U.S. patent application number 11/895557 was filed with the patent office on 2012-05-31 for systems and methods for cutting materials.
Invention is credited to Anubha Bhatla, David J. Brenner, Aparajita Dutta, Guy Y. Garty, Gerhard Randers-Pehrson, Alessio Salerno, Nabil Simaan, Y. Lawrence Yao.
Application Number | 20120132313 11/895557 |
Document ID | / |
Family ID | 39107747 |
Filed Date | 2012-05-31 |
United States Patent
Application |
20120132313 |
Kind Code |
A1 |
Bhatla; Anubha ; et
al. |
May 31, 2012 |
Systems and methods for cutting materials
Abstract
Systems and methods for cutting materials are disclosed herein
In some embodiments, methods of at least partially severing a
capillary vessel can include: focusing a laser on a predetermined
point on the capillary vessel, said capillary vessel containing a
biological sample; and cutting the capillary vessel using a laser
at the predetermined point. In some embodiments, the methods
further can include capturing an image of the capillary vessel and
analyzing the image to determine the predetermined point. In some
embodiments, a beam of the laser can be moved using one or more
galvanometric mirrors. In some embodiments, the methods further can
include cutting a plurality of capillary vessels using the laser.
In some embodiments, the methods can include utilizing a plurality
of lasers, and/or further can include rotating the capillary vessel
while the laser can be cutting the capillary vessel. In some
embodiments, cutting the capillary vessel can include cutting only
a portion of the capillary vessel.
Inventors: |
Bhatla; Anubha; (Secaucus,
NJ) ; Salerno; Alessio; (Montreal, CA) ;
Simaan; Nabil; (New York, NY) ; Yao; Y. Lawrence;
(New York, NY) ; Randers-Pehrson; Gerhard;
(Ossining, NY) ; Garty; Guy Y.; (Dobbs Ferry,
NY) ; Dutta; Aparajita; (Waltham, MA) ;
Brenner; David J.; (New York, NY) |
Family ID: |
39107747 |
Appl. No.: |
11/895557 |
Filed: |
August 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60840245 |
Aug 25, 2006 |
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60942090 |
Jun 5, 2007 |
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60954499 |
Aug 7, 2007 |
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Current U.S.
Class: |
141/1 ;
141/83 |
Current CPC
Class: |
C40B 30/10 20130101;
C40B 60/12 20130101 |
Class at
Publication: |
141/1 ;
141/83 |
International
Class: |
B65B 3/26 20060101
B65B003/26 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made in part with Government support
under Department of Health and Human Services Grant U19A1067773-01.
The Government has certain rights in the invention.
Claims
1. A method of at least partially severing a capillary vessel
comprising: (a) focusing a laser on a predetermined point on the
capillary vessel, said capillary vessel containing a biological
sample; and (b) cutting the capillary vessel using a laser at the
predetermined point.
2. The method of claim 1 further comprising capturing an image of
the capillary vessel and analyzing the image to determine the
predetermined point.
3. The method of claim 1 wherein a beam of the laser is moved using
one or more galvanometric mirrors.
4. The method of claim 1 further comprising cutting a plurality of
capillary vessels using the laser.
5. The method of claim 1 utilizing a plurality of lasers
6. The method of claim 1 further comprising rotating the capillary
vessel while the laser is cutting the capillary vessel.
7. The method of claim 1 wherein cutting the capillary vessel
comprises cutting only a portion of the capillary vessel.
8. The method of claim 1 wherein the laser is set to a
predetermined frequency.
9. The method of claim 1 further comprising positioning the
capillary vessel over a multi-well plate, said plate having a
plurality of wells arranged in an array.
10. The method of claim 1 further comprising emptying at least a
portion of the biological sample from the capillary vessel after
cutting the capillary vessel.
11. The method of claim 10 wherein emptying at least a portion of
the biological sample comprises emptying the portion into a
selected well within the array.
12. An apparatus for at least partially bisecting a capillary
comprising: (a) an imaging element adapted to capture an image of
the capillary; (b) a processor adapted to analyze the image and
determine a cutting point based on the image; and (c) a laser
adapted to cut the capillary at the cutting point.
13. The apparatus of claim 12 wherein the imaging element is a
CCD.
14. The apparatus of claim 12 wherein the imaging element is a
CMOS
15. The apparatus of claim 12 wherein the capillary has an outer
diameter of about 2 mm.
16. The apparatus of claim 12 wherein the laser only partially cuts
the capillary at the cutting point.
17. The apparatus of claim 12 further comprising a multi-well plate
positioned under the capillary when the capillary is cut.
18. The apparatus of claim 12 comprising a plurality of capillaries
wherein the laser is adapted to cut the plurality of
capillaries.
19. The apparatus of claim 12 wherein the imaging element comprises
a microscope and a CCD.
20. The apparatus of claim 12 wherein the capillary contains a
biological sample.
21. The apparatus of claim 20 wherein the capillary is cut by the
laser such that the laser does not damage the biological
sample.
20. A system for at least partially severing a capillary vessel
comprising: (a) an imaging means for capturing an image of the
capillary vessel; (b) a processing means for determining a cutting
point based on the image; and (c) a cutting means for cutting the
capillary at the cutting point.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 60/840,245 filed on Aug. 25, 2006; U.S.
Provisional Application Ser. No. 60/942,090 filed Jun. 5, 2007; and
U.S. Provisional Application Ser. No. 60/954,499 filed Aug. 7,
2007, each of which is incorporated herein by reference in its
entirety.
FIELD
[0003] The present application generally relates to systems,
devices, and methods for minimally-invasive, high-throughput
radiation biodosimetry using commonly available biological
samples.
BACKGROUND
[0004] The Homeland Security Council recently established an
interagency working group (Pellmar T C, Rockwell S, and the
Radiological/Nuclear Threat Countermeasures Working Group: Priority
list of research areas for radiological nuclear threat
countermeasures. Radiat Res 2005; 163:115-23) to assess and
prioritize the nation's needs in terms of a response to a terrorist
attack using radiological or nuclear devices. Biodosimetry assay
automation, biomarkers and devices for biodosimetry, and training
in radiation sciences were among the areas of research identified
as top or high priorities.
[0005] Products for high throughput minimally-invasive biodosimetry
are clearly needed. After a large-scale radiological event, there
will be a major need to assess, within a few days, the radiation
doses received by tens or hundreds of thousands of individuals.
SUMMARY
[0006] Systems and methods for cutting materials are disclosed
herein In some embodiments, methods of at least partially severing
a capillary vessel can include: focusing a laser on a predetermined
point on the capillary vessel, said capillary vessel containing a
biological sample; and cutting the capillary vessel using a laser
at the predetermined point. In some embodiments, the methods
further can include capturing an image of the capillary vessel and
analyzing the image to determine the predetermined point. In some
embodiments, a beam of the laser can be moved using one or more
galvanometric mirrors. In some embodiments, the methods further can
include cutting a plurality of capillary vessels using the laser.
In some embodiments, the methods can include utilizing a plurality
of lasers, and/or further can include rotating the capillary vessel
while the laser can be cutting the capillary vessel. In some
embodiments, cutting the capillary vessel can include cutting only
a portion of the capillary vessel.
[0007] In some embodiments, the laser can be set to a predetermined
frequency. In some embodiments, the methods further can include
positioning the capillary vessel over a multi-well plate, said
plate having a plurality of wells arranged in an array. In some
embodiments, the methods further can include emptying at least a
portion of the biological sample from the capillary vessel after
cutting the capillary vessel. In some embodiments, emptying at
least a portion of the biological sample can include emptying the
portion into a selected well within the array.
[0008] In some embodiments, an apparatus is provided for at least
partially bisecting a capillary, and can include: an imaging
element adapted to capture an image of the capillary; a processor
adapted to analyze the image and determine a cutting point based on
the image; and a laser adapted to cut the capillary at the cutting
point. In some embodiments, the imaging element can include a CCD
and/or a CMOS. In some embodiments, the capillary can have an outer
diameter of about 2 mm. In some embodiments, the laser only
partially cuts the capillary at the cutting point. In some
embodiments, the apparatus further can include a multi-well plate
positioned under the capillary when the capillary is cut. In some
embodiments, the apparatus can include a plurality of capillaries
wherein the laser can be adapted to cut the plurality of
capillaries. In some embodiments, the imaging element can include a
microscope and a CCD. In some embodiments, the capillary can
contain a biological sample. In some embodiments, the capillary can
be cut by the laser such that the laser does not damage the
biological sample.
[0009] In some embodiments, a system for at least partially
severing a capillary vessel can include: an imaging means for
capturing an image of the capillary vessel; a processing means for
determining a cutting point based on the image; and a cutting means
for cutting the capillary at the cutting point.
[0010] Other objects and features will be in part apparent and in
part pointed out hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Those of skill in the art will understand that the drawings,
described below, are for illustrative purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0012] FIG. 1 illustrates a system overview of an embodiment of the
invention.
[0013] FIG. 2. illustrates a biodosimetry workstation in accordance
with one embodiment of the invention.
[0014] FIG. 3 depicts the sample hierarchy in accordance with an
embodiment of the invention.
[0015] FIG. 4 depicts a process flow diagram in accordance with an
embodiment of the invention.
[0016] FIG. 5. shows a flow chart of a biodosimetry workstation in
accordance with an embodiment of the invention.
[0017] FIG. 6 shows a flow chart of a cell harvesting module in
accordance with an embodiment of the invention.
[0018] FIG. 7 illustrates an embodiment of an input module and
centrifuge module in accordance with an embodiment of the
invention.
[0019] FIG. 8 depicts features of a service robot manipulating arm
in accordance with an embodiment of the invention.
[0020] FIG. 9 depicts features of a cell harvesting module in
accordance with an embodiment of the invention.
[0021] FIG. 10 illustrates image segmentation of a capillary as
provided in an embodiment of the invention.
[0022] FIG. 11 depicts a laser system a in accordance with an
embodiment of the invention.
[0023] FIG. 12 illustrates further details of the cell harvesting
module in accordance with an embodiment of the invention.
[0024] FIG. 13 illustrates a method of loading a liquid handling
module in accordance with an embodiment of the invention.
[0025] FIG. 14 illustrates a method of transferring a sample from
the liquid handling module to a robotic incubator in accordance
with an embodiment of the invention.
[0026] FIG. 15 compares radiation-induced micronucleus yields of
conventional methods with yields obtained using systems and methods
of the present invention.
[0027] FIG. 16 illustrates results of dose-response studies of
radiation induced .gamma.-H2AX foci in peripheral blood
lymphocytes.
[0028] FIG. 17 illustrates a capillary tube for collection of whole
blood and separation of mononuclear cells in accordance with an
embodiment of the invention.
[0029] FIG. 18 illustrates a relationship between centrifuge time
required as a function of number of capillaries in each
centrifuge.
[0030] FIG. 19 shows a simplified flow diagram of a micronucleous
assay process in accordance with an embodiment of the
invention.
[0031] FIG. 20 illustrates an embodiment of a centrifuge module in
accordance with an embodiment of the invention.
[0032] FIG. 21 illustrates further details of the centrifuge module
in accordance with an embodiment of the invention.
[0033] FIG. 22 illustrates details of a punctuation unit in
accordance with an embodiment of the invention.
[0034] FIG. 23 illustrates a multi-well plate in accordance with an
embodiment of the invention.
[0035] FIG. 24 illustrates filters attached to the multi-well plate
in accordance with an embodiment of the invention.
[0036] FIG. 25 illustrates a punching mechanism used to detach
membranes from the multi-well plate in accordance with an
embodiment of the invention.
[0037] FIG. 26 illustrates a sealed and laminated membrane with
fluorescent beads.
[0038] FIG. 27 illustrates a liquid handling module in accordance
with an embodiment of the invention.
[0039] FIG. 28 illustrates a steered-image compound microscope in
accordance with an embodiment of the invention.
[0040] FIG. 29 illustrates the method and results of operating the
steered-image compound microscope.
[0041] FIG. 30 illustrates simulated images demonstrating the
effect of a cylindrical lens in an optical beam path as used in an
embodiment of the invention.
[0042] FIG. 31 illustrates use of a dichroic mirror and separate
focusing and imaging cameras in accordance with an embodiment of
the invention.
[0043] FIG. 32 depicts data flow for an embodiment of the
invention.
[0044] FIG. 33 FIG. 28 illustrates a further embodiment of the
steered-image compound microscope in accordance with an embodiment
of the invention.
[0045] FIG. 34 illustrates system process flows for an embodiment
of the invention.
[0046] FIG. 35 illustrates an isometric view of an overall system
layout in accordance with an embodiment of the invention.
[0047] FIG. 36 depicts a multi-purpose robotic gripper used in an
embodiment of the invention.
[0048] FIG. 37 apparatus for contactless automatic cutting of
capillaries in accordance with an embodiment of the invention.
[0049] FIG. 38 illustrates an embodiment of a system implementation
of the invention.
[0050] FIG. 39 shows a prototype.
[0051] FIG. 40 shows a field collection kit.
[0052] FIG. 41 illustrates a capillary having a laser-etched bar
code identifier in accordance with an embodiment of the
invention.
[0053] FIG. 42 depicts a flow diagram of an exemplary method of the
system.
[0054] FIG. 43 illustrates dilution tubes modified to accommodate
capillaries for shipping and centrifugation in accordance with an
embodiment of the invention.
[0055] FIG. 44 illustrates a design model of a centrifuge adapted
for use with an embodiment of the invention.
[0056] FIG. 45 illustrates image segmentation of a capillary
accomplished using an embodiment of the invention.
[0057] FIG. 46 shows a white cloudy band of lymphocytes separated
out from whole blood in a glass Accutube.
[0058] FIG. 47 depicts a flow diagram of an imaging process in
accordance with an embodiment of the invention.
[0059] FIG. 48 illustrates a method and results of operating a
microscope with a 2D scan head in accordance with an embodiment of
the invention.
[0060] FIG. 49 illustrates the effect of centrifuge speed and
elapsed time from blood collection on sample quality.
DETAILED DESCRIPTION
[0061] The need for high throughput rapid biodosimetry can be well
illustrated by reference to the 1987 radiation incident in Goiania,
Brazil, a city with about the same population as Manhattan. In the
first few days after the incident became known, about 130,000
people (roughly 10% of the population) came for screening, of whom
20 required treatment (International Atomic Energy Agency. The
Radiological accident in Goiania. Vienna: International Atomic
Energy Agency; 1988.). In response to a RDD (radiological dispersal
device) event in a US city, one would anticipate a similar
scenario. Tens or hundreds of thousands of individuals will need to
be screened for radiation exposure within a few days due to demand
and the medical necessity to perform radiological triage.
[0062] Mass radiological triage will be critical after a
large-scale event in order to identify, at an early stage, those
individuals who will benefit from medical intervention. In
addition, eliminating and reassuring patients who do not need
medical intervention will be crucial in a highly resource-limited
scenario.
[0063] Regarding those who do require medical intervention, the
best estimate for the LD50/60 in humans is in the 3.5 to 4.5 Gy
range (Anno G H, Young R W, Bloom R M, Mercier J R. Dose response
relationships for acute ionizing radiation lethality. Health Phys
2003; 84:565-75.), but this value can be roughly doubled through
the use of antibiotics, platelet and cytokine treatment (Anno G H,
Young R W, Bloom R M, Mercier J R. Dose response relationships for
acute ionizing radiation lethality. Health Phys 2003; 84:565-75.).
Thus, it is crucial that individuals who actually received
whole-body doses above a predetermined threshold value, for
example, 0.5, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or
2.0 Gy are identified and treated. Some individuals who are in this
dose range will be clearly identifiable through early nausea,
vomiting, and acute fatigue, but not all. For example, worker "C"
at the 1999 radiation accident at Tokai-mura received a
best-estimate whole-body equivalent dose of more than 3 Gy
(Ishigure N, Endo A, Yamaguchi Y, Kawachi K. Calculation of the
absorbed dose for the overexposed patients at the JCO criticality
accident in Tokai-mura. J Radiat Res (Tokyo) 2001; 42
Suppl:S137-48; Hayata I, Kanda R, Minamihisamatsu M, Furukawa M,
Sasaki M S. Cytogenetical dose estimation for 3 severely exposed
patients in the JCO criticality accident in Tokai-mura. J Radiat
Res (Tokyo) 2001; 42 Suppl:S149-55.), was initially almost entirely
asymptomatic, yet developed bone marrow failure (Hirama T, Tanosaki
S, Kandatsu S, Kuroiwa N, Kamada T, Tsuji H, et al. Initial medical
management of patients severely irradiated in the Tokai-mura
criticality accident. Br J Radiol 2003; 76:246-53). Thus, accurate
biodosimetry is crucial in this dose range.
[0064] At higher doses, e.g., between 5 and 12 Gy, there is also a
critical need for biodosimetry. This is because there is only a
quite narrow dose window (approximately 7-10 Gy) in which
bone-marrow transplantation is a useful option (below 7 Gy,
survival rates are good solely with medication, while above 10 Gy
patients will generally have lethal gastrointestinal damage) (Hall
E J. Radiobiology for the radiologist. 5th ed. Philadelphia:
Lippincott, Williams & Wilkins; 2000). Thus, it is important to
ascertain, through biodosimetry, whether a patient's dose is within
this dose window, such that a bone-marrow transplant is a useful
option.
[0065] It should be noted that the dose estimates discussed above
are for adults. Children are likely to be more sensitive to
radiation than adults in terms of their LD50. Fred S S, Smith W W.
Radiation sensitivity and proliferative recovery of hemopoietic
stem cells in weanling as compared to adult mice. Radiat Res 1967;
32:314-26; Reincke U, Mellmann J, Goldmann E. Variations in
radioresistance of rats during the period of growth. Int J Radiat
Biol Relat Stud Phys Chem Med 1967; 13:137-46; Ward B C, Childress
J R, Jessup G L, Jr., Lappenbusch W L. Radiation mortality in the
Chinese hamster, Cricetulus griseus, in relation to age. Radiat Res
1972; 51:599-607. Thus, it is desirable that biodosimetric
information should also be obtainable at lower doses in
children.
[0066] Embodiments of the invention disclosed herein emphasize
extremely high throughput (many thousands of samples per day per
machine), in contrast to current technologies which feature at most
a few hundred samples per day per machine (Offer T, Ho E, Traber M
G, Bruno R S, Kuypers F A, Ames B N. A simple assay for frequency
of chromosome breaks and loss (micronuclei) by flow cytometry of
human reticulocytes. Faseb J 2004; Styles J A, Clark H, Festing M
F, Rew D A. Automation of mouse micronucleus genotoxicity assay by
laser scanning cytometry. Cytometry 2001; 44:153-5).
[0067] A related issue is that of invasive vs.
noninvasive/minimally-invasive biodosimetry. The term "invasive
biodosimetry," as used herein, refers to procedures that require a
qualified health professional, such as the drawing of peripheral
blood through venipuncture. Such a procedure would be a major
bottleneck, in that a health professional can, at most, draw blood
from 15 to 25 individuals per hour. Accordingly, embodiments of the
invention disclosed herein relate to minimally invasive procedures,
such as a capillary blood finger or heel stick. Other embodiments
relate to non-invasive approaches such as the use of exfoliated
cells from a buccal smear (mouthwash), or from urine. Some
embodiments relate to completely self-contained readily-deployable
biodosimetry kits.
[0068] Another issue with regard to biodosimetry is that of
inter-individual variability in radiation sensitivity.
Specifically, it would be highly desirable to be able to recognize
individuals with high radiation sensitivity, a) because they would
constitute a high-risk group which might warrant different and/or
additional follow-up procedures, and because b) particularly at
high doses (>2Gy) the uncertainty in a biodosimetrically-based
dose estimate will predominantly be due to inter-individual
differences (Thierens H, Vral A, de Ridder L. Biological dosimetry
using the micronucleus assay for lymphocytes: interindividual
differences in dose response. Health Phys 1991; 61:623-30). Thus,
embodiments of the invention described herein address this issue.
In some aspects of this embodiment, each biological sample is split
in two, with one of the two split samples being irradiated to a
known dose, before being analyzed. This will allow a positive
control for each individual, so that the effects of
inter-individual variability in radiosensitivity can be taken into
account.
[0069] Another issue is that of lower-dose biodosimetry, for
example, doses of less than 2 Gy, 1.8 Gy, 1.5 Gy, 1.2 Gy, 1 Gy, 0.9
Gy, 08 Gy, 0.7 Gy, 0.6 Gy, 0.5 Gy, 0.4 Gy, 0.3 Gy or 0.1 Gy. While,
such doses are typically below life-threatening, it is likely that
long-term carcinogenic risk as a result of such doses will be
increased. Thus, in the event of a large-scale radiological event,
the dosimetric data generated according to the invention disclosed
herein could form the basis for long-term epidemiological
studies.
[0070] Another consideration is the information required. In many
situations, for example, an appropriate first level of triage might
be a very rapid yes/no answer as to whether a predetermined
threshold dose has been exceeded. In other situations, an actual
dose estimate is important.
[0071] While all the biodosimeters will be calibrated over a wide
dose range, some biodosimeters are more appropriate for lower
doses, some for higher doses, and some are useful over a very wide
range of doses. For example, for an individual who potentially
received an extremely high dose, e.g., 10 Gy; a DSB (.gamma.-H2AX)
approach would be more informative than a micronuclei approach.
[0072] An additional issue is time since exposure. Some
biodosimeters, such as micronuclei in lymphocytes, are very stable
with time, over a period of many weeks. Some biodosimeters are
practical for use only within limited time periods after the
radiation incident. For example, the .gamma.-H2AX biodosimeter,
which reflects the presence of DNA double strand breaks, will be
most useful in the first 36 hours after a radiation event, while
micronuclei in blood reticuloctyes will be most useful from about
24 to 60 hours after radiation exposure. These considerations
strongly imply that different biodosimetric endpoints may be needed
for different situations.
[0073] Thus, some embodiments relate to a multi-input and/or
multi-endpoint high-throughput product, which can be applied in
different situations. In one embodiment, the automated device is
useful for both blood lymphocytes and for reticuloctyes, as well as
for exfoliated cells from urine or buccal smears, and the device
can measure both micronuclei and .gamma.-H2AX foci. In a preferred
embodiment, any combination of endpoints can be applied by using
different predetermined sets of instructions in the
robotically-based system.
[0074] The biomarker should have appropriate specificity, i.e. the
measured response should be specific to radiation, as opposed to a
more general stress response, or a chemical or biological agent
response.
[0075] Current systems for performing radiation biodosimetry have
limited throughputs of a few hundred samples per day. Offer T, Ho
E, Traber M G, Bruno R S, Kuypers F A, Ames B N. A simple assay for
frequency of chromosome breaks and loss (micronuclei) by flow
cytometry of human reticulocytes. Faseb J 2004; Styles J A, Clark
H, Festing M F, Rew D A. Automation of mouse micronucleus
genotoxicity assay by laser scanning cytometry. Cytometry 2001;
44:153-5; Smolewski P, Ruan Q, Vellon L, Darzynkiewicz Z.
Micronuclei assay by laser scanning cytometry. Cytometry 2001;
45:19-26; Dertinger S D, Chen Y, Miller R K, Brewer K J, Smudzin T,
Torous D K, et al. Micronucleated CD71-positive reticulocytes: a
blood-based endpoint of cytogenetic damage in humans. Mutat Res
2003; 542:77-87. Accordingly, embodiments of the invention
described herein relate to systems, devices and methods for
high-throughput, minimally invasive radiation biodosimetry.
[0076] Described herein is a high-throughput biodosimetry device
that, in some embodiments, uses purpose-built robotics and/or
advanced high-speed automated image acquisition and analysis. In
preferred embodiments, throughput is at least about 1,000, 2,000,
3,000, 4,000, 5,000, 7,500, 10,000, 12,500, 15,000, 17,500, 20,000,
22,500, 25,000, 27,500, 30,000, 35,000 40,000, 45,000, 50,000,
60,000, 70,000, 75,000, 80,000, 90,000, or 100,000 samples day,
compared with current maximal throughputs of a few hundred
samples/day. In some embodiments, several endpoints (micronuclei
and/or .gamma.-H2AX foci) and/or several tissues (blood
lymphocytes, reticuloctyes, and/or exfoliated cells from urine or a
buccal smear) can be used. Purpose-built liquid-handling robotics
and advanced high-speed automated image acquisition can be used to
increase throughput.
