Steroid hormone-containing immunosuppressor accurate monitoring treatment kit

Abstract

The invention relates to the technical field of in-vitro diagnostic reagents, in particular to a steroid hormone-containing immunosuppressive agent precise monitoring treatment kit, and discloses a joint detection and correction method for precise monitoring of a steroid hormone-containing immunosuppressive agent. Comprising the following steps: step 1, collecting clinical samples to be detected, and subpackaging each collected sample into two equal parts; 2, carrying out immunological quantitative determination on the first equal part of sample by adopting an immunological kit to obtain an immunological determination value; 3, the second equal sample corresponding to the step 2 is subjected to reference quantitative determination through liquid chromatography-tandem mass spectrometry, a mass spectrometry determination value is obtained, and mass spectrometry quantification uses an isotope labeling internal standard corresponding to each target analyte and is carried out according to a standardized sample pretreatment process. According to the method, high throughput and operation convenience of the immunoassay are guaranteed, meanwhile, the accuracy equivalent to that of mass spectrometry is provided, and a reliable basis is provided for clinical individualized medication.

Description

Technical Field

[0001] This invention relates to a kit for the precise monitoring and treatment of immunosuppressants containing steroid hormones, and particularly to a kit for the precise monitoring and treatment of immunosuppressants containing steroid hormones, belonging to the field of in vitro diagnostic reagent technology. Background Technology

[0002] In the clinical treatment of organ transplantation, autoimmune diseases, and chronic inflammatory diseases, immunosuppressants containing steroid hormones require long-term and precise drug concentration monitoring to guide individualized dosing and avoid toxic side effects. Currently, immunological methods (such as chemiluminescence, radioimmunoassay, and enzyme immunoassay) are mainly used in clinical practice for the quantitative detection of these hormone-based immunosuppressants. These methods have advantages such as high automation, high throughput, and ease of operation. However, the results are easily affected by antibody cross-reactivity, heterophilic antibodies, metabolites, and matrix components, leading to decreased sensitivity and insufficient specificity in the low concentration range. Furthermore, incomplete recognition and quantitative bias often occur between structurally similar steroid hormones. High-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) is recognized as the "gold standard" and is significantly superior to immunological detection in terms of simultaneous identification of multiple targets, quantification at low concentrations, differentiation of metabolites, and specificity. However, the equipment is expensive, the operation is complex, and the maintenance requirements are high, making it unsuitable for all routine high-throughput detection scenarios in laboratories. To compensate for the discrepancies between immunological methods and LC-MS / MS results, some laboratories use both methods in parallel and manually establish quantitative comparison relationships. However, current methods generally suffer from problems such as non-standardized data processing, lack of unified standards for conversion models, reliance on manual experience for update cycles, inability to automatically identify conversion model failures, and difficulty in handling batch-to-batch and pretreatment differences. Existing kits do not provide conversion models that can be directly used for immunological-mass spectrometry correction, nor do they have unified regression methods, quality control acceptance standards, or consistency evaluation procedures. In sample pretreatment, the lack of standardized procedures for steps such as protein precipitation and solid-phase extraction can lead to uncertainties in recovery rates and matrix effects, further increasing the deviation between immunological and mass spectrometry methods. In addition, the imperfect heterophile antibody interference recognition mechanism can also affect the accuracy of immunological results. Therefore, there is an urgent need to improve the kit for precise monitoring and processing of immunosuppressants containing steroid hormones to solve the above-mentioned problems. Summary of the Invention

[0003] The purpose of this invention is to provide a precise monitoring and processing kit for immunosuppressants containing steroid hormones, in order to address the lack of a complete system that can standardize, verify, and traceably convert immunological assay results to LC-MS / MS assay results, including a unified sample pretreatment process, interference suppression mechanism, regression model construction method, bias and consistency evaluation criteria, and automated model management mechanism, which leads to the problem that immunological results cannot reliably replace mass spectrometry results at clinical decision points.

