A SYSTEM AND METHOD THEREOF FOR REAL-TIME AUTOMATIC LABEL-FREE HOLOGRAPHY-ACTIVATED SORTING OF CELLS

Abstract

The present invention relates to an automatic real-time label-free holography-activated sorting of the cell's technique. The technique provides high-discriminative power on the level of the individual cell. The technique includes rapid automated cell processing during cell visualization and flow, with high discriminative power on the level of the individual cell. The technique may be useful in detection of cancer and to identify different stages of oncogenesis.

Claims

1. A method comprising: performing a holographic imaging of a flow of a heterogeneous population of cells to enable label-free quantitative imaging of the flow of cells; automatically processing image data of the holographic imaging to identify a certain type of cells during the flow; and automatically sorting the certain type of cells during flow, thereby obtaining a real-time, automatic, label-free holography-activated sorting of the cells.

2. The method of claim 1, wherein performing a holographic imaging of the flow of cells comprises at least one of (i) performing a digital holographic microscopy and quantitative phase microscopy to measure the quantitative phase profile of the cell being indicative of the optical path delay (OPD) profile of the cell to enable label-free interferometric phase microscopy or (ii) obtaining a plurality of off-axis holograms and performing a quantitative phase reconstruction process.

3. The method of claim 2, wherein automatically processing image data comprises at least one of (i) reconstructing the OPD map for each cell individually; (ii) extracting from each OPD 2D and 3D morphological and quantitative features.

4. (canceled)

5. The method of claim 34, wherein automatically processing image data comprises performing classification based on 2D and 3D morphological quantitative features of the cells during the cell flow.

6. The method of claim 5, wherein automatically processing image data comprises at least one of (i) performing a real-time classification of each cell or (ii) performing classification of unlabeled cancer cells in blood to enable label-free imaging and sorting of cancer cells in blood.

7. The method of claim 6, wherein classifying the cells comprises performing machine learning.

8. The method of claim 6, wherein automatically processing image data comprises performing a sequence of classification, wherein each classification is capable of identifying different types of cells.

9. (canceled)

10. The method of claim 6, further comprising automatically classifying primary and metastatic cancer cells.

11. The method of claim 1, wherein performing a holographic imaging of the flow of cells comprises acquiring at least one single-cell hologram during flow.

12. (canceled)

13. The method of claim 1, wherein automatically sorting the certain type of cells comprises at least one of (i) isolating at least one certain type of cells from other cells in the flow or (ii) automatically sorting the certain type of cells during or following cell visualization.

14. (canceled)

15. The method of claim 1, further comprising at least one of (i) counting cells; or (ii) identifying in the certain type of cells at least one of DNA, RNA, protein or any other metabolite to provide a genetic metabolic profile of a patient.

16. (canceled)

17. The method of claim 15, further comprising analyzing the genetic metabolic profile to enable at least one of a diagnosis or an optimization of a treatment of the patient.

18. A system comprising: a holographic imaging module being configured and operable to image a flow of heterogeneous population of cell; a cell sorting module being configured and operable to sort the flow of cells; and a control unit being configured and operable to receive from said holographic imaging module image data being indicative of the flow of cells; automatically process the image data and identify a certain type of cells during the flow and upon identification of a certain type of cells activating said cell sorting module to enable real-time, automatic, label-free holography-activated sorting of the cells.

19. The system of claim 18, wherein said control unit is configured and operable to at least one of (i) process digital holograms of the cells dynamically and classify the cells during the flow or (ii) calculate an OPD map using a database and extract a plurality of features based on the image phase and amplitude.

20. The system of claim 19, wherein said control unit is configured and operable to classify the cells by using machine learning.

21. (canceled)

22. The system of claim 18, wherein said cell sorting microfluidic module is placed inside the holographic imaging module such that the cell sorting module is viewed through the holographic imaging module.

23. The system of claim 18, wherein said cell sorting module comprises a dielectrophoretic microfluidic module comprises an array of spaced-apart electrodes, when activating said dielectrophoretic microfluidic module comprises operating at least one electrode by alternatively switching on or off one or more relevant electrodes to direct the cells of interest.

24. The system of claim 23, wherein said array of spaced-apart electrodes are positioned on both sides of the dielectrophoretic microfluidic module to define a sorting trajectory for the flow of cells along the dielectrophoretic microfluidic module.

25. The system of claim 23, wherein at least one electrode of said plurality of electrodes is configured and operable to at least one of the following: center and direct the cells along the sorting trajectory into an imaging field of view and a holographic region of interest, push the cells to either side of the dielectrophoretic microfluidic module, increase the distance between cell streams.

26. The system of claim 18, wherein said holographic imaging module comprises a high- or low-coherence off-axis interferometric phase microscope and a microfluidic channel to quantitatively image cells during flow.

27. The system of claim 18, further comprising at least one (i) at least one container for collecting sorted-out cells; or (ii) a plurality of microfluidics pumps.

28. (canceled)

29. The system of claim 27, wherein at least one microfluidics pump of said plurality of microfluidics pumps is configured and operable to direct the sorted-out cells towards said at least one container.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0073] FIG. 1 is a block diagram schematically illustrating the main functional elements of the sorting system of the present invention;

[0074] FIG. 2 is a flow chart schematically illustrating the main functional steps of the method for real-time, automatic, label-free holography-activated sorting of the cells according to some embodiments of the present invention;

[0075] FIG. 3a is a flow chart schematically illustrating a method for label-free imaging and sorting of cancer cells in blood according to some embodiments of the present invention;

[0076] FIG. 3b is a block diagram schematically illustrating a holographic imaging module being configured and operable to acquire and measure the quantitative phase profile of the flowing cells of a sample;

[0077] FIG. 3c is a block diagram schematically illustrating an example of a sorting system according to some embodiments of the present invention;

[0078] FIGS. 4a-4b are block diagrams schematically illustrating a representation of an example of a DEP microfluidic module according to some embodiments of the present invention;

[0079] FIG. 5a presents snapshots of OPD maps taken while the cells are flowing in the microfluidic channel, with the real-time quantitative-imaging visualization and classification results during cell flow;

[0080] FIG. 5b show examples of OPD images used for training for seven types of cells, demonstrating that cells are all driven to the ROI of the classification area and are later sorted by the electrodes;

[0081] FIG. 6 shows histograms of different features extracted from the OPD profiles of cancer and blood cells;

[0082] FIG. 7 shows a scatter plot showing the discrimination between blood and cancer cells for the first three principal components calculated by the PCA analysis;

[0083] FIGS. 8a-8d and 9a-9d show the FOV with a cell flowing into the ROI;

[0084] FIGS. 10a-10b are block diagram schemes of two types of classification processes one step and two-step SVM classifiers; and

[0085] FIGS. 11a-11d show scatter spot in the 2D PCA space for all four classifiers.

