MOBILE ELECTRONIC DEVICES

Related Applications

[0001] This Application claims priority to U.S. Provisional Patent Application No.

61/745,370 filed on December 21, 2012 and U.S. Provisional Patent Application No.

61/799,664 filed on March 15, 2013. Priority is claimed pursuant to 35 U.S.C. § 119. The above-noted Patent Applications are incorporated by reference as if set forth fully herein.

Field of the Invention

[0002] The field of the invention generally relates to blood testing analysis devices and methods. More particularly, the field of the invention relates to a rapid blood testing platform that is used in conjunction with a mobile electronic device such as a mobile phone. The method and device uses the mobile electronic device's integrated camera as a detector for cell counting and colorimetric analysis of samples.

Background

[0003] Blood analysis, including density measurements of white blood cells (WBCs), red blood cells (RBCs), and hemoglobin, is one of the most ordered clinical tests. This battery of tests can provide valuable information for evaluating the overall health condition as well as helping diagnosis of various diseases. For example, an RBC count can be used to diagnose anemia and other conditions affecting RBCs. Likewise, a WBC count can be used to diagnose an infection or see how a patient is handling a particular cancer treatment.

Hemoglobin is a molecule that is contained within RBCs and carries oxygen. A hemoglobin test measures the amount of hemoglobin in the blood and is a good measure of the blood's ability to carry oxygen throughout the body. Typically a blood test requires at least a milliliter of blood sample and the cells are manually counted using a hemocytometer with light microscopy or automatically counted using a hematology analyzer or a flow cytometer in a centralized laboratory. Manual counting is tedious and subject to errors and bias.

Automated counting using e.g., a flow cytometer is highly accurate but requires bulky and expensive instruments, making them less effective for point-of-care diagnostics, especially in resource limited settings. For example, existing hematology analyzers may range in cost, depending on features, between several thousand dollars to hundreds of thousands of dollars. Therefore, a cost-effective, compact and accurate automated blood analyzer that can be used at the point-of-care is highly desirable. Summary

[0004] In one embodiment, a system for analyzing a blood-containing sample with a mobile electronic device having a camera includes a base configured for securement to the mobile electronic device. The base has an attachment region for securing one of a plurality of testing modules to the base. A power source is disposed on the base. At least one lens is contained in the base. The system includes first, second, and third sample holders configured to hold the blood-containing sample. The plurality of testing modules include: a WBC testing module having a housing containing a first light source disposed therein and oriented to side illuminate the first sample holder when loaded into the WBC testing module; a RBC testing module having a housing containing a second light source disposed therein, the second light source configured to illuminate the second sample holder when loaded into the RBC testing module; and a hemoglobin testing module having a housing configured to receive a third sample holder, the housing containing a third light source configured to illuminate the third sample holder. In some embodiments, two of the sample holders may include the same sample holder rather than a different sample holder. For example, the sample holder for the WBC testing module and the RBC testing module may be the same.

[0005] In another embodiment, a method of using the system includes obtaining a blood sample of a subject and incubating the blood sample with a fluorescent stain. The WBC testing module is secured relative to the base. The fluorescent labeled blood sample is loaded into the first sample holder which is then inserted into the WBC testing module. The sample is then illuminated with the first light source of the WBC testing module. An image of fluorescent light emitted from the sample is obtained with the mobile electronic device camera. The image captured on the mobile electronic device is then digitally processed to count the number of fluorescently labeled cells contained in a region of interest in the image.

[0006] In another embodiment, a method of using the system includes obtaining a blood sample of a subject and diluting the blood sample. The RBC testing module is secured relative to the base (e.g. secured directly to the base or through an intermediate support or base like the WBC testing module in some embodiments). The diluted blood sample is loaded into the second sample holder inserted into to the RBC testing module. The cell counting chamber is illuminated with the second light source from the RBC testing module. An image is captured with the mobile electronic device camera. The image captured on the mobile electronic device is digitally processed to count the number of RBCs contained in a region of interest in the image. [0007] In another embodiment, a method of using the system includes obtaining a blood sample of a subject and adding a RBC lysing solution. The hemoglobin testing module is secured relative to the base (e.g. secured directly to the base or through an intermediate support or base like the WBC testing module in some embodiments). The lysed blood sample is loaded into the third sample holder and inserted into the hemoglobin testing module. The third sample holder is illuminated with the third light source from the WBC testing module. An image is captured with the mobile electronic device camera. The image captured on the mobile electronic device is then digitally processed to determine the transmission light intensity of the image captured with the mobile electronic device camera. The concentration of hemoglobin contained within third sample holder is then automatically calculated, wherein the concentration of hemoglobin is based at least in part on the transmission light intensity of the image captured with the mobile electronic device camera. The automatic calculation can take place using software loaded on the mobile electronic device or it may take place remotely, for example, if the transmission data has been transferred to a remote computer/processor.

Brief Description of the Drawings

[0008] FIG. 1A illustrates a perspective view of the system according to one embodiment used in connection with a mobile phone.

[0009] FIG. IB illustrates a mobile phone with a camera contained therein.

[0010] FIG. 1C illustrates another perspective view of the system of FIG. 1A.

[0011] FIG. ID illustrates another perspective view of the system of FIG. 1A.

[0012] FIG. IE illustrates another perspective view of the system of FIG. 1A.

[0013] FIG. IF illustrates an alternative embodiment of the system used in connection with a mobile phone.

[0014] FIG. 2A illustrates a screen shot of a mobile phone containing software for blood analysis that is used in connection with the system of FIG. 1A.

[0015] FIG. 2B illustrates a screen shot of a menu given to a user that has initiated the software program of FIG. 2A.

[0016] FIG. 2C illustrates a screen show of another menu presented to a user after selecting the "New Test" option from FIG. 2B. [0017] FIG. 2D illustrates respective images obtained using the mobile phone as part of a WBC cell counting analysis (top), RBC cell counting analysis (middle), and hemoglobin concentration (bottom).

[0018] FIG. 2E illustrates a screen shot presented to the user according to one

embodiment of a WBC cell counting software program.

