Preventing transfusion related complications in a recipient of a blood
United States Patent: 7,648,699
Issued: January 19, 2010
Raymond P. (Lakewood, CO), Li; Junzhi (Arvada, CO)
Biotechnologies, LLC (Lakewood, CO)
Appl. No.: 11/469,186
Filed: August 31, 2006
This invention is directed toward a
process for reducing transfusion related complications in a recipient of
an allogeneic blood transfusion by adding to the blood to be transfused a
photosensitizer comprising riboflavin, irradiating the blood and
riboflavin with light, transfusing the irradiated blood into a recipient,
and reducing a transfusion related complication by the recipient to cells
in the donor blood. The invention is also directed towards a process for
preventing rejection of a donor organ by a recipient comprising the steps
of transfusing the recipient of the donor organ with treated platelets;
and transplanting the donor organ into the recipient.
Description of the
FIELD OF THE INVENTION
This invention is directed to methods of preventing transfusion related
complications in a recipient of allogeneic donor blood.
Whole blood collected from volunteer donors for transfusion into
recipients is typically separated into components: red blood cells, white
blood cells, platelets, plasma and plasma proteins, using apheresis or
other known methods. Each of these separated blood components may be
stored individually for later use and are used to treat a multiplicity of
specific conditions and disease states. For example, the red blood cell
component is used to treat anemia, the concentrated platelet component is
used to control bleeding, and the plasma protein component is used
frequently as a source of Clotting Factor VIII for the treatment of
In cell separation procedures, there is unusually some small percentage of
other types of cells which are carried over into a separated blood
component. When contaminating cells are carried over into a separated
component of cells in a high enough percentage to cause some undesired
effect, the contaminating cells are considered to be undesirable. White
blood cells, which may transmit infections such as HIV and CMV also cause
other transfusion-related complications such as transfusion-associated
Graft vs. Host Disease (TA-GVHD), alloimmunization and microchimerism.
TA-GVHD is a disease produced by the reaction of immunocompetent T
lymphocytes of the donor that are histoincompatible with the cells of the
recipient into which they have been transplanted. Recipients develop skin
rashes, fever, diarrhea, weight loss, hepatosplenomegaly and aplasia of
the bone marrow. The donor lymphocytes infiltrate the skin,
gastrointestinal tract and liver. Three weeks following transfusion 84% of
patients who develop TA-GVHD die.
Alloimmunization describes an immune response provoked in a recipient by
an alloantigen from a donor of the same species. Alloantigens include
blood group substances (A, B, O) on erythrocytes and histocompatibility
Chimerism, or microchimerism refers to the small numbers of donor cells
found in the recipient's body outside the region of the organ transplant.
It is believed that the presence of these cells may contribute to the long
term development of autoimmune diseases in the transfusion recipient.
Human Leukocyte Antigen (HLA) markers are found on the cell membranes of
many different cell types, including white blood cells. HLA is the major
histocompatibility complex (MHC I) in humans, and contributes to the
recognition of self v. non-self. Recognition by a transfusion recipient's
immune system of differences in HLA antigens on the surface of the
transfused cells may be the first step in the rejection of transplanted
tissues. Therefore, the phenomena of alloimmunization of recipients
against HLA markers on donor blood is a major problem in transfusion
medicine today. This issue arises in recipients of blood products due to
the generation of antibodies against white blood cell HLA antigens in
donor blood by the recipient.
Platelets also contain low levels of these HLA antigens because they bud
from a megakaryocyte cell (a form of white cell) located primarily in the
bone marrow. When a recipient of a whole blood or blood component
transfusion generates antibodies against the HLA antigens on the white
blood cells of the donor blood cells, a consequence is that these
antibodies also lead to recognition and clearance of platelets that carry
this same marker. When this occurs, it becomes necessary to HLA match the
donor and recipient in order to assure that the recipient receiving the
transfusion is able to maintain an adequate number of platelets in
circulation. This is often a complicated, expensive and difficult
procedure but a necessary one, since rapid clearance of the platelets due
to antibody-antigen interaction would otherwise put the recipient at
severe risk of bleeding to death. In cases where recipients are very
heavily transfused with blood or blood products from multiple donors and
antibodies to several different HLA markers are generated, or where no
suitable matched donor for platelets is available, death frequently
results for those patients who become alloimmunized and sustain a bleed.
