CLOSED LOOP MONITORING OF AUTOMATED
MOLECULAR PATHOLOGY SYSTEM
BACKGROUND
[0001] The invention relates generally to automated methods and devices for
iterative staining of biological samples for imaging applications.
[0002] Biological samples are used for analytic and diagnostic purposes, such as
diagnosing diseased tissue at a molecular level. A biological sample, such as tissue
micro arrays (TMA), on which tissue samples are fixed are typically stained with a
morphological stain or biomarker. The stained TMA are then analyzed manually with
a microscope, or an image may be taken of the TMA for subsequent analysis or
comparison. After the first stain is applied and imaged, one or more serial or
successive stains or biomarkers may be applied and the TMA may be analyzed again.
The two or more serial images may then be compared. A single staining cycle may
comprise the steps of flowing a stain (antibody) over the tissue, incubating the stain
for an appropriate time, rinsing away the stain to reduce background fluorescence,
imaging the slide, and bleaching away the stain. As part of the multiplexing
technique developed for fluorescent imaging, sequential staining, rinsing, and
destaining cycles are required. The staining cycle may be then repeated for multiple
stains. For multiplexed applications, the TMA needs to be stained with multiple
molecular probes to investigate protein expression or spatial distribution
quantitatively or qualitatively.
[0003] The staining process is typically performed using time-consuming manual
techniques that are susceptible to error. Thus, the total operation time is the sum of
each of these steps multiplied by the total number of applied stains. Currently, time
for each step is determined based on the amount of fluids required for each step. The
time for each step is fixed independent of which antibody (stain) is being applied.
Thus a very weak stain will be subjected to the same bleaching time as a much
stronger stain, even though a shorter bleaching time may suffice. The reagents used
in the staining process are often expensive and have limited shelf life thereby
requiring special handling techniques
[0004] Therefore, it is desirable to provide a method for optimizing one or more
steps in a staining cycle. It is also desirable to automate the process and reduce
manual intervention to make the process time efficient and reliable.
BRIEF DESCRIPTION
[0005] In one embodiment, an automated device for iterative staining of a
biological sample is provided. The automated device comprises a flow cell in fluid
communication with a staining agent unit and a bleaching agent unit, wherein the flow
cell comprises a surface configured to operatively engage the sample therewith, an
illumination source for illuminating at least a portion of the biological sample, a
monitoring unit operatively coupled to the flow cell and configured for monitoring
one or more optical characteristics of the biological sample before, during, and/or
after the application of at least one of a staining agent and a destaining agent. The
device further comprises a processing unit for determining a figure of merit based on
at least one of the optical characteristics of the biological sample, and a controller unit
in communication with the processing unit and the flow cell, wherein the controller
unit is configured to control the application of at least one of the staining agent and
the destaining agent based at least in part on the figure of merit.
[0006] In another embodiment, a closed loop automated method for staining of a
biological sample is provided. The method comprises providing a biological sample,
staining at least a portion of the biological sample by flowing in a reagent, monitoring
one or more optical characteristics of the biological sample, and calculating a figure
of merit based on at least one of the optical characteristics.
DRAWINGS
[0007] These and other features, aspects, and advantages of the present invention
will become better understood when the following detailed description is read with
reference to the accompanying drawings in which like characters represent like parts
throughout the drawings, wherein:
[0008] FIG. 1 is a schematic view of an example of a closed loop automated
system of the invention; and
[0009] FIG. 2 is a flow chart for example steps of a method for closed loop
monitoring of molecular pathology systems.
DETAILED DESCRIPTION
[0010] Embodiments relate to methods and systems for closed loop monitoring of
an automated molecular pathology system for molecular imaging. The methods and
systems enable optimized operation in molecular imaging. In certain embodiments,
the automated molecular pathology system may operate with minimal operator
intervention by reducing or eliminating the need to transfer samples (e.g., tissue
samples on a slide within the flow cell). The systems and methods may reduce or
eliminate the need to displace samples between the staining component and the
imaging component. Closed loop monitoring minimizes both reagent volume and
reagent dwell time within the system thereby saving on expensive reagents, such as
fluorescence labeled antibodies, and minimizing reagent decomposition or side
reactions.
