Process for preparing a consumable solution, composition of consumable solution, and method for quantifying hemoglobin

Abstract

The present invention relates to a water-based consumable composition, to a process for the preparation thereof, and to a low-cost method for quantifying hemoglobin, integrable into a point-of-care hematology platform applied in the field of biochemistry. Specifically, it uses the spectrophotometry technique with a disposable hemocytometer for the purpose of quantifying hemoglobin. As advantages, in addition to enabling more precise and reliable dosages, it also provides improved transport and greater stabilization of the sample for up to 90 minutes. It further provides high accuracy results at low cost, simple production, non-toxic reagents, additional applicability in veterinary medicine, the possibility of use outside laboratory settings, and integration with other patents of the group (BR 10 2017 008549 0, BR 10 2021 026290 7, BR 10 2021 026289 3), thereby being capable of delivering a complete blood count test.

Description

CONSUMABLE SOLUTION PREPARATION PROCESS, CONSUMABLE SOLUTION COMPOSITION AND HEMOGLOBIN QUANTIFICATION METHOD Field of invention

[0001] This invention patent relates to a low-cost, water-based consumable composition, preparation process, and method for quantifying hemoglobin, which can be integrated into the point-of-care hematology platform applied to biochemistry. Specifically, it utilizes the spectrophotometry technique, utilizing a disposable hemocytometer to quantify hemoglobin. Advantages include, in addition to enabling more accurate and reliable dosages, better transport and greater sample stabilization for up to 90 minutes. Furthermore, it provides highly accurate results, low cost, simple production, non-toxic reagents, additional use in veterinary medicine, the possibility of use in non-laboratory settings, and integration with other patents of the group (BR 10 2017 008549 0, BR 10 2021 026290 7, BR 10 2021 026289 3), thus being capable of providing a complete blood count (CBC) test. Fundamentals of the invention

[0002] As hematology experts know, hemoglobin measurement is part of a complete blood count (CBC), one of the most frequently requested tests by physicians. This test is commonly performed on conventional hematology analyzers, which are characterized by their large footprint and limited mobility and structural requirements for operation. The main reagents used for hemoglobin measurement are potassium cyanide and potassium azide. However, these compounds are harmful to health and generate environmental pollution. Azide, in addition to its toxicity, also has explosive potential, limiting its use in certain contexts.

[0003] In order to define the state of the art, we carried out an in-depth search in national and international patent databases, finding the following relevant prior art.

[0004] HemoCue Apparatus: The invention relates to a method for the quantitative determination of hemoglobin in undiluted, non-hemolyzed whole blood, comprising the steps: providing a disposable capillary with an optical path length of less than 1 mm; filling said cuvette with a sample of unaltered whole blood; performing a first absorption measurement at a wavelength in the range of 490-520 nm directly on the sample in the cuvette; and a second absorption measurement for processing the results of both to determine the hemoglobin concentration in the sample. The processing step comprises compensating for dispersion in the sample, said compensation being dependent on the result of the second absorption measurement. A system for implementing the method is also disclosed.Although test results are provided in less than a second, the sample is stable for a very short period, creating greater technical operational challenges for obtaining analytically satisfactory results. Furthermore, there is no hemolysis of the blood sample, so operational errors during blood collection, which are widespread in the pre-analytical environment, can significantly affect the test result (Patent: W02003056327A1). https: / / patents.google.com / patent / WQ2003056327Al / en').

[0005] SpHb® Device: A patient monitor system is configured to measure and display the patient's hemoglobin concentration, assisting caregivers in providing care or treatment. It also provides automatic control of a fluid, blood, medication, or dialysis administration system. The patient monitor can analyze the displayed hemoglobin concentration and provide alarms and feedback to assist caregivers. Additional measurements can be combined with the hemoglobin concentration measurement to provide useful combined displays for caregivers, such as a plethysmographic variability index versus SpHb display. However, this technique has lower analytical sensitivity compared to the traditional method, which uses a blood sample, and is more appropriate for screening a patient's hemoglobin, rather than as a sole diagnostic method (Patent: US20100099964A1 https: / / patents.google.com / patent / US20100Q99964Al / en').

[0006] DiaSpect™ Method: The present invention (HBX) describes a system and device design for measuring and analyzing properties in suspended liquids, preferably human body fluids, whole blood, and particulate matter. Specifically calibrated light passes through a thin, well-defined layer of the liquid, placed in a non-contact cuvette. The transmitted light is recorded on a spectrophotometer. The data points recorded by the photometer are then processed in a series of steps for corrections and calculation of the desired parameter values / results through different algorithms in the device's microprocessor. The final presentation occurs on a display, and the data are stored in memory and can be communicated with another information receiving unit. This invention also focuses on evaluating the different hemoglobin fractions in a manner similar to the conventional methodology (Patent: US7701581B2 https: / / patents.google.com / patent / US7701581B2 / en').

