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Vox Sang 1991;60:207-213
Photoinactivation of Viruses in Human Fkesh Plasma by Phenothiazine Dyes in Combination with Visible Light Bernd Lambrecht, Harald Mohr, Josef Knuver-Hopf, Heinz Schmitt DRK-Blutspendedienst Niedersachsen, Institut Springe, BRD
Abstract. We developed a photodynamic method to inactivate viruses in human fresh plasma. Single plasma bags were illuminated with visible light in the presence of low doses of phenothiazine dyes like methylene blue or toluidine blue. By this treatment the infectivity of different enveloped viruses including the causative agent of AIDS, HIV-1, was completely removable from the plasma. Non enveloped viruses, however, proved to be more stable. The activities of clotting factors and other plasma proteins were only slightly decreased. There was no indication that the procedure led to important structural modifications of plasma proteins. The dyes are photodynamically active at concentrations much lower than those at which they are therapeutically used as antidots in the treatment of methemoglobinemia.
Introduction The application of suitable inactivation procedures guarantees the virus safety of purified plasma protein preparations like albumin or clotting factors, e.g. heat treatment in the lyophilized state or in solution in the presence of stabilizers, treatment with mixtures of an organic solvent and detergents or with P-propiolactone in combination with UV light [ M I . These procedures have the disadvantages that the additives have to be removed subsequently. Because of practical reasons they have to be carried out in pool preparations derived from large numbers of blood donations. It was our aim to overcome these restrictions. In the present communication we describe a photoinactivation procedure to decontaminate single plasma units in their containers, i.e. plastic bags. This makes it an ideal method to manufacture virus-safe fresh frozen plasma, but also for producing any virus-safe plasma protein preparation. For virus inactivation the plasma is illuminated with visible light in the presence of small amounts of the photosensitizing dyes methylene blue and toluidine blue, respectively. That this procedure inactivates viruses has been known for a long time [7-141. Its practical
use, including for the production of virus-safe human plasma was discussed, but not realized to date [15-181. The so-called ‘photodynamic’ virus inactivation is based on the fact that, like other photoactive compounds, cationic phenothiazine dyes (fig. 1) possess a strong affinity to surface structures of viruses as well as to viral nucleic acids. Illumination with light of appropriate wavelength excites the dye. As a consequence, viral structures connected with the dye are destroyed. Oxygen radicals, among other things, are involved in this process [7-91.
Fig. 1. Structures of phenothiazine dyes.
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Methylene blue and toluidine blue also react with plasma proteins [19-211. Surprisingly, however, we found that the activities of coagulation factors and inhibitors were not influenced very much by the photodynamic treatment. In addition, there was no indication that immunological properties of plasma proteins were altered.
Materials and Methods Fresh Frozen Plasma (FFP) Human fresh plasma was isolated from blood donations within 6 h after phlebotomia and stored deep frozen at -30°C. Prior to use, it was thawed in a water bath at 37°C. Photoactive Dyes Methylene blue for intravenous application (Methylenblau i.v. VITIS) was obtained from Neopharma, Aschau (FRG). It was delivered in ampoules containing 1-ml aliquots of 1% solutions of the dye. Toluidine blue was obtained from Aldrich, Steinheim (FRG) as a powder from which a 1 mM stock solution in deionized water was made. The dye solutions were stored in the dark at 4°C before use. If necessary, dilutions were made with water. Tissue Culture, Virus Cultivation and Assays Culture media and sera were from Gibco, Karlsruhe (FRG). Tissue culture plastic ware was from Nunc, Wiesbaden (FRG). H9, RITA and FL cells were grown in RPMI 1640, MT-4 cells in Click-RPMI and chicken embryo fibroblasts in Dulbecco’s medium. All three media contained 10% fetal calf serum (FCS). The long-term culture of human peripheral blood lymphocytes (PBLs) was carried out as described [22]. The culture medium used was RPMI 1640 containing 5% human serum. The cells were initially activated with 1% phytohemagglutinin-Mfrom Gibco. Vesicular stomatitis virus (VSV) and influenza virus were cultivated in embryonated eggs from day 12-13 and 11-12, respectively. Herpes simplex virus (HSV) was grown in RITA or FL cells. For the multiplication of human immunodeficiency virus (HIV-1). H9 cells and PBLs were used. The infectivity of VSV, HSV and adenoviruses was assayed by the cytopathic effects of serial dilutions of virus-containing samples on FL cells. 150 pl medium (1% FCS) containing about 104 cells was seeded into each cup of a microtiter plate. After adherence of the cells, dilutions from 10-’-10-8 were prepared from the virus-containing samples in phosphate-buffered saline (PBS). Fifty microliters of each dilution was added to each cup. After 5 days the cells were fixed by 5% formaldehyde. The fixation reagent was removed together with the dead cells by decanting. Noninfected adherent cells were stained by a 4% (w/v) cristal violet solution containing 6% acetic acid. For HIV-1 testing MT-4 cells were used. The slow virus growth made it necessary to exchange 50% of the culture medium after 4 days. Because of the nonadherent growing of MT-4 cells the virus titer was determined after 8 days of incubation by microscopic observation. Alternatively an assay was used which is based on the reduction of the soluble tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to a cristalline formazan dye by living cells. This can be dissolved in 1% hydrochlorid acid containing 10% sodium dodecylsulfate. The adsorbance of the dye solution at 590 nm is proportional to the amount of cells not lysed by HIV-1 [23].
