An antibody composition, a detection system and application thereof for detecting acute T lymphoblastic leukemia by full-spectrum flow cytometry

Abstract

The application provides an antibody composition for detecting acute T lymphoblastic leukemia by full-spectrum flow cytometry, a detection system and application thereof, and relates to the technical field of blood disease detection. The antibody composition is two groups of antibodies. The first group is 14 kinds of fluorescein-labeled cell membrane monoclonal antibodies, including anti-CD45, anti-CD99, anti-CD34, anti-CD48, anti-CD2, anti-CD3, anti-CD4, anti-CD5, anti-CD7, anti-CD8a, anti-CD16, anti-CD56, anti-CD94 and anti-TCR gamma delta. The second group is two kinds of intracellular monoclonal antibodies, including anti-nucleus TdT and anti-cytoplasm CD3. The two groups of antibodies are added into the same tube in sequence. The application also provides a flow cytometry sample for detecting acute T lymphoblastic leukemia based on full-spectrum flow cytometry, and a detection analysis method and system, so that the missed diagnosis rate and misdiagnosis rate of T-ALL detection are reduced.

Description

Technical Field

[0001] This invention relates to the field of hematological disease detection technology, and more particularly to the field of full-spectrum flow cytometry detection of acute T-lymphoblastic leukemia. Background Technology

[0002] Acute leukemia is a malignant lymphohematopoietic tumor that seriously endangers human health, and acute lymphoblastic leukemia (ALL) is one of its important types. T-cell acute lymphoblastic leukemia (T-ALL) is a tumor caused by the clonal proliferation of T lymphocyte progenitor cells in the bone marrow, usually accompanied by extramedullary infiltration. Although the incidence of T-ALL is not as high as that of B-cell acute lymphoblastic leukemia (B-ALL), the treatment effect of T-ALL is poor, and the early precursor T-cell acute leukemia (ETP-ALL) subtype has a particularly poor prognosis. T-ALL patients need to be monitored for efficacy after treatment, and accurate detection of minimal residual disease (MRD) is of great clinical significance for treatment decisions and prognostic assessment.

[0003] Flow cytometry (FCM) has become a widely used MRD detection technique in clinical practice due to its advantages such as applicability to almost all cases and stages, high sensitivity, high specificity, simple and rapid operation, and relatively low cost. However, traditional flow cytometry has many technical limitations: interference between fluorescence channels is significant, limiting the simultaneous detection of only about 10 parameters; models with 8 or more colors lack automatic compensation software and microspheres, requiring manual adjustment, which is time-consuming, subjective, and experience-dependent; clinically, to cover the vast majority of cases, multi-tube protocols are usually required, with some parameters needing to be reused in different tubes, resulting in a certain degree of waste of manpower, reagents, and consumables. These limitations are particularly prominent in T-ALL MRD detection.

[0004] MRD detection in T-ALL faces several unique challenges. The immunophenotype of T-ALL is prone to change during treatment; early markers can be gradually lost as treatment progresses, while tumor cells can acquire markers of maturity stages. Furthermore, the immunophenotypes of various mature lymphocyte subsets (including T cell subsets and NK cells) in normal bone marrow overlap to some extent with those of T-ALL tumor cells, interfering with accurate MRD identification. In addition, the clonal evolution of T-ALL and the widespread use of novel treatment methods further increase the heterogeneity of the immunophenotype, making it more difficult to accurately distinguish residual tumor cells from normal cell populations.

[0005] Unlike traditional fluorescence flow cytometry, full-spectrum flow cytometers use multiple detectors to detect the same fluorophore, allowing for the simultaneous use of fluorophores with similar emission spectra and easily achieving multicolor detection schemes. The software uses matrix algorithm technology for single positron tubes to automatically compensate, thereby achieving relative standardization. In the prior art, Chinese patent application CN117310168A discloses an antibody composition for detecting acute leukemia based on full-spectrum flow cytometry, mainly used for broad-spectrum initial screening and lineage typing diagnosis of acute leukemia; CN121431831A discloses a kit for detecting leukemia and lymphoma based on full-spectrum flow cytometry, which can be used for preliminary screening of various hematologic malignancies; CN114487422A discloses a flow cytometry kit for monitoring minimal residual disease in acute B-lymphoblastic leukemia. This kit contains 26 antibodies targeting the developmental patterns of bone marrow B cells and common abnormal expressions in ALL-B. Combined with a full-spectrum flow cytometer, detection can be completed in one tube. However, its antibody composition and analytical logic are specifically targeted at B-lineage cell development patterns and do not involve the T-cell marker system required for T-ALL MRD detection. The above-mentioned prior art is mainly for the initial diagnosis and typing of acute leukemia or lymphoma or for MRD monitoring of B-ALL, rather than specifically for the detection of MRD after T-ALL treatment. Summary of the Invention

[0006] This invention provides a full-spectrum flow cytometry antibody composition and analysis method specifically for the detection of T-ALL minimal residual disease, which solves the problems of insufficient biomarker coverage, inability to effectively exclude interference from NK cells and γδT cells, insufficient detection sensitivity and specificity, and high false negative rate in existing detection methods.

[0007] The technical solution adopted in this invention is:

[0008] In one aspect, this invention provides an antibody composition for detecting acute T-lymphoblastic leukemia using full-spectrum flow cytometry. The antibody composition comprises a first group of antibodies and a second group of antibodies, which are added sequentially to the same tube during application. The first group of antibodies comprises: anti-CD45 antibody, anti-CD99 antibody, anti-CD34 antibody, anti-CD48 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8a antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD94 antibody, and anti-TCRγδ antibody; the second group of antibodies comprises: anti-nuclear TdT antibody and anti-cytoplasmic CD3 antibody.

[0009] This invention employs a single-tube 16-color scheme, integrating all T-cell markers, early markers, and NK-cell screening markers into one tube. This effectively covers the most complete immature T-cell population, reducing the rate of missed diagnoses and false diagnoses, while also saving sample volume and improving detection efficiency.

[0010] In one embodiment of the present invention, each antibody in the antibody composition is a monoclonal antibody. Using monoclonal antibodies ensures highly specific binding to the target antigen, improving the accuracy and repeatability of detection results.

[0011] In one embodiment of the present invention, each antibody in the antibody composition is a fluorescently labeled antibody; in the first group of antibodies, the fluorescent labels of anti-CD45 antibody, anti-CD99 antibody, anti-CD34 antibody, anti-CD48 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8a antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD94 antibody, and anti-TCRγδ antibody are as follows: cFluor V547, cFluor V610, cFluor BYG575, cFluor BYG781, cFluor V670, cFluor V450, cFluor B548, cFluor BYG750, cFluor BYG610, cFluor BYG710, cFluor V420, cFluor BYG667, cFluor R720, cFluor R668; In the second group of antibodies, the fluorescent labels for the anti-TdT antibody and the anti-cytoplasmic CD3 antibody are FITC and cFluor R840, respectively. The above fluorescent label combination is the optimal combination determined after optimization testing using multiple schemes, ensuring excellent staining effects for all fluorescent pigments in each channel, thereby achieving the best clustering results. However, other fluorescent label combinations are also within the scope of this invention. As one implementation method, the fluorescent label for the anti-TdT antibody is FITC, and the fluorescent labels for other antibodies can be interchanged.

[0012] In one embodiment of the present invention, the first group of antibodies is a mixture of the aforementioned 14 antibodies against CD45, CD99, CD34, CD48, CD2, CD3, CD4, CD5, CD7, CD8a, CD16, CD56, CD94, and TCRγδ in equal volume ratios; the second group of antibodies is a mixture of anti-TdT antibody and anti-cytoplasmic CD3 antibody in a 2:3 volume ratio, with the unit volume of the first and second groups of antibodies calculated as equal. This volume ratio configuration, while maintaining essentially equivalent titers, achieves an optimal balance of signal intensity for each biomarker, thereby improving detection sensitivity.

[0013] In another aspect, the present invention provides the use of the antibody composition in the preparation of a reagent for the full-spectrum flow cytometry detection of acute T-lymphoblastic leukemia. As one embodiment, the reagent is a kit. As one embodiment, the kit includes a first container and a second container, each container containing a first group of antibodies and a second group of antibodies from the antibody composition, respectively. The dual-container design of the kit corresponds to the stepwise addition of the two groups of antibodies, ensuring optimal staining effects for membrane antibodies and intracellular antibodies (TdT and cytoplasmic CD3). As one embodiment, the kit further includes one or more of the following: erythrocyte lysis buffer, membrane-permeable agent, buffer solution, and flow cytometry tubes for use with a flow cytometer. The dosage of each reagent can be based on conventional dosages in the art or according to the manufacturer's recommended dosage. These reagents and consumables are commercially available. The membrane-permeable agent is preferably a membrane-permeable agent including solution A and solution B. Each reagent material can be contained in a separate container. The provision of the above-mentioned additional components simplifies the detection procedure and can meet the detection needs of samples with different cell concentrations.

[0014] Another aspect of this invention provides the application of the antibody composition described above in the preparation of flow cytometry samples for the detection of acute T-lymphoblastic leukemia (TTL) using full-spectrum flow cytometry, comprising the following steps: adding the sample to be tested to tube A of a flow cytometry tube, adding the first group of antibodies for incubation; then adding membrane-permeability agent A to tube A for incubation, followed by adding hemolysin for incubation, centrifuging to remove the supernatant; adding membrane-permeability agent B and the second group of antibodies to tube A for incubation, washing, centrifuging to remove the supernatant, resuspending the cells to obtain the flow cytometry sample. The sample to be tested is bone marrow or peripheral blood. The above sample processing steps integrate cell membrane antibody staining, fixation and membrane permeation, and intracellular antibody staining into the same flow cytometry tube A, resulting in a high degree of standardization of the operation process. Dual labeling of the cell membrane and intracellular cells can be achieved without adding additional flow cytometry tubes, significantly simplifying the experimental operation for T-ALL MRD detection.

