Description:
FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)

“A DEVICE, METHOD AND FORMULATION FOR THE DETECTION OF HAEMOGLOBIN, CREATININE, AND GLUCOSE”

By
HUWEL LIFESCIENCES PRIVATE LTD.
Hyderabad, Telangana 502319.

The following specification particularly describes the invention and the manner in which it is to be performed.
Field of the Invention
The invention generally relates to detection of biological samples. More particularly, the present invention relates to a reagent composition for detecting haemoglobin, creatinine, and glucose in a blood sample.

Background of the Invention
Haemoglobin (Hb) is the protein present in red blood cells which is responsible for the transportation of oxygen. The level of haemoglobin in blood is routinely measured in clinical laboratories. According to the World Health Organisation (WHO), the healthy range of Hb levels for men is 13.2–16.6 g/dL and for women is 11.5–15.0 g/dL. The Hb levels for pregnant women must lie in the range of 11.5-15.0 g/dL. The Hb range for children with age group 0–5 years is 11–16 g/dL and for 6–12 years is 11.5–15.5 g/dL. A lower haemoglobin level than the normal range is termed anaemia, and a high haemoglobin level is termed polycythaemia.
Around 25% of the world’s population is affected by Hb-related disorders, causing morbidity and mortality and leading to an economic burden worldwide. A number of clinical conditions cause changes in haemoglobin levels, such as severe dehydration, primary and secondary polycythaemia, and congestive heart failure (all of which lead to increased levels), together with various anaemias (decreased levels). Abnormalities of Haemoglobin (Hb) and creatinine in human leads to various health complications. Around 9.0% of the world’s population is affected by kidney related disorders. Acute kidney injury (AKI) can occur rarely in patients exposed to iodinated contrast and result in contrast-induced AKI (CI-AKI). A key risk factor is the presence of preexisting chronic kidney disease (CKD); therefore, it is important to assess patient risk and obtain kidney function measurement prior to administration. Diabetes mellitus is the most common endocrine disorder of carbohydrate metabolism. Worldwide, it is a leading cause of morbidity and mortality and a major health problem for most developed societies. The prevalence of diabetes continues to increase. Blood glucose monitoring has been established as a valuable tool in the management of diabetes. Hence, regular monitoring of haemoglobin concentrations is strongly recommended. However, early diagnosis and frequent monitoring in economically backward communities, remain challenging due to unaffordable and inaccessible healthcare facilities. In addition, haemoglobin measurement is essential in trauma cases, dialysis, and cancer patients .
In today's techniques for measuring the haemoglobin value in blood often lead to variations in the results of measurement since the analyses are performed on very small blood volumes (approx. 5-20 pl), and therefore the operator's sampling technique is important for the results of the measurement. There are a number of well-known techniques for detecting haemoglobin concentration in the blood sample. It is however important that such techniques should have a reasonable degree of accuracy, since haemoglobin content even in healthy individuals can vary with age, sex, and race. Creatinine is a product of muscle metabolism in the human body and is mainly excreted by glomerular filtration. Blood creatinine comes from both exogenous and endogenous sources. Creatinine, is a breakdown-product of creatinine phosphate in a muscle and is considered as the most important marker for the diagnosis of any abnormality in renal function, chronic kidney disease, thyroid and muscle dysfunctions, since the presence of these abnormalities and dysfunctions, in human subjects, causes abnormal variations in creatinine levels, in the corresponding blood and urine samples.
