Patent Application
20060094109
Automated image analysis systems have been used to reduce subjective errors in cell classification between different operators in manual methods, but such prior art systems without preliminary cell enrichment steps still inherently lack sensitivity. Several automated cell-imaging systems have been described or are commercially available for cell analysis. The system developed by Chromavision, ACIS™ or Automated Cellular Imaging System (Douglass et al., U.S. Pat. No. 6,151,405) uses calorimetric pattern recognition by microscopic examination of prepared cells by size, shape, hue and staining intensity as observed by an automated computer controlled microscope and/or by visual examination by a health care professional. The system uses examination of cells on microscope slides and was designed for tissue sections. The SlideScan™ or MDS™ systems of Applied Imaging Corp. (Saunders et al., U.S. Pat. No. 5,432,054) is described as an automated, intelligent microscope and imaging system that detects cells or “objects” by color, intensity, size, pattern and shape followed by visual identification and classification. In contrast to the ACIS system this system has the ability to detect fluorescent labels, which provides more capability. However, these and other currently available methodologies are not sufficiently sensitive for accurate classification and typing of rare events such as circulating tumor cells in blood.
Epithelial cells are not present in blood under normal circumstances. In patients with epithelial derived cancer (carcinomas) cancer cells can be shed into the blood. These cells are rare in peripheral blood and exhibit a large dynamic range from patient to patient. Tumor cells can be present in blood of carcinoma patients at extremely low frequencies (<10 cells/mL). Flow cytometry and/or fluorescence microscopy are analytical methods frequently used for analyzing the prepared samples. Flow cytometry has the advantage that it is sensitive and reproducible but it lacks the ability to simultaneously assess the immunophenotype and morphological features of the detected cells. Although fluorescence activated cell sorting (FACS) can be used to sort immunophenotypically identified cells it is quite a challenge to sort the rare events and preserve them for cytological evaluation. In addition, the skill level needed for the latter is prohibitive for a clinical assay. Fluorescence microscopy has the disadvantage that considerable and variable cell losses are associated with the preparation of the sample slides for microscopic analysis. However it has the advantage that a cell can be visually confirmed as having features consistent with malignancy.
An analytical system must be capable of accurately identifying as few as 1 cell while still being capable of enumerating as many as 104 cells. The detection of these circulating tumor cells (CTC) is further complicated by their heterogeneity, not only in size and shape, but also in their antigen expression profile such as cytoskeletal proteins that can be present at extremely low or high copy numbers.
Accordingly, the present invention seeks to improve upon the aforementioned methodologies, and to provide simple and efficient means and methods for automated imaging of objects that can be used, for example, in conjunction with high sensitivity immunophenotyping, to permit detection, enumeration and accurate classification of rare target species, such as CTC in blood or other fluids.
The present invention is a cell analyzer that differentiates immunomagnetically cells that are aligned on Nickel lines while passing through a focused laser beam. In one preferred embodiment, a conventional CD player objective was used to focus a laser-diode onto the magnetically aligned cells. An optical focus and tracking system analogous to that used in a CD player was used to scan along the lines. The emitted fluorescence signals were projected onto, and measured by, photomultiplier tubes. The absolute and relative cell populations identified by the instrument system correlated well with the numbers obtained with a standard flow cytometer or hematology analyzer.
In further embodiments, the features of the instrument system were expanded to demonstrate the potential for rare cell analysis by building on its sensitivity to measure immunofluorescence signals. This was accomplished through the addition of the ability to revisit the events of interest, and providing bright field and fluorescent images of these objects.
A further embodiment of the invention includes a scanning mirror to deflect the illumination sources. As the objects of interest pass through illumination, a “rastered” pattern is formed across the objects, which is subsequently detected and transformed into a more detailed image of the object, creating a raster image.
Herein, various terms that are well understood by those of ordinary skill in the art are used. The intended meaning of these terms does not depart from the accepted meaning.
This invention provides devices and methods that permit the application of novel imaging capabilities to such systems as the CellTracks™ cell analysis system as described by Tibbe et al. (Nature Biotech. 17, 1210-13, 1999). Briefly, in a preferred embodiment of the invention, after magnetic collection and enrichment from blood, the magnetically labeled cells are aligned along ferromagnetic lines of nickel (Ni) and are scanned by a laser focused by means of a conventional objective lens such as from a compact disk player. Since the cells have been selectively stained with one or more fluorescent labels, the measured fluorescence emissions and the intensities can be used to identify or classify the cell type.