[0077] Some embodiments relate to a system or device that employs a
micronucleus assay in lymphocytes, with such assays being carried
out in-situ in multi-well plates. Peripheral blood drawn by
venipuncture using a finger or heelstick or a high-throughput laser
skin perforator is used. In some embodiments, pre-programmed
options in timing, liquid handling, and image analysis, the device
are used to measure .gamma.-H2AX foci yields and/or micronucleus
yields in reticuloctyes, thereby providing "same-day answer" dose
estimates. In some embodiments, pre-programmed options in liquid
handling steps are used to measure micronuclei in other
readily-accessible tissues, such as exfoliated cells from urine or
buccal smears. In preferred embodiments, each biological sample is
split in two, with one of the two split samples being irradiated to
a known dose before being analyzed. This allows a positive control
for each individual, providing an internal calibration account for
inter-individual variability in radiosensitivity.
[0078] In some embodiments, a system using 96-well plates provides
a throughput target of 6,000 samples (3,000 individuals) per 15
hour day. In another embodiment, a system using 384-well
plates--provides a throughput target of 30,000 (15,000 individuals)
samples per 15 hour day.
[0079] Other embodiments relate to a blood handling subsystem that
uses either capillary tubes or larger vacutainer tubes. The tubes
can be plastic or glass. In a preferred embodiment, the device uses
capillary tubes.
[0080] Monochrome imaging or color imaging can be used. In some
embodiments, a color image can be split into two or more,
individually processed, monochrome images using dichroic
beamsplitters, for example.
[0081] Some embodiments disclosed herein relate to a workstation
comprising a blood collection module, an irradiation module, a cell
harvesting module, a sample identification and tracking module, a
lymphocyte incubation module, a liquid/plate handling robot, an
image acquisition/processing system, and optionally, an irradiation
module.
[0082] In one embodiment, equipment used for the elements of the
workstation includes:
[0083] VideoScope Gen III High Resolution Intensifier; Photonfocus
MV-D1024 series CMOS High Speed Monochrome Digital Camera System;
Upstate Technical Equipment Co. Inc., East Syracuse, N.Y.; Matrox
Solios XCL Camera Link; Martox, Montreal, QC, Canada: One or more
of each of these items can be used for image capture and read out.
The image intensifier boosts the image intensity to a level
required for fast imaging. CMOS sensors are the fastest imaging
device commercially available that also suit embodiments of the
invention disclosed herein. The Matrox Solios XCL is used to read
and process the image data.
[0084] Mirror/Scanner, Scanlab HurryScan II; Scanlab America
Naperville, Ill.: The galvanometer scanner system is used as the
steering mechanism for the steered-image compound microscope. This
optical scanning system benefits the proposed instrumentation with
its fast speed, especially when compared to the settling times of
bulky mechanical stages. The short switching time improves speed
for promoting high throughput.
[0085] Nikon, CFI60 20X Objective; Cube Changer; Morrell Instrument
Company, Inc., Melville, N.Y.; Mad City, Z-motion 100 micron Piezo
Nano Positioner; Mad City, Piezo--Controller; Mad City labs,
Madison, Wis.; EXFO X-Cite 120 illumination system, (EXFO America
inc, plano TX): This objective lens is the primary lens used for
imaging the cell samples. It is a lens with infinity optics, which
enables other optical elements (mirror/scanner) to be added into an
afocal space, while not distorting the image quality. The piezo
nano positioner is used for precision auto-focusing. The
illumination source for the microscope in a first embodiment is a
high intensity mercury bulb with fiber optic light guide. For
multi-component imaging, the cube changer is used to select which
wavelength is used for excitation and observation of various
fluorochromes.
[0086] Daedal X-Y Mechanical Stage With Compumotor Stepper Motor
Control, Axis N.Y., Fairport, N.Y.: This stepper-motor controlled
X-Y mechanical stage is designated for coarse motions for the
sample arrays being imaged with the microscope. The speed and
resolution of this stage is suited for high throughput.
[0087] Computer, CyberResearch, Inc., New Haven, Conn.: This
computer system is an industrial strength machine with room for
numerous expansion cards for image processing. The computer is
equipped with one terra byte of storage space and a back-up power
supply.
[0088] NanoLED--625 nm; NanoLED Controller; HORIBA Jobin Yvon,
Inc., Edison, N.J.: This LED light source is used for excitation of
the fluorescent beads that are used in the auto-focusing routine on
the microscope.
[0089] Kendro centrifuge(s); Sorvall rotor(s); accessories; Kendro
Laboratory Products, Asheville, N.C.: One or more of each of these
items form the core of the centrifuge module. The rotors are custom
made to have capacity of 48 vacutainer CPT tubes. With the
radiation and control scheme, one rotor load of samples fills a
96-well plate.
[0090] Components to construct the turn table and jack mechanism
for the centrifuge module cylinder with spline joint, timing wheel
and belt, two DC motors with encoders and reduction gears,
precision bearings, multi-channel motion control card, amplifiers
and power supplies, SDP-SI, New Hyde Park, N.Y., ORMEC Systems
Corp. Rochester, N.Y., McMaster-Carr, New Brunswick, N.J.: The turn
table and jack mechanism with the centrifuge and rotors above
complete the centrifuge module. Additional components including
brackets, housing, and supports that are designed and fabricated as
needed.
[0091] Sony DXC-990 Industrial CCD camera, Schneider XENON 17 mm
lens, Mikrotron Inspecta frame grabber, Sony Corporation, Mikrotron
GmbH, Germany: These items build part of the visual servoing system
for the cell harvest module. One set is sufficient for a device.
Additional sets can be added for increased throughput.
[0092] Components to construct the punctuation unit (DC motor with
encoders and reduction gear, precision lead screw and ball-bearing
nut, precision rail and carriage, motion control card, amplifier
and power supply) (SDP-SI, New Hyde Park, N.Y., ORMEC Systems Corp.
Rochester, N.Y., McMaster-Carr, New Brunswick, N.J.) These items
complete the visual servoing system for the cell harvest module.
Additional components including brackets, housing, and supports are
designed and fabricated as needed. Only one set of these items is
required, but additional sets can be added for increased
throughput.
[0093] Adept Cobra i800 SCARA robot and accessories (Adept
Technology, Inc., Livermore, Calif.) SCARA (selective compliance
assembly robot arm) robots provide excellent pick and place
accuracy under very high speed in a simple structure. The Adept
robots pioneered the direct-drive (without reduction gears) SCARC
robot to bring the accuracy and speed to a new level. This robot is
dedicated to interface the centrifuge and cell harvest modules and
a special end-effector is fabricated to load and unload vacutainer
CPT tubes to and from a centrifuge rotor.
[0094] Modified Zymark (now Caliper Life Sciences) Sciclone ALH
3000 including the gantry robot) (Caliper Life Sciences, Hopkinton,
Mass.): This liquid handling system meets most of the current needs
but a number of modifications are made either by working with the
supplier or on site. The width of the system is increased from
approximately 800 mm to 1100 mm to accommodate incorporation of a
pick-and-place robot and plate stacker within the working envelope
of the gantry robot. The two-robot configuration allows task
dedication and thus high throughput. The available EZ-swap dispense
module (attached to the gantry robot) is modified to enable
pneumatically actuated quick change of different end-effector
modules. The liquid handling portion of the system is also modified
to handle the radiated and control samples.
[0095] Allen Bradley (now Rockwell Automation) Programmable Logical
Controller (PLC) with accessories; two computers (Rockwell
Automation, Inc, Milwaukee, Wis., Dell Computer, Austin, Tex.) The
two computers host the motion control cards for the turn table/jack
mechanism and the punctuation unit, respectively. The PLC
implements the sequential control of the entire operation involving
all system components. The PLC interfaces with some components
directly such as the Adept SCARA robot, and with others via digital
input/out capabilities of their motion control cards residing in
the computers.
[0096] Center for Radiological Research Radiation seeds 4 mCi;
(Bebig, Berlin, Germany): Nine of these radioactive seeds are used
as the radiation source in the Strontium-90 irradiator.
[0097] Argon-Ion Laser, (Coherhent Inc., Santa Clara, Calif.): This
Argon-ion laser is used as a light source for the microscope in one
embodiment. It provides a brighter illumination, which is required
for fast imaging, and the light quality is improved over a Mercury
bulb used in an alternative embodiment.
[0098] Laser Optics, (Newport, Irvine, Calif.): Assorted laser
optics are used along the light path for the Argon-ion laser. Also
an assortment of filters, cubes, and mirrors are incorporated into
the steered-image compound microscope.
[0099] Robotioc incubator, (Liconic US, Inc, Woburn, Mass.): This
incubator is used as an atmosphere for the cell samples while they
are being treated and stored prior to the imaging sequence. The
robotic incubator is capable of storing a few hundreds of
multi-well plates and dispensing individual plates
automatically.
[0100] Pick-and-place robot, plate stackers and linear stage from
Matrix Tango Stacker system (without liquid handling subsystem)
(Matrix Technologies, Hudson, N.H.) These items are integrated
within the working envelope of the gantry liquid handling robot,
working in tandem. The linear stage holds 12 plates which are
sufficient. Additional stackers are included to provide total
capacity of 300 plates. The pick-and-place robot also interfaces
with the microscope for image acquisition.
[0101] Sony DXC-990 Industrial CCD camera, Schneider XENON 17 mm
lens, Mikrotron Inspecta frame grabber, (Sony Corporation,
Mikrotron GmbH, Germany) These items build part of the visual
servoing system for the cell harvest module.
[0102] Components to construct the punctuation unit (DC motor with
encoders and reduction gear, precision lead screw and ball-bearing
nut, precision rail and carriage, motion control card, amplifier
and power supply) (SDP-SI, New Hyde Park, N.Y., ORMEC Systems Corp.
Rochester, N.Y., McMaster-Carr, New Brunswick, N.J.) These items
complete the visual servoing system for the cell harvest module.
They share a motion control card.
[0103] OEM barcode printing and applying components from VCode by
Velocity 11, barcode reader, date acquisition and control (DAC)
board and monitoring software computer (Velocity 11, Menlo Park,
Calif., Symbol Technologies, Holtsville, N.Y., National
Instruments, Austin, Tex., Dell Computer, Austin, Tex.) The OEM
components are integrated with the plate stackers and
pick-and-place robot for barcode label applying on microplates for
identification and tracking by the barcode reader. The DAC board
has multi-channel analog-to-digital converter which collects the
current signals from all the actuators in the entire system to
monitor any overshooting as sign of trouble spots. The on-board
digital-to-analog converters connect to the actuator circuits for
emergency stops. Both barcode card and DAC board reside in a
computer.
[0104] VideoScope Gen III High Resolution Intensifier; pco. 12 hs
10-bit CMOS High Speed Monochrome Digital Camera System; pco.2000
14-bit High Performance Monochrome Digital Camera System; Matrox
Odyssey Xpro Camera Link; (The Cooke Corporation, Romulus, Mich.):
These items are used for image capture and read out for multi-color
imaging in one embodiment of the device. The image intensifiers
boost the image intensity to a level required for fast imaging. The
14-bit camera is a CCD camera that will acquire the low
magnification images. The additional CMOS sensor enables
multi-color imaging using two sensors. The Matrox Odyssey Xpro
cards are used to read and process the image data.
[0105] Color Separation Prism, (Redlake, San Diego, Calif.): This
item distributes the optics path in the microscope according to
wavelength and will be used for multi-color imaging.
[0106] Lasette Laser Lancing Device; Lens Shields (box of 250);
(Cell Robotics, Inc., Albuquerque, N. Mex.): This laser lancing
device is used for the perforation of skin to draw capillary blood
samples. This device eliminates injuries and uses disposable
single-use lens shields to prevent cross-contamination.
[0107] Three Sorvall rotors with bundled swing bucket; accessories
(Kendro Laboratory Products, Asheville, N.C.) The rotors are custom
modified to have special swing buckets for capillary tubes. Each
bucket can hold multiple capillary tubes.
[0108] Additional Adept Cobra i800 SCARA robot and accessories
(Adept Technology, Inc., Livermore, Calif.) Together with the SCARA
robot it is used for: 1) transferring capillary tubes from
centrifuge to the tube feeder and 2) transferring cell plug to the
flushing system after tube cutting. The two robots are dedicated to
these tasks with custom designed and fabricated end-effectors.
[0109] Components to construct the tube feeder (feeding tray,
loading unit, monitoring unit, etc.) (Hoppmann Corporation,
Elkwood, Va., SDP-SI, New Hyde Park, N.Y., McMaster-Carr, New
Brunswick, N.J.) These items build part of the tube feeders for
feeding the capillary tubes into the pneumatic transportation
systems. Additional components and supports are fabricated as
needed on site. Two sets of feeders are needed for a second
embodiment of the device: one for samples subject to irradiation,
another for control samples. The tube feeders have the ability of
feeding multiple transportation systems simultaneously.
[0110] Components to construct the pneumatic system for capillary
tube transportation (portable compressed air supplier, air dryer
and filter, transportation pipe network, valves, regulators,
control board, etc.) (Parker Air & Fuel Division, Irvine,
Calif., McMaster-Carr, New Brunswick, N.J.) These items build part
of the pneumatic transportation system for the centrifuged
capillary tubes. Additional components such as fixtures and
brackets are fabricated as needed. One set is sufficient for the
device, but additional sets can be added to increase
throughput.
[0111] Sony DXC-990 Industrial CCD camera, Schneider XENON 17 mm
lens, Mikrotron Inspecta frame grabber, (Sony Corporation,
Mikrotron GmbH, Germany) These items build part of the visual
servoing systems for the visual servoed cutting unit and
irradiation unit, respectively.
[0112] Components to construct the visual servoed irradiation unit
(mechanical barrier, motor, rail, carriage, motion control card,
etc., irradiation source not included) (SDP-SI, New Hyde Park,
N.Y., ORMEC Systems Corp. Rochester, N.Y., McMaster-Carr, New
Brunswick, N.J.) These items build part of the visual servoed
irradiation unit. Additional components including the irradiation
source are fabricated as needed.
[0113] Components to construct the visual servoed cutting unit
(mechanical barrier, cutting tool, motor, rail, carriage, motion
control card, etc.) (SDP-SI, New Hyde Park, N.Y., ORMEC Systems
Corp. Rochester, N.Y., McMaster-Carr, New Brunswick, N.J.) These
items build part of the visual servoed cutting unit. Additional
components and supports are fabricated as needed.
[0114] Components to construct the cell plug flushing system
(liquid reservoir, motor, slides, etc) (Upchurch Scientific, Inc,
Oak Harbor, Wash., SDP-SI, New Hyde Park, N.Y., McMaster-Carr, New
Brunswick, N.J.) These items build part of the cell plug flushing
system for transferring harvested cells into the microplate.
Additional components such as fixtures and brackets are fabricated
as needed. Two sets of this flushing system are needed for the
second embodiment of the device: one for samples subject to
irradiation, another for control samples.
[0115] VideoScope Gen III High Resolution Intensifier; pco. 12 hs
10-bit CMOS High Speed Monochrome Digital Camera System; Matrox
Odyssey Xpro Camera Link; (The Cooke Corporation, Romulus, Mich.):
These items are used for image capture and read out. They provide a
third image sensor to further the multi-color imaging capabilities.
The image intensifier boosts the image intensity to a level
required for fast imaging. The Matrox Odyssey Xpro is used to read
and process the image data.
[0116] Comprehensive RFID Tagging System, (TAGSYS, Doylestown,
Pa.): This system is used as a tracking device for each sample as
the sample is acquired from individuals and is then processed for
imaging.
[0117] Components to construct an additional pneumatic system for
capillary tube transportation (portable compressed air supplier,
air dryer and filter, transportation pipe network, regulators,
valves, control board, etc.) (Parker Air & Fuel Division,
Irvine, Calif., McMaster-Carr, New Brunswick, N.J.) These items
build part of the second pneumatic transportation system for the
centrifuged capillary tubes. Additional components such as fixtures
and brackets are fabricated as needed.
[0118] Sony DXC-990 Industrial CCD camera, Schneider XENON 17 mm
lens, Mikrotron Inspecta frame grabber, (Sony Corporation,
Mikrotron GmbH, Germany) These items build part of the visual
servoing system for an additional irradiation unit.
[0119] Components to construct an additional visual servoed
irradiation unit (mechanical barrier, motor, rail, carriage, motion
control card, etc., irradiation source not included) (SDP-SI, New
Hyde Park, N.Y., ORMEC Systems Corp. Rochester, N.Y.,
McMaster-Carr, New Brunswick, N.J.) These items build part of the
second visual servoed irradiation unit. Additional components
including the irradiation source are fabricated as needed.
[0120] Components to upgrade the liquid handling system and
quick-change end-effectors with .mu.l scale ability and
accessories, (Caliper Life Sciences, Hopkinton, Mass.) The liquid
handling tasks in the first embodiment of the device are mostly in
ml scale. For the second embodiment, the accuracy of the liquid
handling system is improved to handle liquid at the .mu.l-level.
Both liquid handling end-effectors and liquid moving subsystems are
upgraded.
[0121] Two Kendro centrifuges; three Sorvall rotors; accessories;
(Kendro Laboratory Products, Asheville, N.C.) These items form the
core of the centrifuge module. The rotors are custom made to have
capacity of 48 vacutainer CPT tubes. With the radiation and control
scheme, one rotor load of samples will fill a 96-well plate.
[0122] Components to construct the turn table and jack mechanism
for the centrifuge module (cylinder with spline joint, timing wheel
and belt, two DC motors with encoders and reduction gears,
precision bearings, multi-channel motion control card, amplifiers
and power supplies) (SDP-SI, New Hyde Park, N.Y., ORMEC Systems
Corp. Rochester, N.Y., McMaster-Carr, New Brunswick, N.J.) The turn
table and jack mechanism with the centrifuge and rotors above
complete the centrifuge module. Additional components including
brackets, housing, and supports are fabricated as needed.
[0123] Sony DXC-990 Industrial CCD camera, Schneider XENON 17 mm
lens, Mikrotron Inspecta frame grabber, (Sony Corporation,
Mikrotron GmbH, Germany) These items build part of the visual
servoing system for the cell harvest module.
[0124] Components to construct the punctuation unit (DC motor with
encoders and reduction gear, precision lead screw and ball-bearing
nut, precision rail and carriage, motion control card, amplifier
and power supply) (SDP-SI, New Hyde Park, N.Y., ORMEC Systems Corp.
Rochester, N.Y., McMaster-Carr, New Brunswick, N.J.) These items
complete the visual servoing system for the cell harvest module.
Additional components including brackets, housing, and supports are
fabricated as needed.
[0125] Adept Cobra i800 SCARA robot and accessories (Adept
Technology, Inc., Livermore, Calif.) SCARA (selective compliance
assembly robot arm) robots provide excellent pick and place
accuracy under very high speed in a simple structure. The Adept
robots pioneered the direct-drive (without reduction gears) SCARC
robot to bring the accuracy and speed to a new level. This robot is
dedicated to interface the centrifuge and cell harvest modules and
a special end-effector is fabricated to load and unload vacutainer
CPT tubes to and from a centrifuge rotor.
[0126] Modified Zymark (now Caliper Life Sciences) Sciclone ALH
3000 including the gantry robot) (Caliper Life Sciences, Hopkinton,
Mass.) This liquid handling system meets most of our needs but a
number of modifications are made. The width of the system is
increased from approximately 800 mm to 1100 mm to accommodate our
configuration incorporating a pick-and-place robot and plate
stacker within the working envelope of the gantry robot. The
two-robot configuration is designed in favor of task dedication and
thus high throughput. Another important modification is the
available EZ-swap dispense module (attached at the end of the
gantry robot) which is modified to enable pneumatically actuated
quick change of different end-effector modules. The liquid handling
portion of the system is also modified to handle the radiated and
control samples.
[0127] Allen Bradley (now Rockwell Automation) Programmable Logical
Controller (PLC) with accessories; two computers (Rockwell
Automation, Inc, Milwaukee, Wis., Dell Computer, Austin, Tex.) The
two computers host the motion control cards for the turn table/jack
mechanism and the punctuation unit, respectively. The PLC
implements the sequential control of the entire operation involving
all system components. The PLC interfaces with some components
directly such as the Adept SCARA robot, and with others via digital
input/out capabilities of their motion control cards residing in
the computers.
[0128] Pick-and-place robot, plate stackers and linear stage from
Matrix Tango Stacker system (without liquid handling subsystem)
(Matrix Technologies, Hudson, N.H.) These items are integrated
within the working envelope of the gantry liquid handling robot,
working in tandem. The linear stage holds 12 plates which are
sufficient. Additional stackers are included to provide total
capacity of 300 plates. The pick-and-place robot also interfaces
with the microscope for image acquisition.
[0129] Sony DXC-990 Industrial CCD camera, Schneider XENON 17 mm
lens, Mikrotron Inspecta frame grabber, (Sony Corporation,
Mikrotron GmbH, Germany) These items build part of the visual
servoing system for the cell harvest module.
[0130] Components to construct the punctuation unit (DC motor with
encoders and reduction gear, precision lead screw and ball-bearing
nut, precision rail and carriage, motion control card, amplifier
and power supply) (SDP-SI, New Hyde Park, N.Y., ORMEC Systems Corp.
Rochester, N.Y., McMaster-Carr, New Brunswick, N.J.) These items
complete the visual servoing system for the cell harvest
module.
[0131] OEM barcode printing and applying components from VCode by
Velocity 11, barcode reader, date acquisition and control (DAC)
board and monitoring software computer (Velocity 11, Menlo Park,
Calif., Symbol Technologies, Holtsville, N.Y., National
Instruments, Austin, Tex., Dell Computer, Austin, Tex.) The OEM
components are integrated with the plate stackers and
pick-and-place robot for barcode label applying on microplates for
identification and tracking by the barcode reader. The DAC board
has multi-channel analog-to-digital converter which collects the
current signals from all the actuators in the entire system to
monitor any overshooting as sign of trouble spots. The on-board
digital-to-analog converters connect to the actuator circuits for
emergency stops. Both barcode card and DAC board reside in a
computer.
[0132] Three Sorvall rotors with bundled swing bucket; accessories
(Kendro Laboratory Products, Asheville, N.C.) The rotors will be
custom modified to have special swing buckets for capillary tubes.
Each bucket holds multiple capillary tubes.
[0133] Additional Adept Cobra i800 SCARA robot and accessories
(Adept Technology, Inc., Livermore, Calif.) Together with the SCARA
robot, it functions for the following tasks: 1) transferring
capillary tubes from centrifuge to the tube feeder and 2)
transferring cell plug to the flushing system after tube cutting.
The two robots are dedicated to these tasks with custom designed
and fabricated end-effectors.
[0134] Components to construct the tube feeder (feeding tray,
loading unit, monitoring unit, etc.) (Hoppmann Corporation,
Elkwood, Va., SDP-SI, New Hyde Park, N.Y., McMaster-Carr, New
Brunswick, N.J.) These items build part of the tube feeders for
feeding the capillary tubes into the pneumatic transportation
systems. Additional components and supports are fabricated as
needed. Two sets of feeders are needed for the second embodiment of
the device: one for samples subject to irradiation, another for
control samples. The tube feeders are designed to have the ability
of feeding multiple transportation systems simultaneously.
[0135] Components to construct the pneumatic system for capillary
tube transportation (portable compressed air supplier, air dryer
and filter, transportation pipe network, valves, regulators,
control board, etc.) (Parker Air & Fuel Division, Irvine,
Calif., McMaster-Carr, New Brunswick, N.J.) These items build part
of the pneumatic transportation system for the centrifuged
capillary tubes. Additional components such as fixtures and
brackets are fabricated as needed.
[0136] Sony DXC-990 Industrial CCD camera, Schneider XENON 17 mm
lens, Mikrotron Inspecta frame grabber, (Sony Corporation,
Mikrotron GmbH, Germany) These items build part of the visual
servoing systems for the visual servoed cutting unit and
irradiation unit, respectively.
[0137] Components to construct the visual servoed irradiation unit
(mechanical barrier, motor, rail, carriage, motion control card,
etc., irradiation source not included) (SDP-SI, New Hyde Park,
N.Y., ORMEC Systems Corp. Rochester, N.Y., McMaster-Carr, New
Brunswick, N.J.) These items build part of the visual servoed
irradiation unit. Additional components including the irradiation
source are fabricated as needed.
[0138] Components to construct the visual servoed cutting unit
(mechanical barrier, cutting tool, motor, rail, carriage, motion
control card, etc.) (SDP-SI, New Hyde Park, N.Y., ORMEC Systems
Corp. Rochester, N.Y., McMaster-Carr, New Brunswick, N.J.) These
items build part of the visual servoed cutting unit. Additional
components and supports are fabricated as needed.
