Methods to determine the presence and amount of sickle haemoglobin, devices and kits thereof

Abstract

The present disclosure provides methods for determining optical absorbance of a blood sample, methods for identifying the presence or absence of sickle haemoglobin in a blood sample, kits and devices thereof The present methods comprise measuring a first absorbance of the blood sample under a deoxygenated condition at 420-440 nm and measuring a second absorbance of the blood sample under the deoxygenated condition at 541-570 nm. A ratio of the second absorbance to the first absorbance and a difference between the first absorbance and the second absorbance are calculated and employed to identify the presence or absence of sickle haemoglobin and determine the amount of sickle haemoglobin. The methods, kits and devices of the present disclosure are simple, low cost, and provide a rapid way to identify the presence or absence of and amount of sickle haemoglobin in a blood sample in a point-of-care setting.

Description

[0001] “METHODS TO DETERMINE THE PRESENCE AND AMOUNT OF SICKLE HAEMOGLOBIN, DEVICES AND KITS THEREOF” TECHNICAL FIELD

[0002] The present disclosure relates to the methods of determining optical absorbance of a blood sample and methods for identifying the presence or absence of and amount of sickle haemoglobin in a blood sample. The present disclosure also relates to kits and devices for performing the methods.

[0003] BACKGROUND OF THE DISCLOSURE

[0004] Sickle cell disorder is a genetic disorder associated with haemoglobin protein. Sickle cell disorder is inherited in an autosomal recessive pattern and is considered among the most commonly inherited diseases worldwide. Certain point mutations in haemoglobin gene cause Sickle cell disorder.

[0005] Sickle cell disorder is considered a disorder of global importance with economic as well as clinical significance. People affected by this disease are scattered across Sub-Saharan Africa, the Middle East, India, Caribbean, South and Central America, some countries along the Mediterranean Sea, as well as in the United States and Europe. In the United States, 80,000-100,000 individuals are affected by the disorder. Worldwide, more than 300,000 children are estimated to be born annually with sickle cell disease. In India, it is prevalent in Chhattisgarh, Odisha, Maharashtra, Gujarat, Madhya Pradesh, Telangana, Andhra Pradesh and some parts of Tamil Nadu and Kerala.

[0006] Sickle cell disorder can be classified into two categories namely, sickle cell trait (SCT) and sickle cell disease (SCD) based on the zygosity. Since sickle cell disorder is an autosomal recessive disorder, the offspring has to inherit defective genes from both father and mother to be affected by the disease. If the abnormal gene is inherited from both parents, the condition is referred to as SCD (homozygous) and if only one abnormal gene is inherited (heterozygous), then the condition is referred to as SCT. People with SCT are carriers of the abnormal gene and have a 50% probability to pass it to their progeny.Sickle cell diagnostic tests are grouped into two categories namely, screening tests and confirmatory tests. The solubility test is a widely used screening test. It is a simple and cost-effective test that helps to screen a large population to differentiate healthy individuals from individuals with sickle cell disorder. Solubility test involves the addition of blood with freshly prepared reagents and visual inspection for turbidity of the solution. Samples from healthy individuals are transparent, whereas sickle-cell samples appear turbid. This simple test can be performed at point-of-need as it requires very less quality of blood and does not require any sophisticated equipment or power. However, samples tested positive in the screening tests need to be processed further by confirmatory tests to differentiate between SCT and SCD conditions.

[0007] Confirmatory tests help to distinguish between sickle cell trait and sickle cell disease and also provide relative concentration of haemoglobin. Since there is no easily available cure for SCD, disease management and treatment are aimed to improve the anemic condition to reduce complications. Confirmatory test results help in deciding the treatment regime and also monitor the effectiveness of treatment. There are numerous tests available under confirmatory tests, e.g., High-pressure liquid chromatography (HPLC), Isoelectric focusing (IEF), Hb Electrophoresis, and molecular diagnostic test (RFLP). Though these tests are highly specific and provide both qualitative and quantitative results, they are expensive, time-consuming, and require specialized laboratories with trained personnel to operate the device. These confirmatory tests cannot be performed at point-of-care. Hence, there is a need for the development of a portable point-of-care system that can identify the presence or absence of sickle haemoglobin that can be used in low-resource settings.

[0008] India is estimated to have the second largest burden of SCD with an estimate of 42,016 predicted sickle cell anemia births per year (estimated in 2010). Though sickle cell anemia has a widespread geographical distribution, it is highly prevalent among the tribal population. According to a census report in 2011, the tribal population in India is approximately 104 million. It is estimated that between 1 to 40% of the tribal population in India are affected by sickle cell anemia. Since these affected areas are remote, current testing methods (confirmatory test to identify SCT / SCD) involve transporting the blood samples to a centralized laboratory, which is burdensome and time-consuming. Hence there is a strong need for point-of-care, portable, low cost, rapid testing methods, kits, and devices capable of performing identification of sickle haemoglobin. The present disclosure attempts to address this need by providingmethods, kits, and devices capable of operating in low resource settings for measuring the absorbance of blood sample and identifying the presence or absence of sickle haemoglobin based on these absorbance measurements. Confirmatory tests may still be required to distinguish between sickle cell trait and sickle cell disease.

[0009] STATEMENT OF THE DISCLOSURE

[0010] In some embodiments, the present disclosure provides a method for determining optical absorbance of a blood sample, comprising: a) measuring a first absorbance of said blood sample under a deoxygenated condition at 420-440 nm; b) measuring a second absorbance of said blood sample under the deoxygenated condition at 541-570 nm; c) calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; and d) subtracting the second absorbance from the first absorbance to obtain an absorbance difference.

[0011] In some embodiments, the present disclosure provides a method for identifying the presence or absence of sickle haemoglobin in a blood sample: a) measuring a first absorbance of said blood sample under a deoxygenated condition at 420-440 nm; b) measuring a second absorbance of said blood sample under the deoxygenated condition at 541-570 nm; c) calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; d) subtracting the second absorbance from the first absorbance to obtain an absorbance difference in optical absorbance; and e) identifying the presence or absence of sickle haemoglobin based on the absorbance ratio and the absorbance difference.

[0012] In some embodiments, the present disclosure further provides a method of determining a percentage of sickle haemoglobin in a blood sample, comprising: a) measuring a first absorbance of said blood sample under a deoxygenated condition at 420-440 nm; b) measuring a second absorbance of said blood sample under the deoxygenated condition at 541-570 nm; c) calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; d) subtracting the second absorbance from the first absorbance to obtain an absorbance difference; and e) determining a percentage of sickle haemoglobin in the blood sample based on the absorbance ratio obtained and the absorbance difference.

[0013] In some embodiments, provided herein is a kit for performing the methods of the present disclosure, the kit comprising: a) a physiologically acceptable buffer or components thereof; b) a lysing agent; c) a reducing agent; and d) a document comprising instructions to performthe method and a table providing a standard curve for the absorbance ratio and the absorbance difference.

