Reagent composition for screening and / or diagnosing plasma cell tumors and its application

Abstract

The present invention discloses a reagent composition for screening and / or diagnosing plasma cell tumors and its application. The reagent composition of the present invention comprises anti-CD19 antibody, anti-CD56 antibody, anti-CD138 antibody, anti-CD81 antibody, anti-CD38 antibody, anti-CD27 antibody, anti-CD45 antibody, KAPPA antibody and LAMBDA antibody. The reagent composition of the present invention can improve the ability to distinguish tumor plasma cells from normal plasma cells. In addition, the present invention also provides a gate setting method for flow cytometry analysis. Through the gate setting method of the present invention, the abnormal plasma cell ratio in CD56 or CD19 or CD117 or CD81 or CD27 of gate B and the expression ratio of KAPPA and LAMBDA can be combined to achieve accurate and rapid analysis of samples, and to achieve the determination and typing of plasma cell tumors, and effectively avoid false negative judgment results.

Description

Technical Field

[0001] The present invention relates to the technical field of plasma cell tumor detection reagents, and in particular to a reagent composition for screening and / or diagnosing plasma cell tumors and applications thereof. Background Art

[0002] Multiple myeloma (MM) is the most common type of malignant plasma cell disease, also known as myeloma, which can cause the malignant proliferation of monoclonal plasma cells, extensive infiltration and secretion of large amounts of monoclonal immunoglobulin (M protein) and cause extensive bone destruction.

[0003] Multiple myeloma is one of the three most common blood tumors, mostly occurring in the elderly. There are significant differences in gender distribution, with the incidence rate in men being significantly higher than in women. Data show that the incidence rate of MM in my country is approximately 1-1.5 / 100,000. Although lower than the incidence rate abroad, it has been increasing year by year.

[0004] Multiple myeloma causes malignant proliferation and abnormal accumulation of clonal plasma cells in the bone marrow, accompanied by the appearance and deposition of large amounts of monoclonal immunoglobulins. It is a debilitating, incurable malignant disease. Multiple myeloma is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, kidney damage, increased susceptibility to bacterial infections, and suppressed production of normal immunoglobulins. The clinical manifestations are complex and diverse, non-specific, and the misdiagnosis rate is as high as 84%-85%.

[0005] In general, the immunophenotype of normal plasma cells is usually: CD138+, CD38++, CD79a+, CD27+, CIg+, SIg-, CD19 (>75%+), CD45+, CD56–, CD81+, CD20–, CD28–, CD33–, CD117–, and cytoplasmic k / l polyclonal expression. The immunophenotype of common MM is usually: CD138+, CD38++, CD79a+, CD27 (40-50%+), CD81 (55%+), CIg+, SIg-, CD56 (75%++), CD117 (30%+), CD20 (30%+), CD10- / +, CD19–, and cytoplasmic k / l monoclonal expression.

[0006] However, a small number (<30%) of normal plasma cells may also have the abnormal immunophenotype of MM mentioned above: CD38+bri, CD138+bri, CD19-, CD45- / low, CD20+, CD27low, CD81low, CD28+, CD56+, CD200+bri, but the cytoplasmic k / l is still polyclonally expressed. In addition, there are a small number of cases with false negative normal immunophenotypes. Summary of the invention

[0007] To overcome the above disadvantages, the object of the present invention is to provide a reagent composition for screening and / or diagnosing plasma cell tumors and its application. The reagent composition of the present invention is used in combination with CD19 antibody, anti-CD117 antibody, anti-CD56 antibody, anti-CD138 antibody, anti-CD38 antibody, anti-CD27 antibody, anti-CD45 antibody, KAPPA antibody and LAMBDA antibody to obtain the cytoplasmic k / l ratio, which can improve the ability to distinguish between tumor cells and normal plasma cells of MM, and can screen cases with false negative immunophenotypes, thereby improving the accuracy of MM identification.

[0008] The reagent composition for screening and / or diagnosing plasma cell tumors of the present invention comprises: a first antibody composition, wherein the first antibody composition comprises: anti-CD19 antibody, anti-CD117 antibody, anti-CD56 antibody, anti-CD138 antibody, anti-CD81 antibody, anti-CD38 antibody, anti-CD27 antibody, and anti-CD45 antibody;

[0009] The second antibody composition comprises a KAPPA antibody and a LAMBDA antibody.

[0010] Furthermore, the antibody composition is a fluorescein-labeled antibody, and the individual antibodies of the first antibody composition are paired with the following labeled fluorescences in sequence: CD19-ECD, CD117-PC5, CD56-PC7, CD138-APC, CD81-APC700, CD38-APC750, CD27-PB, CD45-KO; the fluorescent label pairing of the second antibody composition is Kappa-FITC and Lambda-PE.

[0011] Furthermore, the ratio of the added amount of each antibody in the first antibody composition and the second antibody composition is CD19-ECD 3-8, CD117-PC5 2-5, CD56-PC7 2-5, CD138-APC 2-6, CD81-APC700 2-4, CD38-APC750 2-5, CD27-PB 2-6, CD45-KO 2-6, Kappa-FITC 1-3, and Lambda-PE 1-3, respectively, and the ratio of the added amount is the volume ratio after each antibody is dissolved in the buffer.

[0012] Furthermore, the added mass ratio of the antibody composition is CD19-ECD 5, CD117-PC5 3, CD56-PC7 3, CD138-APC 3, CD81-APC700 3, CD38-APC750 3, CD27-PB 3, CD45-KO 3, Kappa-FITC1, and Lambda-PE 1.

