Preventing transfusion related complications in a recipient of a blood transfusion

Information

  • Patent Grant
  • 7892535
  • Patent Number
    7,892,535
  • Date Filed
    Thursday, December 3, 2009
    14 years ago
  • Date Issued
    Tuesday, February 22, 2011
    13 years ago
Abstract
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.
Description
FIELD OF THE INVENTION

This invention is directed to methods of preventing transfusion related complications in a recipient of allogeneic donor blood.


BACKGROUND

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 hemophilia.


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 antigens.


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 recipient.


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.





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1 depicts an embodiment of the invention to treat donor cells to be transfused into a recipient with riboflavin and light.



FIG. 2 is a graph showing the effect of treatment with riboflavin and light on the ability of peripheral blood mononuclear cells (PBMNC) to proliferate in response to CD3 and CD3/CD28 stimulation.



FIG. 3 is a graph comparing the production of cytokines by treated and untreated PBMNC.



FIG. 4 is a graph measuring IgG and IgM production by rats transfused with untreated allogeneic platelets for 10 weeks before receiving an allogeneic heart transplant.



FIG. 5 is a graph measuring IgG and IgM production by rats transfused with treated allogeneic platelets for 10 weeks before receiving an allogeneic heart transplant.





DETAILED DESCRIPTION

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” includes isoalloxazines.


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.


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 μM 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.


EXAMPLE 1

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® 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+ 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 μg/mL of anti-CD3 (NA/LE, Pharmingen), 10 μg/mL anti-CD3 and 4 μg/mL anti-CD28 (NA/LE, Pharmingen) or PBS alone were added to wells (50 μl 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 μl 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 μm of the treated or untreated cells at 2×106 cells/ml in RPMI 1640 containing 5% human AB serum, penicillin and streptomycin were added to the wells (replicate 6 wells per group).


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, 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 cell+CD3/CD28) stimulus.


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, 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 cytokine production.


EXAMPLE 2
2.A. The Ability of Treated or Untreated PBMNC to Be Activated in Response to PMA

Phorbol myristic acetate (PMA) is a stimulus that activates WBCs but does not cause proliferation. One of the results of this activation signal is the upregulation onto the surface of the leukocyte the activation antigen CD69. Activation through CD69 does not cause the cell to proliferate. This assay determined whether treatment with riboflavin and light interferes with the ability of the cells to be activated.


As above, WBC were obtained from the leukocyte reduction chamber of a Trima® machine following a double unit platelet collection. The peripheral blood mononuclear cells (PBMNCs) were purified by Ficoll-Hypaque discontinuous gradient centrifugation. These PBMNCs were divided into 2 aliquots and one aliquot was placed in an Extended Life Platelet (ELP) bag containing autologous human plasma and exposed to riboflavin and light. Following the treatment, the PBMNC were collected by centrifugation, washed and then placed in a 50 ml tube filled with RPMI 1640 containing 10% fetal calf serum (FCS). The cells were initially counted and then the following assays were performed:


Stock PMA (Sigma) at 0.5 mg/ml in DMSO was diluted to 1000 ng/ml in PBS. 50 μL of PMA or PBS was transferred to 12×75 mm tubes. Treated and untreated PBMNC were adjusted to 1×106/ml in RPMI-10% Fetal calf serum (R10 medium) and 450 μL of each was transferred to tubes containing either 50 μL of PMA or PBS. The tubes were incubated in a 37° C. water bath for 4 hours. 50 μL of cells were stained with 20 μL of CD8FITC, CD69PE, and CD3PerCP (Becton Dickinson, Fast Immune Kit) and analyzed on the FACScan Flow Cytometer (Becton Dickinson). Fluorescence of the cells containing CD69 and CD8 fluorescent markers was acquired by gating on the CD3+ PerCP positive cells, and quadrant analysis used to assess the level of lymphocyte activation.


The results are shown in Table 2A below. Summarizing this data shows that untreated (−) CD3+ cells (both CD4+ (helper T cells) and CD8+ (cytotoxic T cells)) expressed CD69 upon activation with PMA, while the treated (+) CD3+ cells (both CD4+ and CD8+) did not express CD69 upon activation with PMA. Thus treatment with riboflavin and light resulted in almost 100% inhibition of the ability of PMA to activate cells.














TABLE 2A







Experiment

% CD3+ CD4+
% CD3+ CD8+



No.
Treatment
CD69+ cells
CD69+ cells





















1

39.7
7.7



1
+
0.5
0.0



2

62.9
11.3



2
+
2.8
0.3



3

59.5
15.1



3
+
1.2
0.2










2.B. The Effect of Treatment on the Ability of PBMNC to Proliferate in Response to Mitogens and Allogenenic Stimulator Cells

Other stimuli that have been shown to induce PBMNC to proliferate are mitogens such as phytohaemagglutinin (PHA), which activates T lymphocytes (CD8), and allogeneic stimulator PBMNC. Allogenic stimulator cells are cells from a different donor which initiates an immune response by presenting antigen to responder cells. The ability of treated or untreated PBMNC to proliferate in response to these stimuli was tested. Responder cells proliferate in response to antigen presentation by a stimulator cell.


To measure the proliferative response to PHA, PBMNC were adjusted to 1×106/ml in RPMI-10% fetal calf serum, and Phytohemagglutinin-M(PHA-M) (Gibco) was diluted 1:40 in R10 medium. Equal volumes (100 μL) of each were transferred to triplicate flat bottomed wells of 96 well micro plates (Falcon Primeria). After incubating for 3 days under 10% CO2 the wells were pulsed for 4 hours with luCi of 3H-thymidine (Perkin Elmer/NEN). The cells were harvested on a multi well harvester apparatus (Brandel Scientific), and uptake of the isotope was quantitated on a liquid scintillation counter (Beckman).