[0014] In some embodiments, provided herein is a device (100) for performing the methods of the present disclosure, comprising: a) a first light source (2a) configured to emit light having a wavelength of 420-440 nm; b) a second light source (2b) configured to emit light having a wavelength of 541-570 nm; c) a sample holder (5) facing each of the first light source (2a) and the second light source (2b), wherein each of the first light source (2a) and the second light source (2b) emits light onto a blood sample in the sample holder (5); and d) a detector (6) facing the sample holder (5), the detector (6) configured to detect light transmitted by the blood sample.

[0015] BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

[0016] Figure 1 A shows a plot of absorbance versus time after mixing the blood sample with buffer in the presence of the reducing agent.

[0017] Figure IB shows a plot of absorbance versus time after mixing the blood sample with buffer in the absence of the reducing agent.

[0018] Figure 2 shows a plot of the absorbance ratio and a borderline metric (absorbance difference / absorbance ratio).

[0019] Figure 3A illustrates an exemplary embodiment of the device for performing the methods, according to the present disclosure.

[0020] Figure 3B illustrates another exemplary embodiment of the device for performing the methods, according to the present disclosure.

[0021] Figure 4 is an image of an exemplary device according to the present disclosure.

[0022] DETAILED DESCRIPTION OF THE DISCLOSURE

[0023] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutationsmay be expressly set forth herein for sake of clarity. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “containing” or “has” or “having”, or “including but not limited to” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0024] Reference throughout this specification to “some embodiments”, “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in some embodiments”, “in one embodiment” or “in an embodiment” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0025] The term “subject” or “patient” as used herein refers to a human.

[0026] The term “about” as used herein encompasses variations of + / - 10% and more preferably + / -5%, as such variations are appropriate for practicing the present invention.

[0027] The terms “deoxygenated” and “hypoxic” are used interchangeably throughout this disclosure and refer to a condition where cells (e.g., RBCs) or cellular proteins (e.g., haemoglobin) experience inadequate levels of oxygen.

[0028] Methods

[0029] Previous methods and devices employed to detect the presence or absence of sickle haemoglobin have certain drawbacks. For example, the previous methods require a spectrophotometer to provide accurate absorbance measurements. However, spectrophotometers are bulky, require a power source and glass cuvettes and are not easy touse in a point-of-care setting. Further, glass cuvettes are prone to damage and if not washed properly between the tests, contamination of one sample with another sample can lead to erroneous results. The previous methods also fail to identify if there were any errors in the preparation of the buffer employed for deoxygenation and lysis of blood sample.

[0030] The present inventors surprisingly found that plotting the ratio of absorbance of a blood sample under deoxygenated conditions at 541-570 nm to the absorbance at 420-440 nm (absorbance ratio) versus the difference in absorbance under deoxygenated conditions at 420-440 nm and 541-570 nm (absorbance difference), the presence or absence of sickle haemoglobin can be identified with improved accuracy.

[0031] Accordingly, in some embodiments, the present disclosure provides a method for determining optical absorbance of a blood sample, comprising: a) measuring a first absorbance of said blood sample under a deoxygenated condition at 420-440 nm; b) measuring a second absorbance of said blood sample under the deoxygenated condition at 541-570 nm; c) calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; and d) subtracting the second absorbance from the first absorbance to obtain an absorbance difference.

[0032] In this embodiment, the absorbance of a blood sample collected from a subject is measured under deoxygenated conditions at two different wavelengths: 420-440 nm and 541-570 nm. The absorbance measured at 420-440 nm is referred to herein as the first absorbance. The absorbance measured at 541-570 nm is referred to herein as the second absorbance. Then, a ratio of the second absorbance to the first absorbance (Abs54i-570nm / Abs420-440nm) (referred to herein as the “absorbance ratio”) and the difference between the first absorbance and the second absorbance (Abs420-440nm - Abs54i-570nm) (referred to herein as the “absorbance difference”) is calculated.

[0033] In some embodiments, the first absorbance of a blood sample is measured under deoxygenated conditions at a wavelength selected from 420-440 nm such as at about 420-430 nm, 425-435 nm, 430-440 nm, 433-438 nm, 432-436 nm, 431 nm, 432 nm, 433 nm, 434 nm, 435 nm, 436 nm, 437 nm, 438 nm, 439 nm, or 440 nm. In some embodiments, the first absorbance is measured under deoxygenated conditions at a wavelength of 435 nm.In some embodiments, the second absorbance of a blood sample is measured under deoxygenated conditions at a wavelength selected from 541-570 nm such as at about 541-565 nm, 541-560 nm, 550-570 nm, 550-560 nm, 555-565 nm, 550-570 nm, 555-565 nm, 557-563 nm, 558-562 nm, 540 nm, 545 nm, 550 nm, 555 nm, 556 nm, 557 nm, 558 nm, 559 nm, 560 nm, 561 nm, 562 nm, 563 nm, 564 nm or 565 nm. In some embodiments, the second absorbance is measured under deoxygenated conditions at a wavelength of 560 nm.

[0034] The deoxygenated condition is a condition in which RBCs or haemoglobin experiences inadequate oxygen levels. In some embodiments, the deoxygenated condition comprises mixing the blood sample with a physiologically acceptable buffer comprising a lysing agent and a reducing agent to provide a sample-buffer mixture.

[0035] Accordingly, in some embodiments, the method for determining optical absorbance of a blood sample comprises: (a) mixing said blood sample with a physiologically acceptable buffer comprising a lysing agent and a reducing agent to obtain a sample-buffer mixture; (b) measuring a first absorbance of the sample-buffer mixture at 420-440 nm; (c) measuring a second absorbance of the sample buffer mixture at 541-570 nm; (d) calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; and (e) subtracting the second absorbance from the first absorbance to obtain an absorbance difference.

[0036] In some embodiments, the method for determining optical absorbance of a blood sample comprises measuring a third absorbance at 405-419 nm to determine the level of deoxygenation of the blood sample. If the absorbance at 405-419 nm is higher than the first absorbance measured at 420-440 nm, it indicates that deoxygenation of the blood sample has not been performed properly. Improper deoxygenation could be due to improper mixing of the reducing agent with the buffer or failure to add the reducing agent to the buffer. Thus, the absorbance measurement at 405-419 nm acts as a control to detect manual errors in performing the method such as errors in the preparation of the buffer, addition of the reducing agent to the buffer and the like. This step improves accuracy by substantially reducing errors in the determination of optical absorbance of a sample.

[0037] In some embodiments, the sample-buffer mixture is incubated at room temperature such as about 10-55°C for about 5 minutes to about 45 minutes prior to measuring the absorbance. In some embodiments, the sample-buffer mixture is incubated at a temperature of about 10-50°C, 10-45°C, 10-40°C, 10-35°C, 10-30°C, 15-45°C, 15-40°C, 15-35°C, 15-30°C, 15-25°C, 20-35°C, 20-30°C, 25-30°C and the like for about 5 minutes to about 40 minutes, 5 minutes to about 35 minutes, 5 minutes to about 30 minutes , 5 minutes to about 25 minutes, 5 minutes to about 20 minutes, 5 minutes to about 15 minutes, or 5 minutes to about 10 minutes. In some embodiments, the sample-buffer mixture is incubated at room temperature for about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or 45 minutes. After incubation, the absorbance of the sample-buffer mixture is measured at 420-440 nm and at 541-570 nm.