[0011] In certain embodiments, a closed loop automated method for staining a
biological sample comprises providing a biological sample, staining at least a portion
of the biological sample, monitoring one or more optical characteristics of the
biological sample during staining, and determining a figure of merit based on at least
one of the optical characteristics. The method may further comprises rinsing at least a
portion of the biological sample, monitoring one or more optical characteristics from
the portion of the biological sample during rinsing, and determining a figure of merit
based on at least one of the optical characteristics. The method may also comprise
destaining at least a portion of the biological sample, monitoring one or more optical
characteristics from the portion of the biological sample during destaining, and
determining a figure of merit based on at least one of the optical characteristics. The
biological sample may be incubated for a determined period of time after being
stained to provide sufficient time for the antibodies to bind with the molecules in the
biological sample. The imaging for the staining step may be performed during
incubation period. In one example, monitoring during one or more of staining,
destaining and rinsing comprises acquiring images of the biological sample, and
determining the figure of merit comprises determining a light intensity from the
portion of the biological sample using the acquired images. Monitoring during
rinsing may comprise acquiring images from regions away from the biological
sample. For example, the images may be acquired from regions outside the
biological sample.
[0012] To more clearly and concisely describe the subject matter of the claimed
invention, the following definitions are provided for specific terms, which are used in
the following description and the appended claims. Throughout the specification,
exemplification of specific terms should be considered as non-limiting examples.
[0013] As used herein, the term "biological sample" refers to a sample obtained
from a biological subject, including sample of biological tissue or fluid origin
obtained in vivo or in vitro. Such samples may be, but are not limited to, tissues,
fractions, and cells isolated from mammals including, humans.
[0014] In some embodiments, the biological sample includes tissue sections of
colon, normal breast tissue, prostate cancer, colon adenocarcinoma, breast tissue
microarray, breast TMA, or normal prostrate. A tissue section may include a single
part or piece of a tissue sample, for example, a thin slice of tissue or cells cut from a
tissue sample. In some embodiments, multiple sections of tissue samples may be
taken and subjected to analysis, provided the methods disclosed herein may be used
for analysis of the same section of the tissue sample with respect to at least two
different targets (at morphological or molecular level). In some embodiments, the
same section of tissue sample may be analyzed with respect to at least four different
targets (at morphological or molecular level). In some embodiments, the same section
of tissue sample may be analyzed with respect to greater than four different targets (at
morphological or molecular level). In some embodiments, the same section of tissue
sample may be analyzed at both morphological and molecular levels. A tissue
section, if employed as a biological sample may have a thickness in a range that is
less than about 100 microns, in a range that is less than about 50 microns, in a range
that is less than about 25 microns, or in range that is less than about 10 microns.
[0015] As used herein, the term "probe" refers to an agent including a binder and a
signal generator. In some embodiments, the binder and the signal generator of the
probe are embodied in a single entity (e.g., a radioactive or fluorescent molecule
capable of binding a target). In alternative embodiments, the binder and the signal
generator are embodied in discrete entities (e.g., a primary antibody capable of
binding target and labeled secondary antibody capable of binding the primary
antibody).
[0016] As used herein, the term "binder" refers to a biological molecule that may
non-covalently bind to one or more targets in the biological sample. A binder may
specifically bind to a target. Suitable binders may include one or more of natural or
modified peptides, proteins (e.g., antibodies, affibodies, or aptamers), nucleic acids
(e.g., polynucleotides, DNA, RNA, or aptamers); polysaccharides (e.g., lectins,
sugars), lipids, enzymes, enzyme substrates or inhibitors, ligands, receptors, antigens,
haptens, and the like. A suitable binder may be selected depending on the sample to
be analyzed and the targets available for detection.
[0017] As used herein, the term "signal generator" refers to a molecule capable of
providing a detectable signal using one or more detection techniques (e.g.,
spectrometry, calorimetry, spectroscopy, or visual inspection). Suitable examples of a
detectable signal may include an optical signal, and electrical signal, or a radioactive
signal. In one example, the signal generator may include a lumiphore or a
fluorophore.
[0018] As used herein the term "lumiphore" refers to a chemical compound that
demonstrates luminescence including chemoluminescence, bioluminescence,
phosphorescence, and photoluminescence. Representative examples include, but are
not limited to, luminol, lucigenin, acridans, acridinium esters, and dioxetanes, and
fluorophores.
