A method for quantitatively detecting creatine and creatinine in whole blood

Abstract

The present invention relates to a method for quantitatively detecting creatine and creatinine in whole blood. A hemoglobin detection concentration, a creatine detection concentration, and a creatinine detection concentration of a whole blood sample are obtained by using a combined creatine-creatinine detection test strip. A correction coefficient is determined based on the hemoglobin detection concentration, and the creatine detection concentration and the creatinine detection concentration are corrected. The creatine concentration of the whole blood sample = creatine detection concentration × correction coefficient, and the creatinine concentration of the whole blood sample = creatinine detection concentration × correction coefficient. The method for determining the correction coefficient is as follows: a reference value of the hemoglobin detection concentration is set, and the correction coefficient = (hemoglobin detection concentration / reference value) × correction factor α. If the hemoglobin detection concentration is greater than or equal to the reference value, then the correction factor α < 1; if the hemoglobin detection concentration is less than the reference value, then the correction factor α > 1. The present invention can exclude the influence of endogenous creatine and hemoglobin on the detection of the creatinine concentration in the whole blood sample and obtain more accurate detection results in a short time.

Description

Technical Field

[0001] The present invention relates to a method for quantitatively detecting creatine and creatinine in whole blood. Background Art

[0002] Creatinine is a metabolite of muscle in the human body and is mainly excreted from the body through glomerular filtration. Creatinine is a low-molecular-weight nitrogen-containing compound with a molecular weight of 116 D. Creatinine in blood comes from two sources, exogenous and endogenous. Exogenous creatinine is the product of the metabolism of meat in the body; endogenous creatinine is the product of the metabolism of muscle tissue in the body. Creatinine in blood is mainly excreted by the kidneys in urine. Plasma creatinine comes from muscle tissue, and its concentration is proportional to the muscle mass. Therefore, in acromegaly and gigantism, the plasma creatinine concentration increases; on the contrary, in muscle atrophy diseases, the plasma creatinine concentration decreases. Before and after dialysis treatment, plasma creatinine measurement can be used to select dialysis indications and judge the dialysis treatment effect. The concentration of creatinine in blood is mainly related to the glomerular filtration rate. Therefore, the measurement of plasma creatinine and urinary creatinine excretion is one of the routine clinical renal function tests.

[0003] The determination of creatinine concentration is easily interfered by various endogenous and exogenous factors. Creatine is an intermediate product in creatinine detection. At a certain concentration, endogenous creatine will have a negative interference on the detection of low and high concentrations of creatinine, affecting the accuracy of creatinine detection. When the test sample is whole blood, hemoglobin will affect the test result, and this effect is adverse. Detecting plasma samples can avoid the interference of hemoglobin on the test result, but it requires professional technicians to pre-treat the whole blood sample, and the treatment process is complex and time-consuming. Although the existing creatinine test strips using whole blood samples have added a filter membrane that can remove red blood cells before the sample reaches the reaction layer, the plasma content in different whole blood samples is different, resulting in different amounts of samples finally reaching the reaction layer and causing large deviations in test results.

[0004] Therefore, it is necessary to develop a detection method and detection system for whole blood samples that can obtain more accurate creatinine concentration and creatine concentration, providing a better choice for emergency or on-site detection. Summary of the Invention

[0005] The object of the present invention is to provide a method for quantitatively detecting creatine and creatinine in whole blood with more accurate results.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for quantitatively detecting creatine and creatinine in whole blood. The quantitative detection method is to use a combined creatine-creatinine test strip to detect the hemoglobin detection concentration, creatine detection concentration, and creatinine detection concentration of a whole blood sample. A correction coefficient is determined based on the hemoglobin detection concentration, and the creatine detection concentration and creatinine detection concentration are corrected. The product of the creatine detection concentration and the correction coefficient is the creatine concentration of the whole blood sample, and the product of the creatinine detection concentration and the correction coefficient is the creatinine concentration of the whole blood sample. The method for determining the correction coefficient is as follows: Set a reference value for the hemoglobin detection concentration. The correction coefficient = (hemoglobin detection concentration / reference value) × correction factor α. If the hemoglobin detection concentration is greater than or equal to the reference value, then the correction factor α < 1; if the hemoglobin detection concentration is less than the reference value, then the correction factor α > 1.

[0008] Preferably, if the hemoglobin detection concentration is greater than or equal to the reference value, the correction factor α is 0.9 - 0.97, such as 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97.

