Hemoglobin (Hb) disorders are among the world's most common monogenic diseases Nearly 7% of the world's population carry Hb gene variants. Sickle cell disease (SCD) arises when hemoglobin variant mutations are inherited homozygously (HbSS) or paired with another β-globin gene mutation. Globally, an estimated 400,000 babies are born annually with SCD and 70%-75% are in sub-Saharan Africa (SSA). It is estimated that 50-90% in SSA die by their 5th birthday, 70% of these deaths are preventable. Epidemiological modeling shows that universal screening could save the lives of up to 9,806,000 newborns with SCD by 2050 with 85% born in Sub-Saharan Africa (SSA). Effective management of SCD involves genetic counselling, early diagnosis, and, importantly, newborn screening (NBS).
NBS is a most important public health initiative. SCD NBS performed in centralized laboratories has dramatically dropped SCD mortality in resource-rich countries. NBS requires sensitive detection of certain low level Hb variant from high level Hb variants. For example, among newborns, normal hemoglobin A (HbA) and sickle hemoglobin S (HbS) are at lower levels while high levels fetal hemoglobin (HbF) holds up to 90% of total Hb. In resource-rich countries, standard clinical laboratory technology including high-performance liquid chromatography (HPLC) and isoelectric focusing (IEF) are typically used in testing Hb variant. However, these advanced laboratory techniques require trained personnel and state-of-the-art facilities, which are lacking or in short supply in countries where the prevalence of hemoglobin disorders is the highest.
SCD NBS is challenging in low and middle income countries, where heavy SCD burden exists, due to lack of lab infrastructure and skilled personnel. In a 2019 report, the World Health Organization (WHO) has listed hemoglobin testing as one of the most essential in vitro diagnostic (IVD) tests for primary care use in low and middle income countries. Furthermore, hemoglobin electrophoresis has recently been added to the WHO essential list of IVDs for diagnosing SCD and sickle cell trait. As a result, there is a need in the art for affordable, portable, easy-to-use, accurate, non-capillary flow electrophoresis tests to facilitate decentralized hemoglobin testing in low-resource settings to enable widespread NBS.
Non-limiting and non-exhaustive embodiments of the invention are described with reference to the following drawings. In the drawings, like reference numerals refer to like parts throughout the various figures, unless otherwise specified, wherein:
The disclosed systems and methods detect and diagnose various disease states including hemoglobinopathies, such as sickle cell disease and trait, thalessemias, and the like. Such disease states as sickle cell disease and trait are important to diagnose early in life so treatment can begin and the effects of disease morbidities are reduced. For example, newborns can be screened for sickle cell disease or trait, especially in regions with populations with a high hereditary percentage of sickle cell carriers. Often these same regions with high populations of sickle cell carriers lack medical resources required to perform conventional gold standard laboratory tests to detect and diagnose the disease state.
The disclosed systems and method use electrophoresis in non-capillary flow electrophoresis that generate band data indicative of compounds present in the patient sample. For example, the non-capillary flow electrophoresis device can be a point-of-care (POC) diagnostic device. The patient sample is often blood but could be other patient biologic material as well. The disclosed electrophoresis systems capture one or more images of the electrophoresis strip on which the patient sample is placed and to which an electric field is applied that separates compounds in the patient sample based on their size and electrical charge. The separation of these compounds produces bands that migrate across the electrophoresis strip during the active test. The disclosed systems and methods capture the one or more images of the electrophoresis strip during the active test and oftentimes throughout the active test in a non-capillary flow electrophoresis device. When the band(s) are produced and an image is desired, light is emitted towards the electrophoresis strip. The light is either absorbed by or fluoresced from the band, which is detectable on the captured image(s).