[0019] FIG. 2F illustrates a screen shot presented to the user according to one embodiment of a RBC cell counting software program.

[0020] FIG. 2G illustrates a screen shot presented to the user according to one

embodiment of a hemoglobin concentration software program.

[0021] FIG. 2H illustrates a screen shot presented to the user illustrating WBC cell counting results according to one embodiment.

[0022] FIG. 21 illustrates a screen shot presented to the user illustrating RBC cell counting results according to one embodiment.

[0023] FIG. 2J illustrates a screen shot presented to the user illustrating hemoglobin concentration results according to one embodiment.

[0024] FIGS. 3A-3E illustrate the digital processing operations performed by the mobile electronic device for the WBC cell count and concentration calculations according to one embodiment.

[0025] FIGS. 4A-4E illustrate the digital processing operations performed by the mobile electronic device for the RBC cell count and concentration calculations according to one embodiment.

[0026] FIG. 5 illustrates the digital processing operations performed by the mobile electronic device for the hemoglobin concentration calculation according to one embodiment.

[0027] FIG. 6A illustrates a graph comparing the mobile phone based WBC cell counting results with the standard test results obtained from Sysmex for 30 different blood samples. A linear regression of the experimental data (n = 30) with WBC concentrations ranging from ~ 3,000/μΙ, to 12,000/μΙ, demonstrates a good agreement between the two modalities with a correlation coefficient of - 0.98. The mobile phone based blood analyzer provides an absolute counting error that is less than 7%.

[0028] FIG. 6B illustrates the Bland-Altman analysis results, evaluating the accuracy of the mobile phone blood analyzer for WBC concentration measurements as compared to the standard hematology analyzer. The solid lines show a bias of 230 cells^L and 95% limits of agreement: 955 cells/ μΐ., (upper limit) and -495 cells^L (lower limit). [0029] FIG. 7A illustrates a graph comparing the mobile phone based RBC cell counting results with the standard test results obtained from Sysmex for 12 different blood samples. A linear regression of the experimental data (n = 12) with RBC concentrations ranging from ~ 3 x 10 iL to 5.5 x 10 ⁶ /μΙ, demonstrates a good agreement between the two modalities with a correlation coefficient of - 0.98. The mobile phone blood analyzer counting provides an absolute counting error of less than 5%.

[0030] FIG. 7B illustrates the Bland-Altman analysis results, evaluating the accuracy of the mobile phone blood analyzer for RBC concentration measurements as compared to the standard hematology analyzer. The solid lines show a bias of -2.9xl0 ⁴ cells^L and the 95% limits of agreement of: 2.5xl0 ⁵ cells/ μΐ _^ (upper limit) and -3.2xl0 ⁵ cells^L (lower limit).

[0031] FIG. 8A illustrates a graph comparing the mobile phone cased hemoglobin measurement accuracy as compared to a standard hematology analyzer (Sysmex) for 37 different blood samples. A linear regression of the experimental data (n = 37) with hemoglobin concentrations ranging from ~ 11 g/dL to 17 ^μϊ _^ demonstrates a good agreement between the two modalities with a correlation coefficient of - 0.92. The mobile phone blood analyzer provides an absolute measurement error of less than 5%.

[0032] FIG. 8B illustrates the Bland-Altman analysis results evaluating the accuracy of the mobile blood analyzer for hemoglobin concentration measurements versus the standard hematology analyzer. The solid lines show a bias of 0.036 g/dL and the 95% limits of agreement: - 0.63 g/dL (upper limit) and -0.54g/dL (lower limit).

Detailed Description of the Illustrated Embodiments

[0033] FIGS. 1A-1F illustrates a system 10 for rapidly analyzing blood using a mobile electronic device 12. The mobile electronic device 12 may include portable electronic devices such as tablets (e.g., iPADs), mobile phones (e.g., SMARTPHONES), and the like. Any mobile electronic device 12 that includes a camera 14 as well as a screen 16 or display is potentially usable in connection with the system 10. As best seen in FIGS. 1A, 1C, ID, IE, and IF the system 10 includes a base 20 that is configured for securement to the mobile electronic device 12. In particular, the base 20 is dimensioned and otherwise designed to be removably secured to a side of the mobile electronic device 12 that contains the camera 14. Often, mobile electronic devices 12 such as SMARTPHONES have the camera 14 located on the opposing or "back" side of the display 16. In such as case, the base 20 is configured to attach to the back side so that various modular testing modules (described in more detail below) can be selectively attached to the base 20 whereby the camera 14 is used to analyze a blood sample.

[0034] The base 20 may include one or more flanges 22 that are used to aid in securing the base 20 to the mobile electronic device 12. The flanges 22 may include tabs, rails, clips, or the like that are flexible so that the base attachment 20 can be secured to the mobile electronic device 12 or removed from the same on as needed basis. As seen in FIG. ID, the base 20 includes an aperture 24 that contains a lens 26 therein. The aperture 24 and lens 26 are located in a region of the base 20 such that when the base 20 is secured to the mobile electronic device 12, the aperture 24 and lens 26 are located directly opposite the camera 14. The camera 14 also has its own lens (not shown) located within the body of the mobile electronic device 12. The lens 26 of the base 20 abuts or is located adjacent to the lens of the camera 14. The focal length of the lens 26 may be chosen to define the desired field of view, magnification/de- magnification factor, and the resolution. In one alternative option, different lenses 26 may be inserted into the base 20 depending on the desired application. As best seen in FIG ID, in this embodiment, the base 20 includes an attachment region 28 onto which different modular testing modules 30, 32, 34 can be removable secured thereto. In one preferred embodiment, the attachment region 28 is a universal hub 21 that enables testing modules 30, 32, 34 to be easily exchanged with one another by a user. The hub 21 may take the form of a ring that is formed in the base 20 as seen in FIG. ID. The hub 21 defines the aperture 24 and contains the lens 26 therein. The hub 21 extends generally perpendicularly outward from the base 20 and includes a receiving surface (inner or outer) for a

corresponding interface of the modular testing modules 30, 32, 34. For example, the aperture 24 in the hub 21 may be a cylindrical shape as shown. In one embodiment, as shown in FIG. IF, each testing module 30, 32, 34 has a corresponding extension 39a, 39b, 39c with a shape (e.g., in this example a cylinder-shape) that interfaces with the hub 21 so that the testing module 30, 32, 34 can be inserted into the hub 21 and secured via a friction fit. Of course, the hub 21 may have any number of geometric shapes so long as they testing modules 30, 32, 34 have the corresponding shape.