Since the problem arises from the presence of white cells in the donated
blood products, the elimination of white cells from these products would
be expected to reduce the likelihood and frequency of reactions. Gamma
irradiation of blood products, which kills the cells but does not remove
them from the blood product to be transfused, has not been shown to be
able to prevent alloimmunization reactions. It is likely that this is due
to the fact that the treated cells are still present and capable of
presenting antigens to the recipient's immune system.
Filtration of white blood cells from blood or blood products to be
transfused has been shown to be capable of reducing alloimmunization
reactions. This has been demonstrated based on an extensive clinical study
known as the TRAP study. It was conducted as a multi-institutional study
between 1995-1997 and results were subsequently published in the NEJM in
1997 (Trial to Reduce Alloimmunization to Platelets Study Group. Leukocyte
reduction and ultraviolet B irradiation of platelets to prevent
alloimmunization and refractoriness to platelet transfusions. N Engl J
Med. 1997;337:1861-1869). The data from that study suggested that
leukoreduction significantly decreased the likelihood of alloimmunization
reactions in patients from 13% for non-leukoreduced, untreated products to
3-5% for leukoreduced products. As a result of this work, platelet
products have been routinely filtered by a variety of methods to remove
WBC. The remaining levels of alloimmunization that were observed were
believed to be due to residual white blood cells that were not filtered
out. Even the best WBC filters cannot remove 100% of the white blood cells
and those left behind are potentially able to stimulate antibody
production against the HLA markers on the remaining cells. A decrease in
the occurrence rate from 13% of patients receiving platelets to 3-4% is
significant, but still leaves several tens of thousands of cases occurring
on an annual basis.
In the same TRAP study, treatment of platelet products with ultraviolet B
(UVB) light was evaluated. In the case of the UVB treatment, the results
were equivalent to those obtained through leukoreduction. The work was
consistent with prior studies that showed that UVB treated platelet
products possessed significantly reduced alloimmunization responses (Blundell
et al. Transfusion 1996; 36: 296-302). This was believed to be due to
changes in white cells induced by UVB that cause them to present their
antigens and have those antigens processed differently from non-irradiated
cells by the patient's immune system. The result is that antibody
generation is significantly suppressed for UVB treated products. Although
the results were positive, the UVB treatment described in the TRAP study
was not adopted widely, because the UV dose required to suppress the
alloimmunization response damaged the platelets to an extent which did not
allow the platelets to be stored with adequate maintenance of their
efficacy (Grijzenhout et al. Blood 1994; 84: 3524-3531).
Photosensitizers, or compounds which absorb light of a defined wavelength
and transfer the absorbed energy to an electron acceptor may be a solution
to the above problems, by inactivating undesirable cells contaminating a
blood product without damaging the desirable components of blood.
There are many photosensitizer compounds known in the art to be useful for
inactivating undesirable cells and/or other infectious particles. Examples
of such photosensitizers include porphyrins, psoralens, dyes such as
neutral red, methylene blue, acridine, toluidines, flavine (acriflavine
hydrochloride) and phenothiazine derivatives, coumarins, quinolones,
quinones, anthroquinones and endogenous photosensitizers.
As described above, ways to reduce the risks of transfusion related
complications from white blood cells is either to reduce the number of
white blood cells transfused into a recipient to an extent that no immune
response is generated, and/or to effectively destroy the viability and
capacity of any transfused white blood cells to function post transfusion.
What is not known is whether donor cells which have been subjected to
pathogen reduction treatment with riboflavin and light have modified HLA
surface markers, and therefore will not cause transfusion related
complications such as alloimmunization, GVHD and microchimerism in the
It is to this second aspect that this invention is directed.
