Strategies for inactivation of viruses, bacteria in blood ...



Strategies for inactivation of viral and bacterial contaminants in blood and blood components

Henry O. Ogedegbe, Ph.D., BB, C(ASCP)SC, CC(NRCC)

Department of Environmental Health, Molecular and Clinical Sciences,

Florida Gulf Coast University,

10501 FGCU Blvd. South,

Fort Myers, FL 33965-6565

Tel: 941-590-7486

Fax: 941-590-7474

E-mail: hogedegb@fgcu.edu

Keywords

Viral and bacterial inactivation, photodynamic treatment of red cell concentrates, photochemical treatment of platelet concentrates, prestorage leukocyte reduction, photodynamic treatment, photochemical treatment.

Abstract

The supply of blood in the United States is safer than it has ever been because of the donation process and the extensive laboratory testing that is done on the donated units of blood. However, an approach to improving the safety of blood transfusion therapy is inactivation of infectious pathogens in the blood components. Solvent detergent treatment of pooled plasma has been effective in inactivating lipid enveloped viruses and bacteria in the products. Inactivation of non-lipid enveloped viruses in pooled plasma may be achieved by vapor heating after solvent detergent treatment and before fractionation into factors. Various strategies are currently being investigated for inactivation of viruses and bacteria in blood and blood component such as photodynamic treatment of red blood cell concentrates and photochemical treatment of platelet concentrates. These processes may also inactivate contaminating white blood cells in those products. The goal for the public is the provision of a zero risk blood supply.

Introduction

The provision of a safe and sufficient supply of blood is critical to patient care.1 Volunteers who are carefully screened for health problems, prior to donation, are responsible for maintaining the blood supply in the United States. Eight million volunteers donate over 12.6 million units (including approximately 643,000 autologous donations) of whole blood in the United States each year. The units are transfused to about four million patients each year.2 As a result of the screening of donors and testing of components for specific pathogens, the risk associated with transfusion has been greatly reduced. Donors who might be at risk of transmitting infections are excluded such as those known or suspected to participate in unsafe sex, intravenous drug users, people who have recently had a tattoo or those who have visited places endemic for HIV.3, and those who have lived in Great Britain for longer than 6 months.4 The FDA requires that all donated blood be tested for human immunodeficiency virus (HIV 1 and 2), human T-cell lymphotrophic viruses (HTLV-I and II), hepatitis B surface antigen (HBsAg) and anti-hepatitis C virus (HCV). Cytomegalovirus (CMV) screening is generally done after collection when CMV seronegative products are required. In addition, blood is tested for the syphilis pathogen. At the present time, testing is not routinely done for human parvovirus B19, hepatitis A virus (HAV) hepatitis G virus (HGV) or hepatitis E virus (HEV).5

In spite of all the efforts at prevention of viral and bacterial infections during blood and blood component transfusion, there still remains a risk of transmission of such viruses through single unit blood components from donors in the “window period” of infection as well as from low levels hepatitis B virus carriers.6 The greatest threat to the safety of the blood supply is the donation of blood by seronegative donors during the infectious window period when the donors are undergoing seroconversion.7 Viral mutants that escape screening and failure to produce detectable antibody response may also contribute to the risk.8 Current estimates of the frequency of viral transmission from blood components on a per donor basis are 1 in 100,000 for HCV, 1 in 63,000 for HBV, and 1 in 680,000 for HIV. The aggregate risk of receiving a blood component contaminated with one of the viruses for which sensitive tests are in place has been estimated to be 1 in 34,000 donations.5 It is reported that the average transfusion episode results in exposure to five donors. Consequently, per transfusion episode, the risk of receiving a component contaminated with virus may be as high as 1 in 6,800.5 Improvements in selection and screening of donors have made blood supply safe, however a complementary approach to improving the safety of blood transfusion therapy is inactivation of infectious pathogens in the blood components.5 The goal for the public is a zero risk blood supply and all efforts are directed towards achieving that goal. It is therefore correct to say the blood supply in the US is safer than it has ever been because of the donation process and the extensive laboratory testing of blood.