To measure the proliferative response to allogeneic stimulator PBMNC, 1×107 cells/ml of allogeneic stimulator cells were treated with mitomycin C (Sigma) 33 μg/ml in R10 medium for 30 minutes at 37° C. Mitomycin C was removed by washing 2× with 30 ml of R10 medium. The experimental treated and control PBMNC as well as the allogeneic mitomycin C treated cells were adjusted to 1×106 cells/ml in R10 medium. The treated and control PBMNC were transferred in 100 μL volumes to triplicate flat bottom wells (Falcon Primaria). After adding an equal volume of the mitomycin C allogeneic stimulators the plates were incubated for 5 days at 37° C. under 10% CO2, and cell proliferation assessed by uptake of luCi of 3H-thymidine.


Table 2B below shows that treated PBMNC were unable to proliferate in response to either PHA or allogeneic stimulator cells.











TABLE 2B





Experi-




ment


No.
Untreated cells
Treated cells




















+PHA
−PHA
+PHA
−PHA





1
42349 ± 3947
643 ± 31 
1387 ± 86 
1070 ± 187


2
108946 ± 989 
396 ± 37 
222 ± 56
225 ± 27


3
117373 ± 14215
589 ± 27 
326 ± 4 
336 ± 84


Mean
89556
 543
645
544


±SD
41099
 130
644
459






+Stimulators
−Stimulators
+Stimulators
−Stimulators





1
41598 ± 4697
5019 ± 3391
430 ± 73
 450 ± 191


2
 38089 ± 19733
6813 ± 1880
 322 ± 137
349 ± 88


3
45652 ± 5515
596 ± 143
234 ± 43
251 ± 80


Mean
41780
4143
329
350


±SD
 3784
3200
 98
100









2.C. The Ability of Treated or Untreated PBMNC to Stimulate Proliferation

While treatment with riboflavin and light appears to inhibit the proliferation of treated PBMNCs, there remains the possibility that although the treated donor cells themselves may not proliferate, they may act as stimulator cells to other responder immune cells in a transfusion recipient, causing the recipient's body to mount an immune response to the treated transfused cells, causing ultimate rejection of the cells. This was tested by measuring the ability of the treated and untreated PBMNC to stimulate the proliferation of allogeneic responder PBMNC.


The assay was set up as described in section 2B. above. The experimental treated and control PBMNC as well as the allogeneic responder PBMNC were adjusted to 1×106 cells/ml in R10 medium. The treated and control PBMNC were transferred in 100 μL volumes to triplicate flat bottom wells (Falcon Primaria). After adding an equal volume of the allogeneic responder PBMNC the plates were incubated for 5 days at 37° C. under 10% CO2, and cell proliferation assessed by uptake of luCi of 3H-thymidine as for PHA (see above).


The results in Table 2C below show that the treated cells do not stimulate proliferation of allogeneic responder cells.











TABLE 2C







Experiment
Untreated cells
Treated cells











No.
+Responder
−Responder
+Responder
−Responder





1
55483 ± 3232
436 ± 126
548 ± 221
436 ± 126


2
50690 ± 750 
3028 ± 3323
806 ± 619
3028 ± 3323


3
55295 ± 6149
412 ± 65 
510 ± 90 
412 ± 65 


Mean
53823
1292
621
1292


±SD
 2714
1503
161
1503









EXAMPLE 3

While the results obtained using the in vitro assays above demonstrate that treatment with riboflavin and light inactivates the treated PBMNC, it remains important to confirm these results with an assay that measures the in vivo responsiveness of the treated or untreated PBMNC. One such assay is to measure xenogeneic GVHD responses in mice which have been transfused with human PBMNCs. These mice (Rag−/− double knockout mice) lack T and B lymphocytes as well as natural killer (NK) cells, γc−/− and previous studies have shown that the injection of human WBC into these mice results in xenogeneic GVHD that is characterized by xenoreactive T cells.


Characterization of Donor Cells


White blood cells were obtained from the leukocyte reduction chamber of a Trima® machine following platelet donation from 6 different human donors. The cells were separated into the mononuclear cell fraction using Ficoll-Hypaque discontinuous centrifugation and then placed in a platelet bag containing autologous plasma. Treated cells received treatment with riboflavin and light, while control cells received no treatment.


3.A. Induction and Clinical Observations of Xenogeneic GVHD Mice

Rag2−/−γc−/− double knockout mice were obtained from Taconic (Germantown, N.Y.).


Injection of Cells


The recipient mice received 350 cGy irradiation the night before injection. The number of donor cells either treated or untreated containing 30×106 CD3+ cells was determined and 3 mice were injected intraperitoneally with that number of cells per group. Each injected mouse was assigned a number. Mice receiving treated cells were given the prefix T, mice receiving untreated cells were given the prefix C.