SUMMARY OF THE INVENTION
This invention is directed toward a process for reducing transfusion
related complications in a recipient of an allogeneic blood transfusion by
adding to the blood to be transfused a photosensitizer comprising
riboflavin, irradiating the blood and riboflavin with light, transfusing
the irradiated blood into a recipient, and reducing a transfusion related
complication by the recipient to cells in the donor blood.
Also claimed is a blood product for transfusion into a recipient
comprising inactivated blood or a blood product which has been treated
with riboflavin and light. The treated blood or blood product will not
cause transfusion related complications in the recipient when transfused.
The invention is also directed towards a process for preventing rejection
of a donor organ by a recipient comprising the steps of transfusing the
recipient of the donor organ with treated platelets; and transplanting the
donor organ into the recipient.
Photosensitizers useful in this invention include endogenous
photosensitizers. The term "endogenous" means naturally found in a human
or mammalian body, either as a result of synthesis by the body or because
of ingestion as an essential foodstuff (e.g. vitamins) or formation of
metabolites and/or byproducts in vivo. When endogenous photosensitizers
are used, particularly when such photosensitizers are not inherently toxic
or do not yield toxic photoproducts after photoradiation, no removal or
purification step is required after decontamination, and the
decontaminated product can be directly administered to a recipient in need
of its therapeutic effect.
Examples of such endogenous photosensitizers which may be used in this
invention are alloxazines such as 7,8-dimethyl-10-ribityl isoalloxazine
(riboflavin), 7,8,10-trimethylisoalloxazine (lumiflavin),
7,8-dimethylalloxazine (lumichrome), isoalloxazine-adenine dinucleotide (flavin
adenine dinucleotide [FAD]) and alloxazine mononucleotide (also known as
flavin mononucleotide [FMN] and riboflavine-5-phosphate). The term "alloxazine"
Use of endogenous isoalloxazines as a photosensitizer to pathogen reduce
blood and blood components are described in U.S. Pat. Nos. 6,258,577 and
6,277,337 both issued to Goodrich et al., and are herein incorporated by
reference to the amount not inconsistent.
The process of using endogenous alloxazine and light to reduce the risks
of transfusion related complications from contaminating white blood cells
in blood or blood products are shown in FIG. 1 (see Original Patent).
Whole blood to be transfused into a recipient is collected from a donor.
If desired, the whole blood may be separated into blood components using
any available procedures and/or extracorporeal blood processing machines.
50 .mu.M riboflavin in PBS is added to the whole blood or separated blood
components. The blood product and riboflavin are illuminated at a
wavelength of between about 290-370 nm for a sufficient amount of time to
reduce the number of white blood cells present in the donor blood or blood
product to an extent that no immune response to the donor blood is
generated by the transfusion recipient, and/or to effectively destroy the
viability and capacity of any transfused donor white blood cells to
function in the recipient post transfusion. An illumination time of around
8 minutes appears to be satisfactory. The inactivated blood product is
ready to be transfused into a donor.
The following examples show that allogeneic and xenogeneic donor cells
subjected to a pathogen reduction treatment with riboflavin and light will
not cause transfusion related complications in a donor such as
alloimmunization, TA-GVHD and microchimerism.
The intent of this study was to determine whether human peripheral blood
mononuclear cells (PBMNCs) treated with riboflavin and light (hereinafter
known as treated cells) could be induced to proliferate in vitro when
exposed to a growth stimulus, or whether the treated cells were rendered
inactive by the treatment, and therefore could not be induced to
proliferate. Untreated cells (control) are those human PBMNCs not treated
with riboflavin and light.
For this study, PBMNC were obtained from three human donors, with each
donor set being split into a treated and untreated subset. Each subset was
subsequently tested using the in vitro test methods described below. PBMNC
were isolated from platelets obtained from the donors using a standard
apheresis procedure on a Trima.RTM. apheresis machine (available from
Gambro BCT, Lakewood, Colo., USA). For treatment with riboflavin and
light, the cells were added to ABO-matched platelet-poor plasma (PPP),
which was then mixed with riboflavin and illuminated according to the
procedure shown in FIG. 1.