[0007] Mission®: The apparatus and method for rapidly measuring the hemoglobin content of a blood sample uses a light reflectance measurement of the lysed hemoglobin sample on a membrane. The reflection is inversely proportional to the hemoglobin concentration. A blood sample is placed on a nylon mesh and dispersed within a nylon membrane, both treated with a hemolyzing agent and surfactant and air-dried. As soon as the blood sample comes into contact with the reagent on the membrane, the red blood cells are lysed and the hemoglobin molecules are released and dispersed within the membrane by the action of the surfactant. The hemoglobin apparatus emits light at 522 nm onto the reflective surface of the membrane. The intensity of the reflected light is measured by a detector in less than 29 seconds and converted to hemoglobin concentration (g / dL or mmol / L) by interpolative comparison with stored reflection data indicative of known hemoglobin contents.Although it is an innovative technique, it presents inferior analytical performance compared to the spectrophotometry technique in liquid medium (Patent: US7379167B2 https: / / patents.google.com / patent / US7379167B2 / en').

[0008] INSIGHT® Method: This disclosure relates to the use of a paper medium to measure hemoglobin concentration in blood. In certain embodiments, spectrophotometric techniques are used to measure the transmission of light at specific wavelengths through a paper medium containing a blood sample. The light transmission information is then used to calculate the hemoglobin concentration in the blood. In certain embodiments, the paper medium can be chemically treated to lyse the blood sample before measuring the light transmission information. Although this is a novel technique, humidity can easily affect the values. absorbance readings, being an interferent. Furthermore, stains generated by operator misuse are another known interferent. The authors' data demonstrate only average accuracy of this system with another point-of-care hemoglobin device [HemoCue; up to 3 g / dL difference between the devices] (Patent: US20120257188A1 https: / / patents.google.com / patent / US20120257188Al / en').

[0009] System for measuring total hemoglobin in blood and method of doing so. This invention relates to the measurement of hemoglobin in blood. Chemical interference from medications, other blood components, and incomplete rehydration of reagents can affect accuracy and prolong the reaction time required to determine total hemoglobin in blood. Embodiments of the present invention utilize a disposable body housed within the capillary channel and adapted to receive a blood flow from the inlet, such that air in the disposable body is expelled through a vent. A measurement system is configured to measure a property of the blood flow. This property can be related to the total hemoglobin count. Although it is an innovative technique, this method does not lyse erythrocytes, making it more susceptible to analytical interference and lower accuracy.Therefore, it is more appropriate for screening the patient's hemoglobin, and not as a sole diagnostic method (Patent: W02019059930A1 https: / / patents.google.com / patent / WQ2019059930Al / en').

[0010] Optical sensor for detecting free hemoglobin in a whole blood sample: This invention consists of: a sensor, comprising a translucent plate with a front face and a back face facing away from the front face, wherein the front face is adapted to come into contact with a whole blood sample; a reflective layer on the front face of the translucent plate, the reflective layer being adapted to reflect light striking the reflective layer of the translucent plate; an optical probing device comprising a light source and a detector, wherein the light source is adapted to illuminate at least pores in the translucent plate, wherein the detector is arranged to receive light emerging from the pores in response to illumination by the light source, and wherein the detector is adapted to generate a signal representative of the detected light. The translucent plate has dead-end pores that extend from the front side into the translucent plate towards the back side.Each pore has a corresponding opening on the front of the translucent plate, penetrating the reflective layer. The cross-sectional dimensions of the pore openings are designed to prevent red blood cells from entering the pores while allowing free hemoglobin to enter. Although this is an innovative technique, it is less accurate, more expensive, and more complex than other methodologies. Furthermore, there is no hemolysis of the blood sample, meaning that operational errors during blood collection, which are widespread in the pre-analytical environment, can significantly affect the test result (Patent US11079317B2; https: / / patents.google.com / patent / US11079317B2 / en').

[0011] Process, handheld equipment, and device for in vitro photometric determination of hemoglobin concentration in a one-step diluted blood sample: The present invention allows its use in field anemia screening programs. The equipment comprises: a light source with a wavelength between 500 and 550 nm; a cylindrical sample holder with a diameter between 8 and 20 mm; a photosensor for photometry of the sample; a microprocessor for automatically activating the light source, acquiring the signal obtained by the photosensor, and calculating hemoglobin concentration to display the results on a liquid crystal display. The device also comprises a sealed cylindrical vial, which serves simultaneously as reagent packaging and as a component. optical aspect of the process, allowing photometric reading through its walls. However, this technique has lower analytical sensitivity compared to the traditional method, making it more appropriate for screening a patient's hemoglobin, rather than as a sole diagnostic method. Furthermore, the test operator can easily soil the cuvette used with the blood sample, affecting the analyte reading. Finally, the system as a whole is not straightforward to operate. (Patent: BRPI0802336A2; https: / / patents.google.com / patent / US20110263031 Al / en).

[0012] Device for determining the amount of hemoglobin in a plasma fraction. The present invention relates to a device for determining the amount of hemoglobin in a blood sample. The device comprises a first membrane for filtering the plasma and a second membrane for retaining the filtered sample. A slit in the body allows the energy of the detection system to reach the sample retained in the second membrane, enabling hemoglobin measurement. However, it is more expensive and complex compared to other methodologies. It assesses free hemoglobin in plasma without lysing erythrocytes for analysis. It relies on blood collection (venous or capillary) with a high degree of operator experience to prevent unwanted erythrocyte lysis due to operational errors. It is less technically accurate. It does not present any results when comparing the technique with the conventional methodology (Patent: EP3686597A1; https: / / patents.google.com / patent / EP3686597Al / en).