[0004] To achieve the above objectives, the present invention provides the following technical solution: A precision monitoring kit for steroid hormone-containing immunosuppressants includes a combined detection and correction method for precise monitoring of steroid hormone-containing immunosuppressants, comprising the following steps: Step 1: Collect the clinical samples to be tested and divide each collected sample into two equal parts; Step 2: Use an immunological kit to perform immunological quantitative determination on the first aliquot of the sample to obtain the immunological determination value; Step 3: The second aliquot of the sample corresponding to Step 2 is quantitatively analyzed by liquid chromatography-tandem mass spectrometry to obtain the mass spectrometry values. The mass spectrometry quantification uses an isotope-labeled internal standard corresponding to each target analyte and is performed according to a standardized sample pretreatment procedure. Step 4: Based on the paired data obtained in Step 2 and Step 3, the intercept and slope of the linear transformation model are calculated using the Deming regression method to satisfy the following: any immunoassay value can be converted into the corresponding mass spectrometry equivalent value by the linear transformation rule that "the mass spectrometry equivalent value is equal to the intercept plus the slope multiplied by the immunoassay value". Step 5: In routine testing, perform mass spectrometry verification of samples that meet the triggering conditions according to the preset triggering rules, and incorporate the newly obtained paired data into the model update queue. Reconstruct and save the intercept and slope according to the predetermined cycle or cumulative sample number, so that the immunoassay values ​​can be converted into mass spectrometry equivalent values ​​and the model version and source can be noted in the test report.

[0005] Furthermore, the internal standard used for mass spectrometry quantification is an isotope-labeled internal standard corresponding to each target analyte, used to correct for sample pretreatment recovery, injection differences, and matrix effects.

[0006] Furthermore, the sample pretreatment includes: protein precipitation with an organic solvent to remove protein interference, followed by purification and enrichment of the supernatant by solid-phase extraction to improve the recovery and selectivity of mass spectrometry determination.

[0007] Furthermore, the intercept and slope must meet the following acceptance criteria when they are established: the difference sequence is calculated based on the paired data, the consistency interval is obtained by using the Bland-Altman method, and the proportion of paired samples in the consistency interval is not less than 95 percentage points; at the same time, the average relative bias is calculated and made to be within the allowable total error range, preferably the allowable total error is plus or minus 15 percentage points; in addition, the number of paired samples used when initially establishing the transformation model is not less than 60.

[0008] Furthermore, the kit includes at least: an antibody reagent set for immunological assays, multi-point calibrators matched to clinical sample matrix, low / medium / high quality control materials, interference detection reagents for identifying or blocking heterophile antibodies, and an information interface or electronic file for receiving, storing, or attaching the intercept and slope of the mass spectrometry-immunoassay conversion model. The instruction manual or electronic document accompanying the kit indicates that when the reagent batch is changed or the preset sample size or preset cycle is reached, mass spectrometry calibration should be performed according to the method of claim 1, and the updated intercept and slope should be written back or published.

[0009] Furthermore, the software product includes a software product for automatically converting immunological assay results into mass spectrometry equivalents and managing the conversion model. The software contains executable instructions to perform the following functions: receiving and managing paired datasets; performing Deming regression based on the received data to calculate the intercept and slope of the linear conversion model; calculating the Bland-Altman index, mean bias, and consistency interval, and determining whether the model passes according to preset allowable total error and coverage criteria; versioning the passed model and providing the function of writing version information back to the reagent kit electronic interface or laboratory information system; and automatically determining whether mass spectrometry validation needs to be triggered for a single sample or batch of samples according to preset triggering rules during daily operation.

[0010] Furthermore, the preset triggering rule includes at least one of the following situations: a) Mass spectrometry validation is triggered if the immunoassay value is close to or falls within a preset critical range of the clinical critical decision threshold defined by clinical guidelines or the laboratory; b) Mass spectrometry validation is triggered if the relative change between the current immunoassay value and the previous value of the same patient exceeds a preset threshold set by the laboratory; c) Mass spectrometry validation is triggered if the quality control index for the current test or batch exceeds a preset threshold, or if heterophile antibodies or other interference tests are positive; d) Mass spectrometry validation is triggered by random sampling according to a preset sampling ratio.

