Virus sterilization in platelet concentrates with psoralen and ultraviolet A light in the presence of quenchers H. MARGOLIS-NUNNO, B. WILLIAMS, S . RYWKIN,N. GEACINTOV,AND B. HOROWITZ The virucidal and functional effect of the treatment of platelet concentrates (PCs) with long-wave ultraviolet light (UVA) and the psoralen derivative 4’-aminomethyl4,5’,8-trimethylpsoralen (AMT) was studied. Cell-freevesicular stomatitis virus (VSV) was completely inactivated (26.5 on treatment of PCs with 25 pg per mL (85 ClM) of AMT and with 20.7 J per cm (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 pg 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. TRANSFUSION 1992;32;541-547. Abbrevlations: AMT = 4‘-amlnomethy14,5’,&trimethylpsoralen; GSH = glutathlone; MC540 = merocyanlne540; PC(s) = platelet concentrate(8);8-MOP = 8-methoxypsorelen;SOD = superoxlde dismutase; UVA = ultraviolet A; VSV = veslcular stomatltis virus.
DESPITERECENTLY IMPROVED methods Of donor screening and blood testing, transfused single-donor blood components continue to have a small but defined risk of transmitting hepatitis and acquired immune deficiency syndrome. Viral sterilization of the cellular components of blood is made difficult because of the fragility of cells and the fact that virus is present in both cell-free and cell-associated forms. Toward this end, various photochemical virus inactivation treatments have been described for red cell and platelet preparations. Treatment of whole blood with hematoporphyrin derivatives and visible light (630 nm) inactivated about lo3 infectious units (IDso) of herpes simplex virus type 1 with no significant effect on red cell lysis or osmotic fragility.’ Photodynamic treatment of blood with benzoporphyrin derivativesZor merqanine 540 (MC 540)3*4 inactivated 104 to lo7 ID,, of model viruses under conditions that appeared to maintain red cell integrity. In platelet concentrates ( P a ) , however, conditions proFrom the New York Blood Center, and the Department of Chemistry, New York University, New York, New York. Supported in part by award No. HL41221 from the National Heart, Lung, and Blood Institute. Received for publicafion September 20,1991; revision received January 9, 1992, and accepted January 11, 1992.
ducing both effective virus kill and the retention of the platelet aggregation response could not be attained with MC 540 and visible light.5 By contrast, >lo5.’ ID,, of feline leukemia virus, as well as other viruses and bacteria, was inactivated on treatment of PCs with S-methoxypsoralen (8-MOP) and long-wave ultraviolet light (UVA) under conditions of reduced oxygen tension, and platelet aggregation and other indices of in vitro platelet function were near normal following treatment and upon storage for up to 96 hours.6 Recently, we demonstrated the efficacy of virus inactivation in whole blood and red cell concentrates using aluminum phthalocyanine derivatives and visible light. Both cell-free and cell-associatedvesicular stomatitis virus (VSV) and human immunodeficiency virus were inactivated 2103.6to >10s.6 ID,, by this treatment, with no significant increase in hemolysis after treatment and upon storage. Treatment of PCs with aluminum phthalocyanine tetrasulfonate, however, resulted in a reduced aggregation response to added Currently, we are evaluating methods of virus photoinactivation that are suitable for treating PCs. Because the results of Lin and coworkers6with 8-MOP and UVA appeared promising, we examined the use of the watersoluble psoralen derivative, 4-’aminomethyl-4,5’,8-tri-
MARGOLIS-NUNNO ET AL.
methylpsoralen (AMT). Dodd and colleagues1° also reported that, on treatment of PCs with AMT and UVA, a combination of high levels of virus kill and retention of platelet function was obtained only when plasma concentration was reduced to about 15 percent and the partial pressure of oxygen was reduced to about 20 percent of normal atmospheric pressure. In our study, we describe AMT photoinactivation of viruses in standard blood bank PCs that are suspended in 100-percent plasma. Moreover, because it is inconvenient and potentially harmful to platelets for oxygen concentrations to be decreased, and because doing so can introduce variability, we examined the effects of quenchers of reactive molecular oxygen species on the virus inactivation and functional properties of platelets. Preliminary results from these studies have been rep~rted.~JlJ* Materials and Methods PCS PCs, released after routine blood bank testing, were obtained from the Greater New York Blood Program and were typically 24 to 48 hours old when treated. Prior to treatment, we stored the PCs at 22 to 24°C in the bags (PL 732, Fenwal Laboratories, Deerfield, IL) in which they were received and constantly agitated them on a platelet rotator (Helmer Labs, St, Paul, MN).