Analysis of GVHD Response


Mice were weighed twice per week and observed regularly. Recipient mice that demonstrated a dramatic weight loss (usually >20%) and exhibited lethargy, hunched posture and ruffled fur were considered to have developed a GVHD response and were euthanized. Blood was collected by cardiac puncture using a heparinized syringe. In addition, the spleen, bone marrow from the femurs, liver and any intestinal lymphoid tissue that was observed was collected. The weight of the spleen was determined and then single cell suspensions were prepared from all organs by rubbing the organ on a screen. The liver mononuclear cell population was obtained from the liver cells by centrifuging the cells over a Ficoll-Hypaque discontinuous gradient and collecting the cells at the interface. The blood was centrifuged and the plasma collected and stored at −20° C. The buffy coat cells were collected and the red blood cells were lysed using RBC Lysis solution (Gentra, Minneapolis, Minn.). All mice that did not exhibit a GVHD response were euthanized by day 63 (designated as N/A in table below) and a similar analysis was conducted on all of these recipient mice as well.


Analysis of Cells


Cells were initially stained with PECy5 or PE anti-human CD45 or isotype control and then analyzed for the presence of human CD45+ cells in the organs of the transfused mice. CD45+ is a marker found on all leukocytes. The results are shown in Table 3A below.


















TABLE 3A












% CD45
% CD45



Mouse

Death
Spleen

% CD45
% CD45
bone
Intestinal
% CD45


No.
Treatment
(day)
weight
Hct
spleen
blood
marrow
lymphoid
liver















Donor 1
















T1
Yes
N/A
0.05
57
0.0
0.0
0.0
ND
ND


T2
Yes
N/A
0.02
57
0.0
0.0
0.0
ND
ND


T3
Yes
N/A
0.04
55
0.0
0.0
0.0
ND
ND


C4
No
55
0.59
21
1.9
0.2
0.8
66.8
 6.6


C5
No
N/A
0.03
20
0.1
0.0
0.2
ND
 0.01


C6
No
57
0.79
23
3.0
0.3
0.6
48.4
 3.3







Donor 3
















T7
Yes
N/A
0.04
55
0.0
0.0
0.0
ND
ND


T8
Yes
N/A
0.20
48
0.0
0.0
0.0
ND
ND


T9
Yes
N/A
0.18
50
0.0
0.0
0.0
ND
ND


C10
No
43
ND
68
54.9
3.3
1.7
67.8
35.7


C11
No
19
ND
ND
11.2
ND
ND
ND
ND


C12
No
48
0.04
42
42.1
3.9
7.36
45.8
70.7







Donor 4
















T13
Yes
N/A
0.03
56
0.0
0.0
0.0
ND
ND


T14
Yes
N/A
0.02
56
0.0
0.0
0.0
ND
ND


T15
Yes
N/A
0.02
57
0.0
0.0
0.0
ND
ND


C16
No
61
0.07
25
31.3
4.7
3.2
26.8
 7.2


C17
No
58
0.09
26
36.9
4.3
3.5
82.6
 7.8


C18
No
58
0.02
34
47.6
8.1
16.6
91.3
ND







Donor 5
















T19
Yes
37
ND
ND
0.0
0.0
0.0
ND
ND


T20
Yes
N/A
0.11
52
0.0
0.0
0.0
ND
ND


T21
Yes
N/A
0.19
53
0.0
0.0
0.0
ND
ND


C22
No
42
0.12
 6
15.5
16.7
3.7
91.0
19.1


C23
No
42
0.30
 6
4.7
7.6
1.1
44.5
14.9


C24
No
51
ND
ND
ND
ND
ND
ND
ND







Donor 6
















T25
Yes
N/A
0.04
52
0.0
0.0
0.0
ND
ND


T26
Yes
N/A
0.03
53
0.0
0.0
0.0
ND
ND


T27
Yes
N/A
0.01
52
0.0
0.0
0.0
ND
ND


C28
No
60
0.20
40
38.3
1.5
1.9
43.5
20.9


C29
No
60
0.31
38
39.8
1.0
6.0
 1.4
36.5


C30
No
38
0.68
13
47.6
8.1
16.6
 91.32
ND









CONCLUSION

The clinical evaluation of the mice found that one from 15 recipients per group injected with either untreated or treated cells died of unknown causes. No weight loss or human CD45+ cells were detected in the remaining 14 recipients injected with treated cells. These mice had an average spleen weight of 0.07±0.07 g and an average hematocrit of 53.8±2.8%. In contrast 12 of 14 recipients injected with untreated cells were euthanized because of GVHD symptoms including >20% loss of weight and hunched posture, ruffled fur and lethargy and 13 of 14 recipients had high levels of human cell chimerism. This recipient group had an average spleen weight of 0.27±0.27 g, which is significantly larger (p=0.0138) than that of the treated mice (p value <0.02), and an average hematocrit of 27.9±16.9%, which is also significantly lower than that of the treated mice (p value <0.02).


A summary of the results is shown in the following table:




















No. of
No. of dead
GVHD
Body




survivors at
during study
death
weight loss














Total mice No.
end of study
GVHD
non-GVHD
rate
rate

















Treated Group
15
14
0
1
 0/14
 0/14


Control Group
15
2
12
1
12/14
13/14









3.B. Phenotypic and Functional Analysis of Chimeric Human Cells

If human CD45+ cells were detected and enough cells remained for further study, a second battery of staining was done in which the expression of leukocyte subpopulation markers including CD3 (all T cells), CD4 (T helper cells), CD8 (cytotoxic cells), CD14 (macrophages), CD19 (B cells), and CD56 (NK cells) was measured. The data shown in Table 3B below is expressed as % of total cells.
















