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
1
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
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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
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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 neutrophilchemotactic 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
4
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 pselectin (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. cruziinfected 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
5
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 (UVB) 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
6
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 HIV1 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
7
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 4aminomethyl 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
8
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
9
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.
References
1. Forbes JM, Laurie ML. Blood collections by community blood centers, 1988 through 1992. Transfusion
1994;34:392-395
2. American Association of Blood Banks. Facts about Blood and Blood Banking. September 2000. Available
from URL: http://www.aabb.org/All_About_Blood/FAQs/aabb_faqs.htm#Facts
3. Nusbacher J, Chinvetta J, Naiman R, Buchner B, Reeves J, Scalia V, Herst R. Evaluation of a confidential
method of excluding blood donors exposed to human immunodeficiency virus: studies on hepatitis and
cytomegalovirus markers. Transfusion 1987;27:207-209
4. Ness PM. Transfusion Medicine: an overview and update. Clinical Chemistry 2000;46:1270-1276
5. Corash L. Inactivation of viruses, bacteria, protozoa, and leukocytes in platelet concentrates. Vox Sanguinis
1998;74:173-176
6. Biesert L, Suhartono H. Solvent/detergent treatment of human plasma – a very robust method for virus
inactivation. Validated virus safety of OCTAPLAS. Vox Sanguinis 1988;74: 207-212
7. Schreiber GB, Busch MP, Kleinman SH, korelitz JJ. The risk of transfusion-transmitted viral infections.
New Engl. J Medicine 1996;334:1685-1690
10
8. Willianson LM, Llewelyn NC, Fishern JP, Bellamy MC, Baglin TP, Freeman J, Klinck JR, Ala FA, Smith
N, Neuberger J Wreghitt TG. A randomized trial of solvent/detergent-treated and standard fresh-frozen
plasma in the coagulopathaty of liver disease and liver transplantation. Transfusion 1999;39:1227-1234
9. Tabor E. The epidemiology of virus transmission by plasma derivatives: clinical studies verifying the lack
of transmission of hepatititis B and C viruses and HIV type 1. Transfusion 1999;39:1160-1168
10. Horowitz B, Lazo A, Grossberg H, Page G, Lippin A, Swan G. Virus inactivation by solvent/detergent
treatment and the manufacture of SD-plasma. Vox Sanguines 1998;74,Suppl. 1:203-206
11. Robach JD, Bray RA, Hilyer CD. Longitudinal monitoring of WBC subsets in packed RBC units after
filtration: implication for transfusion transmission of infections. Transfusion 2000;40:500-506
12. Mariani G, Paolantonio TD, Baklaya R, Morfini M, Mannucci PM. Prospective study of the evaluation of
hepatitis C virus infectivity in a high-purity, solvent/detergent-treated factor VIII concentrate: parallel
evaluation of other markers for lipid-enveloped and non-lipid-enveloped viruses. The Ad Hoc Group of the
Foundzione dell’Emofilia. Transfusion 1993;33:814-818
13. Solheim BG, Rollag H, Svennevig JL, Arafa O, Fosse E, Borgerud U. Viral safety of solvent/detergenttreated plasma. Transfusion 2000;40:84-90