[0019] As used herein, the term "fluorophore" refers to a chemical compound,
which when excited by exposure to a particular wavelength of light, emits light (at a
different wavelength. Fluorophores may be described in terms of their emission
profile, or "color." Green fluorophores (for example Cy3, FITC, and Oregon Green)
may be characterized by their emission at wavelengths generally in the range of 515-
540 nanometers. Red fluorophores (for example Texas Red, Cy5, and
tetramethylrhodamine) may be characterized by their emission at wavelengths
generally in the range of 590-690 nanometers. Examples of fluorophores include, but
are not limited to, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine,
derivatives of acridine and acridine isothiocyanate, 5-(2'-
aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS), 4-amino-N-[3-
vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-
anilino-l-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin, coumarin
derivatives, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-aminotrifluoromethylcouluarin
(Coumaran 151), cyanosine; 4',6-diaminidino-2-
phenylindole (DAPI), 5',5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol
Red), 7-diethylamino-3-(4'-isothiocyanatophenyl)4-methylcoumarin, -, 4,4'-
diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, 4, 4'-diisothiocyanatostilbene-
2,2'-disulfonic acid, 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl
chloride), eosin, derivatives of eosin such as eosin isothiocyanate, erythrosine,
derivatives of erythrosine such as erythrosine B and erythrosin isothiocyanate;
ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-
dichlorotriazin-2-yl) aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-
carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), QFITC
(XRITC); fluorescamine derivative (fluorescent upon reaction with amines); IR144;
IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho
cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red, B-phycoerythrin; ophthaldialdehyde
derivative (fluorescent upon reaction with amines); pyrene and
derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate;
Reactive Red 4 (Cibacron .RTM. Brilliant Red 3B-A), rhodamine and derivatives
such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine
rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123,
rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl
chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-
carboxyrhodamine (TAMRA); tetramethyl Rhodamine, tetramethyl rhodamine
isothiocyanate (TRITC); riboflavin; rosolic acid and lathanide chelate derivatives,
quantum dots, cyanines, and squaraines.
[0020] As used herein the term Oxidant" or "oxidizing agent" refers to a
destaining agent that substantially inactivates a lumiphore. In one embodiment, the
destaining agent comprises a bleaching reagent. Representative oxidizing agents
include active oxygen species, hydroxyl radicals, singlet oxygen, hydrogen peroxide,
or ozone such as hydrogen peroxide, potassium permanganate, sodium dichromate,
aqueous bromine, iodine-potassium iodide, or t-butyl hydroperoxide.
[0021] Multiplexing or multiplexed analysis generally refers to analysis of
multiple targets in a biological sample using the same detection mechanism.
[0022] In molecular imaging, a signal generator (such as, fluorophore) may be
excited and the signal (such as, fluorescence signal) obtained may be observed and
recorded in the form of a digital signal (for example, a digitalized image). For
multiplexing, a similar procedure may be repeated for the plurality of different signal
generators (if present) that are bound in the sample using the appropriate fluorescence
filters. In some embodiments, a series of probes may be contacted with the biological
sample in a sequential manner to obtain a multiplexed analysis of the biological
sample. In some embodiments, a series of probe sets (including at most 4 probes in
one set) may be contacted with the biological sample in a sequential manner to obtain
a multiplexed analysis of the biological sample.
[0023] As used herein, the term "figure of merit" includes, but is not limited to a
light intensity, a contrast of image, a Brenner gradient, or a signal to background ratio.
[0024] Monitoring may be performed at two or more processing steps in the
staining cycle. Each staining cycle may comprise staining at least a portion of the
biological sample, rinsing away the stain to reduce background fluorescence,
destaining the stain. The staining cycle may be repeated for multiple stains. During
staining the sample may be incubated for a determined time period. During one of
more steps in the staining cycle, signals generated by corresponding portions of the
biological sample or portions away from the biological sample may be used to
determine the amount of staining, destaining and/or rinsing. In one example,
monitoring comprises imaging. Imaging may be used to obtain signals representative
of the amount of staining, destaining and/or rinsing that takes place in the biological
sample during a staining cycle. In some embodiments, a monitoring unit may
comprises a microscope operatively coupled to a camera. In some embodiments, a
user may utilize the monitoring unit to record images in more than one field on the
sample slide to locate and map multiple stained entities in the sample. The
monitoring unit may be operatively coupled to an image capture window of the flow
cell such that a sample is positioned within a field of view of the imaging unit. In one
example, the monitoring unit may be disposed adjacent the flow cell. The image
capture window may be defined by the substrate (e.g., microscope slide) upon which
the sample is disposed. The image capture window may include an optically
transmissive material on the underside of the slide-receiving member.
[0025] Each of the staining, rinsing and bleaching steps may be accomplished by
flowing a solution containing a particular reagent over the biological sample
positioned within the flow cell. The following parameters may be controlled to
enhance reactivity and, thereby, reduce reagent consumption (1) flow cell internal
volume; (2) flow cell internal temperature; (3) timing of mixing of constituent parts of
the oxidizing solution (e.g., hydrogen peroxide and sodium bicarbonate); (4) extent of
agitation of the solutions as they pass the sample; and (5) bubble removal or
degassing of the flow cell. Appropriate regulation of these parameters also may
reduce sample degradation, permitting a single sample to yield more data.