[0013] Device and method for blood hemoglobin measurement without carboxyhemoglobin interference: The present invention provides a device and method for measuring hemoglobin in blood without carboxyhemoglobin interference. In the present device and method, oxyhemoglobin, deoxyhemoglobin, and methemoglobin are converted to a methemoglobin derivative, i.e., imidazole-methemoglobin. The imidazole-methemoglobin and carboxyhemoglobin in the matrix are collectively quantified by reflectance spectroscopy (spectrometers 6, 7, and 8) of the matrix at 525 nm, the isobesthic point between the imidazole-methemoglobin complex and the carboxyhemoglobin molecule. However, the vertical flow strip is highly influenced by operator action and is easily stained due to moisture or carelessness during handling.Furthermore, the test is not low cost, and is more appropriately used for screening the patient's hemoglobin, rather than as a sole diagnostic method (Patent: W02014006638A1 https: / / patents.google.com / patent / WQ2014006638Al / en).

[0014] Test device and method for determining blood hemoglobin: A test device and method for determining the hemoglobin content of blood are provided. The test device comprises a substantially opaque light-reflecting matrix having a refractive index significantly different from that of blood. In use, a blood test sample is placed in contact with the matrix of said test device, and the light reflectance thereof is measured as an indication of the quantitative amount of hemoglobin present in said sample. However, this is not a low-cost methodology (Patent: US4057394A; https: / / patents.google.com / patent / US4057394A / en).

[0015] Photometric apparatus and method for measuring hemoglobin: In this invention, the cuvette is made of plastic and has a passage slot of predetermined height that allows absorbance measurement with an undiluted blood sample, also allowing cleaning by immersing the body in a liquid cleaning solution. Two absorbance measurements are made: the first, at a wavelength close to 510 nm, in which the The absorbances of oxyhemoglobin and deoxyhemoglobin are approximately equal; in the latter case, these components do not substantially absorb light. In the 510 nm region, absorbance can be measured with a relatively thick slit and an undiluted blood sample in an inexpensive photometer. However, this requires the use of a cleaning solution to allow the cuvette to be reused without interference between analytes. Poor cuvette cleaning by the operator can cause reading errors. The erythrocyte is lysed using saponin as a lysing agent, so that hemoglobin is not converted to methemoglobin and the analyte reading is performed quickly. Furthermore, there is no precision regarding the amount of saponin used in each test, which may interfere with the test (Patent US5064282A; https: / / patents.google.com / patent / US5064282A / en').

[0016] A method and a system for quantitative hemoglobin determination: The invention relates to a method for determining hemoglobin in unaltered whole blood and to a system that can be used for this determination. Some points to be highlighted are: low sample stability; blood analysis must be performed promptly; it is not a low-cost method; lower analytical performance compared to the reference method. This is a system to be used for screening, not for final diagnosis (Patent EP2016390B1; https: / / patents.google.com / patent / EP2016390Bl / ').

[0017] WO2017161363A8 - Point-of-care device for the colorimetric determination of hemoglobin and glucose-6-phosphate dehydrogenase in biological samples. Devices, kits, and assays are provided for testing and monitoring hemoglobin deficiency, anemia, glucose-6-phosphate dehydrogenase deficiency, and glucose-6-phosphate dehydrogenase deficiency in an individual.

[0018] WO2017139478 Al - Devices, systems, and methods for quantifying hemoglobin S concentration. Lateral flow immunoassay devices, systems, and methods are provided for quantifying hemoglobin S in a sample. Point-of-care devices and methods are provided for rapidly and accurately monitoring a subject's hemoglobin S level in a blood sample.

[0019] US6890756B2 - Method of using a cyanide-free lysis solution to emulate a cyanide-containing lysis solution in measuring hemoglobin. A method of emulating a cyanide-free lysis solution performs the measurement of hemoglobin in whole blood using a cyanide-containing lysis solution. Such an illustrative method includes: a) combining a predetermined amount of the whole blood sample with a predetermined amount of a cyanide-free lysis solution to form a mixture; b) developing the solution to a molar absorptivity in the range of 12.4 to 12.6 mM; c) measuring an absorbance level of the solution at a wavelength of 540 nm. In a given composition, the cyanide-free lysis solution includes: a quaternary ammonium salt surfactant, an anionic surfactant, a hemoglobin-binding agent selected from the group consisting of imidazole or hydroxylamine, and an aqueous medium.The molar absorptivity is developed to a value of approximately 12.5 mM, that is, to a level that is substantially identical to the prior art standard, cyanomethemoglobin. By reaching this molar absorptivity level, the calculated results do not need to be corrected for the different molar absorptivities exhibited by imidazolemethemoglobin or hydroxylaminemethemoglobin.

[0020] US2003044995A1 - Cyanide-free reagent and method for detecting hemoglobin. A cyanide-free reagent for detecting hemoglobin is provided. The reagent includes a surfactant, a ligand selected from a nitrate, a nitrate salt, a nitrite, a nitrite salt, and combinations thereof, and a hydrogen ion concentration sufficient to maintain the pH of the reaction medium below 9. Also provided are a method and a kit for detecting hemoglobin in a blood sample using the cyanide-free reagent.

[0021] TW201000895A - Hemoglobin-detecting electrode test strip and device comprising the same The present invention relates to an electrode test strip for detecting hemoglobin, comprising: an insulating substrate; an electrode system; an insulating layer that partially covers the electrode system, such that an uncovered portion of the electrode system forms an electrochemical reaction region and another uncovered portion of the electrode system forms a conductive wire connection region; a reaction layer comprising an electronic mediator and an anionic surfactant, wherein the reaction layer at least partially covers the electrochemical reaction region. The present invention also relates to a device for detecting hemoglobin, comprising the hemoglobin-detecting electrode test strip and an electrochemical sensor.

