Tuberculosis infection T cell spot test method based on sample preservation time and MBS method PBMCs enrichment frequency

Abstract

The invention discloses a tuberculosis infection T cell spot test method based on sample preservation time and MBS method PBMCs enrichment times, and belongs to the technical field of tuberculosis infection detection. The PBMCs are automatically enriched in a fresh whole blood sample (0-4 hours) by adopting an MBS method, a magnetic bead enrichment procedure is selected twice, a T-SPOT.TB test result is highly consistent with a result of a conventional DGC method and a magnetic bead enrichment procedure for four times, and the manual operation time is shorter; the MBS method is adopted to automatically enrich PBMCs for a long-time preserved whole blood sample (longer than 4 h), four magnetic bead enrichment procedures are selected, and the T-SPOT. TB test result is highly consistent with the result of a conventional DGC method and is remarkably superior to that of two times of MBS enrichment. Therefore, different magnetic bead enrichment procedures are adopted for fresh whole blood specimens with different preservation times, so that the detection efficiency can be effectively improved on the basis of ensuring the reliable result, the automation of T-SPOT. TB test can be realized, the manual operation time is shortened, and the detection efficiency is improved. And a more optimized pretreatment scheme and a reliable test result are provided for the T-SPOT.TB test blood sample which is stored for a long time.

Description

Technical Field

[0001] The invention belongs to the technical field of tuberculosis infection detection, and particularly relates to a tuberculosis infection T cell spot test method based on sample storage time and PBMCs enrichment times by an MBS method. Background Art

[0002] Tuberculosis (TB), a global chronic infectious disease caused by infection with Mycobacterium tuberculosis (MTB), poses a serious threat to human health and a major challenge to global public health. TB presents diverse and uncharacteristic clinical manifestations, particularly with the prevalence of latent tuberculosis infection (LTBI). Furthermore, obtaining diagnostically valuable pathogenic specimens is difficult, while traditional laboratory tests such as acid-fast smears and tuberculosis cultures have low positive rates, and molecular biological testing has certain barriers to diagnosis. Therefore, improving and refining rapid TB detection and diagnostic technologies is crucial to achieving the ultimate goal of ending TB.

[0003] The interferon-gamma release assay (IGRA) is an important laboratory-based diagnostic tool for tuberculosis (TB) infection. It measures the release of specific IFN-γ from peripheral blood T cells activated by MTB-specific antigens in vitro, reflecting the presence of infection. Currently, the most commonly used assays include the enzyme-linked immunosorbent spot assay (ELISPOT) and the enzyme-linked immunosorbent assay (ELISA). The ELISPOT assay measures the frequency of IFN-γ-releasing effector T cells, exemplified by the Oxford Immunotec Tuberculosis Infection T Cell Spot Assay (T-SPOT.TB) from the UK, and the ELISA assay measures the level of IFN-γ release, exemplified by the Qiagen QuantiFERON-TB Gold in tube (QFT-GIT) from Germany. Both assays offer advantages in sensitivity and specificity.

[0004] The T-SPOT.TB test is an IGRA assay based on the ELISPOT method. It uses the MTB-specific early secretory target antigen 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) to stimulate peripheral blood mononuclear cells (PBMCs) to measure the MTB immune response at the cellular level. Therefore, the isolation and purification of PBMCs are crucial steps in the T-SPOT.TB test. However, the current conventional density gradient centrifugation (DGC) method for isolating PBMCs requires manual operation, which is cumbersome and time-consuming. It lacks automation and standardization, making it difficult to ensure the stability of test results. It also requires fresh specimens to be collected and processed as soon as possible. Insufficient PBMCs extracted will affect test results, limiting the development of large-scale LTBI screening and the need to expand routine clinical trials.