[0009] Preferably, if the hemoglobin detection concentration is less than the reference value, the correction factor α is 1.02 - 1.12, such as 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12.

[0010] Preferably, the reference value is 125 g / L - 135 g / L, such as 125 g / L, 126 g / L, 127 g / L, 128 g / L, 129 g / L, 130 g / L, 131 g / L, 132 g / L, 133 g / L, 134 g / L, 135 g / L.

[0011] According to some specific and preferred embodiments, the reference value is 130 g / L.

[0012] If the hemoglobin detection concentration is greater than or equal to 130 g / L, the correction coefficient = (hemoglobin detection concentration / 130 g / L) × 0.95;

[0013] If the hemoglobin detection concentration is less than 130 g / L, the correction coefficient = (hemoglobin detection concentration / 130 g / L) × 1.05.

[0014] Preferably, the creatine-creatinine combined test strip is provided with a first detection area, a second detection area and a third detection area. The first detection area of the creatine-creatinine combined test strip includes a diffusion membrane and a reaction membrane stacked from top to bottom. The second detection area and the third detection area of the creatine-creatinine combined test strip respectively include a diffusion membrane, a blood filtration membrane and a reaction membrane stacked from top to bottom. The diffusion membranes of the first detection area, the second detection area and the third detection area are connected as a whole to form a single diffusion membrane.

[0015] The whole blood sample is evenly diffused to the first detection area, the second detection area and the third detection area through the diffusion membrane. A detection reagent capable of binding hemoglobin and showing color is coated on the reaction membrane in the first detection area. The whole blood sample diffused to the second detection area and the third detection area passes through the blood filtration membrane from the diffusion membrane downward to intercept red blood cells to obtain plasma, and the plasma reaches the reaction membrane downward. A reagent A capable of decomposing creatine and showing color is coated on the reaction membrane in the second detection area, and a reagent B capable of decomposing both creatine and creatinine and showing color is coated on the reaction membrane in the third detection area.

[0016] Specifically, the detection reagent is selected from the reagents commonly used in the art for detecting the hemoglobin content in whole blood.

[0017] Specifically, the reagent A includes a reagent capable of decomposing creatine into hydrogen peroxide and a reagent capable of making hydrogen peroxide show color.

[0018] Specifically, the reagent B includes a reagent capable of decomposing both creatine and creatinine into hydrogen peroxide and a reagent capable of making hydrogen peroxide show color.

[0019] According to some embodiments, the formulation of the reagent A is:

[0020]

[0021]

[0022] The formulation of the reagent B is:

[0023]

[0024] Preferably, the formulation of the reagent A is:

[0025]

[0026] The formulation of the reagent B is:

[0027]

[0028] Further preferably, the buffer system is Tris buffer, glycine buffer, citrate buffer, MOPS buffer or phosphate buffer with a pH value of 7.2 - 8.0.

[0029] Further preferably, the surfactant is one or more of polyoxyethylene sorbitan monooleate 80, sodium cholate, polyoxyethylene lauryl ether, and polyethylene glycol p-isooctylphenyl ether.

[0030] Further preferably, the hydrophilic colloid is one or more of polyvinyl alcohol, cellulose ester, polyacrylamide, polyvinylpyrrolidone, and agarose.

[0031] Further preferably, the protective agent is sucrose.

[0032] Further preferably, the chromogen consists of one or more of 4-aminoantipyrine and phenol, p-hydroxybenzoic acid, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, and sodium 3,5-dichlorohydroxybenzenesulfonate.

[0033] Even more preferably, the chromogen consists of 4-aminoantipyrine and sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline. The molar ratio of 4-aminoantipyrine to sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline in the feed is 1.5 - 3:1.

[0034] Preferably, the material of the diffusion membrane includes but is not limited to polyester fiber mesh or nylon fiber mesh.

[0035] Further preferably, the pore size of the polyester fiber mesh is 60 - 100 mesh; the pore size of the nylon fiber mesh is 60 - 100 mesh.

[0036] Preferably, the material of the blood filtration membrane includes but is not limited to fiberglass membrane or polyester fiber.

[0037] Preferably, the reaction membrane is a BioC 1.2μm membrane material.

[0038] Preferably, the preparation method of the reaction membrane is as follows: Add reagent A or reagent B to the solution tank of the membrane processor, and run the machine to process the reaction membrane. The reaction membrane moves forward at a constant speed and evenly dips into reagent A or reagent B and passes through the drying channel, fixing each component in reagent A or reagent B in the corresponding detection area of the reaction membrane and ensuring the uniformity and stability of the substances on the membrane.