[0011] Furthermore, the model update and drift monitoring includes: automatically reconstructing the Deming regression model and generating a new version when the cumulative number of mass spectrometry validation paired samples reaches a laboratory-preset threshold or a preset time period; using a statistical process control algorithm to monitor the continuously obtained average bias or model parameters online, and automatically issuing a reconstruction or manual review alarm if the monitoring algorithm detects statistically significant drift; and writing back the new intercept and slope obtained after reconstruction as a new version and replacing the previous version for subsequent conversion of immunological measurement values ​​to mass spectrometry equivalent values.

[0012] This invention has at least the following beneficial effects: This invention establishes a conversion model by combining immunoassay and LC-MS / MS, enabling accurate conversion of immunoassay values ​​to mass spectrometry equivalents. By integrating unified pretreatment, interference suppression, triggered mass spectrometry verification, and model drift monitoring, it significantly improves the accuracy and consistency of detecting steroid hormone-containing immunosuppressants. While ensuring the high throughput and ease of operation of immunoassay, it provides accuracy comparable to mass spectrometry, offering a reliable basis for personalized clinical medication. Attached Figure Description

[0013] Figure 1 This is a flowchart of the combined detection and correction method for precise monitoring of immunosuppressants containing steroid hormones, as described in this invention. Detailed Implementation

[0014] The following will describe in detail the implementation of this application with reference to the accompanying drawings and embodiments, so that the implementation process of how this application uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0015] like Figure 1 As shown, the steroid hormone-containing immunosuppressant precision monitoring kit provided in this embodiment obtains paired data by dividing each clinical sample into two parts: immunological detection and liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection. Using the mass spectrometry results as a reference, a linear conversion model from immunological assay values ​​to mass spectrometry equivalent values ​​is established using Deming regression. The immunological assay value is denoted as... The mass spectrometry values ​​are recorded as follows: By calculating the intercept With slope Forming a relation This is used to correct for systematic and proportionality biases introduced by the immunization method. The Bland–Altman method is used to calculate the differences during model building. The system specifies the consistency range and requires a coverage rate of no less than 95% and an average relative bias of no more than ±15% to ensure the reliability of the conversion model. A triggered mass spectrometry validation mechanism is also implemented, automatically initiating LC-MS / MS verification when immunoassay results fall into critical threshold ranges, patient results fluctuate abnormally, quality control results are abnormal, or random sampling occurs. Newly added paired data are then included in the model update queue. The system uses statistical process control to manage model parameters. and Drift monitoring is implemented, and the model is rebuilt periodically or by sample size to ensure the continued effectiveness of immunoassay correction. The kit of this invention includes an antibody reagent set, calibrators, quality control materials, and interference inhibitors, and is equipped with an electronic interface for storing model parameters. The software system can perform regression calculations, consistency analysis, model version management, and automatic result conversion, achieving a synergy between high-throughput immunoassay and high-accuracy mass spectrometry detection, making immunosuppressant monitoring more precise and reliable.

