Method for determining a protein content and related apparatus and method

Abstract

The present invention aims to provide a method for determining at least one protein in blood, which method is more precise and at the same time easy to implement. The inventors have used spectroscopy and quantification techniques in an original way to accurately determine the protein content of biological samples, in particular for oxyhemoglobin, methemoglobin, heme bound to serum albumin and hemopexin, and bilirubin. Such a method can be advantageously used in various applications related to heme-related or heme-protein-related disorders, in particular for diagnostic methods, for monitoring treatment, for determining biomarkers or for screening methods. Such a method is also interesting for qualifying blood bags.

Description

Technical Field

[0001] The present invention relates to a method for determining (assaying) the content of at least one protein. The present invention also relates to a method for diagnosing heme-related or heme protein-related disorders. The present invention also relates to a method for identifying a therapeutic target for preventing and / or treating heme-related or heme protein-related disorders. The present invention also relates to a method for identifying a biomarker, which is a diagnostic biomarker for heme-related or heme protein-related disorders, a susceptibility biomarker for liver diseases, a prognostic biomarker for heme-related or heme protein-related disorders, or a predictive biomarker for response to treatment of heme-related or heme protein-related disorders. The present invention also relates to a method for screening compounds that can be used as drugs and that act on known therapeutic targets for preventing and / or treating heme-related or heme protein-related disorders. The method also relates to a method for qualifying or disqualifying a medical bag and a method for monitoring the treatment of heme-related or heme protein-related disorders. The present invention also relates to related computer program products and computer-readable media. Background Art

[0002] Many diseases exhibit symptoms that affect the blood.

[0003] Therefore, there is a desire to perform a chemical analysis of the blood.

[0004] A method for measuring whole blood hemoglobin parameters is known from document US 9 638 686 A1, comprising: providing a light source, guiding light having a spectral range along an optical path from the light source, providing a cuvette module having a sample receiving chamber, providing a pair of first and second light diffusers disposed in the optical path, wherein the cuvette module is disposed between the pair of first and second light diffusers, guiding light from the cuvette module into an optical spectrometer, and processing the electrical signal from the spectrometer into an output signal that can be used to display and report the hemoglobin parameter value and / or total bilirubin parameter value of a whole blood sample.

[0005] A method for measuring whole blood hemoglobin parameters is known from document US 9 638 686 A1, comprising: providing a light source, guiding light having a spectral range along an optical path from the light source, providing a cuvette module having a sample receiving chamber, providing a pair of first and second light diffusers disposed in the optical path, wherein the cuvette module is disposed between the pair of first and second light diffusers, guiding light from the cuvette module into an optical spectrometer, and processing the electrical signal from the spectrometer into an output signal that can be used to display and report the hemoglobin parameter value and / or total bilirubin parameter value of a whole blood sample.

[0006] Document US 2007 / 292963 A1 also proposes a method for determining the presence and concentration of components in serum, including the following steps: providing a serum sample, optically analyzing the serum sample using a spectrometer to provide spectral data based on the optical properties of the serum sample, determining the concentrations of albumin, globulin, and hemoglobin in the serum sample based on a comparison of the spectral data with reference spectra, and outputting the determined concentrations of albumin, globulin, and hemoglobin. This invention can be used to accurately determine the concentrations of albumin, globulin, and hemoglobin in serum, and can form the basis for initial cancer screening and evaluate the patient's response to treatment.

[0007] A method for measuring a substance is also known from document EP 1 211 505 A1. This method performs sample preparation and detection of the substance in the sample in a capillary of a microchip according to a photothermal conversion detection method, so that the amount of substances such as hemoglobin and aluminum phosphide (ALP) in a very small amount of sample obtained from living organism components can be measured simply and easily in a very short time. In addition, this method can reduce the waste caused by measurement. Furthermore, the method of this invention uses a laser with a long wavelength as the excitation light, so that a photothermal conversion detection device can be manufactured and measurement can be performed at low cost. Therefore, the method for measuring a substance in document EP 1 211 505 A1 can be appropriately applied to POC analysis and the like. In addition, the method for measuring a substance of this invention can even simply and easily measure a blood sample containing chyle according to the photothermal detection method. In addition, using the measurement reagent of this invention, the amount of substances such as hemoglobin and ALP in a very small amount of sample obtained from living organism components can be stably measured according to the photothermal conversion detection method.

[0008] However, the methods and devices of these documents are not easy to implement, and the measurements provided may not be as accurate as desired. Summary of the Invention

[0009] The present invention aims to provide a method for determining (assaying) at least one protein in blood, which is more precise and at the same time easy to implement.

[0010] To this end, this specification describes a method for determining the content of at least one protein in a biological sample, several of which proteins include oxyhemoglobin, methemoglobin, heme bound to serum albumin, heme bound to hemopexin (total hemopexin), and bilirubin. The method for determination (hereinafter also referred to as the determination method) at least includes the following steps:

[0011] a) Providing an initial spectrum of the sample within a target wavelength range,

[0012] b) Setting the current spectrum as the initial spectrum,

[0013] c) Iterate the following steps:

[0014] c1) Determine the protein content in the sample, which determination is carried out by using spectral analysis of the current spectrum or / and chemical quantification,

[0015] c2) Based on the determined content of the protein, deduce the overall spectral components within the target wavelength range related to the protein, and

[0016] c3) Remove the deduced spectral components from the current spectrum,

[0017] d) Output the determined protein content,

[0018] Iterate step c) for different proteins successively as long as the contents of oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin and hemopexin, and bilirubin have not been determined.

[0019] According to other advantageous but not mandatory aspects of the present invention, the determination method may incorporate one or several of the following features employed in any technically acceptable combination:

[0020] - Iterate step c) by reducing the value of the second derivative of the protein, where the value of the second derivative of the protein is defined as the maximum amplitude of the second derivative of the spectral components of the protein in the sample with respect to the spectral wavelength within the target wavelength range, or the absolute value of the difference between the minimum and maximum values of the second derivative of the spectral components of the protein in the sample with respect to the spectral wavelength within the target wavelength range. In a similar method, the use of chemical quantification will utilize the value of the absolute value of the difference between the minimum and maximum values of the absorption change (differential spectrum after reaction completion) within the target wavelength range, or the maximum amplitude of the second derivative of the differential spectrum with respect to the wavelength within the target wavelength range. In particular, when two substances contribute to the signal, simulations within the wavelength range of the second derivative are used.

[0021] - Spectral analysis includes providing a reference spectrum related to the protein under consideration and calculating the normalization coefficient between the reference spectrum and the current spectrum.

[0022] - The normalization coefficient is the ratio between the value of the second derivative of the protein under consideration and the value of the second derivative of the reference spectrum, where the value of the second derivative of the reference spectrum is defined as the maximum amplitude of the second derivative of the reference spectrum with respect to the wavelength within the target wavelength range or the absolute value of the difference between the minimum and maximum values of the second derivative of the second derivative of the reference spectrum with respect to the wavelength within the target wavelength range. In particular, when two substances contribute to the signal, simulations within the wavelength range of the second derivative are used.

[0023] - Satisfy at least one of the following properties:

[0024] - Perform spectral analysis on oxyhemoglobin and calculate a normalization coefficient for at least one wavelength selected from three intervals, where the first interval covers wavelengths including those between 415 nanometers and 417 nanometers, the second interval covers wavelengths including those between 542 nanometers and 544 nanometers, and the third interval covers wavelengths including those between 576 nanometers and 578 nanometers, and this interval is preferably the third interval;

[0025] - Perform spectral analysis on methemoglobin and calculate a normalization coefficient for at least one wavelength selected in the range of 350 nanometers to 750 nanometers, and

[0026] - Perform spectral analysis on bilirubin and calculate a normalization coefficient for at least one wavelength selected in the range of 350 nanometers to 750 nanometers.

[0027] - Perform spectral analysis on plasma heme and calculate a normalization coefficient for at least one wavelength selected in the range of 300 nanometers to 750 nanometers using the reference spectra of heme bound to serum albumin and heme bound to hemopexin.

[0028] - Chemically quantify from the group consisting of:

[0029] - Quantify oxyhemoglobin by adding CO,

[0030] - Quantify methemoglobin by adding KCN,

[0031] - Quantify plasma heme by adding CO and sodium dithionite,

[0032] - Quantify heme bound to hemopexin by adding DTN,

[0033] - Quantify total hemopexin by adding heme,

[0034] - Quantify total hemopexin by adding heme and sodium dithionite, and

[0035] - Quantify total hemopexin by adding heme, CO, and sodium dithionite. - In the output step, the determined contents include other protein contents, and these other contents are different from the contents of oxyhemoglobin, methemoglobin, heme bound to serum albumin and hemopexin, and bilirubin. The other contents are the contents of proteins selected from the group consisting of:

[0036] - Carboxyhemoglobin,

[0037] - Ferryl hemoglobin,

[0038] - Other heme target molecules,

[0039] - Myoglobin,

[0040] - Porphyrin,

[0041] - Products of the catabolism of heme by heme oxygenase,

[0042] - Products of bilirubin degradation and oxidation, such as biliverdin or heme box, stercobilin, urobillin,

[0043] - Products of organ dysfunction and cytosis,

[0044] - Metabolic substrates,

[0045] - Cofactors, and

[0046] - Metabolic degradation products.

[0047] - When determining the contents of oxyhemoglobin, methemoglobin, heme bound to serum albumin, and bilirubin in sequence, at least one of oxyhemoglobin, methemoglobin, heme bound to serum albumin, and bilirubin is determined by chemical quantification.

[0048] The specification describes a method for predicting the risk that a subject has a heme-related or heme protein-related disorder, and the prediction method includes at least the following steps:

[0049] - Performing a method step of determining the content of at least one protein in a biological sample of the subject to obtain a determined parameter, and the determination method is as described above, and

[0050] - Predicting the risk that the subject has a heme-related or heme protein-related disorder based on the determined parameter.