[0005] In light of this, Oxford Immunotec, a UK company, has developed the T-Cell Select Kit, a T cell sorting kit based on magnetic beads sorting (MBS) technology. This kit uses superparamagnetic beads coupled to T cell-specific antibodies to capture sample T cells. The cells are then rapidly enriched and purified using the magnetic beads in a fully automated cell sorter. Compared to conventional manual DGC methods, MBS-based T cell sorting offers advantages such as ease of operation, high automation, and ease of standardization. However, it still suffers from low detection efficiency. Summary of the Invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a tuberculosis infection T cell spot test method based on sample storage time and PBMCs enrichment times by MBS method, so as to solve the technical problem of low efficiency of T cell sorting and detection.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention discloses a tuberculosis-infected T cell spot test method based on sample storage time and the number of PBMCs enrichment times by the MBS method, comprising the following steps:

[0009] Step 1: Determine the storage time of the whole blood sample;

[0010] Step 2: If the whole blood sample has been stored for 0 to 4 hours, perform magnetic bead enrichment on the whole blood sample twice using the T-Cell Select magnetic bead sorting method, and then perform a tuberculosis infection T cell spot test; if the whole blood sample has been stored for more than 4 hours, perform magnetic bead enrichment on the whole blood sample four times using the T-Cell Select magnetic bead sorting method, and then perform a tuberculosis infection T cell spot test.

[0011] Preferably, the amount of whole blood sample used is 4 mL.

[0012] Preferably, magnetic bead enrichment is performed using a T-Cell Select kit, which includes a concentration buffer, antibody 1, antibody 2, and a magnetic bead reagent.

[0013] More preferably, the volume ratio of antibody 1, antibody 2 and whole blood sample is 1:1:100.

[0014] Preferably, the volume ratio of antibody 1, antibody 2 and magnetic bead reagent is 2:2:3.

[0015] Preferably, the instrument used in the magnetic bead enrichment process is an automatic cell sorter.

[0016] Further preferably, the magnetic bead enrichment procedure is adsorption, washing and elution.

[0017] More preferably, RPMI-1640 cell culture medium is used for washing.

[0018] Preferably, AIM-V culture medium is used for elution.

[0019] The second aspect of the present invention discloses the use of the above-mentioned tuberculosis-infected T cell spot test method based on sample storage time and PBMCs enrichment times by the MBS method in the preparation of a tuberculosis detection device.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a tuberculosis-infected T-cell spot test method based on sample storage time and the number of PBMCs enrichment times by the MBS method. The method comprises the following steps: PBMCs are separated immediately (0 to 4 hours) from a fresh whole blood sample by density gradient centrifugation (DGC); PBMCs are separated from the whole blood sample by the MBS method after storage for 0 to 4 hours or after 4 hours; the MBS separation procedure is adjusted from the four times recommended by the reagent to two magnetic bead enrichment times; and the consistency of the T-SPOT.TB test results of each treatment group is compared. The results show that, compared with the results of the DGC method, the T-SPOT.TB test results of the fresh (0 to 4 hours) whole blood sample enriched by the MBS method four times and twice have positive coincidence rates of 91.7% and 93.1%, negative coincidence rates of 100%, and total coincidence rates of 98.0% and 97.9%. The T-SPOT.TB assay results for long-term (34-55 hours) storage samples enriched with the MBS method four times and twice showed a positive concordance rate of 90.0% and 81.3%, a negative concordance rate of 95.1% and 100%, and an overall concordance rate of 93.0% and 82.9%. These results demonstrate that the T-SPOT.TB assay results for automated PBMC enrichment using the MBS method for fresh whole blood samples are highly consistent with those of the conventional DGC method, regardless of whether the magnetic bead enrichment procedure is four or two times. The T-SPOT.TB assay results for long-term storage whole blood samples enriched with the MBS method four times were significantly more consistent with the DGC method results than those obtained with two MBS enrichments. Therefore, by adopting different magnetic bead enrichment procedures for fresh whole blood specimens with different storage times, the whole blood specimens stored for 0 to 4 hours are subjected to the magnetic bead enrichment procedure twice, and the whole blood specimens stored for more than 4 hours are subjected to the magnetic bead enrichment procedure four times. On the basis of ensuring the reliability of the results, the detection efficiency can be effectively improved, the automation of the T-SPOT.TB test can be realized, the manual operation time can be shortened, and a more optimized pretreatment scheme and reliable test results can be provided for the long-term stored T-SPOT.TB test blood specimens. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Design a roadmap for the experiments of the present invention;