[0013] Furthermore, the clone number of the anti-CD19 antibody is J3.119, the clone number of the anti-CD117 antibody is 104D2D1, the clone number of the anti-CD56 antibody is N901, the clone number of the anti-CD138 antibody is B-A38, the clone number of the anti-CD81 antibody is JS64, the clone number of the anti-CD38 antibody is LS198-4-3, the clone number of the anti-CD27 antibody is 1A4CD27, and the clone number of the anti-CD45 antibody is J33; the clone number of the KAPPA antibody is GAHK, and the clone number of the LAMBDA antibody is GAHL.

[0014] The second object of the present invention is to provide a kit, which includes the first antibody composition and the second antibody composition, and the kit has a first receiving cavity for receiving the first antibody composition and a second receiving cavity for receiving the second antibody composition.

[0015] The third object of the present invention is to provide a use of the above reagent composition in preparing flow cytometry samples for screening and / or diagnosing plasma cell tumors.

[0016] Furthermore, the process of preparing a flow cytometer sample for screening and / or diagnosing plasma cell tumors comprises the following steps:

[0017] ① Collection and pretreatment of samples to be tested: Take the patient's bone marrow and peripheral blood, lyse the red blood cells, centrifuge, and discard the supernatant;

[0018] ② Preparation of antibody composition reagent: dissolving the first antibody composition and the second antibody composition in a buffer solution respectively to obtain a first antibody composition reagent and a second antibody composition reagent for standby use;

[0019] ③ Preparation of fixative and membrane-breaking agent;

[0020] ④Sample processing:

[0021] Take an appropriate amount of the sample prepared in step ①, add the first antibody composition reagent prepared in step ②, mix well, incubate at room temperature, add a fixative, and incubate in the dark; add an appropriate amount of physiological PBS, centrifuge, discard the supernatant, shake and mix to precipitate cells, add a membrane permeabilizer, and gently blow and mix; add KAPPA antibody and LAMBDA antibody, incubate in the dark; add an appropriate amount of buffer, centrifuge, discard the supernatant, add an appropriate amount of PBS to suspend, and detect on the machine.

[0022] Furthermore, the gates are set according to the following steps when performing flow cytometry testing:

[0023] Set the plasma cell gate A, and use the CD138 / CD45 setting to obtain most plasma cells. In gate A, CD38 / CD81 is plasma cells.

[0024] Set the plasma cell gate K gate and use the CD45 / SS INT setting to obtain plasma cells;

[0025] Set the plasma cell gate E, and in Gate E, use CD19 / CD38 to display B lymphocytes;

[0026] Set the monoclonal plasma cell gate B and use CD56 / CD19 to display monoclonal plasma cells;

[0027] Circle the CD19 positive expression cells in the E gate. If normal, observe the KAPPA and LAMBDA expression ratios of the plasma cells in the B gate; if the abnormal monoclonal plasma cell ratio in CD56 / CD19 is greater than 1% and the KAPPA and LAMBDA expression ratio is greater than 3, it is preliminarily determined to be an abnormal cell; if LAMBDA / KAPPA>3, it is expressed as a LAMBDA type MM abnormal cell, and if KAPPA / LAMBDA>3, it is expressed as a KAPPA type MM abnormal cell. Through the gate setting method of the present invention, the A gate is set using CD138 / CD45, the K gate is set using CD45 / SSINT, and the E gate is set using CD19 / CD38, B lymphocytes can be quickly screened, and then monoclonal plasma cells are obtained by screening using CD56 / CD19. Therefore, it is possible to combine the abnormal monoclonal plasma cell ratio in CD56 / CD19 of the B gate and the KAPPA and LAMBDA expression ratios, and to achieve accurate and rapid analysis of the sample, and to achieve the determination and typing of plasma cell tumors.

[0028] Furthermore, the gating analysis also includes the following steps: for samples with normal phenotype, after using CD81 / CD27 gating analysis, continue to circle CD81-CD27- and set F gate, continue to use Lambda / Kappa gating analysis in F gate, and determine whether the sample is normal based on the analysis results.

[0029] The beneficial effects of the present invention are as follows: by utilizing the antibody composition of the present invention, anti-CD19 antibody, anti-CD117 antibody, anti-CD56 antibody, anti-CD138 antibody, anti-CD81 antibody, anti-CD38 antibody, anti-CD27 antibody, anti-CD45 antibody, KAPPA antibody and LAMBDA antibody are simultaneously combined to obtain a cytoplasmic k / l ratio, thereby improving the ability to distinguish between tumor cells and normal plasma cells of MM; in addition, by adding anti-CD81 antibody and anti-CD27 antibody and setting a gate for CD27-CD81- samples in the present invention, cases with false negative immunophenotypes can be screened, thereby improving the accuracy of MM identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 (a) is a sample flow cytometry gating analysis result of Example 1 of the present invention;

[0031] FIG1( b ) is the immunofixation electrophoresis result of the sample of Comparative Example 1 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0032] FIG2 (a) is a sample flow cytometry gating analysis result of Example 2 of the present invention;

[0033] FIG2 (b) is the immunofixation electrophoresis result of the sample of Comparative Example 2 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0034] FIG3 (a) is a sample flow cytometry gating analysis result of Example 3 of the present invention;

[0035] FIG3 (b) is the immunofixation electrophoresis result of the sample of Comparative Example 3 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0036] FIG4 (a) is a gating analysis result of sample flow cytometry in Example 4 of the present invention;

[0037] FIG4 (b) is the immunofixation electrophoresis result of the sample of Comparative Example 4 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0038] FIG5 (a) is a gating analysis result of sample flow cytometry in Example 5 of the present invention;

[0039] FIG5( b ) is the immunofixation electrophoresis result of the sample of Comparative Example 5 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0040] FIG6 (a) is a gating analysis result of sample flow cytometry in Example 6 of the present invention;

[0041] FIG6 (b) is the immunofixation electrophoresis result of the sample of Comparative Example 6 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0042] FIG. 7 (a) is a gating analysis result of sample flow cytometry in Example 7 of the present invention;