During the active test, one or more targeted wavelengths of light are emitted towards the electrophoresis strip to produce the desired image(s). The targeted wavelengths can be a range of wavelengths in some examples or a particular color or color range of wavelengths. For example, the emitted light can be in a range of 390-430 nanometers (nm), which is in the ultra-violet (UV) wavelength range. In some examples, a second light emission occurs that could be a different wavelength range than the first light emission, which could be white light or another color of light that produces different absorption or fluorescence characteristics in the image(s) of the band(s) on the electrophoresis strip. The image characteristics produced by the absorption or fluorescence of each wavelength of light emitted towards the electrophoresis strip can vary over time throughout the active test and can vary with different wavelengths of light, camera aperture, etc.
Typically, conventional electrophoresis systems run a complete electrophoresis test on the patient sample on the electrophoresis strip then stain the final strip to cause the bands produced during the test to either absorb or fluoresce in a particular way in response to white light. The absorption or reflection can be controlled by the type of stain(s) applied. However, the staining process is lengthy, expensive, and requires sophisticated laboratory equipment. In low resource regions or remote geographic locations without good access to medical facilities, these expensive laboratory tests are impractical or simply unavailable. Such regions need POC diagnostic devices like the disclosed system to accurately detect and diagnose these disease states.
Many of these diseases have a limit of detection (LoD) that is low, which requires the sophisticated laboratory equipment to run the electrophoresis test with high quality cameras and staining processes available. Even then, there are still patient samples that are too low of a concentration of the target compound to be detected in a conventional system. The LOD of a particular compound or disease indicator (e.g., analyte, antibody, label, etc.) can be a low concentration at which the disease is detectable at an acceptable accuracy level. Traditional POC devices could not perform tests at the LOD required to detect certain disease states, such as sickle cell disease or trait, especially in newborns, and thalassemias for example, because they use images of white light emitted towards a strip that has been stained. These images produce edges that are blurred or their shape or visibility has been sacrificed. Such edge, shape, speed (changes in the band position over time), and visibility detection in the images defines whether the captured image includes a band indicating the target compound. For example, when white light is absorbed or reflected from the bands produced at the end of the electrophoresis test, the band image is hard to detect and, in some cases, has an LOD of 20%, which is not sensitive enough to detect diseases like sickle cell disease and trait or thalessemias without sophisticated imaging systems and dyes or staining processes. For example, the position of a band imaged over time indicates speed of migration of the band on the strip. Such position or speed information obtained from images captured during an active electrophoresis test help to identify low concentrations of compounds that are not consistently present throughout the active run. For example, a compound may not appear during a first or final phase of the active run and is only visible during a middle portion of time in the active run. Taking position or speed data of the band associated with the target compound during the middle portion of the active run can detect the compound while taking an image of the final phase of the run in the conventional technique would miss it entirely. The LOD is lower in the disclosed systems and methods because they use light emitted at the targeted wavelength that produces the highest quality absorption or fluorescence qualities in the compound band.
The wavelength of the light emitted towards the electrophoresis strip is selected based on a maximum or optimal absorption or fluorescence characteristic(s) of the compound of interest. For example, the maximum or optimal characteristics could be the wavelength at which the compound band maximally absorbs light or fluoresces the compound. Compounds differ on their absorption and fluorescence properties or characteristics and respond different to various wavelengths of light. Compounds may not absorb or fluoresce at all in response to emission of certain wavelengths of light while the same compound could produce a clear, intense absorption or fluorescence in response to light emitted towards it at a different wavelength. This “imaging wavelength” is the wavelength at which the highest quality image is produced to analyze for detection of the disease state. In some examples, the imaging wavelength is matched to the target compound of interest based on known empirical data or previous tests performed on bands known to have the target compound.
In other examples, the imaging wavelength can also be matched to the target compound of interest based on matching to a control band. The control band(s) can include the target compound or can exclude the target compound. Their purpose is to serve as a relative point of comparison for images of the other bands produced during the active electrophoresis test. The images of the control bands can be compared in intensity, shape, edge shape, speed or clarity, or any other characteristic that either relates to or discerns from a band with an unknown compound or no compound.