Psoralen and quencher solutions We prepared AMT, obtained from HRI Associates (Berkeley, CA), as a 4 mg per mL stock solution in distilled water and stored it frozen ( - 20°C) prior to use. Chemicals used as quenchers were purchased from Sigma Chemical Co. (St. Louis, MO), prepared as concentrated stock solutions in phosphatebuffered saline (except quercetin: see below), and used at the concentrations indicated in the text. They include glutathione (GSH, reduced form), superoxide dismutase (SOD; 3250 units/ mg), mannitol, and a combination of quercetin (prepared as a 10 mM [ l o mmol/L] stock solution dissolved in 0.01 N NaOH) and ascorbic acid (sodium salt).
AMT and W A treatment After adding AMT (and quencher), we distributed 3-mL aliquots of PCs into 8.5-mL capacity polystyrene tubes (Cat. #55495, Sarstedt, Pennsauken, NJ), which transmit about 85 percent of W A light. Treatment time is defined as minutes of W A exposure, as no further virus kill occurs with AMT in the dark. During irradiation and subsequent storage, we gently mixed samples on a tube shaker-rotator (Labquake, PGC Scientific, Gaithersburg, MD). Caps on the polystyrene tubes were opened immediately after treatment and daily thereafter, to restore or maintain atmospheric gas conditions in samples assayed for platelet function following storage. We used a high-intensity, long-wave W lamp with a 100-W mercury flood bulb (Thomas Scientific, Swedesboro, NJ) for irradiation, and the UVA light incident on the sample was 7 to 16 mW per cm2, as measured with a digital 365 nm radiometer (Model 9811-50, Cole-Palmer Co., Chicago, IL). The variation in intensity depended on location under the light, and samples were rotated to ensure a similar exposure. The average
TRANSFUSION Vol. 32. No. 6-1992
intensity was 11.5 mW per cm2. The reported fluences were not corrected to account for the small absorption exhibited by the container in use. We maintained sample temperatures during irradiation at about 24°C by using electric fans and ice. For virus photoinactivation of an entire PC, we used a 250mL capacity freezing container (Cryocyte, PL-269 plastic; Fenwall) for irradiation because of its high transmission of UVA (92 vs. 55% for PL-732 bags). The PC was irradiated from above and agitated (100 rpm) on an orbital shaker (Model 361, Fisher Scientific, Pittsburgh, PA). We maintained the temperature under the W A light at 20 to 22°C by carrying out the irradiation procedure in a 10°C refrigerated chamber. We collected 3-mL samples from a medication injection site at irradiation times indicated in the text and stored them in polystyrene tubes as described above before assessing the functional activity.
Reduced q g e n concentration Where indicated in the text, we exchanged the oxygen in the PCs for nitrogen or a mixture of 95-percent N, and 5percent CO, by displacing the gas above the solution, equilibrating for about 1 minute, and repeating the process three times. One additional gas exchange was performed if the same tube or bag was sampled at an additional time point.
virus studies The inactivation of VSV and Sindbis virus was studied with procedures similar to those described earlier.’**J3 Virus titer, calculated by the Spearman-Karber method,I4 indicated the quantity of virus that infects 50 percent of the tissue culture wells. Briefly, we assessed VSV infectivity by endpoint, 10fold serial dilutions and used each dilution to inoculate eight replicate wells of human A549 cells in 96-well microtiter plates. Virus-induced cytopathology was scored after 72 hours of incubation at 37°C in 5-percent CO,. A similar procedure using primary chicken embryo cells as the indicator cell was performed to assess the infectivity of Sindbis virus. To assess photoinactivation, we added virus to the PC and treated the samples with psoralen and UVA as described. Following treatment, samples were diluted 10-fold into DulbecCO’S minimum Eagle’s medium containing 5-percent fetal calf serum (Whittaker Bioproducts, Walkerville, MD), filtered sterilely with 0.22-JJM filters (Millex GV, Millipore Corp., Bedford, MA), and then frozen at - 70°C or below until assay. When we used the same PC to assess both virus kill and platelet function, we divided the unit before adding virus and treating it.
