U.S. patent application number 10/392636 was filed with the patent office on 2003-09-25 for method and apparatus for selectively targeting specific cells within a mixed cell population.
Invention is credited to Eisfeld, Timothy M., Koller, Manfred R., Palsson, Bernhard O..
Application Number | 20030180902 10/392636 |
Document ID | / |
Family ID | 46203746 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180902 |
Kind Code |
A1 |
Palsson, Bernhard O. ; et
al. |
September 25, 2003 |
Method and apparatus for selectively targeting specific cells
within a mixed cell population
Abstract
This invention provides a method and apparatus for selectively
identifying, and targeting with an energy beam, specific cells
within a mixed cell population, for the purpose of inducing a
response in the targeted cells. Using the present invention, every
detectable cell in a population can be identified and affected,
without substantially affecting non-targeted cells within the
mixture.
Inventors: |
Palsson, Bernhard O.; (La
Jolla, CA) ; Koller, Manfred R.; (San Diego, CA)
; Eisfeld, Timothy M.; (La Jolla, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
46203746 |
Appl. No.: |
10/392636 |
Filed: |
March 18, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10392636 |
Mar 18, 2003 |
|
|
|
09451659 |
Nov 30, 1999 |
|
|
|
6534308 |
|
|
|
|
09451659 |
Nov 30, 1999 |
|
|
|
09049677 |
Mar 27, 1998 |
|
|
|
6143535 |
|
|
|
|
09049677 |
Mar 27, 1998 |
|
|
|
08824968 |
Mar 27, 1997 |
|
|
|
5874266 |
|
|
|
|
Current U.S.
Class: |
435/173.1 ;
435/287.1 |
Current CPC
Class: |
G01N 33/5091 20130101;
C12N 13/00 20130101; C12N 5/0093 20130101; G02B 21/0088 20130101;
C12N 5/0087 20130101; G01N 33/56966 20130101; G02B 21/16 20130101;
C12M 35/02 20130101; C12N 15/1034 20130101; G01N 33/5005 20130101;
G02B 21/365 20130101 |
Class at
Publication: |
435/173.1 ;
435/287.1 |
International
Class: |
C12N 013/00; C12M
001/34 |
Claims
We claim:
1. An apparatus for selectively inducing a response in one or more
targeted cells within a biological specimen, comprising: an
illumination source for illuminating a frame of cells in said
biological specimen; an image capture system that captures an image
of said frame of cells; first commands for determining the
locations of said one or more targeted cells within said image; an
energy source that emits an energy beam sufficient to induce a
response in at least one of said one or more targeted cells within
said frame of cells; and second commands for steering said energy
beam to said locations of said one or more targeted cells.
2. The apparatus of claim 1, wherein said one or more targeted
cells are identified by the presence of, or absence of, a
label.
3. The apparatus of claim 2, wherein said label is directed at the
cell surface.
4. The apparatus of claim 3, wherein said label comprises a
monoclonal antibody.
5. The apparatus of claim 2, wherein said label comprises a
membrane permeable reagent.
6. The apparatus of claim 1, wherein said illumination source
comprises a diffused laser light.
7. The apparatus of claim 1, wherein said illumination source
comprises an arc lamp.
8. The apparatus of claim 1, wherein said illumination source
comprises a light-emitting diode.
9. The apparatus of claim 1, wherein said illumination source
comprises an incandescent lamp.
10. The apparatus of claim 1, wherein said illumination source
comprises a fluorescent lamp.
11. The apparatus of claim 1, wherein said illumination source only
illuminates a sub-population of cells in said biological
specimen.
12. The apparatus of claim 1, wherein said one or more targeted
cells comprises tumor cells.
13. The apparatus of claim 1, wherein said one or more targeted
cells comprises fibroblasts.
14. The apparatus of claim 1, wherein said one or more targeted
cells comprises T-cells.
15. The apparatus of claim 1, wherein said one or more targeted
cells comprises teratoma-forming cells.
16. The apparatus of claim 1, wherein said one or more targeted
cells comprises non-tumor cells.
17. The apparatus of claim 1, wherein said image capture system
comprises a camera.
18. The apparatus of claim 17, wherein said camera comprises a
charge-coupled device.
19. The apparatus of claim 1, wherein said energy source comprises
a laser.
20. The method of claim 19, wherein the wavelength of energy
emitted from said laser is approximately between 100 nanometers and
30 micrometers.
21. The apparatus of claim 1, wherein said first commands comprise
commands for determining the two-dimensional locations of said one
or more targeted cells within said biological specimen.
22. The apparatus of claim 1, wherein said first commands comprise
commands for determining the three-dimensional locations of said
one or more targeted cells within said biological specimen.
23. The apparatus of claim 1, further comprising one or more energy
beam steering devices that are controlled by said second
commands.
24. The apparatus of claim 23, wherein said energy beam steering
devices comprise one or more elements that reflect the energy
beam.
25. The apparatus of claim 23, wherein said energy beam steering
devices comprise one or more elements that refract the energy
beam.
26. The apparatus of claim 23, wherein said energy beam steering
devices comprise one or more elements that diffract the energy
beam.
27. The apparatus of claim 1, wherein said first commands are
executed under the control of a computer.
28. The apparatus of claim 1, wherein said second commands are
executed under the control of a computer.
29. The apparatus of claim 1, wherein said apparatus processes at
least 1, 2, 3, 4, 5, 6, or 7 square centimeters of biological
specimen area per minute.
30. The apparatus of claim 1, wherein said apparatus images at
least 0.25, 0.5, 1, 2, 3 or 4 million cells of said biological
specimen per minute.
31. The apparatus of claim 1, wherein said apparatus can induce a
response in at least 50, 100, 150, 200, 250, 300, 350, or 400
targeted cells per second.
32. The apparatus of claim 1, wherein said response induced in said
one or more targeted cells is cell death.
33. The apparatus of claim 1, wherein said response induced in said
one or more targeted cells comprises activation of a photosensitive
agent.
34. The apparatus of claim 1, wherein said response induced in said
one or more targeted cells comprises optoporation to allow entry of
a substance.
35. The apparatus of claim 34, wherein said substance is genetic
material.
36. The apparatus of claim 1, wherein said response induced in said
one or more targeted cells comprises inactivation of a cell
component.
37. The apparatus of claim 1, wherein said response induced in said
one or more targeted cells comprises controlled movement of said
one or more targeted cells.
38. The apparatus of claim 1, wherein said response is excitation
of fluorescent dye within said one or more targeted cells.
39. The apparatus of claim 1, further comprising an energy
absorbing dye in contact with said one or more targeted cells.
40. The apparatus of claim 1, further comprising third commands
that control an autofocus mechanism linked to said image capture
system.
41. The apparatus of claim 40, wherein said third commands are
executed under the control of a computer.
42. An apparatus for selectively inducing a response in one or more
targeted cells within a biological specimen, comprising: a scanning
lens which focuses on a field-of-view within said biological
specimen, said field-of-view comprising a plurality of frames of
cells; an illumination source that illuminates one or more frames
of cells; an image capture system that captures an image of a first
frame of cells in said field-of-view; first commands for
determining the locations of said one or more targeted cells in
said first frame of cells; an energy source that emits an energy
beam sufficient to induce a response in at least one of said one or
more targeted cells; and second commands for steering said energy
beam to said locations of said one or more targeted cells within
said first frame of cells.
43. The apparatus of claim 42, further comprising: means for
directing the output of said illumination source to a second frame
of cells in said field-of-view; means for capturing an image of
said second frame of cells; third commands for determining the
locations of said one or more targeted cells in said second frame
of cells; and fourth commands for steering said energy beam to said
locations of said one or more targeted cells within said second
frame of cells.
44. The apparatus of claim 42, further comprising means for moving
the position of said biological specimen with respect to said
scanning lens.
45. The apparatus of claim 42, wherein said scanning lens is of
F-theta design with a diameter of at least 8, 10, 12, 14, 16, or 18
mm.
46. An apparatus for identifying the cells of a first population of
labeled cells within a mixture of said first population and a
second population of non-labeled cells, comprising: an illumination
source for illuminating a frame of cells in said mixture; an image
capture system that captures an image of said frame of cells; and
first commands for determining the locations of at least one cell
in said first population of labeled cells by reference to said
image.
47. The apparatus of claim 46, further comprising a memory for
storing said locations of said at least one cell in said first
population of labeled cells.
48. The apparatus of claim 46, further comprising a memory for
storing the quantity of cells in said first population of labeled
cells.
49. The apparatus of claim 46, further comprising an energy source
that emits an energy beam sufficient to induce a response in at
least one cell in said first population of labeled cells.
50. An apparatus for determining a morphological or physiological
characteristic of individual cells in a biological specimen,
comprising: an illumination source for illuminating a frame of
cells in said biological specimen; an image capture system that
captures an image of said frame of cells; first commands for
determining the location of a first targeted cell in said
biological specimen by reference to said image; second commands for
intercepting said first targeted cell with an energy beam
sufficient to interrogate said first targeted cell for the presence
of a morphological or physiological characteristic; and a detector
for measuring the response of said first targeted cell to said
interrogation.
51. The apparatus of claim 50, wherein said first targeted cell is
labeled with a fluorescently-tagged antibody.
52. The apparatus of claim 50, wherein said first targeted cell is
labeled with two labels, wherein the first label is activated by
said illumination source and the second label is activated by said
energy beam.
53. The apparatus of claim 50, wherein said morphological or
physiological characteristic is calcium flux.
54. The apparatus of claim 50, wherein said morphological or
physiological characteristic is cell cycle status.
55. The apparatus of claim 50, wherein said morphological or
physiological characteristic is cell mitotic history.
56. The apparatus of claim 50, wherein said morphological or
physiological characteristic is viability.
57. The apparatus of claim 50, wherein said morphological or
physiological characteristic is membrane integrity.
58. The apparatus of claim 50, wherein said morphological or
physiological characteristic is intracellular pH.
59. The apparatus of claim 50, wherein said morphological or
physiological characteristic is transmembrane potential.
60. The apparatus of claim 50, wherein said morphological or
physiological characteristic is glutathione reductive stage.
61. The apparatus of claim 50, wherein said morphological or
physiological characteristic is thiol activity.
62. The apparatus of claim 50, wherein said morphological or
physiological characteristic is expression of a gene.
63. An apparatus for selectively inducing a response in one or more
targeted cells within a biological specimen, comprising: an
illumination source for illuminating a frame of cells in said
biological specimen; an image capture system that captures an image
of said frame of cells; first commands for determining the
locations of said one or more targeted cells within said image; an
energy source that emits an energy beam sufficient to induce a
response in at least one of said one or more targeted cells within
said frame of cells; and a sensor that detects the amount of energy
emitted by said energy source.