DETAILED DESCRIPTION OF EMBODIMENTS

[0086] FIG. 1 is a flow chart schematically illustrating the main functional elements of the sorting system 100 of the present invention. Sorting system 100 comprises a holographic imaging module 102 being configured and operable to image a flow of heterogeneous population of cell (for example of cells A and B); a cell sorting module 104 being configured and operable to sort the flow of cells; and a control unit 106 being configured and operable to receive from the holographic imaging module image data being indicative of the flow of cells; automatically process the image data and identify a certain type of cells during the flow and upon identification of a certain type of cells activating cell sorting module 104 to enable real-time, automatic, label-free holography-activated sorting of the cells. Holographic imaging module 102 and cell sorting module 104 are connected to control unit 106 by wire or wireless communication. Cell sorting module 104 may be implemented by any cell sorting module such as a DEP microfluidic chip being for example embedded with dielectrophoretic electrodes. Holographic imaging module 102 may be implemented by an interferometric phase microscopy.

[0087] FIG. 2 is a flow chart schematically illustrating the main functional steps of the method 200 for real-time, automatic, label-free holography-activated sorting of the cells according to some embodiments of the present invention. Method 200 comprises performing a holographic imaging of a flow of a heterogeneous population of cells in 202 to enable label-free quantitative imaging of the flow of cells; automatically processing image data of the holographic imaging in 204 to identify a certain type of cells during the flow; and automatically sorting the certain type of cells in 206, thereby obtaining a real-time, automatic, label-free holography-activated sorting of the cells. Automatically processing image data in 204 may comprise performing a real-time classification of each cell. The classification the cells may be performed by using machine learning. In some embodiments, in a learning phase, a training is performed by at least one classifying algorithm selected from a group consisting of Support Vector Machine (SVM), Principal Component Analysis (PCA) algorithm, k-nearest neighbors algorithm, Fisher's linear discriminant, Fisher's nonlinear discriminant, Network Acceleration algorithm (NNA), or any other machine learning algorithm and any combination thereof in a communicable and readable database on the cells in order to generate information data being indicative of the type of cells. The classification of the activation of the DEP module are performed by using the trained machine-learning model.

[0088] Automatically processing image data in 204 may comprise performing a sequence of classification, wherein each classification is capable of identifying another type of cells. Automatically sorting the certain type of cells in 206 may comprise isolating at least one certain type of cells from other cells in the flow.

[0089] The technique of the present invention is not limited to any type of cells to be sorted. The cells may include blood cells, cancer cells, stem cells. For example, the technique of the present invention may be used for the classification of unlabeled cancer cells in blood to enable label-free imaging and sorting of cancer cells in blood. Reference is made to FIG. 3a showing a block diagram 300 schematically illustrating an example of a method for label-free imaging and sorting of cancer cells in blood according to some embodiments of the present invention. In this specific and non-limiting example, the cells are sorted based on the decisions obtained during the cell flow by a label-free quantitative imaging and real-time machine learning classification. The real-time machine learning classification enables to automatically classify cancer cells based on their grade (healthy/primary/metastatic) enabling monitoring cancer progression in cancer patients. The DEP microfluidic module may include a flow chamber containing several DEP electrodes that create between them an altering electrical field, to deflect (negative-DEP) the cells from the electrodes and direct them inside the dielectrophoretic microfluidic module during flow. It should be noted that the negative-DEP and electrode pattern shown in FIG. 3a was the one used for the experiments. This configuration is not limiting and other patterns can also be used to direct and split the cells of interest from the flow. For example, Positive-DEP can also be used to attract the cells to the electrodes. In some embodiments, the method comprises detecting cancer cells and removing them from blood cell background, to further purify CTCs from blood after an initial enrichment.

[0090] Reference is made to FIG. 3b, showing an example of a holographic imaging module 320 being configured and operable to acquire and measure the quantitative phase profile of the flowing cells of a sample S. In this specific and non-limiting example, the holographic imaging module comprises an interferometric phase microscopy (IPM) module based on an off-axis Mach-Zehnder interferometer being configured to rapidly capture OPD maps of flowing cells (e.g. primary and metastatic cancer cells, as well as different types of blood cells ex vivo). As shown in FIG. 3b, the holographic imaging module 320 comprises a Mach-Zehnder imaging interferometer with an off-axis configuration. In this specific and non-limiting example, the off-axis Mach-Zehnder interferometer comprises an illumination source SC (e.g. supercontinuum laser (SC-400-4, Fianium)) being connected to an acousto-optic tunable filter (AOTF-VIS, Fianium), two beam-splitters BS1 and BS2, two mirrors M, four retroreflectors RR, sample holder S, two microscope objectives MO, one tube lens TL common to both MO, and a CMOS camera that records the off-axis image hologram. Using this holographic imaging module 320, it is possible to reconstruct the sample complex wavefront from a single camera exposure, and thus it is suitable for the acquisition of rapid dynamics, such as cells during flow. The holographic imaging module 320 is illuminated by the illumination source SC of the holographic imaging module 420, being for example a supercontinuum laser source SC (e.g. Fianium SC-400-4), coupled to an acousto-optic tunable filter (AOTF), aligned to emit a low-coherence light with bandwidth of 650 nm±4 nm. However, in this connection, it should be noted that the system is not limited to the use of low-coherence light source and high coherence illumination can also be used. The beam is split into a reference and a sample beams by BS1. The sample beam passes through the sample S, containing cells flowing in a microfluidic channel, as shown in FIG. 3b inset. The dashed rectangle illustrates the field of view (FOV) imaged onto the camera. This beam is then magnified by microscope objective MO (e.g. Newport, 40×, 0.85 NA). In parallel, the reference beam propagates through identical microscope objective MO. Both beams are projected through the tube lens TL (f=200 mm) onto a CMOS digital camera (e.g. Thorlabs, DCC1545M). Since low-coherence illumination may be used to minimize spatial noise and parasitic interferences, retroreflectors RRs on micrometers may be utilized to adjust the beam paths of the sample and reference beams, allowing to obtain off-axis interference on the camera. These two beams interfere on the camera at a small off-axis angle and induce straight off-axis fringes. In addition, the chosen spectral bandwidth allowed obtaining acceptable interference fringe modulation on the entire camera sensor.

