HCT detection kit, method for detecting HCT and application thereof

Abstract

The present application relates to a kind of HCT detection kit and the method and application for detecting HCT, belong to biological detection technical field.The kit includes: anti-mouse IgG, bovine serum albumin, polyethylene glycol 8000, ethylenediamine tetraacetic acid disodium, sodium dodecyl sulfate and polyethylene glycol octyl phenyl ether.The present application significantly improves the light transmission effect of the sample to be detected by various reagent anti-interference measures, improves the accuracy and stability of detection.In the detection method, 540nm is selected as the detection wavelength, combined with the optimized light source, sensor and dynamic feedback calibration mechanism, to ensure the high accuracy of detection result, the modified layer on the surface of reaction vessel has low adsorption effect, can reduce sample residue, further improve the repeatability and accuracy of detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to an HCT detection kit, a method for detecting HCT and application. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Hematocrit (HCT) refers to the volume percentage of blood cells in whole blood. The HCT of anemic patients is lower than that of healthy people, which can dilute the concentration of other markers in the plasma. When detecting markers such as C-reactive protein (CRP) in the serum of anemic patients, if the HCT of healthy people is used for calculation, the deviation of plasma volume caused by abnormal HCT will cause the detection value to be falsely low. In some whole blood detection scenarios in the laboratory, the plasma and blood cells separated by centrifugation need to be mixed again, and this mixing process will also cause the HCT to change, resulting in deviation of the detection result.

[0004] In the prior art, common methods for detecting HCT include electrode method (conductivity method), hemoglobin absorbance method (spectrophotometry) and blood cell counting method (impedance method / light scattering method). Among them, the electrode method detects the HCT by calculating the whole blood conductivity based on the difference in conductivity between red blood cells and plasma, but it is affected by the concentration of plasma electrolytes, and the results will be deviated if the patient has electrolyte disorder, and the electrode needs to be calibrated regularly. The hemoglobin absorbance method is based on the absorption characteristics of hemoglobin to light of a specific wavelength, detects the size of the hemoglobin absorbance after processing, and then converts the HCT according to the empirical relationship between hemoglobin and red blood cell size, but the size of red blood cells (MCV) and abnormal morphology (such as thalassemia) will affect the accuracy of the empirical relationship, and factors such as hemolysis, lipemia and hyperbilirubinemia that interfere with absorbance will also cause the results to deviate. The blood cell counting method has higher accuracy, and the principle includes impedance method (counting red blood cells by the change of electric resistance when cells pass through micro-holes, and calculating HCT according to the volume of red blood cells) and light scattering method (accurately calculating HCT by analyzing cell size and complexity through flow cytometry). But it needs large equipment, and the sample processing process is complex and the detection time is long. SUMMARY

[0005] In order to improve the deficiencies of the prior art, the purpose of the present application is to provide an HCT detection kit, a method for detecting HCT and application, which fuses the immunochromatography technology to dynamically correct the HCT, and can compensate for the influence of HCT difference on the concentration of markers.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows.

[0007] In a first aspect, an HCT detection kit comprises: anti-mouse IgG, bovine serum albumin (BSA), polyethylene glycol 8000 (PEG 8000), disodium ethylenediaminetetraacetate (EDTA-2Na), sodium dodecyl sulfate (SDS), and polyethylene glycol octylphenyl ether (Triton X-100).

[0008] In a second aspect, a method for detecting HCT comprises the following steps:

[0009] S1, sodium dodecyl sulfate (SDS), anti-mouse IgG, bovine serum albumin (BSA), polyethylene glycol 8000 (PEG 8000), disodium ethylenediaminetetraacetate (EDTA-2Na), and polyethylene glycol octylphenyl ether (Triton X-100) are prepared into a reagent solution, a whole blood sample to be tested is added to the reagent solution to obtain a sample to be tested, a detection light source with a wavelength of 520-560 nm is used, an auxiliary light source with a wavelength of 850-890 nm is used, and the absorbance A of the sample to be tested is detected. 540 And turbidity I S90 ;

[0010] S2, a lipidemia interference coefficient k is calibrated with multiple lipidemia samples, an absorbance-HCT conversion coefficient a is corrected with a standard product with HCT=4.0, and a turbidity nonlinear compensation factor b is obtained through regression analysis of a high lipidemia sample;

[0011] S3, the detection result obtained in S1 is substituted into formula (1), and the coefficients obtained in S2 are substituted into formula (1) to obtain the hematocrit of the whole blood sample to be tested.