[0139] Components to construct the cell plug flushing system
(liquid reservoir, motor, slides, etc) (Upchurch Scientific, Inc,
Oak Harbor, Wash., SDP-SI, New Hyde Park, N.Y., McMaster-Carr, New
Brunswick, N.J.) These items build part of the cell plug flushing
system for transferring harvested cells into the microplate.
Additional components such as fixtures and brackets are fabricated
as needed. Two sets of this flushing system are needed for the
second embodiment: one for samples subject to irradiation, another
for control samples.
[0140] Components to construct an additional pneumatic system for
capillary tube transportation (portable compressed air supplier,
air dryer and filter, transportation pipe network, regulators,
valves, control board, etc.) (Parker Air & Fuel Division,
Irvine, Calif., McMaster-Carr, New Brunswick, N.J.) These items
build part of the second pneumatic transportation system for the
centrifuged capillary tubes. Additional components such as fixtures
and brackets are fabricated as needed.
[0141] Sony DXC-990 Industrial CCD camera, Schneider XENON 17 mm
lens, Mikrotron Inspecta frame grabber, (Sony Corporation,
Mikrotron GmbH, Germany) These items build part of the visual
servoing system for an additional irradiation unit.
[0142] Components to construct an additional visual servoed
irradiation unit (mechanical barrier, motor, rail, carriage, motion
control card, etc., irradiation source not included) (SDP-SI, New
Hyde Park, N.Y., ORMEC Systems Corp. Rochester, N.Y.,
McMaster-Carr, New Brunswick, N.J.) These items build part of the
second visual servoed irradiation unit. Additional components
including the irradiation source are fabricated as needed.
[0143] Components to upgrade the liquid handling system and
quick-change end-effectors with .mu.l scale ability and
accessories, (Caliper Life Sciences, Hopkinton, Mass.) The liquid
handling tasks in the first embodiment are mostly in ml scale. For
the second embodiment, the accuracy of the liquid handling system
is improved to handle liquid at the .mu.l-level. Both liquid
handling end-effectors and liquid moving subsystems are
upgraded.
[0144] Sony DXC-990 Industrial CCD camera, Schneider XENON 17 mm
lens, Mikrotron Inspecta frame grabber, (Sony Corporation,
Mikrotron GmbH, Germany) These items build part of the visual
servoing system for an additional cutting unit.
[0145] Components to construct an additional visual servoed cutting
unit (mechanical barrier, cutting tool, motor, rail, carriage,
motion control card, etc.) (SDP-SI, New Hyde Park, N.Y., ORMEC
Systems Corp. Rochester, N.Y., McMaster-Carr, New Brunswick, N.J.)
These items build part of the second visual servoed cutting unit.
Additional components and supports are fabricated as needed.
[0146] Components to construct an additional cell plug flushing
system (liquid reservoir, motor, slides, etc.) (Upchurch
Scientific, Inc, Oak Harbor, Wash., SDP-SI, New Hyde Park, N.Y.,
ORMEC Systems Corp. Rochester, N.Y., McMaster-Carr, New Brunswick,
N.J.) These items build part of the second cell plug flushing
system for transferring harvested cells into the microplate.
Additional components such as fixtures and brackets will be
designed are fabricated as needed.
[0147] Computers (precision work station), (Dell, Inc, Round Rock,
Tex.) In order to meet the high throughput requirement, multiple
precision work stations with up-to-date configuration are used in
image analysis tasks. These workstations implement Linux cluster
for parallel computing.
[0148] Other useful materials and supplies for using the device
include, for example, sterile plastic ware, tissue culture flasks,
cell culture media, mitogens, growth supplements, micropipette
tips, filtering units, centrifuge tubes, blood and sterile sample
collection materials, gloves, masks incubator, gases, laboratory
waste disposal containers, liquid nitrogen micropipettors, sterile
tips, cell substrate films, glassware, sterile 96 well plates with
filtration capacity, vacuum manifolds and vacuum system components,
small machine parts, metals and machine shop supplies, electronic
components suitable for integration into small devices, microscopy
supplies, fluorescent stains, fixatives, optimized Chroma
fluorescence filters, calibration kits, small refrigerators, water
baths, washing and cleaning supplies.
[0149] Stainless steel and aluminum alloy materials are used for
fabricating structures and parts for the turn table and jack
mechanism, for the cell harvest module, and for the modification of
the liquid handling system, machine shop, components to modify the
liquid handling system including regulators, pumps, syringes,
valves, and tubing, design and simulation software maintenance, and
other consumables.
[0150] Stainless steel and aluminum alloy materials are used for
fabricating structures and parts for the additional three units of
visual servoing and punctuation units for the cell harvest module,
and for the quick-change end-effector stations, for integrating the
OEM barcode components with the plate stackers and pick-and-place
robot, machine shop, components to modify the liquid handling
system including regulators, pumps, valves, and tubing, design and
simulation software maintenance, and other consumables
[0151] Stainless steel and aluminum alloy materials are used for
fabricating structures and parts for the pneumatic transportation
system, the visual servoed cutting unit, the visual servoed
irradiation unit, the capillary tube feeder, the cell plug flushing
system and for the modification of centrifuge rotors.
[0152] Components to build the liquid handling unit for cell plug
flushing system include regulators, pumps, syringes, valves, and
tubing, design and simulation software maintenance, and other
commercially available components.
[0153] Stainless steel and aluminum alloy materials are used for
fabricating structures and parts for the additional set of
pneumatic transportation system and additional visual servoed
irradiation unit.
[0154] Also used are auxiliary components to modify the liquid
handling system to adapt to the new end-effectors, design and
simulation software maintenance, and other commercially available
components.
[0155] Stainless steel and aluminum alloy materials are used for
fabricating structures and parts for the additional set of visual
servoed cutting unit and additional cell plug flushing system.
[0156] Also used are auxiliary components to build the liquid
handling unit for cell plug flushing system, design and simulation
software maintenance, and other commercially available
components.
[0157] In some embodiments, the automated device described herein
includes of 1) a centrifuge module; 2) a cell recognition/harvest
module, with a lymphocyte/monocyte pre-screening component, 3) a
mini-irradiator module; 4) a plate handling/liquid handling module;
5) an incubator and 6) an image acquisition/processing module.
[0158] The advantages and limitations of various known
biodosimeters are described in (Amundson S A, Bittner M, Meltzer P,
Trent J, Formace A J, Jr. Biological indicators for the
identification of ionizing radiation exposure in humans. Expert Rev
Mol Diagn 2001; 1:211-9). In choosing the biodosimeters for the
embodiments of the invention disclosed herein, criteria considered
were 1) whether a reasonable dose range was covered; 2) whether a
reasonable range of time-since-exposure was covered; 3) sensitivity
and specificity; 4) the extent to which the system has been
characterized in the literature for low throughput studies; 5)
whether the assay is amenable to high-throughput robotically-based
automation; 6) the invasiveness of the assay (at most minimally
invasive, ideally non invasive); and 7) whether the selected
endpoints could share a common platform.
[0159] In light of these considerations, the potential tissues are
lymphocytes or reticuloctyes in blood, and exfoliated buccal cells
from the cheek or exfoliated bladder cells from urine.
Correspondingly, the potential endpoints are micronuclei and
.gamma.-H2AX foci. These endpoints satisfy the above criteria and
share a common multi-well based in-situ scanning platform.
Accordingly, assay protocols have been optimized for application to
fully-automated, high-throughput, multi-well in-situ assays,
for:
[0160] a. micronucleus yields in lymphocytes;
[0161] b. .gamma.-H2AX yields in lymphocytes;
[0162] c. micronucleus yields in blood reticuloctyes; and/or
[0163] d. micronucleus yields in exfoliated bladder cells from
urine, or exfoliated buccal cells.
[0164] System optimization is one using ex-vivo irradiated samples
from healthy human volunteers. Calibration and testing is achieved
using samples from adult and pediatric patients who were subject to
total body irradiation.
[0165] Micronuclei in lymphocytes: This is a well-characterized
endpoint (M. Fenech and A. A. Morley, Measurement of Micronuclei in
Lymphocytes. Mutation Research, 1985. 147(1-2): p. 29-36; Goans R
E, Holloway E C, Berger M E, Ricks R C. Early dose assessment in
criticality accidents. Health Phys 2001; 81:446-9; Goans R E,
Holloway E C, Berger M E, Ricks R C. Early dose assessment
following severe radiation accidents. Health Phys 1997; 72:513-8.)
for radiation dosimetry, and has been used for peripheral blood
(Amundson S A, Bittner M, Meltzer P, Trent J, Formace A J, Jr.
Biological indicators for the identification of ionizing radiation
exposure in humans. Expert Rev Mol Diagn 2001; 1:211-9; Nakamura N,
Miyazawa C, Sawada S, Akiyama M, Awa A A. A close correlation
between electron spin resonance (ESR) dosimetry from tooth enamel
and cytogenetic dosimetry from lymphocytes of Hiroshima atomic-bomb
survivors. Int J Radiat Biol 1998; 73:619-27) and fingerstick
capillary blood (Langlois R G, Nisbet B A, Bigbee W L, Ridinger D
N, Jensen R H. An improved flow cytometric assay for somatic
mutations at the glycophorin A locus in humans. Cytometry 1990;
11:513-21; Prasanna P G, Blakely W F. Premature chromosome
condensation in human resting peripheral blood lymphocytes for
chromosome aberration analysis using specific whole-chromosome DNA
hybridization probes. Methods Mol Biol 2005; 291:49-57). It has
good dose coverage (at least 0.5 to 5 Gy), and the biomarker
remains stable for many weeks post exposure. A disadvantage is that
the lymphocytes need to be cultured, a process which takes
.about.72 hours; however, as described herein, the process can be
made fully automatic. A major advantage of the system is that the
radiation specificity of the assay is excellent. Cellular
proliferation and the scoring of micronuclei/nucleoplasmic bridges
in bi-nucleate cells ensures that what is scored reflects damage to
circulating lymphocytes, as opposed to the background level of
micronuclei present in mono-nuclear lymphocytes. In addition, the
system is amenable to high-throughput automation; in fact, there
have been several attempts at partial automation (Hande M P,
Azizova T V, Geard C R, Burak L E, Mitchell C R, Khokhryakov V F,
et al. Past exposure to densely ionizing radiation leaves a unique
permanent signature in the genome. Am J Hum Genet 2003; 72:1162-70;
Golub T R, Slonim D K, Tamayo P, Huard C, Gaasenbeek M, Mesirov J
P, et al. Molecular classification of cancer: class discovery and
class prediction by gene expression monitoring. Science 1999;
286:531-7; Bittner M, Meltzer P, Chen Y, Jiang Y, Seftor E, Hendrix
M, et al. Molecular classification of cutaneous malignant melanoma
by gene expression profiling. Nature 2000; 406:536-40.), including
the commercially available Metafer system (described below; Golub T
R, Slonim D K, Tamayo P, Huard C, Gaasenbeek M, Mesirov J P, et al.
Molecular classification of cancer: class discovery and class
prediction by gene expression monitoring. Science 1999; 286:531-7).
However the throughput of such systems is at most a few hundred
samples per day, far below what is targeted for high throughput as
described herein.
[0166] .gamma.-H2AX foci in lymphocytes: William Bonner and
colleagues at the NCl were the first to point out (Amundson S A,
Bittner M, Formace A J, Jr. Functional genomics as a window on
radiation stress signaling. Oncogene 2003; 22:5828-33) that
phosphorylation of the histone H2AX (known as .gamma.-H2AX) occurs
at sites of DNA double-strand breaks (DSB). .gamma.-H2AX can be
measured with an antibody raised to the phosphorylated C-terminal
peptide of H2AX, and can be detected with excellent sensitivity
using both flow and in-situ image analysis (Amundson S A, Formace A
J, Jr. Monitoring human radiation exposure by gene expression
profiling: possibilities and pitfalls. Health Phys 2003; 85:36-42;
Formace A J, Jr., Amundson S A, Do K T, Meltzer P, Trent J, Bittner
M. Stress-gene induction by low-dose gamma irradiation. Mil Med
2002; 167:13-5.). Because ionizing radiation is an efficient
inducer of DSB, most the early research on .gamma.-H2AX has been
done with ionizing radiation (Amundson S A, Formace A J, Jr.
Monitoring human radiation exposure by gene expression profiling:
possibilities and pitfalls. Health Phys 2003; 85:36-42; Formace A
J, Jr., Amundson S A, Do K T, Meltzer P, Trent J, Bittner M.
Stress-gene induction by low-dose gamma irradiation. Mil Med 2002;
167:13-5.). The fraction of H2AX that is phosphorylated is
proportional to the number of induced DSB, with about 0.03% of the
H2AX becoming phosphorylated per DSB. A normal human lymphocyte
contains about 6.times.106 H2AX molecules, so about 2000 H2AX
molecules are phosphorylated per DSB, indicating that the signal is
highly amplified (Amundson S A, Bittner M, Formace A J, Jr.
Functional genomics as a window on radiation stress signaling.
Oncogene 2003; 22:5828-33.). The .gamma.-H2AX system well
complements the micronucleus system as a radiation biodosimeter
(Amundson S A, Formace A J, Jr. Gene expression profiles for
monitoring radiation exposure. Radiat Prot Dosimetry 2001;
97:11-6.) because a) cells do not have to be cultured for the
assay, b) the high-sensitivity and automation potential of the
antibody-based assay, c) the .gamma.-H2AX foci appear with their
maximum value within about 30 minutes of irradiation (Amundson S A,
Bittner M, Formace A J, Jr. Functional genomics as a window on
radiation stress signaling. Oncogene 2003; 22:5828-33.), and d) DSB
and thus .gamma.-H2AX are formed linearly with dose from very low
to extremely high (>10 Gy) doses (Amundson S A, Formace A J, Jr.
Monitoring human radiation exposure by gene expression profiling:
possibilities and pitfalls. Health Phys 2003; 85:36-42.). The
disadvantage of the system as a radiation biodosimeter is the
decrease of the signal with time after exposure. From the data
available to this point, it seems clear that the assay is practical
at least up to 24 hours post exposure (Amundson S A, Bittner M,
Meltzer P, Trent J, Formace A J, Jr. Induction of gene expression
as a monitor of exposure to ionizing radiation. Radiat Res 2001;
156:657-61). Studies for .gamma.-H2AX foci in human lymphocytes are
provided in examples below.
[0167] Micronuclei in blood reticuloctyes: During the formative
stages of red blood cell production, chromosomal damage can
manifest in the form of micronuclei. In humans these cells are
generally rapidly cleared in the spleen, but an increased yield of
micronucleated reticulocytes provides an independent means of
assessing radiation exposure over a relatively short (1-3 days)
interval post exposure (Ward B C, Childress J R, Jessup G L, Jr.,
Lappenbusch W L. Radiation mortality in the Chinese hamster,
Cricetulus griseus, in relation to age. Radiat Res 1972;
51:599-607., Smolewski P, Ruan Q, Vellon L, Darzynkiewicz Z.
Micronuclei assay by laser scanning cytometry. Cytometry 2001;
45:19-26., Grace M B, McLeland C B, Gagliardi S J, Smith J M,
Jackson W E, 3rd, Blakely W F. Development and assessment of a
quantitative reverse transcription-PCR assay for simultaneous
measurement of four amplicons. Clin Chem 2003; 49:1467-75; Evans H
J, Neary G J, Williamson F S. The relative biological efficiency of
single doses of fast neutrons and gamma-rays on Vicia faba roots
and the effect of oxygen. Part II. Chromosome damage: the
production of micronuclei. Int J Radiat Biol 1959; 1:216-29; Fenech
M, Holland N, Chang W P, Zeiger E, Bonassi S. The HUman
MicroNucleus Project--An international collaborative study on the
use of the micronucleus technique for measuring DNA damage in
humans. Mutat Res 1999; 428:271-83; Liu R H, Yang J, Lenigk R,
Bonanno J, Grodzinski P. Self-contained, fully integrated biochip
for sample preparation, polymerase chain reaction amplification,
and DNA microarray detection. Anal Chem 2004; 76:1824-31). The
assay reflects damage to cells in the bone marrow, whereas
micronuclei in lymphocytes predominantly reflect damage in the
peripheral circulation. These reticulocytes do not need to be
cultured, as do the lymphocytes, and thus can provide a "same-day
biodosimeter". A gradient-based separation technique can be used
for the blood samples, whereby the reticulocytes are separated.
Accordingly, the reticulocyte assay can share a single sample with
the lymphocyte assays, increasing the information potential of a
given sample.
[0168] Micronuclei in exfoliated buccal or urinary bladder cells:
Cells in renewable tissues outlive their usefulness and are
discarded and replaced continuously. Following exposure to ionizing
radiation during their proliferative pre-differentiation phase,
such cells are exfoliated from their epithelial origin and may
manifest the consequences of chromosomal damage in the form of
micronuclei (Liu R H, Yang J, Lenigk R, Bonanno J, Zenhausern F,
Grodzinski P. Fully integrated microfluidic biochips for DNA
analysis. Int J Comput Eng Sci 2003; 4:145-50; Rogakou E P, Pilch D
R, Orr A H, Ivanova V S, Bonner W M. DNA double-stranded breaks
induce histone H2AX phosphorylation on serine 139. J Biol Chem
1998; 273:5858-68.). Exfoliated cells from the bladder appear in
urine while buccal cells can be collected from the lining of the
oral cavity. These exfoliated cells provide an indication of
exposure to chromosome damaging agents in the form of enhanced
frequencies of micronucleated cells (Pilch D R, Sedelnikova O A,
Redon C, Celeste A, Nussenzweig A, Bonner W M. Characteristics of
gamma-H2AX foci at DNA double-strand breaks sites. Biochem Cell
Biol 2003; 81:123-9; MacPhail S H, Banath. J P, Yu T Y, Chu E H,
Lambur H, Olive P L. Expression of phosphorylated histone H2AX in
cultured cell lines following exposure to X-rays. Int J Radiat Biol
2003; 79:351-8; Wolfram R M, Budinsky A C, Palumbo B, Palumbo R,
Sinzinger H. Radioiodine therapy induces dose-dependent in vivo
oxidation injury: evidence by increased isoprostane 8-epi-PGF(2
alpha). J Nucl Med 2002; 43:1254-8). Here again these cells are not
capable of progressing through the cell-cycle and thus have the
potential to provide a "same-day" radiation biodosimeter.
[0169] An overview of the automated processing steps begins with
loading blood samples into a centrifuge, which will isolate the
cell layer (e.g. lymphocyte) of interest. Next, the cells of
interest are transferred from the centrifuge to an incubator. In
transit, half of each sample receives a prescribed radiation
exposure, providing a positive control. Within the incubator, the
samples are deposited into wells on multiwell plates. A series of
automated liquid processing steps follow, before the cells are
passed on to an imaging stage. Samples are imaged with a unique
steered-image compound microscope specifically designed for high
throughput, through the use of fast sensors and by minimizing
mechanical stage motions. High speed image analysis routines
analyze the images for various indicators of radiation
exposure.
[0170] In some embodiments, the device also analyzes other tissues,
such as buccal cells and exfoliated bladder cells from urine.
[0171] Five basic Modules are summarized below, and illustrated in
FIG. 1. It is noted that specific details of the Modules may vary
in different embodiments. The Modules include: a centrifuge module
1, a cell recognition/harvest module 2, with a lymphocyte/monocyte
pre-screening system; a mini-irradiator module 3; a plate
handling/liquid handling module 4; and an image
acquisition/processing module 5.
[0172] The centrifuge module 1 and cell recognition/harvest module
2 are each served by a turntable-based transport system 6 and an
SCARA robot 7. Modules 3-5 are served by a gantry robot 8 and a
pick-and-place robot 9. Samples are separated in one or more
centrifuges 10 which are mounted on the turntable-based transport
system 6. Preferably, multiple centrifuges 10 operating on the
turntable-based transport system 6 allow multiple batches of blood
samples to simultaneously undergo centrifugation, while at the same
time, a processed batch is conveniently harvested, removed, and
replaced by new blood samples.
[0173] When a centrifuge 10 stops, the rotor (not shown) is lifted
into the cell recognition/harvest module 2. Two or more parallel
CCD camera systems 11 precisely determine the position and
thickness of the separated lymphocyte (or other cell type) layer.
In the embodiment illustrated in FIG. 1, four parallel CCD cameras
11 are shown. The thickness of the lymphocyte (or other cell type)
layer acts as a pre-screening device to immediately identify
individuals who received extremely high doses.
[0174] Next, guided by the CCD cameras 11, a visual servoed
punctuator unit 12 precisely harvests the lymphocytes (or other
cells). Half of the sample is then passed through the
mini-irradiator module 3, the other half is not, and the two
samples are deposited in adjacent wells in a multi-well plate
inside the plate handling/liquid handling module 4. Here, three
quickchange end effectors 13 are designed to perform various liquid
handling steps in the wells, before the cell samples proceed to the
image acquisition/processing module 5.
[0175] Certain differences between a first embodiment and a second
embodiment of the devices are dictated by the available blood
sample volume, and the target throughput. In the second embodiment
of the device, cell samples will be transported to the wells (one
via the irradiator, the other not) while still in their capillary
tubes, at which point the tubes will be cut, the unwanted segments
discarded, and the desired cells flushed into appropriate
wells.
[0176] In one embodiment, illustrated in FIGS. 2 and 3, the system
is designed to process blood samples collected into bar-coded
plastic capillary tubes 31 at the emergency site using a finger or
heel stick. The capillaries are then transported in inserts 32 to
the biodosimetry workstation (FIG. 2). After having been filled
with inserts, centrifuge bucket 33 is loaded into the automation
system by the operator 21 through a safety barrier 22. At this
point the bucket 33 is handled by a robot gripper, 34 of service
robot 23, as illustrated. Samples follow an unmanned series of
operations that automate multiple biological assays in order to
assess the radiation exposure. In some embodiments, a first assay
is effective during the first 1-2 days, post irradiation, and a
second assay is effective at longer times.
[0177] In some embodiments, blood samples enter the automation
system via an input module 24. The service robot 23 moves the
centrifuge bucket(s) 33 from the input module 24 to the centrifuge
25. Referring now to FIG. 4, the centrifuge bucket is loaded into
the centrifuge at step 41. After separation of white blood cells
(WBC) and red blood cells (RBC), the centrifuged samples are
unloaded 41 and transferred 42 to the cell-harvesting module 26 by
the service robot 23 using the robot gripper 34. Using a capillary
gripper (not shown) that prevents the lymphocyte band from being
disrupted, each capillary 31 is removed from the insert 32 and the
samples are then identified one by one using a barcode-reader (not
shown). Upon completion of the identification, the capillary 31 is
imaged 44 by a CCD system in order to detect the separation layer
between RBC and the rest of the sample. Then a laser system is
triggered to cut the capillary thus separating the sample into two
parts one of which is discarded 43, namely the one with RBC.
However, before performing the cut, the sample is imaged by the CCD
system also for outputting an early assessment of radiation
exposure. Referring now to FIG. 6, the thickness of the lymphocyte
band is measured 61 and an alarm signal is given 62 in output if
the value is low.
[0178] One embodiment of an input module 24 is shown in FIG. 7. The
input module 24, in this embodiment, can handle four centrifuge
buckets 33 at the pick location of the service robot 23.
[0179] In this embodiment, four centrifuge buckets are filled with
three inserts, each carrying 44 capillaries, for a total batch of
528 capillaries per centrifugation cycle. When the centrifugation
ends, the service robot 23 transfers the empty centrifuge buckets
back onto a stage 71 of the input module 24 after capillaries have
been transferred to the cell-harvesting module. The stage 71 is
also responsible for simultaneously i) moving the used centrifuge
buckets (without capillaries) out of the system and ii) introducing
a new set of centrifuge buckets (filled with capillaries) by
performing a 180.degree. rotation. This ensures continuity of the
input to the automatic system. The input module 24 serves as a
point of interface with the human user 21 who is separated by the
automation system by a safety barrier 72.
[0180] The service robot (for example, model RS80 SCARA from
Staubli) is responsible for moving the centrifuge buckets 33, one
by one, from the input stage 71 to the centrifuge module 1. As
shown in FIG. 8a, the arm 81 of the service robot 23 can be fitted
with a custom-made link endowed with two custom grippers: a
capillary gripper 82 and a gripper 83 for handling of buckets and
microplates. The capillary gripper 82, as shown in FIGS. 8b and 8c
can be composed of a passive spring-plunger-collet unit 84 and of
an active gear-motor-shaft unit 85. The former is preferably
adapted to grip the capillary 31 without the use of any motor. The
latter is responsible for the rotation of the capillary 31 during
cutting in order to guarantee an even distribution of the power,
thus minimizing thermal effects and contamination generated by the
laser-based cutting.