[0015] Another aspect of the present invention provides a detection system for detecting acute T-lymphoblastic leukemia using full-spectrum flow cytometry. The detection system includes a detection unit, an analysis unit, and a judgment unit. The detection unit includes the flow cytometry sample. The analysis unit performs gating analysis according to the following method: the gating analysis employs a multi-marker combination gating strategy, using T-cell marker combination gating to compensate for missed diagnoses caused by the loss of a single marker after targeted therapy, and comprehensively using logic gates to exclude interference from NK cells and CD4 / CD8 double-negative mature T cells. The analysis method includes the following gating steps:

[0016] Use FSC-A / H to set up the single gate to remove adherent cells, and use FSC / SSC to set up the live gate within the single gate;

[0017] Within the live gate, CD45 / SSC is used to set up gates for each blood cell, and CD45dimCD7bri, TdT, CD99bri, CD34, CD7, cCD3 and TNK logic gates are set up respectively.

[0018] The CD45dimCD7bri gate uses a CD94-positive / CD56-positive NK gate; the TNK gate contains a DP gate, a DN gate, and a CD3-CD94-gate; the DN gate contains a gd-CD94-gate.

[0019] The judgment unit compares the distribution patterns of T cells and NK cell subsets with normal cells based on the expression of markers in each phylum to determine whether minimal residual disease is positive.

[0020] This detection system utilizes logic gates most comprehensively to date to eliminate interference from NK cells and TCRγδT cells, which have the greatest impact on the detection of minimal residual disease in T-ALL, significantly reducing the false negative rate. Compared with traditional flow cytometry, the detection sensitivity has increased from 94.74% to 99.35%, and the specificity has increased from 99.1% to 100%.

[0021] As one embodiment of the present invention, the method for determining minute residual lesions includes at least one of the following:

[0022] Within the TdT gate, CD99bri gate, and CD34 gate, two-dimensional dotted maps of CD7 / cCD3 and CD2 / CD5 are displayed, respectively. If any of the CD7, cCD3, CD2, or CD5 markers are positive, it is judged as a positive minimal residual disease.

[0023] If CD45dimCD7bri cells are not in the NK gate, especially if CD99bri and / or CD48dim and / or CD5 expression is present, it is judged as a positive minimal residual disease.

[0024] Within the DP gate, if positive cells are present, accompanied by an increase or decrease in CD7 and / or cCD3, a decrease in CD45 and / or CD48, an increase in CD99 expression, or an increase in CD34 and / or TdT expression, it is judged as a positive minimal residual disease.

[0025] Within the gd-CD94-gate, the presence of weak CD48 expression and / or strong CD99 expression suggests a positive minimal residual disease.

[0026] Within the CD3-CD94-gate, weak expression of CD48 and / or strong expression of CD99 indicate a positive minimal residual disease.

[0027] The above-mentioned multidimensional judgment criteria complement each other and can comprehensively cover the variations of each subtype and phenotype of T-ALL, effectively preventing missed diagnosis due to the loss of a single biomarker.

[0028] The detection system of this invention organically integrates antibody composition with intelligent analysis unit, realizing the standardization of the entire process from sample detection to result interpretation, which helps to promote the automation and artificial intelligence development of flow cytometry T-ALL detection.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) This invention provides for the first time a full-spectrum flow cytometry antibody combination scheme specifically for the detection of minimal residual disease in T-ALL. Traditional flow cytometry has a limited number of markers that can be detected per tube. Generally, one tube uses early markers CD99, CD34+CD1a and CD4 / CD8 / CD3 / CD56 together. TdT and cCD3 cytoplasmic tubes only use CD3, CD7 and CD56. It cannot combine all early markers and T cell cluster markers in one tube, especially lacking screening antibodies for γδT cells and NK cells. This invention addresses the specific needs of T-ALL MRD detection by employing a single-tube 16-color scheme. It eliminates the redundant backbone antibodies used in traditional two-tube schemes and integrates all T-cell markers (CD2, CD3, CD4, CD5, CD7, CD8a, cCD3), early markers (TdT, CD34, CD99), and NK cell and γδT cell screening markers (CD16, CD56, CD94, TCRγδ, CD48) into a single tube for detection. It adds CD48, a highly efficient marker for T-ALL MRD detection, and also adds TCRgd, CD94, and CD16, enhancing the detection of γδT cells and NK cells. Most importantly, it can identify rare γδT-type T-ALL, achieving comprehensive coverage of key markers for T-ALL MRD detection without increasing detection costs.

[0031] (2) This invention innovatively introduces CD94 for NK cell exclusion. This invention found that the expression rate of CD16 and CD94 in T-ALL is almost zero, while normal CD16-negative and CD56-positive regulatory NK cells express CD94 almost 100%. The combination of the three markers CD16 / CD56 / CD94 can almost perfectly cover all NK cell subsets, effectively solving the misjudgment problem caused by the inability to exclude regulatory NK cells due to the use of CD16 and CD56 alone in the existing scheme.

[0032] (3) This invention innovatively incorporates TCRγδ to achieve systematic screening of γδT cell subsets and rare γδT-type T-ALL. By adding TCRγδ antibody and combining it with CD94 marker, this invention effectively eliminates the interference of normal γδT cells in the CD4 / CD8 double-negative region, while identifying rare but extremely difficult-to-detect TCRγδ-type T-ALL, filling the gap in existing technology in this regard.

[0033] (4) The detection sensitivity and specificity of this invention are significantly improved. Clinically verified, compared with traditional two-tube multi-parameter flow cytometry, this invention improves the detection sensitivity from 10... -4 Increased to 10 -5 The detection sensitivity increased from 94.74% to 99.35%, the specificity increased from 99.1% to 100%, and the positive predictive value and negative predictive value both reached 99.35% and 100%, respectively.

[0034] (5) Improve testing efficiency and reduce overall costs. The single-tube approach increases specimen processing and analysis efficiency by 30% to 40%, reduces consumable costs by 30% to 40%, and reduces the required sample volume, making it especially suitable for pediatric patients with small sample volumes. Attached Figure Description

[0035] Figure 1a and Figure 1b This invention displays the results of full-spectrum flow cytometry gating analysis of bone marrow specimens that have achieved complete remission after treatment for other unrelated diseases, according to a specific embodiment of the invention. Figure 1a The results show the step-by-step phylogenetic results of cell subpopulations in single-cell, live-cell basic phylogenetic, and CD45dimCD7bri, TdT, CD99bri, CD34, TNK, and DN / DP T cells. Figure 1b Further differentiation was made among rare subsets such as γδT and NK cells, and the presence of suspicious abnormal cells was observed in key observation areas where tumor cells frequently appeared in the entire image. In this specimen, CD45dimCD7bri cells were all located within the NK gate, and no microresidual lesion-positive cells were found in the TdT gate, CD99bri gate, CD34 gate, gd-CD94- gate, and CD3-CD94- gate. The 14 key observation areas within the live gate were marked with dashed arrows, indicating that no microresidual lesion-positive cells were found in each area.

[0036] Figure 2a and Figure 2b This invention displays the results of full-spectrum flow cytometry gating analysis of bone marrow specimens from patients with positive T-ALL minimal residual disease after treatment, according to a specific embodiment of the present invention. Figure 2a The results show the basic quality control gating for single cells and live cells, as well as the layer-by-layer gating results for CD45dimCD7bri, TdT, CD99bri, CD34, TNK, and DN / DP T cells. Figure 2b Further differentiation was made among rare subsets such as γδT and NK cells, and the presence of suspicious abnormal cells was observed in key observation areas where tumor cells frequently appeared throughout the image. In this specimen, only a small number of cells were located within the CD45dimCD7bri gate, while a large number of cells outside the NK gate were tumor cells; malignant tumor cells expressing CD7 and cCD3 were observed within the CD99bri and CD34 gates; and tumor cell populations with strong CD7 expression, weak CD48 expression, cCD3 positivity, and strong CD99 expression were observed within the CD3-CD94- gate. In the key observation areas within each live gate, solid arrows indicate areas where tumor cells are visible, and dashed arrows indicate areas where no tumor cells are visible.

[0037] Figure 3a and Figure 3b This invention displays the results of full-spectrum flow cytometry gating analysis of a bone marrow specimen from a patient with positive T-ALL minimal residual disease after treatment, according to a specific embodiment of the present invention. Figure 3a The results show the basic quality control gating for single cells and live cells, as well as the layer-by-layer gating results for CD45dimCD7bri, TdT, CD99bri, CD34, TNK, and DN / DP T cells. Figure 3b Further differentiation was made among rare subsets such as γδT and NK cells, and the presence of suspicious abnormal cells was observed in key observation areas where tumor cells frequently appeared throughout the image. This specimen contained two phenotypically different groups of tumor cells: a red tumor cell population (MRD1) expressing CD56 and strongly expressing CD99, but not expressing CD16, CD94, or CD48; and a pink tumor cell population (MRD2) weakly expressing CD99, but not expressing CD56, CD5, CD16, CD94, or CD48. Both tumor cell populations were located within the DN phylum and the gd-CD94 phylum, reflecting the phenotypic heterogeneity of T-ALL. Key observation areas within the live phylum were marked with solid arrows (MRD1 tumor cells visible) or dashed arrows (no tumor cells seen). MRD2 was only visible in two areas: one with weak CD48 expression and one with strong CD7 expression, and the other with positive CD34 and positive CD7. Detailed Implementation

[0038] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention is described in detail below. It should be understood that the embodiments described below are only for explaining the present invention and are not intended to limit the scope of protection of the present invention. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments described below.

[0039] As used herein, the term "antibody composition" refers to a mixture containing two or more groups of antibodies, wherein each group of antibodies may be contained in different containers and added to the same flow cytometry tube in a specific order during use. The antibody composition of this invention includes a first group of antibodies and a second group of antibodies. The first group of antibodies is an antibody against cell membrane surface antigens, and the second group of antibodies is an antibody against nuclear or cytoplasmic antigens. The two groups of antibodies are added sequentially to the same flow cytometry tube (tube A) for use during detection.

[0040] The term "full-spectrum flow cytometry" as used in this article refers to a flow cytometry technique that uses multiple detectors to simultaneously detect the full spectrum of emission light from the same fluorophore. Unlike traditional fluorescence flow cytometry, this technique changes the hardware from a one-to-one detector-fluorophore relationship to using multiple detectors to detect the same fluorophore. This allows for the simultaneous use of fluorophores with similar emission spectra, easily achieving multi-color detection. On the software side, it achieves relative standardization through automatic compensation using matrix algorithms for single-anode tubes. Representative instruments for full-spectrum flow cytometry include, but are not limited to, the 3-laser 38-channel full-spectrum flow cytometer from Cytek (such as the Cytek NL-CLC model).