[0035] In another preferred embodiment of the invention, which is illustrated in FIGS. 1A, 1C, ID, and IE, only a single testing module, namely, the WBC testing module 32 has a mating surface 38 (e.g., cylindrical projection) that interfaces with the hub 21 of the base 20. In this embodiment, the remaining RBC testing module 30 and the hemoglobin testing module 34 are configured to be mounted atop the WBC testing module 32 as described herein. In this configuration, while the WBC testing module 32 is secured to the base 30 via the hub 21 it is not actively turned on when one of the other modules 30, 34 is used. Rather, the WBC testing module 32 is used as a mount for either the RBC testing module 30 or the hemoglobin testing module 34. As best seen in FIG. IE, the WBC testing module 32 includes ledges 36 onto which the RBC testing module 30 and the hemoglobin testing module 34 can be inserted onto. In this embodiment, the RBC testing module 30 may act as a cover (and is not used) for the WBC testing module 32. Alternatively, a dedicated cover 35 may be used to cover the WBC testing module 32. In this embodiment, the cover 35 engages with one side of the WBC testing module 32 and includes a top portion to "cover" the cell counting chamber as described in more detail below.

[0036] As an alternative to a friction fit between the testing modules 30, 32, 34 and the hub 21, the attachment region 28 may include grooves, notches, slides, threads, tabs, or the like which interface with corresponding or complementary structures in the testing modules 30, 32, 34. For instance, the attachment region 28 may include notches that receive corresponding tabs from testing modules 30, 32, 34 that permit the various testing modules 30, 32, 34 to be secured to and removed from the base 20. Likewise, threads (e.g., female) may be located in a hub 21 that receive corresponding threads (e.g., male) on the testing modules 30, 32, 34. This alternative to a friction fit also applies to the embodiment of FIG. IF

[0037] In one embodiment, testing module 30 is configured to count the number of RBCs in a blood sample. As described below, once the number of RBCs is counted, this can be used by an application or program running on the mobile electronic device 12 to then calculate a concentration of RBCs in the sample. Testing module 32, in this embodiment, is configured to count the number of WBCs in a blood sample. Using a similar process as described for RBCs, the number of WBCs is counted and is used by an application or program running on the mobile electronic device 12 to calculate a concentration of WBCs in a blood sample. In this embodiment, testing module 34 is configured to determine the concentration of hemoglobin in a blood sample. Hemoglobin is released from RBCs by incubating the sample with a RBC lysing solution as explained below. The lysed solution containing released hemoglobin is then illuminated whereby the transmission light intensity is used, as explained below, to arrive at a concentration of hemoglobin within the blood sample.

[0038] Referring to FIGS. 1C, ID, IE, and IF a power source 40 is disposed in the base 20 and is used, in this embodiment, to power the light sources used by the various testing modules 30, 32, 34. The power source 40 may include one or more batteries. For example, the batteries may include two AA batteries. In this embodiment, the power source 40 (batteries) is connected to a terminal 42. In this regard, as each testing module 30, 32, 34 is secured to the base 20, wiring or cabling associated with each testing module 30, 32, 34 can be connected to the terminal 42 to power each light source. While the power source 40 is described in this embodiment as being located within the base 20, in an alternative embodiment, the power source 40 is located within the mobile electronic device 12. In this regard, the testing modules 30, 32, 34 are powered by the power supply of the mobile electronic device 12. For example, the USB or other port on the mobile electronic device 12 can be tapped into for power for running the system 10.

[0039] FIGS. 1A, 1C, ID, IE, and IF illustrate a testing module 32 for counting WBCs. The testing module 32 includes a housing 44 with a set of opposing light sources 46 that are side-oriented to illuminate a cell counting chamber 48 that can be inserted or removed from the housing 44 of the testing module 32. The cell counting chamber 48 is an optically transparent chamber that contains a three-dimensional volume or void that is configured to receive a blood sample. In this regard, the cell counting chamber 48 acts as a sample holder. The cell counting chamber 48, for example, may be made from an optically transparent material such as a polymer or glass. In one embodiment, the cell counting chamber 48 may have a uniform chamber depth (e.g., about 100 μιη) to provide a well-defined sample volume. In this embodiment, the housing 44 of the testing module 32 is designed to slidably receive the counting chamber 48 using slot 37 (as seen in FIG. IE) defined between the ledges 36 and the housing 44 when the counting chamber 48 is slid into the testing module 32, the opposing light sources 46 are butt-coupled to the edges of the counting chamber 48. When the counting chamber 48 is loaded with a blood sample and inserted into the testing module 32, the counting chamber 48 acts as a multimode opto-fluidic waveguide to that the cells contained within the counting chamber 48 are uniformly illuminated.

[0040] The housing 44 of the WBC testing module 32 includes an extension 56 (FIG. ID) that includes the mating surface 38. The extension 56 may include a cylindrical-shaped extension 56 that interfaces with a ring-shaped hub 21. The extension 56 still provides, however, an optical path between the camera 14 and the cell counting chamber 48 when located therein as noted below.

