GB2511362A

GB2511362A - Device

Info

Publication number: GB2511362A
Application number: GB1303718.9A
Authority: GB
Inventors: Tobias Broger
Original assignee: ZHAW ZURCHER HOCHSCHULE fur ANGEWANDTE
Current assignee: ZHAW ZURCHER HOCHSCHULE fur ANGEWANDTE
Priority date: 2013-03-01
Filing date: 2013-03-01
Publication date: 2014-09-03
Also published as: CH707735A2; GB201303718D0

Abstract

A cell analysis apparatus comprises; a slide 10 containing a cell sample treated with at least one fluorescent agent; sample-reading means comprising at least one sample-irradiating means 101, 102, each comprising a radiation source (collimated LEDs) 2, 6, adapted to irradiate the sample; detecting means 103 comprising a remotely controlled focusable lens 15, detector 12, and band pass filter 14, adapted to receive fluorescent radiation emitted from the sample; and a control and data analysis means 1 adapted to control the functional operations of the sample-reading means and to receive, analyse and present the results, the control and data analysis means 1 being provided in a unit remote from the sample and sample-reading means. The device is robust and easily portable. The control/analysis means 1 may be a mobile phone or tablet connected via a USB port or wirelessly.

Description

DEVICE

This disclosure relates to cell cytometry and more particularly to a small, robust device for cell analysis and enumeration.

The detection and enumeration of biological cells is of great importance in various applications such as health monitoring (in humans and animals, e.g. CD4 cell counting, detection of tuberculosis, blood cell counts), biotechnology (e.g. cell analysis in cell growth experiments and production processes), environmental analysis (e.g. waste water microbiology) and food-and beverage industry (e.g. drinking water monitoring, detection of bacteria and parasites in food).

Current practice for the detection and enumeration of biological cells is flow cytometry. In flow cytometry, fluorescently labelled cells are passed, one by one, through a bright laser beam. This results in scattered-and emitted light signals whose detection requires advanced optics and fluidics, senstive photomultipliers, complex electronics and a computer terminal for data storage and analysis. This results in unsatisfactorily complex systems with very limited mobility (heavy weight, high power consumption) and high investment and operational costs. Further, the operation requires highly trained personnel. Because of its complexity, flow cytometry is mainly used under controlled laboratory settings and not for point-of-care or field testing.

In specific fields of application, microscopy with subsequent image analysis for the identification and enumeration of cells is a powerful alternative to flow cytometry. Most of the mentioned disadvantages of flow cytometry do not occur in this so-called image cytometry. In image cytometry typically a cel suspension containing cavity of known volume is imaged by a microscopic system consisting of a light source, a lens system for magnification of the biological cells, an image sensor (CMOS or CCD) for image acquisition and an integrated computer to control the acquisition, performing the image analysis and organizing user interactions.

A proposal for a simple, portable device is found in WO 2011/143075. This device seeks to avoid many of the problems of prior art apparatus and is operated by an integrated computer system resulting in a power requirement of 55W and a total weight of 15kg.

It has now been found that it is possible to provide a simple, robust device for image cytometry, which can be operated remotely by a mobile control and data analysis means, and thus is not dependent on a built-in computer for steering, computing and user interaction purposes.

There is therefore provided a cell analysis apparatus comprising a. a slide 10 containing a cell sample, which sample has been treated with at least one fluorescent agent; b. sample-reading means comprising (i) at least one sample-irradiating means 101, 102, each comprising a radiation source adapted to irradiate the sample (ii) detecting means 103 comprising a remotely focusable lens 15 and adapted to receive fluorescent radiation emitted from the sample; and c. control and data analysis means 1 adapted to control the functional operations of the sample-reading means and to receive, analyse and present the results, the control and data analysis means 1 being provided in a unit remote from the sample and sample-reading means.