All titrations were done in quadruple. Titers are expressed as tissue culture infectious doses (KID,) according to Kaerber [24] and Spearman [25] or plaque forming units (PFU) [26]. virus Inactivation Experiments Virus concentrations in the experiments were 6.25-6.5 for VSV, 4 for HSV,5.5 for adenovirus and 3.25 for HIV, expressed as K I D S o .The titer of influenza was 2.7 x lo’ PFU/ml. Small-scale experiments (volume 0.5-2 ml) to investigate photoinactivation of viruses and the influence of photodynamic treatment on plasma proteins, respectively, were carried out in the presence of at least 80% human plasma, using polystyrene round bottom tubes (Falcon, 12 x 75 mm). In large-scale experiments, plasma (100-200 ml) was spiked with 1-10ml of virus stock solution. At a plasma volume of 100 ml, 400-ml bags from Biotest, FrankfurtlMain (FRG) were used, and at higher volumes, 500-ml bags from Baxter, Munich (FRG). Before illumination the virus-containing plasma was preincubated in the dark for 1h at 4°C in the presence of the photoactive dye. The samples were either illuminated with halogene bulbs (Osram Halostar AR 111, l2V, 75 W) or fluorescent tubes (Osram L 36 W/77). The intensities of light were determined with an eye-corrected selene-illumination meter (Model MX4, BBC Goerz Metrawatt, Niimberg, FRG) in combination with the relevant grey filter. With this equipment light intensities of 150-160,000 Ix at a distance of 60-70 cm for the halogen bulbs (smallscale experiments) were measured. For large-scale experiments with plasma bags 17,000 Ix at a distance of 8 cm could be determined. When halogen bulbs were used, overheating was avoided by placing a waterfilled tissue culture flask between light source and samples. With fluorescent tubes, excess heat was removed with a ventilator. All experiments were carried out 3 times at a minimum. Assays of Plasma Proteins, Immunoelectrophoresis The functional activities of plasma proteins were assayed using commercial test kits. Those for coagulation factors were from Merz & Dade, Dudingen (Switzerland), those for the other plasma proteins from Behring, Marburg (FRG). Crossed immunoelectrophoresis was carried out according to Clarke & Freeman [27], using a goat anti-human antiserum from Sigma, Munich (FRG).
Results The effect of photodynamic treatment depends on the combined action of the photoactive dye and light. This is demonstrated in table 1. Illumination in the absence of dye for 4 h led to only marginal inactivation of VSV in human plasma, as well as incubation in the presence of low concentrations of methylene blue in the dark. At a dye concentration of 50 pM compared to the control, the virus titer was reduced by 1 and at 100pM by approx. 2 loglo steps. In combination with light, however, 1pM was sufficient to achieve a reduction of the virus titers of more than 6 loglo steps. The dependency of the inactivation of various viruses on the concentrations of methylene blue or toluidine blue,
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Photoinactivation of Viruses in Human Plasma
Table 1. Inactivation of VSV in human plasma by high concentrations of methylene blue without illumination, compared with the effect of photodynamic treatment at a low concentration of the dye Dye concentration,
w
Light exposure
Virus inactivation rate 1 1 5 11.8 95 4.8 >lo6
0 1 10 50 100 0 1
Table 3. Inactivation of VSV, HSV, and influenza virus in human plasma by the combined action of toluidine blue with visible light Light 30 min
Dye concentration, FM
-
0
-
1 0 0.01 0.05 0.25 0.5 1.0
+
+ + + + +
Log,, virus titre
vsv
HSV
influenza
5.625 5.75 5.375 4.75 4.375 1.5 0.5 0.5
5.0 5.25 4.5 4.0
7.16 7.33 7.18 7.0 6.76 4.97 3.78 2.0
4.5
2.75 2.25 0.5
Incubation time: 4 h at room temperature. The conditions were identical to those experiments in which methylene blue was used.
Table 2. Inactivation of VSV, HSV, influenza and adenovirus in human plasma by the combined action of methylene blue and visible light Light 30 min
Dye concentration, pM
Loglovirus titre VSV
0 1 0 0.01 0.05 0.25 0.5 1.0 5
10
6.25 6.25 6.25 5.75 5
2.25 10.5
50.5 ND ND
HSV
influenza
adeno
3.5 3.75 4.0 3.25 2.75 1.5 50.5 50.5 ND ND
7.34 7.34 7.43 7.38 7.16 6.54 5.0 1.7 ND ND
5.5 5.5 5.5
ND ND 4.5 4.5 4.5 4.5 3.5
Illumination time: 30 min using a 75-W halogen bulb at a distance of 60cm. Plasma sample size: 0.5 ml. ND =Not done.
respectively, are shown in tables 2 and 3. As in the experiments described before, the volume of the plasma samples was 0.5ml. They were illuminated with a 75-Whalogen bulb at a distance of 60 cm for 30 min. It is evident that different viruses possess different susceptibilities to photoinactivation. The enveloped viruses VSV as well as HSV were significantly inactivated (by 1-2 loglosteps) at a dye concentation of 50 nM whereas for influenza virus which is also enveloped a 5-to 10-fold higher concentration was required. In all three cases, at l p M a reduction of the infectious titer by more than 5 loglosteps was achieved. In contrast, non enveloped viruses proved to be much more insensitive to photoinactivation. A significant reduc-
Table 4 Photoinactivation of HIV-1 in human plasma containing 1FM methylene blue Illumination time min
Log,, virus titer without
with
methylene blue 0 2 5
10 30
2.75 2.5 3.0 2.5 3.0
3.25 50.5 50.5 50.5 50.5
Treatment conditions: see legend to table 2.
tion of the infectivity of adenovirus was not observed until the methylene blue concentration was increased to 10 p M . Under the experimental conditions used also at higher dye concentrations, poliovirus was not inactivated at all [not shown]. It was of special interest to see how the causative agent of AIDS, HIV-1, was influenced by photodynamic treatment. Although no highly concentrated virus preparation was at hand, it is obvious from the data shown in table 4 that this agent is extremely sensitive: in the presence of 1yM of methylene blue the infectivity of the HIV-l-containing plasma samples was reduced below the detection limit within 2 min after exposure to light. This suggests that
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Fig. 2. Kinetics of the photoinactivation of VSV in human plasma. The volume of the samples was 0.5 ml. They were illuminated in the with a presence of l yM methylene blue (B) or toluidine blue (0) 150-W halogen lamp at a distance of 20 cm.