64. A method for selectively inducing a response in one or more
targeted cells within a biological specimen, comprising:
illuminating a frame of cells in said biological specimen;
capturing an image of said frame of cells; determining the
locations of said one or more targeted cells within said image; and
steering an energy beam to the locations of said one or more
targeted cells, wherein said energy beam is sufficient to induce a
response in at least one of said one or more targeted cells.
65. The method of claim 64, wherein determining the locations of
said one or more targeted cells comprises determining the presence
of, or absence of, a label attached to said one or more targeted
cells.
66. The method of claim 64, wherein capturing an image of said
frame of cells comprises capturing an image of labeled cells.
67. The method of claim 64, wherein illuminating said frame of
cells comprises illuminating said frame of cells with an excitation
laser light source.
68. The method of claim 64, wherein said response is selected from
the group consisting of cell death, optoporation, activation of a
photosensitive agent, inactivation of a cell component, controlled
movement, and excitation of a fluorescent reagent.
69. A: method for rapidly inducing a response in cells within a
cell population, said cell population being held in a container,
comprising: illuminating a first field of cells in said cell
population; capturing an image of a first frame of cells within
said first field of cells; determining the locations of first
targeted cells within said first frame of cells by reference to
said image; and steering an energy beam to the locations of said
first targeted cells, wherein said energy beam is sufficient to
induce a response in at least one of said first targeted cells.
70. The method of claim 69, further comprising: capturing an image
of a second frame of cells within said first field of cells;
determining the locations of second targeted cells within said
second frame of cells by reference to said image; and steering an
energy beam to the locations of said second targeted cells within
said second frame of cells, wherein said energy beam is sufficient
to induce a response in at least one of said second targeted
cells.
71. The method of claim 70, further comprising: illuminating a
second field of cells in said cell population; capturing an image
of a first frame of cells within said second field of cells;
determining the locations of first targeted cells within said first
frame of cells by reference to said image; and steering an energy
beam to the locations of said first targeted cells, wherein said
energy beam is sufficient to induce a response in at least one of
said first targeted cells.
72. The method of claim 71, wherein illuminating said second field
of cells comprises moving said container.
73. A method for selectively inducing a response in one or more
targeted cells within a biological specimen, comprising: focusing a
scanning lens on a field-of-view within said biological specimen,
said field-of-view comprising a plurality of frames of cells;
illuminating one or more frames of cells within said field-of-view;
capturing an image of a first frame of cells in said field-of-view;
determining the locations of said one or more targeted cells in
said first frame of cells; emitting an energy beam sufficient to
induce a response in at least one of said one or more targeted
cells; and steering said energy beam to the locations of said one
or more targeted cells within said first frame of cells.
74. The method of claim 73, wherein said response is selected from
the group consisting of cell death, optoporation, activation of a
photosensitive agent, inactivation of a cell component, controlled
movement, and excitation of a fluorescent reagent.
75. The method of claim 73, wherein capturing an image of said
first frame of cells comprises capturing an image of labeled
cells.
76. The method of claim 73, wherein said illuminating one or more
frames of cells comprises illuminating said one or more frames of
cells with an excitation laser light source.
77. A method for inducing a response in one or more targeted cells
within a cell population, comprising: illuminating cells in said
cell population; capturing an image of the illuminated cells;
determining the locations of said one or more targeted cells within
said image; emitting an energy beam sufficient to induce a response
in at least one of said one or more targeted cells; and measuring
the amount of energy contained in said energy beam; and steering
said energy beam to the locations of said one or more targeted
cells.
78. The method of claim 77, comprising measuring the amount of
energy contained in said energy beam prior to steering said energy
beam.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/049,677, filed on Mar. 27, 1998 which is a
continuation-in-part of U.S. patent application Ser. No.
08/824,968, filed on Mar. 27, 1997, now U.S. Pat. No. 5,874,266,
issued on Feb. 23, 1999.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to methods and apparatus for
selectively targeting specific cells within a mixed population of
living cells. In particular, this invention relates to high-speed
methods and apparatus for selectively identifying, and individually
targeting with an energy beam, specific cells within a mixed cell
population to induce a response in the targeted cells.
[0004] 2. Description of the Related Art
[0005] The use of cellular therapies is growing rapidly, and is
therefore becoming an important therapeutic modality in the
practice of medicine. Unlike other therapies, cellular therapies
achieve a long-lasting, and often permanent benefit through the use
of living cells. Hematopoietic stem cell (HSC) (e.g., bone marrow
or mobilized peripheral blood) transplantation is one example of a
practiced, insurance-reimbursed cellular therapy. Many other
cellular therapies are being developed, including immunotherapy for
cancer and infectious diseases, chondrocyte therapy for cartilage
defects, neuronal cell therapy for neurodegenerative diseases, and
stem cell therapy for numerous indications. Many of these therapies
require the removal of unwanted, detrimental cells for full
efficacy to be realized.
[0006] Gene therapy is another active area of developing medicine
that can influence the success of cellular therapy. Given the rapid
advances in the understanding of the human genome, it is likely
that many genes will be available for insertion into cells prior to
transplantation into patients. However, obtaining efficient
targeted delivery of genes into specific cells of interest has
remained a difficult obstacle in the development of these
therapies.
[0007] In the treatment of cancer, it has been found that high-dose
chemotherapy and/or radiation therapy can be used to selectively
kill rapidly dividing cancer cells in the body. Unfortunately,
several other cell types in the body are also rapidly dividing, and
in fact, the dose-limiting toxicity for most anti-cancer therapies
is the killing of HSCs and progenitor cells in the bone marrow. HSC
transplantation was developed as a therapy to rescue the
hematopoietic system following anti-cancer treatments. Upon
infusion, the HSCs and progenitor cells within the transplant
selectively home to the bone marrow and engraft. This process is
monitored clinically through daily blood cell counts. Once blood
counts return to acceptable levels, usually within 20 to 30 days,
the patient is considered engrafted and is released from the
hospital.
[0008] HSC transplants have been traditionally performed with bone
marrow, but mobilized peripheral blood (obtained via leukapheresis
after growth factor or low-dose chemotherapy administration) has
recently become the preferred source because it eliminates the need
to harvest approximately one liter of bone marrow from the patient.
In addition, HSCs from mobilized peripheral blood result in more
rapid engraftment (8 to 15 days), leading to less critical patient
care and earlier discharge from the hospital. HSC transplantation
has become an established therapy for treating many diseases, such
that over 45,000 procedures were performed worldwide in 1997.
[0009] HSC transplantation may be performed using either donor
cells (allogeneic), or patient cells that have been harvested and
cryopreserved prior to administration of high-dose anti-cancer
therapy (autologous). Autologous transplants are widely used for
treating a variety of diseases including breast cancer, Hodgkin's
and non-Hodgkin's lymphomas, neuroblastoma, and multiple myeloma.
The number of autologous transplants currently outnumbers
allogeneic transplants by approximately a 2:1 ratio. This ratio is
increasing further, mainly due to graft-versus-host disease (GVHD)
complications associated with allogeneic transplants. One of the
most significant problems with autologous transplants is the
reintroduction of tumor cells to the patient along with the HSCs,
because these tumor cells contribute to relapse of the original
disease.
[0010] As a tumor grows, tumor cells eventually leave the original
tumor site and migrate through the bloodstream to other locations
in the body. This process, called tumor metastasis, results in the
formation and growth of satellite tumors that greatly increase the
severity of the disease. The presence of these metastatic tumor
cells in the blood and other tissues, often including bone marrow,
can create a significant problem for autologous transplantation. In
fact, there is a very high probability that metastatic tumor cells
will contaminate the harvested HSCs that are to be returned to the
patient following anti-cancer therapy.
[0011] The presence of contaminating tumor cells in autologous bone
marrow and mobilized peripheral blood harvests has been confirmed
in numerous scientific studies. Tumor cell contamination has been
repeatedly observed in patients with T-cell lymphoma, non-Hodgkin's
lymphoma, leukemia, neuroblastoma, lung cancer, breast cancer, etc.
(Brugger et al. 1994; Gulati and Acaba 1993; Kvalheim et al. 1996;
Mapara et al. 1997; Paulus et al. 1997; Shpall and Jones 1994;
Vervoordeldonk et al. 1997). In every study, all or nearly all of
the patient samples analyzed were positive for tumor contamination.
The level of tumor cell burden in these HSC harvests varied widely
depending upon the type and stage of disease. Typical numbers
indicate that tumor cells are present in the range of 3 to 3,000
tumor cells per million hematopoietic cells. Since the transplanted
cell number is on the order of 10 billion hematopoietic cells, the
total number of tumor cells in a transplant varies in the range of
30 thousand to 30 million. The reinfusion of this number of tumor
cells in the HSC transplant following the patient's anti-cancer
therapy is of considerable clinical concern. In fact, animal models
have shown that as few as 25 leukemia cells can establish a lethal
tumor in 50% of mice, and these numbers extrapolate to 3500 cells
in humans (Gulati, Acaba 1993).
[0012] Recent landmark studies have unambiguously shown that
reinfused tumor cells do indeed contribute to disease relapse in
humans (Rill et al. 1994). This was proven by genetically marking
the harvested cells prior to transplant, and then showing that the
marker was detected in resurgent tumor cells in those patients who
relapsed with disease. These data have been confirmed by other
investigators (Deisseroth et al. 1994), indicating that
contaminating tumor cells in HSC transplants represent a real
threat to patients undergoing autologous transplantation.
[0013] Subsequent detailed studies have now shown that the actual
number of tumor cells reinfused in the transplant was correlated
with the risk of relapse for acute lymphoblastic leukemia
(Vervoordeldonk et al. 1997), non-Hodgkin's lymphoma (Sharp et al.
1992; Sharp et al. 1996), mantle cell lymphoma (Andersen et al.
1997), and breast cancer (Brockstein et al. 1996; Fields et al.
1996; Schulze et al. 1997; Vannucchi et al. 1998; Vredenburgh et
al. 1997). One of these studies went even further, showing that the
number of tumor cells infused was inversely correlated with the
elapsed time to relapse (Vredenburgh et al. 1997). These data
suggest that reducing the number of tumor cells in the transplant
will lead to better outcomes for the patient.
[0014] In fact, one clinical study of NHL purging in 114 patients
showed that disease-free survival (after a median 2-year follow-up)
was substantially higher (93%) in the subset of patients that had
all detectable tumor cells purged prior to transplant, as compared
with those in which purging was unsuccessful (54%) (Gribben et al.