[0039] Further preferably, the running speed of the reaction membrane is 100m 2 / h.

[0040] Further preferably, the temperature of the drying channel is 50 °C.

[0041] Further preferably, the processed reaction film is assembled with other layers and the cartridge. The assembly sequence is the lower plate of the cartridge, the reaction film, the blood filtration film, the diffusion film, and the upper plate of the cartridge. The assembled large plate is cut on a strip cutting machine with a width of 10.0 mm. Detection windows are provided on the cartridge, corresponding to the first detection area, the second detection area, and the third detection area respectively. A sample addition port is also provided on the upper plate of the cartridge. All the cut test strips should be sealed with an appropriate amount of desiccant and stored away from light.

[0042] Preferably, a dry biochemical analyzer with a built-in algorithm is used to collect the signal values of the first detection area, the second detection area, and the third detection area, and the detection concentration is calculated and corrected according to the collected signal values.

[0043] The present invention also provides a combined detection reagent strip for creatine and creatinine in whole blood, and the combined detection reagent strip is the creatine-creatinine combined detection test strip in the quantitative detection method.

[0044] The present invention also provides a combined detection system for creatine and creatinine in whole blood. The combined detection system includes a creatine-creatinine combined detection test strip and a biochemical analyzer, and the creatine-creatinine combined detection test strip is the creatine-creatinine combined detection test strip in the quantitative detection method.

[0045] When detecting a whole blood sample, the present invention obtains the hemoglobin detection concentration through the hemoglobin detection window and corrects it according to the built-in algorithm, so as to exclude the interference of hemoglobin on the result. At the same time, the creatine concentration is detected, and the influence of endogenous creatine on the determination of creatinine concentration is excluded according to the internal algorithm of the instrument, so that the accuracy of the test kit is greatly improved. Thus, the creatinine and creatine concentrations of the whole blood sample can be obtained more accurately in a short time. This method can be implemented on a dry chemistry platform, enabling the dry chemistry test kit to play a better role in emergency and on-site detection.

[0046] During detection, the sample to be tested is dropped into the sample adding port. The sample to be tested will quickly and evenly diffuse and penetrate downward through the diffusion membrane. In the first detection area, hemoglobin reacts with the substances in the first detection area of the reaction membrane to produce a color change. Different colors will reflect light to different degrees. The signal receiver of the dry biochemical analyzer will collect the reflected light and convert it into corresponding signal values. The detected hemoglobin concentration of the whole blood sample is given according to different signal values and the built-in algorithm. In the second and third detection areas, the sample to be tested diffuses and penetrates downward onto the entire blood filtering membrane. The red blood cells and plasma are separated through the blood filtering membrane. The plasma penetrates into the reaction membrane, and the red blood cells are intercepted on the blood filtering membrane. Creatine in the plasma reacts with the substances in the second detection area of the reaction membrane to produce a color change. The detected creatine concentration in the sample can be obtained according to the signal values collected by the instrument and the built-in algorithm. Creatine and creatinine in the plasma react with the substances in the third detection area of the reaction membrane to produce a color change. The sum of the concentrations of creatine and creatinine in the sample can be obtained according to the signal values collected by the instrument and the built-in algorithm. The creatinine detection concentration is obtained by subtracting the creatine concentration from the sum of the creatine and creatinine concentrations. According to the hemoglobin concentration, the creatine detection concentration and the creatinine detection concentration are corrected according to the built-in algorithm to exclude the interference of hemoglobin, so as to obtain a more accurate detection result.

[0047] Due to the above technical solution, the present invention has the following advantages compared with the prior art:

[0048] The detection method of the present invention can exclude the influence of endogenous creatine and hemoglobin on the detection of creatinine concentration in whole blood samples, and obtain more accurate creatinine and creatine concentrations in whole blood samples in a short time, providing a better choice for emergency or on-site detection. Description of the Drawings

[0049] Figure 1 Is the correlation result between the corrected creatine result and the biochemical value;

[0050] Figure 2 Is the correlation result between the uncorrected creatine result and the biochemical value;

[0051] Figure 3 Is the correlation result between the corrected creatinine result and the biochemical value;

[0052] Figure 4 Is the correlation result between the uncorrected creatinine result and the biochemical value. Detailed Embodiments

[0053] The technical solution of the present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments. The reagents or raw materials used in this embodiment can be obtained commercially or prepared according to conventional methods in the art.