Fig. 3. Kinetics of the photoinactivation of VSV and HSV in plasma bags at different plasma volumes. The photoactive dye used was methylene blue at a concentration of l yM. The bags were illuminated with = 36-W fluorescent tubes. 0 = VSV, 280ml; 0 = VSV, 100ml; HSV, 250 ml; 0 =HSV, 100 ml.
the susceptibility of HIV-1 to photoinactivation is similar to that of VSV, of which a more concentrated preparation under identical conditions lost 4-5 loglo steps of its infectious titer after 5-10 min (fig. 2). The easy scaleup of the photodynamic virus inactivation procedure is demonstrated in figure 3. In these experiments plasma samples at volumes between 100 and 280 ml in plastic bags were illuminated in the presence of 1K M methylene blue. To ensure an even exposure to light the bags were placed flat on a transparent glass plate and illuminated from beneath with a battery of 36-W fluorescent tubes. Due to the larger plasma volumes and the lower light intensity of the fluorescent tubes compared to the halogen bulb, more time than in the small-scale experiments was requiried to achieve complete virus inactivation. Nevertheless at a plasma volume of 250-280ml the infectious titers of HSV as well as of VSV could be reduced by 4-5 loglosteps within 60 min. When the plasma volume was only 100 ml, an illumination time of about 15 min was sufficient. The following observations were directed to the question of whether and to what degree the photoinactivation procedure influenced the functional activities and the electrophoretic mobilities of plasma proteins. In the latter case, this would give an indication of structural alterations of the proteins. Before testing, 2-ml aliquots of human plasma containing 1pM methylene blue were illuminated with a halogen bulb for 60 min. As table 5 shows, compared to those in untreated plasma most activities of clotting factors and other plasma proteins were reduced by not more than 20%.
Table 5. Activities of coagulation factors and other plasma proteins before and after photoinactivation in the presence of 1yM methylene blue Plasma protein
Activity without
Decrease with
photoinactivation Factor I , mg/ml Factor 11, U/ml Factor V, Ulml Factor VII, U/ml Factor VIII, U/ml Factor IX, U/ml Factor X, U/ml Factor XI, U/ml Factor XII, U/ml Antithrombin 111, % C 1-inactivator, YO Plasminogen
2.20 1.25 0.88 1.20 0.90 1.20 1.20 1.20 1.45 84.10 115.20 90.52
1.65 1.15 0.84 1.10 0.78 1.00 1.05 1.00 1.20 77.60 88.40 90.31
20.4% 8.0%
4.5% 8.3% 13.3% 16.7% 12.5% 16.7% 17.2% 7.7% 23.2% 0%
Illumination time was 60 min using a halogen bulb. The plasma sample volume was 2ml.
No obvious differences in the shapes and positions of the various protein peaks were detectable when the photoinactivated was compared with untreated plasma by crossed immunoelectrophoresis (fig. 4). The same results were obtained when the samples were tested by additional immunoelectrophoretic procedures [not shown].
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Photoinactivation of Viruses in Human Plasma
a
b
Fig. 4 Crossed immunoelectrophoresis of untreated (a) and photoinactivated (b) human plasma illuminated with a halogen lamp in the presence of 1 pM methylene blue for 1 h.
Discussion For technical reasons and because of the lability of some coagulation factors and other proteins it is difficult to decontaminate whole plasma by virus inactivation procedures which are commonly used for purified plasma proteins, e.g. by heat or solvenddetergent treatment. Our data show that photoinactivation using the phenothiazine dyes methylene blue or toluidine blue in combination with visible light represents a practicable alternative. At concentrations which are necessary for photoinactivation (1 pM , i.e. approx. 300 pgA) there should be no toxicity problems for the recipients if the dyes are not removed after treatment of the plasma. Both substances are used therapeutically at dosages which are several hundredfold higher, e.g. in the treatment of methemoglobinemia [2&31]. It is an additional advantage that single units of plasma can easily be inactivated in their original containers because the adsorption maximum of the two dyes is in the range of 620-670 nm [7, 19,321. This means that there is no problem related to the light permeability of the plasma layer and the plastic bag itself. In this regard methylene blue and toluidine blue are similar to hematoporphyrin derivative which has an adsorption maximum at 630 nm and has been used to eradicate viral contaminates from culture media and blood [33]. Merocanine 540 and psoralen are two other photoactive dyes whose application for the decontamination of blood components has been reported [34, 351. But because they need light of shorter wavelengths for excitation, their practical use probably requires more technical expenditures to process the plasma, e.g. the use of continuous centrifuges in combination with special irradiation chambers [36].
The virus-inactivating properties of phenothiazine dyes in combination with visible light have been known for a long time [7-191. As Herpes viruses are also inactivated [8, 101, there is indirect indication of inactivation of cytomegalovirus, as an example of viruses transmittable by blood transfusions. Our own data confirm and extend these earlier findings. In the experiments shown HSV, VSV and influenza virus were rapidly inactivated, as well as the causative agent of AIDS, HIV-1, which seems to be especially sensitive to photoinactivation. Under the experimental conditions used the infectious titer of the HJV-l-containing plasma was reduced below the detection limit within 2min. Thus besides the analytical efforts, which are routinely used to eliminate HIV-containing blood donations, photoinactivation can serve as an additional tool to ensure the safety from HIV-1 (and probably also HIV-2) of fresh plasma and products prepared from it. It was assumed that photoactive dyes like methylene blue penetrate the virus coat and bind to its nucleic acid, especially to guanine residues, i.e. that this is the main target structure of the photodynamic action [8, 37, 381. This explains why enveloped viruses are reliably inactivated, but not why most non enveloped viruses are either not influenced or only under conditions which are protein denaturating, e.g. at high pH values [ll,13,18,33,34,38]. The reason why non enveloped viruses are resistant to photoinactivation is not known to date. It can be speculated that the cores of non enveloped viruses are either more tightly packed than those of enveloped ones and/or that they are protected by their protein coats. This might prevent photoactive compounds from penetrating into the viral core. There is also evidence that it is the lipid envelope rather than the viral nucleic acid to which the dyes preferentially bind [39].