[0026] Accessory devices, such as heating elements or agitation elements (e.g. an
acoustic piezoelectric component) may be operatively coupled to the flow cell. In one
example, the accessory devices may be positioned away from the image capture
window through which a microscope, coupled to a camera, may capture images of the
sample during the various phases of processing.
[0027] The disclosed methods may be performed in a system that comprises a flow
cell configured to enable enhanced access to the sample through an image capture
window. The automated methods for staining a biological sample may be performed
by employing the techniques disclosed in U.S. Patent Application Publication No.
2009/0253163 titled "ITERATIVE STAINING OF BIOLOGICAL SAMPLES"
which is incorporated herein by reference.
[0028] FIG. 1 illustrates a closed loop system for an automated molecular
pathology system. The system comprises a flow cell 10 having an enclosed flow
chamber positioned above a tissue sample. The flow cell 10 may comprise a solid
support-receiving member 12, a gasket 14 with a central opening configured to
receive a tissue sample positioned on a slide 16, a lid 18, an inlet port 20, and an
outlet port 22. The flow cell 10 defines a closed chamber when a slide 16 is
positioned in the slide-receiving member 12 and the gasket 14 is sandwiched between
the slide 16 and the lid 18. As the flow chamber is enclosed inside the flow cell 10,
fluid evaporation and, consequently, reagent loss is minimized. Also, the closed
configuration improves temperature control.
[0029] The solid support-receiving member 12 is compatible with a range of
chemical and temperature variations. In one embodiment, the slide holder may
consist of a base and a pin or tab system for securing the slide in the chamber.
[0030] In some embodiments, the flow cell 10 may be fixed on a microscope stage
for the monitoring process. This allows the sample to be exposed to a series of
reagents without manual intervention thereby eliminating realignment of the sample
on the microscope stage for image acquisition or registration. This is particularly
useful for multiplexed staining and imaging as images acquired after each staining
step may be superimposed to form a composite image. The flow cell 10 may be a
modular unit that is adapted to fit onto a microscope stage. Alternatively, the flow
cell 10 may be an integrated unit including a microscope stage.
[0031] The flow cell 10 may be operatively coupled to a fluid handling unit 13 that
may comprise a staining agent unit (not shown) and a destaining agent unit (not
shown). In one embodiment, the staining agent unit is configured to apply a plurality
of staining agents. The flow cell 10 may further be coupled to an illumination source
15 for illuminating at least a portion of the biological sample disposed in the flow cell
10. Non-limiting examples of the illumination source may include metal halide
source, mercury arc lamp, or a light emitting diode. A shutter (not shown) of the
illumination source 15 may be shut off when the imaging unit is not acquiring images
from the biological sample. Closing the shutter may decrease the amount of photobleaching
of the biological sample. In case of continuous imaging, the shutter of the
illumination source may be kept open throughout the duration of imaging.
[0032] The system may further comprise fluidic and temperature control
subsystems to control fluidic delivery and solution temperature in the internal
chamber of the flow cell 10. In one embodiment, the fluidic control system may
further comprise reservoirs, flow sensors, mixing chambers, and degassers to prepare
one or more reagents prior to injection into the flow cell. The advantage of such a
subsystem is to avoid the need of premixing and storing reagents that may have
limited stability or shelf life. The fluidic control subsystem is in fluidic
communication with the inlet port 20 and outlet port 22 of the flow.
[0033] The premixer, which is positioned upstream of the flow cell 10, may be
based on a chamber design or a tube design. The chamber design may include a small
vessel with inlet and outlet ports and containing a mechanical mixer. In some
embodiments, the solutions are mixed at the molecular level by using a premixer to
intersperse the reactants immediately prior to the reagent is introduced into the flow
cell 10. Mixing times should be sufficiently long to generate the reagent and
sufficiently limited to prevent decomposition.
[0034] The gasket 14 may comprise a central opening configured to receive a
tissue sample positioned on a slide. The gasket 14 may be made of a deformable,
chemically inert, rubber or plastic that retains the liquid applied to the flow chamber.
The central opening of the gasket maybe sized to maximize the field of view of the
image acquisition window.
[0035] The inlet and outlet ports 20 and 22 are preferably placed away from the
image acquisition window. Thus, the inlet and outlet ports may be positioned in the
gasket 14 or upon the lid 18. The inlet and outlet ports 20 and 22 may be similarly
sized so that the in-flow rate and the out-flow rate are coordinated to achieve a desired
rate of flow across the sample.