[0016] Specifically, it includes a combined detection and correction method for the precise monitoring of immunosuppressants containing steroid hormones, comprising the following steps: Step 1: Collect the clinical samples to be tested and divide each collected sample into two equal parts; Step 2: Perform immunological quantitative determination on the first aliquot of the sample using an immunological kit to obtain the immunoassay value, which is recorded as follows: ; Step 3: Perform reference quantification on the second aliquot of the sample corresponding to Step 2 using liquid chromatography-tandem mass spectrometry (LC-MS / MS). After eliminating interference, obtain the true mass spectrometry value. Where i = 1, 2, ..., n are the paired sample numbers; Mass spectrometry quantification uses an isotope-labeled internal standard corresponding to each target analyte and follows a standardized sample pretreatment process (protein precipitation + solid phase extraction). The internal standard used in mass spectrometry quantification is an isotope-labeled internal standard corresponding to each target analyte, used to correct for sample pretreatment recovery, injection differences and matrix effects. Sample pretreatment includes: protein precipitation with organic solvents to remove protein interference, followed by purification and enrichment of the supernatant by solid phase extraction to improve the recovery and selectivity of mass spectrometry determination. To obtain highly accurate quantitative results; Step 4: Based on the paired data obtained in Step 2 and Step 3, the intercept and slope of the linear transformation model are calculated using the Deming regression method to satisfy the following: any immunoassay value can be converted into the corresponding mass spectrometry equivalent value by the linear transformation rule that "the mass spectrometry equivalent value is equal to the intercept plus the slope multiplied by the immunoassay value". Deming regression was used to find the optimal linear relationship between the immunoassay and the mass spectrometry values, avoiding systemic bias caused by potential cross-reactivity and matrix effects in the immunoassay. The model is in the following form: ; In the formula, This is the intercept (systematic offset). Slope (proportional offset). The term represents the residuals, assuming that both the immunoassay and mass spectrometry methods have measurement errors. Deming regression algorithm: Let the sample mean be: Define covariance and variance: In the formula, It represents the variance estimate of immunological assay data and is used to describe the data dispersion of the immunological method itself; It represents the variance estimate of mass spectrometry data and is used to describe the dispersion of mass spectrometry data. It represents the covariance estimate between immunological assays and mass spectrometry assays, which reflects the degree of linear correlation between the two methods and is an important parameter for calculating the slope in Deming regression. The error-to-variance ratio is set as follows: set up ; The formula for calculating the Deming regression slope b is: The intercept 'a' is calculated as follows: Real-time conversion of immunoassay results to mass spectrometry equivalents: After model determination, conventional immunoassay results are converted to mass spectrometry equivalents using the following formula: In the formula, This represents the converted mass spectrometry equivalent value, used to replace direct mass spectrometry detection and improve accuracy. To further ensure model quality, the Bland–Altman method was used to evaluate the consistency of the differences; Define the difference sequence: Calculate the average difference: Standard deviation : The 95% limit of agreement is: Model acceptance criteria: Consistency intervals cover ≥ 95% of paired values; mean relative bias ≤ ±15%; initial modeling sample size n ≥ 60 (the intercept and slope must meet the following acceptance criteria when establishing the model: calculate the difference sequence based on paired data, obtain the consistency interval using the Bland–Altman method, and the proportion of paired samples within the consistency interval is not less than 95%; at the same time, calculate the mean relative bias and keep it within the allowable total error range, preferably the allowable total error is ±15%; in addition, the number of paired samples used when initially establishing the transformation model is not less than 60). Step 5: In routine testing, mass spectrometry validation is triggered according to preset triggering rules (including at least one of the following situations: a) If the immunoassay value is close to or falls within the preset critical range of the clinical critical decision threshold defined by clinical guidelines or the laboratory, mass spectrometry validation is triggered; b) If the relative change between the current immunoassay value and its previous value for the same patient exceeds the laboratory's preset threshold, mass spectrometry validation is triggered; c) If the quality control index for the current test or batch exceeds the preset threshold, or if heterophile antibodies or other interference tests are positive, mass spectrometry validation is triggered; d) Random sampling is performed according to a preset sampling ratio to trigger mass spectrometry validation. For samples that meet the triggering conditions, mass spectrometry validation is performed on samples that meet the triggering conditions, and the newly obtained paired data is added to the model update queue. The intercept and slope are reconstructed and versioned according to a predetermined period or cumulative sample number, so that the immunoassay value can be converted into the mass spectrometry equivalent value and the model version and source used can be noted in the test report. Model updates and drift monitoring include: automatically reconstructing the Deming regression model and generating a new version when the cumulative number of mass spectrometry validation paired samples reaches a laboratory-preset threshold or a preset time period; using a statistical process control algorithm to monitor the continuously obtained average bias or model parameters online, and automatically issuing a reconstruction or manual review alarm if the monitoring algorithm detects statistically significant drift; and writing back the new intercept and slope obtained after reconstruction as a new version to replace the previous version for subsequent conversion of immunological assay values ​​to mass spectrometry equivalent values. Example: The system reconstructs the Deming model based on the cumulative number of samples reaching N (e.g., 100) or the period (e.g., 30 days); The statistical process control monitors the drift of intercept a and slope b. If the drift exceeds the control line, the model is automatically rebuilt and a warning is issued.