CD3 is the signaling complex of the T lymphocyte cell receptor.
Anti-CD3.sup.+ antibody has been shown to induce proliferation of T cells.
CD28 is a low affinity T cell receptor that interacts with B7 (ligand for
CD28). CD28 is considered a co-stimulatory receptor because its signals
are synergistic with those provided by the CD3 receptor in promoting T
cell activation and proliferation. Signals from CD28 to the CD3 receptor
also increase the synthesis of many cytokines. Cytokines are produced
primarily by lymphocytes in response to a stimulus. Production of
cytokines is therefore a measure of white blood cell health.
Preparation of CD3, CD3/CD28 or Control Coated Plates
PBS containing 10 .mu.g/mL of anti-CD3 (NA/LE, Pharmingen), 10 .mu.g/mL
anti-CD3 and 4 .mu.g/mL anti-CD28 (NA/LE, Pharmingen) or PBS alone were
added to wells (50 .mu.l per well) in a 96 well flat bottom plate. The
plates were incubated for at least 90 minutes at room temperature.
Following 2 washes of the wells with PBS, 100 .mu.l of RPMI 1640 media
containing 5% human AB serum, penicillin and streptomycin was added to all
wells and the plates were incubated at room temperature for at least
another 60 minutes. Then 100 .mu.l of the treated or untreated cells at
2.times.10.sup.6 cells/ml in RPMI 1640 containing 5% human AB serum,
penicillin and streptomycin were added to the wells (replicate 6 wells per
1. A. The effect of treatment with riboflavin and light on the ability of
PBMNC to proliferate in response to CD3 and CD3/CD28 stimulation.
As shown in FIG. 2 (see Original Patent), anti-CD3 antibody induced
significant proliferation of untreated (designated as Control cell+CD3 in
FIG. 2) cells in all 3 donors. The combination of anti-CD3 and anti-CD28
antibodies further increased proliferation of untreated (Control
cell+CD3/CD28) cells. Both treated (designated as Tx+medium in FIG. 2) and
untreated (Control cell+medium) cells present in media alone exhibited
minimal proliferation. In contrast, the treated PBMNCs did not proliferate
in response to either anti-CD3 (Tx cell+CD3) or anti-CD3/CD28 antibody (Tx
1. B. The Ability of the Treated or Untreated PBMNC to Produce Cytokines
A comparison of the levels of cytokines present in the supernatants of the
wells after 2 days in culture indicated that both anti-CD3 antibody
(Control cell+CD3) and anti-CD3/CD28 antibodies (Control cell+CD3/CD28)
induced increased cytokine production by the untreated PBMNCs. As shown in
FIG. 3 (see Original Patent), higher levels of some cytokines could be
detected in the wells containing control cells and medium alone (Control
cell+medium). However, the treated cells (Tx cell+CD3; Tx+CD3/CD28 or Tx
cell+medium) did not produce cytokines in any of the wells, even in the
media control. This data demonstrates that the treated leukocytes are
unresponsive in that they do not exhibit any significant proliferation or
Claim 1 of 9 Claims
1. A process for reducing transfusion
related complications in a human recipient due to human donor cells that
may be present in an allogeneic blood transfusion comprising the steps of:
adding to the blood to be transfused a photosensitizer consisting
essentially of riboflavin at a concentration of 50 .mu.M; irradiating the
blood and riboflavin with light at a wavelength between 290-370 nm for
around 8 minutes to reduce transfusion related complications in the
recipient caused by the donor cells; wherein the transfusion related
complications are alloimmunization, Transfusion Associated Graft vs. Host
Disease (TA-GvHD) or microchimerism; transfusing the irradiated blood into
a recipient; and reducing transfusion related complications in the
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