[0043] FIG7 (b) is the immunofixation electrophoresis result of the sample of Comparative Example 7 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0044] FIG8 (a) is a gating analysis result of sample flow cytometry in Example 8 of the present invention;

[0045] FIG8 (b) is the immunofixation electrophoresis result of the sample of Comparative Example 8 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0046] FIG9 (a) is a gating analysis result of sample flow cytometry in Example 9 of the present invention;

[0047] FIG9 (b) is the immunofixation electrophoresis result of the sample of Comparative Example 9 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0048] FIG. 10 ( a ) is a gating analysis result of sample flow cytometry according to Example 10 of the present invention;

[0049] FIG10( b ) is the immunofixation electrophoresis result of the sample of Comparative Example 10 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0050] FIG11( a ) is a gating analysis result of sample flow cytometry according to Example 11 of the present invention;

[0051] FIG11( b ) is the immunofixation electrophoresis result of the sample of Comparative Example 11 of the present invention, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ;

[0052] FIG12 (a) is a flow analysis result of a false negative sample;

[0053] FIG12 (b) shows the flow cytometry analysis result of a false negative sample after setting the gate as CD81-CD27-;

[0054] FIG12( c ) is the result of immunofixation electrophoresis for a false-negative sample, in which the lanes from left to right are ELP, IgG, IgA, IgM, κ and λ. DETAILED DESCRIPTION

[0055] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0056] The present invention improves the ability to distinguish between MM tumor cells and normal plasma cells by combining an antibody composition for MM detection and cytoplasmic k / l analysis.

[0057] In some embodiments, the sample detection using the reagent composition of the present invention is performed according to the following steps:

[0058] 1. Sample collection and pretreatment:

[0059] Take 500 μl of the patient's bone marrow or peripheral blood, add ammonium chloride hemolytic agent in a ratio (1:10), vortex to mix, let stand for 15 minutes; add 5 ml PBS, centrifuge and wash twice for 5 minutes / 1500 rpm, and remove the supernatant.

[0060] 2. Preparation of antibody composition reagents:

[0061] Each fluorescently labeled first antibody composition and second antibody composition is dissolved in a buffer solution.

[0062] In some embodiments, each antibody is a monoclonal antibody, the clone number of the anti-CD19 antibody is J3.119, the clone number of the anti-CD117 antibody is 104D2D1, the clone number of the anti-CD56 antibody is N901, the clone number of the anti-CD138 antibody is B-A38, the clone number of the anti-CD81 antibody is JS64, the clone number of the anti-CD38 antibody is LS198-4-3, the clone number of the anti-CD27 antibody is 1A4CD27, and the clone number of the anti-CD45 antibody is J33; the clone number of the KAPPA antibody is GAHK, and the clone number of the LAMBDA antibody is GAHL.

[0063] In some embodiments, the antibodies of the first antibody composition are paired with the following fluorescent markers in sequence: CD19-ECD, CD117-PC5, CD56-PC7, CD138-APC, CD81-APC700, CD38-APC750, CD27-PB, CD45-KO; the fluorescent marker pairing of the second antibody composition is Kappa-FITC and Lambda-PE.

[0064] The ratio of the added amount of each antibody in the antibody composition of the present invention is the volume ratio after each antibody is dissolved in a phosphate buffer.

[0065] In some embodiments, the ratio of the added amount of each antibody in the first antibody composition and the second antibody composition is CD19-ECD 3-8, CD117-PC5 2-5, CD56-PC7 2-5, CD138-APC 2-6, CD81-APC700 2-4, CD38-APC750 2-5, CD27-PB 2-6, CD45-KO 2-6, Kappa-FITC 1-3, and Lambda-PE 1-3.

[0066] 3. Preparation of fixative and punching solution:

[0067] Preparation of fixative (hereinafter referred to as solution 1):

[0068] In some embodiments, the fixative is commercially available, and its active ingredient is formaldehyde, which is used to fix cells.

[0069] Preparation of membrane-breaking agent (hereinafter referred to as liquid 2): In some embodiments, the membrane-breaking agent is commercially available, and its active ingredient is saponin. The membrane-breaking agent is used to lyse red blood cells.

[0070] 4. Sample processing:

[0071] After washing, the cells (50-100 μl) were added with the first antibody combination reagent (CD19-ECD, CD117-PC5, CD56-PC7, CD138-APC, CD81-APC700, CD38-APC750, CD27-PB, CD45-KO) and mixed, and incubated at room temperature for 15 min; 100 μl of fixative A was added and incubated in the dark for 15 min, washed by centrifugation with 2 ml PBS for 5 min / 1500 rpm, and the supernatant was discarded. The cells were first vortexed to mix, and then 100 μl of membrane permeabilization agent B was added and gently pipetted to mix; cytoplasmic antibodies (Kappa-FITC, Lambda-PE) were added and mixed gently, and incubated in the dark for 30 min at room temperature; 2 ml PBS was centrifuged and washed for 5 min / 1500 rpm, and the supernatant was discarded. 500 μl PBS was added to the cell pellet for suspension and detection.

[0072] 5. MM flow cytometry analysis

[0073] First, find plasma cells, use CD138 / CD45 to set gate A, and most of the obtained cells are plasma cells; CD38 / CD81 is plasma cells in A; CD45 / SS INT is used in gate K to obtain lymphocytes; CD19 / CD38 is used in gate E to display B lymphocytes; circle the CD19 positive cells in gate E, observe the KAPPA and LAMBDA expression ratios, if normal, observe the KAPPA and LAMBDA expression ratios of plasma cells in gate B; use CD56 / CD 19 in gate B to display monoclonal plasma cells; if the proportion of abnormal monoclonal plasma cells in CD56 / CD19 is greater than 1% and the KAPPA and LAMBDA expression ratio is greater than 3, the initial diagnosis is multiple myeloma. If LAMBDA / KAPPA>3, it indicates LAMBDA type MM, and if KAPPA / LAMBDA>3, it indicates KAPPA type MM.