1991). In a recent update of this study, eight-year
freedom-from-relapse was shown to be 83% in the subset of patients
that had all detectable tumor cells purged, as compared to 19% in
patients where purging was unsuccessful (Freedman et al. 1999).
Therefore, the actual number of tumor cells in an HSC transplant,
and the ability to reliably purge them, are of significant and
growing importance in the delivery of HSC transplantation therapies
for cancer patients.
[0015] Due to the known risk of tumor cell contamination in
autologous transplantation, a number of methods have been proposed
for removing contaminating tumor cells from harvested HSC
populations. The basic principle underlying all purging methods is
to remove or kill tumor cells while preserving the HSCs that are
needed for hematopoietic reconstitution in the patient.
[0016] One method based on relatively non-specific adhesion
differences of hematopoietic cells in deep bed filtration has been
described by Dooley, et al. (Dooley et al. 1996). An elutriation
method based on relatively non-specific cell size and density
differences has been described by Wagner, et al. (Wagner et al.
1995). Preferential killing of tumor cells by hyperthermia has been
described by Higuchi, et al. (Higuchi et al. 1991). These
relatively non-specific methods reduce the number of tumor cells
present, but a significant number are known to remain.
[0017] In another method, cytotoxic agents, such as
4-hydroxy-peroxy-cyclophosphamide (4HC), were used to
preferentially kill tumor cells in populations containing HSCs
(Bird et al. 1996). Unfortunately, collateral damage to normal HSCs
was so severe that patient engraftment was delayed by as much as 59
days.
[0018] Another method employed preferential killing of tumor cells
by exposing all cells to photoradiation in the presence of a
light-sensitizing agent (e.g. merocyanide) (Gulliya and Pervaiz
1989). Although more tumor cells were killed than hematopoietic
cells, some tumor cells still remained, and HSC damage was
significant.
[0019] Another method for removing tumor cells from populations of
hematopoietic cells involved immunoconjugating a toxic agent to an
antibody having specificity for the tumor cells. In this system,
antibodies were bound to chemotoxic agents, toxins, or
radionucleides and then contacted with the total cell population.
Unfortunately, not all of the tumor cells were killed by this
treatment (Gribben et al. 1991; Robertson et al. 1992).
[0020] Some companies and physicians have attempted to purge
malignant cells from populations of non-tumor cells using an
immunoaffinity bead-based selection. In this procedure, the total
cell population is contacted by immunoaffinity beads. For example,
a first (positive) CD34-selection procedure enriches HSCs from the
tumor cell-containing hematopoietic cell mixture. In some
instances, a second (negative) immunoaffinity bead-based selection
is also performed using anti-tumor or anti-epithelial cell
antibodies attached to the beads. Although these procedures enrich
HSCs and reduce tumor cell numbers, tumor cells can still be
detected in the final product (Mapara et al. 1999)
[0021] In another protocol, Clarke et al. disclosed the use of
adenovirus-mediated transfer of suicide genes to selectively kill
tumor cells (Clarke et al. 1995). However, it is well known that
virus-mediated gene transfer is far less than 100% efficient, which
would result in a significant number of tumor cells being
unaffected by the protocol.
[0022] Yet another method utilized fluorescence-activated cell
sorting (FACS) to sort HSCs from tumor cells (Tricot et al. 1995).
As is known, flow cytometry sorts cells one at a time and
physically separates one population of cells from a mixture of
cells based upon cell surface markers and physical characteristics.
However, it has been shown that using FACS to separate large cell
populations for clinical applications is not advantageous because
the process is slow, the cell yields can be very low, and purity
greater than .about.98% is rarely achieved.
[0023] Another method utilizing a flow cytometer is described in
U.S. Pat. No. 4,395,397 to Shapiro. In the Shapiro method, labeled
cells are placed in a flow cytometer, and a downstream laser beam
is used to kill the labeled cells in the flowing stream after they
pass by the detector and are recognized as being labeled by the
electronic system. This method suffers from a number of
disadvantages. Firstly, once an unwanted cell has passed through
the detector/laser region there is no way to check that destruction
has been completed successfully. If a tumor cell evades destruction
it will inevitably be reintroduced into the patient. Secondly, the
focal spot diameter of the laser beam is of necessity greater than
the liquid stream cross section. Accordingly, many of the HSCs in
the region of an unwanted cell will also be destroyed by the laser
beam. Also, as described above, the purity obtained by flow
cytometric techniques is not very good due to the random and
dynamic nature of a heterogeneous cell mixture that is flowing in a
fast-moving (1-20 m/sec) stream of liquid.
[0024] Another method that utilizes laser technology is described
in U.S. Pat. No. 4,629,687 to Schindler, et al. In this method,
anchorage-dependent cells are grown on a movable surface, and then
a small laser beam spot is scanned across the moving surface to
illuminate cells one at a time and the information is recorded. The
same laser is then switched to a higher lethal power level, and the
beam is swept over the surface in all areas except where a cell of
interest was recorded during the illumination step. Unfortunately,
this method is slow and only will work on cells that can adhere to
a surface.
[0025] A still further method that utilizes laser technology is
described in U.S. Pat. No. 5,035,693 to Kratzer. In this method,
cells are placed on a moving belt and a small laser beam spot is
scanned across the surface. When a particular cell radiates in
response to the illuminating laser spot, the same laser is quickly
switched to high power in order to kill the cell in a near
simultaneous manner before the scanner has moved appreciably away
from that cell. However, this system has many of the same
disadvantages as the Shapiro method. For example, because the
scanner is continuously moving during the imaging and killing of
cells, the system is highly-dynamic, and therefore less stable and
less accurate than a static system. Also, because the cells are
moving on a belt past the detector in one direction, the method is
not reversible. Thus, if a single tumor cell escapes detection, it
will be reintroduced into the patient.
[0026] Others have used a small laser beam spot to dynamically scan
over a surface to illuminate cells. For example, U.S. Pat. No.
4,284,897 to Sawamura et al. describes the use of galvanometric
mirrors to scan a small laser beam spot in a standard microscope to
illuminate fluorescent cells. U.S. Pat. No. 5,381,224 to Dixon et
al. describes imaging of macroscopic specimens through the use of a
laser beam spot that is raster-scanned with galvanometric mirrors
through an F-theta scanning lens. In U.S. Pat. Nos. 5,646,411,
5,672,880, and 5,719,391 to Kain, scanning of a small laser spot
with galvanometers through an F-theta lens is described. All of
these imaging methods dynamically illuminate a small point that is
moved over the surface to be imaged. In some cases, the surface
being scanned is also moving during imaging.
[0027] Similar methods of scanning a small laser spot have been
described for purposes other than imaging of cells. For example,
U.S. Pat. No. 4,532,402 to Overbeck describes the use of
galvanometers to move a small laser beam spot over a semiconductor
surface for repair of an integrated circuit. Similarly, U.S. Pat.
No. 5,690,846 to Okada et al. describes laser processing by moving
a small laser spot with mirrors through an F-theta scanning lens.
U.S. Pat. No. 5,296,963 to Murakarni et al. describes the use of
galvanometric mirrors to scan a small laser beam spot in a standard
inverted microscope to puncture cells for insertion of genetic
matter.
[0028] Yet another method of scanning a biological specimen is
described in U.S. Pat. No. 5,932,872 to Price. This method uses a
plurality of detectors to simultaneously capture images at a
plurality of focus planes from a constantly moving surface. The
resultant images can be used to choose the best-focus image in
real-time, and can be used to generate a three-dimensional
volumetric image of a specimen.
[0029] Most of the methods described above are based on
administering a tumor cell-removal or tumor cell-killing strategy
to the entire harvested cell population as a whole. In flow
cytometry, cells are sorted on a single cell basis to physically
separate the unwanted tumor cells from HSCs. While each of these
methods has been shown to reduce tumor cell numbers in HSC
transplants, none has demonstrated the ability to remove or kill
all detectable tumor cells. In fact, the majority of patient
transplants still contain detectable tumor cells after these
purging techniques are used. Approximately 30 to 30,000 tumor cells
per transplant still remain, even after multiple-step purging
procedures (Gazitt et al. 1995; Gribben et al. 1991; Mapara et al.
1997; Paulus et al. 1997). Further, all of these methods result in
some degree of HSC loss or damage, which can significantly impact
the success of the HSC transplant by delaying patient engraftment.
In summary, existing purging technologies are inadequate, and there
exists a great unmet clinical need for novel approaches that can
effectively purge all detectable tumor cells from an HSC
transplant. The method and apparatus described herein fulfills this
need.
SUMMARY OF THE INVENTION
[0030] This invention provides a high-speed method and apparatus
for selectively identifying, and individually targeting with an
energy beam, specific cells within a mixed cell population of cells
for the purpose of inducing a response in the targeted cells. Using
the apparatus of the present invention, every detectable target
cell in a mixed cell population can be specifically identified and
targeted, without substantially affecting cells that are not being
targeted.
[0031] Specific cells are identified with the disclosed invention
using several approaches. One embodiment includes a non-destructive
labeling method so that all of the cells of a first population are
substantially distinguishable from the remaining cells of the cell
mixture, the remaining cells comprising the second population. In
this embodiment, a labeled antibody can be used to specifically
mark each cell of the first population, yet not mark cells of the
second population. The labeled cells are then identified within the
cell mixture. A narrow energy beam is thereafter focused on the
first of the targeted cells to achieve a desired response. The next
of the targeted cells is then irradiated, and so on until every
targeted cell has been irradiated.
[0032] In another embodiment, an antibody that selectively binds to
cells of the second population, but not cells of the first
population, is used to identify cells of the first population.
Cells of the first population are identified by the absence of the
label, and are thereafter individually targeted with the energy
beam.
[0033] The nature of the response that is induced by the energy
beam is dependent upon the nature of the energy beam. Responses
that can be induced with an energy beam include necrosis,
apoptosis, optoporation (to allow entry of a substance that is
present in the surrounding medium, including genetic material),
cell lysis, cell motion (laser tweezers), cutting of cell
components (laser scissors), activation of a photosensitive
substance, excitation of a fluorescent reagent, etc.
[0034] A large number of commercially important research and
clinical applications can be envisioned for such an apparatus,
examples of which are presented below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a perspective view of one embodiment of a cell
treatment apparatus and illustrates the outer design of the housing
and display.
[0036] FIG. 2 is a perspective view of one embodiment of a cell
treatment apparatus with the outer housing removed and the inner
components illustrated.
[0037] FIG. 3 is a block diagram of the optical subassembly design
within one embodiment of a cell treatment apparatus.