[0038] In some embodiments, the physiologically acceptable buffer employed in the methods of the present disclosure is selected from a phosphate buffer, a carbonate buffer, a citrate buffer, an acetate buffer, a HEPS buffer or a MOPS buffer. In some embodiments, a phosphate buffer having a concentration of about 1.5 M to 2.5 M is employed in the methods of the present disclosure. Methods to prepare these physiologically acceptable buffers are known in the art. Exemplary methods to prepare a phosphate buffer to perform the present methods are described in the Examples section.

[0039] In some embodiments, the physiologically acceptable buffer employed in the methods of the present disclosure is a phosphate buffer. In some embodiments, the phosphate buffer has a concentration of about 1.5 M to about 2.5 M, including values and ranges therebetween. For example, in some embodiments, the phosphate buffer has a concentration of about 1.5-2.3 M, 1.5-2.2 M, 1.5-2. I M, 1.5-2 M, 1.8-2.5 M, 1.8-2.3 M, 1.8-2.2 M, 2-2.5 M, 2-2.2 M, 1.5 M, 1.7 M, 1.75M, 1.8 M, 1.9 M, 2M, 2.15 M, 2.2 M, 2.4 M, or 2.5 M, including values and ranges thereof. In an exemplary embodiment, the phosphate buffer has a concentration of about 2.0-2.3 M.

[0040] In some embodiments, the lysing agent added to the physiologically acceptable buffer to lyse RBCs is selected from saponin, SDS (sodium dodecyl sulfate), SLS (sodium lauryl sulfate), lipase, Triton-X, and a polysorbate (e.g. TWEEN® 20). In some embodiments, the lysing agent is added at a concentration of about 0.1-5 % w / v of the buffer, including values and ranges therebetween. In some embodiments, the lysing agent is added to the buffer to provide a final concentration of about 0.1-4.5%, 0.1-4%, 0.1-3.5%, 0.1-3 %, 0.1-2.5%, 0.1-2%, 0.3-0.5%, 0.4-0.5%, 0.5-4.5%, 0.5-4%, 0.5-3.5%, 0.5-3%, 0.5-2.5%, 0.5-2%, 1-5%, 1-4.5%, 1-4%, 1-3.5%, 1-3%, 2-5%, 2-4.5%, 2-4%, 2.5-5%, 2.5-4.5%, 2.5-4%, 2.5-3.5%, 3-5%, 3-4.5%, or 3-4% w / v.In some embodiments, the reducing agent added to the physiologically acceptable buffer to induce deoxygenated / hypoxic conditions is selected from sodium metabisulfite, sodium dithionate, disodium disulphate, sulfate tetrasodium, sodium dithionate hydrate, sodium trithionate. In some embodiments, the reducing agent is added to the buffer to provide a concentration of about 4-14% w / v, including values and ranges therebetween, such as about 4-13%, 4-12%, 4-10%, 4-8%, 4-6%, 6-13%, 6-12%, 6-10%, 6-8%, 7-14%, 7-11%, 8-14%, 8-12%, 8-10%, 10-14%, 10-12%, 11-14%, or 12-14%, w / v.

[0041] In some embodiments, the ratio of blood to buffer ranges between 1:10 to 1:10000, such as about 1:10 to 1:8000, 1:10 to 1:5000, 1:10 to 1:2500, 1:10 to 1:1000, 1:10 to 1:750, 1:10 to 1:500, l:10 to 1:250, l:10 to 1:100.

[0042] In some embodiments, the method for determining optical absorbance of a blood sample comprises: (a) mixing said blood sample with a 1.5-2.5 M phosphate buffer comprising saponin and sodium metabisulfite to obtain a sample-buffer mixture; (b) measuring a first absorbance of the sample-buffer mixture at 420-440 nm; (c) measuring a second absorbance of the samplebuffer mixture at 541-570 nm; (d) calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; and (e) subtracting the second absorbance from the first absorbance to obtain an absorbance difference.

[0043] The present disclosure also provides a method of identifying the presence or absence of sickle haemoglobin in a blood sample, comprising: a) measuring a first absorbance of said blood sample under a deoxygenated condition at 420-440 nm; b) measuring a second absorbance of said blood sample under the deoxygenated condition at 541-570 nm; c) calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; d) subtracting the second absorbance from the first absorbance to obtain an absorbance difference; and e) identifying the presence or absence of sickle haemoglobin based on the absorbance ratio and the absorbance difference.

[0044] The measurement of the first and the second absorbance and the deoxygenated conditions are as described above. Further, the physiologically acceptable buffer, the lysing agent, the reducing agent, and their respective concentrations are as described above.In some embodiments, the method of identifying the presence or absence of sickle haemoglobin in a blood sample comprises: (a) mixing said blood sample with a physiologically acceptable buffer comprising a lysing agent and a reducing agent to obtain a sample-buffer mixture; (b) measuring a first absorbance of the sample-buffer mixture at 420-440 nm; (c) measuring a second absorbance of the sample buffer mixture at 541-570 nm; (d) calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; (e) subtracting the second absorbance from the first absorbance to obtain an absorbance difference; and (f) identifying the presence or absence of sickle haemoglobin in the blood sample based on the absorbance ratio and the absorbance difference.

[0045] In some embodiments, the method for identifying the presence or absence of sickle haemoglobin in a blood sample comprises measuring a third absorbance at 405-419 nm to determine the level of deoxygenation of the blood sample. A higher absorbance value at 405-419 nm than the first absorbance measured at 420-440 nm indicates that deoxygenation of the blood sample has not been performed properly due to improper mixing of the reducing agent with the buffer or due to failure to add the reducing agent to the buffer. Thus, the absorbance measurement at 405-419 nm acts as a control to detect manual errors in performing the method such as errors in the preparation of the buffer, addition of the reducing agent to the buffer and the like. This step improves accuracy by substantially reducing errors in the identification of the presence or absence of sickle haemoglobin in a sample.

[0046] In some embodiments, the method for identifying the presence or absence of sickle haemoglobin in a blood sample comprises measuring a fourth absorbance at 520-540 nm to determine the amount of total haemoglobin in the blood sample.

[0047] In some embodiments, the present disclosure also provides a method for determining the amount of sickle haemoglobin in a blood sample. In subjects with sickle haemoglobin, the amount of sickle haemoglobin varies from person to person. Further, to manage the effects of sickle haemoglobin, interventions such as blood transfusion an / or certain medications are given. Therefore, in subjects with sickle haemoglobin, measuring the amount of sickle haemoglobin is required frequently. HPLC, electrophoresis and ELISA are some of the standard techniques that are employed to quantify the amount of haemoglobin; however, these techniques require a proper laboratory set-up and skilled technicians, are time consuming, and are not practical in low resource settings such as rural areas. The ratio of optical absorbance attwo wavelengths and the difference in optical absorbance at two wavelengths according to the present disclosure can be employed to determine the amount of sickle haemoglobin.