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In our own experiments poliovirus was not inactivated, though plasma containing adenovirus lost about 2 loglo steps of infectivity. This indicates that the photoinactivation method may have a similar limitation as the solvent/ detergent procedure which is restricted to lipid-containing viruses [4]. Thus, among those viruses transmittable by blood and plasma products, parvovirus B 19 is probably not inactivated. At present we do not know either whether the hepatitis B virus and non-A, non-B (including hepatitis C virus) are inactivated. But because they belong to the group of enveloped viruses [4,5,40,41], this seems not unlikely. In addition, Alter et al. [42] showed that hepatitis B virus and the causative agent(s) of hepatitis non-A, non-B were effectively inactivated by photodynamic treatment with psoralen and long-wave UV light. The cationic phenothiazine dyes react not only with viral nucleic acids [7, 9,301 and structures of the viral envelope but also with proteins, especially by interaction with histidine residues [20, 21, 431. This touches on the question of whether the photoinactivation procedure modifies plasma proteins to such an extent that their functional activity is lost or that neoantigenic structures are induced. At present, there is no indication that protein structures are modified to a large degree. In crossed immunoelectrophoresis the protein patterns of untreated and photoinactivated plasma samples were undistinguishable. This indicates that the electrophoretic mobilities of most or even all plasma proteins remained unchanged. Of course it will be necessary to investigate in more detail whether neoantigens are formed or not. The other part of the question is concerned with alterations or even loss of the activities of plasma proteins. Indeed, Inada and coworkers [21] found that treatment of fibrinogen with methylene blue in combination with light resulted in a rapid loss of thrombin induced clotting. They used, however, 20-to 100-fold higher concentrations of methylene blue than we did. In our own experiments we found that the illumination of plasma in the presence of 1pM methylene blue led to a decrease of about 20% of the functional activity of fibrinogen; those of other coagulation factors and plasma proteins in general were reduced to a lesser degree. This suggests that the therapeutic value of photodynamically treated fresh plasma is probably identical to that of untreated FFP.
Acknowledgements We thank Prof. H.D. Klenk from the University of Marburg who assayed the influenza virus. Acknowledge is extended to P. Schtifer, C. Schmidt, B. Schroder and U. Grundmann for their technical assistance and to C. Wittich for typing the manuscript.
References 1 Gellis SS, Neefe JR, Stokes J Jr, Strong LE, Janewy CA, Scatchard G: Chemical, clinical and immunological studies on the products of human plasma fractionation: 36. Inactivation of the virus of homologous serum hepatitis in solutionsof normal human serum albumin by means of heat. J Clin Invest 1947;27:239-244. 2 Heimburger H, Schwinn H, Mauler R: Factor VIII concentrate, hepatitis safe: Progress in the treatment of hemophilia. Die Gelben Hefte 1980;20:165-174. 3 Hollinger FB, Dolana G, Thomas W, Gyorkey F: Reduction in risk of hepatitis transmission by heat treatment of human factor VIII concentrate. J Infect Dis 1984;150:250-262. 4 Horowitz B, Wiebe ME, Lippin A, Stryker MH: Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri(n-buty1)phosphate detergent combinations. Transfusion 1985;25:516-522. 5 Edwards CA, Piet MPJ, Chin S, Horowitz B: Tri(n-butyl)phosphateldetergent treatment of licensed therapeutic and experimental blood derivatives. Vox Sang 1987;52:53-59. 6 Stephan W Fractionation of cold sterilized plasma. A new concept of production of non-infectious plasma proteins. Arzneimittel Forschung 1982;32:799-801, 7 Hiatt CW Methods for photoinactivation of viruses; in Concepts in Radiation Cell Biology. New York, Academic Press, 1972;pp 5782. 8 Wallis C, Menick JL: Irreversible photosensitization of viruses. Virology 1964;23:520-527. 9 Schneider JE, Price S, Maidt L, Gutteridge JMC, Floyd RA: Methylene blue plus light mediates 8-hydroxy 2-deoxyguanosine formation in DNA preferently over strand breakage. Nucleic Acids Res 1989;18:631-635. 10 Melnick JL, Wallis C: Photodynamic inactivation of Herpes virus. Ann NY Acad Sci 1977;284:171-181. 11 Hiatt CW, Kaufman E, Helprin JJ, Baron S: Inactivation of viruses by the photodynamic action of toluidine blue. J Immunol 1960;84:480-484. 12 Tomita Y, Prince AM: Photodynamic inactivation of arbor viruses by neutral red and visible light. Proc SOC Exp Biol Med 1963;112:887-890. 13 Wallis C, Melnick JL: Photodynamic inactivation of poliovirus. Virology 1963;21:332-341. 14 Orlob GB: Some effects of photosensitizing dyes on three plant viruses. Virology 1963;21:291-299. 15 Thormar H, Petersen I: Photoinactivation of visna virus. Acta Pathol Microbiol Scand 1964;62:461462. 16 SinkovicsJG, Bertin BS, Howe CD: Some properties of the photodynamically inactivated Rauscher mouse leukemia virus. Cancer Res 1965;25:624-627. 17 Badylak JA, Scherba G, Gustavson DP: Photodynamic inactivation of pseudorabies virus with methylene blue dye, light and electricity. J Clin Microbiol 1983;17:374-376. 18 Gerba CP, Wallis G, Melnick JL: Application of photodynamic oxidation to the disinfection of tapwater, seawater, and sewage contaminated with poliovirus. Photochem Photobiol 1977;26:499-504. 19 Heinmets F, Kingston JR, Hiatt CW Inactivation of viruses in plasma by photosensitized oxidation. Joint report of the Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington D.C. with the Naval Medical Research Institute, Bethesda, 1955.