[0091] Reference is made to FIG. 3c, showing an example of a sorting system 400 of the present invention. Sorting system 400 comprises holographic imaging module 420 of FIG. 3b being configured and operable to acquire and measure the quantitative phase profile of the flowing cells of a sample S, a DEP microfluidic module 430 a control unit 406 and optionally four microfluidics pumps 440. The collected sorted-out cells may be directed to a storage vessel by using the microfluidics pumps 440. In this specific and non-limiting example, the illumination source SC of the holographic imaging module 420 emits a low-coherence light with bandwidth of 650 nm±4 nm. This corresponds to coherence length of approximately 20 μm, still allowing wide-field off-axis interference on the camera sensor, but now with a decreased level of coherent noise. The high-visibility off-axis interference is set to lie only in a portion of the field-of-view (FOV) being defined as the region-of-interest (ROI) in which the quantitative phase profile of the cell is presented and analyzed. The laser beam splits into a reference beam and a sample beam. The sample beam is impinging the DEP microfluidic module 430 and magnified by a 40× microscope objective (Leica, 440, 0.66 NA), with a tube-lens (f=200 mm) and onto a CMOS camera (Thorlabs, DCx1545). The 40× objective is positioned in a turret, on which a 10× microscope objective (Leica, 439, 0.25 NA) is positioned as well, and can be used to align the sorting system 400 while viewing the dielectrophoretic microfluidic module 430 in lower magnification and obtaining a larger FOV. The reference beam goes through an identical 40× microscope objective, for beam curvature matching with the sample beam. Both beams are combined by the second beam splitter and projected through the tube lens on the CMOS camera at a small angle, where an off-axis hologram with straight interference fringes is created. Retroreflectors RR are used to match the optical-path between the sample and reference beams. In this specific and non-limiting example, the dielectrophoretic microfluidic module 430 is placed inside the holographic imaging module 430. The dielectrophoretic microfluidic module 430 is then viewed through a ×40 microscope objective. The image is taken by a digital camera, and control unit 406 performs a computer algorithm decoding the optical phase delay of the imaged cell. 20 features based on the image phase and amplitude may be calculated. During the experiments, these features were calculated earlier on thousands of phase image to train a machine-learning algorithm called support vector machine (SVM) which uses the features to discriminate and classify between the different cell types. The SVM algorithm may classify each cell that pass through the ROI. If the cell is classified as cancer, a command to activate the sorting process is sent by the control unit 106 to switch the relevant electrode to sort the cell out.

[0092] In this specific and non-limiting example, a fluorescence module (in the dashed box) wascombined to verify the label-free classification accuracy. Laser-driven light source (LDLS) is used as the fluorescence excitation source, and a CCD camera is used to record the fluorescence images. Low pass filter (LPF) prevents the fluorescence excitation light from getting to the CMOS camera used for holography. The epi-fluorescence system is composed of a laser-driven light source (Energetic, EQ-99 LDLS) and comprises a 4f system to collimate the light, a fluorescence filter cube (Zeiss, filter set 38) to fit to use with green fluorescence protein (GFP), and a digital CCD camera (Zeiss, AxioCam MRm). The fluorescence imaging system was built to have an external validation for the cell label-free classification results, where only the cancer cells emitted fluorescence. The fluorescence excitation light is focused by the objective to a spot in the FOV, located just before the sorting electrodes. Since the emission light is split by the second beam-splitter, an LPF (BrightLine, FF01-496\LP-25) was placed to remove the excitation light and receive the fluorescence emission light on the camera used for the holographic imaging module.

[0093] At the end of the process all the CTCs are concentrated in a single reservoir and may be analyzed genetically by a next generation PCR to provide patients with the most beneficial treatment for their disease. During the experiments, the following techniques were used: (1) preparation of blood sample using a filtration kit to remove small sized cells; (2) microfluidics system including pumps, microchannel and electrical components for directing the cells to designated reservoirs; (3) a holographic imaging module imaging cells (e.g. IPM); (4) a database that include geometrical and optical parameters for the recognition of different subsets of cells; (5) a control unit being configured and operable to obtain the captured pictures from the holographic imaging module, analyze the pictures for the formation of an OPD map using the database and activate the electrical components in the microfluidic system thus directing cells of interest to a reservoir. Later, the cells in the reservoir can be analyzed genetically.

[0094] In some embodiments, the system further comprising at least one communicable and readable database storing instructions which, when executed by at least one data processor, result in operations comprising: training a machine learning model to identify a certain type of cells in the communicable and readable database in order to generate information data being indicative of at least one cell; and, after the step of training, real time identify the cells by means of the trained machine learning model.

[0095] In some embodiments, control unit 406 comprises a data input utility including a communication module for receiving image data being indicative of the flow of cells, an optional data output utility for generating data relating to identified cell(s), a memory (i.e. non-volatile computer readable medium) for storing database i.e. preselected data indicative of different OPD maps, and a data processing utility adapted for identifying a certain type of cells during the flow. Data processing utility may operate as a classifier or may comprise a classifier module. Memory may be relayed via wireless or wired connection by an external unit to a central database. The database may be implemented with Microsoft Access, Cybase, Oracle, or other suitable commercial database systems.

[0096] In some embodiments control unit 406 is configured in a cloud-based configuration and/or utilize Internet based computing so that parts of processing utility, and/or memory may reside in multiple distinct geographic locations. Upon identification of certain cells, the data processing utility sends signals to the DEP module 430 to direct the cells along a certain trajectory and sort them. Data processing utility may transmit data regarding the activation of the DEP module via the data output utility, via a data communication (e.g. via cellular network) to a communication module of a central computer. The data processing utility may record the received image data in database in memory and/or may query/cross-reference the received image data with OPD data in the database to identify if the cell is a cell of interest. To this end, the preselected data stored in a database may be used to compare the image data with the OPD maps previously used for identifying cells and stored in the learning database. The memory mat thus be configured for storing a learning database i.e. preselected data indicative of cells correlated with OPD maps. The correspondence between the different OPD maps and the different type of cells may be predetermined. For example, a table of correspondence between the different OPD maps and the different type of cells may be stored in a database. Such table may be stored in the memory. Alternatively, storage may be separate from the server(s) (e.g. SAN storage). If separate, the location(s) of the storage may be in one physical location, or in multiple locations and connected through any type of wired or wireless communication infrastructure. The database may rely on any kind of methodology or platform for storing digital data. The database may include for example, traditional SQL databases such as Oracle and MS SQL Server, file systems, Big Data, NoSQL, in-memory database appliances, parallel computing (e.g. Hadoop clusters), etc. If memory is configured as the storage medium of the database, it may include any standard or proprietary storage medium, such as magnetic disks or tape, optical storage, semiconductor storage, etc.