[0048] First Absorbance: Abs at 420-440 nm

[0049] Second Absorbance: Abs at 541-570 nm

[0050] Absorbance Ratio: Second Absorbance / First Absorbance

[0051] Absorbance Difference: First Absorbance - Second Absorbance

[0052] Borderline Metric: Absorbance Difference / Absorbance Ratio

[0053] To determine the amount of sickle haemoglobin, the absorbance ratio is calculated. Based on the absorbance ratio thresholds, the measured sample is assigned to one of five zones. The absorbance ratio thresholds for classification into five zones can vary depending on the buffer concentration, pH, the reducing agent and the like. In an exemplary embodiment, the absorbance ratio thresholds are as follows:

[0054] Zone 1 : 0.00 < Absorbance Ratio < 0.23

[0055] Zone 2: 0.23 < Absorbance Ratio < 0.27

[0056] Zone 3 : 0.27 < Absorbance Ratio < 0.31

[0057] Zone 4: 0.31 < Absorbance Ratio < 0.39

[0058] Zone 5: 0.39 < Absorbance Ratio < 1.00

[0059] The samples falling in Zone 2 and Zone 4 are further classified into one of the two sub-zones based on the borderline metric (see Figure 2). The term “borderline metric” as used herein refers to the Absorbance Difference divided by the Absorbance Ratio (Absorbance Difference / Absorbance Ratio). The samples falling in Zone 2 and Zone 4 are further classified as follows:

[0060] For samples assigned Zone 2 based on the Absorbance Ratio threshold:

[0061] Zone 2a: Borderline Metric > 2.71

[0062] Zone 2b: Borderline Metric < 2.71

[0063] For samples assigned Zone 4 based on the Absorbance Ratio threshold:

[0064] Zone 4a: Borderline Metric > 1.66

[0065] Zone 4b: Borderline Metric < 1.66

[0066] Once the sample is classified into one of the zones, the amount of sickle haemoglobin (%HbS) can be determined as follows:

[0067] Zone 1 : % HbS = 0% (all samples assigned Zone 1 have 0% HbS)

[0068] Zone 2a: % HbS = 0% (all samples assigned Zone 2a have 0% HbS)Zone 2b: % HbS = [(-76.0005 * (Absorbance Difference)2) + (109.15 * (Absorbance Difference)) - 14.846]

[0069] Zone 3: % HbS = [(5.2138 * (Borderline Metric)2) - (17.379 * (Borderline Metric)) + 35.268]

[0070] Zone 4a: % HbS = [(5.3232 * (Borderline Metric)2) - (25.366 * (Borderline Metric)) + 55.484]

[0071] Zone 4b: % HbS = [(-50.485 * (Borderline Metric)2) + (144.65 * (Borderline Metric)) - 32.465]

[0072] Zone 5: % HbS = [(4296.2 * (Absorbance Ratio)2) - (3561.7 * (Absorbance Ratio)) + 807.46]

[0073] The methods of the present disclosure are in vitro methods.

[0074] In some embodiments, the present disclosure provides a method of identifying the presence or absence of sickle cell trait or sickle cell disease in a subject based on the ratio of the second absorbance to the first absorbance and the difference in optical absorbance between the first absorbance and the second absorbance.

[0075] In some embodiments, a method of identifying the presence or absence of sickle cell trait or sickle cell disease in a subject comprises: a) measuring a first absorbance of a blood sample of the subject under a deoxygenated condition at 420-440 nm; b) measuring a second absorbance of said blood sample under the deoxygenated condition at 541-570 nm; c) calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; d) subtracting the second absorbance from the first absorbance to obtain an absorbance difference; and e) identifying the presence or absence of sickle cell trait or sickle cell disease based on the absorbance ratio and the absorbance difference.

[0076] The physiologically acceptable buffer, the lysing agent, the reducing agent, and their respective concentrations are as described above.

[0077] The methods of the present disclosure provide a simple yet very accurate way of measuring the absorbance of a blood sample under deoxygenated conditions which, in turn, identifies the presence or absence of sickle haemoglobin in a blood sample. The present methods employ simple reagents and are thus inexpensive. Further, the methods are easy to perform in a point-of-care setting and can be carried out by employing a simple device. For example, the methods described herein can be carried out by employing a simple point-of-care device based on the present disclosure without the need for bulky, expensive equipment required for current tests. The methods of the present disclosure also obviate the need to transport blood to centralized laboratories. The present methods also work with low quantities of blood (e.g., finger prick) and there is no need to have trained personnel to perform the methods and / or operate the device. The data provided by the present methods may still need to be confirmed by confirmatory tests such as HPLC, electrophoresis, ELISA, and the like.

[0078] Kits

[0079] The present disclosure also provides kits for performing the methods of the present disclosure. In some embodiments, the kit comprises a physiologically acceptable buffer or components thereof, a lysing agent, a reducing agent, and a document comprising instructions to carry out the method and a table providing a standard curve for the ratio of the second absorbance to the first absorbance and the difference in absorbance between the first and the second absorbance. The standard curve can be employed to identify the presence or absence of sickle haemoglobin.

[0080] The physiologically acceptable buffer or components thereof, the lysing agent, the reducing agent, and their respective concentrations are as described above. For example, in some embodiments, the kit comprises a phosphate buffer having a molarity of about 2-3 M or 2.2 -2.4 M. In some embodiments, the kit comprises components of the phosphate buffer, e.g., potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) or sodium dihydrogen phosphate (NaEhPC ) and disodium hydrogen phosphate (ISfeHPCU) in pre-determined amounts. The buffer components can be mixed according to the instructions provided in the document present in the kit to prepare a buffer solution. To prepare the buffer solution, the kit may include sterile distilled water.

[0081] In some embodiments, the lysing agent and the reducing agent are added to the buffer immediately prior to mixing (e.g., about 5 minutes up to 4 hours prior) with a blood sample. In some embodiments, the kit may comprise a preservative such as chloroacetamide or sodium azide.In some embodiments, a buffer in the kit is provided as a ready-to-use (RTU) solution of desired concentration. The RTU solution of the buffer is stable at room temperature as well as 4°C.

[0082] In some embodiments, a buffer in the kit is provided in the lyophilized form. The lyophilized buffer is reconstituted with a pre-determined amount of sterile distilled water prior to performing the method.

[0083] In some embodiments, a concentrated stock solution of a buffer is provided. In some embodiments, a 2X, 4X, 5X, or 10X concentrated stock solution of a buffer is provided in the kit. To dilute this stock solution to a working solution, the kit may include sterile distilled water.

[0084] In some other embodiments, the kit comprises buffer components (e.g., monobasic and dibasic phosphate salts) in solid form. Preferably, pre-determined amounts of these buffer components will be provided and instructions as to how to prepare a buffer solution using these predetermined amounts will be provided. To prepare a buffer solution from solid components, the kit may include sterile distilled water.