[0051] The specification also relates to a method for diagnosing a heme-related or heme protein-related disorder, and the diagnosis method includes at least the following steps:

[0052] - Performing a method step of determining the content of at least one protein in a biological sample of the subject to obtain a determined protein content, and the determination method is as described above, and

[0053] - Diagnosing a heme-related or heme protein-related disorder based on the determined protein content.

[0054] The specification describes a method for identifying therapeutic targets for preventing and / or treating heme-related or heme protein-related disorders, the method comprising the following steps:

[0055] - performing a method step of determining the content of at least one protein in a biological sample of a first subject to obtain a first determined protein content, the determination method being as described above, and the first subject being a subject suffering from a heme-related or heme protein-related disorder,

[0056] - performing a method step of determining the content of at least one protein in a biological sample of a second subject to obtain a second determined protein content, the determination method being as described above, and the second subject being a subject not suffering from a heme-related or heme protein-related disorder, and

[0057] - selecting a therapeutic target based on a comparison of the first and second determined protein contents.

[0058] The specification also relates to a method for defining the stage of a heme-related or heme protein-related disorder, the definition method comprising at least the following steps:

[0059] - performing a method step of determining the content of at least one protein in a biological sample of a subject to obtain a determined protein content, the determination method being as described above, and

[0060] - defining the stage of a heme-related or heme protein-related disorder based on the determined protein content and in combination with other biological parameters (a predictive algorithm for diagnosis).

[0061] The specification also relates to a method for screening compounds that can be used as drugs and that act on known therapeutic targets for preventing and / or treating heme-related or heme protein-related disorders, the method comprising the following steps:

[0062] - performing a method step of determining the content of at least one protein in a biological sample of a first subject to obtain a first determined protein content, the determination method being as described above, and the first subject being a subject suffering from a heme-related or heme protein-related disorder and having received the compound,

[0063] - performing a method step of determining the content of at least one protein in a biological sample of a second subject to obtain a second determined protein content, the determination method being as described above, and the second subject being a subject suffering from a heme-related or heme protein-related disorder and not having received the compound, and

[0064] - selecting a compound based on a comparison of the first and second determined protein contents.

[0065] The specification describes a method for qualifying or disqualifying a medical bag containing a biological sample of a subject, the method comprising:

[0066] - performing a method step for determining the content of at least one protein in the medical bag to obtain a determined protein content, the determination method being as described above, and

[0067] - qualifying or disqualifying the medical bag based on the determined protein content.

[0068] The specification also relates to a method for identifying a biomarker that is a diagnostic biomarker for a heme-related or heme protein-related disorder, a susceptibility biomarker for a heme-related or heme protein-related disorder, a prognostic biomarker for a heme-related or heme protein-related disorder, or a predictive biomarker in response to treatment of a heme-related or heme protein-related disorder, the method comprising the steps of:

[0069] - performing a method step for determining the content of at least one protein in a biological sample of a first subject to obtain a first determined content, the determination method being as described above, and the first subject being a subject suffering from a heme-related or heme protein-related disorder,

[0070] - performing a method step for determining the content of at least one protein in a biological sample of an image of a second subject to obtain a second determined protein content, the determination method being as described above, and the second subject being a subject not suffering from a heme-related or heme protein-related disorder, and

[0071] - selecting a biomarker based on a comparison of the first and second determined protein contents.

[0072] The specification also relates to a method for monitoring treatment of a heme-related or heme protein-related disorder in a subject, the subject suffering from a heme-related or heme protein-related disorder and having received the treatment, the method comprising:

[0073] - performing a method step for determining the content of at least one protein in a biological sample of the subject to obtain a determined protein content, the determination method being as described above, and

[0074] - monitoring the determined protein content so as to monitor treatment of a heme protein-related disease in the subject, the subject suffering from a heme-related or heme protein-related disorder and having received the treatment.

[0075] The description also describes a computer program product, which includes a computer-readable medium having a computer program including program instructions thereon. The computer program can be loaded into a data processing unit, and when the computer program is run by the data processing unit, the computer program is adapted to cause the execution of the steps of the method as described above.

[0076] The description also relates to a computer-readable medium encoded with the computer program as described above.

[0077] The description also relates to a device for determining the content of at least one protein in a biological sample, several of the proteins including oxyhemoglobin, methemoglobin, heme bound to serum albumin, heme-binding or free hemopexin (total concentration), and bilirubin. The device for determination at least includes a spectrophotometer, a quantification material, and an analysis system. The device is adapted to perform a method as described above. Description of the Drawings

[0078] The present invention will be better understood based on the following description, which is given corresponding to the drawings and as illustrative examples without limiting the purpose of the present invention. In the drawings:

[0079] - Figure 1 An example of a device adapted to determine the content of at least one protein in a biological sample is schematically shown;

[0080] - Figure 2 Examples of a system and a computer program product are shown, the system and the computer program product being part of the device and interacting such that calculations for device use can be performed to determine the content of one or more proteins in a sample.

[0081] - Figure 3 It is a flowchart of an example of a method for determining the content of at least one protein in a biological sample.

[0082] - Figure 4 It is an image showing the absorption spectra of various heme complexes.

[0083] - Figure 5 It is an image showing the different second derivatives of different heme complexes, and

[0084] - Figure 6 It is an image showing a calibration measurement based on hemopexin measurement. Detailed Description of the Invention

[0085] Description of the Device

[0086] In Figure 1 the device 10 is shown.

[0087] Device 10 is a device adapted to determine at least one content protein in a biological sample.

[0088] Device 10 includes at least one container 12, a spectrophotometer 14, a quantification material 16, and an analysis system 18.

[0089] At least one container 12 includes a biological sample 20 to be analyzed.

[0090] Biological sample 20 is a sample from a subject.

[0091] The subject is an animal, such as a mammal, like a mouse, a monkey, or a human.

[0092] As used herein and in all aspects of the present invention, the term "sample" means blood, fresh whole blood, peripheral blood, peripheral blood mononuclear cells (PBMC), serum, plasma, or urine.

[0093] Sample 20 includes several proteins, including among others oxyhemoglobin, methemoglobin, heme bound to serum albumin, heme-binding or free hemopexin, and bilirubin.

[0094] Oxyhemoglobin designates the reduced (Fe 2+ ) form of hemoglobin. In this reduced form, hemoglobin is the hemoglobin in which its reduced heme (Fe 2+ ) is bound to oxygen under air (deoxygenated and unbound at low O2 pressure).

[0095] Oxyhemoglobin encompasses oxyhemoglobin A (formula HbAO2), oxyhemoglobin F (formula HbFO2), oxyhemoglobin A2, glycated hemoglobin, and other widespread hemoglobin variants (S, E, C).

[0096] Based on their similar absorption properties between 300 nanometers (nm) and 900 nm, these oxyhemoglobins can be referred to as a single substance.

[0097] Although for healthy adults, about 90% of oxyhemoglobin A can be found, for infants, both oxyhemoglobin A and oxyhemoglobin F can be found.

[0098] Hereinafter, oxyhemoglobin is labeled as HbO2.

[0099] Methemoglobin is a form of hemoglobin in which the iron of the heme is in the oxidized state +3 (Fe 3+ ) and cannot ligate the diatomic ligand of Fe 2+ .

[0100] This means that ferric +3 does not bind to O2, CO, and very weakly to NO. However, this iron can bind strongly to the iron ligand CN - and N3 - and weakly to H2O and OH - .

[0101] Methemoglobin is called metHb. Similar to the ferrous-oxy state, no spectral differences were observed among metHbs caused by different human hemoglobin substances.

[0102] Under steady-state conditions in the presence of O2, the heme (hemin) bound to serum albumin is oxidized to the +3 oxidation state (Fe 3+ ).

[0103] The heme bound to serum albumin can participate in enzyme-catalyzed reactions.

[0104] The heme bound to serum albumin is called heme-SA.

[0105] Under steady-state conditions in the presence of O2, the heme (hemin) bound to hemopexin is oxidized to the +3 oxidation state (Fe 3+ ).

[0106] Due to the very strong six-coordination of iron His-Fe-His, the heme bound to hemopexin does not participate in enzyme-catalyzed reactions.

[0107] The heme bound to serum hemopexin is called heme-Hx.

[0108] It can be noted that, in contrast to metHb, the diatomic ligands of Fe 3+ can bind to heme-SA and heme-Hx only with low affinity values (>> mM).

[0109] Bilirubin is a degradation product of hemoglobin.

[0110] Bilirubin usually circulates in the blood bound to albumin.

[0111] Biological sample 20 is preferably a fluid.

[0112] Spectrophotometer 14 is adapted to measure the spectrum of a solution.

[0113] The term spectrum refers to the absorption spectrum.

[0114] The spectrum is obtained by passing electromagnetic waves through solution 20.

[0115] For example, the electromagnetic waves are generated by a light source.

[0116] The spectrum is the evolution of the absorption of the electromagnetic waves as a function of wavelength.

[0117] In particular, the absorption spectrum can be measured by measuring the transmittance of the solution at each wavelength.

[0118] In particular, a spectrum including wavelengths between 200 nanometers (nm) and 1000 nm, preferably between 300 nm and 700 nm, is obtained.

[0119] The quantification material 16 is a material capable of chemically analyzing at least one component of the sample 20.

[0120] According to Figure 1 an example of, the quantification material 16 includes a syringe 22 and a quantification container 24 with a pierceable stopper 26.

[0121] In Figure 1 it, only three syringes 22 and two quantification containers 24 are shown, but other quantities of syringes 22 and quantification containers 24 can be considered.

[0122] The syringe 22 can be used to pierce the stopper 26 and collect the product contained in the quantification container 24.

[0123] The content of the quantification container 24 is usually a solution of a given chemical material at a given concentration.

[0124] Referring to Figure 2 which further details the analysis system 18, in which the system 18 and the computer program product 28 are shown. The interaction between the computer program product 28 and the system 18 enables a method for determining at least one component to be carried out.

[0125] The system 18 is a computer. In the current case, the system 18 is a notebook computer.