[0054] Example

[0055] The creatine-creatinine combined detection dry chemical test strip used in this example includes a lower cassette plate, a reaction membrane, a blood filtration membrane, a diffusion membrane, and an upper cassette plate. The upper cassette plate is provided with a sample addition port, a first detection window, a second detection window, and a third detection window. The first detection window corresponds to the first detection area, the second detection window corresponds to the second detection area, and the third detection window corresponds to the third detection area.

[0056] The whole blood sample is added through the sample addition port. The sample will quickly and evenly diffuse and infiltrate downward through the diffusion membrane. In the first detection area, hemoglobin reacts with the substances on the reaction membrane to produce a color change. Different colors will reflect light to different degrees. The signal receiver of the dry biochemical analyzer will collect the reflected light and convert it into corresponding signal values. According to different signal values and the built-in algorithm, the hemoglobin detection concentration of the whole blood sample is given; in the second detection area and the third detection area, the whole blood sample infiltrates to the blood filtration membrane, and the red blood cells and plasma are separated through the blood filtration membrane. The red blood cells are intercepted on the blood filtration membrane, and the plasma penetrates to the reaction membrane. Reagent A that can decompose and colorize creatine is coated on the reaction membrane in the second detection area, and reagent B that can decompose and colorize creatine and creatinine is coated on the reaction membrane in the third detection area. Among them, the reaction layer material is: BioC 1.2μm membrane material, and the formula of reagent A is as follows:

[0057]

[0058] The formula of reagent B is as follows:

[0059]

[0060]

[0061] The preparation method of the reaction layer is: add reagent A or reagent B into the solution tank of the membrane processor, and run the machine to process the reaction membrane. Keep the running speed at 100m 2 / h so that the reaction membrane advances uniformly and evenly dips in reagent A or reagent B and is dried at 50°C through the drying channel.

[0062] Assemble the processed reaction layer membrane with other layers. The assembly order is the lower cassette plate, the reaction layer, the blood filtration layer, the diffusion layer, and the upper cassette plate. Cut the assembled large plate on the strip cutter according to a width of 10.0 mm. All the cut test strips should be sealed with an appropriate amount of desiccant and stored away from light.

[0063] Detection method: Cooperate with the optical scene dry biochemical analyzer LP-100 for testing. Insert the IC cards of the same batch into the instrument, and operate according to the operation steps prompted on the screen. Add 30 μL of sample at room temperature and wait for about 4 minutes to record the test results.

[0064] Test samples: 20 fresh clinical whole blood samples with different hemoglobin concentrations.

[0065] The signal values at the reaction end point were collected using a dry biochemical analyzer, and two different results were obtained by whether to perform calibration. In this embodiment, the hemoglobin detection value of 130 g / L was used as the reference value. If the detected hemoglobin concentration was greater than or equal to 130 g / L, the correction coefficient = (detected hemoglobin concentration / 130 g / L) × 0.95; if the detected hemoglobin concentration was less than 130 g / L, the correction coefficient = (detected hemoglobin concentration / 130 g / L) × 1.05. The creatine concentration of the whole blood sample = detected creatine concentration (uncorrected creatine result) × correction coefficient, and the creatinine concentration of the whole blood sample = detected creatinine concentration (uncorrected creatinine result) × correction coefficient.

[0066] The deviations between the two groups of results and the test results of the same samples on the fully automatic biochemical analyzer Beckman AU-680 were compared respectively, and the results are shown in Table 1 and Table 2 below.

[0067] Table 1. Influence of calibration on creatine test results

[0068]

[0069]

[0070] Table 2. Influence of calibration on creatinine test results

[0071]

[0072] Table 1 and Table 2 and Figures 1 to 4 showed that after calibration, the accuracy and correlation with biochemical values of the creatinine test results were significantly improved.