[0041] In the embodiment illustrated in FIGS. 1A, 1C, ID, IE, and IF the opposing light sources 46 include a plurality of LEDs. The wavelength emitted by the opposing light sources 46 is chosen to be at an excitation wavelength or wavelengths that causes

fluorescence of WBCs in response to incubation with a fluorescent stain. WBCs, unlike RBCs, have a nuclei and a nucleic acid stain such as SYT0®16 will bind to the nuclei of WBCs (other fluorescent stains may also be used). The light from the opposing light sources 46 passes through counting chamber 48 excites the fluorescently labeled WBCs. The fluorescent emission from each WBC is collected in a direction perpendicular to the excitation light path. In this regard, fluorescent light travels in the optical path that is formed between the counting chamber 48 and the camera 14. In one embodiment, a removable filter 50 is located in the extension 56/hub 21 (when loaded) and within the optical path between the counting chamber 48 and the camera 14. For example, the removable filter 50 may be located in the extension 56 portion of the housing 44. The removable filter 50 may be made of an inexpensive plastic material that is used to reject scattered excitation light. The filter 50 is removable from the housing 44 because when the other testing modules 30, 34 (RBC and hemoglobin) there is no need for the filter 50.

[0042] To use the WBC testing module 32, a small sample of whole blood is obtained (e.g., only several microliters of blood) from a subject. The sample of blood is then mixed with a buffer solution (e.g., phosphate buffered saline (PBS)) and a stain (e.g., nucleic acid stain). The addition of the buffer and the stain dilutes the sample. After incubation for several minutes, a small portion of the diluted sample (e.g., several microliters) is then loaded into the cell counting chamber 48. The cell counting chamber 48 is then placed flat for several minutes (e.g., two minutes) to allow the cells to sediment. The cell counting chamber 48 is then loaded into the WBC testing module 32 that has been secured to the base 20 that, in turn, is or will be secured to the mobile electronic device 12. The cover 35 can be positioned over the WBC testing module 32 after loading the cell counting chamber 48. With the cell counting chamber 48 loaded into the system, a switch 52 (FIG. ID) located on the WBC testing module 32 is switched on to power the light sources 46. The switch 52 is optional as the WBC testing module 32 may be turned on merely by plugging the power wires into the terminal 42. The WBC testing module 32 has wiring 54 that terminates in a connector that is configured to attach to the terminal 42 for power. The excitation light emitted from the light sources 46 then causes the WBCs contained in the cell counting chamber 48 to fluoresce. The perpendicularly directed fluorescent light is then imaged using the camera 14 of the mobile electronic device 12 as described below.

[0043] As seen in FIGS 1A and 2A, the mobile electronic device 12 is loaded with software 60. The software 60 may take the form of an application or "app" as is commonly used, for example, in software running on SMARTPHONES. The software 60 may run on a number of different operating systems including, without limitation, ANDROID, iOS, and WINDOWS based operating systems. With reference to FIG. 2A, the software 60 may be initiated by tapping an icon 62 or the like on the screen 16 of the mobile electronic device 12. FIG. 2B illustrates a menu 500 invoked by the software 60. In this illustration, the user is presented with the options of New Test, Batch Process, Instructions, Log Out, and Exit. New Test is selected to run a new blood analysis routine (e.g., WBC count, WBC count, hemoglobin concentration). Batch Process can be used to process a number of raw images in parallel and/or in series using the processor(s) of the mobile phone or mobile PC so that the results can be obtained faster, especially if the number of samples is large. The Instructions prompt provides the user instructions on how to perform the blood analysis. This can include, for example, sample preparation instructions as well as instructions on how to use the system 10. Log out and Exit permit the user to end the session with the software 60.

[0044] To initiate the WBC test, a user would select New Test which then brings up menu 502 as seen in FIG. 2C which gives the user the option of performing one of three tests including: (1) white blood cell counting, (2) red blood cell counting, and (3) hemoglobin concentration.

[0045] With reference to FIG. 2D, after the user selects "white blood cell counting" the camera 14 of the mobile electronic device 12 is turned on and an image is obtained (the light source 36 is also powered on at this point). This image contains the fluorescent light emitted by the WBCs in the cell counting chamber 48. The software 60 is then able to count the number of WBCs in a portion of the image which is then used to calculate a concentration of WBCs in the sample.

[0046] With reference to FIGS. 3A-3E, a description of the digital processing steps used by the software 60 to count cells and arrive at a WBC concentration will now be explained. With reference to FIG. 3 A, a raw image of the fluorescent WBCs is obtained. A smaller region of interest (ROI) is defined within the raw image (e.g., central 1000 x 1000 pixels) and cropped. As seen in FIG. 3B, this cropped region is further converted from the RGB (red, green, blue) channel to HSV (hue, saturation, value) space, and the saturation channel is extracted, which provides the maximum intensity contrast between the fluorescent labelled WBCs and the background. Next, as seen in FIG. 3C, an intensity threshold is applied to generate a binary image bitmask. Points of intensity below the threshold value are nulled or ignored. In one embodiment, the threshold may be adjusted by the user. The bitmask is a binary pixel map represented by 0, 1 where only the pixels with "1" values are of interest. As seen in FIG. 3D, cells are identified based on a blob detection scheme in which one can locate and count the labeled cells by using their size and connectivity in the binary image. Blob detection schemes are well known in imaging processing arts and will not be described herein.

[0047] The image volume of the selected ROI is calculated based on the number of pixels, pixel size and the counting chamber height. Then the cell density within the sample can be computed using the following formula:

[0048] C = ( x F) / V (Eq. 1)

[0049] where C is the cell concentration, N is cell count within the ROI, F is the sample dilution factor, and V is the sample volume within the ROI. FIG. 3E illustrates the output to the user of the computed WBC concentration. The concentration is reported to the user as cell numbers/microliter.

[0050] With reference to FIG. 2E, in one embodiment, the user is able to adjust various parameters during the imaging process. For example, the user may be able to adjust the threshold value that is used using the slider 64. In addition, the user may be able to select the crop width and crop height using fields 66. The location of the cropped portion defining the ROI may also be set by the user by defining the center of the width and height using fields 68. After the various parameters have been adjusted a user can analyze begin WBC analysis by depressing the analyze button 69.