The complete separation of the sample-reading function and the control/data receiving & analysis/presentation functions means that the sample-reading means may be particularly small and robust and that the control and data analysis means is a small, robust, portable, low-cost device with a high processing power (such as a mobile phone or tablet). In a particular embodiment, the sample-reading means is a hand-held device, that is, it is sufficiently small and light to be carried and utilised without requiring any supporting means. This makes it ideal for use in remote and difficult locations, where larger devices would be difficult to transport and use.

The cells in or on the sample slide are stationary, that is, they are retained in a sample well or fixed on the surface of the sample slide, and no flow and thus no complex fluidic system is required. The sample well is typically 0.1 mm thick, but other thicknesses (greater and smaller) are possible, depending on use. The sample slide may comprise a simple measuring chamber, optionally containing chemical or biological agents to react with the sample.

The cells in/on the sample slide are treated with at least one fluorescent agent. In a particular embodiment, they are treated with a plurality of such agents, in a particular embodiment with two such agents that fluoresce at different wavelengths. In the case of two agents, radiation may be provided either by two different sources, typically LED (light-emitting diode) sources, having different wavelengths, or by a single source comprising two switchable sources. Typical examples of fluorescent agents include fluorescently-labelled antibodies (e.g. AFC, FITG) or other fluorescent cell stains (e.g. auramin-o, acridine orange, sybr green, propidium iodide) and typical illumination source wavelengths utilized are blue (470nm) and red (640nm). The following discussion will deal with the case in which there is one source, but it is to be understood that two or more are also included.

The light produced by the source is collimated by a collimating means, that is, a system of at least one lens that produces a parallel beam.

The radiation strikes the sample slide and causes the corresponding fluorochrome present on or in the cells to fluoresce. The fluorescent radiation is received at a detecting means. This may be any means that is capable of receiving the fluorescent radiation and generating a corresponding signal to be interpreted by the control and data analysis means. The detecting means comprises a focusable lens. This may be a single lens or a lens system, of any type known to the art (glass and polymeric lenses, lens array or gradient-index (GRIN) lenses) and focusing may be carried out by any convenient means, for example, by a movable lens or lens element, or by an adaptive focus-tunable lens. The focus is changed during the operation, so that the cell sample or fluorescent marks present on the sample slide are either in focus, or fluorescent radiation is observed at multiple levels of the sample (larger than the depth of field of the optical system) and a complete picture is obtained.

The signal generating means may be any suitable device. For example, it may be a CMOS or CCD chip. There may also be present between the lens and the signal generating means a filter that allows only the fluorescent radiation through. In the case of one fluorochrome this may be a band- pass or long-pass filter, whereas in the case of multiple fluorochromes this may be, a multiband-pass filter.

The control and data analysis means (hereinafter "C/A means) is responsible for the functioning of the device and for the reception, analysis of the results of the sample reading process. It controls the functiona operations of the device -as hereinunder further described in the list of operations, there are a number of possible operations, not all of them necessary in all devices or for all uses. It is possible to provide a device with limited functionality. Alternatively, it is possible to provide a wide range of functions, the user having the ability to select the desired ones for any given situation. The C/A device further serves as a user interface to present results. The C/A means is remote from the sample reading means and communication between the two may be by electrical connection or wirelessly. In a particular embodment, the C/A means is a handheld mobile apparatus, such as a smart phone or a tablet and a USB cable is used for communication.

To carry out the necessary tasks, the C/A is equipped with software that allows the C/A device to control the sample reading means, the receipt of results in the desired format and the analysis of the results. The software can be activated by user interaction (for example, by means of a start button on a C/A screen) or autonomously after detecting the presence of a new sample inserted in the sample reading means. Once activated, the C/A performs a number of operations, as listed below. Some of these are optional.