[0085] The lysing agent in the kit can be provided in a solid form or a solution form.

[0086] In some embodiments, the lysing agent in the kit is provided as a ready-to-use (RTU) solution of desired concentration. The RTU solution of the lysing agent is preferably stored at 4°C.

[0087] In some embodiments, a concentrated stock solution of the lysing agent is provided in the kit. In some embodiments, a 10X-50X concentrated stock solution of the lysing agent is provided in the kit. For example, in some embodiments, a 10X, 12.5X, 15X, 20X, 25X, 30X, 40X, or 50X concentrated stock solution of the lysing agent is provided in the kit. To dilute this stock solution to a working solution, the kit may include sterile distilled water.

[0088] In some other embodiments, the kit comprises the lysing agent in a solid form. Preferably, a pre-determined amount of the lysing agent will be provided and instructions as to how to prepare a solution of the lysing agent using the pre-determined amount will be provided. To prepare a solution of the lysing agent, the kit may include sterile distilled water.Preferably, the reducing agent such as sodium metabisulfite is supplied in a solid form as a solution of the reducing agent may not be very stable. Therefore, in some embodiments, the kit comprises the reducing agent in solid form.

[0089] In an exemplary embodiment, a kit comprises a vial containing a solution of phosphate buffer, a vial containing saponin as the lysing agent, a vial containing sodium metabisulfite as the reducing agent, and a document comprising instructions to carry out the method and a table providing a standard curve for the ratio of the second absorbance to the first absorbance and the difference in absorbance between the first and the second absorbance. In this embodiment, saponin and sodium metabisulfite are added to the buffer solution immediately prior (e.g., about 5 minutes up to 4 hours prior) to mixing with a blood sample.

[0090] In another exemplary embodiment, a kit comprises a vial containing a solution of 2.1-2.3 M phosphate buffer, a vial containing saponin, a vial containing sodium metabisulfite, and a document comprising instructions to carry out the method and a table providing a standard curve based on the ratio of the second absorbance to the first absorbance and the difference in absorbance between the first and the second absorbance. In this embodiment, saponin and sodium metabisulfite are added to the buffer solution immediately prior (e.g., about 5 minutes up to 4 hours prior) to mixing with a blood sample.

[0091] In yet another exemplary embodiment, a kit comprises a monobasic salt of sodium phosphate or potassium phosphate, a dibasic salt of sodium phosphate or potassium phosphate, saponin, sodium metabisulfite, and a document comprising instructions to prepare a buffer solution using the monobasic and dibasic phosphate salts, instructions to carry out the method and a standard curve based on the ratio of the second absorbance to the first absorbance and the difference in absorbance between the first and the second absorbance. In this embodiment, the buffer solution can be prepared by a user and stored at room temperature or at 4°C. Saponin and sodium metabisulfite are added to the buffer solution immediately prior (e.g., about 5 minutes up to 4 hours prior) to mixing with a blood sample.

[0092] In some embodiments, the kit also comprises one or more additional components selected from blood collection containers comprising an anti -coagulant, lancets for finger pricking, disposable cuvettes, and the like.Device

[0093] The present disclosure provides a device for performing the method for determining optical absorbance of a blood sample and the method of identifying the presence or absence of sickle haemoglobin in a blood sample.

[0094] In some embodiments, the device comprises:

[0095] a) a first light source configured to emit light having a wavelength of 420-440 nm; b) a second light source configured to emit light having a wavelength of 541-570 nm; c) a sample holder facing each of the first light source and the second light source, wherein each of the first light source and the second light source emits light onto a blood sample in the sample holder; and

[0096] d) a detector facing the sample holder, the detector configured to detect light transmitted by the blood sample.

[0097] Figure 3 A illustrates an exemplary device (100) according to one embodiment for determining optical absorbance and methods, according to the present disclosure.

[0098] The device (100) is configured to perform the methods of the present disclosure. The construction and configuration of the device (100) is now described with reference to Figure 3. The device (100) includes a housing (1). The housing (1) includes a plurality of light sources (2a, 2b, 2c and 2d; collectively referred to as ‘2’ and depicted as ‘2’ in Figure 3) that are configured to emit light of required wavelength. The plurality of light sources (2a, 2b, 2c, 2d) is comprised of a first light source (2a), a second light source (2b), a third light source (2c), and a fourth light source (2d). As illustrated in Figure 3, each of the plurality of light sources (2a, 2b, 2c, 2d) may be placed adjacent to each other in a casing (4). However, in another embodiment, the each of the plurality of light sources (2a, 2b, 2c, 2d) may be placed in separate casings that are positioned adjacent to each other. In yet another embodiment, each of the plurality of light sources (2a, 2b, 2c, 2d) may be mounted to an internal wall of the housing (1). Further, the first light source (2a) is configured to emit light having a wavelength of 420-440 nm. The second light source (2b) is configured to emit light having a wavelength of 541-570 nm. The third light source is configured to emit light having a wavelength of 405-419 nm. The fourth light source is configured to emit light having a wavelength of 520-540 nm. In anembodiment, each of the plurality of light sources (2a, 2b, 2c, 2d) may be a light emitting diode (LED) configured to emit required wavelength of light.

[0099] The device (100) further includes a sample holder (5) facing each of the first light source (2a) and the second light source (2b). The housing (1) is defined with an opening (7) configured to provide access to the sample holder (5) within the housing (1). The sample holder (5) includes a seat (3) configured to facilitate placing of a cuvette containing the blood sample. The cuvette may preferably be a Imm-lOmm cuvette. The plurality of light sources (2a, 2b, 2c, 2d) are positioned and configured such that the light emitted by the pair of light sources (2a, 2b) is emitted onto the blood sample contained in the sample holder (5). The device (100) further includes a detector (6) facing the sample holder (5). The detector (6) may be a photodetector configured to detect amount of light transmitted by the blood sample in the sample holder (5). Further, as illustrated in the Figure 3 A, while the pair of light sources (2a, 2b) may be mounted onto a first internal wall of the housing, the detector (6) may be mounted onto a second internal wall of the housing (1), where the first internal wall and the second internal wall face other, with the sample holder (5) in between.

[0100] In some embodiments (Figure 3B), the device further includes a first collimation lens (8a) and a first aperture (9a) facing the plurality of light sources (2a, 2b, 2c, 2d) and positioned between the plurality of light sources (2a, 2b, 2c, 2d) and the sample holder (5) such that the first collimation lens (8a) is positioned after the plurality of light sources (2a, 2b, 2c, 2d) and the first aperture (9a) is positioned after the first collimation lens (8a). The plurality of light sources (2a, 2b, 2c, 2d) are positioned and configured such that the light emitted by the light sources is emitted onto the blood sample contained in the sample holder (5) through the first collimation lens (8a) and a first aperture (9a). The first collimation lens (8a) is configured to make parallel the light emitted by the plurality of light sources (2a, 2b, 2c, 2d) so that the light beam leaves the first collimator lens (8a) as a parallel beam. The first aperture (9a) is configured to allow the parallel beam of light from the first collimation lens (8a) to pass through and fall onto the blood sample contained in the sample holder (5). The device further includes a second collimation lens (8b) and a second aperture (9b) facing the sample holder (5) and positioned between the sample holder (5) and the detector (6) such that the second collimation lens (8b) is positioned after the sample holder (5) and the second aperture (9b) is positioned after the second collimation lens (8b). The second collimation lens (8b) is configured to make parallel the light transmitted from the blood sample contained in the sample holder (5) so that thetransmitted light leaves the second collimator lens (8b) as a parallel beam. The second aperture (9b) is configured to allow the parallel beam of transmitted light from the second collimation lens (8b) to pass through and fall onto the detector (6).