[0023] Figure 2 Schematic diagram of the seven-tube strip used in the T-Cell Select-MBS method for isolating PBMCs in the present invention; ① refers to the first tube to which the "buffer-antibody-blood-magnetic bead" mixture was added; ②, ③, ④, and ⑤ refer to the second to fifth tubes to which 1 mL of RPMI-1640 cell culture medium was added, respectively; ⑥ refers to the sixth tube, which is empty; and ⑦ refers to the seventh tube to which 550 μL of AIM-V culture medium was added.

[0024] Figure 3This is a flowchart of the magnetic bead enrichment process of an automated cell sorter during the T-Cell Select-MBS method for isolating PBMCs according to the present invention; wherein ① refers to tube 1 in a seven-tube strip, ②, ③, ④, and ⑤ refer to tubes 2, 3, 4, and 5 in the seven-tube strip, respectively, and ⑦ refers to tube 7 in the seven-tube strip.

[0025] Figure 4 The density gradient centrifugation (DGC) method of the present invention is used to separate the lymphocyte subsets of PBMCs; wherein A is the total particle scatter plot, B is the total CD4 + T cell scatter plot, C is memory CD4 + T cells and naive T cells scatter plot, D is B cells scatter plot, E is CD4 without regulatory Treg + T cell scatter plot, F is Th1 cell scatter plot;

[0026] Figure 5 The present invention is a magnetic bead sorting method (MBS) to separate the lymphocyte subsets of PBMCs; wherein A is a total particle scatter plot, B is a total CD4 + T cell scatter plot, C is memory CD4 + T cells and naive T cells scatter plot, D is B cells scatter plot, E is CD4 without regulatory Treg + T cell scatter plot, F is Th1 cell scatter plot. DETAILED DESCRIPTION

[0027] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0028] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0029] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0030] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0031] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0032] The following combination Figure 1 The present invention is further described with reference to specific embodiments. It should be understood that these embodiments are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the contents of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims appended hereto.

[0033] The following examples use conventional instruments and equipment in the art. Experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0034] In the following examples, the sample sources were selected from 300 outpatient and inpatient tuberculosis patients from the First Affiliated Hospital of Air Force Medical University from March 2023 to July 2024, including 167 males and 133 females aged 18 to 65 years. Two tubes of lithium heparin anticoagulated venous blood (4 mL / tube, 8 mL in total) were drawn from each patient for cell separation using the DGC method and the MBS method, respectively. All patients in this study signed an informed consent form, and the study was approved by the Drug Clinical Trial Ethics Committee of the First Affiliated Hospital of Air Force Medical University (Approval No.: QX20201018-1).

[0035] In the following examples, reagents and instruments: T-Cell Select kit (Table 1) and T-SPOT.TB kit (Table 2) were purchased from Oxford Immunotec, UK; Ficoll lymphocyte separation medium, AIM-V culture medium, and RPMI-1640 cell culture medium were purchased from Tianjin Haoyang Huake Biotechnology; fluorescent dye-conjugated monoclonal antibodies FITC-TCRab, BV785-CD19, PE-cy-CD4, BV510-CD8a, PECF594-CD25, APC-cy7-CD45RA, APC-CD127, PE-CCR6, and APC700-CCR7 were purchased from BioLegend, USA; Mag-Sep The 20B fully automatic cell sorter was purchased from Hangzhou Aosheng, with a maximum processing capacity of 20 samples; the ST8R desktop low-temperature centrifuge was purchased from Thermo Fisher Scientific, USA; the BC-30s fully automatic three-differentiation blood cell analyzer was purchased from Shenzhen Mindray; the CO2 incubator was purchased from Astec, Japan; the flow cytometer CytoFLEX was purchased from Beckman Coulter, USA; and the lithium heparin anticoagulant blood collection tubes were purchased from Guangzhou Yangpu Medical Equipment Co., Ltd.