[0012] The formula (1) is: , (1).

[0013] In a third aspect, the application of the above-mentioned method for detecting HCT comprises: correcting the detection result of a marker in blood plasma.

[0014] The present application has the following beneficial effects:

[0015] 1. The present application effectively reduces non-specific antibody binding, protein adsorption, lipid interference, and turbidity interference through various anti-interference measures such as anti-mouse IgG, BSA, and PEG 8000, etc., and simultaneously utilizes the synergistic effect of SDS and Triton X-100 to accelerate the lysis of red blood cell membranes and clean the reaction vessel, significantly improving the light transmission effect of the sample to be tested and improving the accuracy and stability of the detection.

[0016] 2. In the detection method of the present application, the 540nm hemoglobin absorption peak is selected as the detection wavelength, combined with the optimized light source, sensor and dynamic feedback calibration mechanism to ensure the high precision of the detection results. The low adsorption treatment of the surface of the reaction vessel can reduce sample residue, further improving the repeatability and accuracy of the detection.

[0017] 3. In the detection method of the present application, the 540nm hemoglobin absorption peak is selected as the detection wavelength, combined with the optimized light source, sensor and dynamic feedback calibration mechanism to ensure the high precision of the detection results. The low adsorption treatment of the surface of the reaction vessel can reduce sample residue, further improving the repeatability and accuracy of the detection.

[0018] 4. The HCT detection results obtained by the present application can be combined with the immunochromatography technology to correct the detection results of the markers in the plasma, and the influence of the HCT difference on the marker concentration is dynamically compensated through an algorithm, thereby breaking through the limitation of the traditional immunochromatography which only relies on a single optical signal or a fixed calibration curve, and significantly improving the detection reliability of complex samples (such as anemic patient blood and mixed whole blood). BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings, which form a part of this description, are included to provide further understanding of the application, and are incorporated in and constitute a part of this application. The schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application.

[0020] Figure 1 FIG. 1 is a structural schematic diagram of the device in Embodiment 1 of the present application.

[0021] 1, LED light source; 2, optical filter and collimating mirror; 3, absorbance detection module; 4, turbidity detection module; 5, reaction vessel; 6, shell. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0023] It should be noted that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and furthermore, it should be understood that when the terms "comprise" and / or "include" are used in the specification, there is a feature, step, operation, device, component and / or combination thereof.

[0024] One or more embodiments of the present application provide a HCT detection kit, which comprises: anti-mouse IgG, bovine serum albumin (BSA), polyethylene glycol 8000 (PEG 8000), disodium ethylenediaminetetraacetate (EDTA-2Na), sodium dodecyl sulfate (SDS) and polyethylene glycol octylphenyl ether (Triton X-100).

[0025] The detection kit is designed based on the SDS-Hb assay, so that hemoglobin (Hb) in blood reacts with low-concentration sodium dodecyl sulfate (SDS) to generate a brown-red SDS-Hb compound with an absorption peak of 540 nm and an absorption valley of 500 nm;

[0026] The added polyethylene glycol octylphenyl ether (Triton X-100) as a non-ionic surfactant can reduce surface tension and is used to cooperate with SDS to accelerate the lysis of red blood cell membranes, release hemoglobin in less than 3 s and form an SDS-Hb complex, and also can prevent lipid aggregation and stabilize turbidity measurement values;

[0027] The anti-mouse IgG can block the binding sites of non-specific antibodies in the sample, effectively reducing the interference of non-specific antibody binding on the detection results;

[0028] The bovine serum albumin (BSA) can block the adsorption of proteins during the reaction process, avoiding detection errors caused by protein adsorption.

[0029] The polyethylene glycol 8000 (PEG 8000) as a hydrophilic polymer can produce a steric hindrance effect, inhibit chylomicron fusion, further reduce sample turbidity interference, and improve the clarity and stability of the detection signal.