[0051] FIG. 2H illustrates the results screen that reports the concentration of WBCs in the sample. As seen in FIG. 2H, there are additional parameters of channel height (of the cell counting chamber 48), pixel size, and dilution factor that may be adjusted by the user using fields 70a, 70b, 70c. In one embodiment, the user is able to modify one or more of these values. Alternatively, these values may be pre-populated based on the particular system 10 that is being used. For example, a particular system 10 that is being used may include a cell counting chamber 48 which a known height. Likewise, the pixel size relates to the degree of demagnification which is a function of the lens in the camera 14 and the lens 26 in the WBC testing module 32 and/or base 20. This information may be known in advance depending on the type of mobile electronic device 12 that is used and the particular system 10 used.

Finally, the system 10 may accompany instructions that use a known dilution factor (e.g., when the system is sold as part of a kit). All of this information may be pre-populated in fields 70a, 70b, 70c. As seen in FIG. 2H, the calculated concentration is presented to the user. The user is given the option to select a "submit results" button 72 whereby the results and other data related to the test may be communicated to a remote location using the communication functionality of the mobile electronic device 12. The information may include one or more of patient information, calculated WBC concentration, sample ID, sample testing parameters used, image(s) of the tested sample, and the like. The data may be transmitted via a WiFi connection, a wired connection connected to a LAN or WAN, or the cellular/mobile functionality of the mobile electronic device 12. The data may also be transmitted as SMS messages.

[0052] FIGS. 1A, 1C, ID, IE, and IF illustrate a testing module 30 for counting RBCs. The testing module 30 includes an opaque housing 80 or cover that is open at one end that, when mounted to the WBC testing module 32 (already secured to the attachment region 28 of the base 20), places the open end adjacent to the cell counting chamber 48 when loaded therein. Alternatively, as seen in the embodiment of FIG. IF, the testing module 30 may be directly secured to the base 20 in which case the cell counting chamber 48 is inserted into or adjacent to the RBC testing module 30. The open end of the housing 80 is configured and dimensioned to fit within the mounting portion of the WBC testing module 32. For example, the housing 80 may have and end cap 81 that interfaces with one end of the housing 44 of the WBC testing module 32.

[0053] The opposing end of the housing 80 includes a light source 82, for example in the form of an LED or the like. Because the testing module 30 is designed to image unlabeled RBCs in diluted whole blood using bright field illumination, the LED light source 82 can emit white light. The cell counting chamber 48 may be loaded into and removed from the testing module 30 for example using a slot 37 in the WBC testing module 32. In one aspect, the housing 80 has a height so as to place the light source 82 several centimeters (e.g., ~ 4 cm) away from the cell counting chamber 48 so as to uniformly illuminate the sample. The testing module 30 includes an optional switch 84 that is used to selectively turn the light source 82 on and off. The testing module 30 further includes wiring 86 (FIG. 1C) that terminates in a connector that can be connected to the terminal 42 to power the light sources 82.

[0054] To use the RBC testing module 30, a small sample of whole blood is obtained (e.g., less than several microliters of blood is needed) from a subject. The sample of blood is then mixed with a buffer solution (e.g., phosphate buffered saline (PBS)) to dilute the sample. A sample dilution of 1 whole blood with 999 is one illustrative dilution scheme. A small portion of the diluted sample (e.g., several microliters) is then loaded into the cell counting chamber 48. The cell counting chamber 48 may be the same cell counting chamber 48 used for WBC analysis. The RBC testing module 30 is secured to the WBC testing module 32 which is also secured to the base 20 (which is attached to the mobile electronic device 12) and the wiring 86 is coupled to the terminal 42. The cell counting chamber 48 is then loaded into the RBC testing module 30. With the cell counting chamber 48 loaded into the system, a switch 84 located on the RBC testing module 30 is switched on to power the light source 82.

[0055] Referring back to FIG. 2A, the user will initiate the software 60 contained in the mobile electronic device 12 to run the RBC test. After starting the program or application, a user would select "New Test" which then brings up menu 502 which gives the user the option of performing one of three tests including: (1) white blood cell counting, (2) red blood cell counting, and (3) hemoglobin concentration. Here the user would select the "red blood cell counting" option. After the user selects "red blood cell counting" the camera 14 of the mobile electronic device 12 is turned on and an image is obtained. This image contains the bright field image of the RBCs in the diluted solution. The software 60 is then able to count the number of RBCs in a portion of the image which is then used to calculate a concentration of RBCs in the sample.

[0056] With reference to FIGS. 4A-4E, the digital processing steps used by the software 60 to count cells and arrive at a RBC concentration is very similar to the process used to count WBCs with some exceptions. With reference to FIG. 4A, a raw image of the RBCs is obtained. A smaller region of interest (ROI) is defined within the raw image (e.g., central 1000 x 1000 pixels) and cropped. As seen in FIG. 4B, this cropped region is further converted from the RGB (red, green, blue) channel to HSV (hue, saturation, value) space, and the value channel is extracted, which provides better intensity contrast for the RBCs for bright field images. Next, as seen in FIG. 4C, an intensity threshold is applied to generate a binary image bitmask. Points of intensity below the threshold value are nulled or ignored. In one embodiment, the threshold may be adjusted by the user. The bitmask is a binary pixel map represented by 0, 1 where only the pixels with "1" values are of interest. As seen in FIG. 4D, cells are identified based on a blob detection scheme in which one can locate and count the labeled cells by using their size and connectivity in the binary image.

[0057] The image volume of the selected ROI is calculated using the same Eq. 1 described herein based on the number of pixels, pixel size and the counting chamber height. FIG. 4E illustrates the output to the user of the computed RBC concentration. The concentration is reported to the user as cell numbers/microliter. With reference to FIG. 2F, in one embodiment, the user is able to adjust various parameters during the imaging process. For example, the user may be able to adjust the threshold value that is used using the slider 90. In addition, the user may be able to select the crop width and crop height using fields 92. The location of the cropped portion defining the ROI may also be set by the user by defining the center of the width and height using fields 94. After the various parameters have been adjusted a user can analyze begin RBC analysis by depressing the analyze button 96.