TABLE 3B





Source of cells
Mouse No.
% CD3
% CD4
% CD8
% CD56
% CD19
% CD14















Donor 1














Spleen
C4
1.64
0.62
0.88
0.05
0.25
1.9


Intestinal
C4
20.30
8.47
11.07

75.36


Spleen
C6
0.81
0.54
0.57

3.38


Intestinal
C6
8.63
6.09
1.99

32.26







Donor 3














Blood
C10
1.24
0.3
1.16

2.22



Spleen
C10
51.42
14.72
38.90

6.26


Liver
C10
5.68
3.08
4.14

25.90


BM
C10
1.98
0.58
0.58

1.48


Intestinal
C10
6.06
3.24
6.04

37.24


Blood
C12
1.23
0.22
1.14
7.5
3.82


Spleen
C12
33.07
6.52
24.16
6.09
18.03


Liver
C12
2.28
1.82
7.42

24.86


BM
C12
3.32
0.68
3.19

3.43


Intestinal
C12
6.52
1.85
4.86

22.06







Donor 4














Spleen
C16
5.09
3.25
2.09

28.21



BM
C16
0.40
0.34


Intestinal
C16
12.28
3.52
1.06

32.84


Blood
C17
0.90
0.28


Spleen
C17
2.80
3.65
1.96

14.05


Liver
C17
1.05
0.34


BM
C17
0.97

0.39


Intestinal
C17
4.57
3.72
1.68

7.74


Blood
C18
2.71
0.32
0.23


BM
C18
1.00
0.59
0.48

1.48


Intestinal
C18
3.59
0.85
0.85

2.15







Donor 5














Blood
C22
5.84
4.54
1.25
2.33
0.71



Spleen
C22
14.62
11.25
4.67


Liver
C22
18.94
17.12
2.52


BM
C22
2.74
2.98
1.50


Intestinal
C22
43.25
40.27
16.07
5.21
71.2


Blood
C23
7.76
5.74
1.31


Spleen
C23
4.55
2.96
2.41


Liver
C23
13.08
11.30
2.24







Donor 6














Blood
C28
2.8
0.36
1.98

2.42



Spleen
C28
46.6
7.9
39.9

11.0


Liver
C28
14.46
1.50
10.86

19.9


Intestinal
C28
31.38
8.54
18.26

25.22


Blood
C29
9.23
1.79
5.84


Spleen
C29
19.68
5.86
11.96


Liver
C29
3.56
3.08
0.32

29.3


BM
C29
6.70
2.22
5.76


Blood
C30
8.02
6.46
0.98


Spleen
C30
42.96
29.84
27.2


BM
C30
13.96
9.9
6.08


Intestinal
C30
22.84
14.24
10.52

10.22










Results


When examined in a donor by donor fashion, the results indicate that the makeup of donor cells transfused can influence the type of cells that are present in different lymphoid organs and in which organs they will be found. CD3+ cells were found in varying numbers in the different lymphoid compartments and CD19+ cells were primarily found in the intestinal lymphoid tissue and in the liver. In contrast to these findings, no macrophages were found and only a limited number of CD56+ cells in a few mice.


3.C. The Level of Cytokines in the Plasma of Rag2−/−γc−/− Recipients of Treated and Untreated White Blood Cells

Plasma of recipient mice was collected when the mice were euthanized either because they were demonstrating symptoms of GVHD or because the experiment was terminated. The levels of cytokines associated with inflammation and acute phase response were measured using the CBA cytometric bead assay kits available from BD Biosciences. The measurement of cytokines associated with the inflammation response is another approach to determine if recipient mice develop an acute phase response to transplanted human cells and also helps define the nature of the xenogeneic GVHD response.


Results are shown in Table 3C below. As can be seen, human cells treated with riboflavin and light do not cause a significant production of inflammatory cytokines.











TABLE 3C









Cytokine concentration



in plasma (pg/ml)












Mouse No.
Treatment
IL-1β
IL-6
IL-8
IL-12p70










Donor 1












T1
Yes
0.1
0.1
0.1
0.1


T2
Yes
32.5
0.1
2.5
4.8


T3
Yes
0.1
0.1
1.5
0.1


C5
No
0.1
0.1
3.2
4.9


C4
No
0.1
6.3
28.4
8.7


C6
No
0.1
390.6
104
158.8







Donor 3












T7
Yes
41.2
2.5
4.3
7.3


T8
Yes
22.6
0.1
4
7.3


T9
Yes
0.1
0.1
2.9
5


C10
No
205.5
8.0
236.2
78.9


C12
No
396.8
13.2
189.6
149.6







Donor 4












T13
Yes
8.4
0.1
3.3
6.2


T14
Yes
0.1
0.1
2.7
6.3


T15
Yes
12.9
0.1
2.2
4.8


C16
No
0.1
300
105
500


C17
No
0.1
162.6
22.6
17.4


C18
No
40
110
100
11







Donor 5












T20
Yes
8.4
0.1
3.2
5.6


T21
Yes
33.9
0.1
3.7
5.7


C22
No
0.1
0.1
3.8
4.8


C23
No
0.1
0.1
4.1
4.3







Donor 6












T25
Yes
0.1
0.1
2
3.4


T26
Yes
0.1
0.1
2.6
2.8


T27
Yes
21.3
2.2
4.4
8.8


C28
No
65
60
80
11


C29
No
0.1
4.2
3.9
4.3


C30
No
0.1
0.1
3.8
3.5









3.D. The Level of Human Immunoglobulins in the Plasma of Rag2−/−γc−/− Recipients of Untreated Control and Treated White Blood Cells

Another measure of human cell chimerism is to determine the level of human IgG and IgM present in the plasma of the recipient mice using an ELISA assay. IgG and IgM are antibodies produced by B cells in response to an antigen. The results shown in Table 3D below indicate that no human IgG (0.10±0.24 ng/ml) or IgM (0.27±0.68 ng/ml) was detected in the plasma of mice injected with treated cells. High levels of IgG (5980.8±2780.8 ng/ml) or IgM (1389.6±845.3 ng/ml) were detected in the plasma of all recipients in which human cell chimerism was detected (these mice received untreated cells).