[0022] JP2010002401 A - Method and device for measuring hemoglobin. A device for measuring the hemoglobin concentration of a small amount of sample with high precision. A first electrode (working electrode) 1 and a second electrode (reference electrode) 2 are formed on the surface of a silicon substrate 4, and a container 3 is brought into contact with the working electrode 1 and the reference electrode 2. The sample is introduced into the reaction vessel 3, thus establishing continuity between both electrodes 1 and 2 through the sample. The oxidation-reduction potential occurs without external voltage or current and is monitored by a voltmeter 6, and the hemoglobin concentration is estimated by the Nernst equation. Thus, a power source between the two electrodes is unnecessary.

[0023] IN434170A1 - Point-of-care detection of hemoglobin. The present invention provides point-of-care hemoglobin detection. Specifically, the present invention provides compositions, assays, kits, and / or devices for detecting hemoglobin. Furthermore, it provides a liquid-based composition and assay for detecting hemoglobin. This assay does not include any solid support and has less expensive sensors compared to solid-state readers.

[0024] IN-DEL-2012-02074A - Device and method for blood hemoglobin measurement without carboxyhemoglobin interference. The present invention provides a device and a method for determining hemoglobin in blood without carboxyhemoglobin interference. In the present device and method, oxyhemoglobin, deoxyhemoglobin, and methemoglobin are converted to a methemoglobin derivative, i.e., imidazole-methemoglobin. The imidazole-methemoglobin and carboxyhemoglobin in the matrix (2) are collectively quantified by reflectance spectroscopy (spectrometers 6, 7, and 8) of the matrix at 525 nm, the isobestic point between the imidazole-methemoglobin complex and the carboxyhemoglobin molecule.

[0025] CN215866735U - Point-of-Care Testing (POCT) Hematocrit Analyzer and Kit. The utility model provides a POCT (Point-of-Care Testing) hematocrit analyzer and kit comprising at least one cell position; the cell positions are used for placing test tubes and impedance detection; the locking portion is arranged on one side of the tank position and is used to fit a test tube cap; the microporous sheet is arranged between the front tank and the rear tank of the box-type body. The locking portion is arranged The kit is located on one side of the tank and is used to secure the test tube lid. The kit provided by the utility model has an innovative structure and low cost. It can be used as a disposable consumable product and does not require expensive materials that can be cleaned and reused repeatedly.

[0026] CN1313822C - Hemoglobin sensor. The present invention relates to a device and method for measuring hemoglobin in a fluid sample. The device comprises a disposable electrochemical cell, such as a thin-layer electrochemical cell 1, containing a reagent capable of being reduced by hemoglobin. A suitable sample that can be analyzed according to the present invention is whole blood. If the hemoglobin to be analyzed is present in red blood cells, a lysing agent can be added to the sample to release the hemoglobin prior to analysis.

[0027] CN107569237A - Method and device for measuring hemoglobin levels non-invasively. The invention discloses a device for measuring hemoglobin levels non-invasively and provides a dynamic spectrum-based method for detecting hemoglobin content in blood. The device features high accuracy, good stability, low cost, small size, real-time monitoring, and can synchronously obtain photoelectric pulse waves and dynamic spectrum.

[0028] CN107405115B - device and method for detecting hemoglobin and complexes thereof. An electrochemically active device for collecting and retaining a blood sample having at least one two-electrode element connected to an electrically conductive filament is described. The present invention also provides point-of-care biosensors and measurement methods incorporating the inventive devices for detecting and quantitatively measuring the concentrations of hemoglobin (Hb), glycated hemoglobin (GHb), methemoglobin (MetHb), and myoglobin in a reduced-volume blood sample, determining the value of the redox current in the reduced-volume blood sample.

[0029] BR 11 2023 000416 0 - apparatus for determining the hemoglobin or hematocrit level of a circulating liquid. An analyzer having an internal chassis surrounded by a housing includes sample and dilution probes, a mixing housing including first and second mixing chambers, a flow cytometer including a flow cell, and sample and sheath pumps configured to perform first and second plurality of tasks, respectively. The first plurality of tasks includes: aspirating sample into the sample probe, dispensing sample from the sample probe into the first and second mixing chambers, supplying the first sample-dilution fluid mixture to the flow cell, and supplying the second sample-dilution fluid mixture to the flow cell.The second plurality of tasks includes: dispensing the sheath to the flow cell in cooperation with supplying the first sample-dilution fluid mixture to the flow cell, and dispensing the sheath to the flow cell in cooperation with supplying the second sample-dilution fluid mixture to the flow cell.

[0030] BR 11 2023 000158 7 - apparatus and method for determining the hemoglobin or hematocrit level of a circulating liquid. The present invention relates to an apparatus and a method for determining the hematocrit level and / or the hemoglobin level of a circulating liquid in a tubular portion (2), the method comprising: - emitting light beams in the direction of the tubular portion (2) with at least two sources light sources (11; 21), each of the two light sources (11; 21) being configured to emit light beams according to an emission wavelength chosen to correspond to an isosbestic point of hemoglobin; - reception of light signals transmitted through the tubular portion (2) with at least two light sensors (12; 22), each light sensor (12; 22) being associated with one of the two light sources (11; 21); - calculation of the hematocrit rate and / or the hemoglobin rate of the liquid for processing the light signals received by the light sensors (12; 22); characterized in that the emission power of at least one of the light sources (11; 21) is modified during the determination of the hematocrit rate and / or the hemoglobin rate as a function of the hematocrit rate and / or the rate, respectively.