In addition, there is a significant relationship between haemoglobin levels and blood creatinine levels where patients with high creatinine levels tend to be at risk of anemia. Hence, the need for point-of-care (POC) devices for early diagnosis, and monitoring of Haemoglobin and Creatinine, is urgently required that is affordable and accessible in low- and middle-income countries.
The appropriate concentration of glucose was maintained in the blood because it is the main energy source for cells as well as the brain. However, if too much glucose formed from the food in the blood without interacting smoothly with insulin, generated by the pancreas, the excess glucose can cause diabetes, a serious health problem. Hemoglobin (hereinafter also referred to as Hb), particularly hemoglobin A1c (hereinafter also referred to as HbA1c), which is a kind of glycated hemoglobin, reflects an average blood glucose level in the past 1 to 2 months. It is widely used as a screening test and a test item for grasping the blood glucose control status of diabetic patients.
Regarding the detection of blood glucose, the home-use or portable blood glucose meters used recently exist greater error value which is usually criticized by most users, and the most influential factors includes the hematocrit (HCT) of blood samples. The effects caused by the difference between hematocrit includes the difference between the stiffness of blood and the differences between the serum volume; the former factor causes further difference of electron transfer efficiency, and the latter factor causes the difference of testing standard. Hence, many kinds of methods used for testing hematocrit of the blood sample are developed in recent years.
The World Health Organisation (WHO) haemoglobin colour scale was developed in 1995 which is a simple and inexpensive clinical device for diagnosing anaemia in remote areas. The method uses chromatography paper and a reference colour chart. It gives a range of 4–14 g/dL, with a resolution of 2 g/dL, and the intermediate colours are adjusted to in between values. According to many community studies, the method has been found to be useful and convenient for anaemia screening in field conditions, according to many community studies.
There are various prior arts available which talks about the POC devices, but most of the reported POC devices are suffering from their inherent limitations
including complex detection protocol, expensive disposable, or non-disposable strips, provide qualitative results etc. As a result, most of these lab prototypes never reached to the market for commercialization. However, early diagnosis and frequent monitoring in economically backward communities remain challenging due to unaffordable and inaccessible healthcare facilities. Moreover, the results retrieved by the prior arts is not accurate and also not be recommended for all types of patients as any wrong haemoglobin detection will directly affect the treatments for patients, even leading to life-threatening complications. Therefore, it is desirable to have a haemoglobin measuring system that can provide a more accurate, disposable, and user-friendly haemoglobin reading and overcome the deficiencies of the prior art methods. Therefore, there is an urgent need for the point-of-care (POC) devices for early diagnosis, and monitoring of anaemia, which is affordable and accessible in low- and middle-income countries where anaemia is most prevalent. Hence, our innovative device will solve the problem by early diagnosis and monitoring of Haemoglobin and Creatinine that is affordable and accessible in low- and middle-income countries. Using our device creatinine tests before outpatient contrast-enhanced CT scans in the radiology department could minimize the risk of kidney injury. It could also reduce the number of cancelled scans, which is important for patients.