[0074] In the prior art, the typing and detection of MM are mostly achieved through immunofixation electrophoresis. In the present invention, the serum of the blood sample obtained in the above step 1 "collection and pretreatment of samples" is simultaneously used for the detection and typing of MM by immunofixation electrophoresis.

[0075] Electrophoresis is performed using an electrophoresis analyzer (such as HYDRASYS 2SCAN sebia). The agarose film used in the electrophoresis process can be prepared in the laboratory or purchased.

[0076] In some embodiments, the steps of immunofixation electrophoresis of the present invention are:

[0077] Serum dilution—sample loading with sample comb—moisturizing—place agar film—place sample comb—electrophoresis—place dynamic mask rack—add antiserum to paint lanes—incubate—aspirate—dry—stain and decolorize.

[0078] In some embodiments, the HYDRAGEL IF immunoelectrophoresis fixation detection kit developed by Cybill is used for electrophoresis (its medical device registration certificate number is Guoxiezhujin 20172406085). During the electrophoresis, ELP is used as a reference protein to perform a qualitative analysis of the presence or absence of the object to be detected in the sample.

[0079] When performing electrophoresis, ELP was selected as the reference lane, and the IgG, IgA, IgM, κ, and λ bands were detected respectively.

[0080] In some embodiments, when preparing the sample, the serum can be diluted according to the dilution ratio shown in Table 1:

[0081] Table 1 Serum dilution ratio

[0082]

[0083] After the samples to be electrophoresed are prepared, the diluted serum samples are added to the sample wells using a sample comb, and then each sample comb is placed in a moisturizing box with the teeth facing upward for moisturizing.

[0084] After placing the agar gel in the electrophoresis apparatus, insert the sample comb and start electrophoresis.

[0085] Install the dynamic mask and add antiserum: Place the dynamic mask mounting frame on a flat surface, and install the cup holder with an antiserum cup on the dynamic mask mounting frame. Then put in the reagent sample addition reference color card, which should correspond to the analysis program used, and place it on the cup holder in front of the liquid cup hole, press the mask contact, push the mask, and then incubate.

[0086] After incubation, use the smooth side of thick filter paper to spread on the film to absorb excess antibody.

[0087] Drying process: After the absorption of liquid is completed, remove the filter paper and leave the film in place to dry the film.

[0088] Dyeing and decolorization: After drying, the film is dyed and decolorized using acid violet dye solution.

[0089] Different samples were processed according to the above steps 1-5 and flow cytometry analysis was performed. The specific flow cytometry analysis results are shown in Example 1-11.

[0090] Example 1

[0091] In this example, the ratio of the added amount of each antibody composition is CD19-ECD 5, CD117-PC5 3, CD56-PC7 3, CD138-APC 3, CD81-APC700 3, CD38-APC750 3, CD27-PB 3, CD45-KO 3, Kappa-FITC 1, and Lambda-PE 1.

[0092] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG1( a ). The immunophenotype of the sample is: CD45+, CD138+, CD38+, CD117+-, CD19+, CD27-, CD81+, CD56+, LAMBDA / KAPPA>3, which is LAMBDA type MM.

[0093] Comparative Example 1

[0094] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "Sample collection and pretreatment" of Example 1, and the electrophoresis results are shown in Figure 1 (b). In the figure, ELP is a reference spectrum in the experiment, and G, A, M, K, and L refer to IgG antibody, IgA antibody, IgM antibody, KAPPA light chain, and LAMBDA light chain, respectively.

[0095] As can be seen from the results of FIG. 1( b ), IgA antibodies and LAMBDA light chains were detected by immunofixation electrophoresis, and thus, it was determined to be IgA-LAMBDA type multiple myeloma. It can be seen that the flow cytometric analysis results of Example 1 are consistent with the typing and detection results of MM by immunoelectrophoresis of Comparative Example 1.

[0096] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-1:

[0097] Table 2-1 Plasma free light chain detection results of samples in Example 1

[0098] Test items Detection value instruct Reference interval unit Free kappa 6.74 3.30-19.40 mg / L Free λ 1120 ↑ 5.71-26.3 mg / L κ / λ 0.006 ↓ 0.26-1.65

[0099] Therefore, it can be seen that the flow cytometric analysis results of Example 1 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 1, and are consistent with the plasma free light chain detection results.

[0100] Example 2

[0101] In this example, the ratio of the added amount of each antibody composition is CD19-ECD 5, CD117-PC5 3, CD56-PC7 3, CD138-APC 3, CD81-APC700 3, CD38-APC750 3, CD27-PB 3, CD45-KO 3, Kappa-FITC 1, and Lambda-PE 1.

[0102] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG2( a ). The immunophenotype of the sample is: CD45+, CD138+, CD38+, CD117-, CD19+, CD27-, CD81+, CD56+, LAMBDA / KAPPA>3, which is LAMBDA type MM.

[0103] Comparative Example 2

[0104] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 2. The electrophoresis result is shown in FIG. 2 (b).

[0105] As can be seen from the results of Figure 2 (b), IgA antibodies and LAMBDA light chains were detected by immunofixation electrophoresis, and thus it was determined to be IgA-LAMBDA type multiple myeloma. It can be seen that the flow cytometric analysis results of Example 2 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 2.

[0106] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-2:

[0107] Table 2-2 Plasma free light chain detection results of samples in Example 2

[0108] Test items Detection value instruct Reference interval unit Free kappa 9.86 3.30-19.40 mg / L Free λ 48.7 ↑ 5.71-26.3 mg / L κ / λ 0.2 ↓ 0.26-1.65

[0109] Therefore, it can be seen that the flow cytometric analysis results of Example 2 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 2, and are consistent with the plasma free light chain detection results.