During the past 50 years, most US-licensed plasma derivatives have maintained an impressive record of not transmitting HBV, HCV, or HIV.9 The use of solvent/detergent (SD) treatment and ethanol fractionation of clotting factors and immunoglobulin preparations have inactivated transfusion relevant viruses without affecting therapeutic properties of the products. Solvent/detergent treatment kills enveloped viruses and this phenomenon has been confirmed by groups all over the world and the method is now applied worldwide for treatment of plasma products.6,9,10 Treatment with SD irreversibly disrupts the lipid envelope of viruses such as those of HIV, HBV, HCV, HGV, and CMV. It can be used to treat pooled whole plasma, which is capable of providing virus inactivated batched fresh frozen plasma (FFP). Presence of neutralizing antibodies in the plasma of many donors in the pool may contribute to the inactivation of the viruses. However, there are still outbreaks of hepatitis A virus infection among hemophiliacs who receive highly purified immune globulin depleted coagulation factors. The infection by hepatitis A and other non-lipid-enveloped viruses such as human B19 virus is still a potential risk to blood component recipients.

The transfusion of cellular blood products such as red blood cell concentrates (RBCCs) and platelet concentrates (PCs) are associated with many untoward immune reactions. Biologic response modifiers (BRM) or bioactive substances produced in these products are responsible for the observed immune reactions. These substances, or cytokines are synthesized by the WBCs or produced from the granules of disintegrating ones and are capable of inducing febrile nonhemolytic transfusion reactions (FNHTRs) in susceptible patients. In addition to the problems associated with immune reactions, there is the residual risk of transfusion-transmitted disease caused by WBC-associated infectious agents, alloimmunization, and immunosuppression. Specific subsets of peripheral blood WBCs are reservoirs for infectious agents such as CMV and EBV and other pathogenic bacteria. Transfusion transmitted infections particularly from latent herpes viruses such as CMV are of greatest concern with regard to seronegative transfusion recipients who either are immunocompromised or have immature immune systems.11 Various strategies are being developed to sterilize cellular blood products without compromising the integrity of the product to perform their biological and physiological functions of oxygen transport to the tissues as in the case of red blood cells and primary hemostasis as in the case of platelets. The wish is that transfusion of blood and blood components be as safe as possible. It is therefore required that all these products be rid of viruses and bacteria that may contaminate them in vitro and cause in vivo problems post transfusion. Several strategies, devices and methods have been employed to reduce the risk for viral and bacterial contamination of blood products and prevent the production of BRMs. Some of the strategies currently available or under trial include the following methods and processes:

Solvent/detergent treatment of plasma

Solvent/detergent treatment of plasma and plasma proteins is highly efficient against lipid-enveloped viruses, but has little effect on non-lipid-enveloped viruses.12 SD treatment should therefore greatly reduce the risk involved in pooling plasma with respect to HIV, HBV, HCV, and other possible lipid enveloped viruses. In order to inactivate the transfusion-relevant viruses without affecting therapeutic properties of the products, many manufacturers worldwide have successfully applied a mixture of solvent and detergent treatment to clotting factor and immunoglobulin preparations. This method is effective against the enveloped viruses but not against non-enveloped viruses like HAV or human parvovirus B19.13 Vapor heating of the product after SD treatment is effective in inactivating the non-enveloped viruses.14 In a population in epidemiologic equilibrium, neutralizing antibodies can be expected to be present against viruses that are prevalent in the population.15 The solvent, tri(n-butyl)phosphate (TNBP) and the detergent polyoxyethylenesorbitan monooleate (Tween 80) have been found to be superior in their inactivation of viruses than ethyl ether plus Tween 80. Treatment of plasma for 4 hours with 2-percent TNBP at 37 degrees C or 1-percent TNBP and 1-percent Tween 80 at 30 degrees C or with 1-percent TNBP and 1-percent polyoxyethylene ethers (Triton X-45) at 30 degrees C results in rapid and complete inactivation of greater than or equal to 104 tissue culture infectious doses (TCID50) of vesicular stomatitis and Sindbis viruses, which are used as surrogates.16 The recovery of antihemophiliac factor (AHF) after inactivation with TNBP/Tween 80 treatment is found to be over 90%. The recovery of factors VIII, IX, and V are similarly high after treatment with TNBP/Tween 80 and the treated plasma may then be fractionated by standard techniques into desired protein factors or complexes.