In some example systems, light is emitted towards the electrophoresis strip at multiple wavelengths. The multiple wavelengths can produce different responses in absorption or fluorescence of the bands on the electrophoresis strip. Each of those responses can either validate or provide additional data to each other when the bands images are analyzed. For example, light of a wavelength within a range of ˜410 nm in the UV range of 390-430 nm is emitted at a first time and then a second white light is also emitted at a second time. The first time and the second time are temporally spaced apart any suitable amount of time. In another example, the emission occurs at the same time.
In the example in which the light at the imaging wavelength is emitted towards the strip at the first time followed by the light emission at the second wavelength at the second time, when using the disclosed systems for detection of sickle cell disease and anemia, both white light and UV produce images of hemoglobin and marker: UV provides a mode of detection of low levels of sickle cell disease or trait, for example, which has an LOD of ˜4%. In this case, the white light image is used for to separate the marker, allowing for marker-only tracking (no hemoglobin is visible in the red channel of white images) and for calculating the hemoglobin to marker concentration ratio.
Bands with low concentrations of hemoglobin are only visible in UV since hemoglobin absorbs 410 nm, for example, much more than it absorbs white light. For sickle cell disease, for example, the targeted UV light emission allows the sickle cell (hemoglobin-S or HbS) band to absorb light at a detectable level in the captured image. Other wavelengths of light cannot produce the same level or response profile of absorbance of the HbS band.
In detecting HbS in newborns, the required LOD is low—4% or less—and can be masked by presence of fetal Hb or HbF, which has a high or 90% concentration at birth and its concentration is reduced in the first few months of life. Detecting HbS is more difficult in the presence of HbF, especially at early age with high concentrations of HbF. Because of the high concentration of HbF, newborns are particularly hard patients in which to detect HbS.
In some examples, the system captures multiples images of the electrophoresis strip during the active test. These multiple images are combined to produce an enhanced image of the target compound band. To create the enhanced image, the target compound band images can be overlaid to ensure accurate band detection or could be compared to each other to validate data, ensure the edge of the band or the shape is consistent with a compound profile or is consistently developing over time during the test in an expected manner to match it with a target compound profile of the same development or compare it to empirical or threshold data. The enhanced image can be output to a display for a user to visually analyze, in some examples, or could be stored.
Multiple images can provide false colors by combining the images of the light emitted in overlapped wavelength ranges. For example, a UV image can be combined with a white image (e.g., replacing green and blue channels of white with information coming from UV image adapted by histogram matching) to enhance the visibility of the faint blood bands while preserving the familiar appearance of a white-lit image.
In some examples, the disclosed systems and methods can also create a single representation that includes all the band information—the detected characteristics of each imaged band—from all images (or multiple images) captured during the active electrophoresis test. The single representation is a run summary of imaged results of the target compound band throughout the entire the electrophoresis test. Creating the run summary diagram is the first step of the speed profiling algorithm that interprets the decomposition of the patient sample content to different Hb variants based on their electrophoresis speed throughout the active test.
The method then selects an imaging wavelength of light based on the absorption or fluorescence characteristics of the compound 104. The imaging wavelength is the wavelength of light that produces the optimal image characteristics to analyze to detect the compound in the patient sample. Typically, the imaging wavelength causes the absorption or fluorescence characteristics to be enhanced compared to the imaged band's response to light of a wavelength other than the imaging wavelength. The enhanced image typically allows for detection of the band at a lower concentration of the compound in the patient sample or can produce an image that defines the band edge or shape of the band with greater clarity to quantify the compound or to otherwise give detect data relating to the compound. For example, using images captured using UV light, it was observed that the shape of the blood band concentration signal peaks are better modelled by skewed generalized gaussian distribution rather than standard gaussian. Switching to skewed generalized gaussians improved quantification accuracy. Finding that a band is sharp and narrow or diffuse and wide can also be used to determine a test failure since a very wide diffuse band can indicate an issue that renders the results invalid.