[0110] Example 3

[0111] In this example, the ratio of the added amount of each antibody composition is CD19-ECD 3, CD117-PC5 3, CD56-PC7 5, CD138-APC 6, CD81-APC700 3, CD38-APC750 4, CD27-PB 4, CD45-KO 4, Kappa-FITC 2, and Lambda-PE 2.

[0112] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG3 (a). The immunophenotype of the sample in this embodiment is: CD45+, CD138+, CD38+, CD117+, CD19+, CD27-, CD81+, CD56+, LAMBDA / KAPPA>3, which is LAMBDA type MM.

[0113] Comparative Example 3

[0114] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 3. The electrophoresis result is shown in FIG. 3( b ).

[0115] As can be seen from the results of Figure 3 (b), IgG antibodies and LAMBDA light chains were detected by immunofixation electrophoresis, and thus it was determined to be IgG-LAMBDA type multiple myeloma. It can be seen that the flow cytometric analysis results of Example 3 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 3.

[0116] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-3:

[0117] Table 2-3 Plasma free light chain detection results of samples in Example 3

[0118] Test items Detection value instruct Reference interval unit Free kappa 10.2 3.30-19.40 mg / L Free λ 99.8 ↑ 5.71-26.3 mg / L κ / λ 0.1 ↓ 0.26-1.65

[0119] Therefore, it can be seen that the flow cytometric analysis results of Example 3 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 3, and are consistent with the plasma free light chain detection results.

[0120] Example 4

[0121] In this example, the ratio of the added amount of each antibody composition is CD19-ECD 5, CD117-PC5 3, CD56-PC7 4, CD138-APC 4, CD81-APC700 3, CD38-APC750 4, CD27-PB 4, CD45-KO 4, Kappa-FITC 1, and Lambda-PE 1.

[0122] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG4( a ). The immunophenotype of the sample is: CD45+, CD138+, CD38+, CD117+, CD19+, CD27-, CD81++, CD56+, LAMBDA / KAPPA>3, which is LAMBDA type MM.

[0123] Comparative Example 4

[0124] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 4. The electrophoresis result is shown in FIG. 4( b ).

[0125] As can be seen from the results of FIG. 4 ( b ), IgA antibodies and LAMBDA light chains were detected by immunofixation electrophoresis, and thus, it was determined to be IgA-LAMBDA type multiple myeloma. Therefore, it can be seen that the flow cytometric analysis results of Example 4 are consistent with the typing and detection results of MM by immunoelectrophoresis in Comparative Example 4.

[0126] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-4:

[0127] Table 2-4 Plasma free light chain detection results of samples in Example 4

[0128] Test items Detection value instruct Reference interval unit Free kappa 8.65 3.30-19.40 mg / L Free λ 7700 ↑ 5.71-26.3 mg / L κ / λ 0.001 ↓ 0.26-1.65

[0129] Therefore, it can be seen that the flow cytometric analysis results of Example 4 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 4, and are consistent with the plasma free light chain detection results.

[0130] Example 5

[0131] In this example, the ratio of the added amount of each antibody composition is CD19-ECD 8, CD117-PC5 3, CD56-PC7 4, CD138-APC 6, CD81-APC700 3, CD38-APC750 3, CD27-PB 4, CD45-KO 3, Kappa-FITC 3, and Lambda-PE 1.

[0132] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG5( a ). The immunophenotype of the sample is: CD45+, CD138+, CD38+, CD117-, CD19+, CD27-, CD81-, CD56+, KAPPA / LAMBDA>3, which is KAPPA type MM.

[0133] Comparative Example 5

[0134] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 5. The electrophoresis result is shown in FIG. 5( b ).

[0135] As can be seen from the results of Figure 5 (b), IgG antibodies and KAPPA light chains were detected by immunofixation electrophoresis, thus determining it to be IgG-KAPPA type multiple myeloma. Therefore, it can be seen that the flow cytometric analysis results of Example 5 are consistent with the typing and detection results of MM by immunoelectrophoresis in Comparative Example 5.

[0136] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-5:

[0137] Table 2-5 Plasma free light chain detection results of samples in Example 5

[0138] Test items Detection value instruct Reference interval unit Free kappa 710 ↑ 3.30-19.40 mg / L Free λ 8.91 5.71-26.3 mg / L κ / λ 79.69 ↓ 0.26-1.65

[0139] Therefore, it can be seen that the flow cytometric analysis results of Example 5 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 5, and are consistent with the plasma free light chain detection results.

[0140] Example 6

[0141] In this example, the ratio of the added amount of each antibody composition is CD19-ECD 5, CD117-PC5 3, CD56-PC7 4, CD138-APC 3, CD81-APC700 2, CD38-APC750 3, CD27-PB 3, CD45-KO 3, Kappa-FITC 1, and Lambda-PE 2.

[0142] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG6( a ). The immunophenotype of the sample in this embodiment is: CD45+, CD138+, CD38+, CD117-, CD19+, CD27-, CD81++, CD56+, LAMBDA / KAPPA>3, which is LAMBDA type MM.

[0143] Comparative Example 6

[0144] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 6. The electrophoresis result is shown in FIG. 6( b ).

[0145] As can be seen from the results of FIG6( b ), IgA antibodies and LAMBDA light chains were detected by immunofixation electrophoresis, and thus, it was determined to be IgA-LAMBDA type multiple myeloma. Therefore, it can be seen that the flow cytometric analysis results of Example 6 are consistent with the typing and detection results of MM by immunoelectrophoresis of Comparative Example 6.

[0146] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-6:

[0147] Table 2-6 Plasma free light chain detection results of samples in Example 6

[0148] Test items Detection value instruct Reference interval unit Free kappa 16.6 3.30-19.40 mg / L Free λ 95.2 ↑ 5.71-26.3 mg / L κ / λ 0.17 ↓ 0.26-1.65

[0149] Therefore, it can be seen that the flow cytometric analysis results of Example 6 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 6, and are consistent with the plasma free light chain detection results.