[0101] Figure 4 is an image of the device (100) being communicatively coupled to a controller. The controller is communicatively coupled to the detector (6), and is configured to receive, store and analyse data received from the detector (6). As can be seen in the Figure 4, the controller is a smart phone (200) communicatively coupled to the detector (6) of the device (100). The smart phone (200) may be configured to include a processor programmed to implement one or more steps of the methods of the present disclosure. In another embodiment, the device (100) may be connected to a cloud based controller via the smart phone (200). As illustrated in the Figure 4, the device (100) is compact in size and hence, is is a portable device. The device (100) may include a power source [not shown in the Figures] configured to supply power required for operation of the device (100). The power source may be a battery, power bank, a smart phone, and the like. In another embodiment, the device (100) may include a power cord that is connectable to a power source, for deriving power supply required during operation of the device (100).

[0102] In some embodiments, the detector (6) is a photodetector or a photosensor.

[0103] In some embodiments, the sample holder (5) is a cuvette, and preferably a 1mm to 10 mm cuvette.

[0104] In some embodiments, the device (100) is a portable device.

[0105] In some embodiments, the controller is configured to:

[0106] a) determine the first absorbance;

[0107] b) determine the second absorbance;

[0108] c) divide the second absorbance with the first absorbance to obtain an absorbance ratio; and

[0109] d) subtract the second absorbance from the first absorbance to obtain an absorbance difference.In some embodiments, the controller is also configured to determine the third absorbance to determine level of deoxygenation of a blood sample. In some embodiments, the controller is configured to determine the fourth absorbance to determine the amount of total haemoglobin in said blood sample.

[0110] In the embodiments, where the controller is the smart phone (200) communicatively coupled to the detector (6), the amount of haemoglobin in blood samples are displayed on a mobile application of the smart phone (200) instantaneously. The smart phone (200) may be communicatively coupled to the detector (6) by a wired connection, such as by an USB cable. In another embodiment, the smart phone (200) may be communicatively coupled to the detector (6) by a wireless connection like Bluetooth, infrared and the like.

[0111] The device (100) along with the smart phone (200) as a whole system is programmed and run using a mobile application of the smart phone (200). The mobile application employs algorithm for calculating amount of sickle haemoglobin in a blood sample and display such calculation on a mobile application of the smart phone (200).

[0112] It is to be understood that the foregoing descriptive matter is illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

[0113] Descriptions of well-known / conventional methods / steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.EXAMPLES

[0114] The present disclosure is further described with reference to the following example, which is only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

[0115] Example 1: Determination of optical absorbance of blood samples according to the method of the present disclosure

[0116] 2.26 M phosphate buffer (pH7.4) was prepared using K2HPO4 and KH2PO4. Saponin and sodium metabisulphite were added to the buffer to provide a final concentration of 0.42% w / v of saponin and 9.4% w / v of sodium metabisulphite.

[0117] 6 pl of test blood sample was added to 4 mL of the above-prepared buffer, mixed well and incubated at room temperature for 15 minutes. The sample-buffer mixture was loaded into a cuvette of 10mm pathlength and the optical absorbance was measured at 435 nm (first absorbance) and at 560 nm (second absorbance) using the portable device of the present disclosure. The ratio of the second absorbance to the first absorbance and the difference between the first absorbance and the second was calculated by subtracting the second absorbance from the first absorbance.

[0118] Example 2: Identification of the presence or absence of sickle haemoglobin in blood samples according to the method of the present disclosure

[0119] A standard curve for determining the presence or absence of sickle haemoglobin was generated. For this, 6 pl of blood samples containing known amount of sickle haemoglobin were added to 4 mL of buffer, mixed well, and incubated at room temperature for 15 minutes. The samplebuffer mixture was loaded into a cuvette of 10mm pathlength and the optical absorbance was measured at 435 nm (first absorbance) and at 560 nm (second absorbance) using the portable device of the present disclosure. The ratio of the second absorbance to the first absorbance and the difference between the first absorbance and the second were calculated. A standard curve based on the absorbance ratio and the absorbance difference was prepared. After generation of the standard curve, a test blood sample was processed in the similar manner and the presence or absence of sickle haemoglobin in the test blood sample was determined by employing the standard curve generated as described above.Example 3: Measurement of absorbance at 405-419 nm to check proper deoxygenation of the blood sample

[0120] In this experiment, the optical absorbance of blood samples was measured at 415 nm (third absorbance) and 435 nm (first absorbance) in the presence or absence of the reducing agent. Figure 1A shows the absorbance of the blood sample over a period of 16 minutes in the presence of the reducing agent. At all time points, the absorbance at 415 nm is lower than the absorbance at 435 nm.

[0121] Figure IB shows the absorbance of the blood sample over a period of 16 minutes in the absence of the reducing agent. At all time points, the absorbance at 415 nm is higher than the absorbance at 435 nm indicating no or improper deoxygenation of the blood sample.

[0122] Example 4: Identification of the presence or absence of sickle haemoglobin by employing only the ratio of two absorbance versus the ratio of two absorbance and the difference between the two absorbance

[0123] In this experiment, the optical absorbance of 285 samples containing known amount of normal haemoglobin or sickle haemoglobin was measured at 435 nm (first absorbance) and at 560 nm (second absorbance) and the presence or absence of sickle haemoglobin was determined based on the ratio of the second absorbance to the first absorbance. Out of 285, 33 samples were on the borderline of the presence or absence as shown in Table 1 below.

[0124] Table 1

[0125]

[0126] Total Samples i 285i

[0127]

[0128] i% Misclassification i0.350877i

[0129]

[0130] A difference in the first and the second absorbance was calculated by subtracting the second absorbance from the first absorbance. The presence or absence of sickle haemoglobin in the same 285 samples was determined based on the ratio of the second absorbance to the first absorbance and the difference in the second absorbance and the first absorbance. When the absorbance ratio and the absorbance difference both were employed, out of 285, only 9 sampleswere on the borderline of the presence or absence as shown in Table 2 below (compared to 33 borderline samples when only the absorbance ratio was employed).

[0131] Table 2

[0132]

[0133] That is, employing only the ratio of the second absorbance to the first absorbance for identification of sickle haemoglobin resulted in almost 11.58% of all samples falling in the ‘Borderline’ category, whereas employing both the ratio of the second absorbance to the first absorbance and the difference between the first and the second absorbance reduced the classification of samples into ‘Borderline’ to 3.16% of samples.