[0017] Reagent kit composition: Antibody reagent kits for immunological assays, multi-point calibrators matched to clinical sample matrices, low / medium / high-grade quality control products, interference detection reagents for identifying or blocking heterophile antibodies, and information interfaces or electronic files for receiving, storing or accompanying mass spectrometry-immunoassay conversion models of intercept and slope. The instructions or electronic documents accompanying the kit state that when the reagent batch is changed or the preset sample size or preset cycle is reached, mass spectrometry calibration should be performed according to the method of claim 1, and the updated intercept and slope should be written back or published. Through the above-described structure, the kit provides end-to-end calibrability and traceability in the immunoassay process, enabling continuous and dynamic correction of differences between immunoassay and mass spectrometry methods, significantly improving cross-batch consistency and long-term stability. At the same time, the automatic writing and updating of model parameters through the information interface can reduce errors caused by manual operation and improve the intelligence and standardization of the detection system.

[0018] A software product for automatically converting immunological assay results into mass spectrometry equivalents and managing conversion models. The software includes executable instructions to perform the following functions: receiving and managing paired datasets; performing Deming regression based on the received data to calculate the intercept and slope of the linear conversion model; calculating the Bland-Altman index, mean bias, and consistency interval, and determining whether the model passes according to preset allowable total error and coverage criteria; versioning the passed models and providing the function of writing version information back to the reagent kit electronic interface or laboratory information system; and automatically determining whether mass spectrometry validation needs to be triggered for a single sample or batch of samples according to preset trigger rules during daily operation. This software product enables automated and seamless conversion of immunological assay results to mass spectrometry equivalents, reducing biases caused by manual judgment and calculation, and improving the comparability of clinical test results. At the same time, its built-in model validation, version management, and trigger-based mass spectrometry verification mechanisms ensure that the model remains accurate, stable, and controllable during long-term operation, thereby improving the quality management level and clinical application reliability of the entire testing system.

[0019] If certain terms are used in the specification and claims to refer to specific components, those skilled in the art will understand that hardware manufacturers may use different names to refer to the same component. This specification and claims do not distinguish components based on differences in name, but rather on differences in function. The term "comprising" as used throughout the specification and claims is an open-ended term and should be interpreted as "comprising but not limited to." "Approximately" means that within an acceptable margin of error, those skilled in the art can solve the technical problem and substantially achieve the technical effect within a certain margin of error.

[0020] It should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a product or system comprising a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a product or system. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the product or system that includes that element.

[0021] The foregoing description illustrates and describes several preferred embodiments of the present invention. However, as previously stated, it should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the foregoing teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A kit for precise monitoring and treatment of immunosuppressants containing steroid hormones, characterized in that, This includes a combined detection and correction method for the precise monitoring of immunosuppressants containing steroid hormones, comprising the following steps: Step 1: Collect the clinical samples to be tested and divide each collected sample into two equal parts; Step 2: Use an immunological kit to perform immunological quantitative determination on the first aliquot of the sample to obtain the immunological determination value; Step 3: The second aliquot of the sample corresponding to Step 2 is quantitatively analyzed by liquid chromatography-tandem mass spectrometry to obtain the mass spectrometry values. The mass spectrometry quantification uses an isotope-labeled internal standard corresponding to each target analyte and is performed according to a standardized sample pretreatment procedure. Step 4: Based on the paired data obtained in Step 2 and Step 3, the intercept and slope of the linear transformation model are calculated using the Deming regression method to satisfy the following: any immunoassay value can be converted into the corresponding mass spectrometry equivalent value by the linear transformation rule that "the mass spectrometry equivalent value is equal to the intercept plus the slope multiplied by the immunoassay value". Step 5: In routine testing, perform mass spectrometry verification of samples that meet the triggering conditions according to the preset triggering rules, and incorporate the newly obtained paired data into the model update queue. Reconstruct and save the intercept and slope according to the predetermined cycle or cumulative sample number, so that the immunoassay values ​​can be converted into mass spectrometry equivalent values ​​and the model version and source can be noted in the test report.