TABLE 3D









Mice No.























T1
T2
T3
T7
T8
T9
T13
T14
T15
T19
T 20
T21
T25
T26
T27





IgG
0
0
0
0
0
0
0.2
0
0
ND
0
0
0
0
0.9


(ng/ml)


IgM
0
0
0
0
0.1
0
0
0
0
ND
0
2.1
0
2.1
0


(ng/ml)












Mice No.























C4
C5
C6
C10
C11
C12
C16
C17
C18
C22
C23
C24
C28
C29
C30





IgG
5682
0
6134
4241
ND
4102
9087
10202
7283
8973
7034
ND
3478
4221
7314


(ng/ml)


IgM
2189
0
1070
1383
ND
631
685
1500
800
2853
1676
ND
809
1746
2724


(ng/ml)









In vitro studies showed that treatment with riboflavin and light abolished the functional activity of human WBC cells. Consistent with these findings, treated human WBCs did not appear to generate a xenogeneic GVHD response in vivo following injection of these cells into immunodeficient Rag2−/−γc−/− mice recipients. The lack of a xenogeneic GVHD response in the recipient mice also correlated with a lack of human cell chimerism as measured by immunophenotyping. The plasmas of these recipient mice were also found to lack human cytokines or immunoglobulins. These findings indicate that blood cells treated with riboflavin and light are unable to respond in vitro and in vivo and therefore should not induce TA-GVHD in a transfusion recipient.


EXAMPLE 4

This study evaluated the ability of treatment with riboflavin and light to modify the immune response to allogeneic solid organ transplants in rats.


Over a 10 week period, Lewis rats received 8 transfusions (shown by the small arrows in FIGS. 4 and 5) of untreated or treated platelet products containing leukocytes from DA rats. A third group of animals received saline injections. Antibody levels (IgG, IgM) were monitored weekly. At the end of the 10 week period (shown by the large arrow in FIGS. 4 and 5), the transfused animals underwent allogeneic heart transplants with hearts from DA rats to assess the effect pre-transplantation transfusions of platelets with riboflavin and light had on pre-sensitization and transplant rejection.


As can be seen in FIG. 5, the IgM and IgG response in rats that received treated platelets was almost completely abolished compared to animals that received untreated platelets (FIG. 4). In preliminary experiments, (not shown) animals that mounted an IgG response also rejected the subsequent heart transplant.


In summary, treatment with riboflavin and light prevented the development of an Ig response in transplanted animals This inhibition of an Ig response, in particular IgG, shows that pre-transfusion of a solid organ recipient with platelets treated with riboflavin and light helps to prevent alloimmunization to the transplanted allogeneic organ. The lack of rejection of the allogeneic heart transplant in the absence of an IgG response indicates that the pre-treatment may be effective in preventing alloimmune refractoriness to platelets and pre-sensitization to transplants.

Claims
  • 1. A process for reducing rejection of a donor organ by a recipient comprising the steps of: transfusing the recipient of the donor organ with platelets treated with a photosensitizer consisting essentially of riboflavin at a concentration of 50 μM and light at a wavelength between 290-370 nm for around 8 minutes,wherein the recipient is transfused with the treated platelets for a period of time before receiving the donor organ; andtransplanting the donor organ into the recipient.
  • 2. The process of claim 1 wherein the step of transfusing is repeated multiple times before the organ transplant.
  • 3. The process of claim 1 wherein the donor organ to be transplanted into the recipient is allogeneic.
  • 4. The process of claim 1 wherein the donor organ to be transplanted into the recipient is xenogeneic.
  • 5. The process of claim 1 wherein the treated platelets are from an allogeneic donor.
  • 6. The process of claim 1 wherein the treated platelets reduce the development of antibodies to the transplanted organ in the transplant recipient.
  • 7. The process of claim 1 wherein the donor organ is a solid organ.
  • 8. The process of claim 7 wherein the donor organ is a heart.
PRIORITY CLAIM

This application is a divisional of U.S. patent application Ser. No. 11/469,186, filed on Aug. 31, 2006, now allowed, which claims the benefit of U.S. provisional application No. 60/714,682 filed Sep. 7, 2005; and a continuation-in-part of U.S. patent application Ser. No. 10/648,536, filed Aug. 25, 2003, which is a continuation of 10/377,524 filed Feb. 28, 2003 which is a continuation of 09/586,147, filed Jun. 2, 2000, now abandoned.