Objective of the Invention
The main objective of the present invention is to provide a unique and simple user-friendly device for measuring haemoglobin. Another objective of the present invention is to provide an efficient reagent for measuring the haemoglobin in the range of 4g-16g/dL, creatinine in the range of 0.5-10 mg/dL and glucose in the range of 30-300mg/dL. Yet another objective is to provide a facile and efficient process for the preparation of stable reagent. Yet another objective of the present invention is to provide an efficient reagent and method for detecting the glucose will give semiquantitative analysis whether the sample is in the normal range or any deviation indicating diabetes status

Summary of the Invention
As a result of efforts made to solve the above problems, the inventors of the present invention have found that when the method involves the determination of haemoglobin, creatinine and glucose using chemical reagents that give specific colours for various concentrations of the blood samples where only 2-5 µL blood sample is required for the accurate determination of haemoglobin, creatinine and glucose. The scale and the colour attributes are required to determine an accurate haemoglobin, creatinine and glucose value. The invention results in a determination of haemoglobin from 4 g/dL to 16 g/dL, creatinine 0.5mg/dL to 20mg/dL, and glucose 30mg/dL to 300mg/dLand not affected by any interferences present in biological sample, whereby the present invention has been completed.
In one aspect of the present invention is a composition/reagent for detection of haemoglobin in a blood sample, comprising: 0.1-1.0% of dye, a surfactant, 1-5% of oxidant, organic or inorganic acids and solvents.
In another aspect of the present invention is a method for preparing a reagent which facilitates in detecting haemoglobin of a blood sample. The method comprising: preparing a solution of dye (0.1-1%) in organic solvent, preparing a solution of surfactant in water and mixing with solution of dye, adding oxidants, wherein detecting the oxidation so as to detect the presence of haemoglobin in the blood, and adjusting the pH to 5-6 using organic or inorganic acids, where all the above steps are executed simultaneously and in results in generation of one or more colour of the solution corresponding to a haemoglobin level, where each colour corresponds to the particular value of haemoglobin and will be displayed on a smart device application, and where the one or more colour attributes include intensity, brightness, hue, among others, or a combination thereof
In yet another aspect of the present invention is a kit for detecting haemoglobin of a blood sample. The kit mainly includes a vial holder capable of accompanying at least one reagent vial, wherein the reagent has a 0.1-1.0% of dye, a surfactant, 1-5% of oxidant, and organic or inorganic acids and solvents. And a processing unit and a memory, the processing unit operative coupled with the vial holder for reading the reaction of the reagent with a blood sample and also capable of receiving and sending instruction or information from a smart device via an USB connection port. Upon reaction of reagent with the blood sample leads to generation of one or more colour of the solution corresponding to a haemoglobin level, wherein each colour corresponds to the particular value of haemoglobin and will be displayed on a smart device application.

Brief description of the drawings
Figure 1 shows a block diagram of various components of the haemoglobin measure kit in accordance with one embodiment of the present invention.
Figure 2 shows a flow chart of the method for determining haemoglobin, in accordance with one embodiment of the present invention.
Figure 3 shows a graph of training set for algorithm development in accordance with one embodiment of the present invention.
Figure 4 shows a graph of test set for method development in accordance with one embodiment of the present invention.
Figure 5 shows a graph of effect of glucose concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 6 shows a graph of effect of creatinine concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 7 shows a graph of effect of bilirubin concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 8 shows a graph of effect of cholesterol concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 9 shows a graph of effect of sodium chloride concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 10 shows a graph of effect of potassium chloride concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 11 shows a graph of effect of magnesium chloride concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 12 shows a graph of effect of calcium chloride concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 13 shows a graph of effect of zinc chloride concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure14 shows a graph of effect of sodium bicarbonate concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure15 shows a graph of effect of sodium carbonate concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 16 shows a graph of effect of ferric chloride concentration on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 17 shows a graph of stability studies of reagent stored in vials at 2-8degree C in accordance with one embodiment of the present invention.
Figure 18 shows a graph of stability studies of reagent stored in vials at RT in accordance with one embodiment of the present invention.
Figure19 shows a graph of stability studies of reagent stored in bottle at 2-8degree C in accordance with one embodiment of the present invention.
Figure 20 shows a graph of stability studies of reagent stored in bottle at RT in accordance with one embodiment of the present invention.
Figure 21 shows a graph of effect of temperature on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 22 shows a graph of stability studies of reagent stored in bottle at 40degree C in accordance with one embodiment of the present invention.
Figure 23 shows a graph of effect of CTAB on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 24 shows a graph of effect of SDS on haemoglobin measuring in accordance with one embodiment of the present invention.
Figure 25 shows a graph of effect of Tween 20 on haemoglobin measuring in accordance with one embodiment of the present invention
Figure 26 shows a graph of effect of Glycerol on haemoglobin measuring in accordance with one embodiment of the present invention
Figure 27 shows a graph of effect of Triton-X on haemoglobin measuring in accordance with one embodiment of the present invention
Figure 28 shows a graph of testing of creatinine at lower concentration
Figure 29 shows a graph of testing of creatinine using sodium hydroxide
Figure 30 shows a graph of testing of creatinine using sodium methoxide
Figure 31 shows a graph of glucose detection data.