[0038] FIG. 4 is a perspective view of one embodiment of an optical
subassembly within one embodiment of a cell treatment
apparatus.
[0039] FIG. 5 is a side view of one embodiment of an optical
subassembly that illustrates the arrangement of the scanning lens
and the movable stage.
[0040] FIG. 6 is a bottom perspective view of one embodiment of an
optical subassembly.
[0041] FIG. 7 is a top perspective view of the movable stage of the
cell treatment apparatus.
DETAILED DESCRIPTION
[0042] A method and apparatus is described for selectively
identifying, and individually targeting with an energy beam,
specific cells within a mixed cell population for the purpose of
inducing a response in the targeted cells. Generally, the method
first employs a label that acts as a marker to identify and locate
individual cells of a first population of cells within a cell
mixture that is comprised of the first population of cells and a
second population of cells.
[0043] The chosen label can be any that substantially identifies
and distinguishes the first population of cells from the second
population of cells. For example, monoclonal antibodies that are
directly or indirectly tagged with a fluorochrome can be used as
specific labels. Other examples of cell surface binding labels
include non-antibody proteins, lectins, carbohydrates, or short
peptides with selective cell binding capacity. Membrane
intercalating dyes, such as PKH-2 and PKH-26, could also serve as a
useful distinguishing label indicating mitotic history of a cell.
Many membrane-permeable reagents are also available to distinguish
living cells from one another based upon selected criteria. For
example, phalloidin indicates membrane integrity, tetramethyl
rhodamine methyl ester (TMRM) indicates mitochondrial transmembrane
potential, monochlorobimane indicates glutathione reductive stage,
carboxymethyl fluorescein diacetate (CMFDA) indicates thiol
activity, carboxyfluorescein diacetate indicates intracellular pH,
fura-2 indicates intracellular Ca.sup.2+ level, and
5,5',6,6'-tetrachloro-1,1',3,3'-tetrae- thylbenzimidazolo
carbocyanine iodide (JC-1) indicates membrane potential. Cell
viability can be assessed by the use of fluorescent SYTO 13 or YO
PRO reagents. Similarly, a fluorescently-tagged genetic probe (DNA
or RNA) could be used to label cells which carry a gene of
interest, or express a gene of interest. Further, cell cycle status
could be assessed through the use of Hoechst 33342 dye to label
existing DNA combined with bromodeoxyuridine (BrdU) to label newly
synthesized DNA.
[0044] It should be noted that if no specific label is available
for cells of the first population, the method can be implemented in
an inverse fashion by utilizing a specific label for cells of the
second population. For example, in hematopoietic cell populations,
the CD34 or ACC-133 cell markers can be used to label only the
primitive hematopoietic cells, but not the other cells within the
mixture. In this embodiment, cells of the first population are
identified by the absence of the label, and are thereby targeted by
the energy beam.
[0045] After cells of the first population are identified, an
energy beam, such as from a laser, collimated or focused non-laser
light, RF energy, accelerated particle, focused ultrasonic energy,
electron beam, or other radiation beam, is used to deliver a
targeted dose of energy that induces the pre-determined response in
each of the cells of the first population, without substantially
affecting cells of the second population.
[0046] FIG. 1 is an illustration of one embodiment of a cell
treatment apparatus. The cell treatment apparatus 10 includes a
housing 15 that stores the inner components of the apparatus. The
housing includes laser safety interlocks to ensure safety of the
user, and also limits interference by external influences (e.g.,
ambient light, dust, etc.). Located on the upper portion of the
housing 15 is a display unit 20 for displaying captured images of
cell populations during treatment. These images are captured by a
camera, as will be discussed more specifically below. A keyboard 25
and mouse 30 are used to input data and control the apparatus 10.
An access door 35 provides access to a movable stage that holds a
specimen container of cells undergoing treatment.
[0047] An interior view of the apparatus 10 is provided in FIG. 2.
As illustrated, the apparatus 10 provides an upper tray 200 and
lower tray 210 that hold the interior components of the apparatus.
The upper tray 200 includes a pair of intake filters 215A,B that
filter ambient air being drawn into the interior of the apparatus
10. Below the access door 35 is the optical subassembly (not
shown). The optical subassembly is mounted to the upper tray 200
and is discussed in detail with regard to FIGS. 3-6.
[0048] On the lower tray 210 is a computer 225 which stores the
software programs, commands and instructions that run the apparatus
10. In addition, the computer 225 provides control signals to the
treatment apparatus through electrical signal connections for
steering the laser to the appropriate spot on the specimen in order
to treat the cells.
[0049] As illustrated, a series of power supplies 230A,B,C provide
power to the various electrical components within the apparatus 10.
In addition, an uninterruptable power supply 235 is incorporated to
allow the apparatus to continue functioning through short external
power interruptions.
[0050] FIG. 3 provides a layout of one embodiment of an optical
subassembly design 300 within an embodiment of a cell treatment
apparatus 10. As illustrated, an illumination laser 305 provides a
directed laser output that is used to excite a particular label
that is attached to targeted cells within the specimen. In this
embodiment, the illumination laser emits light at a wavelength of
532 nm. Once the illumination laser has generated a light beam, the
light passes into a shutter 310 which controls the pulse length of
the laser light.
[0051] After the illumination laser light passes through the
shutter 310, it enters a ball lens 315 where it is focused into an
SMA fiber optic connector 320. After the illumination laser beam
has entered the fiber optic connector 320, it is transmitted
through a fiber optic cable 325 to an outlet 330. By passing the
illumination beam through the fiber optic cable 325, the
illumination laser 305 can be positioned anywhere within the
treatment apparatus and thus is not limited to only being
positioned within a direct light pathway to the optical components.
In one embodiment, the fiber optic cable 325 is connected to a
vibrating motor 327 for the purpose of mode scrambling and
generating a more uniform illumination spot.
[0052] After the light passes through the outlet 330, it is
directed into a series of condensing lenses in order to focus the
beam to the proper diameter for illuminating one frame of cells. As
used herein, one frame of cells is defined as the portion of the
biological specimen that is captured within one frame image
captured by the camera. This is described more specifically
below.
[0053] Accordingly, the illumination laser beam passes through a
first condenser lens 335. In one embodiment, this first lens has a
focal length of 4.6 mm. The light beam then passes through a second
condenser lens 340 which, in one embodiment, provides a 100 mm
focal length. Finally, the light beam passes into a third condenser
lens 345, which preferably provides a 200 mm focal length. While
the present invention has been described using specific condenser
lenses, it should be apparent that other similar lens
configurations that focus the illumination laser beam to an
advantageous diameter would function similarly. Thus, this
invention is not limited to the specific implementation of any
particular condenser lens system.
[0054] Once the illumination laser beam passes through the third
condenser lens 345, it enters a cube beamsplitter 350 that is
designed to transmit the 532 nm wavelength of light emanating from
the illumination laser. Preferably, the cube beamsplitter 350 is a
25.4 mm square cube (Melles-Griot, Irvine, Calif.). However, other
sizes are anticipated to function similarly. In addition, a number
of plate beamsplitters or pellicle beamsplitters could be used in
place of the cube beamsplitter 350 with no appreciable change in
function.
[0055] Once the illumination laser light has been transmitted
through the cube beamsplitter 350, it reaches a long wave pass
mirror 355 that reflects the 532 nm illumination laser light to a
set of galvanometer mirrors 360 that steer the illumination laser
light under computer control through a scanning lens (Special
Optics, Wharton, N.J.) 365 to the specimen (not shown). The
galvanometer mirrors are controlled so that the illumination laser
light is directed at the proper cell population (i.e. frame of
cells) for imaging.
[0056] The light from the illumination laser is of a wavelength
that is useful for illuminating the specimen. In this embodiment,
energy from a continuous wave 532 nm Nd:YAG frequency-doubled laser
(B&W Tek, Newark, Del.) reflects off the long wave pass mirror
(Custom Scientific, Phoenix, Ariz.) and excites fluorescent tags in
the specimen. In one embodiment, the fluorescent tag is
phycoerythrin. Alternatively, Alexa 532 (Molecular Probes, Eugene,
Oreg.) can be used. Phycoerythrin and Alexa 532 have emission
spectra with peaks near 580 nm, so that the emitted fluorescent
light from the specimen is transmitted via the long wave pass
mirror to be directed into the camera. The use of the filter in
front of the camera blocks light that is not within the wavelength
range of interest, thereby reducing the amount of background light
entering the camera.
[0057] It is generally known that many other devices could be used
in this manner to illuminate the specimen, including, but not
limited to, an arc lamp (e.g., mercury, xenon, etc.) with or
without filters, a light-emitting diode (LED), other types of
lasers, etc. Advantages of this particular laser include high
intensity, relatively efficient use of energy, compact size, and
minimal heat generation. It is also generally known that other
fluorochromes with different excitation and emission spectra could
be used in such an apparatus with the appropriate selection of
illumination source, filters, and long and/or short wave pass
mirrors. For example, allophycocyanin (APC) could be excited with a
633 nm HeNe illumination laser, and fluoroisothiocyanate (FITC)
could be excited with a 488 nm Argon illumination laser. One
skilled in the art could propose many other optical layouts with
various components in order to achieve the objective of this
invention.
[0058] In addition to the illumination laser 305, a treatment laser
400 is present to irradiate the targeted cells once they have been
identified by image analysis. Of course, in one embodiment, the
treatment induces necrosis of targeted cells within the cell
population. As shown, the treatment laser 400 outputs an energy
beam of 523 nm that passes through a shutter 410. Although the
exemplary laser outputs an energy beam having a 523 nm wavelength,
other sources that generate energy at other wavelengths are also
within the scope of the present invention.
[0059] Once the treatment laser energy beam passes through the
shutter 410, it enters a beam expander (Special Optics, Wharton,
N.J.) 415 which adjusts the diameter of the energy beam to an
appropriate size at the plane of the specimen. Following the beam
expander 415 is a half-wave plate 420 which controls the
polarization of the beam. The treatment laser energy beam is then
reflected off a mirror 425 and enters the cube beamsplitter 350.
The treatment laser energy beam is reflected by 90.degree. in the
cube beamsplitter 350, such that it is aligned with the exit
pathway of the illumination laser light beam. Thus, the treatment
laser energy beam and the illumination laser light beam both exit
the cube beamsplitter 350 along the same light path. From the cube
beamsplitter 350, the treatment laser beam reflects off the long
wave pass mirror 355, is steered by the galvanometers 360,
thereafter enters the scanning lens 365, and finally is focused
upon a targeted cell within the specimen.