Following treatment, TNBP may be removed from the protein by gel exclusion chromatography on sephadex G25 prior to fractionation. However, Tween 80 forms large micelles, therefore it cannot be removed from proteins by gel exclusion chromatography on sephadex G25. A partial success at removal may be achieved by differential precipitation of the protein. A different detergent such as sodium cholate will inactivate viruses just as efficiently with about 80% recovery of AHF. Sodium cholate forms small micelles and can be removed by sephadex G25.17 Fresh frozen plasma may also be treated with TNBP and Triton X-100 and then purified; the in vivo effect of the residual SD is reported to be minimal. In clinical transfusion practice, ABO incompatible, HLA-matched, single donor platelets may have to be resuspended in ABO-compatible plasma. The use of SD treated plasma for this purpose would remove the added risk of transfusion transmitted diseases due to the use of another blood component.18

Prestorage filtration

The immunomodulatory effects observed in the recipients of allogeneic blood transfusion have been attributed to the WBCs present in the cellular components transfused to patients. This immunomodulatory effect of transfusion was recognized in the early 1970s, in transplant patients in which it might be beneficial for allograft survival.19 However, this immunomodulatory effect could adversely affect the out come in patients undergoing curative surgery for malignant tumors and cause an increase in the prevalence of postoperative bacterial infection.20,21 The antigen presenting cells and plasma contained in allogeneic transfusion of red cells have been implicated to mediate nonspecific and/or antigen specific immunosupression. Consequently, deranged T-cell mediated immunity, decreased macrophage function and abnormal cytokine synthesis have been noted.22,23 Other alterations in the immune system that have been observed following transfusion of allogeneic blood include decreased number and function of natural killer cells (NK), decreased cytotoxic T cell activity, decreased CD4 cells, increased CD8 cells, increased B cells and HLA-DR expression on lymphocytes. There is also decreased synthesis of IL-2 and interferon gamma (IFN-() and increased production of prostaglandin E2.21,24

A hypothesis for immunomodulation put forth by Blumberg and associates24 is that the stimulation of allogeneic transfusions drives the immune system toward a Th2 type (IL-10, IL-4) response with concomitant downregulation of Th1 (IL-2, IL-12) dependent immune functions. Th2 responses favor humoral immunity and Th1 responses favor antigen presenting cells and T-cell function. This theory might explain the beneficial effects of allogeneic transfusions in renal allotransplantation, Crohn’s disease and women with repetitive spontaneous abortions.24 Thus allogeneic transfusion in such clinical settings favor graft survival, reduces the occurrence of inflammatory bowel disease and enhance the probability of of successful term pregnancy. Immunization against HLA antibodies that may lead to platelet refractoriness has been reported to occur in 50 to 60 percent of multiply transfused patients.25 The number of WBCs per PCs that lead to primary immunization against HLA antigens is 5 x 106 which is considered the critical immunogenic load of WBCs. WBCs are more immunogenic than platelets and refractoriness is initiated by the HLA antigens on the contaminating WBCs.26 Brand et al.27, showed in their study a decreased rate of alloimmunization to random donor platelets when contaminating WBCs were removed before transfusion. The American Association of Blood Banks (AABB) recommends a WBC count less than 5 x 106 cytapheresis concentrate.25,26

While the mechanisms of the immunomodulatory effect of allogeneic blood transfusion remains unresolved,28 it can be stated that allogeneic WBCs, whether lymphocytes, monocytes, or dendritic cells play an important role in mediating this biologic effect.28 A study by Carson et al.29, found that blood transfusion is associated with a 35-percent greater risk of serious bacterial infection and 52-percent greater risk of pneumonia. In a study by van de Watering and associates30, they found that in cardiac surgery patients, who receive more than three blood transfusions, leukocyte depletion by filtration results in a significant reduction in postoperative mortality that may be attributable to the higher incidence of postoperative infections in those patients that receive blood not depleted of contaminating WBCs. An editorial opinion by Blajchman28 concluded that the allogeneic WBC transfusion associated immunomodulatory effect is biologically real and clinically relevant to many allogeneic transfusion recipients.

Also of concern are the released cytokines by WBCs, which may mediate FNHTRs, HLA alloimmunization, transfusion-related acute lung injury (TRALI), transfusion associated graft versus host disease (TA-GVHD), and the transfusion related immune suppression.26 Bioactive substances are released from the WBCs into the red cells and plasma components during storage. These substances are contained in intracellular WBC granules and are released in time-dependent fashion as the WBCs deteriorate and disintegrate. The concentration of histamine, eosinophil cationic protein, eosinophil protein X, myloperoxidase, and plasminogen activator inhibitor 1 are reported to increase by 3- to 25- fold in the supernatant fluid of red cell components between day 0 and day 35 of storage.31 Some of the bioactive substances may be involved in the mediation of and development of immunosuppression. Studies have also reported the generation of cytokines including interleukin (IL) 1 beta, tumor necrosis factor alpha (TNF-(), and IL-8, in the supernatants of stored PCs. The generation of these cytokines may result in FNHTRs.32,33 It has been postulated too that the passive transfer of IL-8 in platelet components may contribute to transfusion related acute lung injury because of the neutrophil-chemotactic and activating functions of the high concentrations of IL-8.33