US Referenced Citations (304)
Number Name Date Kind
683690 Johnson Oct 1901 A
1733239 Roberts Oct 1929 A
1961700 Moehler Jun 1934 A
2056614 Moehler Oct 1936 A
2212330 Thomas Aug 1940 A
2340890 Lang t al Feb 1944 A
2417143 Tishler et al. Mar 1947 A
2786014 Tullis Mar 1957 A
3057865 Bardos et al. Oct 1962 A
3456053 Crawford Jul 1969 A
3629071 Sekhar Dec 1971 A
3683177 Veloz Aug 1972 A
3683183 Vizzini et al. Aug 1972 A
3705985 Manning et al. Dec 1972 A
3776694 Leittl Dec 1973 A
3852032 Urbach Dec 1974 A
3864081 Logrippo Feb 1975 A
3874384 Deindoerfer et al. Apr 1975 A
3894236 Hazelrigg Jul 1975 A
3926556 Boucher Dec 1975 A
3927325 Hungate et al. Dec 1975 A
4061537 Seiler et al. Dec 1977 A
4112070 Harmening Sep 1978 A
4124598 Hearst et al. Nov 1978 A
4139348 Swartz Feb 1979 A
4159320 Opitz Jun 1979 A
4169204 Hearst et al. Sep 1979 A
4173631 Graham et al. Nov 1979 A
4181128 Swartz Jan 1980 A
4196281 Hearst et al. Apr 1980 A
4264601 Trachewsky Apr 1981 A
4267269 Grode et al. May 1981 A
4312883 Baccichetti et al. Jan 1982 A
4321918 Clark, II Mar 1982 A
4321919 Edelson Mar 1982 A
4336809 Clark Jun 1982 A
4381004 Babb Apr 1983 A
4390619 Harmening-Pittiglio Jun 1983 A
4398031 Bender et al. Aug 1983 A
4398906 Edelson Aug 1983 A
4402318 Swartz Sep 1983 A
4407282 Swartz Oct 1983 A
4421987 Herold Dec 1983 A
4424201 Valinsky et al. Jan 1984 A
4428744 Edelson Jan 1984 A
4432750 Estep Feb 1984 A
4456512 Bieler et al. Jun 1984 A
4457918 Holick et al. Jul 1984 A
4464166 Edelson Aug 1984 A
4467206 Taylor et al. Aug 1984 A
4474153 Hanamoto Oct 1984 A
4481167 Ginter et al. Nov 1984 A
4493981 Payne Jan 1985 A
4568328 King Feb 1986 A
4572899 Walker et al. Feb 1986 A
4573960 Goss Mar 1986 A
4573961 King Mar 1986 A
4573962 Troutner Mar 1986 A
4576143 Clark, III Mar 1986 A
4578056 King et al. Mar 1986 A
4585735 Meryman et al. Apr 1986 A
4596547 Troutner Jun 1986 A
4604356 Blake, II Aug 1986 A
4608255 Kahn et al. Aug 1986 A
4609372 Carmen et al. Sep 1986 A
4612007 Edelson Sep 1986 A
4613322 Edelson Sep 1986 A
4614190 Stanco et al. Sep 1986 A
4623328 Hartranft Nov 1986 A
4626431 Batchelor et al. Dec 1986 A
4642171 Sekine et al. Feb 1987 A
4645649 Nagao Feb 1987 A
4648992 Graf et al. Mar 1987 A
4649151 Dougherty et al. Mar 1987 A
4651739 Oseroff et al. Mar 1987 A
4675185 Kandler et al. Jun 1987 A
4683195 Mullis et al. Jul 1987 A
4683202 Mullis Jul 1987 A
4683889 Edelson Aug 1987 A
4684521 Edelson Aug 1987 A
4693981 Wiesehahn et al. Sep 1987 A
4695460 Holme Sep 1987 A
4704352 Miripol et al. Nov 1987 A
4708715 Troutner et al. Nov 1987 A
4726949 Miripol et al. Feb 1988 A
4727027 Wiesehahn et al. Feb 1988 A
4737140 Lee et al. Apr 1988 A
4748120 Wiesehahn May 1988 A
4769318 Hamasaki et al. Sep 1988 A
4775625 Sieber Oct 1988 A
4784852 Johansson Nov 1988 A
4788038 Matsunaga Nov 1988 A
RE32874 Rock et al. Feb 1989 E
4828976 Murphy May 1989 A
4831268 Fisch et al. May 1989 A
4833165 Louderback May 1989 A
4861704 Reemtsma et al. Aug 1989 A
4866282 Miripol et al. Sep 1989 A
4878891 Judy et al. Nov 1989 A
4880788 Moake et al. Nov 1989 A
4915683 Sieber Apr 1990 A
4921473 Lee et al. May 1990 A
4925665 Murphy May 1990 A
4930516 Alfano et al. Jun 1990 A
4946438 Reemtsma et al. Aug 1990 A
4948980 Wedekamp Aug 1990 A
4950665 Floyd Aug 1990 A
4952812 Miripol et al. Aug 1990 A
4960408 Klainer et al. Oct 1990 A
4961928 Holme et al. Oct 1990 A
4978688 Louderback Dec 1990 A
4986628 Lozhenko et al. Jan 1991 A
4992363 Murphy Feb 1991 A
4994367 Bode et al. Feb 1991 A
4998931 Slichter et al. Mar 1991 A
4999375 Bachynsky et al. Mar 1991 A
5011695 Dichtelmuller et al. Apr 1991 A
5017338 Surgenor May 1991 A
5020995 Levy Jun 1991 A
5030200 Judy et al. Jul 1991 A
5039483 Sieber et al. Aug 1991 A
5041078 Matthews et al. Aug 1991 A