[0041] The term "minimal residual disease" (MRD) as used herein refers to a small number of tumor cells remaining in the body after morphological complete remission following treatment. The purpose of MRD testing is to assess treatment efficacy, predict recurrence risk, and guide adjustments to subsequent treatment regimens. In this invention, an MRD positivity criterion is based on the identification of cell populations within each gate that differ from the normal cellular immunophenotype pattern through multi-marker gating analysis, and these cell populations express T-cell-related markers. In some implementations, a recurrence is considered to be defined as a malignant cell proportion exceeding 5% of nucleated cells.

[0042] The term "Group 1 antibodies" as used in this article refers to a group of 14 monoclonal antibodies that contain cell membranes: anti-CD45 antibody, anti-CD99 antibody, anti-CD34 antibody, anti-CD48 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8a antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD94 antibody, and anti-TCRγδ antibody. This group of antibodies is first added to flow cytometry tubes during sample processing to label cell surface antigens.

[0043] As used in this article, "group II antibodies" refers to an antibody group containing two intracellular monoclonal antibodies: an anti-nuclear TdT antibody and an anti-cytoplasmic CD3 antibody. This group of antibodies is added to flow cytometry tubes after membrane permeation treatment to label intracellular antigens. The addition of group II antibodies must occur after membrane permeation treatment to ensure that the antibodies can enter the cells and bind to the target antigens.

[0044] The term "gating" as used in this article refers to the process in flow cytometry data analysis where a specific region is delineated on a scatter plot to select cell subpopulations with specific scattered light or fluorescence characteristics for further analysis. The multi-marker combined gating strategy described in this invention refers to an analytical method that comprehensively utilizes the expression of multiple cellular markers to precisely screen target cell populations through a layer-by-layer gating approach.

[0045] The term "TNK gate" used in this article refers to the logical merging gate that includes CD7-positive cells (CD7 gate) and cytoplasmic CD3-positive cells (cCD3 gate), that is, the total merging gate of T cells and NK cells. The TNK gate contains all T lymphocytes and NK cells in the bone marrow and is the core functional gate for T-ALL MRD analysis.

[0046] The term "CD45dimCD7bri gate" used in this article refers to the gate corresponding to the cell population located in the region of weak CD45 expression (dim) and strong CD7 expression (bright) on a CD45 / CD7 scatter plot. 95% of T-ALL tumor cells aggregate in this region, and NK cells from normal bone marrow are also located in this region. Therefore, the CD45dimCD7bri gate is the most efficient gating site for screening T-ALL MRD.

[0047] The term "NK gate" used in this article refers to the gate corresponding to the cell population located in the CD94-positive / CD56-positive region on a CD94 / CD56 scatter plot, used to distinguish NK cells from T-ALL tumor cells. This invention uses a combination of three markers—CD16, CD56, and CD94—to screen NK cells. The expression rates of CD94 and CD16 in T-ALL are almost zero, while normal CD16-negative, CD56-positive regulatory NK cells express CD94 almost 100%. Therefore, the three markers CD16, CD56, and CD94 can almost perfectly cover all NK cell subsets.

[0048] The term "gd-CD94-gate" used in this article refers to the gate corresponding to the cell population located in the TCRγδ-negative / CD94-negative region on the TCRγδ / CD94 scatter plot. It is set within the DN gate to further exclude interference from normal TCRγδ T cells in CD4 / CD8 double-negative T cells and to identify rare TCRγδ-type T-ALL.

[0049] The term "CD3-CD94-gate" used in this article refers to the gate corresponding to the cell population located in the CD3-negative / CD94-negative region on the CD3 / CD94 scatter plot. It is set within the TNK gate and is used to identify the CD3-negative and CD94-negative T cell population. The presence of weak CD48 expression and / or strong CD99 expression in this population indicates a positive minimal residual disease.

[0050] The term "DP gate" used in this article refers to the gate (double-positive gate) corresponding to cell populations located in CD4-positive / CD8-positive regions on a CD4 / CD8 scatter plot, within the TNK gate. The term "DN gate" refers to the gate (double-negative gate) corresponding to cell populations located in CD4-negative / CD8-negative regions on a CD4 / CD8 scatter plot, within the TNK gate. In normal bone marrow, the DN region is primarily composed of TCRγδT cells, and cases of T-ALL loss of early markers also concentrate in the DN region; therefore, the DN gate is an important analytical area for detecting MRD.

[0051] As used herein, the term "fluorescein labeling" refers to a technique that uses chemical methods to couple fluorescent dyes to antibodies, causing the antibodies to emit fluorescence at a specific wavelength under laser excitation in a flow cytometer, thereby enabling simultaneous detection of multiple parameters. In this invention, the fluorescein labeling schemes for each antibody have been optimized and screened to ensure that the excitation and emission spectra of each fluorescein match the detection channels of the full-spectrum flow cytometer, and to minimize spectral overlap and interference between fluoresceins.

[0052] As used herein, the term "permeabilizing agent" refers to a class of reagents that can permeate the cell membrane, thereby allowing antibodies to enter the cell and bind to nuclear or cytoplasmic antigens. The permeabilizing agent used in this invention comprises two parts: solution A and solution B. Solution A is used to fix cells, and solution B is used to permeabilize the cell membrane and enhance cell permeability. The permeabilizing agent can be a commercially available reagent conventionally used in the art, such as BD Cytofix / Cytoperm Fixation and Permeabilization Solution.

[0053] As used herein, the term "sample to be tested" refers to a biological sample, such as a bone marrow or peripheral blood sample, that is to be subjected to T-ALL MRD testing and is anticoagulated with heparin or EDTA. In some implementations, the cell count per tube is 1 × 10⁻⁶. 6 / pipe to 1×10 7 / tube, preferably with a cell count of approximately 2~7×10 6 One cell per tube, to ensure a sufficient number of cells are obtained for gating analysis.

[0054] This invention provides an antibody composition applicable to the detection of acute T-lymphoblastic leukemia using full-spectrum flow cytometry. The antibody composition comprises a first group of antibodies and a second group of antibodies, which are added sequentially to the same tube during application. The first group of antibodies consists of: anti-CD45 antibody, anti-CD99 antibody, anti-CD34 antibody, anti-CD48 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8a antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD94 antibody, and anti-TCRγδ antibody; the second group of antibodies consists of: anti-nuclear TdT antibody and anti-cytoplasmic CD3 antibody.

[0055] The 16 antibodies selected in this invention have clear technical basis. The first group of 14 cell membrane antibodies can be divided into the following functional markers: (1) Pan-T markers: CD7, CD3, CD2, CD5, of which CD7 and cCD3 are true pan-T markers, appearing throughout the entire process of T cell development; (2) Maturation stage markers: CD4, CD8a, used to classify CD4 single-positive and CD8 single-positive mature T cell subsets, as well as to identify CD4 / CD8 double-positive (DP) and double-negative (DN) cell populations; (3) Early markers: CD99 (with a high initial positive rate in T-ALL). 96.92%), CD34 (overall positive rate of 16.92% in initial treatment); (4) NK cell screening markers: CD56, CD16, CD94, the combination of the three can almost perfectly cover all NK cell subsets; (5) MRD high-sensitivity marker: CD48, which appears in T-ALL with weak expression or negative pattern, is a newly discovered high-efficiency MRD detection marker; (6) γδT cell screening marker: TCRγδ, used to exclude normal γδT cell interference in DN region and identify rare TCRγδ type T-ALL. The second group of two intracellular antibodies includes anti-nuclear TdT antibody (early marker, positive rate of 74.62% in initial treatment) and anti-cytoplasmic CD3 antibody (pan-T marker, positive rate of 99.23% in initial treatment). Both need to be added after membrane rupture treatment, so they are used as the second group of antibodies separately.

[0056] Compared to traditional two-tube 8-10 color flow cytometry protocols, the single-tube 16-color antibody composition of this invention integrates all the key biomarkers required for T-ALL MRD detection into a single tube without increasing detection costs, by eliminating the redundant backbone antibodies (such as CD3, CD7, CD45, CD56, etc.) used in the two-tube protocol. This not only provides more biomarker combination information, improving detection sensitivity and specificity, but also saves sample volume, increases detection efficiency, and reduces consumable costs.

[0057] In one embodiment, each antibody in the antibody composition targets the following antigens: CD45 (a common leukocyte antigen used to identify blood cells and distinguish cell types by fluorescence intensity), CD99 (an early marker with high-frequency and strong expression in T-ALL primitive cells), CD34 (a marker of hematopoietic stem / progenitor cells, positive in some T-ALL cases), CD48 (normal mature T cells express CD48, while T-ALL tumor cells show weakened or negative CD48 expression, forming a detectable difference from normal cells, and is an important differential marker for MRD detection), CD2 (a marker of T cells and NK cells, appearing in the second stage of T cell development), CD3 (cell membrane CD3, a marker of mature T cells), CD4 (… Helper T cell markers include CD5 (a T cell marker, weakly expressed or even negative in the TCRγδT cell subset), CD7 (the earliest appearing and persistent pan-T marker throughout the entire T cell development process, with a positive rate of nearly 100% and strong expression in over 95% of T-ALL cells), CD8a (a cytotoxic T cell marker), CD16 (an NK cell marker, with an expression rate of almost zero in T-ALL cells), CD56 (expressed by NK cells and some T-ALL cells), CD94 (a marker for NK cells and the TCRγδT cell subset, with an expression rate of almost zero in T-ALL cells), and TCRγδ (a TCRγδT cell subset-specific marker, mainly expressed in γδT cells in normal DN regions).

[0058] According to one embodiment of the present invention, each antibody in the antibody composition is a monoclonal antibody. A monoclonal antibody is a highly homogeneous antibody produced by a single B cell clone and targeting a single antigenic epitope. In flow cytometry detection applications, the use of monoclonal antibodies ensures highly specific binding of the antibody to the target antigen, avoiding cross-reactions that may occur with polyclonal antibodies due to recognizing multiple epitopes, thereby improving the accuracy, repeatability, and batch-to-batch consistency of the detection results. All monoclonal antibodies described in this invention are commercially available and should meet the requirements of relevant industry standards.