Adverse effects of transfused cellular component therefore depend not only on the number of residual WBCs in blood components but also on the timing of removal of the WBC.34 The WBCs being reservoirs for infectious agents in blood components, have to be removed in order to rid the blood components of the contaminating infectious agents. Because the number of white cells is a critical factor for the production of cytokines, in platelets and red blood cell concentrates, many investigators have demonstrated that the reduction of white blood cells in these components by WBC filtration are effective in preventing the generation of cytokines.25 A study by Geiger et al.35, to evaluate the effectiveness of bedside polyester WBC reduction filters concluded that some but not all, bedside polyester filters and prestorage polyester filters can remove IL-8, RANTES, C3a and C5a from units of plasma or platelets. Advantages of prestorage reduction in PCs are the abrogation of the accumulation of pyogenic cytokines derived from WBCs during storage, prevention of the release of WBC fragments and microaggregates with immunogenic capacity, and early removal of intact WBCs containing bacteria that may be released during WBC disintegration.36

While the benefits of WBC reduction are clinically beneficial, the exposure of PCs to WBC reduction filters has been shown to have effects beyond the removal of WBCs. A point of concern is whether filtration causes damage to platelets because during the procedure, platelets are exposed to foreign surfaces, which may cause platelet activation with release of internal constituents.37 Negatively charged filter material has been reported to activate blood coagulation proteins and the kallikrein and bradykinin systems.38-40 However, Renaux in a letter41, stated that blood interaction with an active foreign surface is multifaceted with many variables such as dilution, pH and physical properties of the material. Stack et al.42, tested the possibility that the source of cytokines in the transfusion setting is the stored component itself. They concluded from their study that although IL-8 achieved levels in some units of PCs capable of causing physiological changes, the potential adverse effect on transfusion recipients of the infusion of cytokines was inconclusive. On the other hand, and as reported by Gieger et al.35, WBC reduction filters scavenge C3a anaphylotoxin and some cytokines from stored PCs which certainly is beneficial to the recipient of PCs. Devine and associates have reported the removal of p-selectin (CD62)-positive platelets by WBC reduction filtration.43

The WBCs have also been implicated as been responsible for the transmission of many pathogenic infections in blood transfusion therapy. Nonimmune asymptomatic donors may transmit viral, bacterial or parasitic infections to blood recipients. Both Rickettsia rickettsii and Orientia tsutsugamushi have been implicated in cases of transfusion transmission of diseases.44 Some other organisms that have been implicated in transfusion associated sepsis include strains of Serratia marcescens, and Yersinia enterocolitica.45,46 A hypothesis for the transmission of Yersinia enterocolitica is very illustrative of the process of bacteria transmission to recipients of transfusion therapy. The hypothesis states that during Yersinia infection in the blood donor, bacteria are phagocytosed by WBCs, but not killed. After collection of blood from such a donor and component production, the bacteria are present in WBCs for some time, during which time the unit appears sterile. Later when the WBCs disintegrate, the bacteria are released and multiply in the unit.45 There have been 20 documented cases of babesiosis transmitted by blood transfusion and a single reported case of transfusion transmitted Rocky Mountain spotted fever.47 A study by Badon et al.48, could not exclude the possibility of transmission of Lyme disease through blood transfusion. Transfusion associated Chagas’ dusease (TA-CD) is a worldwide problem.49 These parasites may be phagocytosed by macrophages and neutrophils in blood components and invariably infect the recipient of the donated contaminated units of blood. Moraes-Souza and associates in their study provided evidence that white cell reduction filters are effective in reducing the number of parasites in T. cruzi-infected blood and that this efficacy of parasite removal, depends in part on the concentration of parasites in the infected blood.49

The method employed to decrease the risk of transfusion-associated cytomegalovirus (TA-CMV) disease have been to transfuse blood or cellular blood components that are CMV antibody negative or to administer deglycerolized frozen red cells.50 A study by Larsson et al.51, found that all seropositive donors harbored latently infected peripheral blood mononuclear cells. In addition they found that a substantial proportion of seronegative individuals are CMV carriers and might transfer infection. CMV negative blood is recommended for transfusion into neonates and infants and patients who are immunocompromised. Blood contaminated with bacteria may be made relatively safe by prestorage filtration of the products or bedside filtration. Reducing WBC content in red cells to less than 5 x 108 prevents most FNHTRs. For other complicated situations, the WBCs must be reduced to less than 5 x 106. Many third generation filters can achieve this level of WBC reduction and most blood components have less than 1 x 106 WBCs26 after filtration.