[0036] Table 1 T-Cell Select Kit Components

[0037] Reagents volume Concentrated buffer 1×30mL Antibody 1 3×2mL Antibody 2 3×2mL Magnetic bead reagents 1×10mL

[0038] Table 2 T-SPOT.TB kit components

[0039]

[0040] 1. T-SPOT.TB test

[0041] Example 1

[0042] In this embodiment, the blood sample used is a patient's fresh lithium heparin anticoagulated whole blood sample stored at room temperature (10-25° C.) for 0-4 hours.

[0043] 1. Isolation of PBMCs using T-Cell Select-MBS method (secondary enrichment)

[0044] like Figure 2As shown, according to the instructions of the cell processing kit (English name: T-Cell Select Kit; purchased from Oxford Immunotec, UK), 0.2mL of concentrated buffer was diluted with 0.3mL of distilled water (or deionized water, volume ratio of 2:3) and added to the first tube of the seven-tube strip provided with the Mag-Sep 20B fully automated cell sorter. Then, 40μL each of Antibody 1 and Antibody 2 were added and mixed. The mixed blood sample (4mL) from another tube was slowly poured into the first tube of the seven-tube strip, mixed thoroughly, and allowed to stand for 15 minutes. 60μL of magnetic bead reagent was added and mixed to form a "buffer-antibody-blood-magnetic bead" mixture. 1 mL of RPMI-1640 cell culture medium was added to each of the second and third tubes of the seven-tube strip, and 550 μL of AIM-V culture medium was added to the seventh tube. The seven-tube strip was then loaded into the Mag-Sep 20B fully automated cell sorter and the pre-set working program was run to automatically perform the steps of standing, mixing, magnetic bead adsorption, washing, and elution. The "adsorption, washing, and elution" enrichment procedure was repeated twice. The enriched PBMCs were eluted into AIM-V (550 μL) culture medium in the seventh tube of the seven-tube strip. The entire process took 30 min ( Figure 3 ), and obtain PBMCs suspension for later use.

[0045] 2. T-SPOT.TB test

[0046] According to T-SPOT.TB kit ( The kit was purchased from Oxford Immunotec, UK) and the instructions were followed. 50 μL each of AIM-V culture medium (blank control), Mycobacterium tuberculosis-specific mixed polypeptide A, Mycobacterium tuberculosis-specific mixed polypeptide B, and positive control solution (PHA, positive control) were added to four microwells of the microwell culture plate in sequence. 100 μL of the PBMCs standard suspension prepared in step 1 (cell density adjusted to 2.5×10 5 / mL, add appropriate amount of AIM-V culture medium to dilute), place in a 5% CO2 incubator overnight incubation for 16 to 20 hours, take out and wash, add concentrated labeled antibody and incubate for 1 hour and then wash, finally add 50 μL of color development substrate solution, protect from light for color development, wash and dry, and interpret the results according to the number of spots in antigen A well and B well.

[0047] 1) When the number of spots in the blank control well is 0-5, and the number of spots in the antigen A well or antigen B well minus the number of spots in the blank control well is ≥6, the result is "positive"; 2) When the number of spots in the blank control well is 6-10, and the number of spots in the antigen A well or antigen B well is ≥2 times the number of spots in the blank control well, the result is "positive"; 3) If the above criteria are not met, and the number of spots in the positive control well is ≥20 and the number of spots in the negative control well is ≤10, the result is "negative"; 4) When the number of spots in the blank control well is >10 or the number of spots in the positive control well is <20, the result is "indeterminate" or "invalid" and is not included in the statistics.

[0048] Comparative Example 1

[0049] The difference from Example 1 is that the DGC method is used to separate PBMCs.