[0150] Example 7

[0151] In this example, the ratio of the added amount of each antibody composition is CD19-ECD 5, CD117-PC5 5, CD56-PC7 3, CD138-APC 3, CD81-APC700 3, CD38-APC750 3, CD27-PB 3, CD45-KO 3, Kappa-FITC 1, and Lambda-PE 1.

[0152] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG7 (a). The immunophenotype of the sample is: CD45+, CD138+, CD38+, CD117-, CD19+, CD27-, CD81++, CD56+, LAMBDA / KAPPA>3, which is LAMBDA type MM.

[0153] Comparative Example 7

[0154] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 7. The electrophoresis result is shown in FIG. 7( b ).

[0155] As can be seen from the results of Figure 7 (b), IgA antibodies and LAMBDA light chains were detected by immunofixation electrophoresis, and thus it was determined to be IgA-LAMBDA type multiple myeloma. Therefore, it can be seen that the flow cytometric analysis results of Example 7 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 7.

[0156] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-7:

[0157] Table 2-7 Plasma free light chain detection results of samples in Example 7

[0158] Test items Detection value instruct Reference interval unit Free kappa 12.3 3.30-19.40 mg / L Free λ 307.5 ↑ 5.71-26.3 mg / L κ / λ 0.04 ↓ 0.26-1.65

[0159] Therefore, it can be seen that the flow cytometric analysis results of Example 7 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 7, and are consistent with the plasma free light chain detection results.

[0160] Example 8

[0161] In this example, the ratio of the added amount of each antibody composition is CD19-ECD 4, CD117-PC5 5, CD56-PC7 3, CD138-APC 3, CD81-APC700 2, CD38-APC750 3, CD27-PB 3, CD45-KO 3, Kappa-FITC 1, and Lambda-PE 2.

[0162] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG8 (a). The immunophenotype of the sample is: CD45+, CD138+, CD38+, CD117+-, CD19+, CD27-, CD81++, CD56+, LAMBDA / KAPPA>3, which is LAMBDA type MM.

[0163] Comparative Example 8

[0164] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 8. The electrophoresis result is shown in FIG8( b ).

[0165] As can be seen from the results of FIG8( b ), IgA antibodies and LAMBDA light chains were detected by immunofixation electrophoresis, and thus, it was determined to be IgA-LAMBDA type multiple myeloma. Therefore, it can be seen that the flow cytometric analysis results of Example 8 are consistent with the typing and detection results of MM by immunoelectrophoresis of Comparative Example 8.

[0166] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-8:

[0167] Table 2-8 Plasma free light chain detection results of samples in Example 8

[0168] Test items Detection value instruct Reference interval unit Free kappa 8.91 3.30-19.40 mg / L Free λ 2245 ↑ 5.71-26.3 mg / L κ / λ 0.004 ↓ 0.26-1.65

[0169] Therefore, it can be seen that the flow cytometric analysis results of Example 8 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 8, and are consistent with the plasma free light chain detection results.

[0170] Example 9

[0171] The ratio of the added amount of each antibody composition in this example is CD19-ECD 5, CD117-PC5 3, CD56-PC7 2, CD138-APC 3, CD81-APC700 3, CD38-APC750 3, CD27-PB 3, CD45-KO 3, Kappa-FITC 1, and Lambda-PE 1.

[0172] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG9 (a). The immunophenotype of the sample is: CD45+, CD138+, CD38+, CD117-, CD19+, CD27-, CD81+, CD56+, LAMBDA / KAPPA>3, which is LAMBDA type MM.

[0173] Comparative Example 9

[0174] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 9. The electrophoresis result is shown in FIG. 9( b ).

[0175] As can be seen from the results of FIG9( b ), IgA antibodies and LAMBDA light chains were detected by immunofixation electrophoresis, and thus, it was determined to be IgA-LAMBDA type multiple myeloma. Therefore, it can be seen that the flow cytometric analysis results of Example 9 are consistent with the typing and detection results of MM by immunoelectrophoresis of Comparative Example 9.

[0176] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-9:

[0177] Table 2-9 Plasma free light chain detection results of samples in Example 9

[0178] Test items Detection value instruct Reference interval unit Free kappa 7.46 3.30-19.40 mg / L Free λ 295 ↑ 5.71-26.3 mg / L κ / λ 0.03 ↓ 0.26-1.65

[0179] Therefore, it can be seen that the flow cytometric analysis results of Example 9 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 9, and are consistent with the plasma free light chain detection results.

[0180] Example 10

[0181] The ratio of the added amount of each antibody composition in this example is CD19-ECD 5, CD117-PC5 3, CD56-PC7 2, CD138-APC 3, CD81-APC700 3, CD38-APC750 5, CD27-PB 2, CD45-KO 3, Kappa-FITC 1, and Lambda-PE 1.

[0182] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG10( a ). The immunophenotype of the sample is: CD45+, CD138+, CD38+, CD117-, CD19+, CD27-, CD81+, CD56+, KAPPA / LAMBDA>3, which is KAPPA type MM.

[0183] Comparative Example 10

[0184] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 10. The electrophoresis result is shown in FIG. 10( b ).

[0185] As can be seen from the results of Figure 10 (b), IgM antibodies and KAPPA light chains were detected by immunofixation electrophoresis, and thus it was determined to be IgM-KAPPA type multiple myeloma. Therefore, it can be seen that the flow cytometric analysis results of Example 10 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 10.