[0031] The main reagents used for hemoglobin measurement are potassium cyanide and sodium azide. These compounds, however, are harmful to health and generate environmental pollution. Azide, in addition to its toxicity, also has explosive potential, limiting its use in certain contexts. On the other hand, most hemoglobin quantification solutions that do not contain these toxic compounds use Triton X, a reagent that was recently banned in Europe for forming highly toxic compounds that harm nature and the environment. Furthermore, the reagent solutions available on the market are more complex and costly to produce, reducing product viability.

[0032] Another negative aspect is that the point-of-care devices available on the market lack integration with other hematology platforms, meaning they only allow for isolated hemoglobin readings, not complete blood counts. Therefore, they don't allow patients to access complementary analytes. Furthermore, they don't allow for the issuance of a report evaluated by a specialist.

[0033] The "CONSUMABLE SOLUTION COMPOSITION, CONSUMABLE SOLUTION PREPARATION PROCESS, AND HEMOGLOBIN QUANTIFICATION METHOD" covered by this patent was developed to overcome the limitations, disadvantages, and drawbacks of the compositions and preparation process for the hemoglobin quantification method using spectrophotometry. Since the hemoglobin concentration in the solution is proportional to the absorbed light, it is possible to measure the analyte in the sample in a liquid medium using a point-of-care multispectral spectrophotometer, which is responsible for quantifying the absorption, transmission, or refraction of light by the solution.

[0034] The advantages of this solution that promotes efficient hemoglobin measurement using spectrophotometry include: I. Accurate and reliable determination of hemoglobin concentration, with no significant differences (p > 0.05; T-test) when compared to the conventional methodology (Sysmex XE -2100); II. The consumable solution keeps the sample stable for 90 minutes, allowing samples to be stabilized and even transported safely between locations; III. Obtaining highly accurate results through the use of blank analysis, reducing interference that may affect dosage accuracy; IV. Simple and low-cost reaction solution, not requiring a complex laboratory structure; V. Solution without potassium cyanide and / or sodium azide, compounds that are highly toxic to the environment; VI. Rapid quantification of hemoglobin in the blood by inserting an aliquot of the solution into the hemocytometer, with the analysis being able to be performed instantly or in up to 40 min; VII. Consumable solution has stability of at least 1 year and does not require refrigeration to maintain its chemical and functional characteristics. VIII. Unlike all the solutions described, the present reaction solution does not contain Triton X-100 in its composition, a substrate that produces the metabolite 4-tert-octylphenol, considered an endocrine disruptor that is very dangerous for the environment; IX. Possibility of use in point of care devices (low-cost photometers) or conventional spectrophotometers; X. Capable of integration with a hematology platform, since the sample can be inserted into hemocytometers (disposable or not; 0.01 or 0.02 mm in height) so that they can be used as a reading vehicle; XI. Capable of integration with the data system, enabling the release of not only hemoglobin results, but also blood count reports signed by specialists (BR 10 2017 008549 0); XII. Capable of integration with consumable solutions that allow the evaluation of leukogram (BR 10 2021 0262907), erythrogram and platelet count (BR 102021 026289 3), enabling the complete analysis of the patient's blood count data, using a disposable hemocytometer as a reading vehicle; XIII. Use compatible with human blood samples, but also veterinary, presenting satisfactory results when evaluated in blood samples from horses, felines, sheep, goats and dogs, for example; XIV. Use compatible with venous and capillary blood samples, stored or not in collection tubes containing anticoagulant (KjEDTA or K3EDTA); XV. Low cost and easy to use: 10 pL of blood sample is added to the consumable solution. After homogenization by inversion, an aliquot of this solution is transferred to a disposable hemocytometer. An AS7341 sensor is used to read the slide, using the Clear_512x channel (described below), F4 (515 nm), and / or F5 (560 nm); XVI. Compatibility with the AS7341 sensor, which is an 11-channel multispectral spectrophotometer, with 8 covering the visible spectrum. The channels can be applied to wavelengths between 350 and 1000 nm and are independently configurable, including their integration time and gain. XVII. The method used in this point-of-care device is similar to that used by the conventional methodology for hemoglobin quantification, increasing the reliability of the data generated;

[0035] Hemoglobin measurement is part of a complete blood count (CBC), one of the most frequently requested tests by physicians. This test is commonly performed on conventional hematology analyzers, which are characterized by their large footprint and limited mobility. Composed of reagents such as sodium lauryl sulfate (SLS) and monobasic sodium phosphate, the hemoglobin measurement methodology developed allows for the quantification of this analyte in point-of-care devices, reducing time. to obtain the result and speed up medical conduct.

[0036] The main reagents used for hemoglobin measurement are potassium cyanide and sodium azide. These compounds, however, are harmful to health and generate environmental pollution. Azide, in addition to its toxicity, also has explosive potential, limiting its use in certain contexts. Thus, SLS has the advantage of being a safe and biodegradable reagent, producing results equivalent to the aforementioned methods.