[0134] Example 5: Preparation of a kit of the present disclosure

[0135] A kit is provided in accordance with the requirements of the present disclosure. The kit provided comprises the following components:

[0136] a) A phosphate buffer having a concentration of 1.5-2.5 M or components thereof; b) Saponin;

[0137] c) Sodium metabisulfite; and

[0138] d) an instruction manual comprising instructions to perform the method and a table providing a standard curve for the ratio of the absorbance at at 541-570 nm (second absorbance) to the absorbance at 420-440 nm (first absorbance) and the difference in absorbance between the first and the second absorbance.

[0139] The kit may include blood collection devices such as tubes containing an anti -coagulant (e.g., K2EDTA coated collection tubes) or microfluidic devices, lancets, and / or cuvettes for measuring absorbance.

[0140] The kit is used in the following manner to perform the method:

[0141] a) A phosphate buffer is prepared by dissolving pre-determined amounts of K2HPO4 and KH2PO4 in desired amount of sterile water to obtain a phosphatebuffer of 2.26 M. Saponin is added to the phosphate buffer at a final concentration of 0.42% w / v and sodium metabisulfite is added to the phosphate buffer at 9.4% w / v final concentration immediately prior (e.g., about 5 minutes up to 4 hours prior) to mixing with a blood sample. The phosphate buffer comprising saponin and sodium metabisulfite is stored at 4°C for up to 4 hours. b) A subject’s blood sample is collected by finger pricking.

[0142] c) 6 pl of subject’s blood sample is mixed with 4 ml phosphate buffer comprising saponin and sodium metabisulfite.

[0143] d) The sample-buffer mixture is incubated at room temperature for about 5 to 15 minutes.

[0144] e) After incubation, the sample-buffer mixture is loaded into a 10 mm cuvette and absorbance is measured by employing the device of the present disclosure. A first absorbance is measured at a wavelength selected from 420-440 nm and a second absorbance is measured at a wavelength selected from 541-570 nm. f) A ratio of the second absorbance to the first absorbance and a difference between the first absorbance and the second absorbance is calculated. The presence or absence of sickle haemoglobin in the blood sample is determined based on the absorbance ratio and the absorbance difference as described in the instruction manual.

[0145] Table of referral numerals:

[0146]

[0147]

Claims

We Claim:

1. A method for determining optical absorbance of a blood sample, comprising:a. measuring a first absorbance of said blood sample under a deoxygenated condition at 420-440 nm;b. measuring a second absorbance of said blood sample under the deoxygenated condition at 541-570 nm;c. calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio; andd. subtracting the second absorbance from the first absorbance to obtain an absorbance difference.

2. The method as claimed in claim 1, wherein the first absorbance is measured at 435 nm.

3. The method as claimed in claim 1 or 2, wherein the second absorbance is measured at 560 nm.

4. The method as claimed in any one of claims 1-3, comprising measuring a third absorbance at 405-419 nm to determine level of deoxygenation of said blood sample.

5. The method as claimed in any one of claims 1-4, wherein the deoxygenated condition comprises mixing said blood sample with a physiologically acceptable buffer comprising a lysing agent and a reducing agent.

6. The method as claimed in claim 5, wherein the physiologically acceptable buffer is selected from a phosphate buffer, a carbonate buffer, a citrate buffer, an acetate buffer, a HEPS buffer, and a MOPS buffer.

7. The method as claimed in claim 5 or 6, wherein the physiologically acceptable buffer is a phosphate buffer having a concentration of about 1.5 M - 2.5 M.

8. The method as claimed in any one of claims 5-7, wherein the lysing agent is selected from saponin, sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), lipase, Triton-X, and a polysorbate.

9. The method as claimed in any one of claims 5-8, wherein the lysing agent is present at a concentration of about 0.1- 5% w / v of the physiologically acceptable buffer.

10. The method as claimed in any one of claims 5-9, wherein the reducing agent is selected from sodium metabisulfite, sodium dithionate, disodium disulphate, sulfate tetrasodium, sodium dithionate hydrate, and sodium trithionate.

11. The method as claimed in any one of claims 5-10, wherein the reducing agent is present at a concentration of about 4-14 % w / v of the physiologically acceptable buffer.

12. A kit for performing the method as claimed in any one of claims 1-11, comprising: a. a physiologically acceptable buffer or components thereof;b. a lysing agent;c. a reducing agent; andd. a document comprising instructions to perform the method and a table providing a standard curve based on the absorbance ratio and the absorbance difference.

13. The kit as claimed in claim 12, wherein the physiologically acceptable buffer is selected from a phosphate buffer, a carbonate buffer, a citrate buffer, an acetate buffer, a HEPS buffer, and a MOPS buffer or components thereof.

14. The kit as claimed in claim 12 or 13, wherein the lysing agent is selected from saponin, sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), lipase, Triton-X and a polysorbate.

15. The kit as claimed in any one of claims 12-14, wherein the reducing agent is selected from sodium metabisulfite, sodium dithionate, disodium disulphate, sulfate tetrasodium, sodium dithionate hydrate, and sodium trithionate.

16. The kit as claimed in any one of claims 12-15, wherein the physiologically acceptable buffer is a phosphate buffer, the lysing agent is saponin, and the reducing agent is sodium metabisulfite.

17. A device (100) for performing the method as claimed in any one of claims 1-11, comprising:a. a first light source (2a) configured to emit light having a wavelength of 420-440 nm;b. a second light source (2b) configured to emit light having a wavelength of 541- 570 nm;c. a sample holder (5) facing each of the first light source (2a) and the second light source (2b), wherein each of the first light source (2a) and the second light source (2b) emits light onto a blood sample in the sample holder (5); and d. a detector (6) facing the sample holder (5), the detector (6) configured to detect light transmitted by the blood sample.

18. The device (100) as claimed in claim 17, wherein the first light source (2a) and the second light source (2b) is a light emitting diode (LED).

19. The device (100) as claimed in claim 17 or 18, comprising a third light source (2c) configured to emit light having a wavelength of 405-419 nm.

20. The device (100) as claimed in any one of claims 17-19, comprising:a. a first collimation lens (8a) configured to make parallel light emitted by the first and second light source (2a, 2b);b. a first aperture (9a) configured to allow the parallel beam of light from the first collimation lens (8a) to fall onto the blood sample in the sample holder (5); c. a second collimation lens (8b) configured to make parallel light transmitted by the blood sample in the sample holder (5); andd. a second aperture (9b) configured to allow the parallel beam of light from the second collimation lens (8b) to fall onto the detector (6).

21. The device (100) as claimed in any one of claims 17-20, wherein the device (100) is a portable device.

22. The device (100) as claimed in any one of claims 17-21, comprising a power source configured to supply power required for operation of the device (100).

23. The device (100) as claimed in any one of claims 17-22, comprising a controller communicatively coupled to the detector (6), wherein the controller being configured to receive, store and analyse data received from the detector (6).