Prestorage ultraviolet B radiation

Ultraviolet radiation constitutes about 8 percent of the total amount of electromagnetic radiation emitted by the sun, and plays an important role in the development and survival of living biologic cells. Its effect can be harmful and even lethal to cells. However, if it is used in a highly specific way it could modulate immune responses. The immunomudulatory effects of ultraviolet radiation have become a focus in transfusion medicine, bone marrow transplant (BMT) and transplantation immunology. Areas of clinical significance and interest in the fields of transfusion and transplantation that might benefit from the immunomodulatory effects of ultraviolet radiation, include treatment of peripheral blood hematopoeitic progenitor cells (HPCs,) cord blood and bone marrow allografts to decrease the incidence of GVHD, treatment of patient with acute and chronic GVHD, inactivation of occult tumor cells in autologous BM graft, treatment of blood components for the prevention of alloimmunization to WBC antigens and treatment of blood components for virus inactivation. The biologic effects of ultraviolet radiation vary with wavelength and the energy spectrum. Ultraviolet-B (UV-B) radiation is considered biologically the most relevant.52

The production of BRMs in stored PCs is of concern in transfusion therapy. These bioreactive substances include IL-1, TNF-(, and IL-8, and are derived from disintegrating WBCs in the stored platelet concentrate. Fujihara et al.33, have shown in their study that UV-B irradiation of apheresis platelet concentrates before storage, but not gamma radiation was significantly effective in preventing the generation of IL-8. In another study, Johnson et al.53, showed that UV-B irradiation of PCs at doses up to 10,000 mJ per cm2 does not induce significant metabolic or functional derangements following short-term storage. A study by Capon et al54, in which they evaluated several plastic materials used in blood storage for their ability to transmit ultraviolet B light, suggested that platelets could be effectively irradiated with UV-B light in a closed system. In vitro experiments showed a UV-B dose-dependent abrogation of lymphocyte responder and stimulator functions, with concurrent preservation of platelet aggregation responses. Adverse effects attributable to the transfusions were not observed, and variables to be considered included container material, volume, and composition of contents, steady exposure versus agitation and exact UV wavelength.

Ultraviolet B irradiations of platelet concentrates are know to reduce platelet alloimmunization, but the mechanism of the effect is unclear. The speculation is that UV-B may downregulate the expression of surface adhesion molecules on passenger antigen-presenting cells in PCs. A study to determine the effect of blood bank storage and UV-B irradiation on quantitative expression of intracellular adhesion molecule-1 (ICAM-1, or CD54), HLA-DR, CD45, and CD11c on CD14-positive antigen presenting cells was carried out by Fiebig et al.55, They concluded from their study that UV-B exposure nonspecifically affects monocytes in PCs, resulting in downregulation of surface molecules that are important for antigen presentation, as well as in significant cell loss.

Gamma irradiation

The use of irradiated blood products has increased many fold due to the knowledge that gamma irradiation of blood products would reduce the risk of TA-GVHD in patients receiving allogeneic BMT. Blood for such patients must be irradiated to prevent contaminating lymphocytes in the blood product from sensitizing them and causing TA-GVHD. The risk appears to be well defined in BMT patients, patients with congenital immunodeficiency syndrome or Hodgkin’s disease, for intrauterine transfusions, and for blood transfusions from first-degree family members. Most products are irradiated with either cesium 137 (137Cs) or cobalt-60 (60Co) as source of gamma rays. The recommended amount of irradiation is 25 Gy and the range can run as high as 35 Gy.26

Irradiation of blood components eliminates the risk of TA-GVHD but may damage the cellular elements in the transfused components particularly if the cells are stored for prolonged periods in the irradiated state.56 Irradiated units currently have a maximum storage life of 28 days. A study by Samuel et al.57, found that red cell ATP and 2,3 DPG levels were restored in irradiated AS-1 units stored at 4 degrees C for 42 days using a pyruvate-inosine-phosphate-adenine rejuvenating additive. Moroff and associates found that irradiation has only a small effect on the properties of RBCs treated according to the utilized protocols. Longer storage times after irradiation resulted in progressively reduced recovery while long-term survival remained unaffected.58 Blood irradiation protocols may be utilized in intraoperative blood salvage in cancer surgery. Hansen et al.59, have reported that 50 Gy dose far exceeds that needed to inactivate the number of tumor cells observed or expected in wound blood. Jin and associates60 studied the effect of irradiation of blood from persons with sickle cell trait to determine the effects of gamma irradiation on red cells from such donors. They concluded that gamma irradiation of red cells from donors with Hb AS or Hb AA resulted in comparable changes in plasma potassium, red cell ATP, and plasma Hb concentrations, although donors with Hb AS had lower plasma Hb.