[0181] In one embodiment, the bucket/microplate gripper 83 is
composed of a pneumatically-actuated two-jaw unit and two miniature
self-contained photoelectric sensors (not shown), for example two
06 38F from Banner Engineering Corp. Each jaw is composed of two
sections: one for gripping the bucket 33 and one for gripping a
microplate. In a preferred embodiment, the former is a custom-made
jaw that seats into the side slots of the bucket when grip takes
place. The latter is a rubber-padded jaw that grips
microplates.
[0182] The centrifuge 25 is equipped with an electromechanical
clutch that locks the centrifuge rotor in place after it stops
rotating. Optical sensors (not shown) detect the centrifuge rotor
arm in order to provide a reference to the bucket/microplate
gripper 83 when loading bucket 33 in centrifuge 25. The gripper 81
is preferably modular and lightweight.
[0183] In one embodiment, the gripper 81 employs a hollow structure
allowing for the passage of both pneumatic and electrical lines.
The link length can be easily changed without having to change the
mechanical interface with service robot 23. The two grippers,
capillary gripper 82 and bucket/microplate gripper 83, can be
mounted independently on a flange of service robot 23. When a
capillary 31 is gripped, the collet 85 slides onto the capillary
31. In one embodiment a built-in linear actuator of the service
robot 23 performs this operation as a vertical move. The gripping
operation ends when the capillary 31 comes into contact with the
tip of the plunger 86. The plunger 86 prevents loss of lymphocytes
after capillary 31 is cut. The plunger 31 is also provided with
air-conductive channels that allow dispensing positive pressure,
for example, compressed air 87, for transferring lymphocytes into
the microplate well after the capillary 31 has been cut, i.e. after
RBC have been removed.
[0184] After centrifugation, centrifuge buckets 33 are transferred
to the cell-harvesting module 26 by the service robot 23 using the
bucket gripper 83. The cell-harvesting module 26 obtains the
lymphocytes from centrifuged blood samples.
[0185] In the embodiment illustrated in FIG. 9, the cell-harvesting
module consists of a laser system with a galvo head 91, a barcode
reader 92, for example, a Hawkeye 1525 from RVSI, an image sensor
93, and a holder for microplates and centrifuge buckets. The image
sensor 93 provides for image segmentation of a capillary 31, (image
segmentation of a capillary is illustrated in illustrated in FIG.
10), and a custom-made holder for microplates 94 and centrifuge
buckets 33. In one embodiment, the laser system is an Osprey UV
laser system from Quantronix. In some embodiments, the image sensor
can be a CCD camera, such as a SONY XCL-U1000 or a CS3970CL from
Toshiba-Tell, and the like. In some embodiments, the holder can
host three stacks of twenty-one microplates 94, four centrifuge
buckets 33, a microplate reference location, where the wells can be
filled with lymphocytes, and a gravity-based capillary disposal
unit 95.
[0186] The barcode reader 92 identifies the capillary 31
immediately before imaging by the image sensor 93. (In one
embodiment, the capillaries 31 have been registered at the
collection site.) The barcode reader 92 allows tracking of each
sample after it has been transferred to a pre-id microplate. In one
embodiment, shown in FIG. 12, the holder 121 supports i) three
microplate stacks 122, each made of 21 microplates 94, ii) four
centrifuge buckets 33, iii) a microplate reference location 123,
where the wells are filled with lymphocytes, and iv) a
gravity-based disposal unit 95.
[0187] The inputs to the cell harvesting module are centrifuged
capillaries 31 in buckets 33 and sterile automation-compliant
microplates 94. The outputs of the module are cut capillaries,
which are disposed, and microplates 94 containing lymphocytes
transferred from capillaries. In some embodiments, two
software-outputs are the data associated with the barcode-based
identification of the capillaries and the lymphocyte thickness
estimation.
[0188] In one embodiment, the operation of the cell harvesting
module is as follows: The bucket/microplate gripper 83 transfers a
multi-well microplate 94 from a stack 122 to the reference location
123. The capillary gripper 82 is then deployed to service each
capillary 31. The capillary 31 is moved in the field of view of the
barcode reader 92 for identification. After reading, a vertical
move is performed by the service robot 23 and the capillary 31 is
moved in the field of view of an image sensor 93 for detection of
the separation band between RBC and the rest of the sample. Upon
detection of the band, a laser performs a cut while the capillary
31 is rotated by the rotary stage of the capillary gripper 82. In
some embodiments, an estimation of the lymphocyte band thickness is
also performed during imaging using the same machine vision
system.
[0189] Upon cutting the capillary, the RBC-containing portion is
disposed into a disposal unit 95 by means of gravity. In one
embodiment, the cut capillary, containing lymphocytes and plasma,
is moved above the well of the microplate in the reference location
where lymphocytes are dispensed using the capillary gripper 82. The
cut capillary is then disposed into a disposal container 95. During
this operation, the service robot 23 moves downward while the
collet 85 moves upward until the inner wall of the collet 85 is in
contact with the outer surface of the capillary 31. When the
foregoing contact ends, the capillary falls under gravity. The
capillary will then be disposed into a waste container 95. Once a
multi-well plate is fully harvested the service robot transfers it
to the liquid handling module 27.
[0190] The liquid handling module 27, illustrated in FIGS. 13, 35,
38, and 39 preferably provides for the automation of both
micronuclei and .gamma.-H2AX assays for lymphocytes. In some
embodiments, this is accomplished by a sequence of washes and
reagent addition. In the case of a system designed for a throughput
of 6,000 samples per day, using 96-well plates, with an assay
duration time of approximately 72 hours, the incubator should be
capable of storing at least 189 microplates simultaneously. In one
embodiment, a robotic incubator capable of simultaneously hosting
220 microplates is used, namely the Liconic STX220. The system is
integrated with the liquid handling module as shown in FIG. 14. For
example, while running the micronucleus protocol, the service robot
23 places a microplate 94 in the lower left position on the deck of
a liquid handling robot (not shown). After a wash and the addition
of culture medium the microplate 94 is transferred to the incubator
28. The microplate 94 is then transferred back to the liquid
handling robot for the addition of Cytochalasin-B. The microplate
is then transferred into the incubator for the last incubation
cycle. After the latter the protocol continues in the liquid
handling module. In the case of .gamma.-H2AX protocol, the initial
operation consists of moving the microplate to the lower left
position of the liquid handling robot. After a wash and the
addition of the permealization buffer, the microplate 94 is
transferred to a FIFO stacker (not shown) for 20 minutes. The
microplate 94 then moves back to the liquid-handling robot where
blocking reagents are added. The microplate 94 is then transferred
back on to the FIFO stacker for other 30 minutes and then returns
to the liquid-handling robot deck. After, the latter the protocol
continues on the liquid handling robot.
[0191] In one embodiment, a fully-integrated liquid handling
module, such as the commercially available Sciclone ALH 3000, is
composed of a gantry system, an ultrasonic wash-station, a
bulk-dispenser, a positive pressure unit, a filter-to-waste unit, a
fixed-cannula array and a microplate gripper. The gantry system
moves the microplate gripper, the fixed-cannula array (or the
positive pressure unit) and the bulk dispenser at one of the
specified microplate locations on the operation deck. The
filter-to-waste unit collects the result of the well washes. The
ultrasonic wash-station guarantees avoidance of reagent mixing by
washing the metallic fixed-cannula array tips before changing
reagent. The microplate gripper moves the plates or/and their lids
across the deck. The bulk reagent dispenser is capable of
continuously dispensing 10-2,500 ml of a single reagent
simultaneously in eight wells with a coefficient of variation less
than 2-3%.
[0192] The fixed-cannula array guarantees the same coefficient of
variation and it is capable of dispensing up to 25 .mu.l of a
reagent simultaneously in 96 wells. The absence of disposable tips
is intentional in the design of the system, as relying on this type
of consumables could hamper the use of the disclosed system in an
emergency condition. The use of metallic tips makes the system
operation independent on the availability of disposable tips.
[0193] Centrifuge Module: In a preferred embodiment, separation of
human lymphocytes or reticuloctyes is accomplished using the Ficoll
Hypaque density-gradient method. Boyum A. Isolation of mononuclear
cells and granulocytes from human blood. Isolation of monuclear
cells by one centrifugation, and of granulocytes by combining
centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest
Suppl 1968; 97:77-89. In its standard form, the technique employs a
liquid density gradient medium of Ficoll 400 and sodium metrizoate
or sodium diatrizoate solution; this standard procedure uses
anticoagulated blood, which is diluted with a buffered solution,
and then layered onto the medium. This preparation is then
centrifuged to isolate mononuclear cells above the medium, and the
cells harvested by pipetting from the liquid interface.
[0194] For the lymphocyte separation, the assays described herein
can function with 50 .mu.l of whole blood. In one embodiment, the
blood can be transferred into a tube by capillary action, followed
by 50 .mu.l of the lymphocyte separating medium, and centrifuged at
a speed of 40 g for 20 minutes at temperatures between 4.degree. C.
and 37.degree. C. This yielded a good separation of the lymphocytes
in the form of a clearly visible white band of lymphocytes with a
count of 2100/.mu.l of blood and about and 80% purity. The
lymphocyte separating medium, which may be, for example, Ficoll
Hypaque, with a density of 1.114 g/ml, which yielded better counts
of lymphocytes and sharper bands upon separation as compared to a
separation medium with a lower density (1.077 g/ml).
[0195] In one embodiment, the system is designed to use a two-speed
configuration protocol, equivalent to an 8-minute centrifugation at
40 g followed by a 3-minute centrifugation at 160 g.
[0196] In one embodiment the system is designed to use a
centrifugation speed of about 13,000 g and a centrifugation time of
5 minutes.
[0197] In one embodiment, depicted in FIG. 19, peripheral
lymphocytes are separated from whole blood and stimulated to induce
division. During division, the formation of cellular membrane is
blocked resulting in binucleate cells. Chromosomes damaged by
ionizing radiation lag in anaphase and will therefore not be
included in the daughter nuclei during division, resulting in a
small separate "micronucleus", as shown in FIG. 19b. The cells are
then fixed and stained and can be automatically scored.
[0198] A major advantage of the system is that the scoring of
micronuclei in bi-nucleate cells ensures that what is scored
reflects damage to circulating lymphocytes, as opposed to the
background level of micronuclei present in mono-nuclear
lymphocytes. Thus the radiation specificity of the assay is
excellent. This assay also has good dose coverage (at least 0.5 to
5 Gy), and the biomarker remains stable for months or even years
post exposure [Pellmar T C, Rockwell S, and the
Radiological/Nuclear Threat Countermeasures Working Group: Priority
list of research areas for radiological nuclear threat
countermeasures. Radiat Res 2005; 163:115-23]. A downside is that
the lymphocytes need to be cultured, a process which takes about 72
hours during which the cells need to be kept at controlled
temperature (37.degree. C.), CO2 level (5%) and at high humidity.
However, as described herein, the process can be made fully
automatic.
[0199] In an embodiment illustrated in FIG. 17, the procedure is
considerably simplified and improved through the introduction of BD
Vacutainer CPT tubes, which combine a blood collection tube 170
containing a sodium heparin anticoagulant, with a Ficoll Hypaque
density gradient liquid 171 and a polyester gel barrier 172 which
separates the two liquids. The blood separation media takes
advantage of the lower density of mononuclear cells and of
platelets to isolate them from whole blood. The isolation occurs
during centrifugation when the gel portion of the media moves to
form a barrier under the mononuclear cells and platelets,
separating them from the denser blood components below, as shown
here. The result is a very convenient, single tube system for the
collection of whole blood and the separation of mononuclear cells,
and which can be incorporated in a high-throughput robot-based
centrifuge module, described below.
[0200] In a preferred embodiment, a similar approach is used with
capillary tubes instead of vacutainer tubes. Several approaches
have been reported for separating blood in capillary tubes,
including the quantitative buffy coat approach (Levine R A, Hart A
H, Wardlaw S C. Quantitative buffy coat analysis of blood collected
from dogs, cats, and horses. J Am Vet Med Assoc 1986; 189:670-3;
Wardlaw S C, Levine R A. Quantitative buffy coat analysis. A new
laboratory tool functioning as a screening complete blood cell
count. Jama 1983; 249:617-20.), in which a float is put in the tube
to stretch out the linear dimensions of the separated components,
and density-gradient enrichment (Choy W N, MacGregor J T.
Density-gradient enrichment of newly-formed mouse erythrocytes.
Application to the micronucleus test. Mutat Res 1984; 130:159-64;
Kudoh E, Komatu T, Nakaji S, Sugawara K, Kumae T. Investigation of
a new method for separation of neutrophils from a small volume of
human blood. Nippon Eiseigaku Zasshi 1992; 47:650-7.). In using
automated optics to recognize the different bands (FIG. 46), it has
been discovered that a Ficoll Hypaque density gradient liquid
approach (as described above, but without the gel barrier), works
well with capillary tubes. As discussed below, bands of interest
are not drawn off by pipette, but rather the capillary tube is
robotically cut at the appropriate location, and the required band
simply flushed into the appropriate location in the multi-well
plate.
[0201] Multi-Rotor Centrifuge: In some embodiments, in order to
achieve the target throughput, three or more centrifuge rotors,
each with a holding capacity of 48 tubes, are used, as illustrated
in FIG. 20. These centrifuge rotors are operated in a cascading
manner to keep two rotors rotating at any given time and the third
one for cell harvest and loading/unloading of the tubes. The rotor
diameter is preferably about 0.5 meter with each tube in a slotted
bucket (the slot enables the visual servoing control of the cell
harvest process).
[0202] Each rotor in the centrifuge module preferably moves
continuously through a three position loop. From a first position,
the centrifugation process starts and proceeds for about 12
minutes. In a second position, another 12 minute spin is performed
and the rotor stopped. A third position is the cell recognition
position (see below). The rotor transporter is provides for
movement of the centrifuge rotors 201 between the positions as
described above. Preferably a turn-table based transport unit 202
is employed. Thus, a compact, rigid, and light weight device is
provided to permit smooth transport of heavy (about 26 kg)
centrifuge rotors, eliminate vibration in the cell
recognition/harvest module from the large rotating centrifuges, and
allow a closed loop feedback for the entire module to monitor the
status of the rotors. Preferably it prevents scenarios such as a
rotating centrifuge rotor being placed on the cell
recognition/harvest module.
[0203] Accordingly, some embodiments use a transport system
equipped with a turntable. FIG. 20 shows an embodiment of the
centrifuge and cell recognition/harvest modules, wherein three
rotor positions are arranged on a circle. In one embodiment, a
rotary turntable transfer rotors to the next position every 12
minutes--the spin time.
[0204] When a rotor finishes its centrifugation process and arrives
at the cell harvest position, a jack device, completely isolated
from the turn table 202 to eliminate vibration, is used to elevate
the rotor 201 to the level where the lymphocyte layer can be
conveniently monitored and harvested (see cell recognition/harvest
module, below). The jack includes a means of stabilizing the
buckets of the centrifuge during cell harvesting. The centrifuge
driving motor is used as an indexer during the harvesting stage.
For vibration isolation, the cell recognition/harvest module is
also placed on a working table that is isolated from the rotary
turntable. A pick-and-place robot 204 with three degrees of freedom
is used to remove used samples immediately after they are
harvested, and reload tubes containing fresh samples into the empty
buckets. After the cell harvest and loading/unloading processes are
finished, the rotor is transferred again by the turntable to the
first centrifuge position and a new batch started.
[0205] In one embodiment, the pick-and-place robot 204 comprises a
SCARA (Selective Compliance Assembly Robot Arm) robot for the
pick-and-place operations required for loading/unloading of the
tubes. The degrees of freedom needed for the tube manipulation here
are arm rotation, elbow rotation, and the vertical motion for the
end-effector which will grasp the tube. SCARA robots represent a
widely spread and relatively mature technology. Commercially
available SCARA robots include the EPSON B- and E-series, the
Intelligent Actuator IX series, the MekA-Nize Robotics MR series,
and the IBM 7500 series. Because of their unique "elbow" motions,
SCARA robots are ideal for applications which require fast,
repeatable and articulate point-to-point movements such as
loading/unloading, palletizing/de-palletizing and assembly. The
high throughput and high repeatability requirements of the tube
manipulation make the SCARA robot an ideal fit for this
application.
[0206] Cell Recognition/Harvest Module: The automated removal of
lymphocytes from the Vacutainer tubes represents a task that,
traditionally, is done by experienced personnel using pipetting
techniques. The robotic challenge largely arises from the
uncertainties that exist during this process. After centrifugation,
the whole blood sample is separated into multiple layers, and the
position and thickness of the lymphocyte layer separated by the
centrifugation process varies. The thickness of the lymphocyte
layer of course decreases as the sample is withdrawn. In order to
optimize lymphocyte harvesting, and to avoid taking cells from
other layers, it is desirable to always keep the needle tip around
the middle of the lymphocyte layer. This requires real-time
tracking of the harvest tool (a punctuator needle) and the liquid
interfaces. Therefore, for each tube, information about position
and thickness of the lymphocyte layer needs to be quickly acquired
and used as feedback to control the robotic manipulator during the
pipetting process. It is natural to use a CCD-based vision sensor
here, since it mimics the human sense of vision and allows for
non-contact measurement. Visual servoing with better than 100 .mu.m
precision is now a well-established robotic control technique,
integrating vision in feedback control loops (Georgiev A, Allen K
P, Mezouar Y. Microbiotic crystal mounting using computer vision.
In 2003 IEEE/RSJ International Conference on Intelligent Robots and
Systems: (IROS 2003): proceedings: Oct. 27-31, 2003, Las Vegas,
Nev. Piscataway, N.J.: IEEE; 2003. p. 4 v; Mezouar Y, Allen P K.
Visual servoed micropositioning for protein manipulation. In
Proceedings 2002 IEEE/RSJ International Conference on Intelligent
Robots and Systems: IROS 2002, Sep. 30-Oct. 4, 2002, EPFL,
Lausanne, Switzerland. Piscataway, N.J.: IEEE; 2002. p. 1766-77;
Dewan M, Marayong P, Okamura A M, Hager G D. Vision-based
assistance for ophthalmic microsurgery. Medical Image Computing and
Computer-Assisted Intervention--Miccai 2004, Pt 2, Proceedings
2004; 3217:49-57; Kumar R, Kapoor A, Taylor R H. Preliminary
experiments in robot/human cooperative microinjection. In 2003
IEEE/RSJ International Conference on Intelligent Robots and
Systems: (IROS 2003): proceedings: Oct. 27-31, 2003, Las Vegas,
Nev. Piscataway, N.J.: IEEE; 2003. p. 4 v; Lots J F, Lane D M,
Trucco E, Chaumette F. A 2-D visual servoing for underwater vehicle
station keeping. Proceedings of the 2001 IEEE Conference on
Robotics and Automation. Seoul, Korea; 2001. p. 2767-72).
[0207] In some embodiments, to achieve a system throughput of 6,000
samples per day, the cell recognition/harvest module is required to
complete the cell transportation from tubes to a 96-well microplate
in 12 minutes. FIG. 21 illustrates design features of certain
embodiments used to achieve this goal.
[0208] In one embodiment, cell harvesting is performed in a
Vacutainer tube 170 in the rotor 201 immediately after lifting the
rotor from the centrifuge, thereby saving the extra time of taking
170 the tubes out of the rotor 201. Four sets of imaging sensors
211 and punctuating needles 212 are used to harvest cells in
parallel, enabling four tubes to be processed simultaneously. After
cell harvesting is completed at one position, a rotary indexing
motor 213 indexes the rotor to the next position. In this way, the
cell transfer speed required for each punctuator unit 214 is
dramatically reduced to about 1 tube/min, including the time needed
for system cleaning between two indexing positions. With this
arrangement, 96 samples can be completed in 12 minutes. System
cleaning is done by swinging an arm 215 by 90.degree. using a DC
motor 216 and flushing the entire system. When the robot brings in
a rotor after centrifuge, the buckets 217 containing Vacutainers
rest on a holding plate 218 with conforming dents, to provide
better support for punctuating. The plate is indexed with the rotor
by the indexing motor 213.
[0209] For cell pipetting, a high-strength needle 212 is used to
puncture through the rubber lid of the Vacutainer tube 170, and to
aspirate the separated lymphocytes under the guidance of the
imaging sensor 211. As shown in FIG. 22, a DC motor 221 is used in
conjunction with a reducer 222 to supply the driving force. A lead
screw 223 and a preloaded ball-bearing nut 224 provide the linear
motion needed for the punctuation and precise positioning.
[0210] Since the estimated travel is quite long (70 mm) compared to
the very small diameter of the needle, a spring loaded follower 225
provides extra support to the needle.
[0211] A position-based servoing control (Hutchinson S, Hager G D,
Corke P I. A tutorial on visual servo control. Ieee Transactions on
Robotics and Automation 1996; 12:651-70; Hashimoto K. Visual
servoing: Real time control of robot manipulators based on visual
sensory feedback. In World Scientific Series in Robotics and
Automated Systems. Vol 7. Singapore: World Scientific Press; 1993)
is used, which is based on the computation of a Cartesian error,
requiring modeling of the objects and a calibrated camera to obtain
unbiased position estimation. Once the model is established, the
controller is dramatically simplified. This makes position based
control advantageous in applications where the geometric model of
the target can be obtained (Weiss L E, Sanderson A C, Neuman C P.
Dynamic Sensor-Based Control of Robots with Visual Feedback. Ieee
Journal of Robotics and Automation 1987; 3:404-17). In one
embodiment, the tube appears in a semi-definite fashion: with the
same liquid layers in a fixed order, the only uncertainty is the
position and thickness of the layers. Furthermore, the vision
sensor is fixed in position, making for easy calibration.
Considering the fact that many currently available robots already
have an interface for accepting Cartesian velocity or incremental
position commands, this structure provides set-point inputs to the
joint-level controller of the robot (dynamic look-and-move).
[0212] Centrifuge/Band Recognition Modules: In a second embodiment
of the device, the samples in their original hematocrit tubes are
centrifuged, imaged with the image sensor for band recognition, and
transported to the wells (one via the irradiator, the other not)
while still in the hematocrit tubes, at which point the tubes are
cut, the unwanted segments discarded, and the desired cells flushed
into the appropriate well.
[0213] The centrifuges used in a second embodiment of the device
can be physically much smaller, but spin faster than those used in
other embodiments, because the samples are in hematocrit
capillaries rather than in Vacutainer tubes. Hematocrit tubes are
typically spun at 15,000 g for .about.5 minutes in a 24-place
rotor. In this configuration the second embodiment of the device
uses 5 units, 4 spinning and one being robotically loaded and
unloaded, to reach the design throughput.
[0214] In a third embodiment the capillaries are spun at high speed
(13,000 g) in a rotor of larger capacity (about 100-600
capillaries).
[0215] After centrifugation, the sample is picked up and placed in
an imaging station where the lymphocyte or other cell-type band is
identified as before. One of two samples is then ready to be pushed
into the irradiator as described below. Cell harvesting is
completed by simply cutting the hematocrit tube to select the
desired cells. Unwanted segments are discarded and the desired
cells are flushed into the appropriate well.
[0216] This system, in which the sample is flushed directly from
the hematocrit tube to the well, minimizes cell loss during
transport.
[0217] Lymphocyte/Monocyte Pre-Screening Component: Optionally,
once the bands in the Vacutainer/hematocrit tubes have been
recognized by the CCD system, a quick "alarm" is provided should
the lymphocyte/monocyte layer be sufficiently small to indicate a
very large radiation dose. This is not a biodosimetry system per
se, as a time series of lymphocyte counts are required for this
purpose (Goans R E, Holloway E C, Berger M E, Ricks R C. Early dose
assessment in criticality accidents. Health Phys 2001; 81:446-9;
Goans R E, Holloway E C, Berger M E, Ricks R C. Early dose
assessment following severe radiation accidents. Health Phys 1997;
72:513-8.), but it will provide a rapid alarm of an immediately
life threatening situation. For example, after a whole body dose of
5 Gy (which would not necessarily produce major early symptoms for
several weeks (Hirama T, Tanosaki S, Kandatsu S, Kuroiwa N, Kamada
T, Tsuji H, et al. Initial medical management of patients severely
irradiated in the Tokai-mura criticality accident. Br J Radiol
2003; 76:246-53.)), one would expect the lymphocyte count to be
down by typically an order of magnitude after 4 or 5 days (Baranov
A E, Guskova A K, Nadejina N M, Nugis V. Chernobyl experience:
biological indicators of exposure to ionizing radiation. Stem Cells
1995; 13 Suppl 1:69-77.). Thus, alarm criteria are set, in terms of
the width of the separated lymphocyte/monocyte band: Any sample in
which the lymphocyte/monocyte band width is smaller than this
criterion produces in an immediate alarm, allowing that individual
to be identified for potential emergency treatment.