[0097] The inventors conducted experiments as follows: a blood sample is taken from a cancer patient, and undergo CTC enrichment by filtration (e.g. through a ScreenCell Cyto kit), containing a microporous membrane of 6.5-8 μm pores. This commercial kit captures CTCs and removes smaller cells like erythrocytes and most nucleated blood cells. From each 1 mL of blood, this preliminary filtering results in 11,000 white blood cells and 1-10 CTCs within approximately 3 minutes, with an average CTC recovery rate of more than 90%. This enhanced blood sample is then diluted with 10% Nycodenz, a chemical used to increase the buffer density to help the cells flow smoother. The sample is poured into the DEP microfluidic module of and undergo the final sorting process: As illustrated below, the DEP microfluidic module may include a flow chamber containing several twin-electrodes that create between them an altering electrical field, to deflect (negative-DEP) the cells from the electrodes and direct them inside the dielectrophoretic microfluidic module during flow. The field is created through inducing altering voltage on the electrodes using the control unit (e.g. a computer-controlled generator). The voltage is of 3 Vpp and 1 MHz. All the cells are directed to the same region of interest (ROI), there their hologram is captured. If a cancer cell is detected, the control unit (e.g. a computer-based decision) activates the electrodes and deflect the cancer cell to a reservoir of CTCs, away from the rest of the non-cancer cells. Each cell of the parallel flow is directed to its own outlet port to be collected.

[0098] Three types of cancer cells and four types of blood cells were imaged and analyzed. For cancer cells, HT29-GFP cells were used. HT29-GFP are colon adenocarcinoma cells that have been transfected by adenovirus vector to express GFP and neomycin resistance gene. A stable clone was produced by growing the cells with medium supplemented by 600 μg ml.sup.−1 G418 (Sigma, SN. A1720). The other two cancer cell types are a pair of isogenic cancer cell lines: colon adenocarcinoma, SW-480 (CCL-228), and metastatic stage of colon adenocarcinoma from the lymph node, SW-620 (CCL-227). The growth medium used for the cancer cells was Dulbecco's Modified Eagle's Medium (DMEM) (BI, SN. 01-55-1A) supplemented with 10% fetal bovine serum (FBS) (BI, SN. 04-007-1A), 4 mM L-Glutamin (BI, SN. 03-020-1B) and 1% antibiotics (BI, SN. 03-033-1B). The cell lines were incubated under standard cell culture conditions at 37° C. and 5% CO.sub.2 in a humidified incubator until 80% confluence was achieved. Blood was used to isolate four types of blood cells: erythrocytes, lymphocytes, monocytes, and granulocytes. A dilution medium composed of phosphate-buffered saline (PBS) (BI, SN. 02-023-1A) was prepared supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA) (BI, SN. 01-862-1B), 10% FBS and 6% Nycodenz (Alere Technologies AS, SN. 1002424). EDTA prevents bacteria growth inside the sample; FBS keeps the vitality of the cells for a longer time; Nycodenz was added to compare the density of the dilution medium to the cells density to achieve uniform distribution of cells inside the sample while flowing in the microfluidic channel. Since erythrocytes population in the blood is very high (1000:1), the blood was dissolved in the dilution medium with a proportion of 1:250. To isolate different leucocyte populations, 2 ml of blood mixed with 2 ml PBS supplemented with 2 mM EDTA was layered on top of 2 ml of Ficoll-Paque PLUS (GE Healthcare, SN.17-1440-02) and centrifuge them for 20 minutes in 500 g, at room temperature. Then, two layers were extracted: (1) The interphase containing the Peripheral blood mononuclear cells (PBMCs) for the isolation of lymphocytes and monocytes. (2) The lower layer, containing granulocytes and erythrocytes. The interphase was centrifuged with PBS supplemented with 2 mM EDTA (all centrifugations hence are 500 g, 5 minutes in room temperature), the supernatant layer was removed. For the isolation of lymphocytes and monocytes, commercial kits were used (for lymphocytes CD4+: EasySep, SN. 17952, for monocytes: EasySep, SN. 19359) according to the manufacturer's instructions. To remove plasma, the lower layer was added with PBS supplemented with 2 mM EDTA to reach a volume of 10 ml and then centrifuged. The upper phase was discarded, and the last stage was repeated. To lysis the erythrocytes, 4° C. ammonium-chloride solution (STEMCELL, SN. 07850) was added, the mix was incubated on ice for one hour. Afterward, the cells were washed and centrifuged again with PBS supplemented with 2 mM EDTA for the isolation of granulocytes. Each of the samples was examined and counted four times to calculate their concentration, and the samples were found to contain only one type of cells, as expected. These pure samples of blood parts are then remixed to have a desired selected concentration, ranging from 300 to 1000 cells per μl. Alternatively, to cope with blood originated from cancer patients, preliminary enrichment of the blood sample is done by filtering with the ScreenCell Cyto kit. The kit contains a membrane with microspores of 6.5-8 μm. The membrane captures the CTCs and the larger white blood cells and removes smaller cells (e.g., erythrocytes). From 1 ml of full blood, within 3 minutes of filtration, 10.sup.4 cells containing a mixture of white blood cells and CTCs with a ratio of 1-10 CTCs to 11,000 white blood cells, and an average CTCs recovery rate >90% are left.

[0099] To extract the quantitative phase maps from the acquired off-axis image holograms, the off-axis interferometry Fourier-based algorithm was used, including a digital 2-D Fourier transform, filtering one of the cross-correlation terms, and an inverse 2-D Fourier transform, where the argument of the resulting complex-wavefront matrix was the wrapped phase of the sample. Each off-axis hologram obtained by the holographic imaging module undergoes a quantitative phase reconstruction process before entering the classification algorithm. In other words, the ROI is determined inside the FOV manually before flowing starts, in relation to the sorting DEP electrode positions. For each off-axis imaging hologram, the ROI is cut, and the 2D Fourier transform of the ROI is calculated. One of the cross-correlation terms is cut and undergoes a 2D inverse Fourier transform, resulting in the complex wavefront of the light passing through the sample.