[0050] 1. DGC method for isolating PBMCs

[0051] Take a collected blood sample (4 mL) and add an equal volume of RPMI-1640 cell culture medium. Mix thoroughly, then slowly pour the mixture onto the lymphocyte separation medium along the side of a 15 mL conical centrifuge tube to form a clear layer. Centrifuge at 1600 × g for 15 minutes. Aspirate the cloudy layer containing PBMCs, add RPMI-1640 cell culture medium to 11.5 mL, mix thoroughly, centrifuge at 600 × g for 7 minutes, discard the supernatant, and add 11.5 mL of RPMI-1640 cell culture medium to mix thoroughly to obtain a PBMC suspension for later use. Before performing the T-SPOT.TB assay, centrifuge the PBMC suspension at 350 × g for 7 minutes, discard the supernatant, and dilute with an appropriate amount of AIM-V culture medium based on the cell count. Set aside.

[0052] 2. T-SPOT.TB test

[0053] The steps are consistent with those in Example 1.

[0054] Comparative Example 2

[0055] The difference from Example 1 is that PBMCs were isolated using the T-Cell Select-MBS method (enriched 4 times)

[0056] 1. Isolation of PBMCs using T-Cell Select-MBS method (4 enrichments)

[0057] like Figure 2According to the T-CellSelect reagent instructions, 0.2 mL of concentrated buffer was diluted with 0.3 mL of distilled water (or deionized water, 2:3 volume ratio) and added to the first tube of the seven-tube strip supplied with the Mag-Sep 20B fully automated cell sorter. 40 μL each of Antibody 1 and Antibody 2 were then added and mixed thoroughly. Another 4 mL of mixed blood sample was slowly poured into the first tube of the seven-tube strip, mixed thoroughly, and allowed to stand for 15 minutes. 60 μL of magnetic beads were added and mixed to form a "buffer-antibody-blood-magnetic bead" mixture. 1 mL of RPMI-1640 cell culture medium was added to tubes 2 through 5 of the seven-tube strip, and 550 μL of AIM-V culture medium was added to tube 7. The seven-tube strip was then loaded into the Mag-Sep 20B fully automated cell sorter and the pre-set program was run, automatically performing the steps of standing, mixing, magnetic bead adsorption, washing, and elution. According to the reagent supplier's recommendations, the enrichment procedure of "adsorption, washing, and elution" was repeated four times. The enriched PBMCs were eluted into the seventh tube of the seven-tube strip, AIM-V (550 μL) culture medium. The entire process took 45 min ( Figure 3 PBMCs suspension was obtained.

[0058] 2. T-SPOT.TB test

[0059] The steps are consistent with those in Example 1.

[0060] Comparative Example 3

[0061] The difference from Example 1 is that the blood sample used is a lithium heparin anticoagulated whole blood sample from a patient (the same patient as the sample in Example 1) stored at room temperature (10-25° C.) for 34-55 hours.

[0062] Comparative Example 4

[0063] The differences from Example 1 are: 1) the blood sample used is a lithium heparin anticoagulated whole blood sample of a patient (the same patient as the sample in Example 1) stored at room temperature (10-25°C) for 34-55 hours; 2) PBMCs are isolated by the T-Cell Select-MBS method (enrichment 4 times), and the steps are the same as those in Comparative Example 2.

[0064] 2. Method Evaluation

[0065] SPSS 26.0 software was used for statistical analysis. The t-test was used for comparison between the two groups, and the differences were considered statistically significant when P < 0.05. The enumeration data were expressed as n (%). The positive coincidence rate, negative coincidence rate, total coincidence rate and consistency (Kappa value) of the T-SPOT.TB test results between the two PBMCs isolation methods were compared using the χ test. 2The Kappa value > 0.75 indicated good consistency.

[0066] 1. Differences in PBMC cell composition between DGC and MBS methods for separation of fresh whole blood samples

[0067] 1) To compare the PBMC isolation efficiencies of the DGC and MBS methods, fresh whole blood specimens from three healthy adults were extracted using the DGC method (method referenced in Comparative Example 1) and the MBS method (four enrichment steps, method referenced in Comparative Example 2), respectively. The resulting PBMC suspensions were counted using a three-differentiation fully automatic hematology analyzer. The hematology analyzer's calculated cell counts (cells / L) were converted to total cells based on volume for comparison.

[0068] The number of PBMCs extracted by DGC and MBS methods was 3.07±0.66×10 6 and 1.87±0.58×10 6 .