[0218] Flow-Through Low-Activity .sup.90Sr/.sup.90Y Mini-Irradiator
Module: In some embodiments, in order to have a positive control
for each individual, so that the effects of inter-individual
variability in radiosensitivity are accounted for, a simple, small,
low-activity .sup.90Sr/.sup.90Y beta irradiator is used to
irradiate half of each sample with a known dose, e.g., 1.8 Gy,
before it is analyzed. This radioactive source was chosen because
a) it has a very long half life (28 years), and b) it is primarily
a beta emitter, which greatly reduces radiation safety issues, and
c) the beta particles from the .sup.90Y decay are high energy, with
sufficient range to allow uniform dose coverage.
[0219] Mini-Irradiator Module: In a first embodiment of the device,
the basic design splits the cells from each sample as they are
being transferred to 96-well plates. Half the cells from each
individual flow in tubing through a small shielded cavity, where
they are exposed to the radiation from an array of radioactive
seeds arranged around the tube. To achieve the desired throughput,
four parallel channels are used for blood flow, passing through a
single irradiator.
[0220] In some embodiments, a 1.6 mm ID tubing (wall thickness 0.8
mm) is used to transfer the cells to and from the irradiator, at a
velocity of 1.7 mm/sec. At the entrance of the irradiator, the
tubing has a gradual increase in ID to 2.75 mm (with a wall
thickness of 0.4 mm), to slow the cell flow speed through the
irradiator by a factor of 3, to prolong the cell exposure time and
thus reduce the amount of radioactivity required for the sources.
The tube ID is gradually decreased again on exiting the irradiator.
As the transit distance within the irradiator is less than 20 mm,
this will not have a major effect on throughput.
[0221] Given the dose requirement of 1.8 Gy, an estimated four
low-activity 4-mCi 90Sr/90Y seeds are evenly distributed around the
tubing (the seed axis is 2.5 mm from the tube center) and will
produce this dose (Rosenthal P, Weber W, Forster A, Orth O, Kohler
B, Seiler F. Calibration and validation of a quality assurance
system for 90Sr/90Y radiation source trains. Phys Med Biol 2003;
48:573-85.) with a uniformity of better than 90%. These
millimeter-sized 4-mCi .sup.90Sr/.sup.90Y seeds are now in use for
intravascular brachytherapy (Id.), and are readily and
inexpensively available, from Bebig Isotopes or AEA Technology. An
array of nine seeds has been configured which irradiates the blood
passing through four tubes, in a 2.times.2 array. In some
embodiments, the irradiator is calibrated using Fricke dosimeter
solution.
[0222] In terms of radiation safety, at a distance of 10 cm from
four 4 mCi seeds, the dose rate is 40 .mu.Gy/h with no shielding,
entirely from bremsstrahlung (Florkowski T. Shielding for
Radioisotope Bremsstrahlung Sources Sr90 Plus Y90. Int J Appl
Radiat Isot 1964; 15:579-86). Shielding is accomplished using
concentric plastic and lead cylinders, with wall thicknesses of
1/8'' and 1'' respectively, which diminishes the dose rate to well
below radiation safety requirements. Thus, in one embodiment, the
external dimension of the cylindrically-shaped irradiator is 75 mm
(length).times.68 mm (diameter) and it weighs just over 3 kg.
[0223] In the second embodiment of the device, all cell
irradiations are done in sequence within one irradiator containing
five seeds, rather than the four channels employed in the first
embodiment of the device. This simplification is possible because
of the different physical form of the sample, which is in a thin
layer within a previously centrifuged hematocrit capillary moving
inside a tube, rather than blood flowing through a tube. Thus,
instead of a continuous flow, the target cells in the capillary are
brought to the center of the irradiator and remain stationary for a
few seconds. This increases the cell residence time close to the
radioactive seeds, thus increasing the dose rate, and increasing
the throughput. The target cell samples contained in a capillary
are rapidly brought to the center of the Sr irradiator by the
robotic manipulator. The sample stays at a position where the
estimated dose rate is 23 Gy/min for just under 4 seconds to
receive a total estimated dose of 1.8 Gy. The capillary tube is
then displaced by the incoming sample. At this rate, the irradiator
throughput meets a throughput rate of 15,000 individuals/15-hr day.
Because only one channel (rather than four) is used here, the
external dimensions of the irradiator, including shielding, are
slightly smaller than that in a first embodiment of the device.
[0224] In some embodiments the irradiator is not present and no
inter-individual calibration is done.
[0225] Automated Culturing of Lymphocytes: In a first embodiment,
the entire assay will be conducted in-situ in multi-well plates.
Because lymphocytes do not attach well to surfaces, multi-well
plates are used in which the base of each well consists of a
Millipore filter. Use of a filter pore size of 0.65 .mu.m, far
smaller than the lymphocytes, allows efficient medium removal by
aspirating through the filter, whilst ensuring that no cells are
lost during the aspiration. For the 96-well based platform, these
filter-bottomed plates are commercially available from Millipore
and medium removal through the filter works well, without loss of
lymphocytes.
[0226] In another embodiment the aspiration is replaced by applying
positive pressure to the top of the filter, pushing the liquid but
not the lymphocytes through.
[0227] After the cells are fixed, a final aspiration assures that
the lymphocytes will be in an approximate monolayer on the filter
substrate, in the correct geometry for imaging. At this point the
optimal imaging mode is from the top of the well, rather than from
the bottom through the Millipore filters--because of poor optical
transmission through the filter substrate. When imaging the cells,
the microscope objective must be within a few millimeters of the
cells, which can be achieved when imaging through the top by simply
removing the walls of the plate. This can be readily accomplished
as the walls and filter base are not strongly attached to each
other. A robot-friendly automated system for removing the well
walls has been configured and tested.
[0228] Because the entire micronucleus assay is carried out in-situ
in multi-well plates, there are many plate and liquid handling
tasks. Unlike the automated centrifuge and cell harvesting steps,
automated plate and liquid handling are relatively mature
technologies. Commercially available products include the Zymark
Allegro/Staccato, the Qiagen BioRobot, Caliper LS Sciclone and the
Gilson 925/940 workstations. While some components are appropriate
and are adopted here, an entire system from these vendors is not
appropriate for the invention described herein for the following
reasons: 1) these systems are typically designed with "general
purpose" in mind and therefore come with a moderate throughput and
price premium and 2) using components from different vendors to
form a system typically does not lead to an optimal solution and
substantial customer software development is typically required.
Our approach is to develop a dedicated system with optimized
software to achieve high throughput and accuracy.
[0229] As illustrated in FIG. 27, tasks to be performed inside the
plate and liquid handling module 14 fall broadly into two
categories: 1) pick and place tasks associated with plate handling,
and 2) various liquid handling tasks. As seen from a conceptual
design shown in this schematic, a pick-and-place SCARA robot 271, a
linear stage 272 equipped with a vacuum filtration device, and two
plate stackers 273 with drainage function are strategically placed
to handle all the tasks related to plate handling.
[0230] A gantry-type robot 274 is used for liquid handling tasks;
this gives a larger working space to allow the robot to shuttle
between the plate working area (on the linear stage) and the
end-effector (hand) quick-change stations 275. In one embodiment,
the robot has multiple degrees of freedom, and the configuration
gives excellent positioning accuracy, which is important for
multiple channel pipetting and washing.
[0231] In an embodiment, all the components are configured using a
computer aided design (CAD) package and properly assembled inside
the software. The sequence of robotic actions required is animated
in the software to ensure no physical and timing crashes, and to
facilitate any adjustments or repositioning. The high inertial
effect associated with the rotating centrifuge rotor, and also
experienced by the robot holding the rotating rotor, is also
simulated to determine final details. The CAD systems used includes
I-DEAS and ProE, both having strong assembly and animation
capability as well as good dynamic simulation capability.
[0232] As illustrated in FIG. 23, the wet multi-well plate is
placed on a plastic substrate which is coated with a thin (.about.1
micron) layer of non fluorescent water-soluble glue. After drying
for 20 minutes, the walls of the multiwell plate can be simply
lifted away from the glued filter bases, leaving the filter bases
flat, supported by the plastic substrate.
[0233] In one embodiment, the adhesive Elvanol (polyvinyl alcohol,
which is the standard remoistenable postage-stamp adhesive), is
used for this purpose, with no measurable fluorescence, providing a
smooth micron thick layer on a plastic substrate. With the well
walls removed, the cells on each filter can be imaged with optimal
geometry, without imaging through the filter base.
[0234] In one embodiment, in the first procedure, sealing and
transfer are done by filtering a 10% solution of polyvinyl alcohol
(PVA--98% hydrolyzed) through the plate, while it is pressed
against the substrate. After this glue has dried, the well walls
are removed, leaving the filters attached to the substrate (see
FIG. 24 below). To enable efficient filtering the embodiment shown
in FIG. 24 uses Porex.TM. brand sheets as the material for the
substrates. In addition, it was determined that precoating the
porous sheets with glue prior to filtration improved filter
attachment and reduced the time necessary for transfer and
drying.
[0235] In another embodiment, ELISA plate sealers (adhesive strips
used for covering plates during incubation) are used as substrates.
A punching mechanism (for example, MVS Pacific, shown in FIG. 25)
is used to detach the membranes from the plates and to keep them
intact. The plate sealer with the membranes is then laminated with
3-5 mil laminating film using a heat laminator. Prior to detaching,
the membranes are sealed with PVA, as in the first case, to protect
the cells.
[0236] It was determined that beads and stains can withstand the
high temperatures necessary for lamination and the laminating film
does not interfere with the imaging (FIG. 26). In some embodiments,
an adhesive substrate is used for transferring membranes. It does
not require time for drying which is a factor for a high-throughput
system. It does require sealing of the adhesive surface, but this
drawback can be eliminated by using lamination. Previously, the
quality of membrane transfer was better for Porex.TM. sheets, but
in case of adhesive film it was greatly improved by introducing a
punching mechanism.
[0237] In a second embodiment of the device, filter-bottomed
multi-well plates are used but with an added option to increase
throughput by using 384 well plates. In one embodiment, a new
configuration for 384-well plates is used which has a) the same
overall plate size as standard 96-well plates, b) 384 wells, each
with a similar cross sectional area to standard 96-well plates, and
c) 0.65 .mu.m pore Millipore filters as the base of each well. For
these custom plates, the wells are square in cross sectional area,
which allows a higher well area/dish area ratio, and is more
compatible with camera-based imaging than standard circular wells.
The square wells have 4 mm sides, thus a 16 mm.sup.2 cross
sectional area (only slightly smaller than the 25 mm.sup.2 cross
sectional area in standard 96-well plates). As with the commercial
96-well filter-bottomed plates used in the first embodiment, the
filters used in the second embodiment of the device are configured
to be easily detachable from the walls of the wells.
[0238] Acquisition and analysis of microscopic images involves
several discrete steps that must be substantially shortened to
reach a preliminary goal of 6,000 samples/15 hr day, corresponding
to about 10 seconds per well. As shown herein, this goal is
achievable using the unique steered-image compound microscope as
shown in FIG. 28.
[0239] Briefly, two fast galvanometric mirrors, X-steerer 281 and
Y-steerer 282 were placed in the afocal space 283 of an infinity
corrected microscope to steer different small parts of the view
into the camera. This arrangement allows switching from one
high-magnification field of view to another in less than a
millisecond, compared to tens of milliseconds for the same action
using a mechanical stage.
[0240] Commercial microscope stages such as the one used by the
Metafer system are rather slow (70 mm/sec), this is partially due
to the fact that the main bottleneck in those systems is the
generalized image acquisition and quasi-offline analysis and
partially because of the desire to limit the accelerations
experienced by living cells.
[0241] In an embodiment, both requirements are non-existent and a
much faster stage is used. As in the microbeam, in one embodiment,
the motion of the sample is separated into two components: a slower
coarse motion and a rapid fine motion. The coarse motion is
performed by a high speed stage (Parker motion) capable of few-g
accelerations. This motion is used to move between adjacent samples
(9 mm in 50 msec). The fine motion between fields of view within a
single sample is performed, not by moving the sample but rather by
steering light, using fast galvanometric mirrors as shown in FIG.
29. Typical transit times between adjacent fields of view of the
microscope objective were measured at about 100 .mu.sec. Extensive
ray-tracing simulations have shown an effective increase of the
microscope field of view by a factor of 100 (from 100 .mu.m radius
to 1 mm radius), before image degradation becomes noticeable. This
was confirmed qualitatively.
[0242] Focusing
[0243] A major rate limiting step in automated imaging system is
focusing. In order to get good image quality, typically microscope
objective lenses have rather small depth of field and are sensitive
to the roughness of the sample being imaged. The simple solution to
this is to take several images at different object-lens distances,
quantify "fuzziness" and search for the best setting. This process
is very time consuming and therefore unacceptable. In embodiments
of the invention disclosed herein, this concern by placing a weak
cylindrical lens in the optics path. As shown in FIG. 30, by using
an appropriately selected lens, a circular object will be imaged as
circular when in focus and as elliptical when out of focus, the
aspect ratio being proportional to the distance from focus. The
object-lens distance can then be corrected in one step.
[0244] For imaging, one embodiment of the invention disclosed
herein utilizes a complementary metal-oxide-semiconductor ("CMOS")
camera, which has a much faster readout than the, lower noise,
charge-coupled device ("CCD") cameras typically used. The resulting
loss in image quality may be significant for "all purpose" imaging
systems, but is unimportant in for detection of micronuclei.
Analysis of the image is split between the camera and the frame
grabber board to decrease the amount of data transferred to the
controlling computer, the biggest bottleneck in existing imaging
systems. By using a dichroic mirror and two cameras, attached to
the same frame grabber board the system simultaneously "sees" the
nuclear and cytoplasm and rapidly analyzes their overlap obtaining
the number and size of nuclei in each cell.
[0245] To observe micronuclei with diameters down to 0.5 .mu.m, the
pixel size needs to be .about.0.25 .mu.m. Intensified digital
cameras are available with 16 .mu.m effective pixel sizes and are
well suited for this application. Using .times.50 optical
magnification allows one to obtain 0.32 .mu.m pixel resolution. A
1.31 Mega pixel camera with this resolution has a 15.36
mm.times.12.29 mm active area. At .times.50 magnification, this
yields a field of view with an area of 0.075 mm2. Comparing this to
the size of a single well in the plates as described (diameter:
5.74 mm, area: 25.9 mm2), an area ratio is obtained that
corresponds to 345 images to completely image one well surface. So
the task is to move to and image those 345 locations in 10
seconds.
[0246] As a first step in achieving the desired processing rate,
cell samples from a 96-well filtration plate are positioned on the
imaging platform for viewing using the integrated robotics
described above. To position the bottoms of each well under the
microscope lens, a fast X-Y mechanical stage is standardly used. If
a fast X-Y stage is used for 345 movements, taking about 40 msec
per movement, 13.8 seconds per well has already been used, more
than the allotted time, just for positioning. This problem is
solved by replacing this standard configuration of a 50.times.
objective and mechanical stage motion with a novel steered-image
compound microscope. A 10.times. objective (Nikon CFI60) is used,
with a piezo nano-positioner followed by a 5.times. projection lens
to obtain .times.50 magnification. Infinity optics allows the
placement of X-Y steering mirrors (Scanlab) within the afocal space
to guide different parts of the full image down the optic axis to
the camera sensor. Switching between images can thus be
accomplished in much less than one millisecond, using fast steering
mirrors.
[0247] For this image acquisition process, the well surface is
divided into 13 fields of view through the objective lens; the
partitioning consists of a row of two images followed by three rows
of three images and capped by a row of two final images. Each of
these fields is then divided into 25 additional high-magnification
views by the X-Y steering mirrors. The total time for motion using
this scheme is less than one second--a major improvement over the
13.8 seconds per well that is needed for mechanical stage based
motion.
[0248] The second process to be considered is the acquisition and
data transfer of the image. Usually a CCD camera is used for
microscopy. These devices are chosen for their very low leakage,
allowing long exposure times, a factor that is unimportant for the
present application. In fact, the physical structure of CCDs does
not allow rapid readout. Transferring images faster than about 30
frames per second leads to degraded image quality. An emerging
technology that uses CMOS imaging arrays (Loinaz M. Video cameras:
CMOS technology provides on-chip processing. Sensor Review 1999;
19:19-26.) solves this problem. The structure of CMOS devices
allows for fast digitizing and transfer of images to memory. A
camera well-suited is the ultra high speed Photon focus model
MV-D1024E-160 series 8-bit CMOS camera, which can acquire images
and transfer them to a computer at 150 frames per second. Image
analysis is done off-line, as discussed below.
[0249] Due to inevitable non-uniformities in the imaging substrate,
it is necessary to adjust the focus to compensate. There are a
number of autofocusing routines used in current machines, which
depend on focusing random objects which appear in the image. In
order to make the autofocusing more robust, a new approach is used,
involving adding 1-.mu.m diameter fluorescent microspheres to the
sample surface. With 10,000 beads per well, approximately 30 beads
are available per magnified view, which is far fewer beads than
there are 0.6 .mu.m pores in the filter substrate, so the beads
will not interfere with the ability to drain the well through the
pores. The color of the beads is chosen so there is no overlap with
the fluorochromes(s) used in the cellular imaging.
[0250] In a first embodiment of the device, illumination of the
beads is done with a standard mercury lamp. Crimson beads
(Molecular Probes Inc) have an excitation wavelength of 625 nm and
an emission spectrum with a peak wavelength of 645 nm. A two-color
cube allows both wavelengths of the bead emission and the cell
stain emission to pass towards the CMOS-imaging sensor. Prior to
the sensor, barrier filters are alternately introduced into the
optic path to image either the cells (blue light) or to focus on
the beads (crimson). During focusing, the CMOS image is binned in
2.times.2 segments and the images are analyzed to move the
objective in to proper focus. 2.times.2 binned images can be
acquired at 2400 images/sec. Twelve (12) images are expected to be
acquired to obtain the focus, and the entire focusing process is
calculated at about 50 ms, including the settling time of the piezo
nano-positioner of the microscope objective; this is done 13 times
per well.
[0251] In another embodiment of the device the light from the beads
is split off from the light from the cell stain using a dichroic
mirror. The light from the beads then passes to a separate camera
via a cylindrical lens. Using the aspect ratio of the bead image
allows obtaining focus after a single image.
[0252] Data flow factors relevant to the imaging speed are the
motion times for the stage and the steering mirrors, as well as the
camera exposure and read-out limitations. FIG. 32 depicts the data
flow for an embodiment of the invention disclosed herein.
[0253] First, considering that the linear speed for the stage is 50
mm/sec, it takes 40 ms for the stage to move the samples to the
next field (there is a 2 mm step size to the next field) and an
additional 50 ms to focus. Next, it takes 5 ms to grab a CMOS
image, using a mercury-arc illuminator, and 0.1 ms for the X-Y
steering mirrors to point the next partition of the field to the
CMOS sensor, totaling 5.1 ms for this portion of the routine. This
sequence continues until each partition has been imaged.
Afterwards, it takes 90 ms for the next field to be positioned over
the objective lens by the mechanical stage and to be refocused.
When 13 fields have been imaged, it takes 180 ms for the X-Y stage
to move the next well into position above the objective.
[0254] Following a CMOS image grab, the image is transferred at 60
MB/sec, to an asynchronous image analysis computer. This step runs
in parallel with the X-Y steerer moving the next .times.50 view to
the CMOS sensor. In the image analysis section, the images are
scanned for cells with predetermined features and any such
identified cells will be counted. If enough cells are counted, the
X-Y steering loop exits and the mechanical stage moves the next
cell well into an initial position for imaging.
[0255] The goal for this embodiment of the device is to process
6,000 wells per day and this goal is easily achieved with the
proposed monochrome-imaging protocol above. It is possible to
acquire color images with the addition of wavelength filters, and
at the cost of throughput time. In the second embodiment of the
device, color analysis is possible with no loss of throughput.
[0256] To summarize, in terms of high-throughput image acquisition,
new features are incorporated into the first embodiment of the
device. For example, compared to the current state of the art such
the Amersham Ind. Cell 3000 machine. One is the novel steered-image
compound microscope approach, and the other is the use of CMOS
rather than CCD technology. Together, these two advances give more
than an order of magnitude increase in speed compared with known
devices. Another embodiment increases image acquisition speed by
further developing the "steered image compound microscope"
technology introduced in our first embodiment of the device.
[0257] Referring to the FIG. 33, note the second optical channel
with lower magnification. An image taken in this channel is used as
a selector for regions to be observed at high magnification
allowing cherry-picking of the most interesting regions.
[0258] A limitation of the speed of analysis is that, a priori, the
position of the binucleate cells, and so time is spent imaging
areas of no interest. The system described herein overcomes this
problem by first scanning at low magnification to locate regions of
interest to be examined at high magnification after a change of
microscope objectives (Randers-Pehrson G, Geard C R, Johnson G,
Elliston C D, Brenner D J. The Columbia University single-ion
microbeam. Radiat Res 2001; 156:210-4). This approach addresses
this limit using an analogous but much faster technique employing a
dual optical path imaging system, as shown in the previous
schematic. With the 384-well plates described above, there is a
square aspect ratio, convenient for imaging. Twenty-four primary
images are used to capture the well bottom; four of these images
are steered to the .times.20 CMOS imager at each stage position.
There are 25.times.100 views within each .times.20 view. The
primary image is analyzed to find putative binucleate (BN) cells.
Using cluster analysis, a threshold density of BN cells within a
low-magnification view marks the regions for high-magnification
imaging. Imaging only these regions and ignoring regions devoid of
binucleate cells is more efficient because the number of images
grabbed and processed is significantly reduced. A pair of fast
galvanometric mirrors is used to direct the image of only BN cells
into the second optical train which now has a 10.times. projection
lens, resulting in a .times.100 image at the small area, fast
3-CMOS color array for location of micronuclei. An Argon ion laser
significantly enhances the imaging process in the second embodiment
of the device by offering a more collimated, higher power light
source with multiple excitation lines to illuminate multiple
targets, e.g. .gamma.-H2AX and nuclei. The secondary imaging is
done at a rate of 500 frames/sec yielding a measurement time of 2
sec/1000 BN cells.
[0259] The autofocusing routine for the second embodiment of the
device is similar to the one used in the first embodiment of the
device except for the illumination. For the second embodiment of
the device, a red LED emitting at 625 nm is used, an exact
wavelength match for exciting crimson fluorescent beads. Since this
bead illuminator is a separate light source from the Argon ion
laser used for sample illumination, a fast switching mirror
alternates paths, depending on which light source is being used in
the lower magnification channel. This light switch is synchronized
to the barrier filters discussed above.
[0260] Parallel architecture enables a throughput of at least about
30,000 samples per day, using color imaging. The various parallel
processes and the times associated with each are shown in FIG. 34.
Some actions are necessarily done in series, namely, stage motion
and focusing, acquisition of the low magnification image and
acquisition of multiple high magnification images. By selection of
hardware and by careful scheduling of stage motions, all other
activities are performed in parallel with these. Image analysis
occurs in parallel on fast vision processing boards.
[0261] In some embodiments, high-magnification, color images are
acquired using a color-selecting prism and a fast three CMOS camera
array. Each camera is equipped with an on-board binary converter
which speeds up the transfer rate by 8.times.. Also, image
information from each camera is read out over three channels, so
that this step is not a rate-limiting factor. For each well, a
throughput calculation is a summation of the imaging sequence. A
throughput of at least about 30,000 wells per day and is readily
achievable with this parallel color-imaging protocol.
[0262] Image processing needs include rapid identification of
micronuclei associated with binucleate lymphocytes, with high
sensitivity and specificity. Successful computer image analysis
depends on the quality of the image presented for analysis.
Demonstrated herein is successful production of very high contrast
images of the fixed nuclei and micronuclei using a DNA binding
stain such as DAPI or Hoechst 33342. These stains are observed with
epifluorescence imaging with UV light. It is important to make sure
that the substrates and reagents used do not add any background
fluorescence.