[0059] According to one embodiment of the present invention, each antibody in the antibody composition is a fluorescently labeled antibody. In the first group of antibodies, the fluorescent labels of the anti-CD45 antibody, anti-CD99 antibody, anti-CD34 antibody, anti-CD48 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8a antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD94 antibody, and anti-TCRγδ antibody are, in sequence: cFluor V547, cFluor V610, cFluor BYG575, cFluor BYG781, cFluor V670, cFluor V450, cFluor B548, cFluor BYG750, cFluor BYG610, cFluor BYG710, cFluor V420, cFluor BYG667, cFluor R720, and cFluor R668. In the second group of antibodies, the anti-TdT antibody and the anti-cytoplasmic CD3 antibody were labeled with FITC and cFluor R840, respectively.

[0060] The above-described fluorescein labeling scheme is the optimal one determined after optimization testing of various combinations (at least four). It ensures excellent staining effects for all fluoresceins in each channel, resulting in optimal clustering results. Specifically, FITC labeling is preferred for the anti-TdT antibody because the positive rate of TdT BV421 labeling is relatively low, while FITC labeling provides a higher positive rate and clearer signal, thus improving the detection sensitivity of TdT. Fluorescent labeling of other antibodies can be interchanged if their effects are essentially equivalent; however, after extensive experimental verification, the above-described fluorescein combination is the optimal configuration.

[0061] In a preferred embodiment, the fluorophores are selected from Cytek cFluor series fluorophores (including cFluor V series, cFluor B series, cFluor BYG series, and cFluor R series) and FITC. These fluorophores are specifically optimized for full-spectrum flow cytometry and achieve the best detection results when used with a full-spectrum detection system. In another embodiment, while keeping TdT-FITC unchanged, the fluorophore labeling of other antibodies can be appropriately adjusted according to the existing fluorophore configuration in the laboratory. However, the adjustments should be experimentally verified to ensure that the staining effect of each channel is not lower than the optimal scheme described in this invention.

[0062] According to one embodiment of the present invention, the first group of antibodies is a mixture of the above 14 antibodies in equal volume ratios; the second group of antibodies is a mixture of anti-TdT antibody and anti-cytoplasmic CD3 antibody in a volume ratio of 2:3. In one specific embodiment, the total volume of the first group of antibody mixture is 42 µl / tube (3 µl of each antibody), and the total volume of the second group of antibody mixture is 5 µl / tube (2 µl of TdT antibody and 3 µl of cytoplasmic CD3 antibody).

[0063] The above volume ratio configuration is an optimized result that achieves the best balance of signal intensity for each biomarker while ensuring that the titers of each antibody are basically equivalent. The first group of antibodies, consisting of 14 antibodies mixed in equal volumes, simplifies the preparation process and achieves balanced signal intensity when the titers of each antibody are similar. In the second group of antibodies, the volume ratio of anti-TdT antibody to anti-cytoplasmic CD3 antibody is 2:3, which was determined based on the relative titers and staining effects of the two antibodies. In some implementation schemes, when there are differences in the titers of antibodies from different batches or sources, the volume ratios can be appropriately adjusted based on the above to ensure the staining effect of each biomarker. The total amount of the first group of antibodies can be set to 21–84 µl / tube, and the total amount of the second group of antibodies can be set to 3–10 µl / tube, a range that can accommodate test samples with different cell concentrations.

[0064] The antibody composition of this invention can be used for the diagnosis of newly diagnosed cases and for post-treatment follow-up, especially suitable for MRD follow-up detection after targeted therapy (such as CD7-CAR-T therapy). Compared with the traditional two-tube 9-color flow cytometry protocol, the traditional protocol usually uses the following approach: the membrane tube uses a combination of early markers CD99 and CD34 with mature markers CD4 / CD8 / CD3 / CD56, while the cytoplasmic tube only uses a few markers such as CD3, CD7, and CD56 in combination with TdT and cCD3. It cannot integrate all early markers and T cell cluster markers into one tube, especially lacking systematic screening antibodies for γδT cells and NK cells. This invention achieves complete marker coverage in one tube with a full-spectrum flow cytometry 16-color protocol without increasing detection costs, which is impossible with the traditional two-tube protocol.

[0065] It should be noted that although CD1a is a known early marker of the thymic cortex stage of T-ALL, it is not included in the 16-color scheme of this invention for the following reasons: The positive rate of CD1a at the initial treatment stage of T-ALL is approximately 30%, significantly lower than markers such as CD99 (96.92%), CD7 (99.23%), and TdT (74.62%). More importantly, the positive rate of CD1a further decreases during the MRD detection stage after treatment, limiting its actual contribution to MRD detection. Our initial full-spectrum flow cytometry scheme included CD1a, but after testing in the first 50 cases, we found that the positive rate of CD1a was less than 10%, and other markers in these cases could detect MRD; therefore, omitting CD1a would not affect the detection results, so it was replaced with CD48. This invention allocates the fluorescence channels occupied by CD1a to CD48, resulting in a significantly higher contribution (66.45%) to the recognition of T-ALL tumor cells in MRD detection compared to CD1a. Furthermore, this protocol incorporates biomarkers such as CD94, TCRγδ, and CD16 for NK cell and γδT cell screening. These biomarkers are also more valuable than CD1a in improving the specificity of MRD detection. Therefore, the final combination of 16 biomarkers is the optimal approach determined after comprehensively weighing the sensitivity, specificity, and actual contribution of each biomarker in MRD detection.

[0066] This invention provides the application of the above-described antibody composition in the preparation of a reagent for the full-spectrum flow cytometry detection of acute T-lymphoblastic leukemia. The reagent can be a kit comprising a first container and a second container. The first container contains a first group of antibodies (a mixture of 14 cell membrane monoclonal antibodies) of the above-described antibody composition, and the second container contains a second group of antibodies (a mixture of anti-TdT antibody and anti-cytoplasmic CD3 antibody) of the above-described antibody composition.

[0067] The kit employs a dual-container design, storing membrane antibodies (Group 1) and intracellular antibodies (Group 2) separately, corresponding to the sequential addition of the two antibody groups to the flow cytometry tube during sample processing. The technical significance of this design lies in the fact that intracellular antibodies (TdT and cytoplasmic CD3) need to be added after membrane perforation; if stored together with membrane antibodies, antibody stability and labeling efficacy may be affected. Separating the two antibody groups helps ensure the storage stability of each antibody and guarantees the standardization of the operational procedures.

[0068] In one embodiment, the antibodies in the first container are pre-mixed in a volume ratio of 3:3:3:3:3:3:3:3:3:3:3:3:3:3 to simplify the preparation process for the user. In another embodiment, the antibodies in the first container can be stored separately, and the user can mix them on-site according to the above volume ratio to improve flexibility. In a preferred embodiment, the concentration of each antibody stock solution meets relevant industry standards and can be stably stored under recommended storage conditions (e.g., 2–8°C, protected from light).

[0069] According to one embodiment of the present invention, the kit further includes one or more of the following: erythrocyte lysis buffer, membrane permeation agent, buffer solution, and flow cytometry tubes. The first group of antibodies in the kit is prepared at a dosage of 21–84 µl per tube, and the second group of antibodies is prepared at a dosage of 3–10 µl per tube. In one specific embodiment, the recommended dosage of the first group of antibodies is 42 µl / tube, and the recommended dosage of the second group of antibodies is 5 µl / tube.

[0070] The erythrocyte lysis buffer is used to lyse erythrocytes in the sample. Commonly used erythrocyte lysis buffers include, but are not limited to, BD Lysing Buffer 10× Concentrate (BD Biosciences) and Capri Hemolysin (Henan Capri Biotechnology Co., Ltd.). When using, it is usually added at a 1× concentration, with a volume of 2–3 ml / tube, and incubated for 5–30 minutes. The permeabilizing agent includes solution A and solution B. Solution A (fixation solution) is used to fix cells and enhance cell membrane permeability; the recommended volume is 100 µl / tube, and incubation for 5–20 minutes. Solution B (permeabilizing solution) further permeates the cell membrane, allowing antibodies to enter the cell; the recommended volume is 50 µl / tube, and incubation for 10–30 minutes. Commonly used permeabilizing agents include, but are not limited to, BD Cytofix / Cytoperm Fixation and Permeabilization Solution (BD Biosciences, catalog number 554722), Beckman PerFix-nc, and BD IntraSure. The buffer solution is used for washing and resuspending cells. PBS buffer (0.02 mol / L phosphate, pH 7.2–7.6) is commonly used. The washing volume is 2–3 ml / tube, and the resuspending volume is 0.5–1 ml / tube. The flow cytometry tubes are standard flow cytometry tubes compatible with the full-spectrum flow cytometer used.

[0071] The inclusion of these additional components simplifies the testing process, eliminating the need for users to prepare separate reagents and consumables. This facilitates standardization and normalization of the testing procedure, reducing variability in test results caused by differences between different batches of reagents. The preset antibody dosage range (21–84 µl / tube for group 1, 3–10 µl / tube for group 2) accommodates test samples with varying cell concentrations, ensuring sufficient signal intensity while avoiding non-specific staining due to excessive antibody dosage.

[0072] The present invention also provides the application of the above-mentioned antibody composition or the above-mentioned reagent in the preparation of flow cytometry samples for the detection of acute T-lymphoblastic leukemia based on full-spectrum flow cytometry, comprising the following steps: (1) adding the sample to be tested into the flow cytometry tube A to make it into a single-cell suspension, and ensuring that the cell count is 1×10 6 / tube up to 1×10 7 / tube; the sample to be tested is bone marrow or peripheral blood; (2) add the first group of antibodies to tube A, mix well and incubate at room temperature in the dark; (3) add membrane-breaking agent A to tube A after incubation in step (2), and incubate at room temperature in the dark; (4) add 1× hemolysin to tube A after incubation in step (3), and incubate at room temperature in the dark; (5) centrifuge tube A after incubation in step (4) and remove the supernatant; (6) add membrane-breaking agent B and the second group of antibodies to tube A after removing the supernatant in step (5), and incubate at room temperature in the dark; (7) add PBS buffer to tube A after incubation in step (6), centrifuge and remove the supernatant, and resuspend the cells with PBS buffer; (8) perform flow cytometry on the cells resuspended in step (7).