The method then selects an imaging wavelength of light based on the absorption or fluorescence characteristics of the compound 106. The absorption or fluorescence characteristics of the compound are a compound profile that optimizes the image produced when the band is imaged throughout the active test. The determination of the absorption or fluorescence characteristics of the compound 104 and the selection of the imaging wavelength of light 106 can be performed by a remote computing device, server, or system or can be integrated into any of the disclosed systems. The disclosed method then generates an image of a band on an electrophoresis strip during an active run of an electrophoresis test 108. The image can be captured by an imaging device, such as an optical imaging device like a camera, which captures an image of the electrophoresis strip. In some examples, the method captures multiple images timed periodically, randomly, manually, or in a particular sequence or on a specific schedule throughout the active run of the electrophoresis test.
The method also causes emission of light at the imaging wavelength towards an electrophoresis strip with the patient sample. As mentioned above, this occurs during the active electrophoresis test. The light can be emitted by any source that is either integrated within the system or external to the system. The method then determines an absorption characteristic or a fluorescence characteristic of absorbed or fluoresced light, as the case may be from the selected compound, which occurs during the active test 112. The absorption characteristic or fluorescence characteristic can be a relative or absolute value, for example. As explained above, the selected compound may either absorb or fluoresce light at the imaging wavelength. The method then determines the presence of the compound in the patient sample based on the absorption characteristic or the fluorescence characteristic of the absorbed or fluoresced light 114. The compound type or a variant or sub-variant of the compound can be determined from the absorption characteristic, the fluorescence characteristic, the shape of the band, the edge of the band, the position of the band, the speed of the band, or other defining band characteristics. The captured image(s) can be compared against empirical data or compared to image characteristics of a control band in the same active test or other analysis of the data. The data relating to determining the presence of the compound can optionally be output to a data store, an external device, a display, or the like.
Various system parameters can be adjusted in some examples that enhance or provide a different perspective on the captured image(s). For example, the intensity of the light emitted towards the electrophoresis strip is adjusted from a first intensity to a second intensity. The image characteristics of the image(s) captured of the electrophoresis strip when the light at the first intensity is emitted is different than the image characteristics of the image(s) captured of the electrophoresis strip when the light at the second intensity is emitted. That difference value can be calculated and used to determine presence of the compound. If the difference value exceeds a threshold, for example, then the compound is determined to be present.
Turning now to
In the example shown in
Hemoglobin at high concentrations can be detected by both white light and UV light. The acquired data under white light field demonstrates natural red color of hemoglobin. The images captured during the UV light emission is used for detection of low concentration hemoglobin variants and for quantification of individual Hb variants. For example, the shape of the band correlates to a quantification of the concentration of the compound in the patient sample. Data acquired under UV light has enhanced LOD (lower LOD) and higher signal to background noise ratio than white light filed data, which is also shown in
Turning now to
In
The subject matter of embodiments disclosed herein is described here with specificity to meet statutory requirements, but this description is not necessarily intended to limit the scope of the claims. The claimed subject matter may be embodied in other ways, may include different elements or steps, and may be used in conjunction with other existing or future technologies. This description should not be interpreted as implying any particular order or arrangement among or between various steps or elements except when the order of individual steps or arrangement of elements is explicitly described.
This application is a 371 filing of International Patent Application No. PCT/US22/71903, filed Apr. 25, 2022 entitled “IMPROVED DETECTION OF HEMOGLOBIN AND OTHER COMPOUNDS BY ELECTROPHORESIS,” which claims priority and benefit from the U.S. Provisional Patent Application 63/179,118, filed Apr. 23, 2022, and titled, “IMPROVED DETECTION OF HEMOGLOBIN AND OTHER COMPOUNDS BY ELECTROPHORESIS,” which is incorporated herein by reference in its entirety for all purposes.
Filing Document | Filing Date | Country | Kind |
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PCT/US22/71903 | 4/25/2022 | WO |
Number | Date | Country | |
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63179118 | Apr 2021 | US |