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20 Martinez-Carrion M, Turano C, Riva F, Fasella P: Evidence of a critical histidine residue in soluble aspartic aminotransferase. J Biol Chem 1967;242:1426-1430. 21 Inada Y, Hessel B, Blombaeck B: Photooxidation of fibrinogen in the presence of methylene blue and its effect on polymerization. Biochim Biophys Acta 1977;532:161-170. 22 Rosenberg SA, Grimm EA, Lotze MT, Mazumder A: The growth of human lymphocytes in T cell growth factor: Potential applications to tumor immunotherapy; in Pick E, Landy A (eds): Lymphokines. New York, Academic Press, 1982, vol7, pp 213-247. 23 Tada H, Shibo 0, Kuroshima K, Koyama M, Tsukamoto K: An improved colorimetric assay for interleukin-2. J Immunol Methods 1986;93:157-165. 24 Kaerber G: Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn Schmiedebergs Arch Exp Pathol Pharmakol 1931;162:48@483. 25 Spearman C: The method of ‘right and wrong cases’ (‘constant stimuli’) without Gauss’s formulae. Br J Psycho1 1908;2:277-282. 26 Dulbecco R, Vogt M: Plaque formation and isolation of pure lines with poliomyelitis virus. J Exp Med 1954;99:167-182. 27 Clarke MHG, Freeman T Quantitative immunoelectrophoresis of human serum proteins. Clin Sci 1968;35:403-413. 28 Kiese M, Loerchen W, Weger N, Zierer A: Comparative studies on the effects of toluidine blue and methylene blue on the reduction of ferrihaemoglobin in man and dog. Eur J Clin Pharmacol 1972; 4~115-118. 29 Daunderer M: Antidot-Therapie. Toluidinblau bei Methamoglonamie. Fortschr Med 1980;98:462-464. 30 Harvey JW, Keitt AS: Studies on the efficacy and potential hazard of methylene blue therapy in aniline-induced methaemoglobinaemia. Br J Haematol 1983;54:29-41. 31 Osterloh MJ, Olson K: Toxicities of alkyl nitrites. Ann Intern Med 1986;104:727. 32 Kelly JM, van der Putten WJM, McConnell DJ: Laser flash spectorscopy of methylene blue with nucleic acids. Photochem Photobiol 1987;45:167-175. 33 Mathews JL, Newman JT, Sogandares-Bernal F, Judy MM, Skiles H. Leveson JE, Marengo-Rowe AJ, Chanh TC: Photodynamic therapy of viral contaminants with potential for blood banking applications. Transfusion 1988;28:81-83.
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34 Sieber F, O’Brien JM, Krueger GJ, Krueger GJ, Shober SL, Burns WH, Sharkis SJ, Sensenbrenner LL: Antiviral activity of merocyanine 540. Photochem Photobiol 1987;46:707-711. 35 Wiesehahn GP, Creagan RP, Photochemical decontamination of whole blood or blood components. US Patent 1988; No. 4,727,027. 36 Sieber F: Inactivating enveloped viruses with a merocyanine dye. US Patent 1988; No. 4,775,625. 37 Wallis G , Melnick JL: Photodynamic inactivation of animal viruses: A review. Photochem Photobiol1964;4: 159-170. 38 Neyendorff HC, Bartel DL, Levy JG: Development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. Transfusion 1990;30:485-490. 39 OBrien JM, Montgomery RR, Bums 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;116:439-447. 40 Tiollais P, Pourcel C, Dejean A: The hepatitis B virus. Nature 1985;317:489-495. 41 Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M: Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-362. 42 Alter HJ, Morel PA, Dorman BP, Smith GC, Creagan RP, Wiesehahn GP, Corash L, Popper H, Eichberg Jw: Photochemical decontamination of blood components containing hepatitis B and non-A, non-B virus. Lancet 1988;ii:14461450. 43 Fujii Y, Kobashi K, Nakai N: Photo-oxidation of histidine-residues in rat MI- and L-type pyruvate kinases. J Biochem 1984;95:12891296.
Received: September 21,1990 Revised manuscript received: December 6,1990 Accepted: December 10,1990 Dr. Harald Mohr DRK-Blutspendedienst Niedersachsen Institut Springe Eldagsener Strasse 38 D-W-3257 Springe (FRG)
Vox Sang 1991;60:207-213
Photoinactivation of Viruses in Human Fkesh Plasma by Phenothiazine Dyes in Combination with Visible Light Bernd Lambrecht, Harald Mohr, Josef Knuver-Hopf, Heinz Schmitt DRK-Blutspendedienst Niedersachsen, Institut Springe, BRD
Abstract. We developed a photodynamic method to inactivate viruses in human fresh plasma. Single plasma bags were illuminated with visible light in the presence of low doses of phenothiazine dyes like methylene blue or toluidine blue. By this treatment the infectivity of different enveloped viruses including the causative agent of AIDS, HIV-1, was completely removable from the plasma. Non enveloped viruses, however, proved to be more stable. The activities of clotting factors and other plasma proteins were only slightly decreased. There was no indication that the procedure led to important structural modifications of plasma proteins. The dyes are photodynamically active at concentrations much lower than those at which they are therapeutically used as antidots in the treatment of methemoglobinemia.
Introduction The application of suitable inactivation procedures guarantees the virus safety of purified plasma protein preparations like albumin or clotting factors, e.g. heat treatment in the lyophilized state or in solution in the presence of stabilizers, treatment with mixtures of an organic solvent and detergents or with P-propiolactone in combination with UV light [ M I . These procedures have the disadvantages that the additives have to be removed subsequently. Because of practical reasons they have to be carried out in pool preparations derived from large numbers of blood donations. It was our aim to overcome these restrictions. In the present communication we describe a photoinactivation procedure to decontaminate single plasma units in their containers, i.e. plastic bags. This makes it an ideal method to manufacture virus-safe fresh frozen plasma, but also for producing any virus-safe plasma protein preparation. For virus inactivation the plasma is illuminated with visible light in the presence of small amounts of the photosensitizing dyes methylene blue and toluidine blue, respectively. That this procedure inactivates viruses has been known for a long time [7-141. Its practical
use, including for the production of virus-safe human plasma was discussed, but not realized to date [15-181. The so-called ‘photodynamic’ virus inactivation is based on the fact that, like other photoactive compounds, cationic phenothiazine dyes (fig. 1) possess a strong affinity to surface structures of viruses as well as to viral nucleic acids. Illumination with light of appropriate wavelength excites the dye. As a consequence, viral structures connected with the dye are destroyed. Oxygen radicals, among other things, are involved in this process [7-91.
Fig. 1. Structures of phenothiazine dyes.
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Methylene blue and toluidine blue also react with plasma proteins [19-211. Surprisingly, however, we found that the activities of coagulation factors and inhibitors were not influenced very much by the photodynamic treatment. In addition, there was no indication that immunological properties of plasma proteins were altered.