[0030] The disodium ethylenediaminetetraacetate (EDTA-2Na) is used to chelate metal ions to prevent oxidation and maintain Hb stability.

[0031] Optionally, the anti-mouse IgG is selected as goat anti-mouse IgG.

[0032] One or more embodiments of the present application provide a method for detecting HCT, comprising the steps of:

[0033] S1, sodium dodecyl sulfate (SDS), anti-mouse IgG, bovine serum albumin (BSA), polyethylene glycol 8000 (PEG 8000), disodium ethylenediaminetetraacetate (EDTA-2Na) and polyethylene glycol octylphenyl ether (Triton X-100) are prepared into a reagent solution, the whole blood sample to be detected is added to the reagent solution to obtain a sample to be detected, a detection light source with a wavelength of 520-560 nm is used, an auxiliary light source with a wavelength of 850-890 nm is used, and the absorbance of the sample to be detected is detected and turbidity ;

[0034] S2, calibrate the lipemia interference coefficient k with multiple lipemia samples, correct the absorbance-HCT conversion coefficient a with the standard sample of HCT=4.0, and obtain the turbidity nonlinear compensation factor b through regression analysis of the high lipemia sample;

[0035] S3, substitute the detection result obtained in S1 into formula (1), and substitute the coefficients obtained in S2 into formula (1) to obtain the hematocrit of the whole blood sample to be detected;

[0036] The formula (1) is: , (1).

[0037] In the above process, formula (1) is improved from formula (2), and variables k, b and I S90 are added, which significantly improves the accuracy of the HCT result. Formula (2) is as follows:

[0038] , (2).

[0039] Optionally, in S1, the concentrations of the components in the reagent solution are as follows: SDS is 30-40 mg / mL, Triton X-100 is 0.5-0.9 vol.%, PEG 8000 is 30-70 mg / mL, EDTA-2Na is 1.0-2.0 mg / mL, and BSA is 6-10 mg / mL.

[0040] Optionally, in S1, the ratio of the reagent solution to the whole blood sample is (18-19):(1-2).

[0041] Optionally, in S1, an LED light source is used to emit detection light with a wavelength of 520-560 nm, and an auxiliary light source is used to emit auxiliary light with a wavelength of 850-890 nm. The detection light passes through a filter and a collimating mirror and irradiates a reaction vessel containing the sample to be detected. An absorbance detection module opposite the reaction vessel receives transmitted light passing through the reaction vessel to obtain the absorbance A 540 . A turbidity detection module opposite the reaction vessel receives scattered light from the contents of the reaction vessel to obtain the turbidity I S90 .

[0042] Optionally, in S1, the sample to be detected is placed in a reaction vessel for absorbance and turbidity detection. The reaction vessel includes a base made of polymethyl methacrylate (PMMA), and a 150-250 nm thick polysulfobetaine (PSBMA) modified layer is arranged on the contact surface of the base with the sample to be detected. A polyethylene glycol (PEG) graft layer is arranged between the modified layer and the base.

[0043] Optionally, in S2, the method for calibrating the lipidemia interference coefficient k of the plurality of lipidemia samples comprises: setting the value of the lipidemia interference coefficient k as 1; taking a plurality of lipidemia samples with different lipid protein contents, respectively measuring A 540 and I S90 ; using a blood cell meter to measure the actual HCT of the plurality of lipidemia samples, and substituting the A 540 and I S90 data into formula (1) to calculate the HCT calculated value; comparing the actual HCT, and adjusting k by mathematical method to make the calculated value of formula (1) close to the true value.

[0044] Optionally, in S2, the method for correcting the absorbance-HCT conversion coefficient a comprises: setting the value of the absorbance-HCT conversion coefficient a as 1; taking a plurality of samples with different HCT levels, respectively measuring A 540 and the actual HCT, and calculating to obtain the a value of each sample; and then using the average or fitting method to obtain the corrected a.

[0045] Optionally, in S2, the method for obtaining the turbidity nonlinear compensation factor b by regression analysis comprises: setting the value of the turbidity nonlinear compensation factor b as 0; selecting a plurality of high lipidemia samples, respectively measuring A 540 , I S90 and the actual HCT, substituting the corrected k and a into formula (1) to calculate the calculated HCT, and optimizing b by mathematical iteration to reduce the error between the actual HCT and the calculated HCT.