[0060] It should be noted that a small fraction of the illumination
laser light beam passes through the long wave pass mirror 355 and
enters a power meter sensor (Gentec, Palo Alto, Calif.) 445. The
fraction of the beam entering the power sensor 445 is used to
calculate the level of power emanating from the illumination laser
305. In an analogous fashion, a small fraction of the treatment
laser energy beam passes through the cube beamsplitter 350 and
enters a second power meter sensor (Gentec, Palo Alto, Calif.) 446.
The fraction of the beam entering the power sensor 446 is used to
calculate the level of power emanating from the treatment laser
400. The power meter sensors are electrically linked to the
computer system so that instructions/commands within the computer
system capture the power measurement and determine the amount of
energy that was emitted.
[0061] The energy beam from the treatment laser is of a wavelength
that is useful for achieving a response in the cells. In the
example shown, a pulsed 523 nm Nd:YLF frequency-doubled laser is
used to heat a localized volume containing the targeted cell, such
that it is induced to die within a pre-determined period of time.
The mechanism of death is dependent upon the actual temperature
achieved in the cell, as reviewed by Niemz (Niemz 1996).
[0062] A Nd:YLF frequency-doubled, solid-state laser
(Spectra-Physics, Mountain View, Calif.) is used because of its
stability, high repetition rate of firing, and long time of
maintenance-free service. However, most cell culture fluids and
cells are relatively transparent to light in this green wavelength,
and therefore a very high fluence of energy would be required to
achieve cell death. To significantly reduce the amount of energy
required, and therefore the cost and size of the treatment laser, a
dye is purposefully added to the specimen to efficiently absorb the
energy of the treatment laser in the specimen. In the example
shown, the non-toxic dye FD&C red #40 (allura red) is used to
absorb the 523 nm energy from the treatment laser, but one skilled
in the art could identify other laser/dye combinations that would
result in efficient absorption of energy by the specimen. For
example, a 633 nm HeNe laser's energy would be efficiently absorbed
by FD&C green #3 (fast green FCF), a 488 nm Argon laser's
energy would be efficiently absorbed by FD&C yellow #5 (sunset
yellow FCF), and a 1064 nm Nd:YAG laser's energy would be
efficiently absorbed by Filtron (Gentex, Zeeland, Mich.) infrared
absorbing dye. Through the use of an energy absorbing dye, the
amount of energy required to kill a targeted cell can be reduced
since more of the treatment laser energy is absorbed in the
presence of such a dye.
[0063] Another method of achieving thermal killing of cells without
the addition of a dye involves the use of an ultraviolet laser.
Energy from a 355 nm Nd:YAG frequency-tripled laser will be
absorbed by nucleic acids and proteins within the cell, resulting
in thermal heating and death. Yet another method of achieving
thermal killing of cells without the addition of a dye involves the
use of a near-infrared laser. Energy from a 2100 nm Ho:YAG laser or
a 2940 nm Er:YAG laser will be absorbed by water within the cell,
resulting in thermal heating and death.
[0064] Although this embodiment describes the killing of cells via
thermal heating by the energy beam, one skilled in the art would
recognize that other responses can also be induced in the cells by
an energy beam, including photomechanical disruption,
photodissociation, photoablation, and photochemical reactions, as
reviewed by Niemz (Niemz 1996). Also, a photosensitive substance
(e.g., hemtoporphyrin derivative, tin-etiopurpurin, lutetuim
texaphyrin) (Oleinick and Evans 1998) within the cell mixture could
be specifically activated in targeted cells by irradiation.
Additionally, a small, transient pore could be made in the cell
membrane (Palumbo et al. 1996) to allow the entry of genetic or
other material. Further, specific molecules in or on the cell, such
as proteins or genetic material, could be inactivated by the
directed energy beam (Grate and Wilson 1999; Jay 1988). These
mechanisms of inducing a response in a targeted cell via the use of
electromagnetic radiation directed at specific targeted cells are
also intended to be incorporated into the present invention.
[0065] In addition to the illumination laser 305 and treatment
laser 400, the apparatus includes a camera 450 that captures images
(i.e. frames) of the cell populations. As illustrated in FIG. 3,
the camera 450 is focused through a lens 455 and filter 460 in
order to accurately record an image of the cells without capturing
stray background images. A stop 462 is positioned between the
filter 460 and mirror 355 in order to eliminate light that may
enter the camera from angles not associated with the image from the
specimen. The filter 460 is chosen to only allow passage of light
within a certain wavelength range. This wavelength range includes
light that is emitted from the targeted cells upon excitation by
the illumination laser 305, as well as light from a back-light
source 475.
[0066] The back-light source 475 is located above the specimen to
provide back-illumination of the specimen at a wavelength different
from that provided by the illumination laser 305. This LED
generates light at 590 nm, such that it can be transmitted through
the long wave pass mirror to be directed into the camera. This
back-illumination is useful for imaging cells when there are no
fluorescent targets within the frame being imaged. An example of
the utility of this back-light is its use in attaining proper focus
of the system, even when there are only unstained, non-fluorescent
cells in the frame. In one embodiment, the back-light is mounted on
the underside of the access door 35 (FIG. 2).
[0067] Thus, as discussed above, the only light returned to the
camera is from wavelengths that are of interest in the specimen.
Other wavelengths of light do not pass through the filter 460, and
thus do not become recorded by the camera 450. This provides a more
reliable mechanism for capturing images of only those cells of
interest. It is readily apparent to one skilled in the art that the
single filter 460 could be replaced by a movable filter wheel that
would allow different filters to be moved in and out of the optical
pathway. In such an embodiment, images of different wavelengths of
light could be captured at different times during cell processing,
allowing the use of multiple cell labels.
[0068] It should be noted that in this embodiment, the camera is a
charge-coupled device (CCD) and transmits images back to the
computer system for processing. As will be described below, the
computer system determines the coordinates of the targeted cells in
the specimen by reference to the image captured by the CCD
camera.
[0069] Referring now to FIG. 4, a perspective view of an embodiment
of an optical subassembly is illustrated. As illustrated, the
illumination laser 305 sends a light beam through the shutter 310
and ball lens 315 to the SMA fiber optic connector 320. The light
passes through the fiber optic cable 325 and through the output 330
into the condenser lenses 335, 340 and 345. The light then enters
the cube beamsplitter 350 and is transmitted to the long wave pass
mirror 355. From the long wave pass mirror 355, the light beam
enters the computer-controlled galvanometers 360 and is then
steered to the proper frame of cells in the specimen through the
scanning lens 365.
[0070] As also illustrated in the perspective drawing of FIG. 4,
the treatment laser 400 transmits energy through the shutter 410
and into the beam expander 415. Energy from the treatment laser 400
passes through the beam expander 415 and passes through the
half-wave plate 420 before hitting the fold mirror 425, entering
the cube beamsplitter 350 where it is reflected 90.degree. to the
long wave pass mirror 355, from which it is reflected into the
computer controlled galvanometer mirrors 360. After being steered
by the galvanometer mirrors 360 through the scanning lens 365, the
laser energy beam strikes the proper location within the cell
population in order to induce a response in a particular targeted
cell.
[0071] In order to accommodate a very large surface area of
specimen to treat, the apparatus includes a movable stage that
mechanically moves the specimen container with respect to the
scanning lens. Thus, once a specific sub-population (i.e. field) of
cells within the scanning lens field-of-view has been treated, the
movable stage brings another sub-population of cells within the
scanning lens field-of-view. As illustrated in FIG. 5, a
computer-controlled movable stage 500 holds a specimen container
(not shown) to be processed. The movable stage 500 is moved by
computer-controlled servo motors along two axes so that the
specimen container can be moved relative to the optical components
of the instrument. The stage movement along a defined path is
coordinated with other operations of the apparatus. In addition,
specific coordinates can be saved and recalled to allow return of
the movable stage to positions of interest. Encoders on the x and y
movement provide closed-loop feedback control on stage
position.
[0072] The flat-field (F-theta) scanning lens 365 is mounted below
the movable stage. The scanning lens field-of-view comprises the
portion of the specimen that is presently positioned above the
scanning lens by the movable stage 500. The lens 365 is mounted to
a stepper motor that allows the lens 365 to be automatically raised
and lowered (along the z-axis) for the purpose of focusing the
system.
[0073] As illustrated in FIGS. 4-6, below the scanning lens 365 are
the galvanometer-controlled steering mirrors 360 that deflect
electromagnetic energy along two perpendicular axes. Behind the
steering mirrors is the long wave pass mirror 355 that reflects
electromagnetic energy of a wavelength shorter than 545 nm.
Wavelengths longer than 545 nm are passed through the long wave
pass mirror, directed through the filter 460, coupling lens 455,
and into the CCD camera, thereby producing an image of the
appropriate size on the CCD sensor of the camera 450 (See FIGS. 3
and 4). The magnification defined by the combination of the
scanning lens 365 and coupling lens 455 is chosen to reliably
detect single cells while maximizing the area viewed in one frame
by the camera. Although a CCD camera (DVC, Austin, Tex.) is
illustrated in this embodiment, the camera can be any type of
detector or image gathering equipment known to those skilled in the
art. The optical subassembly of the apparatus is preferably mounted
on a vibration-isolated platform to provide stability during
operation as illustrated in FIGS. 2 and 5.
[0074] Referring now to FIG. 7, a top view of the movable stage 500
is illustrated. As shown, a specimen container is mounted in the
movable stage 500. The specimen container 505 rests on an upper
axis nest plate 510 that is designed to move in the
forward/backward direction with respect to the movable stage 500. A
stepper motor (not shown) is connected to the upper axis nest plate
510 and computer system so that commands from the computer cause
forward/backward movement of the specimen container 505.
[0075] The movable stage 500 is also connected to a timing belt 515
that provides side-to-side movement of the movable stage 500 along
a pair of bearing tracks 525A,B. The timing belt 515 attaches to a
pulley (not shown) housed under a pulley cover 530. The pulley is
connected to a stepper motor 535 that drives the timing belt 515 to
result in side-to-side movement of the movable stage 500. The
stepper motor 535 is electrically connected to the computer system
so that commands within the computer system result in side-to-side
movement of the movable stage 500. A travel limit sensor 540
connects to the computer system and causes an alert if the movable
stage travels beyond a predetermined lateral distance.
[0076] A pair of accelerometers 545A,B is preferably incorporated
on this platform to register any excessive bumps or vibrations that
may interfere with the apparatus operation. In addition, a two-axis
inclinometer 550 is preferably incorporated on the movable stage to
ensure that the specimen container is level, thereby reducing the
possibility of gravity-induced motion in the specimen
container.