-optionally, it receives from the sample-reading means information as to the nature of the sample slide and the operation to be performed; -optionally, it controls sample plate movement, should sample plate movement be needed in the particular analysis, for example, to scan more than one field of view; -optionally, it controls the one or more radiation sources powered via cabe, should this be required; -it controls the focus of the detecting means lens, so that the sample or fluorescent marks (e.g. cross-lines, orientation marks, quality control elements) on the sample slide are in focus and/or the entire depth of the sample well is scanned; -it controls and adjusts parameter settings of the light receiving detector (e.g. exposure time, gain, gamma, temperature, hue, brightness, contrast, saturation) -it triggers the sample reading process on the light receiving detector; -it receives and stores the measured data from the detector after the reading process is finished; -it analyses the received data and identifies cells or structures of interest performing image enhancement and image analysis algorithms and, identifies cells or structures of interest and if desired, counts the found objects.

-it calculates and provides the results in a desirable format on its screen, for example graphically or a live streamed image.

-it stores data and/or results -optionally, it sends data and/or results to a central database using the communication ability of the C/A (e.g. via GSM, GPRS, UMTS, HSDAP, LTE or Wireless LAN) The individual steps in the programmed operation are not in themselves new, but the performing of them all in automatic sequence and their incorporation in a mobile, remote C/A device, bringing with it unaccustomed levels of robustness, simplicity of operation and convenience (e.g. easy operable user interface) have never been done before in the field of cell analysis and cytometry.

The software may readily be developed from existing software code by a skilled programmer and can be run on different operation systems.

The operating system of the C/A device may be any suitable system, non-limiting examples including Android, Apple iOS, Windows Phone, Windows, BlackBerry OS, Symbian and Linux Similarly, the software may be programmed in any suitable programming language, non-limiting examples of which include Java, Objective-C, NET, Python, HTML, JavaScript, CSS, C++, C and C#. In a particular embodiment, the operating system is Android and the programming language is Java.

The software controls the communication with the sample-reading means, and the user interactions, and it performs image enhancement and analysis on the received data. In a particular embodiment, the software uses the USB host mode ability of the C/A device (USB On-The-Go) both to power the sample-reading means and to communicate with it. It allows the operator to control the operations by, for example, use of a touch screen to manipulate sliding controls, or to drag an image and thus, for example, cause the slide to move.

In a further embodiment, the software may use the USB accessory mode to communicate with the sample-reading means. In the USB accessory mode the sample-reading means powers the USB bus.

In a further embodiment the software of the C/A device may use a wireless connection to interact with the sample reading means.

In a particular mode of operation, data analysis may include known techniques for image processing, such as image enhancement (e.g. multi-frame super resolution techniques), and cell identification algorithms (e.g. Otsu algorithm for thresholding, Watershed algorithm for segmentation, and object recognition algorithm).

One aspect of the device is the ability to measure more than one fluorochrome present in the cell sample without the need to exchange optical filters. In current image cytometry, mechanical exchange of the emission filter on filter wheels is standard procedure to measure each emission wavelength band (and thus the fluorescence intensity of every fluorochrome) separately. This mechanical filter exchange reduces the system robustness and increases the power consumption and system size.

This is overcome by measuring multiple fluorochromes by means of a multiband-pass filter in combination with a time-controlled on-off switching means of the different excitaton sources. In a particular embodiment using a dualband-pass filter and two excitation sources, up to two fluorescences present in the same sample can be measured. However, it is also possible to use triple-, quad-or pentaband-pass filters, such that up to five fluorescences can be measured without filter movement. In a further embodiment where only one fluorochrome has to be measured, a normal band-pass filter in combination with one radiation source is naturally sufficient.

The device hereinabove described is useful for the examination and measurement of a wide range of cells, for example, those in sputum, blood and urine. It is especially useful for the detection ol tuberculosis cells as described in example 1.

The device is further described with reference to the drawings and the non-limiting examples.

Figure 1 represents a schematic view of a device as hereinabove described.

Figure 2 represents a schematic cross-section of the device of Figure 1.

Figure 3 depicts a typical application user interface.