[0126] More generally, the system 18 is a computer or a computing system, or a similar electronic computing device, adapted to manipulate and / or transform data represented as physical quantities such as electrical quantities in the registers and / or memories of the computing system into other data represented as similar physical quantities in the memories, registers or other such information storage, transmission or display devices of the computing system.

[0127] The system 18 includes a processor 30, a keyboard 32 and a display unit 34.

[0128] The processor 30 includes a data processing unit 36, a memory 38 and a reader 40. The reader 40 is adapted to read a computer-readable medium.

[0129] The computer program product 28 includes a computer-readable medium.

[0130] A computer-readable medium is a medium that can be read by reader 40 of processor 30. A computer-readable medium is a medium suitable for storing electronic instructions and capable of being coupled to a computer system bus.

[0131] Such computer-readable storage media are, for example, magnetic disks, floppy disks, optical disks, CD-ROMs, magneto-optical disks, read-only memory (ROM), random access memory (RAM), electrically programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), magnetic cards or optical cards, or any other type of medium suitable for storing electronic instructions and capable of being coupled to a computer system bus.

[0132] A computer program is stored in a computer-readable storage medium. The computer program includes one or more sequences of stored program instructions.

[0133] The computer program can be loaded into data processing unit 36, and when the computer program is run by data processing unit 36, the computer program is adapted to cause the execution of the calculation steps of a determination method.

[0134] Description of the determination method

[0135] Now, the operation of device 10 will be described by showing an example of the determination method as shown in the Figure 3 flowchart.

[0136] The method includes steps of providing a specified step a), setting a specified step b), iterating a specified step c), and outputting a specified step d).

[0137] In step a), an initial spectrum of sample 20 is provided.

[0138] This spectrum is obtained, for example, by using spectrophotometer 14.

[0139] The spectrum can be measured immediately after collecting sample 20, or after storing the sample under storage conditions.

[0140] For example, sample 20 can be stored at a temperature between 5 °C and 10 °C for several hours (up to 24 h).

[0141] Alternatively, sample 20 can be frozen and stored at a temperature between -80 °C and -20 °C. Freezing of the sample can limit the changes in the simply stored sample (frozen in liquid nitrogen before storage at -80 °C).

[0142] Sample 20 can also be stored with a stabilizer.

[0143] Sample 20 can also be fractionated, diluted, or concentrated. This enables improvement in the detectability of proteins in the sample.

[0144] After dilution, the optical density of sample 20 is less than or equal to 2, preferably less than or equal to 1.

[0145] Sample 20 can also be buffered at a pH less than or equal to 8.

[0146] Buffering is preferably carried out at a pH included between 7.35 and 7.45, and in particular equal to 7.4.

[0147] This is particularly advantageous because metHb depends on pH and has a pKa of 8.

[0148] This buffering is obtained by using a buffer solution.

[0149] For example, the buffer solution is a potassium phosphate solution or a sodium phosphate solution.

[0150] As a specific example, the buffer solution is a solution with a pH of 7.4 and consisting of 50 mM potassium phosphate and 50 mM NaCl.

[0151] The buffer solution can be accommodated in a dosing container 24.

[0152] Alternatively, in another embodiment, the initial spectrum is received by the analysis system 18, and the measurement of the spectrum was previously carried out at another location and in particular at the location where the biological sample 20 was collected.

[0153] At the end of step a), the initial spectrum of sample 20 is obtained.

[0154] In step b), the current spectrum is set to the initial spectrum.

[0155] Step b) enables the initialization for step c).

[0156] Step c) includes an iteration of three steps.

[0157] The first step is step c1), during which the protein content in the sample is determined.

[0158] The protein content refers to the amount of said protein in the sample.

[0159] The protein content is, for example, a mass content, a mass concentration or a molar content.

[0160] The mass content is the mass of the protein.

[0161] For example, the mass content can be expressed in picograms (pg).

[0162] The mass concentration is the ratio of the mass of the protein and the volume of the sample.

[0163] The mass concentration can be expressed in milligrams per liter (mg / L).

[0164] The molar content is the number of moles of protein.

[0165] The molar content is expressed in micromoles / L (μM).

[0166] The first step c1) is carried out by using spectral analysis of the current spectrum by the spectrophotometer 14 and / or chemical quantification of the quantification material 16.

[0167] The second step is step c2), in which the spectral components are associated with the protein based on the determined content.

[0168] Since the spectral components are estimated over a restricted region of wavelengths (less than 3 or the following examples which may be larger), highly specific to the protein or chemical molecule being quantified, its entire spectrum in the UV / visible region, for example, is derived from the purified substance spectrum (reference spectrum) weighted by the intensity ratio between the signal of the substance in the measurement window and its reference.

[0169] For example, the formula "spectral component = reference spectrum × DO sample / DO reference (a specific wavelength or wavelength region can be used or simply the formula [substance]) × reference spectrum" is normalized relative to 1 μM, for example.

[0170] The third step is step c3), in which the derived spectral components are removed from the current spectrum.

[0171] Step c3) consists of subtracting the derived spectral components from the current spectrum to obtain a new current spectrum.

[0172] As long as the contents of the four substances have not been determined, step c) is preferably iterated successively for different proteins by reducing the value of the second derivative.

[0173] The value of the second derivative of the protein is defined as: the maximum amplitude of the second derivative of the spectral component of the protein in the sample with respect to the spectral wavelength within the target wavelength range, or the absolute value of the difference between the minimum and maximum values of the second derivative of the spectral component of the protein in the sample with respect to the spectral wavelength within the target wavelength range.

[0174] For simplicity, only the first definition is used below.

[0175] This value can be measured or obtained by simulation.

[0176] The four substances are HbO2, metHb, bound heme (heme-SA or heme-Hx), and bilirubin.

[0177] It can be noted and considered that, if heme-SA and heme-Hx are considered as two substances, as long as the contents of the five substances are not determined, step c) is iterated.

[0178] Examples of such step c) are detailed in the next part.

[0179] In step d), the determined protein content is output.

[0180] This method is particularly suitable for analyzing various components of a sample such as a biological fluid.

[0181] This method particularly provides the contents of HbO2, metHb, heme-SA, heme-Hx, and bilirubin. As will be explained in other parts, other substances can be output in step d).

[0182] Detailed description of a specific implementation of step C)

[0183] In this case, sample 20, by reducing the order in the second derivative, includes: HbCO, HbO2, metHb, heme-SA, heme-Hx, and bilirubin. Such a specific sample 2 (CO poisoning or CO therapy) can be particularly observed in smokers.

[0184] The determination steps for each of these substances will be described below by providing several techniques for each substance.

[0185] The purpose of this analysis is to remove substances that are easier to define (higher amplitude, narrowest width, specific wavelength window, low or no interference from other spectral substances). Once a substance is determined, after signal normalization, its overall spectrum is subtracted from its pure reference spectrum. Since HbO2 and metHb are the substances that are easiest to identify, the process will start with their determination first.

[0186] Determine the HbCO content

[0187] No specific technique for determining the HbCO content is proposed.

[0188] For example, it can be considered as a spectral technique that uses the maximum absorption peak of HbCO in the initial spectrum (preferably at 420 nm).

[0189] This enables the determination of the HbCO content in sample 20.

[0190] Then this spectral content is removed from the initial spectrum.

[0191] Determine the HbO2 content

[0192] To determine the HbO2 content, two techniques are proposed: a spectral technique and a chemical technique.

[0193] Spectral analysis

[0194] According to this technique, a reference spectrum related to oxyhemoglobin is provided.

[0195] The reference spectrum has, for example, an information library.

[0196] The reference spectrum is obtained from an HbO2 sample, the HbO2 content of which is greater than or equal to 99%.

[0197] Such an HbO2 sample can be obtained from red blood cells of healthy non-smoking subjects. The red blood cells can be washed with physiological saline. The red blood cells are lysed after dilution with water (at least 3-fold H2O volume / RBC volume). The resulting solution is centrifuged at 10,000 - 15,000 g to recover the HbO2 sample in the supernatant. Trace amounts of CO can be removed by photodissociation by exposing the sample kept on ice to a light source under pure O2 and replacing CO with O2. If there are trace amounts of metHb after chemical quantification, they can be removed by several reduction systems (such as ferredoxin / ferredoxin reductase / NADPH) before removal by chromatography. Another option is to saturate the HbO2 sample with CO and then reduce metHb with a slightly excessive amount of sodium dithionite. After removing the dithionite with desalting chromatography, CO is replaced with O2 as described above.

[0198] Optionally, the HbO2 sample can also be purified by a chromatographic column adapted to exclude particles of a specific size and / or for ion exchange. The HbCO spectrum can be obtained by saturating the HbO2 sample with CO gas.

[0199] Then, a normalization coefficient K between the reference spectrum and the current spectrum is calculated HbO2 .

[0200] The normalization coefficient is the ratio of the amplitude of HbO2 in sample 20 and the amplitude of the reference spectrum of HbO2.

[0201] Many definitions can be used to determine this ratio.

[0202] For example, the normalization coefficient is defined as the minimum value of the ratio of the value of the second derivative of HbO2 and the value of the second derivative of the reference spectrum.

[0203] In particular, in this example, the normalization coefficient is defined as the ratio of the value of the second derivative of HbO2 and the value of the second derivative of the reference spectrum. The value of the second derivative of the reference spectrum is defined as the maximum amplitude (negative sign) of the second derivative with respect to the wavelength of the spectrum.

[0204] Mathematically, it can be written as:

[0205]

[0206] Wherein:

[0207] · is an operator that provides the second derivative of quantity A with respect to quantity B,

[0208] ·S C is the current spectrum,

[0209] ·S REF is the reference spectrum,

[0210] ·λ represents the value of the wavelength, and

[0211] · represents the maximum amplitude of A with respect to the wavelength (negative sign).

[0212] The normalization coefficient is calculated for at least one wavelength.

[0213] The wavelength is selected from the group consisting of 416 nanometers (nm), 543 nm, and 577 nm. Preferably, 577 nm is used because the signal is least affected by the spectral contributions of other substances.