[0036] A temperature control unit may comprise a thermoelectric stage 17 for
temperature control and a resistance temperature detector or thermistor for
temperature measurement. For configurations wherein the bottom surface of the
temperature control unit is in direct contact with the fluid, the contacting surface 24
may be made of chemical resistant material, such as stainless steel or titanium. A
frame 26 may also be used to position the components of the temperature control unit.
[0037] In some embodiments, the invention may further comprise a piezo-electric
element connected to the flow chamber and capable of producing vibration within the
flow chamber by conversion of low voltage electrical signals into acoustic energy. In
a preferred embodiment the piezo-electric element maybe composed of a ceramic,
quartz (Si0 2) or barium titanate (BaTi0 3) . The configuration of the piezo-electric
element provides ultrasonic agitation and influences the flow profile of reagents
through the fluid chamber. This is particularly advantageous wherein the desired
staining reaction is diffusion limited and conventional mechanical mixing is
prohibited by the flow cell geometry.
[0038] The monitoring unit 28 may comprise a photomultiplier tube, or a
photodiode, a charge coupling device, a camera and other suitable image-receiving
devices. The monitoring unit 28 may comprise a microscope operatively coupled to
an image-receiving device, such as but not limited to, a camera. Although not
illustrated, the monitoring unit 28 may further comprise optical elements, such as but
not limited to, an objective lens. The image-receiving device is capable of recording
images of the sample while the sample is disposed between the slide and the flow
channel housing. The monitoring unit 28 may also include a positioning device, such
as an actuator operatively coupled to the camera and configured for adjusting a
position of the camera relative to the flow cell. The positioning device may move
objective lens to different fields of interest on the sample, and perform auto-focus
operations relative to the sample.
[0039] In some embodiments, the monitoring unit 28 may be in communication
with a processing unit 30. The processing unit 30 in turn may be in communication
with a controller unit 32. In some embodiments, the monitoring unit 28 and/or
processing unit 30 may be configured for identifying a saturation in light intensity
value during staining, destaining or rinsing steps. Non-limiting examples of the
processing unit 30 may include an image processing unit.
[0040] The controller unit 32 may be in communication with a control input or
user interface (show) 34. The controller unit 32 may be configured for controlling the
application of at least one of the staining agent and the decolorizing agent based at
least in part on the images generated by the imaging system. The controller unit 32
may control the various components of the flow cell system, including for example
the thermal control unit, the premixer, the vibrational unit, and the pumps. Where the
flow cell 10 is incorporated into a combined sample processing and image acquisition
system, the image acquisition components (e.g., microscope or camera) may also be
controlled by a computer.
[0041] The processing unit 30 may be configured to analyze the image acquired by
the monitoring unit 28. In one example, the monitoring unit 28 may acquire images
in real-time. In this example, the image processing unit 30 may process the images in
real-time. The processing unit 30 may determine the light intensity or color from the
acquired images. In one example, for the staining step, the light intensity may be
plotted as a function of time. The controller unit 32 may be intimated when the light
intensity reaches a saturation point. In a closed loop system, the controller unit 32
may transmit a signal to the flow cell 10 indicating the flow cell to stop the staining
step and to move to the next step, such as the rinsing step. Similarly, for bleaching
and rinsing steps, the light intensity may be plotted with respect to time, and as the
light intensity value stabilizes or as the rate of change of light intensity reaches below
a certain rate the corresponding process (bleaching or rinsing) may be stopped, and
the next step in the staining cycle may be started. The rate of change of the light
intensity may be pre-fed in the system using the user interface 34. In one
embodiment, the actual light intensity values or rate of change of light-intensity
values may be compared with the pre-fed values, and if required, the pre-fed values
may be updated and co-related to the corresponding biological sample or the signal
generator. In some embodiments, the monitoring unit 28 and/or the processing unit
30 may be further configured to determine an exposure time for the sample for one or
more steps in the staining cycle.
[0042] Automation may be achieved through computer control of one or more of
the process steps involved in staining cycle, such as but not limited to, addition of
staining reagents and oxidant. Where the flow cell system is incorporated into a
combined sample processing and image acquisition system, the image acquisition
components (e.g., microscope or camera) may also be controlled by software such as
a program written in LabVIEW or C.