[0073] The above has described the present invention in detail, aiming to enable those skilled in the art to understand the content of the present invention and implement it. However, this cannot limit the protection scope of the present invention. Moreover, the present invention is not limited to the above embodiments. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A quantitative detection method for creatine and creatinine in whole blood, characterized in that, The quantitative detection method is to use a creatine-creatinine combined detection test strip to detect the hemoglobin detection concentration, the creatine detection concentration and the creatinine detection concentration of the whole blood sample, determine the correction coefficient by the hemoglobin detection concentration, and correct the creatine detection concentration and the creatinine detection concentration. The product of the creatine detection concentration and the correction coefficient is the creatine concentration of the whole blood sample, and the product of the creatinine detection concentration and the correction coefficient is the creatinine concentration of the whole blood sample. The correction factor is determined by setting a reference value of the hemoglobin test concentration, correction factor = (hemoglobin test concentration / reference value) × correction factor α, The reference value is 125g / L~135g / L. If the hemoglobin test concentration is greater than or equal to the reference value, the correction factor α is 0.9~0.97; If the hemoglobin test concentration is lower than the reference value, the correction factor α is 1.02~1.

12.

2. The quantitative detection method of creatine and creatinine in whole blood according to claim 1, characterized in that, The reference value is 130 g / L. If the hemoglobin concentration is greater than or equal to 130 g / L, the correction factor = (hemoglobin concentration / 130 g / L) × 0.95; If the hemoglobin test concentration is less than 130g / L, the correction factor = (hemoglobin test concentration / 130g / L) × 1.

05.

3. The quantitative detection method according to claim 1 or 2, characterized in that, The creatine-creatinine combined detection test strip is provided with a first detection area, a second detection area and a third detection area. The first detection area of the creatine-creatinine combined detection test strip includes a diffusion membrane and a reaction membrane stacked from top to bottom. The second detection area and the third detection area of the creatine-creatinine combined detection test strip respectively include a diffusion membrane, a blood filter membrane and a reaction membrane stacked from top to bottom. The diffusion membranes of the first detection area, the second detection area and the third detection area are connected as a whole to form a whole diffusion membrane. The whole blood sample is uniformly diffused to the first detection zone, the second detection zone and the third detection zone through the diffusion membrane. The reaction membrane of the first detection zone is coated with a detection reagent that can bind to hemoglobin and develop color. The whole blood sample diffused to the second detection zone and the third detection zone passes through the blood filtration membrane downward from the diffusion membrane to intercept red blood cells to obtain plasma, and the plasma reaches the reaction membrane downward. The reaction membrane of the second detection zone is coated with a reagent A that can decompose creatine and develop color, and the reaction membrane of the third detection zone is coated with a reagent B that can decompose creatine and creatinine and develop color.

4. The quantitative detection method according to claim 3, wherein The formula of the reagent A is: Buffer system 0.01 - 0.5 mol / m 2 Surfactant 0.1 - 100 g / m 2 Hydrophilic colloid 50 - 70 g / m 2 Creatine amidinohydrolase 100 - 8000 KU / m 2 Sarcosine oxidase 10 - 1000 KU / m 2 Peroxidase enzyme 100 - 10000 KU / m 2 Protective agent 1 - 10 g / m 2 Chromogen 0.5 - 20 mmol / m 2 ; The formula of the reagent B is: Buffer system 0.01 - 0.5 mol / m 2 Surfactant 0.1 - 100 g / m 2 Hydrophilic colloid 50 - 70 g / m 2 Creatinine aminohydrolase 1000 - 10000 KU / m 2 Creatine amidinohydrolase 100 - 8000 KU / m 2 Sarcosine oxidase 10 - 1000 KU / m 2 Peroxidase enzyme 100 - 10000 KU / m 2 Protectant 1 - 10 g / m 2 Chromogen 0.5 - 20 mmol / m 2 。 5. The quantitative detection method according to claim 4, wherein The buffer system is a Tris buffer, a glycine buffer, a citric acid buffer, a MOPS buffer or a phosphate buffer with a pH value of 7.2-8.0; The surfactant is one or more of polyoxyethylene sorbitan monooleate 80, sodium cholate, polyoxyethylene lauryl ether, and polyethylene glycol p-isooctylphenyl ether; The hydrophilic colloid is one or more of polyvinyl alcohol, cellulose ester, polyacrylamide, polyvinyl pyrrolidone and agarose; The protective agent is sucrose; The chromogen is composed of one or more of 4-aminoantipyrine and phenol, p-hydroxybenzoic acid, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, and sodium 3,5-dichlorohydroxybenzenesulfonate.

6. The quantitative detection method according to claim 4, wherein A dry biochemical analyzer with a built-in algorithm collects the signal values of the first detection area, the second detection area, and the third detection area, and calculates and corrects the detection concentration according to the collected signal values.

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