14. Barrett PN, Meyer H, Wachtel I, Eibl J, Dorner F. Inactivation of hepatitis A virus in plasma products by
vapor heating. Transfusion 1997;37:215-220
15. Rollag H, Solheim BG, Svennevig JL. Viral safety of blood derivatives by immune neutralization.
In:Solheim BG, ed. S/D-plasma, clinical experience and viral safety. Proceedings of the XXVth Congress of
the International Society of Blood Transfusion. Vox Sanguines 1998;74 (Suppl 1):213-217
16. Piet MP, Chin S, Prince AM, Brotman B, Cundell AM, Horowitz B. The use of tri(butyl)phosphate
detergent mixture to inactivate hepatitis viruses and human immunodeficiency virus in plasma and plasma’s
subsequent fractionation. Transfusion 1990;30:591-598
17. Horowitz B, Wiebe ME, Lippin A, Stryker MH. Inactivation of viruses in labile blood derivatives. I
Disruption of lipid-enveloped viruses by tri(n-butyl)phosphate detergent combinations. Transfusion
1985;25:516-522
18. Tocci LJ, Napychank PA, Cable RG, Snyder EL. The effect of solvent/detergent-treated plasma on stored
platelet concentrates. Transfusion 1993;33:145-149
19. Opelz G, Sengar DP, Mickey MR, Terasaki PI. Effect of blood transfusion on subsequent kidney
transplants. Transplant Proc 1973;5:253-9
20. Bordin,JO, Chiba AK, Carvalho KIL, Takata ET, Falcao RP, Garcia AB, Bordin IAS, Marciel MM,
Andreoni S, Kerbauy J. The effect of unmodified or prestorage white cell-reduced allogeneic red cell
transfusion on the immune responsiveness in orthopedic surgery patients. Transfusion 1999;39:718-723
21. Wang-Rodriguez J, Fry E, Fieberg E, Lee T.H, Busch M, Mannino F, Lane TA. Immune response to blood
transfusion in very-low-birthweight infants. Transfusion 2000;40:25-34
22. Innerhofer P, Luz G, Spotl L, Hobisch-Haagen P, Schobersberger, Fischer M, Nussbaumer W, Lochs A,
Irschick E. Immunologic changes after transfusion of autologus or allogeneic buffy coat-poor white cell
reduced blood to patients undergoing arthroplasty. I. Proliferative T-cell responses and the balance of helper
and suppressor T cells. Transfusion 1999;39:1089-1096
23. Innerhofer P, Wallwczek C, Luz G, Hobisch-Hagen P, Benzer A, Stokl B, Hassenberger G, Nussbaumer W,
Schobersbeeeerger W. Transsfusion of buffy coat depleted blood components and risk of postoperative
infection in orthopedic patients. Transfusion 1999;39:625-632
24. Blumberg N, Heal JM, Immunomodulation by blood transfusion:an evolving scientific and clinical
challenge. American Journal of Medicine 1996:101:299-308
25. Zeller T, Zigsem J, Moog R, Ktretschmer V, Eckstein R, Muller N, Eisenbeisz F. Periodic alternating
interphase positioning to lower WBC contamination of apheresis platlet concentrates: a multicenter
evaluation. Transfusion 2000;40:687-692
11
26. Harmening DM, Modern Blood Banking and Transfusion Practices, 4th ed. 1999. F.A. Davis Company,
Philadelphia.
27. Brand A, van Leeuwen A, Eernisse JG. Platelet immunology with special regard to platelet transfusion
therapy. Exercpta Medica International Congress 1978;415:639
28. Blajchman MA. Transfusion-associated immunomodulation and universal white cell reduction: are we
putting the cart before the horse? Transfusion 1999;39:665-670
29. Carson JL, Altman DG, Duff A, Noveck H, Weinstein MP, Sonnerberg FA, Hudson JI, Provenzano G. Risk
of bacterial infection associated with allogeneic blood transfusion among patients undergoing hip fracture
repair. Transfusion 1999;39:694-700
30. van de Watering LMG, Hermans J, Houbiers JGA, van den Broek PJ, Bouter H, Boer F, Harvey MS,
Huysman HA, Brand A. Beneficial effects of leukocyte depletion of transfused blood on postoperative
complications in patients undergoing cardiac surgery. A randomized clinical trail. Circulation 1998;:97:562568
31. Vamvakas EC, Carven JH. Transfusion and postoperative pneumonia in coronary artery bypass graft
surgery: effect of the length of storage of transfused red cells. Transfusion 1999;39:701-710
32. Fujihara M Takahashi TA, Ogiso C Ikebuchi K, Sekiguchi S. Generation of interlukin 8 in stored apheresis
platelet concentrates and the preventive effect of prestorage ultraviolet B radiation. Transfusion
1997;37:468-475
33. Fujihara M, Ikebuchi K, Wakamoto S, Sekiguchi S. Effects of filtration and gamma radiation on the
accumulation of RANTES and transforming growth factor-1 in apheresis platelet concentrate during
storage. Transfusion 1999;39:498-505
34. Federowicz I, Barrett BB, Andersen JW, Urashima M, Popovsky MA, Anderson KC. Characterization of
reactions after transfusion of cellular blood components that are white cell reduced before storage.