[0100] To compensate for stationary aberrations and field curvatures, a phase map was subtracted from the wrapped phase map of the sample that is extracted from a hologram acquired with no sample present. In other words, to remove aberrations and field curvatures, this complex wavefront is divided by the background wavefront, i.e., the complex wavefront obtained by the same reconstruction process but without the cell present in the ROI. The quantitative phase profile is the angle of the resulting complex wavefront. This phase may be wrapped around 2π. To resolve this phase ambiguity, an unweighted least-squares phase unwrapping algorithm was applied. The resulting unwrapped phase is multiplied by the wavelength and divided by 2π, resulting in the optical phase delay (OPD) map of the sample, and defined as follows:

OPD.sub.c(x, y)=[n.sub.c(x, y)−n.sub.m]×h.sub.c(x, y)  (1)

[0101] where n.sub.m is the RI of the medium, h.sub.c is the thickness profile of the cell, and n.sub.c is the cell integral RI, which is defined as follows:

[00001]$\begin{array}{cc}{\overline{n}}_c\left(x,y\right)=\frac{1}{h_c}\underset{0}{\overset{h_c}{\int }}{n}_c\left(x,y,𝓏\right)d𝓏.& \left(2\right)\end{array}$

[0102] In the resulting OPD profile, the cell area was isolated by a simple threshold, followed by a morphological dilation. In cases of frames with no cell, the classification process was not needed. Therefore, another threshold was applied for the minimum size of the connected component. A maximum size threshold was also applied in cases of attached cells that could not be classified as one object. Partial images of cells on the edges of the FOV were not classified as well. Using the above-described methods, a dataset containing the OPD information was created across the cell areas only and the different parameters that were based directly on the OPD map defined in Eq. (1) were calculated, without decoupling the cellular thickness profile from the refractive index as a prior stage.

[0103] The features that have been extracted from each OPD map divide into two categories: (1) 2D morphological features; and (2) 3D quantitative features. The 2D morphological features are based on the binary image indicating the cell area only. The 3D quantitative features are based on the OPD map across the cell area. These features are presented in Table 1. Table 1 below show 2D (left) and 3D (right) handcrafted features extracted in real time from the OPD profile of the flowing cells. The 3D quantitative features rely on previous works that demonstrate the ability to distinguish between the different stages of the cell lifecycle, as well as other biological phenomena (11,37,38).

TABLE-US-00001 TABLE 1 Cell 2D morphological features Cell 3D quantitative features 1 Diameter Mean 2 Area Energy 3 Major axis length Volume 4 Minor axis length Area to Volume ratio 5 Minor to Major ratio Dry mass 6 Convex Area Variance 7 Eccentricity Kurtosis 8 Circularity Skewness 9 Contrast 10 Entropy 11 Homogeneity 12 Correlation

[0104] The OPD map is an image containing quantitative values that represent the optical thickness of the sample. Since it is quantitative (i.e., contains meaningful optical thickness values on each of the spatial map points), it can be used to calculate both morphological and content-related features of the inspected cell. In earlier work [15, 5], these features were described and as well as the way to use them to discriminate between different types of cells. It should be noted that the algorithm works well for one cell in the ROI simultaneously. For the classification between cancer cells and blood cells, a support vector machine (SVM) algorithm, a common machine-learning algorithm, was used. A dataset from nearly 6,300 static and dynamic OPD images of different cell types (HT29-GFP, SW480, SW620 cancer cells, erythrocytes, monocytes, lymphocytes, and granulocytes) was created for training and testing the algorithm (80% of data was for training and the rest 20% for testing). This classifier receives the reconstructed unwrapped OPD image, extracts 20 features based on 2D morphological features and optical topology features. Eight 2D features are drawn from the binary image (area, diameters, eccentricity, and solidity), the rest of the features are OPD based (mean value, energy, volume, dry mass, variance, kurtosis, skewness, contrast, entropy, homogeneity, and correlation). The algorithm finds a discriminative hyperplane in the features space to distinguish and classify the data points or cells. A radial basis function kernel was used for one-class learning. For dimension reduction and for creating new highly discriminating features based on a linear summation of the extracted original features, principal component analysis (PCA) was then used. Principal component analysis (PCA) is a common method for dimension reduction and for finding highly discriminative features. The PCA method is based on projecting the data onto a lower-dimension subspace, and receiving new features, which are linear combinations of the original features. The first principal component has the largest possible variance of the data, and therefore enables better discrimination between the classes, the second principal component has the second largest variance of the data, and so on.

[0105] The DEP microfluidic module was configured to sort specific selected cells. The dielectrophoretic microfluidic module was designed to use the negative-DEP technique to deflect flowing cells from the electrodes and direct them left or right by applying a voltage at the correct time [9]. A square wave of 1 MHz and 3-5 Volts was applied on the electrodes to exert the DEP phenomenon. The electrodes were controlled by a computer-activated proprietarily developed generator designed for this type of DEP modules. Using these parameters, flow rates up to 20 μl hr.sup.−1 were controlled. The cells flow in and out of the dielectrophoretic microfluidic module may be performed by using four low-pressure pumps (Cetoni, neMESYS 290N) in operating rates of 0.5 μl hr.sup.−1 and up to 20,000 μl hr.sup.−1. In this specific and non-limiting example, one pump was used to insert the cells, one pump was used for washing, one pump was used for collecting the cancer cells, and one pump was used for collecting the non-cancer cells.

[0106] Reference is made to FIGS. 4a-4b showing a schematic representation of an example of a DEP microfluidic module 410 defining a microfluidic channel. In particular, FIG. 4a shows a top view of a DEP microfluidic module 410 comprising an array of spaced-apart electrodes numbered (1)-(4) positioned on both sides of the channel dielectrophoretic microfluidic module 410. As described above, the electrode pattern shown in FIGS. 3a and 4a-4b is a specific and non-limiting example. Electrodes (1)-(4) may have the same or different lengths. In this specific and non-limiting example, electrodes (1) are a pair of electrodes positioned on both sides of the channel of the dielectrophoretic microfluidic module 410 in an intersecting configuration defining a space between them through which cells might flow. Electrodes (1) are configured for focusing the cells into the imaging FOV and the holographic ROI. Electrodes (2) and (4) are also positioned in an intersecting configuration and are configured to push the cells to either side of the DEP microfluidic module 410, per the sorting demands obtained by the control unit (e.g. automatic classifier module). One electrode of the electrodes pair (1) and electrode (4) and the second electrode of the electrodes pair (1) and electrode (2) are positioned along parallel axes respectively on an opposite side of the dielectrophoretic microfluidic module 410 at different distances. Electrode (3) has a special angular configuration being configured to separate between the cells of different types and to increase the distance between cell streams. Electrode (3) define a space with electrode (4) and electrode (2) to enable the cells to flow in between to a different side of the channel. The area marked as FI stands for the fluorescence imaging region (used for validation of the label-free real-time classification process). The electrodes (1)-(4) are positioned to define a sorting trajectory for the flow of cells along the dielectrophoretic microfluidic module 410. A flow path of the sorting trajectory of the cells is indicated by arrows. FIG. 4b shows a non-limiting example of a 3D view of the DEP microfluidic module 410 with the pair electrodes (1), together with an example of the DEP microfluidic module cross-section length and thickness. Each electrode is in-fact a pair of two thin electrodes (e.g. about 2 nm), located at the top and the bottom of the channel. The lines EF illustrate the electric field lines with cells flow near them. The set of electrodes (1) can be always active and used to center and direct the cells into the FOV and ROI. Electrode (3) may also be continually active. Electrodes (2) and (4) may be operated in an alternating manner on a real-time command of the control unit. Inside the FOV, in the ROI region, the OPD was calculated, and each cell was classified to determine whether it is a cancer cell or not. If it was classified as cancer, dielectrophoretic microfluidic module 410 is activated operating at least one electrode by alternatively switching on or off one or more relevant electrodes to tilt the cells of interest out. For example, electrode (2) is switched off, electrode (4) is switched on, to flow the cell to the right, away from the waste flow. After a predetermined number of frames, the electrodes are switched back to guide the cell to the waste by default. The sorting system may be controlled in real-time by a control unit executing a multilevel algorithm. Each holographic frame taken by the camera undergoes the OPD reconstruction algorithm, with the OPD map in the ROI visualized on the computer screen. For the pre-designed samples, all the cells that entered the DEP microfluidic module 410 flow to the ROI, usually one at a time, using the concentration mentioned above of 300-1,000 cells μl.sup.−1. The cells inside the ROI are classified during their flow. If identified as cancer, the control unit is activated by the time it takes the cell to flow and reach the DEP electrodes so that the sorting can happen on time. If a cancer cell arrives at the FI region marked in FIG. 4a, it emits fluorescence light (since only the cancer cells are GFP modified or stained). Recording the fluorescence image in the FI region by the two cameras is used for validating the correctness of the automatic sorting decision of the real-time label-free process.