Evaluation of platelet finction We measured platelet counts and mean platelet volume with a platelet analyzer (Model 810, Baker Diagnostics, Allentown, PA) and measured the extracellular pH of PCs with a standard pH meter (Model 130, Corning Corp., Corning, NY). Platelet aggregation was measured in response to 20 p.g per mL of collagen (Chrono-Log Corp., Havertown, PA) by using a lumiaggregometer (Chrono-Log); it is reported as the percentage of aggregation extent (the total number of units of increase in 3 min) and rate (the number of units of increase/min during the initial aggregation phase), as compared to these values in untreated controls. ATP release in response to 50 kg per mL of collagen (a concentration that gave results similar to 1 unit/ mL thrombin; not shown) was measured simultaneously with aggregation in the lumiaggregometer according to Ingerman-
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VIRUS STERILIZATION IN PLATELETS
AMT concentration (p/ml)
Irradiation tine (min)
AMT concentration (p/ml)
Irradiation time (min) FIG. 1. Treatment of PCs in air with AMT and UVA. PC samples (3 mL) in polystyrene tubes were treated with 11.5 mW per cm2of UVA and AMT in the presence of air for 30 minutes (-0-) or 60 minutes (-O-, -A-, -.-). A) The inactivation of added VSV; B) The and rate (-H-) of aggregation in response to addition of extent (-A-) 20 kg per mL of collagen, expressed as the percent observed in un-
treated controls, after overnight storage.
FIG. 2. Effect of oxygen depletion on AMT and UVA treatment of PCs. PC samples (3 mL) containing 25 kg per mL of AMT were equilibrated with nitrogen (-0-,-A-, -0-)or air (-O-, -A-, -W-) and then irradiated with 11.5 mW per cm2of UVA for the times indicated. A) The inactivation of added VSV; B) The aggregation response after overnight storage. Other details as in Fig. 1.
Wojenski and SilveP with the manufacturer’s (Chrono-Log) reagents. We calculated the ATP released (nmol) by a comparison between the luminescence due to the release of ATP during aggregation and that induced by a 2-nmol ATP standard.
lagen declined progressively with increasing AMT concentration (Fig. 1B; 60 min UVA). At 20 pg per mL of AMT, when VSV inactivation was 2 5 . 5 log,,, the extent and rate of platelet aggregation after overnight storage were 64 and 44 percent of those values in the untreated control.
Results AMT and W A matment in air
AMT and W A treatment under reduced q g e n
In the presence of air, VSV inactivation after treatment of 3-mL PC samples with UVA (11.5 mW/cm2; total duration, 30 or 60 min) and various AMT concentrations (10-30 pg/mL) was determined (Fig. 1A). Complete inactivation of all detectable VSV (25.5 log,,) required 25 pg per mL of AMT when UVA exposure time was 30 minutes (20.7 J/cm2) and 20 pg per mL of AMT when UVA exposure time was 60 minutes (41.4 J/cm2). Platelet aggregation in response to col-
VSV inactivation with AMT (25 pg/mL, 85 phf) proceeded more slowly under conditions of reduced oxygen tension than in the presence of air (Fig. 2A). Under reduced oxygen tension, the inactivation of all detectable VSV ( 2 5 . 5 log,,) required 2 hours of irradiation (82.8 J/cm2). Platelet aggregation in response to collagen, however, was better maintained when irradiation was done at low oxygen tension (Figs. 2B and 3). After overnight storage (Fig. 3A), aggregation in samples that
MARGOLIS-NUNNO ET AL.
AMT* and UVAt treatment of an intact platelet
concentrate# Overnight posttreatment storage
Platelet count UVA (log,o) Immediate Overnight pH ( x 10’lmL) MPW VSVI
0 5.3 26.0 26.0
100/100 93/08 07/81
100/100 100/100 99/97 94/79
7.66 7.66 7.53 7.44
825 028 835 860
6.2 6.1 6.1 6.3
Psoralen derivative 4’-aminomethyl-4,5’,8-trimethylpsoralen.
t Long-wave ultraviolet A light.
The results presented come from a single experiment; however, VSV kill and aggregation results were confirmed in a separate experiment (data not shown). 5 Vesicular stomatitis virus. 11 Mean platelet volume. 1) Expressed as the percentage of aggregation extenthate during initial aggregation as compared to these values in untreated controls.