5089146 Carmen et al. Feb 1992 A
5089384 Hale Feb 1992 A
5092773 Levy Mar 1992 A
5114670 Duffey May 1992 A
5114957 Hendler et al. May 1992 A
5120649 Horowitz et al. Jun 1992 A
5123902 Muller et al. Jun 1992 A
5133932 Gunn et al. Jul 1992 A
5147776 Koerner, Jr. Sep 1992 A
5149718 Meruelo et al. Sep 1992 A
5150705 Stinson Sep 1992 A
5166528 LeVay Nov 1992 A
5184020 Hearst et al. Feb 1993 A
5185532 Zabsky et al. Feb 1993 A
5192264 Fossel Mar 1993 A
5211960 Babior May 1993 A
5216251 Matschke Jun 1993 A
5229081 Suda Jul 1993 A
5232844 Horowitz et al. Aug 1993 A
5234808 Murphy Aug 1993 A
5236716 Carmen et al. Aug 1993 A
5247178 Ury et al. Sep 1993 A
5248506 Holme et al. Sep 1993 A
5250303 Meryman et al. Oct 1993 A
5258124 Bolton et al. Nov 1993 A
5269946 Goldhaber et al. Dec 1993 A
5273713 Levy Dec 1993 A
5281392 Rubinstein Jan 1994 A
5288605 Lin et al. Feb 1994 A
5288647 Zimlich, Jr. et al. Feb 1994 A
5290221 Wolf, Jr. et al. Mar 1994 A
5300019 Bischof et al. Apr 1994 A
5304113 Sieber et al. Apr 1994 A
5318023 Vari et al. Jun 1994 A
5340716 Ullman et al. Aug 1994 A
5342752 Platz et al. Aug 1994 A
5344752 Murphy Sep 1994 A
5344918 Dazey et al. Sep 1994 A
5358844 Stossel et al. Oct 1994 A
5360734 Chapman et al. Nov 1994 A
5366440 Fossel Nov 1994 A
5372929 Cimino Dec 1994 A
5376524 Murphy et al. Dec 1994 A
5378601 Gepner-Puszkin Jan 1995 A
5399719 Wollowitz et al. Mar 1995 A
5418130 Platz et al. May 1995 A
5419759 Naficy May 1995 A
5427695 Brown Jun 1995 A
5433738 Stinson Jul 1995 A
5459030 Lin et al. Oct 1995 A
5466573 Murphy et al. Nov 1995 A
5474891 Murphy Dec 1995 A
5482828 Lin et al. Jan 1996 A
5487971 Holme et al. Jan 1996 A
5494590 Smith et al. Feb 1996 A
5503721 Hearst et al. Apr 1996 A
5512187 Buchholz et al. Apr 1996 A
5516629 Park et al. May 1996 A
5527704 Wolf, Jr. et al. Jun 1996 A
5536238 Bischof Jul 1996 A
5545516 Wagner Aug 1996 A
5547635 Duthie, Jr. Aug 1996 A
5550111 Suhadolnik et al. Aug 1996 A
5556958 Carroll et al. Sep 1996 A
5556993 Wollowitz et al. Sep 1996 A
5557098 D'Silva Sep 1996 A
5559250 Cook et al. Sep 1996 A
5569579 Murphy Oct 1996 A
5571666 Floyd et al. Nov 1996 A
5578736 Wollowitz et al. Nov 1996 A
5585503 Wollowitz et al. Dec 1996 A
5587490 Goodrich, Jr. et al. Dec 1996 A
5593823 Wollowitz et al. Jan 1997 A
5597722 Chapman et al. Jan 1997 A
5607924 Magda et al. Mar 1997 A
5618662 Lin et al. Apr 1997 A
5622867 Livesey et al. Apr 1997 A
5624435 Furumoto et al. Apr 1997 A
5624794 Bitensky et al. Apr 1997 A
5625079 Wollowitz et al. Apr 1997 A
5628727 Hakky et al. May 1997 A
5639376 Lee et al. Jun 1997 A
5639382 Brown Jun 1997 A
5643334 Eckhouse et al. Jul 1997 A
5652096 Cimino Jul 1997 A
5653887 Wahl et al. Aug 1997 A
5654443 Wollowitz et al. Aug 1997 A
5656154 Meryman Aug 1997 A
5656498 Iijima et al. Aug 1997 A
5658530 Dunn Aug 1997 A
5658722 Margolis-Nunno et al. Aug 1997 A
5683661 Hearst et al. Nov 1997 A
5683768 Shang et al. Nov 1997 A
5686436 Van Dyke Nov 1997 A
5688475 Duthie, Jr. Nov 1997 A
5691132 Wollowitz et al. Nov 1997 A
5698524 Mach et al. Dec 1997 A
5698677 Eibl et al. Dec 1997 A
5702684 McCoy et al. Dec 1997 A
5707401 Talmore Jan 1998 A
5709653 Leone Jan 1998 A
5709991 Lin et al. Jan 1998 A
5709992 Rubinstein Jan 1998 A
5712085 Wollowitz et al. Jan 1998 A
5712086 Horowitz et al. Jan 1998 A
5714328 Magda et al. Feb 1998 A
5736313 Spargo et al. Apr 1998 A
5739013 Budowsky et al. Apr 1998 A
5753428 Yuasa et al. May 1998 A
5756553 Iguchi et al. May 1998 A
5769839 Carmen et al. Jun 1998 A
5772960 Ito et al. Jun 1998 A
5783093 Holme Jul 1998 A
5789150 Margolis-Nunno et al. Aug 1998 A
5789151 Bitensky et al. Aug 1998 A
5789601 Park et al. Aug 1998 A
5798238 Goodrich, Jr. et al. Aug 1998 A
5798523 Villeneuve et al. Aug 1998 A
5817519 Zelmanovic et al. Oct 1998 A