[0073] The key technical points of the above specimen processing steps are as follows: Step (2) first, add the first group of 14 cell membrane antibodies to complete the labeling of cell membrane antigens while the cell membrane is intact, ensuring the optimal staining effect of cell membrane markers; Step (3) add membrane permeation agent A for pre-fixation; Step (4) add hemolysin to lyse red blood cells; Step (6) add membrane permeation agent B to make the cell membrane fully permeable before adding the second group of 2 intracellular antibodies (anti-TdT and anti-cytoplasmic CD3) to ensure that the two intracellular antibodies can smoothly enter the cell and bind to the target antigen. This scheme adopts a step-by-step treatment method using membrane permeation agent A and B, which takes into account the needs of cell morphology preservation, red blood cell lysis and intracellular staining. Compared with the traditional single-step membrane permeation scheme, it can better ensure that the cell membrane and intracellular antigens obtain excellent staining effects at the same time.

[0074] According to a specific embodiment of the present invention, in step (1), the volume of sample added to each tube shall not exceed 160 μl (if the patient's peripheral blood cell count is low, the volume may exceed 160 μl if necessary, and the supernatant may be removed by centrifugation and concentration). In step (2), the incubation time may be 10–30 minutes. In step (3), the incubation time may be 5–20 minutes. The amount of membrane-breaking agent A added shall be in accordance with the manufacturer's recommended dosage, usually 100 μl / tube. In step (4), the incubation time may be 5–30 minutes. The amount of 1×hemolysin added is 2–3 ml / tube. In step (5), the centrifugation conditions are usually 1000–2000 rpm (or 300–450 g) for 5 minutes. In step (6), incubation is required for about 10–30 minutes. The amount of membrane-breaking agent B added shall be in accordance with the manufacturer's recommended dosage, usually 50 μl / tube. In step (7), the amount of PBS buffer added for washing is 2-3 ml / tube. The centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes. The amount of PBS buffer added for resuspension is 0.5-1 ml / tube.

[0075] In this invention, "detection sensitivity 10" -5 "This invention refers to the method's ability to detect tumor cells comprising 0.001% of nucleated cells, meaning one tumor cell can be detected per 100,000 nucleated cells. This sensitivity is achieved through the combined effect of the following factors: full-spectrum flow cytometry integrates all 16 biomarkers into a single flow tube, eliminating information loss caused by the inability to directly compare different tubes in traditional two-tube methods; simultaneously, each tube yields at least 300,000 cells (preferably 1 million cells), ensuring a sufficient basis for statistical analysis. In 3140 full-spectrum flow cytometry tests from 2050 individuals, 8 patients (2.58%) had tumor cell proportions of only 0.002%–0.004%, all of which were detected using the method of this invention, while traditional flow cytometry (detection sensitivity 10)..." -4 These cases with low tumor burden could not be identified.

[0076] This invention provides a detection system for detecting acute T-lymphoblastic leukemia using full-spectrum flow cytometry. The detection system includes a detection unit, an analysis unit, and a judgment unit. The detection unit includes flow cytometry samples for detection by full-spectrum flow cytometry, reagent materials from the sample of the individual being tested, and the reagent materials include the antibody composition described in this invention. The analysis unit analyzes the detection results of the detection unit, performing gating analysis according to the following analytical method. The judgment unit compares the displayed distribution patterns of T cells and NK cell subsets with normal cells based on the expression of each marker within the gating system to determine whether minimal residual disease is positive.

[0077] The analysis method of the analysis unit of this invention adopts a multi-marker combination gating strategy. It compensates for missed diagnoses caused by the loss of a single marker after targeted therapy by gating the combination of T cell markers, and uses logic gates to exclude interference from NK cells and CD4 and CD8 double-negative mature T cells. The method includes the following gate setting steps: (1) Using FSC-A / H to set the single gate for removing adhesion cells, and using FSC / SSC to set the live cell gate within the single gate; (2) Using CD45 / SSC to set each blood cell gate within the live gate, and setting the CD45dimCD7bri gate, TdT gate, CD99bri gate, CD34 gate, CD7 gate, cCD3 gate and TNK logic gate respectively; (3) Using CD94 positive / CD56 positive to set the NK gate within the CD45dimCD7bri gate; setting the DP gate, DN gate and CD3-CD94- gate within the TNK gate; setting the gd-CD94- gate within the DN gate; (4) Comparing the distribution patterns of T cells and NK cell subsets with normal cells based on the expression of markers within each gate to determine whether minimal residual disease is positive.

[0078] The technical rationale for the analytical method of this invention is based on the following T cell biological foundations. During normal T cell development, the pan-T markers CD7 and cCD3 appear earliest and persist throughout the entire process; therefore, CD7 and cCD3 are the most commonly used T cell gnostic markers. CD7 is strongly expressed in the primitive / immature stage, weakest in the cortical stage, and moderately strong in the mature stage; in T-ALL, over 95% of cases show strong CD7 expression, therefore CD45dimCD7bri is the region where T-ALL tumor cells most frequently appear. CD4 and CD8 appear simultaneously in the thymic cortex stage, differentiating into two subsets—CD4 single-positive or CD8 single-positive—in the mature stage. Normal DN regions (CD4-negative, CD8-negative) are mainly composed of TCRγδ T cells. When T-ALL loses early markers, these cells also concentrate in the DN region; therefore, it is necessary to increase the use of TCRγδ antibodies and CD94 antibodies to enhance the screening of normal γδ T cells and simultaneously identify the rare TCRγδ type of T-ALL.

[0079] The NK cell exclusion strategy in this invention is a significant innovation. 95% of T-ALL tumor cells reside in the CD45dimCD7bri region, and cells in this region in normal bone marrow are all NK cells. However, NK cells exhibit heterogeneity; approximately 10% of NK cells are CD16-negative, CD56-positive regulatory NK cells. Furthermore, approximately 10%–20% of T-ALL cells express CD56, making the use of CD16 and CD56 alone for NK cell screening limited. This invention employs a combination of three markers—CD16, CD56, and CD94—to establish the NK gating: studies have found that the expression rates of CD16 and CD94 in T-ALL are almost zero, while normal CD16-negative, CD56-positive regulatory NK cells almost 100% express CD94. Therefore, the three markers CD16, CD56, and CD94 can almost perfectly cover all NK cell subsets, effectively solving the misjudgment problem caused by the inability to exclude regulatory NK cells when using CD16 and CD56 alone in existing methods.

[0080] This invention employs a combination of two MRD detection strategies: the Leukemia-Associated Immunophenotyping (LAIP) strategy, which involves the co-expression or combination of antigens not normally present, such as the simultaneous occurrence of strong expression of early markers TdT and / or CD34 and / or CD99 and / or weakened expression of CD48; and the Differential Expression with Normal (DFN) strategy, where the expression intensity or composition of these antigens in the test specimen is altered compared to normal cells in the same region. Currently accepted high-efficiency MRD detection methods combine these two approaches, and the multi-marker combination gating method of this invention systematically embodies this principle.

[0081] In one implementation scheme, in addition to the analysis of the functional gates mentioned above, the following 14 two-dimensional dot plots should also be displayed in the live gate to comprehensively observe the MRD positive signals in each region: CD99 / CD7, CD99 / cCD3, CD99 / CD5, CD48 / CD7, CD48 / cCD3, CD48 / CD5, CD56 / CD94, CD16 / CD56, CD3 / CD5, CD4 / CD8, CD7 / CD2, TCRγδ / CD3, CD7 / TdT, CD34 / CD7. The key areas to observe include: CD99 strongly expressing CD7, CD99 strongly expressing cCD3 positive cells, CD99 strongly expressing CD5 positive or weakly positive cells, CD48 weakly expressing CD7 strongly expressing cells, CD48 weakly expressing cCD3 positive cells, CD48 weakly expressing CD5 positive or weakly positive cells, CD3 negative CD5 positive or weakly positive cells, CD4 positive CD8 positive cells, CD7 positive TdT positive cells, and CD34 positive CD7 positive cells.

[0082] The technical advantages of the analytical method of this invention are as follows: First, by using a combination of multiple T cell markers for gating, even if one marker is lost due to targeted therapy (such as CD7-CAR-T therapy), other markers can still capture tumor cells, effectively compensating for missed diagnoses caused by the loss of a single marker; Second, by using a combination of CD16 / CD56 / CD94 markers to achieve near-perfect NK cell exclusion, significantly reducing false positives caused by misjudgment of NK cells; Third, by using a combination of TCRγδ / CD94 to exclude interference from normal γδ T cells within the DN gate and to identify rare TCRγδ type T-ALL, filling the gap in existing detection methods; Fourth, the multidimensional judgment criteria complement each other, comprehensively covering all subtypes and phenotypic variations of T-ALL, including complex cases such as biclonal T-ALL.

[0083] According to one embodiment of the present invention, the method for determining minimal residual disease includes at least one of the following: (1) Within the TdT gate, CD99bri gate, and CD34 gate, two-dimensional dot plots of CD7 / cCD3 and CD2 / CD5 are displayed respectively. If any marker of CD7, cCD3, CD2, or CD5 is positive, it is determined to be a positive minimal residual disease; (2) Within the CD45dimCD7bri gate, if CD45dimCD7bri cells are not within the NK gate, especially if any expression of CD99bri and / or CD48dim and / or CD5 is present, it is determined to be a positive minimal residual disease. (3) In the DP gate, if positive cells are present, pay attention to whether they are accompanied by enhancement or weakening of CD7 and / or cCD3, weakening of CD45 and / or CD48, enhancement of CD99 expression, and expression of CD34 and / or TdT. If any one of these is present, it is judged as a positive minimal residual disease; (4) In the gd-CD94- gate, if CD48 is weakly expressed and / or CD99 is strongly expressed, it indicates a positive minimal residual disease; (5) In the CD3-CD94- gate, if CD48 is weakly expressed and / or CD99 is strongly expressed, it indicates a positive minimal residual disease.