Materials and Methods Fresh Frozen Plasma (FFP) Human fresh plasma was isolated from blood donations within 6 h after phlebotomia and stored deep frozen at -30°C. Prior to use, it was thawed in a water bath at 37°C. Photoactive Dyes Methylene blue for intravenous application (Methylenblau i.v. VITIS) was obtained from Neopharma, Aschau (FRG). It was delivered in ampoules containing 1-ml aliquots of 1% solutions of the dye. Toluidine blue was obtained from Aldrich, Steinheim (FRG) as a powder from which a 1 mM stock solution in deionized water was made. The dye solutions were stored in the dark at 4°C before use. If necessary, dilutions were made with water. Tissue Culture, Virus Cultivation and Assays Culture media and sera were from Gibco, Karlsruhe (FRG). Tissue culture plastic ware was from Nunc, Wiesbaden (FRG). H9, RITA and FL cells were grown in RPMI 1640, MT-4 cells in Click-RPMI and chicken embryo fibroblasts in Dulbecco’s medium. All three media contained 10% fetal calf serum (FCS). The long-term culture of human peripheral blood lymphocytes (PBLs) was carried out as described [22]. The culture medium used was RPMI 1640 containing 5% human serum. The cells were initially activated with 1% phytohemagglutinin-Mfrom Gibco. Vesicular stomatitis virus (VSV) and influenza virus were cultivated in embryonated eggs from day 12-13 and 11-12, respectively. Herpes simplex virus (HSV) was grown in RITA or FL cells. For the multiplication of human immunodeficiency virus (HIV-1). H9 cells and PBLs were used. The infectivity of VSV, HSV and adenoviruses was assayed by the cytopathic effects of serial dilutions of virus-containing samples on FL cells. 150 pl medium (1% FCS) containing about 104 cells was seeded into each cup of a microtiter plate. After adherence of the cells, dilutions from 10-’-10-8 were prepared from the virus-containing samples in phosphate-buffered saline (PBS). Fifty microliters of each dilution was added to each cup. After 5 days the cells were fixed by 5% formaldehyde. The fixation reagent was removed together with the dead cells by decanting. Noninfected adherent cells were stained by a 4% (w/v) cristal violet solution containing 6% acetic acid. For HIV-1 testing MT-4 cells were used. The slow virus growth made it necessary to exchange 50% of the culture medium after 4 days. Because of the nonadherent growing of MT-4 cells the virus titer was determined after 8 days of incubation by microscopic observation. Alternatively an assay was used which is based on the reduction of the soluble tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to a cristalline formazan dye by living cells. This can be dissolved in 1% hydrochlorid acid containing 10% sodium dodecylsulfate. The adsorbance of the dye solution at 590 nm is proportional to the amount of cells not lysed by HIV-1 [23].
All titrations were done in quadruple. Titers are expressed as tissue culture infectious doses (KID,) according to Kaerber [24] and Spearman [25] or plaque forming units (PFU) [26]. virus Inactivation Experiments Virus concentrations in the experiments were 6.25-6.5 for VSV, 4 for HSV,5.5 for adenovirus and 3.25 for HIV, expressed as K I D S o .The titer of influenza was 2.7 x lo’ PFU/ml. Small-scale experiments (volume 0.5-2 ml) to investigate photoinactivation of viruses and the influence of photodynamic treatment on plasma proteins, respectively, were carried out in the presence of at least 80% human plasma, using polystyrene round bottom tubes (Falcon, 12 x 75 mm). In large-scale experiments, plasma (100-200 ml) was spiked with 1-10ml of virus stock solution. At a plasma volume of 100 ml, 400-ml bags from Biotest, FrankfurtlMain (FRG) were used, and at higher volumes, 500-ml bags from Baxter, Munich (FRG). Before illumination the virus-containing plasma was preincubated in the dark for 1h at 4°C in the presence of the photoactive dye. The samples were either illuminated with halogene bulbs (Osram Halostar AR 111, l2V, 75 W) or fluorescent tubes (Osram L 36 W/77). The intensities of light were determined with an eye-corrected selene-illumination meter (Model MX4, BBC Goerz Metrawatt, Niimberg, FRG) in combination with the relevant grey filter. With this equipment light intensities of 150-160,000 Ix at a distance of 60-70 cm for the halogen bulbs (smallscale experiments) were measured. For large-scale experiments with plasma bags 17,000 Ix at a distance of 8 cm could be determined. When halogen bulbs were used, overheating was avoided by placing a waterfilled tissue culture flask between light source and samples. With fluorescent tubes, excess heat was removed with a ventilator. All experiments were carried out 3 times at a minimum. Assays of Plasma Proteins, Immunoelectrophoresis The functional activities of plasma proteins were assayed using commercial test kits. Those for coagulation factors were from Merz & Dade, Dudingen (Switzerland), those for the other plasma proteins from Behring, Marburg (FRG). Crossed immunoelectrophoresis was carried out according to Clarke & Freeman [27], using a goat anti-human antiserum from Sigma, Munich (FRG).
Results The effect of photodynamic treatment depends on the combined action of the photoactive dye and light. This is demonstrated in table 1. Illumination in the absence of dye for 4 h led to only marginal inactivation of VSV in human plasma, as well as incubation in the presence of low concentrations of methylene blue in the dark. At a dye concentration of 50 pM compared to the control, the virus titer was reduced by 1 and at 100pM by approx. 2 loglo steps. In combination with light, however, 1pM was sufficient to achieve a reduction of the virus titers of more than 6 loglo steps. The dependency of the inactivation of various viruses on the concentrations of methylene blue or toluidine blue,
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Table 1. Inactivation of VSV in human plasma by high concentrations of methylene blue without illumination, compared with the effect of photodynamic treatment at a low concentration of the dye Dye concentration,
w
Light exposure
Virus inactivation rate 1 1 5 11.8 95 4.8 >lo6
0 1 10 50 100 0 1
Table 3. Inactivation of VSV, HSV, and influenza virus in human plasma by the combined action of toluidine blue with visible light Light 30 min
Dye concentration, FM
-
0
-
1 0 0.01 0.05 0.25 0.5 1.0
+
+ + + + +
Log,, virus titre
vsv
HSV
influenza
5.625 5.75 5.375 4.75 4.375 1.5 0.5 0.5
5.0 5.25 4.5 4.0
7.16 7.33 7.18 7.0 6.76 4.97 3.78 2.0
4.5
2.75 2.25 0.5
Incubation time: 4 h at room temperature. The conditions were identical to those experiments in which methylene blue was used.