[0214] In some embodiments, the normalization coefficient takes into account other coefficients.

[0215] For example, the normalization coefficient takes into account the dilution factor of the sample during its collection; for example, the presence of an anticoagulant.

[0216] As a specific implementation, based on the solute volume V S 、collecting volume V C and the hematocrit ratio Htc, the dilution factor FD is obtained. By way of illustration, the following formula can be used:

[0217]

[0218] In a variant or in addition, the normalization coefficient can also take into account the dilution factor of the plasma in PBS.

[0219] Typically, the sample is diluted into a buffer solution between 1 / 2 and 1 / 20. It depends on the signal amplitude, the wavelength path of the optical device, and the volume of the sample required. The aim is to minimize the amount of biological sample used for measurement and, if possible, set the signals of all substances between 0.1 and 1 OD over the entire analysis range.

[0220] The normalization coefficient also takes into account the fact that the substance under consideration may bind to other molecules.

[0221] For the case of HbO2, a fraction binds irreversibly to haptoglobin, meaning that the assessment of the HbO2 spectrum includes contributions from free HbO2 and from HbO2 bound to haptoglobin.

[0222] Haptoglobin can be measured by biochemical reaction with an antibody.

[0223] Free HbO2 is derived based on the fact that different haptoglobin isoforms on average have the binding capacity for two Hb dimers / haptoglobin. Knowing the concentration of haptoglobin in g / L and taking the average MW of approximately 85 kDa, the binding capacity of the heme-based haptoglobin pool in μM of HbO2 can be estimated, and subtracting this value from the total measured HbO2 results in a reasonable estimate of free HbO2. In the presence of HbCO and MetHb, the total hemoglobin concentration (HbCO + HbO2 + MetHb) can be used to calculate free HbO2.

[0224] The normalization coefficient K provides a way to obtain the HbO2 content.

[0225] For example, by using the Beer-Lambert law, the following equation can be obtained:

[0226]

[0227] Where:

[0228] · c is the concentration of HBO2,

[0229] · A is the absorbance of the reference sample,

[0230] · ξ is the extinction coefficient of HBO2, and

[0231] · l is the thickness of the sample.

[0232] A, ξ, and l are known or provided.

[0233] In particular, the extinction coefficient can be found in a table.

[0234] Thus, the above formula enables the determination of the concentration, which then provides a way to obtain the HbO2 content.

[0235] Chemical quantification technique

[0236] HbO2 can be quantified by adding CO.

[0237] Any method can be considered for tracking the reaction resulting from the addition of CO.

[0238] In the context of this example, spectral tracing can be advantageous as long as the device 10 is equipped with a spectrophotometer 14.

[0239] In the case of chemical quantification, the value can be generated by the maximum or minimum of the absorption change after a chemical reaction, or preferably from the absolute value of the difference between the minimum and the maximum. These latter values can also be generated by the second derivative of the absorption change after a chemical reaction.

[0240] Determine the metHb content

[0241] By replacing only oxyhemoglobin with methemoglobin, the similar explanations made previously for the spectral analysis of oxyhemoglobin apply to methemoglobin. These explanations will not be repeated below. Only the specific differences are highlighted.

[0242] The wavelengths used to calculate the normalization coefficient are selected in the range of 350 nm to 700 nm.

[0243] Methemoglobin is quantified by adding KCN.

[0244] Quantification can be achieved by using a KCN solution, the concentration of the anion CN - of which is included between 100 μM and 500 μM, preferably between 150 μM and 250 μM, and more preferably equal to 200 μM.

[0245] The incubation time is, for example, 5 minutes. In fact, at room temperature, the bimolecular rate of CN - binding to metHb is about 100 / M / s, which predicts that more than 99% binding will occur after a few minutes using 200 μM of KCN. The same applies if NaCN is used instead of KCN, especially in terms of concentration.

[0246] Since metHb also reacts with the anion N3 - NaN3 can be used instead of KCN to quantify metHb.

[0247] In this case, quantification can be achieved by using a NaN3 solution, the concentration of the anion N3 - of which is included between 500 μM and 1000 μM, preferably more preferably equal to 500 μM.

[0248] As previously mentioned, spectral techniques can be used to trace the quantification.

[0249] Determine the heme-SA content

[0250] To determine the heme-SA content, two techniques are proposed: spectral techniques and chemical techniques.

[0251] Spectral analysis of the current spectrum

[0252] By replacing HbO2 with hemin-SA only, the similar descriptions made previously for the spectral analysis of HbO2 apply to hemin-SA. These descriptions will not be repeated below. Only the specific differences are highlighted.

[0253] A reference spectrum is obtained for the hemin-SA sample with an excess of human albumin compared to hemin. After waiting for the binding reaction to complete, the spectrum of human albumin is measured before and after adding hemin. The hemin-SA spectrum is obtained after subtracting the initial free albumin spectrum.

[0254] For example, such a hemin-SA sample can be obtained as follows. Hemin can be dissolved in 0.1 N NaOH solution and stored in the dark, and then added to an AS solution filtered at 0.2 μM, with 1 mole of hemin for 5 moles of SA in the final solution. After equilibration for 20 minutes in the absence of light, the solution is obtained.

[0255] Optionally, the buffer solution can be as described in step a).

[0256] The wavelengths used to calculate the normalization coefficient are selected in the range of 200 nm to 800 nm.

[0257] Preferably, this range can include between 300 nm and 700 nm, between 350 and 460 nm, and / or between 600 and 700 nm. In the red part of the spectrum, a first estimate can be made to limit other analyses in the purple part. When hemin-SA is concentrated, the second derivative can give an estimate of its concentration. At lower concentrations, the signal can be simulated by subtracting a certain amount of the reference spectrum to obtain the best fit of a straight line through two points selected between 610 and 600 nm and between 675 and 700 nm respectively; the latter point is restricted to belong to the measured spectrum.

[0258] Chemical quantification technique

[0259] Hemin-SA can be quantified by adding sodium dithionite (DTN) under CO. A reference spectrum of the absorption change caused by DTN reduction of CO: hemin (Fe 3+ )-SA -> hemin (Fe 2+ -CO)-SA is obtained from the blood of patients whose Hx has been depleted of plasma hemin. Due to the plasma biochemical conditions and the state of the binding interaction, this spectrum is slightly different from the spectrum obtained with purified human SA in PBS.

[0260] Determine the heme-Hx content

[0261] Regarding hemin-SA, two techniques can be used to determine the content in hemin-Hx: with or without chemical addition. This also depends on the amount of hemin present in the biological fluid and the binding competition between AS and Hx. In addition to hemin-based therapies, the large amount of natural hemin in plasma is related to the low amount of hemin-Hx after hemin degradation and removal from the circulation. Only hemin-SA is retained because the Hx concentration is about 50 times lower than the AS concentration and the Hx conversion efficiency is insufficient. Then hemin competition for the binding between SA and Hx is only observed at low hemin concentrations. In other words, if the hemin-SA component cannot be removed after separate determination, the in vivo detection of hemin-Hx is related to the concomitant detection of hemin-SA. Therefore, determining hemin-SA at low hemin concentrations may involve similar operations. The determination of both substances requires the use of a linear combination of their respective second derivative spectra, preferably simulated between 350 and 460 nm.

[0262] Spectral analysis

[0263] By replacing only HbO2 with hemin-Hx, the similar explanations made previously for the spectral analysis of HbO2 apply to hemin-Hx. These explanations will not be repeated below. Only the specific differences are highlighted.

[0264] Depending on the technique used, the wavelengths for calculating the normalization coefficient are selected in the range of 300 nm to 700 nm.

[0265] By using a slightly excessive (20%) human hemopexin compared to the hemin in the hemin-Hx sample, a reference spectrum is obtained in a similar method developed for hemin-SA.

[0266] Chemical quantification technique

[0267] Hemin-Hx can be quantified by adding sodium dithionite (DTN) under CO. As described above, the reference spectrum of the absorption change caused by the reduction of CO: hemin (Fe 3+ )-Hx -> hemin (Fe 2+ -CO)-Hx is obtained with purified human Hx in PBS. Hemin-Hx in plasma is determined by simulating the absorption change after reduction with both the hemin-Hx and hemin-SA reference spectra after subtracting the contribution of the reduction of metHb estimated by chemical quantification.

[0268] In a variant, heme-Hx can be quantified by adding dithionite (DTN). This quantification is a powerful tool because, compared to other typical quantifications, it does not require a calibration curve and is based on the binding capacity of the plasma Hx pool. The reduction by DTN in the absence of reagent-consuming oxygen was again obtained with purified human Hx in PBS: the reference spectrum of the absorption change caused by heme(Fe 3+ )-Hx -> heme(Fe 2+ )-Hx. Before adding DTN, the heme(Fe 3+ )-Hx sample was deoxygenated under N2 or Ar (catalase was added to prevent oxidative side reactions). Only this technique can directly measure the heme-Hx content between 520 and 600 nm. In fact, in the deoxygenated state, Hx exhibits two strong and narrow absorption peaks, which can be detected using second-order (2 nd ) derivatives, while other deoxygenated substances (HbFe 2+ , heme Fe 2+ -SA, bilirubin) show broad peaks. The heme-Hx was estimated at the maximum amplitude of the second derivative of plasma supplemented with heme at 561 nm, or preferably using the absolute value of the difference between the maximum (550 - 551 nm) and minimum (561 nm) values of this second derivative. Note that the small contributions of other substances to the second derivative spectrum at the two wavelengths are equal in amplitude and sign, so they do not affect the measurement of heme-Hx after subtraction. Finally, a global simulation can be used between 540 and 575 nm.