Epithelium derived tumor cells in peripheral blood are extremely rare but can be present in the blood of cancer patients. During analysis, the certainty that an event present in a biological sample is an epithelial cell with the assumed characteristics diminishes with the number of events in the analysis gate. Additional and preferably independent information on the individual events aids in the correct classification of the event as an epithelium derived tumor cell. As described hereinbelow, epithelial cells will be immunomagnetically selected from 7.5 mL of blood and magnetically aligned in a sample chamber between a series of parallel thin film nickel lines. The CD head scans along all nickel lines and captures the fluorescence signals of the objects between the lines. Objects that immunophenotypically classify as epithelial tumor cells are revisited for imaging to determine if the identified objects indeed classify as epithelial tumors cells or as debris derived from epithelial cells.
The term “target bioentities” as used herein refers to a wide variety of materials of biological or medical interest and can be distinguished from “non-target” materials that are present in the specimen. Examples include hormones, proteins, peptides, lectins, oligonucleotides, drugs, chemical substances, nucleic acid molecules, (e.g., RNA and/or DNA) and particulate analytes of biological origin, which include bioparticles such as cells, viruses, bacteria and the like. In a preferred embodiment of the invention, rare cells, such as fetal cells in maternal circulation, or circulating cancer cells may be efficiently isolated from non-target cells and/or other bioentities, using the apparatus and methods of the present invention.
The terms “biological specimen” or “biological sample” may be used interchangeably, and refer to a small potion of fluid or tissue taken from a human test subject that is suspected to contain biological entities (or bioentities) of interest, and is to be analyzed. A biological specimen refers to the fluidic portion, the cellular portion, and the portion containing soluble material. Biological specimens or biological samples include, without limit bodily fluids, such as peripheral blood, tissue homogenates, nipple aspirates, colonic lavage, sputum, bronchial (alveolar) lavage, pleural fluids, peritoneal fluids, pericardial fluids, urine, and any other source of cells that is obtainable from a human test subject. An exemplary tissue homogenate may be obtained from the sentinel node in a breast cancer patient. Biological entities refer to objects of interest, as would be understood from the previous description.
The term “determinant”, when used in reference to any of the foregoing target bioentities, refers broadly to chemical mosaics present on macromolecular antigens that often induce an immune response. Determinants may also be used interchangeably with “epitopes”. A “biospecific ligand” or a “biospecific reagent,” used interchangeably herein, may specifically bind determinants. A determinant refers to that portion of the target bioentity involved in, and responsible for, selective binding to a specific binding substance (such as a ligand or reagent), the presence of which is required for selective binding to occur. In fundamental terms, determinants are molecular contact regions on target bioentities that are recognized by agents, ligands and/or reagents having binding affinity therefore, in specific binding pair reactions.
The term “specific binding pair” as used herein includes antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing sequences, Fc receptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin and virus-receptor interactions.
The term “detectably label” is used to herein to refer to any substance whose detection or measurement, either directly or indirectly, by physical or chemical means, is indicative of the presence of the target bioentity in the test sample. Representative examples of useful detectable labels, include, but are not limited to the following: molecules or ions directly or indirectly detectable based on light absorbance, fluorescence, reflectance, light scatter, phosphorescence, or luminescence properties; molecules or ions detectable by their radioactive properties; molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties. Included among the group of molecules indirectly detectable based on light absorbance or fluorescence, for example, are various enzymes which cause appropriate substrates to convert (e.g., from non-light absorbing to light absorbing molecules, or from non-fluorescent to fluorescent molecules). Analysis can be performed using any of a number of commonly used platforms, including multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, CellSpotter® analysis, CellTracks™ analysis, and automated cell analysis.
The phrase “to the substantial exclusion of” refers to the specificity of the binding reaction between the biospecific ligand or biospecific reagent and its corresponding target determinant. Biospecific ligands and reagents have specific binding activity for their target determinant yet may also exhibit a low level of non-specific binding to other sample components.
The present system was designed to identify rare cells. The term “rare cells” is defined herein as cells that are not normally present in biological specimens, but may be present as an indicator of an abnormal condition, such as infectious disease, chronic disease, injury, or pregnancy. Rare cells also refer to cells that may be normally present in biological specimens, but are present with a frequency several orders of magnitude less than cells typically present in a normal biological specimen. The detection and enumeration procedure used herein is further described in U.S. Pat. No. 6,365,362, which is incorporated by reference.