[0043] In some embodiments, one or more of the observing or correlating steps
may be performed using computer-aided means. In embodiments where the signal(s)
from the signal generator may be stored in the form of digital image(s), computeraided
analysis of the image(s) may be conducted. In some embodiments, images
(e.g., signals from the probe(s) and morphological stains) may be overlaid using
computer-aided superimposition to obtain complete information of the biological
sample, for example topological and correlation information.
[0044] In certain embodiments, a closed loop automated method for staining cycle
of a biological sample is provided. The method comprises positioning a biological
sample, such as a tissue section on a microscope slide, in a flow cell, applying a
fluorescent label or a lumiphore to the sample in a manner to allow sufficient contact
time between the lumiphore and the sample which are typically in the range of 30 to
60 minutes depending on the concentration and type of label used, rinsing the
biological sample by applying a wash solution, for example an appropriate buffer
solution to wash away any unbound fluorescent label or lumiphore, acquiring an
image of the labeled sample and determining a saturation in light intensity, and
bleaching. Wherein acquiring the signal comprises acquiring the signal through an
image acquisition window. Imaging may be performed after one or more of staining,
rinsing and bleaching. Alternatively, imaging may be performed after each of
staining, rinsing and bleaching. The method further comprises measuring light
intensity values of the signal generated and correlating the signal with specific
labeling of a biomarker. The imaging may be performed at time intervals that are
greater than exposure time of the sample.
[0045] The imaging may be carried out continuously throughout the staining cycle,
or through a particular step in the staining cycle. In some embodiments, the method
comprises real-time monitoring of at least a portion of the tissue along with a real
time determination of whether the desired state has been reached (i.e. how much
bleaching / staining/rinsing has occurred). In these embodiments, the images may be
continuously acquired images during staining, destaining or rinsing. In other
embodiments, the method comprises intermittent monitoring of the biological sample
for a particular step in the staining cycle. In these embodiments, the images may be
acquired intermittently during staining, destaining or rinsing. For example, imaging
may be performed at a constant time interval for the particular step, such as the
staining step. Alternatively, the imaging may be carried out at a varying time interval
for the particular step, with the time interval decreasing as the time progresses. The
images may be acquired at time intervals greater than an exposure time of the
biological sample to the illumination source. In case of intermittent imaging, the
illumination shutter may be closed when the sample is not being imaged to reduce the
amount of photo-bleaching for the sample.
[0046] In one embodiment, the imaging may be a bright field imaging. The bright
field imaging may enable identification of labels other than the fluorescence imaging.
The imaging may be used to collect data corresponding to color and/or light intensity.
[0047] Embodiments of the invention refer to monitoring one or more optical
characteristics for the one or more steps of the staining cycle. In one embodiment, the
light intensity of the fluorescence from the stained samples may be used to determine
the time period for carrying out the staining/bleaching or rinsing step.
[0048] At step 50, a biological sample is provided. The biological sample is
disposed in a flow cell. At step 52, at least a portion of the biological sample is
stained. Optionally, at step 51, an initial image may be acquired before staining the
sample. Data from the initial image or the background image may be used to
normalize intensity data from the subsequent images. Optionally, at step 53, the
method may include the step of washing the biological sample solution prior to
acquiring the initial image of the sample before staining. For example, after
providing the sample (step 50) a substantially colorless washing solution may be
introduced into the flow channel or in a space defined between the surface on which
the sample is operatively engaged and a slide. The washing solution may be flushed
to condition or prime the sample by bringing the sample and the biological entities
making up the sample into contact with a liquid. The washing step may also provide
a suitable reference to normalize light intensity data.
[0049] The various method steps for applying a staining agent, and applying a
bleaching agent may be accomplished by a variety of washing and/or fluid application
techniques. For example, application of washing solutions, staining agents, and/or
decolorizing agents may be accomplished by a variety of known laboratory
procedures including, but not limited to pipetting; aspiration; mixing; centrifuging;
and combinations of such processes.
[0050] In some embodiments, one or more of the steps may be automated and may
be performed using automated systems. In some embodiments, all the steps of the
staining cycle may be performed using automated systems.
[0051] At step 54, the biological sample is monitored with respect to one or more
optical characteristics of the biological sample. In one embodiment, monitoring
comprises imaging the biological sample. The imaging may be a full field imaging.