24. The device (100) as claimed in claim 23, wherein the controller is configured to: a. determine the first absorbance;b. determine the second absorbance;c. divide the second absorbance with the first absorbance to obtain an absorbance ratio of the second absorbance to the first absorbance; andd. subtract the second absorbance from the first absorbance to obtain an absorbance difference.

25. The device (100) as claimed in claim 23 or 24, wherein the controller is configured to determine the third absorbance to determine level of deoxygenation of said blood sample.

26. A method of identifying the presence or absence of sickle haemoglobin in a blood sample, comprising:a. measuring a first absorbance of said blood sample under a deoxygenated condition at 420-440 nm;b. measuring a second absorbance of said blood sample under the deoxygenated condition at 541-570 nm;c. calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio;d. subtracting the second absorbance from the first absorbance to obtain an absorbance difference; ande. identifying the presence or absence of sickle haemoglobin based on the absorbance ratio and the absorbance difference.

27. The method as claimed in claim 26, wherein the first absorbance is measured at 435 nm.

28. The method as claimed in claim 26 or 27, wherein the second absorbance is measured at 560 nm.

29. The method as claimed in any one of claims 26-28, comprising measuring a third absorbance at 405-419 nm to determine level of deoxygenation of said blood sample.

30. The method as claimed in any one of claims 26-29, comprising measuring a fourth absorbance at 520-540 nm to determine the amount of total haemoglobin.

31. The method as claimed in any one of claims 26-30, wherein the deoxygenated condition comprises mixing said blood sample with a physiologically acceptable buffer comprising a lysing agent and a reducing agent.

32. The method as claimed in claim 31, wherein the physiologically acceptable buffer is selected from a phosphate buffer, a carbonate buffer, a citrate buffer, an acetate buffer, a HEPS buffer, and a MOPS buffer.

33. The method as claimed in claim 31 or 32, wherein the physiologically acceptable buffer is a phosphate buffer having a concentration of about 1.5 M - 2.5 M.

34. The method as claimed in any one of claims 31-33, wherein the lysing agent is selected from saponin, sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), lipase, Triton- X, and a polysorbate.

35. The method as claimed in any one of claims 31-34, wherein the lysing agent is present at a concentration of about 0.1- 5% w / v of the physiologically acceptable buffer.

36. The method as claimed in any one of claims 31-35, wherein the reducing agent is selected from sodium metabisulfite, sodium dithionate, disodium disulphate, sulfate tetrasodium, sodium dithionate hydrate, and sodium trithionate.

37. The method as claimed in any one of claims 31-36, wherein the reducing agent is present at a concentration of about 4-14 % w / v of the physiologically acceptable buffer.

38. The method as claimed in any one of claims 26-37, comprising determining a percentage of sickle haemoglobin in the blood sample based on the absorbance ratio and the absorbance difference.

39. A method of determining a percentage of sickle haemoglobin in a blood sample, comprising:a. measuring a first absorbance of said blood sample under a deoxygenated condition at 420-440 nm;b. measuring a second absorbance of said blood sample under the deoxygenated condition at 541-570 nm;c. calculating a ratio of the second absorbance to the first absorbance to obtain an absorbance ratio;d. subtracting the second absorbance from the first absorbance to obtain an absorbance difference; ande. determining the percentage of sickle haemoglobin in the blood sample based on the absorbance ratio and the absorbance difference.

40. A kit for performing the method as claimed in any one of claims 26-39, comprising:a. a physiologically acceptable buffer or components thereof;b. a lysing agent;c. a reducing agent; andd. a document comprising instructions to perform the method and a table providing a standard curve based on the absorbance ratio and the absorbance difference.

41. The kit as claimed in claim 40, wherein the physiologically acceptable buffer is selected from a phosphate buffer, a carbonate buffer, a citrate buffer, an acetate buffer, a HEPS buffer, and a MOPS buffer or components thereof.

42. The kit as claimed in claim 40 or 41, wherein the lysing agent is selected from saponin, sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), lipase, Triton-X, and a polysorbate.

43. The kit as claimed in any one of claims 40-42, wherein the reducing agent is selected from sodium metabisulfite, sodium dithionate, disodium disulphate, sulfate tetrasodium, sodium dithionate hydrate, and sodium trithionate.

44. The kit as claimed in any one of claims 40-43, wherein the physiologically acceptable buffer is a phosphate buffer, the lysing agent is saponin, and the reducing agent is sodium metabisulfite.

45. A device (100) for performing the method as claimed in any one of claims 26-39, comprising:a. a first light source (2a) configured to emit light having a wavelength of 420-440 nm;b. a second light source (2b) configured to emit light having a wavelength of 541-570 nm;c. a sample holder (5) facing each of the first light source (2a) and the second light source (2b), wherein each of the first light source (2a) and the second light source (2b) emits light onto a blood sample in the sample holder (5); andd. a detector (6) facing the sample holder (5), the detector (6) configured to detect light transmitted by the blood sample.

46. The device (100) as claimed in claim 45, wherein the first light source (2a) and the second light source (2b) is a light emitting diode (LED).

47. The device (100) as claimed in claim 45 or 46, comprising a third light source (2c) configured to emit light having a wavelength of 405-419 nm.

48. The device (100) as claimed in any one of claims 45-47, comprising a fourth light source (2d) configured to emit light having a wavelength of 520-540 nm.

49. The device (100) as claimed in claim 47 or 48, wherein the third light source (2c) and the fourth light source (2d) is a light emitting diode (LED).

50. The device (100) as claimed in any one of claims 45-49, comprising:a. a first collimation lens (8a) configured to make parallel light emitted by the first and second light source (2a, 2b);b. a first aperture (9a) configured to allow the parallel beam of light from the first collimation lens (8a) to fall onto the blood sample in the sample holder (5); c. a second collimation lens (8b) configured to make parallel light transmitted by the blood sample in the sample holder (5); andd. a second aperture (9b) configured to allow the parallel beam of light from the second collimation lens (8b) to fall onto the detector (6).

51. The device (100) as claimed in any one of claims 45-50, wherein the device (100) is a portable device.

52. The device (100) as claimed in any one of claims 45-51, comprising a power source configured to supply power required for operation of the device (100).

53. The device (100) as claimed in any one of claims 45-52, comprising a controller communicatively coupled to the detector (6), wherein the controller being configured to receive, store and analyse data received from the detector (6).

54. The device (100) as claimed in claim 53, wherein the controller is configured to: a. determine the first absorbance;b. determine the second absorbance;c. divide the second absorbance with the first absorbance to obtain an absorbance ratio of the second absorbance to the first absorbance;d. subtract the second absorbance from the first absorbance to obtain an absorbance difference; ande. identify the presence or absence of sickle haemoglobin based on the absorbance ratio and the absorbance difference.

55. The device (100) as claimed in claim 53 or 54, wherein the controller is configured to determine the third absorbance to determine level of deoxygenation of said blood sample.

56. The device (100) as claimed in any one of claims 53-55, wherein the controller is configured to determine the fourth absorbance to determine the amount of total haemoglobin in said blood sample.

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