Detailed description of the Invention
The present invention is to provide a unique device and method for the determination of haemoglobin in clinical samples in the range of 4 g/dL to 16 g/dL, creatinine 0.5 mg/dL to 20 mg/dL, and glucose 30 mg/dL to 300 mg/dL. In the present invention, the system includes a diagnostic device designed for haemoglobin analysis. In another embodiment, the device contains a body to hold the sample vial with cap, mobile and connector for mobile. In some embodiments, the vial is configured to collect a blood sample.
In some embodiments, the device attached to mobile of any type or make with minimum features of display and network which include an information of the sample of corresponding person. In some embodiments, the information member may include patient name, gender, age, any health complications, address, contact details including phone number and email address.
The information of tested member may include the color scale or standardized scale in addition to a unique identifier identifying the color scale, as well as other information, so that a user can determine a range of haemoglobin values if a diagnostic analysis system according to embodiments is not available at the time of analysis. To reiterate, the principle of the method discussed is a color-based image measurement of a haemoglobin sample solution. This is directly proportional to haemoglobin concentration and each colour is specific to the concentration of haemoglobin.
The device comprises of a microcontroller, camera, light source USB facility, on/off switch, power supply etc. The microcontroller should be able to communicate with a camera, Bluetooth/Wi-fi module. The high-resolution images taken by camera is to be sent to the Mobile phone/App connected through the USB cable or Bluetooth or Wi-Fi for further image processing and results to display. USB cameras of various types can be used. USB facilities will be utilized when there is no power supply. Mobile/App can directly take pictures and analyze. LED’s with even light distribution can be controlled and thus, light intensity is adjustable.
In another embodiment, the device contains a camera for capturing the image. The resolution of the camera may be from 2 mega pixel to 10 mega pixels with adjustable lengths from 2 cm to 15 cm. The light sources may be Light-emitting diodes (LED) and will typically have a wattage between 5W-15W and will emit between 300-500 lumens.
In another embodiment, the present invention provides an environmentally friendly process for the preparation of reagent specific for the determination of haemoglobin.
In a preferred embodiment, the process for the preparation of reagent according to the present invention comprises of various surfactant such as tween 20, phosphate buffer (PBS), sodium lauryl sulphate, sodium dodecyl sulphate (SDS), cetyl trimethylammonium bromide (CTAB), triton X-80, and triton X-100.
In a preferred embodiment, the process for the preparation of reagent according to the present invention comprises of various solvents such as acetonitrile, DMSO, DMF, ethanol, methanol, isopropyl alcohol, glycerol, 2-ethoxy ethanol and water.
In yet another preferred embodiment, the process for the preparation of reagent according to the present invention comprises of various dyes such as anisidine, TMB (3,3',5,5'-Tetramethylbenzidine), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), o-dianisidine, O-toluidine, guaiacol, luminol, leucocrystal violet, fluorescein, and phenolphthalein.
In yet another preferred embodiment, the process for the preparation of reagent according to the present invention comprises of various salts such as sodium chloride, potassium chloride, calcium chloride, zinc chloride, and ferric chloride.
In yet another preferred embodiment, the process for the preparation of reagent according to the present invention comprises of various oxidising agents such as hydrogen peroxide (H2O2), sodium hypochlorite, hypochlorous acid, tert-butyl hydroperoxide (TBHP), and cumyl hydroperoxide (CHP).
In yet another preferred embodiment, the process for the preparation of reagent according to the present invention comprises of various acid such as hydrogen chloride, sulfuric acid, acetic acid, formic acid, benzoic acid, and p-toluene sulfonic acid.
In another embodiment image processing algorithm involves several steps such as selection of region of interest (ROI) and segmentation, feature extraction, regression, and training of the algorithm. The accurate results are obtained based on the quality of image which is affected by the of the region of interest, intensity of the light, distance of the camera, vial quality, reagent formulation etc.
The various combination of reagents was used for determination of haemoglobin values. The reagent volumes can vary from 100 µL to 1000 µL. The blood sample can vary from 1 uL-50 µL. The test time varies from 30 seconds to 360 seconds. The temperature can be between 4 oC to 40 oC.
In general, for testing the haemoglobin level associated with a whole blood sample is shown in Fig 1. After the blood is collected in the sample vial, the cap is tightened. After the cap is fixed to the vial, the blood reacts with the reagent in the vial, thereby causing a change in the colour of the prefilled solution. After placing the sample in the vial holder, press the start button. After 2-3 minutes the colour of the solution corresponding to a haemoglobin level will be developed and captured automatically. Thus, based on the automated algorithm each colour corresponds to the particular value of haemoglobin and will be displayed on the mobile application. In this way, the device can provide a simple, user-friendly, disposable, inexpensive system that does not require any skilled person. In addition, device is feasible for self-testing and/or in clinical settings and very simple for use in remote areas and underdeveloped communities.