[0107] The ability to analyze stain-free isolated cells is important for flow-cytometry via quantitative imaging of cells during flow. FIG. 5a presents snapshots of OPD maps taken while the cells are flowing in the microfluidic channel, with the real-time quantitative-imaging visualization and classification results during cell flow. More specifically, FIG. 5a shows snapshot from real-time classification videos, presenting quantitative phase microscopy of live unlabeled cells flowing in a microfluidic channel. Each pair of images depicts the same frame, where the left image is the original off-axis hologram captured by the camera, and the right image is the OPD profile processed in real-time. The classification results are written on the top. Colorbar represents OPD values in meter. The classification result and the confidence score of the classifier prediction are presented on the top. Some of the cells have been classified as a cancer cell, and some of the cells have been classified as a blood cell.

[0108] For classification, the more complex task is classification of white blood cells and cancer cells, since red blood cells and platelets are much easier to detect, since they are very different than cancer cells. A data set of about 4,000 OPD maps of two types of colorectal cancer cells (SW480 and SW620) and four types of blood cells (granulocytes, lymphocytes, monocytes, and erythrocytes) was created. Reference is made to FIG. 5b showing examples of OPD images from the acquired database of quantitative phase images of cancer (SW620, SW480) and blood cells used for training for seven types of cells, demonstrating that cells are all driven to the ROI of the classification area and are later sorted by the electrodes. Colorbar represents OPD values in meter. The machine-learning classifier was tested for separation between cancer and non-cancer cells. Prior to the separation during flow, by running the algorithm on 20% of the dataset, 98.22% accuracy of separation was obtained. OPD values are in 10.sup.−8 m. The Cohen's d values between the two groups (cancer cells and blood cells) per each feature, are shown in FIG. 6. FIG. 6 shows histograms of different features extracted from the OPD profiles of cancer and blood cells. Many features present a very large difference between the two cell types. The blood histograms are characterized by two peaks, noting the difference between red blood cells and white blood cells, where the left peaks are attributed to red blood cells, which are generally smaller. Using the Cohen's d analysis, features displaying d>1.5 are with a large distance between the histograms and are the strongest features to use for classification (e.g., cell diameter, minor axes and area, and also mean OPD, OPD volume, and dry mass [5]). The PCA takes this into account. FIG. 7 shows the discrimination between the two types of cells for the first three principal components (out of ten principal components) calculated by the PCA. FIG. 7 shows a scatter plot showing the discrimination between blood and cancer cells for the first three principal components calculated by the PCA analysis (the actual classification was done based on ten principal components, so the discrimination is even better than shown here).

[0109] Using the holographic imaging module and the DEP flow module of the present invention, electrodes (1), (2) and (3) were turned on, the DEP microfluidic module and syringes were filled with buffer solution (PBS+10% FBS) to remove air and loaded a remodeled blood sample spiked with SW480 cancer cells, which represent the CTCs, in the ratio of 3:5:2 (cancer: white blood: red blood cells). Total cell concentration was 300 cells μl.sup.−1. This low number of cells was used to decrease the probability of having more than one cell at a time in the ROI or clustering. This ratio was chosen to demonstrate the system sorting abilities and is much higher than the ratio of CTCs in blood. For this sorting system, the ROI size was approximately 60 μm×40 μm. The flow was set at rates between 4-7 μl hr.sup.−1, or 45-80 μm sec.sup.−1, per the DEP microfluidic module cross-section (700 μm×35 μm), giving us half to one image of cell per sec. The framerate for this experiment was 8 frames per second (FPS), so each cell had about 2-5 frames in the ROI. The OPD map was imaged and reconstructed of each cell individually and classified to cancer or non-cancer cell using the machine-learning classifier. This automatic decision activated the control unit controlling the electrodes to remove the detected cancer cell from the main flow. For validation only, the SW480 cancer cells were labeled using acridine-orange fluorescence dye, to show if the classifier is precise. The classification process was carried out for each frame in real-time (during cell flow). Electrode switching, once a cancer cell was detected, was active for a defined number of frames as follows: the distance between the ROI and the sorting area near electrode 3 was less than 60 μm or about 1-1.5 seconds for this cell velocity. Since the reaction time of the electrodes was 7 ms, and the images were acquired at 8 FPS, the electrodes were set to stay in the same state after command for 15-20 frames, to allow the cell to be sorted as required. Table 2 below shows the sorting process timing, from an image taken until the sorting electrodes switches. Average values are displayed. SVM features and classification happen only when a cell is present in the ROI.