Table 2. Inactivation of VSV, HSV, influenza and adenovirus in human plasma by the combined action of methylene blue and visible light Light 30 min
Dye concentration, pM
Loglovirus titre VSV
0 1 0 0.01 0.05 0.25 0.5 1.0 5
10
6.25 6.25 6.25 5.75 5
2.25 10.5
50.5 ND ND
HSV
influenza
adeno
3.5 3.75 4.0 3.25 2.75 1.5 50.5 50.5 ND ND
7.34 7.34 7.43 7.38 7.16 6.54 5.0 1.7 ND ND
5.5 5.5 5.5
ND ND 4.5 4.5 4.5 4.5 3.5
Illumination time: 30 min using a 75-W halogen bulb at a distance of 60cm. Plasma sample size: 0.5 ml. ND =Not done.
respectively, are shown in tables 2 and 3. As in the experiments described before, the volume of the plasma samples was 0.5ml. They were illuminated with a 75-Whalogen bulb at a distance of 60 cm for 30 min. It is evident that different viruses possess different susceptibilities to photoinactivation. The enveloped viruses VSV as well as HSV were significantly inactivated (by 1-2 loglosteps) at a dye concentation of 50 nM whereas for influenza virus which is also enveloped a 5-to 10-fold higher concentration was required. In all three cases, at l p M a reduction of the infectious titer by more than 5 loglosteps was achieved. In contrast, non enveloped viruses proved to be much more insensitive to photoinactivation. A significant reduc-
Table 4 Photoinactivation of HIV-1 in human plasma containing 1FM methylene blue Illumination time min
Log,, virus titer without
with
methylene blue 0 2 5
10 30
2.75 2.5 3.0 2.5 3.0
3.25 50.5 50.5 50.5 50.5
Treatment conditions: see legend to table 2.
tion of the infectivity of adenovirus was not observed until the methylene blue concentration was increased to 10 p M . Under the experimental conditions used also at higher dye concentrations, poliovirus was not inactivated at all [not shown]. It was of special interest to see how the causative agent of AIDS, HIV-1, was influenced by photodynamic treatment. Although no highly concentrated virus preparation was at hand, it is obvious from the data shown in table 4 that this agent is extremely sensitive: in the presence of 1yM of methylene blue the infectivity of the HIV-l-containing plasma samples was reduced below the detection limit within 2 min after exposure to light. This suggests that
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Fig. 2. Kinetics of the photoinactivation of VSV in human plasma. The volume of the samples was 0.5 ml. They were illuminated in the with a presence of l yM methylene blue (B) or toluidine blue (0) 150-W halogen lamp at a distance of 20 cm.
Fig. 3. Kinetics of the photoinactivation of VSV and HSV in plasma bags at different plasma volumes. The photoactive dye used was methylene blue at a concentration of l yM. The bags were illuminated with = 36-W fluorescent tubes. 0 = VSV, 280ml; 0 = VSV, 100ml; HSV, 250 ml; 0 =HSV, 100 ml.
the susceptibility of HIV-1 to photoinactivation is similar to that of VSV, of which a more concentrated preparation under identical conditions lost 4-5 loglo steps of its infectious titer after 5-10 min (fig. 2). The easy scaleup of the photodynamic virus inactivation procedure is demonstrated in figure 3. In these experiments plasma samples at volumes between 100 and 280 ml in plastic bags were illuminated in the presence of 1K M methylene blue. To ensure an even exposure to light the bags were placed flat on a transparent glass plate and illuminated from beneath with a battery of 36-W fluorescent tubes. Due to the larger plasma volumes and the lower light intensity of the fluorescent tubes compared to the halogen bulb, more time than in the small-scale experiments was requiried to achieve complete virus inactivation. Nevertheless at a plasma volume of 250-280ml the infectious titers of HSV as well as of VSV could be reduced by 4-5 loglosteps within 60 min. When the plasma volume was only 100 ml, an illumination time of about 15 min was sufficient. The following observations were directed to the question of whether and to what degree the photoinactivation procedure influenced the functional activities and the electrophoretic mobilities of plasma proteins. In the latter case, this would give an indication of structural alterations of the proteins. Before testing, 2-ml aliquots of human plasma containing 1pM methylene blue were illuminated with a halogen bulb for 60 min. As table 5 shows, compared to those in untreated plasma most activities of clotting factors and other plasma proteins were reduced by not more than 20%.
Table 5. Activities of coagulation factors and other plasma proteins before and after photoinactivation in the presence of 1yM methylene blue Plasma protein
Activity without
Decrease with
photoinactivation Factor I , mg/ml Factor 11, U/ml Factor V, Ulml Factor VII, U/ml Factor VIII, U/ml Factor IX, U/ml Factor X, U/ml Factor XI, U/ml Factor XII, U/ml Antithrombin 111, % C 1-inactivator, YO Plasminogen
2.20 1.25 0.88 1.20 0.90 1.20 1.20 1.20 1.45 84.10 115.20 90.52
1.65 1.15 0.84 1.10 0.78 1.00 1.05 1.00 1.20 77.60 88.40 90.31
20.4% 8.0%
4.5% 8.3% 13.3% 16.7% 12.5% 16.7% 17.2% 7.7% 23.2% 0%
Illumination time was 60 min using a halogen bulb. The plasma sample volume was 2ml.
No obvious differences in the shapes and positions of the various protein peaks were detectable when the photoinactivated was compared with untreated plasma by crossed immunoelectrophoresis (fig. 4). The same results were obtained when the samples were tested by additional immunoelectrophoretic procedures [not shown].
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Photoinactivation of Viruses in Human Plasma
a
b
Fig. 4 Crossed immunoelectrophoresis of untreated (a) and photoinactivated (b) human plasma illuminated with a halogen lamp in the presence of 1 pM methylene blue for 1 h.