The biological sample may be imaged to determine an amount of staining for at least
a portion of the biological sample. As the imaging progresses the light intensity of the
biological sample increases. One or more of the optical characteristics may reach a
saturation state. For example, during staining, the light intensity reaches a saturation
level when the staining is completed. The imaging may be carried out to determine
when the light intensity reaches a saturation level. Subsequent to monitoring, a figure
of merit may be calculated based on at least one of the optical characteristics. The
figure of merit may be determined after each monitoring step. The figure of merit
may be chosen based on the nature of the biological sample, marker or dye. In case of
imaging, at step 55, data acquired from the images may be analyzed to determine
when the light intensity reaches its saturation level. The step 55 may be performed
after each imaging step (step 54). If the light intensity has reached a saturation level,
the system may shift to the next step, which is rinsing. At step 56, the method further
comprises rinsing at least a portion of the biological sample. At step 58, the sample is
monitored, for example, imaged to determine an amount of rinsing for at least a
portion of the biological sample. At step 59, data acquired from the images is
analyzed to determine the progress in the step of rinsing. If the desired progress has
occurred, i.e. the background signal has dropped below some threshold in the case of
rinsing, the step of rinsing may be stopped and the next step of the protocol may be
started. In this way each operation is given only as much time as required as opposed
to a globally specified longer time required to work for all cases. In one embodiment,
during rinsing, the imaging may be performed in an area away from the biological
sample to determine the extent of rinsing.
[0052] To minimize effects of photo-bleaching during imaging the images may be
acquired at a determined time interval for real-time monitoring. The determined time
interval for acquiring the images may be greater than the exposure time. Alternatively
or in addition, an anti-fading agent may be added to the biological sample to reduce
any photo-bleaching of the sample.
[0053] At step 60, at least a portion of the biological sample is destained or
bleached. At step 62, while destaining, the sample is monitored to determine an
amount of destaining for at least a portion of the biological sample. In one
embodiment, the sample may be imaged. The imaging may be used to determine
when the biological sample is bleached to a suitable extent.
[0054] As the destaining progresses, the optical characteristic, such as light
intensity decreases and reaches a minimum value upon completion of destaining. At
step 61, data acquired from the biological sample during monitoring is analyzed to
evaluate the extent of destaining. For example, data acquired from the images is
analyzed to determine the progress in the step of destaining. The data may be
analyzed to determine if the light intensity fall off has reached a saturation level. If
the desired progress has occurred, the step of destaining may be stopped and the next
step of the staining cycle may be started. For example, a new antibody may be
introduced in the flow chamber or another biological sample may be provided (step
64). In one example, saturation in intensity falloff may be determined by
continuously imaging the sample during the destaining step.
[0055] The automated destaining step permits the operator to reprobe a single
sample while maintaining the original registration. The addition of the oxidant results
in destaining of the biological sample due to substantial removal of the signal
produced by the lumiphore. Whether the destaining is accomplished by chemically
altering the lumiphore or by detachment, the signal is reduced by at least 80% and
preferably greater than 90%. This reduction in signal may be measured as the poststaining
intensity at a particular wavelength relative to the initial absolute intensity of
the stained biological to adjust for a concomitant reduction in background signal or
autofluorescence resulting from the destaining step.
[0056] Optionally, prior to destaining, the sample may be imaged to identify a
brightest region that needs to be monitored. Next, an initial image may be acquired
and brightest region that needs to be monitored may be identified.
[0057] In one example, time periods for carrying out each of the steps in the
staining cycle may be pre-fed in the system. In this example, the imaging may be
carried out to confirm whether the pre-fed time periods are correct. If the time
periods for one or more of the steps in the staining cycle are different from the pre-fed
time periods, the pre-fed time periods may be updated in the system. These updated
time periods may be later used by the system for similar samples in future. In one
example, the method may include correlating the light intensity values to a specific
labeling of a biomarker.
[0058] In some embodiments, a location of the signal in the biological sample may
be observed. In some embodiments, a localization of the signal in the biological
signal may be observed using morphological stains. In some embodiments relative
locations of two or more signals may be observed. A location of the signal may be
correlated to a location of the target in the biological sample, providing information
regarding localization of different targets in the biological sample. In some
embodiments, an intensity value of the signal and a location of the signal may be
correlated to obtain information regarding localization of different targets in the
biological sample. For examples certain targets may be expressed more in the
cytoplasm relative to the nucleus, or vice versa. In some embodiments, information
regarding relative localization of targets may be obtained by comparing location and
intensity values of two or more signals.
[0059] The method of the invention allows minimization of cycle time over
complete set of stains / antibodies, thereby improving the through. New stains may be
employed using the method of the invention without extensive testing.
[0060] The systems and methods disclosed herein may find applications in various
fields, such as but not limited to analytic, diagnostic, and therapeutic applications in
biology and medicine. In some embodiments, the systems and methods disclosed
herein may find applications in histochemistry, particularly, immunohistochemistry.