[0058] FIG. 21 illustrates the results screen that reports the concentration of RBCs in the sample which is very similar to the WBC test. As seen in FIG. 21, there are additional parameters of channel height (of the cell counting chamber 48), pixel size, and dilution factor that may be adjusted by the user using fields 98a, 98b, 98c. In one embodiment, the user is able to modify one or more of these values. Alternatively, these values may be pre-populated based on the particular system 10 that is being used. The user is presented with a submit results button 100 which may transmit results and other data as described previously herein.

[0059] FIGS. 1A, 1C, ID, IE, and IF illustrate a hemoglobin testing module 34 for determining the concentration of hemoglobin in a blood sample. The testing module 34 includes a housing 104 that includes a cuvette holder 106 that is dimensioned to receive a sample cuvette 108. The sample cuvette 108 is optically transparent and may include conventional type-plastic disposable cuvettes. The sample cuvette 108 acts as a sample holder. While the cuvette 108 illustrated has a square shape, other shapes may also be used. The housing 104 includes a light source 1 10 (FIG. ID) therein that is located adjacent to a pinhole 1 11. The pinhole 11 1 is located in the central region of the cuvette holder 106 such that only a central region of the sample cuvette 108 is illuminated by the light source 1 10 and avoids light scattering from the edges. The pinhole 1 11 may have a size of around 1 mm. For the hemoglobin test, the light source 110 should emit light that is at or near an absorption peak for the hemoglobin molecule. For example, hemoglobin has a peak absorption at around 400-450 nm. A "blue" colored LED light source 110 that emits light at around 430 nm can be used as it falls within this range. The hemoglobin testing module 34 further includes an optional switch 1 12 that is used to turn the light source 110 on and off. A cover 113 is located over the light source 1 10 to aid in eliminating ambient light from entering the housing 104. Wiring 114 electrically connects the light source 110 and switch 1 12 via a connector to the terminal 42 for power. The wiring 1 14 can also be partially contained within the cover 113.

[0060] To use the hemoglobin testing module 34, a small sample of whole blood is obtained (e.g., a few microliters of blood is needed) from a subject. The sample of blood is then mixed with a RBC lysing solution (e.g., HYBRI-MAX, Sigma Aldrich) to lyse the RBCs within the sample. Mixing for several minutes may be used to aid the lysing process. The lysed blood sample is then diluted with PBS buffer solution. A small portion of the diluted sample is then loaded into the cuvette 108. The hemoglobin testing module 34 is secured to the WBC testing module 32 which is already secured to the attachment region 28 of the base 20. The housing 104 of the hemoglobin testing module 34 may have flanges, tabs, boss, or the like that interface with the ledges 36 WBC testing module 32. The hemoglobin testing module 34 includes wiring 1 14 that is coupled to the power terminal 42. The cuvette 108 is then loaded into the hemoglobin testing module 34. With the cuvette 108 loaded into the system, a switch 1 12 located on the hemoglobin testing module 34 is switched on to power the light source 1 10. If no switch is used, the wiring 1 14 may just be connected to the power terminal 42. The pinhole image of the transmitted light through the sample contained in the cuvette 108 can then be captured using the camera 14 of the mobile electronic device 12.

[0061] For the hemoglobin analysis, an internal calibration curve needs to be generated. In this regard, the cuvette 108 is loaded with distilled water and measured in the same manner as noted above with respect to the cuvette 108 containing the lysed blood sample. The "blank" may be analyzed prior to or after testing the actual blood sample.

[0062] With reference to FIG. 5, the digital processing steps used by the software 60 to determine hemoglobin concentration are described. As seen in operation 600 of FIG. 5, an image is obtained of the water sample contained in the cuvette 108. Another image is obtained of the prepared sample contained in the cuvette 108 as seen in operation 602. Next, as seen in operation 604, a common ROI from both images is cropped and converted to grayscale. For example, a 1000 x 1000 pixel area covering about 9 mm ² may be measured. The light intensity of the cropped grayscale images are then determined for both the cuvette 108 containing the sample and the water blank as seen in operation 606. In operation 608, the hemoglobin sample absorbance A can then be calculated using the following equation:

[0064] Where / represents the measured light intensity of the blood-containing sample and I ₀ represents the measured light intensity of the blank or water-containing sample. A calibration curve is generated for the system that is then used to convert absorbance A to a concentration value using the Beer-Lambert law. For example, multiple known

concentrations of hemoglobin can then tested using the system to generate a calibration curve. The calibration curve is typically linear and can be as a linear equation relating absorbance A and hemoglobin concentration. This calibration curve may be generated by the user, or alternatively, the calibration curve may be generated by the manufacturer of the system 10 in which case the user does not have to prepare any sort of calibration process. For example, a user may input the type of mobile electronic device 12 that is used (e.g., Apple iPhone model 5S) along with serial number or model number of the base and associated testing modules. The software 60 can then select or download the appropriate calibration curve.

[0065] FIG. 2G illustrates a screen shot of the screen 16 of the mobile electronic device 12 that illustrates the raw image obtained (in this instance) of a blood-containing sample. In one embodiment, the user is able to adjust various parameters during the imaging process. For example, the user may be able to select the crop width and crop height using fields 116. The location of the cropped portion defining the ROI may also be set by the user by defining the center of the width and height using fields 1 18. After the various parameters have been adjusted a user can analyze begin the hemoglobin analysis by depressing the analyze button 120.

[0066] FIG. 2J illustrates the results screen that reports the concentration hemoglobin in the sample (in units of grams (g) per dL. The user is presented with a submit results button 122 which may transmit results and other data as described previously herein. Also illustrated is a user comments field 124 whereby the user may manually input comments regarding the analysis or testing procedure.

[0067] It should be understood that the results of the tests, namely, the concentration of WBCs, RBCs, and hemoglobin do not necessarily have to be transmitted to a remote location. Instead, the results may be stored on the mobile electronic device 12 using internal memory or other storage functionality. The results may then be used directly at the point-of-care by a healthcare professional or they can be transmitted at a later time to a central database or server.