[0269] According to another example, the final total Hx content can be quantified by adding heme to a biological sample. After heme binding, before measuring Hx, at a pH between 6.5 and 7.4, preferably pH 7.0, a slightly excessive amount of heme relative to the upper standard limit of its biological concentration was added and incubated at room temperature for about 30 min. In this way, this quantification can determine the Hx content in biological fluids in molar concentration (heme binding capacity). Typically, after setting the pH by dilution with buffer (i.e., 1 / 2 dilution), 20 μM heme was added to plasma for Hx quantification. When the heme concentration in plasma increases, it may not be necessary to add heme because Hx is apparently already saturated. Knowing that Hx has only one heme binding site, the Hx concentration was converted to g / L using an average molecular weight of 60 kDa.

[0270] Interestingly, since there is a negative correlation between plasma heme and Hx without upregulation (e.g., in certain inflammatory conditions), measurement of Hx yields an indirect status of heme concentration and vice versa. This means that abnormally high concentrations of Hx and low amounts of heme may indicate specific physiopathological symptoms. Low Hx concentrations may be due to intravascular hemolysis accompanied by Hb degradation and / or abnormal erythropoiesis in the bone marrow. Downregulation is also possible in severe cases of iron overload.

[0271] Determine the bilirubin content

[0272] For the determination of bilirubin content, only spectroscopic analysis has been proposed.

[0273] By replacing only HbO2 with bilirubin, the similar explanations made previously for spectroscopic analysis of HbO2 apply to bilirubin. These explanations will not be repeated below. Only the specific differences are highlighted.

[0274] The reference spectrum is obtained from a bilirubin sample having a bilirubin content of about 10 μM.

[0275] Such a bilirubin sample is a sample of bilirubin with an excess of serum albumin (SA).

[0276] For example, such a bilirubin sample can be obtained as follows. Bilirubin can be dissolved in 0.1 N NaOH solution and stored in the dark, then added to an AS solution filtered at 0.2 μM, and the final solution has 1 mole of bilirubin for 2 moles of SA. After equilibration for 20 minutes in the absence of light, the solution is obtained. Another option is to use plasma samples from blood donors without chronic pathologies having about 50 μM bilirubin (bilirubin in different proportions conjugated or not conjugated with glucuronic acid can be used). The bilirubin spectrum is obtained after subtracting the remaining HbO2 component (the absence of other Hbs or degradation products should be evaluated).

[0277] As described in step a), the solution is preferably buffered in PBS at pH 7.4.

[0278] The wavelengths for calculating the normalization coefficient are selected in the range of 350 nm to 700 nm.

[0279] Preferably, the wavelengths for calculating the normalization coefficient are selected in the range of 440 nm to 540 nm. More preferably, the wavelength used is about 501 nm.

[0280] Illustrate

[0281] Figures 4 to 6 Some steps for the previous determination of content in a specific case of determining hemopexin content are shown diagrammatically.

[0282] Figure 4 shows the spectra of different substances reduced in the absence of O2. HxFe 2+ 's spectrum will be very dominant compared to the other spectra in the second derivative because it shows two very thin bands.

[0283] Figure 5 Shows a method of circumventing such problems of hemopexin. It represents the analysis of the second derivative at 561 nm (first dashed arrow), or the analysis of the second derivative by taking the difference between the maximum value (refer to the second dashed arrow at about 550 - 551 nm) and the minimum value at 561 nm. This makes it possible to eliminate the weak contributions of other substances, which vary little between 550 - 551 nm and 561 nm. Either of these two analyses can be used.

[0284] Figure 6 Shows a calibration graph of the measurement using heme. The precision can be as low as 0.1 g / L and even lower than 0.05 g / L.

[0285] Other elements

[0286] The methods previously described can be extended to many other substances.

[0287] It is worth mentioning that:

[0288] - Carboxyhemoglobin (HbCO),

[0289] - Methemoglobin,

[0290] - Plasma heme, which can be quantified particularly by adding CO and / or sodium dithionite. In the presence of sodium dithionite, the iron atom of heme Fe 3+ is reduced and then binds to CO in the absence of O2 (O2 reacts with sodium dithionite). This makes spectral determination possible,

[0291] - Myoglobin,

[0292] - Porphyrin,

[0293] - Porphobilinogen,

[0294] - Urobilin,

[0295] - Products of the catabolism of heme by heme oxygenase,

[0296] - Bilirubin degradation / oxidation products: biliverdin, heme box,

[0297] - Products of organ dysfunction and cytolysis (coenzyme molecules, heme proteins, cytochrome P450, transaminases), and

[0298] -Metabolic substrates, metabolic degradation products.

[0299] Alternative chemical methods for determining Hb, heme, and Hb degradation products in plasma and other fluid / biological samples

[0300] A similar formalism is used. A "reference spectrum" or "SR" is an absorbance spectrum obtained for a single protein. A reference spectrum is typically obtained prior to practicing the methods of the invention. A differential reference spectrum "SDR" corresponds to the absorbance changes associated with a chemical reaction of a given substance.

[0301] The "calculated spectrum" or "calculated intermediate spectrum" is the spectrum obtained after subtracting from the measured spectrum substance SA its reference spectrum weighted by the "K" normalization factor (the ratio between the signal amplitude of the determined substance and its reference).

[0302] The method is based on the reduction of plasma Fe by sodium dithionite in the presence of CO. 3+ . and only used in CN - Compared to the previous method of the reaction in the presence of , it involves the addition of two additional chemical reactions. For a previously equilibrated sample, the reduction of Fe in the presence of sodium dithionite under CO in the absence of O (which is consumed by dithionite) 3+ The first step is to replace the O2 in the air with CO (CO has an affinity 200 times higher than O2) to reduce the iron atom in the hemoglobin plasma. 2+ Once the bond is formed, Fe 2+ -CO, which is considered irreversible, does not change the chemical properties of iron after adding the chemical reagents required for the determination, and therefore there is no change in the spectral components associated with it. After adding CO, only Fe 3+ The substances remain sensitive to the quantitative methods.

[0303] The sequence of chemical reactions is as follows:

[0304] 1) Add KCN after recording plasma spectra SA as described previously

[0305] 2) Add gaseous CO

[0306] 3) Add sodium dithionite.

[0307] However, it should be noted that steps 1) and 2) can be reversed without changing the final result of the analysis.

[0308] The different steps of the measurement protocol are as follows:

[0309] 1) SA spectra of plasma at pH 7.4 + / - 0.1 buffered air. Dilution factors will be taken into account when calculating heminised material.

[0310] 2) SA after adding CO CO Plasma spectrum: from the spectral changes caused by CO replacing O2 binding to Fe 2+ (SA - SA CO ) Chemically quantify the HbFe that is bound to O2, free and / or complexed with haptoglobin in air (this proposition was removed because the calculation of free Hb gives more haptoglobin). 2+ .

[0311] After adding CO gas or PBS saturated with a known volume of CO, the concentration of CO is between 100 μM and 1 mM (the solubility of gaseous CO at 25 °C is approximately 1 mM at 1 atm). The incubation time is about 1 minute.

[0312] 3) SA of plasma in the presence of CO after adding KCN CO+KCN Spectrum: from the spectral changes caused by the binding of CN - to Fe 3+ (SA CO - SA CO+KCN ) Chemically determine the Fe 3+ globin (formerly called metHb). Other chemical compounds can also be considered for the quantification of NaCN or even NaN3 or KN3 (N3 - is a ligand of Fe 3+ ). The concentration of the cyanide salt is about 0.2 μM, and the concentration of the azide salt is between 0.5 and 1 mM. The incubation time is between 3 and 5 minutes. It should be noted that the affinity of heme for binding to HSA and Hx is too weak to give a binding signal of the iron ligand in this concentration range.

[0313] 4) SA of plasma in the presence of CO and KCN after adding sodium dithionite CO+KCN+DTN Plasma spectrum: from the spectral changes caused by the reduction of Fe 3+ (SA CO+KCN - SA CO+KCN+DTN ) Chemically determine the serum Fe 3+ bound to human albumin (HSA) and serum hemopexin (Hx) in plasma heme. Once the reduced Fe 2+ binds to the CO present in the solution. This signal will consist of several spectral transitions, namely:

[0314] · HSA - heme (Fe 3+ ) -> HSA - heme (Fe 2+ ) - CO,

[0315] · Hx - heme (Fe 3+ ) -> Hx - heme (Fe2+ )-CO and

[0316] metHb(Fe 3+ )-CN - ->Hb(Fe 2+ )-CO, which will be calculated from the previous determination (3) and the reference spectra of the substances involved.

[0317] If metHb quantification is carried out on other samples separately without - CN or - N3, the spectral transition will be metHb(Fe 3+ )->Hb(Fe 2+ )-CO. All quantifications will preferably be carried out at physiological pH 7.4 + / - 0.1 and for the reference spectra. After adding dithionite at a concentration of about 0.5 - 1 mM, the incubation time is about 20 - 30 min. Finally, the signal calculated based on the spectral changes caused by the addition of dithionite (where the contribution of metHb reduction to the signal under CO is subtracted) will be analyzed as a linear combination of the reference spectra to understand the spectral contributions of HSA - heme (Fe 3+ ) and Hx - heme (Fe 3+ ) after reduction in the presence of CO. The total plasma heme will be calculated as the sum of the concentrations of HSA - heme (Fe 3+ ) and Hx - heme (Fe 3+ ). Only HSA - related heme may interact with other molecular targets or dissociate from tissues. Since certain anticoagulants (heparin or EDTA) increase the pH of plasma and may change the heme distribution among scavenger substances, plasma under citrate or simply serum should give a closer state of heme fate (total heme calculation remains the same). Regarding heme - SA, the determination of heme - Hx is based on the pure spectral analysis developed above, or after chemical quantification developed in this section. The concentration of heme - Hx will be inversely proportional to the total plasma heme due to the low conversion rate of Hx after hepatic endocytosis in its heme - complexed form.