2. The combined detection and correction method for accurate monitoring of immunosuppressants containing steroid hormones according to claim 1, characterized in that: The internal standard used for mass spectrometry quantification is an isotope-labeled internal standard corresponding to each target analyte, used to correct for sample pretreatment recovery, injection differences, and matrix effects.

3. The combined detection and correction method for accurate monitoring of immunosuppressants containing steroid hormones according to claim 1, characterized in that: The sample pretreatment includes: protein precipitation with organic solvents to remove protein interference, followed by purification and enrichment of the supernatant by solid-phase extraction to improve the recovery and selectivity of mass spectrometry.

4. The combined detection and correction method for accurate monitoring of immunosuppressants containing steroid hormones according to claim 1, characterized in that: The intercept and slope must meet the following acceptance criteria when they are established: the difference sequence is calculated based on the paired data, the consistency interval is obtained by using the Bland-Altman method, and the proportion of paired samples in the consistency interval is not less than 95%. Simultaneously calculate the average relative bias and keep it within the allowable total error range, preferably with an allowable total error of plus or minus 15 percentage points; In addition, the number of paired samples used when initially establishing the transformation model should not be less than sixty.

5. The precise monitoring and treatment kit for steroid hormone-containing immunosuppressants according to claim 1, characterized in that: The kit includes at least: an antibody reagent set for immunological assays, multi-point calibrators matched to clinical sample matrix, low / medium / high quality control materials, interference detection reagents for identifying or blocking heterophile antibodies, and an information interface or electronic file for receiving, storing or attaching the intercept and slope of the mass spectrometry-immunoassay conversion model. The instruction manual or electronic document accompanying the kit indicates that when the reagent batch is changed or the preset sample size or preset cycle is reached, mass spectrometry calibration should be performed according to the method of claim 1, and the updated intercept and slope should be written back or published.

6. The precise monitoring and treatment kit for immunosuppressants containing steroid hormones according to claim 1, characterized in that, This includes a software product for automatically converting immunological assay results into mass spectrometry equivalents and managing conversion models. The software contains executable instructions to perform the following functions: receiving and managing paired datasets; performing Deming regression based on the received data to calculate the intercept and slope of the linear conversion model; calculating the Bland-Altman index, mean bias, and consistency interval, and determining model pass / fail based on preset allowable total error and coverage criteria; versioning successful models and providing the function of writing version information back to the reagent kit electronic interface or laboratory information system; and automatically determining whether mass spectrometry validation needs to be triggered for individual samples or sample batches based on preset trigger rules during routine operation.

7. The precise monitoring and treatment kit for steroid hormone-containing immunosuppressants according to claim 1, characterized in that: The preset triggering rule includes at least one of the following situations: a) Mass spectrometry validation is triggered if the immunoassay value is close to or falls within a preset critical range of the clinical critical decision threshold defined by clinical guidelines or the laboratory; b) Mass spectrometry validation is triggered if the relative change between the current immunoassay value and the previous value of the same patient exceeds a preset threshold set by the laboratory; c) Mass spectrometry validation is triggered if the quality control index for the current test or batch exceeds a preset threshold, or if heterophile antibodies or other interference tests are positive; d) Mass spectrometry validation is triggered by random sampling according to a preset sampling ratio.

8. The precise monitoring and treatment kit for steroid hormone-containing immunosuppressants according to claim 1, characterized in that: The model update and drift monitoring includes: automatically reconstructing the Deming regression model and generating a new version when the cumulative number of mass spectrometry validation paired samples reaches a preset threshold or a preset time period; using a statistical process control algorithm to monitor the continuously obtained average bias or model parameters online, and automatically issuing a reconstruction or manual review alarm if the monitoring algorithm detects statistically significant drift; and writing back the new intercept and slope obtained after reconstruction as a new version and replacing the previous version for subsequent conversion of immunological measurement values ​​to mass spectrometry equivalent values.

Images