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of those values in controls. Following 48 hours of posttreatment storage (Fig. 3B), the extent and rate of aggregation of samples treated in air declined further (to about 55 and 40%) in comparison to untreated controls, while those of samples treated in nitrogen remained similar to those of controls. To determine whether this AMT and UVA treatment was appropriate for larger PC volumes, we studied the AMT (25 pg/mL) and W A (11.5 mW/cm2) inactivation of VSV added to an intact PC (50 mL) under conditions of reduced oxygen tension (Table 1). The PC was transferred to a PL-269 bag for irradiation because this bag’s transmission of W A light (93%) was much greater than that of the PL-732 bags in which the PCs are stored (55%). Complete inactivation of added VSV was achieved on exposure to W A for 2 hours; it was similar to that observed in 3-mL samples. Variation in virus kill between 3-mL samples and intact units observed at earlier sampling times may be related to variations in oxygen tension. Platelet aggregation, platelet count, mean platelet volume, and pH after 2 or 3 hours of exposure to W A were similar to those in the untreated control when measured after overnight storage following treatment.
Evaluation of quenchers
FIG. 3. Platelet aggregation curves following AMT and W A treatment. Platelet aggregation in response to the addition of 20 p,g per mL of collagen was assessed after overnight (A) or 48-hour (B) storage after treatment of 3-mL samples with 25 pg per mL of AMT and 11.5 mW per cmz of UVA. Left: untreated control; middle: AMTtreated, UVA exposure for 30 minutes in air; right: AMT-treated, UVA exposure for 2 hours in nitrogen.
had been treated with 25 p g per mL of AMT and 2 hours of UVA in nitrogen was essentially the same as in the untreated controls, while in samples irradiated for 30 minutes in air, the extent and rate of aggregation were about 75 and 55 percent
As a means of eliminating the necessity for oxygen removal, we assessed the value of the addition of quenchers of free radicals and singlet oxygen. Treatment with 25 pg per mL of AMT and 30 minutes of W A irradiation in air with or without the addition of quenchers (i.e., 2 mM [2 mmol/L] GSH, 2 mh4 [2 mmol/L] mannitol, 120 pg/mL SOD,or the combination of 40 pJ4 [40 pmollL] quercetin and 100 pJ4 [lo0 pmol/L] sodium ascorbate) inactivated all detectable VSV (26.0 log,,) and Sindbis virus (28.0 log,,; Table 2). The same extent of virus kill was evidenced with 2 hours of W A under reduced oxygen tension. In separate experiments (Table 2, Fig.4), we examined the effects of these treatments on collagen-induced aggregation and ATP release after 3 days of posttreatment storage, the time at which the treatment-induced functional differences in ATF’ release were best distinguished. The presence of 2 mM [2 mmol/L] mannitol greatly improved platelet function when AMT and W A treatment was in air, while GSH, SOD,and a quercetin and ascorbate mixture had little effect; values for ATP release, extent and rate of aggregation, and pH when mannitol was present during air treatment were similar to those obtained with no mannitol under reduced oxygen conditions. Mannitol had to be present during W A exposure, because its addition immediately after irradiation did not improve platelet function following storage (not shown). We studied the effect of duration of AMT and UVA treatment in the presence and absence of 2 mh4 (2mmol/L) mannitol on the aggregation response and ATP release in each of two PCs (Fig. 5). The benefit of including mannitol was readily apparent; aggregation extent and rate and ATP release were close or equivalent to the values in the untreated control after 25 to 30 minutes’ exposure as long as mannitol was present. However, reduced aggregation response and ATP release were noted with a 35-minute exposure to W A . The unit-to-unit variability observed in the aggregation response following overnight storage of PCs (3 mL) treated with 25 p,g per mL of AMT and W A is given in Fig. 6. Statistically similar results were obtained when treatment under reduced oxygen conditions was compared with treatment under normal oxygen conditions in the presence of 2 mM [2 mmol/L] mannitol. Aggregation response was significantly reduced (p < 0.05)
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VIRUS STERILIZATION IN PLATELETS
Table 2. AMT* a n d W A t treatment of platelet concentrates# with a n d without quencher or with nitrogen equilibration prior to treatment After 3 days' posttreatment storage Virus kill (logto) ATP Gas
Air Air Air Air Air
2 mM GSHY 2 mM mannitol 20 kg/mL SOD" 40 p,M quercetln and 100 p,M ascorbate Untreated control
VSVB 26.0 26.0 26.0 26.0 26.0
Sindbis virus 28.0
Aggregation11 95/80 74/43 70161 100185 67/33
2.0 0.75 0.75 2.0 0.5
PH 7.12 6.75 6.75 7.15 6.70
28.0 28.0 28.0
Psoralen derivatlve 4'-aminomethyl-4,5',8-trimethylpsoralen.