[0046] Optionally, in S2, the k, a and b are corrected in sequence for multiple times until the values of k, a and b do not change within the set precision, and the final correction result is obtained.

[0047] One or more embodiments of the present application provide the application of the above-mentioned method for detecting HCT, comprising: correcting the detection result of the marker in the blood plasma.

[0048] Optionally, the HCT value is substituted into the main standard curve of the chromatographic reagent card for calculation.

[0049] Embodiment 1

[0050] A device for detecting HCT, as shown in FIG. 1, comprises LED light sources 1, optical filters and collimating mirrors 2, reaction vessels 5 and absorbance detection modules 3 arranged in a straight line in a housing 6, and turbidity detection modules 4 arranged side by side with the absorbance detection modules 3. Figure 1

[0051] ​The LED light source 1 adopts a multi-wavelength light source, and 540 nm is selected as the detection wavelength, which is the absorption peak of hemoglobin, and efficient detection of hemoglobin can be realized. In this embodiment, the LED light source is a dual-wavelength lamp bead, which can convert 540 nm wavelength and 850 nm wavelength. The 850 nm wavelength is used as an auxiliary light source, and the signal-to-noise ratio and the calibration light path are optimized through the optical filter and the collimating mirror 2, thereby greatly reducing the equipment cost while ensuring the detection accuracy.

[0052] The auxiliary light source is used to provide a light signal complementary to the main detection wavelength, and through joint measurement with the detection light source, background interference (such as lipemia and hemolysis) or light path fluctuation is distinguished, and the detection accuracy is improved.

[0053] The absorbance detection module 3 includes a silicon photodiode, which is used to detect the transmitted light through the reaction dish 5 at a receiving angle of 0°, convert the light signal into an electrical signal, and measure the SDS-Hb absorbance, thereby providing a data basis for subsequent absorbance calculation.

[0054] The turbidity detection module 4 includes an avalanche photodiode, which is used to detect the scattered light of the contents of the reaction dish 5 at a receiving angle of 90°, convert the light signal into an electrical signal, and measure the lipemia turbidity, thereby providing a data basis for subsequent turbidity calculation.

[0055] The reaction dish 5 includes a substrate made of polymethyl methacrylate (PMMA), and a polyethylene glycol (PEG) grafting layer is provided on the substrate. A polysulfobetaine (PSBMA) modified layer is provided on the grafting layer as a contact surface with the sample to be detected.

[0056] The preparation method of the grafting layer includes: soaking the reaction dish 5 in an ethanol solution of benzoin dimethyl ether (a photoinitiator) (the concentration of benzoin dimethyl ether is 1wt%) for 1h, blowing dry, and then irradiating with a UV lamp for 10min to produce active radicals on the surface of PMMA, thereby obtaining an activated PMMA reaction dish; preparing a binary solvent with a volume ratio of ethanol to water of 1:1, adding sulfobetaine monomer to the binary solvent to a concentration of 5wt%, and adding PEG monomer to a concentration of 3wt%, thereby obtaining a grafting liquid; placing the activated PMMA reaction dish in the grafting liquid and irradiating with a UV lamp for 2h, thereby obtaining a grafting layer, and then washing with deionized water and ethanol multiple times for standby.

[0057] The preparation method of the modified layer includes: spraying a 10wt% PSBMA solution on the surface of the grafting layer and then ultraviolet curing, so that the thickness of the modified layer is 200±50nm.

[0058] The purpose of modification and grafting is to improve the low adsorption, prevent the attachment of the sample to be detected on the surface of the reaction dish 5 from affecting the detection result. It has been verified by experiments that the contact angle of the untreated PMMA reaction dish is 35°, the residual amount of SDS-Hb is 12.5 μg / cm 2 ; the contact angle after PEG grafting treatment is <5°, and the residual amount of SDS-Hb is reduced to 2.1 μg / cm 2 ; the contact angle after PSBMA coating treatment is <10°, and the residual amount of SDS-Hb is 1.8 μg / cm 2 , effectively reducing the sample residue on the surface of the reaction dish and improving the repeatability and accuracy of detection. And the PEG coating can be used stably for more than 100 cycles under the conditions of pH 6-8 and temperature <50°C, reducing the use cost. The specific performance is shown in Table 1.