[0077] The specimen chamber has a fan with ductwork to eliminate
condensation on the specimen container, and a thermocouple to
determine whether the specimen chamber is within an acceptable
temperature range. Additional fans are provided to expel the heat
generated by the electronic components, and appropriate filters are
used on the air intakes 215A,B.
[0078] The computer system 225 controls the operation and
synchronization of the various pieces of electronic hardware
described above. The computer system can be any commercially
available computer that can interface with the hardware. One
example of such a computer system is an Intel Pentium II-based
computer running the Microsoft Windows.RTM. NT operating system.
Software is used to communicate with the various devices, and
control the operation in the manner that is described below.
[0079] When the apparatus is first initialized, the computer loads
files from the hard drive into RAM for proper initialization of the
apparatus. A number of built-in tests are automatically performed
to ensure the apparatus is operating properly, and calibration
routines are executed to calibrate the cell treatment apparatus.
Upon successful completion of these routines, the user is prompted
to enter information via the keyboard and mouse regarding the
procedure that is to be performed (e.g. patient name, ID number,
etc.). Once the required information is entered, the user is
prompted to open the access door 35 and load a specimen onto the
movable stage.
[0080] Once a specimen is in place on the movable stage and the
door is closed, the computer passes a signal to the stage to move
into a home position. The fan is initialized to begin warming and
defogging of the specimen. During this time, cells within the
specimen are allowed to settle to the bottom surface. In addition,
during this time, the apparatus may run commands that ensure that
the specimen is properly loaded, and is within the focal range of
the system optics. For example, specific markings on the specimen
container can be located and focused on by the system to ensure
that the scanning lens has been properly focused on the bottom of
the specimen container. After a suitable time, the computer turns
off the fan to prevent excess vibrations during treatment, and cell
treatment processing begins.
[0081] First, the computer instructs the movable stage to be
positioned over the scanning lens so that the first area (i.e.
field) of the specimen to be treated is directly in the scanning
lens field-of-view. The galvanometer mirrors are instructed to move
such that the center frame within the field-of-view is imaged in
the camera. As discussed below, the field imaged by the scanning
lens is separated into a plurality of frames. Each frame is the
proper size so that the cells within the frame are effectively
imaged by the camera.
[0082] The back-light 475 is then activated in order to illuminate
the field-of-view so that it can be brought into focus by the
scanning lens. Once the scanning lens has been properly focused
upon the specimen, the computer system divides the field-of-view
into a plurality of frames so that each frame is analyzed
separately by the camera. This methodology allows the apparatus to
process a plurality of frames within a large field-of-view without
moving the mechanical stage. Because the galvanometers can move
from one frame to the next very rapidly compared to the mechanical
steps involved in moving the stage, this method results is an
extremely fast and efficient apparatus.
[0083] In one preferred embodiment, the apparatus described herein
processes at least 1, 2, 3, 4, 5, 6, or 7 square centimeters of a
biological specimen per minute. In another embodiment, the
apparatus described herein processes at least 0.25, 0.5, 1, 2, 3 or
4 million cells of a biological specimen per minute. In one other
embodiment, the apparatus can preferably induce a response in
targeted cells at a rate of 50, 100, 150, 200, 250, 300, 350, or
400 cells per second.
[0084] Initially, an image of the frame at the center of the
field-of-view is captured by the camera and stored to a memory in
the computer. Instructions in the computer analyze the focus of the
specimen by looking at the size of, number of, and other object
features in the image. If necessary, the computer instructs the
z-axis motor attached to the scanning lens to raise or lower in
order to achieve the best focus. The apparatus may iteratively
analyze the image at several z-positions until the best focus is
achieved. The galvanometer-controlled mirrors are then instructed
to image a first frame, within the field-of-view, in the camera.
For example, the entire field-of-view might be divided into 4, 9,
12, 18, 24 or more separate frames that will be individually
captured by the camera. Once the galvanometer mirrors are pointed
to the first frame in the field-of-view, the shutter in front of
the illumination laser is opened to illuminate the first frame
through the galvanometer mirrors and scanning lens. The camera
captures an image of any fluorescent emission from the specimen in
the first frame of cells. Once the image has been acquired, the
shutter in front of the illumination laser is closed and a software
program (Epic, Buffalo Grove, Ill.) within the computer processes
the image.
[0085] The power sensor 445 discussed above detects the level of
light that was emitted by the illumination laser, thereby allowing
the computer to calculate if it was adequate to illuminate the
frame of cells. If not, another illumination and image capture
sequence is performed. Repeated failure to sufficiently illuminate
the specimen will result in an error condition that is communicated
to the operator.
[0086] Shuttering of illumination light reduces undesirable heating
and photobleaching of the specimen and provides a more repeatable
fluorescent signal. An image analysis algorithm is run to locate
the x-y centroid coordinates of all targeted cells in the frame by
reference to features in the captured image. If there are targets
in the image, the computer calculates the two-dimensional
coordinates of all target locations in relation to the movable
stage position and field-of-view, and then positions the
galvanometer-controlled mirrors to point to the location of the
first target in the first frame of cells. It should be noted that
only a single frame of cells within the field-of-view has been
captured and analyzed at this point. Thus, there should be a
relatively small number of identified targets within this
sub-population of the specimen. Moreover, because the camera is
pointed to a smaller population of cells, a higher magnification is
used so that each target is imaged by many pixels within the CCD
camera.
[0087] Once the computer system has positioned the galvanometer
controlled mirrors to point to the location of the first targeted
cell within the first frame of cells, the treatment laser is fired
for a brief interval so that the first targeted cell is given an
appropriate dose of energy. The power sensor 446 discussed above
detects the level of energy that was emitted by the treatment
laser, thereby allowing the computer to calculate if it was
adequate to induce a response in the targeted cell. If not
sufficient, the treatment laser is fired at the same target again.
If repeated shots do not deliver the required energy dose, an error
condition is communicated to the operator. These targeting, firing,
and sensing steps are repeated by the computer for all targets
identified in the captured frame.
[0088] Once all of the targets have been irradiated with the
treatment laser in the first frame of cells, the mirrors are then
positioned to the second frame of cells in the field-of-view, and
the processing repeats at the point of frame illumination and
camera imaging. This processing continues for all frames within the
field-of-view above the scanning lens. When all of these frames
have been processed, the computer instructs the movable stage to
move to the next field-of-view in the specimen, and the process
repeats at the back-light illumination and auto-focus step. Frames
and fields-of-view are appropriately overlapped to reduce the
possibility of inadvertently missing areas of the specimen. Once
the specimen has been fully processed, the operator is signaled to
remove the specimen, and the apparatus is immediately ready for the
next specimen.
[0089] Although the text above describes the analysis of
fluorescent images for locating targets, one can easily imagine
that the non-fluorescent back-light LED illumination images will be
useful for locating other types of targets as well, even if they
are unlabeled.
[0090] The advantage of using the galvanometer mirrors to control
the imaging of successive frames and the irradiation of successive
targets is significant. One brand of galvanometer is the Cambridge
Technology, Inc. model number 6860 (Cambridge, Mass.). This
galvanometer can reposition very accurately within a few
milliseconds, making the processing of large areas and many targets
possible within a reasonable amount of time. In contrast, the
movable stage is relatively slow, and is therefore used only to
move specified areas of the specimen into the scanning lens
field-of-view. Error signals continuously generated by the
galvanometer control boards are monitored by the computer to ensure
that the mirrors are in position and stable before an image is
captured, or before a target is fired upon, in a closed-loop
fashion.
[0091] In the context of the present invention, the term "specimen"
has a broad meaning. It is intended to encompass any type of
biological sample placed within the apparatus. The specimen may be
enclosed by, or associated with, a container to maintain the
sterility and viability of the cells. Further, the specimen may
incorporate, or be associated with, a cooling apparatus to keep it
above or below ambient temperature during operation of the methods
described herein. The specimen container, if one is used, must be
compatible with the use of the illumination laser, back-light
illuminator, and treatment laser, such that it transmits adequate
energy without being substantially damaged itself.
[0092] Of course, many variations of the above-described embodiment
are possible, including alternative methods for illuminating,
imaging, and targeting the cells. For example, movement of the
specimen relative to the scanning lens could be achieved by keeping
the specimen substantially stationary while the scanning lens is
moved. Steering of the illumination beam, images, and energy beam
could be achieved through any controllable reflective or
diffractive device, including prisms, piezo-electric tilt
platforms, or acousto-optic deflectors. Additionally, the apparatus
can image/process from either below or above the specimen. Because
the apparatus is focused through a movable scanning lens, the
illumination and energy beams can be directed to different focal
planes along the z-axis. Thus, portions of the specimen that are
located at different vertical heights can be specifically imaged
and processed by the apparatus in a three-dimensional manner. The
sequence of the steps could also be altered without changing the
process. For example, one might locate and store the coordinates of
all targets in the specimen, and then return to the targets to
irradiate them with energy one or more times over a period of
time.
[0093] To optimally process the specimen, it should be placed on a
substantially flat surface so that a large portion of the specimen
appears within a narrow range of focus, thereby reducing the need
for repeated auto-focus steps. The density of cells on this surface
can, in principle, be at any value. However, the cell density
should be as high as possible to minimize the total surface area
required for the procedure.
[0094] The following examples illustrate the use of the described
method and apparatus in different applications.
EXAMPLE 1
Autologous HSC Transplantation
[0095] A patient with a B cell-derived metastatic tumor in need of
an autologous HSC transplant is identified by a physician. As a
first step in the treatment, the patient undergoes a standard HSC
harvest procedure, resulting in collection of approximately
1.times.10.sup.10 hematopoietic cells with an unknown number of
contaminating tumor cells. The harvested cells are enriched for HSC
by a commercial immunoaffinity column (Isolex.RTM. 300, Nexell
Therapeutics, Irvine, Calif.) that selects for cells bearing the
CD34 surface antigen, resulting in a population of approximately
3.times.10.sup.8 hematopoietic cells, with an unknown number of
tumor cells. The mixed population is thereafter contacted with
anti-B cell antibodies (directed against CD20 and CD22) that are
conjugated to phycoerythrin. The labeled antibodies specifically
bind to the B cell-derived tumor cells.
[0096] The mixed cell population is then placed in a sterile
specimen container on a substantially flat surface near confluence,
at approximately 500,000 cells per square centimeter. The specimen
is placed on the movable stage of the apparatus described above,
and all detectable tumor cells are identified by reference to
phycoerythrin and targeted with a lethal dose of energy from a
treatment laser. The design of the apparatus allows the processing
of a clinical-scale transplant specimen in under 4 hours. The cells
are recovered from the specimen container, washed, and then
cryopreserved. Before the cells are reinfused, the patient is given
high-dose chemotherapy to destroy the tumor cells in the patient's
body. Following this treatment, the processed cells are thawed at
37.degree. C. and are given to the patient intraveneously. The
patient subsequently recovers with no remission of the original
cancer.