[0318] The differential reference spectrum of Hx after CO reduction at pH 7.4 + / - 0.1 is obtained from the lyophilized protein (Sigma Aldrich). The Hx / heme ratio is about 1.2, i.e., protein excess in the presence of catalase and superoxide dismutase. Then the Hx - heme spectrum (Fe 3+ ) is recorded. After the sample is deoxygenated and equilibrated under CO, after adding iron is reduced in the presence of about 200 μM sodium dithionite a few minutes after addition, finally the Hx - heme (Fe 2+)-CO spectrum. Normalization is based on the extinction coefficient of Hx - heme (Fe 3+ ) at 413 - 414 nm and should be equivalent to the value obtained by calculating the dilution of the heme stock in NaOH in the measurement cell; the concentration of the heme stock is estimated by weighing or diluting in HSA solution (HSA ratio / heme > 2) based on the heme - HAS extinction coefficient (and second derivative) at 403 - 404 nm.

[0319] A differential reference spectrum of HSA after reduction under CO (incubated for 20 - 30 min) is obtained from blood of sickle - cell patients with excess heme (about 10 μM). Since Hx depletion is almost complete (confirmed by quantitative techniques), the spectral component of plasma heme after subtracting the low metHb fraction comes from HSA - heme (Fe 3+ ). In the ultraviolet light between 350 nm and 460 nm, after diluting the plasma in PBS at pH 7.4 + / - 0.1, a differential reference spectrum is obtained using the second derivative normalized from HSA - heme (Fe 3+ ). Due to the biochemical conditions and the conformation of the binding sites in plasma (bilirubin present), the differential reference spectrum of plasma heme - HSA after reduction under CO is slightly different from the differential reference spectrum obtained from human albumin purified in PBS. At high [heme] (heme - Hx depletion), only the differential spectrum from HSA can be used to simulate the signal in the ultraviolet light or / and pre - visible light.

[0320] When measurable (depending on the signal - to - noise ratio), the second - derivative representation of the differential spectrum can also be used to estimate the heme binding between HSA and Hx between 350 and 450 nm. Note that the normalized maximum amplitudes of the second derivatives of heme - HSA and heme - Hx (negative sign between 422 - 424 nm) are close, such that their average can be used to directly estimate the total plasma heme concentration with an accuracy of about + / - 5% (using the differential spectrum, the accuracy is about + / - 10%).

[0321] At high [heme] (heme - Hx depletion), the second derivative of only the differential spectrum from HSA can be used to simulate the signal preferably between 520 and 620 nm in the ultraviolet light or / and in the visible light.

[0322] At this analysis stage, the contributions of the following various substances determined chemically: HbO2, MetHb, HSA - heme, Hx - heme (reference spectrum obtained in air) can be subtracted from the initial spectrum of plasma SA, so that other spectral substances such as total bilirubin (free and conjugated) can be analyzed after signal processing.

[0323] The analytical equations for different assays are as follows (the normalization coefficients related to different quantifications are in bold)

[0324] 1) Determine the HbFe initially bound to O2 by adding CO 2+ :

[0325] SA - SA CO = K CO x SDR CO (SDR CO = SR HbO2 - SR HbCO )

[0326] 2) Quantify MetHbFe by adding KCN 3+ :

[0327] SA CO - SA CO+KCN = K KCN x SDR KCN (SDR KCN = SR MetHb - SR MetHb-CN )

[0328] 3) Perform HSA - and Hx - related plasma heme assays by adding DTN:

[0329] ·SA CO+kcn+DTN - SA CO+kcn = K HSA-血红素 x SDR HSA-血红素Fe2+CO / HSA-血红素Fe3+ + K Hx-血红素 x SDR Hx -血红素Fe2+CO / Hx-血红素Fe3+ - K KCN x SDR HbCO / metHbCN (K KCN is calculated as described above)

[0330] where:

[0331] ·SDR HbCO / metHbCN = SR HbCO - SR MetHb-CN

[0332] ·SDR HSA-血红素Fe2+CO / HSA-血红素Fe3+ = SR HSA-血红素Fe2+CO - SR HSA-血红素Fe3+

[0333] ·SDR Hx-血红素Fe2+CO / Hx-血红素Fe3+ = SR Hx-血红素Fe2+CO - SR Hx-血红素Fe3+

[0334] 4) Perform HSA - and Hx - related plasma heme assays by adding DTN:

[0335] SC CO+kcn+DTN = SA - K CO x SR HbO2 -K KCN x SR MetHb -K HSA-血红素 x

[0336] SR HSA-血红素Fe3+ -K Hx-血红素 x SR Hx-血红素Fe3+

[0337] The SC spectrum will be analyzed to determine other spectral substances such as HbCO and bilirubin.

[0338] Data analysis can involve mathematical processing using a representation of the second derivative to amplify the best - resolved absorption peaks; this is the case for CO - ligated substances (which have a very strong absorption band and narrow width at 420 nm) in this chemical assay. The analysis field covers the entire spectrum of white light and near - UV between 300 and 700 nm, more particularly between 350 nm and 450 nm, where the absorption bands are strongest for heme - containing substances. This method can also be used for the determination of heme / Hb in other body fluids, cells, and tissue extracts from biopsies in humans. Its field of application can be extended to quantification in animals and for cell culture.

[0339] Quantification of heme uses, for example, PBS buffer at pH 7.4 preferably containing human HSA or HAS from animal sources between 100 and 500 μM (the reference spectrum in the presence of heme will be measured and normalized).

[0340] Method for determining hemoproteins by UV / visible spectroscopy

[0341] These methods are based on the same analytical and mathematical methods developed previously. They are based on the addition of Hx substrate, in this case, heme in the plasma to be assayed or any other biological medium. Quantification in plasma will be developed herein.

[0342] The first method is to measure the difference spectrum after the addition of heme. The absorption spectra of the assay medium (SA 基准 ) will be measured separately and in the presence of an excess of heme. Then the dilution factor will be taken into account to calculate the difference spectrum SA 血红素 - SA 基准, to analyze the signals generated by the binding of heme to serum proteins HSA and Hx. The second derivative of the signal will be modeled as a linear combination of the second derivatives of two main protein complexes at λ belonging to [350 nm, 700 nm], preferably at λ belonging to [350 nm, 460 nm]. The high intensity of the second derivative of the Hx-heme spectrum compared to that of HSA-heme makes this method very sensitive for the determination of Hx, even exceeding the binding of heme to HSA.

[0343] However, since the absorbance of the medium to be analyzed is subtracted, the medium can have a heme-binding moiety of heme in a fixed state, especially for plasma with low intravascular hemolysis (Hx is depleted in the case of chronic moderate hemolysis). It is desirable to pre-measure the heme-Hx ratio before adding heme by the above method in order to calculate the total amount of heme-Hx before and after adding heme.

[0344] The second method consists of measuring the plasma spectrum in the same way with an excess of heme, but after adding sodium dithionite to reduce heme and obtain the Hx(Fe 2+ )-heme spectrum. After adding heme to lyophilized Hx and adding dithionite after deoxygenation under N2 (adding SOD and catalase), the Hx(Fe 2+ )-heme spectrum is obtained as described above. In fact, this substance is related to a spectrum with two absorption peaks between 500 nm and 600 nm, one of which is thin and strong at 560 nm. As a result, the second derivative of the plasma sample after adding sodium dithionite is very sensitive to the presence of Hx(Fe 2+ )-heme, while the spectral contributions of other substances present, l'HbFe 2+ , l'HSA(Fe 2+ )-heme and bilirubin in this wavelength range will generally be negligible (broad spectrum and lower intensity). However, in the case of low amounts of Hx, other components quantitatively measured from Hb and bilirubin and the HSA-heme component based on the known amount of heme added can be removed from the signal after adding dithionite in order to more easily measure the Hx content. The latter method relies only on obtaining a single SA 血红素+连二亚硫酸盐 . This analysis is developed in more detail below. Finally, using the same method to determine the Hx(Fe 2+ )-heme substance, the quantification of total plasma heme can be achieved by: adding purified free Hx after diluting the plasma under the same buffer conditions described below to replace the heme-binding steady state interacting with serum components, the strongest scavenger for Hx.

[0345] Both plasma haptoglobin quantification methods rely on the addition of heme to saturate the Hx to be quantified. The incubation pH is an important parameter to be controlled. The incubation pH is between 6.5 and 7.4, especially equal to 7.0, to reduce the affinity of heme for HSA and thus promote Hx binding. Unlike citrate which is closer to physiological pH or serum, the pH of plasma with anticoagulants heparin and EDTA is greater than 7.4. For samples with pH > 7.4, the sample will be diluted 1 / 2 in 50 mM PBS 50 mM phosphate NaCl pH 6.0, and for samples with pH around 7.4, the sample will be diluted in the same pH 6.5 buffer. The final pH of the incubation is close to 7.0. It should be noted that other buffers can be used to buffer the sample at a pH of 7.0. Then heme is gradually added with stirring to obtain a final concentration of approximately 20 μM from a stock solution of 1 mM heme in 0.1 N NaOH prepared on the day of measurement (e.g., Sigma Aldrich). If, for a pathology, the concentration of haptoglobin is abnormally high (>1.2 g / L), the excess of heme may increase. After dilution for more than 20 min, the concentration of the stock is determined by weighing and possibly controlled by UV / visible spectrophotometry after dilution in PBS buffer at pH 7.4 containing HSA.

[0346] The incubation of the plasma sample in the presence of heme lasts for 30 minutes, and then the sample is diluted 1 / 3 in PBS at pH 7.4 for the final measurement, i.e., SA for the first method 血红素 the spectrum and / or SA for the second chemical method after the addition of dithionite 血红素+连二亚硫酸盐 ; thus, the same sample can be used for both types of Hx determination. If there is a significant difference in the presence of Hx ≥ 0.05 g / L measured by the chemical method in the presence of dithionite, it indicates the presence in plasma after removal of Hx-associated plasma heme. It should be noted that plasma aliquots can be diluted in the same way without the addition of heme to measure the reference spectrum (SA 基准 ) of other biomarkers used in the first quantification method and then used to characterize hemolysis.

