Combined microsphere reagent operation detection method based on flow type fluorescence platform

Abstract

The invention discloses a combined microsphere reagent operation detection method based on a flow type fluorescence platform, and belongs to the technical field of medical examination, and the method comprises the following steps: S1, providing a kit containing 2-N bottles of microspheres, each bottle of microspheres containing one or more different specific targets; items contained in different microsphere bottles are not overlapped, and microsphere codes are not repeated; s2, non-microsphere components such as a diluent and a labeled antibody in the microsphere kit are universal reagents and have uniform reaction conditions; s3, acquiring LIS billing detection information of a patient, identifying items needing to be detected, selecting corresponding microsphere bottles, combining according to needs and adding into the same reaction cup, the problem that flow fluorescence can only be used for joint detection and cannot be used for single-item detection is solved, personalized combined detection of flow fluorescent microsphere reagents is effectively achieved, detection requirements of different patients are met, waste of irrelevant reagents is reduced, and the detection efficiency is improved. Meanwhile, the reaction conditions of the microspheres after uniform dilution treatment are consistent, and the accuracy and comparability of results are improved.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to a combined microsphere reagent operation and detection method based on a flow cytometry platform. Background Technology

[0002] In clinical laboratory testing, with the development of personalized medicine, doctors often select specific tests from the Laboratory Information System (LIS) based on the patient's specific symptoms or medical history. However, existing multi-item combined test kits (such as the ToRCH IgM series) typically use fixed combinations, which cannot be flexibly split according to actual needs and can only perform the entire test. This not only increases testing costs but also limits the accuracy and specificity of the tests.

[0003] Traditional techniques such as chemiluminescence immunoassay can only perform single-item detection, resulting in low throughput, inflexible combination, and slow speed. In recent years, flow cytometry microsphere technology has been widely used in the field of in vitro diagnostics due to its coded identification capabilities and suitability for parallel detection of multiple items. Currently, most commercially available flow cytometry microsphere kits have fixed formulations and cannot be combined as needed, still exhibiting the problem of "forced bundling for joint testing and inability to perform individual retests to achieve on-demand detection."

[0004] Based on this, the present invention designs a combined microsphere reagent operation and detection method based on a flow cytometry fluorescence platform to solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a combined microsphere reagent operation and detection method based on a flow cytometry platform, so as to overcome the problem that flow cytometry can only perform joint detection and cannot perform single detection to achieve on-demand detection.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for the operation and detection of combined microsphere reagents based on a flow cytometry fluorescence platform, comprising the following steps: S1. A kit containing 2-N vials of microspheres, each vial containing one or more different specific targets. The targets contained in different vials do not overlap, and the microsphere codes are unique. S2. The diluent, labeled antibody, and other non-microsphere components in the microsphere kit are universal reagents with uniform reaction conditions. S3. Obtain the patient's LIS test order information, identify the items to be tested, select the microspheres in the corresponding reagent kit, and add them to the same reaction cup as needed. S4. Place the reaction cup on the magnetic separation device to adsorb and fix the microspheres by magnetic attraction, and remove the supernatant. S5. Add a fixed volume (e.g., 50 μL) of diluent to the reaction vessel to unify the reaction system; S6. Follow the standard flow cytometry fluorescence detection platform procedures for subsequent reactions, including incubation, cleaning, and fluorescence reading; S7. Establish a detection template that includes all possible combinations of microspheres, but only analyze the detection results corresponding to the items opened in LIS, or only report the results of items that have detected a sufficient number of microspheres.

[0007] Preferably, the magnetic separation device is a magnetic separation module integrated into a chemiluminescence analyzer. The module is used to apply a magnetic field from the side of the reaction cup to achieve the adsorption and fixation of microspheres.

[0008] Preferably, the diluent is 50 μL PBS buffer with a pH of 7.4.

[0009] Preferably, the microspheres are distinguished by fluorescent coding and identified by a flow cytometry detection platform.

[0010] Preferably, the detection template only reports the result of the project when the number of a certain microsphere is greater than a set threshold.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: Firstly, it supports on-demand combination and flexible splitting of multiple tests, improving testing throughput and reagent utilization. Secondly, it can accurately respond to the personalized ordering needs of the LIS system, and is suitable for large-scale screening and re-examination scenarios; Thirdly, the universal reagents and modular microsphere design reduce reagent development costs and experimental complexity, and all reagents have a unified operating procedure and consistent post-processing. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a schematic diagram of four different microspheres used in this invention, taking the ToRCH IgM kit as an example; Figure 2 This is a schematic diagram illustrating the patient's test order requirements in the Laboratory Information System (LIS) according to the present invention; Figure 3 This is a schematic diagram of the microspheres added to the reaction cup according to the LIS test items ordered by the patient in this invention. Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Please see Figure 1-3 This embodiment was implemented on a flow cytometry platform: the combined reagents, using the ToRCH IgM kit, included the following four types of microspheres: Microsphere 1 contains microspheres coupled with Toxo, and the detection item is Toxo IgM; Microsphere 2 contains microspheres coupled with Rub, and the detection item is Rub IgM; Microsphere 3 contains microspheres coupled with CMV, and the detection item is CMV IgM; Microsphere 4 includes microspheres coupled with HSV-1 and microspheres coupled with HSV-2, and the detection items are HSV-1 IgM and HSV-2 IgM.