The optics of the present system includes an illumination source and a detector. As an example, the illumination means may be narrow-spectrum lasers or LEDs. It may also be a broad-spectrum white light source, where light is passed through narrow spectrum filters to achieve the desired wavelengths. The detection components consist of two-dimensional arrays of detector elements. Examples of such a detector array include PMTs and CCDs. However, persons skilled in the art will appreciate that other illumination and detection means may be used in this invention.
A sample preparation process involving immunomagnetic selection, separation, and staining of cells from blood volumes ranging from 1-30 mL precedes the analysis of the sample by the present system. Cells were transferred to a sample chamber (described in U.S. Pat. No. 6,136,182 and US 2002/0109838, incorporated by reference herein) and magnetically aligned between 90 parallel 15 □m-wide Nickel lines on the inside surface of the sample chamber, as described in U.S. Pat. No. 5,985,153, which is incorporated by reference herein. The CD head scans along all Nickel lines with an illuminating means in less than 5 minutes, capturing the fluorescence signals of the objects between the lines with a detection means. Cells maintain their position by magnetic force, which creates the possibility to revisit cells of interest. The imaging techniques used in this invention are based on those described in the commonly owned application PCT/US02/00203, which is incorporated by reference herein.
Low noise, high signals, and minimal fluorescence emission spectral overlap of the fluorescence probes are essential for rare cell detection. The system was equipped with 405 nm, 532 nm and 635 nm lasers for excitation of DAPI, PE and APC respectively. To confirm or reject an identified object as a cell, the object of interest is revisited and a violet laser diode is used to obtain an image of the DAPI stained nucleus (
In summary, the immunophenotype, spatial distributions of the digitized fluorescence signals and the bright field and fluorescence images, measured in the system of the present invention, enables the identification of epithelial cells present at low frequency in blood and further enables sub classification of these cells such as intact, damaged or epithelial cell debris. While in this study carcinoma cells lines were used to demonstrate performance of the instrument, it is clear that the instrument can be used for a variety of cell analyses including the detection of other rare events, such as endothelial cells in blood, fetal cells in maternal blood, bacteria in blood or other fluids. The sample preparation process can be modified to be specific to any target of interest (by ferrofluid selection) and the staining of cells would have to be compatible with the laser wavelength and filter selections established within the system.
The system of the present invention was calibrated using 6 □m Deep Red fluorescent beads (Molecular Probes, Eugene, Oreg.) that were labeled with superparamagnetic ferrofluids (Immunicon, Huntingdon Valley, Pa.) as described in U.S. Pat. No. 6,120,856, which is incorporated by reference herein. The magnetic beads were used at a concentration of 3000/mL and were placed in a sample chamber (320 μl) prior to analyzing.
A semi-automated sample preparation system (CellPrep™, as described in U.S. Ser. No. 10/081,996, incorporated by reference herein) was used to process blood samples. In brief, 7.5 mL of whole blood is incubated with EpCAM (epithelial cell adhesion molecule) labeled immunomagnetic particles (ferrofluids described above). The EpCAM antigen is expressed on cells of epithelial origin, but not on blood cells. A series of incubation, separation, and resuspension steps results in a sample of 320 μL placed in a sample chamber (as described in U.S. Ser. No. 10/074,900, incorporated by reference herein) that is held between two poles of a specially designed permanent magnet fixture (as described in U.S. Pat. No. 6,136,182, incorporated by reference herein). The sample contains immunomagnetically-selected cells labeled fluorescently with anti-cytokeratin conjugated to Phycoerythrin (CK-PE), anti-CD45 conjugated to Allophycocyanin (CD45-APC) and the nucleic acid specific dye DAPI (4,6-diamidino-2-phenylindole). The anti-cytokeratin recognizes low molecular weight keratins 4, 6, 8, 10, 13, and 18, present in cells of epithelial origin. The anti-CD45 identifies leukocytes and DAPI stains the cell nucleus.
The sample chamber consists of a molded polystyrene housing on which a glass coverslip is attached that bears a series of parallel thin film Nickel lines (Metrigraphics, Wilmington, Mass.). The glass top is affixed to the chamber body with an optical grade UV curable adhesive (Dymax, Torrington, Conn.) thus forming the chamber cavity. The dimensions of the chamber, 30 mm×4 mm×2.7 mm, yield an internal fluid volume of 324 □L. The sample is dispensed into the chamber through an entry port and capped off by the operator to seal out air and allow the chamber to be placed on the instrument for analysis. Computer simulation was used to determine the optimal magnet angle and distance of the sample chamber surface to obtain the most uniform field gradient. When the cells are within reach of the field gradient exhibited by the Nickel lines, they are drawn in between the lines where they are held in place for scanning.