[0347] Prepare a sodium dithionite solution in a sealed tube that has low permeability to gas pre-deoxygenated with nitrogen. Then dissolve the reagent in PBS at pH 7.4, deoxygenate under N2, and optionally it can be used for 24 hours at a concentration of approximately 0.1 M after degassing. In the absence of nitrogen, dithionite can be prepared with degassed or non-degassed buffer, however, in this case, the tube containing dithionite must be completely filled with buffer to remove the entire gas phase containing the active substances: O2 and dithionite (remove dithionite without stirring the solution to limit the diffusion of any remaining gas phase into the liquid phase). It is also preferred to deoxygenate the plasma + heme sample or remove the gas phase in the measurement cell before adding dithionite to a final concentration preferably of 0.5 mM. Similarly, it is important to avoid vigorously equilibrating the plasma in the presence of a gas phase containing O2 after adding dithionite, as dithionite is consumed by reacting with O2 contained in the gas phase. After adding dithionite, it is preferred to measure the spectrum without delay within one minute. Relative to the reference spectrum Hx(Fe 2+ )-heme obtained from purified Hx and heme, take the difference between its minimum value at 561 nm or preferably between the minimum value at 561 nm and the maximum value at 551 nm, and measure the concentration of Hx using second derivative by simulating the second derivative between 540 nm and 580 nm.

[0348] The main advantage of these methods is that the analysis is based on a reference spectrum. Therefore, it is not necessary to make a calibration line (unlike the Elisa test). These quantitative methods measure the binding ability of plasma Hx to heme, and compared with the turbidimetric method after precipitation with an antibody, these methods are not affected by changes in Hx glycosylation or the presence of molecular aggregates. The final result of the quantitative method is expressed as μM of Hx and can be converted to g / L of Hx, taking the average molecular weight as 60 kD.

[0349] Applications

[0350] Many applications of this determination method can be considered. Some of them will be developed in this section.

[0351] Applications related to heme-related or heme protein-related disorders are particularly developed, and in particular applications related to diseases that at least as a symptom involve heme-related or heme protein-related disorders. Heme-related or heme protein-related disorders particularly cover hemoglobin-related diseases or RBC diseases. As another specific example, heme-related or heme protein-related disorders particularly cover disorders of intravascular hemolysis.

[0352] However, heme-related or heme protein-related disorders do not cover all blood-related diseases. It is noteworthy that cerebral aneurysms such as subarachnoid hemorrhage are not heme-related or heme protein-related disorders.

[0353] When relevant, these applications are in vitro applications.

[0354] Prediction method

[0355] For this application, a method for predicting the risk that a subject has a heme-related or heme protein-related disorder is proposed.

[0356] The method for prediction includes steps for performing the following method steps: a method for determining the content of at least one protein in a biological sample of a subject or its metabolite / degradation product (such as the natural pigment in the following other applications) to obtain a determined parameter.

[0357] The prediction method further includes a step of predicting the risk that a subject has a heme-related or heme protein-related disorder based on the determined parameter.

[0358] Diagnostic method

[0359] This application corresponds to a method for diagnosing heme-related or heme protein-related disorders.

[0360] The diagnostic method includes steps for performing the following method: a method for determining the content of at least one protein in a biological sample of a subject to obtain a determined protein content.

[0361] The diagnostic method further includes a step of diagnosing a heme-related or heme protein-related disorder based on the determined protein content.

[0362] Therapeutic method

[0363] This application corresponds to a method for treating heme-related or heme protein-related disorders (tracking).

[0364] The treatment method includes steps for performing the following method: a method for determining the content of at least one protein in a biological sample of a subject to obtain a determined protein content (i.e., a steady-state comparison of at least one content molecule before and after treatment).

[0365] The treatment method further includes administering a drug for treating a heme-related or heme protein-related disorder (bone marrow transplantation, gene therapy) based on the determined protein content.

[0366] Method in the definition phase

[0367] In the present application, a method for defining the stage of a heme-related or heme protein-related disorder is proposed.

[0368] The stage of a disease is the different levels of the disease. The stage is usually defined based on the symptoms of the disease.

[0369] The defining method includes performing the steps of the following method: a method for determining the content of at least one protein in a biological sample of a subject to obtain a determined protein content.

[0370] Then, the defining method includes defining the stage of a heme-related or heme protein-related disorder based on the determined protein content and in combination with other biological parameters (a predictive algorithm for diagnosis).

[0371] Method for identifying a target

[0372] The present application corresponds to a method for identifying a therapeutic target for preventing and / or treating a heme-related or heme protein-related disorder.

[0373] The identifying method includes performing the steps of the following method: a method for determining the content of at least one protein in a biological sample of an image of a first subject, where the first subject is a subject suffering from a heme-related or heme protein-related disorder.

[0374] The identifying method further includes performing the steps of the following method: a method for determining the content of at least one protein in a biological sample of a second subject to obtain a second determined protein content, where the second subject is a subject not suffering from a heme-related or heme protein-related disorder.

[0375] The identifying method further includes the step of selecting a therapeutic target based on a comparison of the first determined protein content and the second determined protein content.

[0376] Method for identifying a biomarker

[0377] In the present application, a method for identifying a biomarker is proposed.

[0378] A biomarker can be one of a diagnostic biomarker for a heme-related or heme protein-related disorder, a susceptibility biomarker for a heme-related or heme protein-related disorder, a prognostic biomarker for a heme-related or heme protein-related disorder, or a predictive biomarker in response to treatment of a heme-related or heme protein-related disorder.

[0379] The identification method includes performing the steps of the following method: a method for determining the content of at least one protein in a biological sample of a first subject to obtain a first determined content, where the first subject is a subject suffering from a heme-related or heme protein-related disorder.

[0380] The identification method includes performing the steps of the following method: a method for determining the content of at least one protein in a biological sample of an image of a second subject to obtain a second determined protein content, where the second subject is a subject not suffering from a heme-related or heme protein-related disorder.

[0381] The identification method includes selecting a biomarker based on a comparison of the first determined protein content and the second determined protein content.

[0382] Method for screening compounds

[0383] This application corresponds to a method for screening compounds.

[0384] The compound is a drug.

[0385] The drug acts on a known therapeutic target for preventing and / or treating a heme-related or heme protein-related disorder.

[0386] The screening method includes performing the steps of the following method: a method for determining the content of at least one protein in a biological sample of a first subject to obtain a first determined protein content, where the first subject is a subject suffering from a heme-related or heme protein-related disorder and has received the compound.

[0387] In this context, the term "received" encompasses any mode of administration of the drug.

[0388] The screening method further includes performing the steps of the following method: a method for determining the content of at least one protein in a biological sample of a second subject to obtain a second determined protein content, where the second subject is a subject suffering from a heme-related or heme protein-related disorder and has not received the compound.

[0389] The screening method further includes selecting a compound based on a comparison of the first determined protein content and the second determined protein content.

[0390] Method for qualifying or disqualifying

[0391] This application corresponds to a method for qualifying and disqualifying blood bags. Blood is a general term encompassing red blood cells and plasma.

[0392] A bag is a container.

[0393] The bag contains a biological sample of the subject.

[0394] The method of qualifying or disqualifying includes performing the steps of the following method: a method for determining at least one protein content in a medical bag to obtain a determined protein content.

[0395] The method of qualifying or disqualifying further includes qualifying or disqualifying the medical bag based on the determined protein content.

[0396] For example, if the protein content indicates that the quality of the sample has deteriorated to the extent that the sample can no longer be used, the bag is disqualified.

[0397] The idea of this particular application is to analyze the hemolysis content in the preservation medium, but also to collect red blood cells if there is any doubt and incubate them in isotonic pb at pH 7.4 in the presence of 100 - 500 μM albumin (in vivo) (the pH of the bag decreases with the preservation time and is usually between 6 and 7) to recover all substances that can interact with the membrane and also to measure the most vulnerable components that would reduce the transfusion volume. For plasma bags, the presence of abnormal hemolysis content is detected based on heme and its derived substances, and the total Hx concentration that can be used as a therapeutic agent for treating patients with persistent hemolysis by plasma exchange is also estimated. (Haptoglobin can be measured by adding Hb before deoxygenation and reducing it with dithionite in the presence of catalase; indeed, the spectra of the deoxy-dimer and deoxy-Hb tetramer bound to haptoglobin are different, so adding Hb equivalent to the upper limit of the normal haptoglobin range enables the estimation of the protein using the same method developed in this document). Thus, this application is equivalent to the quality control of therapeutic applications.

[0398] Method for monitoring treatment

[0399] This application corresponds to a method for monitoring the treatment of heme-related or heme-protein-related disorders in a subject, where the subject has a heme-related or heme-protein-related disorder and has received the treatment.

[0400] The monitoring method includes performing the steps of the following method: a method for determining at least one protein content in a biological sample of the subject to obtain a determined protein content.

[0401] The monitoring method further includes monitoring the determined protein content so as to monitor the treatment of heme-related or heme-protein-related disorders in the subject, where the subject has a heme-related or heme-protein-related disorder and has received the treatment.

[0402] Such an application corresponds to many possible applications, some of which will be described below.

[0403] It is worth noting that the method can be used for:

[0404] - Monitoring the treatment of heme arginate porphyries;

[0405] - Monitoring the treatment with purified or recombinant hemopexin;

[0406] - Monitoring the addition of Hb or other blood substitutes for therapeutic use;

[0407] - Monitoring treatments that cause hemolysis, such as extracorporeal membrane oxygenation (ECMO) (extracorporeal circulation, hemodialysis, or cardiac prosthesis);

[0408] - Characterizing plasma exchange, plasmapheresis (removing Hb and heme by adding their scavengers haptoglobin and hemopexin) to calculate the exchange volume (dilution of the patient's plasma), and

[0409] - Using the determination of hemopexin for the pathological interaction with red globules.

[0410] The applicant also conducted experiments on patients with sickle cell disease (SCD) and β-thalassemia using the methods described above. The majority of the patients were being treated with HU, transfusions, or both (other treatments such as chelating agent iron are relevant).