Transfusion 1996;36:21-28
35. Geiger TL, Perrotta PL, Davenport R, Baril L, Snyder EL. Removal of anaphylatoxins C3a and C5a and
chemokines interlukin 8 and RANTES by polyester white cell-reduction and plasma filters. Transfusion
1997; 37:1156-1162
36. Ferrer F, Rivera J, Corral J, Gonzalez-Conejero R, Lozano MI, Vicente V. Evaluation of pooled platelet
concentrates using prestorage versus postorage WBC reduction: impact of filtration timing. Transfusion
2000;40:781-788
37. Novotny VMJ, van Doorn R, Rozier Y, D’Amaro J, Harvey MS, Brand A. Transfusion results of filtered
and subsequently stored random platelet suspensions prepared from buffy coats. Vox San 1992;63:23-30
38. Hume HA, Popovsky MA, Benson K, Glassman AB, Hines D, Oberman HA, Pisciotto PT, Anderson KC.
Hypotensive reactions: a previously uncharacterized complication of platelet transfusion? Transfusion
1996;36:904-909
39. Scott CF, Harvey B, Whitehead J, Coleman RW. Lack of clinically significant contact system activation
during platelet concentrate filtration by leukocyte removal filters. Blood 1998;92:616-622
40. Cyr M, Hume HA, Champagne JD, Sweeney CB, Gervais N, Adam A. Anomaly of the des-Arg9-bradykinin
metabolism associated with severe hypotensive reactions during blood transfusions: a preliminary study.
Transfusion 1999;39:1084-1088
41. Renaux JL. Ex vivo factors affecting contact phase activation in negatively charged medical devices (Letter)
Blood 1999;93:2129-2131
42. Stack G, Snyder EL. Cytokine generation in stored platelets. Transfusion 1994;34:20-25
43. Devine DV, Bradley AJ, Maurer E, Levin E, Chahal S, Serrano K, Gyongyossy-Issa MIC. Effects of
prestorage white cell reduction on platelet aggregate formation and the activation state of platelets and
plasma enzyme systems. Transfusion 1999;39:724-734
12
44. Mettille FC, Salata KF, Belanger KJ, Casleton BG, Kelly DJ. Reducing risk of transfusion-transmitted
rickettsial disease by WBC filtration, using Orientia tsutsugamushi in a model system. Transfusion
2000;40:290-296
45. Hogman CF, Gong J, Hambraeus A, Johansson CS, Erickson L. the role of white cells in the transmission of
Yersinia enterocolitica in blood components. Transfusion 1992;32:654-657
46. Gong J, Hogman CF, Hambraeus A, Johansson CS, Eriksson L. Transfusion-associated Serratia marcescens
infection: studies of the mechanism of action. Transfusion 1993;33:802-808
47. McQuiston JH, Child JE, Chamberland ME, Tabor E. Transmission of tick-borne agents of disease by blood
transfusion: a review of known and potential risk in the United States. Transfusion 2000;40:274-284
48. Badon SJ Fister RD, Cable RG. Survival of Borrelia burgdorferi in blood products. Transfusion
1989;29:581-583
49. Moraes-Souza H, Bordin JO, Bardossy L, Macpherson DW, Blajchman MA. Prevention of transfusionassociated Chagas’ disease: efficacy of white cell-reduction filters in removing trypanosoma cruzi from
infected blood. Transfusion 1995;35:723-726
50. Eisenfeld L, Silver H, McLaughlin J, Klevjer-Anderson P, Mayo D, Anderson J, herson V, Krause P,
Savidakis J, Lazar A. Prevention of transfusion-associated cytomegalovirus infection in neonatal patients by
the removal of white cells from blood. Transfusion 1992;32:205-209
51. Larsson S, Soderberg-Naucler C, Wang FZ, Moller E. Cytomegalovirus DNA can be detected in peripheral
blood mononuclear cells from all seropositive and most seroneagative healthy blood donors over time.