Figure 2 shows the method involving the Image processing algorithm analysis involves selection of region of interest (ROI) and segmentation, feature extraction, regression, and training of the algorithm. The critical aspect of image analysis is the selection of region of interest (ROI) of the captured image for obtaining accurate results. Development of a background noise seems to be one of the inherent issues that often brings further challenges in the post-processing steps. In this context, one can use a binary thresholding technique on the colour intensity (RGB) and hue saturation levels extracted from the ROI. Further manipulation is done on the regions which are not of interest to remove the background noise that may affect the feature extraction. In order to make the best prediction, the extracted features and haemoglobin concentration as independent and dependent variables, respectively were used for regression analysis. Furthermore, one can adapt a dynamic revision of the regression algorithm as new experimental data are available through a convex optimization process.
Training of the algorithm was done with several sets of experimental images obtained from the blood samples with known Hgb values. In this procedure, 80-90% of the images are used for the training set to find the optimal regression equation while the rest of the 10-20% are used for validation of the algorithm. To validate the algorithm, operation was repeated several times to ensure that there are no overlapping training and validation data set. For each iteration, a new regression curve is compared to the previous iteration and found to be minimally varying, which reflects no overfitting during the iterative process. The overall regression equation is quantified by the average over 10-fold operations. From the validation results, it is evident that mean square error for 10-fold operations is minimal, which manifests the higher predictive accuracy of our systems.
To obtain the statistical relevance of the experimental outcomes, Applicant have explored both supervised and unsupervised machine learning approaches in our algorithm for pre-processing, regression analysis, and real-time dynamic adaptation. Pearson correlations, linear regression models, and the ratio of the mean and prediction bounds are employed to estimate the correlation between Hb levels via a pathological haematology analyser Hemocue (HB-201+), and the new device. Sensitivity and specificity of the results were determined collectively for the volunteers irrespective of their age, sex, and ethnicity.
Blood is a fluid that moves through the vessels of a circulatory system, and it comprises of plasma (the liquid portion), blood cells (which come in both red and white varieties), and cell fragments (platelets). There are several components such as gases, salts, proteins, nutrients, waste, and other cellular components. Since, the system uses blood there are chances of several interferences that can affect the ability of invention on the outcomes of accurate haemoglobin values. Hence, the inferences effect of these factors were extensively investigated.
Fig. 5 shows the effect of glucose concentration on haemoglobin measuring. The glucose level up to 0.079 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.079 mg/dL concentration showed the interference effect.
Fig. 6 shows the effect of creatinine concentration on haemoglobin measuring. The creatinine level up to 0.019 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.019 mg/dL concentration showed the interference effect.
Fig. 7 shows the effect of bilirubin concentration on haemoglobin measuring. The creatinine level up to 0.079 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.079 mg/dL concentration showed the interference effect.
Fig. 8 shows the effect of sodium chloride concentration on haemoglobin measuring. The creatinine level up to 0.039 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.039 mg/dL concentration showed the interference effect.
Fig. 9 shows the effect of potassium chloride concentration on haemoglobin measuring. The creatinine level up to 0.039 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.039 mg/dL concentration showed the interference effect.
Fig. 10 shows the effect of magnesium chloride concentration on haemoglobin measuring. The creatinine level up to 0.039 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.039 mg/dL concentration showed the interference effect.
Fig. 11 shows the effect of calcium chloride concentration on haemoglobin measuring. The creatinine level up to 0.079 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.079 mg/dL concentration showed the interference effect.
Fig. 12 shows the effect of zinc chloride concentration on haemoglobin measuring. The creatinine level up to 0.039 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.039 mg/dL concentration showed the interference effect.
Fig. 13 shows the effect of ferric chloride concentration on haemoglobin measuring. The creatinine level up to 0.039 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.039 mg/dL concentration showed the interference effect.
Fig. 14 shows the effect of sodium bicarbonate concentration on haemoglobin measuring. The creatinine level up to 0.039 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.039 mg/dL concentration showed the interference effect.
Fig. 15 shows the effect of sodium carbonate concentration on haemoglobin measuring. The creatinine level up to 0.039 mg/mL didn’t show any interference in determination of haemoglobin while above the 0.039 mg/dL concentration showed the interference effect.
Fig 16-23 shows the Effect of ferric chloride concentration on haemoglobin measuring, stability studies of reagent stored in vials at 2-8degree C, stability studies of reagent stored in vials at RT, stability studies of reagent stored in bottle at 2-8degree C, stability studies of reagent stored in bottle at RT, effect of temperature on haemoglobin measuring, stability studies of reagent stored in bottle at 40degree C and the effect of CTAB on haemoglobin measuring.