[0411] Roughly understanding that when no treatment is given, then all parameters of hemolysis / erythropoiesis abnormalities will be higher, the applicant has been able to detect only a higher Hb content in plasma in SCD and approaching the normal range in β-thalassemia (under moderate or transfusion procedures). In particular, most of this Hb is free and not complexed with haptoglobin in SOD. Therefore, it will be able to exude into tissues and degrade with the accumulation of heme on membranes (e.g., glomeruli), and ultimately cause long-term dysfunction of organs, and also enable the catabolism of a part of NO synthesized by endothelial cells (promoting vasoconstriction). The metHb content can be related to oxidative stress and the level of vasoconstriction (hypertension) after the reaction of NO with oxidized Hb.

[0412] In the case of β-thalassemia, heme is in the majority. Without the support of heme oxygenase (ferritin), the degradation of heme in plasma will also cause pro-oxidant cytotoxic reactions after iron release. If a part of heme is catabolized in the endothelium, there will be potentially beneficial degradation products across heme: biliverdin and bilirubin but especially the activation of the signaling pathway of CO (anti-inflammatory, anti-aggregatory, anti-proliferative, vasoactive...). The accumulation on the membrane will cause necrosis (treatment with heme arginate). The applicant also noted separating the measurement intervals from the biomarkers according to the pathology.

[0413] In beta thalassemia, and especially in transfused patients, significant amounts of heme following depletion of hemopexin were completely unexpected and have been detected thanks to the precision of the aforementioned determination methods.

[0414] Another application of the assay is for monitoring delayed hemolytic transfusion reactions (DHTR), which is a type of transfusion reaction.

[0415] Again, the inventors have shown that the use of this determination method enables a better diagnosis of DHTR to be formed.

[0416] Finally, the applicants showed the steady state of untreated patients by the status of these biomarkers. There were no measurement biases or problems in the management samples that made the data unreliable. Therefore, the tracking of treatment can use these biomarkers in the hope of obtaining a favorable evolution.

[0417] In addition, as an application, it is possible to consider specifically studying Hx-heme complexes. For example, it is possible to consider increasing HO activity in tissues that have receptors for Hx-heme complexes to overcome the presence of oxidative stress. As another example, Hx-heme complexes can be used as an adjuvant in organ conditioning solutions (kidney, liver, etc.) before transplantation to alleviate the harmful effects of local ischemia / reperfusion syndrome. Heme can be used as a biological messenger, especially because its tracking will be possible through the use of quantification of this complex.

[0418] One can also consider HO of heme oxygenase, with activation of antioxidant pathways.

[0419] In summary, a method for determining at least one protein or its metabolite / degradation product (natural pigment) in blood has been proposed which is more accurate while still being easy to implement. This enables many applications to be considered for heme-related or heme protein-related disorders or diseases. This accuracy can be particularly improved by considering one of the following chemical quantitative techniques:

[0420] - Quantification of oxygenated hemoglobin by adding CO,

[0421] - By adding KCN or N3 - To quantify methemoglobin,

[0422] - Quantification of plasma hemoglobin by adding CO and sodium dithionite,

[0423] - Quantification of heme bound to hemopexin by adding DTN,

[0424] - quantification of total hemopexin by adding heme, and

[0425] - Quantify total hemopexin by adding hemin and sodium dithionite.

Claims

1. A method for determining the content of components in a biological sample, wherein the components include oxyhemoglobin, methemoglobin, heme bound to serum albumin, bilirubin, and heme bound to hemopexin, and the method at least includes the following steps: a) Providing an initial spectrum of the sample within a target wavelength range, b) Setting the current spectrum as the initial spectrum, c) Iterating the following steps: c1) Determining the content of the components in the sample, which is determined by spectral analysis and chemical quantification of the current spectrum, c2) Based on the determined content of the components, deriving the overall spectral components within the target wavelength range related to the components, and c3) Removing the derived spectral components from the current spectrum to obtain a new current spectrum, d) Outputting the determined content of the components, As long as the contents of oxyhemoglobin, methemoglobin, heme bound to serum albumin, bilirubin, and heme bound to hemopexin are not determined, step c) is iterated sequentially for different components; Wherein The spectral analysis includes: - Providing a reference spectrum related to the component under consideration, and - Calculating the normalization coefficient between the reference spectrum and the current spectrum; The normalization coefficient is the ratio between the value of the second derivative of the component under consideration and the value of the second derivative of the reference spectrum, and the value of the second derivative of the reference spectrum is defined as the maximum amplitude of the second derivative with respect to the wavelength of the reference spectrum within the target wavelength range, or the absolute value of the difference between the minimum and maximum values of the second derivative of the second derivative of the reference spectrum with respect to the wavelength of the reference spectrum within the target wavelength range; and Wherein, when the chemical quantification is applied, the chemical quantification is selected from the group consisting of: - Quantifying oxyhemoglobin by adding CO, - Quantifying methemoglobin by adding KCN, - Quantifying plasma heme by adding CO and sodium dithionite, - Quantifying heme bound to hemopexin by adding DTN - Quantifying total hemopexin by adding heme, - Quantifying total hemopexin by adding heme and sodium dithionite, and - Quantifying total hemopexin by adding heme, CO, and sodium dithionite.

2. The method according to claim 1, wherein Iterating step c) by reducing the value of the second derivative of the component, and the value of the second derivative of the component is defined as the maximum amplitude of the second derivative of the spectral component of the component in the sample with respect to the spectral wavelength within the target wavelength range, or the absolute value of the difference between the minimum and maximum values of the second derivative of the second derivative of the spectral component of the component in the sample with respect to the spectral wavelength within the target wavelength range.

3. The method according to claim 1, wherein At least one of the following properties is satisfied: - Performing spectral analysis on oxyhemoglobin and calculating the normalization coefficient for at least one wavelength selected from three intervals, the first interval covering wavelengths between 415 nm and 417 nm, the second interval covering wavelengths between 542 nm and 544 nm, and the third interval covering wavelengths between 576 nm and 578 nm; - Perform spectral analysis on methemoglobin and calculate the normalization coefficient for at least one wavelength selected within the range of 350 nanometers to 750 nanometers, and - Perform spectral analysis on bilirubin and calculate the normalization coefficient for at least one wavelength selected within the range of 350 nanometers to 750 nanometers.

4. The method according to claim 3, wherein, Calculate the normalization coefficient for at least one wavelength selected in the third interval.

5. The method according to claim 1, wherein Perform spectral analysis on plasma heme and calculate the normalization coefficient for at least one wavelength selected within the range of 300 nanometers to 750 nanometers using the heme bound to serum albumin and the heme reference spectrum bound to hemopexin.

6. The method according to claim 1, wherein In the step of outputting the determined component content, the determined content includes other contents in the component, which are different from the content of oxyhemoglobin, the content of methemoglobin, the content of heme bound to serum albumin, the content of heme bound to hemopexin, and the content of bilirubin. The other contents are the contents of components selected from the group consisting of: - Carboxyhemoglobin, - Myoglobin, - Porphyrin, - Porphobilinogen, - Urobilin.

7. The method according to claim 1, wherein In the step of outputting the determined component content, the determined content includes other contents in the component, which are different from the content of oxyhemoglobin, the content of methemoglobin, the content of heme bound to serum albumin, the content of bilirubin, and the content of heme bound to hemopexin. The other contents are the contents of the products of the catabolism of heme by heme oxygenase.

8. The method according to claim 1, wherein, In the step of outputting the determined component content, the determined content includes other contents in the component, which are different from the content of oxyhemoglobin, the content of methemoglobin, the content of heme bound to serum albumin, the content of bilirubin, and the content of heme bound to hemopexin. The other contents are the contents of the products of the degradation and oxidation of bilirubin.

9. According to the method of claim 8, wherein The products of the degradation and oxidation of bilirubin are biliverdin or heme box.

10. The method according to claim 1, wherein In the step of outputting the determined component content, the determined content includes other contents in the component, which are different from the content of oxyhemoglobin, the content of methemoglobin, the content of heme bound to serum albumin, the content of bilirubin, and the content of heme bound to hemopexin. The other contents are the contents of the products of organ dysfunction and cytolysis.

11. The method according to claim 1, wherein, In the step of outputting the determined component content, the determined content includes other contents in the component, which are different from the content of oxyhemoglobin, the content of methemoglobin, the content of heme bound to serum albumin, the content of bilirubin, and the content of heme bound to hemopexin. The other contents are the contents of metabolic substrates.

12. The method according to claim 1, wherein, In the step of outputting the determined component content, the determined content includes other contents in the component, which are different from the content of oxyhemoglobin, the content of methemoglobin, the content of heme bound to serum albumin, the content of bilirubin, and the content of heme bound to hemopexin. The other contents are the contents of metabolic degradation products.

13. The method according to any one of claims 1 to 12, wherein, When successively determining the contents of oxyhemoglobin, methemoglobin, heme bound to serum albumin, and bilirubin, at least one of oxyhemoglobin, methemoglobin, heme bound to serum albumin, and bilirubin is determined quantitatively by chemistry.

14. A method for qualifying or disqualifying a medical bag containing a biological sample of a subject, the method comprising: - performing the steps of a method for determining the content of components in the medical bag to obtain a determined component content, the method being as described in any one of claims 1 to 13, - qualifying or disqualifying the medical bag based on the determined component content.

15. The method according to claim 14, wherein, The medical bag is a blood bag.

16. A computer program product including a computer-readable medium having a computer program including program instructions, the computer program being loadable into a data processing unit and, when run by the data processing unit, adapted to cause the execution of the steps of a method as described in any one of claims 1 to 13, or adapted to cause the execution of the steps of a method as described in claim 14 or 15.

17. A computer-readable medium having encoded thereon the computer program as described in claim 16.

18. An apparatus (10) for determining the content of components in a biological sample (20), wherein the components include oxyhemoglobin, methemoglobin, heme bound to serum albumin, heme bound to hemopexin, and bilirubin, the apparatus (10) for determination at least includes a spectrophotometer (14), a quantification material (16), and an analysis system (18), the apparatus (10) being adapted to perform a method as described in any one of claims 1 to 13, or a method as described in claim 14 or 15.

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