In Figures 1 and 2, there is depicted a device having two sample-irradiating means, generally indicated as 101 and I 02, and a detecting means, generally indicated as 103. The device has two LED sources of light, 2 and 6, each of which is provided with a collimating lens 3 and an optical band-pass filter (4,8 respectively). LED source 2 is blue (peak wavelength at 470 nm) and source 6 is red (peak wavelength at 640 nm), and the respective band-pass filters are 470-495 nm and 565-650 nm.

The two LEDs, powered via cable 5, are activated in sequence and illuminate a cell sample on a sample slide 10. This slide contains the sample in a sample well 18, and it is capable of moving along two orthogonal axes, indicated as 7 or about an axis of rotation 70. There are two fluorochromes (16,17 respectively) present on the treated cells, and they are excited at the wavelengths of the LEDs. (The fluorochromes are chosen according to the desired use of the device, as wii be seen in the subsequent examples). The fluorescent radiation is received by a detecting means 103 comprising an objective lens 13, an optical multiband-pass filter 14 (emission bands at 520-560 nm and 655-685 nm) designed to remove all radiation except for those wavelengths emitted from the fluorochromes, a focusable lens 15 and a sensor array 12, which is a CMOS chip (5 MP). This converts the received fluorescent light into a signal, which is sent via a USB cable 11 to the control and data analysis means 1, which, in this case, is a smart phone programmed to control the device. The operating system is Android and the software is written in Java.

In operation, the cell sample treated with the fluorochromes on the sample slide 10 is inserted into the device and the device switched on by means of the smart phone I. The LEDs then emit light on to the sample for a sufficient time to permit the focusing lens IS to focus on the cells and to acquire an image. The data are passed to the smart phone, which performs image optimization and image analysis algorithms and then generates the results in the form of a desired numerical and graphical display. Additionally the results are stored on the smart phone and sent to a central result database exploiting the smart phones communication abilities.

In Figure 2, cells containing a first fluorochrome 16 are excited by light 20 (in a typical example 470-495 nm) from a first LED 2, collimated with a collimating lens 3 and filtered through a band-pass filter 4. The emitted light 23 from the first fluorochrome (typically 520-560 nm) can pass the multiband-pass filter 14 and be detected, whereas other light (e.g. scattered excitation light 22 from cells 19 without fluorochrome (typically 470-495 nm)) is blocked. In a second step, LED 2 is switched off and the second light source 6 is switched on. Cells containing a second fluorochrome 17 are excited by light 21 (typically 565-650 nm) from the second light source 6, collimated with a collimating lens 3 and filtered through a band-pass filter 8. The emitted light 25 from this second fluorochrome (typically 655-685 nm) can also pass the multiband-pass filter 14 and is detected, where unwanted scattered light (565-650 nm) 24 again is blocked by multiband-pass filter 14. In general the multiband-pass filter blocks scattered light 22,24 where from fluorochromes emitted light 23,25 can pass via the filters wavelength bands (represented here figuratively by the two peaks 9).

Each light source serves for the excitation of one specific fluorochrome and results in light intensities (SLEDX) emitted by only one fluorochrome. As a result no calibration is needed and the intensity signals (SLEDX) measured on the CMOS chip 12 are proportional (factors cx, 13) to the concentration of the fluorochromes ([A], [B]): 5LF/)1 = a [A] 5LED2 = P [B] Thus intensity (SLEDX) of each fluorochrome (proportional to the concentration) for every detected cell can be measured and used for data analysis, e.g. to produce intensity histograms or dotplots as commonly known from flow cytometry.

Figure 3 depicts a typical screen of a C/A device running the application user screen for user interaction and data/result presentation. In normal mode, the sample reading is started by pressing an Analyse Sample" button 28 and the automated measuring procedure is initiated. Specific regions of the sample may be examined prior to or after the automated measuring process by using manua controls 27. Alternatively, the live streamed image 29 may be dragged by finger, this causing corresponding movement of the slide i 0 along the axes 7. Focussing, exposure time and gain are controlled using sliders 27. The light sources can be selected using buttons 28 The live-IS streamed image 29 from the light receiving detector 12 is displayed and enables the user to immediately see results 30 and changes on the detected image as a result of adapted control parameters.