TABLE-US-00002 TABLE 2 Step Time [ms] Total time [ms] Image acquiring 45 — Phase 2D FFT 26 71 retrieval Phase unwrapping 2 73 Cell Threshold 0.1 73.1 Segmentation Holes' filling 3.5 76.6 Dilation 5 81.6 Largest object 1.5 83.1 3D mask 3 86.1 No cell check Area size (big/small) 2.7 88.8 Edges 0.4 89.2 Feature's extraction 9 98.2 Classification 10 108.2 Total classification time 63.2 Electrode switching time 7 115.2 Saving frame 1 116.2 Total time 60-120 Frames per second [FPS] 8-16

[0110] Examples from experiments are shown in FIGS. 8a-8d and 9a-9d. FIGS. 8a-8d and 9a-9d show the FOV with a cell flowing into the ROI. The classifier results and score (confidence level) calculated in real-time are shown at the top of each frame. For validation of the label-free sorting decision, the cell continues to flow to the fluorescence area, where only the cancer cell emits light. The fluorescent channel was used for validation only and did not affect the operation of the electrodes, which were solely dictated based on the label-free IPM data. If a cancer cell was detected by the holographic imaging module, then electrodes (2) and (4) are operated alternatively, and the cell flowed to the right, whereas if a blood cell was detected by IPM, it continued to the left part of the DEP microfluidic module. FIGS. 8a-8d show a label-free sorting of an SW480 cancer cell. The large gray images show FOV, and the off-axis hologram acquired. The small window shows the real-time-processed OPD profile (ROI in FIG. 4a). The fluorescence imaging area is marked by a dashed circle. In particular, FIG. 8a shows a classification using the quantitative OPD reconstruction (colorized ROI, the color bar represents the OPD in μm). The automatic classification result is shown at the top. FIG. 8b shows a cell exiting ROI and continues flowing to the electrodes. FIG. 8c shows the fluorescence signal from the validation channel, confirming that this is a cancer cell. FIG. 8d shows that based on the label-free imaging decision only (not using the fluorescence signal), the cell continues to flow to the right side of the DEP microfluidic module, where all cancer cells are sorted. FIGS. 9a-9d show a label-free sorting of a monocyte. The large images show the FOV, and the off-axis hologram acquired. The small window shows the real-time-processed OPD profile (ROI in FIG. 4a). The fluorescence imaging area is marked by a circle. In particular, FIG. 9a shows classification using the quantitative OPD reconstruction (colorized ROI, the color bar represents the OPD in μm). It should be noted that that in FIG. 9b, a sorted cancer cell appears, which is hidden behind the electrode (2) in the previous frame (FIG. 9a). The automatic classification result is shown at the top. FIG. 9b shows the cell in the ROI that continues flowing to the electrodes, along with a cancer cell (top right), which has already been sorted a few frames earlier. FIG. 9c shows no fluorescence signal in the validation channel, confirming that this is not a cancer cell. FIG. 9d shows that based on the label-free imaging decision only (not using the fluorescence signal), the cell continues to flow to the left side of the dielectrophoretic microfluidic module since it is not a cancer cell. For the measured cells, a classification sensitivity, specificity and accuracy of 97.8%, 98.9%, and 99.6% were achieved. After classification, sorting coinciding values were 72.7%, 60.8%, 69.2%, with false-positive (how many blood cells were sorted as cancer) of 12%, and the false-negative (how many cancer cells were sorted as blood cells) of 19%. The throughput got with the loss of cells and settings mentioned was less than 1 cell sec.sup.−1. A label-free classification was thus shown with an accuracy as high as 98.9% for cancer cells from blood cells by using the teachings of the present invention. The classifier of the present invention is also able to classify correctly other types of epithelial cancer cells because of their size and nucleus shape and location in comparison to blood cells.

[0111] The use of OPD for the classifier yields 12 more powerful discriminating features to those of brightfield. Although CTCs are usually larger than PBMC and erythrocytes and brightfield images would suffice, for comparison to granulocytes, the OPD based features have a higher impact on the discrimination. SVM classification based on the OPD maps can be used to discriminate between healthy and cancer cells. In addition, the full OPD image of the cell gives the option to the clinician with other quantitative parameters on the cell, such as its dry mass and phase volume [11]. These data have proven itself useful for detecting abnormalities or introducing more complex analysis methods such as cell tomography [8]. The holographic imaging module is able to produce off-axis holographic videos of up to 15 frames sec.sup.−1. Since one cell per frame is needed, with a flow rate of just a few μl hr.sup.−1, a throughput of 15 cells sec.sup.−1 may be obtained.

[0112] Preliminary purification of the blood based on the size of the cells may be first performed. This preliminary process takes only three minutes and from 1 ml of blood it leaves 1-10 CTC in 11,000 white blood cells. Then, the holographic classification method of the present invention further purifies the sample and detect the single cancer cells based on the OPD profiles. Since the processing rate of the sorting system of the present invention is about 15 cells per second, processing the entire pre-enriched sample may take about 12 minutes, and thus the entire processing of 1 ml of blood, including the pre-enrichment process, may take about 15 min. The framerate is mainly affected form the algorithm running time, but the total throughput is also a function of the maximum cell velocity the DEP allows. With 15 cells sec.sup.−1, flow rates up to 180 μl hr.sup.−1 with 300 cells μl.sup.−1 may be used, but for the electrode to affect the direction of the cells in the mentioned setup, flow rates lower than 10 μl hr.sup.−1 should be used. From Table 2 above, it can be seen that a blind sorting (i.e., without live video) can increase the framerate dramatically. Together with a DEP microfluidic module with a larger ROI and a dedicated set of electrodes, a much higher flow rate and higher cell concentration may be obtained to achieve higher throughput.

[0113] The classifier has been built using SVM, which is a common machine-learning classification algorithm that is based on features extraction. The goal of the SVM algorithm is to find a hyperplane in the features space that distinctly classifies the data points [26]. As shown in FIGS. 10a-10b, two types of SVM classifiers were built and their performance were compared. FIGS. 10a-10b are block diagram schemes of two types of classification processes. FIG. 10a shows a one-step SVM classifier while FIG. 10b shows a two-step SVM classifier. The first classifier is a simple multi-class SVM that classifies all six types of cells in parallel, which is referred as ‘one-step SVM’. The second classifier is combined of three sub-classifiers: (1) SVM 1: classifying between cancer and blood cell; (2) SVM 2: classifying between two types of cancer cells; and (3) SVM 3: classifying between four types of blood cells. The second classifier is referred as ‘two-step SVM’, because its classification process is carried out in two stages: SVM 1 receives the OPD map of the cell and categorizes it in a general manner as ‘cancer’ or ‘blood’ cell. Afterward, a more specific classification is performed by SVMs 2 and 3, which receive their inputs according to SVM 1 result. All models were trained and tested on 80% and 20% of the dataset, respectively. The training was done using the 10-fold cross-validation procedure.