EXAMPLE 2
Allogeneic HSC Transplantation
[0097] In another embodiment, the significant risk and severity of
graft-versus-host disease in the allogeneic HSC transplant setting
can be combated. A patient is selected for an allogeneic transplant
once a suitable donor is found. Cells are harvested from the
selected donor as described in the above example. In this case, the
cell mixture is contacted with phycoerythrin-labeled anti-CD3
T-cell antibodies. Alternatively, specific allo-reactive T-cell
subsets could be labeled using an activated T-cell marker (e.g.
CD69) in the presence of allo-antigen. The cell population is
processed by the apparatus described herein, thereby precisely
defining and controlling the number of T-cells given to the
patient. This type of control is advantageous, because
administration of too many T-cells increases the risk of
graft-versus-host disease, whereas too few T-cells increases the
risk of graft failure and the risk of losing of the known
beneficial graft-versus-leukemia effect. The present invention and
methods are capable of precisely controlling the number of T-cells
in an allogeneic transplant.
EXAMPLE 3
Tissue Engineering
[0098] In another application, the present apparatus is used to
remove contaminating cells in inocula for tissue engineering
applications. Cell contamination problems exist in the
establishment of primary cell cultures required for implementation
of tissue engineering applications, as described by Langer and
Vacanti (Langer and Vacanti 1999). In particular, chondrocyte
therapies for cartilage defects are hampered by impurities in the
cell populations derived from cartilage biopsies. Accordingly, the
present invention is used to specifically remove these types of
cells from the inocula.
[0099] For example, a cartilage biopsy is taken from a patient in
need of cartilage replacement. The specimen is then grown under
conventional conditions (Brittberg et al. 1994). The culture is
then stained with a specific label for any contaminating cells,
such as fast-growing fibroblasts. The cell mixture is then placed
within the apparatus described and the labeled, contaminating cells
are targeted by the treatment laser, thereby allowing the slower
growing chondrocytes to fully develop in culture.
EXAMPLE 4
Stem Cell Therapy
[0100] Yet another embodiment involves the use of embryonic stem
cells to treat a wide variety of diseases. Since embryonic stem
cells are undifferentiated, they can be used to generate many types
of tissue that would find use in transplantation, such as
cardiomyocytes and neurons. However, undifferentiated embryonic
stem cells that are implanted can also lead to a jumble of cell
types which form a type of tumor known as a teratoma (Pedersen
1999). Therefore, therapeutic use of tissues derived from embryonic
stem cells must include rigorous purification of cells to ensure
that only sufficiently differentiated cells are implanted. The
apparatus described herein is used to eliminate undifferentiated
stem cells prior to implantation of embryonic stem cell-derived
tissue in the patient.
EXAMPLE 5
Generation of Human Tumor Cell Cultures
[0101] In another embodiment, a tumor biopsy is removed from a
cancer patient for the purpose of initiating a culture of human
tumor cells. However, the in vitro establishment of primary human
tumor cell cultures from many tumor types is complicated by the
presence of contaminating primary cell populations that have
superior in vitro growth characteristics over tumor cells. For
example, contaminating fibroblasts represent a major challenge in
establishing many cancer cell cultures. The disclosed apparatus is
used to particularly label and destroy the contaminating cells,
while leaving the biopsied tumor cells intact. Accordingly, the
more aggressive primary cells will not overtake and destroy the
cancer cell line.
EXAMPLE 6
Generation of a Specific mRNA Expression Library
[0102] The specific expression pattern of genes within different
cell populations is of great interest to many researchers, and many
studies have been performed to isolate and create libraries of
expressed genes for different cell types. For example, knowing
which genes are expressed in tumor cells versus normal cells is of
great potential value (Cossman et al. 1999). Due to the
amplification methods used to generate such libraries (e.g. PCR),
even a small number of contaminating cells will result in an
inaccurate expression library (Cossman et al. 1999; Schutze and
Lahr 1998). One approach to overcome this problem is the use of
laser capture microdissection (LCM), in which a single cell is used
to provide the starting genetic material for amplification
(Schutze, Lahr 1998). Unfortunately, gene expression in single
cells is somewhat stochastic, and may be biased by the specific
state of that individual cell at the time of analysis (Cossman et
al. 1999). Therefore, accurate purification of a significant cell
number prior to extraction of mRNA would enable the generation of a
highly accurate expression library, one that is representative of
the cell population being studied, without biases due to single
cell expression or expression by contaminating cells. The methods
and apparatus described in this invention can be used to purify
cell populations so that no contaminating cells are present during
an RNA extraction procedure.
EXAMPLE 7
Transfection of a Specific Cell Population
[0103] Many research and clinical gene therapy applications are
hampered by the inability to transfect an adequate number of a
desired cell type without transfecting other cells that are
present. The method of the present invention would allow selective
targeting of cells to be transfected within a mixture of cells. By
generating a photomechanical shock wave at or near a cell membrane
with a targeted energy source, a transient pore can be formed,
through which genetic (or other) material can enter the cell. This
method of gene transfer has been called optoporation (Palumbo et
al. 1996). The apparatus described above can achieve selective
optoporation on only the cells of interest in a rapid, automated,
targeted manner.
[0104] For example, white blood cells are plated in a specimen
container having a solution containing DNA to be transfected.
Fluorescently-labeled antibodies having specificity for stem cells
are added into the medium and bind to the stem cells. The specimen
container is placed within the cell processing apparatus and a
treatment laser is targeted to any cells that become fluorescent
under the illumination laser light. The treatment laser facilitates
transfection of DNA specifically into the targeted cells.
EXAMPLE 8
Selection of Desirable Clones in a Biotechnology Application
[0105] In many biotechnology processes where cell lines are used to
generate a valuable product, it is desirable to derive clones that
are very efficient in producing the product. This selection of
clones is often carried out manually, by inspecting a large number
of clones that have been isolated in some manner. The present
invention would allow rapid, automated inspection and selection of
desirable clones for production of a particular product. For
example, hybridoma cells that are producing the greatest amounts of
antibody can be identified by a fluorescent label directed against
the F.sub.c region. Cells with no or dim fluorescent labeling are
targeted by the treatment laser for killing, leaving behind the
best producing clones for use in antibody production.
EXAMPLE 9
Automated Monitoring of Cellular Responses
[0106] Automated monitoring of cellular responses to specific
stimuli is of great interest in high-throughput drug screening.
Often, a cell population in one well of a well-plate is exposed to
a stimulus, and a fluorescent signal is then captured over time
from the cell population as a whole. Using the methods and
apparatus described herein, more detailed monitoring could be done
at the single cell level. For example, a cell population can be
labeled to identify a characteristic of a subpopulation of cells
that are of interest. This label is then excited by the
illumination laser to identify those cells. Thereafter, the
treatment laser is targeted at the individual cells identified by
the first label, for the purpose of exciting a second label,
thereby providing information about each cell's response. Since the
cells are substantially stationary on a surface, each cell could be
evaluated multiple times, thereby providing temporal information
about the kinetics of each cell's response. Also, through the use
of the large area scanning lens and galvanometer mirrors, a
relatively large number of wells could be quickly monitored over a
short period of time.
[0107] As a specific example, consider the case of alloreactive
T-cells as presented in Example 2 above. In the presence of
allo-antigen, activated donor T-cells could be identified by CD69.
Instead of using the treatment laser to target and kill these
cells, the treatment laser could be used to examine the
intracellular pH of every activated T-cell through the excitation
and emitted fluorescence of carboxyfluorescein diacetate. The
targeted laser allows the examination of only cells that are
activated, whereas most screening methods evaluate the response of
an entire cell population. If a series of such wells are being
monitored in parallel, various agents could be added to individual
wells, and the specific activated T-cell response to each agent
could be monitored over time. Such an apparatus would provide a
high-throughput screening method for agents that ameliorate the
alloreactive T-cell response in graft-versus-host disease. Based on
this example, one skilled in the art could imagine many other
examples in which a cellular response to a stimulus is monitored on
an individual cell basis, focusing only on cells of interest
identified by the first label.
[0108] Although aspects of the present invention have been
described by particular embodiments exemplified herein, the present
invention is not so limited. The present invention is only limited
by the claims appended below.
REFERENCES CITED
[0109] Andersen, N. S., Donovan, J. W., Borus, J. S., Poor, C. M.,
Neuberg, D., Aster, J. C., Nadler, L. M., Freedman, A. S., and
Gribben, J. G.: Failure of immunologic purging in mantle cell
lymphoma assessed by polymerase chain reaction detection in minimal
residual disease. Blood 90: 4212-4221, 1997
[0110] Bird, J. M., Luger, S., Mangan, P., Edelstein, M.,
Silberstein, L., Powlis, W., Ball, J., Schultz, D. J., and
Stadtmauer, E. A.: 4-Hydroperoxycyclophosphamide purged autologous
bone marrow transplantation in non-Hodgkin's lymphoma patients at
high risk of none marrow involvement. BMT 18: 309-313, 1996
[0111] Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C.,
Isaksson, O., and Peterson, L.: Treatment of deep cartilage defects
in the knee with autologous chondrocyte transplantation. N.E.J.Med.
331: 889-895, 1994
[0112] Brockstein, B. E., Ross, A. A., Moss, T. J., Kahn, D. G.,
Hollingsworth, K., and Williams, S. F.: Tumor cell contamination of
bone marrow harvest products: Clinical consequences in a cohort of
advanced-stage breast cancer patients undergoing high-dose
chemotherapy. J.Hematotherapy 5: 617-624, 1996
[0113] Brugger, W., Bross, K. J., Glatt, M., Weber, F.,
Mertelsmann, R., and Kanz, L.: Mobilization of tumor cells and
hematopoietic progenitor cells into peripheral blood of patients
with solid tumors. Blood 83: 636-640, 1994
[0114] Clarke, M. F., Apel, I. J., Benedict, M. A., Eipers, P. G.,
Sumantran, V., Gonzalez-Garcia, M., Doedens, M., Fukunaga, N.,
Davidson, B., Dick, J. E., Minn, A. J., Boise, L. H., Thompson, C.