[0037] In the developed process, sample analysis is performed using spectrophotometry, a methodology based on quantifying the light absorbed by a solution. Considering that the concentration of hemoglobin in the solution is proportional to the absorbed light, it is possible to measure the analyte in the sample. The developed device has an attached multispectral spectrophotometer, responsible for quantifying the absorption, transmission, or refraction of light by the solution.

[0038] Typically, spectrophotometers use a glass, quartz, or acrylic cuvette as a vehicle. However, the process described here uses a disposable hemocytometer (i.e., a chamber designed for cell counting, used in conjunction with an optical microscope). The hemocytometer used in this case consists of a glass slide and a polymethylmethacrylate (PMMA) plate, forming three analysis compartments.

[0039] Alternatively, the hemocytometer can be a disposable B-CHIP device (Incyto®) or equivalent. However, these models are more expensive.

[0040] After adding the sample, the hemocytometer is inserted into the equipment, where the reading is taken by the spectrophotometer. This process allows hemoglobin quantification and subsequent report release in a few minutes, using a small amount (10 pL) of venous or capillary blood.

[0041] Furthermore, this method allows integration with a hematology point-of-care platform, which performs the analysis and release of blood count results through artificial intelligence, machine learning and deep learning techniques (BR 10 2021 026289 3, BR 10 2021 026290 7, BR 10 2017 008549 0).

[0042] Using a small hemocytometer (75 mm x 25 cm) as a vehicle, the test is easily performed using point-of-care devices. These devices are compact and portable. Furthermore, due to the composition of the solution and the other materials required for the test, this test is inexpensive.

[0043] The prior art presents the following problems and technical insufficiencies that were solved by the present invention shown below:

[0044] Use of toxic and / or polluting reagents resolved by the use of a biodegradable compound without harm to human health;

[0045] When measuring hemoglobin, interference occurs from substances such as lipemia, intense hemolysis and platelet agglutination, which are resolved by the presented method of hemoglobin quantification, which allows for the reduction of interferences that could affect the accuracy of the measurement;

[0046] Most point-of-care devices available on the market do not have integration with other hematology platforms, resolved by the proposed hemoglobin quantification method that allows integration with a hematology point-of-care platform;

[0047] The point-of-care devices available on the market do not release a report signed by experts, resolved by the proposed hemoglobin quantification method that allows integration with a hematology point-of-care platform, which performs the analysis and release of the result;

[0048] Point-of-care devices on the market only quantify hemoglobin, excluding other hematological parameters, which is resolved by using a hemocytometer as a vehicle that allows a complete blood count to be performed in the same capsule;

[0049] Most solutions require a robust laboratory infrastructure for their preparation and handling, resolved by the simple preparation of the aqueous solution;

[0050] Use of complex methodology resolved by using a methodology with a conventional spectrophotometer (through a cuvette) or point of care device (disposable hemocytometer associated with the AS7341 sensor), simplifying the method;

[0051] Use of consumable solution with complex operation and low stability solved by the present patent by using simple operation and high stability of the consumable solution (up to 2 years; room temperature, not requiring refrigeration).

[0052] The consumable solutions obtained by current processes do not keep the sample stable for a long time, resolved by the present patent that obtains a process that allows stability for 90 minutes, allowing to stabilize and even transport samples between locations safely.

[0053] This patent presents precision (mean CV < 2.5%) and reliability similar to the conventional methodology (hematological analyzer; Sysmex XE-2100) for hemoglobin quantification, with no statistically significant differences observed between the methodologies (p > 0.05; T-test; Pearson Correlation > 0.9).

[0054] The technology of this patent was obtained after incessant and costly research, described below.

[0055] The research began with the idea of ​​developing a consumable solution that would allow for simple and inexpensive hemoglobin measurement using a disposable hemocytometer and spectrophotometry. The first solutions consisted of a water-based solution and a surfactant, primarily aimed at lysing erythrocytes and releasing hemoglobin. Subsequently, reagents that would convert hemoglobin into methemoglobin were sought. The main reagents used for hemoglobin measurement are potassium cyanide and sodium azide. However, these compounds are harmful to health and cause environmental pollution. Azide, in addition to its toxicity, also has explosive potential, limiting its use in certain contexts. Therefore, the technology of this patent offers the advantage of being a safe and biodegradable reagent, producing results equivalent to the aforementioned methods. Initially, tests with the solution The developed consumable solution was tested using the NanoDrop™ device (standard equipment for spectrophotometric analysis). This test effectively demonstrated the functionality of the consumable solution for hemoglobin quantification, which showed a strong correlation with the data provided by the supporting laboratory (Pearson's correlation = 0.97). Subsequently, tests were performed on the AS7341 sensor, using a glass cuvette as a vehicle. Again, a high correlation between the quantification methodologies was observed (Pearson's correlation = 0.93). Finally, tests using the hemocytometer as a vehicle were performed, and adjustments were made to ensure satisfactory quantification of this analyte (Pearson's correlation = 0.98). Brief description of the drawings

[0056] For a better understanding of this model, the following figures are attached: Figure 1 illustrates the spectral responsivity graph of the AS7341 digital sensor. Description of the invention

[0057] In the developed process, sample analysis is performed using spectrophotometry, a methodology based on quantifying the light absorbed by a solution. Considering that the concentration of hemoglobin in the solution is proportional to the absorbed light, it is possible to measure the analyte in the sample.