Example 1

Detection of acid fast bacilli in sputum smears This example describes the use of the device of Figure 1 for the detection of Mycobacterium tuberculosis on Auramine-O stained sputum smears.

To get a sufficient resolution to allow computer-algorithm aided identification of individual tuberculosis bacteria, the magnification caused by the combination of objective lens 13 and focussing lens 15 was M=3 and thus the observed area on the sample slide ID was smaller than the area of the CMOS image sensor 12. This results in a field-of-view of 1.8 mm2. The spatial resolution was limited by the pixel size of the CMOS image sensor and lies between 0.8-1.5 pm, which is sufficient for the imaging of M. tuberculosis bacteria.

In this example, acid-fast control slides (Doenitz Frolab, Augsburg, Germany) were stained according to the common Auramine-o staining protocol. One positive and one negative control slide were measured. The respective slide was inserted into the device and automated analysis was initiated by pressing the Analyse Sample" button 26 on the smartphone. A total of 18 fields-of-view were observed by rotation 70 of the sample slide 10. The scanning/moving/focussing of the sample was controlled through the C/A 1 and takes less than 60 seconds. Auramine-o absorbs blue light and emits green light, thus fluorescence is only measured at the time LED 2 is on. At the time LED 6 is on, no fluorescence is detected.

The Image enhancement and analysis was performed on all 18 images. On the positive control slide green fluorescent tuberculosis bacteria were successfully identified by the image analysis running on the C/A device I and identified bacteria were presented to the user on the screen for final diagnostic decision-making. On the negative control slide no matching bacteria were found.

In common Auramin-o protocols for microscopic identification of tuberculosis in sputum smears, the microscopist only scans an area of approximately 7-12 mm2 (normally 40 fields cf view using the 40x objective of the microscope). This is factor 3 less than achievable with the abovementioned device. Thus a substantially higher sensitivity is possible. This is especially important in patients with a HIV/tuberculosis co-infection, as they typically have a lower bacterial load in sputum.

Example 2

Counting white blood cells in whole blood This example describes the use of the device of Figure 1 with another fluorochrome proofing that this device can be used for different applications and is not limited to immunofluorescence staining or multiple fluorochromes.

pL EDTA blood was mixed with 100 j.iL of an acridine orange staining solution (containing 0.06 mg/laO mL acridine orange, 1 % Formaldehyde, 0.25% Sodium citrate, 3% Ethylene glycol) and incubated for 5 minutes at room temperature. The acridine orange stains the DNA of all white blood cells. The resulting cell suspension was transferred to the measuring well 18 of a polycarbonate test slide 10. The measuring well has a defined thickness of 0.1 mm. The magnification caused by the combinaton of objective lens 13 and focussing lens 15 was M=0.55 and thus the field-of-view 52mm2. This results in an observed sample volume of 5.2 pL.

The fluorescence of acridine orange bound to DNA was measured by capturing an image during blue light excitation (470-495 nm) resulting in emitted light between 520 and 560 nm. The software counts the white blood cells on the image and provides the diagnostic result in white blood cells per pL. Further green light intensity per cell, size of the cells and circularity of the cells are calculated and can be used to generate intensity or size histograms and dotplots.

Example 3

Diagnosis of insect venom allergy in blood by determining basophile activation This example describes the use of the device of Figure 1 for the measuring of immunostained cells.