Procedure for reagent preparation
The reagent was prepared with dye (0.1-1.0%) in organic solvent (acetonitrile, DMF, DMSO, THF, 2EET, etc.). Solution of surfactant in water was prepared and mixed with the solution A. To this was added, oxidants (1-5%, H2O2, sodium hypochlorite, hypochlorous acid, tert-butyl hydroperoxide (TBHP), cumyl hydroperoxide (CHP) etc). The pH was adjusted to 5-6 using organic or inorganic acids such as hydrogen chloride, sulfuric acid, acetic acid, formic acid, benzoic acid, PTSA, etc.
The haemoglobin test kit is preferably provided or sold together with the reagent vial to allow diabetes patients to prepare the sample fluid and test for haemoglobin using the haemoglobin test kit at home. In this way, the present invention provides a low cost, low bulk haemoglobin test kit that can be used at the point of care and associated with a specific sample fluid source (e.g. a specific patient) without requiring a costly laboratory or clinic set-up to run the tests for haemoglobin.
Whilst there has been described in the foregoing description exemplary embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations in details of design, construction and/or operation may be made without departing from the present invention. Although exemplary modalities of the processes and products described have been presented it is not intended that the scope of protection is limited to the literality thereof. Therefore, the description should not be interpreted as limiting, but merely as examples of particular modalities that keep the inventive concept presented here. A person skilled in the art can readily apply teachings presented here in analogous solutions, arising therefrom, limited only by the scope of the claims of this application.
, C , C , Claims:We Claim:

1. A composition/reagent for detection of haemoglobin, creatinine and blood in a blood sample, comprising: 0.1-1.0% of dye, a surfactant, 1-5% of oxidant, organic or inorganic acids and solvents.

2. The composition/reagent as claimed in claim 1, wherein the dye is chosen from at least one of TMB (3,3',5,5'-Tetramethylbenzidine), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), anisidine, o-dianisidine, O-toluidine, guaiacol, luminol, leucocrystal violet, fluorescein, and phenolphthalein etc.

3. The composition/reagent as claimed in claim 1, wherein the surfactant is chosen from at least one of tween 20, phosphate buffer (PBS), sodium lauryl sulphate, cetyl trimethylammonium bromide (CTAB), Triton X80, and Triton X-100.

4. The composition/reagent as claimed in claim 1, wherein the oxidant is chosen from at least one of H2O2, sodium hypochlorite, hypochlorous acid, tert-butyl hydroperoxide (TBHP), cumyl hydroperoxide (CHP).

5. The composition/reagent as claimed in claim 1, wherein the solvents is chosen from at least one of acetonitrile, DMSO, DMF, ethanol, methanol, isopropyl alcohol, glycerol, 2-ethoxy ethanol, and water, etc.

6. The composition/reagent as claimed in claim 1, wherein the haemoglobin value, a calculated or direct hematocrit value, will not be affected by the interferences such as glucose (max. 0.039 mg/mL), creatinine (0.039 mg/mL), and bilirubin (0.079 mg/mL).

7. The composition/reagent as claimed in claim 1, wherein the haemoglobin value, a calculated or direct hematocrit value, will not be affected by the salt concentrations, for example salts such as sodium, potassium chloride (max. 0.039 mg/mL), magnesium, calcium, zinc chloride (0.039 mg/mL) and ferric chloride (0.079 mg/mL).

8. A method for preparing a reagent which facilitates in detecting haemoglobin of a blood sample, the method comprising:
preparing a solution of dye (0.1-1%) in organic solvent;
preparing a solution of surfactant in water and mixing with solution of dye;
adding oxidants, wherein detecting the oxidation so as to detect the presence of haemoglobin in the blood; and
adjusting the pH to 5-6 using organic or inorganic acids,
wherein all the above steps are executed simultaneously and in results in generation of one or more colour of the solution corresponding to a haemoglobin level, wherein each colour corresponds to the particular value of haemoglobin and will be displayed on a smart device application, and wherein the one or more colour attributes include intensity, brightness, hue, among others, or a combination thereof.

9. The method as claimed in claim 8, wherein the detection of haemoglobin in clinical blood sample in the range of 4 g/dL to 16 g/dL, creatinine 0.5 mg/dL to 20 mg/dL, and glucose 30mg/dL to 300mg/dL using 2-5 µL of blood sample.

10. The method as claimed in claim 8,
wherein the haemoglobin information which includes a haemoglobin value with current status in comparison with WHO recommendation, a calculated or direct haematocrit value, and / or a disease state associated with the haemoglobin value and / or hematocrit value,
wherein determining the haemoglobin information includes image capturing, selection of region of interest, processing, training the algorithm, and testing the new samples for accurate haemoglobin values;
wherein the haemoglobin value, a calculated or direct haematocrit value, will not be affected by the interferences such as glucose (max. 0.039 mg/mL), creatinine (0.039 mg/mL), and bilirubin (0.079 mg/mL),
wherein the haemoglobin value, a calculated or direct haematocrit value, will not be affected by the salt concentrations, for example salts such as sodium, potassium chloride (max. 0.039 mg/mL), magnesium, calcium, zinc chloride (0.039 mg/mL) and ferric chloride (0.079 mg/mL); and
wherein the one or more scale interferences didn’t have any significant effect on the accurate determination of haemoglobin at higher concentration than in physiological conditions.

11. A Kit for detecting haemoglobin, creatinine, and glucose of a blood sample, comprising:
a vial holder capable of accompanying at least one reagent vial, wherein the reagent has a 0.1-1.0% of dye, a surfactant, 1-5% of oxidant, and organic or inorganic acids and solvents; and
a processing unit and a memory, the processing unit operative coupled with the vial holder for reading the reaction of the reagent with a blood sample and also capable of receiving and sending instruction or information from a smart device via an USB connection port,
wherein upon reaction of reagent with the blood sample leads to generation of one or more colour of the solution corresponding to a haemoglobin level, wherein each colour corresponds to the particular value of haemoglobin and will be displayed on a smart device application.