B., Wicha, M., and Nunez, G.: A recombinant bcl-x.sub.S adenovirus
selectively induces apoptosis in cancer cells but not in normal
bone marrow cells. PNAS 92: 11024-11028, 1995
[0115] Cossman, J. C., Annunziata, C. M., Barash, S., Staudt, L.,
Dillon, P., He, W.-W., Ricciardi-Castognoli, P., Rosen, C. A., and
Carter, K. C.: Reed-Sternberg cell genome expression supports a
B-cell lineage. Blood 94: 411-416, 1999
[0116] Deisseroth, A. B., Zu, Z., Claxton, D., Hanania, E. G., Fu,
S., Ellerson, D., Goldberg, L., Thomas, M., Janicek, K., Anderson,
W. F., Hester, J., Korbling, M., Durett, A., Moen, R., Berenson,
R., Heimfeld, S., Hamer, J., Calver, L., Tibbits, P., Talpaz, M.,
Kantarjiam, H., Champlin, R., and Reading, C.: Genetic marking
shows that Ph.sup.+ cells present in autologous transplants of
chronic myelogenous leukemia (CML) contribute to relapse after
autologous bone marrow transplantation in CML. Blood 83: 3068-3076,
1994
[0117] Dooley, D. C., Xiao, M., Wickramasinghe, S. R., Oppenlander,
B. K., and Castino, F.: A novel inexpensive technique for the
removal of breast cancer cells from mobilized peripheral blood stem
cell products. Blood 88: 252a, 1996
[0118] Fields, K. K., Elfenbein, G. J., Trudeau, W. L., Perkins, J.
B., Janssen, W. E., and Moscinski, L. C.: Clinical significance of
bone marrow metastases as detected using the polymerase chain
reaction in patients with breast cancer undergoing high-dose
chemotherapy and autologous bone marrow transplantation.
J.Clin.Oncol. 14: 1868-1876, 1996
[0119] Freedman, A. S., Neuberg, D., Mauch, P., Soiffer, R. J.,
Anderson, K. C., Fisher, D. C., Schlossman, R., Alyea, E. P.,
Takvorian, T., Jallow, H., Kuhlman, C., Ritz, J., Nadler, L. M.,
and Gribben, J. G.: Long-term follow-up of autologous bone marrow
transplantation in patients with relapsed follicular lymphoma.
Blood 94: 3325-3333, 1999
[0120] Gazitt, Y., Reading, C. C., Hoffman, R., Wickrema, A.,
Vesole, D. H., Jagarnath, S., Condino, J., Lee, B., Barlogie, B.,
and Tricot, G.: Purified CD34.sup.+lin-Thy.sup.+ stem cells do not
contain clonal myeloma cells. Blood 86: 381-389, 1995
[0121] Grate, D. and Wilson, C.: Laser-mediated, site-specific
inactivation of RNA transcripts. PNAS 96: 6131-6136, 1999
[0122] Gribben, J. G., Freedman, A. S., Neuberg, D., Roy, D. C.,
Blake, K. W., Woo, S. D., Grossbard, M. L., Rabinowe, S. N., Coral,
F., Freeman, G. J., Ritz, J., and Nadler, L. M.: Immunologic
purging of marrow assessed by PCR before autologous bone marrow
transplantation for B-cell lymphoma. N.E.J.Med. 325: 1525-1533,
1991
[0123] Gulati, S.C. and Acaba, L.: Rationale for purging in
autologous stem cell transplantation. J.Hematotherapy 2: 467-471,
1993
[0124] Gulliya, K. S. and Pervaiz, S.: Elimination of clonogenic
tumor cells from HL-60, Daudi, and U-937 cell lines by laser
photoradiation therapy: Implications for autologous bone marrow
purging. Blood 73: 1059-1065, 1989
[0125] Higuchi, W., Moriyama, Y., Kishi, K., Koike, T., Shibata,
A., Shinada, S., Tada, I., and Miura, A.: Hematopoietic recovery in
a patient with acute lymphoblastic leukemia after an autologous
marrow graft purged by combined hyperthermia and interferon in
vitro. Bone Marrow Transplant. 7: 163-166, 1991
[0126] Jay, D. G.: Selective destruction of protein function by
chromophore-assisted laser inactivation. PNAS 85: 5454-5458,
1988
[0127] Kvalheim, G., Holte, H., Jakobsen, E., and Kvaloy, S.:
Immunomagnetic purging of lymphoma cells from autografts.
J.Hematotherapy 5: 561-562, 1996
[0128] Langer, R. S. and Vacanti, J. P.: Tissue engineering: The
challenges ahead. Sci.Am. 280: 86-89, 1999
[0129] Mapara, M. Y., Kormer, I. J., Hildebrandt, M., Bargou, R.,
Krahl, D., Reichardt, P., and Dorken, B.: Monitoring of tumor cell
purging after highly efficient immunomagnetic selection of CD34
cells from leukapheresis products in breast cancer patients:
Comparison of immunocytochemical tumor cell staining and reverse
transcriptase-polymerase chain reaction. Blood 89: 337-344,
1997
[0130] Mapara, M. Y., Korner, I. J., Lentzsch, S., Krahl, D.,
Reichardt, P., and Dorken, B.: Combined positive/negative purging
and transplantation of peripheral blood progenitor cell autografts
in breast cancer patients: A pilot study. Exp.Hematol. 27: 169-175,
1999
[0131] Niemz, M. H.: Laser-tissue interactions: Fundamentals and
applications. Springer-Verlag, Berlin, 1996
[0132] Oleinick, N. L. and Evans, H. H.: The photobiology of
photodynamic therapy: Cellular targets and mechanisms. Rad.Res.
150: S146-S156, 1998
[0133] Palumbo, G., Caruso, M., Crescenzi, E., Tecce, M. F.,
Roberti, G., and Colasanti, A.: Targeted gene transfer in
eukaryotic cells by dye-assisted laser optoporation.
J.Photochem.Photobiol. 36: 41-46, 1996
[0134] Paulus, U., Dreger, P., Viehmann, K., von Neuhoff, N., and
Schmitz, N.: Purging peripheral blood progenitor cell grafts from
lymphoma cells: Quantitative comparison of immunomagnetic
CD34+selection systems. Stem Cells 15: 297-304, 1997
[0135] Pedersen, R. A.: Embryonic stem cells for medicine.
Sci.Amer. 280: 68-73, 1999
[0136] Rill, D. R., Santana, V. M., Roberts, W. M., Nilson, T.,
Bowman, L. C., Krance, R. A., Heslop, H. E., Moen, R. C., Ihle, J.
N., and Brenner, M. K.: Direct demonstration that autologous bone
marrow transplantation for solid tumors can return a multiplicity
of tumorigenic cells. Blood 84: 380-383, 1994
[0137] Robertson, M. J., Soiffer, R. J., Freedman, A. S., Rabinowe,
S. L., Anderson, K. C., Ervin, T. J., Murray, C., Dear, K.,
Griffin, J. D., Nadler, L. M., and Ritz, J.: Human bone marrow
depleted of CD33-positive cells mediates delayed but durable
reconstitution of hematopoiesis: Clinical trial of MY9 monoclonal
antibody-purged autografts for the treatment of acute myeloid
leukemia. Blood 79: 2229-2236, 1992
[0138] Schulze, R., Schulze, M., Wischnik, A., Ehnle, S., Doukas,
K., Behr, W., Ehret, W., and Schlimok, G.: Tumor cell contamination
of peripheral blood stem cell transplants and bone marrow in
high-risk breast cancer patients. Bone Marrow Transplant. 19:
12231228,1997
[0139] Schutze, K. and Lahr, G.: Identification of expressed genes
by laser-mediated manipulation of single cells. Nature Biotechnol.
16: 737-742, 1998
[0140] Sharp, J. G., Joshi, S. S., Armitage, J. O., Bierman, P.,
Coccia, P. F., Harrington, D. S., Kessinger, A., Crouse, D. A.,
Mann, S. L., and Weisenburger, D. D.: Significance of detection of
occult Non-Hodgkin's Lymphoma in histologically uninvolved bone
marrow by a culture technique. Blood 79: 1074-1080, 1992
[0141] Sharp, J. G., Kessinger, A., Mann, S., Crouse, D. A.,
Armitage, J. O., Bierman, P., and Weisenburger, D. D.: Outcome of
high-dose therapy and autologous transplantation in non-Hodgkin's
lymphoma based on the presence of tumor in the marrow or infused
hematopoietic harvest. J.Clin.Oncol. 14: 214-219, 1996
[0142] Shpall, E. J. and Jones, R. B.: Release of tumor cells from
bone marrow. Blood 83: 623-625, 1994
[0143] Tricot, G., Gazitt, Y., Jagannath, S., Vesole, D., Reading,
C. L., Juttner, C. A., Hoffman, R., and Barlogie, B.:
CD34.sup.+Thy.sup.+lin.sup- .- peripheral blood stem cells (PBSC)
effect timely trilineage engraftment in multiple myeloma (MM).
Blood 86: 293a-0, 1995
[0144] Vannucchi, A. M., Bosi, A., Glinz, S., Pacini, P., Linari,
S., Saccardi, R., Alterini, R., Rigacci, L., Guidi, S., Lombarkini,
L., Longo, G., Mariani, M. P., and Rossi-Ferrini, P.: Evaluation of
breast tumour cell contamination in the bone marrow and
leukapheresis collections by RT-PCR for cytokeratin-19 mRNA.
Br.J.Haematol. 103: 610-617, 1998
[0145] Vervoordeldonk, S. F., Merle, P. A., Behrendt, H.,
Steenbergen, E. J., van den Berg, H., van Wering, E. R., von dem
Borne, A. E. G., van der Schoot, C. E., van Leeuwen, E. F., and
Slaper-Cortenbach, I. C. M.: PCR-positivity in harvested bone
marrow predicts relapse after transplantation with autologous
purged bone marrow in children in second remission of precursor
B-cell acute leukemia. Br.J.Haematol. 96: 395-402, 1997
[0146] Vredenburgh, J. J., Silva, O., Broadwater, G., Berry, D.,
DeSombre, K., Tyer, C., Petros, W. P., Peters, W. P., and Bast, J.,
R.C.: The significance of tumor contamination in the bone marrow
from high-risk primary breast cancer patients treated with
high-dose chemotherapy and hematopoietic support. Biol. Blood
Marrow Transplant. 3: 91-97, 1997
[0147] Wagner, J. E., Collins, D., Fuller, S., Schain, L. R.,
Berson, A. E., Almici, C., Hall, M. A., Chen, K. E., Okarma, T. B.,
and Lebkowski, J. S.: Isolation of small, primitive human
hematpoitic stem cells: Distribution of cell surface cytokine
receptors and growth in SCID-Hu mice. Blood 86: 512-523, 1995
* * * * *