A computer program was written to simulate the movement of magnetically labeled cells in the chamber. The objective was to move all magnetically labeled cells to the upper surface of the chamber but prevent movement to the magnet poles. The distance from the chamber surface to the surface of the magnet must also be short enough to permit viewing with the CD objective that has a working distance of 3.5 mm.
The optical system must provide five functions:
A magnetic actuation support for the CD objective maintains alignment by moving the objective along two axes, along the optical axis for focus and perpendicular to the Nickel lines for tracking. The 532 nm laser reflection from the Nickel lines is detected through a quarter-wave plate and polarizing beamsplitter by tracking and focus detectors, which provide servo signals for actuation of the CD objective. The system aligns the chamber in preparation for scanning and positions the sample so that the CD objective is aligned on the first pair of Nickel lines. The sample is then moved by a stepper motor in the y-direction (0.1 □m stepsize) along the length of the sample chamber while the CD objective and servo electronics maintain proper focus and tracking. At the end of each line, the instrument indexes the sample in the x-direction by a stepper motor (0.1 □m stepsize) to the next pair of adjacent Nickel lines and repeats the y-direction scanning in the opposite direction. The process is repeated until all 90 Nickel line pairs have been scanned. At 10 mm/sec, the time required to scan all 90 lines is 4.5 minutes.
As each cell passes through a laser spot, the fluorescence is measured by the photomultipliers. Each photomultiplier collects light through a filter and a pinhole, which is parallel to the plane of the Nickel lines. Each pinhole eliminates reflected light from the Nickel lines and views only fluorescence from a selected laser. Minimal crosstalk between fluorescent signals is ensured by the spectral and spatial separation provided by each filter and pinhole. The fluorescence signals are converted to analog signals by a 16-bit analog-to-digital converter board. The multiplexed signals are sampled at a sampling frequency of 25 kHz for a speed in the y-direction of 10 mm/sec. For an epithelial cell with a typical diameter of 12 □m, this sampling rate corresponds to 30 data points across the cell.
To revisit and obtain an image of a specific measured event, it has to be relocated on the sample for which the location in x-y coordinates is needed. To obtain positional information in the y-direction, the stage, which moves the sample under the CD objective, has been equipped with a quadrature encoder (Reneshaw, Gloucestershire, UK) that has a resolution of 0.8 microns. The encoder signals are connected to a counter present on the same analog to digital converter board that samples the photomultiplier signals. Fluorescent signals and y-position information are recorded simultaneously during scanning. The discrete line number on which a specific event is recorded and the positional information in the x-direction is also recorded. In summary, the focus and track system operates as follows:
1. find the origin of the first line;
2. lock focus and track;
3. scan the first line Y-stage 5-10 mm/sec;
4. unlock track and focus;
5. move X-stage to the next line;
6. lock focus and track; and
7. scan the next line.
Steps 4-7 are continued for each line until the entire sample has been scanned. The total scan is 90 lines, times 30 mm/line, which results in a 2.7 m scan for each sample.
Images of selected cells are digitized with a CCD array using the CD objective and imaging optics. To avoid the additional cost, low intensity, and short lifetime associated with broadband light sources, fluorescent imaging is accomplished with the lasers. To avoid laser speckle, an angular scanning mirror scans the laser spots, over the cell as the CCD integrates emitted photons. Using this scanning technique, even very dim fluorescent objects are imaged with excellent signal to noise and resolution.
To assess the size and shape of circulating tumor cells, blood samples of cancer patients were prepared with CellPrep™ and analyzed by a fluorescence microscope system. More than 1000 circulating epithelial cells from 8 patients with a variety of cancers were obtained and cell diameters were measured. Circulating epithelial cells within and between patients were heterogeneous in size and shape.
These criteria were used to establish a line spacing of 15 μm. With a chamber width of 2.7 mm and a 15 μm width of the Nickel, a total of 90 lines are on the chamber surface. The traced fluorescence signals of the objects, and images obtained after revisiting the objects, are analyzed to identify events that are smaller and laterally aligned or clustered between the lines.
A large dynamic range of circulating tumor cells (CTC) ranging from 1 to 5,000 cells per 7.5 mL sample was observed in patient samples. Leukocytes carried over through the sample preparation procedure from 57 blood samples ranged from 428-17,718 cells with a mean of 5,203 and a median of 1,857 leukocytes. The 90 lines on the chamber surface provide a linear space of 2.7 meters for capturing cells. Assuming an average tumor cell size of 12 μm and allowing an occupancy of 10%, the chamber capacity is 22,500 tumor cells and is well within the dynamic range of both captured tumor cells and leukocytes.