Analysis of cell or tissue samples from a patient, according to the methods described
herein, may be employed diagnostically (e.g., to identify patients who have a
particular disease, have been exposed to a particular toxin or are responding well to a
particular therapeutic or organ transplant) and prognostically (e.g., to identify patients
who are likely to develop a particular disease, respond well to a particular therapeutic
or be accepting of a particular organ transplant). The methods disclosed herein, may
facilitate accurate and reliable analysis of a plurality (e.g., potentially infinite number)
of targets (e.g., disease markers) from the same biologically sample.
[0061] While only certain features of the invention have been illustrated and
described herein, many modifications and changes will occur to those skilled in the
art. It is, therefore, to be understood that the appended claims are intended to cover
all such modifications and changes as fall within the scope of the invention.

CLAIMS:
1. An automated device for iterative staining of a biological sample
comprising:
a flow cell in fluid communication with a staining agent unit and a destaining
agent unit, wherein the flow cell comprises a surface configured to operatively engage
the sample therewith;
an illumination source for illuminating at least a portion of the biological
sample;
a monitoring unit operatively coupled to the flow cell and configured for
monitoring one or more optical characteristics of the biological sample before, during,
and/or after the application of at least one of a staining agent and a destaining agent;
and
a processing unit for determining a figure of merit based on at least one of the
optical characteristics of the biological sample;
a controller unit in communication with the processing unit and the flow cell,
wherein the controller unit is configured to control the application of at least one of
the staining agent and the destaining agent based at least in part on the figure of merit.
2 . The automated device of claim 1, wherein the monitoring unit
comprises a camera, a charge coupling device, a photomultiplier tube, or a
photodiode.
3 . The automated device of claim 1, wherein the monitoring unit is
operatively coupled to a camera.
4 . The automated device of claim 1, wherein the monitoring unit is
configured to acquire an image via an image acquisition window present in the flow
cell.
5 . The automated device of claim 1, further comprising a display device
for displaying the optical characteristics .
6 . The automated device of claim 1, further comprising a premixer to mix
one or more solutions immediately prior to introducing the solution in the flow cell.
7 . The automated device of claim 1, wherein the staining agent unit is
configured to apply a plurality of staining agents.
8 . A closed loop automated method for staining of a biological sample,
comprising:
providing a biological sample;
staining at least a portion of the biological sample by flowing in a reagent;
monitoring one or more optical characteristics of the biological sample; and
calculating a figure of merit based on at least one of the optical characteristics.
9 . The method of claim 8, comprising monitoring the optical
characteristics until saturation of at least one of the optical characteristics, and ceasing
the reagent flow after saturation of the at least one of the optical characteristics.
10. The method of claim 8, wherein the figure of merit comprises one or
more of a light intensity, a contrast of image, a Brenner gradient, or a signal to
background ratio.
11. The method of claim 8, wherein monitoring comprises acquiring
images from the portion of the biological sample during staining.
12. The method of claim 11, comprising closing an illumination shutter
when not imaging.
13. The method of claim 8, further comprising adding one or more antifading
agents to the biological sample to reduce any photo-bleaching of the biological
sample.
14. The method of claim 8, further comprising:
destaining at least a portion of the biological sample;
monitoring one or more optical characteristics of the biological sample;
calculating a figure of merit based on at least one of the optical characteristics.
15. The method of claim 14, wherein monitoring comprises acquiring
images from the portion of the biological sample during destaining.
16. The method of claim 8, further comprising:
rinsing at least a portion of the biological sample; and
monitoring one or more optical characteristics of the biological sample; and
calculating a figure of merit based on at least one of the optical characteristics.
17. The method of claim 16, wherein monitoring comprises acquiring
images from the portion of the biological sample during rinsing.
18. The method of claim 16, comprising imaging regions away from the
biological sample.
19. The method of claim 8, further comprising acquiring an initial image
of the biological sample prior to staining.
20. The method of claim 19, further comprising rinsing the biological
sample with a buffer solution prior to acquiring the initial image.
21. The method of claim 8, wherein monitoring comprises continuously
acquiring images during staining, destaining or rinsing.
22. The method of claim 21, comprising real-time monitoring of the
biological sample.
23. The method of claim 8, wherein monitoring comprises intermittently
acquiring images during staining, destaining or rinsing.
24. The method of claim 23, wherein intermittently acquiring images
comprises acquiring images at time intervals greater than an exposure time of the
biological sample to an illumination source.
25. The method of claim 8, wherein determining the figure of merit
comprises correlating light intensity values with specific labeling of a biomarker.