There was used cell surface immunostaining for CD2O3c to identify basophils and simultaneous cell surface immunostaining for basophil activation marker CD63 to measure the percentage of activated basophiles. 100 pL EDTA blood of an allergic donor was mixed with 100 pL of stimulation buffer (containing calcium, heparin and lnterleukin-3, BUhlmann Laboratories, Switzerland), 100 pL of Yellow Jacket Wasp Venom (BUhlmann Laboratories, Switzerland), 10 pL of CD2O3c-APC (monoclonal antibody, anti-human, Miltenyl Biotec GmbH, Germany) and 10 pL of CD63-FITC (monoclonal antibody, anti-human, Lucerna-Chem AG, Switzerland). The tube was incubated for minutes at 37 °C and then centrifuged for 4 minLtes at 400 x g and supernatant was decanted.

The cell pellet was re-suspended in 50 pL red blood cell lysis buffer (containing I % formaldehyde, 0.25% sodium citrate, 3 % ethylene glycol) and incubated for 5 minutes at room temperature in the dark until the lysis of red blood cells was competed. The resulting cell suspensicn was transferred to the measuring well 18 of the sample slide 10 and the slide inserted in the device of Figure 1. The automated analysis was initiated by pressing the "Analyse Sample" button 26 on the smartphone 1.

The two fluorochromes (APC and FITC) were consecutively measured. APC fluorescence was measured by capturing a first image during red light excitation (565-650 nm) resulting in emitted light between 655-685 nm. APC positive cells represent all basophile cells in the observed sample.

FITC fluorescence was measured by capturing a second image during blue light excitation (470- 495 nm) resulting in green emitted light between 520 and 560 nm. FITC positive cells represent cells activated by an allergic reaction. In this example a sample volume of 5 pL was observed resulting in a total of 412 CD203c-APC positive basophile cells. 272 out of those basophile cells were detected as CD63-FITC positive activated cells, which equals to 66 % showing a clear respond of the patient's blood to the wasp venom (according to the provider of similar tests measured by flow cytometry the proposed cut-off to distinguish an allergic reaction to venoms lies at 10 % activated basophils)

Claims

Claims: I. A cell analysis apparatus comprising S a. a slide 10 containing a cell sample, which sample has been treated with at least one fluorescent agent; b. sample-reading means comprising (i) at least one sample-irradiating means 101, 102, each comprising a radiation source adapted to irradiate the sample; (H) detecting means 103 comprising a remotely controlled focusable lens 15 adapted to receive fluorescent radiation emitted from the sample; and c. a control and data analysis means 1, adapted to control the functional operations of the sample-reading means and to receive, analyse and present the results, the control and data analyss means I being provided in a unit remote from the sample and sample-reading means.
2. Apparatus according to claim 1, in which the control and data analysis means 1 is a portable communications device, selected from a smart phone and a tablet computer.
3. Apparatus according to claim 1, in which the control and data analysis means 1 communicates with the sample-reading means by means of an electrical connection 11.
4. Apparatus according to claim 1 in which the control and data analysis means 1 communicates wirelessly with the sample-reading means.
5. Apparatus according to claim 1, in which the control and data analysis means 1 comprises software that is adapted to -optionally receive from the sample-reading means information as to the nature of the sample slide and the operation to be performed; -optionally control sample plate movement; -optionally-control the one or more radiation sources; -control and adjust parameter settings of the detecting means; -trigger the sample reading process on the detecting means; -receive and store the measured data from the detecting means after the reading process is finished; -analyse the received data, and utilise image analysis algorithms to identify cells or structures and/or enumerate them; -calculate and provide the results in a desirable format on a screen; -store data and/or results, and -optionally communicate data and/or results to a central database.
6. Apparatus according to claim 1 * in which the detecting means comprises signal-generating means selected from a CMOS and a GOD chip.
7. Apparatus according to claim 1 in which multiple fluorochromes are used and radiation therefrom is measured by means of a niultiband-pass filter in combination with a time-controlled on-off switching means of multiple radiation sources.
8. Apparatus according to claim 7, in which two fluorochromes are used in conjunction with two radiation sources and a dualband-pass filter. Ia