[0084] Of the five judgment criteria mentioned above, criterion (1) mainly targets abnormal T cells in the early marker gates (TdT gate, CD99bri gate, CD34 gate), and uses the LAIP strategy to capture T-ALL cells in the primitive / immature stage; criterion (2) targets abnormal cells in the CD45dimCD7bri region, and uses the DFN strategy to identify tumor cells in the NK cell region but without NK cell phenotype characteristics; criterion (3) targets abnormal cells in the DP region, and identifies mature T-ALL cells that have acquired CD4 and CD8 double positive characteristics; criterions (4) and (5) target abnormal cells in the gd-CD94- gate and CD3-CD94- gate, respectively, and are specifically used to identify T-ALL subgroups that are CD3 negative or TCRγδ negative and CD94 negative, effectively filling the detection blind spots of the traditional scheme in the DN region and CD3-CD94- region.

[0085] In a preferred embodiment, the above five criteria are used in combination to maximize detection sensitivity and specificity. In situations of limited resources, criteria (1) and (2) can be used for initial screening, and further analysis can be determined based on the preliminary results. In a specific embodiment, normal bone marrow specimens (complete remission bone marrow or bone marrow unrelated to disease) are first selected as negative controls for each test to determine the distribution pattern of normal cells within each phylum, serving as a reference for determining MRD positivity.

[0086] The core value of this invention's detection system lies in the organic integration of antibody compositions with intelligent analysis units, achieving standardization throughout the entire process from sample detection to result interpretation. The detection unit prepares flow cytometry samples using the sample processing flow described in this invention (see Example 2), obtaining at least 300,000 cells per tube, preferably 1 million cells, to ensure sufficient statistical analysis basis and detection sensitivity. The analysis unit and judgment unit use preset multi-marker combination gating templates to achieve semi-automated MRD analysis, reducing reliance on operator experience and improving the consistency and reproducibility of detection results.

[0087] In one implementation, the analysis unit and the judgment unit can be integrated with computer-aided analysis software or artificial intelligence analysis algorithms to achieve automated gating and MRD judgment of flow cytometry data. Full-spectrum flow cytometry uses software algorithms for automatic fluorescence compensation without manual adjustment. Building upon this, the present invention further provides a standardized multi-marker combination gating template, solidifying the analysis logic of each T cell subset into a repeatable analysis workflow, which helps promote the artificial intelligence development of T-ALL MRD detection in flow cytometry. Data analysis software suitable for the detection system of the present invention includes, but is not limited to, Kaluza, FCS Express, and FlowJo.

[0088] In a preferred embodiment, the detection system further includes a full-spectrum flow cytometer, preferably a full-spectrum flow cytometer with at least 3 lasers and at least 38 channels, such as the Cytek NL-CLC from Cytek Corporation. In another embodiment, the detection system may also include a standard operating procedure (SOP) document to guide operators in performing each step of specimen processing, instrumentation, and result analysis in a standardized manner.

[0089] According to one embodiment of the present invention, in the detection system, the sample of the individual to be tested is bone marrow or peripheral blood. Bone marrow samples are the preferred sample for T-ALL MRD detection, as they yield a higher proportion of tumor cells and more accurate test results; peripheral blood samples are suitable for patients who have difficulty or cannot tolerate bone marrow aspiration, as well as for situations requiring dynamic monitoring.

[0090] Before testing, bone marrow or peripheral blood samples should be anticoagulated with heparin or EDTA to prevent coagulation. The amount of cells added to the flow cytometry tube is approximately 2 × 10⁻⁶. 6 Cells / tube, cell count range 1×10 6 ~1×10 7 Cells / tube. For peripheral blood samples, if the cell count is low, add a volume exceeding 160 µl of sample first, centrifuge to remove the supernatant, concentrate, and then use. Samples should be processed as soon as possible after collection to ensure cell viability and antigen integrity.

[0091] Understanding the normal T cell development process is crucial background knowledge for comprehending the analytical methods of this invention. Pan-T markers include cCD3, CD7, CD2, and CD5, with CD7 and cCD3 being the true pan-T markers and therefore the most commonly used gating markers. CD7 fluorescence intensity changes during normal T cell development: strong expression in the primitive / immature stage, weakest expression in the cortical stage, and moderate intensity in the mature stage; however, strong CD7 expression is observed in over 95% of T-ALL cases. CD2 and CD5 appear in the second stage of T cell development. CD2 shows little change throughout development, while CD5 expression increases with cell maturation, but is weakly expressed or even negative in the TCRγδ cell subset. Markers for primitive T cells include strong expression of TdT, CD34, CD10, and CD99. T cells in the thymic cortex stage express CD1a, and simultaneously express CD4 and CD8 (i.e., double positive), with weak TCR expression. Mature T lymphocytes are divided into TCRαβ cells (accounting for more than 90% of T cells) and TCRγδ cells (accounting for 3% to 10% of T cells) based on differences in TCR expression.

[0092] The application of CD48 in T-ALL MRD detection is one of the key innovations of this invention. CD48 is a newly discovered biomarker for T-ALL MRD detection. Normal mature T cells express CD48, while T-ALL tumor cells show weakened or negative CD48 expression, forming a detectable difference from normal cells. Therefore, CD48 has significant value for T-ALL MRD detection. However, traditional flow cytometry has a limited number of biomarkers that can be detected, and only a few laboratories include this biomarker. This invention uses full-spectrum flow cytometry to detect 16 biomarkers. By eliminating duplicate backbone antibodies in two-tube protocols, CD48 can be included in the detection protocol without increasing costs, playing an important auxiliary role in MRD detection.

[0093] In summary, the antibody composition and analytical method of this invention are a systematic detection scheme established after long-term clinical practice and extensive experimental verification, based on a thorough understanding of the immunophenotypic characteristics of T-ALL, the normal developmental pattern of T cells, and the specificity and sensitivity of various markers in MRD detection. It represents the current state-of-the-art technology for full-spectrum flow cytometry detection of T-ALL MRD. This invention can be used for detection in newly diagnosed cases as well as for post-treatment follow-up, and is particularly suitable for MRD follow-up detection after targeted therapy (such as CD7-CAR-T therapy).

[0094] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail with reference to specific embodiments, but this should not be construed as limiting the scope of implementation of the present invention.

[0095] Example 1: Preparation of Reagents

[0096] This embodiment describes the formulation of an antibody composition for the full-spectrum flow cytometry detection of acute T-lymphoblastic leukemia.

[0097] Group 1 antibodies: Anti-CD45 antibody (fluorescein-labeled: cFluor V547), anti-CD99 antibody (cFluorV610), anti-CD34 antibody (cFluor BYG575), anti-CD48 antibody (cFluor BYG781), anti-CD2 antibody (cFluorV670), anti-CD3 antibody (cFluor V450), anti-CD4 antibody (cFluor B548), anti-CD5 antibody (cFluor BYG750), anti-CD7 antibody (cFluor BYG610), anti-CD8a antibody (cFluor BYG710), anti-CD16 antibody (cFluor V420), anti-CD56 antibody (cFluor BYG667), anti-CD94 antibody (cFluor R720), and anti-TCRγδ antibody (cFluor V547). A total of 14 fluorescently labeled monoclonal antibodies (R668) were mixed evenly in a volume ratio of 3:3:3:3:3:3:3:3:3:3:3:3:3:3:3 and placed in the first container to obtain the first group of antibody mixtures, with a total volume of 42 µl / tube.

[0098] The second group of antibodies: two fluorescently labeled monoclonal antibodies, anti-nuclear TdT antibody (fluorescein-labeled: FITC) and anti-cytoplasmic CD3 antibody (cFluorR840), were mixed evenly at a volume ratio of 2:3 and placed in the second container to obtain the second group of antibody mixture, with a total volume of 5 µl / tube.

[0099] All of the above antibodies are monoclonal antibodies and are commercially available. The anti-TdT antibody (TdT-FITC) was purchased from Invitrogen, USA; the remaining fluorescein-labeled antibodies were purchased from Cytek Biosciences, USA. All antibodies should meet the relevant industry standards.

[0100] Optional reagents: Hemolysin (1×, Becton Dickinson, USA) in the third container; membrane permeation agent A (Becton Dickinson, USA) in the fourth container; membrane permeation agent B (Becton Dickinson, USA) in the fifth container; PBS buffer (Beckman Coulter, USA) in the sixth container. All of the above reagents are commercially available.

[0101] Table 1. Antibody composition and fluorescent labeling for tube A

[0102] Example 2: Preparation of Flow Cytometry Samples

[0103] In this embodiment, the antibody composition prepared in Example 1 is used to process the sample to be tested, and the sample is prepared for flow cytometry. The specific steps are as follows:

[0104] (1) Based on the cell count results, add heparinized or EDTA-anticoagulated bone marrow or peripheral blood samples to the flow cytometer tube A, ensuring that the added cell count is approximately 2 × 10⁻⁶. 6 Cells / tube (cell count range: 1×10) 6 / tube~1×10 7 / tube). If the sample to be tested is peripheral blood and the cell count is low, a sample volume of more than 160 µl can be added first, centrifuged to remove the supernatant and concentrated before use.

[0105] (2) Add 42 µl of the first group of antibody mixture in the first container to the flow cytometer tube A, mix thoroughly with the cell suspension, and incubate at room temperature (room temperature) in the dark for 15 minutes.

[0106] (3) Add 100 µl of film-breaking agent A solution to the flow cytometer tube A after incubation in step (2) and incubate at room temperature in the dark for 5 minutes.

[0107] (4) Add 3 ml of 1×hemolysin to the flow cytometer tube after incubation in step (3) and incubate at room temperature in the dark for 10 minutes to lyse the red blood cells.

[0108] (5) Place the flow cytometry tube A after incubation in step (4) on a centrifuge and centrifuge at 1500 rpm (about 300-450 g) for 5 minutes to remove the supernatant.

[0109] (6) Add 50 µl of membrane-breaking agent B solution and the second group of antibody mixture (2 µl of anti-TdT-FITC antibody and 3 µl of anti-cytoplasmic CD3-cFluor R840 antibody) to the flow cytometer tube A after removing the supernatant in step (5), and incubate at room temperature in the dark for 15 minutes.

[0110] (7) Add 3 ml of PBS buffer to the flow cytometry tube A after incubation in step (6) and wash thoroughly. After mixing thoroughly, centrifuge at 1500 rpm for 5 minutes to remove the supernatant. Then add 0.5 ml of PBS buffer to resuspend the cells to obtain the processed flow cytometry sample.