[0058] Typically, spectrophotometers use a glass, quartz, or acrylic cuvette as a vehicle. However, the process described here uses a disposable hemocytometer—a chamber designed for cell counting, used in conjunction with an optical microscope.

[0059] The hemocytometer used in the process of the present patent may be composed of a set of a rectangular microscope glass slide (MS) and a rectangular polymethyl methacrylate-PMMA top plate (PS) containing three holes for sample insertion (PS-1), joined by a double-sided microfluidic tape (FDM) from 3M™ Microfluidic Diagnostic Tape, 9972A or similar. In this way, three rectangular spaces (chambers) are generated, enabling analysis of the analyte. Through this hemocytometer model, it is possible to perform three hemoglobin readings per slide, or a complete blood count exam (associated with patents BR 10 2021 026289 3, BR 102021 026290 7, BR 10 2017 008549 0).

[0060] Alternatively, the hemocytometer can be a disposable B-CHIP (Incyto) device or equivalent. Although these models are more expensive and have only two reading chambers, they offer the same analytical performance. The substances used in the aqueous-based composition of this patent have the following technical specifications:

[0061] Monobasic Sodium Phosphate PA ACS; CAS 10049-21-5, PM: 137.99, powder.

[0062] 2 M Sodium Hydroxide Solution; Sodium Hydroxide PA / ACS; CAS: 1310-73-2; Molecular weight: 40.00 g / mol, density 1.08 g / cm 3 (20 °C); colorless liquid

[0063] Sodium lauryl sulfate PA; CAS: 151-21-3; MW: 288.49, powder.

[0064] Deionized water; CAS; 7732-18-5; PM 18.02; colorless liquid. Examples of realization of the invention

[0065] To obtain the consumable solution developed for hemoglobin quantification of the present patent, the following tests were performed to determine the minimum, maximum and preferred ranges of the composition components:

[0066] Test No. 1 Determination of the minimum amount of Monobasic Sodium Phosphate

[0067] The solution was prepared with 3 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined using the spectrophotometer equipment, verifying a difference within the acceptable error standards.

[0068] Test No. 2 Determination of the minimum amount of Monobasic Sodium Phosphate

[0069] The solution was prepared with 2.9 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference above the acceptable error standards.

[0070] Test No. 3 Determination of the maximum amount of Monobasic Sodium Phosphate

[0071] The solution was prepared with 100 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference within the acceptable error standards.

[0072] Test No. 4 Determination of the maximum amount of Monobasic Sodium Phosphate

[0073] The solution was prepared with 101 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference above the acceptable error.

[0074] Note: Test No. 4 was repeated in the absence of Sodium Lauryl Sulfate Solution, observing results similar to those obtained with the presence of its solution.

[0075] Test No. 5 of the minimum quantity of 2 M Sodium Hydroxide Solution

[0076] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 0.2 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment and upon comparison it was found that it was within the acceptable error standards.

[0077] Test No. 6 of the minimum quantity of 2 M Sodium Hydroxide Solution

[0078] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 0.19 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference above the acceptable error.

[0079] Test No. 7 of the maximum amount of 2 M Sodium Hydroxide Solution

[0080] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 24.5 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference within the acceptable error standards.

[0081] Test No. 8 of the maximum amount of 2 M Sodium Hydroxide Solution

[0082] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 25 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference above the acceptable error.

[0083] Test No. 8 was repeated in the absence of Sodium Lauryl Sulfate Solution, observing results similar to those obtained with the presence of its solution.

[0084] Test No. 9 of the minimum quantity of Sodium Lauryl Sulfate Solution

[0085] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 0 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference within the acceptable error standards.

[0086] Test No. 10 of the maximum amount of Sodium Lauryl Sulfate Solution

[0087] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference within the acceptable error standards.

[0088] Test No. 11 of the maximum amount of Sodium Lauryl Sulfate Solution

[0089] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 5.5 g of Sodium Lauryl Sulfate and 250 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference above the acceptable error.

[0090] Test No. 12 of the minimum amount of deionized water

[0091] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 117 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference within the acceptable error standards.

[0092] Test No. 13 of the minimum amount of deionized water

[0093] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 116 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference above the acceptable error.

[0094] Test No. 14 of the maximum amount of deionized water

[0095] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 260 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference within the acceptable error standards.

[0096] Test No. 15 of the maximum amount of deionized water

[0097] The solution was prepared with 3.4 g of Monobasic Sodium Phosphate, 9 mL of 2 M Sodium Hydroxide Solution, 3.5 g of Sodium Lauryl Sulfate and 261 mL of Deionized Water and then the amount of hemoglobin in a blood sample with known hemoglobin was determined in the spectrophotometer equipment, verifying a difference above the acceptable error.

[0098] Test No. 17 which determined the preferred formula of the consumable composition

[0099] The solution was prepared with 3.4 g of monobasic sodium phosphate, 9 mL of 2 M sodium hydroxide solution, 3.5 g of sodium lauryl sulfate, and 250 mL of deionized water. The hemoglobin concentration of an unknown blood sample was then compared with the hemoglobin concentration of a known sample, verifying a difference within acceptable error standards.