Photodynamic sterilization of red cells

Photodynamic treatment (PDT) is a form of experimental cancer therapy that utilizes photosensitizers absorbing light in the visible light spectrum. PDT is an efficient inducer of apoptosis, an active form of cell death61 PDT of cellular components such as red blood cells and platelets is currently being explored as a sterilization technique to reduce risk of transfusion transmitted virus infections. Initial report of this treatment method of whole blood and RBCCs with photodynamically active dyes has been favorable. Studies carried out with hematoporphyrin derivatives, photofrin II and merocyanine 540 showed the virucidal potentials of these compounds when treating culture media and their compatibility with red cell concentrates.62 Other photosensitizers that have been studied include benzoporphyrin derivative, and sappyrin derivatives. Horowitz et al.63, have shown in their study that treatment of a red cell concentrate with 10 (M/L aluminum phthalocyanine tetrasulfonate (AlPcS4) and visible light dosage of 88 to 176 J per cm2 completely inactivated greater than 104 infectious units (TCID50) of both free and cell associated forms of vesicular stomatitis virus (VSV) used as a model and HIV. The integrity of the red cells was not compromised, as there was minimal release of hemoglobin following treatment or on subsequent storage.

This method of treatment with phthalocyanines is probably the only effective inactivation method for RBCs at the present time.63 The photosensitizers used to inactivate viruses and bacteria in red cell concentrates are required to have absorption maximum at a longer wavelength than 600 nm, to prevent the filter effect of hemoglobin. Phthalocyanines (absorption maximum around 670 nm) have been described as very efficient in inactivating various lipid-enveloped viruses with limited RBC damage.64 The principle of the virus inactivation process is based on energy transfer mechanism involving singlet oxygen (type II) while the damage to the RBC is mediated by an energy transfer reaction and an electron transfer reaction (type I).64 The addition of scavengers of type I photodynamic reactions and the use of cremophor to deliver phthalocyanines give protection to the red cells.65,66 Type I quenchers that have been studied include reduced glutathione, manitol, glycerol and superoxide dismutase. Reduced glutathione are known to reduce oxidative and free radical induced cellular damage. Manitol and glycerol are effective quenchers of hydroxyl radicals and superoxide dismutase catalyzes the disproportionation of the superoxide radical anion. Type II quenchers include tryptophan which is a singlet oxygen quencher and sodium azide.62

The use of Pc4 (HOSiPcOSi(CH3)2(CH2)3N(CH3)2) a silicon phthalocyanine, is capable of inactivating HIV-1 in three different forms: cell-free virus, cell associated virus and virus in latently infected cells. Aluminum phthalocynine tetrasulfonate is more water soluble and is efficient in cell free virus inactivation.64 One of the problems with this treatment is the binding of IgG to RBC. Rywkin et al.66, found that sulfhydryl compound are useful in preventing IgG binding to RBCs following phthalocynine photosensitization. Since virus inactivation proceeds at the same rate in the presence and absence of sulfhydryl compounds, their addition to RBCs during treatment should allow crossmatching for transfusion after treatment. The binding of IgG depends on the generation of reactive oxygen species, which are scavenged by sulfhydryl compounds. A study to determine whether PDT could also inactivate parasites in blood such as Plasmodium falciparum found that treatment with Pc4 could make red cell concentrates not only virally safe for transfusion but also safe with respect to transmitting malaria.67 Additional work is required in the areas of biochemical and immunological effects of the treated red cell concentrates on transfused recipients. Ways have to be found to adequately remove the photosensitizers from the components prior to infusion.