[0111] Example 3: Specimen Detection and Clinical Validation

[0112] The specimens processed according to the method in Example 2 were analyzed on a Cytek 38-channel full-spectrum flow cytometer with 3 lasers in the United States. At least 300,000 cells were obtained per tube, preferably 1 million cells. Data analysis was performed using software such as Kaluza.

[0113] Gating procedure: When performing flow cytometry analysis, tube A should be gated as follows:

[0114] (1) Use FSC-A / H to set the single gate to remove adherent cells; use FSC / SSC to set the live cell gate within the single gate to obtain a single live cell.

[0115] (2) CD45 / SSC is used to set up each blood cell gate in the live gate; CD45dimCD7bri gate is set up in the live gate using CD45 weak expression / CD7 strong expression, TdT gate is set up using SSC small / TdT positive, CD99bri gate is set up using CD45 weak expression / CD99 strong expression, CD34 gate is set up using SSC small / CD34 positive, CD7 gate is set up using SSC / CD7 positive, and cCD3 gate is set up using SSC / cytoplasmic CD3 positive; the CD7 gate and cCD3 gate are combined to set up a logic gate, named TNK gate.

[0116] (3) In the CD45dimCD7bri gate, CD94 positive / CD56 positive NK gate is set up; in the TNK gate, CD4 / CD8 is used to set up CD4 positive CD8 positive DP gate and CD4 negative CD8 negative DN gate, and CD3 / CD94 is used to set up CD3-CD94- gate; in the DN gate, TCRγδ / CD94 is used to set up gd-CD94- gate.

[0117] Result Interpretation: Within the multi-marker gating system, the distribution patterns of T cells and NK cell subsets are compared with those of normal cells to identify tumor cells. Specific interpretation principles are as follows:

[0118] ① Within the TdT, CD99bri, and CD34 gates, display two-dimensional dot plots of CD7 / cCD3 and CD2 / CD5, respectively. If any one of these markers is positive, it indicates a positive minimal residual disease (MRD) diagnosis. ② Within the CD45dimCD7bri gate, if CD45dimCD7bri cells are not within the NK gate, especially if CD99bri and / or CD48dim and / or CD5 expression is present, it indicates a positive MRD diagnosis. ③ Within the DP gate, if positive cells are present, note whether they are accompanied by CD7 and... The presence of any one of the following is considered a positive result for minimal residual disease (MRD); ④ The presence of CD48 / CD7 and CD99 / cCD3 within the gd-CD94-gate, with weak CD48 expression and / or strong CD99 expression, indicates a positive MRD; ⑤ The presence of CD48 / CD7 and CD99 / cCD3 within the CD3-CD94-gate, with weak CD48 expression and / or strong CD99 expression, indicates a positive MRD. A recurrence is considered when the proportion of malignant cells exceeds 5% of nucleated cells.

[0119] Clinical Validation: Since February 2024, Hebei Yanda Lu Daopei Hospital has used the full-spectrum flow cytometry antibody composition protocol of this invention for MRD detection of T-ALL, while simultaneously conducting conventional two-tube, nine-color, multi-parameter flow cytometry as a control. From March 2024 to May 2026, a total of 6274 tests were completed from 4094 individuals, of which:

[0120] Full-spectrum flow cytometry group: 3140 tests were performed on 2050 individuals, with a median age of 16 years (range 1–68 years) and a male-to-female ratio of 1536:514; among them, 455 positive cases were performed on 310 individuals, with a median age of 21 years (range 4–68 years) and a male-to-female ratio of 238:72, and a median tumor burden of 1.66% (range 0.002%–94.15%).

[0121] The conventional flow cytometry group consisted of 3,134 tests from 2,044 individuals, with a median age of 18 years (range 1–73 years) and a male-to-female ratio of 1518:526. Among these, 515 tests from 430 individuals were positive cases, with a median age of 19 years (range 4–73 years) and a male-to-female ratio of 330:100. The median tumor burden was 3.45% (range 0.01%–99.31%).

[0122] Simultaneous validation using morphological, genetic, clinical manifestation, and follow-up methods showed that, compared with traditional flow cytometry, the method of this invention increases the detection sensitivity from 10... -4 Increased to 10 -5Of these, 8 patients (2.58%) had tumor cells ranging from 0.002% to 0.004%, which were only detected by full-spectrum flow cytometry. A comparison of detection performance indicators is detailed in Table 2.

[0123] Table 2 Comparison of T-ALL MRD performance between full-spectrum flow cytometry and conventional flow cytometry

[0124] Table 3. Positive rate and contribution percentage of T-ALL MRD markers detected by full-spectrum flow cytometry (310 cases)

[0125] The above results indicate that the expression rates of CD94 and CD16 in T-ALL are almost zero, further confirming the scientific basis for incorporating CD94 into the detection protocol to differentiate NK cells from T-ALL tumor cells. The inclusion of TCRγδ can identify the rare TCRγδ type of T-ALL (accounting for 0.65% in this group), cases that are easily missed in traditional protocols.

[0126] Furthermore, compared to traditional flow cytometry protocols, the full-spectrum flow cytometry protocol of this invention increases the coverage of markers such as CD2, CD16, CD94, and TCRγδ, making the detection of pan-T markers more comprehensive; it significantly enhances the exclusion of NK cells and γδT cells; the efficiency of specimen processing and analysis is improved by 30%–40%, and the cost of consumables is reduced by 30%–40%. Traditional flow cytometry protocols often require manual adjustment and compensation when designed up to nine colors, while the full-spectrum flow cytometry used in this invention completes the compensation by software algorithms, eliminating the need for manual adjustment, reducing reliance on operator training, experience, and subjective judgment, and promoting the artificial intelligence development of flow cytometry T-ALL MRD detection.

[0127] Table 4. Positive rate and contribution percentage of T-ALL MRD markers detected by conventional flow cytometry (430 cases)

[0128] Comparing Tables 3 and 4, it is evident that the full-spectrum flow cytometry protocol outperforms the traditional protocol in terms of pan-T marker coverage (addition of CD2) and exclusion of NK cells and γδT cells (addition of CD94, CD16, and TCRγδ). Notably, the contribution percentage of CD48 in the traditional protocol (87.68%) is higher than that in the full-spectrum protocol (66.45%), which aligns with the "reduced CD48 sensitivity" stated in the invention specification, suggesting that the full-spectrum protocol has stricter interpretation criteria for CD48. CD56 positivity is a challenging symptom to detect; the reduced positivity rate of full-spectrum flow cytometry helps reduce false positives.

[0129] Optimization of the fluorescein regimen: The full-spectrum flow cytometry antibody composition of this invention has undergone continuous optimization. Hebei Yanda Lu Daopei Hospital adopted a preliminary regimen (initially excluding CD48 and TCRγδ, which were gradually added later) since February 2024. Furthermore, the early fluorescein combinations differed—it was later found that the positive rate of TdT BV421 labeling was low, and in April 2026, it was adjusted to TdT-FITC, which is the optimal fluorescein combination described in this invention. TdT is fixed at FITC, and other antibody fluoresceins can be interchanged, but the above-mentioned fluorescein combination is the optimal result determined after optimization testing with multiple regimens (at least four).

[0130] Table 5. Antigen expression in 130 newly diagnosed T-ALL cases (Statistics from Hebei Yanda Lu Daopei Hospital)

[0131] Table 5 shows the antigen expression data of newly diagnosed T-ALL, providing important scientific basis for the gating strategy of the analytical method of this invention. CD7 (total positive rate 99.23%) and cCD3 (total positive rate 99.23%) are the most stable pan-T markers, supporting the construction of TNK logic gates by combining CD7 and cCD3. CD99 (total positive rate 96.92%) and TdT (total positive rate 74.62%) are early markers with high positive rates in newly diagnosed T-ALL, but the positive rates decrease as treatment progresses, so they need to be combined with multiple mature markers for comprehensive judgment. The total positive rate of TCRγδ in newly diagnosed T-ALL is 8.46%, indicating the existence of a certain proportion of TCRγδ type T-ALL. This subtype is difficult to identify by traditional methods, which is one of the important bases for including TCRγδ antibodies in this invention.

[0132] Example 4: Full-spectrum flow cytometry gating analysis of typical cases

[0133] This embodiment uses the antibody composition prepared in Example 1 and the specimen processing method described in Example 2 to perform full-spectrum flow cytometry analysis on three representative bone marrow specimens, demonstrating the application of the analytical method of this invention in actual clinical testing. Specific results are as follows: Figures 1a to 3b As shown in the diagram, 20 bone marrow samples from patients with complete remission from other unrelated diseases were first tested. 16-color full-spectrum flow cytometry was used to detect the normal expression intensity of major markers of T cells and NK cells in the bone marrow, as well as the regular expression distribution patterns of their pairwise combinations. Based on this, tumor cells showed varying degrees of difference from normal cells in the expression patterns of various antigens and antigen combinations, and this difference was used to determine MRD positivity. A relapse was considered when the proportion of malignant cells exceeded 5% of nucleated cells.

[0134] Case 1: Bone marrow specimen in complete remission after treatment for other unrelated diseases (see...) Figure 1a and Figure 1b )

[0135] The tube A was analyzed according to the gating steps described in Example 3. ① A single gate and a live gate were set to obtain single live cells; ② Each blood cell gate was set using CD45 / SSC within the live gate; ③ A CD45dimCD7bri gate was set within the live gate using CD45 weak expression / CD7 strong expression, displaying a two-dimensional dot plot of CD16 / CD56, CD94 / CD56, CD99 / CD7, and CD48 / CD5 within the gate; ④ An NK gate was set within the CD45dimCD7bri gate using CD94 positive / CD56 positive; in this patient, all CD45dimCD7bri cells were within the NK gate, meaning all CD45dimCD7bri cells were NK cells; ⑤ A TdT gate, a CD99bri gate, and a CD34 gate were set within the live gate, displaying C... D7 / cCD3, CD2 / CD5: No positive markers for CD7, cCD3, CD2, or CD5 were observed in any of the gates in this patient, indicating a negative minimal residual disease (MRD) result. ⑥ CD7, cCD3, and TNK logic gates were set up. ⑦ Within the TNK gate, DP, DN, and CD3-CD94- gates were set up; within the DN gate, a gd-CD94- gate was set up, displaying CD48 / CD7 and CD99 / cCD3, with no weak CD48 expression and / or strong CD99 expression observed. ⑧ The CD3-CD94- gate displayed CD48 / CD7 and CD99 / cCD3, also without abnormal expression. ⑨ The live gate displayed 14 two-dimensional dot maps including CD99 / CD7, with no MRD-positive cells observed in any region (indicated by dashed arrows). Overall assessment: This case is negative for MRD.