Cells from the prostate carcinoma tissue culture cell line, PC3, were used at a concentration of 5,000 PC3 cells/mL. Aliquots (10-100 □L) of this cell suspension were spiked into 7.5 mL whole blood samples of normal donors to obtain blood samples with low tumor cell numbers.
Approximately 300 PC3 cells were spiked into 7.5 mL of blood and processed with CellPrep™ as described above. When the sample is dispensed into the sample chamber while being held in the magnetic field, the immunomagnetically labeled cells are drawn to the upper inside surface of the chamber by magnetic forces from the permanent magnets. A bright field image of the aligned PC3 cells is shown in
In the present instrument system, cells are imaged with laser illumination to avoid the additional cost, low intensity, and short lifetime of broadband light sources. However, the long coherence length of lasers and light reflections in the imaging system contribute coherent noise and speckle in the image. Also, the Gaussian intensity profile of the laser beam does not provide uniform illumination of the object. Both of these problems with coherent illumination are overcome by moving the laser spot across the object as the CCD array integrates the light. Movement of the source during exposure washes out the coherent noise in the image. Any angular scanning means, such as a scanning mirror, rotating mirror, electro-optic or acousto-optic deflector, or electro-refractive device could be used to deflect the beam by a small angle, providing motion of the focused laser spot at the object plane. By driving this device with a periodic signal, the spot is scanned back and forth over the object.
One example of this concept is shown in
As each cell passes through a laser spot (illumination means), a corresponding PMT (detection means) measures the fluorescence. Each PMT collects light through a filter and a pinhole, which is conjugate to the plane of the nickel lines. Each pinhole eliminates reflected light from the nickel lines and views only fluorescence from a selected laser. Minimal cross talk between fluorescent signals is insured by the spectral and spatial separation provided by each filter and pinhole.
During the imaging step, the object is moved into a laser spot, which is scanned back and forth by a scanning mirror. The total angular scan range is appropriate for the size of illumination region and focal length of the objective. The scanning mirror reflects the laser beams through a beamsplitter and objective lens to the object plane. The fluorescence or scattered light from the object passes back through the same objective lens and beamsplitter to a second lens and CCD array for imaging. The scanning frequency must be sufficient to provide many full scans during the integration time of the CCD array. If the mirror passes through only a few full scans and one partial scan, the integrated illumination over the object will not be uniform. The CCD should only collect photons during an integral number of full scans, or many full scans plus one partial scan. Then the partial scan contribution becomes a small portion of the total integrated current on each CCD pixel.
After the imaging scans are completed, the optical system will scan more tracks to measure other fluorescent objects with the PMT detectors. Therefore, the static position of the scanning device must return the system to the original optical alignment of the PMT pinholes with the laser beams and nickel line gap. The system shown in
In
In addition to imaging, the scanning technique could improve the resolution of the fluorescent signals from the PMT detectors. As the laser beam in
The scanning mirror in
As the sample is scanned and analyzed, there will be objects that are detected which the instrument operator may wish to revisit. Because the system is capable of obtaining data from the sample object as well as its location, the system can easily return to the location of the object for further analysis of the object. This further analysis allows for confirmation of the object's classification by the system. The imaging subsystem that is used for revisiting objects of interest is shown in
1. the system returns to the position of interest;
2. the LED which is placed off-axis to enhance contrast is turned on;
3. a bright field image is recorded on the CCD array;
4. the mirror starts raster scanning to provide an uniform laser illumination of the region;
5. a fluorescent image is recorded on the CCD array; and
6. the images are pseudo colored and combined.
These steps can be seen in
1. single intact or damaged cells,
2. debris or cell fragments,
3. cell clusters or epithelial cells attached to leukocytes, and
4. leukocytes nonspecifically binding to the epithelial cell marker.
The preferred embodiments of the invention as herein disclosed, are also believed to enable the invention to be employed in fields and applications additional to cancer diagnosis. It will be apparent to those skilled in the art that the improved diagnostic modes of the invention are not to be limited by the foregoing descriptions of preferred embodiments. Finally, while certain embodiments presented above provide detailed descriptions, the following claims are not limited in scope by the detailed descriptions. Indeed, various modifications may be made thereto without departing from the spirit of the following claims.
This application is U.S. National Stage of PCT/US03/13842, which claims the benefit of U.S. Provisional Application No. 60/377,868 filed 3 May 2002. That application is incorporated by reference herein.