[0263] The resulting images are highly compatible with binary
segmentation and standard blob analysis. Several algorithms have
been published in the literature in regard to morphometric
features, such as size, aspect ratio, relative distance and
concavity depth etc., that can be used for identification of the
binucleated cells and micronuclei (Varga D, Johannes T, Jainta S,
Schuster S, Schwarz-Boeger U, Kiechle M, et al. An automated
scoring procedure for the micronucleus test by image analysis.
Mutagenesis 2004; 19:391-7; Lozano A, Marquez J A, Buenfil A E,
Gonsebatt M E. Pattern analysis of cell micronuclei images to
evaluate their use as indicators of cell damage. Engineering in
Medicine and Biology Society, 2003. Proceedings of the 25th Annual
International Conference of the IEEE. Vol 1. Cancun, Mexico: IEEE;
2003. p. 731-4; Bocker W, Streffer C, Muller W U, Yu C. Automated
scoring of micronuclei in binucleated human lymphocytes. Int J
Radiat Biol 1996; 70:529-0.37; Verhaegen F, Vral A, Seuntjens J,
Schipper N R, de Ridder L, Thierens H. Scoring of radiation induced
micronuclei in cytokinesis-blocked human lymphocytes by automated
image analysis. Cytometry 1994; 17:119-27).
[0264] An addition to the traditional image processing approach for
defining the identification criteria, which has been used
extensively (Long X, Cleveland W L, Yao Y L. Effective automatic
recognition of cultured cells in bright field images using Fisher's
linear discriminant preprocessing. In Proceedings of IMECE04: 2004
ASME International Mechanical Engineering Congress. Anaheim,
Calif.; 2004; Long X, Cleveland W L, Yao Y L. Automatic detection
of unstained viable cells in bright field images using a support
vector machine with an improved training procedure. Computers in
Biology and Medicine 2004: accepted; Long X, Cleveland W L, Yao Y
L. A new preprocessing approach for cell recognition. IEEE
Transactions on Information Technology in Biomedicine 2004:
accepted.), is the use of algorithms based on machine learning
techniques (Mitchell T M. Machine Learning. New York: McGraw-Hill;
1997). Machine learning techniques are able to capture complex,
even nonlinear, relationships in high dimensional feature spaces
that are not easily recognized or defined by the human operator.
This technique is used to "teach" the image processing system to
recognize nucleoplasmic bridges, which are an important adjunct for
increasing the specificity of the radiation-induced micronucleus
assay (Fenech M, Bonassi S, Turner J, Lando C, Ceppi M, Chang W P,
et al. Intra- and inter-laboratory variation in the scoring of
micronuclei and nucleoplasmic bridges in binucleated human
lymphocytes. Results of an international slide-scoring exercise by
the HUMN project. Mutat Res 2003; 534:45-64; Fenech M, Chang W P,
Kirsch-Volders M, Holland N, Bonassi S, Zeiger E. HUMN project:
detailed description of the scoring criteria for the
cytokinesis-block micronucleus assay using isolated human
lymphocyte cultures. Mutat Res 2003; 534:65-75,92).
[0265] The classifications defined either explicitly or through a
"learning" technique are programmed into the vision processor for
rapid operation. For example, the Matrox Odyssey Xpro scalable
vision processor board can be used, which is designed for parallel
and pipelined processing. Operating at 120 billion operations/sec,
this board can easily analyze the 300 images acquired for each well
within the ten seconds allotted to achieve the target throughput
for a first embodiment of the device. The board also has the high
speed image transfer capabilities required. As still faster vision
processing boards appear, appropriately upgrading is made, but
image processing is not a bottleneck for a first embodiment of the
device.
[0266] With respect to a second embodiment of the device, there
will be fewer images to analyze in the second embodiment, because
of the cluster techniques as discussed supra in the image
acquisition section, which is an estimated gain by a factor of 2.
As discussed above, the second embodiment of the device also
triples the speed of the image transfer, by using a full camera
link interface. It is also pertinent to note that this device
features improved image quality, due to the use of a 100.times.
lens, rather than the 50.times. lens employed in the first
embodiment of the device.
[0267] In the second embodiment of the device, image processing is
similar to that used with the first embodiment of the device,
except that two (or optionally 3) camera images need to be
processed, corresponding to different colors. As discussed above,
the image processing for the different colors is done in parallel,
rather than in series as in the first embodiment of the device.
Usually one image is used to produce a mask for the image in the
second camera, simplifying the needed calculation. For example, in
the micronucleus assay, an image of the cytoplasm stained in one
color is used to delimit regions that may contain two nuclei and
potentially some micronuclei. Similarly, in the .gamma.-H2AX assay,
the Hoechst stained nuclei produces a colored mask to select
regions in which to count foci.
[0268] With respect to system integration, two levels of control
are implemented: 1) control at the level of components and
subsystems, and 2) sequential (or logical) event control. Component
control is closed loop for most components, with the exception of
the liquid handling system, which is open loop. Sequential control
is implemented by a Programmable Logical Controller.
[0269] Corresponding to the control module, user software is also
implemented at two levels: 1) modular component and subsystem
software, and 2) global monitoring and coordinating. The key
technical strategy is to make the software highly modularized.
Modularization of software not only has the advantage of increasing
code reusability, minimizing software development, but it also
dramatically reduces maintenance costs after commercialization.
[0270] System monitoring and failure avoidance strategies are
contemplated. The system development described so far has aimed at
high reliability. However, because it is also aimed at
high-throughput full automation, system monitoring for early fault
detection and catastrophic failure avoidance in key components is
preferred. The standard approach is by placing sensors at strategic
locations in the system to monitor system performance. However,
such an approach increases system complexity, maintenance needs,
and cost.
[0271] Instead, a novel proposed approach is to make the maximal
use of the sensing capability already existing in the system
components. For instance, the motor current of a rotating
centrifuge unit is a good indicator of any jam and breakage of the
rotor and/or buckets. In any of these events, the current increases
and thus give a sensitive indication for the need of an emergency
stop. The motor current of all the actuators including those for
centrifuges, punctuator unit, stage at the plate working area,
microscope stages, and robotic arms is continuously monitored. For
the liquid handling system as well as the pneumatically-actuated
end effectors, the liquid pressure and pneumatic pressure are
continuously monitored using transducers at strategic
locations.
[0272] High speed identification and tracking are also preferred
for a reliable high throughput system. RFID (radiofrequency ID)
labeling and scanning (Want R. RFID. A key to automating
everything. Sci Am 2004; 290:56-65), which has a number of
practical advantages over barcode systems (Jossi F. Electronic
follow-up: bar coding and RFID both lead to significant
goals--effeciency and safety. Healthc Inform 2004; 21:31-3.), are
used in one embodiment. A commercial RFID scanning system is used
to tag and track both the individual blood/tissue samples, and each
multi-well plate. A commercial RFID database is used to track each
sample during the whole process.
[0273] In summary, the high throughput product described herein is
based on automated assays in situ in multi-well plates. All the
minimally-invasive assays that are used (micronuclei in
lymphocytes, .gamma.-H2AX foci, micronuclei in blood reticuloctyes,
micronuclei in exfoliated bladder or buccal cells) have been chosen
because they are a) well established and b) amenable to automation.
System optimization can be achieved using ex-vivo irradiated
samples from healthy human volunteers. Calibration and testing can
be achieved using samples from adult and pediatric patients who
were subject to total body irradiation.
[0274] In one embodiment, the system described herein comprises
only four main modules adapted to accomplish 0 sample handling, ii)
information logging and iii) imaging: a robotic centrifuge module,
a service robot 352, a cell harvesting module 353 and a
liquid/plate handling robot 354, and a dedicated image
acquisition/processing system. See FIG. 35.
[0275] A Selective Compliant Articulated Robot Arm ("SCARA") is
preferred to automate the blood sample transfer operations among
the modules. To this end the SCARA workspace is augmented by
designing an additional link capable to reach safely into the
workspace of the liquid/plate handling robot 354 as shown in FIG.
35b.
[0276] Robotic Centrifugation Module
[0277] An overview of the automated processing steps begins with
loading blood samples, contained in hematocrit capillaries, which
may be made of polyvinyl chloride (PVC), into a centrifuge which
will isolate the lymphocytes. A challenge here is to meet the
desired throughput and system reliability when handling
capillaries.
[0278] To cope with this problem, as illustrated in FIG. 36, a
novel multi-purpose robotic gripper is designed for i)
centrifuge-buckets and micro-plates handling and multiple handling
of capillaries.
[0279] Cell Harvesting Module
[0280] After centrifugation the samples are transferred to a band
recognition module, where cell harvesting is completed by cutting
the hematocrit tube to select the lymphocytes. Plasma and
lymphocytes are flushed into the appropriate well. A challenge
faced here is the contactless automatic cutting of PVC hematocrit
capillaries. To avoid cross-contamination associated with the use
of non-disposable mechanical cutting tools, a laser-based cutting
system, illustrated in FIG. 37a is preferred for cutting
capillaries (FIG. 37b).
[0281] In an embodiment, an automatic rotary stage is designed and
implemented to allow for even distribution of the laser-delivered
power along the circumference of the cut cross-section, as
illustrated in FIG. 37c. A collet/solenoid-based gripper is
employed for automatic capillary back feed, as illustrated in FIG.
37d.
[0282] Peripheral Blood and Capillary Blood: In some embodiments,
the assays used in the device are blood based. Thus, in one
embodiment, peripheral blood drawn by venipuncture is used. In a
second embodiment, capillary blood is used in order to increase
overall throughput. Currently, the most common source of capillary
blood is a disposable lancing device (Fruhstorfer H. Capillary
blood sampling: the pain of single-use lancing devices. Eur J Pain
2000; 4:301-5; Garvey K, Batki A D, Thomason H L, Holder R, Thorpe
G H. Blood lancing systems for skin puncture. Prof Nurse 1999;
14:643-8, 50-1.); however laser skin perforators, such as the
FDA-approved Lasette P200 (Cell Robotics Inc), have considerable
high-throughput application here, and have high patient
acceptability (Burge M R, Costello D J, Peacock S J, Friedman N M.
Use of a laser skin perforator for determination of capillary blood
glucose yields reliable results and high patient acceptability.
Diabetes Care 1998; 21:871-3).
[0283] In terms of the volume of capillary blood that will be
available, several studies have been made on the volume of blood
that can be obtained from disposable automatic capillary lancing
devices, while causing minimal pain (Rosenthal P, Weber W, Forster
A, Orth O, Kohler B, Seiler F. Calibration and validation of a
quality assurance system for 90Sr/90Y radiation source trains. Phys
Med Biol 2003; 48:573-85; Florkowski T. Shielding for Radioisotope
Bremsstrahlung Sources Sr90 Plus Y90. Int J Appl Radiat Isot 1964;
15:579-86.). A recent study picked out the ITC Tenderlett and the
Roche AccuChek Safe-T-Pro lancets as being safe, reliable, and
causing the least pain (Fruhstorfer H. Capillary blood sampling:
the pain of single-use lancing devices. Eur J Pain 2000; 4:301-5.);
these two devices respectively yielded mean blood volumes of 300
and 200 .mu.l respectively, though with about 10% of the samples
yielding less than 50 .mu.l (Fruhstorfer H. Capillary blood
sampling: the pain of single-use lancing devices. Eur J Pain 2000;
4:301-5.). According to a published study (Burge M R, Costello D J,
Peacock S J, Friedman N M. Use of a laser skin perforator for
determination of capillary blood glucose yields reliable results
and high patient acceptability. Diabetes Care 1998; 21:871-3.), the
Cell Robotics Lasette P200 laser skin perforator can generate blood
volumes of more than 100 .mu.l in 98% of subjects.
[0284] Based on these data, an embodiment is configured to require
no more then 50 .mu.l of blood per sample, with the expectation
that those individuals who produce less than this could be given a
repeat fingersticks or laser perforation. For example, two samples
per individual are drawn for the lymphocyte-based assays, one being
passed through the irradiator in the system to provide a positive
control regarding individual radiosensitivity. However, for each
assay, lower volumes can be adequate and where so, the lower
volumes can and will be used.
[0285] In-situ analysis of micronuclei from lymphocytes in
multi-well plates: The micronucleus assay has been standardized
over many years, as a slide-based non-automated procedure (Fenech
M, Bonassi S, Turner J, Lando C, Ceppi M, Chang W P, et al. Intra-
and inter-laboratory variation in the scoring of micronuclei and
nucleoplasmic bridges in binucleated human lymphocytes. Results of
an international slide-scoring exercise by the HUMN project. Mutat
Res 2003; 534:45-64; Fenech M, Chang W P, Kirsch-Volders M, Holland
N, Bonassi S, Zeiger E. HUMN project: detailed description of the
scoring criteria for the cytokinesis-block micronucleus assay using
isolated human lymphocyte cultures. Mutat Res 2003; 534:65-75;
Fenech M, Holland N, Chang W P, Zeiger E, Bonassi S. The HUman
MicroNucleus Project--An international collaborative study on the
use of the micronucleus technique for measuring DNA damage in
humans. Mutat Res 1999; 428:271-83). To optimize the conditions for
a fully-automated multi-well based system, a first embodiment of
the device employs peripheral blood in 96-well plates. A second
embodiment of the device uses capillary blood from fingersticks, in
384 well plates.
[0286] Sources of biological material: In one embodiment,
peripheral blood is used, as drawn from multiple healthy volunteers
and irradiated ex vivo. Additionally or alternatively, samples are
taken from total body irradiation patients. For demonstration of
the device, blood samples are taken from such patients from the
University of Pittsburgh (adults) and Memorial Sloan Kettering
Cancer Center (children).
[0287] Blood Separation: As discussed above, in the first
embodiment of the device, peripheral blood collection and
separation is based on BD Vacutainer CPT tubes. These tubes contain
an anticoagulant with a Ficoll Hypaque density fluid and a
polyester gel barrier. Centrifugation of these tubes for 25-30 min
results in a single-step very clear separation of mononuclear white
blood cells from plasma and from erythrocytes and neutrophils.
Cells are maintained sterile and lymphocytes are withdrawn from
their band of confinement, seen by eye as a highly turbid band, by
hypodermic syringe. Multiple aliquots are taken.
[0288] Cell Culture: Comparisons are undertaken between standard
culture procedures and culture in 96-well plates, pre-filled with
250 .mu.l of standard culture medium. The standard cultures are
done using .about.1 million cells, compared to about .about.25,000
cells in the 96-well Millipore filtration plates. Under standard
micronucleus assay conditions (Fenech M, Bonassi S, Turner J, Lando
C, Ceppi M, Chang W P, et al. Intra- and inter-laboratory variation
in the scoring of micronuclei and nucleoplasmic bridges in
binucleated human lymphocytes. Results of an international
slide-scoring exercise by the HUMN project. Mutat Res 2003;
534:45-64), cytochalasin B are added to cultures at 44 hr post cell
cycle initiation. In the interest of shortening the assay, earlier
times of addition can be tested, as well as earlier fixation times.
Thus, along with the standard 72 hr time of culture stoppage,
comparisons down to 56 hr can be made. Wash and dilution steps are
minimized.
[0289] Cell Processing: Cells in the culture flasks are processed
by standard procedures (Fenech M, Holland N, Chang W P, Zeiger E,
Bonassi S. The HUman MicroNucleus Project--An international
collaborative study on the use of the micronucleus technique for
measuring DNA damage in humans. Mutat Res 1999; 428:271-83) to
produce microscope slide preparations. However, the number of steps
is minimized where possible and preferably all steps are amenable
to robotic automation. As discussed elsewhere, 96-well Millipore
plates (0.65 .mu.m pores) are used, allowing medium to be drawn off
by vacuum filtration, but without loss of lymphocytes. This is
followed by a wash step with Hanks balanced salt solution and
removal by vacuum filtration, and fixation step using absolute
ethanol. The initial filtration steps result in cells settling
firmly on the membrane and the fixation step enhances their
adherence to the membrane.
[0290] Cell staining: Whereas the long established micronucleus
assay uses light microscopy and Giemsa stain to discern nuclei and
micronuclei, this stain results in unacceptable backgrounds for
automated detection. Instead the highly specific DNA binding
fluorochrome DAPI (4,6-diamino-2-phenylindole) or Hoechst 33342 is
used as the indicator of binucleate cells plus/minus micronuclei.
These fluorochromes are routinely used in cytogenetic studies and,
in fixed cells, are clearly superior to the commonly-used propidium
iodide.
[0291] Preparation of Sample for Imaging: as Discussed in the
description of the plate handling/liquid handling module, there are
some issues involved with automated cell handling of lymphocytes,
in that they do not attach well to surfaces, and thus it is
important to ensure that a) significant numbers of lymphocytes are
not lost during any medium removal/washing steps, and b) that the
lymphocytes are located in a (near) monolayer during the
image-acquisition stage, to ensure optimal imaging. Thus, in some
embodiments, commercially-available Millipore 96-well filter plates
in which the base of each well is a 0.65 .mu.m pore filter are
used. These pores are small enough so that lymphocytes cannot fall
through the pores, nor will fluid pass through the filter, except
when a small differential pressure (vacuum) is applied, in which
case the medium is aspirated through the filters, and the cells
will sit in an approximate monolayer directly on the bottom of the
well. This solves both the problem of medium removal without losing
cells, and of ensuring optimal conditions for the imaging stage.
These 96-well sterile plates are available commercially from
Millipore, and preliminary studies have indicated that this
approach works well.
[0292] Imaging: in some embodiments, analysis tools include:
[0293] 1. A multi-purpose semi-automated slide-based scanning
system (Metafer (Schunck C, Johannes T, Varga D, Lorch T, Plesch A.
New developments in automated cytogenetic imaging: unattended
scoring of dicentric chromosomes, micronuclei, single cell gel
electrophoresis, and fluorescence signals. Cytogenet Genome Res
2004; 104:383-9) developed by MetaSystems), which has been used for
cytogenetically-based radiation dose estimates in highly exposed
Russian nuclear workers (Hande M P, Azizova T V, Geard C R, Burak L
E, Mitchell C R, Khokhryakov V F, et al. Past exposure to densely
ionizing radiation leaves a unique permanent signature in the
genome. Am J Hum Genet 2003; 72:1162-70; Mitchell C R, Azizova T V,
Hande M P, Burak L E, Tsakok J M, Khokhryakov V F, et al. Stable
intrachromosomal biomarkers of past exposure to densely ionizing
radiation in several chromosomes of exposed individuals. Radiat Res
2004; 162:257-63.). This system has also been used for detection of
radiation-induced binucleated post-division lymphocytes with and
without micronuclei (Schunck C, Johannes T, Varga D, Lorch T,
Plesch A. New developments in automated cytogenetic imaging:
unattended scoring of dicentric chromosomes, micronuclei, single
cell gel electrophoresis, and fluorescence signals. Cytogenet
Genome Res 2004; 104:383-9). This system was utilized in the early
first part of the instant studies to compare with results obtained
in multi-well systems.
[0294] 2. An Amersham In Cell 3000 Analyzer system, which is the
current state of the art for automated high-throughput image
analysis of multi-well plates.
[0295] 3. Breadboard versions of the embodiments of the device.
[0296] Deployment of one embodiment includes three stages:
breadboard, low-throughput prototype (6,000 samples per day) and
high-throughput prototype (30,000 samples/day). In order to develop
the breadboard a room was selected in such a way to impose
dimensional constraints that would increase system portability. The
room was equipped with an RS80 SCARA robot from Staubli, an O-Sprey
UV laser system from Quantronix, a Sciclone ALH 3000 liquid
handling robot from Caliper Life Sciences, a 5810RA Robotic
Centrifuge from Eppendorf and an industrial PC from iBASE
technology running RTAI Linux for the low-level control.
[0297] An implementation of the breadboard without the image
acquisition/processing module is illustrated in FIG. 38. A
prototype is illustrated in FIG. 39.
[0298] Current automated imaging systems have limited throughput,
mostly due to their non-specificity, for example the Metafer system
(Metasystems, Germany) can perform rare cell detection, comet
assays, metaphase spreads, location of dicentrics, micronucleus
scoring and more on 100-200 slides per day. This is about 1% of the
desired throughput for a biodosimetry workstation. A need exists
for a dedicated high-throughput imaging system for performing the
micronucleus assay exclusively, seeking creative solutions for
rapid sample manipulation, automated focusing and image acquisition
and analysis, using the experience gained from developing the
automated microbeam workstation. The throughput of an imaging
system according to the present invention is estimated at 5-6
minutes/96-well plate or 20,000-30,000 individual samples/day (FIG.
18).
[0299] Multi-well cultures vs. standard cultures. Comparison can be
made of micronucleus frequencies in both irradiated and
unirradiated lymphocytes, between well-cultured and
standard-cultured cells, along with intra- and inter-plate
comparisons. Ex-vivo irradiated blood is used from a total of 50
healthy donors. Zero, low (0.5 Gy), medium (2 Gy) and high doses (5
and 10 Gy) are used, with every effort made to undertake the
ex-vivo irradiation within a few minutes of the blood being
drawn.
[0300] After the initial developments to establish optimal
handling, culture and observational parameters for multi-well
lymphocyte growth and micronucleus detection, attention can turn to
blood derived from individuals who have been subjected to total
body irradiation (TBI). For these TBI individuals, blood samples
are provided as they become available.
[0301] Samples are expected from approximately 60 TBI adult
patients per year and 12 pediatric patients. All material is coded
and the radiation exposures only made known to the investigators
after the lymphocyte micronucleus assays have been undertaken in
pair wise comparisons for unexposed and exposed samples. To improve
the precision of our dose reconstruction, inter-personal
sensitivity can be accounted for by using information from a sample
exposed to a known dose. The procedure outlined here will be
followed consistently for all TBI samples, and after micronuclei
frequencies have been determined, the code will be broken and
actual exposures compared with experimentally determined
exposures.
[0302] Micronuclei in Lymphocytes: Use of small volumes of
capillary blood in 384-well plates: As discussed above, in the
second embodiment of the device capillary blood from a fingerstick
is used, rather than peripheral blood from venipuncture. This
should increase overall throughput dramatically after a radiation
incident. The most common source of capillary blood is a disposable
lancing device (Fruhstorfer H. Capillary blood sampling: the pain
of single-use lancing devices. Eur J Pain 2000; 4:301-5; Garvey K,
Batki A D, Thomason H L, Holder R, Thorpe G H. Blood lancing
systems for skin puncture. Prof Nurse 1999; 14:643-8, 50-1.),
though laser skin perforators, such as the FDA-approved Lasette
P200 (Cell Robotics Inc), are well-suited, having high patient
acceptability (Burge M R, Costello D J, Peacock S J, Friedman N M.
Use of a laser skin perforator for determination of capillary blood
glucose yields reliable results and high patient acceptability.
Diabetes Care 1998; 21:871-3).
[0303] As discussed above, the device should require no more than
50 .mu.l of blood per sample, with the expectation that those
individuals who produce less than this could be given a second
fingerstick or laser perforation. In fact two groups have
investigated a "micromethod" in which 100 .mu.l of whole blood has
been used for the micronucleus assay after radiation exposure both
in vitro and in vivo (Paillole N, Voisin P. Is micronuclei yield
variability a problem for overexposure dose assessment to ionizing
radiation? Mutat Res 1998; 413:47-56; Gantenberg H W, Wuttke K,
Streffer C, Muller W U. Micronuclei in human lymphocytes irradiated
in vitro or in vivo. Radiat Res 1991; 128:276-81.); in both cases,
essentially identical results were obtained compared with the
standard technique. Likewise two groups have reported successful
results using a the micronucleus test from a capillary fingerstick
(Lee T K, O'Brien K F, Wiley A L, Jr., Means J A, Karlsson U L.
Reliability of finger stick capillary blood for the lymphocyte
micronucleus assay. Mutagenesis 1997; 12:79-81; Xue K X, Ma G J,
Wang S, Zhou P. The in vivo micronucleus test in human capillary
blood lymphocytes: methodological studies and effect of ageing.
Mutat Res 1992; 278:259-64.). Therefore, a 50 .mu.l amount is
expected to be sufficient. In fact as discussed below, the second
embodiment of the machine has been designed explicitly to avoid
losing lymphocytes. Thus, Ficoll Hypaque density fluid can be added
to a 75 .mu.l hematocrit tube, which is centrifuged and imaged (see
Centrifuge/Band Recognition Module, above). The tube is then
robotically transported (with or without a known radiation
exposure) to the multi-well plate, the tube is cut off below the
lymphocytes layer, and all the remaining cells are washed into the
appropriate well. All further cell handling steps take place inside
the well, thus providing a system with essentially no loss of
lymphocytes.