Transfusion 1998;38:271-278
52. Lankford KV, Mosunjac M, Hillyer CD. Effects of UVB radiation on cytokine generation, cell adhesion
molecules, and cell activation markers in T-lymphocytes and peripheral blood HPCs. Transfusion
2000;40:361-367
53. Johnson RB, Napychank PA, Murphy S, Snyder EL. In vitro changes in platelet function and metabolism
following increasing doses of ultraviolet-B irradiation. Transfusion 1993;33:249-255
54. Capon SM, Sacher RA, Deeg HJ. Effective ultraviolet irradiation of platelet concentrates in teflon bags.
Transfusion 1990;30:678-681
55. Fiebig E, Lane TA. Effects of storage and ultraviolet B irradiation on CD14-bearing antigen-presenting cells
(monocytes) in platelet concentrates. Transfusion 1994;34:846-851
56. Davey RJ, McCoy NC, Yu M, Sullivan JA, Spiegel DM, Leitman SF. The effects of prestorage irradiation
on posttransfusion red cell survival. Transfusion 1992;32:525-528
57. Samuel LH, Anderson G, Mintz, PD. Rejuvenation of irradiated AS-1 red cells. Transfusion 1997;37:25-28
58. Moroff G, Holme S, AuBuchon JP, Heaton WA, Sweeney JD, Friedmen LI. Viability and in vitro properties
of AS-1 red cells after gamma irradiation. Transfusion 1999;39:128-134
59. Hansen E, Knuechel R, Altmeppen J, Taeger K. Blood irradiation for intraoperative autotransfusion in
cancer surgery: demonstration of efficient elimination of contaminating tumor cells. Transfusion 1999;
39:608-615
60. Jin YS, Anderson G, Mintz PD. Effects of gamma irradiation on red cells from donors with sickle cell trait.
Transfusion 1997;37:804-804
61. He J, Agarwal ML, Larkin HE, Friedman LR, Xue L, Oleinick NL. The induction of partial resistance to
photodynamic therapy by the protooncogene BCL-2. Photochem. Photobiol. 1996;64:845-852
62. Rywkin S, Lenny L, Goldstein J, Geacintov NE, Margolis-Nunno H. Importance of type I and type II
mechanisms in the photodynamic inactivation of viruses in blood with aluminum phthalocyanine
derivatives. Photochem. Photobiol. 1992;56:463-469
63. Horowitz B, Williams B, Rywkin S, Prince AM, Pascual D, Geacintov N, Valuinsky J. Inactivation of
viruses in blood with aluminum phthalocynine derivatives. Transfusion 1991;31:102-108
13
64. Moor ACE, van der Veen A, Dubbleman TMAR, VanSteveninck J, Brand A. Photodynamic sterilization of
red cells and its effect on contaminating white cells: viability and mechanism of cell death. Transfusion
1999;39:599-607
65. Moor AC, van der Veen A, Wagenaars-van Gompel AE, Dubbleman TM, Vansteveninck J Brand A. Shelflife of photodynamically sterilized red cell concentrates with various numbers of white cells. Transfusion.
1997;37:592-600
66. Rywkin S, Ben-Hur E, Reid ME, Oyen R, Raph H, Horowitz B. Slective protection against IgG binding to
red cells treated with phthalocynines and red light for virus inactivation.Transfusion 1995;35:414-420
67. Lustigman S, Ben-Hur F. Photosensitized inactivation of Plasmodium falciparum in human red cells by
phthalocyanines. Transfusion 1996;36:543-546
68. van Aken WG. What future means to management of blood centers. Vox Sanguinis 1998;74:115-118
69. Grass JA, Hei DJ, Metchette K, Cimino GD, Wiesehahn GP, Corash L, Lin L. Inactivation of leukocytes in
platelet concentrates by photocemical treatment with psolaren plus UVA. Blood 1998;91(6)2180-2188
70. Lin L, Cook DN, Wiesehahn GP, Alfonso R, Behrman B, Cimino GD, Corten L, Damonte PB, Dikeman R,
Dupuis K, Fang YM, Hanson CV, Hearst JE, Lin CY, Londe HF, Metchette K, Nerio AT, Pu JT, Reames
AA, Rheinshmidt M, Tessman J, Isaacs ST, Wollowitz S, Corash L. Photochemical inactivation of viruses
and bacteria in platelet concentrates by use of a novel psolaren and long-wavelength ultraviolet light.