[0136] Case 2: Bone marrow specimens from a patient with positive T-ALL minimal residual disease after treatment (see...) Figure 2a and Figure 2b )

[0137] The tube A was analyzed according to the gate setup steps described in Example 3. Steps ① to ③ are the same as in Case 1; ④ Inside the CD45dimCD7bri gate, an NK gate was set up using CD94-positive / CD56-positive cells. In this case, only a small number of CD45dimCD7bri cells (bright blue) were located inside the NK gate, while a large number of red cells were located outside the NK gate, all of which were tumor cells; ⑤ Inside the live gate, TdT, CD99bri, and CD34 gates were set up respectively: Cells inside the TdT gate did not express T cell markers CD7, cCD3, CD2, and CD5, and no tumor cells were observed; Cells inside the CD99bri and CD34 gates expressed CD7 and cCD3, partially expressed CD2, and did not express CD5, indicating minimal residual disease (malignant tumor cells); ⑥ A TNK gate was set up; ⑦ Inside the TNK gate, The patient had no DP cells; ⑧ A gd-CD94- gate was set within the DN gate, with a small number of tumor cells within the DN gate, mostly CD4-positive and CD8-negative tumor cells (red cell population); ⑨ Tumor cells with strong CD7 expression, weak CD48 expression, cCD3-positive, and strong CD99 expression (red cell population) appeared within the CD3-CD94- gate, indicating a positive minimal residual disease (MRD) diagnosis; ⑩ The live gate showed 14 two-dimensional dot plots, and tumor cells were observed in all regions except for five areas with no tumor cells (dashed arrows): strong CD99 expression, positive or weak CD5 expression, weak CD48 expression, positive or weak CD5 expression, negative CD3, positive or weak CD5 expression, positive CD4, positive CD8, and positive CD7 and TdT. Overall assessment: This case is a positive case for minimal residual disease (MRD).

[0138] Case 3: Bone marrow specimen from a patient with positive T-ALL minimal residual disease after treatment (two tumor cell populations were present, see...) Figure 3a and Figure 3b )

[0139] This case is a typical example of T-ALL heterogeneity, with two groups of tumor cells with different phenotypes (MRD1 and MRD2). The tube A was analyzed according to the gating procedure described in Example 3. Steps ① to ③ are the same as in Case 1; ④ An NK gate was set within the CD45dimCD7bri gate: some CD45dimCD7bri cells (bright blue) were located within the NK gate, and the remaining two groups of tumor cells were: a red tumor cell group (MRD1) expressing CD56, strongly expressing CD99, partially expressing CD5, and not expressing CD16, CD94, or CD48; a pink tumor cell group (MRD2) weakly expressing CD99 and not expressing CD56, CD5, CD16, CD94, or CD48; ⑤ The cells within the TdT and CD99bri gates had the same phenotype, both being the MRD1 cell group, expressing CD7 and cCD3, partially expressing CD5, and not expressing CD2; two groups of tumor cells were observed within the CD34 gate: MRD1 (phenotype same as above) and MRD2 (expressing CD7, CD2, and weakly expressing CD5). (cCD3, not expressing CD5); ⑥ Set up the TNK gate; ⑦ Within the TNK gate, the patient has no DP cells; ⑧ Within the DN gate, a gd-CD94- gate is set up, and both groups of tumor cells are within the DN gate and within the gd-CD94- gate: MRD1 expresses CD7 and cCD3, strongly expresses CD99, and does not express CD48; MRD2 expresses CD7, weakly expresses cCD3 and CD99, and does not express CD48; ⑨ Within the CD3-CD94- gate, the phenotypes of the two groups of tumor cells are the same as above; ⑩ Within the live gate, in the 14 two-dimensional dot plots, MRD1 has no tumor cells except in the two regions of CD3 negative, CD5 positive or weakly positive, and CD4 positive, CD8 positive (dashed arrows), while the rest of the regions are visible (solid arrows); MRD2 is only visible in the two regions of CD48 weak expression, CD7 strong expression, and CD34 positive, CD7 positive. Overall assessment: This case showed positive minimal residual disease and the presence of two groups of tumor cells with different phenotypes, reflecting the phenotypic heterogeneity of T-ALL. The multi-marker gating method of this invention can effectively identify such complex cases.

[0140] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody composition for detecting acute T-lymphoblastic leukemia using full-spectrum flow cytometry, characterized in that, The antibody composition comprises a first group of antibodies and a second group of antibodies. The first group of antibodies consists of: anti-CD45 antibody, anti-CD99 antibody, anti-CD34 antibody, anti-CD48 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8a antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD94 antibody, and anti-TCRγδ antibody. The second group of antibodies consists of: anti-nuclear TdT antibody and anti-cytoplasmic CD3 antibody.

2. The antibody composition according to claim 1, characterized in that, All of the antibodies mentioned are monoclonal antibodies.

3. The antibody composition according to claim 1, characterized in that, All antibodies mentioned are fluorescently labeled antibodies; In the first group of antibodies, the fluorescent labels of anti-CD45 antibody, anti-CD99 antibody, anti-CD34 antibody, anti-CD48 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8a antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD94 antibody, and anti-TCRγδ antibody are as follows: cFluor V547, cFluor V610, cFluor BYG575, cFluor BYG781, cFluor V670, cFluor V450, cFluor B548, cFluor BYG750, cFluor BYG610, cFluor BYG710, cFluor V420, cFluor BYG667, cFluor R720, and cFluor R668. In the second group of antibodies, the anti-TdT antibody and the anti-cytoplasmic CD3 antibody were labeled with FITC and cFluorR840, respectively.

4. The antibody composition according to claim 1, characterized in that, The first group of antibodies is a mixture of anti-CD45 antibody, anti-CD99 antibody, anti-CD34 antibody, anti-CD48 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8a antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD94 antibody and anti-TCRγδ antibody in equal volume ratio; The second group of antibodies is a mixture of anti-TdT antibody and anti-cytoplasmic CD3 antibody in a volume ratio of 2:

3.

5. The use of the antibody composition according to any one of claims 1 to 4 in the preparation of a reagent for the full-spectrum flow cytometry detection of acute T-lymphoblastic leukemia.

6. The application according to claim 5, characterized in that, The reagent is a kit, which includes a first container and a second container. The first container contains the first group of antibodies, and the second container contains the second group of antibodies.

7. The application according to claim 6, characterized in that, The kit also includes one or more of the following: red blood cell lysis buffer, membrane-breaking agent, buffer solution, and flow cytometry tubes.

8. The use of the antibody composition according to any one of claims 1 to 4 or the reagent according to any one of claims 5 to 7 in the preparation of flow cytometry samples for the detection of acute T-lymphoblastic leukemia based on full-spectrum flow cytometry, characterized in that, Includes the following steps: Add the sample to be tested into tube A of the flow cytometer, add the first group of antibodies for incubation; then add membrane-permeability solution A to tube A for incubation, then add hemolysin for incubation, centrifuge to remove the supernatant; add membrane-permeability solution B and the second group of antibodies to tube A for incubation, wash, centrifuge to remove the supernatant, resuspend the cells, and obtain the flow cytometry sample; The sample to be tested is bone marrow or peripheral blood.

9. A detection system for detecting acute T-lymphoblastic leukemia using full-spectrum flow cytometry, characterized in that, The detection system includes a detection unit, an analysis unit, and a judgment unit, wherein: The detection unit includes the flow cytometry sample as described in claim 8; The analysis unit performs gate analysis according to the following analysis method, including the following gate steps: (1) Use FSC-A / H to set the single gate to remove adherent cells, and use FSC / SSC to set the live cell gate within the single gate to obtain a single live cell; (2) CD45 / SSC is used to set up each blood cell gate in the live gate; CD45dimCD7bri gate is set up by CD45 weak expression / CD7 strong expression, TdT gate is set up by SSC small / TdT positive, CD99bri gate is set up by CD45 weak expression / CD99 strong expression, CD34 gate is set up by SSC small / CD34 positive, CD7 gate is set up by SSC / CD7 positive, and cCD3 gate is set up by SSC / cytoplasmic CD3 positive. The CD7 gate and cCD3 gate are combined to set up a logic gate, named TNK gate; (3) In the CD45dimCD7bri gate, CD94 positive / CD56 positive NK gate is set up; in the TNK gate, CD4 / CD8 are used to set up CD4 positive CD8 positive DP gate, CD4 negative CD8 negative DN gate, and CD3 / CD94 is used to set up CD3-CD94- gate; in the DN gate, TCRγδ / CD94 is used to set up gd-CD94- gate; The judgment unit compares the distribution patterns of T cells and NK cell subsets with normal cells based on the expression of the markers in the above-mentioned gate to determine whether minimal residual disease is positive.

10. The detection system according to claim 9, characterized in that, The methods for determining minimal residual lesions include at least one of the following: Within the TdT gate, CD99bri gate, and CD34 gate, CD7 / cCD3 and CD2 / CD5 are displayed, respectively. If any of the CD7, cCD3, CD2, or CD5 markers are positive, it is judged as a positive minimal residual disease. If CD45dimCD7bri cells are not in the NK gate, or if CD99bri and / or CD48dim and / or CD5 expression is present, it is considered a positive minimal residual disease. Within the DP gate, if positive cells are present, accompanied by enhancement or decrease of CD7 and / or cCD3, decrease of CD45 and / or CD48, enhancement of CD99 expression, and expression of CD34 and / or TdT, any one of these is considered a positive minimal residual disease. The gd-CD94-gate shows CD48 / CD7 and CD99 / cCD3. If weak expression of CD48 and / or strong expression of CD99 are present, it indicates a positive minimal residual disease. The CD3-CD94-gate shows CD48 / CD7 and CD99 / cCD3. If weak expression of CD48 and / or strong expression of CD99 are present, it indicates a positive minimal residual disease.

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