[0069] 2) Take 500 μL of PBMCs suspension obtained by DGC method and MBS method respectively, stain with fluorescently labeled lymphocyte surface marker antibodies, and perform flow cytometry to detect lymphocyte subsets, and compare the differences in PBMCs composition obtained by different extraction methods.

[0070] Test results such as Figure 4 and Figure 5 As shown in the figure, it can be seen that the proportion of B cells in PBMCs separated by DGC method and MBS method was 6.60±0.72% and 0.24±0.05% respectively (P<0.01), while there was no significant difference in the proportion of each T cell subset, such as CD4 + T cells, CD4 T cells without regulatory T cells (Treg) + The results showed that compared with PBMCs extracted by DGC method, PBMCs extracted by MBS method significantly removed B cells and could obtain T cells with higher purity.

[0071] 2. Analysis of the consistency between the number of PBMCs isolated from fresh lithium heparin anticoagulated whole blood samples using the DGC and MBS methods with different enrichment times and the T-SPOT.TB test results

[0072] 1) Lithium heparin anticoagulated venous blood was collected from 98 tuberculosis patients, and PBMC suspensions were obtained from the same patients according to the methods of Comparative Example 1 and Comparative Example 2. The PBMC suspensions obtained in Example 1, Comparative Example 1, and Comparative Example 2 were then counted using a three-differentiation fully automatic hematology analyzer. The count results (cells / L) were converted to total cells based on volume for comparison.

[0073] The number of PBMCs extracted from Comparative Example 1 (DGC method) and Comparative Example 2 (MBS method; 4 times enrichment) was 5.47±3.18×10 6 and 2.76±1.11×10 6 (P<0.05).

[0074] Lithium heparin-anticoagulated venous blood was collected from 96 tuberculosis patients, and PBMC suspensions were obtained from the same patients according to the methods of Example 1 and Comparative Example 1. The PBMC suspensions obtained in Example 1 and Comparative Example 1 were then counted using a three-differentiation fully automatic hematology analyzer. The counts (cells / L) calculated by the hematology analyzer were converted to total cells based on volume for comparison.

[0075] The number of PBMCs extracted from Comparative Example 1 (DGC method) and Example 1 (MBS method; 2nd enrichment) was 5.70±5.30×10 6 and 2.63±1.71×10 6 (P<0.05).

[0076] 2) The counted PBMCs suspension was subjected to a T-SPOT.TB test. The result interpretation method was similar to that in Example 1, "T-SPOT.TB test".

[0077] Table 1 Comparison of the consistency of T-SPOT.TB results of PBMCs isolated from fresh whole blood samples by MBS and DGC

[0078]

[0079] The results are shown in Table 1. As can be seen, compared with the DGC method, the T-SPOT.TB assay results for PBMCs extracted from fresh lithium heparinized whole blood samples after four MBS enrichment cycles showed a positive concordance rate of 91.7%, a negative concordance rate of 100%, and an overall concordance rate of 98.0%. The kappa value was 0.94 (95% CI: 0.86-1.02). Compared with the DGC method, the T-SPOT.TB assay results for PBMCs extracted from fresh lithium heparinized whole blood samples after two MBS enrichment cycles showed a positive concordance rate of 93.1%, a negative concordance rate of 100%, and an overall concordance rate of 97.9%. The kappa value was 0.95 (95% CI: 0.88-1.02). Therefore, the T-SPOT.TB assay results for PBMCs extracted from fresh samples using the MBS method were highly consistent with those obtained using the conventional DGC method, regardless of whether the four- or two-pass enrichment procedure was used.

[0080] The above results show that compared with PBMCs extracted by the DGC method, PBMCs extracted by the MBS method (regardless of whether they were enriched twice or four times) significantly removed B cells and reduced the number of cells, while there was no significant difference in the proportion of each T cell subset. The T-SPOT.TB test mainly detects the frequency of effector T cells that release IFN-γ, so it has no obvious effect on the interpretation of T-SPOT.TB results.