[0186] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-10:

[0187] Table 2-10 Plasma free light chain detection results of samples in Example 10

[0188] Test items Detection value instruct Reference interval unit Free kappa 36.7 ↑ 3.30-19.40 mg / L Free λ 10.9 5.71-26.3 mg / L κ / λ 3.37 ↓ 0.26-1.65

[0189] Therefore, it can be seen that the flow cytometric analysis results of Example 10 are consistent with the typing and detection results of MM using immunoelectrophoresis in Comparative Example 10, and are consistent with the plasma free light chain detection results.

[0190] Embodiment 11

[0191] In this example, the ratio of the added amount of each antibody composition is CD19-ECD 5, CD117-PC5 3, CD56-PC7 2, CD138-APC 3, CD81-APC700 3, CD38-APC750 3, CD27-PB 6, CD45-KO 6, Kappa-FITC 1, and Lambda-PE 1.

[0192] This embodiment is the flow cytometric analysis result of a sample treated with the aforementioned antibody composition, as shown in FIG11( a ). The immunophenotype of the sample is: CD45+, CD138+, CD38+, CD117-, CD19+, CD27-, CD81+, CD56+, KAPPA / LAMBDA>3, which is KAPPA type MM.

[0193] Comparative Example 11

[0194] At the same time, immunofixation electrophoresis was used to perform MM typing and detection on the serum of the blood sample obtained in step 1 "sample collection and pretreatment" of Example 11. The electrophoresis result is shown in FIG. 11 (b).

[0195] At the same time, the plasma free light chain of the sample was detected by immunoturbidimetry, and the results are shown in Table 2-11:

[0196] Table 2-11 Plasma free light chain detection results of samples in Example 11

[0197] Test items Detection value instruct Reference interval unit Free kappa 9450 ↑ 3.30-19.40 mg / L Free λ 11.4 5.71-26.3 mg / L κ / λ 828.95 ↓ 0.26-1.65

[0198] As shown in the results of FIG11( b ), IgG antibodies and KAPPA light chains were detected by immunofixation electrophoresis, and thus, it was determined to be IgG-KAPPA type multiple myeloma. The electrophoresis results were consistent with the plasma free light chain detection results, and thus, it can be seen that the flow cytometric analysis results of Example 11 were consistent with the typing and detection results of MM by immunoelectrophoresis of Comparative Example 11, and were consistent with the plasma free light chain detection results.

[0199] Immunoglobulin (Ig) monomers are composed of two light chains and two heavy chains connected by disulfide bonds, wherein the light chain is divided into two types, kappa and lambda, and the normal ratio of the two in the human body is 2:1. Under normal circumstances, the human body produces about 500 mg per day, and its molecular weight is significantly smaller than that of the heavy chain, so the synthesis rate is greater than that of the heavy chain. In addition to combining with the heavy chain to form Ig, 40% of the light chains fail to combine with it and are in a free state, i.e., free light chains (FLC). In the human body, the total amount of kappa light chains is twice that of lambda light chains. Therefore, the present invention combines 8 kinds of antibody compositions such as anti-CD19 antibodies, anti-CD117 antibodies, anti-CD56 antibodies, anti-CD138 antibodies, anti-CD81 antibodies, anti-CD38 antibodies, anti-CD27 antibodies and anti-CD45 antibodies, and uses KAPPA antibodies and LAMBDA antibodies to screen and type MM after membrane permeabilization, which greatly improves the efficiency of detection, and the detection results are reliable.

[0200] In flow cytometry results, the final cell phenotype may sometimes be normal, but in fact there are monoclonal plasma cells in the sample that cannot be detected. Therefore, false negative test results often occur in clinical practice.

[0201] In the antibody composition of the present invention, by compounding CD27 and CD81, the detection rate of monoclonal plasma cells with normal CD19, CD56 and CD117 phenotypes can be significantly increased.

[0202] Example 12

[0203] As shown in FIG. 12 (a), after the sample is processed according to the aforementioned steps 1-4 and analyzed by flow cytometry, the gates are set as follows during flow cytometry analysis: first find plasma cells, use CD45 / SS INT to set gate B; continue to use CD117 / CD19 to circle gate C; use Lambda / Kappa to set gates in gate C; then continue to use CD38 / CD81 to set gates, circle gate E; use CD81 / CD27 to analyze in gate E; use CD138 / CD117 to set gates in gate E; use CD56 / CD19 to set gates in gate E to circle gate F; continue to use Lambda / Kappa to set gates in gate F for analysis.

[0204] According to the above-mentioned flow gating method, one sample was analyzed and the plasma cell immunophenotype obtained was: CD45+, CD138+, CD38+, CD117-, CD19-, CD56+, CD27+, CD81+, KAPPA / LAMBDA=2.02. Therefore, the analysis and detection conclusion of the sample was normal polyclonal plasma cells.

[0205] According to the analysis results of Figure 12 (b), in the circled E gate, the CD81-CD27- gate F was circled, and Lambda / Kappa gating analysis was continued in the F gate. The results showed that KAPPA / LAMBDA=0.07, which proved to be a LAMBDA type monoclonal plasma cell.

[0206] The sample was subjected to immunofixation electrophoresis detection, and the result is shown in FIG12(c), which also proves that IgG-lamada type M protein was found in the sample.

[0207] At the same time, the plasma free light chains of the sample were detected by immunoturbidimetry. The results are shown in Table 2-12. The detection results of the free light chains were consistent with the results of immunoelectrophoresis. This shows that the use of the antibody composition of the present invention, combined with the flow cytometric analysis method of the present invention, can effectively eliminate false negative results, thereby improving the detection accuracy.

[0208] Table 2-12 Plasma free light chain detection results of samples in Example 12

[0209] Test items Detection value instruct Reference interval unit Free kappa 6.1 3.30-19.40 mg / L Free λ 37.8 ↑ 5.71-26.3 mg / L κ / λ 0.16 ↓ 0.26-1.65

[0210] Therefore, it can be shown that the antibody composition of the present invention can effectively improve the detection accuracy of MM and effectively improve the detection rate of samples with false negative immunophenotypes.