[0304] In term of experimental design, device optimization can
proceed as earlier described, with comparisons between the
slide-based approach and 96 well approach and subsequently a 384
well based approach. As discussed in detail above, a custom
384-well plate has been designed for use in some embodiments, with
the same overall plate size as a standard plate, and with only a
slightly smaller well area (16 mm2 vs 25 mm2) than standard 96-well
plates.
[0305] .gamma.-H2AX foci in lymphocytes can provide a "same-day
answer" type biodosimeter. Previous work on .gamma.-H2AX in human
lymphocytes, as described herein. shows the high potential of this
assay. To optimize the assay for use in the system described
herein, the assay was automated, the post-exposure time dependence
of the .gamma.-H2AX foci in human lymphocytes was quantified and
inter-personal variations of sensitivity for this assay was
assessed, in terms of age and smoking status, and intrinsic
radiosensitivity.
[0306] Briefly, the lymphocyte separation is carried out as
described above. In the .gamma.-H2AX assay, lymphocytes are not
cultured, but are immediately delivered to multi-well plates where
they are processed. Current technique is described in several
publications (Rogakou E P, Boon C, Redon C, Bonner W M. Megabase
chromatin domains involved in DNA double-strand breaks in vivo. J
Cell Biol 1999; 146:905-16; Pilch D R, Sedelnikova O A, Redon C,
Celeste A, Nussenzweig A, Bonner W M. Characteristics of gamma-H2AX
foci at DNA double-strand breaks sites. Biochem Cell Biol 2003;
81:123-9.). The speed of the various processes (cell fixation,
processing and generation of antibody fluorochrome signal) can be
maximized, so that the device can provide a final dosimetric answer
within a few hours of the start of the processing.
[0307] Initial studies with ex-vivo irradiated blood will use a
number of doses (0, 0.5, 2, 5, 10 Gy), as well as a number of times
post exposure (0.5 h, 4 h, 24 h and 36 h) in order to quantify the
dose dependent time dependence of the response. Ideally, all 20
dose-time points are covered by blood from each individual, which,
based on preliminary studies should be possible. This allows
further sub-analysis by age and by smoking status. Subsequently,
the blood samples obtained from the TBI patients will allow us to
test and refine the system calibration.
[0308] Micronuclei in blood reticulocytes, as a same day
biodosimeter. Just as the .gamma.-H2AX assay provides a rapid
biodosimeter useful for post-exposure times between 0 and .about.30
h, so the assay for micronuclei in blood reticuloctyes provides a
rapid biodosimeter useful for post irradiation times between
roughly 24 and 48 hours (Lenarczyk M, Slowikowska M G. The
micronucleus assay using peripheral blood reticulocytes from
X-ray-exposed mice. Mutat Res 1995; 335:229-34). The assay can be
modified as needed to allow full automation, and can be quantified
in terms of the post-exposure time dependence of the assay, and the
practicality of using .about.50 .mu.l of blood, from a capillary
fingerstick or laser skin perforator, for the assay.
[0309] Mature erythrocytes are anucleate, but chromosomal damage
events leading to micronuclei can appear in early reticulocytes
after moving into the blood stream from marrow but before passing
through the spleen where they are removed. Micronuclei can be
detected at low frequency in red blood elements, and these
frequencies are enhanced after individual exposure to chromosome
damaging agents (Offer T, Ho E, Traber M G, Bruno R S, Kuypers F A,
Ames B N. A simple assay for frequency of chromosome breaks and
loss (micronuclei) by flow cytometry of human reticulocytes. Faseb
J 2004.), such as ionizing radiation.
[0310] In a first embodiment described here, peripheral blood is
separated into its constituents, with the red blood cells separated
from serum and white blood cells. In a similar fashion to that
outlined above for mononuclear cells, a sample of red blood cells
will be drawn from the lower portion (Pawar V B, Prabhu A.
Isolation of large numbers of fully viable human reticulocytes
using continuous Percoll density gradient. Clin Lab Sci 1991;
4:360-4) of the Vacutainer tube, and smeared by the wedge technique
onto a microscope slide, while the comparison samples will be
placed in the well of a 96-well plate. Unlike the micronucleus
assay in lymphocytes, no cell handling procedures are required, but
rather cells are immediately processed in situ, with rinsing,
vacuum filtration and fixation, prior to staining with DAPI. This
DNA binding specific fluorochrome renders the micronuclei present
in reticulocytes visible as small, bright spherical or
near-spherical encapsulated micron-sized objects.
[0311] Initial protocols studies can be done using unirradiated
blood from healthy human volunteers. Experiments carried out with
this assay using ex-vivo irradiated blood from human volunteers
will be of limited utility, so studies can be undertaken using
reticuloctyes from the total body irradiation (TBI) patients--in
fact using the same blood samples from which the lymphocytes will
be extracted. For each TBI patient, an assay can performed for
micronuclei in reticuloctyes at 24, 48, and 72 hours post
exposure.
[0312] High-throughput automated processing and analysis of
micronuclei in exfoliated bladder cells from urine, and exfoliated
buccal cells is also contemplated. Urine also contains exfoliated
cells shed from the lining of the bladder. Such cells can be
collected and can be shown to express enhanced levels of
micronuclei following the exposure of an individual to DNA damaging
agents (Moore L E, Warner M L, Smith A H, Kalman D, Smith M T. Use
of the fluorescent micronucleus assay to detect the genotoxic
effects of radiation and arsenic exposure in exfoliated human
epithelial cells. Environ Mol Mutagen 1996; 27:176-84; Sarto F,
Finotto S, Giacomelli L, Mazzotti D, Tomanin R, Levis A G. The
micronucleus assay in exfoliated cells of the human buccal mucosa.
Mutagenesis 1987; 2:11-7; Titenko-Holland N, Moore L E, Smith M T.
Measurement and characterization of micronuclei in exfoliated human
cells by fluorescence in situ hybridization with a centromeric
probe. Mutat Res 1994; 312:39-50.). Therefore the expression of
micronuclei in urothelial cells following exposure to ionizing
radiation has the potential to reflect the dose of radiation
received. Such exfoliated cells express their micronuclei in the
mononucleate state and cannot be further cultured. The intent here
is to collect urine from the same group of total body irradiation
(TBI) patients providing peripheral blood for the assessment of
pre- and post TBI micronucleated lymphocytes. In each case the
radiation exposure history of the samples will be blinded at the
time of processing.
[0313] Prior to these studies being undertaken the protocol for
urothelial cell analyses can be established using urine from
healthy volunteers. The standard technique for exfoliated
urothelial cell assays involves centrifuging the sample, washing
and concentrating before placement on a microscope slide and
staining with dyes prior to examination by standard light
microscopy. This approach is not amenable to high throughput, high
speed image analysis, we shall develop a single test tube procedure
whereby the sample is placed in a centrifuge tube with an optical
quality base. After spinning and removal of the supernatant, we
will stain with the DNA specific DAPI fluorochrome prior to
examination for the incidence of cells with micronuclei. DAPI
provides situations with the least amount of background for
subsequent imaging. A robotic handler will place tubes directly
onto the imaging system of our Phase I device.
[0314] Exfoliated buccal cells from the oral cavity can be
collected with a brush or spatula and have also been shown to
express micronuclei in humans after exposure to chromosome damaging
agents including ionizing radiation (Belien J A, Copper M P,
Braakhuis B J, Snow G B, Baak J P. Standardization of counting
micronuclei: definition of a protocol to measure genotoxic damage
in human exfoliated cells. Carcinogenesis 1995; 16:2395-400; Moore
L E, Warner M L, Smith A H, Kalman D, Smith M T. Use of the
fluorescent micronucleus assay to detect the genotoxic effects of
radiation and arsenic exposure in exfoliated human epithelial
cells. Environ Mol Mutagen 1996; 27:176-84; Tolbert P E, Shy C M,
Allen J W. Micronuclei and other nuclear anomalies in buccal
smears: methods development. Mutat Res 1992; 271:69-77). They are
handled in a similar manner to the urothelial cells, and provide an
additional potential indicator of radiation exposure.
[0315] In some embodiments, protocols are optimized and the device
calibrated using unirradiated samples from healthy human
volunteers. For actual testing, the device will use biofluid
(urine, blood, saliva, sweat) samples from patients already being
exposed to total body irradiation (TBI) as part of their medical
therapy, as well as healthy non-irradiated volunteers. For example,
samples of blood and urine from irradiated subjects collected
elsewhere (e.g. at Pittsburgh and MSKCC) will be processed and
constituent parts distributed for measurement and assessment.
Micronuclei, .gamma.-H2AX foci, and functional genomics changes in
blood samples measured. In addition, blood from anonymized healthy
volunteers will be collected and then irradiated ex vivo and
processed as above. Blood and urine samples will also be collected
from patients undergoing total body irradiation prior to
transplantation procedures. 12 cc or 25 cc of blood and 30 cc or 60
cc of urine will be collected. The samples will be sent to Columbia
University Medical Center or to Harvard University School of Public
Health for testing according to the methods and using the device as
described herein. Blood samples a) from healthy volunteers
collected at the NCl through the Department of Transfusion
Medicine, and b) collected from TBI patients at MSKCC and
Pittsburgh, will be also studied for the production of
radiation-induced .gamma.-H2AX foci and for metabolomics
products.
[0316] Blood, urine, saliva and sweat samples (biofluids) will be
collected from patients planned to receive total body irradiation.
Samples will be collected before and after exposure to radiation.
The samples are coded, and the investigators involved in the
measurements will know only the radiation dose, the age, gender,
and smoking status of the subject from which the sample was taken.
The collection of biofluid samples will be collected before and
after the subjects received whole-body irradiation. Blood samples
will be obtained either by venipuncture, through a lancet
fingerstick, or through a laser skin perforator.
[0317] A major challenge posed by local public health authorities
is the actual sample collection in the field from tens of thousands
of individuals. To simplify sample collection, some embodiments
relate to a kit (FIG. 40) containing matched bar-coded capillaries
and data collection cards, a capillary holder as well as anything
else required by the sample collector (gloves, lancets, etc.). The
capillary holder has also been designed such that three holders
exactly fit in one centrifuge bucket, simplifying the input stage
to the biodosimetry workstation. The capillary holder will also be
pre loaded with capillary sealing putty and gelled separation
medium, to provide a simple collection protocol compatible with the
optimal lymphocyte separation requirements (50 .mu.l blood layered
on 50 .mu.l separation medium).
[0318] Having described the invention in detail, it will be
apparent that modifications, variations, and equivalent embodiments
are possible without departing the scope of the invention defined
in the appended claims. Furthermore, it should be appreciated that
all examples in the present disclosure are provided as non-limiting
examples.
EXAMPLES
[0319] The following non-limiting examples are provided to further
illustrate the present invention. It should be appreciated by those
of skill in the art that the techniques disclosed in the examples
that follow represent approaches the inventors have found function
well in the practice of the invention, and thus can be considered
to constitute examples of modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments that are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
Example
Barcoding Capillary Tubes
[0320] One of the major challenges overcome by embodiments of the
invention described herein is traceability. A method and system is
needed that is operable with tens of thousands of samples per day
arriving from multiple collection facilities, and that is able to
correlate between the results of each sample and the identifying
information of the individual. Unambiguous (and foolproof) labeling
of small plastic capillary tubes (outer diameter .about.2 mm) is a
challenge. There are no commercial barcoding or RFID technologies
for capillary tubes. One solution is to laser-etch a 10-digit
barcode on each capillary, as shown in FIG. 41.
[0321] Capillary blood sampling is increasingly used for screening
(for example, blood gas analysis) in a hospital setting, as
compared with venipuncture. This is because capillary blood
sampling is a much easier technique that can be performed by
medical technicians, causes less discomfort to the patient, and has
a much lower frequency of adverse events. Until now, however, there
has not been a practical electronic ID system for capillary tubes
due to their small outer diameter (.about.2 mm), raising the
possibility of misidentified samples.
[0322] Accordingly, a novel barcode system for capillary tubes was
developed based on laser etching of PVC tubes, and the ease of both
writing and reading the barcodes has been confirmed. Briefly, 1-D
barcodes (encoding ten numeric digits or 1011 samples) were marked
on PVC capillary samples using a 350 nm wavelength UV laser system.
The code was approximately 10 mm long and 1.5 mm high. The barcode
was marked using power of 1 Watt and speed 45 ips. Resolution
achieved was 100 .mu.m, and the cycle time was 0.16 seconds. The
barcodes were successfully read by a high-resolution stationary
scanner.
[0323] This will allow the capillary to arrive at the collection
center pre-labeled, reducing the chance of error. It has been shown
that by using this technique, the capillaries can be reliably
labeled and identified using an automated system.
Example
RFID Labels
[0324] RFID labels (Philips ICode I smart IC, part SL1 ICS31; 512
bit, 13.56 MHz, 97 pF) were also successfully tested for
application to PVC hematocrit capillaries. These can be read by a
ACG HF Multi ISO reader with a USB or RS-232 interface. An
evaluation kit to test suitability is used. However, the cost (70
cents/piece) and size of the labels (30.times.7 mm) are less well
suited.
[0325] Although the transport tubes and well plates can be tagged
by bar coding, an RFID solution would be preferable; because the
tubes can be identified easily on entry to the system and the
archival plates can be located and retrieved. Collision issues can
be addressed. Individual wells can be readily identified with
alphanumeric indices and do not need individual tagging.
[0326] A diagram showing an embodiment of the method and system
described herein is shown in FIG. 42.
[0327] Design parameters of some embodiments of the invention
disclosed herein are set forth in the following table.
TABLE-US-00001 Design Parameter Value Centrifuge Module Relative
centrifugation 0-2000 g factor (speed) Centrifugation time 5-15
minutes Capillary Length of Capillary 75-170 mm Volume of Capillary
50 .mu.l of blood + up to 50 .mu.l of separation medium Material
Plastic (PVC). Incubation Module Incubation period for 1-3 hours
(pre-mitotic) and 1-2.5 a single sample days for the micronuclei
Assays micronucleus and pre-mitotic (e.g. .gamma.-H2AX) Throughput
6,000 samples/15 hr-day (phase 1) 30,000 samples/15 hr-day (phase
2) Microplate Number of wells 96-wells (phase 1), 384-wells (phase
2) Footprint Standard multi-well plate (130 mm .times. 85 mm)
Geometry of well cross- Circular (phase 1), square (phase 2)
section Design Off-the-shelf (phase 1), custom-made.(phase 2)
Filter at the base of each 0.6 .mu.m pore from Millipore Corp. well
Maximum volume 300 .mu.l (phase 1) capacity of the well
[0328] In other embodiments, the relative centrifugation factor can
be up to 15,000 g. In one embodiment, smaller capillaries can
used.
Example
Blood Collection Module
[0329] In one embodiment, blood collection is performed by finger
stick and the blood collected into capillaries. Commercially
available glass capillaries from QBC diagnostics were used. These
tubes are internally coated with an anticoagulant and a dye which
is potentially lethal to lymphocytes.
[0330] In one embodiment, plastic capillaries coated with an
anticoagulant are used. Capillaries used for the micronucleus assay
are also coated with a stimulant to reduce the incubation time.
Capillaries used for the pre-mitotic assay are coated with a
fixative, as the lymphocytes must be fixed at the time of
collection to preserve their original state.
[0331] In one embodiment, the cap for these capillaries contains a
gelled separation medium to enhance the lymphocyte separation.
[0332] Though the expected required sample volume is 50 .mu.l (see
below), some embodiments of the system can accommodate capillaries
with a capacity of 50-100 .mu.l in order to take into account
inclusion of lymphocyte separation medium. The centrifugation
parameters and separation method are discussed below.
[0333] Each capillary will be uniquely identifiable for correlating
the sample with a patient. The identification and tracking of
samples is discussed below.
[0334] At the collection point the capillaries will be filled with
blood, capped (thus adding the separation medium) and placed in a
container for shipping to the workstation where it will be dropped
as a whole into the centrifuge for lymphocyte separation. For
shipping/centrifugation containers 50 ml dilution tubes, modified
to accommodate 24 capillaries each, which will be inserted in a
traditional centrifuge as illustrated in FIG. 43.
[0335] In another embodiment a custom insert containing 44
capillary-sized holes will be used. The holes will be pre loaded
with sealing material and gelled separation medium such that
inserting the capillary will add the required amount of separation
medium and seal the capillary bottom. The insert is designed to fit
directly into the centrifuge.
Example
Irradiation Module
[0336] In some embodiments, an irradiation module, based on a
radioactive source array, is used to normalize the radiation
sensitivity of the clients. A new compact x-ray irradiation system
which contains no radioactive sources or components was designed
specifically for irradiating blood in capillaries.
[0337] Briefly, a cylindrical geometry is used with the sample at
the center. There will be a small diameter, cylindrical support for
the anode material which will be plated on its outside surface.
Outside of the anode structure there will be a larger cylinder of
quartz glass which will have an electron emitting material on its
inner surface. There will be vacuum between these cylinders as well
as electrical isolation for the accelerating voltage. The final
cylinder, outside of these, will be an aluminum reflector. In the
intervening space there will be five UV lamps to induce the
necessary electron current.
[0338] In order to get uniform exposure in a capillary, the
attenuation length should be comparable to the capillary diameter;
this dictates the X-ray energy and therefore the anode material. In
one embodiment, copper K X-rays, which have an energy of about 10
keV are used. The cross section for production of K X-rays with
electrons rises from a threshold equal to their energy and reaching
a maximum at three times that, or 30 keV in this case. Because 10
keV X-rays are strongly attenuated by most materials, the anode
support will be made of beryllium.
Example
Lymphocyte Separation Module
[0339] Separation of the lymphocytes from whole blood is done by
centrifugation. An extensive review of existing centrifuges, rotors
and buckets was conducted, and the design requirements were set. A
computer-aided-design (CAD) model of both buckets and rotor of an
existing centrifuge, used for the experiments was developed using
PRO/Engineer.TM. as illustrated in FIG. 44. In this embodiment, the
centrifuge is a Sorvall Legend T which has four buckets, each
containing seven modified 50 ml tubes, with 24 capillaries each (a
total of up to 672 capillaries). The bottom chamber of the
centrifuge contains the motor, control equipment and interface
panels.
[0340] Design requirements of electromagnetic clutches and brakes
were recognized; a review of existing electromagnetic braking
systems was also performed. A preliminary analysis of centrifuge
unloading was performed and several design concepts of robotic
grippers for the capillaries, bucket and rotor were also
considered. Custom-made designs of both single capillary and batch
capillary grippers were compared.
[0341] The expected centrifugation time for the capillaries
represents the throughput limit for our system. Several
centrifugation experiments resulted in an estimate of
centrifugation time of 10 minutes.+-.5 minutes. The centrifuge will
have an adjustable angular velocity, and will be capable of
delivering centrifugal accelerations up to 2000 g.
[0342] In some embodiments, after centrifugation, the lymphocytes
form a thin layer above the compacted red blood cells (RBCs) and
separation medium. The samples will be imaged for lymphocyte band
recognition. To this end, simulations for automatic segmentation of
lymphocyte band on digitized images of actual centrifuged
capillaries were performed using an image recognition software
package (MATLAB). FIGS. 45a and 45c depict the input color images
acquired by a digital camera. These images are based on
centrifugation results with glass hematocrit capillaries.
[0343] Similar investigations will be conducted on centrifuged
plastic hematocrit capillaries in the future. Various methods,
including a gradient based approach, morphological image processing
and color based region segmentation, were investigated to isolate
the boundary of the RBCs that need to be discarded. Among these,
morphological methods on grayscale images produced the best
results. This automatic segmentation program outputs grayscale
images with RBC boundary detection, as shown in FIGS. 45b and 45d.
Based on these observations, very-high quality color images would
be required for color based manipulation and segmentation, while
grayscale image manipulation produces reliable and repeatable
results. In order to increase the computational speed, some
embodiments use a compiled language (C++).
[0344] After centrifugation, lymphocytes contained in each
capillary have to be identified and counted for pre-screening
purposes. To this end, a review of principles and methods of light
scattering for cell identification and counting was conducted.
[0345] Upon completion of the lymphocyte identification and
quantification, the lymphocytes have to be transferred from the
plastic capillary tube to a well plate by a robot for further
analysis. More specifically, the lymphocytes need to be extracted
from each capillary and poured into the wells of the well plate.
Three alternative extractions methods have been identified:
traditional mechanical cutting, punctuation and non-contact laser
cutting. In one embodiment, laser cutting minimizes the possibility
of cross-contamination of the samples. One of the challenges is to
reduce the heat-affected zone so as to decrease the influence of
heat transfer, associated with the cutting, on the blood samples.
To this end, some embodiments use an integrated
laser-cutting/marking system (based on the use of existing devices
from Control Systemation Inc.), capable of cutting capillaries
filled with blood samples and of producing a reduced heat-affected
zone.
Example
Lymphocyte Incubation
[0346] The incubation module and liquid handling systems should be
capable of processing both the pre-mitotic and the micronucleus
assays. The processing time for the pre-mitotic assay is expected
to be between 1 and 3 hours. Processing time for the micronucleus
assay is expected to be 2.5-3 days. During this process, various
reagents need to be added to or drained from the wells. Given the
system requirements, the typical number of plates that will be
simultaneously inside the automatic incubator is 200-300 for all
contemplated embodiments.
[0347] Several design concepts have been analyzed for the
implementation of the incubation module. The use of off-the-shelf
automatic incubators, modified incubators and custom-made
incubators was considered. Existing automatic incubators feature up
to 1000 positions. However, the single-position (i.e., one
microplate at a time) of existing automatic trays strongly hampers
the throughput of these systems. In order to increase the
throughput, design modifications (such as the addition of i) an
internal robot manipulator, ii) one/two linear conveyors, iii)
input/output temperature/humidity/CO2 control buffers and/or two
side-mounted gantry robots) to existing incubators were considered.
To further increase the throughput, concepts for custom-made
robotic incubators, featuring an internal conveyor along with
internal vacuum-to-waste modules were considered.
[0348] With reference to the liquid handling system, several
existing robotic liquid/plate handlers were investigated. Existing
robotic liquid/plate handlers usually consist of a gantry robot and
an'end effector for adding reagents to standard 96 well plates. The
volume range of reagent that can be added per well is typically
1-200 .mu.l, 20-300 .mu.l or 40-1000 .mu.l. For the micronucleus
assay (see Table below), the most suitable volume range for the
dimensioning of the pipette tips of the robotic liquid/plate
handler is 20-300 .mu.l. However, two operations, OP4 and OP10 (see
Table below), might not be performed accurately because the volume
of reagent to be added is equal or below 20 .mu.l. Additionally,
robotic liquid/plate handlers (independently from the pipette tip
volume range) lack crucial built-in automatic functionalities, such
as agitation and draining (negative pressure) and therefore
operations OP2a, OP5, OP6 (partially), OP7, OP8 (partially), and
OP11 are not supported. In order to automate the operations of the
table below that existing robotic liquid/plate handlers do not
perform, the use of multiple vacuum-to-waste units and orbital
shakers is contemplated.
[0349] Some existing robotic liquid/plate handlers can support the
gamma-H2AX protocol, being capable of automating cell
fragmentation, selective extraction, and affinity purification. The
challenge here is to make the robotic liquid/plate handler
compatible for both protocols (micronuclei and pre-mitotic). While
the end-effector is typically equipped with a 96-well plate
gripper, these systems generally lack online motion planning
capabilities. If a faulty condition takes place within the
workspace of the gantry robot (e.g., a plate falls from the tip of
the gantry robot), the system is not capable of automatically
recovering from this faulty condition. This applies also to the
robotic liquid/plate handlers that use an optical sensor to
recognize liquid stocks in the supply vessels, control accessories
on the work surface, control the positioning of the dispensing, and
check if pipette tips/labware match the requested protocol. Even if
the system can detect the error, it is not capable of acting in
order to recover from the faulty condition (e.g., the fallen plate
lies in a position which is not parallel to the XY stage of the
gantry robot). In this regard, the use of a dexterous and
intelligent manipulator, which services the vacuum-to-waste stage,
the robotic liquid/plate handler, the multi-position orbital
shakers, and the incubator, is used.