5827644 Floyd et al. Oct 1998 A
5834198 Famulok et al. Nov 1998 A
5840252 Giertych Nov 1998 A
5843459 Wang et al. Dec 1998 A
5846961 Van Dyke Dec 1998 A
5854967 Hearst et al. Dec 1998 A
5866074 Chapman et al. Feb 1999 A
5869701 Park et al. Feb 1999 A
5871900 Wollowitz et al. Feb 1999 A
5876676 Stossel et al. Mar 1999 A
5899874 Jonsson May 1999 A
5906915 Payrat et al. May 1999 A
5908742 Lin et al. Jun 1999 A
5919614 Livesey et al. Jul 1999 A
5922278 Chapman et al. Jul 1999 A
5935092 Sun et al. Aug 1999 A
5955256 Sowemimo-Coker et al. Sep 1999 A
5955257 Burger et al. Sep 1999 A
5965349 Lin et al. Oct 1999 A
5972593 Wollowitz et al. Oct 1999 A
5976884 Chapman et al. Nov 1999 A
5981163 Horowitz et al. Nov 1999 A
6004741 Wollowitz et al. Dec 1999 A
6004742 Wollowitz et al. Dec 1999 A
6017691 Wollowitz et al. Jan 2000 A
6020333 Berque Feb 2000 A
6060233 Wiggins May 2000 A
6063624 Kandler et al. May 2000 A
6077659 Ben-Hur et al. Jun 2000 A
6087141 Margolis-Nunno et al. Jul 2000 A
6093725 Cook et al. Jul 2000 A
6106773 Miekka et al. Aug 2000 A
6133460 Wollowitz et al. Oct 2000 A
6143490 Cook et al. Nov 2000 A
6171777 Cook et al. Jan 2001 B1
6177441 Cook et al. Jan 2001 B1
6194139 Wollowitz et al. Feb 2001 B1
6197207 Chapman et al. Mar 2001 B1
6214534 Horowitz et al. Apr 2001 B1
6218100 Wollowitz et al. Apr 2001 B1
6258319 Hearst et al. Jul 2001 B1
6258577 Goodrich et al. Jul 2001 B1
6268120 Platz et al. Jul 2001 B1
6270952 Cook et al. Aug 2001 B1
6277337 Goodrich et al. Aug 2001 B1
6410219 Cook et al. Jun 2002 B1
6413714 Margolis-Nunno et al. Jul 2002 B1
6420570 Wollowitz et al. Jul 2002 B1
6433343 Cimino et al. Aug 2002 B1
6455286 Wollowitz et al. Sep 2002 B1
6461567 Hearst et al. Oct 2002 B1
6469052 Wollowitz et al. Oct 2002 B2
6503699 Wollowitz et al. Jan 2003 B1
6514987 Cook et al. Feb 2003 B1
6544727 Hei Apr 2003 B1
6565802 Hanley et al. May 2003 B1
6576201 Woo et al. Jun 2003 B1
6586749 Cimino et al. Jul 2003 B2
6596230 Woo et al. Jul 2003 B1
6680025 Hearst et al. Jan 2004 B2
6686480 Wollowitz et al. Feb 2004 B2
20010053597 Slichter Dec 2001 A1
20020022215 Sobsey et al. Feb 2002 A1
Foreign Referenced Citations (53)
Number Date Country
0066886 Jun 1982 EP
0124363 Apr 1984 EP
0108588 May 1984 EP
0196515 Mar 1986 EP
0184331 Jun 1986 EP
0525138 Dec 1991 EP
0491757 Jul 1992 EP
0510185 Oct 1992 EP
0590514 Apr 1994 EP
0679398 Nov 1995 EP
0754461 Jan 1997 EP
0801072 Oct 1997 EP
2674753 Oct 1992 FR
2715303 Jul 1995 FR
2718353 Oct 1995 FR
2034463 Jun 1980 GB
59020218 Feb 1984 JP
WO8302328 Jul 1983 WO
WO8502116 May 1985 WO
WO8810087 Dec 1988 WO
WO8906702 Jul 1989 WO
WO9000059 Jan 1990 WO
WO9010461 Sep 1990 WO
WO9102529 Mar 1991 WO
WO9208348 May 1992 WO
WO9208349 May 1992 WO
WO9211057 Jul 1992 WO
WO9217173 Oct 1992 WO
WO9300005 Jan 1993 WO
WO9407426 Apr 1994 WO
WO9407499 Apr 1994 WO
WO9502325 Jan 1995 WO
WO9511028 Apr 1995 WO
WO9512973 May 1995 WO
WO9516348 Jun 1995 WO
WO9614740 May 1996 WO
WO9614741 May 1996 WO
WO9707674 Mar 1997 WO
WO9718844 May 1997 WO
WO9722245 Jun 1997 WO
WO9736581 Oct 1997 WO
WO9736634 Oct 1997 WO
WO9822150 May 1998 WO
WO9830545 Jul 1998 WO
WO9831219 Jul 1998 WO
WO9841087 Sep 1998 WO
WO9851147 Nov 1998 WO
WO9911305 Mar 1999 WO
WO0004930 Mar 2000 WO
WO0011946 Mar 2000 WO
WO0128599 Apr 2001 WO
WO0230190 Apr 2002 WO
WO0202153 Oct 2002 WO
Related Publications (1)
Number Date Country
20100080781 A1 Apr 2010 US
Provisional Applications (1)
Number Date Country
60714682 Sep 2005 US
Divisions (1)
Number Date Country
Parent 11469186 Aug 2006 US
Child 12630505 US
Continuations (2)
Number Date Country
Parent 10377524 Feb 2003 US
Child 10648536 US
Parent 09586147 Jun 2000 US
Child 10377524 US
Continuation in Parts (1)
Number Date Country
Parent 10648536 Aug 2003 US
Child 11469186 US