[0100] With these tests, it was concluded that the consumable composition range is as follows: Monobasic Sodium Phosphate: 1.13% to 27.6% by weight; 2 M Sodium Hydroxide Solution: 0.08% to 8.70% by weight Sodium Lauryl Sulfate: 0 to 1.87% by weight; Deionized Water: 88.04% to 94.53% by weight;

[0101] The aforementioned tests concluded that the preferred consumable composition is as follows: Monobasic Sodium Phosphate: 1.28% by weight; 2 M Sodium Hydroxide Solution: 3.38% by weight; Sodium Lauryl Sulfate: 1.32% by weight; Deionized Water: 94.02% by weight.

[0102] The process of preparing the consumable composition takes place in the following sequence: 1. Weighing Monobasic Sodium Phosphate on an analytical balance and transferring it to a beaker with adequate capacity for the volume of solution to be prepared; 2. Adding deionized water to the container containing monobasic sodium phosphate; 3. Stirring at room temperature until the solution is completely homogenized. 4. Addition of 2 M Sodium Hydroxide Solution, followed by stirring at room temperature to homogenize the solution; 5. Weighing Sodium Lauryl Sulfate on an analytical balance; 6. Addition or not of Sodium Lauryl Sulfate to the beaker containing the prepared solution; 7. Homogenize the solution using a magnetic stirrer, at room temperature, until the reagents are completely dissolved.

[0103] The hemoglobin quantification method of the present patent is carried out in the following sequence: 1. Preferential dilution of 10 pL of venous or capillary blood (collected or not in a tube containing KiEDTA or KjEDTA anticoagulant) in 90 pL of the consumable solution followed by homogenization via inversion. A variation in the dilution factor between sample and consumable solution is allowed (1:2 - 1:500); 2. Transfer 10 pL of the consumable solution containing the blood sample to the disposable hemocytometer or equivalent slide set; 3. Introduction to the equipment responsible for reading; 4. The consumable solution promotes the lysis of red blood cells and the conversion of hemoglobin into methemoglobin through the action of Sodium Lauryl Sulfate (SLS), which, as an anionic surfactant, is highly water-soluble and binds to the heme group of hemoglobin, forming a colored complex. (SLS-Hb) which is quantified by a photometric method, proportionally to the concentration of this protein in the blood sample; 5. Hemoglobin quantification occurs in a liquid medium and can be performed between 0 and 40 minutes.

[0104] This method allows integration with a hematology point-of-care platform, which performs the analysis and release of blood count results through artificial intelligence, machine learning and deep learning techniques (BR 102021 026289 3, BR 10 2021 026290 7 and BR 10 2017 008549 0).

Claims

CLAIMS 1. COMPOSITION OF CONSUMABLE SOLUTION, characterized by the following formulation: a) Monobasic Sodium Phosphate: 1.2% to 27.6% by weight; b) 2 M Sodium Hydroxide Solution: 0.08% to 8.7% by weight; c) Sodium Lauryl Sulfate: 0 to 1.92% by weight; and d) Deionized Water: 88.04% to 94.53% by weight.

2. CONSUMABLE SOLUTION COMPOSITION, according to claim 1, characterized by the following preferred formulation: al) Monobasic Sodium Phosphate: 1.28% by weight; bl) 2 M Sodium Hydroxide Solution: 3.38% by weight; cl) Sodium Lauryl Sulfate: 1.32% by weight; and dl) Deionized Water: 94.02% by weight.

3. PROCESS FOR PREPARING CONSUMABLE SOLUTION to obtain the consumable solution used in the hemoglobin determination method, characterized by the following sequence:

1. Weighing Monobasic Sodium Phosphate on an analytical balance and transferring it to a beaker with adequate capacity for the volume of solution to be prepared; 2. Adding deionized water to the container containing monobasic sodium phosphate; 3. Stirring at room temperature until the solution is completely homogenized; 4. Addition of 2 M Sodium Hydroxide Solution, followed by stirring at room temperature to homogenize the solution; 5. Weighing Sodium Lauryl Sulfate on an analytical balance; 6. Addition or not of Sodium Lauryl Sulfate to the beaker containing the prepared solution; and 7. Homogenize the solution using a magnetic stirrer, at room temperature, until the reagents are completely dissolved.

4. HEMOGLOBIN QUANTIFICATION METHOD, characterized by the following sequence; A. Preferential dilution of 10 pL of venous or capillary blood, collected or not in a tube containing KiEDTA or KjEDTA anticoagulant, in 90 pL of the consumable solution and followed by homogenization by inversion and adopting an allowed variation in the dilution factor between sample and consumable solution between (1:2 - 1:500); B. Transfer 10 pL of the consumable solution containing the blood sample to the disposable hemocytometer or equivalent slide set; C. Introduction into the equipment responsible for reading: a coupled point-of-care multispectral spectrophotometer, responsible for quantifying the absorption, transmission or refraction of light by the solution; conventional multispectral spectrophotometer, through the use of a glass or quartz cuvette as a reading vehicle; D. The consumable solution promotes the lysis of red blood cells and the conversion of hemoglobin into methemoglobin through the action of Sodium Lauryl Sulfate (SLS), as it is an anionic surfactant, is highly water-soluble, and binds to the heme group of hemoglobin, forming a colored complex (SLS-Hb) that is quantified by a photometric method, proportionally to the concentration of this protein in the blood sample; and E. Hemoglobin quantification occurs in a liquid medium and can be performed between 0 and 40 minutes.

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