different families, were added to single-donor PLT concentrates containing 3.0 x 10(11) to 6.0 x 10(11) PLTs in approximately 300 mL of 35 percent plasma and 65 percent PLT additive solution (InterSol). After PCT with 150 micromol per L amotosalen and 3 J per cm(2) UVA, residual viral infectivity was assayed by sensitive cell culture or animal systems. RESULTS: Enveloped viruses were uniformly sensitive to inactivation by PCT whereas nonenveloped viruses demonstrated variable inactivation. Log reduction of enveloped viruses for cell-free HIV-1 was >6.2; for cell-associated HIV-1, >6.1; for clinical isolate HIV-1, >3.4; for clinical isolate HIV-2, >2.5; for HBV, >5.5; for HCV, >4.5; for DHBV, >6.2; for BVDV, >6.0; for HTLV-I, 4.2; for HTLV-II, 4.6; for CMV, >5.9; for WNV, >5.5; for SARS-HCoV, >5.8; and for vaccinia virus, >4.7. Log reduction of nonenveloped viruses for human adenovirus 5 was >5.2; for parvovirus B19, 3.5->5.0; for bluetongue virus, 5.6-5.9; for feline conjunctivitis virus, 1.7-2.4; and for simian adenovirus 15, 0.7-2.3. CONCLUSION: PCT inactivates a broad spectrum of pathogenic, blood-borne viruses. Inactivation of viruses in PLT concentrates with amotosalen and UVA offers the potential to prospectively prevent the majority of PLT transfusion-associated viral diseases.     

Direct virus inactivation of tachyplesin I and its isopeptides from horseshoe crab hemocytes.

Direct virus inactivation of tachyplesin I and related isopeptides, which are antimicrobial peptides isolated from the hemocytes of the horseshoe crab (Tachypleus tridentatus and Limulus polyphemus), was examined against several viruses. Vesicular stomatitis virus (VSV) was inactivated by incubation with tachyplesin I and its isopeptides. Influenza A (H1N1) virus was slightly inactivated by tachyplesin I, whereas herpes simplex virus 1 and 2, adenovirus 1, reovirus 2 and poliovirus 1 were resistant to inactivation. The inactivation of VSV by tachyplesin I depended on the concentration, the time and the temperature of incubation. Pretreatment of tachyplesin I with trypsin or lipopolysaccharide of gram-negative bacteria entirely abolished the antiviral activity. Electron microscopy of VSV treated with tachyplesin I showed naked and damaged virions. These data suggest that tachyplesin I directly inactivates the VSV by destroying its envelope subunits.     

光化学处理灭活血浆中细小病毒的初步结果

本文报告了用长波紫外线(UVA)照射结合补骨脂衍生物灭活血浆中指示细小病毒 M13Mpl8的初步结果。当血浆中加入8-MOPSOOμg/ml,UVA 强度为11.5mW/cm~3时,30到120分钟照射可以杀灭10~(5～9)传染剂量/ml 的病毒。照射防护剂可以减轻 UVA 对蛋白质的损伤以提高照射后凝血因子的回收率。2mmol/L 谷胱苷肽或合用2mmol/L 甘露醇可明显提高凝血因子Ⅶ(FW)的回收率。     

Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light

BACKGROUND: A photochemical treatment process has been developed for the inactivation of viruses and bacteria in platelet concentrates. This process is based on the photochemical reaction of a novel psoralen, S- 59, with nucleic acids upon illumination with long-wavelength ultraviolet light (UVA, 320–400 nm). STUDY DESIGN AND METHODS: High levels of pathogens were added to single-donor platelet concentrates containing 3 to 5 × 10(11) platelets in 300 mL of 35-percent autologous plasma and 65-percent platelet additive solution. After treatment with S-59 (150 microM) and UVA (0-3 J/cm2), the infectivity of each pathogen was measured with established biologic assays. In vitro platelet function after photochemical treatment was evaluated during 7 days of storage by using a panel of 14 assays. The in vivo recovery and life span of photochemically treated platelets were evaluated after 24 hours of storage in a primate transfusion model. RESULTS: The following levels of pathogen inactivation were achieved:>10(6.7) plaque-forming units (PFU) per mL of cell-free human immunodeficiency virus (HIV),>10(6.6) PFU per mL of cell-associated HIV,>10(6.8) infectious dose (ID50) per mL of duck hepatitis B virus (a model for hepatitis B virus),>10(6.5) PFU per mL of bovine viral diarrhea virus (a model for hepatitis C virus),>10(6.6) colony-forming units of Staphylococcus epidermidis, and>10(5.6) colony-forming units of Klebsiella pneumoniae. Expression of integrated HIV was inhibited by 0.1 microM S- 59 and 1 J per cm2 of UVA. In vitro and in vivo platelet function were adequately maintained after antiviral and antibacterial treatment. CONCLUSION: Photochemical treatment of platelet concentrates offers the potential for reducing transfusion-related viral and bacterial diseases.