Photochemical sterilization of blood components

Another treatment of blood products especially PCs that has been found effective in inactivating viruses and bacteria is photochemical treatment (PCT) in which psoralens and long wavelength ultraviolet radiation (UV-A 320-400 nm) are used. It has been shown that psoralen combined with UV-A irradiation inactivates more than 5 to 6 logs of various blood borne viruses and in addition most bacteria.68 Psoralens are planar, aromatic molecules that can reversibly bind to nucleic acids by intercalation. Upon illumination with UVA, intercalated psoralens form covalent monoadducts and interstrand crosslinks with RNA and DNA. In the absence of repair, the psoralens-modified genomes of viruses and bacteria are inactivated because replication cannot take place.69 Since platelets and red cells do not have nuclei, they are unaffected by treatment with psoralens and UVA.

Synthetic psoralens such as S-5998 have been shown to be effective in inactivating a wide variety of viruses and bacteria without adversely affecting in vitro or in vivo platelet function.69,70 8-methoxypsoralen (MOP) and long wavelength UVA has been found effective in inactivating cell associated HIV-1 in PCs.71 Psoralen treatment using MOP and long wavelength UVA has also been found very effective in inactivating viruses and bacteria in PCs with minimal adverse effects on the in vitro function of platelets in comparison to untreated control concentrates maintained under current, standard blood bank conditions.72,73 Psoralens being nucleic acid specific, are capable of inactivating contaminating WBCs in RBCCs and PCs. 8-methoxypsoralen coupled with UVA has been found to be effective in inactivating the response of donor T cells against the host in BMT cases.74 This has the potential of preventing GVHD on susceptible BMT patients. Treatment of PCs with MOP and UVA irradiation has been found to effectively reduce the alloantigenicity of class I MHC molecules in recipients.75 Addition of quenchers of reactive oxygen species such as manitol to the platelet concentrates were seen to improve the aggregation response and other in vitro indicators of platelet function.76 A study by Grass et al.69, in which they compared the efficacy of S-59 with that of the two other psoralens, MOP and 4-aminomethyl 4,5’,8-trimethylpsoralen (AMT) found S-59 to be the most efficient psoralen of the three. In the same study, Grass et al.69, showed that PCT with S-59 and UVA inactivates T cells with greater potency than gamma radiation, which is the current method of TA-GVHD prophylaxis in PCs transfusion recipients. Therefore there is great potential for the utilization of PCT as an alternative to gamma radiation for T cell inactivation in PCs. With its proven efficient virucidal activity and potential effects on leukocytes S-59 needs to be investigated further to determine its safety in transfusion therapy. S-59 has entered human clinical trials. Phase 1 and 2 studies with treated, five day old platelets in healthy subjects have shown adequate viability.5 In these studies, full doses of PCT treated PCs were transfused and well tolerated during and after transfusion. Further research is required to elucidate the short and long-term effects of transfusion of PCT treated PCs..

Photochemical treatment of blood products with dimethylmethylene blue (DMMB), a phenothiazine dye with visible light has been found to inactivate several intracellular and extracellular model viruses under conditions which minimally damage or alter the red blood cell properties during 42 days of storage at 1-6 degrees C.77 Treatment with methylene blue (MB) another phenothiazine dye is effective in inactivating extracellular viruses. In a study to determine red cell alteration associated with virucidal MB phototeatment of VSV, Wagner et al.78, reported that red cell surface is altered by the treatment. In their study, Aznar et al.79, found that MB treated plasma and the cryoprecipitates obtained from it may be effective for replacement therapy in cases of von Willebrand disease and deficiencies of factor XIII and fibrinogen. Perrotta and associates80, studied the effects of MB treated plasma on cellular components stored in vitro and concluded that plasma treated by MB photoinactivation can be used for in vitro resuspension and storage of platelets and red cells. The possibility of genotoxicity of MB in two mammalian tests systems was investigated by Wagner et al.81, They concluded that MB is mutagenic in cultured mammalian cells, but results in mouse micronucleus assay suggested that the genotoxicity is not expressed in vivo. The use of DMMB with visible light causes minimal increases in red cell ion permeability and little or no binding of plasma proteins to the red cells membrane.82 MB cannot efficiently inactivate intracellular viruses, results in unwanted binding of plasma proteins to red cell surface and causes a significant increase in ion permeability.78 The potential of intravenously administered DMMB and MB for virus inactivation necessitates the need for further investigation of their usefulness.