ultraviolet A light. The results presented come from slngle experiments. Complete VSV kill under the above treatment conditions w a s confirmed In 3 separate experiments. 5 Vesicular stomatitis virus. II Expressed as the percentage of aggregation extenthate during initial aggregation as compared to these values in untreated controls. Y Glutathione. ** Superoxide dismutase.
FIG. 4. Effect of quenchers on treatment of PCs with AMT and UVA. PC samples (3 mL) containing 25 p.g per mL of AMT were shielded from UVA (A) or were irradiated with UVA at 11.5 mW per cm2 for 30 minutes in air (B-F) or for 2 hours in nitrogen (G). Prior to irradiation, quenchers were added as follows: 2 mM (2 mmol/L) GSH (B), 120 kg per mL of SOD (C), 40 (40kmol/L) quercetin and 100 pM (100 pnol/L) ascorbate (D),or 2 mM (2 mmol/L) mannitol (E). ATP release (upper panel) and platelet aggregation (lower panel) in response to 50 kg per mL of collagen were assessed simultaneously. The untreated control (not shown) was similar to sample A containing AMT and shielded from W A .
in PCs when treatment under oxygenated conditions without mannitol present was compared to either of the above groups.
Discussion Achieving absolute viral safety in blood and blood. components will probably require the implementation of virucidal treatment procedures. The use of psoralen derivatives for treating PCs is encouraged by the work of Lin and colleagues,6 who showed that a variety of viruses and microorganisms could be inactivated with 8MOP and W A under conditions that were largely compatible with the maintenance of the platelet aggregation response and other in vitro platelet properties. Our results are in agreement with this conclusion and serve to
0 4 . , . -
. , . , ! 30
FIG. 5. Effect of mannitol on the time course of AMT and UVA treatment. Platelet aggregation and ATP release in response to the addition of 50 kg per mL of collagen were assessed after overnight storage following treatment of PCs (3 mL) in air for the times indicated with 25 kg per mL of AMT and 11.5 mW per cmz of UVA in the absence (-O-) or presence (-0-) of 2 mM (2 mmol/L) mannitol. Left: the extent of aggregation; middle: the initial rate of aggregation; right: ATP release. Upper and lower panels show the results of two different experiments.
highlight the importance of oxygen removal. In this report, we examine the use of an alternative psoralen derivative, AMT, which has the advantages of enhanced ability to bind to nucleic acid, as a result of its positively charged amino group, and increased water solubility,16 which eliminates the need for organic solvents. Although the rate and extent of virus kill with AMT and UVA are higher under normal atmospheric conditions, the low-
MARGOLIS-NUNNO ET AL.
88.5 f 128
93.9 f 6.1
68.7 f 8.7
32. No. 6-1992
or, with the addition of 2 mM (2 mmol/L) mannitol, for 30 minutes (20.7 J/cm2) in air was optimal. Inactivation of all added virus was complete, and in vitro measurements of platelet function were almost equivalent to those
87.3 f 12.9
82.2 f 12.0
49.4 f 13.2
A i with mamtol
FIG.6. Variability in aggregation response following AMT and UVA treatment. PCs (3 mL) were treated with 25 hg per mL of AMT and 11.5 mW per cm2 of W A . Prior to treatment, air was exchanged for nitrogen (left), or 2 mM (2mmolL) mannitol was added (middle). The duration of exposure to UVA was 2 hours when air was removed or 30 minutes when air was present. Aggregation response to 20 pg per mL of collagen in comparison with the untreated control was assessed following overnight storage. For any one treatment condition, each data point represents a different PC. Averages and standard deviations are provided. Statistical comparisons, given in the text, were by r test.