[0059] Table 1 Performance table of reaction dish

[0060]

[0061] Example 2

[0062] An HCT detection kit, comprising: anti-mouse IgG, bovine serum albumin (BSA), polyethylene glycol 8000 (PEG8000), ethylenediaminetetraacetic acid disodium (EDTA-2Na), sodium dodecyl sulfate (SDS), and polyethylene glycol octylphenyl ether (Triton X-100); the anti-mouse IgG is selected as goat anti-mouse IgG.

[0063] The method for detecting HCT using the above-mentioned HCT detection kit, comprising the steps of:

[0064] S0, take 320 μL of reagent liquid into the reaction dish for blank detection to obtain background absorbance data;

[0065] S1, use the sample arm to take 190 μL of reagent liquid into the mixing well, take 10 μL of whole blood sample to be detected into the mixing well and mix well, so that the whole blood and the reagent liquid are in full contact, the process of red blood cell membrane lysis and SDS-Hb complex formation is started, and the first step reaction liquid is obtained;

[0066] S1.1, take 35 μL of the first step reaction liquid into the reaction dish in the mixing well and perform further reaction, mix well in the reaction dish to obtain the sample to be detected, and detect according to the structure combination of Figure 1 The device in Example 1 is used for detection, the LED light source 1 emits detection light with a wavelength of 520-560 nm, the auxiliary light source emits auxiliary light with a wavelength of 850-890 nm, the detection light passes through the filter and the collimating mirror 2 to irradiate the reaction dish 5 containing the sample to be detected, and the absorbance detection module 3 on the opposite side of the reaction dish 5 receives the transmitted light through the reaction dish 5 to obtain the absorbance The turbidity detection module 4 on the opposite side of the reaction dish receives the scattered light of the contents of the reaction dish to obtain the turbidity ; the suction head recovers 355 μL of waste liquid (a compound after the blood sample reacts with the reagent), and a detection process is completed.

[0067] S2, the lipidemia interference coefficient k is calibrated with multiple lipidemia samples, the absorbance-HCT conversion coefficient a is corrected with the standard sample with HCT=4.0, and the turbidity nonlinear compensation factor b is obtained through regression analysis of the high lipidemia sample;

[0068] S3, the detection result obtained in S1 is substituted into formula (1), and the coefficients obtained in S2 are substituted into formula (1) to obtain the hematocrit of the whole blood sample to be detected;

[0069] (1).

[0070] The preparation method of the reagent liquid in S0 and S1 (taking the preparation of 100 mL of reagent liquid as an example) includes: taking 80 mL of deionized water into a beaker, and under the condition of magnetic stirring, sequentially performing the following operations: adding 3.8 g of SDS, stirring until completely dissolved; then adding 0.5 mL of Triton X-100, stirring and mixing; then adding 5 g of PEG8000, stirring until completely dissolved (if necessary, heating at 37°C to help dissolve), and then cooling; then adding 150 mg of EDTA-2Na, adding NaOH solution to adjust the pH to 8.0 to help dissolve, and stirring and mixing; then adding 1 mL of goat anti-mouse IgG stock solution (which is diluted in the reagent liquid at a ratio of 1:100), gently stirring and mixing, adding 0.8 g of BSA, and stirring again to make it uniform; then adding deionized water to make up to 100 mL, and filtering with a 0.22 μm filter membrane.

[0071] The coefficient obtaining method in S2 includes the calibration method of the lipidemia interference coefficient k, the correction method of the absorbance-HCT conversion coefficient a, and the regression analysis method of the turbidity nonlinear compensation factor b.

[0072] The calibration method of the lipidemia interference coefficient k includes: first, set the value of the lipidemia interference coefficient k to 1; take 200 lipidemia samples with different lipid protein contents, and use the device for detecting HCT in Example 1 to measure A 540 and I S90 ; then use the hemocytometer to measure the actual HCT of the 200 lipidemia samples, substitute the A 540 and I S90 data into formula (1), take a=1 and b=0, and calculate the HCT calculated value; compare the actual HCT, adjust k by mathematical method to make the calculated value of formula (1) closer to the true value; after the above correction, the value of k is corrected from 1 to 0.21.