Heat treatment

The transmission of hepatitis A virus (HAV) has been associated with the use of a number of SD treated factor VIII concentrates and possibly a factor IX concentrate. SD treatment of these products is incapable of eliminating contamination of the products by non-lipid-enveloped viruses. To eliminate the potential contamination of these products by HAV and other non-lipid-enveloped viruses such as human parvovirus B19, heat treatment of the products are recommended. A study by Shapiro et al.83, concluded that vapor-heated factor VII concentrate and vapor-heated factor IX complex are associated with a low risk of viral infection. In a study, Barrett et al.14, demonstrated the efficacy of vapor heating in inactivating high-titer HAV after spiking of plasma products with virus. Double inactivation by SD treatment plus heating at 100 degrees C for 30 minutes after lyophilization has been adopted to improve viral safety of factor VIII and factor IX concentrates especially with respect to non-lipid-enveloped virues.84 Thomas et al.85, evaluated intermediate purity and fibrinogen poor factor VIII concentrates to determine effects of heat treatment on factor VIII, von Willebrand factor (vWf) and other proteins present in the concentrates such as albumin, fibrinogen, fibronectin, IgG, and IgM. They determined in the study that higher-purity factor VIII concentrates withstand heat treatment better than concentrates that contain greater levels of contaminating proteins particularly fibrinogen.

Virus inactivation by pepsin treatment at pH 4.0

Production of immunoglobulin (IgG) preparations is required to be safe and devoid of infecting pathogens. While the products have been relatively safe, there have been a few occurrences of disease transmittance. Inactivation of viruses by pepsin at pH 4 during production of intravenous IgG has been found effective. Omar et al.86, investigated the effectiveness of pepsin treatment at pH 4.0 and concluded that the inactivation process was temperature dependent and selectively influenced by solute composition of IgG solution. Other enzyme methods that have been used to inactivate plasma products include treatment with immobilized trypsin.9

Improvements in blood safety have been achieved during the past decades through careful donor selection and screening. However, residual risk of transfusion-transmitted disease still exists.

Hypericin treatment.

Hypericin is a potent virucidal agent with activity against a wide range of enveloped viruses and retroviruses. The effective virucidal activity emanates from a combination of photodynamic and lipophilic properties. Hypericin binds cell membranes and crosslinks virus capsid proteins. This action results in a loss of infectivity and inability to retrieve the reverse transcriptase enzymatic activity from the virion. Hypericin is devoid of adverse action in most blood components and blood analyses and therefore it is a potential additive to inactivate infective viruses in blood components intended for transfusion.87

Pressure cycling technology

Elevated hydrostatic pressure is a physical process as opposed to the chemical process that does not require any chemicals. The hydrostatic pressure can be applied and released quickly thus enhancing high throughput capability. Industrial application of hydrostatic pressure inactivation of microbes includes food processing and potential vaccine production. At the present time however, this method of virus inactivation is too slow for practical application in blood processing at ambient temperature: 100 hours of 150-megaPascal (Mpa) pressure treatment was the estimated condition for an 8 to 9 log reduction in simian immunodeficiency virus titer. A study by Bradley et al.88, concluded that high-pressure procedures may be useful for the inactivation of viruses in blood and other protein containing components.

Antibody neutralization of viruses

Transfusion-transmitted HAV infection is a rare occurrence. An outbreak of HAV infection has been reported in recipients of SD treated coagulation factors. Antibodies to HAV regularly appear after infection and thus provide immunity to a large proportion of the general population.26 Thus immune antibodies neutralize viral particles in plasma and are of importance in avoiding clinical disease with the non-lipid-enveloped hepatitis A virus and human parvovirus B19.26

Affinity column

Chromatographic processes that are used on some high purity products are effective at removing and/or inactivating non-lipid-enveloped viruses such as hepatitis A virus.17

Monoclonal antibodies to viruses

Wet heating combined with monoclonal antibody purification significantly reduces the risk of coagulation concentrates that are made from large pools of donor plasma.24 This process may be used to inactivate viruses by attaching the antibodies to a solid phase column and passing the plasma through it. The antibodies cause agglutination of the viruses to the solid phase.

Conclusion

Removal of WBCs prior to transfusion therapy is effective in preventing the adverse events associated with transfusion therapy. The ability to inactivate viruses and bacteria in blood and blood components will make blood supply in the US safer than ever. The effectiveness of PDT and PCT in inactivating viruses and bacteria in blood components is proven. These treatments have the added potential of inactivating WBCs which contaminant blood and blood components thus reducing incidences of adverse post transfusion reactions. Studies to determine the long and short-term effects of transfusing PDT and PCT treated products are currently underway. The studies will address questions concerning the toxicity of the chemicals and light used in the treatments and their effects on the physiology and functions of the treated red blood cells and platelets. For now, it is safe to predict that the goal of a zero risk blood supply in the US is one that is achievable.

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