[0068] In the embodiments described above, the base 20 contains a lens 26 that is used with each testing module 30, 32, 34. In an alternative configuration, however, the lens 26 in the base 20 may be omitted and each testing module 30, 32, 34 may be provided with a lens (not shown). The first embodiment, of course, only requires a single lens to be used. While in some embodiments a single lens 26 may be disposed in the base 20 it is also contemplated that additional lenses (e.g., as part of a set of lenses) may be used. In yet another alternative embodiment, instead of having switches 52, 84, 1 12 in each testing module 30, 32, 34 a single switch may be placed in line with the power source 40 to selectively turn on/off the light sources 46, 82, 1 10. Further, as explained herein, no switches may be used. Instead, the user merely plugs the wiring connector into the power terminal 42. Further, the power source may be integrated into the testing modules 30, 32, 34. [0069] In an alternative embodiment, the WBC testing module 32 is used to perform differential WBC measurements. For example, WBCs can be fluorescently labelled with quantum dots conjugated with different markers that can be excited with light (e.g., UV LEDs or the other color LEDs) to simultaneously obtain multi-color images. Based on the color coding scheme, WBC subtypes can then be differentiated from one another. This embodiment may require having a light source 46 that emits light at several wavelengths which can be accomplished using different color LEDs.

[0070] In the embodiment illustrated in FIGS. 1A, 1C, ID, and IE, the WBC testing module 32 is used as a base or support onto which is mounted the RBC testing module 30 and the hemoglobin testing module 34. In other alternative embodiments, the RBC testing module 30 or the hemoglobin testing module 34 may function as the base or support with the remaining other testing modules being mountable thereon. In other embodiments, the RBC testing module 30, the WBC testing module 32, and the hemoglobin testing module 34 may be combined into a single sub-assembly that is modularly secured to the base 20 using a similar attachment region 28 as described herein.

[0071] The system 10 described herein may be sold as part of a kit. For example, the system may include the base 20 along with one or more of the testing modules 30, 32, 34. A complete kit may contain all three modules 30, 32, 34 but other forms of the kit may be sold with less than all three modules 30, 32, 24. The kit may also be sold with various reagents used in sample preparation. For example, the kit may include reagents such as the fluorescent stain (e.g., for nucleic acids), RBC lysing reagents, buffers, and the like. The kit may further include the sample holders (e.g., cell counting chamber 48 and cuvette 108). The kit may be also include a syringe or the like that is used to obtain the blood sample from the patient. The software 60 that is used to perform the calculations may be pre-loaded onto the mobile electronic device 12 or it may be downloaded by the user at, for example, an application store or website.

[0072] Experimental Results

[0073] The performance of the system for analyzing blood with a mobile electronic device was tested by measuring the total white blood cell, red blood cell and hemoglobin concentrations of anonymous human whole blood samples obtained from UCLA Blood and Platelet Center, where the gold standard measurement results for each sample were provided using a commercially available hematology analyzer (Sysmex).

[0074] In the tested design, a Samsung Galaxy SII was used as the starting base mobile phone. This Android phone has an 8 megapixel color camera module and its built-in lens has a focal length of/~ 4 mm. As noted herein, the blood analyzer design described below can also be implemented on different types of mobile electronic devices (e.g., different mobile phones) with small modifications on its base. The tested design includes: a mobile phone base fixed on top of the mobile phone camera unit, which includes two AA batteries and a universal port for three different add-on components. The three separate add-on components include testing modules for WBC imaging/counting, RBC imaging/counting and hemoglobin density measurements, respectively. Each add-on included an inexpensive plano-convex lens installed at its bottom facet and light-emitted-diodes (LEDs) for sample illumination. When the add-on component is attached on the mobile phone base, its lens gets directly in contact with the existing camera lens on the mobile phone to form an imaging system. As noted herein, a single lens may be provided on the base in which case the testing modules would not need separate lenses. LEDs are powered using the two AA batteries on the mobile phone base. All of these opto-mechanical attachments to the mobile phone were built through a 3-D printer which uses ABSplus™ modelling thermoplastic material.

[0075] The WBC testing module images fluorescently labelled WBCs in whole blood using an optofluidic illumination scheme. In this design, fluorescently labelled WBCs in diluted whole blood are loaded into a non-grid cell counting chamber, which has a channel depth of -100 μιη to provide a well-defined sample volume. Eight excitation LEDs (~ 470 nm) are directly butt-coupled to this counting chamber to illuminate the sample volume from its two sides symmetrically. The counting chamber that is filled with blood acts as a multimode opto-fluidic waveguide, uniformly exciting the fluorescent labelled cells inside the counting chamber. The fluorescent emission from each cell is then collected

perpendicular to the excitation light path, where an inexpensive plastic filter is used to reject the scattered excitation light. The fluorescent labelled WBCs are imaged by the mobile phone camera unit through a plano-convex lens that is inserted between the sample and the camera lens. This lens used in the tested WBC add-on has a focal length of/ = 15 mm, which provides an overall system demagnification of M /= 3.75. This optical imaging geometry has a modest spatial resolution; however, within a single image it enables counting of labelled WBCs over a large field-of-view (FOV) of ~ 1 cm ² .

[0076] The tested RBC testing module, on the other hand, is designed to image unlabeled RBCs in diluted whole blood samples using bright field illumination/imaging. The lens used in RBC testing module has a focal length of/ = 4 mm, which provides a system

demagnification factor of M =fjf= 4/4 = 1 and a FOV of ~ 14 mm ² . The RBC testing module used a single white light LED, which is ~ 4 cm away from the counting chamber, to uniformly illuminate the sample generating bright- field images of RBCs. Due to spatial aberrations, only the central region of this image (e.g., ~ 1-2 mm ² ) is used for cell counting purposes. This, however, does not pause a statistical issue since the density of RBCs is rather high, giving a sufficiently large number of cells per field-of-view.