TABLE-US-00002 TABLE Current Micronuclei Assay for
Lymphocytes--Phase 1 biodosimetry Built-in Functionality of
Existing Robotic Liquid/ OP Liquid/Plate Handling Operation Volume
Handled Plate Handlers 1 Pour lymphocytes + plasma into 30-100
.mu.l Not available well. 2a Suck out liquid through filter same as
above Not available 2b and fill with culture medium 75% of well
capacity Available (225 .mu.l for phase 1) 3 Incubate 37 C., humid
air + 5% CO.sub.2 Not available 4 After 44 h add cytochalasin-B (6
in 5-20 .mu.l of saline Available (limit: 20-300 .mu.l) .mu.g/ml) 5
After 28 h, suck out medium through 75% of well capacity Not
available filter 6 Add cold (4.degree. C.) 0.075M KCl and 75% of
well capacity Partially available (only addition suck out medium
through filter of cold (4.degree. C.) 0.075M KCl is immediately.
available) 7 Re-suspend in fixative, agitate to 75% of well
capacity Not available (only fixative prevent clumps and suck out
addition is available) medium through filter 8 Wash cells with
fixative (without 2 .times. 75% of well capacity Partially
available (only fixative formaldehyde) twice, i.e. fill wells
addition is available) and suck medium through filter. 9 Add medium
75% of well capacity Available 10 Stain cells with 10% Giemsa in
5-20 .mu.l of saline Available (limit: 20-300 .mu.l) potassium
phosphate buffer (pH 7.3) and acridine orange (10 .mu.g/ml in
phosphate buffered saline pH 6.9) 11 Aspirate medium through
filter. 75% of well capacity Not available
Example
Transfer of Samples to Permanent Substrate
[0350] The final step in preparation of the samples for viewing by
the image analysis system is to transfer the filter bottoms of the
multi-well plates to a supporting substrate. In some embodiments, a
rewettable adhesive (poly-vinyl alcohol), similar to that used on
postage stamps, can be applied to a solid substrate. The gluing is
accomplished with residual moisture on the filter bottom after the
last wash.
[0351] One embodiment obtains a bond by placing the liquid adhesive
in the wells, drawing it through the filter under vacuum and then
applying the wet plate to a porous substrate to dry. Moreover, a
fully hydrogenated form of the adhesive was used, which is not
re-wetable and therefore will form a more stable surface suitable
for archival storage. Some embodiments will also include an
anti-fade agent to further improve stability. The substrate is a
non-fluorescent semi-rigid expanded plastic from Porex, Inc.
[0352] Another embodiment obtains a bond by using an adhesive film
such as an ELISA plate sealer and a mechanical punch which
transfers the filter bottoms, onto the film. The film is then
sealed by lamination to prevent regions not containing a filter
from remaining sticky.
Example
Imaging Module
[0353] For the imaging, a modified version of the microscope used
for the microbeam endstation (Randers-Pehrson G, Geard C R, Johnson
G, Elliston C D, Brenner D J. The Columbia University single-ion
microbeam. Radiat Res 2001; 156:210-4) is developed, connected to a
high speed camera (150 fps, 1024.times.1024, CMOS camera) and frame
grabber board. The flow diagram of the imaging system is shown in
FIG. 47. The optical path is divided in two by a cold dichroic
mirror (rather than a cube switcher), so that the image of small
red-fluorescing beads is continuously reflected into the focusing
channel, while the main image goes on undisturbed. The tube lens on
the focus channel has an added weak cylinder lens. This allows a
simple, one-step focusing routine; the aspect ratio of the bead
will be proportional to the focus error, allowing fast automated
corrections with a single picture. A fast piezo stage makes the
corrections needed.
[0354] A preferred embodiment improves on standard microscope
design by inserting a 2D scan head, illustrated in FIG. 48 just
above the objective lens. This enables rapid switching between
adjacent fields of view of the microscope, faster than can be done
with a mechanical stage. Extensive simulations have been run to
determine the required mirror size and scan angles. A large mirror
is too slow to rotate and costs time in switching between fields of
view. A small mirror does not collect light efficiently from
external fields of view and require longer exposures in the camera.
The optimal mirror size was found to be approximately 20 mm.
[0355] In these simulations the objective lens design was taken
from Nikon, the auto focus module was replaced with a gap and the
location of the mirrors was taken from the design drawings. FIG.
48c shows the required adjustments to the mirror to compensate for
a movement of the object. As expected, it was observed that the
mirrors are practically decoupled and each one compensates for
deflections in an orthogonal axis.
[0356] In some embodiments, a control software using Visual C++,
which has the flexibility needed for this work is used.
Example
Sample Identification and Tracking
[0357] A review of methods for capillary labeling and tracking was
conducted and promising results were obtained with laser marking of
hematocrit capillaries. Three main factors need to be considered
while considering various technologies for sample identification:
errors, time factor and cost. The workstation should be able to i)
track samples through the complete process, ii) create sufficient
redundancy levels and iii) maintain complete database of sample
data. Bar-coding and radio-frequency identification (RFID), one a
widely used technology and the other, an emerging technology, were
analyzed in detail for applications to the workstation under
development. Results of a comparative analysis among passive,
active RFID and barcodes are summarized in the following table.
TABLE-US-00003 BARCODES PASSIVE RFID ACTIVE RFID Modification
Un-modifiable Modifiable Modifiable of Data Security Minimal
Security From minimal to highly secure Highly Secure Amount of Data
Linear 8-30 Characters 64 kB Up to 8 Mb 2D: 7200 Numbers Cost Low
(<few cents) Medium (>25 cents) Very High (>10-100 S) Life
Span Short unless etched into metal Indefinite 3-5 Yr battery Life
Standards Stable and agreed Evolving to agreed standards Evolving
open standards Reading Line of sight (3-5 ft) No contact or line of
sight (up No contact or line of sight (up to Distance to 50 ft) 100
m or more) Potential Optical barriers e.g. Dirt and Environment or
fields that Limited barriers since broadcast Interference object
between tag and reader affect transmission of RF signal from tag is
strong Multiplicity 1 at a time Hundreds of tags nearly
simultaneously
Example
Dimensioning of Robotic Systems
[0358] At the design level, a series of potential robotic systems
in charge of manipulation and transportation operations of
capillaries and well-plates have been identified. Both hardware and
software specifications and attributes of robotic systems,
conveyors, robot manipulators (modular, gantry, serial, parallel),
linear robots and rotary actuators, are being analyzed.
[0359] Several off-the-shelf robotic systems are under testing in a
CAD environment. CAD models of both modules and workstation layout
have been generated in order to perform "reach and interference"
analyses between robot manipulators and the rest of the workstation
components (incubator, centrifuge, liquid handling system, etc.).
To automate the capillary and plate handling operations, two
robotic systems are used: a robot manipulator, responsible for
capillary/tube/bucket handling-related operations, and a second,
plate-handling, robotic system in charge of the plate
handling-related operations.
[0360] Both the capillary handling robotic system and the plate
handling robotic system are capable of performing online motion
planning, based on encoder, vision, force and proximity sensor
readouts. This allows the system to quickly recover from potential
faulty conditions (e.g., accidental falls of capillary/plate from
the gripper). To this end, an analysis of specifications and
attributes of different types of intelligent serial robot
manipulators has been conducted. The use of different types of
robot manipulators, such as selective compliant assembly robot arms
(SCARAs), five and/or six-degrees-of freedom robots, is currently
under analysis. SCARAs are very suitable for pick and place
operations.
Example
Optimization of Lymphocyte Separation Protocol
[0361] Both assays require separation of lymphocytes from whole
blood. Accordingly, the optimal methodology for extracting
lymphocytes from whole blood has been explored. Tests have been run
using glass capillary tubes (QBC diagnostic AccuTube) to optimize
the lymphocyte separation protocol by centrifugation. The
capillaries can hold up to 100 .mu.l of solution and are internally
coated with sodium heparin (10 .mu.g) and K2EDTA (0.33 mg). The
anticoagulant mixture is optimized for the 100 .mu.l of blood.
[0362] The centrifugation parameters (speed and time) have also
been optimized, as this has a large impact on the design of the
whole system. FIG. 49a shows the number of lymphocytes counted per
.mu.l of blood as a function of the centrifuge speed (5 min
centrifugation time). In order to facilitate the lymphocytes
erythrocytes separation, 50 .mu.l of histopak (1.077 g/ml) has been
added to the capillaries
[0363] Although as many as 80% of the lymphocytes present in the
blood sample can be collected, at low centrifugation speeds the
lymphocyte solution is still contaminated by some erythrocytes FIG.
49b.
[0364] The number of lymphocytes retrieved as a function of elapsed
time from blood collection has been investigated. As expected, the
number of lymphocytes retrieved decreases linearly down to about
15% after 2 days FIG. 49c. This limit is the time allowed between
the blood collection and analysis.
Example
Optimization of Lymphocyte Incubation in 96-Well Plates
[0365] Another important and novel aspect of our design is the
incubation of lymphocytes in filter-based multiwell plates. This is
done to facilitate medium exchange and the addition/removal of
reagents without needing to pellet the lymphocytes each time.
[0366] In a first embodiment of the device the lymphocytes are
incubated in 96 well plates (Multiscreen plates from Millipore),
while in a second embodiment of the device they will be incubated
in custom designed 384 well plates.
[0367] The filter used for this application must be carefully
selected: it must be non-fluorescent, to allow imaging of the
lymphocytes, it must be easily detachable, and the pore size must
be optimal.
[0368] In order to test the effect of pore size, whole blood was
used and red blood cells were selectively lysed using 0.85%
ammonium chloride. 0.45 .mu.m pores tend to get clogged by the
lysed red blood cells, resulting in a very slow removal of liquid
from the wells, though this is not a problem for
centrifuge-separated lymphocytes. On the other hand, 1.2 .mu.m
pores were sufficiently wide to allow the lymphocytes to enter and
become lodged in the pores, and thus could not be extracted for
imaging. Accordingly, 0.65 .mu.m pore-size plates were
selected.
[0369] In one embodiment, the lymphocytes within each capillary
after centrifugation are separated from the red blood cell ("RBC")
pellet and dropped within the microwell. Cultures are set up in
each well with complete medium containing 15% heat inactivated FBS,
PHA (M-form), L-glutamine and antibiotics. After incubation at
37.degree. C. for 44 hours cytochalasin B (in DMSO) was added in
order to block cytokinesis. After 28 hours with the cytochalasin B
at 37.degree. C., cells are treated with hypo and fixed in a
fixative such as, for example, Carnoy's fixative. The liquid
already present within each well is drained out by the application
of a positive pressure before the addition of any fresh reagents.
Finally, the cells are allowed to dry and stained with an agent or
combination of agents such as Acridine Orange and DAPI and viewed
under a fluorescent microscope.
Example
Automated Cytogenetic Imaging
[0370] MetaSystems has automated cytogenetic imaging platforms,
such as the Metafer system (Hande M P, Azizova T V, Geard C R,
Burak L E, Mitchell C R, Khokhryakov V F, et al. Past exposure to
densely ionizing radiation leaves a unique permanent signature in
the genome. Am J Hum Genet. 2003; 72:1162-70; Mitchell C R, Azizova
T V, Hande M P, Burak L E, Tsakok J M, Khokhryakov V F, et al.
Stable intrachromosomal biomarkers of past exposure to densely
ionizing radiation in several chromosomes of exposed individuals.
Radiat Res 2004; 162:257-63.). Briefly described herein is
experience with this platform (Schunck C, Johannes T, Varga D,
Lorch T, Plesch A. New developments in automated cytogenetic
imaging: unattended scoring of dicentric chromosomes, micronuclei,
single cell gel electrophoresis, and fluorescence signals.
Cytogenet Genome Res 2004; 104:383-9.), which provides
cytogenetic-based automated imaging. The Metafer platform features
motorized x, y and z motion, autofocusing, automatic exposure
control, CCD-based image acquisition hardware, and an 80 slide
scanning stage.
[0371] This system has been previously used for automated scoring
of micronuclei (Varga D, Johannes T, Jainta S, Schuster S,
Schwarz-Boeger U, Kiechle M, et al. An automated scoring procedure
for the micronucleus test by image analysis. Mutagenesis 2004;
19:391-7), dicentric chromosome aberrations (Schunck C, Johannes T,
Varga D, Lorch T, Plesch A. New developments in automated
cytogenetic imaging: unattended scoring of dicentric chromosomes,
micronuclei, single cell gel electrophoresis, and fluorescence
signals. Cytogenet Genome Res 2004; 104:383-9.), and analysis of
single-cell gel electrophoresis (Schunck C, Johannes T, Varga D,
Lorch T, Plesch A. New developments in automated cytogenetic
imaging: unattended scoring of dicentric chromosomes, micronuclei,
single cell gel electrophoresis, and fluorescence signals.
Cytogenet Genome Res 2004; 104:383-9.).
[0372] The Metafer system has also been used for high-throughput
scoring of chromosome aberrations (translocations, dicentrics and
inversions) of highly exposed radiation workers, as well as
micronuclei and .gamma.-H2AX foci (Hande M P, Azizova T V, Geard C
R, Burak L E, Mitchell C R, Khokhryakov V F, et al. Past exposure
to densely ionizing radiation leaves a unique permanent signature
in the genome. Am J Hum Genet 2003; 72:1162-70; Mitchell C R,
Azizova T V, Hande M P, Burak L E, Tsakok J M, Khokhryakov V F, et
al. Stable intrachromosomal biomarkers of past exposure to densely
ionizing radiation in several chromosomes of exposed individuals.
Radiat Res 2004; 162:257-63; Balajee A S, Geard C R. Replication
protein A and gamma-H2AX foci assembly is triggered by cellular
response to DNA double-strand breaks. Exp Cell Res 2004;
300:320-34.
[0373] FIG. 50 shows a composite of radiation-induced micronucleus
yields (in human lymphocytes irradiated ex vivo) obtained with the
Metafer automated scanning system, from the MetaSystems group
(Varga D, Johannes T, Jainta S, Schuster S, Schwarz-Boeger U,
Kiechle M, et al. An automated scoring procedure for the
micronucleus test by image analysis. Mutagenesis 2004; 19:391-7.)
(diamonds) and yields obtained using the system and methods
described herein (circles). At a given dose, each data point
corresponds to a different individual, indicating the significance
of inter-personal variation, particularly at high doses (Thierens
H, Vral A, de Ridder L. Biological dosimetry using the micronucleus
assay for lymphocytes: interindividual differences in dose
response. Health Phys 1991; 61:623-30.).
Example
High-Speed Imaging with the Amersham in Cell 3000 Machine
[0374] Studies were performed using the state-of-the-art Amersham
(GE Healthcare) IN Cell Analyzer 3000. This machine is a line
scanning, confocal imaging system, based on standard 96- or
384-well microplates, which was developed specifically for
performing high-throughput cellular assay screening very rapidly
and at high resolution. It is believed to be currently the fastest
such machine on the market. Some key features of the machine are:
two laser line-scanning light sources (krypton: 647 nm, and argon:
364 and 488 nm), high speed, dynamic infrared laser autofocus,
imaging performed by three high-speed, cooled, 12-bit CCD cameras,
field of view using 40.times. objective is 0.75.times.0.75 mm with
a spatial resolution of 1.2 .mu.m, standard image size is
1250.times.1250 pixels, so up to 30 individual fields can be imaged
per well on a 96 well microplate, well scan time for a single
0.75.times.0.75 mm field per well, is 2 min at 2.4 .mu.m
resolution, and robotically-based microplate handling.
[0375] The IN Cell 3000 has dedicated image analysis modules which
perform rapid imaging and quantitative analysis of sub-cellular
components, as well as a "Developer" module allowing users to
"teach" the system to recognize particular structures, such as,
binucleated cells and micronuclei.
[0376] While the IN Cell 3000 has generally performed well within
its limitations, its throughput is limited for the current high
throughput purposes by the fact that it does not have the
capability to "intelligently" scan. Instead it scans the entire
area of interest at high resolution, rather than doing a
preliminary scan at low resolution, and then doing high resolution
scanning only in areas where there are potentially interesting
objects. A second feature that limits its throughput is its use of
CCD rather than CMOS technology.
Example
.gamma.-H2AX in Human Peripheral Human Lymphocytes
[0377] To date, no information has been published on .gamma.-H2AX
foci after irradiation of human lymphocytes. Accordingly, the
dose-response and the inter-person variability of radiation induced
.gamma.-H2AX foci in peripheral blood lymphocytes was studied.
Briefly, blood samples were taken from healthy human volunteers
through the NIH Department of Transfusion Medicine, and irradiated
ex-vivo within a few minutes of being drawn.
[0378] Blood cells were separated both by fractionation on
ficoll-hypaque-metrizoate gradients and using FACS. We Magnetic
separation of lymphocyte subpopulations using antigen specificity
was also explored. Because the blood used in each experiment was
from a different individual, variability between independent
experiments from age-defined donors (<30 y, vs. >50 yrs) was
examined. Each day blood was obtained from one younger and one
older donor, and processed. The experiment was twice repeated with
blood from different donors. The results are shown in FIG. 51,
together with some in-situ images of the .gamma.-H2AX foci (green)
in lymphocytes.
[0379] It should be noted that, in these experiments, assays were
performed 2 hours after radiation exposure. Based on the published
data from Banath J P, Macphail S H, Olive P L. Radiation
sensitivity, H2AX phosphorylation, and kinetics of repair of DNA
strand breaks in irradiated cervical cancer cell lines. Cancer Res
2004; 64:7144-9, significant increases over controls at 24 h after
a 2 Gy exposure were expected.
Example
Robotics
[0380] The Manufacturing Research Laboratory (MRL) provides the
foundation advanced laser-based manufacturing technologies and
industrial manipulators (robots). Published work from the MRL
includes work on robotic dynamics (Yao Y L. Transient lateral
motion of robots in cylindrical part mating. Robotics and Computer
Integrated Manufacturing 1991; 8:103-11; Yao Y L, Korayem M H, Basu
A. Maximum allowable load of flexible manipulators for a given
dynamic trajectory. Robotics and Computer-Integrated Manufacturing
1993; 10:301-9; Yao Y L, Cheng W. Model based motion planning of
robot assembly of non-cylindrical parts. International Journal of
Advanced Manufacturing Technology 1999; 15:683-91.), precision (Yao
Y L, Wu S M. Recursive calibration of industrial manipulators by
adaptive filtering. Journal of Engineering for
Industry-Transactions of the ASME 1995; 117:406-11), and kinematics
(Huang Z, Yao Y L. A new closed-form kinematics of the generalized
3-DOE spherical parallel manipulator. Robotica 1999; 17:475-85;
Abdul Majid M Z, Huang Z, Yao Y L. Workspace analysis of a six-DOE,
three-PPSR parallel manipulator. International Journal of Advanced
Manufacturing Technology 2000; 17:441-9).
[0381] The MRL is examining studying robotic preparation of cDNA
from single cells to develop an instrument that couples the power
of robotics with that of DNA array technology. The breadboard-stage
instrument developed (as described below) incorporates an inverted
research microscope capable of wide-field deconvolution microscopy
as well as a robotic system for manipulation of cells and reagents.
Cells are handled by a robotic pipette arm micromanipulator capable
of changing pipette tips, xyz-positioning and nanoliter scale
liquid handling. The system incorporates pipetting of several
different types of fluids. Pipetting accuracy has been evaluated to
validate that the system achieved sufficient linearity and
accuracy.
Example
High-Speed In-Situ Cellular Image Analysis
[0382] Imaging and Control Program for High Throughput Single-Cell
Identification: At Columbia University's Radiological Research
Accelerator Facility (RARAF), single-cell single particle
irradiation experiments rely on a purpose-built fast cell imaging
analysis approach to recognize the specific cell targets for
irradiation (Randers-Pehrson G, Geard C R, Johnson G, Elliston C D,
Brenner D J. The Columbia University single-ion microbeam. Radiat
Res 2001; 156:210-4). In the basic system, an integrated program
written under the Windows NT operating system controls the video
analysis system and the motion of the stepping motor driven
microscope stage. For each culture dish, the approximate locations
of the attached cells are established using a low-magnification
lens, so that time is not wasted afterwards imaging empty regions
of the dish at high magnification. This preliminary scan consists
of 10 overlapping images arranged to cover the entire active area
of the dish. The area of objects that are brighter than a set
threshold is used to identify and locate cells. These locations are
then translated into defined fields of view for the
high-magnification (40.times.) objective. Then, each field of view
found during the preliminary scan to contain at least one cell is
moved into position, and a high-magnification video image is
grabbed and analyzed.
[0383] This two stage imaging process results in an order of
magnitude increase in throughput. The entire imaging process for a
dish of 2,000 cells takes 4 minutes; 3 minutes are for the
mechanical stage motion time and 1 minute is for reading the CCD.
In the current invention, this same two-stage approach is used for
imaging, though the system is made much faster by using fast
optical scanning mirrors instead of a mechanical stage to switch
between the low- and high-magnification fields of view and by using
CMOS imaging sensors that have a faster read out than conventional
CCD cameras.
[0384] Image Analysis Algorithms: To carry out assays on large
numbers of cells, techniques for high throughput automatic
identification and localization of individual cells have been
developed, based on advanced machine learning techniques (Long X,
Cleveland W L, Yao Y L. Effective automatic recognition of cultured
cells in bright field images using Fisher's linear discriminant
preprocessing. In Proceedings of IMECE04: 2004 ASME International
Mechanical Engineering Congress. Anaheim, Calif.; 2004; Long X,
Cleveland W L, Yao Y L. Automatic detection of unstained viable
cells in bright field images using a support vector machine with an
improved training procedure. Computers in Biology and Medicine
2004: accepted; Long X, Cleveland W L, Yao Y L. A new preprocessing
approach for cell recognition. IEEE Transactions on Information
Technology in Biomedicine 2004: accepted.). The techniques are
highly relevant to the image analysis needs of the current
invention, and are used as described in greater detail below.
[0385] The learning approach uses a feed-forward Artificial Neural
Network in conjunction with an effective preprocessing technique,
Fisher Linear Discriminant (FLD) (Long X, Cleveland W L, Yao Y L. A
new preprocessing approach for cell recognition. IEEE Transactions
on Information Technology in Biomedicine 2004: accepted). In
addition, a more sophisticated variation to the Support Vector
Machine (SVM) approach has been developed, known as Compensatory
Iterative Sample Selection (CISS), to not only identify cells but
to further distinguish viable cells from non-viable cells and other
non-cell objects (Long X, Cleveland W L, Yao Y L. Automatic
detection of unstained viable cells in bright field images using a
support vector machine with an improved training procedure.
Computers in Biology and Medicine 2004: accepted). An important
feature of these algorithms is that they permit supervised
learning. Essentially, the system is taught to distinguish between
cells and non-cells (or viable-cells and other objects) using
images that have been pre-classified by a human expert or other
means. Differences in object appearances and image variations such
as focus, illumination, size and noise are simply accommodated by
training.
Example
Optimization of .gamma.-H2AX Foci Staining Protocol
[0386] The need to detect DNA damage by radiation requires specific
markers that can be easily seen and quantified, and .gamma.-H2AX
foci formation is one such event that can be used in this scenario.
It has been shown that H2AX phosphorylation is specific to sites of
DNA damage and is also indicative of the amount of DNA damage.
However, in order to use .gamma.-H2AX as a quick screening tool, it
must be optimized for sensitivity and rapidity, which is what we
are aiming to achieve.
[0387] The first aspect addressed is the image quality of foci in
cells. Several parameters were tested to optimize the image
quality. For example, light intensity ratios of foci can be
optimized through antibody concentrations during
chemi-luminescence. The goal was to achieve the sharpest image
possible and also to record the relationship between radiation
level and foci counts. For the first experiments, to characterize
the .gamma.H2AX induction, MEF cells in culture were used. In these
cells, an increase of foci number with increasing x-ray dose was
seen (FIG. 51).
[0388] Antibody concentrations were also optimized based on the
contrast between the cell background and fluorescence signal given
by the .gamma.-H2AX foci. Cells that exhibited the largest
intensity ratio were deemed the best for viewing, having the most
distinction between foci and cellular background. Images of cells
treated with various concentrations of both the primary and
secondary antibodies were compared. It was found that the 1:100
dilution for the primary antibody and 1:500 for the secondary
antibody yielded the best intensity ratios. A comparison was also
done using different kinds of blocking agents, and it was found
that even though Superblock (Pierce Biochemicals) yielded faster
results, NFDM (Non fat dried milk) provided clearer foci
images.
[0389] Following these experiments in MEF cells, similar
experiments were performed in human lymphocytes. It was found that
the primary antibody dilution of 1:50 with a secondary antibody
dilution of 1:250 yielded the best brightness of foci. DAPI
concentration of 1.5 .mu.g/ml and 250 ng/ml in mounting medium with
anti-fade were compared and it was found that 250 ng/ml yielded the
best contrast between the foci irradiated with 2Gy .gamma.-rays and
fixed 30 and nuclear membrane.
* * * * *