ering of oxygen tension is important for better maintenance of the platelet aggregation response. For a given level of virus inactivation, Ah4T and W A treatment required about four times as much irradiation time with reduced oxygen tension as with air. We have found that in 3-mL samples, the treatment of PCs with 25 pg per mL (85 pM) of AMT and 11.5 mW per cm2 of UVA for 2 hours (82.8 J/cm2) under reduced oxygen tension
in the untreated control. The treatment conditions (AMT concentration, W A fluence) used throughout this study were sufficient to achieve complete kill of added virus, and they appear to be close to the maximum compatible with platelet function measurements. This became apparent on comparison of platelet aggregation results after 35 versus 25 to 30 minutes of UVA. The large differences observed in platelet function and virus kill following psoralen photoinactivation under normal and reduced oxygen tension are not surprising, as psoralen can react either directly with nucleic acid, even in the absence of oxygen, or photodynamically, that is, in oxygen-dependent reactions. However, the removal of oxygen is cumbersome, and if not carefully controlled, may contribute to unit-to-unit variation. With the hope of eliminating the necessity of oxygen removal, we studied agents known to quench one or more active oxygen species expected to arise during photoinactivation by psoralens in the presence of oxygen. With the addition of mannitol, a hydroxyl radical-quenching agent, platelets treated with 25 pg per mL of AMT and 30 minutes of W A in air had normal or near normal aggregation response and ATP release. Although the hydroxyl radical-quenching properties of mannitol have been shown to protect against DNA breakage caused by UVA in the absence of p~oralen,'~in our studies using the combination of psoralen and UVA, the presence of mannitol had little or no effect on the inactivation of VSV. Thus, the nucleic acid specificity of psoralen and UVA treatment under reduced oxygen conditions may also be attainable when free radical scavengers such as mannitol are present during treatment in air. It is interesting that the benefit observed on the addition of mannitol, an efficient scavenger of free radicals including hydroxyl radicals,'* was not observed on the addition of 2 mM (2 mmol/L) GSH, another efficient radical s~avenger.'~ The difference in platelet protection afforded by those two scavengers of free radicals may reflect differences in cellular uptake or membrane association. No functional benefits to platelets were observed on the addition of 20 pg per mL of SOD, a scavenger of superoxide anion,zoor of a mixture of 100 pit4 (100 p,mol/L) ascorbate and 40 phf (40 p,mol/L) quercetin that scavenges both singlet oxygen and radicals generated by the decomposition of lipid peroxides.z1These results and preliminary data on the treatment of red cell concentrates in the presence of quenchers with aluminum phthalocyanine tetrasulfonate and visible lightZ2are in accord with the idea that singlet oxygen is an important radical involved in virus inactivation, while free radicals (e.g., hydroxyl radical) are most responsible for cell
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VIRUS STERILIZATION IN PLATELETS
damage. Recent published information on the protection by thiols of red cells against photohemolysis on exposure to MC 540 and visible light also supports this conclusion.23 Finally, the results presented in this report should be considered preliminary. The current treatment conditions should not be considered as final. In vitro studies on the inactivation of human pathogenic viruses have yet to be performed, and treated platelets should be evaluated with regard to neoimmunogenicity and in vivo circulatory survival and function. Nonetheless, the current results encourage continuation of these studies. Acknowledgments The authors thank Eleanor Chimezie and Stanley Kirschenbaum for dedication and technical precision in performing viral assays and Desiree Morgan for valuable assistance in the preparation of this manuscript. Special appreciation is extended to Dr. Marjorie Zucker, Dr. Henriette Lackner, and Joyce Javid of New York University Medical Center and to Astra Parts and Susan Wong-Schneider of the New York Blood Center for advice and assistance regarding platelet assay methodology.