[0211] The above implementation modes are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A reagent composition for screening and / or diagnosing plasma cell tumors, characterized in that: include: A first antibody composition, comprising: anti-CD19 antibody, anti-CD117 antibody, anti-CD56 antibody, anti-CD138 antibody, anti-CD81 antibody, anti-CD38 antibody, anti-CD27 antibody, and anti-CD45 antibody; a second antibody composition, wherein the second antibody composition comprises a KAPPA antibody and a LAMBDA antibody, The ratio of the added amount of each antibody in the first antibody composition and the second antibody composition is CD19-ECD3-8, CD117-PC5 2-5, CD56-PC7 2-5, CD138-APC 2-6, CD81-APC700 2-4, CD38-APC750 2-5, CD27-PB 2-6, CD45-KO 2-6, Kappa-FITC 1-3, Lambda-PE 1-3, The reagent composition for screening and / or diagnosing plasma cell tumors is gated according to the following steps when performing flow cytometry detection: Set the plasma cell gate A, and use the CD138 / CD45 setting to obtain most plasma cells. In gate A, CD38 / CD81 is plasma cells. Set the plasma cell gate K gate and use the CD45 / SS INT setting to obtain plasma cells; Set the plasma cell gate E, and in Gate E, use CD19 / CD38 to display B lymphocytes; Set the monoclonal plasma cell gate B and use CD56 / CD19 to display monoclonal plasma cells; Circle the CD19-positive cells in gate E. If they are normal, observe the KAPPA and LAMBDA expression ratios of plasma cells in gate B. If the proportion of abnormal monoclonal plasma cells in CD56 / CD19 is greater than 1% and the KAPPA and LAMBDA expression ratios are greater than 3, they are preliminarily judged as abnormal cells. If LAMBDA / KAPPA>3, it indicates LAMBDA-type MM abnormal cells; if KAPPA / LAMBDA>3, it indicates KAPPA-type MM abnormal cells.

2. The reagent composition for screening and / or diagnosing plasma cell tumors according to claim 1, characterized in that: The added mass ratios of the antibody composition are CD19-ECD 5, CD117-PC5 3, CD56-PC7 3, CD138-APC 3, CD81-APC700 3, CD38-APC750 3, CD27-PB 3, CD45-KO 3, Kappa-FITC 1, and Lambda-PE 1.

3. The reagent composition for screening and / or diagnosing plasma cell tumors according to claim 1, characterized in that: The clone number of the anti-CD19 antibody is J3.119, the clone number of the anti-CD117 antibody is 104D2D1, the clone number of the anti-CD56 antibody is N901, the clone number of the anti-CD138 antibody is B-A38, the clone number of the anti-CD81 antibody is JS64, the clone number of the anti-CD38 antibody is LS198-4-3, the clone number of the anti-CD27 antibody is 1A4CD27, and the clone number of the anti-CD45 antibody is J33; The clone number of the KAPPA antibody is GAHK, and the clone number of the LAMBDA antibody is GAHL.

4. A kit, characterized in that: The kit comprises the first antibody composition and the second antibody composition according to any one of claims 1 to 3, and has a first receiving chamber for receiving the first antibody composition and a second receiving chamber for receiving the second antibody composition.

5. Use of the reagent composition according to any one of claims 1 to 3 in preparing flow cytometry samples for screening and / or diagnosing plasma cell tumors.

6. The use according to claim 5, characterized in that: The process of preparing a flow cytometer sample for screening and / or diagnosing plasma cell tumors comprises the following steps: ① Collection and pretreatment of samples to be tested: Take the patient's bone marrow and peripheral blood, lyse the red blood cells, centrifuge, and discard the supernatant; ② Preparation of antibody composition reagent: dissolving the first antibody composition and the second antibody composition in a buffer solution respectively to obtain a first antibody composition reagent and a second antibody composition reagent for standby use; ③ Preparation of fixative and membrane-breaking agent: ④Sample processing: Take an appropriate amount of the sample prepared in step ①, add the first antibody composition reagent prepared in step ②, mix well, incubate at room temperature, add a fixative, and incubate in the dark; add an appropriate amount of physiological PBS, centrifuge, discard the supernatant, shake and mix to precipitate cells, add a membrane permeabilizer, and gently blow and mix; add KAPPA antibody and LAMBDA antibody, incubate in the dark; add an appropriate amount of buffer, centrifuge, discard the supernatant, add an appropriate amount of PBS to suspend, and detect on the machine.

7. The use according to claim 6, characterized in that: When performing flow cytometry testing, set the gate according to the following steps: Set the plasma cell gate A, and use the CD138 / CD45 setting to obtain most plasma cells. In gate A, CD38 / CD81 is plasma cells. Set the plasma cell gate K gate and use the CD45 / SS INT setting to obtain plasma cells; Set the plasma cell gate E, and in Gate E, use CD19 / CD38 to display B lymphocytes; Set the monoclonal plasma cell gate B and use CD56 / CD19 to display monoclonal plasma cells; Circle the CD19-positive cells in gate E. If they are normal, observe the KAPPA and LAMBDA expression ratios of plasma cells in gate B. If the proportion of abnormal monoclonal plasma cells in CD56 / CD19 is greater than 1% and the KAPPA and LAMBDA expression ratios are greater than 3, they are preliminarily judged as abnormal cells. If LAMBDA / KAPPA>3, it indicates LAMBDA-type MM abnormal cells; if KAPPA / LAMBDA>3, it indicates KAPPA-type MM abnormal cells.

8. The use according to claim 7, characterized in that: The gating analysis also includes the following steps: for samples with normal phenotypes, after using CD81 / CD27 gating analysis, continue to circle CD81-CD27- and set the F gate, continue to use Lambda / Kappa gating analysis in the F gate, and determine whether the sample is normal based on the analysis results.

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