[0114] The holographic imaging module 320 of FIG. 3b was used to quantitatively image all six cell types during flow in a throughput of 15 cells per second, their OPD maps were acquired and classified in real-time, using a standard personal-computer CPU on a MATLAB platform. The experiments work was divided into two main parts: first, homogeneous samples of each cell type were flowed to create a supervised dataset for training and testing the models; second, different heterogeneous samples, containing several cell types together, were flowed to validate the classifier performance in real-time.

[0115] Prior to the analysis of the OPD maps, the segmentation image-processing procedure was applied to track the cell area during flow. Next, the twenty features, mentioned in Table 1, were extracted from the cell OPD area selected by the segmentation process. During the training process, the twenty hand-crafted features were used as an input for PCA analysis in order to extract the best combinations of these features, which were the most useful for classification between various cell types. The best classification results were obtained for the eight, six, ten and thirteen first principal components for SVM 1, SVM 2, SVM 3 and one-step SVM, respectively. FIGS. 11a-11d demonstrate the separation in the 2D PCA space for all four SVM models. The x and y axes represent the two first principal components, which have most of the variance of the data .In practice, the separation between the cell types was even better than shown in this illustration, since more than two principal components were practically used for classification, as elaborated above. The new highly discriminative features provided by PCA were then used as an input for the SVM models. FIGS. 11a-11d show scatter spot in the 2D PCA space for all four classifiers, where the x and y axes represent the two first principal components. In particular, FIG. 11a shows one-step SVM, FIG. 11b shows SVM 1, FIG. 11c shows SVM 2, FIG. 11d shows SVM 3. It should be noted that the actual separations between the groups are even better than presented, since only the first two principal components are used in this illustration, but the actual classifications have used more principal components.

[0116] For comparison, all SVM models were trained separately based the: (1) 2D morphological features; and (2) 2D morphological and 3D quantitative features together. Table 3 below presents the accuracies of these two assays for all SVM models. More specifically, Table 3 below presents accuracy results when using the 2D features and the 3D features for all SVM models. The improvement obtained for each classification method when also using the 3D features in comparison to be using the 2D features only is indicated in bold. As can be seen, the accuracy is higher when considering both the 2D morphological features and the 3D quantitative features for all trained models, demonstrating the advantage of using quantitative phase images for classification rather than simple 2D imaging Table 4 below presents the precision of performing wrong and right classifications with all SVM models combined with PCA on the test set, considering both the 2D morphological features and the 3D quantitative features. Table 4 shows precision of wrong and right predictions for all SVM models on the test set. As seen in Table 3 and Table 4 below, the two-step classifier exhibits the best overall accuracy when examining it on the test set.

TABLE-US-00003 TABLE 3 Total accuracy 2D morphological 2D morphological + Model features 3D features SVM 1 92.88% 98.22%↑ 5.34%  SVM 2 95.6% 96.98% ↑ 1.38%  SVM 3 74.68% 91.77% ↑ 17.09% One-step SVM 78.46%  91.3% ↑ 12.84% Two-step SVM 77.6% 92.56% ↑ 14.96%

TABLE-US-00004 TABLE 4 Right Wrong Model Class prediction prediction SVM 1 Cancer 97.3% 2.7% Not Cancer 99.1% 0.9% SVM 2 SW480 97.5% 2.5% SW620 96.3% 3.7% SVM 3 Granulocyte .sup. 90%  10% Lymphocyte 97.8% 2.2% Erythrocyte 91.2% 8.8% Monocyte 82.6% 17.4%  One-step SVM SW480 97.8% 2.2% SW620 88.6% 11.4%  Granulocyte 88.8% 11.2%  Lymphocyte 98.6% 1.4% Erythrocyte 86.2% 13.8%  Monocyte 82.6% 17.4%  Two-step SVM SW480 97.5% 2.5% SW620 90.1% 9.9% Granulocyte 87.6% 12.4%  Lymphocyte 97.8% 2.2% Erythrocyte 91.2% 8.8% Monocyte 82.6% 17.4% 

[0117] Next, the performance of the one-step and the two-step classifiers were on different samples during flow.

[0118] Next, an even amount of SW480 and SW620 cells was mixed and made them flow in the channel. Table 5 below presents the classification results of a sample of containing a 1:1 mixture of flowing SW480 and SW620 cells of three classifiers: one-step SVM, two-step SVM and SVM 2. As expected, SVM 2 achieved the best results for classifying between the two cancer classes only.

TABLE-US-00005 TABLE 5 One-step Two-steps Class SVM SVM SVM 2 SW480 42.21%  46.5% 50.26%    SW620 10.3% .sup. 29% 49.74%    Granulocyte .sup. 46% 21.75%  0% Lymphocyte   1%   2% 0% Erythrocyte  0.5%  0.5% 0% Monocyte   0% 0.25% 0% Total cancer cells 52.5% 75.5% 100%  Total blood cells 47.5% 24.5% 0%

[0119] Next, a homogeneous sample of granulocytes was used and imaged during flow. Table 6 below shows the classification results of a homogeneous sample of flowing granulocytes of three classifiers: one-step SVM, two-step SVM and SVM 3. Unsurprisingly, SVM 3 achieved the best results, since it classified between blood cells only. Here as well, the two-step SVM achieved better results than the one-step SVM.

TABLE-US-00006 TABLE 6 One-step Two-steps Class SVM SVM SVM 3 SW480 0.59% 1.77% 0% SW620 4.12% 1.64% 0% Granulocyte 78.34%  80.12%  83.04%    Lymphocyte  4.5% 3.29% 4.5%.sup.  Erythrocyte  0.2%  0.3% 0.2%.sup.  Monocyte 12.25%  12.88%  12.25%    Total cancer 4.71% 3.41% 0% Total blood 95.29%  96.59%  100% 

[0120] The average processing times for each step in the algorithm were as follows: (1) 0.028 sec for the reconstruction of the unwrapped phase profile, (2) 0.025 sec for cell segmentation and features extraction, (3) 0.01 sec and 0.095 sec for cell classification by the two-step SVM and the one-step SVM, respectively. Although the two-step classifier included two SVM models, while the one-step classifier included only one, the two-step SVM exhibited faster execution time. Combining all, the total processing times for each off-axis hologram containing 1 megapixel is 0.063 sec and 0.148 sec for the two-step SVM and the one-step SVM, respectively.

[0121] These results demonstrate the ability of the presented automatic algorithm to classify cancer in different cancer stages and white blood cells using flow-cytometry combined with machine learning, using OPD-map-based features. High classification rates for stain-free cells were obtained during real-time flow. The accuracy values and prediction precisions correspond with the separation between the groups presented in the 2D PCA space (see FIGS. 11a-11d, Tables 3-4), where the two-step SVM classifier manifested better classification results than the one-step classifier.