References 1. Matthews JL, Newman JT, Sogandares-Bernal F, et al. Photodynamic therapy of viral contaminants with potential for blood banking applications. Transfusion 1988;28:81-3. 2. Neyndorff HC, Bartel DL, Tufaro F, Levy JG. Development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. Transfusion 1990;30:485-90. 3. Sieber F. Krueger GJ, O’Brien JM, Schober SL, Sensenbrenner LL, Sharkis SJ. Inactivation of Friend erythroleukemia virus and Friend virus-transformed cells by merocyanine 540-mediated photosensitization. Blood 1989;73:345-50. 4. O’Brien JM, Montgomery RR, Burns WH, Gaffney DK, Sieber F. Evaluation of merocyanine 540-sensitized photoirradiation as a means to inactivate enveloped viruses in blood products. J Lab Clin Med 1990;116:439-47. 5 . Prodouz KN, Lyle CD, Keville EA, Budacz AP, Vargo S, Fratantoni JC. Inhibition by albumin of merocyanine 540-mediated photosensitization of platelets and viruses. Transfusion 1991;31:41522. 6. Lin L, Wiesehahn GP, Morel PA, Corash L. Use of 8-me.thoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates. Blood 1989;74:517-25. 7. Horowitz B, Williams B, Rywkin S. et al. Inactivation of viruses in blood with aluminum phthalocyanine derivatives. Transfusion 1991;31:102-8. 8. Horowitz B, Rywkin S, Margolis-Nunno H, et al. Inactivation of viruses in red cell and platelet concentrates with aluminum phthalocyanine (AIPc) sulfonates. Blood Cells 1992;18: 141-50.
9. Margolis-Nunno H, Rywkin S. Williams B, Horowitz B. Photoinactivation of virus in platelet concentrates (abstract). Blood 199O,76(Suppl):403a. 10. Dodd RY, Moroff G, Wagner S, et al. Inactivation of viruses in platelet suspensions that retain their in vitro characteristics: comparison of psoralen-ultraviolet A and merocyanine 540-visible light methods. Transfusion 1991;31:483-90. 11. Margolis-Nunno H, Williams B, Rywkin S, Horowitz B. Photochemical virus sterilization in platelet concentrates with psoralen derivatives (abstract). Thromb Haemost 1991;65:1162. 12. Margolis-Nunno H, Chin S, Geacintw N, Rywkin S. Williams B, Horowitz B. Virus sterilization of plasma and platelet concentrates: enhancement of the specificity of psoralen photoinactivation (abstract). Blood 1991;78(Suppl):3522. 13. Horowitz B, Wiebe ME, Lippin A, Stryker MH. Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-eveloped viruses by tri(n-buty1)phosphate detergent combinations. Transfusion 1985;25:516-22. 14. Spearman C. The method of right and wrong cases (’constant stimuli’) without Gauss’s formulae. Br J Psycho1 1908;2:227-42. 15. Ingerman-Wojenski CM, Silver MJ. A quick method for screening platelet dysfunctions using the whole blood lumi-aggregometer. Thromb Haemost 1984;51:154-6. 16. Isaacs ST, Shen CK, Hearst JE, Rapoport H. Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA. Biochemistry 1977;16:1058-64. 17. Peak MJ, Peak JG. Hydroxyl radical quenching agents protect against DNA breakage caused by both 365-nm UVA and by gamma radiation. Photochem Photobiol 1990;51:649-52. 18. Henderson BW, Miller AC. Effects of scavengers of reactive oxygen and radical species on cell survival following photodynamic treatment in vitro: comparison to ionizing radiation. Radiat Res 1986;108: 196-205. 19. h i c k BA, Nathan CF. Glutathione metabolism as a determinant of therapeutic efficacy: a review. Cancer Res 1984;44:4224-32. 20. Petkau A, Chuaqui CA. Superoxide dismutase as a radioprotector. Radiat Phys Chem 1984;24:307-19. 21. Sorata Y,Takahama U, Kimura M. Cooperation of quercetin with ascorbate in the protection of photosensitized lysis of human erythrocytes in the presence of hematoporphyrin. Photochem Photobiol 1988;48:195-9. 22. Rwykin S, Lenny L, Goldstein J, Geacintov N, Horowitz B. In vivo circulatory surviral of photochemically treated rabbit red blood cells with aluminum phthalocyanine derivatives (abstract). Blood 1991;78(Suppl):3522.. 23. Gaffney DK, O’Brien JM, Sieber F. Modulation by thiols of the merocyanine 540-sensitized photolysis of leukemia cells, red cells, and Herpes simpler virus type 1. Photochem Photobiol 1991;53:8592. Henrietta Margolis-Nunno, PhD, Assistant Member, The New York Blood Center. Bolanle Williams, PhD, Supervisor, The New York Blood Center. Shanti Rywkin. PhD, Research Fellow, The New York Blood Center. Nicholas Geacintov, PhD, Professor, Department of Chemistry, New York University, New York, NY 10003. Bernard Horowitz, PhD, Associate Member, The New York Blood Center, 310 East 67th Street, New York, NY 10021. [Reprint requests]