[0016] The common components (such as diluent, fluorescently labeled secondary antibody, etc.) in all the above projects remain the same, only the types of microspheres encoding and conjugated antigens differ. Patients' testing needs when ordering tests in the Laboratory Information System (LIS) are as follows: Patient 1 was tested for Toxo IgM and CMV IgM; Patient 2 was tested for Rub IgM; Patient 3 was tested for Toxo IgM, Rub IgM, CMV IgM, HSV-1 IgM, and HSV-2 IgM. Patient 4 was tested for HSV-1 IgM and HSV-2 IgM. Patient 5 was tested for CMV IgM, HSV-1 IgM, and HSV-2 IgM.

[0017] In the actual testing process, based on the patient's order in the Laboratory Information System (LIS), the required tests were determined as described above, and the microspheres added to the reaction cup were as follows: Patient 1 was given microspheres 1 and 3; Patient 2 was given microspheres 2; Patient 3 was given microspheres 1, 2, 3, and 4. Patient 4 was given 4 microspheres; Patient 5 was given microspheres 3 and 4.

[0018] The specific operation is as follows: Based on the patient's LIS order items and order results information, take the corresponding microspheres from the four types of microsphere reagent kits and add them to the same reaction cup. Then, place the reaction cup into the magnetic adsorption device to adsorb the microspheres, remove the supernatant of the reaction solution, and add 50μL of diluent to the reaction cup to standardize the reaction system. Subsequently, incubate, label, and read the fluorescence signal according to a unified procedure. The system identifies and analyzes based on the detection template containing all microsphere codes, and only analyzes and reports the results for the microspheres corresponding to the items in the LIS order.

[0019] This embodiment effectively realizes personalized combination detection of flow cytometry fluorescent microsphere reagents, adapts to the testing needs of different patients, reduces waste of irrelevant reagents, and ensures consistent reaction conditions after uniform dilution of microspheres, thereby improving the accuracy and comparability of results.

[0020] In this invention, unless otherwise explicitly specified and limited, the terminology should be interpreted broadly. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0021] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting combined microsphere reagents based on a flow cytometry fluorescence platform, characterized in that, Includes the following steps: S1. A kit containing 2-N vials of microspheres, each vial containing one or more different specific targets. The targets contained in different vials do not overlap, and the microsphere codes are unique. S2. The diluent, labeled antibody, and other non-microsphere components in the microsphere kit are universal reagents with uniform reaction conditions. S3. Obtain the patient's LIS test order information, identify the items to be tested, select the microspheres in the corresponding reagent kit, and add them to the same reaction cup as needed. S4. Place the reaction cup on the magnetic separation device to adsorb and fix the microspheres by magnetic attraction, and remove the supernatant. S5. Add a fixed volume (e.g., 50 μL) of diluent to the reaction vessel to unify the reaction system; S6. Follow the standard flow cytometry fluorescence detection platform procedures for subsequent reactions, including incubation, cleaning, and fluorescence reading; S7. Establish a detection template that includes all possible combinations of microspheres, but only analyze the detection results corresponding to the items opened in LIS, or only report the results of items that have detected a sufficient number of microspheres.

2. The method for handling and detecting combined microsphere reagents based on a flow cytometry fluorescence platform according to claim 1, characterized in that: The magnetic separation device is a magnetic separation module integrated into a flow cytometer. The module is used to apply a magnetic field to the side of the reaction cup to achieve the adsorption and fixation of microspheres.

3. The method for handling and detecting combined microsphere reagents based on a flow cytometry fluorescence platform according to claim 1, characterized in that: The diluent was 50 μL PBS buffer with a pH of 7.

4.

4. The method for handling and detecting combined microsphere reagents based on a flow cytometry fluorescence platform according to claim 1, characterized in that: The microspheres are distinguished by fluorescent coding and identified by a flow cytometry detection platform.

5. The method for handling and detecting combined microsphere reagents based on a flow cytometry fluorescence platform according to claim 1, characterized in that: The detection template only reports the result of the project when the number of a certain microsphere is greater than a set threshold.

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