[0073] The correction method of absorbance-HCT conversion coefficient a includes: setting the value of absorbance-HCT conversion coefficient a as 1; taking 150 samples with different HCT levels (as little as possible interference of lipidemia) to measure A 540 and actual HCT, dividing the value of A 540 by the value of real HCT to obtain the a value of each sample; then using the method of averaging or fitting to obtain the corrected a, and the value of a is corrected from 1 to 2.35 after the above correction process.

[0074] The regression analysis method of turbidity nonlinear compensation factor b includes: setting the value of turbidity nonlinear compensation factor b as 0; selecting 100 high lipidemia samples to measure A 540 , I S90 and real HCT, substituting the corrected k and a into formula (1) to calculate the calculated HCT, and reducing the error between real HCT and calculated HCT by mathematical iteration optimization b; after the above process, the value of b is corrected from 0 to 0.00015.

[0075] The obtained coefficients include: k=0.21; a=2.35; b=0.00015.

[0076] After obtaining the above coefficients, the detection results of the hematology analyzer are analyzed to obtain the statistical analysis data of 200 samples as shown in Table 2, which shows that the accuracy of formula (1) is high and can be used for HCT determination.

[0077] Table 2 Statistical analysis data of 200 samples

[0078]

[0079] The coefficient acquisition process is actually before obtaining the whole blood sample to be measured, and under the premise that a, b and k in formula (1) are determined, the detection results obtained in S1 are substituted into formula (1) to obtain the HCT result.

[0080] Comparative Example 1

[0081] Using 3.8wt% SDS single reagent for detection, the calculation formula is: , to obtain HCT data.

[0082] Result analysis

[0083] (1) The detection results of Example 2 and Comparative Example 1 are shown in Table 3-1 and Table 3-2, in which Sysmex XN-2800 refers to the HCT data obtained using the Sysmex XN-2800 instrument, 3.8% SDS method refers to the HCT data obtained in Comparative Example 1, innovative detection system refers to the HCT data obtained in Example 2, and error refers to the error relative to the HCT data obtained using the Sysmex XN-2800 instrument.

[0084] Table 3-1 Statistical table of detection results of Example 2 and Comparative Example 1

[0085]

[0086] Table 3-2 Statistical table of detection results of Example 2 and Comparative Example 1

[0087]

[0088] The statistical analysis data of Comparative Example 2 and Example 3 are shown in Table 4.

[0089] Table 4 Statistical analysis data table of Table 3

[0090]

[0091] The correlation of Example 2 (innovative system) with the HCT data obtained using the Sysmex XN-2800 instrument is 0.981 (R 2 =0.962), which is significantly better than 0.847 (R 2 =0.717) of the SDS method, and the error is reduced from an average of 8.32% to 0.20%, with a reduction of 97.6%.

[0092] Example 3

[0093] Twenty patients were selected, and serum and whole blood samples were obtained; the detection item was C-reactive protein, and Roche cobas c 701 serum was the gold standard for the control group. The whole blood sample was measured using the immunochromatography reagent method, and the uncorrected result was obtained by direct measurement. Under normal circumstances, the HCT value of healthy people is 37%, and a regression curve is obtained by tracing the gold standard serum sample. When detecting whole blood samples, whole blood = value in plasma curve / (1-0.37), and the HCT value of each sample is obtained through the system during the detection process.

[0094] Corrected whole blood value = value in plasma curve / (1-HCT actual measurement value).

[0095] The 20 clinical verification data and analysis of whole blood detection + HCT correction using an immunochromatographic platform vs. serum gold standard are shown in Table 5.

[0096] Table 5 Whole blood + serum sample detection result statistics table

[0097]

[0098] The statistical analysis data of Comparative Example 2 and Example 3 are shown in Table 6.

[0099] Table 6 Statistical analysis data table of Table 5

[0100]

[0101] It is shown that the HCT correction module enables whole blood detection to reach the level of serum gold standard, providing laboratory-level precision for POCT scenarios.