[0081] 3. Consistency analysis of T-SPOT.TB test results of PBMCs isolated from long-term stored lithium heparin anticoagulated whole blood samples by MBS method

[0082] 1) Lithium heparinized venous blood was collected from 71 tuberculosis patients, and PBMC suspensions were obtained from the same patients according to the methods of Comparative Examples 1 and 4. Lithium heparinized venous blood was collected from 35 tuberculosis patients, and PBMC suspensions were obtained from the same patients according to the methods of Comparative Examples 1 and 3. The counted PBMC suspensions were subjected to the T-SPOT.TB test. The results were interpreted as described in Example 1, "T-SPOT.TB Test."

[0083] Table 2 Comparison of T-SPOT.TB results consistency between MBS-based whole blood specimens and DGC-based fresh blood specimens

[0084]

[0085] The results are shown in Table 2. Compared with the conventional DGC method, the T-SPOT.TB assay results for PBMCs extracted from long-stored specimens after four MBS enrichment cycles had a positive concordance rate of 90.0%, a negative concordance rate of 95.1%, and an overall concordance rate of 93.0%, respectively. The Kappa value was 0.86 (95% CI: 0.78-0.94). Compared with the conventional DGC method, the T-SPOT.TB assay results for PBMCs extracted from long-stored specimens after two MBS enrichment cycles had a positive concordance rate of 81.3%, a negative concordance rate of 100%, and an overall concordance rate of 82.9%, respectively. The Kappa value was 0.43 (95% CI: 0.08-0.77). The concordance between the two results was moderate, which may be related to the decreased T cell activity caused by prolonged storage and insufficient magnetic bead adsorption and enrichment.

[0086] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A tuberculosis-infected T cell spot test method based on sample storage time and the number of PBMCs enrichment by the MBS method, characterized in that: The following steps are involved: Step 1: Determine the storage time of the whole blood sample; Step 2: If the whole blood sample has been stored for 0 to 4 hours, perform magnetic bead enrichment on the whole blood sample twice using the T-Cell Select magnetic bead sorting method, and then perform a tuberculosis infection T cell spot test; if the whole blood sample has been stored for more than 4 hours, perform magnetic bead enrichment on the whole blood sample four times using the T-Cell Select magnetic bead sorting method, and then perform a tuberculosis infection T cell spot test.

2. The tuberculosis-infected T cell spot test method based on sample storage time and PBMCs enrichment times according to claim 1, characterized in that: The volume of whole blood specimen is 4 mL.

3. The tuberculosis-infected T cell spot test method based on sample storage time and PBMCs enrichment times according to claim 1, characterized in that: Magnetic bead enrichment was performed using the T-Cell Select kit, which includes a concentration buffer, antibody 1, antibody 2, and magnetic bead reagents.

4. The tuberculosis-infected T cell spot test method based on sample storage time and PBMCs enrichment times according to claim 3, characterized in that: The volume ratio of antibody 1, antibody 2 and whole blood sample is 1:1:

100.

5. The tuberculosis-infected T cell spot test method based on sample storage time and PBMCs enrichment times according to claim 3, characterized in that: The volume ratio of antibody 1, antibody 2 and magnetic bead reagent is 2:2:

3.

6. The tuberculosis-infected T cell spot test method based on sample storage time and PBMCs enrichment times according to claim 3, characterized in that: The instrument used in the magnetic bead enrichment process is an automatic cell sorter.

7. The tuberculosis-infected T cell spot test method based on sample storage time and PBMCs enrichment times according to claim 6, characterized in that: The magnetic bead enrichment procedure includes adsorption, washing and elution.

8. The tuberculosis-infected T cell spot test method based on sample storage time and PBMCs enrichment times according to claim 7, characterized in that: The cells were washed with RPMI-1640 cell culture medium.

9. The tuberculosis-infected T cell spot test method based on sample storage time and PBMCs enrichment times according to claim 7, characterized in that: Elution was performed using AIM-V culture medium.

10. Use of the tuberculosis-infected T cell spot test method according to any one of claims 1 to 9 based on sample storage time and PBMCs enrichment times by the MBS method in preparing a tuberculosis detection device.

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