Discussion For technical reasons and because of the lability of some coagulation factors and other proteins it is difficult to decontaminate whole plasma by virus inactivation procedures which are commonly used for purified plasma proteins, e.g. by heat or solvenddetergent treatment. Our data show that photoinactivation using the phenothiazine dyes methylene blue or toluidine blue in combination with visible light represents a practicable alternative. At concentrations which are necessary for photoinactivation (1 pM , i.e. approx. 300 pgA) there should be no toxicity problems for the recipients if the dyes are not removed after treatment of the plasma. Both substances are used therapeutically at dosages which are several hundredfold higher, e.g. in the treatment of methemoglobinemia [2&31]. It is an additional advantage that single units of plasma can easily be inactivated in their original containers because the adsorption maximum of the two dyes is in the range of 620-670 nm [7, 19,321. This means that there is no problem related to the light permeability of the plasma layer and the plastic bag itself. In this regard methylene blue and toluidine blue are similar to hematoporphyrin derivative which has an adsorption maximum at 630 nm and has been used to eradicate viral contaminates from culture media and blood [33]. Merocanine 540 and psoralen are two other photoactive dyes whose application for the decontamination of blood components has been reported [34, 351. But because they need light of shorter wavelengths for excitation, their practical use probably requires more technical expenditures to process the plasma, e.g. the use of continuous centrifuges in combination with special irradiation chambers [36].
The virus-inactivating properties of phenothiazine dyes in combination with visible light have been known for a long time [7-191. As Herpes viruses are also inactivated [8, 101, there is indirect indication of inactivation of cytomegalovirus, as an example of viruses transmittable by blood transfusions. Our own data confirm and extend these earlier findings. In the experiments shown HSV, VSV and influenza virus were rapidly inactivated, as well as the causative agent of AIDS, HIV-1, which seems to be especially sensitive to photoinactivation. Under the experimental conditions used the infectious titer of the HJV-l-containing plasma was reduced below the detection limit within 2min. Thus besides the analytical efforts, which are routinely used to eliminate HIV-containing blood donations, photoinactivation can serve as an additional tool to ensure the safety from HIV-1 (and probably also HIV-2) of fresh plasma and products prepared from it. It was assumed that photoactive dyes like methylene blue penetrate the virus coat and bind to its nucleic acid, especially to guanine residues, i.e. that this is the main target structure of the photodynamic action [8, 37, 381. This explains why enveloped viruses are reliably inactivated, but not why most non enveloped viruses are either not influenced or only under conditions which are protein denaturating, e.g. at high pH values [ll,13,18,33,34,38]. The reason why non enveloped viruses are resistant to photoinactivation is not known to date. It can be speculated that the cores of non enveloped viruses are either more tightly packed than those of enveloped ones and/or that they are protected by their protein coats. This might prevent photoactive compounds from penetrating into the viral core. There is also evidence that it is the lipid envelope rather than the viral nucleic acid to which the dyes preferentially bind [39].
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In our own experiments poliovirus was not inactivated, though plasma containing adenovirus lost about 2 loglo steps of infectivity. This indicates that the photoinactivation method may have a similar limitation as the solvent/ detergent procedure which is restricted to lipid-containing viruses [4]. Thus, among those viruses transmittable by blood and plasma products, parvovirus B 19 is probably not inactivated. At present we do not know either whether the hepatitis B virus and non-A, non-B (including hepatitis C virus) are inactivated. But because they belong to the group of enveloped viruses [4,5,40,41], this seems not unlikely. In addition, Alter et al. [42] showed that hepatitis B virus and the causative agent(s) of hepatitis non-A, non-B were effectively inactivated by photodynamic treatment with psoralen and long-wave UV light. The cationic phenothiazine dyes react not only with viral nucleic acids [7, 9,301 and structures of the viral envelope but also with proteins, especially by interaction with histidine residues [20, 21, 431. This touches on the question of whether the photoinactivation procedure modifies plasma proteins to such an extent that their functional activity is lost or that neoantigenic structures are induced. At present, there is no indication that protein structures are modified to a large degree. In crossed immunoelectrophoresis the protein patterns of untreated and photoinactivated plasma samples were undistinguishable. This indicates that the electrophoretic mobilities of most or even all plasma proteins remained unchanged. Of course it will be necessary to investigate in more detail whether neoantigens are formed or not. The other part of the question is concerned with alterations or even loss of the activities of plasma proteins. Indeed, Inada and coworkers [21] found that treatment of fibrinogen with methylene blue in combination with light resulted in a rapid loss of thrombin induced clotting. They used, however, 20-to 100-fold higher concentrations of methylene blue than we did. In our own experiments we found that the illumination of plasma in the presence of 1pM methylene blue led to a decrease of about 20% of the functional activity of fibrinogen; those of other coagulation factors and plasma proteins in general were reduced to a lesser degree. This suggests that the therapeutic value of photodynamically treated fresh plasma is probably identical to that of untreated FFP.
Acknowledgements We thank Prof. H.D. Klenk from the University of Marburg who assayed the influenza virus. Acknowledge is extended to P. Schtifer, C. Schmidt, B. Schroder and U. Grundmann for their technical assistance and to C. Wittich for typing the manuscript.
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34 Sieber F, O’Brien JM, Krueger GJ, Krueger GJ, Shober SL, Burns WH, Sharkis SJ, Sensenbrenner LL: Antiviral activity of merocyanine 540. Photochem Photobiol 1987;46:707-711. 35 Wiesehahn GP, Creagan RP, Photochemical decontamination of whole blood or blood components. US Patent 1988; No. 4,727,027. 36 Sieber F: Inactivating enveloped viruses with a merocyanine dye. US Patent 1988; No. 4,775,625. 37 Wallis G , Melnick JL: Photodynamic inactivation of animal viruses: A review. Photochem Photobiol1964;4: 159-170. 38 Neyendorff HC, Bartel DL, Levy JG: Development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. Transfusion 1990;30:485-490. 39 OBrien JM, Montgomery RR, Bums 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;116:439-447. 40 Tiollais P, Pourcel C, Dejean A: The hepatitis B virus. Nature 1985;317:489-495. 41 Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M: Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-362. 42 Alter HJ, Morel PA, Dorman BP, Smith GC, Creagan RP, Wiesehahn GP, Corash L, Popper H, Eichberg Jw: Photochemical decontamination of blood components containing hepatitis B and non-A, non-B virus. Lancet 1988;ii:14461450. 43 Fujii Y, Kobashi K, Nakai N: Photo-oxidation of histidine-residues in rat MI- and L-type pyruvate kinases. J Biochem 1984;95:12891296.
Received: September 21,1990 Revised manuscript received: December 6,1990 Accepted: December 10,1990 Dr. Harald Mohr DRK-Blutspendedienst Niedersachsen Institut Springe Eldagsener Strasse 38 D-W-3257 Springe (FRG)