[0077] Using the same mobile phone based blood analysis platform, hemoglobin concentration of blood samples was determined based on the measurement of light absorbance through a standard 1 cm cuvette containing lysed blood. According to the Beer- Lambert law, the absorbance of hemoglobin solution is proportional to its concentration. Therefore, to calculate the absorbance due to hemoglobin only, a differential measurement scheme was used using disposable cuvettes, where the transmission intensity of water sample was first measured (as reference), and then of the transmission intensity of a blood sample of interest was measured. A single blue LED (~ 430 nm) was used as the light source in the hemoglobin testing module for hemoglobin density measurements. For each test, a disposable plastic cuvette was filled with the liquid sample and is inserted into the sample holder. A 1 mm diameter pinhole aligned with the central region of the cuvette was located in the sample holder. The LED is in direct contact with this pinhole so that it only illuminates the central region of the cuvette and avoids any scattering from the edges. The sample cuvette is then slid into the hemoglobin testing module, which has an external lens (¾ = 8 mm) installed at the bottom. Then the hemoglobin testing module with the cuvette was clicked onto the mobile phone base and the transmission light intensity was recorded by the mobile phone camera, which is used to calculate the absorbance of the blood sample, revealing the hemoglobin concentration.

[0078] The concentration of RBCs, WBCs, and hemoglobin was obtained using a software program that operated as described herein. Three different tests were thus performed using the three analysis options presented by the software.

[0079] Whole Blood Cell Density Measurements

[0080] Whole blood samples with WBC concentrations ranging from 3,000/μΕ up to 12,000/μΕ were tested. In the sample preparation process for each test, 10 of whole blood sample was mixed with 85 μΐ _^ phosphate buffered saline (PBS) buffer and 5 μΐ _^ of nucleic acid staining (SYT016) at room temperature for ~ 20 minutes in dark. Since the quantum yield of SYT016 is very low when it is not bound to DNA or RNA, there is no extra washing or separation step needed after cell labelling. 10 μΐ _^ of this diluted and labelled whole blood sample was then loaded into the cell counting chamber (channel height of 100 μιη). The counting chamber was placed flat for two (2) minutes for the cells to sediment. Then the mobile phone took the microscopic image of the labelled WBCs using the WBC testing module. The images were digitally processed with using the algorithm described herein. The number of labelled WBCs were counted within a FOV of ~21 mm ² . Typically between around 600 to around 2500 cells per image/test were counted. The mobile phone counted WBCs results were compared with the standard test results obtained with Sysmex hematology analyzer (Sysmex America, Inc., Lincolnshire, Illinois). Using thirty (30) different blood samples, a correlation coefficient of - 0.98 was obtained between the two methods as shown in FIG. 6A. The mobile phone blood analyzer generated an absolute error within 7% of the standard test. A Bland-Altman analysis was also performed on the results (see FIG. 6B), which shows a bias of 230 cells μΐ^ ¹ with 95% limits of agreement of 955 and -495 cells μΐ ¹ for a wide range of WBC concentrations spanning 3,000/μΙ, up to 12,000/μΙ,.

[0081] Red Blood Cell Density Measurements

[0082] Whole blood samples were tested with RBC concentrations ranging from ~ 3 x 10 ⁶ to 6 x 10 ⁶ cells per μ . To prepare the diluted blood sample to be imaged, 1 μΐ., of whole blood was mixed with 999 μΐ., PBS buffer directly. Then 10 μΐ., of diluted whole blood sample was loaded into the same counting chamber, the same counting chamber as the one used for WBC counting experiments. Using the RBC testing module, bright-field images of the RBCs were taken after cell sedimentation. The number of RBCs within a field-of-view of - 1.2 mm ² was counted. Typically, around - 400 to 700 cells per image/test were counted. The mobile phone counted RBC results (n=12) were compared with the standard test results obtained using Sysmex hematology analyzer. As shown in FIG. 7A, the two methods showed a good correlation coefficient of - 0.98. The mobile phone blood analyzer generated an absolute error of 5% compared to the standard test results. A Bland-Altman analysis was performed on the results (see FIG. 7B), which shows a bias of -2.9 x 10 ⁴ cells^L with 95% limits of agreement of -2.5 x 10 ⁵ cells^L and - 3.2 x 10 ⁵ cells^L.

[0083] Hemoglobin Density Measurements

[0084] To measure hemoglobin density of blood samples in g/dL an internal "calibration curve" was generated for the mobile phone attachment. As part of the calibration process, a test cuvette was filled with DI water the transmission light intensity over 9 mm ² (1000 x 1000 pixels) was measured. Then 10 μΐ., of whole blood sample was added to 90 μΐ., RBC lysing buffer solution (HYBRI-MAX, Sigma-Aldrich) to lyse the RBCs. The blood sample was then mixed with lysing buffer for five (5) minutes using a tube rotator. After RBCs were lysed completely, another 900 μΐ., of PBS buffer was added to further dilute the lysed blood sample. Then the diluted blood sample was loaded into another cuvette and the transmission light intensity was measured over the same area. The absorbance value A for each lysed blood sample was calculated using Equation 2 above. The absorbance values for sixty (60) different samples with known hemoglobin concentrations was obtained (measured by Sysmex ) ranging from 1 1 g/dL to 16 g/dL and generated a linear calibration curve:

[0085] A = -0.28 +0.056 (Eq. 3)

[0086] where A is the absorbance value and X is the haemoglobin density of the sample (g/dL). To test a given blood sample, the software on the mobile phone computed its absorbance value (A) first and then further calculated the hemoglobin density based on the calibration equation (Eq. 3).

[0087] To evaluate the accuracy of the mobile phone based hemoglobin measurement platform, blind tests were performed with thirty seven (37) blood samples using the same approach described above. The hemoglobin concentrations obtained from the mobile phone based blood analyzer were compared with the standard test results obtained with Sysmex. FIG. 8A shows the comparison of these two methods, achieving a correlation coefficient of 0.92. The absolute error of the mobile phone measurements is less than 5%. FIG. 8B shows the Bland-Altman analysis for these two methods, revealing a bias of 0.036 g/dL with 95% limits of agreement of 0.63g/dL and -0.54g/dL.

[0088] While embodiments have been shown and described, various modifications may be made without departing from the scope of the inventive concepts disclosed herein. The invention(s), therefore, should not be limited, except to the following claims, and their equivalents.