[0102] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Various modifications and changes can be made by those skilled in the art based on the principles and technical solutions of the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting HCT, characterized in that it comprises the steps of: S1, sodium dodecyl sulfate (SDS), anti-mouse IgG, bovine serum albumin (BSA), polyethylene glycol 8000 (PEG8000), ethylenediaminetetraacetic acid disodium (EDTA-2Na) and polyethylene glycol octylphenyl ether (Triton X-100) are prepared into a reagent solution, a whole blood sample to be tested is added to the reagent solution to obtain a sample to be tested, a detection light source with a wavelength of 520-560 nm is used, an auxiliary light source with a wavelength of 850-890 nm is used, and the absorbance A of the sample to be tested is detected 540 and turbidity I S90 ; S2. Using multiple lipemia samples to calibrate the lipemia interference coefficient k, using a standard sample with HCT=4.0 to correct the absorbance-HCT conversion coefficient a, and using a high-lipemia sample to obtain a turbidity nonlinear compensation factor b through regression analysis; S3. Substituting the detection result obtained in S1 into formula (1) and substituting the coefficients obtained in S2 into formula (1) to obtain the hematocrit of the whole blood sample to be detected; The formula (1) is: , (1).

2. The method of detecting HCT according to claim 1, wherein, The concentrations of the components in the reagent solution are as follows: SDS is 30-40 mg / mL, Triton X-100 is 0.5-0.9 vol.%, PEG 8000 is 30-70 mg / mL, EDTA-2Na is 1.0-2.0 mg / mL, and BSA is 6-10 mg / mL. Alternatively, in S1, the ratio of the reagent solution to the whole blood sample is (18-19):(1-2).

3. The method of claim 1, wherein the HCT is detected by measuring the level of the marker in the sample. In S1, a detection light with a wavelength of 520-560 nm is emitted by an LED light source, an auxiliary light with a wavelength of 850-890 nm is emitted by an auxiliary light source, the detection light is irradiated on a reaction dish containing a sample to be detected after passing through a filter and a collimating mirror, a transmittance light passing through the reaction dish is received by an absorbance detection module on the opposite side of the reaction dish, and the absorbance A is obtained 540 A turbidity detection module on the opposite side of the reaction dish receives a scattering light of the content of the reaction dish, and the turbidity I is obtained S90 .

4. The method of claim 1, wherein the HCT is detected by measuring the level of the marker in the sample. In S1, the sample to be detected is placed in a reaction dish for turbidity detection, and the reaction dish is a substrate made of polymethyl methacrylate, and a 150-250 nm thick polysulfobetaine modified layer is arranged on the contact surface of the substrate with the sample to be detected, and a polyethylene glycol grafted layer is arranged between the modified layer and the substrate.

5. The method of detecting HCT of claim 1, In S2, the method for calibrating the lipemia interference coefficient k of multiple lipemia samples comprises: First, set the value of the lipemia interference coefficient k to 1; Take multiple different lipoprotein content of blood samples, respectively, A 540 and I S90 ; then use the blood cell instrument to measure the actual HCT of the blood sample, and A 540 and I S90 data into formula (1) to calculate the HCT value; compared with the actual HCT, adjust k by mathematical method, so that the calculated value of formula (1) is close to the true value.

6. The method of detecting HCT of claim 1, The method for correcting the absorbance-HCT conversion coefficient a in S2 comprises the following steps: firstly, setting the value of the absorbance-HCT conversion coefficient a as 1; taking a plurality of samples with different HCT levels, respectively measuring the A 540 of each sample; then, using the method of averaging or fitting to obtain the corrected a value. Or, in S2, the method for obtaining the turbidity nonlinear compensation factor b by regression analysis comprises the following steps: setting the value of the turbidity nonlinear compensation factor b as 0; selecting multiple high-fat blood samples, respectively measuring A 540 、 S90 and the true HCT, substituting the corrected k and a into formula (1) to calculate the calculated HCT, and optimizing b by mathematical iteration to reduce the error between the true HCT and the calculated HCT.

7. Use of a method of detecting HCT as claimed in any one of claims 1 to 6, characterised in that, for correcting the detection result of the marker in the blood plasma.

8. The use according to claim 7, characterized in that The HCT value is substituted into the main standard curve of the chromatographic reagent card for calculation.

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