Transfusion 1997;37:423-435
71. Lin L, Londe H, Hansson CV, Wiesehahn G, Isaacs S, Cimino G. Photochemical inactivation of cellassociated human immunodeficiency virus in platelet concentrates. Blood 1993;82:292-297
72. Lin L, Londe H, Janda JM, Hanson CV, Corash L. Photochemical inactivation of pathogenic bacteria in
human platelet concentrates. Blood 1994;83:2698-2706
73. Lin L, Wiesehahn GP, Morel PA, Corash L. Use of 8-methoxypsoralen and long-wavelength ultraviolet
radiation for decontamination of platelet concentrates. Blood 1989;74:517-525
74. Ulrich SE. Photoinactivation of T-cell function with psoralen and UVA radiation suppresses the induction
of experimental murine graft-versus-host disease across major histocompatibility barriers. Journal of
Investigative Dermatology 1991;96:303-308
75. Grana NH, Kaao KJ. Use of 8-methoxypssoralen and ultraviolet –A pretreated platelet concentrates to
prevent alloimmunization against class I major histocompatibility antigens. Blood 1991;77:2530-2537
76. Margolis-Nunno H, Williams B, Rywkin S, Geacintov N, Horowitz B. Virus sterilization in platelet
concentrates with psolaren and ultraviolet A light in the presence of quenchers. Transfusion 1992;32:541547
77. Wagner SJ, Skripchenko A, Roninette D, Mallory DA, Cincotta L. Preservation of red cell properties after
virucidal phototreatment with dimethylene blue. Transfusion 1998;38:729-737
78. Wagner SJ, Storry JR, Mallory DA, Stromberg RR, Benade LE, Friedman LI. Red cell alteration associated
with virucidal methylene blue phototreatment. Transfusion 1993;33:30-36
79. Aznar JA, Molina R, Montoro JM. Factor VIII/von Willebrand factor complex in methylene blue-treated
plasma. Transfusion. 1999;39:748-750
80. Perrotta PL, Baril L, Tead C, Chapman J, Dincecco D, Buchholz DH, Snyder EL. Effects of methylene
blue-treated plasma on red cells and stored platelet concentrates. Transfusion 1999;39:63-639
81. Wagner SJ, Cifone MA, Murli H, Dodd RY, Myhr B. Mammalian genotoxicity assessment of methylene
blue in plasma: implication for virus inactivation. Transfusion 1995;35:407-413
82. Skripchenko AA, Wagner SJ. Inactivation of WBCs in RBC suspensions by photoactive phenothiazine
dyes: comparison of dimethylmethylene blue and MB. Transfusion 2000;40:968-975
83. Shapiro A, Abe T, Aledort LM, Anderle K, Hilgartner MW, Knunschak M, Preston FE, Rivard GE,
Schimpf K. Low risk of viral infection after administration of vapor-heated factor VII concentrate or factor
IX complex in first-time recipients of blood components. International factor safety study group.
Transfusion 1995;35:204-208
14
84. Santagostino E, Mannucci PM, Gringeri A, Azzi A, Morfini M, Musso R, Santoro R, Schiavoni M.
Transmission of parvovirus B19 by coagulation factor concentrates exposed to 100 degrees C heat after
lyophilization. Transfusion 1997;37:517-522
85. Thomas KB, McGrath KM, Taylor M, Young IF, Herrington RW, Schiff P. Effect of virucidal heat
treatment on proteins in human factor VIII concentrates. Transfusion 1988;28:8-13
86. Omar A, Kempf C, Immelmann A, Rentsch M, Morgenthaler, JJ. Virus inactivation by pepsin treatment at
pH 4 of IgG solutions: factors affecting the rate of virus inactivation. Transfusion 1996;36:866-872
87. Lavie G, Mazur Y, Lavie D, Prince AM, Pascual D, Liebes L, Levin B, Meruelo D. Hypericin as an
inactivator of infectious viruses in blood components. Transfusion 1995;35:392-400
88. Bradley DW, Hess RA, Tao F, Sciaba-Lentz L, Remaley AT, Laugharn Jr. JA, Manak M. Pressure cycling
technology: a novel approach to virus inactivation in plasma. Transfusion 2000;40:193-200
15